RESUMEN
BACKGROUND: Somatic mutations in the epidermal growth factor receptor (EGFR) intracellular signalling pathways predict non-response to cetuximab in the treatment of advanced colorectal cancer (aCRC). We hypothesised that common germline variants within these pathways may also play similar roles. METHODS: We analysed 54 potentially functional, common, inherited EGFR pathway variants in 815 patients with aCRC treated with oxaliplatin-fluoropyrimidine chemotherapy plus cetuximab. Primary endpoints were response and skin rash (SR). We had >85% power to detect ORs=1.6 for variants with minor allele frequencies >20%. RESULTS: We identified five potential biomarkers for response and four for SR, although none remained significant after correction for multiple testing. Our initial data supported a role for Ser313Pro in PIK3R2 in modulating response to cetuximab-in patients with KRAS wild-type CRCs, 36.4% with one allele encoding proline responded, as compared with 71.2% homozygous for allele encoding serine (OR 0.23, 95% CI 0.09 to 0.56, p=0.0014), and this association was predictive for cetuximab (pinteraction=0.017); however, independent replication failed to validate this association. No previously proposed predictive biomarkers were validated. CONCLUSIONS: Our study highlights the need to validate potential pharmacogenetic biomarkers. We did not find strong evidence for common germline biomarkers of cetuximab response and toxicity.
Asunto(s)
Cetuximab/uso terapéutico , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Receptores ErbB/genética , Compuestos Organoplatinos/uso terapéutico , Adulto , Anciano , Anciano de 80 o más Años , Antineoplásicos/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Biomarcadores Farmacológicos , Femenino , Frecuencia de los Genes , Mutación de Línea Germinal , Humanos , Masculino , Persona de Mediana Edad , Oxaliplatino , Variantes Farmacogenómicas/genética , Transducción de Señal/genéticaRESUMEN
Familial adenomatous polyposis (FAP) and MUTYH-associated polyposis (MAP) are inherited disorders associated with multiple colorectal adenomas that lead to a very high risk of colorectal cancer. The somatic mutations that drive adenoma development in these conditions have not been investigated comprehensively. In this study we performed analysis of paired colorectal adenoma and normal tissue DNA from individuals with FAP or MAP, sequencing 14 adenoma whole exomes (eight MAP, six FAP), 55 adenoma targeted exomes (33 MAP, 22 FAP) and germline DNA from each patient, and a further 63 adenomas by capillary sequencing (41 FAP, 22 MAP). With these data we examined the profile of mutated genes, the mutational signatures and the somatic mutation rates, observing significant diversity in the constellations of mutated driver genes in different adenomas, and loss-of-function mutations in WTX (9%; p < 9.99e-06), a gene implicated in regulation of the WNT pathway and p53 acetylation. These data extend our understanding of the early events in colorectal tumourigenesis in the polyposis syndromes.
Asunto(s)
Poliposis Adenomatosa del Colon/genética , Poliposis Adenomatosa del Colon/patología , Poliposis Intestinal/genética , Poliposis Intestinal/patología , ADN Glicosilasas/genética , Análisis Mutacional de ADN , Humanos , Síndromes Neoplásicos Hereditarios/genética , TranscriptomaRESUMEN
Inherited defects of base excision repair have not been associated with any human genetic disorder, although mutations of the genes mutM and mutY, which function in Escherichia coli base excision repair, lead to increased transversions of G:C to T:A. We have studied family N, which is affected with multiple colorectal adenomas and carcinoma but lacks an inherited mutation of the adenomatous polyposis coli gene (APC) that is associated with familial adenomatous polyposis. Here we show that 11 tumors from 3 affected siblings contain 18 somatic inactivating mutations of APC and that 15 of these mutations are G:C-->A transversions--a significantly greater proportion than is found in sporadic tumors or in tumors associated with familial adenomatous polyposis. Analysis of the human homolog of mutY, MYH, showed that the siblings were compound heterozygotes for the nonconservative missense variants Tyr165Cys and Gly382Asp. These mutations affect residues that are conserved in mutY of E. coli (Tyr82 and Gly253). Tyrosine 82 is located in the pseudo-helix-hairpin-helix (HhH) motif and is predicted to function in mismatch specificity. Assays of adenine glycosylase activity of the Tyr82Cys and Gly253Asp mutant proteins with 8-oxoG:A and G:A substrates show that their activity is reduced significantly. Our findings link the inherited variants in MYH to the pattern of somatic APC mutation in family N and implicate defective base excision repair in predisposition to tumors in humans.
Asunto(s)
Neoplasias Colorrectales/genética , ADN Glicosilasas , N-Glicosil Hidrolasas/genética , Mutación Puntual , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Neoplasias Colorrectales/enzimología , Secuencia Conservada , Reparación del ADN/genética , ADN de Neoplasias/genética , Evolución Molecular , Femenino , Genes APC , Variación Genética , Humanos , Masculino , Datos de Secuencia Molecular , Linaje , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido NucleicoRESUMEN
Across the globe, long-term care has been under increased pressure throughout the COVID-19 pandemic. This is the first study to examine the experiences and needs of long-term care staff and management during COVID-19, in the Canadian context. Our group conducted online survey research with 70 staff and management working at public long-term care facilities in central Canada, using validated quantitative measures to examine perceived stress and caregiver burden; and open-ended items to explore stressors, ways of coping, and barriers to accessing mental health supports. Findings indicate moderate levels of stress and caregiver burden, and highlight the significant stressors associated with working in long-term care during the COVID-19 pandemic (i.e., rapid changes in pandemic guidelines, increased workload, "meeting the needs of residents and families", fear of contracting COVID-19 and COVID-19 coming into long-term care facilities, and concern over a negative public view of long-term care staff and facilities). A small subset (13.2%) of our sample identified accessing mental health supports to cope with work-related stress, with most participants identifying barriers to seeking help. Novel findings of this research highlight the significant and unmet needs of this high-risk segment of the population.
Asunto(s)
COVID-19 , Cuidados a Largo Plazo , Humanos , Pandemias , Canadá , Carga de TrabajoRESUMEN
Familial adenomatous polyposis (FAP) is characterised by the development of hundreds to thousands of colorectal adenomas and results from inherited or somatic mosaic variants in the APC gene. Index patients with suspected FAP are usually investigated by APC coding region sequence and dosage analysis in a clinical diagnostic setting. The identification of an APC variant which is predicted to alter protein function enables predictive genetic testing to guide the management of family members. This report describes a 4-generation family with a phenotype consistent with FAP, but in which an APC variant had not been identified, despite testing. To explore this further, quantitative PCR (qPCR) was employed to assess APC transcription, demonstrating reduced levels of APC RNA. Next generation sequencing (NGS) identified the APC 5'UTR/ Exon 1 variant, c.-190 G>A, that had been reported previously in an another FAP family with APC allelic imbalance. Quantitative RNA studies and DNA sequencing of the APC promoters/ Exon 1 may be useful diagnostically for patients with suspected FAP when coding region variants cannot be identified.
Asunto(s)
Proteína de la Poliposis Adenomatosa del Colon/genética , Poliposis Adenomatosa del Colon/genética , Mutación , Regiones Promotoras Genéticas , Regiones no Traducidas 5' , Poliposis Adenomatosa del Colon/diagnóstico , Proteína de la Poliposis Adenomatosa del Colon/metabolismo , Humanos , Linaje , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
BACKGROUND: Inherited genetic variants may influence response to, and side-effects from, chemotherapy. We sought to generate a comprehensive inherited pharmacogenetic profile for oxaliplatin and 5FU/capecitabine therapy in advanced colorectal cancer (aCRC). METHODS: We analysed more than 200 potentially functional, common, inherited variants in genes within the 5FU, capecitabine, oxaliplatin and DNA repair pathways, together with four rare dihydropyrimidine dehydrogenase (DPYD) variants, in 2183 aCRC patients treated with oxaliplatin-fluoropyrimidine chemotherapy with, or without, cetuximab (from MRC COIN and COIN-B trials). Primary end-points were response, any toxicity and peripheral neuropathy. We had >85% power to detect odds ratios (ORs) = 1.3 for variants with minor allele frequencies >20%. RESULTS: Variants in DNA repair genes (Asn279Ser in EXO1 and Arg399Gln in XRCC1) were most associated with response (OR 1.9, 95% confidence interval [CI] 1.2-2.9, P = 0.004, and OR 0.7, 95% CI 0.5-0.9, P = 0.003, respectively). Common variants in DPYD (Cys29Arg and Val732Ile) were most associated with toxicity (OR 0.8, 95% CI 0.7-1.0, P = 0.008, and OR 1.6, 95% CI 1.1-2.1, P = 0.006, respectively). Two rare DPYD variants were associated with increased toxicity (Asp949Val with neutropenia, nausea and vomiting, diarrhoea and infection; IVS14+1G>A with lethargy, diarrhoea, stomatitis, hand-foot syndrome and infection; all ORs > 3). Asp317His in DCLRE1A was most associated with peripheral neuropathy (OR 1.3, 95% CI 1.1-1.6, P = 0.003). No common variant associations remained significant after Bonferroni correction. CONCLUSIONS: DNA repair genes may play a significant role in the pharmacogenetics of aCRC. Our data suggest that both common and rare DPYD variants may be associated with toxicity to fluoropyrimidine-based chemotherapy.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Capecitabina/efectos adversos , Neoplasias Colorrectales/tratamiento farmacológico , Dihidrouracilo Deshidrogenasa (NADP)/genética , Fluorouracilo/efectos adversos , Oxaliplatino/efectos adversos , Pruebas de Farmacogenómica , Variantes Farmacogenómicas , Cetuximab/efectos adversos , Toma de Decisiones Clínicas , Neoplasias Colorrectales/enzimología , Neoplasias Colorrectales/genética , Enzimas Reparadoras del ADN/genética , Exodesoxirribonucleasas/genética , Frecuencia de los Genes , Genotipo , Humanos , Farmacogenética , Fenotipo , Medicina de Precisión , Valor Predictivo de las Pruebas , Factores de Riesgo , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X/genéticaRESUMEN
A Bloom's deficient mouse model (Blm(m3/m3)) has been shown to induce colorectal tumorigenesis when crossed with Apc+/Min mice. Here, we investigated whether the Blm(m3/m3) genotype could induce tumorigenesis in extracolonic tissues in tuberous sclerosis 1-deficient (Tsc1+/-) mice that are predisposed to renal cystadenomas and carcinomas. Genotyping of offspring from Tsc1+/- Blm+/m3 intercrosses showed that a approximately 24% excess of Tsc1+/- over Tsc1+/+ mice died before weaning (P = 0.016), although Blm deficiency had no cumulative effect on Tsc1+/- survival. Tsc1+/- Blm(m3/m3) mice had significantly more macroscopic and microscopic renal lesions at 3 to 6 months compared with Tsc1+/- Blm+/m3 mice (P =0.0003 and 0.0203, respectively), and their tumors showed significantly increased levels of somatic loss of heterozygosity (LOH) of the wild-type Tsc1 (Tsc1wt) allele compared with those from Tsc1+/- Blm+/+ mice (P < 0.0001). Tsc1+/- Blm+/m3 mice did not show significantly more renal lesions compared with Tsc1+/- Blm+/+ animals; however, their lesions still showed significantly increased levels of somatic LOH of the Tsc1wt allele (P = 0.03). Ninety-five percent (19 of 20) of lesions from Tsc1+/- Blm+/m3 mice retained the wild-type Blm (Blm(wt)) allele, indicating that the increased somatic LOH at Tsc1 was mediated by Blm haploinsufficiency. Renal lesions from a Blm-deficient background stained positively with anti-phospho-S6 ribosomal protein (Ser240/244), suggesting that these lesions develop through the normal pathway of Tsc-associated tumorigenesis. This work shows the use of the Blm(m3/m3) mice for inducing renal tumorigenesis, and the high levels (approximately 87%) of LOH in the resultant tumors will help facilitate mapping of loci involved in tumor progression.
Asunto(s)
Adenosina Trifosfatasas/deficiencia , Transformación Celular Neoplásica/genética , ADN Helicasas/deficiencia , Neoplasias Renales/genética , Pérdida de Heterocigocidad , Proteínas Supresoras de Tumor/genética , Adenosina Trifosfatasas/genética , Alelos , Animales , ADN Helicasas/genética , Femenino , Genes Supresores de Tumor , Masculino , Ratones , Ratones Endogámicos BALB C , Mutación , RecQ Helicasas , Proteína 1 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de Tumor/deficienciaRESUMEN
Purpose: Duodenal polyposis and cancer are important causes of morbidity and mortality in familial adenomatous polyposis (FAP) and MUTYH-associated polyposis (MAP). This study aimed to comprehensively characterize somatic genetic changes in FAP and MAP duodenal adenomas to better understand duodenal tumorigenesis in these disorders.Experimental Design: Sixty-nine adenomas were biopsied during endoscopy in 16 FAP and 10 MAP patients with duodenal polyposis. Ten FAP and 10 MAP adenomas and matched blood DNA samples were exome sequenced, 42 further adenomas underwent targeted sequencing, and 47 were studied by array comparative genomic hybridization. Findings in FAP and MAP duodenal adenomas were compared with each other and to the reported mutational landscape in FAP and MAP colorectal adenomas.Results: MAP duodenal adenomas had significantly more protein-changing somatic mutations (P = 0.018), truncating mutations (P = 0.006), and copy number variants (P = 0.005) than FAP duodenal adenomas, even though MAP patients had lower Spigelman stage duodenal polyposis. Fifteen genes were significantly recurrently mutated. Targeted sequencing of APC, KRAS, PTCHD2, and PLCL1 identified further mutations in each of these genes in additional duodenal adenomas. In contrast to MAP and FAP colorectal adenomas, neither exome nor targeted sequencing identified WTX mutations (P = 0.0017).Conclusions: The mutational landscapes in FAP and MAP duodenal adenomas overlapped with, but had significant differences to those reported in colorectal adenomas. The significantly higher burden of somatic mutations in MAP than FAP duodenal adenomas despite lower Spigelman stage disease could increase cancer risk in the context of apparently less severe benign disease. Clin Cancer Res; 23(21); 6721-32. ©2017 AACR.
Asunto(s)
Adenoma/genética , Poliposis Adenomatosa del Colon/genética , Carcinogénesis/genética , Neoplasias Duodenales/genética , Adenoma/sangre , Adenoma/patología , Poliposis Adenomatosa del Colon/sangre , Poliposis Adenomatosa del Colon/patología , Adulto , Anciano , Biopsia , ADN Glicosilasas/genética , Análisis Mutacional de ADN , ADN de Neoplasias/sangre , Neoplasias Duodenales/sangre , Neoplasias Duodenales/patología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Proteínas de Neoplasias/genética , Secuenciación del ExomaRESUMEN
Despite the availability of numerous technologies for detecting mutations, only a few have been formatted for automated mutation calling. Here, we evaluate the utility of the Transgenomic Navigator software to facilitate automated detection of aberrant denaturing high performance liquid chromatography (dHPLC) elution profiles. We used dHPLC to identify germline variants in MSH6, NEIL2, NEIL3, and OGG1 in 172 patients with multiple colorectal adenomas. 3,747 dHPLC profiles were analysed with the Navigator software using three levels of analysis, each differing in the degree of operator input. 43.5% (60/138) and 98.3% (59/60) of products with profiles distinct from wild type ('outliers') harboured novel variants under Level 1 and Levels 2/3 analysis conditions, respectively. We also assessed the utility of the software to rapidly detect samples carrying common polymorphisms by analysing regions of the genes that harbour polymorphisms with minor allele frequencies between 8 and 40%, therein analysing 2,784 profiles. We showed that 1573/1612 (97.6%) and 1137/1172 (97.0%) of PCR products were correctly classified as wild-type and variant, respectively (Level 3 analysis conditions). Finally, we assessed the utility of the software to detect novel variants in fragments that also harboured common polymorphisms and showed that 59/61 (96.7%) of products with profiles outlying both the wild type and polymorphism groups harboured novel variants. We conclude that the Navigator software provides an excellent tool for rapid discrimination of aberrant dHPLC elution profiles that harbour sequence variants.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Biología Computacional/métodos , Análisis Mutacional de ADN/métodos , Genoma , Humanos , Desnaturalización de Ácido Nucleico , Polimorfismo Genético , Análisis de Secuencia de ADN , Programas Informáticos , TransgenesRESUMEN
Familial adenomatous polyposis (FAP) and attenuated FAP are autosomal dominant disorders characterised by multiple colorectal adenomas and cancers. Both are caused by inherited mutations in the APC gene, and management includes genetic testing, colonoscopic surveillance, and prophylactic surgery for the relatives of index cases. Among 614 families recorded in six regional registers of polyposis in the UK, we identified 111 with neither dominant transmission nor evidence of APC mutation. Molecular genetic analysis showed that 25 had biallelic mutations of the MYH gene. Since our data show that MYH polyposis can be transmitted as an autosomal recessive trait, a change in genetic counselling, testing, and surveillance is needed.
Asunto(s)
Poliposis Adenomatosa del Colon/genética , ADN Glicosilasas , Genes Recesivos , Mutación , N-Glicosil Hidrolasas/genética , Poliposis Adenomatosa del Colon/epidemiología , Adolescente , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Sistema de Registros , Reino Unido/epidemiologíaRESUMEN
Somatic mosaicism is a frequent phenomenon in mendelian disorders that exhibit a high proportion of new mutations; however, mutant alleles present at low frequency are difficult to detect and characterize. We have previously shown that denaturing high-performance liquid chromatography (DHPLC) can detect TSC1 and TSC2 mutations in tuberous sclerosis patients with low-level somatic mosaicism, even when direct sequencing cannot identify the causative lesion. Characterization of these mutations traditionally involves extensive sequencing of cloned products. To overcome this limitation, we have utilized DHPLC with an in-line fraction collector to isolate low-level heteroduplex peaks that can be directly sequenced to reveal the mutation. We have successfully applied this technique to resolve the mutations 2724-1G>C in TSC1and 1462-28del42bp, 1774del4bp, and N1643K (4947C>G) in TSC2, which were present in only 6.5-17% of the patients' alleles. We have also applied this technique to successfully resolve seven somatic APC mutations in colorectal tumor samples that were previously undetectable by direct PCR product sequencing. This method may simplify many of the currently challenging goals in mutation detection.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Mosaicismo/genética , Mutación/genética , Desnaturalización de Ácido Nucleico/genética , Ácidos Nucleicos Heterodúplex/genética , ADN/sangre , Genes Supresores de Tumor , Humanos , Leucocitos/química , Ácidos Nucleicos Heterodúplex/química , Proteínas/química , Proteínas/genética , Proteínas Represoras/química , Proteínas Represoras/genética , Esclerosis Tuberosa/genética , Proteína 1 del Complejo de la Esclerosis Tuberosa , Proteína 2 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de TumorRESUMEN
Tuberous sclerosis complex (TSC) is caused by mutations in either the TSC1 or TSC2 gene. Both genes are generally considered to act as tumor suppressors that fulfill Knudson's "two-hit hypothesis" and that function within the phosphoinositide 3-kinase-Akt-mammalian target of rapamycin (mTOR) pathway. We previously generated Tsc1(+/-) mice that are predisposed to renal cysts, which develop into cystadenomas and renal cell carcinomas. Here, we identified somatic Tsc1 mutations (second hits) in approximately 80% of cystadenomas and renal cell carcinomas, but only 31.6% of cysts from Tsc1(+/-) mice (P < 0.0003), raising the possibility that haploinsufficiency for Tsc1 plays a role in cyst formation. Consistent with this proposal, many cysts showed little or no staining for phosphorylated mTOR (53%) and phosphorylated S6 ribosomal protein (37%), whereas >90% of cystadenomas and renal cell carcinomas showed strong staining for both markers (P < 0.0005). We also sought somatic mutations in renal lesions from Tsc1(+/-) Blm(-/-) mice that have a high frequency of somatic loss of heterozygosity, thereby facilitating the detection of second hits. We also found significantly less somatic mutations in cysts as compared with cystadenomas and renal cell carcinomas from these mice (P = 0.017). Our data indicate that although activation of the mTOR pathway is an important step in Tsc-associated renal tumorigenesis, it may not be the key initiating event in this process.
Asunto(s)
Enfermedades Renales Quísticas/genética , Enfermedades Renales Quísticas/patología , Neoplasias Renales/genética , Proteínas Quinasas/fisiología , Proteínas Supresoras de Tumor/deficiencia , Proteínas Supresoras de Tumor/genética , Animales , ADN/genética , ADN/aislamiento & purificación , Genotipo , Neoplasias Renales/patología , Pérdida de Heterocigocidad , Ratones , Ratones Noqueados , Mutación , Fosforilación , Serina-Treonina Quinasas TOR , Proteína 1 del Complejo de la Esclerosis TuberosaRESUMEN
The base excision repair gene MYH protects against damage to DNA from reactive oxygen species, which are commonly found in cigarette smoke. Inherited mutations in MYH predispose to colorectal adenomas and carcinomas that show a characteristic pattern of somatic G:C-->T:A mutations in the APC gene. A similar pattern of somatic mutations in the TP53 gene is reported in smoking-related lung cancers. We therefore tested whether germline changes in MYH may also contribute to the development of lung cancer by screening for variants in 276 patients with lung carcinoma and 106 normal controls. No patients harboured truncating mutations in MYH and only a single patient was a carrier for the G382D missense mutation. We identified three common coding region (V22M, Q324H and S501F) and intronic (157+30A>G, 462+35G>A and 1435-40G>C) variants, but none were over-represented in the patient samples, indicating that MYH variants are unlikely to predispose significantly to the risk of lung cancer.
Asunto(s)
ADN Glicosilasas/genética , Variación Genética , Neoplasias Pulmonares/genética , Mutación , Adenocarcinoma/enzimología , Adenocarcinoma/genética , Carcinoma de Células Grandes/enzimología , Carcinoma de Células Grandes/genética , Carcinoma de Pulmón de Células no Pequeñas/enzimología , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Células Pequeñas/enzimología , Carcinoma de Células Pequeñas/genética , Carcinoma de Células Escamosas/enzimología , Carcinoma de Células Escamosas/genética , Estudios de Casos y Controles , ADN de Neoplasias/sangre , ADN de Neoplasias/genética , Exones/genética , Femenino , Predisposición Genética a la Enfermedad/genética , Humanos , Pulmón/fisiología , Neoplasias Pulmonares/enzimología , Masculino , Fenotipo , Reacción en Cadena de la Polimerasa , Factores de RiesgoRESUMEN
We have recently demonstrated that inherited defects of the base excision repair gene MYH predispose to multiple colorectal adenomas and carcinoma. Three affected siblings from a single British family were identified as Y165C/G382D compound heterozygotes and both missense mutations were shown to be functionally compromised. Here, we report the identification of seven further unrelated patients with >100 colorectal adenomas (six with colorectal cancer) and biallelic germline mutations in MYH: four were homozygous for truncating mutations, two were homozygous for Y165C and one was a Y165C/G382D compound heterozygote. As predicted from studies of the bacterial and yeast orthologues of MYH, colorectal tumours from affected individuals displayed a significant excess of somatic G:C-->T:A mutations in APC, as compared to sporadic ( chi(2)=242.96, P<10(-20)) or FAP-associated ( chi(2)=194.85, P<10(-20)) colorectal tumours. The sequence immediately downstream of the somatic G:C-->T:A mutations was predominantly AA, irrespective of the nature of the germline MYH mutations. These findings confirm the role of MYH in colorectal adenoma and carcinoma predisposition.