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    Oral Communication

    Platelet Disorders, von Willebrand Disease and Thrombotic Microangiopathies

    Inherited and Acquired Platelet Disorders

    OC 65.3 - LongRead Nanopore Sequencing reveal novel structural variants in ITGB3, HPS5 and HPS3, allowing genetic diagnosis of Glanzmann's Thrombasthenia and Hermansky-Pudlak Syndrome in four unrelated patients

    Tuesday, June 27, 2023
    15:15 – 15:30 ET
    Room: 510
    Session Available As: On-Demand

      Presenting Author(s)

    • AM

      Ana Marin-Quilez, PhD

      Postdoctoral researcher
      Servicio de Hematología y Oncología Médica, Hospital Universitario Morales Meseguer, Centro Regional de Hemodonación, University of Murcia, IMIB, CIBERER-U765, Murcia, Spain
      Murcia, Murcia, Spain

      • Co-Author(s)

    • Ana Zamora-Cánovas

      Biotechnology
      Servicio de Hematología y Oncología Médica, Hospital Universitario Morales Meseguer, Centro Regional de Hemodonación, Universidad de Murcia, IMIB-Pascual Parrilla, Murcia, Spain.
      Murcia, Murcia, Spain

    • Belen de la morena-Barrio, PhD

      Postdoctoral fellow
      Servicio de Hematología y Oncología Médica, Hospital Universitario Morales Meseguer, Centro Regional de Hemodonación, Universidad de Murcia, IMIB-Pascual Parrilla, CIBERER, Murcia, Spain
      murcia, Murcia, Spain

    • Cristina Sierra Aisa, MD

      Hematology Department
      Hospital Universitario Cruces (Barakalo-Bizkaia)
      Bilbao, Pais Vasco, Spain

    • Christoph Male, MD, MSc

      Head of the Pediatric Hemostasis & Thrombosis Unit
      Department of Paediatrics, Medical University of Vienna, Vienna, Austria.
      Vienna, Wien, Austria

    • JP

      Jose Padilla

      Technician
      Servicio de Hematología y Oncología Médica, Hospital Universitario Morales Meseguer, Centro Regional de Hemodonación, Universidad de Murcia, IMIB-Pascual Parrilla, CIBERER, Murcia, Spain
      Murcia, Murcia, Spain

    • Nuria Fernández-Mosteirín, PhD MD

      Hematology Department
      Hospital Universitario Miguel Servet (Zaragoza)
      Zaragoza, Aragon, Spain

    • MT

      María Trapero-Marugán

      Hematologist
      Hospital Universitario Puerta de Hierro, Madrid
      Madrid, Madrid, Spain

    • PG

      Pedro L Gómez-González

      Laboratory technician
      Servicio de Hematología y Oncología Médica, Hospital Universitario Morales Meseguer, Centro Regional de Hemodonación, Universidad de Murcia, IMIB-Pascual Parrilla, Murcia, Spain.
      Murcia, Murcia, Spain

    • Maria Eugenia de la Morena-Barrio, PhD

      Reseacher
      Servicio de Hematología y Oncología Médica, Hospital Universitario Morales Meseguer, Centro Regional de Hemodonación, Universidad de Murcia, IMIB-Arrixaca, CIBERER, Murcia, Spain
      Murcia, Murcia, Spain

    • AS

      Ana Sánchez-Fuentes, PhD

      Hematologist
      Servicio de Hematología y Oncología Médica, Hospital Universitario Morales Meseguer, Centro Regional de Hemodonación, Universidad de Murcia, IMIB-Pascual Parrilla, CIBERER-U765, Murcia
      Murcia, Murcia, Spain

    • AR

      Agustín Rodríguez-Alén

      MD
      Hematology Department, Hospital Universitario de Toledo, Spain
      Toledo, Castilla-La Mancha, Spain

    • Nuria Revilla, MD, PhD

      Hospital Universitario Fundación Jiménez Díaz, Instituto Investigación Sanitaria FJD (IIS-FJD) Madrid, Spain.
      Madrid, Madrid, Spain

    • RB

      Rocío Benito

      Postdoctoral researcher
      Cancer Research Center - CSIC, University of Salamanca, Instituto de Investigación Biomédica de Salamanca (IBSAL), Spain
      Salamanca, Castilla y Leon, Spain

    • Jose Mª Bastida, PhD, MD

      Hematology Department
      Complejo Asistencial Universitario de Salamanca-IBSAL-USAL (Salamanca)
      Salamanca, Castilla y Leon, Spain

    • ML

      María Luisa Lozano

      Leader of Hematology Service in Murcia
      Servicio de Hematología y Oncología Médica, Hospital Universitario Morales Meseguer, Centro Regional de Hemodonación, Universidad de Murcia, IMIB-Pascual Parrilla, CIBERER, Murcia, Spain
      Murcia, Murcia, Spain

    • Javier Corral, PhD

      PhD
      Servicio de Hematología y Oncología Médica, Hospital Universitario Morales Meseguer, Centro Regional de Hemodonación, Universidad de Murcia, IMIB-Pascual Parrilla, CIBERER, Murcia, Spain
      Murcia, Murcia, Spain

    • José RIVERA Rivera, PhD

      PhD
      Servicio de Hematología y Oncología Médica, Hospital Universitario Morales Meseguer, Centro Regional de Hemodonación, Universidad de Murcia, IMIB-Pascual Parrilla, Murcia, Spain
      Murcia, Murcia, Spain

    Background: Genetic diagnosis of inherited platelet disorders (IPD) was classically addressed by Sanger sequencing (SS) of candidate gene, and nowadays by high-throughput sequencing (HTS). These short-read based sequencing methods fail in many patients.

    Aims: To evaluate long-read Nanopore sequencing of DNA (Nanopore) in IPD patients lacking genetic diagnosis with conventional HTS.

    Methods: We investigated four unrelated patients (P1 to P4) with clinical and platelet phenotype diagnosis of Glanzmann's thrombasthenia (GT)(P1 & P2) and Hermansky-Pudlak syndrome (HPS)(P3&P4) (Table 1). Patient´s DNA was analysed by: 1) HTS-gene panel including ITGB3, ITGA2B and HPS1 to HPS11; 2) Nanopore in a MinION device, following computational enrichment of DNA spanning regions covering GT and HPS genes. Sequences were aligned to hg38 with Minimap2 and structural variants (SV) called with Sniffles.

    Results: By HTS we identified: no variants in P1; the ITGB3 heterozygous splicing variant c.2301+1G>C in P2; the heterozygous variant c.632G>C [p.Gly211Ala] in HPS6 in P3; the heterozygous variant c.2464C>T [p.Arg822*] in HPS3 in P4. Nanopore showed in P1 and P2 the same complex SV affecting exons 2-6 in ITGB3, a 788bp deletion plus 15446bp inversion plus 174bp duplication (SV-Del-Inv-Dup), in homozygosity and heterozygosity in P1 and P2, respectively (Figure 1). In P3 revealed a homozygous 10kb deletion in HPS5, and in P4 an heterozygous 3kb deletion in HPS3. Specific PCR and SS confirmed the SV in patients and allowed family segregation. Thus, genetic diagnosis were: P1, homozygous GT, ITGB3 SV-Del-Inv-Dup; P2, compound heterozygous GT, ITGB3 SV-Del-Inv-Dup & c.2301+1G>C; P3, homozygous HPS-5, HPS5 10kb deletion; P4: compound heterozygous HPS-3, HPS3 3kb deletion & c.2464C>T [p.Arg822*].

    Conclusion(s): The new Nanopore technology overcomes limitations of standard short-read sequencing, allowing genetic diagnosis and characterization at nucleotide resolution of SV in rare IPD. Using Nanopore, we unraveled novel abnormalities in ITGB3, HPS5 and HPS3 and expanded the genetic diversity of GT and HPS.
    PI20/00926(ISCIII&Feder)-PMP21/00052(ISCIII&NG-EU)-SETH-Grant-GEAPC

    Learning Objectives:

    • Sanger and HTS sequencing fail to identify the underlying genetic defect in about 30% of patients with inherited platelet disorders (IPD)
    • The novel Nanopore technology overcomes the limitations in structural variant detection of standard short read sequencing techniques.
    • Using Nanopore, we unravel new abnormalities in ITGB3, HPS5 and HPS3 and highlight the utility of long-read sequencing as an additional diagnostic tool in IPD.