Mitochondrial 12S rRNA variants in 1642 Han Chinese pediatric subjects with aminoglycoside-induced and nonsyndromic hearing loss

In this report, we investigated the frequency and spectrum of mitochondrial 12S rRNA variants in a large cohort of 1642 Han Chinese pediatric subjects with aminoglycoside-induced and nonsyndromic hearing loss. Mutational analysis of 12S rRNA gene in these subjects identified 68 (54 known and 14 novel) variants. The frequencies of known 1555A>G and 1494C>T mutations were 3.96% and 0.18%, respectively, in this cohort with nonsyndromic and aminoglycoside-induced hearing loss. Prevalence of other putative deafness-associated mutation at positions 1095 and 961 were 0.61% and 1.7% in this cohort, respectively. Furthermore, the 745A>G, 792C>T, 801A>G, 839A>G, 856A>G, 1027A>G, 1192C>T, 1192C>A, 1310C>T, 1331A>G, 1374A>G and 1452T>C variants conferred increased sensitivity to ototoxic drugs or nonsyndromic deafness as they were absent in 449 Chinese controls and localized at highly conserved nucleotides of this rRNA. However, other variants appeared to be polymorphisms. Moreover, 65 Chinese subjects carrying the 1555A>G mutation exhibited bilateral and sensorineural hearing loss. A wide range of severity, age-of-onset and audiometric configuration was observed among these subjects. In particular, the sloping and flat-shaped patterns were the common audiograms in individuals carrying the 1555A>G mutation. The phenotypic variability in subjects carrying these 12S rRNA mutations indicated the involvement of nuclear modifier genes, mitochondrial haplotypes, epigenetic and environmental factors in the phenotypic manifestation of these mutations. Therefore, our data demonstrated that mitochondrial 12S rRNA is the hot spot for mutations associated with aminoglycoside ototoxicity.     

Mutation analysis of mitochondrial 12S rRNA gene in Polish patients with non-syndromic and aminoglycoside-induced hearing loss

Mutations in mitochondrial DNA have been reported as associated with non-syndromic and aminoglycoside-induced hearing loss. In the present study, we have performed mutational screening of entire 12S rRNA gene in 250 unrelated patients with non-syndromic and aminoglycoside-induced hearing loss. Twenty-one different homoplasmic sequence variants were identified, including eight common polymorphisms, one deafness-associated mutation m.1555 A>G and three putatively pathogenic variants: m.669 T>C, m.827 A>G, m.961 delT+C(n)ins. The incidence of m.1555 A>G was estimated for 3.6% (9/250); however, where aminoglycoside exposure was taken as a risk factor, the frequency was 5.5% (7/128). Substitution m.669 T>C was identified only in patients with hearing impairment and episode of aminoglycoside exposure, which may suggest that such additional risk factors must appear to induce clinical phenotype. Moreover, two 12S rRNA sequence variants: m.988 G>A and m.1453 A>G, localized at conserved sites and affected RNA secondary structure, may be new candidates for non-syndromic and aminoglycoside-induced hearing loss associated mutations.     

Mitochondrial haplotypes may modulate the phenotypic manifestation of the deafness-associated 12S rRNA 1555A>G mutation

Mitochondrial 12S rRNA 1555A>G mutation is one of the important causes of aminoglycoside-induced and nonsyndromic deafness. Our previous investigations showed that the A1555G mutation was a primary factor underlying the development of deafness but was insufficient to produce deafness phenotype. However, it has been proposed that mitochondrial haplotypes modulate the phenotypic manifestation of the 1555A>G mutation. Here, we performed systematic and extended mutational screening of 12S rRNA gene in a cohort of 1742 hearing-impaired Han Chinese pediatric subjects from Zhejiang Province, China. Among these, 69 subjects with aminoglycoside-induced and nonsyndromic deafness harbored the homoplasmic 1555A>G mutation. These translated to a frequency of ～3.96% for the 1555A>G mutation in this hearing–impaired population. Clinical and genetic characterizations of 69 Chinese families carrying the 1555A>G mutation exhibited a wide range of penetrance and expressivity of hearing impairment. The average penetrances of deafness were 29.5% and 17.6%, respectively, when aminoglycoside-induced hearing loss was included or excluded. Furthermore, the average age-of-onset for deafness without aminoglycoside exposure ranged from 5 and 30 years old, with the average of 14.5 years. Their mitochondrial genomes exhibited distinct sets of polymorphisms belonging to ten Eastern Asian haplogroups A, B, C, D, F, G, M, N, R and Y, respectively. These indicated that the 1555A>G mutation occurred through recurrent origins and founder events. The haplogroup D accounted for 40.6% of the patient’s mtDNA samples but only 25.8% of the Chinese control mtDNA samples. Strikingly, these Chinese families carrying mitochondrial haplogroup B exhibited higher penetrance and expressivity of hearing loss. In addition, the mitochondrial haplogroup specific variants: 15927G>A of haplogroup B5b, 12338T>C of haplogroup F2, 7444G>A of haplogroup B4, 5802T>C, 10454T>C, 12224C>T and 11696G>A of D4 haplogroup, 5821G>A of haplogroup C, 14693A>G of haplogroups Y2 and F, and 15908T>C of Y2 may enhance the penetrace of hearing loss in these Chinese families. Moreover, the absence of mutation in nuclear modifier gene TRMU suggested that TRMU may not be a modifier for the phenotypic expression of the 1555A>G mutation in these Chinese families. These observations suggested that mitochondrial haplotypes modulate the variable penetrance and expressivity of deafness among these Chinese families.     

Mutation analysis of mitochondrial DNA 12SrRNA and tRNASer(UCN) genes in non-syndromic hearing loss patients

Specific mitochondrial DNA (mtDNA) mutations in 12SrRNA and tRNASer(UCN) cause non-syndromic hearing loss (NSHL). In this study, we screened 466 hearing loss (HL) patients, negative for GJB2 mutations, for mutations in the two mtDNA genes and flanking regions. In total, 43 different variants were identified, 31 of which were polymorphisms, one was a mutation (m.1555A-->G), two were known variants of controversial pathological nature (m.827A-->G and m.961delTinsC(n)) and nine were newly identified variants. The frequency of m.1555A-->G in this set of HL patients was 0.3%, which was lower than expected. To assess the putative causative nature of controversial or newly identified variants, the frequencies of these variants were determined in 400 Belgian control subjects, and their effect on the secondary structure and their conservation among different species was determined. Our data provide further support for a polymorphic nature of the controversial m.961delTinsC(n) variant. In addition, two of the newly identified variants, m.636A-->G in the 12SrRNA flanking tRNA(Phe) and m.990T-->C in 12SrRNA, may be new candidates for pathogenic HL variants. If the pathogenic nature of m.636A-->G can be confirmed, this would be the first NSHL mutation in tRNA(Phe).     

Novel Variants in the Putative Peroxisome Proliferator‐activated Receptor γ Promoter and Relationships with Obesity in Men

Yet unidentified variants within the peroxisome proliferator‐activated receptor γ (PPARγ) 2 promoter may explain the inconsistent reports on associations between variants in the coding region and obesity or diabetes. Thus, we examined the putative PPARγ2 promoter (?3371 to +43 bp) for variants in 83 subjects with obesity or type 2 diabetes. We identified eight variants, seven of which were novel, including ?792A>G, ?816C>T, ?882T>C, ?1505G>A, ?1881C>T, ?1884T>A, ?2604T>C, and ?2953A>G. The variants ?816C>T, ?1505G>A, ?1881C>T, and ?2604T>C were in total linkage disequilibrium, and there was a high degree of linkage disequilibrium between several of the novel variants and Pro12Ala. The novel variants were, together with Pro12Ala and 1431C>T, examined for relationships with obesity among 234 men with early‐onset obesity with a BMI at age ～20 years of 33.2 ± 2.5 kg/m² and 323 nonobese men with a BMI of 21.7 ± 2.5 kg/m², who were also reexamined after ～29 years. The prevalence of the identified variants was not significantly different between the two groups, and the variants did not affect changes in BMI over time. In conclusion, the identified novel variants in the PPARγ2 promoter region do not explain the reported discrepancies in the association of previously identified variants with obesity and type 2 diabetes.     

Unique spectrum of SPAST variants in Estonian HSP patients: presence of benign missense changes but lack of exonic rearrangements

Background

Hereditary spastic paraplegia (HSP) is a clinically and genetically heterogeneous disorder that can be an autosomal-dominant, autosomal-recessive, or X-linked disease. The most common autosomal-dominant form of the disease derives from mutations in the SPAST gene.

Methods

The aim of this study was to analyze 49 patients diagnosed with HSP from the Estonian population for sequence variants of the SPAST gene and to describe the associated phenotypes. Healthy control individuals (n = 100) with no family history of HSP were also analyzed. All patient samples were screened using denaturing high performance liquid chromatography (DHPLC) and multiplex ligation-dependent probe amplification (MLPA) assay. Samples with abnormal DHPLC and MLPA profiles were sequenced, with the same regions sequenced in control samples.

Results

Sequence variants of SPAST were identified in 19/49 HSP patients (38.8%), twelve among them had pathogenic mutations. Within the latter group there was one sporadic case. Eight patients had pure, and four - complex HSP. The twelve variants were identified: seven pathogenic (c.1174-1G>C, c.1185delA, c.1276C>T, c.1352_1356delGAGAA, c.1378C>A, c.1518_1519insTC, c.1841_1842insA) and five non-pathogenic (c.131C>T, c.484G>A, c.685A>G, c.1245+202delG, c.1245+215G>C). Only 2 of these mutations had previously been described (c.131C>T, c.1245+202delG). Three mutations, c.1174-1G>C, c.1276 C>T, c.1378C>A, showed intrafamilial segregation.

Conclusion

This study identified new variants of the SPAST gene which included benign missense variants and short insertions/deletions. No large rearrangements were found. Based on these data, 7 new pathogenic variants of HSP are associated with clinical phenotypes.     

Mitochondrial 12S rRNA mutations associated with aminoglycoside ototoxicity

The mitochondrial 12S rRNA is a hot spot for mutations associated with both aminoglycoside-induced and nonsyndromic hearing loss. Of those, the homoplasmic 1555A>G and 1494C>T mutations at the highly conserved decoding region of the 12S rRNA have been associated with hearing loss worldwide. In particular, these two mutations account for a significant number of cases of aminoglycoside ototoxicity. The 1555A>G or 1494C>T mutation is expected to form a novel 1494C-G1555 or 1494U-A1555 base-pair at the highly conserved A-site of 12S rRNA. These transitions make the human mitochondrial ribosomes more bacteria-like and alter binding sites for aminoglycosides. As a result, the exposure to aminoglycosides can induce or worsen hearing loss in individuals carrying one of these mutations. Biochemical characterization demonstrated an impairment of mitochondrial protein synthesis and subsequent defects in respiration in cells carrying the A1555G or 1494C>T mutation. Furthermore, a wide range of severity, age-at-onset and penetrance of hearing loss was observed within and among families carrying these mutations. Nuclear modifier genes, mitochondrial haplotypes and aminoglycosides should modulate the phenotypic manifestation of the 12S rRNA 1555A>G and 1494C>T mutations. Therefore, these data provide valuable information and technology: (1) to predict which individuals are at risk for ototoxicity; (2) to improve the safety of aminoglycoside antibiotic therapy; and (3) eventually to decrease the incidence of hearing loss.     

Very low penetrance of hearing loss in seven Han Chinese pedigrees carrying the deafness-associated 12S rRNA A1555G mutation

Mutations in mitochondrial DNA (mtDNA) have been found to be associated with sensorineural hearing loss. We report here the clinical, genetic and molecular characterizations of seven Han Chinese pedigrees with aminoglycoside-induced and nonsyndromic bilateral hearing loss. Clinical evaluation revealed the variable phenotype of hearing impairment including severity, age-at-onset and audiometric configuration in these subjects. The penetrance of hearing loss in these pedigrees ranged from 3% to 29%, with an average of 13.6%, when aminoglycoside-induced deafness was included. When the effect of aminoglycosides was excluded, the penetrances of hearing loss in these seven pedigrees varied from 0% to 17%, with an average of 5.3%. Sequence analysis of the complete mitochondrial genomes in these pedigrees showed the presence of the deafness-associated 12S rRNA A1555G mutation, in addition to distinct sets of mtDNA polymorphism belonging to East Asian haplogroups B4, D4, D5 and F1, respectively. This suggested that the A1555G mutation occurred sporadically and multiplied through evolution of the mtDNA in China. Despite the presence of several evolutionary conservative variants in protein-encoding genes, there was the absence of functionally significant mutations in tRNA and rRNAs or secondary LHON mutations in these seven Chinese families. These suggest that these mtDNA haplogroup-specific variants may not play an important role in the phenotypic expression of the A1555G mutation in those Chinese families with very low penetrance of hearing loss. However, aminoglycosides appear to be a major modifier factor for the phenotypic manifestation of the A1555G mutation in these Chinese families.     

The coexistence of mitochondrial ND6 T14484C and 12S rRNA A1555G mutations in a Chinese family with Leber's hereditary optic neuropathy and hearing loss

We report here the clinical, genetic and molecular characterization of one three-generation Han Chinese family with Leber's hereditary optic neuropathy (LHON) and hearing loss. Four of 14 matrilineal relatives exhibited the moderate central vision loss at the average age of 12.5 years. Of these, one subject exhibited both LHON and mild hearing impairment. Sequence analysis of the complete mitochondrial genomes in the pedigree showed the presence of homoplasmic LHON-associated ND6 T14484C mutation, deafness-associated 12S rRNA A1555 mutation and 47 other variants belonging to Eastern Asian haplogroup H2. None of other mitochondrial variants was evolutionarily conserved and functional significance. Therefore, the coexistence of the A1555G mutation and T14484C mutations in this Chinese family indicate that the A1555G mutation may play a synergistic role in the phenotypic manifestation of LHON associated ND6 T14484C mutation. However, the incomplete penetrance of vision and hearing loss suggests the involvement of nuclear modifier genes and environmental factors in the phenotypic expression of these mtDNA mutations.     

Mutational analysis of the mitochondrial 12S rRNA gene in Chinese pediatric subjects with aminoglycoside-induced and non-syndromic hearing loss

Mutations in mitochondrial DNA (mtDNA) have been found to be associated with sensorineural hearing loss. We report here a systematic mutational screening of the mitochondrial 12S rRNA gene in 128 Chinese pediatric subjects with sporadic aminoglycoside-induced and non-syndromic hearing loss. We show that aminoglycoside ototoxicity accounts for 48% of cases of hearing loss in this Chinese pediatric population. Of the known deafness-associated mutations in this gene, the incidence of the A1555G mutation is ~13% and ~2.9% in this Chinese pediatric population with aminoglycoside-induced and non-syndromic hearing loss, respectively. Furthermore, mutations at position 961 in the 12S rRNA gene account for ~1.7% and 4.4% of cases of aminoglycoside-induced and non-syndromic hearing loss in this Chinese clinical population, respectively. The T1095C mutation has been identified in one maternally inherited family with aminoglycoside-induced and non-syndromic hearing loss. However, the C1494T mutation was not detected in this clinical population. In addition, three variants, A827G, T1005C and A1116G, in the 12S rRNA gene, localized at highly conserved sites, may play a role in the pathogenesis of aminoglycoside ototoxicity. These data strongly suggest that the mitochondrial 12S rRNA is a hot-spot for deafness-associated mutations in the Chinese population.Z. Li and R. Li contributed equally to this work.     

Detection of Ultra-Rare Mitochondrial Mutations in Breast Stem Cells by Duplex Sequencing

Long-lived adult stem cells could accumulate non-repaired DNA damage or mutations that increase the risk of tumor formation. To date, studies on mutations in stem cells have concentrated on clonal (homoplasmic) mutations and have not focused on rarely occurring stochastic mutations that may accumulate during stem cell dormancy. A major challenge in investigating these rare mutations is that conventional next generation sequencing (NGS) methods have high error rates. We have established a new method termed Duplex Sequencing (DS), which detects mutations with unprecedented accuracy. We present a comprehensive analysis of mitochondrial DNA mutations in human breast normal stem cells and non-stem cells using DS. The vast majority of mutations occur at low frequency and are not detectable by NGS. The most prevalent point mutation types are the C>T/G>A and A>G/T>C transitions. The mutations exhibit a strand bias with higher prevalence of G>A, T>C, and A>C mutations on the light strand of the mitochondrial genome. The overall rare mutation frequency is significantly lower in stem cells than in the corresponding non-stem cells. We have identified common and unique non-homoplasmic mutations between non-stem and stem cells that include new mutations which have not been reported previously. Four mutations found within the MT-ND5 gene (m.12684G>A, m.12705C>T, m.13095T>C, m.13105A>G) are present in all groups of stem and non-stem cells. Two mutations (m.8567T>C, m.10547C>G) are found only in non-stem cells. This first genome-wide analysis of mitochondrial DNA mutations may aid in characterizing human breast normal epithelial cells and serve as a reference for cancer stem cell mutation profiles.     

GJB2 and mitochondrial A1555G gene mutations in nonsyndromic profound hearing loss and carrier frequencies in healthy individuals

This study aimed to assess mutations in GJB2 gene (connexin 26), as well as A1555G mitochondrial mutation in both the patients with profound genetic nonsyndromic hearing loss and healthy controls. Ninety-five patients with profound hearing loss (>90 dB) and 67 healthy controls were included. All patients had genetic nonsyndromic hearing loss. Molecular analyses were performed for connexin 26 (35delG, M34T, L90P, R184P, delE120, 167delT, 235delC and IVS1+1 A-->G) mutations, and for mitochondrial A1555G mutation. Twenty-two connexin 26 mutations were found in 14.7% of the patients, which were 35delG, R184P, del120E and IVS1+1 A-->G. Mitochondrial A1555G mutation was not encountered. The most common GJB2 gene mutation was 35delG, which was followed by del120E, IVS1+1 A-->G and R184P, and 14.3% of the patients segregated with DFNB1. In consanguineous marriages, the most common mutation was 35delG. The carrier frequency for 35delG mutation was 1.4% in the controls. 35delG and del120E populations, seems the most common connexin 26 mutations that cause genetic nonsyndromic hearing loss in this country. Nonsyndromic hearing loss mostly shows DFNB1 form of segregation.     

Cochlear alterations in deaf and unaffected subjects carrying the deafness-associated A1555G mutation in the mitochondrial 12S rRNA gene

The A1555G mutation in the mitochondrial small ribosomal RNA gene (12S rRNA) has been associated with aminoglycoside-induced, nonsyndromic hearing loss. However, the clinical phenotype of A1555G carriers is extremely variable. In the present study, we have performed an audiological evaluation of a group of deaf patients and hearing carriers of mutation A1555G with the aim to assess the prevalence of the mutation and determine the associated cochlear alterations. Fifty-four patients affected of nonsyndromic hearing loss were screened for the presence of the A1555G mitochondrial mutation. Nine of the familial cases (21%) carried the A1555G mutation, whereas the mutation was not found in any of the sporadic cases. The positive cases and some of their family members underwent a clinical study consisting in a clinical evaluation and audiological testing. The phenotype of A1555G patients varied in age of onset and severity of hearing loss, ranging from profound deafness to completely normal hearing. The audiometric alterations showed bilateral hearing loss, being more severe at high frequencies. Otoacoustic emissions were absent in deaf A1555G carriers, and auditory brainstem response indicated a prolonged Wave I, suggesting a cochlear dysfunction without any effect of the auditory nerve. Moreover, all hearing carriers of A1555G also presented alterations in cochlear physiology. In conclusion, the A1555G mitochondrial mutation causes a cochlear form of deafness, characterized by a more severe loss of hearing at high frequencies. Although the expression of the mutation is variable, cochlear alterations are present in all carriers of mutation A1555G.     

The Role of Mitochondrial DNA Mutations in Hearing Loss

Mutations in mitochondrial DNA (mtDNA) are one of the most important causes of hearing loss. Of these, the homoplasmic A1555G and C1494T mutations at the highly conserved decoding site of the 12S rRNA gene are well documented as being associated with either aminoglycoside-induced or nonsyndromic hearing loss in many families worldwide. Moreover, five mutations associated with nonsyndromic hearing loss have been identified in the tRNA^Ser(UCN) gene: A7445G, 7472insC, T7505C, T7510C, and T7511C. Other mtDNA mutations associated with deafness are mainly located in tRNA and protein-coding genes. Failures in mitochondrial tRNA metabolism or protein synthesis were observed from cybrid cells harboring these primary mutations, thereby causing the mitochondrial dysfunctions responsible for deafness. This review article provides a detailed summary of mtDNA mutations that have been reported in deafness and further discusses the molecular mechanisms of these mtDNA mutations in deafness expression.     

Mitochondrial tRNA^Glu A14693G variant may modulate the phenotypic manifestation of deafness-associated 12S rRNA A1555G mutation in a Han Chinese family

Mutations in mitochondrial 12S rRNA gene are one of the most important causes of aminoglycoside-induced and nonsyndromic hearing loss. Here we report the characterization of one Han Chinese pedigree with aminoglycoside-induced and nonsyndromic hearing loss. This Chinese family carrying the 12S rRNA A1555G mutation exhibited high penetrance and expressivity of heating impairment. In particular, penetrances of hearing loss in this family pedigree were 43.8% and 25%, respectively, when aminoglycoside-induced heating loss was included or excluded. Mutational analysis of entire mitochondrial genomes in this family showed the homoplasmic A1555G mutation and a set of variants belonging to haplogroup Y2. Of these, the A14693G variant occurred at the extremely conserved nucleotide （conventional position 54） of the TψC-loop of tRNA^Clu and was absent in 156 Chinese controls. Nucleotides at position 54 of tRNAs are often modified, thereby contributing to the structural formation and stabilization of functional tRNAs. Thus, the structural alteration of tRNA by the A14693G variant may lead to a failure in tRNA metabolism and impair mitochondrial protein synthesis, thereby worsening mitochondrial dysfunctions altered by the A1555G mutation. Therefore, the tRNA^Glu A14693G variant may have a potential modifier role in increasing the penetrance and expressivity of the deafness-associated A1555G mutation in this Chinese pedigree.     

Mutations of <Emphasis Type="Italic">MC4R</Emphasis> gene and its association with economic traits in Qinchuan cattle

MC4R belongs to a seven-transmembrane G-protein-coupled receptor which may regulate body composition and insulin action. Many mutations in the MC4R gene are associated with obesity, energy expenditure and serum triglyceride levels in human and animals. Six mutations in the MC4R gene were identified in our study (-293C>G, -193A>T, -192T>G, -129A>G, -84T>C and 1,069C>G). The -129A>G was significantly associated with live weight (LW) (P < 0.05), Cattle with the genotypes AG and GG had higher LW than genotype AA. The 1,069C>G was significantly associated with LW, carcass weight (CW), backfat thickness and marbling score (MS). Cattle with the genotype GG had higher LW, CW and MS than genotype CC; Cattle with the genotypes GG and CG had higher MS than CC. The results suggested that -129A>G and 1,069C>G SNP of the MC4R gene may be useful as a genetic marker for carcass and meat quality traits in Qinchuan cattle.     

线粒体tRNAThr G15927A突变可能影响耳聋相关的12S rRNA A1555G突变的表型表达

摘要: 对1个中国汉族耳聋家系进行了临床和分子遗传学特征分析。家系中听力下降的母系成员表现为程度不等、听力图形态不同的听力损害, 但同为双侧对称的感觉神经性耳聋。该家系耳聋外显率很高, 包括药物致聋的耳聋外显率为75%, 而非药物致聋的外显率为41.7%。对母系成员进行线粒体DNA(mtDNA)全序列扩增分析, 发现了耳聋相关12S rRNA A1555G同质性突变位点和多态性位点, 属于东亚人群B5b单体型。在这些变异位点中, mtDNA 15927位点的G-A碱基变化破坏tRNA^Thr反密码子结构上十分保守的C-G碱基对, 这可能加重由A1555G突变造成的线粒体功能缺陷。这表明tRNAThrG15927A突变可能增强携带12S rRNA A1555G的中国汉族耳聋家系的外显率和表现度。