Biology subjects

Foulkes, W. D.

Publications and source records attributed to Foulkes, W. D..

3 recordsLinked to original sources

bioRxiv2018

Custom hereditary breast cancer gene panel selectively amplifies target genes for reliable variant calling

Background: Target enrichment coupled with next generation sequencing provide high-throughput approaches for screening several genes of interest. These approaches facilitate screening a panel of genes for mutations associated with inherited breast cancer for research, diagnostic, and genetic counseling applications.\n\nObjective: To evaluate the performance of our custom 13 gene breast cancer panel, based on singleplex PCR, developed by WaferGen BioSystems. The panel was evaluated using patient-derived DNA samples, in terms of target enrichment efficiency, off-target enrichment, uniformity of target capture, effect of GC content of target regions on coverage depth, and concordance with validated variant calls.\n\nResults: At least 90% of target sequence for each gene was captured at 30x or greater. We evaluated uniformity of target capture across samples by calculating the percentage of samples with at least 90% of total target captured at 100x or greater and found 92% (33/36 samples) uniformity for our panel. Off-target enrichment ranges between 7.2% and 22.3%. We found perfect concordance between our custom panel and the Qiagen human breast cancer panel for functionally annotated variant calls in high read depth shared target regions. Altogether, there was agreement between the panels for 779 variants at 41 loci. We also confirmed 10 pathogenic mutations, initially discovered by Sanger sequencing, in the appropriate samples following target enrichment using our custom WaferGen panel.\n\nConclusion: Our custom hereditary breast cancer panel is sensitive to the desired target genes and facilitates deep sequencing for reliable variant calling.

genetics↗

bioRxiv2017

Analysis of DICER1 in familial and sporadic cases of Transposition of the Great Arteries

BackgroundDICER1 plays a major role in development and in generating mature microRNAs that are important in gene expression. We screened for DICER1 mutations in a family with DICER1 syndrome and we discovered a pathogenic mutation in a child with transposition of the great arteries (TGA). In view of a report linking DICER1 knock-out in murine cardiomyocytes to cardiac outflow defects, we investigated the involvement of DICER1 in TGA.\n\nFindingsWe screened 129 germline DNA samples from children with either sporadic or familial forms of TGA for DICER1 mutations using a Fluidigm access array, followed by next-generation sequencing. We identified 16 previously reported variants (5 synonymous, 6 intronic, and 5 missense) and 2 novel variants (1 intronic and 1 missense). We did not find any apparent pathological mutation in our cohort.\n\nConclusionHere we report that DICER1 mutations do not appear to play a major role in TGA.

genetics↗

bioRxiv2016

Resolving the Functional Significance of BRCA1 RING Domain Missense Substitutions

Part 1Development and calibration of suitably accurate functional assays for BRCA1 RING domain and BRCT domain missense substitutions could dramatically accelerate clinical classification of rare missense substitutions observed in that gene. Leveraging data from 68,000 full sequence tests of BRCA1 and BRCA2, plus data from the limited number of already classified BRCA1 RING domain missense substitutions, we used logistic regression and related techniques to evaluate three BRCA1 RING domain assays. These were recently described high throughput yeast 2-hybrid and E3 ubiquitin ligase assays, plus a newly developed mammalian 2-hybrid assay. While there were concerns about the accuracy of the yeast 2-hybrid assay and the indirect nature of the ubiquitin ligase assay, the mammalian 2-hybrid assay had excellent correlation with existing missense substitution classifications. After calibration, this assay contributed to classification of one newly reported BRCA1 missense substitution. In principal, the mammalian 2-hybrid assay could be converted to a high-throughput format that would likely retain suitable accuracy. Part 2How does one achieve clinically applicable classification of the vast majority of all possible sequence variants in disease susceptibility genes? BRCA1 is a high-risk susceptibility gene for breast and ovarian cancer. Pathogenic protein truncating variants are scattered across the open reading frame, but all known missense substitutions that are pathogenic because of missense dysfunction are located in either the amino-terminal RING domain or the carboxy-terminal BRCT domain. Heterodimerization of the BRCA1 and BARD1 RING domains is a molecularly defined obligate activity. Hence, we tested every BRCA1 RING domain missense substitution that can be created by a single nucleotide change for heterodimerization with BARD1 in a Mammalian 2-hybrid (M2H) assay. Downstream of the M2H laboratory assay, we addressed three additional challenges: assay calibration, validation thereof, and integration of the calibrated results with other available data such as computational evidence and patient/population observational data to achieve clinically applicable classification. Overall, we found that about 20% of BRCA1 RING domain missense substitutions are pathogenic. Using a Bayesian point system for data integration and variant classification, we achieved clinical classification of about 89% of observed missense substitutions. Moreover, among missense substitutions not present in the human observational data used here, we find an additional 47 with concordant computational and functional assay evidence in favor of pathogenicity; these are particularly likely to be classified as Likely Pathogenic once human observational data become available.

genetics↗