Protein prenylation is defective in mevalonate kinase deficiency and is an accurate biomarker in peripheral blood mononuclear cells

Abstract

Introduction: Mevalonate kinase deficiency (MKD, which includes HIDS and mevalonic aciduria) is caused by recessive, hypomorphic mutations in the MVK gene encoding mevalonate kinase, a crucial enzyme of the mevalonate pathway. This biochemical pathway is required for the synthesis of isoprenoid lipids that are essential for the post-translational prenylation of proteins, particularly small GTPases such as Rab proteins and Rap1A. Blockade of the mevalonate pathway prevents the synthesis of isoprenoid lipids and results in the cytosolic accumulation of small GTPase proteins in their unprenylated form. It has long been assumed that mutations in MVK lead to defective protein prenylation in patients with MKD, but direct evidence for the accumulation of unprenylated proteins in patients' cells has been lacking. Objectives: To address the question whether protein prenylation is defective in MKD patients' peripheral blood mononuclear cells (PBMCs), we optimized an in vitro prenylation assay that is capable of detecting very low levels of unprenylated Rab proteins in cell lysates and compared it to a western blot approach that specifically recognises the unprenylated form of Rap1A. Methods: We analysed freshly-isolated PBMCs from nine individuals with genetically-confirmed MKD and compared these with PBMCs from parents who were heterozygous for MVK mutations, as well as PBMCs from unaffected controls and 2 individuals each with genetically-confirmed FMF, CAPS or TRAPS. Results: Using the in vitro prenylation assay we observed a very clear accumulation of unprenylated Rab proteins in freshlyisolated PBMCs from eight MKD patients with different compound heterozygous mutations in MVK. In a patient homozygous for the V377I mutation the defect in Rab prenylation was milder but still detectable. Unprenylated Rap1A was also identifiable by western blotting in the MKD patients that had the greatest defect in Rab prenylation, but this western blot approach was considerably less sensitive than the in vitro prenylation assay. Importantly, the defect in Rab and Rap1A prenylation was absent in individuals with FMF, CAPS or TRAPS, and absent in individuals with heterozygous mutations in MVK or healthy volunteers. Conclusion: These findings demonstrate that protein prenylation is indeed defective in individuals with MKD. Furthermore, the accumulation of unprenylated Rab proteins in PBMCs is a sensitive and specific biomarker of MKD that distinguishes this autoinflammatory disease from FMF, CAPS and TRAPS and could therefore be used to aid diagnosis.L6279734992019-06-11 <br />