Identification of a novel WASP splice-site mutation in a patient with thrombocytopenia

Wiskott-Aldrich syndrome (WAS) (MIM #301000) is a rare X-linked primary immunodeficiency due to mutations in Wiskott-Aldrich syndrome protein (WASP) gene, characterized by thrombocytopenia with small platelets, eczema, recurrent infections and an increased incidence of autoimmunity and malignancies. Aim of this report is to describe the functional characterization of a novel WASP mutation. Here we report on the case of a 2-month-old boy presenting with petechiae, thrombocytopenia (23000/mm3MPV 6.9 fL), slightly increased eosinophil count (620 cells/mm3) and reduced CD8+ (6%; 267 cells/mm3) and IgM levels (20.9 mg/dL). Targeted NGS revealed a novel 4 nucleotides deletion from position +3 to +6 of intron 8 (c.777+3_777+6delGACT) of WASP. To characterize the pathogenetic role of this deletion we performed a Polymerase Chain Reaction (PCR) analysis on cDNA obtained from white blood cells using primers annealing on exon 8 (FW) and 9 (Rev). Western Blot was used to evaluate protein expression. In the mature transcript we observed the complete retention of intron 8, suggesting a splicing defect, due to the loss of a splice donor site at the 5ʹ-end of intron 8. By sequencing the PCR product, we identified a premature stop at codon 269. Real-time PCR confirmed that the expression levels of the transcript containing intron 8 in the patient were similar to those observed for the wild-type transcript in healthy controls, although no WAS protein was detectable in peripheral blood mononuclear cells (PBMC) from the patient. We identified and characterized a novel splice site mutation in a patient presenting with petechiae and thrombocytopenia. The young age of the patient did not allow to define the phenotype (classical WAS vs X-linked thrombocytopenia) associated with the mutation.