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Hum Mutat [journal]
- Contents. [Journal Article]
- Hum Mutat 2014 Nov; 35(11):i-iii.
- An Augmented ABCA4 Screen Targeting Non-coding Regions Reveals a Deep Intronic Founder Variant in Belgian Stargardt Patients. [JOURNAL ARTICLE]
- Hum Mutat 2014 Oct 27.
Autosomal recessive Stargardt disease (STGD1) is hallmarked by a large proportion of patients with a single heterozygous causative variant in the disease gene ABCA4. Braun et al. (2013) reported deep intronic variants of ABCA4 in STGD1 patients with one coding variant, prompting us to perform an augmented screen in 131 Belgian STGD1 patients with one or no ABCA4 variant to uncover deep intronic causal ABCA4 variants. This revealed a second variant in 28.6% of cases. Twenty-six percent of these carry the same causal variant c.4539+2001G>A (V4). Haplotyping in V4 carriers showed a common region of 63 kb, suggestive of a founder mutation. Genotype-phenotype correlations suggest a moderate-to-severe impact of V4 on the STGD1 phenotype. In conclusion, V4 occurs in a high fraction of Belgian STGD1 patients and represents the first deep intronic founder mutation in ABCA4. This emphasizes the importance of augmented molecular genetic testing of ABCA4 in Belgian STGD1. This article is protected by copyright. All rights reserved.
- Mutations in COA6 Cause Cytochrome C Oxidase Deficiency and Neonatal Hypertrophic Cardiomyopathy. [JOURNAL ARTICLE]
- Hum Mutat 2014 Oct 22.
COA6/C1orf31 is involved in cytochrome c oxidase (complex IV) biogenesis. We present a new pathogenic COA6 variant detected in a patient with neonatal hypertrophic cardiomyopathy and isolated complex IV deficiency. For the first time, clinical details about a COA6 deficient patient are given and patient fibroblasts are functionally characterized: COA6 protein is undetectable and steady state levels of complex IV and several of its subunits are reduced. The monomeric COX1 assembly intermediate accumulates. Using pulse-chase experiments, we demonstrate an increased turnover of mitochondrial encoded complex IV subunits. While monomeric complex IV is decreased in patient fibroblasts, the CI/CIII2 /CIVn -supercomplexes remain unaffected. Copper supplementation shows a partial rescue of complex IV deficiency in patient fibroblasts. We conclude that COA6 is required for complex IV subunit stability. Furthermore, the proposed role in the copper delivery pathway to complex IV subunits is substantiated and a therapeutic lead for COA6 deficient patients is provided. This article is protected by copyright. All rights reserved.
- Novel SLC2 Variants Contribute to Renal Glucosuria in Chinese Families༚Abnormal Expression and Dysfunction of Variant SLC2. [JOURNAL ARTICLE]
- Hum Mutat 2014 Oct 22.
Familial renal glucosuria (FRG) is characterized by persistent glucosuria despite normal serum glucose and the absence of overt tubular dysfunction. Variants in solute carrier family 5 (sodium-glucose co-transporter), member 2 (SLC5A2) have been reported in FRG patients. However, the functional and expression-related consequences of such variants have been scarcely investigated. In the current study, we studied five FRG families and identified six missense mutations, including four novel variants (c.1051T>C/p.(C351R), c.1400T>C/p.(V467A), c.1420G>C/p.(A474P), c.1691G>A/p.(R564Q); RNA not analyzed) and two variants that had been previously reported (c.294C>A/p.(F98L), c.736C>T/p.(P246S); RNA not analyzed). The probands were either heterozygous or compound heterozygous for SLC5A2 variants and had glucosuria of 5.9-19.6 g/day. Human 293 cells were transfected with plasmid constructs to study the expression and function of SLC5A2 Variants in vitro. Western blotting revealed that the expression levels of SLC5A2-351R-GFP, SLC5A2-467A-GFP, SLC5A2-474P-GFP and SLC5A2-564Q-GFP were significantly decreased compared with wild-type SLC5A2-GFP (37-55%). Confocal microscopy revealed that three variants (c.1400T>C, c.1420G>C, c.1691G>A) resulted in a loss of the punctate membrane pattern typical of wild-type SLC5A2. All Variants had a significantly lower transport capacity in than the wild-type control. The current study provides a starting point to further investigate the molecular mechanism of SLC5A2 in FRG families and provides functional clues for anti-diabetes drugs. This article is protected by copyright. All rights reserved.
- Simultaneous Analysis of Hundreds of Y-chromosomal SNPs for High-resolution Paternal Lineage Classification Using Targeted Semiconductor Sequencing. [JOURNAL ARTICLE]
- Hum Mutat 2014 Oct 22.
Single-nucleotide polymorphisms from the non-recombining part of the human Y chromosome (Y-SNPs) are informative to classify paternal lineages in forensic, genealogical, anthropological, and evolutionary studies. Although thousands of Y-SNPs were identified thus far, previous Y-SNP multiplex tools target only dozens of markers simultaneously, thereby restricting the provided Y-haplogroup resolution and limiting their applications. Here we overcome this shortcoming by introducing a high-resolution multiplex tool for parallel genotyping-by-sequencing of 530 Y-SNPs using the Ion Torrent PGM platform, which allows classification of 432 worldwide Y haplogroups. Contrary to previous Y-SNP multiplex tools, our approach covers branches of the entire Y tree, thereby maximizing the paternal lineage classification obtainable. We used a default DNA input amount of 10 ng per reaction but preliminary sensitivity testing revealed positive results from as little as 100 pg input DNA. Furthermore, we demonstrate that sample pooling using barcodes is feasible, allowing increased throughput for lower per-sample costs. In addition to the wetlab protocol, we provide a software tool for automated data quality control and haplogroup classification. The unique combination of ultra-high marker density and high sensitivity achievable from low amounts of potentially degraded DNA makes this new multiplex tool suitable for a wide range of Y-chromosome applications. This article is protected by copyright. All rights reserved.
- Whole-exome Sequencing Identifies a Variant in TMEM132E Causing Autosomal-Recessive Nonsyndromic Hearing Loss DFNB99. [JOURNAL ARTICLE]
- Hum Mutat 2014 Oct 21.
Autosomal recessive non-syndromic hearing loss (ARNSHL) features a high degree of genetic heterogeneity. Many genes responsible for ARNSHL have been identified or mapped. We previously mapped an ARNSHL locus at 17q12, herein designated DFNB99, in a consanguineous Chinese family. In this study, whole-exome sequencing revealed a homozygous missense mutation (c.1259G>A, p.Arg420Gln) in the gene encoding transmembrane protein 132E (TMEM132E) as the causative variant. Immunofluorescence staining of the Organ of Corti showed Tmem132e highly expressed in murine inner hair cells. Furthermore, knockdown of the tmem132e ortholog in zebrafish affected the mechanotransduction of hair cells. Finally, wild-type human TMEM132E mRNA but not the mRNA carrying the c.1259G>A mutation rescued the Tmem132e knockdown phenotype. We conclude that the variant in TMEM132E is the most likely cause of DFNB99. This article is protected by copyright. All rights reserved.
- Next Generation Sequencing Identifies a Novel Rearrangement in the HBB Cluster Permitting to-the-Base Characterization. [JOURNAL ARTICLE]
- Hum Mutat 2014 Oct 21.
Genetic testing for hemoglobinopathies is required for prenatal diagnosis, understanding complex cases where multiple pathogenic variants may be present or investigating cases of unexplained anemia. Characterization of disease causing variants that range from single base changes to large rearrangements, may require several different labor-intensive methodologies. Multiplex ligation probe amplification analysis is the current method used to detect indels, but the technique does not characterize the breakpoints or detect balanced translocations. Here we describe a next generation sequencing (NGS) method that is able to identify and characterize a novel rearrangement of the HBB cluster responsible for εγδβ thalassemia in an English family. The structural variant involved a 59.0 kb inversion encompassing HBG2 exon 3, HBG1, HBD, HBB and OR51V1, juxtaposed by a deletion of 122.6 kb including 82 bp of the inverted sequence, HBG2 exon 1 and 2, HBE, and the β-locus control region. Identification of reads spanning the breakpoints provided to-the-base resolution of the rearrangement, subsequently confirmed by gap-PCR and Sanger sequence analysis. The same rearrangement, termed Inv-Del English V εγδβ thalassemia (HbVar 2935), was identified in two other unrelated English individuals with a similar hematological phenotype. Our NGS approach should be applicable as a diagnostic tool for other disorders. This article is protected by copyright. All rights reserved.
- MAP4 Dependent Regulation of Microtubule Formation Affects Centrosome, Cilia and Golgi Architecture as A Central Mechanism in Growth Regulation. [JOURNAL ARTICLE]
- Hum Mutat 2014 Oct 17.
Numerous genes are involved in human growth regulation. Recently, autosomal recessive inherited variants in centrosomal proteins have been identified in Seckel syndrome, primary microcephaly or microcephalic osteodysplastic primary dwarfism. Common hallmarks of these syndromic forms are severe short stature and microcephaly. In a consanguineous family with two affected children with severe growth retardation and normocephaly we used homozygosity mapping and next generation sequencing to identify a homozygous MAP4 variant. MAP4 is a major protein for microtubule assembly during mitosis. High expression levels in the somite boundaries of zebrafish suggested a role in growth and body segment patterning. The identified variant affects binding sites of kinases necessary for dynamic instability of microtubule formation. We found centrosome amplifications in mitotic fibroblast cells in vivo and in vitro. These numeric centrosomal aberrations were also present during interphase resulting in aberrant ciliogenesis. Furthermore, affected cells showed a dysfunction of the microtubule dependent assembly of the Golgi apparatus, indicated by a significant lack of compactness of Golgi membranes. These observations demonstrated that MAP4 mutations contribute to the clinical spectrum of centrosomal defects and confirmed the complex role of a centrosomal protein in centrosomal, ciliary and Golgi regulation associated with severe short stature. This article is protected by copyright. All rights reserved.
- Clinical Applications and Implications of Common and Founder Mutations in Indian Subpopulations. [JOURNAL ARTICLE]
- Hum Mutat 2014 Oct 17.
South Asian Indians represent a sixth of the world's population and are a racially, geographically, and genetically diverse people. Their unique anthropological structure, prevailing caste system, and ancient religious practices have all impacted the genetic composition of most of the current-day Indian population. With the evolving socio-religious and economic activities of the sub-sects and castes, endogamous and consanguineous marriages became a commonplace. Consequently, the frequency of founder mutations and the burden of heritable genetic disorders rose significantly. Specifically, the incidence of certain autosomal recessive disorders is relatively high in select Indian subpopulations and communities that share common recent ancestry. Although today clinical genetics and molecular diagnostic services are making inroads in India, the high costs associated with the technology and the tests often keep patients from an exact molecular diagnosis, making more customized and tailored tests, such as those interrogating the most common and founder mutations or those that cater to select sects within the population, highly attractive. These tests offer a quick first-hand affordable diagnostic and carrier screening tool. Here we provide a comprehensive catalog of known common mutations and founder mutations in the Indian population and discuss them from a molecular, clinical, and historical perspective. This article is protected by copyright. All rights reserved.
- Identification of Splicing Defects Caused by Mutations in the Dysferlin Gene. [JOURNAL ARTICLE]
- Hum Mutat 2014 Oct 14.
Missense, iso-semantic and intronic mutations are challenging for interpretation in particular for their impact in mRNA. Various tools such as the Human Splicing Finder system (HSF) could be used to predict the impact on splicing, however no diagnosis result could rely on predictions alone, but requires functional testing. Here we report an in vitro approach to study the impact of DYSF mutations on splicing. It was evaluated on a series of 45 DYSF mutations, both intronic and exonic. We confirmed splicing alterations for all intronic mutations localized in 5' or 3' splice sites. Then, we showed that DYSF missense mutations could also result in splicing defects: mutations c.463G>A and c.2641A>C abolished ESEs and led to exon skipping; mutations c.565C>G and c.1555G>A, disrupted ESE, while concomitantly creating new 5' or 3' splice site leading to exonic out of frame deletions. We demonstrated that 20% of DYSF missense mutations have a strong impact on splicing. This minigene strategy is an efficient tool for the detection of splicing defects in dysferlinopathies, which could allow for a better comprehension of splicing defects due to mutations and could improve prediction tools evaluating splicing defects. This article is protected by copyright. All rights reserved.