Integrating sequence and array data to create an improved 1000 Genomes Project haplotype reference panel

A major use of the 1000 Genomes Project (1000GP) data is genotype imputation in genome-wide association studies (GWAS). Here we develop a method to estimate haplotypes from low-coverage sequencing data that can take advantage of single-nucleotide polymorphism (SNP) microarray genotypes on the same samples. First the SNP array data are phased to build a backbone (or 'scaffold') of haplotypes across each chromosome. We then phase the sequence data 'onto' this haplotype scaffold. This approach can take advantage of relatedness between sequenced and non-sequenced samples to improve accuracy. We use this method to create a new 1000GP haplotype reference set for use by the human genetic community. Using a set of validation genotypes at SNP and bi-allelic indels we show that these haplotypes have lower genotype discordance and improved imputation performance into downstream GWAS samples, especially at low-frequency variants. © 2014 Macmillan Publishers Limited. All rights reserved

Comprehensive Analysis of Genetic Ancestry and Its Molecular Correlates in Cancer

We evaluated ancestry effects on mutation rates, DNA methylation, and mRNA and miRNA expression among 10,678 patients across 33 cancer types from The Cancer Genome Atlas. We demonstrated that cancer subtypes and ancestry-related technical artifacts are important confounders that have been insufficiently accounted for. Once accounted for, ancestry-associated differences spanned all molecular features and hundreds of genes. Biologically significant differences were usually tissue specific but not specific to cancer. However, admixture and pathway analyses suggested some of these differences are causally related to cancer. Specific findings included increased FBXW7 mutations in patients of African origin, decreased VHL and PBRM1 mutations in renal cancer patients of African origin, and decreased immune activity in bladder cancer patients of East Asian origin

Determinants of penetrance and variable expressivity in monogenic metabolic conditions across 77,184 exomes

Hundreds of thousands of genetic variants have been reported to cause severe monogenic diseases, but the probability that a variant carrier develops the disease (termed penetrance) is unknown for virtually all of them. Additionally, the clinical utility of common polygenetic variation remains uncertain. Using exome sequencing from 77,184 adult individuals (38,618 multi-ancestral individuals from a type 2 diabetes case-control study and 38,566 participants from the UK Biobank, for whom genotype array data were also available), we apply clinical standard-of-care gene variant curation for eight monogenic metabolic conditions. Rare variants causing monogenic diabetes and dyslipidemias display effect sizes significantly larger than the top 1% of the corresponding polygenic scores. Nevertheless, penetrance estimates for monogenic variant carriers average 60% or lower for most conditions. We assess epidemiologic and genetic factors contributing to risk prediction in monogenic variant carriers, demonstrating that inclusion of polygenic variation significantly improves biomarker estimation for two monogenic dyslipidemias

Recessive DES cardio/myopathy without myofibrillar aggregates: intronic splice variant silences one allele leaving only missense L190P-desmin

We establish autosomal recessive DES variants p.(Leu190Pro) and a deep intronic splice variant causing inclusion of a frameshift-inducing artificial exon/intronic fragment, as the likely cause of myopathy with cardiac involvement in female siblings. Both sisters presented in their twenties with slowly progressive limb girdle weakness, severe systolic dysfunction, and progressive, severe respiratory weakness. Desmin is an intermediate filament protein typically associated with autosomal dominant myofibrillar myopathy with cardiac involvement. However a few rare cases of autosomal recessive desminopathy are reported. In this family, a paternal missense p.(Leu190Pro) variant was viewed unlikely to be causative of autosomal dominant desminopathy, as the father and brothers carrying this variant were clinically unaffected. Clinical fit with a DES-related myopathy encouraged closer scrutiny of all DES variants, identifying a maternal deep intronic variant within intron-7, predicted to create a cryptic splice site, which segregated with disease. RNA sequencing and studies of muscle cDNA confirmed the deep intronic variant caused aberrant splicing of an artificial exon/intronic fragment into maternal DES mRNA transcripts, encoding a premature termination codon, and potently activating nonsense-mediate decay (92% paternal DES transcripts, 8% maternal). Western blot showed 60-75% reduction in desmin levels, likely comprised only of missense p.(Leu190Pro) desmin. Biopsy showed fibre size variation with increased central nuclei. Electron microscopy showed extensive myofibrillar disarray, duplication of the basal lamina, but no inclusions or aggregates. This study expands the phenotypic spectrum of recessive DES cardio/myopathy, and emphasizes the continuing importance of muscle biopsy for functional genomics pursuit of 'tricky' variants in neuromuscular conditions.Lisa G. Riley, Leigh B. Waddell, Roula Ghaoui, Frances J. Evesson, Beryl B. Cummings, Samantha J. Bryen, Himanshu Joshi, Min-Xia Wang, Susan Brammah, Leonard Kritharides, Alastair Corbett, Daniel G. MacArthur, Sandra T. Coope

Reassessment of Mendelian gene pathogenicity using 7,855 cardiomyopathy cases and 60,706 reference samples

10.1038/gim.2016.90Genetics in Medicine192192-203GEME

Pathogenic abnormal splicing due to intronic deletions that induce biophysical space constraint for spliceosome assembly

A precise genetic diagnosis is the single most important step for families with genetic disorders to enable personalized and preventative medicine. In addition to genetic variants in coding regions (exons) that can change a protein sequence, abnormal pre-mRNA splicing can be devastating for the encoded protein, inducing a frameshift or in-frame deletion/insertion of multiple residues. Non-coding variants that disrupt splicing are extremely challenging to identify. Stemming from an initial clinical discovery in two index Australian families, we define 25 families with genetic disorders caused by a class of pathogenic non-coding splice variant due to intronic deletions. These pathogenic intronic deletions spare all consensus splice motifs, though they critically shorten the minimal distance between the 5' splice-site (5'SS) and branchpoint. The mechanistic basis for abnormal splicing is due to biophysical constraint precluding U1/U2 spliceosome assembly, which stalls in A-complexes (that bridge the 5'SS and branchpoint). Substitution of deleted nucleotides with non-specific sequences restores spliceosome assembly and normal splicing, arguing against loss of an intronic element as the primary causal basis. Incremental lengthening of 5'SS-branchpoint length in our index EMD case subject defines 45-47 nt as the critical elongation enabling (inefficient) spliceosome assembly for EMD intron 5. The 5'SS-branchpoint space constraint mechanism, not currently factored by genomic informatics pipelines, is relevant to diagnosis and precision medicine across the breadth of Mendelian disorders and cancer genomics.Samantha J.Bryen, Himanshu Joshi, Frances J.Evesson, Cyrille Girard, Roula Ghaoui, Leigh B.Waddell ... et al