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目的探讨先天性无虹膜症家系的基因突变位点。方法抽取家系成员的外周血2~5ml,提取DNA;先合成2个多态性微卫星遗传标记(D11S904和D11S935)的引物进行聚合酶链反应(PCR),PCR产物变性后用变性聚丙烯酰胺(PAGE)胶分离,根据带型和家系成员间的关系进行单体型连锁分析,判断家系无虹膜表型是否与PAX6基因相关;PCR扩增PAX6基因的所有外显子,所有PCR产物分别进行单链构象多态性(SSCP)分析,通过患者与正常人带型的差异确定突变发生的外显子,对有差异SSCP带型的PCR产物进行直接DNA测序,找到突变位点。结果该家系先天性无虹膜表型明显与PAX6基因连锁;SSCP分析PAX6基因第9外显子PCR产物,显示患者均有异常带型出现,而家系正常人均无此异常带;测序结果显示突变位点为PAX6基因第9外显子c1080核苷酸C突变为T,使编码精氨酸的密码子突变为终止密码子。结论PAX6基因突变可导致先天性无虹膜。
Objective To investigate the gene mutation sites of congenital aniridia family. Methods Two to five milliliters of peripheral blood from family members were extracted and DNA was extracted. Two primers for two polymorphic microsatellite markers (D11S904 and D11S935) were synthesized for polymerase chain reaction (PCR). The PCR products were denatured, denatured with polyacrylamide (PAGE) gel. According to the relationship between genotype and family members, haplotype linkage analysis was performed to determine whether the family iris phenotype was associated with PAX6 gene. All the exons of PAX6 gene were amplified by PCR, and all the PCR products were separately performed Single-strand conformation polymorphism (SSCP) analysis was performed to determine the exon in which the mutation occurred based on the difference between the patient’s and normal people’s band patterns. Direct DNA sequencing was performed on the PCR products with differential SSCP band patterns to find the mutation sites. Results The congenital non-iris phenotype of this pedigree was linked to PAX6 gene. SSCP analysis of PAX6 gene exon 9 PCR products showed that all the patients had abnormal bands, while normal individuals had no such abnormal bands. Sequencing results showed that the mutations The point is that the nucleotide C of exon 9 of the PAX6 gene is mutated to T and the codon encoding arginine is mutated to a stop codon. Conclusions Mutations in PAX6 gene lead to congenital absence of iris.