The cohort of Chinese pediatric hearing-impaired subjects consisted of 98 subjects with aminoglycoside ototoxicity and 342 subjects, who did not have a history of exposure to aminoglycosides. Of known deafness-associated 12S rRNA mutations, the 1555A > G mutation accounted for 7.5% cases of this Chinese clinical population, while incidences of this mutation were 1.76% and 3.96% in two large cohorts of hearing impaired pediatric Han Chinese subjects from schools of deaf children [
22,
36]. In the present study, the incidences of the 1555A > G mutation were 2.7% and 21.4% cases of nonsyndromic and aminoglycoside-induced hearing loss, respectively. In fact, the incidences of the 1555A > G mutation varied among different ethnic origins. With regard to the subjects with aminoglycoside ototoxicity, the incidences of the 1555A > G mutation were 33% in a small Japanese cohort [
19] 13%, 10.4% and 5% in three Chinese cohorts [
3,
21,
22] and ~17% in the two white cohorts from United States and Spain [
5,
32,
33]. However, the incidence of 1555A > G mutation in nonsyndromic hearing loss was much lower than in those with aminoglycoside ototoxicity. In two white cohorts with nonsyndromic hearing loss, the frequency of the 1555A > G mutation varied from 0.6% to 2.5% [
20,
24], while the incidence of the 1555A > G mutation in several Asian cohorts ranged from 2.9% to 5.3% [
19,
21-
23]. Thus, the large proportion of subjects with aminoglycoside ototoxicity in this cohort may contribute to higher incidence of the 1555A > G mutation than other cohorts. On the other hand, the incidences of the 1494C > T mutation appeared to be lower than those of the 1555A > G mutation. In this cohort, two subjects carrying the 1494C > T mutation had a history of exposure to aminoglycosides. This data appeared to be higher than the previous reports that three familial cases of 1340 sporadic Spanish hearing-impaired subjects carried the 1494C > T mutation [
12] and three cases of 1642 pediatric deaf children [
22]. Therefore, these two known 12S rRNA mutations account for from 4% to 8% cases among these Chinese hearing-impaired populations [
10].
Of other known deafness-mutations, the frequency of the 1095T > C mutation was 0.91% in this cohort. The 1095T > C mutation, whose CI was 92.9%, occurred in one of 449 Chinese controls. This mutation has been found in several genetically-unrelated families with nonsyndromic and aminoglycoside-induced hearing loss [
21,
22,
30,
31]. This T-to-C transition disrupted an evolutionarily conserved base-pair at stem loop of the helix 25 of 12S rRNA [
27]. This nucleotide is also located at the P-site of ribosome, suggesting an important role in the initiation of mitochondrial protein synthesis [
31]. Furthermore, the frequency of mutations at position 961 including 961insC and 961T > C was 2.27% in this pediatric population. Although mutations at this position have been implicated to be associated with hearing loss in different ethnic groups [
21,
22,
32,
33], the lower CI (42.9%) and presence of 4% in the controls indicated that mutations in this position were polymorphisms.
A total of 41 (39 known and 2 novel) variants in 12S rRNA gene were identified in this cohort. Similar to other mtDNA variations, these variants can be grouped into three categories: neutral, adaptive and deleterious [
35]. To identify putative deleterious mutation, these variants were further evaluated using following three criteria: 1). Absent in the 449 Chinese controls; 2). CI is >78%, proposed by Ruiz-Pesini and Wallace [
35]; 3). Potential structural and functional alterations [
22]. Among these variants, 19 variant were absent in the 449 Han Chinese controls, while the frequency of other variants ranged from 0.2% (13 variants such as 789T > C) to 22.7% (709G > A variant) in this Chinese control population. In particular, some of these variants occurring at high frequencies of both control and patient populations were the mitochondrial haplogroup specific variants [
36]. These included the 663A > G variant of haplogroup A, the 827A > G and 1119T > C variants of haplogroup B4, the 709G > A and 1598G > A variants of haplogroup B5, the 1382A > C variant of haplogroup D4, the 681T > C, 752C > T, 1048C > T and 1107T > C variants of D5 haplogroup, the haplogroup F2 specific variant 1005T > C, the 1041A > G variant of haplogroup M9a, and the 1541T > C variant of haplogroup R5b [
38]. Apparently, these haplogroup specific variants were adaptive or neutral but unlikely deleterious.
Phylogenetic analysis showed that CIs of 28 variants were more than 78%. Despite their higher CI, the 14 variants such as 663A > G, 681T > C, 752C > T, 735A > G, 827A > G, 1107T > C, 1382A > C and 1438A > G were present in the controls. On the other hand, the CIs for other 7 variants including 1555A > G and 1494C > T were at least 78% but these variants were absent in 449 Chinese controls. Based on the predicted secondary structure of mitochondrial 12S rRNA [
27,
35], 23 variants were located at the loops and 18 variants occurred in the stems of this rRNA. Among these variants, 11 variants including the 1095T > C disrupted a WC base pairing(s) of 12S rRNA, while 5 variants including the 1555A > G and 1494C > T created a novel WC base-pairing(s) of this rRNA [
28,
29]. In fact, the 1555A > G or 1494C > T mutation made the mitochondrial ribosome more bacteria-like [
4,
11,
14]. Functional characterization demonstrated that the 1555A > G or 1494C > T mutation conferred sensitivity to aminoglycosides [
11,
15,
16,
18]. Thus, individuals carrying either of mutations are predisposed to hearing loss. Indeed, the novel 747A > G variant and the known 839A > G, 1310C > T and 1413T > C variants [
22,
34], which resided at the stems of 12S rRNA, were fitted with three criteria for the pathogenic mutations as described above. Furthermore, the 1027A > G variant, whose location was at a loop in the 12S rRNA and whose CI was 92.9%, was absent in 449 Han Chinese controls. Thus, alterations of the tertiary or quaternary structure of 12S rRNA by these putative variants may lead to significant effects on function, thereby contributing to the deafness phenotype. Genetic and clinical evaluations of these five hearing-impaired Chinese subjects carrying one of 5 putative 12S rRNA mutation were performed. The pedigree FE239 carrying the 1027A > G mutation exhibited suggestively maternally transited hearing loss, while other four pedigrees did not have a typically maternal inheritance of hearing loss. The presence of the known 235DelC/299DelAT mutation in the
GJB2 gene in the subject ZX039-IV-1 carrying the 1413T > C mutation indicated its role in the deafness phenotype. The absence of mutation(s) in the
GJB2 gene in other four subjects suggested the involvement of other modifier factors in the phenotypic manifestation of these putative deafness-associated 12S rRNA variants, as in the case of these families carrying the 1555A > G mutation [
39]. Further genetic and biochemical characterizations were necessary for the understanding the pathophysiology of these putative deafness-associated 12S rRNA mutations. Moreover, approximately 70% of subjects with aminoglycoside-indece hearing loss in this cohort did not carry the pathogenic 12S rRNA 1555A > G and 1494C > T mutations as well as putative deafness-associated 12S rRNA mutations. These data implicated the involvement of other nuclear genes, besides mitochondrial 12S rRNA mutations, in development of hearing loss in these subjects.
2,6,7
