Abstract
IGF2R has been proposed to be a tumor suppressor gene given its antagonist role on cellular growth and evidence of loss of heterozygosity in several cancers, including breast cancer. To investigate whether inherited differences in potentially functional IGF2R variants influence the risk of breast cancer, we sequenced 46 exons of IGF2R to identify novel missense single-nucleotide polymorphisms (SNP) and tested 12 missense SNPs for their associations with breast cancer risk among 1,614 breast cancer cases and 1,960 controls from the Multiethnic Cohort. None of these missense SNPs were significantly associated with breast cancer risk. Our findings provide no evidence that missense SNPs in IGF2R influence breast cancer susceptibility.
Introduction
The insulin-like growth factor-II receptor (IGF2R) is a transmembrane receptor that primarily binds IGF-II, resulting in the degradation of IGF-II by internalization and transport to the lysosomes. By removing IGF-II from the extracellular environment and precluding its activation of the insulin-like growth factor-I receptor (IGF1R), IGF2R is believed to reduce the mitogenic effects of IGF-II (1). Loss of heterozygosity at the IGF2R locus has been reported in breast carcinomas, and somatic missense mutations of the remaining allele have shown alteration in ligand binding (2–4). IGF2R has been proposed to be a tumor suppressor gene given its antagonist role on cellular growth and evidence of loss-of-heterozygosity and loss-of-function mutations in several cancers (5–10), including breast cancer (2–4). To investigate whether inherited differences in potentially functional IGF2R variants influence the risk of breast cancer, we sequenced 46 exons of IGF2R to identify novel missense single-nucleotide polymorphisms (SNP) and tested 12 missense SNPs for their associations with breast cancer risk among 1,614 breast cancer cases and 1,960 controls from the Multiethnic Cohort.
Materials and Methods
Study Subjects
The Multiethnic Cohort study is a large population-based cohort study of more than 215,000 individuals from Hawaii and California. The cohort is composed of predominantly African Americans, Native Hawaiians, Japanese Americans, Latinos, and Whites, who were between the ages of 45 and 75 y when recruited from 1993 to 1996. Further methodologic details of this study are provided elsewhere (11).
The present case-control study was nested in the Multiethnic Cohort, as previously described (12). It includes 1,614 breast cancer cases and 1,960 controls that were frequency matched on age (within 5 y) and race/ethnicity. The majority of these women were postmenopausal at baseline (87% of cases and 82% of controls). This study was approved by the Institutional Review Boards at the University of Hawaii and the University of Southern California.
Sequencing and Validation Genotyping
To identify novel missense SNPs, we successfully sequenced 46 of the 48 exons of IGF2R in 95 advanced breast cancer cases (n = 19 per racial/ethnic group). Two exons (exon 1 and exon 30) did not meet our sequencing criteria of >80% of samples with Phred (base-calling) scores >20 for >80% of the target bases; thus, for these we relied on publicly available SNP information. Further details on sequencing methods are described elsewhere (13). Nineteen IGF2R missense SNPs were identified by sequencing. For validation, these 19 SNPs and 2 additional common missense SNPs (minor allele frequency >0.05) in dbSNP (rs8191754 and rs629849) were genotyped in an independent multiethnic panel of 349 control subjects (n = 69-70 per racial/ethnic group).
Genotyping of Breast Cancer Cases and Controls
Table 1 lists the12 IGF2R missense SNPs that were genotyped in the breast case-control study. Ten SNPs were genotyped using the Sequenom genotyping platform; two SNPs (rs8191754 and rs629849) that failed Sequenom assay design or genotyping were genotyped by the Taq-Man platform. The average concordance rate for ~5% quality control repeats was 99.7% and the average genotyping success was 97.1%. There were no deviations from Hardy-Weinberg equilibrium (P > 0.01 > 1 racial/ethnic group).
Table 1.
Twelve IGF2R missense SNPs used for association testing
| SNP | Exon no. | Position* | Nucleotide change† |
Amino acid change |
PolyPhen prediction‡ |
Minor allele frequency (%) |
||||
|---|---|---|---|---|---|---|---|---|---|---|
| African Americans |
Native Hawaiians |
Japanese Americans |
Latinos | Whites | ||||||
| rs8191746 | 5 | 160365688 | C>T | Pro203Leu | Probably damaging |
2.1 | 0 | 0 | 0 | 0 |
| rs8191754 | 6 | 160368314 | C>G | Leu252Val | Benign | 12.1 | 18.4 | 31.6 | 6.4 | 14.5 |
| rs6413491 | 16 | 160388299 | G>A | Ala724Thr | Benign | 3.9 | 0 | 0 | 0.8 | 0.8 |
| IGF2R_C722T§ | 17 | 160388898 | C>T | Pro772Leu | Probably damaging |
2.9 | 0 | 0 | 0.70 | 0 |
| rs8191808 | 18 | 160389500 | G>C | Val817Leu | Benign | 0 | 0 | 0.7 | 0.7 | 0.7 |
| rs8191844 | 25 | 160402919 | C>G | Thr1184Ser | Possibly damaging |
3.6 | 0 | 0 | 0 | 0 |
| IGF2R_C1194T§ | 25 | 160402949 | C>T | Ser1194Leu | Benign | 0 | 2.2 | 0 | 0 | 0 |
| rs8191859 | 28 | 160405480 | G>A | Gly1315Glu | Possibly damaging |
0 | 0 | 8.0 | 0 | 0 |
| rs629849 | 34 | 160414399 | G>A | Gly1619Arg | Benign | 2.9 | 11.0 | 9.4 | 6.5 | 10.7 |
| IGF2R_C1822T§ | 37 | 160419371 | C>T | Thr1822Met | — | 3.6 | 0 | 0 | 1.43 | 0 |
| rs8191904 | 38 | 160420618 | G>A | Arg1832His | Benign | 4.8 | 0 | 0 | 0 | 0.91 |
| rs8191955 | 48 | 160446006 | C>T | Ala2459Val | Benign | 0 | 2.9 | 0.7 | 0 | 0.7 |
SNP position based on dbSNP reference assembly (Build 36.3).
Minor allele based on all groups combined.
PolyPhen: http://genetics.bwh.harvard.edu/pph/.
Novel missense SNP identified by sequencing.
In silico Analysis
The Polymorphism Phenotype (PolyPhen)4 algorithm (14) was used to predict the potential effect of each missense SNP on IGF2R protein structure and function (Table 1). Predictions are based on sequence, phylogenetic, and structural information to evaluate the degree of damage a variant may have on the structural properties of the protein. A score is assigned to each SNP indicating either “probably damaging,”“possibly damaging,” or “benign” effects on protein function and/or structure.
Statistical Analysis
Odds ratios (OR) and 95% confidence intervals (95% CI) were estimated by unconditional logistic regression to estimate genotype-specific risks, adjusting for age and ethnicity in any analysis that combined racial/ethnic groups. All results were similar when adjusted for established breast cancer risk factors (15). In addition, given that missense SNPs generally have low minor allele frequencies, we examined the association between the aggregate number of variant missense alleles and breast cancer risk to assess whether there was an overrepresentation of variant alleles among cases versus controls. The aggregate number of variant alleles was determined by counting the total number of minor alleles across all 12 missense SNPs (38.5% of controls had >1 variant allele). All reported P values are two-sided.
Results
Nineteen IGF2R missense SNPs were identified by sequencing 46 exons. With genotyping of these 19 SNPs in a multiethnic panel for validation, 3 SNPs were identified to be novel (Supplementary Table S1) because they are not present in the dbSNP database5; 7 SNPs were already present in dbSNP; and 9 SNPs were monomorphic (minor allele frequency <0.01 in all of the five racial/ethnic groups).
Ten validated missense SNPs identified by sequencing and two additional missense SNPs from dbSNP (rs8191746 and rs8191808) were tested in our breast cancer case-control study. There were no associations between these missense SNPs and breast cancer risk (P > 0.06; Table 2). In addition, no association with breast cancer risk was observed for the aggregate number of variant missense alleles in comparison with no variant alleles (OR, 1.07; 95% CI, 0.93-1.25). Similarly, no association was observed for an increment of one variant allele (OR, 1.03; 95% CI, 0.95-1.12). For the aggregate number of variant alleles of the four missense SNPs (rs8191746, IGF2R_C722T, rs8191844, and rs8191859) predicted to have “probably/possibly damaging effects,” no association was observed (OR, 0.97; 95% CI, 0.66-1.42).
Table 2.
Associations between IGF2R missense variants and breast cancer risk
| SNP | Genotype | All |
||
|---|---|---|---|---|
| Cases, n (%) | Controls, n (%) | OR (95% CI)* | ||
| rs8191746 | CC | 1584(99.5) | 1862 (99.4) | 1.00 |
| CT | 8 (0.5) | 11 (0.6) | 0.76 (0.30–1.91) | |
| rs8191754 | CC | 1096 (71.1) | 1354 (71.2) | 1.00 |
| CG/GG | 445 (28.9) | 547 (28.8) | 1.14 (0.95–1.30) | |
| rs6413491 | GG | 1568 (98.6) | 1833 (98.4) | 1.00 |
| GA/AA | 23 (1.4) | 29 (1.6) | 0.85 (0.48–1.48) | |
| IGF2R_C722T | CC | 1572 (99.9) | 1923 (100.0) | 1.00 |
| CT | 1 (0.06) | 1 (0.1) | 1.18 (0.07–18.97) | |
| rs8191808 | CC | 1572 (99.1) | 1846 (99.3) | 1.00 |
| CG | 14 (0.9) | 14 (0.8) | 1.28 (0.60–2.73) | |
| rs8191844 | CC | 1562 (98.3) | 1839 (98.6) | 1.00 |
| CG/GG | 27 (1.7) | 26 (1.4) | 1.12 (0.64–1.96) | |
| IGF2R_C1194T | CC | 1574 (97.9) | 1851 (98.7) | 1.00 |
| CT/TT | 34 (2.1) | 25 (1.3) | 1.40 (0.82–2.39) | |
| rs8191859 | GG | 1557 (98.9) | 1877 (97.4) | 1.00 |
| GA/AA | 17 (1.1) | 50 (2.6) | 0.87 (0.48–1.59) | |
| rs629849 | AA | 1283 (82.8) | 1589 (83.7) | 1.00 |
| AG/GG | 267 (17.2) | 309 (16.3) | 1.06 (0.88–1.28) | |
| IGF2R_C1822T | CC | 1572 (98.7) | 1839 (98.7) | 1.00 |
| CT | 21 (1.3) | 24 (1.3) | 0.92 (0.50–1.68) | |
| rs8191904 | GG | 1551 (99.1) | 1893 (98.4) | 1.00 |
| GA/AA | 14 (0.9) | 31 (1.6) | 0.54 (0.28–1.03) | |
| rs8191955 | CC | 1550 (99.5) | 1910 (99.5) | 1.00 |
| CT | 8 (0.5) | 9 (0.5) | 1.50 (0.55–4.14) | |
Adjusted for age and racial/ethnic group.
Discussion
In summary, our multiethnic study does not support the influence of IGF2R missense SNPs on breast cancer risk. Our study had 80% power to detect a minimum OR of 1.35 and 1.57 for SNPs at 5% and 2% allele frequencies, respectively (α = 0.05, two-sided hypothesis test, log-linear model; ref. 16). In addition, we had 80% power to detect a minimum OR of 1.16 for missense SNPs in aggregate (38.5% of controls had >1 variant allele) under similar parameters. Our study cannot exclude the possibility that common genetic variation in IGF2R may have weak effects on breast cancer risk. Moreover, larger studies, such as in the National Cancer Institute Breast and Prostate Cancer Consortium (17), are indicated to evaluate whether the combined effects of several genes in the IGF pathway are more likely to affect breast cancer susceptibility.
Supplementary Material
Acknowledgments
We thank the participants of this study, who have contributed to a better understanding of the genetic contributions to breast cancer susceptibility.
Grant support: Susan G. Komen Foundation grant BCTR0504392 and National Cancer Institute grants CA 63464 and CA 54281
Footnotes
Note: Supplementary data for this article are available at Cancer Epidemiology Biomakers and Prevention Online (http://cebp.aacrjournals.org/).
Disclosure of Potential Conflicts of Interest No potential conflicts of interest were disclosed.
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