New insights into genotype–phenotype correlation for GLI3 mutations

The phenotypic spectrum of GLI3 mutations includes autosomal dominant Greig cephalopolysyndactyly syndrome (GCPS) and Pallister–Hall syndrome (PHS). PHS was first described as a lethal condition associating hypothalamic hamartoma, postaxial or central polydactyly, anal atresia and bifid epiglottis. Typical GCPS combines polysyndactyly of hands and feet and craniofacial features. Genotype–phenotype correlations have been found both for the location and the nature of GLI3 mutations, highlighting the bifunctional nature of GLI3 during development. Here we report on the molecular and clinical study of 76 cases from 55 families with either a GLI3 mutation (49 GCPS and 21 PHS), or a large deletion encompassing the GLI3 gene (6 GCPS cases). Most of mutations are novel and consistent with the previously reported genotype–phenotype correlation. Our results also show a correlation between the location of the mutation and abnormal corpus callosum observed in some patients with GCPS. Fetal PHS observations emphasize on the possible lethality of GLI3 mutations and extend the phenotypic spectrum of malformations such as agnathia and reductional limbs defects. GLI3 expression studied by in situ hybridization during human development confirms its early expression in target tissues.     

Molecular analysis of non-syndromic preaxial polydactyly: preaxial polydactyly type-IV and preaxial polydactyly type-I

Human GLI3 gene mutations have been identified in several phenotypes of digital abnormality such as Greig cephalopolysyndactyly syndrome, Pallister-Hall syndrome, preaxial polydactyly type-IV (PPD-IV) and postaxial polydactyly. However, the different phenotypes resulting from GLI3 mutations have not yet been properly defined. We have experienced two types of digital abnormality without other complicating developmental defects; a family with foot PPD-IV with syndactyly of the third and fourth fingers, and four sporadic cases with biphalangeal thumb polydactyly (PPD-I). The genes responsible for syndactyly of the third and fourth fingers (syndactyly type-I) and PPD-I have not yet been identified; we therefore examined the involvement of the GLI3 gene in these subtypes of digital abnormality. We found a non-sense mutation in the GLI3 gene in the family with foot PPD-IV accompanied with hand syndactyly of the third and fourth fingers, but no mutations were detected in the GLI3 gene in the four other cases with PPD-I alone. Thus, the phenotype of foot PPD-IV accompanied with hand syndactyly of the third and fourth fingers may result from a GLI3 mutation, whereas the PPD-I phenotype alone is not caused by GLI3 gene defect. These results will help to define the phenotypic spectrum of GLI3 morphopathies, which have been recently proposed.     

GLI3‐related polydactyly: a review

GLI3 mutations are known to be associated with nine syndromes/conditions in which polydactyly is a feature. In this review, the embryology, pathogenesis, and animal models of GLI3‐related polydactyly are discussed first. This is followed by a detailed review of the genotype–phenotype correlations. Based on our review of the literature and our clinical experiences, we recommend viewing GLI3‐related syndromes/conditions as four separate entities; each characterized by a specific pattern of polydactyly. These four entities are: the preaxial polydactyly type IV‐Greig‐acrocallosal spectrum, postaxial polydactyly types A/B, Pallister–Hall syndrome (PHS), and oral‐facial‐digital overlap syndrome. We also provide illustrative clinical examples from our practice including a family with a novel GLI3 mutation causing PHS. The review also introduces the term ‘Forme Fruste’ preaxial polydactyly and gives several conclusions/recommendations including the recommendation to revise the current criteria for the clinical diagnosis of PHS.     

Point mutations in human GLI3 cause Greig syndrome

Greig cephalopolysyndactyly syndrome (GCPS, MIM 175700) is a rare autosomal dominant developmental disorder characterized by craniofacial abnormalities and post-axial and pre-axial polydactyly as well as syndactyly of hands and feet. Human GLI3, located on chromosome 7p13, is a candidate gene for the syndrome because it is interrupted by translocation breakpoints associated with GCPS. Since hemizygosity of 7p13 resulting in complete loss of one copy of GLI3 causes GCPS as well, haploinsufficiency of this gene was implicated as a mechanism to cause this developmental malformation. To determine if point mutations within GLI3 could be responsible for GCPS we describe the genomic sequences at the boundaries of the 15 exons and primer pair sequences for mutation analysis with polymerase chain reaction-based assays of the entire GLI3 coding sequences. In two GCPS cases, both of which did not exhibit obvious cytogenetic rearrangements, point mutations were identified in different domains of the protein, showing for the first time that Greig syndrome can be caused by GLI3 point mutations. In one case a nonsense mutation in exon X generates a stop codon truncating the protein in the C-H link of the first zinc finger. In the second case a missense mutation in exon XIV causes a Pro-->Ser replacement at a position that is conserved among GLI genes from several species altering a potential phosphorylation site.      

Asymptomatic laryngeal malformations are common in patients with Pallister-Hall syndrome

Pallister-Hall syndrome (PHS) comprises hypothalamic hamartoma, polydactyly, pituitary dysfunction, laryngotracheal cleft, imperforate anus, and other anomalies. Some patients with PHS have a bifid epiglottis, a rare malformation. Greig cephalopolysyndactyly syndrome (GCPS) comprises polydactyly with craniofacial malformations without the PHS malformations. Both disorders are caused by mutations in the GLI3 gene. Laryngoscopy on 26 subjects with PHS showed that 15 had a bifid or cleft epiglottis (58%) and none of 14 subjects with GCPS had a cleft epiglottis. The malformed epiglottis was asymptomatic in all of the prospectively evaluated subjects. One additional PHS subject was found to have bifid epiglottis and a posterior laryngeal cleft on autopsy. We conclude that bifid epiglottis is common in PHS but not GCPS. Posterior laryngeal clefts are an uncommon manifestation of PHS and are identified only in severely affected patients. The diagnosis of a bifid epiglottis should prompt a thorough search for other sometimes asymptomatic anomalies of PHS to provide better medical care and recurrence risk assessment for affected individuals and families.     

Preaxial polydactyly caused by Gli3 haploinsufficiency is rescued by Zic3 loss of function in mice

Limb anomalies are important birth defects that are incompletely understood genetically and mechanistically. GLI3, a mediator of hedgehog signaling, is a genetic cause of limb malformations including pre- and postaxial polydactyly, Pallister-Hall syndrome and Greig cephalopolysyndactyly. A closely related Gli (glioma-associated oncogene homolog)-superfamily member, ZIC3, causes X-linked heterotaxy syndrome in humans but has not been investigated in limb development. During limb development, post-translational processing of Gli3 from activator to repressor antagonizes and posteriorly restricts Sonic hedgehog (Shh). We demonstrate that Zic3 and Gli3 expression overlap in developing limbs and that Zic3 converts Gli3 from repressor to activator in vitro. In Gli3 mutant mice, Zic3 loss of function abrogates ectopic Shh expression in anterior limb buds, limits overexpression in the zone of polarizing activity and normalizes aberrant Gli3 repressor/Gli3 activator ratios observed in Gli3+/- embryos. Zic3 null;Gli3+/- neonates show rescue of the polydactylous phenotype seen in Gli3+/- animals. These studies identify a previously unrecognized role for Zic3 in regulating limb digit number via its modifying effect on Gli3 and Shh expression levels. Together, these results indicate that two Gli superfamily members that cause disparate human congenital malformation syndromes interact genetically and demonstrate the importance of Zic3 in regulating Shh pathway in developing limbs.     

Asymptomatic laryngeal malformations are common in patients with Pallister‐Hall syndrome

Pallister‐Hall syndrome (PHS) comprises hypothalamic hamartoma, polydactyly, pituitary dysfunction, laryngotracheal cleft, imperforate anus, and other anomalies. Some patients with PHS have a bifid epiglottis, a rare malformation. Greig cephalopolysyndactyly syndrome (GCPS) comprises polydactyly with craniofacial malformations without the PHS malformations. Both disorders are caused by mutations in the GLI3 gene. Laryngoscopy on 26 subjects with PHS showed that 15 had a bifid or cleft epiglottis (58%) and none of 14 subjects with GCPS had a cleft epiglottis. The malformed epiglottis was asymptomatic in all of the prospectively evaluated subjects. One additional PHS subject was found to have bifid epiglottis and a posterior laryngeal cleft on autopsy. We conclude that bifid epiglottis is common in PHS but not GCPS. Posterior laryngeal clefts are an uncommon manifestation of PHS and are identified only in severely affected patients. The diagnosis of a bifid epiglottis should prompt a thorough search for other sometimes asymptomatic anomalies of PHS to provide better medical care and recurrence risk assessment for affected individuals and families. Am. J. Med. Genet. 94:64–67, 2000. Published 2000 Wiley‐Liss, Inc.     

Genital abnormalities in Pallister-Hall syndrome: Report of two patients and review of the literature

We describe two patients with Pallister-Hall syndrome (PHS) with genital abnormalities: a female with hydrometrocolpos secondary to vaginal atresia and a male with micropenis, hypoplastic scrotum, and bilateral cryptorchidism. Nonsense mutations in GLI3 were identified in both patients. Clinical and molecular findings of 12 previously reported patients who had GLI3 mutations and genital abnormalities were reviewed. Genital features in the male patients included hypospadias, micropenis, and bifid or hypoplastic scrotum, whereas all the females had hydrometrocolpos and/or vaginal atresia. No hotspot for GLI3 mutations has been found. The urogenital and anorectal abnormalities associated with PHS might be related to dysregulation of SHH signaling caused by GLI3 mutations rather than hormonal aberrations. We recommend that clinical investigations of genital abnormalities are considered in patients with PHS, even those without hypopituitarism.     

Gonadal mosaicism in severe Pallister-Hall syndrome

Pallister-Hall syndrome (PHS, MIM #146510) is characterized by central and postaxial polydactyly, hypothalamic hamartoma (HH), bifid epiglottis, imperforate anus, renal abnormalities, and pulmonary segmentation anomalies. It is inherited in an autosomal dominant pattern. Here, we describe a family with two affected children manifesting severe PHS with mental retardation, behavioral problems, and intractable seizures. Both parents are healthy, with normal intelligence, and have no malformations on physical, laryngoscopic, and cranial MRI exam. The atypical presentation of these children and the absence of parental manifestations suggested an autosomal recessive mode of inheritance or gonadal mosaicism. Sequencing of GLI3 revealed a two nucleotide deletion in exon 15 (c.3385_3386delTT) predicting a frameshift and premature stop at codon 1129 (p.F1129X) in the children while both parents have wild type alleles. Genotyping with GLI3 intragenic markers revealed that both children inherited the abnormal allele from their mother thus supporting gonadal mosaicism as the underlying mechanism of inheritance (paternity was confirmed). This is the first reported case of gonadal mosaicism in PHS. The severe CNS manifestations of these children are reminiscent of children with non-syndromic HH who often have progressive mental retardation with behavioral problems and intractable seizures. We conclude that the phenotypic spectrum of PHS can include severe CNS manifestations and that recurrence risks for PHS should include a proviso for gonadal mosaicism, though the frequency cannot be calculated from a single case report. Published 2003 Wiley-Liss, Inc.     

Complex postaxial polydactyly types A and B with camptodactyly,hypoplastic third toe,zygodactyly and other digit anomalies caused by a novel GLI3 mutation

Polydactyly is a phenotypically and genetically highly heterogeneous limb malformation with preaxial and postaxial subtypes and subtypes A and B. Most polydactyly entities are associated with GLI3 mutation. We report on 10 affected individuals from a large Pakistani kindred initially evaluated as a possible new condition. The phenotype is postaxial polydactyly types A and B associated with zygodactyly, postaxial webbing of toes and additional features not previously reported for isolated polydactyly such as camptodactyly, hypoplasia of third toe, and wide space between hallux and second toe. Hypothesizing that the disorder could have resulted from a mutation in a novel gene responsible for polydactyly, we launched a genetic investigation. By linkage mapping and exome sequencing in the most severe case, we identified novel heterozygous frameshift mutation NM_000168.5 (GLI3): c.3635delG (p.(Gly1212Alafs*18)) but did not detect any other possibly deleterious mutation that could explain the unusual features of camptodactyly, hypoplasia of third toe and wide space between first and second toes. Our findings further expand the phenotypic variability of GLI3 polydactyly. We also present a review of GLI3-associated isolated limb anomalies, which indicates that GLI3 mutation leads primarily to two well-established polydactyly types: postaxial types A and B and crossed polydactyly type I. In addition, a variety of other minor digit anomalies generally accompany polydactyly, and there is no straightforward genotype-polydactyly phenotype correlation.     

Pallister-Hall syndrome: unreported skeletal features of a GLI3 mutation

We describe two patients with Pallister-Hall syndrome (PHS), both with evidence of a generalized skeletal dysplasia as typified by upper and lower acromesomelic limb shortening and the previously unreported fibular hypoplasia, radio-ulnar bowing, and proximal epiphyseal hypoplasia. Genomic DNA was only available for sequencing analysis in patient 2 and the mutation, c.3386_3387delTT was detected in exon 14 of the GL13 gene. It is also possible that the findings in patient 1 represent the phenotypic expression of a novel GLI3 mutation. This report further expands the PHS phenotype and raises the possibility of specific GLI3 mutations resulting in more severe skeletal features. It also suggests that PHS should be included in the differential diagnosis of antenatally ascertained acromesomelic limb shortening and bowing with fibular hypoplasia particularly in the presence of polysyndactyly.     

Clinical and molecular delineation of the Greig cephalopolysyndactyly contiguous gene deletion syndrome and its distinction from acrocallosal syndrome

Greig cephalopolysyndactyly syndrome (GCPS) is caused by haploinsufficiency of GLI3 on 7p13. Features of GCPS include polydactyly, macrocephaly, and hypertelorism, and may be associated with cognitive deficits and abnormalities of the corpus callosum. GLI3 mutations in GCPS patients include point, frameshift, translocation, and gross deletion mutations. FISH and STRP analyses were applied to 34 patients with characteristics of GCPS. Deletions were identified in 11 patients and the extent of their deletion was determined. Nine patients with deletions had mental retardation (MR) or developmental delay (DD) and were classified as severe GCPS. These severe GCPS patients have manifestations that overlap with the acrocallosal syndrome (ACLS). The deletion breakpoints were analyzed in six patients whose deletions ranged in size from 151 kb to 10.6 Mb. Junction fragments were found to be distinct with no common sequences flanking the breakpoints. We conclude that patients with GCPS caused by large deletions that include GLI3 are likely to have cognitive deficits, and we hypothesize that this severe GCPS phenotype is caused by deletion of contiguous genes.