CN112877415A

CN112877415A - Marker TRIP13 gene for judging female primary infertility and detection kit thereof

Info

Publication number: CN112877415A
Application number: CN201911209671.8A
Authority: CN
Inventors: 王磊; 桑庆; 张治华
Original assignee: Fudan University; Zhuhai Fudan Innovation Research Institute
Current assignee: Fudan University; Zhuhai Fudan Innovation Research Institute
Priority date: 2019-12-01
Filing date: 2019-12-01
Publication date: 2021-06-01

Abstract

The invention belongs to the technical field of gene detection, and particularly relates to a kit for detecting TRIP13 gene mutation of female primary infertility. The mutation of the human TRIP13 gene is a cause of female infertility caused by ovum immaturity. The detection of the mutation of the TRIP13 gene can be used as a marker gene for judging female infertility caused by ovum immaturity. The TRIP13 gene mutation provided by the invention can be used for evaluating or preparing a screening kit for female infertility caused by ovum immaturity. Whether the TRIP13 gene is mutated or not can be used for guiding whether a corresponding patient is suitable for performing tube infancy in clinic.

Description

Marker TRIP13 gene for judging female primary infertility and detection kit thereof

Technical Field

The invention belongs to the technical field of gene detection, and particularly relates to a marker for judging female primary infertility and a detection kit thereof.

Background

Normal pregnancy and reproduction are important links in maintaining and extending human populations. For female infertility, genes ZP-1, Stag3, FSHR, etc. have been found to be closely related to female infertility (Huang HL et al, Mutant ZP1 in human sterility. N Engl J Med. 2014370 (13):1220-6; de Roux N et al, A family with hypo-gonadotropic hypo and mutations in the gonadolitropin-releasehormonereceiver. N Engl J. 1997337 (22): 947 159602; calret S et al, Mutant conjugation in precursor failure. N Engl Med. 2014370 (10): 943-9). However, none have been clinically applied.

The reasons for female infertility are many, and there are some clinical reports relating to the description of female infertility characterized by egg immaturity (Rudak et al, fertility and fertility, 199054: 292-. 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 found the causative 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) that cause the maturation disorders of human ova. However, the reasons for these two genes can only be explained for some patients, and the reasons for a considerable number of patients remain unknown.

The search of the prior art literature found that only a few research articles surrounding the function of TRIP13 gene. These studies report that the gene plays an important role in the meiosis process of mouse eggs, the knockout leads to the infertility of female mice, and the truncated mutation of TRIP13 gene leads to the Wilms tumor, but the report that the gene is related to the infertility of female mice is not found.

Disclosure of Invention

The invention aims to provide a marker for judging female primary infertility and a detection kit thereof, which are convenient to operate and have clear effect.

In the invention, the nucleic acid sequence of the TRIP13 gene for detecting female primary infertility is shown as SEQ ID NO. 1.

The invention also relates to a method for screening primary infertility caused by ovum immaturity, which is to judge whether a patient is the primary infertility caused by ovum immaturity by detecting whether the TRIP13 gene is mutated. Patients carrying TRIP13 gene mutation showed infertility, which was marked by a failure of the ovum to mature and tube failure.

Therefore, whether the TRIP13 gene is mutated or not can be used as a marker for judging female infertility caused by ovum immaturity.

Specifically, the method for detecting whether the TRIP13 gene is mutated can be implemented by extracting DNA from peripheral blood of a patient and then detecting whether the TRIP13 gene is mutated by using a PCR (polymerase chain reaction) method in combination with DNA sequencing.

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 TRIP13 gene is mutated.

The TRIP13 gene has 13 exons, and PCR amplification primer pairs for detecting the No.1 exon are as follows:

5’-TTCCGGGTCAGGAGGTGG-3’（SEQ ID NO.2）

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

the sequencing primer pair is as above;

the PCR amplification primer pair for detecting the No.2 exon is as follows:

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

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

the sequencing primer pair is as above;

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

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

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

the sequencing primer pair is as above;

the PCR amplification primer pair for detecting the exons 4 and 5 is as follows:

5’-GCAGAATTTATACTTTCCTGTAAT-3’（SEQ ID NO.8）

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

the sequencing primer pair is as above;

the PCR amplification primer pair for detecting the No.6 exon is as follows:

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

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

the sequencing primer pair is as above;

the PCR amplification primer pair for detecting the No.7 exon is as follows:

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

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

the sequencing primer pair is as above;

the PCR amplification primer pair for detecting the No.8 and No.9 exons is as follows:

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

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

the sequencing primer pair is as above;

the PCR amplification primer pair for detecting the No.10 exon is as follows:

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

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

the sequencing primer pair is as above;

the PCR amplification primer pair for detecting the No.11 exon is as follows:

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

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

the sequencing primer pair is as above;

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

5’-ACGCCTCGGCTTGTGTTCCCAG-3’（SEQ ID NO.20）

5’-CCTGTGTTTCGGTGCCAAGGTC-3’（SEQ ID NO.21）

the sequencing primer pair is as above;

the PCR amplification primer pair for detecting the No.13 exon is as follows:

5’-GACGTGTGTGGTGCGCACTCACTG-3’（SEQ ID NO.22）

5’-TTATGAAAGAACACTTTACCGACC-3’（SEQ ID NO.23）

the sequencing primer pair is as above.

The invention also relates to a method for detecting whether the TRIP13 gene is mutated, which comprises the following steps:

for exon 1, primer pairs are used: SEQ ID NO.2 and SEQ ID NO.3, PCR amplified (amplification conditions: 2 min at 92 ℃, 30 sec at 92 ℃,1 min at 57 ℃, 3 sec at 72 ℃ (these three steps are repeated for 35 cycles), 10 min at 72 ℃), then sequenced using the same primers, aligned with the standard sequence of TRIP13 in UCSC (SEQ ID NO. 1), and thereby mutations were found;

similarly to the above conditions, exons 2 to 13 of TRIP13 were amplified with corresponding primers (SEQ ID NO.4 to NO. 23) and sequenced, and then aligned with the standard sequence of TRIP13 (SEQ ID NO. 1) in UCSC to find mutations. Whether the TRIP13 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 TRIP13 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.23 for amplifying the exons 1 to 13 of TRIP13 and reaction mixed liquor; the reaction mixed solution 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 of 10uM concentration. 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 immaturity.

The invention also provides a gene repair target site (namely actually detected mutation sites of a patient, such as 5 mutation sites 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 immaturity in drug treatment, and TRIP13 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 immaturity.

The invention can also guide the clinical patients with the gene mutation whether to be suitable for the test-tube infant operation or not by detecting the mutation of the TRIP13 gene. In routine clinics, infertile couples of unknown cause require test tube babies to attempt to reproduce the offspring. However, if the patient detected a mutation with TRIP13, the patient would fail if he attempted a tube-fed infant. Because the ovum of the patient with the mutation is immature or it is difficult to obtain a valid embryo after fertilization of a partially mature ovum, the subsequent manipulation of the tube baby cannot be completed. Such patients are therefore advised to donate eggs.

Drawings

FIG. 1 shows the distribution of patient mutations over the primary structure of TRIP 13.

Detailed Description

The present invention will be further described with reference to the following specific 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 caused by ovum immaturity come from Shanghai Jiai inheritance and sterility diagnosis and treatment center of the affiliated Red House hospital of Shanghai Compound denier university in China and the affiliated ninth people hospital reproductive center of the Shanghai transportation university in China. Diagnostic criteria are set forth by Rudak E et al (Rudak E., DorJ., Kimchim., Goldman B., Levran D and Mashiach S, analysts of human society from human genome understanding treatment by in vitro transfer, FertilSteril.1990 Aug; 54(2): 292-6). The semen of male is checked normally, female reproductive organ, ovary function and sex hormone of female patient are all normal, more than 5 eggs are obtained each time in more than 2 discharge promoting periods, most of the eggs are in MI stage, and normal meiosis and maturation can not be carried out. 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 the purity of the DNA are detected by an ultraviolet spectrophotometer.

Example 2: detection of mutation of TRIP13 Gene

The invention adopts PCR combined with sequencing to search for TRIP13 gene mutation. The principle is that primer design (primer sequence can be specifically given) and amplification are firstly carried out on 13 exon coding regions of the TRIP13 gene, and TRIP13 gene mutation search is carried out. (DNA samples and reaction working solution (DNA polymerase, dNTP, water and buffer) after mixing, according to PCR program amplification, the product, through purification and further sequencing reaction, ABI3730 sequencing, analysis of results through HLA Fusion software (One lambda, CA, USA, HLA Fusion 3.0).

Example 3: TRIP13 gene mutation and primary infertility caused by ovum immaturity

As a result: we have gathered 4 cases of patients with primary infertility caused by immature ovum due to TRIP13 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 mutation information of TRIP13 Gene of patient

。

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

(1) the TRIP13 gene provided by the invention can be used as a marker gene for predicting female infertility caused by ovum immaturity;

(2) the TRIP13 gene provided by the invention can be used for evaluating or preparing a screening kit for female infertility caused by ovum immaturity;

(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 immaturity;

(4) the TRIP13 gene provided by the invention can be used for guiding patients with the gene mutation clinically, judging the success possibility of test-tube infants and recommending subsequent egg donation.

Sequence listing

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Claims

1. A marker for judging female primary infertility is characterized in that the marker is TRIP13 gene and has a nucleic acid sequence shown as SEQ ID NO.1, and the female primary infertility refers to female infertility caused by ovum immaturity, fertilization and embryo abnormality.

2. A method for screening female primary infertility is characterized in that whether a patient is primary infertility caused by ovum immaturity, fertilization and embryo abnormality is judged by detecting whether TRIP13 gene has mutation, wherein the nucleotide sequence of the TRIP13 gene is shown as SEQ ID NO. 1.

3. The primer for detecting whether the TRIP13 gene is mutated or not is characterized in that the PCR amplification primer pairs for detecting 13 exons of the TRIP13 gene 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, SEQ ID NO.20, SEQ ID NO.21, SEQ ID NO.22, SEQ ID NO. 23.

4. A method for detecting whether a TRIP13 gene is mutated or not is characterized by comprising the following specific steps:

carrying out PCR amplification on 13 exon coding regions in TRIP13 by using corresponding primer pairs of SEQ ID NO.2, SEQ ID NO.3, …, SEQ ID NO.22 and SEQ ID NO.23 respectively, and then carrying out sequencing by using the same primers, wherein the sequencing result is the same as the standard sequence of TRIP13 in UCSC: SEQ ID NO.1 was aligned to find a mutation.

5. A kit for detecting whether the TRIP13 gene is mutated or not is characterized by comprising an amplification primer for amplifying exons 1 to 13 of the TRIP13 gene and a corresponding sequencing primer: SEQ ID NO.2-SEQ ID NO.23, and a reaction mixture comprising DNA polymerase, dNTP and a buffer.

6. The site of TRIP13 gene mutation is applied to research and preparation of drugs for repairing gene mutation target sites, namely drugs for treating primary infertility caused by ovum immaturity, fertilization and embryo abnormality.