Purpose: To carry out the mutation analysis of the KIF21A gene in a four-generation Indian family affected with CFEOM1 and to find out the molecular basis of the most frequent mutations c.2860C>T and c.2861G>A in exon 21 of the KIF21A gene.
Methods: Mutational analysis was carried out by direct automated sequencing of the PCR products from exons 8, 20, and 21 of the KIF21A gene. Allele specific oligo hybridization analysis was carried out to study the segregation of the mutation within the family. Methylation status of the mutated CpG dinucleotide in exon 21 was detected using bisulfite genomic sequencing technique on genomic DNA isolated from blood and sperms.
Results: We found a previously reported missense mutation c.2860C>T (p.954R>W) in exon 21 of the KIF21A gene in our family. This mutation was found in a CpG dinucleotide. Bisulfite genomic sequencing revealed that all the CpG dinucleotides in exon 21 including the one which harbored the two most frequent mutations were methylated both in the genomic DNA from blood and sperms.
Conclusions: CFEOM1 phenotype in our family was caused by a previously reported most frequent missense mutation, c.2860C>T. This mutation occurred at the C residue in a CpG dinucleotide, which was found to be methylated. Previous work has demonstrated that this CpG dinucleotide is a mutational hotspot in the KIF21A gene, and our finding suggests that its high mutability may result, in part, from its methylated state.