Blog

Summary

A long-term autoimmune illness called celiac disease (CD) mainly affects the small intestine, which is where gluten intolerance occurs. Since multiple extra-digestive signs are often present with this disease. CD may begin with extraintestinal symptoms, and related disorders may present at the time of diagnosis as well as the disease progresses. Adopting a gluten-free diet (GFD) prevents the development of related disorders and enhances the overall clinical course. According to these facts, the present work investigated the influence of gluten on the level of the number of autoantibodies associated with CD-related autoimmune illnesses, including anti-pituitary IgG, anti-thyroid peroxidase IgG (anti-TPO IgG), anti-islet IgG, and anti-ganglioside IgG, and analyzed the correlation between anti-tissue transglutaminase IgA(tTG-IgA) and the mentioned autoantibodies. In addition to investigate the association of one type of human leukocyte antigen (HLA) class 1 through the study of its level in serum as a soluble protein named soluble HLAG (sHLAG) and detected the HLAs class 2 type of CD patients regarding DQ2 and DQ8 alleles to determine the disease association with genetic haplotype.

To apply these aims, a total of 200 blood samples were collected in this study during the period from July 2021 until October 2022 in Raparin Children’s Hospital and Rizgary Teaching Hospital in Erbil City – Kurdistan Region of Iraq (KRI), 75 of them were collected from newly diagnoses (ND) of CD patients after they were diagnosed clinically and serologically, their ages ranged from 2 to 56 years. DNA was extracted and sera were separated then kept at -80Cº and -20Cº until used, respectively. After 4 months of GFD treatment, 75 blood samples were gathered from the same patients in addition to the control group which contained 50 healthy donors.

Sera were used to measure the level of tTG-IgA, anti-pituitary IgG, TPO- IgG, anti-islet IgG, anti-ganglioside IgG, and sHLAG level by enzyme-linked immunosorbent assay (ELISA). DNA was extracted; the purity and concentration were determined using the nanodrop technique. Real-time polymerase chain reaction (RT-PCR) was utilized to detect DQ2 and DQ8 alleles of the ND group, following the collection of samples. Graph Pad Prism 8 was used to conduct all statistical analyses

The result of the present study is divided into the finding of autoantibodies, sHLAG, and genetic study to determine homozygosity and heterozygosity of DQ2 and DQ8 alleles. The level of anti-tTG IgA, anti-pituitary IgG , anti-TPO IgG , anti-islet IgG and anti-ganglioside IgG were evidently greater (P < 0.05) in ND group of CD patients than control. Moreover, serum anti-tTG IgA , anti-pituitary IgG , anti-TPO IgG , anti-islet IgG and anti-ganglioside IgG levels were considerably higher (P < 0.05) in the CD patients under GFD group as compared with control. A comparison between CD patients, pre and post-4 months’ treatment with GFD, was conducted concerning the concentrations of autoantibodies (anti-tTG IgA, anti-pituitary IgG, anti-TPO IgG, anti-Islet IgG, and anti-ganglioside IgG). The result exhibited that the mean levels of all tested autoantibodies in CD patients were significantly (p < 0.0001) decreased after 4 months of the GFD regime compared with ND CD patients before starting the regime.

In the ND group, anti-tTG level showed a significant positive correlation with anti-ganglioside (rs=0.238, p = 0.039), however, no significant correlation was observed with other studied autoantibodies. Meanwhile, in GFD, anti-tTG IgA confirmed the significant positive correlation with anti-ganglioside (rs=0. 231, p = 0.046) as well as with anti-pituitary (rs=0.340, p = 0.002). Both anti-TPO and anti-islet did not correlate with anti-tTG IgA levels in the GFD group.

Patients were divided into two age groups: 25 of them were 2–17 years old, and 50 patients were 18–65 years old. Only anti-tTG and anti-pituitary levels differed significantly when compared between children and adults in ND patients while in GFD, the concentrations of anti-tTG, anti-pituitary, and anti-TPO were significantly (P < 0.05) influenced between age groups.Neither ND nor GFD patients reflect any significant alteration according to sex.

The other part of the findings related to sHLAG level in study groups. The level of sHLA-G was significantly (P < 0.05) dropped after 4 months of GFD treatment as compared with the ND patients but remained higher than its level in healthy controls. A weak correlation was recorded between sHLAG and anti-pituitary IgG (rs =0.317, p=0.005) in the ND group. After 4 months of GFD, anti-pituitary IgG was still in a weak correlation with sHLAG (rs= 0.324, p=0.004) while a weak correlation was also recorded between sHLAG and anti-tTG IgA (rs= 0.279, p=0.015). Concerning age and gender, there were no statistically significant (P > 0.05) variations between male and female patients of ND as well as GFD group in sHLAG mean levels.

Eight patients coded for DQ2 and twelve for DQ8, out of the patients included in this investigation. However, fifty-five individuals coded for both DQ2 and DQ8. Regarding the allele polymorphism of the DQ2 gene, DQA1*05 and DQB1*02 were encoded in 100% and 65.097% of patients carried DQ2. DQA1*03 and DQB1*0302 alleles of DQ8 were coded in 100% and 71.641% of patients carrying DQ8, respectively. Patients were categorized into three groups: homozygote, heterozygote, and negative for each gene included in this study, according to the RT-PCR results for the DQ2 and DQ8 alleles. Levels of anti-pituitary in the ND group and its levels in the GFD group in addition to anti-islet level in ND patients and  in GFD patients were significantly increased (p < 0.0001) among the patients with heterozygous DQ2 polymorphism compared to homozygous or negative. Concerning DQ8 categorizations, anti-pituitary levels and anti-TPO levels decreased significantly (p < 0.0001) in the homozygote group compared to the heterozygote and negative in the ND group while anti-islet levels decreased significantly in homozygote and heterozygote compared with the negative group. The same results were recorded regarding the GFD group where anti-pituitary levels, anti-TPO levels and anti-islet levels.

Statistically non-significant (P >0.05) differences were recorded in sHLA-G level in the homozygote group when compared with heterozygote and negative groups of CD patients , whereas significant (P < 0.05) elevation was found in heterozygote group in comparison with negative groups, the same result was obtained after GFD treatment where sHLA-G mean concentration of DQ2 heterozygote group significantly (P < 0.05) elevated in comparison with a negative group in contrast with  sHLA-G level  of DQ2 homozygote group which did not record significant alteration with other groups. Whereas ND and GFD patients did not record significant alteration of sHLA-G level among groups divided based on DQ8 categorizations.

The present study concluded that gluten-free meals have a significant impact on many autoimmune disorders by affecting the disease severity depending on the reduction in the levels of autoantibodies specific for the diagnosis of each disease and emphasized that sHLA-G has an important role in determining the severity of CD, which pertinent to the characters of genes related to this disease. As well as the results revealed that sHLA-G reflects the sequels of this disease after consuming gluten-free meals. Patients coded heterozygosity of the DQ2 allele were more severe than other polymorphisms to develop some type of autoimmune disease.