Combining genome-editing in human IPS cells with Type 2 diabetes genomics to decipher mechanisms for islet dysfunction
Diabetes already affects 415 million people worldwide. In the UK, there will be 5M people with type 2 diabetes (T2D) by 2025, accounting for 1 in 30 prescriptions and £25 billion in annual NHS costs. The focus of the Gloyn group is the translation of genetic association signals for type 2 diabetes and glycaemic traits into mechanisms for beta‐cell dysfunction and diabetes.
There are now over 200 regions of the human genome which robustly influence the risk of developing type 2 diabetes (T2D). Each of these association signals provides an opportunity to understand the causal mechanisms driving T2D pathogenesis. Broad physiological characterisation of T2D-associated variants in humans has elucidated their role in regulation of glucose levels, insulin secretion and/or action, identifying pathways involved in T2D pathogenesis, and demonstrating that most impact primarily on beta-cell function rather than insulin sensitivity. However, until recently efforts to make more detailed statements about biological mechanisms have typically been thwarted by difficulties in defining the precise causal variant, the transcripts through which the signals exert their impact on diabetes risk on account of the vast majority of signals mapping to poorly-annotated intergenic sequence and limited availability of authentic cellular models for mechanistic studies. In recent years next-generation sequencing technology has fuelled advancements in high throughput methodologies to link genetic variants to regulatory annotation through cis-eQTL mapping, transcript annotation (chromatin state) and conformational structure (Capture-C). These advancements have coincided with breakthroughs in genome editing (CRISPR-cas 9) and the availability of authentic cellular models (human IPS cell derived beta-cells) providing for the first time the opportunity to work at genome scale to deliver molecular mechanisms for T2D. The overall objective of this DPhil project is to elucidate the functional mechanisms whereby genetic variants shown to influence T2D predisposition exert their effects at the molecular, cellular and whole-body level. The successful student will use transcriptomic data (RNA-seq, ATAC-seq, whole-genome bisulphite sequencing, Capture-C) from human islets generated by the Gloyn & McCarthy groups to identify association signals for mechanistic studies in authentic human beta-cell models. The selection of the precise variant(s) and transcripts will dictate the specifics of the project but the approaches that are likely to be employed for downstream characterisation will include siRNA and CRISPR-cas 9 mediated gene knockdown in human primary islets, beta-cell and IPS derived cell lines with appropriate transcriptomic and cellular characterisation. There is scope to design projects with both "wet" and "dry" components to provide training in computational biology as well as state-of-the art genome editing. This work is expected to provide powerful insights into the pathophysiology of T2D that will support translational advances in disease management. Recent review articles covering concepts relevant to project ( all open access ) are listed below and can be read in concert with the references listed below to give a flavour of the work currently being conducted in the group.
Wet Lab : Genome-editing (CRISPR-cas 9) in human IPS cells and differentiation down the endocrine lineage. SiRNA mediated gene knockdown / over-expression in human beta-cell lines (EndoC-bh1) and primary cells (including human islets) and assessment of impact on cellular phenotypes (imaging, electrophysiology, secretion) .
Computational : Analysis of RNA-seq, ATAC-seq and Capture-C datasets, integration of genetic and genomic datasets, protein-protein interactions.
As well as the specific training detailed above, students will have access to a wide-range of seminars and training opportunities through the many research institutes and centres based in Oxford. Students are also able to attend the Methods and Techniques course run by the MRC Weatherall Institute of Molecular Medicine. This course runs through the year, ensuring that students have the opportunity to build a broad-based understanding of differing research techniques.
Generic skills training is offered through the Medical Sciences Division's Skills Training Programme. This programme offers a comprehensive range of courses covering many important areas of researcher development: knowledge and intellectual abilities, personal effectiveness, research governance and organisation, and engagement, influence and impact. Students are actively encouraged to take advantage of the training opportunities available to them.
The Department has a successful mentoring scheme, open to graduate students, which provides an additional possible channel for personal and professional development outside the regular supervisory framework. We hold an Athena SWAN Silver Award in recognition of our efforts to support the careers of female students and staff.
|1||Thomsen, S.K. and A.L. Gloyn, Human genetics as a model for target validation: finding new therapies for diabetes. Diabetologia 2017 Jun;60(6):960-970.|
|2||Beer, N.L. and A.L. Gloyn, Genome-edited human stem cell-derived beta cells: a powerful tool for drilling down on type 2 diabetes GWAS biology. F1000Res , 2016.|
|3||Thomsen, S.K., Ceroni A, van de Bunt M, Burrows C, Barrett A, Scharfmann R, Ebner D, McCarthy MI, Gloyn A.L. Systematic Functional Characterization of Candidate Causal Genes for Type 2 Diabetes Risk Variants. Diabetes, 2016. Dec;65(12):3805-3811.|
|4||Gaulton KJ, Ferreira T, Lee Y, Raimondo A, Mägi R, Reschen ME, (…207 authors….) [Gloyn AL, Altshuler D, Boehnke M, Teslovich TM, McCarthy MI, Morris AP]# Genetic fine-mapping and genomic annotation defines causal mechanisms at type 2 diabetes susceptibility loci Nature Genetics 2015 Dec;47(12):1415-25. doi: 10.1038/ng.3437. Epub 2015 Nov 9 [Joint senior authors]#|
|5||van de Bunt M, Manning Fox JE, Dai X, Barret A, Grey C, Li L, Bennet AJ, Johnson RP, Rajotte RV, Gaulton KJ, Dermitzakis ET, MacDonald PE, McCarthy MI, Gloyn AL. Transcript expression data from human islets links regulatory signals from genome-wide association studies for type 2 diabetes and glycemic traits to their downstream effectors PLoS Genetics 2015 Dec 1;11(12):e1005694. doi: 10.1371/journal.pgen.1005694. eCollection 2015 Dec|
|6||Pal A, Potjer TP, Thomsen SK, Ng HJ, Barrett A, Scharfmann R, James TJ, Bishop DT, Karpe F, Godsland IF, Vasen HFA, Newton-Bishop J, Pijl H, McCarthy MI, Gloyn AL, Loss-of-Function Mutations in the Cell-Cycle Control Gene CDKN2A Impact on Glucose Homeostasis in Humans. Diabetes 2015 Nov 5. pii: db150602.|