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Cancer Epidemiology Biomarkers & Prevention Vol. 15, 762-768, April 2006
© 2006 American Association for Cancer Research

Polymorphisms in the hMSH2 Gene and the Risk of Primary Lung Cancer

Chi Young Jung1, Jin Eun Choi2, Jung Min Park2, Myung Hwa Chae2, Hyo-Gyoung Kang2, Kyung Mee Kim2, Su Jeong Lee2, Won Kee Lee3, Sin Kam3, Seung Ick Cha1, Chang Ho Kim1, Sung Beom Han4, Tae Hoon Jung1, Su Han Jeon5 and Jae Yong Park1,2

1 Department of Internal Medicine, 2 Cancer Research Institute, and 3 Department of Preventive Medicine, Kyungpook National University; 4 Department of Internal Medicine, School of Medicine, Keimyung University; and 5 Department of General Surgery, Kyungpook National University Hospital, Daegu, Korea

Requests for reprints: Jae Yong Park, Department of Internal Medicine, Kyungpook National University Hospital, Samduk 2Ga 50, Daegu 700-412, Korea. Phone: 82-53-420-5536; Fax: 82-53-426-2046. E-mail: jaeyong{at}kyungpook.ac.kr


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Polymorphisms in the DNA repair genes may be associated with differences in the capacity to repair DNA damage, and so this can influence an individual's susceptibility to lung cancer. To test this hypothesis, we investigated the association of hMSH2 –118T>C, IVS1+9G>C, IVS10+12A>G, and IVS12–6T>C genotypes and their haplotypes with the risk of lung cancer in a Korean population. The hMSH2 genotypes were determined in 432 lung cancer patients and in 432 healthy controls who were frequency matched for age and gender. The hMSH2 haplotypes were estimated based on a Bayesian algorithm using the Phase program. The presence of at least one IVS10+12G allele was associated with a significantly decreased risk of adenocarcinoma, as compared with the IVS10+12AA genotype [adjusted odds ratio (OR), 0.59; 95% confidence interval (95% CI), 0.40-0.88; P = 0.01], and the presence of at least one IVS12-6C allele was associated with a significantly increased risk of adenocarcinoma, as compared with the IVS12-6TT genotype (adjusted OR, 1.52; 95% CI, 1.02-2.27; P = 0.04). Consistent with the results of the genotyping analysis, the TGGT haplotype with no risk allele was associated with a significantly decreased risk of adenocarcinoma, as compared with the TCAC haplotype with two risk allele [i.e., IVS10+12A and IVS12-6C allele; adjusted OR, 0.49; 95% CI, 0.30-0.78; P = 0.003 and Pc (Bonferroni corrected P value) = 0.012]. The effect of the hMSH2 haplotypes on the risk of adenocarcinoma was statistically significant in the never smokers and younger individuals (adjusted OR, 0.45; 95% CI, 0.27-0.75; P = 0.002 and Pc = 0.004; and adjusted OR, 0.44; 95% CI, 0.23-0.85; P = 0.014 and Pc = 0.028, respectively) but not in the ever-smokers and older individuals. These results suggest that the hMSH2 polymorphisms and their haplotypes may be an important genetic determinant of adenocarcinoma of the lung, particularly in never smokers. (Cancer Epidemiol Biomarkers Prev 2006;15(4):762–8)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Although cigarette smoking is the major cause of lung cancer, only a small fraction of smokers develop the disease, and this suggests that genetic factors contribute to the risk of lung cancer. This genetic susceptibility may result from inherited polymorphisms in the genes involved in the carcinogen metabolism and in the repair of DNA damage (1, 2).

DNA repair systems are of fundamental importance for the maintenance of genomic integrity in the face of replication errors, environmental carcinogens, and the cumulative effects of age, and their inactivation can dramatically increase the susceptibility to cancer (3, 4). In humans, >70 genes are involved in the five major DNA repair pathways: nucleotide excision repair, base excision repair, mismatch repair, homologous recombinational repair, and nonhomologous end joining (3, 4).

Molecular epidemiologic studies have shown considerable interindividual variation in the DNA repair capacity in the general population. Individuals with a suboptimal DNA repair capacity are at an increased risk of cancers such as lung cancer and squamous cell carcinoma of the head and neck (5, 6). Polymorphisms in the DNA repair genes may contribute to the DNA repair capacity variations in the general population. Therefore, it has been hypothesized that inherited polymorphisms in the DNA repair genes may modulate the susceptibility to lung cancer. To test this hypothesis, we have previously studied the contribution of polymorphisms in the DNA repair genes to the risk of lung cancer in a Korean population (7-9).

A highly conserved set of mismatch repair proteins is primarily responsible for the correction of replication errors (base-base or insertion-deletion mismatches) that are caused by DNA polymerase errors (10, 11). Genetic and epigenetic inactivation of the mismatch repair genes has been implicated in the etiology of hereditary nonpolyposis colorectal cancer syndrome and also in a wide variety of sporadic tumors such as colorectal, ovarian, and endometrial cancers (12, 13). However, the pathogenic role of the mismatch repair genes in the environment-induced cancers such as lung cancer has not been well defined (14, 15). In addition to their established role in the repair of postreplicative DNA errors, mismatch repair proteins are also involved in a variety of other vital cellular processes such as the induction of apoptosis in response to exogenous DNA damage (16, 17) and the transcription-coupled nucleotide excision repair of bulky DNA adducts (18, 19). Therefore, a subtle defect in the DNA repair capacity that is caused by functional polymorphisms in the mismatch repair genes that are neither necessary nor sufficient for the development of lung cancer could place some individuals at an increased risk of lung cancer.

The hMSH2 gene is one of the mismatch repair genes and it encodes the human homologue of the bacterial MutS protein, which is responsible for recognizing DNA mismatches (10, 11). To date, several polymorphisms in the hMSH2 gene have been reported (refs. 20-33; Table 1 ). Whereas the functional effects of these polymorphisms are not known, we hypothesized that some of these variants, and particularly their haplotypes, may have an effect on the DNA repair capacity, and this can modulate the susceptibility to lung cancer. To test this hypothesis, we conducted a case-control study to evaluate the association between the hMSH2 genotypes/haplotypes and the risk of lung cancer. Among the identified polymorphisms in the hMSH2 gene, we evaluated the association of the –118T>C, IVS1+9G>C, IVS10+12A>G, and IVS12-6T>C polymorphisms with lung cancer because the other polymorphisms were not detected in a preliminary study that consisted of 27 lung cancer cases and 27 healthy controls.


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  		Table 1. Known polymorphisms in the hMSH2 gene

 

   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Study Population
This case-control study included 432 lung cancer patients and 432 healthy controls. The details of the study population have been described elsewhere (34, 35). In brief, the eligible cases included all the patients who were newly diagnosed with primary lung cancer at Kyungpook National University Hospital, Daegu, Korea from January 2001 to February 2002. There were no age, gender, histologic, or stage restrictions but those patients who had a prior history of cancers were excluded from this study. The cases included 210 (48.6%) squamous cell carcinomas, 141 (32.6%) adenocarcinomas, 73 (16.9%) small-cell carcinomas, and 8 (1.9%) large-cell carcinomas. The control subjects were randomly selected from a pool of healthy volunteers who had visited the general health check-up center at Kyungpook National University Hospital during the same period. The control subjects were frequency matched (1:1) to the cancer cases on the basis of gender and age (±5 years). All the cases and the controls were ethnic Koreans and they resided in Daegu City or in the surrounding regions. A detailed questionnaire was completed for each patient and each control by a trained interviewer. The questionnaire included information on the average number of cigarettes they smoked daily and the number of years the subjects had been smoking. For the smoking status of the subjects, a person who had smoked at least once a day for >1 year during 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 the diagnosis of lung cancer in the case of the patients and 1 year before the date signed on an informed consent form for the blood sample collection in the case of the controls. The cumulative cigarette dose (pack-years) was calculated by using the following formula: pack-years = (packs per day) x (years smoked).

Genotyping
Genomic DNA was extracted from peripheral blood lymphocytes by proteinase K digestion and phenol/chloroform extraction. The hMSH2 –118T>C, IVS1+9G>C, IVS10+12A>G, and IVS12-6T>C genotypes were determined by using a PCR-restriction fragment length polymorphism assay. The PCR primers were designed based on the GenBank reference sequence (accession no. AY601851). The PCR primers for the –118T>C, IVS1+9G>C, IVS10+12A>G, and IVS12-6T>C polymorphisms were 5'-GAAACGCAGCCCTGGAAGCTA(G->A)A-3' (forward) and 5'-AAACCTCCTCACCTCCTGGTTG-3' (reverse); 5'-GACCGGGGCGACTTCTATAC-3' (forward) and 5'-AAAGGAGCCGCGCCACAAGG-3' (reverse); 5'-TACCAACAGGTTTGCAAGA(T->A)C-3' (forward) and 5'-GACTCTACTTTTACCTCGTC-3' (reverse); and 5'-CTTGCTTTCTGATATAATTTGA(T->A)-3' (forward) and 5'-GAAGCAGTTCCAACATTTCA-3' (reverse), respectively. The PCR reactions were done in a total volume of 20 µL that contained 100 ng genomic DNA, 25 pmol/L of each primer, 0.2 mmol/L deoxynucleotide triphosphates, 10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, 1.5 mmol/L MgCl2, and 1 unit of Taq polymerase (Takara Shuzo Co., Otsu, Shiga, Japan). The PCR cycle conditions consisted of an initial denaturation step at 95°C for 5 minutes followed by 35 cycles of 20 seconds at 94°C; 20 seconds at 58°C for -118T>C, 54°C for IVS1+9G>C, 57°C for IVS10+12A>G, and 49°C for IVS12-6T>C; 20 seconds at 72°C; and a final elongation step at 72°C for 10 minutes. The PCR products were digested overnight at 65°C (–118T>C) or 37°C (IVS1+9G>C, IVS10+12A>G, and IVS12-6T>C) with the appropriate restriction enzymes (New England BioLabs, Beverly, MA). The restriction enzymes for the –118T>C, IVS1+9G>C, IVS10+12A>G, and IVS12–6T>C genotypes were Tsp509I, BglI, HpyCH4IV, and DpnII, respectively. The digested PCR products were resolved on 6% acrylamide gels and they were stained with ethidium bromide for visualization under UV light. To ensure quality control, the genotyping analysis was done blind with respect to the case/control status. About 10% of the samples were randomly selected to be genotyped again by a different author and the results were 100% concordant. To confirm the genotyping results, selected PCR-amplified DNA samples (n = 2, respectively, for each genotype) were examined by DNA sequencing, and the results were also 100% concordant.

Statistical Analysis
The cases and controls were compared by using Student's t test for the continuous variables and by using the {chi}2 test for the categorical variables. Hardy-Weinberg equilibrium was tested for with a goodness-of-fit {chi}2 test with 1 degree of freedom to compare the observed genotype frequencies among the subjects with the expected genotype frequencies. The linkage disequilibriums (LD) among the polymorphisms were examined using the Lewontin's standardized coefficient D' (|D'|; ref. 36). The haplotypes and their frequencies were estimated based on a Bayesian algorithm using the Phase program (37).6 Unconditional logistic regression analysis was used to calculate the odds ratios (OR) and 95% confidence intervals (95% CI), with adjustment being made for the possible confounders (gender and family history of lung cancer as a nominal variable; age and pack-years, as continuous variables). In addition to the overall association analysis, we did stratified analyses according to age, gender, smoking status, and tumor histology to further explore the association between the hMSH2 genotypes/haplotypes and the risk of lung cancer in each stratum. To test which polymorphism is more likely to be the main cause of the observed association, we employed logistic regression models in which we allowed for the effects of the four polymorphisms, both individually and jointly. When multiple comparisons were made, the corrected P values (Pc values) were also calculated for multiple testing by using Bonferroni's inequality method. All the analyses were done using Statistical Analysis Software for Windows, version 8.12 (SAS Institute, Gary, NC).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The demographics of the cases and controls enrolled in this study are shown in Table 2 . There were no significant differences between the cases and controls for the mean age or gender distribution, suggesting that the matching based on these two variables was adequate. Cases had a higher prevalence of current smokers than did the controls (P < 0.001) and the number of pack-years in the smokers was significantly higher in the cases than in the controls (39.9 ± 17.9 versus 34.4 ± 17.6 pack-years, respectively; P < 0.001). These differences were controlled for later by the multivariate analyses.


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  		Table 2. Characteristics of the study population

 
The genotype and polymorphic allele frequencies of the four hMSH2 polymorphisms (–118T>C, IVS1+9G>C, IVS10+12A>G, and IVS12–6T>C) among the controls and cases are shown in Table 3 . The genotype distributions of the four polymorphisms among the controls were in Hardy-Weinberg equilibrium. When the overall lung cancer cases were compared with the controls, no significant difference was found in the distributions of the genotypes for any of the polymorphisms studied. However, when the lung cancer cases were categorized by tumor histology, the distribution of the IVS10+12A>G genotypes in the adenocarcinoma cases differed from that of the controls (P = 0.03), and the variant allele frequencies of the IVS10+12A>G and IVS12-6T>C polymorphisms in the adenocarcinomas differed significantly from those of the controls, respectively (0.305 versus 0.392, P = 0.008; and 0.369 versus 0.304, P = 0.044, respectively).


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  		Table 3. Genotype frequencies of hMSH2 polymorphisms in lung cancer cases and controls

 
Table 4 shows the lung cancer risk related to the hMSH2 –118T>C, IVS1+9G>C, IVS10+12A>G, and IVS12–6T>C genotypes, respectively. No significant association was found between all the four polymorphisms and the risk of overall lung cancer. However, when the lung cancer cases were categorized by tumor histology, those individuals with at least one IVS10+12G allele were at a significantly decreased risk of adenocarcinoma compared with the IVS10+12A homozygotes (adjusted OR, 0.59; 95% CI, 0.40-0.88; P = 0.01), and those individuals with at least one IVS12–6C allele were at a significantly increased risk of adenocarcinoma compared with the IVS12–6T homozygotes (adjusted OR, 1.52; 95% CI, 1.02-2.27; P = 0.04).


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  		Table 4. Adjusted ORs (95% CIs) for lung cancer associated hMSH2 genotype

 
The association between the combined genotypes of the -1263A>G and -712C>T polymorphisms and the risk of lung cancer was examined because these two polymorphisms were associated with the risk of lung cancer in a logistic regression analysis for each polymorphism. The distribution of the combined genotypes among the adenocarcinoma cases was significantly different from that among the controls (Table 5 ; P = 0.02). The IVS10+12 AG+GG/IVS12-6 TT genotype was associated with a significantly lower risk of adenocarcinoma compared with the IVS10+12 AA/IVS12-6 TC+CC genotype (adjusted OR, 0.47; 95% CI, 0.28-0.78; P = 0.004 and Pc = 0.012). In addition, the risk of adenocarcinoma was decreased with decreasing number of high-risk genotypes (Ptrend = 0.007).


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  		Table 5. Combined genotype frequencies of hMSH2 IVS10+12A>G and IVS12–6T>C polymorphisms among the cases and controls and their associations with the risk of lung cancer

 
The four hMSH2 polymorphisms were in LD (|D'| value ranged from 0.60 to 0.83), and we observed 14 haplotypes out of the possible 16 (24) haplotypes. For statistical advantage, nine haplotypes that had a frequency of <2% were excluded from any further analysis. The remaining five haplotypes accounted for 92.8% of the chromosomes for the 864 subjects (controls, 93.8%; cases, 91.8%). Table 6 shows the inferred haplotype distributions for the controls and cases, as well as the lung cancer risk as related to the haplotypes. Consistent with genotyping analysis, the TGGT haplotype was associated with a significantly decreased risk of adenocarcinoma compared with the TGAC haplotype (adjusted OR, 0.49; 95% CI, 0.30-0.78; P = 0.003 and Pc = 0.012).


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  		Table 6. Distribution of hMSH2 inferred haplotypes in controls and cases

 
The risk of adenocarcinoma related to the hMSH2 haplotypes was further examined with stratification according to age, gender, and the smoking status. Because the number of adenocarcinoma cases in each stratum was small, the five haplotypes were categorized into three groups according to the presence of two, one, or no risk alleles at the IVS10+12A>G and IVS12–6T>C (i.e., TGAC, TGAT+CGAT, and TGGT+TGCT). As compared with the TGAC haplotype, the combined haplotype (TGGT+TGCT) with no risk allele was associated with a significantly decreased risk of adenocarcinoma in only the younger individuals and the never smokers (adjusted OR, 0.45; 95% CI, 0.27-0.75; P = 0.002 and Pc = 0.004; and adjusted OR, 0.44; 95% CI, 0.23-0.85; P = 0.014 and Pc = 0.028, respectively), whereas it had no significant effect on the risk of adenocarcinoma in the older individuals and the ever-smokers (adjusted OR, 0.86; 95% CI, 0.51-1.44; and adjusted OR, 0.74; 95% CI, 0.49-1.12).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
DNA sequence variations in the hMSH2 gene may have an effect on the DNA repair capacity, thereby causing inter-individual differences in the susceptibility to lung cancer. To test this hypothesis, we evaluated the potential association of the hMSH2 polymorphisms (–118T>C, IVS1+9G>C, IVS10+12A>G, and IVS12–6T>C) with the risk of lung cancer. The hMSH2 IVS10+12A>G and IVS12–6T>C polymorphisms were significantly associated with the risk of adenocarcinoma of the lung. This finding suggests that the hMSH2 IVS10+12A>G and IVS12–6T>C polymorphisms could be used as markers for the genetic susceptibility to adenocarcinoma. Of the three major histologic types of lung cancer, the proportion of adenocarcinoma is increasing worldwide. Thus, identification of the genetic factors that are responsible for the susceptibility to adenocarcinoma is indispensable for establishing novel and efficient ways to prevent this disease.

In the current study, we validated the presence of hMSH2 –118T>C, IVS1+9G>C, IVS10+12A>G, and IVS12–6T>C polymorphisms in a Korean population. However, the other seven polymorphisms among the previously reported polymorphisms listed in Table 1 were not detected in the preliminary study that included 27 healthy controls. These samples included 54 chromosomes, which provides at least a 95% confident level to detect alleles with frequencies >5%. Thus, it is very likely that if these polymorphisms exist, they may not play a major role in the genetic susceptibility to lung cancer in the Korean population (38, 39). The frequency of the –118C allele among healthy Koreans was 0.181, which was similar to that of Japanese (0.202; ref. 26). Frequencies of the IVS1+9C and IVS10+12G alleles among healthy Koreans were 0.20 and 0.39, respectively, which were lower than those of healthy Caucasians (0.47-0.62 and 0.35-0.54, respectively; refs. 20, 21, 23, 25, 29). Frequency of IVS12–6C allele among healthy Koreans was 0.304, which was similar to that of healthy Japanese (0.285; ref. 33) but significantly lower than that of healthy Caucasians (0.05-0.14; refs. 20, 27, 28, 32).

In the current study, the hMSH2 polymorphisms were significantly associated with the risk of adenocarcinoma but they were not associated with the squamous cell carcinoma or small-cell carcinoma. Although the reason for the observed histology-dependent difference in the risk conferred by the hMSH2 polymorphism is unknown, this difference may be attributable to the differences in the pathways of carcinogenesis among the different histologic types of lung cancer. Various lines of evidence have suggested that the histologic type of lung cancer may be determined by the particular initiating agent to which an individual is exposed (40-42). Therefore, certain polymorphisms could confer a greater susceptibility to a particular histologic type of lung cancer (34, 35, 43). Several studies have shown that hMSH2 gene was frequently inactivated in lung cancers, and the hMSH2 inactivation rate was significantly higher in adenocarcinoma than in squamous cell carcinoma (44-46). These observations suggest that the hMSH2 gene may have a pronounced association with the development of adenocarcinoma, and these observations are comparable with our finding that the hMSH2 polymorphisms play an important role in determining the genetic susceptibility to adenocarcinoma.

Another interesting finding of the present study is that the effect of the hMSH2 polymorphisms on the risk of adenocarcinoma was pronounced in the younger individuals and never smokers. Several recent studies suggest that adenocarcinomas arising in never smokers and smokers are caused by different etiologies (i.e., carcinogens other than environmental tobacco smoke play an important role in the pathogenesis of adenocarcinomas in never smokers; refs. 47, 48). In view of this suggestion, our finding that the effect of the hMSH2 polymorphisms on the risk of adenocarcinoma is more pronounced in the never smokers may be due to that the hMSH2 polymorphisms may contribute to the development of adenocarcinoma by influencing on the repair capacity of DNA damages caused by environmental factor(s) other than cigarette smoking and/or endogenous factor(s). This explanation is comparable with the previous studies (45, 46) that showed that the altered hMSH2 protein expression in non–small-cell lung cancers was significantly associated with the adenocarcinoma histology, younger patients, female patients, and the never smokers. However, it is possible that such a finding is attributable to chance because of the relatively small numbers of subjects in the subgroups. Additional studies with more patients will be needed to confirm this finding.

The haplotypes can increase the power to detect disease associations compared with a single polymorphism on account of the higher heterozygosity and tighter LD with the disease-causative variant (49-51). In this study, the IVS1+9G>C polymorphism had no effect on the risk of adenocarcinoma in individual polymorphism analysis, but haplotype analysis showed that the TGGT haplotype carrying the IVS1+9G allele was associated with a significantly decreased risk compared with the TGAC haplotype whereas the TCGT haplotype carrying IVS1+9C allele was not significantly associated with the risk of adenocarcinoma. These results also suggest that haplotype analysis may be a more suitable tool for assessing the disease association than the individual polymorphism.

Several polymorphisms have been reported in the hMSH2 gene (20-33). It has been suggested that one of these polymorphism, IVS12–6T>C, may predispose to some kind of cancer (27, 28, 32, 52, 53). In agreement with the previous studies, the IVS12–6C allele was associated with a significantly increased risk of lung adenocarcinoma in the present study. A characteristic finding in the present study is that the IVS10+12A>G polymorphism as well as the IVS12–6T>C polymorphism was significantly associated with the risk of adenocarcinoma. Because these two polymorphisms were in strong LD, it is not easy to discern the relative contribution of each polymorphism to the observed association. In an attempt to resolve this, we compared three different logistic regression models (each polymorphism alone and both polymorphisms together). The model incorporating both polymorphisms did not fit significantly better than the model with the IVS10+12A>G alone (P = 0.15) but the model with the IVS12–6T>C alone fitted less well than the model incorporating both polymorphisms (P = 0.03). These results suggest that the genetic effect of the IVS10+12A>G polymorphism is stronger than the effect of the IVS12–6T>C polymorphism.

It has been reported that multiple alternatively spliced isoforms of hMSH2 mRNA are present in normal human tissues, some of them would produce truncated proteins that lack exons 2-8 or exon 13 and others were in-frame deletions lacking exon 5 or exons 2-7; the relative expression level of each splicing variant has shown considerable interindividual variation (54-56). Although the functional significance of the IVS10+12A>G and IVS12–6T>C polymorphisms remains to be elucidated, these splicing site polymorphisms may have an influence on the alternative splicing of hMSH2, resulting in interindividual variation in the expression levels of the hMSH2 splicing variants. Some of these variant proteins might cause a dominant-negative effect by interfering with the wild-type hMSH2 protein. An alternative explanation for the association between the hMSH2 polymorphisms and the risk of lung cancer may be due to LD with either another hMSH2 variant or with an adjacent true susceptible gene.

In conclusion, we found that the hMSH2 polymorphisms and their haplotypes were associated with the risk of adenocarcinoma of the lung. The effects of the hMSH2 polymorphisms on the risk of adenocarcinoma were more evident in the younger individuals and the never smokers. These results suggest that the hMSH2 gene may contribute to an inherited predisposition to adenocarcinoma of the lung. It is possible that these findings, particularly those findings from stratified analyses, can be attributed to chance because of the relatively small numbers of cases in the subgroups. Therefore, additional studies with larger sample sizes are required to confirm our findings. Moreover, because the gene-gene interactions and gene-environment interactions often vary between ethnic groups, further studies are needed to clarify the association between the hMSH2 polymorphisms and lung cancer in diverse ethnic populations.


   Footnotes
 
Grant support: The Regional Technology Innovation Program of The Ministry of Commerce, Industry, and Energy grant RTI04-01-01.

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: C.Y. Jung and J.E. Choi contributed equally to this work.

6 Available at http://www.stat.washington.edu/stephens/software.html Back

Received 10/26/05; revised 2/ 1/06; accepted 2/14/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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