Pathogenic variants in NUBPL result in failure to assemble the matrix arm of complex I and cause a complex leukoencephalopathy with thalamic involvement

Abstract

Disorders of the white matter are genetically very heterogeneous including several genes involved in mitochondrial bioenergetics. Diagnosis of the underlying cause is aided by pattern recognition on neuroimaging and by next-generation sequencing. Recently, genetic changes in the complex I assembly factor NUBPL have been characterized by a consistent recognizable pattern of leukoencephalopathy affecting deep white matter including the corpus callosum and cerebellum. Here, we report twin boys with biallelic variants in NUBPL, an unreported c.351 G > A; p.(Met117Ile) and a previously reported pathological variant c. 693 + 1 G > A. Brain magnetic resonance imaging showed abnormal T2 hyperintense signal involving the periventricular white matter, external capsule, corpus callosum, and, prominently, the bilateral thalami. The neuroimaging pattern evolved over 18 months with marked diffuse white matter signal abnormality, volume loss, and new areas of signal abnormality in the cerebellar folia and vermis. Magnetic resonance spectroscopy showed elevated lactate. Functional studies in cultured fibroblasts confirmed pathogenicity of the genetic variants. Complex I activity of the respiratory chain was deficient spectrophotometrically and on blue native gel with in-gel activity staining. There was absent assembly and loss of proteins of the matrix arm of complex I when traced with an antibody to NDUFS2, and incomplete assembly of the membrane arm when traced with an NDUFB6 antibody. There was decreased NUBPL protein on Western blot in patient fibroblasts compared to controls. Compromised NUBPL activity impairs assembly of the matrix arm of complex I and produces a severe, rapidly-progressive leukoencephalopathy with thalamic involvement on MRI, further expanding the neuroimaging phenotype.

Keywords: Complex I assembly; Complex I deficiency; Leukoencephalopathy; NUBPL.

Conflict of interest statement

Declaration of Competing Interest JVH participates in clinical trials of mitochondrial disorders by Stealth Biotherapeutic, Inc. RPS participates in clinical trials of mitochondrial disorders by Bioelectron Therapeutics, Stealth Biotherapeutics, Inc. and a National Institutes of Health funded phase 3 trial of dichloroacetate in pyruvate decarboxylase deficiency.

Copyright © 2019 Elsevier Inc. All rights reserved.

Figures

Figure 1:. Brain magnetic resonance imaging of patients with NUBPL pathogenic variants

Legend: Brain MRI of Twin A (A-D) and Twin B (E-H) at age 4 months (A, B, E, and F) and at age 22 months (C, D, G, and H). At 4 months of age, T2 images (A and E) show similar diffuse hyperintense signal in the white matter (black asterisks) and enlarged thalami (white arrowheads) in both twins. Diffusion-weighted imaging (B and F) demonstrates hypointense signal on ADC maps in the thalami (white arrowheads) indicating cytotoxic edema. Abnormal cortical T2 hyperintense signal (A) and restriction diffusion (B) is present at 4 months in Twin A (white arrows). At 22 months of age, T2 images (C, D, G, and H) show progressive T2 hyperintense signal involving the supratentorial white matter (black asterisks) and cortex (white arrows) with new T2 signal hyperintensity in the cerebellar vermis (dashed white arrows in D and H). There is interval thalamic atrophy (white arrowheads in C and G) with relative sparing of the basal ganglia (black arrows in C and G), hippocampus (black arrowheads in D and H), and occipital lobes (black dashed arrows in D and H).

Figure 2:. Mitochondrial functional testing on blue native gel with in-gel activity staining and the amount of NUBPL protein

Legend: (A) The activities of respiratory chain enzyme complexes I, II, IV, and V are visualized by in-gel activity staining after separation of the complexes on blue native polyacrylamide gel electrophoresis from an isolated inner mitochondrial membrane preparation of the fibroblasts from the patient and a control. The assay shows decreased staining of complex I with normal activities of complexes II, IV and V. NUBPL protein: The NUBPL protein is identified on western blot in a post-600 g supernatant (B), and a mitochondrial preparation obtained by differential centrifugation (C). In controls, the NUBPL protein is present in two sizes as a double band in the whole cell preparation (B), but as a single band in the mitochondrial preparation (C), whereas both bands are absent in the patient. Abbreviation: FB = fibroblasts

Figure 3:. The assembly of complex I is affected in the patient

Legend: The assembly of complex I on an inner mitochondrial membrane preparation is identified on a blue native gel using an antibody against NDUFS2, a protein of the matrix arm (A) and against NDUFB6 a protein of the membrane arm (C). This shows a reduction in the holocomplex without an intermediate accumulating detectable with the antibody against NDUFS2, a component of the matrix arm (A), but with an intermediate detected around 480 kD with an antibody against NDUFB6, a component of the membrane arm. Chloramphenicol treated HepG2 cells are shown as a reference for the location of abnormal assembly intermediates. The amount of the NDUFS3 subunit, which is present in the matrix arm, is strongly reduced as shown on SDS-PAGE followed by western blot (B), whereas the amount of NDUFB8 protein present in the membrane arm is strongly reduced but not absent (D). Citrate synthase is shown as a loading control. The amount of subunits of other respiratory chain complexes is not different from controls (E). Abbreviation: FB = fibroblasts, CAM = chloramphenicol treated.

Figure 4:. High resolution respirometry

Legend: The oxygen consumption rate is measured in high resolution respirometry using a substrate-inhibitor protocol, first identifying the ADP-stimulated rate with sequentially added complex I substrates pyruvate (patient 15.0±2.8 pmol/min.106 cells n=6 vs. controls 30.5±14.0 n=41, p<0.001) (blue) and glutamate (patient 16.0±2.9 vs controls 31.5±14.6, p<0.001) (orange), then is added the complex II substrate succinate (patient 46.6±15.6 vs. control 42.9±14.6, p=0.61) (grey), before uncoupling with carbonylcyanide-p-trifluoromethoxy-phenylhydrazone (FCCP) (patient 56.5±18.9 vs control 83.0±23.7, p=0.016) (yellow). The complex I specific activity is reflected in the difference between the uncoupled rate and the rate after complex I inhibition with rotenone (patient 23.9±11.5 vs controls 21.4±15.2, p<0.001) (green). The complex IV activity rate is estimated from the stimulation with N,N,N’,N’-tetramethyl-p-phenylenediamine dihydrochloride (TMPD) and ascorbate after subtraction of the azide inhibited rate (patient 91.0±58.9 vs controls 70.6±28.3, p=0.44) (dark blue). The increase in activity with the complex II substrate over that of the complex I substrates (ratio of the S/G rate, light blue) was greater than the 95th percentile in controls (patient 2.9±0.6 vs controls 1.6±0.6, p=0.002), whereas the rotenone sensitive rate (green) was just above the 5th percentile of controls.