BRCA1 and BRCA2 germline mutational spectrum and evidence for genetic anticipation in Portuguese breast/ovarian cancer families.

We present the first characterisation of the mutational spectrum of the entire coding sequences and exon-intron boundaries of the BRCA1 and BRCA2 genes as well as large BRCA1 rearrangements in Portuguese families with inherited predisposition to breast/ovarian cancer. Of the 100 probands studied, pathogenic mutations were identified in 22 (24.7%) of 89 breast and/or ovarian cancer families with more than one affected member (15 in BRCA1 and seven in BRCA2), but in none of the 11 patients without family history of cancer. One (6.7%) of the BRCA1 mutations is a large deletion involving exons 11-15. Seven pathogenic point mutations are novel: 2088C>T, 2156delinsCC, and 4255_4256delCT in BRCA1 and 4608_4609delTT, 5036delA, 5583_5584insT, and 8923C>T in BRCA2. The novel 2156delinsCC was identified in three probands from different families and probably represents a founder mutation in our population. We also found a previously reported 3450_3453del4 mutation in three unrelated patients. In addition to the 22 pathogenic mutations, we identified 19 missense mutations of uncertain pathogenic significance, three of them (5241G>C in BRCA1 and IVS6+13C>T and 3731T>C in BRCA2) previously undescribed. The percentage of cases with truncating mutations in BRCA1 and BRCA2 was higher in breast/ovarian cancer (37.0%, mostly BRCA1) and male breast cancer (40%, all BRCA2) families than in families with only female breast cancer (17.5%). Interestingly, we found evidence for genetic anticipation regarding age at diagnosis of both breast and ovarian cancer in those families presenting affected members in more than one generation. These findings should be taken into consideration while planning screening and prophylactic measures in families with inherited predisposition to breast and ovarian cancer.

A quantitative promoter methylation profile of prostate cancer.

PURPOSE: Promoter hypermethylation is an alternative pathway for gene silencing in neoplastic cells and a promising cancer detection marker. Although quantitative methylation-specific PCR (QMSP) of the GSTP1 promoter has demonstrated near perfect specificity for cancer detection in prostate biopsies, we postulated that identification and characterization of additional methylation markers might further improve its high (80-90%) sensitivity. EXPERIMENTAL DESIGN: We surveyed nine gene promoters (GSTP1, MGMT, p14/ARF, p16/CDKN2A, RASSF1A, APC, TIMP3, S100A2, and CRBP1) by QMSP in tissue DNA from 118 prostate carcinomas, 38 paired high-grade prostatic intraepithelial neoplasias (HGPIN), and 30 benign prostatic hyperplasias (BPH). The methylation levels were calculated and were correlated with clinical and pathologic indicators. RESULTS: Only the methylation frequencies of GSTP1 and APC were significantly higher in prostate carcinoma compared with BPH (P < 0.001). Methylation levels of GSTP1, APC, RASSF1A, and CRBP1, differed significantly between prostate carcinoma and HGPIN, and/or HGPIN or BPH (P < 0.0001). With QMSP and empirically defined cutoff values, the combined use of GSTP1 and APC demonstrated a theoretical sensitivity of 98.3% for prostate carcinoma, with 100% specificity. Methylation levels were found to correlate with tumor grade (GSTP1 and APC) and stage (GSTP1, RASSF1A, and APC). CONCLUSIONS: Our data demonstrate the existence of a progressive increase of promoter methylation levels of several cancer-related genes in prostate carcinogenesis, providing additional markers to augment molecular detection of prostate carcinoma. Because methylation levels of GSTP1, APC, and RASSF1A are associated with advanced grade and stage, QMSP might augment the pathologic indicators currently used to predict tumor aggressiveness.

I105V polymorphism and promoter methylation of the GSTP1 gene in prostate adenocarcinoma.

The GSTP1 gene encodes for an enzyme, glutathione S-transferase pi (GSTpi),involved in detoxification of carcinogens. An aminoacid substitution (I105V) in GSTP1 produces a variant enzyme with lower activity and less capability of effective detoxification. This variant GSTP*B allele has been associated with a propensity to develop several neoplasms. Because GSTP1 promoter hypermethylation and inactivation of GSTpi expression is a frequent alteration in prostate carcinoma, we hypothesized that this somatic epigenetic modification could obviate any reduced enzyme activity caused by the germ-line polymorphism. We tested for the GSTP1 genotype in a population of prostate cancer patients, and in a control group composed of patients with benign prostatic hyperplasia (BPH) and healthy blood donors. Tissue samples from the 105 prostate cancer cases (105 adenocarcinomas and 34 prostatic intraepithelial neoplasia lesions), and from 43 BPH patients were tested for GSTP1 hypermethylation by methylation-specific PCR. GSTpi protein expression was assessed by immunohistochemistry. No significant effect on prostate cancer risk was detectable for GSTP1 genotype compared with the control population (odds ratio, 1.02; 95% confidence interval, 0.59-1.75). Moreover, no association was found between this genotype and tumor or BPH methylation status. Patients with unmethylated carcinomas did not disclose significant differences in genotypic distribution compared with the control population. In adenocarcinoma, a strong association (P < 0.00001) between GSTP1 promoter hypermethylation and loss of GSTpi expression was observed; however, this trend was not retained in prostatic intraepithelial neoplasia or BPH lesions. Although the GSTP1 polymorphism is not associated with altered susceptibility to prostate cancer, somatic promoter hypermethylation is an effective, but not the only, cause of decreased GSTpi function.

Polymorphisms of arylamine N-acetyltransferase (NAT1 and NAT2) and larynx cancer susceptibility.

Our aim was to examine the role of NAT1 and NAT2 polymorphisms in human larynx cancer susceptibility. Genotype tests for NAT1 alleles *4, *10 and *11, and NAT2 alleles *4, *5, *6A and *7A, using PCR-RFLP analysis, were performed in 172 healthy Portuguese individuals and 88 patients with squamous cell carcinoma of the larynx. NAT1 and NAT2 genotype frequencies were correlated between patients and control groups, using the chi-square test. Odds ratios and 95% confidence intervals were calculated from 2 x 2 tables with the Fisher's exact model. The statistical analysis of NAT1 and NAT2 genotype frequencies revealed a significant difference of NAT1*10/*11 (p = 0.038) and NAT2*5/*7 (p = 0.003) genotype distribution between cases and controls. We also observed differences concerning tumor location, since NAT1*10/*11 genotype frequency was significantly different when comparing normal control individuals with the glottic subgroup of patients. The present data suggest that NAT1 and NAT2 polymorphisms may be correlated with an increased risk of larynx cancer.

Cyclin D1 A870G polymorphism and amplification in laryngeal squamous cell carcinoma: implications of tumor localization and tobacco exposure.

Altered Cyclin D1 activity, due to gene amplification and/or protein overexpression, is related to the development of several human cancers, including head and neck SCC. This study investigated the relationship between CCND1 A870G gene polymorphism and amplification with the development and progression of laryngeal SCC, considering the implications of tumor localization and tobacco exposure. The study population consisted of 66 larynx cancer patients and 110 healthy individuals. CCND1 A/G polymorphism in exon 4 was genotyped by a PCR-RFLP assay. Cyclin D1 gene amplification was evaluated by a Differential-PCR assay and determined by a quantitative densitometric analysis. Our data on gene amplification did not show any correlation with disease stage, histological tumor differentiation, recurrent disease, disease-specific survival or tumor location. However, GG870 genotype was associated with a shorter disease free interval and a reduced overall survival in laryngeal cancer patients. Moreover, this constitutes the first report of a correlation between cyclin D1 A870G polymorphism and increased susceptibility for laryngeal tumor development at the glottic region, which supports the theory of site-specific prevalence of genetic alterations.