CN115927584A - TACC3 gene for detecting female primary infertility and kit for detecting mutation of gene - Google Patents

Abstract

The invention belongs to the technical field of gene detection, and particularly relates to a kit for detecting the TACC3 gene mutation of female primary infertility. The mutation of the human TACC3 gene can cause the ovum maturation disorder to cause female infertility. The mutation of the TACC3 gene can be used as a marker gene for judging female infertility caused by ovum maturation disorder. The TACC3 gene mutation provided by the invention can be used for evaluating or preparing a screening kit for female infertility caused by ovum maturation disorder. Whether the TACC3 gene provided by the invention is mutated or not can be used for guiding whether a corresponding patient is suitable for tube infancy in clinic.

Description

TACC3 gene for detecting female primary infertility and kit for detecting mutation of gene

Technical Field

The invention belongs to the technical field of gene detection, and particularly relates to a TACC3 gene for detecting female primary infertility and a kit for detecting gene mutation.

Background

Normal pregnancy and reproduction are important links in maintaining and extending human populations. For female infertility, genes such as ZP-1, stag3, FSHR, etc. have been found to be closely related to female infertility (Huang HL et al, mutant ZP1 in facial infertility. N Engl J Med.2014 370 (13): 1220-6 de Roux N et al, A family with hypo-gonadotropin hypo and mutations in the gonadolropin-refining hormon receptor. N Engl J Med.1997 (337) (1597-602 Caburet S et al, mutant maize in prediction genetic improvement. N Engl J. Med.2014.370 (10): 943-9). However, none have been clinically applied.

There are many reasons for female infertility, and there are several clinical reports relating to the description of female infertility characterized by egg immaturity (Rudak et al, fertility and fertility, 1990 54. These patients, by repeated insemination-embryo transfer (tube transfer), fail to achieve successful in vitro fertilization procedures due to the failure to obtain mature egg cells. If relevant 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 responsible for the barrier to maturation of human ova TUBB8 (N Engl J Med.2016Jan 21 (3): 223-32), PATL2 (Am J Hum Genet.2017Oct 5 (4): 609-615), the pathogenic genes responsible for the barrier to fertilization of human ova WEE2 (Am J Hum Genet.2018Apr 5 (4): 649-657), the pathogenic genes responsible for the death of ova PANX1 (Sci Transl Med.2019Mar 27 (485): eaav 8731), the pathogenic genes responsible for the failure of zygote division BTG4 (Am J Hum Genet.2020Jul 2 (1): 24-33) and the pathogenic genes responsible for early stage of birth PADI6 (Am J Hum Genet.2016Sep.744 (3-744), FB43 (Hujrom: 2021Jhum 19) 2022 (2022-2408), rep 3, et al (2022). However, the reasons for these several genes are only partially explained in some patients, and the reasons for a considerable number of patients are still unknown.

The research of the prior art documents shows that the TACC3 gene is involved in the assembly process of mitotic microtubules of cells. The research shows that TACC3 is specifically and highly expressed in the development process of mouse ovum and early embryo, participates in the formation of meiotic ovum spindle, and has no report related to female infertility diseases.

Disclosure of Invention

The invention aims to provide a TACC3 gene which is convenient to operate and has a definite effect and is used for detecting female primary infertility and a kit for detecting the gene mutation.

The nucleic acid sequence of the TACC3 gene for detecting female primary infertility provided by the invention is shown in SEQ ID NO. 1.

The invention relates to a method for screening primary infertility caused by ovum maturation disorder, which judges whether a patient is the primary infertility caused by the ovum maturation disorder by detecting whether mutation occurs in TACC3 gene. The patients carrying TACC3 gene mutation show ovum maturation disorder.

Therefore, whether the TACC3 gene is mutated or not can be used as a marker for judging female infertility caused by ovum maturation disorder.

The method for detecting whether the TACC3 gene has mutation can be specifically characterized in that DNA is extracted from peripheral blood of a patient and then the PCR (polymerase chain reaction) method is combined with DNA sequencing to detect whether the TACC3 gene has mutation.

Wherein the sample to be detected is DNA, and the DNA sample is from peripheral blood of a human to be detected.

Alternatively, the sample to be tested is an RNA, protein, cell or serum sample derived from the peripheral blood of the human to be tested.

The invention relates to a primer for detecting whether a TACC3 gene is mutated or not.

The TACC3 gene has 15 exon coding regions, and PCR amplification primer pairs for detecting the 1 st-2 nd exon coding regions are as follows:

5’-TCCAGAGCAATGAGCACACC-3’(SEQ ID NO.2)

5’-CCAGCCAAGGCCTGATAGAAC-3’(SEQ ID NO.3)

the sequencing primer pair is the same as above;

the PCR amplification primer pair for detecting the coding region of the No.3 exon is as follows:

5’-AAGCGCGTGATGAGCACAAT-3’(SEQ ID NO.4)

5’-ATTGCACCAGGAGGCGAAGT-3’(SEQ ID NO.5)

5’-CCGCCTCTGAGACCCTAGAA-3’(SEQ ID NO.6)

5’-GAACCTACCCGGCCTCAAAG-3’(SEQ ID NO.7)

the sequencing primer pair is the same as above;

the PCR amplification primer pair for detecting the coding region of the exon 4-5 is as follows:

5’-TGAATGTTCACGTGGCCG-3’(SEQ ID NO.8)

5’-AACCACAAGTTTGTGCATGAGT-3’(SEQ ID NO.9)

the sequencing primer pair is as above;

the PCR amplification primer pair for detecting the coding regions of the exons 6-7 is as follows:

5’-AGTTTTGCCCCCGCCTTTC-3’(SEQ ID NO.10)

5’-CTAGCACAGCTCCTTTCCAGT-3’(SEQ ID NO.11)

the sequencing primer pair is as above;

the PCR amplification primer pair for detecting the coding region of the No. 8-9 exons is as follows:

5’-GGGTCTGCGTTTTCTACCACT-3’(SEQ ID NO.12)

5’-CAATTCCACCCCAACCAGCA-3’(SEQ ID NO.13)

the sequencing primer pair is as above;

the PCR amplification primer pair for detecting the coding region of the 10 th-11 th exon is as follows:

5’-CCCACATGGAACTGTGCCTC-3’(SEQ ID NO.14)

5’-CAGAGGCTGCTGCTAGACAT-3’(SEQ ID NO.15)

the sequencing primer pair is as above;

the PCR amplification primer pair for detecting the coding region of the No.12 exon is as follows:

5’-CTTTGCATCCGGCCTAGAAGA-3’(SEQ ID NO.16)

5’-GTTCACAGGTCATCCCATGC-3’(SEQ ID NO.17)

the sequencing primer pair is as above;

the PCR amplification primer pair for detecting the coding region of the No. 13-15 exons is as follows:

5’-GGAAAACGGGAGCTACTGGC-3’(SEQ ID NO.18)

5’-GCTTCATCAAGACCAGAGCG-3’(SEQ ID NO.19)

the sequencing primer pair is as above.

The invention also relates to a method for detecting whether the TACC3 gene is mutated, which comprises the following steps:

for the coding regions of the exons 1 to 2, primer pairs are used: SEQ ID NO.2 and SEQ ID NO.3, PCR amplification (amplification conditions: 2 minutes at 92 ℃, 30 seconds at 92 ℃,1 minute at 57 ℃, 3 seconds at 72 ℃ (these three steps are repeated for 35 cycles), 10 minutes at 72 ℃) is carried out, then sequencing is carried out by using the same primers, and comparison is carried out with a standard sequence (SEQ ID NO. 1) of TACC3 in UCSC, thereby finding mutation;

similar to the above conditions, the coding region of exon 3-15 of TACC3 was amplified with the corresponding primers (SEQ ID NO.4-NO. 19)

Sequencing is carried out, and then, the sequence is aligned with a standard sequence (SEQ ID NO. 1) of TACC3 in UCSC, thereby finding the mutation. And detecting whether the TACC3 gene is mutated or not through PCR and one-generation bidirectional sequencing and comparison with a standard sequence.

The invention also provides a kit for detecting whether the TACC3 gene is mutated. The kit can guide doctors to judge the etiology of diseases, correctly classify the diseases, and inform patients whether the IVF method or the ICSI method is adopted for the operation of test-tube infants and whether the in-vitro insemination-embryo transfer (IVF) or the single sperm injection (ICSI) (test-tube infants) is suitable to be continuously performed.

The kit provided by the invention comprises the amplification primers SEQ ID NO.2-SEQ ID NO.19 for amplifying the coding regions of the exons 1 to 15 of the TACC3 and reaction mixed liquid, wherein the reaction mixed liquid comprises DNA polymerase, dNTP and buffer solution.

The specific using method comprises the following steps: the primers were diluted with water to 10uM working solution. The reaction system is 10ul, wherein 1ul of sample DNA,0.5ul of forward primer, 0.5ul of reverse primer, 5ul of reaction mixture and 3ul of water. After mixing, PCR reactions were performed according to the above conditions, followed by one-generation sequencing.

The kit can be used for screening primary infertility caused by ovum maturation disorder.

The invention also provides a gene repair target site (namely actually detected mutation sites of a patient, for example, 4 mutation sites are listed in table 1, in the actual detection process, new mutation sites may exist by comparing with a standard sequence) for researching the primary infertility caused by ovum maturation disorder drug treatment, and the TACC3 can perform normal functions by repairing the target site, so that the disease can be treated. Namely, the invention can also provide a medicine for researching and repairing a gene mutation target site, namely a medicine for treating primary infertility caused by ovum maturation disorder.

The invention can also guide the clinical patients with the gene mutation whether to carry out the test tube infant operation or not by detecting the mutation of the TACC3 gene. In routine clinics, infertile couples of unknown cause require test tube babies to attempt to reproduce the offspring. However, if a patient is detected to have a mutation in TACC3, the patient will fail if he/she attempts a tube-based infant surgery. Because the phenotype of the patient with this mutation is an obstacle to maturation of the ovum and thus it is difficult to obtain a valid embryo, the manipulation of subsequent tube babies is not accomplished. Such patients are therefore advised to donate eggs.

Drawings

FIG. 1 is a graphical representation of the distribution of patient mutations over the primary structure of TACC 3.

Detailed Description

The present invention will be further described with reference to the following examples. It should be understood that the following examples are only illustrative of the present invention and are not intended to limit the scope of the present invention.

Example 1: collection of samples and extraction of peripheral blood DNA

The primary infertility patients come from Shanghai love heredity and infertility diagnosis and treatment center of Shanghai Red House Hospital affiliated to Shanghai Compound denier university, shanghai transportation university, ninth people Hospital affiliated to the Shanghai transportation university, and the like. Diagnostic criteria are set forth by Rudak E et al (Rudak E., dorJ., kimchim., goldman B., levran D and Mashiach S., and analysts of human society from human genome understanding treatment by in vitro transfer FertilSteril.1990 Aug;54 (2): 292-6). The semen of a male is checked to be normal, female reproductive organs, ovarian functions and sex hormones of a female patient are all normal, more than 5 eggs are taken each time in a ovulation promoting period of more than 2 times, and most of the eggs can not mature. The study of the subject was performed under the premise of informed consent, blood was collected, and an informed consent was signed. All patients enrolled excluded other genital endocrine diseases by medical history. The control population of the experiment is 1000 women with normal fertility, 300ul of the blood is sampled, DNA is extracted according to the kit specification, and the concentration and purity of the DNA are detected by using Nanodrop 2000.

Example 2: detection of mutation of TACC3 Gene

The invention searches for TACC3 gene mutation by combining PCR with sequencing. The principle is that firstly, primer design (a primer sequence can be specifically given) and amplification are carried out on 15 exon coding regions of the TACC3 gene, and mutation of the TACC3 gene is searched. (i.e., DNA sample and reaction working solution (DNA polymerase, dNTP, water and buffer) mixed and mixed, according to the PCR program for amplification, the product, through purification and further sequencing reaction, ABI3730 sequencing, result analysis through HLA Fusion software (One lambda, CA, USA, HLA Fusion 3.0).

Example 3: TACC3 gene mutation and primary infertility caused by ovum maturation disorder

As a result: we have gathered 2 cases of primary infertility patients caused by ovum maturation disorder due to TACC3 gene mutation. The corresponding mutation site information is shown in the following table (table 1). The distribution of the mutation sites is shown in FIG. 1.

TABLE 1 patient TACC3 Gene mutation information

In conclusion, the invention has the following important practical significance:

(1) The TACC3 gene provided by the invention can be used as a marker gene for predicting female infertility caused by ovum maturation disorder;

(2) The TACC3 gene provided by the invention can be used for evaluating or preparing a screening kit for female infertility caused by ovum maturation disorder;

(3) The invention can be used for researching and preparing the medicine for repairing the gene mutation target site, namely the medicine for treating primary infertility caused by ovum maturation disorder;

(4) The TACC3 gene provided by the invention can be used for guiding a patient with the gene mutation clinically, judging the success possibility of a test-tube infant and suggesting subsequent egg donation.

Claims (6)

1. A marker gene for judging female infertility caused by ovum maturation disorder is a TACC3 gene, the nucleic acid sequence of the TACC3 gene is shown as SEQ ID NO.1, and the judgment is based on whether the TACC3 gene is mutated.

2. A method for screening primary infertility caused by ovum maturation disorder is characterized in that whether a patient is the primary infertility caused by ovum maturation disorder is judged by detecting whether mutation occurs in TACC3 gene.

3. The method for screening primary infertility due to ovum maturation disorder according to claim 2, wherein the information about the mutation of TACC3 gene is:

4. the primer for detecting whether the TACC3 gene is mutated or not is characterized in that for 15 exon coding regions of the TACC3 gene, PCR amplification primer pairs for detection sequentially comprise: 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.

5. A method for detecting whether a TACC3 gene is mutated or not is characterized by comprising the following specific steps:

carrying out PCR amplification on 15 exon coding regions in TACC3 by using corresponding primers of SEQ ID NO.2, SEQ ID NO. 3-SEQ ID NO.18 and SEQ ID NO.19 respectively, and then carrying out sequencing by using the same primers, wherein the primers are identical to the standard sequence of TACC3 in UCSC: the alignment of SEQ ID NO.1 was performed to find a mutation.

6. A kit for detecting whether a TACC3 gene is mutated or not is characterized by comprising an amplification primer for amplifying a coding region of exons 1 to 15 of the TACC3 gene and a corresponding sequencing primer: SEQ ID NO.2-SEQ ID NO.19, and a reaction mixture comprising DNA polymerase, dNTP and a buffer.

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