NDUFB8 Mutations Cause Mitochondrial Complex I Deficiency in Individuals with Leigh- like Encephalomyopathy Dorota Piekutowska-Abramczuk, Zahra Assouline,

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NDUFB8 Mutations Cause Mitochondrial Complex I Deficiency in Individuals with Leigh- like Encephalomyopathy Dorota Piekutowska-Abramczuk, Zahra Assouline, Lavinija Mataković, René G. Feichtinger, Eliška Koňařiková, Elżbieta Jurkiewicz, Piotr Stawiński, Mirjana Gusic, Andreas Koller, Agnieszka Pollak, Piotr Gasperowicz, Joanna Trubicka, Elżbieta Ciara, Katarzyna Iwanicka- Pronicka, Dariusz Rokicki, Sylvain Hanein, Saskia B. Wortmann, Wolfgang Sperl, Agnès Rötig, Holger Prokisch, Ewa Pronicka, Rafał Płoski, Giulia Barcia, Johannes A. Mayr The American Journal of Human Genetics Volume 102, Issue 3, Pages 460-467 (March 2018) DOI: 10.1016/j.ajhg.2018.01.008 Copyright © 2018 American Society of Human Genetics Terms and Conditions

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. The American Journal of Human Genetics 2018 102, 460-467DOI: (10.1016/j.ajhg.2018.01.008) Copyright © 2018 American Society of Human Genetics Terms and Conditions

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). The American Journal of Human Genetics 2018 102, 460-467DOI: (10.1016/j.ajhg.2018.01.008) Copyright © 2018 American Society of Human Genetics Terms and Conditions

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. The American Journal of Human Genetics 2018 102, 460-467DOI: (10.1016/j.ajhg.2018.01.008) Copyright © 2018 American Society of Human Genetics Terms and Conditions

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. The American Journal of Human Genetics 2018 102, 460-467DOI: (10.1016/j.ajhg.2018.01.008) Copyright © 2018 American Society of Human Genetics Terms and Conditions

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). The American Journal of Human Genetics 2018 102, 460-467DOI: (10.1016/j.ajhg.2018.01.008) Copyright © 2018 American Society of Human Genetics Terms and Conditions