Using molecular QTLs to identify cell types and causal variants for complex traits

Genetic associations have been discovered for many human complex traits, and yet for most associated loci the causal variants and molecular mechanisms remain unknown. Studies mapping quantitative trait loci (QTLs) for molecular phenotypes, such as gene expression, RNA splicing, and chromatin accessibility, provide rich data that can link variant effects in specific cell types with complex traits. These genetic effects can also now be modeled in vitro by differentiating human induced pluripotent stem cells (iPSCs) into specific cell types, including inaccessible cell types such as those of the brain. In this thesis, I explore a range of approaches for using QTLs to identify causal variants and to link these with molecular functions and complex traits. In Chapter 2, I describe QTL mapping in 123 sensory neuronal cell lines differentiated from human iPSCs. I observed that gene expression was highly variable across iPSC-derived neuronal cultures in specific gene categories, and that a portion of this variability was explained by commonly used iPSC culture conditions, which influenced differentiation efficiency. A number of QTLs overlapped with common disease associations; however, using simulations I showed that identifying causal regulatory variants with a recall-by- genotype approach in iPSC-derived neurons is likely to require large sample sizes, even for variants with moderately large effect sizes. In Chapter 3, I developed a computational model that uses publicly available gene expression QTL data, along with molecular annotations, to generate cell type-specific probability of regulatory function (PRF) scores for each variant. I found that predictive power was improved when the model was modified to use the quantitative value of annotations. PRF scores outperformed other genome-wide scores, including CADD and GWAVA, in identifying likely causal eQTL variants. In Chapter 4, I used PRF scores to identify relevant cell types and to fine map potential causal variants using summary association statistics in six complex traits. By examining individual loci in detail, I showed how the enrichments contributing to a high PRF score are transparent, which can help to distinguish plausible causal variant predictions from model misspecification.Wellcome Trust Sanger Institut

Compound heterozygous mutations in glycyl-tRNA synthetase are a proposed cause of systemic mitochondrial disease

Abstract Background Glycyl-tRNA synthetase (GARS) is an aminoacyl-tRNA synthetase (ARS) that links the amino acid glycine to its corresponding tRNA prior to protein translation and is one of three bifunctional ARS that are active within both the cytoplasm and mitochondria. Dominant mutations in GARS cause rare forms of Charcot-Marie-Tooth disease and distal spinal muscular atrophy. Case presentation We report a 12-year old girl who presented with clinical and biochemical features of a systemic mitochondrial disease including exercise-induced myalgia, non-compaction cardiomyopathy, persistent elevation of blood lactate and alanine and MRI evidence of mild periventricular leukomalacia. Using exome sequencing she was found to harbor compound heterozygous mutations within the glycyl-tRNA synthetase (GARS) gene; c.1904C > T; p.Ser635Leu and c.1787G > A; p.Arg596Gln. Each mutation occurred at a highly conserved site within the anticodon binding domain. Conclusion Our findings suggest that recessive mutations in GARS may cause systemic mitochondrial disease. This phenotype is distinct from patients with previously reported dominant mutations in this gene, thereby expanding the spectrum of disease associated with GARS dysregulation

Intellectual disability associated with a homozygous missense mutation in THOC6

BACKGROUND: We recently described a novel autosomal recessive neurodevelopmental disorder with intellectual disability in four patients from two related Hutterite families. Identity-by-descent mapping localized the gene to a 5.1 Mb region at chromosome 16p13.3 containing more than 170 known or predicted genes. The objective of this study was to identify the causative gene for this rare disorder. METHODS AND RESULTS: Candidate gene sequencing followed by exome sequencing identified a homozygous missense mutation p.Gly46Arg, in THOC6. No other potentially causative coding variants were present within the critical region on chromosome 16. THOC6 is a member of the THO/TREX complex which is involved in coordinating mRNA processing with mRNA export from the nucleus. In situ hybridization showed that thoc6 is highly expressed in the midbrain and eyes. Cellular localization studies demonstrated that wild-type THOC6 is present within the nucleus as is the case for other THO complex proteins. However, mutant THOC6 was predominantly localized to the cytoplasm, suggesting that the mutant protein is unable to carry out its normal function. siRNA knockdown of THOC6 revealed increased apoptosis in cultured cells. CONCLUSION: Our findings associate a missense mutation in THOC6 with intellectual disability, suggesting the THO/TREX complex plays an important role in neurodevelopment

Mutations in SYNGAP1 cause intellectual disability, autism, and a specific form of epilepsy by inducing haploinsufficiency

De novo mutations in SYNGAP1, which codes for a RAS/RAP GTP-activating protein, cause nonsyndromic intellectual disability (NSID). All disease-causing point mutations identified until now in SYNGAP1 are truncating, raising the possibility of an association between this type of mutations and NSID. Here, we report the identification of the first pathogenic missense mutations (c.1084T&gt;C [p.W362R], c.1685C&gt;T [p.P562L]) and three novel truncating mutations (c.283dupC [p.H95PfsX5], c.2212_2213del [p.S738X], and (c.2184del [p.N729TfsX31]) in SYNGAP1 in patients with NSID. A subset of these patients also showed ataxia, autism, and a specific form of generalized epilepsy that can be refractory to treatment. All of these mutations occurred de novo, except c.283dupC, which was inherited from a father who is a mosaic. Biolistic transfection of wild-type SYNGAP1 in pyramidal cells from cortical organotypic cultures significantly reduced activity-dependent phosphorylated extracellular signal-regulated kinase (pERK) levels. In contrast, constructs expressing p.W362R, p.P562L, or the previously described p.R579X had no significant effect on pERK levels. These experiments suggest that the de novo missense mutations, p.R579X, and possibly all the other truncating mutations in SYNGAP1 result in a loss of its function. Moreover, our study confirms the involvement of SYNGAP1 in autism while providing novel insight into the epileptic manifestations associated with its disruption.</p

De novo CCND2 mutations leading to stabilization of cyclin D2 cause megalencephaly-polymicrogyria-polydactyly-hydrocephalus syndrome

Activating mutations in genes encoding phosphatidylinositol 3-kinase (PI3K)-AKT pathway components cause megalencephaly-polymicrogyria-polydactyly-hydrocephalus syndrome (MPPH, OMIM 603387). Here we report that individuals with MPPH lacking upstream PI3K-AKT pathway mutations carry de novo mutations in CCND2 (encoding cyclin D2) that are clustered around a residue that can be phosphorylated by glycogen synthase kinase 3β (GSK-3β). Mutant CCND2 was resistant to proteasomal degradation in vitro compared to wild-type CCND2. The PI3K-AKT pathway modulates GSK-3β activity, and cells from individuals with PIK3CA, PIK3R2 or AKT3 mutations showed similar CCND2 accumulation. CCND2 was expressed at higher levels in brains of mouse embryos expressing activated AKT3. In utero electroporation of mutant CCND2 into embryonic mouse brains produced more proliferating transfected progenitors and a smaller fraction of progenitors exiting the cell cycle compared to cells electroporated with wild-type CCND2. These observations suggest that cyclin D2 stabilization, caused by CCND2 mutation or PI3K-AKT activation, is a unifying mechanism in PI3K-AKT–related megalencephaly syndromes

Multi-ancestry Mendelian randomization of omics traits revealing drug targets of COVID-19 severity

BACKGROUND: Recent omic studies prioritised several drug targets associated with coronavirus disease 2019 (COVID-19) severity. However, little evidence was provided to systematically estimate the effect of drug targets on COVID-19 severity in multiple ancestries. METHODS: In this study, we applied Mendelian randomization (MR) and colocalization approaches to understand the putative causal effects of 16,059 transcripts and 1608 proteins on COVID-19 severity in European and effects of 610 proteins on COVID-19 severity in African ancestry. We further integrated genetics, clinical and literature evidence to prioritise drug targets. Additional sensitivity analyses including multi-trait colocalization and phenome-wide MR were conducted to test for MR assumptions. FINDINGS: MR and colocalization prioritized four protein targets, FCRL3, ICAM5, ENTPD5 and OAS1 that showed effect on COVID-19 severity in European ancestry. One protein target, SERPINA1 showed a stronger effect in African ancestry but much weaker effect in European ancestry (odds ratio [OR] in Africans=0.369, 95%CI=0.203 to 0.668, P = 9.96 × 10(−4); OR in Europeans=1.021, 95%CI=0.901 to 1.157, P = 0.745), which suggested that increased level of SERPINA1 will reduce COVID-19 risk in African ancestry. One protein, ICAM1 showed suggestive effect on COVID-19 severity in both ancestries (OR in Europeans=1.152, 95%CI=1.063 to 1.249, P = 5.94 × 10(−4); OR in Africans=1.481, 95%CI=1.008 to 2.176; P = 0.045). The OAS1, SERPINA1 and ICAM1 effects were replicated using updated COVID-19 severity data in the two ancestries respectively, where alternative splicing events in OAS1 and ICAM1 also showed marginal effects on COVID-19 severity in Europeans. The phenome-wide MR of the prioritised targets on 622 complex traits provided information on potential beneficial effects on other diseases and suggested little evidence of adverse effects on major complications. INTERPRETATION: Our study identified six proteins as showing putative causal effects on COVID-19 severity. OAS1 and SERPINA1 were targets of existing drugs in trials as potential COVID-19 treatments. ICAM1, ICAM5 and FCRL3 are related to the immune system. Across the six targets, OAS1 has no reliable instrument in African ancestry; SERPINA1, FCRL3, ICAM5 and ENTPD5 showed a different level of putative causal evidence in European and African ancestries, which highlights the importance of more powerful ancestry-specific GWAS and value of multi-ancestry MR in informing the effects of drug targets on COVID-19 across different populations. This study provides a first step towards clinical investigation of beneficial and adverse effects of COVID-19 drug targets. FUNDING: No

Integrative GWAS and co-localisation analysis suggests novel genes associated with age-related multimorbidity

Abstract Advancing age is the greatest risk factor for developing multiple age-related diseases. Therapeutic approaches targeting the underlying pathways of ageing, rather than individual diseases, may be an effective way to treat and prevent age-related morbidity while reducing the burden of polypharmacy. We harness the Open Targets Genetics Portal to perform a systematic analysis of nearly 1,400 genome-wide association studies (GWAS) mapped to 34 age-related diseases and traits, identifying genetic signals that are shared between two or more of these traits. Using locus-to-gene (L2G) mapping, we identify 995 targets with shared genetic links to age-related diseases and traits, which are enriched in mechanisms of ageing and include known ageing and longevity-related genes. Of these 995 genes, 128 are the target of an approved or investigational drug, 526 have experimental evidence of binding pockets or are predicted to be tractable, and 341 have no existing tractability evidence, representing underexplored genes which may reveal novel biological insights and therapeutic opportunities. We present these candidate targets for exploration and prioritisation in a web application