CN107475378A

CN107475378A - For detecting the PATL2 genes of women primary infertility and detecting the kit of the gene mutation

Info

Publication number: CN107475378A
Application number: CN201710657304.9A
Authority: CN
Inventors: 王磊; 陈标榜; 桑庆胥; 尧穆健
Original assignee: Fudan University
Current assignee: Fudan University
Priority date: 2017-08-03
Filing date: 2017-08-03
Publication date: 2017-12-15
Anticipated expiration: 2037-08-03
Also published as: CN107475378B

Abstract

The invention belongs to technical field of gene detection, particularly for the kit of the PATL2 gene mutations of detection women primary infertility.Mankind PATL2 genes of the present invention, it is the reason for causing to cause female acyesis because ovum is immature during its mutation.Mutation by detecting PATL2 genes can be used as the marker gene for judging that female acyesis is caused because ovum is immature.It can be used for evaluation using PATL2 gene mutations provided by the invention or prepare because of the kit for screening of the immature cause female acyesis of ovum.Using PATL2 gene mutations provided by the invention whether, available for instruct clinical respective patient whether properly carry out test-tube baby's art.

Description

For detecting the PATL2 genes of women primary infertility and detecting the examination of the gene mutation Agent box

Technical field

The invention belongs to technical field of gene detection, and in particular to a kind of PATL2 bases for being used to detect women primary infertility Cause and the kit for detecting the gene mutation.

Background technology

Normal pregnancy and reproduction are maintenance and the important step for continuing human population.For female acyesis, have at present The genes such as ZP-1, Stag3, FSHR are found closely related with female infertility（Huang HL et al, Mutant ZP1 in familial infertility. 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）.But do not applied in clinic.

The reason for causing female infertility, is a lot, has some clinical reports to be related to women characterized by ovum is immature not The description of pregnant disease（Rudak et al, fertility and sterility,1990 54:292-296;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 are by being repeated insemination-transplantation of embryo （Test-tube baby）, because failing to get ripe egg cell, and successful inseminatio externalis operation can not be completed.If phase can be found Close Disease-causing gene, then it is all significant to disease clinical diagnosis and parting.Recently, we found that cause mankind ovum into First Disease-causing gene TUBB8 (Jan 21 of N Engl J Med. 2016 of ripe obstacle;374(3):223-32).But this The reason for gene is only capable of explaining some patientss, it is still unknown the reason for quite a lot of patient.

Find by prior art documents, only find that a several pieces surround the research article of PATL2 gene functions. These researchs cause Xenopus Oocytes ripe abnormal after reporting this gene overexpression.But so far there are no on Any report of relation between PATL2 gene mutations and human diseases.Its report related to female infertility is not found yet.

The content of the invention

It is an object of the invention to provide a kind of easy to operate, definite effect for detecting women primary infertility PATL2 genes and the kit for detecting the gene mutation.

The nucleotide sequence such as SEQ ID NO.1 institutes proposed by the present invention for being used to detect the PATL2 genes of women primary infertility Show.

The present invention relates to the method that examination causes primary infertility because ovum is immature, exactly by detecting PATL2 genes Whether undergo mutation judge patient whether be ovum it is immature caused by primary infertility.Patient carries PATL2 genes and dashed forward Change shows as infertile, and showing as ovum all the time can not ripe and test-tube baby failure.

It can be seen that PATL2 genes whether undergo mutation can as judge because ovum it is immature cause female acyesis mark.

Whether detection PATL2 genes undergo mutation method, after specifically can extracting DNA from the peripheral blood of patient, use PCR （Polymerase chain reaction）Method combination DNA sequencing, to detect whether PATL2 genes are mutated.

Wherein, the sample of detection is DNA, and the DNA sample comes from the peripheral blood of crowd to be checked.

Or the sample of detection is RNA, albumen, cell or serum sample, the sample comes from the periphery of crowd to be checked Blood.

The present invention relates to the primer whether detection PATL2 genes undergo mutation.

PATL2 genes have 15 extrons, for detect the 1st extron pcr amplification primer thing to for：

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

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

Sequencing primer is to ibid；

For detect the 2nd exon pcr amplification primer thing to for：

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

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

Sequencing primer is to ibid；

For detect the 3rd exon pcr amplification primer thing to for：

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

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

Sequencing primer is to ibid；

For detect the 4th exon pcr amplification primer thing to for：

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

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

Sequencing primer is to ibid；

For detect the 5th, 6 exon pcr amplification primer thing to for：

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

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

Sequencing primer is to ibid；

For detect the 7th, 8 exon pcr amplification primer thing to for：

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

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

Sequencing primer is to ibid；

For detect the 9,10th exon pcr amplification primer thing to for：

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

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

Sequencing primer is to ibid；

For detect the pcr amplification primer thing of o.11 extron to for：

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

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

Sequencing primer is to ibid；

For detect the 12nd exon pcr amplification primer thing to for：

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

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

Sequencing primer is to ibid；

For detect the 13rd exon pcr amplification primer thing to for：

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

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

Sequencing primer is to ibid；

For detect the 14th exon pcr amplification primer thing to for：

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

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

Sequencing primer is to ibid；

For detect the 15th exon pcr amplification primer thing to for：

5’-AGTGCTAACCTATTTGAGGGCA -3’（SEQ ID NO.24）

5’-TGCCATGTCTTATTTTTAGGCACA -3’（SEQ ID NO.25）

Sequencing primer is to ibid.

It is specific as follows the invention further relates to the method whether detection PATL2 genes undergo mutation：

To wherein 1 exon, primer pair is utilized：SEQ ID NO.2, SEQ ID NO.3, enter performing PCR amplification（Amplification condition： 92 DEG C 2 minutes, 92 DEG C 30 seconds, 57 DEG C 1 minute, 72 DEG C 3 seconds（This three step repeats 35 circulations）, 72 DEG C 10 minutes）, so It is sequenced afterwards using identical primer, the standard sequence with PATL2 in UCSC（SEQ ID NO.1）It is compared, so as to send out Now it is mutated；

It is similar to above-mentioned condition, PATL2 2-15 exons, (SEQ ID NO.4- are expanded by corresponding primer NO.25)

It is sequenced, then the standard sequence with PATL2 in UCSC（SEQ ID NO.1）It is compared, so as to find to be mutated.Pass through PCR and generation sequencing, and the comparison with standard sequence, to detect whether PATL2 genes undergo mutation.

The present invention also provides the detection kit whether PATL2 genes undergo mutation.This kit can instruct doctor couple The judgement of the disease cause of disease, disease is correctly classified, so as to inform that patient carries out test-tube baby using IVF methods or ICSI methods Operation, and if appropriate for continue implement IVF-ET cycle art（IVF）Or intracytoplasmic sperm injection art（ICSI）（Test tube baby Youngster）.

Kit provided by the invention, include the amplimer SEQ ID of above-mentioned amplification PATL2 1-15 exons NO.2-SEQ ID NO.25, and reaction mixture；Reaction mixture includes archaeal dna polymerase, dNTP and buffer solution；

Specifically used method is：Primer is diluted with water as the working solution of 10uM concentration.Reaction system is 10ul, wherein, 1ul Sample DNA, 0.5ul forward primers, 0.5ul reverse primers, 5ul reaction mixtures and 3ul water composition.After mixing, by above-mentioned Condition enters performing PCR reaction, then carries out generation sequencing.

Mentioned reagent box can be used for the immature examination for causing primary infertility of detection ovum.

The present invention is also provided for studying the immature gene repair target site for causing primary infertility of drug therapy ovum（I.e. It is actually detected go out patient mutational site, for example 9 mutational sites are listed in table 1, during actually detected, by with Standard sequence compares, and may have new mutational site）, and by the reparation to target site, so that PATL2 can go Make normal function, and then such a disease can be treated.I.e. the present invention, which can also provide to study, is used for repair gene mutation target site Medicine, that is, treat ovum it is immature cause primary infertility medicine.

The present invention can also instruct the clinical patient with this gene mutation, if close by detecting the mutation of PATL2 genes It is suitable to carry out test-tube baby's art.In routine clinical, the Infertile couples of unknown cause need to carry out test-tube baby to attempt to produce offspring. But if patient has found the mutation with PATL2 by detecting, then this patient can fail if test-tube baby's art is attempted. Because the ovum of the patient with this mutation is operation that is jejune, thus can not completing follow-up test-tube baby.Therefore, build Discuss such patient and carry out tax ovum.

Brief description of the drawings

Fig. 1 is the distribution displaying that patient is mutated in PATL2 primary structures.

Embodiment

Below in conjunction with specific embodiment, to further illustrate the present invention.It should be understood that following examples are merely to illustrate this hair Bright rather than limitation the scope of the present invention.

Embodiment 1：The collection of sample and the extracting of peripheral blood DNA

Ovum is immature to cause primary infertility patient to come from the attached red house hospital Shanghai collection love something lost of Chinese Shanghai Fudan University Pass and sterile diagnosis and treatment center, the attached 9th the People's Hospital's reproductive center of Chinese Shanghai university of communications.Diagnostic criteria is by Rudak E etc. People（Rudak E., DorJ.,KimchiM.,Goldman B.,Levran D and Mashiach S, Anomalies of human oocytes from infertile women undergoing treatment by in vitro fertilization. FertilSteril. 1990 Aug; 54(2):292-6）It is proposed.Bridegroom's or husband's side semen efamination is normal, the wife's side Patient female reproductive organ, ovarian function, sex hormone are normal, and more than 2 times decorporation cycles get more than 5 ovums, ovum every time Son is mostly in the GV phases, it is impossible to carries out normal meiosis and maturation.This problem is participated on the premise of informed consent Research, blood is gathered, and sign informed consent form.All patients in group exclude other Procreation models by medical history. The control crowd of this experiment is 1000 normal women of reproductive function, takes sample blood 300ul, is extracted by kit specification DNA, DNA concentration and purity are detected with UV detector.

Embodiment 2：Detect the mutation of PATL2 genes

The present invention combines the searching of sequencing progress PATL2 gene mutations using PCR.Its principle is first to 15 of PATL2 genes Extron carries out design of primers（Primer sequence can be specifically given）And amplification, carry out the searching of PATL2 gene mutations.（I.e. by DNA Sample and reaction working solution（Archaeal dna polymerase, dNTP, water and buffer solution）Mix after mixing, expanded according to PCR programs.Institute Product is obtained, by purifying and further sequencing reaction, is sequenced in ABI3730 sequencings.Interpretation of result is soft by HLA Fusion Part (One lambda, CA, USA, HLA Fusion 3.0) is carried out.

Embodiment 3：PATL2 gene mutations because ovum is immature with causing primary infertility

As a result：It is immature and cause primary infertility patient 5 that we collected PATL2 gene mutations institute reason ovum altogether.It is corresponding prominent Become site information see the table below（Table 1）.Distribution of the mutational site in structure is as shown in Figure 1.

The patient P ATL2 gene mutation information of table 1

In summary, it is of the invention that there is following important practical usage：

（1）The marker gene of female acyesis can be caused because ovum is immature by the use of PATL2 genes proposed by the present invention as prediction；

（2）It can be used for evaluation using PATL2 genes provided by the invention or prepare because of the examination of the immature cause female acyesis of ovum Kit；

（3）Using the present invention, the medicine for preparing repair gene mutation target site can be studied, that is, it is primary to treat the immature cause of ovum The infertile medicine of property；

（4）It can be used for instructing the clinical patient with this gene mutation using PATL2 genes provided by the invention, judge test tube baby The successful possibility of youngster, and suggest carrying out follow-up tax ovum.

SEQUENCE LISTING

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<120>For detecting the PATL2 genes of women primary infertility and detecting the kit of the gene mutation

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atcttgcagc agcagcagca tagtcaaaca ccaagtcccc cagccaagaa gccttggtct 540

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Claims

1. whether PATL2 genes undergo mutation as the application for judging the mark that female acyesis is caused because ovum is immature, described The nucleotide sequence of PATL2 genes is as shown in SEQ ID NO.1.

2. a kind of method that examination causes primary infertility because ovum is immature, it is characterised in that be by detecting PATL2 genes It is no undergo mutation judge patient whether be ovum it is immature caused by primary infertility.

3. for detecting the primer whether PATL2 genes undergo mutation, it is characterised in that for aobvious outside 15 of PATL2 genes Son, the pcr amplification primer thing of detection is to being followed successively by：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, SEQ ID NO.24, SEQ ID NO.25.

4. a kind of method whether detection PATL2 genes undergo mutation, it is characterised in that comprise the following steps that：

To 15 exons in PATL2, corresponding primer pair SEQ ID NO.2, SEQ ID NO.3--SEQ ID are utilized respectively NO.24, SEQ ID NO.25, enter performing PCR amplification, be then sequenced using identical primer, the standard with PATL2 in UCSC Sequence：SEQ ID NO.1 are compared, so as to find to be mutated.

5. a kind of be used to detect the kit whether PATL2 genes undergo mutation, it is characterised in that kit includes amplification The amplimer and corresponding sequencing primer of the 1-15 exons of PATL2 genes：SEQ ID NO.2-SEQ ID NO.25, And reaction mixture, reaction mixture include archaeal dna polymerase, dNTP and buffer solution.