Mitochondrial complex II is a critical enzyme for cellular respiration and despite its key roles in both the Krebs cycle and the electron transport chain, it represents a rare cause of disease in the general population. Given that so few Krebs cycle enzyme deficiencies are reported, bi-allelic mutations affecting these enzymes appear to be typically incompatible with life; all reported cases are recessive and involve isolated deficiencies of fumarate hydratase, SDH and, most recently, aconitase,39
and are associated with a severe neurological phenotype and very poor prognosis, being almost always fatal in the neonatal period. Conversely, OXPHOS disease presentations are comparatively common, can present at any stage throughout life and are associated with vast clinical and genetic heterogeneity. Pathogenic mutations have been identified in numerous OXPHOS genes, but the correlation with clinical phenotype is rarely pathognomonic. Isolated deficiencies involving SDH are the rarest of all OXPHOS deficiencies, accounting for approximately 2% of all mitochondrial disease cases.
Here, we describe two children who presented during infancy with motor manifestations of leukodystrophy and one who also had significant cardiomyopathy, in whom biochemical and histochemical analyses of respiratory chain activities in skeletal muscle uncovered a severe, isolated deficiency of complex II. Mutations in SDHAF1
genes are known to cause mitochondrial complex II deficiency;7
thus, sequencing of these genes was prioritised. This analysis revealed novel compound heterozygous variants of unknown pathological significance within the SDHA
gene (c.1523C>T; p.Thr508Ile and c.1526C>T; p.Ser509Leu) for Patient 1, while analysis of the SDHAF1
genes for Patient 2 revealed only wild-type sequence. A candidate gene sequencing approach led to the discovery of a novel homozygous (c.143A>T; p.Asp48Val) SDHB
variant in Patient 2. Recessive inheritance of the SDHA
variants was confirmed by parental DNA screening. Functional investigations supported the deleterious effect of the putative SDHA
mutations, with SDS-PAGE, BN-PAGE and western blotting confirming decreased levels of SDHA and SDHB protein expression for Patients 1 and 2, respectively; both patients had a marked reduction in stable, fully-assembled complex II.
While there are numerous reports describing SDHB mutations in the context of hereditary and sporadic cancer pathology, this report represents the first case of SDHB mutation in association with a neurological phenotype. Additional functional evidence supporting the pathogenicity of the novel homozygous SDHB mutation was provided through modelling the p.Asp48Val mutation in Saccharomyces cerevisae Despite a very high degree of conservation across the SDHB/SDH2 genes, a lack of homology at the human p.Asp48 SDHB locus and corresponding Saccharomyces cerevisae p.Asn42 residue was noted and addressed in our experimental design. Site-directed mutagenesis was employed to generate a p.Asn42Val mutant, in which a marked decrease in SDH activity was observed. A humanised wild-type yeast model was subsequently generated to investigate whether the lack of homology at the p.Asn42/p.Asp48 locus impacted upon SDH activity; SDH activity in the humanised p.Asp42 and wild-type Sdh2 model were comparable, thus corroborating the pathogenicity of the human p.Asp48Val mutation.
Based on Piccolo modelling, the wild-type human p.Asp48 residue is not involved in direct binding to other SDH residues but is located within the highly conserved 2Fe-2S binding domain of the SDHB protein. While p.Asp48 has no obvious primary interaction with other residues of the complex, we hypothesise that it is critical for efficient binding and onward procession of electrons within the mitochondrial respiratory chain.
We provide functional evidence linking the primary SDHB
defect with specific brain pathology. The abnormal MRI signal intensities in brain are restricted to the white matter and indicative of myelin or glial pathology (ie, leukodystrophy); such anomalies have been described in mitochondrial disease, while the spinal cord changes as seen in Patient 1 have been described in mitochondrial pathologies including leukoencephalopathy with brain stem and spinal cord involvement and lactate elevation (LBSL).40
The accumulation of succinate detectable in the occipito-parietal (clinically affected) region by MRS provides further evidence of a metabolic aetiology; elevated succinate on MRS imaging investigations could be a key diagnostic indicator of SDH deficiency with the elevated metabolite levels reflecting ‘enzymatic cascade stalling’ and MRS should be considered in patients with suspected mitochondrial disease. Succinate inhibits prolyl hydroxylase activity, which stabilises hypoxia-inducible factor (HIF1α) under ‘normal’ anaerobic conditions (eg, exercise or high altitude) and triggers hypoxia-inducible pathways. In the case of SDH deficiency, metabolic stalling generates an accumulation of succinate that mimics this process.41
This report provides mechanistic evidence illustrating a link between specific white matter changes and SDH deficiency. The factors determining localisation of these effects to specific brain regions remain unclear.
In addition to their recognised role in cellular respiration, SDHA, SDHB, SDHC
have all been shown to possess tumour suppressor functionality, with loss of heterozygosity of SDH
gene germline mutations leading to cellular proliferation in paraganglioma and pheochromocytoma,13–15
small cell renal carcinoma44
The reported penetrance of SDHB
mutations is the highest of all SDH genes in familial cases of head and neck paraganglioma and pheochromocytoma, being associated with early-onset tumourigenesis.44
The link between SDH defects and tumourigenesis can be made on more than one level. First, characterised by the Warburg effect, the ability of tumours to switch from aerobic to anaerobic/glycolytic respiration is key for immortalisation.46
Second, succinate-directed activation of the HIF pathway inhibits apoptosis and stimulates angiogenesis.47
Moreover, there is evidence supporting an increase of reactive oxygen species (ROS) in systems with SDH deficiencies; increased ROS is a recognised cause of DNA damage, further compounding the already mounting oncogenic pressures on the cell, increasing the likelihood of transformation to immortalised cell status.41
Proving the pathogenicity of candidate gene mutations is always important, perhaps more so when the genes involved are also tumour suppressors. Having confirmed the SDH mutation as pathogenic, there are further ethical considerations with regard to disclosure of carrier status and prenatal testing. A child born to carrier parents has a 25% risk of a severe neurological phenotype, a 50% risk of ‘elevated cancer susceptibility’ and in the situation where only one parent harbours a SDHB mutation, a 50% risk of elevated cancer susceptibility. The issue of whether clinicians should be able to reveal carrier status as well as ‘clinically affected’ status during prenatal screening is one that should be considered indepth, particularly if there is evidence of oncogenesis in relation to the SDH gene mutation in question.
While the recessive genetic defects in SDHA
identified in the two probands described here have caused severe neurological presentations and complex II deficiency, the fact that the parents of Patients 1 and 2 are heterozygous carriers of germline SDH mutations may place them at an elevated risk of tumourigenesis although neither the SDHA (
p.Ala508Thr and p.Ser509Thr) nor SDHB
(p.Asp48Val) mutations are reported in the Leiden Open Variation Database (http://chromium.liacs.nl/LOVD2/SDH/home.php
), suggesting that these particular mutations have not yet been linked to tumourigenesis. Although there is no indication of cancer susceptibility in either family, both families have been referred for surveillance.
As with the majority of mitochondrial disease presentations, there are no effective cures for SDH deficiency although riboflavin has been shown to alleviate some of the symptoms and delay disease progression.49
Following the diagnosis of SDH deficiency, oral coenzyme Q10 treatment was commenced in Patient 2 and a subjective improvement in strength was reported.
We recommend screening of the SDHA, SDHB and SDHAF1 genes for patients with a biochemically and histochemically characterised isolated complex II deficiency. Identifying the underlying genetic basis of isolated complex II deficiency is vital to ensure that appropriate counselling is available for the family. It facilitates access to cascade screening, and given the increased cancer susceptibility, particularly in relation to SDHB defects, routine surveillance would enable early detection of tumours and appropriate intervention.