Pathological variants in TOP3A cause distinct disorders of mitochondrial and nuclear genome stability

**Background:** TOP3A is a dual-localized topoisomerase that functions in both the nucleus (as part of the BTRR complex involved in DNA repair and chromosome segregation) and mitochondria (where it resolves hemicatenated mtDNA replication products). Pathogenic variants in TOP3A have been associated with Bloom syndrome-like disorder (nuclear genome instability) and, in a single prior case, adult-onset mitochondrial disease. The factors determining which clinical phenotype manifests are poorly understood.

**Methods:** The authors identified 11 individuals from 9 families with bi-allelic TOP3A variants using whole-genome or whole-exome sequencing. Clinical data were collected, and muscle biopsies from 4 patients were analyzed by COX-SDH histochemistry, quadruple immunofluorescence for respiratory chain subunits, long-range PCR for mtDNA deletions, and deep sequencing with the MitoSAlt pipeline to map mtDNA rearrangements. Southern blotting assessed mtDNA structure. Cellular rescue experiments were performed in U2OS Flp-In cells depleted of endogenous TOP3A by siRNA and expressing inducible siRNA-resistant wild-type or variant TOP3A; mtDNA copy number (by qPCR) and topology (by Southern blot) were measured. Recombinant TOP3A proteins (wild-type and 8 missense variants plus a truncation variant) were purified from Sf9 insect cells and assayed for DNA binding (EMSA with 80-nt ssDNA), relaxation of negatively supercoiled pUC19 plasmid, and decatenation of synthetic Lk1 ssDNA catenanes.

**Key Results:** Seven of 11 patients presented in the 5th or 6th decade; 4 women presented in adolescence/early 20s. Common features included bilateral ptosis/ophthalmoplegia (9/11), sensorineural hearing loss (7/11), myopathy (7/11), axonal sensorimotor neuropathy (8/11), cerebellar ataxia (7/11), and cardiac conduction defects requiring pacemaker (5/11). Muscle biopsies showed mosaic COX deficiency and multiple mtDNA deletions by long-range PCR. Deep sequencing revealed abundant major arc deletions with breakpoints near the termination-associated sequence, and in one patient (Pa2), small duplications in the non-coding region. Southern blotting showed high-molecular-weight hemicatenated mtDNA in Pa2 and Pa5-1. In cellular rescue experiments, TOP3A depletion caused ~75% loss of mtDNA copy number, which was fully rescued by wild-type TOP3A and by Leu37Val, Ala95Val, and Met100Val variants, but not by other variants. Hemicatenated mtDNA accumulation was eliminated by wild-type TOP3A but persisted with Leu37Val, Arg558Trp, Ala176Val, and Ser810* variants. In vitro, Leu37Val and Met575Val showed severely impaired DNA binding (EMSA). All variants except Met100Val showed markedly reduced relaxation activity. In decatenation assays, Leu37Val, Met575Val, and Ala176Val showed significant loss of activity, while Ala95Val, Met100Val, Arg103Gln, Arg558Trp, and Ser810* retained activity comparable to wild-type. Compound heterozygous combinations showed intermediate activity.

**Clinical Implications:** This study establishes TOP3A as a mitochondrial disease gene associated with adult-onset progressive external ophthalmoplegia (PEO) and multisystem involvement. The data support a model where variants causing moderate enzymatic impairment lead to mitochondrial disease, while severe loss causes Bloom syndrome with mitochondrial features. The findings underscore the importance of including TOP3A in diagnostic gene panels for mitochondrial disorders and highlight the need to consider dual-localized proteins in genetic diagnostics.