Role of Somatic Mutations and Clonal Thrombopoiesis in Immune Thrombocytopenia

BACKGROUND: Immune thrombocytopenia (ITP) is an autoimmune disorder caused by antibodies directed at self-antigens leading to platelet destruction. Autoantibodies from most patients with ITP are directed to platelet membrane glycoprotein(GP) IIb-IIIa or GPIb-IX. 80% of ITP cases are classified as primary and it's unclear why auto reactive antibodies are formed in these patients. Studies from the United States, Asia and Europe show an increasing incidence of ITP with aging. Thrombopoietin receptor agonists (TPO-RA) interact directly with the TPO receptor on megakaryocytes to stimulate platelet production and can surprisingly produce sustained remissions of chronic ITP in some patients even after discontinuation of treatment. The heterogeneity of the course of ITP and the response to treatment, especially the response to treatment with thrombopoietin receptor agonists (TPO-RA), suggests pathogenic and clinical diversity of ITP. Clonal hematopoiesis is known to occur in elderly patients and has been associated with adverse outcomes including hematologic cancers and cardiovascular disease. We hypothesized that clonal hematopoiesis and associated passenger somatic mutations contribute to loss of immune tolerance in some ITP patients, causing the generation of auto-reactive antibodies and affecting the response to TPO-RA.

METHODS: Platelet RNA was extracted from 13 blood samples obtained from 11 ITP patients using Direct-zol^TM RNA MicroPrep Kit (Zymo Research). 2 patients provided samples during both relapse and remission. cDNA generation was performed from enriched total RNA using SMARTer^R Target RNA Capture for Illumina^R Kit (Clontech). Target-specific biotinylated DNA probes were designed to hybridize to mRNA transcripts for common platelet antigens including (GP) IIb-IIIa and GPIb-IX. cDNA libraries were prepared using the Nextera™ XT DNA Library preparation kit. Following the pooling of libraries they were sequenced on an Illumina Nextseq 500 instrument in Mid-output mode with single-ended reads of 150 bp.

RESULTS: In one of the paired patient specimens, we found substantial differences in platelet populations between relapse and remission on a TPO-RA. Different previously-reported non-disease-causing heterozygous single nucleotide variants (SNVs) were detected during relapse and remission. Expression at a level approximating 50% in remission and a reciprocal expression in relapse suggests that 2 point mutations leading to a missense amino acid change (Glu to Lys at amino acid 671) in GPIIb (ITGA2B) and (Thr to Met at amino acid 161) in GPIb (GP1BA) provoke an immune response (Table 1). Further, the reciprocal expression at these levels is consistent with expression of a somatic mutation in a clonal fashion.

CONCLUSION: We documented that unique platelet clones were expressed during relapse and remission of ITP. These findings demonstrate that clonal expression of platelets bearing somatic mutations in target glycoproteins are associated with ITP disease course and response to therapy. Such somatic mutations are likely to be passenger mutations acquired prior to clonal expansion. Expression of a somatic mutation in a clonal fashion may drive autoimmune response.

Table 1.

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Table 1.

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