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Hum Mutat [journal]
- Impaired Function is a Common Feature of Neuropathy-Associated Glycyl-tRNA Synthetase Mutations. [JOURNAL ARTICLE]
- Hum Mutat 2014 Aug 28.
Charcot-Marie-Tooth disease type 2D (CMT2D) is an autosomal dominant axonal peripheral neuropathy characterized by impaired motor and sensory function in the distal extremities. Mutations in the glycyl-tRNA synthetase (GARS) gene cause CMT2D. GARS is a member of the ubiquitously expressed aminoacyl-tRNA synthetase (ARS) family and is responsible for charging tRNA with glycine. To date, thirteen GARS mutations have been identified in patients with CMT disease. While functional studies have revealed loss-of-function characteristics, only four GARS mutations have been rigorously studied. Here, we report the functional evaluation of nine CMT-associated GARS mutations in tRNA charging, yeast complementation, and subcellular localization assays. Our results demonstrate that impaired function is a common characteristic of CMT-associated GARS mutations. Additionally, one mutation previously associated with CMT disease (p.Ser581Leu) does not demonstrate impaired function, was identified in the general population, and failed to segregate with disease in two newly identified families with CMT disease. Thus, we propose that this variant is not a disease-causing mutation. Together, our data indicate that impaired function is a key component of GARS-mediated CMT disease and emphasize the need for careful genetic and functional evaluation before implicating a variant in disease onset. This article is protected by copyright. All rights reserved.
- Comprehensive Analysis of Pathogenic Deletion Variants in Fanconi Anemia Genes. [JOURNAL ARTICLE]
- Hum Mutat 2014 Aug 28.
Fanconi anemia (FA) is a rare recessive disease resulting from mutations in one of at least 16 different genes. Mutation types and phenotypic manifestations of FA are highly heterogeneous and influence the clinical management of the disease. We analyzed 202 FA families for large deletions, using high-resolution Comparative Genome Hybridization arrays (arrayCGH), Single Nucleotide Polymorphism arrays (SNParrays) and DNA sequencing. We found pathogenic deletions in 88 FANCA, seven FANCC, two FANCD2, and one FANCB families. We find 35% of FA families carry large deletions, accounting for 18% of all FA pathogenic variants. Cloning and sequencing across the deletion breakpoints revealed that 52 FANCA deletion ends, and one FANCC deletion end extended beyond the gene boundaries, potentially affecting neighboring genes with phenotypic consequences. Seventy-five percent of the FANCA deletions are Alu-Alu mediated, predominantly by AluY elements, and appear to be caused by Non-Allelic Homologous Recombination. Individual Alu hotspots were identified. Defining the haplotypes of four FANCA deletions shared by multiple families revealed that three share a common ancestry. Knowing the exact molecular changes that lead to the disease may be critical for a better understanding of the FA phenotype, and to gain insight into the mechanisms driving these pathogenic deletion variants. This article is protected by copyright. All rights reserved.
- Three Different Cone Opsin Gene Array Mutational Mechanisms; Genotype-Phenotype Correlation and Functional Investigation of Cone Opsin Variants. [JOURNAL ARTICLE]
- Hum Mutat 2014 Aug 28.
Mutations in the OPN1LW (L-) and OPN1MW (M-) cone opsin genes underlie a spectrum of cone photoreceptor defects from stationary loss of colour vision to progressive retinal degeneration. Genotypes of 22 families with a range of cone disorders were grouped into three classes: deletions of the Locus Control Region (LCR); missense mutation (p.Cys203Arg) in an L-/M- hybrid gene; and exon 3 single nucleotide polymorphism (SNP) interchange haplotypes in an otherwise normal gene array. Moderate to high myopia was observed in all mutation categories. Individuals with LCR deletions or p.Cys203Arg mutations were more likely to have nystagmus and poor vision, with disease progression in some p.Cys203Arg patients. Three disease-associated exon 3 SNP haplotypes encoding LIAVA, LVAVA or MIAVA, were identified in our cohort. These patients were less likely to have nystagmus but more likely to show progression, with all patients over the age of 40 having marked macular abnormalities. Previously, the haplotype LIAVA has been shown to result in exon 3 skipping. Here we show that haplotypes LVAVA and MIAVA also result in aberrant splicing, with a residual low level of correctly spliced cone opsin. The OPN1LW/OPN1MW:c.532A>G SNP, common to all three disease-associated haplotypes, appears to be principally responsible for this mutational mechanism. This article is protected by copyright. All rights reserved.
- Functional and Clinical Impact of Novel TMPRSS6 Variants in Iron-Refractory Iron-Deficiency Anaemia Patients and Genotype-Phenotype Studies. [JOURNAL ARTICLE]
- Hum Mutat 2014 Aug 25.
Iron-refractory iron-deficiency anaemia (IRIDA) is a rare autosomal-recessive disorder characterized by hypochromic microcytic anaemia, low transferrin saturation and inappropriate high levels of the iron hormone hepcidin. The disease is caused by variants in the transmembrane protease serine 6 (TMPRSS6) gene that encodes the type II serine protease matriptase-2, a negative regulator of hepcidin transcription. Sequencing analysis of the TMPRSS6 gene in 21 new IRIDA patients from 16 families with different ethnic origin reveal 17 novel mutations, including the most frequent mutation in Southern Italy (p.W590R). Eight missense mutations were analysed in vitro. All but the p.T287N variant impair matriptase-2 autoproteotylic activation, decrease ability to cleave membrane HJV and inhibit the HJV-dependent hepcidin activation. Genotype-phenotype studies in IRIDA patients have been so far limited due to the relatively low number of described patients. Our genotype-phenotype correlation analysis demonstrates that patients carrying 2 nonsense mutations present a more severe anaemia and microcytosis and higher hepcidin levels than the other patients. We confirm that TMPRSS6 mutations are spread along the gene and that mechanistically they fully or partially abrogate hepcidin inhibition. Genotyping IRIDA patients help in predicting IRIDA severity and may be useful for predicting response to iron treatment. This article is protected by copyright. All rights reserved.
- An Efficient Pipeline for the Generation and Functional Analysis of Human BRCA2 Variants of Uncertain Significance. [JOURNAL ARTICLE]
- Hum Mutat 2014 Aug 21.
The implementation of next-generation sequence analysis of disease-related genes has resulted in a increasing number of genetic variants with an unknown clinical significance. The functional analysis of these so-called "variants of uncertain significance" (VUS) is hampered by the tedious and time-consuming procedures required to generate and test specific sequence variants in genomic DNA. Here we describe an efficient pipeline for the generation of gene variants in a full-length human gene, BRCA2, using a bacterial artificial chromosome (BAC). This method permits the rapid generation of intronic and exonic variants in a complete gene through the use of an exon-replacement strategy based on simple site-directed mutagenesis and an effective positive-negative selection system in E. coli. The functionality of variants can then be assessed through the use of functional assays, such as complementation of gene-deficient mouse embryonic stem (mES) cells in the case of human BRCA2. Our methodology builds upon an earlier protocol and, through the introduction of a series of major innovations, now represents a practical proposition for the rapid analysis of BRCA2 variants and a blueprint for the analysis of other genes using similar approaches. This method enables rapid generation and reliable classification of VUS in disease-related genes, allowing informed clinical decision-making. This article is protected by copyright. All rights reserved.
- Type I Procollagen C-Propeptide Defects: Study of Genotype-Phenotype Correlation and Predictive Role of Crystal Structure. [JOURNAL ARTICLE]
- Hum Mutat 2014 Aug 21.
The type I procollagen carboxyterminal(C-)propeptides are crucial in directing correct assembly of the procollagen heterotrimers. Defects in these domains have anecdotally been reported in patients with osteogenesis imperfecta (OI) and few genotype-phenotype correlations have been described. To gain insight in the functional consequences of C-propeptide defects, we performed a systematic review of clinical, molecular and biochemical findings in all patients in whom we identified a type I procollagen C-propeptide defect, and compared this with literature data. We report 30 unique type I procollagen C-propeptide variants, 24 of which are novel. The outcome of COL1A1 nonsense and frameshift variants depends on the location of the premature termination codon (PTC). Those located prior to 50-55 nucleotides upstream of the most 3' exon-exon junction lead to nonsense-mediated mRNA decay (NMD) and cause mild OI. Those located beyond this boundary escape NMD, generally lead to production of stable, overmodified procollagen chains, which may partly be retained intracellularly, and are usually associated with severe-to-lethal OI. Proα1(I)-C-propeptide defects, permitting chain association result in more severe phenotypes than those inhibiting chain association. We demonstrate that the crystal structure of the proα1(III)-C-propeptide is a reliable tool to predict phenotypic severity for most COL1A1-C-propeptide missense variants, while for COL1A2-C-propeptide variants the phenotypic outcome is milder than predicted. This article is protected by copyright. All rights reserved.
- Impaired Development Of Neural-Crest Cell Derived Organs and Intellectual Disability Caused By MED13L Haploinsufficiency. [JOURNAL ARTICLE]
- Hum Mutat 2014 Aug 18.
MED13L is a component subunit of the Mediator complex, an important regulator of transcription that is highly conserved across eukaryotes. Here we report MED13L disruption in a translocation t(12;19) breakpoint of a patient with Pierre-Robin syndrome, moderate intellectual disability (ID), craniofacial anomalies, and muscular defects. The phenotype is similar to previously described patients with MED13L haploinsufficiency. Knockdown of MED13L orthologue in zebrafish, med13b, showed early defective migration of cranial neural crest cells (NCCs) that contributed into cartilage structure deformities in the later stage, recapitulating craniofacial anomalies seen in human patients. Notably, we observed abnormal distribution of developing neurons in different brain regions of med13b morphant embryos, which could be rescued upon introduction of full-length human MED13L mRNA. To compare with mammalian system, we suppressed MED13L expression by short-hairpin RNA in ES-derived human neural progenitors, and differentiated them into neurons. Transcriptome analysis revealed differential expression of components of Wnt and FGF signalling pathways in MED13L-deficient neurons. Our finding provides a novel insight into the mechanism of overlapping phenotypic outcome targeting NCCs derivatives organs in patients with MED13L haploinsufficiency, and emphasizes a clinically recognizable syndromic phenotype in these patients. This article is protected by copyright. All rights reserved.
- Morquio A Syndrome-Associated Mutations: A Review of Alterations in the GALNS gene and a New Locus-Specific Database. [JOURNAL ARTICLE]
- Hum Mutat 2014 Aug 18.
Morquio A syndrome (mucopolysaccharidosis IVA) is an autosomal recessive disorder that results from deficient activity of the enzyme N-acetylgalactosamine-6-sulfatase (GALNS) due to alterations in the GALNS gene, which causes major skeletal and connective tissue abnormalities and effects on multiple organ systems. The GALNS alterations associated with Morquio A are numerous and heterogeneous, and new alterations are continuously being identified. To aid detection and interpretation of GALNS alterations, from previously published research we provide a comprehensive and up-to-date listing of 277 unique GALNS alterations associated with Morquio A identified from 1091 published GALNS alleles. In agreement with previous findings, most reported GALNS alterations are missense changes and even the most frequent alterations are relatively uncommon. We found that 48% of patients are assessed as homozygous for a GALNS alteration, 39% are assessed as heterozygous for two identified GALNS alterations, and in 13% of patients only one GALNS alteration is detected. We report here the creation of a locus-specific database for the GALNS gene (http://galns.mutdb.org/) that catalogs all reported alterations in GALNS to date. We highlight the challenges both in alteration detection and genotype-phenotype interpretation caused in part by the heterogeneity of GALNS alterations and provide recommendations for molecular testing of GALNS. This article is protected by copyright. All rights reserved.
- A Novel SHOC2 Variant in Rasopathy. [JOURNAL ARTICLE]
- Hum Mutat 2014 Aug 18.
Rasopathies are a group of genetic disorders caused by germline mutations in multiple genes of the Extracellular signal-Regulated Kinases 1 and 2 (ERK1/2) pathway. The only previously identified missense mutation in SHOC2, a scaffold protein of the ERK1/2 pathway, led to Noonan-like syndrome with loose anagen hair. Here we report a novel mutation in SHOC2(c.519G>A; p.M173I) that leads to a Rasopathy with clinical features partially overlapping those occurring in Noonan and Cardio-Facio-Cutaneous syndromes. Studies to clarify the significance of this SHOC2 variant revealed that the mutant protein has impaired capacity to interact with protein phosphatase 1c (PP1c), leading to insufficient activation of RAF-1 kinase. This SHOC2 variant thus is unable to fully rescue ERK1/2 activity in cells depleted of endogenous SHOC2. We conclude that SHOC2 mutations can cause a spectrum of Rasopathy phenotypes in heterozygous individuals. Importantly, our work suggests that individuals with mild Rasopathy symptoms may be under-diagnosed. This article is protected by copyright. All rights reserved.
- Instability of Trinucleotidic Repeats During Chromatin Remodeling in Spermatids. [JOURNAL ARTICLE]
- Hum Mutat 2014 Aug 18.
Transient DNA breaks and evidence of DNA damage response have recently been reported during the chromatin remodeling process in haploid spermatids, creating a potential window of enhanced genetic instability. We used flow cytometry to achieve separation of differentiating spermatids into four highly purified populations using transgenic mice harboring 160 CAG repeats within exon 1 of the human Huntington disease gene (HTT). Trinucleotic repeat expansion was found to occur immediately following the chromatin remodeling steps, confirming the genetic instability of the process and pointing to the origin of paternal anticipation observed in some TNR diseases. This article is protected by copyright. All rights reserved.