**3. Results**

From 44 LHON families, 32 harbored a primary mutation; the results are shown in Table 1. Among families with a primary mutation, the m.11778G>A represented 53.10% (17/32), m.3460G>A was 21.90% (7/32), and m.14484T>C represented18,75% (6/32). Rare m.10663T>C and m.3635G>A represented 6.25% (2/32) of the families.


**Table 1.** Summary data for examined Leber's hereditary optic neuropathy (LHON) and LHON-like (without primary mutations) families. Age of onset of visual loss vary between families/patients; the peak age of onset is ~20–30 years old. Some families were published previously \* [23]; \*\* [22]; \*\*\* [21].


**Table 1.** *Cont.*

According to the family pedigrees, only 50% (22/44) of cases had a family history of vision loss in the maternal lineage in more than one generation, among which m.11778G>A represented 36% (8/22), m.3460G>A covered 23% (5/22), and the m.14484T>C was 14% (3/22), as well as those without primary mutations (LHON-like cases), which represented 18% (4/22). Rare mutation cases (m.10663T>C and m.3635G>A) were family–inherited. In total, 39% (17/44) of cases were sporadic, among which 13 were cases with only one affected person diagnosed, and four were cases with two affected persons in one generation. Among the sporadic cases, m.11778G>A represented 41% (7/17), m.3460G>A was 12% (2/17), m.14484T>C was 6% (1/17), and the LHON-like cases represented 41% (7/17) of the total.

Summary information on penetrance is shown in Table 2. The penetrance is highly variable between separate families, even with the same primary mutation. The average penetrance among men was 32% (6–100%) and 12% among women (0–58%); these correlate with data previously published [4]. However, there are some families with higher penetrance among females than among males: L24, L26, and L28.

**Table 2.** Summary information about penetrance.


In 12 families with clear-cut LHON phenotypes, no pathogenic mtDNA mutations were found. Analysis of the mtDNA revealed non-synonymous mutations: m.4766A>G, m.13105A>G, m.14002A>G, which have not been noted as associated with LHON or other diseases in the MITOMAP database. All pathogenicity prediction tools indicated low probability that the amino acid substitutions are disease-associated for these mutations. Mutation m.4659G>A has been previously reported as being associated with Parkinson's disease [35], as well as in an Australian LHON pedigree that was heteroplasmic for the m.14484T>C [36]. Polyphen-2 predicted the pathogenicity for this mutation as benign and MutPed 2 showed low probability score, but MutPred 1.2, PROVEAN, and SIFT determined this mutation as deleterious. The results are shown in Table 3.

**Table 3.** Non-synonymous mutations revealed in LHON-like cases. Known primary mutations (m.3460G>A, m.3635G>, m.10663T>C, m.11778G>A, and m.14484T>C) are placed in bold to demonstrate distinction between different prediction algorithms and frequencies in general population for pathogenic mutations. A MutPred 1.2 score > 0.75 and a Mutpred 2 score > 0.50 would sugges<sup>t</sup> pathogenicity.


In several families with primary mutations (L01, L03, L12, L28, L30, L40, L43, and L50) we found out additional, non-synonymous mutations (Table 4). Mutations m.8875T>C, m.14582A>G, m.8400T>C, and m.4639T>C were neutral, and mutation m.9444C>T had a high probability of being pathogenic, according to data from all the pathogenicity prediction tools; for other mutations, we observed divergence of prediction results. Since prediction results for primary pathogenic mutations diverged too (see Table 3), novel non-synonymous nucleotide change was considered potentially pathogenic if it had extremely low frequency in the general population, and if it was predicted by at least three algorithms to have an e ffect on protein function. For mutations m.6261G>A and m.15468C>T, only PolyPhen2 predicted pathogenicity as probably damaging and possible damaging, respectively. However, mutation m.6261G>A had already been reported by Abu-Amero [37] in patients with optic neuropathy, and also as a somatic mutation associated with prostate cancer. Interestingly, the family (L01) with m.6261G>A and m.11778G>A has the same haplogroup, T2, as the case reported by Abu-Amero. Other mutations (m.8412T>C, m.8551T>C, m.9921G>A, m.15077G>A) were predicted as pathogenic by at least by three algorithms, but the first three of them have not been noted as associated with diseases in the MITOMAP database, and mutation m.15077G>A was reported as being associated with maternally-inherited isolated deafness [38].

**Table 4.** Additional, non-synonymous mutations revealed in LHON cases. All these mutations still have no the status of "primary LHON mutations". A MutPred 1.2 score > 0.75 and a Mutpred 2 score > 0.50 would sugges<sup>t</sup> pathogenicity.


Phylogenetic analysis illustrates that Siberian carriers of pathogenic LHON mutations are unrelated and belong to di fferent maternal lines. In rare cases (4/44), m.3460G>A and m.14484T>C belong to East Eurasian M8, M9, and D haploclusters (Figure 1). The classic (m.3460G>A, m.11778G>A, m.14484T>C) and rare LHON-causing mutations occur mostly in the mtDNA background of West Eurasian haploclusters H'V (Figure 2), J'T and U'K (Figure 3).

**Figure 1.** Phylogenetic tree based on the complete mtDNA genome sequences of pedigree probands with pathogenic LHON mutations (M8, M9, and D haploclusters). The non-synonymous coding region variants are denoted by "ns" (known pathogenic mutations designated in bold). Mutations are transitions unless a specific base change was specified; deletions are denoted by "del"; underlined mutations are recurrent.

**Figure 2.** Phylogenetic tree based on the complete mtDNA genome sequences of pedigree probands with pathogenic LHON mutations (H'V haplocluster). The non-synonymous coding region variants are denoted by "ns" (known pathogenic mutations designated in bold). Mutations are transitions unless a specific base change was specified; deletions are denoted by "del"; underlined mutations are recurrent.

**Figure 3.** Phylogenetic tree based on the complete mtDNA genome sequences of pedigree probands with pathogenic LHON mutations (J'T, and U'K haploclusters). The non-synonymous coding region variants are denoted by "ns" (known pathogenic mutations designated in bold). Mutations are transitions unless a specific base change was specified; deletions are denoted by "del"; underlined mutations are recurrent.
