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1.
PLoS One ; 15(7): e0235613, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32634176

RESUMO

Germline variants inactivating the mismatch repair (MMR) genes MLH1, MSH2, MSH6 and PMS2 cause Lynch syndrome that implies an increased cancer risk, where colon and endometrial cancer are the most frequent. Identification of these pathogenic variants is important to identify endometrial cancer patients with inherited increased risk of new cancers, in order to offer them lifesaving surveillance. However, several other genes are also part of the MMR pathway. It is therefore relevant to search for variants in additional genes that may be associated with cancer risk by including all known genes involved in the MMR pathway. Next-generation sequencing was used to screen 22 genes involved in the MMR pathway in constitutional DNA extracted from full blood from 199 unselected endometrial cancer patients. Bioinformatic pipelines were developed for identification and functional annotation of variants, using several different software tools and custom programs. This facilitated identification of 22 exonic, 4 UTR and 9 intronic variants that could be classified according to pathogenicity. This study has identified several germline variants in genes of the MMR pathway that potentially may be associated with an increased risk for cancer, in particular endometrial cancer, and therefore are relevant for further investigation. We have also developed bioinformatics strategies to analyse targeted sequencing data, including low quality data and genomic regions outside of the protein coding exons of the relevant genes.


Assuntos
Reparo de Erro de Pareamento de DNA , Neoplasias do Endométrio/patologia , Endonuclease PMS2 de Reparo de Erro de Pareamento/genética , Proteína 1 Homóloga a MutL/genética , Proteína 2 Homóloga a MutS/genética , Neoplasias Colorretais Hereditárias sem Polipose/genética , Neoplasias Colorretais Hereditárias sem Polipose/patologia , Variações do Número de Cópias de DNA , DNA de Neoplasias/sangue , DNA de Neoplasias/química , DNA de Neoplasias/metabolismo , Neoplasias do Endométrio/genética , Éxons , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Íntrons , Fatores de Risco , Regiões não Traduzidas/genética
2.
PLoS One ; 15(7): e0235038, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32609729

RESUMO

Lynch syndrome (LS) is an autosomal dominant condition caused by pathogenic variants in mismatch repair (MMR) genes that predispose individuals to different malignancies, such as colorectal cancer (CRC) and endometrial cancer. Current guidelines recommended testing for LS in individuals with newly diagnosed CRC to reduce cancer morbidity and mortality in relatives. Economic evaluations in support of such approach, however, are not available in Italy. We developed a decision-analytic model to analyze the cost-effectiveness of LS screening from the perspective of the Italian National Health System. Three testing strategies: the sequencing of all MMR genes without prior tumor analysis (Strategy 1), a sequential IHC and MS-MLPA analysis (Strategy 2), and an age-targeted strategy with a revised Bethesda criteria assessment before IHC and methylation-specific MLPA for patients ≥ than 70 years old (Strategy 3) were analyzed and compared to the "no testing" strategy. Quality Adjusted Life Years (QALYs) in relatives after colonoscopy, aspirin prophylaxis and an intensive gynecological surveillance were estimated through a Markov model. Assuming a CRC incidence rate of 0.09% and a share of patients affected by LS equal to 2.81%, the number of detected pathogenic variants among CRC cases ranges, in a given year, between 910 and 1167 depending on the testing strategy employed. The testing strategies investigated, provided one-time to the entire eligible population (CRC patients), were associated with an overall cost ranging between €1,753,059.93-€10,388,000.00. The incremental cost-effectiveness ratios of the Markov model ranged from €941.24 /QALY to €1,681.93 /QALY, thus supporting that "universal testing" versus "no testing" is cost-effective, but not necessarily in comparison with age-targeted strategies. This is the first economic evaluation on different testing strategies for LS in Italy. The results might support the introduction of cost-effective recommendations for LS screening in Italy.


Assuntos
Neoplasias Colorretais Hereditárias sem Polipose/diagnóstico , Neoplasias Colorretais/diagnóstico , Testes Genéticos/economia , Neoplasias Colorretais/economia , Neoplasias Colorretais/epidemiologia , Neoplasias Colorretais/genética , Neoplasias Colorretais Hereditárias sem Polipose/economia , Neoplasias Colorretais Hereditárias sem Polipose/epidemiologia , Neoplasias Colorretais Hereditárias sem Polipose/genética , Análise Custo-Benefício , Reparo de Erro de Pareamento de DNA , Testes Genéticos/métodos , Humanos , Itália/epidemiologia , Proteína 1 Homóloga a MutL/genética , Linhagem , Probabilidade , Anos de Vida Ajustados por Qualidade de Vida
3.
Clin Chem ; 66(1): 199-206, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-32609854

RESUMO

BACKGROUND: Exome sequencing has become a commonly used clinical diagnostic test. Multiple studies have examined the diagnostic utility and individual laboratory performance of exome testing; however, no previous study has surveyed and compared the data quality from multiple clinical laboratories. METHODS: We examined sequencing data from 36 clinical exome tests from 3 clinical laboratories. Exome data were compared in terms of overall characteristics and coverage of specific genes and nucleotide positions. The sets of genes examined included genes in Consensus Coding Sequence (CCDS) (n = 17723), a subset of genes clinically relevant to epilepsy (n = 108), and genes that are recommended for reporting of secondary findings (n = 57; excludes X-linked genes). RESULTS: The average exome nucleotide coverage (≥20×) of each laboratory varied at 96.49% (CV = 3%), 96.54% (CV = 1%), and 91.68% (CV = 4%), for laboratories A, B, and C, respectively. For CCDS genes, the average number of completely covered genes varied at 12184 (CV = 29%), 11687 (CV = 13%), and 5989 (CV = 37%), for laboratories A, B, and C, respectively. With smaller subsets of genes related to epilepsy and secondary findings, the CV revealed low consistency, with a maximum CV seen in laboratory C for both epilepsy genes (CV = 60%) and secondary findings genes (CV = 71%). CONCLUSIONS: Poor consistency in complete gene coverage was seen in the clinical exome laboratories surveyed. The degree of consistency varied widely between the laboratories.


Assuntos
Exoma/genética , Proteína BRCA1/genética , Epilepsia/genética , Epilepsia/patologia , Éxons , Guias como Assunto , Humanos , Laboratórios Hospitalares/normas , Proteína 1 Homóloga a MutL/genética , Sequenciamento Completo do Exoma
4.
Artigo em Inglês | MEDLINE | ID: mdl-32682592

RESUMO

OBJECTIVE: The aim of this study was to evaluate the expression of DNA repair genes in cases of oral squamous cell carcinoma (OSCC). STUDY DESIGN: Expression of the MLH1, MSH2, MLH3, ATM, MRE11A, XRCC1, and PMS2 genes was evaluated by reverse transcription-quantitative polymerase chain reaction in the OSCC group (32 patients) and the control group (15 patients). The groups were compared by using the Mann-Whitney test, with Bonferroni correction. Associations between gene expression levels and clinical data were explored by using Pearson's and Spearman's correlation coefficients, with P value less than .05 indicating a significant difference. RESULTS: The MLH1, MSH2, MLH3, ATM, MRE11A, XRCC1, and PMS2 genes were downregulated in the OSCC group compared with the control group, with significant values for MLH1 (P < .0001); MSH2 (P = .038); MLH3 (P < .0001); ATM (P < .0001); MRE11A (P < .0001); XRCC1 (P = .0004); and PMS2 (P = .008). Analysis of the correlation between gene expression and clinical data only revealed a significant negative correlation between age and expression of the PMS2 gene. CONCLUSIONS: Expression of the DNA repair genes MLH1, MSH2, MLH3, ATM, MRE11 AMRE11A, XRCC1, and PMS2 was reduced in OSCC.


Assuntos
Carcinoma de Células Escamosas , Neoplasias Bucais , Carcinoma de Células Escamosas/genética , Reparo do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Humanos , Endonuclease PMS2 de Reparo de Erro de Pareamento , Neoplasias Bucais/genética , Proteína 1 Homóloga a MutL/genética , Proteína 1 Homóloga a MutL/metabolismo , Reação em Cadeia da Polimerase , Transcrição Reversa , Proteína 1 Complementadora Cruzada de Reparo de Raio-X
5.
Jpn J Clin Oncol ; 50(6): 635-642, 2020 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-32372090

RESUMO

BACKGROUND: Regular endoscopic surveillance for Lynch syndrome is reported to reduce colorectal cancer (CRC)-related mortality. However, the appropriate surveillance intervals are still unclear. We evaluated the adequacy of annual colonoscopy and investigated the differences in tumor occurrence rates between individual patients. METHODS: In total, 25 patients with Lynch syndrome who underwent colonoscopic surveillance between 2007 and 2016 at the Iwakuni Clinical Center were included. We retrospectively investigated the surveillance frequency and the clinical features associated with tumor development. RESULTS: Colonoscopic surveillance was performed every 397 days on average. A total of 101 tumors, including 8 intramucosal carcinomas and 15 carcinomas, were observed within the study period. Annual colonoscopy detected six malignancies, including a carcinoma requiring surgery. Tumor incidence was associated with tumor existence in the initial colonoscopies (P = 0.018). Patients with a tumor occurrence rate of 0.4 tumors per year during our observation period were significantly more likely to have malignancies detected during regular surveillance than patients who had a lower occurrence rate (P < 0.001). Malignancy occurrence rate was strongly associated with tumor occurrence rate (P < 0.001, R2 = 0.44). CONCLUSIONS: Annual colonoscopic surveillance for Lynch syndrome patients was effective in reducing the risk of CRC progression, but was insufficient to completely avoid surgery. Because the tumor occurrence rate differed substantially between individuals, more intensive surveillance was required for high-risk patients.


Assuntos
Variação Biológica da População , Neoplasias Colorretais Hereditárias sem Polipose/genética , Neoplasias Colorretais/diagnóstico , Adulto , Idoso , Colonoscopia , Neoplasias Colorretais/epidemiologia , Neoplasias Colorretais/etiologia , Neoplasias Colorretais/genética , Neoplasias Colorretais Hereditárias sem Polipose/complicações , Feminino , Humanos , Incidência , Japão/epidemiologia , Masculino , Pessoa de Meia-Idade , Proteína 1 Homóloga a MutL/genética , Proteína 2 Homóloga a MutS/genética , Mutação , Estudos Retrospectivos
6.
PLoS Genet ; 16(5): e1008798, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32469861

RESUMO

Alterations in epigenetic silencing have been associated with ageing and tumour formation. Although substantial efforts have been made towards understanding the mechanisms of gene silencing, novel regulators in this process remain to be identified. To systematically search for components governing epigenetic silencing, we developed a genome-wide silencing screen for yeast (Saccharomyces cerevisiae) silent mating type locus HMR. Unexpectedly, the screen identified the mismatch repair (MMR) components Pms1, Mlh1, and Msh2 as being required for silencing at this locus. We further found that the identified genes were also required for proper silencing in telomeres. More intriguingly, the MMR mutants caused a redistribution of Sir2 deacetylase, from silent mating type loci and telomeres to rDNA regions. As a consequence, acetylation levels at histone positions H3K14, H3K56, and H4K16 were increased at silent mating type loci and telomeres but were decreased in rDNA regions. Moreover, knockdown of MMR components in human HEK293T cells increased subtelomeric DUX4 gene expression. Our work reveals that MMR components are required for stable inheritance of gene silencing patterns and establishes a link between the MMR machinery and the control of epigenetic silencing.


Assuntos
Proteína 1 Homóloga a MutL/genética , Proteínas MutL/genética , Proteína 2 Homóloga a MutS/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Acetilação , Reparo de Erro de Pareamento de DNA , Epigênese Genética , Inativação Gênica , Genes Fúngicos Tipo Acasalamento , Hereditariedade , Histonas/metabolismo , Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/metabolismo , Sirtuína 2/metabolismo , Telômero/genética
7.
Int J Clin Oncol ; 25(9): 1644-1652, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32430733

RESUMO

BACKGROUND: Risk factors for metachronous colorectal cancer (mCRC) in Lynch Syndrome (LS) patients are essential for colorectal cancer (CRC) treatment strategy to perform not only a curative but also preventive surgery. The aim of this study was to evaluate the risk factors for mCRC development in LS patients to define the patient subset that may benefit an extended curative and preventive surgical resection. METHODS: Patient's clinical history, oncological, molecular and follow-up were collected retrospectively from the Hereditary Digestive Tumors Registry at the National Cancer Institute of Milan. The age-related cumulative risk of mCRC was calculated using the Kaplan-Meier method. Factors significantly associated with mCRC were analyzed with a Cox regression model. Overall and specific competitive risks were also calculated. RESULTS: In a total of 1346 CRC patients, 159 (11.8%) developed a mCRC after a mean follow-up of 138 months from the primary tumor. The independent risk factors reported by a multivariate analysis were: pathogenetic variants in MLH1 and MSH2 (HR 2.96 and 1.91, respectively) and history of colorectal adenomas (HR 1.54); whereas female sex and extended surgery were protective (HR 0.59 and 0.79, respectively). CONCLUSIONS: Among a high-risk population for CRC, in particular LS, an extended surgery may be considered in CRC patients with specific risk factors (MLH1 or MSH2 germline pathogenic variants, history of colorectal adenomas) to reduce the risk of mCRC development.


Assuntos
Neoplasias Colorretais Hereditárias sem Polipose/patologia , Neoplasias Colorretais/patologia , Neoplasias Colorretais/cirurgia , Segunda Neoplasia Primária/patologia , Adulto , Idoso , Neoplasias Colorretais/genética , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais Hereditárias sem Polipose/genética , Feminino , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Proteína 1 Homóloga a MutL/genética , Proteína 2 Homóloga a MutS/genética , Segunda Neoplasia Primária/genética , Segunda Neoplasia Primária/mortalidade , Sistema de Registros , Estudos Retrospectivos , Fatores de Risco
8.
Sci Rep ; 10(1): 6655, 2020 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-32313015

RESUMO

Retinopathy continues to progress even when diabetic patients try to control their blood sugar, but the molecular mechanism of this 'metabolic memory' phenomenon remains elusive. Retinal mitochondria remain damaged and vicious cycle of free radicals continues to self-propagate. DNA methylation suppresses gene expression, and diabetes activates DNA methylation machinery. Our aim was to investigate the role of DNA methylation in continued compromised mitochondrial dynamics and genomic stability in diabetic retinopathy. Using retinal endothelial cells, incubated in 20 mM glucose for four days, followed by 5 mM glucose for four days, and retinal microvessels from streptozotocin-induced diabetic rats in poor glycemia for four months, followed by normal glycemia for four additional months, DNA methylation of mitochondrial fusion and mismatch repair proteins, Mfn2 and Mlh1 respectively, was determined. Retinopathy was detected in trypsin-digested microvasculature. Re-institution of good glycemia had no beneficial effect on hypermethylation of Mfn2 and Mlh1 and retinal function (electroretinogram), and the  retinopathy continued to progress. However, intervention of good glycemia directly with DNA methylation inhibitors (Azacytidine or Dnmt1-siRNA), prevented Mfn2 and Mlh1 hypermethylation, and ameliorated retinal dysfunction and diabetic retinopathy. Thus, direct regulation of DNA methylation can prevent/reverse diabetic retinopathy by maintaining mitochondrial dynamics and DNA stability, and prevent retinal functional damage.


Assuntos
Azacitidina/farmacologia , DNA Mitocondrial/genética , Diabetes Mellitus Experimental/terapia , Retinopatia Diabética/terapia , Epigênese Genética , Hiperglicemia/terapia , Mitocôndrias/genética , Animais , Linhagem Celular , DNA (Citosina-5-)-Metiltransferase 1/antagonistas & inibidores , DNA (Citosina-5-)-Metiltransferase 1/genética , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Metilação de DNA , DNA Mitocondrial/metabolismo , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patologia , Retinopatia Diabética/induzido quimicamente , Retinopatia Diabética/genética , Retinopatia Diabética/patologia , Progressão da Doença , Eletrorretinografia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/patologia , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Glucose/efeitos adversos , Humanos , Hiperglicemia/induzido quimicamente , Hiperglicemia/genética , Hiperglicemia/patologia , Masculino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Proteína 1 Homóloga a MutL/genética , Proteína 1 Homóloga a MutL/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Wistar , Retina/efeitos dos fármacos , Retina/metabolismo , Retina/patologia , Transdução de Sinais , Estreptozocina/administração & dosagem
9.
Breast Cancer Res Treat ; 180(2): 511-514, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32040686

RESUMO

BACKGROUND: BRCA germline pathogenic variants represent the most common inherited mechanism predisposing individuals to breast cancer, while germline pathogenic variants in one of the mismatch repair (MMR) genes represent the most common colon cancer-predisposing inherited syndrome, known as the Lynch syndrome (LS). Individuals who harbor pathogenic germline variants for both syndromes are extremely rare. Germline testing is now done routinely for patients with breast cancer and MMR testing is recommended for nearly all patients diagnosed with colon or rectal cancer (Benson et al in NCCN clinical practice guidelines in oncology (NCCN guidelines) colon cancer (Version 4.2019-November 8, 2019). www.NCCN.org, Gradishar et al in NCCN clinical practice guidelines in oncology (NCCN guidelines) breast cancer (Version 3.2019-September 6, 2019).www.NCCN.org). We report a patient with germline mutations in both BRCA2 and the MMR gene MLH1 who developed breast cancer. The breast cancer showed loss of heterozygosity (LOH) in BRCA2 (the molecular hallmark of cancers related to inheritance of a BRCA alteration) and was also deficient in mismatch repair gene protein expression (dMMR), the hallmark of LS-related cancers. We discuss the possible mechanisms of transformation that would explain the finding that the tumor showed both BRCA2 LOH and was dMMR, each of which would generally be considered a gatekeeper event for transformation of normal cells to malignancy. RESULTS: This report describes a patient with molecularly diagnosed breast and ovarian cancer syndrome (BRCA2) and LS. Next generation sequencing (NGS) and immunohistochemical (IHC) testing demonstrated her breast cancer to show BRCA2 LOH and to be dMMR. CONCLUSION: The patient presented represents the first reported case where both next generation sequencing (NGS) for BRCA LOH and MMR IHC testing of her breast cancer were performed and underscores the importance of using NGS including the reported mutational allelic frequency (MAF) and IHC use to predict the likely responsiveness to the recently approved PARP inhibitors and checkpoint inhibitor therapies (Robson et al in N Engl J Med 377:523-533, 2017, Lemery et al in 377(15):1409-1412, https://doi.org/10.1056/NEJMp1709968, 2017), key because the gatekeeper transforming event for tumors related to inherited cancer syndromes is loss of normal tumor suppressor gene (TSG) protein expression.


Assuntos
Proteína BRCA2/genética , Neoplasias da Mama/patologia , Neoplasias Colorretais Hereditárias sem Polipose/patologia , Mutação em Linhagem Germinativa , Perda de Heterozigosidade , Proteína 1 Homóloga a MutL/genética , Neoplasias Ovarianas/patologia , Neoplasias da Mama/genética , Neoplasias Colorretais Hereditárias sem Polipose/genética , Feminino , Predisposição Genética para Doença , Humanos , Imunoterapia/métodos , Pessoa de Meia-Idade , Terapia de Alvo Molecular/métodos , Neoplasias Ovarianas/genética , Prognóstico
10.
Proc Natl Acad Sci U S A ; 117(7): 3535-3542, 2020 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-32015124

RESUMO

MutL proteins are ubiquitous and play important roles in DNA metabolism. MutLγ (MLH1-MLH3 heterodimer) is a poorly understood member of the eukaryotic family of MutL proteins that has been implicated in triplet repeat expansion, but its action in this deleterious process has remained unknown. In humans, triplet repeat expansion is the molecular basis for ∼40 neurological disorders. In addition to MutLγ, triplet repeat expansion involves the mismatch recognition factor MutSß (MSH2-MSH3 heterodimer). We show here that human MutLγ is an endonuclease that nicks DNA. Strikingly, incision of covalently closed, relaxed loop-containing DNA by human MutLγ is promoted by MutSß and targeted to the strand opposite the loop. The resulting strand break licenses downstream events that lead to a DNA expansion event in human cell extracts. Our data imply that the mammalian MutLγ is a unique endonuclease that can initiate triplet repeat DNA expansions.


Assuntos
Proteína 1 Homóloga a MutL/metabolismo , Proteínas MutL/metabolismo , Reparo de Erro de Pareamento de DNA , Dimerização , Endonucleases/química , Endonucleases/genética , Endonucleases/metabolismo , Humanos , Proteína 1 Homóloga a MutL/química , Proteína 1 Homóloga a MutL/genética , Proteínas MutL/química , Proteínas MutL/genética , Expansão das Repetições de Trinucleotídeos
11.
Dis Markers ; 2020: 8360841, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32076465

RESUMO

Lynch syndrome (LS) is the most common hereditary colorectal cancer (CRCs) inherited in an autosomal-dominant manner. Here, we reported a multigeneration Chinese family clinically diagnosed with LS according to the Amsterdam II criteria. To identify the underlying causative gene for LS in this family, whole-exome sequencing (WES) was performed. A germline missense variant (c.2054C>T:p.S685F) in exon 18 of MLH1 was successfully identified by WES. Sanger sequencing verified the results of WES and also confirmed the cosegregation of the MLH1 missense variant in all affected members of the family including two unaffected family members. Bioinformatic tools predicted the identified MLH1 variant as deleterious. Immunohistochemistry (IHC) staining showed loss of MLH1 and PMS2 protein expression. In vitro expression analysis also revealed that the identified MLH1 missense variant (c.2054C>T:p.S685F) results in reduced expression of both MLH1 and PMS2 proteins. Based on the American College of Medical Genetics and Genomics (ACMG) guidelines, the missense mutation c.2054C>T in MLH1 was classified as a "pathogenic" variant. Two unaffected family members were later recommended for colonoscopy and other important cancer diagnostic inspections every 1-2 years as both were at higher risk of LS. In conclusion, our findings widen the genotypic spectrum of MLH1 mutations responsible for LS. This study increases the phenotypic spectrum of LS which will certainly help the clinicians in diagnosing LS in multigeneration families. This study also puts emphasis on the importance of genetic counselling for the benefit of asymptomatic carriers of MMR gene variants who are at higher risk of LS.


Assuntos
Neoplasias Colorretais Hereditárias sem Polipose/genética , Proteína 1 Homóloga a MutL/genética , Proteína 1 Homóloga a MutL/metabolismo , Mutação de Sentido Incorreto , Sequenciamento Completo do Exoma/métodos , Adulto , China , Neoplasias Colorretais Hereditárias sem Polipose/metabolismo , Regulação para Baixo , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Endonuclease PMS2 de Reparo de Erro de Pareamento/metabolismo , Linhagem
12.
Jpn J Clin Oncol ; 50(3): 270-275, 2020 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-31958127

RESUMO

OBJECTIVE: The aim of this study was to investigate a magnetic resonance imaging-based definition of lower uterine segment carcinoma. METHODS: We retrospectively reviewed 587 consecutive patients with endometrial cancer who underwent hysterectomy. Lower uterine segment carcinoma was determined through pathological examination and magnetic resonance imaging assessment. For imaging assessment, the location of the inner lining of the uterus was classified into four equal parts on a sagittal section image. A tumor was defined as lower uterine segment carcinoma when its thickest part was located in the second or the third part from the uterine fundus. Lower uterine segment carcinoma was further divided into lower uterine segment in a narrow sense, upon which diagnosis was exclusively based on pathological findings, and lower uterine segment in a broad sense that were the remaining lower uterine segment carcinomas except lower uterine segment carcinomas in a narrow sense. The relationship between lower uterine segment carcinoma and probable Lynch syndrome was investigated. Patients with loss of MSH2, MSH6, and PMS2 expression or those with tumors with loss of MLH1 and absence of MLH1 promoter methylation were diagnosed as probable Lynch syndrome. RESULTS: Lower uterine segment carcinoma was identified in 59 (10.2%) patients. Twenty-eight (47.5%) patients were categorized as lower uterine segment in a narrow sense and 31 (52.5%) as lower uterine segment in a broad sense. Among them, probable Lynch syndrome was identified in 12 (20.3%) cases. There was no difference in clinical profiles, including the prevalence of probable Lynch syndrome between the two categories. CONCLUSIONS: A magnetic resonance imaging-based expanded definition of lower uterine segment carcinoma is likely to secure characteristics equivalent to a conventional pathology-based definition of lower uterine segment carcinoma. The novel definition of lower uterine segment carcinoma might improve the detection of probable Lynch syndrome.


Assuntos
Neoplasias Colorretais Hereditárias sem Polipose/diagnóstico por imagem , Neoplasias Uterinas/diagnóstico por imagem , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias Colorretais Hereditárias sem Polipose/patologia , Proteínas de Ligação a DNA/genética , Feminino , Humanos , Imagem por Ressonância Magnética , 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 , Regiões Promotoras Genéticas , Neoplasias Uterinas/patologia
13.
Gastroenterology ; 158(5): 1326-1333, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31926173

RESUMO

BACKGROUND & AIMS: Lynch syndrome is caused by variants in DNA mismatch repair (MMR) genes and associated with an increased risk of colorectal cancer (CRC). In patients with Lynch syndrome, CRCs can develop via different pathways. We studied associations between Lynch syndrome-associated variants in MMR genes and risks of adenoma and CRC and somatic mutations in APC and CTNNB1 in tumors in an international cohort of patients. METHODS: We combined clinical and molecular data from 3 studies. We obtained clinical data from 2747 patients with Lynch syndrome associated with variants in MLH1, MSH2, or MSH6 from Germany, the Netherlands, and Finland who received at least 2 surveillance colonoscopies and were followed for a median time of 7.8 years for development of adenomas or CRC. We performed DNA sequence analyses of 48 colorectal tumors (from 16 patients with mutations in MLH1, 29 patients with mutations in MSH2, and 3 with mutations in MSH6) for somatic mutations in APC and CTNNB1. RESULTS: Risk of advanced adenoma in 10 years was 17.8% in patients with pathogenic variants in MSH2 vs 7.7% in MLH1 (P < .001). Higher proportions of patients with pathogenic variants in MLH1 or MSH2 developed CRC in 10 years (11.3% and 11.4%) than patients with pathogenic variants in MSH6 (4.7%) (P = .001 and P = .003 for MLH1 and MSH2 vs MSH6, respectively). Somatic mutations in APC were found in 75% of tumors from patients with pathogenic variants in MSH2 vs 11% in MLH1 (P = .015). Somatic mutations in CTNNB1 were found in 50% of tumors from patients with pathogenic variants in MLH1 vs 7% in MSH2 (P = .002). None of the 3 tumors with pathogenic variants in MSH6 had a mutation in CTNNB1, but all had mutations in APC. CONCLUSIONS: In an analysis of clinical and DNA sequence data from patients with Lynch syndrome from 3 countries, we associated pathogenic variants in MMR genes with risk of adenoma and CRC, and somatic mutations in APC and CTNNB1 in colorectal tumors. If these findings are confirmed, surveillance guidelines might be adjusted based on MMR gene variants.


Assuntos
Adenoma/epidemiologia , Neoplasias Colorretais Hereditárias sem Polipose/epidemiologia , Proteínas de Ligação a DNA/genética , Proteína 1 Homóloga a MutL/genética , Proteína 2 Homóloga a MutS/genética , Adenoma/diagnóstico , Adenoma/genética , Proteína da Polipose Adenomatosa do Colo/genética , Adulto , Colonoscopia , Neoplasias Colorretais Hereditárias sem Polipose/diagnóstico , Neoplasias Colorretais Hereditárias sem Polipose/genética , Reparo de Erro de Pareamento de DNA , Análise Mutacional de DNA , Feminino , Finlândia/epidemiologia , Alemanha/epidemiologia , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Países Baixos/epidemiologia , Estudos Prospectivos , beta Catenina/genética
14.
Gynecol Oncol ; 156(3): 669-675, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31924330

RESUMO

OBJECTIVE: Mismatch repair (MMR) deficiency and Bethesda panel microsatellite instability (MSI) are increasingly analyzed to identify tumors that might benefit from immune checkpoint inhibitors, but tumor heterogeneity is a potential obstacle for such analyses. In ovarian cancer, data on intratumoral heterogeneity of MMR deficiency/MSI are lacking. METHODS: N = 582 ovarian cancers were screened for MMR deficiency by immunohistochemistry (IHC) on a tissue microarray. 10 cases suspect for MMR deficiency were identified among 478 interpretable cancers and repeated IHC on large sections combined with polymerase chain reaction (PCR)-based MSI analysis validated MMR deficiency/MSI in 9 of these tumors. RESULTS: MMR deficiency/MSI was predominantly seen in endmetrioid cancers (8 of 35, 23%) and also in 1 of 358 serous carcinomas (0.3%), but was absent in 34 mucinous carcinomas, 23 clear cell carcinomas, 17 malignant mixed Mullerian tumors (carcinosarcomas), and 11 mixed carcinomas. MMR deficiency involed protein loss of PMS2/MLH1 in 6 cases and of MSH2 and/or MSH6 in 3 cases. 7 MMR deficient cancers were MSI-high (all endometrioid), one was MSI-low (endometrioid) and one cancer with unequivocal MMR protein loss exhibited microsatellite stability (serous). MLH1 promotor methylation was observed in 4 of 5 endometrioid cancers with MLH1 protein loss. Immunostaining of all available cancer-containing tissue blocks (n = 114) of tumors with confirmed MMR deficiency/MSI revealed uniform MMR status throughout the entire tumor mass. CONCLUSIONS: Our data show that MSI is present in a substantial proportion of endometrioid ovarian cancers but can also occur in other tumor subtypes. MMR deficiency/MSI typically involves the entire tumor mass, suggesting that MMR inactivation occurs early in tumorigenesis in a subset of ovarian cancers.


Assuntos
Reparo de Erro de Pareamento de DNA , Enzimas Reparadoras do DNA/deficiência , Neoplasias Ovarianas/enzimologia , Neoplasias Ovarianas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Endometrioide/genética , Enzimas Reparadoras do DNA/genética , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Feminino , Humanos , Imuno-Histoquímica , Instabilidade de Microssatélites , Pessoa de Meia-Idade , Endonuclease PMS2 de Reparo de Erro de Pareamento/deficiência , Endonuclease PMS2 de Reparo de Erro de Pareamento/genética , Proteína 1 Homóloga a MutL/deficiência , Proteína 1 Homóloga a MutL/genética , Proteína 2 Homóloga a MutS/deficiência , Proteína 2 Homóloga a MutS/genética , Análise Serial de Tecidos , Adulto Jovem
15.
Gynecol Oncol ; 157(1): 245-251, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31980219

RESUMO

OBJECTIVES: To apply the Proactive Molecular Risk Classifier for Endometrial Cancer (ProMisE) to a consecutive series of endometrial cancer (EC) patients diagnosed at a tertiary referral center and assign EC specimens to one of four molecular subgroups using immunohistochemistry (IHC) for p53/mismatch repair protein expression and sequencing for Polymerase Epsilon Exonuclease Domain Mutations (POLE-EDM). Mismatch Repair Deficient (MMR-D) cases were more thoroughly investigated to identify underlying somatic or germline genetic defects. METHODS: Hundred-and eight consecutive endometrial cancer patients, diagnosed between March 2017 and April 2019, were subjected to immunohistochemical and molecular analysis, according to ProMisE. IHC for p53 and the mismatch repair proteins (MLH1, PMS2, MSH6 and PMS2) was performed. All patients were also tested for POLE-EDM by Sanger sequencing. In addition, tumor and corresponding normal tissue of cases with abnormal MMR IHC were tested by PCR for microsatellite instability (MSI) (MSI analysis system, Promega). Hypermethylation of MLH1 promotor was tested with (methylation specific) multiplex ligation dependent probe amplification. MMR-D cases were subjected to germline mutation analysis of the mismatch repair genes, using next generation sequencing on MiSeq (Illumina) with the BRCA Hereditary Cancer MASTR Plus, (Multiplicom/Agilent), RNA mutation analysis and MLPA. RESULTS: FIGO classification was stage IA (n = 54), IB (n = 22) II(n = 8), III(n = 18) and IV(n = 6). Of the 33 patients with MMR-D on IHC (31%), 26 showed MLH1 promotor hypermethylation as the probable cause of MMR-D. The remaining 7 patients without MLH1 promotor hypermethylation were referred for germline analysis of Lynch syndrome. Six patients carried a pathogenic germline mutation in one of the mismatch repair genes: MSH6(n = 3), PMS2(n = 1), MLH1(n = 1) and MSH2 (n = 1). Pathogenic POLE-EDM were identified in 7 (6%) patients. Multiple molecular features (POLE-EDM + MMR-D or POLE-EDM + p53 abnormal) were observed in 4 patients (4%). A high concordance between MMR-D and microsatellite instability was observed in our cohort. In cases of a genetic defect in the MMR genes, we do note a large proportion of cases exhibiting microsatellite instability. On the contrary a hypermutation state, as seen in POLE EDM, does not result in accompanied phenotypic changes in MSI status. CONCLUSION: The ProMisE classification proved to be an efficient and easily implementable system. Future research should elucidate the precise biological and prognostic meaning of the cases with multiple molecular markers.


Assuntos
Reparo de Erro de Pareamento de DNA , Enzimas Reparadoras do DNA/genética , Neoplasias do Endométrio/classificação , Idoso , Idoso de 80 Anos ou mais , DNA Polimerase II/genética , DNA Polimerase II/metabolismo , Enzimas Reparadoras do DNA/deficiência , Enzimas Reparadoras do DNA/metabolismo , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/metabolismo , Neoplasias do Endométrio/patologia , Feminino , Humanos , Imuno-Histoquímica , Instabilidade de Microssatélites , Pessoa de Meia-Idade , Proteína 1 Homóloga a MutL/deficiência , Proteína 1 Homóloga a MutL/genética , Proteína 1 Homóloga a MutL/metabolismo , Estadiamento de Neoplasias , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteína Supressora de Tumor p53/genética
16.
Sci Rep ; 10(1): 168, 2020 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-31932604

RESUMO

Chromatin organization influences most aspects of gene expression regulation. The linker histone H1, along with the core histones, is a key component of eukaryotic chromatin. Despite its critical roles in chromatin structure and function and gene regulation, studies regarding the H1 protein-protein interaction networks, particularly outside of Opisthokonts, are limited. The nuclear dimorphic ciliate protozoan Tetrahymena thermophila encodes two distinct nucleus-specific linker histones, macronuclear Hho1 and micronuclear Mlh1. We used a comparative proteomics approach to identify the Hho1 and Mlh1 protein-protein interaction networks in Tetrahymena during growth, starvation, and sexual development. Affinity purification followed by mass spectrometry analysis of the Hho1 and Mlh1 proteins revealed a non-overlapping set of co-purifying proteins suggesting that Tetrahymena nucleus-specific linker histones are subject to distinct regulatory pathways. Furthermore, we found that linker histones interact with distinct proteins under the different stages of the Tetrahymena life cycle. Hho1 and Mlh1 co-purified with several Tetrahymena-specific as well as conserved interacting partners involved in chromatin structure and function and other important cellular pathways. Our results suggest that nucleus-specific linker histones might be subject to nucleus-specific regulatory pathways and are dynamically regulated under different stages of the Tetrahymena life cycle.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Grupo de Alta Mobilidade/metabolismo , Histonas/metabolismo , Proteína 1 Homóloga a MutL/metabolismo , Proteoma/análise , Proteínas de Protozoários/metabolismo , Tetrahymena thermophila/crescimento & desenvolvimento , Sequência de Aminoácidos , Animais , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromatina/genética , Cromatina/metabolismo , Regulação da Expressão Gênica , Proteínas de Grupo de Alta Mobilidade/genética , Proteína 1 Homóloga a MutL/genética , Domínios e Motivos de Interação entre Proteínas , Proteínas de Protozoários/genética , Inanição , Tetrahymena thermophila/genética , Tetrahymena thermophila/metabolismo
17.
Nat Commun ; 11(1): 139, 2020 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-31949146

RESUMO

Mismatch repair deficient (dMMR) gastro-oesophageal adenocarcinomas (GOAs) show better outcomes than their MMR-proficient counterparts and high immunotherapy sensitivity. The hypermutator-phenotype of dMMR tumours theoretically enables high evolvability but their evolution has not been investigated. Here we apply multi-region exome sequencing (MSeq) to four treatment-naive dMMR GOAs. This reveals extreme intratumour heterogeneity (ITH), exceeding ITH in other cancer types >20-fold, but also long phylogenetic trunks which may explain the exquisite immunotherapy sensitivity of dMMR tumours. Subclonal driver mutations are common and parallel evolution occurs in RAS, PIK3CA, SWI/SNF-complex genes and in immune evasion regulators. MSeq data and evolution analysis of single region-data from 64 MSI GOAs show that chromosome 8 gains are early genetic events and that the hypermutator-phenotype remains active during progression. MSeq may be necessary for biomarker development in these heterogeneous cancers. Comparison with other MSeq-analysed tumour types reveals mutation rates and their timing to determine phylogenetic tree morphologies.


Assuntos
Reparo de Erro de Pareamento de DNA , Neoplasias Esofágicas/genética , Heterogeneidade Genética , Neoplasias Gástricas/genética , Adenocarcinoma/genética , Proteínas de Ligação a DNA/genética , Exoma , Genes Neoplásicos/genética , Humanos , Evasão da Resposta Imune , Imunoterapia , Endonuclease PMS2 de Reparo de Erro de Pareamento/genética , Proteína 1 Homóloga a MutL/genética , Proteína 2 Homóloga a MutS/genética , Mutação , Fenótipo , Filogenia
18.
J Med Genet ; 57(1): 62-69, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31391288

RESUMO

BACKGROUND: Pathogenic variants in mismatch repair (MMR) genes (MLH1, MSH2, MSH6 and PMS2) increase risk for Lynch syndrome and related cancers. We quantified tumour characteristics to assess variant pathogenicity for germline MMR genes. METHODS: Among 4740 patients with cancer with microsatellite instability (MSI) and immunohistochemical (IHC) results, we tested MMR pathogenic variant association with MSI/IHC status, and estimated likelihood ratios which we used to compute a tumour characteristic likelihood ratio (TCLR) for each variant. Predictive performance of TCLR in combination with in silico predictors, and a multifactorial variant prediction (MVP) model that included allele frequency, co-occurrence, co-segregation, and clinical and family history information was assessed. RESULTS: Compared with non-carriers, carriers of germline pathogenic/likely pathogenic (P/LP) variants were more likely to have abnormal MSI/IHC status (p<0.0001). Among 150 classified missense variants, 73.3% were accurately predicted with TCLR alone. Models leveraging in silico scores as prior probabilities accurately classified >76.7% variants. Adding TCLR as quantitative evidence in an MVP model (MVP +TCLR Pred) increased the proportion of accurately classified variants from 88.0% (MVP alone) to 98.0% and generated optimal performance statistics among all models tested. Importantly, MVP +TCLR Pred resulted in the high yield of predicted classifications for missense variants of unknown significance (VUS); among 193 VUS, 62.7% were predicted as P/PL or benign/likely benign (B/LB) when assessed according to American College of Medical Genetics and Genomics/Association for Molecular Pathology guidelines. CONCLUSION: Our study demonstrates that when used separately or in conjunction with other evidence, tumour characteristics provide evidence for germline MMR missense variant assessment, which may have important implications for genetic testing and clinical management.


Assuntos
Reparo de Erro de Pareamento de DNA , Mutação de Sentido Incorreto , Neoplasias/genética , Neoplasias Colorretais Hereditárias sem Polipose , Simulação por Computador , Proteínas de Ligação a DNA/genética , Feminino , Predisposição Genética para Doença , Mutação em Linhagem Germinativa , Humanos , Masculino , Instabilidade de Microssatélites , 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/metabolismo
19.
Eur J Med Genet ; 63(3): 103753, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31491536

RESUMO

Hereditary non-polyposis colorectal cancer (HNPCC), also known as Lynch syndrome (LS), is a common cancer-predisposing syndrome. This study aimed to investigate the spectrum of germ-line mutations in Russian LS patients. LS-related mismatch repair (MMR) genes were analyzed in 16 patients, who were forwarded to genetic testing due to strong clinical features of LS and had high-level microsatellite instability (MSI-H) in the tumor (n = 14) or unknown MSI status (n = 2). In addition, 672 consecutive colorectal cancer (CRC) cases were screened for family history; 15 patients were younger than 50 years and reported 2 or more instances of LS-related cancers in 1st- or 2nd-degree relatives. Seven of these cases demonstrated MSI-H and therefore were subjected to DNA germ-line testing. Overall, 17/23 (74%) subjects carried LS-associated gene variants (MLH1: 10; MSH2: 4; MSH6: 2; PMS2: 1), with 2 alleles (MLH1 c.677G > T and MSH2 с.1906G > C) detected twice. Testing for recurrent mutations of 30 consecutive MSI-H CRCs led to the identification of 2 additional subjects with LS. The analysis of all relevant publications identified 28 unrelated LS patients presented in Russian medical literature and 3 unrelated Russian LS subjects described in international journals. Overall, 15/49 (31%) genetic defects revealed in Russian LS patients were represented by six recurrent alleles (MLH1: c.350C > T, c.677G > T, c.1852_1854del; MSH2: c.942+3A > T, c.1861C > T, с.1906G > C). We conclude that the founder effect for LS in Russia is seemingly less pronounced than the one for hereditary breast-ovarian cancer syndrome, however testing for recurrent LS mutations may be considered feasible in some circumstances.


Assuntos
Neoplasias Colorretais Hereditárias sem Polipose/genética , Predisposição Genética para Doença , Mutação em Linhagem Germinativa , Adulto , Alelos , Reparo de Erro de Pareamento de DNA/genética , Proteínas de Ligação a DNA/genética , Feminino , Efeito Fundador , Testes Genéticos , Humanos , Masculino , Instabilidade de Microssatélites , 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 , Mutação , Federação Russa , Análise de Sequência de DNA
20.
Mol Carcinog ; 59(1): 24-31, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31579968

RESUMO

Hexavalent chromium is recognized as a human carcinogen. Our previous studies revealed that lung cancer (LC) in chromate-exposed workers (chromate LC) had molecular features of frequent microsatellite instability (MSI), repression of MLH1 level, and aberrant DNA methylation of several tumor-suppressor genes, including MLH1. In the present study, we quantitatively investigated MLH1-promoter methylation status using bisulfite pyrosequencing of paired tumorous/nontumorous tissues from chromate and nonchromate LCs to determine the effect of chromate exposure on MLH1-promoter methylation. The methylation level of MLH1 promoter was significantly higher in chromate LC tumors (P < .001) than nonchromate LC tumors and, among chromate LC, significantly higher in tumorous tissue than nontumorous tissue (P = .004). Moreover, the methylation level of MLH1 promoter in normal lung tissue tended to be higher in chromate LC than nonchromate LC (P = .062). In addition, LC with reduced levels of MLH1 showed significantly higher methylation levels of MLH1 promoter than LC exhibiting normal MLH1 levels (P = .019). Moreover, immunohistochemical analyses determined that levels of SUV39H1, an H3K9me2-related methyltransferase, were higher in chromate LC than nonchromate LC (P = .076). Furthermore, we evaluated three DNA double-strand break-repair genes (MRE11, RAD50, and DNA-PKcs) as possible targets of MSI by fragment-length polymorphism analysis, revealing the mutation frequency of RAD50 as significantly higher in chromate LC than nonchromate LC (P = .047). These results suggest that chromate exposure might induce MLH1 hypermethylation in LC as a mechanism of chromate-induced carcinogenesis.


Assuntos
Cromatos/efeitos adversos , Metilação de DNA/efeitos dos fármacos , Neoplasias Pulmonares/induzido quimicamente , Neoplasias Pulmonares/genética , Proteína 1 Homóloga a MutL/genética , Idoso , Carcinogênese/induzido quimicamente , Carcinogênese/genética , Reparo de Erro de Pareamento de DNA/efeitos dos fármacos , Humanos , Instabilidade de Microssatélites/efeitos dos fármacos , Pessoa de Meia-Idade , Regiões Promotoras Genéticas/efeitos dos fármacos
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