Interaction of HLA-DRB1* alleles and CTLA4 (+ 49 AG) gene polymorphism in Autoimmune Thyroid Disease

Gene 642 (2018) 430–438

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Research paper

Interaction of HLA-DRB1* alleles and CTLA4 (+ 49 AG) gene polymorphism in Autoimmune Thyroid Disease

T

Ramgopal Sivanadhama, Rathika Chinniaha, Padma Malini Ravia, Murali Vijayanb, Arun Kannanc, ⁎ Kamaludeen Mohamed Nainard, Balakrishnan Karuppiaha, a

Department of Immunology, School of Biological Sciences, Madurai Kamaraj University, Madurai 625021, India Texas Tech University Health Sciences Center, Lubbock, TX 79430, United States c Endocrinology & Diabetology, Madurai Institute of Diabetes and Endocrine Practice and Research, Madurai 625001, India d Abban Hospital, Madurai 625020, India b

A R T I C L E I N F O

A B S T R A C T

Keywords: Autoimmune Thyroid Disease Human leukocyte antigen Hashimoto's thyroiditis Graves' disease DRB1* CTLA4

Autoimmune Thyroid Diseases (AITDs), including Hashimoto's thyroiditis (HT) and Graves' disease (GD), arise by the complex interaction of genes and environmental factors. The aim of present study was to study the susceptible associations of HLA-DRB1* alleles and CTLA4 +49 AG polymorphism in AITD in south India. AITD patients (n = 235; HT = 180; GD = 55) and age/sex matched healthy controls (n, 235) were enrolled to type HLA-DRB1* alleles and ‘CTLA4 +49 AG’ by PCR-SSP and PCR-RFLP methods respectively. Analysis revealed CTLA4 +49 ‘GG’ genotype was increased significantly in patients (PL: p = 8.7 × 10− 8; HT: p = 9.3 × 10− 6; GD: p = 0.006). Decreased frequencies of ‘AA’ genotype was observed in patients (PL: p = 9.4 × 10− 6; HT: p = 0.008; GD: p = 9.0 × 10− 6). Increased frequencies were observed for HLA alleles DRB1*12 (PL: p = 1.42 × 10− 10; HT: p = 5.75 × 10− 8; GD: p = 0.002) and DRB1*11 (PL: p = 0.0025; HT: p = 0.013) in patients. Decreased frequencies for alleles DRB1*10 (PL: p = 0.00002; HT: p = 0.018; GD: p = 1.63 × 10− 5) and DRB1*03 (PL: p = 0.003; HT: p = 0.003) were observed, suggesting a protective association. Combinatorial/Synergistic analysis have revealed an increased frequencies for ‘DRB1*11 + AG’ (PL: p = 0.022), ‘DRB1*12 + AG’ (PL: p = 6.1 × 10− 5; HT: p = 0.0001), ‘DRB1*04 + GG’ (PL: p = 0.003; HT: p = 0.008), ‘DRB1*07 + GG’ (PL: p = 0.009; HT: p = 0.014) and ‘DRB1*12 + GG’ (PL: p = 0.005; HT: p = 0.005) in patients. However, the combinations such as ‘DRB1*10 + AA’ (PL: p = 1.8 × 10− 6; HT: p = 0.003) and ‘DRB1*15 + AA’ (PL: p = 0.006; GD: p = 0.011) were decreased in patients showing a protective association. The ‘GG/G’ of CTLA4 +49AG SNP, HLA-DRB1*11/-DRB1*12 (DR5) alleles and the combinations of DRB1*11/ DRB1*12 alleles with AG/GG genotype and DRB1*04/07/12 alleles with GG genotype may act as synergistic manner to confer the strong susceptibility to AITD in south India.

1. Introduction Autoimmune Thyroid Disease (AITD) is a multi-factorial disease (Stassi and De Maria, 2002). The genetic factors contribute for about 70–80 % and environmental factors for about 20–30 % to the pathogenesis of AITD (Wiersinga, 2016). AITD is an endocrine disorder which includes predominantly Hashimoto's thyroiditis (HT) and Graves' disease (GD). HT is characterised by inflammation and autoantibody formation that leads to hypothyroidism, whereas GD is characterised by production of TSHR (thyroid stimulating hormone receptor) antibodies that accelerate the stimulation and uncontrolled thyroid hormone

secretion by thyrocytes (Weetman and McGregor, 1994). The prevalence of AITD is 5 % in general population and higher in women than men, with approximately a male:female ratio of 1:10 (Whitacre, 2001; Matthias and Werner, 2006). Generally, autoimmune diseases primarily targets women between the age 20 and 50 yrs although elderly may occasionally be affected. For instance, in GD cases, women are seven times more likely to be affected than in men. Although, the reasons are unclear, estrogen hormone is a likely factor since it is linked to stronger immune response. A number of different environmental factors proposed include bacterial (Valtonen et al., 1986) or viral (Jaspan et al., 1996) infections, physiological stress (Kung, 1995), synthetic chemical

Abbreviations: AITD, Autoimmune Thyroid Disease; HT, Hashimoto's thyroiditis; GD, Graves' disease; HLA, human leukocyte antigen; CTLA4, cytotoxic T lymphocyte associated antigen 4; PCR-RFLP, polymerase chain reaction-restriction fragment length polymorphism ⁎ Corresponding author. E-mail address: [email protected] (K. Balakrishnan). https://doi.org/10.1016/j.gene.2017.11.057 Received 15 April 2017; Received in revised form 5 November 2017; Accepted 20 November 2017 Available online 22 November 2017 0378-1119/ © 2017 Elsevier B.V. All rights reserved.

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2. Materials and methods

exposure (Brucker-Davis, 1998) and levels of iodine uptake (McIver and Morris, 1998). However, to date, conclusive evidence for any of these factors is lacking and the molecular mechanism remains largely elusive. The genetic factors involved in the etiopathogenesis are either thyroid specific genes and/or non-thyroid specific genes. In the recent past, several AITD susceptibility genes such as HLA-DRB1*, CTLA4, PTPN22, CD40, CD25, TG and TSHR genes have been identified and confirmed through linkage and association studies (Huber et al., 2008). Further, it has been showed that, the genetic susceptibility to AITD is polygenic, the combinations of two or more genes/gene-products are necessary to predispose to disease. Most recently in a study conducted in the Italian GD patients, three SNPs such as rs13097181, rs763313 and rs6792646 on chromosome 3q showed a risk towards the disease (Lombardi et al., 2016). Strong evidence for a genetic basis to AITD comes from family studies demonstrating that a family history of GD has been reported in approximately 50 % of patients. Twin studies have estimated the concordance rates for disease in identical monozygotic (MZ) twins to be 50–70 % and in non-identical dizygotic (DZ) twins to be 3–25 % for GD (Harvald and Hauge, 1956). The genes encoding human leukocyte antigen (HLA) is present in chromosome 6p21.3 and comprise of 3 Mbp genome. There are 3 classes of HLA (class-I, class-II and class-III). HLA class-II molecules (DR/DQ/DP) are playing a major role in peptide presentation to T cells both in the periphery and during thymic selection. The HLA class-II alleles (gene variants) have been associated with most of the autoimmune diseases (Gough and Simmonds, 2007). It has been reported that, the extended haplotype DRB1*03-DQB1*02-DQA1*05 as susceptible, and another haplotype DRB1*07-DQB1*02-DQA1*02 as protective towards GD (Heward et al., 1998). Several previous studies have reported that HT was shown to be associated with HLA alleles DR3, DR4 and DR5 in different ethnic populations (Supplementary Table 1). However, due to a lack of replication between these studies, efforts in elucidating HLA class-II associations in HT has been much slower, leaving confusion over whether GD and HT share the same susceptibility loci within this region or not. Cytotoxic T-lymphocyte-associated protein-4 (CTLA-4), also called as CD152 is a protein receptor present on T cells and acts as an ‘off switch’ to T cell activity. The gene for CTLA-4 protein in humans is located on chromosome 2q33 (Brunet et al., 1987). In general, the predisposition of autoimmunity with CTLA4 variants is believed to reduce the inhibitory effect of CTLA-4 on T-cell activation, resulting in uncontrolled activation of T-cells (Ban et al., 2003). Previous studies have shown that, the human CTLA4 gene is highly polymorphic and is associated with T cell mediated autoimmune diseases such as autoimmune endocrinopathies that include GD, HT, T1DM, Addison's disease (AD), rheumatoid arthritis (RA) and multiple sclerosis (MS) (Ligers et al., 1999; Vaidya and Simon, 2004). CTLA4 gene harbors ‘A/G’ dimorphism at position + 49 in exon-1 which causes an amino acid exchange (Threonine → Alanine) in the peptide leader sequence of the CTLA-4 protein (Nisticò et al., 1996). However, reports on association of CTLA4 gene with AITD were contradicting (Supplementary Tables 2 and 3). Strong genetic links of HLA and CTLA4 genes with the development of AITD have been consistently shown in a number of world populations. Estimates suggest that these two gene regions may account for around 50 % of the genetic contribution to AITD in the UK population (Vaidya et al., 1999; Nithiyananthan and Gough, 2002). There is no synergistic genetic study available to demonstrate the association of HLA-CTLA4 + 49 AG dimorphism in south India. Thus, the aim of present work is to study the association of HLA class-II gene and CTLA4 + 49 AG SNP in Hashimoto's thyroiditis and Graves' disease. Further, we have analyzed the possible synergistic interactions between HLADRB1* alleles and CTLA4 gene in predisposing HT and GD in south India.

2.1. Subjects The subjects were 235 AITD patients (206 females; mean age 33.73 ± 11.78 yrs; 29 males; mean age 42.89 ± 10.88 yrs) and 235 age/sex matched healthy controls (206 females; mean age of 33.85 ± 12.11 yrs and 29 males; mean age 35.11 ± 12.46 yrs). The AITD patients were clinically subdivided into 180 HT (164 females; mean age 32.15 ± 11.36 yrs; 16 males; mean age 41.31 ± 12.44 yrs) and 55 GD (42 females; mean age 39.92 ± 11.50 yrs and 13 males; mean age 44.84 ± 8.68 yrs). The inclusion and exclusion criteria were followed such as, patients with auto antibody (i.e., Anti-TPO/TSHR Abs) positive and diagnosed recently were included, whereas patients with unknown thyroiditis were excluded from the study. The recruited controls were healthy individuals without any autoimmune diseases. The written informed consent was obtained from all participants according to the study protocol and the Institutional Ethical Committee; Madurai Kamaraj University approved the study. For all subjects, demographic details such as age, gender, age of onset and other medical illnesses were documented in a structured/pre-coded Questionnaire. 2.2. Detection of clinical parameters The quantification of T3, FT4 and TSH serum levels were determined by chemiluminescence method by automated analyzer (Access 2 Beckman Coulter, USA). The anti-TPO antibody (normal value: < 34 IU/mL) was measured by chemiluminescence method and the TSHR antibody (normal value: < 1.5 U/L) was measured by Radio Immuno Assay (RIA) by using “125I Gamma Counter”. Hyperthyroidism was confirmed when the serum T3 (normal value: 80–200 ng/dL) and FT4 (normal value: 0.58–1.64 ng/dL) levels were exceeded the normal range and TSH (Normal range: 0.34–5.60 uIU/mL) level decreased than normal range. Whereas, hypothyroidism was confirmed by lower levels of T3 and FT4 than the normal range, and TSH was greater than normal range. Hashimoto's thyroiditis (HT) was diagnosed by hypothyroidism with anti-TPO antibody positivity, whereas, Graves' disease (GD) was confirmed by hyperthyroidism with TSHR antibody positivity. 2.3. HLA-DRB1* typing Genomic DNA was isolated from peripheral blood by salting out method (Miller et al., 1988). Typing of HLA-DRB1* alleles were performed with sequence specific PCR (PCR-SSP) method as previously described (Olerup and Zetterquist, 1992). PCR amplification was performed using Agilent Thermal Cycler (USA) and the alleles were assigned by detecting the specific bands in a 1.5 % agarose gel stained with ethidium bromide (EtBr) having growth hormone gene as the internal control (Fig. 1). The results were documented by Gel Documentation System (Vilber Lourmat, France). 2.4. Genotyping of CTLA4 exon-1 + 49 AG polymorphism The CTLA4 gene was amplified by PCR with a forward: 5′-GCT CTA CTT CCT GAA GAC CT-3′ and reverse: 5′-AGT CTC ACT CAC CTT TGC AG-3′ primers as described previously (Hajilooi et al., 2014). The PCR reaction was performed in a total volume of 25 μL, using 25 ng of genomic DNA, 10 × PCR buffer (GenetBio, Korea), 10 μm dNTPs (Cinnagen, Iran) and 10 μm of each primers. The optimal PCR conditions consisted of an initial 95 °C denaturation for 5 min followed by 30 cycles of denaturation at 95 °C for 30 s, annealing at 58 °C for 30 s and elongation at 72 °C for 30 s. The 162 bp amplified product was subjected to BseXI (BbvI) restriction enzyme (Thermo Fisher Scientific, USA) digestion for 16 h at 65 °C to identify the allele present at position + 49 of CTLA4 exon-1. The DNA fragments obtained were analyzed in 3 % agarose gel. The undigested ‘A’ allele yielded fragment of 162 bp 431

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Fig. 1. Amplification pattern of HLA-DRB1* by PCRSSP method. Fig. 1 legend: The gel picture shows amplification of 16 samples for DRB1*12. Internal control (growth hormone gene) was used for quality control of amplification. Samples 1, 9, 12, 13 and 15 were positive for DRB1*12.

Table 1 Demographical details of AITD patients and controls. Characteristics

AITD patients (n = 235)

Controls (n = 235)

Age Sex ratio (male: female) Hashimoto's thyroiditis (HT) Grave's disease (GD) Nutrition (veg: non veg) Age at onset

34.87 ± 12.043 1:7

35.11 ± 12.46 1:7

76.6% (180)

76.6% (180)

23.4% (55) 1:11

23.4% (55) 1:11

GD: 35.43 ± 10.46 years HT: 27.94 ± 11.34 years Anti-TPO: 499.29 ± 391.09 IU/mL TSHR Ab: 22.25 ± 24.51 U/L 15.74% (37)

N/A N/A < 34 IU/mL < 1.5 U/L N/A

6.38% (15) 6.38% (15) 0.85% (02)

N/A N/A N/A

2.13% (05)

N/A

18.18% (10)

N/A

Autoantibody Associated complications i. Diabetes ii. Hypertension iii. Rheumatoid arthritis iv. Other complications Ophthalmopathy in GD Fig. 2. PCR-RFLP pattern of CTLA4 + 49 AG SNP. Fig. 2 legend: The 162 bp amplified product was digested with BbvI restriction enzyme. Sample number 1 is negative control; samples 4 and 5 were GG; samples 2, 6, 8 and 9 were AG; samples 3 and 7 were AA.

3. Results 3.1. Demographical details of the study subjects

and digested product of ‘G’ allele resulted in 91 bp and 71 bp fragments (Fig. 2).

The base-line demographic details of AITD patients and controls are presented in Table 1. The mean age of the patients (n = 235) was 34.87 ± 12.043 yrs and the controls (n = 235) was 35.11 ± 12.46 yrs. Out of 235 AITD patients, 76.59 % (n = 180) were HT and 23.04 % (n = 55) were GD. The age-at-onset of HT and GD were 27.9 ± 11.34 yrs and 35.41 ± 10.46 yrs respectively. Among 235 AITD subjects, 15.75 % (n = 37) were affected by other complications such as diabetes (6.38 %; n = 15), hypertension (6.38 %; n = 15), rheumatoid arthritis (0.85 %; n = 02) and other clinical entities (2.13 %; n = 05). Out of 55 GD patients, 18.18 % (10) individuals have ophthalmopathy.

2.5. Statistical analysis Statistical analyses were performed with the SPSS (IBM Corporation, New York, NY, USA) statistical package (Version 20.0) for windows and Epi Info™ (Version 7.1.4.0). Allele frequencies were calculated by direct counting. Differences between means or percentages were tested using chi-squared test and student t-test. Box plot analysis was performed for the Anti-TPO antibody and TSHR antibody linkage. For all the genotype/allele, the odds ratios (OR) and p-values were calculated by the Chi-square test after Yates' correction in 2 × 2 contingency table. In order to correct for multiple comparisons, p-values < 0.05/n were considered significant, where n represents the number of comparisons. All genotypes and allele frequencies were given as percentage frequencies.

3.2. HLA-DRB1* allele frequency The HLA-DRB1* alleles showing susceptible association in patients were DRB1*11 (PL: OR = 3.51, p = 0.0025) and DRB1*12 (PL: OR = 8.62, p = 1.42 × 10− 10; HT: OR = 8.63, p = 4.0 × 10− 12; GD: OR = 10.6, p = 3.9 × 10− 12). In patients, reduced frequencies were observed for alleles DRB1*10 (PL: OR = 0.37, p = 0.00002; HT: OR = 0.47, p = 0.002; GD: OR = 0.12, p = 0.001) and DRB1*03 (PL: OR = 0.50, p = 0.003) revealed protective associations. The 432

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Table 2 HLA-DRB1* allele frequencies in AITD patients and controls. HLA-DRB1*

*01 *03 *04 *07 *08 *09 *10 *11 *12 *13 *14 *15 *16

Controls

2.56 (12) 14.10 (66) 11.53 (54) 12.60 (59) 2.13 (10) – 15.81 (74) 1.70 (08) 1.70 (08) 5.12 (24) 6.41 (30) 25.42 (119) 1.27 (06)

HT + GD vs. controls

HT vs. controls

n

OR; p value

n

OR; p value

n

OR; p value

2.55 (12) 8.29 (39) 14.25 (67) 12.97 (61) 2.97 (14) 0.63 (03) 7.44 (35) 5.53 (26) 12.17 (57) 6.59 (31) 4.46 (21) 21.7 (102) 0.42 (02)

0.99; 0.50; 1.32; 1.03; 1.41; – 0.37; 3.51; 8.62; 1.32; 0.66; 0.74; 0.32;

1.66 (06) 9.16 (33) 14.44 (52) 15.0 (54) 2.22 (08) 0.83 (03) 8.88 (32) 5.27 (19) 11.66 (42) 6.66 (24) 3.05 (11) 20.55 (74) 0.55 (02)

0.64; 0.57; 1.36; 1.27; 1.04; – 0.47; 3.34; 8.63; 1.35; 0.44; 0.68; 0.42;

5.45 (06) 5.45 (06) 13.63 (15) 6.36 (07) 5.45 (06) – 2.72 (03) 6.36 (07) 13.63 (15) 6.36 (07) 9.09 (10) 25.45 (28) –

2.27; 0.31; 1.25; 0.43; 2.75; – 0.12; 4.13; 10.6; 1.28; 1.51; 1.01; –

0.842 0.003 0.215 0.937 0.536 0.00002 0.0025 1.42 × 10− 10 0.398 0.229 0.139 0.284

GD vs. controls

0.525 0.028 0.209 0.317 0.881 0.002 0.006 4.0 × 10− 12 0.406 0.037 0.067 0.484

0.195 0.013 0.618 0.073 0.105 0.0001 0.013 3.9 × 10− 12 0.763 0.405 0.909

p values considered significance whenever p less than or equals to 0.05/13 or 0.003 (Bonferroni correction). Bold-significant p value (Yates corrected).

3.4. Association of autoantibody levels with CTLA4 genotypes

frequencies of remaining alleles were not significantly different between patients and controls (Table 2).

The serum levels of anti-TPO antibody (HT) and TSHR antibody (GD) in relation to CTLA4 genotypes were presented in Table 5. Box plot analysis have revealed a moderate increase of anti-TPO antibody levels in patients with ‘AG’ and ‘GG’ genotypes when compared to patients with ‘AA’ genotype, however, without any statistical significant. Gender based analysis showed no association with anti-TPO antibodies in patients to CTLA4 genotype (Fig. 3). Whereas, the TSHR antibody levels were increased moderately in patients with ‘GG’ genotype than patients with ‘AA’ and ‘AG’ genotypes (GG: 19.405 ± 22.0; AA: 17.69 ± 18.78; AG: 10.36 ± 25.83) in males (Fig. 4). However, in females, the TSHR antibody levels were 10.36 ± 21.77, 6.72 ± 6.3 and 5.61 ± 45.78 respectively for patients with ‘GG’, ‘AG’ and ‘AA’ genotypes. We observed a moderate significant difference in TSHR antibody levels when we compared ‘GG’ genotype with ‘AA’ genotype (p = 0.009) and ‘AG’ genotype with ‘AA’ genotype (p = 0.033) in patients. However, we did not observed any significant association of autoantibody levels with CTLA4 variants after Bonferroni correction.

3.3. Distribution of CTLA4 exon 1 + 49 AG polymorphism The CTLA4 + 49 ‘AA’ genotype/‘A’ allele was wild genotype/allele and considered as reference genotype/allele. Out of 235 patients, the frequencies of homozygous genotype ‘GG’ (PL: OR = 6.90, p = 8.7 × 10− 8; HT: OR = 6.84, p = 2.5 × 10− 6; GD: OR = 7.09, p = 2.9 × 10− 4) and allele ‘G’ (GG + AG) were significantly increased in patients (PL: OR = 2.45, p = 1.04 × 10− 9; HT: OR = 2.52, p = 2.6 × 10− 12; GD: OR = 2.22, p = 0.0005) than the controls. Decreased frequencies of homozygous genotype ‘AA’ (PL: OR = 0.42, p = 9.4 × 10− 6; HT: OR = 0.40, p = 9.7 × 10− 5) and allele ‘A’ (AA + AG) were observed in patients (PL: OR = 0.40, p = 2.29 × 10− 9; HT: OR = 0.39, p = 2.6 × 10− 12; GD: OR = 0.44, p = 0.0005) than the controls (Table 3). The age-stratified analysis have revealed an increased frequency of homozygous ‘GG’ genotype (PL: OR = 6.48, p = 3.8 × 10− 7; HT: OR = 6.21, p = 1.7 × 10− 5; GD: OR = 7.65, p = 2.7 × 10− 4) and ‘G’ allele (PL: OR = 2.52, p = 1.04 × 10− 9; HT: OR = 2.58, p = 4.6 × 10− 12; GD: OR = 2.76, p = 4.6 × 10− 4) in patients of < 50 yrs age. However, the frequency of homozygous ‘AA’ (PL: OR = 0.40, p = 7.8 × 10− 6; HT: OR = 0.38, p = 1.0 × 10− 4) and ‘A’ allele (PL: OR = 0.39, p = 3.57 × 10− 9; HT: OR = 0.38, p = 4.6 × 10− 12; GD: OR = 0.36, p = 4.6 × 10− 4) were significantly decreased in patients < 50 yrs of age (Table 4). We did not found association of CTLA4 allele/genotype in the patients > 50 yrs of age (data not shown).

3.5. Association of age-at-onset with CTLA4 genotypes Fig. 5 represented the age-at-onset in AITD with respect of CTLA4 + 49 AG genotypes. The age-at-onset of ‘GG’ genotype was lower (30 yrs) when compared to ‘AA’ (37 yrs) and ‘AG’ (35 yrs) genotypes. 3.6. Analysis of genetic model of inheritance The genetic model of inheritance analysis based on the dominant model of inheritance (GG + AG vs. AA) have revealed a highly significant positive association with the disease (PL: OR = 2.17, p = 4.9 × 10− 4; HT: OR = 2.46, p = 3.9 × 10− 4). A negative

Table 3 Genotype and allele frequencies of CTLA4 + 49 AG in AITD patients and controls. Genotype/allele

AA (Ala/Ala) AG (Ala/Thr) GG (Thr/Thr) A (Ala) G (Thr)

Controls

61.27 (144) 35.31 (83) 3.40 (08) 78.93 (371) 21.07 (99)

HT + GD vs. controls

HT vs. controls

n

n

40.04 40.00 19.57 60.43 39.57

OR; p value (95) (94) (46) (284) (186)

0.42; 1.22; 6.90; 0.40; 2.45;

9.4 × 10− 6 0.341 8.7 × 10− 8 2.29 × 10− 9 1.04 × 10− 9

38.88 41.66 19.44 59.72 40.27

OR; p value (70) (75) (35) (215) (145)

p values considered significance whenever p less than or equals to 0.05/15 or 0.0033 (Bonferroni correction). Bold-significant p value.

433

GD vs. controls

0.40; 1.30; 6.84; 0.39; 2.52;

9.7 × 10− 5 0.223 2.5 × 10− 6 2.6 × 10− 12 2.6 × 10− 12

n 45.45 34.54 20.00 62.72 37.27

OR; p value (25) (19) (11) (69) (41)

0.52; 0.96; 7.09; 0.44; 2.22;

0.046 0.961 2.9 × 10− 4 0.0005 0.0005

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Table 4 Genotype/allele frequencies of CTLA-4 + 49 AG SNP in AITD patients and controls of age < 50 yrs (n, 210; HT, 167; HD, 43). Genotype/allele

AA (Ala/Ala) AG (Ala/Thr) GG (Thr/Thr) A (Ala) G (Thr)

Controls

60.47 (127) 36.66 (77) 3.80 (08) 78.57 (330) 21.90 (92)

HT + GD vs. controls

HT vs. controls

n

n

38.00 42.38 20.47 59.28 41.66

OR; p value (80) (89) (43) (249) (175)

0.40; 1.26; 6.48; 0.39; 2.52;

7.8 × 10− 6 0.273 3.8 × 10− 7 3.57 × 10− 9 1.04 × 10− 9

37.12 43.11 19.76 58.68 41.31

GD vs. controls OR; p value

(62) (72) (33) (196) (138)

0.38; 1.30; 6.21; 0.38; 2.58;

1.0 × 10− 4 0.243 1.7 × 10− 5 4.6 × 10− 12 4.6 × 10− 12

n 37.20 39.53 23.25 56.97 43.02

OR; p value (16) (17) (10) (49) (37)

0.38; 1.12; 7.65; 0.36; 2.76;

0.008 0.856 2.7 × 10− 4 4.6 × 10− 4 4.6 × 10− 4

p values considered significance whenever p less than or equals to 0.05/15 or 0.0033 (Bonferroni correction). Bold-significant p value. Table 5 Correlation between thyroid auto-antibody levels with CTLA4 genotype. + 49 AG SNP

AA

AG

GG

Anti-TPO Ab (IU/mL)

p value

TSHR Ab (IU/L)

PL (n = 180)

PL (n = 55)

Female (n = 164)

Female (n = 42)

Male (n = 16)

Male (n = 13)

446 ± 402.967 491.5 ± 396.58 311.4 ± 459.08 446 ± 402.967 494.25 ± 390.20 300 ± 139.20 500 ± 367.426 301 ± 370.43 253 ± 410.026

0.795 0.510 0.063 1.000 0.963 0.062 0.059 0.058 0.065

15.26 ± 26.87 17.69 ± 18.78 5.61 ± 45.78 10.2 ± 23.96 10.36 ± 25.83 6.72 ± 6.3 53.8 ± 26.74 19.405 ± 22.0 10.36 ± 21.77

p value

0.021 0.510 0.283 0.033 0.069 0.513 0.009 0.952 0.054

Data are represented as mean ± SD. ‘AA’ genotype considered as reference. p < 0.05/9 or p < 0.005 considered significant (Bonferroni correction).

Fig. 4. Box plot analysis of TSHR antibody levels in Grave's disease patients connection with CTLA4 + 49 AG.

Fig. 3. Box plot analysis of anti-TPO antibody levels in Hashimoto's thyroiditis patients connection with CTLA4 + 49 AG. Fig. 5. Graphical representation of age-at-onset with CTLA4 genotypes.

434

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HT: OR = 0.56, p = 0.017; GD: OR = 0.18, p = 0.023), (iii) ‘DRB1*07 + AA’ (GD: OR = 0.18; p = 0.023) and (iv) ‘DRB1*03 + AA’ (PL: OR = 0.22, p = 4.4 × 10− 4; HT: OR = 0.27, p = 0.004; GD: OR = 0.17, p = 0.017).

Table 6 Analysis of genotypes risk factor in AITD patients. Study model

OR

95% CI

χ2

p value

(AG + GG) vs. AAa

2.17i 2.46ii 1.44iii 0.05i 0.05ii 0.06iii 0.23i 0.25ii 0.19iii 0.44i 0.51ii 0.27iii

1.502–3.139 1.616–3.772 0.679–3.050 0.037–0.093 0.034–0.098 0.024–0.159 0.142–0.385 0.140–0.443 0.071–0.527 0.307–0.642 0.335–0.775 0.126–0.611

16.476 16.900 0.5818 171.60 132.01 37.236 32.680 22.019 9.3889 18.005 9.3444 9.3091

4.9 × 10− 4 3.9 × 10− 4 0.445 8.7 × 10− 12 9.3 × 10− 12 5.0 × 10− 10 7.8 × 10− 10 2.6 × 10− 5 0.002 2.1 × 10− 4 0.002 0.002

GG vs. (AG + AA)

b

c

GG vs. AA

AG vs. (AA + GG)d

4. Discussion The MHC encoded Human Leukocyte Antigens (HLA) are implicated in a number of infections and autoimmune diseases. Associations of components of the HLA class-II have been detected with several autoimmune diseases that include T1DM (DRB1*03, *04), rheumatoid arthritis (DRB1*04, *10, *14), Addison's disease (DRB1*03), systemic lupus erythematosus (DRB1*03, *08, *15) and multiple sclerosis (DRB1*15) (Gough and Simmonds, 2007). HLA alleles have been considered to be one of the major genetic factors involved in the genetic susceptibility to AITD (Cho et al., 2011). Both ethnicity and geographic variations influence the genetic susceptibility to GD. Differential effects of susceptibility and/or protection of HLA alleles (serological and/or PCR-SSP based molecular types) were documented. For example, in Caucasians, a positive and negative association of serologically defined HLA-DR3 and DR7 alleles respectively were shown in GD (Zamani et al., 2000). Further, the influence of PCR-SSP defined HLA haplotypes on the development of GD in non-Caucasian populations such as HLADRB1*04:05-DQB1*04:01 in Asians (Hashimoto et al., 2005) and DRB1*03:02-DQA1*05:01 haplotype in African-Americans (Chen et al., 2000) were reported. In our study, we observed a susceptible association of HLA-DRB1*11 and DRB1*12 (DR5) alleles in AITD. Our study is in concordance with the results observed in AITD patients from Canada, Denmark, Japan, USA and Australia (Farid et al., 1979; Uno et al., 1981; Schleusener et al., 1983; Thomsen et al., 1983; Sheila et al., 1992). Previous reports have documented an increased frequency of DR3 allele in GD (Ban et al., 2002). Studies in Caucasoid populations have shown an association of alleles HLA-DR3, -DR4 and -DR5 with GD (Jenkins et al., 1992; Zamani et al., 2000). In contrast to the previous reports, the present study showed the decreased frequency of HLADRB1*03 allele in AITD patients and rather surprisingly, have suggested a protective association. Similar to our findings, HLA-DR3 was shown to be negatively associated with HT in Denmark population (Thomsen et al., 1983). A significant association was observed particularly in HT for alleles HLA-DR3, -DR4, -DR5, -DQA1*03:01, -DQB1*02:01 and -DQB1*03:01 (Farid et al., 1979; Heward et al., 1998; Petrone et al., 2001; Zeitlin et al., 2008; Kokaraki et al., 2009). HLA alleles DR7 and DR13 showed a protective association in AITD in UK white Caucasians and Koreans (Zeitlin et al., 2008; Cho et al., 2011). In non-Caucasian groups, the predisposition to HT was observed with DR9 allele in Chinese (Wang et al., 1988) and DR8 allele in Koreans (Cho et al., 2011). The HLA-DRB1*03 and DRB1*04 were

p values were considered significant when p less than or equals to 0.05/12 or 0.004 (Bonferroni correction). i AITD pooled. ii Hashimoto's thyroiditis. iii Graves' disease. a Dominant effect of G allele. b Recessive effect of G allele. c Additive effect of G allele. d Co-dominant effect of G allele.

association was observed for the analyses based on recessive (PL: OR = 0.05, p = 8.7 × 10− 12; HT: OR = 0.05, p = 9.3 × 10− 12; GD: OR = 0.06, p = 5.0 × 10− 10), additive (PL: OR = 0.23, − 10 p = 7.8 × 10 ; HT: OR = 0.25, p = 2.6 × 10− 5; GD: OR = 0.19, p = 0.002) and co-dominant (PL: OR = 0.44, p = 2.1 × 10− 4; HT: OR = 0.51, p = 0.002; GD: OR = 0.27, p = 0.002) models (Table 6).

3.7. Synergistic effect of CTLA4 and HLA-DRB1* The synergistic interaction analyses have resulted in the identification of protective and susceptible combinations (Table 7). The susceptible combinations: (i) ‘DRB1*12 + AG’ (PL: OR = 26.60, p = 6.1 × 10− 5; HT: OR = 26.0, p = 1.1 × 10− 4; GD: OR = 28.6, p = 4.6 × 10− 4), (ii) ‘DRB1*12 + GG’ (PL: OR = 12.5, p = 0.004; HT: OR = 16.7, p = 0.0008), (iii) ‘DRB1*15 + GG’ (PL:OR = 10.20, p = 0.00003; HT: OR = 6.13, p = 0.021; GD: OR = 25.8, p = 5.5 × 10− 7), (iv) ‘DRB1*11 + AG’ (PL: OR = 4.529, p = 0.022; HT: OR = 4.54, p = 0.028), (v) ‘DRB1*07 + GG’ (PL: OR = 6.82, p = 0.008; HT: OR = 8.32, p = 0.002) and (vi) ‘DRB1*04 + GG’ (PL: OR = 6.03, p = 0.002; HT: OR = 6.02, p = 0.004; GD: OR = 6.06, p = 0.033). The protective combinations identified were (i) ‘DRB1*10 + AA’ (PL: OR = 0.18, p = 1.8 × 10− 6; HT: OR = 0.24, p = 2.8 × 10− 4), (ii) ‘DRB1*15 + AA’ (PL: OR = 0.54; p = 0.006; Table 7 Synergistic association of DRB1*-CTLA4 in AITD patients and controls. DRB1*-CTLA4

Controls

Susceptible *12-AG *12-GG *15-GG *11-AG *07-GG *04-GG

0.42 0.42 0.85 1.27 0.85 1.27

Protective *10-AA *15-AA *07-AA *03-AA

21.27 31.48 16.59 17.44

(01) (01) (02) (03) (02) (03) (50) (74) (39) (41)

HT + GD vs. controls

HT vs. controls

n

OR; p value

n

OR; p value

10.21 (24) 5.10 (12) 8.08 (19) 5.53 (13) 5.53 (13) 7.23 (17)

26.6; 12.5; 10.2; 4.52; 6.82; 6.03;

6.1 × 10− 5 0.004 0.003 0.022 0.008 0.002

10.00 (18) 6.66 (12) 5.00 (09) 5.55 (10) 6.66 (12) 7.22 (13)

26.0; 16.7; 6.13; 4.54; 8.32; 6.02;

4.68 (11) 20.00 (47) 11.48 (27) 5.10 (12)

0.18; 0.54; 0.65; 0.22;

1.8 × 10− 6 0.006 0.144 4.4 × 10− 4

6.11 (11) 20.55 (37) 13.88 (25) 5.55 (10)

0.24; 0.56; 0.81; 0.27;

All p values were Yates corrected.

435

GD vs. controls n

OR; p value

1.1 × 10− 4 0.0008 0.021 0.028 0.002 0.004

10.90 (06) – 18.18 (10) 5.45 (03) 1.81 (01) 7.27 (04)

28.6; – 25.8; 4.46; 2.15; 6.06;

2.8 × 10− 4 0.017 0.535 0.004

– 18.18 (10) 3.63 (02) 6.63 (02)

– 0.48; 0.072 0.18; 0.023 0.17; 0.017

4.6 × 10− 4 5.5 × 10− 7 0.151 0.918 0.033

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We analyzed the data based on age groups of patients. The analysis revealed a significant increase of ‘GG’ (p < 0.009), ‘AG’ (p < 0.014) genotypes and ‘G’ allele (p < 1.1 × 10− 6) in AITD patients < 50 yrs of age. Whereas, a significantly reduced frequencies were observed for ‘AA’ genotype (p = 7.8 × 10− 6) and ‘A’ allele (3.57 × 10− 9) in the same age group. No such associations were observed in AITD patients > 50 yrs of age. These findings have again suggested a dominant role of CTLA4 gene, particularly ‘GG’ genotype/‘G’ allele, in early onset of disease. The genetic model of inheritance analysis too reveled a strong susceptible association for the dominant model of inheritance (AG + GG vs. AA) in patients (PL: OR = 2.17, p < 4.9 × 10− 4; HT: OR = 2.46, p < 3.9 × 10− 4). However, in recessive model of inheritance (GG vs. AG + AA), a negative association was observed (PL: OR = 0.05, p < 8.7 × 10− 12; HT: OR = 0.05, p < 9.3 × 10− 12; GD: OR = 0.06, p < 5.0 × 10− 10). Thus, it is obvious to conclude that, the homozygous ‘GG’ genotype showed a greater risk for AITD than ‘AA’ and ‘AG’ genotypes. The anti-TPO antibody is considered to be a diagnostically sensitive marker for autoimmune thyroid dysfunction than Tg (thyroglobulin) antibody. The presence of abnormal levels of anti-TPO antibodies in HT and TSHR antibodies in GD is a hallmark of impending autoimmune thyroid disorder (Demers and Spencer, 2003). It was documented that the anti-TPO antibody titers are related to the degree of lymphocytic infiltration of thyroid gland (Yoshida et al., 1978). In our study, significant correlations were observed in patients with CTLA4 ‘GG’ and ‘AG’ genotypes for TSHR levels and the severity of disease. However, the anti-TPO antibody levels of HT patients did not show any significant association with CTLA4 genotype. It has been reported earlier that the CTLA4 +49 AG SNP may predispose to AITD when interacting with other gene loci (Vieland et al., 2008). The findings of present study have revealed the strong disease association of CTLA4 + 49 ‘AG’ genotypic combination with HLA-DRB1*11 (OR = 4.52) and DRB1*12 (OR = 26.6) alleles. The ‘GG’ genotypic combination with DRB1*12 (OR = 12.5), DRB1*04 (OR = 6.03) and DRB1*07 (OR = 6.82) alleles too revealed significant association with AITD. A protective association was observed with ‘AADRB1*03/*10/*15’ combinations. Thus, results of the present study have strongly establish a susceptible association for the combination of ‘GG genotype/G allele’ with HLA-DR5 (DRB1*11 and DRB1*12) allele and a protective association for the combination of ‘AA genotype/A allele’ with DR3, DR10 and DR15 alleles towards AITD in south India. The present study has some limitations: the potential interaction of the CTLA4 + 49 AG SNP and HLA gene polymorphisms studied with other nearby functional variants by further functional immunologic studies to discover the true underlying mechanism of action of these genetic variations. One of the important implications of the identification of genes linked to AITD is its usefulness as a molecular diagnostic marker in population level screening. The identification of subjects genetically susceptible to AITD may be an important step towards the development of strategies for early diagnosis and community based preventive medicine. We believe that our findings will be able to contribute to early diagnosis (predisposition), follow-up prognosis and therapeutic strategies for AITD. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.gene.2017.11.057.

associated with autoimmune atrophic thyroiditis in Brazilians (ZantutWittmann et al., 2004). A recent study based on Immunochip SNPs in young-age-of-onset GD have revealed a strongest association of three SNP markers rs3132486, rs9273012 and rs6457680 that are closely linked to HLA-C, HLA-DQA1 and HLA-DQB1 loci respectively (Brown et al., 2014). Recently, there are reports published to explain association of HLA/CTLA4 with other autoimmune diseases like T1DM, RA, MS, Vitiligo in Indian populations (Mehra et al., 2007; Mohan et al., 2014; Bhatia et al., 2015; Dwivedi et al., 2011; Verma et al., 2017). In the present study, the HLA-DRB1*10 (OR = 0.37) allele showed protective association in AITD. This is in line with our previous findings of protective association of DRB1*10 allele in south Indian patients with nephritic syndrome (Ramanathan et al., 2016), ischemic stroke (Murali et al., 2016) and T2DM (Rathika et al., 2016). Contrary to our results, in Turkey population, a susceptible association was documented for DR10 in GD (Orhan et al., 1993). The genes involved in the immunological synapse may predispose to autoimmune diseases by enabling presentation of self-antigens. The DRβ1-Arg74, which is associated with GD and HT, predisposes to AITD by creating a peptide binding pocket that can more easily accommodate pathogenic TPO and/or TSHR peptides which trigger appropriate T-cell response to thyroid self-antigens. The co-stimulatory molecules can enhance the activation of cells participating in the immunological events (Sawai and DeGroot, 2000; Jacobson et al., 2009). Previous results from our laboratory have documented that, HLADRB1*15 allele was the most predominant allele observed in south Indian populations (Pitchappan et al., 1997; Ravikumar et al., 1999; Balakrishnan et al., 2012; Rathika et al., 2016). Further, a susceptible association of the DRB1*15 allele was documented with multiple sclerosis (Alcina et al., 2012). Except a few reports showing susceptibility in tuberculosis (Brahmajothi et al., 1991) and multiple sclerosis (Alcina et al., 2012), the association of DRB1*15 allele was shown to be either neutral or protective in several diseases in south India (Rathika et al., 2016; Murali et al., 2016). In the present study, we observed a high frequency of DRB1*15 in both patients (n, 102) and controls (n, 119) suggesting a neutral role for DRB1*15 allele in AITD (OR = 0.74). The CTLA4 gene encodes a co-stimulatory molecule, which is expressed on activated T cells that mediate T cell apoptosis and involved in the pathogenesis of AITD (Kouki et al., 2002). The CTLA4 ‘G’ allele was shown to be associated strongly with many autoimmune diseases such as T1DM, GD, HT, rheumatoid arthritis (RA) and multiple sclerosis (MS) (Donner et al., 1997; Seidl et al., 1998; Ligers et al., 1999; Vaidya and Simon, 2004). In a meta-analysis, CTLA4 ‘G’ allele have showed a significant association with the disease in Asian population (Si et al., 2012). In the present study, we observed a significant association of ‘GG’ genotype (OR = 6.90) and ‘G’ allele (OR = 2.45) in south Indian AITD patients. However, we observed a higher frequency of ‘AG’ genotype in GD (OR = 4.03). A study conducted in Chinese children, have documented a susceptible association of ‘G’ allele in GD (Yung et al., 2002). The ‘A’ allele increases the CTLA4 expression and hence increase immunological function (Jacobson and Tomer, 2007). We have documented a protective association of ‘AA’ genotype and ‘A’ allele in both HT and GD. Thus it is clear from these findings that, the autoimmune response may drastically be influenced by an altered CTLA4 allele/ genotype expression. Mechanistically, a polymorphism that reduces CTLA-4 function would be expected to augment T-cell activation leading to autoimmunity (Ban et al., 2003). It was documented that, ‘GG’ genotype is associated with reduced inhibitory function of CTLA-4 on T-cell proliferation that ultimately resulted in GD (Kouki et al., 2002). Individuals with ‘GG’ genotype showed reduced T-cell proliferation under conditions of CTLA-4 blockade when compared to individuals with ‘AA’ genotype (Park et al., 2000). Further, our results revealed a lower age-at-onset for patients with CTLA4 ‘GG’ (< 30 yrs) genotype than ‘AA’ (37 yrs) and ‘AG’ (35 yrs) genotypes. Thus, our findings have established the strong risk of ‘GG’ genotype for early development of AITD in south India.

Competing interest/disclosure None. Acknowledgments The authors were thankful to the various central facilities such as NRCBS, DBT-IPLS, DST-PURSE and Tissue Typing Service (KB) at Madurai Kamaraj University. We sincerely thank all the patients and paramedical staffs of hospitals from where we have collected blood 436

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samples.

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