CN112941164A - Method for detecting TRPC6 gene pathogenic mutation by target gene sequencing - Google Patents
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Abstract
The invention discloses a method for detecting TRPC6 gene pathogenic mutation by using target gene sequencing, which is used for carrying out TRPC6 gene sequencing on children patients with idiopathic nephrotic syndrome by using a second-generation sequencing technology and carrying out related bioinformatics analysis. Designing 13 exon region specific capture probes of TRPC6, hybridizing with a genome DNA library, enriching DNA fragments of a target genome region, sequencing by using an illumina Nextseq second generation sequencer, comparing with a normal Zhuang child, and determining mutation sites through data analysis. Through data analysis, c.172C > T (p.R58W) heterozygous mutation on the exon 2 is found to cause related protein expression and dysfunction, and is considered as pathogenic mutation of Guangxi Zhuang children in China.
Description
[ technical field ] A method for producing a semiconductor device
The invention relates to the technical field of gene sequencing, in particular to a method for detecting TRPC6 gene pathogenic mutation by utilizing target gene sequencing, and particularly relates to a method for detecting TRPC6 gene pathogenic mutation of Guangxi Zhuang child idiopathic nephrotic syndrome in China by utilizing the target gene sequencing technology.
[ background of the invention ]
Nephrotic Syndrome (NS) is a clinical syndrome in which a series of pathophysiological changes are caused by increased permeability of the glomerular filtration membrane to plasma proteins and loss of large amounts of plasma proteins from urine. Primary Nephrotic Syndrome (PNS) of children is a type of immune-mediated glomerular disease which seriously affects the normal growth and development of children and has serious influence on the life health and the quality of life of children. Research on kidney pathogenic genes becomes an industrial hotspot, and the research on the kidney pathogenic genes screens genes playing a key role in the occurrence and development of PNS and provides specific intervention, thereby having important significance for treating PNS. The TRPC6 gene is located in the long arm 21 region to 22 region (11q21-q22) of chromosome 11, contains 13 exons, and the gene mutation of human can express autosomal dominant familial Focal Segmental Glomerulosclerosis (FSGS). TRPC is one of the major members of the Transient Receptor Potential (TRP) family, including TRPC, TRPV, TRPM, TRPP, TRPN, and TRPML 6 protein family members, each of which has many subtypes, and other members except TRPN are expressed in humans. The TRPC comprises 7 subfamilies, namely TRPC1, 2, 3, 4, 5, 6 and 7, the similarity of the TRPC6 and the amino acid sequences of TRPC3 and TRPC 397 is up to 90 percent, and TRPC channels are widely expressed in human bodies and comprise human body vital organs such as a nervous system, a blood system, smooth muscles, ovaries, lungs, spleens, kidneys and the like. Transient receptor potential cation channel protein 6 (TRPC 6) is a Slit Diaphragmatic (SD) protein encoded by TRPC6 gene, SD is formed by the regular interdigitation of adjacent podocyte foot processes, is an important component of glomerular filtration barrier, and plays an important role in maintaining the integrity of glomerular filtration membrane. The multifactorial effects cause glomerular podocyte injury to cause the amalgamation of the foot processes of SD as the most prominent morphological change of kidney disease. TRPC6 is widely expressed in kidney tissues, expressed in glomerular podocytes, mesangial cells, endothelial cells, tubular interstitial vascular walls and epithelial cells, and expressed in kidney blood vessels, with the expression of the podocytes being most important and the primary processes of the podocytes close to SD being most strongly expressed.
The traditional Sanger sequencing method has low sequencing flux, is difficult to meet the sequencing requirement, and needs a high-flux method for detecting the pathogenic genes. Compared with the prior method, the newly developed second-generation sequencing and biological information analysis technology is faster and has higher flux. The target gene capturing and second-generation sequencing technology is a rapid and high-flux sequencing method developed in recent years, can simultaneously sequence a plurality of genes, and has lower cost than whole exon sequencing.
In conclusion, the research on pathogenic mutation and new mutation of TRPC6 has better understanding on the molecular etiology of the Zhuang child primary nephrotic syndrome.
[ summary of the invention ]
Aiming at the defects that the sequencing flux of the traditional Sanger sequencing method in the prior art is low, the sequencing requirement is difficult to meet, and a high-flux method is needed for detecting the pathogenic gene, the invention provides a method for detecting the pathogenic mutation of a TRPC6 gene by utilizing the sequencing of a target gene, in particular to a method for detecting the pathogenic mutation of a TRPC6 gene of the Guangxi Zhuang child idiopathic nephrotic syndrome in China by utilizing the sequencing technology of the target gene, and the method is quick and effective, so that people can better know the molecular etiology of the Zhuang child idiopathic nephrotic syndrome.
A method for detecting TRPC6 gene pathogenic mutation by using target gene sequencing, in particular to a method for detecting TRPC6 gene pathogenic mutation of Guangxi Zhuang child essential nephrotic syndrome in China by using a target gene sequencing technology, which comprises the following steps:
1) selection of study subjects: selecting children with diagnosis meeting the children primary nephrotic syndrome diagnosis standard and excluding glomerulonephritis and secondary nephrotic syndrome, and extracting 2mL of peripheral venous blood of the children with empty stomach (EDTA anticoagulation);
2) extraction of genomic DNA: collecting a sample of venous blood from a nephrotic syndrome patient, extracting genomic DNA with DNeasy blood and tissue DNA extraction kit, and then testing DNA purity by calculating the ratio of absorbance at 260nm to absorbance at 280nm using Invitrogen Qbit spectrophotometer;
3) sequencing the FastTarget target region:
designing a high-quality sequencing primer aiming at a TRPC6 target region, selecting a primer which can obtain a clear single band by using a standard human genome as a template for amplification, mixing the optimized primers into a multiplex PCR primer panel according to the standard of each panel 20 pair primer, and performing amplification by using the standard human genome as the template;
based on a special method of capillary electrophoresis, the number of the finally obtained optimized panel is one twentieth of the number of the primers;
introducing a specific tag sequence compatible with an illumina platform into the tail end of the library through PCR amplification by using a primer with an Index sequence, and reducing the PCR tendency by adopting a PCR program with 11 cycles;
equally mixing all sample Index PCR amplification products, obtaining a final FastTarget sequencing library through tapping recovery, verifying fragment length distribution of the library by an Agilent 2100Bioanalyzer, and performing high-throughput sequencing on an Illumina Miseq platform in a 2 x 150bp/2 x 250bp double-end sequencing mode to obtain FastQ data after the molar concentration of the library is accurately quantified;
4) data screening and biological information analysis:
comparing raw data of each sample with a reference genome (UCSC hg19) by using BWA (Burrows-Wheeler Aligner) software, correcting a primary comparison result obtained by the BWA software by using a GATK standard program, detecting SNV/InDel of each sample by using two modes of VarScan software and a GATK HaplotpypeCaller method, filtering according to a screening scheme recommended by the software, comparing SNV/InDel found by the two detection schemes, merging all samples, comparing all SNV/InDel positions with a database by ANNOVAR, evaluating variation frequency, functional characteristics, conservation and pathogenicity of the SNV/InDel positions, and quickly finding the SNV/InDel position with the most biological significance;
5) determination of serum TRPC6 levels: detecting the concentration of the target protein in the sample by adopting a double-antibody sandwich ELISA method, and operating according to the instruction of the kit;
6) statistical analysis:
searching a site related to a disease, performing statistical analysis by using SPSS 24.0 software according to genotype data and phenotype data of a mutation site of an existing sample by using a correlation analysis method, performing correlation analysis by using plink analysis software, and performing linkage disequilibrium analysis by using Haploview software;
statistical analysis was performed using mathematical tools such as logistic regression, chi-square, rank-sum test, etc.:
the TRPC6 genotype and the allele thereof in the diseased group and the healthy group are counted to calculate the frequency, and Hardy-Weinberg genetic equilibrium law test is carried out;
logistic regression was performed in units of single locus, according to 5 hypothetical genetic models: performing correlation analysis on Dominant (low-frequency allole is Dominant), Recessed (low-frequency allole is Recessive), Log-additive and HOM/HET (co-Dominant model, and homozygous normal is used as reference); chi-square analysis was performed on each locus as well, and 4 analytical models, Codominant co-Dominant, domiant Dominant, Recessed Recessive, Allole alleles were set.
In the invention:
the secondary nephrotic syndrome in the step 1) refers to secondary nephrotic syndrome caused by hepatitis B, systemic lupus erythematosus, purpura and medicines.
DNeasy blood and tissue DNA extraction kit described in step 2) from Qiagen, Germany.
The database in the step 4) refers to dbSNP database, thousand human genomes, ESP6500, ExAC03, ExAC03_ EAS, gnomaD and Hrcr1Kaviar _20150923 database.
The genotype data and phenotype data in step 6) refer to case/control data, sex, age.
Compared with the prior art, the invention has the following advantages:
1. the method for detecting the pathogenic mutation of the TRPC6 gene of the Chinese Guangxi Zhuang child essential kidney disease syndrome by using the target gene sequencing technology is characterized in that 13 exon region specific capture probes of TRPC6 are designed, hybridized with a genome DNA library, DNA fragments of a target genome region are enriched, sequenced by using an illumina Nextseq (the American illumina company) second-generation sequencer, compared with normal Zhuang children, and mutation sites are determined by data analysis.
2. The method for detecting TRPC6 gene pathogenic mutation of Guangxi Zhuang child idiopathic nephrotic syndrome in China by using the target gene sequencing technology successfully discovers pathogenic mutation and new mutation of TRPC6 by combining the established FastTarget target gene capturing technology with the illumina Nextseq second generation sequencing technology, is quick and effective, and can enable people to better know molecular etiology of the Zhuang child idiopathic nephrotic syndrome.
[ description of the drawings ]
FIG. 1 is a graph of TRPC6 gene locus linkage analysis (D 'value) in the example of the present invention, in which D' of data was not labeled.
FIG. 2 is a TRPC6 gene locus linkage analysis (r) in the comparative example of the present invention2Value), r2The site association value is 1.
[ detailed description ] embodiments
The following examples are provided to further illustrate the embodiments of the present invention.
Example (b):
a method for detecting TRPC6 gene pathogenic mutation by using target gene sequencing, in particular to a method for detecting TRPC6 gene pathogenic mutation of Guangxi Zhuang child essential nephrotic syndrome in China by using a target gene sequencing technology, which comprises the following steps:
1 object and method
1.1 venous blood samples of 134 patients with nephrotic syndrome collected from outpatients and wards of the hospital affiliated to the Youjiang national medical institute during the period from 7 months to 2017 months of 2015, wherein the diagnosis accords with the primary nephrotic syndrome diagnosis standard [5] of children, and glomerulonephritis, (hepatitis B), systemic lupus erythematosus, purpura, medicaments and the like) secondary nephrotic syndrome and the like are excluded, wherein 90 patients of male and female patients are 44 patients, the ages of 2.7-15.8 years, the average (9.3 +/-1.4) years, 107 healthy children serve as controls, and the patients are healthy children for the same physical examination, 76 patients of male and female patients, 31 patients, the ages of 3.8-14.2 years, the average (8.5 +/-0.7) years, nephrotic syndrome in family and other kidney diseases. The third generation of the two groups of study objects is Zhuang nationality, and there is no relationship between them. The two groups of differences were not statistically significant in age and gender composition (P > 0.05). According to the principle of informed consent, the parents of the children are signed with informed consent and approved by the ethical committee of the national medical college of the Yangtze river.
1.2 major apparatus and reagents AB 2720 thermocycler (applied biosystems, Waltherm, Mass.); an Albender 5810R centrifuge (Albender GmbH, Germany); spectrophotometers (wilmington, usa); invitrogen quantum spectrophotometer (carlsbad Invitrogen, usa); illumina NextSeq (Illumina, san Diego, Calif.); multiskan FC microplate reader (american sermer feishell science); micropipettes (edwards, germany); nextseq kit V2 (Illumina, san diego, california, usa); gel extraction kit (Beijing Tiangen Biochemical technology Co., Ltd., China); 10 × reaction buffer (Dalianbao bioengineering, Inc., China); HotStarTaq polymerase (Dalianbao bioengineering, Inc., China); TRPC6 enzyme-linked immunosorbent kit (china enzyme-linked biology);
Blood&tissue kit (Hildenhagee, Germany).
1.3 extraction of genomic DNA 2mL of peripheral venous blood (EDTA anticoagulated) from infant was extracted on an empty stomach and genomic DNA was extracted using DNeasy blood and tissue DNA extraction kit (Qiagen, Germany). The DNA purity was then tested using an Invitrogen Qbit spectrophotometer by calculating the ratio of the absorbance at 260nm to the absorbance at 280 nm.
1.4 sequencing FastTarget target region high quality sequencing primers were designed for TRPC6 target region, and primers were selected that could amplify to obtain a clear single band using standard human genome as template. And (3) mixing the optimized primers into a multiplex PCR primer panel according to the standard of each panel 20 pair primer, and carrying out amplification by using a standard human genome as a template. Based on the special method of capillary electrophoresis, the number of the finally obtained optimized panel is about one twentieth of the number of the primers. Specific tag sequences compatible with the illumina platform were introduced to the ends of the library by PCR amplification using primers with Index sequences. The reaction adopts a PCR program with 11 cycles, and the PCR tendency is reduced as much as possible. All sample Index PCR amplification products were mixed in equal amounts and recovered by tapping to obtain the final FastTarget sequencing library, the fragment length distribution of which was verified by Agilent 2100 Bioanalyzer. After the molar concentration of the library is accurately quantified, high-throughput sequencing is finally carried out on an Illumina Miseq platform in a double-end sequencing mode of 2 × 150bp/2 × 250bp, and FastQ data are obtained.
1.5 data screening and biological information analysis Using BWA (Burrows-Wheeler Aligner) software, raw data of each sample was aligned with a reference genome (UCSC hg19), preliminary alignment results obtained by BWA software were corrected using the GATK standard program, so that the processed alignment results could identify SNV and InDel more accurately, SNV/InDel of each sample was detected using VarScan software and the GATK HaplotpypeCaller method, and filtered according to the screening protocol recommended by the software, SNV/InDel found by the above two detection schemes was compared, all samples were combined, all SNV/InDel sites were aligned with dbSNP database, thousand human genome, dbESP database, ESP genome, 6500, ExAC03, ExAC03_ EAS, Hgnomad, Hrcr1Kaviar _20150923 database by ANNOVAR, and the functional frequency of these variations, SNV/InDel sites, and functional frequency of variations were evaluated (Table 2), and the SNV/InDel, Conservation, pathogenicity, etc., rapidly finding the most biologically significant SNV/InDel site (table 1).
1.6 determination of serum TRPC6 levels the concentration of the protein of interest in the samples was determined by a double antibody sandwich ELISA method, according to the kit instructions.
1.7 statistical analysis to find disease-related sites, we generally used correlation analysis method, based on genotype data and phenotype data (such as case/control data, sex, age, etc.) of mutation sites of existing samples, statistical analysis using SPSS 24.0 software, correlation analysis using plink analysis software, linkage disequilibrium analysis using Haploview software. Statistical analysis is performed using mathematical tools such as logistic regression, chi-square, rank sum test, and the like. TRPC6 genotype and its alleles were counted for frequency in the affected and healthy groups and tested by Hardy-Weinberg's law of genetic equilibrium. Logistic regression was performed on a single locus basis by correlation analysis of 5 hypothetical genetic models, Dominant (low frequency allole is Dominant), recessability (low frequency allole is Recessive), Log-additive, HOM/HET (co-Dominant model, with homozygous normal as reference). Chi-square analysis was performed on each locus as well, and 4 analytical models, Codominant co-Dominant, domiant Dominant, Recessed Recessive, Allole alleles were set.
2, results:
2.1 data statistics of sequencing depth and coverage the average sequencing depth of target areas of 134 diseased groups and 107 normal control groups was 216.965-1181.708, 86.50-98.20% of target area >2 × coverage, 82.50-97.10% of target area >10 × coverage, 78.40-95.30% of target area >20 × coverage, and 76.00-94.70% of target area >30 × coverage.
2.2 results of analysis and sequencing of pathogenic mutation TRPC6 show that c.172C > T (p.R58W) heterozygous mutation on No. 2 exons of 1 case is found in a case group, the mutations are not detected in 107 healthy control groups, and through data screening and biological information analysis, the mutation is pathogenic mutation which can cause expression and function abnormality of related proteins and is considered as pathogenic mutation of Guangxi Zhuang children in China.
TABLE 1 results of all samples TRPC6 Low frequency functional mutations
TABLE 2-1 probability of all low frequency samples in the database
TABLE 2-2 probability of all low frequency samples in the database
2.3 Hardy-Weinberg equilibrium test
And (3) judging whether the research sample has population representativeness or not by checking whether the genotype frequency distribution in the researched random sample conforms to Hardy-Weinberg equilibrium law or not. Comparing the difference between observed and expected genotype frequencies, P >0.05 indicates that Hardy-Weinberg equilibrium is being followed, with population representativeness.
2.4 SNP association analysis
2.4.1 SNP site Chi-square test
Chi-square test analysis is carried out on all SNP sites of TRPC6, and the analysis model has co-dominant, recessive and allelic genes. The locus rs117895343, rs7931399, rs12805398, rs59743346, rs7105083, rs12362848, rs12366144, rs10501986, rs17096918, rs3802829 and rs3824934 has a P value of more than 0.05 in different genotypes of a co-dominant, recessive and allelic analysis model, and the difference has no statistical significance in case and control.
2.4.2 SNP locus logistic regression analysis
Logistic regression analysis was performed on all SNP sites of TRPC6, corrected for logistic regression using gender, age. The analysis models comprise Dominant (low-frequency allole is Dominant), Reccess (low-frequency allole is invisible), Log-additive and HOM/HET (co-Dominant model, and homozygosis is used as a reference) for correlation analysis. The loci rs117895343, rs7931399, rs12805398, rs59743346, rs7105083, rs12362848, rs12366144, rs10501986, rs17096918, rs3802829 and rs3824934 are all higher than 0.05 in P value of Dominant, Reccess, Log-additive and HOM/HET analysis models, and the difference has no statistical significance in case and control.
2.4.3 phenotypic and genotypic Association analysis
In addition to the sample-based case/control logistic regression correlation analysis, the sample phenotype information can also be used for correlation analysis of genotypes. In data analysis, we performed correlation analysis of all phenotypic information (except sex, age) with genotype. At the same time, sex, age data were used for correction. We used linear regression for continuous variables for analysis; for categorical variables, logistic regression was used for analysis. The analytical models include Dominant (Dominant for low-frequency allole), Reccess (invisible for low-frequency allole), Log-additive and HOM/HET (co-Dominant). All SNP sites (rs117895343, rs7931399, rs12805398, rs59743346, rs7105083, rs12362848, rs12366144, rs10501986, rs17096918, rs3802829 and rs3824934 sites) of TRPC6 have P values of more than 0.05 in different genotypes of Dominant, reprocess, Log-addive and HOM/HET analysis models, and phenotype and genotype differences have no statistical significance in case and control.
2.4.4 linkage disequilibrium analysis
Linkage disequilibrium analysis was performed using haploview software on sites rs117895343, rs7931399, rs12805398, rs59743346, rs7105083, rs12362848, rs12366144, rs10501986, rs17096918, rs3802829, and rs3824934 located in the TRPC6 gene (fig. 1, fig. 2). Thereby obtaining the haplotype with stronger association. It is found that rs7931399 has strong linkage disequilibrium with rs12805398 and rs59743346 sites (D' is 0.93 and 0.94 respectively, r2Values of 0.69, 0.84), respectively, and rs7931399 is fully linked to both sites of rs7105083 (D' ═ 1.00, r2 ═ 1.00). rs12805398 has strong linkage disequilibrium with rs59743346 and rs7105083 sites (D' is 1.00 and 0.93 respectively, r2Values of 0.83, 0.69), while there is a strong linkage disequilibrium between the rs59743346 and the rs7105083 sites (D' ═ 0.94, r)20.84), rs12362848 and rs12366144 are in strong linkage disequilibrium (D' ═ 1.00, r)20.73); rs17096918 and rs3824934 site has strong linkage disequilibrium (D' ═ 0.96, r)20.92) (fig. 1 and 2).
2.4.5 environmental factor corrected haplotype analysis
Blocks with strong correlation are found through linkage disequilibrium analysis, haplotype analysis is carried out on the blocks, correlation analysis of logistic regression is carried out by using case/control information, and sex and age are used for logistic regression correction. Several haplotypes and frequencies thereof that may exist by performing haplotype analysis on TRPC6 genes rs117895343, rs7931399, rs12805398, rs59743346, rs7105083, rs12362848, rs12366144, rs10501986, rs17096918, rs3802829, and rs3824934 sites are shown in table 3. The TRPC6-1(rs117895343, rs7931399, rs12805398, rs59743346 and rs7105083 sites) haplotypes are mainly GACAAGG haplotypes in the nephrotic syndrome group and the healthy group, and are respectively 91.2% and 87.1%, and the distribution frequency of the haplotypes is not found to have statistical difference between the nephrotic syndrome group and the healthy group; the TRPC6-2(rs12362848 and rs12366144 locus) haplotypes were found to be predominant to the GA haplotype in both nephrotic syndrome group and healthy group, 94.4% and 94.3%, respectively, and no statistical difference in haplotype distribution frequency was found between the two groups. The TRPC6-3(rs17096918, rs3802829 and rs3824934 loci) haplotypes are mainly found in TGC haplotypes in a nephrotic syndrome group and a healthy group, and are respectively 48.0 percent and 51.5 percent, and no statistical difference between the two groups in the distribution frequency of the haplotypes is found.
TABLE 3 Gene haplotype Association analysis results
Note: "case _ F" referred to in the data processing refers to the number and frequency of case sample haplotypes, and "control _ F" refers to the number and frequency of control sample haplotypes.
2.5 TRPC6 serum level comparison Using a non-parametric rank-sum test (Mean-Whitney U) analysis, the TRPC6 serum level in the healthy group was 544.7(264.6,952.6ng/mL, significantly higher than in nephrotic syndrome group 241.20(110.75, 542.35ng/mL (Z4.154, P < 0.001).
Discussion of 3
More and more studies find that TRPC6 gene mutation is the leading genetic cause of delayed autosomal dominant familial FSGS, and patients usually receive medical treatment in early adulthood. Winn equals 2005 and first found that the P112Q mutation of TRPC6 caused a significant increase in TRPC6 protein expression and intracellular calcium ion concentration. Recent studies have successively reported that 15 TRPC6 mutations are involved in the onset of FSGS. Wherein 3 mutations (G109S, P112Q, N125S) are located in the ANK1 region, 2 mutations (M132T, N143S) are located in the ANK2 region, 1 mutation (R175Q) is located in the ANK3 region, 1 mutation (H218L) is located in the ANK4 region, and the other 2 mutations (S270T, L395A) are located at the cytoplasmic N-terminus; the remaining 6 mutations (L780P, K874X, Q889K, R895C, R895L, E897K) are located at the cytoplasmic C-terminus. 10 of these mutations (P112Q, N143S, R895C, E897K, Q889K, M132T, N125S, H218L, R895L, R175Q mutations) all resulted in increased calcium channel activity. Kuang et al found that the TRPC6-254C > G gene variation is associated with enhanced transcription and childhood hormone-resistant nephrotic syndrome. Screening of 80 families of Chinese descent shows that 2 independent families have Q889K missense mutation, and the mutation rate of TRPC6 is 2.5%. It has also been shown that the gene polymorphisms rs3824934(-254C > G) and rs56134796(-218C > T) located in the TRPC6 promoter region do not affect the susceptibility of Indian hormone-resistant nephrotic syndrome patients. Research on 9 diseased members of mexico familial FSGS found that R895C heterozygote mutation of TRPC6 resulted in increased calcium influx, leading to FSGS. Hofstra et al found that the expression of TRPC6 was significantly increased in the glomeruli of Heymann nephritis rat as compared with the normal group, but the susceptibility to MN and the disease prognosis were unrelated to TRPC6 gene polymorphism, and 13 single nucleotide polymorphisms were found by sequencing TRPC6 gene of 101 patients with Idiopathic Membranous Nephropathy (IMN), of which 2 mutations (rs3802829, pro15ser and rs36111323, ala404val) caused amino acid substitution, but there was no statistical significance in the genotype and allele frequency difference from the normal control group. Kidney tissue biopsy was performed by Chen et al on 134 nephritis patients and it was found that the TRPC6(rs3824935C/T, rs17096918C/T, and rs4326755A/G) single nucleotide polymorphisms were not statistically different from the normal control group, wherein the rs17096918C/T, and rs4326755A/G nucleotide polymorphisms were detected only in MN patients. Kistler et al found that elevated expression of TRPC6 at the initial stage of MN podocyte injury protected podocytes, but that disease progression could be promoted with prolonged overexpression time of TRPC 6.
The reports indicate that the TRPC6 mutation is related to the occurrence of nephrotic syndrome of different nationalities and different regional populations. c.172C > T (p.R58W) heterozygous mutations of No. 2 exons in 1 case group are found in 107 healthy control groups, and the mutations are pathogenic mutations through data screening and biological information analysis, can cause related protein expression and function abnormality and are considered as pathogenic mutations of the Guangxi Zhuang children in China. Correlation analysis is carried out on all SNP sites (rs117895343, rs7931399, rs12805398, rs59743346, rs7105083, rs12362848, rs12366144, rs10501986, rs17096918, rs3802829 and rs3824934) of the TRPC6 gene, and all SNP site polymorphisms of the TRPC6 do not show statistical differences in the Guangxi Zhuang child nephrotic syndrome group and the healthy group. Haplotype analysis is carried out on the loci of TRPC6-1(rs117895343, rs7931399, rs12805398, rs59743346 and rs7105083), TRPC6-2(rs12362848 and rs12366144) and TRPC6-3(rs17096918, rs3802829 and rs3824934) respectively, and no haplotype increasing the risk of nephrotic syndrome is found.
In conclusion, the TRPC6 gene is a candidate gene of the children nephrotic syndrome, the gene mutation is related to the primary nephrotic syndrome of children of Guangxi Zhuang nationality in China, compared with the traditional primary sequencing, the target gene sequencing technology has higher flux, higher speed and higher accuracy, has important significance for deeply researching the molecular etiology of diseases, and is helpful for clarifying the occurrence mechanism of the diseases, thereby being helpful for the development of related medicines and achieving the effects of early diagnosis and early treatment.
The above description is intended to describe in detail the preferred embodiments of the present invention, but the embodiments are not intended to limit the scope of the claims of the present invention, and all equivalent changes and modifications made within the technical spirit of the present invention should fall within the scope of the claims of the present invention.
Claims (5)
1. The method for detecting the pathogenic mutation of the TRPC6 gene by utilizing target gene sequencing is characterized by comprising the following steps: the method for detecting the pathogenic mutation of the TRPC6 gene of the Chinese Guangxi Zhuang child essential nephrotic syndrome by using a target gene sequencing technology comprises the following steps:
1) selection of study subjects: selecting children with diagnosis meeting the children primary nephrotic syndrome diagnosis standard and excluding glomerulonephritis and secondary nephrotic syndrome, extracting 2mL of peripheral venous blood of the children with empty stomach, and performing EDTA anticoagulation;
2) extraction of genomic DNA: collecting a sample of venous blood from a nephrotic syndrome patient, extracting genomic DNA with DNeasy blood and tissue DNA extraction kit, and then testing DNA purity by calculating the ratio of absorbance at 260nm to absorbance at 280nm using Invitrogen Qbit spectrophotometer;
3) sequencing the FastTarget target region:
designing a high-quality sequencing primer aiming at a TRPC6 target region, selecting a primer which can obtain a clear single band by using a standard human genome as a template for amplification, mixing the optimized primers into a multiplex PCR primer panel according to the standard of each panel 20 pair primer, and performing amplification by using the standard human genome as the template;
based on a special method of capillary electrophoresis, the number of the finally obtained optimized panel is one twentieth of the number of the primers;
introducing a specific tag sequence compatible with an illumina platform into the tail end of the library through PCR amplification by using a primer with an Index sequence, and reducing the PCR tendency by adopting a PCR program with 11 cycles;
equally mixing all sample Index PCR amplification products, obtaining a final FastTarget sequencing library through tapping recovery, verifying fragment length distribution of the library by an Agilent 2100Bioanalyzer, and performing high-throughput sequencing on an Illumina Miseq platform in a 2 x 150bp/2 x 250bp double-end sequencing mode to obtain FastQ data after the molar concentration of the library is accurately quantified;
4) data screening and biological information analysis:
comparing the original data of each sample with a reference genome UCSC hg19 by using BWA software, correcting a primary comparison result obtained by the BWA software by using a GATK standard program, detecting SNV/InDel of each sample by respectively adopting two modes of VarScan software and a GATK HaplotpypeCaller method, filtering according to a screening scheme recommended by the software, comparing SNV/InDel found by the two detection schemes, merging all samples, comparing all SNV/InDel positions with a database by ANNOVAR for analysis, evaluating the variation frequency, functional characteristics, conservation and pathogenicity of the SNV/InDel positions, and quickly finding the SNV/InDel position with the most biological significance;
5) determination of serum TRPC6 levels: detecting the concentration of the target protein in the sample by adopting a double-antibody sandwich ELISA method, and operating according to the instruction of the kit;
6) statistical analysis:
searching a site related to a disease, performing statistical analysis by using SPSS 24.0 software according to genotype data and phenotype data of a mutation site of an existing sample by using a correlation analysis method, performing correlation analysis by using plink analysis software, and performing linkage disequilibrium analysis by using Haploview software;
statistical analysis was performed using mathematical tools such as logistic regression, chi-square, rank and test:
the TRPC6 genotype and the allele thereof in the diseased group and the healthy group are counted to calculate the frequency, and Hardy-Weinberg genetic equilibrium law test is carried out;
logistic regression was performed in units of single locus, according to 5 hypothetical genetic models: the method comprises the following steps that (1) an allole with low Dominant frequency is Dominant, an allole with low Recessed frequency is Recessive, a Log-additive superposition effect and an HOM/HET co-Dominant model are subjected to association analysis by taking homozygous normal as a reference; chi-square analysis was performed on each locus as well, and 4 analytical models, Codominant co-Dominant, domiant Dominant, Recessed Recessive, Allole alleles were set.
2. The method for detecting the pathogenic mutation of the TRPC6 gene by using the target gene sequencing as claimed in claim 1, wherein the method comprises the following steps: the secondary nephrotic syndrome in the step 1) refers to secondary nephrotic syndrome caused by hepatitis B, systemic lupus erythematosus, purpura and medicines.
3. The method for detecting the pathogenic mutation of the TRPC6 gene by using the target gene sequencing as claimed in claim 1, wherein the method comprises the following steps: DNeasy blood and tissue DNA extraction kit described in step 2) from Qiagen, Germany.
4. The method for detecting the pathogenic mutation of the TRPC6 gene by using the target gene sequencing as claimed in claim 1, wherein the method comprises the following steps: the database in the step 4) refers to dbSNP database, thousand human genomes, ESP6500, ExAC03, ExAC03_ EAS, gnomaD and Hrcr1Kaviar _20150923 database.
5. The method for detecting the pathogenic mutation of the TRPC6 gene by using the target gene sequencing as claimed in claim 1, wherein the method comprises the following steps: the genotype data and phenotype data in step 6) refer to case/control data, sex, age.
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