Author s response to reviews

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1 Author s response to reviews Title: A novel variant on chromosome 6p21.1 is associated with the risk of developing colorectal cancer: a two-stage case-control study in Han Chinese Authors: Chunxiao Xu (mickychunxiao@126.com) Dan Zhou (zdan@zju.edu.cn) Feixia Pan ( @qq.com) Yi Liu (lyzju_2012@163.com) Dandan Zhang (dandanz@zju.edu.cn) Aifen Lin (linaf@enzemed.com) Xiaoping Miao (miaoxp@mail.hust.edu.cn) Yaqin Ni (Aileenyaqin@126.com) Duo Lv (lvduo8905@foxmail.com) Shuai Zhang (ssnn@zju.edu.cn) Xiaobo Li (oboaixil@126.com) Yimin Zhu (zhuym@zju.edu.cn) Maode Lai (lmp@zju.edu.cn) Version: 2 Date: 12 Aug 2016 Author s response to reviews: Aug 13th, 2016 Dear editor, Thank you for your letter with the comments on our manuscript (BCAN-D R1), "A novel variant on chromosome 6p21.1 associates the risk of colorectal cancer: a two-stage case-

2 control study in Han Chinese". The comments are all valuable and helpful for revising and improving our manuscript. Attached please find our point-by-point responses to the reviewer s questions and comments. We have also incorporated these changes into our revised manuscript. All of the authors have declared no competing interests and have agree to submit revised manuscript. We hope that you will find our revised manuscript acceptable for publication in BMC Cancer. If there are further questions, please feel free to contact us. Thank you very much. Best regards, Sincerely, Yimin Zhu, PhD Department of Epidemiology and Biostatistics, Zhejiang University School of Medicine, Hangzhou, Zhejiang, , China zhuym@zju.edu.cn Maode Lai, MD Department of Pathology, Zhejiang University School of Medicine, 866 Yuhangtang Road, Hangzhou, Zhejiang, China. address: lmp@zju.edu.cn Point-by-point responses: The authors have done a good job with the revisions in the previous review cycle. There are a few remaining issues that need to be addressed before publication. Thank you for your time and valuable comments. We have addressed the manuscript with your helpful suggestions. We hope that you will find our revised manuscript acceptable for publication in BMC Cancer. 1. There should be no abbreviations in the abstract. Please remove them.

3 Thank you for your comment. We have remove the abbreviations in the abstract. 2. While most of the paper has been revised, there are still some instances of the past tense being used to refer to this study. The present study must always be referred to in the present tense. (for example, abstract conclusions says "was"). Please check carefully throughout. Thank you for your suggestion. We have check tenses carefully throughout the manuscript. The revised conclusions show as following: Conclusions (abstract): Our findings indicate that the C allele of the novel colorectal cancerassociated variant rs is associated with increased expression levels of HSP90AB1, which is expressed higher in colorectal tumor tissues than in normal tissues. Conclusions (discussion): In conclusion, this study identifies a novel variant of rs , which is associated with the risk of developing CRC in Han Chinese. This association between rs and CRC is probably mediated by the expression of the inflammatory-related gene HSP90AB1, which is upregulated in colorectal tumor tissues. Further studies are warranted to explore the potential mechanism. 3. Background line 26, says "estimated 1,360,000 confirmed diagnoses...". Remove the word confirmed. It cannot be used with "estimated". Thank you for your correction. We have remove the improper word. 4. You use too many abbreviations. Remove the following as abbreviations: SNP, eqtl, NFkB, GWAS, HWE, FDR, LD. These are non-standard and inappropriate as abbreviations. Thank you for your suggestions. Most of the non-standard and inappropriate abbreviations have been removed from the revised manuscript. However, to our knowledge, some of the abbreviations (e.g. SNP = Single Nucleotide Polymorphism and GWAS = Genome-Wide Association Study) are commonly used in published papers in some influential journals [1-3]

4 including BMC Cancer [4, 5]. The abstracts of the reference papers are attached at the end of the letter. 1. Zhu Z, Zhang F, Hu H, Bakshi A, Robinson MR, Powell JE, Montgomery GW, Goddard ME, Wray NR, Visscher PM: Integration of summary data from GWAS and eqtl studies predicts complex trait gene targets. Nature genetics Auton A, Brooks LD, Durbin RM, Garrison EP, Kang HM, Korbel JO, Marchini JL, Mccarthy S, Mcvean GA, Abecasis GR: A global reference for human genetic variation. Nature 2015, 526(7571): Law MH, Bishop DT, Lee JE, Brossard M, Martin NG, Moses EK, Song F, Barrett JH, Kumar R, Easton DF: Genome-wide meta-analysis identifies five new susceptibility loci for cutaneous malignant melanoma. Nature genetics 2015, 47(9): Maturana ELD, Picornell A, Masson-Lecomte A, Kogevinas M, Márquez M, Carrato A, Tardón A, Lloreta J, García-Closas M, Silverman D: Prediction of non-muscle invasive bladder cancer outcomes assessed by innovative multimarker prognostic models. Bmc Cancer 2016, 16(1): Thomsen H, Chen B, Figlioli G, Elisei R, Romei C, Cipollini M, Cristaudo A, Bambi F, Hoffmann P, Herms S: Runs of homozygosity and inbreeding in thyroid cancer. BMC cancer 2016, 16(1):1. 5. Figure legends must define all abbreviations. The reader is not expected to refer to the text for these definitions. Thank you for your suggestion. We have defined all of the abbreviations in Figure legends in the revised manuscript. e.g. The revised Figure legend of Figure 3: Abbreviations: OR= odds ratio; CI = confidence interval; MuTHER = Multiple Tissue Human Expression Resource; TCGA = The Cancer Genome Atlas; SYSCOL = Systems Biology of Colorectal Cancer; T / N = Tumor / Normal.

5 6. The title is strangely worded. It says "..associates the risk of colorectal...". This is meaningless English. Do you mean "...is associated with the risk of developing colorectal..."??? Please rewrote appropriately. Thanks for your correction. The title has been rewritten as following: A novel variant on chromosome 6p21.1 is associated with the risk of developing colorectal cancer: a two-stage case-control study in Han Chinese 7. This is an international journal, and most people may not be aware of the ethnic group of "Han Chinese". The discussion section needs a few sentences to introduce this Ethnic group. Is this group a relatively closed social group? Why is it significant that this is among Han Chinese? Thank you for your comment. We felt that it is necessary to introduce the Ethnic group as you mentioned. China is made up of 56 nationalities. The Han Chinese is the majority ethnic group (approximately 92% of the population) in Mainland China. In order to control the genetic background of study subjects, we recruited Han Chinese only. As you suggested, a few sentences have been added in discussion section as following: All of the study subjects are Han Chinese (an ethnic group native to East Asia), which constitute approximately 92% of the population of Mainland China and about 18% of the entire global human population. Nat Genet May;48(5): doi: /ng Epub 2016 Mar 28. Integration of summary data from GWAS and eqtl studies predicts complex trait gene targets. Zhu Z1, Zhang F1, Hu H2, Bakshi A1, Robinson MR1, Powell JE1,3, Montgomery GW4, Goddard ME5,6, Wray NR1, Visscher PM1,7, Yang J1,7. Author information Abstract Genome-wide association studies (GWAS) have identified thousands of genetic variants associated with human complex traits. However, the genes or functional DNA elements through

6 which these variants exert their effects on the traits are often unknown. We propose a method (called SMR) that integrates summary-level data from GWAS with data from expression quantitative trait locus (eqtl) studies to identify genes whose expression levels are associated with a complex trait because of pleiotropy. We apply the method to five human complex traits using GWAS dataon up to 339,224 individuals and eqtl data on 5,311 individuals, and we prioritize 126 genes (for example, TRAF1 and ANKRD55 for rheumatoid arthritis and SNX19 and NMRAL1 for schizophrenia), of which 25 genes are new candidates; 77 genes are not the nearest annotated gene to the top associated GWAS SNP. These genes provide important leads to design future functional studies to understand the mechanism whereby DNA variation leads to complex trait variation. Nature Oct 1;526(7571): doi: /nature A global reference for human genetic variation Genomes Project Consortium, Auton A, Brooks LD, Durbin RM, Garrison EP, Kang HM, Korbel JO, Marchini JL, McCarthy S, McVean GA, Abecasis GR. Collaborators (843) Abstract The 1000 Genomes Project set out to provide a comprehensive description of common human genetic variation by applying whole-genome sequencing to a diverse set of individuals from multiple populations. Here we report completion of the project, having reconstructed the genomes of 2,504 individuals from 26 populations using a combination of low-coverage wholegenome sequencing, deep exome sequencing, and dense microarray genotyping. We characterized a broad spectrum of genetic variation, in total over 88 million variants (84.7 million single nucleotide polymorphisms (SNPs), 3.6 million short insertions/deletions (indels), and 60,000 structural variants), all phased onto high-quality haplotypes. This resource includes >99% of SNP variants with a frequency of >1% for a variety of ancestries. We describe the distribution of genetic variation across the global sample, and discuss the implications for common disease studies. Nat Genet Sep;47(9): doi: /ng Epub 2015 Aug 3. Genome-wide meta-analysis identifies five new susceptibility loci for cutaneous malignant melanoma.

7 Law MH1, Bishop DT2, Lee JE3, Brossard M4,5, Martin NG6, Moses EK7, Song F8, Barrett JH2, Kumar R9, Easton DF10, Pharoah PD11, Swerdlow AJ12,13, Kypreou KP14, Taylor JC2, Harland M2, Randerson-Moor J2, Akslen LA15,16, Andresen PA17, Avril MF18, Azizi E19,20, Scarrà GB21,22, Brown KM23, Dȩbniak T24, Duffy DL6, Elder DE25, Fang S3, Friedman E20, Galan P26, Ghiorzo P21,22, Gillanders EM27, Goldstein AM23, Gruis NA28, Hansson J29, Helsing P30, Hočevar M31, Höiom V29, Ingvar C32, Kanetsky PA33, Chen WV34; GenoMEL Consortium; Essen-Heidelberg Investigators; SDH Study Group; Q-MEGA and QTWIN Investigators; AMFS Investigators; ATHENS Melanoma Study Group, Landi MT23, Lang J35, Lathrop GM36, Lubiński J24,Mackie RM35,37, Mann GJ38, Molven A16,39, Montgomery GW40, Novaković S41, Olsson H42,43, Puig S44,45, Puig-Butille JA44,45, Qureshi AA46,Radford-Smith GL47,48,49, van der Stoep N50, van Doorn R28, Whiteman DC51, Craig JE52, Schadendorf D53,54, Simms LA47, Burdon KP55, Nyholt DR40,56, Pooley KA10, Orr N57, Stratigos AJ14, Cust AE58, Ward SV7, Hayward NK59, Han J60,61, Schulze HJ62, Dunning AM11, Bishop JA2, Demenais F4,5, Amos CI63, MacGregor S1, Iles MM2. Author information Abstract Thirteen common susceptibility loci have been reproducibly associated with cutaneous malignant melanoma (CMM). We report the results of an international 2-stage meta-analysis of CMM genome-wide association studies (GWAS). This meta-analysis combines 11 GWAS (5 previously unpublished) and a further three stage 2 data sets, totaling 15,990 CMM cases and 26,409 controls. Five loci not previously associated with CMM risk reached genome-wide significance (P < 5 10(-8)), as did 2 previously reported but unreplicated loci and all 13 established loci. Newly associated SNPs fall within putative melanocyte regulatory elements, and bioinformatic and expression quantitative trait locus (eqtl) data highlight candidate genes in the associated regions, including one involved in telomere biology. BMC Cancer Jun 3;16:351. doi: /s Prediction of non-muscle invasive bladder cancer outcomes assessed by innovative multimarker prognosticmodels. López de Maturana E1, Picornell A1, Masson-Lecomte A1, Kogevinas M2,3, Márquez M1, Carrato A4, Tardón A5,3, Lloreta J6, García-Closas M7, Silverman D8, Rothman N8, Chanock S8, Real FX9, Goddard ME10, Malats N11; SBC/EPICURO Study Investigators.

8 Author information Abstract BACKGROUND: We adapted Bayesian statistical learning strategies to the prognosis field to investigate if genome-wide common SNP improve the prediction ability of clinico-pathological prognosticators and applied it to non-muscle invasive bladder cancer (NMIBC) patients. METHODS: Adapted Bayesian sequential threshold models in combination with LASSO were applied to consider the time-to-event and the censoring nature of data. We studied 822 NMIBC patients followed-up >10 years. The study outcomes were time-to-first-recurrence and time-toprogression. The predictive ability of the models including up to 171,304 SNP and/or 6 clinicopathological prognosticators was evaluated using AUC-ROC and determination coefficient. RESULTS: Clinico-pathological prognosticators explained a larger proportion of the time-to-first-recurrence (3.1 %) and time-to-progression (5.4 %) phenotypic variances than SNPs (1 and 0.01 %, respectively). Adding SNPs to the clinico-pathological-parameters model slightly improved theprediction of time-to-first-recurrence (up to 4 %). The prediction of time-to-progression using both clinico-pathological prognosticators and SNP did not improve. Heritability (ĥ (2)) of both outcomes was <1 % in NMIBC. CONCLUSIONS: We adapted a Bayesian statistical learning method to deal with a large number of parameters in prognostic studies. Common SNPs showed a limited role in predicting NMIBC outcomes yielding a very low heritability for both outcomes. We report for the first time a heritability estimate for a disease outcome. Our method can be extended to other disease models. BMC Cancer Mar 16;16:227. doi: /s Runs of homozygosity and inbreeding in thyroid cancer. Thomsen H1, Chen B2, Figlioli G2,3, Elisei R4, Romei C4, Cipollini M3, Cristaudo A4, Bambi F5, Hoffmann P6,7,8, Herms S6,7,8, Landi S3, Hemminki K2,9, Gemignani F3, Försti A2,9. Author information

9 Abstract BACKGROUND: Genome-wide association studies (GWASs) have identified several single-nucleotide polymorphisms (SNPs) influencing the risk of thyroid cancer (TC). Most cancer predisposition genes identified through GWASs function in a co-dominant manner, and studies have not found evidence for recessively functioning disease loci in TC. Our study examines whether homozygosity is associated with an increased risk of TC and searches for novel recessively acting disease loci. METHODS: Data from a previously conducted GWAS were used for the estimation of the proportion of phenotypic variance explained by all common SNPs, the detection of runs of homozygosity (ROH) and the determination of inbreeding to unravel their influence on TC. RESULTS: Inbreeding coefficients were significantly higher among cases than controls. Association on a SNP-by-SNP basis was controlled by using the false discovery rate at a level of q* < 0.05, with 34 SNPs representing true differences in homozygosity between cases and controls. The average size, the number and total length of ROHs per person were significantly higher in cases than in controls. A total of 16 recurrent ROHs of rather short length were identified although their association with TC risk was not significant at a genome-wide level. Several recurrent ROHs harbor genes associated with risk of TC. All of the ROHs showed significant evidence for natural selection (ihs, Fst, Fay and Wu's H). CONCLUSIONS: Our results support the existence of recessive alleles in TC susceptibility. Although regions of homozygosity were rather small, it might be possible that variants within these ROHs affect TC risk and may function in a recessive manner.