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Lynch syndrome (Hereditary Nonpolyposis Colorectal Cancer, HNPCC) is the most common hereditary syndrome predisposing to colorectal cancer, accounting for 1-3% of all colorectal cancer. This multi-organ cancer predisposition syndrome is caused by mutations in the mismatch repair (MMR) genes, especially MLH1 and MSH2, and to lesser extents MSH6 and PMS2, which lead to widespread genetic instability and thus microsatellite instability (MSI). Hereditary cancer often manifests in two or more tumours in a single individual; 35-40% of Lynch syndrome patients have synchronous or metachronous tumours of the two major Lynch syndrome-related cancers: colorectal and endometrial. The main purposes of the work underlying this thesis were to identify persons at risk of Lynch syndrome or other types of hereditary colorectal cancer, to estimate the cancer risks associated with these predispositions and to identify the underlying genetic causes. A population-based cohort of 78 persons with double primary colorectal or colorectal and endometrial cancer was identified. Cancer risks in their 649 first-degree relatives were estimated in relation to tumour MSI status (positive or negative) and age at diagnosis (before or after 50 years of age) in the probands. The overall standardised incidence ratio was 1.69 (95% CI; 1.39-2.03). The highest risks for Lynch syndrome-associated cancers: (colorectal, endometrial, ovarian and gastric) were found in families with young MSI-positive probands, likely representing Lynch syndrome families. Importantly, no overall risk was found in families with old probands, irrespective of MSI status. Blood samples were available from 24 MSI-positive patients for mutation screening of MLH1, MSH2 and MSH6. Sequence variants or rearrangements predicted to affect protein function were found in 16 patients. Six novel variants were found: two large rearrangements, two truncating and two missense mutations. The missense mutations were found to segregate in the families. Studies of allele frequencies, MSI and loss of immunostaning in tumours from family members further supports the hypothesis that these missense changes play a role in Lynch syndrome, as do the non-conservative nature and evolutionary conservation of the amino acid exchanges. Five families had mutations in MLH1, five in MSH2, and six in MSH6. The unexpectedly large impact of MSH6 was in genealogical studies shown to be due to a founder effect. Cumulative risk studies showed that the MSH6 families, despite their late age of onset, have a high lifetime risk for all Lynch syndrome-related cancers, significantly higher in women (89% by age 80 years) than in men (69%). The gender differences are in part due to high endometrial (70%) and ovarian cancer risk (33%) in addition to the high colorectal cancer risk (60%). These findings are of great importance for counselling and surveillance of families with MSH6 mutations. Finally, in a large family with MSI-negative hereditary colorectal cancer for which the MMR genes and APC had been excluded as possible causes, a genome-wide linkage analysis was performed, resulting in a suggested linkage to chromosome 7. Conclusions: Relatives of probands with MSI-positive, double primary colorectal and endometrial cancer diagnosed before the age of 50 years have significantly increased risks of Lynch syndrome-related cancers. MSH6 mutations, which have unusually high impact in this study population due to a founder effect, confer high cumulative risks of cancer despite the generally late age of onset.

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2017-04-17T23:12:48Z No. of bitstreams: 1 AnaRafaelaDeSouzaTimoteo_TESE.pdf: 3659954 bytes, checksum: d2c8b061166b6be5547b4452cf6fed7b (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2017-04-20T21:54:51Z (GMT) No. of bitstreams: 1 AnaRafaelaDeSouzaTimoteo_TESE.pdf: 3659954 bytes, checksum: d2c8b061166b6be5547b4452cf6fed7b (MD5) / Made available in DSpace on 2017-04-20T21:54:51Z (GMT). No. of bitstreams: 1 AnaRafaelaDeSouzaTimoteo_TESE.pdf: 3659954 bytes, checksum: d2c8b061166b6be5547b4452cf6fed7b (MD5) Previous issue date: 2016-12-12 / A S?ndrome de c?ncer de mama e ov?rio heredit?rio corresponde a 10-15% de todos os casos diagnosticados de c?ncer de mama no mundo. A maioria das muta??es germinativas s?o identificadas nos genes BRCA1 e BRCA2, contudo, a aplica??o de pain?is multig?nicos tem aumentado o n?mero de variantes patog?nicas detectadas em outros genes supressores de tumor. De acordo com a vers?o atual do protocolo americano NCCN (National Comprehensive Cancer Network), as muta??es em BRCA1 e BRCA2, TP53 e PTEN conferem alto risco de desenvolver c?ncer de mama, e muta??es em CDH1, CHEK2, PALB2, ATM e BRIP podem aumentar em 20% o risco para o desenvolvimento desta doen?a. Neste estudo foram analisados 157 indiv?duos com hist?rico pessoal e/ou familiar de c?ncer de mama. O DNA gen?mico foi isolado a partir de sangue perif?rico por meio de extra??o ? base de solu??o salina e as amostras foram analisadas usando o sequenciamento de nova gera??o (NGS). Foram identificadas 15 variantes patog?nicas e 4 VUS (Variants of Uncertain Significance) em 27 indiv?duos (27/157; 17%), dos quais tr?s s?o assintom?ticos. Foram identificadas sete novas variantes em 4 genes: BRCA1_c.3409A>G; BRCA2_g.26826_30318del, BRCA2_c.5800C>T; BRCA2_c.5228G>A; BRCA2_c.5305delG; ATM_c.634delT e ATR_c.3043C>T. Sessenta e oito por cento (13/19; 68%) de variantes foi detectada nos genes BRCA1 e BRCA2, enquanto 32% (6/19) foram identificados nos genes de risco moderado ATM (2/19); ATR (1/19); CDH1 (1/19); MLH1 (1/19) e MSH6 (1/19). Os indiv?duos foram separados em dois grupos para a an?lise comparativa: portadores de muta??o nos genes de alto risco e nos genes de risco moderado. Entre os tr?s indiv?duos assintom?ticos, duas variantes est?o presentes nos genes de risco moderado ATM e MLH1. Entre os indiv?duos com c?ncer de mama, dezoito pacientes (18/24; 75%) apresentaram muta??es em genes de alto risco, enquanto seis (6/24; 25%) s?o portadores de muta??es em genes de risco moderado. Ambos os grupos apresentaram alta incid?ncia de c?ncer de mama precocemente (83% dos indiv?duos). O grupo de portadores de muta??o nos genes de alto risco apresentaram maior ocorr?ncia de tumores de alto grau (83% vs 67%, P = 0,0090). No grupo de indiv?duos com muta??es em genes de risco moderado, os tumores apresentaram um fen?tipo mais agressivo com c?ncer bilateral (33% versus 11%, P = 0,0002), ocorr?ncia de met?stases (33% vs 5,6%, P <0,0001) e ?bito (33% vs 5,6%, P <0,0001). Ao todo, 1/3 de variantes foram identificadas em genes de risco moderado em pacientes com c?ncer mais agressivo. Estes resultados refor?am a import?ncia da aplica??o de an?lise multig?nica em indiv?duos em situa??o de risco para c?ncer de mama, especialmente em uma popula??o heterog?nea como brasileira. / Hereditary breast and ovarian cancer (HBOC) corresponds to 10-15% of all diagnosed cases of breast cancer in the world. The majority germline mutations are identified in BRCA1 and BRCA2 genes, however the application of multigene panels has increased the number of pathogenic variations detected in DNA repair genes. According to the current version of NCCN (National Comprehensive Cancer Network) Guideline, mutations in BRCA1, BRCA2, TP53 and PTEN confers high risk to develop breast cancer, and mutations in CDH1, CHEK2, PALB2, ATM and BRIP can increases over than 20% this risk. We analyzed 157 individuals with personal and/or familial breast cancer history. Genomic DNA was isolated from peripheral blood through saline-based extraction and samples were analyzed using next-generation sequencing (NGS). We identified 15 pathogenic variants and 4 VUS (Variants of Uncertain Significance) in 27 individuals (27/157; 17%), in which three are asymptomatic. Seven novel variants in 4 genes were identified: BRCA1_c.3409A>G; BRCA2_g.26826_30318del, BRCA2_c.5800C>T; BRCA2_c.5228G>A; BRCA2_c.5305delG; ATM_c.634delT and ATR_c.3043C>T. Sixty-eight percent (13/19; 68%) of variants was detected in BRCA1 and BRCA2 genes, while 32% (6/19) were identified in moderate risk genes ATM (2/19); ATR (1/19); CDH1 (1/19); MLH1 (1/19) and MSH6 (1/19). The individuals were separated in two groups for comparative analysis: high-risk genes and moderate risk genes. Among three asymptomatic individuals, two present variants in moderate risk genes ATM and MLH1. Among breast cancer individuals, eighteen patients (18/24; 75%) presented mutations in high-risk genes, while six (6/24; 25%) harbored mutations in moderate risk genes. Both groups had a high incidence of early-onset breast cancer, 83%. The group of individuals harboring variants in high-risk genes presented a greater occurrence of high-grade tumors (83% vs. 67%, P= 0.0090). In the group of individuals harboring mutation in moderate risk genes, tumors presented a more aggressive phenotype with bilateral cancer (33% vs. 11%, P= 0.0002), occurrence of metastasis (33% vs. 5.6%, P<0.0001) and incidence of deaths (33% vs. 5.6%, P<0.0001). Altogether, 1/3 of variants were identified in moderate risk genes in patients presenting a more aggressive phenotype. These results reinforce the importance of applying multigene analysis in individuals at-risk for breast cancer, especially in a heterogeneous population as Brazilian.