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.      

Metopic and sagittal synostosis in Greig cephalopolysyndactyly syndrome: five cases with intragenic mutations or complete deletions of GLI3

Greig cephalopolysyndactyly syndrome (GCPS) is a multiple congenital malformation characterised by limb and craniofacial anomalies, caused by heterozygous mutation or deletion of GLI3. We report four boys and a girl who were presented with trigonocephaly due to metopic synostosis, in association with pre- and post-axial polydactyly and cutaneous syndactyly of hands and feet. Two cases had additional sagittal synostosis. None had a family history of similar features. In all five children, the diagnosis of GCPS was confirmed by molecular analysis of GLI3 (two had intragenic mutations and three had complete gene deletions detected on array comparative genomic hybridisation), thus highlighting the importance of trigonocephaly or overt metopic or sagittal synostosis as a distinct presenting feature of GCPS. These observations confirm and extend a recently proposed association of intragenic GLI3 mutations with metopic synostosis; moreover, the three individuals with complete deletion of GLI3 were previously considered to have Carpenter syndrome, highlighting an important source of diagnostic confusion.     

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.     

Phenotype of five patients with Greig syndrome and microdeletion of 7p13

Here we describe five patients with Greig cephalopolysyndactyly syndrome (GCPS), including one pair of monozygotic twin boys with a de novo microdeletion involving the chromosomal band 7p13, where various clinical manifestations, in addition to GCPS, were recognized. Besides the twin pair, all patients are unrelated. Since there is a considerable lack of well-defined clinical delineation of the few patients with microdeletions involving 7p13 with GCPS described so far, we focus on the symptoms that are not typically related to GCPS, such as moderate psychomotor retardation, seizures, muscle fiber anomalies, cardiac anomalies, hyperglycemia, and hirsutism. Our observations suggest that in all cases of atypical GCPS, the presence of a cytogenetically detectable microdeletion or a submicroscopic deletion of 7p13 should be suspected.     

Greig cephalopolysyndactyly syndrome with dysgenesis of the corpus callosum in a Bedouin family

We report on the first known Bedouin family with Greig cephalopolysyndactyly syndrome (MIM 175700). The index patient and his father shared pre- and postaxial polysyndactyly, mild mental retardation, and corpus callosum dysgenesis. Their phenotypic findings were compared with reported cases of both Greig cephalopolysyndactyly (GCPS) and acrocallosal syndromes. This family represents the second report of the rare occurrence of dysgenesis of the corpus callosum in GCPS. © 1996 Wiley-Liss, Inc.     

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.     

MODY type 2 in Greig cephalopolysyndactyly syndrome (GCPS) as part of a contiguous gene deletion syndrome

Maturity-onset diabetes of the young type 2 (MODY2) is a form of monogenic diabetes, characterized by mild fasting hyperglycemia. MODY2 is caused by heterozygous mutations in the GCK gene that encodes the glucokinase enzyme. We describe the clinical features and the underlying genetic defect of MODY2 in a patient with atypical Greig cephalopolysyndactyly syndrome (GCPS). The patient presented with the limb formation and the craniofacial developmental abnormalities typical to GCPS, in addition to mental retardation and epilepsy (assigned as atypical syndrome). Fasting hyperglycemia in the diabetic range, impaired glucose tolerance, and lack of diabetes autoantibodies were compatible with MODY2. In order to delineate the genetic aberrations relevant both to MODY2 and Greig syndrome in this patient, we performed cytogenetic analysis, real-time PCR of the GCK gene, and comparative genomic hybridization (CGH) array. Cytogenetic study has shown a microscopic detectable deletion in the 7p13-15 chromosomal region. Real-time PCR demonstrated a deletion of the GCK gene in the patient but not her parents, and CGH array revealed a deleted region of approximately 12 Mb in the 7p13-15 region. This deleted region included GLI3 and GCK genes (where heterozygous mutations cause GCPS and MODY2, respectively), and many other contiguous genes. Our patient manifests a unique form of MODY2, where GCK gene deletion is part of a large deleted segment in the 7p13-15 chromosomal region.     

Chromosomal localisation of a developmental gene in man: direct DNA analysis demonstrates that Greig cephalopolysyndactyly maps to 7p13

Greig cephalopolysyndactyly syndrome (GCPS) is a rare autosomal dominant form of complex polydactyly. GCPS has been tentatively assigned to chromosome 7 on the basis of association of the condition with balanced translocations involving the short arm of chromosome 7 (7p13) in two families. Seven GCPS pedigrees with no chromosome abnormality were studied, and linkage was demonstrated between GCPS and the DNA sequence coding for the receptor for epidermal growth factor (localised to 7p12-13) (Z = 3.17; O = theta).     

De novo GLI3 mutation in acrocallosal syndrome: broadening the phenotypic spectrum of GLI3 defects and overlap with murine models

Since others have excluded GLI3 in ACS, we suggest that ACS may represent a heterogeneous group of disorders that, in some cases, may result from a mutation in GLI3 and represent a severe, allelic form of GCPS. The finding is important for counselling families with suspected ACS.     

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.     

The clinical atlas of Greig cephalopolysyndactyly syndrome

Greig cephalopolysyndactyly syndrome (GCPS) is a rare multiple congenital anomaly syndrome that is inherited in an autosomal dominant pattern and is caused by haploinsufficiency of the GLI3 gene. The syndrome typically includes preaxial or mixed pre- and postaxial polydactyly and cutaneous syndactyly, ocular hypertelorism, and macrocephaly in its typical forms, but sometimes includes hydrocephalus, seizures, mental retardation, and developmental delay in more severe cases. Patients with milder forms of GCPS can have subtle craniofacial dysmorphic features that are difficult to distinguish from normal variation. This article presents the spectrum of dysmorphic findings in GCPS highlighting some of its key presenting features to familiarize clinicians with the variable expressivity of the condition.     

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.     

Genomic analysis of five chromosome 7p deletion patients with Greig cephalopolysyndactyly syndrome (GCPS)

Chromosomal deletions on chromosome 7p are associated with Greig cephalopolysyndactyly syndrome (GCPS, OMIM 175700) a syndrome affecting the development of the skull, face, and limbs. We have compared data from molecular cytogenetic and genetic analyses with clinical symptoms from five previously published GCPS deletion patients, including a pair of monozygotic twins. The genomic DNA of the probands and their parents, as well as the DNA from monoallelic cell lines of two patients, was analyzed using microsatellite markers. In some cases (e.g. where the microsatellite studies were uninformative) we also used fluorescence in situ hybridization (FISH) with bacterial artificial chromosomes (BAC) probes. The fine mapping results of the deletions and genomic data from chromosome 7, were compared to the clinical symptoms. Common breakpoint sequences or mutation hotspots were not observed. Mutation screening for PGAM2, which is responsible for a form of myopathy with recessive inheritance, was performed in all patients. Loss of heterozygosity for known genes with dominant inheritance, such as the glucokinase gene (GCK), which, when mutated or haploinsufficient, is responsible for maturity-onset diabetes of the young, type II (MODY2, OMIM 125851), was identified and included in a genetic counseling of the patients' families.     

Greig cephalopolysyndactyly syndrome: altered phenotype of a microdeletion syndrome due to the presence of a cytogenetic abnormality

A male had several features of Greig cephalopolysyndactyly syndrome (GCPS) and significant developmental delay. He was found to have a de novo chromosomal deletion of chromosome no. 7 involving p13; this resulted in loss of the zinc finger gene, GLI3, which is the candidate gene in this syndrome. Modification of the CGPS phenotype in a sporadic case emphasizes the importance of searching for a chromosomal origin of this autosomal dominant disorder. Detection of a chromosomal deletion in these patients may be associated with a poor prognosis from the standpoint of cognitive development, and the potential for other structural abnormalities not normally associated with GCPS.     

Retrospective family study of childhood medulloblastoma

Medulloblastoma is the most common malignant central nervous system tumor of childhood and can occur sporadically or in association with inherited cancer susceptibility syndromes such as the nevoid basal cell carcinoma syndrome (NBCCS). To determine whether an association existed between the risk of developing medulloblastoma and undiagnosed syndromes, we retrospectively reviewed clinical data on 33 patients with medulloblastoma from a single institution and compared them with their unaffected relatives (n = 46). Six patients had tumors showing desmoplastic histology. Two of the six met diagnostic criteria for NBCCS. One NBCCS patient had a missense mutation of patched-1 (PTCH1); the other had no identifiable PTCH1 mutation. Two patients with isolated desmoplastic medulloblastoma had an insertion and splice site mutation, respectively, in suppressor of fused (SUFU). All patients with nondesmoplastic medulloblastoma histology received molecular testing for SUFU. None of these patients had an identifiable mutation in PTCH1 or SUFU. We performed a clinical evaluation for Greig cephalopolysyndactyly syndrome (GCPS) in four medulloblastoma families, who exhibited macrocephaly as the only finding consistent with the diagnosis of GCPS. Molecular analysis of GLI3 in these four families was negative. There was a paucity of clinical findings among the majority of medulloblastoma patients in this study group to suggest a definable cancer genetic syndrome. We conclude that clinically recognizable syndromes are uncommon among patients with medulloblastoma, however, PTCH1 and SUFU mutations are present at a low but significant frequency.     

Zoom-in comparative genomic hybridisation arrays for the characterisation of variable breakpoint contiguous gene syndromes

Contiguous gene syndromes cause disorders via haploinsufficiency for adjacent genes. Some contiguous gene syndromes (CGS) have stereotypical breakpoints, but others have variable breakpoints. In CGS that have variable breakpoints, the extent of the deletions may be correlated with severity. The Greig cephalopolysyndactyly contiguous gene syndrome (GCPS-CGS) is a multiple malformation syndrome caused by haploinsufficiency of GLI3 and adjacent genes. In addition, non-CGS GCPS can be caused by deletions or duplications in GLI3. Although fluorescence in situ hybridisation (FISH) can identify large deletion mutations in patients with GCPS or GCPS-CGS, it is not practical for identification of small intragenic deletions or insertions, and it is difficult to accurately characterise the extent of the large deletions using this technique. We have designed a custom comparative genomic hybridisation (CGH) array that allows identification of deletions and duplications at kilobase resolution in the vicinity of GLI3. The array averages one probe every 730 bp for a total of about 14,000 probes over 10 Mb. We have analysed 16 individuals with known or suspected deletions or duplications. In 15 of 16 individuals (14 deletions and 1 duplication), the array confirmed the prior results. In the remaining patient, the normal CGH array result was correct, and the prior assessment was a false positive quantitative polymerase chain reaction result. We conclude that high-density CGH array analysis is more sensitive than FISH analysis for detecting deletions and provides clinically useful results on the extent of the deletion. We suggest that high-density CGH array analysis should replace FISH analysis for assessment of deletions and duplications in patients with contiguous gene syndromes caused by variable deletions.     

CYP3A isoforms in Ewing's sarcoma tumours: an immunohistochemical study with clinical correlation

Ewing's sarcoma is an aggressive malignancy of bone and soft tissue with high incidence of metastasis and resistance to chemotherapy. Cytochrome P450 (CYP) monooxygenases are a family of enzymes that are involved in the metabolism of exogenous and endogenous compounds, including anti‐cancer drugs, and have been implicated in the aggressive behaviour of various malignancies. Tumour samples and clinical information including age, sex, tumour site, tumour size, clinical stage and survival were collected from 36 adult and paediatric patients with Ewing's sarcoma family tumours. Tissue microarrays slides were processed for immunohistochemical labelling for CYP3A4, CYP3A5 and CYP3A7 using liver sections as positive control. The intensity of staining was scored as negative, low or high expression and was analysed statistically for any association with patients' clinical information. Four cases were later excluded due to inadequate viable tissue. CYP3A4 staining was present in 26 (81%) cases with high expression noted in 13 (40%) of 32 cases. High expression was significantly associated with distant metastases (P < 0.05). CYP3A5 and CYP3A7 were expressed in 5 and 13 cases respectively (15.6%, 40.6%). There was no association between the expression of CYP3A isoforms and age, sex, tumour size, or location (pelvic or extra‐pelvic). None of the biomarkers showed any correlation with overall or disease‐free survival. In conclusion, expression of CYP3A isoforms is noted in Ewing's sarcoma tumours and high CYP3A4 expression may be associated with metastasis. Additional studies are needed to further investigate the role of CYP3A4 in the prognosis of these tumours.