Molecular characterization in human neurons of genes associated with severe obesity identified from consanguineous pedigrees.

Obesity is a growing public health problem. While it has undoubtedly been driven by the changing environment, there is a large heritable component underlying the variation in body-weight within the population. Genetic studies point to the brain, in particular a region called the hypothalamus, as having a crucial role in modulating appetite. For example, mutations of single genes in the hypothalamic leptin melanocortin pathway leads to severe, early-onset obesity. Around 10% of severe human obesity currently has a clear identified cause, with the majority of mutations sitting within the genes encoding the melanocortin pathway. Thus, much of the genetic aetiology of severe obesity remains to be uncovered, identification of which could reveal novel pathways and potential therapeutic targets. However, studying the disease in inbred populations increases the chances of finding disease causing mutations. Using this approach, we have identified mutations in two new genes, ADCY3 and ROCK1, that are associated with severe obesity. In this proposal, we will study both these genes in detail. Given the role of the brain in the regulation of body-weight and food intake, we will characterize these genes in human neurons. We need to understand not only the function of these genes in normal cells but also the consequences of loss of function for the neuronal populations in which they are expressed. Although murine models have been hugely helpful in the past to overcome the difficulties of inaccessibility of the human hypothalamus, we believe studying human tissue from the relevant regions is essential to get a fuller understanding of the role of these genes. An existing collaboration with the Cambridge Brain Bank, allows us access to fresh and fixed human donor brain samples. The access to this precious material, coupled with recent developments in single cell sequencing and mapping technologies provides us with a timely opportunity to study where these genes are expressed in the human hypothalamus. Additionally, we have recently acquired the ability to generate, genetically manipulate and differentiate human stem-cells into hypothalamic neurons. This will allow us to directly examine the impact of these obesity associated mutations in the context of a relevant human cell. Using high-throughput DNA sequencing approaches, we will also continue our search for other new obesity candidates. Our current proposal is a collaborative venture between two UK centres of excellence in the study of the genetics of obesity. The group of Froguel, from Imperial College London, brings a large collection of severely obese children from highly consanguineous Pakistani pedigrees; and the group of Coll, O'Rahilly and Yeo, from the MRC Metabolic Diseases Unit at the University of Cambridge, who bring bespoke functional expertise and access to human hypothalamic samples. Our aim is to uncover novel appetite control pathways and reveal new potential therapeutic targets in order to tackle obesity.

Up to 10% of severe early onset obesity has an identified monogenic cause. We showed that studying the disease in consanguineous Pakistani pedigrees enriches for homozygous variations, increasing the likelihood of finding causative mutations. Using this approach, we have identified that homozygous and compound heterozygous loss of function mutations in ADCY3, which encodes for adenylate cyclase 3, and homozygous mutations in the gene ROCK1, which encodes for Rho-kinase 1, are linked to severe obesity. In this proposal, we will study both these genes in detail, as well continue our search for other new obesity candidates. Given the role of the brain in the regulation of body-weight and food intake, we will molecularly characterize these genes in human neurons. Using single nucleus RNAseq, we will identify human neurons expressing ADCY3 and ROCK1. We will characterize the heterogeneity of the neurons and define markers for different populations. We will use RNAscope single molecule FISH to map where both of these genes are expressed within the human hypothalamus. Using CRISPR/Cas9 gene editing, we will 'knock-in' the ADCY3 and ROCK1 mutations that are associated with obesity into human pluripotent stem cells and then differentiate them into hypothalamic neurons. In the cells with ADCY3 mutations, we will study the effect on the primary cilia and leptin responsiveness. In the cells with ROCK1 mutations, we will measure leptin and insulin responsiveness. Using single cell RNAseq, we will examine the transcriptomic consequences of the mutations on the neurons. Using CoDE-seq, an augmented whole-exome sequencing approach that additionally enables the accurate detection of CNVs, we will continue to further identify candidate genes associated with severe obesity. Where the weight of evidence dictates, these will be molecularly characterized as above. Our aim is to uncover novel appetitive control pathways and reveal new potential therapeutic targets to tackle obesity.

This study integrates a fundamental basic science with a biomedical problem - neuroscience and obesity - that will initially impact academic and commercial research communities including post- and under-graduate students working on metabolism, neuroanatomy, physiology and gene expression. In the near term, the project will directly impact those focussing on the hypothalamic control energy balance, but as it develops, it will bring an awareness about the importance of neuroscience to the obesity epidemic to an increasingly broad audience locally, nationally and internationally. Between them, Dr Yeo, Prof O'Rahilly and Prof Froguel have over four decades of experience working in the US and UK, presenting an ongoing opportunity for knowledge transfer from many centres of excellence. Outreach to potential stake-holders including academics, policy-makers and the commercial sector will be maximised.

The work proposed here is, by its very nature, fundamental. However, knowledge generated here will eventually inform new health care messages, interventions and treatment regimes, with beneficiaries that include health care professionals, private and public health institutions (NHS, social services), policy makers (Department of Health, Health Authorities), charities (eg WHO, Diabetes UK, British Heart Foundation), pharmaceutical and biotechnology companies and those at risk of developing obesity-associated disease. We expect the work to identify novel therapeutic targets of human relevance that directly inform clinical studies, benefiting healthcare workers and some of the 74% of adults in the UK are predicted to be over-weight by 2030. Given that the NHS in England spent ~£5.1 billion on obesity-related ill-health in 2014-15, reducing incidence of this condition will have a major economic impact. The work will benefit the commercial private sector because it will lead to new information on human relevant markers that influence metabolic disease.