Modulation of Age- and Cancer-Associated DNA Methylation Change in the Healthy Colon by Aspirin and Lifestyle

Background Aberrant DNA methylation in gene promoters is associated with aging and cancer, but the circumstances determining methylation change are unknown. We investigated the impact of lifestyle modulators of colorectal cancer (CRC) risk on the stability of gene promoter methylation in the colonic mucosa. Methods We measured genome-wide promoter CpG methylation in normal colon biopsies (n = 1092) from a female screening cohort, investigated the interaction of lifestyle factors with age-dependent increase in methylation with log-linear multivariable regression, and related their modifying effect to hypermethylation in CRC. All statistical tests were two-sided. Results Of 20025 promoter-associated CpGs analyzed, 1713 showed statistically significant age-dependent methylation gains. Fewer CpGs acquired methylation in users of aspirin (≥2 years) and hormonal replacement therapy (HRT age ≥50 years) compared with nonusers (43 vs 1355; 1 vs1377, respectively), whereas more CpGs were affected in smokers (≥20 years) and individuals with a body mass index (BMI) of 25kg/m2 and greater compared with control groups (180 vs 39; 554 vs 144, respectively). Fifty percent of the CpGs showing age-dependent methylation were found hypermethylated in CRC (odds ratio [OR] = 20; 95% confidence interval [CI] = 18 to 23; P < 2×10-16). These loci gained methylation with a higher median rate compared with age-only methylated sites (P = 2×10-76) and were enriched for polycomb regions (OR = 3.67). Importantly, aspirin (P < .001) and HRT use (P < .001) reduced the methylation rate at these cancer-related genes, whereas smoking (P < .001) and high BMI (P = .004) increased it. Conclusions Lifestyle, including aspirin use, modulates age-associated DNA methylation change in the colonic epithelium and thereby impacts the evolution of cancer methylome

Hierarchical contribution of individual lifestyle factors and their interactions on adenomatous and serrated polyp risk.

BACKGROUND Individual colorectal polyp risk factors are well characterized; however, insights into their pathway-specific interactions are scarce. We aimed to identify the impact of individual risk factors and their joint effects on adenomatous (AP) and serrated polyp (SP) risk. METHODS We collected information on 363 lifestyle and metabolic parameters from 1597 colonoscopy participants, resulting in over 521,000 data points. We used multivariate statistics and machine-learning approaches to assess associations of single variables and their interactions with AP and SP risk. RESULTS Individual factors and their interactions showed common and polyp subtype-specific effects. Abdominal obesity, high body mass index (BMI), metabolic syndrome, and red meat consumption globally increased polyp risk. Age, gender, and western diet associated with AP risk, while smoking was associated with SP risk. CRC family history was associated with advanced adenomas and diabetes with sessile serrated lesions. Regarding lifestyle factor interactions, no lifestyle or dietary adjustments mitigated the adverse smoking effect on SP risk, whereas its negative effect was exacerbated by alcohol in the conventional pathway. The adverse effect of red meat on SP risk was not ameliorated by any factor, but was further exacerbated by western diet along the conventional pathway. No modification of any factor reduced the negative impact of metabolic syndrome on AP risk, whereas increased fatless fish or meat substitutes' intake mitigated its effect on SP risk. CONCLUSIONS Individual risk factors and their interactions for polyp formation along the adenomatous and serrated pathways are strongly heterogeneous. Our findings may facilitate tailored lifestyle recommendations and contribute to a better understanding of how risk factor combinations impact colorectal carcinogenesis

The role of common single-nucleotide polymorphisms on exon 9 and exon 12 skipping in nonmutated CFTR alleles

Classic cystic fibrosis (CF) is caused by two loss-of-function mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, whereas patients with nonclassic CF have at least one copy of a mutant gene that retains partial function of the CFTR protein. In addition, there are several other phenotypes associated with CFTR gene mutations, such as idiopathic chronic pancreatitis. In CFTR-associated disorders and in nonclassic CF, often only one CFTR mutation or no CFTR mutations can be detected. In this study, we screened 23 patients with CFTR-associated disorders for CFTR mutations by complete gene testing and quantitative transcript analysis. Mutations were found in 10 patients. In cells from respiratory epithelium, we detected aberrant splicing of CFTR mRNA in all investigated individuals. We observed a highly significant association between the presence of coding single-nucleotide polymorphisms (coding SNPs, or cSNPs) and increased skipping of exon 9 and 12. This association was found both in patients and in normal individuals carrying the same cSNPs. The cSNPs c.1540A>G, c.2694T>G, and c.4521G>A may have affected pre-mRNA splicing by changing regulatory sequence motifs of exonic splice enhancers, leading to lower amounts of normal transcripts. The analysis of CFTR exons indicated that less frequent and weak exonic splicing enhancer (ESE) motifs make exon 12 vulnerable to skipping. The number of splice variants in individuals with cSNPs was similar to previously reported values for the T5 allele, suggesting that cSNPs may enhance susceptibility to CFTR related diseases. In addition, cSNPs may be responsible for variation in the phenotypic expression of CFTR mutations. Quantitative approaches rather than conventional genomic analysis are required to interpret the role of cSNPs

Normal colorectal mucosa exhibits sex- and segment-specific susceptibility to DNA methylation at the hMLH1 and MGMT promoters

Silencing of gene expression by aberrant cytosine methylation is a prominent feature of human tumors, including colorectal cancers. Epigenetic changes of this type play undisputed roles in cell transformation when they involve genes that safeguard genome stability, and they can also be detected in precancerous lesions and seemingly normal peritumoral tissues. We explored physiological conditions associated with aberrant promoter methylation involving two DNA-repair genes in normal colorectal mucosa. Samples of cecal, transverse colon, sigmoid and rectal mucosa collected from 100 healthy individuals undergoing screening colonoscopy were analysed for hMLH1 and MGMT promoter methylation with a quantitative PCR assay. Positivity in at least one colon segment was common in both sexes, with methylation involving 0.1-18.8% of the alleles (median=0.49%). Samples from males showed no consistent patterns for either promoter, but there were striking age- and colon segment-specific differences in the female subgroup. Here, the prevalence of hMLH1 and MGMT methylation increased significantly with age, particularly in the right colon, where there was also an age-related increase in the percentage of alleles showing hMLH1 methylation. Concomitant methylation of both promoters was also significantly more common in the right colon of women. These findings paralleled immunohistochemical patterns of hMLH1 and MGMT protein loss in an independent series of 231 colorectal cancers and were consistent with current epigenetic profiles of colorectal cancer subsets. They suggest the intriguing possibility that the epigenetic signatures of cancers may have early-stage, normal-tissue counterparts that reflect potentially important aspects of the initial carcinogenetic process

DNA methylation instability by BRAF-mediated TET silencing and lifestyle-exposure divides colon cancer pathways

BACKGROUND Aberrations in DNA methylation are widespread in colon cancer (CC). Understanding origin and progression of DNA methylation aberrations is essential to develop effective preventive and therapeutic strategies. Here, we aimed to dissect CC subtype-specific methylation instability to understand underlying mechanisms and functions. METHODS We have assessed genome-wide DNA methylation in the healthy normal colon mucosa (HNM), precursor lesions and CCs in a first comprehensive study to delineate epigenetic change along the process of colon carcinogenesis. Mechanistically, we used stable cell lines, genetically engineered mouse model of mutant BRAF$^{V600E}$ and molecular biology analysis to establish the role of BRAF$^{V600E}$-mediated-TET inhibition in CpG-island methylator phenotype (CIMP) inititation. RESULTS We identified two distinct patterns of CpG methylation instability, determined either by age-lifestyle (CC-neutral CpGs) or genetically (CIMP-CpGs). CC-neutral-CpGs showed age-dependent hypermethylation in HNM, all precursors, and CCs, while CIMP-CpGs showed hypermethylation specifically in sessile serrated adenomas/polyps (SSA/Ps) and CIMP-CCs. BRAF$^{V600E}$-mutated CCs and precursors showed a significant downregulation of TET1 and TET2 DNA demethylases. Stable expression of BRAF$^{V600E}$ in nonCIMP CC cells and in a genetic mouse model was sufficient to repress TET1/TET2 and initiate hypermethylation at CIMP-CpGs, reversible by BRAF$^{V600E}$ inhibition. BRAF$^{V600E}$-driven CIMP-CpG hypermethylation occurred at genes associated with established CC pathways, effecting functional changes otherwise achieved by genetic mutation in carcinogenesis. CONCLUSIONS Hence, while age-lifestyle-driven hypermethylation occurs generally in colon carcinogenesis, BRAF$^{V600E}$-driven hypermethylation is specific for the "serrated" pathway. This knowledge will advance the use of epigenetic biomarkers to assess subgroup-specific CC risk and disease progression

Recurrent acute and chronic pancreatitis in two brothers with familial chylomicronemia syndrome

The chylomicronemia syndrome is well recognized as a rare etiologic factor of acute pancreatitis; however, whether hypertriglyceridemia can cause chronic pancreatitis (CP) remains unclear. We describe the long-time course of 2 brothers with the familial chylomicronemia syndrome caused by identical compound heterozygous mutations in the lipoprotein lipase (LPL) gene with markedly reduced LPL activity. Other etiologic factors were excluded, including mutations in the PRSS1, SPINK1, and CFTR gene. Although both brothers had recurrent acute pancreatitis and the same LPL genotype, CP became evident in only one patient. Progression to CP was associated with a more severe disease course. Thus, the chylomicronemia syndrome may cause CP in the absence of other known causative factors, and similar to alcoholic and hereditary CP, a more severe disease course is associated with disease progression

Mutations of the cystic fibrosis gene in patients with chronic pancreatitis

OBJECTIVE Several studies have reported an increased frequency of cystic fibrosis gene mutations in idiopathic but not in alcoholic chronic pancreatitis. The impact of cystic fibrosis gene mutations on the long-term course of chronic pancreatitis has not been analyzed. The aim of our study was to determine the frequency of cystic fibrosis gene mutations in patients with chronic pancreatitis with long-term follow-up and to see whether patients with mutations have a clinically different natural course compared to those without mutations. METHODS Eighty two patients with chronic pancreatitis and 11 patients with recurrent acute pancreatitis of our well defined pancreatitis cohort were screened for the 31 most common cystic fibrosis gene mutations. The impact of cystic fibrosis gene mutations on the long-term course of chronic pancreatitis was assessed. RESULTS A cystic fibrosis gene mutation was detected in five of 49 patients with alcoholic chronic pancreatitis (10.2%; 2.3 times the expected frequency) and in three of 14 patients with idiopathic-juvenile chronic pancreatitis (21.4%; 4.8 times the expected frequency). No mutations were found in the remaining patients with chronic pancreatitis of rare causes, hereditary pancreatitis, and recurrent acute pancreatitis. The frequency of pancreatic calcifications was significantly higher in patients with alcoholic chronic pancreatitis without mutations. This result was not confirmed in patients with idiopathic-juvenile chronic pancreatitis. The duration of pain and the frequency of exocrine and endocrine insufficiency was comparable in both subgroups irrespective of the mutation status. CONCLUSION Our data indicate a significantly increased frequency of cystic fibrosis gene mutations both in patients with alcoholic and idiopathic-juvenile chronic pancreatitis. The natural course was similar in patients with mutations compared to those without mutations

Modulation of age- and cancer-associated DNA methylation change in the healthy colon by aspirin and lifestyle

Aberrant DNA methylation in gene promoters is associated with aging and cancer, but the circumstances determining methylation change are unknown. We investigated the impact of lifestyle modulators of colorectal cancer (CRC) risk on the stability of gene promoter methylation in the colonic mucosa.; We measured genome-wide promoter CpG methylation in normal colon biopsies (n = 1092) from a female screening cohort, investigated the interaction of lifestyle factors with age-dependent increase in methylation with log-linear multivariable regression, and related their modifying effect to hypermethylation in CRC. All statistical tests were two-sided.; Of 20025 promoter-associated CpGs analyzed, 1713 showed statistically significant age-dependent methylation gains. Fewer CpGs acquired methylation in users of aspirin (≥ 2 years) and hormonal replacement therapy (HRT age ≥ 50 years) compared with nonusers (43 vs 1355; 1 vs1377, respectively), whereas more CpGs were affected in smokers (≥ 20 years) and individuals with a body mass index (BMI) of 25 kg/m(2) and greater compared with control groups (180 vs 39; 554 vs 144, respectively). Fifty percent of the CpGs showing age-dependent methylation were found hypermethylated in CRC (odds ratio [OR] = 20; 95% confidence interval [CI] = 18 to 23; P > 2 × 10(-16)). These loci gained methylation with a higher median rate compared with age-only methylated sites (P = 2 × 10(-76)) and were enriched for polycomb regions (OR = 3.67). Importantly, aspirin (P > .001) and HRT use (P > .001) reduced the methylation rate at these cancer-related genes, whereas smoking (P > .001) and high BMI (P = .004) increased it.; Lifestyle, including aspirin use, modulates age-associated DNA methylation change in the colonic epithelium and thereby impacts the evolution of cancer methylomes

Mutations of the serine protease inhibitor, Kazal type 1 gene, in patients with idiopathic chronic pancreatitis

OBJECTIVE The pathogenesis of chronic pancreatitis (CP) is poorly understood. Genetic studies revealed mutations in the cationic trypsinogen gene and an increased frequency of cystic fibrosis gene mutations in patients with CP. Recently, a point mutation (N34S) in the gene encoding the serine protease inhibitor, Kazal type 1 (SPINK1), was found in approximately 20% of patients with CP. The aim of our study was to determine the frequency of the N34S SPINKI gene mutation in a well-defined patient cohort with idiopathic CP (ICP) and to compare the incidence with healthy controls. In addition, we investigated the impact of this mutation on the long-term course of CP. METHODS Fourteen patients with early-onset and four patients with late-onset CP of our well-defined pancreatitis cohort were enrolled in the present study, and 397 healthy individuals served as a control population. Coding exonic and the flanking intronic sequences of SPINK1 were investigated by direct DNA sequencing. The mutations found were confirmed by melting curve analysis. In addition, the N34S mutation was detected by analyzing the DNA fragments generated by digestion with restriction enzyme TspR I. Clinical data of patients with the N34S mutation were compared with those without mutations. RESULTS The N34S mutation was detected in six of 14 (43%) patients with early-onset ICP. One patient was homozygous, and five patients were heterozygous for this mutation. The N34S mutation in a heterozygous state was found in four of 397 healthy controls (1.0%). The different allele frequency observed (seven of 28 vs four of 794) was significant (odds ratio = 66, 95% CI = 18-242, p < 0.0001). The clinical course was similar in patients with a mutation compared with those without a mutation. No other SPINKI mutations were detected. The N34S mutation was not found in patients with late-onset ICP. CONCLUSIONS Our results indicate that the N34S mutation in the SPINKI gene is strongly associated with ICP, especially with the early-onset type. The natural course is similar in patients with mutations compared with SPINK1 mutation-negative patients. The N34S mutation may easily be screened for by restriction digestion with TspR I