*4.5. Haplogroup Analysis*

Our LHON cohort from the Western Siberia region is represented predominantly by West Eurasian haplogroups and includes several East Eurasian haplogroups, namely C5d1 (L24), D4p (L18), D5a2a2 (L25), and M9a1a1c1a (L10).

Rare LHON mutations m.10663T>C and m.3635G>A were found in Russian families from Kazakhstan (the first) and the Novosibirsk region (the second), associated with the European haplogroups J1c4 and J2b1c1, respectively [21,22]. Mutation m.10663T>C was also reported in the background of the haplogroups J1c2c, L2a1, L3'4, and L3f1b [37,45–47], and mutation m.3635G>A was reported in haplogroups R11a, D4g2b, M7b4, F1a, B5b, and M7b [48,49]. The presence of the

same pathogenic mutations on the background of various mitochondrial haplogroups confirms that pathogenic LHON mutations arise de novo, independently from the mtDNA or ethnic backgrounds.

It is known that the clinical impact of mDNA mutations may be modulated by mitochondrial haplogroup background. For example, Hudson et al. performed a multicenter study of 3613 subjects from 159 different families, and showed that the risk of visual failure is greater when m.11778G>A or m.14484T>C mutations are present in specific subgroups of haplogroup J; the same as the m.3460G>A mutation is present in haplogroup K, and the risk of visual failure is significantly lower when m.11778G>A occurs in haplogroup H [50]. Romero et al. supposed that haplogroup D has a protective effect in carriers of LHON mutations. His hypothesis was based on the fact that there was a markedly decreased frequency of haplogroup D in Chilean subjects with LHON, as haplogroup D is one of the most common in the Chilean population [12]. Also, other experimental research serves as proof that cybrids and fibroblasts bearing LHON mutations have different response to neurotoxic agents, depending on haplogroup background [51].

It has been suggested that at the end of the last glaciation, phylogenetically more ancient mutations could have provided their carriers with adaptive advantages during the human population expansion. Today, those mutations contribute to the saving and expression of weakly pathogenic LHON mutations, which appear randomly in different region-specific genetic backgrounds [52,53]. The theory could be tested by further searching of pathogenic, LHON-causing mutations in relation to specific mtDNA backgrounds (phylogenetically ancient set of mutations).

New data collected from future studies regarding mtDNA variations of LHON in Western Siberia might be used to develop a LHON system registry in the Russian Federation. We intend to conduct consecutive experimental research, including the parameters of the pathogenicity of each novel substitution.

#### **5. Limitations of the Study**

The main limitation of the data presented is the absence of physiological tests as proof with respect to the pathogenicity of novel mtDNA substitutions. Additional tests should be done, such as oxygen consumption, ATP and ROS measuring, and electron microscopy study (for example, [54]). In addition, development of the newest editing systems [55] could give us more reliable instruments to test pathogenicity.

**Author Contributions:** Conceptualization, E.S. and R.S.; data curation, I.B., E.S., S.S., S.D., A.N., N.V., I.M., and R.S.; analysis and interpretation of data, E.S., S.S., S.D., A.N., and R.S., writing—review and editing, E.S., S.S., S.D., A.N., I.M., and R.S.

**Funding:** The research was supported by the Russian Science Foundation [No. 17-75-20015] and the Russian Foundation of Basic Research [No. 19-29-04101].

**Conflicts of Interest:** The authors declare no conflict of interest.
