CN117004703A - KPNA7 gene for detecting female primary infertility and kit for detecting mutation of KPNA7 gene - Google Patents

KPNA7 gene for detecting female primary infertility and kit for detecting mutation of KPNA7 gene Download PDF

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CN117004703A
CN117004703A CN202210453449.8A CN202210453449A CN117004703A CN 117004703 A CN117004703 A CN 117004703A CN 202210453449 A CN202210453449 A CN 202210453449A CN 117004703 A CN117004703 A CN 117004703A
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gene
kpna7
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mutation
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王磊
桑庆
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Fudan University
Zhuhai Fudan Innovation Research Institute
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Zhuhai Fudan Innovation Research Institute
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    • C12Q2600/156—Polymorphic or mutational markers

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Abstract

The invention belongs to the technical field of gene detection, and particularly relates to a KPNA7 gene for detecting female primary infertility and a kit for detecting mutation of the gene. The mutation of the human KPNA7 gene can cause female infertility caused by repeated embryo sterility after ovum fertilization. The mutation of the KPNA7 gene can be detected to be used as a marker gene for judging female infertility caused by repeated embryo sterility after ovum fertilization. The KPNA7 gene mutation provided by the invention can be used for evaluating or preparing a screening kit for female infertility caused by repeated embryo sterility after ovum fertilization. The KPNA7 gene provided by the invention is mutated or not, and can be used for guiding a corresponding clinical patient whether to perform a test tube infant operation or not.

Description

KPNA7 gene for detecting female primary infertility and kit for detecting mutation of KPNA7 gene
Technical Field
The invention belongs to the technical field of gene detection, and particularly relates to a KPNA7 gene for detecting female primary infertility and a kit for detecting mutation of the gene.
Background
Normal pregnancy and reproduction are important links in maintaining and continuing the human population. For female infertility, ZP1, stag3, FSHR and other genes have been found to be closely related to female infertility (Huang HL et al, mutant ZP1 in family of characteristics N Engl J Med.2014 370 (13): 1220-6;de Roux N et al, A family with hypogonadotropic hypogonadism and mutations in the gonadotropin-releasing hormone receptor. N Engl J Med. 1997 337 (22): 1597-602;Caburet S et al, mutant cohesion in premature ovarian failure. N Engl J Med.2014 370 (10): 943-9). However, none are clinically applicable.
There are many causes of female infertility, and there are several clinical reports concerning descriptions of female infertility characterized by ovum immaturity (Rudak et al, fertility and sterility, 1990:292-296;Human Reproduction,1995 10:2343-2349; fertility and sterility 1999 71:567-570;Human Reproduction 2001 16 (10): 2136-2138;Human Reproduction 2002 17 (6): 1604-1609;Human Reproduction 2002 17 (10): 2556-2559). These patients failed to complete a successful in vitro insemination procedure by repeated insemination-embryo transfer (test tube infants) due to failure to obtain mature egg cells. If the related pathogenic genes can be found, the method has important significance for clinical diagnosis and typing of diseases. Recently we have found that the pathogenic genes TUBB8 (N Engl J Med. 2016 Jan 21;374 (3): 223-32), PATL2 (Am J Hum Genet. 2017 Oct 5;101 (4): 609-615), the pathogenic genes WEE2 (Am J Hum Genet. 2018 Apr 5;102 (4): 649-657), the pathogenic genes PANX1 (Sci Transl Med. 2019 Mar 27;11 (485): eaav 8731) which lead to ovum death, the pathogenic genes BTG4 (Am J Hum Genet. 2020 Jul 2;107 (1): 24-33) and the early embryo arrest pathogenic genes PADI6 (Am J Hum Genet. 2016-752), FBXO43 (Hum red. 2021 Jul 19;36 (8): 2392-2022), the pathogenic genes Bm Rem 2; 37) which lead to zygotic failure, and early embryo arrest pathogenic genes PADI6 (Am J Hum Genet. 2016-752) are responsible for several reasons, but the reasons are still unknown to some of the patient.
Through the search of the prior art documents, only a few studies have been found around the function of the KPNA7 gene. These studies report that the gene expresses a nuclear transport protein, plays an important role in early embryo development in mice, and that homozygous knockout females have reduced fertility. However, no report has been made so far on the relationship between the KPNA7 gene mutation and human diseases, nor on the relationship with female infertility.
Disclosure of Invention
The invention aims to provide a KPNA7 gene for detecting female primary infertility and a kit for detecting mutation of the gene, which are convenient to operate and have definite effects.
The nucleic acid sequence of the KPNA7 gene for detecting female primary infertility is shown as SEQ ID NO. 1.
The invention relates to a method for screening primary infertility caused by repeated embryo sterility after ovum fertilization, which is to judge whether a patient is primary infertility caused by repeated embryo sterility after ovum fertilization by detecting whether KPNA7 gene is mutated. The patient carried KPNA7 gene mutation and showed infertility, showing that ovum was mature, but repeated embryo arrest and test tube infant failed after fertilization.
Therefore, whether the KPNA7 gene is mutated or not can be used as a marker for judging female infertility caused by repeated embryo sterility after ovum fertilization.
The method for detecting whether the KPNA7 gene is mutated or not specifically comprises extracting DNA from peripheral blood of a patient, and detecting whether the KPNA7 gene is mutated or not by combining PCR (polymerase chain reaction) method with DNA sequencing.
Wherein the sample to be detected is DNA, and the DNA sample is derived from peripheral blood of the population to be detected.
Alternatively, the sample to be tested is an RNA, protein, cell or serum sample from the peripheral blood of the population to be tested.
The invention relates to a primer for detecting whether KPNA7 gene is mutated or not.
The KPNA7 gene has 10 exon coding regions, and the PCR amplification primer pair for detecting the 1 st exon coding region is as follows:
5’- GCTCCACTGTGAGCCCTTTA-3’（SEQ ID NO.2）
5’- TTCCACAGTTGTCTGGCTCC-3’（SEQ ID NO.3）
sequencing primer pairs are the same;
the PCR amplification primer pair for detecting the coding region of the No.2 exon is as follows:
5’- CCGAAAAGGCCTGGGTACAT-3’（SEQ ID NO.4）
5’- CCTCTCCATCCCCCACTAGA-3’（SEQ ID NO.5）
sequencing primer pairs are the same;
the PCR amplification primer pair for detecting the coding region of the No.3 exon is as follows:
5’- ACAGCATGCAAGATGGGACA-3’（SEQ ID NO.6）
5’- TGCCCTCAAGGACAGTGTAG-3’（SEQ ID NO.7）
sequencing primer pairs are the same;
the PCR amplification primer pair for detecting the coding region of the No.4 exon is as follows:
5’- CTTAAGTAGCTCCGGCCCAG-3’（SEQ ID NO.8）
5’- GTACCCTTGCCAGCACTTCT-3’（SEQ ID NO.9）
sequencing primer pairs are the same;
the PCR amplification primer pair for detecting the coding region of the No.5 exon is as follows:
5’- CATCAGGACTGTCCACCCAC-3’（SEQ ID NO.10）
5’- GGTTCTAAGAGGCCAGGCAG-3’（SEQ ID NO.11）
sequencing primer pairs are the same;
the PCR amplification primer pair for detecting the coding region of the No.6 exon is as follows:
5’- ACGAACCTCGTTCTGTGTCC-3’（SEQ ID NO.12）
5’- GCAGATGACAAACCTTGCCC-3’（SEQ ID NO.13）
sequencing primer pairs are the same;
the PCR amplification primer pair for detecting the coding region of the No.7 exon is as follows:
5’- CTGTCAGACAACCATCGGCT-3’（SEQ ID NO.14）
5’- TCTTCAGAATGGAACGCCCC-3’（SEQ ID NO.15）
sequencing primer pairs are the same;
the PCR amplification primer pair for detecting the coding region of the No.8 exon is as follows:
5’- GCTAGAGGCAGGTAGCCAAG-3’（SEQ ID NO.16）
5’- ACACATGGACAGAGGAGGGT-3’（SEQ ID NO.17）
sequencing primer pairs are the same;
the PCR amplification primer pair for detecting the coding region of the No.9 exon is as follows:
5’- CCTTCTGAGTCTTCCCTGTGT-3’（SEQ ID NO.18）
5’- TCCAGGCACCAAGATGTCAC-3’（SEQ ID NO.19）
sequencing primer pairs are the same;
the PCR amplification primer pair for detecting the coding region of the No.10 exon is as follows:
5’- ACTCATTGTCAGGACGGTGT-3’（SEQ ID NO.20）
5’- CCCTGCAGCACTAAGAACCA-3’（SEQ ID NO.21）
the sequencing primer pairs are as above.
The invention also relates to a method for detecting whether KPNA7 gene is mutated, which comprises the following steps:
for the coding region of exon 1, primer pairs were used: SEQ ID No.2 and SEQ ID No.3, PCR amplification (amplification conditions: 92℃for 2 minutes, 92℃for 30 seconds, 57℃for 1 minute, 72℃for 3 seconds (35 cycles repeated in this three step), 72℃for 10 minutes) was performed, and then sequencing was performed using the same primers, and aligned with the standard sequence (SEQ ID No. 1) of KPNA7 in UCSC, thereby finding a mutation;
similar to the above conditions, the coding region of exons 2 to 10 of KPNA7 was amplified by the corresponding primers (SEQ ID NO.4 to 21), sequenced, and aligned with the standard sequence of KPNA7 in UCSC (SEQ ID NO. 1), thereby finding the mutation. Whether the KPNA7 gene is mutated or not is detected by PCR and first generation sequencing and comparison with a standard sequence.
The invention also provides a kit for detecting whether the KPNA7 gene is mutated. The kit can guide doctors to judge the cause of the diseases and correctly classify the diseases, so as to inform patients of whether IVF or ICSI is adopted for the operation of the test tube infants, and whether the operation is suitable for continuous in vitro fertilization-embryo transfer (IVF) or single sperm injection (ICSI) (test tube infants).
The kit provided by the invention comprises the amplification primers SEQ ID NO.2-SEQ ID NO.21 for amplifying the 1-10 exon coding region of KPNA7 and a reaction mixed solution; the reaction mixture comprises DNA polymerase, dNTP and buffer solution;
the specific using method comprises the following steps: the primers were diluted with water to a working solution at a concentration of 10 uM. The reaction system consisted of 10ul of sample DNA,0.5ul of forward primer, 0.5ul of reverse primer, 5ul of reaction mixture and 3ul of water. After mixing, a PCR reaction was performed under the above conditions, followed by a generation of sequencing.
The kit can be used for detecting the screening of primary infertility caused by repeated embryo sterility after ovum fertilization.
The invention also provides a gene repair target site (namely, mutation sites of actually detected patients, such as that 9 patients listed in table 1 carry 5 mutation sites (partial sites are shared by a plurality of patients), and in the actual detection process, new mutation sites possibly exist through comparison with a standard sequence and the target site is repaired, so that KPNA7 can perform normal functions and further can treat the diseases.
The invention can also guide patients with clinical mutation of the KPNA7 gene to whether test tube babies are suitable or not by detecting the mutation of the KPNA7 gene. In conventional clinics, unexplained infertility couples require test tube infants to try to reproduce offspring. However, if a patient finds by examination a bi-allelic mutation carrying KPNA7, then the patient will suffer from a high risk of failed tube babies. Because, although the ovum can be mature in the patient with the mutation, the ovum can start cleavage after fertilization, but an effective embryo is difficult to obtain, so that the subsequent test-tube infant operation cannot be completed. Therefore, such patients are advised to donate eggs.
Drawings
FIG. 1 is a distribution display of patient mutations over KPNA7 primary structures.
Detailed Description
The invention will be further illustrated by the following examples. It should be understood that the following examples are illustrative of the present invention and are not intended to limit the scope of the present invention.
Example 1: sample collection and extraction of peripheral blood DNA
Primary infertility patients come from Shanghai collected love genetic and infertility diagnosis and treatment centers, shanghai ninth people hospital reproductive centers and the like of affiliated gynaecology and obstetrics hospitals of Shanghai double denier university. Diagnostic criteria are set forth by Rudak E et al (Rudak E., dorJ., kimchi M., goldman B., levran D and Mashiach S, anomalies of human oocytes from infertile women undergoing treatment by in vitro reference, fertillSteril 1990 Aug; 54 (2): 292-6). The semen inspection of the male is normal, the female reproductive organs, the ovary functions and the sex hormones of the female are normal, more than 5 ova are taken each time in more than 2 defecation promoting periods, most of the ova cannot be mature, or the mature ova are bad in fertilization or embryo stop is repeated. Taking part in the study of the subject on the premise of informed consent, collecting blood and signing the informed consent. All patients in the group rule out other reproductive endocrine diseases through medical history. The control population of the experiment is 1000 women with normal fertility function, 300ul of blood is sampled, DNA is extracted according to the instruction of the kit, and the concentration and purity of the DNA are detected by an ultraviolet spectrophotometer.
Example 2: detection of mutations in the KPNA7 Gene
The invention adopts PCR combined sequencing to search KPNA7 gene mutation. The principle is that the primer design (the primer sequence can be specifically given) and the amplification are carried out on 10 exon coding regions of the KPNA7 gene, and the KPNA7 gene mutation is searched. (i.e., mixing the DNA sample with reaction working solution (DNA polymerase, dNTP, water and buffer) and amplifying according to PCR procedure. The obtained product is purified and further sequenced by sequencing on ABI 3730. The analysis of the result is performed by HLA Fusion software (One lambda, CA, USA, HLA Fusion 3.0).
Example 3: KPNA7 gene mutation and primary infertility caused by repeated embryo arrest after ovum fertilization
Results: we gathered 9 cases of primary infertility patients caused by repeated embryo arrest after ovum fertilization due to KPNA7 gene mutation. The corresponding mutation site information is shown in the following table (Table 1, part of the sites are common to multiple patients). The structural distribution of the mutation sites is shown in FIG. 1.
TABLE 1 patient KPNA7 Gene mutation information
。
In summary, the invention has the following important practical significance:
(1) The KPNA7 gene provided by the invention can be used as a marker gene for predicting female infertility caused by repeated embryo sterility after ovum fertilization;
(2) The KPNA7 gene provided by the invention can be used for evaluating or preparing a screening kit for female infertility caused by repeated embryo sterility after ovum fertilization;
(3) By utilizing the invention, the medicine for repairing the gene mutation target site, namely the medicine for treating primary infertility caused by repeated embryo infertility after ovum fertilization can be researched and prepared;
(4) The KPNA7 gene provided by the invention can be used for guiding patients with clinical gene mutation, judging the success possibility of test tube infants and suggesting the subsequent egg donation.
Sequence listing
<110> university of double denier
Zhuhai Fudan Innovation Research Institute
<120> KPNA7 Gene for detecting female primary infertility and kit for detecting mutation of the Gene
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gtgtctctga ggcgacagca gaggatggcg gtcagtctgg agctccgaaa ggccaagaaa 120
gatgaacaga ccttaaagag aaggaatatc acgagcttct gccctgacac accttctgaa 180
aaaacagcca aaggggtggc ggtcagcctc actctgggtg aaataatcaa aggtgtgaat 240
agctcagatc cagtcctatg tttccaggcc acccagacag ccaggaaaat gctatcccag 300
gaaaagaacc cccctctgaa actggtcatt gaagcgggcc tcattcccag gatggtggag 360
ttcctgaagt catcacttta cccctgcttg cagtttgagg ctgcctgggc cctgaccaac 420
atcgcttcag ggacttcgga gcagactcgt gccgtggtag aagggggagc catccagccc 480
ttgattgagc tcctgtcttc ctccaacgtg gctgtgtgtg aacaggcagt gtgggctctt 540
ggtaatatag ccggtgatgg cccagagttc agagataacg tcatcacaag caatgccatc 600
ccacatctcc tagccttgat ttcacccacc ctgccgatca catttctgcg gaacatcacg 660
tggaccttgt cgaatctgtg ccgaaacaag aacccatacc cttgcgacac tgcggtgaag 720
cagatactgc cggccctcct tcacctcctg cagcaccagg acagtgaggt tctctcggat 780
gcctgctggg cactgtccta cctcaccgac ggctccaaca agcgcatcgg ccaagtggtt 840
aacacggggg tcctgcccag gctggtagtg ctcatgacca gctcagaact caatgtcttg 900
actccttctc tccgcaccgt ggggaacatt gtcacgggca cagatgagca gacgcagatg 960
gccattgatg cgggtatgct gaacgtgctc ccccagctcc tgcaacacaa caagccctcc 1020
atccagaagg aggcagcctg ggccctgagc aacgtagcag cggggccttg tcaccacatc 1080
cagcagctgc ttgcctacga cgtcttgcct cccttggtgg ctctgctaaa aaacggagaa 1140
tttaaagtcc agaaagaggc tgtctggatg gtggcgaact ttgcaacagg ggccaccatg 1200
gatcagctga tccagctcgt ccactctggg gtcctggagc cactggtgaa tctgctcact 1260
gccccagatg ttaaaattgt tctcatcatc cttgatgtca tctcttgcat cctccaggcg 1320
gcagagaaac ggtctgagaa ggaaaacctg tgtcttctga tagaagaact tggtgggatc 1380
gatagaattg aggctttaca gctgcatgag aaccgtcaaa ttggccagtc ggctttgaac 1440
atcatcgaga agcactttgg tgaggaagaa gatgagagcc aaactttact gagccaagtc 1500
atagaccaag attatgaatt tatagattat gaatgcttag caaaaaaata g 1551
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gctccactgt gagcccttta 20
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ttccacagtt gtctggctcc 20
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ccgaaaaggc ctgggtacat 20
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gtacccttgc cagcacttct 20
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gcagatgaca aaccttgccc 20
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ctgtcagaca accatcggct 20
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Claims (5)

1. The application of the KPNA7 gene as a marker for judging the primary infertility of females caused by repeated embryo sterility after ovum fertilization, wherein the nucleic acid sequence of the KPNA7 gene is shown as SEQ ID NO. 1.
2. A method for screening female primary infertility caused by repeated embryo sterility after ovum fertilization is characterized in that whether a patient is primary infertility caused by repeated embryo sterility after ovum fertilization is judged by detecting whether KPNA7 gene is mutated or not.
3. A primer for detecting whether mutation occurs in KPNA7 gene, characterized in that for 10 exon coding regions of KPNA7 gene, PCR amplification primer pairs for detection are in order: SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No.8, SEQ ID No.9, SEQ ID No.10, SEQ ID No.11, SEQ ID No.12, SEQ ID No.13, SEQ ID No.14, SEQ ID No.15, SEQ ID No.16, SEQ ID No.17, SEQ ID No.18, SEQ ID No.19, SEQ ID No.20, SEQ ID No.21.
4. A method for detecting whether a KPNA7 gene is mutated, comprising the following steps:
and carrying out PCR amplification on 10 exon coding regions in KPNA7 by using corresponding primer pairs SEQ ID NO.2, SEQ ID NO. 3-SEQ ID NO.20 and SEQ ID NO.21 respectively, and then sequencing by using the same primer, wherein the primer pairs are identical to the standard sequence of KPNA7 in UCSC: the SEQ ID NO.1 was aligned so that mutations were found.
5. A kit for detecting whether a KPNA7 gene is mutated, comprising amplification primers for amplifying the coding region of exons 1 to 10 of the KPNA7 gene and corresponding sequencing primers: SEQ ID NO.2-SEQ ID NO.21, and a reaction mixture comprising DNA polymerase, dNTPs and a buffer.
CN202210453449.8A 2022-04-27 2022-04-27 KPNA7 gene for detecting female primary infertility and kit for detecting mutation of KPNA7 gene Pending CN117004703A (en)

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