Differential diagnosis of isolated chronic thrombocytopenia is often challenging and covers a wide range of disorders, including immune thrombocytopenia (ITP), inherited platelet functional disorders (IPFD), onset of bone marrow failure and secondary thrombocytopenias.
To assess the role of next generation sequencing (NGS) in diagnostics of inherited thrombocytopenias; to an optimal diagnostic algorithm for these disorders.
Twenty seven children and adolescents aged from 4 months to 17 years were chosen among patients with chronic isolated thrombocytopenia for NGS study. All patients meet at least one of next criteria: 1. family history of thrombocytopenia or excessive bleeding, 2. excessive bleeding, despite of mild thrombocytopenia, 3. absence of even a transitional response to IVIG and/or steroids, 4. onset of thrombocytopenia from birth. All patients did not have exact diagnosis in spite of performance of spectrum of functional tests (peripheral blood film evaluation, light transmission aggregometry, platelet flow cytometry) and exclusion of secondary thrombocytopenia causes.
DNA analysis was performed using high throughput sequencing on NextSeq (Illumina) platform. Targeted gene panel included 134 genes and was formed based on such databases as Online Mendelian Inheritance in Man (OMIM), Human Gene Mutation Database (HGMD) and published data. SeqCap EZ Target Enrichment System (Roche) was used for enrichment of genomic regions of interest. Median depth of coverage was not less than 100x, coverage of targeted region was 99% with depth of coverage not less than 10x. Conclusion on clinical significance of discovered variants was made based on American College of Medical Genetics and Genomics (ACMG) guideline.
Pathogenic and likely pathogenic variants in targeted genes were found in y 11 (41%) patients. In 5 (18%) cases variants of uncertain significance were identified. The above variants were found in following spectrum of genes: MYH9, RUNX1, ANKRD26, ETV6, WAS, NBEAL2, TUBB1, CYCS, GP1BB, THPO, ETV6. There were no relevant findings in 11 (41%) patients. Variants associated with predisposition to hematological malignancies were identified in 6 cases (3 – ANKRD26, 2 – RUNX1, 1 – ETV6). Only 1 child with pathogenic variant in RUNX1 had the family history of hemoblastosis and in case of ETV6-associated thrombocytopenia diagnosis was made after onset of acute lymphoblastic leukemia.
Among 13 patients with family history of thrombocytopenia pathogenic or likely pathogenic variants were identified in 7 (54%), in 4 (31%) – variants of uncertain significance. Onset of thrombocytopenia not immediately since birth did not exclude inherited cause of disorders in particular for patients with ANKRD26- and ETV6-associated thrombocytopenia. PBQ score ranged from 0 to 15 in our group. Bleeding severity did not correlate with probability of relevant findings in NGS.
Diagnosis of IFPD is often challenging. Functional tests are insufficient for exact diagnosis in many cases. Perspectives in this sphere are associated foremost with high throughput sequencing technologies. Positive family history seems to be a strong predictive factor of finding of causative genetic variants.