Background: Inherited platelet disorders (IPDs) are a heterogeneous group of diseases where genetic alterations cause platelet malfunction and lead to a hemorrhagic tendency. Next-generation sequencing (NGS) arises as an interesting diagnostic tool for these entities whose phenotypes are often ambiguous, allowing simultaneous analysis of multiple candidate genes.

Aims: To assess the utility of NGS in the diagnosis of IPDs.

Methods: Forty-one patients with suspected IPDs were selected between 2019 and 2021 based on platelet function test findings. NGS was performed using a custom panel (Nonacus) that included 83 genes associated to diverse hemostatic disorders (Table 1) chosen according to current published evidence. Sequencing results were processed using Datagenomics platform (Imegen). Variants were classified following to ACMG criteria. Pathogenic variants were confirmed with Sanger sequencing.

Results: Our panel detected 17 variants in 10 genes of 17 patients (Table 2), 11 of them were classified as pathogenic, 2 likely pathogenic and 4 variants of uncertain significance (VUS). No pathogenic variants were found in 24 patients.

NGS served as a confirmatory tool in 4 patients: 2 cases of Glanzmann thrombasthenia and 2 cases of Bernard Soulier Syndrome with variants affecting ITGA2B (GT) and GP1BA and GP9 (BSS). Other 2 suspected cases of GT carried variants in GP1BA classified as likely pathogenic.

In 7 patients where functional studies were inconclusive, our panel detected pathogenic variants in NBEAL2, HPS1, MYH9 and TUBB1 and provided a diagnostic orientation.

Four VUS were found in GP1BA, TBXAS and TBXA2R genes. Further studies are needed to elucidate their role in the pathogenesis of IPDs.

Conclusion(s): NGS is a feasible diagnostic test for IPDs. Our data reflect the importance of combining functional and genetic studies to provide an accurate diagnosis. Interpretation of genetic variants, especially VUS, can be challenging and functional protein models are necessary to determine their clinical significance.

Table 1

Genes included in our custom panel of hemostatic disorders.

Table 2

Detailed report of the 17 variants found in our study. P: pathogenic variant. LP: likely pathogenic variant. VUS: variant of uncertain significance.

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/application-of-next-generation-sequencing-in-the-study-of-inherited-platelet-disorders/