Scaled Multidimensional Assays of Variant Effect Identify Sequence-Function Relationships in Hypertrophic Cardiomyopathy

Context-aware phenotyping of cardiac disease across translational models

Cardiomyopathies are a leading cause of heart failure, arrhythmia and sudden cardiac death, yet patients sharing a diagnosis, and even a causal variant, follow markedly different clinical courses. This thesis argues that disease expression depends on the biological context in which genetic susceptibility acts, and that studying this requires experimental models containing the relevant context. Each chapter adds a layer. The starting point is genetic. Among nearly 470,000 UK Biobank participants, carriers of TNNT2 p.Arg288Cys showed no excess hypertrophic cardiomyopathy after correction for multiple testing and cumulative penetrance below 1% by age 80, while displaying subtle functional differences on cardiac MRI. Dutch probands presented with more severe disease, consistent with ascertainment bias and enrichment for modifiers. The variant is therefore intermediate in effect, insufficient alone but capable of contributing in a permissive context. The question shifts from whether a variant is pathogenic to under which conditions it becomes so. Cellular identity is the first layer. Endothelial and smooth muscle cells from nine canine macrovessels retained location-associated transcriptional programmes through at least three passages, with embryonic positional transcription factors providing the strongest discrimination. An epigenetic mechanism is plausible. Cell source is thus a design decision rather than a practical detail. Local tissue context was added in a fibrin-based co-culture, where endothelial cells self-assembled into lumenised networks alongside hiPSC-derived cardiomyocytes and remained stable for at least three weeks in a commercially available microfluidic device. The positional identity findings simultaneously define its main limitation, since umbilical vein endothelial cells were used. Environmental context was introduced as lipid overload in an isogenic MYH7 p.Arg403Leu pair. A model providing context is of limited value if the readout averages that context away. Across 44,403 single-cell measurements of calcium handling and contraction, within-well variability accounted for 76 to 96% of total variance, and clustering identified a lipid-enriched subpopulation invisible in well averages. Model complexity and measurement resolution are two sides of the same problem. Some layers cannot be engineered. Lifelong exposure, genetic diversity and spontaneous progression are inherent to naturally occurring disease in companion animals. Canine dilated cardiomyopathy returns genetic susceptibility to the foreground with full context attached, with breed-level uniformity supporting bidirectional discovery. In 37 cats with hypertrophic cardiomyopathy, digital pathology combined with Nanopore sequencing revealed structure only after outcome-based reclassification, distinguishing arterial thromboembolism from congestive heart failure. The same morphometric approach across 46 vertebrate species established a physiological reference frame, since a tenfold increase in body mass corresponded to roughly 8% greater cardiomyocyte diameter and no species approached dimensions reported in hypertrophic cardiomyopathy. The contribution is both methodological, favouring continuous measurement analysed with models matched to nested data structures, and conceptual, in showing that genetic findings acquire meaning only within their spatial, metabolic and species-specific context. No single model suffices, but complementary models each supply a different layer.

LRRK2 in Focus: A Global Browser Linking Genetic Diversity to Functional Effects

Background: LRRK2 variants are major contributors to Parkinsons disease (PD). Many pathogenic variants increase kinase activity, underscoring the value of functional assays in nominating therapeutic targets and kinase inhibitors as potential disease-modifying therapies. Objective: To develop an interactive resource that provides functional context and ancestry-specific variant frequencies. Methods: Genotyping and short-read sequencing data were analyzed for 101,678 individuals (61,709 PD, 39,969 controls) from the Global Parkinsons Genetics Program (GP2) and integrated with clinical and in-vitro biochemical kinase activity information. Results: The LRRK2 Browser (http://gp2.org/lrrk2browser) displays ancestry-specific genetic data for 19,596 LRRK2 variants (968 exonic, 14 disease-associated) across 11 populations, and functional data for 171 variants. Clinical annotations include age, age at onset, and family history of PD. Discussion: The publicly available LRRK2 Browser represents an open-access, multi-ancestry resource to support LRRK2 variant interpretation. It aims to enhance the translational potential of genetic and functional data for precision medicine and the implementation of gene-targeted therapies in diverse populations.

Phenotype and outcome of patients with hypertrophic cardiomyopathy MYH7 variants: A longitudinal cohort study.

INTRODUCTION AND OBJECTIVES Hypertrophic cardiomyopathy (HCM) is primarily caused by mutations in the MYH7 and MYBPC3 genes which exhibit diverse clinical expression and prognosis. This study aims to outline the clinical features and long-term cardiovascular outcome of patients with MYH7-related HCM, and to look for clinical and prognostic implications of specific variants. METHODS A retrospective longitudinal analysis was conducted on 30 unrelated HCM families with pathogenic/likely pathogenic (P/LP) MYH7 mutations. A composite endpoint encompassing hospitalisation for heart failure, cardiovascular admissions, or all-cause mortality was evaluated. Kaplan-Meier survival analysis was used to compare outcomes across prevalent variants and genetic profiles. RESULTS Among 118 individuals (30 probands, 88 relatives), 77 were carriers of P/LP MYH7 variants - 69% with HCM (G+/Ph+) at diagnosis and 31% P/LP MYH7 variant carriers only (G+/Ph-). Thirteen different P/LP variants were identified in the 30 families, with four (p.Ile263Thr, p.Ala797Thr, p.Glu1356Lys, p.Arg663His) accounting for two-thirds of cases. HCM patients had a mean age at diagnosis of 40.4±18.1 years and 49% were male. Baseline maximal wall thickness (MWT) was 18.6±0.8 mm, left atrial diameter (LAD) was 41.3±9.4 mm, and 23% exhibited resting left ventricular outflow tract obstruction (LVOTO); 68% had abnormal ECGs, but only one patient had atrial fibrillation (AF). Probands showed significantly larger LAD than relatives (p=0.004). A history of premature familial sudden cardiac death (SCD) was reported in 43% families. During a median follow-up of 9.5 years (IQR 3.4-24.7, range 0.2-46.8 years), 35% of patients reached the composite endpoint, including 16 deaths (1 SCD) and 6 heart failure (HF) -related hospitalisations; 30% of patients developed AF, 17% received an ICD for primary prevention of SCD and 17% had a pacemaker implantation. Older age at diagnosis (p<0.01) and increased LAD (p=0.048) predicted poorer outcomes. The composite endpoint was similar between probands and relatives with HCM (p=0.08) and across the main variants (p=0.06). Overall penetrance was 70%, with only one carrier progressing to mild HCM. ESC HCM Risk SCD scores were similar across variants, both at baseline (p=0.601) and at last follow-up (p=0.286). CONCLUSIONS Most patients with MYH7-related HCM presented with a benign phenotype over the long term. Nonetheless, the risks of AF, SCD, and worsening HF throughout life justify regular monitoring, and the need to look for particular genetic profiles that may potentially help tailored management strategies.

Whole-exome sequencing identifies rare genetic variants in Egyptian patients with hypertrophic cardiomyopathy: a pilot study.

As the first genetic study of hypertrophic cardiomyopathy in an Egyptian population, this work expands the global mutational spectrum, demonstrates the utility of genetic testing for risk stratification and personalised management, and underscores the need to diversify genomic datasets for equitable precision medicine.

Network-based stratification of allele-specific expression reveals patient subgroups in Huntington’s disease

Abstract Motivation Huntington’s disease (HD) exhibits substantial variability in age of onset and disease progression that is not fully explained by CAG repeat length alone. Part of this residual variation is heritable, implicating additional genetic mechanisms. cis-regulatory variation, genetic variants that alter transcription and splicing of nearby genes, represents one such mechanism that can be quantified through allele-specific expression (ASE) analysis. However, methods for integrating ASE profiles into patient stratification frameworks remain underdeveloped, particularly for rare diseases with small cohorts and sparse data. Results We adapt a network-based stratification algorithm, originally developed for somatic tumour mutations, to ASE data. By propagating gene-level ASE imbalance profiles through a protein-protein interaction network, we stratified 20 HD patients into three distinct biological patient subgroups. Differential gene expression analysis highlights neuroinflammatory pathways, including microglial activation, immune cell activation, and cytokine regulation, as key sources of inter-patient heterogeneity, while differential ASE analysis implicates proteasomal and ubiquitin-dependent protein catabolic processes, immune activation, and central nervous system development. Intersection of differentially imbalanced and expressed genes identified FAM181B as a candidate gene with potential eQTL-mediated regulation, supported by independent cis-eQTL evidence for rs3780 in the caudate and putamen, the primary HD-affected striatal regions. FAM181B encodes a nuclear protein expressed in neural tissues acting as an interactor of the Hippo pathway TEAD transcription factors, implicating transcriptional regulatory variation as a potential contributor to molecular heterogeneity between patient subgroups. Differences in cortical and striatal neuropathological scores between clusters, even when adjusted for CAG repeat length, provide clinical support for the biological relevance of the identified subgroups. Availability All analysis code, Docker containers, and conda environments are available at https://github.com/macsbio/HD-ASE-NBS.

AKT1/mTOR/RICTOR risk variants in Indian hypertrophic cardiomyopathy patients