MLPA+Ddpcr: Dual Molecular Strategy to Detect Copy Number Variations (CNVs) in Inborn Error of Immunity (IEI) Genes: A Case Study of the WAS Gene

Introduction IEIs are a heterogeneous group of primarily inherited genetic disorders characterized by impaired immune system function. While most genetic alterations are single nucleotide variants (SNVs), some cases are caused by larger genomic changes such as insertions or deletions that lead to copy number variations (CNVs). To identify potential large deletions in the WAS gene, we developed a molecular strategy combining a novel own design multiplex ligation-dependent probe amplification (MLPA) assay specific to WAS with a droplet digital PCR (ddPCR) assay. Methods Three patients with suspected Wiskott–Aldrich syndrome (WAS) were studied. Initial analysis involved Sanger sequencing. When a CNV was suspected, additional methodologies were incorporated: a WAS-specific MLPA test (covering exons 1, 2, 3, 4, 5, 6, and 8) designed according to the manufacturer’s recommendations and a ddPCR assay targeting designed exons 7, 8, 9, and 12. Results None of the patients showed amplification of individual WAS exons by PCR. MLPA analysis revealed a complete absence of all exons in two patients, while the third patient showed absence of exon 8 by MLPA and absence of exons 7, 8, 9, and 12 by ddPCR. Both methodologies allowed for the analysis of women carriers of this X-linked hereditary pathology, leading to the identification of a carrier. Conclusions These patients clearly demonstrate the importance of having more than one screening strategy for efficient diagnosis and family genetic counseling, mostly in cases where the clinical suspicion is very strong but conventional diagnostic strategies yield negative results. In many IEIs, bioinformatic algorithms in next-generation sequencing studies detect potential CNVs that need to be confirmed. Both MLPA and ddPCR are complementary techniques that would allow for a definitive diagnosis in these cases.