Title: DNA repair gene polymorphisms and risk of chronic atrophic gastritis: a case-control study

Transcription

1 Author's response to reviews Title: DNA repair gene polymorphisms and risk of chronic atrophic gastritis: a case-control study Authors: Bernd Frank (b.frank@dkfz.de) Heiko Müller (h.mueller@dkfz.de) Melanie N Weck (m.weck@dkfz.de) Norman Klopp (klopp@gsf.de) Thomas Illig (illig@helmholtz-muenchen.de) Elke Raum (e.raum@dkfz.de) Hermann Brenner (h.brenner@dkfz.de) Version: 3 Date: 5 August 2011 Author's response to reviews: see over

2 Dear Dr. Riker, Thank you very much for your kind from July 7 and your interest in our manuscript (MS: DNA repair gene polymorphisms and risk of chronic atrophic gastritis: a case-control study). We are grateful for the reviewers constructive suggestions and have prepared a revised version of the manuscript. The manuscript has been modified according to the reviewers suggestions; modifications within the text are highlighted. In the following, you will find detailed explanations to the manuscript changes: Reviewer 1: Phouthone Keohavong In this paper, Frank et al analyzed single nucleotide polymorphisms of DNA repair genes in 533 cases with chronic atrophic gastritis (CAG) and 1054 controls in order to determine whether any association existed between any of these gene variants and risk of CAG. Their finding did not support associations of DNA repair pathway gene single nucleotide polymorphisms with CAG risk. The main strength is that this is a case-control study involving large numbers cases and controls from a homogenous population. On the other hand, besides reporting negative results, it would be more informative to readers if the authors discussed their results further. For instance, some XRCC1 gene polymorphisms have been previously found to be associated with GC risk. Would ethnic/racial or geographical differences play any role? >>> We addressed these issues in the Discussion section (lines ): Remarkably, XRCC1-77T>C was identified as functional polymorphism, diminishing promoter activity and thus increasing the risk of non-small cell lung cancer (NSCLC), while the three non-synonymous XRCC1 SNPs R194W, R280H and R399Q, whose functional characteristics are not determined yet, showed no association with NSCLC risk [15]. As demonstrated by Capellá et al., R399Q in XRCC1 showed an association with an increased risk of severe chronic atrophic gastritis [20]. Contrariwise, a recent meta-analysis indicated R194W to be XRCC1 susceptibility variant for GC [23]. After extensive studies of DNA repair polymorphisms in various cancer sites and ethnic populations, the results still remain inconsistent [17], which may be attributed to both different aetiologies of cancers, and ethnic or geographical disparities [11]..

3 Reviewer 2: Arto Mannermaa The manuscript "DNA repair gene polymorphisms and risk of chronic atrophic gastritis: a case control study represents an interesting investigation on possible association of DNA repair gene polymorphisms to risk of chronic atrophic gastritis. The topic of this manuscript is relevant; DNA damage contributes to carcinogenesis and the genetic risk of CAG caused by DNA repair gene polymorphisms is yet relatively unresolved. The authors have chosen to focus on 11 functional SNPs on seven genes. The material used is well characterized and relatively large. According to authors they had 80% power to detect ORs over 1.44 or under 0.69 for all SNPs. Presumably the authors have been confident with this and have decided to implement the project. The genotyping methods used are valid and properly described. However the choice of SNPs and power analysis need more clarification. This manuscript describes no evidence for DNA repair gene variants to have an effect to CAG risk. The paper is concisely and very clearly written, though short, but the data is not convincing. The major weakness of the manuscript is the lack of convincing evidence for or against the hypothesis - that DNA repair gene polymorphisms affect the risk of CAG. Moreover, there is almost no discussion how the presented evidence is in harmony with what is known of CAG risk in respect to DNA repair in the literature. The manuscript has major fundamental points to revise before it is acceptable for BMC cancer. >>> As results of DNA repair SNPs are still inconsistent, we added the following paragraph and discussed the possible reason for the lack of association, taking XRCC1 polymorphisms as examples (lines ): Remarkably, XRCC1-77T>C was identified as functional polymorphism, diminishing promoter activity and thus increasing the risk of non-small cell lung cancer (NSCLC), while the three non-synonymous XRCC1 SNPs R194W, R280H and R399Q, whose functional characteristics are not determined yet, showed no association with NSCLC risk [15]. As demonstrated by Capellá et al., R399Q in XRCC1 showed an association with an increased risk of severe chronic atrophic gastritis [20]. Contrariwise, a recent meta-analysis indicated R194W to be XRCC1 susceptibility variant for GC [23]. After extensive studies of DNA repair polymorphisms in various cancer sites and ethnic populations, the results still remain inconsistent [17], which may be attributed to both different aetiologies of cancers, and ethnic or geographical disparities [11]..

4 1. Selection of SNPs: The manuscript is built on the putative role of DNA repair mechanisms on CAG risk. The authors do not justify enough their selection of 11 SNPs let alone the seven genes. The manuscript also lacks the discussion of the possibility of missing the real risk altering SNPs. >>> We clarified the section about SNP selection, which now reads as follows (lines ): In line with recent epidemiologic studies that have revealed associations of BER and NER pathway gene variants with risk of glioma, colorectal, prostate, lung and gastric cancers and their precursors [9-20], selection was focussed on SNPs in DNA repair genes. Non-synonymous and putative functional SNPs were of particular interest. Therefore, we searched public literature resources and databases (NCBI PubMed and dbsnp), favouring genes and polymorphisms with previous findings in view of susceptibility to precancerous lesions and different types of cancers. SNP selection included four BER gene variants (APEX1 D148E rs , PARP1-17G>C rs and V762A rs , XRCC1-77T>C rs ), five SNPs in NER genes (ERCC1 N118N rs11615, ERCC2/XPD K751Q rs13181, D312N rs , R156R rs and - 114C>G rs ), and two SNPs in CD3EAP/ASE-1 (rs735482) and PPP1R13L (rs6966) which, together with ERCC1 N118N rs11615, represent the high-risk haplotype on chromosome 19q13.3 [12].. In the Discussion section we addressed the possibility that we may have missed risk altering SNPs (lines ): A possible explanation for the lack of significance may be that a real risk altering SNP (within one of the selected or another DNA repair pathway gene) was not analysed and missed.. 2. Power analysis: The authors have utilized a power analysis to prove the size of the material and selection of SNPs to be appropriate for the analysis. Authors do not present all the details used in the analysis. What was the minor allele frequency used in the analysis? Is it comparable to that of the observed frequencies? >>> Before we started the analyses, we searched the literature (NCBI PubMed) for putative functional SNPs within the DNA repair pathway. To ensure that we have enough power to detect possible associations, we selected SNPs with minor allele frequencies > 10%. These were verified by means of NCBI dbsnp HapMapCEU data. We now have addressed this issue to the Methods section (lines ): For reasons of statistical power, we restricted our analyses to SNPs with minor allele frequencies (MAFs) > 10%, according to dbsnps HapMapCEU data

5 ( MAFs of the chosen SNPs ranged from 13% - 49%. The statistical tests were implemented with SAS (SAS Institute Inc., Cary, USA), and power calculations were employed with the power and sample size software PS [22], applying the observed genotype frequencies, respectively. Two-sided Fisher s exact tests were used to compare carrier frequencies between CAG cases and controls with a type I error probability of α = In the summary the authors suggest further studies with larger sample sets. Also full coverage of genes and their genetic variants in DNA repair machinery should be engaged. >>> This issue was addressed in the Abstract/Conclusions and Conclusions sections (lines 40-43): On the basis of a large number of CAG cases, they do not support associations of DNA repair pathway SNPs with CAG risk, but suggest the need of larger studies to disclose or exclude potential weak associations, or of studies with full coverage of candidate genes. and (lines ): Moreover, full coverage of candidate DNA repair genes, i.e. tagging SNP approaches should be aimed for in future studies.. Reviewer 3: Matthias Kloor In the present case-control study, Frank et al. report the absence of correlation between DNA repair gene polymorphisms and the risk of chronic atrophic gastritis (CAG) in a German cohort (ESTHER study). The authors selected literature-defined SNPs that are located within DNA repair genes and that may have a putative functional consequence. SNPs previously reported to be associated with gastrointestinal premalignant lesions have been included. Genotyping data were then correlated with the presence of serologically defined chronic atrophic gastritis (CAG). The study design is very clear and straight-forward. Using serum pepsinogen I and II levels as parameters for the definition of CAG, no statistically significant correlation between any of the eleven SNPs analyzed and CAG was observed. Specific points: The authors might want to discuss the carcinogenic process in the stomach that includes CAG as a precancerous lesion in more detail. The manuscript could be improved if the potential impact of DNA repair systems at different steps, initiation and progression from normal mucosa towards cancer were outlined more specifically and in the context of other known or assumed risk factors for CAG and gastric cancer.

6 >>> We addressed these issues in the Introduction section (lines 60-68): The multistage model of gastric carcinoma development assumes that carcinogenesis is initiated by host-inflammatory response following infection by the Gram-negative bacterium Helicobacter pylori (H. pylori), and by dietary exposure to salt and nitrate, which cause DNA damage [4-7]. To date, three molecular mechanisms, by which H. pylori may provoke a loss of genomic integrity and promote transformation, are postulated [6, 7]. These include a) mutations in mitochondrial DNA, b) the induction of a transient mutator phenotype, resulting in mutations in the nuclear genome, and c) increased amounts of reactive oxygen species (ROS) in gastric epithelial cells that induce oxidative damage in the DNA coupled to the decrease of repair activity [6, 7].. The authors should discuss their results in direct comparison with the results of the study by Capella et al. who reported associations between atrophic gastritis and DNA repair polymorphisms. >>> We addressed this issue in the Discussion section (lines ): In contrast, Capellá et al. found associations of ERCC2/XPD D312N and K751Q with an increased risk of severe CAG [20]. The discrepant findings may be due to different serological definitions of CAG in the studies [3, 20]. Another possible reason to be considered is random variation, having in mind the much smaller number of cases (n = 246) in the study by Capellá et al.[20].. Reference 15 (now reference 19) is about colorectal adenomas. This should be mentioned in the introduction, page 3 "such as colorectal adenomas or CAG". There is a typing error in this reference (first author). >>> Reference corrected and done (lines 77-78). Editorial request: 1) Ethics - Experimental research that is reported in the manuscript must have been performed with the approval of an appropriate ethics committee. Research carried out on humans must be in compliance with the Helsinki Declaration ( and any experimental research on animals must follow internationally recognized guidelines. A statement

7 to this effect must appear in the Methods section of the manuscript, including the name of the body which gave approval, with a reference number where appropriate. >>> We added the necessary statement to the Methods section (lines ): Briefly, 9,953 women and men aged years were recruited between July 2000 and December 2002 by their general practitioners during a general health check-up in Saarland, a federal state in the south-west of Germany [3]. The study was approved by the ethics committees of the medical faculty of the University of Heidelberg and the medical board of the state of Saarland.. We believe that the reviewers suggestions have been very helpful to improve the manuscript, and we are looking forward to your editorial decision. Again, thank you very much for your review. Yours sincerely Bernd Frank