Celiac disease (CD) is an autoimmune disease affecting approximately 3 million people worldwide (Gallegos and Merkel, 2019). Patients of CD are sensitive to gluten (a protein found in wheat, rye and barely) containing diet which can trigger the immune response in their body leading to intestinal cell damage resulting in malabsorption of essential nutrients, diarrhea, fatigue, weight loss and many other symptoms. Prevalence of CD is not same in all countries, it is more prevalent in European countries as compared to Asia-pacific region. These variations could be due to the difference in genetic factors and per-capta wheat consumption (Cummins and Roberts-Thomson, 2009, Al-Hakami, 2016). Clinical manifestations of CD are wide-ranging from intestinal symptoms to non-intestinal symptoms (Roshanzamir et al., 2021), however, most of the patients exhibit intestinal problems (Lebwohl et al., 2018). Usually, diagnosis of CD is based on the titer of IgA anti-transglutaminase antibodies (IgA anti-tTG) and endoscopic findings (Saadah et al., 2020, Morreale et al., 2017). Confocal endomicroscopy can also be used for the diagnosis of CD (Meriam et al., 2020). Saadah et al. also suggested that serological assessment of deamidated gliadin peptide of IgG can be used in CD diagnosis instead of measuring the IgA anti-tTG (Saadah et al., 2020).
In children, CD can influence the growth and development, however, in adults these side effects are less pronounced. CD is of major concern among all age groups owing to the unavailability of any cure/therapy for this disease. Individuals who are genetically at risk and/or their immune system is sensitive to gluten, can only manage the symptoms by strictly following the gluten free diet (Reviewed in Kamiński et al., 2020). Although, gluten-free diet successfully manages CD and helps in intestinal healing, however, some patients do not respond to this dietary intervention and develop CD related complications like intestinal adenocarcinoma, T-cell lymphoma and refractory sprue(Green and Cellier, 2007). Several environmental and genetic factors are involved in the etiology of CD (Serena et al., 2019). Approximately 40% of the CD patients carry Human leukocyte antigen (HLA) class II haplotype DQ2 or DQ8 (Gnodi et al., 2022). Studies have shown that HLA alter the intestinal microbiome composition and that dysbiosis triggers sensitivity to gluten (Reviewed in Serena et al., 2019). Genome wide-studies have also identified 39 non-HLA genes which are linked with CD risk (Lebwohl et al., 2018). Among these, CCR3 is an important non-HLA gene encoding C-C chemokine receptor type 3 (CCR3 receptor) which is abundantly expressed on immune cells involved in allergic reactions like mast cells, eosinophils, basophils and TH2 cell subsets (Yang et al., 2015). CCR3 receptor plays a vital role in basal trafficking of eosinophils to intestinal mucosa (Humbles et al., 2002). CCR3 receptor and its respective chemokines have been implicated in several diseases like T-cell lymphoma, cutaneous lupus erythematosus (Freutel et al., 2011, Miyagaki et al., 2010). CCR3 receptor also acts as co-receptor for HIV-1 and help it in cell entry (He et al., 1997).
Genetic variations in CCR3 and nearby region can influence its function and increase the risk of CD and other diseases like type 1 diabetes and rheumatoid arthritis. Studies have identified that CCR3 rs6441961 T > C increase the susceptibility for CD (Smyth et al., 2008, Yang et al., 2015, Amundsen et al., 2010, Izzo et al., 2011). This polymorphism is located on 3p21 chromosomal region which contains gene cluster of chemokine receptors. CCR3 also belongs to the chemokine receptor gene cluster and this is the nearest gene located only 44 kb from rs6441961 T > C (Dema et al., 2009). Assessment of genetic risk factors can help in early diagnosis of CD which can prevent long-term complications and reduce patient suffering.
rs6441961 is widely studied in European populations, however, its risk potential for CD in Asian populations needs to be studied. European countries are economically stable and commonly employ high throughput advance techniques like GWAS (genome-wide association studies), WES (whole exome sequencing) and TaqMan genotyping assay for genotyping of rs6441961(Simonyte et al., 2017, Ji et al., 2017, Hussain et al., 2019). Although these techniques are quite specific and sensitive, however, they are costly, therefore; cannot be used for screening of larger populations in resource limited settings like Pakistan. Thus, less expensive and specific genotyping assays should be developed to conduct large scale replicative studies in developing countries. One such technique is tetra-ARMS-PCR which is simple, rapid as well as cost effective. The main objective of this study was to design a tetra-ARMS-PCR genotyping assay for genotyping of CCR3 rs6441961 T > C and find its association with celiac disease in Pakistani Punjabi population.