P033: Mitochondrial copy number in undiagnosed and rare monogenic diseases

Madelyn Klemmensen; Moon Ley Tung; Bharatendu Chandra

doi:10.1016/j.gimo.2025.102877

Introduction Mitochondrial disorders are highly heterogeneous in clinical presentation and are often associated with long diagnostic odysseys. One of the major challenges in establishing a diagnosis of mitochondrial disorder is the lack of clinically reliable biomarkers and heteroplasmy for these disorders. In addition, several genetic disorders could indirectly affect mitochondrial function resulting in secondary mitochondrial dysfunction. Mitochondrial DNA copy number variants (mtDNA CNV) could serve as a surrogate for mitochondrial function in a given cell type, and mtCNV assays can be used to detect mitochondrial dysfunction. These assays have been validated in muscle biopsy tissue in individuals with mitochondrial cytopathies. Given the invasive nature of muscle biopsy, it is not feasible to perform these assays on muscle biopsy as a first-tier test in individuals suspected with mitochondrial disorder. However, skin punch biopsy is a relatively safe and less invasive procedure, and cultured fibroblasts derived from the biopsy tissue are an inexhaustible source for ex-vivo studies in patients with suspected genetic or mitochondrial disorders. In this study, our aim was to look into mtDNA CNV in a rare disease cohort from our institution consisting of individuals with undiagnosed diseases and rare Mendelian diseases with established molecular diagnosis. The purpose of this study was to determine the mtDNA CNV abnormalities in individuals with both undiagnosed and monogenic genetic diseases, while excluding individuals with known primary mitochondrial disorders. In particular, we wanted to test the correlation of mtCNV with Age, clinical phenotype, genotype (for rare disease with established diagnosis, and ancillary mitochondrial assays (GDF-15, muscle biopsy, and mitochondrial functional assays, where available). Methods A total of 28 individuals from our rare diseases biorepository, with available cultured skin fibroblast were included for this study. Three samples were excluded from the final analysis due to poor quality metrics for the extracted DNA. Of the final 25 included for the analysis, 10/25 were individuals with undiagnosed diseases. All the undiagnosed participants had negative mtDNA and nuclear DNA (nuDNA) genome sequencing. Whereas 15/25 individuals were individuals with established monogenic disorders due to genes not implicated in primary mitochondrial disorders (eg, GATA2, SHANK3 and TUBB3). Four apparently healthy fibroblast lines were used as controls. Total genomic DNA (containing mtDNA and nuDNA) was extracted and normalized to 1ng/ul. A quantitative-PCR based assay was performed using two unique primers targeting two mtDNA genes, and another two unique primers targeting two of the nuclear DNA (nuDNA) genes. Results Our results showed that mitochondrial DNA copy number was significantly reduced in all the participants with undiagnosed disease (Median 362.6, IQR 329.7-459.6) and monogenic rare disease (Median 360.7, IQR 233.5-434.2), when compared with controls (Median 967.65, IQR 610.5-1283.15). The lowest average CNV (23.3) was noted to be in a patient with an underlying Tubulinopathy, whereas the highest average CNV (604) was seen in a patient with an underlying monogenic telomere disorder. Interestingly, one of the control fibroblast line had a relatively low average CNV of 300.1, while the remaining three control lines had an average CNV of 920.9, 1014.4, and 1551.9, respectively. Our results did not reveal any significant difference in mitochondrial copy number in relation to sex and age of the individuals with undiagnosed and monogenic rare diseases in this cohort. Conclusion In this study, we found significantly lower mt-DNA CNV in complex undiagnosed diseases and rare monogenic disorders. This could be due to impaired mitochondrial biogenesis as a direct or indirect consequence of the underlying molecular perturbations associated with these disorders.

P033: Mitochondrial copy number in undiagnosed and rare monogenic diseases

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