WO2017000289A1

WO2017000289A1 - Idiopathic azoospermia-related genetic marker

Info

Publication number: WO2017000289A1
Application number: PCT/CN2015/083084
Authority: WO
Inventors: 牟丽莎; 蔡志明
Original assignee: 深圳市第二人民医院
Priority date: 2015-07-01
Filing date: 2015-07-01
Publication date: 2017-01-05
Also published as: CN105492631A; CN105492631B

Abstract

An idiopathic azoospermia-related genetic marker, the genetic marker being located in the coding region of the AR gene, and the nucleotide sequence thereof being represented by SEQ ID No.1, the mutation sites of the sequence being one or more of c. 868 T>C, c. 1484 G>A, c. 1888 C>T, c.569 C>T, c. 616 A>G, and c. 1149 C>T. By means of large-scale sequencing, 6 new mutation sites of the AR gene have been selected from idiopathic azoospermia patients; AR gene wild-type and mutant expression plasmids have been constructed and transfected into cells, and experiments demonstrate that said mutations of the AR gene may cause idiopathic azoospermia; thus, by means of the mutations, diagnosis and detection of idiopathic azoospermia can be implemented.

Description

A genetic marker associated with idiopathic azoospermia

Technical field

The invention relates to the field of male infertility detection, in particular to a genetic marker related to idiopathic azoospermia.

Background technique

About 10% to 15% of couples of childbearing age in the world face infertility problems, half of which are due to male infertility. Primary azoospermia is a very important cause of male infertility, affecting about 1% of adult males. Male infertility factors are characterized by complexity and diversity, including diseases, malnutrition, endocrine disorders, genetic defects and environmental factors. The specific pathogenesis is still unclear, but the results from family case reports and mouse models It can be inferred that genetic factors play a large role. In recent years, with the development of modern molecular biology technology, it has been found that nearly 200 genes are closely related to the occurrence of male infertility; using gene knockout technology, it is found that nearly 400 genes are closely related to mouse spermatogenesis. Mutations, deletions or abnormal expression of genes may be important causes of male infertility. Studies of these mutant genes will contribute to the diagnosis of male infertility and will also help 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 hormones regulated by nuclear transcription factors. AR has four protein structures, including the N-terminal transactivation domain (NTD), the DNA binding domain (DBD), the hinge region (HR), and the carboxyl ligand binding domain (LBD). It binds to androgen, including the ligand binding domain of testosterone (T) and 5α-dihydrotestosterone (DHT), thereby mediating nuclear translocation and AR transcriptional regulation.

In the past few years, some AR mutations or polymorphisms have been identified that can lead to or are associated with genetic diseases such as complete or partial androgen insensitivity syndromes (CAIS and PAIS). However, in-depth study is needed to reveal the relationship between AR mutation and idiopathic azoospermia (IA), and provide evidence and guidance for the diagnosis of idiopathic azoospermia.

Summary of the invention

In order to determine the AR gene mutation in IA patients, we will have 776 patients with IA and 709 patients. The AR gene exon sequencing of the newly born males revealed that the first five missense mutations and one synonymous mutation were associated with the occurrence of IA.

Based on the findings of the present invention, the present invention provides a genetic marker associated with idiopathic azoospermia, which is located in the coding region of the AR gene, the nucleotide sequence of which is set forth in SEQ ID NO: 1, wherein the sequence The mutation site 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 first five mutation sites are missense mutations and the sixth mutation site is a synonymous mutation.

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

The amino acid changes corresponding to the first five mutation sites are p.C290R, p.S495N, p.R630W, p.T190I and p.S206G, ie, the amino acid at position 290 is changed from C to R, and amino acid 495 is S becomes N, the amino acid at position 630 changes from R to W, the amino acid at position 190 changes from T to I, and the amino acid at position 206 changes from S to G. There is no corresponding amino acid change at the 6th mutation site.

As 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.

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

Six new mutation sites of AR gene were screened in patients with idiopathic azoospermia by large-scale sequencing, and AR gene wild type and mutant expression plasmids were constructed and transfected into cells for research. There is a significant difference, indicating that the aforementioned mutation of the AR gene may lead to the occurrence of idiopathic azoospermia, thereby enabling the diagnosis of male infertility (especially idiopathic azoospermia) by the above mutation.

DRAWINGS

Figure 1 is a sequencing map of three missense mutation sites of the AR gene in azoospermia patients;

Figure 2 is an evolutionarily conserved amino acid affected by three missense mutation sites of the AR gene in azoospermia patients;

Figure 3 shows the location of the missense mutations specific to the three IA patients found on the AR gene;

Figure 4 shows the effect of three AR mutants on the expression of the downstream target gene MMTV, NC for the negative control and WT for the wild type.

detailed description

The present invention will be further described in detail below with reference to the accompanying drawings.

1 Experimental content

1.1 Collection of specimens

The applicant collected a total of 1880 patients with azoospermia from June 2007 to October 2011, including 776 patients with idiopathic azoospermia. Our screening criteria were: 1) random examination of sperm in three semen; 2) no obstruction, inflammation and injury in the reproductive system or pelvic; 3) non-nuclear abnormalities and Y chromosome microdeletions, and 709 normal fertile men (at least one child and no IVF, ICSI, IMSI, etc.) Reproductive technology) was studied as a control. Each subject carefully signed an informed consent form, and the study was approved by the hospital ethics committee.

1.2 Exon sequencing

The peripheral blood was extracted from the peripheral blood, and the peripheral blood of the study subject was collected with a sodium citrate anticoagulation tube. The peripheral blood was quickly placed in a -80 ° C refrigerator, and the peripheral blood DNA was extracted with a QIAamp DNA Blood Mini Kit.

Five micrograms of genomic DNA was taken and sent to the China National Genetics Research Center (Shenzhen) for exon capture and sequencing. Among the 9 mutation sites in the AR gene, 7 missense mutations were screened in patients with idiopathic azoospermia. And two synonymous mutations, compared with the data in the dbSNP135 database, the thousand human genome database and the ExAC database, there are five missense mutations and one synonymous mutation is the first mutation we found.

1.3 Verify missense mutations

Using the extracted peripheral blood genomic DNA as a template, three pairs of primers designed and synthesized were used to identify only three missense mutation sites in the AR gene of idiopathic azoospermia (W320, W691, W530) (c.868T> Specific amplification was performed for C, c.1484G>A and c.1888C>T), and the AR sequence was found from the NCBI database (NCBI Gene ID: 367). The primers were synthesized by Invitrogen, and the three pairs of primer pairs synthesized are shown in Table 1.

Table 1 AR mutation site verification primers

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

DNA electrophoresis: Take 3 μl of PCR product in 1% agarose gel well, 140V electrophoresis, 15min, UV gel imaging system to observe the electropherogram, ensure a single band, and the rest of the PCR products were sent to Shanghai Yingjie Jieji Company for sequencing. The PCR product fragment sequences amplified by primer pairs Primer1, Primer2 and Primer3 are shown in SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, respectively, in the Sequence Listing.

1.4 Construction of AR mutant expression plasmid

Using pcDNA3.1-AR (Mou L et al., Identification of Ube2b as a novel tARget of androgen receptor in mouse sertoli cells. Biol Reprod. 2013 Aug 15; 89(2): 32.) as a template, using three pairs of design synthesis The primers were mutated to construct three mutant expression plasmids of AR. Primers were synthesized by Invitrogen, and the three 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 experiment

1.5.1 Plasmid preparation

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

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

pcDNA3.1-AR S495N (S495N group)

pcDNA3.1-AR R630W (R630W group)

1.5.2 HeLa cell culture

HeLa cells were cultured in a medium containing 10% fetal bovine serum DMEM at 37 ° C, 5% CO ₂ and 95% humidity. When the adherent cells were overgrown, they were digested with 0.25% trypsin and subcultured in proportion.

1.5.3 HeLa cells transient transfection

HeLa cells were seeded in 24-well plates, After transfection adherent cells, the method of Reference transfection reagent Lipofectamine ^TM 2000 specification. After transfection for 6 hours, each well was aspirated with a pipette, and 500 μl of new 1640 medium was added to each well to reduce the toxicity of Lipofectamine ^TM 2000 to the cells.

1.5.4 Dual luciferase reporter system for detection of transcription factor activity

1) After 24 hours of transfection, the cells were collected, and each well was aspirated with a pipette, and each well was washed with 1 ml of PBS for 2 times;

2) Dilute 5×Passive Lysis Buffer into 1×Passive Lysis Buffer, and add 100 μl Passive Lysis Buffer to each well with a pipette;

3) lyse the cells on a shaker for 15 min at room temperature, and pipette 5 μl of each cell lysate into a clean 1.5 mL clear EP with a micropipette;

4) Add 19 μl of Luciferase Assay II Buffer to each EP tube in a dark environment, and then measure the illuminance of the firefly with a single tube photometer;

5) In the dark environment, add 19μl STOP Buffer to each EP tube, then use the single-tube photometer to detect the fluorescence of the kidney and kidney, and use the firefly fluorescence to correct the experimental error of the renal and renal fluorescence reduction. The relative activity of the reporter gene was determined.

1.6 Statistical analysis

The statistical methods used were from SPSS 17.0 software. The data of each group were expressed as mean ± standard deviation (x ± s). The mean between groups was compared by independent sample t test, P < 0.05 was considered statistically significant.

2 Experimental results

2.1 Identification of AR gene point mutations in patients with idiopathic azoospermia

The results of exon sequencing of AR gene in 776 patients with idiopathic azoospermia and 709 normal fertile men are shown in Table 3. The 9 mutations in the AR gene are homozygous mutations. The first 7 are Wrong sense Change, the last two are synonymous mutations. Compared with the data in the dbSNP135 database, the 1000 human genome database and the ExAC database, there are 5 missense mutations (c.868T>C, c.1484G>A, c.1888C>T, c.569C>T,c .616A>G) and a synonymous mutation (c.1149C>T) are the first mutations we have discovered. Three missense mutations (c.868T>C, c.1484G>A, c.1888C>T) and one synonymous mutation (c.1149C>T) were absent in 709 normal fertile men. . We further verified these three missense mutations by PCR sequencing. The results are shown in Figure 1.

The homology comparison of AR amino acid sequences in different species showed that the mutation of R630W affected a highly conserved amino acid (Fig. 2). Figure 2 shows the comparison of different proteins using MegAlign (Demonstration System DNASTAR, Inc.) software. , wherein the AR protein number is as follows: human (NP_000035.2), chimpanzee (XP_009437511.1), rhesus (rhesus) (NP_001028083.1), cow (cow) (NP_001231056.1), Rat (NP_036634.1), mouse (NP_038504.1) and chicken (NP_001035179.1). The box in Figure 2 indicates the location of the mutation and * indicates the conserved sequence.

Based on conservative analysis, SIFT and Polyphen 2.0 software analysis showed that mutations in R630W may affect protein function (Table 4). The positions of these three missense mutations are shown in Figure 3.

Table 3 AR point mutations in the idiopathic azoospermia group and the normal group

Note: Patient numbers start with W, numbers 1-7 are missense mutations, and numbers 8 and 9 are synonymous mutations.

Table 4 SIFT and PolyPhen 2.0 software predict the effect of missense mutations on protein function

Note: SIFT score ≤ 0.05: damage, ≥ 0.05: tolerance;

PolyPhen-2: Probably destroyed (probability score > 0.85), possible damage (probability score > 0.15), benign (hold).

2.2 Changes in AR mutant function

In order to assess whether the patient-specific AR missense mutations we have identified affect its regulatory function, we used a dual luciferase reporter gene assay, MMTV-LUC (Mou L et al., Identification of Ube2b as a novel tARget of androgen receptor in Mouse sertoli cells.BiolReprod.2013Aug 15;89(2):32) co-transfected with AR wild type and three mutant plasmids into HeLa cells, respectively. Since AR is a ligand-dependent transcription factor, we transfected Treatment with androgen, the detected firefly fluorescence value / sea cucumber fluorescence value to indicate the activity of the promoter. Consistent with wild-type AR, the AR C290R and S495N mutants also activated the activity of the MMTV promoter, and only the AR R630W mutant was significantly different from the wild type and could not activate the MMTV promoter activity (see Figure 4). These results indicate that the R630W variant inhibits AR transcriptional regulatory activity.

2.3 Clinical information and hormone level detection

Patients with three missense mutations (c.868T>C, c.1484G>A, c.1888C>T) further underwent scrotal ultrasonography, and the results showed that the scrotal testis was small, with uniform echo characteristics and a wide range of low Echo; no solid or cystic lesions were observed. Table 5 summarizes the clinical and hormonal data for these three patients. None of these patients had a family history of male infertility. There were no individual or family history of CAIS or PAIS in all AR mutation patients.

Table 5. Clinical information and hormone levels in patients with 3 missense mutations

3 Discussion

There is increasing evidence that AR is a ligand-dependent transcription factor that regulates the expression of androgen-responsive genes. Androgen and AR are essential for male spermatogenesis and fertility.

Although it has been reported that the AR gene has more than 700 mutations and polymorphisms, only 5 mutations are located in exon 1, and there are varying degrees of spermatogenic disorders or MAIS in patients with azoospermia. The normal genital manifestations of all of these patients, complex clinical presentations and our reports confirm this aspect. At the same time, male AR knockout mice exhibited a typical appearance of female individuals, similar to a human androgen insensitivity syndrome or testicular feminized mutant mice.

In the study of the present invention, we sequenced the coding sequences of 776 IA patients with AR. The R630W mutation was localized to the DNA binding domain of AR, and one patient was found to have this mutation in 776 patients, but not in 709 fertile men and other individuals reported in the public database. Analysis of the R630W mutation using PolyPhen-2 and Sift software predicted an effect on protein structure. In addition, local alignment analysis of the amino acid sequence of AR revealed that the affected arginine residues are highly conserved across multiple species, including chickens. The amino acids of this mutant accessory are very conservative, suggesting that the mutation may have a greater impact on the function of AR.

When we attempt to assess the pathogenic role of AR in infertile patients, a key question to address is whether mutated AR affects its transcriptional regulatory function. As expected, we found that the R630W mutation affects the transcriptional regulation of AR on the MMTV promoter.

In summary, we identified seven missense mutations and two synonymous mutations in the AR gene by massively parallel sequencing. Functional tests have shown that the R630W mutation inhibits the normal transcriptional regulatory function of AR. The above is a further detailed description of the present invention in connection with the specific embodiments, and the specific embodiments of the present invention are not limited to the description. It will be apparent to those skilled in the art that the present invention may be made without departing from the spirit and scope of the invention.

Claims

A genetic marker associated with idiopathic azoospermia, characterized in that it is located in the coding region of the AR gene, and its nucleotide sequence is as shown in SEQ ID NO: 1, wherein the mutation site of the sequence is selected One or more of c.868T>C, c.1484G>A, c.1888C>T, c.569C>T, c.616A>G, and c.1149C>T.
The genetic marker according to claim 1, wherein the mutation site of the sequence is selected from one or more of c.868T>C, c.1484G>A and c.1888C>T.
The genetic marker according to claim 1, wherein the mutation site of the sequence is selected from the group consisting of c.1888C>T.