Diana Ruixi Yang

Genetic deficiency of adenosine deaminase (ADA) results in varying degrees of immunodeficiency, including neonatal onset severe combined immunodeficiency (ADA- SCID) and milder, later onset immunodeficiency. We have determined the molecular basis of disease in a child from a consanguineous mating with ADA- SCID of clinically and biochemically reduced severity, diagnosed at 15 months of age and characterized by retention of more immunologic function than is typical of the fulminant neonatal onset type. The course was notable for an early predominance of bacterial infections and eosinophilia. In contrast to its absence in most ADA- SCIDs, residual ADA activity (1-2% of normal) could be detected in EBV-transformed B cells. Consistent with the increased residual ADA, excretion of the substrate deoxyadenosine and accumulation of the toxic metabolite deoxyATP were less than seen in ADA- SCID patients with fulminant disease. Sequence analysis of cDNA revealed a G853C transversion, predicting a substitution of proline for arginine at codon 253 (Arg253Pro). The parents were heterozygous and the child was homozygous for the mutation, as shown by sequence analysis of amplified genomic DNA. Transient expression of mutant cDNA in Cos cells revealed an electrophoretically abnormal, more negatively charged ADA with 1-2% of normal activity. These observations are consistent with replacement of positively charged arginine by proline, the lower accumulation of toxic metabolites, and the milder phenotype. By contrast, transient expression of a Gly216Arg mutant cDNA, associated, when homozygous, with neonatal onset ADA-SCID, did not reveal ADA activity. Mutations such as Arg253Pro, which retain residual activity of monomeric ADA, should be dominant for ameliorating the phenotype in patients carrying two different allelic mutations. Identification of additional similar mutations may be significant in evaluating the goals for and efficacy of current trials of gene and gene product replacement.

Mutations at the adenosine deaminase (ADA) locus can result in varying degrees of immunodeficiency, including rapidly fulminant severe combined immunodeficiency (SCID) as well as a slowly progressive immunodeficiency not diagnosed until later in childhood. Genetic heterogeneity is a factor in the clinical heterogeneity. We have now identified, by direct sequencing of PCR-amplified genomic DNA, a G to A transition at a CpG dinucleotide predicting a glycine to arginine substitution at codon 20 (G20R). The mutation, in homozygosity, was associated with neonatal-onset rapidly fatal SCID. Consistent with homozygosity, the child was derived from a small isolated inbred community in Newfoundland. The mutation abolishes a site for the restriction enzyme BamHI and can be simply detected by agarose gel electrophoresis following amplification of exon 2 from genomic DNA and digestion with BamHI. The majority of ADA missense mutations can now be detected by similar amplification and enzyme digestion. We demonstrated that the G20R mutation is deleterious since introduction of the mutation into a normal ADA minigene abolished enzyme activity, as determined by transient expression in monkey kidney (Cos) cells. The amino acid substitution occurs in an area of the molecule conserved from Escherichia coli to man and that, as shown by crystallographic analysis, is involved in the binding of Zn2+ at the catalytic site. Although the mutation is in a CpG dinucleotide, known "hotspots" for G to A transitions, it was not found in a series of 43 additional ADA- chromosomes. Identification of mutations in additional ADA- patients with immunodeficiency of varying severity should further define the role that genotype plays in determining the extent of immunologic dysfunction.