*4.2. Penetrance*

We suppose that relatively reduced incidence of LHON in Western Siberia is associated with incomplete penetrance and diagnostic difficulties of atypical (e.g., late-onset) and combined (e.g., multiple sclerosis) forms of LHON. Patients with LHON could have also been wrongly diagnosed as suffering from toxic amblyopia, tobacco–alcohol amblyopia, or optic neuritis [41]. On the other hand, the problem is that patients do not know their family history. Molecular testing for LHON

is not routinely performed in patients with optic atrophy in Russian Federation. Identification and registration of una ffected carriers plays an important role for prevention of disease manifestation. For example, there is strong evidence that smoking is associated with an increased risk of visual failure among LHON carriers—93% penetrance of vision loss in male smokers versus 66% in male non-smokers [17].

Our observations highlight the importance of molecular genetic examination for una ffected carriers. The presence of pathogenic mutations should be tested not only for probands, but for all relatives in the maternal line, Since the proportion of sporadic cases is about 40% according to our published data [4].

#### *4.3. Potentially Pathogenic Mutation m.4659G*>*A*

We found m.4659G>A in one subject without any known primary mutations (L51). This sequence change is located at codon 64 in the functional domain of the ND2 gene and changes an alanine—a hydrophobic amino acid—into threonine—a neutral amino acid. Mutation m.4659G>A has been reported as being associated with LHON in an Australian pedigree that also had heteroplasmic mutations m.14484T>C and m.5460G>A [36]. This family had 10 maternally-related descendants, five of whom had vision loss. Unfortunately, our patient does not know his family history, and we could not confirm maternal inheritance for this mutation. However, m.4659G>A has very low frequency in the general population (0.0011–0.00161), and has a high probability of being pathogenic (Table 3).

#### *4.4. Additional Non-Synonymous Mutations Revealed in LHON Cases*

The phenomenon of co-existence of two pathogenic mutations in one family has already been described. The first case included m.4659G>A, m.5460G>A, and m.14484T>C in an Australian LHON pedigree, described above [36]. In the second case, a Polish family harboring two primary LHON mutations m.3460G>A and m.11778G>A occurred in a haplogroup H background [43]. In the third case, a family harbored two primary LHON mutations, m.11778G>A and m.14484T>C, and both mutations had a synergistic pathogenic e ffect on protein function, as well as a higher degree of heteroplasmy of the m.14484T>C, correlated with an earlier age at onset [44]. Finally, the fourth example is a unique double-mutant ND4L with two concurrent mutations (m.10609T>C and m.10663T>C) in an Arab pedigree from Kuwait [45].

We reported mutations m.6261G>A, m.8412T>C, m.8551T>C, m.9444C>T, m.9921G>A, and m.15077G>A, which could be potentially pathogenic because of their low frequency in the general population, and high probability of pathogenicity according to data from di fferent prediction tools. Two of them, m.6261G>A and m.15077G>A, have already been reported in subjects with optic neuropathy and maternally-inherited isolated deafness, respectively. However, we suppose that additional non-synonymous mutations could either have a synergistically pathogenic or a protective effect. To demonstrate the full significance of novel mutations, a respiratory chain assay would need to be performed. An example is the study [38], where cybrids with m.15077G>A showed normal activities for mitochondrial electron chain enzymatic complexes.
