A novel IBA57 variant is associated with mitochondrial iron-sulfur protein deficiency and necrotizing myelopathy in dogs

Abstract

Introduction: Hereditary necrotizing myelopathy (HNM) in young Kooiker dogs is characterized by progressive ataxia and paralysis with autosomal recessive inheritance. The basic genetic defect is unknown. We investigated the possible cause by a genome-wide analysis using six affected and 17 unrelated unaffected Kooiker dogs and by functional follow-up studies. Method: The HNM locus was mapped by a case-control study using a dense SNP array and confirmed by linkage analysis of two pedigrees. The gene exons in the critical region were analyzed by next-generation sequencing. The functional effect of the candidate canine IBA57 pathogenic variant was biochemically examined in an established HeLa cell culture model in which the endogenous IBA75 gene product was depleted by RNAi. Results: The basic defect was localized in the centromeric 5 Mb region of canine chromosome 14. The most associated SNP co-segregated fully with HNM and reached an LOD score of 6.1. A candidate pathogenic mutation was found in the iron-sulfur cluster assembly gene IBA57 and led to the amino acid substitution R147W. The expression of human IBA57 harboring the canine R147W exchange could only partially restore the biochemical defects of several mitochondrial [4Fe-4S] proteins upon IBA57 depletion, showing that the mutant protein is functionally impaired. Discussion: Pathogenic variants in human IBA57 cause multiple mitochondrial dysfunction syndrome 3 (MMDS3), a neurodegenerative disorder with distant similarities to HNM. The incomplete functional complementation of IBA57-depleted human cells by IBA57-R147W identifies the DNA mutation in affected Kooiker dogs as the genetic cause of HNM. Our findings further expand the phenotypic spectrum of pathogenic IBA57 variants.

Keywords: IBA57-R147W; Kooiker dog; Kooikerhondje; canine; leukodystrophy; lipoyl synthase; respiratory complexes; spinal cord.

FIGURE 1

Genome-wide association analysis of hereditary necrotizing myelopathy in Kooiker dogs. Chi-square p-values for SNP allele number differences between six cases and 17 unaffected dogs were calculated with PLINK and plotted against the chromosome number and position on the chromosome. Chromosome 39 represents the X chromosome. The peak near the telocentric centromere of chromosome 14 represents the six SNPs, which includes BICF2G630517911.

FIGURE 2

Multi-sequence alignment of the IBA57 segment carrying the canine R147W amino acid exchange. Partial IBA57 amino acid sequences from the indicated organisms were aligned using Multalin (Corpet, 1988). The partial conservation of canine residue R147 in IBA57 is highlighted in yellow, and the strict conservation of the preceding R146 is in gray. Homo, Homo sapiens; Tetraodon, Tetraodon nigroviridis; Danio, Danio rerio; Aedes, Aedes aegypti; Drosophila, Drosophila melanogaster; Caenorhab, Caenorhabditis elegans; Citrus, Citrus sinensis; Brassica, Brassica carinata; Aspergillus, Aspergillus fumigatus; Chaetoglobo, Chaetomium globosum; Chaetotherm, Chaetomium thermophilum; Neurospora, Neurospora crassa.

FIGURE 3

IBA57-R147W amino acid exchange compromises mitochondrial [4Fe–4S] proteins. HeLa cells were transfected thrice at a 3-day interval with scrambled control siRNAs (SCR) or with an IBA57-directed siRNA (siIBA57) in combination with complementing (compl.) plasmids encoding RNAi-resistant wild-type or HNM-related IBA57 (smIBA57^wt or smIBA57^R147W, respectively). As a control, cells were mock-transfected. Analyses were performed with cells harvested after the third round of transfection (9 days of IBA57 depletion). (A) Total cell lysates were subjected to immunoblotting and analyzed for the protein levels of the indicated ISC proteins and β-actin as a loading control. (B) Immunoblot signals from part (A) were quantified relative to β-actin levels, and the ratio was normalized to mock-transfected control cells (dashed line). (C, D) Specific activities of mitochondrial aconitase (ACO2), SDH (RCC-II), and COX (RCC-IV) (C) were determined in mitochondria-containing membrane fractions prepared by digitonin-based cell fractionation, related to citrate synthase (CS) activity (D), and normalized to mock-transfected control cells. (E) Total cell lysates were subjected to immunoblotting as in (A) and analyzed for the steady-state levels of mitochondrial proteins and RCC subunits as indicated. (F) Immunoblot signals from (E) were quantified, and the protein per β-actin ratios (see A) were normalized to mock-transfected control cells. Representative blots are shown. All values are given as the mean ± SD (n = 3–4); *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant; dashed lines, 100% value of mock-transfected control cells.

FIGURE 4

IBA57-R147W amino acid exchange compromises lipoyl cofactor formation. HeLa cells were depleted for IBA57 and treated as in Figure 3A. LIAS activity was indirectly determined by immunoblotting for lipoylation (LA) of KGDHc-E2 and PDHc-E2. DLAT (PDHc-E2 protein) was immunostained for comparison. (B) Immunoblot signals from (A) were quantified, and the antigen per β-actin ratio was normalized to mock-transfected control cells (dashed line). Representative blots are shown. All values are given as the mean ± SD (n = 3-4); *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant.