- Email: [email protected]
Lack of association between IL17A and IL17F polymorphisms and related serum levels in north Indians with tuberculosis
GENE-38796; No. of pages: 4; 4C: Gene xxx (2013) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Gene journal homepage: www.elsevier.com/locate/gene
1
Letter to Editor
2
Keywords: Serum IL-17 levels Pulmonary tuberculosis Lymph node tuberculosis IL17A IL17F variant Single nucleotide polymorphisms Mycobacterium tuberculosis
16
We were pleased to read the article entitled “The IL-17F sequence variant is associated with susceptibility to tuberculosis” in your reputed journal (Peng et al., 2013). The authors in order to address the role of IL17 gene polymorphisms in tuberculosis show that a variant in IL17F gene is associated with tuberculosis. The study uses a good sample size and very apt conclusions are drawn. We wish to report here that independently we have also examined the role of rs2275913 and rs763780 single nucleotide polymorphisms of IL17A and IL17F genes respectively on the development of pulmonary (PTB) and lymph node tuberculosis (LNTB) in north Indians and correlated the serum IL-17A and IL-17F levels associated with them. We in our study did not find any association between the IL17F gene polymorphism and TB. To our knowledge, it is the first study to investigate the IL17 gene polymorphisms and their correlation with serum IL-17 levels. This communication is no criticism of the reported work per se but a means to report the different results we obtained in an unrelated ethnic population adding a different vantage point for the role of IL17 sequence variants in tuberculosis. The role of CD4+ T cells and T helper (Th) 1 cytokines are critical and well characterized in the cell mediated response to Mycobacterium tuberculosis (M.tb), but it has emerged that the Th1 axis alone is not enough to counter the infection (Goldsack and Kirman, 2007) and there can be a mixed bag of other factors involved. IL-17 is a novel family of proinflammatory cytokines produced by Th17 cells in response to IL-23 (Kawaguchi et al., 2004). The IL-17 axis is very crucial for initial immune responses in tuberculosis (Khader and Cooper, 2008). To investigate the significance of the abovementioned genetic polymorphisms in the IL17 gene in human tuberculosis susceptibility, genomic DNA was isolated from 215 patients comprising of both PTB
28 29 30 31 32 33 34 Q3 35 36 37 38 39 40 41 42 43
C
E
26 27
R
24 25
R
22 23
N C O
20 21
U
19
E
Dear Editor,
T
15
D
P
14
17 18
(n = 165), LNTB (n = 50) and 130 regional healthy controls (HC). The inclusion and exclusion criteria for patients and HC were as previously described (Abhimanyu et al., 2012). The ethical committee of the V.P. Chest Institute approved the study. The mean age of the PTB cases was 34 years with a range of 18–70 years, while for LNTB cases the mean age was 25 years with a range of 18–49 years. The HC group had a mean age of 28 years with a range of 21–65 years. Logistic regression showed that there was no significant difference in age of the study groups. Amplification refractory mutation system polymerase chain reaction (ARMS-PCR) was used to genotype the sequence variants. We used agarose gel electrophoresis to read the genotypes. The success rate for genotyping was 100% for the both studied variants of IL17A and IL17F for all the study subjects except for the LNTB cases of rs22275913 (IL17A) where the success rate was 86% (7/50 cases could not be typed). We sequenced 10% of the samples for each studied variant by using ABI 3130xL genetic analyzer using BigDye termination chemistry to check for concordance and we observed full concordance between the genotypes obtained by the two used methods. The representative sequencing results for both the studied variants are presented in Fig. 1. We selected a subset of these patients based on their genotype frequencies (to get a meaningful representation of the genotypes) to decipher the effect of genotypes on serum levels of the corresponding cytokine. We obtained serum from 75 PTB, 40 LNTB and 75 HC and measured the serum IL-17A and IL-17F by using enzyme-linked immunosorbent assay using human cytokine ELISA kit (Diaclone, France) which uses a solid phase sandwich ELISA. We correlated the obtained serum levels with the corresponding genotypes. By using, PLINK (http://pngu.mgh.harvard.edu/~purcell/plink/), we carried out Identity by state clustering (IBS) analysis to assess for population stratification. (Genotyping data from twenty-five cytokine SNPs previously reported by us were used as markers). In the genotyping data obtained Hardy– Weinberg equilibrium (HWE) was calculated in PTB, LNTB and HC separately by using Haploview v4.2 (http://www.broad.mit.edu/mpg/ haploview) using default conditions (HW p-value cut off = 0.0010; minimum genotype = 75% and minimum minor allele frequency (MAF = 0.0010)). The samples and variations failing this test were not selected for further analysis. Odds ratio and allelic p values were computed using a 2 × 2 contingency table using GraphPad Prism (version 5.00 for Windows, Graph Pad Software, San Diego California USA, www.graphpad.com) which was also used for one way analysis of variance (ANOVA) with Bonferroni comparison was used to compare the serum cytokine levels between the different genotypes within cases and HC. A two-tailed p value b 0.05 was considered statistically significant. Haploview v4.2 was used for allelic association analysis. Multi-dimensional scaling plot generated using R package, revealed a homogeneous group free from any stratification (submitted to publication). The study groups were not deviating from HWE in cases as well as controls. For both of the studied variants i.e. rs2275913 (IL17A) and rs763780 (IL17F) the alleles and genotypes were evenly distributed among cases and controls in both pulmonary or LNTB cases and it did not show any sign of association (Table 1). The observed mean serum cytokine level for IL-17A although higher in PTB cases as compared to
F
6 5 Q2 7 8 9 10 11 12 13
Lack of association between IL17A and IL17F polymorphisms and related serum levels in north Indians with tuberculosis
O
4
R O
3
Abbreviations: IL-17, interleukin (IL)-17; IFN-γ, Interferon (IFN)-gamma; IL-12, interleukin (IL)-12; M. tuberculosis, Mycobacterium tuberculosis; PTB, pulmonary tuberculosis; LNTB, lymph node tuberculosis; DNA, Deoxy ribonucleic acid; SNPs, single nucleotide polymorphisms; TB, tuberculosis; Th cell, T helper cell; Th17 cells, Interleukin-17-producing T helper cells; ARMS-PCR, Amplification refractory mutation system polymerase chain reaction; ELISA, enzyme-linked immunosorbent assay; HWE, Hardy–Weinberg equilibrium; MAF, Minor allele frequency; OR, odds ratios; CI, confidence intervals. 0378-1119/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2013.06.090
Please cite this article as: Abhimanyu, et al., Lack of association between IL17A and IL17F polymorphisms and related serum levels in north Indians with tuberculosis, Gene (2013), http://dx.doi.org/10.1016/j.gene.2013.06.090
44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 Q4 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95
Letter to Editor
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2
109 110 111 112
Q5 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134
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Funding
146
The study was funded by in-house funding at Vallabhbhai Patel Chest Institute, University of Delhi, Delhi, India. Mr. Abhimanyu is the recipient of Senior Research Fellowship from Indian Council of Medical Research (ICMR) via project 5/8/5/14/ECD-I. MB is the recipient of ICMR Emeritus Medical scientist award vide letter no. 74/1/2012Pers(EMS).
147
Conflict of interest
153
E
the functional axis governing the tuberculosis pathogenesis in the population under study. Further investigations in a large sample size could use these genetic polymorphisms in conjunction to study the effect of these polymorphisms in tuberculosis. In conclusion, we may say that our results, albeit based on a limited sample, indicate that the variants rs2275913 (IL17A) and rs763780 (IL17F) are not associated with susceptibility to tuberculosis and the serum cytokine levels also don't differ between cases and controls as well as the polymorphisms don't affect the levels of serum cytokines in north Indians. We feel that further studies on the IL17 gene polymorphisms are warranted to clear its genetics in governing the susceptibility to TB.
T
C
E
107 108
R
105 106
R
103 104
O
101 102
C
99 100
HC, the difference was not statistically significant (Table 1). When the serum levels were compared between the corresponding genotypes of rs2275913 (IL17A) and rs763780 (IL17F) no significant association was observed (Table 1). The variant rs2275913 (IL17A) was not associated with TB in both north Indians and the Chinese population, but still there were differences in pattern of allelic distribution. While A was the minor allele in our study G was the minor allele in the study on the Chinese population. In the reported association for rs763780 (IL17F), the authors found that C allele was higher in both pulmonary and extrapulmonary groups and the CT genotype was differentially distributed among cases and controls. However, we could not find any such association. The difference between the study on Chinese population (Peng et al., 2013) and our study for IL17F variant rs763780 could be due to ethnicity difference between the two different studied populations. Case–control association studies are valuable tools to address the effect of genetic polymorphisms on disease pathogenesis and this could be a confounded by many factors (Abhimanyu et al., 2012). In our study we have accounted for all such factors as checking of the presence of a population substructure, carrying out multiple corrections and accounted for the Hardy–Weinberg equilibrium. It becomes pertinent here to point out that the current sample size had 84% power to detect an odds ratio of 2.0 with fisher exact test in place. Sample size, though not very low, was definitely a limitation for the study as the current sample size would miss any weak association or rare variant effect if any in the studied population. The non-association in this population could be due to the effect of natural selection, which has resulted in variable distribution of the alleles of IL17A/F gene in the north Indian population as compared to the Chinese population. The serum cytokine level association with genotypes although was not significant but could be accounted for considering that the crucial role of IL-17 is also complemented by the IL-12/IFN-γ axis, where a balance between the two is required for the regulation of inflammatory consequences following M.tb infection (Khader and Cooper, 2008). To that effect, We previously have shown that the polymorphisms in the IL12 gene are associated with risk in the north Indian population (Abhimanyu et al., 2012) and that the polymorphisms in the IL12 gene were associated with varying levels of serum IL-12 of the corresponding SNPs (Abhimanyu et al., 2011). So, it might be postulated that the IL-12 axis rather than the IL-17 axis is
N
97 98
U
96
D
P
Fig. 1. Representative electropherogram obtained for samples. A–C: for rs22275913 (IL17A). All three genotypes were obtained (i.e. GA, GG, AA); D–E: for rs763780 (IL17F) (only two genotypes seen CT, TT here shown as GA, AA these represent results obtained by the reverse primers). This was done for 10% of the samples and full concordance was seen between the genotypes identified by ARMS-PCR and sequencing.
We declare no conflict of interest exists.
136 137 138 139 140 141 142 143 144 145
148 149 150 151 152
154
References
155
Abhimanyu, et al., 2011. Differential serum cytokine levels are associated with cytokine gene polymorphisms in north Indians with active pulmonary tuberculosis. Infect. Genet. Evol. 11, 1015–1022. Abhimanyu, Bose, M., Jha, P., 2012. Indian Genome Variation Consortium. Footprints of genetic susceptibility to pulmonary tuberculosis: cytokine gene variants in north Indians. Indian J. Med. Res. 135, 763–770. Goldsack, L., Kirman, J.R., 2007. Half-truths and selective memory: interferon gamma, CD4(+) T cells and protective memory against tuberculosis. Tuberculosis (Edinb) 87, 465–473. Kawaguchi, M., Adachi, M., Oda, N., Kokubu, F., Huang, S.K., 2004. IL-17 cytokine family. J. Allergy Clin. Immunol. 114, 1265–1273 (quiz 1274). Khader, S.A., Cooper, A.M., 2008. IL-23 and IL-17 in tuberculosis. Cytokine 41, 79–83. Peng, R., Yue, J., Han, M., Zhao, Y., Liu, L., Liang, L., 2013. The IL-17F sequence variant is associated with susceptibility to tuberculosis. Gene 515, 229–232 (Epub 2012).
156 157 158 159 160 161 162 163 164 165 166 167 168 169
Please cite this article as: Abhimanyu, et al., Lack of association between IL17A and IL17F polymorphisms and related serum levels in north Indians with tuberculosis, Gene (2013), http://dx.doi.org/10.1016/j.gene.2013.06.090
N
C
O
Q1 Q6
t1:1 Q1 Table 1 t1:2 Q6 Showing the genotypic, allelic frequencies with association analysis of the variants of IL17 gene and the mean serum cytokine correlation among the genotypes in study subjects. t1:3 a
t1:4
Gene
rsID dbSNP
Subjects
Genotype/allele frequencies
t1:5 t1:6 t1:7 t1:8 t1:9 t1:10 t1:11 t1:12 t1:13 t1:14 t1:15 t1:16 t1:17 t1:18 t1:19 t1:20 t1:21
IL17A
rs22275913
PTB patients (n = 165)
GG 17 (10.3%)
t1:22 t1:23 t1:24
LNTB patients (n = 50) Controls (n = 130)
IL17F
rs763780
PTB patients (n = 165) LNTB patients (n = 50) Controls (n = 130)
Allelic association analysis
G 120 (41.1%)
23 (46%)
20 (40%)
22 (27%)
14 (11%)
63 (48.4%)
42 (32.3%)
91 (37.4%)
CC 0 (0%)
CT 6 (3.8%)
TT 159 (96.2%)
0 (0%)
3 (5.6%)
47 (94.4%)
0 (0%)
4 (2.7%)
126 (97.2%)
C 6 (1.8%) 3 (3%) 4 (1.5%)
Cytokine analysis
Chi-square
Odds ratio (CI)
P value
Subjects
Genotype
A 172 (58.9%)
0.576
1.2 (0.79–1.7)
0.48
PTB (n = 75)
62 (73%)
0.411
R
AA 43 (26.0%)
0 (0%)
GA 86 (52.1%)
R
E
C
T
0.8 (0.45–1.4)
T 324 (98.2%)
0.229
1.4 (0.34–6.0)
97 (97%)
0.662
2.1 (0.34–13)
149 (62.6%)
256 (98.5%)
0.5214
LNTB (n = 40) HC (n = 75)
E
D
IL-17 F PTB (n = 75)
0.63
P
0.416
LNTB (n = 40)
R O HC (n = 75)
Mean serum cytokine level(pg/ml)
Comparison of serum cytokine level among genotypes
P value
IL-17A GA (40) GG (11) AA(24) GA (21) AA(19) GA (40) GG (11) AA(24)
23 17 27 20 19 21 19 21
± ± ± ± ± ± ± ±
2.2 0.8 5.2 1.5 1.9 2.8 1.0 2.2
GA Vs GG GG Vs AA AA Vs GA GA Vs AA
P P P P
GA Vs GG GG Vs AA AA Vs GA
P N 0.05 P N 0.05 P N 0.05
TT (69) CT (6)b TT (37) CT (3)b TT (71) CT (4)b
25 27 16 12 14 16
± ± ± ± ± ±
6.3 3.7 0.79 0.059 0.73 2.61
TT Vs CT
P N 0.05
TT Vs CT
P N 0.05
TT Vs CT
P N 0.05
N N N N
0.05 0.05 0.05 0.05
Letter to Editor
Please cite this article as: Abhimanyu, et al., Lack of association between IL17A and IL17F polymorphisms and related serum levels in north Indians with tuberculosis, Gene (2013), http://dx.doi.org/10.1016/j.gene.2013.06.090
U
PTB = pulmonary tuberculosis; LNTB = lymph node tuberculosis; SNP = single nucleotide polymorphism. a dbSNP, the SNP database (http://www.ncbi.nlm.nih.gov/projects/SNP); two tailed P b 0.05 was considered significant. For cytokine analysis the numbers in parentheses indicate the number of samples tested for that genotype; values are expressed as mean ± standard error. b For rs763780 C was minor allele and the number in parentheses indicate actual number of CT genotype observed in PTB, LNTB and Controls.
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170 171 172
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4
Letter to Editor
⁎Corresponding author. Tel.: +91 11 27667120; 178 fax: +91 11 27666549. 179 E-mail address: [email protected] (M. Bose). 180
Abhimanyu Mridula Bose⁎ Komal Mandira Varma-Basil Department of Microbiology, Vallabhbhai Patel Chest Institute, University of Delhi, New Delhi 110007, India
181
Available online xxxx 182
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183
Please cite this article as: Abhimanyu, et al., Lack of association between IL17A and IL17F polymorphisms and related serum levels in north Indians with tuberculosis, Gene (2013), http://dx.doi.org/10.1016/j.gene.2013.06.090
Contents lists available at SciVerse ScienceDirect
Gene journal homepage: www.elsevier.com/locate/gene
1
Letter to Editor
2
Keywords: Serum IL-17 levels Pulmonary tuberculosis Lymph node tuberculosis IL17A IL17F variant Single nucleotide polymorphisms Mycobacterium tuberculosis
16
We were pleased to read the article entitled “The IL-17F sequence variant is associated with susceptibility to tuberculosis” in your reputed journal (Peng et al., 2013). The authors in order to address the role of IL17 gene polymorphisms in tuberculosis show that a variant in IL17F gene is associated with tuberculosis. The study uses a good sample size and very apt conclusions are drawn. We wish to report here that independently we have also examined the role of rs2275913 and rs763780 single nucleotide polymorphisms of IL17A and IL17F genes respectively on the development of pulmonary (PTB) and lymph node tuberculosis (LNTB) in north Indians and correlated the serum IL-17A and IL-17F levels associated with them. We in our study did not find any association between the IL17F gene polymorphism and TB. To our knowledge, it is the first study to investigate the IL17 gene polymorphisms and their correlation with serum IL-17 levels. This communication is no criticism of the reported work per se but a means to report the different results we obtained in an unrelated ethnic population adding a different vantage point for the role of IL17 sequence variants in tuberculosis. The role of CD4+ T cells and T helper (Th) 1 cytokines are critical and well characterized in the cell mediated response to Mycobacterium tuberculosis (M.tb), but it has emerged that the Th1 axis alone is not enough to counter the infection (Goldsack and Kirman, 2007) and there can be a mixed bag of other factors involved. IL-17 is a novel family of proinflammatory cytokines produced by Th17 cells in response to IL-23 (Kawaguchi et al., 2004). The IL-17 axis is very crucial for initial immune responses in tuberculosis (Khader and Cooper, 2008). To investigate the significance of the abovementioned genetic polymorphisms in the IL17 gene in human tuberculosis susceptibility, genomic DNA was isolated from 215 patients comprising of both PTB
28 29 30 31 32 33 34 Q3 35 36 37 38 39 40 41 42 43
C
E
26 27
R
24 25
R
22 23
N C O
20 21
U
19
E
Dear Editor,
T
15
D
P
14
17 18
(n = 165), LNTB (n = 50) and 130 regional healthy controls (HC). The inclusion and exclusion criteria for patients and HC were as previously described (Abhimanyu et al., 2012). The ethical committee of the V.P. Chest Institute approved the study. The mean age of the PTB cases was 34 years with a range of 18–70 years, while for LNTB cases the mean age was 25 years with a range of 18–49 years. The HC group had a mean age of 28 years with a range of 21–65 years. Logistic regression showed that there was no significant difference in age of the study groups. Amplification refractory mutation system polymerase chain reaction (ARMS-PCR) was used to genotype the sequence variants. We used agarose gel electrophoresis to read the genotypes. The success rate for genotyping was 100% for the both studied variants of IL17A and IL17F for all the study subjects except for the LNTB cases of rs22275913 (IL17A) where the success rate was 86% (7/50 cases could not be typed). We sequenced 10% of the samples for each studied variant by using ABI 3130xL genetic analyzer using BigDye termination chemistry to check for concordance and we observed full concordance between the genotypes obtained by the two used methods. The representative sequencing results for both the studied variants are presented in Fig. 1. We selected a subset of these patients based on their genotype frequencies (to get a meaningful representation of the genotypes) to decipher the effect of genotypes on serum levels of the corresponding cytokine. We obtained serum from 75 PTB, 40 LNTB and 75 HC and measured the serum IL-17A and IL-17F by using enzyme-linked immunosorbent assay using human cytokine ELISA kit (Diaclone, France) which uses a solid phase sandwich ELISA. We correlated the obtained serum levels with the corresponding genotypes. By using, PLINK (http://pngu.mgh.harvard.edu/~purcell/plink/), we carried out Identity by state clustering (IBS) analysis to assess for population stratification. (Genotyping data from twenty-five cytokine SNPs previously reported by us were used as markers). In the genotyping data obtained Hardy– Weinberg equilibrium (HWE) was calculated in PTB, LNTB and HC separately by using Haploview v4.2 (http://www.broad.mit.edu/mpg/ haploview) using default conditions (HW p-value cut off = 0.0010; minimum genotype = 75% and minimum minor allele frequency (MAF = 0.0010)). The samples and variations failing this test were not selected for further analysis. Odds ratio and allelic p values were computed using a 2 × 2 contingency table using GraphPad Prism (version 5.00 for Windows, Graph Pad Software, San Diego California USA, www.graphpad.com) which was also used for one way analysis of variance (ANOVA) with Bonferroni comparison was used to compare the serum cytokine levels between the different genotypes within cases and HC. A two-tailed p value b 0.05 was considered statistically significant. Haploview v4.2 was used for allelic association analysis. Multi-dimensional scaling plot generated using R package, revealed a homogeneous group free from any stratification (submitted to publication). The study groups were not deviating from HWE in cases as well as controls. For both of the studied variants i.e. rs2275913 (IL17A) and rs763780 (IL17F) the alleles and genotypes were evenly distributed among cases and controls in both pulmonary or LNTB cases and it did not show any sign of association (Table 1). The observed mean serum cytokine level for IL-17A although higher in PTB cases as compared to
F
6 5 Q2 7 8 9 10 11 12 13
Lack of association between IL17A and IL17F polymorphisms and related serum levels in north Indians with tuberculosis
O
4
R O
3
Abbreviations: IL-17, interleukin (IL)-17; IFN-γ, Interferon (IFN)-gamma; IL-12, interleukin (IL)-12; M. tuberculosis, Mycobacterium tuberculosis; PTB, pulmonary tuberculosis; LNTB, lymph node tuberculosis; DNA, Deoxy ribonucleic acid; SNPs, single nucleotide polymorphisms; TB, tuberculosis; Th cell, T helper cell; Th17 cells, Interleukin-17-producing T helper cells; ARMS-PCR, Amplification refractory mutation system polymerase chain reaction; ELISA, enzyme-linked immunosorbent assay; HWE, Hardy–Weinberg equilibrium; MAF, Minor allele frequency; OR, odds ratios; CI, confidence intervals. 0378-1119/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2013.06.090
Please cite this article as: Abhimanyu, et al., Lack of association between IL17A and IL17F polymorphisms and related serum levels in north Indians with tuberculosis, Gene (2013), http://dx.doi.org/10.1016/j.gene.2013.06.090
44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 Q4 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95
Letter to Editor
R O
O
F
2
109 110 111 112
Q5 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134
135
Funding
146
The study was funded by in-house funding at Vallabhbhai Patel Chest Institute, University of Delhi, Delhi, India. Mr. Abhimanyu is the recipient of Senior Research Fellowship from Indian Council of Medical Research (ICMR) via project 5/8/5/14/ECD-I. MB is the recipient of ICMR Emeritus Medical scientist award vide letter no. 74/1/2012Pers(EMS).
147
Conflict of interest
153
E
the functional axis governing the tuberculosis pathogenesis in the population under study. Further investigations in a large sample size could use these genetic polymorphisms in conjunction to study the effect of these polymorphisms in tuberculosis. In conclusion, we may say that our results, albeit based on a limited sample, indicate that the variants rs2275913 (IL17A) and rs763780 (IL17F) are not associated with susceptibility to tuberculosis and the serum cytokine levels also don't differ between cases and controls as well as the polymorphisms don't affect the levels of serum cytokines in north Indians. We feel that further studies on the IL17 gene polymorphisms are warranted to clear its genetics in governing the susceptibility to TB.
T
C
E
107 108
R
105 106
R
103 104
O
101 102
C
99 100
HC, the difference was not statistically significant (Table 1). When the serum levels were compared between the corresponding genotypes of rs2275913 (IL17A) and rs763780 (IL17F) no significant association was observed (Table 1). The variant rs2275913 (IL17A) was not associated with TB in both north Indians and the Chinese population, but still there were differences in pattern of allelic distribution. While A was the minor allele in our study G was the minor allele in the study on the Chinese population. In the reported association for rs763780 (IL17F), the authors found that C allele was higher in both pulmonary and extrapulmonary groups and the CT genotype was differentially distributed among cases and controls. However, we could not find any such association. The difference between the study on Chinese population (Peng et al., 2013) and our study for IL17F variant rs763780 could be due to ethnicity difference between the two different studied populations. Case–control association studies are valuable tools to address the effect of genetic polymorphisms on disease pathogenesis and this could be a confounded by many factors (Abhimanyu et al., 2012). In our study we have accounted for all such factors as checking of the presence of a population substructure, carrying out multiple corrections and accounted for the Hardy–Weinberg equilibrium. It becomes pertinent here to point out that the current sample size had 84% power to detect an odds ratio of 2.0 with fisher exact test in place. Sample size, though not very low, was definitely a limitation for the study as the current sample size would miss any weak association or rare variant effect if any in the studied population. The non-association in this population could be due to the effect of natural selection, which has resulted in variable distribution of the alleles of IL17A/F gene in the north Indian population as compared to the Chinese population. The serum cytokine level association with genotypes although was not significant but could be accounted for considering that the crucial role of IL-17 is also complemented by the IL-12/IFN-γ axis, where a balance between the two is required for the regulation of inflammatory consequences following M.tb infection (Khader and Cooper, 2008). To that effect, We previously have shown that the polymorphisms in the IL12 gene are associated with risk in the north Indian population (Abhimanyu et al., 2012) and that the polymorphisms in the IL12 gene were associated with varying levels of serum IL-12 of the corresponding SNPs (Abhimanyu et al., 2011). So, it might be postulated that the IL-12 axis rather than the IL-17 axis is
N
97 98
U
96
D
P
Fig. 1. Representative electropherogram obtained for samples. A–C: for rs22275913 (IL17A). All three genotypes were obtained (i.e. GA, GG, AA); D–E: for rs763780 (IL17F) (only two genotypes seen CT, TT here shown as GA, AA these represent results obtained by the reverse primers). This was done for 10% of the samples and full concordance was seen between the genotypes identified by ARMS-PCR and sequencing.
We declare no conflict of interest exists.
136 137 138 139 140 141 142 143 144 145
148 149 150 151 152
154
References
155
Abhimanyu, et al., 2011. Differential serum cytokine levels are associated with cytokine gene polymorphisms in north Indians with active pulmonary tuberculosis. Infect. Genet. Evol. 11, 1015–1022. Abhimanyu, Bose, M., Jha, P., 2012. Indian Genome Variation Consortium. Footprints of genetic susceptibility to pulmonary tuberculosis: cytokine gene variants in north Indians. Indian J. Med. Res. 135, 763–770. Goldsack, L., Kirman, J.R., 2007. Half-truths and selective memory: interferon gamma, CD4(+) T cells and protective memory against tuberculosis. Tuberculosis (Edinb) 87, 465–473. Kawaguchi, M., Adachi, M., Oda, N., Kokubu, F., Huang, S.K., 2004. IL-17 cytokine family. J. Allergy Clin. Immunol. 114, 1265–1273 (quiz 1274). Khader, S.A., Cooper, A.M., 2008. IL-23 and IL-17 in tuberculosis. Cytokine 41, 79–83. Peng, R., Yue, J., Han, M., Zhao, Y., Liu, L., Liang, L., 2013. The IL-17F sequence variant is associated with susceptibility to tuberculosis. Gene 515, 229–232 (Epub 2012).
156 157 158 159 160 161 162 163 164 165 166 167 168 169
Please cite this article as: Abhimanyu, et al., Lack of association between IL17A and IL17F polymorphisms and related serum levels in north Indians with tuberculosis, Gene (2013), http://dx.doi.org/10.1016/j.gene.2013.06.090
N
C
O
Q1 Q6
t1:1 Q1 Table 1 t1:2 Q6 Showing the genotypic, allelic frequencies with association analysis of the variants of IL17 gene and the mean serum cytokine correlation among the genotypes in study subjects. t1:3 a
t1:4
Gene
rsID dbSNP
Subjects
Genotype/allele frequencies
t1:5 t1:6 t1:7 t1:8 t1:9 t1:10 t1:11 t1:12 t1:13 t1:14 t1:15 t1:16 t1:17 t1:18 t1:19 t1:20 t1:21
IL17A
rs22275913
PTB patients (n = 165)
GG 17 (10.3%)
t1:22 t1:23 t1:24
LNTB patients (n = 50) Controls (n = 130)
IL17F
rs763780
PTB patients (n = 165) LNTB patients (n = 50) Controls (n = 130)
Allelic association analysis
G 120 (41.1%)
23 (46%)
20 (40%)
22 (27%)
14 (11%)
63 (48.4%)
42 (32.3%)
91 (37.4%)
CC 0 (0%)
CT 6 (3.8%)
TT 159 (96.2%)
0 (0%)
3 (5.6%)
47 (94.4%)
0 (0%)
4 (2.7%)
126 (97.2%)
C 6 (1.8%) 3 (3%) 4 (1.5%)
Cytokine analysis
Chi-square
Odds ratio (CI)
P value
Subjects
Genotype
A 172 (58.9%)
0.576
1.2 (0.79–1.7)
0.48
PTB (n = 75)
62 (73%)
0.411
R
AA 43 (26.0%)
0 (0%)
GA 86 (52.1%)
R
E
C
T
0.8 (0.45–1.4)
T 324 (98.2%)
0.229
1.4 (0.34–6.0)
97 (97%)
0.662
2.1 (0.34–13)
149 (62.6%)
256 (98.5%)
0.5214
LNTB (n = 40) HC (n = 75)
E
D
IL-17 F PTB (n = 75)
0.63
P
0.416
LNTB (n = 40)
R O HC (n = 75)
Mean serum cytokine level(pg/ml)
Comparison of serum cytokine level among genotypes
P value
IL-17A GA (40) GG (11) AA(24) GA (21) AA(19) GA (40) GG (11) AA(24)
23 17 27 20 19 21 19 21
± ± ± ± ± ± ± ±
2.2 0.8 5.2 1.5 1.9 2.8 1.0 2.2
GA Vs GG GG Vs AA AA Vs GA GA Vs AA
P P P P
GA Vs GG GG Vs AA AA Vs GA
P N 0.05 P N 0.05 P N 0.05
TT (69) CT (6)b TT (37) CT (3)b TT (71) CT (4)b
25 27 16 12 14 16
± ± ± ± ± ±
6.3 3.7 0.79 0.059 0.73 2.61
TT Vs CT
P N 0.05
TT Vs CT
P N 0.05
TT Vs CT
P N 0.05
N N N N
0.05 0.05 0.05 0.05
Letter to Editor
Please cite this article as: Abhimanyu, et al., Lack of association between IL17A and IL17F polymorphisms and related serum levels in north Indians with tuberculosis, Gene (2013), http://dx.doi.org/10.1016/j.gene.2013.06.090
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PTB = pulmonary tuberculosis; LNTB = lymph node tuberculosis; SNP = single nucleotide polymorphism. a dbSNP, the SNP database (http://www.ncbi.nlm.nih.gov/projects/SNP); two tailed P b 0.05 was considered significant. For cytokine analysis the numbers in parentheses indicate the number of samples tested for that genotype; values are expressed as mean ± standard error. b For rs763780 C was minor allele and the number in parentheses indicate actual number of CT genotype observed in PTB, LNTB and Controls.
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170 171 172
Q1 173 174 175 176 177
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Letter to Editor
⁎Corresponding author. Tel.: +91 11 27667120; 178 fax: +91 11 27666549. 179 E-mail address: [email protected] (M. Bose). 180
Abhimanyu Mridula Bose⁎ Komal Mandira Varma-Basil Department of Microbiology, Vallabhbhai Patel Chest Institute, University of Delhi, New Delhi 110007, India
181
Available online xxxx 182
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Please cite this article as: Abhimanyu, et al., Lack of association between IL17A and IL17F polymorphisms and related serum levels in north Indians with tuberculosis, Gene (2013), http://dx.doi.org/10.1016/j.gene.2013.06.090