ADA deficiency treatment.

In the report "X-ray diffraction to 302 giga-pascals: High-pressure crystal structure of cesium iodide" by H. K. Mao et al. (3 Nov., p. 649), reference 10, to a paper by R. Reichlin et al. [Phys. Rev. Lett. 56, 2858 (1986)], was incorrectly numbered (9) in the text (p. 649, column 3, line 1; p. 650, column 1, line 49).

Absence of adenosine deaminase activity in a mammalian cell line transformed by Rous sarcoma virus.

No detectable adenosine deaminase activity was found in whole cells or 105,000g cytosol preparations of B-mix K-44/6 cells when either [3H]adenosine or [3H]arabinosyladenine was used as substrate. When grown in tissue culture medium supplemented with horse serum these cells provide a deaminase-free system not requiring the use of an adenosine deaminase inhibitor.

S-adenosylhomocysteine hydrolase is an adenosine-binding protein: a target for adenosine toxicity.

When adenosine deaminase activity is inhibited, low concentrations of adenosine are toxic to human lymphoblast mutants that are unable to convert adenosine to intracellular nucleotides. In order to identify the mediator of this cytotoxicity, we searched for a cytoplasmic protein capable of binding adenosine with high affinity. Such a protein was identified in extracts of human lymphoblasts and placenta as the enzyme S-adenosylhomocysteine hydrolase.

Genetic heterogeneity in partial adenosine deaminase deficiency.

Inherited deficiency of the enzyme adenosine deaminase (ADA) results in a syndrome of severe combined immunodeficiency (SCID). Children with ADA- -SCID lack ADA in all cells and tissues. In contrast, a "partial" deficiency of ADA has been described in six immunologically normal children from four different "families." These children lack ADA in their erythrocytes but retain variable amounts of activity in their lymphoid cells. We have examined ADA activity in lymphoid line cells from four of these children, who are unrelated, for evidence of genetic heterogeneity. One child, who is Caucasian, has an enzyme with increased electrophoretic mobility, a diminished isoelectric point (pI 4.8 vs. Nl = 4.9) and very low activity (2.3 vs. Nl = 82.9 +/- 12.9 nmol/mg protein per min); as a second child has an enzyme with normal electrophoretic mobility but increased isoelectric point (pI = 5.0), markedly diminished heat stability at 56 degrees C (t1/2 = 4.2' vs. Nl = 40') and low activity (12.1); a third has an enzyme with only diminished heat stability (t1/2 = 6.5'), no detectable abnormality in charge and almost normal activity (41.9); while the fourth exhibits only diminished ADA activity (25.0) with no striking qualitative abnormalities. Thus, we have found evidence for three different mutations at the structural locus for ADA in three of these individuals, (a) an acidic, low activity heat stable mutation (b) a basic, somewhat higher activity, heat labile mutation, and (c) a relatively normal activity heat labile mutation. In the fourth, there is as yet no compelling evidence for a mutation at the structural locus for ADA and a mutation at a regulatory locus cannot be excluded.

In vivo inactivation of erythrocyte S-adenosylhomocysteine hydrolase by 2'-deoxyadenosine in adenosine deaminase-deficient patients.

The cytotoxic nucleoside 2'-deoxyadenosine is excreted in excessive amounts by individuals with genetic deficiency of adenosine deaminase, and may be in part responsible for the severe combined immune dysfunction from which they suffer. Earlier studies from this laboratory showed that 2'-deoxyadenosine causes the irreversible inactivation of the enzyme S-adenosylhomocysteine hydrolase by an active site-directed, "suicide-like" process. In this communication we have demonstrated similar inactivation of S-adenosylhomocysteine hydrolase in hemolysate and in intact erythrocytes, as well as a striking deficiency of S-adenosylhomocysteine hydrolase activity in the erythrocytes of three adenosine deaminase-deficient patients. In vivo suicide-like inactivation of S-adenosylhomocysteine hydrolase by 2'-deoxyadenosine may contribute to the cytotoxicity of 2'-deoxyadenosine and to the immune dysfunction in adenosine deaminase deficiency.

Immunoreconstitution by peripheral blood leukocytes in adenosine deaminase-deficient severe combined immunodeficiency.

Transplantation of histocompatible allogeneic peripheral blood leukocytes resulted in successful reconstitution of an adenosine deaminase (ADA)-deficient, severe combined immune-deficient patient. Erythrocyte transfusions before the transplant were associated with a rise of serum immunoglobulin concentration to normal without improvement in T cell function. The patient received 5 x 10(7) peripheral blood mononuclear leukocytes/kg obtained from the histocompatible father by leukopheresis. 3 wk after the transplant the lymphocyte count, proportion of E rosetting lymphocytes, and the ADA content of the patient's mononuclear leukocytes became normal while the phytohemagglutinin-stimulated blastogenic responses improved and became normal 52 d after the transplant. Antibody response to diphtheria immunization and response to naturally acquired herpes simplex infection were normal while isohemagglutinins progressively increased. Immunization with a neoantigen, bacteriophage phiX 174, resulted in a small but definite antibody response but no amplification of the response after secondary immunization. A positive reaction to a skin test for Candida albicans developed. Erythrocyte deoxy ATP (dATP) concentration decreased during the course of erythrocyte transfusions. 9 mo after the transplant, the erythrocyte dATP was elevated to twice pretransfusion levels while mononuclear leukocyte dATP varied from normal to elevated during the first 4 mo of the posttransplant period, but remained normal during the last 8 mo. The improvement in immune function persisted during the 12-mo posttransplant observation period while the mononuclear leukocyte ADA concentration stabilized at approximately 0.25 of normal, which is similar to the enzyme activity of the donor cells. This in vivo study supports the hypothesis that lymphoid precursor cells are present in peripheral blood which may partially reconstitute an immune-deficient recipient.

Erythrocyte adenosine deaminase deficiency without immunodeficiency. Evidence for an unstable mutant enzyme.

Inherited deficiency of the purine salvage enzyme adenosine deaminase (ADA) gives rise to a syndrome of severe combined immunodeficiency (SCID). We have studied a 2.5-yr-old immunologically normal child who had been found to lack ADA in his erythrocytes during New York State screening of normal newborns. His erythrocytes were not detectably less deficient in ADA than erythrocytes of ADA(-)-SCID patients. In contrast, his lymphocytes and cultured long-term lymphoid cells contained appreciably greater ADA activity than those from patients with ADA(-)-SCID. This residual ADA activity had a normal molecular weight and K(m) but was markedly unstable at 56 degrees C. His residual erythrocytes-ADA activity also appeared to have diminished stability in vivo. ADA activity in lymphoid line cells of a previously reported erythrocyte-ADA-deficient!Kung tribesman was found to contain 50% of normal activity and to exhibit diminished stability at 56 degrees C. ATP content of erythrocytes from both partially ADA-deficient individuals was detectably greater than normal (12.3 and 6.1 vs. normal of 2.6 nmol/ml packed erythrocytes). However, the dATP content was insignificant compared to that found in erythrocytes of ADA(-)-SCID patients (400-1,000 nmol/ml packed erythrocytes). The New York patient, in contrast to normals, excreted detectable amounts of deoxyadenosine, but this was <2% of deoxyadenosine excreted by ADA(-)-SCID patients. Thus, the residual enzyme in cells other than erythrocytes appears to be sufficient to almost totally prevent accumulation of toxic metabolites.

Adenosine deaminase deficiency with normal immune function. An acidic enzyme mutation.

In most instances, marked deficiency of the purine catabolic enzyme adenosine deaminase results in lymphopenia and severe combined immunodeficiency disease. Over a 2-yr period, we studied a white male child with markedly deficient erythrocyte and lymphocyte adenosine deaminase activity and normal immune function. We have documented that (a) adenosine deaminase activity and immunoreactive protein are undetectable in erythrocytes, 0.9% of normal in lymphocytes, 4% in cultured lymphoblasts, and 14% in skin fibroblasts; (b) plasma adenosine and deoxyadenosine levels are undetectable and deoxy ATP levels are only slightly elevated in lymphocytes and in erythrocytes; (c) no defect in deoxyadenosine metabolism is present in the proband's cultured lymphoblasts; (d) lymphoblast adenosine deaminase has normal enzyme kinetics, absolute specific activity, S20,w, pH optimum, and heat stability; and (e) the proband's adenosine deaminase exhibits a normal apparent subunit molecular weight but an abnormal isoelectric pH. In contrast to the three other adenosine deaminase-deficient healthy subjects who have been described, the proband is unique in demonstrating an acidic, heat-stable protein mutation of the enzyme that is associated with less than 1% lymphocyte adenosine deaminase activity. Residual adenosine deaminase activity in tissues other than lymphocytes may suffice to metabolize the otherwise lymphotoxic enzyme substrate(s) and account for the preservation of normal immune function.

Plasma deoxyadenosine, adenosine, and erythrocyte deoxyATP are elevated at birth in an adenosine deaminase-deficient child.

We have determined concentrations of adenosine, deoxyadenosine, and deoxyATP (dATP) in cord blood from an infant prenatally diagnosed as ADA deficient. Plasma deoxyadenosine and adenosine were already elevated in cord blood (0.7 and 0.5 microM vs. normal of less than 0.07 microM). Elevation of plasma deoxyadenosine has not previously been documented in these children. Erythrocyte dATP content was also elevated at birth (215 nmol/ml packed erythrocytes vs. normal of 2.9). These elevated concentrations of adenosine, deoxyadenosine, and dATP are similar to those we observed in another older adenosine deaminase-deficient patient and may explain the impaired immune function and lymphopenia seen at birth.

Adenosine deaminase deficiency: disappearance of adenine deoxynucleotides from a patient's erythrocytes after successful marrow transplantation.

Accumulation of adenine deoxynucleotides (dATP and dADP) in the erythrocytes of a patient with adenosine deaminase (ADA) deficiency was confirmed. The patient, now 18 mo old, was treated with a bone marrow transplantation from his HLA identical sister at 7 mo of age. Before and after the transplant, his erythrocyte and lymphocyte ADA activities, as well as his erythrocyte nucleotide profiles, were measured. 10 wk after the marrow transplant, no ADA activity could be detected in his erythrocytes, whereas there was a mixture of donor and patient lymphocytes as measured by ADA assays and karyotyping. At the same time, both dATP and dADP had disappeared from his erythrocytes, which were entirely of patient origin. These findings indicate that partial engraftment of donor lymphocytes into an ADA-deficient patient is capable of "correcting" alterations of deoxynucleotide concentrations in the patient's ADA-deficient erythrocytes.

Adenosine deaminase (ADA) deficiency due to deletion of the ADA gene promoter and first exon by homologous recombination between two Alu elements.

In 15-20% of children with severe combined immunodeficiency (SCID), the underlying defect is adenosine deaminase (ADA) deficiency. The goal of this study was to determine the precise molecular defect in a patient with ADA-deficient SCID whom we previously have shown to have a total absence of ADA mRNA and a structural alteration of the ADA gene. By detailed Southern analysis, we now have determined that the structural alteration is a deletion of approximately 3.3 kb, which included exon 1 and the promoter region of the ADA gene. DNA sequence analysis demonstrates that the deletion created a novel, complete Alu repeat by homologous recombination between two existing Alu repeats that flanked the deletion. The 26-bp recombination joint in the Alu sequence includes the 10-bp "B" sequence homologous to the RNA polymerase III promoter. This is the first example of homologous recombination involving the B sequence in Alu repeats. Similar recombination events have been identified involving Alu repeats in which the recombination joint was located between the A and B sequences of the polymerase III split promoter. The nonrandom location of these events suggests that these segments may be hot spots for recombination.

Overproduction of adenine deoxynucleosides and deoxynucletides in adenosine deaminase deficiency with severe combined immunodeficiency disease.

The deoxynucleotide, dATP, is elevated 50- to 1,000-fold above normal in erythrocytes, lymphocytes, and bone marrow from a child with adenosine deaminase deficiency and severe combined immunodeficiency disease. The child, when 17 mo of age, was also excreting approximately 30 mg of deoxyadenosine per day in urine (normal is less than 0.1 mg/day). Urinary excretion of uric acid was decreased. Elevated dATP levels in lymphocytes and bone marrow, and increased urinary excretion of deoxyadenosine, persisted despite hypertransfusion of the child with irradiated erythrocytes from a donor with normal adenosine deaminase. Overproduction of deoxynucleotides by increased salvage of adenosine appears to be the primary metabolic abnormality in patients with adenosine de aminase deficiency.

Adenosine deaminase messenger RNAs in lymphoblast cell lines derived from leukemic patients and patients with hereditary adenosine deaminase deficiency.

Hereditary deficiency of adenosine deaminase (ADA) usually causes profound lymphopenia with severe combined immunodeficiency disease. Cells from patients with ADA deficiency contain less than normal, and sometimes undetectable, amounts of ADA catalytic activity and ADA protein. The molecular defects responsible for hereditary ADA deficiency are poorly understood. ADA messenger RNAs and their translation products have been characterized in seven human lymphoblast cell lines derived as follows: GM-130, GM-131, and GM-2184 from normal adults; GM-3043 from a partially ADA deficient, immunocompetent !Kung tribesman; GM-2606 from an ADA deficient, immunodeficient child; CCRF-CEM and HPB-ALL from leukemic children. ADA messenger (m)RNA was present in all lines and was polyadenylated. The ADA synthesized by in vitro translation of mRNA from each line reacted with antisera to normal human ADA and was of normal molecular size. There was no evidence that posttranslational processing of ADA occurred in normal, leukemic, or mutant lymphoblast lines. Relative levels of specific translatable mRNA paralleled levels of ADA protein in extracts of the three normal and two leukemic lines. However, unexpectedly high levels of ADA specific, translatable mRNA were found in the mutant GM-2606 and GM-3043 lines, amounting to three to four times those of the three normal lines. Differences in the amounts of ADA mRNA and rates of ADA synthesis appear to be of primary importance in maintaining the differences in ADA levels among lymphoblast lines with structurally normal ADA. ADA deficiency in at least two mutant cell lines is not caused by deficient levels of translatable mRNA, and unless there is some translational control of this mRNA, the characteristic cellular ADA deficiency is most likely secondary to synthesis and rapid degradation of a defective ADA protein.

Biochemical and functional abnormalities in lymphocytes from an adenosine deaminase-deficient patient during enzyme replacement therapy.

Biochemical and immunological properties of lymphocytes were measured repetitively over a period of 40 mo during enzyme replacement by transfusion in a child with adenosine deaminase (ADA) deficiency and severe combined immunodeficiency disease. Catalytically defective ADA protein is present in the child's cells. ADA activity in his lymphocytes is 7 nmol/min per 10(8) cells with 51 ng of ADA protein/10(8) cells by radioimmunoassay. ADA activities in normal cord and adult lymphocytes average 193 and 92 nmol/min per 10(8) cells, respectively, with 429 and 223 ng of ADA protein/10(8) cells. Deoxy(d)ATP accumulates in the patient's erythrocytes and lymphocytes. Transfusion of irradiated packed erythrocytes partially corrects the metabolic defects. Frank metabolic relapse occurs if transfusions are discontinued for several months. The amounts of dATP in erythrocytes and lymphocytes averaged 13 and 2 times normal, respectively, during periods when transfusions were administered every 2-4 wk. Deoxyguanosine triphosphate and deoxycytidine triphosphate in lymphocytes were normal on 11 occasions, but deoxyribosylthymine triphosphate was ninefold increased. On 11 occasions dATP was measured in lymphocytes and erythrocytes isolated simultaneously. There was a positive, but statistically insignificant, correlation between amounts of dATP in the two types of cells (r = 0.25,P > 0.1). The absolute peripheral lymphocyte count was correlated with the activity of ADA in circulating erythrocytes and with the response of lymphocytes to phytohemagglutinin (r = 0.64, P < 0.01; r = 0.49, P < 0.05). Response of lymphocytes to stimulation by phytohemagglutinin in vitro and absolute peripheral lymphocyte counts were not significantly correlated with levels of dATP in the erythrocyte or lymphocyte during periods of intensive therapy. Although there was objective improvement during enzyme replacement, the child remained immunodeficient and biochemically abnormal.

Bone marrow transplantation only partially restores purine metabolites to normal in adenosine deaminase-deficient patients.

To delineate the extent to which bone marrow transplantation provides "enzyme replacement therapy", we have determined metabolite concentrations in two patients with adenosine deaminase (ADA) deficiency treated with bone marrow transplants and rendered immunologically normal. 10 yr after engraftment of lymphoid cells, erythrocyte deoxy ATP was markedly decreased compared to the marked elevations of deoxy ATP observed in untreated patients, but was still significantly elevated (62 and 90 vs. normal of 6.0 +/- 6.0 nmol/ml packed erythrocytes). Similarly, deoxyadenosine and adenosine excretion were both markedly diminished compared to that of untreated patients but deoxyadenosine excretion was still clearly increased (20.1 and 38.6 vs. normal of less than 0.2 nmol/mg creatinine) while adenosine excretion was in the upper range of normal (7.0 and 8.1 vs. normal of 5.6 +/- 3.6 nmol/mg creatinine). Mononuclear cell deoxy ATP content was also elevated compared to normal (5.25 and 14.4 vs. 1.2 +/- 0.3). Separated mononuclear cells of bone marrow transplanted patients contain both donor lymphocytes and recipient monocytes. When mononuclear cells were depleted of the cells enriched for donor lymphocytes (i.e. monocyte depleted) was lower than that of the mixed mononuclear cells (2.2 vs. 5.26). Surprisingly, plasma adenosine was as high as in untreated ADA-deficient patients (3.2 and 1.5 vs. untreated of 0.3-3 microM). Consistent with the elevated plasma adenosine and urinary deoxyadenosine, erythrocyte S-adenosyl homocysteine hydrolase activity was diminished (0.88 and 1.02 vs. normal of 5.64 +/- 0.25). Thus, bone marrow transplantation of ADA-deficient patients not only provides lymphoid stem cells, but also partially, albeit incompletely, clears abnormally increased metabolites from nonlymphoid body compartments.

Purine nucleoside metabolism in the erythrocytes of patients with adenosine deaminase deficiency and severe combined immunodeficiency.

Deficiency of erythrocytic and lymphocytic adenosine deaminase (ADA) occurs in some patients with severe combined immunodeficiency disease (SCID). SCID with ADA deficiency is inherited as an autosomal recessive trait. ADA is markedly reduced or undetectable in affected patients (homozygotes), and approximately one-half normal levels are found in individuals heterozygous for ADA deficiency. The metabolism of purine nucleosides was studied in erythrocytes from normal individuals, four ADA-deficiency patients, and two heterozygous individuals. ADA deficiency in intake erythrocytes was confirmed by a very sensitive ammonia-liberation technique. Erythrocytic ADA activity in three heterozygous individuals (0.07,0.08, and 0.14 mumolar units/ml of packed cells) was between that of the four normal controls (0.20-0.37 mumol/ml) and the ADA-deficient patients (no activity). In vitro, adenosine was incorporated principally into IMP in the heterozygous and normal individuals but into the adenosine nucleotides in the ADa-deficient patients. Coformycin (3-beta-D-ribofuranosyl-6,7,8-trihydroimidazo[4,5-4] [1,3] diazepin-8 (R)-ol), a potent inhibitor of ADA, made possible incorporation of adenosine nucleotides in the ADA-deficient patients...

Paradoxical expression of adenosine deaminase in T cells cultured from a patient with adenosine deaminase deficiency and combine immunodeficiency.

T lymphocytes cultured from a patient (T.D.) with adenosine deaminase (ADA) deficiency expressed ADA activity in the normal range, inconsistent with her severe immunodeficiency, metabolic abnormalities, and with the absence of ADA activity in her B lymphocytes and other nucleated hematopoietic cells. ADA from T.D. T cells had normal Km, heat stability, and sensitivity to ADA inhibitors. Examination of HLA phenotype and polymorphic DNA loci indicated that T.D. was neither chimeric nor a genetic mosaic. Amplified and subcloned ADA cDNA from ADA+ T.D. T cells was shown by allele-specific oligonucleotide hybridization to possess the same mutations (Arg101----Trp, Arg211----His) previously found in the ADA-T.D. B cell line GM 2606 (Akeson, A. L., D. A. Wiginton, M. R. Dusing, J. C. States, and J. J. Hutton. 1988. J. Biol. Chem. 263:16291-16296). Our findings suggest that one of these mutant alleles can be expressed selectively in IL-2-dependent T cells as stable, active enzyme. Cultured T cells from other patients with the Arg211----His mutation did not express significant ADA activity, while some B cell lines from a patient with an Arg101----Gln mutation have been found to express normal ADA activity. We speculate that Arg101 may be at a site that determines degradation of ADA by a protease that is under negative control by IL-2 in T cells, and is variably expressed in B cells. Il-2 might increase ADA expression in T cells of patients who possess mutations of Arg101.

Characterization of an adenosine deaminase-deficient human histiocytic lymphoma cell line (DHL-9) and selection of mutants deficient in adenosir kinase and deoxycytidine kinase.

The association of adenosine deaminase (ADA) deficiency with immunodeficiency disease has emphasized the importance of this purine metabolic enzyme for human lymphocyte growth and function. This report describes the natural occurrence of ADA deficiency in a human histiocytic lymphoma cell line, DHL-9. The minimal ADA activity in DHL-9 extracts, 0.028 nmol/min/mg protein, was less than 50% of the activity in two B-lymphoblastoid cell lines from ADA-deficient patients and was resistant to the potent ADA inhibitor deoxycoformycin. A sensitive radioimmunoassay failed to detect immunoreactive ADA in DHL-9 cells. Moreover, in DHL-9 cells, deoxycoformycin did not augment either the growth-inhibitory effects of adenosine and deoxyadenosine or the accumulation of deoxyadenosine triphosphate from deoxyadenosine. When compared to six other human hematopoietic cell lines, DHL-9 had 5.6-fold-higher levels of adenosylhomocysteinase. Chromosome 20, which bears the structural gene for ADA and adenosylhomocysteinase, was diploid and had a normal Giemsa banding pattern. The parental DHL-9 cell line was used for the selection and cloning of secondary mutants deficient in deoxycytidine kinase and adenosine kinase.

Deoxynucleoside overproduction in deoxyadenosine-resistant, adenosine deaminase-deficient human histiocytic lymphoma cells.

Deoxyadenosine toxicity toward lymphocytes may produce immune dysfunction in patients with adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) deficiency. The relationship between endogenous deoxynucleoside synthesis in adenosine deaminase-deficient cells and sensitivity to adenosine and deoxyadenosine toxicity is unclear. The human histiocytic lymphoma cell line (DHL-9) naturally lacks adenosine deaminase, and has minimal levels of thymidine kinase. Dividing DHL-9 cells excrete deoxyadenosine and thymidine into the extracellular space. The present experiments have analyzed nucleoside synthesis and excretion in a mutagenized clone of DHL-9 cells, selected for increased resistance to deoxyadenosine toxicity. The deoxyadenosine-resistant cells excreted both deoxyadenosine and thymidine at a 6-7-fold higher rate than wild-type lymphoma cells. The deoxyadenosine overproduction was accompanied by a reduced ability to form dATP from exogenous deoxyadenosine, and a 2.5-fold increase in ribonucleotide reductase activity. The pace of adenosine excretion, the growth rate, and the levels of multiple other enzymes involved in deoxyadenosine and adenosine metabolism were equivalent in the two cell types. These results suggest that the excretion of deoxyadenosine and thymidine, but not adenosine, is exquisitely sensitive to alterations in the rate of endogenous deoxynucleotide synthesis. Apparently, small changes in deoxynucleotide synthesis can significantly influence cellular sensitivity to deoxyadenosine toxicity.

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.