A Specific CNOT1 Mutation Results in a Novel Syndrome of Pancreatic Agenesis and Holoprosencephaly through Impaired Pancreatic and Neurological Development.

We report a recurrent CNOT1 de novo missense mutation, GenBank: NM_016284.4; c.1603C>T (p.Arg535Cys), resulting in a syndrome of pancreatic agenesis and abnormal forebrain development in three individuals and a similar phenotype in mice. CNOT1 is a transcriptional repressor that has been suggested as being critical for maintaining embryonic stem cells in a pluripotent state. These findings suggest that CNOT1 plays a critical role in pancreatic and neurological development and describe a novel genetic syndrome of pancreatic agenesis and holoprosencephaly.

The Role of Sonic Hedgehog in Human Holoprosencephaly and Short-Rib Polydactyly Syndromes.

The Hedgehog (HH) signalling pathway is one of the major pathways controlling cell differentiation and proliferation during human development. This pathway is complex, with HH function influenced by inhibitors, promotors, interactions with other signalling pathways, and non-genetic and cellular factors. Many aspects of this pathway are not yet clarified. The main features of Sonic Hedgehog (SHH) signalling are discussed in relation to its function in human development. The possible role of SHH will be considered using examples of holoprosencephaly and short-rib polydactyly (SRP) syndromes. In these syndromes, there is wide variability in phenotype even with the same genetic mutation, so that other factors must influence the outcome. SHH mutations were the first identified genetic causes of holoprosencephaly, but many other genes and environmental factors can cause malformations in the holoprosencephaly spectrum. Many patients with SRP have genetic defects affecting primary cilia, structures found on most mammalian cells which are thought to be necessary for canonical HH signal transduction. Although SHH signalling is affected in both these genetic conditions, there is little overlap in phenotype. Possible explanations will be canvassed, using data from published human and animal studies. Implications for the understanding of SHH signalling in humans will be discussed.

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.

Nongenetic risk factors for holoprosencephaly: An updated review of the epidemiologic literature.

Holoprosencephaly (HPE) is a major structural birth defect of the brain that occurs in approximately 1 in 10,000 live births. Although some genetic causes of HPE are known, a substantial proportion of cases have an unknown etiology. Due to the low birth prevalence and rarity of exposure to many potential risk factors for HPE, few epidemiologic studies have had sufficient sample size to examine risk factors. A 2010 review of the literature identified several risk factors that had been consistently identified as occurring more frequently among cases of HPE, including maternal diabetes, twinning, and a predominance of females, while also identifying a number of potential risk factors that had been less widely studied. In this article, we summarize a systematic literature review conducted to update the evidence for nongenetic risk factors for HPE.

Holoprosencephaly: A clinical genomics perspective.

New and rapidly evolving technologies have dramatically impacted the practice of clinical genetics as well as broader areas of medicine. To illustrate this trend from the perspective of a clinical molecular laboratory, we briefly summarize our general experience conducting exome testing for patients with holoprosencephaly (HPE). Though these cases are not representative of HPE more generally (i.e., cases undergoing exome sequencing represent a skewed sample), results include a 22% positive rate from exome testing. Of interest, 29% of reported results involved genes not considered to be classic HPE genes, indicating more evidence that HPE may fall within the severe spectrum of many other genetic conditions.

Extracephalic manifestations of nonchromosomal, nonsyndromic holoprosencephaly.

Nonchromosomal, nonsyndromic holoprosencephaly (NCNS-HPE) has traditionally been considered as a condition of brain and craniofacial maldevelopment. In this review, we present the results of a comprehensive literature search supporting a wide spectrum of extracephalic manifestations identified in patients with NCNS-HPE. These manifestations have been described in case reports and in large cohorts of patients with "single-gene" mutations, suggesting that the NCNS-HPE phenotype can be more complex than traditionally thought. Likely, a complex network of interacting genetic variants and environmental factors is responsible for these systemic abnormalities that deviate from the usual brain and craniofacial findings in NCNS-HPE. In addition to the systemic consequences of pituitary dysfunction (as a direct result of brain midline defects), here we describe a number of extracephalic findings of NCNS-HPE affecting various organ systems. It is our goal to provide a guide of extracephalic features for clinicians given the important clinical implications of these manifestations for the management and care of patients with HPE and their mutation-positive relatives. The health risks associated with some manifestations (e.g., fatty liver disease) may have historically been neglected in affected families.

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.

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.

Neuropathology of holoprosencephaly.

Holoprosencephaly (HPE) is a primary disorder of neural induction and patterning of the rostral neural tube resulting in noncleavage of the forebrain with failure to form two separate distinct hemispheres. The spectrum of HPE is very broad and encompasses various neuropathological phenotypes of different severity. The recent literature has demonstrated that the phenotypic variability of HPE ranges from aprosencephaly-atelencephaly, at the most severe end, to milder forms such as the "middle interhemispheric variant" of HPE at the less severe end of the spectrum. Between them, different intermediate forms demonstrate a continuum in a wide phenotypic spectrum rather than well-defined categories. Although the term "HPE" suggests a disorder affecting only the prosencephalon, other brain structures are involved, underlining the complexity of the malformation. Because of close spatiotemporal interactions and common signaling pathways contributing to the development of both brain and face, concomitant facial and ocular anomalies are associated with brain malformation. In this review, the characteristic neuropathological features of the various forms of HPE are described as well as their associated brain, face, and ocular malformations, to delineate the different phenotypes.

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.

A familial GLI2 deletion (2q14.2) not associated with the holoprosencephaly syndrome phenotype.

Molecular alterations of the GLI2 gene in 2q14.2 are associated with features from the holoprosencephaly spectrum. However, the phenotype is extremely variable, ranging from unaffected mutation heterozygotes to isolated or combined pituitary hormone deficiency, and to patients with a phenotype that overlaps with holoprosencephaly, including abnormal pituitary gland formation/function, craniofacial dysmorphisms, branchial arch anomalies, and polydactyly. Although many point mutations within the GLI2 gene have been identified, large (sub) microscopic deletions affecting 2q14.2 are rare. We report on a family with a 4.3 Mb deletion in 2q14 affecting GLI2 without any dysmorphologic features belonging to the holoprosencephaly spectrum. This family confirms the incomplete penetrance of genomic disturbances affecting the GLI2 gene. However, the family presented here is unique as none of the three identified individuals with a GLI2 deletion showed any typical signs of holoprosencephaly, whereas all patients reported so far were referred for genetic testing because at least one member exhibited holoprosencephaly and related features.

Holoprosencephaly: a survey of the entity, with embryology and fetal imaging.

Structural malformations of the brain are an important cause of childhood mortality and morbidity, with the latter having long-term financial and psychosocial implications for the affected child and family. Holoprosencephaly (HPE) is a severe brain malformation characterized by abnormal cleavage of the prosencephalon in the 5th gestational week. Aprosencephaly and atelencephaly occur earlier because of failure in the formation of the prosencephalon and telencephalon, respectively. The HPE holoprosencephaly spectrum classically includes alobar, semilobar, and lobar forms, although there are no clear-cut defining features. The middle interhemispheric variant (MIH), also known as syntelencephaly, is classified as a variant of HPE holoprosencephaly with midline interhemispheric fusion. Other conditions sometimes included in the spectrum of HPE holoprosencephaly include septo-optic dysplasia (SOD); "minimal" HPE holoprosencephaly , which is associated with subtle craniofacial malformations and mild developmental delay; and microform HPE holoprosencephaly , which by definition excludes brain involvement. The focus of this article will be on the spectrum of findings visible in fetal manifestation of the HPE holoprosencephaly spectrum. Brain embryology; the imaging characteristics, epidemiology, and embryology of HPE; and the more common associated anomalies, particularly those of the face ("the face predicts the brain") are reviewed. Recognition of these anomalies is important for accurate parental counseling, since the prognosis is poor but not invariably lethal; children with the milder forms may live well into their teens with severe developmental delays, endocrine dysfunction, and disrupted homeostasis. Available data on outcome in surviving children are summarized. Illustrative fetal ultrasonographic and magnetic resonance images are presented with clinical, autopsy, and postnatal imaging correlation.

Loss of Tgif function causes holoprosencephaly by disrupting the SHH signaling pathway.

Holoprosencephaly (HPE) is a severe human genetic disease affecting craniofacial development, with an incidence of up to 1/250 human conceptions and 1.3 per 10,000 live births. Mutations in the Sonic Hedgehog (SHH) gene result in HPE in humans and mice, and the Shh pathway is targeted by other mutations that cause HPE. However, at least 12 loci are associated with HPE in humans, suggesting that defects in other pathways contribute to this disease. Although the TGIF1 (TG-interacting factor) gene maps to the HPE4 locus, and heterozygous loss of function TGIF1 mutations are associated with HPE, mouse models have not yet explained how loss of Tgif1 causes HPE. Using a conditional Tgif1 allele, we show that mouse embryos lacking both Tgif1 and the related Tgif2 have HPE-like phenotypes reminiscent of Shh null embryos. Eye and nasal field separation is defective, and forebrain patterning is disrupted in embryos lacking both Tgifs. Early anterior patterning is relatively normal, but expression of Shh is reduced in the forebrain, and Gli3 expression is up-regulated throughout the neural tube. Gli3 acts primarily as an antagonist of Shh function, and the introduction of a heterozygous Gli3 mutation into embryos lacking both Tgif genes partially rescues Shh signaling, nasal field separation, and HPE. Tgif1 and Tgif2 are transcriptional repressors that limit Transforming Growth Factor ß/Nodal signaling, and we show that reducing Nodal signaling in embryos lacking both Tgifs reduces the severity of HPE and partially restores the output of Shh signaling. Together, these results support a model in which Tgif function limits Nodal signaling to maintain the appropriate output of the Shh pathway in the forebrain. These data show for the first time that Tgif1 mutation in mouse contributes to HPE pathogenesis and provide evidence that this is due to disruption of the Shh pathway.

Mutations in SUFU predispose to medulloblastoma.

The sonic hedgehog (SHH) signaling pathway directs the embryonic development of diverse organisms and is disrupted in a variety of malignancies. Pathway activation is triggered by binding of hedgehog proteins to the multipass Patched-1 (PTCH) receptor, which in the absence of hedgehog suppresses the activity of the seven-pass membrane protein Smoothened (SMOH). De-repression of SMOH culminates in the activation of one or more of the GLI transcription factors that regulate the transcription of downstream targets. Individuals with germline mutations of the SHH receptor gene PTCH are at high risk of developmental anomalies and of basal-cell carcinomas, medulloblastomas and other cancers (a pattern consistent with nevoid basal-cell carcinoma syndrome, NBCCS). In keeping with the role of PTCH as a tumor-suppressor gene, somatic mutations of this gene occur in sporadic basal-cell carcinomas and medulloblastomas. We report here that a subset of children with medulloblastoma carry germline and somatic mutations in SUFU (encoding the human suppressor of fused) of the SHH pathway, accompanied by loss of heterozygosity of the wildtype allele. Several of these mutations encode truncated proteins that are unable to export the GLI transcription factor from nucleus to cytoplasm, resulting in the activation of SHH signaling. SUFU is a newly identified tumor-suppressor gene that predisposes individuals to medulloblastoma by modulating the SHH signaling pathway through a newly identified mechanism.

The rare malformation holoprosencephaly: pathogenesis, association with pregestational diabetes and the possible link with food pollutants.

BACKGROUND: Holoprosencephaly is a rare (1/16,000 livebirths) and severe brain malformation occurring during early embryogenesis. The malformation originates from absent or incomplete forebrain division and is associated with altered embryonic patterning. OBJECTIVES: A narrative review to identify and assess the evidence on non-genetic risk factors. RESULTS: Genes involved include sonic hedgehog, Zinc finger protein, SIX homeobox 3. Pregestational diabetes, with periconceptional hyperglycaemia, is the main non-genetic risk factor; increased oxidative stress in neuroectoderm, in particular neural crest cells, appears as the main mechanism. Several widespread pollutants, including inorganic arsenic, PFAS and PCBs, may increase the risk of pregestational diabetes by altering metabolic factors, including lipids and insulin. A scenario "widespread exposures-rare outcomes in susceptible subjects" suggests that exposure to dietary pollutants may increase the risk of pregestational diabetes, hence of holoprosencephaly in susceptible embryos. CONCLUSIONS: This complex pathway is plausible and worth being investigated; moreover, it highlights the importance of assessing risk factors, and the associated uncertainties, in order to support primary prevention strategies for multifactorial malformations.

Aberrant forebrain signaling during early development underlies the generation of holoprosencephaly and coloboma.

In this review, we highlight recent literature concerning the signaling mechanisms underlying the development of two neural birth defects, holoprosencephaly and coloboma. Holoprosencephaly, the most common forebrain defect, occurs when the cerebral hemispheres fail to separate and is typically associated with mispatterning of embryonic midline tissue. Coloboma results when the choroid fissure in the eye fails to close. It is clear that Sonic hedgehog (Shh) signaling regulates both forebrain and eye development, with defects in Shh, or components of the Shh signaling cascade leading to the generation of both birth defects. In addition, other intercellular signaling pathways are known factors in the incidence of holoprosencephaly and coloboma. This review will outline recent advances in our understanding of forebrain and eye embryonic pattern formation, with a focus on zebrafish studies of Shh and retinoic acid pathways. Given the clear overlap in the mechanisms that generate both diseases, we propose that holoprosencephaly and coloboma can represent mild and severe aspects of single phenotypic spectrum resulting from aberrant forebrain development. This article is part of a Special Issue entitled Zebrafish Models of Neurological Diseases.

Risk factors for nonsyndromic holoprosencephaly: a Manitoba case-control study.

Holoprosencephaly (HPE) is one of the most common developmental field defects, occurring in 1 in 250 conceptuses and in 1 in 10,000-20,000 live births. Nearly half of patients with HPE have a recognized syndrome or a single gene defect. However, little is known about the risk factors for the remainder with "nonsyndromic" HPE. In our case-control study, we examine factors associated with nonsyndromic HPE. We identified 47 patients with HPE from the genetics clinic database with an equal number of controls matched for gender and birthdate. Of the 47 patients, 23 were identified as nonsyndromic. No statistically significant differences were noted between the mean maternal and paternal ages of patients and controls. Factors associated with nonsyndromic HPE were: having an Aboriginal mother (unadjusted odds ratio [OR] 3.5, 95% confidence interval [CI] 1.1-11.1), an Aboriginal father (OR 12.8, 95% CI 3.0-55.1), at least one Aboriginal parent (OR 5.0, 95% CI 1.6-16.0), or two Aboriginal parents (OR 8.8, 95% CI 2.0-37.8), the presence of a family history of a midline facial defect (OR 8.2, 95% CI 1.5-45.2), and being of low socioeconomic status (OR 3.0, 95% CI 1.0-9.1). Having an Aboriginal background remained statistically significant after adjusting for low socioeconomic status. Other associations evaluated--history of prior spontaneous abortion, stillbirth, neonatal death, prepregnancy diabetes, infections during pregnancy, alcohol exposure, smoking, and substance abuse--were not significantly associated with nonsyndromic HPE. The use of periconceptional folic acid or vitamins was not associated with a lower risk of nonsyndromic HPE.

Holoprosencephaly in clomiphene-induced pregnancy: a possible association? A case report and literature review.

Clomiphene is widely used for inducing ovulation. Evidence for congenital abnormalities, in particular central nervous system defects (CNS-D) and in babies born from clomiphene-induced pregnancies is conflicting. The authors report a case of holoprosencephalia (HPE) in a fetus delivered from a mother receiving clomiphene.