BRCA1 and BRCA2 mutation testing in Cyprus; a population based study.

This paper presents the largest study in Cyprus evaluating the frequency and distribution of BRCA1/2 mutations in a high risk patient cohort. Deleterious mutations in the BRCA1/2 genes were identified in 68 of the 527 patients tested (13%). It is of interest that a quarter of those tested positive, did not have an extensive family history of breast/ovarian cancer but were diagnosed with early onset breast cancer, ovarian cancer under the age of 60 or triple negative breast cancer. The spectrum of mutations identified in our patient cohort is different compared to other Mediterranean countries. Furthermore, several of the mutations detected are novel and have not been identified in other ethnic populations. This highlights the importance of operating a national reference center for cancer genetic diagnosis which offers services tailored to the needs of the Cypriot population.

Contribution of BRCA1 and BRCA2 germline mutations to the incidence of early-onset breast cancer in Cyprus.

In Cyprus, the prevalence of breast cancer associated with BRCA1 and BRCA2 mutations in young women is unknown. In this study, we present the results of mutational analysis of the BRCA1 and BRCA2 genes in 26 Cypriot women diagnosed with breast cancer by the age of 40. The entire coding regions, including splice sites, of the BRCA1 and BRCA2 genes were sequenced using cycle sequencing. We identified four pathogenic mutations: two in BRCA1 [c.1840A>T (K614X), c.5310delG (5429delG)] and two in BRCA2 [c.3531-3534delCAGC (3758del4), c.8755delG (8984delG)] in six of 26 unrelated patients. The BRCA2 mutation c.3531-3534delCAGC (3758del4) is novel and the BRCA1 mutation c.1840A>T (K614X) is reported for the first time in Cypriot patients. The BRCA2 Cypriot founder mutation c.8755delG (8984delG) was detected in three unrelated patients. Additionally, we identified one novel BRCA1 missense mutation, two novel polymorphisms and three novel intronic variants of which BRCA1 c.4185+3A>G (IVS12+3A>G) may be pathogenic. Of the six BRCA1/2 mutation carriers, only four had a family history. These results show that the prevalence of BRCA1 and BRCA2 mutations in Cypriot women diagnosed with early-onset breast cancer is high. We conclude that Cypriot women with early-onset breast cancer should be offered BRCA1/2 testing irrespective of their family history.

Novel germline mutations in the APC gene of Cypriot patients with familial and sporadic adenomatous polyposis.

Familial adenomatous polyposis (FAP) is one of the two commonest familial syndromes that predispose to colorectal cancer. FAP is caused by mutations in the adenomatous polyposis coli (APC) tumour suppressor gene that has a high penetrance. The disease is characterized by the occurrence of hundreds to thousands of colorectal polyps, which if left untreated give rise to colorectal cancer. In Cyprus, there are no molecular data available as yet on families with FAP. This work presents the results of APC analysis in our population for the first time. The APC gene was analyzed in 33 DNA samples from 20 individuals belonging to four FAP families and 13 patients with sporadic polyposis. We identified three truncating mutations, four missense mutations and 11 polymorphisms. It is of interest that two of the three truncating mutations, 2307delA and Q1242X, are novel, which supports the existence of a unique genetic pool in the Cypriot population. This ethnic molecular study in addition to highlighting population heterogeneity also contributes to phenotype-genotype associations that are essential for the clinical management of FAP families in Cyprus.

Morphological methods in the diagnosis of mitochondrial encephalomyopathies: the role of electron microscopy.

Mitochondrial encephalomyopathies (MEs) encompass a heterogeneous group of disorders that frequently present a diagnostic challenge to clinicians. Historically, MEs were diagnosed by finding ragged red fibers in the muscle biopsy and confirmatory evidence was provided by the presence of numerical and/or ultrastructural abnormalities in mitochondria. In most centers diagnosis involves clinical evaluation and the morphological, histochemical, and biochemical investigation of a skeletal muscle biopsy. However, with the availability of mitochondrial DNA analysis, the necessity and role of morphological methods and, in particular, electron microscopy has been questioned. The aim of this study was to delineate the role of electron microscopy in the diagnosis of MEs.

Correlation between morphology, immunohistochemistry and molecular pathology in hereditary and sporadic breast cancer cases.

Breast cancer still represents a serious health problem and is currently the most frequent malignancy in the female population in developed countries. In Cyprus, there are 300 new cases annually. In the present study, histology, electron microscopy, immunohistochemistry, and Western blot analysis were used to investigate 100 cases of invasive breast carcinoma. In addition, mutation analysis for the BRCA1 gene was carried out in patient DNA from 26 families with multiple cases of breast/ovarian cancers. Of note are the results of molecular biology which show that there are no germline truncating mutations in the BRCA1 gene in these 26 Cypriot breast cancer families. Furthermore, Western blot analysis revealed the presence of multiple BRCA1 bands in homogenates of tumor and normal tissues, and immunoelectron microscopy showed the presence of nuclear staining for BRCA1 antibodies.

BRCA1 germline mutations in Cypriot breast cancer patients from 26 families with family history.

Germline mutations in the BRCA1 gene are causative for a variable number of hereditary breast/ovarian cancers. The data presented in this study are based on genetic analysis of the BRCA1 gene in 49 DNA samples from breast cancer patients with a positive family history. A combination of manual direct DNA sequencing and SSCP analysis was used to screen the entire coding region of BRCA1. Overall 13 variants were detected which included 5 missense mutations, 3 polymorphisms and 5 intronic changes. Further genetic analysis of the 13 variants was carried out using 50 control DNA samples. Our results showed that 12 out of the 13 variants detected in the DNA of the patients group, were also present in the control group. It appears that the Greek Cypriot families studied so far have an unexpectebly low frequency of deleterious mutations in the BRCA1 gene. This is the first report on BRCA1 mutation analysis in Cyprus.

Immunohistochemical versus molecular detection of RAK antigens in breast cancer.

RAK antigens p120, p42, and p25 exhibit molecular and immunological similarity to the proteins encoded by HIV-1 and are expressed by 95% of breast and gynecological cancer cases in women and prostate cancer cases in men. Binding of the monoclonal antibody (MAb) RAK-BrI to cancer RAK antigens has been found to be inhibited by a peptide derived from the variable loop V3 of HIV-1. Since MAb RAK-BrI has been developed against denatured froms of breast cancer proteins, and it binds to a short epitope, GRAF, this MAb does not recognize the native, three-dimensional structure of proteins. Subsequently Western blot, after electrophoretic separation in gels with SDS, has been used to detect these unique cancer markers. The current studies were focused on the immunohistochemical evaluation of the novel marker RAK. Serial sections, 5 microm thick, were cut from frozen or Formalin-fixed, paraffin-embedded tissue blocks and immunostained with MAb RAK-BrI. All of the 53 cases of breast cancer tested RAK positive and no differences were observed in the immunohistochemical staining of lobular and ductal carcinoma cases. In contrast, MAb RAK-BrI antigens were detected in only 3 of 15 cases of macroscopically normal breast removed during mastectomy for breast cancer. It is noteworthy that Western blots of breast samples from the same series demonstrated a high expression of three RAK antigens in 20/20 of invasive breast carcinomas, while there was only a very weak expression of RAK antigens in 2/7 of the macroscopically "normal" breast samples. Due to the suspected viral origin of RAK markers, immunohistochemical staining with MAb RAK-BrI might be a useful tool in the early detection of malignant changes occurring in breast tissues.