Figure 3. Mutation analysis of CRYBB1. A: The sequence chromatogram of a wild type allele shows glutamine (CAG) at codon 223. B: The sequence chromatogram of a mutant allele shows a heterozygous C→T transition that changed glutamine 223 to a stop codon (TAG). C: RFLP analysis illustrates that the mutation, Q223X, introduces a new BfaI site. This mutation-specific BfaI digestion pattern co-segregated with the disease phenotype. Squares and circles symbolize males and females, respectively. The clear and shaded symbols denote unaffected and affected individuals, respectively. D: Multiple sequence alignment of the fourth Greek key motif of CRYBB1 is shown from Homo sapiens (codons 191–233), Mus musculus, Rattus norvegicus, Bos Taurus, Cavia porcellus, Gallus gallus, Danio rerio, and Xenopus tropicalis. The Gln223 residue is highly conserved. “X” indicates premature chain-termination mutations in human CRYBB1 (Q223X).