Neurogenetics J Med Genet: first published as 10.1136/jmedgenet-2020-107383 on 2 April 2021. Downloaded from Original research Whole genome and exome sequencing identify NDUFV2 mutations as a new cause of progressive cavitating leukoencephalopathy Zhimei Liu ,1 Li Zhang,2,3 Changhong Ren,1 Manting Xu ,1 Shufang Li,1 Rui Ban,1 Ye Wu,4 Ling Chen,5 Suzhen Sun,5 Matthias Elstner,6 Masaru Shimura,7 Minako Ogawa- Tominaga ,7 Kei Murayama,7 Tieliu Shi,2,3 Holger Prokisch,1,8,9 Fang Fang1 ► Additional material is ABSTRACT the clinical and neuroradiological entity of PCL is a published online only. To view, Background Progressive cavitating leukoencephalopathy syndrome strongly related to mitochondrial respiratory please visit the journal online chain enzyme deficiency.5 Subsequently, NDUFV1 and (http:// dx. doi. org/ 10. 1136/ (PCL) is thought to result from mutations in nuclear genes jmedgenet- 2020- 107383). affecting mitochondrial function and energy metabolism. To other genes involved in mitochondrial function and date, mutations in two subunits of complex I, NDUFS1 and energy metabolism were also reported to be related to For numbered affiliations see NDUFV1, have been reported to be related to PCL. PCL.6–10 Both NDUFS1 and NDUFV1 encode mito- end of article. Methods Patients underwent clinical examinations, brain chondrial Co I subunits. MRI, skin biopsy and muscle biopsy. Whole-genome or Co I (NADH: quinone oxidoreductase) is by far the Correspondence to largest and most complicated enzyme of the respira- Professor Fang Fang, whole-exome sequencing was performed on the index Department of Neurology, patients from two unrelated families with PCL. The effects of tory chain and is composed of more than 40 subunits, Beijing Children’s Hospital, the mutations were examined through complementation of 14 central subunits and up to 32 accessory subunits, 7 Capital Medical University, the NDUFV2 mutation by cDNA expression. of which are encoded by mitochondrial DNA and the National Center for Children’s 5 6 11 Results The common clinical features of the patients in remaining by nuclear DNA (nDNA). Co I can be Health, Beijing, China; 13910150389@ 163. com this study were recurring episodes of acute or subacute subdivided into three functional modules: the electron Professor Tieliu Shi; developmental regression that appeared in the first years input (N) module that oxidises NADH, the P module tieliushi@ yahoo. com of life, followed by gradual remissions and prolonged that pumps protons across the membrane and the elec- Professor Holger Prokisch; periods of stability. MRI showed leukoencephalopathy with tron output (Q) module that reduces quinone. The N Prokisch@ helmholtz- muenchen. module consists of the subunits encoded by the nDNA de multiple cavities. Three novel NDUFV2 missense mutations were identified in these families. Complex I deficiency was genes NDUFV1, NDUFV2 and NDUFS1.6 11 ZL and LZ contributed equally. confirmed in affected individuals’ fibroblasts and a muscle NDUFV2 is a highly conserved core subunit of http://jmg.bmj.com/ biopsy. Functional and structural analyses revealed that these Co I and functions in the transfer of electrons from Received 9 August 2020 mutations affect the structural stability and function of the NADH to the respiratory chain. To date, only six Revised 21 December 2020 Accepted 10 January 2021 NDUFV2 protein, indicating that defective NDUFV2 function likely pathogenic mutations in NDUFV2 have been is responsible for the phenotypes in these individuals. reported, of which two mutations (c.427C>T, Conclusions Here, we report the clinical presentations, c.580G>A) result in Leigh syndrome,12 two muta- neuroimaging and molecular and functional analyses of tions (c.120+5_120+8delGTAA, c.669_670insG) novel mutations in NDUFV2 in two sibling pairs of two lead to hypertrophic cardiomyopathy and encephalop- on September 27, 2021 by guest. Protected copyright. Chinese families presenting with PCL. We hereby expand athy13–17 and Leigh syndrome,17 while the other two the knowledge on the clinical phenotypes associated with mutations (c.86C>T, c.626A>G) are likely related to mutations in NDUFV2 and the genotypes causative for PCL. Parkinson’s disease.18 19 To date, no leukoencephalop- athy phenotype has been reported. Here, we report the clinical findings, neuroimaging and molecular and functional analyses of novel muta- INTRODUCTION tions in NDUFV2 in four Chinese patients (two sets of Leukoencephalopathy is seen in disorders of toxic, siblings) presenting with PCL. Thus, we expand the acquired vascular or infectious origin as well as in knowledge on the clinical phenotypes of NDUFV2 and inherited disorders. Inherited leukoencephalopathies the genotypes of PCL. © Author(s) (or their are a large group of heterogeneous disorders that result employer(s)) 2021. Re- use in white matter abnormalities.1 2 Cavitating leukoen- permitted under CC BY- NC. No cephalopathy associated with mitochondrial complex I METHODS commercial re- use. See rights 3 and permissions. Published deficiency was first described in 1999, while progres- Participants by BMJ. sive cavitating leukoencephalopathy (PCL) was first Sibling pairs from two families (F-1 II-1 and II-2; proposed in 2005 and is thought to result from muta- F-2 II-1 and II-2) participated in this study, and their To cite: Liu Z, Zhang L, tions in nuclear genes affecting mitochondrial func- pedigrees are shown in figure 1. The families were Ren C, et al. J Med Genet 4 Epub ahead of print: [please tion and energy metabolism. In 2011, Ferreira and recruited by the Department of Neurology, Beijing include Day Month Year]. colleagues first reported that the NDUFS1 gene was Children’s Hospital, Capital Medical University and by doi:10.1136/ related to PCL and associated with respiratory chain the Department of Pediatrics, Peking University First jmedgenet-2020-107383 complex I (Co I) deficiency. The study concluded that Hospital (China). Liu Z, et al. J Med Genet 2021;0:1–7. doi:10.1136/jmedgenet-2020-107383 1 Neurogenetics J Med Genet: first published as 10.1136/jmedgenet-2020-107383 on 2 April 2021. Downloaded from Figure 1 Pedigree of the families. Black/white represents the healthy parents with heterozygous variant in NDUFV2, while the black symbols depict the affected patients with homozygous or compound heterozygous variant in NDUFV2. Next-generation sequencing Mitochondrial respiratory chain (MRC) enzyme assay Targeted next- generation sequencing was performed for each Skin biopsy was performed for II-1 and II-2 of Family 1. The patient, which contained 115 genes involved in vanishing white skeletal muscle used for the MRC enzyme assay was stored at matter disease, Krabbe disease, adrenoleukodystrophy, meta- −80°C before use. Activities of MRC complexes I, II, II+III, chromatic leukodystrophy, Alexander disease and so on. No III, IV and the mitochondrial marker enzyme citrate synthase suspected disease- causing mutations were found. Therefore, (CS) were assayed in the crude post-600 g supernatant of the we further applied whole- genome sequencing (WGS) or whole- muscle sample and in isolated mitochondria from skin fibro- exome sequencing (WES) to the individuals’ DNA extracted blasts using spectrophotometric assays as described previ- from peripheral blood. Family 1 (including the two sibling ously.12 31 32 The enzyme activities of each complex are presented patients and their parents) was investigated by WGS, and Family as the percentage of the normal control mean relative to appro- 2 was investigated by WES. WES coding regions were enriched priate reference enzyme activities, such as CS or MRC complex using the Agilent SureSelect Human All Exon V6 Kit followed by II. Enzyme activity was defined as being decreased at a level of sequencing as 150 bp paired- end runs on an Illumina HiSeq2000. <40% in a cell line or <30% in tissue.33 WGS sequencing libraries were prepared using Illumina TruSeq Nano Kits and sequenced on Illumina HiSeq X sequencers via 2×150 bp reads. Reads were aligned to the human reference Oxygen consumption rate (OCR) measurement OCR was measured in fibroblasts from the two patients (F1 II-1 genome (UCSC Genome Browser build hg19, https://genome. 20 and II-2) with a XF96 Extracellular Flux Analyzer (Seahorse http://jmg.bmj.com/ ucsc. edu/) using Burrows- Wheeler Aligner (BWA, http:// bio- 21 Bioscience, Billerica, Massachusetts, USA). Samples were bwa.sourceforge. net/). ANNOVAR (http:// annovar. openbio- prepared as previously described.12 34 In each run, we measured informatics.org/) software was used for variant filtering and two controls along with the patient samples. The control cell annotation. Rare variants were subsequently filtered by a minor lines used were derived from fHDF (Toyobo Biotech Support allele frequency (MAF) lower than 0.1% in 1000 Genomes 22 Department, Japan). Each control and patient fibroblast cell line Project (http://www. internationalgenome. org/), Exome Aggre- 23 was seeded in at least 14 wells of two XF96 cell culture micro- gation Consortium (ExAC, http:// exac. broadinstitute. org/) plates (Seahorse Bioscience) at a density of 20 000 cells/well in on September 27, 2021 by guest. Protected copyright. and gnomAD24 (https:// gnomad. broadinstitute. org/) databases. 80 µL of growth medium and incubated at 5% CO and 37°C The missense variants in highly conserved amino acid residues 2 25 overnight. The following day, the growth medium was replaced predicted by PolyPhen-2 (http:// genetics. bwh. harvard. edu/ 26 with