【作者】 金瑞林；

【导师】 郑梅玲； 张延丽；

【作者基本信息】 山西医科大学 ， 妇产科学， 2004， 硕士

【摘要】 目的：探讨性反转综合征患者发病的分子遗传学机理；建立一种有效地在大片段中检测突变的方法，为临床诊治提供科学依据。方法：应用聚合酶链反应扩增12例性反转患者SRY基因片段；PCR产物通过限制性酶切—单链构象多态性方法检测未知突变；基因测序以进一步明确突变的内容和性质；应用荧光原位杂交技术证实患者X、Y染色体结构及有无嵌合体存在。结果：2例46XY女性和1例46XX男性患者SRY基因缺失，9例46XY女性患者PCR扩增出现609bp特异性片段；通过限制性酶切-单链构象多态性方法检测发现其中1例患者单链电泳带发生变异；基因序列分析进一步明确该例患者SRY基因HMG盒内相应于第244位核苷酸的位置存在点突变 (A→T)，导致第82位密码子由原来编码天冬酰胺Asn(AAT)变为编码酪氨酸Tyr(TAT)，发生错义突变；荧光原位杂交结果与经典细胞遗传学的染色体核型分析结果完全一致，且未发现嵌合体存在。结论：SRY基因的研究有助于阐明性反转发病的分子遗传学机理及SRY基因在性别决定中的作用；所建立的限制性酶切—单链构象多态性方法可用于实验室检测大片段中的突变。更多还原

【Abstract】 Objective: to explore the mechanism of molecular genetics in the patients with sex reverse syndrome; to develop a efficient method applied to screen for mutation in the long segments, which offers scientific ground for clinical diagnosis and treatment.Methods: The SRY gene segment in the genomic DNA of twelve patients with sex reverse syndrome was amplified by polymerase-chain reaction(PCR); the PCR products were screened mutations using restriction endonuclease fingerprinting-single strand conformation polymorphism(REF-SSCP); mutations were confirmed by DNA sequencing; fluorescence in situ hybridization(FISH) was used to detect X、Y chromosome and mosaicism.Results: SRY gene was deleted in two 46XY female patients and one 46XX male patient, 609-bp segments of SRY gene were amplified in nine 46XY female patients; using restriction endonuclease fingerprinting-Single strand Conformation polymorphism (REF-SSCP), electrophoretic bands of DNA single strand in one patienet differed from that in normal male control; by DNA sequenceing techniques, A novel missense mutation was identified within the HMG-box of SRY gene in this patient. A T is replaced by T in codon 82 at position +244, resulting in a substitution of asparagine(AAT) for tyrosine(TAT), this point mutation has not been descried earlier and be a new mutation; the results by fluorescence in situ hybridization(FISH) were consistent with kayotypes by cytogentics and chromosome mosaicism was not found. Conclusion: the study of SRY gene have provided valuable information for the understanding of molecular mechanism of the patients with sex reversal syndrome. this finding also supports the hypothesis that SRY play an important role in normal sex determination. The present REF-SSCP method is applicable to screen mutations in long segment DNA in laboratory.更多还原