12S rRNA 1555A Research Articles

Genetic correction of TRMU allele restored the mitochondrial dysfunction-induced deficiencies in iPSCs-derived hair cells of hearing-impaired patients.

Sensorineural hearing loss often results from damaged or deficient inner ear hair cells. Mitochondrial 12S rRNA 1555A>G mutation has been associated with hearing loss in many families. The m.1555A>G mutation is a primary factor underlying the development of hearing loss and TRMU allele (c.28G>T, p.Ala10Sser) encoding tRNA thiouridylase interact with m.1555A>G mutation to cause hearing loss. However, the tissue specificity of mitochondrial dysfunction remains elusive and there is no highly effective therapy for mitochondrial deafness. We report here the generation of induced pluripotent stem cells (iPSCs) from lymphoblastoid cell lines derived from members of an Arab-Israeli family (asymptomatic individual carrying only m.1555A>G mutation, symptomatic individual bearing both m.1555A>G and c.28G>T mutations, and control subject). The c.28G>T mutation in iPSC lines from a hearing-impaired subject was corrected by CRISPR/Cas9. These iPSCs were differentiated into otic epithelial progenitor (OEP) cells and subsequent inner ear hair cell (HC)-like cells. The iPSCs bearing m.1555A>G mutation exhibited mildly deficient differentiation into OEP and resultant HC-like cells displayed mild defects in morphology and electrophysiological properties. Strikingly, those HC-like cells harboring m.1555A>G and TRMU c.28G>T mutations displayed greater defects in the development, morphology and functions than those in cells bearing only m.1555A>G mutation. Transcriptome analysis of patients-derived HC-like cells revealed altered expressions of genes vital for mechanotransduction of hair cells. Genetic correction of TRMU c.28G>T mutation yielded morphologic and functional recovery of patient derived HC-like cells. These findings provide new insights into pathophysiology of maternally inherited hearing loss and a step toward therapeutic interventions for this disease.

Screening of A1555G mDNA Variant Using U-TOP™HL Genotyping Kit in Korean Family with Progressive Hearing Loss

Recently, real-time polymerase chain reaction (PCR) using the U-TOP™HL Genotyping Kit has been introduced to detect genetic hearing loss caused by certain type of gene variants popularly found in Korea. The mitochondrial 12S ribosomal ribonucleic acid (rRNA) genes are related to aminoglycoside induced or non-syndromic, sensorineural hearing loss. Among them, 1555A>G is commonly found and reported worldwide. We are presenting the case of a mother and a son, who were screened by real-time PCR using the U-TOP™HL Genotyping Kit and were found both to have the mitochondrial 12s rRNA 1555A>G variant with a different hearing loss phenotype. This report encourages clinicians to use this or similar screen methods for patients with familial hearing loss.

Contribution of the tRNAIle 4317A→G mutation to the phenotypic manifestation of the deafness-associated mitochondrial 12S rRNA 1555A→G mutation

The 1555A→G mutation in mitochondrial 12S rRNA has been associated with aminoglycoside-induced and non-syndromic deafness in many individuals worldwide. Mitochondrial genetic modifiers are proposed to influence the phenotypic expression of m.1555A→G mutation. Here, we report that a deafness-susceptibility allele (m.4317A→G) in the tRNAIle gene modulates the phenotype expression of m.1555A→G mutation. Strikingly, a large Han Chinese pedigree carrying both m.4317A→G and m.1555A→G mutations exhibited much higher penetrance of deafness than those carrying only the m.1555A→G mutation. The m.4317A→G mutation affected a highly conserved adenine at position 59 in the T-loop of tRNAIle We therefore hypothesized that the m.4317A→G mutation alters both structure and function of tRNAIle Using lymphoblastoid cell lines derived from members of Chinese families (three carrying both m.1555A→G and m.4317A→G mutations, three harboring only m.1555A→G mutation, and three controls lacking these mutations), we found that the cell lines bearing both m.4317A→G and m.1555A→G mutations exhibited more severe mitochondrial dysfunctions than those carrying only the m.1555A→G mutation. We also found that the m.4317A→G mutation perturbed the conformation, stability, and aminoacylation efficiency of tRNAIle These m.4317A→G mutation-induced alterations in tRNAIle structure and function aggravated the defective mitochondrial translation and respiratory phenotypes associated with the m.1555A→G mutation. Furthermore, mutant cell lines bearing both m.4317A→G and m.1555A→G mutations exhibited greater reductions in the mitochondrial ATP levels and membrane potentials and increasing production of reactive oxygen species than those carrying only the m.1555A→G mutation. Our findings provide new insights into the pathophysiology of maternally inherited deafness arising from the synergy between mitochondrial 12S rRNA and tRNA mutations.

Newborn Screening of Genetic Mutations in Common Deafness Genes With Bloodspot-Based Gene Chip Array.

This study screens for deafness gene mutations in newborns in the Northwest China population. The 9 sites of 4 common deafness genes (GJB2, GJB3, SLC26A4, and mt 12S rRNA) were detected by bloodspot-based gene chip array in 2,500 newborns. We detected mutations of the 4 genes in 101 (4.04%) newborns; particularly, 0.20% detected the double mutations. In the Hui population, 4.58% of the newborns tested positive for mutations, whereas 4.01% of Han newborns tested positive for mutations. The detective rates are as follows: 1.44% for GJB2 235delC, 1.08% for SLC26A4 IVS7-2A>G, 0.48% for GJB2 299_300delAT, 0.28% for SLC26A4 2168A>G, 0.2% for mt 12S rRNA 1555A>G, and 0.16% for GJB3 538C>T. The 31.25% (5/16) of infants with GJB2 235delC, 50% (3/6) with GJB2 299_300delAT, and 25% (3/12) with SLC26A4 IVS7-2A>G showed abnormal hearing when tested; only 1 double mutation case received the hearing test, and this infant showed abnormality in both ears on the hearing test. High mutation rates in the common deafness genes were detected in newborns in Northwest China. Our study is helpful in understanding the deafness genomic epidemiology and also provides evidence for prenatal and postnatal care as well as policy making on population health in the region.

Modification factors associated with maternally inherited non-syndromic hearing loss

Mutations in the mitochondrial DNA have been certified to be one of the most important causes of maternally inherited sensorineural hearing loss. Among these, mitochondrial 12S rRNA1555A>G, 1494C>T and other mutations are associated with both nonsyndromic and drug induced hearing loss caused by aminoglycosides. Individuals carrying 1555A>G or 1494C>T mutation have a variety of clinical manifestations, which implies that the 1555A>G or 1494C>T mutation is a chief factor underlying the development of deafness but insufficient to produce the clinical phenotype. Therefore other modifier factors, such as aminoglycosides, mitochondrial haplotypes, secondary mutation or nuclear modifier genes, may play an important role in the phenotypic expression of the deafness-associated mitochondrial 12S rRNA1555A>G or 1494C>T mutation. In this review, the modifier factors for the phenotypic expression of deafness-associated mitochondrial 12S rRNA1555A>G or 1494C>T mutations were summarized and proposed the pathogenesis of maternally inherited deafness.

Mutation analysis of common GJB2, SCL26A4 and 12S rRNA genes among 380 deafness patients in northern China

ObjectivesThe molecular etiology of nonsyndromic deafness in Chinese population has not been investigated systematically, our study is aim to investigate the molecular etiology of nonsyndromic deafness patients from Northern China (Heilongjiang province), in order to provide genetic test and counseling to families. Methods380 unrelated patients with hearing loss who attended to the Department of Otolaryngology, The Fourth Affiliated Hospital of Harbin Medical University were enrolled to our study. All patients were diagnosed with nonsyndromic deafness by audiologic evaluation, 202 normal-hearing individuals were taken as controls. Mutations in three common deafness-causing genes (GJB2, SLC26A4 and 12S rRNA) were screened by direct sequencing. ResultsMutations (homozygote or compound heterozygote) in GJB2 accounted for 8.9% (34/380) of the patients, mutations in SLC26A4 accounted for 10.0% (38/380) of the patients screened. Only one case was found to carry 12S rRNA 1555A > G (1/380, 0.26%). Five types of mutations in GJB2 were identified, GJB2 235delC was the most prevalent mutation in our patient group (76/380, 20.0%), followed by 299-300delAT with a frequency of 7.4% (28/380). Two types of mutations in SLC26A4 were detected in our patient group (IVS7-2A > G and 2168A > G). IVS7-2A > G was identified in 27 patients (27/380, 7.1%) and 2168A > G was identified in 14 patients (14/380, 3.7%). ConclusionsOur results demonstrate that 19.2% patients with nonsyndromic deafness were caused by mutations in three common deafness genes (GJB2, SLC26A4 and 12S rRNA) in our northern China patient group. GJB2 235delC was the most prevalent mutation, same as in the most Asian populations. These data enrich the database of deafness mutations and provide the standard for clinical diagnose, treatment and genetic counseling in Northern China population.

Biochemical Evidence for a Nuclear Modifier Allele (A10S) in TRMU (Methylaminomethyl-2-thiouridylate-methyltransferase) Related to Mitochondrial tRNA Modification in the Phenotypic Manifestation of Deafness-associated 12S rRNA Mutation

Nuclear modifier gene(s) was proposed to modulate the phenotypic expression of mitochondrial DNA mutation(s). Our previous investigations revealed that a nuclear modifier allele (A10S) in TRMU (methylaminomethyl-2-thiouridylate-methyltransferase) related to tRNA modification interacts with 12S rRNA 1555A→G mutation to cause deafness. The A10S mutation resided at a highly conserved residue of the N-terminal sequence. It was hypothesized that the A10S mutation altered the structure and function of TRMU, thereby causing mitochondrial dysfunction. Using molecular dynamics simulations, we showed that the A10S mutation introduced the Ser10 dynamic electrostatic interaction with the Lys106 residue of helix 4 within the catalytic domain of TRMU. The Western blotting analysis displayed the reduced levels of TRMU in mutant cells carrying the A10S mutation. The thermal shift assay revealed the Tm value of mutant TRMU protein, lower than that of the wild-type counterpart. The A10S mutation caused marked decreases in 2-thiouridine modification of U34 of tRNALys, tRNAGlu and tRNAGln However, the A10S mutation mildly increased the aminoacylated efficiency of tRNAs. The altered 2-thiouridine modification worsened the impairment of mitochondrial translation associated with the m.1555A→G mutation. The defective translation resulted in the reduced activities of mitochondrial respiration chains. The respiratory deficiency caused the reduction of mitochondrial ATP production and elevated the production of reactive oxidative species. As a result, mutated TRMU worsened mitochondrial dysfunctions associated with m.1555A→G mutation, exceeding the threshold for expressing a deafness phenotype. Our findings provided new insights into the pathophysiology of maternally inherited deafness that was manifested by interaction between mtDNA mutation and nuclear modifier gene.

Common molecular etiology of nonsyndromic hearing loss in 484 patients of 3 ethnicities in northwest China

Conclusions: In the study population in northwest China, a total of 33.06% of deaf patients have inherited hearing impairment caused by GJB2, SLC26A4, and mtDNA 1555A>G mutations. The mutation frequencies of GJB2, SLC26A4, and mtDNA 1555A>G genes were 16.12%, 10.54%, and 6.4%, respectively, in our study cohort. Thus, screening is conventionally performed for GJB2, SLC26A4, and mtDNA 1555A>G in these populations. Objective: This study aimed to investigate the mutations of GJB2, mitochondrial DNA 12S rRNA1555A>G, and SLC26A4 genes in Han Chinese, Hui people, and Tibetan ethnicities in patients with nonsyndromic hearing loss (NSHL) in northwest China. Methods: A total of 484 unrelated subjects with hearing loss who attended special education schools in northwest China were enrolled in this study. Three prominent deafness-related genes, GJB2, SLC26A4, and mtDNA 1555A>G, were screened for mutations in our study cohort. Results: The mutation frequencies of GJB2, SLC26A4, and mtDNA 1555A>G genes were 16.12%, 10.54%, and 6.4%, respectively. The prevalence of GJB2 mutations was 17.52%, 15.35%, and 11.43% in Han Chinese, Hui people, and Tibetan participants, respectively. c.235delC was the most prevalent mutation, accounting for 65.71% of all GJB2 mutant alleles. The prevalence of SLC26A4 mutations was 12.39%, 8.84%, and 8.57% in Han Chinese, Hui people, and Tibetan participants, respectively. The c.919-2 A>G mutation was the most common form, accounting for 60.47% of all SLC26A4 mutant alleles. The prevalence of the homoplasmic mtDNA 1555A>G mutation was 8.97%, 3.72%, and 5.71% in Han Chinese, Hui people, and Tibetan participants, respectively, which represents a statistically significant difference between the Han Chinese and Hui people (χ2 = 5.118, p < 0.05).

External quality survey results of newborn deafness gene mutations in China

To evaluate the external quality survey results of newborn hereditary deafness gene mutations to improve and enhance the testing quality of hereditary deafness gene mutations. Each of 30 voluntarily participating laboratories testing newborn hereditary deafness gene mutations received 15 lots of quality control blood spots in this survey at May 2013, including mitochondria DNA 12SrRNA 1555A>G (201311-201315), SLC26A4 IVS7-2A>G (201321-201325) and GJB2 235delC (201331-201335). The testing results, methods, equipments and reagent information were submitted for analyses of Clinet EQA, Microsoft Excel 2007 and SPSS 13.0. The rates of correction (number of correct results/total number of submitted results) were used for evaluating the performance of laboratories. Among them, 24 laboratories submitted the testing results of mitochondria DNA 12SrRNA 1555A>G and the rate of submission was 80.0% (24/30). The rates of correction for each lot were 95.8% (23/24), 95.8% (23/24), 100% (24/24), 95.8% (23/24) and 95.8% (23/24) respectively and the overall rate was 96.7% (116/120). And 23 laboratories submitted the other two kinds of genetic test results, the rate of submission was 76.7% (23/30); the rates of correction for each lot of SLC26A4 IVS7-2A>G genetic test were 95.7% (22/23), 95.7% (22/23), 100% (23/23), 95.7% (22/23) and 95.7% (22/23) respectively and the overall rate was 96.5% (111/115); the genetic test results of GJB2 235delC were all correct (23/23). In this survey, 2/3 laboratories employed fluorescent polymerase chain reaction (PCR) and the remainder microarray chips. The survey of newborn deafness gene mutation testing results is generally satisfactory. Only mitochondria DNA 12SrRNA and SLC26A4 have some errors. The laboratories of gene testing should improve their quality control system, correct mistakes during the test period without any delay and boost the rate of correction for newborn hereditary deafness gene testing.

Analysis and prenatal diagnosis of deafness-related gene mutations in patients with nonsyndromic hearing loss

To analyze deaf-related genes in patients with nonsyndromic hearing loss (NSHL) and set up a prenatal diagnosis system for such patients. Nine NSHL families were collected. Potential mutations of GJB2 (35delG, 176del16, 235delC, 299delAT), SLC26A4 (2168A> G, IVS7-2A> G), GJB3 (538C> T) and mtDNA (1494C> T, 12S rRNA 1555A> G) were detected by direct sequencing. Maternal blood contamination was excluded prior to the testing. Sixteen patients from 4 families were detected with GJB2 mutations, 8 patients from 2 families were found with SLC26A4 mutations, and 4 patients from 2 families were found with mutations in mtDNA. For 2 patients from one remaining family, no mutations were found with above genes. A diagnostic system for NSHL has been established, which may provide a basis for prenatal diagnosis and genetic counseling to NSHL families.

Analysis of GJB2 gene and mitochondrial DNA A1555G mutations in 16 families with non-syndromic hearing loss

To screen for genetic mutations in families featuring non-syndromic hearing loss. Sixteen families with non-syndromic hearing loss were interviewed to identify medical histories by a questionnaire. Audiological and neurological examinations were conducted for all families. Coding regions of GJB2 and 12S rRNA genes were amplified and sequenced. Of the 17 patients with sensorineural hearing loss, 3 were homozygous mutation for GJB2 235 delC, 1 was 235 delC heterozygous mutation, 1 was 235 delC+299_300 delAT compound heterozygous mutation, and 6 were 79G>A+341G>A heterozygosis in cis mutation. No 1555A>G mutation of mitochondrial DNA (mtDNA) was found in the 16 families. The incidence of mtDNA 12S rRNA 1555A>G mutation in Jiangsu province may be lower than the average across China. Mutations of GJB2 genes may account for as much as 64.7% of non-syndromic hearing loss in this study. Screening for such mutations and genetic counseling may play an important role in the prevention of hereditary hearing loss.

Auditory screening concurrent deafness predisposing genes screening in 10,043 neonates in Gansu province, China

ObjectiveThe aim of our study was to evaluate the effectiveness of combining newborn hearing screening with screening for genetic mutations associated with deafness. MethodsTen thousand forty-three newborn babies, born between December 2009 and April 2011 in Gansu province, China, were screened for hearing loss using the otoacoustic emissions test or automatic auditory brainstem response test and genetic mutations associated with deafness using a standard protocol. ResultsIn the hearing screening, the referral rate for hearing loss in the first-step screening was 14.4% (1409/9786), decreasing significantly to 3.8% (362/9506) upon retesting. After the second-step screening, a total of 537 newborns were lost to follow-up. The genetic screening found that about 2.29% (230/10,043) individuals carried one or more recessive risk alleles or the mitochondrial mutation. Among them, 18 babies had the pathogenic mitochondrial DNA mutation, 92 babies were SLC26A4 heterozygote carriers, one case with both SLC26A4 and 12S rRNA 1555A>G mutation, 117 babies were GJB2 heterozygote carriers, and two babies were GJB2 homozygote carriers. However, 83.5% (192/230) neonates passed the conventional hearing screening among these carriers. ConclusionsIt might be effective to complement the conventional hearing screening with gene screening for the purpose of early diagnosis and discovery of the late-onset hearing loss.

Unique penetrance of hearing loss in a five-generation Chinese family with the mitochondrial 12S rRNA 1555A > G mutation

Conclusions: Analysis of the complete mtDNA genome and X-linkage of this five-generation Chinese family revealed that the 1555A > G mutation may lead to deafness. Objectives: Mutations in mitochondrial DNA (mtDNA) have been found to be associated with sensorineural hearing loss. However, the variable clinical phenotype and incomplete penetrance of mtDNA 1555A > G-induced hearing loss complicate our understanding of this mutation. We aimed to identify whether nuclear genes, mitochondrial haplotypes/variants, and a possible threshold effect are involved in its manifestation in the pedigree. Methods: We performed clinical, genetic, and X-linkage analysis of a five-generation Chinese family in which all the affected individuals were male. Results: Clinical evaluation revealed that affected individuals with or without aminoglycoside exposure developed hearing loss extending gradually from 8000 Hz to 4000 Hz and then to 1000 Hz. Using X-linkage analysis and sequencing, we detected an identical homoplasmic 1555A > G mutation in nine individuals, and a previously unreported variant 14163C > T in mtDNA. The new variant 14163C > T coexisted with the 1555A > G mutation in six affected subjects of our pedigree. The previously unreported variant 14163C > T and aminoglycoside exposure may synergize the development of this deafness.

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.