Inherited Deficiency Of Adenosine Deaminase Research Articles

Adenosine deaminase: functional implications and different classes of inhibitors.

Adenosine deaminase (ADA) is an enzyme of the purine metabolism which catalyzes the irreversible deamination of adenosine and deoxyadenosine to inosine and deoxyinosine, respectively. This ubiquitous enzyme has been found in a wide variety of microorganisms, plants, and invertebrates. In addition, it is present in all mammalian cells that play a central role in the differentiation and maturation of the lymphoid system. However, despite a number of studies performed to date, the physiological role played by ADA in the different tissues is not clear. Inherited ADA deficiency causes severe combined immunodeficiency disease (ADA-SCID), in which both B-cell and T-cell development is impaired. ADA-SCID has been the first disorder to be treated by gene therapy, using polyethylene glycol-modified bovine ADA (PEG-ADA). Conversely, there are several diseases in which the level of ADA is above normal. A number of ADA inhibitors have been designed and synthesized, classified as ground-state and transition-state inhibitors. They may be used to mimic the genetic deficiency of the enzyme, in lymphoproliferative disorders or immunosuppressive therapy (i.e., in graft rejection), to potentiate the effect of antileukemic or antiviral nucleosides, and, together with adenosine kinase, to reduce breakdown of adenosine in inflammation, hypertension, and ischemic injury.

Adult onset immunodeficiency caused by inherited adenosine deaminase deficiency.

Adenosine deaminase (ADA) deficiency is identified here as a cause of adult onset immunodeficiency. Two sisters who noted recurrent, predominantly chest infections in their twenties were found in their thirties to have CD4+ lymphopenia and lymphocyte ADA activity of approximately 5% of the lower limit of normal. Immune function, measured by proliferation of PBMCs in vitro to mitogens and specific Ags, was impaired. Inheritance of a polymorphic marker showed that both patients were heterozygous at the ADA locus. In the paternal allele there was a deletion resulting from homologous recombination between two alu elements that normally flank the first exon and the polymorphic marker. The recombination site was distinct from that in similar deletions described in two infants having severe combined immunodeficiency. This allele is predicted to result in a null phenotype. In the mutant allele inherited from the mother, a C to T transition in a CpG dinucleotide changed the codon for arginine 211, which lies in a conserved sequence close to the active site, to that for cysteine. This mutation has been observed previously in a child in whom the other allele was also a null mutation, but who was diagnosed as having partial ADA deficiency because immune function was apparently normal. The late onset of immunodeficiency in our patients suggests that immune function in children with partial ADA deficiency may deteriorate with time and that ADA deficiency should be regarded as a possible cause of adult onset immune dysfunction of unknown etiology.

IgG antibody response to polyethylene glycol-modified adenosine deaminase in patients with adenosine deaminase deficiency.

Polyethylene glycol (PEG)-modified bovine adenosine deaminase (ADA) is used for replacement therapy of severe combined immunodeficiency disease due to inherited ADA deficiency. We monitored IgG anti-ADA antibody in 17 patients treated by intramuscular injections of PEG-ADA for 1 to greater than 5.5 yr. ELISA-detectable anti-ADA IgG appeared in 10 patients, usually between the third and eighth months of treatment. Anti-ADA levels did not correlate with trough plasma ADA activity, which averaged 1.8-5 times normal blood (erythrocyte) ADA activity, depending on dose (15-60 U/kg per wk). ELISA-detectable anti-ADA antibodies were directed primarily at bovine-specific peptide (rather than PEG-containing) epitopes. Enhanced enzyme clearance, mediated by antibody that directly inhibited native and PEG-modified bovine ADA, and native, but not PEG-modified human ADA, occurred in two patients. In one, tolerance was induced; in the second, twice weekly injections of PEG-ADA compensated for accelerated clearance. We speculate that inhibitory antibodies recognize conserved, relatively PEG-free epitope(s) encompassing the active site, and that in human, but not bovine, ADA a PEG-attachment site "shields" the active site from immune recognition. We conclude that PEG-modification largely prevents the development of high affinity, or high levels of clearing antibodies to bovine ADA, and that PEG-modified human ADA should be further investigated as a possible treatment for ADA deficiency.

One adenosine deaminase allele in a patient with severe combined immunodeficiency contains a point mutation abolishing enzyme activity.

We have cloned and sequenced an adenosine deaminase (ADA) gene from a patient with severe combined immunodeficiency (SCID) caused by inherited ADA deficiency. Two point mutations were found, resulting in amino acid substitutions at positions 80 (Lys to Arg) and 304 (Leu to Arg) of the protein. Hybridization experiments with synthetic oligonucleotide probes showed that the determined mutations are present in both DNA and RNA from the ADA-SCID patient. In addition, wild-type sequences could be detected at the same positions, indicating a compound heterozygosity. Studies with ADA expression clones mutagenized in vitro showed that the mutation at position 304 is responsible for ADA inactivation.

Superoxide radicals, immunodeficiency and xanthine oxidase activity: man is not a mouse!

The importance of intact adenosine deaminase (ADA) activity in the generation of superoxide anion by xanthine oxidase has been disputed in studies using human neutrophils or mouse macrophages. The latter demonstrated a positive correlation between ADA activity and superoxide production during phagocytosis. The immunodeficiency in inherited ADA deficiency was related to a defect in this process. Since there is considerable interspecies variation in the tissue distribution of xanthine oxidase, the metabolism of [8-14C]deoxyadenosine (dAR), the toxic metabolite which accumulates in inherited ADA deficiency, was investigated in human peritoneal macrophages. Evaluation of the distribution of radiolabel in both cell and medium demonstrated that human macrophages with intact ADA metabolize dAR under physiological conditions to deoxyinosine and hypoxanthine exclusively. The hypoxanthine is further metabolized within the cell to ATP and GTP, via IMP. No xanthine or uric acid could be detected, confirming that in human macrophages xanthine oxidase activity is insignificant, as it is in most other human cells and tissues, except liver and intestinal mucosa. Thus production of superoxide radicals in such cells via this route would be impossible, and consequently unaffected either by ADA deficiency or the xanthine oxidase inhibitor allopurinol.

Importance of Platelet-Free Preparations for Evaluating Lymphocyte Nucleotide Levels in Inherited or Acquired Immunodeficiency Syndromes

Low ATP/ADP ratios have been reported consistently for nucleotide levels of mononuclear cells separated from peripheral blood by conventional techniques. We have established that these low values (mean 2.3:1) were not due to cell damage or poor viability, but resulted from heavy platelet contamination, which is unavoidable when heparinized blood is used. The results reflect the low ATP/ADP ratios (mean 1.6:1) characteristic of platelets. Platelet-free extracts from defibrinated blood had very high ATP/ADP ratios (mean 17.4:1). The initial finding of detectable amounts of deoxy-ATP and deoxy-GTP in mononuclear cells from children with two distinct inherited immunodeficiency disorders [adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) deficiency respectively] many have been due to contamination by nucleated erythrocytes as well as platelets in non-defibrinated preparations. Defibrination before nucleotide extraction of mononuclear cells from a patient with T-cell leukaemic/lymphoma treated with the ADA inhibitor deoxycoformycin enabled the demonstration of grossly raised deoxy-ATP levels relative to deoxy-ADP levels (ratio 16.1:1), associated with severe ATP depletion. This reciprocal relationship between ATP and dATP was found by us previously in the erythrocytes in inherited ADA deficiency. These findings underline the importance of extracts uncontaminated by platelets, or nucleated erythrocytes, in the evaluation of lymphocyte nucleotide levels in inherited or acquired immunodeficiency syndromes.

Formation and degradation of deoxyadenosine nucleotides in inherited adenosine deaminase deficiency.

dATP, dADP, and dAMP equalled or exceeded the depleted levels of ATP, ADP, and AMP in erythrocytes from two children with adenosine deaminase (ADA; EC 3.5.4.4) deficiency. dATP and dADP were identified in the mononuclear cells of only one child. The levels of deoxyadenosine compounds fell dramatically after enzyme replacement therapy and were no longer detectable in the urine or in mononuclear cells. Erythrocyte adenosine nucleotide levels showed a corresponding increase. Intact erythrocytes prior to treatment contained adenine, presumed to be from deoxyadenosine degraded during extraction. Adenosine at high concentrations in vitro increased both dATP and ATP levels and decreased intracellular deoxyadenosine levels. There was no significant deamination of either [8-14C]adenosine or deoxyadenosine by intact ADA-deficient erythrocytes. About 90% of adenosine was metabolized to ATP at substrate concentrations from 10-100 microM, compared to 40-60% of deoxyadenosine metabolized to dATP. These studies suggest that (i) high intracellular deoxyadenosine levels may be necessary in vivo to sustain the raised dATP levels in ADA deficiency. (ii) When ADA is inhibited or absent, deoxyadenosine is removed rapidly from the circulation by the human erythrocyte utilizing an adenosine transport system linked to both ADA and adenosine kinase (EC 2.7.1.20).

Reciprocal relationship between erythrocyte ATP and deoxy-ATP levels in inherited ADA deficiency

A reciprocal relationship between erythrocyte ATP and deoxy-ATP levels has been noted in an immunodeficient child with adenosine deaminase (ADA) deficiency during therapy with red cell transfusions. The sum of red cell ATP plus deoxy-ATP equalled the normal complement of ATP prior to any form of therapy. dATP, dADP and dAMP levels were found in the same ratio (10:1:0.1) as the adenine nucleotides ATP, ADP and AMP. Red cell ATP levels were low, not high or normal as found by others in ADA deficiency, but no deoxyadenosine nucleotides could be found in peripheral blood mononuclear cells. Erythrocyte ATP depletion has recently been identified as a serious consequence of anti-leukaemic therapy with ADA inhibitors; it may thus be an important but hitherto unrecognised contributing factor in the clinical expression of inherited ADA deficiency.

Deoxycoformycin-induced response in chronic lymphocytic leukaemia: deoxyadenosine toxicity in non-replicating lymphocytes

The occurrence of severe immunodeficiency disease in children with inherited adenosine deaminase deficiency, and reports of remission induction in T-cell acute lymphoblastic leukaemia with the adenosine deaminase inhibitor deoxycoformycin, prompted a study of the effects of deoxyadenosine on resting peripheral blood lymphocytes (PBL) and chronic lymphocytic leukaemic (CLL) lymphocytes in short-term culture. In the presence of an inhibitor of adenosine deaminase, micromolar concentrations of dAdo caused elevation of deoxyadenosine-5'-triphosphate (dATP) pools and in vitro lysis of non-dividing PBL and CLL lymphocytes. This death of non-replicating cells indicates a mechanism of deoxyadenosine toxicity independent of DNA replication and ribonucleotide reductase inhibition. Similar changes occurred in vivo in a patient with advanced CLL who responded to treatment with deoxycoformycin, 0.1 mg/kg, days 1-5, with a fall in the WCC from 102.0 x 10(9)/1 to 6.8 x 10(9)/l over 21 d. Therapeutic blockade of deoxyadenosine catabolism deserves further investigation both in the treatment of lymphoproliferative disease and as a method lympholytic immunosuppression.