UvA-DARE (Digital Academic Repository) Branched chain amino acids : facts and defects Loupatty, F.J. Link to publication

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1 UvA-DARE (Digital Academic Repository) Branched chain amino acids : facts and defects Loupatty, F.J. Link to publication Citation for published version (APA): Loupatty, F. J. (2007). Branched chain amino acids : facts and defects General rights It is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons). Disclaimer/Complaints regulations If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible. UvA-DARE is a service provided by the library of the University of Amsterdam ( Download date: 24 Dec 2017

2 Branched Chain Amino Acids Facts and Defects ACADEMISCH PROEFSCHRIFT ter verkrijging van de graad van doctor aan de Universiteit van Amsterdam op gezag van de Rector Magnificus prof. dr. J.W. Zwemmer ten overstaan van een door het college voor promoties ingestelde commissie, in het openbaar te verdedigen in de Aula der Universiteit op dinsdag 29 mei 2007, te uur door Ference John Loupatty geboren te Amersfoort

3 Promotiecommissie Promotor Prof. dr. R.J.A. Wanders Co-promotor Dr. M. Duran Overige leden Prof. dr. J.M.F.G. Aerts Prof. dr. G. Brown Prof. dr. W.H. Lamers Prof. dr. A. Sturk Prof. dr. F.A. Wijburg Dr. H.R. Waterham Faculteit der Geneeskunde The work described in this thesis was carried out at the laboratory Genetic Metabolic Diseases, Departements of Clinical Chemistry and Paediatrics, Emma Children s Hospital, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

4 Contents Scope of this Thesis 5 Branched Chain Amino Acids: Facts and Defects 7 3-Methylglutaconic Aciduria Type I is Caused by 19 Mutations in AUH Am J Hum Genet. 2002; 71(6): Direct Non-isotopic Assay of 3-Methylglutaconyl-CoA Hydratase 23 Clin Chem. 2004; 50(8): Identification of a Novel Enoyl-CoA Hydratase Encoded 27 by ECHDC2 Clinical, Biochemical and Molecular Findings in Three 35 Patients with 3-Hydroxyisobutyric Aciduria Mol Genet Metab. 2006; 87(3): Mutations in the Gene Encoding 3-Hydroxyisobutyryl-CoA 41 Hydrolase Results in Progressive Infantile Neurodegeneration Am J Hum Genet. 2007; 80(1):195-9 Summary 46 Vertakte keten aminozuren: Feiten en Fouten 47 Dankwoord 48

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6 Chapter 1 Scope of this Thesis Ference J. Loupatty In recent years tremendous progress has been made with respect to the enzymology of the catabolic pathway of the branched chain amino acids and defects therein. First, the introduction of tandem mass-spectrometry for the analysis of plasma acylcarnitines has facilitated the identification of patients with a defect in the catabolic pathways of branched chain amino acids to a great extent. Second, a number of new enzymes have been identified, including 2-methylbutyryl-CoA dehydrogenase, 2-methyl-3-hydroxybutyryl-CoA dehydrogenase and 3-hydroxyisobutyryl-CoA hydrolase. Finally, several branched chain organic acidurias have been resolved at the molecular level. These developments explain why the number of patients with an inborn error of branched chain amino acid catabolism has increased over the last decade. An update of the current state of knowledge regarding the clinical, biochemical and molecular aspects of branched chain organic acidurias, which includes data presented in the consecutive chapters, is described briefly in chapter 2. Nonetheless, at the start of this project the metabolic and molecular basis of several inborn errors of branched chain amino acid metabolism, including 3-methylglutaconyl-CoA hydratase deficiency, 3-hydroxyisobutyric aciduria and 3-hydroxyisobutyryl-CoA hydrolase deficiency, were still elusive. Therefore, it was the purpose of this study to examine and elucidate the underlying defect in these disorders and to devise new and improved means to identify patients with a branched chain organic aciduria. The results are presented in chapters 3 to 7. In chapter 3 the resolution of the molecular basis of 3-methylglutaconyl-CoA hydratase deficiency is presented. The need to differentiate between 3-methylglutaconic aciduria type I and other forms of 3-methylglutaconic aciduria which are all characterized by normal 3-methylglutaconyl-CoA hydratase activity necessitated the development of a specific assay to measure this enzyme activity. The results are described in chapter 4. In our quest to resolve the molecular basis of 3-methylglutaconic aciduria type I another gene was encountered which encodes a protein with similar enzymatic properties as 3-methylglutaconyl-CoA hydratase. This novel enoyl-coa hydratase was characterized and compared to 3-methylglutaconyl-CoA hydratase (chapter 5). In an effort to elucidate the abnormal metabolite profiles detected in patients suffering from 3-hydroxyisobutyric aciduria, the molecular and biochemical characterization of the human 3-hydroxyisobutyrate dehydrogenase was undertaken (chapter 6). After its initial report in 1982 we have resolved the metabolic and molecular basis of 3-hydroxyisobutyryl-CoA hydrolase deficiency. In addition, through acylcarnitine profiling a second patient with 3-hydroxyisobutyryl- CoA hydrolase deficiency has been identified of which the results are presented in chapter 7. From the Departments of Pediatrics, Emma Children s Hospital, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands (F.J.L.)

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8 Branched Chain Amino Acids: Facts and Defects Ference J. Loupatty Chapter 2 General aspects of BCAAs catabolism The three branched chain amino acids (BCAA) leucine, isoleucine and valine are neutral aliphatic amino acids, each with a methyl branch in the side chain. As the human body is unable to synthesize these amino acids de novo, the BCAAs are essential nutrients. Although they are present in all protein-containing foods, the most prominent sources are dairy products, red meat, whey and egg protein. Most diets provide adequate amounts of these amino acids which is approximately mg per kg body weight per day. 1,2 The BCAAs are indispensable amino acids as stimulants of protein synthesis and as building blocks of proteins. 1,3 BCAAs obtained in excess are immediately catabolized in cells, because amino acids are little stored in free form in mammalian species. BCAAs are metabolized by skeletal muscle, liver, kidney, heart, brain and adipose tissue as an alternative energy source, or used for the biosynthesis of lipids. In humans, the BCAAs are metabolized in the mitochondria via the concerted action of a series of enzymes to simple organic acid intermediates that enter general metabolism (figure 1). The catabolic pathways of the BCAAs can be divided into two sequential series of reactions, referred to as the common pathway and the distal pathway. The process begins with the transport of the BCAAs via a Na + -dependent L-amino acid Figure 1. Catabolic pathways for the branched chain amino acids valine, isoleucine and leucine. The structures and names of the intermediate are denoted whereas the enzymes that catalyze the reactions are indicated by their E.C. numbers. CH 3 CH 3 O HO S HO S S CoA CoA HO CoA O O O O O D-methylmalonyl-CoA L-methylmalonyl-CoA Succinyl-CoA From the Departments of Pediatrics, Emma Children s Hospital, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands (F.J.L.)

9 specific transport protein across the plasma membrane into the cells. Once in the cytoplasm, the BCAAs can enter the mitochondria in two ways. First, the BCAAs can be transaminated in the cytosol by the reversible action of a cytosolic branched chain aminotransferase. The resulting branched chain 2-oxo acids are then transported across the mitochondrial innermembrane via a specific transporter protein into the matrix space. Alternatively, the BCAAs can enter the mitochondria via a neutral amino acid carrier protein, and are then converted to their corresponding branched chain 2- oxo acids by a mitochondrial aminotransferase. Once inside the mitochondria, the branched chain 2-oxo acids are oxidatively decarboxylated by the branched chain 2-oxo acid dehydrogenase (BCKADH) complex to form branched chain acyl-coa thioesters with one carbon atom less than the parent amino- or 2-oxoacids. This reaction is irreversible and considered the ratelimiting step in the catabolic pathway of BCAAs. After these common steps the degradative pathway for each of the BCAAs diverges, in a so-called distal pathway, generating different end products. Moreover, the distal pathways are completely different for the three BCAAs, and comprise enzymes specific for each amino acid. Remarkably, the catabolic pathways of leucine, valine and isoleucine present many analogies to fatty acid oxidation. Isoleucine degradation The major pathway of isoleucine degradation proceeds via metabolites having the S-stereochemical configuration to the common intermediates acetyl- CoA and propionyl-coa. This S-pathway of isoleucine degradation is depicted in figure 2. First, isoleucine is transaminated to 2-oxo-3-methylvaleric acid, followed by oxidative decarboxylation to 2-methylbutyryl-CoA. Next, like any 2-methyl branched chain fatty acid, 2- methylbutyryl-coa undergoes further breakdown by oxidation via a four-step pathway involving dehydrogenation to tiglyl-coa, hydration to 2- methyl-3-hydroxybutyryl-coa, dehydrogenation to 2- methylacetoacetyl-coa, and finally, thiolytic cleavage to produce acetyl-coa plus propionyl-coa. This fourstep sequence constitutes a complete cycle of fatty acid β-oxidation. It is generally accepted that the enzyme crotonase which is involved in the β-oxidation of fatty acids is responsible for the hydratase activity in both the isoleucine and valine oxidation. Human crotonase deficiency has not been reported so far. As isoleucine has an asymmetrical carbon atom it can be converted to its diastereomer, alloisoleucine. The degradation of alloisoleucine proceeds through the minor R-pathway (not discussed here), which results in different end products as compared to the major S-pathway for Figure 2. Catabolic pathway of Isoleucine. The structures and the names of the intermediates are shown in the center with the names of the enzyme on the left and the metabolites that may be elevated due to a deficiency in one of these enzymes shown on the right. (BCKADH complex: branched chain ketoacid dehydrogenase complex; SBCAD: short/branched chain acyl-coa dehydrogenase; SCHMAD: short chain hydroxymethylacyl-coa dehydrogenase)

10 Figure 3. Catabolic pathway of leucine. The structures and the names of the intermediates are shown in the center with the names of the enzyme on the left and the metabolites that may be elevated due to a deficiency in one of these enzymes shown on the right. (BCKADH complex: branched chain ketoacid dehydrogenase complex; IVD: isovaleryl-coa dehydrogenase; MCC: 3- methylcrotonyl-coa carboxylase; 3MGH: 3-methylglutaconyl-CoA hydratase) isoleucine. Three inborn errors of the distal pathway of isoleucine degradation, i.e. 2-methylbutyryl- CoA dehydrogenase deficiency, 2-methyl-3- hydroxybutyryl-coa dehydrogenase deficiency and 2- methylacetoacetyl-coa thiolase deficiency, have now been resolved at both the enzymatic and molecular level and will be discussed below. Leucine degradation The catabolic pathway of leucine is depicted in figure 3. First, leucine is transaminated to 2-oxoisocapronic acid, which is subsequently converted into isovaleryl- CoA. In contrast to 2-methylbutyryl-CoA, isovaleryl- CoA cannot undergo fatty acid β-oxidation as the 3-methyl group blocks β-oxidation. Instead, isovaleryl-coa undergoes a four-step process, involving dehydrogenation to 3-methylcrotonyl-CoA, carboxylation to 3-methylglutaconyl-CoA, hydration to 3-hydroxy-3-methylglutaryl-CoA, and finally a thioester hydrolysis forming acetyl-coa and acetoacetic acid. Leucine degradation is different from the degradation of the other branched chain amino acids, in that it includes a carboxylation step, which requires a biotin cofactor. Four inborn errors of the distal pathway of isoleucine degradation, including: (1) isovaleric acidemia, (2) isolated 3-methylcrotonyl-CoA carboxylase deficiency, (3) 3-methylglutaconyl-CoA hydratase deficiency and (4) 3-hydroxy-3-methylglutaryl-CoA lyase deficiency, have been identified and will be discussed below. Valine degradation The metabolic and molecular basis of valine degradation has always been poorly understood. Now, with the recent resolution of 3-hydroxyisobutyryl-CoA hydrolase deficiency (see chapter 7) 4 and isobutyryl- CoA dehydrogenase deficiency some light has been shed on this enigmatic pathway. The catabolic route of valine is exceptional, because it is believed that part of the pathway between 3-hydroxyisobutyryl-CoA and propionyl-coa proceeds via free acids, thus requiring a specific hydrolase (figure 4). This is in marked contrast to the degradation pathways of the other branched chain amino acids, i.e. leucine and isoleucine, in which the intermediates distal to the 2-oxoacids are all CoA thioesters. Indeed, the hydrolysis of an activated

11 Figure 4. Catabolic pathway of valine. The structures and the names of the intermediates are shown in the center with the names of the enzyme on the left and the metabolites that may be elevated due to a deficiency in on of these enzymes shown one the right. (BCKADH complex: branched chain ketoacid dehydrogenase complex; ACAD8: isobutyryl-coa dehydrogenase; HIBCH: 3-hydroxyisobutyryl-CoA hydrolase; HIBADH: 3-hydroxyisobutyrate dehydrogenase; MMSDH: methylmalonic semialdehyde dehydrogenase acyl group in the heart of a catabolic pathway is not only uncommon, but also energetically unfavourable especially when subsequent steps of the pathway again involve CoA thioester intermediates. The resolution of the aforementioned disorder has provided conclusive evidence which indicates that in humans, part of the catabolic pathway of valine does indeed proceed via free acids. This opens the door to a better understanding of the catabolic pathway of valine and defects therein. Inborn errors of branched chain amino acids metabolism Maple Syrup Urine Disease (MSUD; MIM ) MSUD is caused by a defect in the branched chain 2- oxo acid dehydrogenase (BCKADH) complex which catalyses the second step in branched chain amino acid metabolism This multi enzyme system is composed of three components: (1) a decarboxylase that requires thiamine as a cofactor (E1α and E1β), (2) a dihydrolipoyl acyltransferase (E2), and (3) a dihydrolipoamide dehydrogenase (E3). A deficiency of each of these 10 catalytic subunits can cause MSUD. The diagnosis can be readily made by amino acid analysis and organic acid analysis. The BCAAs are considerably elevated in body fluids which include blood, cerebrospinal fluid and urine, and the presence of alloisoleucine is pathognomonic for MSUD. In addition, a defect in BCKADH will result in the increased excretion of 2- oxo-3-methylvaleric acid, 2-oxoisocaproic acid and 2-oxoisovaleric acid. These organic ketoacids give the urine its intensive and characteristic maple-syrup like odour, hence the name of the disease. Since its initial report by Menkes and associates 11 five distinct clinical and biochemical phenotypes classic, intermediate, intermittent, thiamine-responsive and dihydrolipoamide dehydrogenase deficient MSUD have been recognized. 5,6,8,9,12 However, categorization of the variant cases is often complicated since no uniform criteria are present. Classic MSUD with a neonatal onset of encephalopathy represents a severe, but most common form of this disorder. Affected newborns appear normal at birth, and symptoms often develop between four to

12 seven days of age. Lethargy and poor feeding are usually the first signs followed by weight loss and alternating hypertonia and hypotonia. Ketosis and the maple syrup-like odour now become apparent. The majority of these patients die within the early months of life from recurrent metabolic crises, neurological deterioration and coma if left untreated. The surviving patients often manifest severe neurological damage which includes mental retardation. In classic MSUD BCAAs levels, in particular leucine, are markedly elevated and more than 50 % of the branched chain 2-oxo acids are derived from leucine. BCKADH activities are less than 2% of normal. The intermediate and intermittent forms of MSUD are mild clinical phenotypes which are associated with higher BCKADH activities, i.e % and 5-20 % of normal, respectively. 13 Most patients with the intermediate form were diagnosed between the age of 5 months and seven years when evaluated for developmental delay or seizures. Episodes of ketoacidosis and acute encephalopathy were rare in these patients. Persistent elevated excretion of BCAAs and 2-oxo acids 75% derived from isoleucine were noted. In contrast, patients with the intermittent form of MSUD demonstrated normal BCAAs levels when asymptomatic. Moreover, the hallmark of the intermittent type is its late onset and the episodes of acute behavioural change and unsteady gait, often associated with increased protein consumption or intercurrent illness, that may progress to seizures and coma. Some patients have died during these episodes. A number of patients with thiamine-responsive MSUD have been reported in which the metabolic abnormalities were ameliorated with large doses of thiamine (vitamin B1). In general, these patients do not present with an acute neonatal illness, and their early clinical course resembles that of the intermediate form of MSUD. Dihydrolipoamide dehydrogenase (E3) is not exclusive for the BCKADH, but is also a component of α-ketoglutarate dehydrogenase and pyruvate dehydrogenase. A defect in E3 will result in combined ketoacid dehydrogenase deficiency and the clinical manifestations are similar to the intermediate form of MSUD. However, the organic acid profile of E3 deficiency demonstrates abnormalities of both lactic acidosis and MSUD. Plasma BCAAs are moderately increased as compared to classic MSUD. Dietary restriction of BCAAs has proven a valuable strategy in the treatment of MSUD. However, life-long treatment is an absolute necessity as potentially fatal episodes of metabolic decompensation may occur in MSUD as a result of environmental factors which include infection, fever or any other recurrent illness at any age. Thiamine therapy is advisable in every novel patient to determine thiamine-responsiveness. Marked accumulation of BCAAs and their corresponding ketoacids will cause an acute deterioration of cerebral functions if left untreated. This life-threatening situation requires aggressive and prompt treatment which may include peritoneal dialysis or hemodialysis These aggressive approaches are warranted with plasma leucine in excess of 800 µmol/l. Orthotopic liver transplantation has been performed in several patients with MSUD and turned out to be an effective method in controlling plasma BCAA levels. 14,15 More importantly, these patients remained metabolically and neurologically stable on an unrestricted diet for more than two years. Inborn Errors of Leucine Degradation Isovaleric acidemia (MIM ) Isovaleric acidemia is caused by a deficiency of the enzyme isovaleryl- CoA dehydrogenase. This flavoenzyme catalyses the conversion of isovaleryl-coa to 3-methylcrotonyl- CoA and transfers electrons to the electron transfer flavoprotein. Since its first report by Tanaka and coworkers 16 approximately one hundred cases have been reported 17-19, but many more are known todate. Initially, two distinct forms of isovaleric acidemia were recognized. The acute neonatal form which causes massive metabolic acidosis, encephalopathy and will lead to rapid death, and the chronic form which is characterized by episodes of severe ketoacidotic attacks with asymptomatic intervals. More recently, a third phenotype has been identified in which patients manifest mild biochemical abnormalities or are asymptomatic. 20 The phenotypic abnormalities of isovaleric acidemia are due to the accumulation of isovaleric acid which is toxic to the central nervous system. Interestingly, during episodes of acute metabolic decompensation the concentration of isovaleric acid can reach levels as high as several hundred times normal values, but due to its rapid conjugation to other compounds isovaleric acid itself is not the hallmark of this disorder. In fact, isovalerylcarnitine in conjunction with extremely low carnitine, and isovalerylglycine are the distinctive metabolites elevated in this disorder in plasma and urine, respectively The majority of patients with isovaleric acidemia today are diagnosed through newborn screening by use of acylcarnitine profiling in dried blood spots which reveals increased amounts of C 5 -acylcarnitine, i.e. isovalerylcarnitine. The gene encoding isovaleryl-coa dehydrogenase is mapped to chromosome 15q14-q15 and consists of 12 exons that span 15 kb of genomic DNA. Although, several disease-causing mutations have been documented in the numerous patients with isovaleric acidemia, no genotype-phenotype correlation has been found. The reduction of leucine intake and supplementation with either glycine or carnitine have proven useful strategies in the management of this disorder. Indeed, administration of glycine reduces isovaleric acidemia in neonates by conjugating isovaleric acid, with urinary excretion of the conjugate. 24,25 Isolated 3-methylcrotonyl-CoA carboxylase deficiency (MIM and MIM ) 3-Methylcrotonyl-

13 CoA carboxylase is a heteromeric mitochondrial enzyme comprising biotin-containing α subunits and smaller β subunits, encoded by the MCCA and MCCB gene, respectively. The enzyme catalyses the fourth step in leucine catabolism converting 3- methylcrotonyl-coa to 3-methylglutaconyl-CoA in a reversible ATP-dependent reaction using biotin as source of the carboxyl-group. 3-Methylcrotonyl-CoA carboxylase deficiency is caused either by mutations in the structural genes MCCA and MCCB, or by deficient activities of the enzymes involved in the metabolism of its cofactor biotin. In addition to 3-methylcrotonyl-CoA carboxylase, three other biotin-dependent carboxylases exist in human pyruvate carboxylase, acetyl-coa carboxylase and propionyl-coa carboxylase and a deficit in biotin will result in multiple-carboxylase deficiency. Isolated 3-methylcrotonyl-CoA carboxylase deficiency is inherited as an autosomal recessive trait and the molecular basis of this disorder was resolved in 2001 by two independent groups. 26,27 The characteristic abnormal urinary metabolites in isolated 3-methylcrotonyl-CoA carboxylase deficiency are 3- hydroxyisovaleric acid and 3-methylcrotonylglycine, usually in conjunction with severe secondary carnitine deficiency. Indeed, acyl-coas that accumulate in this disorder are readily transesterified to acylcarnitine species with 3-hydroxyisobutyrylcarnitine characteristically present in blood and urine. This explains the acute carnitine depletion. At present more than fifty patients with an isolated deficiency in 3-methylcrotonyl-CoA carboxylase have been reported. The phenotypic presentation is highly variable. Some patients present in the neonatal period with seizures and muscular hypotonia, whereas others demonstrated severe psychomotor retardation, but most children manifest acute episodes of metabolic decompensation followed by complete recovery. These episodes often arise after an infection or fasting, and include feeding problems, vomiting, failure to thrive, lethargy, apnea, hypotonia and infantile spasms. For definitive diagnosis of isolated 3-methylcrotonyl- CoA carboxylase deficiency, one should demonstrate deficient 3-methylcrotonyl-CoA carboxylase activity with normal enzyme activities of at least one of the other carboxylases. 3-methylglutaconic aciduria type I (MIM ) 3- Methylglutaconic aciduria type I represents the isolated deficiency of 3-methylglutaconyl-CoA hydratase. Sixteen patients with 3-methylglutaconic aciduria type I have been reported Whereas the clinical manifestations in some patients is confined to just a mildly delayed speech development, several other patients have been reported with severe (leuko)encephalopathy in both the neonatal period and late adulthood. No episodes of acidosis have been reported. In addition, three patients with 3-methylglutaconyl-CoA hydratase deficiency were initially misdiagnosed as suffering from multiple sclerosis. 28,30,31 In these patients 12 magnetic resonance imaging of the brain has shown widespread involvement of the cerebral white matter and bilateral involvement of the cerebellar peduncles, closely resembling the brain abnormalities observed in multiple sclerosis. Three additional forms of 3- methylglutaconic aciduria have been recognized all characterized by normal hydratase activities. Moreover, patients with type I additionally excrete increased amounts of 3-hydroxyisovaleric acid and therefore, 3- hydroxyisovaleric acid is an important parameter for differentiation between isolated 3-methylglutaconyl- CoA hydratase deficiency and the other types. More importantly, the advent of a specific enzymatic assay to determine 3-methylglutaconyl-CoA hydratase activity has greatly facilitated the identification of patients with 3-methylglutaconic aciduria type I (see chapter 4). 40 In 1995 Nakagawa purified an AU-specific RNAbinding protein which was believed to be involved in the turnover of mrna In addition, this protein demonstrated enoyl-coa hydratase activity. In fact, we have shown that this protein is responsible for the 3-methylglutaconyl-CoA hydratase activity in leucine degradation and that mutations in the AUH gene cause 3-methylglutaconic aciduria type I (see chapter 3) hydroxy-3-methylglutaryl-CoA lyase deficiency (MIM ) More than forty patients with 3-hydroxy- 3-methylglutaryl-CoA lyase deficiency have been reported and most of them clinically present within the first year of life This disorder is often a lifethreatening condition in the neonatal period. The key clinical features include severe infantile hypoglycemia, metabolic acidosis, hepatomegaly, lethargy or coma and a characteristic absence of ketosis. The neurological complications in this disorder due to hypoketotic hypoglycemia and acidosis may result in a severe and permanent handicap. Patients may also manifest Reye syndrome-like episodes characterized by increased levels of ammonia and abnormal liver function tests, fever, hepatomegaly, altered levels of consciousness and seizures. 3-Hydroxy-3-methylglutaryl-CoA lyase catalyses the ultimate step of both leucine degradation and ketogenesis, and therefore plays an important role in ketone body production. Nonetheless, the diagnosis is often made from the typical abnormal organic acid profile dominated by leucine metabolites. Indeed, the pattern of urinary organic acids includes 3-hydroxy- 3-methylglutaric acid, 3-methylglutaconic acid, 3- methylglutaric acid and 3-hydroxyisovaleric acid. In common with other (branched chain) organic acidurias, patients with 3-hydroxy-3-methylglutaryl-CoA lyase deficiency usually show an elevated acyl-carnitine to free carnitine ratio, with the most prominent carnitine species in this disorder being 3-methylglutarylcarnitine. Ultimately, the diagnosis is best confirmed by measuring lyase activity 48,49 in conjunction with molecular analysis. Indeed, pathogenic mutations in the encoding HL gene have been identified, but revealed considerable genetic heterogeneity.

14 Inborn Errors of Isoleucine Degradation 2-methylbutyryl-CoA dehydrogenase deficiency (MIM ) 2-Methylbutyryl-CoA dehydrogenase deficiency, also referred to as short/branched chain acyl-coa dehydrogenase (SBCAD) deficiency, has been described as an autosomal recessive disorder of L-isoleucine catabolism. Although 18 individuals have been diagnosed with SBCAD deficiency up till now, the clinical presentation of this disorder is still not well defined ranging from completely asymptomatic to severe neurological handicaps The lack of clinical symptoms in most patients should not give the paediatrician a false sense of security, as environmental factors are often crucial determinants of the clinical course of inborn errors of metabolism. Moreover, in other (branched chain) organic acidurias acute decompensation and death have occurred with the first severe catabolic episode. More importantly, those patients who are clinically symptomatic often demonstrated neurological involvement with epilepsy being the key feature. This may indicate that when SBCAD deficiency manifests itself clinically, the brain and the spinal cord are primarily affected. Although the underlying cause of the central nervous system dysfunction in SBCAD deficiency remains unclear, it is noteworthy that in most branched chain organic acidurias neurological handicaps appear to be common. 2-Methylbutyryl-CoA dehydrogenase catalyses the conversion of 2-methylbutyryl-CoA to tiglyl-coa and a deficiency of this enzyme results in the accumulation of its precursors. It appears that the major fate of 2-methylbutyryl-CoA that accumulates in this disorder is not deacylation to 2-methylbutyric acid but rather transesterification with glycine to form 2-methylbutyrylglycine. Indeed, SBCAD deficiency is characterized by persistently increased urinary excretion of 2-methylbutyrylglycine. In addition, eight additional patients with SBCAD deficiency were detected through prospective newborn screening using tandem mass spectrometry as their plasma acylcarnitine analysis revealed elevated levels of C 5 -acylcarnitine, i.e. 2-methylbutyrylcarnitine. The molecular basis of this inborn error of metabolism has been resolved by Andresen and colleagues. 2-Methylbutyryl-CoA dehydrogenase is encoded by the ACADSB gene located at chromosome 10q26.13 and contains 11 exons. The gene encodes a mitochondrial precursor protein which is cleaved upon mitochondrial import and predicted to yield a mature peptide of approximately 44 kda. At present, a total of nine distinct mutations have been reported in eighteen patients. 2-methyl-3-hydroxybutyryl-CoA dehydrogenase deficiency (MIM ) 2-Methyl-3-hydroxybutyryl- CoA dehydrogenase deficiency has been described as a X-linked neurodegenerative disorder with psychomotor regression. Since its initial report in 2000 by Zschocke and co-workers 56 nine additional 13 affected individuals, including two female patients, have been described These female patients demonstrated a less severe phenotype without loss of developmental milestones which has been attributed to lyonization (X-chromosome inactivation) in most but not all body tissues. Although considerable clinical heterogeneity is observed among all patients, this disorder is characterized by normal early development followed by a progressive loss of acquired mental and motor skills, hypotonia, loss of vision and seizures. The onset of the regression appears to be variable ranging from the neonatal period to as late as five years of age. Furthermore, virtually all patients developed neurological symptoms associated with changes in MRI. The most common MRI finding was a pronounced atrophy of the brain, usually manifesting itself as frontotemporal atrophy. 2-Methyl-3-hydroxybutyryl- CoA dehydrogenase deficiency is biochemically characterized by moderately elevated urinary excretion of tiglylglycine and 2-methyl-3-hydroxybutyric acid. Furthermore, a number of additional metabolites have been found, including elevated CSF or blood lactate, increased urinary amounts of 3-hydroxyisobutyric acid, 2-ethylhydracrylic acid and 3-methylglutaconic acid using urinary organic acid analysis. In addition, analysis of plasma acylcarnitine levels may show a mild increase in C 5:1 -acylcarnitine and C 5 - hydroxyacylcarnitine, representing tiglylcarnitine and 2-methyl-3-hydroxybutyrylcarnitine, respectively. Molecular analysis in eight patients with this disorder revealed only three mutations in the encoding HADH2 gene, all being missense mutations. 61 One missense mutation (c.388c>t; R130C), was identified in five of the eight patients, which is remarkable in a severe X-linked disorder. Expression studies of the mutant cdnas demonstrated that two missense mutations generated a fully inactive protein, whereas some residual activity (2-3% of the wildtype enzyme) was found for the third. There are speculations on an alternative role of the HADH2 protein, possibly associated with other mechanisms of neurological regression. β-ketothiolase deficiency (MIM ) β-ketothiolase deficiency is an autosomal recessive disorder of ketone body metabolism and isoleucine catabolism and is caused by a deficiency in the mitochondrial acetoacetyl- CoA thiolase. 64 This enzyme catalyzes the final step in isoleucine degradation which is the conversion of 2- methylacetoacetyl-coa into acetyl-coa and propionyl- CoA. In ketone body metabolism its substrate is acetoacetyl-coa rather than 2-methylacetoacetyl- CoA. The major clinical manifestations of this disorder are intermittent episodes of acute ketoacidosis which typically occur in a previously healthy child. The age of onset is often between the age of six and twenty four months and is usually triggered by a febrile illness. However, there is considerable clinical heterogeneity in β-ketothiolase deficiency. 53,65-67 Vomiting and dehydration often occur and the patient

15 becomes progressively lethargic, sometimes leading to coma. In contrast, some patients have died or had severe neurological sequelae as a result of recurrent or prolonged episodes of ketoacidosis. Occasionally an affected but asymptomatic sibling or even parent is identified within the same family. During an acute episode patients excrete massive amounts of ketone bodies (3-hydroxybutyric acid and acetoacetic acid), 2-methylacetoacetic acid, 2-methyl-3-hydroxybutyric acid and tiglylglycine. Remarkably, rather than 2- methylacetoacetic acid the most prominent metabolites in ß-ketothiolase deficiency are tiglylglycine and 2-methyl-3-hydroxybutyric acid. ß-Ketothiolase deficiency is caused by mutations in the ACAT1 (acetyl- CoA acetyltransferase 1) gene mapped to chromosome 11q22-q23. 66,68 Over 40 mutations have been identified which revealed a biochemical phenotype-genotype correlation in this disorder. Those patients whose mutation(s) resulted in residual activities of the thiolase protein often demonstrated no increased excretion of tiglylglycine even during ketoacidotic episodes and (borderline) normal urine organic acid and plasma acylcarnitine profiles when in remission. However, no phenotype-genotype correlation has been found. Prompt diagnosis and aggressive treatment will often lead to a rapid and full recovery. In general, treatment with a low protein diet can prevent the recurrence of ketoacidotic episodes and improve the long-term outcome of this disorder. Inborn Errors of Valine Degradation Isobutyryl-CoA dehydrogenase deficiency (MIM ) Isobutyryl-CoA dehydrogenase deficiency was first reported in and to date only nine patients have been reported. In 2000, Andresen et al demonstrated that acyl-coa dehydrogenase (ACAD) 8 is in fact isobutyryl-coa dehydrogenase, after which the molecular basis of isobutyryl-coa dehydrogenase deficiency was solved by the identification of diseasecausing mutations in the ACAD8 gene. 51,70-72 Although significant clinical symptoms, which included dilated cardiomyopathy, failure to thrive and carnitine deficiency, were observed in the first patient reported, the isobutyryl-coa dehydrogenase deficient newborns identified by screening either remained asymptomatic or presented with mild clinical abnormalities. Examination of the urinary organic acid profile of these patients showed marginally elevated excretion of isobutyrylglycine. In addition, acylcarnitine profiling has demonstrated elevated levels of C 4 -acylcarnitines and an increased C 4 /C 2 -acylcarnitine ratio in this disorder. As mentioned below (see diagnosis of branched chain organic acidurias ), tandem-ms does not differentiate between isomers and therefore C 4 - acylcarnitine could represent either butyryl-coa or isobutyryl-coa. Accumulation of butyrylcarnitine is suggestive of short chain acyl-coa dehydrogenase (SCAD) deficiency, an enzymatic defect in fatty acid 14 β-oxidation that may cause severe clinical symptoms. Surprisingly, although disease-causing mutations were found in the ACAD8 gene in the aforementioned patients, SCAD gene analysis revealed heterozygosity for the prevalent c.625g>a susceptibility variation all individuals investigated. This variant of the SCAD gene is frequent in the European population and is considered to confer increased susceptibility to clinical SCAD deficiency in combination with either environmental factors or other genetic factors, such as perhaps mutations in the ACAD8 gene. However, more patients with isobutyryl-coa dehydrogenase deficiency should be identified and subsequently investigated to clarify if isobutyryl-coa dehydrogenase deficiency and the SCAD c.625g>a variation are associated. In addition, several reports have described patients with increased excretion of C4-acylcarnitine and homozygosity for the c.625g>a variant. However, isobutyryl-coa dehydrogenase deficiency has not been excluded and therefore it could prove prudent to analyse the ACAD8 gene and isobutyryl-coa dehydrogenase activity in these patients. 3-hydroxyisobutyryl-CoA hydrolase deficiency (MIM ) 3-Hydroxyisobutyryl-CoA hydrolase deficiency is very rare with only two reported patients sofar. 4 Both male patients clinically presented with poor feeding, hypotonia, developmental delay of motor skills and neurological regression in infancy. Whereas one patient demonstrated episodes of ketoacidosis and Leigh-like changes in the basal ganglia, the other patient presented with dysmorphic features and died at an early age. The biochemical basis of 3- hydroxyisobutyryl-coa hydrolase deficiency was solved in the early 1980s after the finding of elevated urinary levels of S-2-carboxypropyl-cysteine and S- 2-carboxypropyl-cysteamine. 73,74 These derivatives are formed through the addition of cysteine and cysteamine across the carbon-carbon double bond of methacrylyl-coa. Normal enoyl-coa hydratase activities in the patient s fibroblasts suggested a deficiency in 3-hydroxyisobutyryl-CoA hydrolase which was subsequently confirmed enzymatically. In contrast, no unusual sulphur containing amino acids were identified in the urine of the second patient. It was the detection of persistently elevated hydroxy-c 4 - carnitine species in blood that suggested a deficiency of 3-hydroxyisobutyryl-CoA hydrolase. Indeed, enzymatic analysis demonstrated the complete absence of 3-hydroxyisobutyryl-CoA hydrolase activity. The molecular basis of this disorder was only recently resolved by the identification of disease-causing mutations in the encoding HIBCH gene (chapter 7). 4 3-hydroxyisobutyric aciduria (MIM ) 3- Hydroxyisobutyric aciduria is a rare metabolic anomaly in valine degradation and today only thirteen patients have been described in literature These patients usually present with dysmorphic features including

16 microcephaly, a small triangular face, low set ears and long philtrum. Patients may show widely different phenotypes ranging from mild vomiting attacks with normal brain and cognitive development, to delayed motor development, profound mental impairment and early death. Patients with 3-hydroxyisobutyric aciduria usually excrete elevated amounts of 3- hydroxyisobutyric acid (3-HIBA) in urine, ranging from 60 to 390 mmol/mol of creatinine (normal < 40 mmol/mol of creatinine) when in stable condition and increasing up to 10,000 mmol/mol creatinine during acute ketoacidotic episodes. In half of the reported cases elevated lactate levels were observed. Other, less frequently observed abnormal metabolites in urine are 3-hydroxypropionic acid, 2-ethyl-3-hydroxypropionic acid, 2-hydroxybutyric acid, S-(2-carboxypropyl)- cysteine and 3-hydroxyisovaleric acid. Although the responsible defect has not been identified yet, a deficiency of 3-hydroxyisobutyrate dehydrogenase has been suggested as the most likely enzyme defect in patients with 3-hydroxyisobutyric aciduria. Methylmalonic semialdehyde dehydrogenase deficiency (MIM ) So far, only a single patient with methylmalonic semialdehyde dehydrogenase deficiency has been described in literature Routine newborn screening demonstrated a marked hypermethioninemia. The male patient was hospitalized at the age of three weeks with an unexplained episode of diarrhoea and vomiting. Subsequent urinary organic acid analysis revealed extremely elevated levels of 3-hydroxyisobutyric acid. Further investigations also demonstrated increased urinary excretion of ß- alanine, 3-hydroxypropionic acid, and both isomers of 3-aminoisobutyric acids, suggesting methylmalonic semialdehyde dehydrogenase deficiency. Molecular analysis of the human MMSDH gene revealed homozygosity for a missense mutation (c.1336g>a; G446R) and the deletion of exon ,87 No expression studies were performed with the mutant cdna. Inborn Errors of Valine-Isoleucine Degradation Propionic acidemia (MIM ) and methylmalonic aciduria (MIM ) Propionic acidemia and methylmalonic aciduria are the most frequent forms of branched chain organic acidurias (reviewed in ). These autosomal recessive disorders are caused by deficiencies of propionyl-coa carboxylase and methylmalonyl-coa mutase, respectively. Propionyl- CoA carboxylase is a heteromeric mitochondrial enzyme comprising biotin-containing α subunits and smaller β subunits, encoded by the PCCA and PCCB genes, respectively. 91,96 Propionic acidemia is caused either by mutations in the structural genes PCCA or PCCB, or by deficient activities of the enzymes involved in the metabolism of its cofactor biotin. In addition to propionyl-coa carboxylase, three other biotin-dependent carboxylases exist in human 3-15 methylcrotonyl-coa carboxylase (see above), pyruvate carboxylase, and acetyl-coa carboxylase and a deficit in biotin will result in multiple-carboxylase deficiency. Methylmalonic aciduria includes disorders of methylmalonate and cobalamin metabolism. Isolated methylmalonic aciduria is caused by mutations in the MUT gene, which encodes methylmalonyl-coa mutase. Remarkably, patients with propionic acidemia or methylmalonic aciduria manifest similar clinical and biochemical abnormalities and therefore these disorders are discussed here simultaneously. Patients with propionic acidemia persistently demonstrate increased urinary excretion of methylcitric acid often in combination with 3-hydroxypropionic acid and other propionyl-coa metabolites which include propionylglycine, tiglylglycine, 2-methyl- 3-hydroxybutyric acid and 2-methylacetoacetate. Episodes of ketoacidosis occur frequently and are accompanied by the excretion of abnormal, but highly characteristic ketones such as 3-oxovaleric acid and 3- hydroxyvaleric acid. Methylmalonic aciduria will show large amounts of methylmalonic acid in conjunction with the propionyl-coa metabolites which are the result of a secondary inhibition of propionyl-coa carboxylase. Plasma acylcarnitine analysis in both propionic acidemia and methylmalonic aciduria shows elevation of propionylcarnitine as the predominant acylcarnitine species. Free carnitine is reduced and the ratio of propionylcarnitine to free carnitine is greatly increased. There are two vitamin B12 responsive forms of methylmalonic aciduria, denoted CblA and B; both involving defects in adenosylcobalamin metabolism. Methylmalonic aciduria with homocystinuria is caused by cobalamin C/D defects which impair the conversion of vitamin B12 into its two metabolically active forms, methylcobalamin and adenosylcobalamin. This disease usually presents with a different predominantly neurological clinical picture distinct from isolated methylmalonic aciduria. In most cases of propionic acidemia and methylmalonic aciduria the onset of the disease is in the neonatal period in which the patient rapidly deteriorates for no apparent reason. After an initial unremarkable interval the symptoms proceed from vomiting, poor feeding and weight loss to progressive neurologic sequelae which include mental retardation, motor dysfunction, (epileptic) seizures, hypotonia and lethargy. If left untreated, the patient s condition could progress to coma with brain oedema and respiratory arrest, and ultimately permanent brain damage or death. Normochloremic metabolic acidosis, ketonuria, hyperuricemia, hyperammonemia, thrombocytopenia, leukopenia and anemia are typically found which often results in the false diagnosis of sepsis. In the late onset forms, the clinical manifestations range from acute encephalopathy to intermittent or chronic symptoms which include intermittent ataxia, abnormal behaviour and feeding with the selective refusal of protein-

17 rich foods, anorexia, recurrent vomiting, failure to thrive and (neuro)developmental delay. The recurrent episodes are often triggered by stress and between these intervals the patient may appear normal. Management of patients with propionic acidemia and methylmalonic aciduria is difficult. Therapy includes a low-protein high-energy diet and carnitine supplementation. Some patients with methylmalonic aciduria respond to pharmacological doses of vitamin B12. Therefore a trial with vitamin B12 supplementation should always be made in newly diagnosed methylmalonic aciduria patients. Propionic acidemia is theoretically amenable to biotin treatment. No biotin-responsive propionic acidemia patient has been reported thus far, however. Given the poor long-term prognosis, liver transplantation has been attempted recently as an alternative therapy, but this approach has not been proven efficient. Biochemical Diagnosis of Branched Chain Organic Acidurias Among the organic acidurias genetic defects in the degradation of branched chain amino acids are most frequent. Two thirds of the cases manifest themselves during the neonatal period, most of them with an acute onset. Prompt diagnosis of organic acidurias depends on their early identification of children with suspect clinical symptoms. Once an inborn error of metabolism is suspected from the clinical presentation, the diagnosis should be corroborated further through laboratory analysis. First, the biochemical evaluation of patients with a potential metabolic disorder should be aimed at the identification of either abnormal levels of a physiological compound(s) or abnormal metabolite(s) in body fluids. Indeed, accumulation of intermediates prior to an obstruction in a metabolic pathway will provide the paediatrician with clues for the underlying enzymatic defect. The rapid differential diagnosis is best accomplished by determination of an abnormal pattern of urinary organic acids determined by gas chromatography mass spectrometry. The major fates of the acyl-coa thioesters that accumulate as a result of a metabolic block in branched chain organic acidurias are a) hydrolysis to the corresponding organic acid and free coenzyme A, and b) transesterification with glycine and/or carnitine to form acylglycines and/or acylcarnitines, respectively. Although the acylglycines can be detected by routine urinary organic acid analysis some patients may have been overlooked as a result of the insufficient sensitivity of acylglycine detection and the inadequate interpretation of the acylglycine profile. In contrast, organic acids are often easily detected. During acute episodes, patients often excrete massive amounts of accumulated metabolites. However, the elevation, if any, may be very modest when stable. Thus, a diagnosis could be missed when an interval sample is analyzed. In recent years acylcarnitine profiling by means of 16 tandem mass spectrometry has become an increasingly powerful tool in the diagnosis of inborn errors of metabolism. 17,20,50,53,68,70,95, Indeed, this technique has proven crucial in the identification of additional patients with a branched chain organic aciduria, including SBCAD deficiency and 3-hydroxyisobutyryl- CoA hydrolase deficiency. Moreover, acylcarnitine profiling has the additional advantage that it can be applied on dried bloodspots obtained for routine newborn screening. This can ultimately lead to a very early diagnosis of many of the branched chain organic acidurias. The acyl-coa thioesters that accumulate as a result of a metabolic block are in equilibrium with their corresponding acylcarnitines. These carnitine species are present in plasma and readily excreted in urine. However, routine combined high performance liquid chromatography - tandem mass spectrometric analysis as used in many presentday laboratories does not differentiate between isomers and, for example, a C 5 -acylcarnitine species could represent either 2-methylbutyryl-, isovaleryl-, valeryl- or pivaloylcarnitine or even a combination of these isomers, whereas hydroxy-c 5 -acylcarnitine could represent either 3-hydroxyisovaleryl- or 2- methyl-3-hydroxybutyrylcarnitine. Consequently, the contribution of this technique in the diagnosis of inborn errors of metabolism, such as branched chain organic acidurias, is rather limited. Nevertheless, in conjunction with a carefully interpreted urinary organic acid profile one should be able to determine the exact diagnosis. Ultimately, enzymatic studies have to be performed to identify the enzymatic defect, which then must be followed by molecular analysis of the gene involved. Implications for therapy Accumulated intermediates proximal to the enzyme deficiency are usually considered toxic and thus the cause of the primary pathological effects. In addition, much of the physiological abnormalities may result from either endogenous compensatory mechanisms, inhibition of general metabolism, or the reduced availability of pivotal compounds such as free coenzyme A and reducing equivalents. As branched chain amino acids are essential amino acids, an increased flux through the catabolic pathway can only be accomplished via dietary intake or via endogenous breakdown of protein. Thus, treatment of patients with an inborn error of BCAAs degradation should be aimed at removal of toxic compounds, i.e. minimizing the flux through the catabolic pathway in both the acute and chronic state. In the critically ill, immediate treatment should be initiated even if a definitive diagnosis may not yet be established. Within 72 hours, the results of amino acid and organic acid analysis should be available, allowing diagnostic confirmation in most cases. Prompt and aggressive treatment before the confirmation of a diagnosis may be life-saving and could prevent or

18 reduce the neurological sequelae of some of these disorders. First, the patient should be stabilized by protein restriction, administration of fluids and intravenous glucose. Insulin could be added to utilize its anabolic effect. If warranted, (hemo)dialysis should be instituted immediately without waiting a response to the dietary manipulation or medication, providing maximal supportive care simultaneously. Altogether, this will often lead to a rapid and complete recovery provided that treatment is initiated without delay. Although this approach is very effective on short terms, protein degradation will escape the restraint of limited endogenous catabolism within 3 to 5 days and the preceding improvement will arrest or, for worse, the patient will deteriorate. Fortunately, the introduction of formulas deficient in given amino acid enables one to keep the level of the amino acid in question within acceptable limits, while maintaining amino acid homeostasis. This will result in improved growth and fewer and less severe episodes of catabolic decompensation associated with fasting or infection, provided that a high carbohydrate intake is maintained. Thus, selective amino acid restriction in conjunction with a high carbohydrate or lipid intake is an important strategy in the long-term management of many branched chain organic acidurias. Other eminent therapeutic approaches for long-term removal of toxic metabolites include the administration of detoxifying agents. Treatment with either glycine or carnitine could increase the elimination of acyl-coas as urinary acylglycines or acylcarnitines and, thus, prevent the formation of toxic compounds. In addition, organic acidurias often cause elevated amounts of acyl- CoAs, acylglycines and acylcarnitine species, which affect the availability of free coenzyme A, glycine and carnitine. As acylcarnitine species are not reabsorbed in the renal tube, a further decrement in the amount of carnitine is often observed, which could impair substrate transport across the mitochondrial membrane and subsequent energy production. Administration of exogenous carnitine corrects this deficit and may also shift the acyl-coa:coa ratio to free coenzyme A. In addition, provision of pantothenate (vitamin B5) could compensate for the fact that at least part of intracellular CoA will be trapped in the form of acyl-coa esters. Once we have gained more experience with the natural history of branched chain organic acidurias, the role of therapy in these inborn error of metabolism will become clearer. References 1. 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19 al Am J Hum Genet 62: Ozand PT, al Aqeel A, Gascon G, Brismar J, Thomas E, Gleispach H J Inherit Metab Dis 14: Gibson KM, Breuer J, Nyhan WL Eur J Pediatr 148: Wysocki SJ, Hahnel R J Inherit Metab Dis 9: Wanders RJ, Zoeters PH, Schutgens RB, de Klerk JB, Duran M, et al Clin Chim Acta 189: Wanders RJ, Schutgens RB, Zoeters PH Clin Chim Acta 171: Matern D, He M, Berry SA, Rinaldo P, Whitley CB, et al Pediatrics 112: Andresen BS, Christensen E, Corydon TJ, Bross P, Pilgaard B, et al Am J Hum Genet 67: Gibson KM, Burlingame TG, Hogema B, Jakobs C, Schutgens RB, et al Pediatr Res 47: Korman SH Mol Genet Metab 89: Madsen PP, Kibaek M, Roca X, Sachidanandam R, Krainer AR, et al Hum Genet 118: Korman SH, Andresen BS, Zeharia A, Gutman A, Boneh A, Pitt JJ Clin Chem 51: Zschocke J, Ruiter JP, Brand J, Lindner M, Hoffmann GF, et al Pediatr Res 48: Perez-Cerda C, Garcia-Villoria J, Ofman R, Sala PR, Merinero B, et al Pediatr Res 58: Poll-The BT, Wanders RJ, Ruiter JP, Ofman R, Majoie CB, et al Mol Genet Metab 81: Sass JO, Forstner R, Sperl W Brain Dev 26: Sutton VR, O Brien WE, Clark GD, Kim J, Wanders RJ J Inherit Metab Dis 26: Ofman R, Ruiter JP, Feenstra M, Duran M, Poll-The BT, et al Am J Hum Genet 72: Ensenauer R, Niederhoff H, Ruiter JP, Wanders RJ, Schwab KO, et al Ann Neurol 51: Olpin SE, Pollitt RJ, McMenamin J, Manning NJ, Besley G, et al J Inherit Metab Dis 25: Fukao T, Yamaguchi S, Kano M, Orii T, Osumi T, Hashimoto T Prog Clin Biol Res 375: Fukao T, Scriver CR, Kondo N Mol Genet Metab 72: Fukao T, Yamaguchi S, Orii T, Hashimoto T Hum Mutat 5: Ozand PT, Rashed M, Gascon GG, al Odaib A, Shums A, et al Brain Dev 16 Suppl: Pasquali M, Monsen G, Richardson L, Alston M, Longo N Am J Med Genet C Semin Med Genet 142: Roe CR, Cederbaum SD, Roe DS, Mardach R, Galindo A, Sweetman L Mol Genet Metab 65: Pedersen CB, Bischoff C, Christensen E, Simonsen H, Lund AM, et al Pediatr Res 60: Sass JO, Sander S, Zschocke J J Inherit Metab Dis 27: Nguyen TV, Andresen BS, Corydon TJ, Ghisla S, Abd-El Razik N, et al Mol Genet Metab 77: Brown GK, Hunt SM, Scholem R, Fowler K, Grimes A, et al Pediatrics 70: Truscott RJ, Malegan D, McCairns E, Halpern B, Hammond J, et al Biomed Mass Spectrom 8: Loupatty FJ, van der Steen A, Ijlst L, Ruiter JP, Ofman R, et al Mol Genet Metab 87: Sasaki M, Yamada N, Fukumizu M, Sugai K Brain Dev 28: Shield JP, Gough R, Allen J, Newbury-Ecob R Clin Dysmorphol 10: Sasaki M, Iwata H, Sugai K, Fukumizu M, Kimura M, Yamaguchi S Brain Dev 23: Yoshida I Ryoikibetsu Shokogun Shirizu: Sasaki M, Kimura M, Sugai K, Hashimoto T, Yamaguchi S Pediatr Neurol 18: Boulat O, Benador N, Girardin E, Bachmann C J Inherit Metab Dis 18: Chitayat D, Meagher-Villemure K, Mamer OA, O Gorman A, Hoar DI, et al J Pediatr 121: Ko FJ, Nyhan WL, Wolff J, Barshop B, Sweetman L Pediatr Res 30: Gibson KM, Lee CF, Bennett MJ, Holmes B, Nyhan WL J Inherit Metab Dis 16: Roe CR, Struys E, Kok RM, Roe DS, Harris RA, Jakobs C Mol Genet Metab 65: Gray RG, Pollitt RJ, Webley J Biochem Med Metab Biol 38: Zhang YX, Tang L, Hutchinson CR J Bacteriol 178: Deodato F, Boenzi S, Santorelli FM, Dionisi-Vici C Am J Med Genet C Semin Med Genet 142: Feliz B, Witt DR, Harris BT Arch Pathol Lab Med 127: e Ohura T, Miyabayashi S, Narisawa K, Tada K Hum Genet 87: Ugarte M, Perez-Cerda C, Rodriguez-Pombo P, Desviat LR, Perez B, et al Hum Mutat 14: Acquaviva C, Benoist JF, Pereira S, Callebaut I, Koskas T, et al Hum Mutat 25: Kasahara M, Horikawa R, Tagawa M, Uemoto S, Yokoyama S, et al Pediatr Transplant 10: Kobayashi A, Kakinuma H, Takahashi H Pediatr Int 48: Dionisi-Vici C, Deodato F, Roschinger W, Rhead W, Wilcken B J Inherit Metab Dis 29: Lamhonwah AM, Barankiewicz TJ, Willard HF, Mahuran DJ, Quan F, Gravel RA Proc Natl Acad Sci U S A 83: Heldt K, Schwahn B, Marquardt I, Grotzke M, Wendel U Mol Genet Metab 84: Wei CC, Lin WD, Tsai FJ, Wu JY, Peng CT, Tsai CH Acta Paediatr Taiwan 45: Lin JF, Chiu PC, Hsu HY, Lin SM, Chen YY, Hsieh KS Acta Paediatr Taiwan 45: Koeberl DD, Millington DS, Smith WE, Weavil SD, Muenzer J, et al J Inherit Metab Dis 26: Ogier de Baulny H, Saudubray JM Semin Neonatol 7: Wiley V, Carpenter K, Wilcken B Acta Paediatr Suppl 88:

20 Chapter 3 3-Methylglutaconic Aciduria Type I is Caused by Mutations in AUH Ference J. Loupatty, Lodewijk IJlst, Jos P.N. Ruiter, Marinus Duran, Willy Lehnert and Ronald J.A. Wanders 3-Methylglutaconic aciduria type I is an autosomal recessive disorder clinically characterized by various symptoms ranging from delayed speech development to severe neurological handicap. This disorder is caused by a deficiency of 3-methylglutaconyl-CoA hydratase, one of the keyenzymes of leucine degradation. This results in elevated urinary levels of 3-methylglutaconic acid, 3-methylglutaric acid and 3-hydroxyisovaleric acid. By heterologous expression in E. coli, we show that 3-methylglutaconyl-CoA hydratase is encoded by the AUH gene, whose product previously had been reported as an AU-specific RNA-binding protein. Mutation analysis of AUH in two patients revealed a nonsense mutation (R197X) and a splice site mutation (IVS8-1G>A) demonstrating that mutations in AUH cause 3-methylglutaconic aciduria type I. 3-Methylglutaconic aciduria is a group of metabolic disorders characterized by increased urinary excretion of 3-methylglutaconic acid and 3-methylglutaric acid. At present, four distinct forms have been recognized. Type I (MIM ) represents the isolated 3- methylglutaconyl-coa hydratase deficiency. This mitochondrial enzyme catalyses the fifth step in leucine catabolism, as shown in figure 1, which is the conversion of 3-methylglutaconyl-CoA to 3-hydroxy- 3-methylglutaryl-CoA (HMG-CoA). Type II (MIM ), also referred to as Barth syndrome, is an X-linked mitochondrial cardiomyopathy associated with neutropenia and growth retardation and caused by mutations in the gene encoding tafazzin (TAZ). Type III (MIM ) or Costeff syndrome, is an autosomal recessive disorder caused by mutations in the OPA3 gene which leads to bilateral optic atrophy. Type IV or unspecified 3-methylglutaconic aciduria (MIM ) comprises a heterogeneous group of patients with progressive neurological symptoms. The hydratase activity in Barth syndrome, Costeff syndrome and type IV 3-methylglutaconic aciduria is normal and the excretion of 3-methylglutaconic acid and 3-methylglutaric acid is secondary. Amongst the four types, patients with the type I excrete the highest levels of 3-methylglutaconic acid and 3-methylglutaric acid. Furthermore, these patients additionally excrete increased amounts of 3-hydroxyisovaleric acid and therefore, 3-hydroxyisovaleric acid is an important parameter for differentiation between the isolated 3-methylglutaconyl-CoA hydratase deficiency and the other types. The phenotypic presentation of 3- methylglutaconyl-coa hydratase deficiency varies from mild, including delayed speech development and hyperchloremic acidosis associated with gastroesophageal reflux to a much more severe phenotype, including seizures and cerebellar abnormalities. Although the hydratase was purified partially in the late 1950s 1, the gene encoding the protein had not been identified. Several mitochondrial enzymes with enoyl-coa hydratase activity have been characterized. The first one, called crotonase, is most active towards short chain enoyl-coas and participates in the β-oxidation of short-chain fatty acids. Studies performed by Hilz H et al. showed that an enzyme other than crotonase is responsible for the hydratase step in the degradation of leucine. 1 Indeed, our preliminary studies showed that crotonase is not active with HMG-CoA when measured in the reverse direction. A second enzyme harbouring hydratase activity is mitochondrial trifunctional protein (MTP), which also catalyses dehydrogenation and thiolytic cleavage of long-chain fatty acids. Together with very long-chain acyl-coa dehydrogenase, this From the Departments of Clinical Chemistry and Pediatrics, Emma Children s Hospital, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands (F.J.L.;J.P.N.R.;L.IJ.;M.D.;R.J.A.W.); University Children s Hospital Freiburg, Germany (W.L) 19 HOOC S-CoA CH3 O 3-Methylglutaconyl-CoA HOOC L-Leucine 2-Ketoisocaproic acid Isovaleryl-CoA 3-Methylcrotonyl-CoA OH S-CoA CH3 O 3-Hydroxy-3-methylglutaryl-CoA Acetoacetic acid + Acetyl-CoA 3-Hydroxyisovaleric acid 3-Methylglutaric acid 3-Methylglutaconic acid 3-Methylglutaconyl-CoA Hydratase Figure 1. Catabolism of L-leucine. The names of the intermediates in the pathway for catabolism of leucine are shown on the left with solid arrows indicating enzymatic reactions. The structures of 3-methylglutaconyl-CoA and HMG-CoA are depicted on the left. The names of the metabolites that are elevated due to 3-methylglutaconyl-CoA hydratase (italic) deficiency are shown by dashed arrows to the right.

21 protein is the main enzyme in the long-chain fatty acid β-oxidation. It is unlikely, however, that this enzyme is involved in the degradation of leucine, because patients with MTP deficiency (MIM , ) have no abnormal urinary excretion of 3-methylglutaconic acid. Nakagawa and co-workers purified an AU-specific RNA binding protein, designated AUH, using an affinity column displaying A+U-rich elements. 2 The protein contained a hydratase motif and shared 32 % sequence identity with crotonase. The enoyl-coa hydratase activity with butenoyl-coa (C 4:1 -CoA) as substrate was thousand-fold lower when compared to crotonase. The three-dimensional structure of human AUH has been determined at 2.2 Å resolution and predicted a high affinity for short chain substrates. 3 To investigate whether AUH has 3-methylglutaconyl- CoA hydratase activity we expressed AUH as a fusion protein with maltose binding protein (MBP). To this end, the ORF of AUH was amplified from human cdna by use of the primers 3AUHfEcoRI 5 -ATA TGA ATT CAA CAT GGC GGC CGC GGT GG-3 and 1097AUHrXbaI 5 -ATA TTC TAG ACA TAT AGT GGA TCC GAA AGA C-3, digested with EcoRI and XbaI, and subsequently cloned into the EcoRI and XbaI restriction sites of the pmal-c2x vector (New England Biolabs). The cloned PCR products were sequenced to assess the integrity of the PCR process. Next, the MBP- AUH was expressed in E. coli and affinity-purified to homogeneity according to the manufacturer s protocol (fig. 2). kda T P Figure 2. Expression and purification of MBP-AUH fusion protein in E. coli. Total E. coli extract (T) and affinity-purified fusion protein (P) were analyzed by 10 % SDS-PAGE gel electrophoresis and stained with Coomassie Brilliant blue. 20 The 3-methylglutaconyl-CoA hydratase activity of the expressed protein was measured in the reverse direction using HMG-CoA as substrate. The reaction mixture contained 50 mm Tris ph 7.4, 10 mm EDTA, 1 mg/ml BSA, 0.1 mm HMG-CoA and was started by addition of purified protein. The formation of 3-methylglutaconyl- CoA was followed spectrophotometrically at 260 nm using a molar extinction coefficient of 6200 mol/l/ cm. The short-chain enoyl-coa hydratase activity was measured using butenoyl-coa as a substrate as described. 4 For comparison, both activities were also measured using bovine liver crotonase obtained from Sigma. As reported previously for AUH, we also found very low hydratase activity with C 4:1 -CoA. In addition, we found that AUH has high 3-methylglutaconyl-CoA hydratase activity. Indeed, the HMG/C 4:1 activityratio was more than 20. In contrast, crotonase has no detectable 3-methylglutaconyl-CoA hydratase activity (table 1). Therefore, AUH is a strong candidate for the underlying enzymatic defect of 3-methylglutaconic aciduria type I. Table 1. Hydratase activity measurements of AUH and crotonase with HMG-CoA and butenoyl-coa (C4:1-CoA) as substrate (nmol/min/mg) Hydratase activity MBP-AUH Bovine liver crotonase HMG-CoA 110 < 1 C4:1-CoA * 10 5 HMG-CoA/C4:1-CoA 23 < 10-6 To date, eleven patients have been described with isolated 3-methylglutaconyl-CoA hydratase deficiency The hydratase deficiency in these patients has been determined by use of an overall enzyme assay measuring three steps of leucine degradation, from 3-methylcrotonyl-CoA to acetoacetic acid. 11 We reinvestigated fibroblasts from two patients 5,6 with our novel enzyme assay using the same conditions as described above, but the reaction was started with 0.3 mg/ml fibroblast homogenate. The reaction mixture was incubated for 30 minutes at 37 C, stopped with 0.2 M hydrochloric acid and subsequently neutralized with sodium hydroxide. Formation of 3-methylglutaconyl- CoA was quantified on HPLC using a reverse-phase C18-column (Supelcosil LC-18-DB, 250 mm X 10 mm, 5 µm, Supelco). Separation of the acyl-coas was achieved by elution with a linear gradient of methanol (10% to 37.6% in 22 min) in potassium phosphate buffer (50 mm, ph 5.3). Both patients showed clear deficiency of 3-methylglutaconyl-CoA hydratase (patients <0.1; controls 2.8 ± 0.6 nmol/min/mg protein), whereas the crotonase activity was normal for both patients. To determine whether 3-methylglutaconic aciduria type I is caused by mutations in AUH, the ORF of AUH was amplified from human cdna in two overlapping fragments using the following M13-tagged primers: fragment A: -28AUHf-21M tgt aaa acg acg gcc

22 C1 C2 P Figure 3. Mutation analysis of AUH in patient with 3- methylglutaconic aciduria type I. PCR fragment B amplified from cdna of two control subjects (C1 and C2) and patient 1 (P) showing the 42 bp deletion (exon 9) as a consequence of a IVS8-1G>A mutation. References agt TCG CAG GTC CAC GCC GTA AAC-3 and 680AUHrM cag gaa aca gct atg acc CGC GTG GCA ATC GCT GTG TC-3 ; fragment B: 505AUHf- 21M tgt aaa acg acg gcc agt GCT ATT CTT CCA GTG CCA AC-3 and 1097AUHrM cag gaa aca gct atg acc CAT ATA GTG GAT CCG AAA GAC-3. PCR fragments obtained from patients and two control subjects were sequenced using Big Dye Terminators and analyzed on an Applied Biosystems 377A automated DNA sequencer following the manufacturer s protocols (PE Applied Biosystems, Foster City). The first patient was the third son of consanguineous parents of Afghan origin with retardation in speech development. A delayed motor development became evident in retrospect. 5 The amplification product of fragment B was approximately 50 bp smaller than found in controls (figure 3). Sequence analysis revealed that this was due to skipping of exon 9. The splice acceptor and donor site neighboring exon 9 conform to the AG/ GT rule. To identify the underlying mutation causing missplicing we amplified splice sites flanking exon 9 from genomic DNA using the following primers: In8AUHf-21M13 5 tgt aaa acg acg gcc agt GAC ATG CCT CTT TGA AGC AC 3 and In9AUHrM13 5 cag gaa aca gct atg acc GCA AGG GTA ATC TTG CTC AG 3. In patient 1 we identified a homozygous IVS8-1G>A mutation, which disrupts the consensus sequence of the splice acceptor site. Exon 9 encodes α-helix H9 3, which crystal structure analysis suggests to be a constituent of the active-site pocket, and is also necessary for subunit interaction and trimer stability. The second patient is the younger of two affected brothers of healthy nonconsanguineous Moroccan parents. He had no physical abnormalities, only his speech development was retarded. 6 Sequence analysis at the cdna level showed an apparently homozygous nonsense mutation c.589c>t. Homozygosity for the nonsense mutation was confirmed at the genomic level. The R197X nonsense mutation causes the translation to be terminated before glutamate 209, which together with glutamate 189 forms the catalytic group of the active-site pocket. To conclude, our data show that mutations in AUH cause 3-methylglutaconic aciduria type I. 1. Hilz H, Knappe J, Ringelmann E, Lynen F (1958) Methylglutaconase, eine neue hydratase, die am stoffwechsel verzweigter carbonsäuren beteiligt ist. Biochem Z 329: Nakagawa J, Waldner H, Meyer-Monard S, Hofsteenge J, Jeno P, Moroni C (1995) AUH, a gene encoding an AU-specific RNA binding protein with intrinsic enoyl-coa hydratase activity. Proc Natl Acad Sci 92: Kurimoto K, Fukai S, Nureki O, Muto Y, Yokoyama S (2001) Crystal structure of human AUH protein, a single-stranded RNA binding homolog of enoyl-coa hydratase. Structure 9: Wanders RJA, IJlst L, Poggi F, Bonnefont JP, Munnich A, Brivet M, Rabier D, Saudubray JM (1992) Human trifunctional protein deficiency: a new disorder of mitochondrial fatty acid oxidation. Biochem Biophys Res Commun 188: Ensenauer R, Muller CB, Schwab KO, Gibson KM, Brandis M, Lehnert W (2000) 3-Methylglutaconyl-CoA hydratase deficiency: a new patient with speech retardation as the leading sign. J Inherit Metab Dis 23: Duran M, Beemer FA, Tibosch AS, Bruinvis L, Ketting D, Wadman SK (1982) Inherited 3-methylglutaconic aciduria in two brothers- Another defect of leucine metabolism. J Pediatr 101: Gibson KM, Lee CF, Wappner RS (1992) 3-Methylglutaconylcoenzyme-A hydratase deficiency: a new case. J Inherit Metab Dis 15: Gibson KM, Wappner RS, Jooste S, Erasmus E, Mienie LJ, Gerlo E, Desprechins B, De Meirleir L (1998) Variable clinical presentation in three patients with 3-methylglutaconylcoenzyme A hydratase deficiency. J Inherit Metab Dis 21: Narisawa K, Gibson KM, Sweetman L, Nyhan WL (1986) Deficiency of 3-methylglutaconyl-coenzyme A hydratase in two siblings with 3-methylglutaconic aciduria. J Clin Invest 77: Shoji Y, Takahashi T, Sawaishi Y, Ishida A, Matsumori M, Shoji Y, Enoki M, Watanabe H, Takada G (1999) 3-Methylglutaconic aciduria type I: clinical heterogeneity as a neurmetabolic disease. J Inherit Metab Dis 22: Narisawa K, Gibson KM, Sweetman L, Nyhan WL (1989) 3-Methylglutaconyl-CoA hydratase, 3-methylcrotonyl- CoA carboxylase and 3-hydroxy3-methylglutaryl-CoA lyase deficiencies: a coupled enzyme assay useful for their detection. Clin Chim Acta 184:

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24 Chapter Direct Non-isotopic Assay of 3-Methylglutaconyl-CoA Hydratase 4 Ference J. Loupatty, Jos P.N. Ruiter, Lodewijk IJlst, Marinus Duran and Ronald J.A. Wanders Our objective was to develop a specific and sensitive assay for the determination of 3-methylglutaconyl- CoA hydratase activity. The 3-methylglutaconyl-CoA hydratase activity in fibroblast homogenates was measured in the reverse direction using 3-hydroxy-3-methylglutaryl-CoA as a substrate. The formation of 3-methylglutaconyl-CoA was quantified by use of reversed-phase HPLC with UV detection at 260 nm. Baseline separation of substrate and product was achieved. Little variation of 3-methylglutaconyl-CoA hydratase activity was observed between ph 7.0 and 9.0. The assay was linear in time up to 60 min using 50 µg protein/ml. The intraassay and interassay coefficients of variation were 4.0% (n=10) and 4.8 % (n=11), respectively. The detection limit was 20 pmol/min/ mg protein. Mean (SD) control 3-methylglutaconyl-CoA hydratase activity was 2.1 (0.7) nmol/min/ mg protein (range ; n=13). Patients with proven 3-methylglutaconic aciduria type I had 3- methylglutaconyl-coa hydratase activities below the detection limit. This rapid and nonradioactive enzyme assay allows the specific and sensitive determination of 3-methylglutaconyl-CoA hydratase activity in cultured human skin fibroblasts. The enzyme assay can be used for the reliable diagnosis of 3-methylglutaconic aciduria type I and their differentiation from other forms of 3-methylglutaconic aciduria. 3-Methylglutaconic aciduria (3MGA) type I (MIM ) is biochemically characterized by increased excretion of 3-methylglutaconic acid, 3-methylglutaric acid and 3-hydroxyisovaleric acid in urine. Affected individuals display a range of clinical manifestations varying from mildly delayed speech development to severe neurological involvement. 1,2 3MGA type I is an autosomal recessive disorder caused by a deficiency of 3-methylglutaconyl-CoA hydratase (3MGH, E.C ). Three additional forms of 3-methylglutaconic aciduria have been recognized (type II, Barth syndrome, MIM ; type III, Costeff syndrome, MIM ; type IV, unspecified, MIM ) all characterized by normal hydratase activities. 3 Recently, the gene encoding 3MGH was identified by two independent groups. 4,5 As shown in figure 1, this mitochondrial enzyme catalyzes the penultimate step in leucine catabolism, which is the reversible conversion of 3- methylglutaconyl-coa to 3-hydroxy-3-methylglutaryl- CoA (HMG-CoA). Eleven patients have been described with isolated 3- methylglutaconyl-coa hydratase deficiency. 1,2,5-10 The hydratase deficiency in these patients was determined by use of a radioactive enzyme assay measuring three consecutive steps of leucine degradation, from 3- methylcrotonyl-coa to acetoacetic acid. 11 However, this procedure lacks specificity and is laborious because of the need to purify the coupling enzyme 3- methylcrotonyl-coa carboxylase (E.C ) from bovine liver. Furthermore, the assay is impractical because it involves the use of radiochemicals. The need to differentiate 3MGA type I patients from others with 3-methylglutaconic aciduria requires the availability of a sensitive and specific enzyme assay. Based on the knowledge that in general hydratase reactions are readily reversible and 3- methylglutaconyl-coa is not commercially available, we studied the 3MGH activity in the reverse direction, using HMG-CoA as a substrate. The formation of 3- methylglutaconyl-coa was quantified by reversedphase high performance liquid chromatography with HOOC S-CoA CH3 O 3-Methylglutaconyl-CoA HOOC L-Leucine 2-Ketoisocaproic acid Isovaleryl-CoA 3-Methylcrotonyl-CoA OH S-CoA CH3 O 3-Hydroxy-3-methylglutaryl-CoA Acetoacetic acid + Acetyl-CoA 3-Hydroxyisovaleric acid 3-Methylglutaric acid 3-Methylglutaconic acid 3-Methylglutaconyl-CoA Hydratase Figure 1. Catabolism of L-leucine. The names of the intermediates in the pathway for catabolism of leucine are shown on the left with solid arrows indicating enzymatic reactions. The structures of 3-methylglutaconyl-CoA and HMG-CoA are depicted on the left. The names of the metabolites that are elevated due to 3-methylglutaconyl-CoA hydratase (italic) deficiency are shown by dashed arrows to the right. From the Departments of Clinical Chemistry and Pediatrics, Emma Children s Hospital, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands (F.J.L.;J.P.N.R.;L.IJ.;M.D.;R.J.A.W.) 23

25 UV detection. The described assay allows the precise measurement of residual 3MGH activity in cultured human skin fibroblasts. We obtained potassium dihydrogen phosphate (KH 2 PO 4 ), phosphoric acid (H 3 PO 4 ), Tris, EDTA, hydrochloric acid, potassium hydroxide, 2- morpholinoethanesulfonic acid (MES) and acetonitrile (chromatography grade) from Merck. Bovine serum albumin essentially fatty acid free (BSA), 3-hydroxy- 3-methylglutaryl-coenzyme A (HMG-CoA) and bicinchoninic acid were purchased at Sigma. Skin fibroblasts were obtained from two subjects with proven 3MGA type I 1,6, five subjects with proven Barth syndrome 12, two subjects with 3-methylglutaconic aciduria type IV 13 and thirteen control subjects with no evidence of an inborn error of branched chain amino acid oxidation or mitochondrial fatty acid oxidation. All patient cells were initially obtained in the process of diagnosing inborn errors of metabolism. Fibroblasts were grown and harvested as described elsewhere. 14 For determination of the intraassay (within-run) and interassay (between-day) variation, six cell cultures of three different control subjects were harvested, pooled, divided in 20 pellets and stored frozen at -80 C. Cell pellets were suspended in 200 µl of phosphatebuffered saline by repeated pipetting and sonicated three times on ice for 15 seconds at 8 W at 45 sec intervals. Protein was determined by the bicinchoninic acid assay (Sigma) using BSA as standard. The assay mixture contained, in a final volume of 100 µl: 100 mmol/l TRIS, ph 8.0, 10 mmol/l EDTA, 1g/L BSA, 100 µmol/l HMG-CoA and mg/l of fibroblast protein. After incubation at 37 C for 60 min the reaction was terminated by addition of 10 µl of 2 mol/ L HCl. The samples were homogenized and the assay tubes placed on ice. After 5 min, the homogenates were brought to a ph 6 with (2 mol/l KOH)/ (1 mol/l MES ph 6) and centrifuged for 10 min at 4 C at rpm. Finally, 100 µl of the supernatant was transferred to a HPLC vial. HPLC separation was performed at ambient temperature with a Perkin Elmer pump (PE series 200) and a Gilson 234 auto-sampling injector. A frit C-402X (Upchurch scientific), a 4.6 mm x 20 mm SUPELCOSIL LC-18-DB (5µM) guard column (Supelco) and a 4.6 mm x 250 mm column filled with the same packing material were used. Compounds were detected with a UV detector (SPD-10A VP UV-VIS, Shimadzu) at 260 nm. For gradient elution, we used a binary solvent system. Solvent A was 100 mmol/l KH 2 PO 4 adjusted to ph 4 with 100 mmol/l H 3 PO 4, filtered before use through a 0.45 µm nitrocellulose membrane under reduced pressure. Solvent B was a 4:1 mixture (by volume) of solvent A, and acetonitrile. Both solvents were degassed for 15 min in an ultrasonic bath (Branson 3510). 50 µl of sample was injected and the acyl-coas were eluted at a flow rate of 1 ml/min by a linear gradient of 20 % B to 100 % B in 15 min. Peak areas of interest were integrated using Chromeleon software package (Dionex). We first developed a HPLC-based method to allow baseline separation between HMG-CoA and 3- methylglutaconyl-coa. Separation of acyl-coa esters using reversed-phase HPLC is dependent on the salt concentration and the ph of the eluent. 15 We found that a linear gradient of acetonitrile in potassium phosphate buffer (100 mmol/l, ph 4.0) allowed baseline separation between substrate and product. Typical chromatograms for incubations with fibroblast extracts of a control subject and a 3MGA type I patient are shown in figure 2. Using acyl-coa standards we were able to identify free CoA (9.0 min) and HMG-CoA and acetyl-coa which coeluted at 11.8 min. Comparison of the chromatograms of a 3MGA type I patient and a control subject suggested the extra peak at 16.5 min to be 3-methylglutaconyl-CoA. Indeed, after hydrolysis and derivatization of its contents, the presence of 3-methylglutaconate was identified on the basis of its retention time and confirmed by mass spectrometry (results not shown) as described by Röschinger et al. 16 Thus, the acyl-coa which eluted at 16.5 min was the product 3-methylglutaconyl-CoA. HMG-CoA is converted into 3-methylglutaconyl- CoA by the action of 3MGH and its activity can be quantified by determining the amount of 3- methylglutaconyl-coa formed. The acyl-coas present in the reaction mixture are susceptible to Signal output (mv) A B C Retention time (min) Figure 2. Separation of HMG-CoA and 3-methylglutaconyl-CoA on HPLC. HPLC chromatograms of incubations with fibroblast extracts of a control subject (upper, 30.1 µg protein/assay), and a 3MGA type I patient with less than 1% residual 3MGH activity (lower, 22.4 µg protein/assay). Compounds were detected with a UV detector at 260 nm. Peaks: A, free CoA (CoASH); B, coelution of the substrate 3-hydroxy-3-methylglutaryl-CoA and acetyl-coa; C, the product 3-methylglutaconyl-CoA. 24

26 acetyl-coa according to the following equation, which expresses the 3MGH activity as nmol/min/mg protein: Specific activity 3MGH (nmol/min/mg protein) Figure 3. Effect of the ph on 3-methylglutaconyl-CoA hydratase (3MGH) activity. enzymatic and chemical hydrolysis, but HMG-CoA and 3-methylglutaconyl-CoA were equally hydrolyzed (results not shown). Therefore, the 3MGH activity was calculated as the ratio of product to the sum of product and substrate. However, since HMG-CoA is a substrate for other enzymes, calculating the 3MGH activity in such manner would result in an overestimation of the 3MGH activity. Preliminary studies revealed that, under the incubation conditions described above, < 5 % of HMG- CoA was converted into acetyl-coa by the action of HMG-CoA lyase (results not shown). Second, HMG- CoA could be converted into mevalonic acid by the action of HMG-CoA reductase (E.C ) using NADPH as a coenzyme. However, since NADPH was not added, the action of the reductase on HMG-CoA under the assay conditions was negligible. Therefore the 3MGH activity was calculated as the ratio of the amount of 3-methylglutaconyl-CoA to the sum of the amounts of 3-methylglutaconyl-CoA, HMG-CoA and ph 1 3MG CoA ( 1 3MG CoA) 2 ( HMG CoA acetyl CoA) T P where ε1 = 22.6 cm 2 /µmol 17, ε2 = 16.0 cm 2 /µmol 17, T = incubation time (min), P = the amount of protein (mg), and the acyl-coas are expressed as peak areas. The activity of 3-methylglutaconyl-CoA hydratase in human fibroblast extracts showed little variation as a function of ph in the range from 7.0 to 9.0 (figure 3) as reported by others using the coupled enzyme assay. 18 For the standard assay we selected a ph of 8.0. The assay was linear with an incubation time up to at least 60 min with 50 mg fibroblast protein/l assay (figure 4). For the standard assay, an incubation time of 60 min and a protein content of mg fibroblast protein/l assay was chosen. Using optimized assay conditions, we reinvestigated fibroblasts from two patients with established 3MGA type I 1,6 and detected no formation of 3- methylglutaconyl-coa in either case. Inclusion of 1 % control homogenate in a homogenate of a 3MGA type I patient, corresponding to a 3MGH activity of 20 pmol/ min/mg protein, could readily be detected. Hence, the 3MGH activity in both patients was < 20 pmol/min/mg protein. The intraassay variation, estimated by assaying 10 of the pooled control pellets in a single experiment, was 4.0 % [mean (SD) = 2.26 (0.09) nmol/min/mg protein). The interassay variation was 4.8 % [mean (SD) = 2.2 (0.1) nmol/min/mg protein], n = 10 days Measurement of 3MGH activity in thirteen control fibroblast homogenates revealed a mean (SD) 3MGH activity of 2.1 (0.7) nmol/min/mg protein (range ). The mean (SD) 3MGH activity in fibroblasts of five Barth syndrome patients was 2.8 (0.8) nmol/min/mg protein (range ). The two patients with proven 3MGA type IV demonstrated 3MGH activities of 1.59 and 1.02 nmol/min/mg protein. These values for Methylglutaconyl-CoA (nmol) Time (min) A Methylglutaconyl-CoA (nmol) B Fibroblast protein (µg/ml assay) 200 Figure 4. A, Effect of time on 3-methylglutaconyl-CoA hydratase activity; Fibroblast extracts containing 50 µg protein/ml assay were incubated at 37 C. The values are the mean ± SD of three independent experiments. B, Effect of protein on 3-methylglutaconyl-CoA hydratase activity. Fibroblast extracts were incubated at 37 C for 60 min. 25

27 3MGH activity were within the control range. Hence, the 3MGH activity in cultured fibroblast homogenates of patients with Barth syndrome or 3-methylglutaconic aciduria type IV is normal. This corresponds with the finding that normal 3MGH activity has been found in Barth syndrome and 3MGA type IV using the coupled enzyme assay. 3 In conclusion, we present a sensitive and specific enzymatic assay for 3-methylglutaconyl-CoA hydratase that enables the rapid enzymatic diagnosis of 3- methylglutaconic aciduria type I in cultured human skin fibroblasts without the involvement of radiochemicals. Baseline separation between the substrate HMG- CoA and the product 3-methylglutaconyl-CoA was achieved. Our novel procedure allows detection of 1% residual 3MGH activity in 3MGA type I patients. So far, all 3MGA type I patients demonstrated a 3MGH activity below this limit. Our method could be useful for studies of genotype / phenotype correlation as well as studies for the enzymatic characterization of mutated proteins as expressed in E.coli. References 1. Duran M, Beemer FA, Tibosch AS, Bruinvis L, Ketting D, Wadman SK. Inherited 3-methylglutaconic aciduria in two brothers--another defect of leucine metabolism. J Pediatr 1982;101: Shoji Y, Takahashi T, Sawaishi Y, Ishida A, Matsumori M, Shoji Y et al. 3-Methylglutaconic aciduria type I: clinical heterogeneity as a neurometabolic disease. J Inherit Metab Dis 1999;22: Gibson KM, Sherwood WG, Hoffman GF, Stumpf DA, Dianzani I, Schutgens RB et al. Phenotypic heterogeneity in the syndromes of 3-methylglutaconic aciduria. J Pediatr 1991;118: IJlst L, Loupatty FJ, Ruiter JP, Duran M, Lehnert W, Wanders RJ. 3-Methylglutaconic aciduria type I is caused by mutations in AUH. Am J Hum Genet 2002;71: Ly TB, Peters V, Gibson KM, Liesert M, Buckel W, Wilcken B et al. Mutations in the AUH gene cause 3-methylglutaconic aciduria type I. Hum Mutat 2003;21: Ensenauer R, Muller CB, Schwab KO, Gibson KM, Brandis M, Lehnert W. 3-Methylglutaconyl-CoA hydratase deficiency: a new patient with speech retardation as the leading sign. J Inherit Metab Dis 2000;23: Hou JW, Wang TR. 3-Methylglutaconic aciduria presenting as Reye syndrome in a Chinese boy. J Inherit Metab Dis 1995;18: Jooste S, Erasmus E, Mienie LJ, de Wet WJ, Gibson KM. The detection of 3-methylglutarylcarnitine and a new dicarboxylic conjugate, 3-methylglutaconylcarnitine, in 3-methylglutaconic aciduria. Clin Chim Acta 1994;230: Gibson KM, Lee CF, Wappner RS. 3-Methylglutaconylcoenzyme-A hydratase deficiency: a new case. J Inherit Metab Dis 1992;15: Arbelaez A, Castillo M, Stone J. MRI in 3-methylglutaconic aciduria type 1. Neuroradiology 1999;41: Narisawa K, Gibson KM, Sweetman L, Nyhan WL. 3- Methylglutaconyl-CoA hydratase, 3-methylcrotonyl-CoA carboxylase and 3-hydroxy-3-methylglutaryl-CoA lyase deficiencies: a coupled enzyme assay useful for their detection. Clin Chim Acta 1989;184: Valianpour F, Wanders RJ, Overmars H, Vreken P, Van Gennip AH, Baas F et al. Cardiolipin deficiency in X-linked cardioskeletal myopathy and neutropenia (Barth syndrome, MIM ): a study in cultured skin fibroblasts. J Pediatr 2002;141: Gibson KM, Nyhan WL, Sweetman L, Narisawa K, Lehnert W, Divry P et al. 3-Methylglutaconic aciduria: a phenotype in which activity of 3-methylglutaconyl-coenzyme A hydratase is normal. Eur J Pediatr 1988;148: Wanders RJ, Denis S, Ruiter JP, Schutgens RB, van Roermund CW, Jacobs BS. Measurement of peroxisomal fatty acid betaoxidation in cultured human skin fibroblasts. J Inherit Metab Dis 1995;18 Suppl 1: Corkey BE. Analysis of acyl-coenzyme A esters in biological samples. Methods Enzymol 1988;166: Roschinger W, Muntau AC, Duran M, Dorland L, IJlst L, Wanders RJ, Roscher AA. Carnitine-acylcarnitine translocase deficiency: metabolic consequences of an impaired mitochondrial carnitine cycle. Clin Chim Acta 2000;298: Lazarow PB. Rat liver peroxisomes catalyze the beta oxidation of fatty acids. J Biol Chem 1978;253: Narisawa K, Gibson KM, Sweetman L, Nyhan WL, Duran M, Wadman SK. Deficiency of 3-methylglutaconyl-coenzyme A hydratase in two siblings with 3-methylglutaconic aciduria. J Clin Invest 1986;77:

28 Chapter 5 Identification of a Novel Enoyl-CoA Hydratase Encoded by ECHDC2 Ference J. Loupatty, Rob Ofman, Jos P.N. Ruiter, Lodewijk IJlst, Marinus Duran and Ronald J.A. Wanders In this study we describe the identification of a novel enoyl-coa hydratase encoded by ECHDC2 and present new data on human 3-methylglutaconyl-CoA hydratase (E.C ), the enzyme which is deficient in patients with 3-methylglutaconic aciduria type I. By heterologous expression in Escherichia coli, we show that the ECHDC2 protein has 3-methylglutaconyl-CoA, crotonyl- CoA and octenoyl-coa hydratase activities, but no tiglyl-coa or 3-hydroxyisobutyryl-CoA hydratase activities. Comparison of the enzymatic characteristics of the ECHDC2 protein with 3-methylglutaconyl-CoA hydratase showed that both enzymes have highly similar substrate specificities. Subcellular fractionation studies revealed that both enzymes are localized exclusively in the mitochondria. Using mono-specific antibodies we studied the expression of the ECHDC2 protein and 3-methylglutaconyl-CoA hydratase in different types of tissue in humans, rats and mice. 3-Methylglutaconyl-CoA hydratase is predominantly expressed in brain, kidney, heart, and moderately expressed in liver, muscle, testis and skin fibroblasts. The ECHDC2 protein is expressed in kidney, liver and heart, but is not expressed in brain and cultured human skin fibroblasts. As the ECHDC2 protein is not expressed in human skin fibroblasts, whereas 3-methylglutaconyl-CoA hydratase is, a deficiency of the ECHDC2 protein should be considered in patients with normal 3-methylglutaconyl-CoA hydratase activity in human skin fibroblasts, but with elevated urinary excretion of 3-methylglutaconic acid. 3-Methylglutaconic aciduria type I (3MGA I, MIM ) is an autosomal recessive disorder caused by a deficiency of 3-methylglutaconyl-CoA hydratase (3MGH, E.C ). Affected individuals display a range of clinical manifestations varying from mildly delayed speech development to severe neurological involvement. As shown in figure 1, 3MGH catalyzes the penultimate step in leucine catabolism, which is the reversible conversion of 3-methylglutaconyl-CoA into 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA). The absence of 3-methylglutaconyl-CoA hydratase activity will result in elevated excretion of 3-methylglutaconic acid, 3-methylglutaric acid and 3-hydroxyisovaleric acid in the urine of affected individuals. We have recently shown that crotonase (E.C ), also referred to as enoyl-coa hydratase, harbors 3-methylglutaconyl-CoA hydratase activity, although the catalytic efficiency of the enzyme is much higher with crotonyl-coa as compared to 3- methylglutaconyl-coa. 1 Crotonase plays a key role in fatty acid metabolism by catalyzing the second step in the classical β-oxidation cycle. The enzyme is most active towards short-chain substrates and acts with decreasing efficiency on 2-enoyl-CoAs from crotonyl- CoA to hexadecenoyl-coa, which is only hydrated at a rate of 1-2 % of the rate of crotonyl-coa. 2 In 1996, Wieringa and colleagues described the quaternary structure of human crotonase as a dimer of trimers with each subunit containing 261 amino acids. 3,4 The active site is primarily contained within one subunit, but some amino residues that line the binding pocket are contributed by a neighboring subunit in the hexamer. On the basis of the three-dimensional structure of crotonase, a catalytic mechanism has been proposed with the key players being Glu144 and Glu In an effort to resolve the molecular basis of 3MGA I we screened the database of the National Center for Biotechnology Information (NCBI) for HOOC S-CoA CH3 O 3-Methylglutaconyl-CoA HOOC L-Leucine 2-Ketoisocaproic acid Isovaleryl-CoA 3-Methylcrotonyl-CoA OH S-CoA CH3 O 3-Hydroxy-3-methylglutaryl-CoA Acetoacetic acid + Acetyl-CoA 3-Hydroxyisovaleric acid 3-Methylglutaric acid 3-Methylglutaconic acid 3-Methylglutaconyl-CoA Hydratase Figure 1. Catabolism of L-leucine. The names of the intermediates in the pathway for catabolism of leucine are shown on the left with solid arrows indicating enzymatic reactions. The structures of 3-methylglutaconyl-CoA and HMG-CoA are depicted. The names of the metabolites that are elevated due to 3-methylglutaconyl-CoA hydratase (italic) deficiency are shown by dashed arrows to the right. From the Departments of Clinical Chemistry and Pediatrics, Emma Children s Hospital, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands (F.J.L.;R.O.;J.P.N.R.;L.IJ.;M.D.;R.J.A.W.) 27

29 3-methylglutaconyl-CoA hydratase using crotonase (ECHS1; E.C ) as query which resulted in several potential candidates. Molecular and biochemical investigations performed by our group demonstrated that one of the candidate genes, AUH, encodes 3-methylglutaconyl-CoA hydratase (3MGH). More importantly, mutations in this AUH gene cause 3- methylglutaconic aciduria type I. 1 Additionally, we also found a homologue, referred to as ECHDC2, belonging to the crotonase superfamily. In the present study, we describe the molecular cloning and characterization of AUH and ECHDC2 which encodes a novel enoyl-coa hydratase. We demonstrate that the ECHDC2 enzyme harbors considerable 3-methylglutaconyl-CoA hydratase activity and is located in the mitochondria, similar to 3MGH. As 3-methylglutaconyl-CoA hydratase activity is essential in the catabolism of leucine, the tissue distribution of 3MGH and the ECHDC2 protein is of considerable interest. To this end, antibodies were raised against both proteins. Immunoblot analysis revealed tissue-specific expression of 3MGH and ECHDC2 in humans, rats and mice. Therefore, we suggest that the ECHDC2 protein is a therapeutic target for patients with 3-methylglutaconic aciduria type I. Experimental procedures General. E.coli INV-αF and BL21 cells were purchased from Invitrogen (Carlsbad, USA). The Amylose Resin Column, the restriction enzymes EcoRI, BamHI and SalI, the pmal-c2x and pgex-4t-1 vectors were obtained from New England Biolabs (Herts, United Kingdom). I-BLOCK was from Tropix (Bedford, MA), goat anti-rabbit IgG-alkaline phosphatase conjugate from Santa Cruz Biotechnology (CA, USA). All other reagents were purchased at Sigma (St. Louis, MO). For sequence analysis, the tools provided by the National Center for Biotechnology Information (available at and the ExPASy Molecular Biology Server (available at expasy.ch) were used. BLAST search with human crotonase The human_ EST database of the National Center for Biotechnology Information (NCBI) was screened for sequences homologous to human crotonase (ECHS1; AAH08906) using a translated BLAST search. The following criteria were used for evaluation; (1) considerable similarity of the translated sequence, (2) conservation of the regions that define the active site pocket, (3) conservation of the catalytic group of crotonase (Glu144 and Glu164), and (4) localization of the candidate proteins to the mitochondria. Potential candidates were also evaluated using a CLUSTALW multiple sequence alignment program (available at Potential mitochondrial targeting sequences were evaluated with the signal prediction program MitoProt (available at 28 Cloning and expression of human AUH and human ECHDC2 in E.coli. The complete open reading frames (ORFs) of the candidate genes were amplified from human liver cdna by use of an EcoRI-tagged forward primer 5 -atatgaattcaacatggcggccgcggtgg- 3 and a XbaI-tagged reverse primer 5 - atattctagacatatagtggatccgaaagac-3 for the AUH gene, and an EcoRI-tagged forward primer 5 -atatgaattcatgctgcgcgttctgtgcctc- 3 and a XbaI-tagged reverse primer 5 - atattctagactggccacaaatcttccttc-3 for the ECHDC2 gene. Next, the PCR products were digested with EcoRI and XbaI, and subsequently cloned into the EcoRI and XbaI restriction sites of the pmal-c2x vector, to express the 3MGH and ECHDC2 as fusion proteins with maltose binding protein (MBP). The ORFs were sequenced to exclude errors introduced by PCR after which the constructs were transformed to the E.coli strain INV-αF in accordance with the manufacturer s protocol. Finally, the MBP-3MGH and MBP-ECHDC2 were expressed in E.coli and affinitypurified to homogeneity using an Amylose Resin Column following the instructions of the manufacturer and stored at 80 C in 2 mm DTT, 10 % glycerol (w/ v), PBS. In addition, the assessed constructs were also digested with EcoRI and SalI and cloned into the EcoRI and SalI restriction sites of the pgex-4t-1 vector. Next, the constructs were transformed to the E.coli strain BL21, expressed as GST-fusion proteins. The pellets were stored at 80 C. Generation of antisera. Female New Zealand white rabbits were primed with 100 µg of the highly purified MBP-fusion protein in 750 µl PBS mixed with an equal volume of Freund s complete adjuvant. The emulsion, divided in two equal parts, was injected both subcutaneously and intramuscular. After 4 weeks the immunization was followed by a boost injection of 100 µg of purified MBP-fusion protein mixed with an equal volume of Freund s incomplete adjuvant. Two more boost injections were given at 4-week intervals. One week after each boost injection a 15-ml blood sample was taken, and serum was prepared. Density gradient analysis. Kidneys were obtained from male Swiss mice, 4-6 months of age, and homogenized in 5 mm morpholinopropanesulphonic acid buffer, ph 7.4, containing 250 mm sucrose, 2 mm EDTA and 0.1 % ethanol. A post-nuclear supernatant was produced by centrifugation of the homogenate at 600 x g for 10 min at 4 C and subfractionated by equilibrium density gradient centrifugation in a linear Nycodenz gradient as described. 6 Glutamate dehydrogenase, catalase, phosphoglucoisomerase, β- hexosaminidase and esterase were used as markers for mitochondria, peroxisomes, cytoplasm, lysosomes and microsomes, respectively. The activity of these enzymes was determined as described elsewhere. 7,8 Protein concentration was measured according to Bradford 9.

30 Immunoblot analysis. Samples were separated by 12% SDS-PAGE and transferred onto nitrocellulose by semidry blotting. Nonspecific binding sites were blocked for one hour in block buffer (PBS containing 1 g/l Tween-20, supplemented with 2 g/l I-BLOCK). Primary (polyclonal anti-mbp-3mgh and polyclonal anti-mbp-echdc2) and secondary (goat antirabbit IgG-alkaline phosphatase conjugate) antibody incubations were performed in block buffer. Polyclonal antibodies raised against purified MBP-3MGH and MBP-ECHDC2 were used at a 1: dilution. Antigen-antibody complexes were visualized with goat anti-rabbit IgG-alkaline phosphatase conjugate using alkaline phosphatase staining in a buffer containing 0.1 M Tris-HCl (ph 9.5), 0.1 M NaCl, 5 mm MgCl 2, 0.33 g/ L 4-nitro blue tetrazolium chloride, 0.17 g/l 5-bromo- 4-chloro-3-indolyl-phosphate (disodium salt). As a control for transfer of protein, each blot was reversibly stained with Ponceau S immediately after blotting and prior to the incubation with antibodies. Enzyme assay. Enoyl-coenzyme A hydratase activities were measured spectrophotometrically at 260 nm and a molar extinction coefficient of 6200 L mol/ cm. 3-Hydroxy-3-methylglutaryl-CoA, butenoyl- CoA, octenoyl-coa, 2-methyl-butenoyl-CoA and 3- hydroxyisobutyryl-coa were used as substrates. The reaction mixture contained 50 mm Tris ph 7.4, 10 mm EDTA, 1 mg/ml BSA, 0.2 mm substrate and was started by addition of purified fusion proteins. The Michaelis- Menten constants (K m ) and maximal velocity (V max ) were determined for the highly purified MBP-fusion proteins from Line weaver-burke plots. Results BLAST search. We searched the NCBI database by performing a translated BLAST search using human crotonase as the query which resulted in the identification of the ECHDC2 protein (accession no. AAH44574) and the AU-specific RNA-binding enoyl- CoA hydratase (3MGH; accession no. CAA56260) as candidates. As mentioned in the introduction this latter protein is in fact 3-methylglutaconyl-CoA hydratase (E.C ). Both candidate proteins satisfied the criteria mentioned in the experimental procedures. First, as determined by the CLUSTALW multiple alignment program, the ECHDC2 protein and 3MGH were 31% and 27% identical to the protein sequence of human crotonase. Furthermore, both candidate proteins shared 46% sequence identity. Second, as depicted in figure 2, the counterparts of the catalytic group of crotonase (Glu144 and Glu164) were conserved as Glu189 and Glu209 in 3MGH, and as Glu142 and Glu162 in the ECHDC2 protein. Also the regions that line the active site pocket, i.e. the oxyanion hole, were conserved. Finally, as predicted by the MitoProt program both proteins display a mitochondrial leader sequence and are likely located to the mitochondria. Taken together, ECHS MAALRVLLSCVRGPLRPPVRCPAWRP FASGA 3 M G H 1 M A A A V A A A P G A L G S L H A G G A R L V A AC S A W L C P G L R L P G S L A G R R A G P A I W A Q G WV P A A G G ECHDC MLRVLCLLRPWRPLRARGCASDG AAGG E C H S N F E Y I I A E K R G KN N T V G L I Q L N R P K A L N A L C D G L I D E L N Q AL K T F E E 3 M G H 6 1 P A P K R G Y S S E M K T E D E L R V R H L E E EN R G I V V L G I N R A Y G K N S L S K N L I K M L S K AV D A L K S ECHDC SEIQVRALAGPDQGITEILMNRPSARNALGNVFVSELLETLAQLRE E C H S D P A V G A I V L T G G D K-A F A A G A D I K EM Q N L S F Q D C--Y S S K F L K H W D H L T Q V K K PV I A A V N 3 M G H D K K V R T I I I R S E V P G I F C A G A D L K ER A K M S S S E V G P F V S K I R A V I N D I A N L P V PT I A A I D E C H D C D R Q V R V L L F R S G V K G V F C A G A D L K ER E Q M S E A E V G V F V Q R L R G L M D D I A A F P A PT I A A M D E C H S G Y A F G G G C E L A M M C D I I Y A G E K A Q FA Q P E I L I G T I P G A G G T Q R L T R A V G K S L A ME M V L T G 3 M G H G L A L G G G L E L A L A C D I R V A A S S A K MG L V E T K L A I I P G G G G T Q R L P R A I G M S L A KE L I F S A E C H D C G F A L G G G L E L A L A C D L R V A A S S A V MG L I E T T R G L L P G A G G T Q R L P R C L G V A L A KE L I F T G E C H S D R I S A Q D A K Q A G L V S K I C P----V ET L V E E A I Q C A E K I A S N S K I V V A M A K E S V NA A F E M T 3 M G H R V L D G K E A K A V G L I S H V L E Q N Q E G DA A Y R K A L D L A R E F L P Q G P V A M R V A K L A I NQ G M E V D E C H D C R R L S G T E A H V L G L V N H A V A Q N E E G DA A Y Q R A R A L A Q E I L P Q A P I A V R L G K V A I DR G T E V D ECHS1 252 LTEGSKLEKKLFYSTFATDDRKEGMTAFVEKRKANFKDQ 3MGH 301 LVTGLAIEEACYAQTIPTKDRLEGLLAFKEKRPPRYKGE ECHDC2 254 IASGMAIEGMCYAQNIPTRDRLEGMAAFREKRTPKFVGK Figure 2. Comparison of human enoyl-coa hydratases. Amino acid sequence of crotonase (ECHS1; GenBank /EBI Data Bank accession number NP_004083), 3-methylglutaconyl-CoA hydratase (3MGH; GenBank /EBI Data Bank accession number NP_001689) and the ECHDC2 protein (GenBank /EBI Data Bank accessionnumber AAH44574) are aligned with identities between the hydratases indicated by black boxes,similarities indicated by grey boxes. Amino acids are numbered on the left. Arrows above the alignment indicate the positions of the conserved glutamate residues of the catalytic group. 29

31 in addition to 3MGH, the ECHDC2 protein is a strong candidate for a mitochondrial enoyl-coa hydratase. The ECHDC2 protein harbors 3-methylglutaconyl- CoA hydratase activity. To investigate if the ECHDC2 protein harbors enoyl-coa hydratase activity, and to further characterize the substrate specificity of 3MGH, both proteins were expressed as fusion proteins with the maltose binding protein (MBP) in E.coli and purified to apparent homogeneity using affinity chromatography. By SDS-PAGE analysis (figure 3), the purified MBPfusion proteins of ECHDC2 and 3MGH migrated with a monomer molecular weight of ~ 80 kda, which corresponds to their predicted molecular weight activity was detected using 2-methyl-butenoyl-CoA or 3-hydroxyisobutyryl-CoA as a substrate. These data indicate that the ECHDC2 gene encodes a novel enzyme with enoyl-coa hydratase activities similar to 3-methylglutaconyl-CoA hydratase. Generation of specific antibodies raised against both enoyl-coa hydratases are specific. The similarity in the observed substrate specificities between 3MGH and ECHDC2 makes it impossible to differentiate between the two enzymes using our standard hydratase assay in any given tissue. To solve this problem, we generated antibodies against the MBP-fusion proteins and their specificity was determined using E.coli lysates overexpressing GST-ECHDC2 or GST-3MGH. Western blot analysis (figure 4) of GST-ECHDC2 using the ECHDC2 antiserum revealed a single immunoreactive protein with an approximate molecular mass of 55 kda, which is in good agreement with the calculated Figure 3. Expression and purification of MBP-enoyl-CoA hydratase fusion proteins in E.coli. Total E.coli extract (T) and affinity-purified fusion protein (P) were analyzed by 10% SDS- PAGE and stained with Coomassie brilliant blue. of 74,086 and 78,567 Da, respectively. The kinetic properties of these highly purified fusion proteins were determined using different acyl-coas as substrates. Table 1 shows the kinetic parameters (K m and V max ) of the proteins with 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA), butenoyl-coa (C 4:1 -CoA) and octenoyl- CoA (C 8:1 -CoA) as substrates. Clearly, both enzymes show considerable hydratase activity towards HMG-CoA and butenoyl-coa, whereas no enoyl-coa hydratase Figure 4. Specificity of anti-bodies. Each lane corresponds to 50 ng of GST-fusion protein. Lane a is GST-fusion protein 3- methylglutaconyl-coa hydratase. Lane b is GST-fusion protein ECHDC2 protein. molecular weight of 57,583 Da. Moreover, the antiserum demonstrated no detectable immunoreactive response towards GST-3MGH. In contrast, immunoblot analysis using the 3MGH antiserum identified a cross-reacting species with an approximate size of ~ 50 kda in both lysates, which is probably due to nonspecific binding of the antiserum. More importantly, however, only western blot analysis of GST-3MGH using this antiserum revealed an Table 1. Substrate specificity of heterologously expressed MBP fusion proteins. Enzyme assays were performed as described in Experimental Procedures, and the results are the means of three independent measurements. Substrates used were 3-hydroxy- 3-methylglutaryl-CoA (HMG-CoA), butenoyl-coa (C4:1-CoA) and octenoyl-coa (C8:1-CoA). MBP-ECHDC2 MBP-3MGH Km Vmax Vmax/Km Km Vmax Vmax/Km substrate μm nmol/min/mg μm nmol/min/mg HMG-CoA C4:1-CoA C8:1-CoA n.d. n.d. n.d. not detectable 30

32 immunoreactive protein with an approximate size of 62 kda, which is in agreement with the calculated molecular mass of 62,064 Da for GST-3MGH. These data show that both the 3MGH and ECHDC2 antisera are specific for detecting either 3-methylglutaconyl- CoA hydratase or the ECHDC2 protein, respectively. ECHDC2 and 3MGH are localized to the mitochondria. We verified the subcellular localization of ECHDC2 and 3MGH by performing a density gradient centrifugation of a mouse kidney homogenate (figure 5). The distinct activity patterns for the marker enzymes, catalase (peroxisomes), glutamate dehydrogenase (mitochondria), β-hexosaminidase (lysosomes), esterase (microsomes) and phosphoglucoisomerase (cytosol) indicated a good resolution between the organelles (figure 5). Western blot analysis using the antisera identified a single immunoreactive protein with an approximate size of 36 kda and 31 kda for the human 3-methylglutaconyl-CoA hydratase and the ECHDC2 protein, respectively. The distribution profile of both proteins corresponded to the mitochondrial marker glutamate dehydrogenase, confirming that both enzymes are exclusively located in the mitochondria. Tissue-specific expression of ECHDC2 and 3MGH human, mouse and rat. As 3-methylglutaconyl-CoA hydratase activity is essential in the catabolism of leucine, and both hydratases are localized to the mitochondria, the tissue distribution of 3MGH and the ECHDC2 protein is of considerable interest. Western blot analysis demonstrated tissue-specific distribution of stably expressed 3MGH and ECHDC2 in humans, rats and mice (figure 6). The tissue distribution of 3MGH appears to be similar among humans, rats and mice. 3MGH is Figure 5. Subcellular distribution of enoyl-coa hydratases in mouse kidney. A post-nuclear supernatant of kidneys from nine male mice was prepared and subjected to equilibrium density-gradient centrifugation using a linear g/l Nycodenz gradient as described in experimental procedures. After centrifugation, the gradient was fractionated, and marker enzymes activities were measured in all 21 fractions: (II) b-hexosaminidase (lysosomes); (III) catalase (peroxisomes); (IV) phosphoglucoisomerase (cytosol); (V) glutamate dehydrogenase (mitochondria); (VI) esterase (microsomes); and (I) total protein concentration. Results are percentages of total activity observed (II-VI) or mg/ml protein (I). (VII) Immunoblot analysis of gradient fractions with antibodies raised against crotonase (ECHS1), 3-methylglutaconyl-CoA hydratase (3MGH) and the ECHDC2 protein (ECHDC2). 31

33 predominantly expressed in kidney, heart and brain of these species. Only in rat liver there is considerable expression of 3MGH as compared to human and mouse. Furthermore, there is moderate expression of 3MGH in the liver, muscle and testis of humans and rats. Analysis of the various tissues with antibodies raised against the ECHDC2 protein demonstrated that this protein is highly expressed in liver and kidney, whereas it is moderately expressed in the heart of humans, mice and rats. More importantly, no expression of the ECHDC2 protein could be detected in the brain of human, mouse and rat, in contrast to 3MGH which was highly expressed in this type of tissue. In addition, we also demonstrated that in human skin fibroblasts 3MGH is expressed, but the ECHDC2 protein not. Figure 6. Tissue distribution of 3-methylglutaconyl-CoA hydratase (A) and the ECHDC2 protein (B) in various human, rat and mouse tissues. Each lane corresponds to 50 µg of extract protein from the indicated tissues brain (B), kidney (K), liver (L), heart (H), muscle (M) and testis (T). Lane with human skin fibroblasts (HF) corresponds to 100 µg of extract protein. Discussion In this study we describe the molecular cloning of the human ECHDC2 gene encoding a novel mitochondrial enoyl-coa hydratase, the ECHDC2 protein. In addition, we present new data on human 3-methylglutaconyl- CoA hydratase (3MGH). The amino acid sequences of 3MGH and the ECHDC2 protein displayed features typical of other well-defined members of the crotonase superfamily, including the regions that define the active site pocket. Members of the crotonase superfamily catalyze a broad range of metabolic reactions, but share a common structural solution to a mechanistic problem. 5 In this family the common theme is the need to stabilize an enolate anion intermediate derived from an acyl-coenzyme A substrate. In fact, stabilization of this intermediate is accomplished by two structurally conserved peptide NH groups, in a so-called oxyanion hole, that provide hydrogen bonds to the carbonyl moieties of the acyl-coenzyme A substrates. Amongst this superfamily are enzymes that display hydratase, 32 dehalogenase, isomerase and thioesterase activities. In addition, both the ECHDC2 protein and 3MGH described in this paper also displayed two glutamate residues that are conserved in all currently reported enoyl-coa hydratases. Therefore, it appears that these two glutamate residues determine enoyl-coa hydratase activity. Indeed, members of this superfamily with only one or none of the conserved glutamate residues demonstrated no enoyl-coa hydratase activity but have isomerase, racemase or dehalogenase activity. 5,10 More importantly, both the ECHDC2 protein and 3MGH demonstrated hydratase activities towards 3-hydroxy- 3-methylglutaryl-CoA and butenoyl-coa. However, at present, the relationship between structure and enzymatic activity in the crotonase superfamily is still too ill-defined to allow predictions about the potential physiological function of the protein based on the amino acid sequence. No hydratase activity was found using 2-methylbutenoyl-CoA or 3-hydroxyisobutyryl-CoA as a substrate, which strongly indicates that the ECHDC2 protein is not involved in the catabolic pathways of the branched-chain amino acids isoleucine and valine, respectively. In fact, the ECHDC2 protein demonstrated considerable 3-methylglutaconyl-CoA hydratase activity, raising the interesting question if the ECHDC2 protein could be involved in the catabolic pathway of leucine, which is generally believed to be mitochondrial. For this reason, the subcellular localization of 3MGH and the ECHDC2 protein becomes a topic of considerable interest. Our data clearly show that both enzymes are located to the mitochondria, indicating that the ECHDC2 protein can relay 3-methylglutaconyl-CoA metabolism. Interestingly, all reported patients with 3- methylglutaconic aciduria type I clinically present with various neurological handicaps, whereas no abnormalities in peripheral organs are observed. Immunoblot analysis revealed that both the ECHDC2 protein and 3MGH have a tissue-specific distribution. Indeed, 3MGH is predominantly expressed in kidney, heart and brain, and moderately expressed in skin fibroblasts, liver, muscle and testis. Whereas the ECHDC2 protein is expressed in kidney, liver and heart, but no expression of this enzyme could be detected in brain and skin fibroblasts. Given that 3MGH and the ECHDC2 protein are expressed in peripheral tissues, a deficiency of 3MGH in these organs in principle can be corrected by the enzymatic actions of the ECHDC2 protein. Yet, in human brain there is no expression of the ECHDC2 protein. Hence, in the brain of patients with 3-methylglutaconic aciduria type I there is a total block of 3-methylglutaconyl-CoA hydratase activity, which will lead to neurological problems as seen in patients with this deficiency. Three additional forms of 3-methylglutaconic aciduria have been recognized (Barth syndrome, MIM ; Costeff syndrome, MIM ; and type IV, MIM ). Yet, all of these additional types are

34 characterized by normal 3-methylglutaconyl-CoA hydratase activity in human skin fibroblasts. As the ECHDC2 protein is not expressed in this tissue type, a deficiency of the ECHDC2 protein should be considered in patients with normal 3-methylglutaconyl-CoA hydratase activity in human skin fibroblasts, but with elevated urinary excretion of 3-methylglutaconic acid. Fortunately, as the ECHDC2 protein is expressed in other human tissues, such as liver, kidney and muscle, a biopsy of these tissues could provide a solution or diagnosis. In conclusion, although the physiological function of the ECHDC2 protein remains unresolved, it is important to note that the ECHDC2 protein could, in theory, relay 3-methylglutaconyl-CoA metabolism. Hence, upregulation of the ECHDC2 protein in human brain could be beneficial in patients with 3- methylglutaconic aciduria type I. References 1. Ijlst, L., Loupatty, F. J., Ruiter, J. P., Duran, M., Lehnert, W., and Wanders, R. J. (2002) Am. J. Hum. Genet. 71, Wanders, R. J., Vreken, P., den Boer, M. E., Wijburg, F. A., van Gennip, A. H., and Ijlst, L. (1999) J. Inherit. Metab Dis. 22, Engel, C. K., Mathieu, M., Zeelen, J. P., Hiltunen, J. K., and Wierenga, R. K. (1996) EMBO J. 15, Engel, C. K., Kiema, T. R., Hiltunen, J. K., and Wierenga, R. K. (1998) J. Mol. Biol. 275, Holden, H. M., Benning, M. M., Haller, T., and Gerlt, J. A. (2001) Acc. Chem. Res. 34, Wanders, R. J., van Roermund, C. W., Schor, D. S., ten Brink, H. J., and Jakobs, C. (1994) Biochim. Biophys. Acta 1227, Wanders, R. J., van Roermund, C. W., de Vries, C. T., van den, B. H., Schrakamp, G., Tager, J. M., Schram, A. W., and Schutgens, R. B. (1986) Clin. Chim. Acta 159, Wanders, R. J., Kos, M., Roest, B., Meijer, A. J., Schrakamp, G., Heymans, H. S., Tegelaers, W. H., van den, B. H., Schutgens, R. B., and Tager, J. M. (1984) Biochem. Biophys. Res. Commun. 123, Bradford, M. M. (1976) Anal. Biochem. 72, Wong, B. J. and Gerlt, J. A. (2004) Biochemistry 43,

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36 Chapter 6 Clinical, Biochemical and Molecular Findings in Three Patients with 3-Hydroxyisobutyric Aciduria Ference J. Loupatty, Annemarie van der Steen, Lodewijk IJlst, Jos P.N. Ruiter, Rob Ofman, Matthias R. Baumgartner, Diana Balhausen, Seji Yamaguchi, Marinus Duran and Ronald J.A. Wanders 3-Hydroxyisobutyric aciduria is a rare entity and affected individuals display a range of clinical manifestations including dysmorphic features and neurodevelopmental problems in the majority of patients. Here we present two novel patients with 3-hydroxyisobutyric aciduria. To our knowledge, these are the eleventh and twelfth cases of 3-hydroxyisobutyic aciduria reported. It is believed that a deficiency in 3-hydroxyisobutyrate dehydrogenase is the most likely cause of this disorder. Measurement of 3-hydroxyisobutyrate dehydrogenase activity in fibroblasts homogenates of the two newly identified patients and a previously reported patient, however, revealed similar activities as in control fibroblasts. Since other enzymes with overlapping substrate specificity could conceal abnormal 3-hydroxyisobutyrate dehydrogenase activity, we cloned a candidate human cdna for 3-hydroxyisobutyrate dehydrogenase (HIBADH). By heterologous expression in Escherichia coli, we showed that the product of the HIBADH gene indeed displays 3-hydroxyisobutyrate dehydrogenase activity. Mutation analysis of the corresponding gene in the patients suffering from 3-hydroxyisobutyric aciduria revealed no mutations. We conclude that HIBADH is not the causative gene in 3-hydroxyisobutyric aciduria. 3-Hydroxyisobutyric aciduria (MIM ) is a rare organic aciduria and to date only ten patients have been described in literature. 1-7 These patients usually present with dysmorphic features including a small triangular face, low set ears, long philtrum and microcephaly. Patients may show widely different phenotypes ranging from mild vomiting attacks with normal brain and cognitive development, to delayed motor development, profound mental impairment and early death. Patients with 3-hydroxyisobutyric aciduria usually excrete elevated amounts of 3-hydroxyisobutyric acid (3- HIBA) in urine, ranging from 60 to 390 mmol/mol of creatinine (normal < 40 mmol/mol of creatinine) when in stable condition and increasing up to 10,000 mmol/ mol creatinine during acute ketoacidotic episodes. 1 In half of the reported cases elevated lactate levels were observed. 1-7 Other, less frequent, abnormal metabolites in urine are 3-hydroxypropionic acid, 2-ethyl-3- hydroxypropionic acid, 2-hydroxybutyric acid, S-(2- carboxypropyl)-cysteine and 3-hydroxyisovaleric acid. The biochemical and molecular basis of 3- hydroxyisobutyric aciduria is poorly understood. 3-hydroxyisobutyric acid is an intermediate in the generally accepted pathway of valine oxidation (figure 1). Oral administration of valine in patients with 3- hydroxyisobutyric aciduria led to a major increase in the excretion of 3-HIBA and reproduced the clinical signs of ketoacidosis, suggesting that the 3-hydroxyisobutyric aciduria as observed in these patients is indeed due to a block in valine oxidation. 1 Although the responsible defect has not been identified, a deficiency of 3- hydroxyisobutyrate dehydrogenase has been suggested as the most likely enzyme defect in patients with 3- hydroxyisobutyric aciduria. 4 In 1957, Robinson and Coon were the first to isolate and characterize 3- hydroxyisobutyrate dehydrogenase from pig kidney and this NAD + -dependent enzyme has further been purified Figure 1. Catabolism of L-valine. The names of the intermediates in the pathway for catabolism of L-valine are shown on with solid arrows indicating enzymatic reactions. The structures of 3-hydroxyisobutyrate and methylmalonic semialdehyde are depicted. 3-Hydroxyisobutyrate dehydrogenase is HIBADH. From the Departments of Clinical Chemistry and Pediatrics, Emma Children s Hospital, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands (F.J.L.;J.P.N.R.;L.IJ.;M.D.;R.J.A.W.); Division of Metabolism and Molecular Pediatrics, University Children s Hospital, Zurich, Switzerland (M.R.B.;D.B.); Department of Pediatrics, Shimane Medical University, Japan (S.Y.) 35

37 and characterized from Pseudomonas aeruginosa, Candida rugosa, rabbit liver and rat liver Only, the rat 3-hydroxyisobutyrate dehydrogenase gene has been cloned until now. In this report, we present two novel patients manifesting dysmorphic features and developmental delay, in who urinary organic acid analysis revealed abnormal excretion of 3-hydroxyisobutyric acid. To our knowledge, these are the eleventh and twelfth cases of 3-hydroxyisobutyic aciduria reported. In an effort to clarify the abnormal metabolite profiles detected in patients suffering from 3-hydroxyisobutyric aciduria, we undertook the molecular and biochemical characterization of the human 3-hydroxyisobutyrate dehydrogenase in cultured fibroblasts derived from patients with 3-hydroxyisobutyric aciduria and control subjects. Case reports Patient 1. This boy is the first child of unrelated healthy Swiss parents. The pregnancy was uneventful until ultrasound displayed intrauterine growth retardation at 33 weeks of gestation. He was born by C-section at 39 weeks of gestation because of pathological cardiotocogram. APGAR was 9/10/10. His weight was 2360 g (150 g below the 10 th percentile), length was 44.5 cm (2 cm below the 10 th percentile) and head circumference 32 cm (0.8 cm below the 10 th percentile). He was mildly dysmorphic with unusually prominent eye brows and lashes and displayed mild muscular hypotonia. No hepatomegaly was noted. Echocardiography showed persistent pulmonary hypertension. In the first days of life he repeatedly had hypoglycemia ( mmol/ L) and developed lactic acidosis with peak lactate concentrations of up to 20 mmol/l. Plasma lactate/ pyruvate and 3-hydroxybutyrate/acetoacetate ratios were elevated (43 and 4, respectively). While the tendency to hypoglycemia resolved over time, lactate remained elevated (5-10 mmol/l). There was no hyperammonemia and no ketonuria. A plasma amino acid profile displayed elevated alanine (940 μmol/ L) and mildly elevated branched chain amino acids. Acylcarnitine analysis showed low free carnitine and analysis of urinary organic acids repeatedly displayed elevated concentrations of 3-hydroxyisobutyric acid (340 mmol/mol creatinine) and 2-ethyl-3-hydroxypropionic acid (260 mmol/mol creatinine) combined with lactic aciduria suggesting a defect at the level of 3- hydroxyisobutyrate dehydrogenase. On carnitine and a low protein diet supplemented with valine free amino acid mixture he stabilized, but remained with feeding difficulties and made only little developmental progress. Organic acid excretion normalized. Lactate remained mildly elevated ( 5 mmol/l) but no further hypoglycemia was noted. At the age of 7 months he was hospitalized because of persistent diarrhea and vomiting. While hospitalized he suffered an acute deterioration due to a gram positive sepsis which was 36 complicated by pericarditis and cardiac decompensation from which he slowly recovered. Echocardiography showed excentric hypertrophy of the right ventricle and persistent pulmonary hypertension (pulmonary artery pressure about half systemic pressure). A therapy with an endothelin receptor antagonist (Bosentan, Tracleer ) was started. A muscle biopsy performed at the age of 14 months showed reduced activities of respiratory chain complex I and IV (NADH-CoQ-oxidoreductase 0.1 U/ U citrate synthase [controls ] and cytochrome c oxidase 0.8 U/U citrate synthase [controls ], respectively). Furthermore, mitochondrial proliferation and ragged red fibers were observed upon enzyme histochemistry suggestive of a respiratory chain defect. A search for mitochondrial DNA mutations, insertions and deletions was performed with a normal result. His diet low in protein was changed to a diet enriched with fat (60% of total energy), and a therapy with riboflavin and coenzyme Q10 was started. Under this therapy plasma lactate ( 5 mmol/l) and urinary organic acids were essentially unchanged with intermittent mild elevations of 3-hydroxyisobutyric acid (10 and 30 mmol/mol creatinine). He had no further hypoglycemia or metabolic decompensation and his cardiomyopathy and pulmonary hypertension remained stable. However, despite some developmental progress, he remains severely retarded. At 21 months of age he shows severe failure to thrive (weight and length 1.7 cm below the 3 rd percentile) and microcephaly (head circumference 3 cm below the 3 rd percentile). Patient 2. This male patient is the second child of nonconsanguineous parents of Swiss origin. His 5 years older brother is healthy. In the 20th week of gestation ultrasound showed significant growth retardation. The child was born at 36 5/7 weeks of gestation with APGAR 9/9/10. He was small for date with weight 1450 g (550 g below the 10 th percentile), length 40 cm (4 cm below the 10 th percentile) and head circumference 31 cm (10 th - 50 th percentile). Due to icterus neonatorum he received phototherapy for 3 days. The child was very weak and therefore was fed with bottle milk. A herniotomy and orchidopexy on the left side was performed at 6 weeks. During operation it was noted that the left vas deferens was missing. There was also agenesis of the left kidney. Mild dysmorphic features including ptosis of the left eye lid, clinodactyly of the fifth finger and toe, and a syndactyly of the 2 nd and 3 rd toes were present. Smith-Lemli-Opitz syndrome was excluded. At the age of 10 weeks the patient was hospitalized because of a severe failure to thrive (weight 2140 g). He showed hyperaminoaciduria with prominent glycine and proline. Urinary organic acids analysis displayed repeated elevation of 3-hydroxyisobutyric acid (50 mmol/mol creatinine) and lactate (450 mmol/mol creatinine). Plasma amino acid analysis was unremarkable. Plasma lactate was only mildly elevated ( 5 mmol/l). Acylcarnitine analysis showed low free carnitine. A defect of 3-hydroxyisobutyrate

38 dehydrogenase was suspected and a therapy with a low protein diet supplemented with a valine-free amino acid mixture and carnitine was initiated at 4 months of age. Under this regimen the metabolic parameters normalized. Motor development was mildly delayed ( independent walking at 20 months). Mild spasticity of the lower extremities was observed. Development of speech was delayed with a remarkable deep voice. The diet was slowly released and then stopped at the age of 2 years. Off diet mild elevations of 3-hydroxyisobutyrate ( 20 mmol/mol creatinine) were again present. At 33 months of age all measures are still below the 3 rd percentile (weight 10.4 kg, length 86.3 cm, and head circumference 46.6 cm). Patient 3. The clinical details of this patient were described previously by Sasaki and co-workers. 4 Materials and methods Cell culture. Primary skin fibroblasts were obtained from the three subjects with 3-hydroxyisobutyric aciduria mentioned above and four control subjects. Fibroblasts were grown in HAM F10 medium, supplemented with 100 U/L penicillin G sodium, 100 U/L streptomycin sulfate, 250 mg/l amphotericin B and 100 ml/l fetal calf serum (Life technologies). Cultures were grown to confluence in 162 cm2 disposable culture flasks (Costar) and maintained at 37 C in an atmosphere of 5% CO 2-95% air with a relative humidity of 98%. Next, the cells were harvested with trypsin-edta (Life technologies) and the trypsin was inactivated by dilution in culture medium. Finally, the cells were washed twice with phosphate-buffered saline and stored as pellets frozen at -80 C. Enzymatic assay for 3-hydroxyisobutyrate dehydrogenase. S-3-Hydroxyisobutyric acid was prepared from its methyl ester (Sigma-Aldrich) as described by Rougraff and co-workers. 11 Quantitative analysis of S-3-hydroxyisobutyrate was done using GC/ MS as described by Röschinger and co-workers Hydroxyisobutyrate dehydrogenase activity was measured spectrophotometrically. The reaction mixture contained 100 mmol/l Tris-CHES-CAPS buffer ph 9.5, 1 g/l Triton X-100, 100 mmol/l hydrazine ph 9.5, 1 mmol/l NAD + and 0.9 mg/ml fibroblast homogenate or recombinant protein. After a preincubation of five minutes the reaction was started by the addition of 3- hydroxyisobutyric acid at a final concentration of 2.5 mmol/l. The production of NADH was followed in time on a Cobas-Fara centrifugal spectrophotometer at 340 nm using a molar extinction coefficient of 6220 L.mol/cm. Protein was determined by the bicinchoninic acid assay (Sigma) using BSA as standard. Cloning and expression of HIBADH. First strand cdna was synthesized from RNA isolated from normal cultured human skin fibroblasts as described by IJlst 37 and co-workers. 14 The open reading frame (ORF) of HIBADH was amplified from human cdna by PCR using the primers: +1HIBADHfBamHI: 5 -a tat gga tcc ATG GCA GCC TCC TTA CGG CTC and +1124HIBADHrSalI: 5 - a tat gtc gac GGT TCC CAA CAG TGT CCG TG. The primer set introduced a 5 BamHI and 3 SalI restriction site (underlined). The PCR product was ligated into the pgem-t vector (Promega, Madison, USA) and sequenced to exclude errors introduced by Taq polymerase. The ORF of HIBADH was subsequently released from the pgem-t vector as a BamHI-SalI fragment and ligated in-frame with the maltose binding protein (MBP) ORF into the BamHI and SalI restriction sites of pmal-c2x (Promega, Madison, USA). pmal- C2X plasmids containing the HIBADH ORF were transformed into E.coli INV-α chemo-competent cells (Invitrogen, Carlsbad, California, USA). Cells were grown from a 100-fold diluted fresh overnight culture for 4 hours in Luria-Bertani medium containing 100 µg/ ml ampicillin and 2 g/l glucose and grown to an OD Isopropyl-1-thio-β-D-galactopyranoside was added to a final concentration of 1 mmol/l to induce the production of the MBP-HIBADH fusion protein. Cells were lysed by sonication (nine times on ice for 15 seconds at 8 W at 45 second intervals) and the fusion proteins were affinity purified using an Amylose Resin Column following the instructions of the manufacturer (New England Biolabs, Ipswich, Massachusetts, USA). Mutation analysis of human HIBADH on genomic DNA. Genomic DNA was extracted from cultured human skin fibroblasts using the Wizard Genomic DNA purification Kit according to the instructions of the manufacturer (Promega, Madison, USA). Eight sets of HIBADH specific primers with 21M13 or M13rev extensions were used for amplification of the eight exons and their flanking intronic sequences. The sequences of these primers on request. The PCR reaction mixture contained 0.5 μmol/l of each primer (Eurogentec, Liege, Belgium), 10 mmol/l Tris/HCl ph 8.4, 50 mmol/l KCl, 1.5 mmol/l MgCl 2, 0.1 g/l BSA, 0.2 mmol/l dntp (Pharmacia, New York, USA) and 2.5 U Taq DNA polymerase (Promega, Madison, USA). With the following exceptions; for exon 8 the reaction mixture contained 2.0 mmol/l MgCl 2, and the above mixture was supplemented with 5 % DMSO for exons 1 and 5. Two different PCR programs were used to amplify HIBADH from genomic DNA. For amplification of exon 1 and 5 a touchdown approach was used for increased specific primer annealing and started with 2 min of denaturation at 96 C, followed by 10 cycles during which the annealing temperature was lowered with 1 C per cycle from 65 C to 55 C. Every cycle was initiated with 30 s of denaturation at 96 C, 30 s of annealing and 30 s of extension at 72 C. These ten touchdown cycles were followed by 23 cycles including

39 30 s at 94 C, 30 s at 55 C and 30 s at 72 C, with a final extension step of 5 min at 72 C. For the remaining exons the amplification program started with 2 min of denaturation at 96 C, followed by 5 cycles including 30 s of denaturation at 96 C, 30 s of annealing at 55 C and 1 min extension at 72 C. These five cycles were followed by 25 cycles with 30 s at 94 C, 30 s at 55 C and 1 min at 72 C, with a final extension step of 7 minutes at 72 C. With the exception that for exon 8 the extension step was 30 s instead of 1 min. Sequence analysis of these PCR fragments using Big Dye fluorescent-labeled M13 primers was performed on an Applied Biosystems 377A automated DNA sequencer following the manufacturer s protocols (Perkin-Elmer Applied Biosystems, Massachusetts, USA). Results and Discussion Here we present two patients biochemically characterized by increased urinary excretion of 3- hydroxyisobutyric acid and 2-ethyl-3-hydroxypropionic acid. As our patients showed no elevated urinary levels of 3-aminoisobutyrate the differential diagnosis of methylmalonate-semialdehyde dehydrogenase deficiency was excluded. In addition, our patients showed lactic acidosis. The phenotypic presentation of our patients includes minor dysmorphic features, microcephaly, episodes of vomiting and developmental delay. As the biochemical and clinical manifestations are common in patients with 3-hydroxyisobutyric aciduria, our patients were diagnosed with this condition. As mentioned above, a deficiency of 3- hydroxyisobutyrate dehydrogenase has been suggested as the most likely enzyme defect in patients with 3- hydroxyisobutyric aciduria. 4 However, measurement of 3-hydroxyisobutyrate dehydrogenase activity in the patients showed normal activities (patient 1: 4.6; patient 2: 4.2; index patient: 3.7 nmol/min/mg protein) as compared to controls (4.7 ± 1.2 nmol/min/mg protein (mean ± SD); range ). These data suggest that the 3-hydroxyisobutyrate dehydrogenase activity in cultured human skin fibroblasts of three patients with 3-hydroxyisobutyric aciduria is not affected. However, the specificity of the enzymatic assay has not been established, e.g. by immuno-precipitation with antibodies raised against 3-hydroxyisobutyrate dehydrogenase, and thus the presence of other enzymes with overlapping substrate specificity could potentially conceal a deficient activity of 3-hydroxyisobutyrate dehydrogenase. Another possibility for the normal activity found in the patients fibroblasts is that the defect is not expressed in cultured human skin fibroblasts due to the presence of different isoforms of 3-hydroxyisobutyrate dehydrogenase with a tissue-specific expression. The elevated metabolites found in the plasma and urine of patients with 3-hydroxyisobutyric aciduria could originate from organs in which the amino acid metabolism is high, e.g. liver and muscle. As a consequence, these types of tissue are potentially the best sources for diagnosis at the enzymatic level. As these tissues were not available we decided to study the defect on the genomic level. HIBADH has been characterized in different organisms and was cloned from a single mammalian species, i.e. the rat, but the human 3-hydroxyisobutyrate dehydrogenase gene has not been cloned. In the Genbank database we identified a human candidate gene (HIBADH; Genbank accession no. NM152740) located on chromosome 7p15.2 and encoding a protein with 70% identity to the rat 3-hydroxyisobutyrate dehydrogenase protein. To investigate whether this protein displays 3-hydroxyisobutyrate dehydrogenase activity we expressed it as a fusion protein with maltose binding protein in E. coli and affinity-purified the fusion protein according to the manufacturer s protocol. The purified fusion protein indeed displayed 3-hydroxyisobutyrate dehydrogenase activity. The K m values of 3-hydroxyisobutyric acid and NAD + were A HIBADH activity (µmol/min/mg protein) y = x R 2 = /V /S hydroxyisobutyric acid (mm) B HIBADH activity (µmol/min/mg protein) /V 4 2 y = x R = /S NAD + (mm) Figure 2. Km-curves for the purified MBP-fusion HIBADH protein. A). The effect of the 3-hydroxyisobutyric acid concentration on 3-hydroxyisobutyrate dehydrogenase activity in the presence of 1 mmol/l NAD +. Km for 3-HIBA was 0.12 mm as determined from the Lineweaver Burke plot (inset). B) The effect of the NAD + concentration on 3-hydroxyisobutyrate dehydrogenase activity in the presence of 2.5 mmol/l 3-hydroxyisobutyric acid. Km for NAD + was 0.5 mm as determined from the Lineweaver Burke plot (inset). 38

40 determined for the purified enzyme from Lineweaver- Burke double-reciprocal plots and were 0.12 mm and 0.5 mm respectively (figure 2). Finally, all the eight exons of HIBADH and their flanking intronic sequences were analyzed, but no mutations were found in the three patients with 3- hydroxyisobutyric aciduria. Hence, we conclude that mutations in HIBADH are not common in patients with 3-hydroxyisobutyric aciduria. The finding of normal 3-hydroxyisobutyrate dehydrogenase activity in the patients cells raises the question whether 3-hydroxyisobutyric aciduria is due to a secondary deficiency. It has been suggested that accumulation of 3-hydroxyisobutyrate is the result of an increased NADH/NAD + ratio inhibiting the NAD + - dependent 3-hydroxisobutyrate dehydrogenase. 11,15 This occurs when the respiratory chain is blocked due to a genetic deficiency of one or more of the individual complexes thus leading to an impaired oxidation of NADH and consequently, an elevated NADH/NAD + -ratio. This is supported by the fact that fifty percent of the patients with 3-hydroxyisobutyric aciduria excreted elevated amounts of lactic acid in their urine. In line with these observations, our first patient is a further example of a respiratory chain deficiency (in particular a complex I defect) leading to impaired oxidation of NADH and secondary inhibition of 3-hydroxyisobutyrate. 15 Thus the presence of large quantities of urinary 3-hydroxyisobutyrate together with lactic acidemia should raise suspicion of a respiratory chain deficiency. In summary, although we did identify the human gene encoding 3-hydroxyisobutyrate dehydrogenase, this gene appeared not to be defective in patient with 3-hydroxyisobutyric aciduria, leaving the underlying cause of this defect unknown. References 1 F.J.Ko, W.L.Nyhan, J.Wolff, B.Barshop, and L.Sweetman, 3-Hydroxyisobutyric aciduria: an inborn error of valine metabolism, Pediatr.Res. 30 (1991) D.Chitayat, K.Meagher-Villemure, O.A.Mamer, A.O Gorman, D.I.Hoar, K.Silver, and C.R.Scriver, Brain dysgenesis and congenital intracerebral calcification associated with 3- hydroxyisobutyric aciduria, J.Pediatr. 121 (1992) O.Boulat, N.Benador, E.Girardin, and C.Bachmann, 3- hydroxyisobutyric aciduria with a mild clinical course, J.Inherit. Metab Dis. 18 (1995) M.Sasaki, M.Kimura, K.Sugai, T.Hashimoto, and S.Yamaguchi, 3-Hydroxyisobutyric aciduria in two brothers, Pediatr.Neurol. 18 (1998) M.Sasaki, H.Iwata, K.Sugai, M.Fukumizu, M.Kimura, and S.Yamaguchi, A severely brain-damaged case of 3- hydroxyisobutyric aciduria, Brain Dev. 23 (2001) J.P.Shield, R.Gough, J.Allen, and R.Newbury-Ecob, 3- Hydroxyisobutyric aciduria: phenotypic heterogeneity within a single family, Clin.Dysmorphol. 10 (2001) Mienie L.J. and Erasmus E. Biochemical studies on a patient with a possible 3-hydroxyisobutyrate dehydrogenase deficiency. 5th international congress on inborn errors of metabolism W.G.ROBINSON and M.J.Coon, The purification and properties of beta-hydroxyisobutyric dehydrogenase, J.Biol.Chem. 225 (1957) J.W.Hawes, E.T.Harper, D.W.Crabb, and R.A.Harris, Structural and mechanistic similarities of 6-phosphogluconate and 3- hydroxyisobutyrate dehydrogenases reveal a new enzyme family, the 3-hydroxyacid dehydrogenases, FEBS Lett. 389 (1996) J.W.Hawes, D.W.Crabb, R.J.Chan, P.M.Rougraff, R.Paxton, and R.A.Harris, Mammalian 3-hydroxyisobutyrate dehydrogenase, Methods Enzymol. 324 (2000) P.M.Rougraff, R.Paxton, M.J.Kuntz, D.W.Crabb, and R.A.Harris, Purification and characterization of 3-hydroxyisobutyrate dehydrogenase from rabbit liver, J.Biol.Chem. 263 (1988) P.M.Rougraff, B.Zhang, M.J.Kuntz, R.A.Harris, and D.W.Crabb, Cloning and sequence analysis of a cdna for 3-hydroxyisobutyrate dehydrogenase. Evidence for its evolutionary relationship to other pyridine nucleotide-dependent dehydrogenases, J.Biol. Chem. 264 (1989) W.Roschinger, A.C.Muntau, M.Duran, L.Dorland, L.IJlst, R.J.Wanders, and A.A.Roscher, Carnitine-acylcarnitine translocase deficiency: metabolic consequences of an impaired mitochondrial carnitine cycle, Clin.Chim.Acta 298 (2000) L.IJlst, R.J.Wanders, S.Ushikubo, T.Kamijo, and T.Hashimoto, Molecular basis of long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency: identification of the major disease-causing mutation in the alpha-subunit of the mitochondrial trifunctional protein, Biochim.Biophys.Acta 1215 (1994) M.J.Bennett, W.G.Sherwood, K.M.Gibson, and A.B.Burlina, Secondary inhibition of multiple NAD-requiring dehydrogenases in respiratory chain complex I deficiency: possible metabolic markers for the primary defect, J.Inherit.Metab Dis. 16 (1993)

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42 Chapter Mutations in the Gene Encoding 3-Hydroxyisobutyryl-CoA Hydrolase Results in Progressive Infantile Neurodegeneration 7 Ference J. Loupatty, Peter T. Clayton, Jos P.N. Ruiter, Rob Ofman, Lodewijk IJlst, Garry K. Brown, David R. Thorburn, Robert A. Harris, Marinus Duran, Carlos DeSousa, Steve Krywawych, Simon J.R. Heales and Ronald J.A. Wanders Only a single patient with 3-hydroxyisobutyryl-CoA hydrolase deficiency has been described in literature and the molecular basis of this inborn error of valine catabolism has remained unsolved thus far. Here, we present a second patient with 3-hydroxyisobutyryl-CoA hydrolase deficiency who was identified through blood spot acylcarnitine analysis which showed persistently increased levels of hydroxy-c 4 -carnitine. Both patients manifested hypotonia, poor feeding, motor delay and subsequent neurological regression in infancy. Additional features in the newly identified patient included episodes of ketoacidosis and Leigh-like changes in the basal ganglia on the MRI scan. In cultured skin fibroblasts of both patients, the 3-hydroxyisobutyryl-CoA hydrolase activity was deficient, and virtually no 3-hydroxyisobutyryl-CoA hydrolase protein could be detected by western blotting. Molecular analysis in both patients uncovered mutations in the HIBCH gene, including one missense mutation which is in a conserved part of the protein and two mutations affecting splicing. A carefully interpreted acylcarnitine profile will allow more patients with 3-hydroxyisobutyryl-CoA hydrolase deficiency to be diagnosed. The catabolic pathway for the branched-chain amino acid valine is exceptional, because it is believed that part of the pathway between 3-hydroxyisobutyryl-CoA and propionyl-coa proceeds via free acids, thus requiring a specific hydrolase. This is in marked contrast to the degradation pathways of the other branched-chain amino acids, i.e. leucine and isoleucine, in which the intermediates distal to the 2-oxo-acids are all CoA thioesters. Indeed, the hydrolysis of an activated acyl group in the heart of a catabolic pathway is not only uncommon, but also energetically unfavorable especially when subsequent steps of the pathway again involve CoA thioester intermediates. Nevertheless, although the evidence for the need of a specific hydrolase in the catabolic pathway of valine was poor, a strong support for this hypothesis came from one case report documenting 3-hydroxyisobutyryl-CoA hydrolase (HIBCH) deficiency (MIM ) published in However, the molecular basis of 3-hydroxyisobutyryl- CoA hydrolase deficiency has remained unsolved. Brown and collegues 1 described a male infant (patient 1) born to consanguineous parents, who clinically manifested various physical malformations (dysmorphic facial features, multiple vertebral anomalies, tetralogy of Fallot and - at post-mortem examination - agenesis of the cingulate gyrus and corpus callosum), as well as failure to thrive and marked hypotonia. More importantly, however, the urine of this patient persistently demonstrated increased levels of S-2-carboxypropyl-cysteamine and S-2-carboxypropyl-cysteine. 1,2 As the latter compound can be formed by the condensation between cysteine and methacrylyl-coa, an intermediate in valine oxidation, the abnormal levels suggested a defect in the valine catabolic pathway. In valine catabolism (fig. 1), methacrylyl-coa is converted to 3-hydroxyisobutyryl- CoA by crotonase, but fibroblast and liver homogenates of patient 1 showed normal hydratase activities towards methacrylyl-coa (as well as crotonyl-coa and tiglyl-coa) when compared to controls. This finding suggested that if a defect in the catabolic pathway of valine were to exist, it would be distal to this hydration reaction. Indeed, through a coupled enzyme assay using methacrylyl-coa and an excess of the enzyme crotonase, Brown and co-workers demonstrated a 20 % residual activity of 3-hydroxyisobutyryl-CoA hydrolase in cultured skin fibroblasts. 1 No additional patients with 3-hydroxyisobutyryl-CoA hydrolase deficiency have been reported so far. In this report we describe the second patient with From the Departments of Clinical Chemistry and Pediatrics, Emma Children s Hospital, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands (F.J.L.;J.P.N.R.;R.O.;L.IJ.;M.D.;R.J.A.W.); University College London Institute of Child Health with Great Ormond Street Hospital for Children, London WC1, UK (P.T.C.;C.D.;S.K.); University College London Institute of Neurology with the National Hospital for Neurology and Surgery, Queen Square, London WC1, UK (S.J.R.H.); Genetics Unit, Department of Biochemistry, University of Oxford, Oxford, UK (G.K.B.); Murdoch Children s Research Institute, Royal Children s Hospital (D.R.T.) and Department of Pediatrics, University of Melbourne, Melbourne, Australia (D.R.T.); Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, USA (R.A.H.) 41

43 thiamine or a combination of vitamin C, vitamin E and ubiquinone. As he recovered from his episode of acute encephalopathy, he developed dystonia predominantly affecting the right arm and leg. A CT scan performed during the episode of acute encephalopathy revealed generalised oedema of the brain with loss of grey / white differentiation in the basal ganglia. In addition, an MRI scan (fig. 2) detected signal abnormalities in the globus pallidus and the midbrain with asymmetrical involvement of the cerebral peduncles (right greater than left). No structural abnormalities of the brain were observed. As the neuroimaging was reminiscent of Leigh s disease, we investigated the activities of pyruvate dehydrogenase and pyruvate carboxylase in cultured Figure 1. Catabolism of L-valine. The names of the intermediates in the pathway for catabolism of L-valine are shown on the left with solid arrows indicating enzymatic reactions. The structures of methacrylyl-coa, 3-hydroxyisubutyryl-CoA and 3-hydroxyisobutyrate are depicted. The names of the metabolites that are increased as result of 3-hydroxyisobutyryl-CoA hydrolase deficiency are shown by dashed arrows to the right. HIBCH deficiency (patient 2), who was the first child of healthy non-consanguineous Caucasian parents and was born at term following an uneventful pregnancy. Initially he fed poorly but otherwise appeared well until 4 months of age when his parents noted head bobbing. This was followed by a delay in motor milestones, then ataxia and a loss of skills. He was able to roll over at 5 months but lost this ability at 10 months. He was reaching out and grasping objects at 4 months but gradually lost this skill at 13 months. From the age of 9 months he started to have transient absences and episodes of eye rolling. At the age of 10 months he had a febrile illness during which he became irritable and more wobbly. He lost the ability to finger feed. Examination at 11 months revealed an alert interactive child who had no nystagmus but constant titubation of the head. He had marked truncal ataxia and was unable to sit unsupported. Tone, power and reflexes were normal in the upper limbs; tone was reduced, particularly distally, in the lower limbs. He had marked dysmetria and tremor on reaching out. At the age of 14 months following two days of coryza and lethargy, he became acutely unwell with a reduced level of consciousness and metabolic acidosis, and required intubation and ventilatory support. Blood ph was 7.29 with a base deficit of 15.8 mmol/l. Blood lactate and ammonia were normal. Echocardiography demonstrated no anomalies. Examination following recovery from this illness showed drowsiness, variable nystagmus, sluggish papillary responses, pooling of secretions in the mouth, fasciculation of the tongue, titubation of the head, and upper and lower limb hypotonia with preservation of reflexes. He showed no improvement on treatment with 42 Figure 2. MRI scans of patient 2 MRI scan undertaken when patient 2 was 14 months old. T2-weighted images. There is signal abnormality in the brain regions indicated by the white arrows: in the globi pallidi (upper image) and in the midbrain with asymmetrical involvement of the cerebral peduncles (R > L) (lower image). The appearances were considered likely to represent a neurometabolic disorder. No structural abnormality was noted.

44 human skin fibroblasts and measured the activities of respiratory chain complexes in a muscle biopsy of patient 2, as described elsewhere. 3-7 Normal activities were found for pyruvate carboxylase and pyruvate dehydrogenase. The activities of the respiratory chain complexes, expressed as a ratio to citrate synthase, revealed a marked reduction of complex I (patient 0.089, controls ) and a borderline reduction in complex IV (patient 0.013, controls ). These results were obtained on a muscle biopsy obtained 3 months after the major episode of metabolic decompensation. A second muscle biopsy obtained 3 months later showed normal activities of all four respiratory chain complexes. Urine organic acid analysis during ketotic episodes showed excessive excretion of 3-hydroxybutyrate and acetoacetate, and moderate excretion of lactate, 2-hydroxyisovalerate, 2-oxoisocaproic acid, dicarboxylic acids (C 6, C 8, C 10 ) and methylmalonate with normal methylcitrate. In contrast, he excreted normal levels of urinary organic acids when well. Normal values were found for non- fasting blood 3-hydroxybutyrate (<0.05mM), and blood and CSF lactate. Urine amino acid analysis was normal, except on one occasion when it showed increased ratios to creatinine of several amino acids (including glycine, alanine and taurine) plus one unidentified ninhydrin positive compound. Remarkably, elevated levels of hydroxy-c 4 - carnitine were consistently found in blood spots of patient 2. Its concentration ranged from µm, controls <0.4 µm. Elevation of hydroxy-c 4 - carnitine may occur in ketosis but the elevated blood concentration persisted in this patient even when he was not ketotic. Tandem mass spectrometric analysis does not distinguish between 3-hydroxy-n-butyrylcarnitine or 3-hydroxyisobutyryl-carnitine. Either of these hydroxy-c 4 -carnitine species could be increased as a consequence of a specific enzyme defect, i.e. short chain 3-hydroxyacyl-CoA dehydrogenase deficiency (SCHAD; MIM ) or 3-hydroxyisobutyryl-CoA hydrolase deficiency, respectively. Patients with SCHAD deficiency uniformly suffer from hyperinsulinism 8, a phenotype very different from that observed in patient 2. As expected, a normal activity of short chain 3- hydroxyacyl-coa dehydrogenase was found in the fibroblasts of this patient as well as of patient 1 (data not shown), suggesting 3-hydroxyisobutyryl-CoA hydrolase deficiency. Therefore, we investigated fibroblasts of patients 1 and 2 for 3-hydroxyisobutyryl-CoA hydrolase activity using a direct enzyme assay based on the use of the physiological substrate S-3-hydroxyisobutyryl- CoA. The thioester was synthesized from methyl S-3-hydroxyisobutyrate as described elsewhere. 3-Hydroxyisobutyryl-CoA hydrolase activity was subsequently measured spectrophotometrically. The reaction mixture contained in a total volume of 250 µl, 100 mm Tris.HCl ph 8.0, 1 mm EDTA, 1 g/l Triton X-100, 0.1 mm DTNB and mg/ml 43 fibroblast homogenate. After a preincubation of ten minutes the reaction was started by the addition of 3- hydroxyisobutyryl-coa at a final concentration of 0.2 mm. The reduction of DTNB was followed in time on a Cobas-Fara centrifugal analyzer (Roche) at 412 nm using a molar extinction coefficient of L/mol/ cm. Using this newly developed assay, an activity of 6.4 ± 1.6 nmol/min/mg protein (mean ± SD) was found in control fibroblasts (n=8) whereas in fibroblasts from both patients no 3-hydroxyisobutyryl-CoA hydrolase activity could be detected. A human cdna (HIBCH) encoding a protein with 3-hydroxyisobutyryl-CoA hydrolase activity has been described previously. 9 This HIBCH gene maps to chromosome 2q32.2 and has an open reading frame of 1161 base pairs encoding a protein of 386 amino acid residues with a calculated molecular weight of 43 kda. Immunoblot analysis using an antibody against 3- hydroxyisobutyryl-coa hydrolase revealed the absence of the HIBCH protein in patient 1 (fig. 3). Fibroblast lysates of patient 2 demonstrated an apparently lower expression of the HIBCH protein as compared to controls. To establish that 3-hydroxyisobutyryl-CoA hydrolase deficiency is caused by mutations in HIBCH, we analyzed the gene at the genomic level and the mrna level in both patients. We identified a homozygous IVS3-9T>G mutation in patient 1, consistent with parental consanguity. The IVS3-9T>G mutation was absent from 210 control chromosomes. However, this T>G transversion only slightly weakens the consensus sequence for a splice acceptor site, since a pyrimidine is preferred over a purine at this position in the consensus sequence. Hence, the consequence of this missense mutation on the splicing efficiency was investigated by analyzing HIBCH HIBCH> HL C1 C2 P1 P2 Figure 3. Immunoblot analysis of 3-hydroxyisobutyryl-CoA hydrolase in fibroblast lysates and human liver homogenates. 25 µg of human liver (HL) protein and equal amounts of fibroblast protein (100µg) of two control subjects (C1 and C2), the index patient (P1) and the newly identified patient (P2) were subjected to SDS-PAGE and transferred onto nitrocellulose by semidry blotting. Polyclonal antibodies raised against purified rat liver 3-hydroxyisobutyryl-CoA hydrolase were used at a 1:5000 dilution. Antigen-antibody complexes were visualized with goat anti-rabbit IgG-alkaline phosphatase conjugate. As a control for transfer of protein, each blot was reversibly stained with Ponceau S prior to the incubation with antibodies.