Abstract 4139615: X-linked LAMP2B p.Val397Ile variant leads to late-onset Danon disease in females with hypertrophic cardiomyopathy

Introduction: Danon disease is an X-linked dominant disorder in lysosome-associated membrane protein 2B (LAMP2B) that presents with cardiomyopathy and in males, intellectual disability. Dysfunction of LAMP2B, a lysosomal transmembrane protein in muscle, prevents macroautophagy and causes intracytoplasmic accumulation of cellular debris. Loss-of-function mutations in LAMP2B manifest as full Danon disease while missense mutations drive milder phenotypes. There is currently no effective therapy. Existing literature reports two transmembrane missense mutations, which presented as late-onset cardiac hypertrophy, but do not explore mutation pathogenicity. In our work, we investigate the mechanism of a novel transmembrane missense variant p.Val397Ile in two unrelated females who presented with late-onset hypertrophic obstructive cardiomyopathy with patient myectomy sample. Methods: Myectomy heart tissue was analyzed via Western blot and RT-qPCR for protein and RNA levels. In vitro overexpression of reference and alternate missense alleles was conducted in HEK293 cells, then analyzed by Western blot, RT-qPCR, and immunofluorescence. Immunofluorescence image analysis compared LAMP2B signal intensity and distribution between variant and wild-type proteins. Results: Patient heart tissue showed increased LAMP2 degradation and decreased autophagy markers by Western blot. EndoH treatment to investigate N- linked glycosylation patterns further revealed decreased total LAMP2 in patient samples. RT-qPCR showed increased RNA expression in the variant allele. LAMP2 overexpression in HEK293 cells resulted in reduced LAMP2B molecular weight and subcellular mislocalization. Fluorescent image segmentation revealed increased aggregation of variant LAMP2B around nuclei and surrounding structures compared to wild-type. Collectively, these results suggest arrested protein maturation in the Golgi apparatus and increased RNA expression to compensate for reduced functional protein. Conclusion: Our study demonstrated that the LAMP2B missense variant c.1189G>A p.Val397Ile is implicated with increased degradation of LAMP2B protein, reduced protein levels of autophagy markers, and elevated LAMP2B RNA expression in cardiomyocytes. Our in vitro studies suggest that missense mutations may cause myocardial dysfunction via abnormal LAMP2B maturation and localization. Our work enhances mechanistic understanding of LAMP2B-related pathogenicity and may inform future therapeutic strategies for Danon disease.