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Succinic Semialdehyde Dehydrogenase Deficiency (SSADH) Symposium March 31, 2016 Inborn Errors of Metabolism: Metabolomics Gerard T. Berry, MD Professor of Pediatrics, Harvard Medical School Director, Metabolism Program, Boston Children’s Hospital No conflicts of interest to disclose Classification of Genetic Diseases A) • Chromosomal • Single Gene Defect - Autosomal Dominant - Autosomal Recessive - X-Linked • Nucleotide Repeat Defects • Parental Imprinting Defects • MicroRNA mutations • Complex Genetic disease (Polygenic or Multifactorial) B) • Mitochondrial DNA Disorders (“Heteroplasmic Cytopathies”) Most metabolic disorders are due to gene defects inherited as autosomal recessive traits The defective or missing gene products, the proteins, are usually enzymes but may be transport or structural proteins. List of Disorders Carbohydrate • Galactosemia • Glycogen Storage Disease Amino Acid • Phenylketonuria • Maple Syrup Urine Disease • Nonketotic Hyperglycinemia • Homocystinuria Urea Cycle/Ammonia • Ornithine Transcarbamylase Def. Vitamin/Cofactor • Biotinidase Deficiency Lysosomal Storage Diseases • Gaucher Disease • Tay-Sachs Disease • Hurler Disease • Neuronal Ceroid Lipofuscinosis • Cystinosis Disorders, cont. Peroxisomal • X-Linked Adrenoleukodystrophy • Zellweger Disease Transport • Cystinuria • GLUT2 Deficiency Purine and Pyrimidine • Lesch-Nyhan Disease • Dihydropyrimidine dehydrogenase deficiency Metal • Wilson Disease • Menkes Disease Organic Acid • Methylmalonic Acidemia • Propionic Acidemia • Glutaric Aciduria, type 1 Fatty Acid • MCAD Deficiency • Trifunctional Protein and LCHAD Deficiency Disorders, Cont. Isoprenoid/Sterol Pathways • Smith-Lemli-Opitz Disease Neurogenetics • Canavan Disease • Alexander Disease Purine and Pyrimidine • Lesch-Nyhan Disease • Dihydropyrimidine dehydrogenase deficiency Cell Signaling • Ataxia-Telangiectasia Mitochondrial Oxidative Metabolism • Respiratory Chain Defects • Pyruvate Dehydrogenase Complex Deficiency Classifications of Disorders • Small molecule diseases • Storage diseases • Organelles: – Lysosomal diseases – Mitochondrial diseases – Peroxisomal diseases NEWBORN SCREENING Guthrie specimen -Heel stick -Filter paper -Dried blood -Transport to central lab NEWBORN SCREENING BY TANDEM MASS SPECTROMETRY The Assay Acylcarnitine Profile Amino Acid Profile MCADD PKU LCHADD, VLCADD Propionic Acidemia Methylmalonic Acidemia Isovaleric Acidemia CPT II, MADD Glutaric Aciduria I & II 3-MCCD MSUD, Citrullinemia, Argininosuccinic Aciduria, Tyrosinemia General Modes of Presentation • Acute life-threatening illness • Chronic failure to thrive/ poor growth/ developmental delay picture • Chronic progressive psychomotor retardation/ loss of developmental milestones/ progressive neurologic disease • Liver disease and renal tubular dysfunction • Cardiomyopathy • Dysmorphic features or evidence of organ dysgenesis in the neonate • Psychosis/ psychiatric disease in the older patient Important Metabolic Tests When You Suspect a Metabolic Disorder • • • • • • • Blood glucose Serum electrolytes Plasma ammonium Plasma lactate Blood lactate and pyruvate (special PCA tube) Plasma amino acid quant (1ml GTT) CSF glucose, lactate, amino acids and neurotransmitter metabolites • Blood specimen for workup of hypoglycemia (5ml RTT) • Plasma acylcarnitine analysis (1ml GTT) • Urine organic acids Scriver et al., MMBID 2001; 1-2:2199. Scriver et al., MMBID 2001; 1-2:2198. Glutaric Aciduria, Type 1 • • • • • Congenital Macrocephaly Coma Dystonia Developmental Delay Subdural Hygroma / Hematoma Glutaric Acidemia, type 1 Enzyme defect: glutaryl-CoA + FAD glutaryl-CoA Dehydrogenase (GCDH) GCDH crotonyl-CoA + CO2 + FADH2 Gene: GCDH on chromosome 19p13.2 Frequency: very rare (except in Canadian Indians and Pennsylvania Old-Order Amish) Inheritance: autosomal recessive Nonketotic Hyperglycinemia • • • • • • • Seizures Coma Severe Hypotonia with Areflexia Hiccups Developmental Delay / MR Progressive Neurodegeneration Death Nonketotic Hyperglycinemia Enzyme defect: glycine cleavage system (GCS) GCS Glycine + THF + NAD + 5,10-CH2-THF + NH3 + CO2 + NADH pyridoxal phosphate lipoate Gene: P protein on chromosome 9p22 H protein on chromosome 16q24 T protein on chromosome 3p21.2-p21.1 Lipoic acid biosynthetic defects Frequency: very rare (1/12,000 in Finland) Inheritance: autosomal recessive Phenylketonuria Enzyme defect: phenylalanine hydroxylase (PAH) PAH phenylalanine + O2 tyrosine + H2O tetrahydrobiopterin(BH4) Gene: PAH on chromosome 12q22-q24.1 Frequency: 1/12,000 Inheritance: autosomal recessive Useful Thought Small molecule diseases may present as acute lifethreatening illnesses Large molecule disorders often present as storage diseases Neuronal Ceroid Lipofuscinosis • Developmental Delay / MR • Seizures • Blindness • Spastic Quadriplegia • Death Neuronal Ceroid Lipofuscinosis, Juvenile Type (JNCL); Batten Disease Enzyme defect: lysosomal membrane protein, CLN3 Gene: CLN3 on chromosome 16p12.1 Frequency: in Finland, 1/21,000 Inheritance: autosomal recessive 10 year old male with history of a decline in school performance over the past year. Parents brought him for a check-up because he has been bumping into objects for the past 6 weeks. P.E. reveals bilateral pale optic disks. MRI of the head shows bilateral occipito-parietal white matter disease. X-Linked Adrenoleukodystrophy • • • • • • Poor Vision Personality Change Dementia Spastic Quadriplegia Adrenal Insufficiency Death X-linked Adrenoleukodystrophy Protein defect: ALDP Peroxisomal membrane ABC protein, ALDP, necessary for conversion of very long-chain fatty acids to very long chain acyl-Co esters Gene: ALDP on chromosome Xq28 Frequency: 1/20,000 to 1/50,000 males Inheritance: X-linked recessive X-Linked Adrenoleukodystrophy Very Long Chain Fatty Acids (VLCFA) Very Long Chain Fatty Acyl-CoA Esters Laboratory Findings • Elevated Serum VLCFA • +/- Cortisol Deficiency Scriver et al., MMBID 2001; 1-2:3272. Scriver et al., MMBID 2001; 1-2:3273. Psychosis/ Psychiatric Disease in the Older Patient • Examples – Hypercalcemia – Homocystinuria – OTC Deficiency – Citrullinemia – Storage diseases • E.g. Tay-Sachs disease variant Normal Bulls eye maculopathy Scriver et al., MMBID 2001; 1-2:2171. Clinical Presentations of cblC Deficiency Prenatal: IUGR, ? Malformations, HYDROPS, CM Neonatal: neonatal crisis, HUS-like syndrome, “acrodermatitis acidopathica”, macular scarring (TORCH) Infancy: FTT/growth failure, hypotonia +/siezures, hematological disturbances, visual disturbances Childhood/Adolescence: psychiatric symptoms (dementia, regression, confusion), renal disease Adult: progressive encephalopathy, spinal cord syndrome (paraplegia) MMAA (cblA,?cblH) ? Protection Cytosol L-methylmalonyl-CoA Methymalonyl-CoA Racemase Methymalonyl-CoA Mutase Adenosylcobalamin MMCR Cbl(I?) lysosome ? cblC ? Methionine Synthase Reductase (Mitochondrial Isoform) Cbl(II?) ? Transport into the Mitochondria Cbl(III) Mitochondrial Matrix MMAB cblB D-methylmalonyl-CoA OH-Cbl(III) cblF Succinyl-CoA ? Cbl(II?) Cbl(II?) ? cblD (Variant 1) ? cblD (Variant 2) Cbl(II?) Methionine Synthase Reductase cblE Methylcobalamin Cbl(I) Methionine Methionine Synthase (cblG) Homocysteine Cerebral folate deficiency • First report in 1994 by Wevers in adult man with slowly progressive cerebellar syndrome, pyramidal tract dysfunction and hearing loss • Low CSF folate despite normal plasma folate • Low immunoreactive soluble FR1 protein, suggestive of diminished expression/secretion of FR1 Cerebral folate deficiency • • • • • • • • Onset age in infancy Unrest, irritability, sleep disturbance Psychomotor retardation Cerebellar ataxia Spastic paraplegia Dyskinesia Seizure Deceleration of head growth Cerebral folate deficiency • Neuroimaging: atrophy of frontotemporal regions periventricular demyelination slowly progressive brain atrophy Secondary cerebral folate deficiency • • • • • In all cases of Kearns-Sayre syndrome In some mitochondrial disorders In some cases of Rett syndrome Aicardi-Goutieres syndrome Some disorders of biopterin metabolism i.e., AADC, dihydropteridine reductase Metabolomics The latest omic’s to surface or was it the first, now only bigger? ACMG presentation Sarah Elsea, PhD et al Baylor College of Medicine Global-MAPS Metabolomics • Clinical metabolomic profiling is a novel platform that allows for parallel testing of hundreds of metabolites in a single plasma specimen analysis. It uses a stateof-the-art mass spectrometry platform, and the resulting spectra are compared against a library of ~2,500 human metabolites. On average, 886 small molecules are detected in a given sample with a core group of 404 analytes found in all specimens tested to date. The analytes detected encompass numerous classes of important small molecule biomarkers such as acylcarnitines, amino acids, bile acids, carbohydrates, lipids and nucleotides. Elsea et al Global-MAPS Metabolomics • Using this platform on >100 patients from our clinic with confirmed diagnosis of inborn errors of metabolism (IEM), we have successfully identified over 25 disorders including amino acid, organic acid, fatty acid oxidation, vitamin cofactor, pyrimidine biosynthesis, creatine biosynthesis, and urea cycle disorders. • • • • • • • • • • • • • • • • • • • • Elsea et al Global-MAPS Metabolomics 3-MCC deficiency arginemia citrate transporter deficiency cobalamin-related disorders citrullinemia glutaric academia type 1 holocarboxylase synthetase deficiency HMG CoA lyase deficiency homocystinuria isovaleric acidemia lysinuric protein intolerance MCAD deficiency methylmalonic acidemia maple syrup urine disease ornithine transcarbamylase deficiency propionic acidemia phenylketonuria mitochondrial neurogastrointestinal encephalopathy (thymidine phosphorylase deficiency) VLCAD sarcosinemia trimethyllysine hydroxylase deficiency Elsea et al Global-MAPS Metabolomics • Classic pathognomonic analytes were among the most significantly elevated analytes detected. Metabolomic data in many cases afforded a much richer view of a patient's metabolic disturbance by identifying: (1) elevated metabolites located far upstream of the genetic defect, (2) treatment related compounds, including commonly tested therapeutic drug monitoring analytes, and (3) spectrally unique analytes that are not yet associated with a biochemical phenotype. • For the undifferentiated genetic phenotypes such as intellectual disability, autism or seizures, often many different tests involving different sample types are needed to ascertain a diagnosis. This can lead to prohibitive costs and ongoing diagnostic odysseys. Global-MAPS Metabolomics Interesting Findings • Increased plasma 3-methoxytyrosine in aromatic Lamino acid decarboxylase (AADC) deficiency • Increased plasma 2-pyrrolidinone in aminobutyrate aminotransferase (ABAT) deficiency • Increased plasma succinyladenosine in adenylosuccinate lyase (ADSL) deficiency • Increased plasma urocanate and imidazole propionic acid in urocanase deficiency METABOLOMICS CORE Co-PIs: David Koeller (OHSU) & Tom Metz (PNNL) Metabolomics followed by definitive quantification Initial hit from metabolomics In collaboration with Joslin Diabetes Center Definitive quantification Metabolomics followed by definitive quantification Initial hit from metabolomics In collaboration with University of California, Irvine Definitive quantification Enzyme kinetics assays for metabolic disorders and conditions Disease related metabolic pathway GALACTOSE METABOLISM Specific assays are developed for: GALK: galactokinase, GALT: galactose-1-phosphate uridyltransferase, GALE: UDP galactose-4′-epimerase. Development of enzyme assays/ Evaluation of patient samples Definitive quantification of analytes in various samples Custom assay development Definitive quantification of metabolite in tissue or blood In vivo whole body metabolism study using isotopes IV injection of isotopically labelled substrate Breath test plasma sample Measurement of the metabolites over time References • • • • • • • • • • • The Metabolic & Molecular Bases of Inherited Disease, Valle D, Beaudet AL, Vogelstein B, Kinzler KW, Antonarakis SE, Ballabio A, Gibson K, Mitchell G. Valle D, Beaudet A.L., Vogelstein B, Kinzler K.W., Antonarakis S.E., Ballabio A, Gibson K, Mitchell G Eds. New York, NY: McGraw-Hill; 2014. http://ommbid.mhmedical.com. Inborn Metabolic Diseases, Saudubray J-M, van den Berghe G, Walter JH, eds., SpringerVerlag, Inc., 5th Edition, 2012. Harris H: Garrod’s Inborn Errors on Metabolism, Oxford University Press, 1965. Cederbaum S, Berry GT. Inborn Errors of Carbohydrate, Ammonia, Amino Acid, and Organic Acid Metabolism, Chapter 22. In: Avery’s Diseases of the Newborn (Ninth Edition). Gleason CA, Devaskar SU, eds., Elsevier Saunders Company, Philadelphia, PA, 2011. Thomas JA, Greene CL, Berry GT. Lysosomal storage, peroxisomal, and glycosylation disorders and Smith-Lemli-Opitz syndrome in the neonate, Chapter 23. In: Avery’s Diseases of the Newborn (Ninth Edition). Gleason CA, Devaskar SU, eds., Elsevier Saunders Company, Philadelphia, PA, 2011. Sahai I, Levy HL. Newborn screening, Chapter 27. In: Avery’s Diseases of the Newborn (Ninth Edition). Gleason CA, Devaskar SU, eds., Elsevier Saunders Company, Philadelphia, PA, 2011. Venditti CP and Berry GT. Treatment of Acute Metabolic Emergencies in the Newborn period. In: Current Pediatric Therapy 18th edition. Burg FD, Ingelfinger JR, Polin RA, Gershon AA, eds., W.B. Saunders Company, Philadelphia, PA, 2006. Venditti CP and Berry GT. Inborn Errors of Metabolism and the Liver. In: Nutrition in Pediatrics, Part 2, Chapter 46, 4th edition. Walker, WA, ed. B.C. Decker, Inc., Hamilton, Ontario, Canada 2007 Neurology of Hereditary Metabolic Diseases of Children, Lyon G, Kolodny E, Pastores GM, McGraw-Hill, 3rd Edition, 2006. Atlas of Metabolic Diseases, Nyhan WL, Barshop BA and Ozand PT, A Hodder Arnold Publication; Second Edition, 2005. Zschocke J, Hoffmann GF. Vademecum Metabolicum: Manual of Metabolic Paediatrics. Milupa, Schattauer, 3rd edition, 2011. References Miller MJ, Kennedy AD, Eckhart AD, Burrage LC, Wulff JE, Miller LA, Milburn MV, Ryals JA, Beaudet AL, Sun Q, Sutton VR, Elsea SH. "Untargeted metabolomic analysis for the clinical screening of inborn errors of metabolism." J Inherit Metab Dis. 2015 April 15;38:1029-39. Pubmed PMID: 25875217 Atwal PS, Donti TR, Cardon AL, Bacino CA, Sun Q, Emrick L, Reid Sutton V, Elsea SH. "Aromatic Lamino acid decarboxylase deficiency diagnosed by clinical metabolomic profiling of plasma." Mol Genet Metab. 2015 June;115:91-4. Pubmed PMID: 25956449 The End Thank you Clary Clish, Ph.D. Co-Director, Broad Institute/ Gerard T. Berry, M.D. Co-Director, Boston Children’s Hospital, Harvard University Undiagnosed Diseases Network Metabolomics Core Facility Undiagnosed Diseases Network Metabolomics Core Facility Projects: • Establishing the normal relative range of metabolites in serum, plasma, CSF and urine in healthy individuals of different ages, gender and ethnicity • Validating and quantifying the metabolites identified via metabolomics METABOLOMICS CORE FACILITY BROAD INSTITUTE & BOSTON CHILDREN’S HOSPITAL From: BWH MGH BCH Plasma Metabolomics analyses Plasma Identification of key metabolites, metabolic pathways tissue CSF urine cultured cells Definitive quantification of specific metabolites Enzyme assays/transport studies for relevant pathway In vivo whole body metabolism study using isotopes Genetic testing for a putative genetic disease • Whole exome/genome sequencing • Nuclear genome mutation identified: Sanger sequencing confirmation suggests putative gene defect • Known gene and protein product Send plasma, tissues, cells to CLIA-certified lab for final diagnosis • Genetic defect and metabolism NOT known – Plasma to Broad for metabolomic analysis – Plasma, urine, CSF, tissues, cells to Metabolism Core Lab for analyte quantification, enzyme analysis, transport study (also samples from isotope kinetic whole body metabolism studies in CTSU) – Send appropriate samples to CLIA certified lab if testing is available Hypothesis: the Undiagnosed Diseases Network (UDN) Metabolomics approach for the establishment of a diagnosis is also suitable for SIDS and SUDP Putative genetic disease genetic testing Whole exome/genome sequencing No mutation identified Transcriptome and proteome analyses of tissues and cells Epigenetic studies of tissues Mitochondrial DNA mutation analysis Nuclear genome mutation identified Sanger sequencing confirmation suggests putative gene defect Mitochondrial DNA mutation identified Mootha Lab 1. Tissue mutant mtDNA burden 2. Nuclear modifier gene search Genetic testing for a putative genetic disease • Sanger sequencing confirmation suggests putative gene defect – Unknown gene and protein product • Other genomic lesion (non-mitochondrial) – Plasma, urine, CSF, tissue, cells to Harvard/MIT lab with special research interest – Plasma to Broad for metabolomic analysis – Plasma, urine, CSF, tissues, cells to Metabolism Core Lab for analyte quantification, enzyme analysis, transport study (also samples from isotope kinetic whole body metabolism studies in CTSU) – Send appropriate samples to CLIA certified lab if testing is available Genetic testing for a putative genetic disease Putative genetic disease genetic testing Whole exome/genome sequencing No mutation identified Transcriptome and proteome analyses of tissues and cells Epigenetic studies of tissues Mitochondrial DNA mutation analysis Nuclear genome mutation identified Sanger sequencing confirmation suggests putative gene defect Mitochondrial DNA mutation identified Mootha Lab 1. Tissue mutant mtDNA burden 2. Nuclear modifier gene search Genetic testing for a putative genetic disease • Whole exome/genome sequencing: No mutation identified – Transcriptome and proteome analyses of tissues and cells – Epigenetic studies of tissues – Plasma to Broad for metabolomic analysis – Plasma, urine, CSF, tissues, cells to Metabolism Core Lab for analyte quantification, enzyme analysis, transport study (also samples from isotope kinetic whole body metabolism studies in CTSU) – Send appropriate samples to CLIA certified lab if testing is available Genetic testing for a putative genetic disease Putative genetic disease genetic testing Whole exome/genome sequencing No mutation identified Transcriptome and proteome analyses of tissues and cells Epigenetic studies of tissues Mitochondrial DNA mutation analysis Nuclear genome mutation identified Sanger sequencing confirmation suggests putative gene defect Mitochondrial DNA mutation identified Mootha Lab 1. Tissue mutant mtDNA burden 2. Nuclear modifier gene search Genetic testing for a putative genetic disease • Mitochondrial DNA mutation analysis – Mitochondrial DNA mutation identified 1. Tissue mutant mtDNA burden 2. Nuclear modifier gene search