Cranial Facial Diagnostic test for FASD
Diagnostic test for FAS performed by Dr. David Wargowski MD
from The University of Wisconsin School of Medicine and Public Health

In order for an individual to be diagnosed with fetal alcohol syndrome (FAS), they must exhibit all of the four cardinal symptoms. These symptoms include the consumption of alcohol of the mother during pregnancy, deformities in facial structures, growth deficits and abnormal development of the central nervous system.[1]

The early diagnosis of FAS is important in implementing possible treatments and preventative measures that may inhibit the adverse effects of intrauterine consumption of ethanol. Although there are substantial advantages in early FAS detection, there are currently no definitive diagnostic techniques which can be implemented.[1] Current diagnosis of FAS relies mainly on post-natal examinations as well as testimonies of the mother, which can be altered through social desirability and recall bias.[1],[2] Researchers have been searching for a consistent assessment of FAS by investigating genetic prevalence, detectable changes in gene expression and molecular mechanisms associated with the disorder.

Current Diagnostic Techniques

Facial Malformations
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Key facial-cranial malformations that are characteristic of FAS

Cranial Facial Malformations

One of the most apparent symptoms of FAS is the development of abnormal facial structures. Currently, health care professionals are trained to diagnosis FAS by recognizing deformities such as a smooth philtrum, short palpebral fissures and a thin upper lip.[1] Two of these three structural deformities are required under existing diagnostic guidelines in order to be formally diagnosed with FAS.[3] The cause of these malformations can be traced to the inhibition of neural crest cells to develop correctly.[6]

Although, detecting abnormalities in facial structures has been a traditional diagnostic tool for FAS, some individuals affected with FAS may show the cognitive and behavioural symptoms without exhibiting any facial malformations. [1] This complication makes facial and cranial features an unreliable method in determining FAS patients and strengthens the need for a more conclusive test.

Fatty acid Ethyl Esters
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Metabolism of Ethanol produces fatty acid ethyl esters

Detection Fatty Acid Ethyl Ester

When ethanol is introduced to the fetal circulation, enzymes such as fatty acid ethyl ester synthase, catalyze the esterification of ethanol and fatty acids to form fat soluble fatty acid ethyl esters (FAEE).[1],[9] These FAEE accumulate in the fetus and can be identified in the meconium, the first bowel movement of the infant.[1] Although this method is reliable, detection can only occur during later periods of pregnancy, at which the application of preventive measures is less effective. [4]

Ultrasound identification of developmental aberrations

An ultrasound preformed on a pregnant woman can help support a diagnosis of FAS in fetuses that are at high probability for developing FAS. The timing of the ultrasound during the pregnancy will alter which developmental disorders are occurring. During the second trimester, structural anomalies such cardiac and renal irregularities can be detected while in the third trimester inhibited growth of the fetus can be observed.[1],[5] However, an ultrasound procedure does not meet the standard that would allow it to be completely indicative of a clinical diagnosis of FAS.[2]

Genetic factors

Although the development of FAS is by definition is dependent on the maternal consumption of alcohol, studies have shown that the genetic composition of both the developing fetus as well as the mother play an important role in modulating the teratogenic effects of ethanol.[6] A study performed by Streissguth and Dehaene (1993) showed that in twins affected by intrauterine alcohol exposure, monozygotic twins had more similar FAS symptoms than genetically different dizygotic twins. Using clinical diagnostic and cognitive test to measure the levels of FAS phenotypes between twins, the researchers concluded that genetic factors have a large impact on the expression of FAS symptoms.[7]

Alcohol dehydrogenase 1B gene

Alcohol Metabolism
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Metabolism of Ethanol to Acetate by Alcohol Dehydrogenase and Aldehyde dehydrogenase

Alcohol dehydrogenase (ADH) has been a reoccurring target for FAS research due to its role in the metabolism of ethanol into acetaldehyde. Among the different isoforms of ADH found throughout the body, different polymorphisms of each are found to dominant in particular populations.[8] The focus of many studies have been on two of the three polymorphisms of the ADH1B isoform, ADH1B*1 and ADH1B*3. The ADH1B*1 polymorphism has an arginine at position 369 and has lower enzymatic activity relative to the ADH1B*3 polymorphism, which has a cysteine at position 369.[8]

Most correlative studies have indicated that infant and maternal ADH1B*1 homozygosity are associated with an increase in birth defects while having at least one ADH1B*3 allele decreased the severity of FAS phenotypes.8 Such defects included lower birth weights, and smaller head circumference and facial measurements. However a smaller portion of studies have found results contrary to this theory, stating a maternal heterogeneous genotype for these two polymorphisms (ADH1B*1/ADH1B*3) are associated with an increased risk of developing characteristic FAS features, physical facial features and low birth weight.[8]

CYP17 gene

The cytochrome P-450c17alpha gene (CYP17) encodes for a catalytic protein required in the branching steps involved in synthesizing steroid hormones.[10],[11] Polymorphisms of this gene have been associated with an increase in the negative effects of fetal intrauterine alcohol exposure. The differences between these polymorphisms on intrauterine growth restrictions is believed to be mediated by its effect on the production of androgens.[11] Research by Delpisheh et al. (2008) has shown that in mothers that drank alcohol during pregnancy, homozygostity of the A1 polymorphism correlated with a significant decrease in intrauterine growth of the develop fetus compared with heterozygotic and A2 homozygotic mothers.

Epigenetic factors

Paternal alcohol use has been implicated with changes in epigenetic enzymatic activities which have been theorized to play a role in embryonic defects.[12] Ethanol has been hypothesised to inhibit the transcription and function of DNA methyltransferases which methylate CpG dinucleotides. These methylations are responsible for silencing specific genes.[6],[12] If this silencing mechanism were to be compromised in the imprinted regions of sperm DNA, the ethanol impaired regions could be transferred to the individual’s offspring thus affecting fetal development.[12] A study by Ouko et al. (2009) correlated an increase of alcohol consumption in men with increased demethylation of two normally methylated regions, H19 and IG-DMR in sperm cells.[6],[12] Other studies have linked hypermethylation of the H19 and IG-DMR regions of DNA to normal fetal development.[12]

Changes in Gene expression

Matrix Metalloprotein

Neuronal cell migration is an essential part of the development of the nervous system and is facilitated through the balance between matrix metalloprotease (MMP) and matrix metalloprotease inhibitor activity.[4],[14] MMP act to degrade the extracellular matrix in order for cells to be able to properly migrate and develop. Hard et al. (2005) have shown that gene expression of many genes including MMP inhibitors are down regulated in mouse models of FAS. The down-regulation of these MMP inhibitors could serves as an unbiased biomarker for the development of FAS in at-risk human fetuses.[15]

Cell-cell adhesion molecules

Intergrins and Laminins are molecules responsible for cell-cell adhesion, a process by which cells recognize and interact with nearby cells to form complex networks. This process is important to the development and function of the nervous system because of its role in neuron migration, and the formation of synapses, axon bundles, and networks of gial cells.[16] Research from Vangipuram et al. (2008) showed that ethanol exposure had an effect on the expression of cell-cell adhesion molecules in human embryonic brain tissue in vitro. Specifically, α-laminin-1, β-laminins, phosphoprotins1, collagen type 4 α-2, sarclycan epsilon and β integrins 3 and 5 were all found to be up-regulated in the alcohol-exposed cells. Monitoring the regulation of these genes in infants can be a potential way to detect the development of FAS.[4]

Molecular Biomarkers

Retinoic Acid rescue of Ethanol induced
symptoms of FAS in zebra fish
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Development of the forebrain, hind brain, eyes and hatching
glands become hindered in ethanol treated Zebrafish embryos.
Recovery of the development of these structures can be observed in
the addition of retinoic acid to ethanol treatments.
Marrs et al. (2010)

Alpha fetoprotein

There is a complex array of proteins found in the amniotic fluid during pregnancy which can be used to identify abnormal development. One such protein is Alpha Fetoprotein, which plays an important role in binding and delivering of polyunsaturated fatty acids from the maternal circulation to fetal tissue. It is also involved in the development of the nervous system.[19]

The use of alpha fetoprotein measurements within the amniotic fluid would be a promising technique to detect FAS as other defects such as neural tube defects, tumours, and Down’s syndrome are currently detected through alpha fetoprotein levels found in the amniotic fluid.[19], [20]

Retinoic acid

The biosynthesis of retinoic acid from retinoids plays an important role in apoptosis, differentiation, growth and development of cells.[17], [18] In particular, the development of nervous system through primary neurogenesis, hindbrain and anterior spinal cord development is facilitated by retinoid and retinoic acid signalling pathways.[17] This pathway has also been implicated in activities in the adult brain such as learning and motor functions. Synaptic plasticity, which measured through long term potentiation (LTP) and long term depression (LTD), is dependent on retinoic acid signalling due to its role in synaptic remodelling, neurogenesis and synaptic turnover.[17]

Inhibition of the retinoic acid production in pregnant women by ethanol consumption may be a possible biomarker for FAS diagnosis. Marrs et al. (2010) was the first to demonstrate retinoic acid recovery of normal phenotype from ethanol induced FAS symptoms in zebrafish. In their experiment, ethanol was introduced during gastrulation phase of embryonic development and another treatment group was simultaneously treated with retinoic acid. Reductions in the development of the swim bladder, the forebrain and the hatching glands were used to quantify FAS symptoms.[18] A last treatment group of retinoic acid in the absence of ethanol exposure showed similar embryonic development as the control and retinoic acid + ethanol treatment. This last result supports the notion that ethanol is acting on the retinoic acid pathway. Since the retinoic acid treatment did not cause an increase in growth unless ethanol is also present suggests that retinoic acid is being supplemented in the developing embryo with ethanol and that retinoic acid increases growth up to normal development.[18]

The retinoic acid signalling pathway and ethanol metabolism are also linked in that the oxidative enzyme ADH involved in ethanol metabolism is also involved in the conversion of retinal to retinoic acid. One theory suggests that ethanol acts as a competitive antagonist to retinal-ADH interactions which inhibit the retinoic acid pathway.[8], [18]

Quantification of swim bladders in Zebrafish Model of FAS
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The swim bladders of control, ethanol, ethanol+retinoic acid, and retinoic acid treatment were measured.
Marrs et al. (2010) found significant recovery of normal development in ethanol + retinoic acid treatment.
1. Pruett, D., Hubbard Waterman, E., Caughey, A.B. (2013). Fetal Alcohol Exposure: Consequences, Diagnosis, and Treatment. Obstetrical and Gynecological survey, 68(1), 62-69
2. Landgraf, M.N., Nothacker, M., Kopp, I.B., & Heinen, F. (2013). The Diagnosis of Fetal Alcohol syndrome. Dtsch Arztebl Int, 110(42), 703-10
3. Landgraf, M.N., Nothacker, M., Kopp, I.B., & Heinen, F. (2013). The Diagnosis of Fetal Alcohol syndrome. Dtsch Arztebl Int, 110(42), 703-10.
4. Ismail, S., Buckley, S., Budacki, R., Jabbar, A., & Gallicano, G.I. (2010). Screening, Diagnosing and Prevention of Fetal Alcohol Syndrome: Is This Syndrome Treatable? Dev Neurosci, 32, 91-100.
5. Cook, J.D. (2003). Biochemical markers of alcohol use in pregnant women. Clinical Biochemistry, 36, 9-19.
6. Ornoy, A., & Ergaz, Z., (2010). Alcohol Abuse in Pregnant Women: Effects on the Fetus and Newborn, Mode of Action and Maternal Treatment. Int. J. Environ. Res. Public Health, 7, 364-379.
7. Streissguth, A.P., & Dehaene, P. (1993) Fetal Alcohol Syndrome in twins of alcoholic mothers: concordance of diagnosis and IQ. Am J Med genet, 47, 857-861.
8. Green, R.F. & Stoler, J.M. (2007). Alcohol dehydrogenase 1B genotype and fetal alcohol syndrome: a HuGE minireview. Am J Obstet Gynecol, 197(1), 12-25.
9. Gorski, N. P., Hufford, C., Weiss, R. D., Lewandrowski, K. B., Villa, E. M., Dombkowski, D. M., et al. (1996). Reduced Fatty Acid Ethyl Ester Synthase Activity in the White Blood Cells of Alcoholics. Alcoholism: Clinical and Experimental Research, 20(2), 268-274.
10. Sharp, L., Cardy, A., Cotton, S., & Little, J. (2004). CYP17 gene polymorphisms: prevalence and associations with hormone levels and related factors. a HuGE review. Am J Epidemiol, 16(8), 729-40.
11. Delpisheh, A., Topping, J., Reyad, M., Tang, A., & Brabin, B. J. (2008). Prenatal alcohol exposure, CYP17 gene polymorphisms and fetal growth restriction. European Journal of Obstetrics & Gynecology and Reproductive Biology,138(1), 49-53.
12. Ouko, L. A., Shantikumar, K., Knezovich, J., haycock, P., Schnugh, D. J., & Ramsay, M. (2009). Effect of Alcohol Consumption on CpG Methylation in the Differentially Methylated Regions of H19 and IG-DMR in Male Gametes—Implications for Fetal Alcohol Spectrum Disorders. Alcoholism: Clinical and Experimental Research, 33(9), 1615-1627.
13. Michael, S. K., & Weinberg, J. (2011). FOCUS ON: EPIGENETICS AND FETAL ALCOHOL SPECTRUM DISORDERS. Alcohol Research and Health, 34(1), 29-37.
14. Kleinerjr, D., & Stetlerstevenson, W. (1993). Structural biochemistry and activation of matrix metalloproteases.Current Opinion in Cell Biology, 5(5), 891-897.
15. Hard, M. L., Abdolell, M., Robinson, B. H., & Koren, G. (2005). Gene-expression analysis after alcohol exposure in the developing mouse. J Lab Clin Med, 145(1), 47-54.
16. Togashi, H., Sakisaka, T., & Takai, Y. (2009). Cell adhesion molecules in the central nervous system. Cell Adhesion & Migration, 3(1), 29-35.
17. Das, B. C., Ray, S. K., Karki, R., Thapa, P., Evans, T., Verma, A., et al. (2014). Retinoic Acid Signaling Pathways in Development and Diseases. Bioorganic & Medicinal Chemistry, 22, 673-683.
18. Marrs, J. A., Clendenon, S. G., Ratcliffe, D. R., Fielding, S. M., Liu, Q., & Bosron, W. F. (2010). Zebrafish fetal alcohol syndrome model: effects of ethanol are rescued by retinoic acid supplement.Alcohol, 44, 707-715.
19. Panova, I. G., Tatikolov, A. S., Poltavtseva, R. A., & Sukhikh, G. T. (2011). α-Fetoprotein in Human Fetal Vitreous Body. Bulletin of Experimental Biology and Medicine, 150(4), 420-421.
20. Datta, S., Turner, D., Singh, R., Ruest, L. B., Pierce, W. M., & Knudsen, T. B. (2008). Fetal alcohol syndrome (FAS) in C57BL/6 mice detected through proteomics screening of the amniotic fluid. Birth Defects Research Part A: Clinical and Molecular Teratology, 82(4), 177-186.

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