This case control study included 100 patients of CD and 31 healthy controls from January 2015 to August 2016. Cases were taken from Department of Paediatrics and Endocrinology of SS Hospital BHU. The power of the study is 80%. Samples were collected on the basis of feasibility and availability. All patients and controls were informed the purpose of the study, and their consent was obtained before collecting the blood samples. All agreed samples was taken during the given time period. This study was approved by Institute of Medical Sciences, Human Research Ethics Committee, Banaras Hindu University, Varanasi, India (No.Dean/2014-15/EC/1047).

The present study took only symptomatic patients diagnosed as CD based on elevated anti-tTg antibody, history, HLA and histopathology as defined by diagnostic criteria of European Society of Paediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN) described by Husby S et al., [11].

In all patients and controls 3 mL blood was taken in EDTA vial for HLA DQ study by PCR and 4 mL blood was taken in plain vial for autoantibodies analysis.

Autoantibodies analysis: The autoantibodies like Anti-Tissue Transglutaminase (Anti-tTg), Antinuclear Antibody (ANA), Anti-TPO and Anti-scleroderma 70 Ab were performed by Enzyme-Linked Immunosorbent Assay (ELISA) method. The autoantibodies were done by using kits of following company:

The principle of the ELISA kit is based on indirect non competitive enzyme immunoassay for the quantitative and semi-quantitative determination of antibodies in human serum. The well of the solid phase were coated with a balanced mixture of autoantigen of nuclear antigens. The wells were Washed with 300 μL wash buffer and dispensed 100 μL of calibrators, negative control and diluted patient sample with diluents buffer (in ratio 1:101) into appropriate wells. Incubated for 30 minutes, fluids were aspirated from wells and wells were washed 3 times with washed buffer. 100 μL of Horse-Radish Peroxidase (HRP) conjugate was dispensed into all wells and incubated for 30 minutes. Fluid was aspirated from wells and wells were washed. 100 μL of TMB substrate was dispensed into all wells and incubated for 10 minutes in the dark. 50 μL stop solution was dispensed into all wells and absorbance (OD) was read at 450 nm and reference wavelength at 620 nm.

HLA DQ typing: HLA DQ typing was done in 44 cases of CD, 20 cases of CD with T1DM, 22 cases of CD with autoimmune thyroid disease and 31 healthy controls. HLA DQ typing was done by Sequence Specific Oligonucleotide (SSO) probe hybridisation method on instrument called as Mr. SPOT, BAG health Care, Germany and supplied by Shiva Scientific, New Delhi. DNA sample was extracted by salt-out and phenol chloroform method. A final reaction volume of 20 μL for the Polymerase Chain Reaction (PCR) was constituted, which contained 10 μL of master mix (biotynylated primers, dNTPs, taq polymerase, reaction buffer, 0.05% sodium azide), 5 μL of MgCl₂ (6 mm containing 0.001% Proclin R 300) and 5 μL of DNA (15-30 ng/μL). The Sequence specific amplification with biotinylated primers was carried out on an Biorad Thermal cycler under the following conditions: 96°C for 2 min, 10 cycles of 96°C for 15 s, 65°C for 60 s, followed by 20 cycles of 96°C for 10 s, 61°C for 50 s and 72°C for 30 s. Whole amplified product was inserted into heating bar of Mr. SPOT machine for SSO hybridization. Process as indicated on the screen was followed.

The resulting coloured dots in the bottom of each well are photographed by Mr. SPOT and the image is transferred into the Histo Match software installed on the PC user. Image analysis programme of the Histo Match software determines the intensity of each spot in the array and compares the intensity of background and displays the results.

All data were analysed using Statistical Package for Social Sciences (SPSS, Chicago, Illinos, USA), version 16. Pearson’s Chi-square (χ²) and Relative Risk (RR) test were used to compare differences between the frequencies as per the requirement. A p-value <0.05 was considered significant for all analysis.

CD was more common in between 6 months to 10 years of age (52%). As a whole 70% patients were upto 20 years of age and 25% were young adults between 21 to 40 years of age and only 5% patients were above 41 years of age [Table/Fig-1].

Sex wise distribution of patients showed that majority patients were female (60 cases) and 40 were males. Female to male ratio was 1.5:1.

Among the clinical manifestations analysed in 100 cases of CD showed that failure to thrive and weight loss were the most common manifestations seen in 79% cases followed by chronic recurrent diarrhea (71%), anaemia (66%), short stature (62%) and pain in abdomen (56%).

Anti-tTg was positive in all cases (100%). Anti-scleroderma 70 antibody was significantly positive in cases (p=0.026) as compared to control [Table/Fig-2].

DQβ1 typing in CD and control showed that DQβ1*02 was found in significantly higher percentage (65.9%) than controls (0%). High resolution typing showed that 45.5% had DQβ1*02:01 (p<0.001) and 20.5% had DQβ1*02:02 (p<0.007). Contrary to this, DQβ1*06:01 was significantly reduced in CD patients (p<0.001) suggesting that DQβ1*06:01 has protective role. DQβ1*03:03 was also reduced in CD patients but it was not significant (p=0.053) [Table/Fig-3].

DQα1 typing revealed DQα1*05:01 and 05:05 was significantly increased in CD patients (p<0.001 and p<0.001). Contrary to this DQα1*01:01, DQα1*01:03 and DQα1*01:04 were significantly reduced in CD patients as compared to controls suggesting its protective role [Table/Fig-4].

It was noticed that DQβ1*02:01 genotype analysis revealed that DQβ1*02:01 homozygosity was very common (45.5%) in CD patients followed by DQβ1*02:02 (18.2%) DQα1*05:01 (36.4%) [Table/Fig-5].

Correlation of DQβ1 with T1DM in CD patients showed again increased frequency of DQβ1*02:01 (70%) and DQβ1*02:02 (65%) which was statistically significant (p<0.001 and p<0.001) [Table/Fig-6].

Similarly, in DQα1, DQα1*05:01 (60% vs. 0%, p<0.001) and DQα1*05:05 (50% vs. 3.2%, p<0.001) were significantly more common in CD with T1DM patients as compared to controls. DQα1*01:03 and DQα1*01:04 were significantly reduced in CD patients with T1DM [Table/Fig-7].

Similarly, anti-TPO Ab positive cases also had significantly increased expression of DQβ1*02:01 (63.6% vs. 0% p<0.001) DQβ1*02:02 (36.4% vs. 0%, p<0.001) [Table/Fig-8].

DQα1*05:01 (63.6% vs. 0%) and DQα1*05:05 (40.9% vs. 3.2%) were also significantly increased (p<0.001 and 0.001) in anti-TPO positive cases [Table/Fig-9] contrary to this DQα1*01:01, DQα1*01:03 and DQα1*01:04 were significantly reduced in anti-TPO positive cases.

In present study, we found that majority of patients (70%) were children between 6 months-20 years of age and 52% children were below 10 years of age only 30% patients were adults. Similar to our findings the US based study by Llorente-Alonso MJ et al., reported that 67% patients of CD were children and 14.3% were adults [12]. However, Freeman HJ et al., from USA found bimodal peak of CD, first peak in children (8-12 years) and second peak during third to fourth decades of life [13]. While one of the studies from India reported increasing prevalence of CD in adults [14].

In the present study, we found female predominance (60%) showing a female to male ratio of 1.5:1. Similar to our study Elli L et al., of Spain also reported the female predominance in CD with female to male ratio of 2.3:1 [15]. Megiorni F et al., from America also reported that CD is twice more frequent in females than males [16]. They proposed that female predominance was due to increased frequency of HLA DQ 2 haplotype in females.

Major clinical manifestation of CD patients were weight loss and failure to thrive (79%) followed by chronic diarrhea (71%), short stature (62%), anaemia (66%) and pain in abdomen. Our study is in accordance with other Indian study [17,18]. Some studies from India reported short stature in 58%, delayed puberty in 31%, rickets in 8%, carpopedal spasm in 6% and gastrointestinal symptoms in 86% [19].

There was strong association of T1DM with CD (40%) in present series. More or less similar observations were noted by Philip R et al., from Australia (40%) and Bakker SF et al., from Italy (42%) [19,20]. One study from India found prevalence of T1DM in 25% cases in CD [19]. As a whole prevalence rates of T1DM from both cross-sectional and longitudinal studies range from 1.6-16.4% worldwide [21].

Reports on association of autoimmune thyroid diseases in CD patients are variable. In the present study we found that 22% patients had autoimmune hypothyroid diseases while Hakanen M et al., found much higher incidence of 38% [22]. While Velluzzi F et al., and Casta MG et al., reported incidence of about 29.7% and 30.5% [23,24]. Contrary to this Ventura A et al. reported low frequency of autoimmune thyroid diseases (14.4%) [25].

We also studied the ANA in CD patients which was detected in 10% cases only. In a study carried out by da Rosa Utiyama SR et al., studied in 56 patients with CD and 118 first degree relative and 101 healthy controls, ANA was detected in 9% while Caglar E et al., reported ANA positivity in 12% patients with CD [26,27].

Anti-scleroderma 70 antibody was detected in 25% patients with CD. There are variable reports in literature. Gomez-Puerta JA et al., studied 6 cases of CD and found all had anti-scl 70 antibody [28], while one of the Indian study found anti-TPO antibodies positivity in 53% and clinical hypothyroidism in 28% CD cases [19]. In our study, all anti-TPO Ab positive cases had autoimmune hypothyroidism but anti-scl 70 Ab positive cases and ANA positive cases did not have any classical presentation of scleroderma or SLE.

Author found that most frequent HLA DQ genotype in CD patient is DQ2 heterodimer composed of DQβ1*02, DQα1*05. DQβ1*02:01 was seen in 45.5% (p<0.001) while DQβ1*02:02 in 20.5% (p=0.007) which collectively forming 66% in total CD patients. Risk ratio was 1.833 and 1.257 respectively for 02:01 and 02:02 which indicate that their presence increases the risk for CD.

In DQα1 we found predominant allele was DQα1*05:01 (45.5%) and DQα1*05:05 (40.9%) in CD patients. Its association with CD was significant (p<0.001). RR for DQα1*05 was 1.833 and 20.769.

Vidales MC et al., studied 136 cases of CD found frequency of DQα1*05:01 in 59.2% and DQα1*02:01 in 26% while DQα1*01:04 and DQα1*03:02 were found in very low frequency (6.7%) [29]. In DQβ1 most frequent alleles were DQβ1*02:01 (47.1%) and DQβ1*02:02 (25.4%) and DQβ1*03:01 (12.5%) which is very much close to our observation.

Study conducted in western India found high prevalence of DQ2 (95%) and DQ8 (94%) among celiac patients as compared to controls (12%). DQ2.5 (DQα1*0501-DQβ1*0201) and DQ2 (DQβ1*02) haplotypes were common in CD patients (70%) [30]. However in this study, about 66% patient had DQ2 and 13.6% had DQ8 haplotypes.

Piccini B et al., also reported that 64% patients had DQ2 heterodimer and 13.5% had DQ8 haplotype that influences development of CD [31]. Megiorni F et al., also supported our observation and reported that 90-95% of CD patients carry DQ2.5 heterodimer encoded by DQβ1*03:02 in combination of DQα1*03 variant [32]. It was very interesting to note that 63% patients were homozygous for DQβ1*02 gene and 36.4% were homozygous for DQα1*05:01 suggesting a link between homozygosity for their alleles and increased risk for CD. Similar to our work some other reports also reported that DQ2.5 homozygotes are several times more likely to have CD than the DQ2.5 heterozygotes [33]. These homozygotes also have elevated risk for severe complication of the disease.

Major histocompatibility complex class II HLA DQ2 and DQ8 confers greatest disease susceptibility. The prevalence of autoimmune disease e.g., T1DM and autoimmune thyroid diseases are increased in CD patients. HLA DQ typing in CD with T1DM and without T1DM was analysed. It was found that DQβ1*02:01 (70%), DQβ1*02:02 (65%), DQα1*05:05 (50%) and DQα1*05:01 (60%) were significantly more common in CD associated with T1DM than CD alone. Barker JM et al., reported that DR3-DQ2 with T1DM carried a 33% risk for the presence of anti-tTg antibody and CD [34].

Camarca ME et al., reported that approximately 90% of the individuals with T1DM have either DQ2 or DQ8 compared to 40% of the general population [35]. Some of the studies from Northern Spain and USA also found increased frequency of DQβ1*02:01 in T1DM [36,37]. Some of the studies from India Singh S et al., also found increased frequency of DQβ1*02:01 in about 80% patients of T1DM [38]. HLA DQ genotype were also studied in anti-TPO positive CD patients and found that anti-TPO positive CD patients have genotype compatible with CD e.g., DQβ1*02:01 (63.6%), DQβ1*02:02 (36.4%), DQα1*05:01 (63.6%), DQα1*05:05 (40.9%).

The coexistence of CD and autoimmune thyroid diseases is thought to be partly due to common genetic predisposition. Lorini R et al., and Badenhoop K et al., proposed that DQ2 and DQ8 haplotypes are over represented in many autoimmune diseases including thyroid diseases and graves’ disease [39,40].

Smaller sample size may limit the result validation. Due to logistic reason, number of controls could not be matched with the number of cases. Further the specificity and sensitivity of such gene typing needs to be carried out further.

The present study suggests that CD is an autoimmune disease where the genetics have important role. HLA DQ antigen contributes more than 65% cases of CD hence it may be kept in diagnostic criteria of CD. HLA DQβ1*02 and HLA DQα1*05 alleles predisposes for development of other autoimmune diseases like T1DM and autoimmune thyroid disease. The involvement of genes other than HLA can be studied in the future to know the exact prevalence of these genes in CD.