PROTECTIVE EFFECTS OF THE ALCOHOL DEHYDROGENASE-ADH1B ALLELE IN CHILDREN EXPOSED TO ALCOHOL DURING PREGNANCY

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1 PROTECTIVE EFFECTS OF THE ALCOHOL DEHYDROGENASE-ADH1B ALLELE IN CHILDREN EXPOSED TO ALCOHOL DURING PREGNANCY SANDRA W. JACOBSON, PHD, LUCINDA G. CARR, PHD, JULIE CROXFORD, BSC, ROBERT J. SOKOL, MD, TING-KAI LI, MD, AND JOSEPH L. JACOBSON, PHD Objectives To examine alcohol use for mothers with and without an ADH1B*3 allele and the moderating effects of the maternal and child ADH1B*3 allele on a broad range of infant and 7.5-year outcomes. Study design Blood samples from 263 black mother/child pairs (217 mothers and 239 children) were analyzed to determine incidence of the ADH1B allele and the relation of the maternal allele to pregnancy drinking assessed at every prenatal clinic visit. Moderating effects of ADH1B were examined by dichotomizing the moderator variable and performing regression analyses on the 2 groups. Results Pregnancy drinking at conception was less frequent in the presence of the ADH1B*3 allele, and virtually no adverse effects were found in children whose mothers had at least one ADH1B*3 allele. By contrast to the maternal allele, we found no consistent pattern of greater vulnerability in children lacking the ADH1B*3 allele. Conclusions These data are consistent with the hypothesis that the maternal ADH1B*3 allele provides some protection to the fetus from prenatal alcohol exposure. (J Pediatr 2006;148:30-7) F etal alcohol syndrome (FAS) is characterized by intrauterine or postnatal growth restriction, central nervous system impairment, and a distinctive craniofacial dysmorphology. 1 Fetal alcohol spectrum disorder (FASD) refers to the range of alcoholrelated neurodevelopmental disorders from the most severely affected FAS cases to nonsyndromal individuals with confirmed heavy prenatal alcohol exposure who exhibit subtler neurobehavioral deficits than those seen with FAS. Of children born to women who drink heavily during pregnancy, only 4% to 10% develop full FAS. 2,3 Although maternal age has been identified as a moderator of fetal alcohol effects, there has been relatively little systematic empirical investigation of other factors that may determine which children are affected. To date, genetic polymorphisms of 1 enzyme, the class I alcohol dehydrogenase, ADH1B, which plays an important role in the rate of alcohol metabolism, were shown to impact on FASD vulnerability. 4-6 The major pathway of alcohol elimination involves its oxidation to acetaldehyde and further oxidation to acetate in the liver. Alcohol dehydrogenase is the principal enzyme that catalyzes ethanol oxidation to acetaldehyde, which then is oxidized to acetate by the AA BAC CPT FAS FASD FD FTII Absolute alcohol Blood alcohol concentration Continuous Performance Test Fetal alcohol syndrome Fetal alcohol spectrum disorder Freedom from distractibility Fagan Test of Infant Intelligence MAST MDI RT TRF VExP WISC-III Michigan Alcoholism Screening Test Mental Development Index Reaction time Teacher Report Form Visual Expectancy Paradigm Wechsler Intelligence Scale for Children, Third Edition See editorial, p 5. From the Departments of Psychiatry and Behavioral Neurosciences, Obstetrics and Gynecology, and Psychology, Wayne State University School of Medicine, Detroit, Department of Medicine and Pharmacology, Indiana University School of Medicine, Bloomington, and the National Institute on Alcohol Abuse and Alcoholism. Supported by grants RO1-AA06966, R01-AA09524, and P50-AA07606 from the National Institute on Alcohol Abuse and Alcoholism, with supplemental support from a Minority Biomedical Research Support grant SO6-RR08167 from the National Institutes of Health, and a grant from the Joseph Young, Sr., Fund from the State of Michigan. Submitted for publication Apr 8, 2005; last revision received Jul 25, 2005; accepted Aug 1, Reprint requests: Sandra W. Jacobson, PhD, Department of Psychiatry and Behavioral Neurosciences, Wayne State University School of Medicine, 2751 E. Jefferson, Room 460, Detroit, MI sjacobs@med.wayne.edu /$ - see front matter Copyright ª 2006 Elsevier Inc. All rights reserved /j.jpeds

2 enzyme aldehyde dehydrogenase. Expression of 3 possible alleles at the ADH1B locus* results in enzymes that differ in activity, with ADH1B*2 and the ADH1B*3 alleles associated with more rapid alcohol metabolism. 7 A high frequency of the ADH1B*3 allele appears to be unique to African Americans, occurring at a rate of about 22%. 8 Given the kinetic characteristics of the isozyme encoded by ADH1B*3, 9 the rate of alcohol oxidation should be greater for individuals with that allele than for individuals with only the ADH1B*1 allele. Our study was designed to replicate and extend birth weight and Bayley Scale findings detected in a previous study of black mothers and infants 5 by examining the degree to which the maternal ADH1B*3 allele may moderate the effects of prenatal alcohol exposure on fetal growth and a broad range of cognitive and behavioral outcomes examined in the Detroit Longitudinal Study in infancy and at 7.5 years. A second aim was to test the hypothesis that the presence of this allele in the child might exert a similar protective effect. We also examined the hypothesis that the observed protective effects on the fetus may be mediated by reduced levels of drinking in mothers with an ADH1B*3 allele. Participants METHODS The sample consisted of 263 black mother/child pairs. Maternal allele data were genotyped for 217 pairs and child allele data for 239. The mothers were recruited into the study between September 1986 and April 1989 during their first prenatal visit. Moderate- and heavy-drinking women were overrepresented in the sample by including all women reporting alcohol consumption at conception averaging at least 0.5 oz absolute alcohol (AA)/day (about 1 standard drink per day). A random sample of approximately 5% of the lower level drinkers and abstainers was also invited to participate. To reduce the risk that alcohol might be confounded with cocaine exposure, 53 heavy cocaine ($2 days/week) light alcohol (<7 drinks/week) users were also included. Infant exclusionary criteria included birth weight <1500 g, gestational age <32 weeks, major chromosomal anomalies, neural tube defects, or multiple births. The children were evaluated at 6.5, 12, and 13 months and 7.5 and 14 years. Assessment of Prenatal Exposure At each prenatal clinic visit, the mother was interviewed using the timeline follow-back interview. 10 Volume was recorded for each type of beverage consumed each day, converted to AA, and averaged across clinic visits. At the first visit, the mother was also asked to recall her day-by-day drinking during a typical week at time of conception, and she was administered the 25-item Michigan Alcoholism Screening Test (MAST). 11 A positive MAST score ($5) indicates that *Until a recent change in nomenclature (Human Genome Organization Gene Nomenclature Committee, 2001), the ADH1B alleles were known as ADH2*1, ADH2*2, and ADH2*3, respectively. the respondent has experienced psychosocial or physical problems related to alcohol abuse. Smoking during pregnancy was reported at the first antenatal visit as mean number of cigarettes/day. Detailed drug use data were also obtained at each clinic visit. 10 To limit attrition, mothers and children were transported to the laboratory by a community-based outreach worker. Each child received a gift, and the mother received a small remuneration and a photo of her child. All maternal and child assessments were conducted by a research team member blind with respect to maternal alcohol and drug use. Eighty-nine percent of the children were followed up at 7.5 years. Procedures were approved by the Wayne State University Human Investigation Committee. Written informed consent was obtained from the mother/primary caregiver at recruitment and the infant and 7.5-year assessments and oral assent from the child at 7.5 and 14 years. ADH Genotype Determination Five drops of blood were taken by a phlebotomist from the mother by finger-stick puncture and from the child by finger-stick or venipuncture at either the 7.5- or 14-year visits and placed on diagnostic filter paper for ADH genotyping at the Indiana University School of Medicine. ADH1B genotypes were determined using enzymatic amplification of genomic DNA followed by hybridization with allele-specific oligonucleotides. 12 Assessment During Infancy In this article we examined the degree to which associations of prenatal alcohol exposure with developmental outcomes are modified by the ADH1B polymorphism, focusing on those outcomes that were related to prenatal alcohol in the 263 children for whom maternal or child allele data were available. The following outcomes were examined in these analyses. Weight and head circumference were assessed at birth. The Mental Development Index (MDI) from the Bayley Scales of Infant Development 13 was administered at 13 months. Processing speed was assessed with the Fagan Test of Infant Intelligence (FTII) 14 at 6.5 and 12 months and crossmodal transfer 15 at 12 months. In the FTII, the infant is administered 10 problems consisting of 2 identical photos and then a novel photo paired with the familiar one. In the cross-modal transfer test, the stimuli consist of 6 pairs of acrylic objects. One member of each pair is shielded from view and presented to the infant for tactile familiarization. The infant is then shown the pair of objects. The normative response in both tests is to look longer at the novel stimulus. Processing speed in both tests is computed by dividing the total looking time to the stimuli by the number of looks. Shorter looks are indicative of more rapid information processing, related to faster infant reaction times, 16 and predict higher childhood IQ. 15,17 The Visual Expectancy Paradigm (VExP), 18 which measures latency to shift gaze in response to the appearance of a visual stimulus on a video screen, is the first test developed to measure reaction time (RT) in infancy. After Protective Effects Of The Alcohol Dehydrogenase-ADH1B Allele In Black Children Exposed To Alcohol During Pregnancy 31

3 a brief baseline of random presentations, 60 stimuli appear in a predictable left-right alternating sequence. The infant s eye movements are recorded on videotape, which are subsequently scored to determine RT, defined as latency between the onset of the stimulus and the time the infant s eye begins to move toward the stimulus. Percent quick responses is tabulated by combining number of fast ( ms) RTs with the number of anticipations (< 200 ms) and dividing by total number of events fixated. Like processing speed on FTII and Cross Modal transfer, RT on the VExP is predictive of childhood IQ. 19 Complexity of play, a measure of symbolic development predictive of childhood verbal IQ, was assessed at 12 months. 20 After 10 minutes of free play, suggestion and modeling are used to elicit higher levels of play than those spontaneously exhibited by the infant. Elicited play level is defined as the highest level imitated by the infant. Assessment at 7.5 Years Working memory was assessed with 2 subtests from the Wechsler Intelligence Scale for Children, Third Edition (WISC-III), Digit Span and Arithmetic, which comprise the freedom from distractibility (FD) factor. FD is believed to measure the ability to concentrate or remain attentive. Focused attention was assessed with the Digit Cancellation test, 21 in which the child is asked to cross out all the 3 s and 7 s listed on a page of random digits as quickly as possible. The task is then repeated with an auditory interference, strings of numbers simultaneously heard through headphones. Category Fluency, based on the Verbal Fluency subtest of the McCarthy Scales of Children s Abilities, was used to measure executive function/working memory. The child is asked to list as many items from a given category (eg, things to eat) as possible within 20 seconds. Often considered a test of focused attention, in this cohort Digit Cancellation loaded with the working memory dimension of attention. 22 Sustained attention was assessed on the Continuous Performance Test (CPT). 23 Maintenance of attention in the presence of distractions was assessed by asking the child to press a button whenever the letter X appeared on the screen immediately preceded by the letter A. In Magnitude Estimation the child must judge which of 2 numbers is larger, a judgment that is easier if the 2 numbers differ substantially (eg, 1 and 9) than when they are closer in magnitude (eg, 4 and 5), and whether a triple arrow (///) is pointed left or right when displayed together at various distances from a single arrow (/). As in the classic Sternberg 24 memory scan paradigm, the slope of RT plotted in relation to item difficulty yields a measure of processing efficiency with a flatter slope indicative of more efficient information processing. Teacher Behavior Ratings The child s classroom teacher completed the Achenbach Teacher Report Form (TRF), 25 which assesses social competence and behavior problems. The teacher also completed the Barkley-DuPaul ADHD Rating Scale. 26 Potential Confounders Nineteen control variables were considered as potential confounders: (a) demographics marital status, parity, and education of the child s primary caregiver; maternal age at delivery; child sex; number of children in the household; and disruption of care giving (separation from the mother for at least 4 months); (b) other prenatal exposures maternal smoking, cocaine, or marijuana use during pregnancy; (c) child-rearing environment the Home Observation for Measurement of the Environment Inventory 27 ; care giver s Peabody Picture Vocabulary Test Revised 28 ; perceived life stress on the Life Events Scale 29 ; (d) current use of alcohol, hard drugs, marijuana, and cigarettes by the care giver; and (e) child s age at the laboratory visit. Examiner effects were included for tests involving child/examiner interaction, such as the Bayley MDI, elicited play, and the WISC-III FD. Data Analysis The alcohol and drug use measures were transformed to log X 1 1 to reduce the influence of extreme outliers. The relation between prenatal alcohol exposure and child outcomes was examined by multiple regression analysis. Because a control variable cannot be the true cause of an observed deficit unless it is related both to the exposure and outcome, association with either exposure or outcome can be used as a criterion for identification of potential confounders. Selection in relation to outcome has the additional advantage of including covariates unrelated to exposure, which can increase precision. Any control variable that was related even weakly (at P<.10) to a given developmental outcome was controlled statistically as a potential confounder in all analyses of effects on that outcome. Each outcome measure was examined in hierarchical multiple regression analyses on the basis of maternal drinking averaged across pregnancy. A prenatal alcohol effect was inferred only if the relation of pregnancy drinking to developmental outcome was significant (at P<.05), after adjustment for the effects of the potential confounders. In the tables, Pearson r refers to the unadjusted relation of prenatal alcohol exposure to child outcome; the standardized b coefficient, to the relation after adjusting for potential confounding variables. The infant and 7.5-year outcomes found to be related to prenatal alcohol exposure for the sample as a whole were reexamined to assess the moderating role of the ADH1B allele. Following McClelland and Judd, 30 first the mothers and subsequently the children were each divided into 2 groups on the basis of the presence or absence of at least one ADH1B*3 allele. Regressions were run separately for each of these subgroups using the covariates selected in relation to the outcomes for the entire sample of 263 families for whom allele data were available. RESULTS The sample was predominantly lower social class and economically disadvantaged (Table I). The number of 32 Jacobson et al The Journal of Pediatrics January 2006

4 Table I. Sample characteristics (N = 263) N Mean or % SD Range Primary caregiver Infancy Socioeconomic status y Maternal age at delivery Education (years) Marital status (% married) Parity Primary caregiver 7.5 years Socioeconomic status y Education (years) Marital status (% married) Prenatal exposure (consumers only) Absolute alcohol per day (oz) At conception During pregnancy Smoking (cigarettes/day) Marijuana (days/month) Cocaine (days/month) Child Sex (% male) Bayley Scales of Infant Development MDI Psychomotor Development Index WISC-III Full Scale IQ Verbal IQ Performance IQ Frequency distribution of ADH1B * 1 and ADH1B * 3 alleles Mother ADH1B * 1/ADH1B * ADH1B * 1/ADH1B * ADH1B * 3/ADH1B * Total Child ADH1B * 1/ADH1B * ADH1B * 1/ADH1B * ADH1B * 3/ADH1B * Total yhollingshead Four Factor Index of Social Status (1975). moderate and heavy drinkers declined across pregnancy. Although the frequency of drinking among the non-abstaining women decreased by more than 50% (from 1.9 to 0.9 days per week) during pregnancy, t (210) = 15.44, P<.001, the number of drinks consumed per occasion declined by only 20% (from 4.5 to 3.6 on average), t (210) = 5.91, P<.001. Thus, many women continued to drink at concentrated doses that place the fetus at risk. The main illicit drugs used by the women during pregnancy were cocaine and marijuana. About one third (34.4%) of the women reported using cocaine. Fifty-three (20.2%) were heavy cocaine users who drank low levels of alcohol (<0.5 oz AA/day) during pregnancy. Almost one third (30.4%) reported using marijuana, but only 4.9% smoked it at least once per week. Most of the women (60.5%) smoked cigarettes, and 22.4% were heavy smokers (1 pack/day or more). Very few women reported using opiates (2.3%) or depressants (1.3%) at least once per week during pregnancy. Alcohol consumption was moderately related to smoking (r = 0.30, P<.001) and weakly related to cocaine (r =.25, P<.001) but not to marijuana use (r = 20.03). Most women (82.4%) were negative on the MAST and thus did not present with a history of alcohol abuse problems. At 7.5 years, 9.8% of the primary care givers reported using cocaine, and 5.1% were heavy users (at least once/week); 31.8% used marijuana with 15.0% reporting heavy use; 52.4% smoked, and 12.3% were heavy smokers. Protective Effects Of The Alcohol Dehydrogenase-ADH1B Allele In Black Children Exposed To Alcohol During Pregnancy 33

5 Table II. Pregnancy drinking patterns by presence of ADH1B * 3 allele Absent (N = 140) At conception Present (N = 77) t Absent (N = 140) During pregnancy Present (N = 77) t Oz absolute alcohol/day * Drinks/occasion Frequency (drinking days/week) * MAST score Values are means. *P <.05. Allele Frequency and Alcohol Use Table I presents the genotype distribution at the ADH1B*3 locus for the mothers and children. As in previous studies, ADH1B*1/*1 was the predominant genotype for both the mothers and children. The frequencies of the ADH1B*3 allele were 18.9% in the maternal sample and 20.3% in the children, which are consistent with the percentages expected for the black population. Women lacking an ADH1B*3 allele reported drinking almost twice as much alcohol at time of conception compared with those for whom at least one allele was present (Table II). Although the mean number of drinks per occasion was not different, the number of drinking days during pregnancy was greater for those without this allele. The presence of the ADH1B*3 allele variant was unrelated to drinking across pregnancy and to MAST positive score, but of 8 women with MAST scores $ 10, only one had this allele. No group differences in ADH1B*3 frequencies were found in relation to use of cocaine, marijuana, or cigarettes smoked during pregnancy, all Fs < Impact of Maternal ADH1B*3 Allele on Infant Outcomes In examining the moderating role of the ADH1B polymorphism, the association of prenatal alcohol with smaller head circumference was seen only in infants whose mothers lacked the ADH1B*3 allele (Table III). The presence of the maternal ADH1B*3 allele protected against the alcoholrelated deficit in Bayley MDI performance, confirming the results of McCarver et al. 5 In addition, except for birth weight and processing speed on the FTII, the negative effects of alcohol exposure were seen only in infants whose mothers lacked the ADH1B*3 allele. These effects were seen for all of the other developmental outcomes, including elicited play on the Belsky scale, processing speed on the cross modal transfer test, and reaction time on the Visual Expectancy Paradigm. Impact of maternal ADH1B*3 Allele on 7.5-year Outcomes COGNITIVE EFFECTS. Moderating effects of the maternal ADH1B*3 allele were seen at 7.5 years in measures involving focused attention/working memory and information processing efficiency (Table III). On the Digit Cancellation test, prenatal alcohol exposure was associated with poorer performance during the auditory interference condition for those children whose mothers lacked an ADH1B*3 allele. Prenatal alcohol was also associated with poorer Category Fluency, which assesses the ability to monitor information retrieved from long-term memory for conformity with prescribed rules, only for the children whose mothers lacked the ADH1B*3 allele. Prenatal alcohol exposure was associated with less efficient magnitude estimation processing on the number and arrows tasks in the absence of this allele. The pattern of effect was similar for performance on the WISC-III FD factor and slower RT during the more challenging AX condition on the CPT. EFFECTS ON SOCIAL AND EMOTIONAL FUNCTION. Among the children whose mothers lacked an ADH1B*3 allele, prenatal alcohol exposure was associated with poorer teacher ratings on 3 TRF problem syndromes social problems, attention, aggressive behavior and the TRF externalizing and total problems scores, as well as greater teacher-rated inattention and impulsivity and a higher ADHD score on the Barkley- DuPaul ADHD Scale (Table III). Impact of Child ADH1B*3 Allele on Infant and 7.5-year Outcomes INFANT EFFECTS. The negative effect of prenatal alcohol exposure on head circumference at birth was moderated by the absence of the child s ADH1B*3 allele (Table IV). However, unlike the impact of the maternal polymorphism, the impact of prenatal alcohol on the Bayley MDI was not moderated by the child s ADH1B*3 allele. The negative impact of alcohol exposure on processing speed was seen in children who lacked the allele variant on the FTII. By contrast, effects of prenatal alcohol on elicited symbolic play and VExP proportion quick responses were seen only in infants who had at least ADH1B*3 allele. 7.5-YEAR EFFECTS. Four of the effects of prenatal alcohol exposure on child cognitive outcome Category Fluency, RT on the CPT AX task, and magnitude estimation slope for number and arrows were seen only in the absence of the child ADH1B*3 allele. By contrast, a pattern of greater vulnerability was seen in relation to the teacher-reported 34 Jacobson et al The Journal of Pediatrics January 2006

6 Table III. Relation of pregnancy drinking to infant and child outcomes moderated by maternal ADH1B * 3 allele Absent Present N r b N r b Infant outcomes Birth size Weight z y 2.19 Head circumference z Bayley Scales MDI z 2.24 z Elicited symbolic play z 2.18 * Processing speed FTII k *.31 y Cross-modal z Visual expectancy paradigm Reaction time z.46 y Proportion quick z responses 7.5 years Cognitive outcomes Freedom from * distractibility { Digit cancellation z.23 y (interference) Category fluency y 2.23 y CPT RT (AX task) * Magnitude estimation (slope) Number z 2.30 z Arrows y.22 y Achenbach Teacher Report Form Social problems y.24 y Attention problems y.21 y Aggressive behavior y.22 y Delinquent behavior * Externalizing y.23 y Total problems ADHD Rating Scale Impulsivity z.29 z Inattention z.22 y ADHD classification z.30 z *P <.10. yp <.05. zp <.01. P <.001. kmean for 6.5 and 12 months. {WISC-III. Table IV. Relation of pregnancy drinking to infant and child outcomes moderated by child ADH1B * 3 allele Absent Present N r b N r b Infant outcomes Birth size Weight z 2.14 * y 2.16 Head circumference z Bayley Scales MDI * y 2.16 Elicited symbolic play * z 2.34 y Processing speed FTII k y.20 y Cross-modal Visual expectancy paradigm Reaction time y Proportion quick y 2.34 y * 2.41 * responses 7.5 years Cognitive outcomes Freedom from distractibility { Digit cancellation * (interference) Category fluency * 2.22 y CPT RT (AX task) * Magnitude estimation (slope) Number y.21 y Arrows z 2.28 z Achenbach Teacher Report Form Social problems y.20 y Attention problems z.35 z Aggressive behavior *.16 * *.21 * Delinquent behavior z.37 z Externalizing * y.28 y Total problems ADHD Rating Scale Impulsivity y.21 y y.24 * Inattention z.32 y ADHD classification z.28 z y.27 y *P <.10. yp <.05. zp <.01. P <.001. kmean for 6.5 and 12 months. {WISC-III. behavior measures of attention and delinquent problems, externalizing, and total problems scores on the TRF and inattention on the ADHD Rating Scale in the children for whom the ADH1B*3 allele was present. One effect, that of alcohol on social problems on the TRF, was seen only when the allele was absent, and the allele did not appear to modify the effects of prenatal alcohol exposure on impulsivity or ADHD classification. Protective Effects Of The Alcohol Dehydrogenase-ADH1B Allele In Black Children Exposed To Alcohol During Pregnancy 35

7 Thus, by contrast to the maternal ADH1B*3 allele, there was no consistent pattern of diminished vulnerability during infancy in the children who were born with an ADH1B*3 allele. Although the pattern for the child cognitive outcomes is similar to that seen for the maternal ADH1B*3 allele, it is reversed for the child behavior outcomes, where the effects are seen primarily in the children with at least one ADH1B*3 allele. Moreover, even where TRF effects were seen in children with the ADH1B*3 allele, we noted that 3 of the 4 most heavily exposed children (oz AA/day $ 1.0) were born to mothers who lacked the ADH1B*3 allele. DISCUSSION Several factors have recently been identified as determinants that alter teratogenic risk. In the early research on FASD, the marked individual differences in vulnerability to prenatal alcohol exposure were generally attributed to differences in nutritional adequacy or fetal genetic predisposition. Our prenatal alcohol studies in Detroit and Cape Town, South Africa, have provided extensive evidence that both older maternal age and a history of maternal drinking problems are important factors that enhance fetal vulnerability to a broad range of alcohol-related deficits. 31,32 The data reported in this article have confirmed a third important effect modifier of fetal teratogenesis initially suggested by McCarver et al 5 the maternal ADH1B*3 allele. Our findings extend the evidence of the protective effects of this allele on birth size and infant Bayley MDI performance reported by McCarver et al 5 by demonstrating protective effects on a broad range of alcohol-related cognitive and behavioral outcomes in infancy and at 7.5 years. Moreover, these data are consistent with Viljoen et al s 6 report that the ADH1B*2 variant of this allele in the Cape Coloured (mixed ancestry) population in South Africa, which is also associated with more rapid alcohol metabolism, is protective against FAS. By contrast to these 3 studies, Stoler et al 33 found greater effects of prenatal alcohol exposure on growth restriction and facial features of FAS in black neonates born to mothers with the ADH1B*3 allele. However, the vast majority of the mothers in the study were abstinent; in the preceding 4- week period, only 10 black women (1.4%) reported drinking more than 1 drink/day, the lowest level at which most alcohol effects have been detected 34 ; and alcohol exposure was not related to infant outcome, after control for maternal genotype, smoking, and weight gain. McCarver et al 5 reported that the child ADH1B*3 allele also protected against 1 of the 4 infant end points they examined. By contrast, we found no consistent pattern of greater vulnerability in the children with the ADH1B*3 allele across the large number of endpoints we examined. Although the genetic basis for alcohol metabolism differs in mice, Chernoff 35 also observed that alcohol teratogenesis was a function of maternal rather than fetal genotype in his study of mice. Viljoen et al s 6 South African findings suggest a protective role of the ADH1B*2 allele within either the mother or the fetus, but in their sample all 4 children with FAS with this allele were born to the 4 mothers who also had this allele (Viljoen, April 11, 2005, written personal communication), making it impossible to determine whether the child ADH1B*2 allele alone provided any additional protection. Thus, by contrast to the maternal allele, none of the studies to date provide clear evidence for protection by the child ADH1B allele in relation to developmental outcomes. The mechanism for variation in offspring susceptibility to intrauterine alcohol exposure is yet unknown. Because McCarver et al s 5 cohort was selected on the basis of maternal alcohol consumption and genotype, the design did not permit analysis of the degree to which the protective effect of the ADH1B*3 allele might be mediated indirectly by a reduction in maternal drinking. Animal studies have demonstrated that peak blood alcohol concentration (BAC) is an important determinant of extent of fetal alcohol damage. For example, ingestion of a given dose of alcohol over a short time period generates a higher peak BAC and greater neuronal 36,37 and behavioral impairment 38 than a larger dose ingested more gradually over several days. In a recent human study, Khaole et al 39 examined the relation of drinking, alcohol metabolic rate, and risk for an FAS birth outcome using free-choice drinking and consequent breath alcohol concentrations among drinking women who previously had given birth to a child with FAS and drinking women whose children did not have FAS. Although alcohol elimination rates did not differ between the 2 groups, women who previously had given birth to a child with FAS drank more alcohol, thereby attaining higher peak breath alcohol concentrations. Based on the animal and Khaole et al 39 findings, we assume that a high peak BAC is necessary for adverse alcoholrelated effects to emerge. In our Detroit study, alcohol intake by the mothers who lacked an ADHB1*3 allele differed in terms of mean alcohol use at conception. The mothers exhibited a decrease in frequency of drinking episodes but no significant reduction in alcohol intake per occasion. While this decrease in frequency of drinking may relate to an enhanced rate of alcohol metabolism, the precise mechanism is not yet known. It has been hypothesized that the increased acetaldehyde levels that presumably result from the more kinetically active variant of ADH1B causes an unpleasant physiological state that may serve to discourage drinking. 4 Because the adverse effects associated with fetal alcohol exposure depend principally on higher peak BAC attained per drinking occasion, it seems most likely that the protective effect of this allele is due to the more rapid metabolism of alcohol that it confers on the mother, which presumably results in a reduction of the peak BAC attained during a given drinking episode. It had initially been assumed that the ADH1B*3 allele would enhance the teratogenicity of alcohol by increasing levels of acetaldehyde, which was hypothesized to be harmful to the fetus. Our data support McCarver et al s 5 conclusion that the protective effects of this gene are likely secondary to the high turnover rate of the enzyme variant it encodes, which would provide more efficient alcohol metabolism at high BACs. 36 Jacobson et al The Journal of Pediatrics January 2006

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