Holoprosencephaly: the Maastricht experience.

Holoprosencephaly (HPE) is a developmental field defect with impaired cleavage of the embryonic forebrain as the cardinal feature. The prevalence is about 1 in 11.000-20.000 in live births and 1 in 250 during embryogenesis. In most cases, craniofacial abnormalities are associated and reflect in 80% of cases the degree of severity. The severity is of marked variability and ranges from cyclopia to minimal craniofacial dysmorphism, such as mild microcephaly with a single central incisor. The etiology of HPE is very heterogeneous and comprises environmental factors (e.g. maternal diabetes) and genetic causes. Approximately 50% of HPE cases are associated with a cytogenetic abnormality (the most common of which is trisomy 13) or a monogenic syndrome. Based on recurrent cytogenetic abnormalities, there are at least 12 genetic loci that likely contain genes implicated in the pathogenesis of HPE. Currently, four human HPE genes are known: SHH at 7q36, ZIC2 at 13q32, SIX3 at 2p21 and TGIF at 18p11.3. Over the past 13 years, 16 patients with HPE have been observed at the Department of Clinical Genetics at Maastricht. Some of them are briefly presented in order to emphasize the spectral nature of HPE and the etiological heterogeneity. One patient appeared to have a partial 18p deletion due to a maternal cryptic translocation t(1:18) and, in addition, a SHH mutation. The mildest affected patient presented with microcephaly and a single maxillary incisor; she had a submicroscopic 7q deletion. Finally, we propose a protocol of etiological work-up of HPE cases.     

Microform holoprosencephaly in mice that lack the Ig superfamily member Cdon

Holoprosencephaly (HPE), the most common developmental defect of the forebrain and midface, is caused by a failure to delineate the midline in these structures. Despite the identification of several HPE genes, its genetic basis is largely unknown. Furthermore, the phenotype of affected individuals is highly variable, even within pedigrees. Facial defects in HPE range from cyclopia and proboscis in severe cases to solitary median maxillary central incisor in individuals with microforms of HPE. Cdon (also known as Cdo), an Ig superfamily member, is a component of a cell surface receptor that positively regulates skeletal myogenesis. Cdon is also highly expressed in the frontonasal and maxillary processes (FNP and MXP, respectively) of the developing mouse embryo, structures that contain signaling centers that pattern the face. We report here that mice homozygous for targeted mutations of Cdon display the hallmark facial defects associated with microforms of HPE. This is the first example of a mouse mutant with this phenotype, and this finding implicates a new family of receptors in development of the facial midline and suggests a potential role for Cdon in the pathogenesis and expressivity of HPE in humans.     

The role of sonic hedgehog in normal and abnormal craniofacial morphogenesis.

There is growing evidence that implicates a role for Sonic hedgehog (SHH) in morphogenesis of the craniofacial complex. Mutations in human and murine SHH cause midline patterning defects that are manifested in the head as holoprosencephaly and cyclopia. In addition, teratogens such as jervine, which inhibit the response of tissues to SHH, also produce cyclopia. Thus, the loss of SHH signaling during early stages of neural plate patterning has a profound influence of craniofacial morphogenesis. However, the severity of these defects precludes analyses of SHH function during later stages of craniofacial development. We have used an embryonic chick system to study the role of SHH during these later stages of craniofacial development. Using a combination of surgical and molecular experiments, we show here that SHH is essential for morphogenesis of the frontonasal and maxillary processes (FNP and MXPs), which give rise to the mid- and upper face. Transient loss of SHH signaling in the embryonic face inhibits growth of the primordia and results in defects analogous to hypotelorism and cleft lip/palate, characteristics of the mild forms of holoprosencephaly. In contrast, excess SHH leads to a mediolateral widening of the FNP and a widening between the eyes, a condition known as hypertelorism. In severe cases, this widening is accompanied by facial duplications. Collectively, these experiments demonstrate that SHH has multiple and profound effects on the entire spectrum of craniofacial development, and perturbations in SHH signaling are likely to underlie a number of human craniofacial anomalies.     

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.     

Mouse Shh is required for prechordal plate maintenance during brain and craniofacial morphogenesis

In humans, holoprosencephaly (HPE) is a common birth defect characterized by the absence of midline cells from brain, facial, and oral structures. To understand the pathoetiology of HPE, we investigated the involvement of mammalian prechordal plate (PrCP) cells in HPE pathogenesis and the requirement of the secreted protein sonic hedgehog (Shh) in PrCP development. We show using rat PrCP lesion experiments and DiI labeling that PrCP cells are essential for midline development of the forebrain, foregut endoderm, and ventral cranial mesoderm in mammals. We demonstrate that PrCP cells do not develop into ventral cranial mesoderm in Shh^−/− embryos. Using Shh^−/− and chimeric embryos we show that Shh signal is required for the maintenance of PrCP cells in a non-cell autonomous manner. In addition, the hedgehog (HH)-responding cells that normally appear during PrCP development to contribute to midline tissues, do not develop in the absence of Shh signaling. This suggests that Shh protein secreted from PrCP cells induces the differentiation of HH-responding cells into midline cells. In the present study, we show that the maintenance of a viable population of PrCP cells by Shh signal is an essential process in development of the midline of the brain and craniofacial structures. These findings provide new insight into the mechanism underlying HPE pathoetiology during dynamic brain and craniofacial morphogenesis.     

Defining a holoprosencephaly locus on human chromosome 14q13 and characterization of potential candidate genes

Holoprosencephaly (HPE) is the most common developmental field defect in patterning of the human prosencephalon and associated craniofacial structures. The genetics is complex, with 12 loci defined on 11 chromosomes. We defined a locus for HPE (HPE8) on human chromosome 14q13 between markers D14S49 and AFM205XG5, by mapping deletion intervals of affected subjects with proximal chromosome 14q interstitial cytogenetic deletions. A 35-BAC contig was built by chromosome walking. By annotation of the 2.82-Mb minimal critical region, we identified 28 possible genes. Seven genes were expressed in human fetal brain: NPAS3, SNX6, C14ORF11, C14ORF10, PAX9, NKX2.1, and C14ORF19, the last an apparent gene fragment. Molecular embryology, animal modeling, and human mutation studies were reported elsewhere for PAX9 and NKX2.1. We focused on three genes, SNX6, NPAS3, and C14ORF11, as potential candidates for HPE. Genomic structure, human expression patterns, protein cellular localization, and embryonic expression patterns of orthologous murine genes were determined, showing that the three genes have properties similar to those of known HPE genes.     

Craniofacial and central nervous system malformations induced by triamcinolone acetonide in nonhuman primates: II. Craniofacial pathogenesis

This study further defines the craniofacial malformations induced by triamcinolone acetonide in the rhesus monkey. Ten timed-mated pregnant rhesus monkeys (Macaca mulatta) received intramuscular injections of 10 mg/kg TAC on days 23, 25, 27, 29, and 31 of gestation. Results of previous experiments with rhesus and bonnet monkeys and baboons indicated that specific craniofacial and brain malformations could be induced with TAC during this period of pregnancy (Hendrickx et al., '80). Stage-matched TAC-treated and control embryos (stages 17-18 and 22) and age-matched TAC-treated and control fetuses (50, 60, and 70 days gestation) were removed by hysterotomy. Stage 17-18 TAC embryos appeared grossly normal but histologic evaluation revealed a shortened anlage of the posterior cranial base. Stage 22 TAC embryos and all TAC fetuses exhibited craniofacial dysmorphia and encephalocele. The developing sphenoid was the earliest affected and most severely malformed bone. Its defects included reduced anterioposterior and transverse dimensions, reduced orbitosphenoid and alisphenoid, abnormal pituitary fossa, and reduced dorsum and tuberculum sellae. In addition, shortening of the posterior cranial base and decreased cranial base angle was a consistent finding in the treated embryos and fetuses. Decreased ossification and remodeling in the facial bones and abnormal position due to the malformed sphenoid occurred.     

Genesis of alcohol-induced craniofacial dysmorphism

The initial diagnosis of fetal alcohol syndrome (FAS) in the United States was made because of the facial features common to the first cohort of patients. This article reviews the development of an FAS mouse model whose craniofacial features are remarkably similar to those of affected humans. The model is based on short-term maternal treatment with a high dosage of ethanol at stages of pregnancy that are equivalent to Weeks 3 and 4 of human gestation. At these early stages of development, alcohol's insult to the developing face is concurrent with that to the brain, eyes, and inner ear. That facial and central nervous system defects consistent with FAS can be induced by more "realistic" alcohol dosages as illustrated with data from an oral alcohol intake mouse model in which maternal blood alcohol levels do not exceed 200 mg/dl. The ethanol-induced pathogenesis involves apoptosis that occurs within 12 hrs of alcohol exposure in selected cell populations of Day 7, 8, and 9 mouse embryos. Experimental evidence from other species also shows that apoptosis underlies ethanol-induced malformations. With knowledge of sensitive and resistant cell populations at specific developmental stages, studies designed to identify the basis for these differing cellular responses and, therefore, to determine the primary mechanisms of ethanol's teratogenesis are possible. For example, microarray comparisons of sensitive and resistant embryonic cell populations have been made, as have in situ studies of gene expression patterns in the populations of interest. Studies that illustrate agents that are effective in diminishing or exacerbating ethanol's teratogenesis have also been helpful in determining mechanisms. Among these agents are antioxidants, sonic hedgehog protein, retinoids, and the peptides SAL and NAP.     

Midline and laterality defects: left and right meet in the middle

The aim of this review is to summarize some of the recent advances in molecular embryology that help to explain the pathogenesis of holoprosencephaly (HPE), or its related malformation in model organisms, cyclopia, and laterality defects in humans, derived from detailed analysis of similar malformations in animal models. Recently, defects in several developmental pathways including those operated by the Sonic hedgehog and Nodal signaling factors have been implicated as causes of HPE or laterality defects in humans. Here we summarize the findings in animal models that indicate that both defects can be explained by mechanisms that relate to the proper development of the axial midline in vertebrates. Published 2001 John Wiley & Sons, Inc.     

Mouse Zic5 deficiency results in neural tube defects and hypoplasia of cephalic neural crest derivatives

Zic family genes encode zinc finger proteins, which are homologues of the Drosophila pair-rule gene odd-paired. In the present study, we characterized the fifth member of the mouse Zic family gene, mouse Zic5. Zic5 is located near Zic2, which is responsible for human brain malformation syndrome (holoprosencephaly, or HPE). In embryonic stages, Zic5 was expressed in dorsal part of neural tissues and limbs. Expression of Zic5 overlapped with those of other Zic genes, most closely with Zic2, but was not identical. Targeted disruption of Zic5 resulted in insufficient neural tube closure at the rostral end, similar to that seen in Zic2 mutant mice. In addition, the Zic5-deficient mice exhibited malformation of neural-crest-derived facial bones, especially the mandible, which had not been observed in other Zic family mutants. During the embryonic stages, there were delays in the development of the first branchial arch and extension of the trigeminal and facial nerves. Neural crest marker staining revealed fewer neural crest cells in the dorsal cephalic region of the mutant embryos without significant changes in their migration. When mouse Zic5 was overexpressed in Xenopus embryos, expression of a neural crest marker was enhanced. These findings suggested that Zic5 is involved in the generation of neural crest tissue in mouse development. ZIC5 is also located close to ZIC2 in humans, and deletions of 13q32, where ZIC2 is located, lead to congenital brain and digit malformations known as the "13q32 deletion syndrome". Based on both their similar expression pattern in mouse embryos and the malformations observed in Zic5-deficient mutant mice, human ZIC5 might be involved in the deletion syndrome.     

Human developmental disorders and the Sonic hedgehog pathway

Sonic hedgehog (Shh) is a morphogen that is crucial for normal development of a variety of organ systems, including the brain and spinal cord, the eye, craniofacial structures, and the limbs. Mutations in the human SHH gene and genes that encode its downstream intracellular signaling pathway cause several clinical disorders. These include holoprosencephaly (HPE, the most common anomaly of the developing forebrain), nevoid basal cell carcinoma syndrome, sporadic tumors, including basal cell carcinomas, and three distinct congenital disorders: Greig syndrome Pallister–Hall syndrome, and isolated postaxial polydactyly. These conditions caused by abnormalities in the SHH pathway demonstrate the crucial role of SHH in complex developmental processes, and molecular analyses of these disorders provide insight into the normal function of the SHH pathway in human development.     

The genomic structure, chromosome location, and analysis of the human DKK1 head inducer gene as a candidate for holoprosencephaly

Holoprosencephaly (HPE) is the most common developmental defect of the brain and face in humans. Here we report the analysis of the human ortholog of dkk-1 as a candidate gene for HPE. We determined the genomic structure of the human gene DKK1 and mapped it to chromosome 10q11.2. Functional analysis of four missense mutations identified in HPE patients revealed preserved activity in head induction assays in frogs suggesting a limited role for this gene in HPE pathogenesis.     

Structure of the human Lanosterol Synthase gene and its analysis as a candidate for holoprosencephaly (HPE1)

Holoprosencephaly (HPE) is the most common birth defect of the brain in humans. It involves various degrees of incomplete separation of the cerebrum into distinct left and right halves, and it is frequently accompanied by craniofacial anomalies. The HPE1 locus in human chromosome 21q22.3 is one of a dozen putative genetic loci implicated in causing HPE. Here, we report the complete gene structure of the human lanosterol synthase (LS) gene, which is located in this interval, and present its mutational analysis in HPE patients. We considered LS an excellent candidate HPE gene because of the requirement for cholesterol modification of the Sonic Hedgehog protein for the correct patterning activity of this HPE-associated protein. Despite extensive pedigree analysis of numerous polymorphisms, as well as complementation studies in yeast on one of the missense mutations, we find no evidence that the LS gene is in fact HPE1, implicating another gene located in this chromosomal region in HPE pathogenesis.     

Mutations in Hedgehog Acyltransferase (Hhat) Perturb Hedgehog Signaling, Resulting in Severe Acrania-Holoprosencephaly-Agnathia Craniofacial Defects

Holoprosencephaly (HPE) is a failure of the forebrain to bifurcate and is the most common structural malformation of the embryonic brain. Mutations in SHH underlie most familial (17%) cases of HPE; and, consistent with this, Shh is expressed in midline embryonic cells and tissues and their derivatives that are affected in HPE. It has long been recognized that a graded series of facial anomalies occurs within the clinical spectrum of HPE, as HPE is often found in patients together with other malformations such as acrania, anencephaly, and agnathia. However, it is not known if these phenotypes arise through a common etiology and pathogenesis. Here we demonstrate for the first time using mouse models that Hedgehog acyltransferase (Hhat) loss-of-function leads to holoprosencephaly together with acrania and agnathia, which mimics the severe condition observed in humans. Hhat is required for post-translational palmitoylation of Hedgehog (Hh) proteins; and, in the absence of Hhat, Hh secretion from producing cells is diminished. We show through downregulation of the Hh receptor Ptch1 that loss of Hhat perturbs long-range Hh signaling, which in turn disrupts Fgf, Bmp and Erk signaling. Collectively, this leads to abnormal patterning and extensive apoptosis within the craniofacial primordial, together with defects in cartilage and bone differentiation. Therefore our work shows that Hhat loss-of-function underscrores HPE; but more importantly it provides a mechanism for the co-occurrence of acrania, holoprosencephaly, and agnathia. Future genetic studies should include HHAT as a potential candidate in the etiology and pathogenesis of HPE and its associated disorders.     

High prevalence of defective human embryos at the early postimplantation period

Thirty-seven cases of human embryos at the early postimplantation period were procured after induced abortion and examined histologically. Their developmental stages ranged from Carnegie stages 6 to 11, and their standard ages ranged from 14 to 24 days after fertilization. Five cases (13.5%) were grossly abnormal, and seven (18.9%) were degenerating partially or in toto. Gross abnormalities included distorted embryonic disc, disorganized neural groove or tube, and neural tube dysraphism. The high prevalence rate of defective embryos at the early postimplantation period supports the clinical finding that a substantial proportion of human conceptions are eliminated from an early stage of pregnancy, often without the knowledge of the mother. The fate of undifferentiated pathological embryos is uncertain and remains to be determined.     

Fetal alcohol syndrome: an examination of craniofacial dysmorphology in Macaca nemestrina

The purpose of this investigation was to evaluate the craniofacial features in nonhuman primate models exposed in utero to moderate and high weekly binge doses of ethanol. While the high-dosed animal did have unusual craniofacial dysmorphology, she did not exhibit the typical facial pattern seen in human fetal alcohol syndrome. The high-dosed specimen displayed a scaphocephalic head shape secondary to synostosis of the sagittal suture. The brain of this monkey was grossly abnormal and microcephalic.     

6.5-mm human embryo with a single nasal placode: cyclopia or hypotelorism?

A macroscopic study on the missing elements in cyclopia (a single eye or closely approximated eyes with all intergrades in a single orbit) with or without proboscis and hypotelorism was performed on 12 human fetuses and 2 human fetal skulls. In addition, microscopic investigations were carried out on the orbital contents of the cyclops with a single eye without proboscis, crown-heel length (C-HL) 37 cm, and on the 6.5-mm-crown-rump length (C-RL) human embryo with a single nasal placode localized in front of two eye cups. In the embryo and in all 14 fetal cases the midfacial region was more-or-less deficient. In the two cyclopia cases without proboscis the nasal placode(s) had not developed at all during the embryonic period. In cases with proboscis, consisting of a single tube localized above both eyes, and in the hypotelorismic specimens, there could only have been a single nasal placode during development: a situation evident in the 6.5-mm-C-RL human embryo. In this holoprosencephalic embryo the single nasal placode was undulated, as if formed from two fused nasal placodes, and flanked by the prospective areas for the lateral nasal processes. Caudally, it was bordered by the maxillary processes. In view of the position of the single placode in this embryonic face, as described above, it is most likely that this is a preliminary stage of hypotelorism. Moreover, both medial nasal processes with the internasal groove in between, i.e., the interplacodal area, were missing.(ABSTRACT TRUNCATED AT 250 WORDS)     

Physical mapping of the holoprosencephaly critical region in 18p11.3.

Holoprosencephaly (HPE) is a common developmental defect that results in a spectrum of craniofacial malformations. HPE is genetically heterogeneous, some cases being associated with deletions of the short arm of chromosome 18. In order to map the putative HPE gene located on 18p (HPE4) more precisely, six patients with various cytogenetic 18p deletions and clinical features of HPE have been characterized by using a combination of somatic cell hybrid analysis and FISH. By using a set of 27 chromosome 18p-specific markers, the deletion in each patient was characterized. The HPE minimal critical region on 18p was defined on a molecular level, localizing the HPE4 gene to 18p11.3.