Long-read sequencing identifies the first retrotransposon insertion and resolves structural variants causing antithrombin deficiency

Abstract

The identification of inherited antithrombin deficiency (ATD) is critical to prevent potentially life-threatening thrombotic events. Causal variants in SERPINC1 are identified for up to 70% of cases, the majority being single-nucleotide variants and indels. The detection and characterization of structural variants (SVs) in ATD remain challenging due to the high number of repetitive elements in SERPINC1. Here, we performed long-read whole-genome sequencing on 10 familial and 9 singleton cases Page 5 of 48 Thrombosis and Haemostasis For Peer Review with type I ATD proven by functional and antigen assays, that were selected from a cohort of 340 patients with this rare disorder because genetic analyses were either negative, ambiguous, or not fully characterized. We developed an analysis workflow to identify disease-associated SVs. This approach resolved, independently of its size or type, all 8 SVs detected by MLPA, and identified for the first time a complex rearrangement previously misclassified as a deletion. Remarkably, we identified the mechanism explaining ATD in 2 out of 11 cases with previous unknown defect: the insertion of a novel 2.4Kb SINE-VNTR-Alu retroelement, which was characterized by de novo assembly and verified by specific PCR amplification and sequencing in the probands and affected relatives. The nucleotide-level resolution achieved for all SVs allowed breakpoint analysis, which revealed repetitive elements and microhomologies supporting a common replication-based mechanism for all the SVs. Our study underscores the utility of long-read sequencing technology as a complementary method to identify, characterize and unveil the molecular mechanism of disease-causing SVs involved in ATD, and enlarges the catalogue of genetic disorders caused by retrotransposon insertions.