Purpose: To identify PAX6 mutations in patients from four Japanese families with aniridia.
Methods: Polymerase chain reaction (PCR)-single stand conformational polymorphism (SSCP) analysis (SSCA) was performed in probands of the families, and restriction analysis using MaeIII or AvaI was carried out in other affected family members.
Results: PCR-SSCA demonstrated in the proband from one family an extra-band in the PCR product for PAX6 exon 8. Base sequence analysis revealed that the patient is a heterozygote for a C to T transition mutation at codon 203. DNAs from the patient and another affected member in the same family were cut with MaeIII into two fragments, while non-affected members in the family showed only one MaeIII fragment, the result confirmed the mutation. In another family, PCR-SSCA revealed an extra-band in the PCR product for exon 9. Sequencing detected a C-->T substitution at codon 240 in the patient, the mutation resulted in loss of an AvaI site. AvaI cleavage analysis confirmed the mutation in the patient. The two transition mutations observed in the two families also predict the conversion of arginine to a stop codon (R203X and R240X, respectively) around the homeodomain (HD), leading to the truncation of the PAX6 protein within its glycine-rich region. No abnormal SSCP bands or abnormal restriction fragments were detected in patients from the other two families.
Conclusions: The two mutations sites identified in the two families, one at codon 203 and the other at codon 240, are those most frequently observed among 118 previously reported PAX6 mutations. This indicates that the two mutations are two hot-spots in the gene.