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Genetic investigations on ancient human remains affected by rheumatological pathologies are a research field of particular interest for identifying origins and the etiopathology of diseases, especially those having an autoimmune background such as rheumatoid arthritis (RA). We wish to demonstrate how reliable studies concerning this topic require collaboration between multiple disciplines, usually starting from paleopathologic observations up to immunogenetic screening, even involving analytical chemistry. Here, we focused our investigation on the skeleton of Cardinal Carlo de'Medici (1595–1666) for whom RA and psoriatic arthritis (PsA) were postulated after paleopathologic examination. RA susceptibility is linked to specific HLA alleles belonging to DRB1*04 locus, such as DRB1*0401, while Cw*0602 and DRB1*07 predispose to PsA. Thus, we genotyped the Cardinal's remains to search for RA or PsA “risk genes”. Ancient DNA is often subjected to hydrolysis followed by fragmentation. For this reason, all immunogenetic tests were preceded by an original RP-HPLC-FL method able to inform on the ancient DNA preservation and the extent of contamination, with the purpose of avoiding the risk of false positive results. After DNA isolation from a piece of bone from the Cardinal, PCR-SSP and reverse-SSO hybridization assays were applied to perform genomic HLA-typing. RP-HPLC-FL analysis revealed a good preservation of DNA without contamination by exogenous genomes. Molecular tests assigned to the Cardinal the genotype DRB1*0401/*1102 for HLA-DRB locus and Cw*04/*12 for HLA-C locus, data that support a genetic predisposition for RA but not for PsA. This multidisciplinary study has allowed us: (i) to ascertain that the remains undoubtledy belonged to the specific subject, Cardinal Carlo de'Medici; (ii) to sustain that the subject suffered from RA rather then that PsA, and (iii) to state that RA was already widespread in Europe at the Renaissance age, despite some authors claiming that the disease was introduced to the Old Continent from America after colonization during the 18 th  century

Immunogenetics and Hplc Analyses Contribute to Understanding the Etiopathology of Rheumatoid Arthritis through Studies on Ancient Human Remains