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Rare ImmunodeficiencyUnanticipated Benefits from the Study of a Adenosine Deaminase Deficiency:

Michael R. Blackburn and Linda F. Thompson

http://www.jimmunol.org/content/188/3/933doi: 10.4049/jimmunol.1103519

2012; 188:933-935; ;J Immunol 

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Adenosine Deaminase Deficiency: UnanticipatedBenefits from the Study of a Rare ImmunodeficiencyMichael R. Blackburn* and Linda F. Thompson†

The serendipitous discovery of adenosine deaminase(ADA) deficiency in two patients with cellular im-munodeficiency in 1972 by Dr. Eloise Giblett and

colleagues (1) ushered in a new era in the investigation of themolecular mechanisms underlying primary immunodeficiencydisorders. This finding led to the eventual development ofnovel therapies not only for ADA deficiency but also for otherimmunodeficiency disorders and certain leukemias.

In the early 1970s, several primary immunodeficiency dis-eases, including SCID, X-linked agammaglobulinemia, andWiskott-Aldrich syndrome, were well known to pediatric im-munologists and presumed to be caused by single gene defectsbased on patterns of inheritance. However, the gene defectsresponsible for these devastating disorders were unknown.In those days, the only “cure” for severe immunodeficiencydiseases was bone marrow transplantation (BMT) from a his-tocompatible donor. In the case of one of two patients de-scribed by Giblett et al., routine HLA typing of familymembers failed to identify suitable donors. Thus, the patient’sphysicians sent blood samples to Dr. Giblett at the KingCounty Central Blood Bank. It was hoped that she could shedlight on the relationships among the family members of thepatient by examining isozyme patterns for the enzyme ADA.Much to her surprise, starch gel electrophoresis indicated thatthe RBCs of the patient were completely devoid of ADAenzyme activity! The parents showed detectable, but reduced,ADA activity, suggesting an autosomal recessive mode of in-heritance. Subsequently, a second patient with severe cellularimmunodeficiency was studied and also found to be ADAdeficient. These were completely unexpected findings, as noprecedent existed for ADA deficiency in humans or for ADA’srole in either the development or the function of the immunesystem.

ADA is part of the purine salvage pathway that includesthe enzyme hypoxanthine-guanine phosphoribosyltransferase(HPRT). Mutations in the HPRT gene were known to causethe neurologic disorder Lesch-Nyhan syndrome and its asso-

ciated gouty arthritis (2), but this pathway was not thoughtto be important for the immune system. Giblett and col-leagues proposed that the two patients might have rare mutantalleles for the ADA gene. Alternatively, it was speculated thatthey might have a short chromosomal deletion encompassingthe ADA gene and a nearby critical immune response gene.In either case, Giblett et al. concluded the following: “SinceADA anenzymia and the inherited diseases of cellular im-munity are extremely rare, their coexistence in two unrelatedpatients seems very unlikely to be fortuitous.”

Measurements of purine metabolites in the body fluids ofADA-deficient patients showed elevated levels of adenosine(3), one of the two substrates for ADA. Investigators quicklyshowed that adenosine could slow the growth of lymphoidcell lines and the mitogen-induced proliferation of primarylymphocytes (3). In 1975, Giblett and colleagues (4) reporteda patient with an isolated T cell immunodeficiency wholacked activity of purine nucleoside phosphorylase, an enzymesituated between ADA and HPRT in the purine salvage path-way, providing convincing evidence of the critical importanceof normal purine metabolism for a functioning immune sys-tem. Although it was originally reported that ATP was ele-vated in the RBCs of ADA-deficient patients (5), more sen-sitive HPLC separation schemes in the laboratories of Drs.Mary Sue Coleman and Amos Cohen (6, 7) revealed that 29-deoxyadenosine 59-triphosphate (dATP) levels were elevatedas well. This finding confirmed an earlier speculation by Dr.Dennis Carson et al. (8) that deoxyadenosine, the other sub-strate of ADA, rather than adenosine, was the toxic metabolitein this disease. Subsequent experimentation showed thatdeoxyadenosine is converted first to 29-deoxyadenosine 59-monophosphate and finally to dATP by the high levels ofdeoxynucleoside kinases in the thymus. A likely pathogenicmechanism is dATP-triggered cytochrome c release from mi-tochondria, which triggers an apoptotic cascade, leadingto failure of T cell development (9). Interestingly, an under-standing of this pathway led to the development of novel andsuccessful chemotherapeutic approaches for treating hairy cellleukemia (10).

Both ADA and purine nucleoside phosphorylase are ex-pressed in virtually every cell in the body and had been con-sidered “housekeeping” genes. Thus, an immediate questionwas why the effects of ADA deficiency were focused upon theimmune system. This question led to a systematic evaluationof the expression of purine-metabolizing enzymes in varioushuman tissues and to the discovery that ADA was found atvery high levels in the thymus, suggesting that this organ hadevolved a mechanism to prevent the buildup of ADA sub-

*Department of Biochemistry and Molecular Biology, The University of Texas Med-ical School at Houston, Houston, TX 77030; and †Immunobiology and Cancer Pro-gram, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104

Address correspondence and reprint requests to Dr. Linda F. Thompson, Immunobi-ology and Cancer Program, Oklahoma Medical Research Foundation, 825 NE 13thStreet, Oklahoma City, OK 73104. E-mail address: Linda-Thompson@omrf.org

Abbreviations used in this article: ADA, adenosine deaminase; BMT, bone marrowtransplantation; dATP, 29-deoxyadenosine 59-triphosphate; HPRT, hypoxanthine-gua-nine phosphoribosyltransferase; PEG, polyethylene glycol.

Copyright � 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00

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strates. ADA is needed because the high rate of cell death inthe thymus following T cell selection events provides a sourceof DNA that is degraded to deoxyadenosine. This, coupledwith high levels of deoxynucleoside kinases, explains why thethymi of ADA-deficient patients accumulate such high levelsof dATP (8).

In addition to the normal supportive therapy given topatients with SCID, ADA-deficient patients were initiallytreated with packed RBC transfusions as a sort of “enzyme-replacement” therapy (5). Many patients showed significantimprovement in immune function as a result, especially thosewith residual ADA enzyme activity. The breakthrough in thetreatment of these patients came with the development ofpolyethylene glycol (PEG)-modified bovine ADA by the bio-technology company Enzon. PEG-ADA (Adagen) was thefirst FDA-approved PEG-modified protein drug. Its use as atherapy for ADA-deficient patients was championed by Dr.Michael Hershfield at Duke (11). Many patients who do nothave suitable bone marrow donors have been able to lead rea-sonably normal lives as a result of treatment with PEG-ADA.Today, a number of protein-based drugs that are modified bypegylation to improve stability and decrease immunogenicityare on the market. These include Neulasta (Amgen) for thetreatment of leukemia, IFN-b for the treatment of chronichepatitis C, and uricase for the treatment of refractory gout(12).

ADA deficiency also played a prominent role in the devel-opment of gene therapy. It was the perfect disease for thisfledgling field. As mentioned above, it was already known thatpatients with SCID could be cured by BMT from a histocom-patibledonor. Itwas alsoknown thatpatientswithonly10–12%of normal ADA enzyme activity had normal immune systems(13). Thus, it was logical to predict that autologous BMT withgenetically modified bonemarrow cells would be of therapeuticvalue even if normal levels of gene expression could not beattained. However, initial attempts were unsuccessful becausethe small numbers of genetically modified cells were notmaintained after transplantation (14). Nevertheless, this ap-proach was successful in patients with X-linked SCID becausethe genetically modified cells had a selective advantage andeventually overgrew the remaining unmodified cells (15). Thisrealization led to the hypothesis that gene therapy for ADAdeficiency was unsuccessful because patients were maintainedon PEG-ADA as a sort of standard of care. This treatment re-moved the selective advantage that ADA gene-corrected cellswould enjoy in an otherwise ADA-deficient host. Indeed, whentreatment protocols were modified to remove the PEG-ADA,gene therapy for this disorder was successful, although it usuallytook a year or more for the number of gene-corrected T cellsto reach maximal levels (16).

As with many human diseases, immunologists developedmouse models to have an experimental system in which theconsequences of ADA deficiency could be studied and newtreatment strategies evaluated. Much to the surprise of theinvestigators who made ADA-deficient mice, these mice diedin the immediate perinatal period—not of immunodeficiency,but of liver failure (17, 18). At the time of death, the effect ofADA deficiency on thymus development was relatively mod-est. To sidestep this problem, a strain of mice was developedthat was globally ADA deficient except for that controlledwith a placenta-specific promoter (19). Thus, they had ADA

during fetal development and became ADA deficient only af-ter birth. Surprisingly, they had normal liver function, show-ing that ADA was needed in the liver during fetal develop-ment, but not thereafter. Equally surprising, these mice diedof respiratory failure at z3 wk of age (20). However, theycould be maintained on PEG-ADA indefinitely. When ADAwas suboptimal, they developed immunodeficiency, as ex-pected (21). These mice have proved useful for examining themechanisms of ADA-deficient SCID (9). In addition, owingto the accumulation of adenosine, these animals have served asa biological screen for disorders associated with aberrant aden-osine receptor signaling (22). In the past 20 y, it has becomeincreasingly apparent that adenosine regulates many impor-tant aspects of physiology through binding to four distinct,seven-transmembrane–spanning G protein-coupled adenosinereceptors (23). Although adenosine is usually immunosup-pressive and anti-inflammatory, work in ADA-deficient micehelped uncover novel roles for adenosine in promoting theprogression of chronic diseases, including asthma, chronic ob-structive pulmonary disease, and pulmonary fibrosis (22). Inaddition, these mice helped to define a novel role for adeno-sine signaling in certain manifestations of sickle cell disease(24).

In conclusion, the discovery of ADA deficiency as a cause ofSCID was groundbreaking for several reasons. First, it was thefirst immunodeficiency disease for which the molecular defectwas identified, making possible both a prenatal and a postnatalmolecular diagnosis. Second, it underscored the importanceof normal purine metabolism for the development of the im-mune system. Understanding the mechanisms of ADA-de-ficient SCID led to the development of ADA inhibitors anddeoxyadenosine analogs for the treatment of hairy cell leuke-mia (10). PEG-ADA became the first PEG-modified proteinto be used as a therapeutic and opened the door for the de-velopment of additional PEG-modified proteins that are inwide clinical use today. ADA deficiency was the first inheriteddisease to be treated by gene therapy. Finally, ADA-deficientmice became an invaluable tool for the study of adenosinereceptor signaling in chronic lung diseases and sickle cell dis-ease. Thus, the history of investigations of ADA deficiency,initiated by the startling absence of ADA bands on EloiseGiblett’s starch gel, illustrates the potential impact of seren-dipitous discoveries in science and medicine and the unantic-ipated rewards that can arise from the study of patients withrare diseases.

DisclosuresThe authors have no financial conflicts of interest.

References1. Giblett, E. R., J. E. Anderson, F. Cohen, B. Pollara, and H. J. Meuwissen. 1972.

Adenosine-deaminase deficiency in two patients with severely impaired cellularimmunity. Lancet 2: 1067–1069.

2. Seegmiller, J. E., F. M. Rosenbloom, and W. N. Kelley. 1967. Enzyme defect as-sociated with a sex-linked human neurological disorder and excessive purine syn-thesis. Science 155: 1682–1684.

3. Thompson, L. F., and J. E. Seegmiller. 1980. Adenosine deaminase deficiencyand severe combined immunodeficiency disease. In Advances in Enzymology,Vol. 51. A. Meister, ed. John Wiley and Sons, Chichester, England, p. 167–210.

4. Giblett, E. R., A. J. Ammann, D. W. Wara, R. Sandman, and L. K. Diamond.1975. Nucleoside-phosphorylase deficiency in a child with severely defective T-cellimmunity and normal B-cell immunity. Lancet 1: 1010–1013.

934 PILLARS OF IMMUNOLOGY

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5. Polmar, S. H., E. M. Wetzler, R. C. Stern, and R. Hirschhorn. 1975. Restoration ofin-vitro lymphocyte responses with exogenous adenosine deaminase in a patientwith severe combined immunodeficiency. Lancet 2: 743–746.

6. Coleman, M. S., J. Donofrio, J. J. Hutton, L. Hahn, A. Daoud, B. Lampkin, andJ. Dyminski. 1978. Identification and quantitation of adenine deoxynucleotides inerythrocytes of a patient with adenosine deaminase deficiency and severe combinedimmunodeficiency. J. Biol. Chem. 253: 1619–1626.

7. Cohen, A., R. Hirschhorn, S. D. Horowitz, A. Rubinstein, S. H. Polmar, R. Hong,and D. W. Martin, Jr. 1978. Deoxyadenosine triphosphate as a potentially toxicmetabolite in adenosine deaminase deficiency. Proc. Natl. Acad. Sci. USA 75: 472–476.

8. Carson, D. A., J. Kaye, and J. E. Seegmiller. 1977. Lymphospecific toxicity inadenosine deaminase deficiency and purine nucleoside phosphorylase deficiency:possible role of nucleoside kinase(s). Proc. Natl. Acad. Sci. USA 74: 5677–5681.

9. Van De Wiele, C. J., J. G. Vaughn, M. R. Blackburn, C. A. Ledent, M. Jacobson,H. Jiang, and L. F. Thompson. 2002. Adenosine kinase inhibition promotes sur-vival of fetal adenosine deaminase-deficient thymocytes by blocking dATP accumu-lation. J. Clin. Invest. 110: 395–402.

10. Golomb, H. M. 2011. Fifty years of hairy cell leukemia treatments. Leuk.Lymphoma 52(Suppl 2): 3–5.

11. Hershfield, M. S., R. H. Buckley, M. L. Greenberg, A. L. Melton, R. Schiff,C. Hatem, J. Kurtzberg, M. L. Markert, R. H. Kobayashi, A. L. Kobayashi, andA. Abuchowski. 1987. Treatment of adenosine deaminase deficiency with poly-ethylene glycol-modified adenosine deaminase. N. Engl. J. Med. 316: 589–596.

12. Jevsevar, S., M. Kunstelj, and V. G. Porekar. 2010. PEGylation of therapeuticproteins. Biotechnol. J. 5: 113–128.

13. Jenkins, T., A. R. Rabson, G. T. Nurse, and A. B. Lane. 1976. Deficiency ofadenosine deaminase not associated with severe combined immunodeficiency. J.Pediatr. 89: 732–736.

14. Kohn, D. B., M. S. Hershfield, D. Carbonaro, A. Shigeoka, J. Brooks,E. M. Smogorzewska, L. W. Barsky, R. Chan, F. Burotto, G. Annett, et al. 1998.T lymphocytes with a normal ADA gene accumulate after transplantation oftransduced autologous umbilical cord blood CD341 cells in ADA-deficient SCIDneonates. Nat. Med. 4: 775–780.

15. Cavazzana-Calvo, M., S. Hacein-Bey, G. de Saint Basile, F. Gross, E. Yvon, P. Nusbaum,F. Selz, C. Hue, S. Certain, J. L. Casanova, et al. 2000. Gene therapy of human severecombined immunodeficiency (SCID)-X1 disease. Science 288: 669–672.

16. Aiuti, A., F. Cattaneo, S. Galimberti, U. Benninghoff, B. Cassani, L. Callegaro,S. Scaramuzza, G. Andolfi, M. Mirolo, I. Brigida, et al. 2009. Gene therapy forimmunodeficiency due to adenosine deaminase deficiency. N. Engl. J. Med. 360:447–458.

17. Wakamiya, M., M. R. Blackburn, R. Jurecic, M. J. McArthur, R. S. Geske,J. Cartwright, Jr., K. Mitani, S. Vaishnav, J. W. Belmont, R. E. Kellems, et al. 1995.Disruption of the adenosine deaminase gene causes hepatocellular impairment andperinatal lethality in mice. Proc. Natl. Acad. Sci. USA 92: 3673–3677.

18. Migchielsen, A. A. J., M. L. Breuer, M. A. van Roon, H. te Riele, C. Zurcher,F. Ossendorp, S. Toutain, M. S. Hershfield, A. Berns, and D. Valerio. 1995.Adenosine-deaminase-deficient mice die perinatally and exhibit liver-cell degenera-tion, atelectasis and small intestinal cell death. Nat. Genet. 10: 279–287.

19. Blackburn, M. R., S. K. Datta, and R. E. Kellems. 1998. Adenosine deaminase-deficient mice generated using a two-stage genetic engineering strategy exhibita combined immunodeficiency. J. Biol. Chem. 273: 5093–5100.

20. Blackburn, M. R., J. B. Volmer, J. L. Thrasher, H. Zhong, J. R. Crosby, J. J. Lee,and R. E. Kellems. 2000. Metabolic consequences of adenosine deaminase defi-ciency in mice are associated with defects in alveogenesis, pulmonary inflammation,and airway obstruction. J. Exp. Med. 192: 159–170.

21. Blackburn, M. R., M. Aldrich, J. B. Volmer, W. Chen, H. Zhong, S. Kelly,M. S. Hershfield, S. K. Datta, and R. E. Kellems. 2000. The use of enzyme therapyto regulate the metabolic and phenotypic consequences of adenosine deaminasedeficiency in mice. Differential impact on pulmonary and immunologic abnor-malities. J. Biol. Chem. 275: 32114–32121.

22. Zhou, Y., D. J. Schneider, and M. R. Blackburn. 2009. Adenosine signaling and theregulation of chronic lung disease. Pharmacol. Ther. 123: 105–116.

23. Gessi, S., S. Merighi, K. Varani, and P. A. Borea. 2011. Adenosine receptors inhealth and disease. Adv. Pharmacol. 61: 41–75.

24. Mi, T., S. Abbasi, H. Zhang, K. Uray, J. L. Chunn, L. W. Xia, J. G. Molina,N. W. Weisbrodt, R. E. Kellems, M. R. Blackburn, and Y. Xia. 2008. Excessadenosine in murine penile erectile tissues contributes to priapism via A2B adeno-sine receptor signaling. J. Clin. Invest. 118: 1491–1501.

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