CN104531683B - A kind of genetic marker related to Non-obstructive Azoospermia - Google Patents

A kind of genetic marker related to Non-obstructive Azoospermia Download PDF

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CN104531683B
CN104531683B CN201410444435.5A CN201410444435A CN104531683B CN 104531683 B CN104531683 B CN 104531683B CN 201410444435 A CN201410444435 A CN 201410444435A CN 104531683 B CN104531683 B CN 104531683B
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dax
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obstructive azoospermia
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牟丽莎
刁瑞英
谢妮
黄卫人
蔡志明
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Shenzhen Second Peoples Hospital
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Abstract

The invention discloses a kind of genetic marker related to Non-obstructive Azoospermia, its nucleotide sequence is the sequence of the genes of DAX 1, and c.152G the mutational site of the sequence is selected from>A、c.312C>G、c.725C>T、c.766G>C、c.1153G>T、c.1279A>G and c.162G>One or more of A.1 genes of DAX, 7 new mutational sites have been filtered out in Non-obstructive Azoospermia patient by large scale sequencing, build the wild types of DAX 1 and mutant expression plasmid is transfected into intracellular studied, it is found through experiments that its function has notable difference, the generation of Non-obstructive Azoospermia may be caused by showing DAX 1 above-mentioned mutation, realize that the diagnosis to male sterility (especially Non-obstructive Azoospermia) detects so as to be mutated by this.

Description

A kind of genetic marker related to Non-obstructive Azoospermia
Technical field
The application is related to male sterility detection field, more particularly to Non-obstructive Azoospermia detection field.
Background technology
The couple at child-bearing age that the whole world there are about 10%-15% faces infertile problem, and wherein half is due to male sterility. Primary Azoospermia be cause one of male sterility it is extremely important the reason for, about influence 1% adult male.Male sterility Pathogenic factors has complexity and multifarious feature, including disease, malnutrition, endocrine disturbance, gene defect and environment Factor etc., its specific pathogenesis is still not clear, but may infer that from the achievement in research of family's Case report and mouse model Inherent cause has played great role.In recent years, with the development of modern molecular biology technique, it has been discovered that nearly 200 bases Because the generation with male sterility is closely related;Using gene Knockout, it is found that nearly 400 genes occur with mouse sperm close Cut is closed, mutation, missing or the abnormal expression of these genes, it may be possible to the major reason that male sterility occurs.Most base Because abnormal expression can be damaged Development in Puberty, then because Hypothalamus-pituitary has important facilitation to sexual gland or its gene The factor lack, finally cause male sterility.The sex reversion congenital adrenal hypoplasia gene 1 of susceptible-dose (Dosage-sensitive sex reversal,adrenal hypoplasia critical region,on Chromosome X gene 1, DAX-1) be the gene belonged on hypothalamic pituitary gonadal axis, its albumen encoded be core by The member of body family.DAX-1 rises to pituitary gonadotropic cell and adrenocortical development as a kind of transcription factor and weighed very much The effect wanted.
Mankind DAX-1 genes are located at X chromosome p21, are cloned successfully in 1994.The albumen of the gene code is by 470 Amino acid is constituted, and is a member in nuclear receptor family, is mainly expressed in hypothalamus, hypophysis, adrenal gland and sexual gland, to hypophysis Gonadotroph and adrenocortical development play a very important role.Increasing research shows that Congenital adrenal is sent out Educate bad (Adrenal hypoplasia congenital, AHC) and low promoting sexual gland hormone sexual inadequacy The morbidity of (Hypogonadotropic hypogonadism, HH) is relevant with DAX-1 mutation.Because DAX-1 genes are located on Xp DSS region, the sex reversal (womanlike) of 46, XY males is often accompanied by when the double copy in the area, so originally DAX-1 is considered as ovum Nest determines gene.But, subsequent condition knocks out result of study and shows that it plays an important role in regulation spermatogenesis, after knockout Male mice show as hypogonadism, testicualr development exception, dyszoospermia etc..Holter et al. research shows, male Hormone receptor (Androgen receptor, AR) is a DAX-1 new action target spot, and DAX-1 passes through directly mutual with AR Effect suppresses AR transcriptional activity.
The content of the invention
The present invention provides a kind of genetic marker related to Non-obstructive Azoospermia.
The present invention provides a kind of genetic marker related to Non-obstructive Azoospermia, and its nucleotide sequence is DAX-1 bases The sequence of cause, the mutational site of the sequence is selected from c.152 G>A、c.312 C>G、c.725 C>T、c.766 G>C、c.1153 G>T、c.1279 A>G and c.162 G>One or more of A.
Preceding 6 mutation are missense mutation, and the 7th mutation is same sense mutation.
Above-mentioned mutation is the 152nd site in DAX-1 gene orders, 312 sites, 725 sites, 766 sites, 1153 respectively Site, 1279 sites and 162 sites.
A kind of genetic marker related to Non-obstructive Azoospermia, its nucleotide sequence is the sequence of DAX-1 genes, institute The mutational site for stating sequence is selected from c.152 G>A、c.312 C>G、c.725 C>T、c.766 G>C、c.1153 G>T and c.1279 A>One or more of G.
The mutational site is c.1153 G>T.
The primer pair of the described genetic marker of detection, draws selected from primer 1, primer 2, primer 3 or primer 4 Thing pair, wherein, the sequence such as SEQ ID NO of the sense primer of the primer pairs of primer 1:Shown in 1, the sequence of primer downstream Row such as SEQ ID NO:Shown in 2;The sequence such as SEQ ID NO of the sense primer of the primer pairs of primer 2:Shown in 3, under it Swim the sequence such as SEQ ID NO of primer:Shown in 4;The sequence such as SEQ ID NO of the sense primer of the primer pairs of primer 3:5 It is shown, the sequence of primer such as SEQ ID NO downstream:Shown in 6;The sequence of the sense primer of the primer pairs of primer 4 is such as SEQ ID NO:Shown in 7, the sequence of primer such as SEQ ID NO downstream:Shown in 8.
A kind of detection kit of Non-obstructive Azoospermia, genetic marker that can be described in specific recognition.The heredity Mark especially c.1153 G>T is mutated.
A kind of application of described genetic marker on Non-obstructive Azoospermia detection kit is prepared.
The beneficial effects of the invention are as follows:DAX- has been filtered out in Non-obstructive Azoospermia patient by large scale sequencing The 1 new mutational site of gene 7, build DAX-1 wild types and mutant expression plasmid be transfected into it is intracellular studied, pass through Experiment finds that its function has notable difference, and the generation of Non-obstructive Azoospermia may be caused by showing DAX-1 above-mentioned mutation, from And the diagnosis to male sterility (especially Non-obstructive Azoospermia) can be realized by the mutation and is detected.
Brief description of the drawings
Fig. 1 is 6 missense mutation site Sequencing chromatograms of azoospermia patients DAX-1 genes;
Fig. 2 is the result figure of co-immunoprecipitation detection DAX-1 wild types and mutant and AR combination;
Fig. 3 is the result figure to the AR downstream target genes MMTV influences expressed after DAX-1 mutant interacts with AR.
Embodiment
The present invention is described in further detail below by embodiment combination accompanying drawing.
1 experiment content
1.2.1 the collection of sample
The applicant collects 1880 azoospermia patients, wherein non-obstructivity altogether from June, 2007 in October, 2011 Azoospermia patient is 776, and screening criteria is:1) without sperm in three seminal fluid of casual inspection;2) reproductive system or pelvic cavity without Obstruction, inflammation and damage;3) it is micro-deleted without chromosome abnormalities and Y chromosome, 706 normal fertile males (are at least separately given birth to one The Issues of Human Assisted Reproductive Technologies such as individual child and non-row IVF, ICSI, IMSI) studied as control.Each subject is conscientious Informed consent form is signed, the examination and approval that this research passes through Hospital Ethical Committee.
1.2.2 extron is sequenced
Extract peripheral blood and extract genomic DNA, with the peripheral blood of sodium citrate anticoagulant tube collection research object, put rapidly It is standby in -80 DEG C of refrigerators, extract peripheral blood DNA with QIAamp DNA Blood Mini Kit kits.
Taking out 5ug genomic DNAs send Hua Da cara gene (Shenzhen) to carry out exon trapping, sequencing, in non-obstruction Property azoospermia patients in filter out 7 new mutational sites in DAX-1 genes, wherein 6 missense mutation and 1 it is synonymous prominent Become, with the data in dbSNP135 databases and thousand human genome databases compared with pair, do not find that this 7 kinds are mutated, and 706 This 7 kinds of new variations are not found yet in example normal male.
1.2.3 missense mutation is verified
Peripheral blood genomic DNA using extraction is template, using 4 pairs of primer pairs of design synthesis only in non-obstructivity without essence 6 missense that sub- disease patient (W024, W033, W251, W505, W520, W566, W570, W628, W698) DAX-1 genes are present Mutational site carries out specific amplification, and DAX-1 sequences are checked in from ncbi database.Primer is synthesized by Invitrogen companies, 4 kinds of synthesized primers are shown in Table 1.Numbering of the DAX-1 genes in ncbi database is Gene ID:190.Using primer 1 The sequence of the PCR primer of primer pair such as SEQ ID NO:Shown in 23;Using the primer pairs of primer 2 PCR primer sequence such as SEQ ID NO:Shown in 24;Using the sequence such as SEQ ID NO of the PCR primer of the primer pairs of primer 3:Shown in 25;Using The sequence of the PCR primer of the primer pairs of primer 4 such as SEQ ID NO:Shown in 26.
Primer is verified in the DAX-1 point mutation of table 1
PCR amplification conditions are:98 DEG C of pre-degeneration 2min, are then carried out 35 and are followed with 98 DEG C of 10s, 60 DEG C of 30s, 72 DEG C of 45s Ring, last 72 DEG C of extensions 5min.
DNA electrophoresis:3 μ l PCR primers are taken in 1% Ago-Gel hole, 140V electrophoresis, 15min, ultraviolet gel imaging System is taken pictures observation electrophoretogram, it is ensured that single band, and remaining PCR primer serves extra large Invitrogen Corp.'s sequencing.
1.2.4 plasmid construction
1.2.4.1 the structure of DAX-1 wild type expression plasmids
1.2.4.1.1 the acquisition of people's DAX-1 gene cDNA encodings area full length sequence
Human Testis tissue RNA reverse transcriptions are synthesized into cDNA, and as template, design contains Hind III and two kinds of BamH1 The primer of restriction enzyme site, with the 3 ' pcDNA3.1 (+) with HA Polypeptide tags for carrier, builds wild type DAX-1 expression Plasmid.Primer sequence with restriction enzyme site is:
Upstream:5'-CCCAAGCTTATGGCGGGCGAGAAC-3'(SEQ ID NO:9)
Downstream:5'-CGCGGATCCCGTATCTTTGTACAG-3'(SEQ ID NO:10)
Wherein the restriction enzyme sites of Hind III are contained at the end of sense primer 5 ', and BamH1 restriction enzyme sites are contained at the end of anti-sense primer 5 '.
1.2.4.2 the structure of DAX-1 mutant expression plasmids:
So that correct pcDNA3.1-DAX1 is sequenced as template, using 6 pairs of point mutation primers of design synthesis, DAX- is built 16 kinds of mutant expression plasmids.Primer is synthesized by Invitrogen companies, and 6 pairs of synthesized primers are shown in Table 2.
The DAX-16 of table 2 is to rite-directed mutagenesis primer
1.2.5 it is double to entangle the plain enzyme Reporter Gene Experiments of light
1.2.5.1 plasmid prepares
Wild type DAX-1 expression vectors:PcDNA3.1-DAX-1 WT (WT groups)
Saltant type DAX-1 expression vectors:PcDNA3.1-DAX-1 R51K (R51K groups)
PcDNA3.1-DAX-1 C104W (C104W groups)
PcDNA3.1-DAX-1 A242V (A242V groups)
PcDNA3.1-DAX-1 E256Q (E256Q groups)
PcDNA3.1-DAX-1 V385L (V385L groups)
PcDNA3.1-DAX-1 I427V (I427V groups)
1.2.5.2 HeLa cell culture
HeLa cells are used and trained containing 10% hyclone DMEM culture mediums under 37 DEG C, 5%CO2 and 95% damp condition Support.Attached cell is when covering with bottom of bottle, with carrying out Secondary Culture in proportion after 0.25% Trypsin Induced.
1.2.5.3 HeLa cell transient transfections
HeLa cells are inoculated in 24 orifice plates, after being transfected after cell attachment, method refers to transfection reagent LipofectamineTM2000 specifications.Transfect after 6h, inhaled with liquid-transfering gun and abandon every hole nutrient solution, new 1640 are added per hole The μ l of culture medium 500, reduce toxicity of the Lipofectamine 2000 to cell.
1.2.5.4 it is double to entangle the plain enzyme reporting system detection transcription factor activity of light
1) cell is collected after transfection 24h, is inhaled with liquid-transfering gun and abandon every hole nutrient solution, PBS 1ml are added per hole and are washed 2 times;
2) 5 × Passive Lysis Buffer are diluted to 1 × Passive Lysis Buffer, with liquid-transfering gun to every Hole adds 100 μ l Passive Lysis Buffer;
3) the cell lysis 15min on shaking table at room temperature, every μ l of hole cell pyrolysis liquid 5 are drawn with micropipette rifle extremely respectively In one transparent EP of clean 1.5mL;
4) used after adding the Buffer of 19 μ l Luciferase Assay II into each EP pipes successively in light protected environment Single-tube luminometer detection firefly entangles illuminance;
5) examined after adding 19 μ l STOP Buffer into each EP pipes again successively in light protected environment with single-tube luminometer Survey sea pansy and entangle illuminance, light is entangled with firefly to correct sea pansy and entangles light reduction experimental error.Determine the relative activity of reporter gene.
1.2.6 co-immunoprecipitation experiment
1.2.6.1 plasmid prepares
People's AR expression plasmids:pcDNA3.1-AR
Wild type DAX-1 expression vectors:PcDNA3.1-DAX-1 WT (WT groups)
Saltant type DAX-1 expression vectors:PcDNA3.1-DAX-1 R51K (R51K groups)
PcDNA3.1-DAX-1 C104W (C104W groups)
PcDNA3.1-DAX-1 A242V (A242V groups)
PcDNA3.1-DAX-1 E256Q (E256Q groups)
PcDNA3.1-DAX-1 V385L (V385L groups)
PcDNA3.1-DAX-1 I427V (I427V groups)
1.2.6.2 HeLa cell culture
HeLa cells are used containing 10% hyclone DMEM in high glucose culture medium under 37 DEG C, 5%CO2 and 95% damp condition Culture.Attached cell is when covering with bottom of bottle, with carrying out Secondary Culture in proportion after 0.25% Trypsin Induced.
1.2.6.3 the extraction of total protein of cell
With the DMEM complete medium culture HeLa cells containing 10% hyclone, 37 DEG C, 5%CO2 training are positioned over Support in case, according to the regular growth cultural method culture in aseptic operating platform.By the HeLa cells in exponential phase with 3 × 105/ hole density is inoculated in 6 well culture plates, after after second day cell attachment, according to transfection reagent LipofectamineTM 2000 Transfection procedure, by the HA-DAX1 WT of recombinant plasmid pcDNA3.1 (+) -3 ' and 6 kinds of mutant mut1-mut6 (experimental group, Expression vector (2ug) difference cotransfection 2ug) with the HA of pcDNA3.1 (+) -3 ' unloaded (control group, 2ug) and hAR is thin to HeLa Born of the same parents.Inhaled after transfection 48h and abandon culture medium, according to the extraction step of the mammalian proteins extracts kit of Qiagene companies with 1 × PBS solution is washed 2 times, and the PBS for adding 1ml precoolings is scraped under gently extension with cell, is transferred to 1.5ml Ep pipes, 450 × g, 4 DEG C 5min is centrifuged, supernatant is discarded and is placed on ice, with 100ul Lysis Buffer (Benzonase containing 0.1U Nuclease, 1ul 100 × Protease Inhibitor) it is resuspended after precipitation, 4 DEG C of rotation 5min, 14,000 × g, 4 DEG C of centrifugation 10min, by supernatant It is transferred in new centrifuge tube.
1.2.6.4 co-immunoprecipitation
The albumen that experimental group and control group are extracted is divided into 4 points, and a copy of it is added as Input samples 6.25ul 5 × SDS Loading Buffer (DTT containing 5%1mM), 98 DEG C of processing 10min, -80 DEG C of preservations after mixing.It is remaining Lower 3 portions of supernatants add 475ul Lysis Buffer or Co-IP Buffer, are supplemented to every pipe cumulative volume for 500ul, it After be separately added into 2ug AR antibody, 2ug HA antibody, 2ug IgG;4 DEG C of rotation 1h;Often pipe addition 50ul is pretreated 4 DEG C of rotations of ProteinA/G Beads are stayed overnight.
DEG C brief centrifugation 20s of 12,000 × g, 4 after 4 DEG C of rotations overnight, abandons supernatant;Add 600ul Lysis Buffer weights Outstanding, 12,000 × g, 4 DEG C of centrifugation 1min washing Beads (being inhaled without pipette tips, flicked) are washed 3 times, suctioned out with rifle altogether Supernatant, but should not blot, note that Beads can not be drawn onto;15ul Lysis Buffer are added into Beads and isometric 2XSDS sample-loading buffers, 98 DEG C of processing 10min, are that antigen antibody complex is disintegrated down from Beads by albumen after mixing, It can be used to protein electrophoresis.Sds page is prepared, per hole applied sample amount 15ul, after conventional electrophoretic by protein delivery extremely On nitrocellulose filter, with TBS-T buffer solutions (the 0.02M Tris-Cl, pH 7.6,0.137M containing 5% skimmed milk power NaCl, 0.1% Tween-20) room temperature close 1 hour after, discard confining liquid.Addition is a certain amount of to be suitably diluted in containing 5% degreasing Primary antibody (the anti-human HA of mouse and the rabbit-anti people's AR monoclonal antibodies, 1 of identifying purpose albumen in the TBS-T buffer solutions of milk powder:2000 is dilute Release), 4 DEG C are overnight;Discard primary antibody, TBS-T buffer solution for cleaning 3 times, every time 5 minutes.A certain amount of appropriate be diluted in is added to take off containing 5% Secondary antibody (1 in the TBS-T buffer solutions of fat milk powder:10000 dilutions), it is incubated at room temperature 1 hour;Secondary antibody is discarded, TBS-T buffer solutions are clear Wash 3 times, every time 5 minutes.ECL chemical luminous substrates are added dropwise on film, are then exposed with X-ray, develop fixed automatically through sheet-punching machine Shadow.
1.2.7 statistical analysis
Statistical method used comes from SPSS17.0 softwares, and every group of data are with mean ± standard deviationRepresent, Average relatively uses independent samples t test, P between group<0.05 is statistically significant.
2 experimental results
2.1 the identification of Patients with Non-obstructive Azoospermia DAX-1 point mutations
Extron sequencing to 776 Patients with Non-obstructive Azoospermia and the DAX-1 genes of 706 normal fertile males Interpretation of result, as shown in table 3:7 new mutations of DAX-1 genes are homozygous mutation, and mutational site is respectively:c.152 G>A (p.R51K)、c.312 C>G(p.C104W)、c.725 C>T(p.A242V)、c.766 G>C(p.E256Q)、c.1153 G>T (p.V385L)、c.1279 A>G (p.I427V) and c.162 G>A (p.L54L), preceding 6 position missense mutation, last 1 is same Justice mutation, with the data in dbSNP135 databases and thousand human genome databases compared with pair, finds no these and is mutated, and These new variations (as shown in table 3) are not found yet in 706 normal fertile males.Further PCR checkings missense mutation Result it is as shown in Figure 1.
Situation of the DAX-1 point mutation of table 3 in Non-obstructive Azoospermia group and normal group
Note:Patient number is started with W, and only numbering 7 is same sense mutation.
2.2 DAX-1 wild types and mutant and the combinations of AR in the cell
In order to further detect the interaction situation after DAX-1 mutation with AR, using Immunoprecipitation, in HeLa Corotation collects cell extraction total protein after entering hAR and DAX-1 wild types and mutant expression plasmid, transfection 48h respectively in cell, The Western Blot of the co-immunoprecipitation and anti-AR and anti-HA that are anti-AR and anti-HA detect the phase of albumen Interaction and expression, as a result show compared with DAX-1 wild types, and only DAX-1V385L mutant and AR combination weakens.
The influence that 2.3 DAX-1 mutant are expressed AR downstream target genes MMTV after being interacted with AR
DAX-1 interacts to regulate and control the expression of downstream gene as co-factor and AR, in order to further detect DAX-16 The influence expressed after mutant interacts with AR AR downstream target genes MMTV is planted, the plain enzyme reporter gene reality of light is entangled using double Test, AR expression plasmids and MMTV-LUC distinguished into cotransfection into HeLa cells with DAX-1 wild types and 6 kinds of mutant plasmids, Handled after belonging to the ligand-dependent transcriptional factor, therefore transfection due to AR with androgen, the firefly detected is entangled into light Value/sea cucumber entangles light value to represent the activity of promoter.It is consistent with wild type DAX-1, DAX-1R51K, C104W, A242V, E256Q, I427V mutant can also substantially suppress the activity of MMTV promoters, and only DAX-1V385L mutant has with wild type Significant difference, it is impossible to suppress the activity of MMTV promoters, as shown in Figure 3.
3 discuss
DAX-1 gene mutations are mainly shown as adrenal cortex hypoplasia, with or without low promoting sexual gland hormone Adenasthenia.Due to most of patients onset symptoms typical case, make a definite diagnosis and be not easy to.DAX-1 genes are orphan nuclear receptor, on kidney Expressed in gland cortex, sexual gland, hypothalamus and hypophysis.DAX-1 genes encode 470 amino acid altogether containing 2 extrons.1994 Muscatelli etc. reports that DAX-1 single gene mutations are relevant with AHC and HH mutation first, and Shanghai Ruijin Hospital 2007 is at home This patient is reported first.The occurrence frequency of DAX-1 gene mutation types is followed successively by individually or lacked jointly with contiguous gene Lose (about 40%), frameshift mutation (about 28%), nonsense mutation (about 18%) and missense mutation (about 14%).Most mutational site positions In the ligand binding region of supposition, its functional transcription is caused to be damaged.This in 7 DAX-1 point mutations experimental studies have found that have 6 Individual is missense mutation, and 1 is same sense mutation, and compared with the data in dbSNP135 databases and thousand human genome databases It is right, find no these mutation.Holter et al. research shows that androgen receptor AR is a DAX-1 new effect Target spot, DAX-1 passes through the transcriptional activity with AR direct interactions suppression AR.This experiment is by building DAX-1 wild types and dashing forward Variant expression plasmid, functions of the research DAX-1 in cell.By sequencing result and functional study analyze DAX-1 wild types and Mutant expression plasmid is successfully constructed, and the function of DAX-1 V385L mutant has significant difference compared with DAX-1 wild types.
DAX-l has expression in the tissue such as hypothalamus, hypophysis, adrenal gland and sexual gland.SABC is confirmed:From the 11.5th It starts, and DAX-l is expressed in embryo mouse forebrain, is expressed in anterior pituitary within the 13.5th day, is expressed in diencephalon veutro within the l4.5 days, Expressed in the ventromedial nucleus of hypothalamus within the l8 days.And research confirms that the different times of mouse gonad development have DAX-l's Expression:DAX-1 is expressed when urogenital ridge occurs in embryo mouse within the 9th day;After Sex Differentiation, the DAX-l in male mice testis Level it is first rapid decline, then gradually rise, peaked in Sertoli cells and Leydig cells within the l2 days, thus it is speculated that DAX-l may have the effect of the male specific gene of regulation and control in testicualr development late period;But it is then different in female mice, the Still there is expression at l3.5 days in sexual gland, and continue to adult, but act on unknown.People DAX-1 genes are located at Xp2l, people and mouse DAX- 1 genetic homology is 65%.It is atypical lonely nuclear receptor family member, lacks DNA binding domain, the lonely core of vertebrate by Belong to the 0th class in body family.DAX-l also has a conservative LBD, and its N-terminal overlay region is the conserved sequence of a zinc finger, can Combined with the hairpin structure on DNA.Nuclear receptor typically each has conservative LBD, by that can activate the target of nuclear receptor with ligand binding Gene.DAX-l is probably nuclear receptor family earlier occur in evolutionary process, and LBD undergos mutation in evolution process, lose with The ability of ligand binding, thus while DAX-l has LBD, but not yet finds its part at present.
This experiment is by the collection to clinical data, using large scale sequencing technology in Non-obstructive Azoospermia patient The mutational site of DAX-1 genes is screened, function test result shows DAX-1 V385L mutant and DAX-1 wild types in cell There were significant differences for interior function, points out this mutational site to there is correlation with non-obstructivity azoospermatism, and DAX-1 may It is the clinical potential diagnosis and treatment target spot of male sterility one.
4 conclusions
4.1 filter out 6 missense of the DAX-1 genes only in Patients with Non-obstructive Azoospermia by large scale sequencing It is mutated, its corresponding amino acid change is:R51K, C104W, A242V, E256Q, V385L, I427V, with dbSNP135 databases Compared with the data in thousand human genome databases pair, find no these mutation.
There were significant differences with the combinations of AR in the cell for 4.2 DAX-1 wild types and DAX-1V385L mutant.
4.3 DAX-1 V385L mutant and the expression after AR interactions to AR downstream target genes MMTV and DAX-1 are wild Raw type, which is compared, significant difference.
Above content is to combine specific embodiment further description made for the present invention, it is impossible to assert this hair Bright specific implementation is confined to these explanations.For general technical staff of the technical field of the invention, do not taking off On the premise of from present inventive concept, some simple deduction or replace can also be made.

Claims (3)

1. a kind of genetic marker related to Non-obstructive Azoospermia, its nucleotide sequence is the coded sequence of DAX-1 genes, Numbering of the DAX-1 genes in ncbi database is Gene ID:190, the nucleotide sequence includes mutational site c.1153G>T。
2. a kind of primer pair of the genetic marker described in test right requirement 1 is in the heredity mark being prepared by described in claim 1 The application of the kit of note detection Non-obstructive Azoospermia, the sequence such as SEQ ID NO of the sense primer of the primer pair:5 It is shown, the sequence such as SEQ ID NO of anti-sense primer:Shown in 6.
3. application of the genetic marker on Non-obstructive Azoospermia detection kit is prepared described in a kind of claim 1.
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