CN112226440B

CN112226440B - A kind of pathogenic mutation of hereditary primary infertility and its detection reagent

Info

Publication number: CN112226440B
Application number: CN202011207067.4A
Authority: CN
Inventors: 霍然; 凌秀凤; 曹琦琦; 赵纯; 王丛静; 张军强; 张笑兰
Original assignee: Nanjing Medical University
Current assignee: Nanjing Medical University
Priority date: 2020-11-03
Filing date: 2020-11-03
Publication date: 2021-07-09
Anticipated expiration: 2040-11-03
Also published as: CN112226440A

Abstract

The invention discloses a pathogenic mutation of hereditary primary infertility and a detection reagent thereof. A mutated NLRP5 gene causing hereditary primary infertility, said mutation being a homozygous frameshift mutation NLRP5 c.2945delT p.Leu982fs. Application of the reagent for detecting the mutated NLRP5 gene or the mutated NLRP5 protein of the present invention in the preparation of auxiliary diagnostic reagents or detection equipment for female primary infertility. The invention provides a new pathogenic site of an important maternal pathogenic gene NLRP5, and provides a new molecular biology basis for the diagnosis of the disease.

Description

Pathogenic mutation of hereditary primary infertility and detection reagent thereof

Technical Field

The invention belongs to the field of biological medicine, and relates to a pathogenic mutation of hereditary primary infertility and a detection reagent thereof.

Background

Infertility has become a common disease in modern society, and WHO data shows that infertility is now the third disease second to tumor and cardiovascular and cerebrovascular diseases. Data released by the national committee of birth show that the infertility rate of couples of childbearing age in China has risen to 12% -15% in recent years, wherein the number of female patients exceeds 2500 thousands, so that the exploration of female infertility causes is particularly urgent. Assisted reproduction technology is an infertility treatment means which is widely adopted at present, but a part of infertility cases still find no etiology and no effective treatment method clinically, and a considerable part of female patients continuously experience repeated ART cycle failure, and the common characteristic of the female patients is early embryonic development disorder. The genetic factors and mechanisms behind this clinical phenotype have not been known. Therefore, finding and identifying possible genetic factors is particularly important for clinical diagnosis and treatment.

The pathogenic mechanism of early embryo dysgenesis is mainly studied by means of animal models, and the pathogenic gene and genetic pattern of human are rarely reported. An important objective of the study of early embryonic developmental disorders is the molecular diagnosis of female primary infertility that results therefrom. In view of the fact that the related research of infertility caused by human embryo dysgenesis is lacked, the research method for finding brand-new pathogenic genes and sites of embryo dysgenesis is particularly important. The high-throughput second-generation sequencing technology can efficiently capture genetic information of a large number of genes, is the most effective method for searching pathogenic genes of complex genetic diseases and mutation thereof, and has the advantages that the traditional technology cannot reach.

The NLR family pyrin domain linking 5 gene is located at the 19 # chromosome long arm 19q13.43 position, the gene contains 15 exons, and the encoded NLRP5 protein is an oocyte specific expression protein containing 1200 amino acids. At present, researches report that NLRP5 is an important maternal protein and is important for the development of mice and human early embryos and the initiation of life individuals, but the pathogenic mutation site of the NLRP5 still needs to be further confirmed.

The primary infertility caused by heredity seriously harms the reproductive health of the people. The excavation of new pathogenic mutation is beneficial to further exploring the molecular genetic cause of hereditary primary infertility, is a practical need of fully utilizing reproductive genetic disease resources of China and benefiting patients with infertility, and is one of the most important research directions in the genome era.

This patent is directed to exploring the genetic etiology of primary infertility, thereby aiding understanding of pathogenesis, aiding clinical diagnosis, prenatal diagnosis, and transgenic therapy.

Disclosure of Invention

The present invention aims to provide a pathogenic mutation of genetic primary infertility against the above-mentioned disorders.

Another object of the present invention is to provide the use of such pathogenic mutations.

It is still another object of the present invention to provide a reagent for detecting the pathogenic mutation.

The purpose of the invention can be realized by the following technical scheme:

a mutant NLRP5 gene leading to hereditary primary infertility, said mutation being a homozygous frameshift mutation NLRP5c.2945delT p.Leu982fs; the gene number of the wild type NLRP5 gene in the Ensemble database is as follows: the mutation of the gene ENSG00000171487 and NLRP5 is in the 11 th exon at the physical position, the base T deletion of 56552446 causes frame shift mutation in the subsequent sequence, and the other parts are the same as the wild type.

The mutant NLRP5 protein coded by the mutant NLRP5 gene is characterized in that the gene transcript number of the wild NLRP5 protein in an Ensemble database is ENST00000390649.3, the mutant NLRP5 protein is subjected to frameshift mutation at amino acid No. 982, a stop codon is met after 2 new amino acids are translated, translation of the protein is stopped, the protein sequence is truncated, an exon originally coded by the LRR rich in leucine repeat sequence does not code the protein any more after mutation, and other parts of the exon are the same as the wild type.

The application of the mutant NLRP5 gene or the mutant NLRP5 protein as a detection target in preparing an auxiliary diagnostic reagent or detection equipment for female primary infertility.

Preferably, the auxiliary diagnostic reagent is selected from one or more of a primer or a primer pair, a probe, a nucleic acid chip or an antibody.

In a preferred embodiment of the present invention, the detection device is a detection platform of a gene chip containing the NLRP5 gene for detecting mutation.

The application of the reagent for detecting the mutant NLRP5 gene or the mutant NLRP5 protein in the invention in preparing an auxiliary diagnostic reagent or detection equipment for female primary infertility.

Preferably, the reagent is selected from one or more of a primer or a primer pair, a probe, a nucleic acid chip or an antibody.

A female primary infertility auxiliary diagnostic kit comprises a reagent for detecting the physical position chr1956552446 nucleotide site of NLRP5 gene; or a reagent for detecting the 982 th amino acid site of the NLRP5 protein.

Preferably, the reagent is one or more of a primer or primer pair, a probe, an antibody, or a nucleic acid chip.

Preferably, the reagent is a gene chip hybridization probe based on deep sequencing as a platform; the sequence of a hybridization probe for detecting the chr1956552446 bit nucleotide position is shown as SEQ ID N0.31.

Has the advantages that:

animal experiments show that NLRP5 maternal protein plays a crucial role in early embryo. Therefore, the research aiming at the NLRP5 gene and the female primary infertility and early embryo disorder is particularly necessary. The c.2945delT p.Leu982fs mutation of NLRP5 is selected as a candidate site of primary infertility, and is screened by the applicant on the basis of years of clinical medical and genetic researches.

A gene may correspond to multiple different transcripts, and the RNA and protein encoded by different transcripts may vary. In the patent, the applicant screens out the optimal transcript of the NLRP5 gene according to the experience of long-term genetic research, and designs corresponding probes according to different transcripts, so that the screening benefit reaches the highest, and finally obtains the pathogenic mutation NLRP5c.2945delT p.Leu982fs causing early embryonic development disorder.

The clinical manifestations of primary infertility are heterogeneous, and some pathogenic genes such as PADI6 and TLE6 are known at present, but many unknown pathogenic genes still exist. The invention provides a new pathogenic site of an important maternal pathogenic gene NLRP5, and provides a new molecular biological basis for the diagnosis of the disease.

Drawings

FIG. 1: family picture

FIG. 2: embryo photograph of early embryo development disorder patient and normal control

FIG. 3: NLRP5 mutant sequencing map

FIG. 4: inter-species conservation assay

FIG. 5: wild type and NLRP5 protein three-dimensional structure carrying mutation

FIG. 6: embryonic development map of mouse

FIG. 7: immunofluorescent staining of mouse embryos

FIG. 8: cell experiment co-immunoprecipitation results

Detailed Description

Example 1

The NLRP5 gene mutation of a primary female infertility family is detected.

The experimental method comprises the following steps:

1. the collection of the family clinical resources and the establishment of a genetic resource library:

the ancestor in the family is a patient (IV-2), and the female is diagnosed in a maternal-child health care institute of Nanjing city two years ago and is diagnosed with primary infertility; the fertility of sister and brother is normal; the parents have been married but the mothers have passed. (family diagram is shown in FIG. 1). The clinical data mainly includes personal medical history, family history, birth history, etc. Clinical data were collected from patients (IV-2) in this family and blood samples from the father and two fertile sisters. And blood genomic DNA of the patient (IV-4) and the father (III-1) and the sister (IV-4, IV-6) was extracted using a blood genomic DNA extraction kit (Qiagen, Hilden, Germany).

2. Pathogenic mutations in this family were explored by means of high-throughput next generation sequencing:

2.1 design and order of Capture chips:

2.1.1NLRP5 Gene and transcript sequence information:

the gene capturing chip is a full exome capturing chip and can detect all known genes at present, wherein the known genes comprise all known early embryo block related pathogenic genes which are researched at present. The gene number of the NLRP5 gene referred to by us in the Ensemble database is ENSG00000171487 (note: the number is from Ensemble database, www.ensembl.org, and the detailed information of the gene code and the gene sequence can be input).

2.1.2 selection of transcripts:

specific transcripts are selected aiming at different genes, each gene contains a plurality of transcripts, and when the transcripts are selected, the transcripts with NLRP5 encoding proteins are considered firstly, if one gene has a plurality of transcripts encoding proteins, the transcripts corresponding to the proteins with the most amino acids are selected firstly, and if the amino acids of the plurality of transcripts are the same, the transcripts with the most bases are further selected. Based on the above principle, the number of the transcript of the NLRP5 gene screened by the method is ENST00000390649.3 (note: the number is from Ensemble database, www.ensembl.org, and the transcript code can be input to search the detailed information and the sequence of the transcript).

2.1.2 design of hybridization probes:

the design standard of the hybridization probe is (1) the probe covers the target area of all candidate genes, namely the exon area and the splicing part of the exon and the intron (100 bp respectively at the upstream and the downstream of the exon); (2) and (3) removing repeated sequences, namely removing the high-frequency repeated sequences appearing in the genome and the repeated fragments with the frequency of 2-5 times lower than that of the human genome, so as to avoid capturing other homologous genes, increase false positives and reduce the detection efficiency. Applicants aligned the target regions of all candidate genes to human genomic DNA sequences, removing 2.5% of the repeat sequences altogether; (3) in the process of designing the probe, specific integration is carried out on adjacent exons, and the integration standard of the adjacent probes is that when the sum of the integrated target regions of the adjacent exons (namely the upstream 100bp of the former exon to the downstream 100bp of the latter exon) is less than 600bp, the adjacent exons are integrated into one probe so as to complete the capture of a plurality of pairs of exon regions by one pair of probes; (4) when the designed probe sequence is smaller than 250bp, on the basis that the two ends of the designed probe sequence respectively contain 100bp introns at the upstream and the downstream, the introns with the same bp number are continuously added, so that the size of the probe reaches 250 bp. According to the design principle, the probe sequence designed aiming at the NLRP5 gene is shown as SEQ ID N0.1-SEQ ID NO. 31.

2.2 target region capture and deep sequencing:

firstly, genome DNA is fragmented, an A is marked at the end of the DNA, the DNA is connected with an Illumina PE joint-oligonucleotide mixture, and a connection product is enriched through PCR to obtain a DNA library. Then the DNA library is hybridized with the known pathogenic gene capturing chip, eluted and purified to obtain the coding sequence. Finally, paired ends were created and the target sequence was sequenced on the HiSeqTM 2000(Illumina, Inc., San Diego, Calif., USA) platform.

2.3, performing bioinformatics analysis on the sequencing data, and screening out candidate pathogenic genes:

2.3.1 Mosaik software (http:// bioinformatics. bc. edu/marthlab/Mosaik) was used to process the raw sequencing data (paired end data) of Gollumina, resulting in a.bam type file. The bam file is input into a GATK, a single nucleotide variant (single nucleotide variant) and small insertions or deletions (insertions/deletions) are detected by using the GATK, quality evaluation is carried out simultaneously, downstream bioinformatics analysis is facilitated, and finally a vcf type file is generated.

2.3.2 Included the sequencing results of patients

dbSNP144(http:// hgdownload. cse. ucsc. edu/goldenPath/hgl9/database/snpl44.txt. gz.), the HapMa program (ftp:// ftp. ncbi. nlm. nih. gov/hapmap), 1000Genome Project (ftp:// ftp. l. 000genome. ebi. ac. uk/voll/ftp), the Yanhuang database ((http:// yh. Genome. org. cnj) and the Exome Variant Server (http:// EVS. gs. washing. edu/EVS /) are screened in five Single Nucleotide Polymorphism (SNP) databases, all known SNP sites are filtered;

2.3.3 comparing and analyzing the gene sequences corresponding to the sequencing results of the patients, and preferentially analyzing the insertion/deletion mutation, the nonsense mutation and the missense mutation, wherein the results can be divided into three types including the known mutation, the new mutation of the known gene and the mutation of the new gene.

2.4 through Sanger sequencing verification, the pathogenic gene is identified:

the PCR method is respectively used for amplifying the screened mutation sites and adjacent DNA sequences in corresponding families, and the used Primer sequences are designed by adopting Primer 3(http:// frodo. wi. mit. edu /) Primer design software. The PCR reaction system (20. mu.L system) used was 5. mu.L buffer, 25mM MgCl 22. mu.L, DNA L. mu.L, forward primer F1. mu.L, reverse primer R1. mu.L, 10mM dNTP 0.4. mu.L, Taq enzyme 0.1. mu.L, ddH2O 10.5.5. mu.L. PCR program of 98 deg.C for 5min,35 cycles (98 deg.C for 10s, 60 deg.C for 15s, 72 deg.C for 1min), 72 deg.C for 7min, 4 deg.C for 5 min. Detecting by 2% agarose gel electrophoresis, cutting PCR product gel under an ultraviolet gel cutting instrument, and purifying. All PCR products were sequenced with forward and reverse primers, respectively, and the sequencing results were further analyzed, using NCBI online comparison tool BLAST (http:// BLAST. NCBI. nlm. nih. gov /), to exclude false positive results, and to screen out mutation sites co-segregating in families. Wherein the primer sequence for detecting the chr 1956552373-56552493 nucleotide site is SEQ ID N0.31.

The experimental results are as follows:

1. family clinical data:

after the clinical specialist in reproductive medicine has performed detailed and comprehensive clinical examination on proband (IV-2) in the family, the clinical diagnosis of primary infertility is made for the patient, and IVF treatment is recommended, and the specific ovum obtaining condition and embryo culture condition are shown in Table 1 and FIG. 2.

TABLE 1 endocrine examination results and ovum acquisition situation of patients with early embryo developmental disorder (patient IV-2)

2. The genetic detection result of the family is as follows:

after whole exome sequencing and bioinformatics analysis of family members, we found that this patient carried the suspected homozygous deletion mutation NLRP5c.2945delt p.l982fs. The sequencing results are shown in FIG. 3. The homozygous deletion mutation NLRP5c.2945delT p.L982fs existing in the patient is positioned on chromosome 19, and the physical position is that 1 base T is deleted at chr 19-56552446; at the RNA level, 2945 th base of the NLRP5 gene coding RNA is deleted; protein level: the NLRP5 gene encodes protein with 982 th amino acid frame shift mutation and advanced stop codon, and the relevant deletion mutation of the gene is never found in primary infertility patients.

According to the screening process designed by us, by means of the gene chip and deep sequencing technology designed by us, we successfully prove that the detected NLRP5c.2945delT p.L982fs is a new pathogenic site of the disease.

Example 2:

biological analyses were carried out on the pathogenic mutation NLRP5c.2945delt p.l982fs detected in example 1.

The experimental results are as follows:

1. conservation analysis:

conservative evaluation and prediction are carried out on the screened NLRP5 p.L982fs mutation in a plurality of species of human, mice, orangutan, sheep, dogs and horses by using an NCBI homoGene database (http:// www.ncbi.nlm.nih.gov/HomoloGene), the site is found to be conserved in the evolutionary process, and the mutation of the site is laterally proved to possibly cause more serious pathological diseases, and the result is shown in figure 3.

2. Protein crystal structure change study:

the structures of NLRP5 wild-type protein and mutant protein carrying c.2945delT p.Leu982fs are respectively predicted by adopting SWISS MODEL (http:// swissmodel.expasy.org /) prediction software, and the change of the protein structure caused by mutation is evaluated. The prediction result of the protein crystal structure shows that c.2945delT p.L982fs mutation exposes a hydrophobic region of an LRR unit in which 982 is positioned, the folding of the protein is influenced or aggregation is caused, the LRR has the function of recognizing ligand, and the early termination mutation of leu982 can destroy the recognition of the ligand, and the result is shown in figure 4.

3. Mouse experimental results:

we have several aspects from the following: comparison NLRP5^WTAnd NLRP5^MUDissimilarity of mRNA injected group mouse embryos:

1) mouse embryo development rate: we performed NLRP5 on mouse zygotes^WTAnd NLRP5^MUIn vitro culture after mRNA injection compares embryo development rate, and NLRP5 is found^WTAnd NLRP5^MUThe mouse embryos of the mRNA injection group do not have obvious difference from the development stage to the 2 cell stage, but the subsequent 4 cell, morula and blastocyst stage NLRP5^MUThe mRNA injection group showed a significant decrease in the development rate, and many embryo blocks were not developing further at the 2-cell stage, which is similar to the results of the patient's embryo development as shown in FIG. 5.

2) Alteration of NLRP5 protein localization in mouse embryos: we compared NLRP5^WTAnd NLRP5^MUAfter mRNA injectionLocalization in mouse 2-cell embryos, NLRP5 was found^MUAfter mRNA injection, the mutated NLRP5 protein no longer specifically localized to the subcortical space, but appeared to be cytoplasmic in distribution, a result that is also consistent with the context of the structural prediction part, see figure 6.

4. Cell experiment results:

effect of mutations on binding of NLRP5 to other SCMC members:

we used MYC-NLRP5 separately^WTOr MYC-NLRP5^MU293T cell lines were transfected with V5-KHDC3L, FLAG-TLE6, and HA-OOEP high expression plasmids, and the mutations were found by co-immunoprecipitation experiments to attenuate the interaction of NLRP5 with SCMC components TLE6, OOEP, KHDC3L (FIG. 8). This result suggests that NLRP5 mutation may lead to SCMC formation failure.

In conclusion, we confirmed the specific damage of NLRP5c.2945delT p.L982fs mutation to embryonic development from multiple aspects through experimental conclusion of genetics and animal experiments, and clarified the association with primary infertility. The NLRP5 mutation site or the NLRP5 protein can be applied to preparation of a primary infertility detection reagent or detection equipment.

Sequence listing

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tatccaacaa cagcctgggg aacgaaggtg taaatctact gtgtcgatcc atgaggcttc 60

cccactgtag tctgcagagg ctgatgtgag tctggcttgc tcccctgcaa ggacttccta 120

<210> 32

<211> 120

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 32

tcacgatctt ctttcccatg tgtgtgcccc acaggctgaa tcagtgccac ctggacacgg 60

ctggctgtgg ttttcttgca cttgcgctta tgggtaactc atggctgacg cacctgagcc 120

<210> 33

<211> 120

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 33

ttagcatgaa ccctgtggaa gacaatggcg tgaagcttct gtgcgaggtc atgagagaac 60

catcttgtca tctccaggac ctggagtgag tttcccatgg gcgttgggtc aactctatca 120

<210> 34

<211> 120

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 34

gaggcagact ctctctattc cccgcctctt gcaggttggt aaagtgtcat ctcaccgccg 60

cgtgctgtga gagtctgtcc tgtgtgatct cgaggagcag acacctgaag agcctggatc 120

<210> 35

<211> 120

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 35

tcacggacaa tgccctgggt gacggtgggg ttgctgcgct gtgcgaggga ctgaagcaaa 60

agaacagtgt tctggcgaga ctcgggtaac ttcctggggc gcctctttgc gggccgggct 120

<210> 36

<211> 120

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 36

ttggggttat tttctgggtg tcctgaccct gcaggttgaa ggcatgtgga ctgacttctg 60

attgctgtga ggcactctcc ttggcccttt cctgcaaccg gcatctgacc agtctaaacc 120

<210> 37

<211> 120

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 37

tggtgcagaa taacttcagt cccaaaggaa tgatgaagct gtgttcggcc tttgcctgtc 60

ccacgtctaa cttacagata attgggtaag tcgccagcaa ttgtcttctg agatacagac 120

<210> 38

<211> 120

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 38

ggagaagagt cgaaagcaac atctcagtaa cgagtcctct ctctgcctcc ccaggctgtg 60

gaaatggcag taccctgtgc aaataaggaa gctgctggag gaagtgcagc tactcaagcc 120

<210> 39

<211> 120

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 39

ccgagtcgta attgacggta gttggcattc ttttgatgaa gatgaccggt actggtggaa 60

aaactgaaga tacggaaacc tgccccactc acacccatct gatggaggaa ctttaaacgc 120

Claims

1. a mutation NLRP5 gene that causes early embryonic developmental disorder is characterized in that described mutation is a homozygous frameshift mutation NLRP5 c.2945delT p.Leu982fs; the gene numbering of wild-type NLRP5 gene in the Ensemble database is: ENSG00000171487, the physical position of the NLRP5 gene mutation is in exon 11, which is the 56552446-base T deletion, and the subsequent sequence causes a frameshift mutation, and other parts are the same as the wild type.

2. The mutated NLRP5 protein encoded by the mutated NLRP5 gene of claim 1, characterized in that the gene transcript number of the wild-type NLRP5 protein in the Ensemble database is ENST00000390649.3, and the mutated NLRP5 protein is at the 982nd amino acid shift. Code mutation, the stop codon is encountered after the translation of 2 new amino acids, the protein translation is terminated, the protein sequence is truncated, and the exon that originally encoded the leucine-rich repeat sequence after the mutation no longer encodes the protein, and the other parts are the same as Same as wild type.

3. Use of the mutated NLRP5 gene of claim 1 or the mutated NLRP5 protein of claim 2 as a detection target in the preparation of an auxiliary diagnostic reagent or detection device for female primary infertility.

4. The application according to claim 3, wherein the auxiliary diagnostic reagent is selected from one or more of primers or primer pairs, probes, nucleic acid chips or antibodies.

5 . The application according to claim 3 , wherein the detection device is a detection platform containing a gene chip for detecting the mutated NLRP5 gene. 6 .

6. Application of the reagent for detecting the mutated NLRP5 gene of claim 1 or the mutated NLRP5 protein of claim 2 in the preparation of auxiliary diagnostic reagents or detection equipment for early female embryonic development disorders.

7. The application according to claim 6, wherein the reagent is selected from one or more of primers or primer pairs, probes, nucleic acid chips or antibodies.

8. An auxiliary diagnostic test kit for early embryonic developmental disorders in women, the test kit comprising: the reagent for detecting the NLRP5 gene physical position chr19 56552446 nucleotide site; or the reagent for detecting the 982nd amino acid site of the NLRP5 protein.

9. The kit according to claim 8, wherein the reagent is selected from one or more of primers or primer pairs, probes, antibodies, or nucleic acid chips.

10. test kit according to claim 8, is characterized in that described reagent is the gene chip hybridization probe that is platform based on deep sequencing; The hybridization probe sequence of detecting chr19 56552446 nucleotide site is such as SEQ ID NO. .31 shown.