A significant association of the CTLA4 gene variants with the risk of autoimmune Graves’ disease in ethnic Kashmiri population

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A significant association of the CTLA4 gene variants with the risk of autoimmune Graves’ disease in ethnic Kashmiri population Faheem Shehjara, Dil-Afrozeb, Riaz A Misgara, Sajad A Malika, Bashir A Lawaya, a b

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Department of Endocrinology, Sher-I-Kashmir Institute of Medical Sciences (SKIMS), Srinagar, J&K, India Immunology and Molecular Medicine, SKIMS, Srinagar, J&K, India

A R T I C LE I N FO

A B S T R A C T

Keywords: CTLA4 Single nucleotide polymorphism Restriction fragment length polymorphism Graves’ disease

Graves’ disease (GD) is the commonest cause of hyperthyroidism in populations with adequate iodine intake. It results from an abnormality in the immune system, which produces unique antibodies causing over production of thyroid hormones and glandular hyperplasia in individuals with genetic susceptibility. The Cytotoxic Lymphocyte Associated Antigen-4 (CTLA4) gene product serves the important function of immunomodulation, thereby helping in maintenance of peripheral self-tolerance. Studies on the association of the CTLA4 SNPs with GD have shown variations in the results from different populations. Since no such study has been carried out in ethnic Kashmiri population, we aimed to study a possible association of the CTLA4 SNPs (+49 A/G, −318C/T, CT 60 A/G and −1661 A/G) with GD. A total of 285 individuals (135 patients with GD and 150 healthy individuals) were genotyped using PCR-RFLP method and the results showed statistically significant differences in genotypic and allelic frequencies of cases and controls for + 49 A/G SNP (p= < 0.001; OR = 5.14; CI = 2.17–12.19) and CT 60 A/G SNP (p = < 0.001; OR = 6.9; CI = 2.8–16.6), while −318C/T and −1661 A/G SNPs showed no significant association. We also studied the mRNA expression of the CTLA4 in patients with GD and healthy individuals by Real-Time PCR and found a decreased expression of the CTLA4 mRNA in PBMCs of patients with GD as compared to healthy controls with a −3.71-fold change. We conclude that the CTLA4 + 49 A/G and CT 60 A/G SNPs have a significant association with the risk of GD development in Kashmiri population and CTLA4 mRNA expression is significantly decreased in GD.

1. Introduction Graves’ disease (GD) is a multifactorial organ specific autoimmune disorder characterized by abnormal functioning of the thyroid gland. GD presents as, hyperthyroidism, diffuse goiter, Graves' opthalmopathy (GO) and occasionally Graves' dermopathy [1,2]. There are increased levels of circulating antibodies against thyroid-stimulating hormone receptor (TSHR) in patients with GD [3]. GD affects about 0.5% of the world population [2] but its incidence has not been documented in Indian population,. In our population (Kashmir region, North India), there is no data available but our observational study shows fairly a good strength of GD patients as we could register 135 cases from a single tertiary care hospital in a span of 2 years. The pathogenesis of GD is not completely understood, certain findings indicate a complex interaction between environmental, genetic, endogenous and local factors to be involved in its pathogenesis [4–6]. Although environmental factors, such as infection and stress, are among the important factors in the process of GD development in susceptible individuals, a study in twins

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has shown that genetic factors account for ~ 80% role in predisposition to GD [7]. Further, linkage and association studies describe both thyroid-specific genes and genes involved in immune regulation to be involved in the pathogenesis of GD [8]. The immune- regulatory genes involved in GD pathogenesis are also shared by some other autoimmune diseases these genes are HLA-DR, CD40, CTLA-4, PTPN22, thyroglobulin and TSH receptor [9]. Recently we reported an association between Interleukin-1β [10] and FoxP3 [11] polymorphisms with GD in Kashmiri population. Among these genes the most extensively explored is Cytotoxic T-lymphocyte-associated antigen-4 (CTLA4) gene. The CTLA4 gene, located on chromosome 2q33, encodes a co-stimulatory molecule, which down regulates T-cell activation [12]. The CTLA-4 gene product serves the important function of immunomodulation, thereby helping in the maintenance of peripheral self-tolerance. An association between certain autoimmune diseases and the molecular variants of CTLA4 has been reported [13–16]. GD is associated with various functional genetic variants within the CTLA4 gene which include the + 49 A/G (rs231775) single nucleotide

Corresponding author at: Dept. of Endocrinology, Sher-I-Kashmir Institute of Medical Sciences, Srinagar, Kashmir, India. E-mail address: [email protected] (B.A. Laway).

https://doi.org/10.1016/j.cellimm.2019.103995 Received 19 July 2019; Received in revised form 8 October 2019; Accepted 19 October 2019 0008-8749/ © 2019 Elsevier Inc. All rights reserved.

Please cite this article as: Faheem Shehjar, et al., Cellular Immunology, https://doi.org/10.1016/j.cellimm.2019.103995

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50 ng genomic DNA template, 1X PCR buffer (Biotools, B & M Labs, S.A. Madrid-Spain) with 2 mM MgCl2, 0.4 μmol of each primer (SigmaAldrich Co. LLC·USA), 0.20 mM dNTPs (Biotools, B & M Labs, S.A. Madrid-Spain), and 1 U DNA polymerase (Biotools, B & M Labs, S.A. Madrid-Spain). For PCR amplification, the standard program was used as follows: one initial denaturation step at 94 °C for 4 min, followed by 30 cycles of denaturation for 45 s at 94 °C, 30 cycles annealing for 45sec at 57 °C (CTLA4 + 49A/G) (rs231775), 60 °C (CTLA4 −318C/T) (rs5742909) and 59 °C (CTLA4 CT60 A/G) (rs3087243) , and 30 cycles of extension for 30sec at 72 °C, followed by a final elongation cycle at 72 °C for 7 min (Supplementary Table 1). For RFLP, 10 µl of PCR products of CTLA4 + 49A/G (rs231775), CTLA4 −318C/T (rs5742909), CTLA-4 CT60AG (rs3087243) and CTLA-4 −1661A > G (rs4553808) polymorphisms were digested with enzymes Bbv1 (Fermentas Thermo Fisher Scientific Inc. Massachusetts, USA) (1 U at 65°Cfor 16 h), Mse1 (Fermentas Thermo Fisher Scientific Inc. Massachusetts, USA) (1 U at 37 °C for 3 h), Mae11 (Fermentas Thermo Fisher Scientific Inc. Massachusetts, USA) (1 U at 65°Cfor 16 h) and Mse1 (Fermentas Thermo Fisher Scientific Inc. Massachusetts, USA) (1 U at 37 °C for 3 h) respectively. DNA amplicons, as well as the digested products (Supplementary Table 2), were electrophoresed through 3% agarose gel (Genie, Bangalore, India) for resolution. The genotypes of 20% of the samples were reassessed in double-blind manner by two independent researchers, to confirm the results.

polymorphism (SNP) (that leads to substitution of alanine in place of threonine in codon 17 of the signal peptide of CTLA-4) [9–15] and (AT) n dinucleotide polymorphism at the 3´ untranslated region [10,12–14]. Among the CTLA-4 promoter polymorphisms, a cytosine-to-thymidine substitution at position −318 [C/T ( −3 1 8)] represents the most frequently evaluated polymorphic marker in genetic association analyses [9]. Some studies have shown an association between promoter SNP (−318C/T) (rs5742909) and GD while others do not. GD was associated with this SNP in Germans, Canadians and Koreans [10,11], whereas in Hong Kong Chinese patients and UK and US Caucasians no such association was found [5,12,13]. CT 60 (rs30807243) is one of the functional polymorphisms of CTLA4. The G allele of CT 60, for which the expression of soluble CTLA4 (sCTLA4) is lower than that with the A allele [14]. CTLA4 −1661 A/G has been shown to be associated with many autoimmune diseases. The CTLA4 −1661 A/G dimorphism alters the potential response element for myocyte enhancer factor 2 (MEF2). There are two separate mechanisms of down regulation of the T cell responses by CTLA4. First one is CTLA4-mediated negative signaling in response to T cell receptor activation [15]. This occurs in the early stages of an immune response; CTLA4 and B7 are scarcely expressed and utilizes the cytoplasmic tail of CTLA4. The other mechanism involves competition between CTLA4 and CD28 for B7 binding. The levels of CTLA4 expressed on surface dictate this phenomenon [16]. The interaction between CTLA4 and B7 has an indispensable role in maintaining the peripheral immune tolerance status, and therefore in autoimmunity [17]. Considering the importance of the CTLA4 gene polymorphisms in GD development and the variable results of association between the SNPs of CTLA4 and GD in different ethnic populations, it could be concluded that role of these polymorphism vary considerably from population to population. Since no such study has been reported from our ethnically conserved population, it is of great importance to conduct this study. Keeping in view the above mentioned points it is obvious that CTLA4 is one of the most important genes associated with autoimmune diseases, hence we selected four common SNPs of CTLA4 and analyzed their role in the development of GD using a conventional PCR-RFLP approach followed by the comparative expression of CTLA4 mRNA between patients with GD and healthy individuals using Real-Time PCR.

2.3. Analysis of CTLA4 mRNA expression Newly diagnosed 30 GD untreated cases were selected for mRNA expression according to differential status of genotypes of CTLA4. Total RNA was extracted by using TRIZOL (Sigma Aldrich, USA) from 30 GD patients and 30 healthy control PBMCs.. Integrity of the mRNA was checked on 1% agarose gel and quantified at 260/280 ratio. RNA was converted to cDNA by using first strand cDNA synthesis kit according to manufactures protocol (Fermentas, USA). Dilution of the cDNA was performed to get uniform quantity of cDNA in all samples. For CTLA4 primer sequence was as follows forward CTLA4 5′-CTACCTGGGCATA GGCAACG-3′ and reverse 5′CCCCGAACTAACTGCTGCAA-3 [18] and for glyceraldehyde 3-phosphate dehydrogenase, the primers were as follows: forward 5′-GATCCGCATAATCTGCATGGT-3′ and reverse 5′-GATCCGCATAATCTGCATGGT-3′. Quantitative Real time PCR (Agilent Biotechnologies, Germany) was performed for the detection of CTLA4 mRNA by recruiting Applied Biosystems Inc StepOne software v2.0. PCR was performed containing Maxima® SYBR Green qPCR Master Mix (2X) and all the samples (unknown and standards) were run in triplicates and accompanied by non template control (NTC). Thermal cycling conditions included 40 cycles at 95 °C for 15 s, 60 °C for 1 min/ cycle. The melting curves of all final real time PCR products were analyzed for determination of genuine products and contamination of non specific products and primer dimer. All amplified products of real time polymerase chain reaction were subjected for separation on 2% agarose gel electrophoresis for ensuring the correct amplification products. Delta CT (ΔCT) method was used to check the CTLA-4 mRNA expression in patients with GD and healthy individuals by normalization against glyceraldehyde 3-phosphate dehydrogenase used as a reference gene.

2. Material and methods 2.1. Participants A total of 135 patients with GD (33 men and 102 women) between the age of 14 and 68 years (average = 37.97 years) were enrolled in this study. Diagnosis of GD was established on the basis of guidelines by American thyroid association that included clinical parameters of thyrotoxicosis (history of marked weight loss, presence of diffuse goiter, presence of skin or nail changes), diffusely increased 99mTechnetium pertechnetate uptake on thyroid scintigraphy and positive TSH receptor antibody. Information was collected on the patient's age at onset, history of smoking, size of thyroid gland, presence of thyroid eye disease, serum thyroid hormone levels and the titers of TSHR antibodies. A group of 150 age and sex matched healthy volunteers (42 men and 108 women), who were euthyroid and had no personal or family history of Graves' disease or other autoimmune diseases, served as controls. Informed consent was obtained from each individual who participated in the study.

2.4. Statistical analysis For statistical analysis, the genotype and allelic frequency distributions of polymorphisms in the control and Graves' disease patient groups were compared using the χ2 test. When the assumption of the χ2 test was violated (i.e., when one cell had an expected count of < 1, or > 20% of the cells had an expected count of < 5), Fisher's exact test was used. Odds ratios (ORs) with 95% confidence intervals (CIs) were determined for the disease susceptibility of specific genotypes and alleles. Results were considered statistically significant when the

2.2. Sample collection and molecular analysis Five milliliter of peripheral blood from the patients with GD and healthy individuals was collected in EDTA vials. HiPurA™ Blood Genomic DNA Miniprep Purification Kit (HiMedia Laboratories Pvt. Ltd., Mumbai, India) was used for isolating the DNA. Polymerase chain reaction (PCR) was carried out in a final volume of 25 μl containing 2

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probability of findings occurring by chance was < 5% (P < 0.05). The statistical analysis was done with SPSS v 20 and online software via http://vassarstats.net.

group than in the control group and was found significantly associated with the risk of GD when compared with CT60 A allele (OR-2.21 (1.56– 3.13), P < 0.001). CTLA4 CT60 GG genotype showed significant association only with smoking (P = 0.01), while other clinical parameters showed no significant association with CTLA4 CT60 (rs3087243) genotype (P > 0.05) (Table 4). The distribution of CTLA4 −318 (rs5742909) CC, CT and TT genotypes in patients with GD was 91.85%, 8.15% and 0% respectively, while in controls the distribution of CTLA4 −318 (rs5742909) CC, CT and TT was 92.67%, 7.33% and 0% respectively (Table 1). There were no statistically significant differences in the case and control genotype frequencies (OR-1.12 (95% CI 0.47–2.68), P = 0.82). Also, no statistically significant differences were observed in allelic distribution (OR1.12 (0.48–2.62), P = 0.83). Further, there was no correlation between the genotypes of CTLA4 −318 (rs5742909) C/T and the clinical parameters (Table 3). The distribution of CTLA4 −1661 A/G (rs4553808) AA, AG and GG genotypes in patients with GD was 85.93%, 9.63% and 4.44% respectively, while in controls the distribution of CTLA4 −1661 A/G (rs4553808) AA, AG and GG was 88%, 10.67% and 1.33% respectively (Table 1). There were no statistically significant differences in the case and control genotype frequencies (OR-0.92 (95% CI 0.42–2.0), P = 1). Also, no statistically significant differences were observed in allelic distribution (OR-1.12 (0.48–2.62), P = 0.83) (Table 1). Further, there was no correlation between the genotypes of CTLA4 −1661 A/G (rs4553808) and the clinical parameters. The genotypic association of all four SNPs with GD is represented in bar diagrams (Supplementary Fig. 1). The CTLA4 −318 & CTLA4 −1661 A/G didn’t show any significant association under different models (Table 2), whereas CTLA4 + 49 A/G showed significant association in co-dominant (p = 0.0001), dominant (p = 0.04) and recessive (p = 0.0001) models. Similarly, CTLA4 CT60 A/G also showed significant association in co-dominant (p = 0.0001), dominant (p = 0.0002) and recessive (p = 0.0001) models (Table 2). The lack of association for −318C/T & −1661 A/G is not definitive because these variants are rare in our population and there is little

3. Results A total of 285 individuals (135 GD cases and 150 healthy controls) were included in our study. The patients consisted of 102 females and 33 males (Female/Male ratio = 3.09) and the control subjects consisted of 108 females and 42 males (Female/Male ratio = 2.57). Mean age in patient group was ± 37.97 years. No significant gender or age-related differences were observed between the groups (P > 0.05) (Supplementary Table 3). Among 135 patients with GD, ophthalmopathy was positive in 22 (16.30%) patients. Hardy–Weinberg equilibrium (HWE) analysis was performed. All SNPs were in HWE in the controls (CTLA4 − 318C/T, P = 0.64; CTLA4 + 49A/G, P = 0.15; CTLA4 CT60A/G, P = 0.10). The distribution of CTLA4 + 49 (rs231775) AA , AG and GG genotypes in patients with GD was 37.78%, 41.48% and 20.74% respectively, while in healthy controls the genotypic distribution of CTLA4 + 49 (rs231775) AA , AG and GG was 50%, 44.67% and 5.33% respectively (Table 1). The CTLA4 + 49 GG genotype was associated with an increased risk of GD development compared with CTLA4 + 49 AA genotype (OR-5.14 (95% CI 2.17–12.19), p < 0.001). CTLA4 + 49 GG genotype did not show any significant association with various clinical parameters such as age, gender, Orbitopathy, family history and dwelling (P > 0.05) while smoking showed significant association with the + 49 GG genotype (P = 0.04) (Table 3). The distribution of CTLA4 CT60 (rs3087243) AA, AG and GG genotypes in patients with GD was 27.41%, 51.85% and 20.74% respectively while in controls the distribution of CTLA4 CT60 AA, AG and GG was 48.67%, 46% and GG 5.33% respectively (Table 1). The CTLA-4 CT60 AG and GG genotypes were associated with an increased risk for GD development compared with the CTLA4 CT60 AA genotype (OR-2.0 (95% CI 1.19–3.35), P = 0.009) and (OR 6.9 (2.8–16.6), P < 0.001) respectively. CTLA4 CT60 G allele occurred more frequently in GD Table 1 Genotypic frequencies of CTLA-4 polymorphisms in GD cases and controls. Genotype CTLA-4 −318C/T CC CT Allele (2 N) C T CTLA-4 + 49A/G AA AG GG Allele (2 N) A G CTLA-4 CT60 A/G AA AG GG Allele (2 N) A G CTLA-4 −1661A/G AA AG GG Allele (2 N) A G

GD Casesn = 135 (%)

Controlsn = 150 (%)

OR (95% CI); P

124(91.85) 11(08.15) 270 259 (95.93) 11 (04.07)

139(92.67) 11(07.33) 300 289(96.33) 11(03.67)

1.0 (Reference) 1.12(0.47–2.68);0.82

51(37.78) 56(41.48) 28(20.74) 270 158(58.52) 112(41.48)

75(50.00) 67(44.67) 08(05.33) 300 217(72.33) 83(27.67)

1.0 (Reference) 0.91(0.74–2.03);0.44 5.14(2.17–12.19); < 0.001

37(27.41) 70(51.85) 28(20.74) 270 144(53.33) 126(46.67)

73(48.67) 69(46) 08(5.33) 300 215(71.67) 85(28.33)

1.0 (Reference) 2.00(1.19–3.35);0.009 6.9 (2.8–16.6); < 0.001

116(85.93) 13(9.63) 06(4.44) 270 245(90.74) 25(9.26)

132(88)16(10.67) 02(1.33) 300 280(93.33) 20(6.67)

3

1.12(0.48–2.62); 0.83

1.85(1.30–2.63); < 0.001

1.0 (Reference) 2.21(1.56– 3.13); < 0.001

1.0 (Reference) 0.92(0.42–2.0); 1 3.4(0.67–17.2); 0.15 1.0 (Reference) 1.42 (0.77– 2.63); 0.27

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Table 2 Genotypic distribution of gene polymorphism under different models. SNP

Model

Genotype

Cases

Controls

p-Value

OR

CTLA-4 −318C/T

Co-Dominant

CC CT TT CC CT + TT CT + CC TT AA AG

124(91.85) 11(08.15)0

139(92.67) 11(07.33)0

Ref 0.82

1.12 (0.46–2.67)

124(91.85) 11(08.15) 135 (1 0 0) 0 51(37.78) 56(41.48) 28(20.74)

139(92.67) 11(07.33) 150 (1 0 0) 0 75(50) 67(44.67) 08(5.33)

Ref 0.82 –

1.12 (0.46–2.67) –

Ref 0.45 0.0001

1.22 (0.74–2.03) 5.14 (2.17–12.19)

AA AG + GG AG + AA GG AA AG GG AA AG + GG AG + AA GG AA AG GG AA AG + GG AG + AA GG

51(37.78) 84(62.22) 107(79.26) 28(35) 37(27.41) 70(51.85) 28(20.74) 37(27.41) 98 (75.59) 107 (79.26) 28(20.74) 116(85.93) 13(9.63) 06(4.44) 116(85.93) 19(14.07) 129(95.56) 06(4.44)

75(50) 75(50) 142(94.67) 08(20) 73(48.67) 69(46) 08(5.33) 73(48.67) 77 (51.33) 142 (94.67 08(5.33) 132(88) 16(10.67) 02(1.33) 132(88) 18(12) 148(98.67) 02(1.33)

Ref 0.04 Ref 0.0001 Ref 0.009 < 0.0001 Ref 0.0002 Ref 0.0001 Ref 1 0.15 Ref 0.72 Ref 0.15

Dominant Recessive CTLA-4 + 49A/G

Co-Dominant

Dominant Recessive CTLA-4 CT60 A/G

Co-Dominant

Dominant Recessive CTLA-4 −1661A/G

Co-Dominant

Dominant Recessive

1.64 (1.04–2.64) 4.64 (2.03–10.59) 2.0 (1.19–3.35) 6.90(2.86–16.64) 2.5 (1.52–4.12) 4.64 (2.0–10.60) 0.9 (0.42–2.0) 3.4(0.67–17.24) 1.2 (0.60–2.39) 3.45 (0.68–17.35)

However, there was no statistically significant difference in ΔCT values of GD patients with respect to the CTLA-4 SNPs (Table 6). The association of clinicopathological variables and CTLA4 mRNA expression in Graves’ disease showed no statistical significance with any of the clinical parameters studied (Table 7)

power to detect an association. CTLA4 mRNA expression in peripheral blood of GD patients and Healthy controls by qRT-PCR: In the present study, the analysis of total CTLA4 mRNA was detected and quantified in 30 GD patients and 30 healthy control PBMCs. The fold change in expression of CTLA4 mRNA expression in GD compared to normal PBMCs was calculated by Delta CT method. The mRNA expression was checked by ΔCT method with expression ratio of (amount of CTLA-4 mRNA/amount of GAPDH mRNA) by applying the formula ΔCT = (CT GAPDH- CT CTLA-4 mRNA). We found a decreased expression of CTLA-4 mRNA in PBMCs of patients with GD as compared to healthy controls with a −3.71-fold change (Table 5).

4. Discussion To our knowledge this is the first analysis investigating CTLA4 polymorphisms in patients with GD in our population. The main finding of this study is that CTLA4 + 49 GG genotype and CTLA4 CT60 GG genotype were significantly associated with the risk of GD.

Table 3 Association between CTLA-4 −318C/T and CTLA-4 + 49A/G polymorphisms and various clinical and laboratory features of GD patients. Cases (n = 135) Parameter CTLA-4 −318C/T

CTLA-4 + 49A/G

CC(1 2 4)

CT(11)

p-value

AA(51)

AG(56)

GG(28)

p-value

91(91.92) 33(91.67)

08(08.08) 03(08.33)

1

40(40.04) 11(30.56)

37(37.37) 19(52.78)

22(22.22) 06(16.67)

0.0047

94(92.16) 30(90.91)

08(07.84) 03(09.09)

0.07

41(40.19) 11(33.33)

42(41.17) 13(39.39)

19(18.63) 09(27.27)

0.70

28(90.32) 96(92.31)

03(09.68) 08(07.69)

0.00

08(25.80) 29(27.88)

20(64.52) 50(48.08)

03(9.68) 25(24.04)

0.51

19(86.36) 105(92.92)

03(13.64) 08(07.08)

0.38

09(40.91) 42(37.17)

13(59.09) 43(38.05)

0 28(24.78)

0.67

18(90.00) 106(92.17)

02(10.00) 09(07.83)

1

0(0) 51(44.35)

14(70) 42(36.52)

06(30.00) 22(19.13)

0.04

94(94.95) 30(83.33)

05(05.05) 06(16.67)

0.03

34(34.35) 17(47.22)

40(40.40) 16(44.44)

25(25.25) 03(8.34)

0.60

Age ≤40 years(99) > 40 years(36) Gender Females(1 0 2) Males(33) Family History Positive(31) Negative(1 0 4) Orbitopathy Positive(22) Negative(1 1 3) Smoking Yes(20) No(1 1 5) Dwelling Rural(99) Urban(36)

4

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Table 4 Association between CTLA-4 CT60 A/G polymorphisms and various clinical and laboratory features of GD patients.

Table 7 The association of clinico-pathological variables and CTLA-4 mRNA expression in Graves’ disease:

Cases (n = 135) Parameter

CTLA-4 mRNA Expression (mean ± SD)

Number

p -value

≤40 years > 40 years

3.43 ± 0.63 3.72 ± 0.65

23 07

0.8

Male Female

3.46 ± 0.65 3.42 ± 0.63

07 23

0.5

3.43 ± 0.65 3.46 ± 0.65

26 04

0.58

3.57 ± 0.30 3.43 ± 0.63

20 10

0.48

3.40 ± 0.61 3.43 ± 0.63

08 22

0.8

3.44 ± 0.65 3.43 ± 0.63

05 25

0.06

Parameters CTLA-4 CT60 A/G AA(37)

AG(70)

GG(28)

Age

p-value

Age ≤40 years(99) > 40 years(36) Gender Females(1 0 2) Males(33) Family History Positive(31) Negative(1 0 4) Orbitopathy Positive(22) Negative(1 1 3) Smoking Yes(20) No(1 1 5) Dwelling Rural(99) Urban(36)

32(32.32) 05(13.89)

50(50.50) 20(55.56)

17(17.17) 11(30.55)

0.05

31(30.39) 06(18.18)

50(49.02) 20(60.61)

21(20.59) 07(21.21)

0.36

08(25.80) 29(27.88)

20(64.52) 50(48.08)

03(09.68) 25(24.04)

0.16

06(27.27) 31(27.43)

13(59.09) 57(50.44)

03(13.64) 25(22.13)

0.63

Sex

Smoking status Non-Smoker Smoker Dwelling Rural Urban Family History Yes No Orbitopathy Yes No

0.01 00(00.00) 37(32.17)

14(70.00) 56(48.70)

06(30.00) 22(19.13)

28(28.28) 09(25.00)

48(48.48) 22(61.11)

23(23.24) 05(13.89)

0.36

CI = 1.30–2.63) respectively, indicating that there is a five-fold risk of developing GD in individuals with + 49 (rs231775)GG genotype as compared to individuals with AA genotype, and hence the carriers of the rs231775 G allele have a strong risk for GD predisposition (Table 1). Despite the fact that function of CTLA-4 protein is probably unaffected by exon 1 + 49 (rs231775) SNP, it might have an impact on the level or pattern of protein expression. The G allele is related to diminished control of T cell multiplication and in this way adds to the pathogenesis of GD and apparently of other immune system diseases [52]. Furthermore, this change from alanine (Ala) to threonine (Thr) in the 17-codon 49-site AG of CTLA4 gene results in error-prone handling of CTLA4 in the endoplasmic reticulum, leading to impaired glycosylation reaction and reduced expression of CTLA-4 protein on the T cell surface [42,53]. Due to this reduced expression of CTLA4 the down regulation of T cell function will be impaired, resulting in hyperactivity which may ultimately lead to development of GD [31–41]. This study is consistent with those of Veeramuthumari et al. (South Indian), Chong et al. (Chinese), Yung et al. (Chinese) and Ting et al. (Taiwanese), in these mentioned studies the authors have shown that ‘G’ allele of + 49 AG (rs231775) SNP is associated with an increased risk of developing GD [54–57]. In our study, the frequencies of CT60 (rs3087243) GG genotype and G allele were significantly higher in patients with GD as compared to healthy controls. The statistical analysis indicated that the difference genotypic and allelic frequencies of CTLA4 CT60 (rs3087243) polymorphism in cases and controls to be significant (p < 0.001; OR = 6.9; CI = 2.8–16.6) and (p < 0.001; OR = 2.21; CI = 1.56– 3.13) respectively, indicating that there is almost seven-fold risk of developing GD in individuals with CT60 (rs3087243) GG genotype as compared to individuals with CT60 (rs3087243) AA genotype, therefore the carriers of CT60 (rs3087243) G allele have a strong risk for GD predisposition (Table 1). The CT60 SNP is potentially important because it may be associated with an alteration in the ratio of splice forms of the CTLA4 gene and this ratio may affect disease susceptibility, as -reported by Ueda et al. They recognized various SNPs in the region and have identified that the most related marker was SNP CT60 (rs3087243) [14]. Albeit found downstream of poly (A) termination site, this SNP is fascinating, in light of the fact that it may influence the termination of the transcript and therefore splicing of CTLA-4; in abovementioned study, the CT60 genotype was related with contrasts in proportion of two alternative RNA splicing forms: a full-length form, flCTLA-4, and a soluble form, sCTLA4. Subjects with the protective CT60 AA genotype had a higher percentage of sCTLA4 than those with

Table 5 CTLA-4 mRNA expression in peripheral blood samples of GD patients and healthy controls. Gene

CTLA-4

ΔCt Controls (n = 30)

ΔCt GD (n = 30)

ΔΔCt

Fold (2)

Change pvalue

5.45 ± 0.59

3.56 ± 0.63

−3.71

0.007

Table 6 CTLA-4 mRNA expression of GD patients in relation to CTLA-4 + 49 and CTLA4 CT60 genotypes Genotypes

CTLA-4 + 49/CT60 AA/AA AG/AG GG/GG

CTLA-4mRNA Expression (mean ± SD)

Number

p-value

3.56 ± 1.15 3.15 ± 0.33 3.97 ± 1.15

16 08 06

1(Ref.) 0.33 0.46

Cytotoxic T-lymphocyte associated protein 4 is a trans-membrane regulatory protein encoded by the CTLA4 gene and is responsible for down regulating the function of activated T cells [19]. Many single nucleotide polymorphisms have been described in the CTLA4 gene, including C/T –318 (rs5742909) [20], A/G + 49 A/G (rs231775) [21], (AT)n repeat in the 3′- UTR [22], and three SNPs CT60 (rs3087243A/ G), JO31 and JO30 in the 6.1-kb 3′ non-coding region have been shown to be associated with many autoimmune disorders [14,23–29] SNPs + 49AG and CT60 have been most widely studied in many AIDs [15]. These two SNPs are associated with thyroid antibody production [45,46], GD relapse [44], Graves’ ophthalmopathy [47,48] and susceptibility to GD [22,49] and HD [50,51]. In the present case-control study, we investigated the association between three SNPs of CTLA4 gene and GD in Kashmiri population. We found statistically significant differences in the genotypic and allelic frequencies between cases and controls in case of CTLA-4 + 49A/G (rs231775) SNP (p < 0.001; OR = 5.14; CI = 2.17–12.19) and (p < 0.001; OR = 1.85; 5

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small population size and this augments the need for the study to be replicated in larger cohort in order to ascertain our results.

the susceptible GG genotype. The soluble form of CTLA-4 is shown to be translated, secreted, and present in human serum. It can bind the CD80/ 86 molecules, and recombinant sCTLA-4 causes suppression of T-cell proliferation in vitro, so it can be proposed that decrease of this form could prompt an inefficacious restriction of the immune response, predisposing towards an autoimmune disorder. More functional studies are required to elucidate the role of soluble form of CTLA4 in the immunoregulation [14]. The CT60 SNP, which is exhibited to be mostly associated with GD and HT in the Caucasian population [14], is at the 3′-UTR of the CTLA-4 gene. The association between the CT60 SNP and the autoimmune disorders, which have been established, differed among patients from various ethnicities. A relation between CT60 and SLE was reported by Torres et al. in Spanish population [43]. Ban et al., proposed that CT60 SNP in the CTLA4 gene may play a part in the development of AITD in the Japanese population [22]. An association between the onset of GD and the GG genotype and G allele in CT60 was shown by Weng et al. in Taiwanese population [49]. Contrary to our report, the investigation of Pastuszak et al. demonstrated no relationship between CT60 AG and GD patients in the Polish population [58]. More recently Fang et al. demonstrated that GG genotype of CT60 might be related to the onset of GD in the Han population of Southern China [5]. Likewise, the frequency of the G allele was significantly associated with GD in our study, which is in agreement with the findings in other ethnic groups from several researchers [49,58–60]. These studies reporting the association between G allele and GD further support our hypothesis that the CT60 of CTLA4 appears to be associated with the increased risk of developing GD in our population. In the clinico-pathological assessment of the CT60 SNP, CTLA-4 CT60 GG genotype showed statistically significant association only with smoking (P < 0.05), while other clinical parameters showed no significant association (Table 1). In case of CTLA4 promoter −318C/T (rs5742909) SNP, we didn’t observe any statistically significant difference in the CC and CT genotypes between patients with GD and healthy controls, additionally TT genotype was completely absent in both cases and controls in our study group. The statistical analysis indicated that the genotypic and allelic frequencies of CTLA4-318 (rs5742909) C/T polymorphism in cases and controls were statistically insignificant. One study focusing on this polymorphism and CTLA4 protein expression showed that the CTLA4 −318 T allele is associated with higher promoter activity and thus reduced autoimmunity [61]. However, most published studies do not show any association between CTLA4 −318CT and GD [27,30,55,62]. The homozygous TT genotype is absent in our data set and either absent or rare in most of the other populations [23,27,63]. Therefore, this SNP might not be important for susceptibility to AITD including GD. We also investigated CTLA4 mRNA expression in patients with GD and healthy controls. We observed that the CTLA4 mRNA expression was significantly decreased in GD PBMCs as compared to those of healthy controls. However, we did not find any significant association of CTLA4 SNP genotypes with the expression of CTLA4 mRNA in patients with GD. We investigated CTLA4 mRNA expression using qRTPCR. The total CTLA4 mRNA was expressed at significantly lower levels (−3.71-fold) in PBMCs of patients with GD as compared to those of healthy controls (P = 0.007) (Table 5). The diminished mRNA expression of CTLA4 in GD may indicate inadequate T regulatory function of this molecule in GD. The majority of CTLA4 molecules occur within the cytoplasm and can be quickly mobilized from intracellular store compartments to the site of T cell receptor engagement on the cell surface [64]. Hence, low concentrations of intracellular CTLA4 may correlate with decreased cell surface expression of CTLA4 and therefore with reduced negative control of T cell proliferation, ultimately leading to T cell hyper responsiveness and predisposition to the AITDs including GD. We conclude that CTLA4 + 49 A/G and CT60 A/G SNPs have a significant association with the risk of GD development in Kashmiri population and CTLA4 mRNA expression is significantly decreased in GD. Certain limitations in our study include small sample size due to

5. Conclusions We conclude that CTLA4+49 A/G and CT60 A/G SNPs have a significant association with the risk of GD development in Kashmiri population and CTLA4 mRNA expression is significantly decreased in GD. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements We are grateful to the Head and Staff of Department of Endocrinology, SKIMS, who helped us in the sample procurement. Ethical approval. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/ or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Funding The study was supported by the research grant received from the SKIMS. Informed consent Informed consent was obtained from all individual participants included in the study. Furthermore parental consent was obtained for all participants under the age of 16. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.cellimm.2019.103995. References [1] O. Khalilzadeh, M. Anvari, A. Esteghamati, F. Momen-Heravi, M. Mahmoudi, A. Rashidi, H.M. Amiri, M. Ranjbar, S. Tabataba-Vakili, A. Amirzargar, The interleukin-1 family gene polymorphisms and Graves’ disease, Ann. Endocrinol. 71 (2010) 281–285, https://doi.org/10.1016/j.ando.2010.01.005. [2] A.P. Weetman, Graves’ Disease, N. Engl. J. Med. 343 (2000) 1236–1248, https:// doi.org/10.1056/NEJM200010263431707. [3] M. Giménez-Barcons, R. Colobran, A. Gómez-Pau, A. Marín-Sánchez, A. Casteràs, G. Obiols, R. Abella, J. Fernández-Doblas, M. Tonacchera, A. Lucas-Martín, et al., Graves’ Disease TSHR-stimulating antibodies (TSAbs) induce the activation of immature thymocytes: a clue to the riddle of TSAbs generation? J. Immunol. 194 (2015) 4199–4206, https://doi.org/10.4049/jimmunol.1500183. [4] T.H. Brix, K. Christensen, N.V. Holm, B. Harvald, L. Hegedüs, A population-based study of Graves’ disease in Danish twins, Clin. Endocrinol. (Oxf) 48 (1998) 397–400. [5] W. Fang, Z. Zhang, J. Zhang, Z. Cai, H. Zeng, M. Chen, J. Huang, Association of the CTLA4 gene CT60/rs3087243 single-nucleotide polymorphisms with Graves’ disease, Biomed. Rep. (2015), https://doi.org/10.3892/br.2015.493. [6] R. Płoski, K. Szymański, T. Bednarczuk, The Genetic Basis of Graves’ Disease, Curr. Genom. 12 (2011) 542–563, https://doi.org/10.2174/138920211798120772. [7] Y. Tomer, Mechanisms of autoimmune thyroid diseases: from genetics to epigenetics, Annu. Rev. Pathol. Mech. Dis. 9 (2014) 147–156, https://doi.org/10.1146/ annurev-pathol-012513-104713. [8] D.C. Eschler, A. Hasham, Y. Tomer, Cutting edge: the etiology of autoimmune thyroid diseases, Clin. Rev. Allergy Immunol. 41 (2011) 190–197, https://doi.org/ 10.1007/s12016-010-8245-8. [9] F. Shehjar, D. Afroze, R.A. Misgar, S.A. Malik, B.A. Laway, Association of polymorphic variants of IL-1β and IL-1RN genes in the development of Graves’ disease in Kashmiri population (North India), Hum. Immunol. 79 (2018) 228–232, https:// doi.org/10.1016/j.humimm.2018.02.006. [10] F. Shehjar, D. Afroze, R.A. Misgar, S.A. Malik, B.A. Laway, Association of FoxP3 promoter polymorphisms with the risk of Graves’ disease in ethnic Kashmiri population, Gene (2018), https://doi.org/10.1016/j.gene.2018.06.023. [11] J.L. Riley, The CD28 family: a T-cell rheostat for therapeutic control of T-cell

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