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.     

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.     

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.      

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.     

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.     

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.     

Alu-related 5q35 microdeletions in Sotos syndrome

Haploinsufficiency of the NSD1 gene due to 5q35 microdeletions or intragenic mutations causes Sotos syndrome (SoS). In 46 of the 49 Japanese deletion cases, common deletion breakpoints were located at two flanking low copy repeats (LCRs), implying that non-allelic homologous recombination (NAHR) between LCRs is the major mechanism for the common deletion. In the other three cases of atypical deletions, the mechanism(s) of deletions remains unanswered. We characterized the atypical microdeletions using fluorescence in situ hybridization (FISH), quantitative real-time polymerase chain reaction (qPCR), and Southern blot hybridization. All the deletion breakpoints in the three cases were successfully determined at the nucleotide level. Two deletions are 1.07 Mb (SoS19) and 1.23 Mb (SoS109) in size, and another consisted of two deletions with sizes of 28 kb and 0.72 Mb, intervened by an intact 29-kb segment (SoS44). All deletions were smaller than a typical 1.9-Mb common deletion. Alu elements were identified in both deletion breakpoints in SoS19, one of deletion breakpoints in SoS109, and both deletion breakpoints of the proximal 28-kb deletion in SoS44. Alu-mediated NAHR is strongly suggested at least in two of atypical deletions. Finally, qPCR is a very useful method to determine deletion breakpoints even in repeat-related regions.     

Rett syndrome from quintuple and triple deletions within the MECP2 deletion hotspot region

Rett syndrome results from mutations in the X-linked methyl-CpG-binding protein 2 (MECP2) gene, which are nearly always lethal in males and lead to regression and reduced life expectancy in females. Herein we report one propositus with five tandem deletions and a second propositus with three tandem deletions within MECP2 exon 4 that encode truncated protein products resulting in classic Rett syndrome. These deletion breakpoints and single deletions in 3 other patients were all found within a 185-bp region along with 64 of 69 other reported deletion breakpoints in the MECP2 gene. Illegitimate recombination resulting in deletion at a substantial proportion of the shared MECP2 sites is enhanced by repeated guanosine (G) DNA sequences in the antisense direction, consistent with reports at other gene loci that polypurine (multiple guanosine or adenosine (A)) basepairs enhance sequence deletion. Multiple deletions at the same poly G recombination sites confirm the existence of deletion hotspots in this gene region with numerous repeated antisense sites that are enriched 26- to 161-fold. Deletion by illegitimate recombination within a single allele can occur during mitotic or meiotic cell cycles. Although prone to disease-causing deletion, this region is unique in humans and highly conserved among mammals for the last 75 000 000 years to maintain the MECP2 gene's critical function.     

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.     

Phenotypic variability in Angelman syndrome: comparison among different deletion classes and between deletion and UPD subjects

Angelman syndrome (AS) can result from either a 15q11-q13 deletion (del), paternal uniparental disomy (UPD), imprinting, or UBE3A mutations. Here, we describe the phenotypic and behavioral variability detected in 49 patients with different classes of deletions and nine patients with UPD. Diagnosis was made by methylation pattern analysis of exon 1 of the SNRPN-SNURF gene and by microsatellite profiling of loci within and outside the 15q11-q13 region. There were no major phenotypic differences between the two main classes (BP1-BP3; BP2-BP3) of AS deletion patients, except for the absence of vocalization, more prevalent in patients with BP1-BP3 deletions, and for the age of sitting without support, which was lower in patients with BP2-BP3 deletions. Our data suggest that gene deletions (NIPA1, NIPA2, CYF1P1, GCP5) mapped to the region between breakpoints BP1 and BP2 may be involved in the severity of speech impairment, since all BP1-BP3 deletion patients showed complete absence of vocalization, while 38.1% of the BP2-BP3 deletion patients were able to pronounce syllabic sounds, with doubtful meaning. Compared to UPD patients, deletion patients presented a higher incidence of swallowing disorders (73.9% del x 22.2% UPD) and hypotonia (73.3% del x 28.57% UPD). In addition, children with UPD showed better physical growth, fewer or no seizures, a lower incidence of microcephaly, less ataxia and higher cognitive skills. As a consequence of their milder or less typical phenotype, AS may remain undiagnosed, leading to an overall underdiagnosis of the disease.     

Identification of novel deletions of 15q11q13 in Angelman syndrome by array-CGH: molecular characterization and genotype-phenotype correlations

Angelman syndrome (AS) is a neurodevelopmental disorder characterized by mental retardation, absent speech, ataxia, and a happy disposition. Deletions of the 15q11q13 region are found in approximately 70% of AS patients. The deletions are sub-classified into class I and class II based on their sizes of approximately 6.8 and approximately 6.0, respectively, with two different proximal breakpoints and a common distal breakpoint. Utilizing a chromosome 15-specific comparative genomic hybridization genomic microarray (array-CGH), we have identified, determined the deletion sizes, and mapped the breakpoints in a cohort of 44 cases, to relate those breakpoints to the genomic architecture and derive more precise genotype-phenotype correlations. Interestingly four patients of the 44 studied (9.1%) had novel and unusually large deletions, and are reported here. This is the first report of very large deletions of 15q11q13 resulting in AS; the largest deletion being >10.6 Mb. These novel deletions involve three different distal breakpoints, two of which have been earlier shown to be involved in the generation of isodicentric 15q chromosomes (idic15). Additionally, precise determination of the deletion breakpoints reveals the presence of directly oriented low-copy repeats (LCRs) flanking the recurrent and novel breakpoints. The LCRs are adequate in size, orientation, and homology to enable abnormal recombination events leading to deletions and duplications. This genomic organization provides evidence for a common mechanism for the generation of both common and rare deletion types. Larger deletions result in a loss of several genes outside the common Angelman syndrome-Prader-Willi syndrome (AS-PWS) critical interval, and a more severe phenotype.     

Variable phenotype in Greig cephalopolysyndactyly syndrome: clinical and radiological findings in 4 independent families and 3 sporadic cases with identified GLI3 mutations

Greig cephalopolysyndactyly (GCPS) (OMIM 175700) is an autosomal dominant disorder characterized by a distinct combination of craniofacial, hand and foot malformations. In this report, clinical and radiological findings of 12 patients with GCPS derived from 4 independent families and 3 sporadic cases with documented GLI3 mutations are presented with particular emphasis on inter- and intrafamilial variability. In a particularly instructive family in which 9 members of 4 generations could be studied clinically and molecularly, a missense mutation (R625W) is transmitted and shows a partially penetrant pattern. In a branch of the family, the GCPS phenotype skips a generation via a normal female carrier without clinical signs providing evidence that GCPS does not always manifest full penetrance as generally supposed.     

Molecular and clinical characterization of two patients with Prader-Willi syndrome and atypical deletions of proximal chromosome 15q

Prader-Willi syndrome (PWS) is caused by the disturbed expression of genes from the imprinted region of 15q11-q13, but the specific contributions of individual genes remain unknown. Most paternal PWS deletions are bracketed by recurrent breakpoints BP1 or BP2 and BP3. Atypical deletions are very rare. In the present work, we describe the molecular analysis of two patients with atypical deletions using microsatellite analysis, methylation-specific MLPA, and microarray CGH. A deletion of about 2 Mb in Patient 1 started at BP2 and ended in the middle of the typically deleted region within the UBE3A gene. The deletion in Patient 2 started 1.3 Mb distal from BP2 within the C15ORF2 gene, extended over 9.5 Mb, and ended within the AVEN gene in proximal 15q14. In Patient 1 both deletion breakpoints involved repetitive regions, which precluded cloning of the junction and pointed to non-allelic homologous recombination as a possible mechanism of this rearrangement. The breakpoints in Patient 2 were sequenced, and their structure suggested non-homologous end joining as the most likely cause of this deletion. The phenotype of both patients did not depart significantly from the typical clinical picture of PWS, although some symptoms in Patient 2 were also reminiscent of the phenotype of individuals with the recently described 15q13.3 microdeletion syndrome. Our findings support previous observations of relatively mild phenotypic effects resulting from deletions that extend distally from the PWS region and observations of the modest effects of different types of genetic defects on the spectrum and severity of symptoms in PWS.     

Williams syndrome: from genotype through to the cognitive phenotype

Williams syndrome, due to a contiguous gene deletion at 7q11.23, is associated with a distinctive facial appearance, cardiac abnormalities, infantile hypercalcemia, and growth and developmental retardation. The deletion is approximately 1.5Mb and includes approximately 17 genes. Large repeats containing genes and pseudogenes flank the deletion breakpoints, and the mutation mechanism commonly appears to be unequal meiotic recombination. Elastin hemizygosity is associated with supravalvular aortic stenosis and other vascular stenoses. LIM Kinase 1 hemizygosity may contribute to the characteristic cognitive profile. The relationship of the other deleted genes to phenotypic features is not known. People with Williams syndrome tend to be over friendly-though anxious-and lack social judgement skills. They exhibit an uneven cognitive-linguistic profile together with mild to severe mental retardation. Analysis of the cognitive phenotype based on analyses of the mental processes underlying overt behavior demonstrates major differences between normal and WS subjects although for some areas, such as face processing, WS subjects can achieve near normal scores. Cognitive analysis of patients with small deletions in 7q11.23 which include elastin and LIM Kinase 1 have revealed varying results and it is premature to draw genotype-phenotype correlations.     

Boy with syndactylies, macrocephaly, and severe skeletal dysplasia: not a new syndrome, but two dominant mutations (GLI3 E543X and COL2A1 G973R) in the same individual

An unusual combination of syndactylies, macrocephaly, and severe skeletal dysplasia was observed in a newborn infant. A history of digital anomalies in the father and grandfather lead to the diagnosis of dominantly inherited Greig cephalopolysyndactyly syndrome (GCPS, MIM #175700). Having explained the digital findings and macrocephaly, the skeletal changes were thought to fit best congenital spondyloepiphyseal dysplasia (SEDC MIM #183900), a type II collagen disorder. Molecular analysis confirmed the presence of two dominant mutations in the propositus: a GLI3 mutation (E543X), which was present also in the father and grandfather, and a de novo COL2A1 mutation leading to a G973R substitution. Thus, this boy combined the syndactyly-macrocephaly phenotype of Greig cephalosyndactyly syndrome with a severe form of spondyloepiphyseal dysplasia caused by the structural defect in type II collagen. The diagnostic difficulties posed by the combination of two genetic disorders and the contribution of molecular diagnostics are well illustrated by this case.