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BACKGROUND & AIMS: Small intestinal neuroendocrine tumor (SI-NET) is a rare disease, but its incidence has increased over the past 4 decades. Understanding the genetic risk factors underlying SI-NETs can help in disease prevention and may provide clinically beneficial markers for diagnosis. Here the results of the largest genome-wide association study of SI-NETs performed to date with 405 cases and 614,666 controls are reported. METHODS: Samples from 307 patients with SI-NETs and 287,137 controls in the FinnGen study were used for the identification of SI-NET risk-associated genetic variants. The results were also meta-analyzed with summary statistics from the UK Biobank (n = 98 patients with SI-NET and n = 327,529 controls). RESULTS: We identified 6 genome-wide significant (P < 5 × 10-8) loci associated with SI-NET risk, of which 4 (near SEMA6A, LGR5, CDKAL1, and FERMT2) are novel and 2 (near LTA4H-ELK and in KIF16B) have been reported previously. Interestingly, the top hit (rs200138614; P = 1.80 × 10-19) was a missense variant (p.Cys712Phe) in the LGR5 gene, a bona-fide marker of adult intestinal stem cells and a potentiator of canonical WNT signaling. The association was validated in an independent Finnish collection of 70 patients with SI-NETs, as well as in the UK Biobank exome sequence data (n = 92 cases and n = 392,814 controls). Overexpression of LGR5 p.Cys712Phe in intestinal organoids abolished the ability of R-Spondin1 to support organoid growth, indicating that the mutation perturbed R-Spondin-LGR5 signaling. CONCLUSIONS: Our study is the largest genome-wide association study to date on SI-NETs and reported 4 new associated genome-wide association study loci, including a novel missense mutation (rs200138614, p.Cys712Phe) in LGR5, a canonical marker of adult intestinal stem cells.
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Neoplasias Intestinais , Tumores Neuroendócrinos , Adulto , Humanos , Tumores Neuroendócrinos/genética , Tumores Neuroendócrinos/patologia , Mutação de Sentido Incorreto , Estudo de Associação Genômica Ampla , Neoplasias Intestinais/genética , Neoplasias Intestinais/patologia , Receptores Acoplados a Proteínas G/genética , Cinesinas/genéticaRESUMO
Background: Papillary thyroid cancer (PTC) is the predominant subtype of thyroid cancer (THCA), and it can cluster in families with an autosomal dominant (AD) inheritance pattern. The aim of this study was to identify novel genes and mechanisms underlying PTC susceptibility. Methods: Our previous investigation of 17 AD PTC families led us to conduct a deeper analysis on one family (Family Q) with whole-genome sequencing data from 3 PTC-affected individuals. In addition, 323 sporadic THCA cases from Avatar data and 12 familial adenomatous polyposis (FAP) individuals with secondary THCA were screened for pyruvate dehydrogenase phosphatase regulatory (PDPR) variants. CRISPR-Cas9 was used to create PDPR-deficient THCA (TPC1) and transformed normal thyroid cell lines (N-Thyori3-1) to study the metabolic consequences of PDPR loss. Results: We found truncating PDPR splice donor variants (NM_017990.4:c.361 + 1G>C) in all affected PTC Family Q members, and another PDPR splice donor variant (NM_017990.4:c.443 + 1G>C) in a sporadic PTC case. In addition, an ultra-rare missense variant was found in an FAP-PTC patient. The PDPR-deficient cells presented with elevated phosphorylation of pyruvate dehydrogenase and altered glucose metabolism, implying that PDPR plays an essential part in regulating glucose metabolism in thyroid cells. Conclusions: Our finding of novel truncating germline variants in PDPR in Family Q and additional cohorts suggests a role for PDPR loss in PTC predisposition. Also, somatic and RNA sequencing from the thyroid carcinoma (Firehouse Legacy) data showed that PDPR gene expression is much lower in THCA tumor tissue compared with matching normal tissue. Thus, PDPR appears to have a loss of function effect on THCA tumorigenesis.
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The 3' untranslated regions (UTRs) modulate gene expression levels by regulating mRNA stability and translation. We previously showed that the replacement of the negative regulatory elements from the 3'UTR of glial cell line-derived neurotrophic factor (GDNF) resulted in increased endogenous GDNF expression while retaining its normal spatiotemporal expression pattern. Here, we have developed a methodology for the generation of in vivo hyper- and hypomorphic alleles via 3'UTR targeting using the CRISPR/Cas9 system. We demonstrate that CRISPR/Cas9-mediated excision of a long inhibitory sequence from Gdnf native 3'UTR in mouse zygotes increases the levels of endogenous GDNF with similar phenotypic alterations in embryonic kidney development as we described in GDNF constitutive and conditional hypermorphic mice. Furthermore, we show that CRISPR/Cas9-mediated targeting of 3'UTRs in vivo allows the modulation of the expression levels of two other morphogens, Gdf11 and Bdnf. Together, our work demonstrates the power of in vivo 3'UTR editing using the CRISPR/Cas9 system to create hyper- and hypomorphic alleles, suggesting wide applicability in studies on gene function and potentially, in gene therapy.
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Intestinal epithelial organoids recapitulate many of the in vivo features of the intestinal epithelium, thus representing excellent research models. Morphology of the organoids based on light-microscopy images is used as a proxy to assess the biological state of the intestinal epithelium. Currently, organoid classification is manual and, therefore, subjective and time consuming, hampering large-scale quantitative analyses. Here, we describe Tellu, an object-detector algorithm trained to classify cultured intestinal organoids. Tellu was trained by manual annotation of >20,000 intestinal organoids to identify cystic non-budding organoids, early organoids, late organoids and spheroids. Tellu can also be used to quantify the relative organoid size, and can classify intestinal organoids into these four subclasses with accuracy comparable to that of trained scientists but is significantly faster and without bias. Tellu is provided as an open, user-friendly online tool to benefit the increasing number of investigations using organoids through fast and unbiased organoid morphology and size analysis.
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Mucosa Intestinal , Intestinos , Organoides , AlgoritmosRESUMO
Growth factors secreted by stromal fibroblasts regulate the intestinal epithelium. Stroma-derived epidermal growth factor (EGF) family ligands are implicated in epithelial regeneration and tumorigenesis, but their specific contributions and associated mechanisms remain unclear. Here, we use primary intestinal organoids modeling homeostatic, injured and tumorigenic epithelia to assess how the fibroblast-derived EGF family ligands neuregulin 1 (NRG1) and epiregulin (EREG) regulate the intestinal epithelium. NRG1 was expressed exclusively in the stroma, robustly increased crypt budding and protected intestinal epithelial organoids from radiation-induced damage. NRG1 also induced regenerative features in the epithelium, including a fetal-like transcriptome, suppression of the Lgr5+ stem cell pool and remodeling of the epithelial actin cytoskeleton. Intriguingly, unlike EGF and EREG, NRG1 failed to support the growth of pre-tumorigenic intestinal organoids lacking the tumor suppressor Apc, commonly mutated in human colorectal cancer (CRC). Interestingly, high expression of stromal NRG1 was associated with improved survival in CRC cohorts, suggesting a tumor-suppressive function. Our results highlight the power of stromal NRG1 in transcriptional reprogramming and protection of the intestinal epithelium from radiation injury without promoting tumorigenesis.
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Fator de Crescimento Epidérmico , Mucosa Intestinal , Neuregulina-1 , Humanos , Carcinogênese/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Fibroblastos/metabolismo , Mucosa Intestinal/metabolismo , Ligantes , Neuregulina-1/metabolismo , Reprogramação CelularRESUMO
The human mismatch repair (MMR) gene MSH2 is the second most frequently mutated hereditary nonpolyposis colorectal cancer (HNPCC) susceptibility locus. Given that missense mutations account for 17% of all identified alterations in this gene, the study of their pathogenicity is of increasing importance. Previously, we showed that pathogenic MSH2 missense mutations typically impaired the repair activity of the protein. In this study, we took advantage of its crystal structure and attempted to correlate the mismatch binding and ATP-catalyzed mismatch release activities with the location of 18 nontruncating MSH2 mutations. We observed that the MMR-deficient mutations situated in the amino-terminal connector and lever domains of MSH2 (V161D, G162R, G164R, L173P, L187P, C333Y, and D603N) affected protein stability, whereas mutations in the ATPase domain (A636P, G674A, C697F, I745_I746del, and E749 K) mainly caused defects in mismatch binding or release. Of the MMR-proficient variants, four (T33P, A272 V, G322D, and V923E) showed slightly reduced mismatch binding and/or release efficiencies compared to wild-type (WT) protein, while two variants (N127S and A834 T) showed no defects in the assays. Similar to our biochemical data, the mutations that affected protein stability were associated with an absence of the protein in tumors in immunohistochemical (IHC) analyses. In contrast, the protein with the mutation E749 K, which abrogates MMR but not protein stability, is well expressed in tumors. In conclusion, pathogenic missense mutations in MSH2 may interfere with different mechanisms that tend to cluster in separate protein domains with varying effects on protein stability, which could be taken into account when interpreting IHC data.
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Neoplasias Colorretais Hereditárias sem Polipose/genética , Reparo de Erro de Pareamento de DNA/genética , Proteína 2 Homóloga a MutS/genética , Mutação de Sentido Incorreto , Células Cultivadas , Proteínas de Ligação a DNA/genética , Predisposição Genética para Doença , Variação Genética , Humanos , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Proteínas Recombinantes/genéticaRESUMO
Hereditary non-polyposis colorectal cancer (HNPCC) is associated with germline mutations in mismatch repair (MMR) genes. Inherited missense mutations, however, complicate the diagnostics because they do not always cause unambiguous predisposition to cancer. This leads to variable and contradictory interpretations of their pathogenicity. Here, we establish evidence for the functionality of the 2 frequently reported variations, MSH2 N127S and G322D, which have been described both as pathogenic and non-pathogenic in literature and databases. We report the results of 3 different functional analyses characterizing the biochemical properties of these protein variants in vitro. We applied an immunoprecipitation assay to assess the MSH2-MSH6 interaction, a bandshift assay to study mismatch recognition and binding, and a MMR assay for repair efficiency. None of the experiments provided evidence on reduced functionality of these proteins as compared to wild-type MSH2. Our data demonstrate that MSH2 N127S and G322D per se are not sufficient to trigger MMR deficiency. This together with variable clinical phenotypes in the mutation carriers suggest no or only low cancer risk in vivo.
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Neoplasias Colorretais Hereditárias sem Polipose/genética , Reparo de Erro de Pareamento de DNA , Mutação em Linhagem Germinativa , Proteína 2 Homóloga a MutS/genética , Asparagina , Ácido Aspártico , Neoplasias do Sistema Biliar/genética , Western Blotting , Proteínas de Ligação a DNA/genética , Neoplasias do Endométrio/genética , Feminino , Predisposição Genética para Doença , Glicina , Humanos , Imunoprecipitação , Neoplasias Pancreáticas/genética , Serina , IncertezaRESUMO
Hereditary nonpolyposis colorectal cancer (HNPCC) is an autosomal dominantly inherited cancer predisposition syndrome caused by germ line mutations in DNA mismatch repair genes, predominantly MLH1 and MSH2, with large genomic rearrangements accounting for 5% to 20% of all mutations. Although crucial to the understanding of cancer initiation, little is known about the second, somatic hit in HNPCC tumorigenesis, commonly referred to as loss of heterozygosity. Here, we applied a recently developed method, multiplex ligation-dependent probe amplification, to study MLH1/MSH2 copy number changes in 16 unrelated Swiss HNPCC patients, whose cancers displayed microsatellite instability and loss of MLH1 or MSH2 expression, but in whom no germ line mutation could be detected by conventional screening. The aims of the study were (a) to determine the proportion of large genomic rearrangements among Swiss MLH1/MSH2 mutation carriers and (b) to investigate the frequency and nature of loss of heterozygosity as a second, somatic event, in tumors from MLH1/MSH2 germ line deletion carriers. Large genomic deletions were found to account for 4.3% and 10.7% of MLH1 and MSH2 mutations, respectively. Multiplex ligation-dependent probe amplification analysis of 18 cancer specimens from two independent sets of Swiss and Finnish MLH1/MSH2 deletion carriers revealed that somatic mutations identical to the ones in the germ line occur frequently in colorectal cancers (6 of 11; 55%) and are also present in extracolonic HNPCC-associated tumors. Chromosome-specific marker analysis implies that loss of the wild-type allele predominantly occurs through locus-restricted recombinational events, i.e., gene conversion, rather than mitotic recombination or deletion of the respective gene locus. (Cancer Res 2006; (66)2: 659-64).
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Proteínas de Transporte/genética , Neoplasias Colorretais Hereditárias sem Polipose/genética , Conversão Gênica , Perda de Heterozigosidade , Proteína 2 Homóloga a MutS/genética , Proteínas Nucleares/genética , Proteínas Adaptadoras de Transdução de Sinal , Adulto , Idoso , Transformação Celular Neoplásica , Análise Mutacional de DNA , Feminino , Finlândia , Deleção de Genes , Dosagem de Genes , Perfilação da Expressão Gênica , Predisposição Genética para Doença , Mutação em Linhagem Germinativa , Humanos , Masculino , Repetições de Microssatélites , Pessoa de Meia-Idade , Proteína 1 Homóloga a MutL , SuíçaRESUMO
Germline mutations in the gene encoding tumor suppressor kinase LKB1 lead to gastrointestinal tumorigenesis in Peutz-Jeghers syndrome (PJS) patients and mouse models; however, the cell types and signaling pathways underlying tumor formation are unknown. Here, we demonstrated that mesenchymal progenitor- or stromal fibroblast-specific deletion of Lkb1 results in fully penetrant polyposis in mice. Lineage tracing and immunohistochemical analyses revealed clonal expansion of Lkb1-deficient myofibroblast-like cell foci in the tumor stroma. Loss of Lkb1 in stromal cells was associated with induction of an inflammatory program including IL-11 production and activation of the JAK/STAT3 pathway in tumor epithelia concomitant with proliferation. Importantly, treatment of LKB1-defcient mice with the JAK1/2 inhibitor ruxolitinib dramatically decreased polyposis. These data indicate that IL-11-mediated induction of JAK/STAT3 is critical in gastrointestinal tumorigenesis following Lkb1 mutations and suggest that targeting this pathway has therapeutic potential in Peutz-Jeghers syndrome.
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Transformação Celular Neoplásica , Interleucina-11/metabolismo , Neoplasias Intestinais/metabolismo , Janus Quinase 1/metabolismo , Janus Quinase 2/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Serina-Treonina Quinases/deficiência , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Neoplasias Gástricas/metabolismo , Proteínas Quinases Ativadas por AMP , Animais , Interleucina-11/genética , Neoplasias Intestinais/genética , Neoplasias Intestinais/patologia , Janus Quinase 1/genética , Janus Quinase 2/genética , Camundongos , Camundongos Knockout , Mutação , Proteínas de Neoplasias/genética , Fator de Transcrição STAT3/genética , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologiaRESUMO
The human genome contains frequent single-basepair variants that may or may not cause genetic disease. To characterize benign vs. pathogenic missense variants, numerous computational algorithms have been developed based on comparative sequence and/or protein structure analysis. We compared computational methods that use evolutionary conservation alone, amino acid (AA) change alone, and a combination of conservation and AA change in predicting the consequences of 254 missense variants in the CDKN2A (n = 92), MLH1 (n = 28), MSH2 (n = 14), MECP2 (n = 30), and tyrosinase (TYR) (n = 90) genes. Variants were validated as either neutral or deleterious by curated locus-specific mutation databases and published functional data. All methods that use evolutionary sequence analysis have comparable overall prediction accuracy (72.9-82.0%). Mutations at codons where the AA is absolutely conserved over a sufficient evolutionary distance (about one-third of variants) had a 91.6 to 96.8% likelihood of being deleterious. Three algorithms (SIFT, PolyPhen, and A-GVGD) that differentiate one variant from another at a given codon did not significantly improve predictive value over conservation score alone using the BLOSUM62 matrix. However, when all four methods were in agreement (62.7% of variants), predictive value improved to 88.1%. These results confirm a high predictive value for methods that use evolutionary sequence conservation, with or without considering protein structural change, to predict the clinical consequences of missense variants. The methods can be generalized across genes that cause different types of genetic disease. The results support the clinical use of computational methods as one tool to help interpret missense variants in genes associated with human genetic disease.
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Proteínas Adaptadoras de Transdução de Sinal/genética , Genes p16 , Proteína 2 de Ligação a Metil-CpG/genética , Monofenol Mono-Oxigenase/genética , Proteína 2 Homóloga a MutS/genética , Mutação de Sentido Incorreto , Proteínas Nucleares/genética , Algoritmos , Evolução Molecular , Humanos , Proteína 1 Homóloga a MutL , Homologia de Sequência de AminoácidosRESUMO
Western-style diets (WD) high in fat and scarce in fiber and vitamin D increase risks of colorectal cancer. Here, we performed a long-term diet study in mice to follow tumorigenesis and characterize structural and metabolic changes in colon mucosa associated with WD and predisposition to colorectal cancer. WD increased colon tumor numbers, and mucosa proteomic analysis indicated severe deregulation of intracellular bile acid (BA) homeostasis and activation of cell proliferation. WD also increased crypt depth and colon cell proliferation. Despite increased luminal BA, colonocytes from WD-fed mice exhibited decreased expression of the BA transporters FABP6, OSTß, and ASBT and decreased concentrations of secondary BA deoxycholic acid and lithocholic acid, indicating reduced activity of the nuclear BA receptor FXR. Overall, our results suggest that WD increases cancer risk by FXR inactivation, leading to BA deregulation and increased colon cell proliferation. Cancer Res; 77(12); 3352-63. ©2017 AACR.
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Ácidos e Sais Biliares/metabolismo , Transformação Celular Neoplásica/patologia , Dieta Ocidental/efeitos adversos , Homeostase/fisiologia , Mucosa Intestinal/patologia , Animais , Western Blotting , Proliferação de Células , Cromatografia Líquida , Colo/patologia , Modelos Animais de Doenças , Eletroforese em Gel Bidimensional , Feminino , Ensaios de Triagem em Larga Escala , Mucosa Intestinal/metabolismo , Masculino , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase , Distribuição Aleatória , Receptores Citoplasmáticos e NuclearesRESUMO
A majority of families with hereditary nonpolyposis colorectal cancer (HNPCC) are attributable to germline mutations in three DNA mismatch repair (MMR) genes, MLH1, MSH2 and MSH6. However, the clinical phenotype appears to reflect a complex interplay between the predisposing mutation and putative constitutional and somatic modifiers. Certain MMR gene mutations predispose to combined occurrence of cutaneous sebaceous gland neoplasms and visceral malignancies, which is known as Muir-Torre syndrome (MTS) and regarded as a phenotypic variant of HNPCC. The sebaceous tumors associated with MTS appear in many patients before visceral malignancies providing important predictability of HNPCC-related integral cancers in mutation carriers. Since most sebaceous skin tumors are, however, sporadic, the contribution of non-truncating mutations found in skin cancer patients is difficult to interpret and genetic assessment of MTS requires a functional test. Here, we studied the repair efficiency of the two MSH2 missense mutations, L187P and C697F, found in HNPCC families including a few mutation carriers with sebaceous skin tumors. Both mutations were completely deficient in an MMR assay, which together with tumor findings suggested their predisposing role in both internal and skin malignancies in the families.
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Neoplasias Colorretais Hereditárias sem Polipose/genética , Reparo do DNA , Proteína 2 Homóloga a MutS/genética , Predisposição Genética para Doença , Humanos , Proteína 2 Homóloga a MutS/fisiologia , Mutação de Sentido Incorreto , Fenótipo , SíndromeRESUMO
AMP-activated protein kinase (AMPK) inhibits several anabolic pathways such as fatty acid and protein synthesis, and identification of AMPK substrate specificity would be useful to understand its role in particular cellular processes and develop strategies to modulate AMPK activity in a substrate-specific manner. Here we show that SUMOylation of AMPKα1 attenuates AMPK activation specifically towards mTORC1 signalling. SUMOylation is also important for rapid inactivation of AMPK, to allow prompt restoration of mTORC1 signalling. PIAS4 and its SUMO E3 ligase activity are specifically required for the AMPKα1 SUMOylation and the inhibition of AMPKα1 activity towards mTORC1 signalling. The activity of a SUMOylation-deficient AMPKα1 mutant is higher than the wild type towards mTORC1 signalling when reconstituted in AMPKα-deficient cells. PIAS4 depletion reduced growth of breast cancer cells, specifically when combined with direct AMPK activator A769662, suggesting that inhibiting AMPKα1 SUMOylation can be explored to modulate AMPK activation and thereby suppress cancer cell growth.
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Proteínas Quinases Ativadas por AMP/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas Inibidoras de STAT Ativados/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Complexos Multiproteicos/genética , Fosforilação , Proteínas de Ligação a Poli-ADP-Ribose , Proteínas Inibidoras de STAT Ativados/genética , Transdução de Sinais , Sumoilação , Serina-Treonina Quinases TOR/genéticaRESUMO
Colorectal cancer (CRC) is the second most common cause of cancer-related deaths in the Western world and interactions between genetic and environmental factors, including diet, are suggested to play a critical role in its etiology. We conducted a long-term feeding experiment in the mouse to address gene expression and methylation changes arising in histologically normal colonic mucosa as putative cancer-predisposing events available for early detection. The expression of 94 growth-regulatory genes previously linked to human CRC was studied at two time points (5 weeks and 12 months of age) in the heterozygote Mlh1(+/-) mice, an animal model for human Lynch syndrome (LS), and wild type Mlh1(+/+) littermates, fed by either Western-style (WD) or AIN-93G control diet. In mice fed with WD, proximal colon mucosa, the predominant site of cancer formation in LS, exhibited a significant expression decrease in tumor suppressor genes, Dkk1, Hoxd1, Slc5a8, and Socs1, the latter two only in the Mlh1(+/-) mice. Reduced mRNA expression was accompanied by increased promoter methylation of the respective genes. The strongest expression decrease (7.3 fold) together with a significant increase in its promoter methylation was seen in Dkk1, an antagonist of the canonical Wnt signaling pathway. Furthermore, the inactivation of Dkk1 seems to predispose to neoplasias in the proximal colon. This and the fact that Mlh1 which showed only modest methylation was still expressed in both Mlh1(+/-) and Mlh1(+/+) mice indicate that the expression decreases and the inactivation of Dkk1 in particular is a prominent early marker for colon oncogenesis.
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Proteínas Adaptadoras de Transdução de Sinal/genética , Colo/metabolismo , Neoplasias do Colo/genética , Dieta Hiperlipídica , Regulação Neoplásica da Expressão Gênica , Mucosa/metabolismo , Proteínas Nucleares/genética , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Animais , Proteínas de Transporte de Cátions/genética , Colo/patologia , Neoplasias do Colo/diagnóstico , Neoplasias Colorretais Hereditárias sem Polipose/diagnóstico , Neoplasias Colorretais Hereditárias sem Polipose/genética , Metilação de DNA , Modelos Animais de Doenças , Proteínas de Homeodomínio/genética , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Camundongos , Camundongos da Linhagem 129 , Camundongos Knockout , Transportadores de Ácidos Monocarboxílicos , Mucosa/patologia , Proteína 1 Homóloga a MutL , Proteínas Nucleares/deficiência , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína 1 Supressora da Sinalização de Citocina , Proteínas Supressoras da Sinalização de Citocina/genética , Fatores de TempoRESUMO
Mutations in the tumor suppressor gene LKB1 are important in hereditary Peutz-Jeghers syndrome, as well as in sporadic cancers including lung and cervical cancer. LKB1 is a kinase-activating kinase, and a number of LKB1-dependent phosphorylation cascades regulate fundamental cellular and organismal processes in at least metabolism, polarity, cytoskeleton organization, and proliferation. Conditional targeting approaches are beginning to demonstrate the relevance and specificity of these signaling pathways in development and homeostasis of multiple organs. More than one of the pathways also appear to contribute to tumor growth following Lkb1 deficiencies based on a number of mouse tumor models. Lkb1-dependent activation of AMPK and subsequent inactivation of mammalian target of rapamycin signaling are implicated in several of the models, and other less well characterized pathways are also involved. Conditional targeting studies of Lkb1 also point an important role of LKB1 in epithelial-mesenchymal interactions, significantly expanding knowledge on the relevance of LKB1 in human disease.
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Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Quinases Proteína-Quinases Ativadas por AMP , Animais , Modelos Animais de Doenças , Desenvolvimento Embrionário/genética , Epitélio/metabolismo , Marcação de Genes , Homeostase/genética , Humanos , Mesoderma/metabolismo , Camundongos , Neoplasias/genética , Neoplasias/metabolismo , Especificidade de Órgãos/genética , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Síndrome de Peutz-Jeghers/enzimologia , Síndrome de Peutz-Jeghers/genética , Transdução de Sinais , Células Estromais/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
Inherited pathogenic mutations in the mismatch repair (MMR) genes, MSH2, MLH1, MSH6, and PMS2 predispose to Lynch syndrome (LS). However, the finding of a variant or variants of uncertain significance (VUS) in affected family members complicates the risk assessment. Here, we describe a putative LS family carrying VUS in both MSH2 (c.2768T>A, p.Val923Glu) and MSH6 (c.3563G>A, p.Ser1188Asn). Two colorectal cancer (CRC) patients were studied for mutations and identified as carriers of both variants. In spite of a relatively high mean age of cancer onset (59.5 years) in the family, many CRC patients and the tumor pathological data suggested that the missense variation in MSH2, the more common susceptibility gene in LS, would be the predisposing alteration. However, MSH2 VUS was surprisingly found to be MMR proficient in an in vitro MMR assay and a tolerant alteration in silico. By supplying evidence that instead of MSH2 p.Val923Glu the MSH6 p.Ser1188Asn variant is completely MMR-deficient, the present study confirms the previous findings, and suggests that MSH6 (c.3563G>A, p.Ser1188Asn) is the pathogenic mutation in the family. Moreover, our results strongly support the strategy to functionally assess all identified VUS before predictive gene testing and genetic counseling are offered to a family.
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Neoplasias Colorretais Hereditárias sem Polipose/genética , Neoplasias Colorretais Hereditárias sem Polipose/patologia , Proteínas de Ligação a DNA/genética , Proteína 2 Homóloga a MutS/genética , Mutação/genética , Adulto , Idoso , Reparo de Erro de Pareamento de DNA/genética , Feminino , Humanos , Técnicas Imunoenzimáticas , Masculino , Instabilidade de Microssatélites , Pessoa de Meia-Idade , Linhagem , PrognósticoRESUMO
BACKGROUND & AIMS: Inherited deleterious mutations in mismatch repair genes MLH1, MSH2, and MSH6 predispose to hereditary nonpolyposis colorectal cancer. A major diagnostic challenge is the difficulty in evaluating the pathogenicity of missense mutations. Previously we showed that most missense variants in MSH6 do not impair MMR capability and are associated with no or low cancer susceptibility, whereas in MLH1, functional studies distinguished nontruncating mutations with severe defects from those not or slightly impaired in protein expression or function. The present study was undertaken to evaluate the pathogenicity of inherited missense mutations in MSH2. METHODS: Fifteen mutated MSH2 proteins including 14 amino acid substitutions and one in-frame deletion were tested for expression/stability, MSH2/MSH6 interaction, and repair efficiency. The genetic and biochemical data were correlated with the clinical data. Comparative sequence analysis was performed to assess the value of sequence homology as a tool for predicting functional results. RESULTS: None of the studied MSH2 mutations destroyed the protein or abolished MSH2/MSH6 interaction, whereas 12 mutations impaired the repair capability of the protein. Comparative sequence analysis correctly predicted functional studies for 13 of 14 amino acid substitutions. CONCLUSIONS: Interpretation was pathogenic for 12, nonpathogenic for 2, and contradictory for 1 mutation. The pathogenicity could not be distinguished unambiguously by phenotypic characteristics, although correlation between the absence of staining for MSH2 and pathogenicity of the missense mutation was notable. Unlike in MSH6 and MLH1, the pathogenicity of missense mutations in MSH2 was always associated with impaired repair capability of the mutated protein.