WO2014153753A1

WO2014153753A1 - Gene mutant and use thereof

Info

Publication number: WO2014153753A1
Application number: PCT/CN2013/073365
Authority: WO
Inventors: 张建国; 方明艳; 王海荣; 杜萨特科瓦•佩特拉; 王威; 王俊
Original assignee: 深圳华大基因科技有限公司
Priority date: 2013-03-28
Filing date: 2013-03-28
Publication date: 2014-10-02
Also published as: HK1213573A1; CN105263944A

Abstract

Provided are an isolated nucleic acid, an isolated polypeptide, a method for screening a biological sample of maturity onset diabetes of the susceptible young, a system for screening a biological sample of maturity onset diabetes of the susceptible young, and a kit for screening a biological sample of maturity onset diabetes of the susceptible young, wherein the isolated nucleic acid has c.703C＞T mutation in comparison with SEQ ID NO: 1, or has at least one mutation selected from the group consisting of c.338G＞T and c.358A＞G compared with SEQ ID NO: 3.

Description

Gene Mutants and Their Applications Priority Information

No technical field

The present invention relates to gene mutants and uses thereof. In particular, the present invention relates to an isolated nucleic acid, an isolated polypeptide, a method of screening a biological sample of adult-onset diabetes in a susceptible young person, a system for screening a biological sample of adult-onset diabetes in a susceptible young person, and A kit for screening biological samples of adult onset diabetes in susceptible young people. Background technique

Maturity onset diabetes of the young (MODY) is a group of monogenic diseases with high genetic and clinical phenotype heterogeneity. In 1975, Fajans and Tattersall analyzed and named the series according to the series since 1950, and summarized the following clinical features: (1) involving 3 or more generations of family members, autosomal dominant, not related to human leukocyte antigen (HLA) (2) There are usually more than 2 patients in the family who are ill before the age of 25; (3) The penetrance rate is high, which can exceed 90%; (4) The disease progresses slowly, and can be asymptomatic or only exhibits glucose tolerance during adolescence. Reduced; (5) General ketoacidosis, at least for 2 years after the onset of insulin treatment.

At this stage, finding a disease-causing gene is of great significance for the diagnosis or treatment of the disease. Although some of the pathogenic genes of adult-onset diabetes mellitus have been found in young people, such as the GCK, HNF1A, HNF4A, and HNF1B genes, there are still a number of unknown pathogenic gene loci.

Therefore, the current research on adult-onset diabetes in young people remains to be deepened. Summary of the invention

The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, it is an object of the present invention to provide a method for efficiently screening biological samples of adult onset diabetes in susceptible young people.

The present invention was accomplished based on the inventors' work in that the inventors identified new mutations in the causative genes of adult onset diabetes in young adults by high-throughput exome sequencing combined with candidate gene mutation verification.

According to a first aspect of the invention, the invention proposes an isolated nucleic acid. According to an embodiment of the invention,

The nucleic acid has a C.703 OT mutation compared to SEQ ID NO: 1, or the nucleic acid has at least one mutation selected from the group consisting of c.338G>T and c.358A>G compared to SEQ ID NO:3. In the examples of the present invention, the inventors identified HNF1B and HNF1A gene mutants, which are closely related to the onset of adult-onset diabetes in young people, thereby detecting the presence or absence of these new mutants in biological samples. It can effectively detect whether biological samples are susceptible to adult-onset diabetes in young people.

According to a second aspect of the invention, the invention provides an isolated polypeptide. According to an embodiment of the invention, SEQ ID NO: 2, the isolated polypeptide has a p. Arg235Trp mutation; or compared to SEQ ID NO: 4, the isolated polypeptide has at least one mutation selected from the group consisting of: p.Trpl l3Leu and p.Lysl20Glu . By detecting whether or not the polypeptide is expressed in the biological sample, it is possible to effectively detect whether the biological sample is susceptible to adult-onset diabetes in young people.

According to a third aspect of the invention, the invention provides a method of screening a biological sample of adult onset diabetes in a susceptible young person. According to an embodiment of the invention, the method comprises the steps of: extracting a nucleic acid sample from a biological sample; determining a nucleic acid sequence of the nucleic acid sample; having a nucleic acid sequence of the nucleic acid sample or a complementary sequence thereof, having SEQ ID NO: 1 An at least one mutation selected from the group consisting of C. 703 OT mutations or having SEQ ID NO: 3 and having at least one mutation selected from the group consisting of c.338G>T and c.358A>G is an adult-onset type in a susceptible young person of the biological sample. Instructions for diabetes. By screening a biological sample of adult-onset diabetes in a susceptible young person according to an embodiment of the present invention, a biological sample of adult-onset diabetes in a susceptible young person can be effectively screened.

According to a fourth aspect of the invention, the invention provides a system for screening biological samples of adult onset diabetes in susceptible young people. According to an embodiment of the present invention, the system includes: a nucleic acid extraction device for extracting a nucleic acid sample from the biological sample; a nucleic acid sequence determining device, the nucleic acid sequence determining device being 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 judging device, the judging device being coupled to the nucleic acid sequence determining device, based on the nucleic acid sequence of the nucleic acid sample or a complementary sequence thereof, Having a C.703OT mutation compared to SEQ ID NO: 1, or having at least one mutation selected from c.338G>T and c.358A>G compared to SEQ ID NO: 3, determining whether the biological sample is easy Adults with age-onset diabetes. With this system, the above-described method of screening biological samples of adult-onset diabetes among susceptible young people can be effectively carried out, thereby effectively screening biological samples of adult-onset diabetes in susceptible young people.

According to a fifth aspect of the present invention, the present invention provides a kit for screening a biological sample of adult onset diabetes in a susceptible young person. According to an embodiment of the present invention, the kit comprises: an agent suitable for detecting at least one of the HNF1B and HNFL4 gene mutants, wherein the HN ^B gene mutant has C.703OT compared to SEQ ID NO: 1. Mutation; compared to SEQ ID NO: 3, the HNFL4 gene mutant has at least one mutation selected from the group consisting of: c.338G>T and c.358A>G. Using the kit of the embodiment of the present invention, it is possible to efficiently screen biological samples of adult-onset diabetes in susceptible young people.

The additional aspects and advantages of the invention will be set forth in part in the description which follows. DRAWINGS

The above and/or additional aspects and advantages of the present invention will become apparent and readily understood from

Figure 1 is a schematic view showing a system for screening a biological sample of adult-onset diabetes mellitus in a susceptible young person, and a component thereof, according to an embodiment of the present invention, wherein

A is a line for screening biological samples of adult-onset diabetes in susceptible young people according to an embodiment of the present invention. Schematic diagram,

B is a schematic diagram of a nucleic acid extraction device according to an embodiment of the present invention,

C is a schematic diagram of a nucleic acid sequence determining device according to an embodiment of the present invention;

Figure 2: shows the family diagram of the MODY patient family CZ01 and CZ02 according to one embodiment of the present invention, wherein

A is a family diagram of the CZ01 family according to an embodiment of the present invention,

B is a family diagram of a CZ02 family according to an embodiment of the present invention;

Fig. 3 is a view showing a Sanger sequencing verification peak of a HNF1B gene mutation site in a patient and a normal human in a family of CZ01 in a family of MODY patients according to an embodiment of the present invention;

Figure 4: shows a Sanger sequencing verification peak of the HNF1A gene mutation site in patients and their normal humans in the family of CZ02 in the MODY patient family according to one embodiment of the present invention;

Figure 5: shows a Sanger sequencing validation peak map of the HNFL4 gene mutation site in MODY sporadic patients and off-normal subjects in accordance with one embodiment of the present invention. Detailed description of the invention

Embodiments of the invention are described in detail below, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the drawings are intended to be illustrative only and not to limit the invention.

HNF1B and HNF1A gene mutants

According to a first aspect of the invention, the invention proposes an isolated nucleic acid. According to an embodiment of the invention, the nucleic acid has a C.703OT mutation compared to SEQ ID NO: 1 or, in contrast to SEQ ID NO: 3, the nucleic acid has at least one mutation selected from the group consisting of: c.338G> T and c.358A>G. It should be noted that the isolated nucleic acid of the present invention encodes a HNF1B or HNF1A mutant, which may also be referred to as a "nucleic acid encoding a HNF1B or HNF1A mutant", that is, the nucleic acid may be understood to correspond to a gene encoding a HNF1B or HNF1A mutant. The nucleic acid substance, that is, the type of the nucleic acid is not particularly limited, and may be any polymer containing deoxyribonucleotides and/or ribonucleotides corresponding to the coding genes of HNF1B and HNF1A, including but not limited to DNA, RA. Or cDNA. According to a specific example of the present invention, the nucleic acid encoding the HNF1B and HNF1A mutants described above is DNA. According to an embodiment of the present invention, the inventors identified a novel mutant of the HA^JB p HNFM gene, which is closely related to the onset of adult-onset diabetes in young people, thereby detecting the mutant in a biological sample. Whether it exists, can effectively detect whether the biological sample is susceptible to adult-onset diabetes in young people, or can detect whether the organism is prevalent in the organism, and can effectively predict whether the organism is susceptible to adulthood in young people. Onset diabetes.

These nucleic acids encoding HNF1B and HNF1A mutants are new in the inventors of the present application by high-throughput exome sequencing combined with candidate gene mutation verification, and identified new genes in the pathogenic genes of adult onset diabetes in young people. Mutation, and the C.703OT mutation on the HN B gene is not seen in the prior art, and the c.338G>T, c.358A>G mutation on the HNFL4 gene is associated with adult onset diabetes in young people. Report.

The nucleotide sequence of the cDNA of the wild type HNF1B gene is shown below (1674 nt):

TDTSSISTLTNMSSSKQCPLQAW (SEQ ID NO: 2).

The nucleotide sequence of the cDNA of the wild type HNFIA gene is shown below (1896 nt):

NGQSHLLPSNHSVIETFISTQMASSSQ ( SEQ ID NO: 4 ),

The HNF1B gene mutant discovered by the inventors has a C.703OT (exon 3 ) mutation compared to SEQ ID NO: 1, that is, the 703th C in the cDNA of the HNF1B gene mutant of the present invention relative to the wild type HNF1B gene. Mutation to T (in exon 3), whereby the encoded product has a p.Arg235Trp (R235W) mutation compared to hepatocyte nuclear factor 1 β (HNF IB) (SEQ ID NO: 2) That is, its 235 amino acid is mutated from Arg to Trp.

Furthermore, the HNFJ4 gene mutant discovered by the inventors has at least one mutation of c. 338G>T (exon 2 ) and c.358A>G (exon 2 ) compared to SEQ ID NO: 3, ie, relative to wild type The HNF1A gene, in the cDNA of the HNF1A gene mutant of the present invention, has a G mutation at position 338 of T (in exon 2), and an A mutation at position 358 is G (in exon 2), whereby the encoded product thereof Hepatocyte nuclear factor 1 α (HNF1A) ( SEQ ID NO: 4 ) has p.Trpl l3Leu ( W113L ) or p丄ysl20Glu (K120E) mutation, ie its 113 amino acid is mutated from Trp to Leu Or its 120 amino acid is mutated from Lys to Glu.

The inventors of the present invention first proposed that the HNF1B C.703OT mutation may cause clinical manifestations of hyperglycemia in individuals without renal function defects, and the c.338G>T and c.358A>G mutations of the H HNF1A gene cause the patient to appear in young people. Adult onset diabetes symptoms.

According to a second aspect of the invention, the invention proposes an isolated polypeptide. According to an embodiment of the invention, with SEQ

In contrast to ID NO: 2, the isolated polypeptide has a p.Arg235Trp mutation; or compared to SEQ ID NO: 4, the isolated polypeptide has at least one mutation selected from the group consisting of p.Trpl l3Leu and p丄ysl20Glu. According to some specific examples of the present invention, the polypeptide having the p.Arg235Trp mutation is encoded by the aforementioned isolated nucleic acid encoding the HNF1B mutant, and the polypeptide having the p.Trpl l3Leu and p丄ysl20Glu mutations is the HNF1A mutant encoded by the foregoing. Nucleic acid encoded. By detecting whether the polypeptide is expressed in the biological sample, it is possible to effectively detect whether the biological sample is susceptible to adult onset diabetes in young people, or to detect the presence of these polypeptides in the organism, and can effectively predict the growth. Whether the object is susceptible to adult-onset diabetes in young people. Method for screening biological samples of adult onset diabetes in susceptible young people

According to a third aspect of the invention, the invention provides a method of screening a biological sample of adult onset diabetes in a susceptible young person. According to an embodiment of the present invention, the method of screening a biological sample of adult onset diabetes in a susceptible young person may comprise the following steps:

First, a nucleic acid sample is extracted from a biological sample. According to an 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 a mutation in the biological samples HNF1B and HNF1A 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 the group consisting of human blood, skin, and subcutaneous tissue. Thereby, sampling and detection can be conveniently performed, thereby further improving the efficiency of screening biological samples of adult-onset diabetes in susceptible young people. According to an embodiment of the present invention, the term "nucleic acid sample" as used herein shall be understood broadly, and may be any sample capable of reflecting the presence or absence of a mutation in HA^JB pHNFM in a biological sample, for example, may be directly extracted from a biological sample. Whole genome DNA, which may also be part of the whole genome comprising the HNF1B and HNF1A coding sequences, may be total RA extracted from a biological sample, or may be a NA NA extracted from a biological sample. According to an embodiment of the invention, the nucleic acid sample is whole genome DNA. Thereby, the source range of the biological sample can be expanded, and various information of the biological sample can be simultaneously determined, thereby improving the efficiency of screening biological samples of adult-onset diabetes in susceptible young people. In addition, according to an embodiment of the present invention, extracting a nucleic acid sample from a biological sample for using RA as a nucleic acid sample may further include: extracting an RNA sample from the biological sample, preferably the RA sample is mRNA; and based on the obtained RNA sample, The transcription reaction, obtaining a cDNA sample, and the obtained cDNA sample constitutes a nucleic acid sample. Thereby, the efficiency of screening a biological sample of adult-onset diabetes in a susceptible young person using R A as a nucleic acid sample can be further improved.

Next, after the nucleic acid sample is obtained, the nucleic acid sample can be analyzed to enable determination of the nucleic acid sequence of the resulting nucleic acid sample. According to an embodiment 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 a particular embodiment of the invention, the nucleic acid sequence of the nucleic acid sample can be determined by sequencing methods. The method and apparatus that can be used for sequencing according to embodiments of the present invention are not particularly limited. According to embodiments of the present invention, second generation sequencing techniques can be employed, and third generation and fourth generation or more advanced sequencing techniques can also be employed. According to a specific example of the present invention, the nucleic acid sequence can be sequenced using at least one selected from the group consisting of Hiseq2000, SOLiD, 454, and a single molecule sequencing device. Therefore, combined with the latest sequencing technology, high sequencing depth can be achieved for a single site, detection sensitivity and accuracy are greatly improved, and thus the high-throughput and deep sequencing characteristics of these sequencing devices can be utilized to further improve nucleic acid samples. The efficiency of the test analysis. Thereby, the accuracy and accuracy of subsequent analysis of the sequenced 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, constructing a nucleic acid sequencing library for the obtained nucleic acid sample; and sequencing the obtained nucleic acid sequencing library to obtain a plurality of The sequencing results of the sequencing data. According to some embodiments of the invention, the resulting nucleic acid sequencing library can be sequenced using at least one selected from the group consisting of Hiseq2000, SOLiD, 454, and a single molecule sequencing device. In addition, according to an embodiment of the present invention, nucleic acid samples can be screened and enriched for HNF1B and HNFJ4 exons, which can be used in the process of constructing a sequencing library, or constructing a test before constructing a sequencing library. The sequence library is followed. According to an embodiment of the present invention, constructing the nucleic acid sequencing library for the nucleic acid sample further comprises: performing PCR amplification on the nucleic acid sample using at least one selected from the group consisting of HN B and HNFM gene exon-specific primers; Amplification product, construct a nucleic acid sequencing library. Thus, the HNF1B and HNF1A exons can be enriched by PCR amplification, thereby further improving the efficiency of screening biological samples of adult-onset diabetes in susceptible young people. According to an embodiment of the present invention, the sequence of the HNF1B HNFM gene exon-specific primer is not particularly limited, and according to a preferred embodiment of the present invention, the HN B gene exon-specific primer may have SEQ ID NOs: 5-6 The nucleotide sequence shown, the HNF1A gene exon-specific primer may have a nucleotide sequence as shown in SEQ ID NOS: 7-8. The inventors have surprisingly found that by using the primers shown in SEQ ID NOS: 5-6, amplification of the exon 3 sequence of HA^JB, particularly C.703OT, can be performed in the PCR reaction system significantly and efficiently. The primers shown in SEQ ID NOS: 7-8 can be used to significantly and efficiently amplify the exon 2 sequence of HNF1A, particularly c.338G>T and c.358A>G. It is to be noted that these nucleotide sequences shown by SEQ ID NOS: 5-8 were unexpectedly obtained by the inventors of the present invention after exerting laborious labor.

For methods and procedures for constructing sequencing libraries for nucleic acid samples, those skilled in the art can appropriately select according to different sequencing technologies. For details of the processes, refer to the procedures provided by manufacturers of sequencing instruments such as Illumina, for example. Illumina Corporation Multiplexing Sample Preparation Guide (Part #1005361; Feb 2010) or Paired-End SamplePrep Guide (Part # 1005063; Feb 2010), which is incorporated herein by reference. According to an embodiment of the present invention, the method and apparatus for extracting a nucleic acid sample from a biological sample are also not particularly limited, and can be carried out using a commercially available nucleic acid extraction kit.

It should be noted that the term "nucleic acid sequence" as used herein should be understood in a broad sense, which may be the complete nucleic acid sequence information obtained after assembling the sequencing data obtained by sequencing the nucleic acid sample, or may be direct 釆The sequencing data obtained by sequencing the nucleic acid sample is used as the nucleic acid sequence as long as the nucleic acid sequence contains the coding sequences corresponding to HNF1B and HNF1A.

Finally, after determining the nucleic acid sequence of the nucleic acid sample, the corresponding reference sequence of the nucleic acid sequence of the obtained nucleic acid sample is aligned, and when the obtained nucleic acid sequence has at least one of the aforementioned three mutations, the biological sample is indicated Adults with age-onset diabetes. Specifically, when the obtained nucleic acid sample is obtained by amplification using an HN B gene exon-specific primer, the nucleic acid sequence of the nucleic acid sample is compared with the sequence of SEQ ID NO: 1, if the obtained nucleic acid is A C.703OT mutation in the sequence indicates that the biological sample is susceptible to adult onset diabetes in young people. When the obtained nucleic acid sample is obtained by amplification with an exon-specific primer of the HNFL4 gene, the nucleic acid sequence of the nucleic acid sample is aligned with the sequence of SEQ ID NO: 3, if it has c in the obtained nucleic acid sequence At least one mutation of .338G>T and c.358A>G indicates that the biological sample is susceptible to adult-onset diabetes in young people. When obtained nuclear The acid sample is obtained by amplification of the exon-specific primers of the HNFL4 and HN B genes, and the nucleic acid sequence of the nucleic acid sample is compared with the sequences of SEQ ID NO: 1 and SEQ ID NO: 3, respectively. The resulting nucleic acid sequence has at least one mutation of c.703C>T, c.338G>T and c.358A>G, indicating that the biological sample is susceptible to adult onset diabetes in young people. Thus, by screening a biological sample of adult-onset diabetes in a susceptible young person according to an embodiment of the present invention, a biological sample of adult-onset diabetes in a susceptible young person can be effectively screened. According to an embodiment of the present invention, the method and apparatus for aligning a nucleic acid sequence with SEQ ID NO: 1 and SEQ ID NO: 3 are not particularly limited, and may be operated by any conventional software, according to the specific For example, SOAPALIGNER/SOAP2 can be used for comparison.

It is to be noted that the use of the "method of screening biological samples of adult-onset diabetes among susceptible young people" according to an embodiment of the present invention is not particularly limited, and for example, it can be used as a screening method for non-diagnostic purposes. System and kit for screening biological samples of adult-onset diabetes in susceptible young people

According to a fourth aspect of the present invention, the present invention provides a system capable of effectively performing the above-described method of screening a biological sample of adult-onset diabetes in a susceptible young person.

Referring to Fig. 1, a system 1000 for screening a biological sample of adult-onset diabetes in a susceptible young person according to an embodiment of the present invention comprises: a nucleic acid extraction device 100, a nucleic acid sequence determining device 200, and a judging device 300.

According to an embodiment of the invention, the nucleic acid extraction device 100 is for extracting a nucleic acid sample from a biological sample. As described above, according to an embodiment of the present invention, the type of the nucleic acid sample is not particularly limited, and for RA as a nucleic acid sample, the nucleic acid extraction device further includes an RA extraction unit 101 and a reverse transcription unit 102, wherein The unit 101 is for extracting an RA sample from a biological sample, and the reverse transcription unit 102 is connected to the RA extraction unit 101 for performing a reverse transcription reaction on the RA sample to obtain a cDNA sample, and the obtained cDNA sample constitutes a nucleic acid sample.

According to an embodiment of the present invention, the nucleic acid sequence determining device 200 is coupled to the nucleic acid extraction device 100 for analyzing a nucleic acid sample to determine a nucleic acid sequence of the nucleic acid sample. As indicated previously, the nucleic acid sequence of the nucleic acid sample can be determined using sequencing methods. Thus, according to an embodiment of the present invention, the nucleic acid sequence determining apparatus 200 may further include: a library construction unit 201 and a sequencing unit 202. The library construction unit 201 is configured to construct a nucleic acid sequencing library for the nucleic acid sample; the sequencing unit 202 is connected to the library construction unit 201 for sequencing the nucleic acid sequencing library to obtain a sequencing result composed of a plurality of sequencing data. As described above, the HNF1B and HNF1A exons can be amplified by PCR to further improve the efficiency of screening biological samples of adult-onset diabetes in susceptible young people. Thus, the library construction unit 201 may further include a PCR amplification module (not shown) in which at least one selected from the group consisting of HNF1B and HNF1A gene exon-specific primers is provided in order to utilize At least one of the HNF1B and HNF1A gene exon-specific primers, the nucleic acid sample is subjected to PCR amplification, and according to a specific embodiment of the present invention, the HNF1B gene exon-specific primer has SEQ ID NOs: 5-6 The nucleotide sequence shown, the HNF1A gene exon-specific primer has the nucleotide sequence shown in SEQ ID NOS: 7-8. According to an embodiment of the present invention, the sequencing unit 202 may include at least one selected from the group consisting of HISEQ2000, SOLiD, 454, and a single molecule sequencing device. Therefore, combined with the latest sequencing technology, a high 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 these sequencing devices can be utilized to further improve the The efficiency of detection and analysis of nucleic acid samples. Thereby, the accuracy and accuracy of subsequent analysis of the sequenced data are improved. According to an embodiment of the present invention, the determining device 300 is coupled to the nucleic acid sequence determining device 200, and is adapted to align the nucleic acid sequences of the nucleic acid samples so as to be based on the nucleic acid sequences of the nucleic acid samples and the SEQ ID NO: 1 and SEQ ID NO: Distinguish whether the biological sample is susceptible to adult-onset diabetes in young people. Specifically, based on the nucleic acid sequence of the nucleic acid sample or its complementary sequence, it has a C.703OT mutation compared to SEQ ID NO: 1, or has a selection from c.338G>T and c compared to SEQ ID NO:3. At least one mutation of 358A>G determines whether the biological sample is susceptible to adult-onset diabetes in young people. As described above, according to one embodiment of the present invention, the nucleic acid sequence of the nucleic acid sample or the complement thereof has a C.703 OT mutation selected from SEQ ID NO: 1, or has a selection compared to SEQ ID NO: 3. At least one mutation from c.338G>T and c.358A>G is indicative of adult-onset diabetes in a biologically susceptible young person. As described above, according to an embodiment of the present invention, the apparatus for aligning the nucleic acid sequence with SEQ ID NO: 1 and SEQ ID NO: 3 is not particularly limited and may be operated by any conventional software, for example, according to Specific examples of the present invention can be aligned using SOAPALIGNER/SOAP2.

Thus, with the system, the above-described method for screening a biological sample of adult-onset diabetes in a susceptible young person can be effectively carried out, thereby effectively screening a biological sample of adult-onset diabetes in a susceptible young person.

According to a fifth aspect of the present invention, the present invention provides a kit for screening a biological sample of adult onset diabetes in a susceptible young person. According to an embodiment of the present invention, the kit for screening a biological sample of adult onset diabetes in a susceptible young person comprises: an agent suitable for detecting at least one of the HNF1B and HNF1A gene mutants, wherein SEQ ID NO The HA^JB gene mutant has a C.703OT mutation compared to SEQ ID NO: 3, and the HNFL4 gene mutant has at least one mutation selected from the group consisting of c.338G>T and c. 358A>G. With the kit according to the embodiment of the present invention, it is possible to effectively screen biological samples of adult-onset diabetes in a susceptible young person. As used herein, the term "agent suitable for detecting at least one of the HNF1B and HNF1A gene mutants" should be understood broadly, that is, an agent that detects at least one of the HA^JB and HNFM-encoding genes, or It is an agent that detects at least one of the HNF1B and HNF1A mutants, and for example, an antibody that recognizes a specific site can be used. According to an embodiment of the invention, the reagent is a nucleic acid probe. Thereby, biological samples of adult-onset diabetes in susceptible young people can be efficiently screened.

It should be noted that the features and advantages described in the method section for screening biological samples of adult-onset diabetes in susceptible young people are also applicable to screening biological samples of adult-onset diabetes in susceptible young people. The system or kit will not be described here.

In addition, it should be noted that the method, system and kit for screening biological samples of adult-onset diabetes in susceptible young people according to embodiments of the present invention are the inventors of the present application undergoing arduous creative labor and optimization work. Only completed. The solution of the present invention will be explained below in conjunction with the embodiments. Those skilled in the art will understand that the following examples are merely illustrative of the invention and should not be construed as limiting the scope of the invention. In the examples, the specific techniques or conditions are not indicated, according to the techniques or conditions described in the literature in the field (for example, refer to J. Sambrook et al., Huang Peitang et al., Molecular Cloning Experimental Guide, Third Edition, Science Press) or in accordance with the product manual. The reagent or instrument used is not marked Ming manufacturers are all conventional products that can be obtained through the market, for example, they can be purchased from Illumina.

Example 1 Whole exome sequencing to determine pathogenic genes and mutation sites

1. Sample collection:

The inventor collected two families of Czech MODY patients, CZ01 and CZ02. Among them, Figure 2 shows the family diagrams of the CZ01 and CZ02 families. 2A is a family diagram of the CZ01 family, and FIG. 2B is a family diagram of the CZ02 family. As shown in Figure 2, O indicates normal women; Qin indicates that it is impossible to determine whether a woman is ill; ^LJ indicates a normal male; the figure indicates a male who is unable to determine whether the disease is ill; the country indicates a male patient; indicates a female patient; indicates a deceased male Patient; ^ indicates a deceased female patient; ^ indicates a deceased normal male; ^ indicates a deceased normal female; the arrow indicates a proband. Among them, CZ01 family has 3 generations of survival, a total of 8 members, including 5 patients; CZ02 family has 3 generations of survival, a total of 6 members, including 2 patients.

In addition, the inventors conducted a thorough and detailed physical examination of all patients in CZ01. The results showed that all patients had typical clinical features of diabetes. Among them, no renal dysfunction was detected in the ultrasound examination of the proband, and the mother and grandfather passed. Repeated nephropathy was found to have a horseshoe kidney. Since the renal dysfunction reported in the clinical phenotype is a dominant feature, the HNF1B gene was not detected before the exon was sequenced.

As shown in Figure 2, CZ01 family, proband, female, 26 years old, body mass index 21.2. The patient first detected diabetes symptoms at 17 years of age, late insulin therapy and oral hypoglycemic agents. More than three patients in the family have developed before the age of 25, involving 3 or more generations of family members, which are autosomal dominant; the penetrance rate is high, which can exceed 90%.

CZ02 family, proband, female, 21 years old, body mass index 28.6. The patient first detected the symptoms of diabetes at the age of 17 and was treated with oral hypoglycemic agents. Family members of the family with 3 or more generations are autosomal dominant; the penetrance rate is high, which can exceed 90%.

The pathological diagnosis of the two probands is: adult onset diabetes in young people.

The inventors collected DNA samples from three patients and three normal persons surviving the above CZ01 family, two patients surviving in the CZ02 family, and four normal human DNA samples.

2. The whole exome group is sequenced to determine the pathogenic genes and mutation sites.

The inventors used the Agilent Sureselect 44M Kit in combination with Solexa high-throughput sequencing technology to perform the above-mentioned probands in the family of CZ01 patients and their diseased mothers and probands in the families of normal fathers and CZ02 patients, and their affected mothers and normal fathers. Exon sequencing, the specific steps are as follows:

2.1 Sample preparation

Peripheral blood of the probands of CZ01 and CZ02 patients and their diseased mothers and normal fathers were taken separately, genomic DNA was extracted by conventional salting out method, and the concentration and purity of DNA were measured by spectrophotometer. The OD260/OD280 of the genomic DNA of the specimen is located between 1.7 and 2.0, the concentration is not less than 200 ng^l, and the total amount is not less than 30 μ§.

2.2 Library construction and sequencing

Randomly interrupted each genomic DNA sample using a sonicator (CovarisS2, Massachusetts, USA) Fragments of about 200-300 bp were subsequently ligated to the ends of the fragments to prepare libraries (see the Illumina/Solexa standard library instructions provided at http://www.illumina.com/, according to the manufacturer's instructions. This is incorporated herein by reference in its entirety. The library was purified and linearized by Ligation-mediated PCR (LM-PCR) and purified by SureSelect Biotiny lated RA Library (BAITS). After linear amplification by LM-PCR, the library was tested and qualified. Sequencing to obtain raw sequencing data. Among them, the Illumina Hiseq 2000 was sequenced according to the Illumina standard clustering and sequencing protocol. The read length was 90 bp, and the average sequencing depth of the sample was 170x.

2.3 Variation detection, annotation and database comparison

The raw sequencing data obtained above was processed using Illumina basecalling Software 1.7, and after filtration and decontamination, SOAPaligner/SOAP2 was used (see: Li R, Li Y, Kristiansen K, et al, SOAP: short oligonucleotide alignment program. Bioinformatics 2008 , 24(5): 713-714; Li R, Yu C, Li Y, ea al, SOAP2: an improved ultrafast tool for short read alignment. Bioinformatics 2009, 25(15): 1966-1967, by reference Incorporating into the reference genome UCSC NCBI37/hgl9, in order to obtain a unique aligned sequence aligned to the genome. Then use SOAPsnp (see: Li R, Li Y, Fang X, Yang H, et al, SNP detection for massively parallel whole-genome resequencing. Genome Res 2009, 19(6): 1124-1132, by reference Incorporate herein to determine the genotype of the target region.

After sequencing results, the most likely pathologically relevant mutations were investigated for non-synonymous mutations, splice acceptor/donor site mutations, coding region insertions, and deletion mutations. As a result, the inventors found through sequencing analysis that an average of 67,745 single nucleotide polymorphisms (SNPs) and 4,784 insertions/deletions were found in three sequencing samples in the CZ01 family, and three sequencing in the home line CZ02. On average, 69,624 single nucleotide polymorphisms (SNPs) and 4840 insertions/deletions were found in the sample. Then through the dbSNP132 database (http://www.ncbi.nlm.nih.gov/projects/SNP/snp_summary.cgi), Exome Variant Server database (http://evs.gs.washington.edu/EVS/)<thousands Filtration of public databases such as the Human Genome Database (www.1000genomes.org), the HapMa database (http:〃hapmap.ncbi.nlm.nih.gov), and the LuCamp database of diabetes genetic variants (http://www.lucamp.org), Remove all known variants with a gene allele frequency greater than 0.005 in the database. The remaining non-synonymous/splicing site mutations and microinteger deletions are preferentially selected according to the following characteristics: (a) Selection of isogenic homologous and mutant mutations shared between the two patients, filtering out mutations present in normal humans There are 343 SNP mutation sites and 39 Indel mutations in the CZ01 family; there are 336 SNP mutation sites and 45 Indel mutations in the CZ02 family; (b) Among these mutation sites, the MODY known genes are preferentially searched. Mutation, the C.703OT mutation of the HNF1B gene was found in the CZ01 family; the c.338G>T mutation of the HNF1A gene was found in the CZ02 family.

Since the exome sequencing has a certain degree of false positives, the inventors then used the Sanger sequencing method to verify the two SNP mutations identified above in the family CZ01 and CZ021. The results show that fflV ^ The C.703OT mutation of the B gene and the c.338G>T mutation of the HNFL4 gene are all true mutations. Thus, HNF1B and HNF1A genes are involved in the pathogenesis-related genes of adult-onset diabetes mellitus in young adults. C.703OT (HNF1B), c.338G>T (HNFJ4) mutation is a cause of adult-onset diabetes in young adults. Disease mutation. Example 2 Sanger method sequencing to verify the pathogenic genes of adult onset diabetes in young people

6 of the CZ01 family of the MODY patients (3 female patients and 3 normal family members) 6 of the CZ02 (2 female patients and 4 normal family members) (see Figure 2), 1 scatter The HNF1B and HNF1A genes were detected in the patients and 95 normal off-campus normal subjects. Primers were designed for the exon 2 sequence of exon 3 HNFL4 gene of HNF1B gene, and then HNF1B gene was obtained by PCR amplification, product purification and sequencing. Sequence No. 2 of the exon 2 of the exon HNFL4 gene, whether it is a mutant or a wild type according to the determined sequence, confirms the correlation between HNF1B and HNF1A and adult onset diabetes in young people. The specific method steps are as follows:

1, DNA extraction

The peripheral venous blood of 3 patients and 3 normal family members, 2 patients in CZ02, 4 normal family members, 1 sporadic patient, and 95 normal off-campus normal persons in the above-mentioned family CZ01 were collected. The genomic DNA in the peripheral blood leukocytes was extracted by conventional salting out method, and the concentration and purity of the extracted DNA were measured by a spectrophotometer. The OD260/OD280 of each specimen genomic DNA was between 1.7 and 2.0, and the concentration was not Less than 200 ng/μl, the total amount is not less than 6 g, whereby 108 DNA samples are obtained and used.

2. Primer design and PCR reaction

First, with reference to the human genome sequence database GRCh37.1/hgl9, the exon 3 of the HNF1B gene having the nucleotide sequence shown by SEQ ID NOS: 5-6 is designed, and has the SEQ ID NOs: 7-8 The HNF1A gene exon 2 specific primer of the nucleotide sequence is shown in the following table.

HNF1B and HNF1A gene exon specificity

Next, the PCR reaction system of each genomic DNA sample was prepared and subjected to a PCR reaction according to the following ratios. For the exon 2 sequence of HNF1B gene exon 3 HNFL4 gene, each PCR reaction system of each genomic DNA sample was prepared according to the following ratios (different mutation sites use the same reaction system):

Reaction system: 10 μΐ

DNA 1

Total volume 10

Then, each PCR reaction system of the exon 2 of the HNF1B gene exon 2 HNFL4 gene obtained is subjected to PCR reaction according to the following reaction conditions (different mutation sites are used for the same reaction conditions): Reaction conditions:

10 cycles

Thus, PCR amplification products were obtained from 3 patients in the above-mentioned family CZ01 and 3 normal persons in the family, 2 patients in CZ02, 4 normal persons in the family, 1 sporadic patient, and 95 normal off-campus normal persons. .

3. Sequencing

PCR amplification of 3 patients and 3 family members in the CZ01 obtained in step 2, 2 patients in CZ02, 4 normal subjects in the family, sporadic patients and 95 normal off-campus normal subjects For the products, each PCR reaction system of each genomic DNA sample was prepared for DNA sequencing according to the following ratios (different mutation sites, the same reaction system, BigDye® Terminator v3.1 Cycle Sequncing kit, Applied Biosystems):

Then, each PCR reaction system for preparing the exon 2 of the HNF1B exon HNFJ4 gene obtained was subjected to a PCR reaction under the following reaction conditions and then subjected to sequencing using DNA sequencer ABI Prism 3130x1 (Applied Biosystems, Warrington, UK) (different The mutation site uses the same reaction conditions): Reaction conditions: 96 °C 1 min

96 °c 10 s

55 °c 5 s J" 25

63 °c 4 min

72 °c 7 min

15 . c oo

The results showed that the c.703C>T mutation of HNF1B gene was co-segregated in the CZ01 family, and the c.338G>T mutation of HNF1A gene was co-segregated in the CZ02 family.

Next, based on the sequencing results, the HN B HNFM gene coding sequence alignment was performed on each of the above samples. The inventors found that patients in their families CZ01 and CZ02 and their normal families had co-segregation in c.703C>T (HNF1B) and c.338G>T (HNF1A), respectively, and patients in families CZ01 and CZ02 carried C. 703OT and c.338G>T mutations, and c.358A>G mutation (HNF1A) was found in a sporadic patient. HNF1B gene was found in 95 out-of-home normal humans without C.703OT mutation; 95 out-of-home normals None of the HNF1A genes were found to have c.338G>T and c.358A>G mutations. Thus, the C.703OT mutation of the HNF1B gene and the c.338G>T and c.358A>G mutations of the HNF1A gene were found to be young. A pathogenic mutation in adult onset diabetes.

Among them, Figure 3 shows the Sanger sequencing verification peak of the HA^JB gene mutation site in the CZ01 family of MODY patients, and Figure 4 shows the HNF1A gene mutation site in the CZ02 family and normal subjects. The Sanger sequencing verification peak map, Figure 5 shows the Sanger sequencing verification peak of the HNF1A gene mutation site in MODY spora patients and off-normal subjects. As can be seen from Figures 3-5, the HNF1B gene of the CZ01 family has a C.703OT mutation, and the normal person in the family does not have the mutation; the HNFL4 gene of the CZ02 family has a c.338G>T mutation, and the family The normal person does not have this mutation; the HNFJ4 gene of the sporadic patient has a c.358A>G mutation, and the normal human outside the family does not have the mutation at the corresponding site.

Thus, the inventors performed mutations on the sequence of exon 3 of HNF1B gene and exon 2 of HNF1A gene in patient family members, and found that three patients of family CZ01 had C.703OT (HNF1B) mutation, and two of family CZ02. The patient had a c.338G>T (HNF1A) mutation; a c.358A>G (HNF1A) mutation was found in the sporadic patients; the mutation was not found in 95 out of normal families from the same region as the patient. Thus, it was further demonstrated that the C.703OT mutation of the HNF1B group and the c.338G>T and c.358A>G mutation of the HNF1A gene are pathogenic mutations in adult onset diabetes in young people.

It should be noted that hepatocyte nuclear factor l α (HNF1A) belongs to the POU-homology domain family, which is activated by the N-terminal myosin dimer region and the C-terminus. The region and the DNA binding region are constructed. The gene is located on chromosome 12q24.2, with a total length of 3241 bp, wherein the protein coding region is 1 896 bp long. The HNF1A DNA binding region contains a POU-homology domain sequence, and the dimeric region forms a homodimer and HNF1B forms a heterodimer to collectively regulate target gene expression. Hepatocyte nuclear factor 1 β (HNF1B) is a transcription factor that plays an important role in the formation of pancreatic cells and the maintenance of blood glucose homeostasis. HNF1B is an HNF1A analog expressed in vertebrate, highly homologous to HNF1A, and its biological activity is significantly lower than that of HNF1A. When HNF1A expression is down-regulated, HNF1B can be compensated for. This study may be applied to, but is not limited to, HNF1B gene mutation screening and clinical phenotypic analysis in special patients with no renal structural/dysfunction in diabetic patients; on the other hand, HN B gene mutations will be evaluated in Czech young people. The relationship between susceptibility to adult-onset diabetes.

In conclusion, the inventors further demonstrated that the HA^JB and HNFL4 genes are pathogenicity-related genes for adult-onset diabetes in young adults, the C.703OT mutation of the HNF1B gene, and the c.338G>T and c.358A of the HNF1A gene. The G mutation is a causative mutation in adult-onset diabetes in young people. Example 3 Sanger Method Sequencing Verification of Pathogenic Mutations in Adult-onset Diabetes in Young People

The pathogenic mutations of C.703OT (HNF1B). c.338G>T (HNF1A) and c.358A>G (HNF1A) on the HNF1B and HNF1A genes obtained above were verified, that is, the exon 3 of HNF1B gene was utilized. The exon 2 primer sequence of the HNFL4 gene was subjected to the corresponding method of Example 2, and the genomic DNA samples of 95 out-of-home normal humans obtained in Example 2 were subjected to HNF1B m exon 2 of the exon 2 HNFL4 gene. Proton mutation site detection. As a result, the inventors found that the above mutations were not found in 95 normal off-campus normal persons.

Thus, the inventors further demonstrated that the C.703OT mutation of the HA^ £ gene, the c.338G>T and the c.358A>G mutation of the HNFL4 gene are pathogenic mutations in adult onset diabetes in young people. Industrial applicability

The isolated nucleic acid, the isolated polypeptide of the present invention, the method, system and kit for screening biological samples of adult onset diabetes in susceptible young people can be effectively applied to detect whether a biological sample is susceptible to adult onset in young people Type 2 diabetes, and the results obtained are accurate and reliable. Although specific embodiments of the invention have been described in detail, those skilled in the art will understand. Various modifications and alterations of those details are possible in light of the teachings of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "illustrative embodiment", "example", "specific example", or "some examples", etc. Particular features, structures, materials or features described in the examples or examples are included in at least one embodiment or example of the invention. In the present specification, the schematic representation of the above terms does not necessarily mean the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in a suitable manner in any one or more embodiments or examples.

Claims

claims

1. An isolated nucleic acid, characterized by:

Compared with SEQ ID NO: 1, the nucleic acid has a C.703OT mutation; or

Compared with SEQ ID NO: 3, the nucleic acid has at least one mutation selected from the following: c.338G>T and c.358A>G, optionally, the nucleic acid is DNA.

2. An isolated polypeptide, characterized by:

Compared with SEQ ID NO: 2, the isolated polypeptide has a p.Arg235Trp mutation; or

Compared to SEQ ID NO: 4, the isolated polypeptide has at least one mutation selected from the group consisting of: p.Trpl 13Leu and p.Lysl20Glu,

Optionally, the polypeptide is encoded by the nucleic acid of claim 1.

3. A method for screening biological samples for adult-onset diabetes in susceptible young people, characterized by including the following steps:

extract a nucleic acid sample from the biological sample;

Determine the nucleic acid sequence of the nucleic acid sample;

The nucleic acid sequence of the nucleic acid sample or its complementary sequence, compared with SEQ ID NO: 1, has a mutation selected from the group consisting of C.703OT, or compared with SEQ ID NO: 3, has a mutation selected from the group consisting of c.338G>T and c.358A> at least one mutation of G that is indicative of adult-onset diabetes in a susceptible young adult in said biological sample,

Optionally, the biological sample is at least one selected from human blood, skin, hair and muscle,

Optionally, the nucleic acid sample is whole genome DNA.

4. The method of claim 3, wherein extracting nucleic acid samples from the biological samples further includes:

Extracting an RA sample from the biological sample, preferably the RA sample is mRA; and

Based on the RA sample, a cDNA sample is obtained through a reverse transcription reaction, and the cDNA sample constitutes the nucleic acid sample.

5. The method of claim 3, wherein determining the nucleic acid sequence of the nucleic acid sample further includes:

Construct a nucleic acid sequencing library for the nucleic acid sample; and

Sequencing the nucleic acid sequencing library to obtain a sequencing result composed of multiple sequencing data,

Optionally, the nucleic acid sequencing library is sequenced using at least one selected from the group consisting of Hiseq2000, SOLiD, 454 and a single molecule sequencing device,

Optionally, for the nucleic acid sample, constructing a nucleic acid sequencing library further includes:

Using at least one selected from the group consisting of HNF1B and HNF1A gene exon-specific primers, the nucleic acid sample is subjected to PCR amplification; and

Construct the nucleic acid sequencing library based on the obtained amplification product, Optionally, the HNF1B gene exon-specific primer has a nucleotide sequence as shown in SEQ ID NO: 5-6, and the HNF1A gene exon-specific primer has a nucleotide sequence as SEQ ID NO: 7-8 The nucleotide sequence shown.

6. A system for screening biological samples for adult-onset diabetes in susceptible young people, characterized by comprising: a nucleic acid extraction device, the nucleic acid extraction device being used to extract nucleic acid samples from the biological samples;

Nucleic acid sequence determination device, the nucleic acid sequence determination device is connected to the nucleic acid extraction device, and is used to analyze the nucleic acid sample to determine the nucleic acid sequence of the nucleic acid sample;

A judgment device, the judgment device is connected to the nucleic acid sequence determination device, so that based on the nucleic acid sequence of the nucleic acid sample or its complementary sequence, it has a C.703OT mutation compared with SEQ ID NO: 1, or has a C.703OT mutation compared with SEQ ID NO: 1. 3 Determine whether the biological sample is susceptible to adult-onset diabetes in young adults compared to having at least one mutation selected from c.338G>T and c.358A>G.

7. The system according to claim 6, wherein the nucleic acid extraction device further includes: an RA extraction unit, the RA extraction unit is used to extract an RA sample from the biological sample; and a reverse transcription unit, The reverse transcription unit is connected to the RA extraction unit and is used to perform a reverse transcription reaction on the RNA sample to obtain a cDNA sample, and the cDNA sample constitutes the nucleic acid sample.

8. The system according to claim 6, wherein the nucleic acid sequence determination device further includes: a library construction unit, the library construction unit is used to construct a nucleic acid sequencing library for the nucleic acid sample; and a sequencing unit, The sequencing unit is connected to the library construction unit and is used to sequence the nucleic acid sequencing library to obtain a sequencing result composed of multiple sequencing data,

Optionally, the library building blocks further include:

A PCR amplification module, the PCR amplification module is provided with at least one selected from the group consisting of HNF1B and HNF1A gene exon-specific primers, so as to utilize at least one type of HNF1B and HNFL4 gene exon-specific primers, Perform PCR amplification of the nucleic acid sample,

Optionally, the HNF1B gene exon-specific primer has a nucleotide sequence as shown in SEQ ID NO: 5-6, and the HNF1A gene exon-specific primer has a nucleotide sequence as SEQ ID NO: 7-8 The nucleotide sequence shown is,

Optionally, the sequencing unit includes at least one selected from the group consisting of HISEQ2000, SOLiD, 454 and single molecule sequencing devices.

9. A kit for screening biological samples for adult-onset diabetes in susceptible young people, characterized by containing:

A reagent suitable for detecting at least one of HNF1B and HNF1A gene mutants, wherein

Compared with SEQ ID NO: 1, the fflV^JB gene mutant has a C.703OT mutation;

Compared with SEQ ID NO: 3, the HNF1A gene mutant has at least one mutation selected from the following: c.338G>T and c.358A>G,

Optionally, the reagent is a nucleic acid probe.