Page 191 - 2019_10 resto del Mondo_web

Exome sequencing in platelet secretion defects
Platelet candidate gene filtering approaches
Candidate gene discovery was carried out by two inde- pendent filtering approaches: by identification of variants in platelet candidate genes and by selecting singletons (Online Supplementary Figure S1). In the former approach, we selected from PSD patients all rare, potentially delete- rious variants located in the coding regions of 329 candi- date platelet genes listed by Leo et al.15 This prioritizing strategy revealed 37 gene defects, of which six were novel (Online Supplementary Table S2). Since this variant prioritiz- ing strategy yielded multiple SNV for the following patients, C729 (5 SNV), C732 (4 SNV), C739 (4 SNV), C740 (7 SNV), and C831 (4 SNV), we used the ACMG variant pathogenicity classification,31 which revealed 14 gene defects classified as variants of uncertain significance (VUS) in eight patients. To provide functional analysis of these genes, we assessed their expression patterns in platelets using the HPM, which integrates mass spectrom- etry analysis of different human tissues and cell types as part of the human proteome project.32 This evaluation identified potential gene defects in seven PSD patients, with the genes involved being: EXOC1 (C732), DIAPH1 (C739), STXBP5L and PRKACG (C740), PTPN12 (C749), VWF (C831), PRKCD (C1075), PTPN7 and PRKCD (C1107).
Singleton filtering approach
Given that the first approach failed to identify gene defects in six patients, we decided to apply another filter- ing strategy based on the isolation of singletons. To this end, we selected from all 14 patients private variants, which were rare and possibly deleterious and we obtained 2,875 SNV in 2,162 genes. To prioritize these SNV for their putative role in PSD, we performed functional anno- tation using the Database for Annotation, Visualization and Integrated Discovery (DAVID).33 Significantly associ- ated Gene Ontology (GO) annotations were found for gene clusters in the following functional categories: bio- logical process - extracellular matrix organization for 48 genes (P=2.1x10-7, Bonferroni P=9.9x10-4); cellular compo- nent - basal lamina containing 10 genes (P=5.7x10-6, Bonferroni P=4.4x10-3); molecular function - extracellular matrix structural constituent comprising 22 genes (P=5.6x10-6, Bonferroni P=8.3x10-3). In addition, Kyoto Encylopedia of Genes and Genomes (KEGG) pathway analysis (www.genome.jp/kegg/pathway.html) revealed once again a cluster of 26 genes with functional annotation associated with extracellular matrix-receptor interactions (P=2.9x10-6, Bonferroni P=7.9x10-4). The extracellular matrix functional category can be defined as any material produced by cells and secreted into the surrounding medi- um, includiing collagen, laminin, fibronectin proteins and glycosaminoglycans (http://www.uniprot.org/keywords /?query=Extracellular%20matrix), indicating that our priori- tizing method had indeed identified genes potentially affected in PSD.
Functional overlap between the above-mentioned gene clusters was achieved by enriching for variants present in genes exhibiting GO terms such as platelets and secretion, platelets and granules, platelets and signaling.
In this way, we identified 70 potential gene defects, of which 68 were missense variants. We also found a STOP gain variant in the PHF14 gene (c.G298T, p.E100X) in patient C749 and a frameshift deletion in the TBXAS1 gene (c.151_152delGT, p.V51fs) present in patient C831.
Importantly, all 37 missense variants identified by filtering for gene defects in platelet candidate genes were also found in the list of singletons, which together produced a list of 107 candidate gene defects presented in Online Supplementary Table S2.
Similar to the previous filtering strategy, the singleton approach revealed an excess of potential gene defects in several patients (Online Supplementary Table S2). To be able to assign causality, a further reduction in the number of SNV was necessary. To this end, we once again used the ACMG variant pathogenicity classification,31 which result- ed in the identification of 22 putative gene defects classi- fied as VUS in ten patients with primary PSD. However, only 13 of these variants were located in genes expressed in human platelets according to the HPM32 (Table 2). In summary, this variant prioritization approach provided candidate gene defects for four patients, C696, C708, C797 and C847, for whom the previous strategy was inef- fective. It is interesting to note that several of these gene defects were missing from the list of Leo et al.,15 indicating that these genomic loci could potentially become novel candidate genes associated with PSD.
Family analysis of patient C740
Only one notable pedigree, case C740, was investigated. The distribution of the PSD phenotype and BSS in his rel- atives are reported in Figure 2 (father C1300, mother C1301, and two sisters C1302 and 1304). WES was per- formed in all four individuals and the variant filtering steps were based on MAF ≤1%, selecting SNV with potentially damaging consequences and assuming disease transmis- sion present in affected and absent in unaffected family members (Online Supplementary Figure S2). Upon classifica- tion according to the ACMG,31 four SNV were confirmed in a heterozygous state in PSD-affected C740 and father C1300, suggesting an autosomal dominant transmission of the disease. Two of the SNV, p.D1144N in the STXBP5L gene and p.P83H in the KCNMB3 gene, classified as VUS (Table 3) may be involved in the secretion process, thus being the most probable gene defects responsible for the PSD phenotype in this family.
Discussion
In this pilot study, we performed WES in 14 unrelated Italian patients diagnosed with primary PSD and 16 healthy controls. We selected a group with a common phenotype characterized by impaired platelet aggregation and secretion with two or more stimuli as assessed with lumi-aggregometer and a normal platelet content of the granules, confirming the diagnosis of PSD. In our previous study, we demonstrated that a PSD was present in almost one fifth of patients with a mild bleeding diathesis.5
To identify causal genes underlying these defects, we carried out two prioritizing approaches, which were based on the identification of rare, potentially deleterious variants present in 329 platelet candidate genes listed by Leo et al.15 or by selecting singletons (Online Supplementary Figure S1). These strategies revealed a number of plausible candidate gene defects explaining the phenotypic defects of primary PSD. For instance, patient C740 carries a mis- sense variant p.D1144N in the STXBP5L gene (Table 2). In a recent report, another missense variant was identified in this gene as being potentially causal in platelet secretion
haematologica | 2019; 104(10)
2087