RESUMO
MSTO1 encodes a cytosolic mitochondrial fusion protein, misato homolog 1 or MSTO1. While the full genotype-phenotype spectrum remains to be explored, pathogenic variants in MSTO1 have recently been reported in a small number of patients presenting with a phenotype of cerebellar ataxia, congenital muscle involvement with histologic findings ranging from myopathic to dystrophic and pigmentary retinopathy. The proposed underlying pathogenic mechanism of MSTO1-related disease is suggestive of impaired mitochondrial fusion secondary to a loss of function of MSTO1. Disorders of mitochondrial fusion and fission have been shown to also lead to mitochondrial DNA (mtDNA) depletion, linking them to the mtDNA depletion syndromes, a clinically and genetically diverse class of mitochondrial diseases characterized by a reduction of cellular mtDNA content. However, the consequences of pathogenic variants in MSTO1 on mtDNA maintenance remain poorly understood. We present extensive phenotypic and genetic data from 12 independent families, including 15 new patients harbouring a broad array of bi-allelic MSTO1 pathogenic variants, and we provide functional characterization from seven MSTO1-related disease patient fibroblasts. Bi-allelic loss-of-function variants in MSTO1 manifest clinically with a remarkably consistent phenotype of childhood-onset muscular dystrophy, corticospinal tract dysfunction and early-onset non-progressive cerebellar atrophy. MSTO1 protein was not detectable in the cultured fibroblasts of all seven patients evaluated, suggesting that pathogenic variants result in a loss of protein expression and/or affect protein stability. Consistent with impaired mitochondrial fusion, mitochondrial networks in fibroblasts were found to be fragmented. Furthermore, all fibroblasts were found to have depletion of mtDNA ranging from 30 to 70% along with alterations to mtDNA nucleoids. Our data corroborate the role of MSTO1 as a mitochondrial fusion protein and highlight a previously unrecognized link to mtDNA regulation. As impaired mitochondrial fusion is a recognized cause of mtDNA depletion syndromes, this novel link to mtDNA depletion in patient fibroblasts suggests that MSTO1-deficiency should also be considered a mtDNA depletion syndrome. Thus, we provide mechanistic insight into the disease pathogenesis associated with MSTO1 mutations and further define the clinical spectrum and the natural history of MSTO1-related disease.
Assuntos
Proteínas de Ciclo Celular/genética , Doenças Cerebelares/genética , Proteínas do Citoesqueleto/genética , DNA Mitocondrial , Doenças Mitocondriais/genética , Distrofias Musculares/genética , Mutação , Adolescente , Adulto , Atrofia , Células Cultivadas , Doenças Cerebelares/diagnóstico por imagem , Doenças Cerebelares/patologia , Doenças Cerebelares/fisiopatologia , Criança , Variações do Número de Cópias de DNA , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Doenças Mitocondriais/diagnóstico por imagem , Doenças Mitocondriais/patologia , Doenças Mitocondriais/fisiopatologia , Músculos/patologia , Distrofias Musculares/diagnóstico por imagem , Distrofias Musculares/patologia , Distrofias Musculares/fisiopatologia , Fenótipo , Adulto JovemRESUMO
The urea cycle protects the central nervous system from ammonia toxicity by converting ammonia to urea. N-acetylglutamate synthase (NAGS) catalyzes formation of N-acetylglutamate, an essential allosteric activator of carbamylphosphate synthetase 1. Enzymatic activity of mammalian NAGS doubles in the presence of L-arginine, but the physiological significance of NAGS activation by L-arginine has been unknown. The NAGS knockout (Nags-/-) mouse is an animal model of inducible hyperammonemia, which develops hyperammonemia without N-carbamylglutamate and L-citrulline supplementation (NCG + Cit). We used adeno associated virus (AAV) based gene transfer to correct NAGS deficiency in the Nags-/- mice, established the dose of the vector needed to rescue Nags-/- mice from hyperammonemia and measured expression levels of Nags mRNA and NAGS protein in the livers of rescued animals. This methodology was used to investigate the effect of L-arginine on ureagenesis in vivo by treating Nags-/- mice with AAV vectors encoding either wild-type or E354A mutant mouse NAGS (mNAGS), which is not activated by L-arginine. The Nags-/- mice expressing E354A mNAGS were viable but had elevated plasma ammonia concentration despite similar levels of the E354A and wild-type mNAGS proteins. The corresponding mutation in human NAGS (NP_694551.1:p.E360D) that abolishes binding and activation by L-arginine was identified in a patient with NAGS deficiency. Our results show that NAGS deficiency can be rescued by gene therapy, and suggest that L-arginine binding to the NAGS enzyme is essential for normal ureagenesis.