Recessive germline SDHA and SDHB mutations causing leukodystrophy and isolated mitochondrial complex II deficiency

Abstract

Background: Isolated complex II deficiency is a rare form of mitochondrial disease, accounting for approximately 2% of all respiratory chain deficiency diagnoses. The succinate dehydrogenase (SDH) genes (SDHA, SDHB, SDHC and SDHD) are autosomally-encoded and transcribe the conjugated heterotetramers of complex II via the action of two known assembly factors (SDHAF1 and SDHAF2). Only a handful of reports describe inherited SDH gene defects as a cause of paediatric mitochondrial disease, involving either SDHA (Leigh syndrome, cardiomyopathy) or SDHAF1 (infantile leukoencephalopathy). However, all four SDH genes, together with SDHAF2, have known tumour suppressor functions, with numerous germline and somatic mutations reported in association with hereditary cancer syndromes, including paraganglioma and pheochromocytoma.

Methods and results: Here, we report the clinical and molecular investigations of two patients with histochemical and biochemical evidence of a severe, isolated complex II deficiency due to novel SDH gene mutations; the first patient presented with cardiomyopathy and leukodystrophy due to compound heterozygous p.Thr508Ile and p.Ser509Leu SDHA mutations, while the second patient presented with hypotonia and leukodystrophy with elevated brain succinate demonstrated by MR spectroscopy due to a novel, homozygous p.Asp48Val SDHB mutation. Western blotting and BN-PAGE studies confirmed decreased steady-state levels of the relevant SDH subunits and impairment of complex II assembly. Evidence from yeast complementation studies provided additional support for pathogenicity of the SDHB mutation.

Conclusions: Our report represents the first example of SDHB mutation as a cause of inherited mitochondrial respiratory chain disease and extends the SDHA mutation spectrum in patients with isolated complex II deficiency.

Figure 1

(B) Axial T2-weighted MR image of Patient 1’s brain demonstrates high signal intensity in frontal (arrow) and peritrigonal (arrow head) white matter sparing subcortical U-fibres, normal basal ganglia whilst the sagittal T2-weighted spinal image (C) demonstrates extensive high signal intensity in spinal cord grey matter (arrow heads).

Figure 2

(A–C) H&E staining, cytochrome c oxidase (COX) histochemistry and succinate dehydrogenase (SDH) histochemistry respectively, for Patient 1, indicating a marked decrease in the activity of SDH (C) compared with a control SDH reaction (D). (E–H) Images from the muscle biopsy of Patient 2 including H&E staining (E), COX histochemistry reactivity (F), SDH histochemistry reactivity (G) and the Oil Red O stain (H), the last highlighting a very subtle increase in intrafibre lipid.

Figure 3

Identification of pathogenic SDHA and SDHB mutations. (A) Compound heterozygous c.1523C>T (p.Thr508Ile) and c.1526C>T (p.Ser509Leu) SDHA mutations were identified in Patient 1, with parental DNA screening supporting recessive inheritance. Both mutations affect highly conserved p.Thr508 and p.Ser509 residues in the SDHA-encoded subunit A of succinate dehydrogenase (SDH). (B) A novel homozygous c.143A>T (p.Asp48Val) SDHB mutation was identified in Patient 2, with recessive inheritance supported by parental DNA screening. Multiple sequence alignment of this region of the SDHB subunit confirms that p.Asp48Val mutation affects an evolutionary conserved residue.

Figure 4

Investigation of complex activities and protein expression in Patients 1 and 2 and controls. (A) BN-PAGE analysis of mitochondria isolated from cultured patient and control fibroblasts revealed a reduction of assembled complex II. SDS-PAGE analysis, probed with antibodies against porin (loading control) and the flavoprotein subunit of succinate dehydrogenase (SDH), revealed an almost complete obliteration of SDHA expression for Patient 1, corroborating the pathogenicity of the c.1523C>T (p.Thr508Ile) and c.1526C>T (p.Ser509Leu) SDHA variants. (B) BN-PAGE analysis of enriched mitochondria from patient and control muscle revealed a reduction of complex II assembly, with normal complex I assembly. SDS-PAGE analysis shows a gross decrease in iron–sulphur (SDHB) and a slightly reduction in SDHA subunit expression, with normal levels of TOM20 and porin.

Figure 5

Complementation studies in yeast confirming pathogenicity of the p.Asn42Val SDHB mutation. (A) ClustalW alignment of the yeast and human SDHB homologues confirm that although the amino acid sequences are highly conserved, the p.Asp48 residue in human SDHB is not conserved in yeast, with the corresponding SDH2 residue being p.Asn42. (B) Oxidative growth phenotype. The strain BY4741 sdh2Δ was transformed with either pFL38 empty vector, pFL38 carrying the wild-type SDH2, the humanised wild-type (sdh2^N42D) allele or the pathological (sdh2^N42V) allele. Equal amounts of serial dilutions of cells from exponentially grown cultures (10⁵, 10⁴, 10³, 10² and 10¹ cells) were spotted onto yeast nitrogen base (YNB) plates supplemented with 2% glucose or with 2% ethanol. The growth was scored after 5 days of incubation at 28°C. (C) Respiration was measured in cells grown in YNB supplemented with 0.6% glucose. ^aAllele carried by the vector introduced into the null Δsdh2 strain; ^bexpressed as nmol O₂/min/mg dry weight. Each value is the mean of three independent experiments. (D) Assessment of succinate dehydrogenase (SDH) activity. Enzyme activity was measured in mitochondria isolated from cells grown to late exponential phase at 28°C in YNB supplemented with 0.6% glucose. The values of the sdh2 mutants are expressed as percentage of the activities obtained in the sdh2Δ/SDH2. Values are means of three independent experiments in duplicate.