Long-read sequencing resolves structural variants inSERPINC1causing antithrombin deficiency and identifies a complex rearrangement and a retrotransposon insertion not characterized by routine diagnostic methods

Abstract

The identification and characterization of structural variants (SVs) in clinical genetics have remained historically challenging as routine genetic diagnostic techniques have limited ability to evaluate repetitive regions and SVs. Long-read whole-genome sequencing (LR-WGS) has emerged as a powerful approach to resolve SVs. Here, we used LR-WGS to study 19 unrelated cases with type I Antithrombin Deficiency (ATD), the most severe thrombophilia, where routine molecular tests were either negative, ambiguous, or not fully characterized. We developed an analysis workflow to identify disease-associated SVs and resolved 10 cases. For the first time, we identified a germline complex rearrangement involved in ATD previously misclassified as a deletion. Additionally, we provided molecular diagnoses for two unresolved individuals that harbored a novel SINE-VNTR-Alu retroelement insertion that we fully characterized by de novo assembly and confirmed by PCR amplification in all affected relatives. Finally, the nucleotide-level resolution achieved for all the SVs allowed breakpoint analysis, which revealed a replication-based mechanism for most of the cases. Our study underscores the utility of LR-WGS as a complementary diagnostic method to identify, characterize, and unveil the molecular mechanism of formation of disease-causing SVs, and facilitates decision making about long-term thromboprophylaxis in ATD patients.

The clinical and biochemical hallmarks generally associated with GLUT1DS may be caused by defects in genes other than SLC2A1

“…It should be added that the pathogenic variant found in SLC9A6 in patient P34 might affect the recycling pathway of several proteins, including GLUT1 12 . RNA‐Seq in combination with whole genome sequencing (using short‐read or long‐read technologies) might help improve our understanding in this respect 13–15 …”

“…12 RNA-Seq in combination with whole genome sequencing (using short-read or long-read technologies) might help improve our understanding in this respect. [13][14][15] HPO terms are very useful for harmonising clinical features, in delineating longitudinal disease phenotypes, and in integrating phenotypic data into diagnostic workflows. 16 However, and despite the important overlap between the HPOs of GLUT1DS associated with SLC2A1 pathogenic variants and variants in the other genes here described, those associated with the former are rather distinct.…”

Long‐read nanopore DNA sequencing can resolve complex intragenic duplication/deletion variants, providing information to enable preimplantation genetic diagnosis

“…These instruments have therefore been used for both the targeted follow-up of complex alleles 5,6 and structural variant discovery. 7 Here, we describe the use of a low-throughput long-read nanopore device, the "Flongle", to delineate the molecular breakpoint of an apparent heterozygous RB1 exon 23 deletion, at nucleotide resolution. We assess the accuracy of the nanopore platform and highlight the importance of retrospectively characterising incompletely defined sequence variants.…”

“…By contrast, “third‐generation” single molecule sequencers can generate long sequence reads that unambiguously define structural variants by spanning low‐complexity regions. These instruments have therefore been used for both the targeted follow‐up of complex alleles 5,6 and structural variant discovery 7 …”

“…These instruments have therefore been used for both the targeted follow‐up of complex alleles 5 , 6 and structural variant discovery. 7 …”