RESUMEN
PURPOSE: Deficiency of adenosine deaminase 2 (DADA2) is an autosomal recessive disorder that manifests with fever, early-onset vasculitis, strokes, and hematologic dysfunction. This study aimed to identify disease-causing variants by conventional Sanger and whole exome sequencing in two families suspected to have DADA2 and non-confirmatory genotypes. ADA2 enzymatic assay confirmed the clinical diagnosis of DADA2. Molecular diagnosis was important to accurately identify other family members at risk. METHODS: We used a variety of sequencing technologies, ADA2 enzymatic testing, and molecular methods including qRT-PCR and MLPA. RESULTS: Exome sequencing identified heterozygosity for the known pathogenic variant ADA2: c.1358A>G, p.Tyr453Cys in a 14-year-old female with a history of ischemic strokes, livedo, and vasculitis. No second pathogenic variant could be identified. ADA2 enzymatic testing in combination with quantitative RT-PCR suggested a loss-of-function allele. Subsequent genome sequencing identified a canonical splice site variant, c.-47+2T>C, within the 5'UTR of ADA2. Two of her unaffected siblings were found to carry the same two pathogenic variants. A homozygous 800-bp duplication comprising exon 7 of ADA2 was identified in a 5-year-old female with features consistent with Diamond-Blackfan anemia (DBA). The duplication was missed by Sanger sequencing of ADA2, chromosomal microarray, and exome sequencing but was detected by MLPA in combination with long-read PCR sequencing. The exon 7 duplication was also identified in her non-symptomatic father and younger sister. CONCLUSIONS: ADA2 pathogenic variants may not be detected by conventional sequencing and genetic testing and may require the incorporation of additional diagnostic methods. A definitive molecular diagnosis is crucial for all family members to make informed treatment decisions.