CN106170562B

CN106170562B - Kit for detecting genetic marker related to idiopathic azoospermia

Info

Publication number: CN106170562B
Application number: CN201580000582.6A
Authority: CN
Inventors: 牟丽莎; 蔡志明
Original assignee: Shenzhen Second Peoples Hospital
Current assignee: Shenzhen Second Peoples Hospital
Priority date: 2015-07-01
Filing date: 2015-07-01
Publication date: 2021-04-20
Anticipated expiration: 2035-07-01
Also published as: WO2017000287A1; CN106170562A

Abstract

A kit for detecting a genetic marker associated with idiopathic azoospermia, the kit comprising a primer pair, wherein the genetic marker is located in a coding region of an AR gene and has a nucleotide sequence shown as SEQ ID NO:1, wherein mutation sites of the sequence are selected from one or more of c.868T > C, c.1484G > A, c.1888C > T, c.569C > T, c.616A > G and c.1149C > T; the upstream primer and the downstream primer of the primer pair are respectively positioned at the upstream and the downstream of the mutation site. 6 new mutation sites of the AR gene are screened out in patients with idiopathic azoospermia through large-scale sequencing, and research shows that the mutation of the AR gene can cause the idiopathic azoospermia, so that the diagnosis and detection of the idiopathic azoospermia can be realized by detecting the mutation by using the kit disclosed by the invention.

Description

Kit for detecting genetic marker related to idiopathic azoospermia

Technical Field

The invention relates to the field of male infertility detection, in particular to a kit for detecting genetic markers related to idiopathic azoospermia.

Background

About 10% to 15% of all women of the world's reproductive age face the problem of being unable to survive, half of which are due to male sterility. Primary azoospermia is a very important cause of male infertility, affecting approximately 1% of adult males. The male infertility pathogenesis has the characteristics of complexity and diversity, including diseases, malnutrition, endocrine disturbance, gene defects, environmental factors and the like, the specific pathogenesis of the male infertility pathogenesis is not clear, but the genetic factors can be deduced from family case reports and research results of mouse models to play a great role. In recent years, with the development of modern molecular biology techniques, it has been found that nearly 200 genes are closely related to the occurrence of male infertility; by adopting a gene knockout technology, nearly 400 genes are found to be closely related to the generation of mouse sperms, and mutation, deletion or expression abnormality of the genes can be an important reason for the generation of male infertility. The study of these mutant genes will help in the diagnosis of male infertility and also help in the future to prevent genetic defects from being brought to the next generation during in vitro fertilization.

The androgen receptor (AR, NCBI Gene ID:367) is an important steroid hormone receptor that plays a key role in male sexual differentiation and maintenance of normal spermatogenesis. AR belongs to a family of steroid hormone actions regulated by nuclear transcription factors. AR has 4 protein structures including an N-terminal transactivation domain (NTD), a DNA Binding Domain (DBD), a Hinge Region (HR) and a carboxy Ligand Binding Domain (LBD). It binds to the ligand binding domains of androgens, including testosterone (T) and 5 α -Dihydrotestosterone (DHT), thereby mediating nuclear translocation and the transcriptional regulatory function of the AR.

Over the past few years, several AR mutations or polymorphisms have been identified that can lead to or be associated with genetic diseases such as complete or partial androgen insensitive syndrome (CAIS and PAIS). However, intensive research is required to reveal the relationship between AR mutations and Idiopathic Azoospermia (IA), providing basis and guidance for the diagnosis of idiopathic azoospermia.

Disclosure of Invention

To determine the presence of mutations in the AR gene in IA patients, we sequenced the AR gene exons from 776 IA patients and 709 normal-bearing males, and found that the first 5 missense mutations and 1 synonymous mutation were associated with IA development.

Based on the discovery of the invention, the invention provides a genetic marker related to idiopathic azoospermia, which is positioned in a coding region of an AR gene, and the nucleotide sequence of the genetic marker is shown as SEQ ID NO. 1, wherein mutation sites of the sequence are selected from one or more of c.868T > C, c.1484G > A, c.1888C > T, c.569C > T, c.616A > G and c.1149C > T.

The first 5 mutation sites are missense mutations and the 6 th mutation site is a synonymous mutation.

The mutations are at position 868, 1484, 1888, 569, 616, 1279 and 1149, respectively, of the coding region of the AR gene sequence shown in SEQ ID NO. 1.

The amino acid changes corresponding to the first 5 mutation sites are p.C290R, p.S495N, p.R630W, p.T190I and p.S206G, namely, the 290 th amino acid is changed from C to R, the 495 th amino acid is changed from S to N, the 630 th amino acid is changed from R to W, the 190 th amino acid is changed from T to I, and the 206 th amino acid is changed from S to G. No corresponding amino acid changes were found at the 6 th mutation site.

In a preferred embodiment of the present invention, the mutation site of the above sequence is selected from one or more of c.868T > C, c.1484G > A and c.1888C > T.

In a preferred embodiment of the present invention, the mutation site of the above sequence is selected from c.1888C > T.

The invention also provides a kit for detecting the genetic marker related to idiopathic azoospermia, wherein the kit comprises a primer pair, the genetic marker is positioned in the coding region of an AR gene, and the nucleotide sequence of the genetic marker is shown as SEQ ID NO. 1, wherein the mutation site of the sequence is selected from one or more of c.868T > C, c.1484G > A, c.1888C > T, c.569C > T, c.616A > G and c.1149C > T; the upstream primer and the downstream primer of the primer pair are respectively positioned at the upstream and the downstream of the mutation site.

In a preferred embodiment of the present invention, the primer pair is selected from primer1, primer2 and primer3 primer pairs,

the sequence of the upstream primer of the primer1 primer pair is shown as SEQ ID NO. 2, and the sequence of the downstream primer is shown as SEQ ID NO. 3;

the sequence of the upstream primer of the primer2 primer pair is shown as SEQ ID NO. 4, and the sequence of the downstream primer is shown as SEQ ID NO. 5;

the sequence of the upstream primer of the primer3 primer pair is shown as SEQ ID NO. 6, and the sequence of the downstream primer is shown as SEQ ID NO. 7.

As a preferred embodiment of the present invention, the kit further comprises dNTPs, Taq DNA polymerase, Mg²⁺And PCR reaction buffer solution.

6 new mutation sites of the AR gene are screened from patients with idiopathic azoospermia through large-scale sequencing, AR gene wild type and mutant expression plasmids are constructed and transfected into cells for research, and experiments show that obvious differences exist in functions of the AR gene wild type and mutant expression plasmids, so that the mutation of the AR gene can possibly cause the idiopathic azoospermia, and the diagnosis and the detection of the male infertility (especially the idiopathic azoospermia) can be realized through the mutation. The invention designs an upstream primer and a downstream primer respectively at the upstream and the downstream of the mutation site for detecting the mutation site, thereby realizing the diagnosis and detection of idiopathic azoospermia.

Drawings

FIG. 1 is a3 missense mutation sites sequencing map of the AR gene of a patient with azoospermia;

FIG. 2 is the evolutionarily conserved amino acids affected by 3 missense mutation sites of the AR gene of a patient with azoospermia;

FIG. 3 is the location of 3 IA patient-specific missense mutations found on the AR gene;

FIG. 4 shows the effect of 3 AR mutants on MMTV expression of downstream target genes, NC for negative control and WT for wild type.

Detailed Description

The present invention will be described in further detail with reference to the following detailed description and accompanying drawings.

1 content of the experiment

1.1 Collection of specimens

The applicant collects 1880 azoospermia patients from 6 months to 10 months in 2011, wherein the azoospermia patients are 776 patients, and the screening standard is as follows: 1) randomly checking three times of semen without sperms; 2) non-obstructive, inflammatory and traumatic to the reproductive system or pelvic cavity; 3) without karyotypic abnormalities and microdeletion of the Y chromosome, 709 normal fertile males (with at least one child born and without human assisted reproductive technologies such as IVF, ICSI, IMSI) were studied as controls. Each subject underwent earnest informed consent and the study was approved by the review of the hospital ethics committee.

1.2 exon sequencing

Extracting peripheral Blood to extract genome DNA, collecting peripheral Blood of a study object by using a sodium citrate anticoagulation tube, quickly placing the peripheral Blood in a refrigerator at the temperature of-80 ℃ for standby, and extracting the peripheral Blood DNA by using a QIAamp DNA Blood Mini Kit.

Taking 5 micrograms of genomic DNA, sending the genomic DNA to a Shenzhen research center (Shenzhen) for exon capture and sequencing, and screening 9 mutation sites in the AR gene in idiopathic azoospermia patients, wherein 7 missense mutations and 2 synonymous mutations are compared with data in a dbSNP135 database, a thousand-human genome database and an ExAC database, and 5 missense mutations and 1 synonymous mutation are new mutations discovered for the first time by us.

1.3 validation of missense mutations

The extracted peripheral blood genome DNA was used as a template, and 3 primer pairs designed and synthesized were used to specifically amplify only 3 missense mutation sites (c.868T > C, c.1484G > A and c.1888C > T) existing in AR genes of idiopathic azoospermia patients (W320, W691, W530), and AR sequences were found from NCBI database (NCBI Gene ID: 367). Primers were synthesized by Invitrogen corporation, and the 3 primer pairs synthesized are shown in Table 1.

TABLE 1 verification primers for AR mutation sites

The PCR amplification conditions were: pre-denaturation at 98 ℃ for 2min, followed by 35 cycles at 98 ℃ for 10s, 60 ℃ for 30s, and 72 ℃ for 45s, and final extension at 72 ℃ for 5 min.

DNA electrophoresis: 3 mul of PCR product is put in a 1% agarose gel hole, 140V electrophoresis is carried out, 15min is carried out, an ultraviolet gel imaging system is used for photographing and observing an electrophoretogram, a single strip is ensured, the rest PCR products are sent to Weijiji corporation Shanghai for sequencing, and the sequences of the PCR product fragments amplified by the Primer pairs Primer1, Primer2 and Primer3 are respectively shown as SEQ ID NO 8, SEQ ID NO 9 and SEQ ID NO 10 in the sequence list.

1.4 construction of AR mutant expression plasmids

3 kinds of mutant expression plasmids of AR were constructed using 3 pairs of synthetic mutation primers designed and synthesized using pcDNA3.1-AR (Mou L et al, Identification of Ube2b as a novel tARget of android receiver in mouse sertoli cells, biol report.2013 Aug 15; 89(2):32.) as a template. Primers were synthesized by Invitrogen corporation, and the 3 pairs of point mutation primers synthesized are shown in Table 2.

TABLE 2 AR 3 pairs of site-directed mutagenesis primers

1.5 Dual luciferase reporter Gene experiments

1.5.1 plasmid preparation

Wild-type AR expression vector: pcDNA3.1-AR WT (WT group)

Mutant AR expression vectors: pcDNA3.1-AR C290R (group C290R)

pcDNA3.1-AR S495N (S495N group)

pcDNA3.1-AR R630W (R630W group)

1.5.2 HeLa cell culture

HeLa cells were cultured in DMEM medium containing 10% fetal bovine serum at 37 ℃ with 5% CO₂And culturing under 95% humidity conditions. Adherent cells were subcultured proportionally after digestion with 0.25% trypsin when they had grown to the bottom of the flask.

1.5.3 transient transfection of HeLa cells

Inoculating HeLa cells into a 24-pore plate, and transfecting after the cells adhere to the wall by using a reference transfection reagent Lipofectamine^TM2000, specification. After 6h of transfection, transfer was performedRemoving culture solution from each well by liquid gun, adding 500 μ l of new 1640 culture medium into each well, and reducing Lipofectamine^TM2000 toxicity to cells.

1.5.4 dual-luciferase reporter system for detecting activity of transcription factor

1) After 24h of transfection, cells are collected, culture solution in each hole is removed by a pipette, and 1ml of PBS is added into each hole for washing for 2 times;

2) diluting 5 × Passive lysine Buffer to 1 × Passive lysine Buffer, and adding 100 μ l Passive lysine Buffer to each well with a pipette;

3) lysing the cells on a shaker for 15min at room temperature, and aspirating 5 μ l of cell lysate per well into a clean 1.5mL clear EP with a micropipette gun, respectively;

4) sequentially adding 19 mu l of Luciferase Assay II Buffer into each EP tube in a dark environment, and detecting the fluorescence illumination of the firefly by using a single-tube photometer;

5) and sequentially adding 19 mu l of STOP Buffer into each EP tube in a dark environment, detecting the fluorescence illumination of the renilla by using a single-tube photometer, and correcting the fluorescence of the renilla to reduce experimental errors by using the fluorescence of the firefly. The relative activity of the reporter gene is determined.

1.6 statistical analysis

The statistical method used was from the SPSS17.0 software, each group of data is expressed as mean ± standard deviation (x ± s), the comparison of the mean between groups was performed using independent sample t-tests, and P <0.05 is statistically significant.

2 results of the experiment

2.1 identification of Point mutations in AR genes in patients with idiopathic azoospermia

Results of exon sequencing of the AR gene were analyzed in 776 idiopathic azoospermia patients and 709 normal fertile males, as shown in table 3: the 9 mutations of the AR gene were all homozygous, the first 7 missense mutations and the last 2 synonymous mutations. In comparison with the data in dbSNP135 database, thousand human genome database and ExAC database, there were 5 missense mutations (c.868T > C, c.1484G > A, c.1888C > T, c.569C > T, c.616A > G) and 1 synonymous mutation (c.1149C > T) which we first discovered. There were 3 missense mutations (c.868T > C, c.1484G > A, c.1888C > T) and 1 synonymous mutation (c.1149C > T) absent in 709 normal fertile males. We further verified these 3 missense mutations by PCR sequencing, the results are shown in figure 1.

Homology comparisons of AR amino acid sequences in different species showed that mutation of R630W affected a highly conserved amino acid (fig. 2), and fig. 2 was obtained by aligning different proteins using the MegAlign (mutation System DNASTAR, Inc.) software, where the AR proteins are numbered as follows: human (human) (NP _000035.2), chimpanzee (chimpanzee) (XP _009437511.1), rhesus (rhesus) (NP _001028083.1), cow (cow) (NP _001231056.1), rat (rat) (NP _036634.1), mouse (mouse) (NP _038504.1), and chicken (chicken) (NP _ 001035179.1). Boxes in figure 2 indicate the position of the mutation and conserved sequences.

Based on the results of the conservative analysis, SIFT and Polyphen 2.0 software analysis, it was shown that mutations in R630W may affect the function of the protein (table 4). The positions of these 3 missense mutations are shown in FIG. 3.

TABLE 3 cases of AR Point mutations in the idiopathic azoospermia and Normal groups

Note: the patient numbers begin with W, numbers 1-7 are missense mutations, and numbers 8, 9 are synonymous mutations.

Table 4 SIFT and Polyphen 2.0 software predicts the effect of missense mutations on protein function

Note: SIFT score is less than or equal to 0.05: destruction is greater than or equal to 0.05: tolerance;

polyphen-2: probably corrupt (probability score >0.85), probably corrupt (probability score >0.15), benign (retention).

2.2 changes in function of AR mutants

To assess whether the patient-specific AR missense mutations we found affected their regulatory function, we used a dual-luciferase reporter assay in which MMTV-LUC (Mou L et al, Identification of Ube2b as a novel tARget of an organic receptor in mouse serum cells, biol. Reprod.2013Aug 15; 89(2):32) was co-transfected into HeLa cells with the AR wild-type and 3 mutant plasmids, respectively, and since AR is a ligand-dependent transcription factor, we treated with androgen after transfection and the fluorescence of firefly/Stichopus japonicus detected was used to express promoter activity. Consistent with the wild-type AR, the AR C290R and S495N mutants also activated the activity of MMTV promoter, and the AR R630W mutant alone was significantly different from the wild-type, and failed to activate the activity of MMTV promoter (see fig. 4). These results indicate that the R630W variant inhibits AR transcriptional regulatory activity.

2.3 clinical information and hormone level detection

Patients with 3 missense mutations (c.868t > C, c.1484g > a, c.1888c > T) were further subjected to scrotal ultrasound and showed small scrotal testes, uniform echogenic properties and extensive hypoechogenicity; no solid or cystic lesions were observed. Table 5 summarizes clinical and hormone level data for these three patients. None of these patients had a family history of male infertility. There is no personal or family history of CAIS or PAIS in all patients with AR mutations.

TABLE 5 clinical information and hormone level measurements of patients with 3 missense mutations

Discussion of 3

There is increasing evidence that AR is a ligand-dependent transcription factor that regulates the expression of androgen-responsive genes. Androgens and AR are critical to male spermatogenic function and fertility.

Although more than 700 mutations and polymorphisms in the AR gene have been reported, only the 5 mutation is located in exon 1 and there are varying degrees of spermatogenic dysfunction or MAIS in azoospermic patients. This aspect is confirmed by the normal, complex clinical manifestations of the external genitalia and our reports in all these patients. At the same time, male AR knockout mice display the typical appearance of female individuals, similar to a human androgen insensitive syndrome or testicular feminized variant mouse.

In the present study, we sequenced the coding sequences of 776 patients with IA AR. The R630W mutation was localized to the DNA binding domain of the AR, and was found in one of 776 patients, but not in 709 fertile males and other individuals reported in public databases. Analysis of the R630W mutation using the Polyphen-2 and Sift software predicted an effect on protein structure. Furthermore, local alignment analysis of the amino acid sequence of AR indicates that the affected arginine residues are highly conserved across multiple species, including chickens. The amino acids of this mutant attachment are well conserved, suggesting that mutations may have a major impact on AR function.

When we attempted to assess the pathogenic effects of AR on sterile patients, a key issue to solve was whether the mutated AR affected its transcriptional regulatory function. As expected, we found that the R630W mutation affected the transcriptional regulatory function of AR on MMTV promoter.

In summary, we determined seven missense mutations and two synonymous mutations of the AR gene by massively parallel sequencing techniques. Functional tests show that the R630W mutation inhibits the normal transcriptional regulation function of AR. The foregoing is a more detailed description of the present invention that is presented in conjunction with specific embodiments, and the practice of the invention is not to be considered limited to those descriptions. For those skilled in the art to which the invention pertains, several simple deductions or substitutions can be made without departing from the spirit of the invention, and all shall be considered as belonging to the protection scope of the invention.

Claims

1. Use of a kit for detecting a genetic marker associated with idiopathic azoospermia for the preparation of a kit for detecting a genetic marker associated with idiopathic azoospermia, said kit comprising a primer pair, said genetic marker being located in a coding region of an AR gene and having a nucleotide sequence as set forth in SEQ ID No. 1, wherein the mutation site of said sequence is selected from the group consisting of c.1888C > T; the upstream primer and the downstream primer of the primer pair are respectively positioned at the upstream and the downstream of the mutation site, the sequence of the upstream primer is shown as SEQ ID NO. 6, and the sequence of the downstream primer is shown as SEQ ID NO. 7.

2. Use according to claim 1, wherein the kit further comprises dNTPs, Taq DNA polymerase, Mg²⁺And PCR reaction buffer solution.