Adenosine deaminase deficiency (ADA) is an autosomal recessive form of severe combined immunodeficiency. The purpose of this research is to delineate the etiology of reduced ADA mRNA levels in ADA-deficient patient JH and to explore the effects of nonsense mutations upon ADA mRNA levels. Northern blotting of poly A+ enriched mRNA prepared from an EBV- transformed cell line derived from patient JH did not reveal any detectable ADA mRNA but the amount of actin mRNA in JH's cells was normal. Sequencing of JH's ADA cDNA prepared from poly A+ mRNA revealed the following alterations from the published sequence: (1) an amino acid substitution at codon 8 in exon 1 which destroys a Taq restriction site and changes asp to asn, (2) a missense mutation at nt position 334 codon 80 which changes lys to arg, and finally (3) a 5 bp deletion at nt positions 1050-54 in exon 10 which results in a frameshift and a premature stop codon corresponding to a 321 of the mature ADA protein. The above mutations have been verified by sequencing PCR-amplified genomic DNA from JH. There have been only two previously described ADA mutations which lack ADA mRNA; both of these (unrelated) individuals have a deletion in ADA gene encompassing the promoter region and exon 1. JH is the first ADA-deficient patient in which there is no ADA mRNA detectable by Northern analysis and in whom the etiology appears to be the presence of a premature stop codon. Previously published work for other genes has shown that nonsense and frameshift mutations which result in premature stop codons may have varying effects on mRNA levels. We propose to determine whether JH's reduced ADA mRNA levels are due to transcriptional dysfunction, cytoplasmic mRNA instability, heteronuclear mRNA processing or a combination of these. Transcriptional function of the ADA genes from patient JH will be investigated by nuclear run-ons in both JH EBV-transformed cell lines and also in Cos cells transfected with ADA promoter/cDNA constructs. Cytoplasmic ADA mRNA stability will be measured by pulse-chase experiments with 32P-UTP or by using actinomycin D. Processing of heteronuclear mRNA will be examined by transfecting Cos cells with the relevant portions of JH's ADA gene containing the 5 bp deletion and the flanking intronic regions. Site-directed mutagenesis and tRNA suppressor analysis will be employed to prove whether the 5 bp deletion or the premature termination codons are responsible for the reduced ADA mRNA levels. Data from these experiments may shed light upon ADA RNA processing.
