Polymorphisms in hMLH1 and risk of early-onset lung cancer in a southeast Chinese population

Lung Cancer (2008) 59, 164—170

available at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/lungcan

Polymorphisms in hMLH1 and risk of early-onset lung cancer in a southeast Chinese population Yu An a,1, Guangfu Jin b,1, Haifeng Wang c, Yi Wang a, Hongliang Liu a, Rui Li a, Haijian Wang a, Ji Qian a, Weiwei Sun c, Yi Wang c, Hongxia Ma b, Ruifeng Miao b, Zhibin Hu b, Li Jin a, Qingyi Wei d, Hongbing Shen b, Wei Huang c,∗, Daru Lu a,∗∗ a

State Key Laboratory of Genetic Engineering and MOE Key Laboratory of Contemporary Anthropology, School of Life Sciences, Fudan University, Handan Road, Shanghai 200433, China b Department of Epidemiology and Biostatistics, Cancer Research Center of Nanjing Medical University, Nanjing, China c Department of Genetics, Chinese National Human Genome Center at Shanghai, Shanghai 201203, China d Department of Epidemiology, The University of Texas MD Anderson Cancer Center, Houston, TX, United States Received 25 April 2007; received in revised form 27 July 2007; accepted 6 August 2007

KEYWORDS hMLH1; Genetic polymorphisms; Lung cancer; Susceptibility; Molecular epidemiology

Summary DNA mismatch repair (MMR) plays an important role in maintaining genome stability. Defects in MMR genes have been involved in several types of sporadic and hereditary cancers. hMLH1 is considered one of central members of the MMR pathway. We conducted a hospitalbased case—control study to investigate associations of common variations in the hMLH1 gene and risk of lung cancer. A total of 500 cases and 517 controls were genotyped for seven SNPs in hMLH1. Overall, the rs1799977 I219V polymorphism was marginally associated with the risk of lung cancer (P = 0.055). This association was much stronger in younger patients (P = 0.01; odds ratio, 5.28; 95% CI 1.45—19.21) and lung squamous cell carcinoma (P = 0.006; odds ratio, 3.65; 95% CI 1.44—9.24). These findings indicate that the hMLH1 rs1799977 polymorphism may contribute to the etiology of early-onset lung cancer as well as some specific subtype of lung cancer. Larger association studies are warranted to validate our findings and mechanistic studies are needed to elucidate the underlying molecular mechanisms of the association. © 2007 Elsevier Ireland Ltd. All rights reserved.

1. Introduction ∗

Corresponding author. Corresponding author. Tel.: +86 21 65642799; fax: +86 21 65642799. E-mail addresses: [email protected] (W. Huang), [email protected] (D. Lu). 1 These authors contributed equally to this work. ∗∗

Lung cancer is the most common cancer in the world today in terms of its incidence in both sexes in both developing countries and developed countries. Global cancer statistics indicated that in 2002 there were 1.35 million of new cases and 1.18 million of deaths for lung cancer and that the ratio

0169-5002/$ — see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.lungcan.2007.08.003

Polymorphisms in hMLH1 and risk of early-onset lung cancer in a southeast Chinese population of mortality to incidence was 0.87 due to extreme poor outcome [1,2]. Yang and colleagues predicted that there would be 120,000 new lung cancer cases between 2000 and 2005. The total number of lung cancer cases increase 26.9% in men and 28.4% in women in China [2]. Tobacco smoke is a major risk factor for lung cancer; however, not all smokers develop lung cancer [3]. Accumulating studies have indicated that host genetic susceptibility plays a role in the development of lung cancer, one of typical multifactor diseases resulting from complex interactions between many genetic and environmental factors. DNA mismatch repair (MMR) is a highly conserved process that removes nucleotides that have been misincorporated into the newly synthesized DNA strand during replication [4]. Double-strand break repair through recommendation is also modulated by MMR [5]. Recent studies have implicated that MMR components as DNA damage sensors are involved in cell cycle regulation and the p53-dependent apoptotic response to a variety of DNA damage [6]. MMR-defective cells display various forms of genomic instability [7]. Mice that carry MMR gene deficiencies display increased levels of microsatellite instability (MSI) and increased susceptibility to cancer [8]. In humans, defects in MMR genes have been implicated in several types of sporadic and hereditary cancers [9]. Therefore, MMR can be one of possible genetic susceptibility factors in lung cancer etiology. The key proteins in MMR are highly conserved from bacteria to mammals. In eukaryotes, there are multiple homologs of key bacterial MMR proteins which form heterodimers to have discrete roles in MMR-related processes [10,11]. There are three different heterodimeric MutL-related complexes in humans that all contain hMLH1. Therefore, hMlh1p is considered a central member of this group because heterodimers play a major role in postreplicative MMR. The hMLH1 locus is located on chromosme 3p21.3, consists of 19 exons, and encodes a 756 amino acid protein. hMLH1 is a common subunit to all three complexes, and its deficiency lead to a sever disease phenotype [12—17]. Recent reports have shown that hMLH1 not only physically interacts with other components of MMR but also interacts with other signaling molecules such as BRCA1 [18], PCNA [19], ATM [20], P53 [21,22] and other enzymes such as BLM (DNA helicase) [23], Exo1p [24] and MED1 [25,26]. These interaction defects are associated with cancer predisposition [28,29]. hMlh1p-Pms2p subunits have been shown to bind and hydrolyze ATP in a reaction that drives conformational changes thought to be important for MMR [27]. Polymorphisms of hMLH1 in individuals may have an effect on the DNA repair capacity and therefore on risk of cancer. But the polymorphisms of hMLH1 in Chinese populations are rarely studied. In the present study, we investigated the association between genetic variants of hMLH1 and risk of lung cancer in a southeast Chinese population.

2. Materials and methods 2.1. Study population and genomic DNA extraction As described previously, this study recruited 552 lung cancer patients between July 2002 and November 2004 at both the

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Cancer Hospital of Jiangsu province and the First Affiliated Hospital of Nanjing Medical University, Nanjing, China. We only included those patients who had clear histological diaglosis, which resulted in 500 eligible cases [30]. We also selected 517 Cancer-free controls randomly from 10,500 individuals participated in a community-based screening program conducted in Jiangsu province during the same time period [30]. These control subjects had no history of cancer and were frequency-matched to the cases on age, sex and residential area (urban or rural areas). All subjects were genetically unrelated ethnic Han Chinese and were from the city of Nanjing and surrounding regions in southeast China. A standardized structured questionnaire was used to obtain information on demographic characteristics, environmental exposure history including family history of lung cancer and tobacco smoking intake. Those who had smoked less than one cigarette per day and shorter than 1-year in their lifetime were defined as non-smokers, otherwise, they were considered as ever-smokers. Those smokers who had stopped for more than 1-year were considered as former-smokers. Smoking pack-years as (cigarettes perday/20) × years smoked were calculated to estimate the cumulative smoking exposure. About 5 ml of peripheral blood was obtained from all individuals. Genomic DNA was extracted from blood samples using phenol—chloroform extraction method. The study was approved by the Institutional Review Boards of Nanjing Medical University.

2.2. SNP selection and genotyping We identified seven candidate SNPs of the hMLH1 gene using the NCBI SNP database (Table 1). These SNPs almost equally covered the whole gene with the density of about one marker per 5 kb. Those SNPs with minor allele frequency (MAF) ≥0.05 and a non-synonymous SNP (rs1799977) that could cause missense mutations were included. All SNPs were genotyped by using the Illumina platform at Chinese National Human Genome Center at Shanghai. Quality control was performed by validation of the SNPs randomly selected as described previously [30].

2.3. Statistical analysis The agreement of Hardy—Weinberg equilibrium (HWE) was evaluated using Haploview (http://www.broad.mit. edu/mpg/haploview/download.php). Genotype and allele frequencies among the respective groups were compared by Fisher’s exact test. Logistic regression analysis was used to estimate the odds ratios (ORs) and 95% confidence interval (CIs) for the association between genotypes and lung cancer risk. Age, sex and smoking package-year and family history of lung cancer were included for multivariate adjustment. Stratified analysis was conducted to investigate modification of ORs by age status, gender, smoking intake, family history of lung cancer and histological characteristics. The above statistical tests were analyzed by SPSS software (10.0 version) and the significance level was set at 0.05 with two tails for each analysis.

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Y. An et al.

Table 1

Primary information of genotyped SNPs of hMLH1

Gene and locus

rs1800734 rs1540354 rs4647250 rs1799977 rs1558528 rs2286939 rs4678925

hMLH1 3p21.3

a b

NCBI rs #

Location 

5 flanking Intron3 Intron5 Exon8 Intron9 Intron11 Intron13

NCBI location in NT 022517

Base change

MAFa

P for HWEb

%Genotyped

36974950 36984493 36989102 36996994 37002044 37018510 36974950

A>G A>T C>T A>G A>C C>T A>G

0.427 0.311 0.057 0.016 0.057 0.057 0.052

0.90 0.65 0.81 0.47 0.84 0.84 0.92

99.3 99.3 99.1 98.0 99.3 99.3 90.1

Minor allele frequencies. Hardy—Weinberg equilibrium test.

3. Result Table 2 shows the distribution of selected characteristics between lung cancer patients and controls. We did not observe statistically significant difference in the distributions of age (t-test P = 0.64) and sex (exact 2 test, P = 1.00) between the cases and controls. The mean (±S.D.) age of the subjects was 59.98 ± 10.3 for controls and 59.27 ± 10.4 for cases. As the previous studies, smoking status and packyear of smoking was associated with higher risk of lung cancer in our study (P value < 0.0001 for both). Family history of cancer among first-degree relatives also significantly increased lung cancer risk (P = 0.004). Among the cases with histological types of lung cancer, 466 (93.2%) were classi-

fied as non-small-cell lung cancer (229 adenocarcinoma, 141 squamous cell carcinoma, and 96 large cell, mixed cell carcinomas, or undifferentiated carcinoma), and only 34 (6.8%) as small-cell lung cancer. For all SNPs, we tested departure from HWE in both cases and controls respectively by exact 2 test with the Haploview software (Table 1). None of the seven SNPs deviated from HWE in cases and controls in this population. Success rates of genotyping for individual polymorphism averaged as 97.8% (Table 1). In general, we did not observe a strong main effect for hMLH1 SNPs (Table 3). However, there was a marginally significant difference of allele frequencies for rs1799977 I219V (P = 0.055) (Table 4). Logistic regression analysis revealed that subjects carrying the rs1799977

Table 2

Distribution of demographic features in lung cancer cases and controls

Variable

Controls (n = 517)

Cases (n = 500)

No.

No.

Age (years) Mean

(%)

59.98 ± 10.3

P value* (%) 0.64†

59.27 ± 10.4

Sex Male Female

400 117

77.4 22.6

386 114

77.2 22.8

Smoking status Non-smokers Former-smokers Current-smokers

249 52 216

48.2 10.0 41.8

165 108 227

33.0 21.6 45.3

Pack-years of smoking 0 1—20 >20

249 74 194

48.2 14.0 37.4

165 88 247

33.0 17.6 49.4

Family history of cancer No Yes

430 87

83.2 16.8

378 122

75.6 24.4

229 141 96 34

45.8 28.2 19.2 6.8

Histological types Adenocarcinomas Squamous cell Other carcinomas Small cell

1.000

<0.0001

<0.0001 0.003

This table had been reproduced according to Li’s article (in press) with same population information (doi:10.1016/j.lungcan.2007.06.016). * P-value based on Fisher’s exact 2 test. † Indepent samples t-test.

Polymorphisms in hMLH1 and risk of early-onset lung cancer in a southeast Chinese population Table 3

Distribution of the genotype in selected SNPs of hMLH1

Genotype

Case

rs1800734 rs1540354 rs1558528 rs2286939 rs4647250 rs4678925 rs1799977 *

167

P value*

Control

Wild

Hetero

Variant

Wild

Hetero

Variant

163 242 440 440 440 403 479

243 205 59 59 58 47 20

94 53 1 1 1 2 1

169 244 465 465 464 427 493

258 224 49 49 49 43 11

90 49 3 3 3 1 0

0.83 0.71 0.30 0.30 0.34 0.63 0.09

P value based on Fisher’s exact 2 test.

G had a 2.01-fold increased risk of lung cancer (95% CI 0.98—4.23) compared with the rs1799977A allele. Comparing with rs1799977AA wild-type genotype, those carrying rs1799977AG/GG had a non-significant 2-fold elevated risk (95% CI 0.95—4.26) (Table 4). We have evaluated the associations of all genotypes with lung cancer risk stratified by selected variables and histological types. No significant difference was observed except for rs4647250, rs1558528 and rs2286939 which had a marginal association with risk of squamous cell type (P = 0.04 for all of three) (Data did not show here). Because the nonsynonymous rs1799977 I219V might alter protein function, we further evaluated the associations of I219V genotypes with lung cancer risk stratified by selected variables and histological types. As summarized in Table 5, there were associations observed among younger individuals (<60) with significantly increased risk of lung cancer (P = 0.01, OR 5.28, 95% CI 1.45—19.21) and squamous cell carcinoma (P = 0.006 and OR 3.65 with 95% CI 1.44—9.24) with adjustment for age, sex, and pack-years of smoking and family history of lung cancer. The higher ORs for carriers of hMLH1 rs1799977 G allele in non-smokers indicated that it was one of indicators of disease susceptibility or factors in lung cancer. Haplotype frequencies for of hMLH1 SNPs did not differ significantly between cases and controls in this eastern China population. However, those haplotypes carrying hMLH1 rs1799977 Val was associated with marginal risk (data did not show here).

Table 4

4. Discussion As a key member of MMR genes, hMLH1 plays a critical role in human cancers including lung carcinogenesis. In this study, we have investigated the association of polymorphisms of hMLH1 with risk of lung cancer. We found boardline significance between hMLH1 rs1799977 I219V and risk of lung cancer. A significant difference in the frequency distribution of a non-synonymous alternation of I219V between younger cases and controls (age < 60) and also in lung squamous cell carcinoma (SQC) had been observed. As shown in Table 1, we have selected seven SNPs from dbSNP database (http://www.ncbi.nlm.nih.gov), which covered evenly the hMLH1 with the density of one SNP per about 5 kb. All SNPs in this study population had a MAF > 5% except a non-synonymous SNP (rs1799977). When the HapMap public SNP database became richer density, we compared our SNPs selection with ones from the database of HapMap (HapMap Data Rel 21/PhaseII July 06). The tagSNPs (MAF > 0.05, r2 > 0.8) from the HapMap database (rs1540354 and rs2286939) are included in our selection. Furthermore, we also compared these selected tagSNP with those selected from the Environmental Genome Project (EGP) database (http://egp.gs.washington.edu/finished genes.html), from which the tagSNPs rs1540354, rs1800734 and rs4647224 were selected. Among these, rs1540354 and rs1800734 were included in final analysis because rs4647224 is in complete linkage disequilibrium (LD) with rs2286939 (r2 = 1). These

Logistic regression analysis of associations between the genotypes in rs1799977 and lung cancer risk

Genotype

rs1799977 AA AG GG A G AG + GG vs. AA

Controls (n = 517)

Cases (n = 500)

No.

(%)

No.

(%)

493 11 0 997 11 11/493

97.8 2.18 0 98.9 1.1

479 20 1 978 21 21/479

95.8 4 0.2 97.9 2.1

P valuea

Crude OR (95% CI)b

Adjusted OR (95% CI)c

0.085 1.00 1.87 (0.89—3.95) 0.055 0.07

1.00 1.97 (0.94—4.12)

1.00 2.01 (0.98—4.23) 2.00 (0.95—4.26)

P-value based on Fisher’s exact 2 test. Logistic regression analysis with hMLH1 AA or A allele as reference group without adjustment. c Logistic regression analysis with hMLH1 AA or A allele as reference group and adjusted for sex, age, pack-years of smoking, family history. a

b

168 Table 5

Y. An et al. Stratified analyses between the hMLH1 rs1799977 genotypes and lung cancer risk

Variables

hMLH1 rs1799977 Cases/control

Crude OR (95% CI)a

Adjusted OR (95% CI)b

AA

AG + GG

AA

AG + GG

AG + GG

Age (years) <60 ≥60

227/284 252/209

13/3 8/8

1.00 1.00

5.42 (1.53—19.26) 0.83 (0.31—2.25)

5.28 (1.45—19.21) 0.56 (0.19—1.63)

Sex Male Female

371/378 108/115

15/9 6/2

1.00

1.70 (0.73—3.93) 2.27 (0.40—13.01)

1.63 (0.71—3.88) 2.11 (0.38—11.72)

Pack-years of smoking 0 1—20 >20

160/239 82/68 237/186

7/5 4/2 10/4

1.00 1.00 1.00

2.09 (0.65—6.70) 1.66 (0.30—9.33) 1.96 (0.61—6.36)

2.05 (0.60—7.06) 1.56 (0.27—8.82) 1.53 (0.45—5.21)

Family history of cancer No Yes

363/409 116/84

15/9 6/2

1.00 1.00

1.88 (0.81—4.34) 2.17 (0.43—11.03)

1.94 (0.83—4.52) 1.62 (0.29—8.92)

Histological types Adenocarcinomas Squamous cell Other carcinomas Small cell

223/493 130/493 93/493 33/493

6/11 11/11 3/11 1/11

1.00 1.00 1.00 1.00

1.12 3.79 2.04 1.36

1.17 3.65 1.92 1.74

(0.38—3.27) (1.61—8.94) (0.55—7.49) (0.17—10.84)

(0.40—3.45) (1.44—9.24) (0.50—7.41) (0.21—14.33)

a Data were calculated by unconditional logistic regression with the hMLH1 AA genotype as reference group without adjustment for other factors. b Data were calculated by unconditional logistic regression with the hMLH1 AA genotype as reference group with adjusted for sex, age, pack-years of smoking and family history.

results indicated that the density of SNPs we investigated provides a good coverage across the hMLH1 gene. These data were all analyzed based on pairwise LD by publicly available Haploview software with default settings (r2 = 0.8 and MAF = 0.05). These SNPs formed one LD block determined by the Gabriel et al. approach [31] (figure did not show). Among them, rs4647250, rs1558528, and rs2286939 had a complete LD with each other (both D and r2 are 1). It can be the reason why they share the same statistical results. According to the data released from the HapMap database (HapMap Data Rel 21a/Phase II January 07), we reconstructed the LD plot in Haploview with MAF > 0.01 as threshold and they formed a large LD block in hMLH1 gene (figure did not show). Sun and colleagues have examined hMLH1−93G>A polymorphism (rs1800734) in Korea for its association with lung cancer risk in a case—control study and found that it was significantly associated with the risk of squamous cell carcinoma with a gene-smoking interaction [38]. In our study, the frequency of this polymorphism hMLH1−93A was 0.57, which was the same as Sun’s study. But we did not find any association of the hMLH1−93G > A polymorphism with risk of lung cancer. The hMLH1 I219V variant had been identified as a SNP in European and North-American populations. In the previous study, the substitution of an amino acid (V—I), reversing the substitution of the hMLH1 I219V, has been reported to reduce the expression of the hMLH1 protein [32]. Also, Kim and colleagues have observed that tumors with homozygous normal alleles AA of hMLH1 I219V had a higher expression

of the hMLH1 protein than that in tumors with variant alleles GG in Korean populations of 330 healthy individuals, 107 sporadic colorectal cancer patients, and 107 of their firstdegree relatives [16]. In recent report, Brennan and his team reported an reduced risk for carriers of 219Val (G) in hMLH1 (OR, 0.69; 95% CI 0.48—0.98) in 299 lung cancer cases and 317 controls from six countries of central and eastern Europe [32]. Krajinovic and colleagues have identified the variant G of hMLH1 I219V exerted its influence on childhood acute lymphocytic lymphoma (ALL) outcome when combined with CYP1A1*2A variants (OR, 5.6; 95% CI 1.9—16.8) [33]. However, Sun and colleagues had not identified any significant difference of genotype distribution between 150 sporadic colorectal cancers (SCRC) and 150 healthy controls in a Chinese population for hMLH1 I219V, although their data on the genotypes frequency of hMLH1 655 G were the same as the previous publication from Korea with 6% [34]. In this study, we have shown an allele frequency of hMLH1 655 G with 2% and the genotype frequency has a bordering difference between cases and controls in an eastern Chinese population. We also observed the hMLH1 655 G may be associated with the young-onset of lung cancer, especially in histological squamous cell type. The result seems to be conflicting with Brennan’s work [32]; however, it may be explained by genetic variation in different ethnic groups of the different study populations. We also performed functional prediction of the substitution of hMLH1 I219V on the protein by SIFT, and the results indicated that this polymorphism may be

Polymorphisms in hMLH1 and risk of early-onset lung cancer in a southeast Chinese population tolerated by the hMLH1 protein (http://blocks.fhcrc.org/pauline/SIFT.html) [35]. According to the crystal structure analysis, hMLH1 I219V located in linkage of two conserved regions in all MutL homologues and near the conserved motifs of the ATP binding pocket of the hMLH1 protein [27]. We can hypothesize that the function of this site may not be lethal for the activation of binding to ATP but it can also influence the stability of binding sites or easy entry of ATP into the binding pocket. It needs to be confirmed by further functional studies. Some non-synonymous SNPs may alter their protein functions, but their effects on cancer risk are difficult to predict, while some non-synonymous SNPs might be tolerated by the protein, but tiny and additive effects might erode cancer risk in populations due to cumulative environmental carcinogen exposure. Therefore, the further study of the role of the variants of rs1799977 G in the hMLH1 gene in lung cancer risk is warranted. The normal expression of MMR genes seems to reflect the overall MMR capacity to cumulative environmental carcinogen exposure. Xinarianos et al. have shown that 58.6 and 57.8% of lung cancer tumor specimens had reduced expression levels of the hMLH1 and hMSH2 proteins, respectively [13,14]. Strom et al. found an association between reduced expression of hMLH1 and increased risk of head and neck cancer (OR 4.4; 95% CI 2.1—9.1). Positive correlation between age and expression of MMR genes may reflect the need to repair damage due to cumulative environmental carcinogen exposure [36]. Therefore, we believe the subgroup with hMLH1 rs1799977 G allele may increase the risk of lung cancer in the younger individuals. To date, over 70% of all lung cancer cases usually are diagnosed in individuals over the age of 65, but the number of people who are diagnosed with lung cancer at age 50 or earlier is increasing [37]. So the exact mechanisms of age-related increases in gene expression need to be addressed in the future study. In summary, the present study with a moderate sample size benefited from the high call rate in the process of genotyping. Although this case—control study has some inherent biases, potential confounding factors may be minimized by frequency matching on age and sex between the cases and controls. Our post-power calculation suggested a power of 80% when we set expected allele frequency as 6% as published in previous literature, and our study has a sufficient power to detect OR of 2.0 as calculated by free software Epi Info 3.4.1 (http://www.cdc.gov/epiinfo/). Due to the low-actual allele frequency of the hMLH1 rs1799977 G allele, however, our statistical power is still very limited. Finally, no biological conclusions can be made based on our observed data, even though we did provide some speculations. Despite these limitations, the present study is one of the largest investigations on the role of hMLH1 in the etiology of lung cancer in a southeast Chinese population. Since relatively few epidemiologic studies have included southeast Chinese populations, our study of moderate sample size first addressed the association of hMLH1 I219V with the early-onset of lung cancer. However, larger population based case—control study are needed to provide further evaluation. Furthermore, other genes included in MMR should be also investigated, which may find additional etiological roles of the MMR pathway in lung cancer.

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Acknowledgements This work was supported in part by the China National Key Basic Research Program Grants 2002CB512902 (to D. Lu & H. Shen), 2002BA711A10 and 2004CB518605 (to W. Huang), National Outstanding Youth Science Foundation of China 30425001 (to H. Shen), and National ‘‘211’’ Environmental Genomics Grant (to D. Lu).

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