Mitochondrial DNA variation of Leber’s Hereditary Optic Neuropathy (LHON) in Western Siberia

Leber’s hereditary optic neuropathy (LHON) is a form of disorder caused by pathogenic mutations in a mitochondrial DNA. LHON is maternally inherited disease, which manifests mainly in young adults, affecting predominantly males. Clinically LHON has a manifestation as painless central vision loss, resulting in early onset of disability. Epidemiology of LHON has not been fully investigated yet. In this study, we report 44 genetically unrelated families with LHON manifestation. We performed whole mtDNA genome sequencing and provided genealogical and molecular genetic data on mutations and haplogroup background of LHON patients in the Western Siberia population. Known “primary” pathogenic mtDNA mutations (MITOMAP) were found in 32 families: m.11778G>A represents 53,10% (17/32), m.3460G>A – 21,90% (7/32), m.14484T>C – 18,75% (6/32), and rare m.10663T>C and m.3635G>A represent 6,25% (2/32). We describe potentially pathogenic m.4659G>A in one subject without known pathogenic mutations, and potentially pathogenic m.9444C>T, m.6261G>A, m.9921G>A, m.8551T>C, m.8412T>C, m.15077G>A in families with known pathogenic mutations confirmed. We suppose these mutations could contribute to the pathogenesis of optic neuropathy development. Our results indicate that haplogroup affiliation and mutational spectrum of the Western Siberian LHON cohort substantially deviate from those of European populations.


Introduction
Leber's hereditary optic neuropathy is a form of hereditary disorder caused by pathogenic mutations in a mitochondrial DNA. These mutations are non-synonymous and affect genes coding for different subunits of complex I of the mitochondrial respiratory chain. The occurrence of such kind of mutations in mtDNA subunits leads to dysfunction of the electron transport, increased reactive oxygen species production and defective ATP synthesis ( Consequently, the worldwide prevalence of LHON varies in different populations and is unknown for the majority of them. According to broad epidemiologic studies, it is estimated between 1:30000 and 1:50000 (   42 mitochondrial genomes obtained through this study were deposited in GenBank with accession numbers XX000000-XX000000. Two genomes, EU807741.1 and EU807742.1, had been accessed earlier (Brown, Zhadanov et al. 2001).

Penetrance analysis
We determined penetrance as the proportion of affected individuals from all maternally related family members using family pedigrees (Puomila, Hamalainen et al. 2007). Values for both men and women were calculated separately. Pedigrees are available in supplementary materials.

Analysis of pathogenicity for non-synonymous mutations
To make sure that the revealed non-synonymous mtDNA mutations are not sequencing errors or hot points for the general population, and to find out the disease-associated To assess the possible pathogenicity of these mutations and to predict whether a protein sequence variation affects protein function, we used the following web applications: MutPred 1.

Results
From 44 LHON families, 32 harbored a primary mutation; the results are shown in Table   1 Table 2. The average penetrance among men was 32% (6-100%) and among women -12% (0-58%). Our data correlate with data previously published by (Meyerson, Van Stavern et al. 2015). However, there are particular families with higher penetrance among females than among males: L24, L26, L28. The penetrance is highly variable between separate families, even with the same primary mutation as shown in pedigrees in supplementary materials.  Table 3.

Summary information on penetrance is shown in
In several families with primary mutations (L01, L03, L12, L28, L30, L40, L43) we found out additional non-synonymous mutations, as shown in Table 4. Mutations m.8875T>C, m.14582A>G, m.8400T>C, m.4639T>C were neutral and mutation m.9444C>T had high probability to be pathogenic according to data of 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 the majority of patients, and 58-84% of patients with DOA report visual impairment by age 11 (Fraser, Biousse et al. 2010). In our 11 cases ages of onset were between 13 -36 years. We are going to study these cases for the mutation spectrum of common pathogenic genes for DOA in future.

Penetrance
We suppose that the reasons for low prevalence of LHON disease in Western Siberia are associated with incomplete penetrance and diagnostic difficulties of atypical (e.g. late onset) and

Potentially pathogenic mutation m.4659G>A
We found out m.4659G>A in one subject without any known primary mutations (L51).
This sequence change is located at codon 120 in the functional domain of the CO1 gene and changes an alanine, a hydrophobic amino acid, into threonine -a neutral amino acid. Mutation m.4659G>A has been reported as associated with LHON in an Australian pedigree that also had heteroplasmic mutation m.14484T>C and m.5460G>A (Mackey and Howell 1992). This family had 10 maternally related descendants, five of whom with vision loss. Unfortunately, our patient does not know his family history and we couldn't confirm maternal inheritance for this mutation.
However, m.4659G>A has very low frequency in the general population (0,0011) and high probability to be pathogenic according to data of different prediction tools (Table 3).

Additional non-synonymous mutations revealed in LHON cases
Phenomenon of co-existence of two pathogenic mutations in one family has already been described. In the first case, m.14484T>C, m.4659G>A and m.5460G>A in an Australian LHON pedigree, described above (Mackey and Howell 1992

Conclusion
These data may be insufficient for evaluating the prevalence of LHON. However, the project is still ongoing, and all the data collected might be used to develop a LHON registry in Russian Federation.    Figure 1, 2, 3. Phylogenetic trees based on complete mtDNA genome sequences of pedigreeprobands with pathogenic mutations. The non-synonymous coding-region variants are denoted by "ns" (known pathogenic mutations designated "in bold"), the insertions and deletions are denoted by "ins" and "del," respectively, underlined mutations are recurrent.