Fetal Description of the Pancreatic Agenesis and Holoprosencephaly Syndrome Associated to a Specific CNOT1 Variant.

Holoprosencephaly (HPE) is a clinically and genetically heterogeneous disease, which can be associated with various prenatal comorbidities not always detectable on prenatal ultrasound. We report on the case of a foetus carrying a semi-lobar HPE diagnosed at ultrasound, for which a fetal autopsy and a whole exome sequencing were performed following a medical termination of pregnancy. Neuropathological examination confirmed the semi-lobar HPE and general autopsy disclosed a total pancreas agenesis. Whole exome sequencing found the CNOT1 missense c.1603C>T, p.(Arg535Cys), occurring de novo in the foetus. The same variant was previously reported in 5 unrelated children. All individuals had HPE, and 4 out of 5 presented endo- and exocrine pancreatic insufficiency or total pancreas agenesis. CNOT1 encodes a subunit of the CCRN4-NOT complex, expressed at the early stage of embryonic development. This report is the first fetal description of the phenotype associating HPE and pancreatic agenesis linked to the recurrent CNOT1 missense c.1603C>T, p.(Arg535Cys). This finding strengthens the hypothesis of a specific recurrent variant associated with a particular phenotype of HPE and pancreas agenesis. The fetal autopsy that revealed the pancreas agenesis was crucial in guiding the genetic diagnosis and enabling accurate genetic counselling.

Patterns of congenital anomalies among individuals with trisomy 13 in Texas.

Few population-based studies have analyzed patterns of co-occurring birth defects among those with trisomy 13. We evaluated the frequency of all possible combinations of any one, two, three, or four additional co-occurring birth defects among 736 individuals with trisomy 13 using data from the Texas Birth Defects Registry for deliveries during 1999-2014. We calculated the observed-to-expected ratio for each combination, adjusting for the known tendency for birth defects to cluster non-specifically. To address potential ascertainment differences among live births and non-live births, we repeated analyses specifically among live births. The combination of defects with the largest observed-to-expected ratio was microcephalus, reduction deformities of brain (e.g., holoprosencephaly), anomalies of nose, and polydactyly. As expected, most of the highest 30 observed-to-expected ratios involved combinations with documented features of trisomy 13, including defects of the scalp (e.g., aplasia cutis) and heart. Results were similar among sensitivity analyses restricted to live births. Our findings may help further delineate the phenotypic spectrum for trisomy 13 and may inform future research related to improving screening and counseling for the condition.

The hedgehog co-receptor BOC differentially regulates SHH signaling during craniofacial development.

The Hedgehog (HH) pathway controls multiple aspects of craniofacial development. HH ligands signal through the canonical receptor PTCH1, and three co-receptors: GAS1, CDON and BOC. Together, these co-receptors are required during embryogenesis to mediate proper HH signaling. Here, we investigated the individual and combined contributions of GAS1, CDON and BOC to HH-dependent mammalian craniofacial development. Notably, individual deletion of either Gas1 or Cdon results in variable holoprosencephaly phenotypes in mice, even on a congenic background. In contrast, we find that Boc deletion results in facial widening that correlates with increased HH target gene expression. In addition, Boc deletion in a Gas1 null background partially ameliorates the craniofacial defects observed in Gas1 single mutants; a phenotype that persists over developmental time, resulting in significant improvements to a subset of craniofacial structures. This contrasts with HH-dependent phenotypes in other tissues that significantly worsen following combined deletion of Gas1 and Boc Together, these data indicate that BOC acts as a multi-functional regulator of HH signaling during craniofacial development, alternately promoting or restraining HH pathway activity in a tissue-specific fashion.

Rare hypomorphic human variation in the heptahelical domain of SMO contributes to holoprosencephaly phenotypes.

Holoprosencephaly (HPE) is the most common congenital anomaly affecting the forebrain and face in humans and occurs as frequently as 1:250 conceptions or 1:10,000 livebirths. Sonic Hedgehog signaling molecule is one of the best characterized HPE genes that plays crucial roles in numerous developmental processes including midline neural patterning and craniofacial development. The Frizzled class G-protein coupled receptor Smoothened (SMO), whose signaling activity is tightly regulated, is the sole obligate transducer of Hedgehog-related signals. However, except for previous reports of somatic oncogenic driver mutations in human cancers (or mosaic tumors in rare syndromes), any potential disease-related role of SMO genetic variation in humans is largely unknown. To our knowledge, ours is the first report of a human hypomorphic variant revealed by functional testing of seven distinct nonsynonymous SMO variants derived from HPE molecular and clinical data. Here we describe several zebrafish bioassays developed and guided by a systems biology analysis. This analysis strategy, and detection of hypomorphic variation in human SMO, demonstrates the necessity of integrating the genomic variant findings in HPE probands with other components of the Hedgehog gene regulatory network in overall medical interpretations.

Cdon mutation and fetal alcohol converge on Nodal signaling in a mouse model of holoprosencephaly.

Holoprosencephaly (HPE), a defect in midline patterning of the forebrain and midface, arises ~1 in 250 conceptions. It is associated with predisposing mutations in the Nodal and Hedgehog (HH) pathways, with penetrance and expressivity graded by genetic and environmental modifiers, via poorly understood mechanisms. CDON is a multifunctional co-receptor, including for the HH pathway. In mice, Cdon mutation synergizes with fetal alcohol exposure, producing HPE phenotypes closely resembling those seen in humans. We report here that, unexpectedly, Nodal signaling is a major point of synergistic interaction between Cdon mutation and fetal alcohol. Window-of-sensitivity, genetic, and in vitro findings are consistent with a model whereby brief exposure of Cdon mutant embryos to ethanol during gastrulation transiently and partially inhibits Nodal pathway activity, with consequent effects on midline patterning. These results illuminate mechanisms of gene-environment interaction in a multifactorial model of a common birth defect.

A common birth defect known as holoprosencephaly affects how the brain and face of a fetus develop in the womb. In many cases, the condition is so severe that the fetus dies before, or shortly after, birth. Mutations in certain genes that control how the fetus develops are associated with holoprosencephaly. For example, mutations in components of the Hedgehog and Nodal signaling pathways, which transmit information that help cells to become specialized, increase the risk that a fetus will develop holoprosencephaly. Environmental factors, such as exposure to alcohol in the womb, are also thought to contribute to this condition. A gene known as Cdon is a component of the Hedgehog signaling pathway. In 2012, a team of researchers reported that mice with a mutation in the Cdon gene exposed to alcohol in the womb develop symptoms similar to holoprosencephaly in humans. Here, Hong et al. ­ including some of the researchers involved in the previous work ­ set out to understand how Cdon and alcohol work together to cause holoprosencephaly in the mutant mice. First, the team exposed pregnant mice to alcohol at different times during gestation to find out when their young were sensitive to developing holoprosencephaly. This showed that the young mice were most sensitive in early pregnancy when the Nodal pathway was active in their growing bodies. Further experiments found that alcohol and mutations in Cdon change Nodal signaling in cells. Together, these findings demonstrate that exposure to alcohol in the womb works together with the mutant form of Cdon via the Nodal signaling pathway, rather than the Hedgehog pathway, to cause holoprosencephaly in mice. The causes of many common birth defects are complex and difficult to distinguish at the level of individual cases. The work of Hong et al. illuminates how multiple risk factors during pregnancy, which may not create any problems on their own, may work together to produce birth defects in the fetus. The findings also offer new ways to understand how exposure to alcohol in the womb affects the fetus. Ultimately, understanding how birth defects form could lead to new strategies to prevent them in the future.

A case of rare isolated agnathia and literature review.

Agnathia is a rare congenital malformation with unknown etiology characterized by absence of the mandible, microstomia, and tongue aplasia, often found to have other anomalies including holoprosencephaly. The purpose of this paper was to describe the symptoms and imaging of a case of isolated agnathia and to conduct a comprehensive literature review of reported patients with isolated agnathia. Case reports of isolated agnathia are very rare, with most infants as stillborn. We report a child's management of isolated agnathia with microstomia and tongue aplasia. A literature review was performed with focus on diagnosis, airway, and feeding management of isolated agnathia. Polyhydramnios was a common pregnancy complication reported in 25 out of the 39 patients in the case study. Five infants were stillborn, while 23 died within the neonatal period. Of the deceased infants within the neonatal period, 19 died within minutes to hours while four died within days to weeks. There are nine patients with agnathia that survived past infancy. The results of this study suggest that isolated agnathia is a rare malformation which requires a multi-disciplinary approach for airway and feeding management.

Compound heterozygous splicing CDON variants result in isolated ocular coloboma.

Ocular coloboma is caused by failure of optic fissure closure during development and recognized as part of the microphthalmia, anophthalmia, and coloboma (MAC) spectrum. While many genes are known to cause colobomatous microphthalmia, relatively few have been reported in coloboma with normal eye size. Genetic analysis including trio exome sequencing and Sanger sequencing was undertaken in a family with two siblings affected with bilateral coloboma of the iris, retina, and choroid. Pathogenic variants in MAC genes were excluded. Trio analysis identified compound heterozygous donor splice site variants in CDON, a cell-surface receptor known to function in the Sonic Hedgehog pathway, c.928 + 1G > A and c.2650 + 1G > T, in both affected individuals. Heterozygous missense and truncating CDON variants are associated with dominant holoprosencephaly (HPE) with incomplete penetrance and Cdon-/- mice display variable HPE and coloboma. A homozygous nonsense allele of uncertain significance was recently identified in a consanguineous patient with coloboma and a second molecular diagnosis. We report the first compound heterozygous variants in CDON as a cause of isolated coloboma. CDON is the first HPE gene identified to cause recessive coloboma. Given the phenotypic overlap, further examination of HPE genes in coloboma is indicated.

Disrupted Hypothalamo-Pituitary Axis in Association With Reduced SHH Underlies the Pathogenesis of NOTCH-Deficiency.

CONTEXT: In human, Sonic hedgehog (SHH) haploinsufficiency is the predominant cause of holoprosencephaly, a structural malformation of the forebrain midline characterized by phenotypic heterogeneity and incomplete penetrance. The NOTCH signaling pathway has recently been associated with holoprosencephaly in humans, but the precise mechanism involving NOTCH signaling during early brain development remains unknown. OBJECTIVE: The aim of this study was to evaluate the relationship between SHH and NOTCH signaling to determine the mechanism by which NOTCH dysfunction could cause midline malformations of the forebrain. DESIGN: In this study, we have used a chemical inhibition approach in the chick model and a genetic approach in the mouse model. We also reported results obtained from the clinical diagnosis of a cohort composed of 141 holoprosencephaly patients. RESULTS: We demonstrated that inhibition of NOTCH signaling in chick embryos as well as in mouse embryos induced a specific downregulation of SHH in the anterior hypothalamus. Our data in the mouse also revealed that the pituitary gland was the most sensitive tissue to Shh insufficiency and that haploinsufficiency of the SHH and NOTCH signaling pathways synergized to produce a malformed pituitary gland. Analysis of a large holoprosencephaly cohort revealed that some patients possessed multiple heterozygous mutations in several regulators of both pathways. CONCLUSIONS: These results provided new insights into molecular mechanisms underlying the extreme phenotypic variability observed in human holoprosencephaly. They showed how haploinsufficiency of the SHH and NOTCH activity could contribute to specific congenital hypopituitarism that was associated with a sella turcica defect.

[Clinical features and genetic analysis of a fetus with holoprosencephaly].

OBJECTIVE: To analyze the clinical features and pathogenesis of a fetus with holoprosencephaly. METHODS: The findings of prenatal ultrasonography was reviewed. Following elective abortion, whole exome sequencing (WES) was carried out to identify potential pathogenic variant. Copy number variants (CNVs) of the abortus and its parents were detected by low-depth high-throughput sequencing. The parents were also analyzed by chromosomal karyotyping. RESULTS: Prenatal ultrasound suggested that the fetus had holoprosencephaly. WES revealed that it had approximately 33 Mb deletion at chromosome 13 involving ZIC2, a haploid dose sensitive gene. The results of low-depth high-throughput sequencing confirmed that the fetus carried a de novo 32.32 Mb deletion at 13q31.1-34. Karyotyping analysis has excluded gross chromosomal aberration in both parents. CONCLUSION: The fetus was diagnosed with holoprosencephaly, which may be attributable to the 13q31.1-34 deletion involving the ZIC2 gene.

Comorbidity of congenital heart defects and holoprosencephaly is likely genetically driven and gene-specific.

Comorbidity of holoprosencephaly (HPE) and congenital heart disease (CHD) in individuals with genetic variants in known HPE-related genes has been recurrently observed. Morphogenesis of the brain and heart from very early stages are regulated by several biological pathways, some of them involved in both heart and brain development as evidenced by genetic studies on model organisms. For instance, downregulation of Hedgehog or Nodal signaling pathways, both known as major triggers of HPE, has been shown to play a role in the pathogenesis of CHD, including structural defects and left-right asymmetry defects. In this study, individuals with various types of HPE were investigated clinically and by genomic sequencing. Cardiac phenotypes were assessed in 434 individuals with HPE who underwent targeted sequencing. CHDs were identified in 8% (n = 33) of individuals, including 10 (30%) cases of complex heart disease. Only four individuals (4/33) had damaging variants in the known HPE genes STAG2, SIX3, and SHH. Interestingly, no CHD was identified in the 37 individuals of our cohort with pathogenic variants in ZIC2. These findings suggest that CHD occurs more frequently in HPE-affected individuals with or without identifiable genetic variants, and this co-occurrence may be genetically driven and gene-specific.

Lobar holoprosencephaly with craniofacial defects in a Friesian calf: A case report.

BACKGROUND: Holoprosencephaly is a forebrain deformity that results from varying degrees of separation failure of cerebral hemispheres. The condition is classified based on the degree of non-separation of the hemispheres which, in turn, determines its severity. Holoprosencephaly is usually accompanied by craniofacial defects whose severity tends to reflect the extent of brain deformities. In humans, holoprosencephaly is one of the commonest congenital brain anomalies but in animals, reported cases are scarce. The condition has multifactorial aetiology that involves interactions between several genetic and environmental factors. CASE PRESENTATION: A 4-day-old female Friesian calf with a deformed face was reported to the Faculty of veterinary medicine and surgery, Egerton University. The calf and the dam were sired by the same bull. On clinical and radiographic examination, the calf had a short snout that curved dorsally with bilateral cleft lip, right-sided cleft jaw and a largely absent primary palate. Anatomopathological examination revealed brain deformities which included ventral fusion of frontal lobes of cerebral hemispheres, large merged lateral ventricles without septum pellucidum and fornix, hypoplastic corpus callosum, high degree of non-separation between diencephalic structures, poorly developed hippocampal formation and hypoplastic olfactory lobe, optic chiasma, and nerve. CONCLUSION: The case was confirmed as lobar holoprosencephaly based on characteristic anatomopathological findings. The aetiology of the defects in the present case could not be determined though they are thought to be either a result of recessive inheritance or exposure to teratogenic steroid alkaloids through materials fed to the dam during early pregnancy.

Holoprosencephaly in Kabuki syndrome.

Kabuki syndrome is a rare, multi-systemic disorder of chromatin regulation due to mutations in either KMT2D or KDM6A that encode a H3K4 methyltransferase and an H3K27 demethylase, respectively. The associated clinical phenotype is a direct result of temporal and spatial changes in gene expression in various tissues including the brain. Although mild to moderate intellectual disability is frequently recognized in individuals with Kabuki syndrome, the identification of brain anomalies, mostly involving the hippocampus and related structures remains an exception. Recently, the first two cases with alobar holoprosencephaly and mutations in KMT2D have been reported in the medical literature. We identified a de novo, pathogenic KMT2D variant (c.6295C > T; p.R2099X) using trio whole-exome sequencing in a 2-year-old female with lobar holoprosencephaly, microcephaly and cranio-facial features of Kabuki syndrome. This report expands the spectrum of brain anomalies associated with Kabuki syndrome underscoring the important role of histone modification for early brain development.

Loss-of-Function Variants in PPP1R12A: From Isolated Sex Reversal to Holoprosencephaly Spectrum and Urogenital Malformations.

In two independent ongoing next-generation sequencing projects for individuals with holoprosencephaly and individuals with disorders of sex development, and through international research collaboration, we identified twelve individuals with de novo loss-of-function (LoF) variants in protein phosphatase 1, regulatory subunit 12a (PPP1R12A), an important developmental gene involved in cell migration, adhesion, and morphogenesis. This gene has not been previously reported in association with human disease, and it has intolerance to LoF as illustrated by a very low observed-to-expected ratio of LoF variants in gnomAD. Of the twelve individuals, midline brain malformations were found in five, urogenital anomalies in nine, and a combination of both phenotypes in two. Other congenital anomalies identified included omphalocele, jejunal, and ileal atresia with aberrant mesenteric blood supply, and syndactyly. Six individuals had stop gain variants, five had a deletion or duplication resulting in a frameshift, and one had a canonical splice acceptor site loss. Murine and human in situ hybridization and immunostaining revealed PPP1R12A expression in the prosencephalic neural folds and protein localization in the lower urinary tract at critical periods for forebrain division and urogenital development. Based on these clinical and molecular findings, we propose the association of PPP1R12A pathogenic variants with a congenital malformations syndrome affecting the embryogenesis of the brain and genitourinary systems and including disorders of sex development.

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.

Holoprosencephaly or severe hydrocephalus: T1 sequence tells the story.

Intracranial lipoma is a relatively rare benign lesion. Many are incidental findings; however, some others may present with headache, hydrocephalus or other neurological symptoms; thus, correct diagnosis of this condition is important. These lesions are of high signal intensity on T2-weighted MRI and especially those close to cerebrospinal fluid (CSF) spaces, can easily be overlooked in the background of high signal intensity of CSF. Here, we present a case of tectal lipoma, with subsequent severe hydrocephalus and absence of septum pellucidum which was initially misinterpreted as a form of holoprosencephaly, due to inadequate attention to T1-weighted images.

A CCR4-NOT Transcription Complex, Subunit 1, CNOT1, Variant Associated with Holoprosencephaly.

Holoprosencephaly is the incomplete separation of the forebrain during embryogenesis. Both genetic and environmental etiologies have been determined for holoprosencephaly; however, a genetic etiology is not found in most cases. In this report, we present two unrelated individuals with semilobar holoprosencephaly who have the identical de novo missense variant in the gene CCR4-NOT transcription complex, subunit 1 (CNOT1). The variant (c.1603C>T [p.Arg535Cys]) is predicted to be deleterious and is not present in public databases. CNOT1 has not been previously associated with holoprosencephaly or other brain malformations. In situ hybridization analyses of mouse embryos show that Cnot1 is expressed in the prosencephalic neural folds at gestational day 8.25 during the critical period for subsequent forebrain division. Combining human and mouse data, we show that CNOT1 is associated with incomplete forebrain division.

A Specific CNOT1 Mutation Results in a Novel Syndrome of Pancreatic Agenesis and Holoprosencephaly through Impaired Pancreatic and Neurological Development.

We report a recurrent CNOT1 de novo missense mutation, GenBank: NM_016284.4; c.1603C>T (p.Arg535Cys), resulting in a syndrome of pancreatic agenesis and abnormal forebrain development in three individuals and a similar phenotype in mice. CNOT1 is a transcriptional repressor that has been suggested as being critical for maintaining embryonic stem cells in a pluripotent state. These findings suggest that CNOT1 plays a critical role in pancreatic and neurological development and describe a novel genetic syndrome of pancreatic agenesis and holoprosencephaly.

A forebrain undivided: Unleashing model organisms to solve the mysteries of holoprosencephaly.

Evolutionary conservation and experimental tractability have made animal model systems invaluable tools in our quest to understand human embryogenesis, both normal and abnormal. Standard genetic approaches, particularly useful in understanding monogenic diseases, are no longer sufficient as research attention shifts toward multifactorial outcomes. Here, we examine this progression through the lens of holoprosencephaly (HPE), a common human malformation involving incomplete forebrain division, and a classic example of an etiologically complex outcome. We relate the basic underpinning of HPE pathogenesis to critical cell-cell interactions and signaling molecules discovered through embryological and genetic approaches in multiple model organisms, and discuss the role of the mouse model in functional examination of HPE-linked genes. We then outline the most critical remaining gaps to understanding human HPE, including the conundrum of incomplete penetrance/expressivity and the role of gene-environment interactions. To tackle these challenges, we outline a strategy that leverages new and emerging technologies in multiple model systems to solve the puzzle of HPE.

The solitary median maxillary central incisor (SMMCI) syndrome: Associations, prenatal diagnosis, and outcomes.

Solitary median maxillary central incisor (SMMCI) syndrome is a complex disorder consisting of multiple, developmental defects involving midline structures of the head, which includes the cranial bones, the maxilla, and its container dentition (specifically the central incisor tooth germ), together with other midline structures of the body. SMMCI may appear as an isolated trait or in association with other midline developmental anomalies. We describe the case of a patient with SMMCI. He presented with a solitary median maxillary incisor, short stature, corpus callosum anomalies and a microform of holoprosencephaly (HPE), diabetes insipidus, and neurodevelopmental delay. The diagnosis was performed postnatally based on clinical features, radiological imaging, and a comprehensive genetic study. SMMCI can be diagnosed during the prenatal or neonatal periods or during infancy. Evaluation of the superior maxillary bone is important for prenatal diagnosis. Direct evaluation through bidimensional ultrasound or the use of multiplanar ultrasound or tridimensional reconstruction should be performed in cases of brain or face malformations. Early diagnosis can contribute to improved prenatal assessment and postnatal management.