To elucidate the molecular basis of visual impairment, we have performed a mutational analysis of the mitochondrial genome in these families. First, we examined three commonly known LHON-associated mtDNA mutations (G3460A, G11778A, and T14484C) by PCR-amplification and subsequent restriction enzyme
10-DAB analysis of PCR fragments derived from each proband of those families. We failed to detect the presence of the G3460A, G11778A, and T14484C mutations. We then performed a PCR-amplification of fragments spanning the entire mitochondrial genome and subsequent DNA sequence analysis in these probands. As shown in Fig. 2, the T to C transition at position 14502 (T14502C) in ND6 gene, which resulted in the substitution of an isoleucine for valine (I58V) at amino
acid position 58, has been found in those subjects. As shown in Fig. 3, the isoleucine at position 58 in ND6 is highly conserved among 20 organisms [23]. Indeed, this mutation has been associated with LHON in Chinese families [14] and [24] and with idiopathic cardiomyopathy in Japanese families [25]. Further sequence analysis, as shown in Fig. 2, confirmed the presence of the homoplasmic T14502C mutation in matrilineal relatives of these families but not in other members of these families. The allele frequency analysis of the T14502C mutation showed that one (18-year-old female) of 167 unrelated Chinese control subjects carried the T14502C mutation.
