Sensitive Detection of Colorectal Cancer in Peripheral Blood by Septin 9 DNA Methylation Assay

Background

Colorectal cancer (CRC) is the second leading cause of cancer deaths despite the fact that detection of this cancer in early stages results in over 90% survival rate. Currently less than 45% of at-risk individuals in the US are screened regularly, exposing a need for better screening tests. We performed two case-control studies to validate a blood-based test that identifies methylated DNA in plasma from all stages of CRC.

Methodology/Principal Findings

Using a PCR assay for analysis of Septin 9 (SEPT9) hypermethylation in DNA extracted from plasma, clinical performance was optimized on 354 samples (252 CRC, 102 controls) and validated in a blinded, independent study of 309 samples (126 CRC, 183 controls). 168 polyps and 411 additional disease controls were also evaluated. Based on the training study SEPT9-based classification detected 120/252 CRCs (48%) and 7/102 controls (7%). In the test study 73/126 CRCs (58%) and 18/183 control samples (10%) were positive for SEPT9 validating the training set results. Inclusion of an additional measurement replicate increased the sensitivity of the assay in the testing set to 72% (90/125 CRCs detected) while maintaining 90% specificity (19/183 for controls). Positive rates for plasmas from the other cancers (11/96) and non-cancerous conditions (41/315) were low. The rate of polyp detection (>1 cm) was ∼20%.

Conclusions/Significance

Analysis of SEPT9 DNA methylation in plasma represents a straightforward, minimally invasive method to detect all stages of CRC with potential to satisfy unmet needs for increased compliance in the screening population. Further clinical testing is warranted.     

IGFBP3 Methylation Is a Novel Diagnostic and Predictive Biomarker in Colorectal Cancer

Background and Aim

Aberrant hypermethylation of cancer-related genes has emerged as a promising strategy for the development of diagnostic, prognostic and predictive biomarkers in human cancer, including colorectal cancer (CRC). The aim of this study was to perform a systematic and comprehensive analysis of a panel of CRC-specific genes as potential diagnostic, prognostic and predictive biomarkers in a large, population-based CRC cohort.

Patients and Methods

Methylation status of the SEPT9, TWIST1, IGFBP3, GAS7, ALX4 and miR137 genes was studied by quantitative bisulfite pyrosequencing in a population-based cohort of 425 CRC patients.

Results

Methylation levels of all genes analyzed were significantly higher in tumor tissues compared to normal mucosa (p<0.0001); however, cancer-associated hypermethylation was most frequently observed for miR137 (86.7%) and IGFBP3 (83%) in CRC patients. Methylation analysis using the combination of these two genes demonstrated greatest accuracy for the identification of colonic tumors (sensitivity 95.5%; specificity 90.5%). Low levels of IGFBP3 promoter methylation emerged as an independent risk factor for predicting poor disease free survival in stage II and III CRC patients (HR = 0.49, 95% CI: 0.28–0.85, p = 0.01). Our results also suggest that stage II & III CRC patients with high levels of IGFBP3 methylation do not benefit from adjuvant 5FU-based chemotherapy.

Conclusion

By analyzing a large, population-based CRC cohort, we demonstrate the potential clinical significance of miR137 and IGFBP3 hypermethylation as promising diagnostic biomarkers in CRC. Our data also revealed that IGFBP3 hypermethylation may serve as an independent prognostic and predictive biomarker in stage II and III CRC patients.     

Genome-Scale Discovery of DNA-Methylation Biomarkers for Blood-Based Detection of Colorectal Cancer

Background

There is an increasing demand for accurate biomarkers for early non-invasive colorectal cancer detection. We employed a genome-scale marker discovery method to identify and verify candidate DNA methylation biomarkers for blood-based detection of colorectal cancer.

Methodology/Principal Findings

We used DNA methylation data from 711 colorectal tumors, 53 matched adjacent-normal colonic tissue samples, 286 healthy blood samples and 4,201 tumor samples of 15 different cancer types. DNA methylation data were generated by the Illumina Infinium HumanMethylation27 and the HumanMethylation450 platforms, which determine the methylation status of 27,578 and 482,421 CpG sites respectively. We first performed a multistep marker selection to identify candidate markers with high methylation across all colorectal tumors while harboring low methylation in healthy samples and other cancer types. We then used pre-therapeutic plasma and serum samples from 107 colorectal cancer patients and 98 controls without colorectal cancer, confirmed by colonoscopy, to verify candidate markers. We selected two markers for further evaluation: methylated THBD (THBD-M) and methylated C9orf50 (C9orf50-M). When tested on clinical plasma and serum samples these markers outperformed carcinoembryonic antigen (CEA) serum measurement and resulted in a high sensitive and specific test performance for early colorectal cancer detection.

Conclusions/Significance

Our systematic marker discovery and verification study for blood-based DNA methylation markers resulted in two novel colorectal cancer biomarkers, THBD-M and C9orf50-M. THBD-M in particular showed promising performance in clinical samples, justifying its further optimization and clinical testing.     

Somatic Mutations,Allele Loss,and DNA Methylation of the Cub and Sushi Multiple Domains 1 (CSMD1) Gene Reveals Association with Early Age of Diagnosis in Colorectal Cancer Patients

Background

The Cub and Sushi Multiple Domains 1 (CSMD1) gene, located on the short arm of chromosome 8, codes for a type I transmembrane protein whose function is currently unknown. CSMD1 expression is frequently lost in many epithelial cancers. Our goal was to characterize the relationships between CSMD1 somatic mutations, allele imbalance, DNA methylation, and the clinical characteristics in colorectal cancer patients.

Methods

We sequenced the CSMD1 coding regions in 54 colorectal tumors using the 454FLX pyrosequencing platform to interrogate 72 amplicons covering the entire coding sequence. We used heterozygous SNP allele ratios at multiple CSMD1 loci to determine allelic balance and infer loss of heterozygosity. Finally, we performed methylation-specific PCR on 76 colorectal tumors to determine DNA methylation status for CSMD1 and known methylation targets ALX4, RUNX3, NEUROG1, and CDKN2A.

Results

Using 454FLX sequencing and confirming with Sanger sequencing, 16 CSMD1 somatic mutations were identified in 6 of the 54 colorectal tumors (11%). The nonsynonymous to synonymous mutation ratio of the 16 somatic mutations was 15∶1, a ratio significantly higher than the expected 2∶1 ratio (p = 0.014). This ratio indicates a presence of positive selection for mutations in the CSMD1 protein sequence. CSMD1 allelic imbalance was present in 19 of 37 informative cases (56%). Patients with allelic imbalance and CSMD1 mutations were significantly younger (average age, 41 years) than those without somatic mutations (average age, 68 years). The majority of tumors were methylated at one or more CpG loci within the CSMD1 coding sequence, and CSMD1 methylation significantly correlated with two known methylation targets ALX4 and RUNX3. C:G>T:A substitutions were significantly overrepresented (47%), suggesting extensive cytosine methylation predisposing to somatic mutations.

Conclusions

Deep amplicon sequencing and methylation-specific PCR reveal that CSMD1 alterations can correlate with earlier clinical presentation in colorectal tumors, thus further implicating CSMD1 as a tumor suppressor gene.     

Detection of Methylated Septin 9 in Tissue and Plasma of Colorectal Patients with Neoplasia and the Relationship to the Amount of Circulating Cell-Free DNA

Background

Determination of methylated Septin 9 (mSEPT9) in plasma has been shown to be a sensitive and specific biomarker for colorectal cancer (CRC). However, the relationship between methylated DNA in plasma and colon tissue of the same subjects has not been reported.

Methods

Plasma and matching biopsy samples were collected from 24 patients with no evidence of disease (NED), 26 patients with adenoma and 34 patients with CRC. Following bisulfite conversion of DNA a commercial RT-PCR assay was used to determine the total amount of DNA in each sample and the fraction of mSEPT9 DNA. The Septin-9 protein was assessed using immunohistochemistry.

Results

The percent of methylated reference (PMR) values for SEPT9 above a PMR threshold of 1% were detected in 4.2% (1/24) of NED, 100% (26/26) of adenoma and 97.1% (33/34) of CRC tissues. PMR differences between NED vs. adenoma and NED vs. CRC comparisons were significant (p<0.001). In matching plasma samples using a PMR cut-off level of 0.01%, SEPT9 methylation was 8.3% (2/24) of NED, 30.8% (8/26) of adenoma and 88.2% (30/34) of CRC. Significant PMR differences were observed between NED vs. CRC (p<0.01) and adenoma vs. CRC (p<0.01). Significant differences (p<0.01) were found in the amount of cfDNA (circulating cell-free DNA) between NED and CRC, and a modest correlation was observed between mSEPT9 concentration and cfDNA of cancer (R² = 0.48). The level of Septin-9 protein in tissues was inversely correlated to mSEPT9 levels with abundant expression in normals, and diminished expression in adenomas and tumors.

Conclusions

Methylated SEPT9 was detected in all tissue samples. In plasma samples, elevated mSEPT9 values were detected in CRC, but not in adenomas. Tissue levels of mSEPT9 alone are not sufficient to predict mSEPT9 levels in plasma. Additional parameters including the amount of cfDNA in plasma appear to also play a role.     

KRAS Mutations in Primary Colorectal Cancer Tumors and Related Metastases: A Potential Role in Prediction of Lung Metastasis

Background

KRAS mutations in colorectal cancer primary tumors predict resistance to anti-Epidermal Growth Factor Receptor (EGFR) monoclonal antibody therapy in patients with metastatic colorectal cancer, and thus represent a true indicator of EGFR pathway activation status.

Methodology/Principal Findings

KRAS mutations were retrospectively studied using polymerase chain reactions and subsequent sequencing of codons 12 and 13 (exon 2) in 110 patients with metastatic colorectal tumors. These studies were performed using tissue samples from both the primary tumor and their related metastases (93 liver, 84%; 17 lung, 16%). All patients received adjuvant 5-Fluorouracil-based polychemotherapy after resection of metastases. None received anti-EGFR therapy. Mutations in KRAS were observed in 37 (34%) of primary tumors and in 40 (36%) of related metastases, yielding a 94% level of concordance (kappa index 0.86). Patients with primary tumors possessing KRAS mutations had a shorter disease-free survival period after metastasis resection (12.0 vs 18.0 months; P = 0.035) than those who did not. A higher percentage of KRAS mutations was detected in primary tumors of patiens with lung metastases than in patients with liver metastases (59% vs 32%; p = 0.054). To further evaluate this finding we analyzed 120 additional patients with unresectable metastatic colorectal cancer who previously had their primary tumors evaluated for KRAS mutational status for clinical purposes. Separately, the analysis of these 120 patients showed a tendency towards a higher degree of KRAS mutations in primary tumors of patients with lung metastases, although it did not reach statistical significance. Taken together the group of 230 patients showed that KRAS was mutated significantly more often in the primary tumors of patients with lung metastases (57% vs 35%; P = 0.006).

Conclusions/Significance

Our results suggest a role for KRAS mutations in the propensity of primary colorectal tumors to metastasize to the lung.     

Pre-Diagnostic Leukocyte Genomic DNA Methylation and the Risk of Colorectal Cancer in Women

Background

Abnormal one-carbon metabolism may lead to general genomic (global) hypomethylation, which may predispose an individual to the development of colorectal neoplasia.

Methods

We evaluated the association between pre-diagnostic leukocyte genomic DNA methylation level and the risk of colorectal cancer in a nested case-control study of 358 colorectal cancer cases and 661 matched controls within the all-female cohort of the Nurses’ Health Study (NHS). Among control subjects, we further examined major plasma components in the one-carbon metabolism pathway in relation to genomic DNA methylation level. Liquid chromatography/tandem mass spectrometry was used to examine leukocyte genomic DNA methylation level. We calculated odds ratios (ORs) and 95% confidence intervals (95% CIs) using logistic regression.

Results

Overall genomic DNA methylation level was not associated with the risk of colorectal cancer (p for trend, 0.45). Compared with women in the lowest quintile of methylation, the multivariate OR of colorectal cancer risk was 1.32 (95% CI, 0.82–2.13) for those in the highest quintile. We did not find significant associations between major plasma components of one-carbon metabolism or risk factors for colorectal cancer and genomic DNA methylation level (all p for trend >0.05). Also, neither one-carbon metabolism-related plasma components nor well-known risk factors for colorectal cancer modified the association between genomic DNA methylation level and the risk of colorectal cancer (all p for interaction >0.05).

Conclusions

We found no evidence that hypomethylation of leukocyte genomic DNA increases risk of colorectal cancer among women. Additional studies are needed to investigate the association between pre-diagnostic genomic DNA methylation level and colorectal cancer risk among diverse populations.     

Aberrant Gene Promoter Methylation Associated with Sporadic Multiple Colorectal Cancer

Background

Colorectal cancer (CRC) multiplicity has been mainly related to polyposis and non-polyposis hereditary syndromes. In sporadic CRC, aberrant gene promoter methylation has been shown to play a key role in carcinogenesis, although little is known about its involvement in multiplicity. To assess the effect of methylation in tumor multiplicity in sporadic CRC, hypermethylation of key tumor suppressor genes was evaluated in patients with both multiple and solitary tumors, as a proof-of-concept of an underlying epigenetic defect.

Methodology/Principal Findings

We examined a total of 47 synchronous/metachronous primary CRC from 41 patients, and 41 gender, age (5-year intervals) and tumor location-paired patients with solitary tumors. Exclusion criteria were polyposis syndromes, Lynch syndrome and inflammatory bowel disease. DNA methylation at the promoter region of the MGMT, CDKN2A, SFRP1, TMEFF2, HS3ST2 (3OST2), RASSF1A and GATA4 genes was evaluated by quantitative methylation specific PCR in both tumor and corresponding normal appearing colorectal mucosa samples. Overall, patients with multiple lesions exhibited a higher degree of methylation in tumor samples than those with solitary tumors regarding all evaluated genes. After adjusting for age and gender, binomial logistic regression analysis identified methylation of MGMT2 (OR, 1.48; 95% CI, 1.10 to 1.97; p = 0.008) and RASSF1A (OR, 2.04; 95% CI, 1.01 to 4.13; p = 0.047) as variables independently associated with tumor multiplicity, being the risk related to methylation of any of these two genes 4.57 (95% CI, 1.53 to 13.61; p = 0.006). Moreover, in six patients in whom both tumors were available, we found a correlation in the methylation levels of MGMT2 (r = 0.64, p = 0.17), SFRP1 (r = 0.83, 0.06), HPP1 (r = 0.64, p = 0.17), 3OST2 (r = 0.83, p = 0.06) and GATA4 (r = 0.6, p = 0.24). Methylation in normal appearing colorectal mucosa from patients with multiple and solitary CRC showed no relevant difference in any evaluated gene.

Conclusions

These results provide a proof-of-concept that gene promoter methylation is associated with tumor multiplicity. This underlying epigenetic defect may have noteworthy implications in the prevention of patients with sporadic CRC.     

Promoter methylation analysis of WNT/β-catenin pathway regulators and its association with expression of DNMT1 enzyme in colorectal cancer

Background

Aberrant DNA methylation as the most important reason making epigenetic silencing of genes is a main mechanism of gene inactivation in patients with colorectal cancer. In this study, we decided to identify promoter methylation status of ten genes encoding WNT negative regulators, and measure the expression of DNMT1 enzyme in colorectal cancer samples.

Results

Aberrant methylation of APC gene was statistically significant associated with age over 50 (p = 0.017), DDK3 with male (p < 0.0001), SFRP4, WIF1, and WNT5a with increasing tumor stage (p = 0.004, p = 0.029, and p = 0.004), SFRP4 and WIF1 with tumor differentiation (p = 0.009 and p = 0.031) and SFRP2 and SFRP5 with histological type (p = 0.001 and p = 0.025). The increasing number of methylated genes correlated with the expression levels of the DNMT1 mRNA.

Conclusions

The rate of gene promoter methylation of WNT pathway regulators is high in colorectal cancer cells. Hyper-methylation is associated with increased expression of the DNMT1 enzyme.     

Association between Common Germline Genetic Variation in 94 Candidate Genes or Regions and Risks of Invasive Epithelial Ovarian Cancer

Background

Recent studies have identified several single nucleotide polymorphisms (SNPs) in the population that are associated with variations in the risks of many different diseases including cancers such as breast, prostate and colorectal. For ovarian cancer, the known highly penetrant susceptibility genes (BRCA1 and BRCA2) are probably responsible for only 40% of the excess familial ovarian cancer risks, suggesting that other susceptibility genes of lower penetrance exist.

Methods

We have taken a candidate approach to identifying moderate risk susceptibility alleles for ovarian cancer. To date, we have genotyped 340 SNPs from 94 candidate genes or regions, in up to 1,491 invasive epithelial ovarian cancer cases and 3,145 unaffected controls from three different population based studies from the UK, Denmark and USA.

Results

After adjusting for population stratification by genomic control, 18 SNPs (5.3%) were significant at the 5% level, and 5 SNPs (1.5%) were significant at the 1% level. The most significant association was for the SNP rs2107425, located on chromosome 11p15.5, which has previously been identified as a susceptibility allele for breast cancer from a genome wide association study (P-trend = 0.0012). When SNPs/genes were stratified into 7 different pathways or groups of validation SNPs, the breast cancer associated SNPs were the only group of SNPs that were significantly associated with ovarian cancer risk (P-heterogeneity = 0.0003; P-trend = 0.0028; adjusted (for population stratification) P-trend = 0.006). We did not find statistically significant associations when the combined data for all SNPs were analysed using an admixture maximum likelihood (AML) experiment-wise test for association (P-heterogeneity = 0.051; P-trend = 0.068).

Conclusion

These data suggest that a proportion of the SNPs we evaluated were associated with ovarian cancer risk, but that the effect sizes were too small to detect associations with individual SNPs.     

Detection of Methylated SEPT9 in Plasma Is a Reliable Screening Method for Both Left- and Right-Sided Colon Cancers

Background

Methylated Septin 9 (SEPT9) is a sensitive biomarker for colorectal cancer (CRC) from peripheral blood. However, its relationship to cancer localization, guaiac-based fecal occult blood test (gFOBT) and carcinoembryonic antigen (CEA) have not been described.

Methodology/Principal Findings

Plasma samples were collected for SEPT9 analysis from patients with no evidence of disease (NED) (n = 92) before colonoscopy and CRC (n = 92) before surgical treatment. DNA was isolated and bisulfite-converted using Epi proColon kit 2.0. Qualitative determination was performed using Epi proColon 2.0 RT-PCR assay. Samples for gFOBT and CEA analysis were collected from NED (n = 17 and 27, respectively) and CRC (n = 22 and 27, respectively). SEPT9 test was positive in 15.2% (14/92) of NED and 95.6% (88/92) of CRC, including 100% (67/67) from stage II to stage IV CRC and 84% (21/25) of stage I CRC when a sample was called positive if 1 out of 3 PCR replicates was positive. In a second analysis (2 out of 3 PCR replicates) specificity improved to 99% (91/92) of NEDs, at a sensitivity of 79.3% (73/92) of SEPT9 positives in CRC. gFOBT was positive in 29.4% (5/17) of NED and 68.2% (15/22) of CRC and elevated CEA levels were detected in 14.8% (4/27) of NED and 51.8% (14/27) of CRC. Both SEPT9 (84.8%) and CEA (85.2%) showed higher specificity than gFOBT (70.6%). SEPT9 was positive in 96.4% (54/56) of left-sided colon cancer (LSCC) cases and 94.4% (34/36) of right-sided colon cancer (RSCC) cases. gFOBT was positive in 83.3% (10/12) of cases with LSCC and 50% (5/10) of cases with RSCC, elevated CEA was detected 60% (9/15) of LSCC and 41.7% (5/12) of RSCC.

Conclusions/Significance

The high degree of sensitivity and specificity of SEPT9 in plasma makes it a better method to detect CRC than gFOBT and CEA, even for the more difficult to detect RSCC.     

Attaching and Effacing Escherichia coli Downregulate DNA Mismatch Repair Protein In Vitro and Are Associated with Colorectal Adenocarcinomas in Humans

Background

Mucosa-associated Escherichia coli are frequently found in the colonic mucosa of patients with colorectal adenocarcinoma, but rarely in healthy controls. Chronic mucosal E. coli infection has therefore been linked to colonic tumourigenesis. E. coli strains carrying eae (encoding the bacterial adhesion protein intimin) attach intimately to the intestinal mucosa and are classed as attaching and effacing E. coli (AEEC). Enteropathogenic Escherichia coli (EPEC) are the most common form of AEEC identified in man. EPEC utilise a type III secretion system to translocate effector proteins into host cells and infection induces wide-ranging effects on the host cell proteome. We hypothesised that EPEC infection could influence molecular pathways involved in colorectal tumourigenesis.

Methodology/Principal Findings

When co-cultured with human colorectal cell lines, EPEC dramatically downregulated the expression of key DNA mismatch repair proteins MSH2 and MLH1 in an attachment specific manner. Cytochrome c staining and TUNEL analysis confirmed that this effect was not a consequence of apoptosis/necrosis. Ex vivo human colonic mucosa was co-cultured with EPEC and probed by immunofluorescence to locate adherent bacteria. EPEC entered 10% of colonic crypts and adhered to crypt epithelial cells, often in the proliferative compartment. Adenocarcinoma and normal colonic mucosa from colorectal cancer patients (n = 20) was probed by immunofluorescence and PCR for AEEC. Mucosa-associated E. coli were found on 10/20 (50%) adenocarcinomas and 3/20 (15%) normal mucosa samples (P<0.05). AEEC were detected on 5/20 (25%) adenocarcinomas, but not normal mucosa samples (P<0.05).

Significance/Conclusions

The ability of EPEC to downregulate DNA mismatch repair proteins represents a novel gene-environment interaction that could increase the susceptibility of colonic epithelial cells to mutations and therefore promote colonic tumourigenesis. The potential role of AEEC in colorectal tumourigenesis warrants further investigation.     

The potential role of Alu Y in the development of resistance to SN38 (Irinotecan) or oxaliplatin in colorectal cancer

Background

Irinotecan (SN38) and oxaliplatin are chemotherapeutic agents used in the treatment of colorectal cancer. However, the frequent development of resistance to these drugs represents a considerable challenge in the clinic. Alus as retrotransposons comprise 11% of the human genome. Genomic toxicity induced by carcinogens or drugs can reactivate Alus by altering DNA methylation. Whether or not reactivation of Alus occurs in SN38 and oxaliplatin resistance remains unknown.

Results

We applied reduced representation bisulfite sequencing (RRBS) to investigate the DNA methylome in SN38 or oxaliplatin resistant colorectal cancer cell line models. Moreover, we extended the RRBS analysis to tumor tissue from 14 patients with colorectal cancer who either did or did not benefit from capecitabine + oxaliplatin treatment. For the clinical samples, we applied a concept of ‘DNA methylation entropy’ to estimate the diversity of DNA methylation states of the identified resistance phenotype-associated methylation loci observed in the cell line models. We identified different loci being characteristic for the different resistant cell lines. Interestingly, 53% of the identified loci were Alu sequences- especially the Alu Y subfamily. Furthermore, we identified an enrichment of Alu Y sequences that likely results from increased integration of new copies of Alu Y sequence in the drug-resistant cell lines. In the clinical samples, SOX1 and other SOX gene family members were shown to display variable DNA methylation states in their gene regions. The Alu Y sequences showed remarkable variation in DNA methylation states across the clinical samples.

Conclusion

Our findings imply a crucial role of Alu Y in colorectal cancer drug resistance. Our study underscores the complexity of colorectal cancer aggravated by mobility of Alu elements and stresses the importance of personalized strategies, using a systematic and dynamic view, for effective cancer therapy.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1552-y) contains supplementary material, which is available to authorized users.     

Genetic Variant rs7758229 in 6q26–q27 Is Not Associated with Colorectal Cancer Risk in a Chinese Population

Background

A recent genome-wide association study has identified a new genetic variant rs7758229 in SLC22A3 for colorectal cancer susceptibility in a Japanese population, but it is unknown whether this newly identified variant is associated with colorectal cancer in other populations, including the Chinese population.

Methods

We examined the associations between rs7758229 and colorectal cancer risk among 1,147 cases and 1,203 controls matched by age and sex. Logistic regression model was used to assess the associations.

Results

No significant association was found between rs7758229 and colorectal cancer risk (OR = 0.95, 95%CI  = 0.84–1.09, P = 0.463). Similar results were observed in the stratification of tumor location (OR  = 0.94, 95%CI = 0.80–1.11, P = 0.481 for colon cancer, and OR  = 0.96, 95%CI  = 0.82–1.13, P = 0.621 for rectum cancer).

Conclusions

Our findings did not support an association between rs7758229 in 6q26-q27 and the risk of colorectal cancer in a Chinese population.     

Human Papillomavirus Genotype Distribution in Czech Women and Men with Diseases Etiologically Linked to HPV

Background

The HPV prevalence and genotype distribution are important for the estimation of the impact of HPV-based cervical cancer screening and HPV vaccination on the incidence of diseases etiologically linked to HPVs. The HPV genotype distribution varies across different geographical regions. Therefore, we investigated the type-specific HPV prevalence in Czech women and men with anogenital diseases.

Methods

We analyzed 157 squamous cell carcinoma samples, 695 precancerous lesion samples and 64 cervical, vulvar and anal condylomata acuminate samples. HPV detection and typing were performed by PCR with GP5+/6+ primers, reverse line blot assay and sequencing.

Results

Thirty different HPV genotypes were detected in our study, HPV 16 being the most prevalent type both in precancerous lesions (45%) and squamous cell carcinomas (59%). In benign lesions, HPV 6 (72%) was the most common type. Altogether, 61% of carcinoma samples and 43% of precancerous lesion samples contained HPV 16 and/or 18. The presence of HPV types related to the vaccinal ones (HPV 31, 45, 33, 52, 58) were detected in 16% of carcinoma samples and 18% of precancerous lesion samples. HPV 16 and/or 18 were present in 76% of cervical cancer samples, 33% of CIN1, 43% CIN2 and 71% of CIN3 samples. HPV types 6 and/or 11 were detected in 84% samples of condylomata acuminate samples.

Conclusions

The prevalence of vaccinal and related HPV types in patients with HPV-associated diseases in the Czech Republic is very high. We may assume that the implementation of routine vaccination against HPV would greatly reduce the burden of HPV-associated diseases in the Czech Republic.     

Periconceptional Maternal Folic Acid Use of 400 μg per Day Is Related to Increased Methylation of the IGF2 Gene in the Very Young Child

Background

Countries worldwide recommend women planning pregnancy to use daily 400 µg of synthetic folic acid in the periconceptional period to prevent birth defects in children. The underlying mechanisms of this preventive effect are not clear, however, epigenetic modulation of growth processes by folic acid is hypothesized. Here, we investigated whether periconceptional maternal folic acid use and markers of global DNA methylation potential (S-adenosylmethionine and S-adenosylhomocysteine blood levels) in mothers and children affect methylation of the insulin-like growth factor 2 gene differentially methylation region (IGF2 DMR) in the child. Moreover, we tested whether the methylation of the IGF2 DMR was independently associated with birth weight.

Methodology/Principal Findings

IGF2 DMR methylation in 120 children aged 17 months (SD 0.3) of whom 86 mothers had used and 34 had not used folic acid periconceptionally were studied. Methylation was measured of 5 CpG dinucleotides covering the DMR using a mass spectrometry-based method. Children of mother who used folic acid had a 4.5% higher methylation of the IGF2 DMR than children who were not exposed to folic acid (49.5% vs. 47.4%; p = 0.014). IGF2 DMR methylation of the children also was associated with the S-adenosylmethionine blood level of the mother but not of the child (+1.7% methylation per SD S-adenosylmethionine; p = 0.037). Finally, we observed an inverse independent association between IGF2 DMR methylation and birth weight (−1.7% methylation per SD birthweight; p = 0.034).

Conclusions

Periconceptional folic acid use is associated with epigenetic changes in IGF2 in the child that may affect intrauterine programming of growth and development with consequences for health and disease throughout life. These results indicate plasticity of IGF2 methylation by periconceptional folic acid use.