Mutations in PATCHED-1, the receptor for SONIC HEDGEHOG, are associated with holoprosencephaly

Holoprosencephaly (HPE) is the most commonly occurring congenital structural forebrain anomaly in humans. HPE is associated with mental retardation and craniofacial malformations. The genetic causes of HPE have recently begun to be identified, and we have previously shown that HPE can be caused by haploinsufficiency for SONIC HEDGEHOG ( SHH). We hypothesize that mutations in genes encoding other components of the SHH signaling pathway could also be associated with HPE. PATCHED-1 (PTCH), the receptor for SHH, normally acts to repress SHH signaling. This repression is relieved when SHH binds to PTCH. We analyzed PTCH as a candidate gene for HPE. Four different mutations in PTCHwere detected in five unrelated affected individuals. We predict that by enhancing the repressive activity of PTCH on the SHH pathway, these mutations cause decreased SHH signaling, and HPE results. The mutations could affect the ability of PTCH to bind SHH or perturb the intracellular interactions of PTCH with other proteins involved in SHH signaling. These findings further demonstrate the genetic heterogeneity associated with HPE, as well as showing that mutations in different components of a single signaling pathway can result in the same clinical condition.     

Cdon Mutation and Fetal Ethanol Exposure Synergize to Produce Midline Signaling Defects and Holoprosencephaly Spectrum Disorders in Mice

Holoprosencephaly (HPE) is a remarkably common congenital anomaly characterized by failure to define the midline of the forebrain and midface. HPE is associated with heterozygous mutations in Sonic hedgehog (SHH) pathway components, but clinical presentation is extremely variable and many mutation carriers are unaffected. It has been proposed that these observations are best explained by a multiple-hit model, in which the penetrance and expressivity of an HPE mutation is enhanced by a second mutation or the presence of cooperating, but otherwise silent, modifier genes. Non-genetic risk factors are also implicated in HPE, and gene–environment interactions may provide an alternative multiple-hit model to purely genetic multiple-hit models; however, there is little evidence for this contention. We report here a mouse model in which there is dramatic synergy between mutation of a bona fide HPE gene (Cdon, which encodes a SHH co-receptor) and a suspected HPE teratogen, ethanol. Loss of Cdon and in utero ethanol exposure in 129S6 mice give little or no phenotype individually, but together produce defects in early midline patterning, inhibition of SHH signaling in the developing forebrain, and a broad spectrum of HPE phenotypes. Our findings argue that ethanol is indeed a risk factor for HPE, but genetically predisposed individuals, such as those with SHH pathway mutations, may be particularly susceptible. Furthermore, gene–environment interactions are likely to be important in the multifactorial etiology of HPE.     

Heterozygous mutations in SIX3 and SHH are associated with schizencephaly and further expand the clinical spectrum of holoprosencephaly

Schizencephaly (SCH) is a clinically and etiologically heterogeneous cerebral malformation presenting as unilateral or bilateral hemispheric cleft with direct connection between the inner and outer liquor spaces. The SCH cleft is usually lined by gray matter, which appears polymicrogyric implying an associated impairment of neuronal migration. The majority of SCH patients are sporadic, but familial SCH has been described. An initial report of heterozygous mutations in the homeobox gene EMX2 could not be confirmed in 52 patients investigated in this study in agreement with two independent SCH patient cohorts published previously. SCH frequently occurs with additional cerebral malformations like hypoplasia or aplasia of the septum pellucidum or optic nerve, suggesting the involvement of genes important for the establishment of midline forebrain structures. We therefore considered holoprosencephaly (HPE)-associated genes as potential SCH candidates and report for the first time heterozygous mutations in SIX3 and SHH in a total of three unrelated patients and one fetus with SCH; one of them without obvious associated malformations of midline forebrain structures. Three of these mutations have previously been reported in independent patients with HPE. SIX3 acts directly upstream of SHH, and the SHH pathway is a key regulator of ventral forebrain patterning. Our data indicate that in a subset of patients SCH may develop as one aspect of a more complex malformation of the ventral forebrain, directly result from mutations in the SHH pathway and hence be considered as yet another feature of the broad phenotypic spectrum of holoprosencephaly.     

<Emphasis Type="Italic">Sonic hedgehog</Emphasis> mutations identified in holoprosencephaly patients can act in a dominant negative manner

Sonic hedgehog (SHH) plays an important instructional role in vertebrate development, as exemplified by the numerous developmental disorders that occur when the SHH pathway is disrupted. Mutations in the SHH gene are the most common cause of sporadic and inherited holoprosencephaly (HPE), a developmental disorder that is characterized by defective prosencephalon development. SHH HPE mutations provide a unique opportunity to better understand SHH biogenesis and signaling, and to decipher its role in the development of HPE. Here, we analyzed a panel of SHH HPE missense mutations that encode changes in the amino-terminal active domain of SHH. Our results show that SHH HPE mutations affect SHH biogenesis and signaling at multiple steps, which broadly results in low levels of protein expression, defective processing of SHH into its active form and protein with reduced activity. Additionally, we found that some inactive SHH proteins were able to modulate the activity of wt SHH in a dominant negative manner, both in vitro and in vivo. These findings show for the first time the susceptibility of SHH driven developmental processes to perturbations by low-activity forms of SHH. In conclusion, we demonstrate that SHH mutations found in HPE patients affect distinct steps of SHH biogenesis to attenuate SHH activity to different levels, and suggest that these variable levels of SHH activity might contribute to some of the phenotypic variation found in HPE patients. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. S. Singh, R. Tokhunts and V. Baubet contributed equally to this work.     

Identification of novel mutations in SHH and ZIC2 in a South American (ECLAMC) population with holoprosencephaly

Holoprosencephaly (HPE) is genetically heterogeneous with four genes, SIX3, SHH, TGIF, and ZIC2 that have been identified to date and that are altered in 12% of patients. To analyze this prevalence in a South American population-based sample (57 HPE cases in 244,511 live and still births or 1 in 4300), we performed a mutational study of these genes in 30 unrelated children (26 newborns and 4 non-newborns) with HPE being ascertained by ECLAMC (Latin American Collaborative Study of Congenital Malformations). We identified three novel mutations: two were missense mutations of the SHH gene (Cys183-->Phe; His140-->Pro); the third mutation was a 2-bp deletion in the zinc-finger region of the ZIC2 gene. These molecular results explained 8% (2/26 newborn samples) of the HPE cases in this South American population-based sample, a proportion similar to our previously published data from a collection of cases.     

Single median maxillary central incisor: new data and mutation review

BACKGROUND: Single median maxillary central incisor (SMMCI) is a rare anomaly that may occur alone or associated with other conditions, frequently as part of the holoprosencephaly (HPE) spectrum. However, it has been suggested that SMMCI alone, or associated with some midline defects, may be considered a different entity from HPE (OMIM: 147250). Families with SMMCI, without HPE cases, are difficult to counsel for the risk of HPE in future generations because the same midline defects described as part of the "SMMCI syndrome" can also be part of the HPE spectrum. METHODS: We screened five cases of SMMCI for mutations in three HPE genes, SHH, TGIF, and SIX3. RESULTS: A missense mutation c.686C>T was found in the gene SIX3 of one patient, which did not differ from the accepted 20% of known HPE gene mutations among all HPE cases. Our results and an extensive literature review of gene mutations in patients with SMMCI showed that 27/28 of them were in HPE genes: SHH (n = 21), SIX3 (n = 3), TGIF (n = 1), GLI2 (n = 1), and PTCH (n = 1), and only one in the SALL4 gene. CONCLUSIONS: The clinical findings in patients with SMMCI without HPE in families with mutations in HPE genes cannot be distinguished from the findings reported in the SMMCI syndrome. Therefore, persons with SMMCI and their relatives should be carefully investigated for related midline disorders, especially of the HPE spectrum, and all known HPE genes screened.     

Missense substitutions in the GAS1 protein present in holoprosencephaly patients reduce the affinity for its ligand,SHH

Holprosencephaly (HPE) is the most common disorder of the developing forebrain in humans, and is characterized by varying degrees of abnormal union of the cerebral hemispheres. These defects are typically co-associated with midline craniofacial anomalies. The combination of forebrain and craniofacial defects that comprise HPE can present along a broad and variable phenotypic spectrum. Both the SHH and NODAL signaling pathways play important roles in the pathogenesis of this disorder. Disruption of these pathways by chromosomal rearrangements, mutations in pathway-related genes and/or biochemical alterations are proposed to contribute to HPE in a large number of patients. Additional factors that are not yet fully delineated are also very likely to be involved in the pathogenesis and phenotypic heterogeneity of the disorder. Genetic loss of GAS1, a cell membrane receptor and positive regulator of SHH, has been demonstrated to contribute to the HPE phenotypic spectrum in animal models. We have evaluated the coding and flanking sequence of GAS1 in 394 patients who have clinical findings within the HPE phenotypic spectrum, and now report five novel missense sequence variants among five unrelated HPE probands. Finally, we tested the effect of these variants (as well as previously reported GAS1 variants) on the ability of GAS1 to bind to SHH. Here, we demonstrate that sequence variants in GAS1 can impair its physical interaction with SHH, suggesting a decrease in the SHH downstream signaling cascade as a pathogenic mechanism of disease.     

Functional characterization of sonic hedgehog mutations associated with holoprosencephaly

Mutations of the developmental gene Sonic hedgehog (SHH) and alterations of SHH signaling have been associated with holoprosencephaly (HPE), a rare disorder characterized by a large spectrum of brain and craniofacial anomalies. Based on the crystal structure of mouse N-terminal and Drosophila C-terminal hedgehog proteins, we have developed three-dimensional models of the corresponding human proteins (SHH-N, SHH-C) that have allowed us to identify within these two domains crucial regions associated with HPE missense mutations. We have further characterized the functional consequences linked to 11 of these mutations. In transfected HEK293 cells, the production of the active SHH-N fragment was dramatically impaired for eight mutants (W117R, W117G, H140P, T150R, C183F, L271P, I354T, A383T). The supernatants from these cell cultures showed no significant SHH-signaling activity in a reporter cell-based assay. Two mutants (G31R, D222N) were associated with a lower production of SHH-N and signaling activity. Finally, one mutant harboring the A226T mutation displays an activity comparable with the wild-type protein. This work demonstrates that most of the HPE-associated SHH mutations analyzed have a deleterious effect on the availability of SHH-N and its biological activity. However, because of the lack of correlation between genotype and phenotype for SHH-associated mutations, our study suggests that other factors intervene in the development of the spectrum of HPE anomalies.     

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.     

A Sonic Hedgehog Missense Mutation Associated with Holoprosencephaly Causes Defective Binding to GAS1

Holoprosencephaly (HPE) is a common birth defect predominantly affecting the forebrain and face and has been linked to mutations in the sonic hedgehog (SHH) gene. HPE is genetically heterogeneous, and clinical presentation represents a spectrum of phenotypes. We have previously shown that Gas1 encodes a cell-autonomous Hedgehog signaling enhancer. Combining cell surface binding, in vitro activity, and explant culture assays, we provide evidence that SHH contains a previously unknown unique binding surface for its interaction with GAS1 and that this surface is also important for maximal signaling activity. Within this surface, the Asn-115 residue of human SHH has been documented to associate with HPE when mutated to lysine (N115K). We provide evidence that HPE associated with this mutation can be mechanistically explained by a severely reduced binding of SHH to GAS1, and we predict a similar result if a mutation were to occur at Tyr-80. Our data should encourage future searches for mutations in GAS1 as possible modifiers contributing to the wide spectrum of HPE.Holoprosencephaly (HPE)² is a developmental defect of the brain and face estimated to affect 1 in 250 conceptuses (1). Clinical presentation represents a spectrum of phenotypes, ranging from the most severe (alobar), where embryos have cyclopia and the prosencephalon fails to divide into hemispheres, to relatively mild defects (microform HPE) such as maxillary central incisor fusion, midfacial hypoplasia and clefting, and the presence of a single nostril (2). The use of mice as a model has proven invaluable for investigating the molecular and genetic causes of HPE. We have previously reported that microform HPE develops in growth arrest-specific gene 1 (Gas1) mutant mice (3, 4). Additionally, we determined that the 37-kDa, cell surface-presented, and glycosylphosphatidylinositol-anchored GAS1 protein binds to the secreted cell-cell signaling protein Sonic hedgehog (SHH) and that it functions as a cell-autonomous enhancer of SHH signaling activity (3, 5, 6). Consistently, the Gas1 mutant phenotype is more severe when an allele of Shh is removed, supporting a genetic interaction between the two genes (3, 4). Given the strong evidence that mutations in Shh can cause HPE in mice and humans (7–11), we investigated the hypothesis that some of these mutations cause defective SHH signaling due to a failed interaction with GAS1.Here we identify specific residues on SHH that are required for maximal binding to GAS1 and show, in both cell culture and explant culture assays, that these mutant SHH proteins have decreased signaling activity due to their defective interaction with GAS1. Significantly, one of these mutations has been associated with autosomal dominant HPE in a human family (9). These results lead us to propose that human embryos carrying this mutation may develop HPE due to a failed GAS1-SHH protein interaction.     

Mutations in CDON, encoding a hedgehog receptor, result in holoprosencephaly and defective interactions with other hedgehog receptors

Holoprosencephaly (HPE), a common human congenital anomaly defined by a failure to delineate the midline of the forebrain and/or midface, is associated with diminished Sonic hedgehog (SHH)-pathway activity in development of these structures. SHH signaling is regulated by a network of ligand-binding factors, including the primary receptor PTCH1 and the putative coreceptors, CDON (also called CDO), BOC, and GAS1. Although binding of SHH to these receptors promotes pathway activity, it is not known whether interactions between these receptors are important. We report here identification of missense CDON mutations in human HPE. These mutations diminish CDON's ability to support SHH-dependent gene expression in cell-based signaling assays. The mutations occur outside the SHH-binding domain of CDON, and the encoded variant CDON proteins do not display defects in binding to SHH. In contrast, wild-type CDON associates with PTCH1 and GAS1, but the variants do so inefficiently, in a manner that parallels their activity in cell-based assays. Our findings argue that CDON must associate with both ligand and other hedgehog-receptor components, particularly PTCH1, for signaling to occur and that disruption of the latter interactions is a mechanism of HPE.     

A novel mutation of the human 7-dehydrocholesterol reductase gene reduces enzyme activity in patients with holoprosencephaly

Defects in cholesterol biosynthesis genes are recognized as a leading cause for holoprosencephaly (HPE). Previous reports suggest that mutations of human 7-dehydrocholesterol reductase (Dhcr7), which catalyzes the final step of cholesterol biosynthesis, may cause HPE [Clin. Genet. 53 (1998) 155]. To determine whether Dhcr7 mutations are involved in HPE pathogenesis, we analyzed the sequence of exon 9, which contains both a catalytic domain and a mutational hot spot. We examined 36 prematurely terminated fetuses with HPE at their gestation ages in the range from 21 to 33 weeks by single strand conformation polymorphism analysis and DNA sequencing. A novel missense mutation was identified: G344D. Dhcr7 enzyme assays using overexpressed recombinant mutant proteins revealed altered enzyme activity. Mutant G344D harbored less than 50% of enzyme activity compared with the control. Two previously reported mutations, R404C and G410S, abolished enzyme activity. These results suggest that mutation of the Dhcr7 gene is involved in HPE pathogenesis.     

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.     

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.     

Truncating loss-of-function mutations of <Emphasis Type="Italic">DISP1</Emphasis> contribute to holoprosencephaly-like microform features in humans

Defective function of the Sonic Hedgehog (SHH) signaling pathway is the most frequent alteration underlying holoprosencephaly (HPE) or its various clinical microforms. We performed an extensive mutational analysis of the entire human DISP1 gene, required for secretion of all hedgehog ligand(s) and which maps to the HPE 10 locus of human chromosome 1q41, as a HPE candidate gene. Here, we describe two independent families with truncating mutations in human DISP1 that resemble the cardinal craniofacial and neuro-developmental features of a recently described microdeletion syndrome that includes this gene; therefore, we suggest that DISP1 function contributes substantially to both of these signs in humans. While these clinical features are consistent with common HPE microforms, especially those linked to defective signaling by Sonic Hedgehog, we have insufficient evidence so far that functionally abnormal DISP1 alleles will commonly contribute to the more severe features of typical HPE.