testing Untargeted Metabolomic Profiling in Inborn Errors ofprevention Metabolism research V. Reid Sutton, MD education Professor, Molecular & Human Genetics Baylor College of Medicine Medical Director, Biochemical Genetics Laboratory solutions Disclosure A fixed portion of my salary is paid by Baylor Genetics Laboratories but compensation is not tied to laboratory revenue Outline • Common practice and limitations of current routine testing for IEMs • Global Metabolomic Assisted Pathway Screen (Global MAPS®) - Methods • Validation for common IEMs • Confirmation of DNA variant pathogenecity • Discovery of Novel Biomarkers CURRENT RECOMMENDATIONS FOR INTELLECTUAL DISABILITY EVALUATION AAN Recommendations for Intellectual Disability (2011) • Screening for inborn errors of metabolism (IEMs) in children with GDD/ID has a yield of between 0.2% and 4.6%, depending on the presence of clinical indicators and the range of testing performed (Class III). • Testing for congenital disorders of glycosylation has a yield of up to 1.4%, and testing for creatine disorders has a yield of up to 2.8% (Class III). van Karnebeek CDM et al., Mol Genet & Metab 111:428-38, 2014 Current Challenges • For undifferentiated phenotypes, such as intellectual disability, seizures, recurrent vomiting, failure to thrive etc. many different tests are needed • Various fluid types (blood, urine, cerebrospinal fluid) may be needed for diagnosis • Cost for multiple tests may be prohibitive and many are rare, so no good way to tier testing Methods/Tests • HPLC – amino acids • GC/MS – organic acids • MS/MS – Acylcarnitines – Newborn screening – Individual specialized tests • Purines & Pyrimidines • Creatine & guanidinoacetate • Pyridoxine responsive seizure panels • Bile acids • CSF Neurotransmitters • Etc! Rationale for Metabolomic Approach Biochemistry DNA Advantages Any sample type RNA Condensed & information rich Proteins Actionable OH O H O O OH O H HO OH N H OH 2 Biochemicals Bridge between glucose cholesterol threonine genome & phenotype Mechanistic Insight into Phenotype 9 METHODS Metabolon, a global leader in metabolomics Pioneering the emerging field of global biochemical pathway analysis for biomarker discovery and the development of innovative diagnostic tests • Founded in 2000 • Over 150 employees worldwide with expertise in biochemistry, mass spectrometry and software development • 54,000 sq. ft. facility in Research Triangle Park, NC and Sacramento • CLIA-certified lab onsite UHPLC-MS/MS (+ESI) Early/Polar Library Search Biochemical UHPLC-MS/MS (+ESI) Late/Lipid RT, Mass, MS/MS Extraction Data Reduction UHPLC-MS/MS (-ESI) Compound ID QA/QC UHPLC (HILIC)-MS/MS Pathway Visualization: Metabolync™ plugin to Cytoscape developed to Statistical Analysis overlay analyte findings onto metabolic pathways Biomarkers Mechanistic Understanding Drug MoA Interpretation Cellular Characteristics UHPLC-MS/MS (+ESI) Early/Polar Library Search Biochemical UHPLC-MS/MS (+ESI) Late/Lipid RT, Mass, MS/MS Extraction Data Reduction UHPLC-MS/MS (-ESI) Compound ID QA/QC UHPLC (HILIC)-MS/MS Enrich 50-1500 Da small molecule analytes Statistical Analysis Biomarkers Mechanistic Understanding Drug MoA Interpretation Cellular Characteristics UHPLC-MS/MS (+ESI) Early/Polar Library Search Biochemical UHPLC-MS/MS (+ESI) Late/Lipid RT, Mass, MS/MS Extraction Data Reduction UHPLC-MS/MS (-ESI) Compound ID QA/QC UHPLC (HILIC)-MS/MS •Q exactive MS/MS - Orbitrap based MS/MS (Thermo) •Accurate to 1 ppm vs 100 ppm for quadrupole analyzers Statistical Analysis •Cost ~2X quadrupole Biomarkers Mechanistic Understanding Drug MoA Interpretation Cellular Characteristics UHPLC-MS/MS (+ESI) Early/Polar Library Search Biochemical UHPLC-MS/MS (+ESI) Late/Lipid RT, Mass, MS/MS Extraction Data Reduction UHPLC-MS/MS (-ESI) Compound ID QA/QC UHPLC (HILIC)-MS/MS •Library of ~2500 human metabolites Statistical Analysis Biomarkers Mechanistic Understanding Drug MoA Interpretation Cellular Characteristics UHPLC-MS/MS (+ESI) Early/Polar Library Search Biochemical UHPLC-MS/MS (+ESI) Late/Lipid RT, Mass, MS/MS Extraction Data Reduction UHPLC-MS/MS (-ESI) Compound ID QA/QC UHPLC (HILIC)-MS/MS Pathway Visualization: Metabolync™ plugin to Cytoscape developed to Statistical Analysis overlay analyte findings onto metabolic pathways Biomarkers Mechanistic Understanding Drug MoA Interpretation Cellular Characteristics Metabolon’s Approach to Metabolomics - a Comprehensive Survey of the Metabolome 17 Extensive Metabolite Library High resolution biochemistry surveys central metabolism & Baylor Genetics peripheral pathways that 18 drive attributes of phenotype. te a n o r u c u l g - o r d y h e -d P-4 D G e n i m ta u l g isoleucine leucine 2.4.1.18 - -N l y s o b i r o h p s o h -p ' 5 - l y s o b i r o h p s o h -p ' 5 e s o n n a -m -D y x o e -d 6 2.6.1.42 e n i m ta u l g e n i m a s o c u l -g l ty e c -a N 1 2.4.1.1 e n i c u e l o s i o l l -a L valine e d i m a n i c y l g l y m r fo glycineamide te a h p s o h -p 1 e n i m a s o c u l g l ty e c -a P-N D U te a m ta u l g 2.7.7.23 isovalerylalanine ) R A G (F ) R A (G e n i m a l y s o b i r o h p s o h -p 5 2.7.7.44 5.4.2.3 te a r e l a v o s i y x o r d y -h 3 6.3.5.3 2.1.2.2 6.3.4.13 2.4.2.14 3.1.4.3 2.7.7.64 N IO T A L Y S CO Y L G 2.6.1.42 3.1.3.25 5.3.3.- l to i s o n i te a r e l a v o x -o -2 l y th e -m 3 2.6.1.42 1.4.3.2 l to i s o n i l y d ti a h p s o Ph GDE te a h p s o h p o n o -m 2 e n i m a s o c u l g l ty e c -a N 2.6.1.42 l to i s o n -i o y m e s o c u l P-g D U te a n o c a s e m te a n o r u c u l g 1.1.1.271 4.2.1.47 E T RBA CO S A te a h p s o h -p 6 te a r e l a v o s i y x o r d y -h 4 ) te a r a m fu l y th e (m te a h p s o h -p 1 4.6.1.13 2.7.1.64 2.4.1.17 -1 (n n e g o c y l g - l y th e -m 4 SO I Glycogen PP PR M IS L BO A T E M 1.2.1.- te a o r p a c o s i y x o r d y -h a h p l a ? residue) E T A N E H T O T N PA te a r e l a v l y th e -m -3 y x o r d y -h 2 te a r e l a v o x -o 2 te ta o r o ? te a r e l a v o x -o -2 l y th e -m 3 e s o c P-fu D G e s o n n a P-m D G e n i m a s o c u l g l ty e c -a N te a r ty u b o x -o -2 l y th e -m 3 - l y o m a b r a -c N 2.7.1.59 te a r e l a v o s i y x o r d y -h a h p l a ? M) A G (F e n i d i m a n i c y l g l y m r -fo -N l y s o b i r o h p s o h -p ' 5 l to i s o n -i o l l y c s B5) IT (V 3.1.3.25 - -1 l y th e -m 3 te a h p s o h p l y o m a b r a c te ta r a p s a te ta o r o o r d y h i -d 5 , 4 te a m ta u l g l y r e l a v o s i te a n i c c u s l y th e m e n i m ta u l g - -1 l y th e -m 2 PP h -T l ty u b y x o r d y h nd CoA nd a - -1 l y th e -m 2 3.1.3.64 2.7.1.43 A o -C l y r ty u b l y th e -m )-2 (R PP h -T l ty u b y x o r d y h 2.3.1.4 5.1.3.2 glucurono- N E G CO Y L G M IS L BO A T E M PP h -T l y p o r p y x o r d y h -P 4 l to i s o n i e n to c a -l 3 , 6 2.7.7.9 BCKD - 5 , -3 l to i s o n i l y d ti a h p s o h p GTP BCKD 6.3.5.5 2.1.3.2 3.5.2.3 1.3.5.2 3.1.3.64 2.7.1.137 3.1.3.B4 2.7.1.67 2.7.7.13 2.7.7.22 2.4.1.1 BCKD isovaleryglucuronide isovalerylsarcosine 2.7.7.8 e n i e th te n a p , te a h p s o h p i d M IS L BO A T E M te a n e th to n a p 3.5.1.92 6.3.3.1 l to i s o n i T PG F e s to c a l a P-g D U 1.3.99.- te a n o r u c u l g 2.7.7.12 - S-(2 )P2 5 , (3 PI 1.13.99.1 3.1.1.19 )- l y o n ta u b l y th e -m S-(2 )- l y o n ta u b l y th e -m S-(3 te a h p s o h p s i -b 3 , 1 te a h p s o h -p -6 e n i m a s o c u l g 2.7.1.1 glucosamine 2.4.1.1 n i tr x e d to l a m te a l y d ti o r o 2.4.2.10 glucose ) l y n o i p o r p l y th e dihydrolipoamide -E e d i m a o p i l o r d y h i d ) l y d i r -u ' (5 s i -B P4 , P1 ) (n te a o n ta p e h o s i y x o r d y -h 3 te a h p s o h -p 1 -E e d i m a o p i l o r d y h i -d te a h p s o h -p -1 e s o n n a m te a h p s o h p a tr te 3.1.3.57 2.7.1.33 ROS 3.6.1.19 te a h p s o h -p -1 e s o c fu A o -C l y l y r c a l y th -e 2 te a r ty u b l y th e -m 2 3.1.2.20 A o -C l y r ty u b l y th e -m 2 te a r e l a v o s i e s to c a l a g te a r ty u b o s i A o -C l y r ty u b o s I A o -C l y r e l a v o s i 3.1.2.20 3.1.2.20 5.3.3.- 4.1.1.23 1.1.1.19 te a h p s o h -p 1 e s o a x e h to l a m 2.7.7.10 3.6.1.17 1.2.1.3 2.7.1.48 2.7.4.6 3.5.99.6 2.6.1.16 2.7.4.14 )P (3 PI )P (4 PI 2.7.1.150 l to i s o n i e n i th te n a p e l zo a d i m i o n i m -a -5 l y x o b i r o h p s o h p ' 5 5.4.2.2 5.4.2.8 2.4.1.1 uracil MP U UDP UTP RNA te a h p s o h p s i -b 4 , 1 te a n e th to n a p o h p s o h -p ' 4 4.2.1.17 ) R I (A 2.4.2.3 uridine 3.1.3.5 3.6.1.6 2.7.4.10 2.7.7.6 L O IT S O IN 2.7.1.52 te a n o l u g E S O CT A L A G 3.6.1.5 3.6.1.5 M IS L BO A T E M e s o ta n e p to l a m e n i te s y c 2.4.2.9 5.3.1.8 2.7.1.6 e s o c fu te a h p s o h -p -6 e s o n n a m te a h p s o h -p -6 e s to c u fr M IS L BO A T E M 2.3.1.7 1.3.8.1 1.3.99.12 1.3.99.- 1.3.8.7 2.3.1.13 2.3.1.13 1.3.8.7 1.3.99.12 1.3.8.1 2.3.1.7 1.3.8.7 1.3.8.4 2.3.1.13 2.3.1.7 1.3.1.2 e s to a g ta A o -C l y y r c a r d y h l y th -e 2 ET I D / T U G 2.7.1.33 l o r e c y l g l y to i m l a p i -d 2 , 1 te a r ta c a l a g 6.3.2.5 3.5.4.5 6.3.4.2 2.4.1.1 2.7.4.68 ) d i c a c i c u (m 3.1.3.66 2.7.1.154 2.7.1.154 3.1.3.36 3.1.1.25 3.1.1.17 3.1.3.56 e s to c a l a g 3.2.1.23 e s to c a l 4.1.1.21 2.7.1.48 2.7.4.14 2.7.4.6 2.7.1.7 2.7.1.1 3.1.2.4 te a r ta c a l a g e s o a tr te to l a m l to ti c a l a g e n i c y l g l y r ty u b l y th e -m 2 e n ti i n r a c l y r ty u b l y th e -m 2 CTP ) d i c a c i c u (m 2.7.1.1 ) l to i c l u (d e n i d ti y c MP -C ' 5 CDP 2.7.1.159 2.7.1.127 1.1.1.21 ) 5 (C e n to c a -l 4 , -1 o n o l u g mannose