GENE-40627; No. of pages: 6; 4C: Gene xxx (2015) xxx–xxx
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Research paper
Association of KEAP1 and NFE2L2 polymorphisms with temporal lobe epilepsy and drug resistant epilepsy Zhou Liu a,b,1, Xiaojian Yin b,1, Lingying Liu c, Hua Tao a, Haihong Zhou b, Guoda Ma a, Lili Cui a, You Li a, Shuyan Zhang d, Zhi'en Xu b, LiFen Yao e, Zhiyou Cai f, Bin Zhao a,b,⁎, Keshen Li a,⁎⁎ a
Guangdong Key Laboratory of Age-Related Cardiac and Cerebral Diseases, Affiliated Hospital of Guangdong Medical University, Zhanjiang, China Department of Neurology, Affiliated Hospital of Guangdong Medical University, Zhanjiang, China Department of Neurology, Chenzhou No. 1 People's Hospital, Chenzhou, China. d Department of Neurology, The Fourth Affiliated Hospital of Harbin Medical University, Harbin, China e Department of Neurology, The First Affiliated Hospital, Harbin Medical University, Harbin, Heilongjiang, China f Department of Neurology, Renmin Hospital, Hubei University of Medicine, Hubei, China b c
a r t i c l e
i n f o
Article history: Received 1 February 2015 Received in revised form 20 June 2015 Accepted 22 June 2015 Available online xxxx Keywords: Drug resistant epilepsy (DRE) Kelch-like ECH-associated protein 1 (KEAP1) Nuclear erythroid 2-related factor 2 (NFE2L2) Polymorphism Temporal lobe epilepsy (TLE)
a b s t r a c t Background and aims: Temporal lobe epilepsy (TLE) is a prevalent form of epilepsy. TLE contributes to the majority of drug resistant epilepsy (DRE) cases and is associated with genetic factors. Kelch-like ECH-associated protein 1 (KEAP1)/Nuclear erythroid 2-related factor 2 (known as NFE2L2 or Nrf2) association has been implicated in neuroprotection due to induction of antioxidant enzymes. The association of one single KEAP1 gene nucleotide polymorphism (SNP) and nine NFE2L2 gene SNPs with TLE and DRE were examined to determine whether these SNPs influenced the risk of TLE and DRE in a Han population. Subjects and methods: A total of 184 TLE patients (including 72 DRE patients) and 183 controls were included in this analysis. The SNaPshot Multiplex kit was used to assess the genotypes. Results: A NFE2L2 gene haplotype was identified as a risk factor for TLE (OR = 7.11, 95% CI 1.53–32.98). Additionally, rs2706110 G N A in the NFE2L2 gene and rs1048290 C N G in the KEAP1 gene showed a significant risk for and a protective effect against DRE, respectively. Conclusion: Our findings suggest that variations in NFE2L2 gene increase the risk of TLE and DRE but that variations in KEAP1 gene play a protective role for DRE. © 2015 Published by Elsevier B.V.
1. Introduction Temporal lobe epilepsy (TLE) is the most common form of partial epilepsy referred for epilepsy surgery; it is also responsible for the majority of drug-resistant epilepsy (DRE) cases (Semah et al., 1998; Tellez-Zenteno and Hernandez-Ronquillo, 2012). It is becoming Abbreviations: AD, Alzheimer's disease; ALS, amyotrophic lateral sclerosis; ABCG2, ATP-binding cassette transporter protein; bal acc, balanced accuracy; CI, confidence interval; HER2CA, constitutively active human epidermal growth factor receptor 2; CV, cross validation; CYP3A4, cytochrome P450 3A4; DRE, drug resistant epilepsy; NDRE, drug responsive epilepsy group; SNP, gene nucleotide polymorphism; GSTA2, glutathione S-transferase alpha 2; GSTP1, glutathione S-transferase pi-1; HWE, Hardy–Weinberg equilibrium; HO-1, heme oxygenase 1; ILAE, International League Against Epilepsy; KEAP1, Kelch-like ECH-associated protein 1; LD, linkage disequilibrium; MRP1, multidrug resistance protein 1; MRP5, multidrug resistance protein 5; MDR, Multifactor Dimensionality Reduction; (NFE2L2 or Nrf2), Nuclear erythroid 2-related factor 2; OR, odds ratio; OS, oxidative stress; PD, Parkinson's disease; TLE, temporal lobe epilepsy. ⁎ Correspondence to: B. Zhao, Guangdong Key Laboratory of Age-Related Cardiac and Cerebral Diseases, Affiliated Hospital of Guangdong Medical University, Zhanjiang, China. ⁎⁎ Corresponding author. 1 Dr. Zhou Liu and Dr. Xiaojian Yin contributed equally to this work.
increasingly clear that both genetic factors (Hwang and Hirose, 2012) and environmental conditions contribute to its complex pathogenesis. Oxidative stress (OS) is a disturbance in the pro-oxidant–antioxidant balance that damages all components of the cell, including proteins, lipids, and DNA. OS-mediated excitotoxicity (Sun et al., 2010) and hippocampal neuron death (Liu et al., 2010) are considered possible mechanisms behind the pathogenesis of epilepsy (Aguiar et al., 2012). Nuclear factor erythroid 2-related factor 2 (NFE2L2, also known as Nrf2) protein is a transcription factor that regulates the expression of many antioxidant pathway genes. Nrf2 protein is localized to the cytoplasm through interactions with its inhibitor protein, Kelch like-ECHassociated protein 1 (KEAP1), which functions as an adaptor for Cul3based E3 ubiquitin ligase to regulate proteasomal degradation of Nrf2 (Kobayashi et al., 2004). In response to oxidative stress, Nrf2 protein dissociates from KEAP1 protein, translocates into the nucleus and binds to the antioxidant response elements (ARE) located in the upstream promoter region of many antioxidative genes, thereby initiating their transcription (Itoh et al., 1997). The expression of Nrf2 protein target genes are regulated by mutations in KEAP1 gene (Teshiba et al., 2013) or NFE2L2 gene (Yu et al., 2013). Furthermore, the activated
http://dx.doi.org/10.1016/j.gene.2015.06.055 0378-1119/© 2015 Published by Elsevier B.V.
Please cite this article as: Liu, Z., et al., Association of KEAP1 and NFE2L2 polymorphisms with temporal lobe epilepsy and drug resistant epilepsy, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.06.055
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KEAP1–Nrf2/ARE pathway has been shown play a protective role in brain tissue (Meng et al., 2014). The relationship between TLE and NFE2L2 has attracted increasing attention. For example, NFE2L2-knockout mice were shown to be more vulnerable to kainate-induced seizures (Kraft et al., 2006). Activated Nrf2–ARE signal pathway in hippocampus suppressed the progression of amygdala kindling, and also ameliorated the cognitive impairment and oxidative stress induced by epileptic seizure (Wang et al., 2014). In a study of the relationship between the KEAP1–NFE2L2 genes and the risk of Parkinson's disease (PD) (von Otter et al., 2010a), an NFE2L2 haplotype was found to be associated with a reduced risk of PD. However, no association has been demonstrated between NFE2L2 or KEAP1 gene polymorphisms and epilepsy.
Table 1 Demographics and clinical characteristics. Variables
TLE (n = 184)
Control (n = 183)
P value
Mean age (years) Male/female
27.2 ± 14.4 94/90
49.1 ± 15.6 90/93
b0.001 0.72
Variables
DRE (n = 72)
NDRE (n = 112)
P value
Mean age (years) Age at onset (years) Male/female Hippocampal sclerosis History of febrile seizure History of status epilepticus
26.9 ± 15.0 17.6 ± 13.5 35/37 4 18 7
27.4 ± 14.1 21.3 ± 13.2 59/53 5 14 7
0.83 0.07 0.59 0.5 0.03 0.34
Data are presented as absolute numbers or mean ± SD. P-values are calculated with chisquared test for categorical parameters and t test for continuous parameters.
2. Material and methods 2.1. Study population
sequence detector for capillary electrophoresis. The experimental results were analyzed with GeneMapper 4.0 (Applied Biosystems Co., Ltd.).
Our study consecutively recruited 184 unrelated TLE patients of Han Chinese descent from the Department of Neurology of the Affiliated Hospital of Guangdong Medical College and the Department of Neurology of the First Affiliated Hospital of Harbin Medical University. Patients' diagnoses were based on the 1989 ILAE classification (Roger et al., 1989). None of the patients had a mass lesion, malformations of cortical development, or traumatic brain injury. The only accepted MRI sign was hippocampal sclerosis (HS) based on the characteristic MRI pattern of abnormalities. The TLE patients were divided into drug resistant epilepsy (DRE) group (n = 72) and drug responsive epilepsy group (NDRE) (n = 112) based on the International League Against Epilepsy (ILAE) consensus criteria (Kwan et al., 2010). Patients with extratemporal epilepsies, mental retardation, and systemic diseases were excluded. Patients with histories of ischemic cerebrovascular diseases, cardiogenic cerebral infarctions, cerebral hemorrhage, coronary artery diseases, autoimmune diseases, systemic inflammatory diseases, blood diseases, and malignant tumors were excluded from the study. A total of 183 healthy control subjects were recruited from the Health Examination Center of the Affiliated Hospital of Guangdong Medical College; the healthy subjects were comparable in sex and race to the TLE subjects. Written informed consent was obtained from all of the enrolled participants, and this study was approved by the Ethics Committee of the Affiliated Hospital of Guangdong Medical College and the First Affiliated Hospital of Harbin Medical University.
2.3. Magnetic resonance imaging (MRI)
2.2. DNA isolation and genotyping
3.1. Baseline characteristics
DNA isolation and genotyping were performed using previously published protocols (Li et al., 2013). Genomic DNA was extracted from venous blood using the EZ-10 Spin Column Whole Blood Genomic DNA Isolation Kit (Sangon Biotech, Shanghai, China), according to manufacturer's instruction. The genotypes were analyzed using the SNaPshot Multiplex Kit (Applied Biosystems Co., Ltd., Foster City, CA, USA). The primers used are listed in Table 1. SNaPshot reactions were executed in a 10 μL final volume containing 5 μL of the SNaPshot Multiplex Kit (ABI), 1 μL primer mix, 2 μL water, and 2 μL templates consisting of the multiplex PCR products from the different genes. The SNaPshot response procedures included (1) initial denaturation at 96 °C for 1 min; (2) denaturation at 96 °C for 10 s; (3) annealing at 52 °C for 5 s; (4) extension at 60 °C for 30 s; and (5) for a total of 28 cycles. Amplified samples were stored at 4 °C. Extension products were purified by a 1-h incubation with 1 U of shrimp alkaline phosphatase (Takara:Otsu, Shiga, Japan) at 37 °C, followed by incubation at 75 °C for 15 min to inactivate the enzyme. The purified products (0.5 μL) were mixed with 9 μL of Hi–Di and 0.5 μL of the Liz120 size standard (Applied Biosystems Co., Ltd.). The samples were incubated at 95 °C for 5 min and then loaded onto an ABI 3130XL DNA
The baseline characteristics of all of the participants in the study are summarized in Table 2. Of the 367 participants, 184 were patients with TLE and 183 were healthy controls. There was a significant difference in the distribution of age, while cases and controls were well matched by sex (Table 1). The TLE patients were divided into two groups: 72 patients with DRE and 112 patients with NDRE. There were no statistically significant differences between the DRE and NDRE groups in terms of age, sex, and history of status epilepticus. There were a higher proportion of patients with a history of febrile seizure in the DRE group compared with the NDRE group.
All MR studies were performed on a 1.5 T MRI-scanner (General Electric, Sigma 1.5, Waukesha, WI, USA) including sagittal T1, coronal FLAIR and T2. Details of the sequence were as follows: repetition time (TR) 4800 ms, echo time (TE) 120 ms, NEX 1, field of view 240 mm × 240 mm, 512 × 512 matrix. 2.4. Statistical analysis Chi-squared tests and t-tests were performed with SPSS software, version 19.0 (IBM, Armonk, NY, USA). The Hardy–Weinberg equilibrium (HWE), linkage disequilibrium (LD) and haplotypes were analyzed using SNPStats (http://bioinfo.iconcologia.net/SNPstats) (Sole et al., 2006). Co-dominant, dominant and recessive genetic models of inheritance were chosen to evaluate the associations between each SNP and TLE or DRE. The odds ratio (OR) and 95% confidence intervals (95% CI) were determined for each SNP in three genetic models adjusted by age and sex; the significance level was set to a P-value of 0.05. Additionally, gene–gene interactions were evaluated using Multifactor Dimensionality Reduction (MDR 3.0.2). QUANTO (version 1.2.4) software was used to calculate statistic power. The power was computed at the 0.05 significance level for the two-sided test, assumed an odds ratio of 1.5. 3. Results
3.2. Polymorphisms of the NFE2L2 and KEAP1 genes and the risk of TLE and DRE The genotype frequencies of NFE2L2 and KEAP1 polymorphisms in the study subjects are provided in Table 2. No deviations from the Hardy–Weinberg equilibrium for the examined polymorphisms were found in the distributions of genotypes between the patients and controls or the DRE and NDRE groups (data not shown). None of the genotypes alone significantly affected the risk of TLE. However, significant
Please cite this article as: Liu, Z., et al., Association of KEAP1 and NFE2L2 polymorphisms with temporal lobe epilepsy and drug resistant epilepsy, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.06.055
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Table 2 Relationship between the frequencies of the NFE2L2 and KEAP1 genotypes and alleles and the risk of TLE and DRE. TLE (n = 184) NFE2L2 rs7557529 Codominant AA AG GG Dominant AA GG + AG Recessive AG + AA GG rs35652124 Codominant AA AG GG Dominant AA GG + AG Recessive AG + AA GG rs6706649 Codominant GG AG AA Dominant GG AG + AA Recessive GG + AG AA rs6721961 Codominant CC AC AA Dominant CC AC + AA Recessive CC + AC AA rs2886161 Codominant AA AG GG Dominant AA AG + GG Recessive AA + AG GG rs1806649 Codominant GG AG AA Dominant GG AG + AA Recessive GG + AG AA rs2001350 Codominant AA AG GG
Control (n = 183)
OR (95%CI)
P value
DRE (n = 72)
NDRE (n = 112)
OR (95%CI)
P value
57 86 41
60 91 32
1 0.79 (0.44–1.42) 1.59 (0.75–3.41)
0.15
20 36 16
37 50 25
1 1.32 (0.66–2.64) 1.20 (0.52–2.77)
0.74
57 127
60 123
1 0.96 (0.55–1.66)
0.88
20 52
37 75
1 1.28 (0.67–2.45)
0.46
143 41
151 32
1 1.83 (0.93–3.60)
0.08
56 16
87 25
1 1.02 (0.50–2.08)
0.96
60 84 40
52 86 45
1 0.71 (0.38–1.30) 0.74 (0.36–1.51)
0.51
23 34 15
37 50 25
1 1.07 (0.54–2.12) 0.95 (0.41–2.17)
0.95
60 124
52 131
1 0.72 (0.41–1.27)
0.25
23 49
37 75
1 1.03 (0.54–1.95)
0.93
144 40
138 45
1
0.79
57 15
87 25
1 0.91 (0.44–1.87)
0.79
157 27 0
153 29 1
1 0.77 (0.38–1.57) NA (0.00–NA)
0.57
64 8 0
93 19 0
1 0.61 (0.25–1.48) NA (0.00–NA)
0.26
157 184
153 182
1 0.75 (0.37–1.52)
0.42
64 8
93 19
1 0.61 (0.25–1.48)
0.26
1 182
0 184
1 NA (0.00–NA)
0.42
72 0
112 0
1 NA (0.00–NA)
NA
89 82 13
86 85 12
1 0.93 (0.55–1.58) 1.30 (0.45–3.76)
0.82
32 34 6
57 48 7
1 1.27 (0.68–2.35) 1.56 (0.48–5.08)
0.64
89 95
86 97
1 0.97 (0.58–1.62)
0.91
32 40
57 55
1 1.30 (0.72–2.37)
0.38
171 13
171 12
1 1.35 (0.48–3.77)
0.56
66 6
105 7
1 1.39 (0.45–4.35)
0.57
60 85 39
51 87 45
1 0.67 (0.36–1.24) 0.69 (0.34–1.41)
0.41
23 34 15
37 51 24
1 1.05 (0.53–2.08) 0.99 (0.43–2.27)
0.98
60 124
51 132
1 0.68 (0.38–1.20)
0.18
23 49
37 75
1 1.03 (0.54–1.95)
0.93
145 39
138 45
1 0.89 (0.49–1.61)
0.7
57 15
88 24
1 0.96 (0.46–1.99)
0.91
146 36 2
154 26 3
1 1.40 (0.69–2.84) 0.92 (0.09–9.13)
61 9 2
85 27 0
1 0.47 (0.20–1.07) NA (0.00–NA)
0.03
146 38
154 29
1 1.35 (0.68–2.68)
61 11
85 27
1 0.57 (0.26–1.24)
0.15
0.39
182 2
180 3
1 0.87 (0.09–8.63)
70 2
112 0
1 NA (0.00–NA)
0.05
0.9
98 67 19
87 85 11
1 0.79 (0.46–1.36) 1.61 (0.61–4.30)
35 28 9
63 39 10
1 1.30 (0.68–2.47) 1.63 (0.60–4.39)
0.54
0.65
0.33
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Please cite this article as: Liu, Z., et al., Association of KEAP1 and NFE2L2 polymorphisms with temporal lobe epilepsy and drug resistant epilepsy, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.06.055
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Table 2 (continued) TLE (n = 184) Dominant AA AG + GG Recessive AA + AG GG rs10183914 Codominant GG AG AA Dominant GG AG + AA Recessive GG + AG AA rs2706110 Codominant GG AG AA Dominant GG AG + AA Recessive GG + AG AA KEAP1 rs1048290 Codominant CC CG GG Dominant CC CG + GG Recessive CC + CG GG
Control (n = 183)
OR (95%CI)
P value
DRE (n = 72)
NDRE (n = 112)
OR (95%CI)
P value
98 86
87 96
1 0.89 (0.53–1.49)
0.66
35 57
63 49
1 1.36 (0.75–2.49)
0.31
165 19
172 11
1 1.80 (0.70–4.64)
0.22
63 9
102 10
1 1.46 (0.56–3.80)
0.44
159 23 2
156 27 0
1 0.95 (0.43–2.07) NA (0.00–NA)
0.75
63 8 1
96 15 1
1 0.82 (0.33–2.06) 1.37 (0.08–22.95)
0.89
159 25
156 27
1 0.98 (0.45–2.13)
0.96
63 9
96 16
1 0.86 (0.36–2.07)
0.73
182 2
183 0
1 NA (0.00–NA)
0.46
71 1
111 1
1 1.40 (0.08–23.42)
0.82
92 77 15
92 80 11
1 1.03 (0.60–1.76) 1.30 (0.47–3.59)
29 36 7
63 41 8
1 1.94 (1.03–3.66) 1.99 (0.65–6.05)
0.11 0.09
92 92
92 91
1 1.07 (0.64–1.78)
0.81
29 43
63 49
1 1.95 (1.06–3.58)
0.03
169 15
172 11
1 1.28 (0.48–3.43)
0.63
65 7
104 8
1 1.44 (0.50–4.20)
0.5
50 94 40
46 97 40
1 0.70 (0.38–1.32) 1.16 (0.54–2.45)
0.27
27 31 14
23 63 26
1 0.41 (0.20–0.84) 0.45 (0.19–1.06)
0.04
50 134
46 137
1 0.82 (0.45–1.48)
0.5
27 45
23 89
1 0.42 (0.22–0.82)
0.01
144 40
143 40
1 1.46 (0.78–2.73)
0.23
58 27
86 23
1 0.78 (0.37–1.64)
0.51
0.88
Table 3 Haplotypes of NFE2L2 that influence the risk of TLE and DRE.
1 2 3 4 5 6 7 8 9 10
1 2 3 4 5 6 7 8 9 10
rs7557529–rs35652124–rs6706649–rs6721961–rs2886161– rs1806649–rs2001350–rs10183914–rs2706110
TLE
Control
OR (95% CI)
P value
AGGCGGAGG GAGAAGGGA AAACAGAGG GAGCAAAAG GAGAAGGGG GAGCAGAGA GAGCAAAGG GAGAAGAGG AAGCAGAGG AGGCGGGGA
41.80% 21.70% 7.10% 6.50% 4.30% 4.00% 4.40% 2.20% 3.00% 1.90%
43.20% 21.20% 8.50% 5.80% 3.00% 2.70% 2.30% 4.20% 1.40% 1.60%
1 1.17 (0.71–1.93) 0.76 (0.36–1.57) 1.25 (0.55–2.86) 1.64 (0.57–4.75) 1.31 (0.43–4.01) 1.93 (0.61–6.08) 0.44 (0.14–1.34) 7.11 (1.53–32.98) 1.14 (0.26–5.02)
– 0.55 0.46 0.6 0.36 0.64 0.26 0.15 0.013 0.86
rs7557529–rs35652124–rs6706649–rs6721961–rs2886161– rs1806649–rs2001350–rs10183914–rs2706110
DRE
NDRE
OR (95% CI)
P value
AGGCGGAGG GAGAAGGGA AAACAGAGG GAGCAAAAG GAGCAAAGG GAGAAGGGG GAGCAGAGA AAGCAGAGG GAGAAGAGG AGGCGGGGA
41.50% 18.50% 8.00% 6.00% 6.00% 5.10% 2.60% 3.10% 2.10% 1.80%
41.80% 21.70% 7.10% 6.50% 4.40% 4.30% 4.00% 3.00% 2.20% 1.90%
1 1.44 (0.81–2.57) 0.72 (0.27–1.88) 1.01 (0.41–2.46) 0.37 (0.09–1.42) 0.62 (0.21–1.85) 2.43 (0.76–7.84) 0.87 (0.23–3.31) 1.06 (0.22–5.16) 0.95 (0.20–4.62)
– 0.21 0.5 0.99 0.15 0.39 0.14 0.84 0.95 0.95
The haplotypes with frequencies of b0.01 were not included in haplotype association analysis.
Please cite this article as: Liu, Z., et al., Association of KEAP1 and NFE2L2 polymorphisms with temporal lobe epilepsy and drug resistant epilepsy, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.06.055
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differences were observed in the DRE group compared with the NDRE group in a dominant model of rs2706110 (OR = 1.95, 95% CI = 1.06– 3.58, P = 0.03) and codominant model and dominant model of rs1048290 (OR = 0.41, 95% CI = 0.20–0.84, P = 0.04; OR = 0.42, 95% CI = 0.22–0.82, P = 0.01, respectively). Moreover, power analysis showed that with our sample size, we would have 40% power for rs2706110, 47% power for rs1048290 to detect a genotype relative risk with an odds ratio of 1.5 at a significance level of 0.05. Furthermore, we performed a haplotype-based association analysis of nine polymorphisms of NFE2L2 gene. The haplotype AAGC AGAGG was associated with an increased risk of TLE (OR = 7.11, 95% CI = 1.53–32.98). However, no significant associations were observed between these haplotypes and DRE (Table 3). 3.3. Multi-dimensionality reduction (MDR) analysis MDR analysis showed that the rs2001350 and rs1048290 polymorphisms represented the best one factor models for TLE and DRE respectively. rs7557529/rs1048290 and rs2886161/rs2706110 represented the best model two factor models for TLE and DRE, respectively. Finally, the rs7557529/rs2001350/rs1048290 and rs35652124/rs6721961/ rs1048290 polymorphisms represented the best three factor models for TLE and DRE, respectively. However, the P-values were not significant for all factor models (Table 4). 4. Discussion The present study explored the association between polymorphisms in antioxidant transcription factor NFE2L2 gene and its inhibitor protein KEAP1 gene with TLE and DRE. The results showed that one NFE2L2 haplotype (AAGCAGAGG) was associated with an increased risk of TLE, the AG + AA genotypes of NFE2L2 rs2706110 under the dominant mode increased the risk of DRE, and the CG genotype under the codominant mode and the CG + GG genotypes of KEAP1 rs1048290 under the dominant mode decreased the risk of DRE. To the best of our knowledge, this is the first study to investigate an association between SNPs in NFE2L2 and KEAP1 with TLE and DRE. However, the association between SNPs in NFE2L2 and KEAP1 with other nervous system diseases (NSD) has been explored. Consistent with the present study, no significant associations between SNPs in NFE2L2 and KEAP1 with NSD have been found. Malin's two studies investigating 11 NFE2L2 SNPs (rs16865105, rs7557529, rs35652124, rs6706649, rs6721961, rs2886161, rs1806649, rs2001350, rs10183914, rs2706110, and rs13035806) and 3 KEAP1 SNPs (rs1048287, rs11085735, and rs1048290) did not find an association between the polymorphisms and either the risk of Parkinson's disease (PD) (von Otter et al., 2010a) or Alzheimer's disease (AD) (von Otter et al., 2010b). An Italian study investigated three NFE2L2 SNPs (rs35652124, rs6706649, and rs6721961) found that the polymorphisms did not differ between patients with amyotrophic lateral sclerosis (ALS) and controls (LoGerfo et al., 2014). Although promoter polymorphism rs6721961 of the C allele enhanced promoter activity (Marzec et al., 2007), the T allele was
Table 4 Summary of the MDR analysis.
TLE
DRE
Model
CV consistency
Bal acc CV testing
P value
rs2001350 rs7557529, rs1048290 rs7557529, rs2001350, rs1048290 rs1048290 rs2886161, rs2706110 rs35652124, rs6721961, rs1048290
10/10 9/10 8/10 6/10 3/10 9/10
0.55 0.5 0.52 0.52 0.45 0.59
0.54 0.96 0.75 0.86 0.74 0.43
Abbreviations: bal acc, balanced accuracy; CV, cross validation; MDR, Multifactor Dimensionality Reduction.
5
associated with a low extent of Nrf2 protein expression (Hartikainen et al., 2012), and the AGC polymorphisms (rs35652124, rs6706649, and rs6721961) increased NFE2L2 promoter activity (Marczak et al., 2012). The current study showed that a haplotype of NFE2L2 (AAGCAG AGG, rs7557529, rs35652124, rs6706649, rs6721961, rs2886161, rs1806649, rs2001350, rs10183914, and rs2706110) included the AGC, while rs10183914 included the G allele; these polymorphisms increased the risk of TLE. Moreover, evidences proved that rs10183914 G allele combined with AGC may influence the risk of AD and TLE. Malin found that GAGCAAAG (rs7557529, rs35652124, rs6706649, rs6721961, rs2886161, rs1806649, rs2001350, and rs10183914) was associated with an increased risk of PD in Swedish populations (von Otter et al., 2010a) and Italian populations (von Otter et al., 2014), also including rs10183914 G allele and AGC. However, the association could not be replicated in a meta-analysis (von Otter et al., 2014). Conflicting results are common in TLE SNPs researches (Cavalleri et al., 2005), because of differences of study design, statistical analysis, and interpretation of findings in association studies. We did not find an association between the polymorphisms in NFE2L2 and KEAP1 with TLE, but we did determine that rs2706110 of NFE2L2 and rs1048290 of KEAP1 affected the risk of DRE. Moreover, the AG + AA genotypes of rs2706110 under the dominant mode increased the DRE risk. However, several studies reported that this polymorphism was not associated with ALS (Bergström et al., 2014), PD (von Otter et al., 2010a) and AD (von Otter et al., 2010b). But, the rs2706110 AA genotype was associated with an increased risk of breast cancer (Hartikainen et al., 2012). Another SNP related with DRE (rs1048290) is located in exon 4 of KEAP1. The CG genotype under the codominant mode and the CG + GG genotypes under the dominant mode decreased the risk of DRE. In agreement with this finding, a recent study demonstrated that haplotype CGG (rs1048287, rs11085735, and rs1048290) in KEAP1 was associated with later sporadic ALS (LSALS) onset (Bergström et al., 2014), and the CG genotype has been significantly associated with progression-free survival in endometrial cancer (Wong et al., 2011). Under basal conditions, two molecules of Keap1 bind with one molecule of Nrf2, which functions as an adapter for Cul3/Rbx1 E3 ligase for Nrf2 ubiquitination. Ubiquitinated Nrf2 is degraded in the proteasome. When Keap1 conformational changes are make by oxidative or electrophilic stress, Nrf2 and Cul3 dissociate from Keap1, E3 ligase activity subsequently declines, Nrf2 ubiquitination stops. Nrf2 is thus stabilized and translocates to the nucleus, to be available for rapid Nrf2/ARE-dependent gene transcription (Taguchi et al., 2011). Although there is no direct evidence in regard to the possible participation of Nrf2 in DRE, one study reported that Nrf2 and constitutively active human epidermal growth factor receptor 2 (HER2CA) cooperatively up-regulated the mRNA expression of various drug-resistant and detoxifying enzymes (Kang et al., 2014). Because the NFE2L2 and KEAP1 genes may exert synergistic effects on TLE and DRE, we evaluated gene–gene interactions using the MDR method. However, no significant interactions among the SNPs analyzed in this study were identified. However, for the some potential limitations we cannot definitely exclude the possibility of false positive results. First, there is a possibility of the low statistical power. In the present sample size, the statistical powers were 40% for rs2706110, 47% for rs1048290, for detecting statistical differences to the stratification of DRE. Secondly, p-values from 0.05 to 0.01 are usually inadequate conservative to support our findings. Therefore, we still need the validation cohort study for further confirm our results, further investigation with a larger and more ethnically diverse population of TLE, DRE patients is warranted to support our preliminary conclusions.
Declaration of interest The authors declare no conflict of interest.
Please cite this article as: Liu, Z., et al., Association of KEAP1 and NFE2L2 polymorphisms with temporal lobe epilepsy and drug resistant epilepsy, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.06.055
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Funding This work was supported by funding from the National Nature Science Foundation of China (grant numbers 31171219, 81271213, 81271214, 81471294 and 81400986), the Medical Research Foundation of Guangdong Province (grant number B2013298) and the Science and Technology Planning Project of Zhanjiang (grant number 2014A01031). References Aguiar, C.C.T., Almeida, A.B., Araújo, P.V.P., Abreu, R.N.D.C.d., Chaves, E.M.C., Vale, O.C.d., Macêdo, D.S., Woods, D.J., Fonteles, M.M.d.F., Vasconcelos, S.M.M., 2012. Oxidative stress and epilepsy: literature review. Oxidative Med. Cell. Longev. 795259. http:// dx.doi.org/10.1155/2012/795259. Bergström, P., von Otter, M., Nilsson, S., Nilsson, A.-C., Nilsson, M., Andersen, P.M., Hammarsten, O., Zetterberg, H., 2014. Association of NFE2L2 and KEAP1 haplotypes with amyotrophic lateral sclerosis. Amyotroph. Lateral Scler. Frontotemporal Degener. 15, 130–137. Cavalleri, G.L., Lynch, J.M., Depondt, C., Burley, M.-W., Wood, N.W., Sisodiya, S.M., Goldstein, D.B., 2005. Failure to replicate previously reported genetic associations with sporadic temporal lobe epilepsy: where to from here? Brain 128, 1832–1840. Hartikainen, J.M., Tengström, M., Kosma, V.-M., Kinnula, V.L., Mannermaa, A., Soini, Y., 2012. Genetic polymorphisms and protein expression of NRF2 and sulfiredoxin predict survival outcomes in breast cancer. Cancer Res. 72, 5537–5546. Hwang, S.K., Hirose, S., 2012. Genetics of temporal lobe epilepsy. Brain Dev. 34, 609–616. Itoh, K., Chiba, T., Takahashi, S., Ishii, T., Igarashi, K., Katoh, Y., Oyake, T., Hayashi, N., Satoh, K., Hatayama, I., Yamamoto, M., Nabeshima, Y., 1997. An Nrf2/small Maf heterodimer mediates the induction of phase II detoxifying enzyme genes through antioxidant response elements. Biochem. Biophys. Res. Commun. 236, 313–322. Kang, H.J., Yi, Y.W., Hong, Y.B., Kim, H.J., Jang, Y.J., Seong, Y.S., Bae, I., 2014. HER2 confers drug resistance of human breast cancer cells through activation of NRF2 by direct interaction. Sci. Rep. 4, 7201. Kobayashi, A., Kang, M.-I., Okawa, H., Ohtsuji, M., Zenke, Y., Chiba, T., Igarashi, K., Yamamoto, M., 2004. Oxidative stress sensor Keap1 functions as an adaptor for Cul3-based E3 ligase to regulate proteasomal degradation of Nrf2. Mol. Cell. Biol. 24, 7130–7139. Kraft, A.D., Lee, J.M., Johnson, D.A., Kan, Y.W., Johnson, J.A., 2006. Neuronal sensitivity to kainic acid is dependent on the Nrf2-mediated actions of the antioxidant response element. J. Neurochem. 98, 1852–1865. Kwan, P., Arzimanoglou, A., Berg, A.T., Brodie, M.J., Allen Hauser, W., Mathern, G., Moshe, S.L., Perucca, E., Wiebe, S., French, J., 2010. Definition of drug resistant epilepsy: consensus proposal by the ad hoc Task Force of the ILAE Commission on Therapeutic Strategies. Epilepsia 51, 1069–1077. Li, Y., Liao, F., Yin, X.J., Cui, L.L., Ma, G.D., Nong, X.X., Zhou, H.H., Chen, Y.F., Zhao, B., Li, K.S., 2013. An association study on ADAM10 promoter polymorphisms and atherosclerotic cerebral infarction in a Chinese population. CNS Neurosci. Ther. 19, 785–794. Liu, J., Wang, A., Li, L., Huang, Y., Xue, P., Hao, A., 2010. Oxidative stress mediates hippocampal neuron death in rats after lithium–pilocarpine-induced status epilepticus. Seizure 19, 165–172. LoGerfo, A., Chico, L., Borgia, L., Petrozzi, L., Rocchi, A., D'Amelio, A., Carlesi, C., Caldarazzo Ienco, E., Mancuso, M., Siciliano, G., 2014. Lack of association between nuclear factor
erythroid-derived 2-like 2 promoter gene polymorphisms and oxidative stress biomarkers in amyotrophic lateral sclerosis patients. Oxidative Med. Cell. Longev. 2014, 432626. Marczak, E.D., Marzec, J., Zeldin, D.C., Kleeberger, S.R., Brown, N.J., Pretorius, M., Lee, C.R., 2012. Polymorphisms in the transcription factor NRF2 and forearm vasodilator responses in humans. Pharmacogenet. Genomics 22, 620. Marzec, J.M., Christie, J.D., Reddy, S.P., Jedlicka, A.E., Vuong, H., Lanken, P.N., Aplenc, R., Yamamoto, T., Yamamoto, M., Cho, H.-Y., 2007. Functional polymorphisms in the transcription factor NRF2 in humans increase the risk of acute lung injury. FASEB J. 21, 2237–2246. Meng, H., Guo, J., Wang, H., Yan, P., Niu, X., Zhang, J., 2014. Erythropoietin activates Keap1–Nrf2/ARE pathway in rat brain after ischemia. Int. J. Neurosci. 124, 362–368. Roger, J., Dreifuss, F., Martinez-Lage, M., Munari, C., Porter, R., Seino, M., Wolf, P., 1989. Proposal for revised classification of epilepsies and epileptic syndromes. Epilepsia 30, 389–399. Semah, F., Picot, M.C., Adam, C., Broglin, D., Arzimanoglou, A., Bazin, B., Cavalcanti, D., Baulac, M., 1998. Is the underlying cause of epilepsy a major prognostic factor for recurrence? Neurology 51, 1256–1262. Sole, X., Guino, E., Valls, J., Iniesta, R., Moreno, V., 2006. SNPStats: a web tool for the analysis of association studies. Bioinformatics 22, 1928–1929. Sun, Z.W., Zhang, L., Zhu, S.J., Chen, W.C., Mei, B., 2010. Excitotoxicity effects of glutamate on human neuroblastoma SH-SY5Y cells via oxidative damage. Neurosci. Bull. 26, 8–16. Taguchi, K., Motohashi, H., Yamamoto, M., 2011. Molecular mechanisms of the Keap1– Nrf2 pathway in stress response and cancer evolution. Genes Cells 16, 123–140. Tellez-Zenteno, J.F., Hernandez-Ronquillo, L., 2012. A review of the epidemiology of temporal lobe epilepsy. Epilepsy Res. Treat. 2012, 630853. Teshiba, R., Tajiri, T., Sumitomo, K., Masumoto, K., Taguchi, T., Yamamoto, K., 2013. Identification of a KEAP1 germline mutation in a family with multinodular goitre. PLoS One 8, e65141. http://dx.doi.org/10.1371/journal.pone.0065141. von Otter, M., Landgren, S., Nilsson, S., Celojevic, D., Bergstrom, P., Hakansson, A., Nissbrandt, H., Drozdzik, M., Bialecka, M., Kurzawski, M., Blennow, K., Nilsson, M., Hammarsten, O., Zetterberg, H., 2010a. Association of Nrf2-encoding NFE2L2 haplotypes with Parkinson's disease. BMC Med. Genet. 11, 36. von Otter, M., Landgren, S., Nilsson, S., Zetterberg, M., Celojevic, D., Bergström, P., Minthon, L., Bogdanovic, N., Andreasen, N., Gustafson, D.R., 2010b. Nrf2-encoding NFE2L2 haplotypes influence disease progression but not risk in Alzheimer's disease and age-related cataract. Mech. Ageing Dev. 131, 105–110. von Otter, M., Bergström, P., Quattrone, A., De Marco, E.V., Annesi, G., Söderkvist, P., Wettinger, S.B., Drozdzik, M., Bialecka, M., Nissbrandt, H., 2014. Genetic associations of Nrf2-encoding NFE2L2 variants with Parkinson's disease—a multicenter study. BMC Med. Genet. 15, 131. Wang, W., Wu, Y., Zhang, G., Fang, H., Wang, H., Zang, H., Xie, T., 2014. Activation of Nrf2– ARE signal pathway protects the brain from damage induced by epileptic seizure. Brain Res. 1544, 54–61. Wong, T.F., Yoshinaga, K., Monma, Y., Ito, K., Niikura, H., Nagase, S., Yamamoto, M., Yaegashi, N., 2011. Association of keap1 and nrf2 genetic mutations and polymorphisms with endometrioid endometrial adenocarcinoma survival. Int. J. Gynecol. Cancer 21, 1428–1435. Yu, B., Chen, J., Liu, D., Zhou, H., Xiao, W., Xia, X., Huang, Z., 2013. Cigarette smoking is associated with human semen quality in synergy with functional NRF2 polymorphisms. Biol. Reprod. 89, 5.
Please cite this article as: Liu, Z., et al., Association of KEAP1 and NFE2L2 polymorphisms with temporal lobe epilepsy and drug resistant epilepsy, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.06.055
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Research paper
Association of KEAP1 and NFE2L2 polymorphisms with temporal lobe epilepsy and drug resistant epilepsy Zhou Liu a,b,1, Xiaojian Yin b,1, Lingying Liu c, Hua Tao a, Haihong Zhou b, Guoda Ma a, Lili Cui a, You Li a, Shuyan Zhang d, Zhi'en Xu b, LiFen Yao e, Zhiyou Cai f, Bin Zhao a,b,⁎, Keshen Li a,⁎⁎ a
Guangdong Key Laboratory of Age-Related Cardiac and Cerebral Diseases, Affiliated Hospital of Guangdong Medical University, Zhanjiang, China Department of Neurology, Affiliated Hospital of Guangdong Medical University, Zhanjiang, China Department of Neurology, Chenzhou No. 1 People's Hospital, Chenzhou, China. d Department of Neurology, The Fourth Affiliated Hospital of Harbin Medical University, Harbin, China e Department of Neurology, The First Affiliated Hospital, Harbin Medical University, Harbin, Heilongjiang, China f Department of Neurology, Renmin Hospital, Hubei University of Medicine, Hubei, China b c
a r t i c l e
i n f o
Article history: Received 1 February 2015 Received in revised form 20 June 2015 Accepted 22 June 2015 Available online xxxx Keywords: Drug resistant epilepsy (DRE) Kelch-like ECH-associated protein 1 (KEAP1) Nuclear erythroid 2-related factor 2 (NFE2L2) Polymorphism Temporal lobe epilepsy (TLE)
a b s t r a c t Background and aims: Temporal lobe epilepsy (TLE) is a prevalent form of epilepsy. TLE contributes to the majority of drug resistant epilepsy (DRE) cases and is associated with genetic factors. Kelch-like ECH-associated protein 1 (KEAP1)/Nuclear erythroid 2-related factor 2 (known as NFE2L2 or Nrf2) association has been implicated in neuroprotection due to induction of antioxidant enzymes. The association of one single KEAP1 gene nucleotide polymorphism (SNP) and nine NFE2L2 gene SNPs with TLE and DRE were examined to determine whether these SNPs influenced the risk of TLE and DRE in a Han population. Subjects and methods: A total of 184 TLE patients (including 72 DRE patients) and 183 controls were included in this analysis. The SNaPshot Multiplex kit was used to assess the genotypes. Results: A NFE2L2 gene haplotype was identified as a risk factor for TLE (OR = 7.11, 95% CI 1.53–32.98). Additionally, rs2706110 G N A in the NFE2L2 gene and rs1048290 C N G in the KEAP1 gene showed a significant risk for and a protective effect against DRE, respectively. Conclusion: Our findings suggest that variations in NFE2L2 gene increase the risk of TLE and DRE but that variations in KEAP1 gene play a protective role for DRE. © 2015 Published by Elsevier B.V.
1. Introduction Temporal lobe epilepsy (TLE) is the most common form of partial epilepsy referred for epilepsy surgery; it is also responsible for the majority of drug-resistant epilepsy (DRE) cases (Semah et al., 1998; Tellez-Zenteno and Hernandez-Ronquillo, 2012). It is becoming Abbreviations: AD, Alzheimer's disease; ALS, amyotrophic lateral sclerosis; ABCG2, ATP-binding cassette transporter protein; bal acc, balanced accuracy; CI, confidence interval; HER2CA, constitutively active human epidermal growth factor receptor 2; CV, cross validation; CYP3A4, cytochrome P450 3A4; DRE, drug resistant epilepsy; NDRE, drug responsive epilepsy group; SNP, gene nucleotide polymorphism; GSTA2, glutathione S-transferase alpha 2; GSTP1, glutathione S-transferase pi-1; HWE, Hardy–Weinberg equilibrium; HO-1, heme oxygenase 1; ILAE, International League Against Epilepsy; KEAP1, Kelch-like ECH-associated protein 1; LD, linkage disequilibrium; MRP1, multidrug resistance protein 1; MRP5, multidrug resistance protein 5; MDR, Multifactor Dimensionality Reduction; (NFE2L2 or Nrf2), Nuclear erythroid 2-related factor 2; OR, odds ratio; OS, oxidative stress; PD, Parkinson's disease; TLE, temporal lobe epilepsy. ⁎ Correspondence to: B. Zhao, Guangdong Key Laboratory of Age-Related Cardiac and Cerebral Diseases, Affiliated Hospital of Guangdong Medical University, Zhanjiang, China. ⁎⁎ Corresponding author. 1 Dr. Zhou Liu and Dr. Xiaojian Yin contributed equally to this work.
increasingly clear that both genetic factors (Hwang and Hirose, 2012) and environmental conditions contribute to its complex pathogenesis. Oxidative stress (OS) is a disturbance in the pro-oxidant–antioxidant balance that damages all components of the cell, including proteins, lipids, and DNA. OS-mediated excitotoxicity (Sun et al., 2010) and hippocampal neuron death (Liu et al., 2010) are considered possible mechanisms behind the pathogenesis of epilepsy (Aguiar et al., 2012). Nuclear factor erythroid 2-related factor 2 (NFE2L2, also known as Nrf2) protein is a transcription factor that regulates the expression of many antioxidant pathway genes. Nrf2 protein is localized to the cytoplasm through interactions with its inhibitor protein, Kelch like-ECHassociated protein 1 (KEAP1), which functions as an adaptor for Cul3based E3 ubiquitin ligase to regulate proteasomal degradation of Nrf2 (Kobayashi et al., 2004). In response to oxidative stress, Nrf2 protein dissociates from KEAP1 protein, translocates into the nucleus and binds to the antioxidant response elements (ARE) located in the upstream promoter region of many antioxidative genes, thereby initiating their transcription (Itoh et al., 1997). The expression of Nrf2 protein target genes are regulated by mutations in KEAP1 gene (Teshiba et al., 2013) or NFE2L2 gene (Yu et al., 2013). Furthermore, the activated
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Please cite this article as: Liu, Z., et al., Association of KEAP1 and NFE2L2 polymorphisms with temporal lobe epilepsy and drug resistant epilepsy, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.06.055
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KEAP1–Nrf2/ARE pathway has been shown play a protective role in brain tissue (Meng et al., 2014). The relationship between TLE and NFE2L2 has attracted increasing attention. For example, NFE2L2-knockout mice were shown to be more vulnerable to kainate-induced seizures (Kraft et al., 2006). Activated Nrf2–ARE signal pathway in hippocampus suppressed the progression of amygdala kindling, and also ameliorated the cognitive impairment and oxidative stress induced by epileptic seizure (Wang et al., 2014). In a study of the relationship between the KEAP1–NFE2L2 genes and the risk of Parkinson's disease (PD) (von Otter et al., 2010a), an NFE2L2 haplotype was found to be associated with a reduced risk of PD. However, no association has been demonstrated between NFE2L2 or KEAP1 gene polymorphisms and epilepsy.
Table 1 Demographics and clinical characteristics. Variables
TLE (n = 184)
Control (n = 183)
P value
Mean age (years) Male/female
27.2 ± 14.4 94/90
49.1 ± 15.6 90/93
b0.001 0.72
Variables
DRE (n = 72)
NDRE (n = 112)
P value
Mean age (years) Age at onset (years) Male/female Hippocampal sclerosis History of febrile seizure History of status epilepticus
26.9 ± 15.0 17.6 ± 13.5 35/37 4 18 7
27.4 ± 14.1 21.3 ± 13.2 59/53 5 14 7
0.83 0.07 0.59 0.5 0.03 0.34
Data are presented as absolute numbers or mean ± SD. P-values are calculated with chisquared test for categorical parameters and t test for continuous parameters.
2. Material and methods 2.1. Study population
sequence detector for capillary electrophoresis. The experimental results were analyzed with GeneMapper 4.0 (Applied Biosystems Co., Ltd.).
Our study consecutively recruited 184 unrelated TLE patients of Han Chinese descent from the Department of Neurology of the Affiliated Hospital of Guangdong Medical College and the Department of Neurology of the First Affiliated Hospital of Harbin Medical University. Patients' diagnoses were based on the 1989 ILAE classification (Roger et al., 1989). None of the patients had a mass lesion, malformations of cortical development, or traumatic brain injury. The only accepted MRI sign was hippocampal sclerosis (HS) based on the characteristic MRI pattern of abnormalities. The TLE patients were divided into drug resistant epilepsy (DRE) group (n = 72) and drug responsive epilepsy group (NDRE) (n = 112) based on the International League Against Epilepsy (ILAE) consensus criteria (Kwan et al., 2010). Patients with extratemporal epilepsies, mental retardation, and systemic diseases were excluded. Patients with histories of ischemic cerebrovascular diseases, cardiogenic cerebral infarctions, cerebral hemorrhage, coronary artery diseases, autoimmune diseases, systemic inflammatory diseases, blood diseases, and malignant tumors were excluded from the study. A total of 183 healthy control subjects were recruited from the Health Examination Center of the Affiliated Hospital of Guangdong Medical College; the healthy subjects were comparable in sex and race to the TLE subjects. Written informed consent was obtained from all of the enrolled participants, and this study was approved by the Ethics Committee of the Affiliated Hospital of Guangdong Medical College and the First Affiliated Hospital of Harbin Medical University.
2.3. Magnetic resonance imaging (MRI)
2.2. DNA isolation and genotyping
3.1. Baseline characteristics
DNA isolation and genotyping were performed using previously published protocols (Li et al., 2013). Genomic DNA was extracted from venous blood using the EZ-10 Spin Column Whole Blood Genomic DNA Isolation Kit (Sangon Biotech, Shanghai, China), according to manufacturer's instruction. The genotypes were analyzed using the SNaPshot Multiplex Kit (Applied Biosystems Co., Ltd., Foster City, CA, USA). The primers used are listed in Table 1. SNaPshot reactions were executed in a 10 μL final volume containing 5 μL of the SNaPshot Multiplex Kit (ABI), 1 μL primer mix, 2 μL water, and 2 μL templates consisting of the multiplex PCR products from the different genes. The SNaPshot response procedures included (1) initial denaturation at 96 °C for 1 min; (2) denaturation at 96 °C for 10 s; (3) annealing at 52 °C for 5 s; (4) extension at 60 °C for 30 s; and (5) for a total of 28 cycles. Amplified samples were stored at 4 °C. Extension products were purified by a 1-h incubation with 1 U of shrimp alkaline phosphatase (Takara:Otsu, Shiga, Japan) at 37 °C, followed by incubation at 75 °C for 15 min to inactivate the enzyme. The purified products (0.5 μL) were mixed with 9 μL of Hi–Di and 0.5 μL of the Liz120 size standard (Applied Biosystems Co., Ltd.). The samples were incubated at 95 °C for 5 min and then loaded onto an ABI 3130XL DNA
The baseline characteristics of all of the participants in the study are summarized in Table 2. Of the 367 participants, 184 were patients with TLE and 183 were healthy controls. There was a significant difference in the distribution of age, while cases and controls were well matched by sex (Table 1). The TLE patients were divided into two groups: 72 patients with DRE and 112 patients with NDRE. There were no statistically significant differences between the DRE and NDRE groups in terms of age, sex, and history of status epilepticus. There were a higher proportion of patients with a history of febrile seizure in the DRE group compared with the NDRE group.
All MR studies were performed on a 1.5 T MRI-scanner (General Electric, Sigma 1.5, Waukesha, WI, USA) including sagittal T1, coronal FLAIR and T2. Details of the sequence were as follows: repetition time (TR) 4800 ms, echo time (TE) 120 ms, NEX 1, field of view 240 mm × 240 mm, 512 × 512 matrix. 2.4. Statistical analysis Chi-squared tests and t-tests were performed with SPSS software, version 19.0 (IBM, Armonk, NY, USA). The Hardy–Weinberg equilibrium (HWE), linkage disequilibrium (LD) and haplotypes were analyzed using SNPStats (http://bioinfo.iconcologia.net/SNPstats) (Sole et al., 2006). Co-dominant, dominant and recessive genetic models of inheritance were chosen to evaluate the associations between each SNP and TLE or DRE. The odds ratio (OR) and 95% confidence intervals (95% CI) were determined for each SNP in three genetic models adjusted by age and sex; the significance level was set to a P-value of 0.05. Additionally, gene–gene interactions were evaluated using Multifactor Dimensionality Reduction (MDR 3.0.2). QUANTO (version 1.2.4) software was used to calculate statistic power. The power was computed at the 0.05 significance level for the two-sided test, assumed an odds ratio of 1.5. 3. Results
3.2. Polymorphisms of the NFE2L2 and KEAP1 genes and the risk of TLE and DRE The genotype frequencies of NFE2L2 and KEAP1 polymorphisms in the study subjects are provided in Table 2. No deviations from the Hardy–Weinberg equilibrium for the examined polymorphisms were found in the distributions of genotypes between the patients and controls or the DRE and NDRE groups (data not shown). None of the genotypes alone significantly affected the risk of TLE. However, significant
Please cite this article as: Liu, Z., et al., Association of KEAP1 and NFE2L2 polymorphisms with temporal lobe epilepsy and drug resistant epilepsy, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.06.055
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Table 2 Relationship between the frequencies of the NFE2L2 and KEAP1 genotypes and alleles and the risk of TLE and DRE. TLE (n = 184) NFE2L2 rs7557529 Codominant AA AG GG Dominant AA GG + AG Recessive AG + AA GG rs35652124 Codominant AA AG GG Dominant AA GG + AG Recessive AG + AA GG rs6706649 Codominant GG AG AA Dominant GG AG + AA Recessive GG + AG AA rs6721961 Codominant CC AC AA Dominant CC AC + AA Recessive CC + AC AA rs2886161 Codominant AA AG GG Dominant AA AG + GG Recessive AA + AG GG rs1806649 Codominant GG AG AA Dominant GG AG + AA Recessive GG + AG AA rs2001350 Codominant AA AG GG
Control (n = 183)
OR (95%CI)
P value
DRE (n = 72)
NDRE (n = 112)
OR (95%CI)
P value
57 86 41
60 91 32
1 0.79 (0.44–1.42) 1.59 (0.75–3.41)
0.15
20 36 16
37 50 25
1 1.32 (0.66–2.64) 1.20 (0.52–2.77)
0.74
57 127
60 123
1 0.96 (0.55–1.66)
0.88
20 52
37 75
1 1.28 (0.67–2.45)
0.46
143 41
151 32
1 1.83 (0.93–3.60)
0.08
56 16
87 25
1 1.02 (0.50–2.08)
0.96
60 84 40
52 86 45
1 0.71 (0.38–1.30) 0.74 (0.36–1.51)
0.51
23 34 15
37 50 25
1 1.07 (0.54–2.12) 0.95 (0.41–2.17)
0.95
60 124
52 131
1 0.72 (0.41–1.27)
0.25
23 49
37 75
1 1.03 (0.54–1.95)
0.93
144 40
138 45
1
0.79
57 15
87 25
1 0.91 (0.44–1.87)
0.79
157 27 0
153 29 1
1 0.77 (0.38–1.57) NA (0.00–NA)
0.57
64 8 0
93 19 0
1 0.61 (0.25–1.48) NA (0.00–NA)
0.26
157 184
153 182
1 0.75 (0.37–1.52)
0.42
64 8
93 19
1 0.61 (0.25–1.48)
0.26
1 182
0 184
1 NA (0.00–NA)
0.42
72 0
112 0
1 NA (0.00–NA)
NA
89 82 13
86 85 12
1 0.93 (0.55–1.58) 1.30 (0.45–3.76)
0.82
32 34 6
57 48 7
1 1.27 (0.68–2.35) 1.56 (0.48–5.08)
0.64
89 95
86 97
1 0.97 (0.58–1.62)
0.91
32 40
57 55
1 1.30 (0.72–2.37)
0.38
171 13
171 12
1 1.35 (0.48–3.77)
0.56
66 6
105 7
1 1.39 (0.45–4.35)
0.57
60 85 39
51 87 45
1 0.67 (0.36–1.24) 0.69 (0.34–1.41)
0.41
23 34 15
37 51 24
1 1.05 (0.53–2.08) 0.99 (0.43–2.27)
0.98
60 124
51 132
1 0.68 (0.38–1.20)
0.18
23 49
37 75
1 1.03 (0.54–1.95)
0.93
145 39
138 45
1 0.89 (0.49–1.61)
0.7
57 15
88 24
1 0.96 (0.46–1.99)
0.91
146 36 2
154 26 3
1 1.40 (0.69–2.84) 0.92 (0.09–9.13)
61 9 2
85 27 0
1 0.47 (0.20–1.07) NA (0.00–NA)
0.03
146 38
154 29
1 1.35 (0.68–2.68)
61 11
85 27
1 0.57 (0.26–1.24)
0.15
0.39
182 2
180 3
1 0.87 (0.09–8.63)
70 2
112 0
1 NA (0.00–NA)
0.05
0.9
98 67 19
87 85 11
1 0.79 (0.46–1.36) 1.61 (0.61–4.30)
35 28 9
63 39 10
1 1.30 (0.68–2.47) 1.63 (0.60–4.39)
0.54
0.65
0.33
(continued on next page) (continued on next page)
Please cite this article as: Liu, Z., et al., Association of KEAP1 and NFE2L2 polymorphisms with temporal lobe epilepsy and drug resistant epilepsy, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.06.055
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Table 2 (continued) TLE (n = 184) Dominant AA AG + GG Recessive AA + AG GG rs10183914 Codominant GG AG AA Dominant GG AG + AA Recessive GG + AG AA rs2706110 Codominant GG AG AA Dominant GG AG + AA Recessive GG + AG AA KEAP1 rs1048290 Codominant CC CG GG Dominant CC CG + GG Recessive CC + CG GG
Control (n = 183)
OR (95%CI)
P value
DRE (n = 72)
NDRE (n = 112)
OR (95%CI)
P value
98 86
87 96
1 0.89 (0.53–1.49)
0.66
35 57
63 49
1 1.36 (0.75–2.49)
0.31
165 19
172 11
1 1.80 (0.70–4.64)
0.22
63 9
102 10
1 1.46 (0.56–3.80)
0.44
159 23 2
156 27 0
1 0.95 (0.43–2.07) NA (0.00–NA)
0.75
63 8 1
96 15 1
1 0.82 (0.33–2.06) 1.37 (0.08–22.95)
0.89
159 25
156 27
1 0.98 (0.45–2.13)
0.96
63 9
96 16
1 0.86 (0.36–2.07)
0.73
182 2
183 0
1 NA (0.00–NA)
0.46
71 1
111 1
1 1.40 (0.08–23.42)
0.82
92 77 15
92 80 11
1 1.03 (0.60–1.76) 1.30 (0.47–3.59)
29 36 7
63 41 8
1 1.94 (1.03–3.66) 1.99 (0.65–6.05)
0.11 0.09
92 92
92 91
1 1.07 (0.64–1.78)
0.81
29 43
63 49
1 1.95 (1.06–3.58)
0.03
169 15
172 11
1 1.28 (0.48–3.43)
0.63
65 7
104 8
1 1.44 (0.50–4.20)
0.5
50 94 40
46 97 40
1 0.70 (0.38–1.32) 1.16 (0.54–2.45)
0.27
27 31 14
23 63 26
1 0.41 (0.20–0.84) 0.45 (0.19–1.06)
0.04
50 134
46 137
1 0.82 (0.45–1.48)
0.5
27 45
23 89
1 0.42 (0.22–0.82)
0.01
144 40
143 40
1 1.46 (0.78–2.73)
0.23
58 27
86 23
1 0.78 (0.37–1.64)
0.51
0.88
Table 3 Haplotypes of NFE2L2 that influence the risk of TLE and DRE.
1 2 3 4 5 6 7 8 9 10
1 2 3 4 5 6 7 8 9 10
rs7557529–rs35652124–rs6706649–rs6721961–rs2886161– rs1806649–rs2001350–rs10183914–rs2706110
TLE
Control
OR (95% CI)
P value
AGGCGGAGG GAGAAGGGA AAACAGAGG GAGCAAAAG GAGAAGGGG GAGCAGAGA GAGCAAAGG GAGAAGAGG AAGCAGAGG AGGCGGGGA
41.80% 21.70% 7.10% 6.50% 4.30% 4.00% 4.40% 2.20% 3.00% 1.90%
43.20% 21.20% 8.50% 5.80% 3.00% 2.70% 2.30% 4.20% 1.40% 1.60%
1 1.17 (0.71–1.93) 0.76 (0.36–1.57) 1.25 (0.55–2.86) 1.64 (0.57–4.75) 1.31 (0.43–4.01) 1.93 (0.61–6.08) 0.44 (0.14–1.34) 7.11 (1.53–32.98) 1.14 (0.26–5.02)
– 0.55 0.46 0.6 0.36 0.64 0.26 0.15 0.013 0.86
rs7557529–rs35652124–rs6706649–rs6721961–rs2886161– rs1806649–rs2001350–rs10183914–rs2706110
DRE
NDRE
OR (95% CI)
P value
AGGCGGAGG GAGAAGGGA AAACAGAGG GAGCAAAAG GAGCAAAGG GAGAAGGGG GAGCAGAGA AAGCAGAGG GAGAAGAGG AGGCGGGGA
41.50% 18.50% 8.00% 6.00% 6.00% 5.10% 2.60% 3.10% 2.10% 1.80%
41.80% 21.70% 7.10% 6.50% 4.40% 4.30% 4.00% 3.00% 2.20% 1.90%
1 1.44 (0.81–2.57) 0.72 (0.27–1.88) 1.01 (0.41–2.46) 0.37 (0.09–1.42) 0.62 (0.21–1.85) 2.43 (0.76–7.84) 0.87 (0.23–3.31) 1.06 (0.22–5.16) 0.95 (0.20–4.62)
– 0.21 0.5 0.99 0.15 0.39 0.14 0.84 0.95 0.95
The haplotypes with frequencies of b0.01 were not included in haplotype association analysis.
Please cite this article as: Liu, Z., et al., Association of KEAP1 and NFE2L2 polymorphisms with temporal lobe epilepsy and drug resistant epilepsy, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.06.055
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differences were observed in the DRE group compared with the NDRE group in a dominant model of rs2706110 (OR = 1.95, 95% CI = 1.06– 3.58, P = 0.03) and codominant model and dominant model of rs1048290 (OR = 0.41, 95% CI = 0.20–0.84, P = 0.04; OR = 0.42, 95% CI = 0.22–0.82, P = 0.01, respectively). Moreover, power analysis showed that with our sample size, we would have 40% power for rs2706110, 47% power for rs1048290 to detect a genotype relative risk with an odds ratio of 1.5 at a significance level of 0.05. Furthermore, we performed a haplotype-based association analysis of nine polymorphisms of NFE2L2 gene. The haplotype AAGC AGAGG was associated with an increased risk of TLE (OR = 7.11, 95% CI = 1.53–32.98). However, no significant associations were observed between these haplotypes and DRE (Table 3). 3.3. Multi-dimensionality reduction (MDR) analysis MDR analysis showed that the rs2001350 and rs1048290 polymorphisms represented the best one factor models for TLE and DRE respectively. rs7557529/rs1048290 and rs2886161/rs2706110 represented the best model two factor models for TLE and DRE, respectively. Finally, the rs7557529/rs2001350/rs1048290 and rs35652124/rs6721961/ rs1048290 polymorphisms represented the best three factor models for TLE and DRE, respectively. However, the P-values were not significant for all factor models (Table 4). 4. Discussion The present study explored the association between polymorphisms in antioxidant transcription factor NFE2L2 gene and its inhibitor protein KEAP1 gene with TLE and DRE. The results showed that one NFE2L2 haplotype (AAGCAGAGG) was associated with an increased risk of TLE, the AG + AA genotypes of NFE2L2 rs2706110 under the dominant mode increased the risk of DRE, and the CG genotype under the codominant mode and the CG + GG genotypes of KEAP1 rs1048290 under the dominant mode decreased the risk of DRE. To the best of our knowledge, this is the first study to investigate an association between SNPs in NFE2L2 and KEAP1 with TLE and DRE. However, the association between SNPs in NFE2L2 and KEAP1 with other nervous system diseases (NSD) has been explored. Consistent with the present study, no significant associations between SNPs in NFE2L2 and KEAP1 with NSD have been found. Malin's two studies investigating 11 NFE2L2 SNPs (rs16865105, rs7557529, rs35652124, rs6706649, rs6721961, rs2886161, rs1806649, rs2001350, rs10183914, rs2706110, and rs13035806) and 3 KEAP1 SNPs (rs1048287, rs11085735, and rs1048290) did not find an association between the polymorphisms and either the risk of Parkinson's disease (PD) (von Otter et al., 2010a) or Alzheimer's disease (AD) (von Otter et al., 2010b). An Italian study investigated three NFE2L2 SNPs (rs35652124, rs6706649, and rs6721961) found that the polymorphisms did not differ between patients with amyotrophic lateral sclerosis (ALS) and controls (LoGerfo et al., 2014). Although promoter polymorphism rs6721961 of the C allele enhanced promoter activity (Marzec et al., 2007), the T allele was
Table 4 Summary of the MDR analysis.
TLE
DRE
Model
CV consistency
Bal acc CV testing
P value
rs2001350 rs7557529, rs1048290 rs7557529, rs2001350, rs1048290 rs1048290 rs2886161, rs2706110 rs35652124, rs6721961, rs1048290
10/10 9/10 8/10 6/10 3/10 9/10
0.55 0.5 0.52 0.52 0.45 0.59
0.54 0.96 0.75 0.86 0.74 0.43
Abbreviations: bal acc, balanced accuracy; CV, cross validation; MDR, Multifactor Dimensionality Reduction.
5
associated with a low extent of Nrf2 protein expression (Hartikainen et al., 2012), and the AGC polymorphisms (rs35652124, rs6706649, and rs6721961) increased NFE2L2 promoter activity (Marczak et al., 2012). The current study showed that a haplotype of NFE2L2 (AAGCAG AGG, rs7557529, rs35652124, rs6706649, rs6721961, rs2886161, rs1806649, rs2001350, rs10183914, and rs2706110) included the AGC, while rs10183914 included the G allele; these polymorphisms increased the risk of TLE. Moreover, evidences proved that rs10183914 G allele combined with AGC may influence the risk of AD and TLE. Malin found that GAGCAAAG (rs7557529, rs35652124, rs6706649, rs6721961, rs2886161, rs1806649, rs2001350, and rs10183914) was associated with an increased risk of PD in Swedish populations (von Otter et al., 2010a) and Italian populations (von Otter et al., 2014), also including rs10183914 G allele and AGC. However, the association could not be replicated in a meta-analysis (von Otter et al., 2014). Conflicting results are common in TLE SNPs researches (Cavalleri et al., 2005), because of differences of study design, statistical analysis, and interpretation of findings in association studies. We did not find an association between the polymorphisms in NFE2L2 and KEAP1 with TLE, but we did determine that rs2706110 of NFE2L2 and rs1048290 of KEAP1 affected the risk of DRE. Moreover, the AG + AA genotypes of rs2706110 under the dominant mode increased the DRE risk. However, several studies reported that this polymorphism was not associated with ALS (Bergström et al., 2014), PD (von Otter et al., 2010a) and AD (von Otter et al., 2010b). But, the rs2706110 AA genotype was associated with an increased risk of breast cancer (Hartikainen et al., 2012). Another SNP related with DRE (rs1048290) is located in exon 4 of KEAP1. The CG genotype under the codominant mode and the CG + GG genotypes under the dominant mode decreased the risk of DRE. In agreement with this finding, a recent study demonstrated that haplotype CGG (rs1048287, rs11085735, and rs1048290) in KEAP1 was associated with later sporadic ALS (LSALS) onset (Bergström et al., 2014), and the CG genotype has been significantly associated with progression-free survival in endometrial cancer (Wong et al., 2011). Under basal conditions, two molecules of Keap1 bind with one molecule of Nrf2, which functions as an adapter for Cul3/Rbx1 E3 ligase for Nrf2 ubiquitination. Ubiquitinated Nrf2 is degraded in the proteasome. When Keap1 conformational changes are make by oxidative or electrophilic stress, Nrf2 and Cul3 dissociate from Keap1, E3 ligase activity subsequently declines, Nrf2 ubiquitination stops. Nrf2 is thus stabilized and translocates to the nucleus, to be available for rapid Nrf2/ARE-dependent gene transcription (Taguchi et al., 2011). Although there is no direct evidence in regard to the possible participation of Nrf2 in DRE, one study reported that Nrf2 and constitutively active human epidermal growth factor receptor 2 (HER2CA) cooperatively up-regulated the mRNA expression of various drug-resistant and detoxifying enzymes (Kang et al., 2014). Because the NFE2L2 and KEAP1 genes may exert synergistic effects on TLE and DRE, we evaluated gene–gene interactions using the MDR method. However, no significant interactions among the SNPs analyzed in this study were identified. However, for the some potential limitations we cannot definitely exclude the possibility of false positive results. First, there is a possibility of the low statistical power. In the present sample size, the statistical powers were 40% for rs2706110, 47% for rs1048290, for detecting statistical differences to the stratification of DRE. Secondly, p-values from 0.05 to 0.01 are usually inadequate conservative to support our findings. Therefore, we still need the validation cohort study for further confirm our results, further investigation with a larger and more ethnically diverse population of TLE, DRE patients is warranted to support our preliminary conclusions.
Declaration of interest The authors declare no conflict of interest.
Please cite this article as: Liu, Z., et al., Association of KEAP1 and NFE2L2 polymorphisms with temporal lobe epilepsy and drug resistant epilepsy, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.06.055
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Funding This work was supported by funding from the National Nature Science Foundation of China (grant numbers 31171219, 81271213, 81271214, 81471294 and 81400986), the Medical Research Foundation of Guangdong Province (grant number B2013298) and the Science and Technology Planning Project of Zhanjiang (grant number 2014A01031). References Aguiar, C.C.T., Almeida, A.B., Araújo, P.V.P., Abreu, R.N.D.C.d., Chaves, E.M.C., Vale, O.C.d., Macêdo, D.S., Woods, D.J., Fonteles, M.M.d.F., Vasconcelos, S.M.M., 2012. Oxidative stress and epilepsy: literature review. Oxidative Med. Cell. Longev. 795259. http:// dx.doi.org/10.1155/2012/795259. Bergström, P., von Otter, M., Nilsson, S., Nilsson, A.-C., Nilsson, M., Andersen, P.M., Hammarsten, O., Zetterberg, H., 2014. Association of NFE2L2 and KEAP1 haplotypes with amyotrophic lateral sclerosis. Amyotroph. Lateral Scler. Frontotemporal Degener. 15, 130–137. Cavalleri, G.L., Lynch, J.M., Depondt, C., Burley, M.-W., Wood, N.W., Sisodiya, S.M., Goldstein, D.B., 2005. Failure to replicate previously reported genetic associations with sporadic temporal lobe epilepsy: where to from here? Brain 128, 1832–1840. Hartikainen, J.M., Tengström, M., Kosma, V.-M., Kinnula, V.L., Mannermaa, A., Soini, Y., 2012. Genetic polymorphisms and protein expression of NRF2 and sulfiredoxin predict survival outcomes in breast cancer. Cancer Res. 72, 5537–5546. Hwang, S.K., Hirose, S., 2012. Genetics of temporal lobe epilepsy. Brain Dev. 34, 609–616. Itoh, K., Chiba, T., Takahashi, S., Ishii, T., Igarashi, K., Katoh, Y., Oyake, T., Hayashi, N., Satoh, K., Hatayama, I., Yamamoto, M., Nabeshima, Y., 1997. An Nrf2/small Maf heterodimer mediates the induction of phase II detoxifying enzyme genes through antioxidant response elements. Biochem. Biophys. Res. Commun. 236, 313–322. Kang, H.J., Yi, Y.W., Hong, Y.B., Kim, H.J., Jang, Y.J., Seong, Y.S., Bae, I., 2014. HER2 confers drug resistance of human breast cancer cells through activation of NRF2 by direct interaction. Sci. Rep. 4, 7201. Kobayashi, A., Kang, M.-I., Okawa, H., Ohtsuji, M., Zenke, Y., Chiba, T., Igarashi, K., Yamamoto, M., 2004. Oxidative stress sensor Keap1 functions as an adaptor for Cul3-based E3 ligase to regulate proteasomal degradation of Nrf2. Mol. Cell. Biol. 24, 7130–7139. Kraft, A.D., Lee, J.M., Johnson, D.A., Kan, Y.W., Johnson, J.A., 2006. Neuronal sensitivity to kainic acid is dependent on the Nrf2-mediated actions of the antioxidant response element. J. Neurochem. 98, 1852–1865. Kwan, P., Arzimanoglou, A., Berg, A.T., Brodie, M.J., Allen Hauser, W., Mathern, G., Moshe, S.L., Perucca, E., Wiebe, S., French, J., 2010. Definition of drug resistant epilepsy: consensus proposal by the ad hoc Task Force of the ILAE Commission on Therapeutic Strategies. Epilepsia 51, 1069–1077. Li, Y., Liao, F., Yin, X.J., Cui, L.L., Ma, G.D., Nong, X.X., Zhou, H.H., Chen, Y.F., Zhao, B., Li, K.S., 2013. An association study on ADAM10 promoter polymorphisms and atherosclerotic cerebral infarction in a Chinese population. CNS Neurosci. Ther. 19, 785–794. Liu, J., Wang, A., Li, L., Huang, Y., Xue, P., Hao, A., 2010. Oxidative stress mediates hippocampal neuron death in rats after lithium–pilocarpine-induced status epilepticus. Seizure 19, 165–172. LoGerfo, A., Chico, L., Borgia, L., Petrozzi, L., Rocchi, A., D'Amelio, A., Carlesi, C., Caldarazzo Ienco, E., Mancuso, M., Siciliano, G., 2014. Lack of association between nuclear factor
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