X-Linked Myotubular Myopathy and Mitochondrial Function in Muscle and Liver Samples.

X-linked myotubular myopathy (XLMTM) is a rare congenital myopathy that commonly manifests with liver involvement. In most XLMTM cases, disease-causing variants have been identified in the myotubularin gene (MTM1) on chromosome Xq28, which encodes myotubularin protein (MTM1). The impairment of mitochondrial respiratory chain (MRC) enzyme activity in muscle has been observed in the XLMTM mouse model. Though several reports mentioned possible mechanisms of liver involvement in XLMTM patients and animal models, the precise underlying mechanisms remain unknown, and there is no report focused on mitochondrial functions in hepatocytes in XLMTM. We encountered two patients with XLMTM who had liver involvement. We measured MRC enzyme activities in two muscle biopsy specimens, and one liver specimen from our patients to investigate whether MTM1 variants cause MRC dysfunction and whether mitochondrial disturbance is associated with organ dysfunction. MRC enzyme activities decreased in skeletal muscles but were normal in the liver. In our patients, the impaired MRC enzyme activity found in muscle is consistent with previously reported mechanisms that the loss of MTM1-desmin intermediate filament and MTM1-IMMT (a mitochondrial membrane protein) interaction led to the mitochondrial dysfunction. However, our study showed that liver involvement in XLMTM may not be associated with mitochondrial dysfunction.

Identification of a novel MT-ND3 variant and restoring mitochondrial function by allotopic expression of MT-ND3 gene.

Mitochondrial diseases are caused by nuclear, or mitochondrial DNA (mtDNA) variants and related co-factors. Here, we report a novel m.10197G > C variant in MT-ND3 in a patient, and two other patients with m.10191 T > C. MT-ND3 variants are known to cause Leigh syndrome or mitochondrial complex I deficiency. We performed the functional analyses of the novel m.10197G > C variant that significantly lowered MT-ND3 protein levels, causing complex I assembly and activity deficiency, and reduction of ATP synthesis. We adapted a previously described re-engineering technique of delivering mitochondrial genes into mitochondria through codon optimization for nuclear expression and translation by cytoplasmic ribosomes to rescue defects arising from the MT-ND3 variants. We constructed mitochondrial targeting sequences along with the codon-optimized MT-ND3 and imported them into the mitochondria. To achieve the goal, we imported codon-optimized MT-ND3 into mitochondria in three patients with m.10197G > C and m.10191 T > C missense variants in the MT-ND3. Nuclear expression of the MT-ND3 gene partially restored protein levels, complex I deficiency, and significant improvement of ATP production indicating a functional rescue of the mutant phenotype. The codon-optimized nuclear expression of mitochondrial protein and import inside the mitochondria can supplement the requirements for ATP in energy-deficient mitochondrial disease patients.