Ethnic differences in poly(ADP-ribose) polymerase pseudogene genotype distribution and association with lung cancer risk

Jun Gu1, Margaret R. Spitz1, Fang Yang1 and Xifeng Wu1,2,3

1 Department of Epidemiology, Box 189, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030 and
2 School of Public Health, The University of Texas Health Science Center at Houston, Houston, TX, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Poly(ADP-ribose) polymerase (PADPRP) is a nuclear DNA-binding enzyme that can modulate chromatin structure close to DNA replication, recombination and repair regions. Two-allele polymorphism on the PADPRP chromosome 13 pseudogene has been studied in several ethnic subpopulations, and the association of each allele with different types of cancer has been investigated. To study the frequency of the allele in the context of lung cancer, we performed a PCR assay for the PADPRP polymorphism in 288 lung cancer patients and 292 matched controls and examined the frequency of the alleles in different ethnic groups. Our results showed that the allele distribution was significantly different among members of different ethnic groups. Specifically, the A allele was dominant in Mexican-American and Caucasian groups but not in the African-American group. The frequencies of the B allele in Mexican-American, Caucasian and African-American controls were 0.184, 0.218 and 0.606, respectively, with the Caucasian cases and controls showing an almost identical lower B allele frequency (0.199 in cases versus 0.218 in controls), and the African-American cases and controls showing an almost identical but considerably higher frequency (0.578 in cases versus 0.606 in controls). In contrast, the Mexican-American cases and controls exhibited a considerable difference in the B allele frequency (0.306 in cases versus 0.184 in controls). When we combined subjects with the AB or BB genotype into a susceptible genotype group and compared them with the AA group using univariate analysis, the susceptible genotype was not shown to be associated with a risk of lung cancer in either the Caucasian or African-American subpopulation but was significantly associated with an increased risk (2.29-fold) of lung cancer in the Mexican-American group. When lung cancer was categorized by histologic type, no elevated risk was noted for squamous cell carcinoma in any ethnicity. However, in Mexican-Americans, susceptible genotypes were associated with significantly increased risks of adenocarcinoma (3.21-fold) and large cell carcinoma (10.79). Our study and others have demonstrated that the PADPRP polymorphism may modify an individual's susceptibility to certain cancers. Assessment of the interaction between genetic constitution and environmental exposure might expand our understanding of carcinogenesis and enhance our ability to evaluate the populational cancer risk.

Abbreviations: CI, confidence interval; OR, odds ratio; PADPRP, poly(ADP-ribose) polymerase


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Poly(ADP-ribose) polymerase (PADPRP) is a DNA-binding protein that can modulate chromatin structure close to DNA replication, recombination and repair regions in the cell (17). The PADPRP gene has been assigned to 1q42, and two processed pseudogenes have also been identified in chromosomes 13 and 14 (8). In addition, a simple two-allele (the common A allele and the less common B allele) polymorphism, localized to chromosome 13q33-qter, has been observed in tumor cells (9). However, the relationship between the PADPRP genetic polymorphism and cancer risk is controversial. Some groups have shown that the frequency of the B allele is increased in patients with certain cancers, including multiple myeloma, prostate cancer, the B-cell tumor Burkitt's lymphoma and lung cancer (912); however, others have not confirmed this finding (13). This apparent discrepancy may actually stem from underlying variation in the genetic background of members of different ethnic groups (14). This is illustrated by the observations of Bhatia et al. (9), who showed that the frequency of the B allele in germline DNA was 35% in African-Americans without cancer, compared with 14% in Caucasians without cancer. Cao et al. (11) also found that the B allele was more prevalent in African-American patients with multiple myeloma than in controls without cancer, but that this was not the case for Caucasian patients and controls. We observed previously that the B allele prevalence was 0.598 in African-Americans but only 0.196 in Mexican-Americans (15). Lyn et al. (10) found that there was no relationship between this genetic polymorphism and lung cancer risk. However, a drawback to their study was that the study population consisted of only nine patients. Previously, we examined the polymorphism and lung cancer risk in African-Americans and Mexican-Americans and found that this polymorphism was associated with lung cancer risk (15). In the study we describe here, we extended our original research by recruiting more subjects for these two minority populations and including Caucasian case and control populations. In this way, we attempted to further examine the prevalence of the PADPRP pseudogene genotype in three ethnic groups and to confirm the ethnic differential relationship between a potentially susceptible genotype and lung cancer risk.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Study populations
A total of 580 individuals (288 with histologically confirmed, previously untreated lung cancer and 292 controls) were analyzed for this study. The Caucasian patients were recruited from The University of Texas M.D. Anderson Cancer Center, and minority patients were also recruited from various county, community and Veterans affairs hospitals in the Houston, Galveston and San Antonio metropolitan areas. Caucasian controls were recruited from the largest health maintenance organization in the Houston metropolitan areas. Mexican-American and African-American controls were also recruited from community centers and cancer screening programs. All controls were matched to cases by age, sex and ethnicity. Caucasian subjects were also matched by smoking status. After informed consent was obtained, all subjects were interviewed by well-trained interviewers, who obtained information about sociodemographic and other risk factors. At the end of the interview, blood samples were collected by venipuncture into sodium-heparinized vacutainers for molecular genetic analysis.

Polymorphism analysis
DNA was extracted from the peripheral blood lymphocytes of each individual using a standard method. Techniques described by Lyn et al. (10) were used to amplify the polymorphic area in the chromosome 13 PADPRP pseudogene. The following primers were used: 5'-AAG AAG CCA ACA TCT GAG CT-3' and 5'-TTT CCT TGT CAT CCT TCA GC-3'. These primers have mismatches at their 3' ends and internal mismatches that prevent the amplification of related PADPRP sequences on chromosomes 1 and 14. PCR was performed in 25 µl reaction mixtures containing 1.5 mM MgCl2, 0.2 mM dNTPs, 1 µM primers, 1 µg of template DNA, 1.5 U of Taq polymerase and PCR buffer (20 mM Tris–HCl pH 8.4 and 50 mM KCl; Boehringer Mannheim, Indianapolis, IN). After an initial denaturation at 94°C for 4 min, the DNA was amplified by 10 cycles of 45 s at 94°C, 60 s at 60°C and 90 s at 72°C, followed by 25 cycles of 30 s at 94°C, 40 s at 62°C and 60 s at 72°C, and a final extension step of 5 min at 72°C. The PCR products were then resolved on a 2.1% agarose gel (at 5 V/cm) containing ethidium bromide. The PADPRP A allele fragment was 595 bp; the B allele was 402 bp.

Statistical analysis
Statistical analysis was performed separately for each ethnic group. Pearsons {chi}2 test was used to compare the distribution of host characteristics and PADPRP pseudogene genotypes between cases and controls. The B allele frequencies were calculated and compared under different forms of stratification. The odds ratio (OR) was calculated to estimate PADPRP susceptible genotypes, smoking status and lung cancer risk using the method of Woolf (16). A person who had smoked at least 100 cigarettes in his or her lifetime was regarded as a smoker. A former smoker was defined as a person who had stopped smoking at least 1 year before diagnosis in the case of patients and 1 year before the study began in the case of controls. Light and heavy smokers were determined by <30 and >=30 pack-years, respectively, which is the median value of the pack-year for the overall controls. Logistic regression analysis was performed to estimate risks, which were then adjusted for multiple factors using STATA statistical software (17).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Characteristics of study population
There were 123 cases and 101 controls in the Caucasian group, 67 cases and 87 controls in the Mexican-American group, and 98 cases and 104 controls in the African-American group (Table IGo). There were no significant differences in the age and sex distributions between the cases and controls in the three ethnic groups. There was also no significant difference in the smoking status of cases and controls in the Caucasian group, since Caucasian controls were also matched to cases by smoking status. In the Mexican-American and African-American groups, however, there were significantly more smokers among the cases than the controls. Mexican-American and African-American cases who were former smokers also reported significantly fewer years of cessation than did their controls.


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Table I. Distribution of select host characteristics by case-control status in lung cancer patients and controls
 
Distribution of select host characteristics by chromosome 13 PADPRP pseudogene genotypes and B allele frequency
The distribution of chromosome 13 PADPRP pseudogene genotypes and B allele frequency by disease status is shown in Table IIGo for each ethnic group and sex. Regardless of sex, the B allele frequency was higher in African-Americans than it was in either the Caucasian or Mexican-American groups. Specifically, the frequencies of the B allele in the Caucasian, Mexican-American and African-American groups were 0.218, 0.183 and 0.606 in the controls and 0.199, 0.306 and 0.578 in the case groups, respectively. Only Mexican-American cases exhibited a significantly higher B allele frequency than their counterpart controls (0.306 versus 0.183), but this overall difference was actually attributable to the high frequency of the B allele among male cases.


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Table II. Distribution of chromosome 13 PADPRP pseudogene genotypes and B allele frequency by lung cancer case control status and ethnicity
 
Lung cancer risk estimates associated with chromosome 13 PADPRP pseudogene genotype, sex and smoking status
Since the number of individuals with the BB genotype was small, we combined those with the AB genotype into one susceptible group and compared it with the group with the AA genotype for each ethnic subgroup. The combined AB and BB genotype was associated with increased risk for lung cancer only in the Mexican-American group. Specifically, univariate analysis showed a 2.29-fold increased risk of lung cancer in the Mexican-American group [95% confidence interval (CI) = 1.18, 4.43]. The OR (95% CI) after adjustment for age, sex and smoking status was 2.17 (1.06, 4.44). However, the risk was especially high in Mexican-American males [OR = 2.59 (1.10, 6.10)], though not in the females [OR = 1.00 (0.24, 4.24)] (data not shown). When lung cancer was categorized by histologic type, no elevated risk was noted for squamous cell carcinoma in any ethnicity (Table IIIGo). However, in Mexican-Americans, susceptible genotypes were associated with significantly increased risks for adenocarcinoma and large cell carcinoma with ORs (95% CI) of 3.21 (1.14, 9.08) and 10.79 (1.16, 100.19), respectively. In African-Americans, the risk was elevated for adenocarcinoma with an OR of 3.82 (0.99, 14.73).


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Table III. Association of susceptible genotypes and lung cancer risk by histologic type
 
In addition, a strong association between PADPRP susceptibility genotype and cigarette smoking was observed on stratified analysis in Mexican-Americans (Table IVGo). In particular, compared with subjects who had the AA genotype but had never smoked, the ORs (95% CI) for having the PADPRP susceptibility genotype, being a light smoker (smoked <30 pack-years), and having both risk factors combined in the Mexican-Americans were 0.86 (0.15, 4.97), 2.00 (0.58, 6.91) and 7.80 (2.22, 27.37), respectively. The ORs (95% CI) for having the PADPRP susceptibility genotype, being a heavy smoker (smoked >=30 pack-years), and having both risk factors combined in the Mexican-Americans were 0.86 (0.15, 4.97), 24.00 (6.14, 93.75) and 57.00 (10.03, 323.97), respectively. The joint effect was greater than multiplicative.


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Table IV. Association of susceptible genotypes (AB+BB), smoking status, and lung cancer risk for each ethnic group
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The aim of this study was to further explore ethnic differences in the prevalence of the B allele and the likelihood that ethnic differences play a role in determining the risk of lung cancer. The overall study population included 288 additional subjects (167 patients and 121 controls) and an additional group of Caucasian subjects.

We observed significant ethnic differences in the frequency of the B allele in the control populations. The frequency was highest in African-Americans (0.606), followed by Caucasians (0.218) and Mexican-Americans (0.184), frequencies that were similar to 0.56 in African-Americans and 0.20 in Caucasians, reported by Doll et al. (13), but higher than 0.35 and 0.14, reported by Bhatia et al. (9).

Although an association between this genetic polymorphism and lung cancer risk has been reported in the context of lung cancer, the association seems to be largely restricted to African-Americans (9,10). For example, Bhatia et al. (9) observed a 2-fold increase in the frequency of the B allele in African-American patients with lung cancer (0.70) as compared with the frequency in a non-cancer population (0.35). In contrast, although Lyn et al. (10) found that the B allele frequency was 0.61 in African-American patients with lung cancer, they did not observe that the B allele was associated with an increased risk in their study population. However, all these studies involved small numbers of subjects, which diminishes the meaningfulness of the findings.

Few data are available on the potential role of PADPRP in the development of lung cancer in various ethnic groups. We therefore also examined the relationship between the PADPRP genetic polymorphism and lung cancer risk in Mexican-Americans, African-Americans and Caucasians. However, we found that there was no association between an increased B allele frequency and lung cancer in Caucasians, which was consistent with the findings from other studies (9,10). Although we noted that the frequency of the B allele was significantly higher in African-American controls (0.606) than in Mexican-American controls (0.184), the frequency of the allele in African-American patients with lung cancer was not higher than that in African-American controls. Only in Mexican-Americans, especially males, was there a statistically significant increase in the B allele frequency in the patients with lung cancer as compared with controls, suggesting an association between the genetic polymorphism and an increased lung cancer risk in this subpopulation. When lung cancer was analyzed by histologic types, we found that there was no association between specific histologic types and the PADPRP polymorphism in Caucasians. There were no associations between squamous cell carcinoma or small cell carcinoma and the polymorphism in any of the three ethnic groups. However, adenocarcinoma and large cell carcinoma were significantly associated with the susceptible genotype for Mexican-Americans. A 4-fold elevated risk was also noted for adenocarcinoma in African-Americans. These results suggest that there is a relationship between the PADPRP polymorphism and a predisposition to lung cancer, but one that depends on the racial group and histologic type. As to the gender difference in the association of lung cancer risk and susceptibile genotype in Mexican-Americans, it might be due to a specificity for histologic type. In Mexican-Americans, adenocarcinoma was present in 37.25% of male cases compared with only 21.43% of female cases. Squamous cell carcinoma was present in 21.57% of male cases compared with 35.71% of female cases.

We also observed a strong gene–environment interaction in terms of the PADPRP susceptibility genotype and smoking status in Mexican-Americans. Specifically, the risk for lung cancer was dramatically increased in those Mexican-Americans who had both the PADPRP susceptibility genotype and smoking exposure. However, as to the underlying mechanism, it is not clear whether the association results from expression of the PADPRP chromosome 13 gene or from linkage disequilibium with some other polymorphism. One gene of interest is the xeroderma pigmentosum complementation group G genes, also known as ERCC5, which is located at 13q33-34. Since this gene is involved in nucleotide excision repair, it may contribute to the development of lung tumorigenesis.

In conclusion, our data showed that the frequency of the B allele differed among the three ethnic groups studied. They also showed that the B allele of the chromosome 13 PADPRP pseudogene can be used as a marker of lung cancer, though mainly among Mexican-Americans.


    Acknowledgments
 
We thank Ms Beth Notzon for editorial assistance, and Ms Susan Honn for recruiting study subjects. This study was supported by Grants CA 55769 and CA 68437 from the National Cancer Institute and Devereaux Award from Cancer Research Foundation of America.


    Notes
 
3 To whom correspondence should be addressed Email: xwu{at}notes.mdacc.tmc.edu Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received August 6, 1998; revised March 24, 1999; accepted April 27, 1999.