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Cancer Epidemiology Biomarkers & Prevention Vol. 14, 1319-1321, May 2005
© 2005 American Association for Cancer Research

Short Communication

Association of an Exon 1 Polymorphism in the IGFBP3 Gene with Circulating IGFBP-3 Levels and Colorectal Cancer Risk: The Multiethnic Cohort Study

Loïc Le Marchand1, Laurence N. Kolonel1, Brian E. Henderson2 and Lynne R. Wilkens1

1 Etiology Program, Cancer Research Center of Hawaii, University of Hawaii, Honolulu, Hawaii and 2 Department of Preventive Medicine, Keck School of Medicine, University of Southern California, Los Angeles, California

Requests for reprints: Loïc Le Marchand, Etiology Program, Cancer Research Center of Hawaii, University of Hawaii, 1236 Lauhala Street, Suite 407, Honolulu, HI 96813. Phone: 808-586-2988; Fax: 808-586-2082. E-mail: loic{at}crch.hawaii.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Laboratory and seroepidemiologic studies have suggested that insulin-like growth factor binding protein-3 (IGFBP-3), the main binding protein for IGF-I, may be protective against colorectal cancer. We investigated the association of two polymorphisms (A-202C and G2133C) in the IGFBP3 gene with plasma IGF hormone levels among 887 randomly selected participants in the Multiethnic Cohort study. We found that these two genetic variants were in strong linkage disequilibrium and were both inversely associated with plasma IGFBP-3. However, the effect on plasma IGFBP-3 levels was stronger for the G2133C variant than the A-202C variant. Thus, we assessed the colorectal cancer risk associated with the G2133C in a case-control study of 817 cases and 1,995 controls nested within the Multiethnic Cohort study. Under the assumption of dominant genetic model, carriers of the 1233C allele were at 32% increased risk of colorectal cancer [95 % confidence interval (95% CI) for the odds ratio (OR), 1.07-1.62] and that this effect seemed stronger for the rectum (OR for the C allele, 1.95; 95% CI, 1.35-2.83) than the colon (OR, 1.16; 95% CI, 0.92-1.45). These data suggest that the exon 1 G2133C missense variant in IGFBP3 may be a susceptibility factor for colorectal cancer.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Plasma levels of insulin-like growth factor binding protein-3 (IGFBP-3), the main binding protein for IGF-I, have been inversely associated with colorectal cancer risk in two nested case-control studies (1, 2). IGFBP-3 may be protective by reducing cell proliferation and inducing apoptosis, independently of decreasing the amount of bioactive IGF-I (3). However, it is not clear whether associations of colorectal cancer with IGF hormones are causal or merely correlates of other aspects of the western lifestyle (e.g., overnutrition and physical inactivity) that increase colorectal cancer risk. In an attempt to confirm the specificity of the relationship between IGFBP-3 and colorectal cancer, we investigated the association of two polymorphisms in the IGFBP3 gene with IGF hormone levels and assessed the colorectal cancer risk associated with the more promising of the two in a large case-control study nested within the Multiethnic Cohort study.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
The design and baseline characteristics of the Multiethnic Cohort have been described in detail elsewhere (4). In short, participants are Hawaii and Los Angeles residents who entered the cohort from 1993 to 1996 by completing a 26-page mail questionnaire about demographic factors, lifestyle (including diet and smoking), medical history, medication use, and family history of common cancer. The cohort included 96,810 men and 118,441 women ages 45 to 75 years at cohort creation in 1993. Twenty-six percent were Japanese American, 23% White, 22% Latino, 16% African American, 7% Native Hawaiian, and 6% other ethnic/racial origin. Colorectal cancer cases were identified through the Rapid Reporting System of the Hawaii Tumor Registry and through quarterly linkages to the Los Angeles County Cancer Surveillance Program, two cancer registries that are members of the Surveillance, Epidemiology and End Results program of the National Cancer Institute. This was complemented by annual linkages to the State of California's cancer registry. All incident colorectal cancer cases occurring since January 1995 in the five main ethnic groups were contacted for inclusion in a case-control study and donation of a blood sample, as were a set of controls, randomly selected from the Multiethnic Cohort participants after stratification by sex and race/ethnicity (5). Samples were collected at the subject's home, processed within 8 hours and stored at 80°C. The participation rate was 74% among cases and 66% among controls.

In an IGF hormone substudy, plasma total IGFBP-3 and IGF-I were measured on a random sample of ~1,000 controls (100 in each sex, race/ethnic group) by ELISA (6). DNA was extracted from blood lymphocytes of the subjects using a standard method (Mini Kit, Qiagen, Valencia, CA). IGFBP3 genotyping used the method published by Deal et al. (7) for the A-202C promoter variant (rs2854744) and a modification of the protocol of Eggerman et al. (8) for the exon 1 G2133C (G32A) variant (rs2854746). Primers used for the latter variant were as follows: 5'-TGCAGGCGTCATGCAG-3' and 5'-CAGCTCCGCGCACAC-3'. This gives a 193-bp PCR product. Because the region is GC rich, 1 mol/L Betaine was added to the PCR reaction. The PCR conditions were an initial denaturation at 94°C for 5 minutes followed by 40 cycles at 94°C for 30 seconds, 64°C for 30 seconds, 72°C for 1 minute with an extension at 72°C for 10 minutes. The PCR product was digested with AciI (New England BioLabs, Beverly, MA) at 37°C overnight. The digests were run on a 4.5% MetaPhor agarose gel. The G allele gives fragments of 85, 28, and 23 bp and many smaller fragments, whereas the C allele gives fragments of 66, 19, 28, and 23 bp plus the smaller fragments. Only the 85- and 66-bp fragments are visible on the gel. The authenticity of this assay was confirmed by DNA sequencing.

In the IGF hormone cross-sectional study, analysis of covariance was used to test for differences in mean plasma levels of IGFBP-3 and the IGF-I/IGFBP-3 molar ratio by genotype, adjusting for sex, race, age, and saturated fat intake, which were determinants of IGFBP-3 levels in this population (6). IGF hormone, genotype, and covariate information was available on 887 subjects for G2133C and 876 subjects for A-202C. The hormone measurements were log transformed to meet the model assumption. Linkage disequilibrium was tested by the D' and R2 statistics. In the nested case-control study, unconditional logistic regression was used to compute odds ratios (OR) and 95% confidence intervals (95% CI) for genotype. All available cases and additional controls were genotyped, resulting in 817 cases and 1995 controls with genotype and covariate information. A gene-dosage effect was tested by inclusion of a trend variable assigned 1, 2, or 3 according to the number of variant alleles present (0, 1, or 2, respectively). The likelihood ratio test was used to test for interaction among certain variables with respect to colorectal cancer. The test compares a main effect, no interaction model with a fully parameterized model containing all possible interaction terms for the variables of interest.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The subject characteristics have been described earlier for the nested case-control study (5), as well as for the IGF hormone substudy (6). Thirty-four percent of the colorectal cancer cases were Japanese American, 20% African American, 20% Latino, 19% Caucasian, and 7% Native Hawaiian. Fifty-six percent were males and the mean age ± SD at blood draw was 67.2 ± 7.9 years. Seventy-three percent of the cases had colon cancer and 27% rectal cancer.

The two variants genotyped in the IGF hormone substudy showed high linkage disequilibrium with the following Ds: Whites, 0.96; African Americans, 0.98; Japanese Americans, 0.94; Latinos, 0.91; and Native Hawaiians, 0.92. The corresponding ethnic-specific R2 values were as follows: 0.83, 0.37, 0.89, 0.76, and 0.74, suggesting that recombination may have occurred in African Americans.

Table 1 presents the relationships between genotype and plasma hormone levels in the IGF hormone substudy. For both polymorphisms, the variant alleles were associated with lower mean levels for plasma IGFBP-3 and the molar ratio IGF-I/IGFBP-3. These data are consistent with previous findings for the A-202C variant (7) and are consistent with the variants possibly increasing risk of colorectal cancer.


View this table:
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  		Table 1. Geometric means (95% CIs) for plasma IGFBP-3 and IGF-I/IGFBP-3 by IGFBP3 variant genotypes

 
Next, we simultaneously introduced both variants into the multiple regression of genotype on plasma IGFBP-3 levels and found that only the P for the G2133C variant remained of borderline statistical significance (P = 0.07), whereas the –202C variant showed no significance (P = 0.79). We interpreted this finding as suggesting that the effect of the exon 1 missense variant on hormone levels was greater than that of the –202C variant and that, consequently, the former was the stronger candidate for disease association. Thus, only the G2133C variant was genotyped in cases and additional controls for the nested case-control study.

Based on the controls, the frequency for the 2133C allele was 0.22 for Japanese, 0.56 in Caucasians, 0.66 in African Americans, 0.62 in Latinos, and 0.46 in Native Hawaiians. The genotype distributions were consistent with Hardy-Weinberg equilibrium in each ethnic group, except Latinos (P = 0.02) and Hawaiians (P = 0.01).

Table 2 shows the ORs for colorectal cancer by genotype for the G2133C polymorphism. A statistically significant association was found with a dominant genetic model for the C allele (P = 0.008). The age-, sex-, and race/ethnicity-adjusted OR for the presence of the C allele was 1.32 (95% CI, 1.07-1.62). This effect was only slightly modified by further adjustment for other covariates (Table 2) or after exclusion of Latinos and Hawaiians (OR, 1.29; 95% CI, 1.01-1.15). The ORs for the C allele among Japanese, Caucasian, Hawaiian, African American, and Latino subjects were 1.34 (95% CI, 0.98-1.83), 0.97 (95% CI, 0.61-1.52), 1.42 (95% CI, 0.75-2.70), 1.69 (95% CI, 0.86-3.30), and 1.37 (95% CI, 0.81-2.31), respectively. The effect of the C allele was similar in both sexes and for early-stage (in situ/localized) and late-stage (regional/distant) tumors (data not shown). However, the effect seemed greater for the rectum (OR, 1.95; 95% CI, 1.35-2.83) than the colon (OR, 1.16; 95% CI, 0.92-1.45). No interaction was suggested between IGFBP3 and body mass index or height.


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  		Table 2. Colorectal cancer ORs (95% CIs) for the IGFBP3 G2133C variant

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we found that the IGFBP3 2133C variant allele was associated with lower circulating IGFBP-3 hormone levels and an increased risk of colorectal cancer. This increased risk was suggested in four of the five ethnic groups studied and seemed stronger for rectal cancer.

IGFBP-3 binds 70% to 80% of IGF-I and thus limits availability of this crucial growth factor to cells. IGFBP-3 also exhibits independent antiproliferative and proapoptotic activities (3) and has been shown to inhibit the development of colon tumors in experimental animals (9). Two nested case-control studies observed a marked reduction in colorectal cancer risk with plasma IGFBP-3 (1, 2). However, these findings were not confirmed in subsequent studies that used a different type of ELISA assay to measure plasma IGFBP-3. Indeed, these studies observed a positive association with colorectal cancer (10-12). A recent meta-analysis of these published data estimated the colorectal cancer OR comparing the 75th to the 25th percentile of circulating IGFBP-3 to be 0.77 (95% CI, 0.36-1.66; ref. 13). Thus, the data on IGFBP-3 levels and colorectal cancer remain inconsistent. The independent association of colorectal cancer with inherited genetic variants that affect IGFBP-3 levels, presumably over the lifetime, may clarify this relationship.

In agreement with our data, the A-202C transition polymorphism has previously been associated with lower plasma levels of IGFBP-3 (6, 14); it was not associated with colorectal cancer or breast cancer in recent studies (14, 15). In addition, we found that the exon 1 G32A missense variant was in strong linkage disequilibrium with the –202C variant, was more strongly related to plasma IGFBP-3 levels, and was associated with a 30% increase in colorectal cancer risk. Confirmation of these findings and a systematic characterization of the association between colorectal cancer and SNPs and haplotypes recently identified in this gene are warranted.


   Acknowledgments
 
We thank Jana Koerte and Jennifer Yamamoto for assistance with data analysis and Wendy Chang, Annette Jones, and Ann Seifried for their help with genotyping.


   Footnotes
 
Grant support: National Cancer Institute, U.S. Department of Health and Human Services grants R01 CA 63464 and R01 CA54281.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: The content of this article is solely the responsibility of the authors and does not necessarily represent the official views or policies of these institutions.

Received 11/16/04; revised 2/15/05; accepted 3/10/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Ma J, Pollak MN, Giovannucci E, et al. Prospective study of colorectal cancer risk in men and plasma levels of insulin-like growth factor (IGF)-I and IGF-binding protein-3. J Natl Cancer Inst 1999;91:620–5.[Abstract/Free Full Text]
  2. Giovannucci E, Pollak MN, Platz EA, et al. A prospective study of plasma insulin-like growth factor-1 and binding protein-3 and risk of colorectal neoplasia in women. Cancer Epidemiol Biomarkers Prev 2000;9:345–9.[Abstract/Free Full Text]
  3. Firth SM, Baxter RC. Cellular actions of the insulin-like growth factor binding proteins. Endocr Rev 2002;23:824–54.[Abstract/Free Full Text]
  4. Kolonel LN, Henderson BE, Hankin JH, et al. A multiethnic cohort in Hawaii and Los Angeles: baseline characteristics. Am J Epidemiol 2000;151:346–57.[Abstract/Free Full Text]
  5. Le Marchand L, Wilkens LR, Kolonel LN, Henderson BE. The MTHFR C677T polymorphism and colorectal cancer: The Multiethnic Cohort Study. Cancer Epidemiol Biomarkers Prev 2005;14:1198–203.[Abstract/Free Full Text]
  6. DeLellis K, Rinaldi S, Kaaks RJ, Kolonel LN, Henderson B, Le Marchand L. Determinants of plasma insulin-like growth factor related proteins in the Multiethnic Cohort. Cancer Epidemiol Biomarkers Prev 2004;13:1444–51.[Abstract/Free Full Text]
  7. Deal C, Ma J, Wilkin F, et al. Novel promoter polymorphism in insulin-like growth factor-binding protein-3: correlation with serum levels and interaction with known regulators. J Clin Endocrinol Metab 2001;86:1274–80.[Abstract/Free Full Text]
  8. Eggermann K, Wollmann HA, Tomiuk J, Ranke MB, Kaiser P, Eggermann T. Screening for mutations in the promoter and the coding region of the IGFBP1 and IGFBP3 genes in Silver-Russell Syndrome patients. Hum Hered 1999;49:123–8.[CrossRef][Medline]
  9. Kirman I, Poltoratskaia N, Sylla P, Whelan RL. Insulin-like growth factor-binding protein 3 inhibits growth of experimental colocarcinoma. Surgery 2004;136:205–9.[CrossRef][Medline]
  10. Kaaks R, Toniolo P, Akhmedkhanov A, et al. Serum C-peptide, insulin-like growth factor (IGF)-I, IGF-binding proteins, and colorectal cancer in women. J Natl Cancer Inst 2000;92:1592–600.[Abstract/Free Full Text]
  11. Probst-Hensch NM, Yuan JM, Stanczyk FZ, Gao YT, Ross RK, Yu MC. IGF-I, IGF-2 and IGFBP-3 in prediagnostic serum: association with colorectal cancer in a cohort of Chinese men in Shanghai. Br J Cancer 2001;85:1695–9.[CrossRef][Medline]
  12. Palmqvist R, Hallmans G, Rinaldi S, et al. Plasma insulin-like growth factor 1, insulin-like growth factor binding protein 3, and risk of colorectal cancer: a prospective study in Northern Sweden. Gut 2002;50:642–6.[Abstract/Free Full Text]
  13. Renehan AG, Zwahlen M, Minder C, O-Dwyer ST, Shalet SM, Egger M. Insulin-like growth factor (IGF-I)-I, IGF binding protein-3, and cancer risk: systematic review and meta-regression analysis. Lancet 2004;363:1346–53.[CrossRef][Medline]
  14. Schernhammer ES, Hankinson SE, Hunter DJ, Blouin MJ, Pollak MN. Polymorphic variation at the –202 locus in IGFBP3: influence on serum levels of insulin-like growth factors, interaction with plasma retinol and vitamin D and breast cancer risk. Int J Cancer 2003;107:60–4.[CrossRef][Medline]
  15. Slattery ML, Samowitz W, Curtin K, et al. Associations among IRS1, IRS2, IGF1, and IGFBP3 genetic polymorphisms and colorectal cancer. Cancer Epidemiol Biomarkers Prev 2004;13:1206–14.[Abstract/Free Full Text]



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