CN105255902B

CN105255902B - Early-onset diabetes gene mutant and application thereof

Info

Publication number: CN105255902B
Application number: CN201510742283.1A
Authority: CN
Inventors: 帕它木·莫合买提; 哈木拉提·吾甫尔; 戴兰兰; 伊力哈木江·依马木; 热沙来提·阿不都瓦衣特; 张建国; 方明艳; 木哈达斯·吐尔逊依明; 托兰古丽·买买提库尔班; 祖力卡提阿依·阿布都拉
Original assignee: Xinjiang Medical University
Current assignee: Xinjiang Medical University
Priority date: 2015-11-04
Filing date: 2015-11-04
Publication date: 2020-01-10
Anticipated expiration: 2035-11-04
Also published as: CN105255902A

Abstract

The invention discloses an early-onset diabetes gene mutant, the nucleotide sequence of which is shown as SEQ ID NO. 1. The invention also discloses an early-onset diabetes gene mutant encoding protein, and the amino acid sequence of the early-onset diabetes gene mutant encoding protein is shown as SEQ ID NO. 2. The invention also discloses a system for screening biological samples susceptible to early-onset diabetes, and the invention discovers a novel pathogenic gene of early-onset diabetes (mody). Based on the method, the carrier can be used for early screening the carrier of the pathogenic mutant gene of the early-onset diabetes (mody), and then early intervention treatment is carried out before the carrier is attacked; it can also be used for molecular diagnosis of patients with early-onset diabetes (mody) and differential diagnosis of related diseases. The technology has the advantages of rapidness, accuracy, high efficiency, simplicity, high early diagnosis rate and the like, and the detection result can provide scientific basis for early diagnosis, differential diagnosis and development of the treatment drugs of the early-onset diabetes (mody).

Description

Early-onset diabetes gene mutant and application thereof

Technical Field

The invention belongs to the technical field of medical molecular biology, and particularly relates to an early-onset diabetes gene mutant, an early-onset diabetes gene mutant encoding protein, and application of the early-onset diabetes gene mutant.

Background

MODY is a monogenic dominant disease, which is considered to be a special subtype of T2DM and is now classified as a special type of diabetes mellitus, MODY should be diagnosed as having a disease onset before the age of ① 25, at least 2 patients in the ② family, ③ has at least three autosomal dominant inheritance, and ④ oral hypoglycemic drugs are effective for at least 5 years or have normal plasma C-peptide levels.

With the development of technologies such as genetic linkage analysis, candidate gene cloning, association analysis and sequencing, 13 more specific genes related to the onset of MODY were found by the beginning of 2013, and they are (in the time sequence found): liver nuclear factor 4 alpha gene (hepatic nuclear factor 4 alpha, HNF 4 alpha; MODY1), glucose kinase gene (glucokinase, GCK; MODY2), liver nuclear factor1 alpha gene (hepatic nuclear factor1 alpha, HNF1 alpha; MODY3), insulin promoter factor1 gene (insulin promoter factor1, IPF 1; MODY4), liver nuclear factor1 beta gene (hepatic nuclear factor1 beta, HNF1 beta; MODY5), neurogenic differentiation factor1 gene (neurogenic differentiation factor1, NEUROD 1; MODY6) and later reported gene of gene, beta-cell adenosine triphosphate-sensitive potassium channel genes (KCNJ11, MODY 13). These genes play an important role in the expression of genes related to sugar metabolism in adulthood, and also influence the development of organs and the differentiation of cells in human embryonic period. The different genetic subtypes of MODY generated by the gene mutation have obvious differences in clinical, physiological phenotype and genetic characteristics. Because of their different clinical manifestations, the clinical treatment methods are different, and the clinical effects are different.

In recent years, MODY has been drawing attention from various researchers as one of the special types of diabetes mellitus due to single gene mutation. Because MODY has the characteristics of early onset age, autosomal dominant inheritance, high penetrance rate and the like, and is beneficial to the collection of multi-generation families, the MODY family provides an ideal research object for the research of molecular genetic etiology and pathogenesis of diabetes, and the MODY etiology is determined from the gene molecular biology level, thereby being beneficial to understanding the disease process, judging the prognosis of patients and carrying out targeted treatment on the patients. And the MODY family exome sequencing related research has not yet seen related research reports at home and abroad.

Whole genome exon sequencing (Exome sequencing) is considered to be an effective method for searching for rare monogenic disease-causing genes.

The majority of the single-gene disease mutation sites exist in exon regions, a few samples are selected, the SNP specific to a case is found by scanning all exons and combining with a bioinformatics analysis technology, and the pathogenic genes and the mutation sites of the single-gene disease can be found by verification. The exome sequencing technology can be applied to the research of discovering the monogenic disease with unknown pathogenic genes and can also be applied to the research of new variation sites of the monogenic disease pathogenic genes with definite pathogenic genes. Some researchers carry out exome sequencing on the monogenic disease with definite pathogenic genes, and the feasibility and the application value of the exome sequencing method in determining the monogenic disease pathogenic genes are proved. In 2009, 8 months, the Nature journal published a study on 12 human exome sequencing [4], which selected 12 sequencing subjects for exome sequencing, wherein 8 people's DNA map was confirmed by HapMap program, and 4 people were also freimann-Sheldon syndrome (Freeman syndrome) patients, a rare genetic disease caused by MYH3 gene variation. The disease-causing gene MYH3 was accurately identified from the DNA of 4 patients with Feremann-Scherdon syndrome by a multi-step classification assay after filtering out common and individual mutations.

In 9 months 2009, Choi et al discovered the site of pathogenic mutations in the new coding region of patients with congenital chloride-deprived diarrhea using whole exome sequencing techniques, and corrected previous misdiagnoses. Studies have shown the efficient use of whole exome sequencing for the discovery and clinical diagnosis of disease-associated genes.

In 11 months 2009, the Nature Genetics journal published a report of scientifically revealing a monogenic genetic disease of unknown etiology, namely, a miller syndrome pathogenic gene, using a whole exome sequencing technology. After researchers selected four patients with Miller syndrome from three independent families, performed exome sequencing on them, and compared the obtained data with public SNP database and previously measured exome data of 8 HapMap individuals, and filtered out common variation and individual variation, scientists found that there were two unknown mutation sites in all four patients, which were located on the DHODH gene.

In addition, several new mutant genes of human genetic diseases were discovered by exome sequencing technology, including sensory/ataxia neuropathy (sensory/motor neuropathy with ataxia), cleeric type cutaneous heterochromosis neutropenia (cleeric-type phikuidoderma with neutropenia), familial exudative vitreoretinopathy (familial exudative vitreoretinopathy), recessive non-syndromic deafness (recessive non-syndromic), talipes equinovarus (talipes equinovarus), atrial septal defect (atrial temporal defect), lobin sequence (robinine sequence), persistent left superior vena cava (persistent vena cava), and the like. Most of the new gene mutations found were nonsynonymous mutations, but insertion/deletion gene mutations were also found when the external control personnel were subjected to target gene sequencing by further using ordinary Sanger sequencing. Johnston et al discovered pathogenic mutation sites by sequencing the target regions of family members without probands. These studies demonstrate the superior ability of region-of-interest capture sequencing technologies, including exon sequencing, to discover new pathogenic sites of human disease.

Research shows that if the mutagenic gene of the disease is determined, the therapeutic means of the disease will be relatively clear. Especially diseases caused by single gene mutation. Mutations in several monogenes have been associated with the onset of type 2 diabetes. The clinical phenotype typing and the individual treatment of the diabetic patients caused by single gene mutation have been reported. For example, diagnosed patients with HNFl α diabetes are very sensitive to sulfonylurea treatment. The beta cell hypofunction of the patients is caused by the reduction of transcription factors in the glucose metabolism process, and sulfonylureas can act on K + channels to stimulate insulin secretion, so the sulfonylureas can be effectively treated.

Therefore, the etiology of the disease is known from the level of molecular biology, the progress of the disease can be better predicted, and more reasonable treatment can be carried out on a patient, so that the ideal state of blood sugar is kept. Therefore, when the exon sequencing technology is used for finding the pathogenic mutant gene of the Uygur family of MODY, the individual treatment of MODY patients is possible.

In view of the current state of diabetes diagnosis, type 2 diabetes has a "false positive" in its diagnosis. The specific type of diabetes that many patients may not have been definitively diagnosed due to the limited diagnostic means is classified as type 2 diabetes. In fact, with the progress of understanding of diseases and the progress of diagnostic techniques, a considerable part of type 2 diabetes, which has been considered to be polygenic, has been proved to be caused by genetic heterogeneity, i.e., it is controlled by a single major gene, such as MODY type, mitochondrial gene diabetes, etc. Diagnostic bias is one of the major reasons for poor therapeutic efficacy at present. Therefore, accurate diagnosis and even individualized diagnosis of diabetic patients are of great practical significance for improving the curative effect of diabetes and reducing a large number of complications, so as to improve the life quality of the patients and reduce the long-term economic burden of the patients.

Disclosure of Invention

The invention aims to provide an early-onset diabetes gene mutant, which solves the problems in the prior art.

Another objective of the invention is to provide a protein coded by the early-onset diabetes gene mutant.

Another object of the present invention is to provide a method for detecting early onset diabetes.

The first technical scheme adopted by the invention is that the nucleotide sequence of the early-onset diabetes mellitus gene mutant is shown as SEQ ID NO. 1.

The second technical scheme adopted by the invention is that the amino acid sequence of the early-onset diabetes gene mutant coding protein is shown as SEQ ID NO. 2.

The third technical scheme adopted by the invention is that the system for screening the biological sample susceptible to early-onset diabetes comprises the following steps:

a nucleic acid extraction device for extracting a nucleic acid sample from the biological sample;

a nucleic acid sequence determining device connected with the nucleic acid extracting device and used for analyzing the nucleic acid sample so as to determine the nucleic acid sequence of the nucleic acid sample;

a judging means connected to the nucleic acid sequence determining means to judge whether or not the biological sample is susceptible to early-onset diabetes based on whether or not the nucleic acid sequence of the nucleic acid sample has a c.10006A > G mutation as compared with the sequence of the existing wild-type early-onset diabetes gene.

Further, the nucleic acid extraction apparatus further comprises: an RNA extraction unit for extracting an RNA sample from the biological sample; and a reverse transcription unit connected to the RNA extraction unit for performing a reverse transcription reaction on the RNA sample to obtain a cDNA sample, the cDNA sample constituting the nucleic acid sample.

Further, the nucleic acid sequence determination apparatus further comprises: a library construction unit for constructing a nucleic acid sequencing library for the nucleic acid sample; and the sequencing unit is connected with the library construction unit and used for sequencing the nucleic acid sequencing library so as to obtain a sequencing result consisting of a plurality of sequencing data.

Further, the library construction unit further comprises: the PCR amplification module is internally provided with an early-onset diabetes gene specific primer so as to carry out PCR amplification on the nucleic acid sample by using the specific primer, the early-onset diabetes gene specific primer comprises a forward primer and a reverse primer, and the sequence of the forward primer is shown as Seq ID.3; the sequence of the reverse primer is shown in Seq ID.4.

The fourth technical scheme adopted by the invention is that the kit for screening the biological sample susceptible to the early-onset diabetes comprises a reagent suitable for detecting the early-onset diabetes genetic mutant; the reagent is a nucleic acid probe or a primer; the nucleic acid probe or the primer has nucleotide sequences shown in SEQ ID NO.3 and SEQ ID NO. 4.

According to a fifth technical scheme adopted by the invention, a construct comprises the early-onset diabetes mellitus gene mutant.

The sixth technical means of the present invention is a recombinant cell obtained by transforming a recipient cell with the above-described construct.

The seventh technical scheme adopted by the invention is that the method for constructing the drug screening model comprises the following steps: at least a part of cells of the animal express the early-onset diabetes gene mutant.

The invention has the beneficial effects that: the invention discovers a novel pathogenic gene of early-onset diabetes (mody). Based on the method, the carrier can be used for early screening the carrier of the pathogenic mutant gene of the early-onset diabetes (mody), and then early intervention treatment is carried out before the carrier is attacked; it can also be used for molecular diagnosis of patients with early-onset diabetes (mody) and differential diagnosis of related diseases. The technology has the advantages of rapidness, accuracy, high efficiency, simplicity, high early diagnosis rate and the like, and the detection result can provide scientific basis for early diagnosis, differential diagnosis and development of the treatment drugs of the early-onset diabetes (mody).

Drawings

FIG. 1 is a diagram showing a validation peak of sanger of the present invention, in which a heterozygous mutation is present at the 201 st base;

FIG. 2 is a diagram showing a validation peak of sanger according to the present invention, in which there is no mutation at the 201 st base;

FIG. 3 is a family diagram of the present invention;

FIG. 4 is a schematic diagram of the structure of a system for screening a biological sample susceptible to early onset diabetes in accordance with the present invention;

FIG. 5 is a schematic view of the structure of the nucleic acid isolation apparatus according to the present invention;

FIG. 6 is a schematic diagram of the structure of a library building block of the present invention.

Detailed Description

The present invention will be described in detail with reference to the following embodiments.

Example 1 early-onset diabetic Gene mutants

According to a first aspect of the present invention, the present invention provides an early-onset diabetic gene mutant nucleic acid. According to an embodiment of the invention, the nucleic acid has a c.10006A > G mutation compared to the sequence of an existing wild-type early-onset diabetes gene. Compared with the existing wild type early-onset diabetes protein sequence, the serine S of the coded protein of the PCR amplification product at the 3336 th site is mutated into the glycine G, the nucleotide sequence of the coded protein of the PCR amplification product is shown as SEQ ID NO.2, and the p.S3336G mutation exists.

Example 2 detection of early onset diabetes

1. Sample preparation

Collecting peripheral blood of family members, extracting genomic DNA in peripheral blood leukocytes, determining OD value of DNA by using Nanodrop8000, extracting 1-2 μ l of sample on Nanodrop8000, and recording sample concentration and OD_260/280And OD_260/230Ratio, etc. OD of genomic DNA of each specimen thus obtained₂₆₀/OD₂₈₀All are between 1.8 and 2.0, the concentration is between 50 and 200ng/ul, and the total amount is not less than 3 mug.

2. Disease mutant gene detection

Respectively detecting MDN1 genes of members in a family, including 6 patients and 7 normal families thereof, designing primers according to the sequence of MDN1, obtaining related sequences of MDN1 by PCR amplification, product purification and sequencing, and verifying the correlation between MDN1 and early-onset diabetes (mody) according to whether the sequence determination result belongs to a mutant type or a wild type. The method comprises the following specific steps:

1) DNA extraction:

the peripheral venous blood of 10 patients and 13 normal families thereof are extracted with genome DNA according to the method in the technical scheme, and the DNA content is determined by Nanodrop 8000.

2) Primer design and PCR reaction

Primer design is referenced to the human genome sequence database hg 19/built 36.3, see below.

a) The primer sequence is as follows:

TABLE 1 primer sequences

b) Reaction system: 25 μ L

c) Reaction conditions are as follows:

TABLE 2 PCR reaction conditions

3) And (3) directly carrying out sanger sequencing on the PCR amplification product obtained in the step (2).

The mutation site of MDN1 gene is screened in patient family members, and all patients carry corresponding heterozygous mutation, and the normal family members do not carry the pathogenic mutation. Therefore, the MDN1 gene is considered to be a pathogenic gene of early-onset diabetes.

And the existing wild early-onset diabetes gene sequence (MDN1-005ENST 00000369393: (http:// grch37.ensembl.org/Homo_sapiens/Transcript/Sequence_cDNA？db＝core；g＝ ENSG00000112159；r＝6:90352218-90529442；t＝ENST00000369393) In comparison, if the nucleotide sequence of the PCR amplification product has T at the 10006 th base>Mutation of C, i.e. presence of mutation point C10006T>C, the nucleotide sequence of the PCR amplification product is shown as SEQ ID NO. 1; and the sequence of the protein is compared with the existing wild early-onset diabetes (MDN1-005ENST 00000369393: (http://grch37.ensembl.org/Homo_sapiens/Transcript/ Sequence_Protein？db＝core；g＝ENSG00000112159；r＝6:90352218-90529442；t＝ ENST00000369393) In comparison, serine S at the 3336 th site of the encoded protein of the PCR amplification product is mutated into glycine G, and the nucleotide sequence of the encoded protein of the PCR amplification product is shown in SEQ ID No.2, namely, if p.S3336G mutation exists, the biological sample belongs to early-onset diabetes.

Example 3 System for screening biological samples susceptible to early onset diabetes

As shown in FIGS. 4 to 6, the system for screening a biological sample susceptible to early onset diabetes comprises a nucleic acid isolation apparatus 100, a nucleic acid sequence determination apparatus 200, and a determination apparatus 300.

According to an embodiment of the present invention, the nucleic acid extraction apparatus 100 is used to extract a nucleic acid sample from a biological sample. As described above, according to the 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 further includes an RNA extraction unit 101 and a reverse transcription unit 102, wherein the extraction unit 101 is used for extracting 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 to obtain a cDNA sample, and the obtained cDNA sample constitutes the nucleic acid sample.

According to an embodiment of the present invention, the nucleic acid sequence determining apparatus 200 is connected to the nucleic acid extracting apparatus 100, and is configured to analyze the nucleic acid sample to determine the nucleic acid sequence of the nucleic acid sample. As indicated above, sequencing methods can be used to determine the nucleic acid sequence of a nucleic acid sample. Thus, according to one embodiment of the present invention, the nucleic acid sequence determination apparatus 200 may further include: a library construction unit 201 and a sequencing unit 202. The library construction unit 201 is used for constructing a nucleic acid sequencing library aiming at a nucleic acid sample; the sequencing unit 202 is connected to the library construction unit 201 and is configured to sequence the nucleic acid sequencing library to obtain a sequencing result consisting of a plurality of sequencing data. As described above, the early-onset diabetes exons can be enriched by PCR amplification, further improving the efficiency of screening biological samples susceptible to early-onset diabetes. Thus, the library construction unit 201 may further comprise a PCR amplification module (not shown) in which early-onset diabetes exon-specific primers are arranged to perform PCR amplification on said nucleic acid sample using the early-onset diabetes exon-specific primers, according to an embodiment of the present invention, AQP5 exon-specific primers have the amino acid sequence as shown in SEQ ID NO:3 and 4. Therefore, by combining the latest sequencing technology, the higher sequencing depth can be achieved for a single site, and the detection sensitivity and accuracy are greatly improved, so that the characteristics of high throughput and deep sequencing of the sequencing devices can be utilized to further improve the efficiency of detecting and analyzing the nucleic acid sample. Thereby improving the accuracy and precision of the subsequent analysis of the sequencing data.

According to the embodiment of the present invention, the determining means 300 is connected to the nucleic acid sequence determining means 200, and is adapted to compare the nucleic acid sequences of the nucleic acid samples so as to determine whether the biological sample is susceptible to the early onset diabetes based on the difference between the nucleic acid sequences of the nucleic acid samples and the existing wild-type early onset diabetes gene. Specifically, it is judged whether or not the biological sample is susceptible to the early-onset diabetes based on whether or not the nucleic acid sequence of the nucleic acid sample has a c.10006A > G mutation as compared with the sequence of the existing wild-type early-onset diabetes gene. According to an embodiment of the present invention, SOAP software may be used for the comparison. Thus, the method for screening a biological sample susceptible to early onset diabetes can be effectively carried out by using the system, and a biological sample susceptible to early onset diabetes can be effectively screened.

EXAMPLE 4 kit for screening biological samples susceptible to early onset diabetes

The invention provides a kit for screening a biological sample susceptible to early-onset diabetes. According to an embodiment of the present invention, the kit for screening a biological sample susceptible to early onset diabetes comprises: a reagent suitable for detecting an early-onset diabetic gene mutant, wherein the mutant has a c.10006A > G mutation as compared with the sequence of the existing wild-type early-onset diabetic gene. With the kit according to the embodiment of the present invention, a biological sample susceptible to early-onset diabetes can be effectively screened. As used herein, the term "reagent suitable for detecting a mutant early-onset diabetes gene" is to be understood in a broad sense, i.e., a reagent for detecting a gene encoding early-onset diabetes and a reagent for detecting a polypeptide of a mutant early-onset diabetes, for example, an antibody recognizing a specific site can be used. According to one embodiment of the invention, the reagent is a nucleic acid probe or primer, preferably, the nucleic acid probe or primer has a nucleotide sequence as shown in SEQ ID NO 3-4. Thus, a biological sample susceptible to early onset diabetes can be efficiently screened.

Example 5 constructs and recombinant cells

The invention also provides a construct. According to an embodiment of the present invention, the construct comprises the isolated nucleic acid encoding the mutant early-onset diabetes gene as described above, i.e., the mutant early-onset diabetes gene of the present invention. Therefore, the recombinant cell obtained by transforming the receptor cell with the construct of the invention can be effectively used for screening the medicine for treating early-onset diabetes. The type of the recipient cell is not particularly limited, and may be, for example, an escherichia coli cell or a mammalian cell, and the recipient cell is preferably derived from a mammal.

The term "construct" as used in the present invention refers to a genetic vector comprising a specific nucleic acid sequence and capable of transferring the nucleic acid sequence of interest into a host cell to obtain a recombinant cell. According to an embodiment of the present invention, the form of the construct is not particularly limited. 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), and a virus, and is preferably a plasmid. The plasmid is used as a genetic carrier, has the characteristics of simple operation, capability of carrying larger fragments and convenience for operation and treatment. The form of the plasmid is not particularly limited, and may be a circular plasmid or a linear plasmid, and may be either single-stranded or double-stranded. The skilled person can select as desired. The term "nucleic acid" used in the present invention may be any polymer containing 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 be DNA, as DNA is more stable and easier to manipulate than RNA.

Example 6 recombinant cells

According to an embodiment of the invention, the recombinant cell is obtained by transforming a recipient cell with the construct described above. Thus, the recombinant cells of the invention are capable of expressing the early-onset diabetic gene mutant carried by the construct. According to some embodiments of the present invention, using the recombinant cells of the present invention, a drug for treating early-onset diabetes can be effectively screened. According to the embodiment of the present invention, the kind of the recipient cell is not particularly limited, and may be, for example, an escherichia coli cell, a mammalian cell, and preferably, the recipient cell is derived from a non-human mammal.

Example 7 application example

2.1 sample Collection

The early onset diabetes (mody) family contains 40 members, 6 patients, and the family is shown in figure 3. 4 mody patients and 1 normal control were selected as high throughput sequencing study samples in the home series. Peripheral blood samples of all members of the family (except the deceased families) were collected, anticoagulated with EDTA, and stored at-80 ℃. All blood samples were submitted to informed consent and approved by the ethical committee.

2.2DNA extraction

Extracting DNA from a peripheral blood sample by using a whole blood DNA extraction kit;

1.1) transfer 200ul of whole blood (without clot) to a new 1.5mL centrifuge tube.

1.2) 300ul ATL buffer was added.

1.3) 20ul proteinase K was added.

1.4) add 300ul of AL buffer and shake on Vortex for 15s (ensure Mix is well mixed).

1.5) incubation at 56 ℃ for 30min (if a thermomixer is used, mix; if the culture is incubated in a water bath, the culture needs to be shaken up by hand every 5 minutes or so).

1.6) taking out the sample after incubation, cooling to room temperature, adding 200ul of absolute ethyl alcohol (freezing at 4 ℃ or-20 ℃), Vortex 15s, and standing for 3min at room temperature.

2.2.2. Exon capture and sequencing

We collected a 4-generation Uygur family of early onset diabetes (mody), with the syndrome of the early onset mainly manifested as relatively insufficient insulin secretion and hyperglycemia. All family member patients present with the same autosomal dominant inheritance of disease in the clinically manifest family. We selected 4 patients (2-9; 2-15; 2-6; 2-3) and one normal (2-7) in the family for exome sequencing and data analysis as follows.

First, the inventors sequenced Exome sequences of two patients in this family using the NimbleGen SeqCap EZ exosome (44M) in combination with Solexa high throughput sequencing technology as follows:

1) genomic DNA was randomly fragmented into fragments of about 200-300bp, followed by ligation of adaptors at both ends of the fragments to prepare hybrid libraries (see http:// www.illumina.com/Illumina/Solexa standard library building instructions provided).

2) The Library was purified, hybridized with Biotinylated DNA Library by ligation-mediated PCR (LM-PCR) linear amplification, enriched, and subjected to LM-PCR linear amplification for on-machine sequencing (FIGS. 1 and 2). The sequencing platform is Illumina Hiseq 2000, the reading length is 90bp, and the average sequencing depth of each sample is at least 50.

3) The raw data obtained after sequencing was processed by Illumina based Software 1.7, filtered to remove contamination, using SOAPaligner 2.20(Li R, Li Y, Kristiansen K, et al, SOAP: short oligonucleotide alignment program. bioinformatics 2008,24(5): 713-714; li R, YuC, Li Y, et al, SOAP2: an improved ultra fast tool for short alignment. bioinformatics 2009,25(15): 1966-. The genotype of the target region was determined by SOAPsnp (Li R, Li Y, Fang X, Yang H, et al, SNP detection for mapping parallel gene re-sequencing. genome Res2009,19(6): 1124-1132.).

As a result, in these samples, respectively, it was found that: samples 2-6 had 118982 Single Nucleotide Polymorphisms (SNPs) and an insertion/deletion at 7533; 113938 Single Nucleotide Polymorphisms (SNPs) and an insertion/deletion at 7421 in samples 2-15; 112059 Single Nucleotide Polymorphisms (SNPs) and 7433 insertions/deletions in samples 2-3; 112489 Single Nucleotide Polymorphisms (SNPs) and 7548 insertions/deletions in samples 2-4; 107686 Single Nucleotide Polymorphisms (SNPs) and an insertion/deletion at 7182 in samples 2-9; 116872 Single Nucleotide Polymorphisms (SNPs) and an insertion/deletion at 7463 in samples 2-7; the results were then passed through four public databases (dbSNP (v135):http:// hgdownload.cse.ucsc.edu/goldenPath/hg19/database/snp135.txt.gz.；1000human:ftp:// ftp.1000genomes.ebi.ac.uk/vol1/ftpor ftp:// ftp-trace. ncbi. nih. gov/1000 genes/ftp; filtration of hapmap:// ftp. ncbi. nlm. nih. gov/hapmap, YH database: http:// YH. genomics. org. cn) removed all known variations with allele frequencies in the database greater than 0.005.

Then, based on the genetic pattern being autosomal dominant and assuming full penetrance, rare heterozygous mutations present in 4 patients, absent in the normal control, were selected as candidate genes for validation among mody13 family members; only heterozygosity mutations (c.A10006G, p.S3336G) in the MDN1 gene were co-segregating and predicted as deleterious mutations in various prediction software (polyphen 2; LRT; MutationTaster; MutationAnassosser; FATHMM; GERP + +; Pho; SiPhy).

Thus, MDN1 is the causative gene of early-onset diabetes.

Claims

1. The application of the early-onset diabetes gene mutant in preparing and screening products for early-onset diabetes is characterized in that the nucleotide sequence of the early-onset diabetes gene mutant is shown as SEQ ID NO. 1; the amino acid sequence of the early-onset diabetes gene mutant coding protein is shown as SEQ ID NO. 2.

2. The use of claim 1, wherein the product is a device for screening a biological sample susceptible to early-onset diabetes, comprising:

a judging means connected to the nucleic acid sequence determining means to judge whether the biological sample is a sample susceptible to early onset diabetes based on whether the nucleic acid sequence of the nucleic acid sample has a c.10006A > G mutation compared with the sequence of the existing wild-type early onset diabetes gene.

3. The use according to claim 2, wherein the nucleic acid extraction device comprises: an RNA extraction unit for extracting an RNA sample from the biological sample; and a reverse transcription unit connected to the RNA extraction unit for performing a reverse transcription reaction on the RNA sample to obtain a cDNA sample, the cDNA sample constituting the nucleic acid sample.

4. The use according to claim 2, wherein the nucleic acid sequence determination means comprises: a library construction unit for constructing a nucleic acid sequencing library for the nucleic acid sample; and the sequencing unit is connected with the library construction unit and used for sequencing the nucleic acid sequencing library so as to obtain a sequencing result consisting of a plurality of sequencing data.

5. The use of claim 4, wherein the library construction unit further comprises: a PCR amplification module, wherein the PCR amplification module is provided with an early-onset diabetes gene specific primer so as to carry out PCR amplification on the nucleic acid sample by using the specific primer, the early-onset diabetes gene specific primer comprises a forward primer and a reverse primer, and the sequence of the forward primer is shown as Seq ID NO. 3; the sequence of the reverse primer is shown as Seq ID NO. 4.

6. The use according to claim 1, wherein the product is a kit comprising reagents suitable for detecting early-onset diabetic gene mutants; the reagent is a nucleic acid probe or a primer; the nucleic acid probe or primer is a nucleotide sequence shown in SEQ ID NO.3 and SEQ ID NO. 4.