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1.
Artigo em Inglês | MEDLINE | ID: mdl-31636762

RESUMO

Background: We previously reported that in pathogenic mismatch repair (path_MMR) variant carriers, the incidence of colorectal cancer (CRC) was not reduced when colonoscopy was undertaken more frequently than once every 3 years, and that CRC stage and interval since last colonoscopy were not correlated. Methods: The Prospective Lynch Syndrome Database (PLSD) that records outcomes of surveillance was examined to determine survival after colon cancer in relation to the time since previous colonoscopy and pathological stage. Only path_MMR variants scored by the InSiGHT variant database as class 4 or 5 (clinically actionable) were included in the analysis. Results: Ninety-nine path_MMR carriers had no cancer prior to or at first colonoscopy, but subsequently developed colon cancer. Among these, 96 were 65 years of age or younger at diagnosis, and included 77 path_MLH1, 17 path_MSH2, and 2 path_MSH6 carriers. The number of cancers detected within < 1.5, 1.5-2.5, 2.5-3.5 and at > 3.5 years after previous colonoscopy were 9, 43, 31 and 13, respectively. Of these, 2, 8, 4 and 3 were stage III, respectively, and only one stage IV (interval 2.5-3.5 years) disease. Ten-year crude survival after colon cancer were 93, 94 and 82% for stage I, II and III disease, respectively (p < 0.001). Ten-year crude survival when the last colonoscopy had been < 1.5, 1.5-2.5, 2.5-3.5 or > 3.5 years before diagnosis, was 89, 90, 90 and 92%, respectively (p = 0.91). Conclusions: In path_MLH1 and path_MSH2 carriers, more advanced colon cancer stage was associated with poorer survival, whereas time since previous colonoscopy was not. Although the numbers are limited, together with our previously reported findings, these results may be in conflict with the view that follow-up of path_MMR variant carriers with colonoscopy intervals of less than 3 years provides significant benefit.

2.
J Med Genet ; 2019 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-31494577

RESUMO

INTRODUCTION: Lynch syndrome (LS) and constitutional mismatch repair deficiency (CMMRD) are hereditary cancer syndromes associated with mismatch repair (MMR) deficiency. Tumours show microsatellite instability (MSI), also reported at low levels in non-neoplastic tissues. Our aim was to evaluate the performance of high-sensitivity MSI (hs-MSI) assessment for the identification of LS and CMMRD in non-neoplastic tissues. MATERIALS AND METHODS: Blood DNA samples from 131 individuals were grouped into three cohorts: baseline (22 controls), training (11 CMMRD, 48 LS and 15 controls) and validation (18 CMMRD and 18 controls). Custom next generation sequencing panel and bioinformatics pipeline were used to detect insertions and deletions in microsatellite markers. An hs-MSI score was calculated representing the percentage of unstable markers. RESULTS: The hs-MSI score was significantly higher in CMMRD blood samples when compared with controls in the training cohort (p<0.001). This finding was confirmed in the validation set, reaching 100% specificity and sensitivity. Higher hs-MSI scores were detected in biallelic MSH2 carriers (n=5) compared with MSH6 carriers (n=15). The hs-MSI analysis did not detect a difference between LS and control blood samples (p=0.564). CONCLUSIONS: The hs-MSI approach is a valuable tool for CMMRD diagnosis, especially in suspected patients harbouring MMR variants of unknown significance or non-detected biallelic germline mutations.

3.
Sci Rep ; 9(1): 9020, 2019 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-31227763

RESUMO

The cancer-predisposing syndrome caused by biallelic mutations in NTHL1 may not be a solely colorectal cancer (CRC) and polyposis syndrome but rather a multi-tumor recessive disease. The presence of ≤10 adenomas in several mutation carriers suggests a possible causal role of NTHL1 in hereditary or early-onset nonpolyposis CRC. The involvement of NTHL1 in serrated/hyperplastic polyposis remains unexplored. The aim of our study is to elucidate the role of NTHL1 in the predisposition to personal or familial history of multiple tumor types, familial/early-onset nonpolyposis CRC, and serrated polyposis. NTHL1 mutational screening was performed in 312 cancer patients with personal or family history of multiple tumor types, 488 with hereditary nonpolyposis CRC, and 96 with serrated/hyperplastic polyposis. While no biallelic mutation carriers were identified in patients with personal and/or family history of multiple tumor types or with serrated polyposis, one was identified among the 488 nonpolyposis CRC patients. The carrier of c.268C>T (p.Q90*) and 550-1G>A was diagnosed with CRC and meningioma at ages 37 and 45 respectively, being reclassified as attenuated adenomatous polyposis after the cumulative detection of 26 adenomas. Our findings suggest that biallelic mutations in NTHL1 rarely cause CRC, a personal/familial multi-tumor history, or serrated polyposis, in absence of adenomas.

4.
Hum Mutat ; 40(11): 1910-1923, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31243857

RESUMO

Technological advances have allowed the identification of new adenomatous and serrated polyposis genes, and of several candidate genes that require additional supporting evidence of causality. Through an exhaustive literature review and mutational screening of 177 unrelated polyposis patients, we assessed the involvement of MCM9, FOCAD, POLQ, and RNF43 in the predisposition to (nonserrated) colonic polyposis, as well as the prevalence of NTHL1 and MSH3 mutations among genetically unexplained polyposis patients. Our results, together with previously reported data and mutation frequency in controls, indicate that: MCM9 and POLQ mutations are not associated with polyposis; germline RNF43 mutations, with a prevalence of 1.5-2.5% among serrated polyposis patients, do not cause nonserrated polyposis; MSH3 biallelic mutations are highly infrequent among European polyposis patients, and the prevalence of NTHL1 biallelic mutations among unexplained polyposes is ~2%. Although nonsignificant, FOCAD predicted deleterious variants are overrepresented in polyposis patients compared to controls, warranting larger studies to provide definite evidence in favor or against their causal association with polyposis predisposition.

5.
Artigo em Inglês | MEDLINE | ID: mdl-30858900

RESUMO

Background: Recent epidemiological evidence shows that colorectal cancer (CRC) continues to occur in carriers of pathogenic mismatch repair (path_MMR) variants despite frequent colonoscopy surveillance in expert centres. This observation conflicts with the paradigm that removal of all visible polyps should prevent the vast majority of CRC in path_MMR carriers, provided the screening interval is sufficiently short and colonoscopic practice is optimal. Methods: To inform the debate, we examined, in the Prospective Lynch Syndrome Database (PLSD), whether the time since last colonoscopy was associated with the pathological stage at which CRC was diagnosed during prospective surveillance. Path_MMR carriers were recruited for prospective surveillance by colonoscopy. Only variants scored by the InSiGHT Variant Interpretation Committee as class 4 and 5 (clinically actionable) were included. CRCs detected at the first planned colonoscopy, or within one year of this, were excluded as prevalent cancers. Results: Stage at diagnosis and interval between last prospective surveillance colonoscopy and diagnosis were available for 209 patients with 218 CRCs, including 162 path_MLH1, 45 path_MSH2, 10 path_MSH6 and 1 path_PMS2 carriers. The numbers of cancers detected within < 1.5, 1.5-2.5, 2.5-3.5 and at > 3.5 years since last colonoscopy were 36, 93, 56 and 33, respectively. Among these, 16.7, 19.4, 9.9 and 15.1% were stage III-IV, respectively (p = 0.34). The cancers detected more than 2.5 years after the last colonoscopy were not more advanced than those diagnosed earlier (p = 0.14). Conclusions: The CRC stage and interval since last colonoscopy were not correlated, which is in conflict with the accelerated adenoma-carcinoma paradigm. We have previously reported that more frequent colonoscopy is not associated with lower incidence of CRC in path_MMR carriers as was expected. In contrast, point estimates showed a higher incidence with shorter intervals between examinations, a situation that may parallel to over-diagnosis in breast cancer screening. Our findings raise the possibility that some CRCs in path_MMR carriers may spontaneously disappear: the host immune response may not only remove CRC precursor lesions in path_MMR carriers, but may remove infiltrating cancers as well. If confirmed, our suggested interpretation will have a bearing on surveillance policy for path_MMR carriers.

6.
Cancer Lett ; 447: 86-92, 2019 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-30677446

RESUMO

Somatic epigenetic inactivation of the DNA repair protein O6-methylguanine DNA methyltransferase (MGMT) is frequent in colorectal cancer (CRC); however, its involvement in CRC predisposition remains unexplored. We assessed the role and relevance of MGMT germline mutations and epimutations in familial and early-onset CRC. Mutation and promoter methylation screenings were performed in 473 familial and/or early-onset mismatch repair-proficient nonpolyposis CRC cases. No constitutional MGMT inactivation by promoter methylation was observed. Of six rare heterozygous germline variants identified, c.346C > T (p.H116Y) and c.476G > A (p.R159Q), detected in three and one families respectively, affected highly conserved residues and showed segregation with cancer in available family members. In vitro, neither p.H116Y nor p.R159Q caused statistically significant reduction of MGMT repair activity. No evidence of somatic second hits was found in the studied tumors. Case-control data showed over-representation of c.346C > T (p.H116Y) in familial CRC compared to controls, but no overall association of MGMT mutations with CRC predisposition. In conclusion, germline mutations and constitutional epimutations in MGMT are not major players in hereditary CRC. Nevertheless, the over-representation of c.346C > T (p.H116Y) in our familial CRC cohort warrants further research.


Assuntos
Neoplasias Colorretais/genética , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , Células Germinativas/fisiologia , Mutação em Linhagem Germinativa/genética , Proteínas Supressoras de Tumor/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Metilação de DNA/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Masculino , Pessoa de Meia-Idade , Regiões Promotoras Genéticas/genética , Adulto Jovem
7.
J Med Genet ; 56(8): 521-525, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30580288

RESUMO

IMPORTANCE: Genetic testing of hereditary cancer using comprehensive gene panels can identify patients with more than one pathogenic mutation in high and/or moderate-risk-associated cancer genes. This phenomenon is known as multilocus inherited neoplasia alleles syndrome (MINAS), which has been potentially linked to more severe clinical manifestations. OBJECTIVE: To determine the prevalence and clinical features of MINAS in a large cohort of adult patients with hereditary cancer homogeneously tested with the same gene panel. PATIENTS AND METHODS: A cohort of 1023 unrelated patients with suspicion of hereditary cancer was screened using a validated panel including up to 135 genes associated with hereditary cancer and phakomatoses. RESULTS: Thirteen (1.37%) patients harbouring two pathogenic mutations in dominant cancer-predisposing genes were identified, representing 5.7% (13/226) of patients with pathogenic mutations. Most (10/13) of these cases presented clinical manifestations associated with only one of the mutations identified. One case showed mutations in MEN1 and MLH1 and developed tumours associated with both cancer syndromes. Interestingly, three of the double mutants had a young age of onset or severe breast cancer phenotype and carried mutations in moderate to low-risk DNA damage repair-associated genes; two of them presented biallelic inactivation of CHEK2. We included these two patients for the sake of their clinical interest although we are aware that they do not exactly fulfil the definition of MINAS since both mutations are in the same gene. CONCLUSIONS AND RELEVANCE: Genetic analysis of a broad cancer gene panel identified the largest series of patients with MINAS described in a single study. Overall, our data do not support the existence of more severe manifestations in double mutants at the time of diagnosis although they do confirm previous evidence of severe phenotype in biallelic CHEK2 and other DNA repair cancer-predisposing genes.

8.
Br J Cancer ; 119(8): 978-987, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30283143

RESUMO

BACKGROUND: Constitutional MLH1 epimutations are characterised by monoallelic methylation of the MLH1 promoter throughout normal tissues, accompanied by allele-specific silencing. The mechanism underlying primary MLH1 epimutations is currently unknown. The aim of this study was to perform an in-depth characterisation of constitutional MLH1 epimutations targeting the aberrantly methylated region around MLH1 and other genomic loci. METHODS: Twelve MLH1 epimutation carriers, 61 Lynch syndrome patients, and 41 healthy controls, were analysed by Infinium 450 K array. Targeted molecular techniques were used to characterise the MLH1 epimutation carriers and their inheritance pattern. RESULTS: No nucleotide or structural variants were identified in-cis on the epimutated allele in 10 carriers, in which inter-generational methylation erasure was demonstrated in two, suggesting primary type of epimutation. CNVs outside the MLH1 locus were found in two cases. EPM2AIP1-MLH1 CpG island was identified as the sole differentially methylated region in MLH1 epimutation carriers compared to controls. CONCLUSION: Primary constitutional MLH1 epimutations arise as a focal epigenetic event at the EPM2AIP1-MLH1 CpG island in the absence of cis-acting genetic variants. Further molecular characterisation is needed to elucidate the mechanistic basis of MLH1 epimutations and their heritability/reversibility.


Assuntos
Neoplasias Colorretais Hereditárias sem Polipose/genética , Neoplasias Colorretais/genética , Metilação de DNA/genética , Epigênese Genética/genética , Predisposição Genética para Doença/genética , Proteína 1 Homóloga a MutL/genética , Sequência de Bases , Neoplasias Colorretais/epidemiologia , Ilhas de CpG/genética , Feminino , Haplótipos/genética , Humanos , Masculino , Mutação/genética , Regiões Promotoras Genéticas/genética , Análise de Sequência de DNA
9.
Hum Mutat ; 39(9): 1214-1225, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29900613

RESUMO

The causal association of NUDT1 (=MTH1) and OGG1 with hereditary colorectal cancer (CRC) remains unclear. Here, we sought to provide additional evidence for or against the causal contribution of NUDT1 and OGG1 mutations to hereditary CRC and/or polyposis. Mutational screening was performed using pooled DNA amplification and targeted next-generation sequencing in 529 families (441 uncharacterized MMR-proficient familial nonpolyposis CRC and 88 polyposis cases). Cosegregation, in silico analyses, in vitro functional assays, and case-control associations were carried out to characterize the identified variants. Five heterozygous carriers of novel (n = 1) or rare (n = 4) NUDT1 variants were identified. In vitro deleterious effects were demonstrated for c.143G>A p.G48E (catalytic activity and protein stability) and c.403G>T p.G135W (protein stability), although cosegregation data in the carrier families were inconclusive or nonsupportive. The frequency of missense, loss-of-function, and splice-site NUDT1 variants in our familial CRC cohort was similar to the one observed in cancer-free individuals, suggesting lack of association with CRC predisposition. No OGG1 pathogenic mutations were identified. Our results suggest that the contribution of NUDT1 and OGG1 germline mutations to hereditary CRC and to polyposis is inexistent or, at most, negligible. The inclusion of these genes in routine genetic testing is not recommended.


Assuntos
Polipose Adenomatosa do Colo/genética , Neoplasias Colorretais Hereditárias sem Polipose/genética , DNA Glicosilases/genética , Enzimas Reparadoras do DNA/genética , Monoéster Fosfórico Hidrolases/genética , Polipose Adenomatosa do Colo/patologia , Neoplasias Colorretais Hereditárias sem Polipose/patologia , Reparo do DNA/genética , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Variação Genética/genética , Genótipo , Mutação em Linhagem Germinativa/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mutação com Perda de Função/genética , Masculino , Mutação de Sentido Incorreto/genética , Estresse Oxidativo , Isoformas de Proteínas/genética
10.
Mol Cancer ; 17(1): 23, 2018 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-29448935

RESUMO

Germline mutations in BUB1 and BUB3 have been reported to increase the risk of developing colorectal cancer (CRC) at young age, in presence of variegated aneuploidy and reminiscent dysmorphic traits of mosaic variegated aneuploidy syndrome. We performed a mutational analysis of BUB1 and BUB3 in 456 uncharacterized mismatch repair-proficient hereditary non-polyposis CRC families and 88 polyposis cases. Four novel or rare germline variants, one splice-site and three missense, were identified in four families. Neither variegated aneuploidy nor dysmorphic traits were observed in carriers. Evident functional effects in the heterozygous form were observed for c.1965-1G>A, but not for c.2296G>A (p.E766K), in spite of the positive co-segregation in the family. BUB1 c.2473C>T (p.P825S) and BUB3 c.77C>T (p.T26I) remained as variants of uncertain significance. As of today, the rarity of functionally relevant mutations identified in familial and/or early onset series does not support the inclusion of BUB1 and BUB3 testing in routine genetic diagnostics of familial CRC.


Assuntos
Polipose Adenomatosa do Colo/genética , Proteínas de Ciclo Celular/genética , Neoplasias Colorretais Hereditárias sem Polipose/genética , Mutação em Linhagem Germinativa , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas Serina-Treonina Quinases/genética , Fuso Acromático/genética , Proteínas de Ciclo Celular/química , Humanos , Modelos Moleculares , Linhagem , Proteínas de Ligação a Poli-ADP-Ribose/química , Conformação Proteica , Proteínas Serina-Treonina Quinases/química
12.
Gastroenterology ; 154(1): 181-194.e20, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28912018

RESUMO

BACKGROUND & AIMS: Although there is a genetic predisposition to colorectal cancer (CRC), few of the genes that affect risk have been identified. We performed whole-exome sequence analysis of individuals in a high-risk family without mutations in genes previously associated with CRC risk to identify variants associated with inherited CRC. METHODS: We collected blood samples from 3 relatives with CRC in Spain (65, 62, and 40 years old at diagnosis) and performed whole-exome sequence analyses. Rare missense, truncating or splice-site variants shared by the 3 relatives were selected. We used targeted pooled DNA amplification followed by next generation sequencing to screen for mutations in candidate genes in 547 additional hereditary and/or early-onset CRC cases (502 additional families). We carried out protein-dependent yeast growth assays and transfection studies in the HT29 human CRC cell line to test the effects of the identified variants. RESULTS: A total of 42 unique or rare (population minor allele frequency below 1%) nonsynonymous genetic variants in 38 genes were shared by all 3 relatives. We selected the BRF1 gene, which encodes an RNA polymerase III transcription initiation factor subunit for further analysis, based on the predicted effect of the identified variant and previous association of BRF1 with cancer. Previously unreported or rare germline variants in BRF1 were identified in 11 of 503 CRC families, a significantly greater proportion than in the control population (34 of 4300). Seven of the identified variants (1 detected in 2 families) affected BRF1 mRNA splicing, protein stability, or expression and/or function. CONCLUSIONS: In an analysis of families with a history of CRC, we associated germline mutations in BRF1 with predisposition to CRC. We associated deleterious BRF1 variants with 1.4% of familial CRC cases, in individuals without mutations in high-penetrance genes previously associated with CRC. Our findings add additional evidence to the link between defects in genes that regulate ribosome synthesis and risk of CRC.


Assuntos
Neoplasias Colorretais/genética , Mutação em Linhagem Germinativa/genética , Fatores Associados à Proteína de Ligação a TATA/genética , Adulto , Idoso , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Feminino , Predisposição Genética para Doença/genética , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , Espanha
13.
Gut ; 67(7): 1306-1316, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-28754778

RESUMO

BACKGROUND: Most patients with path_MMR gene variants (Lynch syndrome (LS)) now survive both their first and subsequent cancers, resulting in a growing number of older patients with LS for whom limited information exists with respect to cancer risk and survival. OBJECTIVE AND DESIGN: This observational, international, multicentre study aimed to determine prospectively observed incidences of cancers and survival in path_MMR carriers up to 75 years of age. RESULTS: 3119 patients were followed for a total of 24 475 years. Cumulative incidences at 75 years (risks) for colorectal cancer were 46%, 43% and 15% in path_MLH1, path_MSH2 and path_MSH6 carriers; for endometrial cancer 43%, 57% and 46%; for ovarian cancer 10%, 17% and 13%; for upper gastrointestinal (gastric, duodenal, bile duct or pancreatic) cancers 21%, 10% and 7%; for urinary tract cancers 8%, 25% and 11%; for prostate cancer 17%, 32% and 18%; and for brain tumours 1%, 5% and 1%, respectively. Ovarian cancer occurred mainly premenopausally. By contrast, upper gastrointestinal, urinary tract and prostate cancers occurred predominantly at older ages. Overall 5-year survival for prostate cancer was 100%, urinary bladder 93%, ureter 85%, duodenum 67%, stomach 61%, bile duct 29%, brain 22% and pancreas 0%. Path_PMS2 carriers had lower risk for cancer. CONCLUSION: Carriers of different path_MMR variants exhibit distinct patterns of cancer risk and survival as they age. Risk estimates for counselling and planning of surveillance and treatment should be tailored to each patient's age, gender and path_MMR variant. We have updated our open-access website www.lscarisk.org to facilitate this.


Assuntos
Neoplasias do Colo/epidemiologia , Neoplasias Colorretais Hereditárias sem Polipose/complicações , Neoplasias Colorretais Hereditárias sem Polipose/mortalidade , Neoplasias Pancreáticas/epidemiologia , Neoplasias Urogenitais/epidemiologia , Fatores Etários , Idoso , Neoplasias Colorretais Hereditárias sem Polipose/patologia , Bases de Dados Factuais , Feminino , Humanos , Incidência , Masculino , Estudos Prospectivos
14.
Artigo em Inglês | MEDLINE | ID: mdl-29046738

RESUMO

BACKGROUND: We have previously reported a high incidence of colorectal cancer (CRC) in carriers of pathogenic MLH1 variants (path_MLH1) despite follow-up with colonoscopy including polypectomy. METHODS: The cohort included Finnish carriers enrolled in 3-yearly colonoscopy (n = 505; 4625 observation years) and carriers from other countries enrolled in colonoscopy 2-yearly or more frequently (n = 439; 3299 observation years). We examined whether the longer interval between colonoscopies in Finland could explain the high incidence of CRC and whether disease expression correlated with differences in population CRC incidence. RESULTS: Cumulative CRC incidences in carriers of path_MLH1 at 70-years of age were 41% for males and 36% for females in the Finnish series and 58% and 55% in the non-Finnish series, respectively (p > 0.05). Mean time from last colonoscopy to CRC was 32.7 months in the Finnish compared to 31.0 months in the non-Finnish (p > 0.05) and was therefore unaffected by the recommended colonoscopy interval. Differences in population incidence of CRC could not explain the lower point estimates for CRC in the Finnish series. Ten-year overall survival after CRC was similar for the Finnish and non-Finnish series (88% and 91%, respectively; p > 0.05). CONCLUSIONS: The hypothesis that the high incidence of CRC in path_MLH1 carriers was caused by a higher incidence in the Finnish series was not valid. We discuss whether the results were influenced by methodological shortcomings in our study or whether the assumption that a shorter interval between colonoscopies leads to a lower CRC incidence may be wrong. This second possibility is intriguing, because it suggests the dogma that CRC in path_MLH1 carriers develops from polyps that can be detected at colonoscopy and removed to prevent CRC may be erroneous. In view of the excellent 10-year overall survival in the Finnish and non-Finnish series we remain strong advocates of current surveillance practices for those with LS pending studies that will inform new recommendations on the best surveillance interval.

15.
Int J Cancer ; 141(7): 1365-1380, 2017 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-28577310

RESUMO

In a proportion of patients presenting mismatch repair (MMR)-deficient tumors, no germline MMR mutations are identified, the so-called Lynch-like syndrome (LLS). Recently, MMR-deficient tumors have been associated with germline mutations in POLE and MUTYH or double somatic MMR events. Our aim was to elucidate the molecular basis of MSH2-deficient LS-suspected cases using a comprehensive analysis of colorectal cancer (CRC)-associated genes at germline and somatic level. Fifty-eight probands harboring MSH2-deficient tumors were included. Germline mutational analysis of MSH2 (including EPCAM deletions) and MSH6 was performed. Pathogenicity of MSH2 variants was assessed by RNA analysis and multifactorial likelihood calculations. MSH2 cDNA and methylation of MSH2 and MSH6 promoters were studied. Matched blood and tumor DNA were analyzed using a customized next generation sequencing panel. Thirty-five individuals were carriers of pathogenic or probably pathogenic variants in MSH2 and EPCAM. Five patients harbored 4 different MSH2 variants of unknown significance (VUS) and one had 2 novel MSH6 promoter VUS. Pathogenicity assessment allowed the reclassification of the 4 MSH2 VUS and 6 probably pathogenic variants as pathogenic mutations, enabling a total of 40 LS diagnostics. Predicted pathogenic germline variants in BUB1, SETD2, FAN1 and MUTYH were identified in 5 cases. Three patients had double somatic hits in MSH2 or MSH6, and another 2 had somatic alterations in other MMR genes and/or proofreading polymerases. In conclusion, our comprehensive strategy combining germline and somatic mutational status of CRC-associated genes by means of a subexome panel allows the elucidation of up to 86% of MSH2-deficient suspected LS tumors.


Assuntos
Neoplasias Colorretais Hereditárias sem Polipose/genética , Reparo de Erro de Pareamento de DNA/genética , Proteínas de Ligação a DNA/genética , Mutação em Linhagem Germinativa , Proteína 2 Homóloga a MutS/deficiência , Proteína 2 Homóloga a MutS/genética , DNA Glicosilases/genética , Metilação de DNA , Análise Mutacional de DNA , Proteínas de Ligação a DNA/deficiência , Molécula de Adesão da Célula Epitelial/genética , Exodesoxirribonucleases/genética , Sequenciamento de Nucleotídeos em Larga Escala , Histona-Lisina N-Metiltransferase/genética , Humanos , Perda de Heterozigosidade , Regiões Promotoras Genéticas/genética , Proteínas Serina-Treonina Quinases/genética
16.
Fam Cancer ; 16(4): 501-507, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28365877

RESUMO

The clinical spectrum of germline mismatch repair (MMR) gene variants continues increasing, encompassing Lynch syndrome, Constitutional MMR Deficiency (CMMRD), and the recently reported MSH3-associated polyposis. Genetic diagnosis of these hereditary cancer syndromes is often hampered by the presence of variants of unknown significance (VUS) and overlapping phenotypes. Two PMS2 VUS, c.2149G>A (p.V717M) and c.2444C>T (p.S815L), were identified in trans in one individual diagnosed with early-onset colorectal cancer (CRC) who belonged to a family fulfilling clinical criteria for hereditary cancer. Clinico-pathological data, multifactorial likelihood calculations and functional analyses were used to refine their clinical significance. Likelihood analysis based on cosegregation and tumor data classified the c.2444C>T variant as pathogenic, which was supported by impaired MMR activity associated with diminished protein expression in functional assays. Conversely, the c.2149G>A variant displayed MMR proficiency and protein stability. These results, in addition to the conserved PMS2 expression in normal tissues and the absence of germline microsatellite instability (gMSI) in the biallelic carrier ruled out a CMMRD diagnosis. The use of comprehensive strategies, including functional and clinico-pathological information, is mandatory to improve the clinical interpretation of naturally occurring MMR variants. This is critical for appropriate clinical management of cancer syndromes associated to MMR gene mutations.


Assuntos
Neoplasias Colorretais/genética , Endonuclease PMS2 de Reparo de Erro de Pareamento/genética , Mutação de Sentido Incorreto , Idade de Início , Estudos de Casos e Controles , Reparo de Erro de Pareamento de DNA , Feminino , Frequência do Gene , Síndrome Hereditária de Câncer de Mama e Ovário/genética , Humanos , Masculino , Instabilidade de Microssatélites , Endonuclease PMS2 de Reparo de Erro de Pareamento/metabolismo , Linhagem
17.
Sci Rep ; 7: 37984, 2017 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-28050010

RESUMO

Next generation sequencing panels have been developed for hereditary cancer, although there is some debate about their cost-effectiveness compared to exome sequencing. The performance of two panels is compared to exome sequencing. Twenty-four patients were selected: ten with identified mutations (control set) and fourteen suspicious of hereditary cancer but with no mutation (discovery set). TruSight Cancer (94 genes) and a custom panel (122 genes) were assessed alongside exome sequencing. Eighty-three genes were targeted by the two panels and exome sequencing. More than 99% of bases had a read depth of over 30x in the panels, whereas exome sequencing covered 94%. Variant calling with standard settings identified the 10 mutations in the control set, with the exception of MSH6 c.255dupC using TruSight Cancer. In the discovery set, 240 unique non-silent coding and canonic splice-site variants were identified in the panel genes, 7 of them putatively pathogenic (in ATM, BARD1, CHEK2, ERCC3, FANCL, FANCM, MSH2). The three approaches identified a similar number of variants in the shared genes. Exomes were more expensive than panels but provided additional data. In terms of cost and depth, panels are a suitable option for genetic diagnostics, although exomes also identify variants in non-targeted genes.


Assuntos
Predisposição Genética para Doença , Testes Genéticos/métodos , Síndromes Neoplásicas Hereditárias/diagnóstico , Benchmarking , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mutação , Síndromes Neoplásicas Hereditárias/genética , Sequenciamento Completo do Exoma/métodos
18.
Sci Rep ; 7: 39348, 2017 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-28051113

RESUMO

We wanted to implement an NGS strategy to globally analyze hereditary cancer with diagnostic quality while retaining the same degree of understanding and control we had in pre-NGS strategies. To do this, we developed the I2HCP panel, a custom bait library covering 122 hereditary cancer genes. We improved bait design, tested different NGS platforms and created a clinically driven custom data analysis pipeline. The I2HCP panel was developed using a training set of hereditary colorectal cancer, hereditary breast and ovarian cancer and neurofibromatosis patients and reached an accuracy, analytical sensitivity and specificity greater than 99%, which was maintained in a validation set. I2HCP changed our diagnostic approach, involving clinicians and a genetic diagnostics team from panel design to reporting. The new strategy improved diagnostic sensitivity, solved uncertain clinical diagnoses and identified mutations in new genes. We assessed the genetic variation in the complete set of hereditary cancer genes, revealing a complex variation landscape that coexists with the disease-causing mutation. We developed, validated and implemented a custom NGS-based strategy for hereditary cancer diagnostics that improved our previous workflows. Additionally, the existence of a rich genetic variation in hereditary cancer genes favors the use of this panel to investigate their role in cancer risk.


Assuntos
Detecção Precoce de Câncer/métodos , Testes Genéticos/métodos , Técnicas de Diagnóstico Molecular/métodos , Síndromes Neoplásicas Hereditárias/diagnóstico , Síndromes Neoplásicas Hereditárias/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Sensibilidade e Especificidade
19.
Gut ; 66(3): 464-472, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-26657901

RESUMO

OBJECTIVE: Estimates of cancer risk and the effects of surveillance in Lynch syndrome have been subject to bias, partly through reliance on retrospective studies. We sought to establish more robust estimates in patients undergoing prospective cancer surveillance. DESIGN: We undertook a multicentre study of patients carrying Lynch syndrome-associated mutations affecting MLH1, MSH2, MSH6 or PMS2. Standardised information on surveillance, cancers and outcomes were collated in an Oracle relational database and analysed by age, sex and mutated gene. RESULTS: 1942 mutation carriers without previous cancer had follow-up including colonoscopic surveillance for 13 782 observation years. 314 patients developed cancer, mostly colorectal (n=151), endometrial (n=72) and ovarian (n=19). Cancers were detected from 25 years onwards in MLH1 and MSH2 mutation carriers, and from about 40 years in MSH6 and PMS2 carriers. Among first cancer detected in each patient the colorectal cancer cumulative incidences at 70 years by gene were 46%, 35%, 20% and 10% for MLH1, MSH2, MSH6 and PMS2 mutation carriers, respectively. The equivalent cumulative incidences for endometrial cancer were 34%, 51%, 49% and 24%; and for ovarian cancer 11%, 15%, 0% and 0%. Ten-year crude survival was 87% after any cancer, 91% if the first cancer was colorectal, 98% if endometrial and 89% if ovarian. CONCLUSIONS: The four Lynch syndrome-associated genes had different penetrance and expression. Colorectal cancer occurred frequently despite colonoscopic surveillance but resulted in few deaths. Using our data, a website has been established at http://LScarisk.org enabling calculation of cumulative cancer risks as an aid to genetic counselling in Lynch syndrome.


Assuntos
Neoplasias Colorretais Hereditárias sem Polipose/epidemiologia , Neoplasias Colorretais Hereditárias sem Polipose/genética , Neoplasias do Endométrio/epidemiologia , Neoplasias Ovarianas/epidemiologia , Vigilância da População , Adolescente , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Criança , Colonoscopia , Neoplasias Colorretais Hereditárias sem Polipose/diagnóstico por imagem , Neoplasias Colorretais Hereditárias sem Polipose/mortalidade , Proteínas de Ligação a DNA/genética , Bases de Dados Factuais , Neoplasias do Endométrio/mortalidade , Feminino , Expressão Gênica , Heterozigoto , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , Endonuclease PMS2 de Reparo de Erro de Pareamento/genética , Proteína 1 Homóloga a MutL/genética , Proteína 2 Homóloga a MutS/genética , Neoplasias Ovarianas/mortalidade , Estudos Prospectivos , Taxa de Sobrevida , Adulto Jovem
20.
Gut ; 66(9): 1657-1664, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-27261338

RESUMO

OBJECTIVE: Today most patients with Lynch syndrome (LS) survive their first cancer. There is limited information on the incidences and outcome of subsequent cancers. The present study addresses three questions: (i) what is the cumulative incidence of a subsequent cancer; (ii) in which organs do subsequent cancers occur; and (iii) what is the survival following these cancers? DESIGN: Information was collated on prospectively organised surveillance and prospectively observed outcomes in patients with LS who had cancer prior to inclusion and analysed by age, gender and genetic variants. RESULTS: 1273 patients with LS from 10 countries were followed up for 7753 observation years. 318 patients (25.7%) developed 341 first subsequent cancers, including colorectal (n=147, 43%), upper GI, pancreas or bile duct (n=37, 11%) and urinary tract (n=32, 10%). The cumulative incidences for any subsequent cancer from age 40 to age 70 years were 73% for pathogenic MLH1 (path_MLH1), 76% for path_MSH2 carriers and 52% for path_MSH6 carriers, and for colorectal cancer (CRC) the cumulative incidences were 46%, 48% and 23%, respectively. Crude survival after any subsequent cancer was 82% (95% CI 76% to 87%) and 10-year crude survival after CRC was 91% (95% CI 83% to 95%). CONCLUSIONS: Relative incidence of subsequent cancer compared with incidence of first cancer was slightly but insignificantly higher than cancer incidence in patients with LS without previous cancer (range 0.94-1.49). The favourable survival after subsequent cancers validated continued follow-up to prevent death from cancer. The interactive website http://lscarisk.org was expanded to calculate the risks by gender, genetic variant and age for subsequent cancer for any patient with LS with previous cancer.


Assuntos
Neoplasias do Colo , Neoplasias Colorretais Hereditárias sem Polipose , Proteínas de Ligação a DNA/genética , Proteína 1 Homóloga a MutL/genética , Proteína 2 Homóloga a MutS/genética , Adulto , Idoso , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Neoplasias Colorretais Hereditárias sem Polipose/epidemiologia , Neoplasias Colorretais Hereditárias sem Polipose/genética , Neoplasias Colorretais Hereditárias sem Polipose/patologia , Reparo de Erro de Pareamento de DNA/genética , Progressão da Doença , Europa (Continente)/epidemiologia , Feminino , Variação Genética , Mutação em Linhagem Germinativa , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Medição de Risco/métodos , Medição de Risco/estatística & dados numéricos , Análise de Sobrevida
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