Pathogenic Bi-allelic Mutations in NDUFAF8 Cause Leigh Syndrome with an Isolated Complex I Deficiency

Abstract

Leigh syndrome is one of the most common neurological phenotypes observed in pediatric mitochondrial disease presentations. It is characterized by symmetrical lesions found on neuroimaging in the basal ganglia, thalamus, and brainstem and by a loss of motor skills and delayed developmental milestones. Genetic diagnosis of Leigh syndrome is complicated on account of the vast genetic heterogeneity with >75 candidate disease-associated genes having been reported to date. Candidate genes are still emerging, being identified when "omics" tools (genomics, proteomics, and transcriptomics) are applied to manipulated cell lines and cohorts of clinically characterized individuals who lack a genetic diagnosis. NDUFAF8 is one such protein; it has been found to interact with the well-characterized complex I (CI) assembly factor NDUFAF5 in a large-scale protein-protein interaction screen. Diagnostic next-generation sequencing has identified three unrelated pediatric subjects, each with a clinical diagnosis of Leigh syndrome, who harbor bi-allelic pathogenic variants in NDUFAF8. These variants include a recurrent splicing variant that was initially overlooked due to its deep-intronic location. Subject fibroblasts were found to express a complex I deficiency, and lentiviral transduction with wild-type NDUFAF8-cDNA ameliorated both the assembly defect and the biochemical deficiency. Complexome profiling of subject fibroblasts demonstrated a complex I assembly defect, and the stalled assembly intermediates corroborate the role of NDUFAF8 in early complex I assembly. This report serves to expand the genetic heterogeneity associated with Leigh syndrome and to validate the clinical utility of orphan protein characterization. We also highlight the importance of evaluating intronic sequence when a single, definitively pathogenic variant is identified during diagnostic testing.

Keywords: NDUFAF8; complex I deficiency; mitochondrial disease; molecular diagnosis.

Figure 1

Neuroimaging of Subjects Harbouring Bi-Allelic Pathogenic NDUFAF8 Variants (A and B) Subject 1 at 5 months of age. (C and D) Subject 2 at 2 years, 9 months of age. (E and F) Subject 3 at 1 year, 2 months of age. Sagittal T1-weighted imaging (T1WI) (A, C, and E) demonstrates dysmorphic corpus callosum (arrows): For subjects 1 and 2, no splenium is present (A and C), and only a small posterior part of the splenium is present in subject 3 (E). Sagittal T1WI (C and E) reveals signal abnormality in the dorsal brainstem corresponding to areas with restricted diffusion for subjects 2 and 3. Coronal T2-weighted imaging (B) of subject 1 demonstrates bilateral periventricular cysts and absent septum pellucidum (arrows). Axial T2-weighted imaging (D) depicts right frontal gray matter heterotopia in subject 2 (arrow). Axial diffusion-weighted imaging (F) shows bilateral symmetrical diffusion restriction in the putamina, thalami, and hippocampal tails in subject 3 (arrows).

Figure 2

Bi-Allelic NDUFAF8 Variants Are Identified in Three Unrelated Subjects Respiratory chain enzyme analysis reveals a marked isolated complex I deficiency in subject 1’s skeletal muscle biopsy (shaded blue) compared to controls (shaded red) (A); this finding is recapitulated in the fibroblasts from subject 1 (B). Mean enzyme activities of muscle controls (n = 25) and fibroblast controls (n = 10) are set to 100%, with error bars representing standard deviation. The asterisk denotes a significant loss of enzyme activity. (C) Quadruple immunofluorescent histochemical analysis of skeletal muscle biopsy from subject 1 demonstrates reduced levels of NDUFB8 (complex I) in the majority of single muscle fibers relative to those of the marker protein (porin), and expression of COX1 (complex IV) is preserved. Each dot represents a single muscle fiber. Black dashed lines represent the SD limits for the classification of the fibers. The X- and Y-axes represent the expression of NDUFB8 and COX-1: normal (< −1), intermediate +ve (−1 to −2 SD), intermediate −ve (−2 to −3 SD), and deficient (> −3 SD). The mean expression level of normal fibers is denoted a value of 0. Dots that fall outwith the solid box at -3 SD are strongly deficient fibres. Dots are color coded according to their mitochondrial mass (very low, blue; normal, beige; very high, red). (D) Family pedigrees of subjects 1, 2, and 3 and corresponding recessive NDUFAF8 variants; compound heterozygous c.45_52dup (p.Phe18Serfs^∗32) and c.195+271C>T (p.?) NDUFAF8 variants in subject 1; compound heterozygous c.1A>G (p.?) and c.195+271C>T (p.?) NDUFAF8 variants in subject 2 and a homozygous c.165C>G (p.Phe55Leu) NDUFAF8 variant in subject 3. (E) Sequencing chromatograms depict the c.45_52dup and c.195+271C>T variants present in genomic DNA from S1. (F) cDNA studies using RNA derived from subject 1 fibroblasts show that only the c.45_52dup allele is present at the mRNA level, supporting the possibility of degradation of the transcript associated with the c.195+271C > T variant. S1 = subject 1; C = wild-type control. Variant nomenclature is according to GenBank accession NM_001086521.1

Figure 3

BN-PAGE and Complementation Studies (A and B) Mitochondria isolated from cultured skin fibroblasts (A) and skeletal muscle (B) from subject 1 and age-matched control subjects were solubilized in n-dodecyl β-d-maltoside (DDM) and subjected to BN-PAGE and immunoblotting analysis using antibodies directed to various OXPHOS complexes (complex I: NDUFB8; complex II: SDHA; complex III: Core2; complex IV: COX1; complex V: ATP5A). A striking reduction in assembled complex I was apparent in both the skeletal muscle biopsy and fibroblast cell line from subject 1 in comparison to controls. C, Wild-type NDUFAF8 cDNA was generated and introduced into control and subject cell lines via lentiviral expression under the EF1α promoter. Enriched mitochondria were solubilized in DDM before BN-PAGE analysis. Immunoblotting using antibodies against the complex I subunit NDUFA9 (top) and the complex II subunit SDHA (bottom) as a loading control revealed less complex I in subject cell lines than in control cell lines. After transduction with NDUFAF8 cDNA, complex I levels were restored. D, SDS-PAGE and western blotting of whole fibroblast cell lysates from subject 1 transduced with wild-type NDUFAF8 cDNA (+FAF8) clearly demonstrates increased levels of NDUFAF5 relative to the empty vector (-FAF8) or untreated subject 1 fibroblasts (S1p), comparable to those observed in the healthy age matched control, C. E, Comparison of the complex 1 activity exhibited by subject 1 fibroblasts following transduction with either the empty vector (–FAF8) or wild-type NDUFAF8 mRNA vector (+FAF8) cell lines corroborates functional rescue of the biochemical phenotype. Experimental data shown are derived from the results of three technical replicate assays; error bars are at 1 standard deviation. P value calculated as by Student’s t test.

Figure 4

Complexome Profiling of Fibroblasts from Subject 1 Confirms Complex I Assembly Defect Enriched mitochondrial membranes derived from control (A) and subject 1 (B) fibroblasts were solubilized with digitonin, separated on native gradient gels, cut into 60 pieces and digested with trypsin. Peptides were analyzed by quantitative mass spectrometry. Intensity-based absolute quantification (IBAQ) values of subject set were normalized to the sum of all values from control dataset. For visualization in a heatmap, values were normalized to maximum appearance between both samples. There is a reduction in the functional respirasome (complex I/III₂/IV) (purple box) in subject fibroblasts and stalled assembly intermediates corresponding to P_D-a (blue box) and a partly-assembled Q module (orange box), compared to control fibroblast data. There is also an increase in “free” complex III (gray box). Complex I subunits are presented according to their ascribed module, N-module (N), Q-module (Q), proximal P-module (P_P-a containing ND1, P_P-b containing ND2/3/6), distal P-module (P_D-a containing ND4, P_D-b containing ND5) and assembly factors (A). Color-matched dashed boxes are used to denote the corresponding regions of interest in the control data (Panel A). Subcomplex denotations are according to Guerrero-Castillo and colleagues C. Complexome data schematic summarizes the consequence of defective NDUFAF8 on complex I assembly. Complex I subcomplexes are color-coded and named consistent with Panels A and B.