American Journal of Medical Genetics Part C (Seminars in Medical Genetics) 142C:64 –76 (2006) A R T I C L E Biochemical Findings in Common Inborn Errors of Metabolism MARZIA PASQUALI,* GAVIN MONSEN, LEAH RICHARDSON, MARTHA ALSTON, AND NICOLA LONGO The application of tandem mass spectrometry (MS/MS) to newborn screening has led to the detection of patients with a wider spectrum of inborn errors of metabolism. A definitive diagnosis can often be established early enough to start treatment before symptoms appear. Here, we review common biochemical findings in disorders caused by deficiency of 3-methylcrotonyl-CoA carboxylase, isobutyryl-CoA dehydrogenase, 2-methyl-3hydroxybutyryl-CoA dehydrogenase, 3-ketothiolase, 2-methylbutyryl-CoA dehydrogenase, and medium chain acyl CoA dehydrogenase. The diagnosis of these disorders requires biochemical confirmation by measurement of plasma acylcarnitine profile, urine organic acids, and urine acylglycine profiles followed by measurement of enzyme activity or detection of causative mutations. Early treatment can improve the outcome of these disorders. ß 2006 Wiley-Liss, Inc. KEY WORDS: newborn screening; tandem mass spectrometry; organic acidemias; urine acylglycine; urine acylcarnitine How to cite this article: Pasquali M, Monsen G, Richardson L, Alston M, Longo N. 2006. Biochemical findings in common inborn errors of metabolism. Am J Med Genet Part C Semin Med Genet 142C:64–76. INTRODUCTION Inborn errors of metabolism affect the capabilities of the organism to convert nutrients into one another or to use them for energy production. They are due to impaired activity of enzymes, Marzia Pasquali is an Associate Professor of Pathology at the University of Utah in Salt Lake City and Medical Director of the Biochemical Genetics and Newborn Screening at ARUP Laboratories. She has a strong interest in the development of new testing for the diagnosis of inborn errors of metabolism. Gavin Monsen is a Senior Technologist in the Biochemical Genetics Section of ARUP Laboratories. Leah Richardson is a Technical Specialist for the ARUP Laboratories Supplemental Newborn Screening Laboratory. Martha Alston is a Technologist in the Biochemical Genetics Section of ARUP Laboratories. Nicola Longo is a Professor of Pediatrics and Director of the Metabolic Service at the University of Utah. His research covers inherited disorders of fatty acid oxidation and the development of new therapies for metabolic disorders. *Correspondence to: Marzia Pasquali, Ph.D., Biochemical Genetics and Newborn Screening Laboratories, ARUP Laboratories, 500 Chipeta Way, Salt Lake City, Utah 84108. E-mail: email@example.com DOI 10.1002/ajmg.c.30086 ß 2006 Wiley-Liss, Inc. transporters, or cofactors resulting in accumulation of abnormal metabolites (substrates) proximal to the metabolic block or lack of necessary products. Abnormal byproducts can be produced when alternative pathways are utilized to transform the accumulated metabolites whose concentration renders them substrates of enzymes not usually involved in their metabolism. The measurement of some of these metabolites or their byproducts after conjugation with carnitine is the basis of expanded newborn screening by tandem mass spectrometry (MS/MS). Two main classes of metabolites are currently measured by expanded newborn screening programs using MS/MS: amino acids and acylcarnitine conjugates. The level of one or more amino acids increases in disorders of amino acid metabolism, when the metabolic block is close enough to the actual amino acid, resulting in an abnormal amino acid profile in the newborn blood spot (Table I). Other disorders in the intermediary metabolism of amino acids cause organic acidemias, detected because the abnormal metabolite is conjugated with carnitine to facilitate its excretion and to balance the Coen- zyme A pool (Table II). In disorders of fatty acid oxidation, fatty acids are physiologically conjugated with carnitine and the acylcarnitines produced accumulate in case of a metabolic block (Table III). Work is in progress to allow identification of other metabolites or to measure the activities of individual enzymes after the addition of suitable substrates [Li et al., 2004]. We are only using a small portion of the capabilities of the instrumentation currently available and many more diseases will become amenable to screening as our knowledge advances. MS/MS allows the presymptomatic identification of several metabolic disorders before irreversible organ damage has occurred. The vast majority of MS/MS allows the presymptomatic identification of several metabolic disorders before irreversible organ damage has occurred. inborn errors of metabolism are inherited as autosomal recessive traits, although there Proline Phenylalanine Methionine Leucine Citrulline Arginine Abnormal metabolite Arginase deficiency Liver disease Hyperalimentation Citrullinemia type 1 Citrullinemia type 2 Arginino succinic aciduria Pyruvate carboxylase deficiency (French form) Lysinuric protein intolerance Liver disease Hyperalimentation Maple syrup urine disease Hyperalimentation Homocystinuria Methionine adenosyltransferase deficiency Glycine N-methyltransferase deficiency S-adenosylhomocysteine hydrolase deficiency Tyrosinemia type 1 Liver disease Hyperalimentation Prematurity Phenylketonuria Biopterin syntesis defects Dihydrobiopterin reductase Liver disease Hyperalimentation Prematurity Hyperprolinemia type 2 Hyperprolinemia type 1 Lactic acidosis Liver disease Hyperalimentation Possible causes Plasma amino acids, urine organic acids Plasma amino acids, urine neopterin profile, blood DHPR activity Plasma amino acids and total plasma homocysteine, urine organic acids Plasma amino acids, urine organic acids Plasma ammonia, plasma amino acids, urine orotic acid, urine organic acids (blood lactate and pyruvate) Plasma ammonia, plasma amino acids, urine orotic acid Additional tests Pyridoxine (for type2) (Type 1 is considered benign) Diet low in phenylalanine Mental retardation, microcephaly Febrile seizures, seizures, developmental delays Diet low in branched chain amino acids, thiamine Diet low in methionine, pyridoxine, folic acid, betaine Low-protein diet, benzoate, phenylbutyrate. High-protein (arginine), low carbohydrate diet in citrin deficiency Refusal to feed, lethargy progressing to coma, hyperammonemia Refusal to feed, lethargy progressing to coma, brain edema Usually asymptomatic in the neonatal period, mental retardation, thrombosis, cataracts Low-protein diet, benzoate, phenylbutyrate Treatment Mental retardation, spasticity, microcephaly Signs/symptoms TABLE I. Amino Acid Abnormalities Identifiable by Newborn Screening ARTICLE AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS): DOI 10.1002/ajmg.c 65 AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS): DOI 10.1002/ajmg.c Tyrosine Tyrosinemia type 1 Tyrosinemia type 2 Tyrosinemia type 3 Transient tyrosinemia of the newborn Liver disease Prematurity Hyperalimentation Plasma amino acids and urine organic acids. Liver failure (Type 1), rickets (Type 1), photophobia (Type 2), keratosis (Type 2), delays (Types 2,3) Diet low in phenylalanine and tyrosine (Types 1–3), NTBC (Type 1) 66 are exceptions (ornithine transcarbamylase deficiency, 2-methyl-3-hydroxybutyrylCoA dehydrogenase deficiency, and others that are X-linked). Parents of affected children are usually asymptomatic, and their child is often the first with the disease in the family. Symptoms of inborn errors of metabolism usually appear early in infancy, although several cases become symptomatic in late childhood or adulthood. Signs and symptoms include failure to thrive, seizures, mental retardation, organs failure, and even death. With the application of MS/MS for newborn screening, the capability of identifying simultaneously several analytes and the increased sensitivity have led to the detection of an increased number of patients. In asymptomatic patients, the confirmation of diagnosis relies on specific tests such as ionexchange chromatography for amino acids analysis, gas-chromatography-mass spectrometry (GC/MS) for organic acids and acylglycines analyses, and MS/MS with (LC-MS/MS) or without liquid chromatographic separation for acylcarnitines profile. The combination of these tests, run on different specimen types, is the key in the confirmation of abnormal newborn screening results, especially for patients with borderline results. In this review, we will detail the most common findings for selected metabolic disorders, outlining the procedures that we follow to confirm or exclude specific diagnoses. A number of disorders (carnitine cycle, long-chain fatty acid oxidation, tyrosinemias, argininemia, glutaric, propionic, methylmalonic, and isovaleric acidemias) are covered in other chapters of this issue and will not be repeated in this more general review. ORGANIC ACIDEMIAS Organic acidemias include a large spectrum of disorders involving many metabolic pathways. A number of these disorders are on the catabolic pathways of branched-chain amino acids (Fig. 1), although in most cases, they do not result in specific abnormalities detectable by amino acid analysis. ARTICLE Accumulation of specific acylcarnitine species results from a metabolic block at any site in the pathway. Table II lists the common abnormalities detected by newborn screening in organic acidemias. Confirmation of screening results should be performed, as a rule of thumb, on a different specimen type than the screening test (e.g., urine) and possibly use a different methodology. For confirmation of a possible organic acidemia, usually urine organic acids and urine acylglycine profile are the primary and most informative tests; plasma acylcarnitine profile is also widely used, but should be ordered in conjunction with the urine studies, while urine acylcarnitine profile might give complementary information, useful to characterize borderline patients. For confirmation of a possible organic acidemia, usually urine organic acids and urine acylglycine profile are the primary and most informative tests; plasma acylcarnitine profile is also widely used, but should be ordered in conjunction with the urine studies, while urine acylcarnitine profile might give complementary information, useful to characterize borderline patients. 3-Methylcrotonyl-CoA Carboxylase Deficiency (OMIM 210200 and 210210) 3-Methylcrotonyl-CoA carboxylase is a biotin-dependent mitochondrial enzyme involved in the catabolism of leucine [Baumgartner et al., 2001; Gallardo et al., 2001], converting 3-methylcrotonylCoA to 3-methylglutaconyl-CoA (Fig. 1). The heteromeric mitochondrial enzyme is composed of a larger a subunit that binds biotin, bicarbonate, and ATP and a smaller b subunit containing the High C5:1, C3, C5-OH High C6-DC, C5-OH High C5-OH, C5:1 High C5-OH High C5-DC High C4 High C5 High C3 Abnormal acylcarnitine 3-Methylcrotonyl CoA carboxylase (MCC) deficiency (leucine metabolism) Maternal MCC deficiency Biotinidase deficiency Prematurity 3-Ketothiolase deficiency (isoleucine metabolism) 2-methyl-3-hydroxybutyryl-CoA dehydrogenase deficiency 3-OH 3-CH3 glutaryl CoA Lyase deficiency Prematurity Ketosis Holocarboxylase synthase deficiency Biotinidase deficiency Propionic acidemia Methylmalonic acidemia Prematurity Diet low in vitamin B12 Isovaleric acidemia (leucine metabolism) 2-methylbutyryl-CoA dehydrogenase deficiency (isoleucine metabolism) Medications (antibiotics) Isobutyryl CoA dehydrogenase deficiency (valine metabolism) SCAD deficiency Glutaric acidemia type 1 Kidney disease Possible causes Carnitine Special diet, carnitine, vigorous treatment of fever and infections Carnitine, low-protein diet Low-protein diet, fasting avoidance, carnitine Fasting avoidance, carnitine, IV glucose, vigorous treatment of infections Biotin Failure to thrive, carnitine deficiency, cardiomyopathy Macrocephaly, brain atrophy, hypotonia, dystonia, degeneration of basal ganglia Developmental delays, metabolic acidosis, hypoglycemia Metabolic acidosis, vomiting, headaches, occasional hyperammonemia Hypoglycemia, mental retardation, epilepsy Plasma ammonia, basic metabolic panel, plasma amino acids, plasma acylcarnitine profile, urine organic acids Comprehensive metabolic panel, plasma ammonia, plasma amino acids, plasma acylcarnitine profile, urine organic acids Comprehensive metabolic panel, plasma ammonia, plasma amino acids, plasma acylcarnitine profile, urine organic acids, serum biotinidase, fibroblast studies Vomiting, ketoacidosis, dehydration, coma, skin rash, alopecia Special diet, carnitine, glycine Plasma ammonia, basic metabolic panel, plasma amino acids, plasma acylcarnitine profile, urine organic acids, serum biotinidase Plasma ammonia, basic metabolic panel, plasma amino acids, plasma acylcarnitine profile, urine organic acids and acylglycine profile Plasma acylcarnitine profile, plasma amino acids, urine organic acids, acylglycine and acylcarnitine profile Plasma acylcarnitine profile, urine organic acids and acylcarnitine profile Plasma acylcarnitine profile, urine organic acids and acylglycine profile, serum biotinidase activity Metabolic acidosis, hyperammonemia, coma Therapy Special diet, carnitine, vitamin B12, biotin, other vitamins Signs/symptoms Metabolic acidosis, hyperammonemia, coma, hypotonia or hypertonia Additional tests TABLE II. Organic Acidemias Identifiable by Newborn Screening ARTICLE AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS): DOI 10.1002/ajmg.c 67 High C4, C5, C5DC, C6, C8, C10, C16 High C16, C16:1, C18, C18:1, low free carnitine High free carnitine/(C16 þ C18) Low free carnitine (C0) and C2 High C16-OH, C16:1-OH, C18-OH, C18:1-OH High C4 High C14:1, C14 High C6, C8, C10:1 Abnormal acylcarnitine Medium chain acyl CoA dehydrogenase (MCAD) deficiency Diet (special care formulas) Medications Very long chain acyl CoA dehydrogenase (VLCAD) deficiency Ketosis Short chain acyl CoA dehydrogenase (SCAD) deficiency Isobutyryl CoA dehydrogenase deficiency Long chain 3-OH acyl CoA dehydrogenase deficiency Sepsis Ketosis Primary carnitine deficiency Maternal primary carnitine deficiency Prematurity Infant of vegan mother Medications Carnitine palmitoyl transferase I deficiency Sepsis Carnitine supplementation Carnitine palmitoyl transferase 2 (CPT2) deficiency Carnitine acylcarnitine translocase (CACT) deficiency Medications Multiple acyl CoA dehydrogenase (MADD) deficiency Toxins Possible causes Frequent feedings, low-fat diet with MCT oil, low-dose carnitine Frequent feedings, low-fat diet with MCToil Frequent feedings, low-fat diet with MCT oil, carnitine Hypoglycemia, cardiomyopathy, sudden death Hypoglycemia, cardiomyopathy, hypotonia, sudden death Hypoglycemia Hypoglycemia, cardiac arrest, cardiomyopathy, dysmorphism Plasma carnitine, plasma acylcarnitine profile, urine organic acids, 3-OH-fatty acids, DNA testing Plasma carnitine levels, carnitine transport in fibroblasts Plasma carnitine, plasma acylcarnitine profile, urine organic acids Plasma carnitine, plasma acylcarnitine profile, urine organic acids, DNA testing, enzyme/transporter assay in fibroblasts Plasma carnitine, plasma acylcarnitine profile, urine organic acids, fibroblast studies Hypoglycemia, metabolic acidosis, recurrent vomiting, hepatomegaly Frequent feedings, low-fat diet, carnitine Hypotonia (?) Quantitative plasma acylcarnitine profile, urine organic acids, enzyme assay in fibroblasts, DNA testing Plasma acylcarnitine profile, urine organic acids, enzyme assay in fibroblasts, DNA testing Frequent feedings, low-fat diet with MCT oil, carnitine, riboflavin Carnitine Frequent feedings, low-fat diet with MCT oil, carnitine Therapy Hypoglycemia, cardiomyopathy, sudden death Signs/symptoms Frequent feedings, low-fat diet, carnitine Plasma acylcarnitine profile, urine organic acids and acylglycine profile, DNA testing Hypoglycemia, sudden death Additional tests TABLE III. Abnormalities of Fatty Acid Oxidation and the Carnitine Cycle Identifiable by Newborn Screening 68 AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS): DOI 10.1002/ajmg.c ARTICLE ARTICLE AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS): DOI 10.1002/ajmg.c Valine Leucine Isoleucine Cytosol 69 Mitochondrial matrix α-Ketoisovaleric acid α-Ketoisocaproic acid α-Keto-β-methylvaleric acid Branched Chain KetoacidDehydrogenase (BCKD) (Thiamine) Isobutyryl-CoA 2-Methylbutyryl-CoA Isobutyryl-CoA Dehydrogenase Metacrylyl-CoA 2-Methylbutyryl-CoA Dehydrogenase Tiglyl-CoA Hydratase (Crotonase) 3-OH-Isobutyryl-CoA 3-OH-Isobutyryl-CoA Deacylase 3-OH-Isobutyric Acid 3-OH-Isobutyric acid Dehydrogenase Methylmalonic acid semialdehyde Methylmalonic semialdehyde Dehydrogenase Propionyl-CoA Hydratase (Crotonase) 2-Methyl-3-OHbutyryl-CoA 3-Methyl-3-OH-ButyrylCoA Dehydrogenase 2-Methylacetoacetyl-CoA Isovaleryl-CoA IsovalerylCoA Dehydrogenase 3-Methylcrotonyl-CoA CO2 MethylcrotonylCoA Carboxylase (Biotin) 3-Methylglutaconyl-CoA 3-MethylglutaconlylCoA Hydratase 3-OH-3-Methylglutaryl-CoA 3-OH 3-methyl glutaryl-CoA lyase 3-Ketothiolase Acetyl-CoA Mevalonic acid Cholesterol Acetoacetic acid + Acetyl-CoA Figure 1. Catabolic pathways of branched chain amino acids. Intermediate metabolites feed into the citric cycle or into the respiratory chain. Enzymatic blocks can occur at any point in these pathways. binding site for 3-methylcrotonyl-CoA [Holzinger et al., 2001; Sweetman and Williams, 2001]. The two subunits are encoded by two different genes, MCCA (3q25-q27) and MCCB (5q12-q13.1), respectively. Deficiency of 3-methylcrotonyl-CoA carboxylase is inherited as an autosomal recessive trait and heterogeneous mutations in the MCCA and MCCB genes have been reported in affected patients [Dantas et al., 2005]. The phenotype is extremely variable with patients presenting in infancy with severe neurological involvement and developmental delays, to patients with recurrent attacks of metabolic decompensation followed by complete recovery to asymptomatic adults [Bartlett et al., 1984; Bannwart et al., 1992; Visser et al., 2000; Koeberl et al., 2003a; Baykal et al., 2005; Dantas et al., 2005]. Signs and symptoms include ketoacidosis, hypoglycemia, seizures, hyperammonemia, and coma. In other patients, failure to thrive is the predominant sign. There is no clear correlation between genotype and phenotype and modifying genes and environmental factors must have a major influence on the phenotype of 3methylcrotonyl-CoA carboxylase deficiency [Dantas et al., 2005]. Patients with 3-methylcrotonylCoA carboxylase deficiency can be identified through newborn screening by MS/MS because of an increased concentration of a hydroxylated five carbon atoms species (C5-OH) corresponding to 3-hydroxyisovaleryl-carnitine [Roschinger et al., 1995; van Hove Patients with 3-methylcrotonyl-CoA carboxylase deficiency can be identified through newborn screening by MS/MS because of an increased concentration of a hydroxylated five carbon atoms species (C5-OH) corresponding to 3-hydroxyisovaleryl-carnitine. et al., 1995; Koeberl et al., 2003a]. Newborn screening has also identified asymptomatic affected mothers of heterozygous babies [Koeberl et al., 2003a]. Some of these mothers had a history of recurrent vomiting requiring IV fluids after minor illnesses or high-protein meals [Koeberl et al., 2003a]. Urine organic acid analysis is the most informative test to confirm 3methylcrotonylglycinuria. Affected patients show a markedly increased excretion of 3-hydroxyisovaleric acid and the presence of 3-methylcrotonylglycine (Fig. 2A) that increases during episodes of acute decompensation. Urine acylglycine analysis might help in cases where 3-methylcrotonylglycine is only mildly elevated (Fig. 2B). Quantitative analysis of acylcarnitine profiles in plasma and urine provides further supporting evidence, showing a marked increase in C5OH carnitine and the absence of other acylcarnitine species seen in other disorders such as 3-ketothiolase deficiency, 3-hydroxy-3methylglutaryl-CoA lyase deficiency, or multiple carboxylase deficiency (Fig. 3). An isolated increase in C5OH carnitine can be seen with biotinidase deficiency that can be easily excluded by measurement of serum enzyme activity. The diagnosis is confirmed by measuring enzyme activity in cultured fibroblasts. Holocarboxylase synthase deficiency can also cause 3-methylcrotonylglycinuria and is excluded by measuring 70 AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS): DOI 10.1002/ajmg.c ARTICLE organic acids and urine acylglycine profiles and normal levels of ethylmalonic acid [Sass et al., 2004]. Therapy with carnitine supplements has reversed clinical symptoms in the first patient reported. 2-Methyl-3-hydroxybutyryl-CoA Dehydrogenase Deficiency (OMIM 300438) Figure 2. Urine organic acid profiles from patients with 3-methylcrotonyl-CoA carboxylase deficiency. 3-Hydroxyisovaleric acid and 3-methylcrotonylglycine are the key metabolites in urine. Note variable excretion of 3-methylcrotonylglycine (compare 3A and 3B) and consistently elevated excretion of 3-hydroxyisovaleric acid. Isolated elevations of 3-hydroxyisovaleric acid can also be seen in patients with biotinidase deficiency that should be excluded in all patients with 3-methylcrotonyl-CoA carboxylase deficiency. IS, internal standard. activity of another carboxylase in the same cells. Although the clinical presentation of 3-methylcrotonylglycinuria is variable and several patients have been asymptomatic, all patients should be considered at risk for developing life threatening Reye-like episodes in case of metabolic stress, such as protein load, infections, or prolonged fasting [Nyhan et al., 2005]. Isobutyryl-CoA Dehydrogenase Deficiency Isobutyryl-CoA dehydrogenase catalyzes the conversion of isobutyryl-CoA to methylacrylyl-CoA (Fig. 1) in the metabolism of valine. Its deficiency has been reported only recently in a patient presenting with failure to thrive, anemia, and dilated cardiomyopathy [Roe et al., 1998]. This patient had profound carnitine deficiency and responded well to carnitine supplements with reversal of cardiomyopathy and normalization of growth parameters [Roe et al., 1998]. All subsequent patients were prospectively identified by newborn screening and the clinical significance of this disease is still uncertain [Sass et al., 2004]. Heterogeneous mutations in the ACAD8 (OMIM 604773) gene on 11q25 have been identified in patients with isobutyryl-CoA dehydrogenase deficiency [Nguyen et al., 2002; Sass et al., 2004]. Increased concentrations of isobutyryl (C4)-carnitine in blood and urine and of isobutyrylglycine in urine are the biochemical findings characteristics of this disease. Newborn screening by MS/MS may identify newborn with isobutyryl-CoA dehydrogenase deficiency by their increased C4-carnitine level [Koeberl et al., 2003b] as in the more common Short chain acyl-CoA dehydrogenase (SCAD) deficiency. The presumptive diagnosis relies on detection of isobutyrylglycine in urine 2-Methyl-3-hydroxybutyryl-CoA dehydrogenase (MHBD, OMIM 300256) deficiency is an X-linked disorder of isoleucine degradation [Zschocke et al., 2000; Ensenauer et al., 2002; Sutton et al., 2003; Garcia-Villoria et al., 2005] characterized by a progressive loss of mental and motor skills with developmental regression, seizures, and rigidity sometimes triggered by illnesses. The metabolic block occurs in the conversion of 2-methyl-3-hydroxybutyryl-CoA to 2methylacetoacetyl-CoA (Fig. 1), one step before the last enzyme (3-ketothiolase) of the isoleucine degradation pathway. Different missense mutations in the HADH2 gene, located on Xp11.2, have been identified in a series of five patients with this disease [Ofman et al., 2003]. Females can be affected by this disorder due to skewed X-inactivation, although their phenotype is usually milder of that seen in males [Perez-Cerda et al., 2005]. The abnormal metabolites in urine or whole blood and plasma of patients with MHBD deficiency are essentially the same as those observed in patients with 3-ketothiolase deficiency: 2-methyl-3hydroxybutyrate and tiglylglycine in urine, C5:1 (tiglyl) carnitine and C5-OH (2methyl-3-hydroxybutyryl) carnitine in whole blood and plasma. Newborn screening by MS/MS can identify the two characteristic acylcarnitine species (C5:1, C5-OH). The diagnosis must be confirmed with urine organic acids and enzyme assay in cultured fibroblasts, since in some cases, urine organic acids are indistinguishable from those of a patient with 3-ketothiolase deficiency, although the clinical presentation is different. Dietary treatment with an isoleucine restricted, high-carbohydrate diet may improve the condition and, if initiated early, may prevent neuro- ARTICLE AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS): DOI 10.1002/ajmg.c Figure 3. Plasma acylcarnitine profiles obtained by MS/MS in patients with 3methylcrotonyl-CoA carboxylase deficiency. Note the great variability in C5OH (3-hydroxyisovaleryl-) carnitine. * ¼ Internal standards (m/z 311 ¼ d9-C5; m/z 347 ¼ d3-C8; m/z 375 ¼ d3-C10; m/z 403 ¼ d3-C12; m/z 437 ¼ d9-C14; m/z 459 ¼ d3-C16; m/z 487 ¼ d3-C18). degeneration [Zschocke et al., 2000; Olpin et al., 2002; Sutton et al., 2003]. 3-Ketothiolase Deficiency (OMIM 203750) 3-Ketothiolase (mitochondrial acetoacetyl-CoA thiolase, T2) is an enzyme 3-Ketothiolase (mitochondrial acetoacetyl-CoA thiolase, T2) is an enzyme involved in isoleucine catabolism and ketone bodies metabolism. Its deficiency is characterized by intermittent episodes of severe ketoacidosis, usually with normoglycemia or hyperglycemia (for which they can be confused with diabetes mellitus) that can result in hyperventilation, dehydration, lethargy, coma, and death. involved in isoleucine catabolism and ketone bodies metabolism. Its deficiency is characterized by intermittent episodes of severe ketoacidosis, usually with normoglycemia or hyperglycemia (for which they can be confused with diabetes mellitus) that can result in hyperventilation, dehydration, lethargy, coma, and death. Episodes are usually 71 associated with severe vomiting and are triggered by infections or other illnesses. Analysis of urine organic acids by GC/ MS during acute episodes reveals high excretion of 2-methyl-3-hydroxybutyrate, 2-methylacetoacetate, and tiglylglycine with large amounts of 3hydroxy-butyrate and acetoacetate. Analysis of acylcarnitines by MS/MS in whole blood or in plasma shows increased concentrations of C5OH (2methyl-3-hydroxybutyryl) carnitine, and C5:1 (tiglyl) carnitine (Fig. 4). The diagnosis is confirmed by enzyme assay in cultured fibroblasts. Therapy consists in mild protein restriction to limit the intake of isoleucine, avoidance of fasting, supplementation with carnitine, avoidance of prolonged fasting, and prompt treatment of illnesses that can precipitate acute attacks. The gene coding for this enzyme, ACAT1, maps to 11q22.3-23.1 and heterogenous mutations have been reported in patients with 3-ketothiolase deficiency [Fukao et al., 2001, 2002; Fukao and Yamaguchi, 2002; Mrazova et al., 2005]. Two groups of patients have been identified based on the presence of null mutations in both alleles or of mutations leaving some residual activity in at least one of the mutant alleles [Fukao et al., 2003; Zhang et al., 2004]. Although there is no correlation between phenotype and genotype, there are differences in the biochemical profiles under stable conditions between the two groups [Fukao et al., 2001]. In the group of patients with two null alleles the typical organic acids and acylcarnitine profile persists even in stable conditions. By contrast, tiglylglycine, 2-methyl-3-hydroxybutyrate, and 2-methyl-3-hydroxybutyrylcarnitine are not always detected in the group of patients with residual 3-ketothiolase activity [Fukao et al., 2003]. Newborn screening by MS/MS can identify infants with 3-ketothiolase deficiency with severe mutations, but it might fail to detect infants with the ‘‘milder’’ mutation(s). The outcome of 3-ketothiolase deficiency is favorable with early diagnosis, dietary therapy, and appropriate treatment of ketoacidosis [Fukao et al., 2001]. 72 AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS): DOI 10.1002/ajmg.c Figure 4. MS/MS plasma acylcarnitine profile in a patient with b-ketothiolase deficiency. Note the simultaneous elevation of C5:1 and C5OH carnitine. * ¼ Internal Standards (m/z 311 ¼ d9-C5; m/z 347 ¼ d3-C8; m/z 375 ¼ d3-C10; m/z 403 ¼ d3C12; m/z 437 ¼ d9-C14; m/z 459 ¼ d3-C16; m/z 487 ¼ d3-C18). ARTICLE Chinese origin [Matern et al., 2003], we have identified patients of different ethnicity. The gene (ACADSB) is located on chromosome 10q25-26 and mutations have been reported in affected patients [Andresen et al., 2000; Matern et al., 2003]. Homozygosity for a methionine to valine change at position 356 of the mature protein (M356V) has been found in several of the Hmong patients studied, suggesting a founder effect [Matern et al., 2003]. A newborn screening result with elevated C5-carnitine could indicate also isovaleric acidemia (isovalerylcarnitine is a 5-carbon atom species) and should be followed up with urine organic acids and urine acylglycine analyses in order to identify the specific metabolic defect. 2-Methylbutyryl-CoA Dehydrogenase Deficiency 2-Methylbutyryl-CoA dehydrogenase (SBCAD, short branched chain acylCoA dehydrogenase, OMIM No. 600301) catalyzes the conversion of 2-methylbutyryl-CoA to tiglyl-Coa in the catabolic pathway of isoleucine (Fig. 1). The deficiency of this enzyme has been described only recently [Andresen et al., 2000; Gibson et al., 2000] and the phenotype is not yet well defined, ranging from completely asymptomatic patients to muscle weakness, cerebral palsy, developmental delays, lethargy, hypoglycemia, and metabolic acidosis. Biochemically, 2-methylbutyryl-CoA dehydrogenase deficiency is characterized by increased urinary excretion of 2methylbutyrylglycine (Fig. 5B), without an increase in isovalerylglycine, and increased whole blood and plasma concentrations of 2-methylbutyryl(C5) carnitine (Fig. 5A). Diagnosis can be confirmed by enzyme assay in fibroblasts or DNA studies. Therapy consists of protein restriction and carnitine supplements. Newborn screening by MS/MS can identify patients with 2-methylbutyrylCoA dehydrogenase deficiency by elevated C5-carnitine. Although the majority of patients described belong to the Hmong population, an ethnic group of Figure 5. Urine acylcarnitine profile (A) and urine acylglycine profile (B) from a patient with 2-methylbutyryl-CoA dehydrogenase deficiency. A: Note the extreme elevation of C5 carnitine.* ¼ Internal standards (m/z 311 ¼ d9-C5; m/z 403 ¼ d3-C12; m/z 459 ¼ d3-C16; m/z 487 ¼ d3-C18). B: PG ¼ propionylglycine; IBG, isobutyrylglycine; BG, butyrylglycine; 2MBG, 2-methylbutyrylglycine; IVG, isovalerylglycine. The internal standards are deuterated glycine conjugates (d3-PG; d7-IBG; d3-BG; d92MBG; d9-IVG). ARTICLE AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS): DOI 10.1002/ajmg.c FATTY ACID OXIDATION DISORDERS Fatty acid oxidation plays a major role in energy production in conditions of increased demand, such as during a prolonged fasting, illness, and/or muscular exertion. In these cases, the depletion of glycogen stores and the falling glucose levels activate lipid mobilization from fat stores and its utilization by the liver, the heart, and the muscle. Fatty acid b-oxidation occurs in mitochondria and it involves at least 20 steps, some of which catalyzed by enzymes with overlapping chain length specificity (Fig. 6). While short and medium chain fatty acids are believed to freely cross the mitochondrial membrane, long chain fatty acids (C16 and C18) enter the mitochondrial matrix after conjugation with carnitine. Measurement of these Fatty acid b-oxidation occurs in mitochondria and it involves at least 20 steps, some of which catalyzed by enzymes with overlapping chain length specificity. While short and medium chain fatty acids are believed to freely cross the mitochondrial membrane, long chain fatty acids (C16 and C18) enter the mitochondrial matrix after conjugation with carnitine. acylcarnitine conjugates allows newborn screening for several of these disorders. Abnormalities in beta-oxidation can result in hypoketotic hypoglycemia, myopathy, cardiomyopathy, sudden infant death syndrome (SIDS). Analysis of urine organic acids and plasma acylcarnitine is part of the routine evaluation of a patient presenting with hypoglycemia. In urine organic acids, excess excretion of dicarboxylic acids, especially unsaturated, deriving from microsomal o-oxidation of fatty acids not metabolized by mitochondria, C16:0 palmitoylCoA without excess 3-hydroxybutyrate is a nonspecific sign of defective b-oxidation. Other abnormal organic acids and acylcarnitine species allow the identification of the specific defect in the boxidation cascade. Many disorders of fatty acid oxidation are episodic, not only clinically but also biochemically. This means that a normal organic acid or acylcarnitine result obtained from an asymptomatic patient does not necessarily exclude a fatty acid oxidation disorder. Newborn screening by MS/ MS allows the identification of patients with fatty acid oxidation disorders, often before symptoms occur. Often, the newborn screening blood spot is the most informative sample for detecting these disorders. Medium Chain Acyl-CoA Dehydrogenase (MCAD) Deficiency (OMIM 201450) Medium chain acyl-CoA dehydrogenase deficiency (MCAD) is the most common disorder of fatty acid oxidation, with an estimated frequency of 1:6,000 to 1:10,000 Caucasian births [Roe and Ding, 2001; S-CoA VLCAD: C14-C20 Acyl-CoA LCAD: C12-C18 FAD Acyl-CoA dehydrogenases FADH2 MCAD: C4-C12 O SCoA 2,3-Enoyl-CoA O H2O Hydratases S-CoA SCAD: C4-C6 TFP: C12-C18 Crotonase: C4>C14 L-3-hydroxyacyl-CoA NAD+ LCHAD (TFP): C12-C18 Hydroxyacyl-CoA dehydrogenases NADH+H+ SCHAD: C4>C16 S-CoA 3-Ketoacyl-CoA TFP: C6-C16 O O HS-CoA MKAT: C4-C12 Thiolases OH O C14:0 myristoyl-CoA O β-ketothiolase: C4 S-CoA Acyl-CoA + Acetyl-CoA TCA cycle muscle (n-2) S-CoA O 73 li v er Ketogenesis New Cycle Figure 6. Mitochondrial b-oxidation of fatty acids. Fatty acids are progressively shortened by two carbon units at each cycle by a series of enzymes with overlapping specificity. At the end of each cycle, the residual fatty acyl chain (two carbon atoms shorter) enters another cycle for further shortening. The relative specificity of each enzyme toward a carbon length chain is indicated. VLCAD, very long chain acyl CoA dehydrogenase; LCAD, long chain acyl CoA dehydrogenase; MCAD, medium chain acyl CoA dehydrogenase; SCAD, short chain acyl CoA dehydrogenase; TFP, tri functional protein; LCHAD, long chain 3-hydroxy acyl CoA dehydrogenase; SCHAD, short chain 3-hydroxy acyl CoA dehydrogenase; MKAT, medium chain 3-keto acyl CoA thiolase; TCA, tri-carboxylic acid (Krebs). 74 AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS): DOI 10.1002/ajmg.c Medium chain acyl-CoA dehydrogenase deficiency is the most common disorder of fatty acid oxidation, with an estimated frequency of 1:6,000 to 1:10,000 Caucasian births. Nyhan et al., 2005]. The symptoms of the disease are variable, from completely The symptoms of the disease are variable, from completely asymptomatic patients to hypoglycemia, lethargy, coma, sudden death and they are usually triggered by prolonged fasting or illness. The gene encoding medium chain acyl-CoA dehydrogenase, ACADM, maps to 1p21 and is composed of 12 exons [Zhang et al., 1992]. Among patients presenting with clinical symptoms, 98% carry at least one copy of the common mutation K304E, with 80% being homozygous for this mutation [Gregersen et al., 1991; Yokota et al., 1991; Andresen et al., 1997, 2001; Pollitt and Leonard, 1998]. There is a wide spectrum of the second causative mutation in these patients [McKinney et al., 2004]. In newborns detected prospectively by newborn screening, the Y42H mutation has been found frequently in association with the common mutation K304E [Andresen et al., 2001; Zschocke ARTICLE et al., 2001; O’Reilly et al., 2004]. This mutation (Y42H) has not yet been reported in patients with clinical symptoms [Andresen et al., 2001; Zschocke et al., 2001] and is associated with lower levels of diagnostic metabolites in blood and urine [Waddell et al., 2006]. Patients with MCAD deficiency are identified by MS/MS newborn screening because of the characteristic acylcarnitine profile, with increased concentration of C6-(hexanoyl), C8-(octanoyl), and C10:1-(decenoyl) carnitine and elevated C8/C2 and C8/C10 ratios (Fig. 7). Urine organic acids and urine acylglycines analyses during metabolic crisis, show increased excretion of dicarboxylic acids (adipic, suberic, sebacic) saturated asymptomatic patients to hypoglycemia, lethargy, coma, sudden death and they are usually triggered by prolonged fasting or illness [Roe and Ding, 2001]. Although the majority of patients present in the first year of life, clinical symptoms can occur at any time and as many as 20% of patients die prior to diagnosis [Iafolla et al., 1994]. The treatment consists in avoidance of fasting, low-fat diet, carnitine supplementation, and institution of an emergency plan in case of intercurrent illness or other metabolic stress. Early diagnosis through newborn screening and early initiation of treatment leads to Early diagnosis through newborn screening and early initiation of treatment leads to improved outcome. improved outcome [Wilson et al., 1999; Carpenter et al., 2001]. Figure 7. Plasma acylcarnitine profiles in patients with MCAD deficiency. Note the consistent pattern with elevation of C6, C8, and C10:1 carnitines, although the C8 concentration is much higher in 7B. * ¼ Internal Standards (m/z 311 ¼ d9-C5; m/z 347 ¼ d3-C8; m/z 375 ¼ d3-C10; m/z 403 ¼ d3-C12; m/z 437 ¼ d9-C14; m/z 459 ¼ d3-C16; m/z 487 ¼ d3-C18). 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