CN110577953B - Gene mutant and application thereof - Google Patents
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- CN110577953B CN110577953B CN201810595667.9A CN201810595667A CN110577953B CN 110577953 B CN110577953 B CN 110577953B CN 201810595667 A CN201810595667 A CN 201810595667A CN 110577953 B CN110577953 B CN 110577953B
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Abstract
The invention discloses nucleic acid, gene mutation and application thereof. Wherein the nucleic acid has at least one mutation type selected from the group consisting of: c.379G > A, c.137T > A/c.980G > A or c.494C > A/c.980G > A. By detecting the presence or absence of these mutations in a biological sample, it is possible to effectively detect whether or not the biological sample is suffering from immunodeficiency.
Description
Technical Field
The invention relates to a gene mutant and application thereof. In particular, the invention relates to nucleic acids, genetic mutations, proteins, systems for screening biological samples for immunodeficiency disorders, agents for screening for treatment of immunodeficiency disorders, kits for screening biological samples for immunodeficiency disorders, biological models, constructs, and recombinant cells.
Background
Common Variant Immunodeficiency Disease (CVID) and selective IgA deficiency (SIgAD) are the most common Primary Immunodeficiency Disease (PID). The proportion of these two diseases is one quarter of the worldwide PID. CVID is mainly characterized by a reduction in serum immunoglobulins, especially IgA, igE and IgG, with a morbidity of about 1/50000.CVID clinical manifestations are highly heterogeneous, patients can develop recurrent mucosal infections, and 20% -30% of these are accompanied by autoimmune diseases, splenomegaly and malignancy. CVID inheritance patterns are variable, and it is now found that both normally-stained stealth inheritance and autosomal dominant inheritance are present. SIgAD is similar to CVID in terms of genetic patterns, either autosomal recessive or autosomal dominant, or sporadic onset. The incidence rate of SIgAD has race specificity, the incidence rate of caucasian is about 1/223-1/1000, and Japanese is 1/18500; the prevalence of SIgAD in China is 1/4100.SIgAD patients may have no symptoms for a long period of time, and many patients only have upper respiratory tract infection; some patients develop various concomitant diseases, particularly autoimmune diseases (about 50%) and allergic diseases; some patients are accompanied by mental retardation and paresthesia, and are closely related to primary epilepsy; some patients also have asthma, and about 10% of patients with European asthma are diagnosed with SIgAD. Studies found that some SIgAD patients developed CVID and that CVID patients were found in other SIgAD families, evidence that CVID and SIgAD may be caused by the same genetic mutation.
Meanwhile, an exome refers to a collection of all exon regions of the human genome, comprising flanking sequences of protein coding and untranslated regions, which encompass most of the functional variations associated with the individual phenotype. The exome sequencing technology refers to a method for capturing and enriching exome sequences of human genome by utilizing a special probe and then performing high-throughput sequencing and bioinformatics analysis by utilizing a second generation sequencing technology. The human whole genome has 180,000 exons, accounting for 1% of the whole genome sequence, while 85% of the pathogenic mutations (particularly rare pathogenic variations) are located in this region. Therefore, for rare diseases such as CVID and SIgAD, the exome sequencing analysis is more economical and efficient than the whole genome sequencing analysis. Furthermore, with the same budget, the depth of exon sequencing is higher, which is more favorable for finding new pathogenic genes. The novel pathogenic genes discovered by the exome sequencing technology can be verified by adopting target gene association analysis in patients with large sample size and normal control, and more pathogenic sites can be discovered at lower sequencing cost.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, an object of the present invention is to propose a method capable of effectively screening biological samples suffering from immunodeficiency diseases.
The human Mendelian genetic database (OMIM) currently records 13 CVID subtypes, 13 pathogenic genes (CR 2, IRF2BP2, ICOS, NFKB1, IL21, LRBA, IKZF1, NFKB2, CD81, MS4A1, CD19, TNFRSF13B, and TNFRSF 13C). OMIM only records one pathogenic gene TNFRSF13B of SIgAD. However, although several CVIDs and one SIgAD causative gene have been found, the cause of the disease in many patients cannot be explained. Therefore, to improve early diagnosis and therapeutic effects of CVID and SIgAD, it is important to find more new pathogenic genes of CVID and SIgAD as soon as possible.
Based on the findings of the facts and problems described above, the inventors cloned a new pathogenic gene of CVID and SIgAD, RAD50, and found that CVID would occur if RAD50 of a biological sample had homozygosity c.379G > A or compound heterozygosity c.137T > A/c.1400G > A by high throughput exome sequencing; SIgAD can occur if a biological sample develops a complex heterozygous c.494C > A/c.980G > A for RAD 50.
The c.379G > A referred to in the present application means that the 379 th base of the coding region of the RAD50 gene is changed by homozygote G- > A; the 137T > A/c.0G > A means that the 137 th basic group of the coding region of the RAD50 gene is changed by heterozygous T- > A and the 980 th basic group of the coding region is changed by heterozygous G- > A; and c.494C > A/c.0G > A means that the 494 th base of the coding region of the RAD50 gene is changed by heterozygous C- > A and the 980 th base of the coding region is changed by heterozygous G- > A.
According to a first aspect of the invention, the invention proposes a nucleic acid. According to an embodiment of the invention, the nucleic acid has at least one type of mutation compared to the wild-type RAD50 gene selected from the group consisting of: c.379G > A, c.137T > A/c.980G > A or c.494C > A/c.980G > A. According to the embodiments of the present invention, the inventors cloned a novel CVID and SIgAD pathogenic gene RAD50 gene, and determined that mutation of RAD50 gene to at least one of the above was closely related to the onset of primary immunodeficiency diseases (CVID and SIgAD), so that by detecting the presence or absence of these mutants in biological samples, it was possible to effectively detect whether or not the biological samples were suffering from immunodeficiency diseases, particularly CVID and SIgAD.
According to a second aspect of the invention, the invention proposes a genetic mutation. According to an embodiment of the invention, the nucleic acid has at least one type of mutation compared to the wild-type RAD50 gene selected from the group consisting of: c.379G > A, c.137T > A/c.980G > A or c.494C > A/c.980G > A. According to the embodiment of the invention, the inventor discovers a novel CVID and SIgAD pathogenic gene RAD50 gene and determines that the mutation of the RAD50 gene is closely related to the incidence of immunodeficiency diseases, especially primary immunodeficiency diseases such as CVID and SIgAD, and the like, so that whether the biological sample suffers from immunodeficiency diseases, especially whether the biological sample suffers from primary immunodeficiency diseases such as CVID and SIgAD, and the like can be effectively detected by detecting whether the mutation of the RAD50 gene occurs in the biological sample at the position.
According to a third aspect of the present invention, the present invention provides a protein. According to an embodiment of the invention, the protein has at least one of the following types of mutations compared to the amino acid sequence of the protein expressed by the wild-type RAD50 gene: (1) Isoleucine at amino acid sequence position 46 is mutated to valine and arginine at position 327 is mutated to histidine ammonia, the protein having the amino acid sequence as set forth in SEQ ID NO:1, and a polypeptide sequence shown in the specification; (2) Amino acid sequence 127, and from valine to isoleucine homozygous mutation, said protein having the amino acid sequence as set forth in SEQ ID NO:2, and a polypeptide sequence represented by the following formula (2); (3) Proline at amino acid sequence 165 is mutated to histidine and arginine at position 327 is mutated to histidine, the protein having the amino acid sequence as set forth in SEQ ID NO:3, and a polypeptide having the amino acid sequence shown in 3. The inventors cloned a novel CVID and SIgAD pathogenic gene-RAD 50 gene, and determined that mutation of RAD50 gene according to the embodiments of the present invention is closely related to primary immunodeficiency diseases, especially CVID and SIgAD pathogenesis, so that whether a biological sample suffers from primary immunodeficiency diseases, especially CVID and SIgAD can be effectively detected by detecting whether proteins expressing these mutated RAD50 genes exist in the biological sample.
According to a fourth aspect of the invention, the present invention provides the use of a reagent for detecting a nucleic acid as described hereinbefore or a mutation in a gene as described hereinbefore or a protein as described hereinbefore in the preparation of a kit. According to an embodiment of the invention, the agent is used for diagnosing primary immunodeficiency. According to the embodiment of the invention, the reagent can effectively screen biological samples with immunodeficiency diseases, especially CVID, SIgAD and other primary immunodeficiency diseases, and further can be used for preparing a kit for screening biological samples with primary immunodeficiency diseases, especially CVID and SIgAD primary immunodeficiency diseases.
According to a fifth aspect of the invention, the invention proposes the use of a biological model for screening drugs. According to an embodiment of the invention, the biological model carries at least one of: (1) the nucleic acid described above; (2) the aforementioned gene mutation; (3) expressing the aforementioned protein. By "a biological model carries the nucleic acid as described above" it is meant that the biological model of the invention carries a gene having at least one type of mutation as compared to the wild-type RAD50 gene selected from the group consisting of: the nucleic acid sequence of the RAD50 gene mutant of c.379G > A, c.137T > A/c.920G > A or c.284C > A/c.460G > A or the nucleic acid sequence of the gene mutants comprising both; "biological model carrying the aforementioned genetic mutation" means that the biological model of the present invention carries a RAD50 gene having at least one type of mutation selected from the group consisting of: the RAD50 gene mutation of c.379G > A, c.137T > A/c.980G > A or c.494C > A/c.980G > A or both. By "the biological model carries the aforementioned protein" it is meant that the biological model of the invention carries a mutation of at least one type selected from the group consisting of: a protein expressed by a RAD50 gene mutant of c.379G > A, c.137T > A/c.920G > A or c.284C > A/c.460G > A or a protein expressed by a gene comprising both of the above-mentioned various gene mutations. The biological model according to the embodiment of the present invention can be effectively used as a model for the related study of immunodeficiency diseases, particularly primary immunodeficiency diseases such as CVID and SIgAD.
According to a sixth aspect of the present invention, the present invention provides a medicament for the treatment of immunodeficiency disorders. According to an embodiment of the present invention, the medicament contains: an agent which specifically alters the nucleic acid as described above or the gene mutation as described above or the protein as described above. Specific changes herein mean that the mutated nucleic acid or the site of gene mutation or the mutated protein can be restored to the original wild state or other non-pathogenic state without having a substantial effect on other sequences of the genome of the individual. The medicine according to the embodiment of the invention can specifically prevent or treat immunodeficiency diseases, especially primary immunodeficiency diseases such as CVID, SIgAD and the like.
According to a seventh aspect of the invention, the invention provides a method of screening a biological sample for immunodeficiency disease. According to an embodiment of the invention, the method comprises the steps of: extracting a nucleic acid sample from a biological sample; determining the nucleic acid sequence of the nucleic acid sample; a nucleic acid sequence of the nucleic acid sample, or a complement thereof, having at least one type of mutation as compared to the wild-type RAD50 gene selected from the group consisting of: c.379G > A, c.137T > A/c.920G > A or c.494C > A/c.920G > A are indicative of an immunodeficiency disorder in the biological sample. By the method for screening the biological samples with immunodeficiency diseases, which is provided by the embodiment of the invention, the biological samples with immunodeficiency diseases, especially primary immunodeficiency diseases such as CVID, SIgAD and the like, can be effectively screened.
According to an eighth aspect of the invention, the invention provides a system for screening a biological sample for immunodeficiency disease. According to an embodiment of the invention, the system comprises: a nucleic acid extraction device for extracting a nucleic acid sample from the biological sample; a nucleic acid sequence determination device, coupled to the nucleic acid extraction device, for analyzing the nucleic acid sample to determine a nucleic acid sequence of the nucleic acid sample; a judgment means connected to the nucleic acid sequence determination means so that the nucleic acid has at least one type of mutation selected from the group consisting of: c.379G > A, c.137T > A/c.980G > A or c.494C > A/c.980G > A, and determining whether said biological sample has immunodeficiency disease. By utilizing the system, the method for screening the biological samples with the immunodeficiency diseases, especially the primary immunodeficiency diseases such as CVID and SIgAD, can be effectively implemented, so that the biological samples with the immunodeficiency diseases, especially the primary immunodeficiency diseases such as CVID and SIgAD, can be effectively screened.
According to a ninth aspect of the invention, the invention provides a kit for screening a biological sample for immunodeficiency disease. According to an embodiment of the invention, the kit contains: a reagent suitable for detecting at least one of the RAD50 gene mutants, wherein the nucleic acid has at least one type of mutation compared to the wild-type RAD50 gene selected from the group consisting of: c.379G > A, c.137T > A/c.980G > A or c.494C > A/c.980G > A. By using the kit according to the embodiment of the invention, biological samples suffering from immunodeficiency diseases, especially primary immunodeficiency diseases such as CVID, SIgAD and the like can be effectively screened.
According to a tenth aspect of the invention, the invention proposes a construct. According to an embodiment of the invention, the construct comprises a nucleic acid as described above or a mutation of said gene. It should be noted that by "the construct comprises a nucleic acid as described above" it is meant that the construct of the invention comprises a nucleic acid having at least one type of mutation selected from the group consisting of: c.379G > A, c.137T > A/c.980G > A or c.494C > A/c.980G > A or a nucleic acid sequence comprising both of the above-mentioned various gene mutants. By "the construct comprises the aforementioned genetic mutation" it is meant that the construct of the invention comprises a RAD50 gene having at least one type of mutation compared to the wild-type RAD50 gene selected from the group consisting of: c.379G > A, c.137T > A/c.980G > A or c.494C > A/c.980G > A or both contain the above-mentioned various gene mutations. Thus, the recombinant cells obtained by transforming the receptor cells with the construct of the present invention can be effectively used as a model for the related study of immunodeficiency diseases, particularly primary immunodeficiency diseases such as CVID and SIgAD.
According to an eleventh aspect of the invention, the invention also provides a recombinant cell. According to an embodiment of the invention, the recombinant cell is obtained by transforming a recipient cell with a construct as described above or a protein as described above. According to some embodiments of the invention, the recombinant cells of the invention can be effectively used as models for the relevant study of immunodeficiency diseases, in particular primary immunodeficiency diseases such as CVID and SIgAD.
Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
Drawings
The foregoing and/or additional aspects and advantages of the invention will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:
FIG. 1 is a schematic diagram of a system for screening biological samples for immunodeficiency disease according to an embodiment of the present invention,
FIG. 2 is a schematic diagram of a system for screening a biological sample for immunodeficiency disease (further comprising a nucleic acid extraction device) according to an embodiment of the invention,
FIG. 3 is a schematic diagram of a system for screening a biological sample for immunodeficiency disease (further comprising a nucleic acid sequence determining apparatus) according to an embodiment of the present invention;
Fig. 4: 3 CVID and SIgAD family maps of new pathogenic sites are detected, wherein families 1 and 2 are CVID families, the pathogenic sites of which are determined by an exon sequencing technology, according to one embodiment of the invention; family 3 is SIgAD family, the pathogenic site is determined by scanning RAD50 gene, square represents male, circle represents female, diamond represents gender unknown, unfilled color in the graph represents normal person, black in the graph represents patient, arrow indicates that the patient is first-evidence, and oblique line indicates that the tested person has gone out;
fig. 5: shows the RAD50 gene Sanger sequencing results for CVID families 1 and 2, according to one embodiment of the present invention;
fig. 6: shows that the protein products of the two transcripts of the RAD50 gene (ENST 00000265335 and ENST 00000453394) are widely involved in different physiological and biochemical pathways according to one embodiment of the present invention;
fig. 7: shows a RAD50 gene homology comparison analysis (BLAST result) according to one embodiment of the present invention;
fig. 8: there is shown a cartoon model of RAD50 protein, according to one embodiment of the present invention, wherein,
a is the position on the exon of the identified mutation according to the examples of the invention,
b is the position of the mutation in the protein domain according to an embodiment of the invention,
C is the position of the mutation in the spatial structure of the protein according to an embodiment of the invention,
d is the position of the mutation in the nuclease complex MRN according to an embodiment of the invention;
fig. 9: shows that according to one embodiment of the invention, cell experiments verify that RAD50 mutations affect DNA repair, wherein,
a is a patient P1 chromosome break, fusion (indicated by arrow) according to an embodiment of the invention,
b is a comparison of the analysis of chromatin abnormalities in the G0 and G2 phases for patient P1 according to an embodiment of the invention with a control group (30 normal controls and 6A-T patients),
c is an analysis of MN length usage of RAD50 patients and control groups (normal control, MRE11 patients, NBS patients and DNA ligase 4 deficient patients) according to an embodiment of the present invention.
Reference numerals: a system 1000 for screening a biological sample for immunodeficiency disease, a nucleic acid extraction device 100, a nucleic acid sequence determination device 200, a judgment device 300, an rna extraction unit 101, a reverse transcription unit 102, a dna extraction unit 103, a target nucleic acid capture enrichment unit 104, a library construction unit 201, and a sequencing unit 202.
Detailed Description
Embodiments of the present invention are described in detail below. The following examples are illustrative only and are not to be construed as limiting the invention.
It should be understood by those skilled in the art that the wild-type RAD50 gene sequence as used herein is based on the sequence of the wild-type RAD50 gene in the human genome, but when the wild-type RAD50 gene is present in other species, the sequence may be different, and the corresponding position in the wild-type RAD50 gene of the species may be obtained by comparing the wild-type RAD50 gene of the species with the human wild-type RAD50 gene.
Nucleic acid
According to a first aspect of the invention, the invention proposes a nucleic acid. According to an embodiment of the invention, the nucleic acid has at least one type of mutation compared to the wild-type RAD50 gene selected from the group consisting of: c.379G > A, c.137T > A/c.980G > A or c.494C > A/c.980G > A. It should be noted that the nucleic acid encoding the RAD50 mutant of the present invention may also be referred to as "nucleic acid encoding the RAD50 mutant", that is, the nucleic acid may be understood as a nucleic acid substance corresponding to a gene encoding the RAD50 mutant, that is, the type of the nucleic acid is not particularly limited, and may be any polymer comprising deoxyribonucleotides and/or ribonucleotides corresponding to a gene encoding the RAD50, including, but not limited to, DNA, RNA, or cDNA. According to a specific example of the invention, the nucleic acid encoding a RAD50 mutant as described above is DNA. According to the embodiments of the present invention, the inventors cloned a novel CVID and SIgAD pathogenic gene-RAD 50 gene, and determined that these RAD50 gene mutants are closely related to the onset of primary immunodeficiency diseases such as CVID and SIgAD, so that by detecting the presence of these mutants in a biological sample, whether the biological sample suffers from primary immunodeficiency diseases, particularly CVID and SIgAD, can be effectively detected.
For purposes of the present specification and claims, reference to a nucleic acid will be understood by those skilled in the art to include virtually any one, or both, of the complementary double strands. For convenience, in the present description and claims, although only one strand is shown in most cases, the other strand complementary thereto is actually disclosed. For example, reference to SEQ ID NO 10-SEQ ID NO 15 actually includes the complement thereof. One skilled in the art will also appreciate that one strand may be used to detect another strand and vice versa.
The inventors found that the RAD50 gene mutant has at least one type of mutation selected from the group consisting of: the 379 th basic group of the RAD50 gene coding region is subjected to homozygous G- > A change (c.379G > A); the 137 th basic group of the RAD50 gene coding region is changed by heterozygous T- > A and the 980 th basic group of the coding region is changed by heterozygous G- > A (c.137T > A/c.980G > A); the 494 th base of the coding region of RAD50 gene is changed by heterozygous C- > A and the 980 th base of the coding region is changed by heterozygous G- > A (c.494C > A/c.980G > A).
The inventor firstly proposes that the RAD50 gene is a pathogenic gene of CVID and SIgAD, and firstly discovers that the mutation of c.379G > A or c.137T > A/c.980G > A of the RAD50 gene can cause patients to suffer from CVID; c.494C > A/c.980G > A of the RAD50 gene may lead to SIgAD in patients.
The cDNA and/or genomic DNA sequences of the wild-type genes described above can be obtained from the following web sites:
the RAD50 genomic DNA sequence acquisition website is as follows:
http://grch37.ensembl.org/Homo_sapiens/Transcript/Summarydb＝core；g＝ENSG00000113522；r＝5:131892630-131979752；t＝ENST00000378823。
according to a specific embodiment of the invention, the wild type RAD50 gene exon 2 region has the nucleotide sequence as set forth in SEQ ID NO:10, wherein the underlined base site is a site at which mutation is likely to occur.
ACCATCATTGAATGTCTAAAATATATTTGTACTGGAGATTTCCCTCCTGGAACCAAAGGAAATACATTTGTACACGATCCCAAG(SEQ ID NO：10)。
The wild-type RAD50 gene exon 4 has the nucleotide sequence shown in SEQ ID NO:11, wherein the underlined base site is a site at which mutation is likely to occur.
GCATGGTGAAAAGGTCAGTCTGAGCTCTAAGTGTGCAGAAATTGACCGAGAAATGATCAGTTCTCTTGGGGTTTCCAAGGCTGTGCTAAATAATGTCATTTTCTGTCATCAAGAAGATTCTAATTGGCCTTTAAGTGAAGGAAAGGCTTTGAAGCAAAAGTTTGATGAGATTTTTTCAGCAACAAG(SEQ ID NO：11)。
The wild-type RAD50 gene number 7 exon region has the sequence shown in SEQ ID NO:12, wherein the underlined base site is a site at which mutation is likely to occur.
GTTTTTCAAGGGACTGATGAGCAACTAAATGACTTATATCACAATCACCAGAGAACAGTAAGGGAGAAAGAAAGGAAATTGGTAGACTGTCATCGTGAACTGGAAAAACTAAATAAAGAATCTAGGCTTCTCAATCAGGAAAAATCAGAACTGCTTGTTGAACAGG(SEQ ID NO：12)。
Gene mutation
According to a second aspect of the invention, the invention proposes a genetic mutation. According to an embodiment of the invention, the nucleic acid has at least one type of mutation compared to the wild-type RAD50 gene selected from the group consisting of: c.379G > A, c.137T > A/c.980G > A or c.494C > A/c.980G > A. In other words, the gene mutation is located at least one of the following positions of the nucleotide sequence of the RAD50 gene, i.e. compared to the wild-type RAD50 gene: the gene mutation is located at 8 positions of the No. 2 exon region of the RAD50 gene and is changed into A from T, and the gene mutated No. 2 exon region of the RAD50 gene has the nucleotide sequence shown in SEQ ID NO:13, a nucleotide sequence shown in seq id no; the gene mutation is positioned at 14 th and 129 th positions of the exon 4 of the RAD50 gene, and is changed from G into A and C into A, and the exon 4 of the RAD50 gene after the gene mutation has the nucleotide sequence shown in SEQ ID NO:14, a nucleotide sequence shown in seq id no; the gene mutation is located at 95 th position of the No. 7 exon region of the RAD50 gene and is changed into A from G, and the gene mutated No. 7 exon region of the RAD50 gene has the nucleotide sequence shown in SEQ ID NO:15, and a nucleotide sequence shown in seq id no. According to the embodiments of the present invention, the inventors have found a novel CVID and SIgAD pathogenic gene-RAD 50 gene, and have determined that the occurrence of mutation of the RAD50 gene according to the embodiments of the present invention is closely related to the occurrence of primary immunodeficiency diseases (CVID and SIgAD), so that by detecting whether the above mutation of the RAD50 gene occurs at the above site in a biological sample, whether the biological sample suffers from primary immunodeficiency diseases, particularly CVID and SIgAD, can be effectively detected. Wherein, SEQ ID NO:13 to 15 are shown below, and the underlined base sites are sites where mutation occurs.
ACCATCAATGAATGTCTAAAATATATTTGTACTGGAGATTTCCCTCCTGGAACCAAAGGAAATACATTTGTACACGATCCCAAG(SEQ ID NO：13)。
GCATGGTGAAAAGATCAGTCTGAGCTCTAAGTGTGCAGAAATTGACCGAGAAATGATCAGTTCTCTTGGGGTTTCCAAGGCTGTGCTAAATAATGTCATTTTCTGTCATCAAGAAGATTCTAATTGGCATTTAAGTGAAGGAAAGGCTTTGAAGCAAAAGTTTGATGAGATTTTTTCAGCAACAAG(SEQ ID NO：14)。
GTTTTTCAAGGGACTGATGAGCAACTAAATGACTTATATCACAATCACCAGAGAACAGTAAGGGAGAAAGAAAGGAAATTGGTAGACTGTCATCATGAACTGGAAAAACTAAATAAAGAATCTAGGCTTCTCAATCAGGAAAAATCAGAACTGCTTGTTGAACAGG(SEQ ID NO：15)。
Proteins
According to a third aspect of the present invention, the present invention provides a protein. According to an embodiment of the invention, the protein has at least one of the following types of mutations compared to the amino acid sequence of the protein expressed by the wild-type RAD50 gene: (1) Isoleucine at amino acid sequence 46 is mutated to valine and arginine at position 327 is mutated to histidine, the protein having the amino acid sequence as set forth in SEQ ID NO:1, and a polypeptide sequence shown in the specification; (2) Valine at amino acid sequence 127 is mutated to isoleucine and this site is a homozygous mutation, and the protein has the amino acid sequence as set forth in SEQ ID NO:2, and a polypeptide sequence represented by the following formula (2); (3) Proline at amino acid sequence 165 is mutated to histidine and arginine at position 327 is mutated to histidine, the protein having the amino acid sequence as set forth in SEQ ID NO:3, and a polypeptide having the amino acid sequence shown in 3. The inventors have found a novel CVID and SIgAD pathogenic gene-RAD 50 gene, and have determined that mutation of RAD50 gene according to the embodiments of the present invention is closely related to the onset of immunodeficiency diseases, especially primary immunodeficiency diseases such as CVID and SIgAD, so that whether a biological sample suffers from immunodeficiency diseases, especially primary immunodeficiency diseases such as CVID and SIgAD, can be effectively detected by detecting the presence or absence of proteins expressing these mutated RAD50 genes in the biological sample.
According to an embodiment of the invention, the wild-type RAD50 gene expressed protein has the sequence as set forth in SEQ ID NO:16, wherein the underlined amino acid position is a position at which mutation is likely to occur.
MSRIEKMSILGVRSFGIEDKDKQIITFFSPLTILVGPNGAGKTTIIECLKYICTGDFPPGTKGNTFVHDPKVAQETDVRAQIRLQFRDVNGELIAVQRSMVCTQKSKKTEFKTLEGVITRTKHGEKVSLSSKCAEIDREMISSLGVSKAVLNNVIFCHQEDSNWPLSEGKALKQKFDEIFSATRYIKALETLRQVRQTQGQKVKEYQMELKYLKQYKEKACEIRDQITSKEAQLTSSKEIVKSYENELDPLKNRLKEIEHNLSKIMKLDNEIKALDSRKKQMEKDNSELEEKMEKVFQGTDEQLNDLYHNHQRTVREKERKLVDCHRELEKLNKESRLLNQEKSELLVEQGRLQLQADRHQEHIRARDSLIQSLATQLELDGFERGPFSERQIKNFHKLVRERQEGEAKTANQLMNDFAEKETLKQKQIDEIRDKKTGLGRIIELKSEILSKKQNELKNVKYELQQLEGSSDRILELDQELIKAERELSKAEKNSNVETLKMEVISLQNEKADLDRTLRKLDQEMEQLNHHTTTRTQMEMLTKDKADKDEQIRKIKSRHSDELTSLLGYFPNKKQLEDWLHSKSKEINQTRDRLAKLNKELASSEQNKNHINNELKRKEEQLSSYEDKLFDVCGSQDFESDLDRLKEEIEKSSKQRAMLAGATAVYSQFITQLTDENQSCCPVCQRVFQTEAELQEVISDLQSKLRLAPDKLKSTESELKKKEKRRDEMLGLVPMRQSIIDLKEKEIPELRNKLQNVNRDIQRLKNDIEEQETLLGTIMPEEESAKVCLTDVTIMERFQMELKDVERKIAQQAAKLQGIDLDRTVQQVNQEKQEKQHKLDTVSSKIELNRKLIQDQQEQIQHLKSTTNELKSEKLQISTNLQRRQQLEEQTVELSTEVQSLYREIKDAKEQVSPLETTLEKFQQEKEELINKKNTSNKIAQDKLNDIKEKVKNIHGYMKDIENYIQDGKDDYKKQKETELNKVIAQLSECEKHKEKINEDMRLMRQDIDTQKIQERWLQDNLTLRKRNEELKEVEEERKQHLKEMGQMQVLQMKSEHQKLEENIDNIKRNHNLALGrQKGYEEEIIHFKKELREPQFRDAEEKYREMMIVMRTTELVNKDLDIYYKTLDQAIMKFHSMKMEEINKIIRDLWRSTYRGQDIEYIEIRSDADENVSASDKRRNYNYRVVMLKGDTALDMRGRCSAGQKVLASLIIRLALAETFCLNCGIIALDEPTTNLDRENIESLAHALVEIIKSRSQQRNFQLLVITHDEDFVELLGRSEYVEKFYRIKKNIDQCSEIVKCSVSSLGFNVH(SEQ ID NO：16)。
According to an embodiment of the invention, the protein has at least one of the following position mutations compared to the amino acid sequence of the protein expressed by the wild-type RAD50 gene:
(1) Isoleucine at amino acid sequence 46 is mutated to valine and arginine at position 327 is mutated to histidine, the protein having the amino acid sequence as set forth in SEQ ID NO:1, wherein the underlined amino acid site is a site where mutation occurs, the mutation of the amino acid according to the specific embodiment of the present invention can cause a patient to suffer from a variant immunodeficiency disease;
MSRIEKMSILGVRSFGIEDKDKQIITFFSPLTILVGPNGAGKTTIVECLKYICTGDFPPGTKGNTFVHDPKVAQETDVRAQIRLQFRDVNGELIAVQRSMVCTQKSKKTEFKTLEGVITRTKHGEKVSLSSKCAEIDREMISSLGVSKAVLNNVIFCHQEDSNWPLSEGKALKQKFDEIFSATRYIKALETLRQVRQTQGQKVKEYQMELKYLKQYKEKACEIRDQITSKEAQLTSSKEIVKSYENELDPLKNRLKEIEHNLSKIMKLDNEIKALDSRKKQMEKDNSELEEKMEKVFQGTDEQLNDLYHNHQRTVREKERKLVDCHHELEKLNKESRLLNQEKSELLVEQGRLQLQADRHQEHIRARDSLIQSLATQLELDGFERGPFSERQIKNFHKLVRERQEGEAKTANQLMNDFAEKETLKQKQIDEIRDKKTGLGRIIELKSEILSKKQNELKNVKYELQQLEGSSDRILELDQELIKAERELSKAEKNSNVETLKMEVISLQNEKADLDRTLRKLDQEMEQLNHHTTTRTQMEMLTKDKADKDEQIRKIKSRHSDELTSLLGYFPNKKQLEDWLHSKSKEINQTRDRLAKLNKELASSEQNKNHINNELKRKEEQLSSYEDKLFDVCGSQDFESDLDRLKEEIEKSSKQRAMLAGATAVYSQFITQLTDENQSCCPVCQRVFQTEAELQEVISDLQSKLRLAPDKLKSTESELKKKEKRRDEMLGLVPMRQSIIDLKEKEIPELRNKLQNVNRDIQRLKNDIEEQETLLGTIMPEEESAKVCLTDVTIMERFQMELKDVERKIAQQAAKLQGIDLDRTVQQVNQEKQEKQHKLDTVSSKIELNRKLIQDQQEQIQHLKSTTNELKSEKLQISTNLQRRQQLEEQTVELSTEVQSLYREIKDAKEQVSPLETTLEKFQQEKEELINKKNTSNKIAQDKLNDIKEKVKNIHGYMKDIENYIQDGKDDYKKQKETELNKVIAQLSECEKHKEKINEDMRLMRQDIDTQKIQERWLQDNLTLRKRNEELKEVEEERKQHLKEMGQMQVLQMKSEHQKLEENIDNIKRNHNLALGRQKGYEEEIIHFKKELREPQFRDAEEKYREMMIVMRTTELVNKDLDIYYKTLDQAIMKFHSMKMEEINKIIRDLWRSTYRGQDIEYIEIRSDADENVSASDKRRNYNYRVVMLKGDTALDMRGRCSAGQKVLASLIIRLALAETFCLNCGIIALDEPTTNLDRENIESLAHALVEIIKSRSQQRNFQLLVITHDEDFVELLGRSEYVEKFYRIKKNIDQCSEIVKCSVSSLGFNVH(SEQ ID NO：1)。
(2) Valine at amino acid sequence 127 is mutated to isoleucine and this site is a homozygous mutation, and the protein has the amino acid sequence as set forth in SEQ ID NO:2, wherein the underlined amino acid site is a site where mutation occurs, the mutation of the amino acid according to the specific embodiment of the present invention can cause a patient to suffer from a variant immunodeficiency disease;
MSRIEKMSILGVRSFGIEDKDKQIITFFSPLTILVGPNGAGKTTIIECLKYICTGDFPPGTKGNTFVHDPKVAQETDVRAQIRLQFRDVNGELIAVQRSMVCTQKSKKTEFKTLEGVITRTKHGEKISLSSKCAEIDREMISSLGVSKAVLNNVIFCHQEDSNWPLSEGKALKQKFDEIFSATRYIKALETLRQVRQTQGQKVKEYQMELKYLKQYKEKACEIRDQITSKEAQLTSSKEIVKSYENELDPLKNRLKEIEHNLSKIMKLDNEIKALDSRKKQMEKDNSELEEKMEKVFQGTDEQLNDLYHNHQRTVREKERKLVDCHRELEKLNKESRLLNQEKSELLVEQGRLQLQADRHQEHIRARDSLIQSLATQLELDGFERGPFSERQIKNFHKLVRERQEGEAKTANQLMNDFAEKETLKQKQIDEIRDKKTGLGRIIELKSEILSKKQNELKNVKYELQQLEGSSDRILELDQELIKAERELSKAEKNSNVETLKMEVISLQNEKADLDRTLRKLDQEMEQLNHHTTTRTQMEMLTKDKADKDEQIRKIKSRHSDELTSLLGYFPNKKQLEDWLHSKSKEINQTRDRLAKLNKELASSEQNKNHINNELKRKEEQLSSYEDKLFDVCGSQDFESDLDRLKEEIEKSSKQRAMLAGATAVYSQFITQLTDENQSCCPVCQRVFQTEAELQEVISDLQSKLRLAPDKLKSTESELKKKEKRRDEMLGLVPMRQSIIDLKEKEIPELRNKLQNVNRDIQRLKNDIEEQETLLGTIMPEEESAKVCLTDVTIMERFQMELKDVERKIAQQAAKLQGIDLDRTVQQVNQEKQEKQHKLDTVSSKIELNRKLIQDQQEQIQHLKSTTNELKSEKLQISTNLQRRQQLEEQTVELSTEVQSLYREIKDAKEQVSPLETTLEKFQQEKEELINKKNTSNKIAQDKLNDIKEKVKNIHGYMKDIENYIQDGKDDYKKQKETELNKVIAQLSECEKHKEKINEDMRLMRQDIDTQKIQERWLQDNLTLRKRNEELKEVEEERKQHLKEMGQMQVLQMKSEHQKLEENIDNIKRNHNLALGRQKGYEEEIIHFKKELREPQFRDAEEKYREMMIVMRTTELVNKDLDIYYKTLDQAIMKFHSMKMEEINKIIRDLWRSTYRGQDIEYIEIRSDADENVSASDKRRNYNYRVVMLKGDTALDMRGRCSAGQKVLASLIIRLALAETFCLNCGIIALDEPTTNLDRENIESLAHALVEIIKSRSQQRNFQLLVITHDEDFVELLGRSEYVEKFYRIKKNIDQCSEIVKCSVSSLGFNVH(SEQ ID NO：2)。
(3) Proline at amino acid sequence 165 is mutated to histidine and arginine at position 327 is mutated to histidine, the protein having the amino acid sequence as set forth in SEQ ID NO:3, wherein the underlined amino acid position is the position at which the mutation occurred, wherein the mutation of the amino acid according to the embodiment of the invention is capable of causing the patient to suffer from a selective IgA deficiency:
MSRIEKMSILGVRSFGIEDKDKQIITFFSPLTILVGPNGAGKTTIIECLKYICTGDFPPGTKGNTFVHDPKVAQETDVRAQIRLQFRDVNGELIAVQRSMVCTQKSKKTEFKTLEGVITRTKHGEKVSLSSKCAEIDREMISSLGVSKAVLNNVIFCHQEDSNWHLSEGKALKQKFDEIFSATRYIKALETLRQVRQTQGQKVKEYQMELKYLKQYKEKACEIRDQITSKEAQLTSSKEIVKSYENELDPLKNRLKEIEHNLSKIMKLDNEIKALDSRKKQMEKDNSELEEKMEKVFQGTDEQLNDLYHNHQRTVREKERKLVDCHHELEKLNKESRLLNQEKSELLVEQGRLQLQADRHQEHIRARDSLIQSLATQLELDGFERGPFSERQIKNFHKLVRERQEGEAKTANQLMNDFAEKETLKQKQIDEIRDKKTGLGRIIELKSEILSKKQNELKNVKYELQQLEGSSDRILELDQELIKAERELSKAEKNSNVETLKMEVISLQNEKADLDRTLRKLDQEMEQLNHHTTTRTQMEMLTKDKADKDEQIRKIKSRHSDELTSLLGYFPNKKQLEDWLHSKSKEINQTRDRLAKLNKELASSEQNKNHINNELKRKEEQLSSYEDKLFDVCGSQDFESDLDRLKEEIEKSSKQRAMLAGATAVYSQFITQLTDENQSCCPVCQRVFQTEAELQEVISDLQSKLRLAPDKLKSTESELKKKEKRRDEMLGLVPMRQSIIDLKEKEIPELRNKLQNVNRDIQRLKNDIEEQETLLGTIMPEEESAKVCLTDVTIMERFQMELKDVERKIAQQAAKLQGIDLDRTVQQVNQEKQEKQHKLDTVSSKIELNRKLIQDQQEQIQHLKSTTNELKSEKLQISTNLQRRQQLEEQTVELSTEVQSLYREIKDAKEQVSPLETTLEKFQQEKEELINKKNTSNKIAQDKLNDIKEKVKNIHGYMKDIENYIQDGKDDYKKQKETELNKVIAQLSECEKHKEKINEDMRLMRQDIDTQKIQERWLQDNLTLRKRNEELKEVEEERKQHLKEMGQMQVLQMKSEHQKLEENIDNIKRNHNLALGRQKGYEEEIIHFKKELREPQFRDAEEKYREMMIVMRTTELVNKDLDIYYKTLDQAIMKFHSMKMEEINKIIRDLWRSTYRGQDIEYIEIRSDADENVSASDKRRNYNYRVVMLKGDTALDMRGRCSAGQKVLASLIIRLALAETFCLNCGIIALDEPTTNLDRENIESLAHALVEIIKSRSQQRNFQLLVITHDEDFVELLGRSEYVEKFYRIKKNIDQCSEIVKCSVSSLGFNVH(SEQ ID NO：3)。
Organisms carrying proteins according to embodiments of the invention suffer from immunodeficiency diseases, in particular from primary immunodeficiency diseases such as CVID and SigAD.
Application of reagent in preparation of kit
According to a fourth aspect of the invention, the present invention provides the use of a reagent for detecting a nucleic acid as described hereinbefore or a mutation in a gene as described hereinbefore or a protein as described hereinbefore in the preparation of a kit. According to an embodiment of the invention, the agent is used for diagnosing primary immunodeficiency. According to the embodiment of the invention, the reagent can be used for effectively screening biological samples suffering from immunodeficiency diseases, especially primary immunodeficiency diseases such as CVID and SIgAD, and further preparing a kit for screening biological samples suffering from primary immunodeficiency diseases, especially primary immunodeficiency diseases such as CVID and SIgAD.
According to an embodiment of the invention, the reagent comprises at least one of an antibody, a probe, a primer and a mass spectrometry detection reagent specific for at least one of the nucleic acid, the gene mutation and the protein. The reagent according to the specific embodiment of the invention can specifically and highly sensitively screen the nucleic acid or the gene mutation or the protein, and further specifically and highly sensitively screen biological samples suffering from immunodeficiency diseases, especially primary immunodeficiency diseases such as CVID and SIgAD, and further can be effectively used for preparing the kit for screening the biological samples suffering from immunodeficiency diseases, especially primary immunodeficiency diseases such as CVID and SIgAD.
According to a specific embodiment of the invention, the immunodeficiency is a primary immunodeficiency.
According to a specific embodiment of the invention, the primary immunodeficiency disease is a variant immunodeficiency disease or a selective IgA deficiency.
According to a specific embodiment of the invention, the RAD50 gene mutant has one mutation selected from the group consisting of: c.379G > A or c.137T > A/c.980G > A, the primary immunodeficiency disease is variant immunodeficiency disease (CVID). The inventors found that the occurrence of at least one of the above mutations in the coding region of the RAD50 gene can confirm that the organism suffers from CVID.
According to a specific embodiment of the invention, the RAD50 gene mutant has a mutation c.494c > a/c.980g > a compared to the wild-type RAD50 gene, and the primary immunodeficiency disease is a selective IgA deficiency (SIgAD). The inventors found that the occurrence of the above mutation in the coding region of the RAD50 gene confirmed that the organism had SIgAD.
According to a specific embodiment of the invention, the gene encoding the protein has at least one of the following types of mutations compared to the amino acid sequence of the protein expressed by the wild-type RAD50 gene: isoleucine at amino acid sequence position 46 is mutated to valine and arginine at position 327 is mutated to histidine; or valine at amino acid sequence 127 is mutated to isoleucine and the site is homozygously mutated, and the primary immunodeficiency disease is variant immunodeficiency disease. The inventors found that the occurrence of at least one of the above mutations in the RAD50 gene-expressed protein can confirm that the organism suffers from CVID.
According to a specific embodiment of the present invention, the coding gene of the protein has the following mutation of proline to histidine at position 165 and arginine to histidine at position 327 of the amino acid sequence 165 compared to the amino acid sequence of the protein expressed by the wild-type RAD50 gene, and the primary immunodeficiency disease is a selective IgA deficiency. The inventors found that the above mutation of the RAD50 gene-expressed protein can confirm that the organism suffers from SIgAD.
Use of biological model in screening drugs
According to a fifth aspect of the invention, the invention proposes the use of a biological model for screening drugs. According to an embodiment of the invention, the biological model carries at least one of: (1) the nucleic acid described above; (2) the aforementioned gene mutation; (3) expressing the aforementioned protein. By "a biological model carries the nucleic acid as described above" it is meant that the biological model of the invention carries a gene having at least one type of mutation as compared to the wild-type RAD50 gene selected from the group consisting of: nucleic acid sequence of RAD50 gene mutant of c.379G > A, c.137T > A/c.980G > A or c.494C > A/c.980G > A; "biological model carrying the aforementioned genetic mutation" means that the biological model of the present invention carries a RAD50 gene having at least one type of mutation selected from the group consisting of: RAD50 gene mutation of c.379G > A, c.137T > A/c.980G > A or c.494C > A/c.980G > A. By "the biological model carries the aforementioned protein" it is meant that the biological model of the invention carries at least one mutation compared to the wild-type RAD50 gene selected from the group consisting of: proteins expressed by RAD50 gene mutants of c.379G > A, c.137T > A/c.980G > A or c.494C > A/c.980G > A. The biological model according to the embodiment of the present invention can be effectively used as a model for primary immunodeficiency diseases, particularly, CVID and SIgAD related studies.
According to a specific embodiment of the invention, the biological model is a cellular model or an animal model.
Medicine for treating immunodeficiency
According to a sixth aspect of the present invention, the present invention provides a medicament for the treatment of immunodeficiency disorders. According to an embodiment of the present invention, the medicament contains: an agent which specifically alters the nucleic acid as described above or the gene mutation as described above. It should be noted that the specific change herein refers to a change that can restore the mutated nucleic acid or the site of the gene mutation to the original wild state or other non-pathogenic state without substantially affecting other sequences in the genome of the individual. The medicine according to the embodiment of the invention can specifically prevent or treat immunodeficiency diseases, especially primary immunodeficiency diseases such as CVID, SIgAD and the like.
According to an embodiment of the invention, the reagent is a reagent based on a gene editing or nucleic acid synthesis method.
According to an embodiment of the invention, the immunodeficiency disease is a primary immunodeficiency disease.
According to an embodiment of the invention, the primary immunodeficiency diseases include variant immunodeficiency diseases and selective IgA deficiency
According to an embodiment of the invention, the nucleic acid or the gene is mutated to c.379g > a or c.137t > a/c.980g > a, and the primary immunodeficiency disease is a variant immunodeficiency disease. The inventors have found that mutations in drugs which specifically alter at least one of the above can prevent or treat CVID.
According to a specific embodiment of the invention, said nucleic acid or said gene is mutated to c.494C > A/c.980G > A, and said primary immunodeficiency disorder is a selective IgA deficiency. The inventors found that drugs used to specifically alter the above mutations could prevent or treat SIgAD.
Method for screening biological samples for immunodeficiency disorders
According to a seventh aspect of the invention, the invention provides a method of screening a biological sample for a primary immunodeficiency disorder. According to an embodiment of the present invention, the method of screening a biological sample for a primary immunodeficiency disorder may include the steps of: according to an embodiment of the invention, the method comprises the steps of: extracting a nucleic acid sample from a biological sample; determining the nucleic acid sequence of the nucleic acid sample; a nucleic acid sequence of the nucleic acid sample, or a complement thereof, having at least one type of mutation as compared to the wild-type RAD50 gene selected from the group consisting of: c.379G > A, c.137T > A/c.920G > A or c.494C > A/c.920G > A are indicative of an immunodeficiency disorder in the biological sample. By the method for screening the biological samples with immunodeficiency diseases, which is provided by the embodiment of the invention, the biological samples with immunodeficiency diseases, especially primary immunodeficiency diseases such as CVID, SIgAD and the like, can be effectively screened.
First, a nucleic acid sample is extracted from a biological sample. According to the embodiment of the present invention, the type of the biological sample is not particularly limited as long as a nucleic acid sample reflecting the presence or absence of mutation of the RAD50 gene of the biological sample can be extracted from the biological sample. According to an embodiment of the present invention, the biological sample may be at least one selected from human blood, skin, subcutaneous tissue. Thus, sampling and detection can be conveniently performed, so that the efficiency of screening biological samples suffering from primary immunodeficiency diseases can be further improved. The term "nucleic acid sample" as used herein is to be understood in a broad sense according to embodiments of the present invention and may be any sample that reflects the presence or absence of mutations in the RAD50 gene in a biological sample, such as whole genomic DNA extracted directly from a biological sample, or a portion of the whole genome comprising the coding sequence of the RAD50 gene, or total RNA extracted from a biological sample, or mRNA extracted from a biological sample. According to one embodiment of the invention, the nucleic acid sample is whole genomic DNA. Thus, the source range of the biological sample can be enlarged, and various information of the biological sample can be determined at the same time, so that the efficiency of screening the biological sample with the primary immunodeficiency disease can be improved. In addition, according to an embodiment of the present invention, for using RNA as a nucleic acid sample, extracting the nucleic acid sample from the biological sample may further include: extracting an RNA sample from the biological sample, preferably the RNA sample is mRNA; and obtaining a cDNA sample by reverse transcription reaction based on the obtained RNA sample, the obtained cDNA sample constituting a nucleic acid sample. Thus, the efficiency of screening biological samples for primary immunodeficiency disease using RNA as a nucleic acid sample can be further improved.
Next, after the nucleic acid sample is obtained, the nucleic acid sample may be analyzed, so that the nucleic acid sequence of the obtained nucleic acid sample can be determined. According to the embodiments of the present invention, the method and apparatus for determining the nucleic acid sequence of the obtained nucleic acid sample are not particularly limited. According to particular embodiments of the invention, the nucleic acid sequence of a nucleic acid sample may be determined by a sequencing method. Methods and apparatuses that may be used to perform sequencing according to embodiments of the present invention are not particularly limited. According to embodiments of the present invention, second generation sequencing techniques may be employed, as well as third generation and fourth generation or more advanced sequencing techniques. According to a specific example of the present invention, the nucleic acid sequence may be sequenced using at least one selected from the group consisting of BGIseq500, hiseq2000, SOLiD, 454 and single molecule sequencing devices. Therefore, by combining the latest sequencing technology, a higher sequencing depth can be achieved for a single site, and the detection sensitivity and accuracy are greatly improved, so that the high-throughput and deep sequencing characteristics of the sequencing devices can be utilized, and the efficiency of detecting and analyzing the nucleic acid sample is further improved. Thus, the accuracy and precision of subsequent analysis of the sequencing data can be improved. Thus, according to an embodiment of the present invention, determining the nucleic acid sequence of the nucleic acid sample may further comprise: first, a nucleic acid sequencing library is constructed for the obtained nucleic acid sample; and sequencing the obtained nucleic acid sequencing library so as to obtain a sequencing result composed of a plurality of sequencing data. According to some embodiments of the invention, the resulting nucleic acid sequencing library may be sequenced using at least one selected from the group consisting of BGIseq500, hiseq2000, SOLiD, 454, and single molecule sequencing devices. In addition, according to embodiments of the invention, nucleic acid samples may be screened for enrichment of RAD50 gene exons, either prior to construction of the sequencing library, during construction of the sequencing library, or after construction of the sequencing library. Exon targeted sequence enrichment systems such as: and (3) enriching target fragments by using a Huada autonomous exon capturing chip, aglientSureSelect, nimblegen and other exons or target region capturing platforms. According to one embodiment of the invention, constructing a nucleic acid sequencing library for a nucleic acid sample further comprises: performing PCR amplification on the nucleic acid sample using at least one primer selected from the group consisting of exon-specific primers of the RAD50 gene; and constructing a nucleic acid sequencing library for the obtained amplification product. Thus, the RAD50 gene exons can be enriched by PCR amplification, so that the efficiency of screening biological samples suffering from primary immunodeficiency diseases can be further improved. According to an embodiment of the present invention, the sequence of the RAD50 gene exon specific Primer is not particularly limited, and may be obtained, for example, by referring to human genome sequence database GRCh37.1/hg19, using Primer3.0 on-line design, for example, referring to UCSC (http:// genome. UCSC. Edu /), designing and synthesizing the Primer of the candidate gene using Primer3 (version 0.4.0, http:// Primer3.Ut. Ee /) (synthesized by Biotechnology), and verifying the Primer specificity using Primer-BLAST (http:// www.ncbi.nlm.nih.gov/tools/Primer-BLAST /). According to a preferred embodiment of the invention, the RAD50 gene exon 2 specific primer has the nucleotide sequence set forth in SEQ ID NO:4-5, said RAD50 gene exon 4-specific primer having a nucleotide sequence as set forth in SEQ ID NO:6-7, said RAD50 gene exon 7 specific primer having the nucleotide sequence set forth in SEQ ID NO: 8-9. According to some specific examples of the invention, the RAD50 gene exon specific primers have the nucleotide sequence set forth in SEQ ID NO:6-7, said RAD50 gene exon specific primer having the nucleotide sequence set forth in SEQ ID NO:4-5, said RAD50 gene exon specific primer having the nucleotide sequence set forth in SEQ ID NO:8-9, said RAD50 gene exon specific primer having the nucleotide sequence set forth in SEQ ID NO: 6-7. The inventors have surprisingly found that by using SEQ ID NO:4-9, can significantly and effectively complete the amplification of the exon sequence where the RAD50 gene mutation is located in a PCR reaction system. It should be noted that these SEQ ID NOs in the following tables: 4-9 is unexpectedly obtained after the inventors of the present invention have paid a laborious effort.
Regarding the method and procedure for constructing a sequencing library for a nucleic acid sample, the person skilled in the art may make appropriate selections according to different sequencing techniques, and regarding the details of the procedure, reference may be made to the manufacturer of the sequencing instrument, for example to the procedure provided by Illumina company, for example to Illumina company Multiplexing Sample Preparation Guide (Part #1005361; feed 2010) or Paired-End SamplePrep Guide (Part #1005063; feed 2010), which are incorporated herein by reference. The method and apparatus for extracting a nucleic acid sample from a biological sample according to the embodiments of the present invention are also not particularly limited, and may be performed using a commercially available nucleic acid extraction kit.
The term "nucleic acid sequence" as used herein is to be understood in a broad sense, and may be the complete nucleic acid sequence information obtained after assembling the sequencing data obtained by sequencing the nucleic acid sample, or may be the nucleic acid sequence directly using the sequencing data (reads) obtained by sequencing the nucleic acid sample, as long as the nucleic acid sequence contains the coding sequence corresponding to the RAD50 gene.
Finally, after determining the nucleic acid sequence of the nucleic acid sample, the corresponding reference sequences of the nucleic acid sequences of the obtained nucleic acid sample are aligned, and when at least one of the aforementioned mutations is present in the obtained nucleic acid sequence, an immune deficiency is indicated in the biological sample. Thus, by the method for screening a biological sample having an immunodeficiency disease according to an embodiment of the present invention, a biological sample having an immunodeficiency disease can be effectively screened. The method and apparatus for aligning nucleic acid sequences with corresponding wild-type gene sequences according to embodiments of the present invention are not particularly limited, and may be performed using any conventional software, and according to embodiments of the present invention, may be performed using SOAPALIGNER/SOAP 2.
The use of the "method for screening a biological sample for immunodeficiency disease" according to the embodiment of the present invention is not particularly limited, and may be used as a screening method for non-diagnostic purposes, for example.
System for screening biological samples for immunodeficiency disorders
According to an eighth aspect of the present invention, the present invention provides a system capable of effectively performing the above-described method of screening a biological sample for immunodeficiency disease.
Referring to fig. 1, the system 1000 for screening a biological sample for immunodeficiency disease according to an embodiment of the present invention includes: nucleic acid extraction device 100, nucleic acid sequence determination device 200, and judgment device 300.
According to an embodiment of the present invention, the nucleic acid extraction apparatus 100 is used for extracting a nucleic acid sample from a biological sample. As described above, referring to fig. 2A, according to an embodiment of the present invention, the type of the nucleic acid sample is not particularly limited, and for using RNA as the nucleic acid sample, the nucleic acid extraction apparatus 100 further includes an RNA extraction unit 101 and a reverse transcription unit 102, wherein the extraction unit 101 is used to extract the RNA sample from the biological sample, and the reverse transcription unit 102 is connected to the RNA extraction unit 101 for performing a reverse transcription reaction on the RNA sample so as to obtain a cDNA sample, and the obtained cDNA sample constitutes the nucleic acid sample. According to another embodiment of the present invention, the nucleic acid extraction apparatus 100 described with reference to FIG. 2B further includes: a DNA extraction unit 103 for extracting a DNA sample from the biological sample, and a target nucleic acid capture enrichment unit 104 for enriching a capture target nucleic acid from the biological sample, the DNA sample and the target nucleic acid constituting the nucleic acid sample. According to another embodiment of the present invention, the nucleic acid extraction apparatus 100 described with reference to FIG. 2C further includes: a DNA extraction unit 103 for extracting a DNA sample from the biological sample, the DNA sample constituting the nucleic acid sample. According to another embodiment of the present invention, the nucleic acid extraction apparatus 100 described with reference to FIG. 2D further includes: a target nucleic acid capture enrichment unit 104 for enriching a capture target nucleic acid from the biological sample, the target nucleic acid constituting the nucleic acid sample.
According to an embodiment of the present invention, the nucleic acid sequence determination means 200 is connected to the nucleic acid extraction means 100 for analyzing the nucleic acid sample in order to determine the nucleic acid sequence of the nucleic acid sample. As indicated previously, the nucleic acid sequence of a nucleic acid sample can be determined using sequencing methods. Thus, referring to FIG. 3, according to one embodiment of the invention, the nucleic acid sequence determination apparatus 200 may further include: library construction unit 201 and sequencing unit 202. The library construction unit 201 is used for constructing a nucleic acid sequencing library for a nucleic acid sample; the sequencing unit 202 is connected to the library construction unit 201 for sequencing the nucleic acid sequencing library so as to obtain a sequencing result composed of a plurality of sequencing data. As described above, the efficiency of screening biological samples for immunodeficiency disease can be further improved by PCR amplification, enrichment of RAD50 gene exons. Thus, the library construction unit 201 may further include a PCR amplification module (not shown in the drawing) in which at least one selected from the group consisting of RAD50 gene exon-specific primers is provided, so that the nucleic acid sample is PCR amplified using at least one of the RAD50 gene exon-specific primers. According to the embodiment of the invention, the sequence of the RAD50 gene Exon specific primer is not particularly limited, and can be obtained by referring to the Human genome sequence database GRCh37.1/hg19 and adopting Primer3.0 online design, and can also be obtained by adopting a Aglient SureSelect Exon targeted sequence enrichment system (SureSelect_Human_all_Exon_V4 and SureSelect_Human_all_Exon_V6) to enrich target fragments. According to a preferred embodiment of the invention, the RAD50 gene exon 2 specific primer has the nucleotide sequence set forth in SEQ ID NO:4-5, said RAD50 gene exon 4-specific primer having a nucleotide sequence as set forth in SEQ ID NO:6-7, said RAD50 gene exon 7 specific primer having the nucleotide sequence set forth in SEQ ID NO: 8-9. According to some specific examples of the invention, the RAD50 gene exon specific primer has the nucleotide sequence set forth in SEQ ID NO:6-7, said RAD50 gene exon specific primer having a nucleotide sequence as set forth in seq id NO:4-5, said RAD50 gene exon specific primer having the nucleotide sequence set forth in SEQ ID NO:8-9, said RAD50 gene exon specific primer having the nucleotide sequence set forth in SEQ ID NO: 6-7. According to an embodiment of the invention, the sequencing unit 202 may comprise at least one selected from the group consisting of BGISEQ500, HISEQ2000, SOLiD, 454 and single molecule sequencing devices. Therefore, by combining the latest sequencing technology, a higher sequencing depth can be achieved for a single site, and the detection sensitivity and accuracy are greatly improved, so that the high-throughput and deep sequencing characteristics of the sequencing devices can be utilized, and the efficiency of detecting and analyzing the nucleic acid sample is further improved. Thus, the accuracy and precision of subsequent analysis of the sequencing data are improved.
According to an embodiment of the present invention, referring to FIG. 1, the judging means 300 is connected to the nucleic acid sequence determining means 200 and adapted to align the nucleic acid sequences of the nucleic acid samples so as to judge whether the biological sample suffers from immunodeficiency disease based on the differences between the nucleic acid sequences of the nucleic acid samples and the corresponding wild type gene sequences. Specifically, based on the nucleic acid sequence of the nucleic acid sample or its complement, there is at least one type of mutation as compared to the wild-type RAD50 gene selected from the group consisting of: c.379G > A, c.137T > A/c.980G > A or c.494C > A/c.980G > A mutation, and determining whether said biological sample has an immunodeficiency disorder. As previously mentioned, the apparatus for aligning nucleic acid sequences with corresponding wild-type gene sequences according to embodiments of the present invention is not particularly limited and may be operated using any conventional software, for example, according to embodiments of the present invention, the alignment may be performed using SOAPALIGNER/SOAP 2.
Thus, the method for screening a biological sample for immunodeficiency disease can be effectively carried out by using the system, so that the biological sample for immunodeficiency disease can be effectively screened.
Kit for screening biological samples suffering from immunodeficiency diseases
According to a ninth aspect of the invention, the invention provides a kit for screening a biological sample for immunodeficiency disease. According to an embodiment of the present invention, the kit for screening a biological sample for immunodeficiency disease includes: a reagent suitable for detecting at least one of the RAD50 gene mutants, wherein the nucleic acid has at least one mutation selected from the group consisting of: c.379G > A, c.137T > A/c.980G > A or c.494C > A/c.980G > A. By using the kit according to the embodiment of the invention, biological samples suffering from immunodeficiency diseases can be effectively screened. As used herein, the term "reagent adapted to detect at least one of the RAD50 gene mutants" is to be understood in a broad sense, i.e.as a reagent for detecting at least one of the genes encoding the RAD50 mutants, as well as a reagent for detecting at least one of the RAD50 protein mutants, e.g.antibodies recognizing specific sites may be used.
According to a specific embodiment of the invention, the immunodeficiency is a primary immunodeficiency.
According to a specific embodiment of the invention, the primary immunodeficiency disease is a variant immunodeficiency disease or a selective IgA deficiency.
According to a specific embodiment of the invention, the RAD50 gene mutant has one type of mutation compared to the wild-type RAD50 gene selected from the group consisting of: c.379G > A or c.137T > A/c.980G > A, the primary immunodeficiency disease is variant immunodeficiency disease (CVID). The inventors found that the occurrence of at least one of the above mutations in the coding region of the RAD50 gene can confirm that the organism suffers from CVID.
According to a specific embodiment of the invention, the RAD50 gene mutant has a mutation selected from the group consisting of: c.494C > A/c.980G > A, the primary immunodeficiency disorder is a selective IgA deficiency (SIgAD). The inventors found that the occurrence of the above mutation in the coding region of the RAD50 gene confirmed that the organism had SIgAD.
According to one embodiment of the invention, the reagent comprises at least one of an antibody, a probe, a primer and a mass spectrometry detection reagent specific for a RAD50 gene mutant or an expressed RAD50 gene mutant protein. Thus, a biological sample suffering from immunodeficiency can be efficiently screened.
Construct and recombinant cell
According to a tenth aspect of the invention, the invention also proposes a construct. According to an embodiment of the invention, the construct comprises a nucleic acid as described previously. It should be noted that by "the construct comprises a nucleic acid as described above" it is meant that the construct of the invention comprises a nucleic acid having at least one type of mutation selected from the group consisting of: c.379G > A, c.137T > A/c.980G > A or c.494C > A/c.980G > A or a nucleic acid sequence comprising both of the above-mentioned various gene mutants. By "the construct comprises the aforementioned genetic mutation" it is meant that the construct of the invention comprises a RAD50 gene having at least one type of mutation compared to the wild-type RAD50 gene selected from the group consisting of: c.379G > A, c.137T > A/c.980G > A or c.494C > A/c.980G > A or both contain the above-mentioned various gene mutations. Thus, the recombinant cells obtained by transforming the receptor cells with the construct of the present invention can be effectively used as a model for the related study of immunodeficiency diseases, particularly primary immunodeficiency diseases such as CVID and SIgAD. The type of the receptor cell is not particularly limited, and may be, for example, an E.coli cell or a mammalian cell, and preferably the receptor cell is derived from a mammal.
The term "construct" as used in the present invention refers to a genetic vector which comprises a specific nucleic acid sequence and is capable of transferring the nucleic acid sequence of interest into a host cell to obtain a recombinant cell. The form of the construct is not particularly limited according to the embodiments of the present invention. According to an embodiment of the present invention, it may be at least one of a plasmid, a phage, an artificial chromosome, a Cosmid (Cosmid), a virus, preferably a plasmid. As a genetic carrier, the plasmid has the characteristics of simple operation and capability of carrying larger fragments, and is convenient to operate and process. The form of the plasmid is not particularly limited either, and may be a circular plasmid or a linear plasmid, i.e., may be single-stranded or double-stranded. Those skilled in the art can make selections as desired. The term "nucleic acid" as used in the present invention may be any polymer comprising deoxyribonucleotides or ribonucleotides, including but not limited to modified or unmodified DNA, RNA, the length of which is not subject to any particular limitation. For constructs used to construct recombinant cells, it is preferred that the nucleic acid is DNA, as DNA is more stable relative to RNA and is easy to handle.
According to an eleventh aspect of the invention, the invention also provides a recombinant cell. According to an embodiment of the invention, the recombinant cell is obtained by transforming a recipient cell with a construct as described above or by expressing a protein as described above. Thus, the recombinant cells of the invention are capable of efficiently expressing at least one of the RAD50 gene mutants carried by the construct or at least one of the proteins expressed by the RAD50 gene mutations. According to some embodiments of the invention, the recombinant cells of the invention can be effectively used as models for the relevant study of immunodeficiency diseases, in particular primary immunodeficiency diseases such as CVID and SIgAD. According to an embodiment of the present invention, the kind of the recipient cell is not particularly limited, and may be, for example, an E.coli cell, a mammalian cell, preferably the recipient cell is derived from a non-human mammal.
The features and advantages described in the method section herein before for screening a biological sample for immunodeficiency disease are equally applicable to a system or kit for screening a biological sample for immunodeficiency disease and are not described in detail herein.
In addition, it should be noted that the method, system and kit for screening a biological sample having immunodeficiency disease according to the embodiments of the present invention are completed by the inventor of the present application through hard creative work and optimization work.
The invention will now be described with reference to specific examples, which are intended to be illustrative only and are not to be construed as limiting the invention. Unless otherwise indicated, the technical means employed in the examples are conventional means well known to those skilled in the art, and may be carried out with reference to the third edition of the guidelines for molecular cloning experiments or related products, and the reagents and products employed are also commercially available. The various processes and methods not described in detail are conventional methods well known in the art, the sources of the reagents used, the trade names and those necessary to list the constituents are all indicated at the first occurrence, and the same reagents used thereafter, unless otherwise indicated, are the same as those indicated at the first occurrence.
Example 1 determination of pathogenic Gene and mutation site by exome sequencing
1. Sample collection:
the inventor collected 2 CVID families (see fig. 1), corresponding foreigners P1 and P2. P1 is Iran from close family; p2 is swedish. Patient specific phenotypic information is shown in table 1.
Table 1: CVID patients P1, P2 and SIgAD patient P4 phenotype information
| Patient numbering | P1 | P2 | P4 |
| Population group | Iran' s | Swedish (Swedish) | Swedish (Swedish) |
| Sex (sex) | Female | Female | Man's body |
| Age of onset/age of genetic diagnosis | 1.5/28 | 12/46 | 5/40 |
| Disease of the human body | CVID | CVID | SIgAD |
| IgG(mg/dl) | 195↓ | 265↓ | 2130↑ |
| IgG1 | 40↓ | 137↓ | 1490↑ |
| IgG2 | 140 | 85↓ | 150 |
| IgG3 | 10↓ | 18↓ | 159↑ |
| IgA(mg/dl) | 5↓ | <2↓ | 1 |
| IgM(mg/dl) | 20↓ | 4.4↓ | 320 |
| IgE(IU/ml) | 18 | <2↓ | - |
| Specific antibody production | Damaged by | Damaged by | Damaged by |
| Repeated infection | + | + | - |
| Allergy to humans | + | + | - |
| Growth retardation | + | - | - |
| Neurological dysfunction | - | - | + |
| Small head deformity | + | - | - |
| Delay of sexual development | + | - | - |
| Abnormal metabolism of skin pigment | + | - | - |
| Radiation sensitivity | + | + | - |
| Immunodeficiency | + | + | + |
| Therapeutic agent | IVIg+ prophylactic antibiotics | SCIg+ preventive antibiotics | - |
| Current status quo | IVIg dependent | Dependent on SCIg | Death from hip fracture at 64 years old |
Remarks: the detection item is lower than the normal contrast level; the ∈detection item is higher than the normal control level;
positive results of +detection item; -the test item results negative.
2. Exome sequencing to determine pathogenic genes and mutation sites
The inventor screens candidate variation of 2 CVID families through an exon sequencing technology, filters non-pathogenic nucleotide variation through bioinformatics analysis (VEP, public frequency database, hazard prediction, homologous sequence comparison and equivalent annotation), further reduces the number of candidate variation by utilizing homozygote localization analysis aiming at a close family (family 1), designs a primer for the candidate variation, carries out PCR amplification, purifies a product, carries out candidate variation detection on members in the family and normal control groups, carries out coseparation analysis, and confirms the relationship between the gene mutation and the disease. Based on the exome sequencing results, target gene association analysis was performed by collecting other disease family samples (48 CVID patients, 791 SIgAD patients) and normal control samples (1118), and pathogenic mutations were found in the other CVID family and two SIgAD family patients. The specific method comprises the following steps:
DNA extraction: collecting 10ml of peripheral venous blood, performing anticoagulation by sodium citrate, extracting genome DNA by a conventional phenol-chloroform method, measuring the DNA concentration and OD value by a spectrophotometer, and diluting to 100 ng/. Mu.l;
2. exome sequencing: performing exome sequencing on the foreigners (P1 and P2) of the 2 CVID families to obtain nucleotide variation data;
1) Breaking the genomic DNA into random fragments and creating a random fragment library;
2) Enriching the target fragment by adopting a Aglient SureSelect Exon targeting sequence enrichment system (SureSelect_Human_all_Exon_V4 and SureSelect_Human_all_Exon_V6);
3) High-throughput sequencing is carried out by using a second-generation high-throughput sequencer Hiseq 2000;
3. bioinformatics analysis yields candidate genes and loci:
1) After the original data are obtained by sequencing the exome, removing the joint sequence and the low-quality sequence, comparing the sequence with a reference genome (GRCh 37) by using comparison software bwa-men (v 0.7.10-r 789), and obtaining nucleotide variation data by using GTAK (v 3.3-0) based on the comparison result;
2) Annotating nucleotide variation data including VEP (v 77), public frequency databases (thousand person database, exAC database, etc.), hazard prediction (SIFT, polyphen2_hdiv, polyphen2_ HVAR, LRT, mutationTaster, mutationAssessor, FATHMM, GERP ++, phyloP, siPhy, gerp, phastCons, and GWAWA); based on the annotation information, filtering the sites with synonymous mutation and mutation frequency greater than 0.01;
3) Homozygote positioning is carried out on the rest loci of the CVID patient P1, so that the number of candidate variation is further reduced; reserving a homozygous mutation site and a compound heterozygous mutation site for the CVID patient P2;
4) Determining candidate genes and mutation according to the functions of the genes with the mutation sites;
4. primer design: candidate gene Genomic DNA standard sequences were derived from UCSC (http:// genome. UCSC. Edu /), primers for candidate genes were designed using Primer3 (version 0.4.0, http:// Primer3.Ut. Ee /) and synthesized (biological engineering Co., ltd.) and Primer-BLAST (http:// www.ncbi.nlm.nih.gov/tools/Primer-BLAST /) was used to verify Primer specificity. The primer sequences were as follows:
p1 (homozygote c.379G > A)
Forward primer: 5'-AGCAATAGAATAGATACACTGAAGGTT-3' (SEQ ID NO: 6)
Reverse primer: 5'-TGGACTACAAAGTCTATTTAAGGGTTA-3' (SEQ ID NO: 7)
P2 (Compound hybrid c.137T > A/c.480G > A)
For c.137T > A:
forward primer: 5'-TGGTAAACTTCTGTGGTTCTCTT-3' (SEQ ID NO: 4)
Reverse primer: 5'-TGGGGCTGACTTTCATTTCT-3' (SEQ ID NO: 5)
For c.980g > a:
forward primer: 5'-TGTACATTAAAGCTTTTTATTTTGGTG-3' (SEQ ID NO: 8)
5. Reverse primer: 5'-CATCATAGTAGGAAAAACGACCA-3' (SEQ ID NO: 9) candidate nucleotide variation mutation detection for patient and corresponding in-family members:
1) And (3) PCR amplification: amplifying the extracted DNA sample by using the designed primer;
(1) PCR reaction system: 25 μl of
(2) PCR reaction conditions for exon 2:
exon 4 PCR reaction conditions:
exon 7 PCR reaction conditions:
exon 8 PCR reaction conditions:
exon 21 PCR reaction conditions:
2) PCR product analysis: the PCR product is subjected to 6% non-denaturing polyacrylamide gel electrophoresis (proper voltage is selected according to the size of amplified fragments), and a silver nitrate solution is used for dyeing and developing; analyzing the electropherogram;
3) After a sample with single band type and higher concentration is purified by the PCR product, the PCR amplification product is analyzed by adopting the automatic gene analyzer 3500 of the American ABI company to analyze the Sequencing chart according to the operation of a BigDye 3.1Sequencing kit (ABI company) kit, and the mutation is analyzed by comparing the Sequencing chart with a standard sequence. Identifying pathogenic genes and pathogenic mutation, and finding that P1 is the 379 th basic group of the RAD50 gene coding region to generate homozygous G- > A change (figure 5A), P2 is the 137 th basic group of the RAD50 gene coding region to generate heterozygous T- > A change and 980 th basic group of the coding region to generate heterozygous G- > A change (figure 5B);
(1) PCR product purification reaction system:
(2) PCR product purification reaction conditions: heat preservation at 37 ℃ for 120 min, 80 ℃ for 15 min and 4 DEG C
(3) The sequencing PCR reaction was 1/16 (10. Mu.l):
(4) sequencing PCR reaction conditions: (96 ℃ C. 30 seconds- & gt 55 ℃ C. 15 seconds- & gt 60 ℃ C. 3 minutes). Times.33 cycles- & gt 4 ℃ C. Heat preservation
(5) Sequencing product purification: the sequencing PCR product was purified by ethanol/EDTA/NaAc (sodium acetate) method, 10. Mu.l reaction system, 96 well plate. The specific operation steps are as follows:
a) Mu.l of a mixture of 125mM EDTA and 1. Mu.l of 3M NaAc was added to each well;
b) Adding 25 μl of 100% absolute ethanol into each hole, sealing with aluminum foil paper, shaking, mixing for 4 times, and standing at room temperature for 15 min;
c) Centrifuging at 2800 Xg for 30 min in a centrifuge, immediately inverting the 96-well plate, centrifuging to 185 Xg, and stopping centrifuging (centrifuging for 1 min from centrifuge start to 185 Xg stop);
d) Adding 35 μl of 70% ethanol into each well, centrifuging at 4deg.C for 15 min at 1650 Xg, immediately inverting 96-well plate, centrifuging to 185 Xg, and stopping centrifuging (from start of centrifuge to 185 Xg stop, centrifuging for 1 min);
e) Repeating the step d once, and standing at room temperature;
f) After ethanol is volatilized, 10 μl Hi-Di Formamide is added into each well to dissolve DNA;
g) After the sample is completely dissolved, placing the sample at 96 ℃ for denaturation for 5 minutes, rapidly placing the sample in ice for cooling for 15 minutes, and loading the sample into a machine;
4) Sequencer (ABI 3500DNA Analyzer) operation;
6. Mutation analysis: after Sanger sequencing is carried out on the PCR amplified products, comparing standard sequences, and identifying whether the 379 th basic group of the coding region of the RAD50 gene of the patient P1 is subjected to G-A homozygous change, and whether the RAD50 gene of the patient P2 is subjected to c.137T > A heterozygous mutation and c.630G > A heterozygous change simultaneously;
7. collecting other CVID family samples (48) and normal control samples (1118), scanning the RAD50 gene, and finding that the pathogenic mutation of another CVID family patient is c.1114C > T of RAD 50; in view of the fact that SIgAD and CVID may have the same pathogenic gene, the inventors also collected 791 SIgAD patients, scanned their RAD50, and found pathogenic mutations for two SIgAD patients, composite heterozygous c.494c > a/c.980g > a and heterozygous c.3230g > a, respectively.
The inventors found that P1 makes a homozygous mutation c.379G > A in exon 4 of RAD50, which results in the substitution of valine (Val) at amino acid sequence 127 with isoleucine (Ile). Sanger sequencing found that the patient's mother carried heterozygous c.379G > A (the patient's father had gone), and that the patient's normal siblings had no such mutation (FIG. 5A). The pathogenic mutation of P2 is complex heterozygous, heterozygous mutation c.137T > A occurs in exon 2 and heterozygous mutation c.980G > A occurs in exon 7 of RAD50, which respectively results in substitution of isoleucine at amino acid sequence 46 with valine and substitution of arginine at 327 with histidine (FIG. 5B). The inventors collected 48 CVID patients, 791 SIgAD patients and 1118 normal controls, scanned for RAD50 genes from these samples, preserving sites with a frequency of less than one thousandth. The inventors found that the pathogenic mutation in patient P4 was a complex heterozygous for the pathogenic mutation in patient SIgAD family (fig. 4, family 3), c.284 c > a (p.165P > H) on exon 4 and c.630 g > a (p.327 r > H) on exon 7. RAD50 is a component of the nuclease complex MRE11/RAD50/NBS1 (MRN). MRN increases DNA double strand break damage repair efficiency by recruiting and activating ATM proteins. Patients with defects in ATM, MER11 or NBS1 were found to develop a range of symptoms of antibody deficiency including SIgAD, CVID and hyperimmune M syndrome. RAD50 has two major transcripts (ENST 00000265335 and ENST 00000453394). The protein products of these two transcripts are widely involved in a number of different physiological and biochemical pathways including DNA double strand break damage repair pathways, maintenance of telomerase activity, DNA single strand break damage repair pathways, meiotic recombination, and the like (fig. 6). The mutation sites found above have an effect on both transcripts. All mutation sites were found to be highly conserved across multiple species by alignment across species homology sequences (fig. 7); p.127V > I is located on the surface of protein RAD50, potentially disrupting its binding capacity to the ATPase-DNA binding domain (FIG. 8); isoleucine at position 46 of wild-type RAD50 was centered in the first alpha helical groove and hydrogen-bonded to position 26 (threonine), 34 (leucine), 156 (phenylalanine), 1229 (alanine), 1267 (isoleucine) of the protein sequence, respectively, so the p.46i > N mutation would disrupt these interactions, altering the tertiary structure and stability of the RAD50 protein product (fig. 8); the c.494c > a mutation results in a positively charged protein sequence at position 165, with a strongly polar histidine being replaced by an uncharged, non-polar proline, which is located next to the N-terminal nucleotidyl hydrolase domain, which is thought to play an important role in RAD50 binding to MER11 (fig. 8); arginine at position 327 (uncharged) is located in the N-terminal coiled-coil domain, replaced with positively charged histidine, a mutation which may be a minor allelic mutation.
Example 2 verification of mutation of the novel Gene RAD50 into the pathogenic Gene
1. Observing the abnormal conditions of the cell intervals G2 and G0, wherein the experimental procedure of the abnormal condition of the G2-phase chromatin is as follows:
1) Heparinized blood cells of patients and control groups were cultured using RPMI-1640 cell culture medium supplemented with 10% calf serum, 1% L-glutamine, penicillin (100U/mL) and streptomycin (100 g/mL), and lymphocyte proliferation was induced using phytohemagglutinin (1. Mu.g/mL);
2) The medium system was placed at 37℃with 5% CO 2 4 days in a wet environment;
3) Exposing the culture medium to gamma-ray (100 cGy) radiation for 4 hours;
4) Adding colchicamide (0.15 mug/mL) to the irradiated culture medium to fix the metaphase;
5) The medium was placed in a centrifuge (1200 RPM) for 10 minutes and lymphocytes were taken;
6) Lymphocytes were treated with sodium chloride solution for 15 minutes and fixed with fresh fixative (methanol/glacial acetic acid: 3/1);
7) Repeating the step 6;
8) Staining was performed for 5 min using Giemsa (2%);
9) Observing and counting the number of breaks of chromosome haplotypes and isocratic monomers of the patient and the control;
the steps of the G2-phase chromatin abnormality experiment are as follows:
1) Heparinized blood cells of patients and controls were cultured using RPMI-1640 cell culture medium supplemented with 10% calf serum, 1% l-glutamine, penicillin (100U/mL) and streptomycin (100 g/mL);
2) Exposing the cells to gamma-ray (300 cGy) radiation;
3) Stimulation with phytohemagglutinin and placing at 37deg.C, 5% CO 2 Culturing in the environment for 44 hours;
4) Adding cytochalasin B (6 mug/mL) to the medium;
5) After 92 hours hypotonic stimulation was performed using sodium chloride solution and fixation was repeated 3 times (methanol/glacial acetic acid: 3/1);
6) Staining was performed using Giemsa (2%);
7) Taking 500 cells with double nuclei for counting the occurrence frequency of each micronucleus respectively from patients and controls;
the inventors observed chromatin abnormalities (chromosome haplotypes and break-off numbers of the isochrome monomers) in patients and control groups after gamma irradiation, and found that P1 lymphocytes from CVID patients had a frequency of chromatin abnormalities as high as 92.5%, whereas normal control lymphocytes had a frequency of chromatin abnormalities of 40% (fig. 9a, b).
2. After observing the radiation, the relative activity of the patient and control cells:
1) Peripheral blood lymphocytes of CVID patients and control groups are adopted, and EBV is used for infection to obtain a B cell line;
2) Cloning experiments: inoculating a proper amount of irradiated (1.0 Gy) cells into a culture medium, culturing for 10-13 days, staining with MTT, counting the clone number of the cells larger than 32, and calculating the cell survival fraction;
The inventors observed the cloning efficiency of patient B lymphocytes after irradiation, and found that the average survival rate of P2 cells in CVID patients was 31%, which is a moderate level.
3. Analysis of the use of microhomology sequences (MH) in immune cell VDJ rearrangement: extracting DNA from the white blood cells for multiplex PCR, and amplifying S [ mu ] -S [ alpha ] fragments in B memory cells; judging exchange recombination connection through alignment with S [ mu ] and S [ alpha ] sequences of a germ line; the use of the minor homologous sequences was judged by comparison with the S.mu. -S.alpha.fragment of the control group.
The inventor observes the use condition of tiny homologous sequences (MH) when B lymphocyte VDJ rearrangements, and finds that the frequency of MH length of more than 10bp used by patients with RAD50 defects is more than 40%, and only 5% -15% of the normal control; the patients with RAD50 defects used a higher proportion of MH than MRE11 patients and NBS patients (fig. 9C).
Example 3 detection of the presence of RAD50 Gene mutation in DNA samples
1) Designing primers aiming at CVID and SIgAD pathogenic genes RAD50, and performing PCR amplification;
2) Purifying the PCR product and performing Sanger sequencing analysis;
3) Identifying whether the RAD50 coding region carries any of the following mutations: homozygous mutation of c.379g > a, complex heterozygous mutation of c.137t > a/c.480 g > a or complex heterozygous mutation of c.494c > a/c.460 g > a=. Wherein the c.379G > A homozygous mutation and the c.137T > A/c.980G > A complex heterozygous mutation result in CVID.
The mutation occurs in exons 2, 4 and 7 of a gene RAD50, and the nucleotide sequence of PCR amplification is shown as SEQ ID NO: 13-15, the corresponding normal gene sequence is SEQ ID NO:10 to 12.
Example 4 in vitro kit for detecting CVID and SIgAD pathogenic gene RAD50 mutation
The kit comprises:
1) Primers for amplifying the CVID and SIgAD pathogenic genes RAD50 exons;
2) Enzyme amplified by PCR and corresponding buffer solution;
3) The kit comprises the following components:
the kit contains amplified exons and all forward (F) and reverse (R) primers (SEQ ID NO: 4-SEQ ID NO:9RAD50-2F,2R; RAD50-4F,4R; RAD50-7F, 7R) of sequences at the junction of the exons and the introns, and the primers are used in pairs.
4) The using method comprises the following steps:
a) Adding the PCR primer, taq DNA polymerase and other reagents into the DNA sample to be detected, and performing PCR reaction;
b) The PCR reaction products were purified and Sanger sequenced, and the resulting sequences were aligned with the standard sequence of the gene to determine the presence or absence of mutation.
In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiments or examples. Furthermore, the particular features, structures, or material characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
While embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that: many changes, modifications, substitutions and variations may be made to the embodiments without departing from the spirit and principles of the invention, the scope of which is defined by the claims and their equivalents.
Claims (8)
1. A nucleic acid, characterized in that,
the nucleic acid has a mutation selected from the group consisting of:
137T > A and c.980G > A; or (b)
c.494C > A and c.980G > A;
wherein the nucleic acid is DNA.
2. A protein, characterized in that,
compared to the amino acid sequence of a protein expressed by the wild-type RAD50 gene, the protein has the following mutations:
(1) Isoleucine at amino acid sequence 46 is mutated to valine and arginine at position 327 is mutated to histidine, the protein having the amino acid sequence as set forth in SEQ ID NO:1, and a polypeptide sequence shown in the specification; or (b)
(2) Proline at amino acid sequence 165 is mutated to histidine and arginine at position 327 is mutated to histidine, the protein having the amino acid sequence as set forth in SEQ ID NO:3, and a polypeptide having the amino acid sequence shown in 3.
3. Use of a reagent for detecting the nucleic acid of claim 1 or the protein of claim 2 for the preparation of a kit for diagnosing immunodeficiency disorders.
4. The use of claim 3, the reagent comprising at least one of an antibody, a probe, a primer, and a mass spectrometry detection reagent specific for at least one of the nucleic acid and the protein;
the immunodeficiency is primary immunodeficiency;
the primary immunodeficiency diseases include variant immunodeficiency diseases and selective IgA defects.
5. Use of a biological model for screening a drug, said biological model carrying at least one of the following:
(1) The nucleic acid of claim 1;
(2) The protein of claim 2.
6. The use according to claim 5, wherein the biological model is a cellular model or an animal model.
7. A medicament for treating immunodeficiency disorders, said medicament comprising: an agent that specifically alters the nucleic acid of claim 1.
8. The drug according to claim 7, wherein the agent is an agent based on a gene editing or nucleic acid synthesis method;
the immunodeficiency is primary immunodeficiency;
the primary immunodeficiency diseases include variant immunodeficiency diseases and selective IgA defects;
the nucleic acid or the gene is mutated to c.137T > A and c.980G > A, the primary immunodeficiency disease being a variant immunodeficiency disease;
The nucleic acid or the gene is mutated to c.284C > A and c.480G > A, and the primary immunodeficiency disorder is a selective IgA deficiency.
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