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


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.

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
BMC Med Genomics ; 8: 2, 2015 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-25739810


BACKGROUND: A clinical overlap exists between mosaic Neurofibromatosis Type 2 and sporadic Schwannomatosis conditions. In these cases a molecular analysis of tumors is recommended for a proper genetic diagnostics. This analysis is challenged by the fact that schwannomas in both conditions bear a somatic double inactivation of the NF2 gene. However, SMARCB1-associated schwannomas follow a four-hit, three-step model, in which both alleles of SMARCB1 and NF2 genes are inactivated in the tumor, with one of the steps being always the loss of a big part of chromosome 22 involving both loci. CASE PRESENTATION: Here we report a 36-year-old woman who only presented multiple subcutaneous schwannomas on her right leg. To help discriminate between both possible diagnoses, an exhaustive molecular genetic and genomic analysis was performed on two schwannomas of the patient, consisting in cDNA and DNA sequencing, MLPA, microsatellite multiplex PCR and SNP-array analyses. The loss of a big part of chromosome 22 (22q12.1q13.33) was identified in both tumors. However, this loss involved the NF2 but not the SMARCB1 locus. SNP-array analysis revealed the presence of the same deletion breakpoint in both schwannomas, indicating that this alteration was actually the first NF2 inactivating hit. In addition, a distinct NF2 point mutation in each tumor was identified, representing independent second hits. In accordance with these results, no deletions or point mutations in the SMARCB1 gene were identified. None of the mutations were present in the blood. Two of the patient's children inherited chromosome 22 deleted in schwannomas of the mother, but in its wild type form. CONCLUSIONS: These results conclusively confirm the segmental mosaic NF2 nature of the clinical phenotype presented.

Perna (Membro) , Técnicas de Diagnóstico Molecular , Neurilemoma/genética , Neurofibromatose 2/diagnóstico , Neurofibromatose 2/genética , Adulto , Sequência de Bases , Proteínas Cromossômicas não Histona/genética , Proteínas de Ligação a DNA/genética , Diagnóstico Diferencial , Feminino , Genes da Neurofibromatose 2 , Humanos , Repetições de Microssatélites/genética , Neurilemoma/diagnóstico , Polimorfismo de Nucleotídeo Único , Proteína SMARCB1 , Fatores de Transcrição/genética
Eur J Cancer ; 50(13): 2241-50, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24953332


BACKGROUND AND AIMS: Individuals with tumours showing mismatch repair (MMR) deficiency not linked to germline mutations or somatic methylation of MMR genes have been recently referred as having 'Lynch-like syndrome' (LLS). The genetic basis of these LLS cases is unknown. MUTYH-associated polyposis patients show some phenotypic similarities to Lynch syndrome patients. The aim of this study was to investigate the prevalence of germline MUTYH mutations in a large series of LLS patients. METHODS: Two hundred and twenty-five probands fulfilling LLS criteria were included in this study. Screening of MUTYH recurrent mutations, whole coding sequencing and a large rearrangement analysis were undertaken. Age, sex, clinical, pathological and molecular characteristics of tumours including KRAS mutations were assessed. RESULTS: We found a prevalence of 3.1% of MAP syndrome in the whole series of LLS (7/225) and 3.9% when only cases fulfilling clinical criteria were considered (7/178). Patients with MUTYH biallelic mutations had more adenomas than monoallelic (P=0.02) and wildtype patients (P<0.0001). Six out of nine analysed tumours from six biallelic MUTYH carriers harboured KRAS-p.G12C mutation. This mutation was found to be associated with biallelic MUTYH germline mutation when compared with reported series of unselected colorectal cancer cohorts (P<0.0001). CONCLUSIONS: A proportion of unexplained LLS cases is caused by biallelic MUTYH mutations. The obtained results further justify the inclusion of MUTYH in the diagnostic strategy for Lynch syndrome-suspected patients.

Neoplasias Colorretais Hereditárias sem Polipose/genética , DNA Glicosilases/genética , Mutação em Linhagem Germinativa , Feminino , Predisposição Genética para Doença , Humanos , Masculino , Pessoa de Meia-Idade , Prevalência , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas p21(ras) , Proteínas ras/genética
Eur J Hum Genet ; 21(7): 769-73, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23188051


Neurofibromatosis type 2 (NF2) is an autosomal-dominant disorder affecting about 1:33 000 newborns, mainly characterized by the development of tumors of the nervous system and ocular abnormalities. Around 85% of germline NF2 mutations are point mutations. Among them, ∼25% affect splicing and are associated with a variable disease severity. In the context of our NF2 Multidisciplinary Clinics, we have identified a patient fulfilling clinical criteria for the disease and exhibiting a severe phenotype. The patient carries a deep intronic mutation (g. 74409T>A, NG_009057.1) that produces the insertion of a cryptic exon of 167pb in the mature mRNA between exons 13 and 14, resulting in a truncated merlin protein (p.Pro482Profs*39). A mutation-specific antisense phosphorodiamidate morpholino oligomer was designed and used in vitro to effectively restore normal NF2 splicing in patient-derived primary fibroblasts. In addition, merlin protein levels were greatly recovered after morpholino treatment, decreasing patient's fibroblasts in vitro proliferation capacity and restoring cytoeskeleton organization. To our knowledge, this is the first NF2 case caused by a deep intronic mutation in which an in vitro antisense therapeutic approximation has been tested. These results open the possibility of using this approach in vivo for this type of mutation causing NF2.

Morfolinos/administração & dosagem , Neurofibromatose 2/genética , Neurofibromatose 2/terapia , Neurofibromina 2/genética , Adolescente , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Mutação em Linhagem Germinativa/genética , Humanos , Íntrons/genética , Morfolinos/genética , Morfolinos/uso terapêutico , Neurofibromatose 2/patologia , RNA Antissenso/genética