Identification and molecular characterization of six novel mutations in the UDP-N-acetylglucosamine-1-phosphotransferase gamma subunit (GNPTG) gene in patients with mucolipidosis III gamma.
TL;DR: RT‐PCR analysis of the c.610–2A>G transition demonstrated that the change altered splicing, leading to the production of two distinct aberrantly spliced forms, supporting the view that the mutational spectrum of the GNPTG gene is strongly influenced by the properties of the local DNA sequence environment.
Abstract: Mucolipidosis type III (MLIII) is an autosomal recessive disorder affecting lysosomal hydrolase trafficking. In a study of 10 patients from seven families with a clinical phenotype and enzymatic diagnosis of MLIII, six novel GNPTG gene mutations were identified. These included missense (p.T286M) and nonsense (p.W111X) mutations and a transition in the obligate AG-dinucleotide of the intron 8 acceptor splice site (c.610–2A>G). Three microdeletions were also identified, two of which (c.611delG and c.640_667del28) were located within the coding region whereas one (c.609+28_610-16del) was located entirely within intron 8. RT-PCR analysis of the c.610–2A>G transition demonstrated that the change altered splicing, leading to the production of two distinct aberrantly spliced forms, viz. the skipping of exon 9 (p.G204_K247del) or the retention of introns 8 and 9 (p.G204VfsX28). RT-PCR analysis, performed on a patient homozygous for the intronic deletion (c.609+28_610-16del), failed to detect any GNPTG RNA transcripts. To determine whether c.609+28_610-16del allele-derived transcripts were subject to nonsense-mediated mRNA decay (NMD), patient fibroblasts were incubated with the protein synthesis inhibitor anisomycin. An RT-PCR fragment retaining 43 bp of intron 8 was consistently detected suggesting that the 33-bp genomic deletion had elicited NMD. Quantitative real-time PCR and GNPTG western blot analysis confirmed that the homozygous microdeletion p.G204VfsX17 had elicited NMD resulting in failure to synthesize GNPTG protein. Analysis of the sequences surrounding the microdeletion breakpoints revealed either intrinsic repetitivity of the deleted region or short direct repeats adjacent to the breakpoint junctions. This is consistent with these repeats having mediated the microdeletions via replication slippage and supports the view that the mutational spectrum of the GNPTG gene is strongly influenced by the properties of the local DNA sequence environment.
Citations
More filters
University of California, Los Angeles1, Icahn School of Medicine at Mount Sinai2, National Institutes of Health3, Broad Institute4, National University of Singapore5, North Carolina Central University6, University of Ottawa7, National Institute for Health Research8, University of Lübeck9, Harvard University10, Heidelberg University11, MedStar Washington Hospital Center12, Duke University13, Cleveland Clinic14, University of Pennsylvania15, Technische Universität München16, Stanford University17
TL;DR: The results suggest genetic influences on a diverse set of both known and novel biological processes that contribute to CAD risk, and highlight potential novel targets for further mechanistic studies and therapeutic interventions.
Abstract: The majority of the heritability of coronary artery disease (CAD) remains unexplained, despite recent successes of genome-wide association studies (GWAS) in identifying novel susceptibility loci. Integrating functional genomic data from a variety of sources with a large-scale meta-analysis of CAD GWAS may facilitate the identification of novel biological processes and genes involved in CAD, as well as clarify the causal relationships of established processes. Towards this end, we integrated 14 GWAS from the CARDIoGRAM Consortium and two additional GWAS from the Ottawa Heart Institute (25,491 cases and 66,819 controls) with 1) genetics of gene expression studies of CAD-relevant tissues in humans, 2) metabolic and signaling pathways from public databases, and 3) data-driven, tissue-specific gene networks from a multitude of human and mouse experiments. We not only detected CAD-associated gene networks of lipid metabolism, coagulation, immunity, and additional networks with no clear functional annotation, but also revealed key driver genes for each CAD network based on the topology of the gene regulatory networks. In particular, we found a gene network involved in antigen processing to be strongly associated with CAD. The key driver genes of this network included glyoxalase I (GLO1) and peptidylprolyl isomerase I (PPIL1), which we verified as regulatory by siRNA experiments in human aortic endothelial cells. Our results suggest genetic influences on a diverse set of both known and novel biological processes that contribute to CAD risk. The key driver genes for these networks highlight potential novel targets for further mechanistic studies and therapeutic interventions.
187 citations
TL;DR: The generation of recombinant single-chain antibody fragments against M6P residues and of new mouse models of MLII and MLIII will have considerable impact to provide deeper insight into the cell biology of lysosomal dysfunctions and the pathomechanisms underlying these lysOSomal disorders.
103 citations
TL;DR: A novel exonic SNP (c.303G>A; E101E) was identified which is predicted to create an SFRS1 (SF2/ASF) binding site that may be of potential functional/clinical relevance.
Abstract: Mutational analysis of the GNPTAB gene was performed in 46 apparently unrelated patients with mucolipidosis IIα/β or IIIα/β, characterized by the mistargeting of multiple lysosomal enzymes as a consequence of a UDP-GlcNAc-1-phosphotransferase defect. The GNPTAB mutational spectrum comprised 25 distinct mutant alleles, 22 of which were novel, including 3 nonsense mutations (p.Q314X, p.R375X, p.Q507X), 5 missense mutations (p.I403T, p.C442Y, p.C461G, p.Q926P, p.L1001P), 6 microduplications (c.749dupA, c.857dupA, c.1191_1194dupGCTG, c.1206dupT, c.1331dupG, c.2220_2221dupGA) and 8 microdeletions (c.755_759delCCTCT, c.1399delG, c.1959_1962delTAGT, c.1965delC, c.2550_2554delGAAAA, c.3443_3446delTTTG, c.3487_3490delACAG, c.3523_3529delATGTTCC). All micro-duplications/deletions were predicted to result in the premature termination of translation. A novel exonic SNP (c.303G>A; E101E) was identified which is predicted to create an SFRS1 (SF2/ASF) binding site that may be of potential functional/clinical relevance. This study of mutations in the GNPTAB gene, the largest yet reported, extends our knowledge of the mutational heterogeneity evident in MLIIα/β/MLIIIα/β.
42 citations
TL;DR: The GNPTAB and GNPTG genes encode the α/β-precursor and the γ-subunit of N-acetylglucosamine (GlcNAc)-1-phosphotransferase, respectively, the key enzyme for the generation of mannose 6phosphate targeting signals on lysosomal enzymes.
Abstract: Mutations in the GNPTAB and GNPTG genes cause mucolipidosis (ML) type II, type III alpha/beta, and type III gamma, which are autosomal recessively inherited lysosomal storage disorders. GNPTAB and GNPTG encode the α/β-precursor and the γ-subunit of N-acetylglucosamine (GlcNAc)-1-phosphotransferase, respectively, the key enzyme for the generation of mannose 6-phosphate targeting signals on lysosomal enzymes. Defective GlcNAc-1-phosphotransferase results in missorting of lysosomal enzymes and accumulation of non-degradable macromolecules in lysosomes, strongly impairing cellular function. MLII-affected patients have coarse facial features, cessation of statural growth and neuromotor development, severe skeletal abnormalities, organomegaly, and cardiorespiratory insufficiency leading to death in early childhood. MLIII alpha/beta and MLIII gamma are attenuated forms of the disease. Since the identification of the GNPTAB and GNPTG genes, 564 individuals affected by MLII or MLIII have been described in the literature. In this report, we provide an overview on 258 and 50 mutations in GNPTAB and GNPTG, respectively, including 58 novel GNPTAB and seven novel GNPTG variants. Comprehensive functional studies of GNPTAB missense mutations did not only gain insights into the composition and function of the GlcNAc-1-phosphotransferase, but also helped to define genotype-phenotype correlations to predict the clinical outcome in patients.
40 citations
TL;DR: The exomes of four members of a family presenting with spondylo‐epiphyseal dysplasia and retinitis pigmentosa were sequenced and a six‐base‐pair deletion in GNPTG was identified, the gene implicated in mucolipidosis type IIIγ, demonstrating the clinical utility of next‐generation sequencing to diagnose rare genetic diseases.
Abstract: Linkage analysis with subsequent candidate gene sequencing is typically used to diagnose novel inherited syndromes. It is now possible to expedite diagnosis through the sequencing of all coding regions of the genome (the exome) or full genomes. We sequenced the exomes of four members of a family presenting with spondylo-epiphyseal dysplasia and retinitis pigmentosa and identified a six-base-pair (6-bp) deletion in GNPTG, the gene implicated in mucolipidosis type IIIγ. The diagnosis was confirmed by biochemical studies and both broadens the mucolipidosis type III phenotype and demonstrates the clinical utility of next-generation sequencing to diagnose rare genetic diseases.
31 citations
References
More filters
TL;DR: Suggestions are presented for reporting complex mutations in a unified manner for efficient and accurate reporting, testing, and curation of the growing number of disease mutations and useful polymorphisms being discovered in the human genome.
Abstract: Consistent gene mutation nomenclature is essential for efficient and accurate reporting, testing, and curation of the growing number of disease mutations and useful polymorphisms being discovered in the human genome. While a codified mutation nomenclature system for simple DNA lesions has now been adopted broadly by the medical genetics community, it is inherently difficult to represent complex mutations in a unified manner. In this article, suggestions are presented for reporting just such complex mutations.
1,744 citations
19 Jan 1997
TL;DR: An improved splice site predictor for the genefinding program Genie is presented and it is shown that Genie shows significant improvements in the sensitivity and specificity of gene structure identification.
Abstract: We present an improved splice site predictor for the genefinding program Genie. Genie is based on a generalized Hidden Markov Model (GHMM) that describes the grammar of a legal parse of a multi-exon gene in a DNA sequence. In Genie, probabilities are estimated for gene features by using dynamic programming to combine information from multiple content and signal sensors, including sensors that integrate matches to homologous sequences from a database. One of the hardest problems in genefinding is to determine the complete gene structure correctly. The splice site sensors are the key signal sensors that address this problem. We replaced the existing splice site sensors in Genie with two novel neural networks based on dinucleotide frequencies. Using these novel sensors, Genie shows significant improvements in the sensitivity and specificity of gene structure identification. Experimental results in tests using a standard set of annotated genes showed that Genie identified 86% of coding nucleotides correctly with a specificity of 85%, versus 80% and 84% in the older system. In further splice site experiments, we also looked at correlations between splice site scores and intron and exon lengths, as well as at the effect of distance to the nearest splice site on false positive rates.
1,550 citations
TL;DR: The acquisition and loss of mRNA-associated proteins accompanies the transition from the pioneer round to subsequent rounds of translation, and from translational competence to substrate for nonsense-mediated mRNA decay.
Abstract: Studies of nonsense-mediated mRNA decay in mammalian cells have proffered unforeseen insights into changes in mRNA–protein interactions throughout the lifetime of an mRNA. Remarkably, mRNA acquires a complex of proteins at each exon–exon junction during pre-mRNA splicing that influences the subsequent steps of mRNA translation and nonsense-mediated mRNA decay. Complex-loaded mRNA is thought to undergo a pioneer round of translation when still bound by cap-binding proteins CBP80 and CBP20 and poly(A)-binding protein 2. The acquisition and loss of mRNA-associated proteins accompanies the transition from the pioneer round to subsequent rounds of translation, and from translational competence to substrate for nonsense-mediated mRNA decay.
1,175 citations
TL;DR: Cet article pass en revue les connaissances actuelles sur l'orientation des enzymes lysosomales vers leur cible and discute les anomalies de cette voie.
Abstract: Cet article pass en revue les connaissances actuelles sur l'orientation des enzymes lysosomales vers leur cible et discute les anomalies de cette voie qui ont ete etablies jusqu'ici
444 citations
TL;DR: The Mutation Analyzer (Mutalyzer) sequence variation nomenclature checker is developed for automated analysis and correction of sequence variant descriptions using reference sequences from any organism.
Abstract: Unambiguous and correct sequence variant descriptions are of utmost importance, not in the least since mistakes and uncertainties may lead to undesired errors in clinical diagnosis. We developed the Mutation Analyzer (Mutalyzer) sequence variation nomenclature checker (www.lovd.nl/mutalyzer; last accessed 13 September 2007) for automated analysis and correction of sequence variant descriptions using reference sequences from any organism. Mutalyzer handles most variation types: substitution, deletion, duplication, insertion, indel, and splice-site changes following current recommendations of the Human Genome Variation Society (HGVS). Input is a GenBank accession number or an uploaded reference sequence file in GenBank format with user-modified annotation, an HGNC gene symbol, and the variant (single or in a batch file). Mutalyzer generates variant descriptions at DNA level, the level of all annotated transcripts and the deduced outcome at protein level. To validate Mutalyzer's performance and to investigate the sequence variant description quality in locus-specific mutation databases (LSDBs), more than 11,000 variants in the PAH, BIC BRCA2, and HbVar databases were analyzed, showing that 87%, 25%, and 38%, respectively, were error-free and following the recommendations. Low recognition rates in BIC and HbVar (38% and 51%, respectively) were due to lack of a well-annotated genomic reference sequence (HbVar) or noncompliance to the guidelines (BRCA2). Provided with well-annotated genomic reference sequences, Mutalyzer is very effective for the curation of newly discovered sequence variation descriptions and existing LSDB data. Mutalyzer will be linked to the Leiden Open source Variation Database (LOVD) (www.LOVD.nl; last accessed 13 September 2007) and is the first module of a sequence variant effect prediction package.
389 citations