Christopher D. Molteno

Validation of a new biomarker of fetal exposure to alcohol

OBJECTIVE To test the sensitivity and specificity of fatty acid ethyl esters (FAEEs) extracted from meconium to identify alcohol-using pregnant women with a sensitive and specific methodology, gas chromatography-tandem mass spectroscopy (GC/MS/MS). Study design Twenty-seven samples of meconium were obtained from infants from the mixed race community in Cape Town, South Africa, who were enrolled in a longitudinal neurobehavioral study. Maternal alcohol use was reported prospectively during pregnancy. FAEEs were isolated from meconium and quantitated by GC/MS/MS. RESULTS Ethyl oleate was the FAEE that correlated most strongly with maternal self-reported drinking, especially with the average ounces of absolute alcohol ingested per drinking day. Ethyl oleate was most strongly related to drinking in the second and third trimesters (Pearson r=.55 and.40, respectively). At a threshold of 1.5 average ounces of absolute alcohol ingested per drinking day, the area under the receiving operator characteristic curve was.92 (95% confidence interval, 0.74-0.97). Using a cut-off value of 32 ng/g, sensitivity was 84.2% and specificity was 83.3%. CONCLUSIONS Ethyl oleate concentration in meconium assayed by GC/MS/MS provides a highly sensitive and specific indicator of maternal alcohol use during pregnancy.

Impaired delay and trace eyeblink conditioning in school-age children with fetal alcohol syndrome.

BACKGROUND Classical eyeblink conditioning (EBC) involves contingent temporal pairing of a conditioned stimulus (e.g., tone) with an unconditioned stimulus (e.g., air puff). Impairment of EBC has been demonstrated in studies of alcohol-exposed animals and in children exposed prenatally at heavy levels. METHODS Fetal alcohol syndrome (FAS) was diagnosed by expert dysmorphologists in a large sample of Cape Coloured, South African children. Delay EBC was examined in a new sample of 63 children at 11.3 years, and trace conditioning in 32 of the same children at 12.8 years. At each age, 2 sessions of 50 trials each were administered on the same day; 2 more sessions the next day, for children not meeting criterion for conditioning. RESULTS Six of 34 (17.6%) children born to heavy drinkers were diagnosed with FAS, 28 were heavily exposed nonsyndromal (HE), and 29 were nonexposed controls. Only 33.3% with FAS and 42.9% of HE met criterion for delay conditioning, compared with 79.3% of controls. The more difficult trace conditioning task was also highly sensitive to fetal alcohol exposure. Only 16.7% of the FAS and 21.4% of HE met criterion for trace conditioning, compared with 66.7% of controls. The magnitude of the effect of diagnostic group on trace conditioning was not greater than the effect on short delay conditioning, findings consistent with recent rat studies. Longer latency to onset and peak eyeblink CR in exposed children indicated poor timing and failure to blink in anticipation of the puff. Extended training resulted in some but not all of the children reaching criterion. CONCLUSIONS These data showing alcohol-related delay and trace conditioning deficits extend our earlier findings of impaired EBC in 5-year-olds to school-age. Alcohol-related impairment in the cerebellar circuitry required for both forms of conditioning may be sufficient to account for the deficit in both tasks. Extended training was beneficial for some exposed children. EBC provides a well-characterized model system for assessment of degree of cerebellar-related learning and memory dysfunction in fetal alcohol exposed children.

An FMRI study of number processing in children with fetal alcohol syndrome.

BACKGROUND Number processing deficits are frequently seen in children exposed to alcohol in utero. METHODS Functional magnetic resonance imaging was used to examine the neural correlates of number processing in 15 right-handed, 8- to 12-year-old children diagnosed with fetal alcohol syndrome (FAS) or partial FAS (PFAS) and 18 right-handed, age- and gender-matched controls from the Cape Coloured (mixed ancestry) community in Cape Town, South Africa, using Proximity Judgment and Exact Addition tasks. RESULTS Control children activated the expected fronto-parietal network during both tasks, including the anterior horizontal intraparietal sulcus (HIPS), left posterior HIPS, left precentral sulcus, and posterior medial frontal cortex. By contrast, on the Proximity Judgment task, the exposed children recruited additional parietal pathways involving the right and left angular gyrus and posterior cingulate/precuneus, which may entail verbally mediated recitation of numbers and/or subtraction to assess relative numerical distances. During Exact Addition, the exposed children exhibited more diffuse and widespread activations, including the cerebellar vermis and cortex, which have been found to be activated in adults engaged in particularly challenging number processing problems. CONCLUSIONS The data suggest that, whereas control children rely primarily on the fronto-parietal network identified in previous studies to mediate number processing, children with FAS/PFAS recruit a broader range of brain regions to perform these relatively simple number processing tasks. Our results are consistent with structural neuroimaging findings indicating that the parietal lobe is relatively more affected by prenatal alcohol exposure and provide the first evidence for brain activation abnormalities during number processing in children with FAS/PFAS, effects that persist even after controlling statistically for group differences in total intracranial volume and IQ.

Facial dysmorphism across the fetal alcohol spectrum.

OBJECTIVE: Classic facial characteristics of fetal alcohol syndrome (FAS) are shortened palpebral fissures, smooth philtrum, and thin upper vermillion. We aim to help pediatricians detect facial dysmorphism across the fetal alcohol spectrum, especially among nonsyndromal heavily exposed (HE) individuals without classic facial characteristics. METHODS: Of 192 Cape Coloured children recruited, 69 were born to women who reported abstaining from alcohol during pregnancy. According to multifaceted criteria, the remainder were allocated clinically to the FAS (n = 22), partial FAS (n = 26) or nonsyndromal HE (n = 75) categories. We used dense surface modeling and signature analyses of 3-dimensional facial photographs to determine agreement between clinical categorization and classifications induced from face shape alone, to visualize facial differences, and to consider predictive links between face shape and neurobehavior. RESULTS: Face classification achieved significant agreement with clinical categories for discrimination of nonexposed from FAS alone (face: 0.97–1.00; profile: 0.92) or with the addition of partial FAS (face: 0.90; profile: 0.92). Visualizations of face signatures delineated dysmorphism across the fetal alcohol spectrum and in half of the nonsyndromal HE category face signature graphs detected facial characteristics consistent with prenatal alcohol exposure. This subgroup performed less well on IQ and learning tests than did nonsyndromal subjects without classic facial characteristics. CONCLUSIONS: Heat maps and morphing visualizations of face signatures may help clinicians detect facial dysmorphism across the fetal alcohol spectrum. Face signature graphs show potential for identifying nonsyndromal heavily exposed children who lack the classic facial phenotype but have cognitive impairment.

Diffusion Tensor Imaging of the Cerebellum and Eyeblink Conditioning in Fetal Alcohol Spectrum Disorder

BACKGROUND Prenatal alcohol exposure is related to a wide range of neurocognitive effects. Eyeblink conditioning (EBC), which involves temporal pairing of a conditioned with an unconditioned stimulus, has been shown to be a potential biomarker of fetal alcohol exposure. A growing body of evidence suggests that white matter may be a specific target of alcohol teratogenesis, and the neural circuitry underlying EBC is known to involve the cerebellar peduncles. Diffusion tensor imaging (DTI) is a magnetic resonance imaging (MRI) technique that has proven useful for assessing central nervous system white matter integrity. This study used DTI to examine the degree to which the fetal alcohol-related deficit in EBC may be mediated by structural impairment in the cerebellar peduncles. METHODS Thirteen children with fetal alcohol spectrum disorder (FASD) and 12 matched controls were scanned using DTI and structural MRI sequences. The DTI data were processed using a voxelwise technique, and the structural data were used for volumetric analyses. Prenatal alcohol exposure group and EBC performance were examined in relation to brain volumes and outputs from the DTI analysis. RESULTS Fractional anisotropy (FA) and perpendicular diffusivity group differences between alcohol-exposed and nonexposed children were identified in the left middle cerebellar peduncle. Alcohol exposure correlated with lower FA and greater perpendicular diffusivity in this region, and these correlations remained significant even after controlling for total brain and cerebellar volumes. Conversely, trace conditioning performance was related to higher FA and lower perpendicular diffusivity in the left middle peduncle. The effect of prenatal alcohol exposure on trace conditioning was partially mediated by lower FA in this region. CONCLUSIONS This study extends recent findings that have used DTI to reveal microstructural deficits in white matter in children with FASD. This is the first DTI study to demonstrate mediation of a fetal alcohol-related effect on neuropsychological function by deficits in white matter integrity.

Fetal alcohol exposure, iron-deficiency anemia, and infant growth.

OBJECTIVES. Our goals were to determine whether prenatal alcohol exposure is associated with an increased incidence of iron-deficiency anemia in infancy and to compare effects of fetal alcohol exposure and iron-deficiency anemia on infant growth. We also tested whether effects of fetal alcohol exposure on growth are mediated or moderated by iron-deficiency anemia. METHODS. A total of 96 infants born to mothers from the Coloured (mixed ancestry) community in Cape Town, South Africa, were recruited prenatally; 42 mothers drank heavily during pregnancy, and 54 abstained or drank small amounts of alcohol. Growth was assessed at birth and 6.5 and 12 months, and iron-deficiency anemia was assessed at 6.5 or 12 months. RESULTS. Infants whose mothers binge drank during pregnancy (≥4 drinks per occasion) were 3.6 times more likely to be diagnosed with iron-deficiency anemia at 12 months than were infants whose mothers did not binge drink. Prenatal alcohol exposure was associated with reduced weight at birth, 6.5 months, and 12 months and with shorter length at 6.5 and 12 months. Iron-deficiency anemia was related to reduced 12-month weight and head circumference and to slower growth velocity between 6 and 12 months. The effects of prenatal alcohol on weight were not mediated by iron-deficiency anemia; however, they were seen primarily in infants with iron-deficiency anemia. CONCLUSIONS. The association of maternal binge drinking with an increased incidence of iron-deficiency anemia may reflect disruption of accumulation of fetal iron stores or postnatal deficiencies in iron uptake, absorption, or intake. Moreover, iron deficiency seems to exacerbate the prenatal alcohol effects on growth.

Differences in cortico‐striatal‐cerebellar activation during working memory in syndromal and nonsyndromal children with prenatal alcohol exposure

Although children with heavy prenatal alcohol exposure may exhibit the distinctive facial dysmorphology seen in full or partial fetal alcohol syndrome (FAS/PFAS), many lack that dysmorphology. This study examined the functional organization of working memory in the brain in three groups of children—those meeting diagnostic criteria for FAS or PFAS, heavily exposed (HE) nonsyndromal children, and healthy controls. A verbal n‐back task (1‐back and 0‐back) was administered to 47 children (17 with FAS/PFAS, 13 HE, and 17 controls) during fMRI. Intra‐group one‐sample t‐tests were used to identify activity regions of interest central to verbal working memory including the dorsal prefrontal cortex (dPFC), inferior frontal gyrus, caudate/putamen, parietal cortex, and cerebellar Crus I/lobule VI and lobule VIIB‐IX. Whereas groups did not differ in task sensitivity, fMRI analyses suggested different patterns of sub‐network recruitment across groups. Controls primarily recruited left inferior frontal gyrus (Brocas area). By contrast, HE primarily recruited an extensive set of fronto‐striatal regions, including left dPFC and left caudate, and the FAS/PFAS group relied primarily on two cerebellar subregions and parietal cortex. This study is, to our knowledge, the first to demonstrate differential recruitment of critical brain regions that subserve basic function in children with different fetal alcohol spectrum disorders compared to controls. The distinct activation patterns seen in the two exposed groups may be related to substantial differences in alcohol dose/occasion to which these groups were exposed in utero. Hum Brain Mapp, 2013.

Biobehavioral markers of adverse effect in fetal alcohol spectrum disorders

Identification of children with fetal alcohol spectrum disorders (FASD) is difficult because information regarding prenatal exposure is often lacking, a large proportion of affected children do not exhibit facial anomalies, and no distinctive behavioral phenotype has been identified. Castellanos and Tannock have advocated going beyond descriptive symptom-based approaches to diagnosis to identify biomarkers derived from cognitive neuroscience. Classical eyeblink conditioning and magnitude comparison are particularly promising biobehavioral markers of FASD—eyeblink conditioning because a deficit in this elemental form of learning characterizes a very large proportion of alcohol-exposed children; magnitude comparison because it is a domain of higher order cognitive function that is among the most sensitive to fetal alcohol exposure. Because the neural circuitry mediating both these biobehavioral markers is well understood, they have considerable potential for advancing understanding of the pathophysiology of FASD, which can contribute to development of treatments targeted to the specific deficits that characterize this disorder.

A tensor-based morphometry analysis of regional differences in brain volume in relation to prenatal alcohol exposure

Reductions in brain volumes represent a neurobiological signature of fetal alcohol spectrum disorders (FASD). Less clear is how regional brain tissue reductions differ after normalizing for brain size differences linked with FASD and whether these profiles can predict the degree of prenatal exposure to alcohol. To examine associations of regional brain tissue excesses/deficits with degree of prenatal alcohol exposure and diagnosis with and without correction for overall brain volume, tensor-based morphometry (TBM) methods were applied to structural imaging data from a well-characterized, demographically homogeneous sample of children diagnosed with FASD (n = 39, 9.6–11.0 years) and controls (n = 16, 9.5–11.0 years). Degree of prenatal alcohol exposure was significantly associated with regionally pervasive brain tissue reductions in: (1) the thalamus, midbrain, and ventromedial frontal lobe, (2) the superior cerebellum and inferior occipital lobe, (3) the dorsolateral frontal cortex, and (4) the precuneus and superior parietal lobule. When overall brain size was factored out of the analysis on a subject-by-subject basis, no regions showed significant associations with alcohol exposure. FASD diagnosis was associated with a similar deformation pattern, but few of the regions survived FDR correction. In data-driven independent component analyses (ICA) regional brain tissue deformations successfully distinguished individuals based on extent of prenatal alcohol exposure and to a lesser degree, diagnosis. The greater sensitivity of the continuous measure of alcohol exposure compared with the categorical diagnosis across diverse brain regions underscores the dose dependence of these effects. The ICA results illustrate that profiles of brain tissue alterations may be a useful indicator of prenatal alcohol exposure when reliable historical data are not available and facial features are not apparent.

The factor structure of the Edinburgh postnatal depression scale in a South African peri-urban settlement.

The factor structure of the Edinburgh Postnatal Depression scale (EPDS) and similar instruments have received little attention in the literature. The researchers set out to investigate the construct validity and reliability of the EPDS amongst impoverished South African women. The EPDS was translated into isiXhosa (using Brislins back translation method) and administered by trained interviewers to 147 women in Khayelitsha, South Africa. Responses were subjected to maximum likelihood confirmatory factor analysis. A single factor structure was found, consistent with the theory on which the EPDS was based. Internal consistency was satisfactory (a = 0.89).