Abstract
Introduction
The inherited platelet disorders (IPD) are a heterogeneous group of rare diseases including quantitative and/or qualitative platelet defects. Classically, patients with IPD are first functionally tested to know the possible defect before sequencing a single or a few genes. Phenotipyc diagnostic of IPD often requires light transmission aggregometry, quantitative analysis of receptors by flow cytometry and fluorescence and electron microscopy. This diagnostic strategy is complex, poorly standardised and time consuming. In addition, the phenotype can seldom guide the singles candidates genes for conventional Sanger squencing. Therefore, many patients remain without a accurate diagnosis of their IPD. Next generation sequencing (NGS) enables the simultaneous analysis of large groups of candidate genes in IPD and may be useful for rapid genetic diagnosis.
The aim of this study was to design and validate a NGS panel for IPD.
Patients & Methods
We describe a strategy for rapid genetic diagnosis of IPD with Illumina sequencing of 60 candidates genes previously associated with IPD (table1). The baits were designed to tile 400 kb of gDNA sequence corresponding to the exons and splice sites in all known transcripts of the candidate genes identified. The bait library was tested by enriching the candidate IPD genes from 50 ng DNA obtained and sequencing by Nextera Rapid Custom Enrichment system. Results were analysed by Variant Studio system and Sequencing Analysis Viewer.
A total of 21 patients were studied. For the validation process, DNA samples of 9 unrelated patients with IPD and their mutation known were used: two patients with Glanzmann Thrombasthenia (ITGA2B, p.Ala989Thr, p.Val982Met and p.Glu538Stop; ITGB3, p.Leu222Pro and p.Tyr216Cys), one Hermansky-Pudlak Sd. (HPS1, p.Glu204 Stop), another with Bernard-Soulier Sd. (GPIX, p.Phe71Stop), a case of Congenital Amegakaryocytic Thrombocytopenia (MPL, p.Arg102Cys), and 2 patients with Chediak Higashi Sd. (LYST, p.Gly3725Arg and p.Cys258Arg). Once validated, the NGS panel was used for genetic diagnostic of 8 patients with suspected IPD.
Results
Eleven mutations, previously identified in another center by conventional sequencing, were detected by our panel NGS (100% success in the validation process). We then tested this strategy for patients with suspected of IPD without diagnosis: I. a 13 years old girl with agenesis of the corpus callosum, facial dysmorphia, renal agenesis and thrombocytopenia was diagnosed of Thrombocytopenia FLNA-related and Periventricular Nodular Heterotopia (PNHV)[mutation in the FLNA was detected (p.Thr1232Ile)]. II. A two years old patient with severe thrombocytopenia and recurrent infections was diagnosed of Wiskott-Aldrich Sd (WAS, p.Arg268Gly fs Stop40). III. A patient with deafness, macrothrombocytopenia, and Döhle bodies was diagnosed by MYH9 deletion (MYH9; p.Asp1925Thr fs Stop23). IV. Six members of a family (2 of them with symptoms of mucocutaneous bleeding, and macrothrombocytopenia), in which an insertion in NBAL2 (p.Gly1142Arg fs Stop49) gene was found. Therefore, Gray Platelet Sd was diagnosed. Moreover, one patient with aspirin-like syndrome showed a P2RY12 mutation (p.Val279Met). Finally, mother and son with mild Hemophilia A (F8; p.Gln2208Arg) were detected.
Conclusions
This NGS panel enables a rapid genetic diagnostic of IPD.
The use of NGS-based strategy is a feasible tool for the diagnosis of IPD that could be added to the screening of these disorders.
Five mutations have not previously been described in the literature.
Table 1: Sixty candidates' genes previously associated with IPD: Inherited Platelet Disorders Genes = 60 Cytoskeletal Assembly and Structural Proteins GP1BA, GP1BB, GP5, A2M, GP9, VWF, ITGA2, ITGA2B, ITGB3, ABCA1, ANO6,FERMT3, ACTN1, MASTL Disorders of agonist platelet receptors P2RX1, P2RY1, P2RY12, TBXA2R, TBXAS1, ADRA2A, GP6, CD36 o GP4, DTNBP1 Disorders signal transduction GNAI3, GNAQ, GNAS, PLA2G7, PLCB2PTS, GGCX, DPAGT1, DHCR24 Disorders of platelet granules NBEAL2, GFI1B, PLAU, HPS1, HPS3, HPS4, HPS5, HPS6, LYST, MLPH, BLOC1S3, BLOC1S6, AP3B1, VIPAS39, VPS33B, RAB27A, MYO5A, USF1 Thrombocytopenias and syndromes WAS, MYH9, FLNA, FLI1, STIM1, HOXA11, ANKRD26, MPL, RBM8A RUNX1, GATA1
Disclosures
No relevant conflicts of interest to declare.
De novo transcriptome based on next-generation sequencing reveals candidate genes with sex-specific expression in Arapaima gigas (Schinz, 1822), an ancient Amazonian freshwater fish
