BRCA1 185delAG MUTATION CAN BE EASILY DETECTED BY AN ADAPTED ALLELE-SPECIFIC PCR

BRCA1 gene accounts for a majority of hereditary breast and ovarian cancers. Germinal deleteriousmutations within this gene are directly responsible for the disease, with a lifetime risk of cancer for mutations carriers ofabout 80%. While outbred and western populations usually show a heterogeneous profile of unique and familialmutations, in isolated and eastern European populations some recurrent mutations can be afforded the most responsibilityfor familial hereditary cases. In Ashkenazi Jewish and most Slavic eastern population, the BRCA1 185delAG is one of themost frequent mutations. Therefore, rapid screening by PCR-based methods can be useful in oncogenetic diagnosis. Herewe present implementation of an adapted allele-specific PCR for the detection of 185delAG, with wide applications indiagnosis and genotyping for large population groups

LIMITS AND PITFALLS OF SYBR GREEN DETECTION IN QUANTITATIVE PCR

Both PCR and Real-Time qPCR are nowadays widely applicable techniques in diagnosis and research. InReal-Time qPCR, current detection methods are based on changes in fluorescence, which are proportional to the increaseof target. Fidelity of Real-Time RT-PCR is associated with its “true” specificity, sensitivity, reproducibility androbustness and a most important point in succeed a RT-qPCR is to choose the right detection chemistry. SYBR Green andTaqMan assays produce comparably dynamic range and sensitivity, while SYBR Green detection can be more precisethan the TaqMan probe detection if reaction specificity is perfected. When amplifying by the SYBR Green detectionsystem, we observed variations between genes in the specificity and reliability, proving that a unique amplificationsystem doesn’t exist, and that each experimental system has its own ideal conditions needing to be optimized. Therefore,we focused on the great dangers in evaluating SYBR Green quantitative results without complete optimization

Sanger sequencing of MMR genes in a one-plate system

Both incidence and mortality of colorectal cancer (CRC) in Romania have shown a continuous increase during the last decades. Hereditary Non-Polyposic Colorectal Cancer (HNPCC), also known as Lynch syndrome, is mainly attributable to mismatch repair (MMR) genes MSH2, MSH6, and MLH1. Individuals carrying germ-line mutations of these genes present high lifetime risk of colorectal and other cancers, compared to non-carriers. Oncogenetics is developed worldwide nowadays, for identifying hereditary predisposition to cancer and offering appropriate clinical follow-up to patients and mutation carriers in Lynch families. Molecular oncogenetic diagnosis in Lynch syndrome is based on complete Sanger sequencing of entire MMR genes, which is time and resources consuming, therefore needing an appropriate and adapted optimization. Conventional sequencing requires a sufficient number of available samples to be processed simultaneously, which increases the waiting time for diagnostic results. Complete analysis for only one patient meets difficult technical problems due to the complex co-amplification of all gene regions of interest within the same conditions, therefore increasing the costs and reducing the cost-effectiveness of the test. Here we present an original and robust technical protocol for sequencing the entire MSH2, MSH6, and MLH1 coding sequence for one patient in a single PCR plate. Our optimized and verified system overcomes all technical problems and offers a quick, robust, and cost-effective possibility to personalize molecular oncogenetic diagnosis in Lynch syndrome

Identification of rare intronic variants defining novel BRCA haplotypes

Sanger DNA sequencing is widely used nowadays to identify germ-line alterations of cancer predisposition genes. Molecular diagnosis of hereditary risk of breast and ovarian cancer (HBOC) is mainly targeting BRCA1 and BRCA2, as carriers of deleterious mutations in any of these genes are at significantly higher risk of developing cancer than general population. Usual diagnosis performs a pre-screening step, followed by systematic sequencing of exons and exon/intron boundaries. Unfortunately, not all sequence variations found by sequencing are pathogenic. About one half of identified variants are of unclear clinical significance. Common single nucleotide polymorphisms (SNPs) are also usually detected, either heterozygous or homozygous. When systematically detected, SNPs can be used to define haplotypes, which are particularly useful in population disease studies or ethnogenomics. Frequent BRCA1 SNPs are well known, as well as associated haplotypes. Here we present some rare intronic variants defining novel BRCA1 haplotypes

ÉTUDE COMPARATIVE SUR L’ÉVOLUTION DE L’ALCOOLÉMIE AU COURS DU TEMPS

La consommation excessive d’alcool éthylique est connue depuis longtemps comme très dangereuse pour la santé humaine. De plus, l’alcool au volant représente, après la vitesse excessive, la deuxième cause d’accidents mortels sur la route. Quoique autorisé au volant à faibles doses dans certains pays, l’alcool met toujours en danger la vie du chauffeur et de ses passagers. Le taux d’alcool dans le sang, pour la même quantité d’éthanol consommée, varie en fonction de différents facteurs physiologiques. Il existe à l’heure actuelle de nombreuses modalités d’estimer l’évolution de l’alcoolémie au cours du temps. Nous avons essayé de démontrer que cette évolution présente une forte variabilité, en fonction du sexe, de l’age et de la physiologie individuell

Multiplex-PCR generates false positives in detection of the BRCA1 185delAG recurrent mutation

reast and ovarian cancer are common pathologies in women, with increasing incidences worldwide. In hereditary breast and ovarian cancer (HBOC) families, a large percentage of cases are attributable to hereditary factors compatibles with dominant autosomal transmission of a major tumour suppressor gene with incomplete penetrance. Screening for BRCA1 mutations is now standard practice for HBOC cases in western world, and permits medical follow- up and genetic counselling. Over 300 BRCA1 germinal mutations are stored in the Breast Cancer Information Core (BIC) mutation database. 185delAG, an Ashkenazi founder mutation, is a recurrent BRCA1 mutation in eastern European populations. Several screening methods were proposed for detection of 185delAG. We demonstrate that one screening methods generates false positives by unspecific amplifications, therefore being inappropriate for molecular diagnosis

EXPRESSION IN VIVO DU GÈNE GFP SOUS LA FORME D’UNE PROTÉINE DE FUSION

La protéine GFP (Green Fluorescent Protein) se caractérise par sa capacité à émettre sous l’action des rayons UV une lumière verte de basse énergie, ce qui la rend particulièrement utile en biotechnologies. Nous avons procédé à la production de la GFP in vivo par Eschericha coli sous forme d’une protéine chimère de fusion avec le peptide de la –Galactosidase. Pour ce, nous avons cloné un fragment d’ADN comportant la phase codante de la GFP à partir d’un vecteur dans lequel il ne peut être exprimé vers un autre vecteur permettant lui cette expression

Description and interpretation of various SNPs identified by BRCA2 gene sequencing

Molecular diagnosis for hereditary breast and ovarian cancer (HBOC) involves systematic DNA sequencing of predisposition genes like BRCA1 or BRCA2. Deleterious mutations within such genes are responsible for developing the disease, but other sequence variants can also be identified. Common Single Nucleotide Polymorphisms (SNPs) are usually present in human genome, defining alleles whose frequencies widely vary in different populations. Either intragenic or intronic, silent or generating aminoacid substitutions, SNPs cannot be afforded themselves a predisposition status. However, prevalent SNPs can be used to define gene haplotypes, with also various frequencies. Since some mutation can easily be assigned to haplotypes (such is the case for BRCA1 gene), SNPs can therefore provide usual information in interpreting gene mutations effects on hereditary predisposition to cancer. Here we describe 10 BRCA2 SNPs identified by complete gene sequencin

SNP GENOTYPING BY TAQMAN ALLELE DISCRIMINATION TECHNIQUE

Breast cancer is the most frequent neoplasm in women worldwide and the principal cause of deaths by cancer, the majority being by metastatic disease. About half of breast tumors are hormone dependent, and in post- menopause women the preferred first line treatment uses third generation aromatase inhibitors. Aromatase is encoded by CYP19 gene on 15q21.1, and there is strong evidence that mutations in this gene affect its expression, with direct consequences on cancer phenotype and response to treatment. Several single nucleotide polymorphisms have been studied on CYP19A1 transcription variant, notably rs727479, rs10046, rs4646 and rs700518. We implemented a Taqman- based allele discrimination assay for the rapid investigation of the 4 SNPs in CYP19A1. We genotyped 22 metastatic breast cancer patients by the technique described

Optimization of heteroduplex analysis for the detection of BRCA mutations and SNPs

BRCA1 and BRCA2 are tumour suppressor genes whose mutant phenotypes predispose to breast and ovarian cancer. Screening for mutations in these genes is now standard practice for hereditary breast and ovarian cancer (HBOC) cases in Europe, and permits medical follow-up and genetic counselling adapted to the needs of individuals in such families. Currently, most laboratories performing diagnostic analysis of the BRCA genes use PCR of exons and intron-exon boundaries coupled to a pre-screening step to identify anomalous amplicons. The techniques employed for the detection of mutations and SNPs have evolved over time and vary in sensitivity, specificity and cost-effectiveness. As a variant for pre-screening techniques, we chose the recently developed Surveyor® heteroduplex cleavage method as a sensitive and specific technique to reveal anomalous amplicons of the BRCA genes, using only basic laboratory equipment and agarose gel electrophoresis. Here we present the detection of either mutations or SNPs within the BRCA1 exon 7, using heteroduplex analysis (HA) by mismatch-specific endonuclease, confirmed by dideoxy sequencing