Familial cylindromatosis (turban tumour syndrome) gene localised to chromosome 16q12-q13: evidence for its role as a tumour suppressor gene.

The human skin is a complex organ composed of the surface epidermis, the subjacent dermis (in which blood vessels, lymphatics and nerves are located) and the skin appendages. The latter include hair follicles, sebaceous glands (which secrete lipids that may serve as a permeability barrier, emollient or antimicrobial agent), apocrine glands (which secrete scents) and eccrine glands (which produce sweat for temperature control). Hereditary cylindromatosis (MIM 123850) is a rare autosomal dominant disease characterised by the development of multiple neoplasms originating from the skin appendages. These neoplasms have been termed cylindromas due to their characteristic microscopic architecture and are believed to exhibit apocrine or eccrine differentiation. We have carried out a genome search using two families with this disease, which has provided strong evidence for linkage of cylindromatosis to loci on chromosome 16q12-q13. Using markers close to the cylindromatosis gene, consistent loss of the wild-type allele was observed in 19 tumours from four individuals in the two families, indicating that the gene is likely to be a tumour suppressor gene.

Familial male breast cancer is not linked to the BRCA1 locus on chromosome 17q.

Breast cancer in men is about a hundredfold less common than in women and this has hindered research into its genetic basis. We have examined 22 families with at least one case of male breast cancer for linkage to the hereditary breast and ovarian cancer locus, BRCA1, on chromosome 17q. We found strong evidence against linkage to BRCA1 (lod score-16.63) and the best estimate of the proportion of linked families was 0% (95% CI 0-18%). Our results indicate that there is a gene(s) other than BRCA1 which predisposes to early-onset breast cancer in women and which confers a higher risk of male breast cancer. Identification of additional pedigrees that include cases of male breast cancer may therefore facilitate the mapping and isolation of this gene.

A germline mutation in the androgen receptor gene in two brothers with breast cancer and Reifenstein syndrome.

Breast cancer in men is rare--among the risk factors that have been identified are a family history of breast cancer and evidence of androgen insufficiency. We report a family in which two brothers who both developed breast cancer had clinical and endocrinological evidence of androgen resistance. Sequence analysis revealed a mutation in the androgen receptor gene on the X chromosome, within the region encoding the DNA binding domain. This is the first report of a germline mutation in a member of the steroid/thyroid hormone receptor superfamily associated with the development of cancer.

Instability of short tandem repeats (microsatellites) in human cancers.

The allele sizes of polymorphic microsatellite repeats in DNA from human cancers were compared to normal DNA from the same patients. In 16 out of 196 paired samples (8%), we found evidence of an extra allele of a different size in the tumour which was not present in the normal DNA. Sequence analysis confirmed that the extra allele originates from the appropriate locus and that the size change is attributable to alteration in the number of repeat units. This form of instability was more common in tri- and tetranucleotide repeats than in dinucleotide repeats. In any single tumour sample only one repeat in the set examined was abnormal, the remainder showing identical patterns in normal and tumour DNA or evidence of allele loss. The pattern of instability in diverse types of cancer differs from that reported in colorectal neoplasms.

BRCA2 mutations in primary breast and ovarian cancers.

The second hereditary breast cancer gene, BRCA2, was recently isolated. Germline mutations of this gene predispose carriers to breast cancer, and, to a lesser extent, ovarian cancer. Loss of heterozygosity (LOH) at the BRCA2 locus has been observed in 30-40% of sporadic breast and ovarian tumours, implying that BRCA2 may act as a tumour suppressor gene in a proportion of sporadic cases. To define the role of BRCA2 in sporadic breast and ovarian cancer, we screened the entire gene for mutations using a combination of techniques in 70 primary breast carcinomas and in 55 primary epithelial ovarian carcinomas. Our analysis revealed alterations in 2/70 breast tumours and none of the ovarian carcinomas. One alteration found in the breast cancers was a 2-basepair (bp) deletion (4710delAG) which was subsequently shown to be a germline mutation, the other was a somatic missense mutation (Asp3095Glu) of unknown significance. Our results suggest that BRCA2 is a very infrequent target for somatic inactivation in breast and ovarian carcinomas, similar to the results obtained for BRCA1.

Breast cancer genetics: what we know and what we need.

Breast cancer results from genetic and environmental factors leading to the accumulation of mutations in essential genes. Genetic predisposition may have a strong, almost singular effect, as with BRCA1 and BRCA2, or may represent the cumulative effects of multiple low-penetrance susceptibility alleles. Here we review high- and low-penetrance breast-cancer-susceptibility alleles and discuss ongoing efforts to identify additional susceptibility genes. Ultimately these discoveries will lead to individualized breast cancer risk assessment and a reduction in breast cancer incidence.

Localization of a breast cancer susceptibility gene, BRCA2, to chromosome 13q12-13.

A small proportion of breast cancer, in particular those cases arising at a young age, is due to the inheritance of dominant susceptibility genes conferring a high risk of the disease. A genomic linkage search was performed with 15 high-risk breast cancer families that were unlinked to the BRCA1 locus on chromosome 17q21. This analysis localized a second breast cancer susceptibility locus, BRCA2, to a 6-centimorgan interval on chromosome 13q12-13. Preliminary evidence suggests that BRCA2 confers a high risk of breast cancer but, unlike BRCA1, does not confer a substantially elevated risk of ovarian cancer.

AutoCSA, an algorithm for high throughput DNA sequence variant detection in cancer genomes.

UNLABELLED: The undertaking of large-scale DNA sequencing screens for somatic variants in human cancers requires accurate and rapid processing of traces for variants. Due to their often aneuploid nature and admixed normal tissue, heterozygous variants found in primary cancers are often subtle and difficult to detect. To address these issues, we have developed a mutation detection algorithm, AutoCSA, specifically optimized for the high throughput screening of cancer samples. AVAILABILITY: http://www.sanger.ac.uk/genetics/CGP/Software/AutoCSA.

Hereditary predisposition to breast cancer.

The breast cancer susceptibility gene on chromosome 17q, BRCA1, has now been isolated. Mutations in this gene have been detected in many families with a predisposition to breast cancer. Most of these mutations result in truncation and presumed inactivation of the BRCA1 protein. A large number of distinct mutations have been reported, although some families have identical mutations, probably due to a founder effect. Certain evidence suggests that mutations positioned towards the 5' end of the gene carry a higher risk of ovarian cancer than those at the 3' end. BRCA1 is infrequently somatically mutated in sporadic breast or ovarian cancer. The BRCA2 gene has been localized to chromosome 13q12-q13. BRCA2 carries a risk of breast cancer similar to that of BRCA1, but is associated with a lower risk of ovarian cancer and a higher risk of male breast cancer. Additional breast cancer susceptibility genes probably exist, but may be difficult to locate by conventional methods.

Cancer classification with DNA microarrays is less more?

The dissection of cancer and the underlying molecular processes that are defective in cancer cells has become an important tool in the fight against this disease. DNA microarrays can provide detailed information of the expression pattern of thousands of genes in tumours. But how much of this data is useful and is some superfluous? Can array data be used to identify a handful of critical genes that will lead to a more-detailed taxonomy of tumours and can this or similar array data be used to predict clinical outcome? Primary tumours will give us the statistical power to draw these conclusions, but can cancer cell lines be used as models to point us in the right direction?

Consistent loss of the wild type allele in breast cancers from a family linked to the BRCA2 gene on chromosome 13q12-13.

A small proportion of breast cancer is attributable to the inheritance of dominant, high penetrance susceptibility genes. One of these genes, BRCA2, has recently been localised by genetic linkage analysis to chromosome 13q12-13. This is a region known to exhibit loss of heterozygosity in 20-40% sporadic breast cancers. In this study, we have examined cancers from a family showing strong evidence of linkage to BRCA2. LOH was seen in seven out of eight informative cancers. In all cases the allele lost was the wild type allele that does not segregate with the disease in the family. The data suggest that both alleles of BRCA2 are inactivated in cancers, the pattern expected of a recessive oncogene or tumour suppressor gene.

Expression of a human liver cDNA encoding a UDP-glucuronosyltransferase catalysing the glucuronidation of hyodeoxycholic acid in cell culture.

A cDNA encoding a human liver UDPGT (HLUG 25) transcribed and translated in vitro showed that the encoded protein was synthesized as a precursor and was cleaved and glycosylated when dog pancreatic microsomes were present during translation. The UDPGT cDNA was transiently expressed in mammalian cell culture (COS-7 cells) resulting in the biosynthesis of a polypeptide of 52 kDa. This expressed UDPGT glycoprotein catalysed the glucuronidation of hyodeoxycholic acid forming an ether glucuronide. These results suggest that this UDPGT isoenzyme may be responsible for the glucuronidation of 6 alpha-hydroxy bile acids in human liver.

Gene expression microarray analysis in cancer biology, pharmacology, and drug development: progress and potential.

With the imminent completion of the Human Genome Project, biomedical research is being revolutionised by the ability to carry out investigations on a genome wide scale. This is particularly important in cancer, a disease that is caused by accumulating abnormalities in the sequence and expression of a number of critical genes. Gene expression microarray technology is gaining increasingly widespread use as a means to determine the expression of potentially all human genes at the level of messenger RNA. In this commentary, we review developments in gene expression microarray technology and illustrate the progress and potential of the methodology in cancer biology, pharmacology, and drug development. Important applications include: (a) development of a more global understanding of the gene expression abnormalities that contribute to malignant progression; (b) discovery of new diagnostic and prognostic indicators and biomarkers of therapeutic response; (c) identification and validation of new molecular targets for drug development; (d) provision of an improved understanding of the molecular mode of action during lead identification and optimisation, including structure-activity relationships for on-target versus off-target effects; (e) prediction of potential side-effects during preclinical development and toxicology studies; (f) confirmation of a molecular mode of action during hypothesis-testing clinical trials; (g) identification of genes involved in conferring drug sensitivity and resistance; and (h) prediction of patients most likely to benefit from the drug and use in general pharmacogenomic studies. As a result of further technological improvements and decreasing costs, the use of microarrays will become an essential and potentially routine tool for cancer and biomedical research.

Drug and xenobiotic glucuronidation catalysed by cloned human liver UDP-Glucuronosyltransferases stably expressed in tissue culture cell lines.

Two human UDP-Glucuronosyltransferase (UGT) cDNA clones were stably integrated into V79 chinese hamster fibroblast cells and the functional enzymes were expressed in this heterologous environment. More than 100 drugs and xenobiotics were used as substrates for glucuronidation, catalysed by the cloned UGTs to determine the chemical structures accepted as substrates. UGT HP1 exhibited a limited specificity for planar phenolic compounds, whereas UGT HP4 was more promiscuous in acceptance of non-planar phenols, anthraquinones, flavones, aliphatic alcohols, aromatic carboxylic acids, steroids and many drugs of varied structure. These conclusions are illustrated here by using a series of alkyl- and halophenols. This work indicates the considerable potential value in use of these recombinant cell lines to study human drug glucuronidation.

COSMIC 2005.

The Catalogue Of Somatic Mutations In Cancer (COSMIC) database and web site was developed to preserve somatic mutation data and share it with the community. Over the past 25 years, approximately 350 cancer genes have been identified, of which 311 are somatically mutated. COSMIC has been expanded and now holds data previously reported in the scientific literature for 28 known cancer genes. In addition, there is data from the systematic sequencing of 518 protein kinase genes. The total gene count in COSMIC stands at 538; 25 have a mutation frequency above 5% in one or more tumour type, no mutations were found in 333 genes and 180 are rarely mutated with frequencies <5% in any tumour set. The COSMIC web site has been expanded to give more views and summaries of the data and provide faster query routes and downloads. In addition, there is a new section describing mutations found through a screen of known cancer genes in 728 cancer cell lines including the NCI-60 set of cancer cell lines.

Mutations in the RSK2(RPS6KA3) gene cause Coffin-Lowry syndrome and nonsyndromic X-linked mental retardation.

We describe three families with X-linked mental retardation, two with a deletion of a single amino acid and one with a missense mutation in the proximal domain of the RSK2(RPS6KA3) (ribosomal protein S6 kinase, 90 kDa, polypeptide 3) protein similar to mutations found in Coffin-Lowry syndrome (CLS). In two families, the clinical diagnosis had been nonsyndromic X-linked mental retardation. In the third family, although CLS had been suspected, the clinical features were atypical and the degree of intellectual disability much less than expected. These families show that strict reliance on classical clinical criteria for mutation testing may result in a missed diagnosis. A less targeted screening approach to mutation testing is advocated.

Somatic mutations in human cancer: insights from resequencing the protein kinase gene family.

All cancers arise due to the accumulation of mutations in critical target genes that, when altered, give rise to selective advantage in the cell and its progeny that harbor them. Knowledge of these mutations is key in understanding the biology of cancer initiation and progression, as well as the development of more targeted therapeutic strategies. We have undertaken a systematic screen of all annotated protein kinases in the human genome for mutations in a series of cancers including breast, non-small-cell lung, and testicular cancer. Our results show a wide diversity in mutation prevalence within and between tumor types. We have identified a mutator phenotype in human breast previously undescribed. The results presented from sequencing the same 1.3 million base pairs through several tumor types suggest that most of the observed mutations are likely to be passenger events rather than causally implicated in oncogenesis. However, this work does provide evidence for the likely existence of multiple, infrequently mutated kinases.

Absence of methylation of CpG dinucleotides within the promoter of the breast cancer susceptibility gene BRCA2 in normal tissues and in breast and ovarian cancers.

Germline mutations of the BRCA2 gene on chromosome 13q12-q13 predispose to the development of early-onset breast cancer and ovarian cancer. Loss of heterozygosity detected using chromosome 13q markers in the vicinity of BRCA2 is observed in most cancers arising in carriers of germline BRCA2 mutations and also in 30-50% of sporadic breast and ovarian cancers. However, somatic mutations of BRCA2 are extremely rare in sporadic cancers. We have examined the hypothesis that expression of the BRCA2 gene may be suppressed in sporadic breast cancers by a mechanism that is associated with increased methylation of cytosine residues in the promoter region. Using a HpaII/MspI digestion-polymerase chain reaction based assay, the presence of 5-methylcytosine in three CpG dinucleotides within the BRCA2 promoter was assessed in 18 breast or ovarian cancer cell lines, in an SV40 large T antigen immortalized cell line derived from normal breast epithelial cells, in 64 primary sporadic breast cancers and peripheral blood leucocytes from these cases and in a number of other normal human tissues. Methylation was not detected in any of the tissues examined, suggesting that this mechanism of transcriptional repression is unlikely to explain the absence of somatic mutations in sporadic cancers.

Rapid detection of DNA sequence variants by conformation-sensitive capillary electrophoresis.

The identification of novel sequence variants, which may be either disease-causing mutations or silent polymorphisms, in large numbers of samples is becoming the rate-limiting step in associating diseases with specific genes. This is particularly true in light of the imminent arrival of the complete reference sequence of the human genome. A number of techniques have been developed to analyze DNA samples for sequence variants rapidly. We describe a new technique, capillary-based conformation-sensitive gel electrophoresis (capillary CSGE) that transfers mutation detection from acrylamide gel to capillary electrophoresis. Capillary CSGE was able to detect 7/7 short insertion/deletions and 16/22 base substitutions in a series of random single-nucleotide polymorphisms and known variants in the lipoprotein lipase and BRCA2 genes. This technique has the potential to screen many megabases of DNA in a single day.