An interactive genome browser of association results from the UK10K cohorts project.

UNLABELLED: High-throughput sequencing technologies survey genetic variation at genome scale and are increasingly used to study the contribution of rare and low-frequency genetic variants to human traits. As part of the Cohorts arm of the UK10K project, genetic variants called from low-read depth (average 7×) whole genome sequencing of 3621 cohort individuals were analysed for statistical associations with 64 different phenotypic traits of biomedical importance. Here, we describe a novel genome browser based on the Biodalliance platform developed to provide interactive access to the association results of the project. AVAILABILITY AND IMPLEMENTATION: The browser is available at http://www.uk10k.org/dalliance.html. Source code for the Biodalliance platform is available under a BSD license from http://github.com/dasmoth/dalliance, and for the LD-display plugin and backend from http://github.com/dasmoth/ldserv

Multi-level evidence of an allelic hierarchy of USH2A variants in hearing, auditory processing and speech/language outcomes.

Language development builds upon a complex network of interacting subservient systems. It therefore follows that variations in, and subclinical disruptions of, these systems may have secondary effects on emergent language. In this paper, we consider the relationship between genetic variants, hearing, auditory processing and language development. We employ whole genome sequencing in a discovery family to target association and gene x environment interaction analyses in two large population cohorts; the Avon Longitudinal Study of Parents and Children (ALSPAC) and UK10K. These investigations indicate that USH2A variants are associated with altered low-frequency sound perception which, in turn, increases the risk of developmental language disorder. We further show that Ush2a heterozygote mice have low-level hearing impairments, persistent higher-order acoustic processing deficits and altered vocalizations. These findings provide new insights into the complexity of genetic mechanisms serving language development and disorders and the relationships between developmental auditory and neural systems

The UK10K project identifies rare variants in health and disease

M. Kivimäki työryhmäjäsen.The contribution of rare and low-frequency variants to human traits is largely unexplored. Here we describe insights from sequencing whole genomes (low read depth, 7x) or exomes (high read depth, 80x) of nearly 10,000 individuals from population-based and disease collections. In extensively phenotyped cohorts we characterize over 24 million novel sequence variants, generate a highly accurate imputation reference panel and identify novel alleles associated with levels of triglycerides (APOB), adiponectin (ADIPOQ) and low-density lipoprotein cholesterol (LDLR and RGAG1) from single-marker and rare variant aggregation tests. We describe population structure and functional annotation of rare and low-frequency variants, use the data to estimate the benefits of sequencing for association studies, and summarize lessons from disease-specific collections. Finally, we make available an extensive resource, including individual-level genetic and phenotypic data and web-based tools to facilitate the exploration of association results.Peer reviewe

Mutations in the histone methyltransferase gene KMT2B cause complex early-onset dystonia.

Histone lysine methylation, mediated by mixed-lineage leukemia (MLL) proteins, is now known to be critical in the regulation of gene expression, genomic stability, cell cycle and nuclear architecture. Despite MLL proteins being postulated as essential for normal development, little is known about the specific functions of the different MLL lysine methyltransferases. Here we report heterozygous variants in the gene KMT2B (also known as MLL4) in 27 unrelated individuals with a complex progressive childhood-onset dystonia, often associated with a typical facial appearance and characteristic brain magnetic resonance imaging findings. Over time, the majority of affected individuals developed prominent cervical, cranial and laryngeal dystonia. Marked clinical benefit, including the restoration of independent ambulation in some cases, was observed following deep brain stimulation (DBS). These findings highlight a clinically recognizable and potentially treatable form of genetic dystonia, demonstrating the crucial role of KMT2B in the physiological control of voluntary movement.Funding for the project was provided by the Wellcome Trust for UK10K (WT091310) and DDD Study. The DDD study presents independent research commissioned by the Health Innovation Challenge Fund [grant number HICF-1009-003] - see www.ddduk.org/access.html for full acknowledgement. This work was supported in part by the Intramural Research Program of the National Human Genome Research Institute and the Common Fund, NIH Office of the Director. This work was supported in part by the German Ministry of Research and Education (grant nos. 01GS08160 and 01GS08167; German Mental Retardation Network) as part of the National Genome Research Network to A.R. and D.W. and by the Deutsche Forschungsgemeinschaft (AB393/2-2) to A.R. Brain expression data was provided by the UK Human Brain Expression Consortium (UKBEC), which comprises John A. Hardy, Mina Ryten, Michael Weale, Daniah Trabzuni, Adaikalavan Ramasamy, Colin Smith and Robert Walker, affiliated with UCL Institute of Neurology (J.H., M.R., D.T.), King’s College London (M.R., M.W., A.R.) and the University of Edinburgh (C.S., R.W.)

Unravelling the genetic relationships between auditory processing and speech and language

Auditory processing disorder is a common developmental disorder affecting about 10% of children. It is characterised by poor perception of speech sounds, especially in background noise environments, despite normal hearing sensitivity, which can lead to poor performance in school with a negative impact on education and everyday life. Previous studies have shown that auditory processing skills have a substantial genetic component, however, it is not clear which genes or molecular mechanisms are involved. In this thesis three different genetic approaches are applied (monogenic, common disease-common variant and common disease-rare variant) to assess the effect of candidate genes on neurodevelopmental measures, including hearing and language phenotypes, in a population cohort (ALSPAC) of more than 14,000 children. To complement these analyses, a reverse phenotype to genotype approach is used, focussing on a surrogate measure of auditory processing difficulties in ALSPAC children, to identify potential high impact coding variants that may explain these difficulties. Given previous work, these genetic investigations focus upon candidate genes related to Usher syndrome, a recessive disorder leading to hearing and vision loss resulting from dysfunctional neurosensory cells in the inner ear and retina (hair cells and photoreceptor cells respectively). Analyses indicate that there is no one single risk variant, but a complex mix of variation across Usher genes (such as USH2A, PCDH15, CLRN1, and ADGRV1) might explain some of the APD risk. The phenotype to genotype analysis across coding regions further shows that rare pathogenic variants with large effect in other genes (such as GRHL3, DIAPH1, FAT4 and IFT88) can contribute to risk of APD in simplex cases. These results provide insights into the genetic landscape underlying APD and offer candidate genes and variants for further investigation and validation. Furthermore, the results highlight allelic heterogeneity where multiple variants present in the same Usher gene (USH2A) can display different, but related hearing phenotypes. In a wider context, this study also highlights the viability of using related/surrogate phenotypes for genetic discovery in a large sample when deep phenotyping of APD is unavailable

Challenges and novel approaches for investigating molecular mediation

Understanding mediation is useful for identifying intermediates lying between an exposure and an outcome which, when intervened upon, will block (some or all of) the causal pathway between the exposure and outcome. Mediation approaches used in conventional epidemiology have been adapted to understanding the role of molecular intermediates in situations of high-dimensional omics data with varying degrees of success. In particular, the limitations of observational epidemiological study including confounding, reverse causation and measurement error can afflict conventional mediation approaches and may lead to incorrect conclusions regarding causal effects. Solutions to analysing mediation which overcome these problems include the use of instrumental variable methods such as Mendelian randomization, which may be applied to evaluate causality in increasingly complex networks of omics data

Integrated approaches to elucidate the genetic architecture of congenital heart defects

Congenital heart defects (CHD) are structural anomalies affecting the heart, are found in 1% of the population and arise during early stages of embryo development. Without surgical and medical interventions, most of the severe CHD cases would not survive after the first year of life. The improved health care for CHD patients has increased CHD prevalence significantly, and it has been estimated that the population of adults with CHD is growing ~5% per year. Understanding the causes of CHD would greatly help improve our knowledge of the pathophysiology, family counseling and planning and possibly prevention and treatment in the future. Several lines of evidence from humans and animal models have supported a substantial genetic component for CHD. However, gene discovery in CHD has been difficult due to the extreme locus heterogeneity and the lack of a distinct genotype–phenotype correlation. Currently, genetic causes are identified in fewer than 20-­‐30% of the cases, most of which are syndromic while the isolated CHD cases remain largely without explanation. The aim of my thesis was to identify novel or known CHD genes enriched for rare coding genetic variants in isolated CHD cases and learn about the relative performance of different study designs. High-throughput next generation sequencing (NGS) was used to sequence all coding genes (whole exome) coupled with various analytical pipelines and tools to identify candidate genes in different family-based study designs. Since there is no general consensus on the underlying genetic model of isolated CHD, I developed a suite of software tools to enable different family-based exome analyses of de novo and inherited variants (chapter 2) and then piloted these tools in several gene discovery projects where the mode of inheritance was already known to identify previously described and novel pathogenic genes, before applying them to an analysis of families with two or more siblings with CHD. Based on the tools developed in chapter 2, I designed a two-stage study to investigate isolated parent-offspring trios with Tetralogy of Fallot (chapter 3). In the first stage, I used whole exome sequence data from 30 trios to identify genes with de novo coding variants. This analysis identified six de novo loss-of-function and 13 de novo missense variants. Only one gene showed recurrent de novo mutations in NOTCH1, a well known CHD gene that has mostly been associated with left ventricle outflow tract malformations (LVOT). Besides NOTCH1, the de novo analysis identified several possibly pathogenic novel genes such as ZMYM2 and ARHGAP35, that harbor de novo loss-of-function variants (frameshift and stop gain, respectively). In the second stage of the study, I designed custom baits to capture 122 candidate genes for additional sequencing using NGS in a larger sample size of 250 parent-offspring trios with isolated Tetralogy of Fallot and identified six de novo variants in four genes, half of them are loss-of-function variants. Both of NOTCH1 and its ligand JAG1 harbor two additional de novo mutations (two stop gains in NOTCH1 and one missense and a splice donor in JAG1). The analysis showed a strongly significant over-representation of de novo loss-of-function variants in NOTCH1 (P=3.8 ×10-9). Additionally, when compared with 1,080 control trios, NOTCH1 exhibit significant burden of inherited rare missense variant (minor allele frequency < 1% in 1000 genomes) (Fisher exact test, P= 8.8 × 10^­‐05) in about 10% of the isolated Tetralogy of Fallot patients. I also modified the transmission disequilibrium test (TDT) to detect any distortion of rare coding allele transmission from healthy parent to their affected children. This modified TDT test identified ARHGAP35 gene, which exhibits an over-­‐transmission of rare missense variants in children (P=0.025). Although, the p value does not reach a genome-­‐wide significant level after correcting for multiple tests, ARHGAP35 gene has also a de novo stop gain variant in one trio from the primary cohort and recently shown to play a role in cardiomyocyte fate which make it an interesting novel ToF candidate gene for future studies. To assess alternative family-based study design in CHD, I combined the analysis from 13 isolated parent-offspring trios with 112 unrelated index cases of isolated atrioventricular septal defects (AVSD) in chapter 4. Initially, I started with a case/control analysis to test the burden of rare missense variants in cases compared with 5,194 ethnically matching controls and identified the gene NR2F2 (Fisher exact test P=7.7×10-07, odds ratio=54). The de novo analysis in the AVSD trios identified two de novo missense variants in the same gene. NR2F2 encodes a pleiotropic developmental transcription factor, and decreased dosage of NR2F2 in mice has been shown to result in abnormal development of atrioventricular septa. The results from luciferase assays show that all coding sequence variants observed in patients significantly alter the activity of NR2F2 target promoters. My work has identified both known and novel CHD genes enriched for rare coding variants using next-generation sequencing data. I was able to show how using single or combined family-based study designs is an effective approach to study the genetic causes of isolated CHD subtypes. Despite the extreme heterogeneity of CHD, combining NGS data with the proper study design has proved to be an effective approach to identify novel and known CHD genes. Future studies with considerably larger sample sizes are required to yield deeper insights into the genetic causes of isolated CHD.National Guard Health Affairs, Riyadh, Saudi Arabi