Expression of the Sonic hedgehog (SHH ) gene during early human development and phenotypic expression of new mutations causing holoprosencephaly.

Holoprosencephaly (HPE), the most common developmental defect of the forebrain and the face, is genetically heterogeneous. One of the genes involved, Sonic hedgehog ( SHH ), on 7q36, has been identified as the first HPE-causing gene both in mouse and humans. In order to delineate the phenotype of specific SHH mutations, we described the expression of the SHH gene during early human embryogenesis and investigated the phenotype of novel SHH mutations. In situ hybridization studies were performed on paraffin-embedded human embryo sections at three different development stages. These studies show that SHH is expressed in the notochord, the floorplate, the brain, the zone of polarizing activity and the gut. We also report on the phenotype of four novel mutations identified in 40 HPE families (two in isolated HPE and two in familial HPE). Expressivity ranged from alobar HPE to microcephaly and hypoplasia of the pituitary gland in one family, and from HPE to an asymptomatic form in another family. No SHH mutation was found in six polymalformed cases combining HPE with other defects, such as skeletal, limb, cardiac, anal and/or renal anomalies. This study confirms the genetic heterogeneity of HPE, and further demonstrates that SHH mutations are associated with a broad spectrum of cerebral midline defects.     

The mutational spectrum of the sonic hedgehog gene in holoprosencephaly: SHH mutations cause a significant proportion of autosomal dominant holoprosencephaly.

Holoprosencephaly (HPE) is a common developmental anomaly of the human forebrain and midface where the cerebral hemispheres fail to separate into distinct left and right halves. We have previously reported haploinsufficiency for Sonic Hedgehog ( SHH ) as a cause for HPE. We have now performed mutational analysis of the complete coding region and intron-exon junctions of the SHH gene in 344 unrelated affected individuals. Herein, we describe 13 additional unrelated affected individuals with SHH mutations, including nonsense and missense mutations, deletions and an insertion. These mutations occur throughout the extent of the gene. No specific genotype-phenotype association is evident based on the correlation of the type or position of the mutations. In conjunction with our previous studies, we have identified a total of 23 mutations in 344 unrelated cases of HPE. They account for 14 cases of familial HPE and nine cases of sporadic HPE. Mutations in SHH were detected in 10 of 27 (37%) families showing autosomal dominant transmission of the HPE spectrum, based on structural anomalies. Interestingly, three of the patients with an SHH mutation also had abnormalities in another gene that is expressed during forebrain development. We suggest that the interactions of multiple gene products and/or environmental elements may determine the final phenotypic outcome for a given individual and that variations among these factors may cause the wide variability in the clinical features seen in HPE.     

BOC is a modifier gene in holoprosencephaly

Holoprosencephaly (HPE), a common developmental defect of the forebrain and midface, has a complex etiology. Heterozygous, loss‐of‐function mutations in the sonic hedgehog (SHH) pathway are associated with HPE. However, mutation carriers display highly variable clinical presentation, leading to an “autosomal dominant with modifier” model, in which the penetrance and expressivity of a predisposing mutation is graded by genetic or environmental modifiers. Such modifiers have not been identified. Boc encodes a SHH coreceptor and is a silent HPE modifier gene in mice. Here, we report the identification of missense BOC variants in HPE patients. Consistent with these alleles functioning as HPE modifiers, individual variant BOC proteins had either loss‐ or gain‐of‐function properties in cell‐based SHH signaling assays. Therefore, in addition to heterozygous loss‐of‐function mutations in specific SHH pathway genes and an ill‐defined environmental component, our findings identify a third variable in HPE: low‐frequency modifier genes, BOC being the first identified.     

Holoprosencephaly: from Homer to Hedgehog

Holoprosencephaly (HPE), a common developmental defect affecting the forebrain and face, is etiologically heterogeneous and exhibits wide phenotypic variation. Graded degrees of severity of the brain malformation are also reflected in the highly variable craniofacial malformations associated with HPE. In addition, individuals with microforms of HPE, who usually have normal cognition and normal brain imaging, are at risk for having children with HPE. Some obligate carriers for HPE may not have any phenotypic abnormalities. Recurrent chromosomal rearrangements in individuals with HPE suggest loci containing genes important for brain development, and abnormalities in these genes may result in HPE. Recently, Sonic Hedgehog (SHH) was the first gene identified as causing HPE in humans. Proper function of SHH depends on cholesterol modification. Other candidate genes that may be involved in HPE include components of the SHH pathway, elements involved in cholesterol metabolism, and genes expressed in the developing forebrain.     

Mutations in holoprosencephaly

Holoprosencephaly (HPE) is the most common developmental defect of the forebrain and midface in humans. In holoprosencephaly the cerebral hemispheres of the brain fail to separate into distinct left and right hemispheres. This malformation is due to the improper specification and formation of the forebrain during early development. When one considers the great number and kinds of genetic interactions that must occur to properly pattern the developing forebrain, it is not surprising that HPE is extremely heterogeneous. In addition to teratogenic agents, several genes are implicated as the cause of HPE. At least 12 different loci have been associated with HPE and now several distinct human genes for holoprosencephaly have been identified. These genes include Sonic Hedgehog (SHH), ZIC2, SIX3, and TG-interacting factor (TGIF). Here we present an overview of the presently known genes causing human holoprosencephaly. We discuss their functional role in development of the forebrain and summarize the mutations and polymorphisms that have been identified within them. Hum Mutat 16:99-108, 2000. Published 2000 Wiley-Liss, Inc.     

Language skills and neuropsychological performance in patients with SHH mutations and a holoprosencephaly-like phenotype

Here, we evaluate linguistic skills and neuropsychological performance in a sample of patients with SHH mutations and a holoprosencephaly (HPE)-like phenotype, a minor form of classic HPE. Our findings suggest that patients with SHH mutations and a HPE-like phenotype have normal cognitive ratios and significant language impairment. Imaging evaluation by magnetic resonance imaging (MRI) was normal in three patients and in one there was hypoplasia of the anterior commissure and the presence of a temporal cyst, apparently not related to the clinical findings.     

Modeling the complex etiology of holoprosencephaly in mice

Holoprosencephaly (HPE) is a common developmental defect caused by failure to define the midline of the forebrain and/or midface. HPE is associated with heterozygous mutations in Nodal and Sonic hedgehog (SHH) pathway components, but clinical presentation is highly variable, and many mutation carriers are unaffected. It is therefore thought that such mutations interact with more common modifiers, genetic and/or environmental, to produce severe patterning defects. Modifiers are difficult to identify, as their effects are context-dependent and occur within the complex genetic and environmental landscapes that characterize human populations. This has made a full understanding of HPE etiology challenging. We discuss here the use of mice, a genetically tractable model sensitive to teratogens, as a system to address this challenge. Mice carrying mutations in human HPE genes often display wide variations in phenotypic penetrance and expressivity when placed on different genetic backgrounds, demonstrating the existence of silent HPE modifier genes. Studies with mouse lines carrying SHH pathway mutations on appropriate genetic backgrounds have led to identification of both genetic and environmental modifiers that synergize with the mutations to produce a spectrum of HPE phenotypes. These models favor a scenario in which multiple modifying influences—both genetic and environmental, sensitizing and protective—interact with bona fide HPE mutations to grade phenotypic outcomes. Despite the complex interplay of HPE risk factors, mouse models have helped establish some clear concepts in HPE etiology. A combination of mouse and human cohort studies should improve our understanding of this fascinating and medically important issue.     

A broad range of ophthalmologic anomalies is part of the holoprosencephaly spectrum

Holoprosencephaly (HPE) is the most common disorder of the developing forebrain in humans, and is characterized by failed or incomplete cleavage of the cerebral hemispheres and deep brain structures. HPE includes wide phenotypic variability, with a continuum of both brain and craniofacial anomalies. While "classic" eye findings, including the spectrum of midline anomalies ranging from cyclopia to hypotelorism, as well as chorioretinal coloboma and microphthalmia, have been frequently described in patients with HPE, other subtle eye anomalies may also occur. In our study we prospectively analyzed a small cohort of 10 patients in whom we identified mutations in SHH, SIX3, ZIC2, or FGF8, the latter of which is a very recently described HPE-associated gene. We found that 9 of 10 patients had at least two ophthalmologic anomalies, including refractive errors, microcornea, microphthalmia, blepharoptosis, exotropia, and uveal coloboma. These findings contribute to the understanding of the phenotypic variability of the HPE spectrum, and highlight findings in one medically important but often incompletely investigated system.     

SHH mutation is associated with solitary median maxillary central incisor: a study of 13 patients and review of the literature

Solitary median maxillary central incisor (SMMCI) or single central incisor is a rare dental anomaly. It has been reported in holoprosencephaly (HPE) cases with severe facial anomalies or as a microform in autosomal dominant HPE (ADHPE). In our review of the literature, we note that SMMCI may also occur as an isolated finding or in association with other systemic abnormalities. These anomalies include short stature, pituitary insufficiency, microcephaly, choanal atresia, midnasal stenosis, and congenital nasal pyriform aperture stenosis. SMMCI can also be a feature of recognized syndromes or associations or a finding in patients with specific chromosomal abnormalities. We performed a molecular study on a cohort of 13 SMMCI patients who did not have HPE. We studied two genes, Sonic Hedgehog (SHH) and SIX3, in which mutations have been reported in patients showing SMMCI as part of the HPE spectrum. A new missense mutation in SHH (I111F), segregating in one SMMCI family, was identified. Our results suggest that this mutation may be specific for the SMMCI phenotype since it has not been found in the HPE population or in normal controls. Published 2001 Wiley-Liss, Inc.     

Functions of TGIF homeodomain proteins and their roles in normal brain development and holoprosencephaly

Holoprosencephaly (HPE) is a frequent human forebrain developmental disorder with both genetic and environmental causes. Multiple loci have been associated with HPE in humans, and potential causative genes at 14 of these loci have been identified. Although TGIF1 (originally TGIF, for Thymine Guanine-Interacting Factor) is among the most frequently screened genes in HPE patients, an understanding of how mutations in this gene contribute to the pathogenesis of HPE has remained elusive. However, mouse models based on loss of function of Tgif1, and the related Tgif2 gene, have shed some light on how human TGIF1 variants might cause HPE. Functional analyses of TGIF proteins and of TGIF1 single nucleotide variants from HPE patients, combined with analysis of forebrain development in mouse embryos lacking both Tgif1 and Tgif2, suggest that TGIFs regulate the transforming growth factor ß/Nodal signaling pathway and sonic hedgehog (SHH) signaling independently. Although, some developmental processes that are regulated by TGIFs may be Nodal-dependent, it appears that the forebrain patterning defects and HPE in Tgif mutant mouse embryos is primarily due to altered signaling via the Shh pathway.     

Previously undescribed nonsense mutation in SHH caused autosomal dominant holoprosencephaly with wide intrafamilial variability

Holoprosencephaly (HPE) is the most common developmental defect of the forebrain and midface in humans, with a frequency of 1/16,000 live births. Different genes are implicated in the pathogenesis of HPE; these include SHH, ZIC2, SIX3, TGIF, and human DKK1. We describe here a family with recurrence of autosomal dominant HPE in different members showing a wide clinical variability. The mother presents a single central maxillary incisor and mild hypotelorism as signs of the diseases, while three of her sons were affected by HPE. By direct sequencing and restriction analysis of exon 2 of the SHH gene, we have identified a previously undescribed nonsense mutation at codon 128 (W128X). The identification of this mutation allowed us to give a prenatal diagnosis in this family and confirms a wide intrafamilial variability in the phenotypic spectrum.     

Holoprosencephaly: molecular study of a California population

Holoprosencephaly (HPE) is a common developmental anomaly of the forebrain and midface in which the cerebral hemispheres fail to separate into distinct left and right halves. HPE is extremely heterogeneous. In addition to teratogenic agents, several genes are implicated in the cause of HPE. Using samples from a population-based birth defects registry in California, we performed a mutational analysis of the known HPE genes Sonic Hedgehog (SHH), ZIC2, and SIX3, in addition to two HPE candidate genes, TG-interacting factor (TGIF), and Patched (PTC), on a group of sporadic HPE patients. This is the first molecular study of HPE in a population-based sample of patients. Among these patients, a deletion in the homeodomain of SIX3 and several polymorphisms in SIX3 and TGIF were identified. No sequence changes were detected in SHH, ZIC2, and PTC. Our results suggest that mutations in the currently recognized HPE genes may explain <5% of all sporadic HPE cases.     

Molecular screening of SHH, ZIC2, SIX3, and TGIF genes in patients with features of holoprosencephaly spectrum: Mutation review and genotype-phenotype correlations

Holoprosencephaly (HPE; 1 out of 16,000 live births; 1 out of 250 conceptuses) is a complex brain malformation resulting from incomplete cleavage of the prosencephalon, affecting both the forebrain and the face. Clinical expressivity is variable, ranging from a single cerebral ventricle and cyclopia to clinically unaffected carriers in familial dominant autosomic HPE. The disease is genetically heterogeneous, but additional environmental agents also contribute to the etiology of HPE. In our cohort of 200 patients, 34 heterozygous mutations were identified, 24 of them being novel ones: 13 out of 17 in the Sonic hedgehog gene (SHH); 4 out of 7 in ZIC2; and 7 out of 8 in SIX3. The two mutations identified in TGIF have already been reported. Novel phenotypes associated with a mutation have been described, such as abnormalities of the pituitary gland and corpus callosum, colobomatous microphthalmia, choanal aperture stenosis, and isolated cleft lip. This study confirms the great genetic heterogeneity of the disease, the important phenotypic variability in HPE families, and the difficulty to establish genotype-phenotype correlations.     

Solitary median maxillary central incisor syndrome: clinical case with a novel mutation of sonic hedgehog

Solitary median maxillary central incisor (SMMCI) is a rare dental anomaly. It is usually considered as a minor manifestation of holoprosencephaly (HPE). Some reported families had severe cases of HPE in some members and SMMCI in others. Mutations of Sonic Hedgehog (SHH) have been documented in these families. SMMCI has also been found as an isolated finding or together with other anomalies such as microcephaly, short stature, endocrine pathology, and choanal atresia. We describe a patient with SMMCI and a novel SHH mutation: Val332Ala.     

Holoprosencephaly-Polydactyly syndrome: in search of an etiology

Holoprosencephaly-Polydactyly (HPS) or Pseudotrisomy 13 syndrome are names conferred to clinically categorize patients whose phenotype is congruent with Trisomy 13 in the context of a normal karyotype. The literature suggests that this entity may be secondary to submicroscopic deletions in holoprosencephaly (HPE) genes; however, a limited number of investigations have been undertaken to evaluate this hypothesis. To test this hypothesis we studied a patient with HPE, polydactyly, and craniofacial dysmorphologies consistent with the diagnosis of Trisomy 13 whose karyotype was normal. We performed mutational analysis in the four main HPE causing genes (SHH, SIX3, TGIF, and ZIC2) and GLI3, a gene associated with polydactyly as well as fluorescent in situ hybridization (FISH) to search for microdeletions in these genes and two candidate HPE genes (DISP1 and FOXA2). No mutations or deletions were detected. A whole genome approach utilizing array Comparative Genomic Hybridization (aCGH) to screen for copy number abnormalities was then taken. No loss or gain of DNA was noted. Although a single case, our results suggest that coding mutations in these HPE genes and copy number anomalies may not be causative in this disorder. Instead, HPS likely involves mutations in other genes integral in embryonic development of the forebrain, face and limbs. Our systematic analysis sets the framework to study other affected children and delineate the molecular etiology of this disorder.     

Phenotypic and molecular variability of the holoprosencephalic spectrum

Since 1996, a European network has been organized from Rennes, France and holoprosencephalic files were collected for clinical and molecular study. Familial instances of typical and atypical holoprosencephaly (HPE) were found in 30% of cases. All affected children had psychomotor delay with microcephaly, often associated with endocrine, digestive, and respiratory abnormalities, and thermal dysregulation. Among 173 subjects in the molecular study, 28 heterozygous mutations were identified (16%): 15 SHH mutations, 6 ZIC2 mutations, 5 SIX3 mutations, and 2 TGIF mutations.     

Functional analysis of mutations in TGIF associated with holoprosencephaly

Holoprosencephaly (HPE) is the most common structural malformation of the forebrain and face in humans. Our current understanding of the pathogenesis of HPE attempts to integrate genetic susceptibility, evidenced by mutations in the known HPE genes, with the epigenetic influence of environmental factors. Mutations or deletions of the human TGIF gene have been associated with HPE in multiple population cohorts. Here we examine the functional effects of all previously reported mutations, and describe four additional variants. Of the eleven sequence variations in TGIF, all but four can be demonstrated to be functionally abnormal. In contrast, no potentially pathogenic sequence alterations were detected in the related gene TGIF2. These results provide further evidence of a role for TGIF in HPE and demonstrate the importance of functional analysis of putative disease-associated alleles.     

Array‐CGH analysis indicates a high prevalence of genomic rearrangements in holoprosencephaly: An updated map of candidate loci

Holoprosencephaly (HPE) is the most frequent malformation of the brain. To date, 12 different HPE loci and 8 HPE genes have been identified from recurrent chromosomal rearrangements or from the sequencing of genes from Nodal and SHH pathways. Our cohort of HPE patients presents a high genetic heterogeneity. Point mutations were found in SHH, ZIC2, SIX3, and TGIF genes in about 20% of cases (with 10% in SHH). Deletions in these same genes were found in 7.5% of the patients and 4.4% presented with other subtelomeric gain or losses. Consequently, the molecular basis of HPE remains unknown in 70% of our cohorts. To detect new HPE candidate genes, we used array‐CGH to refine the previous karyotype based HPE loci map. We analyzed 111 HPE patients with high‐performance Agilent oligonucleotidic arrays and found that 28 presented anomalies involving known or new potential HPE loci located on different chromosomes but with poor redundancy. This study showed an impressive rate of 19 patients among 111 with de novo chromosomal anomalies giving evidence that microrearrangements could be a major molecular mechanism in HPE. Additionally, this study opens new insights on HPE candidate genes identification giving an updated HPE candidate loci map. Hum Mutat 30:1–8, 2009. © 2009 Wiley‐Liss, Inc.