Fetal Alcohol Spectrum Disorder Phenotypes Research Articles

Use of Telemedicine for the Physical Examination of Children With Fetal Alcohol Spectrum Disorders.

The fetal alcohol spectrum disorders (FASD) are among the most prevalent causes of neurodevelopmental disorders. The diagnosis is based on assessment of prenatal alcohol exposure, specific physical features identified with a dysmorphology examination, and neurobehavioral assessment. Prompt diagnosis of affected children is necessary to provide early intervention services in a timely manner; however, the availability of diagnostic expertise is limited. We propose telemedicine (TM) as a valid and reliable mode by which the physical phenotype of FASD can be accurately assessed. We compared face-to-face (F2F) physical examinations of the 3 key facial features and the resulting physical phenotype of the fetal alcohol syndrome (FAS) and partial FAS (pFAS), as well as 12 additional physical features seen more frequently in children with FAS than in the general population in 61 individuals with 2 different TM methods. These included a Transportable Examination Station system using a precision camera and a laptop and a Zoom secure connection system (ZOOM), using a smart phone and a tablet. We measured the percentages of agreement and the Cohen's K coefficient for each comparison. Agreements for most physical features and for the physical phenotype of FAS and pFAS were in the "almost perfect" range with some exceptions in the "substantial" range. Imprecision in measurement and subjectivity underlie lower agreement for some features, both F2F and using TM. We identified the optimal conditions for the F2F examinations in order to assure reliability using TM. TM is a valid and reliable method for the examination of the physical features of FAS that may contribute to greater access to an early diagnosis of FASD in children prenatally exposed to alcohol and/or with characteristic neurobehavioral deficits.

Murine Models for the Study of Fetal Alcohol Spectrum Disorders: An Overview.

Prenatal alcohol exposure is associated to different physical, behavioral, cognitive, and neurological impairments collectively known as fetal alcohol spectrum disorder. The underlying mechanisms of ethanol toxicity are not completely understood. Experimental studies during human pregnancy to identify new diagnostic biomarkers are difficult to carry out beyond genetic or epigenetic analyses in biological matrices. Therefore, animal models are a useful tool to study the teratogenic effects of alcohol on the central nervous system and analyze the benefits of promising therapies. Animal models of alcohol spectrum disorder allow the analysis of key variables such as amount, timing and frequency of ethanol consumption to describe the harmful effects of prenatal alcohol exposure. In this review, we aim to synthetize neurodevelopmental disabilities in rodent fetal alcohol spectrum disorder phenotypes, considering facial dysmorphology and fetal growth restriction. We examine the different neurodevelopmental stages based on the most consistently implicated epigenetic mechanisms, cell types and molecular pathways, and assess the advantages and disadvantages of murine models in the study of fetal alcohol spectrum disorder, the different routes of alcohol administration, and alcohol consumption patterns applied to rodents. Finally, we analyze a wide range of phenotypic features to identify fetal alcohol spectrum disorder phenotypes in murine models, exploring facial dysmorphology, neurodevelopmental deficits, and growth restriction, as well as the methodologies used to evaluate behavioral and anatomical alterations produced by prenatal alcohol exposure in rodents.

Ethanol and cannabinoids interact to alter behavior in a zebrafish fetal alcohol spectrum disorder model.

Recent work suggests that endocannabinoids (eCBs) may signal through the sonic hedgehog signaling pathway. We therefore hypothesized that combined ethanol and eCB exposure during defined stages of zebrafish embryogenesis will produce deficits comparable to human fetal alcohol spectrum disorder (FASD). Zebrafish embryos were exposed to ethanol or cannabinoid agonists alone or in combination at defined developmental stages and assessed for changes in brain morphology or expression of marker genes such as pax6a. Juvenile fish were then assessed for risk-taking/anxiety-like behavior using the novel tank dive test. Either chronic or acute exposure to high doses of the CB1R agonist ACEA resulted in FASD phenotypes. However, acute subthreshold doses of CB1R agonist alone, or combined with 0.5% ethanol, did not induce morphological phenotypes, but did induce dysmorphogenesis when combined with acute 1% ethanol. Phenotypes were rescued using the CB1R antagonist SR141716A. In addition, JZL195, a dual inhibitor of FAAH and MAGL, two degradative enzymes for eCBs, induced FASD phenotypes in the presence of subthreshold ethanol, confirming the activation of common signaling pathways by ethanol and eCBs. We next analyzed the effects of ethanol and CB1R agonist on juvenile zebrafish behavior and show that ACEA or ethanol alone did not alter behavior, but combined ACEA and ethanol increased risk-taking behavior. These studies demonstrate that pathological and behavioral phenotypes associated with FASD are induced by exposure to CB1R agonists and suggest that combined exposure to lower levels of alcohol and marijuana may be capable of inducing FASD-like morphological and behavioral impairments.

Developmental ethanol exposure impairs locomotor movement in Japanese medaka (Oryzias latipes) larvae targeting epigenome

Evidence indicated ethanol exposure during development disrupts brain functions that induces fetal alcohol spectrum disorder (FASD) phenotypes with behavioral abnormalities. We aimed to investigate whether prenatal ethanol exposure has any potential impact on behavior of a FASD fish model. Fertilized Japanese medaka (Oryzias latipes) eggs were exposed to 100–300 mM ethanol or 2 mM 5-azacytidine (5-azaC), 0–2 day post fertilization (dpf), in embryo-rearing medium (ERM). Survived embryos were maintained in clean ERM and used either for gene expression analysis on 2- and 6-dpf or allowed to hatch for behavioral study. Photomotor response of 3–4 day post hatch larvae were tracked for 3 h with light-dark transitions. It was observed that larval swimming was phototactic; enhanced in presence of light, declined in dark. Phototactic response was also observed in larvae prenatally exposed to ethanol or 5-azaC; however, the total distance swum by these larvae compared to controls declined. Further analysis indicated that, in light phases, total swimming activity and average swimming speed were reduced in larvae prenatally exposed to ethanol (300 mM) or 5-azaC. Expression analysis of baz1a and baz2a in embryos indicated developmental regulation. Ethanol (100–300 mM) or 5-azaC (2 mM) were able to modulate downregulation of both baz1a and baz2a mRNAs only in 6 dpf embryos of 300 mM ethanol and 5-azaC (2 mM) groups. These studies indicated that prenatal exposure to ethanol or 5-azaC was able to disrupt movements and thus swimming behavior in FASD phenotypes probably due to delayed remodeling of genome and epigenome.

Epigenetic Mechanisms in Developmental Alcohol-Induced Neurobehavioral Deficits.

Alcohol consumption during pregnancy and its damaging consequences on the developing infant brain are significant public health, social, and economic issues. The major distinctive features of prenatal alcohol exposure in humans are cognitive and behavioral dysfunction due to damage to the central nervous system (CNS), which results in a continuum of disarray that is collectively called fetal alcohol spectrum disorder (FASD). Many rodent models have been developed to understand the mechanisms of and to reproduce the human FASD phenotypes. These animal FASD studies have provided several molecular pathways that are likely responsible for the neurobehavioral abnormalities that are associated with prenatal alcohol exposure of the developing CNS. Recently, many laboratories have identified several immediate, as well as long-lasting, epigenetic modifications of DNA methylation, DNA-associated histone proteins and microRNA (miRNA) biogenesis by using a variety of epigenetic approaches in rodent FASD models. Because DNA methylation patterns, DNA-associated histone protein modifications and miRNA-regulated gene expression are crucial for synaptic plasticity and learning and memory, they can therefore offer an answer to many of the neurobehavioral abnormalities that are found in FASD. In this review, we briefly discuss the current literature of DNA methylation, DNA-associated histone proteins modification and miRNA and review recent developments concerning epigenetic changes in FASD.

Comparing Executive Functioning in Children and Adolescents With Fetal Alcohol Spectrum Disorders and ADHD: A Meta-Analysis

Objective: Fetal alcohol spectrum disorders (FASD) and Attention-Deficit/Hyperactivity Disorder (ADHD) are associated with a range of neurocognitive impairments. Executive functioning deficits are a hallmark feature of both disorders. Method: The present meta-analysis was undertaken to disentangle the behavioral phenotype of FASD and ADHD by quantitatively differentiating executive functioning differences between these two groups. The current meta-analysis reviews 15 studies comparing children and adolescents with FASD and ADHD to typically developing (TD) samples, on a variety of executive function measures. Results: Results indicate that when compared with TD samples, FASD and ADHD samples demonstrate significant executive function deficits (d = 0.82 and d = 0.55, respectively). In addition, FASD samples experience significantly greater deficits when compared with ADHD samples (d = 0.25). Results were moderated by IQ and socioeconomic status. Conclusion: These findings further our understanding of the cognitive differences between FASD and ADHD samples and have the potential to influence future basic research, assessment, and intervention.

Fetal Alcohol Spectrum Disorder: Potential Role of Endocannabinoids Signaling.

One of the unique features of prenatal alcohol exposure in humans is impaired cognitive and behavioral function resulting from damage to the central nervous system (CNS), which leads to a spectrum of impairments referred to as fetal alcohol spectrum disorder (FASD). Human FASD phenotypes can be reproduced in the rodent CNS following prenatal ethanol exposure. Several mechanisms are expected to contribute to the detrimental effects of prenatal alcohol exposure on the developing fetus, particularly in the developing CNS. These mechanisms may act simultaneously or consecutively and differ among a variety of cell types at specific developmental stages in particular brain regions. Studies have identified numerous potential mechanisms through which alcohol can act on the fetus. Among these mechanisms are increased oxidative stress, mitochondrial damage, interference with the activity of growth factors, glia cells, cell adhesion molecules, gene expression during CNS development and impaired function of signaling molecules involved in neuronal communication and circuit formation. These alcohol-induced deficits result in long-lasting abnormalities in neuronal plasticity and learning and memory and can explain many of the neurobehavioral abnormalities found in FASD. In this review, the author discusses the mechanisms that are associated with FASD and provides a current status on the endocannabinoid system in the development of FASD.

DNA methyltransferase expressions in Japanese rice fish (Oryzias latipes) embryogenesis is developmentally regulated and modulated by ethanol and 5-azacytidine

We aimed to investigate the impact of the epigenome in inducting fetal alcohol spectrum disorder (FASD) phenotypes in Japanese rice fish embryogenesis. One of the significant events in epigenome is DNA methylation which is catalyzed by DNA methyltransferase (DNMT) enzymes. We analyzed DNMT enzyme mRNA expressions in Japanese rice fish development starting from fertilized eggs to hatching and also in embryos exposed for first 48h of development either to ethanol (300mM) or to 5-azacytidine (5-azaC; 2mM), an inhibitor of DNMT enzyme activity. As observed in FASD phenotypes, 5-azaC exposure was able to induce microcephaly and craniofacial cartilage deformities in Japanese rice fish. Moreover, we have observed that expression of DNMTs (dnmt1, dnmt3aa, and dnmt3bb.1) are developmentally regulated; high mRNA copies were found in early stages (1–2day-post-fertilization, dpf), followed by gradual reduction until hatched. In ethanol-treated embryos, compared to controls, dnmt1 mRNA is in reduced level in 2dpf and in enhanced level in 6dpf embryos. While dnmt3aa and 3bb.1 remained unaltered. In contrast, embryos exposed to 5-azaC have an enhanced level of dnmt1 and dnmt3bb.1 mRNAs both in 2 and 6dpf embryos while dnmt3aa is enhanced only in 6dpf embryos. Moreover, endocannabinoid receptor 1a (cnr1a) mRNA which was found to be reduced by ethanol remained unaltered and cnr1b and cnr2 mRNAs, which were remained unaltered by ethanol, were increased significantly by 5-azaC in 6dpf embryos. This study indicates that the craniofacial defects observed in FASD phenotypes are the results of dysregulations in DNMT expressions.

Gene-specific disruption of endocannabinoid receptor 1 (cnr1a) by ethanol probably leads to the development of fetal alcohol spectrum disorder (FASD) phenotypes in Japanese rice fish (Oryzias latipes) embryogenesis

The present study was designed to investigate the probable roles played by cannabinoid (CB) receptors in fetal alcohol spectrum disorder (FASD) induction in Japanese rice fish (Oryzias latipes). Searching of public databases (GenBank, Ensembl) indicated that the Japanese rice fish genome includes three human ortholog CB receptor genes (cnr1a, cnr1b and cnr2). Quantitative real-time PCR (qPCR) and whole mount in situ hybridization (WMISH) techniques were used to analyze the expression of these cnr genes during Japanese rice fish embryogenesis and also in response to developmental ethanol exposure. qPCR analyses showed that the expression of all three CB receptor genes were developmentally regulated and only cnr2 showed maternal expression. The mRNA concentrations of these genes were found to be enhanced after 3dpf and attained maximal levels either prior to or after hatching. WMISH technique indicated that all three cnr genes were expressed in the head region of hatchlings. During development, ethanol selectively attenuated the expression of cnr1a mRNA only. Blocking of cnr1a mRNA by CB1 receptor antagonists rimonabant (10–20μM) or AM251 (0.2–1μM) 0–2dpf were unable to induce any FASD-related phenotypic features in embryos or in hatchlings. However, continuous exposure of the embryos (0–6dpf) to AM251 (1μM) was able to reduce the hatching efficiency of the embryos. Our data indicated that in Japanese rice fish, ethanol disrupted the expression of only cnr1a in a concentration-dependent manner that induced delay in hatching and might be responsible for the development of FASD phenotypes.

Evaluation of Panax ginseng ginsenosides as potential therapeutic agent of fetal alcohol spectrum disorders targeting CB1 receptors

Identification of endocannabinoid receptor 1 subtype a (cnr1a) as a potential target of alcohol in Japanese medaka embryogenesis prompted us to use this model in discovering anti-alcoholic drugs from the natural products that may attenuate fetal alcohol spectrum disorder (FASD), a birth defect observed in the babies of mothers who ingested alcohol during pregnancy. FASD phenotypes are identified by growth retardation with central nervous system (CNS) malformation and dysfunction, and distinctive patterns of craniofacial, cardiovascular, and limb defects. The molecular mechanism(s) by which ethanol perturbs fetal development is unknown. Also, prevention of FASD, other than women abstaining from drinking alcohol during pregnancy, is not known. We have demonstrated that developmental exposure of fertilized medaka eggs to ethanol has generated several phenotypic end points in the neurocranium and cadriovasculature which are analogous to human FASD phenotypes. Moreover, the expression of cnr1a gene is reduced by developmental ethanol exposure. Further we have shown that ginseng root extract is able to partially rescue the embryos from the ethanol-induced developmental disorders. We will evaluate whether ginsenoside extracted from Panax ginseng is able to attenuate FASD in medaka embryos.

Developmental regulation of neuroligin genes in Japanese ricefish (Oryzias latipes) embryogenesis maintains the rhythm during ethanol-induced fetal alcohol spectrum disorder

Although prenatal alcohol exposure is the potential cause of fetal alcohol spectrum disorder (FASD) in humans, the molecular mechanism(s) of FASD is yet unknown. We have used Japanese ricefish (Oryzias latipes) embryogenesis as an animal model of FASD and reported that this model has effectively generated several phenotypic features in the cardiovasculature and neurocranial cartilages by developmental ethanol exposure which is analogous to human FASD phenotypes. As FASD is a neurobehavioral disorder, we are searching for a molecular target of ethanol that alters neurological functions. In this communication, we have focused on neuroligin genes (nlgn) which are known to be active at the postsynaptic side of both excitatory and inhibitory synapses of the central nervous system. There are six human NLGN homologs of Japanese ricefish reported in public data bases. We have partially cloned these genes and analyzed their expression pattern during normal development and also after exposing the embryos to ethanol. Our data indicate that the expression of all six nlgn genes in Japanese ricefish embryos is developmentally regulated. Although ethanol is able to induce developmental abnormalities in Japanese ricefish embryogenesis comparable to the FASD phenotypes, quantitative real-time PCR (qPCR) analysis of nlgn mRNAs indicate unresponsiveness of these genes to ethanol. We conclude that the disruption of the developmental rhythm of Japanese ricefish embryogenesis by ethanol that leads to FASD may not affect the nlgn gene expression at the message level.

Asian Ginseng (Panax ginseng) Potentiates Ethanol-Induced Cardiovascular Dysfunction in Medaka Embryogenesis (Oryzias latipes)

Alcohol is a teratogen, induces fetal alcohol spectrum disorder (FASD) which has serious central nervous system (CNS), cardiovascular, and craniofacial defects affecting the entire lifetime of an individual. Prevention of FASD, other than women abstaining from drinking alcohol during pregnancy, is not known. The synthetic drugs recommended for the treatment of alcoholism cannot be used by women during pregnancy which led us to an investigation of natural products. Due to ethical constraints, FASD studies in humans are very limited and several animal models are used instead to understand the molecular mechanisms. We have observed that developmental ethanol exposure of medaka (Oryzias latipes) embryos generates features which are analogous to human FASD phenotypes [1]. We hypothesize that ethanol metabolism generates oxidative stress which can disrupt embryonic development of medaka. In the present investigation, we have used root extracts of Asian ginseng (Panax ginseng) as a preventive agent of FASD. Fertilized medaka eggs within 4h post fertilization (hpf) were exposed to methanolic extracts (50 – 100 µg/mL) of ginseng root (PG) or ethanol (300 mM) either alone or in combination. After 48h of treatment the viable embryos were transferred to clean hatching solution and on 6 dpf the embryos were examined for vessel circulation followed by mRNA analyses of enzymes related to ethanol metabolism and oxidative stress. It was observed that ethanol (300 mM) alone was able to disrupt vessel circulation and co-treatment of PG (50 – 100 µg/mL) with ethanol enhanced the effect; PG (100 µg/mL) alone had no effect. mRNA analysis of alcohol metabolizing enzymes or oxidative stress-related enzymes did not show any significant alterations under any of these treatment conditions. It is therefore concluded that potentiation of ethanol-induced cardiovascular deformities in medaka by PG may be mediated through a different mechanism rather than oxidative stress. Acknowledgements: Thanks go to the United States Department of Agriculture, Agriculture Research Service, Specific Cooperative Agreement No. 1UO1FD004246.

Asian Ginseng (Panax ginseng) Attenuates Ethanol-induced Neurocranial Cartilage Deformities in a Fish Model of Fetal Alcohol Spectrum Disorder

Fetal Alcohol spectrum disorder (FASD) is an important clinical problem resulting from prenatal alcohol exposure. It has serious central nervous system (CNS), cardiovascular, and craniofacial defects. Prevention of FASD, other than women abstaining from drinking alcohol during pregnancy, is not known. The synthetic drugs recommended for the treatment of alcoholism cannot be used by women during pregnancy which led us to investigate on natural products. Many herbs including Asian ginseng (Panax ginseng) have therapeutic potencies on alcoholism. Recently, ginseng has been shown to inhibit ethanol-induced teratogenesis in mouse embryos in vitro. We therefore tested the potency of Asian ginseng (PG) as an antialcoholic agent using medaka (Oryzias latipes) as an animal model. Previously we have demonstrated that medaka embryos exposed to ethanol developed neurocranial cartilage deformities which are analogous to the craniofacial abnormalities observed in human FASD phenotypes [1]. Moreover, the chondrification in cartilages, specifically trabecular cartilages (TC), was inhibited by ethanol [2]. In the present experiments we have tested whether PG can attenuate ethanol-induced TC deformities in medaka. Fertilized medaka eggs in standard laboratory conditions (16L: 8D, 25 0C) were exposed to PG root extract (0–2mg/ml) for 0–2 day post fertilization (dpf). The calculated IC50 of PG as determined on 10 dpf is 355±1g/ml (n=3, r2=0.9001). In separate experiments embryos were exposed to sub-lethal concentrations of PG (50 and 200µg/ml) with or without ethanol (100 and 300 mM) either for 0–2 dpf or 1–3 dpf and the developmental abnormalities were assessed in embryos and hatchlings. It was observed that PG dose-dependently prevent TC deformity in hatchlings and reduce thrombus formation in embryos; however, embryo mortality were enhanced. It is therefore concluded that PG is able to attenuate some of the ethanol- induced birth defects in medaka even though the embryo mortality was aggravated. Acknowledgements: This research is supported in part by the United States Department of Agriculture, Agricultural Research Service, Specific Cooperative Agreement No 586408–2-0009. The project described was supported by grant number 5P20RR021929 from the National Center for Research Resources. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Center for Research Resources or the National Institutes of Health.