Newborn Screening Historical, Ethical, Technological Aspects

1. Newborn ScreeningHistorical, Ethical, Technological Aspects Nutrition 526 November 9, 2009 Cristine M Trahms, MS, RD Beth Ogata, MS, RD Lisa Sniderman King, CGC

2. First screen must be taken 24-48 hours of life regardless of feeding status or weight Blood Sample on Guthrie Filter Paper Card

3. Supersaturated Quantity Insufficient for Testing Specimen Not Dried Before Mailing Scratched or Abraded Serum Rings Diluted, Discolored, or Contaminated Clotted or Layered No Blood Unsatisfactory Specimens (Provided by the New York State Department of Health)

4. Criteria for Newborn Screening • important condition • acceptable treatment available • facilities for diagnosis and treatment • difficult to recognize early • suitable screening test • natural history known • cost effective to diagnose and treat Wilson & Jungner, 1968

5. MS/MS -High Impact and High Throughput • One disease, one test is not cost effective • Many diseases, one test is cost effective • MS/MS allows for rapid, simultaneous analysis and detection of many disorders of amino acid, organic acid, and fatty acid metabolism.

6. What is MS/MS ? CH3 R - COO CH3+N - CH2 - CH - CH2 - COO - C4H9 CH3 CH3 R - COO CH3+N - CH2 - CH - CH2 - COO - C4H9 CH3 Mass Spectrometer 2 Collision Cell  Mass Spectrometer 1 3/16” blood spot deproteinization derivatization +CH2 - CH - CH2 - COOH (m/z 85) Data of product ions with a mass of 85 only Data of all compounds within selected range (250 - 500 m/z) Data system correlates m/z 85 to its precursor ion’s mass and records the abundance of all precursors (parents of m/z 85)

7. Tandem Mass Spectrometry (MS/MS) • Compounds analyzed are amino acids & acylcarnitines • Amino acids – PKU, MSUD, Homocystinuria • Acylcarnitine {Carnitine (vehicle) +fatty acid} for identification of organic acidurias and fatty acid oxidation disorders.

8. Amino Acid Disorders • AA that are not used to make proteins are recycled by their specific metabolic pathways. Enzymatic deficiencies in these pathways lead to various clinical phenotypes. • PKU – Phenylketonuria : severe perm MR • MSUD – Maple syrup urine disease: dd, hallucinations, ataxia • HCY – Homocystinuria: connective tissue damage – joints, heart, dd, psychiatric dist. • CIT – Citrullinemia: risk of hyperammonemiadd, coma, death • ASA – Argininosuccinic acidemia: brittle hair, liver dis, dd • TYR I – Tyrosinemia type I: acute or chronic liver disease, liver cancer, neurologic pain crises. • Diagnosed by plasma amino acids, and/or urine amino acids, and/or urine organic acids (takes 2-5 days)

9. Organic Acid Disorders • Organic acids are breakdown products of protein and fatty acid metabolism. Defects in their breakdown lead to (generally) • Vomiting, metabolic acidosis, elevated ammonia in crises • Dd, motor delay, ataxia, heart/kidney/pancreatic problems • IVA - Isovaleric acidemia • GA I – Glutaric acidemia type I • HMG – 3-OH 3-CH3 glutaric aciduria • MCD – Multiple carboxylase deficiency • MUT – Methylmalonic acidemia (mutase def) • 3MCC – 3-Methylcrotonyl-CoA carboxylase deficiency • Cbl A,B – Methylmalonic acidemia • PROP – Propionic acidemia • BKT – Beta-ketothiolase deficiency • Diagnosed by urine organic acids and/or plasma acylcarnitines.

10. Fatty Acid Oxidation Disorders • Fatty acid disorders lead to impaired energy production. • Hypoglycemia, cardiomyopathy, muscle weakness can be seen • MCAD – Medium-chain acyl-CoA dehydrogenase deficiency • VLCAD – Very long-chain acyl-CoA dehydrogenase deficiency • LCHAD – Long-chain L-3-OH acyl-CoA dehydrogenase deficiency • TFP – Trifunctional protein deficiency • CUD – Carnitine uptake defect • Diagnosed by plasma acylcarnitines and urine organic acids can be helpful.

11. * 100% MCAD * C8 * * * C2 Intensity * C6 C10:1 C16 * * * * * * MS/MS Plasma Acylcarnitines 100% Control C2 Intensity * internal standards

12. VLCAD profile * VLCAD Free Carnitine * * * * * C12 C14:1 C16 C18:1 C16 * C2 * C12 C8 * C4 * C3 * * Normal Free Carnitine * internal standards

13. MS/MS Plasma Amino Acids

14. Acylcarnitine – VLCAD Deficiency

15. Which Disorders to Screen For? • NBS mandates are under state control • Some states screened for 3 diseases, others 40+ • 2002 Maternal and Child Health Bureau commissioned ACMG • Analyze literature • Develop consensus on which disorders • Recommend a core panel to create uniform NBS across all states.

20. Historical Harm (?) • Early PKU screening led to cases of overly restricted phe and/or implementation of diet prior to confirmation of diagnosis • Today, diagnosis is quite rapid • 40 years ago it took much longer so more potential for harm • However, no published evidence of wide-spread physical/medical harm • BUT the cases do underscore need for expertise and resources for mgmt

21. Whom do we see? • Patients who need active management • Symptomatic at diagnosis • Strong evidence of pathology if untreated • Examples: PKU, classic gal, MSUD, PA etc.

22. Whom do we see? • Patients with disorders known to pose risk but reduced penetrance • ie. probably not everyone needs to be treated • HPHE, MCAD • Both are/have mild ends of the spectrum that have only been identified through NBS • MCAD mutation c.199 C>T • Never seen in patients picked up clinically

23. Whom do we see? • Patients who may not need any management • Disorders considered extremely rare but seen in large numbers via NBS programs • Reported cases have significant morbidity • NBS pickups are mostly mild • 3MCC, SCAD • Biochemical phenotype

24. Proceeding with Caution(Reasons to be Thoughtful) Proceeding with caution  Not screening • Core diseases vs secondary targets/unintended targets • What is reported vs withheld? • Will we p/u untreatable conditions? • What is the impact of false positives on families? • No long-term outcome data – consider research paradigm • Consider infrastructure needed for f/u

25. Other Benefits to Screening For disorders in which proven, effective treatment is not available, or very new. • Consider non-medical benefits: • Avoid the diagnostic odyssey • Allow for reproductive decision making before future children are born • Allow for early access to clinical trials for new therapies • Emotional preparation for disease

26. What Are We Screening For?

27. What Are We Measuring?

28. Emma • 13 months old • Normal pregnancy and delivery • Healthy • Normal eating pattern, no allergies or intolerances • Feb 2008: Vomited Saturday and 4-5 times throughout the weekend • No fever • Sleeping for extended periods – parents concerned but previous fever had same pattern. • Parents gave Pedialyte

29. Emma • 4 ½ y brother, parents sick on Sunday/Monday. Same symptoms • Monday night 9:30 checked on E • Raspy breathing – thought respiratory problem but not worried • Tuesday morning 11am she was found motionless in her crib and pronounced dead at the scene

30. Emma • Autopsy revealed fatty changes to liver • Coroner requested newborn screening blood spot be sent for acylcarnitine profile • Diagnostic for Very Long Chain Acyl-Co A Dehydrogenase Deficiency (VLCAD)

31. VLCAD • Disorder of long chain fatty acid breakdown • C14, C14:1 C16, C18 • Normal beta oxidation occurs in mitochondria

32. Fatty Acid Oxidation • During times of fasting, fatty acids are primary substrate for energy production in liver, cardiac muscle and skeletal muscle • Brain uses ketones (produced by normal b-oxidation)

33. Fatty Acid Oxidation http://www.genomeknowledge.org/figures/saturatedbetao.jpg

34. VLCAD Enzyme • VLCAD enzyme sits on inner mitochondrial membrane • Catalyzes first step of b-oxidation for C14-C20 • Defect leads to • impaired energy production during times of fasting stress • Accumulation of toxic long-chain acyl-CoA intermediates within mitochondria • Steatosis (fatty accumulation/degeneration) seen in hepatic, cardiac and skeletal muscle

35. VLCAD Presentations • Hypertrophic cardiomyopathy, with hypoglycemia and skeletal myopathy, lethargy, failure to thrive • Usually present birth-5 months • Hypoglycemia, hepatomegaly, muscle weakness without cardiac manifestations • Late infancy – older childhood • Muscle weakness/pain, rhabdomyolysis with exercise or illness. No hypoglycemia or cardiac • Teens to adulthood

36. VLCAD Treatment • Diet low in long-chain fats (Portagen, Monogen = 87%, 90% of fats as MCT) • Additional medium chain fats (MCT oil, walnut oil) • Carnitine 100 mg/kg/day • Avoidance of fasting • Treating illness with IV glucose support

37. VLCAD Diagnosis • Newborn screening • Plasma acylcarnitine profile • Urine organic acids (should be normal) • DNA sequencing

38. Zach Testing • Family referred to genetics by coroner • Parents requested testing for older brother • Acylcarnitine ordered • DNA sequencing of ACADVL gene ordered

39. Normal acylcarnitine profile

40. C14:1  C14  C16 - nl C16:1- nl Acylcarnitine – Zach 5 y.o.

41. Zach Testing • Reported: mild elevation of C14 and C14:1 with low free carnitine. VLCAD cannot be ruled out • Recommend supplementing with carnitine and retest in 1 week • Family left for Disneyland • DNA testing results back before AC repeat

42. Zach Testing • Zach’s DNA testing reveal he is affected. • Family seen in BCG clinic, started on treatment. • Consent to obtain NBS blood spot obtained

43. Acylcarnitine – Zach 5 y.o. C14:1  C14  C16 - nl C16:1- nl C18 - nl

44. Acylcarnitine – Zach newborn C16  C14:1  C14  C18  C16:1 

45. Zach Clinical picture • 5 y.o • Healthy • No symptoms of muscle weakness • CPK = 315U/L (35-230) • No hepatomegaly • AST= 49 (5-41) • ALT= 23 • Bilirubin conj, unconj = normal (0.0, 0.4) • No evidence of cardiac involvement • Has had several viral illnesses in his lifetime without difficulty • Once on carnitine, AC profile was classic for VLCAD

46. Newborn Screening – A Team Effort • The Presumptive Positive Phase DOH NBS Laboratory Personnel Mike Glass, Sheila Weiss, John Thompson, Carol-Nucup-Villaruz, Charlene Adams, Jessica Dolle, many laboratory technologists • The Diagnostic Confirmation Phase CHRMC Diagnostic Laboratory Personnel Sihoun Hahn, Rhona Jack, Lisa Sniderman King, Cindy Gordon, Nancy McDowell Laura Mitchell, Diane Rebholz, Malcolm Reider Monica Jensen, Ngoc-Diep Pham, Min Zhang

47. Newborn Screening – A Team Effort • The Clinical Follow-up Phase Clinic Personnel All previously screened disorders: (PKU, MCAD, gal, btd, msud, hcys): • UW: Ron Scott, Cris Trahms, Beth Ogata, Janie Heffernan, Jan Garretson, Stefanie Uhrich, Angie Fox All expanded screening disorders (FAOs, OAs…etc): • CHRMC: Lawrence Merritt, Michael Raff, Sihoun Hahn, Sue Hale, Kelly McKean, Melissa Edwards, Lisa Sniderman King, Penny Schubert

48. What Happens After a Positive • NBS Lab notifies all clinical f/u and key laboratory personnel of referral. • Laboratory technologists prioritize samples and collate results • Multiple tests (AA, OA, AC) on each kiddo • Interpreted together once all are completed • Uniform, concise reporting

49. 2008 Cases • 58 total cases since Jan • 51 since expanded NBS started (July 21) • 8 true positives for targeted disorders (MCAD and PKU) • 1 true positive for secondary disorder (CblC) • Elev C3 1° targets MMA/PA/CblA,B • 38 FP/FPA – targeted disorder ruled out • False Positive Active • Persistent elevations in ‘normal’ baby • Carriers (ie. further testing needed) • Benign forms (D/G galactosemia) • These are active because they require genetic counseling or lab repeats • They are reclassified as FP when case is closed • False Positive • 1 mom with low free carnitine • Several D/G galactosemia • A few VLCAD carriers • 10 pending (waiting for samples) • 2 Unknown

50. True Positive Pending False Positive Unknown 2008 Cases