Whole exome sequencing is an efficient, sensitive and specific method for determining the genetic cause of short-rib thoracic dystrophies.

Short-rib thoracic dystrophies (SRTDs) are congenital disorders due to defects in primary cilium function. SRTDs are recessively inherited with mutations identified in 14 genes to date (comprising 398 exons). Conventional mutation detection (usually by iterative Sanger sequencing) is inefficient and expensive, and often not undertaken. Whole exome massive parallel sequencing has been used to identify new genes for SRTD (WDR34, WDR60 and IFT172); however, the clinical utility of whole exome sequencing (WES) has not been established. WES was performed in 11 individuals with SRTDs. Compound heterozygous or homozygous mutations were identified in six confirmed SRTD genes in 10 individuals (IFT172, DYNC2H1, TTC21B, WDR60, WDR34 and NEK1), giving overall sensitivity of 90.9%. WES data from 993 unaffected individuals sequenced using similar technology showed two individuals with rare (minor allele frequency <0.005) compound heterozygous variants of unknown significance in SRTD genes (specificity >99%). Costs for consumables, laboratory processing and bioinformatic analysis were

From linked marker to gene.

An area of increasing importance in human genetics research is the strategy of 'reverse genetics' or 'positional cloning', whereby a gene associated with a genetic disease is isolated on the basis of its approximate chromosomal position. This article and the accompanying centrepage diagram describe the main features of this strategy and outline the techniques involved; they are designed to be used for teaching purposes and as a general reference for those from other fields of genetics research.

Medulloblastomas of the desmoplastic variant carry mutations of the human homologue of Drosophila patched.

Inactivating mutations in the PTCH gene, a human homologue of the Drosophila segment polarity gene patched, have been identified recently in patients with nevoid basal cell carcinoma syndrome. These patients are predisposed to various neoplasias including basal cell carcinomas and medulloblastomas (MBs). To determine the involvement of PTCH in sporadic MBs, which represent the most frequent malignant brain tumors in children, we screened for PTCH alterations in an unselected panel of 64 biopsy samples from 62 patients and four continuous MB cell lines, all derived from patients with sporadic MBs. Using single-strand conformational polymorphism analysis, we screened exons 2-22 and detected nonconservative PTCH mutations in 3 of 11 samples from sporadic cases of the desmoplastic variant of MB but none in 57 MBs with classical (nondesmoplastic) histology. In two of the tumors with mutations and in two additional desmoplastic cases, loss of heterozygosity was found at 9q22. These findings suggest that PTCH represents a tumor suppressor gene involved in the development of the desmoplastic variant of MB.

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.

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 (Chawengsaksophak et al., 1997). 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.

No evidence for the H133Y mutation in SONIC HEDGEHOG in a collection of common tumour types.

The human homologue of the Drosophila segment polarity gene patched is mutated in the cancer predisposition syndrome naevoid basal cell carcinoma syndrome (NBCCS), as well as in several types of tumour associated with the disorder. It was recently reported that a single recurrent mutation in the SONIC HEDGEHOG gene, which encodes the PATCHED ligand, was found in one of 43 basal cell carcinomas (BCCs), one of 14 medulloblastomas and one of six breast carcinomas analysed (Oro et al., 1997). We have searched extensively for this same mutation in a large collection of BCCs, medulloblastomas and carcinomas of the breast, ovary and colorectum and have failed to detect the mutation in any sample analysed.

DHPLC analysis of patients with Nevoid Basal Cell Carcinoma Syndrome reveals novel PTCH missense mutations in the sterol-sensing domain.

Nevoid Basal Cell Carcinoma Syndrome (NBCCS) is an autosomal dominant disorder characterised by multiple basal cell carcinomas, palmar and plantar pitting, odontogenic keratocysts of the jaws and bilamellar calcification of the falx. Mutations in the PTCH gene are responsible for NBCCS but most studies have found mutations in less than half of the cases tested. We used denaturing high performance liquid chromatography (DHPLC) to screen for PTCH mutations in 28 NBCCS cases, most of whom had been previously evaluated by single stranded conformation polymorphism analysis but found to be negative. Protein truncating (n = 10) and missense or indel (n = 4) mutations were found in 14/28 (50%) cases and one additional case carried an unclassified variant, c.2777G>C. Thirteen of the variants were novel. The mutation frequency was similar in inherited and de novo cases. Three of the missense and indel mutations were in the sterol-sensing domain, and one was in the sixth transmembrane domain.

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 role of hedgehog signalling in tumorigenesis.

It has long been known from work in both Drosophila and vertebrate systems that the hedgehog signalling pathway is pivotal to embryonic development, but the past 5 years has seen an increase in our understanding of how members of this pathway are crucial to the processes of tumorigenesis. This important link was firmly established with the discovery that mutations in the gene encoding the hedgehog receptor molecule patched are responsible for both familial and sporadic forms of basal cell carcinoma (BCC), as well as a number of other tumour types. It is now known that a number of key members of the hedgehog cascade are involved in tumorigenesis, and dysregulation of this pathway appears to be a key element in the aetiology of a range of tumours.

De novo mutations of the Patched gene in nevoid basal cell carcinoma syndrome help to define the clinical phenotype.

The demonstration that mutations in the Patched (PTCH) gene cause nevoid basal cell carcinoma syndrome (NBCCS) has led to the identification of the exact molecular lesion in a percentage of individuals with the syndrome. In addition, it has been possible to determine, through molecular analysis of parents and other relatives of these individuals, if the mutation is inherited or has arisen de novo. We have previously reported 28 mutations in individuals with NBCCS, and here we present an additional 4 novel mutations. We have also analyzed relatives of a number of the individuals in whom we have found mutations. In total we have identified 8 individuals who carry a de novo mutation in the PTCH gene. In 5 of these cases, clinical and radiological examination had not unequivocally ruled out a diagnosis in one of the parents. This helps to define the clinical phenotype and suggests that diagnostic criteria in this complex syndrome may require review.

Nevoid basal cell carcinoma syndrome: review of 118 affected individuals.

One hundred eighteen cases of nevoid basal cell carcinoma syndrome (NBCCS, Gorlin's syndrome or basal cell nevus syndrome) are presented in this study. In aiming to ascertain all the affected families in Australia, we have examined the largest series to date. Relative frequencies of associated complications are presented and compared with those of the recent English survey by Evans et al. [J Med Genet 30:460-464, 1993]. The frequencies of most manifestations are similar. However, one major difference is that the multiple basal cell carcinomas are manifest from an earlier age in the Australian population, which probably reflects greater exposure to ultraviolet radiation. Of the 64 families ascertained, 37 represented simplex cases, and, accordingly, the apparent new mutation rate is surprisingly high (14-81%) given the lack of impact of NBCCS on reproductive capabilities. There is some evidence to suggest that this may be attributable to anticipation.

ATF3 and p15PAF are novel gatekeepers of genomic integrity upon UV stress.

After genotoxic stress, normal cells trigger DNA repair or, if unable to repair, undergo apoptosis to eradicate the cells that bear the risk of becoming tumorigenic. Here we show that repression of the transcription factor, activating transcription factor 3 (ATF3), after ultraviolet (UV)-mediated genotoxic stress impairs the DNA repair process. We provide evidence that ATF3 directly regulates the proliferating cell nuclear antigen (PCNA)-associated factor KIAA0101/p15(PAF). We further show that the expressions of ATF3 and p15(PAF) is sufficient to trigger the DNA repair machinery, and that attenuation of their expression alters DNA repair mechanisms. We show that the expression of p15(PAF) compensates for a lack of ATF3 expression, thereby constituting a major effector of ATF3 in the DNA repair process. In addition, we provide evidence that p15(PAF) expression is required for the correct function of PCNA during DNA repair, as prevention of their interaction significantly alters DNA repair mechanisms. Finally, defective DNA repair, because of the downregulation of p15(PAF) expression, rendered the cells more sensitive to UV-induced cell death. Therefore, our results suggest ATF3 and p15(PAF) as novel gatekeepers of genomic integrity after UV exposure.

Molecular basis of the nevoid basal cell carcinoma syndrome.

Many genes originally identified because of their role in embryonic development are also important in control of cell growth and differentiation postnatally. Mutations in some of these genes have been shown to contribute to carcinogenesis. The nevoid basal cell carcinoma syndrome is an autosomal dominant disorder that predisposes to basal cell carcinomas of the skin as well as widespread developmental defects. The gene for this disorder, NBCCS, maps to chromosome 9q22.3, and loss of heterozygosity at this site in both sporadic and hereditary basal cell carcinomas suggests that it functions as a tumor suppressor. NBCCS was positionally cloned and shown to be the human homologue of the Drosophila gene, patched. Drosophila patched is part of the hedgehog signaling pathway, important in determining embryonic patterning and cell fate in multiple structures of the developing embryo. That mutations in human patched result in dysregulation of several genes known to play a role in both organogenesis and carcinogenesis may explain both the birth defects and the cancer predisposition seen in the nevoid basal cell carcinoma syndrome.

The effects of splice site mutations in patients with naevoid basal cell carcinoma syndrome.

We have previously identified the human homologue of the Drosophila patched gene and have described, in this gene, mutations that give rise to naevoid basal cell carcinoma syndrome (NBCCS). Here, we have analysed the effects of three splice site mutations within human PATCHED (PTCH) by the reverse transcription/polymerase chain reaction method in cultured patient lymphocyte cell lines. Two alterations, a point mutation in intron 7 and an insertion in intron 10, lead to premature truncation of the PATCHED protein. Another point mutation in intron 17 results in the skipping of exon 18 and the subsequent in-frame deletion of 46 amino acids. Additionally, in all lymphocyte and keratinocyte cell lines examined, exon 10 of PTCH is alternatively spliced leading to an in-frame deletion of 52 amino acids.

Fine genetic mapping of the gene for nevoid basal cell carcinoma syndrome.

Nevoid basal cell carcinoma syndrome (NBCCS, or Gorlin syndrome) is a cancer predisposition syndrome characterized by multiple basal cell carcinomas and diverse developmental defects. The gene responsible for NBCCS, which is most likely to be a tumor suppressor gene, has previously been mapped to 9q22.3-q31 in a 12-cM interval between the microsatellite marker loci D9S12.1 and D9S109. Combined multipoint and haplotype analyses of additional polymorphisms in this region in our collection of Australasian pedigrees have further refined the localization of the gene to between the markers D9S196 and D9S180, an interval reported to be approximately 2 cM.

Immunochemical analysis of normal and mutant forms of human pyruvate dehydrogenase.

Pyruvate dehydrogenase (PDH) deficiency has been described in many patients with primary lactic acidosis. However, there are very few cases in which a structural defect in the complex has been clearly demonstrated. Measurement of the activity of the PDH complex in cultured human cells has proved unreliable, and a combination of structural and functional studies are required to make a definitive diagnosis. For this reason, an immunochemical strategy has been developed to complement direct enzyme assay in the detection and further characterization of PDH deficiency. We illustrate the usefulness of this approach by describing defects in the alpha-subunit of the pyruvate decarboxylase component of the PDH complex in two patients with primary lactic acidosis. In one patient, there is no immunologically cross-reacting material corresponding to this subunit. In the second patient, there appears to be an intrinsic structural defect in the subunit which restricts dephosphorylation (and hence activation) of the inactive phosphorylated complex.

+P5 (D1S3309E), a novel target binding site for the Wilms' tumour suppressor 1 (WT1) gene, maps to human chromosome 1q21-->22.

The Wilms' tumor suppressor 1 gene (WT1) encodes a zinc finger transcription factor critical for normal urogenital development. We have previously isolated a DNA fragment, +P5 (D1S3309E), to which all WT1 protein isoforms bind. Using PCR of a human x rodent somatic cell hybrid mapping panel, together with two-color fluorescence in situ hybridisation of +P5-containing cosmids and previously localised human chromosome 1q cosmids, we have mapped the +P5 fragment to chromosome 1q21-->q22.

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.