NDUFB8 Mutations Cause Mitochondrial Complex I Deficiency in Individuals with Leigh-like Encephalomyopathy

Abstract

Respiratory chain complex I deficiency is the most frequently identified biochemical defect in childhood mitochondrial diseases. Clinical symptoms range from fatal infantile lactic acidosis to Leigh syndrome and other encephalomyopathies or cardiomyopathies. To date, disease-causing variants in genes coding for 27 complex I subunits, including 7 mitochondrial DNA genes, and in 11 genes encoding complex I assembly factors have been reported. Here, we describe rare biallelic variants in NDUFB8 encoding a complex I accessory subunit revealed by whole-exome sequencing in two individuals from two families. Both presented with a progressive course of disease with encephalo(cardio)myopathic features including muscular hypotonia, cardiac hypertrophy, respiratory failure, failure to thrive, and developmental delay. Blood lactate was elevated. Neuroimaging disclosed progressive changes in the basal ganglia and either brain stem or internal capsule. Biochemical analyses showed an isolated decrease in complex I enzymatic activity in muscle and fibroblasts. Complementation studies by expression of wild-type NDUFB8 in cells from affected individuals restored mitochondrial function, confirming NDUFB8 variants as the cause of complex I deficiency. Hereby we establish NDUFB8 as a relevant gene in childhood-onset mitochondrial disease.

Keywords: Leigh syndrome; NADH dehydrogenase; complex I; lactic acidosis; mitochondria; oxidative phosphorylation; respiratory chain.

Figure 1

Mutations Identified in NDUFB8 in Two Families (A) Compound heterozygous NDUFB8 mutations identified in two families. (B) Gene structure of NDUFB8 and localization of mutations. (C) Multiple alignment of NDUFB8 protein sequences from different species by Clustal Omega.

Figure 2

Brain Magnetic Resonance Imaging in Individual P1 Top row: MR brain examination at the age of 3 months; axial T2-weighted images; symmetrical increased signal intensity in the midbrain (A, black arrow), bilateral putamina and thalami (B, black arrows), sparing of the caudate heads (B, white arrow). Axial post contrast T1-weighted image (C, black arrows); patchy putamina and thalami enhancement. Bottom row: follow-up MRI at the age of 6 months: profound supratentorial brain and brainstem atrophy with large bilateral, chronic subdural hematomas (D, black arrow) and cystic lesions in the midbrain (E, black arrow) and in the bilateral putamina and thalami (F, black arrows).

Figure 3

Biochemical Investigations in Fibroblasts (A) Blue native gel electrophoresis of individual P2. Abbreviations: CI, complex I; CII, complex II; CIII, complex III; CIV, complex IV; CV, complex V; FCCP, carbonyl cyanide-4-(trifluoromethoxy)-phenylhydrazone. (B) Western blot analysis of fibroblasts from individual P1, which were complemented by lentiviral transduction with wild-type NDUFB8. C1–C3 represent control fibroblasts. 10 μg protein were loaded. VDAC1 (porin) was used as a loading control. An antibody against the NDUFS4 subunit of complex I was used. (C and D) Densitometric analysis of this western blot. Error bars indicate the standard error of the mean. (E) Blue native gel electrophoresis of mitochondrial membranes prepared from fibroblasts of P1, P2, and controls solubilized by laurylmaltoside. Normalization of the complex I was observed after lentiviral transduction with wild-type NDUFB8 (NDUFB8-T) in P1.

Figure 4

Microrespirometry and Flow Cytometry Analysis in Fibroblasts (A) Oxygen consumption of fibroblasts of individual P1 and P2 compared to a control either with or without lentiviral transduction with wild-type NDUFB8 (NDUFB8-T). At least 16 wells were analyzed per each sample, and at least two replicates were performed for each sample. Error bars indicate the standard deviation. (B and C) Results of representative flow cytometry analysis of fibroblasts from individual P1, P1 transduced with wild-type NDUFB8, P2, and a control are shown. An antibody against the NDUFS4 subunit was used as marker for complex I (B), an antibody against VDAC1 (porin) as a control for mitochondria (C). Positive staining of primary antibodies was evaluated with suitable isotype controls. Mean fluorescence intensity (MFI) of complex I: Control = 8.59; P1 = 2.52; P1-NDUFB8-T = 7.59, P2 = 2.84. MFI of porin: Control = 22.15; P1 = 15.71; P1-NDUFB8 = 16.16, P2 = 22.36. Flow cytometry analysis of each sample was performed at least two times.

Figure 5

Immunofluorescence Staining of Complex I and Porin Fibroblasts, transduced fibroblasts (NDUFB8-T) of individual P1, P2, and a control cell line were stained with antibodies against complex I subunit NDUFS4 (Abcam, 1:100) in green and VDAC1 (Abcam; 1:400), mitochondrial marker protein in red. The merge of staining is shown; all images were taken with the same microscope settings as used for the control (scale bar = 10 μm).