Affiliations of authors: G. Severi, P. Boyle, Division of Epidemiology and Biostatistics, European Institute of Oncology, Milan, Italy; G. G. Giles, D. R. English, Cancer Epidemiology Centre, The Cancer Council Victoria, Melbourne, Australia; M. C. Southey, A. Tesoriero (Department of Pathology), J. L. Hopper (Centre for Genetic Epidemiology), The University of Melbourne, Melbourne; W. Tilley, P. Neufing, Flinders Cancer Centre, Flinders University and Flinders Medical Centre, Adelaide, Australia; H. Morris, Hanson Institute, Adelaide; M. R. E. McCredie, Department of Preventive and Social Medicine, Dunedin Medical School, University of Otago, Dunedin, New Zealand.
Correspondence to: John L. Hopper, Ph.D., The University of Melbourne, Centre for Genetic Epidemiology, Level 2, 723 Swanston St., Carlton, Victoria 3053, Australia (e-mail: j.hopper{at}unimelb.edu.au).
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ABSTRACT |
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INTRODUCTION |
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The reverse argument has been used to support the claim that the ELAC2 gene is a prostate cancer susceptibility gene. ELAC2 was recently implicated in prostate cancer risk because mutations in this gene were reported to segregate with the disease in multiple-case families (7,8) and because two common polymorphisms (Ser217Leu and Ala541Thr) were apparently associated with a modestly increased risk (9). The Ser217Leu polymorphism appeared to act in a recessive fashion and Ala541Thr in a dominant fashion. The apparent association of these polymorphisms with prostate cancer was a major factor in the claim by some investigators that ELAC2 should be renamed HPC2. However, to date, only one study (10) has confirmed this association, and four larger studies (1114) have found no evidence for the association. A possible reason for the inconsistent findings among the seven studies is that in three studies (9,10,12), the control subjects were excluded if they had an elevated prostate-specific antigen (PSA) level, and in two others (11,14), both case patients and control subjects were selected on the basis of elevated PSA levels. If there was a relationship between PSA levels and the ELAC2 polymorphisms, then excluding control subjects with elevated PSA levels would decrease the prevalence of the genotype(s) associated with higher PSA levels and perhaps provide spurious evidence of an association of the polymorphism with prostate cancer risk. Another issue is statistical power; given their sample sizes, no single study reported to date could exclude odds ratios (ORs) of prostate cancer risk associated with Ser217Leu and Ala541Thr polymorphisms (acting recessively and dominantly) of 1.5 and 2.5, respectively, or less with 80% power at the .05 level of significance.
We sought to resolve the question of whether the Ser217Leu and Ala541Thr polymorphisms in ELAC2 are associated with risk of prostate cancer, possibly through an association with PSA level, by using a population-based casecontrol study in which the polymorphisms were measured in case patients and control subjects and plasma PSA levels were measured in control subjects. To help resolve the question, we also report a meta-analysis of all published studies of the association between the two ELAC2 polymorphisms and prostate cancer, including the present study.
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SUBJECTS AND METHODS |
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A population-based casecontrol study of prostate cancer was carried out in Melbourne, Sydney, and Perth, Australia, from 1994 through 1997, with data from population-complete state cancer registers (15); men from Sydney were not included in the current study. Eligible case patients were identified as those men diagnosed before the age of 70 years with histopathologically confirmed carcinoma of the prostate, excluding tumors with Gleason scores of less than 5. Random samples of 100%, 50%, and 25%, respectively, of the case patients diagnosed in the age groups younger than 60 years, 6064 years, and 6569 years were asked to participate in this study. Eligible control subjects were identified through government electoral rolls (registration by adults for voting is compulsory in Australia) and were frequency matched to the age distribution of the case patients in a ratio of one control subject to one case patient.
All consenting subjects were administered a questionnaire to record family history of cancer and other known or potential risk factors for prostate cancer. We defined a family history of prostate cancer as having at least one first-degree relative diagnosed with the disease. We interviewed 1052 control subjects and 1040 case patients from Melbourne and Perth [50% and 65%, respectively, of those eligible (15)]. Blood samples were collected from 745 control subjects (71%) and 862 case patients (83%). The probability of obtaining a blood sample (data not shown) was independent of age, educational level, and ever being a smoker (all P>.05), but it was greater for case patients than for control subjects (P<.001). It was also greater for men born in Australia than for those born elsewhere (P<.001) and for men with a family history of prostate cancer than for men without such a history (P = .02). The strengths of the effects of country of birth and family history, in terms of ORs of blood sampling, were similar and not statistically different in case patients and control subjects (all P>.4). In case patients, blood sampling was not statistically significantly associated with tumor stage or grade (both P>.06). The great majority of subjects were born in Australia, the British Isles, or Western Europe and were of Caucasian descent; 14 control subjects (2%) and nine case patients (1%) were born in Asia. Statistical analyses were carried out on the 732 control subjects (98% of control subjects with blood samples) and the 825 case patients (96% of case patients with blood samples) for whom both genotypes were measured and complete information on essential variables (i.e., age and family history of prostate cancer) was available.
Genotyping
Buffy coats were prepared by centrifugation of 10 mL of fresh blood (collected in tubes containing EDTA) at 2000g for 15 minutes. Plasma was removed, and the interphase (buffy coat) layer was removed and stored at -70 °C. Genomic DNA was extracted from the stored buffy coats with a QiAmp Blood Midi kit (Qiagen, Valencia, CA). We used only the internal primers of the nested polymerase chain reaction (PCR) approach described by Rebbeck et al. (9) to amplify the regions of interest in ELAC2. The PCRs and subsequent restriction fragment length polymorphism (RFLP) analyses were performed under standard conditions (9), and products were visualized on 3% agarose gels. RFLP genotype data were confirmed in several ways. PCR products identified to be encoding the Thr541 polymorphism via RFLP analysis were manually sequenced to confirm the genotype. All samples homozygous for Leu217 were redigested to confirm the result (and rule out incomplete digestion). A parallel analysis of a random selection of DNA representing 5% of all DNA in the study was performed via manual sequencing.
Measurement of Plasma PSA Levels
Plasma was separated by centrifugation of 10 mL of fresh blood (collected in tubes containing EDTA) at 2000g for 15 minutes and stored in 0.5-mL aliquots at -70 °C. Plasma PSA levels were measured by a Microparticle Enzyme Immunoassay (AxSYM analyzer; Abbott Laboratories, Abbott Park, IL). The coefficient of variation of the assay at 0.4 ng/mL was 9.5%.
Statistical Analyses
Estimates of allele frequencies and tests of deviation from HardyWeinberg equilibrium were carried out using standard procedures based on asymptotic likelihood theory (16). Casecontrol analyses were conducted with unconditional logistic and polytomous regression (17), adjusting for reference age (age at diagnosis for case patients and age at selection for control subjects), year, study center (Melbourne; Perth), country of birth (Australia; other), and family history (none; any first-degree relative diagnosed with prostate cancer). Study center and calendar year were included because the diagnostic zeal for prostate cancer screening using PSA varied between centers and over time. Unconditional logistic regression was also used to test for factors associated with blood sampling. Linear regression was used to study the variation of the logarithm of PSA level by genotype, adjusting for age.
We identified the studies to include in the meta-analysis by searching MEDLINE for the keywords "prostate cancer," "ELAC2," "HPC2," and "polymorphisms" for publications listed up to mid-August 2002. Meta-analyses were conducted with standard methods for combining the crude estimates of ORs based on the weighted sum of the log(ORs) with the inverse of the variance as weight (18). Homogeneity in ORs across studies was tested by calculating the weighted (inverse of variance) sum of the squared differences between the log(OR) and the log(pooled OR) estimates, assuming that this statistic follows a chi-square distribution with n 1 degrees of freedom (where n = number of studies). Homogeneity in the genotype frequencies across studies was tested by fitting a factor for each study in unconditional logistic regression models separately for case patients and control subjects and by using the likelihood ratio test.
Statistical analyses were carried out with S-plus software (19). All statistical tests were two-sided.
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RESULTS |
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We next examined the association between the two polymorphisms and prostate cancer risk. Table 2 shows that, when all control subjects were used as a reference group, no statistically significant association of either polymorphism with prostate cancer was observed, either overall or in subgroups with or without a family history of prostate cancer. In addition, no association with genotypes defined by combinations of the two polymorphisms was detected. We did observe a marginally statistically significantly lower risk of prostate cancer in men without a family history of prostate cancer who were homozygous for Leu217 (OR = 0.64, 95% CI = 0.42 to 0.97; P = .04). However, the patterns of risk by genotype were similar in men with or without a family history of prostate cancer (all P
.06) and in men with moderate- or high-grade tumors (data not shown). There was little difference between crude and adjusted risk estimates (data not shown).
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Meta-Analysis
We carried out a meta-analysis of all eight [the seven previous (814) and this one] studies of the association between the Ser217Leu and Ala541Thr polymorphisms and prostate cancer to obtain pooled estimates of association and test for their statistical significance, to test for heterogeneity across studies in terms of ORs and genotype frequencies, and perhaps to provide insight into the reasons for any inconsistencies. The more recent studies have involved substantially larger sample sizes [typically about 1000 subjects (11,13,14) compared with 500600 subjects in earlier studies (810,12)] and therefore had more precision in risk estimates and gave point estimates closer to unity and CIs that overlapped unity (Figs. 1 and 2
). The three studies (810) that found evidence for a nominally statistically significant association (as indicated by a 95% CI that did not include unity), which included the first two studies to be published (8,9), were among the smaller studies and had the least information (as indicated by having the widest 95% CIs). The pooled OR estimates for prostate cancer risk from the meta-analysis (Fig. 1
) were 1.04 (95% CI = 0.85 to 1.26) for men homozygous for Leu217 compared with men heterozygous for Leu217 or homozygous for Ser217 [recessive model, data not available from (9) and (13); P = .7], 1.06 (95% CI = 0.93 to 1.20) for men homozygous or heterozygous for Leu217 compared with men homozygous for Ser217 [dominant model, data not available from (8)]; P = .4), and 1.18 (95% CI = 0.98 to 1.42) for men heterozygous or homozygous for Thr541 (P = .08) compared with men homozygous for Ala541. There was some evidence for at least marginally statistically significant heterogeneity among the studies for the ORs of prostate cancer associated with being homozygous for Leu217 (P = .1) and for being heterozygous or homozygous for Thr541 (P = .02).
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To test for such an association, we measured plasma PSA levels in control subjects, but we found no evidence for an association between either polymorphism and PSA level. Fig. 3 shows PSA levels by genotypes of the Ser217Leu and Ala541Thr polymorphisms. The geometric mean PSA level was 1.15 ng/mL for Ser217 homozygotes; after adjusting for age, the geometric means were 11% (95% CI = -3% to 28%; P = .2) higher for Ser217 heterozygotes and 6% (95% CI = -25% to 18%; P = .7) lower for Leu217 homozygotes. The geometric mean PSA level was 1.19 ng/mL for Ala541 homozygotes; after adjusting for age, the geometric mean was 6% (95% CI = -17% to 36%) higher for the Thr541 heterozygotes and homozygotes combined. After adjusting for study center, country of birth, and family history, the estimates changed only marginally, the standard errors and hence CIs were essentially unchanged and, in all instances, the differences in mean PSA levels between genotypes were not statistically significant.
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DISCUSSION |
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When interpreting the eight studies in the meta-analysis, some issues related to the sampling of case patients and control subjects need to be considered. First, some studies used case patients sampled from one or more sources, such as multiple-case families (8,10,1214), urology clinics (9,12), or PSA screening programs (11), whereas this study and one other (13) used a population register. Consequently, the prostate cancer phenotype is likely to be heterogeneous across studies; if so, this heterogeneity may be masking the existence of a small genetically defined subtype at increased risk. Second, some studies used control subjects sampled through hospitals (9), PSA screening programs (1012,14), and blood donors (13), and some subjects were even of unstated provenance (8), whereas we used a population register. Third, some of the sampling designs of these studies are potentially problematic because they are prone to several types of selection bias, and their genotype distributions are not necessarily generalizable to the general population. Some designs could also have given different risk estimates, depending on whether or not PSA levels were strongly related to the genotype of one or both of these ELAC2 polymorphisms. However, our data (Fig. 3) indicate that such a relationship between PSA levels and these two ELAC2 polymorphisms is unlikely.
The most plausible reason for the differences between study findings is chance. Despite the different study designs and response rates, the data in Fig. 2 show that the larger studies generally reported similar genotype frequencies for case patients and control subjects. Variations in frequencies between studies were not inconsistent with sampling errors (for studies with 200800 subjects and genotype frequencies of 10%, the widths of the 95% CIs vary from 8% to 4%). Across all studies, the frequency of Leu217 homozygotes was similar across case patients and control subjects, as was the frequency of Thr541 heterozygotes and homozygotes combined across case patients. In contrast, the frequency of the Thr541 genotype was not homogeneous across control subjects (Fig. 2
). Given that the selection criteria for controls differed across studies, this raised the possibility that PSA level might differ by genotype and hence explain discrepancies between study findings. However, our estimates of prostate cancer risk by genotype were not changed by excluding control subjects with elevated plasma PSA levels. Therefore, it is unlikely that increased risks of twofold or more, as originally reported (8,9), pertain to these polymorphisms.
Several criteria have recently been proposed for assessing associations between genetic polymorphisms and disease (20); the claim was that studies "ideally . . . should have large sample sizes, small P values, report associations that make biological sense and alleles that affect the gene product in a physiologically meaningful way," although the latter condition ignores the possible influence of linkage disequilibrium. The three studies (810) that reported a nominally significant association between the two ELAC2 polymorphisms and prostate cancer satisfied the criterion based on a small P value, but those studies now appear to be outliers because of the low frequencies of the putative risk alleles, especially Thr541, in control subjects. Given the lack of compelling evidence from other investigations of multiple-case prostate cancer families for the existence of mutations in ELAC2 that confer a high risk of the disease (1114) and the lack of evidence from our study and the meta-analysis that the common polymorphisms are associated with any substantial increased risk for prostate cancer, it is premature to refer to ELAC2 as HPC2.
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NOTES |
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We thank the many urologists who kindly assisted us by providing information and access to their patients. We express our gratitude to the many men who participated.
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Manuscript received August 13, 2002; revised March 20, 2003; accepted March 28, 2003.
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