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Meta-analysis of MTHFR polymorphisms and pancreatic cancer susceptibility
CHAPTER 13
Meta-analysis of MTHFR polymorphisms and pancreatic cancer susceptibility L.V.K.S. Bhaskar1, L. Saikrishna2 2 1
Sickle Cell Institute Chhattisgarh, Raipur, India; Department of Zoology, Visvodaya Government Degree College, Venkatagiri, India
Abstract Methylenetetrahydrofolate reductase (MTHFR) deficiency is the most common determinant of serum homocysteine and folate status that is associated with increased risk for many cancers. This MTHFR deficiency in pancreatic cancer (PC) pathogenesis has been attributed to genetic polymorphism of MTHFR 677C>T and 1298A>C, but the evidence is still unclear. We conducted a meta-analysis of studies evaluating the effect of these polymorphisms on PC. We searched databases such as PubMed, Embase, and Google Scholar to retrieve studies that investigated MTHFR 677C>T and 1298A>C polymorphism for PC risk. For MTHFR 677C>T analysis, a total of eight publications with 1680 cases and 2520 controls were included; and for MTHFR 1298A>C analysis, six studies with 1421 cases and 1641 controls were included. Genotypes retrieved were subjected to meta-analysis using a random- or fixed-effects model with odds ratios and 95% confidence intervals as effect measures. Our met-analysis in all genetic models showed that MTHFR polymorphisms were not associated with the risk for PC. MTHFR polymorphisms revealed significant heterogeneity between studies for MTHFR 677C>T (P ¼ .003) but not for 1298A>C (P ¼ .495). Furthermore, there was no evidence for publication bias for these polymorphisms. Because the distribution of MTHFR genetic polymorphism differs among ethnic groups, the results of this meta-analysis cannot be extrapolated to patients belonging to any other ethnic group.
Keywords: Meta-analysis; MTHFR; Pancreatic cancer; SNPs.
List of abbreviations CI Confidence interval Hcy Homocysteine HWE HardyeWeinberg equilibrium MTHFR Methylenetetrahydrofolate reductase OR Odds ratio PC Pancreatic cancer RBC Red blood cell SNP Single nucleotide polymorphism G.P. Nagaraju, S. Ahmad (eds.) Theranostic Approach for Pancreatic Cancer ISBN 978-0-12-819457-7 https://doi.org/10.1016/B978-0-12-819457-7.00013-X
Copyright © 2019 Elsevier Inc. All rights reserved.
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Introduction Pancreatic cancer (PC) is the second leading cause of cancer-related deaths worldwide [1]. Whipple procedure or surgery remains the only possible treatment option for PC. However, because of the lack of specific symptoms and improved screening and diagnostic methods, only 10e20% of patients are diagnosed at a stage amenable to resection and possible therapy. As a result, PC is correlated with high mortality burden and poor prognosis [2]. Many risk factors are associated with PC. Nonmodifiable risk factors such as age, familial cancer syndromes, Afro-American race, hereditary, other forms of chronic pancreatitis, diabetes, and non-O blood group are associated with PC. Smoking, obesity, dietary factors such as a nonvegetarian diet, and toxins are some modifiable risk factors contributing to PC [3]. Folic acid has a major role in the biosynthesis of essential mediators for DNA methylation. Hence, folate deficiency leads to abnormal DNA integrity and epigenetic methylation patterns [4]. Several lines of evidence indicated an inverse relation between dietary folate intake and PC risk [5e7]. A large meta-analysis investigation that examined the effect of the interaction of serum concentration of homocysteine (Hcy), folate, and vitamin B12 and 5,10-methylenetetrahydrofolate reductase (MTHFR) polymorphisms demonstrated that an elevated serum Hcy level and folate deficiency are associated with increased overall risk for cancer [8]. As an important regulator of folate metabolism, the MTHFR enzyme has an important role in converting Hcy to methionine. The MTHFR gene harbors two important genetic variations (677C>T and 1298A>C) that contribute to inactivation of this enzyme [9]. Several studies examined the association between MTHFR gene polymorphisms and PC risk; however, findings have been inconsistent [10e13]. We performed a meta-analysis to investigate the relation between MTHFR gene polymorphisms and PC susceptibility.
Materials and methods Literature search strategy To select eligible studies on MTHFR polymorphisms and PC, electronic databases including PubMed, Embase, and Google Scholar were implemented with the following search terms: “pancreatic cancer,” “MTHFR,” “677C>T,” “1298A>C,” “rs1801133,” “rs1801131,” “SNP,” “polymorphism,” and “genetic variant.” All relevant studies published until Sep. 2018 were included in the meta-analysis. Studies met the following criteria to be included in the analysis: (1) original observational study of human participants; (2) an association study between 677C>T and/or 1298A>C
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of the MTHFR gene and PC; (3) case-control study; (4) availability of genotype data; and (6) studies were published in English. All studies were independently reviewed by two researchers and the following data were extracted: (1) first author name; (2) publication year; (3) population ethnicity/country of origin; and (4) genotype frequencies in PC and control. The current meta-analysis was conducted according to guidelines issued in the Preferred Reporting Items for Systematic Reviews and Meta-analysis 2009. The literature search strategy and process for selection of papers for this meta-analysis are shown in Fig. 13.1.
Statistical analysis The HardyeWeinberg equilibrium (HWE) test was calculated using chi-square test. Studies that deviated from the HWE of genotype frequencies in controls were excluded from the analysis. Heterogeneity across individual studies was assessed by Cochran Q test and I2 statistic. If there was no evidence of heterogeneity, a fixed-effects model was used to estimate the pooled odds ratio (OR) and corresponding 95% confidence intervals (95% CIs); otherwise, a random-effects model was used. The ORs and 95% CIs were calculated to assess the association between two SNPs of the MTHFR gene and PC risk. Four genetic models were performed in the current meta-analysis (using MTHFR 677C>T as an example): (1) allele model, T allele versus C allele; (2) dominant model (CTþTT versus CC); (3) recessive model (TT vs. CTþCC); and (4) codominant model (TT versus CC; CT versus CC; CT versus TT). Begg’s funnel plot and Egger’s test were used to evaluate publication bias, which was presented
Figure 13.1 Flowchart of study selection procedure. MTHFR, methylenetetrahydrofolate reductase.
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using the funnel plot of precision by the log of OR. To examine the influence of each study on robustness of the method used for meta-analysis, a sensitivity analysis was performed. MetaGenyo, a Web tool, was used to calculate results of the meta-analysis [14].
Results Study characteristics A total of eight eligible studies met inclusion criteria [10e13,15e18]. For the MTHFR 677C>T analysis, a total of eight publications with 1680 cases and 2520 controls were included; and for the MTHFR 1298A>C analysis, six studies with 1421 cases and 1641 controls were included. The genotype distributions of all studies were consistent with the HWE. The strategy adopted for selecting articles for this meta-analysis is shown in Fig. 13.1. Characteristics of selected studies are listed in Table 13.1.
Quantitative synthesis Among studies performed on MTHFR 677C>T in PC, there was significant heterogeneity (P ¼ .003, I2 ¼ 63.8%); hence, the random-effects model was used to estimate pooled ORs (Fig. 13.2). The meta-analysis results showed that there was no statistically significant association between MTHFR 677C>T and PC risk in four genetic models (T allele versus C allele, P ¼ .344; dominant model, P ¼ .405; recessive model, P ¼ .281) (Table 13.2, Fig. 13.2). With regard to the MTHFR 1298A>C polymorphism, heterogeneity was not observed among studies (P ¼ .495, I2 ¼ 0%); hence the fixed-effects model was used to estimate pooled ORs (Fig. 13.2). MTHFR 1298A>C also was not associated with PC risk in all-genetic models (C allele versus A allele, P ¼ .882; dominant model, P ¼ .634; recessive model, P ¼ .609) (Table 13.2, Fig. 13.2).
Sensitivity analysis and publication bias The sensitivity analysis performed by sequentially omitting individual studies demonstrated that no individual study exerted an excessive influence on the pooled effect of the MTHFR 677C>T and 1298A>C polymorphisms on PC risk (Fig. 13.3). In all genetic models, the shape of funnel plots seemed symmetrical for both MTHFR 677C>T and MTHFR 1298A>C polymorphisms, which suggested the absence of publication bias (Fig. 13.4). Statistical evidence of publication bias was obtained through
Table 13.1 Characteristics of studies included for meta-analysis. Author
Cases
Control
MTHFR 677C>T
Ethnicity
CC
CT
TT
CC
CT
TT
HardyeWeinberg P value
Wang et al., 2005 [18] Matsubayashi et al., 2005 [11] Li et al., 2005 [15]a Li et al., 2005 [15]b Li et al., 2005 [15]c Suzuki et al., 2008 [17] Nisevic et al., 2008 [16] Mazzuca et al., 2015 [12] Nakao et al., 2016 [13] Chittiboyina et al., 2018 [10]
East Asian Caucasian Caucasian Hispanic African East Asian Caucasian Caucasian East Asian Caucasian
31 145 150 10 11 57 43 32 127 61
79 115 117 10 6 80 52 51 161 73
53 43 36 1 0 20 5 14 72 25
135 134 149 5 12 291 42 76 124 24
149 135 138 5 4 366 50 98 194 26
53 36 20 3 0 128 8 28 82 5
0.270 0.823 0.108 0.444 0.568 0.475 0.191 0.687 0.701 0.585
AA
AC
CC
AA
AC
CC
124 133 129 12 11 49 240 87
37 134 145 5 6 45 107 60
2 36 29 0 0 5 13 12
243 144 133 9 12 103 285 24
86 140 137 6 4 80 102 24
8 21 40 1 0 19 13 7
MTHFR 1298A>C
Wang et al., 2005 [18] Matsubayashi et al., 2005 [11] Li et al., 2005 [15]a Li et al., 2005 [15]b Li et al., 2005 [15]c Mazzuca et al., 2015 [12] Nakao et al., 2016 [13] Chittiboyina et al., 2018 [10]
East Asian Caucasian Caucasian Hispanic African Caucasian East Asian Caucasian
0.905 0.093 0.613 1.000 0.568 0.548 0.305 0.795
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(A) MTHFR 677C>T Study
Odds Ratio
Control Experimental Events Total Events Total
Wang et al., 2005 Matsubayashi et al., 2005 Li et al., 2005a Li et al., 2005b Li et al., 2005c Suzuki et al., 2008 Nisevic et al., 2008 Mazzuca et al., 2015 Nakao et al., 2016 Chittiboyina et al., 2018
132 158 153 11 6 100 57 65 233 98
Random effects model
163 303 303 21 17 157 100 97 360 159 1680
OR
95%-CI W(random)
337 305 307 13 16 785 100 202 400 55
2.85 [1.82; 4.45] 0.85 [0.62; 1.18] 0.96 [0.70; 1.32] 0.69 [0.17; 2.81] 1.64 [0.36; 7.38] 1.03 [0.72; 1.48] 0.96 [0.55; 1.68] 1.23 [0.74; 2.04] 0.82 [0.61; 1.12] 1.24 [0.67; 2.32]
11.4% 13.9% 14.0% 2.7% 2.4% 13.2% 9.4% 10.3% 14.3% 8.5%
2520
1.11 [0.87; 1.43]
100%
202 171 158 8 4 494 58 126 276 31
Heterogeneity: I-squared=63.8%, tau-squared=0.0894, p=0.0032
0.2
0.5
1
2
5
(B) MTHFR 1298A>C Study
Wang et al., 2005 Matsubayashi et al., 2005 Li et al., 2005a Li et al., 2005b Li et al., 2005c Mazzuca et al., 2015 Nakao et al., 2016 Chittiboyina et al., 2018 Fixed effect model
Odds Ratio
Experimental Control Events Total Events Total 39 170 174 5 6 50 120 72
163 303 303 17 17 99 360 159
94 161 177 7 4 99 115 31
1421
OR
95%-CI W(fixed)
337 305 310 16 16 202 400 55
0.81 [0.53; 1.25] 1.14 [0.83; 1.57] 1.01 [0.74; 1.40] 0.54 [0.13; 2.25] 1.64 [0.36; 7.38] 1.06 [0.66; 1.72] 1.24 [0.91; 1.69] 0.64 [0.35; 1.19]
12.4% 22.6% 22.5% 1.1% 1.0% 10.0% 24.3% 6.0%
1641
1.04 [0.89; 1.21]
100%
Heterogeneity: I-squared=0%, tau-squared=0, p=0.4949
0.2
0.5
1
2
5
A: MTHFR 677 TT+CT vs. CC; B: MTHFR 1298 CC+AC vs. AA; OR: Odds ratio
Figure 13.2 Forest plot representing pooled results of odds ratios (ORs) for association between MTHFR gene variants and pancreatic cancer risk. (A) MTHFR 677 TTþCT versus CC. (B) MTHFR 1298 CCþAC versus AA. CI, confidence interval.
Egger’s test. P values for both MTHFR 677C>T and MTHFR 1298A>C polymorphisms are listed in Table 13.2.
Discussion PC symptoms are rarely noticeable in the early stages and adversely affect the gastrointestinal system and patients’ quality of life [19]. It is widely acknowledged that the etiology of PC emerges from the dynamic interaction between genetic and environmental factors [20]. Because familial and sporadic PC share the same molecular pathogenesis, study of genee environment interactions has been invoked as a premise to illuminate the etiological complexity of PC oncogenesis [21]. Although treatment options
Table 13.2 Meta-analysis of association between MTHFR gene variants and pancreatic cancer susceptibility. Test of heterogeneity Test of association Comparison model
c2
P value
I2
Statistics model
Odds ratio (95% confidence interval)
P value
Egger’s test P value
33.39 24.83 23.70 32.26 16.05 17.81
<0.001 0.003 0.003 <0.001 0.042 0.037
73.0 63.8 66.3 75.2 50.1 49.5
Random Random Random Random Random Random
1.10 1.11 1.22 1.25 1.21 1.06
(0.90e1.36) (0.87e1.43) (0.85e1.74) (1.79e1.99) (0.89e1.65) (0.85e1.32)
0.344 0.405 0.281 0.334 0.229 0.229
0.942 0.542 0.454 0.785 0.421 0.266
9.68 6.39 10.10 10.14 8.56 4.70
0.207 0.495 0.120 0.119 0.200 0.696
27.7 0 40.6 40.9 29.8 0
Fixed Fixed Fixed Fixed Fixed Fixed
1.01 1.04 0.93 0.94 0.90 1.06
(0.89e1.14) (0.89e1.21) (0.69e1.25) (0.69e1.29) (0.66e1.23) (0.90e1.24)
0.882 0.634 0.609 0.712 0.520 0.504
0.299 0.436 0.299 0.295 0.387 0.420
MTHFR 677C>T
T allele versus C allele TTþCT versus CC TT versus CCþCT TT versus CC TT versus CT CT versus CC MTHFR 1298A>C
C allele versus A allele CCþAC versus AA CC versus ACþAA CC versus AA CC versus AC AC versus AA
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(A) MTHFR 677C>T Study
Odds Ratio
OR
95%-CI
Omitting Wang et al., 2005 Omitting Matsubayashi et al., 2005 Omitting Li et al., 2005a Omitting Li et al., 2005b Omitting Li et al., 2005c Omitting Suzuki et al., 2008 Omitting Nisevic et al., 2008 Omitting Mazzuca et al., 2015 Omitting Nakao et al., 2016 Omitting Chittiboyina et al., 2018
0.95 1.10 1.07 1.05 1.05 1.05 1.06 1.04 1.12 1.04
[0.82; 1.10] [0.95; 1.28] [0.92; 1.25] [0.92; 1.21] [0.91; 1.20] [0.91; 1.22] [0.92; 1.22] [0.90; 1.20] [0.96; 1.30] [0.91; 1.20]
Fixed effect model
1.05 [0.92; 1.20] 0.8
1
1.25
(B) MTHFR 1298A>C Odds Ratio
Study
OR
95%-CI
Omitting Wang et al., 2005 Omitting Matsubayashi et al., 2005 Omitting Li et al., 2005a Omitting Li et al., 2005b Omitting Li et al., 2005c Omitting Mazzuca et al., 2015 Omitting Nakao et al., 2016 Omitting Chittiboyina et al., 2018
1.07 1.01 1.04 1.05 1.03 1.03 0.98 1.07
[0.91; 1.26] [0.85; 1.20] [0.88; 1.24] [0.90; 1.22] [0.89; 1.20] [0.88; 1.21] [0.82; 1.17] [0.92; 1.25]
Fixed effect model
1.04 [0.89; 1.21] 0.8
1
1.25
A: MTHFR 677 TT+CT vs. CC; B: MTHFR 1298 CC+AC vs. AA; OR: Odds ratio
Figure 13.3 Forest plot representing corresponding pooled odds ratio (OR) of removing each study. (A) MTHFR 677 TTþCT versus CC. (B) MTHFR 1298 CCþAC versus AA. CI, confidence interval.
such as surgery, radiation, chemotherapy, and chemoradiation have progressed continuously to control symptoms, there is no sure way to prevent PC. Understanding the genetics of PC may enable us to identify high-risk individuals and open the door to designing novel therapeutic strategies. Hyperhomocysteinemia has been reported in patients with neoplastic diseases. 5-Methyltetrahydrofolate serves as the methyl group donor for the
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(A) 0.0
MTHFR 677C>T
Standard Error
0.2
0.4
0.6
0.5
1.0 Odds Ratio
2.0
5.0
1.0 Odds Ratio
2.0
5.0
(B) 0.0
MTHFR 1298A>C
Standard Error
0.2
0.4
0.6
0.5
A: MTHFR 677C>T; B: MTHFR 1298A>C; OR: Odds ratio
Figure 13.4 Begg’s funnel plot of ORs for association between MTHFR gene variants and pancreatic cancer risk. (A) MTHFR 677C>T. (B) MTHFR 1298A>C. OR, odds ratio.
remethylation of Hcy to methionine. Because folate supplies the substrate for the MTHFR reaction, reduced serum folate and dysfunctional MTHFR appeared to be key factors in hyperhomocysteinemia. This was further supported by the development of acute pancreatitis in animals fed with ethionine (inhibitor of methylation)-supplemented or choline (methyl donor)-deficient diets [22]. Furthermore, a prospective study of Swedish
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women and men suggested that higher methionine intake may reduce the risk for PC [23]. MTHFR functional polymorphisms have been studied in different ethnicities for their possible association with PC. Wang et al. [18] first observed MTHFR 677C>T mutant genotypes more frequently (ORs of 2.6 and 5.1 for the CT and TT genotypes, respectively) in East Asian PC patients compared with controls. Furthermore, a twofold increased risk for PC for individuals with the MTHFR 677 CT/TT was reported in Caucasians [15]. Subsequent case-control studies failed to show a significant association between MTHFR gene polymorphisms and PC [10e13,16]. However, a study showed that the MTHFR 677C>T variant allele resulted in lower red blood cell (RBC) folate levels compared with the referent wild-type genotype [10]. In contrast, individuals with variants genotypes of the MTHFR 1298A>C single nucleotide polymorphism (SNP) had higher mean RBC folate concentrations compared with the wild-type genotype [10]. The opposite consequences of MTHFR 677C>T and 1298A>C polymorphisms on RBC folate levels was also documented earlier [24]. This indicated that the effects of MTHFR polymorphisms on total Hcy levels in various populations remain controversial [25]. Our metaanalysis revealed that there is no association between MTHFR polymorphisms and PC risk. Meta-analysis of MTHFR polymorphisms revealed significant heterogeneity between studies for MTHFR 677C>T but not for MTHFR 1298A>C. Furthermore, there was no evidence for publication bias for these polymorphisms. Consistent with our results, two previous meta-analyses provided no evidence of association between MTHFR polymorphisms and PC risk [26,27]. Another meta-analysis showed an obvious association between MTHFR 677C>T polymorphism and PC risk in East Asians but not Caucasians [28]. This meta-analysis provided no evidence for an association between PC risk and MTHFR polymorphisms (677C>T and 1298A>C). The metaanalysis had several potential limitations. First, the study was conducted on MTHFR polymorphisms, which are not extensively studied in PC. Second, information on red cell folate concentrations or serum Hcy levels was unavailable in these studies. Third, the information on lifestyle factors influencing folate status, such as smoking, alcohol consumption, and dietary folate intake, were unavailable for included studies. Because most of the studies included in this meta-analysis were of Caucasians and East Asians and the distribution of MTHFR genetic polymorphism differs among ethnic groups [29,30], the results cannot be extrapolated to patients belonging to any other ethnic group.
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Meta-analysis of MTHFR polymorphisms and pancreatic cancer susceptibility L.V.K.S. Bhaskar1, L. Saikrishna2 2 1
Sickle Cell Institute Chhattisgarh, Raipur, India; Department of Zoology, Visvodaya Government Degree College, Venkatagiri, India
Abstract Methylenetetrahydrofolate reductase (MTHFR) deficiency is the most common determinant of serum homocysteine and folate status that is associated with increased risk for many cancers. This MTHFR deficiency in pancreatic cancer (PC) pathogenesis has been attributed to genetic polymorphism of MTHFR 677C>T and 1298A>C, but the evidence is still unclear. We conducted a meta-analysis of studies evaluating the effect of these polymorphisms on PC. We searched databases such as PubMed, Embase, and Google Scholar to retrieve studies that investigated MTHFR 677C>T and 1298A>C polymorphism for PC risk. For MTHFR 677C>T analysis, a total of eight publications with 1680 cases and 2520 controls were included; and for MTHFR 1298A>C analysis, six studies with 1421 cases and 1641 controls were included. Genotypes retrieved were subjected to meta-analysis using a random- or fixed-effects model with odds ratios and 95% confidence intervals as effect measures. Our met-analysis in all genetic models showed that MTHFR polymorphisms were not associated with the risk for PC. MTHFR polymorphisms revealed significant heterogeneity between studies for MTHFR 677C>T (P ¼ .003) but not for 1298A>C (P ¼ .495). Furthermore, there was no evidence for publication bias for these polymorphisms. Because the distribution of MTHFR genetic polymorphism differs among ethnic groups, the results of this meta-analysis cannot be extrapolated to patients belonging to any other ethnic group.
Keywords: Meta-analysis; MTHFR; Pancreatic cancer; SNPs.
List of abbreviations CI Confidence interval Hcy Homocysteine HWE HardyeWeinberg equilibrium MTHFR Methylenetetrahydrofolate reductase OR Odds ratio PC Pancreatic cancer RBC Red blood cell SNP Single nucleotide polymorphism G.P. Nagaraju, S. Ahmad (eds.) Theranostic Approach for Pancreatic Cancer ISBN 978-0-12-819457-7 https://doi.org/10.1016/B978-0-12-819457-7.00013-X
Copyright © 2019 Elsevier Inc. All rights reserved.
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Introduction Pancreatic cancer (PC) is the second leading cause of cancer-related deaths worldwide [1]. Whipple procedure or surgery remains the only possible treatment option for PC. However, because of the lack of specific symptoms and improved screening and diagnostic methods, only 10e20% of patients are diagnosed at a stage amenable to resection and possible therapy. As a result, PC is correlated with high mortality burden and poor prognosis [2]. Many risk factors are associated with PC. Nonmodifiable risk factors such as age, familial cancer syndromes, Afro-American race, hereditary, other forms of chronic pancreatitis, diabetes, and non-O blood group are associated with PC. Smoking, obesity, dietary factors such as a nonvegetarian diet, and toxins are some modifiable risk factors contributing to PC [3]. Folic acid has a major role in the biosynthesis of essential mediators for DNA methylation. Hence, folate deficiency leads to abnormal DNA integrity and epigenetic methylation patterns [4]. Several lines of evidence indicated an inverse relation between dietary folate intake and PC risk [5e7]. A large meta-analysis investigation that examined the effect of the interaction of serum concentration of homocysteine (Hcy), folate, and vitamin B12 and 5,10-methylenetetrahydrofolate reductase (MTHFR) polymorphisms demonstrated that an elevated serum Hcy level and folate deficiency are associated with increased overall risk for cancer [8]. As an important regulator of folate metabolism, the MTHFR enzyme has an important role in converting Hcy to methionine. The MTHFR gene harbors two important genetic variations (677C>T and 1298A>C) that contribute to inactivation of this enzyme [9]. Several studies examined the association between MTHFR gene polymorphisms and PC risk; however, findings have been inconsistent [10e13]. We performed a meta-analysis to investigate the relation between MTHFR gene polymorphisms and PC susceptibility.
Materials and methods Literature search strategy To select eligible studies on MTHFR polymorphisms and PC, electronic databases including PubMed, Embase, and Google Scholar were implemented with the following search terms: “pancreatic cancer,” “MTHFR,” “677C>T,” “1298A>C,” “rs1801133,” “rs1801131,” “SNP,” “polymorphism,” and “genetic variant.” All relevant studies published until Sep. 2018 were included in the meta-analysis. Studies met the following criteria to be included in the analysis: (1) original observational study of human participants; (2) an association study between 677C>T and/or 1298A>C
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of the MTHFR gene and PC; (3) case-control study; (4) availability of genotype data; and (6) studies were published in English. All studies were independently reviewed by two researchers and the following data were extracted: (1) first author name; (2) publication year; (3) population ethnicity/country of origin; and (4) genotype frequencies in PC and control. The current meta-analysis was conducted according to guidelines issued in the Preferred Reporting Items for Systematic Reviews and Meta-analysis 2009. The literature search strategy and process for selection of papers for this meta-analysis are shown in Fig. 13.1.
Statistical analysis The HardyeWeinberg equilibrium (HWE) test was calculated using chi-square test. Studies that deviated from the HWE of genotype frequencies in controls were excluded from the analysis. Heterogeneity across individual studies was assessed by Cochran Q test and I2 statistic. If there was no evidence of heterogeneity, a fixed-effects model was used to estimate the pooled odds ratio (OR) and corresponding 95% confidence intervals (95% CIs); otherwise, a random-effects model was used. The ORs and 95% CIs were calculated to assess the association between two SNPs of the MTHFR gene and PC risk. Four genetic models were performed in the current meta-analysis (using MTHFR 677C>T as an example): (1) allele model, T allele versus C allele; (2) dominant model (CTþTT versus CC); (3) recessive model (TT vs. CTþCC); and (4) codominant model (TT versus CC; CT versus CC; CT versus TT). Begg’s funnel plot and Egger’s test were used to evaluate publication bias, which was presented
Figure 13.1 Flowchart of study selection procedure. MTHFR, methylenetetrahydrofolate reductase.
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using the funnel plot of precision by the log of OR. To examine the influence of each study on robustness of the method used for meta-analysis, a sensitivity analysis was performed. MetaGenyo, a Web tool, was used to calculate results of the meta-analysis [14].
Results Study characteristics A total of eight eligible studies met inclusion criteria [10e13,15e18]. For the MTHFR 677C>T analysis, a total of eight publications with 1680 cases and 2520 controls were included; and for the MTHFR 1298A>C analysis, six studies with 1421 cases and 1641 controls were included. The genotype distributions of all studies were consistent with the HWE. The strategy adopted for selecting articles for this meta-analysis is shown in Fig. 13.1. Characteristics of selected studies are listed in Table 13.1.
Quantitative synthesis Among studies performed on MTHFR 677C>T in PC, there was significant heterogeneity (P ¼ .003, I2 ¼ 63.8%); hence, the random-effects model was used to estimate pooled ORs (Fig. 13.2). The meta-analysis results showed that there was no statistically significant association between MTHFR 677C>T and PC risk in four genetic models (T allele versus C allele, P ¼ .344; dominant model, P ¼ .405; recessive model, P ¼ .281) (Table 13.2, Fig. 13.2). With regard to the MTHFR 1298A>C polymorphism, heterogeneity was not observed among studies (P ¼ .495, I2 ¼ 0%); hence the fixed-effects model was used to estimate pooled ORs (Fig. 13.2). MTHFR 1298A>C also was not associated with PC risk in all-genetic models (C allele versus A allele, P ¼ .882; dominant model, P ¼ .634; recessive model, P ¼ .609) (Table 13.2, Fig. 13.2).
Sensitivity analysis and publication bias The sensitivity analysis performed by sequentially omitting individual studies demonstrated that no individual study exerted an excessive influence on the pooled effect of the MTHFR 677C>T and 1298A>C polymorphisms on PC risk (Fig. 13.3). In all genetic models, the shape of funnel plots seemed symmetrical for both MTHFR 677C>T and MTHFR 1298A>C polymorphisms, which suggested the absence of publication bias (Fig. 13.4). Statistical evidence of publication bias was obtained through
Table 13.1 Characteristics of studies included for meta-analysis. Author
Cases
Control
MTHFR 677C>T
Ethnicity
CC
CT
TT
CC
CT
TT
HardyeWeinberg P value
Wang et al., 2005 [18] Matsubayashi et al., 2005 [11] Li et al., 2005 [15]a Li et al., 2005 [15]b Li et al., 2005 [15]c Suzuki et al., 2008 [17] Nisevic et al., 2008 [16] Mazzuca et al., 2015 [12] Nakao et al., 2016 [13] Chittiboyina et al., 2018 [10]
East Asian Caucasian Caucasian Hispanic African East Asian Caucasian Caucasian East Asian Caucasian
31 145 150 10 11 57 43 32 127 61
79 115 117 10 6 80 52 51 161 73
53 43 36 1 0 20 5 14 72 25
135 134 149 5 12 291 42 76 124 24
149 135 138 5 4 366 50 98 194 26
53 36 20 3 0 128 8 28 82 5
0.270 0.823 0.108 0.444 0.568 0.475 0.191 0.687 0.701 0.585
AA
AC
CC
AA
AC
CC
124 133 129 12 11 49 240 87
37 134 145 5 6 45 107 60
2 36 29 0 0 5 13 12
243 144 133 9 12 103 285 24
86 140 137 6 4 80 102 24
8 21 40 1 0 19 13 7
MTHFR 1298A>C
Wang et al., 2005 [18] Matsubayashi et al., 2005 [11] Li et al., 2005 [15]a Li et al., 2005 [15]b Li et al., 2005 [15]c Mazzuca et al., 2015 [12] Nakao et al., 2016 [13] Chittiboyina et al., 2018 [10]
East Asian Caucasian Caucasian Hispanic African Caucasian East Asian Caucasian
0.905 0.093 0.613 1.000 0.568 0.548 0.305 0.795
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(A) MTHFR 677C>T Study
Odds Ratio
Control Experimental Events Total Events Total
Wang et al., 2005 Matsubayashi et al., 2005 Li et al., 2005a Li et al., 2005b Li et al., 2005c Suzuki et al., 2008 Nisevic et al., 2008 Mazzuca et al., 2015 Nakao et al., 2016 Chittiboyina et al., 2018
132 158 153 11 6 100 57 65 233 98
Random effects model
163 303 303 21 17 157 100 97 360 159 1680
OR
95%-CI W(random)
337 305 307 13 16 785 100 202 400 55
2.85 [1.82; 4.45] 0.85 [0.62; 1.18] 0.96 [0.70; 1.32] 0.69 [0.17; 2.81] 1.64 [0.36; 7.38] 1.03 [0.72; 1.48] 0.96 [0.55; 1.68] 1.23 [0.74; 2.04] 0.82 [0.61; 1.12] 1.24 [0.67; 2.32]
11.4% 13.9% 14.0% 2.7% 2.4% 13.2% 9.4% 10.3% 14.3% 8.5%
2520
1.11 [0.87; 1.43]
100%
202 171 158 8 4 494 58 126 276 31
Heterogeneity: I-squared=63.8%, tau-squared=0.0894, p=0.0032
0.2
0.5
1
2
5
(B) MTHFR 1298A>C Study
Wang et al., 2005 Matsubayashi et al., 2005 Li et al., 2005a Li et al., 2005b Li et al., 2005c Mazzuca et al., 2015 Nakao et al., 2016 Chittiboyina et al., 2018 Fixed effect model
Odds Ratio
Experimental Control Events Total Events Total 39 170 174 5 6 50 120 72
163 303 303 17 17 99 360 159
94 161 177 7 4 99 115 31
1421
OR
95%-CI W(fixed)
337 305 310 16 16 202 400 55
0.81 [0.53; 1.25] 1.14 [0.83; 1.57] 1.01 [0.74; 1.40] 0.54 [0.13; 2.25] 1.64 [0.36; 7.38] 1.06 [0.66; 1.72] 1.24 [0.91; 1.69] 0.64 [0.35; 1.19]
12.4% 22.6% 22.5% 1.1% 1.0% 10.0% 24.3% 6.0%
1641
1.04 [0.89; 1.21]
100%
Heterogeneity: I-squared=0%, tau-squared=0, p=0.4949
0.2
0.5
1
2
5
A: MTHFR 677 TT+CT vs. CC; B: MTHFR 1298 CC+AC vs. AA; OR: Odds ratio
Figure 13.2 Forest plot representing pooled results of odds ratios (ORs) for association between MTHFR gene variants and pancreatic cancer risk. (A) MTHFR 677 TTþCT versus CC. (B) MTHFR 1298 CCþAC versus AA. CI, confidence interval.
Egger’s test. P values for both MTHFR 677C>T and MTHFR 1298A>C polymorphisms are listed in Table 13.2.
Discussion PC symptoms are rarely noticeable in the early stages and adversely affect the gastrointestinal system and patients’ quality of life [19]. It is widely acknowledged that the etiology of PC emerges from the dynamic interaction between genetic and environmental factors [20]. Because familial and sporadic PC share the same molecular pathogenesis, study of genee environment interactions has been invoked as a premise to illuminate the etiological complexity of PC oncogenesis [21]. Although treatment options
Table 13.2 Meta-analysis of association between MTHFR gene variants and pancreatic cancer susceptibility. Test of heterogeneity Test of association Comparison model
c2
P value
I2
Statistics model
Odds ratio (95% confidence interval)
P value
Egger’s test P value
33.39 24.83 23.70 32.26 16.05 17.81
<0.001 0.003 0.003 <0.001 0.042 0.037
73.0 63.8 66.3 75.2 50.1 49.5
Random Random Random Random Random Random
1.10 1.11 1.22 1.25 1.21 1.06
(0.90e1.36) (0.87e1.43) (0.85e1.74) (1.79e1.99) (0.89e1.65) (0.85e1.32)
0.344 0.405 0.281 0.334 0.229 0.229
0.942 0.542 0.454 0.785 0.421 0.266
9.68 6.39 10.10 10.14 8.56 4.70
0.207 0.495 0.120 0.119 0.200 0.696
27.7 0 40.6 40.9 29.8 0
Fixed Fixed Fixed Fixed Fixed Fixed
1.01 1.04 0.93 0.94 0.90 1.06
(0.89e1.14) (0.89e1.21) (0.69e1.25) (0.69e1.29) (0.66e1.23) (0.90e1.24)
0.882 0.634 0.609 0.712 0.520 0.504
0.299 0.436 0.299 0.295 0.387 0.420
MTHFR 677C>T
T allele versus C allele TTþCT versus CC TT versus CCþCT TT versus CC TT versus CT CT versus CC MTHFR 1298A>C
C allele versus A allele CCþAC versus AA CC versus ACþAA CC versus AA CC versus AC AC versus AA
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(A) MTHFR 677C>T Study
Odds Ratio
OR
95%-CI
Omitting Wang et al., 2005 Omitting Matsubayashi et al., 2005 Omitting Li et al., 2005a Omitting Li et al., 2005b Omitting Li et al., 2005c Omitting Suzuki et al., 2008 Omitting Nisevic et al., 2008 Omitting Mazzuca et al., 2015 Omitting Nakao et al., 2016 Omitting Chittiboyina et al., 2018
0.95 1.10 1.07 1.05 1.05 1.05 1.06 1.04 1.12 1.04
[0.82; 1.10] [0.95; 1.28] [0.92; 1.25] [0.92; 1.21] [0.91; 1.20] [0.91; 1.22] [0.92; 1.22] [0.90; 1.20] [0.96; 1.30] [0.91; 1.20]
Fixed effect model
1.05 [0.92; 1.20] 0.8
1
1.25
(B) MTHFR 1298A>C Odds Ratio
Study
OR
95%-CI
Omitting Wang et al., 2005 Omitting Matsubayashi et al., 2005 Omitting Li et al., 2005a Omitting Li et al., 2005b Omitting Li et al., 2005c Omitting Mazzuca et al., 2015 Omitting Nakao et al., 2016 Omitting Chittiboyina et al., 2018
1.07 1.01 1.04 1.05 1.03 1.03 0.98 1.07
[0.91; 1.26] [0.85; 1.20] [0.88; 1.24] [0.90; 1.22] [0.89; 1.20] [0.88; 1.21] [0.82; 1.17] [0.92; 1.25]
Fixed effect model
1.04 [0.89; 1.21] 0.8
1
1.25
A: MTHFR 677 TT+CT vs. CC; B: MTHFR 1298 CC+AC vs. AA; OR: Odds ratio
Figure 13.3 Forest plot representing corresponding pooled odds ratio (OR) of removing each study. (A) MTHFR 677 TTþCT versus CC. (B) MTHFR 1298 CCþAC versus AA. CI, confidence interval.
such as surgery, radiation, chemotherapy, and chemoradiation have progressed continuously to control symptoms, there is no sure way to prevent PC. Understanding the genetics of PC may enable us to identify high-risk individuals and open the door to designing novel therapeutic strategies. Hyperhomocysteinemia has been reported in patients with neoplastic diseases. 5-Methyltetrahydrofolate serves as the methyl group donor for the
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(A) 0.0
MTHFR 677C>T
Standard Error
0.2
0.4
0.6
0.5
1.0 Odds Ratio
2.0
5.0
1.0 Odds Ratio
2.0
5.0
(B) 0.0
MTHFR 1298A>C
Standard Error
0.2
0.4
0.6
0.5
A: MTHFR 677C>T; B: MTHFR 1298A>C; OR: Odds ratio
Figure 13.4 Begg’s funnel plot of ORs for association between MTHFR gene variants and pancreatic cancer risk. (A) MTHFR 677C>T. (B) MTHFR 1298A>C. OR, odds ratio.
remethylation of Hcy to methionine. Because folate supplies the substrate for the MTHFR reaction, reduced serum folate and dysfunctional MTHFR appeared to be key factors in hyperhomocysteinemia. This was further supported by the development of acute pancreatitis in animals fed with ethionine (inhibitor of methylation)-supplemented or choline (methyl donor)-deficient diets [22]. Furthermore, a prospective study of Swedish
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women and men suggested that higher methionine intake may reduce the risk for PC [23]. MTHFR functional polymorphisms have been studied in different ethnicities for their possible association with PC. Wang et al. [18] first observed MTHFR 677C>T mutant genotypes more frequently (ORs of 2.6 and 5.1 for the CT and TT genotypes, respectively) in East Asian PC patients compared with controls. Furthermore, a twofold increased risk for PC for individuals with the MTHFR 677 CT/TT was reported in Caucasians [15]. Subsequent case-control studies failed to show a significant association between MTHFR gene polymorphisms and PC [10e13,16]. However, a study showed that the MTHFR 677C>T variant allele resulted in lower red blood cell (RBC) folate levels compared with the referent wild-type genotype [10]. In contrast, individuals with variants genotypes of the MTHFR 1298A>C single nucleotide polymorphism (SNP) had higher mean RBC folate concentrations compared with the wild-type genotype [10]. The opposite consequences of MTHFR 677C>T and 1298A>C polymorphisms on RBC folate levels was also documented earlier [24]. This indicated that the effects of MTHFR polymorphisms on total Hcy levels in various populations remain controversial [25]. Our metaanalysis revealed that there is no association between MTHFR polymorphisms and PC risk. Meta-analysis of MTHFR polymorphisms revealed significant heterogeneity between studies for MTHFR 677C>T but not for MTHFR 1298A>C. Furthermore, there was no evidence for publication bias for these polymorphisms. Consistent with our results, two previous meta-analyses provided no evidence of association between MTHFR polymorphisms and PC risk [26,27]. Another meta-analysis showed an obvious association between MTHFR 677C>T polymorphism and PC risk in East Asians but not Caucasians [28]. This meta-analysis provided no evidence for an association between PC risk and MTHFR polymorphisms (677C>T and 1298A>C). The metaanalysis had several potential limitations. First, the study was conducted on MTHFR polymorphisms, which are not extensively studied in PC. Second, information on red cell folate concentrations or serum Hcy levels was unavailable in these studies. Third, the information on lifestyle factors influencing folate status, such as smoking, alcohol consumption, and dietary folate intake, were unavailable for included studies. Because most of the studies included in this meta-analysis were of Caucasians and East Asians and the distribution of MTHFR genetic polymorphism differs among ethnic groups [29,30], the results cannot be extrapolated to patients belonging to any other ethnic group.
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