Cong Ri-chang, Song Shhu-juan, Liu Ying-zhi
Department of Ophthalmology, Chaoyang Central Hospital, Chaoyang 122000, China.
Zhonghua Yan Ke Za Zhi. 2006 Dec;42(12):1113-7.
The aim of this study was to determine the genetic mutation locus of congenital aniridia.
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.
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).
Mutation of PAX6 gene can result in the occurrence of congenital aniridia.
本研究旨在确定先天性无虹膜的基因突变位点。
采集一个先天性无虹膜家系所有成员的外周静脉血（2 - 5毫升）。提取DNA，然后合成多态性微卫星遗传标记的引物，接着进行聚合酶链反应（PCR）分析。采用聚丙烯酰胺凝胶电泳（PAGE）分析变性后的PCR产物，并根据条带与家庭成员之间的关系进行单倍型连锁分析，以确定无虹膜表型与PAX6基因之间的关联。通过PCR扩增人PAX6基因的14个外显子，并采用单链构象多态性（SSCP）检测等位基因特异性变异。通过比较患者与未患病成员之间条带的差异，检测出突变的外显子。对具有不同SSCP条带的PCR产物进行DNA自动测序，检测突变位点。
PAX6突变与该家系中先天性无虹膜的发生相关。通过单链构象多态性分析发现，该家系所有先天性无虹膜患者中均出现了一条与PAX6基因第9外显子相对应的额外条带，而在未患病成员中未检测到。测序发现在第1080位核苷酸处发生了C到T的突变，该突变将精氨酸密码子（CGA）转换为终止密码子（TGA）。
PAX6基因突变可导致先天性无虹膜的发生。
