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Hum Mutat [journal]
- Identification and Functional Analysis of a SLC33A1: c.339T>G (p.Ser113Arg) Variant in the Original SPG42 Family. [JOURNAL ARTICLE]
- Hum Mutat 2014 Nov 17.
Using whole exome sequencing, we surveyed all the potential pathogenic variants in an SPG42 family and found five SNPs and four indels that are shared by two patients and lie in the mapped region. Two variants, SLC33A1 p.Ser113Arg and VEPH1 p.Gln433His, co-segregated with the disease. However, VEPH1 p.Gln433His was predicted to be tolerated, thus leaving SLC33A1 p.Ser113Arg as the most plausible causal variant in this family. We found that the phosphorylated SMAD1/5/8 (P-SMAD1/5/8) and BMP receptor type 1A (BMPR1A) were substantially upregulated in fibroblasts derived from an SPG42 individual. Slc33a1 knockdown zebrafish, which exhibited defects in morphology and axon outgrowth, also showed a significant elevation in the level of P-smad1/5/8. While the phenotypes in slc33a1 knockdown zebrafish could be rescued by human wild-type SLC33A1 mRNA, this rescuing effect was diminished by co-injected mutant mRNA encoding p.Ser113Arg, indicating that p.Ser113Arg variant acts in a dominant-negative manner. Importantly, pharmacological blockade of BMPR1 activity by dorsomorphin could efficiently rescue the phenotypic defects in slc33a1 knockdown zebrafish. These results indicate that SLC33A1 can negatively regulate BMP signaling. This article is protected by copyright. All rights reserved.
- Identification and in vivo functional characterization of novel compound heterozygous BMP1 variants in osteogenesis imperfecta. [JOURNAL ARTICLE]
- Hum Mutat 2014 Nov 17.
Osteogenesis imperfecta (OI) comprises a heterogeneous group of disorders that are characterized by susceptibility to bone fractures, and range in severity from a subtle increase in fracture frequency to death in the perinatal period. Most patients have defects in type I collagen biosynthesis with autosomal dominant inheritance; but, many autosomal recessive genes have been reported. We applied whole exome sequencing to identify mutations in a Korean OI patient who had an umbilical hernia, frequent fractures, a markedly short stature, delayed motor development, scoliosis, and dislocation of the radial head, with a bowed radius and ulna. We identified two novel variants in the BMP1 gene: c.808A>G and c.1297G>T. The former variant caused a missense change p.(Met270Val) and the latter variant caused the skipping of exon 10. The hypo-functional nature of the two variants was demonstrated in a zebrafish assay. This article is protected by copyright. All rights reserved.
- ECHS1 Mutations Cause Combined Respiratory Chain Deficiency Resulting in Leigh Syndrome. [JOURNAL ARTICLE]
- Hum Mutat 2014 Nov 13.
The human ECHS1 gene encodes the short chain enoyl coenzyme A (CoA) hydratase, the enzyme that catalyzes the second step of β-oxidation of fatty acids in the mitochondrial matrix. We report on a 21-month-old boy with ECHS1 deficiency resulting in Leigh syndrome. The patient presented with hypotonia, metabolic acidosis, and developmental delay. A combined respiratory chain deficiency was also observed. Targeted exome sequencing of 776 mitochondria-associated genes encoded by nuclear DNA identified compound heterozygous mutations in ECHS1. ECHS1 protein expression was severely depleted in the patient's skeletal muscle and patient-derived myoblasts; a marked decrease of enzyme activity was also evident in patient-derived myoblasts. Immortalized patient-derived myoblasts that expressed exogenous wild-type ECHS1 exhibited the recovery of the ECHS1 activity, indicating that the gene defect was pathogenic. Mitochondrial respiratory complex activity was also mostly restored in these cells, suggesting that there was an unidentified link between deficiency of ECHS1 and respiratory chain. Here we describe the patient with ECHS1 deficiency; these findings will advance our understanding not only the pathology of mitochondrial fatty acid β-oxidation disorders, but also the regulation of mitochondrial metabolism. This article is protected by copyright. All rights reserved.
- Two Siblings with Homozygous Pathogenic Splice Site Variant in Mitochondrial Asparaginyl-tRNA Synthetase (NARS2). [JOURNAL ARTICLE]
- Hum Mutat 2014 Nov 11.
A homozygous missense mutation (c.822G>C) was found in the gene encoding the mitochondrial asparaginyl-tRNA synthetase (NARS2) in two siblings born to consanguineous parents. These siblings presented with different phenotypes: one had mild intellectual disability and epilepsy in childhood whereas the other had severe myopathy. Biochemical analysis of the oxidative phosphorylation (OXPHOS) complexes in both siblings revealed a combined complex I and IV deficiency in skeletal muscle. In-gel activity staining after BN-PAGE confirmed the decreased activity of complex I and IV, and, in addition, showed the presence of complex V subcomplexes. Considering the consanguineous descent, homozygosity mapping and whole exome sequencing were combined revealing the presence of one single missense mutation in the shared homozygous region. The c.822G>C variant affects the 3' splice site of exon 7, leading to skipping of the whole exon 7 and a part of exon 8 in the NARS2 mRNA. In EBV-transformed lymphoblasts a specific decrease in the amount of charged mt-tRNA(Asn) was demonstrated as compared to controls. This confirmed the pathogenic nature of the variant. To conclude, the reported variant in NARS2 results in a combined OXPHOS complex deficiency involving complex I and IV, making NARS2 a new member of disease associated aaRS2. This article is protected by copyright. All rights reserved.
- Varisnp, A Benchmark Database For Variations from dbSNP. [JOURNAL ARTICLE]
- Hum Mutat 2014 Nov 11.
For development and evaluation of methods for predicting the effects of variations, benchmark datasets are needed. Some previously developed datasets are available for this purpose, but newer and larger benchmark sets for benign variants have largely been missing. VariSNP datasets are selected from dbSNP. These subsets were filtered against disease-related variants in the ClinVar, UniProtKB/Swiss-Prot and PhenCode databases, to identify neutral or non-pathogenic cases. All variant descriptions include mapping to reference sequences on chromosomal, genomic, coding DNA and protein levels. The datasets will be updated with automated scripts on a regular basis and are freely available at https://structure.bmc.lu.se/VariSNP. This article is protected by copyright. All rights reserved.
- Variants in CUL4B are Associated with Cerebral Malformations. [JOURNAL ARTICLE]
- Hum Mutat 2014 Nov 11.
Variants in CUL4B are a known cause of syndromic X-linked intellectual disability. Here, we describe an additional 25 patients from eleven families with variants in CUL4B. We identified nine different novel variants in these families and confirmed the pathogenicity of all non-truncating variants. Neuroimaging data, available for 15 patients, showed the presence of cerebral malformations in ten patients. The cerebral anomalies comprised malformations of cortical development, ventriculomegaly and diminished white matter volume. The phenotypic heterogeneity of the cerebral malformations might result from the involvement of CUL-4B in various cellular pathways essential for normal brain development. Accordingly, we show that CUL-4B interacts with WDR62, a protein in which variants were previously identified in patients with microcephaly and a wide range of MCD. This interaction might contribute to the development of cerebral malformations in patients with variants in CUL4B. This article is protected by copyright. All rights reserved.
- ProKinO: A Unified Resource for Mining the Cancer Kinome. [JOURNAL ARTICLE]
- Hum Mutat 2014 Nov 10.
Protein kinases represent a large and diverse family of evolutionarily related proteins that are abnormally regulated in human cancers. Although genome sequencing studies have revealed thousands of variants in protein kinases, translating "big" genomic data into biological knowledge remains a challenge. Here we describe an ontological framework for integrating and conceptualizing diverse forms of information related to kinase activation and regulatory mechanisms in a machine readable, human understandable form. We demonstrate the utility of this framework in analyzing the cancer kinome, and in generating testable hypotheses for experimental studies. Through the iterative process of aggregate ontology querying, hypothesis generation and experimental validation, we identify a novel mutational hotspot in the αC-β4 loop of the kinase domain and demonstrate the functional impact of the identified variants in epidermal growth factor receptor (EGFR) constitutive activity and inhibitor sensitivity. We provide a unified resource for the kinase and cancer community, ProKinO, housed at http://vulcan.cs.uga.edu/prokino. This article is protected by copyright. All rights reserved.
- Functional Classification of BRCA2 DNA Variants by Splicing Assays in a Large Minigene with 9 Exons. [JOURNAL ARTICLE]
- Hum Mutat 2014 Nov 10.
Numerous pathogenic DNA variants impair the splicing mechanism in human genetic diseases. Minigenes are optimal approaches to test variants under the splicing viewpoint without the need of patient samples. We aimed to design a robust minigene construct of the breast cancer gene BRCA2 in order to investigate the impact of variants on splicing. BRCA2 exons 19 to 27 (MGBR2_ex19-27) were cloned in the new vector pSAD. It produced a large transcript of the expected size (2174 nucleotides) and exon structure (V1-ex19-27-V2). Splicing assays showed that 18 (17 splice-site and 1 silencer variants) out of 40 candidate DNA variants induced aberrant patterns. Twenty-four anomalous transcripts were accurately detected by fluorescent-RT-PCR that were generated by exon-skipping, alternative site usage and intron-retention events. Fourteen variants induced major anomalies and were predicted to disrupt protein function so they could be classified as pathogenic. Furthermore, minigene mimicked previously reported patient RNA outcomes of seven variants supporting the reproducibility of minigene assays. Therefore, a relevant fraction of variants are involved in breast cancer through splicing alterations. MGBR2_ex19-27 is the largest reported BRCA2 minigene and constitutes a valuable tool for the functional and clinical classification of sequence variations. This article is protected by copyright. All rights reserved.
- Functional Characterization and Classification of Frequent LDL Receptor Variants. [JOURNAL ARTICLE]
- Hum Mutat 2014 Nov 6.
Familial Hypercholesterolemia (FH) is an autosomal dominant disorder mostly caused by mutations in the low-density lipoprotein receptor (LDLR) gene leading to increased risk for premature cardiovascular diseases. According to functional studies, LDLR mutations may be classified into five classes. The main objective of this study was to characterize seven LDLR variants previously detected in FH patients. Analysis by flow cytometry and confocal microscopy of LDLR activity demonstrate that all the studied variants are pathogenic. Among the mutations located in β-propeller, p.Trp577Gly and p.Ile624del were classified as class 2 whereas p.Arg416Trp and p.Thr454Asn as class 5. p.Phe800Glyfs*129 (located in the cytoplasmic domain), p.Cys155Tyr (located in the binding domain) and p.Asn825Lys (inside FxNPxY motif) were classified as class 2, 3 and 4, respectively. The results also show that LDLR activity of these class 4 and 5 variants is not completely abolished, showing a milder phenotype. We have also determined that statin response is more efficient lowering total cholesterol in heterozygous patients carrying p.Ile624del (class 2) compared to p.Arg416Trp and p.Thr454Asn (class 5) variants. In conclusion, these findings emphasize the importance of characterizing LDLR pathogenic variants to provide an indisputable FH diagnosis and to gain insight into the statin response depending on the LDLR class mutation. This article is protected by copyright. All rights reserved.
- Preliminary Functional Assessment and Classification of DEPDC5 Variants Associated with Focal Epilepsy. [JOURNAL ARTICLE]
- Hum Mutat 2014 Nov 4.
The mechanistic target of rapamycin (mTOR) complex 1 (TORC1) senses nutrient availability to regulate eukaryotic anabolic metabolism. In response to limiting concentrations of amino acids, TORC1 kinase activity is inhibited through the GATOR-1 complex. Mutations in DEPDC5, that encodes one of the components of the GATOR-1 complex, have recently been associated with different forms of focal epilepsy. Here we investigate the effects of 10 DEPDC5 variants identified in individuals with focal epilepsy and 2 DEPDC5 variants identified in serous ovarian tumours, on TORC1 signaling and GATOR-1 complex formation. According to our functional assessment, 3 variants clearly disrupted the DEPDC5-dependent inhibition of TORC1. We did not obtain functional evidence to support pathogenicity in the remaining cases. The observed functional differences between the DEPDC5 variants might underlie some of the clinical differences observed in the individuals carrying the different variants. This article is protected by copyright. All rights reserved.