Fetal Alcohol Spectrum Disorders

1. NEONATAL SCREENING FORPRENATAL ALCOHOL EXPOSUREDaphne ChanMotherisk Laboratory for Drug Exposure

2. Fetal Alcohol Spectrum Disorders • Range of outcomes resulting from maternal alcohol use --> 100% preventable • Incidence & cost of treatment in Canada unknown • About 1 to 3 live births per 1000 affected • Estimated $1.4 million (U.S.) per person affected • Early diagnosis and intervention leads to significant improvements in development and overall quality of life • Only a small fraction of affected individuals are identified and treated • Difficult to diagnosis • Maternal Hx required for Dx of CNS disorders

3. Detection of Prenatal Alcohol Exposure • Biological mother sometimes unavailable • Medical-legal issues • Maternal self-reporting • Denial, under-reporting • Maternal biomarkers • Variable sensitivity and specificity TRUE FETAL EXPOSURE Neonatal Screening Test

4. Neonatal Screening Test • Biological markers indicative of fetal exposure • Objective and independent of maternal history • Ideal scenario: Hair + meconium analyses

5. ETHANOL METABOLISM Oxidative ACETALDEHYDE ADH and MEOS (CYP 2E1) FATTY ACYL CoA ETHANOL Microsomal FAEE Synthase FAEE FATTY ACIDS Cytosolic FAEE Synthase Non-Oxidative POTENTIAL BIOMARKERS

6. FATTY ACID ETHYL ESTERS (FAEE) • Significant FAEE accumulation in organs and tissues commonly damaged by chronic alcoholism • Brain, heart, liver, pancreas, adipose tissue • FAEE synthase activity detected in human and mouse placentae, and FAEE accumulation in mouse fetal tissues • Biomarker with short and long term clinical utility • Positive blood test 24 hrs post alcohol consumption • Postmortem markers for premortem ethanol intake • Recent development of FAEE hair screening test • Recently detected in the meconium of neonates exposed heavily to alcohol in utero

7. Meconium Analysis Protocol Parental Consent / Physician’s or CAS referral Collect clinical information (e.g. questionnaire, including self-reported drug use history) Review maternal and neonatal records Collect meconium sample (>1g) directly from newborn’s diaper into specimen container Store frozen and ship to Motherisk Lab on dry ice Extract FAEE from meconium Analyze by gas chromatography

8. FAEE Detected In Meconium • Derived from endogenous FA or FA acquired from diet • New FAEE* included into screening profile E14 E16 E12 E18:2 E17 (IS) E18:1 E16:1* E18:3* E20:4* E22:6* E18

9. Development of FAEE asBiomarkers in Meconium • Selective FAEE analysis - ethyl linoleate (C18:2) [Bearer et al. 1999] • FAEE spectrum analysis - profile of common esters [Moore et al. 2001] • Existence of basal FAEE levels ? • Positive cut-off not clearly defined ? • Clinical sensitivity and specificity ?

10. Population Baseline Of Meconium Fatty Acid Ethyl Esters Among Infants Of Non-Drinking Women In Jerusalem And Toronto D. Chan; B. Bar-oz*; B. Pellerin; C. Paciorek; J. Klein; B. Kapur; D. Farine**; G. Koren. Division of Clinical Pharmacology/Toxicology, The Hospital for Sick Children, Toronto, Canada; *Department of Neonatology, Hadassah University Hospital, Jerusalem, Israel; **Department of Obstetrics, Mount Sinai Hospital, Toronto, Canada.

11. Rationale • Ethanol is a metabolite of normal physiological metabolism. However, a well defined baseline and positive cut-off that accounts for the endogenous presence of FAEE does not exist for the meconium screening test in clinical practice to date.

12. Objective • To determine basal FAEE levels in the meconium of neonates without prenatal alcohol exposure from 2 distinct populations Study Populations • Mount Sinai Hospital (Toronto) • A large urban teaching hospital that serves a culturally and ethnically diverse population • Hadassah University Hospital (Jerusalem) • Chosen as a negative control group as it represents a true alcohol-abstaining population because of cultural and religious reasons

13. STUDY DESIGN Expecting mother recruited upon admission to delivery ward Toronto (n = 104 mothers) Jerusalem (n = 104 mothers) Obtain Informed Consent (Verbal or Written) Questionnaire (Demographics, Diet, Drug & Alcohol Hx) Transcription of Maternal and Neonatal Health Records Meconium Sample Collection for Analysis (n = 206) Jerusalem (n = 104) Toronto (n = 102) 15 social drinkers excluded 84 mother-child pair included into baseline analysis 99 mother-child pair included into baseline analysis 3 excluded due to dirty matrix 3 excluded due to dirty matrix 2 social drinkers excluded

14. Results:

15. SUMMARY OF RESULTS • FAEE species distribution was similar in Jerusalem and Toronto • Social drinkers (< 1 drink per month during pregnancy) led to the accumulation of FAEE within normal baseline levels • Additional presence of longer chain FAEE (E16 +) in neonates exposed to alcohol • Lauric (E12), myristic (E14), and palmitic (E16:0) acid ethyl esters predominate baseline meconium

16. DETERMINATION OF POSITIVE CUT-OFF • Important in clinical practice to distinguish between true fetal exposure and natural endogenous production • Calculations of clinical sensitivity, specificity, and predictive values • SENS = TP/TP + FN • SPEC = TN/TN + FP • + PV = TP/TP + FP • - PV = TN/TN+FN

17. DETERMINATION OF POSITIVE CUT-OFF • Positive cut-off was varied from the LOD (I.e. the “presence” of FAEE constituted a positive test) at intervals to 2 nmol/g (I.e. the lowest level detected from a TP case) • SENS = 100%; - PV = 100% • SPEC increased from 12 to 91% (+PV from 4 to 25%) • Repeat calculations excluding ethyl laurate and myristate from the total FAEE sum • SPEC increased from 45 to 98% (+PV from 6 to 63%) • [ ]s of E12 and E14 ethyl esters in baseline and cases were insignificantly different

18. CONCLUSIONS • FAEE exists at low levels in the meconium of neonates without prenatal alcohol exposure • There is a characteristic pattern of FAEE distribution in baseline meconium (predominantly short chain FAEE), which was similarly observed in two culturally and genetically distinct populations • Neonates born to minimally/ socially drinking mothers were indistinguishable from baseline • Significant improvement in specificity after exclusion of ethyl laurate and myristate suggested the role of these esters in constituting the background noise

19. Future Directions • Prospective study with a larger cohort of “true positives” • To verify sensitivity and specificity • Development of FAEE screening test in hair • Provincial/ national epidemiological study • Incidence of FASD in Canada • Prevalence of heavy drinking during pregnancy • Basis for more effective public health initiatives • Predictive potential of screening test? • Immediate: Correlation between laboratory result and pregnancy/ fetal outcomes • Longitudinal: Follow-up of neurobehavioral development and other social parameters

20. Remember…… FASD are 100% preventable Neonatal screening for prenatal alcohol exposure An alternative Harm Reduction approach to treat the mother, her child, and her future pregnancies