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.     

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.     

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.     

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.     

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.     

<Emphasis Type="Italic">SONIC HEDGEHOG</Emphasis> mutations causing human holoprosencephaly impair neural patterning activity

Holoprosencephaly (HPE) is a common forebrain malformation associated with mental retardation and craniofacial anomalies. Multiple lines of evidence indicate that loss of ventral neurons is associated with HPE. The condition is etiologically heterogeneous, and abnormalities in any of several genes can cause human HPE. Among these genes, mutations in SONIC HEDGEHOG ( SHH) are the most commonly identified single gene defect causing human HPE. SHH mediates a number of processes in central nervous system development and is required for the normal induction of ventral cell types in the brain and spinal cord. Although a number of missense mutations in SHH have been identified in patients with HPE, the functional significance of these mutations has not yet been determined. We demonstrate that two SHH mutations that cause human HPE result in decreased in vivo activity of SHH in the developing nervous system. These mutant forms of SHH fail to regulate genes properly that are normally responsive to SHH signaling and do not induce ventrally expressed genes. In addition, the immunoreactivity of the mutant proteins is altered, suggesting that the conformation of the SHH protein has been disrupted. These studies are the first demonstration that mutations in SHH associated with human HPE perturb the in vivo patterning function of SHH in the developing nervous system.     

Molecular evaluation of foetuses with holoprosencephaly shows high incidence of microdeletions in the HPE genes

Holoprosencephaly (HPE), the most common structural malformation of the forebrain in humans, can be detected early during pregnancy using prenatal ultrasonography . Among foetuses with a normal karyotype, 14% have mutations in the four main HPE genes (SHH, ZIC2, SIX3 and TGIF). Genomic rearrangements have now been implicated in many genetic diseases, so we hypothesized that microdeletions in the major HPE genes may also be common in HPE foetuses with severe phenotype or other associated malformations. We screened the DNA obtained from 94 HPE foetuses with a normal karyotype for the presence of microdeletions involving the four major HPE genes (SHH, ZIC2, SIX3 and TGIF). Thirteen of the foetuses had a point mutation in one of the 4 genes and 81 had no known mutations. Quantitative multiplex PCR of short fluorescent fragments (QMPSF) analysis was used for rapid determination of HPE genes copy numbers and the identified microdeletions were confirmed by real time quantitative PCR, or fluorescent in situ hybridization (FISH) (if a cell line was available). Microdeletions were detected in 8 of 94 foetuses (8.5%) (2 in SHH, 2 in SIX3, 3 in ZIC2 and 1 in TGIF genes), and only among the 81 foetuses with a normal karyotype and no point mutations. These data suggest that microdeletions in the four main HPE genes are a common cause of prenatal HPE, as well as point mutations, and increase the total diagnosis rate close to ≈22.3% of foetuses with normal karyotype. Detection can be achieved by the QMPSF testing method that proved to be efficient for testing several genes in a single assay. Databases: SHH - OMIM: 600725; GenBank: NM_000193.2, ZIC2 - OMIM: 603073; GenBank: AF104902.1, SIX3 - OMIM: 603714; GenBank: NM_005413.1, TGIF - OMIM: 602630; GenBank: NM_003244.2, On-line Mendelian Inheritance in Man (OMIM), http://www.ncbi.nlm.nih.gov/omim/, UCSC (http://www.genome.ucsc.edu/), Ensembl (http://www.ensembl.org/), Database of genomic variants (http://projects.tcag.ca/variation/genomeView.html)     

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.     

Holoprosencephaly: clinical, anatomic, and molecular dimensions

Holoprosencephaly is addressed under the following headings: alobar, semilobar, and lobar holoprosencephaly; arrhinencephaly; agenesis of the corpus callosum; pituitary abnormalities; hindbrain abnormalities; syntelencephaly; aprosencephaly/atelencephaly; neural tube defects; facial anomalies; median cleft lip; minor facial anomalies; single maxillary central incisor; holoprosencephaly-like phenotype; epidemiology; genetic causes of holoprosencephaly; teratogenic causes of holoprosencephaly; SHH mutations; ZIC2 mutations; SIX3 mutations; TGIF mutations; PTCH mutations; GLI2 mutations; FAST1 mutations; TDGF1 mutations; and DHCR7 mutations.     

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.     

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.     

<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.     

Haploinsufficiency of Six3 fails to activate Sonic hedgehog expression in the ventral forebrain and causes holoprosencephaly

Holoprosencephaly (HPE), the most common forebrain malformation, is characterized by an incomplete separation of the cerebral hemispheres. Mutations in the homeobox gene SIX3 account for 1.3% of all cases of human HPE. Using zebrafish-based assays, we have now determined that HPE-associated Six3 mutant proteins function as hypomorphs. Haploinsufficiency of Six3 caused by deletion of one allele of Six3 or by replacement of wild-type Six3 with HPE-associated Six3 mutant alleles was sufficient to recapitulate in mouse models most of the phenotypic features of human HPE. We demonstrate that Shh is a direct target of Six3 in the rostral diencephalon ventral midline (RDVM). Reduced amounts of functional Six3 protein fail to activate Shh expression in the mutant RDVM and ultimately lead to HPE. These results identify Six3 as a direct regulator of Shh expression and reveal a crossregulatory loop between Shh and Six3 in the ventral forebrain.     

Clinical characterization of individuals with deletions of genes in holoprosencephaly pathways by aCGH refines the phenotypic spectrum of HPE

Holoprosencephaly (HPE) is the most common developmental forebrain anomaly in humans. Both environmental and genetic factors have been identified to play a role in the HPE phenotype. Previous studies of the genetic bases of HPE have taken a phenotype-first approach by examining groups of patients with HPE for specific mutations or deletions in known or candidate HPE genes. In this study, we characterized the presence or absence of HPE or a microform in 136 individuals in which microarray-based comparative genomic hybridization (aCGH) identified a deletion of one of 35 HPE loci. Frank holoprosencephaly was present in 11 individuals with deletions of one of the common HPE genes SHH, ZIC2, SIX3, and TGIF1, in one individual with a deletion of the HPE8 locus at 14q13, and in one individual with a deletion of FGF8, whereas deletions of other HPE loci and candidate genes (FOXA2 and LRP2) expressed microforms of HPE. Although individuals with deletions of other HPE candidates (DISP1, LSS, HHIP, SMO, BMP4, CDON, CDC42, ACVR2A, OTX2, and WIF1) had clinically significant features, none had frank HPE or a microform. A search for significant aCGH findings in individuals referred for testing for HPE revealed a novel association of a duplication involving GSK3B at 3q13.33 with HPE or a microform, seen in two unrelated individuals.     

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.     

Expression of the sonic hedgehog gene in human embryos with neural tube defects

BACKGROUND: To estimate the rate of malformations observed during early human development, a series of 38,913 first-trimester abortions were studied. Neural tube defects (NTD) were found in 57 cases. METHODS: A histological study of serial sections performed in 25 embryos revealed a spectrum of axial structure abnormalities. Expression of the SHH gene was studied by in situ hybridization in one case of CRS and in two cases of SB. RESULTS: A cervical notochord duplication was always found in craniorachischisis (CRS, n = 8), but not in spina bifida (SB, n = 10) or diplomyelia (split cord malformation, n = 3). In the embryo with CRS, expression of SHH was found in both domains, corresponding to the duplicated part of the notochord, whereas a single signal was observed in the nonduplicated part. This expression was associated at the cervical level of the open neural tube with a broad SHH expression domain and with two or even three domains in its lumbar region, suggesting multiple functional floor plates. Similarly, in two embryos with SB, two domains of SHH expression were found in the ventral neural tube. CONCLUSIONS: Our findings suggest that notochord splitting in the cervical region might be involved in the pathogenesis of CRS. Interestingly, similar notochord abnormality and altered expression of the shh gene are observed in Lp mice with NTD. This suggests that the Lp gene could be a candidate gene for human CRS. Further studies are needed to establish the primary event responsible for the notochord splitting and for the abnormal expression of the SHH gene in the floor plate in embryos with CRS and SB.