Rapid diagnosis of trisomy 18 of maternal origin by quantitative fluorescent polymerase chain reaction analysis following tissue culture failure for conventional cytogenetic analysis in a fetus with holoprosencephaly, ventricular septal defect, arthrogryposis of bilateral wrists and aplasia of the thumbs.

OBJECTIVE: We present rapid diagnosis of trisomy 18 of maternal origin by quantitative fluorescent polymerase chain reaction (QF-PCR) analysis following tissue culture failure for conventional cytogenetic analysis in a fetus with holoprosencephaly (HPE), ventricular septal defect (VSD), arthrogryposis of bilateral wrists and aplasia of the thumbs. CASE REPORT: A 22-year-old, primigravid woman was referred for first-trimester ultrasound screening at 13 weeks of gestation, and the fetus was found to have HPE and VSD. The pregnancy was subsequently terminated at 14 weeks of gestation, and a malformed fetus was delivered with cebocephaly, arthrogryposis of bilateral wrists and aplasia of the thumbs. The umbilical cord and placental tissues were collected for genetic analysis. However, tissue culture failure for conventional cytogenetic analysis occurred because of contamination. QF-PCR analysis using the polymorphic DNA markers of D18S1369 (18q12.2) and D18S1361 (18q22.3) confirmed trisomy 18 of maternal origin. CONCLUSION: QF-PCR analysis is useful for rapid confirmation of trisomy 18 and the parental origin when tissue culture failure for conventional cytogenetic analysis occurs in pregnancy suspicious of fetal trisomy 18.

Cilia, ciliopathies and hedgehog-related forebrain developmental disorders.

Development of the forebrain critically depends on the Sonic Hedgehog (Shh) signaling pathway, as illustrated in humans by the frequent perturbation of this pathway in holoprosencephaly, a condition defined as a defect in the formation of midline structures of the forebrain and face. The Shh pathway requires functional primary cilia, microtubule-based organelles present on virtually every cell and acting as cellular antennae to receive and transduce diverse chemical, mechanical or light signals. The dysfunction of cilia in humans leads to inherited diseases called ciliopathies, which often affect many organs and show diverse manifestations including forebrain malformations for the most severe forms. The purpose of this review is to provide the reader with a framework to understand the developmental origin of the forebrain defects observed in severe ciliopathies with respect to perturbations of the Shh pathway. We propose that many of these defects can be interpreted as an imbalance in the ratio of activator to repressor forms of the Gli transcription factors, which are effectors of the Shh pathway. We also discuss the complexity of ciliopathies and their relationships with forebrain disorders such as holoprosencephaly or malformations of cortical development, and emphasize the need for a closer examination of forebrain defects in ciliopathies, not only through the lens of animal models but also taking advantage of the increasing potential of the research on human tissues and organoids.

Cellular identities in an unusual presentation of cyclopia in a chick embryo.

Cyclopia is a congenital anomaly characterized by the presence of a single or partially divided eye in a single orbit at the body midline. This condition is usually associated with other severe facial malformations, such as the absence of the nose and, on rare occasions, the presence of a proboscis located above the ocular structures. The developmental origin of cyclopia in vertebrates is the failure of the embryonic prosencephalon to divide properly during the formation of the two bilateral eyes. Although the developmental origin of the cyclopia-associated proboscis is not clear, it has been suggested that this unique structure results from the disrupted morphogenesis of the olfactory placodes, the main organizers of the developing nose. In this study, we report a spontaneous congenital case of cyclopia with a proboscis-like appendage in a chick embryo. By means of both conventional histology and immunohistochemical methods, we have analyzed this anomaly in detail to suggest an alternative identity for the anatomical embryonic features of cyclopic vertebrate embryos displaying a proboscis. Our findings are discussed in the context of previously reported cases of cyclopia, and provide additional insight into this complex congenital malformation.

Fetal brain MRI findings and neonatal outcome of common diagnosis at a tertiary care center.

Fetal Magnetic Resonance Imaging (MRI) is increasingly used in prenatal evaluations. OBJECTIVE: Identify common brain malformations on fetal MRI and evaluate perinatal course. METHODS: Fetal consultations from 10/2016 to 12/2017 reviewed. RESULTS: Hundred consultations were requested; 94 were completed. Findings included: posterior fossa malformations (19%), agenesis/dysgenesis of corpus callosum (15%), congenital aqueductal stenosis (CAS) (14%), ventriculomegaly (11%), isolated cortical malformations (8.5%), and holoprosencephaly (6%). Posterior fossa malformations were more likely to be associated with genetic conditions and cardiac malformations. Patients with CAS all required intensive care unit admission. Overall, few patients with congenital brain malformations required feeding or respiratory support at discharge. None had seizures as neonates except two with early epileptic encephalopathy syndromes. CONCLUSIONS: Even though long term neurological prognosis is poor for many conditions including high lifetime risk of epilepsy, most are discharged with no feeding or respiratory support. Seizures are rarely seen in the neonatal period.

The wide spectrum of ultrasound diagnosis of holoprosencephaly.

AIM: Holoprosencephaly (HPE) is the most common brain malformation. A wide spectrum of anatomical variants are characterized by a lack of midline separation of the cerebral hemispheres. The aim of this study was to assess the ultrasound diagnostic criteria for HPE. MATERIAL AND METHOD: A database of 175 fetuses with central nervous system anomalies identified by ultrasound was collected retrospectively from 2006 to 2016 in this multicenter, retrospective, observational study. Among them 18 cases (10.2%) with HPE were identified. RESULTS: The prevalence of HPE was 2.5:10.000 with the sex distributionmale:female of 1:1.6. Six cases were alobar subtype, 3 were semilobar, 7 were lobar and 2 were middle interhemispheric variant. In the second trimester, we consider that the abnormal fusion of the lateral ventricles and the absence of the cavum septum pellucidum are the most important landmarks for HPE. Facial abnormalities varied considerably. CONCLUSION: This study illustrates the heterogeneity of HPE with different cerebral and facial appearances.

Holoprosencephaly from conception to adulthood.

Holoprosencephaly (HPE) consists of a spectrum of malformations related to incomplete separation of the prosencephalon. There is a wide clinical variability depending on the HPE subtype seen on imaging. Early postnatal lethality is common, however a significant fraction of newborns diagnosed with HPE will survive into childhood and even adulthood. Here we will review the clinical management of HPE during different ages from the prenatal period to adulthood.

Holoprosencephaly in the genomics era.

Holoprosencephaly (HPE) is the direct consequence of specific genetic and/or environmental insults interrupting the midline specification of the nascent forebrain. Such disturbances can lead to a broad range of phenotypic consequences for the brain and face in humans. This malformation sequence is remarkably common in utero (1 in 250 human fetuses), but 97% typically do not survive to birth. The precise molecular pathogenesis of HPE in these early human embryos remains largely unknown. Here, we outline our current understanding of the principal driving factors leading to HPE pathologies and elaborate our multifactorial integrated genomics approach. Overall, our understanding of the pathogenesis continues to become simpler, rather than more complicated. Genomic technologies now provide unprecedented insight into disease-associated variation, including the overall extent of genetic interactions (coding and noncoding) predicted to explain divergent phenotypes.

ZIC2 in Holoprosencephaly.

The ZIC2 transcription factor is one of the most commonly mutated genes in Holoprosencephaly (HPE) probands. HPE is a severe congenital defect of forebrain development which occurs when the cerebral hemispheres fail to separate during the early stages of organogenesis and is typically associated with mispatterning of the embryonic midline. Recent study of genotype-phenotype correlations in HPE cases has defined distinctive features of ZIC2-associated HPE presentation and genetics, revealing that ZIC2 mutation does not produce the craniofacial abnormalities generally thought to characterise HPE but leads to a range of non-forebrain phenotypes. Furthermore, the studies confirm the extent of ZIC2 allelic heterogeneity and that pathogenic variants of ZIC2 are associated with both classic and middle interhemispheric variant (MIHV) HPE which arise from defective ventral and dorsal forebrain patterning, respectively. An allelic series of mouse mutants has helped to delineate the cellular and molecular mechanisms by which one gene leads to defects in these related but distinct embryological processes.

Prenatal diagnosis of holoprosencephaly associated with Smith-Lemli-Opitz syndrome (SLOS) in a 46,XX fetus.

OBJECTIVE: To show the importance of measuring cholesterol precursor levels in amniotic fluid in all pregnancies with ultrasound features (such as holoprosencephaly) suggestive of Smith-Lemli-Opitz syndrome (SLOS), after exclusion of chromosomal anomalies. CASE REPORT: A 28-year-old woman, gravida 1 para 0, performed chorionic villus sampling for fetal karyotyping at 13 weeks of gestation due to positive combined first trimester screening in a fetus with increased nuchal translucency and suspected holoprosencephaly. The result was normal - 46,XX. The diagnosis of alobar holoprosencephaly was confirmed at 15 weeks of gestation, and cardiac and limb defects were also identified. Thus, a syndromic cause was considered, specifically a chromosomal microdeletion syndrome or a monogenic entity such as SLOS. The latter was confirmed by measuring 7-dehydrocholesterol (7DHC) and 8-dehydrocholesterol (8DHC) in amniotic fluid. Molecular analysis of DHCR7 gene identified a homozygous mutation in intron 8, c.964-1G>C, providing molecular confirmation for this diagnosis. CONCLUSION: The differential diagnosis of holoprosencephaly is broad. Identification of the cause of holoprosencephaly aids in establishing the prognosis and is essential to ascertain the mode of inheritance for adequate genetic counseling.

Gli2 gene-environment interactions contribute to the etiological complexity of holoprosencephaly: evidence from a mouse model.

Holoprosencephaly (HPE) is a common and severe human developmental abnormality marked by malformations of the forebrain and face. Although several genetic mutations have been linked to HPE, phenotypic outcomes range dramatically, and most cases cannot be attributed to a specific cause. Gene-environment interaction has been invoked as a premise to explain the etiological complexity of HPE, but identification of interacting factors has been extremely limited. Here, we demonstrate that mutations in Gli2, which encodes a Hedgehog pathway transcription factor, can cause or predispose to HPE depending upon gene dosage. On the C57BL/6J background, homozygous GLI2 loss of function results in the characteristic brain and facial features seen in severe human HPE, including midfacial hypoplasia, hypotelorism and medial forebrain deficiency with loss of ventral neurospecification. Although normally indistinguishable from wild-type littermates, we demonstrate that mice with single-allele Gli2 mutations exhibit increased penetrance and severity of HPE in response to low-dose teratogen exposure. This genetic predisposition is associated with a Gli2 dosage-dependent attenuation of Hedgehog ligand responsiveness at the cellular level. In addition to revealing a causative role for GLI2 in HPE genesis, these studies demonstrate a mechanism by which normally silent genetic and environmental factors can interact to produce severe outcomes. Taken together, these findings provide a framework for the understanding of the extreme phenotypic variability observed in humans carrying GLI2 mutations and a paradigm for reducing the incidence of this morbid birth defect.

Hedgehog receptor function during craniofacial development.

The Hedgehog signalling pathway plays a fundamental role in orchestrating normal craniofacial development in vertebrates. In particular, Sonic hedgehog (Shh) is produced in three key domains during the early formation of the head; neuroectoderm of the ventral forebrain, facial ectoderm and the pharyngeal endoderm; with signal transduction evident in both ectodermal and mesenchymal tissue compartments. Shh signalling from the prechordal plate and ventral midline of the diencephalon is required for appropriate division of the eyefield and forebrain, with mutation in a number of pathway components associated with Holoprosencephaly, a clinically heterogeneous developmental defect characterized by a failure of the early forebrain vesicle to divide into distinct halves. In addition, signalling from the pharyngeal endoderm and facial ectoderm plays an essential role during development of the face, influencing cranial neural crest cells that migrate into the early facial processes. In recent years, the complexity of Shh signalling has been highlighted by the identification of multiple novel proteins that are involved in regulating both the release and reception of this protein. Here, we review the contributions of Shh signalling during early craniofacial development, focusing on Hedgehog receptor function and describing the consequences of disruption for inherited anomalies of this region in both mouse models and human populations.

The chick embryo as a model for the effects of prenatal exposure to alcohol on craniofacial development.

Prenatal exposure to ethanol results in fetal alcohol spectrum disorder (FASD), a syndrome characterised by a broad range of clinical manifestations including craniofacial dysmorphologies and neurological defects. The characterisation of the mechanisms by which ethanol exerts its teratogenic effects is difficult due to the pleiotropic nature of its actions. Different experimental model systems have been employed to investigate the aetiology of FASD. Here, I will review studies using these different model organisms that have helped to elucidate how ethanol causes the craniofacial abnormalities characteristic of FASD. In these studies, ethanol was found to impair the prechordal plate-an important embryonic signalling centre-during gastrulation and to negatively affect the induction, migration and survival of the neural crest, a cell population that generates the cartilage and most of the bones of the skull. At the cellular level, ethanol appears to inhibit Sonic hedgehog signalling, alter levels of retionoic acid activity, trigger a Ca(2+)-CamKII-dependent pathway that antagonises WNT signalling, affect cytoskeletal dynamics and increase oxidative stress. Embryos of the domestic chick Gallus gallus domesticus have played a central role in developing a working model for the effects of ethanol on craniofacial development because they are easily accessible and because key steps in craniofacial development are particularly well established in the avian embryo. I will finish this review by highlighting some potential future avenues of fetal alcohol research.

The human brain and face: mechanisms of cranial, neurological and facial development revealed through malformations of holoprosencephaly, cyclopia and aberrations in chromosome 18.

The study of inborn genetic errors can lend insight into mechanisms of normal human development and congenital malformations. Here, we present the first detailed comparison of cranial and neuro pathology in two exceedingly rare human individuals with cyclopia and alobar holoprosencephaly (HPE) in the presence and absence of aberrant chromosome 18 (aCh18). The aCh18 fetus contained one normal Ch18 and one with a pseudo-isodicentric duplication of chromosome 18q and partial deletion of 18p from 18p11.31 where the HPE gene, TGIF, resides, to the p terminus. In addition to synophthalmia, the aCh18 cyclopic malformations included a failure of induction of most of the telencephalon - closely approximating anencephaly, unchecked development of brain stem structures, near absence of the sphenoid bone and a malformed neurocranium and viscerocranium that constitute the median face. Although there was complete erasure of the olfactory and superior nasal structures, rudiments of nasal structures derived from the maxillary bone were evident, but with absent pharyngeal structures. The second non-aCh18 cyclopic fetus was initially classified as a true Cyclops, as it appeared to have a proboscis and one median eye with a single iris, but further analysis revealed two eye globes as expected for synophthalmic cyclopia. Furthermore, the proboscis was associated with the medial ethmoid ridge, consistent with an incomplete induction of these nasal structures, even as the nasal septum and paranasal sinuses were apparently developed. An important conclusion of this study is that it is the brain that predicts the overall configuration of the face, due to its influence on the development of surrounding skeletal structures. The present data using a combination of macroscopic, computed tomography (CT) and magnetic resonance imaging (MRI) techniques provide an unparalleled analysis on the extent of the effects of median defects, and insight into normal development and patterning of the brain, face and their skeletal support.

Holoprosencephaly: signaling interactions between the brain and the face, the environment and the genes, and the phenotypic variability in animal models and humans.

Holoprosencephaly (HPE) is the most common developmental defect of the forebrain characterized by inadequate or absent midline division of the forebrain into cerebral hemispheres, with concomitant midline facial defects in the majority of cases. Understanding the pathogenesis of HPE requires knowledge of the relationship between the developing brain and the facial structures during embryogenesis. A number of signaling pathways control and coordinate the development of the brain and face, including Sonic hedgehog, Bone morphogenetic protein, Fibroblast growth factor, and Nodal signaling. Mutations in these pathways have been identified in animal models of HPE and human patients. Because of incomplete penetrance and variable expressivity of HPE, patients carrying defined mutations may not manifest the disease at all, or have a spectrum of defects. It is currently unknown what drives manifestation of HPE in genetically at-risk individuals, but it has been speculated that other gene mutations and environmental factors may combine as cumulative insults. HPE can be diagnosed in utero by a high-resolution prenatal ultrasound or a fetal magnetic resonance imaging, sometimes in combination with molecular testing from chorionic villi or amniotic fluid sampling. Currently, there are no effective preventive methods for HPE. Better understanding of the mechanisms of gene-environment interactions in HPE would provide avenues for such interventions.

The ubiquitin E3 ligase NOSIP modulates protein phosphatase 2A activity in craniofacial development.

Holoprosencephaly is a common developmental disorder in humans characterised by incomplete brain hemisphere separation and midface anomalies. The etiology of holoprosencephaly is heterogeneous with environmental and genetic causes, but for a majority of holoprosencephaly cases the genes associated with the pathogenesis could not be identified so far. Here we report the generation of knockout mice for the ubiquitin E3 ligase NOSIP. The loss of NOSIP in mice causes holoprosencephaly and facial anomalies including cleft lip/palate, cyclopia and facial midline clefting. By a mass spectrometry based protein interaction screen we identified NOSIP as a novel interaction partner of protein phosphatase PP2A. NOSIP mediates the monoubiquitination of the PP2A catalytic subunit and the loss of NOSIP results in an increase in PP2A activity in craniofacial tissue in NOSIP knockout mice. We conclude, that NOSIP is a critical modulator of brain and craniofacial development in mice and a candidate gene for holoprosencephaly in humans.

Biparietal diameter-to-crown-rump length disproportion in first-trimester fetuses with holoprosencephaly.

OBJECTIVES: To determine whether the biparietal diameter measurement is altered in first-trimester fetuses with holoprosencephaly. METHODS: Cases of holoprosencephaly were collected retrospectively from 4 fetal medicine centers, and first-trimester biparietal diameter measurements were reviewed. The diagnosis of holoprosencephaly was established sonographically by the detection of abnormal choroid plexus morphologic characteristics (absent "butterfly" sign) and the identification of a monoventricular cerebral cavity on axial views of the fetal brain. The proportion of fetuses with biparietal diameter measurements below the 5th percentile for crown-rump length was determined. RESULTS: Among 45 cases of holoprosencephaly reviewed, 43 had information on both biparietal diameter and crown-rump length measurements. The biparietal diameter was below the 5th percentile for crown-rump length in 14 (32.6%) fetuses. Chromosomal analysis was available in 41; no statistically significant difference in biparietal diameter measurement between those with associated chromosomal anomalies and those without anomalies was noted. A supplementary analysis using head circumference measurement showed an even greater proportion of fetuses with holoprosencephaly with measurements below the 5th percentile for crown-rump length (18 of 42 [42.9%]). CONCLUSIONS: One-third of first-trimester fetuses with a sonographic diagnosis of holoprosencephaly had a biparietal diameter that was smaller than expected for crown-rump length. In this subset of fetuses, the evaluation of intracranial anatomy for signs of holoprosencephaly may be more difficult to perform due to the smaller size of the brain. Therefore, the detection of a biparietal diameter below the 5th percentile as expected from crown-rump length on the first-trimester scan may be a warning sign of holoprosencephaly and should prompt a detailed examination of the intracranial anatomy.

Biparietal diameter at 11 to 13 weeks' gestation in fetuses with holoprosencephaly.

OBJECTIVE: Studies have suggested that fetuses with holoprosencephaly have smaller head size, demonstrated as early as the first trimester. However, the majority of these cases were diagnosed in the second or third trimesters. The aim of this study was to investigate biparietal diameter (BPD) measured at 11 to 13 weeks' gestation in fetuses with holoprosencephaly. METHODS: This was a retrospective study in which BPD was measured at 11 to 13 weeks in 34 fetuses with prenatal diagnosis of holoprosencephaly and 7775 unaffected controls. BPD values were converted into multiples of the expected median (MoM) after adjustment for crown-rump length and maternal characteristics. RESULTS: The median gestational age at the BPD recording was 12.6 (interquartile range 12.3-13.0) weeks. The nuchal translucency was increased (≥3mm) in 58.8% of the cases. Aneuploidy was confirmed in 73.5% of the cases; the commonest was trisomy 13 (50.0%). BPD values at 11 to 13 weeks were below the 5(th) centile in 32.4% of cases and below the 50(th) centile in 67.6%. BPD MoM values were significantly smaller than in the control group (median: 0.98; interquartile range: 0.90-1.06 vs 1.00; 0.96-1.04 MoM, p = 0.03). CONCLUSION: Fetuses with holoprosencephaly have a smaller BPD in the first trimester. This property may be useful in early diagnosis.

Rescue of holoprosencephaly in fetal alcohol-exposed Cdon mutant mice by reduced gene dosage of Ptch1.

Holoprosencephaly (HPE) is a commonly occurring developmental defect in which midline patterning of the forebrain and midface is disrupted. Sonic hedgehog (SHH) signaling is required during multiple stages of rostroventral midline development, and heterozygous mutations in SHH pathway components are associated with HPE. However, clinical presentation of HPE is highly variable, and carriers of heterozygous mutations often lack apparent defects. It is therefore thought that such mutations must interact with more common modifiers, genetic and/or environmental. We have modeled this scenario in mice. Cdon mutant mice have a largely subthreshold defect in SHH signaling, rendering them sensitive to a wide spectrum of HPE phenotypes by additional hits that are themselves insufficient to produce HPE, including transient in utero exposure to ethanol. These variable HPE phenotypes may arise in embryos that fail to reach a threshold level of SHH signaling at a specific developmental stage. To provide evidence for this possibility, here we tested the effect of removing one copy of the negative regulator Ptch1 from Cdon(-/-) embryos and compared their response to ethanol with that of Cdon(-/-);Ptch1(+/+) embryos. Ptch1 heterozygosity decreased the penetrance of HPE in this system by >75%. The major effect of reduced Ptch1 gene dosage was on penetrance, as those Cdon(-/-);Ptch1(+/-) embryos that displayed HPE did not show major differences in phenotype from Cdon(-/-);Ptch1(+/+) embryos with ethanol-induced HPE. Our findings are consistent with the notion that even in an etiologically complex model of HPE, the level of SHH pathway activity is rate-limiting. Furthermore, the clinical outcome of an individual carrying a SHH pathway mutation will likely reflect the sum effect of both deleterious and protective modifier alleles and their interaction with non-genetic risk factors like fetal alcohol exposure.

Cyclopia: from Greek antiquity to medical genetics.

Cyclops are among the best-known monsters of Greek mythology, also mentioned in art and literature. According to the most recent scientific knowledge, the malformations caused by defective development of the anterior brain and midline mesodermal structures include cyclopia (synophthalmos), ethmocephaly, cebocephaly and arrhinencephaly. These severe forebrain lesions often are accompanied by severe systemic malformations, and affected infants rarely survive. Neither true cyclopia nor synophthalmos are compatible with life because an anomalous development of the brain is involved. Thus, it is difficult to assume that ancient Greeks drew their inspiration from an adult patient suffering from cyclopia. Cyclops appear for the first time in literature in Homer's Odyssey (8th-7th century BC) and one of them, Polyphemus, is blinded by the hero of the epic poem. The description of the creature is identical with patients suffering from cyclopia; eyes are fused and above the median eye there is a proboscis, which is the result of an abnormal development of the surface ectodermal structures covering the brain. The next literature appearance of Cyclops is at the end of 7th century BC in "Theogonia", written by Hesiodus. Another interesting description is made by Euripides in his satyr play entitled 'Cyclops' (5th century BC). In conclusion, though it is not certain whether Homer's description of Cyclops was based on his personal experience or the narration of his ancestors, there is no doubt that the ophthalmological disease, cyclopia, was named after this mythical creature.