Biochemical Genetics and Genomics in Pediatrics

1. Biochemical Genetics and Genomics in Pediatrics Steven F. Dobrowolski Medical Director Clinical Biochemical Genetics Service Children’s Hospital of Pittsburgh

2. Biochemical Genetics • Identify Genetic Disease via the Biochemical Phenotype • Inborn Errors of Metabolism • Organic Acidopathies • Amino Acidopathies • Storage Disorders • Energy Metabolism • Peroxisomal Biogenesis Defects • Sterols and cholesterol • Dyslipidemias

3. Characteristics of Inborn Errors • Most are recessive, some X-linked • All are rare • PAH deficient Phenylketonuria 1:3,000 -1:120,000 • 2,4 Dienoyl-CoAReductase Deficiency 3 cases world-wide • Presentation varies over full spectrum • Smith-Lemli-Opitz:clefting, cardiac malformation, 2-3 syndactyly of toes • Hurler Syndrome: normal at birth, coarsening, short stature, MR • CarnitinePalmitoyltransferase2: adult onset, myopathic phenotype • Pyruvate Carboxylase Def: metabolic acidosis, brain malformation, death

4. Guthrie R, Sussi A, A simple phenylalanine method for detecting phenylketonuria in large populations of newborn infants, Pediatrics 32 (1963) 318–322.

5. . 172.1 Normal Amino Acid Profile 1.20e6 1.10e6 1.00e6 9.00e5 188.1 8.00e5 7.00e5 Intensity, cps 6.00e5 5.00e5 4.00e5 146.1 227.1 3.00e5 238.1 222.1 2.00e5 150.1 244.1 209.1 260.1 1.00e5 182.1 249.1 162.1 263.1 134.1 217.1 197.1 254.1 0.00 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 m/z, amu Amino Acids by ESI-MS/MS Proline Leucine D3-Leucine Alanine D5-Phenylalanine Tyrosine Phenylalanine D4-Alanine 13C6 Tyrosine D3 Methionine

6. PKU where it all began….. d3-Leu d5-Phe Leu d4-Ala Phe Ala 13C6-Tyr Tyr d3-Met Met Phe PKU

7. Academic Pedigree Robert Guthrie (father of newborn screening) • Edwin W Naylor • (Bob’s Fellow, My mentor, applied MS/MS to NBS at Magee) • Steven F Dobrowolski • your humble speaker

8. Tools of Clinical Biochemical Genetics • Tandem Mass Spectrometry • Carnitine Cycle • Fatty Acid Oxidation Defects • Mitochondrial disease • GC-MS • Organic acidemias • Energy pathways • Amino Acid Analysis • Urea cycle • Cysteine, methionine, and re-methylation disorders • N-link and O-link Glycosylation • Congenital disorders of glycosylation • 1980 5 subtypes, today at least 75 novel disorders of diverse phenotypes

9. Congenital Disorders of Glycosylation 7/2012 2012 50 45 40 6-6-12 35 N-linked 30 O-Mannose Muscular Dystrophy 7-1-12 GPI-Anchor 25 O-GalNAc 20 15 10 5 1980

10. Proximal N-linked Glycan Synthesis Freeze HH. Nature Reviews Genetics 2006;7:537–51

11. Distal N-linked Glycan Synthesis Freeze HH. op. cit.

12. sialotransferrins SerumTransferrin IEF Patterns

13. ESI-MS and MALDI Profiles for CDG testing A Normal Intensity B PMM2-CDG

14. Urine glycan MALDI profile or Glu4 or Glu3Man1

15. When to consider CDG • Developmental delay/early onset dementia • Early onset/intractable seizures • Stroke-like episodes • Hypotonia • Muscular dystrophy phenotype • Peripheral neuropathy • MRI - generalized atrophy +/-cerebellarhypoplasia • Retinitis pigmentosa/optic atrophy/eye anomalies • Dilated cardiomyopathy

16. When to consider CDG • Endocrinopathies • Anemia • Feeding difficulty/failure to thrive • Protein-losing enteropathy • Hepatic dysfunction • Coagulopathy • Immune dysfunction • Ichthyosis/excessively dry skin/cutis laxa • Skeletal abnormalities

17. Dysmorphic features • Suprapubic fat pad in infancy • Usually disappear by 5 y/o

18. Skin features Atrophic skin dimpling Excessively dry skin Ichthyosis

20. Organic Acid Analysis in Pediatrics • Amino Acid Catabolism (branch chains, lysine) • Krebs Cycle Disorders • Respiratory Chain Disorders • Glycerol Metabolism • Liver and Peripheral Ketone Body Metabolism • Purine & Pyrimidine Disorders • Glutathione and the γ-Glutamyl Cycle

21. Branched Chain A.A. Catabolism

22. Organic Acids in Normal Urine Pretty busy isn’t it!!!!

23. Gas Chromatography Keto-lactic Acidosis

24. MS-Coupled Gas Chromatography Profile generated Profile from library

25. Co-Elution of Critical Analytes

26. Urea Cycle Nyhan Pathway Atlas of Metabolic Diseases. Nyhan and Ozand

27. Matched to Orotic Acid Profile

28. MS Output

29. ESI-MS/MS • Acyl-Carnitine Precursor 85 Ion Scan • Fatty Acid Oxidation Defects • Carnitine Cycle Defects • Organic Acidemias • Mitochondial and Energy metabolism

31. Pleasant Routine Normal Profile

32. Medium Chain Acyl-CoA Dehydrogenase Deficiency

33. GlutaricAcidemia Type 1

34. When it all gets you no where • Ideopathic Epilepsy • Clearly genetic by family Hx but no diagnosis. • Phenotype fits no recognized pattern • Lab results uninformative • Genomics in the Pediatric Clinic

35. Case Study, Exome Sequencing • Mixed intractable epilepsy at 7th week with complex partial seizures • Short stature • Weight <3rd percentile • Microcephaly • Significant developmental delay • Hypotonia • Nonverbal • Cortical visual impairment.

36. Exome Assessment • Proband, both biological patients SMC1A 2394dupA in proband, neither parent • Sanger confirmation in pedigree • SMC1A <5% Cornelia de Lange Syndrome • Dominant new mutation, common scenario as fitness in Cornelia de Lange is effectively zero

37. Classical Phenotype

38. Genomics in Pediatric Clinic • Single gene test when appropriate • 90% surety to order single gene test • SNP array for structural chromosome defects • Exome