Carol Wicking

Mutations of the Human Homolog of Drosophila patched in the Nevoid Basal Cell Carcinoma Syndrome

The nevoid basal cell carcinoma syndrome (NBCCS) is an autosomal dominant disorder characterized by multiple basal cell carcinomas (BCCs), pits of the palms and soles, jaw keratocysts, a variety of other tumors, and developmental abnormalities. NBCCS maps to chromosome 9q22.3. Familial and sporadic BCCs display loss of heterozygosity in this region, consistent with the gene being a tumor suppressor. A human sequence (PTC) with strong homology to the Drosophila segment polarity gene, patched, was isolated from a YAC and cosmid contig of the NBCCS region. Mutation analysis revealed alterations of PTC in NBCCS patients and in related tumors. We propose that a reduction in expression of the patched gene can lead to the developmental abnormalities observed in the syndrome and that complete loss of patched function contributes to transformation of certain cell types.

A Mammalian patched Homolog Is Expressed in Target Tissues of sonic hedgehog and Maps to a Region Associated with Developmental Abnormalities

Drosophila patched is a segment polarity gene required for the correct patterning of larval segments and imaginal discs during fly development and has a close functional relationship with hedgehog. We have isolated a complete human PATCHED cDNA sequence, which encodes a putative protein of 1296 amino acids, and displays 39% identity and 60% similarity to the Drosophila PATCHED protein. Hydropathy analysis suggests that human PATCHED is an integral membrane protein with a pattern of hydrophobic and hydrophilic stretches nearly identical to that of Drosophila patched. In the developing mouse embryo, patched is initially detected within the ventral neural tube and later in the somites and limb buds. Expression in the limb buds is restricted to the posterior ectoderm surrounding the zone of polarizing activity. The results show that patched is expressed in target tissues of sonic hedgehog, a murine homolog of Drosophila hedgehog suggesting that patched/hedgehog interactions have been conserved during evolution. Human PATCHED maps to human chromosome 9q22.3, the candidate region for the nevoid basal cell carcinoma syndrome. Patched expression is compatible with the congenital defects observed in the nevoid basal cell carcinoma syndrome.

A Genome-Wide Association Study Identifies Five Loci Influencing Facial Morphology in Europeans

Inter-individual variation in facial shape is one of the most noticeable phenotypes in humans, and it is clearly under genetic regulation; however, almost nothing is known about the genetic basis of normal human facial morphology. We therefore conducted a genome-wide association study for facial shape phenotypes in multiple discovery and replication cohorts, considering almost ten thousand individuals of European descent from several countries. Phenotyping of facial shape features was based on landmark data obtained from three-dimensional head magnetic resonance images (MRIs) and two-dimensional portrait images. We identified five independent genetic loci associated with different facial phenotypes, suggesting the involvement of five candidate genes—PRDM16, PAX3, TP63, C5orf50, and COL17A1—in the determination of the human face. Three of them have been implicated previously in vertebrate craniofacial development and disease, and the remaining two genes potentially represent novel players in the molecular networks governing facial development. Our finding at PAX3 influencing the position of the nasion replicates a recent GWAS of facial features. In addition to the reported GWA findings, we established links between common DNA variants previously associated with NSCL/P at 2p21, 8q24, 13q31, and 17q22 and normal facial-shape variations based on a candidate gene approach. Overall our study implies that DNA variants in genes essential for craniofacial development contribute with relatively small effect size to the spectrum of normal variation in human facial morphology. This observation has important consequences for future studies aiming to identify more genes involved in the human facial morphology, as well as for potential applications of DNA prediction of facial shape such as in future forensic applications.

Murine Wnt-11 and Wnt-12 have temporally and spatially restricted expression patterns during embryonic development

The Wnt gene family encodes a set of signalling molecules implicated in the development of a wide range of organisms. We have recently cloned partial cDNA sequences of murine Wnt-11 and Wnt-12. Here, we describe the spatio-temporal expression patterns of both genes during mouse embryogenesis. Wnt-11 expression is first detected within the truncus arteriosus from 8.25 dpc. By 9.5 dpc, Wnt-11 expression is detected in the somites at the medial junction of the dermatome and the myotome. Wnt-11 transcripts are also detected in limb bud mesenchyme from the time the bud is first visible. Wnt-12 is detected in the apical ectodermal ridge from 10.5 dpc. The implications of these expression patterns are discussed.

The hedgehog signalling pathway in tumorigenesis and development.

The hedgehog signalling pathway is responsible for the embryonic patterning of a range of tissues, and it is now known that dysregulation of this pathway can result in the formation of several tumour types. This cascade is regulated at the cell surface by the opposing actions of the patched and smoothened molecules which together form a receptor complex for hedgehog. The discovery that inactivation of the human patched gene is responsible for familial and sporadic forms of basal cell carcinoma firmly established a role for dysregulation of hedgehog signalling in tumorigenesis. Other key members of this pathway have also been shown to be involved in tumour formation, as have more distal downstream targets of hedgehog signalling. Since it appears that tumorigenesis results from constitutive activation of hedgehog responsive genes, the identification of novel downstream targets of hedgehog signalling in given cell types is likely to increase our understanding of the molecular processes underlying tumour formation.

Novel genes regulated by Sonic Hedgehog in pluripotent mesenchymal cells

Sonic Hedgehog is a secreted morphogen involved in patterning a wide range of structures in the developing embryo. Disruption of the Hedgehog signalling cascade leads to a number of developmental disorders and plays a key role in the formation of a range of human cancers. The identification of genes regulated by Hedgehog is crucial to understanding how disruption of this pathway leads to neoplastic transformation. We have used a Sonic Hedgehog (Shh) responsive mouse cell line, C3H/10T1/2, to provide a model system for hedgehog target gene discovery. Following activation of cell cultures with Shh, RNA was used to interrogate microarrays to investigate downstream transcriptional consequences of hedgehog stimulation. As a result 11 target genes have been identified, seven of which are induced (Thrombomodulin, GILZ, BF-2, Nr4a1, IGF2, PMP22, LASP1) and four of which are repressed (SFRP-1, SFRP-2, Mip1-γ, Amh) by Shh. These targets have a diverse range of putative functions and include transcriptional regulators and molecules known to be involved in regulating cell growth or apoptosis. The corroboration of genes previously implicated in hedgehog signalling, along with the finding of novel targets, demonstrates both the validity and power of the C3H/10T1/2 system for Shh target gene discovery.

The IFT-A complex regulates Shh signaling through cilia structure and membrane protein trafficking

Mutations in mouse intraflagellar transport–A complex genes alter Sonic hedgehog signaling because of their effects on cilia structure and on trafficking of membrane proteins into cilia.

CDX2 , a human homologue of Drosophila caudal , is mutated in both alleles in a replication error positive colorectal cancer

The Cdx2 gene is one of three murine homologues of the Drosophila homeobox gene caudal. Mice heterozygous for a null mutation in Cdx2 exhibit a variable phenotype including tail abnormalities, stunted growth and a homeotic shift of vertebrae. Most strikingly, however, 90% of heterozygous mice were reported to develop multiple intestinal adenomatous polyps, most notably in the proximal colon (). These observations led us to propose that mutation of CDX2 may be involved in the genesis of some human colorectal tumours. A survey of DNA from 85 colorectal tumours revealed that one with extensive microsatellite instability (RER+ phenotype) has mutations in both alleles of CDX2. Both mutations occur in coding regions which contain repetitive elements and are consistent with those found in RER+ tumours.

Rab23, a negative regulator of hedgehog signaling, localizes to the plasma membrane and the endocytic pathway.

The regulation of hedgehog signaling by vesicular trafficking was exemplified by the finding that Rab23, a Rab‐GTPase vesicular transport protein, is mutated in open brain mice. In this study, the localization of Rab23 was analyzed by light and immunoelectron microscopy after expression of wild‐type (Rab23‐GFP), constitutively active Rab23 (Rab23Q68L‐GFP), and inactive Rab23 (Rab23S23N‐GFP) in a range of mammalian cell types. Rab23‐GFP and Rab23Q68L‐GFP were predominantly localized to the plasma membrane but were also associated with intracellular vesicular structures, whereas Rab23S23N‐GFP was predominantly cytosolic. Vesicular Rab23‐GFP colocalized with Rab5Q79L and internalized transferrin‐biotin, but not with a marker of the late endosome or the Golgi complex. To investigate Rab23 with respect to members of the hedgehog signaling pathway, Rab23‐GFP was coexpressed with either patched or smoothened. Patched colocalized with intracellular Rab23‐GFP but smoothened did not. Analysis of patched distribution by light and immunoelectron microscopy revealed it is primarily localized to endosomal elements, including transferrin receptor‐positive early endosomes and putative endosome carrier vesicles and, to a lesser extent, with LBPA‐positive late endosomes, but was excluded from the plasma membrane. Neither patched or smoothened distribution was altered in the presence of wild‐type nor mutant Rab23‐GFP, suggesting that despite the endosomal colocalization of Rab23 and patched, it is likely that Rab23 acts more distally in regulating hedgehog signaling.

Characterisation of human patched germ line mutations in naevoid basal cell carcinoma syndrome

Mutations in the human patched gene have recently been detected in patients with naevoid basal cell carcinoma syndrome. We have characterised a further 5 novel germ line mutations in patients presenting with multiple odontogenic keratocysts. Four mutations cause premature stop codons and one mutation results in an amino-acid substitution towards the carboxyl terminus of the predicted patched protein. No obvious genotype-phenotype correlations could be interpreted, consistent with previous studies.