先天性无虹膜症家系的基因突变位点研究

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。

Study of genetic mutation locus in a family with congenital aniridia

Objective The aim of this study was to determine the genetic mutation locus of congenital aniridia. Methods Peripheral vein blood (2-5 ml) was collected from all members of a congenital aniridia family. DNA was extracted, then, the primers of polymorphic microsatellite genetic markers were synthesized, followed by polymerase chain reaction (PCR) analysis. Polyacrylamide gel electrophoresis (PAGE) was used to analyze the denatured PCR products and haplotype linkage analysis was applied according to the relationship between bands and family members to determine the association between the phenotype of aniridia and PAX6 gene. Fourteen exons of human PAX6 gene were amplified by PCR and allele specific variations were detected by single strand conformation polymorphism (SSCP). By comparing the difference of bands between patients and the unaffected members, the mutated exons was detected. The mutated locus was detected by direct DNA automated sequencing of the PCR products with different SSCP bands. Results PAX6 mutation was linked to the occurrence of aniridia in this family. An extra band corresponding to exon 9 in PAX6 was found by single strand conformational polymorphism analysis in all aniridia patients in this family, but not detected in the unaffected members. A mutation of C to T was detected by sequencing at the nucleotide 1080 that converts the Arg codon (CGA) to a termination codon (TGA). Conclusion Mutation of PAX6 gene can result in the occurrence of congenital aniridia.