Figure 4. Mutation analysis of GJA8. Sequence chromatograms of the wild-type allele (A) demonstrate a nucleotide sequence encoding a serine (Ser) at codon 276, and the chromatograms of the mutant allele (B) demonstrate a C to T transition resulting in the substitution of serine (Ser) by phenylalanine (Phe). Confirmation of segregation of the S276F mutation was given by the PCR-RFLP method (C). M is the DNA ladder; Lane 1 shows the digestions of the PCR products amplified from samples of the patients by Hpy188I. Lane 2 and 4 show the PCR products amplified from samples of one patient or the unaffected member in the pedigree. Lane 3 illustrates the digestions of the PCR products amplified from samples of the unaffected family member by Hpy188I. The mutant primer results in the gain of Hpy188I sites producing digested fragments of 215 and 24 bp with wild-type GJA8 alleles, and the mutation in the GJA8 gene leads to an abolition of this site, remaining undigested (239 bp).