Genetic regulation of mouse liver metabolite levels.

We profiled and analyzed 283 metabolites representing eight major classes of molecules including Lipids, Carbohydrates, Amino Acids, Peptides, Xenobiotics, Vitamins and Cofactors, Energy Metabolism, and Nucleotides in mouse liver of 104 inbred and recombinant inbred strains. We find that metabolites exhibit a wide range of variation, as has been previously observed with metabolites in blood serum. Using genome-wide association analysis, we mapped 40% of the quantified metabolites to at least one locus in the genome and for 75% of the loci mapped we identified at least one candidate gene by local expression QTL analysis of the transcripts. Moreover, we validated 2 of 3 of the significant loci examined by adenoviral overexpression of the genes in mice. In our GWAS results, we find that at significant loci the peak markers explained on average between 20 and 40% of variation in the metabolites. Moreover, 39% of loci found to be regulating liver metabolites in mice were also found in human GWAS results for serum metabolites, providing support for similarity in genetic regulation of metabolites between mice and human. We also integrated the metabolomic data with transcriptomic and clinical phenotypic data to evaluate the extent of co-variation across various biological scales

Genetics of Type 2 Diabetes and Metabolic Syndrome: From Genome Wide Linkage Scan and Candidate Genes to Genome Wide Association Studies

Type 2 diabetes (T2D) and metabolic syndrome (MetS) are major health problems associated with cardiovascular disease. Both diseases are influenced by a combination of genetic and environmental factors. The aim of this thesis was to identify genetic risk factors for T2D, particularly T2D associated with obesity, and for MetS. To achieve this goal, we 1) followed-up a region linked to obese T2D in an earlier genome wide linkage study; 2) studied IRS1 as a candidate gene for T2D and MetS and 3) studied the role of 28 genes, earlier identified in candidate gene- and genome wide association studies (GWAS) as susceptibility genes for T2D, in MetS. In study I, extensive fine mapping by genotyping 501 haplotype tag SNPs in 27 genes within the 15 Mb region linked to obese T2D on chromosome 18p was undertaken in Botnia 2 family material. Nominal association (p 9 000 individuals. We found no association between T2D and G972R nor any of the 20 tag SNPs capturing 85% of the common variation in IRS1. These findings argue against any major involvement of common polymorphisms in IRS1 in the development of T2D. In studies IV and V, we investigated the role of candidate or susceptibility genes for T2D or components of MetS in the development of MetS. In the family based Botnia prospective study (study IV) we found that polymorphisms in PPARG and ADRB1 predicted development of MetS, indicating a role of altered free fatty acid metabolism in the pathogenesis of MetS. In study V, polymorphisms in candidate genes for T2D (TCF7L2, WFS1, IGF2BP2) and obesity (FTO) predicted development of MetS and the risk to develop MetS seemed to be driven by associations with the previously reported phenotypes. These data do not support the view that the different components of MetS share a common genetic background

Genes and type 2 diabetes: polymorphisms of the EIF2AK3 gene and its relationship to type 2 diabetes mellitus

MDAims/ Hypothesis: Wolcott- Rallison syndrome (WRS) is a rare autosomal recessively inherited Mendelian disorder. It is characterised by a short trunk compared to arm span, multiple epiphyseal dysplasia, multiple fractures, hepatosplenomegaly and renal insufficiency in addition to insulin dependent diabetes. The onset of diabetes in WRS families is mainly below the age of 6 months and is characterised by permanent severe non-autoimmune insulin deficiency. Mutations of the gene encoding eukaryotic translational initiation factor 2 - alpha kinase 3 (EIF2AK3) were found to account for diabetes in WRS. The aim of our study was to determine whether common polymorphisms in the EIF2AK3 gene (Candidate gene association study) could be associated with type 2 diabetes. Methods: Direct sequencing was performed on all 17 exons/coding regions and intron/exon boundaries of EIF2AK3 gene in 48 diabetes and control subjects. Single Nucleotide Polymorphisms (SNPs) tagging the common haplotypes (tag SNPs) were identified and 11 SNPs were genotyped initially in 2,835 subjects with type 2 diabetes, 3,538 control subjects in the British Irish, Bangladeshi and South Indian Populations and 522 families (n= 1,722) in the British Irish and South Indian Populations. Results: We identified 19 SNPs by direct sequencing. There was no association (all p>0.05) between the SNPs and type 2 diabetes in the case–control study and in the family study. In the one marker, rs7605713, that showed a nominal significance in Warren 2 European samples, further replication studies in the Dundee samples (3,334 diabetes cases and 3,456 controls) proved to be negative thereby avoiding a false positive result. The results also showed several of the SNPs had different minor allele frequencies between the British/Irish Caucasians as compared to the South Asians. Conclusions/interpretation: Common variations in the EIF2AK3 gene were not associated with type 2 diabetes in the British Irish and the South Asian population

Investigation of the molecular basis of inherited developmental conditions in high risk population isolates

The Amish communities of Ohio (USA) are a distinct group of endogamous, rural-living Anabaptist Christians. An ancestral bottleneck, caused by migratory events in the 17th century and subsequent rapid population expansion, has led to the enrichment of a number of inherited conditions within these communities. This provides significantly enhanced power to identify genes responsible for rare monogenic disorders, as well traits with more complex inheritance patterns. The studies detailed in this thesis aims to provide diagnoses to individuals and their families for the underlying genetic causes responsible for the difficulties they experience and contributes to a long-running, non-profit community clinical-genetic research programme called the Windows of Hope (WoH). Forming part of a wider Amish Hearing Loss Program the studies described in chapter three document the discovery of the genetic causes of hearing loss for eight Amish families. Through a combination of targeted gene sequencing, genome-wide SNP mapping and exome sequencing this study identified a variant in the Gap junction beta-2 (GJB2) gene, not previously reported in the Amish, as the cause of non-syndromic hearing loss in six families. Additionally, one family initially thought to be affected by a neurodevelopment disorder which included syndromic hearing loss, was found to possess two distinct genetic disorders; a 16p11.2 microdeletion, responsible for the developmental delay, and a homozygous GJB2 variant, responsible for the hearing loss. Finally, this chapter proposes two novel hearing loss genes and details the functional work undertaken to assess the pathogenicity of one of these genes (SLC15A5). This work provided important diagnoses for many families and acquired significant information regarding the spectrum and frequency of hearing loss-associated gene variants across distinct Amish communities. Chapter four details work undertaken to define the clinical phenotype and molecular basis of a novel complex autosomal recessive neurological disorder. Work undertaken by one of our collaborators, Dr Zineb Ammous, was instrumental in precisely defining the clinical phenotype of this disorder. A combination of genome-wide SNP mapping and exome sequence identified a sequence variant in Smad Nuclear Interacting Protein 1 (SNIP1), which encodes an evolutionary-conserved transcriptional regulator, as the likely underlying genetic cause. Due to its role as a transcription regulator whole transcriptome sequencing was undertaken to determine the impact of this gene mutation. This work provided important information regarding the specific biological role of SNIP1 and identified gene expression pathways of direct relevance to the clinical phenotype, highlighting therapeutic approaches likely to benefit affected individuals. Additionally, this study determined that SNIP1-associated syndrome is one of the most common conditions across many Amish communities. In recent years the WoH Project has accumulated extensive single nucleotide polymorphisms (SNP) and exome sequencing datasets from patients and individuals from the Amish community. Chapter five outlines a pilot, proof-of-principle study undertaken to explore this data with the aim characterising the architecture of the Amish genome. The interrogation of 26 exomes identified the presence of 12 pathogenic variants known to cause autosomal recessive (AR) diseases that have not yet been reported in the Amish but are likely to be present. Additionally, a PLEXseq sequencing approach was implemented to determine the prevalence of 165 pathogenic variants in 171 unaffected Amish individuals. The findings indicated diverse carrier frequencies within the different Amish communities and contributed to the consolidation of two genes responsible for ultra-rare inherited AR diseases (CEP55, MNS1). By developing approaches to improve knowledge of the specific causes of inherited diseases in the community, this work has laid the foundation for the development of a new genetic-based approach to diagnostic testing in the community. This thesis, and the wider programme of work of Windows of Hope, occupies a privileged positioned at the interface between scientific research and clinical care. The findings described here have made a significant contribution to our understanding of the pathomolecular cause of a number of rare inherited disorders by increasing our knowledge of the nature and spectrum of inherited disease within the Amish laying the foundations to aid the future discovery of new disease genes and improving clinical outcomes by enabling focussed clinical diagnostic and management strategies to be implemented

Genetic studies of abdominal MRI data identify genes regulating hepcidin as major determinants of liver iron concentration

Background & Aims: Excess liver iron content is common and is linked to hepatic and extrahepatic disease risk. We aimed to identify genetic variants influencing liver iron content and use genetics to understand its link to other traits and diseases. Methods: First, we performed a genome-wide association study (GWAS) in 8,289 individuals in UK Biobank with MRI quantified liver iron, and validated our findings in an independent cohort (n=1,513 from IMI DIRECT). Second, we used Mendelian randomisation to test the causal effects of 29 predominantly metabolic traits on liver iron content. Third, we tested phenome-wide associations between liver iron variants and 770 anthropometric traits and diseases. Results: We identified three independent genetic variants (rs1800562 (C282Y) and rs1799945 (H63D) in HFE and rs855791 (V736A) in TMPRSS6) associated with liver iron content that reached the GWAS significance threshold (p<5x10-8). The two HFE variants account for ~85% of all cases of hereditary haemochromatosis. Mendelian randomisation analysis provided evidence that higher central obesity plays a causal role in increased liver iron content. Phenome-wide association analysis demonstrated shared aetiopathogenic mechanisms for elevated liver iron, high blood pressure, cirrhosis, malignancies, neuropsychiatric and rheumatological conditions, while also highlighting inverse associations with anaemias, lipidaemias and ischaemic heart disease. Conclusion: Our study provides genetic evidence that mechanisms underlying higher liver iron content are likely systemic rather than organ specific, that higher central obesity is causally associated with higher liver iron, and that liver iron shares common aetiology with multiple metabolic and non-metabolic diseases

Towards precision medicine for hypertension: a review of genomic, epigenomic, and microbiomic effects on blood pressure in experimental rat models and humans

Compelling evidence for the inherited nature of essential hypertension has led to extensive research in rats and humans. Rats have served as the primary model for research on the genetics of hypertension resulting in identification of genomic regions that are causally associated with hypertension. In more recent times, genome-wide studies in humans have also begun to improve our understanding of the inheritance of polygenic forms of hypertension. Based on the chronological progression of research into the genetics of hypertension as the "structural backbone," this review catalogs and discusses the rat and human genetic elements mapped and implicated in blood pressure regulation. Furthermore, the knowledge gained from these genetic studies that provide evidence to suggest that much of the genetic influence on hypertension residing within noncoding elements of our DNA and operating through pervasive epistasis or gene-gene interactions is highlighted. Lastly, perspectives on current thinking that the more complex "triad" of the genome, epigenome, and the microbiome operating to influence the inheritance of hypertension, is documented. Overall, the collective knowledge gained from rats and humans is disappointing in the sense that major hypertension-causing genes as targets for clinical management of essential hypertension may not be a clinical reality. On the other hand, the realization that the polygenic nature of hypertension prevents any single locus from being a relevant clinical target for all humans directs future studies on the genetics of hypertension towards an individualized genomic approach

Host Gene Expression and Genetic Susceptibility to Dengue Virus Infection in Vietnamese Children

Dengue has been a public health problem in over 120 tropical and subtropical countries. It is estimated that there are approximately 3.6 billion of the world’s population at risk of dengue virus (DENV) infection, and about 70-500 million DENV infections annually. Every year, about 36 million cases of dengue fever, of which about 2.1 million cases are dengue haemorrhagic fever and dengue shock syndrome (DHF/DSS) resulting in 21,000 deaths among children and young adults, are reported. The disease pathogenesis is still poorly understood. Effective antiviral drug, vaccine and animal model for the disease are not available. We conducted two global gene expression microarray studies and a genome-wide association study in order to identify prognostic markers of severe dengue in Vietnamese patients and to identify genetic markers that confer susceptibility to dengue and DSS. The results show that acute DENV infection is associated with over-presentation of transcripts that are related to interferon-inducible transcripts and canonical gene ontology terms that included response to virus, immune response, innate immune response and inflammatory response. Pathway and network analysis identified STAT1, STAT2, STAT3, IRF7, IRF9, IRF1, CEBPB and SP1 as key transcriptional factors mediating the early response. Interestingly, the only differences in the transcriptional signatures of early DSS and uncomplicated dengue cases were the greater abundance of several neutrophil-associated transcripts in patients who progressed to DSS. Host genetic study identified several single nucleotide polymorphisms (SNPs) that show strong evidence for association with dengue or DSS. None of the SNPs was significant at suggestive genome-wide level but ontology analysis of genes implicated by associated SNPs suggests biologically plausible functional relationships between the genes. This thesis provides insights into host gene response in DENV infection and also host genetic susceptibility to dengue which in turn could be useful hint for future studies in the field

The South Asian genome

Genetics of disease Microarrays Variant genotypes Population genetics Sequence alignment AllelesThe genetic sequence variation of people from the Indian subcontinent who comprise one-quarter of the world's population, is not well described. We carried out whole genome sequencing of 168 South Asians, along with whole-exome sequencing of 147 South Asians to provide deeper characterisation of coding regions. We identify 12,962,155 autosomal sequence variants, including 2,946,861 new SNPs and 312,738 novel indels. This catalogue of SNPs and indels amongst South Asians provides the first comprehensive map of genetic variation in this major human population, and reveals evidence for selective pressures on genes involved in skin biology, metabolism, infection and immunity. Our results will accelerate the search for the genetic variants underlying susceptibility to disorders such as type-2 diabetes and cardiovascular disease which are highly prevalent amongst South Asians.Whole genome sequencing to discover genetic variants underlying type-2 diabetes, coronary heart disease and related phenotypes amongst Indian Asians. Imperial College Healthcare NHS Trust cBRC 2011-13 (JS Kooner [PI], JC Chambers)

A Path to Implement Precision Child Health Cardiovascular Medicine.

Congenital heart defects (CHDs) affect approximately 1% of live births and are a major source of childhood morbidity and mortality even in countries with advanced healthcare systems. Along with phenotypic heterogeneity, the underlying etiology of CHDs is multifactorial, involving genetic, epigenetic, and/or environmental contributors. Clear dissection of the underlying mechanism is a powerful step to establish individualized therapies. However, the majority of CHDs are yet to be clearly diagnosed for the underlying genetic and environmental factors, and even less with effective therapies. Although the survival rate for CHDs is steadily improving, there is still a significant unmet need for refining diagnostic precision and establishing targeted therapies to optimize life quality and to minimize future complications. In particular, proper identification of disease associated genetic variants in humans has been challenging, and this greatly impedes our ability to delineate gene-environment interactions that contribute to the pathogenesis of CHDs. Implementing a systematic multileveled approach can establish a continuum from phenotypic characterization in the clinic to molecular dissection using combined next-generation sequencing platforms and validation studies in suitable models at the bench. Key elements necessary to advance the field are: first, proper delineation of the phenotypic spectrum of CHDs; second, defining the molecular genotype/phenotype by combining whole-exome sequencing and transcriptome analysis; third, integration of phenotypic, genotypic, and molecular datasets to identify molecular network contributing to CHDs; fourth, generation of relevant disease models and multileveled experimental investigations. In order to achieve all these goals, access to high-quality biological specimens from well-defined patient cohorts is a crucial step. Therefore, establishing a CHD BioCore is an essential infrastructure and a critical step on the path toward precision child health cardiovascular medicine