Malignant hyperthermia (MH) is a pharmacogenetic condition of skeletal muscle that manifests in hypermetabolic responses upon exposure to volatile anaesthetics. This condition is caused primarily by pathogenic variants in the calcium-release channel RYR1, which disrupts calcium signalling in skeletal muscle. However, our understanding of MH genetics is incomplete, with no variant identified in a significant number of cases and considerable phenotype diversity. In this study, we applied a transcriptomic approach to investigate the genome-wide gene expression in MH-susceptible cases using muscle biopsies taken for diagnostic testing. Baseline comparisons between muscle from MH-susceptible individuals (MHS, n = 8) and non-susceptible controls (MHN, n = 4) identified 822 differentially expressed genes (203 upregulated and 619 downregulated) with significant enrichment in genes associated with oxidative phosphorylation (OXPHOS) and fatty acid metabolism. Investigations of 10 OXPHOS target genes in a larger cohort (MHN: n = 36; MHS: n = 36) validated the reduced expression of ATP5MD and COQ6 in MHS samples, but the remaining 8 selected were not statistically significant. Further analysis also identified evidence of a sex-linked effect in SDHB and UQCC3 expression, and a difference in ATP5MD expression across individuals with MH sub-phenotypes (trigger from in vitro halothane exposure only, MHS_h (n = 4); trigger to both in vitro halothane and caffeine exposure, MHS_hc (n = 4)). Our data support a link between MH-susceptibility and dysregulated gene expression associated with mitochondrial bioenergetics, which we speculate plays a role in the phenotypic variability observed within MH.

Exertional heat illness (EHI) is an occupational health hazard for athletes and military personnel–characterised by the inability to thermoregulate during exercise. The ability to thermoregulate can be studied using a standardised heat tolerance test (HTT) developed by The Institute of Naval Medicine. In this study, we investigated whole blood gene expression (at baseline, 2 h post-HTT and 24 h post-HTT) in male subjects with either a history of EHI or known susceptibility to malignant hyperthermia (MHS): a pharmacogenetic condition with similar clinical phenotype. Compared to healthy controls at baseline, 291 genes were differentially expressed in the EHI cohort, with functional enrichment in inflammatory response genes (up to a four-fold increase). In contrast, the MHS cohort featured 1019 differentially expressed genes with significant down-regulation of genes associated with oxidative phosphorylation (OXPHOS). A number of differentially expressed genes in the inflammation and OXPHOS pathways overlapped between the EHI and MHS subjects, indicating a common underlying pathophysiology. Transcriptome profiles between subjects who passed and failed the HTT (based on whether they achieved a plateau in core temperature or not, respectively) were not discernable at baseline, and HTT was shown to elevate inflammatory response gene expression across all clinical phenotypes.

Variants in six pre-mRNA processing factors cause autosomal dominant Retinitis Pigmentosa (adRP). The RP9 gene encodes a seventh splicing factor, and in 2002, we published RP9 variants c.410A>T; p.(H137L) and c.509A>G; p.(D170G) as likely causes of adRP in a large multigenerational RP9 -linked family and a single case, respectively. It has since been suggested these variants might be artefacts due to simultaneous amplification of the RP9P pseudogene, and no further pathogenic variants have been reported. We therefore rescreened two members of the RP9 -linked family by genome sequencing. Examination of the 2 Mb locus defined by crossovers in the original family revealed no other plausible causative variants. Alignment of both short and long-read sequences confirmed that p.(H137L) is in the RP9 gene, not the pseudogene. Screening for p.(H137L) in 1961 RP/Rod-cone dystrophy (RCD) cases from the Leeds patient cohort and UK 100,000 Genomes Project (100kGP) database revealed four further carriers. Including the original family, this variant was therefore present in 5/1962 RP/RCD probands, and is absent from gnomAD, constituting statistically significant enrichment in RP cases. Long-read sequencing of p.(H137L) in available carriers showed this is a UK founder allele. The RP9 p.(D170G) allele was also confirmed as gene, not pseudogene, derived, but is present in 22 individuals in the 100kGP cohort, none with RP, as well as >200 individuals in gnomAD and Biobank, suggesting it is non-pathogenic. In conclusion, RP9 p.(H137L) is strongly associated with RP and remains the only plausible variant accounting for the condition in a large multi-generation adRP family.

Malignant hyperthermia (MH) is a potentially fatal hypermetabolic reaction to general anaesthesia arising from skeletal muscle calcium dysregulation. Previous studies of resting cells support an association between MH susceptibility, mitochondrial dysfunction, and defects in fatty acid metabolism, which are understood to be downstream consequences of calcium dysregulation. We hypothesised that in mouse models of MH susceptibility, genotypes associated with higher cytoplasmic calcium concentrations would have a proportionally higher mitochondrial oxygen consumption rate (OCR). We aimed to test this and validate a cell-based assay system. A high-throughput mitochondrial assay was used to compare OCR between myotubes derived from control and three different genotypes of mice containing ryanodine receptor 1 variants (p.G2435R heterozygous and homozygous, p.T4826I heterozygous) that confer susceptibility to MH. Baseline comparisons showed effects of genotype on OCR (P<0.0001), with Ryr1 p.G2435R homozygous myotubes having the highest basal normalised OCR (P<0.01). Ryr1 p.G2435R homozygous required a greater proportion of basal respiration to produce adenosine triphosphate (ATP), and had a higher proton leak and greater non-mitochondrial OCR (P<0.01). All genotypes except Ryr1 p.G2435R homozygous were primarily dependent on the glucose/pyruvate pathway for achieving their maximal OCR upon uncoupling. The high-throughput method used produced data consistent with findings in skeletal muscle fibres, but with a greater sensitivity to genotypic effects. This validates the use of cultured myotubes in lieu of muscle fibres in studying mitochondrial bioenergetics in models of MH, and indicates that mitochondrial bioenergetics are not directly affected by myoplasmic calcium concentrations in young MH mice.

Understanding the genetic and immune heterogeneity in hepatocellular carcinoma (HCC) is crucial as treatments advance towards personalisation. This study characterises the genetic and immune heterogeneity within a population of 56 tumours from 28 patients, 10 of whom have multifocal disease, obtained from patients undergoing surgery at the Leeds Teaching Hospital NHS Trust. These samples represent the most common aetiological groups found in the UK: alcohol-related liver disease, non-alcoholic fatty liver disease and hepatitis C. Whole exome sequencing was performed on DNA from these tumour alongside matched background liver samples to determine tumour-specific variants. Tissue sections were stained for both proliferation and immune cell markers. Tumours across aetiological groups showed significant levels of variation, with high inter-individual variation. Analyses of multifocal tumours revealed significant discordance in genetic and immune cell profiles, both between multi-centric primary and metastatic tumours. This work emphasises the genetic and immuneheterogeneity in HCC across HCC subtypes, between and within individuals, highlighting mechansisms for therapeutic resistance and the need for personalised medicine.

Introduction/Objectives: Understanding the genetic and immune heterogeneity in hepatocellular carcinoma (HCC) is critical for accurate pathological characterisation and treatment planning, as therapies advance towards personalisation. Tumour mutational burden (TMB) has been proposed as a biomarker of response to immune checkpoint blockade but its relationship with immune infiltration in HCC remains unclear, particularly across different aetiologies and within multifocal disease. Methods: This study characterises both the inter- and intra-patient heterogeneity in HCC, integrating whole-exome sequencing with detailed histopathologic and immune cell profiling, to identify implications for diagnosis and response to treatment. Samples of 54 tumours from 26 patients undergoing surgery at the Leeds Teaching Hospital NHS Trust were examined, including 10 patients with multifocal disease. The cohort includes the major UK aetiological groups. Whole-exome sequencing of tumours and matched background liver identified somatic variants. Histopathological and immunohistochemical evaluations were performed to assess tumour features and characterise the tumour immune microenvironment. Results: Marked inter-individual heterogeneity was observed across aetiological groups with variation in tumour mutation burden, genes containing somatic mutations and immune infiltration. Multifocal tumours frequently demonstrated discordant genetic and immune profiles irrespective of clonal relatedness. NeoTMB was not associated with recurrence following surgery, whereas recurrent cases demonstrated significantly higher intratumoural FoxP3+ T-cell frequencies. Conclusions: This study demonstrates substantial genetic and immune heterogeneity in HCC, including marked discordance between multifocal tumours within the same patient. Tumour mutation burden was not related to immune cell infiltration or risk of recurrence after surgery, whereas elevated intratumoural FoxP3+ regulatory T-cell frequencies were associated with recurrence. These findings suggest that the immunological composition of the tumour microenvironment may be more relevant to clinical outcome than mutational burden alone and highlight the importance of comprehensive tumour profiling to inform personalised therapeutic strategies.