Robust Diagnostic Genetic Testing Using Solution Capture Enrichment and a Novel Variant‐Filtering Interface

Watson, Christopher M.; Crinnion, Laura A.; Morgan, Joanne; Harrison, Sally M.; Diggle, Christine P.; Adlard, Julian; Lindsay, Helen; Camm, Nick; Charlton, Ruth; Sheridan, Eamonn; Bonthron, David T.; Taylor, Graham R.; Carr, Ian

doi:10.1002/humu.22490

Cited by 38 publications

(36 citation statements)

References 17 publications

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“…Identified variants were annotated using Alamut Batch standalone v.1.4.4, (database v.2016.03.04) and filtered using the AgileExomeFilter (http://dna.leeds.ac.uk/agile), essentially as described previously [21]. …”

Section: Methodsmentioning

confidence: 99%

A tubulin alpha 8 mouse knockout model indicates a likely role in spermatogenesis but not in brain development

et al. 2017

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Tubulin alpha 8 (Tuba8) is the most divergent member of the highly conserved alpha tubulin family, and uniquely lacks two key post-translational modification sites. It is abundantly expressed in testis and muscle, with lower levels in the brain. We previously identified homozygous hypomorphic TUBA8 mutations in human subjects with a polymicrogyria (PMG) syndrome, suggesting its involvement in development of the cerebral cortex. We have now generated and characterized a Tuba8 knockout mouse model. Homozygous mice were confirmed to lack Tuba8 protein in the testis, but did not display PMG and appeared to be neurologically normal. In response to this finding, we re-analyzed the human PMG subjects using whole exome sequencing. This resulted in identification of an additional homozygous loss-of-function mutation in SNAP29, suggesting that SNAP29 deficiency, rather than TUBA8 deficiency, may underlie most or all of the neurodevelopmental anomalies in these subjects. Nonetheless, in the mouse brain, Tuba8 specifically localised to the cerebellar Purkinje cells, suggesting that the human mutations may affect or modify motor control. In the testis, Tuba8 localisation was cell-type specific. It was restricted to spermiogenesis with a strong acrosomal localization that was gradually replaced by cytoplasmic distribution and was absent from spermatozoa. Although the knockout mice were fertile, the localisation pattern indicated that Tuba8 may have a role in spermatid development during spermatogenesis, rather than as a component of the mature microtubule-rich flagellum itself.

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Section: Methodsmentioning

confidence: 99%

A tubulin alpha 8 mouse knockout model indicates a likely role in spermatogenesis but not in brain development

et al. 2017

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“…19,42 TEM in mice and zebrafish was prepared as previously reported, 11 and video microscopy was performed as previously reported. 44,[47][48][49] cDNA Cloning CCDC114 and CCDC151 were cloned by nested PCR from human bronchial epithelial cell cDNA (ScienCell, cat no.…”

Section: Cilia Transmission Electron Microscopy and Motilitymentioning

confidence: 99%

CCDC151 Mutations Cause Primary Ciliary Dyskinesia by Disruption of the Outer Dynein Arm Docking Complex Formation

Hjeij

Onoufriadis

Watson

et al. 2014

The American Journal of Human Genetics

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A diverse family of cytoskeletal dynein motors powers various cellular transport systems, including axonemal dyneins generating the force for ciliary and flagellar beating essential to movement of extracellular fluids and of cells through fluid. Multisubunit outer dynein arm (ODA) motor complexes, produced and preassembled in the cytosol, are transported to the ciliary or flagellar compartment and anchored into the axonemal microtubular scaffold via the ODA docking complex (ODA-DC) system. In humans, defects in ODA assembly are the major cause of primary ciliary dyskinesia (PCD), an inherited disorder of ciliary and flagellar dysmotility characterized by chronic upper and lower respiratory infections and defects in laterality. Here, by combined high-throughput mapping and sequencing, we identified CCDC151 loss-of-function mutations in five affected individuals from three independent families whose cilia showed a complete loss of ODAs and severely impaired ciliary beating. Consistent with the laterality defects observed in these individuals, we found Ccdc151 expressed in vertebrate left-right organizers. Homozygous zebrafish ccdc151(ts272a) and mouse Ccdc151(Snbl) mutants display a spectrum of situs defects associated with complex heart defects. We demonstrate that CCDC151 encodes an axonemal coiled coil protein, mutations in which abolish assembly of CCDC151 into respiratory cilia and cause a failure in axonemal assembly of the ODA component DNAH5 and the ODA-DC-associated components CCDC114 and ARMC4. CCDC151-deficient zebrafish, planaria, and mice also display ciliary dysmotility accompanied by ODA loss. Furthermore, CCDC151 coimmunoprecipitates CCDC114 and thus appears to be a highly evolutionarily conserved ODA-DC-related protein involved in mediating assembly of both ODAs and their axonemal docking machinery onto ciliary microtubules.

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“…7 ESP5400 dataset) and in silico variant effect predictions using Alamut Batch v1.1.5 (http://www.interactive-biosoftware.com/software/ alamut/overview). Interpretation of the annotated variant dataset was aided using AgileExomeFilter (http://dna.leeds.ac.uk/agile) [6] and the variant-decision support software Alamut Visual. To assess exon copy number variation, FishingCNV v2.1 (http://sourceforge.net/ projects/fishingcnv/) was used to compare the sequenced read depth of BHD individuals to a pooled reference control comprising 65 BHD disease-free patients.…”

Section: 'C' Allele Is Notated '1' and The 'T' Allele As '2' (Displaymentioning

confidence: 99%

Identification of a mutation in the ubiquitin-fold modifier 1-specific peptidase 2 gene, UFSP2, in an extended South African family with Beukes hip dysplasia

et al. 2015

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Background. Beukes hip dysplasia (BHD) is an autosomal dominant disorder of variable penetrance that was originally identified in a large South African family of European origin. BHD is characterised by bilateral dysmorphism of the proximal femur, which results in severe degenerative osteoarthropathy. Previous studies mapped the disorder to a 3.34 Mb region on chromosome 4q35. Objective. To fine-map the BHD locus and identify the disease-causing mutation by direct sequencing. Results. The linked BHD allele was refined to 1.33 Mb, reducing the number of candidate genes from 25 to 16. Analysis of protein coding and invariant splice-site sequences in three distantly related individuals identified a single-candidate disease-causing variant c.868T>C within exon 8 of the ubiquitin-fold modifier 1 (Ufm1)-specific peptidase 2 gene, UFSP2. The presence of this unique mutation was confirmed in all 17 affected members of the BHD family who were genotyped. The mutation segregated with the BHD phenotype in the extended family with a two-point (single marker) LOD score of 10.4 (θ = 0.0 and 80% penetrance). The mutation predicts the substitution of a highly conserved amino acid, p.Tyr290His, in the encoded protein. In vitro functional assays performed using purified recombinant wild-type and mutant UFSP2 protein demonstrated that the BHD mutation abolishes UFSP2-mediated C-terminal cleavage of its substrate, Ufm1. Conclusion. We report a unique UFSP2 mutation that segregates with the BHD phenotype. The predicted amino acid substitution inactivates UFSP2 proteolytic function, thus implicating the ubiquitin-fold modifier 1 cascade in this form of severe hip osteoarthropathy. The facile polymerase chain reaction-based assay we describe could be used to confirm the diagnosis of BHD, or for presymptomatic testing of members of the extended BHD family.

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Robust Diagnostic Genetic Testing Using Solution Capture Enrichment and a Novel Variant‐Filtering Interface

Cited by 38 publications

References 17 publications

A tubulin alpha 8 mouse knockout model indicates a likely role in spermatogenesis but not in brain development

A tubulin alpha 8 mouse knockout model indicates a likely role in spermatogenesis but not in brain development

CCDC151 Mutations Cause Primary Ciliary Dyskinesia by Disruption of the Outer Dynein Arm Docking Complex Formation

Identification of a mutation in the ubiquitin-fold modifier 1-specific peptidase 2 gene, UFSP2, in an extended South African family with Beukes hip dysplasia

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