O03: Hydroxocobalamin (OHCbl) dose intensification can prevent visual deterioration and improve neurological and biochemical outcomes in CBLC deficiency

Introduction: Large deletions in multi-copy mitochondrial DNA (mtDNA) are associated with chronic progressive external ophthalmoplegia (CPEO), Kearns-Sayre syndrome (KSS), and Pearson syndrome (PS), collectively known as single large-scale mtDNA deletion syndromes (SLSMDs).CPEO and KSS present with a spectrum of symptoms including ptosis, ophthalmoplegia, and mitochondrial myopathy, whereas PS is characterized by early-onset anemia or pancytopenia in infancy, along with multisystem involvement.SLSMDs often arise from sporadic single large deletions in the mitochondrial genome and are present in a heteroplasmic state.While the level of heteroplasmy and tissue distribution of the deletions are reported to be determining factors for disease severity, a clear mechanism behind the genotype-phenotype correlation has not been previously documented.Methods: To study the functional changes in muscles with a mtDNA deletion, we retrospectively reviewed clinical cases that included both electron transport chain (ETC) functional testing and mtDNA genome sequencing results that were assayed from the same muscle specimen.In ETC assays, the activities of all ETC complexes except complex V ATP synthase were measured using specific electron donors and acceptors.Citrate synthase (CS) activity was also determined and used as a calibrator of individual differences in mitochondrial copy number or tissue preparation.Each respiratory chain enzyme activity was normalized to CS activity.The mtDNA deletions were detected by next generation sequencing (NGS) analysis of long-range PCR products of the mitochondrial genome and confirmed using either Southern blot (SB) or multiplex ligation-dependent amplification (MLPA) analysis.The deletion breakpoints were based on NGS results and the heteroplasmy levels were estimated by SB or MLPA.Patient medical histories and other available testing results were also included in the analysis.Results: In this study, we reviewed 973 consecutive cases with both ETC functional testing and mtDNA genome sequencing results.Among these, 39/973 (4.0%) cases exhibited single large mtDNA deletions.We further identified 14/973 (1.4%) cases with clinical indications consistent with the CPEO/KSS spectrum.Each of these participants presented with ptosis and/or ophthalmoplegia, along with additional CPEO/KSS-related features.No cases with PS were identified, aligning with the fact that this syndrome typically does not initially present with muscle involvement.Of the 14 CPEO/KSS cases, 7 exhibited deficient ETC complex enzyme activity (defined as <30% based on modified Walker minor criteria) before or after CS activity correction.Notably, all 7 participants had low level heteroplasmy (<10%) for the mtDNA deletions.Among them, 2 showed defects in a single respiratory chain complex, while the other 5 had defects across multiple complexes.Complex IV was most commonly impacted, being reduced in 5 samples, while other complexes were impaired in at least 2 samples.There was no observed correlation between the specific affected complexes, level of heteroplasmy, or mtDNA deletion size.As expected, the activity of nuclear-encoded complex II remained intact in all samples.Conclusion: This study demonstrates that the approach of long-range PCR plus NGS can detect low-heteroplasmy mtDNA deletions.Furthermore, muscle functional studies of ETC enzyme activities can provide further support for mtDNA deletion syndromes.These results show that mitochondrial function can be significantly compromised even when mtDNA deletion heteroplasmy percentage is well below the expected deficient thresholds, providing strong support for the pathogenicity of the detected deletions.These findings emphasize the need for comprehensive molecular and functional evaluations in making a SLSMD diagnosis.