Transcript Berry-Metabolomics - SSADH Association

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
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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
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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
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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
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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
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cblC
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Methionine Synthase Reductase
(Mitochondrial Isoform)
Cbl(II?)
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Transport into the
Mitochondria
Cbl(III)
Mitochondrial Matrix
MMAB
cblB
D-methylmalonyl-CoA
OH-Cbl(III) cblF
Succinyl-CoA
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Cbl(II?)
Cbl(II?)
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cblD
(Variant 1)
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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
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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
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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.
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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.
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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
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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