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Deoxyguanosine Triphosphate As a Possible Toxic Metabolite in the Immunodeficiency Associated with Purine Nucleoside Phosphorylase Deficiency

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Deoxyguanosine Triphosphate As a Possible Toxic Metabolite in the Immunodeficiency Associated with Purine Nucleoside Phosphorylase Deficiency RAPID PUBLICATIONS Deoxyguanosine Triphosphate as a Possible Toxic Metabolite in the Immunodeficiency Associated with Purine Nucleoside Phosphorylase Deficiency ANMOS COHEN, LORRAINE J. GUDAS, ARTHUR J. AMNIANN, GERARD E. J. STAAL, and DAVID V. MARTIN, JR., Departmlenits of MIedicinie, Biochemiiistry anid Biophysics, atnd Pediatrics, Uniiversity of California at San Franicisco, San Francisco, California 94143, anid thle Department of Aledical Enzymology, Academic Hospital, Utrecl t, The Nethierlanids A B S T R A C T Puiriine nucleoside phosphorviase deoxyguanosine to deoxyGTP, which acts as a po- (PNP) deficiency is associated with a severe defect tenit inhibitor of maimmalian ribonucleotide reductase. in thvmnus-derived (T)-lymphocyte function comlbinied with normal bone marrow-derived (B)-lvmphocvte INTRODUCTION ftinction. To investigate the role of this enzvme deficiency in the resulting immune dysfunction, wve Deficiencies in either adenosine deamliinase (ADA)' or mleastured the levels of ribonucleoside and deoxv- purinie nucleoside phosphorylase (PNP), two enzymnes riboniucleoside triphosphates in erythrocvtes from twvo wvhich act sequentially in the purine salvage pathway unirelated PNP-deficient, T-lymphocvte-deficient pa- (Fig. 1), are associated with immunodeficiencv tients. Both PNP-deficient patients have abnormally diseases (1, 2). WVhereas ADA deficiency results in high levels of deoxyguanosine triphosphate (deoxy- combined bone marrow-derived (B)- anid thvmus- GTP) in their erythrocytes (5 and 8 nmol/ml packed derived (T)-lvmphocvte deficienicy, PNP-deficient pa- ervthrocvtes). In contrast, normal controls and adeno- tients exhibit T-cell dysfunction with normiial B- sinie deaminase-deficient, immunodeficient patients lymphocyte function. WVhile there are a number of do not have detectable amounts of deoxvGTP (<0.5 proposed mechanismiis to explain the associationi of nmol/nml packed erythrocytes). We propose that lymphotoxicitv with ADA deficiency (3-7), the cauise deoxvguanosine, a substrate of PNP. is the potentially of the T-cell deficiency associated with the absenice lvImphotoxic metabolite in PNP deficiency. The of PNP activity has received less attenitioni. It was mechanismii of toxicitv involves phosphorvlation of postulated that an accumulationi of the PNP sub- strate, inosine, inhibited ADA activity cauising finie- Dr. Cohen is supported by a Program Project grant, Clini- tional ADA deficiency (8). Subseqiuenitly, orotic cal Pharmiiacologv-Pharmacokinetics, from the National Insti- aciduria wvas f0und in two PNP-deficienit patients, tutes of Health. Dr. Gudas was supported by a fellowship from the Nationial Institute of Child Health and Humani Development. Dr. MIartin is an Investigator, Howard Hughes 'Abbreviationis used in this paper: ADA, adenosinc Medical Instittute. Reprint requests should be addressed to deamiiniase; deox\vATP, deoxyadenosine triphosphate; deoxy- Dr. Martin at University of California at San Francisco, CTP, deoxveytidine triphosphate; deoxyGTP, deoxyguano- Departmeint of Medicine, M-1093, San Francisco, Calif: sinle triphosphate; GTP, guanosinie triphlosphate; HGPRT, 94143. hvpoxanthine-guanine phosphoribosyl tranisferase; HPLC, Received for puiblication 27 December 1977 anid ini revised high-performance li(qtuid chromatography; PNP, purine foirm 10 Febrnaril 1978. nuicleoside phosphorvlase. J. Clin. Invest. © The Ainiericant Socictql fOr Clinical Iinvestigatioti, Inc., 0021-973817810.501-1405 s1.00 140.5 dGTP the imimunwiio(leficient patients ha(I b)een transftiused (r was reeeix i ug (Irligs l)efore study. dATP j Preparatiooi oj' samlies. Whole I)100() (2-:3 ml) \was t t withdrawn fromil pIatienits inito lhepariniize(l s\ ringes aod t DEOXYGUANOSINE PNP promptly cenitriflugerl (10,000 g for 2 imin) at ambient GUANINE teml)erature. The p)lasma was removed, ain(l an eual DEOXDEOXYADNOSINEADAYADENOSINE \ _DEOXYINOSINE(GUANOSINE) HYPOXANTHINE volume of ice-coldc _2 N perehlioric acid was added to the (ADENOSINE )- ( INOSINE) erythrocyte pellet. The chilled mixttire was againl cenltri- fugedl (10,000 g for 2 min) to remove the precipitated pro- XNUCLEOTIDASSE teini, and the stiperinate was neuitralizedl to pH 6 to 8 with .5 N KOH, to precipitate potassitiuim perchiorate. After chillinlg (DEOXY) NUCLEOSIDE on ice, the supernatant soluitionis were centrifuiged to re- MONOPHOSPHATES move the l)otassiun perchiorate, frozeni, anid \when niee cs- sary, shiippe(d to Sani Franciseo, Calif for analysis. FIGURE 1 Enzymes involved in purine nucleoside m11etab- Highi performnatice li(u/nid chromlatographyp. High per- olisill. formance li(juiid chromatography was performed uitilizing an Altex miieteriing system with a 20-,il UV analyzer (Altex Scientific Inc., Berkeley, Calif:), as described previously (10). indicatinig that pyrimidine starvation imiight exist in some Eitzi)lmatic detern iiniationi of deoxyGTP. The concentra- of the tissues of the patients (9). However, oral uridine tion of deoxyGTP in erythrocyte extracts was determiinled therapy did not significantly improve their immune by the method of' Solter and Handschumacher (15), uitiliz- funletion but did eliminate the orotic aciduria (9). ing the dependence of the DNA polymerase reiaetioni ulpo]n the presence of the triphosphates of all fouir deoxyribonllte1o- Recently, strikingly high levels of' deoxyadeniosine sides. The reactions were carried out for 60 min in the triphosphate (deoxyATP) were found in erythrocytes presnciee of satuiratinig amounts of (deoxycytidine triplos- from several ADA-deficient, immunodeficient pa- phate ((deox\C'TP) (2 tLMI), deoxyATP (2 ,uM) and [3H]TTP tients (10). Since deoxyATP is a known inhibitor (0.6 uC'i, 2 ,uM). The reactioni was dependent uponi, and linear of ribonucleotide reductase it was that with, the volume of adde(d erythrocyte extract. deoxvGTP (11), suggested standar(Is ad(led to normal erythrocyte extracts could not he an aecumuilation of deoxyATP in the patients' lympho- (letected at conieentrationis corresponding to less than 0.5 cytes is the cause of the immunodeficiency in ADA- nmol/m l of packed erythrocvtes. deficient childreni (10). Deoxyguanosine is one of' the four PNP substrates RESULTS which accumulate in the urine of PNP-deficient children (12). Human lymphocytes are capable of' The levels of nucleoside triphosphates in erythrocyte phosphorylating deoxyguanosine to deoxyguanosine extracts from ADA- and PNP-deficient patients, and triphosphate (deoxyGTP) (13), which like deoxyATP from normiial controls, were determined with high- can inhibit mammalian ribonucleotide reduetase (11). performiiance liquid chromatography (HPLC). Erythro- Thus, we measured deoxyGTP levels in erythrocytes cytes from PNP-deficient patients contain a significant from PNP-deficient patients and normal controls, and amount of deoxyGTP, which is not present in erythro- we now demonstrate that deoxyGTP is present in cytes from either normal controls or ADA-deficienit erythrocytes from PNP-deficient patients but not in patienits (Fig. 2). In contrast, erythrocytes from ADA- normal control erythrocytes. deficient patients contain deoxyATP, whiclh is not presenit in either normal controls or PNP-deficienit erythrocytes (10) (Fig. 2). METHODS The concentrations of nucleoside triphosphates in Materials. Nucleotide and deoxynucleotide standards erythrocytes from normal controls, ADA-deficienit, were purchased from Sigma Chemnical Co., St. Louis, Mo. PNP-deficienit, and HGPRT-deficient patients are sum- DNA-dependent DNA plymerase and calf thymus DNA marized in Table I. deoxyGTP levels are more than were purchased from Miles Laboratories Inc., Elkhart, 10-fold higher in erythrocyte extracts from the two Ind. [3H]TTP (60 Ci/mmol) was purchased from New England Nuclear, Boston, Mass. All other reagents were of PNP-deficient patients, compared to the other con- the hiighest grades commercially available. trols and patients. While the ATP levels of all the Patients. The two immunodeficient children, patient 1 (2) extracts tested are comparable, guanosine triphosphate and patient 2 (14), with inherited deficiencies of PNP (GTP) concentrations are two-four times lower in have been previously described. The ADA-deficient child with combined B- and T-lymphocyte deficiency has also extracts from either PNP-deficient or HGPRT-deficient been described previously (10) as has the adult male patients, compared to normal controls. This decrease deficient in erythrocyte hypoxanthine-guanine phosphori- in GTP concentration is presumably the result of im- bosyl transferase (HGPRT) (12). The control subjects in- paired guanine salvage capability caused by the ab- cluded normal children and adults and two immunodeficient sence of either PNP or children. One immunodeficient child has a severe com- HGPRT; however, other bined immunodeficiency while the other has only a T- mechanisms are certainly possible. lymphocyte deficiency, but both children have normal To confirm the presence of deoxyGTP in PNP- activities of ADA and PNP in their erythrocytes. None of deficient erythrocytes, we assayed deoxyGTP in the 1406 A. Cohen, L. J. Gudas, A. J. Ammann, G. E. J. Staal, and D. W. Martin, Jr. ATP TABLE I A GTP NORMAL Ribonucleoside-Triphosphate and Deoxyribonucleoside- Triphosphate Concentrations in Erythrocyte Extracts from Controls, PNP-, ADA-, and HGPRT- Deficient Patients Subjects ATP dATP GTP dGTP t?mol/ll packed erythrocytes Controls 1,150+100 <20 74±20 <0.5 PNP-deficient (patient 1) 1,260 <20 26 5 PNP-deficient dGTP (patient 2)
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