CN101403008A - Gene diagnosis reagent kit for detecting adults progeria syndrome - Google Patents

Abstract

The invention discloses a genetic diagnosis kit which detects the early-aging syndromes of adults and belongs to the biotechnical field. A method which diagnoses the early-aging syndromes of the adults includes the following steps of: (1) gathering the blood, body fluid or tissue samples of individuals to be tested and extracting the DNA; (2) taking the DNA as a template and carrying out PRC reaction to PRC primers which are designed in accordance with the mutation sites of WRN genes to obtain products of the PRC reaction; (3) carrying out sequencing analysis to the obtained PRC products and comparing the obtained sample sequences with normal genetic sequences to determine whether mutation exists; and (4) judging whether the individuals to be tested have the early-aging syndromes caused by the mutation of the WRN genes according to the results. The invention has the advantages of providing the mutation of the WRN genes among the Chinese population for the first time and showing the correlation between the mutated genes and WS symptoms. The detection method is simple and low in cost, with direct and reliable results, and can be applied to the large-scale screening and diagnosis of the 2806insA mutation of WRN genes of Werner syndrome.

Description

A genetic diagnostic kit for detecting adult progeria syndrome

technical field

The present invention relates to a gene diagnosis kit for detecting adult progeria syndrome, more specifically a kit for detecting mutation of WRN gene 2806insA, the kit and method can be used for auxiliary diagnosis and new drug development of the disease, Belongs to the field of biotechnology.

Background technique

Werner syndrome (Werner syndrome, WS), also known as adult progeria (Adult progeria), adult premature aging syndrome (Adult premature aging syndrome), is a rare autosomal recessive genetic disease. The clinical manifestations of WS mainly occur in connective tissue, endocrine system, immune system, nervous system, etc. Including: short stature, premature hair growth, skin fibrosis, scleroderma, cataract, subcutaneous tissue calcification, diabetes, hypothyroidism, hyperlipidemia, systemic lupus erythematosus, dementia, schizophrenia, etc. The development of WS patients is basically normal in childhood, and various symptoms begin to appear after the age of 18. Due to the thin subcutaneous fat and skin fibrosis, special facial features are formed, including pseudo-exophthalmos, beak-shaped nose, and radial grooves around the mouth , teeth protruding, lower jaw retracted, known as the "bird-like face". WS patients may be accompanied by various tumors, including soft tissue sarcoma, osteosarcoma, melanoma, thyroid cancer, and meningioma. Due to the early multisystem aging manifestations of WS patients, their life expectancy is greatly shortened, with an average life expectancy of only 47 years.

The study of the pathogenesis of WS has an important reference role in elucidating the mechanism of human aging and aging-related diseases, so WS is called "the natural gene model of human aging". In 1992, GOTO et al. located the WRN gene (WRN) at 8p11.1-21.1. The full length of WRN is 5.2 kb, including 35 exons, encoding 1432 amino acids. WRN protein is a DNA helicase, a member of the RecQ family, and it contains seven helicase motifs, two of which are ATP-binding proteins. The important role of DNA helicase is to form the unwinding complex during DNA replication and participate in DNA unwinding and DNA damage repair. Structural analysis shows that WRN protein includes a RecQ-type uncoiling domain in the middle of the protein and a nuclease domain at the N-terminus. WRN protein has both 3'-5' helicase activity and exonuclease activity. Various DNA structures generated during DNA metabolism are substrates of WRN protein.

The 1369-1402 amino acid residues at the C-terminal of WRN protein have the function of nucleic acid localization. In addition, the 949-1092 amino acid residues in the RecQ helicase domain also have nucleic acid positioning functions. WRN protein is mainly located in the nucleolus, and WRN protein gathers at the site of DNA damage in the nucleus when DNA is damaged. There are two domains at the C-terminus of the WRN protein: RecQ helicase conserved region (RQC) and helicase, ribonuclease, C-terminal conserved structure (helicase, RNaseD, C-terminal conserved Region, HRDC) . Studies have confirmed that WRN protein interacts with substrate DNA or protein through these two domains. Mutations of the WRN gene were found in WS patients, and a total of 50 mutations were found by 2006. These mutations include missense mutations, frame-shift mutations, nonsense mutations, etc., and are distributed in various exons. The WRN gene has obvious heterogeneity, and the mutation forms of different races are completely different. At present, the reports on Werner syndrome in the Chinese population are all case reports, and there is a lack of research on genetic diagnosis. The research on the hotspots of WRN gene mutations in the Chinese population is still blank.

After searching the existing domestic and foreign literature, there is no report on the 2806insA mutation of the WRN gene in Werner syndrome. The discovery of the WRN gene in the Chinese population will not only promote the development of basic research on the pathogenesis of the disease, but will also greatly promote the development of Werner syndrome. The development of genetic diagnosis and treatment. Through prenatal screening and newborn WRN gene mutation screening, the birth rate of children with Werner syndrome can be reduced, the life behavior of patients carrying the disease-causing gene can be guided, the occurrence of diseases can be prevented, and social pressure can be greatly relieved. In addition, WRN gene research will play an important role in the research of longevity and aging. Kits for in vitro detection of the 2806insA mutation of the Werner syndrome-related gene WRN are indispensable for the research and development of these disciplines.

Contents of the invention

The first technical problem to be solved by the present invention is to provide a method for diagnosing progeria syndrome (Werner syndrome) in adults, by detecting whether the test sample of suspected patients has the 2806insA mutation described in the present invention and confirms the patient Whether Werner syndrome, provide reference for clinical diagnosis and treatment.

The second technical problem to be solved by the present invention is to provide a kit for the 2806insA mutation of adult progeria syndrome (Werner syndrome).

The third technical problem to be solved by the present invention is to provide the application of 2806insA mutation of adult progeria syndrome (Werner syndrome) in the diagnosis of diseases related to Werner syndrome.

To achieve the above object, the present invention adopts the following technical solutions:

A method for diagnosing progeria syndrome in adults, comprising the steps of:

(1) Collect blood, body fluid or tissue samples of the individual to be tested, and extract DNA;

(2) using the DNA as a template, performing a PCR reaction with PCR primers designed for the mutation site of the WRN gene to obtain a PCR reaction product;

(3) Perform sequencing analysis on the obtained PCR product, compare the obtained sample sequence with the normal gene sequence, and confirm whether there is a mutation;

(4) According to the above results, it is judged whether the individual to be tested has Werner syndrome caused by WRN gene mutation.

In the present invention, conventional methods for detecting point mutations in the art can be used to detect the mutation site of the WRN gene, such as PCR sequencing.

The PCR primer used in the present invention can utilize Primer Premier 5.0 to design according to known nucleotide sequence, is usually 15-30 bases, and GC content is about 45-50%, under appropriate temperature and terminal specific binding. In the present invention, the sequence of WRN gene refers to Genbank GeneID: NM_000553.

The PCR primers designed for the mutation site of the WRN gene are:

WRN2806-F: 5'-CCCACGGAGGGTTTCTATCT-3' (SEQ ID No. 1)

WRN2806-R: 5'-GAACCATTGGCAGTGCTGTC-3' (SEQ ID No. 2)

A kit for detecting the 2806insA mutation of adult progeria syndrome, the components and content of the kit are as follows, and stored at -20°C:

20ul 10X PCR buffer (Pharmacia),

4ul 10mM dNTP mixture (Pharmacia),

2ul (5unit/ul) Taq DNA polymerase (Takara),

Each 10ul (10pmol/ul) F1 (SEQ ID NO.1) and R1 (SEQ ID NO.2) primers,

1.5ml pure water.

For example, one embodiment of the present invention provides a kit for detecting WRN gene mutations. The container is filled with components for detecting WRN gene 2806 mutations. Manufacturing, use and sales information of products. For example, a kit for directly detecting the WRN gene mutation site in a sample after PCR amplification may contain a set of amplification primers, dNTPs, DNA polymerase and its buffer for PCR reactions, reagents required for sequencing reactions, etc. one or more species. Those skilled in the art know that the above components are only illustrative, for example, the primers can use the above-mentioned pair of WRN2806-F and WRN2806-R primers, and the DNA polymerase used in the PCR reaction can be used Enzyme for PCR amplification.

The present invention collects meningioma patients through the neurosurgery outpatient clinic. On the premise that the patients and their families are voluntary, after signing the informed consent, 5-10ml blood samples are collected, and a database of inpatient medical records is established to record in detail the patient's condition, the incidence of disease in the family, and contact information. . Genomic DNA was then extracted by phenol-chloroform extraction method, quantified, stored at -20°C, and each copy of DNA accurately corresponded to the registered patient's clinical data. Primers were designed according to Primer Premier 5.0, including the 2806 site of the WRN gene and the sequences on both sides, for PCR amplification. Direct sequencing of PCR products: the sequencing primers are the same as the PCR amplification primers, and ABI 3730 DNA sequencer is used for forward and reverse sequencing. The obtained sequence was compared with the standard sequence in Genbank to determine 2806 mutation sites. Translate according to the normal reading frame to determine the mutation site of WRN gene.

Taking patients with petroclival meningioma as the research object, through the screening of the exons of the WRN gene coding region in 3 family members of the disease and 20 normal controls, it was found that a patient had a new mutation of the WRN gene in exon 8 . The patient is a compound heterozygote. This mutation was not found in 20 cases of control group, indicating that this mutation point is related to WRN gene.

The insertion of bases leads to frameshift mutations, which change the entire DNA coding frame downstream of the insertion point, and stop codons appear prematurely, resulting in premature termination of polypeptide chain synthesis, shortening the length of the peptide chain, and becoming inactive polypeptide fragments. Since the WRN protein is a member of the RecQ family of DNA helicases, the frameshift mutations caused by the insertion of bases ultimately affect DNA unwinding and DNA damage repair.

The present invention also provides the application of the WS mutation gene in the diagnosis and treatment of WS. By detecting whether the mutation of the WRN gene exists in the sample to be tested from the patient, the cause and type of the patient's systemic disease can be judged, and further provide clinical diagnosis and treatment. for reference. In addition, in further clinical treatment, after the detection result shows that the mutation of the WRN gene has occurred, the normal gene can be introduced into cells carrying the mutant gene and expressed therein, and it can recombine with the endogenous mutant gene, thereby Gene therapy is available.

The advantage of the present invention is: the present invention finds that WS patients have the 2806insA mutation site by screening the WRN gene of 3 WS family members and 20 normal control members. The mutation was not detected in the screening of 20 normal people, indicating that the mutation site is closely related to the WRN gene. The present invention provides for the first time a mutation of the WRN gene in the Chinese population, and illustrates the correlation between the mutation gene and WS. Simultaneously, the present invention proposes the cause and detection method for judging the occurrence of WS syndrome by detecting whether there is a WRN gene mutation in the patient. The method is simple, low in cost, and the detection result is direct and reliable. large-scale screening and diagnosis.

The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, so that the public has a deeper understanding of the content of the invention, and is not a limitation of the present invention. All equivalent replacements in the field performed according to the disclosure of the present invention belong to this invention. protection scope of the invention.

Description of drawings

Fig. 1 is a partial sequencing result of exon 18 of the WRN gene by the method of the present invention, the upper part is the mutant sequence, the lower part is the normal control sequence, and the arrow points to the mutation site.

Detailed ways

Example 1: Detection method for WRN gene 2806 heterozygous mutation

1. Research object

Meningioma patients were collected through the neurosurgery outpatient clinic. On the premise that the patients and their family members signed the informed consent, 5-10ml blood samples were collected, and the inpatient medical record database was established to record the patient's condition, family history and contact information in detail. This study has been approved by the institutional ethics committee.

2. Preparation of Genomic DNA

first day

1. In the presence of the anticoagulant EDTA, centrifuge the collected 5-10ml human peripheral blood at 2500rpm for 30 minutes to remove serum;

2. Add 0.2% NaCl solution to bring the total volume to 50 ml. Gently shake the solution 5-6 times and place it on ice for 15 minutes;

3. Centrifuge at 2500rpm for 30 minutes to collect the precipitate;

4. Wash with 0.2% NaCl solution in a similar manner to the previous one;

5. In the thus obtained precipitate, add 10 mM Tris-HCl (pH 8.0) and 10 mM EDTA (4 ml) to suspend the precipitate;

6. Add 10% SDS, 25mg/ml proteinase K and 10mg/ml RNaseA to the suspension, the addition amount is 4ml, 16ul and 20ul respectively, then mix the suspension gently upside down;

7. Incubate the suspension overnight at 37°C.

the next day

1. Add 4ml of phenol/Tris solution, mix the mixture by inverting the mixture up and down;

2. Centrifuge at 3000rpm for 10 minutes to remove the water layer;

3. Mix the aqueous layer with 4ml of phenol/chloroform solution, then inversely mix and centrifuge at 3000rpm for 10 minutes;

4. Remove the water layer, extract twice with chloroform to obtain the water phase, add 1/10, 3M NaAC (pH5.2), and twice the amount of cold absolute ethanol to it to precipitate the DNA;

5. Wash with 70% ethanol to obtain genomic DNA;

6. Dissolving the thus obtained DNA, genomic DNA, in TE, and quantitatively measuring the absorbance of the mixture at 260 nm;

7. Correct the concentration of the DNA working solution to 50ng/ul, and store in a -20°C refrigerator.

3. PCR amplification of WRN gene 2806

1. Primer sequence

Upstream primer: WRN2806-F: 5'-CCCACGGAGGGTTTCTATCT-3'

Downstream primer: WRN2806-R: 5'-GAACCATTGGCAGTGCTGTC-3'

2. PCR reaction system

Table 1: PCR reaction system

Table 2: PCR reaction process of WRN gene 2806 fragment

3. Sequencing: After PCR, the product was directly sequenced, which was entrusted to Invitrogen of the United States to complete.

4. Mutation Analysis

Sequence comparison analysis was performed using SeqmanTM software at the end of the DNAStat5.0 (Lasergene Inc.) software package. Compare the sequence obtained by sequencing with the standard sequence retrieved by NCBI to find the mutant sequence and find the mutation site.

The results are shown in Figure 1. After the insertion of the base, a frameshift mutation is caused, which makes the DNA coding frame downstream of the insertion point all changed, and the stop codon appears prematurely, causing the premature termination of the polypeptide chain synthesis, shortening the length of the peptide chain, and becoming inactive. polypeptide fragments. Since the WRN protein is a member of the RecQ family of DNA helicases, the frameshift mutations caused by the insertion of bases ultimately affect DNA unwinding and DNA damage repair.

Example 2: Kit for the detection of WRN gene 2806 heterozygous mutation

1. Components: Contains a pair of primers that can amplify the 2806 heterozygous mutation site of the WRN gene, and corresponding reagents for sequencing. The components and contents are as follows, and stored at -20°C:

20ul 10X PCR buffer (Pharmacia),

4ul 10mM dNTP mixture (Pharmacia),

2ul (5unit/ul) Taq DNA polymerase (Takara),

Each 10ul (10pmol/ul) F1 (SEQ ID NO.1) and R1 (SEQ ID NO.2) primers (self-made),

1.5ml pure water (homemade).

Two. the using method of described test kit mainly comprises the following steps:

(1) Extract the DNA of the blood sample to be tested, and use a pair of WRN2806-F and WRN2806-R primers shown in the sequence table SEQ ID No.1 and SEQ ID No.2 to carry out PCR amplification reaction;

(2) The PCR reaction product was directly sequenced after purification, and the obtained sequence was compared with the standard sequence in Genebank to determine the existence of the mutation site.

(3) Translate according to the normal reading frame to determine whether the amino acid mutation site exists.

For the specific method, refer to the detailed description in the previous embodiments.

The kit can detect the mutation site of the WRN gene simply and quickly, so as to be applied to the mutation detection, diagnosis and treatment of WS cases.

sequence listing

<110> Beijing Institute of Neurosurgery

  Beijing Tiantan Hospital Affiliated to Capital Medical University

<120> A genetic diagnostic kit for detecting adult premature aging syndrome

<130>

<160>2

<170>PatentIn version 3.5

<210>1

<211>20

<212>DNA

<213> Synthetic, Primer 1

<400>1

cccacggagg gtttctatct 20

<210>2

<211>20

<212>DNA

<213> artificial synthesis, primer 2

<400>2

gaaccattgg cagtgctgtc 20

Claims (3)

1. A method for diagnosing adult progeria, comprising the steps of: (1) Collect blood, body fluid or tissue samples of the individual to be tested, and extract DNA; (2) using the DNA as a template and performing a PCR reaction with PCR primers designed for the mutation site of the WRN gene to obtain a PCR reaction product; (3) Perform sequencing analysis on the obtained PCR product, compare the obtained sample sequence with the normal gene sequence, and confirm whether there is a mutation; (4) According to the above results, it is judged whether the individual to be tested has adult progeria syndrome caused by WRN gene mutation. 2. The application of 2806insA mutation in adult progeria syndrome in the diagnosis of Werner syndrome-related diseases. 3. A test kit for detecting progeria syndrome in adults, characterized in that: the composition and content of the test kit are as follows, stored at -20°C: 20ul 10X PCR buffer (Pharmacia), 4ul 10mM dNTP mixture (Pharmacia), 2ul (5unit/ul) Taq DNA polymerase (Takara), Each 10ul (10pmol/ul) F1 (SEQ ID NO.1) and R1 (SEQ ID NO.2) primers, 1.5ml pure water.

Images