Unveiling Genetic Insights: Promises and Challenges of Next-Generation Sequencing in Diagnosing Inherited Thrombocytopenia
Authors: Moreno Carnonell Marta , Ortiz López Alicia , Silva Pablo , Casares Diana , Fernández Villalobos Manuel Jorge , Ochoa Zemeño Diego , Martinez-Laperche Carolina , Pascual Izquierdo Maria Cristina 
Published: December 2024

Introduction: Inherited Thrombocytopenia (IT) is a group of inherited disorders characterized by reduced platelet counts and often abnormal platelet functionality due to mutations in various genes, including those encoding surface receptors, cytoskeletal, and signalling proteins. The IT heterogeneity makes causative gene identification challenging. However, identifying genetic abnormalities is crucial for understanding diverse clinical presentations and potential associations with hematological malignancies. Over the past decade, advances in high-throughput sequencing technologies, particularly next-generation sequencing (NGS), have revolutionized the molecular diagnosis of IT, expanding the spectrum of knowledge and aiding in the fight against underdiagnosis. Our aim is to assess the efficacy of our targeted NGS panel for cases of suspected IT.
Methods: This study retrospectively included 98 patients with suspected IT, defined as a sustained and unexplained platelet count of less than 110 x109/L in the absence of any other cytopenia, between January 2020 and April 2024. Patients under 18 years old, patients being followed up in other centres, and/or with intermittent thrombocytopenia (normal platelet counts in some measurements) were excluded.
Genomic DNA was purified from peripheral blood samples using the Maxwell® RSC Whole Blood DNA Kit (Promega). The NGS study employed a custom capture panel encompassing 102 genes (Twist, USA). Paired-end sequencing (2 × 150 bp) was performed using the Illumina MiSeq platform (Illumina, San Diego, CA, USA), achieving an average depth of 100X. Seqone software was used for variant annotation, providing infrastructure and interface for bioinformatic analysis. The study included pathological, probably pathological, and uncertain significance variants according to ACMG classification. The study was conducted in accordance with the principles of the Declaration of Helsinki.
Results: A genetic variant of interest was found in 54% (53/98) of patients. 52 distinct variants were identified in 23 of the genes included in our panel. Considering hemorrhagic manifestation, mutations in VWF were associated with greater clinical expression (3/5, with one case of CNS bleeding), followed by ITGA2B (2/4) and GNE, NBEAL, SLNF, GFI1B and MYH9 (one patient each).
Twenty-eight percent of the patients received treatment before the suspicion/confirmation of IT (steroids, immunoglobulins, and/or thrombopoietin receptor agonists-TPO-RAs-) having been considered to have immune thrombocytopenia. None of the patients who were finally diagnosed with IT responded to treatment, except for one patient treated with TPO-RAs (ITGA2. With no response in a patient carrier of a NBEAL variant).
Conclusions: Expanded knowledge of IT suggests that prognosis and severity are related to the molecular pattern, so making suspicion, evaluation of hemorrhagic risk and early diagnosis are vital, especially to avoid unnecessary treatment. Advances in NGS have enabled, in our experience, the genetic characterization of more than 50% of cases, a percentage consistent with findings in the literature.
For patients without identified genetic mutation, some may not have IT, while others may have alterations in genes that have yet to be discovered. Personal and family medical history, physical examination, and basic laboratory tests remain essential for diagnostic guidance. Nonetheless, we continue to strive for increased sensitivity in molecular studies for these patients. Novel approaches, such as family segregation studies through whole-exome sequencing in multiple relatives, could provide additional insight.