非梗阻性原发无精与少精症男性DAZ基因突变研究

目的:探讨DAZ基因突变对男性生殖的影响及表型特征。方法:筛选无明显诱因的男性非梗阻性无精及少精患者,首先排除染色体核型异常病例,进一步在AZFa、AZFb、AZFc和AZFd区域均匀选择缺失高发位点排除缺失,设130例正常男性与10例女性做对照,对DAZ1～4基因进行突变性研究。包括DAZ1～4共缺失性研究和基因内点突变研究。选择同时存在于DAZ1～4基因内部的STS位点sY587,结合其PCR扩增产物中DraI酶切位点的差异判断DAZ1～4共缺失情况;选择DAZ1～4高度同源保守的1～6外显子、接头以及上下游调控区域,采用PCR-SSCP、限制性酶切(RFLP)、PCR产物测序以及特异性基因型nest相结合方法对外显子进行突变检测;设sY14(SRY)基因作为阳性内参照。结果:经筛选得到80例先天非梗阻性无精与20例少精病例,所有患者及男性对照sY14(SRY)检测均为阳性,女性对照均为阴性;共发现16例DAZ共缺失患者,发生频率为20%;无精患者11例,DAZ1～4共缺失及DAZ1/DAZ2共缺失分别为1例和10例;少精患者5例,DAZ1～4共缺失及DAZ1/DAZ2共缺失分别为2例和3例;分别在3个不同患者的DAZ1的第2、4内含子发现3个变异位点,DAZ基因的调控序列、外显子及接头处未发现突变,而在130例正常对照中未见缺失及变异发生。结论:DAZ基因缺失是引起男性生殖异常的主要原因;共缺失的部位及长度与表型的严重程度未见相关性;基因组比较确定DAZ1第2内含子第64碱基处发杂合变异G→A为新的变异,是否是新的SNP位点、是否能够造成无精或少精需要进一步研究证实;DAZ基因突变发生情况也有待进一步探讨。

Detection of DAZ Gene Mutations in Patients with Non-obstructive Azoospermia or Oligozoospermia

Objective:To investigate the effect and correlation between the mutation of DAZ gene and male spermatogenesis.Methods:The mutations in DAZ 1-4 genes in 80 males with idiopathic non-obstructive azoopsermia and 20 oligozoospermia without hot spots deletion in AZFa,AZFb,AZFc,and AZFd were investigated.All cases of chromosomal karyotypes were normal,and 130 normal males and 10 females were selected as controls.Common STS of sY587 was picked out to detect co-deletion of DAZ1-4 by DraI RELP,PCR-RELP-SSCP and direct sequencing were applied to probe mutation in high homology and conservative region of DAZ1-4 genes,and these including exon1-6 and its exon/intorn junctions and up-down regulation areas.sY14(SRY)was used as an internal control.Results:SY14(SRY)was positive in all males and negative in 10 female controls.co-deletion of DAZ1-4 were found in 16 patients with the frequency of 20% and 11 oligozoospermia patients were involved:1 in DAZ1-4 and 10 in DAZ1/DAZ2,and 5 oligozoospermia were identified which were 2 in DAZ1-4 and 3 in DAZ1/DAZ2,and no deletion of DAZ3/DAZ4 was identified.A new heterozygous variation G to A had been detected in the 64bp of intron 2 of DAZ1 gene.Conclusion:It is supported that DAZ mutations is the main cause of male reproductive abnormality.No internal correlation between co-deletion position and length and severity of phenotype can be identified.More evidences are needed to verified the variation G to A in intron 2 of DAZ1 as a new SNP or as a inducement causing azoospermia or oligospermia.