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Exome sequencing in platelet secretion defects
not discriminatory, genotyping using next-generation DNA sequencing (NGS) could be a comprehensive and cost-effective strategy for the diagnosis of platelet func- tion disorders.11-13 Indeed, NGS-based approaches, based on whole-exome sequencing (WES) or custom gene pan- els, proved to be successful for the diagnosis of inherited platelet defects.11,13,14 Leo et al. applied WES to study 329 candidate genes involved in platelet function defects and identified gene variants in patients with defects in Gi sig- naling and with platelet secretion abnormalities.15 WES was also successful in identifying causal mutations in the RASGRP2 gene, which encodes a protein required for sig- naling and platelet activation,16,17 and in identifying a causal mutation displaying autosomal dominant inheri- tance located in the THBD gene.18 However, a standard- ized pipeline or procedure linking the identified gene defects to the specific sub-phenotype of diverse platelet function disorders is still lacking.
Given the positive experience acquired with the use of WES in identifying potentially pathogenic genetic variants in platelet function defects, the use of NGS-based diagnos- tics provides a great opportunity to improve causal gene identification and understand the underlying clinical phe- notype.19-22 For this reason, we decided to apply exome sequencing in a well-characterized group of patients with primary PSD and clinically relevant bleeding.5 The aim of our pilot study was to test whether WES could be an ade- quate diagnostic tool for causal gene discovery in a hetero- geneous group of platelet function defects such as primary PSD.
Methods
Study population
Fourteen unrelated patients with a diagnosis of primary PSD were enrolled from among 360 individuals with suspected platelet function disorders referred to our outpatient clinic at Ospedale Maggiore Policlinico (Milan, Italy).
The patients’ inclusion criteria were: (i) European ancestry; (ii) platelet count >120x109/L; (iii) impaired platelet ATP secretion after stimulation with two or more agonists measured by lumi- aggregometry; (iv) normal expression of platelet glycoprotein (GP) Ib/IX/V and GPIIb/IIIa to exclude Bernard-Soulier syndrome and Glanzmann thrombasthenia; (v) absence of any other known platelet disorder; and (vi) absence of von Willebrand disease. Four family members of one patient (C740) were also included and studied.
All studied subjects abstained from taking drugs that affect platelet function for 2 weeks before blood sampling. All platelet function results were compared with our internal normal ranges.
The study was approved by the local Ethical Committee of the Ospedale Maggiore Policlinico and carried out according to the Declaration of Helsinki. All participants signed informed consent.
Platelet phenotyping
Personal and family histories and results of blood tests including a complete blood count, prothrombin time and activated partial thromboplastin time determined by standard methods, von Willebrand factor antigen and von Willebrand factor ristocetin cofactor determined by an automated latex enhanced immunoas- say (Instrumentation Laboratory, Milan, Italy)23 were collected (Online Supplementary Methods). The bleeding severity score (BSS) was calculated for each patient according to Tosetto et al.24 (normal values: children <2; men <5; women <6).
Blood samples were drawn into trisodium citrate for coagula- tion, von Willebrand factor measurement, and platelet function studies and into K–EDTA for DNA extraction25 and blood cell counts.
Platelet aggregation and ATP secretion induced by ADP (4 and 20 μM), collagen (2 μg/mL), thrombin receptor activator peptide- 14 (10 μM), and thromboxane A2 analog U46619 (1 μM) were measured in platelet-rich plasma by lumi-aggregometry (Chrono- log 560, Mascia Brunelli, Milan, Italy).26 Platelet-rich plasma was prepared as previously reported.27 Intraplatelet ADP, ATP, sero- tonin, and fibrinogen content were measured as previously report- ed28,29 (Online Supplementary Methods).
Whole-exome sequencing
Individual exomes were enriched using a SeqCap EZ Human Exome Library Kit v2.0 (Roche NimbleGen) and paired-end sequencing was carried out on the HiSeq2000 (Illumina, San Diego, CA, USA) at the Beijing Genomics Institute (www.bgi.com).
The Short Oligonucleotide Analysis Package aligner (soap2.21)30 was used to align reads to the reference human genome (hg19/GRCh37) and produce individual binary alignment map (BAM) files. The Genome Analysis Tool Kit was used for quality recalibration, duplicate read marking, insertions/deletions (indels) realignment, and BAM sorting to produce a merged, sample-level variant calling file (VCF) (Online Supplementary Methods).
Variant filtering and candidate gene discovery
Variant filtering and candidate gene discovery were performed on the project level, merged VCF file containing 14 unrelated Italian PSD patients and 16 healthy controls by using two different filtering strategies: selection of singletons and filtering for the sin- gle nucleotide variants (SNV) reported by Leo et al.15 (Online Supplementary Methods).
Variant pathogenicity was assigned according to the American College of Medical Genetics and Genomics (ACMG) pathogenic- ity classification.31 Platelet gene expression was evaluated using the Human Proteome Map (HPM).32 (Online Supplementary Methods).
Results
Clinical characteristics of patients with platelet secretion defects
Of 360 patients with suspected platelet disorders inves- tigated at our center, 14 unrelated patients (12 females and 2 males; median age 23 years) fulfilled the study inclusion criteria (Table 1). The patients’ BSS ranged between 0 and 15 and 64% of the cases resulted abnormal (Table 1). Prothrombin time, activated partial thromboplastin time, plasma fibrinogen, and von Willebrand factor levels were within the normal ranges (data not shown). Platelet count was normal in all PSD patients (median 258 x109/L, mini- mum-maximum 120-357; normal values 150-450), except for patient C749 who had a slightly low platelet count (120 x109/L).
Platelet functions studies
Platelet aggregation was lower than the normal range in the majority of the patients with all agonists tested (Figure 1A) and rapidly reversible in 60% of the cases when induced by ADP (4 μM). Platelet ATP secretion was absent after stimulation by ADP (4 μM) in all patients and lower than the normal range in response to the other agonists in the majority of cases (Figure 1B). In particular, platelet
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