Mannose-binding lectin-2 genetic variation and stomach cancer risk Andrea Baccarelli 1 * , Lifang Hou 2 , Jinbo Chen 2 , Jolanta Lissowska 3 , Emad M. El-Omar 4 , Paolo Grillo 1 , Sara M. Giacomini 1 , Meredith Yaeger 5 , Toralf Bernig 6 , Witold Zatonski 3 , Joseph F. Fraumeni, Jr 2 , Stephen J. Chanock 5,6 and Wong-Ho Chow 2 1 EPOCA Epidemiology Research Centre, Maggiore Hospital IRCCS Foundation, University of Milan, Milan, Italy 2 Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Bethesda, MD 3 Division of Cancer Epidemiology and Prevention, Cancer Centre and M. Sklodowska-Curie Institute of Oncology, Warsaw, Poland 4 Department of Medicine and Therapeutics, Institute of Medical Sciences, University of Aberdeen, Aberdeen, United Kingdom 5 Core Genotyping Facility, Advanced Technology Center, National Cancer Institute, National Institutes of Health, Bethesda, MD 6 Section on Genomic Variation, Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD Deficiency of the mannose-binding lectin (MBL) protein, an anti- gen-recognition molecule involved in systemic and mucosal innate immunity, is determined by variant alleles in MBL2 gene pro- moter and exon-1 regions. We conducted a population-based study on 305 stomach cancer cases and 427 controls in Warsaw, Poland to determine whether MBL2 gene variants predispose to stomach cancer. Single nucleotide polymorphisms (SNPs) in MBL2 were determined by TaqMan TM . The 5 tested MBL2 variants are in complete linkage disequilibrium and comprise 6 different haplo- types. The risk of stomach cancer was increased in subjects carry- ing the H/H promoter genotype (OR 5 1.8, 95%CI 1.1–2.9; p 5 0.020) relative to L/L carriers, after adjustment for age, gender, education and smoking. Carrying at least one D exon-1 allele was associated with nonsignificant excess risk (OR 5 1.5, 95% CI 0.9– 2.4; p 5 0.081). In haplotype analysis, the HYD haplotype was associated with increased risk of stomach cancer when compared with HYA, the most common haplotype (OR 5 1.9, 95% CI 1.1– 3.2; p 5 0.021). In diplotype analysis, subjects carrying the YA/D haplotype combination showed the highest risk (OR 5 3.0, 95% CI 1.2–7.1; p 5 0.015), compared with YA/YA. Further analyses to examine the joint effect of MBL2 and IL-1B polymorphisms, previously shown to predispose to stomach cancer, indicated that the combination of at-risk IL-1B genotypes (CT or TT at location -511) and HYD MBL2 haplotype was associated with a 3.5-fold risk (OR 5 3.5, 95% CI 1.6–7.6; p 5 0.001). Our findings suggest that the codon 52 D MBL2 variant causing a cysteine > arginine replacement, but not B and C variants producing glycine substitu- tions, is specifically associated with gastric cancer risk. ' 2006 Wiley-Liss, Inc. Key words: stomach cancer; mannose-binding lectin; immunologic deficiency syndromes; haplotypes Stomach cancer is the second leading cause of cancer-related death in the world, accounting for 8.6% of cancer diagnoses and 10.4% of cancer deaths. 1 Central and Eastern European popula- tions, including Poland, have the highest incidence and mortality rates for this tumour among Caucasians. 1 Although H. pylori infection is the main risk factor, only a small proportion (<3%) of the infected individuals develop stomach cancer. 2,3 There is an evidence that genes involved in immune and inflammatory response may contribute to differences in susceptibility to H. pylori-related stomach cancer. 4,5 The mannose-binding lectin (MBL) protein, coded by the MBL2 human gene, has been recently identified as a key component in systemic and mucosal innate immunity. 6,7 MBL is able to bind to a range of microbes and subsequently kill them by activating the complement system and promoting complement-independent opso- nophagocytosis. 8 MBL deficiency is considered as one of the most common human immunodeficiencies, 8 determined by variant alleles in MBL2 exon-1 and promoter regions that disrupt MBL oligomeri- zation and complement-activation capacity. 9 Defective MBL2 var- iants have been associated with infectious and autoimmune diseases, such as sepsis in neutropenic patients, meningococcal dis- ease, pneumonia, systemic lupus erythematosus, early-onset rheu- matoid arthritis and celiac disease. 8,10,11 In addition, the risk of acute lymphoblastic leukaemia appeared increased in children car- rying defective MBL2 variants, suggesting modulation of an infec- tive cause that remains to be identified. 12 In previous investigations, we and other groups showed a signif- icantly increased risk of stomach cancer and precancerous gastric lesions in subjects carrying proinflammatory gene polymorphisms of the IL-1 cluster. 5,13–15 Between 2000 and mid-2005, 26 studies on the association of IL-1 polymorphisms and gastric cancer were published, of which 21 found significant positive associations, although the specific genotypes differed somewhat between Cau- casian and Asian populations. 16 We were, therefore, motivated to assess the relationship between other immune-response or inflammatory genes and the risk of stomach cancer. Herein, we report the findings for MBL2 variants and stomach cancer risk in a population-based case-con- trol study in Warsaw, Poland. Material and methods Study design The design of the population-based case-control study of stom- ach cancer has been described in detail previously. 13,17 Briefly, Warsaw residents aged 21–79 years, who were newly diagnosed with stomach cancer (ICD-O 151 or ICD-O-2 C16) between 1994 and 1996, were identified by collaborating physicians in each of the 22 hospitals serving the study area. All diagnoses were patho- logically confirmed. Controls were randomly selected among Warsaw residents from a computerized registry of all legal resi- dents in Poland, the Polish Electronic System of Residence Evi- dency (PESEL), and were frequency-matched to cases by sex and age in 5-year groups. The system was updated monthly, and com- pleteness of registration was estimated to be nearly 100%. The study protocol was approved by the institutional review boards of the National Cancer Institute (NCI), National Institutes of Health, Bethesda, Maryland, USA and M. Sklodowska-Curie Institute of Oncology, Warsaw, Poland. After written informed consent was obtained, detailed information on lifetime tobacco use, alcohol consumption, family history of stomach cancer, child- hood living conditions, demographic background, history of Grant sponsor: NIH. *Correspondence to: Exposure, Epidemiology and Risk Program, Har- vard School of Public Health, 401 Park Drive, Landmark Center, Suite 412F West, P.O. Box 15698, Boston, MA 02215, USA. Fax: 11-617-384- 8745. E-mail: abaccare@hsph.harvard.edu Received 19 December 2005; Accepted 24 March 2006 DOI 10.1002/ijc.22075 Published online 23 May 2006 in Wiley InterScience (www.interscience. wiley.com). Abbreviations: 95% CI, 95% confidence interval; EM algorithm, expec- tation-maximization algorithm; IL-1, interleukin-1; FPRP, false-positive report probability; MBL, mannose-binding lectin protein; MBL2, mannose- binding lectin-2 gene; NIH, National Institutes of Health; OR, odds ratio; SNP, single nucleotide polymorphism. Int. J. Cancer: 119, 1970–1975 (2006) ' 2006 Wiley-Liss, Inc. Publication of the International Union Against Cancer