CN106701925B - Cataract pathogenic gene detection kit and detection method thereof - Google Patents

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 hereditary diseases, in particular to a cataract pathogenic gene detection kit and a detection method thereof.

Background technique

Cataract is the opacity of the lens caused by congenital or acquired factors. The prevalence of congenital cataract in the world is 0.01%~0.06%, and it is about 0.05% in my country. The prevention of visual impairment caused by congenital cataracts is an important part of the World Health Organization (WHO) international project "End Avoidable Blindness by 2020".

Congenital cataracts have obvious clinical heterogeneity. According to the clinical phenotype, they can be divided into pan cataract, membranous cataract, nuclear cataract, circumnuclear cataract, anterior pole cataract, posterior pole cataract, suture cataract, punctate cataract, disc The pathogenesis of cataract is complex, involving factors such as heredity, metabolic disease, intrauterine infection and spontaneity, of which about 30%-50% are related to heredity, collectively referred to as hereditary cataract. The hereditary cataract inheritance modes include autosomal dominant, autosomal recessive, and sex chromosome-linked inheritance, among which autosomal dominant inheritance is the main one. According to statistics, among familial cataract patients in my country, autosomal dominant cataract accounts for 73% of the total number of familial cataracts.

The development of the lens is a very complex process involving many genes. Mutations in any one of these genes may cause the lens to become cloudy, which is what we call cataracts. To understand the genes (proteins) that play different functions at the cellular level by studying hereditary cataracts is the most effective way for humans to understand such genes and even their gene families. Therefore, in terms of scientific theoretical research, hereditary cataract is a good hereditary model disease for elucidating the function of human genes.

In order to search for the causative gene causing cataract, molecular genetic research methods are often used including functional cloning (such as candidate gene screening and protein analysis), positional cloning, locational candidate gene cloning (such as family-based linkage analysis and allele sharing analysis) )Wait. So far, 30 genes have been identified that are related to pure hereditary cataract, collectively referred to as hereditary cataract pathogenic genes, which can be roughly classified into four categories: crystal protein 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), etc.

Hereditary congenital cataracts have a high degree of genetic and clinical heterogeneity, that is, different gene mutations can cause cataracts with the same phenotype, and cataracts with different phenotypes can be caused by the same gene mutation. Due to the large number of causative genes of hereditary cataract, and there is no certain correlation between genotype and phenotype, its clinical genetic testing is quite challenging.

With the rapid development of biotechnology, especially exome sequencing and whole-genome sequencing, it provides a fast method for the screening and discovery of pathogenic gene mutations in hereditary diseases, but the cost is relatively expensive. Therefore, this type of technology is often applied after excluding known disease-causing genes to reduce the cost risk of experiments, which would otherwise be too cost-effective. Target sequence capture sequencing technology can realize high-throughput screening of many known pathogenic gene mutations, and has the advantages of fast and efficient, but high cost. In terms of scientific rigor, the screening of suspected pathogenic gene mutations by the above new technologies still requires further verification by the Sanger method of gold-labeled DNA sequencing. Therefore, in the era of precision medicine, the establishment of a rapid, economical and efficient genetic detection system for hereditary cataracts not only helps to identify known causative genes in cataract families as soon as possible, but also helps to discover new mutation sites. The causative genes provide reliable pedigree material and the formulation of methodological decisions for subsequent research. In clinical application, a corresponding cataract gene diagnosis kit can be developed to provide scientific genetic counseling and prenatal genetic diagnosis for those with a family history of cataract.

At present, as far as we know, the research on hereditary cataract causative genes is mainly limited to scientific research, and there is no clinical product for hereditary cataract causative gene detection; and the patents related to cataract causative gene screening are only for single There are no invention patent applications for multiple genetic cataract pathogenic gene mutation detection kits for hereditary cataract pathogenic gene mutation detection methods or kits.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a rapid, economical and efficient cataract pathogenic gene detection kit and its detection method.

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

The theoretical basis of the present invention is that hereditary cataract has high-frequency pathogenic genes or mutation hot spots, and the specific operation steps to obtain the high-frequency mutation spectrum of cataract pathogenic genes are as follows: From the disease gene CRYBB2 to July 2014, 211 domestic and foreign literatures were reported on hereditary cataract pathogenic genes and their mutations, 299 families or probands were counted, involving 30 related pathogenic genes, distributed on 16 chromosomes (chr.1, 2, 3, 4, 6, 9, 10, 11, 12, 13, 16, 17, 19, 20, 21, 22), identified 221 single mutations (Uni-mutation), distributed in 72 exons, accounting for 34.6% (72/205) of the total exons of the gene; then, the occurrence spectrum of pathogenic genes was analyzed, and the results showed that 90.97% of the cases were related to mutations in 16 pathogenic genes, which occurred The spectrum from high to low is 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%); FYCO1 and GCNT2 are only involved in autosomal recessive inheritance; Exon mutation frequency, the results show that 57.19% of mutations are located in 11 exons of 8 genes, 65.22% of mutations are located in 15 exons of 12 genes, and 70.23% of mutations are located in 18 of 13 genes. 80.27% of the mutations were located in 26 exons of 18 genes. The visual diagram of the statistical analysis results is shown in Figure 1, and the present invention defines the 26 exons of the 18 genes as the high-frequency mutation exons of the cataract pathogenic gene.

The present invention provides a cataract pathogenic gene detection kit, which mainly detects the protein coding region of the high-frequency mutation exon of the cataract pathogenic gene defined above and its splice site connection region, that is, the cataract pathogenic gene In the high-frequency mutation region, the pathogenic gene mutation in 80% of the hereditary cataract families can be detected.

The cataract pathogenic gene detection kit provided by the present invention, the gene detection kit includes 27 pairs of PCR primers. Artificial synthesis; the specific information of the 27 pairs of PCR primers is shown in Table 1.

Table 1. 26 pairs of primer information and PCR conditions provided by the present invention

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

Table 2 Composition of cataract pathogenic gene detection kit

The kit provided by the invention is used for a cataract pathogenic gene detection method, and the method mainly includes three parts:

1) Use the cataract pathogenic gene detection kit of the present invention to amplify the DNA fragment of the target gene by PCR; 2) DNA sequencing analysis of the PCR product to find the mutation site;

3) Identify whether the target gene mutation site is a pathogenic mutation site.

The specific operation steps of the cataract pathogenic gene detection method of the kit are as follows:

1) Prepare PCR reaction solution (50 μl) according to Table 3, mix well and place it on the PCR machine;

Table 3 Composition of PCR reaction system (100 μl)

2) Carry out PCR amplification reaction under the following conditions:

94°C/4min pre-denaturation; 94°C/30sec, 57°C-61°C (annealing temperature varies according to different PCR primers, see Appendix 1 for details)/30sec, 72°C/1min, 30 cycles; 72°C/4min; Store at 4°C.

3) Identification of PCR products by agarose gel electrophoresis: Take 9ul of PCR products, add 1ul of 10× loading buffer, run 1% agarose gel electrophoresis, and check the specificity and size of PCR products under the UV imaging system. match.

4) DNA sequencing analysis of PCR products to find variation sites: PCR products are separated and purified by agarose gel electrophoresis and then DNA sequenced by ABI3730XL automatic DNA sequencer. The sequencing results are compared with the normal DNA sequence Blast to screen for variation site.

5) Identification of pathogenic mutation sites: If the mutation site is a known pathogenic gene mutation site, it is determined to be a pathogenic mutation; if the mutation site is a new mutation site, it is excluded by SNP and disease table. Co-segregation of type and variant sites, comparison of protein sequence homology, and application of some biological protein variant function prediction software analysis, including PolyPhen2 (Polymorphism Phenotyping v2) and SIFT (Sorting Intolerant From Tolerant) new missense to coding regions Mutations are used for functional prediction, and HSF (Human Splicing Finder) is used to predict changes in splice junctions on introns.

In addition to the gene detection kit provided by the present invention, according to the high-frequency mutation region of the cataract pathogenic gene provided by the present invention, the application of existing biological technologies, such as DNA chip technology and target sequence capture second-generation sequencing technology, can easily develop related Cataract pathogenic gene detection reagents and methods. Therefore, the high-frequency mutation region of the cataract pathogenic gene provided by the present invention is also suitable for the above-mentioned technology, but is not limited to the above-mentioned technology.

The present invention also provides 16 new mutation sites of cataract pathogenic genes, as shown in Table 5 and accompanying drawing 2 . According to the new mutation site provided by the present invention, the existing technology, such as DNA sequencing based on PCR amplification technology, restriction fragment length polymorphism (restriction fragment length polymorphism, RFLP) analysis, single-strand conformation polymorphism detection ( SSCP), allele-specific PCR, real-time quantitative PCR, and high-resolution melting curve (HRM) analysis, etc., can easily develop corresponding quick detection reagents and methods for the new mutation site information provided by the present invention. Therefore, the 16 high-level mutations of cataract pathogenic genes provided by the present invention are also suitable for the above-mentioned technology, but are not limited to the above-mentioned technology.

In order to illustrate the advantages and beneficial effects of the present invention, some related prior art situations are listed first: Hansen L et al. used PCR to amplify all exons of 17 cataract pathogenic genes in 28 Danish cataract families, and the results of DNA sequencing showed that the detection rate was 71% (Invest Ophthalmol Vis Sci. 2009, 50, 3291-3303); Sun W et al used PCR to amplify all exons of 12 cataract-causing genes in 25 Chinese cataract families, DNA Sequencing results showed that the detection rate was 40% (Mol Vis. 2011, 17, 2197-2206); Ponnam SP et al. used SSCP (single strand conformation polymorphism) technology to screen 10 cataract causative genes in 40 Indian cataract families. For all exons, the detection rate is only 10% (Mol Vis. 19, 1141-1148 2013); Sun W et al. used exome sequencing technology to screen 18 Chinese cataract families, and the detection rate was 67.6% ( PLoS One. 2014, 9, e100455); Ma AS et al. used target sequence capture next-generation sequencing technology to screen 32 cataract-causing genes in 46 congenital cataract families, and the detection rate was 70% (Hum Mutat. 2016, 37, 371-384).

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

Since the present invention establishes a cataract pathogenic gene detection system based on the hot spot region of cataract pathogenic gene mutation, only by screening 26 exons of 18 cataract pathogenic genes, the pathogenic genes of 80% of hereditary cataract families can be detected mutation. Therefore, the present invention can obviously reduce the workload and cost of cataract pathogenic gene detection on the basis of not reducing the detection rate, and has the advantages of rapidity, economy and efficiency.

In terms of basic research, the cataract pathogenic gene detection kit and the detection method provided by the present invention can quickly check the known pathogenic genes of cataract families, discover new mutation sites, expand the cataract pathogenic gene mutation spectrum, and further It can provide reliable and valuable genetic resources for screening new cataract pathogenic genes; in clinical application, the kit and the method system provided by the present invention are used for cataract pathogenic gene detection, which can provide scientific genetic information for cataract family history Counseling and prenatal diagnosis.

Description of drawings

Figure 1 Selection of high-frequency mutation regions of cataract causative genes: The black dots indicate the hot spot mutation regions selected in this study, including 26 exons of 18 genes, and these regions cover 80.27% of cataract pedigree causative genes reported in the literature Mutation; the size of the dot is related to the frequency of mutation in that exon, the higher the mutation frequency, the larger the diameter of the dot. ; It can be expected that 36.11% (26/72) of the exon region can cover more than 80% of the mutations.

Figure 2: DNA sequencing map of novel mutation sites in cataract-causing genes.

Detailed ways

Example 1: High-frequency mutation spectrum of cataract-causing genes

In order to obtain the high-frequency mutation spectrum of cataract causative genes, that is, the hot spot regions of causative genes and their exons, we searched PubMed for 210 related literatures on human purely hereditary cataract causative genes and their mutations (since the discovery of the first in 1997). A human hereditary cataract causative gene CRYBB2 since July 2014), a total of 299 families or probands have been reported, involving 30 related causative genes distributed on 16 chromosomes (chr.1, 2, 3). , 4, 6, 9, 10, 11, 12, 13, 16, 17, 19, 20, 21, 22), identified 221 single mutations (Uni-mutation), distributed in 72 exons, accounting for gene 34.6% (72/208) of total exons. All mutations in 30 cataract-causing genes were counted in the unit of family or proband; then, the frequency of exon mutations was summarized, sorted from high to bottom, and the high-frequency mutation spectrum of cataract-causing genes was drawn. Results See Figure 1 for details; finally, the present invention selects 26 exons distributed in 18 genes as mutation hotspot regions (see Table 1 for details) for subsequent detection and analysis of cataract pathogenic genes.

Example 2: Cataract causative gene detection method

According to the selected cataract pathogenic gene mutation hotspot region in Example 1, use biological software such as Primer and Generunner to design and synthesize corresponding PCR primers; take the proband gDNA as a template, PCR amplify the corresponding protein coding region and the splice junction region The DNA fragments, PCR primers and PCR conditions are shown in Table 1. After purification of the PCR products, direct DNA sequencing was performed using an ABI 3730XL automated sequencer (Automated Sequencer PE Biosystems, Foster City, CA).

Through DNA sequence alignment, analysis and screening of pathogenic gene mutation sites. If the mutation site is a known mutation site, it is determined to be a pathogenic mutation; if the mutation site is new, it is excluded by SNP, disease phenotype and mutation site co-segregation, protein sequence homology comparison, And apply some biological protein variant function prediction software analysis, including PolyPhen2 (Polymorphism Phenotyping v2) and SIFT (Sorting Intolerant FromTolerant) for functional prediction of new missense mutations in the coding region, and HSF (Human Splicing Finder) for introns. Changes in upper splice junctions are predicted.

For the newly identified mutations, 2-3 microsatellite markers with high information were selected in the upstream and downstream of the mutant gene, and the DNA region of the microsatellite locus was amplified by PCR. Gel electrophoresis was used for separation, DNA fragment size was analyzed with GeneMarker 2.4.0 software, pedigree was drawn with Cyrillic 2.1 software, and haplotypes were constructed to further confirm new pathogenic gene mutation sites.

Example 3: Collection of blood samples from hereditary cataract families or sporadic cases and isolation and purification of genomic DNA

In order to verify whether there is a high-frequency mutation spectrum of cataract pathogenic genes and discover new pathogenic gene mutations, the present invention collected 43 cataract probands (31 from autosomal dominant families, 8 from families with no family history and 4 sporadic cases) and blood samples from 234 family members; in addition, 112 normal human blood samples were collected as controls. Genomic DNA (gDNA) was extracted and purified from whole blood samples according to the instructions of the Wizard Genomic DNA Purification Kit (Promega, Beijing, China).

Example 4: Application of cataract pathogenic gene detection kit

Taking the genomic DNA of Example 3 as the object, the cataract pathogenic gene detection method established in Example 2 was applied. As a result, 7 known mutation sites (Table 4) and 16 new mutation sites (Table-5 and Figure-2) were found. Statistical analysis showed that pathogenic mutations were detected in 60.5% (26/43) of the probands/families, which was lower than the reported detection rate of 80.27% (240/299, X2=7.99, p=0.005) . For autosomal dominant pedigrees, the detection rate was 80% (24/30), which was not statistically different from the 83.57% (234/280) detection rate based on literature reports (X2=0.489, p=0.484 ). For families with no family history and sporadic cases, the detection rate was only 16.67% (2/12). Therefore, the kit and the detection method provided by the present invention are particularly suitable for the detection of the causative gene of an autosomal dominant cataract family.

Table 4. Identification of 7 known causative gene mutation loci in 10 families with congenital cataract

Table 5 Identification of 16 new mutation sites of cataract pathogenic genes in congenital cataract families

Remarks: D= damaging; PD = probably damaging; MPA = mostprobablyaffecting splicing

The above descriptions are only preferred embodiments of the present invention, and all equivalent changes and modifications made according to the scope of the patent application of the present invention shall fall within the scope of the present invention.

SEQUENCE LISTING

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Claims (2)

1. a cataract pathogenic gene detection kit, is characterized in that: described test kit comprises 27 pairs of PCR primers, is used for PCR amplification cataract pathogenic gene high frequency mutation region, and its primer DNA sequence is respectively as SEQ ID NO. 1-54; The high-frequency mutation region of the cataract pathogenic gene includes 26 exons of 18 cataract pathogenic genes, which can detect the pathogenic genes of the hereditary cataract family; The pathogenic genes for the detection of hereditary cataract families are: GJA8, GJA3, CRYGD, CRYAA, CRYBB2, CRYBB1, CRYBA1, CRYAB, CRYGC, HSF4, BFSP2, EPHA2, PITX3, FYCO1, MIP, CRYGS. 2. a kind of cataract pathogenic gene detection kit according to claim 1, is characterized in that: described kit comprises the following components: 5U/ul 100uL Taq enzyme, 10 × Taq contains Mg ²⁺ buffer 2.0mL , 1.6mL of 2.5mM dNTP mixture, 27 pairs of upstream and downstream primers according to claim 1, 10pM for each primer, 1.0mL of 10× loading buffer.

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