A kindred with an autosomal dominant form of chronic hemolytic anemia has been found to have a 40- to 70-fold elevation in erythrocyte adenosine deaminase (ADA) activity in association with depletion of red blood cell (RBC) ATP pools. ADA activities in B lymphoblasts, skin fibroblasts, and granulocytes were normal. There were no alterations in the kinetic properties of partially purified proband ADA. We have shown by Western blot analysis that the elevation in ADA activity is accompanied by a corresponding increase in the amount of immunoreactive ADA protein. Southern blot analysis of proband DNA ruled out gene amplification and revealed no gross insertions, deletions, or rearrangements in the ADA gene. Northern blot analysis demonstrated a marked increase in the amount of ADA mRNA in proband and sibling reticulocytes compared to high reticulocyte controls. ADA mRNA levels in B lymphoblasts from the proband, sibling, and GM558 cell line were normal. Cloning and sequencing of proband reticulocyte cDNA revealed normal ADA mRNA sequence. No polymorphisms were detected among the seven clones studied. RNase mapping of the 5'- and 3'-non-coding sequences confirmed the quantitative increase in reticulocyte ADA mRNA and verified that these regions were normal in length and sequence. Southern blot analysis of DNA from four affected and three unaffected family members revealed two restriction fragment length polymorphisms (RFLPs) which segregate with the ADA allele from the unaffected grandfather. Both RFLPs are present in the unaffected grandchild and absent in the affected grandchild. These findings are consistent with a cis- mutation within the ADA gene, but they do not rule out a trans- mutation affecting some non-ADA regulatory factor. We conclude that erythrocyte-specific ADA overproduction is associated with increased amounts of structurally normal ADA mRNA. This increase may result from either increased transcription of the ADA gene or altered post-transcriptional processing resulting in increased stability of the RNA transcript. Further elucidation of the defect should provide valuable insights into the normal tissue-specific regulation of the ADA gene and the mechanisms by which erythroid cells regulate gene expression during differentiation.
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