CN117597132A

CN117597132A - Treatment of liver disease with inhibitors of ring finger protein 213 (RNF 213)

Info

Publication number: CN117597132A
Application number: CN202280032349.6A
Authority: CN
Inventors: L·A·洛塔; N·维尔韦杰; M·A·R·费雷拉; A·巴拉斯
Original assignee: Regeneron Pharmaceuticals Inc
Current assignee: Regeneron Pharmaceuticals Inc
Priority date: 2021-03-02
Filing date: 2022-03-01
Publication date: 2024-02-23

Abstract

The present disclosure provides methods of treating a subject having a liver disease, and methods of identifying a subject at increased risk of having a liver disease.

Description

Treatment of liver disease with inhibitors of ring finger protein 213 (RNF 213)

Reference to sequence Listing

The present application includes an electronically submitted sequence listing of 13,777 kilobytes in size for a text file created at 28 of 2 nd year 2022 under the name 18923805902 SEQ. The sequence listing is incorporated herein by reference.

Technical Field

The present disclosure relates generally to methods of treating a subject with liver disease with an inhibitor of ring finger protein 213 (RNF 213), and identifying a subject at increased risk of having liver disease.

Background

Chronic liver disease and cirrhosis are the leading causes of morbidity and mortality in the united states, and lead to 38,170 deaths (1.5% of total deaths) in 2014 (Kochanek et al, nat' l. In the united states, the most common causes of cirrhosis are alcoholic liver disease, chronic hepatitis c, and nonalcoholic fatty liver disease (NAFLD), which accounts for 80% of subjects awaiting liver transplantation during 2004 to 2013 (Wong et al, gastroenterology,2015,148,547-555). NAFLD has an estimated prevalence of between 19% and 46% in the United states (Browning et al, hepatology,2004,40,1387-1395; lazo et al, am. J. Epidemic, 2013,178,38-45; and Williams et al, gastroenterology,2011,140,124-131), and increases over time (Younossi et al, clin. Gastroenterol. Hepatology, 2011,9,524-530), possibly associated with an increase in obesity rate, which is a major risk factor (Cohen et al, science,2011,332,1519-1523). Although significant progress has been made in the treatment of hepatitis c, no evidence-based treatment is currently available for alcoholic or non-alcoholic liver disease and cirrhosis.

Ring finger protein 213 (RNF 213) is a protein containing a C3HC4 ring finger domain, which is a special type of zinc finger that binds two zinc atoms and is thought to be involved in mediating protein-protein interactions. The protein also contains an AAA domain that is associated with ATPase activity. RNF213 is also known as E3 ubiquitin protein ligase and is involved in a non-canonical Wnt signaling pathway in angiogenesis and vasculogenesis, wherein RNF213 functions by mediating ubiquitination and degradation of FLNA and NFATC2 downstream of RSPO3, thereby inhibiting the non-canonical Wnt signaling pathway and promoting vascular degeneration. In addition, RNF213 is a susceptibility gene for smoke disease (MMD), a cerebrovascular disease characterized by arterial occlusion and abnormal angiogenesis. In addition, RNF213 plays a role in lipid metabolism, regulating lipid toxicity, fat storage, and lipid droplet formation. RNF213 ^-/- MGI mice showed body weight and leptinDecreased food intake, increased glucose, and impaired insulin.

Disclosure of Invention

The present disclosure provides methods of treating a subject having liver disease comprising administering an RNF213 inhibitor to the subject.

The present disclosure also provides methods of treating a subject having fatty liver disease comprising administering an RNF213 inhibitor to the subject.

The disclosure also provides methods of treating a subject having hepatocellular carcinoma comprising administering an RNF213 inhibitor to the subject.

The present disclosure also provides methods of treating a subject having cirrhosis, the methods comprising administering an RNF213 inhibitor to the subject.

The disclosure also provides methods of treating a subject having liver fibrosis, the methods comprising administering an RNF213 inhibitor to the subject.

The disclosure also provides methods of treating a subject having simple steatosis, steatohepatitis, or nonalcoholic steatohepatitis (NASH), the method comprising administering an RNF213 inhibitor to the subject.

The present disclosure also provides a method of treating a subject with a therapeutic agent that treats or inhibits liver disease, wherein the subject is afflicted with liver disease, the method comprising the steps of: determining whether the subject has an RNF213 predictive loss of function or missense variant nucleic acid molecule encoding a human RNF213 polypeptide by: obtaining or having obtained a biological sample from a subject; and genotyping the biological sample to determine whether the subject has a genotype comprising an RNF 213-predictive loss of function or missense variant nucleic acid molecule; and when the subject is an RNF213 reference, then administering or continuing to administer therapeutic agents to treat or inhibit liver disease to the subject at standard dosages, and administering an RNF213 inhibitor to the subject; and when the patient is heterozygous for the RNF213 predicted loss of function or missense variant, administering to the subject or continuing to administer to the subject a therapeutic agent that treats or inhibits liver disease in an amount equal to or less than the standard dose, and administering to the subject an RNF213 inhibitor; wherein the presence of a genotype with an RNF213 predictive loss of function or missense variant nucleic acid molecule encoding a human RNF213 polypeptide indicates that the subject is at reduced risk for liver disease.

The present disclosure also provides a method of identifying a subject at increased risk of having a liver disease, wherein the method comprises: determining or having determined the presence or absence of an RNF213 predictive loss of function or missense variant nucleic acid molecule encoding a human RNF213 polypeptide in a biological sample obtained from the subject; wherein: when the subject is RNF213 reference, then the subject is at increased risk of having liver disease; and when the subject is heterozygous for the RNF213 predicted loss of function or missense variant or homozygous for the RNF213 predicted loss of function or missense variant, the subject is at reduced risk of suffering from liver disease.

The present disclosure also provides a therapeutic agent for treating or inhibiting liver disease, for treating liver disease in a subject having: a genomic nucleic acid molecule encoding a nucleotide sequence of an RNF213 polypeptide, wherein the nucleotide sequence comprises: guanine at a position corresponding to position 102,917 according to SEQ ID NO. 2 or its complement; a cytosine at a position corresponding to position 102,391 according to SEQ ID NO. 3 or a complement thereof; or thymine at a position corresponding to position 103,226 according to SEQ ID NO. 4 or a complement thereof; an mRNA molecule having a nucleotide sequence encoding a human RNF213 polypeptide, wherein the nucleotide sequence comprises: guanine at a position corresponding to position 11,887 according to SEQ ID NO. 12 or its complement; guanine at a position corresponding to position 12,036 according to SEQ ID NO. 13 or its complement; guanine at a position corresponding to position 2,685 according to SEQ ID NO. 14 or its complement; guanine at a position corresponding to position 1,050 according to SEQ ID NO. 15 or its complement; guanine at a position corresponding to position 438 according to SEQ ID NO. 16 or its complement; guanine at a position corresponding to position 112 according to SEQ ID NO. 17 or its complement; guanine at a position corresponding to position 84 according to SEQ ID NO. 18 or its complement; a cytosine at a position corresponding to position 11,655 according to SEQ ID NO. 19 or a complement thereof; a cytosine at a position corresponding to position 11,804 according to SEQ ID NO. 20 or a complement thereof; a cytosine at a position corresponding to position 2,453 according to SEQ ID NO. 21 or a complement thereof; cytosine at a position corresponding to position 818 according to SEQ ID NO. 22 or its complement; or a cytosine at a position corresponding to position 206 according to SEQ ID NO. 23 or a complement thereof; or a cDNA molecule having a nucleotide sequence encoding a human RNF213 polypeptide, wherein the nucleotide sequence comprises: guanine at a position corresponding to position 11,887 according to SEQ ID NO. 31 or its complement; guanine at a position corresponding to position 12,036 according to SEQ ID NO. 32 or its complement; guanine at a position corresponding to position 2,685 according to SEQ ID NO. 33 or its complement; guanine at a position corresponding to position 1,050 according to SEQ ID NO. 34 or its complement; guanine at a position corresponding to position 438 according to SEQ ID NO. 35 or its complement; guanine at a position corresponding to position 112 according to SEQ ID NO. 36 or its complement; guanine at a position corresponding to position 84 according to SEQ ID NO. 37 or its complement; a cytosine at a position corresponding to position 11,655 according to SEQ ID NO. 38 or a complement thereof; a cytosine at a position corresponding to position 11,804 according to SEQ ID NO. 39 or a complement thereof; a cytosine at a position corresponding to position 2,453 according to SEQ ID NO. 40 or a complement thereof; cytosine at a position corresponding to position 818 according to SEQ ID NO. 41 or its complement; or a cytosine at a position corresponding to position 206 according to SEQ ID NO. 42 or its complement.

The present disclosure also provides RNF213 inhibitors for use in treating liver disease in a subject having: a genomic nucleic acid molecule having a nucleotide sequence encoding a human ring finger protein 213 polypeptide, wherein the nucleotide sequence comprises: guanine at a position corresponding to position 102,917 according to SEQ ID NO. 2 or its complement; a cytosine at a position corresponding to position 102,391 according to SEQ ID NO. 3 or a complement thereof; or thymine at a position corresponding to position 103,226 according to SEQ ID NO. 4 or a complement thereof; an mRNA molecule having a nucleotide sequence encoding a human RNF213 polypeptide, wherein the nucleotide sequence comprises: guanine at a position corresponding to position 11,887 according to SEQ ID NO. 12 or its complement; guanine at a position corresponding to position 12,036 according to SEQ ID NO. 13 or its complement; guanine at a position corresponding to position 2,685 according to SEQ ID NO. 14 or its complement; guanine at a position corresponding to position 1,050 according to SEQ ID NO. 15 or its complement; guanine at a position corresponding to position 438 according to SEQ ID NO. 16 or its complement; guanine at a position corresponding to position 112 according to SEQ ID NO. 17 or its complement; guanine at a position corresponding to position 84 according to SEQ ID NO. 18 or its complement; a cytosine at a position corresponding to position 11,655 according to SEQ ID NO. 19 or a complement thereof; a cytosine at a position corresponding to position 11,804 according to SEQ ID NO. 20 or a complement thereof; a cytosine at a position corresponding to position 2,453 according to SEQ ID NO. 21 or a complement thereof; cytosine at a position corresponding to position 818 according to SEQ ID NO. 22 or its complement; or a cytosine at a position corresponding to position 206 according to SEQ ID NO. 23 or a complement thereof; or a cDNA molecule having a nucleotide sequence encoding a human RNF213 polypeptide, wherein the nucleotide sequence comprises: a guanine or at a position corresponding to position 11,887 of its complement according to SEQ ID NO. 31; guanine at a position corresponding to position 12,036 according to SEQ ID NO. 32 or its complement; guanine at a position corresponding to position 2,685 according to SEQ ID NO. 33 or its complement; guanine at a position corresponding to position 1,050 according to SEQ ID NO. 34 or its complement; guanine at a position corresponding to position 438 according to SEQ ID NO. 35 or its complement; guanine at a position corresponding to position 112 according to SEQ ID NO. 36 or its complement; guanine at a position corresponding to position 84 according to SEQ ID NO. 37 or its complement; a cytosine at a position corresponding to position 11,655 according to SEQ ID NO. 38 or a complement thereof; a cytosine at a position corresponding to position 11,804 according to SEQ ID NO. 39 or a complement thereof; a cytosine at a position corresponding to position 2,453 according to SEQ ID NO. 40 or a complement thereof; cytosine at a position corresponding to position 818 according to SEQ ID NO. 41 or its complement; or a cytosine at a position corresponding to position 206 according to SEQ ID NO. 42 or its complement.

Detailed Description

Various terms relating to aspects of the present disclosure are used throughout the specification and claims. Unless otherwise indicated, such terms are to be given their ordinary meaning in the art. Other specifically defined terms are to be construed in a manner consistent with the definitions provided herein.

Unless explicitly stated otherwise, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Therefore, in the claims or the specification, when a method claim does not explicitly state that the steps are limited to a particular order, it is in no way intended that the order be inferred. This applies to any possible non-expressed interpretation base including logical matters with respect to arrangement of steps or operational flow, ordinary meanings derived from grammatical organization or punctuation, or numbering or types of aspects described in the specification.

As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

As used herein, the term "about" means that the recited values are approximations, and that the minor variations do not significantly affect the practice of the disclosed embodiments. Where numerical values are used, the term "about" means that the numerical values can vary by + -10% and still be within the scope of the disclosed embodiments unless the context indicates otherwise.

As used herein, the term "comprising" may be replaced with "consisting of … …" or "consisting essentially of … …" in particular embodiments, as desired.

As used herein, the term "isolated" with respect to a nucleic acid molecule or polypeptide means that the nucleic acid molecule or polypeptide is in a condition different from its natural environment, e.g., away from blood and/or animal tissue. In some embodiments, the isolated nucleic acid molecule or polypeptide is substantially free of other nucleic acid molecules or other polypeptides, particularly other nucleic acid molecules or polypeptides of animal origin. In some embodiments, the nucleic acid molecule or polypeptide may be in a highly purified form, i.e., greater than 95% pure or greater than 99% pure. The term "isolated" as used in this context does not exclude the presence of the same nucleic acid molecule or polypeptide in alternative physical forms, such as dimers or alternatively phosphorylated or derivatized forms.

As used herein, the terms "nucleic acid," "nucleic acid molecule," "nucleic acid sequence," "polynucleotide," or "oligonucleotide" may include polymeric forms of nucleotides of any length, may include DNA and/or RNA, and may be single-stranded, double-stranded, or multi-stranded. One strand of a nucleic acid also refers to its complement.

As used herein, the term "subject" includes any animal, including mammals. Mammals include, but are not limited to, farm animals (e.g., horses, cows, pigs), companion animals (e.g., dogs, cats), laboratory animals (e.g., mice, rats, rabbits), and non-human primates. In some embodiments, the subject is a human. In some embodiments, the person is a patient under care of a doctor.

Rare variants in the RNF213 gene associated with reduced risk of a subject for liver disease have been identified in accordance with the present disclosure. For example, genetic alterations have been observed to change the adenine nucleotide at position 102,917 in the human RNF213 reference (see SEQ ID NO: 1) to guanine, or to change the guanine nucleotide at position 102,391 in the human RNF213 reference (see SEQ ID NO: 1) to cytosine, or to change the cytosine nucleotide at position 103,226 in the human RNF213 reference (see SEQ ID NO: 1) to thymine, indicating that a person with such alterations may have a reduced risk of liver disease. It is believed that neither the RNF213 gene nor the variant of the protein has any known association with liver disease. In summary, the genetic analysis described herein surprisingly shows that RNF213 genes, in particular variants in RNF213 genes, are associated with a reduced risk of developing liver disease. Thus, subjects at increased risk of developing liver disease (e.g., fatty liver disease (including Alcoholic Fatty Liver Disease (AFLD) and NAFLD), hepatocellular carcinoma, cirrhosis, liver fibrosis, simple steatosis, steatohepatitis, non-alcoholic steatohepatitis (NASH), and essential liver disease) referenced as RNF213 may be treated to prevent liver disease, reduce symptoms thereof, and/or inhibit the development of symptoms. Thus, the present disclosure provides methods for identifying or stratifying the risk of such subjects for liver disease, or diagnosing subjects as having an increased risk of liver disease, using the identification of such variants in a subject, such that a subject at risk or a subject having active disease may be treated accordingly.

It was further observed in accordance with the present disclosure that the total burden of certain variations in RNF213 is associated with a lower risk of developing liver disease (e.g., fatty liver disease (including AFLD and NAFLD), hepatocellular carcinoma, cirrhosis, liver fibrosis, simple steatosis, steatohepatitis, nonalcoholic steatohepatitis (NASH), and essential liver disease). Thus, it is believed that people with liver disease may be treated with molecules that inhibit RNF 213. Thus, the present disclosure provides methods of using the identification of such variants in a subject and the overall load with such variants to identify or stratify the risk of such subjects for liver disease, or to diagnose a subject as having liver disease, such that a subject at risk or a subject having active disease can be treated.

For purposes of this disclosure, any particular person may be classified as having one of three RNF213 genotypes: i) RNF213 reference; ii) heterozygous for RNF213 predictive loss of function or missense variants; or iii) homozygous for the RNF213 predictive loss of function or missense variant. When a person does not have a copy of an RNF 213-predictive loss of function or missense variant nucleic acid molecule, the person is an RNF213 reference. When a person has a single copy of an RNF 213-predictive loss-of-function or missense variant nucleic acid molecule, the person heterozygotes the RNF 213-predictive loss-of-function or missense variant. As used herein, an RNF 213-predictive loss-of-function variant nucleic acid molecule is any RNF213 nucleic acid molecule (e.g., genomic nucleic acid molecule, mRNA molecule, or cDNA molecule) encoding an RNF213 polypeptide having partial loss of function, complete loss of function, predictive partial loss of function, or predictive complete loss of function. Humans having an RNF213 polypeptide with partial loss of function (or predicted partial loss of function) are suballelic to RNF 213. The RNF213 predicted loss of function or missense variant nucleic acid molecule may be any nucleic acid molecule encoding RNF213 Glu3915Gly, glu3964Gly, glu822Gly, glu350Gly, glu146Gly, glu37Gly, glu28Gly, val3838Leu, val3887Leu, val745Leu, val273Leu, or Val69 Leu. In some embodiments, the RNF 213-predictive loss of function or missense variant nucleic acid molecule encodes RNF213 Glu3915Gly or Val3838Leu. When a human has two copies of an RNF213 predictive loss of function or missense variant nucleic acid molecule, the human is homozygous for the RNF213 predictive loss of function or missense variant.

For subjects genotyped or identified as RNF213 reference, such subjects have an increased risk of liver disease (e.g., fatty liver disease (including AFLD and NAFLD), hepatocellular carcinoma, cirrhosis, liver fibrosis, simple steatosis, steatohepatitis, nonalcoholic steatohepatitis (NASH), and substantial liver disease). For subjects genotyped or determined to be RNF213 reference or heterozygous for RNF213 predicted loss of function or missense variants, such subjects may be treated with an RNF213 inhibitor.

In any of the embodiments described herein, the RNF 213-predictive loss of function or missense variant nucleic acid molecule may be any RNF213 nucleic acid molecule (e.g., genomic nucleic acid molecule, mRNA molecule, or cDNA molecule) encoding an RNF213 polypeptide having partial loss of function, complete loss of function, predictive partial loss of function, or predictive complete loss of function. For example, the RNF213 predicted loss of function or missense variant nucleic acid molecule may be any nucleic acid molecule encoding RNF213Glu3915Gly, glu3964Gly, glu822Gly, glu350Gly, glu146Gly, glu37Gly, glu28Gly, val3838Leu, val3887Leu, val745Leu, val273Leu, or Val69 Leu. In some embodiments, the RNF 213-predictive loss of function or missense variant nucleic acid molecule encodes RNF213Glu3915Gly or Val3838Leu.

In any of the embodiments described herein, an RNF213 polypeptide can be any RNF213 polypeptide having partial loss of function, complete loss of function, predicted partial loss of function, or predicted complete loss of function. In any of the embodiments described herein, the RNF213 polypeptide can be any RNF213 polypeptide described herein, including, for example, RNF213 Glu3915Gly, glu3964Gly, glu822Gly, glu350Gly, glu146Gly, glu37Gly, glu28Gly, val3838Leu, val3887Leu, val745Leu, val273Leu, or Val69Leu. In some embodiments, the RNF213 polypeptide is RNF213 Glu3915Gly or Val3838Leu.

In any of the embodiments described herein, the liver disease is a fatty liver disease, including AFLD and NAFLD, hepatocellular carcinoma, cirrhosis, liver fibrosis, simple steatosis, steatohepatitis, NASH, or a substantial liver disease. In some embodiments, the liver disease is fatty liver disease. In some embodiments, the liver disease is AFLD. In some embodiments, the liver disease is NAFLD. In some embodiments, the liver disease is hepatocellular carcinoma. In some embodiments, the liver disease is cirrhosis. In some embodiments, the liver disease is liver fibrosis. In some embodiments, the liver disease is simple steatosis. In some embodiments, the liver disease is steatohepatitis. In some embodiments, the liver disease is NASH. In some embodiments, the liver disease is a substantial liver disease.

In some embodiments, the liver disease is liver injury, liver fat deposition, liver inflammation, toxic liver disease, immune liver disease, or elevated alanine Aminotransferase (ALT). In some embodiments, the liver disease is liver injury. In some embodiments, liver injury is measured by an increase in liver enzymes. In some embodiments, the liver disease is liver fat deposition. In some embodiments, liver fat deposition is identified by imaging, biopsy, or other procedure. In some embodiments, the liver disease is liver inflammation. In some embodiments, liver inflammation is identified by biopsy, imaging, or other procedures. In some embodiments, the liver disease is a toxic liver disease. In some embodiments, the liver disease is an immune liver disease. In some embodiments, the liver disease is ALT elevation. In some embodiments, liver disease is due to accumulation of metals, proteinaceous materials, bile acids, or other irritating or pro-inflammatory substances. In some embodiments, liver disease is due to accumulation of metals, such as iron. In some embodiments, liver disease is due to accumulation of proteinaceous matter, e.g., in alpha 1 antitrypsin deficiency. In some embodiments, liver disease is due to accumulation of bile acids. In some embodiments, liver disease is due to accumulation of irritants. In some embodiments, liver disease is due to accumulation of pro-inflammatory substances.

Symptoms of liver disease include, but are not limited to, hepatomegaly, fatigue, pain in the right upper abdomen, abdominal swelling (ascites), increased blood vessels under the surface of the skin, enlarged breasts in men, splenomegaly, redness of the palms, yellowing of the skin and eyes (jaundice), itching, dark urine, pale stool, nausea or vomiting, loss of appetite, and susceptibility to bruising. Liver disease detection may include blood detection, liver imaging, and liver biopsy. If a subject has at least one known risk factor (e.g., a genetic factor, such as a pathogenic mutation) such that an individual having the risk factor is at significantly higher risk of developing liver disease than an individual without the risk factor, the individual is at increased risk of developing liver disease. Risk factors for liver disease are also well known and include, for example, excessive drinking, obesity, high cholesterol, high blood triglyceride levels, polycystic ovary syndrome, sleep apnea, type 2 diabetes, hypothyroidism (hypothyroidism), hypopituitarism (hypopituitarism), and metabolic syndrome, including elevated blood lipids.

The disclosure also provides methods of treating a subject having fatty liver disease (e.g., AFLD or NAFLD) comprising administering an RNF213 inhibitor to the subject.

The present disclosure also provides methods of treating a subject having cirrhosis, the method comprising administering an RNF213 inhibitor to the subject.

The disclosure also provides methods of treating a subject having simple steatosis, steatohepatitis, or NASH, the method comprising administering an RNF213 inhibitor to the subject.

In some embodiments, the RNF213 inhibitors comprise inhibitory nucleic acid molecules. Examples of inhibitory nucleic acid molecules include, but are not limited to, antisense nucleic acid molecules, small interfering RNAs (siRNAs), and short hairpin RNAs (shRNAs). Such inhibitory nucleic acid molecules may be designed to target any region of the RNF213 nucleic acid molecule, such as an mRNA molecule. In some embodiments, the inhibitory nucleic acid molecule hybridizes to a sequence within an RNF213 genomic nucleic acid molecule or an mRNA molecule and reduces expression of an RNF213 polypeptide in a cell of the subject. In some embodiments, the RNF213 inhibitors comprise antisense RNAs that hybridize to an RNF213 genomic nucleic acid molecule or an mRNA molecule and reduce expression of an RNF213 polypeptide in a cell of a subject. In some embodiments, the RNF213 inhibitors comprise siRNA that hybridizes to an RNF213 genomic nucleic acid molecule or an mRNA molecule and reduces expression of an RNF213 polypeptide in a cell of the subject. In some embodiments, the RNF213 inhibitors comprise shRNA that hybridizes to an RNF213 genomic nucleic acid molecule or an mRNA molecule and reduces expression of an RNF213 polypeptide in a cell of a subject.

In some embodiments, the antisense nucleic acid molecule comprises or consists of any of the nucleotide sequences represented by SEQ ID NOS: 82-20602. In some embodiments, the siRNA molecule comprises or consists of any of the nucleotide sequences represented by SEQ ID NOS: 20603-64588 (sense strand and antisense strand) (e.g., sense strand is, e.g., SEQ ID NO:20603 and the corresponding antisense strand is SEQ ID NO:20604; sense strand is, e.g., SEQ ID NO:20605 and the corresponding antisense strand is SEQ ID NO:20606; etc.).

The inhibitory nucleic acid molecules disclosed herein can comprise RNA, DNA, or both RNA and DNA. The inhibitory nucleic acid molecule may also be linked or fused to a heterologous nucleic acid sequence, e.g., in a vector, or a heterologous marker. For example, an inhibitory nucleic acid molecule disclosed herein can be located within a vector or as an exogenous donor sequence comprising the inhibitory nucleic acid molecule and a heterologous nucleic acid sequence. The inhibitory nucleic acid molecule may also be linked or fused to a heterologous marker. The label may be directly detectable (e.g., a fluorophore) or indirectly detectable (e.g., a hapten, an enzyme, or a fluorophore quencher). Such labels may be detected by spectroscopic, photochemical, biochemical, immunochemical or chemical means. Such labels include, for example, radiolabels, pigments, dyes, chromogens, spin labels, and fluorescent labels. The label may also be, for example, a chemiluminescent substance; a metalliferous material; or enzymes, wherein enzyme-dependent secondary signal generation occurs. The term "label" may also refer to a "tag" or hapten that can be selectively bound to a conjugate molecule such that the conjugate molecule is used to generate a detectable signal when subsequently added with a substrate. For example, biotin may be used as a label with avidin or streptavidin conjugate of horseradish peroxide (HRP) to bind to the label and examined for the presence of HRP using a calorimetric substrate (e.g., tetramethylbenzidine (TMB)) or a fluorogenic substrate. Exemplary labels that can be used as a tag to facilitate purification include, but are not limited to myc, HA, FLAG or 3 xglag, 6XHis or polyhistidine, glutathione-S-transferase (GST), maltose binding protein, epitope tag, or Fc portion of an immunoglobulin. Many labels include, for example, particles, fluorophores, haptens, enzymes and their calorimetric, fluorescent and chemiluminescent substrates, and other labels.

The disclosed inhibitory nucleic acid molecules can comprise, for example, nucleotides or non-natural or modified nucleotides, such as nucleotide analogs or nucleotide substitutes. Such nucleotides include nucleotides containing modified base, sugar or phosphate groups, or nucleotides incorporating non-natural moieties in their structure. Examples of non-natural nucleotides include, but are not limited to, dideoxynucleotides, biotinylation, amination, deamination, alkylation, benzylation, and fluorophore-labeled nucleotides.

The inhibitory nucleic acid molecules disclosed herein may also comprise one or more nucleotide analogs or substitutions. Nucleotide analogs are nucleotides that contain modifications to the base, sugar or phosphate moiety. Modifications to the base moiety include, but are not limited to, natural and synthetic modifications of: A. c, G and T/U, as well as different purine or pyrimidine bases, such as pseudouridine, uracil-5-yl, hypoxanthine-9-yl (I) and 2-aminoadenine-9-yl. Modified bases include, but are not limited to: 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy and other 8-substituted adenine and guanine, 5-halo (e.g., 5-bromo), 5-trifluoromethyl and other 5-substituted uracil and cytosine, 7-methylguanine, 7-methyladenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaadenine and 3-deazaadenine.

Nucleotide analogs may also include modifications of the sugar moiety. Modifications to the sugar moiety include, but are not limited to, natural modifications of ribose and deoxyribose. Sugar modifications include, but are not limited to, the following modifications at the 2' position: OH; f, performing the process; o-, S-or N-alkyl; o-, S-or N-alkenyl; o-, S-or N-alkynyl; or O-alkyl-O-alkyl, wherein alkyl, alkenyl and alkynyl groups may be substituted or unsubstituted C _1-10 Alkyl or C _2-10 Alkenyl and C _2-10 Alkynyl groups. Exemplary 2' sugar modifications also include, but are not limited to, -O [ (CH) ₂ ) _n O] _m CH ₃ 、-O(CH ₂ ) _n OCH ₃ 、-O(CH ₂ ) _n NH ₂ 、-O(CH ₂ ) _n CH ₃ 、-O(CH ₂ ) _n -ONH ₂ and-O (CH) ₂ ) _n ON[(CH ₂ ) _n CH ₃ )] ₂ Wherein n and m are independently 1 to about 10. Other modifications at the 2' position include, but are not limited to, C _1-10 Alkyl, substituted lower alkyl, alkylaryl, arylalkyl, O-alkylaryl or O-arylalkyl, SH, SCH ₃ 、OCN、Cl、Br、CN、CF ₃ 、OCF ₃ 、SOCH ₃ 、SO ₂ CH ₃ 、ONO ₂ 、NO ₂ 、N ₃ 、NH ₂ Heterocyclylalkyl, heterocyclylaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleavageA group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, as well as other substituents having similar properties. Similar modifications can also be made at other positions on the sugar, particularly the 3 'position of the sugar and the 5' position of the 5 'terminal nucleotide on the 3' terminal nucleotide or 2'-5' linked oligonucleotide. Modified sugars may also include those containing modifications at the bridging epoxy, e.g., CH ₂ And S. Nucleotide sugar analogs may also have sugar mimics, such as cyclobutyl moieties in place of the pentofuranosyl sugar.

Nucleotide analogs can also be modified at the phosphate moiety. Modified phosphate moieties include, but are not limited to, modified phosphate moieties that can be modified such that the bond between two nucleotides contains the following: phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates (including 3 '-alkylene phosphonates and chiral phosphonates), phosphinates, phosphoramidates (including 3' -phosphoramidates and aminoalkyl phosphoramidates), phosphorothioates, phosphorothioate alkyl phosphonates, phosphorothioate alkyl phosphotriesters and borane phosphates. These phosphate or modified phosphate linkages between two nucleotides may be through a 3'-5' linkage or a 2'-5' linkage, and the linkages may contain reversed polarity such as 3'-5' to 5'-3' or 2'-5' to 5'-2'. Also included are various salts, mixed salts, and free acid forms. Nucleotide substitutions also include Peptide Nucleic Acids (PNAs).

In some embodiments, the antisense nucleic acid molecule is a spacer (gapmer) in which the first 1 to 7 nucleotides at the 5 'and 3' ends each have a 2 '-methoxyethyl (2' -MOE) modification. In some embodiments, the first five nucleotides of the 5' and 3' ends each have a 2' -MOE modification. In some embodiments, the first 1 to 7 nucleotides of the 5 'and 3' ends are RNA nucleotides. In some embodiments, the first five nucleotides at the 5 'and 3' ends are RNA nucleotides. In some embodiments, each backbone linkage between nucleotides is a phosphorothioate linkage.

In some embodiments, the siRNA molecule has a terminal modification. In some embodiments, the 5' end of the antisense strand is phosphorylated. In some embodiments, non-hydrolyzable 5 '-phosphate analogues, such as 5' - (E) -vinyl-phosphonate, are used.

In some embodiments, the siRNA molecule has a backbone modification. In some embodiments, modified phosphodiester groups linked to successive ribonucleosides have been shown to enhance stability and in vivo bioavailability of siRNA. The non-ester groups (-OH, =o) of the phosphodiester linkage may be substituted with sulfur, boron or acetate to give phosphorothioate, borophosphate and phosphonoacetate linkages. In addition, substitution of phosphodiester groups with phosphotriesters can promote cellular uptake of siRNA and remain on serum components by eliminating their negative charge. In some embodiments, the siRNA molecule has a sugar modification. In some embodiments, the sugar is deprotonated (a reaction catalyzed by exonucleases and endonucleases), whereby the 2' -hydroxyl group can act as a nucleophile and attack adjacent phosphorus in the phosphodiester bond. Such alternatives include 2' -O-methyl, 2' -O-methoxyethyl and 2' -fluoro modifications.

In some embodiments, the siRNA molecule has a base modification. In some embodiments, the base may be substituted with modified bases such as pseudouridine, 5' -methylcytidine, N6-methyladenosine, inosine, and N7-methylguanosine.

In some embodiments, the siRNA molecule is conjugated to a lipid. Lipids can be conjugated to the 5 'or 3' end of siRNA to increase their in vivo bioavailability by allowing them to bind to serum lipoproteins. Representative lipids include, but are not limited to, cholesterol and vitamin E, as well as fatty acids such as palmitate and tocopherol.

In some embodiments, the representative siRNA has the formula:

sense: mN 2/mN/i 2FN/mN/i2FN/mN/i2FN/mN/i2FN/mN

i2FN/*mN*/32FN/

Antisense: 52 FN/i 2FN/mN/i2FN/mN/i2FN/mN/i2FN/mN/i2FN/mN

i2FN/mN/i2FN/mN*N*N

Wherein: "N" is a base; "2F" is a 2' -F modification; "m" is a 2' -O-methyl modification and "I" is an internal base; and "×" is phosphorothioate backbone linkage.

The present disclosure also provides vectors comprising any one or more of the inhibitory nucleic acid molecules disclosed herein. In some embodiments, the vector comprises any one or more of the inhibitory nucleic acid molecules and heterologous nucleic acids disclosed herein. The vector may be a viral or non-viral vector capable of transporting the nucleic acid molecule. In some embodiments, the vector is a plasmid or cosmid (e.g., circular double stranded DNA in which segments of DNA may be ligated). In some embodiments, the vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Expression vectors include, but are not limited to, plasmids, cosmids, retroviruses, adenoviruses, adeno-associated viruses (AAV), plant viruses such as cauliflower mosaic virus and tobacco mosaic virus, yeast Artificial Chromosomes (YACs), epstein-Barr (EBV) -derived episomes, and other expression vectors known in the art.

The present disclosure also provides compositions comprising any one or more of the inhibitory nucleic acid molecules disclosed herein. In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the composition comprises a carrier and/or excipient. Examples of carriers include, but are not limited to, poly (lactic acid) (PLA) microspheres, poly (D, L-lactic-co-glycolic acid) (PLGA) microspheres, liposomes, micelles, reverse micelles, lipid helices, and lipid microtubules. The carrier may include a buffered saline solution, such as PBS, HBSS, and the like.

In some embodiments, the RNF213 inhibitors comprise a nuclease agent that induces one or more nicks or double-strand breaks at the recognition sequence or a DNA-binding protein that binds to the recognition sequence within the RNF213 genomic nucleic acid molecule. The recognition sequence may be located within the coding region of the RNF213 gene, or within regulatory regions that affect gene expression. The recognition sequence for the DNA binding protein or nuclease agent can be located in an intron, exon, promoter, enhancer, regulatory region, or any non-protein coding region. The recognition sequence may include or be near the start codon of the RNF213 gene. For example, the recognition sequence may be located about 10, about 20, about 30, about 40, about 50, about 100, about 200, about 300, about 400, about 500, or about 1,000 nucleotides from the start codon. As another example, two or more nuclease agents may be used, each of which targets a nuclease recognition sequence that includes or is near the start codon. As another example, two nuclease agents may be used, one targeting a nuclease recognition sequence comprising or near the start codon and one targeting a nuclease recognition sequence comprising or near the stop codon, wherein cleavage of the nuclease agent may result in a deletion of the coding region between the two nuclease recognition sequences. Any nuclease agent that can induce a nick or double-strand break in the desired recognition sequence can be used in the methods and compositions disclosed herein. Any DNA binding protein that binds to the desired recognition sequence can be used in the methods and compositions disclosed herein.

Suitable nuclease agents and DNA binding proteins for use herein include, but are not limited to, zinc finger proteins or Zinc Finger Nuclease (ZFN) pairs, transcription activator-like effector (TALE) proteins or transcription activator-like effector nucleases (TALENs), or Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas) systems. The length of the recognition sequences can vary and include, for example, a recognition sequence of about 30-36bp for zinc finger proteins or ZFN pairs, about 15-18bp for each ZFN, about 36bp for TALE proteins or TALENs, and about 20bp for CRISPR/Cas guide RNAs.

In some embodiments, the CRISPR/Cas system can be used to modify an RNF213 genomic nucleic acid molecule within a cell. The methods and compositions disclosed herein can employ a CRISPR-Cas system for site-directed cleavage of RNF213 nucleic acid molecules by utilizing CRISPR complexes, comprising guide RNAs (grnas) complexed with Cas proteins.

Cas proteins typically comprise at least one RNA recognition or binding domain that can interact with gRNA. Cas proteins may also comprise nuclease domains (e.g., dnase or rnase domains), DNA binding domains, helicase domains, protein-protein interaction domains, dimerization domains, and other domains. Suitable Cas proteins include, for example, wild-type Cas9 proteins and wild-type Cpf1 proteins (e.g., fnCpf 1). The Cas protein may have full cleavage activity to create a double-strand break in the RNF213 genomic nucleic acid molecule, or it may be a nickase that creates a single-strand break in the RNF213 genomic nucleic acid molecule. Additional examples of Cas proteins include, but are not limited to, cas1B, cas2, cas3, cas4, cas5e (CasD), cas6e, cas6f, cas7, cas8a1, cas8a2, cas8b, cas8c, cas9 (Csn 1 or Csx 12), cas10d, casF, casG, casH, csy1, csy2, csy3, cse1 (CasA), cse2 (CasB), cse3 (CasE), cse4 (CasC), csc1, csc2, csa5, csn2, csm3, csm4, csm5, csm6, cmr1, cmr3, cmr4, cmr5, cmr6, csb1, csb2, csb3, csx17, csx14, csx10, csx16, ax, x3, csx1, csx15, csf1, csf2, csf3, and the like modifications of these and the like. Cas proteins may also be operably linked to heterologous polypeptides as fusion proteins. For example, the Cas protein may be fused to a cleavage domain, an epigenetic modification domain, a transcriptional activation domain, or a transcriptional repression domain. Cas proteins may be provided in any form. For example, the Cas protein may be provided as a protein, such as a Cas protein complexed with a gRNA. Alternatively, the Cas protein may be provided in the form of a nucleic acid molecule encoding the Cas protein, e.g., RNA or DNA.

In some embodiments, targeted genetic modification of the RNF213 genomic nucleic acid molecule can be produced by contacting a cell with a Cas protein and one or more grnas that hybridize to one or more gRNA recognition sequences within a target genomic locus in the RNF213 genomic nucleic acid molecule. For example, the gRNA recognition sequence may be located within the region of SEQ ID NO. 1. The gRNA recognition sequence may also include or be near a position corresponding to position 102,917, position 102,391 or position 103,226 according to SEQ ID NO. 1. For example, the gRNA recognition sequence can be located about 1000, about 500, about 400, about 300, about 200, about 100, about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 15, about 10, or about 5 nucleotides from a position corresponding to position 102,917, position 102,391, or position 103,226 according to SEQ ID No. 1. The gRNA recognition sequence may include or be near the start codon of the RNF213 genomic nucleic acid molecule or the stop codon of the RNF213 genomic nucleic acid molecule. For example, the gRNA recognition sequence can be located about 10, about 20, about 30, about 40, about 50, about 100, about 200, about 300, about 400, about 500, or about 1,000 nucleotides from the start codon or the stop codon.

The gRNA recognition sequence within the target genomic locus in the RNF213 genomic nucleic acid molecule is located near the Protospacer Adjacent Motif (PAM) sequence, which is a 2-6 base pair DNA sequence immediately following the Cas9 nuclease targeted DNA sequence. The canonical PAM is the sequence 5'-NGG-3', where "N" is any nucleobase followed by two guanine ("G") nucleobases. gRNA can transport Cas9 to any location in the genome for gene editing, but editing does not occur at any site other than the site where Cas9 recognizes PAM. In addition, 5'-NGA-3' can be used as high-efficiency non-classical PAM of human cells. Typically, PAM is about 2-6 nucleotides downstream of the gRNA-targeted DNA sequence. PAM may flank the gRNA recognition sequence. In some embodiments, the gRNA recognition sequence may be flanked at the 3' end by PAM. In some embodiments, the gRNA recognition sequence may be flanked at the 5' end by PAM. For example, the cleavage site of the Cas protein may be about 1 to about 10, about 2 to about 5, or 3 base pairs upstream or downstream of the PAM sequence. In some embodiments (e.g., when Cas9 from streptococcus pyogenes(s) or closely related Cas9 is used), the PAM sequence of the non-complementary strand may be 5' -NGG-3', where N is any DNA nucleotide and immediately 3' of the gRNA recognition sequence of the non-complementary strand of the target DNA. Thus, the PAM sequence of the complementary strand will be 5' -CCN-3', where N is any DNA nucleotide and is immediately 5' of the gRNA recognition sequence of the complementary strand of the target DNA.

gRNA is an RNA molecule that binds to and targets Cas protein to a specific location within the RNF213 genomic nucleic acid molecule. An exemplary gRNA is a gRNA effective to direct Cas enzyme binding to or cleavage of an RNF213 genomic nucleic acid molecule, wherein the gRNA comprises a DNA targeting segment that hybridizes to a gRNA recognition sequence within the RNF213 genomic nucleic acid molecule that comprises or closely corresponds to: position 102,917, position 102,391 or position 103,226 according to SEQ ID NO. 1. For example, the gRNA can be selected to hybridize to a gRNA recognition sequence located about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 100, about 200, about 300, about 400, about 500, or about 1,000 nucleotides from a position corresponding to position 102,917, position 102,391, or position 103,226 according to SEQ ID NO. 1. Other exemplary grnas comprise a DNA targeting segment that hybridizes to a gRNA recognition sequence present within an RNF213 genomic nucleic acid molecule that includes or is near an initiation codon or a stop codon. For example, the gRNA can be selected to hybridize to a gRNA recognition sequence located about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 100, about 200, about 300, about 400, about 500, or about 1,000 nucleotides from the start codon or at about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 100, about 200, about 300, about 400, about 500, or about 1,000 nucleotides from the stop codon. Suitable grnas may comprise from about 17 to about 25 nucleotides, from about 17 to about 23 nucleotides, from about 18 to about 22 nucleotides, or from about 19 to about 21 nucleotides. In some embodiments, the gRNA may comprise 20 nucleotides.

Examples of suitable gRNA recognition sequences located within the human RNF213 reference gene are set forth in Table 1 as SEQ ID NOS.62-81.

Table 1: guide RNA recognition sequences near RNF213 variation

Chain	gRNA recognition sequences	SEQ ID NO:
			+	GTGGACCGATTTGCAGTACAGGG	62
-	GTGCTTTTTCCGTCCGGCAATGG	63
			+	CACGTGGTACCATTGCCGGACGG	64
-	GAATCTGTAACGGCAGATGAAGG	65
			-	TTGTTCCCGGAACGGTGAGAAGG	66
+	GGACCCTTGCTGCTACGAAAAGG	67
			+	ATCCAATTCCCCGCGGAGCATGG	68
-	GTCGCCAACCTCGGTGGGCGCGG	69
			+	CTCCACAATGGCGTCGGCCTCGG	70
-	AGGTCACGGTGAAACTCATCTGG	71
			+	AGGGATTTACTACCGGCTTCCGG	72
+	AGTCGGTAAGAATGAACAAGGGG	73
			-	TCCCGGATGACTCACCATAGAGG	74
+	CCCTTGCTGCTACGAAAAGGTGG	75
			+	TTTGCGGGGCAGGATTCCCGAGG	76
+	ACAATGGCGTCGGCCTCGGAGGG	77
			-	ACTCACTTCTTGGACGGTCCAGG	78
-	CTCCGAGGCCGACGCCATTGTGG	79
			+	CACAATGGCGTCGGCCTCGGAGG	80
+	AATTCCCCGCGGAGCATGGCTGG	81

The Cas protein and the gRNA form a complex, and the Cas protein cleaves the target RNF213 genomic nucleic acid molecule. The Cas protein may cleave the nucleic acid molecule at a site within or outside of the nucleic acid sequence present in the target RNF213 genomic nucleic acid molecule to which the DNA targeting segment of the gRNA will bind. For example, the formation of a CRISPR complex (comprising a gRNA that hybridizes to a gRNA recognition sequence and is complexed with a Cas protein) can result in cleavage of one or both strands in or near (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from) a nucleic acid sequence present in an RNF213 genomic nucleic acid molecule to which a DNA targeting segment of the gRNA is to be bound.

Such methods can produce, for example, RNF213 genomic nucleic acid molecules in which the region of SEQ ID NO. 1 is disrupted, the start codon is disrupted, the stop codon is disrupted or the coding sequence is disrupted or deleted. Optionally, the cell may be further contacted with one or more additional grnas that hybridize to additional gRNA recognition sequences within the target genomic locus in the RNF213 genomic nucleic acid molecule. Cleavage by the Cas protein may result in two or more double strand breaks or two or more single strand breaks by contacting the cell with one or more additional grnas (e.g., a second gRNA that hybridizes to a second gRNA recognition sequence).

In some embodiments, the method of treatment further comprises detecting the presence or absence of an RNF213 predictive loss of function or missense variant nucleic acid molecule encoding a human RNF213 polypeptide in a biological sample from the subject. As used throughout this disclosure, an "RNF213 predicted loss of function or missense variant nucleic acid molecule" is any RNF213 nucleic acid molecule (e.g., genomic nucleic acid molecule, mRNA molecule, or cDNA molecule) encoding an RNF213 polypeptide having partial loss of function, complete loss of function, predicted partial loss of function, or predicted complete loss of function.

The present disclosure also provides methods of treating a subject with a therapeutic agent that treats or inhibits liver disease, wherein the subject is suffering from liver disease. In some embodiments, the method comprises determining whether the subject has an RNF213 predicted loss of function or missense variant nucleic acid molecule encoding a human RNF213 polypeptide by: a biological sample from the subject is obtained or has been obtained, and a genotyping assay is performed or has been performed on the biological sample to determine whether the subject has a genotype that includes RNF213 predicted loss of function or missense variant nucleic acid molecules. When the subject is an RNF213 reference, the subject is administered or continues to be administered a therapeutic agent that treats or inhibits liver disease at a standard dose, and an RNF213 inhibitor is administered to the subject. When the subject is heterozygous for the RNF 213-predictive loss of function or missense variant, the subject is administered or continues to be administered a therapeutic agent that treats or inhibits liver disease in an amount equal to or less than the standard dose, and the RNF213 inhibitor is administered to the subject. The presence of a genotype with an RNF 213-predictive loss of function or missense variant nucleic acid molecule encoding a human RNF213 polypeptide indicates that the subject is at reduced risk for liver disease. In some embodiments, the subject is an RNF213 reference. In some embodiments, the subject is heterozygous for the RNF 213-predictive loss of function or missense variant.

In some embodiments, the method comprises determining that the subject has a plurality of RNF213 predicted loss of function or missense variant genomic nucleic acid molecules produced from the mRNA molecules, the RNF213 predicted loss of function or missense variant mRNA molecules, and/or the total load of the RNF213 predicted loss of function or missense variant cDNA molecules by: genotyping assays are performed or have been performed on biological samples obtained from subjects to determine the overall load of the subjects. When the subject has a smaller total load, the subject is at a higher risk of developing liver disease, and standard doses of therapeutic agents for treating or inhibiting liver disease are administered or continued to be administered to the subject. When the subject has a greater total load, the subject is at a lower risk of developing liver disease, and the subject is administered or continues to be administered a therapeutic agent that treats or inhibits liver disease at or below standard doses. The greater the total load, the lower the risk of liver disease.

For subjects that are genotyped or determined to be RNF213 reference or heterozygous for RNF213 predicted loss of function or missense variants, such subjects may be treated with an RNF213 inhibitor as described herein.

Detecting the presence or absence of an RNF213 predictive loss of function or missense variant nucleic acid molecule in a biological sample from a subject and/or determining whether the subject has an RNF213 predictive loss of function or missense variant nucleic acid molecule can be performed by any of the methods described herein. In some embodiments, these methods can be performed in vitro. In some embodiments, these methods may be performed in situ. In some embodiments, these methods can be performed in vivo. In any of these embodiments, the nucleic acid molecule may be present within a cell obtained from the subject.

In some embodiments, when the subject is an RNF213 reference, the therapeutic agent that treats or inhibits liver disease is also administered to the subject at a standard dose. In some embodiments, when the subject is heterozygous for an RNF213 predictive loss of function or missense variant, the subject is also administered a therapeutic agent that treats or inhibits liver disease at a dose equal to or below the standard dose.

In some embodiments, the method of treatment further comprises detecting the presence or absence of an RNF213 predictive loss of function polypeptide in a biological sample from the subject. In some embodiments, when the subject does not have an RNF213 predictive loss of function polypeptide, the subject is also administered a therapeutic agent that treats or inhibits liver disease at a standard dose. In some embodiments, when the subject has an RNF213 predicted loss of function polypeptide, the subject is also administered a therapeutic agent that treats or inhibits liver disease at a dose equal to or lower than the standard dose.

The present disclosure also provides methods of treating a subject with a therapeutic agent that treats or inhibits liver disease, wherein the subject is suffering from liver disease. In some embodiments, the method comprises determining whether the subject has an RNF213 predicted loss of function polypeptide by: a biological sample is obtained or has been obtained from the subject, and an assay is performed or has been performed on the biological sample to determine whether the subject has an RNF213 predicted loss of function polypeptide. When the subject does not have an RNF213 predictive loss of function polypeptide, the subject is administered or continues to be administered a therapeutic agent that treats or inhibits liver disease at a standard dose, and the GPAM inhibitor is administered to the subject. When the subject has an RNF 213-predictive loss of function polypeptide, the subject is administered or continues to be administered a therapeutic agent that treats or inhibits liver disease in an amount equal to or less than a standard dose, and an RNF213 inhibitor is administered to the subject. The presence of RNF213 predicted loss of function polypeptide indicates a reduced risk of the subject for liver disease. In some embodiments, the subject has an RNF213 predicted loss of function polypeptide. In some embodiments, the subject does not have an RNF213 predicted loss of function polypeptide.

Detecting the presence or absence of an RNF213 predicted loss of function polypeptide in a biological sample from a subject and/or determining whether the subject has an RNF213 predicted loss of function polypeptide may be performed by any of the methods described herein. In some embodiments, these methods can be performed in vitro. In some embodiments, these methods may be performed in situ. In some embodiments, these methods can be performed in vivo. In any of these embodiments, the polypeptide may be present within a cell obtained from the subject.

Examples of therapeutic agents that treat or inhibit liver disease include, but are not limited to: disulfiram, naltrexone, acamprosate, prednisone (Prednisone), prednisone, azathioprine, penicillamine, trientine, deferoxamine, ciprofloxacin, norfloxacin, ceftriaxone, ofloxacin, amoxicillin-clavulanic acid, menaquinone, bumetanide, furosemide, hydrochlorothiazide, chlorothiazide, amiloride (Amiloride), triamterene, spironolactone, octreotide, atenolol, metoprolol, nadolol, propranolol, timolol and Carvedilol.

Other examples of liver disease therapeutic agents (e.g., for chronic hepatitis c treatment) include, but are not limited to, ribavirin (ribavirin), peralprevir (paritaprevir), simeprevir (Olysio), glatiramir (grazoprevir), ledipasvir (ledipasvir), obetavir (ombatavir), ebaasvir (elbasvir), daclatasvir (daclatasvir) (daklina), daratavir (dasabavir), ritonavir (ritonavir), sofafrevir (sofosbuvir), valpatavir (velpatavir), fu Xirui (voxiprevir), ganciclovir (glaconaprevir), pirenzaprevir (pirentivir), peginterferon alpha-2 a, peginteralpha-2 b and interferon alpha-2 b.

Other examples of liver disease therapeutic agents (e.g., for non-alcoholic fatty liver disease) include, but are not limited to, weight loss inducers such as orlistat (orlistat) or sibutramine (sibutramine); insulin sensitizers, such as Thiazolidinediones (TZDs), metformin, and glinides; lipid lowering agents such as statins, fibrates, and omega-3 fatty acids; 23 orresponsonin, such as vitamin E, betaine, N-acetylcysteine, lecithin, silymarin and beta-carotene; anti-TNF agents, such as pentoxifylline; probiotics such as vsl#3; and cytoprotective agents, such as ursodeoxycholic acid (UDCA). Other suitable treatments include ACE inhibitors/ARBs, fructooligosaccharides and incretin analogues.

Other examples of liver disease therapeutic agents (e.g., for NASH) include, but are not limited to(obeticholic acid), span Long Se (senserertib), elafeunol (elafebriranor), cenicriviroc (Cenicriviroc), gr_md_02, mgl_3196, IMM124E, arachidyl cholic acid (arachol) ^TM ) GS0976, enlicarson (Emricasan), fuxibat (Volixibat), NGM282, GS9674, topirofem (Tropifanor), MN_001, LMB763, BI_1467335, MSDC_0602, PF_05221304, DF102, sha Luoge column bundle (Saroglitazar), BMS986036, ranilano (Lanibrator), song Ma Lutai (Semaglutide), nitazoxanide, GRI_0621, EYP001, VK2809, nalmefene (Nalmefene), LIK066, MN_3995, endocibat (Elobixibat) Namulcoson, fu Lei Lushan anti (Formalumab), SAR425899, sotagliflozin, EDP_305, ai Sabu Tet (Isosabate), jicabene (Gemcabene), TERN_101, KBP_042, PF_06865571, DUR928, PF_06835919, NGM313, BMS_986171, namacizumab, CER_209, ND_L02_s0201, RTU_1096, DRX_065, IONIS_DGAT2Rx, INT_767, NC_001, seladepar, PXL770, TERN_201, NV556 AZD2693, sp_1373, VK0214, hepastem, TGFTX4, RLBN1127, gkt_137831, RYI _018, CB4209-CB4211 and jh_0920.

In some embodiments, the dose of a therapeutic agent that treats or inhibits liver disease may be reduced by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% (i.e., less than the standard dose) for a subject or a subject heterozygous for the RNF213 predicted loss of function or missense variant as compared to the subject or a human subject (which may receive the standard dose) as a reference to RNF 213. In some embodiments, the dosage of the therapeutic agent to treat or inhibit liver disease may be reduced by about 10%, about 20%, about 30%, about 40%, or about 50%. In addition, the dosage of therapeutic agent that treats or inhibits liver disease may be administered less frequently in subjects or subjects heterozygous for the RNF213 predicted loss of function or missense variant than in subjects or subjects referred to as RNF 213.

Administration of a therapeutic agent and/or RNF213 inhibitor to treat or inhibit liver disease may be repeated after one, two, three, five, one, two, three, one, five, six, seven, eight, two or three months. Repeated administration may be the same dose or different doses. The administration may be repeated one, two, three, four, five, six, seven, eight, nine, ten or more times. For example, according to certain dosage regimens, a subject may receive a long period of therapy, e.g., 6 months, 1 year, or more.

Administration of therapeutic agents and/or RNF213 inhibitors to treat or inhibit liver disease may be by any suitable route including, but not limited to, parenteral, intravenous, oral, subcutaneous, intra-arterial, intracranial, intrathecal, intraperitoneal, topical, intranasal, or intramuscular. The pharmaceutical compositions for administration are desirably sterile and substantially isotonic and manufactured under GMP conditions. The pharmaceutical composition may be provided in unit dosage form (i.e., a single administration dose). The pharmaceutical compositions may be formulated using one or more physiologically and pharmaceutically acceptable carriers, diluents, excipients or auxiliaries. The formulation depends on the chosen route of administration. The term "pharmaceutically acceptable" means that the carrier, diluent, excipient or adjuvant is compatible with the other ingredients of the formulation and not substantially deleterious to the recipient thereof.

As used herein, the terms "treatment", "treatment" and "prevention" refer to eliciting a desired biological response, such as a therapeutic effect and a prophylactic effect, respectively. In some embodiments, after administration of the agent or a composition comprising the agent, the therapeutic effect comprises one or more of: reduction/alleviation of liver disease, reduction/alleviation of the severity of liver disease (e.g., reduction or inhibition of progression of liver disease), reduction/alleviation of symptoms and liver disease-related effects, delay of onset of symptoms and liver disease-related effects, alleviation of the severity of symptoms of liver disease-related effects, alleviation of the severity of acute episodes, diminishment of the number of symptoms and liver disease-related effects, diminishment of latency to symptoms and liver disease-related effects, diminishment of secondary symptoms, diminishment of secondary infections, prevention of recurrence of liver disease, diminishment of the number or frequency of recurrent episodes, increasing latency to symptom onset intervals, increasing duration of progression, expedited remission, induced remission, enhanced remission, accelerated recovery, or increased efficacy of alternative therapeutics or decreased resistance to alternative therapeutics, and/or increased survival time of the affected host animal. The prophylactic effect can include avoiding/inhibiting or delaying progression/progression of liver disease (e.g., avoiding/inhibiting or delaying, in whole or in part) after administration of the therapeutic regimen, as well as increasing survival time of the affected host animal. Treatment of liver disease encompasses treatment of a subject who has been diagnosed as having any form of liver disease in any clinical stage or manifestation, delay of onset or evolution or exacerbation or worsening of symptoms or signs of liver disease, and/or prevention and/or lessening of the severity of liver disease.

The present disclosure also provides methods of identifying a subject at increased risk for developing liver disease. In some embodiments, the methods comprise determining or having determined the presence or absence of an RNF213 predictive loss of function or missense variant nucleic acid molecule (e.g., genomic nucleic acid molecule, mRNA molecule, and/or cDNA molecule) encoding a human RNF213 polypeptide in a biological sample obtained from a subject. In the absence of RNF213 predictive loss of function or missense variant nucleic acid molecules (i.e., the genotype of the subject is classified as RNF213 reference) in the subject, the subject is at increased risk of developing liver disease. When the subject has an RNF 213-predictive loss of function or missense variant nucleic acid molecule (i.e., the subject is heterozygous or homozygous for the RNF 213-predictive loss of function or missense variant), then the subject is at reduced risk of suffering from liver disease.

A single copy of a nucleic acid molecule having an RNF213 predictive loss of function or a missense variant is more capable of protecting a subject from liver disease than a copy of a nucleic acid molecule not having an RNF213 predictive loss of function or a missense variant. Without being bound to any particular theory or mechanism of action, it is believed that a single copy of an RNF213 predicted loss of function or missense variant nucleic acid molecule (i.e., heterozygous for an RNF213 predicted loss of function or missense variant) is capable of protecting a subject from liver disease, and that two copies of an RNF213 predicted loss of function or missense variant nucleic acid molecule (i.e., homozygous for an RNF213 predicted loss of function or missense variant) are more capable of protecting a subject from liver disease than a subject having a single copy. Thus, in some embodiments, a single copy of an RNF 213-predictive loss of function or missense variant nucleic acid molecule may not be fully protective, but may partially or incompletely protect a subject from liver disease. While not wishing to be bound by any particular theory, there may be other factors or molecules involved in liver disease that are still present in subjects with a single copy of RNF213 predictive loss of function or missense variant nucleic acid molecules, thus resulting in less than complete protection against liver disease.

Determining whether a subject has an RNF 213-predictive loss of function or missense variant nucleic acid molecule in a biological sample from the subject and/or determining whether a subject has an RNF 213-predictive loss of function or missense variant nucleic acid molecule can be performed by any of the methods described herein. In some embodiments, these methods can be performed in vitro. In some embodiments, these methods may be performed in situ. In some embodiments, these methods can be performed in vivo. In any of these embodiments, the nucleic acid molecule may be present within a cell obtained from the subject.

The present disclosure also provides methods of identifying a subject at increased risk of having liver disease, wherein the method comprises determining or having determined that the subject has one or more RNF213 predictive loss of function or missense variant genomic nucleic acid molecules, mRNA molecules, or cDNA molecules described herein, and/or one or more RNF213 predictive loss of function or missense variant polypeptides described herein. The greater the overall load of the subject, the lower the risk of developing liver disease. The smaller the overall load of the subject, the higher the risk of developing liver disease.

In some embodiments, the method may further comprise determining that the subject has a predicted loss of function or missense variant RNF213 genomic nucleic acid molecule, an mRNA molecule, or a cDNA molecule produced from an mRNA molecule, and/or a predicted loss of function or missense variant RNF213 polypeptide associated with reduced risk of liver disease. The total load is the sum of all variants in the RNF213 gene, which can be performed in association analysis with liver disease. In some embodiments, the subject is homozygous for one or more predictive loss of function or missense variant RNF213 nucleic acid molecules associated with reduced risk of developing liver disease. In some embodiments, the subject is heterozygous for one or more predictive loss of function or missense variant RNF213 nucleic acid molecules associated with reduced risk of suffering from liver disease. The results of the association analysis indicate that loss of function and missense variation of RNF213 are associated with reduced risk of liver disease. In some embodiments, when the subject is identified as having an increased risk of developing liver disease based on its total load, the subject is further treated as described herein with a therapeutic agent and/or an RNF213 inhibitor that treats or inhibits liver disease.

In some embodiments, the total load of a subject with any one or more RNF213 predicted loss of function or missense variant nucleic acid molecules represents a weighted sum of a plurality of any predicted loss of function or missense variant nucleic acid molecules. In some embodiments, the total load is calculated using at least about 2, at least about 3, at least about 4, at least about 5, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 100, at least about 120, at least about 150, at least about 200, at least about 250, at least about 300, at least about 400, at least about 500, at least about 1,000, at least about 10,000, at least about 100,000, or at least about or more than about 1,000,000 genetic variants present in or around RNF213 (up to 10 Mb), wherein the genetic load is the number of alleles multiplied by an estimate of the association of each allele with liver disease or related outcome (e.g., weighted polygenic load score). This may include any genetic variation that approaches the RNF213 gene (up to 10Mb around the gene) that shows a non-zero association with liver-related traits in genetic association analysis, regardless of its genome annotation. In some embodiments, the subject has a lower or reduced risk of having liver disease when the subject has a total load above a desired threshold fraction. In some embodiments, the subject is at a higher or increased risk of having liver disease when the subject has a total load below a desired threshold fraction.

In some embodiments, the total load may be divided into five quantiles, such as a highest five quantile, a middle five quantile, and a lowest five quantile, where the highest five quantile of the total load corresponds to the lowest risk group and the lowest five quantile of the total load corresponds to the highest risk group. In some embodiments, the subject with the greater total load includes the highest weighted total load, including but not limited to the first 10%, the first 20%, the first 30%, the first 40% or the first 50% of the total load of the population of subjects. In some embodiments, the genetic variants include genetic variants associated with liver disease in the first 10%, first 20%, first 30%, first 40% or first 50% of the range of relevant p-values. In some embodiments, each identified genetic variant comprises a genetic variant associated with liver disease having a p-value of no greater than about 10 ^-2 About 10 ^-3 About 10 ^-4 About 10 ^-5 About 10 ^-6 About 10 ^-7 About 10 ^-8 About 10 ^-9 About 10 ^-10 About 10 ^-11 About 10 ^-12 About 10 ^-13 About 10 ^-14 Or about 10 ^-15 . In some embodiments, the identified genetic variants include p-values less than 5×10 ^-8 Is a genetic variant associated with liver disease. In some embodimentsThe identified genetic variants include genetic variants associated with liver disease in high risk subjects having the following ratio (OR) as compared to the remainder of the reference population: about 1.5 or greater, about 1.75 or greater, about 2.0 or greater, or about 2.25 or greater for the first 20% of distribution; or about 1.5 or greater, about 1.75 or greater, about 2.0 or greater, about 2.25 or greater, about 2.5 or greater, or about 2.75 or greater. In some embodiments, the ratio (OR) may range from about 1.0 to about 1.5, from about 1.5 to about 2.0, from about 2.0 to about 2.5, from about 2.5 to about 3.0, from about 3.0 to about 3.5, from about 3.5 to about 4.0, from about 4.0 to about 4.5, from about 4.5 to about 5.0, from about 5.0 to about 5.5, from about 5.5 to about 6.0, from about 6.0 to about 6.5, from about 6.5 to about 7.0, OR greater than 7.0. In some embodiments, the high risk subjects include subjects with a total load of the lowest ten, five, or three digits in the reference population. The threshold value of the total load is determined according to the nature of the intended actual application and the risk differences that will be considered significant for said actual application.

In some embodiments, when the subject is identified as having an increased risk of having a liver disease, the subject is further treated as described herein with a therapeutic agent that treats or inhibits liver disease and/or an RNF213 inhibitor. For example, when the subject is an RNF213 reference, and thus has an increased risk of developing liver disease, an RNF213 inhibitor is administered to the subject. In some embodiments, a therapeutic agent that treats or inhibits liver disease is also administered to such subjects. In some embodiments, when the subject is heterozygous for a RNF 213-predictive loss of function or missense variant, the subject is administered a therapeutic agent that treats or inhibits liver disease at a dose equal to or below the standard dose, and an RNF213 inhibitor is also administered thereto. In some embodiments, the subject is an RNF213 reference. In some embodiments, the subject is heterozygous for the RNF 213-predictive loss of function or missense variant. Furthermore, when the subject has a lower overall load of RNF 213-predictive loss of function or missense variant nucleic acid molecules and thus has an increased risk of liver disease, a therapeutic agent for treating or inhibiting liver disease is administered to the subject. In some embodiments, when the total load of a subject with RNF 213-predictive loss of function or missense variant nucleic acid molecule is low, the therapeutic agent for treating or inhibiting liver disease is administered to the subject at a dose equal to or greater than the standard dose administered to subjects with RNF 213-predictive loss of function or missense variant nucleic acid molecule that are higher in total load.

The disclosure also provides methods of detecting the presence or absence of an RNF 213-predictive loss of function or missense variant genomic nucleic acid molecule in a biological sample from a subject, and/or the presence or absence of an RNF 213-predictive loss of function or missense variant mRNA molecule in a biological sample from a subject, and/or the presence or absence of an RNF 213-predictive loss of function or missense variant cDNA molecule produced from an mRNA molecule in a biological sample from a subject. It will be appreciated that the sequence of genes within a population and the mRNA molecules encoded by such genes may vary due to polymorphisms (e.g., single nucleotide polymorphisms). The sequences of RNF213 variant genomic nucleic acid molecules, RNF213 variant mRNA molecules and RNF213 variant cDNA molecules provided herein are merely exemplary sequences. Other sequences of RNF213 variant genomic nucleic acid molecules, variant mRNA molecules and variant cDNA molecules are also possible.

The biological sample may be derived from any cell, tissue or biological fluid from the subject. Biological samples may include any clinically relevant tissue, such as bone marrow samples, tumor biopsies, fine needle aspirates, or body fluid samples, such as blood, gingival crevicular fluid, plasma, serum, lymph fluid, ascites fluid, cyst fluid, or urine. In some cases, the sample comprises an oral swab. The biological samples used in the methods disclosed herein may vary based on the assay format, the nature of the detection method, and the tissue, cells, or extract used as the sample. Biological samples may be processed differently depending on the assay employed. For example, when detecting any RNF213 variant nucleic acid molecules, a preliminary treatment designed to isolate or enrich a biological sample for genomic DNA may be employed. A variety of techniques may be used for this purpose. When detecting the level of any RNF213 variant mRNA molecule, different techniques can be used to enrich the biological sample for mRNA molecules. Various methods of detecting the presence or level of an mRNA molecule or the presence of a particular variant genomic DNA locus can be used.

In some embodiments, detecting a human RNF213 predicted loss of function or missense variant nucleic acid molecule in a subject comprises assaying or genotyping a biological sample obtained from the subject to determine whether the RNF213 genomic nucleic acid molecule in the biological sample and/or the RNF213 mRNA molecule in the biological sample and/or the RNF213 cDNA molecule produced by the mRNA molecule in the biological sample comprises one or more variations that result in loss of function (partial or complete) or are predicted to result in loss of function (partial or complete).

In some embodiments, a method of detecting the presence or absence of an RNF213 predictive loss of function or missense variant nucleic acid molecule (e.g., a genomic nucleic acid molecule, an mRNA molecule, and/or a cDNA molecule produced from an mRNA molecule) in a subject comprises assaying a biological sample obtained from the subject. The assay determines whether a nucleic acid molecule in a biological sample comprises a particular nucleotide sequence.

In some embodiments, the nucleotide sequence comprises: guanine (for genomic nucleic acid molecules) at a position corresponding to position 102,917 according to SEQ ID NO. 2; guanine at a position corresponding to: position 11,887 according to SEQ ID NO. 12, position 12,036 according to SEQ ID NO. 13, position 2,685 according to SEQ ID NO. 14, position 1,050 according to SEQ ID NO. 15, position 438 according to SEQ ID NO. 16, position 112 according to SEQ ID NO. 17 or position 84 (for mRNA molecules) according to SEQ ID NO. 18; or guanine at a position corresponding to: position 11,887 according to SEQ ID NO. 31, position 12,036 according to SEQ ID NO. 32, position 2,685 according to SEQ ID NO. 33, position 1,050 according to SEQ ID NO. 34, position 438 according to SEQ ID NO. 35, position 112 according to SEQ ID NO. 36 or position 84 (for cDNA molecules) according to SEQ ID NO. 37.

In some embodiments, the nucleotide sequence comprises: cytosine (for genomic nucleic acid molecules) at a position corresponding to position 102,391 according to SEQ ID NO. 3; cytosine at a position corresponding to: position 11,655 according to SEQ ID NO. 19, position 11,804 according to SEQ ID NO. 20, position 2,453 according to SEQ ID NO. 21, position 818 according to SEQ ID NO. 22, or position 206 (for mRNA molecules) according to SEQ ID NO. 23; cytosine at a position corresponding to: position 11,655 according to SEQ ID NO. 38, position 11,804 according to SEQ ID NO. 39, position 2,453 according to SEQ ID NO. 40, position 818 according to SEQ ID NO. 41 or position 206 according to SEQ ID NO. 42 (for CDNA molecules).

In some embodiments, the nucleotide sequence comprises thymine (for genomic nucleic acid molecules) at a position corresponding to position 103,226 according to SEQ ID NO. 4.

In some embodiments, the nucleotide sequence comprises: guanine at a position corresponding to position 102,917 according to SEQ ID NO. 2 or its complement; a cytosine at a position corresponding to position 102,391 according to SEQ ID NO. 3 or a complement thereof; or thymine at a position corresponding to position 103,226 according to SEQ ID NO. 4 or a complement thereof.

In some embodiments, the nucleotide sequence comprises: guanine at a position corresponding to position 11,887 according to SEQ ID NO. 12 or its complement; guanine at a position corresponding to position 12,036 according to SEQ ID NO. 13 or its complement; guanine at a position corresponding to position 2,685 according to SEQ ID NO. 14 or its complement; guanine at a position corresponding to position 1,050 according to SEQ ID NO. 15 or its complement; guanine at a position corresponding to position 438 according to SEQ ID NO. 16 or its complement; guanine at a position corresponding to position 112 according to SEQ ID NO. 17 or its complement; guanine at a position corresponding to position 84 according to SEQ ID NO. 18 or its complement; a cytosine at a position corresponding to position 11,655 according to SEQ ID NO. 19 or a complement thereof; a cytosine at a position corresponding to position 11,804 according to SEQ ID NO. 20 or a complement thereof; a cytosine at a position corresponding to position 2,453 according to SEQ ID NO. 21 or a complement thereof; cytosine at a position corresponding to position 818 according to SEQ ID NO. 22 or its complement; or a cytosine at a position corresponding to position 206 according to SEQ ID NO. 23 or its complement.

In some embodiments, the nucleotide sequence comprises: guanine at a position corresponding to position 11,887 according to SEQ ID NO. 31 or its complement; guanine at a position corresponding to position 12,036 according to SEQ ID NO. 32 or its complement; guanine at a position corresponding to position 2,685 according to SEQ ID NO. 33 or its complement; guanine at a position corresponding to position 1,050 according to SEQ ID NO. 34 or its complement; guanine at a position corresponding to position 438 according to SEQ ID NO. 35 or its complement; guanine at a position corresponding to position 112 according to SEQ ID NO. 36 or its complement; guanine at a position corresponding to position 84 according to SEQ ID NO. 37 or its complement; a cytosine at a position corresponding to position 11,655 according to SEQ ID NO. 38 or a complement thereof; a cytosine at a position corresponding to position 11,804 according to SEQ ID NO. 39 or a complement thereof; a cytosine at a position corresponding to position 2,453 according to SEQ ID NO. 40 or a complement thereof; cytosine at a position corresponding to position 818 according to SEQ ID NO. 41 or its complement; or a cytosine at a position corresponding to position 206 according to SEQ ID NO. 42 or its complement.

In some embodiments, the biological sample comprises cells or cell lysates. Such methods may also include, for example, obtaining a biological sample from the subject comprising an RNF213 genomic nucleic acid molecule or an mRNA molecule, and if comprising mRNA, optionally reverse transcribing the mRNA into cDNA. Such assays may include, for example, determining identity of these positions of a particular RNF213 nucleic acid molecule. In some embodiments, the method is an in vitro method.

In some embodiments, the determining step, detecting step, or genotyping assay comprises sequencing at least a portion of the nucleotide sequence of the RNF213 genomic nucleic acid molecule, the RNF213 mRNA molecule, or the RNF213cDNA molecule in the biological sample, wherein the sequenced portion comprises one or more variations that result in loss of function (partial or complete) or are predicted to result in loss of function (partial or complete).

In some embodiments, the determining step, detecting step, or genotyping assay comprises sequencing at least a portion of: a nucleotide sequence of an RNF213 genomic nucleic acid molecule in a biological sample, wherein the sequencing portion comprises a position corresponding to position 102,917 according to SEQ ID No. 2 or the complement thereof; a nucleotide sequence of an RNF213 mRNA molecule in a biological sample, wherein the sequencing portion comprises positions corresponding to: position 11,887 according to SEQ ID NO. 12 or a complement thereof; position 12,036 of SEQ ID NO. 13 or its complement; position 2,685 according to SEQ ID NO. 14 or a complement thereof; position 1,050 according to SEQ ID NO. 15 or its complement; position 438 according to SEQ ID NO. 16 or a complement thereof; position 112 according to SEQ ID NO. 17 or a complement thereof; or position 84 according to SEQ ID NO. 18 or a complement thereof; and/or a nucleotide sequence of an RNF213cDNA molecule produced from mRNA in a biological sample, wherein the sequenced portion comprises positions corresponding to: position 11,887 according to SEQ ID NO. 31 or a complement thereof; position 12,036 according to SEQ ID NO. 32 or a complement thereof; position 2,685 according to SEQ ID NO. 33 or a complement thereof; position 1,050 according to SEQ ID NO. 34 or its complement; position 438 according to SEQ ID NO. 35 or its complement; position 112 according to SEQ ID NO. 36 or a complement thereof; or position 84 according to SEQ ID NO. 37 or its complement. When the sequenced portion of the RNF213 nucleic acid molecule in the biological sample comprises: guanine at a position corresponding to position 102,917 according to SEQ ID NO. 2; guanine at a position corresponding to: position 11,887 according to SEQ ID NO. 12, position 12,036 according to SEQ ID NO. 13, position 2,685 according to SEQ ID NO. 14, position 1,050 according to SEQ ID NO. 15, position 438 according to SEQ ID NO. 16, position 112 according to SEQ ID NO. 17, or position 84 according to SEQ ID NO. 18; or guanine at a position corresponding to: when position 11,887 according to SEQ ID NO. 31, position 12,036 according to SEQ ID NO. 32, position 2,685 according to SEQ ID NO. 33, position 1,050 according to SEQ ID NO. 34, position 438 according to SEQ ID NO. 35, position 112 according to SEQ ID NO. 36 or position 84 according to SEQ ID NO. 37, then the RNF213 nucleic acid molecule in the biological sample is an RNF213 predicted loss of function or missense variant nucleic acid molecule.

In some embodiments, the determining step, detecting step, or genotyping assay comprises sequencing at least a portion of the nucleotide sequence of the RNF213 genomic nucleic acid molecule in the biological sample, wherein the sequenced portion comprises a position corresponding to position 102,391 according to SEQ ID No. 3 or the complement thereof; a nucleotide sequence of an RNF213 mRNA molecule in a biological sample, wherein the sequencing portion comprises positions corresponding to: position 11,655 according to SEQ ID NO. 19 or a complement thereof; position 11,804 according to SEQ ID NO. 20 or a complement thereof; position 2,453 according to SEQ ID NO. 21 or a complement thereof; position 818 according to SEQ ID NO. 22 or a complement thereof; or position 206 according to SEQ ID NO. 23 or a complement thereof; and/or a nucleotide sequence of an RNF213 cDNA molecule produced from mRNA in a biological sample, wherein the sequenced portion comprises positions corresponding to: position 11,655 according to SEQ ID NO. 38 or a complement thereof; position 11,804 according to SEQ ID NO. 39 or a complement thereof; position 2,453 according to SEQ ID NO. 40 or a complement thereof; position 818 according to SEQ ID NO. 41 or a complement thereof; or position 206 according to SEQ ID NO. 42 or its complement. When the sequenced portion of the RNF213 nucleic acid molecule in the biological sample comprises: cytosine at a position corresponding to position 102,391 of SEQ ID NO. 3; cytosine at a position corresponding to: position 11,655 according to SEQ ID NO. 19, position 11,804 according to SEQ ID NO. 20, position 2,453 according to SEQ ID NO. 21, position 818 according to SEQ ID NO. 22 or position 206 according to SEQ ID NO. 23; or cytosine at a position corresponding to: when position 11,655 according to SEQ ID NO. 38, position 11,804 according to SEQ ID NO. 39, position 2,453 according to SEQ ID NO. 40, position 818 according to SEQ ID NO. 41 or position 206 according to SEQ ID NO. 42, then the RNF213 nucleic acid molecule in the biological sample is an RNF213 predicted loss of function or missense variant nucleic acid molecule.

In some embodiments, the determining step, detecting step, or genotyping assay comprises sequencing at least a portion of the nucleotide sequence of the RNF213 genomic nucleic acid molecule in the biological sample, wherein the sequenced portion comprises a position corresponding to position 103,226 according to SEQ ID No. 4 or the complement thereof. When the sequenced portion of the RNF213 nucleic acid molecule in the biological sample comprises thymine at a position corresponding to position 103,226 according to SEQ ID NO. 4, then the RNF213 nucleic acid molecule in the biological sample is an RNF213 predicted loss of function or missense variant nucleic acid molecule.

In some embodiments, the determining step, detecting step, or genotyping assay comprises sequencing at least a portion of the nucleotide sequence of the RNF213 genomic nucleic acid molecule in the biological sample, wherein the sequenced portion comprises a position corresponding to: position 102,917 according to SEQ ID NO. 2 or a complement thereof; position 102,391 according to SEQ ID NO. 3 or a complement thereof; or according to position 103,226 of SEQ ID NO. 4. When the sequenced portion of the RNF213 nucleic acid molecule in the biological sample comprises: when the guanine at a position corresponding to position 102,917 according to SEQ ID NO. 2, the cytosine at a position corresponding to position 102,391 according to SEQ ID NO. 3, or the thymine at a position corresponding to position 103,226 according to SEQ ID NO. 4, then the RNF213 nucleic acid molecule in the biological sample is an RNF213 predicted loss of function or missense variant nucleic acid molecule.

In some embodiments, the determining step, detecting step, or genotyping assay comprises sequencing at least a portion of the nucleotide sequence of the RNF213 mRNA molecule in the biological sample, wherein the sequenced portion comprises positions corresponding to: position 11,887 according to SEQ ID NO. 12 or its complement; position 12,036 according to SEQ ID NO. 13 or a complement thereof; position 2,685 according to SEQ ID NO. 14 or a complement thereof; position 1,050 according to SEQ ID NO. 15 or its complement; position 438 according to SEQ ID NO. 16 or a complement thereof; position 112 according to SEQ ID NO. 17 or a complement thereof; position 84 according to SEQ ID NO. 18 or a complement thereof; position 11,655 according to SEQ ID NO. 19 or a complement thereof; position 11,804 according to SEQ ID NO. 20 or a complement thereof; position 2,453 according to SEQ ID NO. 21 or a complement thereof; position 818 according to SEQ ID NO. 22 or a complement thereof; or position 206 according to SEQ ID NO. 23 or a complement thereof. When the sequenced portion of the RNF213 nucleic acid molecule in the biological sample comprises: guanine at a position corresponding to position 11,887 according to SEQ ID NO. 12; guanine at a position corresponding to position 12,036 according to SEQ ID NO. 13; guanine at a position corresponding to position 2,685 according to SEQ ID NO. 14; guanine at a position corresponding to position 1,050 according to SEQ ID NO. 15; guanine at a position corresponding to position 438 according to SEQ ID NO. 16; guanine at a position corresponding to position 112 according to SEQ ID NO. 17; guanine at a position corresponding to position 84 according to SEQ ID NO. 18; cytosine at a position corresponding to position 11,655 according to SEQ ID NO. 19; cytosine at a position corresponding to position 11,804 according to SEQ ID NO. 20; cytosine at a position corresponding to position 2,453 according to SEQ ID NO. 21; cytosine at a position corresponding to position 818 according to SEQ ID NO. 22; or a cytosine at a position corresponding to position 206 according to SEQ ID NO. 23; the RNF213 nucleic acid molecule in the biological sample is an RNF213 predicted loss of function or missense variant nucleic acid molecule.

In some embodiments, the determining step, detecting step, or genotyping assay comprises sequencing at least a portion of the nucleotide sequence of an RNF213 cDNA molecule produced from an mRNA molecule in the biological sample, wherein the sequenced portion comprises positions corresponding to: position 11,887 according to SEQ ID NO. 31 or a complement thereof; position 12,036 according to SEQ ID NO. 32 or a complement thereof; position 2,685 according to SEQ ID NO. 33 or a complement thereof; position 1,050 according to SEQ ID NO. 34 or its complement; position 438 according to SEQ ID NO. 35 or its complement; position 112 according to SEQ ID NO. 36 or a complement thereof; position 84 according to SEQ ID NO. 37 or a complement thereof; position 11,655 according to SEQ ID NO. 38 or a complement thereof; position 11,804 according to SEQ ID NO. 39 or a complement thereof; position 2,453 according to SEQ ID NO. 40 or a complement thereof; position 818 according to SEQ ID NO. 41 or a complement thereof; or position 206 according to SEQ ID NO. 42 or its complement. When the sequenced portion of the RNF213 nucleic acid molecule in the biological sample comprises: guanine at a position corresponding to position 11,887 according to SEQ ID NO. 31; guanine at a position corresponding to position 12,036 according to SEQ ID NO. 32; guanine at a position corresponding to position 2,685 according to SEQ ID NO. 33; guanine at a position corresponding to position 1,050 according to SEQ ID NO. 34; guanine at a position corresponding to position 438 according to SEQ ID NO. 35; guanine at a position corresponding to position 112 according to SEQ ID NO. 36; guanine at a position corresponding to position 84 according to SEQ ID NO. 37; cytosine at a position corresponding to position 11,655 according to SEQ ID NO. 38; cytosine at a position corresponding to position 11,804 according to SEQ ID NO. 39; cytosine at a position corresponding to position 2,453 according to SEQ ID NO. 40; cytosine at a position corresponding to position 818 according to SEQ ID NO. 41; or a cytosine at a position corresponding to position 206 according to SEQ ID NO. 42; the RNF213 nucleic acid molecule in the biological sample is an RNF213 predicted loss of function or missense variant nucleic acid molecule.

In some embodiments, the determining step, the detecting step, or the genotyping assay comprises: a) Contacting the biological sample with a primer that hybridizes to a portion of a nucleotide sequence of a genomic nucleic acid molecule of RNF 213: a genomic nucleic acid molecule in proximity to a position corresponding to position 102,917 according to SEQ ID NO. 2; an mRNA molecule that closely corresponds to the position: position 11,887 according to SEQ ID NO. 12, position 12,036 according to SEQ ID NO. 13, position 2,685 according to SEQ ID NO. 14, position 1,050 according to SEQ ID NO. 15, position 438 according to SEQ ID NO. 16, position 112 according to SEQ ID NO. 17 or position 84 according to SEQ ID NO. 18; and/or a cDNA molecule that approximates the position corresponding to: position 11,887 according to SEQ ID NO. 31, position 12,036 according to SEQ ID NO. 32, position 2,685 according to SEQ ID NO. 33, position 1,050 according to SEQ ID NO. 34, position 438 according to SEQ ID NO. 35, position 112 according to SEQ ID NO. 36 or position 84 according to SEQ ID NO. 37; b) Extending the primer at least through the following positions of the nucleotide sequence of RNF 213: a genomic nucleic acid molecule corresponding to position 102,917 according to SEQ ID NO. 2; an mRNA molecule corresponding to: position 11,887 according to SEQ ID NO. 12, position 12,036 according to SEQ ID NO. 13, position 2,685 according to SEQ ID NO. 14, position 1,050 according to SEQ ID NO. 15, position 438 according to SEQ ID NO. 16, position 112 according to SEQ ID NO. 17 or position 84 according to SEQ ID NO. 18; and/or a cDNA molecule corresponding to: position 11,887 according to SEQ ID NO. 31, position 12,036 according to SEQ ID NO. 32, position 2,685 according to SEQ ID NO. 33, position 1,050 according to SEQ ID NO. 34, position 438 according to SEQ ID NO. 35, position 112 according to SEQ ID NO. 36 or position 84 according to SEQ ID NO. 37; and c) determining whether the extension product of the primer comprises a guanine at a position corresponding to: position 102,917 according to SEQ ID NO. 2, position 11,887 according to SEQ ID NO. 12, position 12,036 according to SEQ ID NO. 13, position 2,685 according to SEQ ID NO. 14, position 1,050 according to SEQ ID NO. 15, position 438 according to SEQ ID NO. 16, position 112 according to SEQ ID NO. 17 or position 84 according to SEQ ID NO. 18; or guanine at a position corresponding to: position 11,887 according to SEQ ID NO. 31, position 12,036 according to SEQ ID NO. 32, position 2,685 according to SEQ ID NO. 33, position 1,050 according to SEQ ID NO. 34, position 438 according to SEQ ID NO. 35, position 112 according to SEQ ID NO. 36 or position 84 according to SEQ ID NO. 37.

In some embodiments, the determining step, the detecting step, or the genotyping assay comprises: a) Contacting the biological sample with a primer that hybridizes to a portion of the nucleotide sequence of RNF 213: a genomic nucleic acid molecule in proximity to a position corresponding to position 102,391 according to SEQ ID NO. 3; an mRNA molecule that closely corresponds to the position: position 11,655 according to SEQ ID NO. 19, position 11,804 according to SEQ ID NO. 20, position 2,453 according to SEQ ID NO. 21, position 818 according to SEQ ID NO. 22 or position 206 according to SEQ ID NO. 23; and/or a cDNA molecule that approximates the position corresponding to: position 11,655 according to SEQ ID NO. 38, position 11,804 according to SEQ ID NO. 39, position 2,453 according to SEQ ID NO. 40, position 818 according to SEQ ID NO. 41 or position 206 according to SEQ ID NO. 42; b) Extending the primer at least through the following positions of the nucleotide sequence of RNF 213: a genomic nucleic acid molecule corresponding to position 102,391 according to SEQ ID NO. 3; an mRNA molecule corresponding to: position 11,655 according to SEQ ID NO. 19, position 11,804 according to SEQ ID NO. 20, position 2,453 according to SEQ ID NO. 21, position 818 according to SEQ ID NO. 22 or position 206 according to SEQ ID NO. 23; and/or a cDNA molecule corresponding to: position 11,655 according to SEQ ID NO. 38, position 11,804 according to SEQ ID NO. 39, position 2,453 according to SEQ ID NO. 40, position 818 according to SEQ ID NO. 41 or position 206 according to SEQ ID NO. 42; and c) determining whether the extension product of the primer comprises: cytosine at a position corresponding to position 102,391 according to SEQ ID NO. 3; cytosine at a position corresponding to position 11,655 according to SEQ ID NO. 19, cytosine at a position corresponding to position 11,804 according to SEQ ID NO. 20, cytosine at a position corresponding to position 2,453 according to SEQ ID NO. 21, cytosine at a position corresponding to position 818 according to SEQ ID NO. 22 or cytosine at a position corresponding to position 206 according to SEQ ID NO. 23; or corresponds to the cytosine at position 11,655 according to SEQ ID NO:38, to the cytosine at position 11,804 according to SEQ ID NO:39, to the cytosine at position 2,453 according to SEQ ID NO:40, to the cytosine at position 818 according to SEQ ID NO:41, to the cytosine at position 206 according to SEQ ID NO: 42.

In some embodiments, the determining step, the detecting step, or the genotyping assay comprises: a) Contacting the biological sample with a primer that hybridizes to a portion of the nucleotide sequence of the RNF213 genomic nucleic acid molecule that is proximal to a position corresponding to position 103,226 according to SEQ ID No. 4; b) Extending the primer at least through a position in the nucleotide sequence of the RNF213 genomic nucleic acid molecule corresponding to position 103,226 according to SEQ ID No. 4; and c) determining whether the extension product of the primer comprises thymine at a position corresponding to position 103,226 of SEQ ID NO. 4.

In some embodiments, the determining step, the detecting step, or the genotyping assay comprises: a) Contacting the biological sample with a primer that hybridizes to a portion of the nucleotide sequence of the RNF213 genomic nucleic acid molecule that closely corresponds to: position 102,917 according to SEQ ID NO. 2, position 102,391 according to SEQ ID NO. 3 or position 103,226 according to SEQ ID NO. 4; b) Extending the primer at least through a position in the nucleotide sequence of the RNF213 genomic nucleic acid molecule corresponding to: position 102,917 according to SEQ ID NO. 2, position 102,391 according to SEQ ID NO. 3 or position 103,226 according to SEQ ID NO. 4; and c) determining whether the extension product of the primer comprises: guanine at a position corresponding to position 102,917 according to SEQ ID NO. 2, cytosine at a position corresponding to position 102,391 according to SEQ ID NO. 3 or thymine at a position corresponding to position 103,226 according to SEQ ID NO. 4.

In some embodiments, the determining step, the detecting step, or the genotyping assay comprises: a) Contacting the biological sample with a primer that hybridizes to a portion of the nucleotide sequence of the RNF213 mRNA molecule near a position corresponding to: position 11,887 according to SEQ ID NO. 12, position 12,036 according to SEQ ID NO. 13, position 2,685 according to SEQ ID NO. 14, position 1,050 according to SEQ ID NO. 15, position 438 according to SEQ ID NO. 16, position 112 according to SEQ ID NO. 17, or position 84 according to SEQ ID NO. 18, position 11,655 according to SEQ ID NO. 19, position 11,804 according to SEQ ID NO. 20, position 2,453 according to SEQ ID NO. 21, position 818 according to SEQ ID NO. 22, or position 206 according to SEQ ID NO. 23. b) Extending a primer at least through a position in the nucleotide sequence of the RNF213 mRNA molecule corresponding to: position 11,887 according to SEQ ID NO. 12, position 12,036 according to SEQ ID NO. 13, position 2,685 according to SEQ ID NO. 14, position 1,050 according to SEQ ID NO. 15, position 438 according to SEQ ID NO. 16, position 112 according to SEQ ID NO. 17, or position 84 according to SEQ ID NO. 18, position 11,655 according to SEQ ID NO. 19, position 11,804 according to SEQ ID NO. 20, position 2,453 according to SEQ ID NO. 21, position 818 according to SEQ ID NO. 22, or position 206 according to SEQ ID NO. 23. And c) determining whether the extension product of the primer comprises: guanine at a position corresponding to position 11,887 according to SEQ ID NO. 12, guanine at a position 12,036 according to SEQ ID NO. 13, guanine at a position 2,685 according to SEQ ID NO. 14, guanine at a position 1,050 according to SEQ ID NO. 15, guanine at a position 438 according to SEQ ID NO. 16, guanine at a position 112 according to SEQ ID NO. 17, guanine at a position 84 according to SEQ ID NO. 18, cytosine at a position 11,655 according to SEQ ID NO. 19, cytosine at a position 11,804 according to SEQ ID NO. 20, cytosine at a position 2,453 corresponding to SEQ ID NO. 21, cytosine at a position 818 according to SEQ ID NO. 22 or cytosine at a position 206 according to SEQ ID NO. 23.

In some embodiments, the determining step, the detecting step, or the genotyping assay comprises: a) Contacting the biological sample with a primer that hybridizes to a portion of the nucleotide sequence of the RNF213 cDNA molecule near a position corresponding to: position 11,887 according to SEQ ID NO. 31, position 12,036 according to SEQ ID NO. 32, position 2,685 according to SEQ ID NO. 33, position 1,050 according to SEQ ID NO. 34, position 438 according to SEQ ID NO. 35, position 112 according to SEQ ID NO. 36, position 84 according to SEQ ID NO. 37, position 11,655 according to SEQ ID NO. 38, position 11,804 according to SEQ ID NO. 39, position 2,453 according to SEQ ID NO. 40, position 818 according to SEQ ID NO. 41 or position 206 according to SEQ ID NO. 42. b) Extending the primer at least through a position in the nucleotide sequence of the RNF213 CDNA molecule corresponding to: position 11,887 according to SEQ ID NO. 31, position 12,036 according to SEQ ID NO. 32, position 2,685 according to SEQ ID NO. 33, position 1,050 according to SEQ ID NO. 34, position 438 according to SEQ ID NO. 35, position 112 according to SEQ ID NO. 36, position 84 according to SEQ ID NO. 37, position 11,655 according to SEQ ID NO. 38, position 11,804 according to SEQ ID NO. 39, position 2,453 according to SEQ ID NO. 40, position 818 according to SEQ ID NO. 41 or position 206 according to SEQ ID NO. 42. And c) determining whether the extension product of the primer comprises: guanine at a position corresponding to position 11,887 according to SEQ ID NO. 31, guanine at a position 12,036 according to SEQ ID NO. 32, guanine at a position 2,685 according to SEQ ID NO. 33, guanine at a position 1,050 according to SEQ ID NO. 34, guanine at a position 438 according to SEQ ID NO. 35, guanine at a position 112 according to SEQ ID NO. 36, guanine at a position 84 according to SEQ ID NO. 37, cytosine at a position 11,655 according to SEQ ID NO. 38, cytosine at a position 11,804 according to SEQ ID NO. 39, cytosine at a position 2,453 corresponding to SEQ ID NO. 40, cytosine at a position 818 according to SEQ ID NO. 41 or cytosine at a position 206 according to SEQ ID NO. 42.

In some embodiments, the determining comprises sequencing the entire nucleic acid molecule. In some embodiments, only RNF213 genomic nucleic acid molecules are analyzed. In some embodiments, only RNF213 mRNA is analyzed. In some embodiments, only RNF213 cdnas obtained from RNF213 mRNA are analyzed.

In some embodiments, the determining step, the detecting step, or the genotyping assay comprises: a) Amplifying at least a portion of a nucleic acid molecule encoding a human RNF213 polypeptide, wherein the amplified portion comprises: i) Guanine at a position corresponding to position 102,917 according to SEQ ID NO. 2 or its complement; ii) guanine at a position corresponding to position 11,887 according to SEQ ID NO. 12 or its complement; guanine at a position corresponding to position 12,036 according to SEQ ID NO. 13 or its complement; guanine at a position corresponding to position 2,685 according to SEQ ID NO. 14 or its complement; guanine at a position corresponding to position 1,050 according to SEQ ID NO. 15 or its complement; guanine at a position corresponding to position 438 according to SEQ ID NO. 16 or its complement; guanine at a position corresponding to position 112 according to SEQ ID NO. 17 or its complement; guanine at a position corresponding to position 84 according to SEQ ID NO. 18 or its complement; and/or iii) guanine at a position corresponding to position 11,887 according to SEQ ID NO. 31 or a complement thereof; guanine at a position corresponding to position 12,036 according to SEQ ID NO. 32 or its complement; guanine at a position corresponding to position 2,685 according to SEQ ID NO. 33 or its complement; guanine at a position corresponding to position 1,050 according to SEQ ID NO. 34 or its complement; guanine at a position corresponding to position 438 according to SEQ ID NO. 35 or its complement; guanine at a position corresponding to position 112 according to SEQ ID NO. 36 or its complement; guanine at a position corresponding to position 84 according to SEQ ID NO. 37 or its complement; b) Labeling the amplified nucleic acid molecules with a detectable label; c) Contacting the labeled nucleic acid molecule with a support comprising a change-specific probe, wherein the change-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleic acid sequence of an amplified nucleic acid molecule comprising: i) Guanine at a position corresponding to position 102,917 according to SEQ ID NO. 2 or its complement; ii) guanine at a position corresponding to position 11,887 according to SEQ ID NO. 12 or its complement; guanine at a position corresponding to position 12,036 according to SEQ ID NO. 13 or its complement; guanine at a position corresponding to position 2,685 according to SEQ ID NO. 14 or its complement; guanine at a position corresponding to position 1,050 according to SEQ ID NO. 15 or its complement; guanine at a position corresponding to position 438 according to SEQ ID NO. 16 or its complement; guanine at a position corresponding to position 112 according to SEQ ID NO. 17 or its complement; guanine at a position corresponding to position 84 according to SEQ ID NO. 18 or its complement; and/or iii) guanine at a position corresponding to position 11,887 according to SEQ ID NO. 31 or a complement thereof; guanine at a position corresponding to position 12,036 according to SEQ ID NO. 32 or its complement; guanine at a position corresponding to position 2,685 according to SEQ ID NO. 33 or its complement; guanine at a position corresponding to position 1,050 according to SEQ ID NO. 34 or its complement; guanine at a position corresponding to position 438 according to SEQ ID NO. 35 or its complement; guanine at a position corresponding to position 112 according to SEQ ID NO. 36 or its complement; or guanine at a position corresponding to position 84 according to SEQ ID NO. 37 or its complement; and d) detecting the detectable label.

In some embodiments, the determining step, the detecting step, or the genotyping assay comprises: a) Amplifying at least a portion of a nucleic acid molecule encoding a human RNF213 polypeptide, wherein the amplified portion comprises: i) A cytosine at a position corresponding to position 102,391 according to SEQ ID NO. 3 or a complement thereof; ii) cytosine at a position corresponding to position 11,655 according to SEQ ID NO. 19 or a complement thereof; a cytosine at a position corresponding to position 11,804 according to SEQ ID NO. 20 or a complement thereof; a cytosine at a position corresponding to position 2,453 according to SEQ ID NO. 21 or a complement thereof; cytosine at a position corresponding to position 818 according to SEQ ID NO. 22 or its complement; or a cytosine at a position corresponding to position 206 according to SEQ ID NO. 23 or a complement thereof; and/or iii) a cytosine at a position corresponding to position 11,655 according to SEQ ID NO. 38 or a complement thereof; a cytosine at a position corresponding to position 11,804 according to SEQ ID NO. 39 or a complement thereof; a cytosine at a position corresponding to position 2,453 according to SEQ ID NO. 40 or a complement thereof; cytosine at a position corresponding to position 818 according to SEQ ID NO. 41 or its complement; or cytosine at a position corresponding to position 206 according to SEQ ID NO. 42; b) Labeling the amplified nucleic acid molecules with a detectable label; c) Contacting the labeled nucleic acid molecule with a support comprising a change-specific probe, wherein the change-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleic acid sequence of an amplified nucleic acid molecule comprising: i) A cytosine at a position corresponding to position 102,391 according to SEQ ID NO. 3 or a complement thereof; ii) cytosine at a position corresponding to position 11,655 according to SEQ ID NO. 19 or a complement thereof; a cytosine at a position corresponding to position 11,804 according to SEQ ID NO. 20 or a complement thereof; a cytosine at a position corresponding to position 2,453 according to SEQ ID NO. 21 or a complement thereof; cytosine at a position corresponding to position 818 according to SEQ ID NO. 22 or its complement; or a cytosine at a position corresponding to position 206 according to SEQ ID NO. 23 or a complement thereof; and/or iii) a cytosine at a position corresponding to position 11,655 according to SEQ ID NO. 38 or a complement thereof; a cytosine at a position corresponding to position 11,804 according to SEQ ID NO. 39 or a complement thereof; a cytosine at a position corresponding to position 2,453 according to SEQ ID NO. 40 or a complement thereof; cytosine at a position corresponding to position 818 according to SEQ ID NO. 41 or its complement; or a cytosine at a position corresponding to position 206 according to SEQ ID NO. 42 or its complement; and d) detecting the detectable label.

In some embodiments, the determining step, the detecting step, or the genotyping assay comprises: a) Amplifying at least a portion of a nucleic acid molecule encoding a human RNF213 polypeptide, wherein the amplified portion comprises thymine at a position corresponding to position 103,226 according to SEQ ID No. 4 or a complement thereof; b) Labeling the amplified nucleic acid molecules with a detectable label; c) Contacting the labeled nucleic acid molecule with a support comprising a change-specific probe, wherein the change-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleic acid sequence of an amplified nucleic acid molecule comprising thymine at a position corresponding to position 103,226 according to SEQ ID No. 4 or a complement thereof; and d) detecting the detectable label.

In some embodiments, the determining step, the detecting step, or the genotyping assay comprises: a) Amplifying at least a portion of a nucleic acid molecule encoding a human RNF213 polypeptide, wherein the amplified portion comprises: guanine at a position corresponding to position 102,917 according to SEQ ID NO. 2 or its complement; a cytosine at a position corresponding to position 102,391 according to SEQ ID NO. 3 or a complement thereof; or thymine at a position corresponding to position 103,226 according to SEQ ID NO. 4 or a complement thereof; b) Labeling the amplified nucleic acid molecules with a detectable label; c) Contacting the labeled nucleic acid molecule with a support comprising a change-specific probe, wherein the change-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleic acid sequence of an amplified nucleic acid molecule comprising: guanine at a position corresponding to position 102,917 according to SEQ ID NO. 2 or its complement; a cytosine at a position corresponding to position 102,391 according to SEQ ID NO. 3 or a complement thereof; or thymine at a position corresponding to position 103,226 according to SEQ ID NO. 4 or a complement thereof; and d) detecting the detectable label.

In some embodiments, the determining step, the detecting step, or the genotyping assay comprises: a) Amplifying at least a portion of a nucleic acid molecule encoding a human RNF213 polypeptide, wherein the amplified portion comprises: guanine at a position corresponding to position 11,887 according to SEQ ID NO. 12 or its complement; guanine at a position corresponding to position 12,036 according to SEQ ID NO. 13 or its complement; guanine at a position corresponding to position 2,685 according to SEQ ID NO. 14 or its complement; guanine at a position corresponding to position 1,050 according to SEQ ID NO. 15 or its complement; guanine at a position corresponding to position 438 according to SEQ ID NO. 16 or its complement; guanine at a position corresponding to position 112 according to SEQ ID NO. 17 or its complement; guanine at a position corresponding to position 84 according to SEQ ID NO. 18 or its complement; a cytosine at a position corresponding to position 11,655 according to SEQ ID NO. 19 or a complement thereof; a cytosine at a position corresponding to position 11,804 according to SEQ ID NO. 20 or a complement thereof; a cytosine at a position corresponding to position 2,453 according to SEQ ID NO. 21 or a complement thereof; cytosine at a position corresponding to position 818 according to SEQ ID NO. 22 or its complement; or a cytosine at a position corresponding to position 206 according to SEQ ID NO. 23 or a complement thereof; b) Labeling the amplified nucleic acid molecules with a detectable label; c) Contacting the labeled nucleic acid molecule with a support comprising a change-specific probe, wherein the change-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleic acid sequence of an amplified nucleic acid molecule comprising: guanine at a position corresponding to position 11,887 according to SEQ ID NO. 12 or its complement; guanine at a position corresponding to position 12,036 according to SEQ ID NO. 13 or its complement; guanine at a position corresponding to position 2,685 according to SEQ ID NO. 14 or its complement; guanine at a position corresponding to position 1,050 according to SEQ ID NO. 15 or its complement; guanine at a position corresponding to position 438 according to SEQ ID NO. 16 or its complement; guanine at a position corresponding to position 112 according to SEQ ID NO. 17 or its complement; guanine at a position corresponding to position 84 according to SEQ ID NO. 18 or its complement; a cytosine at a position corresponding to position 11,655 according to SEQ ID NO. 19 or a complement thereof; a cytosine at a position corresponding to position 11,804 according to SEQ ID NO. 20 or a complement thereof; a cytosine at a position corresponding to position 2,453 according to SEQ ID NO. 21 or a complement thereof; cytosine at a position corresponding to position 818 according to SEQ ID NO. 22 or its complement; or a cytosine at a position corresponding to position 206 according to SEQ ID NO. 23 or a complement thereof; and d) detecting the detectable label.

In some embodiments, the determining step, the detecting step, or the genotyping assay comprises: a) Amplifying at least a portion of a nucleic acid molecule encoding a human RNF213 polypeptide, wherein the amplified portion comprises: guanine at a position corresponding to position 11,887 according to SEQ ID NO. 31 or its complement; guanine at a position corresponding to position 12,036 according to SEQ ID NO. 32 or its complement; guanine at a position corresponding to position 2,685 according to SEQ ID NO. 33 or its complement; guanine at a position corresponding to position 1,050 according to SEQ ID NO. 34 or its complement; guanine at a position corresponding to position 438 according to SEQ ID NO. 35 or its complement; guanine at a position corresponding to position 112 according to SEQ ID NO. 36 or its complement; guanine at a position corresponding to position 84 according to SEQ ID NO. 37 or its complement; a cytosine at a position corresponding to position 11,655 according to SEQ ID NO. 38 or a complement thereof; a cytosine at a position corresponding to position 11,804 according to SEQ ID NO. 39 or a complement thereof; a cytosine at a position corresponding to position 2,453 according to SEQ ID NO. 40 or a complement thereof; cytosine at a position corresponding to position 818 according to SEQ ID NO. 41 or its complement; or a cytosine at a position corresponding to position 206 according to SEQ ID NO. 42 or its complement; b) Labeling the amplified nucleic acid molecules with a detectable label; c) Contacting the labeled nucleic acid molecule with a support comprising a change-specific probe, wherein the change-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleic acid sequence of an amplified nucleic acid molecule comprising: guanine at a position corresponding to position 11,887 according to SEQ ID NO. 31 or its complement; guanine at a position corresponding to position 12,036 according to SEQ ID NO. 32 or its complement; guanine at a position corresponding to position 2,685 according to SEQ ID NO. 33 or its complement; guanine at a position corresponding to position 1,050 according to SEQ ID NO. 34 or its complement; guanine at a position corresponding to position 438 according to SEQ ID NO. 35 or its complement; guanine at a position corresponding to position 112 according to SEQ ID NO. 36 or its complement; guanine at a position corresponding to position 84 according to SEQ ID NO. 37 or its complement; a cytosine at a position corresponding to position 11,655 according to SEQ ID NO. 38 or a complement thereof; a cytosine at a position corresponding to position 11,804 according to SEQ ID NO. 39 or a complement thereof; a cytosine at a position corresponding to position 2,453 according to SEQ ID NO. 40 or a complement thereof; cytosine at a position corresponding to position 818 according to SEQ ID NO. 41 or its complement; or a cytosine at a position corresponding to position 206 according to SEQ ID NO. 42 or a complement thereof; and d) detecting the detectable label.

In some embodiments, the nucleic acid molecule is an mRNA and the determining step further comprises reverse transcribing the mRNA into cDNA prior to the amplifying step.

In some embodiments, the determining step, the detecting step, or the genotyping assay comprises: contacting a nucleic acid molecule in a biological sample with a change-specific probe comprising a detectable label, wherein the change-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of an amplified nucleic acid molecule comprising: i) Guanine at a position corresponding to position 102,917 according to SEQ ID NO. 2 or its complement; ii) guanine at a position corresponding to: position 11,887 according to SEQ ID NO. 12 or a complement thereof; position 12,036 according to SEQ ID NO. 13 or a complement thereof; position 2,685 according to SEQ ID NO. 14 or a complement thereof; position 1,050 according to SEQ ID NO. 15 or its complement; position 438 according to SEQ ID NO. 16 or a complement thereof; position 112 of SEQ ID NO. 17 or a complement thereof; position 84 according to SEQ ID NO. 18 or a sequence complementary thereof; and/or iii) guanine at a position corresponding to: position 11,887 according to SEQ ID NO. 31 or a complement thereof; position 12,036 according to SEQ ID NO. 32 or a complement thereof; position 2,685 according to SEQ ID NO. 33 or a complement thereof; position 1,050 according to SEQ ID NO. 34 or its complement; position 438 according to SEQ ID NO. 35 or its complement; position 112 according to SEQ ID NO. 36 or a complement thereof; or position 84 according to SEQ ID NO. 37 or a complement thereof; and detecting the detectable label.

In some embodiments, the determining step, the detecting step, or the genotyping assay comprises: contacting a nucleic acid molecule in a biological sample with a change-specific probe comprising a detectable label, wherein the change-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of an amplified nucleic acid molecule comprising: i) A cytosine at a position corresponding to position 102,391 according to SEQ ID NO. 3 or a complement thereof; ii) cytosine at a position corresponding to: position 11,655 according to SEQ ID NO. 19 or a complement thereof; position 11,804 according to SEQ ID NO. 20 or a complement thereof; position 2,453 according to SEQ ID NO. 21 or a complement thereof; position 818 according to SEQ ID NO. 22 or a complement thereof; or position 206 according to SEQ ID NO. 23 or a complement thereof; and/or iii) cytosine at a position corresponding to: position 11,655 according to SEQ ID NO. 38 or a complement thereof; position 11,804 according to SEQ ID NO. 39 or a complement thereof; position 2,453 according to SEQ ID NO. 40 or a complement thereof; position 818 according to SEQ ID NO. 41 or a complement thereof; or position 206 according to SEQ ID NO. 42 or its complement; and detecting the detectable label.

In some embodiments, the determining step, the detecting step, or the genotyping assay comprises: contacting a nucleic acid molecule in a biological sample with a change-specific probe comprising a detectable label, wherein the change-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of an amplified nucleic acid molecule comprising thymine at a position corresponding to position 103,226 according to SEQ ID No. 4 or a complement thereof; and detecting the detectable label.

In some embodiments, the determining step, the detecting step, or the genotyping assay comprises: contacting a nucleic acid molecule in a biological sample with a change-specific probe comprising a detectable label, wherein the change-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of an amplified nucleic acid molecule comprising: guanine at a position corresponding to position 102,917 according to SEQ ID NO. 2 or its complement; a cytosine at a position corresponding to position 102,391 according to SEQ ID NO. 3 or a complement thereof; or thymine at a position corresponding to position 103,226 according to SEQ ID NO. 4 or a complement thereof; and detecting the detectable label.

In some embodiments, the determining step, the detecting step, or the genotyping assay comprises: contacting a nucleic acid molecule in a biological sample with a change-specific probe comprising a detectable label, wherein the change-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of an amplified nucleic acid molecule comprising: guanine at a position corresponding to position 11,887 according to SEQ ID NO. 12 or its complement; guanine at a position corresponding to position 12,036 according to SEQ ID NO. 13 or its complement; guanine at a position corresponding to position 2,685 according to SEQ ID NO. 14 or its complement; guanine at a position corresponding to position 1,050 according to SEQ ID NO. 15 or its complement; guanine at a position corresponding to position 438 according to SEQ ID NO. 16 or its complement; guanine at a position corresponding to position 112 according to SEQ ID NO. 17 or its complement; guanine at a position corresponding to position 84 according to SEQ ID NO. 18 or its complement; a cytosine at a position corresponding to position 11,655 according to SEQ ID NO. 19 or a complement thereof; a cytosine at a position corresponding to position 11,804 according to SEQ ID NO. 20 or a complement thereof; a cytosine at a position corresponding to position 2,453 according to SEQ ID NO. 21 or a complement thereof; cytosine at a position corresponding to position 818 according to SEQ ID NO. 22 or its complement; or a cytosine at a position corresponding to position 206 according to SEQ ID NO. 23 or a complement thereof; and detecting the detectable label.

In some embodiments, the determining step, the detecting step, or the genotyping assay comprises: contacting a nucleic acid molecule in a biological sample with a change-specific probe comprising a detectable label, wherein the change-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of an amplified nucleic acid molecule comprising: guanine at a position corresponding to position 11,887 according to SEQ ID NO. 31 or its complement; guanine at a position corresponding to position 12,036 according to SEQ ID NO. 32 or its complement; guanine at a position corresponding to position 2,685 according to SEQ ID NO. 33 or its complement; guanine at a position corresponding to position 1,050 according to SEQ ID NO. 34 or its complement; guanine at a position corresponding to position 438 according to SEQ ID NO. 35 or its complement; guanine at a position corresponding to position 112 according to SEQ ID NO. 36 or its complement; guanine at a position corresponding to position 84 according to SEQ ID NO. 37 or its complement; a cytosine at a position corresponding to position 11,655 according to SEQ ID NO. 38 or a complement thereof; a cytosine at a position corresponding to position 11,804 according to SEQ ID NO. 39 or a complement thereof; a cytosine at a position corresponding to position 2,453 according to SEQ ID NO. 40 or a complement thereof; cytosine at a position corresponding to position 818 according to SEQ ID NO. 41 or its complement; or a cytosine at a position corresponding to position 206 according to SEQ ID NO. 42 or its complement; and detecting the detectable label.

Altering specific polymerase chain reaction techniques can be used to detect mutations in nucleic acid sequences, such as SNPs. Because the DNA polymerase will not extend when there is a mismatch with the template, modified specific primers can be used.

In some embodiments, the nucleic acid molecule in the sample is mRNA, and the mRNA is reverse transcribed to cDNA prior to the amplification step. In some embodiments, the nucleic acid molecule is present in a cell obtained from the subject.

In some embodiments, the assay comprises contacting the biological sample with a primer or probe, e.g., a change-specific primer or change-specific probe, that specifically hybridizes under stringent conditions to an RNF213 variant genomic sequence, variant mRNA sequence, or variant cDNA sequence, but not to a corresponding RNF213 reference sequence, and determining whether hybridization has occurred.

In some embodiments, the assay comprises RNA sequencing (RNA-Seq). In some embodiments, the assay further comprises reverse transcription of the mRNA into cDNA, for example, by reverse transcriptase polymerase chain reaction (RT-PCR).

In some embodiments, the methods utilize probes and primers of sufficient nucleotide length to bind to a target nucleotide sequence and specifically detect and/or identify polynucleotides comprising RNF213 variant genomic nucleic acid molecules, variant mRNA molecules, or variant cDNA molecules. Hybridization conditions or reaction conditions can be determined by the operator to achieve this result. The nucleotide length may be any length sufficient for the detection method selected, including any of the assays described or exemplified herein. Such probes and primers can specifically hybridize to a target nucleotide sequence under high stringency hybridization conditions. Probes and primers can have complete nucleotide sequence identity to consecutive nucleotides within a target nucleotide sequence, but probes that differ from the target nucleotide sequence and retain the ability to specifically detect and/or identify the target nucleotide sequence can be designed by conventional methods. Probes and primers can have about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% sequence identity or complementarity to the nucleotide sequence of the target nucleic acid molecule.

In some embodiments, to determine whether an RNF213 nucleic acid molecule (genomic nucleic acid molecule, mRNA molecule, or cDNA molecule) or its complement in a biological sample contains a nucleotide sequence comprising: i) Guanine (genomic nucleic acid molecule) at a position corresponding to position 102,917 according to SEQ ID NO. 2; ii) guanine at a position corresponding to: position 11,887 according to SEQ ID NO. 12, position 12,036 according to SEQ ID NO. 13, position 2,685 according to SEQ ID NO. 14, position 1,050 according to SEQ ID NO. 15, position 438 according to SEQ ID NO. 16, position 112 according to SEQ ID NO. 17 or position 84 (for mRNA molecules) according to SEQ ID NO. 18; or iii) guanine at a position corresponding to: according to position 11,887 of SEQ ID NO. 31, position 12,036 of SEQ ID NO. 32, position 2,685 of SEQ ID NO. 33, position 1,050 of SEQ ID NO. 34, position 438 of SEQ ID NO. 35, position 112 of SEQ ID NO. 36 or position 84 of SEQ ID NO. 37 (for a CDNA molecule), a biological sample may be subjected to an amplification method using a primer pair comprising a first primer pair derived from a 5' flanking sequence adjacent to guanine at a position corresponding to: a second primer according to position 102,917 of SEQ ID NO. 2, position 11,887 of SEQ ID NO. 12, position 12,036 of SEQ ID NO. 13, position 2,685 of SEQ ID NO. 14, position 1,050 of SEQ ID NO. 15, position 438 of SEQ ID NO. 16, position 112 of SEQ ID NO. 17, position 84 of SEQ ID NO. 18, position 11,887 of SEQ ID NO. 31, position 12,036 of SEQ ID NO. 32, position 2,685 of SEQ ID NO. 33, position 1,050 of SEQ ID NO. 34, position 438 of SEQ ID NO. 35, position 112 of SEQ ID NO. 36 or position 84 of SEQ ID NO. 37, and from a 3' flanking sequence adjacent to guanine at a position corresponding to: position 102,917 according to SEQ ID NO. 2, position 11,887 according to SEQ ID NO. 12, position 12,036 according to SEQ ID NO. 13, position 2,685 according to SEQ ID NO. 14, position 1,050 according to SEQ ID NO. 15, position 438 according to SEQ ID NO. 16, position 112 according to SEQ ID NO. 17, position 84 according to SEQ ID NO. 18, position 11,887 according to SEQ ID NO. 31, position 12,036 according to SEQ ID NO. 32, position 2,685 according to SEQ ID NO. 33, position 1,050 according to SEQ ID NO. 34, position 438 according to SEQ ID NO. 35, position 112 according to SEQ ID NO. 36 or position 84 according to SEQ ID NO. 37 to produce an amplicon indicating the presence of a SNP at a position encoding guanine at a position corresponding to: 102,917 according to SEQ ID No. 2, 11,887 according to SEQ ID No. 12, 12,036 according to SEQ ID No. 13, 2,685 according to SEQ ID No. 14, 1,050 according to SEQ ID No. 15, 438 according to SEQ ID No. 16, 112 according to SEQ ID No. 17, 84 according to SEQ ID No. 18, 11,887 according to SEQ ID No. 31, 12,036 according to SEQ ID No. 32, 2,685 according to SEQ ID No. 33, 1,050 according to SEQ ID No. 34, 438 according to SEQ ID No. 35, 112 according to SEQ ID No. 36 or 84 according to SEQ ID No. 37. In some embodiments, the length of the amplicon can range from the combined length of the primer pair plus one nucleotide base pair to any length of amplicon that can be produced by a DNA amplification scheme. This distance can range from one nucleotide base pair to the limit of the amplification reaction, or about twenty-thousand nucleotide base pairs. Optionally, the primer pair flanks a region comprising a position comprising a guanine at a position corresponding to: position 102,917 according to SEQ ID NO. 2, position 11,887 according to SEQ ID NO. 12, position 12,036 according to SEQ ID NO. 13, position 2,685 according to SEQ ID NO. 14, position 1,050 according to SEQ ID NO. 15, position 438 according to SEQ ID NO. 16, position 112 according to SEQ ID NO. 17, position 84 according to SEQ ID NO. 18, position 11,887 according to SEQ ID NO. 31, position 12,036 according to SEQ ID NO. 32, position 2,685 according to SEQ ID NO. 33, position 1,050 according to SEQ ID NO. 34, position 438 according to SEQ ID NO. 35, position 112 according to SEQ ID NO. 36 or position 84 according to SEQ ID NO. 37, and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides on each side of the position corresponding to the following positions: position 102,917 according to SEQ ID No. 2, position 11,887 according to SEQ ID No. 12, position 12,036 according to SEQ ID No. 13, position 2,685 according to SEQ ID No. 14, position 1,050 according to SEQ ID No. 15, position 438 according to SEQ ID No. 16, position 112 according to SEQ ID No. 17, position 84 according to SEQ ID No. 18, position 11,887 according to SEQ ID No. 31, position 12,036 according to SEQ ID No. 32, position 2,685 according to SEQ ID No. 33, position 1,050 according to SEQ ID No. 34, position 438 according to SEQ ID No. 35, position 112 according to SEQ ID No. 36 or position 84 according to SEQ ID No. 37.

In some embodiments, to determine whether an RNF213 nucleic acid molecule (genomic nucleic acid molecule, mRNA molecule, or cDNA molecule) or its complement in a biological sample contains a nucleotide sequence comprising: i) Cytosine (genomic nucleic acid molecule) at a position corresponding to position 102,391 according to SEQ ID NO. 3; ii) cytosine at a position corresponding to: position 11,655 according to SEQ ID NO. 19, position 11,804 according to SEQ ID NO. 20, position 2,453 according to SEQ ID NO. 21, position 818 according to SEQ ID NO. 22 or position 206 (for mRNA molecules) according to SEQ ID NO. 23; or iii) cytosine at a position corresponding to: according to position 11,655 of SEQ ID NO. 38, position 11,804 of SEQ ID NO. 39, position 2,453 of SEQ ID NO. 40, position 818 of SEQ ID NO. 41, or position 206 of SEQ ID NO. 42 (for a CDNA molecule), a biological sample may be subjected to an amplification method using a primer pair comprising a first primer pair derived from a 5' flanking sequence adjacent to a cytosine at a position corresponding to: position 102,391 according to SEQ ID NO. 3, position 11,655 according to SEQ ID NO. 19, position 11,804 according to SEQ ID NO. 20, position 2,453 according to SEQ ID NO. 21, position 818 according to SEQ ID NO. 22, position 206 according to SEQ ID NO. 23, position 11,655 according to SEQ ID NO. 38, position 11,804 according to SEQ ID NO. 39, position 2,453 according to SEQ ID NO. 40, position 818 according to SEQ ID NO. 41 or position 206 according to SEQ ID NO. 42, and a second primer derived from a 3' flanking sequence adjacent to a cytosine at a position corresponding to: position 102,391 according to SEQ ID NO. 3, position 11,655 according to SEQ ID NO. 19, position 11,804 according to SEQ ID NO. 20, position 2,453 according to SEQ ID NO. 21, position 818 according to SEQ ID NO. 22, position 206 according to SEQ ID NO. 23, position 11,655 according to SEQ ID NO. 38, position 11,804 according to SEQ ID NO. 39, position 2,453 according to SEQ ID NO. 40, position 818 according to SEQ ID NO. 41 or position 206 according to SEQ ID NO. 42 to produce an amplicon indicating the presence of a SNP at a position encoding a cytosine at a position corresponding to: position 102,391 according to SEQ ID NO. 3, position 11,655 according to SEQ ID NO. 19, position 11,804 according to SEQ ID NO. 20, position 2453 according to SEQ ID NO. 21, position 818 according to SEQ ID NO. 22, position 206 according to SEQ ID NO. 23, position 11,655 according to SEQ ID NO. 38, position 11,804 according to SEQ ID NO. 39, position 2,453 according to SEQ ID NO. 40, position 818 according to SEQ ID NO. 41 or position 206 according to SEQ ID NO. 42. In some embodiments, the length of the amplicon can range from the combined length of the primer pair plus one nucleotide base pair to any length of amplicon that can be produced by a DNA amplification scheme. This distance can range from one nucleotide base pair to the limit of the amplification reaction, or about twenty-thousand nucleotide base pairs. Optionally, the primer pair flanks a region comprising a position comprising a cytosine at a position corresponding to: position 102,391 according to SEQ ID NO. 3, position 11,655 according to SEQ ID NO. 19, position 11,804 according to SEQ ID NO. 20, position 2,453 according to SEQ ID NO. 21, position 818 according to SEQ ID NO. 22, position 206 according to SEQ ID NO. 23, position 11,655 according to SEQ ID NO. 38, position 11,804 according to SEQ ID NO. 39, position 2,453 according to SEQ ID NO. 40, position 818 according to SEQ ID NO. 41 or position 206 according to SEQ ID NO. 42, and at least 1, 2,3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides on each side of the position comprising a cytosine corresponding to the position: position 102,391 according to SEQ ID NO. 3, position 11,655 according to SEQ ID NO. 19, position 11,804 according to SEQ ID NO. 20, position 2,453 according to SEQ ID NO. 21, position 818 according to SEQ ID NO. 22, position 206 according to SEQ ID NO. 23, position 11,655 according to SEQ ID NO. 38, position 11,804 according to SEQ ID NO. 39, position 2,453 according to SEQ ID NO. 40, position 818 according to SEQ ID NO. 41 or position 206 according to SEQ ID NO. 42.

In some embodiments, to determine whether an RNF213 nucleic acid molecule (genomic nucleic acid molecule, mRNA molecule, or cDNA molecule) or its complement in a biological sample comprises a nucleotide sequence (genomic nucleic acid molecule) comprising thymine at a position corresponding to position 103,226 according to SEQ ID No. 4, the biological sample may be subjected to an amplification method using a primer pair comprising a first primer derived from a 5 'flanking sequence adjacent to thymine at a position corresponding to position 103,226 of SEQ ID No. 4 and a second primer derived from a 3' flanking sequence adjacent to thymine at a position corresponding to position 103,226 according to SEQ ID No. 4 to produce an amplicon indicative of the presence of a SNP at a position encoding thymine at a position corresponding to position 103,226 according to SEQ ID No. 4. In some embodiments, the length of the amplicon can range from the combined length of the primer pair plus one nucleotide base pair to any length of amplicon that can be produced by a DNA amplification scheme. This distance can range from one nucleotide base pair to the limit of the amplification reaction, or about twenty-thousand nucleotide base pairs. Optionally, the primer pair flanks comprise a region comprising thymine at a position corresponding to position 103,226 according to SEQ ID NO. 4 and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides on each side of the position corresponding to thymine at position 103,226 according to SEQ ID NO. 4.

Similar amplicons can be generated from mRNA and/or cDNA sequences. The PCR primer pairs can be derived from known sequences, for example, by using a computer program intended for the purpose, such as the PCR primer analysis tool of Vector NTI version 10 (Informax inc., bethesda Md.); primerSelect (DNASTAR Inc., madison, wis.); and Primer3 (version 0.4.0.copyrgt, 1991,Whitehead Institute for Biomedical Research,Cambridge,Mass.). In addition, the sequences can be scanned visually and the primers identified manually using known guidelines.

Illustrative examples of nucleic acid sequencing techniques include, but are not limited to, chain terminator (Sanger) sequencing and dye terminator sequencing. Other methods involve nucleic acid hybridization methods other than sequencing, which involve the use of labeled primers or probes (fluorescence in situ hybridization (FISH)) for purified DNA, amplified DNA, and immobilized cell preparations. In some methods, the target nucleic acid molecule can be amplified prior to or concurrent with detection. Illustrative examples of nucleic acid amplification techniques include, but are not limited to, polymerase Chain Reaction (PCR), ligase Chain Reaction (LCR), strand displacement amplification reaction (SDA), and nucleic acid sequence-based amplification reaction (NASBA). Other methods include, but are not limited to, ligase chain reaction, strand displacement amplification reaction, and thermophilic SDA (tSDA).

In hybridization techniques, stringent conditions may be employed such that probes or primers specifically hybridize to their targets. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target sequence to a degree that is, for example, at least 2-fold, at least 3-fold, at least 4-fold, or more (relative to background), including more than 10-fold (relative to background), detectably greater than hybridization to other non-target sequences. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target nucleotide sequence to a degree that is at least 2-fold greater than hybridization to other nucleotide sequences. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target nucleotide sequence to a degree that is at least 3-fold greater than hybridization to other nucleotide sequences. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target nucleotide sequence to a degree that is at least 4-fold detectably greater than hybridization to other nucleotide sequences. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target nucleotide sequence to a degree that is detectably greater than 10-fold over the other nucleotide sequences (against background). Stringent conditions are sequence-dependent and will be different in different circumstances.

Suitable stringency conditions for promoting DNA hybridization (e.g., 6 Xsodium chloride/sodium citrate (SSC), followed by washing at about 45℃with 2 XSSC at 50 ℃) are known and can be found in Current Protocols in Molecular Biology, john Wiley&Sons, n.y. (1989), 6.3.1-6.3.6. In general, stringent conditions for hybridization and detection will be those in which: salt concentration at pH 7.0 to 8.3 is less than about 1.5M Na ⁺ Ions, typically about 0.01 to 1.0M Na ⁺ Ion concentration (or other salt), and temperature is at least about 30 ℃ for short probes (e.g., 10 to 50 nucleotides) and at least about 60 ℃ for longer probes (e.g., greater than 50 nucleotides). Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide. Optionally, the wash buffer may comprise about 0.1% to about 1% sds. The duration of hybridization is typically less than about 24 hours, typically about 4 to about 12 hours. The washing time will last for a length of time at least sufficient to reach equilibrium.

The disclosure also provides methods of detecting the presence of a human RNF213 predictive of a loss of function polypeptide comprising assaying a biological sample obtained from a subject to determine whether the RNF213 polypeptide in the subject contains one or more variations that render the polypeptide either loss of function (partial or complete) or predictive of loss of function (partial or complete). The RNF213 predicted loss of function polypeptide may be any RNF213 variant polypeptide described herein. In some embodiments, the method detects the presence of RNF213 Glu3915Gly, glu3964Gly, glu822Gly, glu350Gly, glu146Gly, glu37Gly, glu28Gly, val3838Leu, val3887Leu, val745Leu, val273Leu, or Val69 Leu. In some embodiments, the method detects the presence of RNF213 Glu3915Gly or Val3838 Leu.

In some embodiments, the method comprises assaying a sample obtained from the subject to determine whether the RNF213 polypeptide in the sample comprises glycine at a position corresponding to: position 3,915 according to SEQ ID NO. 50, position 3,964 according to SEQ ID NO. 51, position 822 according to SEQ ID NO. 52, position 350 according to SEQ ID NO. 53, position 146 according to SEQ ID NO. 54, position 37 according to SEQ ID NO. 55 or position 28 according to SEQ ID NO. 56. In some embodiments, the method comprises assaying a sample obtained from the subject to determine whether the RNF213 polypeptide in the sample comprises leucine at a position corresponding to: position 3,838 according to SEQ ID NO. 57, position 3,887 according to SEQ ID NO. 58, position 745 according to SEQ ID NO. 59, position 273 according to SEQ ID NO. 60 or position 69 according to SEQ ID NO. 61.

In some embodiments, the detecting step comprises sequencing at least a portion of the polypeptide comprising a position corresponding to: position 3,915 according to SEQ ID No. 50 or SEQ ID No. 43, position 3,964 according to SEQ ID No. 51 or SEQ ID No. 44, position 822 according to SEQ ID No. 52 or SEQ ID No. 45, position 350 according to SEQ ID No. 53 or SEQ ID No. 46, position 146 according to SEQ ID No. 54 or SEQ ID No. 47, position 37 according to SEQ ID No. 55 or SEQ ID No. 48, or position 28 according to SEQ ID No. 56 or SEQ ID No. 49. In some embodiments, the detecting step comprises sequencing at least a portion of the polypeptide comprising a position corresponding to: position 3,838 according to SEQ ID NO. 57 or SEQ ID NO. 43, position 3,887 according to SEQ ID NO. 58 or SEQ ID NO. 44, position 745 according to SEQ ID NO. 59 or SEQ ID NO. 45, position 273 according to SEQ ID NO. 60 or SEQ ID NO. 46, or position 69 according to SEQ ID NO. 61 or SEQ ID NO. 47.

In some embodiments, the detecting step comprises an immunoassay for detecting the presence of a polypeptide comprising a position corresponding to: position 3,915 according to SEQ ID No. 50 or SEQ ID No. 43, position 3,964 according to SEQ ID No. 51 or SEQ ID No. 44, position 822 according to SEQ ID No. 52 or SEQ ID No. 45, position 350 according to SEQ ID No. 53 or SEQ ID No. 46, position 146 according to SEQ ID No. 54 or SEQ ID No. 47, position 37 according to SEQ ID No. 55 or SEQ ID No. 48, or position 28 according to SEQ ID No. 56 or SEQ ID No. 49. In some embodiments, the detecting step comprises an immunoassay for detecting the presence of a polypeptide comprising a position corresponding to: position 3,838 according to SEQ ID NO. 57 or SEQ ID NO. 43, position 3,887 according to SEQ ID NO. 58 or SEQ ID NO. 44, position 745 according to SEQ ID NO. 59 or SEQ ID NO. 45, position 273 according to SEQ ID NO. 60 or SEQ ID NO. 46, or position 69 according to SEQ ID NO. 61 or SEQ ID NO. 47.

In some embodiments, the subject is at increased risk of having liver disease when the subject does not have an RNF213 predictive loss of function polypeptide. In some embodiments, the subject is at reduced risk for liver disease when the subject has an RNF213 predicted loss of function polypeptide.

The disclosure also provides isolated nucleic acid molecules that hybridize to an RNF213 variant genomic nucleic acid molecule, an RNF213 variant mRNA molecule, and/or an RNF213 variant cDNA molecule (e.g., any of the genomic variant nucleic acid molecules, mRNA variant molecules, and cDNA variant molecules disclosed herein). In some embodiments, the isolated nucleic acid molecule hybridizes to a portion of an RNF213 nucleic acid molecule comprising positions corresponding to: position 102,917 according to SEQ ID No. 2, position 11,887 according to SEQ ID No. 12, position 12,036 according to SEQ ID No. 13, position 2,685 according to SEQ ID No. 14, position 1,050 according to SEQ ID No. 15, position 438 according to SEQ ID No. 16, position 112 according to SEQ ID No. 17, position 84 according to SEQ ID No. 18, position 11,887 according to SEQ ID No. 31, position 12,036 according to SEQ ID No. 32, position 2,685 according to SEQ ID No. 33, position 1,050 according to SEQ ID No. 34, position 438 according to SEQ ID No. 35, position 112 according to SEQ ID No. 36 or position 84 according to SEQ ID No. 37.

In some embodiments, the isolated nucleic acid molecule hybridizes to a portion of an RNF213 nucleic acid molecule comprising positions corresponding to: position 102,391 according to SEQ ID NO. 3, position 11,655 according to SEQ ID NO. 19, position 11,804 according to SEQ ID NO. 20, position 2,453 according to SEQ ID NO. 21, position 818 according to SEQ ID NO. 22, or position 206 according to SEQ ID NO. 23, position 11,655 according to SEQ ID NO. 38, position 11,804 according to SEQ ID NO. 39, position 2,453 according to SEQ ID NO. 40, position 818 according to SEQ ID NO. 41, or position 206 according to SEQ ID NO. 42.

In some embodiments, the isolated nucleic acid molecule hybridizes to a portion of an RNF213 nucleic acid molecule comprising a position corresponding to position 103,226 according to SEQ ID NO. 4.

In some embodiments, such isolated nucleic acid molecules comprise, at least about 5, at least about 8, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000, at least about 2000, at least about 3000, at least about 4000, or at least about 5000 nucleotides. In some embodiments, such isolated nucleic acid molecules comprise or consist of at least about 5, at least about 8, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, or at least about 25 nucleotides. In some embodiments, the isolated nucleic acid molecule comprises or consists of at least about 18 nucleotides. In some embodiments, the isolated nucleic acid molecule comprises or consists of at least about 15 nucleotides. In some embodiments, the isolated nucleic acid molecule comprises or consists of about 10 to about 35, about 10 to about 30, about 10 to about 25, about 12 to about 30, about 12 to about 28, about 12 to about 24, about 15 to about 30, about 15 to about 25, about 18 to about 30, about 18 to about 25, about 18 to about 24, or about 18 to about 22 nucleotides. In some embodiments, the isolated nucleic acid molecule comprises or consists of about 18 to about 30 nucleotides. In some embodiments, the isolated nucleic acid molecule comprises or consists of at least about 15 nucleotides to at least about 35 nucleotides.

In some embodiments, such isolated nucleic acid molecules hybridize under stringent conditions to RNF213 variant nucleic acid molecules (e.g., genomic nucleic acid molecules, mRNA molecules, and/or cDNA molecules). Such nucleic acid molecules may be used, for example, as probes, primers, altered specificity probes, or altered specificity primers described or exemplified herein, and include, but are not limited to, primers, probes, antisense RNAs, shrnas, and sirnas, each of which is described in more detail elsewhere herein, and may be used in any of the methods described herein.

In some embodiments, the isolated nucleic acid molecule hybridizes to at least about 15 consecutive nucleotides of a nucleic acid molecule that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to an RNF213 variant genomic nucleic acid molecule, an RNF213 variant mRNA molecule, and/or an RNF213 variant cDNA molecule. In some embodiments, the isolated nucleic acid molecule comprises or consists of about 15 to about 100 nucleotides or about 15 to about 35 nucleotides. In some embodiments, the isolated nucleic acid molecule comprises or consists of about 15 to about 100 nucleotides. In some embodiments, the isolated nucleic acid molecule comprises or consists of about 15 to about 35 nucleotides.

In some embodiments, the isolated change-specific probe or change-specific primer comprises at least about 15 nucleotides, wherein the change-specific probe or change-specific primer comprises a nucleotide sequence complementary to a portion of a nucleotide sequence encoding a human RNF213 polypeptide, wherein the portion comprises a position corresponding to: position 102,917 according to SEQ ID NO. 2 or a complement thereof; position 11,887 according to SEQ ID NO. 12 or a complement thereof; position 12,036 according to SEQ ID NO. 13 or a complement thereof; position 2,685 according to SEQ ID NO. 14 or a complement thereof; position 1,050 according to SEQ ID NO. 15 or its complement; position 438 according to SEQ ID NO. 16 or a complement thereof; position 112 according to SEQ ID NO. 17 or a complement thereof; position 84 according to SEQ ID NO. 18 or a complement thereof; position 11,887 according to SEQ ID NO. 31 or a complement thereof; position 12,036 according to SEQ ID NO. 32 or a complement thereof; position 2,685 according to SEQ ID NO. 33 or a complement thereof; position 1,050 according to SEQ ID NO. 34 or its complement; position 438 according to SEQ ID NO. 35 or its complement; position 112 according to SEQ ID NO. 36 or a complement thereof; or position 84 according to SEQ ID NO. 37 or its complement. In some embodiments, the altering a specific probe or altering a specific primer comprises a nucleotide sequence complementary to a portion of the nucleotide sequence comprising a position corresponding to: position 102,916-102,918 according to SEQ ID NO. 2 or a complement thereof; positions 11,886-11,888 according to SEQ ID NO. 12 or the complement thereof; positions 12,035-12,037 according to SEQ ID NO. 13 or its complement; positions 2,684-2,686 according to SEQ ID NO. 14 or the complement thereof; positions 1,049-1,051 according to SEQ ID NO. 15 or the complement thereof; positions 437-439 of SEQ ID NO. 16 or the complement thereof; positions 111-113 of SEQ ID NO. 17 or a complement thereof; positions 83-85 according to SEQ ID NO. 18 or a complement thereof; positions 11,886-11,888 according to SEQ ID NO. 31 or the complement thereof; position 12,035-12,037 according to SEQ ID NO. 32 or a complement thereof; positions 2,684-2,686 according to SEQ ID NO. 33 or the complement thereof; position 1,049-1,051 according to SEQ ID NO. 34 or the complement thereof; positions 437-439 according to SEQ ID NO. 35 or the complement thereof; or positions 111-113 according to SEQ ID NO. 36 or a complement thereof; or positions 83-85 according to SEQ ID NO. 37 or a complement thereof.

In some embodiments, the isolated change-specific probe or change-specific primer comprises at least about 15 nucleotides, wherein the change-specific probe or change-specific primer comprises a nucleotide sequence complementary to a portion of a nucleotide sequence encoding a human RNF213 polypeptide, wherein the portion comprises a position corresponding to: position 102,391 according to SEQ ID NO. 3 or a complement thereof; position 11,655 according to SEQ ID NO. 19 or a complement thereof; position 11,804 according to SEQ ID NO. 20 or a complement thereof; position 2,453 according to SEQ ID NO. 21 or a complement thereof; position 818 according to SEQ ID NO. 22 or a complement thereof; position 206 according to SEQ ID NO. 23 or a complement thereof; position 11,655 according to SEQ ID NO. 38 or a complement thereof; position 11,804 according to SEQ ID NO. 39 or a complement thereof; position 2,453 according to SEQ ID NO. 40 or a complement thereof; position 818 according to SEQ ID NO. 41 or a complement thereof; or position 206 according to SEQ ID NO. 42 or its complement. In some embodiments, the altering a specific probe or altering a specific primer comprises a nucleotide sequence complementary to a portion of the nucleotide sequence, wherein the portion comprises a position corresponding to: position 102,391-102,393 according to SEQ ID NO. 3 or a complement thereof; positions 11,655-11,657 according to SEQ ID NO. 19 or its complement; positions 11,804-11,806 according to SEQ ID NO. 20 or a complement thereof; positions 2,453-2,455 according to SEQ ID NO. 21 or its complement; positions 818-820 according to SEQ ID NO. 22 or a complement thereof; positions 206-208 according to SEQ ID NO. 23 or a complement thereof; positions 11,655-11,657 according to SEQ ID NO. 38 or its complement; positions 11,804-11,806 according to SEQ ID NO. 39 or a complement thereof; positions 2,453-2,455 according to SEQ ID NO. 40 or its complement; positions 818-820 according to SEQ ID NO. 41 or a complement thereof; or positions 206-208 according to SEQ ID NO. 42 or its complement.

In some embodiments, the isolated change-specific probe or change-specific primer comprises at least about 15 nucleotides, wherein the change-specific probe or change-specific primer comprises a nucleotide sequence complementary to a portion of a nucleotide sequence encoding a human RNF213 polypeptide, wherein the portion comprises a position corresponding to position 103,226 according to SEQ ID No. 4 or the complement thereof.

In some embodiments, the altering specific probe and altering specific primer comprise DNA. In some embodiments, the altering specific probe and altering specific primer comprise RNA.

In some embodiments, the probes and primers described herein (including altering specific probes and altering specific primers) have nucleotide sequences that specifically hybridize to any of the nucleic acid molecules disclosed herein, or the complement thereof. In some embodiments, the probes and primers specifically hybridize under stringent conditions to any of the nucleic acid molecules disclosed herein.

In some embodiments, primers (including altering specific primers) may be used in second generation sequencing or high throughput sequencing. In some cases, the primers may be modified, including altering the specific primers. In particular, the primers may comprise various modifications used in different steps such as large-scale parallel signature sequencing (MPSS), polymerase clone sequencing (Polony sequencing), and 454 pyrosequencing. Modified primers can be used in several steps of the process, including biotinylated primers in the cloning step and fluorescently labeled primers in the bead loading step and detection step. Polymerase clone sequencing is typically performed using a library of double-ended sequencing tags, wherein each DNA template molecule is about 135bp in length. Biotinylated primers were used in the bead loading step and emulsion PCR. Fluorescent-labeled degenerate nonamer oligonucleotides were used in the detection step. The adaptors may contain 5' -biotin tags for immobilization of the DNA library onto streptavidin coated beads.

The probes and primers described herein can be used to detect nucleotide variations within any of the RNF213 variant genomic nucleic acid molecules, RNF213 variant mRNA molecules, and/or RNF213 variant cDNA molecules disclosed herein. The primers described herein can be used to amplify an RNF213 variant genomic nucleic acid molecule, an RNF213 variant mRNA molecule or an RNF213 variant cDNA molecule or a fragment thereof.

The present disclosure also provides a primer pair comprising any one of the above primers. For example, if one of the 3' ends of a primer hybridizes to adenine (rather than guanine) at a position in a particular RNF213 nucleic acid molecule corresponding to position 102,917 according to SEQ ID NO. 1, the presence of an amplified fragment will indicate the presence of an RNF213 reference genomic nucleic acid molecule. In contrast, if one of the 3' ends of the primer hybridizes to guanine (rather than adenine) at a position in a particular RNF213 nucleic acid molecule corresponding to position 102,917 according to SEQ ID NO. 2, the presence of an amplified fragment will indicate the presence of an RNF213 variant genomic nucleic acid molecule. In some embodiments, the nucleotide of the primer complementary to guanine at a position corresponding to position 102,917 according to SEQ ID NO. 2 may be at the 3' end of the primer.

If the 3' end of one of the primers hybridizes to adenine at a position corresponding to position 11,887 according to SEQ ID NO. 5 (but not guanine at position 11,887) in a particular RNF213 nucleic acid molecule, the presence of an amplified fragment will indicate the presence of an RNF213 reference mRNA molecule. In contrast, if the 3' end of one of the primers hybridizes to guanine (instead of adenine) at a position in a particular RNF213 mRNA molecule corresponding to position 11,887 according to SEQ ID NO. 12, the presence of an amplified fragment will indicate the presence of an RNF213 variant mRNA molecule. In some embodiments, the nucleotide of the primer complementary to the guanine at a position corresponding to position 11,887 according to SEQ ID NO. 12 may be located at the 3' end of the primer.

If the 3' end of one of the primers hybridizes to adenine at a position corresponding to position 12,036 according to SEQ ID NO. 6 (but not guanine at position 12,036) in a particular RNF213 nucleic acid molecule, the presence of an amplified fragment will indicate the presence of an RNF213 reference mRNA molecule. In contrast, if the 3' end of one of the primers hybridizes to guanine (instead of adenine) at a position in a particular RNF213 mRNA molecule corresponding to position 12,036 according to SEQ ID NO. 13, the presence of an amplified fragment will indicate the presence of an RNF213 variant mRNA molecule. In some embodiments, the nucleotide of the primer complementary to the guanine at a position corresponding to position 12,036 according to SEQ ID NO. 13 may be located at the 3' end of the primer.

If the 3' end of one of the primers hybridizes to adenine at a position corresponding to position 2,685 according to SEQ ID NO. 7 (but not guanine at position 2,685) in a particular RNF213 nucleic acid molecule, the presence of an amplified fragment will indicate the presence of an RNF213 reference mRNA molecule. In contrast, if the 3' end of one of the primers hybridizes to a guanine (rather than an adenine) at a position corresponding to position 2,685 according to SEQ ID NO. 14 in a particular RNF213 mRNA molecule, the presence of the amplified fragment will indicate the presence of an RNF213 variant mRNA molecule. In some embodiments, the nucleotide of the primer complementary to the guanine at a position corresponding to position 2,685 according to SEQ ID NO. 14 may be located at the 3' end of the primer.

If the 3' end of one of the primers hybridizes to adenine at the position corresponding to position 1,050 according to SEQ ID NO. 8 (but not guanine at position 1,050) in a particular RNF213 nucleic acid molecule, the presence of amplified fragments will indicate the presence of RNF213 reference mRNA molecules. In contrast, if the 3' end of one of the primers hybridizes to guanine (instead of adenine) in a particular RNF213 mRNA molecule at a position corresponding to position 1,050 according to SEQ ID NO. 15, the presence of an amplified fragment will indicate the presence of an RNF213 variant mRNA molecule. In some embodiments, the nucleotide of the primer complementary to the guanine corresponding to position 1,050 according to SEQ ID NO. 15 may be located at the 3' end of the primer.

If the 3' end of one of the primers hybridizes to adenine at position 438 corresponding to position 438 according to SEQ ID NO. 9 (but not guanine at position 438) in a particular RNF213 nucleic acid molecule, the presence of an amplified fragment will indicate the presence of an RNF213 reference mRNA molecule. In contrast, if the 3' end of one of the primers hybridizes to guanine (instead of adenine) at a position in a particular RNF213 mRNA molecule corresponding to position 438 according to SEQ ID NO. 16, the presence of an amplified fragment will indicate the presence of an RNF213 variant mRNA molecule. In some embodiments, the nucleotide of the primer complementary to the guanine at position 438 corresponding to SEQ ID NO. 16 may be located at the 3' end of the primer.

If the 3' end of one of the primers hybridizes to adenine at the position corresponding to position 112 according to SEQ ID NO. 10 (but not guanine at position 112) in a particular RNF213 nucleic acid molecule, the presence of an amplified fragment will indicate the presence of an RNF213 reference mRNA molecule. In contrast, if the 3' end of one of the primers hybridizes to guanine (instead of adenine) at a position in a particular RNF213 mRNA molecule corresponding to position 112 according to SEQ ID NO. 17, the presence of an amplified fragment will indicate the presence of an RNF213 variant mRNA molecule. In some embodiments, the nucleotide of the primer complementary to guanine at a position corresponding to position 112 according to SEQ ID NO. 17 may be located at the 3' end of the primer.

If the 3' end of one of the primers hybridizes to adenine at position 84 (but not guanine at position 84) in a particular RNF213 nucleic acid molecule corresponding to position 84 according to SEQ ID NO. 11, the presence of an amplified fragment will indicate the presence of an RNF213 reference mRNA molecule. In contrast, if the 3' end of one of the primers hybridizes to guanine (instead of adenine) at a position in a particular RNF213 mRNA molecule corresponding to position 84 according to SEQ ID NO. 18, the presence of an amplified fragment will indicate the presence of an RNF213 variant mRNA molecule. In some embodiments, the nucleotide of the primer complementary to the guanine at a position corresponding to position 84 according to SEQ ID NO. 18 may be located at the 3' end of the primer.

If the 3' end of one of the primers hybridizes to adenine at a position corresponding to position 11,887 according to SEQ ID NO. 24 (but not guanine at position 11,887) in a particular RNF213 nucleic acid molecule, the presence of an amplified fragment will indicate the presence of the RNF213 reference CDNA molecule. In contrast, if the 3' end of one of the primers hybridizes to a guanine (rather than an adenine) in a particular RNF213 cDNA molecule at a position corresponding to position 11,887 according to SEQ ID NO. 31, the presence of an amplified fragment will indicate the presence of an RNF213 variant cDNA molecule. In some embodiments, the nucleotide of the primer complementary to the guanine at a position corresponding to position 11,887 according to SEQ ID NO. 31 may be located at the 3' end of the primer.

If the 3' end of one of the primers hybridizes to adenine at a position corresponding to position 12,036 according to SEQ ID NO. 25 (but not guanine at position 12,036) in a particular RNF213 nucleic acid molecule, the presence of an amplified fragment will indicate the presence of the RNF213 reference CDNA molecule. In contrast, if the 3' end of one of the primers hybridizes to a guanine (rather than an adenine) in a particular RNF213 CDNA molecule at a position corresponding to position 12,036 according to SEQ ID NO. 32, the presence of an amplified fragment will indicate the presence of an RNF213 variant CDNA molecule. In some embodiments, the nucleotide of the primer complementary to the guanine at a position corresponding to position 12,036 according to SEQ ID NO. 32 may be located at the 3' end of the primer.

If the 3' end of one of the primers hybridizes to adenine at a position corresponding to position 2,685 according to SEQ ID NO. 26 (but not guanine at position 2,685) in a particular RNF213 nucleic acid molecule, the presence of an amplified fragment will indicate the presence of the RNF213 reference CDNA molecule. In contrast, if the 3' end of one of the primers hybridizes to a guanine (rather than an adenine) in a particular RNF213 CDNA molecule at a position corresponding to position 2,685 according to SEQ ID NO. 33, the presence of an amplified fragment will indicate the presence of an RNF213 variant CDNA molecule. In some embodiments, the nucleotide of the primer complementary to the guanine at a position corresponding to position 2,685 according to SEQ ID NO. 33 may be located at the 3' end of the primer.

If the 3' end of one of the primers hybridizes to adenine at the position corresponding to position 1,050 according to SEQ ID NO. 27 (but not guanine at position 1,050) in a particular RNF213 nucleic acid molecule, the presence of the amplified fragment will indicate the presence of the RNF213 reference cDNA molecule. In contrast, if the 3' end of one of the primers hybridizes to guanine (rather than adenine) at the position corresponding to position 1,050 according to SEQ ID NO. 34 in a particular RNF213 CDNA molecule, the presence of an amplified fragment will indicate the presence of an RNF213 variant CDNA molecule. In some embodiments, the nucleotide of the primer complementary to the guanine corresponding to position 1,050 according to SEQ ID NO. 34 may be located at the 3' end of the primer.

If the 3' end of one of the primers hybridizes to adenine at position 438 corresponding to position 28 according to CDNA SEQ ID NO. 28 in a particular RNF213 nucleic acid molecule (instead of guanine at position 438), the presence of an amplified fragment will indicate the presence of the RNF213 reference CDNA molecule. In contrast, if the 3' end of one of the primers hybridizes to a guanine (rather than an adenine) at a position in a particular RNF213 CDNA molecule corresponding to position 438 according to SEQ ID NO. 35, the presence of an amplified fragment will indicate the presence of an RNF213 variant CDNA molecule. In some embodiments, the nucleotide of the primer complementary to the guanine at position corresponding to position 438 according to SEQ ID NO. 35 may be located at the 3' end of the primer.

If the 3' end of one of the primers hybridizes to adenine at a position corresponding to position 112 according to SEQ ID NO. 29 (but not guanine at position 112) in a particular RNF213 nucleic acid molecule, the presence of an amplified fragment will indicate the presence of an RNF213 reference CDNA molecule. In contrast, if the 3' end of one of the primers hybridizes to a guanine (rather than an adenine) in a particular RNF213 cDNA molecule at a position corresponding to position 112 according to SEQ ID NO:36, the presence of an amplified fragment will indicate the presence of an RNF213 variant cDNA molecule. In some embodiments, the nucleotide of the primer complementary to the guanine at a position corresponding to position 112 according to SEQ ID NO. 36 may be located at the 3' end of the primer.

If the 3' end of one of the primers hybridizes to adenine at position corresponding to position 84 according to SEQ ID NO. 30 (but not guanine at position 84) in a particular RNF213 nucleic acid molecule, the presence of an amplified fragment will indicate the presence of an RNF213 reference CDNA molecule. In contrast, if the 3' end of one of the primers hybridizes to a guanine (rather than an adenine) at a position in a particular RNF213 cDNA molecule corresponding to position 84 according to SEQ ID NO. 37, the presence of an amplified fragment will indicate the presence of an RNF213 variant cDNA molecule. In some embodiments, the nucleotide of the primer complementary to guanine at a position corresponding to position 84 according to SEQ ID NO. 37 may be located at the 3' end of the primer.

The present disclosure also provides primer pairs comprising any of the primers described above. For example, if the 3' end of one of the primers hybridizes to a guanine (rather than a cytosine) in a particular RNF213 nucleic acid molecule at a position corresponding to position 102,391 according to SEQ ID NO. 1, the presence of an amplified fragment will indicate the presence of an RNF213 reference genomic nucleic acid molecule. In contrast, if the 3' end of one of the primers hybridizes to a cytosine (rather than a guanine) in a particular RNF213 nucleic acid molecule at a position corresponding to position 102,391 according to SEQ ID NO. 3, the presence of an amplified fragment will indicate the presence of an RNF213 variant genomic nucleic acid molecule. In some embodiments, the nucleotide of the primer complementary to the cytosine at the position corresponding to position 102,391 according to SEQ ID NO. 3 may be located at the 3' end of the primer.

If the 3' end of one of the primers hybridizes to a guanine at a position in a particular RNF213 nucleic acid molecule corresponding to position 11,655 according to SEQ ID NO. 5 (rather than to a cytosine at position 11,655), the presence of an amplified fragment will indicate the presence of an RNF213 reference mRNA molecule. In contrast, if the 3' end of one of the primers hybridizes to a cytosine (rather than a guanine) in a particular RNF213 mRNA molecule at a position corresponding to position 11,655 according to SEQ ID NO. 19, the presence of the amplified fragment will indicate the presence of an RNF213 variant mRNA molecule. In some embodiments, the nucleotide of the primer complementary to the cytosine at the position corresponding to position 11,655 according to SEQ ID NO. 19 may be located at the 3' end of the primer.

If the 3' end of one of the primers hybridizes to a guanine at a position corresponding to position 11,804 according to SEQ ID NO. 6 (but not to a cytosine at position 11,804) in a particular RNF213 nucleic acid molecule, the presence of the amplified fragment will indicate the presence of the RNF213 reference mRNA molecule. In contrast, if the 3' end of one of the primers hybridizes to a cytosine (rather than a guanine) in a particular RNF213 mRNA molecule at a position corresponding to position 11,804 according to SEQ ID NO. 20, the presence of the amplified fragment will indicate the presence of an RNF213 variant mRNA molecule. In some embodiments, the nucleotide of the primer complementary to the cytosine at the position corresponding to position 11,804 according to SEQ ID NO. 20 may be located at the 3' end of the primer.

If the 3' end of one of the primers hybridizes to a guanine at a position corresponding to position 2,453 according to SEQ ID NO. 7 (but not to a cytosine at position 2,453) in a particular RNF213 nucleic acid molecule, the presence of the amplified fragment will indicate the presence of the RNF213 reference mRNA molecule. In contrast, if the 3' end of one of the primers hybridizes to a cytosine (rather than a guanine) in a particular RNF213 mRNA molecule at a position corresponding to position 2,453 according to SEQ ID NO. 21, the presence of the amplified fragment will indicate the presence of an RNF213 variant mRNA molecule. In some embodiments, the nucleotide of the primer complementary to the cytosine at the position corresponding to position 2,453 according to SEQ ID NO. 21 may be located at the 3' end of the primer.

If the 3' end of one of the primers hybridizes to a guanine at a position in a particular RNF213 nucleic acid molecule corresponding to position 818 according to SEQ ID NO. 8 (rather than a cytosine at position 818), the presence of an amplified fragment will indicate the presence of an RNF213 reference mRNA molecule. In contrast, if the 3' end of one of the primers hybridizes to a cytosine (rather than a guanine) in a particular RNF213 mRNA molecule at a position corresponding to position 818 according to SEQ ID NO. 22, the presence of the amplified fragment will indicate the presence of the RNF213 variant mRNA molecule. In some embodiments, the nucleotide of the primer that is complementary to the cytosine at position 818 corresponding to position 818 according to SEQ ID NO. 22 may be located at the 3' end of the primer.

If the 3' end of one of the primers hybridizes to a guanine at a position corresponding to position 206 according to SEQ ID NO. 9 (rather than to a cytosine at position 206) in a particular RNF213 nucleic acid molecule, the presence of an amplified fragment will indicate the presence of an RNF213 reference mRNA molecule. In contrast, if the 3' end of one of the primers hybridizes to a cytosine (rather than a guanine) in a particular RNF213 mRNA molecule at a position corresponding to position 206 according to SEQ ID NO. 23, the presence of the amplified fragment will indicate the presence of an RNF213 variant mRNA molecule. In some embodiments, the nucleotide of the primer complementary to the cytosine at position corresponding to position 206 according to SEQ ID NO. 23 may be located at the 3' end of the primer.

If the 3' end of one of the primers hybridizes to a guanine at a position corresponding to position 11,655 according to SEQ ID NO. 24 (but not to a cytosine at position 11,655) in a particular RNF213 nucleic acid molecule, the presence of an amplified fragment will indicate the presence of the RNF213 reference CDNA molecule. In contrast, if the 3' end of one of the primers hybridizes to a cytosine (rather than a guanine) in a particular RNF213 CDNA molecule at a position corresponding to position 11,655 according to SEQ ID NO. 38, the presence of the amplified fragment will indicate the presence of an RNF213 variant CDNA molecule. In some embodiments, the nucleotide of the primer complementary to the cytosine at the position corresponding to position 11,655 according to SEQ ID NO. 38 may be located at the 3' end of the primer.

If the 3' end of one of the primers hybridizes to a guanine at a position corresponding to position 11,804 according to SEQ ID NO. 25 (rather than to a cytosine at position 11,804) in a particular RNF213 nucleic acid molecule, the presence of an amplified fragment will indicate the presence of the RNF213 reference CDNA molecule. In contrast, if the 3' end of one of the primers hybridizes to a cytosine (rather than a guanine) in a particular RNF213 CDNA molecule at a position corresponding to position 11,804 according to SEQ ID NO. 39, the presence of an amplified fragment will indicate the presence of an RNF213 variant CDNA molecule. In some embodiments, the nucleotide of the primer complementary to the cytosine at the position corresponding to position 11,804 according to SEQ ID NO. 39 may be located at the 3' end of the primer.

If the 3' end of one of the primers hybridizes to a guanine at a position corresponding to position 2,453 according to SEQ ID NO. 26 (but not to a cytosine at position 2,453) in a particular RNF213 nucleic acid molecule, the presence of an amplified fragment will indicate the presence of the RNF213 reference CDNA molecule. In contrast, if the 3' end of one of the primers hybridizes to a cytosine (rather than a guanine) in a particular RNF213 CDNA molecule at a position corresponding to position 2,453 according to SEQ ID NO. 40, the presence of an amplified fragment will indicate the presence of an RNF213 variant CDNA molecule. In some embodiments, the nucleotide of the primer complementary to the cytosine at the position corresponding to position 2,453 according to SEQ ID NO. 40 may be located at the 3' end of the primer.

If the 3' end of one of the primers hybridizes to a guanine at a position in a particular RNF213 nucleic acid molecule corresponding to position 818 according to SEQ ID NO. 27 (rather than a cytosine at position 818), the presence of an amplified fragment will indicate the presence of an RNF213 reference CDNA molecule. In contrast, if the 3' end of one of the primers hybridizes to a cytosine (rather than a guanine) in a particular RNF213 cDNA molecule at a position corresponding to position 818 according to SEQ ID NO. 41, the presence of an amplified fragment will indicate the presence of an RNF213 variant cDNA molecule. In some embodiments, the nucleotide of the primer complementary to the cytosine at position 818 corresponding to position 818 according to SEQ ID NO. 41 may be located at the 3' end of the primer.

If the 3' end of one of the primers hybridizes to a guanine at a position corresponding to position 206 according to SEQ ID NO. 28 (rather than to a cytosine at position 206) in a particular RNF213 nucleic acid molecule, the presence of an amplified fragment will indicate the presence of an RNF213 reference CDNA molecule. In contrast, if the 3' end of one of the primers hybridizes to a cytosine (rather than a guanine) in a particular RNF213 cDNA molecule at a position corresponding to position 206 according to SEQ ID NO. 42, the presence of an amplified fragment will indicate the presence of an RNF213 variant cDNA molecule. In some embodiments, the nucleotide of the primer complementary to the cytosine at position corresponding to position 206 according to SEQ ID NO. 42 may be located at the 3' end of the primer.

The present disclosure also provides primer pairs comprising any of the primers described above. For example, if the 3' end of one of the primers hybridizes to adenine (rather than thymine) at a position in a particular RNF213 nucleic acid molecule corresponding to position 103,226 according to SEQ ID NO. 1, the presence of an amplified fragment will indicate the presence of an RNF213 reference genomic nucleic acid molecule. In contrast, if the 3' end of one of the primers hybridizes to thymine (instead of adenine) at a position in a particular RNF213 nucleic acid molecule corresponding to position 103,226 according to SEQ ID NO. 4, the presence of an amplified fragment will indicate the presence of an RNF213 variant genomic nucleic acid molecule. In some embodiments, the nucleotide of the primer complementary to thymine at the position corresponding to position 103,226 according to SEQ ID NO. 4 may be located at the 3' end of the primer.

In the context of the present disclosure, "specifically hybridizes" means that a probe or primer (e.g., a change in specificity probe or a change in specificity primer) does not hybridize to a nucleic acid sequence encoding an RNF213 reference genomic nucleic acid molecule, an RNF213 reference mRNA molecule, and/or an RNF213 reference cDNA molecule.

In some embodiments, the probe (e.g., a change-specific probe) comprises a label. In some embodiments, the label is a fluorescent label, a radiolabel, or biotin.

The present disclosure also provides a support comprising a substrate to which any one or more of the probes disclosed herein are attached. A solid support is a solid substrate or support to which molecules, such as any of the probes disclosed herein, can bind. One form of solid support is an array. Another form of solid support is an array detector. Array detectors are solid supports to which a variety of different probes are coupled in an array, grid, or other organized pattern. One form of solid substrate is a microtiter dish, e.g., of the standard 96-well type. In some embodiments, porous glass slides may be employed that typically contain an array per well.

The nucleotide sequence of the RNF213 reference genome nucleic acid molecule is shown in SEQ ID NO. 1. Referring to SEQ ID NO. 1, position 102,917 is adenine. Referring to SEQ ID NO. 1, position 102,391 is guanine. Referring to SEQ ID NO. 1, position 103,226 is cytosine.

There is a variant genomic nucleic acid molecule of RNF213 in which adenine at position 102,917 is replaced with guanine. The nucleotide sequence of the RNF213 reference genome nucleic acid molecule is shown in SEQ ID NO. 2.

Another variant genomic nucleic acid molecule of RNF213 is present, wherein the guanine at position 102,391 is substituted with cytosine. The nucleotide sequence of the RNF213 variant genome nucleic acid molecule is shown in SEQ ID NO. 3.

Another variant genomic nucleic acid molecule of RNF213 is present in which the cytosine at position 103,226 is replaced with thymine. The nucleotide sequence of the RNF213 variant genome nucleic acid molecule is shown in SEQ ID NO. 4.

The nucleotide sequence of the RNF213 reference mRNA molecule is shown in SEQ ID NO. 5. Referring to SEQ ID NO. 5, position 11,887 is adenine. Referring to SEQ ID NO. 5, position 11,655 is guanine. The nucleotide sequence of the other RNF213 reference mRNA molecule is shown in SEQ ID NO. 6. Referring to SEQ ID NO. 6, position 12,036 is adenine. Referring to SEQ ID NO. 6, position 11,804 is guanine. The nucleotide sequence of the other RNF213 reference mRNA molecule is shown in SEQ ID NO. 7. Referring to SEQ ID NO. 7, position 2,685 is adenine. Referring to SEQ ID NO. 7, position 2,453 is guanine. The nucleotide sequence of the other RNF213 reference mRNA molecule is shown in SEQ ID NO. 8. Referring to SEQ ID NO. 8, position 1,050 is adenine. Referring to SEQ ID NO. 8, position 818 is guanine. The nucleotide sequence of the other RNF213 reference mRNA molecule is shown in SEQ ID NO. 9. Referring to SEQ ID NO. 9, position 438 is adenine. Referring to SEQ ID NO. 9, position 206 is guanine. The nucleotide sequence of the other RNF213 reference mRNA molecule is shown in SEQ ID NO. 10. Referring to SEQ ID NO. 10, position 112 is adenine. The nucleotide sequence of the other RNF213 reference mRNA molecule is shown in SEQ ID NO. 11. Referring to SEQ ID NO. 11, position 84 is adenine.

There is a variant mRNA molecule of RNF213 in which the adenine at position 11,887 is substituted with guanine. The nucleotide sequence of the RNF213 variant mRNA molecule is shown in SEQ ID NO. 12.

There is another variant mRNA molecule of RNF213 in which adenine at position 12,036 is replaced with guanine. The nucleotide sequence of the RNF213 variant mRNA molecule is shown in SEQ ID NO. 13.

There is another variant mRNA molecule of RNF213 in which adenine at position 2,685 is replaced with guanine. The nucleotide sequence of the RNF213 variant mRNA molecule is shown in SEQ ID NO. 14.

There is another variant mRNA molecule of RNF213 in which adenine at position 1,050 is replaced with guanine. The nucleotide sequence of the RNF213 variant mRNA molecule is shown in SEQ ID NO. 15.

There is another variant mRNA molecule of RNF213 in which adenine at position 438 is replaced with guanine. The nucleotide sequence of the RNF213 variant mRNA molecule is shown in SEQ ID NO. 16.

There is another variant mRNA molecule of RNF213 in which adenine at position 112 is replaced with guanine. The nucleotide sequence of the RNF213 variant mRNA molecule is shown in SEQ ID NO. 17.

There is another variant mRNA molecule of RNF213 in which adenine at position 84 is replaced with guanine. The nucleotide sequence of the RNF213 variant mRNA molecule is shown in SEQ ID NO. 18.

There is a variant mRNA molecule of RNF213 in which the adenine at position 11,655 is substituted with cytosine. The nucleotide sequence of the RNF213 variant mRNA molecule is shown in SEQ ID NO. 19.

There is another variant mRNA molecule of RNF213 in which the adenine at position 11,804 is substituted with cytosine. The nucleotide sequence of the RNF213 variant mRNA molecule is shown in SEQ ID NO. 20.

There is another variant mRNA molecule of RNF213 in which the adenine at position 2,453 is substituted with cytosine. The nucleotide sequence of the RNF213 variant mRNA molecule is shown in SEQ ID NO. 21.

There is another variant mRNA molecule of RNF213 in which adenine at position 818 is replaced with cytosine. The nucleotide sequence of the RNF213 variant mRNA molecule is shown in SEQ ID NO. 22.

There is another variant mRNA molecule of RNF213 in which adenine at position 206 is replaced with cytosine. The nucleotide sequence of the RNF213 variant mRNA molecule is shown in SEQ ID NO. 23.

The nucleotide sequence of the RNF213 reference cDNA molecule is shown in SEQ ID NO. 24. Referring to SEQ ID NO. 24, position 11,887 is adenine. Referring to SEQ ID NO. 24, position 11,655 is guanine. The nucleotide sequence of the other RNF213 reference cDNA molecule is shown in SEQ ID NO. 25. Referring to SEQ ID NO. 25, position 12,036 is adenine. Referring to SEQ ID NO. 25, position 11,804 is guanine. The nucleotide sequence of the other RNF213 reference cDNA molecule is shown in SEQ ID NO. 26. Referring to SEQ ID NO. 26, position 2,685 is adenine. Referring to SEQ ID NO. 26, position 2,453 is guanine. The nucleotide sequence of the other RNF213 reference cDNA molecule is shown in SEQ ID NO. 27. Referring to SEQ ID NO. 27, position 1,050 is adenine. Referring to SEQ ID NO. 27, position 818 is guanine. The nucleotide sequence of the other RNF213 reference cDNA molecule is shown in SEQ ID NO. 28. Referring to SEQ ID NO. 28, position 438 is adenine. Referring to SEQ ID NO. 28, position 206 is guanine. The nucleotide sequence of the other RNF213 reference cDNA molecule is shown in SEQ ID NO. 29. Referring to SEQ ID NO. 29, position 112 is adenine. The nucleotide sequence of the other RNF213 reference cDNA molecule is shown in SEQ ID NO. 30. Referring to SEQ ID NO. 30, position 84 is adenine.

There is a variant cDNA molecule of RNF213 in which adenine at position 11,887 is replaced with guanine. The nucleotide sequence of the RNF213 variant cDNA molecule is shown in SEQ ID NO. 31.

There is another variant cDNA molecule of RNF213 in which adenine in position 12,036 is replaced with guanine. The nucleotide sequence of the RNF213 variant cDNA molecule is shown in SEQ ID NO. 32.

There is another variant cDNA molecule of RNF213 in which adenine in position 2,685 is replaced with guanine. The nucleotide sequence of the RNF213 variant cDNA molecule is shown in SEQ ID NO. 33.

There is another variant cDNA molecule of RNF213 in which adenine in position 1,050 is replaced with guanine. The nucleotide sequence of the RNF213 variant cDNA molecule is shown in SEQ ID NO. 34.

There is another variant cDNA molecule of RNF213 in which adenine at position 438 is replaced with guanine. The nucleotide sequence of the RNF213 variant cDNA molecule is shown in SEQ ID NO. 35.

There is another variant cDNA molecule of RNF213 in which adenine at position 112 is replaced with guanine. The nucleotide sequence of the RNF213 variant cDNA molecule is shown in SEQ ID NO. 36.

There is another variant cDNA molecule of RNF213 in which adenine at position 84 is replaced with guanine. The nucleotide sequence of the RNF213 variant cDNA molecule is shown in SEQ ID NO. 37.

There is a variant cDNA molecule of RNF213 in which the adenine at position 11,655 is replaced with cytosine. The nucleotide sequence of the RNF213 variant cDNA molecule is shown in SEQ ID NO. 38.

There is another variant cDNA molecule of RNF213 in which adenine in position 11,804 is replaced with cytosine. The nucleotide sequence of the RNF213 variant cDNA molecule is shown in SEQ ID NO. 39.

There is another variant cDNA molecule of RNF213 in which adenine in position 2,453 is replaced with cytosine. The nucleotide sequence of the RNF213 variant cDNA molecule is shown in SEQ ID NO. 40.

There is another variant cDNA molecule of RNF213 in which adenine at position 818 is replaced with cytosine. The nucleotide sequence of the RNF213 variant cDNA molecule is shown in SEQ ID NO. 41.

There is another variant cDNA molecule of RNF213 in which adenine at position 206 is replaced with cytosine. The nucleotide sequence of the RNF213 variant cDNA molecule is shown in SEQ ID NO. 42.

Genomic nucleic acid molecules, mRNA molecules, and cDNA molecules may be from any organism. For example, the genomic nucleic acid molecule, mRNA molecule, and cDNA molecule may be human or orthologs from another organism (e.g., a non-human mammal, rodent, mouse, or rat). It will be appreciated that the sequence of genes within a population may vary due to polymorphisms (e.g., single nucleotide polymorphisms). The examples provided herein are merely exemplary sequences. Other sequences are also possible.

Also provided herein are functional polynucleotides that can interact with the disclosed nucleic acid molecules. Examples of functional polynucleotides include, but are not limited to, antisense molecules, aptamers, ribozymes, triplex forming molecules, and external guide sequences. The functional polynucleotides may act as influencing, inhibiting, modulating and stimulating agents for a specific activity possessed by the target molecule, or the functional polynucleotides may possess entirely new activities independent of any other molecule.

The isolated nucleic acid molecules disclosed herein can include RNA, DNA, or both RNA and DNA. The isolated nucleic acid molecule may also be linked or fused to a heterologous nucleic acid sequence (e.g., in a vector) or a heterologous marker. For example, the isolated nucleic acid molecules disclosed herein can be in a vector or as an exogenous donor sequence comprising the isolated nucleic acid molecule and a heterologous nucleic acid sequence. The isolated nucleic acid molecule may also be linked or fused to a heterologous label. The label may be directly detectable (e.g., a fluorophore) or indirectly detectable (e.g., a hapten, an enzyme, or a fluorophore quencher). Such labels may be detected by spectroscopic, photochemical, biochemical, immunochemical or chemical means. Such labels include, for example, radiolabels, pigments, dyes, chromogens, spin labels, and fluorescent labels. The label may also be, for example, a chemiluminescent substance; a metalliferous material; or enzymes, wherein enzyme-dependent secondary signal generation occurs. The term "label" may also refer to a "tag" or hapten which can selectively bind to a conjugated molecule such that the conjugated molecule is used to generate a detectable signal when subsequently added with a substrate. For example, biotin may be used as a label with an avidin or streptavidin conjugate of horseradish peroxidase (HRP) to bind the label and examined for the presence of HRP using a calorimetric substrate (e.g., tetramethylbenzidine (TMB)) or a fluorogenic substrate. Exemplary labels that can be used as a tag to facilitate purification include, but are not limited to myc, HA, FLAG or 3 xglag, 6Xhis or polyhistidine, glutathione-S-transferase (GST), maltose binding protein, epitope tag, or Fc portion of an immunoglobulin. A variety of labels include, for example, particles, fluorophores, haptens, enzymes, and their calorimetric, fluorescent and chemiluminescent substrates, and other labels.

The disclosed nucleic acid molecules can include, for example, nucleotides or non-natural or modified nucleotides, such as nucleotide analogs or nucleotide substitutes. Such nucleotides include nucleotides containing modified base, sugar or phosphate groups, or nucleotides incorporating non-natural moieties in their structure. Examples of non-natural nucleotides include, but are not limited to, dideoxynucleotides, biotinylated, aminated, deaminated, alkylated, benzylated, and fluorophore-labeled nucleotides.

The nucleic acid molecules disclosed herein may also comprise one or more nucleotide analogs or substitutions. Nucleotide analogs are nucleotides that contain modifications to the base, sugar or phosphate moiety. Modifications to the base moiety include, but are not limited to, A, C, G and T/U, natural and synthetic modifications of different purine or pyrimidine bases (e.g., pseudouridine, uracil-5-yl, hypoxanthine-9-yl (I), and 2-aminoadenine-9-yl). Modified bases include, but are not limited to, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thio, 8-thioalkyl, 8-hydroxy and other 8-substituted adenine and guanine, 5-halo (e.g., 5-bromo), 5-trifluoromethyl and other 5-substituted uracil and cytosine, 7-methylguanine, 7-methyladenine, 8-azaguanine, 8-azaadenine, 7-deaza, 3-deaza and 3-deaza.

Nucleotide analogs may also include modifications to the sugar moiety. Modifications to the sugar moiety include, but are not limited to, natural modifications of ribose and deoxyribose. Sugar modifications include, but are not limited to, the following modifications at the 2' position: OH; f, performing the process; o-, S-or N-alkyl; o-, S-or N-alkenyl; o-, S-or N-alkynyl; or O-alkyl-O-alkyl, wherein alkyl, alkenyl and alkynyl groups may be substituted or unsubstituted C _1-10 Alkyl or C _2-10 Alkenyl and C _2-10 Alkynyl groups. Exemplary 2' sugar modifications also include, but are not limited to, -O [ (CH) ₂ ) _n O] _m CH ₃ 、-O(CH ₂ ) _n OCH ₃ 、-O(CH ₂ ) _n NH ₂ 、-O(CH ₂ ) _n CH ₃ 、-O(CH ₂ ) _n -ONH ₂ and-O (CH) ₂ ) _n ON[(CH ₂ ) _n CH ₃ )] ₂ Wherein n and m are from 1 to about 10. Other modifications at the 2' position include, but are not limited to, C _1-10 Alkyl, substituted lower alkyl, alkylaryl, arylalkyl, O-alkylaryl or O-arylalkyl, SH, SCH ₃ 、OCN、Cl、Br、CN、CF ₃ 、OCF ₃ 、SOCH ₃ 、SO ₂ CH ₃ 、ONO ₂ 、NO ₂ 、N ₃ 、NH ₂ A heterocycloalkyl group, a heterocycloalkyl aryl group, an aminoalkylamino group, a polyalkylamino group, a substituted silyl group, an RNA cleavage group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. Similar modifications can also be made at other positions on the sugar, particularly the 3 'position of the sugar on the 3' terminal nucleotide or in the 2'-5' linked oligonucleotide and the 5 'position of the 5' terminal nucleotide. Modified sugars may also include those containing modifications at the bridging epoxy, e.g., CH ₂ And S. Nucleotide sugar analogs may also have sugar mimics, such as cyclobutyl moieties in place of pentofuranylSugar.

The present disclosure also provides vectors comprising any one or more of the nucleic acid molecules disclosed herein. In some embodiments, the vector comprises any one or more of the nucleic acid molecules disclosed herein and a heterologous nucleic acid. The vector may be a viral or non-viral vector capable of transporting a nucleic acid molecule. In some embodiments, the vector is a plasmid or cosmid (e.g., circular double stranded DNA into which additional DNA segments may be ligated). In some embodiments, the vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Expression vectors include, but are not limited to, plasmids, cosmids, retroviruses, adenoviruses, adeno-associated viruses (AAV), plant viruses such as cauliflower mosaic virus and tobacco mosaic virus, yeast Artificial Chromosomes (YACs), epstein-Barr (EBV) -derived episomes, and other expression vectors known in the art.

Desirable regulatory sequences for expression in mammalian host cells may include, for example, viral elements that direct expression of high levels of polypeptides in mammalian cells, such as promoters and/or enhancers derived from retrovirus LTR, cytomegalovirus (CMV) (e.g., CMV promoter/enhancer), simian virus 40 (SV 40) (e.g., SV40 promoter/enhancer), adenoviruses (e.g., adenovirus major late promoter (AdMLP)), polyomaviruses, and mammalian strong promoters (e.g., native immunoglobulin and actin promoters). Methods for expressing polypeptides in bacterial cells or fungal cells (e.g., yeast cells) are also well known. The promoter may be, for example, a constitutively active promoter, a conditional promoter, an inducible promoter, a time limited promoter (e.g., a developmentally regulated promoter), or a spatially limited promoter (e.g., a cell-specific or tissue-specific promoter).

The percent identity (or percent complementarity) between specific stretches of nucleotide sequences within a nucleic acid molecule or amino acid sequences within a polypeptide can be determined using the BLAST program (basic local alignment search tool) and the PowerBLAST program (Altschul et al, j. Mol. Biol.,1990,215,403-410; zhang and Madden, genome res.,1997,7,649-656) or by using the Gap program (Wisconsin sequence analysis package, version 8,Genetics Computer Group,University Research Park,Madison Wis for Unix), using default settings using the algorithm of Smith and Waterman (adv. Appl. Math.,1981,2,482-489). In this context, if reference is made to a percentage of sequence identity, a higher percentage of sequence identity is preferred over a lower percentage of sequence identity.

The present disclosure also provides compositions comprising any one or more of the isolated nucleic acid molecules, genomic nucleic acid molecules, mRNA molecules, and/or cDNA molecules disclosed herein. In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the composition comprises a carrier and/or excipient. Examples of carriers include, but are not limited to, poly (lactic acid) (PLA) microspheres, poly (D, L-lactic-co-glycolic acid) (PLGA) microspheres, liposomes, micelles, reverse micelles, lipid helices, and lipid microtubules. The carrier may include a buffered saline solution, such as PBS, HBSS, and the like.

As used herein, the phrase "corresponding to" or grammatical variations thereof when used in the context of numbering a particular nucleotide or nucleotide sequence or position refers to the numbering of a specified reference sequence (e.g., SEQ ID NO:1, SEQ ID NO:5, or SEQ ID NO: 24) when comparing the particular nucleotide or nucleotide sequence to the reference sequence. In other words, the residue (e.g., nucleotide or amino acid) number or residue (e.g., nucleotide or amino acid) position of a particular polymer is specified relative to a reference sequence, rather than by the actual numerical position of the residue within the particular nucleotide or nucleotide sequence. For example, a particular nucleotide sequence may be aligned to a reference sequence by introducing gaps to optimize residue matching between the two sequences. In these cases, the numbering of residues in a particular nucleotide or nucleotide sequence is relative to the reference sequence to which it is aligned, although gaps exist.

For example, a nucleic acid molecule comprising a nucleotide sequence encoding a human RNF213 polypeptide, wherein the nucleotide sequence comprises a guanine at a position corresponding to position 102,917 according to SEQ ID NO. 2 means that if the nucleotide sequence of the RNF213 genomic nucleic acid molecule is aligned with the sequence of SEQ ID NO. 2, the RNF213 sequence has a guanine residue at a position corresponding to position 102,917 of SEQ ID NO. 2. The same applies to mRNA molecules comprising a nucleotide sequence encoding a human RNF213 polypeptide, wherein the nucleotide sequence comprises a guanine at a position corresponding to position 11,887 according to SEQ ID NO. 12, and cDNA molecules comprising a nucleotide sequence encoding a human RNF213 polypeptide, wherein the nucleotide sequence comprises a guanine at a position corresponding to position 11,887 according to SEQ ID NO. 31. In other words, these phrases refer to nucleic acid molecules encoding RNF213 polypeptides, wherein the genomic nucleic acid molecule has a nucleotide sequence comprising a guanine residue homologous to a guanine residue at position 102,917 of SEQ ID NO. 2 (or wherein the mRNA molecule has a nucleotide sequence comprising a guanine residue homologous to a guanine residue at position 11,887 of SEQ ID NO. 12, or wherein the cDNA molecule has a nucleotide sequence comprising a guanine residue homologous to a guanine residue at position 11,887 of SEQ ID NO. 31). Herein, when referring to genomic nucleic acid molecules, such sequences are also referred to as "RNF 213 sequences with Glu3915Gly changes" or "RNF 213 sequences with Glu3915Gly changes" (or when referring to mRNA molecules "RNF 213 sequences with a11887G changes" or "RNF 213 sequences with a11887G changes", and when referring to cDNA molecules "RNF 213 sequences with a11887G changes" or "RNF 213 sequences with a11887G changes"). The same procedure can be performed for all other molecules disclosed herein.

As described herein, the position within the RNF213 genomic nucleic acid molecule corresponding to position 102,917 according to SEQ ID NO. 2 can be identified, for example, by sequence alignment between the nucleotide sequence of a particular RNF213 nucleic acid molecule and the nucleotide sequence of SEQ ID NO. 2. There are a variety of calculation algorithms available for sequence alignment to identify nucleotide positions corresponding to position 102,917 in, for example, SEQ ID NO. 2. For example, sequence alignment may be performed using NCBI BLAST algorithm (Altschul et al, nucleic Acids Res.,1997,25,3389-3402) or CLUSTALW software (Sievers and Higgins, methods mol. Biol.,2014,1079,105-116). However, sequences may also be aligned manually.

The amino acid sequence of RNF213 reference polypeptide is shown as SEQ ID NO. 43 (isoform 1), SEQ ID NO. 44 (isoform 2), SEQ ID NO. 45 (isoform 3), SEQ ID NO. 46 (isoform 4), SEQ ID NO. 47 (isoform 5), SEQ ID NO. 48 (isoform 6) and SEQ ID NO. 49 (isoform 7). With reference to SEQ ID NO. 43 (isoform 1), the RNF213 reference polypeptide is 5,207 amino acids in length. Referring to SEQ ID NO. 43, position 3,915 is glutamic acid. Referring to SEQ ID NO. 43, position 3,838 is valine. With reference to SEQ ID NO. 44 (isoform 2), the RNF213 reference polypeptide is 5,256 amino acids in length. Referring to SEQ ID NO. 44, position 3,964 is glutamic acid. Referring to SEQ ID NO. 44, position 3,887 is valine. With reference to SEQ ID NO. 45 (isoform 3), the RNF213 reference polypeptide is 2,114 amino acids in length. Referring to SEQ ID NO. 45, position 822 is glutamic acid. With reference to SEQ ID NO. 46 (isoform 4), the RNF213 reference polypeptide is 1,642 amino acids in length. Referring to SEQ ID NO. 46, position 350 is glutamic acid. Referring to SEQ ID NO. 46, position 273 is valine. With reference to SEQ ID NO. 47 (isoform 5), the RNF213 reference polypeptide is 5,256 amino acids in length. Referring to SEQ ID NO. 47, position 146 is glutamic acid. Referring to SEQ ID NO. 44, position 69 is valine. With reference to SEQ ID NO. 48 (isoform 6), the RNF213 reference polypeptide is 37 amino acids in length. With reference to SEQ ID NO. 49 (isoform 7), the RNF213 reference polypeptide is 28 amino acids in length.

There is a set of variant RNF213 polypeptides in which the glutamic acid at the above positions of the RNF213 reference polypeptide (see SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48 and SEQ ID NO: 49) is replaced with glycine. Referring to SEQ ID NO. 50 (Glu 3915Gly; isoform 1), position 3,915 is glycine. Referring to SEQ ID NO. 51 (Glu 3964Gly; isoform 2), position 3,964 is glycine. Referring to SEQ ID NO. 52 (Glu 822Gly; isoform 3), position 822 is glycine. Referring to SEQ ID NO. 53 (Glu 350Gly; isoform 4), position 350 is glycine. Referring to SEQ ID NO. 54 (Glu 146Gly; isoform 5), position 146 is glycine. Referring to SEQ ID NO. 55 (Glu 37Gly; isoform 6), position 37 is glycine. Referring to SEQ ID NO. 56 (Glu 28Gly; isoform 7), position 28 is glycine.

Another group of RNF213 variant polypeptides exists in which valine at the above positions of the RNF213 reference polypeptide (see SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48 and SEQ ID NO: 49) is replaced with leucine. Reference SEQ ID NO. 57 (Val 3838Leu; isoform 1), position 3,838 is leucine. See SEQ ID NO. 58 (Val 3887Leu; isoform 2), position 3,887 is leucine. Reference SEQ ID NO. 59 (Val 745Leu; isoform 3), position 745 is leucine. Referring to SEQ ID NO. 60 (Val 273Leu; isoform 4), position 273 is leucine. Reference is made to SEQ ID NO. 61 (Val 69Leu; isoform 5), position 69 being leucine.

The nucleotide and amino acid sequences listed in the appended sequence listing are shown using the standard alphabetical abbreviations for nucleotide bases and the three letter codes for amino acids. The nucleotide sequence follows standard convention starting from the 5 'end of the sequence and proceeding (i.e., left to right in each row) to the 3' end. Only one strand of each nucleotide sequence is shown, but it is understood that the complementary strand is included by any reference to the displayed strand. The amino acid sequence follows the standard convention of starting from the amino terminus of the sequence and proceeding (i.e., left to right in each row) to the carboxy terminus.

The present disclosure also provides a therapeutic agent for treating or inhibiting liver disease, for treating liver disease in a subject (or for preparing a medicament for treating liver disease), wherein the subject has any one of a genomic nucleic acid molecule, an mRNA molecule, and/or a cDNA molecule encoding a human RNF213 polypeptide described herein. The therapeutic agent for treating or inhibiting liver disease may be any of the therapeutic agents described herein for treating or inhibiting liver disease.

In some embodiments, the subject comprises: i) A genomic nucleic acid molecule having a nucleotide sequence encoding a human RNF213 polypeptide, wherein the nucleotide sequence comprises a guanine at a position corresponding to position 102,917 according to SEQ ID No. 2 or the complement thereof; ii) an mRNA molecule having a nucleotide sequence encoding a human RNF213 polypeptide, wherein the nucleotide sequence comprises guanines at positions corresponding to: position 11,887 according to SEQ ID NO. 12 or a complement thereof; position 12,036 according to SEQ ID NO. 13 or a complement thereof; position 2,685 according to SEQ ID NO. 14 or a complement thereof; position 1,050 according to SEQ ID NO. 15 or its complement; position 438 according to SEQ ID NO. 16 or a complement thereof; position 112 according to SEQ ID NO. 17 or a complement thereof; or position 84 according to SEQ ID NO. 18 or a complement thereof; or iii) a cDNA molecule having a nucleotide sequence encoding a human RNF213 polypeptide, wherein the nucleotide sequence comprises guanine corresponding to the following positions: position 11,887 according to SEQ ID NO. 31 or a complement thereof; position 12,036 according to SEQ ID NO. 32 or a complement thereof; position 2,685 according to SEQ ID NO. 33 or a complement thereof; position 1,050 according to SEQ ID NO. 34 or its complement; position 438 according to SEQ ID NO. 35 or its complement; position 112 according to SEQ ID NO. 36 or a complement thereof; or position 84 according to SEQ ID NO. 37 or a complement thereof; or iv) an RNF213 polypeptide comprising glycine at a position corresponding to: position 3,915 according to SEQ ID NO. 50, position 3,964 according to SEQ ID NO. 51, position 822 according to SEQ ID NO. 52, position 350 according to SEQ ID NO. 53, position 146 according to SEQ ID NO. 54, position 37 according to SEQ ID NO. 55 or position 28 according to SEQ ID NO. 56.

In some embodiments, the subject comprises: a genomic nucleic acid molecule having a nucleotide sequence encoding a human RNF213 polypeptide, wherein the nucleotide sequence comprises a guanine at a position corresponding to position 102,917 according to SEQ ID No. 2 or the complement thereof; an mRNA molecule having a nucleotide sequence encoding a human RNF213 polypeptide, wherein the nucleotide sequence comprises a guanine at a position corresponding to position 11,887 according to SEQ ID No. 12 or the complement thereof; a cDNA molecule having a nucleotide sequence encoding a human RNF213 polypeptide, wherein the nucleotide sequence comprises a guanine at a position corresponding to position 11,887 according to SEQ ID No. 31 or the complement thereof; or an RNF213 polypeptide comprising a glycine at a position corresponding to position 3,915 according to SEQ ID No. 50.

In some embodiments, the subject comprises: i) A genomic nucleic acid molecule having a nucleotide sequence encoding a human RNF213 polypeptide, wherein the nucleotide sequence comprises a cytosine at a position corresponding to position 102,391 according to SEQ ID No. 3 or a complement thereof; ii) an mRNA molecule having a nucleotide sequence encoding a human RNF213 polypeptide, wherein the nucleotide sequence comprises cytosines at positions corresponding to: position 11,655 according to SEQ ID NO. 19 or a complement thereof; position 11,804 according to SEQ ID NO. 20 or a complement thereof; position 2,453 according to SEQ ID NO. 21 or a complement thereof; position 818 according to SEQ ID NO. 22 or a complement thereof; or position 206 according to SEQ ID NO. 23 or a complement thereof; iii) A CDNA molecule having a nucleotide sequence encoding a human RNF213 polypeptide, wherein the nucleotide sequence comprises cytosines at positions corresponding to: position 11,655 according to SEQ ID NO. 38 or a complement thereof; position 11,804 according to SEQ ID NO. 39 or a complement thereof; position 2,453 according to SEQ ID NO. 40 or a complement thereof; position 818 according to SEQ ID NO. 41 or a complement thereof; or position 206 according to SEQ ID NO. 42 or its complement; or iv) an RNF213 polypeptide comprising leucine at a position corresponding to: position 3,838 according to SEQ ID NO. 57, position 3,887 according to SEQ ID NO. 58, position 745 according to SEQ ID NO. 59, position 273 according to SEQ ID NO. 60 or position 69 according to SEQ ID NO. 61.

In some embodiments, the subject comprises: a genomic nucleic acid molecule having a nucleotide sequence encoding a human RNF213 polypeptide, wherein the nucleotide sequence comprises a cytosine at a position corresponding to position 102,391 according to SEQ ID No. 3 or a complement thereof; an mRNA molecule having a nucleotide sequence encoding a human RNF213 polypeptide, wherein the nucleotide sequence comprises a cytosine at a position corresponding to position 11,655 according to SEQ ID No. 19 or the complement thereof; a cDNA molecule having a nucleotide sequence encoding a human RNF213 polypeptide, wherein the nucleotide sequence comprises a cytosine at a position corresponding to position 11,655 according to SEQ ID No. 38 or a complement thereof; or an RNF213 polypeptide comprising leucine at a position corresponding to position 3,838 according to SEQ ID No. 57.

In some embodiments, the subject comprises a genomic nucleic acid molecule having a nucleotide sequence encoding a human RNF213 polypeptide, wherein the nucleotide sequence comprises thymine at a position corresponding to position 103,226 according to SEQ ID No. 4 or a complement thereof.

The present disclosure also provides RNF213 inhibitors for use in treating liver disease (or for the preparation of a medicament for treating liver disease) in a subject, wherein the subject has any one of a genomic nucleic acid molecule, an mRNA molecule, and/or a cDNA molecule encoding a human RNF213 polypeptide described herein. The RNF213 inhibitor may be any of the RNF213 inhibitors described herein.

In some embodiments, the subject comprises: i) A genomic nucleic acid molecule having a nucleotide sequence encoding a human RNF213 polypeptide, wherein the nucleotide sequence comprises a guanine at a position corresponding to position 102,917 according to SEQ ID No. 2 or the complement thereof; ii) an mRNA molecule having a nucleotide sequence encoding a human RNF213 polypeptide, wherein the nucleotide sequence comprises guanines at positions corresponding to: position 11,887 according to SEQ ID NO. 12 or a complement thereof; position 12,036 according to SEQ ID NO. 13 or a complement thereof; position 2,685 according to SEQ ID NO. 14 or a complement thereof; position 1,050 according to SEQ ID NO. 15 or its complement; position 438 according to SEQ ID NO. 16 or a complement thereof; position 112 according to SEQ ID NO. 17 or a complement thereof; or position 84 according to SEQ ID NO. 18 or a complement thereof; or iii) a cDNA molecule having a nucleotide sequence encoding a human RNF213 polypeptide, wherein the nucleotide sequence comprises guanine at positions corresponding to: position 11,887 according to SEQ ID NO. 31 or a complement thereof; position 12,036 according to SEQ ID NO. 32 or a complement thereof; position 2,685 according to SEQ ID NO. 33 or a complement thereof; position 1,050 according to SEQ ID NO. 34 or its complement; position 438 according to SEQ ID NO. 35 or its complement; position 112 according to SEQ ID NO. 36 or a complement thereof; or position 84 according to SEQ ID NO. 37 or a complement thereof; or iv) an RNF213 polypeptide comprising glycine at a position corresponding to: position 3,915 according to SEQ ID NO. 50, position 3,964 according to SEQ ID NO. 51, position 822 according to SEQ ID NO. 52, position 350 according to SEQ ID NO. 53, position 146 according to SEQ ID NO. 54, position 37 according to SEQ ID NO. 55 or position 28 according to SEQ ID NO. 56.

In some embodiments, the subject comprises: i) A genomic nucleic acid molecule having a nucleotide sequence encoding a human RNF213 polypeptide, wherein the nucleotide sequence comprises a cytosine at a position corresponding to position 102,391 according to SEQ ID No. 3 or a complement thereof; ii) an mRNA molecule having a nucleotide sequence encoding a human RNF213 polypeptide, wherein the nucleotide sequence comprises a cytosine at a position corresponding to: position 11,655 according to SEQ ID NO. 19 or a complement thereof; position 11,804 according to SEQ ID NO. 20 or a complement thereof; position 2,453 according to SEQ ID NO. 21 or a complement thereof; position 818 according to SEQ ID NO. 22 or a complement thereof; or position 206 according to SEQ ID NO. 23 or a complement thereof; iii) A CDNA molecule having a nucleotide sequence encoding a human RNF213 polypeptide, wherein the nucleotide sequence comprises cytosines at positions corresponding to: position 11,655 according to SEQ ID NO. 38 or a complement thereof; position 11,804 according to SEQ ID NO. 39 or a complement thereof; position 2,453 according to SEQ ID NO. 40 or a complement thereof; position 818 according to SEQ ID NO. 41 or a complement thereof; or position 206 according to SEQ ID NO. 42 or its complement; or iv) an RNF213 polypeptide comprising leucine at a position corresponding to: position 3,838 according to SEQ ID NO. 57, position 3,887 according to SEQ ID NO. 58, position 745 according to SEQ ID NO. 59, position 273 according to SEQ ID NO. 60 or position 69 according to SEQ ID NO. 61.

All patent documents, websites, other publications, accession numbers and the like cited above or below are incorporated by reference in their entirety for all purposes to the same extent as if each individual item were specifically and individually indicated to be so incorporated by reference. If different versions of a sequence are associated with accession numbers at different times, then that is meant to be the version associated with accession numbers at the date of the effective submission of the present application. Valid date of submission means the actual date of submission or the earlier date of submission of the priority application, if applicable, with reference to the accession number. Also, if different versions of a publication, web site, etc. are published at different times, it is intended that the most recently published version at the effective filing date of the application is meant, unless otherwise indicated. Any feature, step, element, embodiment, or aspect of the disclosure may be used in combination with any other feature, step, element, embodiment, or aspect unless explicitly stated otherwise. Although the present disclosure has been described in detail by way of illustration and example for purposes of clarity and understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims.

The following examples are provided to describe embodiments in more detail. They are intended to illustrate but not limit the claimed embodiments. The following examples are presented to those of ordinary skill in the art to provide a disclosure and description of how the compounds, compositions, articles, devices, and/or methods described herein are prepared and evaluated, and are intended to be merely exemplary and are not intended to limit the scope of any claims. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in degrees celsius or at ambient temperature, and pressure is at or near atmospheric pressure.

Examples

Example 1: loss of function of the gene encoding RNF213 is associated with lower ALT, AST and prevention of liver disease

To determine the genetic factors responsible for chronic liver disease, 597,856 european pedigree participants in the UK Biobank cohort (UKB), geisinger Health System MyCode Community Health Initiative cohort study (GHS) and Mount Sinai's BioMe Personalized Medicine cohort (Sinai) were analyzed for estimated genotype data, exome sequence data, and electronic health records. Discovery analyses were performed in UKB and GHS to identify new genetic variants and genes associated with liver injury, as measured by aspartate Aminotransferase (AST) and alanine Aminotransferase (ALT), which are widely used indicators of liver injury. Subsequently, statistically significant findings were evaluated in UKB, GHS, SINAI, the University of Pennsylvania Penn Medicine BioBank (UPENN-PMBB) and Malmo Diet and Cancer Study (MDCS) for relation to various liver diseases.

In order to find the protective gene for liver disease, a two-step method was used. In a first step, whole genome analytical studies are performed on circulating AST and ALT levels to identify genes having common protein-encoding variants associated with AST or ALT at whole genome significance levels. In a second step, determining the association between the load of the rare loss-of-function allele in the step 1 gene and ALT or AST; triangulating evidence that the identified genes have causal relationships.

In step 1, a genome-wide meta-analysis of AST and ALT was performed using an estimated dataset of 11,914,698 variants from over 500,000 European individuals of blood family, identifying 784 genome-wide important regions. One of the important loci in the genome-wide domain comprises the gene RNF213 and contains a number of related coding variants that drive the associated signal at this locus. The strongest variant of AST is the missense variant in RNF213 rs61740658 (np_001243000.2, glu3915 gly). The strongest variant of ALT is the intron variant rs36103733 (17:80364093:C: T), in perfect linkage disequilibrium with the missense variant rs35332090 (NP-001243000.2, val3838Leu) in RNF 213. After fine mapping using FINEMAP software, 2 additional coding variants were identified in the 95% reliable causal variant set: rs12944385 (np_001243000.2, lys4732 glu) with an ALT posterior probability of 0.20 and an AST posterior probability of 0.42, and rs72849841 (np_066005.2, pro729 leu) with an AST posterior probability of 0.55. The associated statistics for these variants are listed in table 2.

Table 2: several common coding variants in RNF213 are associated with liver enzymes. Results are shown in standard deviation units

RR represents the number of individuals in the population study that do not carry the alternative allele; RA represents the number of individuals carrying one or more heterozygous alternative alleles; AA represents the number of individuals carrying one or more homozygous alternative alleles; the substitution allele is the allele that causes loss of function or amino acid change encoded according to the HGVS proposal, for 17:80364093:c:t, c is the reference allele, T is the substitution and effect allele that reports summary statistics. SD represents standard deviation units; AAF represents the substitution allele frequency.

In step 2, the load of rare (AAF < 1%) predicted loss of function (pLOF) variants in RNF213 genes was estimated to correlate with ALT and AST using exome sequence data. In this analysis, circulating AST and ALT levels were significantly reduced in 1,773 carriers of the ploff variant in RNF213, as shown in table 3.

Table 3: the load lost by RNF213 functional variants was significantly correlated with reduced circulating ALT and AST levels

* The correlation was also corrected for common fine map signals of ALT or AST, respectively, to demonstrate statistical independence from the results described in table 1. RR represents the number of individuals in the population study that do not carry the alternative allele; RA represents the number of individuals carrying one or more heterozygous alternative alleles; AA represents the number of individuals carrying one or more homozygous alternative alleles; alternative alleles are alleles which are encoded according to the HGVS proposal and cause loss of function or amino acid changes; SD represents standard deviation units; AAF represents alternative allele frequencies; plofs indicate the loss of predictive function.

The association between load and AST or ALT for the loss of function variant is driven by multiple loss of function variants in RNF213 (see table 4). These results indicate that the commonly encoded alleles described in table 3 cause loss of RNF213 function.

Table 4: list of predicted loss of function variants in RNF213 included in the analysis-Chr: position: ref: alt represents the position of the genetic variant on chromosome (Chr), and human basis

Reference (ref) and substitution (alt) alleles on genome reference alliance construction 38

Protein changes follow the recommendations of the human genome variation association and correspond to each Ensembl transcript identity, with hgvsp (protein change) given in the case of protein coding variants and hgvsc (cDNA change) given in the case of splice variants. AAF represents the substitution allele frequency.

Table 5 shows that loss of function alleles in RNF213 are closely related to the prevention of liver disease diagnosis, including essential liver disease, alcoholic, non-alcoholic liver disease, liver fibrosis and cirrhosis. These results indicate that loss of RNF213 function can prevent chronic liver disease.

Table 5: in a meta-analysis of UKB, GHS, SINAI, MDCS and UPENN-PMBB, association between RNF213 plofand most significant ALT-associated common coding variants Val3838Leu and various clinical diagnoses of liver disease

RR represents the number of individuals in the population study that do not carry the alternative allele; RA represents the number of individuals carrying one or more heterozygous alternative alleles; AA represents the number of individuals carrying one or more homozygous alternative alleles; alternative alleles are alleles which are encoded according to the HGVS proposal and cause loss of function or amino acid changes; alternative alleles are alleles which are encoded according to the HGVS proposal and cause loss of function or amino acid changes; OR represents the ratio of the ratios; AAF represents the substitution allele frequency.

Participation queue

Genetic association studies were performed in the United Kingdom Biobank (UKB) queue (Sudlow et al, PLoS Med.,2015,12, e 1001779) and the discover EHR queue (Carey et al, genet. Med.,2016,18,906-13) from Geisinger Health System (GHS) MyCode Community Health Initiative. UKB is a group-based cohort study of 40 to 69 year old people recruited by 22 detection centers in the united kingdom between 2006-2010. Over 430,000 european pedigree participants from UKB with available whole genome sequencing and clinical phenotype data were included. GHS MyCode study Community Health Initiative is a health system-based cohort of patients recruited from the middle and eastern parts of the united states, pennsylvania (Central and Eastern Pennsylvania (USA)) in 2007-2019. Over 130,000 european ancestry participants from GHS with available whole genome sequencing and clinical phenotype data were included. The association with liver results also included a Mount Sinai BioMe Biobank queue (SINAI, cell,2019,177,58-69), the University of Pennsylvania Penn Medicine BioBank (UPENN-PMBB; (Park et al 2020, doi:10.1038/s 41436-019-0625-8)), and Malmo Diet and Cancer Study (MDCS), a prospective observation queue based on Swedish population recruited between 1991 and 1996 (Berglund et al, 1993, doi:10.1111/j.1365-2796.1993.Tb00647. X).

Phenotypic definition

Clinical laboratory measurements of ALT or AST are extracted from Electronic Health Records (EHRs) of GHS participants. The median of two or more measurements of all participants was calculated. In UKB, ALT and AST were measured by IFCC (international union of clinical chemistry) analysis at Beckman Coulter AU5800 at study baseline visit; hb1Ac was measured by HPLC using Bio-Rad VARIANT II Turbo. Prior to genetic association analysis, successive phenotype values are converted by an inverse standard normal function, applied within each pedigree group and individually in men and women. Disease outcome is defined according to international disease classification (International Classification of Diseases) ninth and tenth edition (ICD-9 and ICD-10) using EHR and self-reporting, if available, and is consolidated into a single variable, as described in table 6.

Table 6: definition of liver disease outcome in UKB, GHS, SINAI UPENN-PMBB and MALMO

* If the participants were diagnosed with "any liver disease" outcome codes (as defined in the table), or if their ALT was elevated (male >33U/L, female > 25U/L), they were excluded from the control population.

ICD10 represents the 10 th revision of the International disease and related health issue statistical Classification (International Statistical Classification of Diseases and Related Health Problems); UKB. OPCS4 represents the census and investigation office (Office of Population Censuses and Surveys, OPCS) intervention and program classification, 4 th edition, used by UK Biobank (UKB); UKB.f.20002 represents a self-reported non-cancer disease code used in UKB. UKB.f.20004 represents a self-reporting medical procedure used in UKB.

Genotype data

High coverage whole exome sequencing was performed as described earlier (Science, 2016,354: aaf6814; and Nature,2020,586,749-756) and summarized below. The NimbleGen probe (VCRome; for a portion of the GHS queue) or modified versions of the xGen design available from Integrated DNA Technologies (IDT; for the rest of GHS and other queues) was used for target sequence capture of the exome. A unique 6 base pair (bp) barcode (VCRome) or 10bp barcode (IDT) was added to each DNA fragment during library preparation to facilitate multiplex exome capture and sequencing. Equal amounts of samples were pooled prior to exome capture. Sequencing was performed using 75bp paired-end reads on Illumina v4 HiSeq 2500 (for part of GHS queue) or NovaSeq (for the rest of GHS and other queues) instruments. The depth of coverage of sequencing (i.e., the number of sequence reads covering each nucleotide in the genomic target region) is sufficient to provide greater than 20x coverage of 85% of the target bases in 96% of the VCRome samples, and 20x coverage of 90% of the target bases in 99% of the IDT samples. The data processing steps include sample multiplexing (de-multiplexing) using Illumina software, alignment with GRCh38 human genome reference sequences, including generating binary alignment and mapping files (BAMs), processing the BAM files (e.g., marker repeat reads and other read mapping evaluations). Variant calls were performed using the GLNexus system (DOI: 10.1101/343970). Variant mapping and annotation was defined based on GRCh38 human genome reference sequence and the Ensembl v85 gene using snpoff software. The snpoff predictions involving protein-encoded transcripts with annotated initiation and termination were then combined into a single functional impact prediction by selecting the most deleterious functional effect class for each gene. The scale of these annotations (from most deleterious to least deleterious) is frameshift, stop add, stop lose, splice acceptor, splice donor, stop lose, in-frame insertion deletion, missense, other annotations. Predicted LOF genetic variants include: a) an insertion or deletion resulting in a frame shift, b) an insertion, deletion or single nucleotide variant resulting in the introduction of a premature stop codon or loss of transcription start or stop site, and c) a variant of a donor or acceptor splice site. Missense variants were classified according to computer predictive algorithms using SIFT (Adzhubei et al, nat. Methods,2010,7,248-9) and polyphen2_hvar (Adzhubei et al, nat. Methods,2010,7,248-9), LRT (Chun et al, genome res.,2009,19,1553-61) and mutationTaster (Schwarz et al, nat. Methods,2010,7,575-6) to predict harmfulness for possible functional effects. For each gene, the substitution allele frequency (AAF) and functional annotation of each variant was determined to be contained in these 7 gene load exposures: 1) A plofvariant with AAF < 1%; 2) Predicted detrimental by the 5/5 algorithm, AAF <1% of plofs or missense variants; 3) Predicted detrimental by the 5/5 algorithm, AAF <0.1% plofs or missense variants; 4) Predicted detrimental by at least 1/5 algorithm, AAF <1% plofs or missense variants; 5) Predicted detrimental by at least 1/5 algorithm, AAF <0.1% plofs or missense variants; 6) AAF <1% plofor any missense; 7) AAF <0.1% plofor any missense variant.

Gene load correlation analysis of rare loss-of-function variations

By fitting the needle using REGENIE v1.0 (doi: "doi.org/10.1101/2020.06.19.162354" on the "Web")The linear (for quantitative traits) or firth bias correction logic (for binary traits) regression model of the multigenic score adjustment to the near genomic relatedness matrix examined the association between load and phenotype of rare predicted loss of function or missense variants in a given gene. Analysis of stratification by blood lineage and age ² Gender, age and gender and age ² The gender interactive terms, experimental batch related covariates, 10 common variant-derived principal components, and 20 rare variant-derived principal components were adjusted. The cross-queue results for each variant-phenotype association were combined using a fixed-effect inverse variance weighted meta-analysis. In the gene stress test, all individuals are labeled as heterozygotes if they carry one or more qualified rare variants (based on frequency and functional annotation as described above), and as homozygotes if they carry any qualified variants in a homozygous state. This "composite genotype" is then used to test for relevance.

Gwas and fine mapping independent signals of common variants

By conducting whole genome association studies, related common variants were identified, including over 1200 ten thousand common to low frequency genetic variants estimated using the haplotype reference group (Haplotype Reference Consortium). In the GHS study, interpolation was performed separately in samples genotyped with Illumina human Omni expression exome array (Illumina Human Omni Express Exome array, omni collection) and global screening array (GSA collection), respectively. Dose data from the interpolation variants are then combined in the two GHS sets to obtain a combined dataset for correlation analysis. Full genome association analysis was performed in GHS and UKB, respectively, by fitting a full genome regression model using REGENIE (Mbotchou et al 2020, doi: 10.1101/2020.06.19.162354). Within each queue, the analysis is stratified by blood family and by age, as described above for stress testing ² Gender, age and gender and age ² The gender interactive terms, experimental batch related covariates and 10 common variant derived principal components were adjusted. Then combining from UKB and GHS analysis by inverse variance weighting element analysis To obtain whole genome component analysis in the European subset of the discovery cohort. To identify a conditionally independent genetic association signal driven by common variants, FINEMAP was used at p<5×10 ^-08 Is mapped finely in the genomic region containing the genetic variants associated with each trait of interest (Benner et al 2016, doi: 10.1093/bioinformation/btw 018). Genetic data from the precise set of individuals contained in the whole genome association analysis is used to estimate linkage disequilibrium. The fine mapping was performed in meta-analysis of european pedigree GHS and UKB queues, respectively. The fine mapping identifies individual common variant signals and assigns causal-related posterior probabilities to variants associated with a given individual signal. For each locus of the fine map, a set of causal variants that is 95% authentic (i.e., the minimum set of variants that captures 95% causal correlation posterior probability) was identified. A whistle variant is defined as the variant with the highest causal-related posterior probability at each given independent signal.

Various modifications to the described subject matter, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Each reference cited in this application (including, but not limited to, journal articles, U.S. and non-U.S. patents, patent application publications, international patent application publications, gene bank accession numbers, etc.) is incorporated by reference in its entirety and for all purposes.

Claims

1. A method of treating a subject having a liver disease, the method comprising administering to the subject an inhibitor of ring finger protein 213 (RNF 213).

2. A method of treating a subject having fatty liver disease, the method comprising administering to the subject an inhibitor of ring finger protein 213 (RNF 213).

3. The method of claim 2, wherein the fatty liver disease is Alcoholic Fatty Liver Disease (AFLD) or non-alcoholic fatty liver disease (NAFLD).

4. A method of treating a subject having hepatocellular carcinoma, the method comprising administering to the subject an inhibitor of ring finger protein 213 (RNF 213).

5. A method of treating a subject having cirrhosis, the method comprising administering to the subject an inhibitor of ring finger protein 213 (RNF 213).

6. A method of treating a subject having liver fibrosis, the method comprising administering to the subject an inhibitor of ring finger protein 213 (RNF 213).

7. A method of treating a subject having simple steatosis, steatohepatitis, or nonalcoholic steatohepatitis (NASH), the method comprising administering to the subject a ring finger protein 213 (RNF 213) inhibitor.

8. The method of any one of claims 1 to 7, wherein the RNF213 inhibitor comprises an antisense nucleic acid molecule, small interfering RNA (siRNA), or short hairpin RNA (shRNA) that hybridizes to RNF213 mRNA.

9. The method of any one of claims 1 to 7, wherein the RNF213 inhibitor comprises a Cas protein and a guide RNA (gRNA) that hybridizes to a gRNA recognition sequence within an RNF213 genomic nucleic acid molecule.

10. The method of claim 9, wherein the Cas protein is Cas9 or Cpf1.

11. The method of claim 9 or claim 10, wherein the gRNA recognition sequence comprises or is adjacent to a position corresponding to: position 102,917 according to SEQ ID NO. 1, position 102,391 according to SEQ ID NO. 1 or position 103,226 according to SEQ ID NO. 1.

12. The method of claim 9 or claim 10, wherein the gRNA recognition sequence is located about 1000, about 500, about 400, about 300, about 200, about 100, about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 15, about 10, or about 5 nucleotides from positions corresponding to: position 102,917 according to SEQ ID NO. 1, position 102,391 according to SEQ ID NO. 1 or position 103,226 according to SEQ ID NO. 1.

13. The method of claim 9 or claim 10, wherein a Protospacer Adjacent Motif (PAM) sequence is about 2 to about 6 nucleotides downstream of the gRNA recognition sequence.

14. The method of any one of claims 9 to 13, wherein the gRNA comprises about 17 to about 23 nucleotides.

15. The method of any one of claims 9 to 13, wherein the gRNA recognition sequence comprises a nucleotide sequence according to any one of SEQ ID NOs 62-81.

16. The method of any one of claims 1 to 15, further comprising detecting the presence or absence of an RNF213 predictive loss of function or missense variant nucleic acid molecule encoding a human RNF213 polypeptide in a biological sample from the subject.

17. The method of claim 16, wherein when the subject is an RNF213 reference, the subject is also administered a standard dose of therapeutic agent for treating or inhibiting liver disease.

18. The method of claim 16, wherein when the subject is heterozygous for an RNF213 predictive loss of function or missense variant, the subject is further administered a therapeutic agent that treats or inhibits liver disease at a dose equal to or lower than standard dose.

19. The method of any one of claims 16 to 18, wherein the RNF 213-predicted loss-of-function or missense variant nucleic acid molecule is a nucleic acid molecule encoding Glu3915Gly, glu3964Gly, glu822Gly, glu350Gly, glu146Gly, glu37Gly, glu28Gly, val3838Leu, val3887Leu, val745Leu, val273Leu, or Val69 Leu.

20. The method of any one of claims 16-18, wherein the RNF213 predicted loss of function or missense variant nucleic acid molecule is a nucleic acid molecule encoding Glu3915Gly or Val3838 Leu.

21. The method of claim 19, wherein the RNF213 predicted loss of function or missense variant nucleic acid molecule is:

a genomic nucleic acid molecule having a nucleotide sequence comprising: guanine at a position corresponding to position 102,917 according to SEQ ID NO. 2; cytosine at a position corresponding to position 102,391 according to SEQ ID NO. 3; or thymine at a position corresponding to position 103,226 according to SEQ ID NO. 4;

an mRNA molecule having a nucleotide sequence comprising: guanine at a position corresponding to position 11,887 according to SEQ ID NO. 12, guanine at a position 12,036 according to SEQ ID NO. 13, guanine at a position 2,685 according to SEQ ID NO. 14, guanine at a position 1,050 according to SEQ ID NO. 15, guanine at a position 438 according to SEQ ID NO. 16, guanine at a position 112 according to SEQ ID NO. 17, guanine at a position 84 according to SEQ ID NO. 18, cytosine at a position 11,655 according to SEQ ID NO. 19, cytosine at a position 11,804 according to SEQ ID NO. 20, cytosine at a position 2,453 corresponding to SEQ ID NO. 21, cytosine at a position 818 according to SEQ ID NO. 22 or cytosine at a position 206 according to SEQ ID NO. 23. Or (b)

A cDNA molecule produced from an mRNA molecule, wherein the cDNA molecule has a nucleotide sequence comprising: guanine at a position corresponding to position 11,887 according to SEQ ID NO. 31, guanine at a position 12,036 according to SEQ ID NO. 32, guanine at a position 2,685 according to SEQ ID NO. 33, guanine at a position 1,050 according to SEQ ID NO. 34, guanine at a position 438 according to SEQ ID NO. 35, guanine at a position 112 according to SEQ ID NO. 36, guanine at a position 84 according to SEQ ID NO. 37, cytosine at a position 11,655 according to SEQ ID NO. 38, cytosine at a position 11,804 according to SEQ ID NO. 39, cytosine at a position 2,453 corresponding to SEQ ID NO. 40, cytosine at a position 818 according to SEQ ID NO. 41 or cytosine at a position 206 according to SEQ ID NO. 42.

22. The method of any one of claims 16 to 21, wherein the detecting step is performed in vitro.

23. The method of any one of claims 16 to 22, wherein the detecting step comprises sequencing at least a portion of the nucleotide sequence of the RNF213 genomic nucleic acid molecule in the biological sample, wherein the sequenced portion comprises a position corresponding to: position 102,917 according to SEQ ID NO. 2 or a complement thereof; position 102,391 according to SEQ ID NO. 3 or a complement thereof; or position 103,226 according to SEQ ID NO. 4 or a complement thereof;

Wherein when the sequencing portion of the RNF213 genomic nucleic acid molecule in the biological sample comprises: guanine at a position corresponding to position 102,917 according to SEQ ID NO. 2; cytosine at a position corresponding to position 102,391 according to SEQ ID NO. 3; or corresponds to thymine at position 103,226 according to SEQ ID NO. 4, then the RNF213 genomic nucleic acid molecule in the biological sample is an RNF213 predicted loss of function or missense variant genomic nucleic acid molecule.

24. The method of any one of claims 16 to 22, wherein the detecting step comprises sequencing at least a portion of the nucleotide sequence of the RNF213 mRNA molecule in the biological sample, wherein the sequenced portion comprises a position corresponding to: position 11,887 according to SEQ ID NO. 12 or a complement thereof; position 12,036 according to SEQ ID NO. 13 or a complement thereof; position 2,685 according to SEQ ID NO. 14 or a complement thereof; position 1,050 according to SEQ ID NO. 15 or its complement; position 438 according to SEQ ID NO. 16 or a complement thereof; position 112 according to SEQ ID NO. 17 or a complement thereof; or position 84 according to SEQ ID NO. 18 or a complement thereof; position 11,655 according to SEQ ID NO. 19 or a complement thereof; position 11,804 according to SEQ ID NO. 20 or a complement thereof; position 2,453 according to SEQ ID NO. 21 or a complement thereof; position 818 according to SEQ ID NO. 22 or a complement thereof; or position 206 according to SEQ ID NO. 23 or a complement thereof;

Wherein when the sequencing portion of the RNF213 mRNA molecule in the biological sample comprises: guanine at a position corresponding to position 11,887 according to SEQ ID NO. 12; guanine at a position corresponding to position 12,036 according to SEQ ID NO. 13; guanine at a position corresponding to position 2,685 according to SEQ ID NO. 14; guanine at a position corresponding to position 1,050 according to SEQ ID NO. 15; guanine at a position corresponding to position 438 according to SEQ ID NO. 16; guanine at a position corresponding to position 112 according to SEQ ID NO. 17; guanine at a position corresponding to position 84 according to SEQ ID NO. 18; cytosine at a position corresponding to position 11,655 according to SEQ ID NO. 19; cytosine at a position corresponding to position 11,804 according to SEQ ID NO. 20; cytosine at a position corresponding to position 2,453 according to SEQ ID NO. 21; cytosine at a position corresponding to position 818 according to SEQ ID NO. 22; or a cytosine at a position corresponding to position 206 according to SEQ ID NO. 23; the RNF213 mRNA molecule in the biological sample is an RNF213 predicted loss of function or missense variant mRNA molecule.

25. The method of any one of claims 16 to 22, wherein the detecting step comprises sequencing at least a portion of the nucleotide sequence of the RNF213 cDNA molecule produced from an mRNA molecule in the biological sample, wherein the sequenced portion comprises positions corresponding to: position 11,887 according to SEQ ID NO. 31 or a complement thereof; position 12,036 according to SEQ ID NO. 32 or a complement thereof; position 2,685 according to SEQ ID NO. 33 or a complement thereof; position 1,050 according to SEQ ID NO. 34 or its complement; position 438 according to SEQ ID NO. 35 or its complement; position 112 according to SEQ ID NO. 36 or a complement thereof; or position 84 according to SEQ ID NO. 37 or a complement thereof; position 11,655 according to SEQ ID NO. 38 or a complement thereof; position 11,804 according to SEQ ID NO. 39 or a complement thereof; position 2,453 according to SEQ ID NO. 40 or a complement thereof; position 818 according to SEQ ID NO. 41 or a complement thereof; or position 206 according to SEQ ID NO. 42 or its complement;

Wherein when the sequencing portion of the RNF213 cDNA molecule in the biological sample comprises: guanine at a position corresponding to position 11,887 according to SEQ ID NO. 31; guanine at a position corresponding to position 12,036 according to SEQ ID NO. 32; guanine at a position corresponding to position 2,685 according to SEQ ID NO. 33; guanine at a position corresponding to position 1,050 according to SEQ ID NO. 34; guanine at a position corresponding to position 438 according to SEQ ID NO. 35; guanine at a position corresponding to position 112 according to SEQ ID NO. 36; guanine at a position corresponding to position 84 according to SEQ ID NO. 37; cytosine at a position corresponding to position 11,655 according to SEQ ID NO. 38; cytosine at a position corresponding to position 11,804 according to SEQ ID NO. 39; cytosine at a position corresponding to position 2,453 according to SEQ ID NO. 40; cytosine at a position corresponding to position 818 according to SEQ ID NO. 41; or a cytosine at a position corresponding to position 206 according to SEQ ID NO. 42; the RNF213 cDNA molecule in the biological sample is a RNF213 predictive loss of function or missense variant cDNA molecule.

26. The method of any one of claims 16 to 22, wherein the detecting step comprises:

a) Contacting the biological sample with a primer that hybridizes to a portion of the nucleotide sequence of the RNF213 genomic nucleic acid molecule that corresponds approximately to: position 102,917 according to SEQ ID NO. 2, position 102,391 according to SEQ ID NO. 3 or position 103,226 according to SEQ ID NO. 4;

b) Extending the primer at least through a position in the nucleotide sequence of the RNF213 genomic nucleic acid molecule corresponding to: position 102,917 according to SEQ ID NO. 2, position 102,391 according to SEQ ID NO. 3 or position 103,226 according to SEQ ID NO. 4; and

c) Determining whether the extension product of the primer comprises: guanine at a position corresponding to position 102,917 according to SEQ ID NO. 2, cytosine at a position corresponding to position 102,391 according to SEQ ID NO. 3 or thymine at a position corresponding to position 103,226 according to SEQ ID NO. 4.

27. The method of any one of claims 16 to 22, wherein the detecting step comprises:

a) Contacting the biological sample with a primer that hybridizes to a portion of the nucleotide sequence of the RNF213 mRNA molecule that corresponds approximately to a position: position 11,887 according to SEQ ID NO. 12, position 12,036 according to SEQ ID NO. 13, position 2,685 according to SEQ ID NO. 14, position 1,050 according to SEQ ID NO. 15, position 438 according to SEQ ID NO. 16, position 112 according to SEQ ID NO. 17, position 84 according to SEQ ID NO. 18, position 11,655 according to SEQ ID NO. 19, position 11,804 according to SEQ ID NO. 20, position 2,453 according to SEQ ID NO. 21, position 818 according to SEQ ID NO. 22 or position 206 according to SEQ ID NO. 23.

b) Extending the primer at least through a position in the nucleotide sequence of the RNF213 mRNA molecule corresponding to: position 11,887 according to SEQ ID NO. 12, position 12,036 according to SEQ ID NO. 13, position 2,685 according to SEQ ID NO. 14, position 1,050 according to SEQ ID NO. 15, position 438 according to SEQ ID NO. 16, position 112 according to SEQ ID NO. 17, position 84 according to SEQ ID NO. 18, position 11,655 according to SEQ ID NO. 19, position 11,804 according to SEQ ID NO. 20, position 2,453 according to SEQ ID NO. 21, position 818 according to SEQ ID NO. 22 or position 206 according to SEQ ID NO. 23. And

c) Determining whether the extension product of the primer comprises: guanine at a position corresponding to position 11,887 according to SEQ ID NO. 12, guanine at a position 12,036 according to SEQ ID NO. 13, guanine at a position 2,685 according to SEQ ID NO. 14, guanine at a position 1,050 according to SEQ ID NO. 15, guanine at a position 438 according to SEQ ID NO. 16, guanine at a position 112 according to SEQ ID NO. 17, guanine at a position 84 according to SEQ ID NO. 18, cytosine at a position 11,655 according to SEQ ID NO. 19, cytosine at a position 11,804 according to SEQ ID NO. 20, cytosine at a position 2,453 according to SEQ ID NO. 21, cytosine at a position 818 according to SEQ ID NO. 22 or cytosine at a position 206 according to SEQ ID NO. 23.

28. The method of any one of claims 16 to 22, wherein the detecting step comprises:

a) Contacting the biological sample with a primer that hybridizes to a portion of the nucleotide sequence of the RNF213 cDNA molecule near a position corresponding to: position 11,887 according to SEQ ID NO. 31, position 12,036 according to SEQ ID NO. 32, position 2,685 according to SEQ ID NO. 33, position 1,050 according to SEQ ID NO. 34, position 438 according to SEQ ID NO. 35, position 112 according to SEQ ID NO. 36, position 84 according to SEQ ID NO. 37, position 11,655 according to SEQ ID NO. 38, position 11,804 according to SEQ ID NO. 39, position 2,453 according to SEQ ID NO. 40, position 818 according to SEQ ID NO. 41 or position 206 according to SEQ ID NO. 42.

b) Extending the primer at least through a position in the nucleotide sequence of the RNF213 cDNA molecule corresponding to: position 11,887 according to SEQ ID NO. 31, position 12,036 according to SEQ ID NO. 32, position 2,685 according to SEQ ID NO. 33, position 1,050 according to SEQ ID NO. 34, position 438 according to SEQ ID NO. 35, position 112 according to SEQ ID NO. 36, position 84 according to SEQ ID NO. 37, position 11,655 according to SEQ ID NO. 38, position 11,804 according to SEQ ID NO. 39, position 2,453 according to SEQ ID NO. 40, position 818 according to SEQ ID NO. 41 or position 206 according to SEQ ID NO. 42. And

c) Determining whether the extension product of the primer comprises: guanine at a position corresponding to position 11,887 according to SEQ ID NO. 31, guanine at a position 12,036 according to SEQ ID NO. 32, guanine at a position 2,685 according to SEQ ID NO. 33, guanine at a position 1,050 according to SEQ ID NO. 34, guanine at a position 438 according to SEQ ID NO. 35, guanine at a position 112 according to SEQ ID NO. 36, guanine at a position 84 according to SEQ ID NO. 37, cytosine at a position 11,655 according to SEQ ID NO. 38, cytosine at a position 11,804 according to SEQ ID NO. 39, cytosine at a position 2,453 corresponding to SEQ ID NO. 40, cytosine at a position 818 according to SEQ ID NO. 41 or cytosine at a position 206 according to SEQ ID NO. 42.

29. The method of any one of claims 23 to 28, wherein the detecting step comprises sequencing the entire nucleic acid molecule.

30. The method of any one of claims 16 to 22, wherein the detecting step comprises:

a) Amplifying at least a portion of the genomic nucleic acid molecule encoding the human RNF213 polypeptide, wherein the portion comprises: guanine at a position corresponding to position 102,917 according to SEQ ID NO. 2 or its complement; a cytosine at a position corresponding to position 102,391 according to SEQ ID NO. 3 or a complement thereof; or thymine at a position corresponding to position 103,226 according to SEQ ID NO. 4 or a complement thereof;

b) Labeling the amplified nucleic acid molecules with a detectable label;

c) Contacting the labeled nucleic acid molecule with a support comprising a change-specific probe, wherein the change-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleic acid sequence of the amplified nucleic acid molecule, the nucleic acid sequence of the amplified nucleic acid molecule comprising: guanine at a position corresponding to position 102,917 according to SEQ ID NO. 2 or its complement; a cytosine at a position corresponding to position 102,391 according to SEQ ID NO. 3 or a complement thereof; or thymine at a position corresponding to position 103,226 according to SEQ ID NO. 4 or a complement thereof; and

d) Detecting the detectable label.

31. The method of any one of claims 16 to 22, wherein the detecting step comprises:

a) Amplifying at least a portion of the mRNA molecule encoding the human RNF213 polypeptide, wherein the portion comprises: guanine at a position corresponding to position 11,887 according to SEQ ID NO. 12 or its complement; guanine at a position corresponding to position 12,036 according to SEQ ID NO. 13 or its complement; guanine at a position corresponding to position 2,685 according to SEQ ID NO. 14 or its complement; guanine at a position corresponding to position 1,050 according to SEQ ID NO. 15 or its complement; guanine at a position corresponding to position 438 according to SEQ ID NO. 16 or its complement; guanine at a position corresponding to position 112 according to SEQ ID NO. 17 or its complement; guanine at a position corresponding to position 84 according to SEQ ID NO. 18 or its complement; a cytosine at a position corresponding to position 11,655 according to SEQ ID NO. 19 or a complement thereof; a cytosine at a position corresponding to position 11,804 according to SEQ ID NO. 20 or a complement thereof; a cytosine at a position corresponding to position 2,453 according to SEQ ID NO. 21 or a complement thereof; cytosine at a position corresponding to position 818 according to SEQ ID NO. 22 or its complement; or a cytosine at a position corresponding to position 206 according to SEQ ID NO. 23 or a complement thereof;

b) Labeling the amplified nucleic acid molecules with a detectable label;

c) Contacting the labeled nucleic acid molecule with a support comprising a change-specific probe, wherein the change-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleic acid sequence of the amplified nucleic acid molecule, the nucleic acid sequence of the amplified nucleic acid molecule comprising: guanine at a position corresponding to position 11,887 according to SEQ ID NO. 12 or its complement; guanine at a position corresponding to position 12,036 according to SEQ ID NO. 13 or its complement; guanine at a position corresponding to position 2,685 according to SEQ ID NO. 14 or its complement; guanine at a position corresponding to position 1,050 according to SEQ ID NO. 15 or its complement; guanine at a position corresponding to position 438 according to SEQ ID NO. 16 or its complement; guanine at a position corresponding to position 112 according to SEQ ID NO. 17 or its complement; guanine at a position corresponding to position 84 according to SEQ ID NO. 18 or its complement; a cytosine at a position corresponding to position 11,655 according to SEQ ID NO. 19 or a complement thereof; a cytosine at a position corresponding to position 11,804 according to SEQ ID NO. 20 or a complement thereof; a cytosine at a position corresponding to position 2,453 according to SEQ ID NO. 21 or a complement thereof; cytosine at a position corresponding to position 818 according to SEQ ID NO. 22 or its complement; or a cytosine at a position corresponding to position 206 according to SEQ ID NO. 23 or a complement thereof; and

d) Detecting the detectable label.

32. The method of any one of claims 16 to 22, wherein the detecting step comprises:

a) Amplifying at least a portion of the cDNA molecule encoding the human RNF213 polypeptide, wherein the portion comprises: guanine at a position corresponding to position 11,887 according to SEQ ID NO. 31 or its complement; guanine at a position corresponding to position 12,036 according to SEQ ID NO. 32 or its complement; guanine at a position corresponding to position 2,685 according to SEQ ID NO. 33 or its complement; guanine at a position corresponding to position 1,050 according to SEQ ID NO. 34 or its complement; guanine at a position corresponding to position 438 according to SEQ ID NO. 35 or its complement; guanine at a position corresponding to position 112 according to SEQ ID NO. 36 or its complement; guanine at a position corresponding to position 84 according to SEQ ID NO. 37 or its complement; a cytosine at a position corresponding to position 11,655 according to SEQ ID NO. 38 or a complement thereof; a cytosine at a position corresponding to position 11,804 according to SEQ ID NO. 39 or a complement thereof; a cytosine at a position corresponding to position 2,453 according to SEQ ID NO. 40 or a complement thereof; cytosine at a position corresponding to position 818 according to SEQ ID NO. 41 or its complement; or a cytosine at a position corresponding to position 206 according to SEQ ID NO. 42 or its complement;

b) Labeling the amplified nucleic acid molecules with a detectable label;

c) Contacting the labeled nucleic acid molecule with a support comprising a change-specific probe, wherein the change-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleic acid sequence of the amplified nucleic acid molecule, the nucleic acid sequence of the amplified nucleic acid molecule comprising: guanine at a position corresponding to position 11,887 according to SEQ ID NO. 31 or its complement; guanine at a position corresponding to position 12,036 according to SEQ ID NO. 32 or its complement; guanine at a position corresponding to position 2,685 according to SEQ ID NO. 33 or its complement; guanine at a position corresponding to position 1,050 according to SEQ ID NO. 34 or its complement; guanine at a position corresponding to position 438 according to SEQ ID NO. 35 or its complement; guanine at a position corresponding to position 112 according to SEQ ID NO. 36 or its complement; guanine at a position corresponding to position 84 according to SEQ ID NO. 37 or its complement; a cytosine at a position corresponding to position 11,655 according to SEQ ID NO. 38 or a complement thereof; a cytosine at a position corresponding to position 11,804 according to SEQ ID NO. 39 or a complement thereof; a cytosine at a position corresponding to position 2,453 according to SEQ ID NO. 40 or a complement thereof; cytosine at a position corresponding to position 818 according to SEQ ID NO. 41 or its complement; or a cytosine at a position corresponding to position 206 according to SEQ ID NO. 42 or its complement; and

d) Detecting the detectable label.

33. The method of claim 32, wherein the nucleic acid molecule in the sample is mRNA and the mRNA is reverse transcribed into cDNA prior to the amplifying step.

34. The method of any one of claims 16 to 22, wherein the detecting step comprises:

contacting the genomic nucleic acid molecule in the biological sample with a change-specific probe comprising a detectable label, wherein the change-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of the amplified nucleic acid molecule comprising: guanine at a position corresponding to position 102,917 according to SEQ ID NO. 2 or its complement; a cytosine at a position corresponding to position 102,391 according to SEQ ID NO. 3 or a complement thereof; or thymine at a position corresponding to position 103,226 according to SEQ ID NO. 4 or a complement thereof; and

detecting the detectable label.

35. The method of any one of claims 16 to 22, wherein the detecting step comprises:

contacting the mRNA molecules in the biological sample with a change-specific probe comprising a detectable label, wherein the change-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of the amplified nucleic acid molecule comprising: guanine at a position corresponding to position 11,887 according to SEQ ID NO. 12 or its complement; guanine at a position corresponding to position 12,036 according to SEQ ID NO. 13 or its complement; guanine at a position corresponding to position 2,685 according to SEQ ID NO. 14 or its complement; guanine at a position corresponding to position 1,050 according to SEQ ID NO. 15 or its complement; guanine at a position corresponding to position 438 according to SEQ ID NO. 16 or its complement; guanine at a position corresponding to position 112 according to SEQ ID NO. 17 or its complement; guanine at a position corresponding to position 84 according to SEQ ID NO. 18 or its complement; a cytosine at a position corresponding to position 11,655 according to SEQ ID NO. 19 or a complement thereof; a cytosine at a position corresponding to position 11,804 according to SEQ ID NO. 20 or a complement thereof; a cytosine at a position corresponding to position 2,453 according to SEQ ID NO. 21 or a complement thereof; cytosine at a position corresponding to position 818 according to SEQ ID NO. 22 or its complement; or a cytosine at a position corresponding to position 206 according to SEQ ID NO. 23 or a complement thereof; and

Detecting the detectable label.

36. The method of any one of claims 16 to 22, wherein the detecting step comprises:

contacting the cDNA molecule produced from an mRNA molecule in the biological sample with a change-specific probe comprising a detectable label, wherein the change-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of the amplified nucleic acid molecule, the nucleotide sequence of the amplified nucleic acid molecule comprising: guanine at a position corresponding to position 11,887 according to SEQ ID NO. 31 or its complement; guanine at a position corresponding to position 12,036 according to SEQ ID NO. 32 or its complement; guanine at a position corresponding to position 2,685 according to SEQ ID NO. 33 or its complement; guanine at a position corresponding to position 1,050 according to SEQ ID NO. 34 or its complement; guanine at a position corresponding to position 438 according to SEQ ID NO. 35 or its complement; guanine at a position corresponding to position 112 according to SEQ ID NO. 36 or its complement; guanine at a position corresponding to position 84 according to SEQ ID NO. 37 or its complement; a cytosine at a position corresponding to position 11,655 according to SEQ ID NO. 38 or a complement thereof; a cytosine at a position corresponding to position 11,804 according to SEQ ID NO. 39 or a complement thereof; a cytosine at a position corresponding to position 2,453 according to SEQ ID NO. 40 or a complement thereof; cytosine at a position corresponding to position 818 according to SEQ ID NO. 41 or its complement; or a cytosine at a position corresponding to position 206 according to SEQ ID NO. 42 or its complement; and

Detecting the detectable label.

37. A method of treating a subject with a therapeutic agent that treats or inhibits liver disease, wherein the subject is afflicted with liver disease, the method comprising the steps of:

determining whether the subject has a human ring finger protein 213 (RNF 213) polypeptide encoding RNF213 predicted loss of function or missense variant nucleic acid molecule by:

obtaining or having obtained a biological sample from the subject; and

genotyping or has been performed on the biological sample to determine whether the subject has a genotype comprising the RNF213 predictive loss of function or missense variant nucleic acid molecule; and

when the subject is an RNF213 reference, then administering or continuing to administer the therapeutic agent that treats or inhibits liver disease to the subject at a standard dose, and administering an RNF213 inhibitor to the subject; and

administering or continuing to administer the therapeutic agent that treats or inhibits liver disease to the subject in an amount equal to or less than a standard dose when the subject is heterozygous for the RNF213 predicted loss of function or missense variant, and administering an RNF213 inhibitor to the subject;

wherein the presence of a genotype with the RNF 213-predictive loss of function or missense variant nucleic acid molecule encoding the human RNF213 polypeptide indicates that the subject is at reduced risk of developing liver disease.

38. The method of claim 37, wherein the subject is an RNF213 reference and the therapeutic agent for treating or inhibiting liver disease is administered or continued to be administered to the subject at a standard dose and an RNF213 inhibitor is administered.

39. The method of claim 37, wherein the subject is heterozygous for an RNF 213-predictive loss of function or missense variant and the subject is administered or continued to administer the therapeutic agent for treating or inhibiting liver disease in an amount equal to or less than a standard dose and an RNF213 inhibitor is administered.

40. The method of any one of claims 37-39, wherein the RNF213 prediction loss of function or missense variant nucleic acid molecule is a nucleic acid molecule encoding Glu3915Gly, glu3964Gly, glu822Gly, glu350Gly, glu146Gly, glu37Gly, glu28Gly, val3838Leu, val3887Leu, val745Leu, val273Leu, or Val69 Leu.

41. The method of any one of claims 37-39, wherein the RNF213 predicted loss of function or missense variant nucleic acid molecule is a nucleic acid molecule encoding Glu3915Gly or Val3838 Leu.

42. The method of claim 40, wherein the RNF213 predicted loss of function or missense variant nucleic acid molecule is:

A genomic nucleic acid molecule having a nucleotide sequence comprising: guanine at a position corresponding to position 102,917 according to SEQ ID NO. 2, cytosine at a position corresponding to position 102,391 according to SEQ ID NO. 3 or thymine at a position corresponding to position 103,226 according to SEQ ID NO. 4;

43. The method of any one of claims 37 to 42, wherein the genotyping assay comprises sequencing at least a portion of the nucleotide sequence of the RNF213 genomic nucleic acid molecule in the biological sample, wherein the sequenced portion comprises a position corresponding to: position 102,917 according to SEQ ID NO. 2 or a complement thereof; position 102,391 according to SEQ ID NO. 3 or a complement thereof; or position 103,226 according to SEQ ID NO. 4 or a complement thereof;

Wherein when the sequencing portion of the RNF213 genomic nucleic acid molecule in the biological sample comprises: when corresponding to guanine at position 102,917 according to SEQ ID NO. 2, cytosine at position 102,391 according to SEQ ID NO. 3 or thymine at position 103,226 according to SEQ ID NO. 4, then the RNF213 genomic nucleic acid molecule in the biological sample is an RNF213 predicted loss of function or missense variant genomic nucleic acid molecule.

44. The method of any one of claims 37 to 42, wherein the genotyping assay comprises sequencing at least a portion of the nucleotide sequence of the RNF213 mRNA molecule in the biological sample, wherein the sequenced portion comprises a position corresponding to: position 11,887 according to SEQ ID NO. 12 or a complement thereof; position 12,036 according to SEQ ID NO. 13 or a complement thereof; position 2,685 according to SEQ ID NO. 14 or a complement thereof; position 1,050 according to SEQ ID NO. 15 or its complement; position 438 according to SEQ ID NO. 16 or a complement thereof; position 112 according to SEQ ID NO. 17 or a complement thereof; or position 84 according to SEQ ID NO. 18 or a complement thereof; position 11,655 according to SEQ ID NO. 19 or a complement thereof; position 11,804 according to SEQ ID NO. 20 or a complement thereof; position 2,453 according to SEQ ID NO. 21 or a complement thereof; position 818 according to SEQ ID NO. 22 or a complement thereof; or position 206 according to SEQ ID NO. 23 or a complement thereof;

Wherein when the sequencing portion of the RNF213 mRNA molecule in the biological sample comprises: when the biological sample is predicted to be an mRNA of a missense variant 213 or an mRNA of an RNF213 molecule when the biological sample is a mRNA corresponding to guanine at position 11,887 according to SEQ ID NO:12, guanine at position 12,036 according to SEQ ID NO:13, guanine at position 2,685 according to SEQ ID NO:14, guanine at position 1,050 according to SEQ ID NO:15, guanine at position 438 according to SEQ ID NO:16, guanine at position 112 according to SEQ ID NO:17, guanine at position 84 according to SEQ ID NO:18, cytosine at position 11,655 according to SEQ ID NO:19, cytosine at position 11,804 according to SEQ ID NO:20, cytosine at position 2,453 corresponding to SEQ ID NO:21, cytosine at position 818 according to SEQ ID NO:22 or cytosine at position 206 according to SEQ ID NO: 23.

45. The method of any one of claims 37 to 42, wherein the genotyping assay comprises sequencing at least a portion of the nucleotide sequence of the RNF213 cDNA molecule in the biological sample, wherein the sequenced portion comprises a position corresponding to: position 11,887 according to SEQ ID NO. 31 or a complement thereof; position 12,036 according to SEQ ID NO. 32 or a complement thereof; position 2,685 according to SEQ ID NO. 33 or a complement thereof; position 1,050 according to SEQ ID NO. 34 or its complement; position 438 according to SEQ ID NO. 35 or its complement; position 112 according to SEQ ID NO. 36 or a complement thereof; position 84 according to SEQ ID NO. 37 or a complement thereof; position 11,655 according to SEQ ID NO. 38 or a complement thereof; position 11,804 according to SEQ ID NO. 39 or a complement thereof; position 2,453 according to SEQ ID NO. 40 or a complement thereof; position 818 according to SEQ ID NO. 41 or a complement thereof; or position 206 according to SEQ ID NO. 42 or its complement;

Wherein when the sequencing portion of the RNF213cDNA molecule in the biological sample comprises: when guanine at a position corresponding to position 11,887 according to SEQ ID NO. 31, guanine at a position corresponding to position 12,036 according to SEQ ID NO. 32, guanine at a position corresponding to position 2,685 according to SEQ ID NO. 33, guanine at a position corresponding to position 1,050 according to SEQ ID NO. 34, guanine at a position corresponding to position 438 according to SEQ ID NO. 35, guanine at a position corresponding to position 112 according to SEQ ID NO. 36, guanine at a position corresponding to position 84 according to SEQ ID NO. 37, cytosine at a position corresponding to position 11,655 according to SEQ ID NO. 38, cytosine at a position corresponding to position 11,804 according to SEQ ID NO. 39, cytosine at a position corresponding to position 2,453 according to SEQ ID NO. 40, cytosine at a position 818 according to SEQ ID NO. 41 or cytosine at a position corresponding to position 206 according to SEQ ID NO. 42, then the biological sample is a loss of function of the RNA 213 or the RNF 213-error variant cDNA.

46. The method of any one of claims 37 to 42, wherein the genotyping assay comprises:

47. The method of any one of claims 37 to 42, wherein the genotyping assay comprises:

c) Determining whether the extension product of the primer comprises: guanine at a position corresponding to position 11,887 according to SEQ ID NO. 12, guanine at a position 12,036 according to SEQ ID NO. 13, guanine at a position 2,685 according to SEQ ID NO. 14, guanine at a position 1,050 according to SEQ ID NO. 15, guanine at a position 438 according to SEQ ID NO. 16, guanine at a position 112 according to SEQ ID NO. 17, guanine at a position 84 according to SEQ ID NO. 18, cytosine at a position 11,655 according to SEQ ID NO. 19, cytosine at a position 11,804 according to SEQ ID NO. 20, cytosine at a position 2,453 corresponding to SEQ ID NO. 21, cytosine at a position 818 according to SEQ ID NO. 22 or cytosine at a position 206 according to SEQ ID NO. 23.

48. The method of any one of claims 37 to 42, wherein the genotyping assay comprises:

c) Determining whether the extension product of the primer comprises: guanine at a position corresponding to position 11,887 according to SEQ ID NO. 31, guanine at a position 12,036 according to SEQ ID NO. 32, guanine at a position 2,685 according to SEQ ID NO. 33, guanine at a position 1,050 according to SEQ ID NO. 34, guanine at a position 438 according to SEQ ID NO. 35, guanine at a position 112 according to SEQ ID NO. 36, guanine at a position 84 according to SEQ ID NO. 37, cytosine at a position 11,655 according to SEQ ID NO. 38, cytosine at a position 11,804 according to SEQ ID NO. 39, cytosine at a position 2,453 according to SEQ ID NO. 40, cytosine at a position 818 according to SEQ ID NO. 41 or cytosine at a position 206 according to SEQ ID NO. 42.

49. The method of any one of claims 43 to 48, wherein the genotyping assay comprises sequencing the entire nucleic acid molecule.

50. The method of any one of claims 37 to 42, wherein the genotyping assay comprises:

b) Labeling the amplified nucleic acid molecules with a detectable label;

d) Detecting the detectable label.

51. The method of any one of claims 37 to 42, wherein the genotyping assay comprises:

b) Labeling the amplified nucleic acid molecules with a detectable label;

d) Detecting the detectable label.

52. The method of any one of claims 37 to 42, wherein the genotyping assay comprises:

b) Labeling the amplified nucleic acid molecules with a detectable label;

d) Detecting the detectable label.

53. The method of claim 52, wherein the nucleic acid molecule in the sample is mRNA and the mRNA is reverse transcribed into cDNA prior to the amplifying step.

54. The method of any one of claims 37 to 42, wherein the genotyping assay comprises:

contacting the nucleic acid molecule in the biological sample with a change-specific probe comprising a detectable label, wherein the change-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of the amplified nucleic acid molecule comprising: guanine at a position corresponding to position 102,917 according to SEQ ID NO. 2 or its complement; a cytosine at a position corresponding to position 102,391 according to SEQ ID NO. 3 or a complement thereof; or thymine at a position corresponding to position 103,226 according to SEQ ID NO. 4 or a complement thereof; and

detecting the detectable label.

55. The method of any one of claims 37 to 42, wherein the genotyping assay comprises:

contacting the nucleic acid molecule in the biological sample with a change-specific probe comprising a detectable label, wherein the change-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of the amplified nucleic acid molecule comprising: guanine at a position corresponding to position 11,887 according to SEQ ID NO. 12 or its complement; guanine at a position corresponding to position 12,036 according to SEQ ID NO. 13 or its complement; guanine at a position corresponding to position 2,685 according to SEQ ID NO. 14 or its complement; guanine at a position corresponding to position 1,050 according to SEQ ID NO. 15 or its complement; guanine at a position corresponding to position 438 according to SEQ ID NO. 16 or its complement; guanine at a position corresponding to position 112 according to SEQ ID NO. 17 or its complement; guanine at a position corresponding to position 84 according to SEQ ID NO. 18 or its complement; a cytosine at a position corresponding to position 11,655 according to SEQ ID NO. 19 or a complement thereof; a cytosine at a position corresponding to position 11,804 according to SEQ ID NO. 20 or a complement thereof; a cytosine at a position corresponding to position 2,453 according to SEQ ID NO. 21 or a complement thereof; cytosine at a position corresponding to position 818 according to SEQ ID NO. 22 or its complement; or cytosine at a position corresponding to position 206 according to SEQ ID NO. 23; and

Detecting the detectable label.

56. The method of any one of claims 37 to 42, wherein the genotyping assay comprises:

contacting the cDNA molecule in the biological sample with a change-specific probe comprising a detectable label, wherein the change-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of the amplified nucleic acid molecule, the nucleotide sequence of the amplified nucleic acid molecule comprising: guanine at a position corresponding to position 11,887 according to SEQ ID NO. 31 or its complement; guanine at a position corresponding to position 12,036 according to SEQ ID NO. 32 or its complement; guanine at a position corresponding to position 2,685 according to SEQ ID NO. 33 or its complement; guanine at a position corresponding to position 1,050 according to SEQ ID NO. 34 or its complement; guanine at a position corresponding to position 438 according to SEQ ID NO. 35 or its complement; guanine at a position corresponding to position 112 according to SEQ ID NO. 36 or its complement; guanine at a position corresponding to position 84 according to SEQ ID NO. 37 or its complement; a cytosine at a position corresponding to position 11,655 according to SEQ ID NO. 38 or a complement thereof; a cytosine at a position corresponding to position 11,804 according to SEQ ID NO. 39 or a complement thereof; a cytosine at a position corresponding to position 2,453 according to SEQ ID NO. 40 or a complement thereof; cytosine at a position corresponding to position 818 according to SEQ ID NO. 41 or its complement; or a cytosine at a position corresponding to position 206 according to SEQ ID NO. 42 or its complement; and

Detecting the detectable label.

57. The method of any one of claims 37-56, wherein the nucleic acid molecule is present in a cell obtained from the subject.

58. The method of any one of claims 37-57, wherein the RNF213 inhibitor comprises an antisense nucleic acid molecule, small interfering RNA (siRNA), or short hairpin RNA (shRNA) that hybridizes to RNF213 mRNA.

59. The method of any one of claims 37-57, wherein the RNF213 inhibitor comprises a Cas protein and a guide RNA (gRNA) that hybridizes to a gRNA recognition sequence within an RNF213 genomic nucleic acid molecule.

60. The method of claim 59, wherein the Cas protein is Cas9 or Cpf1.

61. The method of claim 59 or claim 60, wherein the gRNA recognition sequence comprises or nearly corresponds to the position: position 102,917 according to SEQ ID NO. 1, position 102,391 according to SEQ ID NO. 1 or position 103,226 according to SEQ ID NO. 1.

62. The method of claim 59 or claim 60, wherein the gRNA recognition sequence is located about 1000, about 500, about 400, about 300, about 200, about 100, about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 15, about 10, or about 5 nucleotides from positions corresponding to: position 102,917 according to SEQ ID NO. 1, position 102,391 according to SEQ ID NO. 1 or position 103,226 according to SEQ ID NO. 1.

63. The method of claim 59 or claim 60, wherein a Protospacer Adjacent Motif (PAM) sequence is about 2 to 6 nucleotides downstream of the gRNA recognition sequence.

64. The method of any one of claims 59 to 63, wherein the gRNA comprises about 17 to about 23 nucleotides.

65. The method of any one of claims 59 to 64, wherein the gRNA recognition sequence comprises a nucleotide sequence according to any one of SEQ ID NOs 62-81.

66. A method of identifying a subject at increased risk of having a liver disease, wherein the method comprises:

determining or having determined the presence or absence of a ring finger protein 213 (RNF 213) predictive loss of function or missense variant nucleic acid molecule encoding a human RNF213 polypeptide in a biological sample obtained from the subject;

wherein:

when the subject is RNF213 reference, then the subject is at increased risk of having liver disease; and is also provided with

When the subject is heterozygous for the RNF213 predicted loss of function or missense variant or homozygous for the RNF213 predicted loss of function or missense variant, the subject is at reduced risk of suffering from liver disease.

67. The method of claim 66, wherein said RNF213 predicted loss of function or missense variant nucleic acid molecule is a nucleic acid molecule encoding Glu3915Gly, glu3964Gly, glu822Gly, glu350Gly, glu146Gly, glu37Gly, glu28Gly, val3838Leu, val3887Leu, val745Leu, val273Leu, or Val69 Leu.

68. The method of claim 66, wherein said RNF213 predicted loss of function or missense variant nucleic acid molecule is a nucleic acid molecule encoding Glu3915Gly or Val3838 Leu.

69. The method of claim 67, wherein the RNF213 predicted loss of function or missense variant nucleic acid molecule is:

70. The method of any one of claims 66-69, wherein the determining step is performed in vitro.

71. The method of any one of claims 66-70, wherein the determining step comprises sequencing at least a portion of a nucleotide sequence of the RNF213 genomic nucleic acid molecule in the biological sample, wherein the sequenced portion comprises a position corresponding to: position 102,917 according to SEQ ID NO. 2 or a complement thereof; position 102,391 according to SEQ ID NO. 3 or a complement thereof; or position 103,226 according to SEQ ID NO. 4 or a complement thereof;

72. The method of any one of claims 66-70, wherein the determining step comprises sequencing at least a portion of the nucleotide sequence of the RNF213 mRNA molecule in the biological sample, wherein the sequenced portion comprises a position corresponding to: position 11,887 according to SEQ ID NO. 12 or a complement thereof; position 12,036 according to SEQ ID NO. 13 or a complement thereof; position 2,685 according to SEQ ID NO. 14 or a complement thereof; position 1,050 according to SEQ ID NO. 15 or its complement; position 438 according to SEQ ID NO. 16 or a complement thereof; position 112 according to SEQ ID NO. 17 or a complement thereof; or position 84 according to SEQ ID NO. 18 or a complement thereof; position 11,655 according to SEQ ID NO. 19 or a complement thereof; position 11,804 according to SEQ ID NO. 20 or a complement thereof; position 2,453 according to SEQ ID NO. 21 or a complement thereof; position 818 according to SEQ ID NO. 22 or a complement thereof; or position 206 according to SEQ ID NO. 23 or a complement thereof;

Wherein when the sequencing portion of the RNF213 mRNA molecule in the biological sample comprises: guanine at a position corresponding to position 11,887 according to SEQ ID NO. 12, guanine at a position 12,036 according to SEQ ID NO. 13, guanine at a position 2,685 according to SEQ ID NO. 14, guanine at a position 1,050 according to SEQ ID NO. 15, guanine at a position 438 according to SEQ ID NO. 16, guanine at a position 112 according to SEQ ID NO. 17, guanine at a position 84 according to SEQ ID NO. 18, cytosine at a position 11,655 according to SEQ ID NO. 19, cytosine at a position 11,804 according to SEQ ID NO. 20, cytosine at a position 2,453 corresponding to SEQ ID NO. 21, cytosine at a position 818 according to SEQ ID NO. 22 or cytosine at a position 206 according to SEQ ID NO. 23. The RNF213 mRNA molecule in the biological sample is an RNF213 predicted loss of function or missense variant mRNA molecule.

73. The method of any one of claims 66-70, wherein the determining step comprises sequencing at least a portion of the nucleotide sequence of the RNF213 cDNA molecule in the biological sample, wherein the sequenced portion comprises a position corresponding to: position 11,887 according to SEQ ID NO. 31 or a complement thereof; position 12,036 according to SEQ ID NO. 32 or a complement thereof; position 2,685 according to SEQ ID NO. 33 or a complement thereof; position 1,050 according to SEQ ID NO. 34 or its complement; position 438 according to SEQ ID NO. 35 or its complement; position 112 according to SEQ ID NO. 36 or a complement thereof; position 84 according to SEQ ID NO. 37 or a complement thereof; position 11,655 according to SEQ ID NO. 38 or a complement thereof; position 11,804 according to SEQ ID NO. 39 or a complement thereof; position 2,453 according to SEQ ID NO. 40 or a complement thereof; position 818 according to SEQ ID NO. 41 or a complement thereof; or position 206 according to SEQ ID NO. 42 or its complement;

Wherein when the sequencing portion of the RNF213cDNA molecule in the biological sample comprises: guanine at a position corresponding to position 11,887 according to SEQ ID NO. 31, guanine at a position 12,036 according to SEQ ID NO. 32, guanine at a position 2,685 according to SEQ ID NO. 33, guanine at a position 1,050 according to SEQ ID NO. 34, guanine at a position 438 according to SEQ ID NO. 35, guanine at a position 112 according to SEQ ID NO. 36, guanine at a position 84 according to SEQ ID NO. 37, cytosine at a position 11,655 according to SEQ ID NO. 38, cytosine at a position 11,804 according to SEQ ID NO. 39, cytosine at a position 2,453 corresponding to SEQ ID NO. 40, cytosine at a position 818 according to SEQ ID NO. 41 or cytosine at a position 206 according to SEQ ID NO. 42; the RNF213cDNA molecule in the biological sample is a RNF213 predictive loss of function or missense variant cDNA molecule.

74. The method of any one of claims 66 to 70, wherein the determining step comprises:

75. The method of any one of claims 66 to 70, wherein the determining step comprises:

76. The method of any one of claims 66 to 70, wherein the determining step comprises:

b) Extending the primer at least through a position in the nucleotide sequence of the RNF213 cDNA molecule corresponding to: position 11,887 according to SEQ ID NO. 31, position 12,036 according to SEQ ID NO. 32, position 2,685 according to SEQ ID NO. 33, position 1,050 according to SEQ ID NO. 34, position 438 according to SEQ ID NO. 35, position 112 according to SEQ ID NO. 36, position 84 according to SEQ ID NO. 37, position 11,655 according to SEQ ID NO. 38, position 11,804 according to SEQ ID NO. 39, position 2,453 according to SEQ ID NO. 40, position 818 according to SEQ ID NO. 41, position 206 according to SEQ ID NO. 42; and

77. The method of any one of claims 71 to 76, wherein the determining step comprises sequencing the entire nucleic acid molecule.

78. The method of any one of claims 66 to 70, wherein the determining step comprises:

b) Labeling the amplified nucleic acid molecules with a detectable label;

d) Detecting the detectable label.

79. The method of any one of claims 66 to 70, wherein the determining step comprises:

b) Labeling the amplified nucleic acid molecules with a detectable label;

d) Detecting the detectable label.

80. The method of any one of claims 66 to 70, wherein the determining step comprises:

b) Labeling the amplified nucleic acid molecules with a detectable label;

d) Detecting the detectable label.

81. The method of claim 80, wherein the nucleic acid molecule in the sample is mRNA and the mRNA is reverse transcribed into cDNA prior to the amplifying step.

82. The method of any one of claims 66 to 70, wherein the detecting step comprises:

detecting the detectable label.

83. The method of any one of claims 66 to 70, wherein the detecting step comprises:

Detecting the detectable label.

84. The method of any one of claims 66 to 70, wherein the detecting step comprises:

Detecting the detectable label.

85. The method of any one of claims 66-84, wherein the subject is an RNF213 reference and a therapeutic agent that treats or inhibits liver disease is administered to the subject at a standard dose and an RNF213 inhibitor is administered.

86. The method of any one of claims 66-84, wherein the subject is heterozygous for an RNF 213-predictive loss of function or missense variant and a therapeutic agent for treating or inhibiting liver disease is administered to the subject in an amount equal to or less than a standard dose and an RNF213 inhibitor is administered.

87. A therapeutic agent for treating or inhibiting liver disease, for treating liver disease in a subject having:

a genomic nucleic acid molecule having a nucleotide sequence encoding a human ring finger protein 213 (RNF 213) polypeptide, wherein the nucleotide sequence comprises: guanine at a position corresponding to position 102,917 according to SEQ ID NO. 2 or its complement; a cytosine at a position corresponding to position 102,391 according to SEQ ID NO. 3 or a complement thereof; or thymine at a position corresponding to position 103,226 according to SEQ ID NO. 4 or a complement thereof;

An mRNA molecule having a nucleotide sequence encoding a human RNF213 polypeptide, wherein the nucleotide sequence comprises: guanine at a position corresponding to position 11,887 according to SEQ ID NO. 12 or its complement; guanine at a position corresponding to position 12,036 according to SEQ ID NO. 13 or its complement; guanine at a position corresponding to position 2,685 according to SEQ ID NO. 14 or its complement; guanine at a position corresponding to position 1,050 according to SEQ ID NO. 15 or its complement; guanine at a position corresponding to position 438 according to SEQ ID NO. 16 or its complement; guanine at a position corresponding to position 112 according to SEQ ID NO. 17 or its complement; guanine at a position corresponding to position 84 according to SEQ ID NO. 18 or its complement; a cytosine at a position corresponding to position 11,655 according to SEQ ID NO. 19 or a complement thereof; a cytosine at a position corresponding to position 11,804 according to SEQ ID NO. 20 or a complement thereof; a cytosine at a position corresponding to position 2,453 according to SEQ ID NO. 21 or a complement thereof; cytosine at a position corresponding to position 818 according to SEQ ID NO. 22 or its complement; or a cytosine at a position corresponding to position 206 according to SEQ ID NO. 23 or a complement thereof; or (b)

A cDNA molecule having a nucleotide sequence encoding a human RNF213 polypeptide, wherein the nucleotide sequence comprises: guanine at a position corresponding to position 11,887 according to SEQ ID NO. 31 or its complement; guanine at a position corresponding to position 12,036 according to SEQ ID NO. 32 or its complement; guanine at a position corresponding to position 2,685 according to SEQ ID NO. 33 or its complement; guanine at a position corresponding to position 1,050 according to SEQ ID NO. 34 or its complement; guanine at a position corresponding to position 438 according to SEQ ID NO. 35 or its complement; guanine at a position corresponding to position 112 according to SEQ ID NO. 36 or its complement; guanine at a position corresponding to position 84 according to SEQ ID NO. 37 or its complement; a cytosine at a position corresponding to position 11,655 according to SEQ ID NO. 38 or a complement thereof; a cytosine at a position corresponding to position 11,804 according to SEQ ID NO. 39 or a complement thereof; a cytosine at a position corresponding to position 2,453 according to SEQ ID NO. 40 or a complement thereof; cytosine at a position corresponding to position 818 according to SEQ ID NO. 41 or its complement; or a cytosine at a position corresponding to position 206 according to SEQ ID NO. 42 or its complement.

88. A ring finger protein 213 (RNF 213) inhibitor for use in treating liver disease in a subject having:

a genomic nucleic acid molecule having a nucleotide sequence encoding a human ring finger protein 213 polypeptide, wherein the nucleotide sequence comprises: guanine at a position corresponding to position 102,917 according to SEQ ID NO. 2 or its complement; a cytosine at a position corresponding to position 102,391 according to SEQ ID NO. 3 or a complement thereof; or thymine at a position corresponding to position 103,226 according to SEQ ID NO. 4 or a complement thereof;

89. The RNF213 inhibitor of claim 88, wherein the inhibitor is an antisense nucleic acid molecule, small interfering RNA (siRNA), or short hairpin RNA (shRNA) that hybridizes to RNF213 mRNA.

90. The RNF213 inhibitor of claim 88, comprising a Cas protein and a guide RNA (gRNA) that hybridizes to a gRNA recognition sequence within the RNF213 genomic nucleic acid molecule.

91. The RNF213 inhibitor of claim 90, wherein the Cas protein is Cas9 or Cpf1.

92. The RNF213 inhibitor of claim 90 or claim 91, wherein the gRNA recognition sequence comprises or is proximal to: position 102,917 according to SEQ ID NO. 1, position 102,391 according to SEQ ID NO. 1 or position 103,226 according to SEQ ID NO. 1.

93. The RNF213 inhibitor of claim 90 or claim 91, wherein the gRNA recognition sequence is located about 1000, about 500, about 400, about 300, about 200, about 100, about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 15, about 10, or about 5 nucleotides from positions corresponding to: position 102,917 according to SEQ ID NO. 1, position 102,391 according to SEQ ID NO. 1 or position 103,226 according to SEQ ID NO. 1.

94. The RNF213 inhibitor of claim 90 or claim 91, wherein a Protospacer Adjacent Motif (PAM) sequence is about 2 to about 6 nucleotides downstream of the gRNA recognition sequence.

95. The RNF213 inhibitor of any one of claims 90 to 94, wherein the gRNA comprises about 17 to about 23 nucleotides.

96. The RNF213 inhibitor of any one of claims 90 to 95, wherein the gRNA recognition sequence comprises a nucleotide sequence according to any one of SEQ ID NOs 62-81.