Deficiency Of Adenosine Deaminase Activity Research Articles

Disruption of the adenosine deaminase (ADA) gene using a dicistronic promoterless construct: Production of an ADA-deficient homozygote ES cell line

In man, deficiency of ADA activity is associated with an autosomal recessive form of severe combined immunodeficiency (SCID), a disease with profound defects both cellular and humoral immunity. Current treatments of ADA deficient patients include bone marrow transplantation, enzyme replacement and somatic gene therapy. The mechanism of the selective immune cell pathogenesis in ADA-SCIDS is, however, still poorly understood. Thus, the generation of an ADA deficient mouse model will be of considerable benefit to understand better the pathophysiology of the disorder and to improve the gene therapy treatments. We have disrupted the adenosine deaminase (ADA) gene in embryonic stem cells using a new efficient promoter trap gene-targeting approach. To this end, a dicistronic targeting construct containing a promoterless IRES beta geo cassette was used. This cassette allows, via the internal ribosomal entry site (IRES), the direct cap-independent translation of the beta geo reporter gene which encodes a protein with both beta-galactosidase and neomycin activities. After indentification of targeted clones by Southern blot, successful inactivation of the ADA gene was first confirmed by producing, from our heterozygote clones, an homozygote cell line. This line shows no ADA activity as judged by zymogram analysis. Second, we have been able to detect in the targeted clones, a specific beta galactosidase activity using a sensitive fluorogenic assay. The targeted ES cell clones are currently being injected into blastocysts to create an ADA deficient mouse model.

Effect of chloroadenosine on phosphoinositide turnover in human T lymphoblasts activated through the TcR/CD3 complex.

The inherited deficiency of adenosine deaminase activity in humans is associated with a severe combined immunodeficiency disease with marked lymphopenia and loss of the proliferative response of lymphocytes to phytohaemagglutinin. The inhibition of ribonucleoside diphosphate reductase by dATP, a mechanism initially proposed to explain the immune dysfunction, cannot account for the earliest effects of dAdo on the proliferative response of the cells (reviewed in 1). This prompted us to study the consequences of adenosine deaminase deficiency on the early biochemical steps of lymphocyte activation. Indeed, we and others have recently demonstrated that the mitogen-induced hydrolysis of membrane phosphoinositides is markedly impaired in conditions of adenosine deaminase deficiency (2,3). In particular, we could demonstrate a significant inverse correlation between the cellular level of dATP and the hydrolysis of phosphoinositol bisphosphate (PIP2) estimated by [32P]phosphatidic acid formation, [3H]inositol phosphate generation and assay of inositol trisphosphate by radiocompetition. This suggested that phosphorylated derivatives of adenosine analogs might affect the activity of phospholipase C. In the present work, we used the same model of T-cell activation to study the effect of chloroadenosine (ClAdo), an adenosine analog known as an A2 receptor agonist (linked to the adenylate cyclase pathway), but which has recently been shown to be phosphorylated into C1ATP (4).

Characterization of normal and mutant adenosine deaminase messenger RNAs by translation and hybridization to a cDNA probe.

Using both in vitro translation and hybridization to an adenosine deaminase (ADA) cDNA probe, ADA mRNA has been characterized in B lymphoblast lines established from seven ADA-deficient children, two parents of an ADA-deficient child, and three normal people. All ADA-deficient lines except GM-2825A, including those with less than 1% of normal catalytic activity, had normal or greater amounts of hybridizable, 1.6 kilobase in size, ADA mRNA. Immunoreactive ADA protein of normal size was produced by in vitro translation of the mRNAs. Deficiency of ADA activity in these lines appears secondary to synthesis of structurally altered proteins rather than to a quantitative deficiency in ADA mRNA. The GM-2825A line contains electrophoretically abnormal species of RNA which hybridize to the cDNA probe. Deficiency of ADA activity in this line appears at least in part secondary to a structural defect in the ADA mRNA or its precursors.

Adenosine metabolism in phytohemagglutinin-stimulated human lymphocytes.

The association of a human genetic deficiency of adenosine deaminase activity with combined immunodeficiency prompted a study of the effects of adenosine and of inhibition of adenosine deaminase activity on human lymphocyte transformation and a detailed study of adenosine metabolism throughout phytohemagglutinin-induced blastogenesis. The adenosine deaminase inhibitor, coformycin, at a concentration that inhibited adenosine deaminase activity more than 95%, or 50 muM adenosine, did not prevent blastogenesis by criteria of morphology or thymidine incorporation into acid-precipitable material. The combination of coformycin and adenosine, however, substantially reduced both the viable cell count and the incorporation of thymidine into DNA in phytohemagglutinin-stimulated lymphocytes. Incubation of lymphocytes with phytohemagglutinin for 72 h produced a 12-fold increase in the rate of deamination and a 6-fold increase in phosphorylation of adenosine by intact lymphocytes. There was no change in the apparent affinity for adenosine with either deamination or phosphorylation. The increased rates of metabolism, apparent as early as 3 h after addition of mitogen, may be due to increased entry of the nucleoside into stimulated lymphocytes. Increased adenosine metabolism was not due to changes in total enzyme activity; after 72 h in culture, the ratios of specific activities in extracts of stimulated to unstimulated lymphocytes were essentially unchanged for adenosine kinase, 0.92, and decreased for adenosine deaminase, 0.44. As much as 38% of the initial lymphocyte adenosine deaminase activity accumulated extracellularly after a 72-h culture with phytohemagglutinin. In phytohemagglutinin-stimulated lymphocytes, the principal route of adenosine metabolism was phosphorylation at less than 5 muM adenosine, and deamination at concentrations greater than 5 muM. In unstimulated lymphocytes, deamination was the principal route of adenosine metabolism over the range of adenosine concentrations studied (0.5-250 muM). These studies demonstrate the dependence of both the unstimulated and stimulated lymphocyte on adenosine and may account for the observed sensitivity of mitogen-stimulated lymphocytes to the toxic effects of exogenously supplied adenosine in the presence of the adenosine deaminase inhibitor coformycin. A single case of immunodeficiency disease has been reported in association with purine nucleoside phosphorylase deficiency. The catabolism of guanosine was also found to be enhanced in stimulated normal lymphocytes; phosphorolysis of guanosine to guanine by intact lymphocytes increased six fold after 72-h culture with phytohemagglutinin. The specific activity of purine nucleoside phosphorylase in extracts, with guanosine as substrate, was essentially the same in stimulated and unstimulated lymphocytes after 72 h of culture.

Molecular form of adenosine deaminase in severe combined immunodeficiency

The specific activity of adenosine deaminase was reduced to approximately 0.5% of normal in splenic tissue obtained from a patient with severe combined immunodeficiency. Sedimentation analysis of splenic homogenate from this patient revealed a major peak of adenosine deaminase activity which corresponded with respect to the sedimentation coefficient of one of three molecular species observed in control spleens but had markedly reduced activity. These findings suggest that the molecular heterogeneity of human adenosine deaminase is under the control of a single genetic locus and that the deficiency of adenosine deaminase activity in severe combined immunodeficiency is not due to a genetic deletion.