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Hum Mutat [journal]
- MitImpact: an Exhaustive Collection of Pre-computed Pathogenicity Predictions of Human Mitochondrial Non-synonymous Variants. [JOURNAL ARTICLE]
- Hum Mutat 2014 Dec 17.
Mitochondrial DNA carries a tiny, but fundamental portion of the eukaryotic genetic code. As its nuclear counterpart, it is susceptible to point mutations. Their level of pathogenicity has been assessed for the newly discovered mutations only, leaving some degree of uncertainty on the potential impact of the unknown mutations. Here we present Mitochondrial mutation Impact (MitImpact), a queryable lightweight web interface to a reasoned collection of structurally and evolutionary annotated pathogenicity predictions, obtained by assembling pre-computed with on-the-fly-computed sets of pathogenicity estimations, for all the possible mitochondrial missense variants. It presents itself as a resource for fast and reliable evaluation of gene-specific susceptibility of unknown and verified amino acid changes. MitImpact is freely available at http://bioinformatics.css-mendel.it/ (tools section). ©2014 Wiley Periodicals, Inc.
- Novel mutations in the DYNC1H1 tail domain refine the genetic and clinical spectrum of dyneinopathies. [JOURNAL ARTICLE]
- Hum Mutat 2014 Dec 16.
The heavy chain 1 of cytoplasmic dynein (DYNC1H1) is responsible for movement of the motor complex along microtubules and recruitment of dynein components. Mutations in DYNC1H1 are associated with spinal muscular atrophy (SMA), motor and sensory neuropathy (HMSN), cortical malformations, or a combination of these. Combining linkage analysis and whole exome sequencing, we identified a novel dominant defect in the DYNC1H1 tail domain (c.1792C>T, p.Arg598Cys) causing axonal HMSN. Mutation analysis of the tail region in 355 patients identified a de novo mutation (c.791G>T, p.Arg264Leu) in an isolated SMA patient. Her phenotype was more severe than previously described, characterized by multiple congenital contractures and delayed motor milestones, without brain malformations. The mutations in DYNC1H1 increase the interaction with its adaptor BICD2. This relates to previous studies on BICD2 mutations causing a highly similar phenotype. Our findings broaden the genetic heterogeneity and refine the clinical spectrum of DYNC1H1, and have implications for molecular diagnostics of motor neuron diseases. This article is protected by copyright. All rights reserved.
- Loss of Function Variants in Human PNPLA8 Encoding Calcium-Independent Phospholipase A2 γ Recapitulate the Mitochondriopathy of the Homologous Null Mouse. [JOURNAL ARTICLE]
- Hum Mutat 2014 Dec 16.
Mitochondriopathies are a group of clinically heterogeneous genetic diseases caused by defects in mitochondrial metabolism, bioenergetic efficiency, and/or signaling functions. The large majority of proteins involved in mitochondrial function are encoded by nuclear genes, with many yet to be associated with human disease. We performed exome sequencing on a young girl with a suspected mitochondrial myopathy that manifested as progressive muscle weakness, hypotonia, seizures, poor weight gain, and lactic acidosis. She was compound heterozygous for two frameshift mutations, p.Asn112HisfsX29 and p.Leu659AlafsX4, in the PNPLA8 gene, which encodes mitochondrial calcium independent phospholipase A2 γ (iPLA2 γ). Western blot analysis of affected muscle displayed the absence of PNPLA8 protein. iPLA2 s are critical mediators of a variety of cellular processes including growth, metabolism, and lipid second messenger generation, exerting their functions through catalyzing the cleavage of the acyl groups in glycerophospholipids. The clinical presentation, muscle histology and the mitochondrial ultrastructural abnormalities of this proband are highly reminiscentofPnpla8 null mice. Although other iPLA2 -related diseases have been identified, namely infantile neuroaxonal dystrophy and neutral lipid storage disease with myopathy, this is the first report of PNPLA8-related disease in a human. We suggest PNPLA8 join the increasing list of human genes involved in lipid metabolism associated with neuromuscular diseases due to mitochondrial dysfunction. This article is protected by copyright. All rights reserved.
- Benefits and Burdens of Using a SNP Array in Pregnancies at Increased Risk for the Common Aneuploidies. [JOURNAL ARTICLE]
- Hum Mutat 2014 Dec 15.
We present the nature of pathogenic SNP array findings in pregnancies without ultrasound (US) abnormalities and show the additional diagnostic value of SNP array as compared to rapid aneuploidy detection (RAD) and karyotyping. 1330 prenatal samples were investigated with a 0.5 Mb SNP array after the exclusion of the most common aneuploidies. In 2.7% (36/1330) of the cases pathogenic chromosome aberrations were found; a microscopically detectable abnormality in 0.7% and a submicroscopic aberration in 2%. Our results show that in addition to the age or screening related aneuploidy risk, in pregnancies without US abnormalities, there is a risk of 1:148 (9/1330) for a (sub)microscopic abnormality associated with an early-onset often severe disease, 1:222 (6/1330) for a submicroscopic aberration causing an early-onset disease, 1:74 (18/1330) for carrying a susceptibility locus for a neurodevelopmental disorder, and 1:443 (3/1330) for a late-onset disorder (HNPP in all 3 cases). These risk figures are important for adequate pre-test counseling so that prospective parents can make informed individualized choices between targeted prenatal testing and broad testing with SNP array. Based on our results, we believe that if invasive testing is performed, SNP array should be the preferred cytogenetic technique irrespective of the indication. This article is protected by copyright. All rights reserved.
- Functional Analysis of FOXE3 Mutations Causing Dominant and Recessive Ocular Anterior Segment Disease. [JOURNAL ARTICLE]
- Hum Mutat 2014 Dec 15.
Mutations in FOXE3 are associated with both recessive and dominant inheritance of severe anterior ocular malformations and glaucoma. However, functional analyses of putative pathogenic mutations have not been performed. We tested the hypothesis that variations in FOXE3 activity underlie the different modes of inheritance and disease phenotype. In band shift assays, three recessive mutants showed loss-of-function, one retained DNA binding activity, whereas two dominant mutants showed altered activity. All six mutants showed reduced transactivation function compared to wild-type, and modelling the heterozygous state resulted in an intermediate level of activity providing no evidence for dominant negative action. Our in vitro data are consistent with loss-of-function below a dosage sensitive threshold as a mechanism of action for recessive mutations, but indicate an altered mutant protein function rather than a haploinsufficient mechanism for dominant mutations. This study provides the first functional evidence demonstrating that FOXE3 mutations identified in patients impair protein function with differential effects. This article is protected by copyright. All rights reserved.
- Somatic MMR Gene Mutations as a Cause for MSI-H Sebaceous Neoplasms in Muir-Torre Syndrome-Like Patients. [JOURNAL ARTICLE]
- Hum Mutat 2014 Dec 15.
Sebaceous neoplasms are a major clinical feature of Muir-Torre syndrome (MTS) associated with visceral malignancies, especially colorectal and endometrial tumors. The diagnosis of MTS relies largely on the MSI phenotype in tumors, suggesting germline mutations in DNA mismatch repair (MMR) genes responsible for the inherited disease. We hypothesized that in some MSI-H sebaceous tumors, acquired rather than inherited mutations in MMR genes could be involved. Using next generation sequencing (NGS), we screened MMR gene mutations in 18 MSI-H sebaceous tumors. We found mutations in 17 samples (94%). Indeed, 12/17 (71%) were shown to carry acquired somatic mutations and among 12 samples, seven were shown to be associated with additional somatic alterations like loss of heterozygosity (LOH) or multiple mutations, suggesting somatic second hits. Our findings strongly suggest that somatic MMR deficiency is responsible for a proportion of MSI-H sebaceous tumors. This article is protected by copyright. All rights reserved.
- Flexible, Scalable and Efficient Targeted Resequencing on a Benchtop Sequencer for Variant Detection in Clinical Practice. [JOURNAL ARTICLE]
- Hum Mutat 2014 Dec 12.
The release of benchtop next generation sequencing (NGS) instruments has paved the way to implement the technology in clinical setting. The need for flexible, qualitative and cost-efficient workflows is high. We used singleplex-PCR for highly efficient target enrichment, allowing us to reach the quality standards set in Sanger sequencing-based diagnostics. For the library preparation a modified NexteraXT protocol was used, followed by sequencing on a MiSeq instrument. With an innovative pooling strategy, high flexibility, scalability and cost-efficiency was obtained, independent of the availability of commercial kits. The approach was validated for ∼250 genes associated with monogenic disorders. An overall sensitivity (>99%) similar to Sanger sequencing was observed in combination with a positive predictive value of >98%. The distribution of coverage was highly uniform, guaranteeing a minimal number of gaps to be filled with alternative methods. ISO15189-accreditation was obtained for the workflow. A major asset of the singleplex PCR-based enrichment is that new targets can be easily implemented. Diagnostic labs have validated assays available ensuring that the proposed workflow can easily be adopted. While our platform was optimized for constitutional variant detection of monogenic disease genes, it is now also used as a model for somatic mutation detection in acquired diseases. This article is protected by copyright. All rights reserved.
- Mutations in CCDC11, Which Encodes a Coiled-coil Containing Ciliary Protein, Causes situs inversus Due to Dysmotility of Monocilia in the Left-Right Organizer. [JOURNAL ARTICLE]
- Hum Mutat 2014 Dec 11.
In vertebrates, establishment of left-right (LR) asymmetry is dependent on cilia-driven fluid flow within the LR organizer. Mutations in CCDC11 disrupt LR asymmetry in humans, but how the gene functions in LR patterning is presently unknown. We describe a patient with situs inversus totalis carrying homozygous loss-of-function mutations in CCDC11. We show that CCDC11 is an axonemal protein in respiratory cilia, but is largely dispensable for their structure and motility. To investigate the role of CCDC11 in LR development, we studied the zebrafish homolog of the gene. Like in human respiratory cilia, loss of Ccdc11 causes minor defects in the motility of zebrafish kidney cilia, although the protein localizes to their axonemes and base. By contrast, Ccdc11 localizes exclusively to the basal bodies of cilia within Kupffer's vesicle (KV), the organ of laterality of teleost fishes, and within the spinal canal. Moreover the rotational motion of the cilia in these tissues of ccdc11 deficient embryos was strongly impaired. Our findings demonstrate that CCDC11 has a conserved essential function in cilia of the vertebrate LR organizer. To the best of our knowledge, this is the first ciliary component which has a differential localization and function in different kinds of motile cilia. This article is protected by copyright. All rights reserved.
- Inhibition of RAS Activation Due to a Homozygous Ezrin Variant in Patients with Profound Intellectual Disability. [JOURNAL ARTICLE]
- Hum Mutat 2014 Dec 11.
Gain-of-function alterations in several components and modulators of the Ras-MAPK pathway lead to dysregulation of the pathway and cause a broad spectrum of autosomal dominant developmental disorders, collectively known as RASopathies. These findings demonstrate the importance of tight multilevel Ras regulation to safeguard signalling output and prevent aberrant activity. We have recently identified ezrin as a novel regulatory element required for Ras activation. Homozygosity mapping and exome sequencing have now revealed the first presumably disease-causing variant in the coding gene EZR in two siblings with a profound intellectual disability. Localization and membrane targeting of the altered ezrin protein appeared normal but molecular modelling suggested protein interaction surfaces to be disturbed. Functional analysis revealed that the altered ezrin protein is no longer able to bind Ras and facilitate its activation. Furthermore, expression of the altered ezrin protein in different cell lines resulted in abnormal cellular processes, including reduced proliferation and neuritogenesis, thus revealing a possible mechanism for its phenotype in humans. To our knowledge this is the first report of an autosomal recessively inherited loss-of-function mutation causing reduced Ras activity and thus extends and complements the pathogenicity spectrum of known Ras-MAPK pathway disturbances. This article is protected by copyright. All rights reserved.
- Mutations in SNRPB, Encoding Components of the Core Splicing Machinery, Cause Cerebro-Costo-Mandibular Syndrome. [JOURNAL ARTICLE]
- Hum Mutat 2014 Dec 11.
Cerebro-costo-mandibular syndrome (CCMS) is a developmental disorder of as yet unknown etiology characterized by the association of Pierre Robin sequence and posterior rib defects. Exome sequencing and Sanger sequencing in five unrelated CCMS patients revealed five heterozygous variants in the small nuclear ribonucleoprotein polypeptides B and B1 (SNRPB) gene. This gene includes three transcripts, namely transcripts 1 and 2 encoding components of the core spliceosomal machinery (SmB' and SmB) and transcript 3 undergoing nonsense-mediated mRNA decay. All variants were located in the premature termination codon (PTC)-introducing alternative exon of transcript 3. Quantitative RT-PCR analysis revealed a significant increase in transcript 3 levels in leukocytes of CCMS individuals compared to controls. We conclude that CCMS is due to heterozygous mutations in SNRPB, enhancing inclusion of a SNRPB PTC-introducing alternative exon and show that this developmental disease is caused by defects in the splicing machinery.