LIMR Genetics: Investigating the functional effect of novel genes and genetic variants in malignant hyperthermia using model systems

Summary

Malignant hyperthermia (MH) is an inherited condition, where patients exposed to anaesthetic drugs are susceptible to a dramatic hyperthermic and hypermetabolic response that can contribute to a significant proportion of post-operative morbidity and deaths. This is thought to result primarily from skeletal muscle Ca2+ dysregulation. A genetic diagnosis is still not possible for a large proportion of patients. Currently an invasive muscle biopsy and functional response, the in vitro contracture test (IVCT), has to be performed. Historically MH was described as an autosomal dominant condition due to RYR1 mutations. But the genetic basis now appears more complex, with variants in additional genes causing or contributing to the condition, indicating a functional threshold required for patients to become susceptible (1).

To address the incomplete understanding of the genetic basis of MH susceptibility, and functionally characterise variants of uncertain significance (VUS), CRISPR-Cas9 gene-editing will be used to replace patients' VUS with wild type sequence in hiPSC derived from fibroblasts, to edit VUS into a control hiPSC cell line, and to generate cells harbouring combinations of genetic variants. Edited cells will be differentiated into a more appropriate model muscle cell type, myotubes, and functional assessment made by live cell imaging techniques, determining responses to different compounds relevant to volatile anaesthesia and Ca2+ handling. This will identify and quantify the mechanistic alterations in Ca2+ flux between cellular compartments. Additional mechanisms such as mitochondrial and oxidative stress will also be assessed (2).

The project aim is therefore to apply the functional data generated to determine the pathogenicity of the sequence variants alone and in combinations, and their contribution to the MH susceptible phenotype. This project is based in the Leeds MH Unit which is the UK reference centre and holds the largest collection of patient-derived phenotypic data and material worldwide including DNA, cells, IVCT, exome, and RNASeq data. A highly motivated graduate will have the opportunity to work closely with both clinical and research staff in this internationally renowned unit.

Techniques used in this project

Approaches used in this project are a range of standard and specialised molecular, biochemical, and cellular techniques currently being undertaken in the MH Unit, including: stem cell culture, CRISPR-Cas9 gene editing, muscle cell differentiation, protein and transcript quantification, live cell imaging, statistical analysis. Episomal, one-step programming will be used to transform the patient fibroblasts to hiPSC’s following the protocol described by Howden et al (3). Both the WT controls and patient hiPSC’s will then be transformed into myogenic progenitor cells following the protocol described by Chal et al (4). Defined media conditions will be used to drive myogenesis, to create viable myotubes for calcium imaging experiments.

References

1. Miller et al (2018) Genetic epidemiology of malignant hyperthermia in the UK. British Journal of Anaesthesia 121(4): 944-952
2. Chang et al (2019) Permeabilised skeletal muscle reveals mitochondrial deficiency in malignant hyperthermia-susceptible individuals. British Journal of Anaesthesia 122(5): 613-621
3. Howden et al (2018) Simultaneous reprogramming and gene editing of human fibroblasts. Nature Protocols 13(5): 875-898
4. Chal et al (2016) Generation of human muscle fibres and satellite-like cells from human pluripotent stem cells in vitro. Nature Protocols 11(10): 1833-1850

This project is part of the International PhD Academy: Medical Research

In line with the bespoke nature of our International PhD Academy a modified PhD project can be proposed dependent on students interests and background.

A degree in biological sciences, dentistry, medicine, midwifery, nursing, psychology or a good honours degree in a subject relevant to the research topic. A Masters degree in a relevant subject may also be required in some areas of the Faculty. For entry requirements for all other research degrees we offer, please contact us.

Applicants whose first language is not English must provide evidence that their English language is sufficient to meet the specific demands of their study. The minimum requirements for this programme in IELTS and TOEFL tests are: • British Council IELTS - score of 7.0 overall, with no element less than 6.5 • TOEFL iBT - overall score of 100 with the listening and reading element no less than 22, writing element no less than 23 and the speaking element no less than 24.