Long-Read Sequencing Identifies the First Retrotransposon Insertion and Resolves Structural Variants Causing Antithrombin Deficiency.

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de la Morena-Barrio, Belén  ORCID logo  https://orcid.org/0000-0002-6696-7762
Stephens, Jonathan 
de la Morena-Barrio, María Eugenia  ORCID logo  https://orcid.org/0000-0001-7426-4947
Stefanucci, Luca 
Padilla, José 

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 with type I ATD proven by functional and antigen assays, who 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 eight SVs detected by multiple ligation-dependent probe amplification, 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 2.4 kb SINE-VNTR-Alu retroelement, which was characterized by de novo assembly and verified by specific polymerase chain reaction 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.

Antithrombin III Deficiency, Antithrombins, Humans, Nucleotides, Retroelements
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Thromb Haemost
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Georg Thieme Verlag KG
Medical Research Council (MR/P02002X/1)
British Heart Foundation (RE/18/1/34212)
Funding has also been provided by the National Institute for Health Research England