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.     

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.     

Novel compound heterozygous mutations in GPT2 linked to microcephaly,and intellectual developmental disability with or without spastic paraplegia

We here describe novel compound heterozygous missense variants, NM_133443:c.[400C>T] and NM_133443:[1435G>A], in the glutamic‐pyruvic transaminase 2 (GPT2) gene in a large consanguineous family with two affected siblings diagnosed with microcephaly intellectual disability and developmental delay (IDD). In addition to these clinical phenotypes, the male sibling has spastic paraplegia, and the female sibling has epilepsy. Their four extended family members have IDD and microcephaly. Both of these variants, c.400C>T (p.R134C) and c.1435G>A (p.V479M), reside in the pyridoxal phosphate‐dependent aminotransferase domain. The missense variants affect highly conserved amino acids and are classified to be disease‐causing by meta‐SVM. The candidate variants were not found in the Exome Aggregation Consortium (ExAC) dataset or in dbSNP. Both GPT2 variants have an allele frequency of 0% (0/ ∼ 600) in the whole‐exome sequenced Turkish cohort. Upon Sanger sequencing, we confirmed these mutations in all affected family members and showed that the index patient and his affected sister inherited one mutant allele from each unaffected parent. To the best of our knowledge, this is the first family in which a novel compound heterozygous variant in the GPT2 gene was identified.

     

Five novel mutations of GALNS in Korean patients with mucopolysaccharidosis IVA

Mucopolysaccharidosis IVA (MPS IVA; OMIM #253000) is caused by the deficiency of N‐acetylgalactosamine‐6‐sulfate sulfatase (GALNS), a lysosomal enzyme involved in the catabolism of keratan and chondroitin sulfate. In this study, we examined biochemical and genetic data from 6 Korean patients presenting with classic MPS IVA by measuring GALNS activity in peripheral blood leukocytes and skin fibroblasts. We initially identified Korean patients with MPS IVA by clinical, biochemical, and genetic analyses. We performed PCR‐direct sequencing to identify molecular defects of the GALNS gene in patients and assessed the mutational statuses of family members as well as 50 healthy unrelated subjects. In silico analyses were performed to check for novel mutations. The mean age of the six female patients was 8.0 ± 5.2 years (range: 2–17 years), and were all found to have severe reductions of GALNS enzyme. A total of 12 mutant alleles were identified, corresponding to 7 different mutations. Five novel mutations were c.218A>G (p.Y73C), c.451C>A (p.P151T), c.725C>G (p.S242C), c.752G>A (p.R251Q), and c.1000C>T (p.Q334X). Two other mutations were c.1156C>T (p.R386C) and c.1243‐1G>A. Two mutations, c.451C>A and c.1000C>T, accounted for 58% of all mutations in this sample. © 2013 Wiley Periodicals, Inc.     

Clinical and molecular analysis in Papillon–Lefèvre syndrome

Papillon–Lefèvre syndrome (PLS; MIM#245000) is a rare recessive autosomal disorder characterized by palmar and plantar hyperkeratosis, and aggressively progressing periodontitis leading to premature loss of deciduous and permanent teeth. PLS is caused by loss‐of‐function mutations in the CTSC gene, which encodes cathepsin C. PLS clinical expressivity is highly variable and no consistent genotype–phenotype correlation has been demonstrated yet. Here we report the clinical and genetic features of five PLS patients presenting a severe periodontal breakdown in primary and permanent dentition, hyperkeratosis over palms and soles, and recurrent sinusitis and/or tonsillitis. Mutation analysis revealed two novel homozygous recessive mutations (c.947T>C and c.1010G>C) and one previous described homozygous recessive mutation (c.901G>A), with parents carrying them in heterozygous, in three families (four patients). The fourth family presented with the CTSC c.628C>T mutation in heterozygous, which was inherited maternally. Patient carrying the CTSC c.628C>T mutation featured classical PLS phenotype, but no PLS clinical characteristics were found in his carrier mother. All mutations were found to affect directly (c.901G>A, c.947T>C, and c.1010G>C) or indirectly (c.628C>T, which induces a premature termination) the heavy chain of the cathepsin C, the region responsible for activation of the lysosomal protease. Together, these findings indicate that both homozygous and heterozygous mutations in the cathepsin C heavy chain domain may lead to classical PLS phenotype, suggesting roles for epistasis or gene–environment interactions on determination of PLS phenotypes.     

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.     

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.     

Novel insights on GTPBP3-associated hypertrophic cardiomyopathy

About 100 genes have been associated with cardiomyopathies with genotype–phenotype correlations often hard to establish. Genetic testing may help to confirm the genetic diagnosis and assess the risk of inheritance in the family. A 25-year old male with hypertrophic cardiomyopathy and fasciculoventricular accessory pathway was referred for genetic testing by his cardiologist. Targeted PRKAG2 screening and whole-exome sequencing were performed, followed by Sanger sequencing segregation analysis in the family. The PRKAG2 gene screening was negative. Whole-exome sequencing revealed the following four variants in the patient: c.181G>C (p.Ala61Pro) and c.1199C>T (p.Thr400Met) in the GTPBP3 gene, as well as c.752C>T (p.Thr251Ile) and c.1760C>T (p.Pro587Leu) in the POLG gene. Family segregation analysis showed that the patient's mother is a carrier of variant c.181G>C and the patient's paternal grandmother is a carrier of variant c.1199C>T in the GTPBP3 gene, which is in accordance with an autosomal recessive model of inheritance of the disease. Both variants in the POLG are found paternally inherited in the patient's healthy half-brother, thus are not considered disease-causing. GTPBP3 variants have been reported in patients with hypertrophic cardiomyopathy, associated with combined oxidative phosphorylation deficiency 23. These novel variants represent the probable cause of the observed clinical symptoms in the patient.     

Ex vivo splicing assays of mutations at noncanonical positions of splice sites in USHER genes

Molecular diagnosis in Usher syndrome type 1 and 2 patients led to the identification of 21 sequence variations located in noncanonical positions of splice sites in MYO7A, CDH23, USH1C, and USH2A genes. To establish experimentally the splicing pattern of these substitutions, whose impact on splicing is not always predictable by available softwares, ex vivo splicing assays were performed. The branch‐point mapping strategy was also used to investigate further a putative branch‐point mutation in USH2A intron 43. Aberrant splicing was demonstrated for 16 of the 21 (76.2%) tested sequence variations. The mutations resulted more frequently in activation of a nearby cryptic splice site or use of a de novo splice site than exon skipping (37.5%). This study allowed the reclassification as splicing mutations of one silent (c.7872G>A (p.Glu2624Glu) in CDH23) and four missense mutations (c.2993G>A (p.Arg998Lys) in USH2A, c.592G>A (p.Ala198Thr), c.3503G>C [p.Arg1168Pro], c.5944G>A (p.Gly1982Arg) in MYO7A), whereas it provided clues about a role in structure/function in four other cases: c.802G>A (p.Gly268Arg), c.653T>A (p.Val218Glu) (USH2A), and c.397C>T (p.His133Tyr), c.3502C>T (p.Arg1168Trp) (MYO7A). Our data provide insights into the contribution of splicing mutations in Usher genes and illustrate the need to define accurately their splicing outcome for diagnostic purposes. Hum Mutat 31:1–9, 2010. © 2010 Wiley‐Liss, Inc.     

Genetic and cellular studies of oxidative stress in methylmalonic aciduria (MMA) cobalamin deficiency type C (cblC) with homocystinuria (MMACHC)

Methylmalonic aciduria (MMA) cobalamin deficiency type C (cblC) with homocystinuria (MMACHC) is the most frequent genetic disorder of vitamin B₁₂ metabolism. The aim of this work was to identify the mutational spectrum in a cohort of cblC-affected patients and the analysis of the cellular oxidative stress and apoptosis processes, in the presence or absence of vitamin B₁₂. The mutational spectrum includes nine previously described mutations: c.3G>A (p.M1L), c.217C>T (p.R73X), c.271dupA (p.R91KfsX14), c.331C>T (p.R111X), c.394C>T (p.R132X), c.457C>T (p.R153X), c.481C>T (p.R161X), c.565C>A (p.R189S), and c.615C>G (p.Y205X), and two novel changes, c.90G>A (p.W30X) and c.81+2T>G (IVS1+2T>G). The most frequent change was the known c.271dupA mutation, which accounts for 85% of the mutant alleles characterized in this cohort of patients. Owing to its high frequency, a real-time PCR and subsequent high-resolution melting (HRM) analysis for this mutation has been established for diagnostic purposes. All cell lines studied presented a significant increase of intracellular reactive oxygen species (ROS) content, and also a high rate of apoptosis, suggesting that elevated ROS levels might induce apoptosis in cblC patients. In addition, ROS levels decreased in hydroxocobalamin-incubated cells, indicating that cobalamin might either directly or indirectly act as a scavenger of ROS. ROS production might be considered as a phenotypic modifier in cblC patients, and cobalamin supplementation or additional antioxidant drugs might suppress apoptosis and prevent cellular damage in these patients. Hum Mutat 30:1–9, 2009. © 2009 Wiley-Liss, Inc.     

Ten novel HMGCL mutations in 24 patients of different origin with 3‐hydroxy‐3‐methyl‐glutaric aciduria

3‐Hydroxy‐3‐methylglutaric aciduria is a rare autosomal recessive genetic disorder that affects ketogenesis and L‐leucine catabolism. The clinical acute symptoms include vomiting, convulsions, metabolic acidosis, hypoketotic hypoglycaemia and lethargy. To date, 33 mutations in 100 patients have been reported in the HMGCL gene. In this study 10 new mutations in 24 patients are described. They include: 5 missense mutations: c.109G>A, c.425C>T, c.521G>A, c.575T>C and c.598A>T, 2 nonsense mutations: c.242G>A and c.559G>T, one small deletion: c.853delC, and 2 mutations in intron regions: c.497+4A>G and c.750+1G>A. Two prevalent mutations were detected, 109G>T (E37X) in 38% of disease alleles analyzed and c.504_505delCT in 10% of them. Although patients are mainly of European origin (71%) and mostly Spanish (54%), the group is ethnically diverse and includes, for the first time, patients from Pakistan, Palestine and Ecuador. We also present a simple, efficient method to express the enzyme and we analyze the possible functional effects of missense mutations. The finding that all identified missense mutations cause a >95% decrease in the enzyme activity, indicates that the disease appears only in very severe genotypes.” © 2009 Wiley‐Liss, Inc.     

Recurrence of reported CDH23 mutations causing DFNB12 in a special cohort of South Indian hearing impaired assortative mating families – an evaluation

Mutations in CDH23 are known to cause autosomal‐recessive nonsyndromic hearing loss (DFNB12). Until now, there was only one study describing its frequency in Indian population. We screened for CDH23 mutations to identify prevalent and recurring mutations among South Indian assortative mating hearing‐impaired individuals who were identified as non‐DFNB1 (GJB2 and GJB6). Whole‐exome sequencing was performed in individuals found to be heterozygous for CDH23 to determine whether there was a second pathogenic allele. In our study, 19 variants including 6 pathogenic missense mutations were identified. The allelic frequency of pathogenic mutations accounts to 4.7% in our cohort, which is higher than that reported previously; three mutations (c.429+4G>A, c.2968G>A, and c.5660C>T) reported in the previous Indian study were found to recur. DFNB12 was found to be the etiology in 3.4% of our cohort, with missense mutation c.2968G>A (p.Asp990Asn) being the most prevalent (2.6%). These results suggest a need to investigate the possibility for higher proportion of CDH23 mutations in the South Indian hearing‐impaired population.     

Type I hyperprolinemia: genotype/phenotype correlations

Type I hyperprolinemia (HPI) is an autosomal recessive disorder associated with cognitive and psychiatric troubles, caused by alterations of the Proline Dehydrogenase gene (PRODH) at 22q11. HPI results from PRODH deletion and/or missense mutations reducing proline oxidase (POX) activity. The goals of this study were first to measure in controls the frequency of PRODH variations described in HPI patients, second to assess the functional effect of PRODH mutations on POX activity, and finally to establish genotype/enzymatic activity correlations in a new series of HPI patients. Eight of 14 variants occurred at polymorphic frequency in 114 controls. POX activity was determined for six novel mutations and two haplotypes. The c.1331G>A, p.G444D allele has a drastic effect, whereas the c.23C>T, p.P8L allele and the c.[56C>A; 172G>A], p.[Q19P; A58T] haplotype result in a moderate decrease in activity. Among the 19 HPI patients, 10 had a predicted residual activity <50%. Eight out of nine subjects with a predicted residual activity ≥50% bore at least one c.824C>A, p.T275N allele, which has no detrimental effect on activity but whose frequency in controls is only 3%. Our results suggest that PRODH mutations lead to a decreased POX activity or affect other biological parameters causing hyperprolinemia. Hum Mutat 31:961–965, 2010. © 2010 Wiley‐Liss, Inc.     

Relationship between programmed cell death‐1 polymorphisms and clearance of hepatitis B virus

Programmed cell death‐1 (PD‐1) plays a critical role in regulating T‐cell function during hepatitis B virus (HBV) infection. This study investigated the relationship between the polymorphisms of PD‐1 gene and the susceptibility to HBV infection. Single nucleotide polymorphisms (SNPs) in PD‐1 gene at positions +7146 G>A (guanine to adenine substitution) and +7209 C>T (cytosine to thymine substitution) were analysed using a polymerase chain reaction–restriction fragment length polymorphism (PCR‐RFLP) method in 220 subjects with chronic hepatitis B infection and 165 spontaneous clearance of HBV subjects. However, no statistically significant differences were found in the genotype distributions of the PD‐1 +7146 G>A and PD‐1 +7209 C>T polymorphisms among chronic hepatitis B and spontaneous clearance subjects. According to stratified analyses, borderline significance was observed between PD‐1 +7146 GA genotype and risk of HBV chronicity in the subgroup of male gender (OR = 1.88, 95% 0.95–3.71; P = 0.07). Our findings demonstrate for the first time that the PD‐1 +7146 G>A and PD‐1 +7209 C>T polymorphisms have not been any major role in genetic susceptibility to chronicity of HBV infection, at least in the population studied here. Independent studies are needed to validate our findings in a larger series, as well as in patients of different ethnic origins.     

Characterization of 11 novel mutations in the X‐linked chronic granulomatous disease (CYBB gene)

The most frequent form of chronic granulomatous disease (CGD) is caused by inactivation of the CYBB gene, which encodes the gp91‐phox subunit of phagocyte NADPH oxidase. This defect prevents phagocytes from producing reactive oxygen species and thus from eradicating bacterial and fungal infections. We investigated 16 unrelated male patients with suspected X‐linked CGD and gp91‐phox deficiency. A mutation was found in the CYBB gene of all 16 patients, and 11 of these mutations were novel. Eleven patients (69%) had a point mutation (84G>A in two unrelated patients, and 177C>G, 217C>T, 388C>T, 676C>T, 691C>T, 868C>T, 919A>C, 1384G>T and T1514G in one case each, yielding W28X, C59W, R73X, R130X, R226X, Q231X, R290X, T307P, E462X, L505R gp‐91phox). One patient had an in‐frame deletion removing two amino acids (R54 and A55). Finally, insertions or duplications were found in four patients (from +1 to +31 bases). Overall, 12 (75%) of the mutations led to the production of a truncated protein. No clear correlation was found between clinical manifestations and genomic/biochemical alterations. Thirteen mothers could be tested, and all were carriers. Hum Mutat 18:163, 2001. © 2001 Wiley‐Liss, Inc.     

Schuurs‐Hoeijmakers syndrome in a patient from India

Schuurs‐Hoeijmakers syndrome (SHMS), or Autosomal Dominant Mental Retardation Syndrome type 17 (MRD17) is a rare form of intellectual disability with distinct facial features. A recurrent de novo heterozygous c.607C>T, p.Arg203Trp mutation in the PACS1 gene accounts for all reported cases except for one patient with a de novo heterozygous c.608G>A, p.Arg203Trp mutation. Ethnic background is known to affect the clinical manifestation of dysmorphic syndromes. Here we describe the first Indian patient with Schuurs‐Hoeijmakers syndrome (SHMS) with a de novo heterozygous NM_018026.3 (PACS1):c.607C>T (p.Arg203Trp) variant. He is the only child with SHMS with a cleft lip. Thus our report expands the phenotypic spectrum of SHMS and establishes its occurrence across populations.