CN116536325B - Mutant of PKD1 gene and application thereof - Google Patents

Mutant of PKD1 gene and application thereof Download PDF

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CN116536325B
CN116536325B CN202310316642.1A CN202310316642A CN116536325B CN 116536325 B CN116536325 B CN 116536325B CN 202310316642 A CN202310316642 A CN 202310316642A CN 116536325 B CN116536325 B CN 116536325B
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nucleic acid
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kidney disease
polycystic kidney
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CN116536325A (en
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姜楠
郭佃强
刘晶
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Qingdao Women and Childrens Hospital
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Abstract

The invention relates to the technical field of genes, in particular to a PKD1 gene mutant and application thereof. The nucleic acid has a c.12284_12287dupTGTG mutation compared to the wild-type PKD1 gene of SEQ ID NO. 1. Use of a reagent for detecting a nucleic acid and/or polypeptide having a c.12284_12287dupTGTG mutation compared to a wild-type PKD1 gene having the sequence of SEQ ID No.1 in the preparation of a kit or device for screening or diagnosing Autosomal Dominant Polycystic Kidney Disease (ADPKD). The invention provides a new mutant gene for the existing gene field, and further researches and confirms the new application of the mutant gene in polycystic kidney disease.

Description

Mutant of PKD1 gene and application thereof
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a PKD1 gene mutant and application thereof.
Background
Polycystic kidney disease (polycystic kidney disease, PKD) is a type of hereditary kidney disease caused by a mutation in a gene, and is further classified into autosomal dominant polycystic kidney disease (autosomal dominant polycystic kidney disease, ADPKD) and autosomal recessive polycystic kidney disease (autosomal recessive polycystic kidney disease, ARPKD) by its genetic means. The main pathological features of the disease are progressive enlargement and increase of kidney cysts, damage to normal kidney structures, and finally lead to end stage renal disease (end stage renal disease, ESRD), which patients can only support life by dialysis or kidney transplantation. ADPKD is the most common monogenic hereditary nephropathy, with a incidence of up to 1/400-1/1000, and patients mostly develop in adulthood, showing that kidneys appear cysts of different sizes and grow progressively, compressing normal kidney tissue, and about half of patients progress to end stage renal disease (end stage renal disease, ESRD) by 60 years of age, relying only on dialysis or kidney transplantation to sustain life. The genetic profile of ADPKD is serial passage, independent of sex, with a risk of progeny re-occurrence of 50%. ADPKD is also commonly associated with various extra-renal manifestations, such as cysts of the liver, pancreas, seminal vesicles, and arachnoid, with a prevalence of intracranial aneurysms about five times higher than normal populations, with significant high morbidity and mortality. About 85% of ADPKD patients are caused by mutations carrying the PKD1 gene, and 15% are caused by mutations carrying the PKD2 gene.
The PKD1 gene (OMIM 601313) is located in the 16p13.3 region of chromosome, contains 46 exons in total, has a coding sequence length 12912 bp and is a polycystic protein 1 (PC1) consisting of 4303 amino acid residues and mainly located in primary cilium, cell membrane tight junctions, desmosomes and focal adhesion sites of cells. The PKD2 gene (OMIM 173910) is located in the 4q22.1 region of chromosome and contains 15 exons, the coding sequence is 2907bp long, and the product is polycystic protein 2 (PC2) consisting of 968 amino acid residues, mainly located in primary cilia, centrosome and endoplasmic reticulum and is also a non-selective calcium ion transport channel. Mutations in the PKD1 gene and PKD2 gene can lead to abnormalities in downstream series of cellular signaling pathways, including intracellular calcium disorders, abnormal activation of cAMP pathways and Wnt signaling pathways, abnormal proliferation and apoptosis of cells, abnormal regulation of cell cycle and cellular energy metabolism, effects of immune cells and inflammatory mediators, and abnormal epigenetic regulation. Up to now, the PKDB database has more than 2000 PKD1 gene mutations, mainly point mutations, and the deletion, repetition or rearrangement of large fragments accounts for only a very small number (3% -4%) of causative agents.
The current research on PKD1 genes is in need of a deep search for mutations in PKD1 genes that lead to polycystic kidney disease, and accordingly, corresponding diagnostic and therapeutic protocols can be developed. For example, the influence of PKD1 gene on polycystic kidney disease has been further studied, and prevention and treatment of polycystic kidney disease have been further studied from the gene level.
Disclosure of Invention
Aiming at the problems, the invention provides a mutant of PKD1 gene and application thereof, which mainly aims to fill up part of the blank of PKD1 gene research, and further research on the causative agent of polycystic kidney disease and explore the cause and the treatable scheme.
In order to solve the problems, the invention adopts the following technical scheme:
the first aspect of the invention relates toNucleic acids, including fragments of interest comprising,
the target fragment has at least one of a c.12284_12287dupTGTG mutation, a c.3136_3137delGA mutation, and a c.9758t > C mutation compared to the wild-type PKD1 gene having the sequence of SEQ ID No. 1.
On the basis of the foregoing, the type of the nucleic acid includes DNA, RNA or cDNA, and the type of the nucleic acid is not particularly limited, and should be regarded as a nucleic acid within the scope of the present invention as long as it has a specific mutation compared with the wild-type PKD1 gene.
A second aspect of the invention relates toA polypeptide having at least one of a p.w4096cfs.62 mutation, a p.d1046lfs.54 mutation, and a p.leu3253pro mutation compared to the protein encoded by the wild type PKD1 gene having the sequence of SEQ ID No. 2. The mutant PKD1 gene encodes a product having at least one of a p.W4096Cfs.times.62 mutation, a p.D1046Lfs.times.54 mutation, and a p.Leu3253Pro mutation, as compared with a wild-type protein (equivalent to a wild-type polypeptide, as explained later) (SEQ ID NO: 2).
The nucleic acid sequence of the wild PKD1 gene shown in SEQ ID NO:1 and the amino acid sequence of the protein encoded by the wild PKD1 gene shown in SEQ ID NO:2 can be all queried through a common website for querying NCBI and other gene information (https:// www.ncbi.nlm.nih.gov/CCDS/cccdsbrowse/cgiREQUEST=CCDS & DATA=CCDS 32369.1); wherein, the wild type PKD1 gene nucleic acid with the sequence of SEQ ID NO.1 has 12912 bases (NM_ 001009944.3), and the polypeptide coded by the wild type PKD1 gene with the sequence of SEQ ID NO. 2 has 4303 amino acids (NP_ 001009944.3). However, the wild type nucleic acid and amino acid sequence are not limited to the examples listed, and the sites where the mutations are located and the mutation conditions are used as the basis for the same or equivalent judgment scheme.
A third aspect of the invention relates toUse of a biological model for screening a drug, wherein the biological model carries at least one of the following:
a. nucleic acid having at least one of a c.12284_12287dupTGTG mutation, a c.3136_3137delGA mutation, a c.9758t > C mutation compared to the wild type PKD1 gene having the sequence of SEQ ID No. 1;
b. a polypeptide having at least one of a p.w4096cfs.62 mutation, a p.d1046lfs.54 mutation, and a p.leu3253pro mutation compared to the protein encoded by the wild type PKD1 gene having the sequence of SEQ ID No. 2.
One form of the biological model is a cellular model. One application mode of the cell model is to perform large-scale drug screening, the cell model can be acted by the drug to verify whether the drug has the effect of inhibiting corresponding mutation, and further verify whether the drug can treat corresponding diseases through inhibiting mutation according to the effect, for example, the cell model can be used for simulating the disease environment of polycystic kidney disease, and then the model can be used for further verifying the effect of part of drugs in vitro. In the present strip, the screening factors mainly considered are specific to the corresponding mutation, the specific treatment object of the drug is not limited in particular, and the application mode is mainly used in the drug research and development process. Wherein the medicine is a medicine for treating polycystic kidney disease; polycystic kidney disease is mainly caused by the aforementioned mutations. More specifically, the medicine is a medicine for preventing and treating autosomal dominant polycystic kidney disease. Therefore, the biological model can be used for screening substances with unknown action effects, and further research on whether the substances have the expectations for treating polycystic kidney disease is facilitated.
A fourth aspect of the invention relates toUse of a reagent for detecting a nucleic acid and/or polypeptide in the preparation of a kit or apparatus for screening or diagnosing polycystic kidney disease; wherein,,
the nucleic acid has at least one (any one or a combination of more) of a c.12284_12287dupTGTG mutation, a c.3136_3137delGA mutation, a c.9758t > C mutation compared to the wild-type PKD1 gene; the polypeptide has at least one (any one or a combination of more) of p.W4096Cfs.times.62 mutation, p.D1046Lfs.times.54 mutation and p.Leu3253Pro mutation compared with the protein encoded by the wild type PKD1 gene. Screening polycystic kidney disease is mainly disease risk screening, and is convenient for early intervention; the diagnosis is to make an auxiliary diagnosis for the people who have the illness, and certainly whether the illness is caused is based on the actual change of the organism of the patient and the corresponding standard specification, and not based on subjective cognition of the person.
The reagents include at least one of antibodies, probes, primers, and mass spectrometry detection reagents specific for the nucleic acid having a mutation or the polypeptide having a mutation. The reagent comprises a product for specifically detecting nucleic acid and a product for specifically detecting polypeptide, wherein the product can be at least any one of an antibody, a probe, a primer and a mass spectrum detection reagent, and can also be other reagents with similar functions. Alternatively, the kit may be in the form of a kit similar to existing products, and the apparatus may be a sequence detection apparatus. The primer and the probe may be selected from any one, or may be used in combination as required.
More specifically, the primer and probe include (adaptively selected according to the detection object)
Primer pair 1: f: CATCCTGGTAGGTGACTGCG, R: TGAGTCGGTCAAACTGGGTG the number of the individual pieces of the plastic,
primer pair 2: f: GCGGTCTTCAAGCTCTCAGT, R: CCAACAGACAGGGAAACCGA the number of the individual pieces of the plastic,
primer pair 3: f: GTTCCATGTTCCCACTCCGT, R: GGCTCCATTCCCAGTACTCC;
the probe 1 sequence is: TGCGGCTGTGGGGCGCCCTAC the number of the individual pieces of the plastic,
the probe 2 sequence is: GTGCTGGTGGACTCGGCCGTG the number of the individual pieces of the plastic,
the probe 3 sequence is: GTGGCTGAGCTGCAGCGTGGC.
Preferably, the polycystic kidney disease is autosomal dominant polycystic kidney disease. The prepared corresponding detection products can be used for screening and diagnosing polycystic kidney disease, can be used for screening people susceptible to polycystic kidney disease more accurately, can be used for diagnosing polycystic kidney disease patients, have higher detection precision and accuracy, and have better effects in the corresponding detection process.
A fifth aspect of the invention relates toA kit for screening a biological sample for polycystic kidney disease comprising a reagent capable of detecting a mutant PKD1 gene;
the PKD1 gene mutant has at least one of a c.12284_12287dupTGTG mutation, a c.3136_3137delGA mutation, a c.9758T > C mutation compared to a wild-type PKD1 gene having the sequence SEQ ID NO.1, or at least one of a p.W4096Cfs 62 mutation, a p.D1046Lfs 54 mutation, a p.Leu3253Pro mutation compared to a protein encoded by a wild-type PKD1 gene having the sequence SEQ ID NO. 2. In this case, the technology of the present invention should be regarded as being applied whenever the corresponding product is applied to an agent capable of detecting either one of the aforementioned two PKD1 gene mutants.
The reagent is a nucleic acid probe or primer. The primer and probe include (adaptively selected according to the detection object)
Primer pair 1: f: CATCCTGGTAGGTGACTGCG, R: TGAGTCGGTCAAACTGGGTG the number of the individual pieces of the plastic,
primer pair 2: f: GCGGTCTTCAAGCTCTCAGT, R: CCAACAGACAGGGAAACCGA the number of the individual pieces of the plastic,
primer pair 3: f: GTTCCATGTTCCCACTCCGT, R: GGCTCCATTCCCAGTACTCC;
the probe 1 sequence is: TGCGGCTGTGGGGCGCCCTAC the number of the individual pieces of the plastic,
the probe 2 sequence is: GTGCTGGTGGACTCGGCCGTG the number of the individual pieces of the plastic,
the probe 3 sequence is: GTGGCTGAGCTGCAGCGTGGC.
Preferably, the polycystic kidney disease biological sample is an autosomal dominant polycystic kidney disease sample; the main sources of such samples are suspected patients, such as peripheral blood, skin, subcutaneous tissue, etc. of the patient. The corresponding probes and primers have higher detection precision and accuracy, and can be rapidly and noninvasively inspected. The kit can be used for detecting risk cases, and also can be used for carrying out large-scale risk investigation in certain scenes so as to screen out risk groups as early as possible.
A sixth aspect of the invention relates toA construct comprising a nucleic acid comprising a fragment of interest having a c.12284_12287dupTGTG mutation, a c.3136_3137delGA mutation, a c.9758t compared to the wild type PKD1 gene of SEQ ID No.1>At least one of the C mutations. The construct can also be used as a test model for the effect of a drug during the pharmaceutical process, for example by introducing the construct into a recipient cell, so that the recipient cell expresses a protein having the aforementioned mutation.
A seventh aspect of the invention relates toA recombinant cell obtained by transforming a recipient cell with the aforementioned construct. The recombinant cells according to the embodiments of the present invention can be used as a cell model for scientific research or commercial drug research, such as drug screening or pathogenesis research of PKD1 genes.
It should be noted that, the mutation sites and sequences given above are all referred to by the content of the Burrows Wheeler sequencing platform, and those skilled in the art will understand that, due to the update of the database or the difference of the database, the mutation sites and sequences may be slightly different or changed, and these differences or changes may be found by the standard given the content of the database, and these differences or changes are also included in the protection scope of the present invention.
The beneficial effects of the invention are as follows:
provides a new mutant gene for the existing gene field, and further researches the application of the mutant gene. The relation between the mutation related to the invention and the polycystic kidney disease is determined, and a new treatment scheme is provided for the treatment of the polycystic kidney disease according to the relation, and a means for diagnosing and treating the polycystic kidney disease is provided for in particular.
Drawings
FIGS. 1-3 are family maps of polycystic kidney disease patients 1-3, respectively;
FIGS. 4-6 are high throughput sequencing results of the pre-existing PKD1 gene c.12284_12287dupTGTG variation, c.3136_3137delGA variation, c.9758T > C variation in 1-3 families of polycystic kidney disease patients, respectively;
FIGS. 7 to 9 are graphs showing the results of Sanger sequencing of the mutation sites of the 1-3 family members PKD1 gene c.12284_12287dupTGTG, c.3136_3137delGA and c.9758T > C of polycystic kidney disease patients, respectively.
Description of the embodiments
The invention will now be further illustrated by means of specific examples in connection with the accompanying drawings.
Example 1: introduction and application cases of mutants
This example relates to an isolated nucleic acid encoding a mutant of the PKD1 gene having the c.12284-12287 dupTGTG, c.3136-3137 delGA, c.9758T > C mutation compared to SEQ ID NO. 1. The nucleic acid encoding the PKD1 gene mutant refers to a nucleic acid substance corresponding to the gene encoding the PKD1 gene mutant, that is, the type of the nucleic acid is not limited, and may be any polymer comprising deoxyribonucleotides and/or ribonucleotides corresponding to the gene encoding the PKD1 gene mutant, including but not limited to DNA, RNA or cDNA.
The nucleic acid actually comprises any one or two complementary double strands. For convenience, in the present solution, although only one strand is shown, the other strand complementary thereto is actually disclosed, and any coverage should be considered as being within the scope of the present invention. For example, reference to SEQ ID NO.1 actually includes its complement. One skilled in the art will also appreciate that one strand may be used to detect another strand and vice versa.
The nucleic acid for encoding PKD1 gene mutant is mutation on pathogenic genes of polycystic kidney disease determined by the inventor through a method of combining whole exome sequencing technology with mutation verification. Although there are reports of genes for polycystic kidney disease, the inventors have confirmed for the first time that the PKD1 gene has c.12284_12287dupTGTG, c.3136_3137delGA, c.9758T > C mutation sites associated with polycystic kidney disease, which have not been mentioned in the prior art. The cDNA sequence of the wild PKD1 gene is shown as SEQ ID NO.1, and contains 12912 bases in total. The protein coded by the wild PKD1 gene contains 4303 amino acids, and the amino acid sequence of the protein is shown as SEQ ID NO. 2.
The present embodiments also relate to a method of screening a biological sample susceptible to polycystic kidney disease. The method comprises the following steps:
s1, extracting a nucleic acid sample from a biological sample (the sample in the step can also be directly provided by a detection party).
The type of the biological sample is not particularly limited as long as a nucleic acid sample reflecting the presence or absence of mutation of the PKD1 gene of the biological sample can be extracted from the biological sample; the biological sample may be at least one selected from human blood, skin, subcutaneous tissue, preferably peripheral blood. Therefore, the sampling and detection can be conveniently carried out, so that the efficiency of screening biological samples prone to polycystic kidney disease can be further improved; the term "nucleic acid sample" as used in this section is to be understood in a broad sense, and may be any sample that reflects the presence or absence of mutation in the PKD1 gene in a biological sample, for example, whole genomic DNA directly extracted from a biological sample, a portion of the whole genome comprising the coding sequence of the PKD1 gene, total RNA extracted from a biological sample, or mRNA extracted from a biological sample. Thus, the source range of the biological sample can be enlarged, and various information of the biological sample can be determined at the same time, so that the efficiency of screening the biological sample which is liable to polycystic kidney disease can be improved. In addition, for using RNA as a nucleic acid sample, extracting the nucleic acid sample from the biological sample further comprises: extracting an RNA sample from the biological sample, preferably the RNA sample is mRNA; and obtaining a cDNA sample by reverse transcription reaction based on the obtained RNA sample, the obtained cDNA sample constituting a nucleic acid sample. Thus, the efficiency of screening a biological sample susceptible to polycystic kidney disease using RNA as a nucleic acid sample can be further improved.
S2, after obtaining a nucleic acid sample, analyzing the nucleic acid sample so as to determine the nucleic acid sequence of the obtained nucleic acid sample; the method and apparatus for determining the nucleic acid sequence of the obtained nucleic acid sample are not particularly limited;
the nucleic acid sequence of the nucleic acid sample may be determined by a sequencing method. The method and apparatus for sequencing are not particularly limited, and second generation sequencing techniques, as well as third generation and fourth generation or more advanced sequencing techniques may be employed; sequencing the nucleic acid sequence using at least one selected from the group consisting of seq2000, sol, 454, ABI3730XL and single molecule sequencing device; therefore, by combining the latest sequencing technology, higher 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 the sequencing devices can be utilized to further improve the efficiency of detecting and analyzing the nucleic acid sample, and the accuracy and the precision of the subsequent analysis of sequencing data can be improved; thus, determining the nucleic acid sequence of the nucleic acid sample may further comprise: first, a nucleic acid sequencing library is constructed for the obtained nucleic acid sample; and sequencing the obtained nucleic acid sequence library so as to obtain a data result consisting of a plurality of sequencing data; the term "nucleic acid sequence" used in this section should be construed broadly, and may be the complete nucleic acid sequence information obtained after assembling the sequencing data obtained by sequencing the nucleic acid sample, or may be the nucleic acid sequence obtained by directly using the sequencing data (reads) obtained by sequencing the nucleic acid sample, as long as the nucleic acid sequence contains the coding sequence corresponding to the PKD1 gene.
S3, after determining the nucleic acid sequence of the nucleic acid sample, comparing the nucleic acid sequence of the obtained nucleic acid sample with the sequence of SEQ ID NO.1, if the obtained nucleic acid sequence has the c.12284_12287dupTGTG mutation and the c.3136_3137delGA mutation, the biological sample is indicated to be susceptible to polycystic kidney disease (meanwhile, the method can also be judged that a kit for screening a biological sample of polycystic kidney disease, a reagent for detecting nucleic acid and/or polypeptide is also used in preparing the kit or the device).
Thus, by the method for screening a biological sample susceptible to polycystic kidney disease according to the embodiments of the present invention, a biological sample susceptible to polycystic kidney disease can be effectively screened; the method and apparatus for aligning the nucleic acid sequence with SEQ ID NO.1 is not particularly limited, and may be performed using any conventional software. Unless otherwise indicated, the technical means employed in the examples are conventional means familiar to those skilled in the art, and the reagents and products employed are also commercially available. The various processes and methods not described in detail are conventional methods well known in the art, the sources of the reagents used, the trade names and those necessary to list the constituents are all indicated at the first occurrence, and the same reagents used thereafter, unless otherwise indicated, are the same as those indicated at the first occurrence.
Example 2: determination of polycystic kidney disease pathogenic genes and mutation sites
Sample collection object:
the inventors collected 3 autosomal dominant polycystic kidney disease families, +.about.normal men, +.about.normal women, +.about.diseased women, ■ diseased men.
First person 1 and family: a forerunner 1 with polycystic kidney disease has the advantages that a plurality of anechoic areas are visible in double kidney parenchyma through ultrasonic examination, the left side of the forerunner is about 4.0x3.2cm, the right side of the forerunner is about 7.4x4.9cm, the wall of the forerunner is thin and smooth, the internal sound permeability of the forerunner is good, the father of the forerunner is obvious, the appearance is abnormal, the envelope is complete, the kidney parenchyma and kidneys Dou Huisheng disappear, the kidneys are full of liquid dark areas with different sizes, the liquid dark areas are honeycomb-shaped, part of liquid dark areas are mutually fused, the left side of the largest dark area is 6.3x3.4cm, the right side of the largest dark area is 5.5x4.2cm, the aggregate system is pressed and deformed, the double kidneys are subjected to multi-echo light clusters, the right side of the person is about 1.2x0.7cm, the left side of the person is about 1.5x1.1cm, the forerunner is waiting for kidney transplant surgery at present, and the forerunner is paler has similar symptoms. The forerunner family is shown in fig. 1;
forensic 2 and family: the forerunner 2 with polycystic kidney disease has the advantages that the size of double kidneys is normal, the structure is normal, a plurality of cystic anechoic areas are visible in the parenchyma of the double kidneys, the size is about 2.0x1.2cm, the wall is thin and smooth, the boundary is clear, sound transmission is feasible, the collecting system is not separated, and the mother of the forerunner has similar symptoms through the ultrasonic examination. The forerunner family is shown in fig. 2;
forensics 3 and family: the forerunner 3 with polycystic kidney disease has the advantages that the double kidneys are enlarged, the shape is full, the kidney cortex echoes are uneven, diffuse and multiple cystic fluid dark areas are seen in the forerunner, the right kidney is about 6.0x3.7cm, the left kidney is about 4.5x4.1cm, the separation between the pith and the skin is unclear, the central collecting system is not obviously dispersed, when the wife of the forerunner is pregnant for 26 weeks, the ultrasound prompts about 3.3x2.1cm for the right kidney of the middle abdominal fetus, the left kidney is about 3.4x1.8cm, the double kidney parenchyma echoes are slightly enhanced, the forerunner mother has no appearance of polycystic kidney disease (specific reason is not detailed), the father and the sister of the forerunner have Sanger sequencing, the long son and the postnatal girl, and the result shows that the forerunner carries c.9758T > C heterozygous variation. The forerunner family is shown in fig. 3;
the forensics and their families of the study signed informed consent.
Sample collection: 5ml of vein peripheral blood of a sample is taken, EDTA is added for anticoagulation, 2ml of the DNA is extracted by using Qiagen Blood DNA mini kit (Qiagen), and the concentration of Qubit (Qubit dsDNA HS Assay Kit, invitrogen) is measured and then stored at-20 ℃ for later use.
2. Whole exome sequencing: firstly, genome DNA is fragmented by adopting a Covaris ultrasonic breaker, and the end repair, A addition, joint addition and amplification operations are carried out on the broken product by utilizing a VAHTS Universal DNA Library Prep Kit for Illumina V (Vazyme) library construction kit, so as to complete the construction of a pre-library. Then, the pre-library is processed by KAPA HyperExome human whole exon sequence capture kit of Roche company, and the target enrichment target area is targeted by a probe (TGCGGCTGTGGGGCGCCCTAC and/or GTGCTGGTGGACTCGGCCGTG and/or GTGGCTGAGCTGCAGCGTGGC) hybridization capture method, so as to obtain a final library; carrying out concentration and fragment distribution analysis on the library by using a Qubit and QIAGEN QIAxcel Advanced full-automatic nucleic acid analysis system; and (5) quantifying the qualified library by using a quantification kit. Finally, the sequencing reaction was completed on a Huada DNBSEQ-T7 gene sequencer.
3. And (3) data processing: after sequencing, the sequences passing through the quality control are aligned to human genome reference sequences by BWA software; identifying mutation sites in a target sequence by adopting GATK software, annotating the mutation sites to a public mutation database by adopting Annovar annotation software, and predicting the influence degree of mutation on protein functions according to the frequency of the mutation sites in normal people, sequence conservation, amino acid change caused by mutation and the position of the mutation sites in a protein structure; and then combining the clinical phenotype of the sample, and carrying out pathogenicity interpretation on the mutation according to ACMG variation classification standards and guidelines. The method comprises the following steps: after converting the raw sequencing data into fastq files, reads were aligned to human reference genome GRCh 37/hg 19 using BWA software to generate bam files. And (3) carrying out local re-alignment on the generated bam file by adopting GATK series software, removing repeated sequences and carrying out mutation annotation. The low frequency mutation sites were screened using the mutation frequency database gnomAD,1000G,ExAC. And carrying out pathogenicity prediction analysis on the mutation by utilizing a plurality of protein function prediction software such as SIFT, polyphen, a mutation master, a recovery and the like. The candidate gene mutation sites of different species were conservatively analyzed using AlignX software (Invitrogen). The pathogenic mutation sites were evaluated in combination with various databases such as dbSNP, OMIM, HGMD, clinVar. The variability was analyzed for pathogenicity ratings according to the american society of medical genetics and genomics (American College of Medical Genetics and Genomics, ACMG) guidelines.
Sequencing the whole exome of precursor 1, showing that the 45 th exon of the PKD1 gene of the tested person has a heterozygous mutation c.12284_12287dupTGTG, which is a frame shift mutation and can lead to early termination of the synthesis of the polypeptide chain of the coded protein (p.W4096Cfs 62) (PVS 1); this variation is not reported in the normal population gene database (allele frequency (%): gnomeAD:; 1000 Genome:; exAC:) (pm2_pp); the subject clinically exhibited bilateral polycystic kidney (PP 4). According to the classification guidelines for variation of ACMG (The American College of Medical Genetics and Genomics, american society of medical genetics and genomics), the variation is a pathogenic variation (ACMG: PVS+2PP). The pathogenic mechanism may be: premature termination of the synthesis of the encoded protein polypeptide chain ultimately results in premature termination of translation of the PC1 protein, resulting in a truncated protein of only 4094 amino acids. The PC1 protein consists of an N-terminal extracellular region, a transmembrane region (comprising 11 transmembrane glycoproteins, which exert a signalling effect) and an intracellular region (several potential phosphorylation sites and a C-terminal helix-helix domain forming a complex with the PC2 protein) 3 part. Truncated proteins, due to their lack of a transmembrane region and a helix-helix domain at the C-terminal portion, presumably lead to abnormal signaling and intracellular localization of the PC1 protein, and are unable to bind to the PC2 protein to form complexes, thereby losing normal function, further affecting subsequent abnormal signaling, causing a series of changes in cell biological behavior, causing abnormal proliferation of renal epithelial cells, abnormal secretion, enlargement of cysts, and ultimately leading to the occurrence of polycystic kidney disease in the family.
Sequencing the whole exome of precursor 2, showing that there is a heterozygous variation c.3136_3137delGA in exon 13 of the subject PKD1 gene, which is a frame shift mutation, resulting in premature termination of encoded protein polypeptide chain synthesis (p.d1046lfs 54) (PVS 1); this variation is not reported in the normal population gene database (allele frequency (%): gnomeAD:; 1000 Genome:; exAC:) (pm2_pp); the clinical symptoms of the subject were in line with autosomal dominant hereditary polycystic kidney disease (PP 4). According to the classification guidelines for variation of ACMG (The American College of Medical Genetics and Genomics, american society of medical genetics and genomics), the variation is a pathogenic variation (ACMG: PVS+2PP). That is, the 1046 th amino acid is changed from aspartic acid to leucine, and the 54 th amino acid after the mutation position generates a new stop codon, which causes premature termination of PC1 protein translation, shortened protein length and reduced activity.
Sequencing analysis of the whole exome of precursor 3 shows that 1 heterozygous mutation c.9758T > C of the PKD1 gene of precursor is located in the 29 th exon, and the mutation is missense mutation, so that the 3253 th amino acid of the protein is changed from leucine to proline (p.Leu3253Pro); the variation is very low in frequency in the normal population gene database (allele frequency (%): gnomeAD:; 1000 Genome:; exAC:) (PM 2); a variety of statistical methods predict this variation as a pathogenic variation (Polyphen-2: deleterious, SIFT: deleterious, mutioTaster: deleterious) (PP 3). According to the classification guidelines for variation of ACMG (The American College of Medical Genetics and Genomics, american society of medical genetics and genomics), the variation is an ambiguous variation (ACMG: PM+PP).
Sanger sequencing validation: site verification was performed on the mutations found using Sanger sequencing. Taking 20ng of DNA (such as peripheral blood genome DNA), and carrying out PCR reaction by using specific primers of a site to be detected according to the operation flow of TaKaRa LA PCR ™ Kit Ver.2.1 (TaKaRa);
specific primer sequences:
primer pair 1: f: CATCCTGGTAGGTGACTGCG, R: TGAGTCGGTCAAACTGGGTG the number of the individual pieces of the plastic,
primer pair 2: f: GCGGTCTTCAAGCTCTCAGT, R: CCAACAGACAGGGAAACCGA the number of the individual pieces of the plastic,
primer pair 3: f: GTTCCATGTTCCCACTCCGT, R: GGCTCCATTCCCAGTACTCC.
The product was analyzed by agarose gel electrophoresis and recovered and purified using a Nucleospin cube Gel and PCR Clean-up (MACHEREY-NAGEL) cut gel. The recovered product was diluted to 10 ng/. Mu.L, and the Sequencing PCR reaction and purification were performed according to the BigDye Terminator v3.1cycle Sequencing Kit (Applied Biosystems) protocol. 10 mu L of Hi-Di (Applied Biosystems) is added to each well, denatured for 5min, taken out, placed on ice for cooling, transferred into a 96-well plate for on-machine use, and subjected to sequencing analysis on an ABI3730XL (Applied Biosystems) platform.
5. Conclusion: the heterozygous mutation of c.12284_12287dupTGTG and c.3136_3137delGA in the PKD1 gene was detected by using the whole exon sequencing technique and was causative of the ADPKD family, while a heterozygous mutation c.9758t > C of the PKD1 gene was also found in another ADPKD patient. The research results broaden the gene spectrum of ADPKD, strengthen the knowledge of clinicians on the disease, provide experience for screening and diagnosing clinical ADPKD, and provide basis for prenatal diagnosis. Therefore, the genetic research on ADPKD is purposefully carried out, the pathogenic genes and the pathogenic mechanism thereof are defined, the genetic consultation and the individual control of ADPKD patients are of potential clinical significance, the research direction and the new theoretical basis are provided for the early diagnosis and the effective treatment of the ADPKD, and a new molecular target is provided for the research and the development of specific medicaments for treating the ADPKD in practice.
It will be apparent to those skilled in the art that various modifications to the above embodiments may be made without departing from the general spirit and concepts of the invention. Which fall within the scope of the present invention. The protection scheme of the invention is subject to the appended claims.

Claims (3)

1. The use of a reagent for detecting a nucleic acid and/or polypeptide in the preparation of a kit for screening or diagnosing autosomal dominant polycystic kidney disease; wherein:
the nucleic acid has a c.12284_12287dupTGTG mutation compared to the wild-type PKD1 gene having the sequence of SEQ ID No. 1;
the polypeptide has p.W4096Cfs 62 mutation compared with the polypeptide coded by the wild PKD1 gene and having the sequence of SEQ ID NO. 2.
2. The use according to claim 1, wherein said reagent comprises at least one of a probe, a primer specific for at least one of said nucleic acid and said polypeptide.
3. The use according to claim 2, wherein the primers and probes are respectively:
primer pair 1: f: CATCCTGGTAGGTGACTGCG, R: TGAGTCGGTCAAACTGGGTG the number of the individual pieces of the plastic,
the probe 1 sequence is: TGCGGCTGTGGGGCGCCCTAC.
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