HIBCH mutations can cause Leigh-like disease with combined deficiency of multiple mitochondrial respiratory chain enzymes and pyruvate dehydrogenase

Mitochondrial disorders affect approximately 1 in 5000 births, and are clinically, biochemically and genetically heterogeneous [1]. Combined deficiency of multiple respiratory chain (RC) enzymes is one of the most frequent findings in children with suspected mitochondrial disease, representing approximately 30% of cases in whom a biochemical abnormality is identified. Approximately 50% of patients with multiple RC deficiencies have impaired replication or maintenance of the mitochondrial DNA (mtDNA), leading to progressive depletion of mtDNA [2] or accumulation of multiple mtDNA deletions. The remaining ~50% of cases have heterogeneous underlying causes, including mitochondrial or nuclear-encoded defects of mitochondrial protein synthesis [3] and the multiple mitochondrial dysfunctions syndrome, in which the activity of PDHc is also impaired [4‐6]. Defects in mtDNA repair, maintenance or translation result in combined deficiency of complexes I, III and IV (i.e. complexes that contain mtDNA-encoded subunits) whereas the multiple mitochondrial dysfunctions syndrome usually affects complexes containing iron-sulphur (Fe-S) clusters (complexes I, II and III) as well as PDHc.

Neurological features of cerebral organic acidurias (disorders of degradation of the carbon skeleton of amino acids) can be clinically and radiologically indistinguishable from mitochondrial encephalomyopathies caused by primary RC deficiencies; seizures, neurological regression and bilateral symmetrical basal ganglia lesions may occur in both groups of disorders [7‐10]. 3-Hydroxy-isobutyryl-CoA hydrolase (HIBCH) is a mitochondrial enzyme that catalyses the fifth step of valine catabolism, the conversion of 3-hydroxy-isobutyryl-CoA to 3-hydroxy-isobutyrate (Figure 1a). HIBCH deficiency has previously been reported in only two patients [11, 12]. We now describe two new genetically confirmed cases (siblings), one of whom presented with combined defects of multiple RC enzymes and the pyruvate dehydrogenase complex (PDHc). This potentially represents a new disease mechanism mimicking the multiple mitochondrial dysfunctions syndrome, namely degradation of multiple enzymes resulting from accumulation of a toxic metabolite methacrylyl-CoA that is postulated to reduce mitochondrial enzyme activities by reacting with exposed thiol groups.

Two brothers with a progressive neurodegenerative disorder are reported. Patient 1 had combined mitochondrial enzyme defects involving PDHc and multiple RC enzymes (complexes I, II + III and IV, Table 1) whereas these were normal in Patient 2. However, the remarkably similar clinical features in both brothers implied that they had the same underlying condition. The presence of persistently elevated plasma hydroxy-C4-carnitine in Patient 2 (Figure 1b, Table 1) suggested the possibility of HIBCH deficiency, which was confirmed by enzyme assay in cultured skin fibroblasts and molecular genetic analysis. Fibroblast HIBCH activity was below detectable limits in both patients (Table 1), and sequence analysis revealed a homozygous missense mutation c.950G <A (p.Gly317Glu) in the HIBCH gene (Figure 4). The causal nature of this mutation is indicated by the fact that this mutation 1) concerns a highly conserved amino acid; 2) is predicted in silico to be deleterious and probably damaging; 3) is the only putative mutation in the coding region of the HIBCH gene encoding HIBCH, an enzyme which was fully deficient in the patients; and 4) has not been observed previously in genome databases, including 200 ethnically matched control alleles. Finally, the mutation segregated with the Leigh-like disease in this family and was heterozygous in the unaffected parents and the maternal aunt and uncle who had a different neurological phenotype. Although we performed extensive metabolic investigations in Patient 1 which did not reveal any other metabolic disturbances, we cannot completely exclude the small possibility that a second autosomal recessive disorder may have been present, which could have accounted for the more severe biochemical defects observed in this patient.

HIBCH deficiency has been reported in only two cases previously including Patient 3 [11, 12]. Table 1 summarises the clinical and biochemical findings in all 4 cases. Consistent features of this condition appear to be hypotonia, poor feeding and developmental regression with seizures starting in infancy (Table 1) and symmetrical involvement of the globi pallidi seen on brain MRI. Images were no longer available from Patient 1 for review, but acute changes suggestive of cytotoxic oedema, shown by imaging changes of restricted diffusion with subsequent evolution to mature scarring, were seen in Patient 2 and also in Patient 3 (previously reported in [12]). The subthalamic regions were also involved in Patient 3. The predilection for the basal ganglia in HIBCH deficiency is intriguing, and the pathomechanism(s) underlying the basal ganglia lesions, and in particular the selective involvement of the globi pallidi and subthalamic nuclei, are not clear. It is possible that the lesions arise from localised cerebral energy failure and subsequent neuronal cell death, as in other mitochondrial diseases including Leigh syndrome (subacute necrotizing encephalomyelopathy) and Kearns-Sayre syndrome [7, 8]. Alternatively, the underlying pathological mechanisms and the imaging changes may more closely resemble those seen in other organic acidurias, such as glutaric aciduria type I and methylmalonic and propionic acidurias, which remain poorly understood [9, 10]. However one hypothesis suggests that one mechanism involves reaction of acrylyl-CoA and glutaconyl-CoA with thiol groups [18] and glutathione depletion has been noted in methylmalonic acidaemia (MMA) [19]. It is also interesting to note that there is some evidence for secondary RC defects as a possible mechanism of basal ganglia damage in these organic acidaemias [20, 21], and one study demonstrated competitive inhibition of PDHc by propionyl-CoA, as well as inhibition of complex III and the α-ketoglutarate dehydrogenase complex [22].

Most cases of multiple RC deficiencies reported in the literature are caused by defects of maintenance or translation of the mtDNA. The former group includes defects of nuclear genes involved in replication of the mtDNA and defects of nucleoside salvage necessary to provide dNTP building blocks for mtDNA synthesis [23]. In the previously reported Patient 3, mtDNA copy number was normal in skeletal muscle (Table 1) and cultured myoblasts and skin fibroblasts (data not shown), excluding the possibility of mtDNA depletion as the cause of the mild RC defects observed in this patient. Disorders of mitochondrial translation may be primary mtDNA defects, such as deletions of mtDNA or point mutations involving the mitochondrial transfer RNA genes, or nuclear gene defects [3]. In a small number of cases, a combined deficiency of multiple RC enzymes associated with PDHc deficiency has been reported, the so-called multiple mitochondrial dysfunctions syndrome. Recently, mutations in two Fe-S cluster assembly proteins were shown to cause multiple mitochondrial enzyme defects including PDHc deficiency. Mutations in NFU1, required late in the maturation of Fe-S clusters, caused a syndrome of fatal infantile encephalopathy and/or pulmonary hypertension in Spanish and Mexican families with combined defects of PDHc, complex II, lipoic acid synthase and the glycine cleavage system [5, 6]. Patients with mutations in BOLA3, which is also needed for Fe-S and lipoic acid biosynthesis, have a similar biochemical phenotype [6, 24], but the precise pattern of RC deficiencies in these defects differs according to the specific Fe-S cluster synthesis pathway that is impaired [6].

With respect to the pathological mechanism underlying HIBCH deficiency, methacrylyl-CoA, a proximal metabolite in the valine degradation pathway (Figure 1a), could well play a causative role. Cysteine and cysteamine conjugates of methacrylyl-CoA were shown to accumulate in multiple tissues, particularly liver, kidney and brain, in the first patient reported with HIBCH deficiency [11]. Cysteine is a non-essential amino acid bearing a thiol side chain that is frequently involved in enzymatic reactions and is susceptible to oxidation, leading to formation of the disulphide cystine. Cystine plays an important structural role in many proteins; disulphide bonds are important in crosslinking proteins, serving to increase rigidity and conferring resistance to proteolytic degradation. Methacrylyl-CoA could react with mitochondrial enzymes containing essential cysteine residues, including PDHc and RC enzymes [25‐29], thereby reducing their activities. Depletion of cysteine also could lead to reduced activity of mitochondrial enzymes containing Fe-S clusters (e.g. RC complexes I, II and III and the Krebs cycle enzyme aconitase), since cysteine provides sulphide for Fe-S clusters. The ability of thiol groups to undergo redox reactions confers antioxidant properties to cysteine, most importantly in the tripeptide glutathione (composed of cysteine, glycine and glutamic acid) and also in mitochondrial thioredoxin. Thioredoxin contains two cysteine residues in its active centre that undergo reversible oxidation/reduction in response to mitochondrial redox status [30]. Depletion of mitochondrial pools of cysteine, glutathione and thioredoxin by methacrylyl-CoA could lead to oxidative damage, with further impairment of RC enzyme function. Indeed, we found mildly reduced muscle glutathione in Patient 3, who also had mildly reduced activities of RC complexes I and IV in muscle (Table 1), and significantly decreased expression of complex IV subunit I in fibroblasts measured by quantitative immunocytochemistry (Figure 3). These effects are likely to vary according to the levels of oxidative stress, which may explain the variable RC and PDHc defects seen in the 3 patients with HIBCH deficiency (Table 1). We have previously demonstrated reversibility of RC defects in Patient 3, who had normal activities of complexes I-IV in a biopsy performed 3 months after the abnormal biopsy shown in Table 1 with mildly reduced complex I and IV levels (Loupatty et al. 2007 [12]). A reduced plasma concentration of glutathione has been found in both mitochondrial disorders and organic acidaemias [31, 32]. Complex I dysfunction was linked to Parkinson’s disease (PD) following the observation of a high incidence of PD in subjects who had recreationally misused 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a known inhibitor of complex I [33] and glutathione depletion has been noted to precede complex I deficiency in PD [34]. Increased oxidation of six complex I cysteine residues was demonstrated in glutathione-depleted mouse brains, an oxidative stress model of murine PD [35]. Methacrylyl-CoA acid could further disrupt enzymatic reactions taking place in mitochondria by irreversibly binding cofactors such as CoA and lipoic acid. Lack of CoA would be expected to inhibit the Krebs cycle, whilst lipoate is an essential component of PDHc.

Toxic damage to mitochondrial enzymes has previously been demonstrated in ethylmalonic encephalopathy. In affected patients mutations of the ETHE1 sulphur dioxygenase lead to accumulation of sulphide, which affects the function of complex IV and the short chain acyl-CoA dehydrogenase involved in mitochondrial fatty acid oxidation [36, 37]. By contrast, complex IV activity appears to be only mildly reduced in HIBCH deficiency, with relatively greater effects on complex I and PDHc observed in Patient 1 in this study.

These cases illustrate the clinical utility of acylcarnitine analysis in the differential diagnosis of suspected mitochondrial disease. The older brother Patient 1 had died before the introduction of blood acylcarnitine analysis into routine metabolic clinical practice, but the persistently elevated hydroxy-C4-carnitine levels in his younger brother Patient 2 eventually led to the correct diagnosis of HIBCH deficiency. Acylcarnitine analysis is a useful investigation in other cases of suspected mitochondrial disease, since it may help to direct diagnostic genetic investigations, for example succinyl and propionyl carnitines may be elevated in patients with succinyl-CoA ligase deficiency caused by SUCLA2 or SUCLG1 mutations [38, 39].

In conclusion, HIBCH deficiency should be considered in the differential diagnosis for patients with multiple RC defects, including those with associated PDHc deficiency. Moreover, the presence of hydroxy-C4-carnitine in the acylcarnitine profile should alert the clinician to the possibility of this condition. Our results stress the importance of acylcarnitine analysis in patients with suspected mitochondrial disease.