Nucleoside Phosphorylase Deficiency Research Articles

Purine Nucleoside Phosphorylase Deficiency: A Mutation Update

Purine nucleoside phosphorylase (PNPase) deficiency is an autosomal recessive disorder affecting purine degradation and salvage pathways. Clinically, patients typically present with severe immunodeficiency, neurological dysfunction, and autoimmunity. Biochemically, PNPase deficiency may be suspected in the presence of hypouricemia. We report biochemical and genetic data on a cohort of seven patients from six families identified as PNPase deficient. In all patients, inosine, deoxyinosine, guanosine, and deoxyguanosine were elevated in urine, and mutation analysis revealed seven different mutations of which three were novel. The mutation c.770A>G resulted in the substitution p.His257Arg. A second novel mutation c.257A>G (p.His86Arg) was identified in two siblings and a third novel mutation, c.199C>T (p.Arg67X), was found in a 2-year-old female with delayed motor milestones and recurrent respiratory infections. A review of the literature identified 67 cases of PNPase deficiency from 49 families, including the cases from our own laboratory. PNPase deficiency was confirmed in 30 patients by genotyping and 24 disease causing mutations, including the three novel mutations described in this paper, have been reported to date. In five of the seven patients, plasma uric acid was found to be within the pediatric normal range, suggesting that PNPase deficiency should not be ruled out in the absence of hypouricemia.

Primary immunodeficiencies (PIDs) presenting with cytopenias

Autoimmune manifestations are increasingly being recognized as a component of several forms of primary immunodeficiencies (PID). Defects in purging of self-reactive T and B cells, impaired Fas-mediated apoptosis, abnormalities in development and/or function of regulatory T cells, and persistence of immune activation as a result of inability to clear infections have been shown to account for this association. Among autoimmune manifestations in patients with PID, cytopenias are particularly common. Up to 80% of patients with autoimmune lymphoproliferative syndrome (ALPS) have autoantibodies, and autoimmune hemolytic anemia and immune thrombocytopenia have been reported in 23% and 51% of ALPS patients, and may even mark the onset of the disease. ALPS-associated cytopenias are often refractory to conventional treatment and represent a therapeutic challenge. Autoimmune manifestations occur in 22% to 48% of patients with common variable immunodeficiencies (CVIDs), and are more frequent among CVID patients with splenomegaly and granulomatous disease. Finally, autoimmune cytopenias have been reported also in patients with combined immunodeficiency. In particular, autoimmune hemolytic anemia is very common among infants with nucleoside phosphorylase deficiency. While immune suppression may be beneficial in these cases, full resolution of the autoimmune manifestations ultimately depends on immune reconstitution, which is typically provided by hematopoietic cell transplantation.

Nucleotide Degradation Products in Cerebrospinal Fluid (CSF) in Inherited and Acquired Pathologies

CSF purines were grossly elevated compared with controls only in adenylosuccinate lyase (ADSL) deficiency and TB meningitis. The former representing low permeability, the latter severe damage to the normal blood/brain barrier. By contrast, the similarity to controls, with no difference between Lesch–Nyhan disease (LND) or LND variants, would exclude hypoxia as a factor in the severe neurological deficits in LND. Similar findings in purine nucleoside phosphorylase (PNP) deficiency (although nucleosides replace the normal bases) likewise exclude hypoxia in the aetiology of the albeit milder neurological deficits.

Inhibition of the enzyme purine nucleoside phosphorylase (PNP) reduces refractoriness to transfused platelets in a dog model

To better define the role of T-cells in alloimmunization, we induced purine nucleoside phosphorylase (PNP) deficiency in a dog platelet transfusion model. Short-term administration of eight different drug schedules using several combinations of the PNP inhibitor 8-aminoguanosine and/or deoxyguanosine did not induce significant toxicity in four treated animals as demonstrated by blood chemistries, cell counts, and autologous platelet recovery and survival measurements. However, continuous long-term daily administration of these agents produced significant renal and/or hepatic toxicity leading to death in five of six animals. Modification of the drug schedule to early short-term administration of both deoxyguanosine and 8-aminoguanosine, followed by long-term intermittent doses of only 8-aminoguanosine, was not only well tolerated by all the animals but also resulted in significant immunosuppression. Overall, six of nine evaluable dogs (67%) treated with some combination of PNP inhibitors did not become refractory to eight weekly transfusions of platelets from a single random donor dog, P less than 0.005, compared to untreated controls, only 3/21 (14%) not immunized. Furthermore, in four evaluable recipients, discontinuation of the 8-aminoguanosine while continuing platelet transfusions from their original donors did not result in refractoriness. In addition, these four recipients were also unable to recognize platelets from two other random donors. This suggests that both specific and non-specific tolerance to foreign platelet antigens had been induced by PNP-inhibitor therapy. Other evidence for the efficacy of this immunosuppressive treatment was the almost normal post-transfusion recovery and survival of donor platelets, both during and after treatment. This suggests failure to form even low levels of platelet alloantibodies in the immunosuppressed recipients. In contrast, B-cell immunity to soluble antigens was intact as demonstrated by a normal antibody response to keyhole limpet haemocyanin (KLH) antigen.

Genetic control of red-cell nucleoside transport and its association with the B blood-group locus and nucleoside phosphorylase activity in sheep.

Nucleoside transport in sheep red cells is controlled by two allelomorphic genes, the gene for nucleoside transport deficiency (NuI) being dominant to that for the functional presence of carrier-mediated nucleoside transport activity (Nui). Sheep are also polymorphic with respect to their red-cell nucleoside phosphorylase (NP) activity, some having high activities and others low activities of this enzyme. The gene for high activity (NPH) is incompletely dominant to that for low activity (NPL). Inheritance data indicate that the Nu locus is genetically linked to that for the B blood-group system and, in addition, exerts a pleiotropic effect on NP activity, Nu permeability stabilizing the heat-labile NPL gene product. Nu-permeable cells have a higher ATP content than Nu-impermeable red cells, and within the Nu-impermeable subgroup, NP deficiency causes a further reduction in red cell ATP concentration. It is concluded that the nucleoside inosine supplements glucose as a physiological energy substrate in sheep red cells.

Purine nucleoside phosphorylase (PNP) deficiency leading to accumulation of lymphocytes in S-phase.

Freshly isolated mononuclear cells of a patient with purine nucleoside phosphorylase (PNP) deficiency contained 7-11% cells in S-phase. During treatment with deoxycytidine and tetrahydrouridine these cells disappeared from peripheral circulation, indicating that the in vivo accumulation of S-phase cells is caused by a shortage in deoxycytidine triphosphate. In vitro it was not possible to cause a blockade in S-phase by culturing normal or PNP-deficient lymphocytes in the presence of deoxyguanosine.

Decreased methionine synthesis in purine nucleoside-treated T and B lymphoblasts and reversal by homocysteine.

Purine nucleosides, which accumulate in adenosine deaminase and purine nucleoside phosphorylase deficiency, are toxic to lymphoid cells. Since adenine nucleosides inhibit S-adenosylhomocysteine hydrolase, they could potentially decrease intracellular methionine synthesis. To test this hypothesis, we measured methionine synthesis by the use of [14C]formate as a radioactive precursor in cultured human T and B lymphoblasts treated with varying concentrations of purine nucleosides; 2'-deoxycoformycin and 8-aminoguanosine were added to inhibit adenosine deaminase and purine nucleoside phosphorylase, respectively. In the T lymphoblasts methionine synthesis was inhibited approximately 50% by 10 microM of 2'-deoxyadenosine, adenine arabinoside, or 2'-deoxyguanosine. By contrast, in the B lymphoblasts methionine synthesis was considerably less affected by these nucleosides, with 50% inhibition occurring at 100 microM of 2'-deoxyadenosine and adenine arabinoside; 100 microM of 2'-deoxyguanosine yielded less than 10% inhibition. Adenosine and guanosine were considerably less potent inhibitors of methionine synthesis in both the T and B lymphoblasts. An adenosine deaminase-deficient and a purine nucleoside phosphorylase-deficient cell line, both of B cell origin, exhibited sensitivities to the nucleosides similar to those of the normal B cell lines. In both the T and B cell lines homocysteine reversed the methionine synthesis inhibition induced by the adenine nucleosides and guanosine and largely reversed that induced by 2'-deoxyguanosine. Methionine synthesis from homocysteine generates free tetrahydrofolate from 5-methyltetrahydrofolate, the main intracellular storage form of folate. We conclude that purine nucleoside toxicity may be partly mediated through (a) decreased intracellular methionine synthesis, and (b) altered folate metabolism.

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.

Evidence for active purine nucleoside cycles in human mononuclear cells and cultured fibroblasts.

Several aspects of purine metabolism were studied in peripheral blood mononuclear cells and fibroblasts from a patient with purine nucleoside phosphorylase deficiency and compared to cells from normal controls. Intact cells were incubated with radioactive purine bases and all purine metabolites were extracted and analyzed. Incubation of purine nucleoside phosphorylase-deficient cells with [3H]hypoxanthine and [3H]guanine resulted in the accumulation of large proportions of the incorporated radioactivity into inosine (60-80%) and to lesser extent into guanosine (15-30%), respectively, whereas normal cells accumulated only minor amounts of inosine and guanosine. This observation indicates that purine nucleoside phosphorylase, together with hypoxanthine-guanine phosphoribosyltransferase and nucleoside monophosphate phosphatase, participate in remarkably active inosine and guanosine cycles. These purine nucleoside cycles may play a role in the regulation of intracellular purine nucleotide levels. The absence of these cycles in purine nucleoside phosphorylase-deficient patients may be detrimental to the differentiation of lymphocytes.

Proposed explanation for S-adenosylhomocysteine hydrolase deficiency in purine nucleoside phosphorylase and hypoxanthine-guanine phosphoribosyltransferase-deficient patients.

We have examined the basis for the recently reported, but unexplained deficiency of S-adenosylhomocysteine hydrolase (AdoHcyase) in the erythrocytes of patients with genetic deficiencies of purine nucleoside phosphorylase and hypoxanthine-guanine phosphoribosyltransferase. We found that a hemolysate from a patient with purine nucleoside phosphorylase deficiency had only 7% of control AdoHcyase activity, conforming the original observation. Of the purine nucleosides known to accumulate in nucleoside phosphorylase-deficient patients, inosine alone caused the phosphate-dependent, irreversible inactivation of purified human placental AdoHcyase, and of AdoHcyase in intact erythrocytes and cultured lymphoblastoid cells. Hypoxanthine did not inactivate purified AdoHcyase, but potentiated the effect of inosine in intact hypoxanthine-guanine phosphoribosyltransferase-deficient human lymphoblastoid cells. This presumably resulted from the ability of hypoxanthine to shift the equilibrium of the nucleoside phosphorylase reaction, preventing inosine breakdown. This could account for the partial AdoHcyase deficiency reported in hypoxanthine-guanine phosphoribosyltransferase-deficient patients. We have also demonstrated the AdoHycase-catalyzed synthesis of S-inosylhomocysteine from inosine and L-homocysteine, a reaction which may occur in nucleoside phosphorylase-deficient patients.

Suppression of cellular immunity due to inhibition of purine nucleoside phosphorylase by allopurinol-riboside.

Recently, children with purine nucleoside Phosphorylase (PNP) deficiency have been reported to show defective T cell immunity but intact B cell immunity (1). These findings suggest that inhibition of PNP may result in suppression of T cell functions and an inhibitor of PNP may also be a useful immunosuppressive agent.

Purine Nucleoside Phosphorylase Deficiency: A Molecular Model for Selective Loss of T Cell Function

Abstract Absence of purine nucleoside phosphorylase (NP) is associated with severe T cell immune deficiency and normal B cell function. Patients with this enzyme defect accumulate inosine, guanosine, and their respective deoxycompounds, all of which are substrates for NP. We have evaluated the effect of these four NP substrates on PHA-stimulated lymphocytes and lymphoblastoid cell lines with B and T cell characteristics. Inosine and deoxyinosine had little inhibitory effect on human lymphocytes, whereas guanosine and deoxyguanosine inhibited DNA and protein synthesis in both PHA-stimulated human lymphocytes and hyman lymphoblastoid cells. In all experiments, deoxyguanosine was more toxic than guanosine. Human lymphoblastoid cells with T cell characteristics (T-LCL) were found to be particularly sensitive to the presence of deoxyguanosine. At low µM concentrations 3H-thymidine and 3H-leucine incorporation into the T-LCL was markedly decreased. At a concentration of 10 µM, no cell growth occurred and 50% of the cells were killed. Partial reversal of the toxic effect of deoxyguanosine on the T-LCL could be achieved with simultaneous addition of deoxycytidine. Human lymphoblastoid cells with B cell characteristics (B-LCL) were not inhibited at these low deoxyguanosine levels but cell growth and incorporation of 3H-thymidine and 3H-leucine were moderately decreased at higher deoxyguanosine concentrations. In a B-LCL established from a patient with NP deficiency, neither DNA nor protein synthesis was inhibited by guanosine or deoxyguanosine at concentrations that affected other B cell lines. The extreme sensitivity of the T lymphoblasts and relative resistance of B lymphoblasts to the toxicity of deoxyguanosine or one of its metabolic products may explain the selective loss of T cell function in patients with NP deficiency.

Adenosine deaminase and nucleoside phosphorylase activity in patients with immunodeficiency syndromes

Red cell adenosine deaminase (ADA) and nucleoside phosphorylase (NP) activities were measured in 62 patients with various immunodeficiency syndromes and in 67 adult and 37 infant controls. NP activity was found to be within the normal range (1206 ± 144 U/gHb in adults, 1266 ± 270 U/gHb in infants) in all but 4 patients who had NP deficiency and abnormal T-cell but normal B-cell function. The mean ADA activity was 36 U/gHb (with 95% confidence interval of 22.5 – 58.1 U/gHb) for normal adult controls ( n = 67) and 35 U/gHb (95% confidence interval 21.6 – 60.8 U/gHb) for infant controls ( n = 37). Red blood cell ADA activity of the patients showed great variability: Normal activity was found in patients with X-linked agammaglobulinemia, ataxia telangiectasia, and Wiskott-Aldrich syndrome. Six of 17 patients with combined immunodeficiency syndrome (CID) had ADA deficiency, and 7 CID patients had significantly elevated red cell ADA activity. Two identical twins with common-variable immunodeficiency showed red cell ADA activity of five to six times that of the normal controls; however, ADA activity of white cells and cultured skin fibroblasts was normal. Family members of these twin sisters had normal red cell ADA activity. It is not known if the elevated ADA activity observed in some patients with immune deficiency is directly related to the abnormal immune function.

A patient with nucleoside phosphorylase deficiency, selective T-cell deficiency, and autoimmune hemolytic anemia

A patient with nucleoside phosphorylase deficiency, selective T-cell deficiency, and autoimmune hemolytic anemia

Immunotherapy and immunopathologic studies in a patient with nucleoside phosphorylase deficiency

The results of immunotherapy and immunopathologic studies are described in a patient with nucleoside phosphorylase deficiency, normal B cell function, and severe absence of T cell function. Treatment for 9 months with bovine thymosin F5 at a dose of 1 mg/kg/week resulted in an increase in the percentage of E rosettes and the total number of T cells, and a return of the PHA and MLC response to greater than 50% of normal control values. With the development of systemic type 1 reactions following thymosin administration, therapy was discontinued. Immunologic studies returned to previous abnormal values. Treatment with oral uridine in a dose of 100 mg/kg/day for 9 months resulted in an increase in the percentage of E rosettes, an increase in the number of T cells, and an improved lymphocyte response to PHA. The MLC remained abnormal. Although the patient remained clinically well for 8 months, she then developed varicella which rapidly progressed to death, in spite of treatment with zoster immune plasma and adenine arabinoside. At autopsy, depletion of T cell-dependent areas in lymphoid tissue was observed. However, numerous plasma cells were present in which the cytoplasm stained for immunoglobulin. The thymus was reduced in size but had a suggestion of corticomedullary differentiation, and numerous Hassals corpuscles were observed. The results in this patient emphasize the susceptibility of nucleoside phosphorylase-deficient individuals to certain viral infections and suggest that additional immunotherapeutic and/or biochemical treatment is needed.

NUCLEOSIDE-PHOSPHORYLASE DEFICIENCY, AUTOIMMUNE HAEMOLYTIC ANAE MIA AND SELECTIVE T-CELL DEFICIENCY

Recently three patients with a severely defective T-cell immunity and no measurable activity of purine nucleoside phosphorylase (NP) were described. We had the occasion to observe a 22 months old girl with NP deficiency who was referred to us because of haemolytic anaemia which proved to be due to IgG and IgM incomplete warm autoantibodies against red cells. A CMV infection was diagnosed and a bilateral infiltration on chest roentgenograra was evident 4 weeks after admission. The NP activity of the red cells and granulocytes was absent. The parents are both heterozygous for the NP deficiency as well as both grandmothers, whose mothers are half-sisters. T-cell deficiency was demonstrated by negative skin tests to PHA, Varidase and Candida, by absence of E-rosette forming cells, total impairment of in vitro mitogenic response and mixed-lymphocyte reaction. The percentage of surface Ig-bearing lymphocytes and all serum immunoglobulin levels were normal. The clinical history will be fully discussed.

Cellular immunodeficiency associated with nucleoside phosphorylase deficiency: Immunologic and biochemical studies

Biological and immunological studies were performed on a child with severely impaired T-cell function, normal B-cell function, and deficiency of nucleoside phosphorylase. Adenosine deaminase levels in erythrocytes, lymphocytes, and fibroblasts of the patient and her immediate family were normal. Nucleoside phosphorylase activity could not be detected in the three cell types studied in the patient and was present at approximately half the normal level in the parents. A healthy sister had a normal level of the enzyme. No inhibitor was found in the patient's erythrocytes. These findings suggest that erythrocytes or lymphocytes may be used for carrier detection and amniotic cells may be used for intrauterine diagnosis of the deficiency. Cultured EB-virus-infected B cells retained the deficiency thus furnishing a lymphoid cell line for further investigation of the disease. No improvement in immunologic function was obtained after infusion with blood products nor could nucleoside phosphorylase be detected in circulating lymphocytes following such infusions.

HUMAN LYMPHOCYTE TRANSFORMATION FOLLOWING INHIBITION OF PURINE NUCLEOSIDE PHOSPHORYLASE

The genetic absence of purine nucleoside phosphorylase (NP) is associated with an isolated T-cell immune deficiency in humans. NP is responsible for the conversion of inosine to hypoxanthine and guanosine to guanine. We have synthesized a non-metabolizable compound, 6-hydroxy-9-p-aminobenzylpurine (HABP), an inhibitor of NP that has a Ki of 250 μM. The presence of HABP reduced the incorporation of 3H-thymidine into DNA in phytohemagglutinin stimulated human lymphocytes from healthy volunteers to 30% of control values. Patients with NP deficiency have increased levels of inosine and guanosine in their plasma. The addition of inosine to the culture medium of stimulated lymphocytes at 4 mM had no inhibitory effect on DNA synthesis as measured by thymidine incorporation. The addition of 2 mM guanosine, however, reduced thymidine incorporation to 4% of control lymphocytes. The inhibitory effect of guanosine could be partially corrected by the addition of hypoxanthine to the medium at 500 μM concentrations. The addition of uridine to the medium at concentrations to 1 mM did not reverse the inhibition induced by guanosine. These studies indicate that the T-cell abnormality observed in NP deficiency can be mimicked by two related mechanisms, 1) by inhibiting NP in normal lymphocytes, or 2) by increased concentration of guanosine in the culture medium.

NUCLEOSIDE PHOSPHORYLASE DEFICIENCY: IMMUNOLOGIC AND ELECTROPHORETIC EVIDENCE FOR ENZYME HETEROGENEITY

An absence of purine nucleoside phosphorylase (NP) activity in humans is associated with T-cell dysfunction. NP catalyses inosine to hypoxanthine and guanosine to guanine in the purine salvage pathway. Two families with children affected with NP deficiency were studied by immunological and biochemical methods. Monospecific rabbit antibody was prepared against purified human NP to test for cross-reacting material (CRM). In studies using RBC hemolysates, one patient had no detectable enzyme activity and no CRM to the NP antibody. Her parents had 1/2 normal NP activity, less than normal CRM and an NP isozyme pattern with multiple slow-moving bands.In a second family, two affected siblings had 0.5% residual enzyme, and 1/2 normal quantities of CRM. Each parent had an electrophoretic pattern for NP which was different from each other and different from the original family. The father had a normal amount of CRM and the mother 1/2 normal quantities of CRM.Our studies document the existence of three separate mutations in two families which effect the structure of NP and which is associated with immune deficiency.

A patient with purine nucleoside phosphorylase deficiency: Enzymological and metabolic aspects

1. 1. Enzymological and metabolic data in a patient with nucleoside phosphorylase (NP) deficiency are described. 2. 2. Incubation of intact NP-deficient red cells with [ 14C]adenosine showed a rapid uptake and conversion to inosine. Almost no radioactivity was incorporated in the adenosine nucleotides and no hypoxanthine labeling could be detected. 3. 3. Incubation with [ 14C]inosine resulted in a rapid conversion to IMP in the normal intact red cells but in an accumulation of inosine in the medium with the erythrocytes of the patient, proving again that a NP deficiency is present. 4. 4. The high PRPP level found may result from impaired consumption due to lack of substrates for the salvage enzyme HGPRT. 5. 5. Incubation with [ 14C] hypoxanthine and [ 14C]adenine showed that normal HGPRT and APRT activities were present in the NP-deficient red cells. 6. 6. In serum and urine of the patient the levels of inosine and guanosine were considerably increased, while the serum and urinary levels of uric acid were very low. In the two deceased sisters NP deficiency was also strongly suggested by analyses of the serum purines, of stored deep frozen samples.