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 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.

Keywords

long-read sequencing - antithrombin deficiency - structural variants - SVA retrotransposon

Author Contributions

B.M.-B., W.H.O., J.C., and A.S.-J. designed the study. M.M.B., L.S., J.P., A.M., N.G., F.L.R., and V.V. helped with the study design. B.M.-B., M.M.B., J.P., and A.M. performed laboratory experiments and analyzed the experimental data. J.S. performed sample preparation and executed long-read sequencing. A.S.-J. developed the analysis workflow for long-read sequencing, applied this to data processing, and performed the computational and statistical analyses. B.M.-B. performed computational analyses and variant validation. J.J.L.C. and F.V. provided valuable insight into microarray and NGS data analysis. A.U., M.F., M.P., and P.M. recruited participants and collected the clinical data and samples. B.M.-B., W.H.O., J.C., and A.S.-J. wrote the manuscript. All authors read and approved the final version of the manuscript.

Data and Code Availability

The workflow developed for the detection of structural variants is publicly available at http://github.com/who-blackbird/magpie.

Patient Consent

All included subjects gave their written informed consent to enter the study.

Ethical Approval

This study was approved by the Ethics Committee of Morales Meseguer Hospital and the East of England Cambridge South National Institutional Review Board (13/EE/0325). The research conforms to the principles of the Declaration of Helsinki and their later amendments.

Publication History

Received: 16 September 2021

Accepted: 10 January 2022

Article published online:
28 June 2022

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