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doi:10.1093/brain/awm023 Brain (2007), 130, 606 ^ 609 SCIENTIF IC COMMENTARY Mutations in SUCLA2: a tandem ride back to the Krebs cycle Mitochondria are small membrane-bound intracellular organelles that are the principal source of adenosine triphosphate (ATP), the high-energy phosphate molecule required by all human cells. ATP is produced by the mitochondrial respiratory chain which is linked to oxidative phosphorylation. Thirteen essential respiratory chain polypeptides are synthesized from small circles of DNA present within each mitochondrion (the mitochondrial genome, mtDNA). Qualitative defects of mtDNA are a major cause of human disease: both point mutations and deletions of mtDNA cause a biochemical defect which often affects skeletal muscle and the nervous system, presenting with neurological features (Zeviani and Di Donato, 2004). MtDNA is inherited exclusively down the maternal line, and pathogenic mtDNA mutations are either maternally inherited or sporadic, affecting at least 1 in 5000 (Schaefer et al., 2004). More recently, autosomal recessive mitochondrial disorders have gained increasing prominence (Table 1). Mutations have been described in nuclear genes coding for respiratory chain proteins or assembly factors, and nuclear-encoded intra-mitochondrial translation factors have recently entered the lime-light (Coenen et al., 2004; Valente et al., 2007). However, a new class of genetic disease has emerged as an important cause of human pathology, ranking along-side the primary mtDNA mutations in terms of their frequency. These diseases are also due to nuclear gene mutations, and all affect proteins that maintain a healthy mitochondrial genome. Genetic linkage in families with autosomal dominant chronic progressive external ophthalmoplegia (ad-PEO) led the way to pathogenic mutations in three nuclear genes: POLG which codes for the intra-mitochondrial DNA polymerase (pol g), PEO1 which codes for the mtDNA helicase Twinkle, and SLC25A4 which codes for the adenine nucleotide translocase ANT1 (Kaukonen et al., 1996; Spelbrink et al., 2001; Van Goethem et al., 2001). Subsequent candidate gene sequencing identified the first mutation in POLG2 which codes for the pol g accessory subunit (Longley et al., 2006). In all of these dominant disorders, the clinical phenotype is due to the formation of multiple deletions of the mtDNA molecule which develop during life. POLG appears to be a major disease gene, with a plethora of clinical presentations [recently described in Brain (Horvath et al., 2006; Tzoulis et al., 2006)], some of which are autosomal recessive (Naviaux and Nguyen, 2004; Ferrari et al., 2005), and cause a ‘quantitative’ defect of mtDNA: mtDNA depletion. MtDNA depletion or the loss of mtDNA copies has been linked to five additional genes: TK2, DGUOK, TP, MPV17 and SUCLA2. Each gene is associated with an emerging clinical phenotype, although there is some overlap. Mutations in POLG, DGUOK and MPV17 cause an encephalopathy and liver failure in early childhood (Mandel et al., 2001; Naviaux and Nguyen, 2004; Spinazzola et al., 2006), with POLG being the principal cause of the Alpers–Huttenlocher syndrome (Nguyen et al., 2006). Mutations in TK2 typically present with a progressive myopathy in childhood (Saada et al., 2003), or spinal muscular atrophy without liver involvement (Oskoui et al., 2006), and mutations in TP cause thymidine phosphorylase (TP) deficiency and mitochondrial neurogastro-intestinal encephalomyopathy (MNGIE) (Nishino et al., 1999). TP, TK2 and DGUOK code for proteins involved in the essential processes regulating the concentration of nucleotide building-blocks of mtDNA (dNTPs) within mitochondria. Being unable to synthesize their own nucleotides, mitochondria are critically dependent on cytosolic dNTPs, which are partly regulated by TP, and also on the recycling of nucleotides within the mitochondrial matrix by thymidine kinase (TK) and dexoyguanosine kinase (DGUOK) (Marti et al., 2003). Although the function of the inner mitochondrial membrane protein MPV17 remains unclear (Spinazzola et al., 2006), a common theme is emerging, with mtDNA depletion arising through the dysregulation of intra-mitochondrial nucleotide pools. But what about SUCLA2, which codes for the b subunit of the mitochondrial matrix enzyme succinyl-CoA synthase (SCS-A)? SCS-A forms part of the tricarboxylic acid (TCA) cycle first described by Hans Krebs in 1937. At first sight this seems an unlikely member of the pack, but two articles in this volume of Brain add to the single previously described family with mutated SUCLA2, expanding the phenotype into adult life, and firmly establishing SUCLA2 as gene involved in mtDNA maintenance. Homozygous mutations in SUCLA2 were first identified in two cousins from a consanguineous Israeli-Muslim family with a presumed recessive mitochondrial encephalomyopathy (Elpeleg et al., 2005). Both had a biochemical defect affecting three respiratory chain ß 2007 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial use License (http://creativecommons.org/lisences/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distributed, and reproduction in medium, provided the original work is properly cited. Scientific Commentary Brain (2007), 130, 606 ^ 609 607 Table 1 Nuclear genes causing mitochondrial disease Nuclear genetic disorders of the mitochondrial respiratory chain, mutations in structural subunits: Leigh syndrome (complex I deficiencyçmutations in NDUFS1, NDUFS4, NDUFS7, NDUFS8, NDUFV1. Complex II deficiency, SDHA) Cardiomyopathy and encephalopathy (complex I deficiency, mutations in NDUFS2) Optic atrophy and ataxia (complex II deficiencyçmutations in SDHA) Hypokalaemia and lactic acidosis (complex III, mutations in UQCRB) Nuclear genetic disorders of the mitochondrial respiratory chain, mutations in assembly factors: Leigh syndrome (mutations in SURF I and the mRNA binding protein LRPPRC) Hepatopathy and ketoacidosis (mutations in SCO1) Cardiomyopathy and encephalopathy (mutations in SCO2) Leucodystrophy and renal tubulopathy (mutations in COX10) Hypertrophic cardiomyopathy (mutations in COX15) Encephalopathy, liver failure, renal tubulopathy (with complex III deficiency, mutations in BCS1L) Encephalopathy (with complex V deficiency, mutations in ATP12) Nuclear genetic disorders of the mitochondrial respiratory chain, mutations in translation factors: Leigh syndrome, Liver failure and lactic acidosis (mutations in EFG1) Lactic acidosis, developmental failure and dysmophism (mutations in MRPS16) Myopathy and sideroblastic anemia (mutations in PUS1) Leukodystrophy and polymicrogyria (mutations in EFTu) Nuclear genetic disorders associated with multiple mtDNA deletions or mtDNA depletion: Autosomal progressive external ophthalmoplegia (mutations in POLG, POLG2, PEO1 and SLC25A4) Mitochondrial neurogastrointestinal encephalomyopathy (thymidine phosphorylase deficiencyçmutations in TP) Alpers^Huttenlocher syndrome (mutations in POLG and MPV) Infantile myopathy/spinal muscular atrophy (mutations in TK2) Encephalomyopathy and liver failure (mutations in DGUOK ) Hypotonia, movement disorder and/or Leigh syndrome with methylmalonic aciduria (mutations in SUCLA2) Others Co-enzyme Q10 deficiency (mutations in COQ2) Barth syndrome (mutations in TAZ) Cardiomyopathy and lactic acidosis (Mitochondrial phosphate carrier deficiency, mutations in SLC25A3) complexes containing mtDNA encoded subunits (I, III and IV), associated with mtDNA depletion. Microsatellite markers spaced every 10 cM (10 million, or mega-bases, Mb) across the whole genome revealed regions of shared homozygosity spanning 20 Mb, where the cousins had identical pairs of alleles. Most mitochondrial proteins are synthesized in the cytoplasm with an additional peptide sequence at the N-(amino)-terminal, which targets the protein to mitochondria. Of the 113 predicted genes within the homozygous region, only three coded for proteins with a mitochondrial target predicted in silico. One of these was SUCLA2, which contained a complex rearrangement (both a deletion and an insertion) of the gene. The affected cousins were homozygous for the mutation, which is predicted to alter the way that the coding (exonic) and non-coding (intronic) regions of the gene are spliced to make the mature messenger RNA coding for SCS-A. SCS-A consists of two different protein subunits (a heterodimer). The a subunit is shared with SCS-G, which uses GTP as a substrate, with the nucleotide specificity being determined by the b subunit (Johnson et al., 1998). Both SCS-G and SCS-A are widely expressed in the liver, kidney and heart, but SCS-A is the dominant enzyme in the central nervous system (Lambeth et al., 2004), possibly explaining the tissue-selectivity seen in the original family (Elpeleg et al., 2005). Twelve patients are described in the first paper (page 853), all with ancestors originating from the Faroe islands, with five linked geneologically (Ostergaard et al., 2007). Clinically the disorder presented at birth or early infancy with hypotonia and motor delay, followed by failure to thrive leading to gastrostomy feeding. All subjects developed a hyperkinetic movement disorder and severe sensorineural deafness requiring cochlear implantation in some. Seven patients died at between 8 months and 21 years of age, usually from respiratory infections, with a 10-year-old being the oldest of five surviving children. Fourteen cases were described in the second paper (page 862) (Carrozzo et al., 2007), including three from southern Italy, with one being of Rumanian extraction. The clinical course in both cohorts was strikingly similar, although careful scrutiny reveals some overlap, with the same 10 patients being described in both papers (Table 2). This makes a total number of 16 new cases described in this edition of Brain. Brain imaging revealed abnormalities in the putamen and caudate nuclei, global atrophy in the more advanced cases and dysmyelination in some. The extent and severity differed both between subjects and between the two studies, probably reflecting the age of each subject and the different imaging modalities used. Proton magnetic resonance spectroscopy in one case showed a high lactate level in 608 Brain (2007), 130, 606 ^ 609 Scientific Commentary Table 2 Correspondence between the patients with SUCLA2 mutations described by Ostergaard et al., 2007 and Carrozzo et al., 2007 Carrozzo et al., 2007 Table 1, patient number Ostergaard et al., 2007 Table 1, patient initials 1 2 3 4 5 6 7 8 9 10 11 12 13 14 ^ ^ ^ ^ ^ ^ BC EH AH JR TO DJ FT BN TD RJ LD BD ^ ¼ not described in that paper. the basal ganglia, providing a clue to the underlying metabolic disturbance. This was reflected by high lactate levels in plasma and CSF in some. Rather surprisingly, epilepsy is not a prominent feature of this encephalopathy. In both studies, all of the patients had abnormal respiratory chain activity in skeletal muscle, with multiple complex defects and depletion of mtDNA (ranging between 15 and 41% of control values across both cohorts). Given the tendency to carry out less invasive procedures on infants, it is important to note that similar investigations on cultured fibroblasts were normal in some patients. The presumed recessive inheritance and ethic homogeneity led one group (Ostergaard et al., 2007) to map the whole human genome with single nucleotide polymorphisms (SNPs) using 10 000 SNPs on a microarray to look for regions of shared homozygosity. This lead to a 1.4 Mb locus by a classical ‘reverse genetic’ approach, refining 25 000 genes to a short list of 10 which included SUCLA2. The other group (Carrozzo et al., 2007) took a ‘forward genetic’ approach which led them, in tandem, to the same gene. They noted high levels of methylmalonic acid in plasma and urine in all patients, which pointed to enzymes in the relevant metabolic pathway and the underlying genetic defect (see figure 1 Carrozzo et al., 2007 or figure 5 in Ostergaard et al., 2007). Both groups identified the same homozygous SUCLA2 mutations in all of the Faroese patients (IVS4 þ 1G4A and c.534 þ 1G4A are actually the same mutation, described by different nomenclature). This mutation was predicted to alter exon splicing and introduce a premature stop codon, further shortening the mRNA transcript. Analysis of the cDNA (formed in vitro from the processed mRNA) revealed exon skipping, although it is intriguing that the two groups identified different transcripts in cultured skin fibroblasts from the same underlying mutation. Further work at the protein level will clarify the principal molecular mechanism, which could vary from tissue to tissue. Both groups determined the carrier frequency in Faroese control subjects, and report a reassuringly similar value (2 vs 3% of the population, which is not significantly different, Fisher’s exact P ¼ 0.27), explaining the high prevalence of the disease in this geographically isolated population (1 in 2500). The patients from southern Italy harboured different mutations, both predicted to alter the protein sequence. SCS-A activity was measured in one patient and was low, and the amount of the SUCLA2 encoded b subunit was reduced in muscle from the Italian and Faroese patients (Carrozzo et al., 2007). The description of additional cases with mutations affecting the coding sequence and a different splice site confirms the role of SUCLA2 as an important human disease gene, and identifies another cause of methylmalonic acidaemia. The methylmalonic acid accumulates because a build up of substrate limits its conversion to succinyl-CoA via methylmalonyl-CoA (see figure 1 Carrozzo et al., 2007 or figure 5 in Ostergaard et al., 2007). So far, the clinical presentation seems consistent, despite the different underlying molecular mechanisms, and measuring urine organic acids provides a non-invasive out-patient-based screening test for the condition. In the future, cases are likely to be detected by tandem mass spectrometry in neonates based on the profile of abnormal carnitine esters seen in their patients (Carrozzo et al., 2007). However, developing an effective treatment depends on our understanding of the disease mechanism—and the fundamental question still remains: how does a Krebs cycle enzyme cause mtDNA depletion? SCS-A is tightly associated with nucleoside diphosphate kinase (NDPK) (Kowluru et al., 2002), which catalyses the exchange of phosphates between tri- and di-phosphoribonucleosides, and is critically involved in the salvage dNTPs during mtDNA synthesis (Bradshaw and Samuels, 2005). Precisely how the two enzymes interact is not clear, but the fundamental role of NDPK in rate-limiting mtDNA replication, with its limited expression in brain (Milon et al., 1997), strongly implicates this interaction as being fundamentally important. This is encouraging, because new treatments are under development for related disorders of intra-mitochondrial nucleoside metabolism, including bone marrow transplantation (Hirano et al., 2006; Lara et al., 2006). Measuring nucleosides levels within different cellular compartments will hopefully clarify the role of NDPK in patients with SUCLA2 mutations. Unfortunately, although some cases of methylmalonic aciduria are vitamin B12 responsive, anecdotally this treatment did not help the Faroese group (Ostergaard et al., 2007). The SUCLA2 story provides further support for ongoing genetic-mapping studies of rare recessive disorders, encouraging us to think ‘outside the box’ when searching Scientific Commentary for molecular mechanisms of disease. Moreover, mutations in SUCLA2 provide key evidence that SCS-A is ‘moonlighting’—beyond its conventional role described by Hans Krebs. 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