Hilmi Ozcelik

MADR2 Maps to 18q21 and Encodes a TGFβ–Regulated MAD–Related Protein That Is Functionally Mutated in Colorectal Carcinoma

The MAD-related (MADR) family of proteins are essential components in the signaling pathways of serine/threonine kinase receptors for the transforming growth factor beta (TGFbeta) superfamily. We demonstrate that MADR2 is specifically regulated by TGFbeta and not bone morphogenetic proteins. The gene for MADR2 was found to reside on chromosome 18q21, near DPC4, another MADR protein implicated in pancreatic cancer. Mutational analysis of MADR2 in sporadic tumors identified four missense mutations in colorectal carcinomas, two of which display a loss of heterozygosity. Biochemical and functional analysis of three of these demonstrates that the mutations are inactivating. These findings suggest that MADR2 is a tumor suppressor and that mutations acquired in colorectal carcinomas may function to disrupt TGFbeta signaling.

Discovery of novel hypermethylated genes in prostate cancer using genomic CpG island microarrays.

Background Promoter and 5′ end methylation regulation of tumour suppressor genes is a common feature of many cancers. Such occurrences often lead to the silencing of these key genes and thus they may contribute to the development of cancer, including prostate cancer. Methodology/Principal Findings In order to identify methylation changes in prostate cancer, we performed a genome-wide analysis of DNA methylation using Agilent human CpG island arrays. Using computational and gene-specific validation approaches we have identified a large number of potential epigenetic biomarkers of prostate cancer. Further validation of candidate genes on a separate cohort of low and high grade prostate cancers by quantitative MethyLight analysis has allowed us to confirm DNA hypermethylation of HOXD3 and BMP7, two genes that may play a role in the development of high grade tumours. We also show that promoter hypermethylation is responsible for downregulated expression of these genes in the DU-145 PCa cell line. Conclusions/Significance This study identifies novel epigenetic biomarkers of prostate cancer and prostate cancer progression, and provides a global assessment of DNA methylation in prostate cancer.

ras mutation and expression of the ras-regulated genes osteopontin and cathepsin L in human esophageal cancer

As part of our ongoing studies to characterize molecular alterations in a well‐defined series of surgically resected esophageal cancers, we examined the expression of 2 ras‐regulated genes, whose products (osteopontin and cathepsin L) previously were shown to be associated with tumor invasion and metastasis. RNA was extracted from primary esophageal tumors (adenocarcinomas, 19; squamous‐cell carcinomas, 6) and matched histologically normal esophageal mucosa from the distant resection margin. Northern analysis was used to quantitate RNA, relative to an 18S rRNA control, and immunohistochemistry to assess the tissue distribution of osteopontin. In addition, H‐, K‐ and N‐ras mutations were studied in the same tissues using PCR and hybridization with allele (mutant)‐specific oligonucleotide probes. We demonstrated a K‐ras mutation (codon 12, GTT) in one esophageal adenocarcinoma. The ras‐regulated gene osteopontin was over‐expressed in 100% of squamous‐cell carcinomas and in 58% of adenocarcinomas relative to matched normal esophageal mucosa. Patterns of immunoreactivity for osteopontin protein also varied between squamous‐cell carcinomas (tumor cell staining) and adenocarcinomas (predominantly tumor‐infiltrating macrophages). Expression of cathepsin L also varied with esophageal tumor histology, with over‐expression in 58% of primary esophageal adenocarcinomas and 33% of squamous‐cell cancers. Int. J. Cancer 72:739–745, 1997.

HER-2/neu Status and Tumor Morphology of Invasive Breast Carcinomas in Ashkenazi Women with Known BRCA1 Mutation Status in the Ontario Familial Breast Cancer Registry

The prevalence of BRCA1 germline mutations is greater in the Ashkenazi Jewish population than in the general North American population. The Ontario Familial Breast Cancer Registry collects clinical and family history data in familial breast carcinoma cases, and unselected Ashkenazi breast carcinomas, and acts as a tumor tissue repository.

BRCA1 and BRCA2 mutation carriers in the Breast Cancer Family Registry: an open resource for collaborative research

The Breast Cancer Family Registry is a resource for interdisciplinary and translational studies of the genetic epidemiology of breast cancer. This resource is available to researchers worldwide for collaborative studies. Herein, we report the results of testing for germline mutations in BRCA1 and BRCA2. We have tested 4,531 probands for mutations in BRCA1 and 4,084 in BRCA2. Deleterious mutations in BRCA1 and BRCA2 were identified for 9.8% of probands tested [233/4,531 (5.1%) for BRCA1 and 193/4,084 (4.7%) for BRCA2]. Of 1,385 Ashkenazi Jewish women tested for only the three founder mutations, 17.4% carried a deleterious mutation. In total, from the proband and subsequent family testing, 1,360 female mutation carriers (788 in BRCA1, 566 in BRCA2, 6 in both BRCA1 and BRCA2) have been identified. The value of the resource has been greatly enhanced by determining the germline BRCA1 and BRCA2 mutation statuses of nearly 6,000 probands.

Methodological issues in detecting gene-gene interactions in breast cancer susceptibility: a population-based study in Ontario

BackgroundThere is growing evidence that gene-gene interactions are ubiquitous in determining the susceptibility to common human diseases. The investigation of such gene-gene interactions presents new statistical challenges for studies with relatively small sample sizes as the number of potential interactions in the genome can be large. Breast cancer provides a useful paradigm to study genetically complex diseases because commonly occurring single nucleotide polymorphisms (SNPs) may additively or synergistically disturb the system-wide communication of the cellular processes leading to cancer development.MethodsIn this study, we systematically studied SNP-SNP interactions among 19 SNPs from 18 key genes involved in major cancer pathways in a sample of 398 breast cancer cases and 372 controls from Ontario. We discuss the methodological issues associated with the detection of SNP-SNP interactions in this dataset by applying and comparing three commonly used methods: the logistic regression model, classification and regression trees (CART), and the multifactor dimensionality reduction (MDR) method.ResultsOur analyses show evidence for several simple (two-way) and complex (multi-way) SNP-SNP interactions associated with breast cancer. For example, all three methods identified XPD-[Lys751Gln]*IL10-[G(-1082)A] as the most significant two-way interaction. CART and MDR identified the same critical SNPs participating in complex interactions. Our results suggest that the use of multiple statistical approaches (or an integrated approach) rather than a single methodology could be the best strategy to elucidate complex gene interactions that have generally very different patterns.ConclusionThe strategy used here has the potential to identify complex biological relationships among breast cancer genes and processes. This will lead to the discovery of novel biological information, which will improve breast cancer risk management.

Meta-analysis of two ERCC2 (XPD) polymorphisms, Asp312Asn and Lys751Gln, in breast cancer.

The excision repair cross-complementing group 2 gene (ERCC2) plays a key role in DNA repair. Several polymorphisms in the ERCC2 gene have been described, including the commonly occurring Lys751Gln and Asp312Asn polymorphisms. Studies investigating the association of these polymorphisms with breast cancer risk produced controversial results. To evaluate these associations presented in diverse populations, we have conducted a meta-analysis based on 40 studies from 33 publications in PubMed which included analyses of Lys751Gln (14,545 cases, 15,352 controls) and Asp312Asn polymorphisms (16,254 cases, 14,006 controls). Overall findings of both polymorphisms have implicated null effects (ORxa0=xa01.01–1.03) when the analyses were limited to the statistically powerful (≥80%) studies. Although modestly increased statistically significant breast cancer risk was detected in the underpowered studies (≤80%), removal of outliers resulted in null associations. Ethnic stratification showed non-significant and relatively null associations for both polymorphisms with breast cancer risk for the overall Caucasians as well as North American and the European sub-populations. Although statistically increased and decreased risks were observed for the homogenous populations of African-Americans (Lys751Gln, OR 1.25, 95% CI 1.03–1.53, Pxa0=xa00.03) and Asians (Asp312Asn, ORs: 0.53–0.55, P values: 0.02–0.03), respectively, this may be the result of small sample size. Analyses of the homogeneous adduct studies, with relatively large sample size, exhibited increased risk for Lys751Gln (OR 1.20, 95% CI (1.02–1.41), Pxa0=xa00.03) and Asp312Asn (OR 1.17 95% CI 1.02–1.34, Pxa0=xa00.03) under the dominant genetic model. In conclusion, our results suggest null associations of both polymorphisms in the overall and the Caucasian subgroups, although some effects can be suggested for relatively smaller minority studies. Increased risk effect was more visible when the adduct studies are considered, suggesting the role of these polymorphisms in the presence of exposure to DNA damaging agents.

Heteroduplex and protein truncation analysis of the BRCA1 185delAG mutation

Abstract We describe a heteroduplex analysis for the detection of the 185delAG mutation in the BRCA1 gene. The protein truncation test (PTT) has previously been used to identify many of the mutations in BRCA1 that result in premature termination of the protein. However, we were not able to detect the 185delAG mutation by PTT and suggest that heteroduplex analysis may complement PTT for analysis of BRCA1 mutations. This simple technique may be useful for studies on the prevalence and the penetrance of the 185delAG mutation.

Discovery, Validation and Characterization of Erbb4 and Nrg1 Haplotypes Using Data from Three Genome-Wide Association Studies of Schizophrenia

Schizophrenia is one of the most common and complex neuropsychiatric disorders, which is contributed both by genetic and environmental exposures. Recently, it is shown that NRG1-mediated ErbB4 signalling regulates many important cellular and molecular processes such as cellular growth, differentiation and death, particularly in myelin-producing cells, glia and neurons. Recent association studies have revealed genomic regions of NRG1 and ERBB4, which are significantly associated with risk of developing schizophrenia; however, inconsistencies exist in terms of validation of findings between distinct populations. In this study, we aim to validate the previously identified regions and to discover novel haplotypes of NRG1 and ERBB4 using logistic regression models and Haploview analyses in three independent datasets from GWAS conducted on European subjects, namely, CATIE, GAIN and nonGAIN. We identified a significant 6-kb block in ERBB4 between chromosome locations 212,156,823 and 212,162,848 in CATIE and GAIN datasets (pu200a=u200a0.0206 and 0.0095, respectively). In NRG1, a significant 25-kb block, between 32,291,552 and 32,317,192, was associated with risk of schizophrenia in all CATIE, GAIN, and nonGAIN datasets (pu200a=u200a0.0005, 0.0589, and 0.0143, respectively). Fine mapping and FastSNP analysis of genetic variation located within significantly associated regions proved the presence of binding sites for several transcription factors such as SRY, SOX5, CEPB, and ETS1. In this study, we have discovered and validated haplotypes of ERBB4 and NRG1 in three independent European populations. These findings suggest that these haplotypes play an important role in the development of schizophrenia by affecting transcription factor binding affinity.

p53 protein accumulation and mutations in normal and benign breast tissue

Mutations in the p53 gene are amongst the most common molecular changes detected in breast cancer, and there are several reports suggesting that changes in p53 may contribute to the pathogenesis of this disease. In a previous case‐control study, we demonstrated that p53 protein accumulation detected by immunohistochemistry in normal or benign breast tissue was associated with a 2.5‐fold increase in the risk of subsequent breast cancer. In this study, we investigated whether p53 gene mutations were present in the 29 p53 immunopositive normal or benign breast tissue samples and in 15 p53 immunonegative normal or benign breast tissue samples selected randomly from the original study. DNA was extracted from paraffin sections and underwent PCR‐SSCP analysis for exons 4 to 10. PCR products that showed abnormal mobility were excised and sequenced. Sixteen (59.2%) of the 27 immunopositive breast tissue samples and 4 (26.7%) of the 15 immunonegative samples had p53 sequence changes. There was no obvious association between the occurrence of these alterations and any specific histopathologic features. Ten cases showed p53 mutations, and they were all missense base substitutions of the transition type. Thirteen other gene changes occurred in 11 breast tissue samples and consisted of 8 silent (no amino acid change), 4 intronic alterations, and 1 indeterminate alteration. One individual had both a mutation and a silent change. In summary, p53 gene alterations can occur in normal or benign breast tissue, but resolution of their role in the pathogenesis of breast cancer will require long‐term follow‐up studies involving comparisons of breast cancer occurrence in patients with and without p53 mutations as well as functional assays to determine their significance. Int. J. Cancer 87:73–78, 2000.