Carcinogenesis Carcinogenesis Advance Access published February 7, 2014
ABCC4 Copy Number Variation is Associated with Susceptibility to Esophageal Squamous Cell Carcinoma
Carcinogenesis
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Manuscript ID: Manuscript Type: Date Submitted by the Author:
Original Manuscript
30-Dec-2013
Sun, YuLin; Cancer Institute & Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, State Key Laboratory of Molecular Oncology Shi, Ni; Cancer Institute & Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, State Key Laboratory of Molecular Oncology Lu, Haizhen; Cancer Institute & Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Department of Pathology Zhang, JinQiang; Cancer Institute & Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, State Key Laboratory of Molecular Oncology Ma, YuLong; Yangquan Cancer Prevention and Treatment Institute, Department of Oncological Surgery Qiao, YuanYuan; Navy General Hospital, Center of Basic Medical Sciences Mao, YongHong; Yangquan Cancer Prevention and Treatment Institute, Department of Oncological Surgery Jia, Kun; Cancer Institute & Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Department of Pathology Han, LiFen; Yangquan Cancer Prevention and Treatment Institute, Department of Oncological Surgery Liu, Fang; Cancer Institute & Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, State Key Laboratory of Molecular Oncology Li, HongXia; Shanxi Population and Family Planning Science Research Institute, Null Lin, Zhengwei; Cancer Institute & Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, State Key Laboratory of Molecular Oncology Li, XinMin; David Geffen School of Medicine, University of California, Department of Pathology & Laboratory Medicine Zhao, XiaoHang; Cancer Institute & Hospital, State Key Laboratory of Molecular Oncology
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Complete List of Authors:
CARCIN-2013-00981.R1
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Keywords:
ABCC4, Copy number variation, Esophageal squamous cell carcinoma, susceptible gene
Downloaded from http://carcin.oxfordjournals.org/ at UB Kaiserslautern on May 21, 2014
Journal:
Page 1 of 61
ABCC4 Copy Number Variation is Associated with Susceptibility to Esophageal Squamous Cell Carcinoma
YULIN SUN,1,# NI SHI,1,# HAIZHEN LU,4 JINQIANG ZHANG,1 YULONG MA,3 YUANYUAN QIAO,2 YONGHONG MAO,3 KUN JIA,1 LIFEN HAN,3 FANG LIU,1 HONGXIA Li,5 ZHENGWEI LIN,1 XINMIN LI,6 XIAOHANG ZHAO1,2 State Key Laboratory of Molecular Oncology, 4Department of Pathology, Cancer
Institute & Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100021, China; 2Center of Basic Medical Sciences, Navy General
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Hospital, Beijing 100048, China; 3Department of Oncological Surgery, Yangquan
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Cancer Prevention and Treatment Institute, Yangquan 045000, Shanxi Province, China; 5Shanxi Population and Family Planning Science Research Institute, Taiyuan
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030006, Shanxi Province, China; 6Department of Pathology & Laboratory Medicine,
#
These authors are contributed equally.
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USA
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David Geffen School of Medicine, University of California, Los Angels, CA 90095,
RUNNING TITLE: ABCC4 CNV is associated with ESCC risk To whom correspondence should be addressed: Dr. Xiaohang Zhao, Phone: +86-10-67709015; Fax: +86-10-87778360; [email protected] and Dr. Xinmin Li,
Phone: 310-825-3664; Fax: 310-825-3570; [email protected] Keywords: ABCC4; Copy number variation; Esophageal squamous cell carcinoma; susceptible gene 1
Downloaded from http://carcin.oxfordjournals.org/ at UB Kaiserslautern on May 21, 2014
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Carcinogenesis
Carcinogenesis
Word count (excluding references and Figure legends): 5000 words Total number of figures and tables: 6
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Abstract Esophageal squamous cell carcinoma (ESCC) is the eighth most common cause of cancer-related death worldwide. However, previous genome-wide single nucleotide polymorphism (SNP) association analyses have not explained the high heritability associated with ESCC. In this study, we performed genome-wide copy number variation (CNV) analysis on 128 discordant sibling pairs to identify novel genes that contribute to ESCC susceptibility. A total of 57,774 individual CNVs were identified, and an interactive network of common CNV-associated genes was constructed, which showed that several ABC transporter genes contain CNVs in ESCC patients.
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Independent validation of a CNV at 13q32.1 in 1,048 northern Chinese Han subjects
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demonstrated that the amplification of ABCC4 significantly correlated with ESCC risk (odds ratio: 3.36 [1.65-7.93], P=0.0013). Immunohistochemistry staining suggested
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that high copy numbers correlated with increased protein levels. High expression of
vi
ABCC4 was an independent poor prognostic factor for ESCC (relative risk: 1.73
ew
[1.10-2.73], P=0.0181). The CNV region showed strong enhancer activity. Furthermore, inhibition of ABCC4 protein in ESCC cells decreased cell proliferation and motility via the inhibition of COX-2, PGE2 receptors and c-Myc expression; AKT, ERK and CREB phosphorylation; and β-catenin nuclear translocation in ESCC cells. In conclusion, the CNV at 13q32.1 is associated with ESCC susceptibility, and a gene within this locus, ABCC4, activates the oncogenic pathways in ESCC and thus facilitates cancer cell development and progression. A direct genetic contribution of ESCC risk through CNV common variants was determined in this study, and ABCC4 3
Downloaded from http://carcin.oxfordjournals.org/ at UB Kaiserslautern on May 21, 2014
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Carcinogenesis
Carcinogenesis
might therefore have predictive and therapeutic potential for ESCC.
Summary The genome-wide copy number variation analysis found that the amplification of ABCC4 at 13q32.1 correlated with esophageal cancer risk. Functional study showed that high expression of ABCC4 predicted poor clinical outcomes which activated the Downloaded from http://carcin.oxfordjournals.org/ at UB Kaiserslautern on May 21, 2014
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oncogenic pathways and facilitated tumorigenesis.
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Introduction Esophageal cancer is the eighth most commonly diagnosed cancer and the sixth leading cause of cancer death worldwide(1). In China, esophageal cancer ranks as the fourth cause of cancer-related death based on the GLOBOCAN 2008 estimates (http://globocan.iarc.fr/). Within this particular subgroup, esophageal squamous cell carcinoma (ESCC) is the most frequent type of esophageal malignancy. An area of China with a particularly high incidence of ESCC is located along the borders of the Hebei, Henan and Shanxi Provinces, which surround the Taihang mountain range. In these high-incidence areas, ESCC has been reported to have high heritability
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(40%-93%)(2,3). Furthermore, we previously performed a genetic epidemiological
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investigation involving 10% of the population of Yangquan City, located in the Shanxi Province, which revealed both a crude mortality rate from esophageal cancer in this
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population of 40.12 per 100,000 person-years and a positive familial aggregation(4).
vi
A subsequent segregation analysis of these data indicated a Mendelian autosomal
esophageal cancer in this population(5).
ew
recessive inheritance pattern of a major gene that influences susceptibility to
Recently, the genetic components underlying the etiology of ESCC have been explored with the use of genome-wide association studies (GWAS) of both Japanese(6,7) and Chinese(8-11) populations. However, the risk variants that were identified from these GWAS, such as ALDH2 and PLCE1, among others, can explain only a small fraction (per-allele odds ratios [OR], 1.3-1.5) of the estimated three to ten-fold familial relative risk (RR) of esophageal cancer in first-degree relatives of 5
Downloaded from http://carcin.oxfordjournals.org/ at UB Kaiserslautern on May 21, 2014
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Carcinogenesis
Carcinogenesis
affected individuals in high-risk areas of China(2,4). Therefore, a role for DNA structural variations as a source of the unexplained heritability has been proposed(12). Copy number variation (CNV) is an important form of genomic structure variation. CNV is defined as the addition or deletion of the number of copies of a particular segment of DNA (>1 kb in length) relative to a reference genome sequence(13). CNVs can cause phenotypic alterations via several mechanisms, including the regulation of gene expression by altering the copy number of an entire gene that is susceptible to dosage effects or by altering proximal or distant DNA regulatory regions that can influence transcription of a neighboring gene(14). Recently, common
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CNVs (Minor Allele Frequency≥5%) were found to contribute to certain genetic and
er
developmental diseases, including neuroblastoma, breast cancer and ovarian cancer(15-17). Therefore, analysis of CNVs that are associated with cancer might
Re
identify novel susceptibility genes that contribute to the mechanisms underlying
vi
tumorigenesis.
ew
Here, we conducted a GWAS of CNVs in 128 discordant sibling pairs (DSPs, patient and his or her healthy sibling) from the Yangquan area. After constructing an interactive network of common CNV-associated genes, one locus was validated in an independent replication of samples that consisted of 1,048 northern Chinese Han subjects, and the functional relevance of this CNV was further explored.
Materials and Methods Study population 6
Downloaded from http://carcin.oxfordjournals.org/ at UB Kaiserslautern on May 21, 2014
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All relevant aspects of the study were approved by the Institutional Review Board of the Cancer Hospital and Institute, Chinese Academy of Medical Sciences (CAMS), Beijing, China. The genome-wide discovery sample consisted of 128 DSPs, who were native residents recruited from the Yangquan area, Shanxi Province, China from Jan, 1999 to Oct, 2008; all participants signed an informed consent form prior to their inclusion. Two senior pathologists independently assessed each participant to diagnose ESCC in all DSPs. Demographic and lifestyle information was collected from all participants by interview (Supplementary Note). The basic characteristics of the subjects involved in the GWAS are summarized in Supplementary Table 1. The
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replication sample set consisted of 521 patients with ESCC (median age, 61 yrs; range,
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36-91 yrs), and 527 healthy controls (median age, 58 yrs; range, 35-90 yrs) were recruited from northern China from Oct, 2008 to Feb, 2011; the basal characteristics
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of the replication sample are listed in Supplementary Table 2. All of the healthy
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pathological abnormalities in the esophagus.
vi
control subjects underwent an endoscopic examination to exclude the possibility of
Genome-wide genotyping Genomic DNA was extracted from the peripheral blood of 128 DSPs using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). Single nucleotide polymorphism (SNP) genotyping was performed using the Illumina HumanHap 610-Quad BeadChip (Illumina Inc., San Diego, CA) according to the manufacturer’s instructions. Normalized bead intensity data were analyzed using the Illumina BeadStudio 3.2 7
Downloaded from http://carcin.oxfordjournals.org/ at UB Kaiserslautern on May 21, 2014
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Carcinogenesis
Carcinogenesis
software genotyping module, which converts fluorescence intensity into SNP genotypes. Individuals with sample call rates
ABCC4 Copy Number Variation is Associated with Susceptibility to Esophageal Squamous Cell Carcinoma
Carcinogenesis
r Fo
Manuscript ID: Manuscript Type: Date Submitted by the Author:
Original Manuscript
30-Dec-2013
Sun, YuLin; Cancer Institute & Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, State Key Laboratory of Molecular Oncology Shi, Ni; Cancer Institute & Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, State Key Laboratory of Molecular Oncology Lu, Haizhen; Cancer Institute & Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Department of Pathology Zhang, JinQiang; Cancer Institute & Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, State Key Laboratory of Molecular Oncology Ma, YuLong; Yangquan Cancer Prevention and Treatment Institute, Department of Oncological Surgery Qiao, YuanYuan; Navy General Hospital, Center of Basic Medical Sciences Mao, YongHong; Yangquan Cancer Prevention and Treatment Institute, Department of Oncological Surgery Jia, Kun; Cancer Institute & Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Department of Pathology Han, LiFen; Yangquan Cancer Prevention and Treatment Institute, Department of Oncological Surgery Liu, Fang; Cancer Institute & Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, State Key Laboratory of Molecular Oncology Li, HongXia; Shanxi Population and Family Planning Science Research Institute, Null Lin, Zhengwei; Cancer Institute & Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, State Key Laboratory of Molecular Oncology Li, XinMin; David Geffen School of Medicine, University of California, Department of Pathology & Laboratory Medicine Zhao, XiaoHang; Cancer Institute & Hospital, State Key Laboratory of Molecular Oncology
er
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Complete List of Authors:
CARCIN-2013-00981.R1
ew
vi
Re
Keywords:
ABCC4, Copy number variation, Esophageal squamous cell carcinoma, susceptible gene
Downloaded from http://carcin.oxfordjournals.org/ at UB Kaiserslautern on May 21, 2014
Journal:
Page 1 of 61
ABCC4 Copy Number Variation is Associated with Susceptibility to Esophageal Squamous Cell Carcinoma
YULIN SUN,1,# NI SHI,1,# HAIZHEN LU,4 JINQIANG ZHANG,1 YULONG MA,3 YUANYUAN QIAO,2 YONGHONG MAO,3 KUN JIA,1 LIFEN HAN,3 FANG LIU,1 HONGXIA Li,5 ZHENGWEI LIN,1 XINMIN LI,6 XIAOHANG ZHAO1,2 State Key Laboratory of Molecular Oncology, 4Department of Pathology, Cancer
Institute & Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100021, China; 2Center of Basic Medical Sciences, Navy General
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Hospital, Beijing 100048, China; 3Department of Oncological Surgery, Yangquan
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Cancer Prevention and Treatment Institute, Yangquan 045000, Shanxi Province, China; 5Shanxi Population and Family Planning Science Research Institute, Taiyuan
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030006, Shanxi Province, China; 6Department of Pathology & Laboratory Medicine,
#
These authors are contributed equally.
ew
USA
vi
David Geffen School of Medicine, University of California, Los Angels, CA 90095,
RUNNING TITLE: ABCC4 CNV is associated with ESCC risk To whom correspondence should be addressed: Dr. Xiaohang Zhao, Phone: +86-10-67709015; Fax: +86-10-87778360; [email protected] and Dr. Xinmin Li,
Phone: 310-825-3664; Fax: 310-825-3570; [email protected] Keywords: ABCC4; Copy number variation; Esophageal squamous cell carcinoma; susceptible gene 1
Downloaded from http://carcin.oxfordjournals.org/ at UB Kaiserslautern on May 21, 2014
1
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Carcinogenesis
Carcinogenesis
Word count (excluding references and Figure legends): 5000 words Total number of figures and tables: 6
Downloaded from http://carcin.oxfordjournals.org/ at UB Kaiserslautern on May 21, 2014
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Abstract Esophageal squamous cell carcinoma (ESCC) is the eighth most common cause of cancer-related death worldwide. However, previous genome-wide single nucleotide polymorphism (SNP) association analyses have not explained the high heritability associated with ESCC. In this study, we performed genome-wide copy number variation (CNV) analysis on 128 discordant sibling pairs to identify novel genes that contribute to ESCC susceptibility. A total of 57,774 individual CNVs were identified, and an interactive network of common CNV-associated genes was constructed, which showed that several ABC transporter genes contain CNVs in ESCC patients.
Pe
Independent validation of a CNV at 13q32.1 in 1,048 northern Chinese Han subjects
er
demonstrated that the amplification of ABCC4 significantly correlated with ESCC risk (odds ratio: 3.36 [1.65-7.93], P=0.0013). Immunohistochemistry staining suggested
Re
that high copy numbers correlated with increased protein levels. High expression of
vi
ABCC4 was an independent poor prognostic factor for ESCC (relative risk: 1.73
ew
[1.10-2.73], P=0.0181). The CNV region showed strong enhancer activity. Furthermore, inhibition of ABCC4 protein in ESCC cells decreased cell proliferation and motility via the inhibition of COX-2, PGE2 receptors and c-Myc expression; AKT, ERK and CREB phosphorylation; and β-catenin nuclear translocation in ESCC cells. In conclusion, the CNV at 13q32.1 is associated with ESCC susceptibility, and a gene within this locus, ABCC4, activates the oncogenic pathways in ESCC and thus facilitates cancer cell development and progression. A direct genetic contribution of ESCC risk through CNV common variants was determined in this study, and ABCC4 3
Downloaded from http://carcin.oxfordjournals.org/ at UB Kaiserslautern on May 21, 2014
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Carcinogenesis
Carcinogenesis
might therefore have predictive and therapeutic potential for ESCC.
Summary The genome-wide copy number variation analysis found that the amplification of ABCC4 at 13q32.1 correlated with esophageal cancer risk. Functional study showed that high expression of ABCC4 predicted poor clinical outcomes which activated the Downloaded from http://carcin.oxfordjournals.org/ at UB Kaiserslautern on May 21, 2014
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oncogenic pathways and facilitated tumorigenesis.
er
Pe ew
vi
Re
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 4 of 61
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Introduction Esophageal cancer is the eighth most commonly diagnosed cancer and the sixth leading cause of cancer death worldwide(1). In China, esophageal cancer ranks as the fourth cause of cancer-related death based on the GLOBOCAN 2008 estimates (http://globocan.iarc.fr/). Within this particular subgroup, esophageal squamous cell carcinoma (ESCC) is the most frequent type of esophageal malignancy. An area of China with a particularly high incidence of ESCC is located along the borders of the Hebei, Henan and Shanxi Provinces, which surround the Taihang mountain range. In these high-incidence areas, ESCC has been reported to have high heritability
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(40%-93%)(2,3). Furthermore, we previously performed a genetic epidemiological
er
investigation involving 10% of the population of Yangquan City, located in the Shanxi Province, which revealed both a crude mortality rate from esophageal cancer in this
Re
population of 40.12 per 100,000 person-years and a positive familial aggregation(4).
vi
A subsequent segregation analysis of these data indicated a Mendelian autosomal
esophageal cancer in this population(5).
ew
recessive inheritance pattern of a major gene that influences susceptibility to
Recently, the genetic components underlying the etiology of ESCC have been explored with the use of genome-wide association studies (GWAS) of both Japanese(6,7) and Chinese(8-11) populations. However, the risk variants that were identified from these GWAS, such as ALDH2 and PLCE1, among others, can explain only a small fraction (per-allele odds ratios [OR], 1.3-1.5) of the estimated three to ten-fold familial relative risk (RR) of esophageal cancer in first-degree relatives of 5
Downloaded from http://carcin.oxfordjournals.org/ at UB Kaiserslautern on May 21, 2014
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Carcinogenesis
Carcinogenesis
affected individuals in high-risk areas of China(2,4). Therefore, a role for DNA structural variations as a source of the unexplained heritability has been proposed(12). Copy number variation (CNV) is an important form of genomic structure variation. CNV is defined as the addition or deletion of the number of copies of a particular segment of DNA (>1 kb in length) relative to a reference genome sequence(13). CNVs can cause phenotypic alterations via several mechanisms, including the regulation of gene expression by altering the copy number of an entire gene that is susceptible to dosage effects or by altering proximal or distant DNA regulatory regions that can influence transcription of a neighboring gene(14). Recently, common
Pe
CNVs (Minor Allele Frequency≥5%) were found to contribute to certain genetic and
er
developmental diseases, including neuroblastoma, breast cancer and ovarian cancer(15-17). Therefore, analysis of CNVs that are associated with cancer might
Re
identify novel susceptibility genes that contribute to the mechanisms underlying
vi
tumorigenesis.
ew
Here, we conducted a GWAS of CNVs in 128 discordant sibling pairs (DSPs, patient and his or her healthy sibling) from the Yangquan area. After constructing an interactive network of common CNV-associated genes, one locus was validated in an independent replication of samples that consisted of 1,048 northern Chinese Han subjects, and the functional relevance of this CNV was further explored.
Materials and Methods Study population 6
Downloaded from http://carcin.oxfordjournals.org/ at UB Kaiserslautern on May 21, 2014
r Fo
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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All relevant aspects of the study were approved by the Institutional Review Board of the Cancer Hospital and Institute, Chinese Academy of Medical Sciences (CAMS), Beijing, China. The genome-wide discovery sample consisted of 128 DSPs, who were native residents recruited from the Yangquan area, Shanxi Province, China from Jan, 1999 to Oct, 2008; all participants signed an informed consent form prior to their inclusion. Two senior pathologists independently assessed each participant to diagnose ESCC in all DSPs. Demographic and lifestyle information was collected from all participants by interview (Supplementary Note). The basic characteristics of the subjects involved in the GWAS are summarized in Supplementary Table 1. The
Pe
replication sample set consisted of 521 patients with ESCC (median age, 61 yrs; range,
er
36-91 yrs), and 527 healthy controls (median age, 58 yrs; range, 35-90 yrs) were recruited from northern China from Oct, 2008 to Feb, 2011; the basal characteristics
Re
of the replication sample are listed in Supplementary Table 2. All of the healthy
ew
pathological abnormalities in the esophagus.
vi
control subjects underwent an endoscopic examination to exclude the possibility of
Genome-wide genotyping Genomic DNA was extracted from the peripheral blood of 128 DSPs using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). Single nucleotide polymorphism (SNP) genotyping was performed using the Illumina HumanHap 610-Quad BeadChip (Illumina Inc., San Diego, CA) according to the manufacturer’s instructions. Normalized bead intensity data were analyzed using the Illumina BeadStudio 3.2 7
Downloaded from http://carcin.oxfordjournals.org/ at UB Kaiserslautern on May 21, 2014
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Carcinogenesis
Carcinogenesis
software genotyping module, which converts fluorescence intensity into SNP genotypes. Individuals with sample call rates
