VPB1251 - Identification and evaluation of novel variants associated with platelet function disorders by NGS-based high-throughput sequencing

Background: Inherited platelet disorders (IPD) are classified as rare diseases. New IPD-associated genes and variants are continuously reported to databases, but strong evidence is sometimes lacking. Coherent classification of the underlying genetic cause is of high relevance for appropriate prognosis and genetic counseling.

Aims: To diagnose patients with IPD. Clinical signs and platelet function analyses were complemented by next generation sequencing (NGS).

Methods: NGS-based analysis using an in-silico panel applied to whole-exome data (Nextera XT-DNA-Library Kit, Illumina; Nextera-xGen-Exome-Research-Panel). The ISTH Platelet Disorder TIER1 genes (v.ISTH_2020.1) are included in our analysis. Data analysis was performed by GensearchNGS (PhenoSystems). Genetic variants were called for minor allele frequency < 0.02, exon distance (±20 bp into intron), and quality (variant frequency < 0.15, coverage ≥10X). Fourty-one patients that were evaluated in 2020 (n=28) and 2021 (n=13) in specialized centers due to a bleeding diathesis and considered having a familial, syndromal or functionally diagnosed platelet disorder with early onset, were included.

Results: In 15 patients we detected single nucleotide variants in: ACTN1; ANKRD26; GFI1B; GP1BA; FLNA; ITGA2B; ITGB3; JAK2; NBEAL2; WASP; and THPO. According to ACMG criteria three variants were defined as pathogenic, three as likely pathogenic, and six as uncertain significance (VUS). In three cases segregation analyses allowed us to classify so far unreported variants as likely pathogenic. In the remaining six VUS, segregation analyses and functional studies were suggested and are still pending. A special constellation was detected in one patient carrying two heterozygous VUS in JAK2 and THPO, respectively. The JAK2 missense variant is inherited from the mother and the THPO missense variant from the father. These two variants in combination could contribute to the patient’s phenotype, however, further characterization is still on-going.

Conclusion(s): We identified the genetic cause of a broad range of clinically conspicuous platelet disorders by NGS with a positive testing ratio of 37%.