Expanding the molecular and phenotypic spectrum of truncating MT-ATP6 mutations

Abstract

Objective: To describe the clinical and functional consequences of 1 novel and 1 previously reported truncating MT-ATP6 mutation.

Methods: Three unrelated probands with mitochondrial encephalomyopathy harboring truncating MT-ATP6 mutations are reported. Transmitochondrial cybrid cell studies were used to confirm pathogenicity of 1 novel variant, and the effects of all 3 mutations on ATPase 6 and complex V structure and function were investigated.

Results: Patient 1 presented with adult-onset cerebellar ataxia, chronic kidney disease, and diabetes, whereas patient 2 had myoclonic epilepsy and cerebellar ataxia; both harbored the novel m.8782G>A; p.(Gly86*) mutation. Patient 3 exhibited cognitive decline, with posterior white matter abnormalities on brain MRI, and severely impaired renal function requiring transplantation. The m.8618dup; p.(Thr33Hisfs*32) mutation, previously associated with neurogenic muscle weakness, ataxia, and retinitis pigmentosa, was identified. All 3 probands demonstrated a broad range of heteroplasmy across different tissue types. Blue-native gel electrophoresis of cultured fibroblasts and skeletal muscle tissue confirmed multiple bands, suggestive of impaired complex V assembly. Microscale oxygraphy showed reduced basal respiration and adenosine triphosphate synthesis, while reactive oxygen species generation was increased. Transmitochondrial cybrid cell lines studies confirmed the deleterious effects of the novel m.8782 G>A; p.(Gly86*) mutation.

Conclusions: We expand the clinical and molecular spectrum of MT-ATP6-related mitochondrial disorders to include leukodystrophy, renal disease, and myoclonic epilepsy with cerebellar ataxia. Truncating MT-ATP6 mutations may exhibit highly variable mutant levels across different tissue types, an important consideration during genetic counseling.

Figure 1. Pedigrees and brain MRI findings

(A.a) Family pedigree chart of patient 1 (P1) harboring m.8782 G>A; p.(Gly86*) mutation. Brain MRI showing cerebellar atrophy (red arrowhead, A.b) and multiple deep and periventricular white matter changes (red arrowheads, A.c). (B.a) Family pedigree chart of patient 2 (P2) harboring m.8782G>A; p.(Gly86*) mutation. Brian MRI showing reduced brain volume, marked global cerebellar, and brainstem atrophy (red arrowhead, B.b) and multiple deep and periventricular white matter changes (red arrowheads, B.c). (C.a) Family pedigree chart of patient 3 (P3) harboring m.8618dup; p.(Thr33Hisfs*32) mutation. Brain MRI showing severe cerebellar atrophy (red arrowhead, C.b) and posterior white matter abnormalities (red arrowheads, C.c). Electron microscopy showing several mitochondria with simplified internal structure (C.d). High magnification (C.e) showing a mitochondria with aberrant cristae formation. Scale bar represents 200 nm. Filled symbols indicate affected individuals. Symbols with diagonal strikethrough indicate deceased. Arrows indicate probands. Mutation load detectable in different tissues: B = blood; F = fibroblasts; M = muscle; U = urine.

Figure 2. One-dimensional and 2-dimensional BNGE

(A and B) Immunovisualization of complex V in 1-dimensional BNGE of enriched mitochondria fractions extracted from fibroblasts and muscles and solubilized with DDM. Control (C), patient 1 (P1), patient 2 (P2), and patient 3 (P3) are shown. Three F1 x, y, and z subcomplexes are present in fibroblasts of P1 and P3, whereas only 2 of 3 subassemblies are present in the muscle of P3 and P2. See discussion for details. Anti-ATP5A and anti-COXIV used to visualize complex V and complex IV, respectively. (C and D) Denaturing 2-dimensional BNGE of enriched mitochondria fractions extracted from fibroblasts and muscle and solubilized with DDM confirmed the presence of ATP synthase subcomplexes in P1, P2, and P3. Residual ATP6 protein is found incorporated into the fully assembled complex V in P1, P2, and P3. (E) Western blot of muscle samples show reduced ATP6 protein in patients (P2, P3) compared with the control. (F) Densitometry analysis of (E) performed in 2 different experiments. Values are normalized to controls. Error bars represent SEM. ATP = adenosine triphosphate; BNGE = blue-native gel electrophoresis; CIV = complex IV; COXIV = cytochrome c oxidase IV; CTR = control; CV = complex V; DDM = n-dodecyl-β-d-maltoside; MW = molecular weight; SDS-PAGE = sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SEM = standard error of the mean; VDAC1 = voltage dependent anion channel 1.

Figure 3. Microscale oxygraphy and reactive oxygen species measurement

(A) Representative graph illustrating the protocol used to measure mitochondrial respiration in fibroblasts using a XF96 extracellular flux analyzer (Seahorse Bioscience). The data represent the outcome of an experimental run before normalization. Bar charts showing (B) basal respiration, (C) maximal Respiratory capacity, and (D) ATP production. Data are the average of 3 biological replicates, n = 30–50 measurements. OCR were normalized to the number of cells and expressed as percentage of control. Error bars represent SEM, ***p < 0.0005, ****p < 0.0001 by unpaired Student t test. (E) Linear regression of the time course analysis of reactive oxygen species production measured by DCFDA in cultured fibroblasts. Error bars represent SEM, statistical analysis performed by paired t test. Color code as in A. ANT = antimycin; FCCP = carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone; ns = nonsignificant; OCR = oxygen consumption rates; OLIGO = oligomycin; ROT = rotenone; SEM = standard error of the mean.