Genetic Epidemiology of Mitochondrial Pathogenic Variants Causing Nonsyndromic Hearing Loss in a Large Cohort of South Indian Hearing Impaired Individuals

Mitochondria play a critical role in the generation of metabolic energy in the form of ATP. Tissues and organs that are highly dependent on aerobic metabolism are involved in mitochondrial disorders including nonsyndromic hearing loss (NSHL). Seven pathogenic variants leading to NSHL have so far been reported on two mitochondrial genes: MT‐RNR1 encoding 12SrRNA and MT‐TS1 encoding tRNA for Ser^(UCN). We screened 729 prelingual NSHL subjects to determine the prevalence of MT‐RNR1 variants at position m.961, m.1555A>G and m.1494C>T, and MT‐TS1 m.7445A>G, m.7472insC m.7510T>C and m.7511T>C variants. Mitochondrial pathogenic variants were found in eight probands (1.1%). Five of them were found to have the m.1555A>G variant, two others had m.7472insC and one proband had m.7444G>A. The extended relatives of these probands showed variable degrees of hearing loss and age at onset. This study shows that mitochondrial pathogenic alleles contribute to about 1% prelingual hearing loss. This study will henceforth provide the reference for the prevalence of mitochondrial pathogenic alleles in the South Indian population, which to date has not been estimated. The m.1555A>G variant is a primary predisposing genetic factor for the development of hearing loss. Our study strongly suggests that mitochondrial genotyping should be considered for all hearing impaired individuals and particularly in families where transmission is compatible with maternal inheritance, after ruling out the most common variants.     

Human Mitochondrial Cytochrome b Variants Studied in Yeast: Not All Are Silent Polymorphisms

Variations in mitochondrial DNA (mtDNA) cytochrome b (mt‐cyb) are frequently found within the healthy population, but also occur within a spectrum of mitochondrial and common diseases. mt‐cyb encodes the core subunit (MT‐CYB) of complex III, a central component of the oxidative phosphorylation system that drives cellular energy production and homeostasis. Despite significant efforts, most mt‐cyb variations identified are not matched with corresponding biochemical data, so their functional and pathogenic consequences in humans remain elusive. While human mtDNA is recalcitrant to genetic manipulation, it is possible to introduce human‐associated point mutations into yeast mtDNA. Using this system, we reveal direct links between human mt‐cyb variations in key catalytic domains of MT‐CYB and significant changes to complex III activity or drug sensitivity. Strikingly, m.15257G>A (p.Asp171Asn) increased the sensitivity of yeast to the antimalarial drug atovaquone, and m.14798T>C (p.Phe18Leu) enhanced the sensitivity of yeast to the antidepressant drug clomipramine. We demonstrate that while a small number of mt‐cyb variations had no functional effect, others have the capacity to alter complex III properties, suggesting they could play a wider role in human health and disease than previously thought. This compendium of new mt‐cyb‐biochemical relationships in yeast provides a resource for future investigations in humans.     

Single‐fiber studies for assigning pathogenicity of eight mitochondrial DNA variants associated with mitochondrial diseases

Whole mitochondrial DNA (mtDNA) sequencing is now systematically used in clinical laboratories to screen patients with a phenotype suggestive of mitochondrial disease. Next Generation Sequencing (NGS) has significantly increased the number of identified pathogenic mtDNA variants. Simultaneously, the number of variants of unknown significance (VUS) has increased even more, thus challenging their interpretation. Correct classification of the variants' pathogenicity is essential for optimal patient management, including treatment and genetic counseling. Here, we used single muscle fiber studies to characterize eight heteroplasmic mtDNA variants, among which were three novel variants. By applying the pathogenicity scoring system, we classified four variants as “definitely pathogenic” (m.590A>G, m.9166T>C, m.12293G>A, and m.15958A>T). Two variants remain “possibly pathogenic” (m.4327T>C and m.5672T>C) but should these be reported in a different family, they would be reclassified as “definitely pathogenic.” We also illustrate the contribution of single‐fiber studies to the diagnostic approach in patients harboring pathogenic variants with low level heteroplasmy.     

Comprehensive functional assessment of MLH1 variants of unknown significance

Lynch syndrome is associated with germline mutations in DNA mismatch repair (MMR) genes. Up to 30% of DNA changes found are variants of unknown significance (VUS). Our aim was to assess the pathogenicity of eight MLH1 VUS identified in patients suspected of Lynch syndrome. All of them are novel or not previously characterized. For their classification, we followed a strategy that integrates family history, tumor pathology, and control frequency data with a variety of in silico and in vitro analyses at RNA and protein level, such as MMR assay, MLH1 and PMS2 expression, and subcellular localization. Five MLH1 VUS were classified as pathogenic: c.[248G>T(;)306G>C], c.[780C>G;788A>C], and c.791‐7T>A affected mRNA processing, whereas c.218T>C (p.L73P) and c.244G>A (p.T82A) impaired MMR activity. Two other VUS were considered likely neutral: the silent c.702G>A variant did not affect mRNA processing or stability, and c.974G>A (p.R325Q) did not influence MMR function. In contrast, variant c.25C>T (p.R9W) could not be classified, as it associated with intermediate levels of MMR activity. Comprehensive functional assessment of MLH1 variants was useful in their classification and became relevant in the diagnosis and genetic counseling of carrier families. Hum Mutat 33:1576–1588, 2012. © 2012 Wiley Periodicals, Inc.     

Identification of recurrent pathogenic alleles using exome sequencing data: Proof-of-concept study of Russian subjects

Whole exome sequencing (WES) is a powerful tool for the cataloguing of population-specific genetic diseases. Within this proof-of-concept study we evaluated whether analysis of a small number of individual exomes is capable of identifying recurrent pathogenic alleles. We considered 106 exomes of subjects of Russian origin and revealed 13 genetic variants, which occurred more than twice and fulfilled the criteria for pathogenicity. All these alleles turned out to be indeed recurrent, as revealed by the analysis of 1045 healthy Russian donors. Eight of these variants (NAGA c.973G>A, ACADM c.985A>C, MPO c.2031-2A>C, SLC3A1 c.1400T>C, LRP2 c.6160G>A, BCHE c.293A>G, MPO c.752T>C, FCN3 c.349delC) are non-Russian-specific, as their high prevalence was previously demonstrated in other European populations. The remaining five disease-associated alleles appear to be characteristic for subjects of Russian origin and include CLCN1 c.2680C>T (myotonia congenita), DHCR7 c.453G>A (Smith-Lemli-Opitz syndrome), NUP93 c.1162C>T (steroid-resistant nephrotic syndrome, type 12), SLC26A2 c.1957T>A (multiple epiphyseal dysplasia) and EIF3F c.694T>G (mental retardation). These recessive disease conditions may be of particular relevance for the Russian Federation and other countries with a significant Slavic population.     

Chinese newborn screening for the incidence of G6PD deficiency and variant of G6PD gene from 2013 to 2017

Glucose‐6‐phosphate dehydrogenase (G6PD) deficiency is one of the most common X‐linked enzymopathies caused by G6PD gene variant. We aimed to provide the characteristics of G6PD deficiency and G6PD gene variant distribution in a large Chinese newborn screening population. We investigated the prevalence of G6PD in China from 2013 to 2017. Then, we examined G6PD activity and G6PD gene in representative Chinese birth cohort to explore the distribution of G6PD gene variant in 2016. We then performed multicolor melting curve analysis to classify G6PD gene variants in 10,357 neonates with activity‐confirmed G6PD deficiency, and DNA Sanger sequencing for G6PD coding exons if hot site variants were not found. The screened population, organizations, and provinces of G6PD deficiency were increased from 2013 to 2017 in China. The top five frequency of G6PD gene variants were c.1376G>T, c.1388G>A, c.95A>G, c.1024C>T, and c.871G>A and varied in different provinces, with regional and ethnic features, and four pathogenic variant sites (c.152C>T, c.290A>T, c.697G>C, and c.1285A>G) were first reported. G6PD deficiency mainly occurs in South China, and the frequency of G6PD gene variant varies in different regions and ethnicities.     

Fine Time Scaling of Purifying Selection on Human Nonsynonymous mtDNA Mutations Based on the Worldwide Population Tree and Mother–Child Pairs

A high‐resolution mtDNA phylogenetic tree allowed us to look backward in time to investigate purifying selection. Purifying selection was very strong in the last 2,500 years, continuously eliminating pathogenic mutations back until the end of the Younger Dryas (～11,000 years ago), when a large population expansion likely relaxed selection pressure. This was preceded by a phase of stable selection until another relaxation occurred in the out‐of‐Africa migration. Demography and selection are closely related: expansions led to relaxation of selection and higher pathogenicity mutations significantly decreased the growth of descendants. The only detectible positive selection was the recurrence of highly pathogenic nonsynonymous mutations (m.3394T>C‐m.3397A>G‐m.3398T>C) at interior branches of the tree, preventing the formation of a dinucleotide STR (TATATA) in the MT‐ND1 gene. At the most recent time scale in 124 mother–children transmissions, purifying selection was detectable through the loss of mtDNA variants with high predicted pathogenicity. A few haplogroup‐defining sites were also heteroplasmic, agreeing with a significant propensity in 349 positions in the phylogenetic tree to revert back to the ancestral variant. This nonrandom mutation property explains the observation of heteroplasmic mutations at some haplogroup‐defining sites in sequencing datasets, which may not indicate poor quality as has been claimed.     

Genetic and bioinformatics analysis of four novel GCK missense variants detected in Caucasian families with GCK‐MODY phenotype

Heterozygous loss‐of‐function mutations in the glucokinase (GCK) gene cause maturity‐onset diabetes of the young (MODY) subtype GCK (GCK‐MODY/MODY2). GCK sequencing revealed 16 distinct mutations (13 missense, 1 nonsense, 1 splice site, and 1 frameshift‐deletion) co‐segregating with hyperglycaemia in 23 GCK‐MODY families. Four missense substitutions (c.718A>G/p.Asn240Asp, c.757G>T/p.Val253Phe, c.872A>C/p.Lys291Thr, and c.1151C>T/p.Ala384Val) were novel and a founder effect for the nonsense mutation (c.76C>T/p.Gln26*) was supposed. We tested whether an accurate bioinformatics approach could strengthen family‐genetic evidence for missense variant pathogenicity in routine diagnostics, where wet‐lab functional assays are generally unviable. In silico analyses of the novel missense variants, including orthologous sequence conservation, amino acid substitution (AAS)‐pathogenicity predictors, structural modeling and splicing predictors, suggested that the AASs and/or the underlying nucleotide changes are likely to be pathogenic. This study shows how a careful bioinformatics analysis could provide effective suggestions to help molecular‐genetic diagnosis in absence of wet‐lab validations.     

Investigation of Killer Cell Immunoglobulin‐Like Receptors KIR2DL2 and KIR2DL3 Diversity and Identification of Ten Novel KIR2DL3 Alleles in the Chinese Han Population

KIR2DL2 and KIR2DL3 are important inhibitory receptors that recognize a subset of HLA‐C allelic products carrying Ser77 and Asn80. In this study, we have determined KIR2DL2 and KIR2DL3 diversity in the Chinese Han population by a PCR sequence‐based typing. Based on sequencing, the coding regions of 166 Chinese Han individuals, seven new polymorphic sites (238G>A, 405G>A, 476A>G, 550G>A, 608G>A, 789T>C, 947T>C) were found. KIR2DL2*00301, *00101, KIR2DL3*00101,*00201,*013, *015 and ten new KIR2DL3 variants (KIR2DL3*00105, 00106, 00107, 00108, 019, 020, 021, 022, 023 and 024) were identified, of which KIR2DL3*00101 was the most frequent allele. Compared with the sequences of KIR2DL3*00101, all sequences of 2DL3*00105, 2DL3*00106, 2DL3*00107 and 2DL3*00108 had one nucleotide substitution(789T>C, 261C>T, 489G>A and 405G>A),but none resulted in amino acid change. An A>G substitution was observed in nucleotide position 476 in 2DL3*019, 608 G>A in 2DL3*020, 824T>C in 2DL3*021 and 238 G>A in 2DL3*023. In addition, 2DL3*022 probably arose from 2DL3*00201 with a nucleotide substitution G>A at 550. There were more HLA‐C1 positive individuals than HLA‐C2. In conclusion, the data of allelic polymorphism for KIR2DL2 and KIR2DL3 were obtained in the Chinese Han population and ten novel KIR2DL3 alleles were identified.     

Mutation spectra and founder effect of TMC1 in patients with non‐syndromic deafness in Xiamen area,China

To analyze the spectrum and founder effect of TMC1 mutations in patients with non‐syndromic deafness in the Xiamen area. Sporadic pedigrees were detected by targeted next‐generation sequencing, and 110 unrelated patients from Xiamen Special Education School were analyzed through Sanger sequencing for the TMC1 gene. In total, 53 SNPs were designed to analyze the haplotypes of the TMC1 c.2050G>C mutation. The probands of three families were found to be homozygous for TMC1 c.2050G>C, and their parents were all heterozygous for the TMC1 c.2050G>C mutation. In 110 unrelated patients from Xiamen Special Education School, four were found to carry compound heterozygotes of TMC1 c.2050G>C, which were compound heterozygotes of c.804G>A, c.1127T>C, c.1165C>T, and c.1396_1398delAAC, respectively. Three types of TMC1 polymorphisms (c.45C>T, c.1713C>T, c.2208+49C>T) and two heterozygotes of novel variants (c.1764‐4C>A, c.2073G>A[p.K691K]) were found in the remaining 100 patients. In total, four novel variants were detected in this study. These mutations and variants were not detected in 100 normal samples. The haplotypes of the probands of families with TMC1 c.2050G>C were identical. There were unique hotspots and spectra of TMC1 mutations in the Xiamen deaf population. Haplotype analysis is useful to understand the founder effect of the hot spot mutation.     

Hydroxymethylbilane Synthase Gene Mutations and Polymorphisms in Brazilian Families with Acute Intermittent Porphyria

Acute intermittent porphyria (AIP), an autosomal dominant disorder, is caused by a deficiency of hydroxymethylbilane synthase (HMBS). In the present study, we sought to establish a correlation between HMBS activity with the presence of mutations and polymorphisms. Enzyme activity was measured in red blood cells of four Brazilian unrelated AIP families (n = 124) and in blood donors (n = 80). The HMBS mutations in AIP family members were studied by PCR‐SSCP followed by direct sequencing. Six intragenic SNPs (1345 G>A, 1500 T>C, 2377 C>A, 2478 A>G, 3581 A>G, and 7064 C>A) were determined by PCR‐RFLP. Abnormal SSCP patterns in exons 7, 9, 12, and 15 were observed. DNA sequencing analysis revealed one nonsense mutation, R149X, two missense mutations, G111R and L338P, and one deletion, CT 730–731. All mutation carriers had lower enzyme activity. All polymorphisms, except 2377 C>A and 7064 C>A, showed no significant differences compared with previous reports. Mutation screening allowed the detection of the missense mutation, L338P, and the 730_731delCT deletion, two as yet unreported mutations in Brazilian AIP patients. Our findings also showed a high frequency of 2478 A>G and 3581 A>G polymorphism combinations suggesting that these polymorphisms contributed to enzymatic activity reduction in our study population.     

Molecular characterization of a large group of Mucopolysaccharidosis type IIIC patients reveals the evolutionary history of the disease

Mucopolysaccharidosis type IIIC (MPSIIIC) is a severe, rare autosomal recessive disorder caused by variants in the heparan‐α‐glucosaminide N‐acetyltransferase (HGSNAT) gene which result in lysosomal accumulation of heparan sulfate. We analyzed clinical presentation, molecular defects and their haplotype context in 78 (27 novel) MPSIIIC cases from 22 countries, the largest group studied so far. We describe for the first time disease‐causing variants in the patients from Brazil, Algeria, Azerbaijan, and Iran, and extend their spectrum within Canada, Colombia, Turkey, and the USA. Six variants are novel: two missense, c.773A>T/p.N258I and c.1267G>T/p.G423W, a nonsense c.164T>A/p.L55*, a splice‐site mutation c.494?1G>A/p.[P165_L187delinsQSCYVTQAGVRWHHLGSLQALPPGFTPFSYLSLLSSWNC,P165fs], a deletion c.1348delG/p.(D450fs) and an insertion c.1479dupA/p.(Leu494fs). The missense HGSNAT variants lacked lysosomal targeting, enzymatic activity, and likely the correct folding. The haplotype analysis identified founder mutations, p.N258I, c.525dupT, and p.L55* in the Brazilian state of Paraiba, c.493+1G>A in Eastern Canada/Quebec, p.A489E in the USA, p.R384* in Poland, p.R344C and p.S518F in the Netherlands and suggested that variants c.525dupT, c.372?2G>A, and c.234+1G>A present in cis with c.564‐98T>C and c.710C>A rare single‐nucleotide polymorphisms, have been introduced by Portuguese settlers in Brazil. Altogether, our results provide insights into the origin, migration roots and founder effects of HGSNAT disease‐causing variants, and reveal the evolutionary history of MPSIIIC.     

Prevalence of Mutations in Deafness‐Causing Genes in Cochlear Implanted Patients with Profound Nonsyndromic Sensorineural Hearing Loss in Shandong Province,China

The mutations of GJB2, SLC26A4, and mtDNA12SrRNA are the most common inherited causes of nonsyndromic sensorineural hearing loss (NSHL) in China, yet previous genetic screenings were mainly carried on patients with moderate‐to‐profound impairment. We aimed to detect the mutation frequencies in NSHL population within a more specified range of severity. Patients with profound NSHL who had undergone cochlear implantation in the Shandong Provincial Hospital (Shandong, China) were recruited. The majority (n = 472) were between 0.7 and 6 years old, and the remaining (n = 63) were between 6 and 70 years old. In total, 115 mutation alleles of the three genes were screened with SNP scan assay. Of the patients, 19.44% (104/535) were found to have GJB2 mutations, and the most common allele was c.235delC, followed by c.299_300delAT and c.109G>A. SLC26A4 mutations were detected in 13.46% patients (72/535), and the most common allele was c.919‐2A>G (IVS7‐2A>G), followed by c.1174A>T and c.2168A>G. Seven patients (1.31%) carried mutations in mtDNA12SrRNA, with the alleles of m.1555A>G and m.1494C>T. We found the allele frequency of c.109G>A (GJB2) was relatively lower in the profound NSHL population in comparison to the moderate‐to‐profound ones, and the c.1174A>T (SLC26A4) relatively higher. It suggests those mutations may be connected with the degree of deafness, which needs more observations and analyses to support.     

Complete Mitochondrial Genome Analysis and Clinical Documentation of a Five‐Generational Indian Family with Mitochondrial 1555A>G Mutation and Postlingual Hearing Loss

Hearing loss is the most common sensory disorder and is genetically heterogeneous. Apart from nuclear gene mutations, a number of inherited mitochondrial mutations have also been implicated. The m.1555A>G mutation in the mitochondrial MT‐RNR1 gene is reported as the most common mutation causing nonsyndromic hearing loss in various ethnic populations. We report here for the first time the clinical, genetic and molecular characterisation of a single large five‐generational Tamil‐speaking South Indian family with maternally inherited nonsyndromic postlingual hearing loss. Molecular analysis led to identification of m.1555A>G in 28 maternal relatives with variable degree of phenotypic expression. The penetrance of hearing loss among the maternal relatives in this family was 55%. Sequence analysis of the complete mitochondrial genome in 36 members of this pedigree identified 25 known variants and one novel variant co‐transmitted along with m.1555A>G mutation. The mtDNA haplotype analysis revealed that the maternal relatives carry the R*T₂ haplotype similar to Europeans and South Asians. Sequencing of the coding exon of GJB2 nuclear gene did not show any pathogenic mutations. The results suggest that other nuclear or environmental modifying factors could have played a role in the differential expression of mutation m.1555A>G in postlingual hearing loss in this family.     

Phenotypes and cellular effects of GJB1 mutations causing CMT1X in a cohort of 226 Chinese CMT families

The aim of this study is to explore the phenotypic and genotypic features of X‐linked Charcot–Marie‐Tooth (CMT) disease in the mainland of China and to study the cellular effects of six novel Gap junction protein beta‐1 variants. We identified 25 missense and 1 non‐sense mutations of GJB1 in 31 unrelated families out of 226 CMT families. The frequency of GJB1 mutations was 13.7% of the total and 65% of intermediate CMT. Six novel GJB1 variants (c.5A>G, c.8G>A, c.242T>C, c.269T>C, c.317T>C and c.434T>G) were detected in six unrelated intermediate CMT families. Fluorescence revealed that HeLa cells transfected with EGFP‐GJB1‐V74M, EGFP‐GJB1‐L81P or EGFP‐GJB1‐L90P had diffuse endoplasmic reticulum staining, HeLa cells transfected with EGFP‐GJB1‐L106P had diffuse intracellular staining, and HeLa cells transfected with EGFP‐GJB1‐N2S had cytoplasmic and nuclear staining. The distribution of Cx32 in HeLa cells transfected with EGFP‐GJB1‐F145C was similar to that of those transfected with wild‐type (WT). These six variants resulted in a higher percentage of apoptosis than did WT as detected by flow cytometry and Hoechst staining. In conclusion, mutation screening should be first performed in intermediate CMT patients, especially those with additional features. The novel GJB1 variants c.5A>G, c.8G>A, c.242T>C and c.269T>C are considered pathogenic, and c.317T>C and c.434T>G are classified as probably pathogenic.