Newborn ScreeningHistorical, Ethical, Technological Aspects
Nutrition 526November 9, 2009
Cristine M Trahms, MS, RDBeth Ogata, MS, RDLisa Sniderman King, CGC
Blood Sample on GuthrieFilter Paper Card
First screen must be taken 24-48 hours of life regardless of feeding status or weight
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
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
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.
Mass Spectrometer 1
Data system correlates m/z 85 to its precursor ion’s mass and records the abundance of all precursors (parents of m/z 85)
CH3 R - COO CH3 +N - CH2 - CH - CH2 - COO - C4H9
CH3
Mass Spectrometer 2
3/16” blood spotdeproteinization
derivatization
+CH2 - CH - CH2 - COOH(m/z 85)
3.78e4 cps+Precursor (85): 0.65 min (10 scans) from AC 012700-07
% % MultiView -- © 1996, SCIEX, a division of MDS Health Group.% % Original concept:% Dr. Ron Bonner% Dr. Lyle Burton % % Development:% Dr. Lyle Burton % Yves Legault% Shengping Ma% % With the help of:% Dr. Victoria Barclay % Scott Champ% Rob McDermid%
260.0
295.0
316.2
347.0
372.0
403.2
426.4 442.6
459.2
482.2
300 350 400 450m/z, amu
5000
10000
15000
20000
25000
30000
35000
Inte
nsity
, cps
Data of all compoundswithin selected range
(250 - 500 m/z)
Data of product ionswith a mass of 85 only
Collision Cell
CH3 R - COO CH3 +N - CH2 - CH - CH2 - COO - C4H9
CH3
What is MS/MS ?
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.
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 hyperammonemiadd, 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)
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.
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.
+Prec (85.10): 0.401 to 1.202 min from Sample 9 (BADER) of AC 101003 DATA.wiff (Turbo Spray... Max. 3.5e4 cps.
260 280 300 320 340 360 380 400 420 440 460 480 500m/z, amu
5%
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35%
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45%
50%
55%
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85%
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95%
100%
Re
l. Int. (%
)
*
* * * *
*
C2
100%
Inte
nsity
* internal standards
Control
+Prec (85.10): 0.401 to 1.202 min from Sample 8 (PATE) of AC 101003 DATA.wiff (Turbo Spray) Max. 3.8e4 cps.
260 280 300 320 340 360 380 400 420 440 460 480 500m/z, amu
5%
10%
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25%
30%
35%
40%
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55%
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80%
85%
90%
95%
100%
Re
l. Int. (%
)
Inte
nsity
100%
*
* * *
*
*
MCAD
C2
C16
C8
C10:1C6
MS/MS Plasma Acylcarnitines
Normal
C2*C3
*C4*
C8
*C12
*
C16*
VLCAD
Free Carnitine
*
* *
* *
*
C14:1C12 C18:1C16
* internal standards
VLCAD profileFree Carnitine
MS/MS Plasma Amino Acids
Acylcarnitine – VLCAD Deficiency
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.
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
Whom do we see?1. Patients who need active
management Symptomatic at diagnosis Strong evidence of pathology if
untreated Examples: PKU, classic gal, MSUD, PA
etc.
Whom do we see?2. 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
Whom do we see?3. 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
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
Other Benefits to ScreeningFor 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
What Are We Screening For?
9 OA 5 FAO 6 AA 3 Hb Pathies
6 Others
CORE PANELIVAGA IHMGMCDMUT3MCCCbl A,BPROPBKT
MCADVLCADLCHADTFPCUD
PKUMSUDHCYCITASATYR I
Hb SSHb S/ßThHb S/C
CHBIOTCAHGALTHEARCF
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 C16OH C14,C14:1,C16:C18,C18:1,C18:1OH, C18OH C16OH:C16
CUD C0 All carnitines (low)
PA C3 C2 C3/C0, C3/C2, C3/C16
MMAs C3 C4DC C3/C0, C3/C2, C3/C16
IVA C5 C5/C2, C5/C0, C5/C3
GA-1 C5DC C5DC/C8, C5DC/C16, C5DC/C5OH
BKT C5:1 C5OH C5OH/C8
HMG C5OH C6DC C5OH/C8
HCSD C5OH C3 C5OH/C8
CIT Cit Cit/arg
ASA Cit Asa Cit/arg, cit/phe, cit/tyr, asa/arg
TYR Suac Tyr
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
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
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)
VLCAD Disorder of long chain fatty acid
breakdown C14, C14:1 C16, C18 Normal beta oxidation occurs in
mitochondria
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)
Fatty Acid Oxidation
http://www.genomeknowledge.org/figures/saturatedbetao.jpg
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
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
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
VLCAD TreatmentVLCAD Treatment
VLCAD DiagnosisVLCAD Diagnosis
Newborn screening Plasma acylcarnitine profile Urine organic acids (should be
normal) DNA sequencing
Zach Testing
Family referred to genetics by coroner Parents requested testing for older
brother Acylcarnitine ordered DNA sequencing of ACADVL gene
ordered
Normal acylcarnitine profile
Acylcarnitine – Zach 5 y.o.
C14:1
C16:1- nl
C14 C16 - nl
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
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
Acylcarnitine – Zach 5 y.o.
C14:1
C16:1- nl
C14 C16 - nl
C18 - nl
Acylcarnitine – Zach newborn
C14:1
C14
C16
C16:1 C18
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
Newborn Screening – A Team Effort
1. The Presumptive Positive PhaseDOH NBS Laboratory Personnel
Mike Glass, Sheila Weiss, John Thompson, Carol-Nucup-Villaruz, Charlene Adams, Jessica Dolle, many laboratory technologists
2. The Diagnostic Confirmation PhaseCHRMC Diagnostic Laboratory Personnel
Sihoun Hahn, Rhona Jack, Lisa Sniderman King, Cindy Gordon, Nancy McDowell Laura Mitchell, Diane Rebholz, Malcolm ReiderMonica Jensen, Ngoc-Diep Pham, Min Zhang
Newborn Screening – A Team Effort
3. The Clinical Follow-up PhaseClinic 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
What Happens After a Positive
1. NBS Lab notifies all clinical f/u and key laboratory personnel of referral.
2. Laboratory technologists prioritize samples and collate results
Multiple tests (AA, OA, AC) on each kiddo
Interpreted together once all are completed
Uniform, concise reporting
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
2008 CasesNewborn Screening Cases per Month
02468
1012141618202224
Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec
Month
Cas
es
True Positive
False Positive
Pending
Unknown