CN106701925B

CN106701925B - Cataract pathogenic gene detection kit and detection method thereof

Info

Publication number: CN106701925B
Application number: CN201611120811.0A
Authority: CN
Inventors: 阳菊华; 陈晓乐; 朱益华
Original assignee: Fujian Medical University
Current assignee: Fujian Medical University
Priority date: 2016-12-08
Filing date: 2016-12-08
Publication date: 2020-02-07
Anticipated expiration: 2036-12-08
Also published as: CN106701925A

Abstract

The invention provides a cataract virulence gene detection kit and a detection method thereof, wherein the kit comprises 27 pairs of PCR primers, and the sequences of the PCR primers are respectively shown in SEQ ID NO. 1-54. The invention establishes a cataract pathogenic gene detection method based on a high-frequency mutation region of cataract pathogenic genes, and 80 percent of pathogenic genes of hereditary cataract families can be detected only by screening 26 exons of 18 cataract pathogenic genes. The invention can obviously reduce the workload and the cost of the cataract pathogenic gene detection on the basis of not reducing the detection rate, has the advantages of rapidness, economy, high efficiency and the like, finds 16 different cataract gene pathogenic mutation sites through the detection of the kit, and provides convenience for diagnosis and treatment and prevention intervention of patients clinically.

Description

Cataract pathogenic gene detection kit and detection method thereof

Technical Field

The invention relates to the field of genetic diagnosis of genetic diseases, in particular to a cataract pathogenic gene detection kit and a detection method thereof.

Background

The cataract is lenticular opacity caused by congenital or acquired factors, wherein the prevalence rate of the congenital cataract in the world is 0.0 l% -0.06%, and is about 0.05% in China. Prevention of vision impairment due to congenital cataract is an important component of World Health Organization (WHO) international project "elimination to avoid blindness in 2020".

Congenital cataract presents obvious clinical heterogeneity, full cataract, membranous cataract, nuclear cataract, circumnuclear cataract, anterior pole cataract, posterior pole cataract, suture-shaped cataract, punctate cataract, discoid cataract, coral type and the like can be divided according to clinical phenotypes, the pathogenesis of the congenital cataract is complex, and the congenital cataract relates to genetic and metabolic diseases, intrauterine infection, spontaneity and other factors, wherein about 30-50% of the congenital cataract is related to heredity and is collectively called genetic cataract. The genetic mode of hereditary cataract includes autosomal dominant, autosomal recessive, sex chromosome linkage inheritance, etc., wherein the autosomal dominant inheritance is the main. According to statistics, the total number of the familial cataract in China is 73% of the autosomal dominant hereditary cataract.

Lens development is a very complex process involving a number of genes, any of which may cause lenticular opacity after mutation, a process known as cataract. The identification of genes (proteins) with different functions at the cellular level by studying genetic cataract is the most effective way for human to identify the genes and gene families. Therefore, from the research of scientific theory, the hereditary cataract is a good hereditary pattern disease for clarifying the human gene function.

In order to find a causative gene causing cataract, molecular genetic research methods including functional cloning (e.g., candidate gene screening and protein analysis), positional cloning, mapping of candidate gene cloning (e.g., family-based linkage analysis and allele-sharing analysis), and the like are often employed. To date, 30 genes have been identified to be associated with simple hereditary cataract, collectively known as hereditary cataract-causing genes, which can be roughly classified into four major groups: crystallin genes (CRYAA, CRYAB, CRYBAI/A3, CRYBA2, CRYBA4, CRYBB1, CRYBB2, CRYBB3, CRYGB, CRYGC, CRYGD, CRYGS), cytoskeleton or membrane protein genes (GJA3, GJA8, MIP, LIM2, BFSP1, BFSP2, EPHA2, TMEM114, CHMP4B, VIM), transcription factor regulatory genes (HSF4, PITX3) and other related genes (GCNT2, FOXE3, WFS1, UNC45B, TDRD7, FYCO1) and the like.

Genetic congenital cataracts are highly heterogeneous in genetics and clinic, i.e., different genetic mutations can lead to cataracts of the same phenotype, and cataracts of different phenotypes can be caused by the same genetic mutation. Because of the numerous genetic cataract disease-causing genes and the absence of a certain correlation between genotype and phenotype, clinical gene detection is challenging.

With the rapid development of biotechnology, especially exome sequencing, whole genome sequencing and the like, a rapid means is provided for screening and finding genetic disease pathogenic gene mutation, but the cost is relatively expensive. Therefore, such techniques are often applied after elimination of known disease causing genes to reduce the cost risk of the experiment, otherwise the cost performance is too high. The target sequence capturing and sequencing technology can realize high-throughput screening of a plurality of known pathogenic gene mutations, has the advantages of rapidness, high efficiency and the like, but has higher cost. In terms of scientific rigor, the suspected pathogenic gene mutation screened by the new technology needs further verification by a gold-labeled DNA sequencing Sanger method. Therefore, in the precise medical age, the establishment of a rapid, economic and efficient genetic cataract pathogenic gene detection system is not only helpful for rapidly screening known pathogenic genes of cataract families and discovering new mutation sites, but also can provide reliable family materials for discovering new pathogenic genes and the formulation of subsequent research method decisions. In clinical application, the kit can be developed into a corresponding cataract gene diagnosis kit, and provides scientific genetic counseling and prenatal gene diagnosis for patients with family history of cataract.

At present, as far as we know, the research of the genetic cataract pathogenic gene is mainly limited to scientific research, and no genetic cataract pathogenic gene detection product exists clinically; the patent related to the screening of the cataract pathogenic gene is searched only aiming at a single genetic cataract pathogenic gene mutation detection method or kit, and the patent application of the invention aiming at a plurality of genetic cataract pathogenic gene mutation detection kits is not found.

Disclosure of Invention

The invention aims to provide a rapid, economic and efficient cataract pathogenic gene detection kit and a detection method thereof.

In order to achieve the purpose, the invention adopts the following technical scheme:

the theoretical basis of the invention is that genetic cataract exists in a high-frequency pathogenic gene or mutation hot spot region, and the operation steps for specifically obtaining the high-frequency mutation spectrum of the cataract pathogenic gene are as follows: first, searching for 211 related documents reporting genetic cataract disease-causing genes and mutations thereof in China and abroad since the discovery of the first human genetic cataract disease-causing gene CRYBB2 in 1997 to 2014 7, 299 families or probands are counted, and 30 related disease-causing genes are related, distributed in 16 chromosomes (chr.1, 2, 3, 4, 6, 9, 10, 11,12, 13, 16, 17, 19, 20, 21,22), 221 single mutations (Uni-mutation) are identified, distributed in 72 exons and occupy 34.6% of the total exons of the genes (72/205); subsequently, the occurrence spectra of the disease-causing genes were analyzed, and it was found that 90.97% of the number of cases were associated with 16 mutations of the disease-causing genes, which were GJA8 and CRYGD (11.04%), GJA3 and CRYAA (10.03%), CRYBB2(7.69%), CRYBA1(6.35%), MIP (5.02%), EPHA2, CRYGC, CRYAB and HSF4(4.01%), FYCO1(3.34%), CRYBB1(3.01%), BFSP2(2.68%), GCNT2 and PITX3 (2.34%); wherein FYCO1 and GCNT2 are involved only in autosomal recessive inheritance; finally, further analysis of the mutation frequency of each exon of the disease-causing gene revealed that 57.19% of the mutations were located on 11 exons of 8 genes, 65.22% of the mutations were located on 15 exons of 12 genes, 70.23% of the mutations were located on 18 exons of 13 genes, and 80.27% of the mutations were located on 26 exons of 18 genes. The statistical analysis result is shown in figure 1, and the invention defines 26 exons of the 18 genes as the high-frequency mutation exons of the cataract pathogenic gene.

The gene detection kit provided by the invention is mainly used for detecting the protein coding region of the high-frequency mutation exon of the cataract pathogenic gene and the splicing site connecting region thereof, namely the high-frequency mutation region of the cataract pathogenic gene, so that 80% of genetic cataract families can be detected.

The gene detection kit provided by the invention comprises 27 pairs of PCR primers, and is designed and artificially synthesized by adopting biological software such as Primer, Generuner and the like according to the DNA sequence of the high-frequency or hot spot mutation region of the cataract pathogenic gene; the specific information of 27 pairs of PCR primers is shown in Table 1.

TABLE 1 26 primer information and PCR conditions thereof provided by the present invention

The components of the cataract pathogenic gene detection kit provided by the invention are as follows 2:

TABLE 2 cataract disease gene detection kit composition

The kit provided by the invention is used for a method for detecting cataract pathogenic genes, and the method mainly comprises three parts:

1) the DNA fragment of the target gene is amplified by PCR by using the cataract pathogenic gene detection kit; 2) DNA sequencing analysis of the PCR product to find out the variation site;

3) and identifying whether the target gene variation site is a pathogenic mutation site.

The method for detecting the cataract disease-causing gene of the kit comprises the following specific operation steps:

1) preparing PCR reaction solution (50 μ l) according to the following table 3, mixing uniformly and placing on a PCR instrument;

TABLE 3 PCR reaction System (100. mu.l) composition

2) The PCR amplification reaction was performed under the following conditions:

pre-denaturation at 94 ℃/4 min; 94 ℃/30sec, 57 ℃ -61 ℃ (the annealing temperature varies according to different PCR primers, specifically referring to attached table 1)/30sec,72 ℃/1min, 30 cycles; 72 ℃/4 min; storing at 4 ℃.

3) And (3) identifying the PCR product by agarose gel electrophoresis: 9ul of PCR product was added to 1ul of 10 Xloading buffer, and the specificity and size of the PCR product were determined by electrophoresis on 1% agarose gel and UV imaging.

4) DNA sequencing analysis of PCR products shows that the variation sites: and separating and purifying the PCR product by agarose gel electrophoresis, performing DNA sequencing by an ABI3730XL automatic DNA sequencer, comparing and analyzing a sequencing result with a normal DNA sequence Blast, and screening a mutation site.

5) Identification of pathogenic mutation sites: if the mutation site is a known pathogenic gene mutation site, determining the mutation site as a pathogenic mutation; if the variation site is a new variation site, the new missense mutation of the coding region is functionally predicted by SNP exclusion, co-segregation of disease phenotype and variation site, protein sequence homology comparison, and application of some biological protein variation function prediction software analysis, including Polyphen2(Polymorphism genotyping v2) and SIFT (sodium interactive From Tolerat), and HSF (human partitioning Finder) for the change of Splicing point on intron.

Besides the gene detection kit provided by the invention, the related cataract pathogenic gene detection reagent and method can be simply developed by applying the existing biological technology, such as DNA chip technology and target sequence capture second-generation sequencing technology, to the high-frequency mutation region of the cataract pathogenic gene provided by the invention. Therefore, the high-frequency mutation region of the cataract-causing gene provided by the present invention is also suitable for the above-mentioned techniques, but not limited to the above-mentioned techniques.

The invention also provides 16 new mutation sites of cataract pathogenic gene, which are shown in Table 5 and figure 2. According to the new mutation site provided by the invention, by applying the prior art, such as DNA sequencing based on PCR amplification technology, Restriction Fragment Length Polymorphism (RFLP) analysis, single-strand conformation polymorphism detection (SSCP), allele specific PCR, real-time quantitative PCR, high-resolution melting curve (HRM) analysis and the like, corresponding rapid detection reagent and method can be easily developed aiming at the new mutation site information provided by the invention. Therefore, the 16 high-new mutations of the cataract-causing genes provided by the invention are also suitable for the above-mentioned techniques, but not limited to the above-mentioned techniques.

To illustrate the advantages and benefits of the present invention, a number of relevant prior art situations are listed: hansen L et al PCR-amplified all exons of 17 cataract-causing genes in 28 Danish cataract families, and the DNA sequencing result showed that the detection rate is 71% (Invest Ophthalmol Vis Sci 2009, 50, 3291-; all exons of 12 cataract-causing genes are amplified by Sun W and the like in 25 Chinese cataract families by adopting PCR, and the detection rate is 40% as shown by a DNA sequencing result (Mol Vis. 2011, 17, 2197-; ponnam SP et al screened 10 full exons of cataract-causing genes in 40 Indian cataract families with SSCP (single strand transformation polymorphism) technique, with a detection rate of only 10% (Mol Vis. 19, 1141 and 11482013); sun W et al screened 18 Chinese cataract families by exome sequencing technology with a detection rate of 67.6% (PLoS one. 2014, 9, e 100455); ma AS and others screen 32 cataract-causing genes in 46 congenital cataract families by adopting a target sequence capture second-generation sequencing technology, and the detection rate is 70% (Hum Mutat, 2016, 37, 371-.

Compared with the prior art, the invention has the advantages and beneficial effects that:

because the invention establishes the cataract pathogenic gene detection system based on the cataract pathogenic gene mutation hotspot region, 80 percent of genetic cataract families can be detected by screening 26 exons of 18 cataract pathogenic genes. Therefore, the method can obviously reduce the workload and the cost of detecting the cataract pathogenic gene on the basis of not reducing the detection rate, and has the advantages of rapidness, economy, high efficiency and the like.

In the aspect of basic research, the cataract pathogenic gene detection kit and the detection method thereof can realize the quick investigation of the known pathogenic genes of the cataract family, discover new mutation sites, expand the mutation spectrum of the cataract pathogenic genes and provide reliable and precious genetic resources for screening the new cataract pathogenic genes; in clinical application, the kit and the method system thereof provided by the invention are used for detecting the cataract pathogenic gene, and can provide scientific genetic counseling and prenatal diagnosis for the family history of cataract.

Drawings

FIG. 1 selection of high-frequency mutation region of cataract-causing gene: black dots indicate the hot spot mutation region of the study selection, including 26 exons on 18 genes, which covers 80.27% of cataract family pathogenic gene mutation reported in the literature; the size of the spot is related to the frequency of mutations in this exon, the higher the mutation frequency, the larger the spot diameter. (ii) a It is expected that 36.11% (26/72) of the exon regions may cover more than about 80% of the mutations.

FIG. 2: DNA sequencing map of new mutation site of cataract pathogenic gene.

Detailed Description

Example 1: high-frequency mutation spectrum of cataract-causing gene

In order to obtain a high-frequency mutation spectrum of cataract-causing genes, namely, a disease-causing gene and exon mutation hotspot region thereof, we searched PubMed to include 210 related documents of human simple hereditary cataract-causing genes and mutation thereof (from the discovery of the first human hereditary cataract-causing gene CRYBB2 in 1997 to 7 months in 2014), 299 families or probands are reported in total, 30 related disease-causing genes are related, and 16 single mutations (Uni-mutation) are identified, wherein the single mutations are distributed in 72 exons and account for 34.6% of the total exons of the genes (72/208). Counting all mutations on 30 cataract-causing genes by taking a family or proband as a unit; then, summarizing frequency statistics according to exon mutation, sorting from top to bottom, drawing a high-frequency mutation spectrum of cataract pathogenic genes, and detailing the result in an attached figure 1; finally, the invention selects 26 exons distributed in 18 genes as mutation hot spot regions (see table 1 for details) to carry out subsequent detection and analysis of cataract-causing genes.

Example 2: cataract pathogenic gene detection method

Designing and synthesizing a corresponding PCR Primer by using biological software such as Primer, Generuner and the like according to the mutation hot spot region of the cataract pathogenic gene selected in the example 1; PCR amplification of DNA fragments corresponding to the protein coding region and the splicing region was performed using proband gDNA as a template, and the PCR primers and PCR conditions are shown in Table 1. After purification of the PCR products, DNA was directly sequenced using an ABI3730XL full-automatic Sequencer (Automated sequence PE Biosystems, Foster City, Calif.).

And analyzing and screening the pathogenic gene variation sites by DNA sequence comparison. Determining the mutation site as a pathogenic mutation if the mutation site is a known mutation site; if the mutation site is new, the new missense mutation of the coding region is predicted functionally by SNP exclusion, co-segregation of disease phenotype and mutation site, protein sequence homology comparison, and application of some biological protein variation function prediction software analysis, including Polyphen2(Polymorphism genotyping v2) and SIFT (sodium interactive random Tolerant), and HSF (human partitioning Finder) for the change of splice point on intron.

For the screened new mutation, 2-3 STR microsatellite markers containing high information are respectively selected at the upstream and downstream of the mutant gene, a DNA region of the microsatellite locus is amplified by PCR, amplified products are separated by capillary gel electrophoresis on an ABI3730 automatic sequencer, the size of the DNA fragment is analyzed by GeneMarker2.4.0 software, a family map is drawn by Cyrilic 2.1 software, a haplotype is constructed, and the new pathogenic gene mutation locus is further confirmed.

Example 3: collection of blood sample specimen of hereditary cataract family or sporadic case and separation and purification of genome DNA

In order to verify whether a high-frequency mutation spectrum of cataract pathogenic genes exists and discover new pathogenic gene mutation, the invention collects blood sample specimens of 43 cataract probands (31 from autosomal dominant inheritance families, 8 from non-family history families and 4 sporadic cases) and 234 family members thereof; in addition, 112 samples of normal human blood were collected as controls. Extraction and Purification of Genomic DNA (gDNA) from whole blood samples was performed according to Wizard Genomic DNA Purification Kit (Promega, Beijing, China) Kit instructions.

Example 4: application of cataract pathogenic gene detection kit

The method for detecting a cataract-causing gene established in example 2 was applied to the genomic DNA obtained in example 3. As a result, 7 known mutation sites (Table 4) and 16 new mutation sites (Table-5 and FIG. 2) were found. The statistical analysis results show that: pathogenic mutations were detected in 60.5% (26/43) probands/pedigree, which was 80.27% lower than the literature reported detection rate (240/299, X2=7.99, p = 0.005). And for the autosomal dominant genealogy, the detection rate is 80% (24/30), and has no statistical difference from the detection rate of 83.57% (234/280) based on literature reports (X2=0.489, p = 0.484). For familial and sporadic cases without family history, the detection rate is only 16.67% (2/12). Therefore, the kit and the detection method thereof provided by the invention are particularly suitable for detecting the autosomal dominant genetic cataract family pathogenic gene.

TABLE 4 identification of 7 known sites of disease-causing gene mutation in 10 congenital cataract families

TABLE 5 identification of 16 New mutation sites of cataract-causing genes in the family of congenital cataract

Remarks D = damaging, PD = basic damaging, MPA = motoprobable influencing spraying

The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.

SEQUENCE LISTING

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Claims

1. A cataract pathopoiesia gene detection kit is characterized in that: the kit comprises 27 pairs of PCR primers for PCR amplification of a high-frequency mutation region of a cataract pathogenic gene, wherein the DNA sequences of the primers are respectively shown as SEQ ID NO. 1-54;

the high-frequency mutation region of the cataract pathogenic gene comprises 26 exons of 18 cataract pathogenic genes and can detect pathogenic genes of hereditary cataract families;

the pathogenic genes for detecting the genetic cataract family are as follows: GJA8, GJA3, CRYGD, CRYAA, CRYBB2, CRYBB1, CRYBA1, CRYAB, CRYGC, HSF4, BFSP2, EPHA2, PITX3, FYCO1, MIP, CRYGS.

2. The cataract reagent kit as claimed in claim 1, which is characterized in that: the kit comprises the following components: 5U/uL 100uL Taq enzyme, 10 XTaq containing Mg²⁺2.0mL of buffer, 1.6mL of 2.5mM dNTP mixture, 27 pairs of the upstream and downstream primers of claim 1, 10pM for each primer, and 1.0mL of 10 Xloading buffer.