Research Projects

Common Variable Immunodeficiency (CVID) is a common primary immunodeficiency (PID) with variable clinical manifestations and age of onset. The etiology of CVID is also variable, with a large proportion of patients not having an identifiable genetic defect. Epigenetics plays an important role in the pathogenesis of the remaining 80% of cases, with DNA methylation being one potential epigenetic regulator. Age-specific alterations in the pattern of DNA methylation may play a significant role in the development of different stages of B cells. This study aims to identify DNA methylation patterns in patients with CVID at various stages of B cell development and functional characterization through RNA sequencing. Thirty patients fulfilling the European society of Immune Deficiencies (EsID) 2014 criteria for diagnosis and 30 unrelated, age, and gender-matched healthy controls will be included. Peripheral blood mononuclear cells (PBMCs) will be isolated from patient samples, and staining of Naïve, unswitched memory, and switched memory B cells will be done using FACs grade antibodies. DNA will be subjected to bisulfite treatment followed by high throughput sequencing of bisulfite treated DNA. Differentially methylated CVID relevant genes will be validated through targeted bisufite sequencing. CpG specific primers will be designed through bisulfite primer seeker, and bisulfite treated DNA from specific B cell subsets will be amplified using regular PCR. RNA will be extracted from sorted B cells using RNA purification kit, and an RNA library will be prepared following the Illumina Trueseq protocol.