Ping Hu

Missense substitutions in the GAS1 protein present in holoprosencephaly patients reduce the affinity for its ligand, SHH

Holprosencephaly (HPE) is the most common disorder of the developing forebrain in humans, and is characterized by varying degrees of abnormal union of the cerebral hemispheres. These defects are typically co-associated with midline craniofacial anomalies. The combination of forebrain and craniofacial defects that comprise HPE can present along a broad and variable phenotypic spectrum. Both the SHH and NODAL signaling pathways play important roles in the pathogenesis of this disorder. Disruption of these pathways by chromosomal rearrangements, mutations in pathway-related genes and/or biochemical alterations are proposed to contribute to HPE in a large number of patients. Additional factors that are not yet fully delineated are also very likely to be involved in the pathogenesis and phenotypic heterogeneity of the disorder. Genetic loss of GAS1, a cell membrane receptor and positive regulator of SHH, has been demonstrated to contribute to the HPE phenotypic spectrum in animal models. We have evaluated the coding and flanking sequence of GAS1 in 394 patients who have clinical findings within the HPE phenotypic spectrum, and now report five novel missense sequence variants among five unrelated HPE probands. Finally, we tested the effect of these variants (as well as previously reported GAS1 variants) on the ability of GAS1 to bind to SHH. Here, we demonstrate that sequence variants in GAS1 can impair its physical interaction with SHH, suggesting a decrease in the SHH downstream signaling cascade as a pathogenic mechanism of disease.

Heterozygous mutations in SIX3 and SHH are associated with schizencephaly and further expand the clinical spectrum of holoprosencephaly.

Schizencephaly (SCH) is a clinically and etiologically heterogeneous cerebral malformation presenting as unilateral or bilateral hemispheric cleft with direct connection between the inner and outer liquor spaces. The SCH cleft is usually lined by gray matter, which appears polymicrogyric implying an associated impairment of neuronal migration. The majority of SCH patients are sporadic, but familial SCH has been described. An initial report of heterozygous mutations in the homeobox gene EMX2 could not be confirmed in 52 patients investigated in this study in agreement with two independent SCH patient cohorts published previously. SCH frequently occurs with additional cerebral malformations like hypoplasia or aplasia of the septum pellucidum or optic nerve, suggesting the involvement of genes important for the establishment of midline forebrain structures. We therefore considered holoprosencephaly (HPE)-associated genes as potential SCH candidates and report for the first time heterozygous mutations in SIX3 and SHH in a total of three unrelated patients and one fetus with SCH; one of them without obvious associated malformations of midline forebrain structures. Three of these mutations have previously been reported in independent patients with HPE. SIX3 acts directly upstream of SHH, and the SHH pathway is a key regulator of ventral forebrain patterning. Our data indicate that in a subset of patients SCH may develop as one aspect of a more complex malformation of the ventral forebrain, directly result from mutations in the SHH pathway and hence be considered as yet another feature of the broad phenotypic spectrum of holoprosencephaly.

Holoprosencephaly in a family segregating novel variants in ZIC2 and GLI2.

Holoprosencephaly (HPE) is the most common malformation of the human forebrain. Typical manifestations in affected patients include a characteristic pattern of structural brain and craniofacial anomalies. HPE may be caused by mutations in over 10 identified genes; the inheritance is traditionally viewed as autosomal dominant with highly variable expressivity and incomplete penetrance. We present the description of a family simultaneously segregating two novel variants in the HPE‐associated genes, ZIC2 and GLI2, as well as the results of extensive population‐based studies of the variant region in GLI2. This is the first time that multiple HPE‐associated variants in these genes have been reported in one family, and raises important questions about how clinicians and researchers should view the inheritance of conditions such as HPE.

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.

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.

In-depth investigations of adolescents and adults with holoprosencephaly identify unique characteristics

PurposeWith improved medical care, some individuals with holoprosencephaly (HPE) are surviving into adulthood. We investigated the clinical manifestations of adolescents and adults with HPE and explored the underlying molecular causes.MethodsParticipants included 20 subjects 15 years of age and older. Clinical assessments included dysmorphology exams, cognitive testing, swallowing studies, ophthalmic examination, and brain magnetic resonance imaging. Genetic testing included chromosomal microarray, Sanger sequencing for SHH, ZIC2, SIX3, and TGIF, and whole-exome sequencing (WES) of 10 trios.ResultsSemilobar HPE was the most common subtype of HPE, seen in 50% of the participants. Neurodevelopmental disabilities were found to correlate with HPE subtype. Factors associated with long-term survival included HPE subtype not alobar, female gender, and nontypical facial features. Four participants had de novo pathogenic variants in ZIC2. WES analysis of 11 participants did not reveal plausible candidate genes, suggesting complex inheritance in these cases. Indeed, in two probands there was a history of uncontrolled maternal type 1 diabetes.ConclusionIndividuals with various HPE subtypes can survive into adulthood and the neurodevelopmental outcomes are variable. Based on the facial characteristics and molecular evaluations, we suggest that classic genetic causes of HPE may play a smaller role in this cohort.

Comparison of mutation findings in ZIC2 between microform and classical holoprosencephaly in a Brazilian cohort.

BACKGROUND Holoprosencephaly is the most frequent congenital malformation of the forebrain in humans. It is anatomically classified by the relative degree of abnormal formation and separation of the developing central nervous system. Mutations of ZIC2 are the second most common heterozygous variations detected in holoprosencephaly (HPE) patients. Mutations in most known HPE genes typically result in variable phenotypes that rage from classic alobar HPE to microforms represented by hypotelorism, solitary central maxillary incisor (SCMI), and cleft lip/palate, among others. Patients with HPE owing to ZIC2 mutations have recently been described by a distinct phenotype compared with mutations in other HPE causative genes. METHODS We report the comparison of ZIC2 molecular findings by Sanger bidirectional DNA sequencing and ad hoc genotyping in a cohort of 105 Brazilian patients within the clinical spectrum of HPE, including classic and microform groups. RESULTS We detected a total of five variants in the ZIC2 gene: a common histidine tract expansion c.716_718dup (p.His239dup), a rare c.1377_1391del_homozygous (p.Ala466_470del, or Ala 15 to 10 contraction), a novel intronic c.1239+18G>A variant, a novel frameshift c.1215dupC (p.Ser406Glnfs*11), and a c.1401_1406dup (p.Ala469_470dup, or alanine tract expansion to 17 residues). CONCLUSIONS From these patients, only the latter two mutations found in classic HPE are likely to be medically significant. In contrast, variants detected in the microform group are not likely to be pathogenic. We show conclusively that the histidine tract expansion is a polymorphic alteration that demonstrates considerable differences in allele frequencies across different ethnic groups. Therefore, careful population studies of rare variants can improve genotype-phenotype correlations. Birth Defects Research (Part A) 2012.

Loss-of-function mutations in FGF8 can be independent risk factors for holoprosencephaly

The utilization of next generation sequencing has been shown to accelerate gene discovery in human disease. However, our confidence in the correct disease-associations of rare variants continues to depend on functional analysis. Here, we employ a sensitive assay of human FGF8 variants in zebrafish to demonstrate that the spectrum of isoforms of FGF8 produced by alternative splicing can provide key insights into the genetic susceptibility to human malformations. In addition, we describe novel mutations in the FGF core structure that have both subtle and profound effects on ligand posttranslational processing and biological activity. Finally, we solve a case of apparent digenic inheritance of novel variants in SHH and FGF8, two genes known to functionally coregulate each other in the developing forebrain, as a simpler case of FGF8 diminished function.

Holoprosencephaly in the genomics era

Holoprosencephaly (HPE) is the direct consequence of specific genetic and/or environmental insults interrupting the midline specification of the nascent forebrain. Such disturbances can lead to a broad range of phenotypic consequences for the brain and face in humans. This malformation sequence is remarkably common in utero (1 in 250 human fetuses), but 97% typically do not survive to birth. The precise molecular pathogenesis of HPE in these early human embryos remains largely unknown. Here, we outline our current understanding of the principal driving factors leading to HPE pathologies and elaborate our multifactorial integrated genomics approach. Overall, our understanding of the pathogenesis continues to become simpler, rather than more complicated. Genomic technologies now provide unprecedented insight into disease‐associated variation, including the overall extent of genetic interactions (coding and noncoding) predicted to explain divergent phenotypes.

Molecular analysis of the Noggin (NOG) gene in holoprosencephaly patients

Holoprosencephaly (HPE) is the most common structural anomaly of the human forebrain. Various genetic and teratogenic causes have been implicated in its pathogenesis. A recent report in mice described Noggin (NOG) as a candidate gene involved in the etiogenesis of microform HPE. Here, we present for the first time genetic analysis of a large HPE cohort for sequence variations in NOG. On the basis of our study, we conclude that mutations in the coding region of NOG are rare, and play at most an uncommon role in human HPE.