Recessive Mutations in NDUFA2 Cause Mitochondrial Leukoencephalopathy

Perrier Stefanie (Orcid ID: 0000-0002-6881-7573)

(Short Report)

Stefanie Perrier1†, Laurence Gauquelin1,2†, Martine Tétreault3,4, Luan Tran1,2,5,6, Neil Webb3,7,8, Myriam Srour1,2,6, John J. Mitchell2,5, Catherine Brunel-Guitton7, Jacek Majewski3,4, Valynne Long9, Stephanie Keller10, Michael J. Gambello9, Cas Simons11, Care4Rare Canada Consortium, Adeline Vanderver12,13, and Geneviève Bernard1,2,5,6* 1 Department of Neurology and Neurosurgery, McGill University, Montreal, QC, Canada. 2 Department of Pediatrics, McGill University, Montreal, QC, Canada. 3 Department of Human Genetics, McGill University, Montreal, QC, Canada. 4 McGill University and Genome Quebec Innovation Centre, Montreal, QC, Canada. 5 Department of Medical Genetics, Montreal Children’s Hospital, McGill University Health Center, Montreal, QC, Canada. 6 Child Health and Human Development Program, Research Institute of the McGill University Health Centre, Montreal, QC, Canada. 7 Division of Medical Genetics, Department of Pediatrics, CHU Sainte-Justine and Université de Montréal, Montreal, QC, Canada. 8 Montreal Neurological Institute, McGill University, Montreal, QC, Canada. 9 Department of Human Genetics, Division of Medical Genetics, Emory University School of Medicine, Atlanta, GA, USA. 10 Department of Pediatrics, Division of Pediatric Neurology, Emory University School of Medicine, Atlanta, GA, USA. 11 Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Queensland, Australia. 12 Department of Neurology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA. 13 Division of Neurology, Children’s Hospital of Philadelphia, Philadelphia, PA, USA.

† Both authors contributed equally.

*Correspondence to: Dr. Geneviève Bernard Research Institute of the McGill University Health Centre 1001 boul Décarie EM02224 (CHHD Mail Drop Point #EM03211 (Cubicle C)) Montréal, QC H4A3J1, Canada.

This is the author manuscript accepted for publication and has undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/cge.13126

Conflict of Interest: The authors declare no conflicts of interest.

Acknowledgements

The authors are grateful to the patients and their families for their participation.

This work was performed under the Care4Rare Canada Consortium, funded by

Genome Canada, the Canadian Institutes of Health Research, the Ontario Genomics

Institute, Ontario Research Fund, Génome Québec, Children’s Hospital of Eastern

Ontario Foundation and Fondation Leuco Dystrophies. This study was supported by grants from MitoCanada, Fondation les Amis d’Eliott and Réseau de Médecine

Génétique Appliquée. We wish to acknowledge the McGill University and Génome

Québec Innovation Centre for use of their high throughput sequencing platform. GB has received a Research Scholar Junior 1 award from the Fonds de Recherche du Québec en Santé (2012-2016) and the New Investigator Salary Award from the Canadian

Institutes of Health Research (2017-2022). SP is supported by the Max Stern

Scholarship from McGill University, and the Research Institute of the McGill University

Health Centre Desjardins Studentship in Child Health Research. MT is supported by a post-doctoral fellowship from the Canadian Institutes of Health Research. JJM receives research support from the Harpur Foundation. AV is supported by the Kamens family.

Abstract

Deficiencies of mitochondrial respiratory chain complex I frequently result in

leukoencephalopathy in young patients, and different mutations in the genes encoding

its subunits are still being uncovered. We report two patients with cystic

leukoencephalopathy and complex I deficiency with recessive mutations in NDUFA2, an

accessory subunit of complex I. The first patient was initially diagnosed with a primary

systemic carnitine deficiency associated with a homozygous variant in SLC22A5, but also exhibited developmental regression and cystic leukoencephalopathy, and an additional diagnosis of complex I deficiency was suspected. Biochemical analysis confirmed a complex I deficiency, and whole exome sequencing revealed a homozygous mutation in NDUFA2 (c.134A>C, p.Lys45Thr). Review of a biorepository of patients with unsolved genetic leukoencephalopathies who underwent whole exome or genome sequencing allowed us to identify a second patient with compound heterozygous mutations in NDUFA2 (c.134A>C, p.Lys45Thr; c.225del,

This article is protected by copyright. All rights reserved. p.Asn76Metfs*4). Only one other patient with mutations in NDUFA2 and a different phenotype (Leigh syndrome) has previously been reported. This is the first report of cystic leukoencephalopathy caused by mutations in NDUFA2.

Keywords

Complex I deficiency, Leukodystrophy, Leukoencephalopathy, NDUFA2, Whole exome sequencing

Introduction

The first enzyme complex of the mitochondrial respiratory chain oxidative phosphorylation system, NADH-ubiquinone oxidoreductase, is most commonly implicated in deficiencies of the respiratory chain (1). Mitochondrial diseases due to complex I deficiency result in a broad range of clinical symptoms, frequently involving the central nervous system (2). Various pathogenic mutations have been identified in genes encoding complex I subunits, which are commonly associated with isolated complex I deficiency (MIM 252010) (3). Human complex I is composed of 44 different subunits, including 14 core subunits responsible for energy transduction through the transfer of electrons and translocation of protons (4). The remaining 30 accessory subunits have several other suspected functions such as assisting in enzyme assembly,

NADH-ubiquinone oxidoreductase subunit A2 (NDUFA2) is an accessory subunit located in the distal matrix arm of complex I, and structural analysis has revealed similarities to thioredoxin-related proteins (6). The exact role of NDUFA2 has yet to be defined, but proposed functions include complex I iron-sulfur cluster assembly and upkeep, and use of redox processes for assembly or regulation of enzymatic activity

(6,7). Only one patient has been previously described with a homozygous splice site mutation in NDUFA2 (c.208+5G>A) (MIM 602137) causing abnormal splicing of exon 2.

This patient exhibited a clinical presentation of Leigh syndrome (MIM, 256000) (8).

Here, we present for the first time two patients with cystic leukoencephalopathy associated with novel pathogenic variants in NDUFA2 identified by whole exome sequencing (WES).

Materials and Methods

Patient Recruitment and Ethics Approval

Using MRI pattern recognition, a first patient with unsolved cystic leukoencephalopathy suggestive of complex I deficiency was identified. Biochemical analysis confirmed complex I deficiency, and a homozygous NDUFA2 mutation was identified through WES. Review of a large biorepository of patients with genetically

sequencing allowed us to identify a second patient with a compatible clinical and

radiological phenotype and recessive mutations in NDUFA2. Written informed consent

from all subjects or their legal representatives was obtained. This study was approved

by the ethics committees of the Montreal Children’s Hospital and the Children’s Hospital

of Philadelphia.

Biochemical and Genetic Analysis

Detailed methods for the biochemical and genetic analyses are provided in the

Supporting Information (Appendix S1, Tables S1-S2). The novel disease-causing

variants have been listed in the Single Nucleotide Polymorphism Database (dbSNP;

https://www.ncbi.nlm.nih.gov/projects/SNP/; rs757982865 and rs863224084), and

phenotypic information has been submitted to ClinVar

(https://www.ncbi.nlm.nih.gov/clinvar/; SCV000584198 and SCV000584199) and

PhenomeCentral (https://phenomecentral.org).

Results

Patient 1, a girl, was born at term after an uneventful pregnancy. Her parents,

first cousins from Pakistan, had a first child that died at age 3 years following a febrile

illness and seizures, as well as three other clinically unaffected children (Fig. S1).

This article is protected by copyright. All rights reserved. Patient 1 presented at 8 months with encephalopathy, hepatomegaly and hyperammonemia. A diagnosis of primary systemic carnitine deficiency was made based on very low plasma carnitine levels (free carnitine 1 nmol/ml, normal 38±21). This condition is associated with a broad clinical spectrum, including an infantile metabolic presentation characterized by episodes of decompensation with poor feeding and lethargy, typically triggered by a febrile illness or fasting. Hepatomegaly, hypoketotic hypoglycemia, and hyperammonemia can be seen with the episodes of decompensation (9). Despite appropriate treatment, patient 1 exhibited developmental regression until age 12 months, after which she stabilized. Her examination was significant for severe spasticity and other upper motor neuron signs, predominantly in the legs, with cerebellar features and generalized dystonia. She had moderate intellectual disability. She was 4 years old when molecular testing confirmed the diagnosis of primary systemic carnitine deficiency due to a homozygous mutation in the

SLC22A5 gene (c.12C>G, p.Tyr4*) (MIM 212140). This variant was previously reported by Wang et al. in a female who presented at 6 months with an episode of encephalopathy, hepatomegaly, hyperammonemia, and hypoketotic hypoglycemia following a febrile illness (10). At 6 years, patient 1 developed focal epilepsy, and at 9 years, she became wheelchair-bound. The first brain MRI, obtained at age 2, revealed periventricular and deep white matter T2 hypersignal with cystic changes. The corpus

MRI was performed at age 12 and appeared stable (Fig. 1.a-f).

Since such white matter anomalies had never been described in primary systemic carnitine deficiency, further investigation for an explanation was pursued.

Complex I deficiency was suspected and patient fibroblasts were tested for OXPHOS enzymatic activities, revealing a decrease in complex I enzymatic activity, compatible with a complex I deficiency (Table S3.a-c). Through BN-PAGE, a technique commonly used to assess complex I assembly (11), an assembly defect was confirmed by the presence of a decreased complex I band and a visible subcomplex band at 750 kDa

(Fig. 2). An additional genetic diagnosis was then sought and WES revealed a homozygous mutation in NDUFA2 (c.134A>C, p.Lys45Thr). The variant was confirmed by Sanger sequencing, and co-segregation analysis revealed inheritance from heterozygous carrier parents.

Another patient with recessive mutations in NDUFA2 (c.134A>C, p.Lys45Thr; c.225del, p.Asn76Metfs*4) and a very similar clinical presentation was identified from a

bioregistry of patients with unsolved genetic leukoencephalopathies who underwent

WES. The variants were confirmed by Sanger sequencing, and co-segregation analysis

revealed maternal inheritance of the c.134A>C (p.Lys45Thr) variant and paternal

inheritance of the c.225del (p.Asn76Metfs*4) variant (Fig. S1). Patient 2, a female

previously described as LD_0821 by Vanderver et al. (12), was born at term to a non-

This article is protected by copyright. All rights reserved. consanguineous Asian-Indian couple. She presented at 8 months with fever, failure to thrive and developmental regression. She required nasogastric feeds. Her examination was significant for severe irritability, no purposeful hand movements, and upper motor neuron signs. The first brain MRI was obtained at 8 months and showed periventricular and subcortical confluent white matter T2 hypersignal with cystic changes. The posterior limb of the internal capsule, middle cerebellar peduncle and cerebellar white matter were also involved. Spectroscopy showed a large lipid/lactate peak and a low N- acetylaspartate (NAA) peak. Brain MRI performed at 13 months revealed a significant progression of the white matter anomalies with diffuse volume loss (Fig. 1.g-l). The patient is currently 4 years, small and microcephalic. She can speak in short sentences and walk with a walker, but uses a wheelchair for long distances.

The effects of the novel NDUFA2 variants were assessed by various in silico prediction algorithms, where the deletion mutation (c.225del), which has not been previously reported in ExAc or other databases, was expected to produce a truncated protein and represents a loss of function allele. The missense mutation (c.134A>C) was predicted to be damaging (with a minor allele frequency of 0.0008 (one heterozygous carrier on ExAc) and position on a highly conserved residue). Additionally, this variant likely derives from a common ancestor as both patients share a common haplotype in the surrounding region (Appendix S1).

We report two patients with novel recessive variants in NDUFA2 who show

degeneration of the cerebral white matter, causing neurological deficits.

Leukoencephalopathy has been reported as associated with variants in several genes

encoding complex I, and typically presents aggressively in young patients with

symptoms of developmental delay and neurological deficits (3,13). Only one other

patient has been described with variants in NDUFA2, and exhibited symptoms

associated with Leigh disease (8). These new reports of allelic variants in NDUFA2 are

noteworthy as they could contribute to the understanding of complex I function, and to

the consequences of variants in the genes encoding its subunits. To expand, in this

case, the presence of the subcomplex at approximately 750 kDa (as revealed by BN-

PAGE) likely indicates an assembly-related defect at an advanced stage of complex I

assembly (14,15).

Our first patient is a testament to the importance of continued testing to

determine an etiology for symptoms that may not be explained by an initial diagnosis.

The patient’s first diagnosis of primary systemic carnitine deficiency (associated with a

previously reported homozygous mutation in SLC22A5) could not account for white

matter abnormalities seen on MRI, and further genetic testing revealed the homozygous

mutation in NDUFA2. Thus, additional genetic investigation explained the unrelated

counselling to the family.

In summary, this is the first report of recessive mutations in NDUFA2 leading to

cystic leukoencephalopathy. Moreover, our manuscript emphasizes the importance of

using extreme caution when atypical clinical features are seen in a patient with a known

genetic disease not to falsely attribute them to a widened disease spectrum.

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Figure Legends

weighted (a, b), axial T2-weighted (c, d), and axial fluid-attenuated inversion recovery

(FLAIR) (e, f) images showing periventricular and deep white matter abnormal signal and cystic changes (arrowheads). (g-l) Brain MRI of Patient 2 obtained at age 13

months. Axial T1-weighted (g, h), axial T2-weighted (i, j), and coronal FLAIR (k) images

showing confluent white matter abnormal signal and cystic changes (arrowheads) with

sparing of the basal ganglia, thalamus and corpus callosum. Single-voxel magnetic

resonance spectroscopy (MRS) in the right centrum semiovale (l) depicting a large

lactate peak (arrow).

Fig. 2. Mitochondrial Complex Activity Staining. Blue native–polyacrylamide gel

electrophoresis (BN-PAGE) followed by immunoblotting to determine the levels of

mitochondrial complexes (CI, CIII, CIV) in fibroblasts of Patient 1. Immunodetection of

each complex was achieved through incubation with antibodies NDUFA9 (CI), CORE1

(CIII), and COX4 (CIV). MWM, molecular weight marker; C, control; P1, Patient 1.