Haplotypic characterization of BRCA1 c.5266dupC, the prevailing mutation in Brazilian hereditary breast/ovarian cancer.

Specific pathogenic mutations associated with breast cancer development can vary between ethnical groups. One example is BRCA1 c.5266dupC that was first described as a founder mutation in the Ashkenazi Jewish population, but was later also found in other populations. In Brazil, this mutation corresponds to 20% of pathogenic BRCA1 variants reported. Our objective was to investigate the haplotype component of a group of Brazilian families who inherited c.5266dupC in the BRCA1 gene and to verify the ancestry contribution from European, African, and Amerindian origins. Fourteen probands carrying c.5266dupC and 16 relatives (carriers and non-carriers) were investigated. The same haplotype was observed segregating within all the families analyzed, revealing no recombinants in a region of 0.68 Mb. Ancestry analysis demonstrated that the European component was predominant among probands. The BRCA1 c.5266dupC analysis indicates that there was a founder effect in the Brazilian population.

From yeast to humans: Understanding the biology of DNA Damage Response (DDR) kinases.

The DNA Damage Response (DDR) is a complex network of biological processes that protect cells from accumulating aberrant DNA structures, thereby maintaining genomic stability and, as a consequence, preventing the development of cancer and other diseases. The DDR pathway is coordinated by a signaling cascade mediated by the PI3K-like kinases (PIKK) ATM and ATR and by their downstream kinases CHK2 and CHK1, respectively. Together, these kinases regulate several aspects of the cellular program in response to genomic stress. Much of our understanding of these kinases came from studies performed in the 1990s using yeast as a model organism. The purpose of this review is to present a historical perspective on the discovery of the DDR kinases in yeast and the importance of this model for the identification and functional understanding of their mammalian orthologues.

Screening for germline mutations in mismatch repair genes in patients with Lynch syndrome by next generation sequencing.

Lynch syndrome (LS) is an autosomal dominant disorder, with high penetrance that affects approximately 3% of the cases of colorectal cancer. Affected individuals inherit germline mutations in genes responsible for DNA mismatch repair, mainly at MSH2, MLH1, MSH6 and PMS2. The molecular screening of these individuals is frequently costly and time consuming due to the large size of these genes. In addition, PMS2 mutation detection is often a challenge because there are 16 different pseudogenes identified until now. In the present work we evaluate a molecular screening strategy based in next generation sequencing (NGS) in order to optimize the mutation detection in LS patients. We established 16 multiplex PCRs for MSH2, MSH6 and MLH1 and 5 Long-Range PCRs for PMS2, coupled with NGS. The strategy was validated by screening 66 patients who filled Bethesda and Amsterdam criteria for LS from health institutions of Brazil. The mean depth of coverage for MSH2, MSH6, MLH1 and PMS2 genes was 7.988, 36.313, 11.899 and 4.772 times, respectively. Ninety-four variants were found in exons and flanking intron/exon regions for the four MMR genes. Twenty-five were pathogenic or VUS and found in 32 patients (7 in MSH2, 5 in MSH6, 12 in MLH1 e 1 in PMS2). All variants were confirmed by Sanger sequencing. The strategy was efficient to reduce time consuming and costs to identify genetic changes at these MMR genes, reducing in three times the number of PCR reactions performed per patient and was efficient in identifying variants at PMS2 gene.

Detecção de Variantes nos genes MSH2, MSH6, MLH1 e PMS2 utilizando Sequenciamento de Nova Geração / [Detection of Variants in the MSH2, MSH6, MLH1 and PMS2 using Nova Sequencing Generation]

A Síndrome de Lynch (SL) é uma herança autossômica dominante de alta penetrância, que atinge aproximadamente 3% dos pacientes com câncer colorretal. Os indivíduos acometidos apresentam mutações germinativas em um dos genes responsáveis pelo reparo de DNA por mal pareamento (Mismatch Repair- MMR): MSH2, MLH1, MSH6 ou PMS2. Por serem genes longos, a identificação de mutações nestes genes se torna demorada e com custo elevado. Desta maneira, o projeto teve como objetivo padronizar uma estratégia de identificação de variantes que permitisse reduzir os custos e tempo de rastreamento molecular dos genes MMR. Para isso, foram padronizadas 16 reações de PCRs-multiplex para os genes MSH2, MLH1 e MSH6 e 5 reações de PCRs de longo alcance para o gene PMS2. Estes produtos foram sequenciados utilizando a tecnologia de Nova Geração (NGS), através do equipamento HiSeq2500. A estratégia foi validada através do sequenciamento pelo método de Sanger dos genes de MMR em 66 pacientes, provenientes de quatro centros distintos do Brasil (INCARJ, HCPA- RS, HJUBB- PA e ACCAM- SP), e que preencheram os critérios de Bethesda para SL. As profundidades médias de cobertura obtidas para os genes MSH2, MSH6, MLH1 e PMS2 foram de 7.988, 36.313, 11.899 e 4.772 vezes, respectivamente. Foram identificadas 98 alterações em éxons e íntrons dos quatro genes, sendo que 25 eram patogênicas ou VUS (7 em MSH2, 5 em MSH6, 12 em MLH1 e 1 em PMS2) e foram encontradas em 32 pacientes. A estratégia padronizada foi eficaz na identificação de variantes dos genes MMR, permitiu reduzir em três vezes o número de reações de PCR realizadas por amostra e em 2,15 vezes o custo de rastreamento molecular para os quatro genes MMR. Dos fragmentos sequenciados, 3,1% apresentaram profundidade média de cobertura <30X e não foram eficientes na detecção de sítios variáveis nos genes MMR

Lynch Syndrome (LS) is an autosomal dominant inheritance of high penetrance that affects approximately 3% of the cases of colorectal cancer. Individuals affected with this syndrome inherit germline mutations in one of the genes responsible for the Mismatch Repair: MSH2, MLH1, MSH6 or PMS2. These large numbers of genes turns mutations' identification time consuming and costly. The project aimed to establish a molecular screening method in order to optimize the cost-effectiveness for screening these genes. We performed 16 Multiplex PCRs for MSH2, MSH6 and MLH1 genes and 5 Long-Range PCRs for PMS2 gene, coupled with Next Generation Sequencing (NGS) technologies. Our strategy was validated by the screening of sixty-six patients who filled Bethesda criteria for LS from different hospitals of Brazil (INCA-RJ, HCPA-RS, HJUBB-PA and ACCAM-SP). The depth of coverage for MSH2, MSH6, MLH1 and PMS2 genes was 7.988, 36.313, 11.899 and 4.772 times, respectively. Ninety-eight alterations were found in exons and introns regions for the four MMR genes. From this, 25 were pathogenic or VUS found in 32 patients (7 in MSH2, 5 in MSH6, 12 in MLH1 e 1 in PMS2). The strategy was efficient to identify genetic changes at the MMR genes, since allowed to reduce to 3 times the number of PCR reactions performed per patient and to reduce in 2,15 times the costs to molecular screening of the four MMR genes. Approximately 3% of the amplicons sequenced had a depth of coverage <30X and were not efficient in variation detection at MMR genes

Screening of the BRCA1 gene in Brazilian patients with breast and/or ovarian cancer via high-resolution melting reaction analysis.

The aim of this study was to evaluate the profile of BRCA1 mutations among cancer-affected Brazilian women from the Midwest region of Minas Gerais state with clearly defined risk factors for hereditary breast and ovarian cancer (HBOC) syndrome. In this Brazilian region, the first Center for Hereditary Cancer Control began operation in 2011, and 90% of patients receive assistance from the public health service. Eighteen patients at high risk for HBOC were subjected to molecular analysis. Primers were designed for 22 coding exons of the gene; DNA was extracted; and real-time PCR followed by high-resolution melting reaction was performed. The amplicons were sequenced to confirm the identified profiles. Only exon 11 was directly sequenced due its length. Multiplex ligation-dependent probe amplification (MLPA) was performed for those patients in whom no pathogenic mutations were found. Among the 14 alterations identified in this study, the c.5263_5264insC pathogenic mutation was present in two patients (11.1%). Four alterations showed no clinical relevance; one exhibited inconclusive clinical relevance according to the examined databases; and eight alterations presented a divergent classification between the databases. No deletions or duplications were found using the MLPA technique. The HRM methodology was highly sensitive in identifying variants in the BRCA1 gene and can dramatically reduce the amount of sequencing required to identify germline mutations in BRCA genes, enabling cheaper tests and increasing their availability to Brazilian women assisted by the public health service.