Genetic dysfunction of MT-ATP6 causes axonal Charcot-Marie-Tooth disease

Abstract

Objective: Charcot-Marie-Tooth (CMT) disease is the most common inherited neuromuscular disorder, affecting 1 in 2,500 individuals. Mitochondrial DNA (mtDNA) mutations are not generally considered within the differential diagnosis of patients with uncomplicated inherited neuropathy, despite the essential requirement of ATP for axonal function. We identified the mtDNA mutation m.9185T>C in MT-ATP6, encoding the ATP6 subunit of the mitochondrial ATP synthase (OXPHOS complex V), at homoplasmic levels in a family with mitochondrial disease in whom a severe motor axonal neuropathy was a striking feature. This led us to hypothesize that mutations in the 2 mtDNA complex V subunit encoding genes, MT-ATP6 and MT-ATP8, might be an unrecognized cause of isolated axonal CMT and distal hereditary motor neuropathy (dHMN).

Methods: A total of 442 probands with CMT type 2 (CMT2) (270) and dHMN (172) were screened for MT-ATP6/8 mutations after exclusion of mutations in known CMT2/dHMN genes. Mutation load was quantified using restriction endonuclease analysis. Blue-native gel electrophoresis (BN-PAGE) was performed to analyze the effects of m.9185T>C on complex V structure and function.

Results: Three further probands with CMT2 harbored the m.9185T>C mutation. Some relatives had been classified as having dHMN. Patients could be separated into 4 groups according to their mutant m.9185T>C levels. BN-PAGE demonstrated both impaired assembly and reduced activity of the complex V holoenzyme.

Conclusions: We have shown that m.9185T>C in MT-ATP6 causes CMT2 in 1.1% of genetically undefined cases. This has important implications for diagnosis and genetic counseling. Recognition that mutations in MT-ATP6 cause CMT2 enhances current understanding of the pathogenic basis of axonal neuropathy.

Figure 1. Pedigrees of 4 unrelated families harboring the m.9185T>C mutation in MT-ATP6

Filled symbols indicate individuals with Charcot-Marie-Tooth type 2 (CMT2), dark gray shaded symbols indicate individuals with upper motor neuron signs only, light gray shaded symbols indicate individuals with unknown phenotype, square symbols indicate male gender, round symbols indicate female gender, diamond symbols indicate gender unknown, numbers in symbols indicate multiple individuals, symbols with slashes indicate deceased, a small square with a slash indicates still birth, a small triangle indicates miscarriage, asterisks indicate affected individuals not examined by the authors, and % = percentage m.9185T>C mutant load detected in each patient. b = blood; u = urine; f = fibroblasts; m = muscle; yo = year old.

Figure 2

Photograph demonstrating distal lower limb muscle wasting and pes cavus in a patient with the m.9185T>C mutation in MT-ATP6 (family A, patient III-8)

Figure 3. Blue-native polyacrylamide gel electrophoresis was performed on muscle tissue from 4 patients with the m.9185T>C mutation and 1 patient with the m.8993T>G mutation

P = patient; C = control; V (Fo + F₁) = complex V holoenzyme; F₁ = F₁ catalytic site of complex V only. *Reduced complex V activity in patient compared with control muscle tissue (demonstrated by reduced band density). **Impaired complex V assembly in patient compared with control muscle tissue (demonstrated by multiple bands indicative of abnormal assembly intermediates). */**Both impaired complex V activity and assembly in patient compared to control muscle tissue (demonstrated by coexistence of reduced band density and multiple bands indicative of abnormal assembly intermediates). (A) Patient with the pathogenic mutation m.8993T>G (positive control). (B–D) Family A, patients III-5, III-6, and IV-2, respectively. (E) Family B, patient III-6.