Allele and Genotype Frequencies of the Polymorphic Methylenetetrahydrofolate Reductase and Lung Cancer in ther Jordanian Population: a Case Control Study

Background: Methylenetetrahydrofolate reductase ( MTHFR ) is involved in amino acid synthesis and DNA function. Two common polymorphisms are reported, C677T and A1298C , that are implicated in a number of human diseases, including cancer. Objective: The association between MTHFR C677T and A1298C genotype and haplotype frequencies in risk for lung cancer (LC) was investigated in the Jordanian population. Materials and Methods: A total of 98 LC cases were studied for MTHFR C677T and A1298C polymorphisms, compared to 89 controls taken from the general population, employing the PCR-RFLP technique. Results: The frequency of the genotypes of MTHFR C677T among Jordanians was: CC, 59.6%, CT, 33%; and TT, 7.4% among LC cases and 49.4%, 40.2% and 10.3% among controls. No significant association was detected between genetic polymorphism at this site and LC. At MTHFR A12987C, the genotype distribution was AA, 29.5%; AC, 45.3%, and CC 25.3% among LC cases and 36.8%, 50.6% and 12.6% among controls. Carriers of the CC genotype were more likely to have LC (OR=2.5; 95%CI: 1.04-6; p=0.039) as compared to AA carriers. Smokers and males with the CC genotype were 9.9 and 6.7 times more likely to have LC, respectively (OR smokers =9.9; 95%CI: 1.2-84.5, p=0.018; OR men =6.6; 95%CI: 1.7-26.2, p=0.005). Haplotype analysis of MTHFR polymorphism at the two loci showed differential distribution of the CC haplotype (677C-1298C) between cases and controls. The CC haplotype was associated with an increased risk for lung cancer (OR=1.6; 95% CI: 1.03-2.4, p=0.037). Conclusions: The genetic polymorphism of MTHFR at 1298 and the CC haplotype (risk is apparently lower with the C allele at position 677 ) may modulate the risk for LC development among the Jordanian population. Risk associated with the 1298C allele is increased in smokers and in males. The results indicate that a critical gene involved in folate metabolism plays a modifying role in lung cancer risk, at least in the Jordanian population.


Introduction
Lung cancer (LC) is the most common cancer in the world with an estimated 1.8 million new cases in 2012. It is also the most common cause of death from cancer, with 1.59 million deaths. Incidence depends on gender and ethnicity. LC is the most frequent cancer in men and the fourth in women. The majority of the cases are in the developing countries (55%) and the lowest rates are estimated in Middle and Western Africa. Patients with LC suffer from high fatality with the ratio of mortality to incidence is 0.87 irrespective of geographical location ranging from 0.75 (USA) to 0.94 (Africa) (Ferlay et al., 2012).
In Jordan, cancer is the second most frequent cause Folate metabolism is thought to play a critical role in carcinogenesis through its involvement in DNA methylation and repair. Methylenetetrahydrofolate reductase (MTHFR) is an important enzyme involved in folate metabolism (Cicek et al., 2004;Lucock, 2004). It converts 5,10-methylenetetrahydrofolate irreversibly to 5-methyltetrahydrofolate (5-MeTHF) which in turn donates its methyl group to homocysteine in the generation of S-adenosylmethionine (SAM). SAM is a major source of methyl groups used for DNA methylation. MTHFR maintains circulating levels of 5-MeTHF and methionine, and prevents the accumulation of homocysteine (Lucock, 2004).
A number of molecular epidemiological studies have been conducted to investigate the associations of the MTHFR C677T and A1298C polymorphisms with lung cancer predisposition. However, the results remain conflicting rather than conclusive (Mao et al., 2008;Boccia et al., 2009;Zhang et al., 2012).
Genetic polymorphism in MTHFR may be related to the risk for lung cancer, but this may not hold true for all populations (Mao et al., 2008;Boccia et al., 2009;Zhang et al., 2012). The aim of this study was to determine if MTHFR C677T and A1298C polymorphism is/are associated with an altered risk of developing lung cancer among Jordanians.

Study design and population
In this case-control study, paraffin-embedded tissues from histologically confirmed lung cancer cases from (98) unrelated Jordanian patients previously diagnosed with lung cancer were collected from Jordan University Hospital (JUH). Histologically, 88 cases were non-smallcell carcinoma mainly squamous cell carcinoma (N=35) and adenocarcinoma (N=30). The remaining 10 cases were small-cell carcinoma.
The control group consisted of (89) healthy individuals from the same geographical area as the lung cancer patients who consented to analysis of their biological samples. The study protocol was approved by the Institutional Review Board (IRB) at JUH. The study was exempted by the IRB from obtaining a formal written consent from patients for paraffin-embedded samples.
Not all samples were successfully analyzed for the two SNPs due to sample depletion, failure of PCR or failure of endonuclease digestion. Four cases had data at 1298 but not at 677, and three cases had data at 677 but not at 1298. One control had data at 1298 but not at 677, and two controls had data at 677 but not at 1298.

MTHFR genotyping
DNA from paraffin-embedded blocks of patient and control samples was extracted using an FFPE kit (Qiagen, Germany) according to the manufacturer's instructions. Genotyping was performed for both polymorphisms, C677T and A1298C, by two separate polymerase chain reaction (PCR) reactions using a thermal cycler (model PTC-100; Bio-Rad-MJ Research, Watertown, MA). Amplification of the C677T region was performed using the forward primer TGAAGGAGAAGGTGTCTGCGGGA (NT_021937.19: 7,861,134 to 7,861,112) and the reverse primer AGGACGGTGCGGTGAGAGTG (NT_021937.19: 7,860,937 to 7,860,956) yielding a 198-bp band, whereas for the A1298C region, the forward primer CAAGGAGGAGCTGCTGAAGA (NT_021937.19: 7,859,255 to 7,859,236), and the reverse primer CCACTCCAGCATCACTCACT (NT_021937.19: 7,859,128 to 7,859,147), were used yielding a 128bp band. For both amplifications, the PCR conditions were described by Yi et al. (2002) 8 minutes of initial denaturation at 95 o C, followed by 40 cycles of 95 o C for 60 seconds, 63 o C for 60 seconds, and 72 o C for 60 seconds, with a final extension at 72 o C for 7 minutes.
The PCR products of C677T and A1298C were separately digested with the HinfI and MboII restriction enzymes (Promega ® ), respectively. Resulting fragments were visualized using ethidium bromide staining and 3% agarose (Promega ® ) gel electrophoresis. The digestion fragment sizes for the C677T genotypes were: a single 198-bp band for CC, 198 bp, 175 bp and 23 bp for CT, and 175 bp and 23 bp for TT. For A1298C genotypes, the fragments were 72 bp and 28 bp (2 fragments) for AA, 28 bp, 72 bp, and 100 bp for AC, and 100 bp and 28 bp for CC.
Findings of the PCR-RFLP were validated by: 1) inclusion of a negative control in all PCR analyses; negative controls contain all PCR components except DNA template; 2) repeat analysis of approximately 15% of all samples; concordance between repeated samples was 100%; and 3) direct DNA sequencing of 30 randomly selected PCR-RFLP digests using BigDye Terminator Cycle Sequencing on 3730xl DNA sequencer (Macrogene ® Co., Korea).

Haplotype analysis
The interaction between genetic polymorphism at the two loci was assessed by evaluating the combinedgenotype effects by haplotype analysis. We analyzed the haplotype frequencies of the two SNPs for lung cancer cases and compared them with those of controls. Haplotype frequencies were calculated using Golden Helix Tree ® software and linkage disequilibrium was represented by D prime (D'). The Golden Helix software is enabled to estimate the haplotype frequencies even with presence of some missing data of one or two of the SNPs. Similar findings were obtained utilizing Multiallelic Interallelic Disequilibrium Analysis Software (University of Southampton, Highfield, Southampton, UK)

Statistical analysis
Statistical analysis was performed using SPSS © software (version 11.0; SPSS, Inc, Chicago, IL). Data was expressed as mean ± SD or as counts and percentages. Data was subdivided between two groups (LC and controls). Allele and genotype frequencies for different alleles among Jordanian population were estimated from the results of the above PCR-RFLP test. This estimation was according to formulas reported previously (Brooker, 2005). Age was compared between the two groups by independent student t-test or Mann Whitney as appropriate. Age was compared between the different genotypes by ANOVA or Kruskal Wallis. Data of categorical nature (gender, smoking behavior, genotypes and haplotypes) were compared with tissue type by chi square or Fisher exact test as appropriate (Graph Pad Software Inc). Odds ratios (OR) and 95% confidence intervals (95% CI) were calculated as a measure of association between MTHFR genotypes/alleles/haplotypes and LC. Genotype and allele frequency were analyzed for concordance to the Hardy-Weinberg equilibrium. Multiple logistic regressions was utilized to discern the relationship between significant variables and the susceptibility to lung cancer. We conducted multivariate logistic regression analysis for variables which demonstrated significance (p<0.05) in bivariate analysis. Adjusted odds ratio (aOR) and 95% confidence intervals (CI) for the probability of LC control were calculated for each predictor to explain the strength of association. A p-value below 0.05 was considered statistically significant throughout the population comparisons.

Genetics
The distribution of MTHFR C677T and A1298C genotypes and their alleles are presented in Table 1. Neither C677T genotypes nor alleles was statistically different in lung cancer cases and controls (table 1). However, there were statistically significant differences in the genotype frequency of MTHFR A1298C between LC cases and the controls. The frequency of the MTHFR C allele was higher in patients with LC compared with healthy controls (Table 1). The CC genotype was associated with a 2.3 to 2.5-fold higher risk of LC (p=0.031); the AC genotype was associated with a 1.1-fold higher risk, albeit not statistically significant (p=0.74). Subjects with the C allele were 1.5 time more likely to suffer from lung cancer (p=0.057).
When comparing age between cases and control within each genotype, no significant differences were observed [(AA control vs AA LC , p=0.084); (AC control vs AC LC , p=0.8); (CC control vs CC LC , p=0.32)].
There were statistically significant differences in 1298 genotypes of MTHFR among smokers (LC-smokers: 20 AA, 37 AC, 18 CC vs controls-smokers: 11 AA, 10 AC, 1 CC, p= 0.044). There was no statistically significant difference in any of 1298 genotypes of MTHFR among non-smokers. 1298CC genotype of MTHFR presented 9.9 times higher risk of LC in smokers as compared with the AA carriers (Table 2).

Logistic regression
Backward stepwise logistic regression with conditional likelihood was used to find out the best subset of variables to predict lung cancer. The logistic regression was specified for significance level of 0.05 for entry and at level of 0.1 for removal. The different models were compared by conditional logistic regression. The Wald statistic was used in comparing the fitness of combined models. As a further check, -2 log-likelihood ratio statistic was utilized. Only when the two statistics agree, then the predictor is decided to be useful for the model. The following variables were evaluated as predictors of lung cancer: gender; smoking; age; and A1298C genotypes (CC vs others). The Hosmer-Lemeshow statistic indicated that the model adequately fits the data (p=0.685). The Nagelkerke's R 2 of the prediction model was 0.383. As a further check, the model was built using forward stepwise methods. The two methods chose the same variables. Smoking behavior and the 12988CC genotype remained statically significant with adjusted odds ratio of 12.1 (95% CI: 6.0-25; p<0.001) and 3.2 (95% CI: 1.3-8.2; p=0.015), respectively.

Discussion
The mechanism of lung carcinogenesis is, like other cancers, still not fully understood. Among the lifestyle related causes of lung cancer, smoking is the primary risk factor for lung cancer. However, lung cancer develops in less than 20% of people who smoke throughout their life (Shields, 2002) This suggests that other factors including genetic susceptibility and nutritional habits contribute to carcinogenesis of lung cancer (Perera, 1998). Examination of genetic polymorphisms may explain individual differences in cancer risk.
Folate metabolism is thought to play a critical role in carcinogenesis through its involvement in both DNA methylation and repair. Previous studies revealed higher risk of breast and esophagus cancer but not colorectal cancer with less functional forms of MTHFR (Ergul et al., 2003;Stolzenberg-Solomon et al., 2003;Pardini et al., 2011). The MTHFR gene is located at the end of the short arm of chromosome 1 (1p36.3) (Goyette et al., 1998). The two common functional polymorphisms have been defined in the MTHFR gene -one is C677T (rs1801133;NT_021937.19: 7,861,110) and another is A1298C (rs1801131; NT_021937.19: 7,859,208).
MTHFR C677T polymorphism results in substitution of alanine by valine at amino acid 222 in exon 4 (Frosst et al., 1995;Sharp and Little, 2004). Studies indicated that subjects with the TT or CT genotype have lower levels of enzyme activity, 30% and 65%, respectively, relative to enzyme activity in subjects carrying the CC genotype (Frosst et al., 1995;Kono and Chen, 2005;Brockton, 2006). Additionally, this single nucleotide polymorphism (SNP) decreases the thermal stability of this enzyme (Cicek et al., 2004). It has been reported that this substitution may lower levels of 5-methyltetrahydrofolate, and increase plasma homocysteine levels (Frosst et al., 1995;Ma et al., 1997;Bagley and Selhub, 1998).
Prior to our investigation, based on Zhang et al, the studies that investigated MTHFR C677T and A1298C were distributed by ethnicity as follows: 5 studies recruited Caucasians (USA, The Netherlands, Poland and France); 9 studies examined individuals of Asian descent (China, Taiwan, Korea, and Japan); the last study was from Turkey (Zhang et al., 2012). This is the first study conducted in Jordan and the in the Arab world. Jordanians are mostly descended from people of villagers and Bedouin descent originating in the Arabian Peninsula (Lowi, 1995); thus, ethnically, the Jordanians represent a mixed stock. Most of the population is Arab (approximately 98%) with 1% of the population, Armenian, and another 1%, Circassian.  There are also Kurd, Druze, and Chechen minorities (Central Intelligence Agency, 2012; The Royal Hashemite Court, 2012). In 2008, Mao et al. performed a meta-analysis based on eight studies and did not find a relationship between the MTHFR polymorphisms and the risk of lung cancer (Mao et al., 2008). In 2009, another meta-analysis aggregated with ten studies and also did not find a significant relationship between the polymorphisms and lung cancer risk, but an increased risk for individuals with low folate intake (Boccia et al., 2009). In 2012, a meta-analysis of 14 publications found no significant association between the MTHFR C677T polymorphism and LC risk irrespective of race, Stratified analysis by histological type indicated significantly increased nonsmall-cell lung cancer (NSCLC) risk among the T allele carriers (T-allele vs C-allele: OR=1.11, 95%CI=1.03-1.19; TT vs CC: OR=1.24, 95%CI=1.09-1.41; TC vsCC: OR=1.11, 95%CI=1.03-1.20 and TT+TC vs CC: OR=1.09, 95%CI=1.03-1.15) (Hou et al., 2012). A more recent meta-analysis of 20 studies was published in 2013 that evaluated the association between MTHFR C677CT polymorphism and lung cancer risk (8 Caucasians and 12 Asians) (Liu et al., 2013). Seven studies suggested that MTHFR C677T polymorphism was associated with an increased lung cancer risk. After stratification by ethnicity (Asian and Caucasian), a significant association between MTHFR C677T polymorphism and lung cancer risk was observed in Asian subjects (OR=1.31, 95% CI =1.01-1.71, p=0.045, TT vs CC), while no significant association was found among Caucasians (OR=1.16; 95% CI =0.91-1.49,p=0.224, TT vs CC) (Liu et al., 2013). The association between lung cancer and MTHFR C677T polymorphism has been recently confirmed (Rai, 2014).
In our study, none of the 677 genotypes or allele types was distributed differentially between cases and controls. The lack of association persisted even after stratification by gender or smoking status (Table 2 and 3). Logistic regression (data not presented) confirmed the lack of association.
The discrepancies in the results may be explained by differences in ethnicity (Cheng et al., 2012), dietary intake, exposure to environmental carcinogens and sample size. Folate intake has been suggested to modulate cancer risk associated with MTHFR genetic polymorphism (Chen et al., 1996;Ma et al., 1997). Kiyohora et al have proposed that excessive folate supplementation increases folic acid levels, reduce homocysteine levels and then restore normal methionine levels, particularly in these individuals with the TT genotype of the MTHFR polymorphism (Paul et al., 2004;Kiyohara et al., 2011).
MTHFR A1298C polymorphism results in an amino acid substitution of alanine to glutamate at codon 429 of the protein in exon 7 (van der Put et al., 1998;Weisberg et al., 1998). The enzyme activity in vitro is decreased in homozygous variants (CC) and, to a lesser extent, in heterozygotes compared with those without the variant (van der Put et al., 1998). Studies of the relationship between A1298C genotype and plasma folate and homocysteine are inconsistent (Weisberg et al., 1998;Friedman et al., 1999;Chango et al., 2000;Lievers et al., 2001;Chen et al., 2002). Enzyme activity in vitro for compound heterozygotes (i.e., heterozygotes for C677T and for A1298C) is unclear (Lievers et al., 2001). A less active form of MTHFR leads to lower S-adenosyl-Lmethionine levels and consequently to hypomethylation; this phenomenon would be expected to increase the risk of some cancers (Stern et al., 2000). It is expected that 1298CC carriers are at higher risk for cancer development.
In our study, we also investigated the frequencies of the MTHFR A1298C genotypes in Jordanian patients with LC and the healthy controls. Contrary to the apparent lack of association between MTHFR C677T and the risk of LC, the MTHFR A1298C polymorphism was found to be significantly related with the risk of LC. There was significant interaction between genetic polymorphism at 1298 and both smoking status and subjects' gender. Carriers of the 1298CC genotype as compared with the AA carriers presented 9.9, 6.7 and 7.3 times higher risk of LC in smokers, men, "men who were smokers", respectively. Because of the unequal distribution of gender and smoking among cases and controls, logistic regression was conducted, and confirmed the association between 1298CC and susceptibility to lung cancer. The interaction between A1298C and smoking maybe explained by the reported decreased circulating folate levels due to low folate intake in smokers (Dastur et al., 1972;Witter et al., 1982).
The results for the A1298C variant are consistent with some but not all published studies. A case-control study evaluated the role of the MTHFR A1298C polymorphisms in 462 lung cancer cases and 379 controls in a Japanese population. The 1298CC genotype was only associated with increased risk among ever-smokers (aOR= 3.2, 95% CI= 1.2-8; p=0.02) non-smokers (aOR=2.83, 95% CI= 1.02-7.8; p=0.05) and excessive drinkers (aOR=7.24, 95% CI=1.89-27.7, p<0.001) (Kiyohara et al., 2011). The 1298CC genotype was associated with a significantly increased risk of lung cancer in women in one study (Shi et al., 2005).
A Turkish study involved 64 lung cancer patients and 61 controls. Lung cancer risk was 1.5 times higher in 1298CC genotype though it was not statistically significant. Numerous other studies worldwide have reported lack of association of MTHFR A1298C polymorphism with the risk of lung carcinoma Mao et al., 2008;Boccia et al., 2009;Liu et al., 2013) The physical distance between the two SNPs is short (1.9 kb), and thus one may expect that LD is evident. There is evidence of linkage disequilibrium between the C677T and the A1298C variants of the MTHFR gene with the possibility that both SNPs may be dependent on each other from the genetic and functional point of view (Chen et al., 2002;Shi et al., 2003). The interdependence of the two loci may imply that haplotype analysis plays an important role in the identification of genetic variations between cases and controls in addition to the customary analysis of SNPs. Although not stated, we calculated D' and r 2 among different ethnicities: Chinese (Controls: D'=0.13, r 2 =0.004) (Li et al., 2011); Indians (Controls: D'=0.56, r 2 =0.08) (Chandy et al., 2010); and three sets of Americans: non-Hispanic whites (Controls: D'=0.24, r 2 =0.043; LC: D'=0.25, r 2 =0.045) (Shen et al., 2001); African Americans (Controls: D'=1, r 2 =0.03) (Keku et al., 2002); and mixed-population Americans (Controls: D'=1, r 2 =0.23) (Curtin et al., 2004). A study from central Europe involved 2250 lung cancer cases, 811 upper aerodigestive tract cases and 2899 controls reported relatively strong linkage disequilibrium (D'= -0.99, r 2 =0.21) (Hung et al., 2007). Our results indicated that the two loci showed relatively strong LD among cases and controls ([D'] = [0.56 (controls), 0.84 (LC)]).
The most frequent haplotypes of MTHFR among Caucasians were reported as CA (37%); CC (31%); and TA (32%) (Ogino and Wilson, 2003). A more recent study on Czek subjects reported CA (33.6%); CC (33.7%); and TA (31.9%) as the most common haplotypes (Pardini et al., 2011). Current study reports a similar trend, albeit a slightly lower TA distribution (24.7%) and higher TC (5%). Because of rarity of TT/CC (677TT and 1298CC) and TT/AC among Jordanians and worldwide, the TC haplotype should be rare as well (Ogino and Wilson, 2003). The estimated TC haplotype among Caucasians-general populations was reported in less than 1% of the general public (0.3-0.8%) (Ogino and Wilson, 2003;Pardini et al., 2011). Some studies lacked the TC haplotype (Meisel et al., 2001;Terrazzino et al., 2006). Still, there may be an increased frequency of the very rare TC haplotype in some parts of the United Kingdom, Canada (Ogino and Wilson, 2003) and USA (Shen et al., 2001). Utilizing published data, we calculated the TC haplotype among other ethnicities and found it rare as well (non-Hispanic white Americans 13.2% (Shen et al., 2001);Chinese 9.4 % (Li et al., 2011) and African Americans 0% (Keku et al., 2002)). Interestingly, the haplotype analysis based on the two investigated MTHFR polymorphisms (C677T and A1298C) showed that haplotype CC was more common in Jordanians with lung cancer than controls (OR=1.6, 95% CI= 1.03-2.4, p=0.037). Examining the literature revealed few studies that investigated the effect of MTHFR haplotypes on susceptibility to lung cancer (Shen et al., 2005;Hung et al., 2007). The TA haplotype, rather than the CC haplotype, was associated with in increased risk of lung cancer in a Central European study (OR=1.16, 95% CI=1.04-1.29) (Hung et al., 2007) and a Chinese study (OR= 1.67, 95% CI: 1.10-2.52) (Shen et al., 2005). In the Chinese study the CC haplotype was associated with increased risk though not statistically significant.
In conclusion, the findings of the current study suggest that genetic polymorphism of MTHFR at A1298C and its haplotype analysis at 677and 1298 modulates the risk of LC in the Jordanian population. To our knowledge, this is being reported for the first time among this population.