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Usefulness of an Academic High-throughput Sequencing Panel to Detect Copy Number Variations in Patients with Syndromic Thrombocytopenia

1IBSAL, IBMCC, Universidad de Salamanca, CSIC, Centro de Investigación del Cáncer (CIC), Salamanca, Spain, 2Departamento de Hematología – Complejo Asistencial Universitario de Salamanca (CAUSA), Instituto de Investigación Biomédica de Salamanca (IBSAL), Universidad de Salamanca (USAL), Salamanca, Spain, 3Servicio de Pediatría - Hospital Clínico Universitario de Valladolid, Valladolid, Spain, 4Servicio de Oncología y Hematología Pediátricas - Hospital Materno-Infantil Gregorio Marañón, Madrid, Spain, 5Servicio de Hematología - Hospital de Jerez, Cádiz, Spain, 6Servicio de Hematología y Hemoterapia - Hospital Virgen de la Salud, Toledo, Spain, 7Servicio de Hematología – Complejo Asistencial de Burgos, Burgos, Spain, 8Servicio de Pediatría – Complejo Asistencial de Zamora, Zamora, Spain, 9Servicio de Hematología y Oncología Médica – Hospital Universitario Morales Meseguer, Centro Regional de Hemodonación, Universidad de Murcia, IMIB-Arrixaca, CB15/00055-CIBERER, Murcia, Spain

Abstract Number: LPB0108

Meeting: ISTH 2021 Congress

Theme: Diagnostics and OMICs » Laboratory Diagnostics

Background: High-throughput sequencing (HTS) has allowed to increase the molecular diagnosis in patients with inherited thrombocytopenia (IT), mainly caused by single-nucleotide variants (SNVs). Syndromic IT could be caused by structural variants like Copy Number Variations (CNV), which are not usually detected by gene panels.

Aims: To design and apply an academic HTS panel for identifying CNVs in patients with syndromic IT.

Methods: A custom HTS panel was designed using the SureDesign Studio (Agilent) including 85 genes with relevance in ITs. Libraries were prepared following the SureSelectQXT-Target-Enrichment protocol (Agilent), and finally sequenced on an Illumina NextSeq platform. Data were processed using an in-house pipeline. CNV analysis was made using “Depth-Of-Coverage” method, assuming a linear correlation between the coverage depth and the number of copies. Lower reading depth than expected represented losses or deletions, while higher depth represented gains or amplifications. Molecular findings were validated with aCGH or MLPA.

Results: Clinical and biological characteristics are showed in Table 1. Patient 1 and 2 (P1, P2) showed a specific syndromic phenotype, while clinical suspicion of P3 and P4 was not clarified. In P1, thrombocytopenia-absent radius syndrome was previously diagnosed and HTS allowed us to identify a low frequency SNP which affects the 5’UTR region of RBM8A (c.-21 G>A; rs139428292) and a deletion on chromosome 1q. In P2, Di George syndrome (DGS) was clinically diagnosed and deletion of GP1BB and TBX1 confirmed a del22q. In P3, a syndromic thrombocytopenia was suspected and a deletion of GP1BB and TBX1 was detected, which led to confirm the diagnosis of incomplete DGS. Finally, In P4 with syndromic thrombocytopenia, a deletion on chromosome 21q which involved RUNX1 was identified (Figure 2).

Case Age, gender / BS Clinical symptoms Laboratory phenotype Suspected / final IT CNVs
1 45 y, female /
2: heavy menstrual bleeding		 Bicuspid aortic valve and cow’s milk intolerance, bilateral radioulnar agenesis, hip dysplasia, other skeletal and facial malformations P=84×103/mm3
MPV=13,8 fL
BF: large platelets		 Thrombocytopenia with absent radius syndrome RBM8A deletion,
del1q, and SNP which affects 5’UTR region
2 13 y, male /
0		 Hypertelorism, prognathism, dental mal-occlusion, short stature, psychomotor retardation, bicuspid aortic valve, umbilical hernia P=135×103/mm3
VPM=13.7 fL
BF: large platelets		 DiGeorge syndrome GP1BB and TBX1 deletion, del22q
3 14 y, male /
0		 Hypertelorism, short stature, learning difficulties, hearing loss, infections, situs solitus P=79×103/mm3
MPV=12.8 fL
BF: large platelets		 Syndromic thrombocytopenia / Incomplete DGS GP1BB and TBX1 deletion, del22q
4 1 y, male /
2: epistaxis and ecchymosis		 Bilateral sensorineural deafness, corpus callosum hypoplasia, cardiac and facial malformations P=35×103/mm3
MPV=8 fL
BF: dysmorphic cells		 Syndromic thrombocytopenia RUNX1 deletion
del21q
Abbreviators: P: platelet counts; MPV: mean platelet volume; BF: blood film; DGS: DiGeorge syndrome; BS: bleeding score according to ISTH-BAT.

Clinical and biological phenotype of patients with syndromic IT in whom CNVs have been analyzed.

Representation of CNVs identified in 4 patients with syndromic ITs.

Conclusions: This academic HTS panel has allowed us to detect SNVs and CNVs. CNV analysis is particularly useful in genetic diagnosis of syndromic IT in which no conventional variants have been detected.

To cite this abstract in AMA style:

Santos-Mínguez S, Miguel-García C, Ramírez-Maldonado V, Serramito-Gómez I, Marín-Quilez A, Matías-Martín J, González-Porras JR, González-García H, Huerta-Aragonés J, Blázquez-Goñi C, Rodríguez-Alén A, García-Diaz C, Urbaneja-Rodríguez E, Hortal Benito-Sendín A, Revilla N, Palma-Barqueros V, Rivera J, Hernández-Rivas JM, Bastida JM, Benito R. Usefulness of an Academic High-throughput Sequencing Panel to Detect Copy Number Variations in Patients with Syndromic Thrombocytopenia [abstract]. Res Pract Thromb Haemost. 2021; 5 (Suppl 2). https://abstracts.isth.org/abstract/usefulness-of-an-academic-high-throughput-sequencing-panel-to-detect-copy-number-variations-in-patients-with-syndromic-thrombocytopenia/. Accessed September 21, 2023.

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