CN105112501B - A kind of detection kit of Non-obstructive Azoospermia - Google Patents
A kind of detection kit of Non-obstructive Azoospermia Download PDFInfo
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Abstract
The invention discloses a kind of detection kit of Non-obstructive Azoospermia, primer pair including detecting the genetic marker related to Non-obstructive Azoospermia, the nucleotide sequence of genetic marker is the sequence of the genes of DAX 1, and 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；The primer pair is selected from primer 1, primer 2, primer 3 or the primer pairs of primer 4.1 genes of DAX, 7 new mutational sites have been filtered out in Non-obstructive Azoospermia patient by large scale sequencing, research shows that DAX 1 above-mentioned mutation may cause the generation of Non-obstructive Azoospermia, so as to detect the mutation by using the kit of the present invention to realize that the diagnosis to male sterility (especially Non-obstructive Azoospermia) detects.
Description
The application is the applying date for September in 2014 2 days, Application No. 201410444435.5, entitled a kind of with non-stalk
The divisional application of the application of the related genetic marker of resistive 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 about influenceed the reason for causing one of male sterility extremely important 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 are 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 closely related with the generation of male sterility；Using gene Knockout, it is found that it is close nearly 400 genes occur with mouse sperm
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) gene that belongs on hypothalamic pituitary gonadal axis, its albumen encoded be core by
The member of body family.DAX-1 is weighed very much as a kind of transcription factor to pituitary gonadotropic cell and adrenocortical developed
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 forms, 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 spermatogenesis is adjusted, 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
C.152G the sequence of cause, the mutational site of the sequence are 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.
Preceding 6 mutation are missense mutation, and the 7th mutation is same sense mutation.
Above-mentioned mutation is in the 152nd site of 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 are the sequences of DAX-1 genes, institute
C.152G the mutational site for stating sequence is selected from>A、c.312C>G、c.725C>T、c.766G>C、c.1153G>T and c.1279A>G
One or more of.
C.1153G the mutational site is>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 is especially c.1153G>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, builds DAX-1 wild types and mutant expression plasmid is transfected into and studied into the cell, passes 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 it can realize that the diagnosis to male sterility (especially Non-obstructive Azoospermia) detects by the mutation.
Brief description of the drawings
Fig. 1 is 6 missense mutation site Sequencing chromatograms of azoospermia patients DAX-1 genes；
Fig. 2 is co-immunoprecipitation detection DAX-1 wild types and the result figure of mutant and AR combination；
Fig. 3 is DAX-1 mutant and the result figure of the influence after AR interactions to AR downstream target genes MMTV expression.
Embodiment
The present invention is described in further detail below by embodiment combination accompanying drawing.
1.2 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) casual inspection is three times in seminal fluid without sperm；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
Peripheral blood extraction genomic DNA is extracted, with the peripheral blood of sodium citrate anticoagulant tube collection research object, is 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
Using the peripheral blood genomic DNA of extraction as template, using 4 pairs of primer pairs of design synthesis only in non-obstructivity without essence
6 missense existing for sub- disease patient (W024, W033, W251, W505, W520, W566, W570, W628, W698) DAX-1 genes
Mutational site carries out specific amplification, and DAX-1 sequences check 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, then carry out 35 with 98 DEG C of 10s, 60 DEG C of 30s, 72 DEG C of 45s and follow
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, which is taken pictures, observes electrophoretogram, it is ensured that single band, remaining PCR primer serve 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 contain Hind III and two kinds of BamH1 as template, design
The primer of restriction enzyme site, with the 3 ' pcDNA3.1 (+) with HA Polypeptide tags for carrier, the DAX-1 expression of structure wild type
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-1 is built
6 kinds of mutant expression plasmids.Primer is synthesized by Invitrogen companies, and synthesized 6 pairs of primers are shown in Table 2.
The DAX-1 6 of table 2 is to rite-directed mutagenesis primer
1.2.5 luciferase reporter gene is tested
1.2.5.2 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)
1.2.5.3 HeLa cell culture
HeLa cells are trained with 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.
1.2.5.4 HeLa cell transient transfections
HeLa cells are inoculated in 24 orifice plates, transfected after cell attachment, method refers to transfection reagent
LipofectamineTM2000 specifications.After transfecting 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.
1.2.5.5 Dual-luciferase reportor systerm detects transcription factor activity
1) cell is collected after transfecting 24h, is inhaled with liquid-transfering gun and abandons 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) list is used after adding the Buffer of 19 μ l Luciferase Assay II into each EP pipes successively in light protected environment
Tube photometer detects firefly fluorescence illumination；
5) examined after adding 19 μ l STOP Buffer into each EP pipes again successively in light protected environment with single-tube luminometer
Sea pansy fluorescence illumination is surveyed, experimental error is reduced to correct sea pansy fluorescence with firefly fluorescence.Determine the relative activity of reporter gene.
1.2.6 co-immunoprecipitation experiment
1.2.6.2 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)
1.2.6.3 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.
1.2.6.4 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 second day cell attachment, according to transfection reagent LipofectamineTM 2000
Transfection procedure, by recombinant plasmid pcDNA3.1 (+) -3 ' HA-DAX1 WT and 6 kinds of mutant mut1-mut6 (experimental group, 2ug)
With pcDNA3.1 (+) -3 ' HA zero loads (control group, 2ug) and hAR expression vector (2ug) difference cotransfection to HeLa cells.Turn
Inhaled after dye 48h and abandon culture medium, it is molten with 1 × PBS according to the extraction step of the mammalian proteins extracts kit of Qiagene companies
Liquid 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 of centrifugations
5min, discard supernatant and be placed on ice, with 100ul Lysis Buffer (Benzonase containing 0.1U Nuclease, 1ul 100
× Protease Inhibitor) precipitation is resuspended, after 4 DEG C rotate 5min, 14,000 × g, 4 DEG C of centrifugation 10min, supernatant is shifted
Into new centrifuge tube.
1.2.6.5 co-immunoprecipitation
The albumen of experimental group and control group extraction is divided into 4 points, a copy of it adds 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, and it is 500ul to be supplemented to every pipe cumulative volume, it
After be separately added into 2ug AR antibody, 2ug HA antibody, 2ug IgG；4 DEG C of rotation 1h；Often it is pretreated to add 50ul for pipe
4 DEG C of rotations of ProteinA/G Beads are 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
It is outstanding, 12,000 × g, 4 DEG C of centrifugation 1min washings Beads (not having to pipette tips to inhale, flicked), wash 3 times, suctioned out with rifle altogether
Supernatant, but not blot, pay attention to that Beads can not be drawn onto；15ul Lysis Buffer and isometric are added into Beads
2XSDS sample-loading buffers, 98 DEG C of processing 10min, are that antigen antibody complex disintegrates 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.Add and a certain amount of be suitably diluted in containing 5% defatted milk
Identifying purpose albumen in the TBS-T buffer solutions of powder primary antibody (the anti-human HA of mouse and rabbit-anti people's AR monoclonal antibodies, 1:2000 is dilute
Release), 4 DEG C are overnight；Discard primary antibody, TBS-T buffer solution for cleaning 3 times, 5 minutes every time.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, it is fixed to develop 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,
The comparison of average uses independent samples t test, P between group<0.05 is statistically significant.
2 experimental results
The identification of 2.1 Patients with Non-obstructive Azoospermia DAX-1 point mutations
The extron of 776 Patients with Non-obstructive Azoospermia and the DAX-1 genes of 706 normal fertile males is sequenced
Interpretation of result, as shown in table 3：7 new mutations of DAX-1 genes are homozygous mutation, and mutational site is respectively：c.152G>A
(p.R51K)、c.312C>G(p.C104W)、c.725C>T(p.A242V)、c.766G>C(p.E256Q)、c.1153G>T
(p.V385L)、c.1279A>G (p.I427V) and c.162G>A (p.L54L), preceding 6 position missense mutation, last 1 is synonymous
Mutation, compared with the data in dbSNP135 databases and thousand human genome databases pair, find no these mutation, and
These new variations (as shown in table 3) are not found yet in 706 normal fertile males.Further PCR checkings missense mutation
As a 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 is mutated with AR, using Immunoprecipitation, in HeLa
Corotation enters hAR and DAX-1 wild types and mutant expression plasmid respectively in cell, and cell extraction total protein is collected after transfecting 48h,
Do the phase of anti-AR and anti-HA co-immunoprecipitation and anti-AR and anti-HA Western Blot detection albumen
Interaction and expression, as a result show compared with DAX-1 wild types, only DAX-1 V385L mutant and AR combination weakens.
2.3 DAX-1 mutant and the influence after AR interactions to AR downstream target genes MMTV expression
DAX-1 interacts to regulate and control the expression of downstream gene, in order to further detect DAX-1 6 as co-factor and AR
Kind mutant and the influence after AR interactions to AR downstream target genes MMTV expression, it is real using luciferase reporter gene
Test, AR expression plasmids and MMTV-LUC distinguished into cotransfection into HeLa cells with DAX-1 wild types and 6 kinds of mutant plasmids,
Because AR belongs to the ligand-dependent transcriptional factor, therefore handled after transfecting with androgen, the firefly fluorescence that will be detected
Value/sea cucumber fluorescent value represents the activity of promoter.It is consistent with wild type DAX-1, DAX-1 R51K, C104W, A242V,
E256Q, I427V mutant can also substantially suppress the activity of MMTV promoters, and only DAX-1 V385L 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, companion 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 contain 2 extrons, encode 470 amino acid altogether.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 pass 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 successfully constructs, 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 confirms：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 since 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 function that 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, lack DNA binding domain, vertebrate orphan core 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 the target that nuclear receptor can be activated with ligand binding
Gene.DAX-l is probably to occur nuclear receptor family earlier 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 find 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, prompts this mutational site correlation to be present 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 6 missense that the DAX-1 genes only in Patients with Non-obstructive Azoospermia are filtered out by large scale sequencing are dashed forward
Become, its corresponding amino acid change is：R51K, C104W, A242V, E256Q, V385L, I427V, with dbSNP135 databases and
Data in thousand human genome databases find no these mutation compared to.
There were significant differences with the combinations of AR in the cell for 4.2 DAX-1 wild types and DAX-1 V385L 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 detection kit of Non-obstructive Azoospermia, it is characterised in that the kit includes detection and non-obstruction
Property the related genetic marker of azoospermia primer pair, the nucleotide sequence of the genetic marker 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、
c.1279 A>G and c.162 G>One or more of A；The primer pair, selected from primer 1, primer 2, primer
The primer pairs of 3 or primer 4, 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 such as SEQ downstream
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, the sequence of primer such as SEQ downstream
ID NO:Shown in 4；
The sequence such as SEQ ID NO of the sense primer of the primer pairs of primer 3:Shown in 5, the sequence of primer such as SEQ downstream
ID NO:Shown in 6；
The sequence such as SEQ ID NO of the sense primer of the primer pairs of primer 4:Shown in 7, the sequence of primer such as SEQ downstream
ID NO:Shown in 8.
2. detection kit according to claim 1, it is characterised in that the nucleotide sequence of the genetic marker is
The sequence of DAX-1 genes, the mutational site of the sequence are 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.
3. detection kit according to claim 2, it is characterised in that the mutational site is c.1153 G>T.
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| WO2017000288A1 (en) * | 2015-07-01 | 2017-01-05 | 深圳市第二人民医院 | Primer pair for detecting idiopathic azoospermia-related genetic marker |
| CN105506110A (en) * | 2015-12-31 | 2016-04-20 | 杭州艾迪康医学检验中心有限公司 | Method and primer for detecting 8th and 25th whole exons of TEX11 gene |
| CN107227370A (en) * | 2017-07-24 | 2017-10-03 | 胡松 | Chromogene primer pair and probe combinations and chromosomal nucleic acid detection kit |
| CN109943631A (en) * | 2019-03-21 | 2019-06-28 | 吉林省银丰生物工程技术有限公司 | Non-obstructive Azoospermia auxiliary diagnosis gene detecting kit |
| CN113278689B (en) * | 2021-04-20 | 2022-05-03 | 厦门市妇幼保健院（厦门市计划生育服务中心） | Application of ATG4D as target in diagnosis and treatment of non-obstructive azoospermia |
| CN114395621B (en) * | 2022-02-28 | 2023-05-02 | 上海市第一人民医院 | Application of ADAD2 gene in preparation of diagnosis kit for detecting non-obstructive azoospermia |
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| CN102296112A (en) * | 2011-08-09 | 2011-12-28 | 南京医科大学 | Seminal plasma miRNA marker associated with human non-obstructive azoospermia and application thereof |
| CN102399898A (en) * | 2011-12-09 | 2012-04-04 | 南京医科大学 | Single nucleotide polymorphism (SNP) marker related with clinically cryptogenic non-obstructive azoospermia aided diagnosis and application of SNP marker |
| CN103290006A (en) * | 2013-06-25 | 2013-09-11 | 夏彦恺 | A clinically unexplained NOA-related mitochondrial DNA SNP marker and applications thereof |
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| CN102296112A (en) * | 2011-08-09 | 2011-12-28 | 南京医科大学 | Seminal plasma miRNA marker associated with human non-obstructive azoospermia and application thereof |
| CN102399898A (en) * | 2011-12-09 | 2012-04-04 | 南京医科大学 | Single nucleotide polymorphism (SNP) marker related with clinically cryptogenic non-obstructive azoospermia aided diagnosis and application of SNP marker |
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| CN105112501A (en) | 2015-12-02 |
| CN104531683B (en) | 2017-11-07 |
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