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Haematologica 2019 Volume 104(10):2084-2090
Platelet Biology & its Disorders
Complications of whole-exome sequencing for causal gene discovery in primary platelet secretion defects
Marcin M. Gorski,1,2 Anna Lecchi,1 Eti A. Femia,1 Silvia La Marca,1 Andrea Cairo,1 Emanuela Pappalardo,1,2 Luca A. Lotta,1 Andrea Artoni1 and Flora Peyvandi1,2
1Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, Milan and 2Università degli Studi di Milano, Department of Pathophysiology and Transplantation and Fondazione Luigi Villa, Milan, Italy
ABSTRACT
Primary platelet secretion defects constitute a heterogeneous group of functional defects characterized by reduced platelet granule secretion upon stimulation by different agonists. The clinical and laboratory het- erogeneity of primary platelet secretion defects warrants a tailored approach. We performed a pilot study in order to develop DNA sequence analysis pipelines for gene discovery and to create a list of candidate causal genes for platelet secretion defects. Whole-exome sequencing analysis of 14 unrelated Italian patients with primary secretion defects and 16 controls was performed on Illumina HiSeq. Variant prioritization was carried out using two filtering approaches: identification of rare, potentially damaging variants in platelet candidate genes or by selecting singletons. To corrobo- rate the results, exome sequencing was applied in a family in which platelet secretion defects and a bleeding diathesis were present. Platelet candidate gene analysis revealed gene defects in 10/14 patients, which included ADRA2A, ARHGAP1, DIAPH1, EXOC1, FCGR2A, ITPR1, LTBP1, PTPN7, PTPN12, PRKACG, PRKCD, RAP1GAP, STXBP5L, and VWF. The analysis of singletons identified additional gene defects in PLG and PHACTR2 in two other patients. The family analysis confirmed a missense variant p.D1144N in the STXBP5L gene and p.P83H in the KCNMB3 gene as potentially causal. In summary, exome sequencing revealed potential causal variants in 12 of 14 patients with primary platelet secretion defects, high- lighting the limitations of the genomic approaches for causal gene identifi- cation in this heterogeneous clinical and laboratory phenotype.
Introduction
Disorders of platelet function are characterized by highly variable mucocuta- neous bleeding manifestations and excessive hemorrhage following surgical proce- dures or trauma.1-4 Primary platelet secretion defects (PSD) are the most common platelet functional defects5 and display both clinical and laboratory heterogeneity.6 From a clinical standpoint, PSD may be associated with a mild to severe bleeding tendency.7 Thus, given the heterogeneous nature of PSD, laboratory testing is lim- ited to specialized laboratories and accurate mechanistic diagnosis remains chal- lenging.
Platelet aggregation and secretion studies with lumi-aggregometry, in which dense granule secretion is assessed in parallel with traditional light transmission aggregom- etry, provide evidence for platelet dysfunction.8,9 PSD is characterized by reduced or absent δ-granule secretion upon stimulation by one or more platelet aggregation ago- nists either at low or high doses.8,9 However, lumi-aggregometry, the gold standard technique for platelet function studies, is not always predictive of the molecular mechanisms, rendering the mechanistic differentiation of primary PSD difficult.
Multiple inherited alterations of platelet function have been described, including forms with different patterns of inheritance.2,4,10 When the laboratory phenotype is
Correspondence:
FLORA PEYVANDI
flora.peyvandi@unimi.it
Received: August 21, 2018. Accepted: February 22, 2019. Pre-published: February 28, 2019.
doi:10.3324/haematol.2018.204990
Check the online version for the most updated information on this article, online supplements, and information on authorship & disclosures: www.haematologica.org/content/104/10/2084
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