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

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 ? Collision Cell Mass Spectrometer 2
3/16” blood spot
deproteinization
derivatization
Mass Spectrometer 1
Mass Spectrometer 2
Collision Cell
CH3 R - COO
CH3 +N - CH2 - CH - CH2 - COO - C4H9
CH3
CH3 R - COO
CH3 +N - CH2 - CH - CH2 - COO - C4H9
CH3
+CH2 - CH - CH2 - COOH
(m/z 85) 
Data of all compounds
within selected range
( m/z)
Data of product ions
with a mass of 85 only
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. MS/MS Plasma Acylcarnitines
Intensity
100% *
MCAD C2
C16 C8
C10:1 C6 *
100%
Control C2
Intensity
* internal standards

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

13. MS/MS Plasma Amino Acids

14. Acylcarnitine – VLCAD Deficiency
Cutoff is 0.65

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.

19. So why the need for intense deliberation, why not screen for everything?

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.
Detecting these babies is the primary goal of the NBS program. A portion of these kids will die within the first week of life despite NBS if sample not collected promptly.

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
For MCAD, biochemical parameters overlap between symptomatic and ‘probable mild’ cases, making decision-making about management challenging. Most clinics implement some type of treatment protocol. MCAD treatment is relatively benign though. Possibly in the future, there will be enough evidence that patients with 199C>T mutation are at very low risk of hypoglycemia.

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
Should abnormalities of unclear significance be reported or withheld? Harm may come to kids who are treated to ultimately don’t need to be. What about conditions that are not treatable?
Consider expanding newborn screening within a research paradigm to create outcome data
Consider the manpower and expertise needed to manage these families

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?
9 OA
5 FAO
6 AA
3 Hb Pathies
6 Others
CORE PANEL
IVA GA I HMG MCD MUT 3MCC Cbl A,B PROP BKT
MCAD VLCAD LCHAD TFP CUD
PKU MSUD HCY CIT ASA TYR I
Hb SS Hb S/ßTh Hb S/C
CH BIOT CAH GALT HEAR CF

27. Cit/arg, cit/phe, cit/tyr, asa/arg
What Are We Measuring?
Disorder
1° Marker
2° Marker
Ratios
VLCADD
C14:1
C14,C16,C18,C18:1
C14:1/C16
LCHADD
C16OH
C14,C14:1,C16:C18,C18:1,C18:1OH, C18OH
C16OH:C16
TFP
CUD C0
All carnitines (low) PA C3 C2
C3/C0, C3/C2, C3/C16
MMAs
C4DC
IVA C5
C5/C2, C5/C0, C5/C3
GA-1
C5DC
C5DC/C8, C5DC/C16, C5DC/C5OH
BKT
C5:1
C5OH
C5OH/C8
HMG
C6DC
HCSD
CIT
Cit
Cit/arg
ASA
Asa
Cit/arg, cit/phe, cit/tyr, asa/arg
TYR
Suac
Tyr

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
With each pass through the cycle, the fatty acid is shortened by 2 carbon atoms as Acetyl-CoA, which is send to the TCA (tricarboxylic acid) cycle to be converted to energy . A fatty acid will cycle through as many times as necessary to generate these 2-carbon fragments. The enzymes along the path are somewhat specific for each chain length.

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
VLCAD is included in the newborn screening program in all but two states now.

38. Zach Testing Family referred to genetics by coroner
Parents requested testing for older brother
Acylcarnitine ordered
DNA sequencing of ACADVL gene ordered
Reported: free carnitine low, mild elevation of C14 and C14:1, VLCAD can’t be ruled out.

39. Normal acylcarnitine profile

40. Acylcarnitine – Zach 5 y.o.
C16:1- nl
C14 
C16 - nl
Reported: free carnitine low, mild elevation of C14 and C14:1, VLCAD can’t be ruled out.

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
Reported: free carnitine low, mild elevation of C14 and C14:1, VLCAD can’t be ruled out.

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
Reported: free carnitine low, mild elevation of C14 and C14:1, VLCAD can’t be ruled out.

43. Acylcarnitine – Zach 5 y.o.
Reported: free carnitine low, mild elevation of C14 and C14:1, VLCAD can’t be ruled out.
C14:1 
C14 
C16 - nl
C16:1- nl
C18 - nl

44. Acylcarnitine – Zach newborn
Cutoff is 0.65
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
Reported: free carnitine low, mild elevation of C14 and C14:1, VLCAD can’t be ruled out.

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. 2008 Cases
True Positive
False Positive
Pending
Unknown