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Dissecting the role of the mtDNA OriB variant in diabetes and other multifactorial diseases (Professor Karl Morten)

PROJECT TITLE

Dissecting the role of the mtDNA OriB variant in diabetes and other multifactorial diseases

SUPERVISORS

DESCRIPTION OF PROJECT

Mitochondria are cellular components that generate the energy needed for all life processes.  Each contains “mtDNA”, a blueprint for the proteins needed to make energy.  We have demonstrated that a common mitochondrial DNA (mtDNA) variant that maps to the centre of a novel origin of mtDNA replication, OriB (henceforth the “OriB variant”) is associated with dilated cardiomyopathy (DCM), type 2 diabetes (T2D) and other multi-factorial disorders.  The OriB variant per se underlies the association because founder effects have been excluded. Other investigators showed that cells with one of the prodiabetic mitochondrial haplotypes harbouring this variant manifest increased apoptosis and mitophagy, fragmented mitochondria with reduced mass, impaired respiratory function and increased reactive oxygenic species (ROS) production compared with haplotype D4 which is diabetes resistant. .

These haplotypes differ by multiple single nucleotide polymorphisms (snps) so the functional differences cannot be attributed to any single snp.

We aim to unpick this uncertainty using paired cybrid cell lines which are are isogenic apart from the presence or absence of the OriB variant (henceforth “OriB” and “WT”) on a rhabdomyosarcoma and an osteosarcoma background.

AIMS to use isogenic cell lines to determine the role of the OriB variant in functional differences that likely underlie mtDNA susceptibility to diabetes.

We previously showed that the variant appears to impair oxygen consumption and generates a unique Raman spectrum in the rhabdomyosarcoma background.  We will confirm these findings in the osteosarcoma cells.  We will use high content imaging to compare mitophagy, mitochondrial mass and morphology in OriB and WT cells under varying glucose concentrations. We will use Clariostar to measure ROS production oxygen consumption and mitochondrial membrane potential.    Raman microspectroscopy will be utilised with different isotopes to study the impact of the variant on cell metabolism in real time  down to the level of individual mitochondrial cytochromes .

Will use next generation sequencing to quantify length variation in the variant and RNAseq to probe effects on abundance of mitochondrial transcripts. We will attempt to make human macrophage and other immune cell lines rho- (mtDNA free) with the aim of placing the OriB variant on an immune cell background. Access to state of the art metabolomics discovery platforms and Raman microscopy is available at the Rosalind Franklin Institute

In the long term we will extend this work using next generation whole genome sequencing and RNA seq of clinical samples to explore whether biallelic inheritance could explain phenotypic differences, by identifying candidate interactions with nuclear encoded genes

TRAINING OPPORTUNITIES

This multi-disciplinary project involves one WRH group and the NHS Specialist Mitochondrial Disorders Service. We will use high-throughput biology approaches, likely including the facilities of the Target-Discovery-Institute. The student will be part of a network bridging genetics, immunometabolism, and mitochondrial-medicine with collaborations at the Rosalind Franklin Institute to study metabolism with access to state of art Raman instrumentation. They will be encouraged to publish, present at national/international conferences and seminars, and training. The project integrates a broad range of molecular, biochemical, and imaging techniques (including primary cell culture, western blot and immunoprecipitation, high-throughput fluorescence microscopy, and flow cytometry), complemented by advanced genomic approaches such as whole-genome sequencing and single-cell transcriptomics.

Funding Information

The position is not currently funded and therefore the candidate will need to secure funding.

HOW TO APPLY

To apply for this research degree, please click here.