Characterization of a WAS splice-site variant in a patient with Wiskott-Aldrich syndrome

Abstract

Wiskott-Aldrich syndrome (WAS) (MIM #301000) is a rare X-linked primary immunodeficiency due to mutations in the WAS gene, characterized by thrombocytopenia with small platelets, eczema, recurrent infections, and an increased incidence of autoimmunity and malignancies. A wide spectrum of mutations has been identified in the WAS gene responsible for a broad variety of clinical phenotypes. By using targeted next-generation sequencing (t-NGS), we identified in a 2-month-old boy with thrombocytopenia and immunological alterations a 4-nucleotide deletion from position +3 to +6 of intron 8 (c.777 + 3_777 + 6delGAGT) of WAS, currently classified on ClinVar as a variant of uncertain significance. The in-vitro characterization of the variant revealed the complete retention of intron 8 in the mature transcript, suggesting a splicing defect due to the loss of a splice donor site at the 5'-end of intron 8. By sequencing the polymerase chain reaction product, we identified a premature stop at codon 269; thus, consequently, no Wiskott-Aldrich syndrome protein (WASp) was detectable in peripheral blood mononuclear cells from the patient. Due to the total absence of a full-length WASp, it is expected that the patient will develop the severe form of the disease, although further monitoring is needed to better define his phenotype.

Keywords: WASP; Wiskott-Aldrich; gene panel; next-generation sequencing (NGS); splice-site mutation.

Figure 1

(A) Schematic representation of WASP gene with the new identified mutation in intron 8 (EX: exon; del: deletion). (B) Gel electrophoresis of the PCR products obtained from cDNA extracted from white blood cells using primers annealing on exon 8 (Fw) and exon 9 (Rev). neg: negative control; CTR2: male, young; CTR1: female, adult.

Figure 2

(A) Results of Sanger sequencing analysis performed on the PCR product from the affected patient showing the retention of intron 8 into the mature transcript. (B) Prediction of the effect of retention of intron 8 on protein traslation; *indicates the putative premature stop codon at position 269.

Figure 3

Quantitative analysis of transcript levels using RT-qPCR. (A) RT-qPCR was performed to compare the expression levels of the transcript containing retained intron 8 (INT8) in the proband (dark bar) to wild-type controls (light bars) and the carrier mother (striped bar). Primer pairs INT8Fw and INT8Rv were used to specifically amplify the transcript with retained intron 8, while the expression levels were normalized to the internal region of the WAS gene spanning exons 5–7 (Ex5–Ex7) for consistency across all samples. The results showed significantly higher expression of the INT8 transcript in the proband compared to controls and the carrier mother. The amplification observed in healthy controls, where intron 8 is normally spliced out, likely represents either background nonspecific amplification or low-level splicing artifacts. Error bars represent the mean ± standard deviation of three independent experiments. Statistical significance was assessed using ordinary one-way ANOVA (*p < 0.05; **p < 0.01). (B) Comparison of Ct values obtained using primer pairs INT8Fw/INT8Rv and Ex8Fw/Ex9Rv for the proband and Ex8Fw/Ex9Rv for controls and the mother. These primer sets amplify different regions of the WAS gene, specifically targeting the INT8 transcript or the canonical transcript. The Ct values were similar in magnitude across the samples, suggesting comparable levels of transcript abundance. However, the Ct values were not normalized against a common housekeeping gene, and the comparison assumes equal amplification efficiencies for the primer pairs. These efficiencies were validated through standard curve analyses, confirming their equivalence.

Figure 4

Western blot analysis on proteins extracted from PBMCs; neg: protein lysate from human fibroblasts; CTR1: female, adult; CTR2: male, young. The expression of WASP was quantified by densitometry analysis relative to GAPDH with ImageJ software (CTR1: 1,20; CTR2: 2,05; mother: 1,55).