ARTICLE OPEN ACCESS Early-onset Parkinson disease caused by a mutation in CHCHD2 and mitochondrial dysfunction Richard G. Lee, BSc (Hons),* Maryam Sedghi, MSc,* Mehri Salari, MD, Anne-Marie J. Shearwood, MSc, Correspondence Maike Stentenbach, BSc, Ariana Kariminejad, MD, Hayley Goullee, BSc (Hons), Oliver Rackham, PhD, Dr. Tajsharghi [email protected] Nigel G. Laing, PhD, Homa Tajsharghi, PhD,§ and Aleksandra Filipovska, PhD§ or Dr. Filipovska [email protected] Neurol Genet 2018;4:e276. doi:10.1212/NXG.0000000000000276 Abstract Objective Our goal was to identify the gene(s) associated with an early-onset form of Parkinson disease (PD) and the molecular defects associated with this mutation. Methods We combined whole-exome sequencing and functional genomics to identify the genes asso- ciated with early-onset PD. We used fluorescence microscopy, cell, and mitochondrial biology measurements to identify the molecular defects resulting from the identified mutation. Results Here, we report an association of a homozygous variant in CHCHD2, encoding coiled-coil- helix-coiled-coil-helix domain containing protein 2, a mitochondrial protein of unknown function, with an early-onset form of PD in a 26-year-old Caucasian woman. The CHCHD2 mutation in PD patient fibroblasts causes fragmentation of the mitochondrial reticular mor- phology and results in reduced oxidative phosphorylation at complex I and complex IV. Although patient cells could maintain a proton motive force, reactive oxygen species production was increased, which correlated with an increased metabolic rate. Conclusions Our findings implicate CHCHD2 in the pathogenesis of recessive early-onset PD, expanding the repertoire of mitochondrial proteins that play a direct role in this disease. *These authors contributed equally to the study and share first authorship. §These authors share senior authorship and are co-corresponding authors. From the Centre for Medical Research (R.G.L., A.-M.J.S., M. Stentenbach, H.G., O.R., N.G.L., H.T., A.F.), University of Western Australia and the Harry Perkins Institute for Medical Research, Nedlands, Western Australia, Australia; Department of Genetics (M. Sedghi), University of Isfahan, Isfahan; Functional Neurosurgery Research Center (M. Salari), Shohada Tajrish Neurosurgical Center of Excellence, Shahid Beheshti University of Medical Sciences, Tehran, Iran; Kariminejad-Najmabadi Pathology and Genetics Center (A.K.), Tehran, Iran; School of Molecular Sciences (O.R., A.F.), The University of Western Australia, Crawley; Department of Diagnostic Genomics (N.G.L.), PathWest, QEII Medical Centre, Nedlands, Western Australia, Australia; and Division Biomedicine and Public Health (H.T.), School of Health and Education, University of Skovde, Sweden. Funding information and disclosures are provided at the end of the article. Full disclosure form information provided by the authors is available with the full text of this article at Neurology.org/NG. The Article Processing Charge was funded by the Australian Research Council. This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND), which permits downloading and sharing the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal. Copyright © 2018 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology. 1 Glossary BCA = Bicinchoninic acid; DHE = dihydroethidium; ETC = electron transport chain; MICOS = mitochondrial contact site and cristae organizing system; NGS = next-generation sequencing; OXPHOS = oxidative phosphorylation; PBS = phosphate- buffered saline; PD = Parkinson disease; RCR = respiratory control ratio; ROS = reactive oxygen species; WES = whole-exome sequencing. Parkinson disease (PD, MIM168600) is the most common Cell culture and transfections movement disorder of aging and the second-most com- Dermal fibroblast cultures from the patient were established mon neurodegenerative disease, after Alzheimer disease by standard protocols, after written informed consent was (MIM104300).1,2 PD has been associated with mutations in obtained. Cultured fibroblasts from a healthy age-matched multiple genes including PARK2, PINK1, PARK7, ATP13A2, individual were used as a control. Detailed methods are pro- – SCNA, LRRK2, VPS35, EIF4G1, DNAJC13, and CHCHD2.3 6 vided in supplementary information. Recent whole-exome sequencing (WES) of Japanese patients Mitochondrial isolation and cell lysis with autosomal dominant or sporadic PD identified a hetero- Mitochondria were isolated from fibroblasts as previously zygous CHCHD2 missense change (p.Thr61Ile) in a family described.8 Cell lysates were prepared using a buffer con- with autosomal dominant late-onset PD.5 Subsequently, the taining the following: 150 mM NaCl, 0.1% (vol/vol) Triton identical variant was identified in a Chinese family with auto- X-100, and 50 mM Tris-HCl (pH 8.0). Protein concentra- somal dominant PD.6 Additional CHCHD2 variants, including tion was determined using a bicinchoninic acid (BCA) p.Ala32Thr, p.Pro34Leu, and p.Ile80Val, have been described assay. in 4 western European familial patients with PD.7 The patho- mechanism of the CHCHD2 variants in these studies is, how- Fluorescence microscopy ever, unclear as is the physiologic role of CHCHD2. Detailed methods are described in the supplementary mate- rial, links.lww.com/NXG/A85. Here, we identify a new CHCHD2 variant in a patient with autosomal recessive early-onset PD and establish the patho- Long-range PCR and mitochondrial DNA copy genic mechanism of this variant. We have shown that the number quantitative PCR mutation results in a fragmented mitochondrial morphology Detailed methods are described in the supplementary mate- and reduced electron transport chain (ETC) activity. rial. The sequences of all primers used for this study are detailed in table e-1, links.lww.com/NXG/A85. Methods SDS-PAGE and immunoblotting Mitochondria (25 μg) isolated from control and patient Standard protocol approvals, registrations, fibroblasts were separated on 4%–12% Bis-Tris gels (Invi- and patient consents trogen) and transferred onto a polyvinylidene difluoride The study was approved by the ethical standards of the rel- (PVDF) membrane (Bio-Rad). All antibodies used are evant institutional review board, the Ethics Review Com- detailed in the supplementary information. mittee in the Gothenburg Region (Dn1: 842-14), and the Human Research Ethics Committee of the University of Mitochondrial protein synthesis Western Australia. Informed consent was obtained from De novo mitochondrial protein synthesis was analyzed in patients included in this study after appropriate genetic fibroblasts using 35S-radiolabeling of mitochondrially enco- counseling. Blood samples were obtained from patients, their ded proteins in the presence of emetine as previously de- parents, and siblings. scribed.9 The cells were suspended in phosphate-buffered saline (PBS) and 20 μg of protein was resolved on a 12.5% Clinical evaluation SDS-PAGE gel, and radiolabelled proteins were visualized Medical history was obtained, and physical examination was on film. performed as part of routine clinical workup. Respiration Genetic analysis Respiration was measured in fibroblasts as previously de- Next-generation sequencing (NGS) whole-exome and/or tar- scribed.10 The full methods are detailed in supplementary geted neuromuscular panel sequencing (WES or neuromus- information. cular sub-exomic sequencing [NSES]) was performed on the patient’sDNA.Confirmatory bidirectional Sanger sequencing Cell function measurements was performed in patients with PD and their family members JC-1, mitochondrial mass, dihydroethidium (DHE), and MTS (see e-Methods, links.lww.com/NXG/A85). assay full methods are detailed in supplementary information. 2 Neurology: Genetics | Volume 4, Number 5 | October 2018 Neurology.org/NG Statistical analysis Mutation Database and ClinVar, and the most recent literature All data are reported as mean ± SEM. Statistical differences allowed the identification of homozygous CHCHD2 (chro- were determined using a two-tailed Student t test. JC-1, DHE, mosome 7) and TOP1MT (chromosome 8) variants. A novel mitochondrial mass, and MTS data are expressed as a percent homozygous missense mutation in exon 2 of CHCHD2 of average control fibroblast values for the respective media (c.211G>C) (p.Ala71Pro) was identified. The size of the ho- treatment. mozygous region covering CHCHD2 variant on chromosome 7 was 158Mb. In addition, a homozygous missense variant of Data availability TOP1MT (c.661G>A, ENST00000523676, transcript ID: Study data for the primary analyses presented in this report NM_001258447.1), changing aspartic acid to asparagine are available upon reasonable request from the corresponding (p.Asp221Asn), was identified. No rare, likely pathogenic and senior author. heterozygous or homozygous variants in the PD-associated genes, including ATP13A2, CHCHD10, DNAJC13, EIF4G1, LRRK2, PARK2, PARK7, PARK15, PINK1, SNCA,andVPS35, Results were identified in the exome sequencing data. The appearance of CHCHD2 and TOP1MT variants was examined in available Clinical characteristics of the patient family members by sequencing analysis. Sanger sequencing A 30-year-old right-handed Caucasian woman (V:2) was born confirmed segregation of both variants compatible with a re- to healthy consanguineous parents aged 55 and 59 years cessive