High prevalence of m.1555A > G in patients with hearing loss in the Baikal Lake region of Russia as a result of founder effect

Mitochondrial forms account approximately 1–2% of all nonsyndromic cases of hearing loss (HL). One of the most common causative variants of mtDNA is the m.1555A > G variant of the MT-RNR1 gene (OMIM 561000). Currently the detection of the m.1555A > G variant of the MT-RNR1 gene is not included in all research protocols. In this study this variant was screened among 165 patients with HL from the Republic of Buryatia, located in the Baikal Lake region of Russia. In our study, the total contribution of the m.1555A > G variant to the etiology of HL was 12.7% (21/165), while the update global prevalence of this variant is 1.8% (863/47,328). The m.1555A > G variant was notably more prevalent in Buryat (20.2%) than in Russian patients (1.3%). Mitogenome analysis in 14 unrelated Buryat families carrying the m.1555A > G variant revealed a predominant lineage: in 13 families, a cluster affiliated with sub-haplogroup A5b (92.9%) was identified, while one family had the D5a2a1 lineage (7.1%). In a Russian family with the m.1555A > G variant the lineage affiliated with sub-haplogroup F1a1d was found. Considering that more than 90% of Buryat families with the m.1555A > G variant belong to the single maternal lineage cluster we conclude that high prevalence of this variant in patients with HL in the Baikal Lake region can be attributed to a founder effect.

The m.1555A > G variant of the MT-RNR1 gene in the homoplasmic state was detected in 21 out of 165 studied patients with HL (Fig. 1).All 165 patients with HL were previously tested for the presence of pathogenic variants in the GJB2 gene associated with autosomal-recessive deafness, type 1A (DFNB1A, OMIM 220290) 18 .No causative variants in the GJB2 gene, including del(GJB6-D13S1830), del(GJB6-D13S1854) and del(GJB2-D13S175) were found in patients with the m.1555A > G variant in the MT-RNR1 gene.The average age of onset of HL in patients with the m.1555A > G variant of the MT-RNR1 gene was 2.7 years.In 85.8% of patients (n = 18) with this variant, bilateral profound sensorineural HL was confirmed.In the other 14.2% patients (n = 3), the severe degree of the HL was detected.In 23.8% of patients (n = 5) with the m.1555A > G variant we found a history of aminoglycoside use (Table S1).In this study, the total contribution of the m.1555A > G variant of the MT-RNR1

Discussion
In the present study, we carried out molecular-genetic screening for the m.1555A > G variant of the MT-RNR1 gene in mitochondrial DNA in 165 patients with HL from the Republic of Buryatia, located in the Baikal Lake region of Russia.The m.1555A > G variant of the MT-RNR1 gene contributed to the etiology of HL in 12.7% of the examined patients (21 out of 165).Notably, a high proportion of Buryat patients (20.2%) had the m.1555A > G variant compared to Russian patients (1.3%).It should be noted that this variant was not identified in the neighboring regions of South Siberia among Tuvinian (0/220) and Altaian (0/93) patients with HL 19,20 .However, the m.1555A > G variant has been identified in the northern parts of the Eastern Siberia, among Yakut (1/108) and Even (4/23) patients with HL [21][22][23] .This variant also has been found in Russian patients with HL from European  21,24 .In general, the prevalence of this variant in Russia is estimated at 1.18% (11/928) (Table S2) [20][21][22][23][24] .
Some authors suggest that the relatively high prevalence of the m.1555A > G variant in Asia (2.48%) may be associated with a wider use of aminoglycoside antibiotics in countries of this region [25][26][27]29 . On he other hand, the relatively low prevalence of the m.1555A > G variant in Europe (0.97%) and America (0.92%) may be associated with underestimation of mitochondrial forms of HL (Table S2).As of now in the DNA-testing of the hereditary HL, including tests using NGS technologies, the pathogenic m.1555A > G variant of the MT-RNR1 gene is not included in all research protocols 30 .In general, despite the relatively low global prevalence of the m.1555A > G variant (from 0.22% in Australia to 2.48% in Asia), it is found across all continents (Fig. 3).The wide distribution of the m.1555A > G variant is probably due to its de novo emergence in different regions of the world, as there are known sporadic cases of the m.1555A > G variant 31 .
Despite to the probability of the independent origin of the m.1555A > G variant in the different regions of the world, the extremely high prevalence of this variant has been identified in Spain (36.3%, 1200/3302) (Fig. 3, Table S2) [45][46][47][48][49] .Increased frequencies of the m.1555A > G variant only in this region of Europe suggest other factors contributing to spread of this pathogenic variant.However, the phylogenetic analysis of the complete mtDNA variation demonstrates that the MT-RNR1 gene is unlikely to be a hotspot region, because other known variants m.827A > G, m.961 T > C, and m.1005 T > C in this gene define sub-haplogroups, which are spread among Asians (B4b' d' e'j, A5b and F2) and Native Americans (B4b' d' e'j) 33 .In this case, the high proportion of the m.1555A > G variant in Spain, compared with other European countries, could possibly be related to exposure to exacerbating environmental factors and aminoglycoside treatment 39 .However, no significant differences in nutrition, living conditions and treatment methods were found between Spain and neighboring European countries 39 .Although different haplogroups have been identified among the m.1555A > G variant carriers in Spain, the majority of these carriers belonged to the haplogroup H (76%) 39 .The authors suggest that increased frequency of the m.1555A > G variant in patients with one haplogroup H (with three of the six studied haplotypes were specific for the m.1555A > G variant) in Spain may be due to a founder effect 39 .However, they emphasize, that haplogroup H is not specific to Spain, as this mitochondrial lineage is dominate in Europe, which does not exclude the possibility of an independent origin of the m.1555A > G variant on this major mitochondrial background 39 .Re-evaluation studies that further dissected mtDNA haplogroup H in Iberia confirmed that the previously reported overrepresentation of haplogroup H (38 from 50 individuals) among Spanish families affected by HL due to the m.1555A > G variant is primarily associated with sub-haplogroup H3 (15 from 38).This is believed to be the result of a significant, likely ancient, founder event associated with Franco-Cantabrian refuge area was the source of late-glacial expansions of hunter-gatherers that repopulated much of Central and Northern Europe approximately 15,000 years ago 50 .S2).
In general, this study confirms that 92.9% of Buryat families affected by HL due to the m.1555A > G variant share a common cluster consisting of several subclades (A5b, A5b1, and A5b1b) of the mitochondrial lineage affiliated with sub-haplogroup A5b.This finding suggests a single origin of this pathogenic variant from a common ancestor in the majority of detected cases in the Baikal Lake region of Russia.

Brief information about studied region
The Republic of Buryatia includes 21 districts and two cities (Ulan-Ude and Severobaikalsk) (https:// egov-burya tia.ru, accessed on 15 September 2022), with an area of 351.3 thousand km 2 .This region of the Russian Federation borders with Mongolia.The population of the Republic of Buryatia is 978,600 people, with an average density of 2.78 people/km 2 .The major ethnic groups are Buryats (30.1%) and Russians (59.4%) (https:// burst at.gks.ru/ vpn20 20, accessed on 25 January 2023).The Buryats are a Mongolic-speaking people and one of the largest indigenous groups of Siberia.Buryats share many customs with Mongols, including nomadic herding and using portable dwellings-yurts.The majority of the Buryat population lives in the Republic of Buryatia, Irkutsk Oblast' and Zabaykalsky Krai of Russia.Buryats also live in the northeastern part of Mongolia and China (Inner Mongolia).

Clinical and audiological analysis
For each patient, a medical history was collected, including the information on previous illnesses, allergological history, injuries and/or surgeries, the use of ototoxic drugs and the exposure to industrial noise.The hearing thresholds were determined by pure-tone audiometry, using a clinical tonal audiometer "AA222" ("Interacoustics", Middelfart, Denmark), according to the current clinical standards.Air-conduction and bone conduction thresholds were obtained at 0.125, 0.25, 0.5, 1, 2, 4 and 8 kHz.Severity of hearing loss was defined by pure tone average (PTA 0.5,1,2,4 kHz ), as mild (25-40 dB), moderate (41-70 dB), severe (71-90 dB) or profound (above 90 dB).

Detection of the m.1555A > G variant in the MT-RNR1 gene
DNA was extracted using the phenol-chloroform method from the blood leukocytes.Detection of the m.1555A > G variant in the MT-RNR1 gene was performed by PCR-RFLP analysis using the previously described oligonucleotide primer, and restriction ferment HaeIII 9 .The presence of the m.1555A > G variant in the MT-RNR1 gene was verified by Sanger sequencing using the original sequence of oligonucleotide primers: F-AAA CGC TTA GCC TAG CCA CA, R-GCT ACA CTC TGG TTC GTC CA, selected using the Primer-BLAST program 88 .

Analysis of mtDNA haplogroups
Sequencing of the mitochondrial genome by next generation sequencing (NGS) was performed using Illumina NextSeq 500.Haplogroups were determined in accordance with PhyloTree.orgmtDNA nomenclature-mtDNA tree Build 17 89 .

Figure 1 .
Figure 1.Detection of the m.1555A > G variant of the MT-RNR1 gene and its contribution to the etiology of HL in the Republic of Buryatia.(A)-Detection of the m.1555A > G variant of the MT-RNR1 gene in 3% agarose gel by PCR-RFLP analysis with use of HaeIII: M-marker PUc19/MspI, lanes 1, 2, 4-6, 8-normal (wt), lanes 3 and 7-m.1555A > G (original electrophoregram presented in supplementary Fig. S1); (B)-Sanger sequencing of the MT-RNR1 gene fragment; (C)-The contribution of the m.1555A > G variant of the MT-RNR1 gene in patients with HL is calculated for all patients (21 out of 165 patients), the proportion of the m.1555A > G variant of the MT-RNR1 gene, depending on ethnicity is calculated for unrelated families.

Figure 2 .
Figure 2. Topology of mtDNA haplogroups in 15 families with the m.1555A > G variant of the MT-RNR1 gene.

Figure 3 .
Figure 3.The worldwide prevalence of the m.1555A > G variant of the MT-RNR1 gene among 47,328 patients with HL.Note: The full data is presented in the supplementary information (TableS2).

Table 1 .
The prevalence of mitochondrial HL caused by the m.1555A > G variant of the MT-RNR1 gene in the Republic of Buryatia.The district with the highest prevalence is highlighted in bold; *-patient with m.1555A > G variant, who was born in Chita, is not included in the genetic-epidemiological analysis.