Genetic Effects on Transcriptome Profiles in Colon Epithelium Provide Functional Insights for Genetic Risk Loci.

BACKGROUND & AIMS: The association of genetic variation with tissue-specific gene expression and alternative splicing guides functional characterization of complex trait-associated loci and may suggest novel genes implicated in disease. Here, our aims were as follows: (1) to generate reference profiles of colon mucosa gene expression and alternative splicing and compare them across colon subsites (ascending, transverse, and descending), (2) to identify expression and splicing quantitative trait loci (QTLs), (3) to find traits for which identified QTLs contribute to single-nucleotide polymorphism (SNP)-based heritability, (4) to propose candidate effector genes, and (5) to provide a web-based visualization resource. METHODS: We collected colonic mucosal biopsy specimens from 485 healthy adults and performed bulk RNA sequencing. We performed genome-wide SNP genotyping from blood leukocytes. Statistical approaches and bioinformatics software were used for QTL identification and downstream analyses. RESULTS: We provided a complete quantification of gene expression and alternative splicing across colon subsites and described their differences. We identified thousands of expression and splicing QTLs and defined their enrichment at genome-wide regulatory regions. We found that part of the SNP-based heritability of diseases affecting colon tissue, such as colorectal cancer and inflammatory bowel disease, but also of diseases affecting other tissues, such as psychiatric conditions, can be explained by the identified QTLs. We provided candidate effector genes for multiple phenotypes. Finally, we provided the Colon Transcriptome Explorer web application. CONCLUSIONS: We provide a large characterization of gene expression and splicing across colon subsites. Our findings provide greater etiologic insight into complex traits and diseases influenced by transcriptomic changes in colon tissue.

Association of diabetes and diabetes treatment with incidence of breast cancer.

AIMS: The aim of this study was to evaluate the association of diabetes and diabetes treatment with risk of postmenopausal breast cancer. METHODS: Histologically confirmed incident cases of postmenopausal breast (N = 916) cancer were recruited from 23 Spanish public hospitals. Population-based controls (N = 1094) were randomly selected from primary care center lists within the catchment areas of the participant hospitals. ORs (95 % CI) were estimated using mixed-effects logistic regression models, using the recruitment center as a random effect term. Breast tumors were classified into hormone receptor positive (ER+ or PR+), HER2+ and triple negative (TN). RESULTS: Diabetes was not associated with the overall risk of breast cancer (OR 1.09; 95 % CI 0.82-1.45), and it was only linked to the risk of developing TN tumors: Among 91 women with TN tumors, 18.7 % were diabetic, while the corresponding figure among controls was 9.9 % (OR 2.25; 95 % CI 1.22-4.15). Regarding treatment, results showed that insulin use was more prevalent among diabetic cases (2.5 %) as compared to diabetic controls (0.7 %); OR 2.98; 95 % CI 1.26-7.01. They also showed that, among diabetics, the risk of developing HR+/HER2- tumors decreased with longer metformin use (ORper year 0.89; 95 % CI 0.81-0.99; based on 24 cases and 43 controls). CONCLUSION: This study reinforces the need to correctly classify breast cancers when studying their association with diabetes. Given the low survival rates in women diagnosed with TN breast tumors and the potential impact of diabetes control on breast cancer prevention, more studies are needed to better characterize this association.

Dietary flavonoids, lignans and colorectal cancer prognosis.

Flavonoids and lignans are polyphenol classes with anticarcinogenic activities against colorectal cancer (CRC). However, very limited epidemiological evidence exists on their effects on CRC prognosis. This study aimed to evaluate the association between flavonoid and lignan intakes with the risk of CRC recurrence and overall survival in CRC patients. The study followed incident histologically confirmed CRC cases in Barcelona (Spain). Validated dietary questionnaires and lifestyle information were collected at recruitment. An ad hoc food composition database on flavonoids and lignans was compiled by using data from the US Department of Agriculture and Phenol-Explorer databases. Adjusted hazards ratios (HR) and 95% confidence intervals (CIs) were estimated using multivariable Cox models. After 8.6 years of mean follow-up, 133 of 409 (32.5%) participants died and 77 of 319 (24.1%) had a CRC recurrence. Total flavonoids were associated neither with CRC recurrence (HR comparing extreme tertiles 1.13, 95% CI 0.64-2.02; P-trend 0.67) nor with overall survival (HR(T3vsT1) 1.06, 95% CI 0.69-1.65; P-trend 0.78) in the multivariable models. No associations were also observed with either total lignans or any flavonoid subclass intake. In conclusion, the results of the current study do not support a role of flavonoid and lignan intake in the CRC prognosis.

Association between habitual dietary flavonoid and lignan intake and colorectal cancer in a Spanish case-control study (the Bellvitge Colorectal Cancer Study).

BACKGROUND: Flavonoid-rich foods, such as fruits, vegetables, and tea, may have a protective effect upon colorectal cancer. However, current epidemiological evidence for a protective effect of flavonoid intake upon colorectal cancer is promising but not conclusive. OBJECTIVE: To examine the relation between dietary flavonoid and lignan intakes and the risk of colorectal cancer within a Spanish population. DESIGN: Data from the Bellvitge Colorectal Cancer Study, a case-control study (424 cases with incident colorectal cancer and 401 hospital-based controls), were used. A reproducible and validated food frequency questionnaire was administered in personal interviews. An ad hoc food composition database on flavonoids and lignans was compiled, mainly using data from the US Department of Agriculture and Phenol-Explorer databases. Adjusted odds ratios (ORs) and 95 % confidence intervals (CIs) were estimated using unconditional logistic regression models. RESULTS: An inverse association was found between intake of total flavonoids (OR, 0.59; 95 % CI, 0.35-0.99 for the highest vs. the lowest quartile; p for trend = 0.04), lignans (OR, 0.59; 95 % CI, 0.34-0.99; p for trend = 0.03), and some individual flavonoid subgroups (flavones, proanthocyanidins) and the risk of colorectal cancer. Separate analyses by cancer site showed similar results. CONCLUSIONS: Intake of total dietary flavonoids (particularly certain flavonoid subgroups) and lignans was inversely associated with colorectal cancer risk in a Spanish population.

Polymorphisms affecting micro-RNA regulation and associated with the risk of dietary-related cancers: a review from the literature and new evidence for a functional role of rs17281995 (CD86) and rs1051690 (INSR), previously associated with colorectal cancer.

In this review, we focus on the genetic variations (single nucleotide polymorphisms, SNPs) known to occur in microRNAs and in their binding sites and the susceptibility to cancers of the gastro-intestinal (GI) tract in humans. Since the sequence complementarity and the thermodynamics of binding play an essential role in the interaction of miRNA with its target mRNA, sequence variations in the miRNA-binding seed regions or in miRNA genes (either within pre-, pri-, or mature miRNA regions) should reinforce, weaken, or disrupt the miRNA-mRNA interaction and affect the expression of mRNA targets. Indirect evidences supporting these hypotheses are reported in the literature, essentially coming from case-control association studies. Several studies have been published on the association between miR-SNPs or SNPs within their binding sites and the risk of oesophageal, gastric, or colorectal cancer. Unfortunately, functional studies are lacking. Besides reviewing the available literature, we present here for the first time two SNPs (rs17281995 in CD86 and rs1051690 in INSR) previously associated with the risk of CRC in a Czech population are also associated with the risk in a Spanish population. Moreover, we show for the first time that both these alleles regulate differentially the amount of a reporter gene (luciferase) in an in vitro assay on HeLa cells. These findings suggest that both these SNPs may have a functional role in regulating the expression of CD-86 and INSR proteins acting at the level of the 3'UTR. More functional studies are needed in order to better understand the role of polymorphic regulatory sequences at the 3'UTR of genes.

Interleukin-4 and interleukin-4 receptor polymorphisms and colorectal cancer risk.

Interleukin-4 (IL-4) and interleukin-4 receptor (IL-4R) modulate inflammation and are associated with the colorectal adenoma-carcinoma progression and the metastatic capacity. IL-4 also causes a dose-dependent reduction of proliferation in colorectal cancer cells. The aim of the study was to evaluate whether genetic variants within IL4 and IL4R could affect the individual risk to develop colorectal cancer. We genotyped all the polymorphisms coding for an aminoacidic change in IL4R and we used a haplotype-tagging SNP approach for IL4. We carried out a case-control association study by genotyping, with the 5' nuclease assay, two common SNPs within IL4 (-588C>T, Ex1-168G>A) and five SNPs within IL4R (I75V, C431R, S436L, S503P, Q576R) in 377 cases of colorectal cancer and 326 controls from Spain. No statistically significant association between the SNPs investigated and colorectal cancer risk was found, as main effects. When the sub-analyses were carried out, the homozygotes for IL4 -588C>T or for Ex1-168G>A showed an increased risk for colon cancer only, with the odds ratios of 4 (95% CI 0.97-16.6; P-interaction=0.016 and 4.66 (95% CI 1.16-18.77; P-interaction=0.023), respectively. Moreover, women showed a significant increased risk associated to the IL4 rare alleles and this was clearly greater than that in men (for Ex1-168G>A: OR=1.96; 95% CI=1.11-3.47; P-interaction=0.006). However, when sub-groups are analysed, the findings should be taken with caution for the weakening of the statistical power.

Tumor thymidylate synthase 1494del6 genotype as a prognostic factor in colorectal cancer patients receiving fluorouracil-based adjuvant treatment.

PURPOSE: The purpose of this study was to analyze the value of germline and tumor thymidylate synthase (TS) genotyping as a prognostic marker in a series of colorectal cancer patients receiving adjuvant fluorouracil (FU) -based treatment. PATIENTS AND METHODS: One hundred twenty-nine colorectal cancer patients homogeneously treated with FU plus levamisole or leucovorin in the adjuvant setting were included. TS enhancer region, 3R G > C single nucleotide polymorphism (SNP), and TS 1494del6 polymorphisms were assessed in both fresh-frozen normal mucosa and tumor. Mutational analyses of TS and allelic imbalances were studied in all primary tumors and in 18 additional metachronic metastases. TS protein immunostaining was assessed in an expanded series of 214 tumors. Multivariate Cox models were adjusted for stage, differentiation, and location. RESULTS: Tumor genotyping (frequency of allelic loss, 26%) showed that the 3R/3R genotype was associated with a better outcome (hazard ratio [HR] = 0.38; 95% CI, 0.16 to 0.93; P = .020 for the recessive model). 3R G > C SNP genotyping did not add prognostic information. Tumor TS 1494del6 allele (frequency of allelic loss, 36%) was protective (for each allele with the deletion, based on an additive model, HR = 0.42; 95% CI, 0.22 to 0.82; P = .0034). Both polymorphisms were in strong linkage disequilibrium (D' = 0.71, P < .001), and the 3R/-6 base pair (bp) haplotype showed a significant overall survival benefit compared with the most prevalent haplotype 2R/+6bp (HR = 0.42; 95% CI, 0.20 to 0.85; P = .017). No TS point mutation was detected in primary tumors or metastases. TS protein immunostaining was not associated with survival or any of the genotypes analyzed. CONCLUSION: Tumor TS 1494del6 genotype may be a prognostic factor in FU-based adjuvant treatment of colorectal cancer patients.

A comprehensive analysis of phase I and phase II metabolism gene polymorphisms and risk of colorectal cancer.

OBJECTIVES: Sporadic colorectal cancer (CRC) is considered a multifactorial disease where multiple exposures interact with the individual genetic background resulting in risk modulation. We performed an association study aimed to investigate the role of single nucleotide polymorphisms (SNPs) within genes of phase I (CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2D6, CYP2E1, CYP2C9, CYP2C19, CYP3A4, ADH2, EPHX1) and phase II of the xenobiotic metabolism (ALDH2, COMT, GSTA2, GSTA4, GSTM1, GSTM3, GSTP1, GSTT2, MTHFR, NAT1, NAT2, NQO1, MnSOD2, SULT1A1, TPMT). METHODS: We genotyped 377 cases and 326 controls, by use of an oligonucleotide micro-array and the arrayed primer extension technique (APEX). RESULTS: N-acetyl-transferase 1 'rapid' phenotype and CYP1A2 -164C>A carriers were associated with increased risk of CRC, confirming data reported in previous studies. Interestingly, homozygotes for allele 48G within CYP1B1, a variant with an increased activity towards several substrates including sex hormones, were at increased risk (OR=2.81, 95% CI 1.32-5.99). Moreover, CYP1A1 SNPs T461N and -1738A>C were associated with a reduced risk of cancer (OR=0.52; 95% CI 0.31-0.88 and OR=0.69, 95% CI 0.50-0.94 for carriers, respectively). CONCLUSIONS: The present data suggest a role for CYP1B1 and CYP1A1 as new candidate genes in the etiology of CRC and confirm the carcinogenic role of aromatic amines metabolism for colorectum.

A TP53 polymorphism is associated with increased risk of colorectal cancer and with reduced levels of TP53 mRNA.

We undertook a case-control study to examine the possible associations of the TP53 variants Arg > Pro at codon 72 and p53PIN3, a 16 bp insertion/duplication in intron 3, with the risk of colorectal cancer (CRC). The p53PIN3 A2 allele (16 bp duplication) was associated with an increased risk (OR 1.55, 95% CI 1.10-2.18, P = 0.012), of the same order of magnitude as that observed in previous studies for other types of cancer. The Pro72 allele was weakly associated with CRC (OR = 1.34, 95% CI 0.98-1.84, P = 0.066). The possible functional role of p53PIN3 was investigated by examining the TP53 mRNA transcripts in 15 lymphoblastoid cell lines with different genotypes. The possibility that the insertion/deletion could lead to alternatively spliced mRNAs was excluded. However, we found reduced levels of TP53 mRNA associated with the A2 allele. In conclusion, the epidemiological study suggests a role for p53PIN3 in tumorigenesis, supported by the in vitro characterization of this variant.

Common genetic evolutionary pathways in familial adenomatous polyposis tumors.

Cancer cells progress through the accumulation of genetic alterations. Familial adenomatous polyposis (FAP) tumors provide an excellent model to unravel the molecular steps underlying malignant transformation. Global genomic damage was assessed in 56 adenomas and 3 carcinomas from six FAP patients and compared with that of sporadic adenomas and carcinomas. Evolutive trees were traced after application of maximum likelihood clustering and split decomposition methods to the analysis of comprehensive genetic profiles generated by diverse molecular approaches: arbitrarily primed PCR, comparative genomic hybridization, and flow cytometry. Overall, genomic damage as assessed by arbitrarily primed PCR was lower in familial adenomas than in sporadic adenomas and carcinomas. Comparative genomic hybridization data also show a low number of alterations in the majority of FAP adenomas. Tumors of the same patient were likely to share specific genetic alterations and may be grouped into one or two clusters. Putative common pathways were also identified, which included tumors of up to three different patients. According to our data, FAP tumors accumulate specific genetic alterations and in a preferred order that is characteristic of each individual. Moreover, the particular genetic background and environmental conditions of a FAP patient restrain the molecular evolution portrait of synchronous tumors.

Association of common polymorphisms in inflammatory genes interleukin (IL)6, IL8, tumor necrosis factor alpha, NFKB1, and peroxisome proliferator-activated receptor gamma with colorectal cancer.

Animal models and epidemiological observations suggest that a continuous inflammatory condition predisposes to colorectal cancer (CRC), but the roles of different elements participating in inflammatory responses have been little investigated in relation to CRC. We have studied the association between single nucleotide polymorphisms in the interleukin (IL)-6 (-174 G>C), IL8 (-251T>A), tumor necrosis factor alpha (-308G>A), and PPARG (Pro12Ala) genes and the risk of CRC in a group of 377 cases and 326 controls from Barcelona, Spain. These genes are known to be important for inflammation of the colorectum and common allelic variants have been shown to have a biological effect. The PPARG Ala12 and IL8-251A genotypes are associated with reduced risk of disease (0.56, 95% CI, 0.37-0.85, P = 0.0056, and 0.70, 95% CI, 0.50-0.99, P = 0.043, respectively), whereas the IL6-174C genotype is associated with increased risk (1.53, 95% CI, 1.12-2.09, P = 0.0073). We also studied a single nucleotide polymorphism in intron 11 of the NFKB1 gene (rs1020759), which probably lacks any functional role, and found no significant association with the disease. This is the first report that IL6, IL8, and PPARG genes are important in relation to inflammation-related risk of sporadic CRC.