A validated single-cell-based strategy to identify diagnostic and therapeutic targets in complex diseases [study of 13 diseases]

We conducted prospective clinical studies to validate the importance of CD4+ T cells in 13 diseases from the following ICD-10-CM chapters: Neoplasms (breast cancer, chronic lymphocytic leukemia); endocrine, nutritional and metabolic diseases (type I diabetes, obesity); diseases of the circulatory system (atherosclerosis); diseases of the respiratory system (acute tonsillitis, influenza, seasonal allergic rhinitis, asthma); diseases of the digestive system (Crohn’s disease [CD], ulcerative colitis [UC]); and diseases of the skin and subcutaneous tissue (atopic eczema, psoriatic diseases). Study participants were recruited by clinical specialists based on diagnostic criteria defined by organizations representing each specialist’s discipline. Age and gender matched healthy controls (n = 127 and 39, respectively) were recruited in the Southeast region of Sweden from outpatient clinics at the University Hospital, Linköping; Ryhov County Hospital, Jönköping, a primary health care center in Jönköping; and a medical specialist unit for children in Värnamo. Study participants represented both urban and rural populations with an age range of 8–94 years. Patients with type I diabetes and obesity had an age range of 8–18 years. 12 patients had more than one diagnosis. Total RNA was extracted using the AllPrep DNA/RNA Micro kit (Qiagen, Hilden, Germany; cat. no. 80284) according to the manufacturer’s instructions. RNA concentration and integrity were evaluated using the Agilent RNA 6000 Nano Kit (Agilent Technologies, Santa Clara, California, USA; cat. no. 5067-1511) on an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, California, USA). Microarrays were then further computationally processed as described in One-Color Microarray-Based Gene Expression Analysis Low Input Quick Amp Labeling protocol (Agilent Technologies, Santa Clara, California, USA).