Impact of Sonic Hedgehog-dependent sphenoid bone defect on craniofacial growth.

OBJECTIVES: The main objective of this study was to evaluate how an apparently minor anomaly of the sphenoid bone, observed in a haploinsufficient mouse model for Sonic Hedgehog (Shh), affects the growth of the adult craniofacial region. This study aims to provide valuable information to orthodontists when making decisions regarding individuals carrying SHH mutation. MATERIALS AND METHODS: The skulls of embryonic, juvenile and adult mice of two genotypes (Shh heterozygous and wild type) were examined and measured using landmark-based linear dimensions. Additionally, we analysed the clinical characteristics of a group of patients and their relatives with SHH gene mutations. RESULTS: In the viable Shh+/ - mouse model, bred on a C57BL/6J background, we noted the presence of a persistent foramen at the midline of the basisphenoid bone. This particular anomaly was attributed to the existence of an ectopic pituitary gland. We discovered that this anomaly led to premature closure of the intrasphenoidal synchondrosis and contributed to craniofacial deformities in adult mice, including a longitudinally shortened skull base. This developmental anomaly is reminiscent of that commonly observed in human holoprosencephaly, a disorder resulting from a deficiency in SHH activity. However, sphenoid morphogenesis is not currently monitored in individuals carrying SHH mutations. CONCLUSION: Haploinsufficiency of Shh leads to isolated craniofacial skeletal hypoplasia in adult mouse. This finding highlights the importance of radiographic monitoring of the skull base in all individuals with SHH gene mutations.

DISP1 deficiency: Monoallelic and biallelic variants cause a spectrum of midline craniofacial malformations.

PURPOSE: DISP1 encodes a transmembrane protein that regulates the secretion of the morphogen, Sonic hedgehog, a deficiency of which is a major cause of holoprosencephaly (HPE). This disorder covers a spectrum of brain and midline craniofacial malformations. The objective of the present study was to better delineate the clinical phenotypes associated with division transporter dispatched-1 (DISP1) variants. METHODS: This study was based on the identification of at least 1 pathogenic variant of the DISP1 gene in individuals for whom detailed clinical data were available. RESULTS: A total of 23 DISP1 variants were identified in heterozygous, compound heterozygous or homozygous states in 25 individuals with midline craniofacial defects. Most cases were minor forms of HPE, with craniofacial features such as orofacial cleft, solitary median maxillary central incisor, and congenital nasal pyriform aperture stenosis. These individuals had either monoallelic loss-of-function variants or biallelic missense variants in DISP1. In individuals with severe HPE, the DISP1 variants were commonly found associated with a variant in another HPE-linked gene (ie, oligogenic inheritance). CONCLUSION: The genetic findings we have acquired demonstrate a significant involvement of DISP1 variants in the phenotypic spectrum of midline defects. This underlines its importance as a crucial element in the efficient secretion of Sonic hedgehog. We also demonstrated that the very rare solitary median maxillary central incisor and congenital nasal pyriform aperture stenosis combination is part of the DISP1-related phenotype. The present study highlights the clinical risks to be flagged up during genetic counseling after the discovery of a pathogenic DISP1 variant.

Local retinoic acid signaling directs emergence of the extraocular muscle functional unit.

Coordinated development of muscles, tendons, and their attachment sites ensures emergence of functional musculoskeletal units that are adapted to diverse anatomical demands among different species. How these different tissues are patterned and functionally assembled during embryogenesis is poorly understood. Here, we investigated the morphogenesis of extraocular muscles (EOMs), an evolutionary conserved cranial muscle group that is crucial for the coordinated movement of the eyeballs and for visual acuity. By means of lineage analysis, we redefined the cellular origins of periocular connective tissues interacting with the EOMs, which do not arise exclusively from neural crest mesenchyme as previously thought. Using 3D imaging approaches, we established an integrative blueprint for the EOM functional unit. By doing so, we identified a developmental time window in which individual EOMs emerge from a unique muscle anlage and establish insertions in the sclera, which sets these muscles apart from classical muscle-to-bone type of insertions. Further, we demonstrate that the eyeballs are a source of diffusible all-trans retinoic acid (ATRA) that allow their targeting by the EOMs in a temporal and dose-dependent manner. Using genetically modified mice and inhibitor treatments, we find that endogenous local variations in the concentration of retinoids contribute to the establishment of tendon condensations and attachment sites that precede the initiation of muscle patterning. Collectively, our results highlight how global and site-specific programs are deployed for the assembly of muscle functional units with precise definition of muscle shapes and topographical wiring of their tendon attachments.

Synonymous variants in holoprosencephaly alter codon usage and impact the Sonic Hedgehog protein.

Synonymous single nucleotide variants (sSNVs) have been implicated in various genetic disorders through alterations of pre-mRNA splicing, mRNA structure and miRNA regulation. However, their impact on synonymous codon usage and protein translation remains to be elucidated in clinical context. Here, we explore the functional impact of sSNVs in the Sonic Hedgehog (SHH) gene, identified in patients affected by holoprosencephaly, a congenital brain defect resulting from incomplete forebrain cleavage. We identified eight sSNVs in SHH, selectively enriched in holoprosencephaly patients as compared to healthy individuals, and systematically assessed their effect at both transcriptional and translational levels using a series of in silico and in vitro approaches. Although no evidence of impact of these sSNVs on splicing, mRNA structure or miRNA regulation was found, five sSNVs introduced significant changes in codon usage and were predicted to impact protein translation. Cell assays demonstrated that these five sSNVs are associated with a significantly reduced amount of the resulting protein, ranging from 5% to 23%. Inhibition of the proteasome rescued the protein levels for four out of five sSNVs, confirming their impact on protein stability and folding. Remarkably, we found a significant correlation between experimental values of protein reduction and computational measures of codon usage, indicating the relevance of in silico models in predicting the impact of sSNVs on translation. Considering the critical role of SHH in brain development, our findings highlight the clinical relevance of sSNVs in holoprosencephaly and underline the importance of investigating their impact on translation in human pathologies.

Disrupted Hypothalamo-Pituitary Axis in Association With Reduced SHH Underlies the Pathogenesis of NOTCH-Deficiency.

CONTEXT: In human, Sonic hedgehog (SHH) haploinsufficiency is the predominant cause of holoprosencephaly, a structural malformation of the forebrain midline characterized by phenotypic heterogeneity and incomplete penetrance. The NOTCH signaling pathway has recently been associated with holoprosencephaly in humans, but the precise mechanism involving NOTCH signaling during early brain development remains unknown. OBJECTIVE: The aim of this study was to evaluate the relationship between SHH and NOTCH signaling to determine the mechanism by which NOTCH dysfunction could cause midline malformations of the forebrain. DESIGN: In this study, we have used a chemical inhibition approach in the chick model and a genetic approach in the mouse model. We also reported results obtained from the clinical diagnosis of a cohort composed of 141 holoprosencephaly patients. RESULTS: We demonstrated that inhibition of NOTCH signaling in chick embryos as well as in mouse embryos induced a specific downregulation of SHH in the anterior hypothalamus. Our data in the mouse also revealed that the pituitary gland was the most sensitive tissue to Shh insufficiency and that haploinsufficiency of the SHH and NOTCH signaling pathways synergized to produce a malformed pituitary gland. Analysis of a large holoprosencephaly cohort revealed that some patients possessed multiple heterozygous mutations in several regulators of both pathways. CONCLUSIONS: These results provided new insights into molecular mechanisms underlying the extreme phenotypic variability observed in human holoprosencephaly. They showed how haploinsufficiency of the SHH and NOTCH activity could contribute to specific congenital hypopituitarism that was associated with a sella turcica defect.

MAU2 and NIPBL Variants Impair the Heterodimerization of the Cohesin Loader Subunits and Cause Cornelia de Lange Syndrome.

The NIPBL/MAU2 heterodimer loads cohesin onto chromatin. Mutations in NIPBL account for most cases of the rare developmental disorder Cornelia de Lange syndrome (CdLS). Here we report a MAU2 variant causing CdLS, a deletion of seven amino acids that impairs the interaction between MAU2 and the NIPBL N terminus. Investigating this interaction, we discovered that MAU2 and the NIPBL N terminus are largely dispensable for normal cohesin and NIPBL function in cells with a NIPBL early truncating mutation. Despite a predicted fatal outcome of an out-of-frame single nucleotide duplication in NIPBL, engineered in two different cell lines, alternative translation initiation yields a form of NIPBL missing N-terminal residues. This form cannot interact with MAU2, but binds DNA and mediates cohesin loading. Altogether, our work reveals that cohesin loading can occur independently of functional NIPBL/MAU2 complexes and highlights a novel mechanism protective against out-of-frame mutations that is potentially relevant for other genetic conditions.

Targeted panel sequencing establishes the implication of planar cell polarity pathway and involves new candidate genes in neural tube defect disorders.

Neural tube defect disorders are developmental diseases that originate from an incomplete closure of the neural tube during embryogenesis. Despite high prevalence-1 out of 3000 live births-their etiology is not yet established and both environmental and genetic factors have been proposed, with a heritability rate of about 60%. Studies in mouse models as well as in human have further suggested a multifactorial pattern of inheritance for neural tube defect disorders. Here, we report results obtained from clinical diagnosis and NGS analysis of a cohort composed of 52 patients. Using a candidate gene panel approach, we identified variants in known genes of planar cell polarity (PCP) pathway, although with higher prevalence than previously reported. Our study also reveals variants in novel genes such as FREM2 and DISP1. Altogether, these results confirm the implication of the PCP genes and involve the FRAS/FREM2 complex and Sonic Hedgehog signaling as novel components in the appearance of NTDs.

Integrated clinical and omics approach to rare diseases: novel genes and oligogenic inheritance in holoprosencephaly.

Holoprosencephaly is a pathology of forebrain development characterized by high phenotypic heterogeneity. The disease presents with various clinical manifestations at the cerebral or facial levels. Several genes have been implicated in holoprosencephaly but its genetic basis remains unclear: different transmission patterns have been described including autosomal dominant, recessive and digenic inheritance. Conventional molecular testing approaches result in a very low diagnostic yield and most cases remain unsolved. In our study, we address the possibility that genetically unsolved cases of holoprosencephaly present an oligogenic origin and result from combined inherited mutations in several genes. Twenty-six unrelated families, for whom no genetic cause of holoprosencephaly could be identified in clinical settings [whole exome sequencing and comparative genomic hybridization (CGH)-array analyses], were reanalysed under the hypothesis of oligogenic inheritance. Standard variant analysis was improved with a gene prioritization strategy based on clinical ontologies and gene co-expression networks. Clinical phenotyping and exploration of cross-species similarities were further performed on a family-by-family basis. Statistical validation was performed on 248 ancestrally similar control trios provided by the Genome of the Netherlands project and on 574 ancestrally matched controls provided by the French Exome Project. Variants of clinical interest were identified in 180 genes significantly associated with key pathways of forebrain development including sonic hedgehog (SHH) and primary cilia. Oligogenic events were observed in 10 families and involved both known and novel holoprosencephaly genes including recurrently mutated FAT1, NDST1, COL2A1 and SCUBE2. The incidence of oligogenic combinations was significantly higher in holoprosencephaly patients compared to two control populations (P < 10-9). We also show that depending on the affected genes, patients present with particular clinical features. This study reports novel disease genes and supports oligogenicity as clinically relevant model in holoprosencephaly. It also highlights key roles of SHH signalling and primary cilia in forebrain development. We hypothesize that distinction between different clinical manifestations of holoprosencephaly lies in the degree of overall functional impact on SHH signalling. Finally, we underline that integrating clinical phenotyping in genetic studies is a powerful tool to specify the clinical relevance of certain mutations.

A de novo variant in ADGRL2 suggests a novel mechanism underlying the previously undescribed association of extreme microcephaly with severely reduced sulcation and rhombencephalosynapsis.

Extreme microcephaly and rhombencephalosynapsis represent unusual pathological conditions, each of which occurs in isolation or in association with various other cerebral and or extracerebral anomalies. Unlike microcephaly for which several disease-causing genes have been identified with different modes of inheritance, the molecular bases of rhombencephalosynapsis remain unknown and rhombencephalosynapsis presents mainly as a sporadic condition consistent with de novo dominant variations. We report for the first time the association of extreme microcephaly with almost no sulcation and rhombencephalosynapsis in a fœtus for which comparative patient-parent exome sequencing strategy revealed a heterozygous de novo missense variant in the ADGRL2 gene. ADGRL2 encodes latrophilin 2, an adhesion G-protein-coupled receptor whose exogenous ligand is α-latrotoxin. Adgrl2 immunohistochemistry and in situ hybridization revealed expression in the telencephalon, mesencephalon and rhombencephalon of mouse and chicken embryos. In human brain embryos and fœtuses, Adgrl2 immunoreactivity was observed in the hemispheric and cerebellar germinal zones, the cortical plate, basal ganglia, pons and cerebellar cortex. Microfluorimetry experiments evaluating intracellular calcium release in response to α-latrotoxin binding showed significantly reduced cytosolic calcium release in the fœtus amniocytes vs amniocytes from age-matched control fœtuses and in HeLa cells transfected with mutant ADGRL2 cDNA vs wild-type construct. Embryonic lethality was also observed in constitutive Adgrl2-/- mice. In Adgrl2+/- mice, MRI studies revealed microcephaly and vermis hypoplasia. Cell adhesion and wound healing assays demonstrated that the variation increased cell adhesion properties and reduced cell motility. Furthermore, HeLa cells overexpressing mutant ADGRL2 displayed a highly developed cytoplasmic F-actin network related to cytoskeletal dynamic modulation. ADGRL2 is the first gene identified as being responsible for extreme microcephaly with rhombencephalosynapsis. Increased cell adhesion, reduced cell motility and cytoskeletal dynamic alterations induced by the variant therefore represent a new mechanism responsible for microcephaly.

Recent advances in understanding inheritance of holoprosencephaly.

Holoprosencephaly (HPE) is a complex genetic disorder of the developing forebrain characterized by high phenotypic and genetic heterogeneity. HPE was initially defined as an autosomal dominant disease, but recent research has shown that its mode of transmission is more complex. The past decade has witnessed rapid development of novel genetic technologies and significant progresses in clinical studies of HPE. In this review, we recapitulate genetic epidemiological studies of the largest European HPE cohort and summarize the novel genetic discoveries of HPE based on recently developed diagnostic methods. Our main purpose is to present different inheritance patterns that exist for HPE with a particular emphasis on oligogenic inheritance and its implications in genetic counseling.

Regulation of downstream neuronal genes by proneural transcription factors during initial neurogenesis in the vertebrate brain.

BACKGROUND: Neurons arise in very specific regions of the neural tube, controlled by components of the Notch signalling pathway, proneural genes, and other bHLH transcription factors. How these specific neuronal areas in the brain are generated during development is just beginning to be elucidated. Notably, the critical role of proneural genes during differentiation of the neuronal populations that give rise to the early axon scaffold in the developing brain is not understood. The regulation of their downstream effectors remains poorly defined. RESULTS: This study provides the first overview of the spatiotemporal expression of proneural genes in the neuronal populations of the early axon scaffold in both chick and mouse. Overexpression studies and mutant mice have identified a number of specific neuronal genes that are targets of proneural transcription factors in these neuronal populations. CONCLUSION: Together, these results improve our understanding of the molecular mechanisms involved in differentiation of the first neuronal populations in the brain.

Mutational Spectrum in Holoprosencephaly Shows That FGF is a New Major Signaling Pathway.

Holoprosencephaly (HPE) is the most common congenital cerebral malformation in humans, characterized by impaired forebrain cleavage and midline facial anomalies. It presents a high heterogeneity, both in clinics and genetics. We have developed a novel targeted next-generation sequencing (NGS) assay and screened a cohort of 257 HPE patients. Mutations with high confidence in their deleterious effect were identified in approximately 24% of the cases and were held for diagnosis, whereas variants of uncertain significance were identified in 10% of cases. This study provides a new classification of genes that are involved in HPE. SHH, ZIC2, and SIX3 remain the top genes in term of frequency with GLI2, and are followed by FGF8 and FGFR1. The three minor HPE genes identified by our study are DLL1, DISP1, and SUFU. Here, we demonstrate that fibroblast growth factor signaling must now be considered a major pathway involved in HPE. Interestingly, several cases of double mutations were found and argue for a polygenic inheritance of HPE. Altogether, it supports that the implementation of NGS in HPE diagnosis is required to improve genetic counseling.

Evolutionary Conservation of the Early Axon Scaffold in the Vertebrate Brain.

The early axon scaffold is the first axonal structure to appear in the rostral brain of vertebrates, paving the way for later, more complex connections. Several early axon scaffold components are conserved between all vertebrates; most notably two main ventral longitudinal tracts, the tract of the postoptic commissure and the medial longitudinal fascicle. While the overall structure is remarkably similar, differences both in the organization and the development of the early tracts are apparent. This review will bring together extensive data from the last 25 years in different vertebrates and for the first time, the timing and anatomy of these early tracts have been directly compared. Representatives of major vertebrate clades, including cat shark, Xenopus, chick, and mouse embryos, will be compared using immunohistochemistry staining based on previous results. There is still confusion over the nomenclature and homology of these tracts which this review will aim to address. The discussion here is relevant both for understanding the evolution of the early axon scaffold and for future studies into the molecular regulation of its formation.

Homozygous STIL mutation causes holoprosencephaly and microcephaly in two siblings.

Holoprosencephaly (HPE) is a frequent congenital malformation of the brain characterized by impaired forebrain cleavage and midline facial anomalies. Heterozygous mutations in 14 genes have been identified in HPE patients that account for only 30% of HPE cases, suggesting the existence of other HPE genes. Data from homozygosity mapping and whole-exome sequencing in a consanguineous Turkish family were combined to identify a homozygous missense mutation (c.2150G>A; p.Gly717Glu) in STIL, common to the two affected children. STIL has a role in centriole formation and has previously been described in rare cases of microcephaly. Rescue experiments in U2OS cells showed that the STIL p.Gly717Glu mutation was not able to fully restore the centriole duplication failure following depletion of endogenous STIL protein indicating the deleterious role of the mutation. In situ hybridization experiments using chick embryos demonstrated that expression of Stil was in accordance with a function during early patterning of the forebrain. It is only the second time that a STIL homozygous mutation causing a recessive form of HPE was reported. This result also supports the genetic heterogeneity of HPE and increases the panel of genes to be tested for HPE diagnosis.

Notch signaling and proneural genes work together to control the neural building blocks for the initial scaffold in the hypothalamus.

The vertebrate embryonic prosencephalon gives rise to the hypothalamus, which plays essential roles in sensory information processing as well as control of physiological homeostasis and behavior. While patterning of the hypothalamus has received much attention, initial neurogenesis in the developing hypothalamus has mostly been neglected. The first differentiating progenitor cells of the hypothalamus will give rise to neurons that form the nucleus of the tract of the postoptic commissure (nTPOC) and the nucleus of the mammillotegmental tract (nMTT). The formation of these neuronal populations has to be highly controlled both spatially and temporally as these tracts will form part of the ventral longitudinal tract (VLT) and act as a scaffold for later, follower axons. This review will cumulate and summarize the existing data available describing initial neurogenesis in the vertebrate hypothalamus. It is well-known that the Notch signaling pathway through the inhibition of proneural genes is a key regulator of neurogenesis in the vertebrate central nervous system. It has only recently been proposed that loss of Notch signaling in the developing chick embryo causes an increase in the number of neurons in the hypothalamus, highlighting an early function of the Notch pathway during hypothalamus formation. Further analysis in the chick and mouse hypothalamus confirms the expression of Notch components and Ascl1 before the appearance of the first differentiated neurons. Many newly identified proneural target genes were also found to be expressed during neuronal differentiation in the hypothalamus. Given the critical role that hypothalamic neural circuitry plays in maintaining homeostasis, it is particularly important to establish the targets downstream of this Notch/proneural network.

Dynamic expression of Notch-dependent neurogenic markers in the chick embryonic nervous system.

The establishment of a functional nervous system requires a highly orchestrated process of neural proliferation and differentiation. The evolutionary conserved Notch signaling pathway is a key regulator of this process, regulating basic helix-loop-helix (bHLH) transcriptional repressors and proneural genes. However, little is known about downstream Notch targets and subsequently genes required for neuronal specification. In this report, the expression pattern of Transgelin 3 (Tagln3), Chromogranin A (Chga) and Contactin 2 (Cntn2) was described in detail during early chick embryogenesis. Expression of these genes was largely restricted to the nervous system including the early axon scaffold populations, cranial ganglia and spinal motor neurons. Their temporal and spatial expression were compared with the neuronal markers Nescient Helix-Loop-Helix 1 (Nhlh1), Stathmin 2 (Stmn2) and HuC/D. We show that Tagln3 is an early marker for post-mitotic neurons whereas Chga and Cntn2 are expressed in mature neurons. We demonstrate that inhibition of Notch signaling during spinal cord neurogenesis enhances expression of these markers. This data demonstrates that Tagln3, Chga and Cntn2 represent strong new candidates to contribute to the sequential progression of vertebrate neurogenesis.

Novel genes upregulated when NOTCH signalling is disrupted during hypothalamic development.

BACKGROUND: The generation of diverse neuronal types and subtypes from multipotent progenitors during development is crucial for assembling functional neural circuits in the adult central nervous system. It is well known that the Notch signalling pathway through the inhibition of proneural genes is a key regulator of neurogenesis in the vertebrate central nervous system. However, the role of Notch during hypothalamus formation along with its downstream effectors remains poorly defined. RESULTS: Here, we have transiently blocked Notch activity in chick embryos and used global gene expression analysis to provide evidence that Notch signalling modulates the generation of neurons in the early developing hypothalamus by lateral inhibition. Most importantly, we have taken advantage of this model to identify novel targets of Notch signalling, such as Tagln3 and Chga, which were expressed in hypothalamic neuronal nuclei. CONCLUSIONS: These data give essential advances into the early generation of neurons in the hypothalamus. We demonstrate that inhibition of Notch signalling during early development of the hypothalamus enhances expression of several new markers. These genes must be considered as important new targets of the Notch/proneural network.

NODAL and SHH dose-dependent double inhibition promotes an HPE-like phenotype in chick embryos.

Holoprosencephaly (HPE) is a common congenital defect that results from failed or incomplete forebrain cleavage. HPE is characterized by a wide clinical spectrum, with inter- and intrafamilial variability. This heterogeneity is not well understood and it has been suggested that HPE involves a combination of multiple gene mutations. In this model, several mutated alleles or modifying factors are presumed to act in synergy to cause and determine the severity of HPE. This could explain the various clinical phenotypes. Screening for HPE-associated genes in humans suggests the involvement of NODAL or SHH signaling, or both. To test this multigenic hypothesis, we investigated the effects of chemical inhibition of these two main HPE signaling pathways in a chick embryo model. SB-505124, a selective inhibitor of transforming growth factor-B type I receptors was used to inhibit the NODAL pathway. Cyclopamine was used to inhibit the SHH pathway. We report that both inhibitors caused HPE-like defects that were dependent on the drug concentration and on the developmental stage at the time of treatment. We also investigated double inhibition of NODAL and SHH pathways from the onset of gastrulation by using subthreshold inhibitor concentrations. The inhibitors of the NODAL and SHH pathways, even at low concentration, acted synergistically to promote an HPE-like phenotype. These findings support the view that genetic heterogeneity is important in the etiology of HPE and may contribute to the phenotypic variability.

New findings for phenotype-genotype correlations in a large European series of holoprosencephaly cases.

BACKGROUND: Holoprosencephaly (HPE) is the most common forebrain defect in humans. It results from incomplete midline cleavage of the prosencephalon. METHODS: A large European series of 645 HPE probands (and 699 relatives), consisting of 51% fetuses and 49% liveborn children, is reported. RESULTS: Mutations in the four main genes involved in HPE (SHH, ZIC2, SIX3, TGIF) were identified in 25% of cases. The SHH, SIX3, and TGIF mutations were inherited in more than 70% of these cases, whereas 70% of the mutations in ZIC2 occurred de novo. Moreover, rearrangements were detected in 22% of the 260 patients screened by array comparative genomic hybridisation. 15 probands had two mutations providing additional support for the 'multiple-hit process' in HPE. There was a positive correlation between the severity of the brain malformation and facial features for SHH, SIX3, and TGIF, but no such correlation was found for ZIC2 mutations. The most severe HPE types were associated with SIX3 and ZIC2 mutations, whereas microforms were associated with SHH mutations. The study focused on the associated brain malformations, including neuronal migration defects, which predominated in individuals with ZIC2 mutations, and neural tube defects, which were frequently associated with ZIC2 (rachischisis) and TGIF mutations. Extracraniofacial features were observed in 27% of the individuals in this series (up to 40% of those with ZIC2 mutations) and a significant correlation was found between renal/urinary defects and mutations of SHH and ZIC2. CONCLUSIONS: An algorithm is proposed based on these new phenotype-genotype correlations, to facilitate molecular analysis and genetic counselling for HPE.