Sandra M. Bell

Kirsten ras mutations in patients with colorectal cancer: The 'RASCAL II' study

Researchers worldwide with information about the Kirsten ras (Ki-ras) tumour genotype and outcome of patients with colorectal cancer were invited to provide that data in a schematized format for inclusion in a collaborative database called RASCAL (The Kirsten ras in-colorectal-cancer collaborative group). Our results from 2721 such patients have been presented previously and for the first time in any common cancer, showed conclusively that different gene mutations have different impacts on outcome, even when the mutations occur at the same site on the genome. To explore the effect of Ki-ras mutations at different stages of colorectal cancer, more patients were recruited to the database, which was reanalysed when information on 4268 patients from 42 centres in 21 countries had been entered. After predetermined exclusion criteria were applied, data on 3439 patients were entered into a multivariate analysis. This found that of the 12 possible mutations on codons 12 and 13 of Kirsten ras, only one mutation on codon 12, glycine to valine, found in 8.6% of all patients, had a statistically significant impact on failure-free survival (P = 0.004, HR 1.3) and overall survival (P = 0.008, HR 1.29). This mutation appeared to have a greater impact on outcome in Dukes’ C cancers (failure-free survival, P = 0.008, HR 1.5; overall survival P = 0.02, HR 1.45) than in Dukes’ B tumours (failure-free survival, P = 0.46, HR 1.12; overall survival P = 0.36, HR 1.15). Ki-ras mutations may occur early in the development of pre-cancerous adenomas in the colon and rectum. However, this collaborative study suggests that not only is the presence of a codon 12 glycine to valine mutation important for cancer progression but also that it may predispose to more aggressive biological behaviour in patients with advanced colorectal cancer.

Identification of Microcephalin, a Protein Implicated in Determining the Size of the Human Brain

Primary microcephaly (MIM 251200) is an autosomal recessive neurodevelopmental condition in which there is a global reduction in cerebral cortex volume, to a size comparable with that of early hominids. We previously mapped the MCPH1 locus, for primary microcephaly, to chromosome 8p23, and here we report that a gene within this interval, encoding a BRCA1 C-terminal domain-containing protein, is mutated in MCPH1 families sharing an ancestral 8p23 haplotype. This gene, microcephalin, is expressed in the developing cerebral cortex of the fetal brain. Further study of this and related genes may provide important new insights into neocortical development and evolution.

Prognostic value of p53 overexpression and c-Ki-ras gene mutations in colorectal cancer

BACKGROUND Mutations in Ki-ras codon 12 and the p53 gene are common abnormalities in colorectal cancer. The occurrence of p53 overexpression and/or Ki-ras codon 12 mutations were analyzed in 100 colorectal adenomas to determine if they were related to patient survival. METHODS p53 overexpression was identified by immunohistochemistry, and Ki-ras codon 12 mutations were detected using the polymerase chain reaction and a restriction enzyme digestion method. RESULTS p53 overexpression was identified in 45% of tumors, with a higher frequency identified in DNA aneuploid and left-sided tumors than in DNA diploid and right-sided tumors. Mutations in Ki-ras codon 12 were identified in 24% of carcinomas. Individually, mutations in Ki-ras codon 12 or p53 overexpression were not prognostic indicators of survival. However, a statistically significant difference in survival was identified when these two oncogenic abnormalities were analyzed together. The median survival of patients whose tumors contained both oncogenic abnormalities was less than half of that of patients with either alteration alone or without either abnormality. CONCLUSIONS Screening for multiple genetic abnormalities in colorectal cancers excised at surgery may prove to be a useful tool in determining prognosis.

Reduced expression of oestrogen receptor β in invasive breast cancer and its re-expression using DNA methyl transferase inhibitors in a cell line model

To gain insights into the possible role of oestrogen receptor (ER) β in breast carcinogenesis, immunohistochemical analysis of ER β was performed on 512 breast specimens encompassing normal (n = 138), pure ductal carcinoma in situ (n = 16), invasive cancers (n = 319), lymph node metastases (n = 31), and recurrences (n = 8). Real‐time polymerase chain reaction (PCR) was used to investigate the methylation status of the ER β gene in the ER β negative breast cancer cell lines SkBr3 and MDA‐MB‐435. A gradual reduction in, but not a complete loss of, ER β expression was observed during the transition from normal and pre‐invasive lesions to invasive cancers, where ER β was lost in 21% of cases. This was more pronounced in invasive ductal than in lobular carcinomas, a significantly higher proportion of which were ER β‐positive (74% compared with 91%, respectively, p = 0.0004). Examination of paired primary cancers with their axillary lymph node metastases showed that if ER β was present in the primary tumour, it persisted in the metastasis. Treatment of ER β‐negative cell lines with DNA methyl transferase inhibitors restored ER β expression, providing experimental evidence that silencing of ER β in breast carcinomas could be due to promoter hypermethylation. These results suggest that loss of ER β expression is one of the hallmarks of breast carcinogenesis and that it may be a reversible process involving methylation. Copyright

Rapid detection of allele loss in colorectal tumours using microsatellites and fluorescent DNA technology.

In order to investigate allele loss in colorectal tumours we have developed a rapid technique which overcomes most of the problems associated with radioactive Restriction Fragment Length Polymorphism (RFLP) analysis of allele loss. We utilise microsatellite length polymorphisms which are highly informative and are closely linked to loci of interest. Sequences containing microsatellites can be amplified from normal and tumour DNA pairs by a polymerase chain reaction (PCR) in which one of the primers is fluorescently labelled. This enables us to detect the products on polyacrylamide gels run on an automated DNA sequencer using dedicated software, by which results are automatically quantitated in terms of peak size, height, and area. Using this technique we have analysed 26 normal tissue: cancer pairs for allele loss at two loci linked to the adenomatous polyposis coli (APC) gene on chromosome 5q. Repeated assays yielded identical results for each pair. Allele loss was found in 10 out of 25 informative samples (40%).

Mutations in Microcephalin Cause Aberrant Regulation of Chromosome Condensation

Microcephalin (MCPH1) is a gene mutated in primary microcephaly, an autosomal recessive neurodevelopmental disorder in which there is a marked reduction in brain size. PCC syndrome is a recently described disorder of microcephaly, short stature, and misregulated chromosome condensation. Here, we report the finding that MCPH1 primary microcephaly and PCC syndrome are allelic disorders, both having mutations in the MCPH1 gene. The two conditions share a common cellular phenotype of premature chromosome condensation in the early G2 phase of the cell cycle, which, therefore, appears to be a useful diagnostic marker for individuals with MCPH1 gene mutations. We demonstrate that an siRNA-mediated depletion of MCPH1 is sufficient to reproduce this phenotype and also show that MCPH1-deficient cells exhibit delayed decondensation postmitosis. These findings implicate microcephalin as a novel regulator of chromosome condensation and link the apparently disparate fields of neurogenesis and chromosome biology. Further characterization of MCPH1 is thus likely to lead to fundamental insights into both the regulation of chromosome condensation and neurodevelopment.

Methylation and colorectal cancer.

Statistics rate colorectal adenocarcinoma as the most common cause of cancer death on exclusion of smoking‐related neoplasia. However, the reported accumulation of genetic lesions over the adenoma to adenocarcinoma sequence cannot wholly account for the neoplastic phenotype. Recently, heritable, epigenetic changes in DNA methylation, in association with a repressive chromatin structure, have been identified as critical determinants of tumour progression. Indeed, the transcriptional silencing of both established and novel tumour suppressor genes has been attributed to the aberrant cytosine methylation of promoter‐region CpG islands. This review aims to set these epigenetic changes within the context of the colorectal adenoma to adenocarcinoma sequence. The role of cytosine methylation in physiological and pathological gene silencing is discussed and the events behind aberrant cytosine methylation in ageing and cancer are appraised. Emphasis is placed on the interrelationships between epigenetic and genetic lesions and the manner in which they cooperate to define a CpG island methylator phenotype at an early stage in tumourigenesis. Finally, the applications of epigenetics to molecular pathology and patient diagnosis and treatment are reviewed. Copyright

An siRNA-based functional genomics screen for the identification of regulators of ciliogenesis and ciliopathy genes

Defects in primary cilium biogenesis underlie the ciliopathies, a growing group of genetic disorders. We describe a whole-genome siRNA-based reverse genetics screen for defects in biogenesis and/or maintenance of the primary cilium, obtaining a global resource. We identify 112 candidate ciliogenesis and ciliopathy genes, including 44 components of the ubiquitin–proteasome system, 12 G-protein-coupled receptors, and 3 pre-mRNA processing factors (PRPF6, PRPF8 and PRPF31) mutated in autosomal dominant retinitis pigmentosa. The PRPFs localize to the connecting cilium, and PRPF8- and PRPF31-mutated cells have ciliary defects. Combining the screen with exome sequencing data identified recessive mutations in PIBF1, also known as CEP90, and C21orf2, also known as LRRC76, as causes of the ciliopathies Joubert and Jeune syndromes. Biochemical approaches place C21orf2 within key ciliopathy-associated protein modules, offering an explanation for the skeletal and retinal involvement observed in individuals with C21orf2 variants. Our global, unbiased approaches provide insights into ciliogenesis complexity and identify roles for unanticipated pathways in human genetic disease.

c-Ki-ras gene mutations in dysplasia and carcinomas complicating ulcerative colitis.

One hundred and nine samples comprising carcinomas, adenomas, dysplastic, inflamed and normal mucosa from patients with sporadic colon cancer and ulcerative colitis (UC) were analysed for c-Ki-ras mutations. DNA was extracted from archival paraffin-embedded material, amplified using the polymerase chain reaction (PCR) and the PCR products analysed using restriction enzyme digestion. Forty-two per cent (14/33) of the sporadic carcinoma controls contained Ki-ras codon 12 mutations in contrast to 24% (8/33) of ulcerative colitis carcinomas. A significantly higher c-Ki-ras mutation rate was observed in rectal carcinomas (72%) in comparison to colonic carcinomas (28%) in control patients (P less than 0.04), while the opposite was observed in UC patients. The difference between the incidence of c-Ki-ras mutations in rectal carcinomas in UC (9%) and in sporadic rectal carcinomas (72%) was also significant (P less than 0.01). This lower prevalence rate and different site distribution of c-Ki-ras mutations in UC carcinomas compared to sporadic carcinomas suggests that specific genetic differences may underlie the causation of carcinomas arising in these situations.

Human ASPM participates in spindle organisation, spindle orientation and cytokinesis

BackgroundMutations in the A bnormal Sp indle M icrocephaly related gene (ASPM) are the commonest cause of autosomal recessive primary microcephaly (MCPH) a disorder characterised by a small brain and associated mental retardation. ASPM encodes a mitotic spindle pole associated protein. It is suggested that the MCPH phenotype arises from proliferation defects in neural progenitor cells (NPC).ResultsWe show that ASPM is a microtubule minus end-associated protein that is recruited in a microtubule-dependent manner to the pericentriolar matrix (PCM) at the spindle poles during mitosis. ASPM siRNA reduces ASPM protein at the spindle poles in cultured U2OS cells and severely perturbs a number of aspects of mitosis, including the orientation of the mitotic spindle, the main determinant of developmental asymmetrical cell division. The majority of ASPM depleted mitotic cells fail to complete cytokinesis. In MCPH patient fibroblasts we show that a pathogenic ASPM splice site mutation results in the expression of a novel variant protein lacking a tripeptide motif, a minimal alteration that correlates with a dramatic decrease in ASPM spindle pole localisation. Moreover, expression of dominant-negative ASPM C-terminal fragments cause severe spindle assembly defects and cytokinesis failure in cultured cells.ConclusionsThese observations indicate that ASPM participates in spindle organisation, spindle positioning and cytokinesis in all dividing cells and that the extreme C-terminus of the protein is required for ASPM localisation and function. Our data supports the hypothesis that the MCPH phenotype caused by ASPM mutation is a consequence of mitotic aberrations during neurogenesis. We propose the effects of ASPM mutation are tolerated in somatic cells but have profound consequences for the symmetrical division of NPCs, due to the unusual morphology of these cells. This antagonises the early expansion of the progenitor pool that underpins cortical neurogenesis, causing the MCPH phenotype.