ADA2 deficiency due to a novel structural variation in 22q11.1

Abstract

To the Editor, Deficiency of the adenosine deaminase 2 (ADA2) gene (OMIM#607575) (DADA2) (OMIM#615688) is a rare recessive autoinflammatory disease caused by bi-allelic loss-of-function ADA2 mutations, showing a wide range of clinical manifestations, including cerebral stroke events and several kind of immune dysregulation. Interestingly, patients with typical DADA2 and complete enzymatic impairment, may present with only one or no ADA2 mutation, thus, suggesting involvement of null alleles. Here, we report about a genetic study, approved by the Ethics Committee of the G. Gaslini Institute, in a DADA2 patient born of consanguineous parents, without mutations in the coding portion of the ADA2 gene. Briefly, at 3 months, the proband presented fever, hypertension, severe myocarditis, livedo reticularis, hypogammaglobulinemia and was responsive to steroid treatment. At 5 years, two episodes of stroke occurred upon steroid withdrawal. ADA2 enzymatic activity was absent in the patient s circulating monocytes and plasma whereas parents showed a level lower than healthy donors (HD) (Figure 1A). The patient consistently responded to anti-tumor necrosis factor treatment (etanercept). CNV analysis of 30× whole genome sequencing data focused on the ADA2 locus at chromosome 22q11, thus, allowing to assess a homozygous tandem duplication of the genomic region encompassing exons 3 and 4, generated by breakpoints at chr22:17676374 and chr22:17689269, in introns 4 and 2, respectively (NM_001282228.1). Ultimately, 12 895 base pairs resulted to be duplicated, thus, leading to a gene transcript containing 1967 instead of 1536 nucleotides, with a frameshift from codon V252 and a premature stop codon after 11 amino acid residues (p.V252Gfs*11) (Figure 1B). PCR reactions, properly designed to amplify the junction fragments from both genomic DNA and cDNA samples, and subsequent Sanger sequencing of the amplification product, confirmed such a finding (Figure 1C-F). The human chromosome 22 contains a cluster of low-copy repeats (LCRs) that can mediate non-allelic homologous recombination (NAHR), resulting in altered gene dosage like in the DiGeorge/Velocardiofacial syndrome (DGS/VCFS) and Cat-eye syndrome (CES). Mapping long and short interspersed repetitive elements, as well simple and low complexity repeats to the ADA2 gene locus has allowed to exclude that any LCR22 could have been responsible for the present duplication in favor of the involvement of two Alu sequences, AluSg and AluSx1, belonging to the SINE family of transposable elements. Indeed, sequence comparison at introns 2 and 4 breakpoints resulted in 88% of identity (Figure 1G), consistent with the relatively small size of the microduplicated segment and non-random enrichment of Alu elements already reported in regions adjacent to frequently duplicated sequences in human 22q11.2. As the proband is homozygous, the 12.9Kb duplication at the ADA2 locus must have occurred, as meiotic/germline or mitotic/postzygotic event, in one of the common ancestors of the asymptomatic consanguineous parents from either unequal cross-over between homologous chromosomes, unequal exchange between sister chromatids, or another Alu repeat sequences driven rearrangement. The structural variation identified here represents the first demonstration of a pathological NAHR mechanism acting in DADA2. Indeed, deletions of variable lengths have already been observed to encompass the ADA2 locus and to associate with DADA2 symptoms, although breakpoints are uncharacterized yet. Noteworthy, a timely identification of the ADA2 enzymatic defect in the presence of a clinical phenotype clearly consistent with DADA2 should lead to a careful search for CNVs at the ADA2 locus, especially in genetically undefined patients often at risk for acute events.