79. Deep intronic mutation in patients with adenosine deaminase 2 deficiency

Background : Deficiency of adenosine deaminase 2 (DADA2) is an autosomal recessive inflammatory disease caused by loss-of-function mutations in the ADA2 gene. Most patients with DADA2 exhibit systemic vasculopathy consistent with polyarteritis nodosa, but large phenotypic variability has been reported. Patients with DADA2 are deficient of ADA2 activity in plasma. We sometimes, however, experience cases in which no apparent mutations are found in the ADA2 gene, even in these definitive diagnosed cases. In this report, we present two siblings who have a deep intronic mutation which thought to result in aberrant splicing and nonsense mediated mRNA decay (NMD). Methods: Now 24 and 21 years old siblings with DADA2 were enrolled. The older brother had repeated episodes of cerebral infarction and hemorrhage from the age of 10. He was diagnosed with DADA2 at 21 years of age due to deficiency of ADA2 activity in plasma. The younger brother revealed persistent elevation of CRP and an episode of cerebral ischemia at the age of 18, then he was diagnosed with DADA2 as same as his older brother. Results: An analysis of whole exon sequencing in these siblings and their parents revealed E328K mutation in ADA2 gene, pathogenic mutation reported in past, in the allele derived from the mother. But no mutation other than SNPs were found in the allele derived from the father. Large deletion nor insertion was also not suggested. Then we analysed mRNA expression of ADA2 gene by deep sequencing and revealed aberrant splicing. As a result of whole genome analysis, an unreported mutation was found in the deep intron. The analysis in silico had suggested aberrant splicing due to this deep intronic mutation. mRNA analysis, using RNA extracted from the peripheral blood mononuclear cells, revealed a clear extra band in the sibling's cells, but not in normal controls. NMD inhibition by cycloheximide made the extra band clearer. In a minigene system, the antisense oligo, that suppress aberrant splicing by this deep intronic mutation, reduced ...