Next-Generation Sequencing of HLA Loci Identifies Predisposing and Protective Factors for Immune-Mediated Thrombotic Thrombocytopenic Purpura in a Japanese Population

Background Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is an autoimmune disorder caused by neutralizing anti-ADAMTS13 autoantibodies. This condition leads to life-threatening microvascular thrombi in systemic organs due to an imbalance between ADAMTS13 and von Willebrand factor. While several European groups reported that HLA-DRB1*11 was one of the strongest genetic risk factors for iTTP, no large-scale studies have analyzed which HLA alleles convey greater risk for iTTP in genetically isolated Japanese. Therefore, we analyzed ten HLA loci using next-generation sequencing. Materials and Methods We performed a prospective cohort study among Japanese patients with iTTP. The diagnosis of iTTP was suspected in cases of thrombocytopenia and hemolytic anemia with unknown cause, and was confirmed by both severe deficiency of ADAMTS13 (below 10% of normal) and the presence of anti-ADAMTS13 inhibitor. Fifty-six patients with iTTP from 19 institutes were enrolled in this study from August 2017 to March 2019, and 52 patients were finally eligible. An EDTA-treated serum sample was obtained from each patient at enrollment, and was analyzed via next-generation sequencing to identify the HLA alleles at following loci: A, B, C, DRB1, DRB3/4/5, DQA1, DQB1, and DPB1. Then, we compared allele frequencies at each locus in iTTP patients with those in a healthy Japanese population as reported by Nakajima et al in 2001. Additionally, we collected data on laboratory tests and clinical response to treatment during the acute phase. The HLA allele frequency of patients with iTTP and controls were compared by Fisher's exact test, and Bonferroni correction was applied to observed P-values. In this study, we multiplied the P-value by the number of alleles in each locus whose frequency was more than 1% in control to calculate the corrected P-value (Pc). This study was approved by the IRB of each referring institute. Results The male/female ratio of enrolled patients was 22/30. The median age was 58 years (range: 1-81). Based on the underlying disease, iTTP patients were classified as having primary TTP (n=38), autoimmune disease (n=12), malignancy (n=1), or acute pancreatitis (n=1). Plasma levels of ADAMTS13 activity were less than 3% in all cases, and the median concentration of ADAMTS13 autoantibodies was 2.5 Bethesda units/mL (range: 0.5-113). All patients showed severe thrombocytopenia (median platelet count: 12×109/L) and hemolytic anemia (median hemoglobin level: 8.