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目的通过遗传物理图谱对Y染色体大片段缺失的断裂位点进行精确的定位,研究Y染色体无精子症因子(azoospermia factor,AZF)区域微缺失与无精症的关系。方法应用多重PCR在4组反应管中对AZFa区的序列标签位点(sequence tagged site,STS)即sY82,sY84,sY86,AZFb区的sY124,sY127,sY128,sY133,sY134,sY143,AZFc区的sY239,sY242,sY254,sY255和AZFd区的sY145,sY152共15个STS位点进行扩增,以性别决定因子为内控,根据多重PCR结果对sY82,sY86,sY85,sY84进行单独扩增。结果对照组中的15个STS位点及单独扩增的sY85中均有特异性扩增产物,而Y染色体异常患者样品仅有sY82,sY86扩增产物,其余呈阴性。从而将患者的近着丝粒端的断裂位点定位于sY86与sY85之间。结论本研究为该患者的Y染色体大片段缺失断裂位点的精确定位提供了直接的分子生物学证据,建立了近着丝粒端的缺失图谱,证明了该患者无精的原因为AZF基因缺失。
OBJECTIVE: To determine the precise location of the deletion site of the large Y chromosome fragment by genetic mapping, and to study the relationship between azoospermia factor (AZF) region deletion and azoospermia. Methods The multiplexed PCR was used to detect the sequence tagged site (STS) of AZFa region in sY82, sY84, sY86 and sY124, sY127, sY128, sY133, sY134, sY143, AZFc sY239, sY242, sY254, sY255 and sY155 and sY152 in sY145 and sY152. A total of 15 STS sites of sY239, sY242, sY254, sY255 and AZFd were amplified with sex determinants. The sY82, sY86, sY85 and sY84 were individually amplified according to the multiplex PCR results. Results There were specific amplification products in 15 STS sites and sY85 in the control group. However, only the products of sY82 and sY86 were detected in the patients with Y chromosome abnormality, and the others were negative. The site of the proximal centromeric end of the patient is thus located between sY86 and sY85. Conclusion This study provided direct molecular biological evidence for the precise location of the large deletion fragment of the Y chromosome in this patient and established a deletion map of the proximal centromere. It was demonstrated that AZF gene deletion was a cause of azoospermia in this patient.