Allele-Specific Expression of APC in Adenomatous Polyposis Families

BACKGROUND & AIMS: Germline mutations in the APC gene cause of most cases of familial adenomatous polyposis (FAP) and a lesser proportion of attenuated FAP (AFAP). Systematic analysis of APC at the RNA level could provide insight into the pathogenicity of identified mutations and the molecular basis of FAP/AFAP in families without identifiable mutations. Here, we analyzed the prevalence of imbalances in the allelic expression of APC in polyposis families with germline mutations in the gene and without detectable mutations in APC and/or MUTYH. METHODS: Allele-specific expression (ASE) was determined by single nucleotide primer extension using an exon 11 polymorphism as an allele-specific marker. In total, 52 APC-mutation-positive (36 families) and 24 APC/MUTYH-mutation-negative (23 families) informative patients were analyzed. Seventy-six controls also were included. RESULTS: Of the APC-mutation-positive families, most of those in whom the mutation was located before the last exon of the gene (12 of 14) had ASE imbalance, which is consistent with a mechanism of nonsense-mediated decay. Of the APC/MUTYH-mutation-negative Families, 2 (9%) had ASE imbalance, which might cause the disease. Normal allele expression was restored shortly after lymphocytes were cultured with puromycin, supporting a 'nonsense-mediated' hypothesis. CONCLUSIONS: ASE analysis might be used to determine the pathogenesis of some cases of FAP and AFAP in which APC mutations are not found. ASE also might be used to prioritize the order in which different areas of APC are tested. RNA-level studies are important for the molecular diagnosis of FAP

Frequent polymorphic changes but not mutations of TRAIL receptors DR4 and DR5 in mantle cell lymphoma and other B-cell lymphoid neoplasms

Background and objectives: tumor necrosis factor related apoptosis-inducing ligand (TRAIL) receptors DR4 and DR5 have been mapped to chromosome 8p21-22, a region frequently deleted in different lymphoid neoplasms. Design and methods: to investigate the potential alterations of these genes in lymphoid neoplasms, we examined the presence of gene mutations in exons 3, 4, and 9 in 69 cases with mantle cell lymphoma (MCL), 16 with chronic lymphocytic leukemia (CLL), 12 with follicular lymphomas (FL) and 17 with large B-cell-lymphomas (DLBCL), as well as in 4 lymphoid cell lines carrying the t(11;14) translocation, and 91 healthy blood donors. Results: three CLL and three MCL cases had 8p deletions. Two nucleotide changes in or near the intron 3 splice consensus sequence and a silent change were found. These rare changes were also present in the germ-line of the patients. The DR4 death domain A1322G polymorphism was significantly more frequent in MCL [odds ratio (OR) = 5.9; 95% confidence interval (CI), 1.92-18.1] and CLL (OR = 4.5; CI, 1.18-17) patients than in a sex and age-adjusted healthy population. In contrast, the DR4 exon 4 C626G polymorphism was associated with a significant overall decreased risk for MCL (OR = 0.3; CI, 0.12-0.8). No mutations or cancer-associated polymorphic changes were found in DR5 domains. Interpretation and conclusions: these findings indicate that mutations of DR4 and DR5 are uncommon in lymphoid neoplasms but DR4 polymorphic alleles may contribute to the pathogenesis of these malignancies

Statin use and the risk of colorectal cancer in a population-based electronic health records study

There is extensive debate regarding the protective effect of 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors (statins) on colorectal cancer (CRC). We aimed to assess the association between CRC risk and exposure to statins using a large cohort with prescription data. We carried out a case-control study in Catalonia using the System for Development of Primary Care Research (SIDIAP) database that recorded patient diseases history and linked data on reimbursed medication. The study included 25 811 cases with an incident diagnosis of CRC between 2010 and 2015 and 129 117 frequency-matched controls. Subjects were classified as exposed to statins if they had ever been dispensed statins. Analysis considering mean daily defined dose, cumulative duration and type of statin were performed. Overall, 66 372 subjects (43%) were exposed to statins. There was no significant decrease of CRC risk associated to any statin exposure (OR = 0.98; 95% CI: 0.95-1.01). Only in the stratified analysis by location a reduction of risk for rectal cancer was observed associated to statin exposure (OR = 0.87; 95% CI: 0.81-0.92). This study does not support an overall protective effect of statins in CRC, but a protective association with rectal cancer merits further research

Serum 25-hydroxyvitamin D3 levels and vitamin D receptor variants in melanoma patients from the Mediterranean area of Barcelona

BACKGROUND: Serum 25-hydroxyvitamin D3 (Vitamin D) insufficiency and single-nucleotide polymorphisms (SNPs) on its receptor, Vitamin D receptor (VDR), have been reported to be involved in melanoma susceptibility in populations mostly from northern countries. OBJECTIVE: To investigate 25-hydroxyvitamin D3 levels and VDR SNPs in melanoma patients from sunny area of Barcelona, two studies were carried out. The first study evaluated the levels of Vitamin D at time of melanoma diagnosis and the second one analyzed the association between VDR genetic variants and risk of having a high nevus number, the strongest phenotypic risk factor for melanoma. METHODS: The levels of 25-hydroxyvitamin D3 in 81 melanoma patients at diagnosis were measured. In a second group of melanoma patients, including 150 with low and 113 with high nevus number, 11 VDR SNPs were analyzed for their association with nevus number. RESULTS: In the first study, 68% of patients had insufficient levels of 25-hydroxyvitamin D3 (<25 ng/ml). Autumn-winter months and fair phototype were associated with 25-hydroxyvitamin D3 insufficiency; after multivariate analysis, season of sampling remained the only independent predictor of 25-hydroxyvitamin D3 levels. In the second study, VDR variant rs2189480 (P = 0.006) was associated with risk of high nevus number whereas rs2239179 (P = 0.044) and rs7975128 (P = 0.0005) were protective against high nevus number. After Bonferroni adjustment only rs7975128 remained significant. In stratified analysis, SNP rs7975128 was found protective against multiple melanomas (P = 0.021). CONCLUSION: This study showed that even in Barcelona, a sunny Mediterranean area, 25-hydroxyvitamin D3 levels were sub-optimal in the majority of melanoma patients at diagnosis. The involvement of VDR in nevi and, in turn, in melanoma susceptibility has also been suggested. Larger studies are needed to confirm our findings

Evaluating the Potential of Polygenic Risk Score to Improve Colorectal Cancer Screening

Background: Colorectal cancer has high incidence and associ-ated mortality worldwide. Screening programs are recommended for men and women over 50. Intermediate screens such as fecal immunochemical testing (FIT) select patients for colonoscopy with suboptimal sensitivity. Additional biomarkers could improve the current scenario. Methods: We included 2,893 individuals with a positive FIT test. They were classified as cases when a high-risk lesion for colorectal cancer was detected after colonoscopy, whereas the control group comprised individuals with low-risk or no lesions. 65 colorectal cancer risk genetic variants were geno-typed. Polygenic risk score (PRS) and additive models for risk prediction incorporating sex, age, FIT value, and PRS were generated. Results: Risk score was higher in cases compared with controls [per allele OR = 1.04; 95% confidence interval (CI), 1.02-1.06; P = 65), compared with those in the first decile (<= 54; OR = 2.22; 95% CI, 1.59-3.12; P < 0.0001). The model combining sex, age, FIT value, and PRS reached the highest accuracy for identifying patients with a high-risk lesion [cross-validated area under the ROC curve (AUROC): 0.64; 95% CI, 0.62-0.66]. Conclusions: This is the first investigation analyzing PRS in a two-step colorectal cancer screening program. PRS could improve current colorectal cancer screening, most likely for higher at-risk subgroups. However, its capacity is limited to predict colorectal cancer risk status and should be complemented by additional biomarkers.Impact: PRS has capacity for risk stratification of colorectal cancer suggesting its potential for optimizing screening strategies alongside with other biomarkers

Torque Teno Sus Virus (TTSuV) in Cell Cultures and Trypsin

Torque teno sus virus (TTSuV), a member of the family Anelloviridae, is a single-stranded, circular DNA virus, widely distributed in swine populations. Presently, two TTSuV genogroups are recognized: Torque teno sus virus 1 (TTSuV1) and Torque teno sus virus 2 (TTSuV2). TTSuV genomes have been found in commercial vaccines for swine, enzyme preparations and other drugs containing components of porcine origin. However, no studies have been made looking for TTSuV in cell cultures. In the present study, a search for TTSuV genomes was carried out in cell culture lineages, in sera used as supplement for cell culture media as well as in trypsin used for cell disaggregation. DNA obtained from twenty-five cell lineages (ten from cultures in routine multiplication and fifteen from frozen ampoules), nine samples of sera used in cell culture media and five batches of trypsin were examined for the presence of TTSuV DNA. Fifteen cell lineages, originated from thirteen different species contained amplifiable TTSuV genomes, including an ampoule with a cell lineage frozen in 1985. Three cell lineages of swine origin were co-infected with both TTSuV1 and TTSuV2. One batch of trypsin contained two distinct TTSuV1 plus one TTSuV2 genome, suggesting that this might have been the source of contamination, as supported by phylogenetic analyses of sequenced amplicons. Samples of fetal bovine and calf sera used in cell culture media did not contain amplifiable TTSuV DNA. This is the first report on the presence of TTSuV as contaminants in cell lineages. In addition, detection of the viral genome in an ampoule frozen in 1985 provides evidence that TTSuV contamination is not a recent event. These findings highlight the risks of TTSuV contamination in cell cultures, what may be source for contamination of biological products or compromise results of studies involving in vitro multiplied cells

Rescue of a porcine anellovirus (torque teno sus virus 2) from cloned genomic DNA in pigs

Anelloviruses are a group of single-stranded circular DNA viruses infecting humans and other animal species. Animal models combined with reverse genetic systems of anellovirus have not been developed. We report here the construction and initial characterization of full-length DNA clones of a porcine anellovirus, torque teno sus virus 2 (TTSuV2), in vitro and in vivo. We first demonstrated that five cell lines, including PK-15 cells, are free of TTSuV1 or TTSuV2 contamination, as determined by a real-time PCR and an immunofluorescence assay (IFA) using anti-TTSuV antibodies. Recombinant plasmids harboring monomeric or tandem-dimerized genomic DNA of TTSuV2 from the United States and Germany were constructed. Circular TTSuV2 genomic DNA with or without introduced genetic markers and tandem-dimerized TTSuV2 plasmids were transfected into PK-15 cells, respectively. Splicing of viral mRNAs was identified in transfected cells. Expression of TTSuV2-specific open reading frame 1 (ORF1) in cell nuclei, especially in nucleoli, was detected by IFA. However, evidence of productive TTSuV2 infection was not observed in 12 different cell lines transfected with the TTSuV2 DNA clones. Transfection with circular DNA from a TTSuV2 deletion mutant did not produce ORF1 protein, suggesting that the observed ORF1 expression is driven by TTSuV2 DNA replication in cells. Pigs inoculated with either the tandem-dimerized clones or circular genomic DNA of U.S. TTSuV2 developed viremia, and the introduced genetic markers were retained in viral DNA recovered from the sera of infected pigs. The availability of an infectious DNA clone of TTSuV2 will facilitate future study of porcine anellovirus pathogenesis and biology

Population-based multicase-control study in common tumors in Spain (MCC-Spain): rationale and study design

Introduction: We present the protocol of a large population-based case-control study of 5 common tumors in Spain (MCC-Spain) that evaluates environmental exposures and genetic factors. Methods: Between 2008-2013, 10,183 persons aged 20-85 years were enrolled in 23 hospitals and primary care centres in 12 Spanish provinces including 1,115 cases of a new diagnosis of prostate cancer, 1,750 of breast cancer, 2,171 of colorectal cancer, 492 of gastro-oesophageal cancer, 554 cases of chronic lymphocytic leukaemia (CLL) and 4,101 population-based controls matched by frequency to cases by age, sex and region of residence. Participation rates ranged from 57% (stomach cancer) to 87% (CLL cases) and from 30% to 77% in controls. Participants completed a face-to-face computerized interview on sociodemographic factors, environmental exposures, occupation, medication, lifestyle, and personal and family medical history. In addition, participants completed a self-administered food-frequency questionnaire and telephone interviews. Blood samples were collected from 76% of participants while saliva samples were collected in CLL cases and participants refusing blood extractions. Clinical information was recorded for cases and paraffin blocks and/or fresh tumor samples are available in most collaborating hospitals. Genotyping was done through an exome array enriched with genetic markers in specific pathways. Multiple analyses are planned to assess the association of environmental, personal and genetic risk factors for each tumor and to identify pleiotropic effects. Discussion: This study, conducted within the Spanish Consortium for Biomedical Research in Epidemiology & Public Health (CIBERESP), is a unique initiative to evaluate etiological factors for common cancers and will promote cancer research and prevention in Spain.The study was partially funded by the “Accion Transversal del Cancer”, approved on the Spanish Ministry Council on the 11th October 2007, by the Instituto de Salud Carlos III-FEDER (PI08/1770, PI08/0533, PI08/1359, PS09/00773, PS09/01286, PS09/01903, PS09/02078, PS09/01662, PI11/01403, PI11/01889, PI11/00226, PI11/01810, PI11/02213, PI12/00488, PI12/00265, PI12/01270, PI12/00715, PI12/00150), by the Fundación Marqués de Valdecilla (API 10/09), by the ICGC International Cancer Genome Consortium CLL, by the Junta de Castilla y León (LE22A10-2), by the Consejería de Salud of the Junta de Andalucía (PI-0571), by the Conselleria de Sanitat of the Generalitat Valenciana (AP 061/10), by the Recercaixa (2010ACUP 00310), by the Regional Government of the Basque Country by European Commission grants FOOD-CT- 2006-036224-HIWATE, by the Spanish Association Against Cancer (AECC) Scientific Foundation, by the The Catalan Government DURSI grant 2009SGR1489