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.     

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.     

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.     

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.     

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.     

MLPA screening reveals novel subtelomeric rearrangements in holoprosencephaly

Holoprosencephaly (HPE) is the most common developmental brain anomaly in human, associated with a wide spectrum of presentations. The etiology is heterogeneous, due to environmental and genetic factors. Out of 12 cytogenetic candidate loci previously reported, eight were subtelomeric, including the loci in which two of the four major HPE genes were identified (SHH and TGIF). Recently, we reported that these two genes could be mutated or microdeleted. Therefore, we hypothesized that subtelomeres screening in HPE patients could refine the known subtelomeric candidate loci and identify novel ones. In this study, 181 samples, 72 fetuses and 109 live-born infants, with HPE and a normal karyotype, and 10 patients deleted for SHH or TGIF (3.5 Mb from telomeres) were screened for subtelomeric rearrangements using the multiplex ligation probe-dependent amplification (MLPA) method with two kits. Quantitative PCR was performed when discrepancies were observed between these two kits. We found that known SHH and TGIF microdeletions on 7q and 18p, encompassed their subtelomeric region (3.5 Mb) and were often associated with cryptic gains. Out of the 181 samples, we detected rearrangements in known candidate HPE loci (1q, 20p, and 21q) as well as in other novel subtelomeric locations (1p, 5q, 8p, 17q, 18q, 22q, and Xq) and in the subcentromeric 15q. We also found associations between cryptic subtelomeric gain and loss that may be inherited from a parental balanced translocation, which is helpful for genetic counseling. These findings reinforce the multihit origin for HPE and contribute to the explanation of the wide phenotypic spectrum described in this developmental disorder.     

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.     

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.     

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.     

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.     

Ectopic sonic hedgehog signaling impairs telencephalic dorsal midline development: implication for human holoprosencephaly

Holoprosencephaly (HPE) is the most common developmental anomaly of the human forebrain, and in its severe form, the cerebral hemispheres fail to completely separate into two distinct halves. Although disruption of ventral forebrain induction is thought to underlie most HPE cases, a subset of HPE patients exhibits preferential dysgenesis of forebrain dorsal midline structures with unknown etiology. In this study, we show that Sonic hedgehog (Shh) lacking cholesterol moiety in one allele (ShhN/+) in mice can elicit ectopic Shh signaling in early telencephalon to induce ventral progenitor marker expression in the cortical region and impair telencephalic dorsal midline development. Prolonged ectopic ShhN signaling impaired Bmp and Wnt signaling from the dorsal patterning center through upregulation of Fgf8, leading to augmented cell proliferation, decreased cell death and impaired roof plate morphogenesis. Accordingly, ShhN/+ mutant telencephalic dorsal midline structures, including cortical hem, hippocampus and choroid plexus, either failed to form or were hypoplastic. Strikingly, ShhN/+ mutants displayed a spectrum of phenotypic features such as failure of anterior cerebral hemisphere to divide, hydrocephalus and cleft palate which have been observed in a human patient with milder HPE predicted to produce SHHN protein due to a truncation mutation in one SHH allele. We propose that elevated ectopic Shh signaling can impair dorsal telencephalic midline morphogenesis, and lead to non-cleavage of midline structures mimicking human HPE with dorsal midline defects.     

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.     

Point mutations in a distant sonic hedgehog cis-regulator generate a variable regulatory output responsible for preaxial polydactyly

Precise spatial and temporal control of developmental genes is crucial during embryogenesis. Regulatory mutations that cause the misexpression of key developmental genes may underlie a number of developmental abnormalities. The congenital abnormality preaxial polydactyly, extra digits, is an example of this novel class of mutations and is caused by ectopic expression of the signalling molecule Sonic Hedgehog (SHH) in the developing limb bud. Mutations in the long-distant, limb-specific cis-regulator for SHH, called the ZRS, are responsible for the ectopic expression which underlies the abnormality. Here, we show that populations of domestic cats which manifest extra digits, including the celebrated polydactylous Hemingway's cats, also contain mutations within the ZRS. The polydactylous cats add significantly to the number of mutations previously reported in mouse and human and to date, all are single nucleotide substitutions. A mouse transgenic assay shows that these single nucleotide substitutions operate as gain-of-function mutations that activate Shh expression at an ectopic embryonic site; and that the sequence context of the mutation is responsible for a variable regulatory output. The plasticity of the regulatory response correlates with both the phenotypic variability and with species differences. The polydactyly mutations define a new genetic mechanism that results in human congenital abnormalities and identifies a pathogenetic mechanism that may underlie other congenital diseases.     

Minimal evidence for a direct involvement of twisted gastrulation homolog 1 (TWSG1) gene in human holoprosencephaly

Holoprosencephaly (HPE) is the most common disorder of human forebrain and facial development. Presently understood etiologies include both genetic and environmental factors, acting either alone, or more likely, in combination. The majority of patients without overt chromosomal abnormalities or recognizable associated syndromes have unidentified etiologies. A potential candidate gene, Twisted Gastrulation Homolog 1 (TWSG1), was previously suggested as a contributor to the complex genetics of human HPE based on (1) cytogenetic studies of patients with 18p deletions, (2) animal studies of TWSG1 deficient mice, and (3) the relationship of TWSG1 to bone morphogenetic protein (BMP) signaling, which modulates the primary pathway implicated in HPE, Sonic Hedgehog (SHH) signaling. Here we present the first analysis of a large cohort of patients with HPE for coding sequence variations in TWSG1. We also performed fine mapping of 18p for a subset of patients with partial 18p deletions. Surprisingly, minimal evidence for alterations of TWSG1 was found, suggesting that sequence alterations of TWSG1 are neither a common direct cause nor a frequent modifying factor for human HPE pathologies.     

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

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

Mutations in the C-terminal domain of Sonic Hedgehog cause holoprosencephaly

Holoprosencephaly (HPE) is the most common brain anomaly in humans, involving abnormal formation and septation of the developing central nervous system. Among the heterogeneous causes of HPE, mutations in the Sonic Hedgehog (SHH) gene have been shown to result in an autosomal dominant form of the disorder. Here we describe a total of five different mutations in the processing domain encoded by exon 3 of SHH in familial and sporadic HPE. This is the first instance in humans where SHH mutations in the domain responsible for autocatalytic cleavage and cholesterol modification of the N-terminal signaling domain of the protein have been observed.      

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.