Developmental delay is present in all live born HPE patients, and seems in agreement with the severity of the brain malformation. Approximately half of the patients with HPE develop epilepsy. Hydrocephaly can occur during pre- or postnatal development. Many others signs like mental retardation, hypotonia, weakness, spasticity, dystonia and abnormal movements are described.

Microcephaly, hypotelorism or hypertelorism, midline or lateral cleft lip and/or palate, flat nose, iris coloboma, single maxillary central incisors and hypoplasia of the pyriform aperture are often noticed.

Diabetes insipidus, growth hormone deficiency, adrenal hypoplasia, hypogonadism, or thyroid hypoplasia are very frequent in HPE because the midline malformation affects the development of the hypothalamus and the pituitary gland.

Feeding and swallowing difficulties are frequent and generally correlate with the grade of HPE, leading to a requirement for gastrostomy tubes in about two thirds of patients with alobar or semilobar HPE 13. These complications can be due to axial hypotonia, cleft lip and/or palate, gastro esophageal reflux, choking, slowness in eating and frequent pauses and often lead to failure to thrive.

Instability of temperature, heart and/or breath rate.

This pathology was first described as autosomal dominant (MIM# 142945), with an incomplete penetrance and a variable expression 42. The penetrance in autosomal dominant HPE is estimated to be 80%, but recent data also suggest a multigenic and multihit origin 43.

The first genes positively implicated in HPE were identified from recurrent chromosomal rearrangements. Other genes were described by studying the SHH signaling pathway actors or the Nodal/TGFβ pathway, summarized in Figure 1.

Sonic hedgehog(SHH) (MIM# 600725; 7q36; HPE3) 4445

SHH was isolated from the human critical region HPE3 on chromosome 7q36. It plays a critical role in early forebrain and central nervous system development. SHH is expressed in the human embryo in the notochord, the floorplate of the neural tube, the posterior limb buds and the gut 46. The SHH protein is a secreted intercellular signaling molecule, which is synthesized as a precursor that undergoes autocatalytic cleavage into N-terminal domain (SHH-N) and C-terminal domain (SHH-C). During the autoprocessing reaction, a cholesterol moiety is covalently attached to the C-terminus of SHH-N 47. Mouse and chicken embryos cultured in vitro and exposed to cyclopamine, an alkaloid blocker of Sonic hedgehog signaling, exhibit craniofacial anomalies and display phenotypes of HPE 48. Homozygous Shh mutant mice present with cyclopia and often die during embryonic development, whereas the heterozygous mouse appears normal 49.

SHH is the major gene implicated in holoprosencephaly (12.7% of HPE cases: 50% of overall point mutations and 38% of overall large deletions) 5051. Some of the mutants were functionally characterized by studying the production of the active SHH-N fragment and the signaling activity in a reporter cell-based assay 5253. In families with SHH mutation, an incomplete penetrance and extremely wide phenotypic variability are observed 7. Large deletions in 7q36, resulting in the loss of not only the SHH gene but also the HLXB9 gene have been observed in patients presenting HPE and Curarrino (sacral agenesia). Nevertheless, one study reported that only 50% of patients with del(7)(q36), including SHH, have HPE 43. HPE can also be caused by apparently balanced translocations near the SHH locus, which might be as much as 200 kb away from the gene, due to position effect 45.

ZIC2 (MIM# 603073; 13q32; HPE5) 54

The second gene identified was ZIC2, which encodes a member of a transcription factor family that includes the Drosophila odd-paired gene (opa), and contains zinc finger DNA binding motifs similar to the GLI protein binding domains. ZIC2 plays an important role in neurulation. Zic2 knockdown mice show a strong holoprosencephaly phenotype in which the cerebral hemispheres are fused, and structures derived from the dorsal midline of forebrain are missing or reduced 55.

ZIC2 is the second HPE gene by order of involvement (9.2% of HPE cases: 31% of overall point mutations and 38% of overall large deletions). Small mutations identified include a significant part of complex mutations that are not just base substitutions but up to 30 base-pairs deletions, insertions or duplications, probably because of the gene sequence composition. Large deletions in 13q, including ZIC2, were narrowed by Comparative Genomic Hybridisation (CGH) array, which allows an inkling of correlations between the size of the rearrangement and the phenotype: one fetus with a typical HPE strictly limited to cerebral anomalies was found to have a deletion of 1, 5 Mb, while another fetus presenting an HPE in a polymalformative context carried a 30 Mb deletion.

Most of the time, alterations in ZIC2 correlate with a normal face and, in some cases, are associated with neural tube defects or syntelencephaly (MIHF). Spina bifida or anencephaly have been also described as being associated with HPE in the same patient or in the same family by several authors 7856.

SIX3 (MIM# 603714; 2p21; HPE2) 47

SIX3 is an homeobox-containing gene which is homologous to the Drosophila sine oculis gene; it is involved in head midline and eye formation.

Mice with reduced levels of Six3 expression present a failure of forebrain and eye development 57.

Point mutations and large deletions in SIX3 represent 3% and 1% of HPE cases respectively. They are generally found in severe phenotypes. New phenotypes like atelencephaly – absence of telencephalon – or syntelencephaly – failure of separation of the posterior frontal and parietal right and left lobes 1458 – may be associated; these phenotypes can be considered as a continuum of the HPE spectrum, based on embryological arguments and molecular results involving SIX3 59.

TGIF (MIM# 602630; 18p11.3; HPE4) 60.

TGIF (TG-interacting factor) is another homeodomain transcription factor that inhibits signaling through Nodal/Transforming Growth Factor beta (TGFβ) pathways by blocking the action of SMAD proteins. Moreover, TGIF is a repressor of retinoic acid regulated gene transcription (Figure 1).

Mutations in TGIF account for 1% of studied cases and large deletions for nearly 1%. These data suggest that TGIF alterations explain only a very small proportion of HPE cases, and TGIF involvement in HPE can be discussed since only 10% of patients carrying a 18p deletion (including the TGIF gene) present HPE 6162 and mouse models with disrupted entire Tgif do not present an HPE phenotype 63. However, recent studies report that heterozygous and homozygous deletions of the third exon of Tgif in mice can result in a defined spectrum of brain developmental defects including exencephaly, microcephaly, holoprosencephaly, and abnormalities in embryonic brain ventricle formation and cleavage 64. The variable expression observed in families with one TGIF mutation makes genotype-phenotype correlations more difficult: both severe forms and labial/palate cleft are reported to be associated with mutations in this gene. Several hypotheses can be produced to explain these observations. First, it is possible that the TGIF2 gene (in 20q11.2-q12) provides redundancy for TGIF activity in the forebrain, even if Tgif2 does not have the same activity as Tgif in the neural tube in chick 65. Additional studies will be required to elucidate the role of TGIF in prosencephalic neural development and HPE.

PATCHED1 (MIM# 601309; 9q22) 66

PATCHED-1 (PTCH) is the receptor for SHH, and normally acts to repress SHH signaling. This repression is relieved when SHH binds to PTCH. Four different mutations of the gene were described in five unrelated HPE-affected individuals. These mutations may affect the ability of PTCH to bind SHH, or perturb the intracellular interactions of PTCH with other proteins involved in SHH signaling. They could subsequently enhance the repressive activity of PTCH on the SHH pathway and so decrease SHH signaling.

GLI2 (MIM# 165230; 2q14)

GLI2 is one of three vertebrate transcription factors implicated as obligatory mediators of SHH signal transduction. Mutations in this gene are preferentially involved in a distinctive phenotype (within the HPE spectrum) whose main features include defective anterior pituitary formation and pan-hypopituitarism 67, not necessarily with obvious abnormalities of the prosencephalon cleavage.

TDGF1/CRIPTO (MIM# 187395; 3p21.31)

TDGF1 (Teratocarcinoma Derived Growth Factor), also called CRIPTO, is an EGF-CFC family member and an obligate co-receptor involved in NODAL signaling, a developmental program implicated in midline, forebrain, and left-right axis development in model organisms. CFC1 (CRYPTIC), another member of this family, has been demonstrated to be required for proper laterality development in humans.

Zebrafish rescue assays indicate a role for TDFG1 in midline and forebrain development. One mutation in TDGF1 (3p21.3) has been described in a patient with midline anomalies of the forebrain 68.

Numerous candidate loci and candidate genes have been suggested for HPE (Table 4) (Figure 1). Among these genes, some were already investigated in HPE.

FOX-H1 (FAST1) (MIM# 603621; 8q24.3) is a co-transcriptional factor of SMAD2 and SMAD4, and intervenes in TGF-β, activin and nodal signaling pathways. These factors control early development in vertebrates and are vital for specification of the anterior-posterior axis. Recent studies on Fox-H1 null mutant mice showed that embryos failed to pattern the anterior-posterior axis, form the node, prechordal mesoderm, notochord and definitive endoderm. These mutants presented aberrant anterior head structures and abnormal cardiac development.

Mutations in FOX-H1 in HPE with cardiac malformations were only described in an abstract 69; these variants would affect the DNA-binding domain or the SMAD interacting domain.

The lanosterol synthase gene (MIM# 600909; 21q22.3; HPE1) is located in the HPE1 critical region, where partial monosomy 21q was described in several HPE cases (.) 7071727374. Lanosterol synthase catalyzes a key step in the biosynthesis of cholesterol, essential in the maturation of the SHH protein. A previous study reported no mutation in a small cohort of 30 patients 75, but further investigations are needed before definitely ruling out this candidate.

CFC1 (CRYPTIC) (MIM# 605194; 2q21.1) is a member of the EGF-CFC family. EGF-CFC genes encode extra cellular proteins that act as essential cofactors for Nodal, a member of the transforming growth factor beta (TGF-beta) family, and play key roles in intercellular signaling pathways during vertebrate embryogenesis (germ-layer formation, anterior-posterior axis orientation and left-right axis specification) 7677787980. CFC1 could be considered as a candidate gene for HPE, even if loss-of-function mutations in human CFC1 (encoding the CRYPTIC protein) were identified only in patients with heterotaxic phenotypes (randomized organ positioning).

SIL(SCL-interrupting locus) (MIM# 181590; 1p32), was considered by Karkera et al. 81 as a candidate HPE gene, as Sil mutants displayed prominent midline neural tube defects including delay or failure of neural tube closure and holoprosencephaly. SIL mutations were searched in a panel of HPE patients, but only several common polymorphisms were identified, suggesting that SIL is not a common factor in HPE pathogenesis in humans.

The human DKK1 gene (MIM# 605189; 10q11.2) was tested by Roessler et al. 82, as a result of the cyclopia observed in frogs lacking dkk-1. The Dkk gene family has been recently described as coding for secreted proteins of a novel class of proteins that act during development to bind and sequester members of the Bmp and Wnt families, establishing developmental zones free of the effects of these powerful morphogenes 83. Search for mutations in a cohort of 100 HPE patients revealed four missense mutations with preserved activity in head induction assays in frogs, suggesting a limited role for this gene in HPE pathogenesis.

TMEM1 (Transmembrane protein 1) (MIM# 602103; 21q22.3; HPE1) was designated EHOC-1 for epilepsy, holoprosencephaly candidate-1 as it was isolated from contigs of the candidate region HPE1. The protein is predicted to present multiple putative transmembrane domains that share partial homology with transmembrane proteins including sodium channel proteins 84.

FOXA2 (MIM# 600288; 20p11); in zebrafish, the winged-helix transcription factor FoxA2 is involved in floor plate development and differentiation in conjunction with Cyclops (Nodal) signaling, and FoxA2 may be one component of the regulatory circuit controlling expression in the floor plate 8586. Moreover, Collignon et al. 87 showed that some mice manifest left-right axis malformations when doubly heterozygous for null mutations in FoxA2 and in Nodal.

DISP1 (MIM# 607502; 1q42; HPE10) 88899091. The encoded protein DISPATCHED (DISP1) presents twelve transmembrane domains and shares structural homology with the Patched Drosophila and vertebrates' gene in the form of a sterol-sensing domain. Disp functions to release cholesterol-anchored Hh and could be involved in multimeric Shh formation in membrane rafts 92.

EAPP/C14ORF11 (MIM# 609486; 14q13; HPE8) was defined as a HPE potential candidate gene on chromosome 14 93.

TECT1/TECTONIC (MIM# 609863; 12q24.1). In mouse embryos, tectonic is expressed in regions that participate in hedgehog signaling. It is first expressed during gastrulation stages in the ventral node, and then in the gut endoderm, limb buds, notochord, somites, neural tube, and floor plate. Tectonic modulated hedgehog signaling downstream of smoothened and Rab23, and is required for maximal hedgehog activation 94.

CHRD (MIM# 603475; 3q27) and NOG (MIM# 602991; 17q22). Chordin is a key developmental protein that dorsalizes early vertebrate embryonic tissues by binding to ventralizing TGF-beta-like bone morphogenetic proteins and sequestering them in latent complexes. Its dorsalizing effects are counteracted by BMP1 759596.

The Noggin gene was first discovered as an important factor in brain and nerve development. The secreted polypeptide noggin binds and inactivates members of the TGF-beta superfamily signaling proteins, such as bone morphogenetic protein 4 97.

Some chordin/noggin double-null embryos present holoprosencephaly, with a single nasal pit, a cyclopic eye, and agnathia, and resembled embryos lacking sonic hedgehog. At embryonic day 12.5, double-mutant embryos were recovered with more severe phenotypes resembling aprosencephaly. In double-mutant embryos dissected at embryonic day 8.5, forebrain reduction was clearly evident. Chordin and noggin are not necessary for establishing the anterior visceral endoderm but are required for subsequent elaboration of anterior pattern 98. BMP antagonists chordin and noggin compensate for each other during early mouse development. When both gene products are removed, antero-posterior, dorso-ventral, and left-right patterning are all affected.

LPR2 (Low Density Lipoprotein Receptor-Related Protein 2) (MIM# 600073; 2q24–q31) is also called glycoprotein 330 or megalin. Homozygous knockout mice manifest abnormalities in epithelial tissues 99. In brain, impaired proliferation of neuroepithelium produces a holoprosencephalic syndrome, characterized by lack of olfactory bulbs, forebrain fusion, and a common ventricular system.

SMOH (MIM# 601500; 7q32.2)

Smoothened mediate with Patched the cellular response to the Hedgehog secreted protein signal; the binding of Sonic hedgehog to its receptor Patched prevents normal inhibition by PTCH of Smoothened (SMOH).

HHIP (Human Hedgehog Interacting protein) (MIM# 606178; 4q31.22) is involved in the attenuation of hedgehog signaling since ectopic expression of Hip in transgenic mice results in severe skeletal defects similar to those observed in Indian hedgehog mutants 100.

BMP4 (MIM# 112262; 14q22.2) is a vital regulatory molecule that functions throughout development in mesoderm induction, tooth development, limb formation, bone induction, and fracture repair. In expression studies in mouse, it was demonstrated that BMP4 activates the expression of Msx1, leading to incisor tooth development 101. In chick embryos, the first signs of left-right asymmetry are detected in Hensen's node, essentially by left-sided Sonic hedgehog expression, and Bmp4 is necessary to maintain Shh asymmetry within the node 102.

NODAL (MIM# 601265; 10q22.1) is a member of the TGF-beta gene family and is expressed during mouse gastrulation 76. Nodal has a left-sided expression pattern that is disrupted in mouse models of LR axis development 76. Moreover, Collignon et al. 87 showed that some mice manifest left-right axis malformations when doubly heterozygous for null mutations in Nodal and in FoxA2.

SMAD2/4 (MIM# 601366; 18q21). SMAD proteins mediate TGF-beta signaling to regulate cell growth and differentiation. TGF-beta induces activation and nuclear translocation of SMAD2 which forms complexes with SMAD4 and regulate transcription of target genes.

CDO/CDON (MIM# 608707; 11q23-24) is another HPE candidate gene since mice lacking the transmembrane protein Cdo/Cdon, previously implicated in myogenesis, display HPE with strain-specific severity and without limb defects, modeling human HPE and implicating modifier genes as a cause of variability 103.

The Twisted gastrulation(TWSG 1) gene (MIM# 605049; 18p11.3; HPE4) is located near the TGIF gene. The TWSG protein is able to either enhance or inhibit signaling by the bone morphogenetic protein (BMP) subfamily of TGF-β type factors. All Twsg1 mutant mice, irrespective of genetic background, exhibit deletions of neural arches in the cervical vertebrae, and C57BL/6 ones present pronounced forebrain defects including rostral truncations, holoprosencephaly, cyclopia, agnathia 104.

HPE seems nowadays to be a multihit pathology, that may require two or more events involving several genes and/or environmental factors 43. Indeed, some of mutations are found in a heterozygous state with a variable phenotypic penetrance in the same family, and double heterozygous mutations have already been identified (in SHH and in ZIC2 44, in SHH and in TGIF 61, in GLI2 and in ZIC2, and two different missense mutations in ZIC2 in the same fetus. Moreover, a number of HPE cases consist of an association between several rearrangements detected by MLPA in different chromosomal regions and, in particular, a duplication associated with a deletion, like (7pdup;7qdel) or (8pdup;7qdel)105. The finding of such multiple rearrangements in a same patient suggests a balanced translocation in one of the normal parents and reinforces the multigenic and multihit origin. This multihit hypothesis can also offer explanation for the wide phenotypic spectrum described for a same mutation in a same family, the most severe form being due to the additive effect of several events. This conception can be illustrated by the finding of a TGIF nonsense mutation in a fetus presenting a semilobar HPE with cebocephaly, turricephaly, microcephaly and flat face detected by ultrasound, but also in her father who presented only mild signs like hypotelorism and lateral cleft lip, although this stop mutation was predicted to encode a truncated version of the TGIF protein missing two repression domains 106. Animal models support this double heterozygosity hypothesis, since HPE is due to digenic inheritance in mouse models 43.

However the number of instances with double hits clearly identified and confirmed by functional studies remains relatively restricted to a few cases for the time being, unlike classic recessive disorders. HPE still behaves as an apparent autosomal dominant disorder with reduced penetrance and variable expression, associated with family-specific genetic alterations. These deleterious mutations give a susceptibility for developing HPE, upon which other factors (either genetic either environmental) modulate expression 107. The genetic background would have an influence on the incidence of brain abnormalities, as it was described in the mutant mice with deletion of the third exon of Tgif, suggesting that genetic modifiers functionally interact with the mutant protein during embryonic brain development 64. Whether or not these factors constitute a second hit remains to be debated.