CN116042798A - TUBA4A gene for detecting female primary infertility and kit for detecting mutation of gene - Google Patents
TUBA4A gene for detecting female primary infertility and kit for detecting mutation of gene Download PDFInfo
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Abstract
The invention belongs to the technical field of gene detection, and particularly relates to a kit for detecting TUBA4A gene mutation of female primary infertility. The mutation of the human TUBA4A gene can lead to ovum maturation disorder and early embryo stagnation to cause female infertility. The mutation of TUBA4A gene can be used as a marker gene for judging female infertility caused by ovum maturation disorder and early embryo stagnation. The TUBA4A gene mutation provided by the invention can be used for evaluating or preparing a screening kit for female infertility caused by ovum maturation disorder and early embryo low quality. The TUBA4A gene provided by the invention is used for guiding whether a corresponding clinical patient is suitable for performing a tube infant operation or not.
Description
Technical Field
The invention belongs to the technical field of gene detection, and particularly relates to a TUBA4A 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, ZP-1, 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.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 (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 a number of 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 gene causing human ovum fertilization disorder WEE2 (Am J Hum Genet.2018 Apr 5;102 (4): 649-657), the pathogenic gene causing ovum death PANX1 (Sci Transl Med.2019 Mar 27;11 (485): eaav 8731), the pathogenic gene causing zygotic division failure BTG4 (Am J Hum Genet.2020 Jul 2;107 (1): 24-33) and the early embryo arrest pathogenic gene PADI6 (Am J Hum Genet.2016 Sep 1;99 (3): 744-752), FBXO43 (Hum red.1 Jul 19;36 (8): 92-2022), MOS (2022) and so on. However, these several genes can only explain the cause of some patients, and the cause of a considerable number of patients remains unknown.
According to the search of the prior art documents, the TUBA4A gene defect and the research article related to the neurological diseases are found, and no report related to female infertility is available. Our research shows that TUBA4A encodes an alpha-tubulin subtype, which is specifically and highly expressed during fertilization of mouse ova and early embryo development. The TUBA4A variation resulted in a severe decrease in the rate of first polar body discharge of the mouse ovum and a significantly shorter spindle length, indicating that α -tubulin TUBA4A may play an important role in the human ovum meiosis spindle assembly process.
Disclosure of Invention
The invention aims to provide a TUBA4A 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 TUBA4A gene for detecting female primary infertility has a nucleic acid sequence shown in SEQ ID NO. 1.
The invention relates to a method for screening primary infertility caused by ovum maturation disorder and early embryo stagnation, which is to judge whether a patient is primary infertility caused by ovum maturation disorder and early embryo stagnation by detecting whether TUBA4A gene is mutated or not. The TUBA4A gene mutation carried by the patient is shown as infertility, ovum maturation disorder or repeated embryo stop after ovum fertilization and failure of test tube infants.
Therefore, whether the TUBA4A gene is mutated or not can be used as a marker for judging female infertility caused by ovum maturation disorder and early embryo stagnation.
Specifically, the method for detecting whether or not the TUBA4A gene is mutated can be used to detect whether or not the TUBA4A gene is mutated by extracting DNA from peripheral blood of a patient and then combining the DNA sequencing by a PCR (polymerase chain reaction) method.
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 TUBA4A gene is mutated or not.
The TUBA4A gene has 4 exon coding regions, and the PCR amplification primer pair for detecting the 1 st exon coding region is as follows:
5’-CCTATAAGGGCGGTGCGG-3’(SEQ ID NO.2)
5’-ACTATACGGCTCGGCCAAAG-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’-GGTGGAAGAGAGACTCGCAA-3’(SEQ ID NO.4)
5’-TGCAGCTTCAAGTACGGCT-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’-AGTCCTTCCATGCATCTGGC-3’(SEQ ID NO.6)
5’-TCACCTGGCTCCATAAAGCG-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’-AAACTGTTCTTTCTCGTTCTTGCC-3’(SEQ ID NO.8)
5’-ATACCAGTGCACAAACGCC-3’(SEQ ID NO.9)
5’-GGCGTTTGTGCACTGGTAT-3’(SEQ ID NO.10)
5’-GGGAGATGACCTGACTTTTA-3’(SEQ ID NO.11)
the sequencing primer pairs are as above.
The invention also relates to a method for detecting whether the TUBA4A gene is mutated, which comprises the following steps:
for the coding region of exon 1, primer pairs were used: SEQ ID No.2, SEQ ID No.3, PCR amplification (amplification conditions: 92 ℃ C. For 2 minutes, 92 ℃ C. For 30 seconds, 57 ℃ C. For 1 minute, 72 ℃ C. For 3 seconds (35 cycles repeated in this three step), 72 ℃ C. For 10 minutes), then sequencing with the same primers, alignment with the TUBA4A standard sequence in UCSC (SEQ ID No. 1), and thus mutation was found;
similar to the above conditions, the coding region of exons 2-4 of TUBA4A was amplified with the corresponding primer (SEQ ID NO. 4-11), then sequenced using the same primer and aligned with the standard sequence of TUBA4A in UCSC (SEQ ID NO. 1), thereby finding the mutation. The TUBA4A gene was tested for mutation by PCR and first generation sequencing, and alignment with the standard sequence.
The invention also provides a kit for detecting whether the TUBA4A 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.11 for amplifying the coding region of the 1-4 exon of TUBA4A 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 ovum maturation disorder and screening primary infertility caused by early embryo stagnation.
The invention also provides a gene repair target site (namely, the mutation sites of a patient which are actually detected, such as 14 mutation sites listed in table 1, and a new mutation site possibly exists in the actual detection process through comparison with a standard sequence) for researching drug treatment on the primary infertility caused by ovum maturation disorder and early embryo stagnation, and the TUBA4A can perform normal functions through repairing the target site, so that the disease can be treated. Namely, the invention can also provide a medicament for researching the target site of repairing gene mutation, namely a medicament for treating the ovum maturation disorder and primary infertility caused by early embryo stagnation.
The invention can also guide patients with the mutation of the TUBA4A gene to judge whether the patients are suitable for test tube babies or not by detecting the mutation of the TUBA4A gene. In conventional clinics, unexplained infertility couples require test tube infants to try to reproduce offspring. However, if the patient finds a mutation with TUBA4A by examination, the patient will fail if tube babies are tried. Because the phenotype of the patient with this mutation is egg maturation disorder or early embryo arrest, and thus it is difficult to obtain an effective embryo, the follow-up test tube infant procedure cannot be completed. Therefore, such patients are advised to donate eggs.
Drawings
FIG. 1 is a distribution display of patient mutations over TUBA4A primary structure.
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 are from Shanghai Jieya and sterile diagnosis and treatment centers, shanghai traffic university auxiliary ninth people's hospitals, shanxi province women and young health care hospital reproductive centers and the like of Shanghai compound denier university auxiliary red house hospitals. 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 mutation of TUBA4A Gene
The invention adopts PCR combined sequencing to search TUBA4A gene mutation. The principle is that the primer design (the primer sequence can be specifically given) and the amplification are carried out on 4 exon coding regions of TUBA4A genes, and the TUBA4A 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: TUBA4A gene mutation and primary infertility caused by ovum maturation disorder and early embryo arrest
Results: we gathered 14 cases of primary infertility patients caused by ovum maturation disorder and early embryo arrest due to TUBA4A gene mutation. The corresponding mutation site information is shown in the following table (Table 1). The structural distribution of the mutation sites is shown in FIG. 1.
TABLE 1 TUBA4A Gene mutation information for patients
In summary, the invention has the following important practical significance:
(1) The TUBA4A gene provided by the invention can be used as a marker gene for predicting female infertility caused by ovum maturation disorder and early embryo stagnation;
(2) The TUBA4A gene provided by the invention can be used for evaluating or preparing a screening kit for female infertility caused by ovum maturation disorder and early embryo stagnation;
(3) The invention can be used for researching and preparing medicines for repairing gene mutation target sites, namely medicines for treating ovum maturation disorder and primary infertility caused by early embryo stagnation;
(4) The TUBA4A gene provided by the invention can be used for guiding a patient with clinical mutation of the gene, judging the success possibility of a test tube infant and suggesting the subsequent egg donation.
Claims (6)
1. A marker gene for judging female infertility caused by ovum maturation disorder and early embryo stagnation is characterized by being TUBA4A gene, wherein the nucleic acid sequence of the TUBA4A gene is shown as SEQ ID NO.1, and the judgment basis is whether the TUBA4A gene is mutated or not.
2. A method for screening the primary infertility caused by ovum maturation disorder and early embryo stagnation is characterized in that whether a patient is the primary infertility caused by ovum maturation disorder and early embryo stagnation is judged by detecting whether the TUBA4A gene is mutated.
3. The method of claim 2, wherein the information for the mutation of the TUBA4A gene is as follows:
4. a primer for detecting whether mutation is generated in the TUBA4A gene, characterized in that, for the 4 exon coding regions of the TUBA4A gene, the 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.
5. A method for detecting whether a mutation occurs in the TUBA4A gene, comprising the steps of:
the 4 exon coding regions in TUBA4A are respectively amplified by PCR by using corresponding primer pairs SEQ ID NO.2, SEQ ID NO. 3-SEQ ID NO.10 and SEQ ID NO.11, and then sequenced by using the same primers, and the sequences are the same as the standard sequences of TUBA4A in UCSC: the SEQ ID NO.1 was aligned so that mutations were found.
6. A kit for detecting the occurrence of a mutation in the TUBA4A gene, the kit comprising amplification primers for amplifying the coding region of exons 1-4 of the TUBA4A gene and corresponding sequencing primers: SEQ ID NO.2-SEQ ID NO.11, and a reaction mixture comprising DNA polymerase, dNTPs and a buffer.
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