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Exome sequencing in platelet secretion defects
Table 3. Putative causal variants identified by whole-exome sequencing in the family of patient C740 (Online Supplementary Figure S2).
Gene dbSNP Nucl. Amino C740 C1300 C1301 C1302 C1304 MAF MAF MAF SIFT Poly Mutation CADD Plt Assess
change acid change
SLC2A7 rs35776221 c.C670T p.R224C het STXBP5L rs139176240 c.G3430A p.D1144N het KCNMB3 rs61734056 c.C248A p.P83H het LCN1 rs117638349 c.G298C p.G100R het
1000G ExAC ESP
het - - - 0.006 0.01 0.008 D
het - - - - 0.0004 0.0001 B
het - - - - 1.50E-050.0001 D
het - - - 0.006 0.008 0.004 D
phen2 Taster Cscore Exp. (**) (*)
D D 27 - VUS
D D 25 + VUS
DD27-VUS
D B 23 - VUS
dbSNP: Database of Single Nucleotide Polymorphisms v.138; MAF: minor allele frequency (MAF from European populations is shown); 1000G: the 1000 Genomes Project; ExAC: the Exome Aggregation Consortium; ESP: the Exome Sequencing Project; SIFT: Sorting Intolerant From Tolerant; PolyPhen2: Polymorphism Phenotyping v.2; Mutation Taster, prediction scores, D: dam- aging; B: benign; CADD C score: Combined Annotation Dependent Depletion score;41 VUS: variant of uncertain significance. (*) Platelet gene expression evaluated by the Human Proteome Map (HPM) (http://www.humanproteomemap.org);32 (**) Assess. – Assessment of variant pathogenicity assigned according to the American College of Medical Genetics and Genomics pathogenicity classification.31
plex and heterogeneous nature of primary PSD. This indi- cates that an in-depth functional analysis of platelet recep- tor and signaling pathways will be necessary to discrimi- nate differences in clinical and laboratory phenotypes of affected individuals.
Study limitations
Following a positive experience with the application of WES to identify gene defects underlying inherited platelet function disorders,19-22 we chose to investigate primary PSD using the same technique, hoping that a genomic approach could be effective in identifying causal variants in a heterogeneous clinical and phenotype such as primary PSD. However, exome sequencing followed by two inde- pendent variant prioritization approaches yielded incon- clusive results. The primary reason for this is undoubtedly the heterogeneous clinical and laboratory phenotype of primary PSD, which may have led to the identification of genes not necessarily associated with the disease. For instance, 20 missense variants were detected in the TTN gene in 11 PSD patients, of which eight are VUS. However, TTN is one of the most frequently mutated genes in the human genome,38 implying that the variations found in this gene are probably due to the size of its cod- ing regions (363 exons).
Another limitation of this study was perhaps the choice of the variant prioritization strategy. We applied a gener- ally accepted filtering method based on the selection of rare (MAF ≤1%), potentially damaging variants. This approach revealed a great abundance of variants for most patients, which required further selection based on the ACMG pathogenic classification of SNV (Table 2). This revealed 34 putative gene defects classified as VUS in 12 patients with primary PSD, of which 24 were located in genes expressed in human platelets according to the HPM (Table 2). However, it is possible that many potentially causal SNV, which were classified as likely benign or benign, were excluded due to lack of supporting evidence or because the gene defects may only manifest at the level of megakaryocyte development or platelet matura- tion.
In addition, some of the functional defects might have been located in the non-coding parts of the genome such as promoters, intronic sequences or enhancers, which were not covered by exome sequencing. Finally, since the identification of gross chromosomal aberration such as copy number variations from the WES data remains a technical challenge, it is likely that these structural vari-
Figure 2. Pedigree of patient C740. Black and white symbols indicate subjects affected by platelet secretion defects and unaffected family members, respec- tively. The arrow indicates the proband C740. BSS: bleeding severity score.
ants would not have been detected. Although several bioinformatics methods have been developed for copy number variation analysis from WES data, they require uniform coverage and high resolution of the sequencing data across all exons/coding regions as well as a special- ized bioinformatics pipeline of data analysis validated against the whole-genome data.39 For this reason, whole- genome sequencing is the only sure means for identifying the copy number variations alongside SNV and small indels.
In conclusion, we carried out exome sequencing in 14 patients with primary PSD and 16 healthy controls, fol- lowed by two variant prioritization strategies. Our analy- sis identified potential gene defects in 12 patients, imply- ing that the NGS-based diagnostic strategies for causal gene identification in such a heterogeneous clinical and laboratory phenotype as primary PSD may be ineffective. In this case, a well-defined, common disease phenotyping and properly established pipeline for variant analysis are necessary. The difficulty in assigning causality can be overcome by genetic screening of affected and unaffected family members, which allows the identification of gene defects that segregate with the clinical phenotype, or by functional studies.
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