Adenosine deaminase deficiency with late onset of recurrent infections: Response to treatment with polyethylene glycol-modified adenosine deaminase

Adenosine deaminase deficiency with late onset of recurrent infections: Response to treatment with polyethylene glycol-modified adenosine deaminase Yael Levy, MD, Michael S. Hershfield, MD, Cristina Fernandez-Mejia, Stephen H. Polmar, PhD, MD, Diane Scudiery, Melvin Berger, PhD, MD, a n d Ricardo U. Sorensen, MD From the Departments of Pediatrics and Pathology, Case Western Reserve University, Cleveland, Ohio, the Departments of Medicine and Biochemistry, Duke University Medical Center, Durham, North Carolina, the Departments of Pediatrics, Microbiology, and Immunology, Washington University School of Medicine, St. Louis, Missouri, and Enzon, Inc., South Plainfield, New Jersey We report a 5-year-old girl with adenosine deaminase (ADA) deficiency who was asymptomatic during the first years of life. At 3 years of age, she d e v e l o p e d chronic and recurrent sinopulmonary infections, and at 41/2years of age she had one major infection with Streptococcus pneumoniae (bacteremla and septic arthritis of the hip). Immunologic evaluation at 5 years of age revealed persistent lymphopenia, decreased helper-suppressor T cell ratios, and low proliferative responses to mitogens. The IgG, IgM, and IgA levels were normal; the IgG2 level was low normal or below normal. The patient had specific antibodies against toxoids and viral antigens but failed to produce antibodies against Haemophilus Influenzae type b and p n e u m o c o c c a l polysaccharides. Although no symptoms of allergy were present, she had persistent eosinophilia and elevated IgE levels. The patient had 0.6% of normal ADA activity in erythrocytes and approximately 1% of normal ADA activity in peripheral blood mononuclear cells. Beginning at 6 years of age, she was treated with weekly injections of polyethylene glycol-modified bovine ADA. This treatment was well tolerated and effectively reversed the biochemical consequence of ADA deficiency. Concomitantly, she improved clinically and her T lymphocyte numbers and blastogenic responses to mltogens in vitro b e c a m e normal. The late onset of clinical symptoms and relatively benign clinical course in this patient emphasize the need to consider ADA deficiency in a broad spectrum of immunodeficient children. (J PEDIATR1988;113:312-7)

Supported in part by grants (Nos. RR00080, RR00036, DK20902, and CA38353) from the National Institutes of Health, U.S. Public Health Service. Submitted for publication Sept. 28, 1987; accepted March 8, 1988. Reprint requests: R. U. Sorensen, MD, Department of Pediatrics, Rainbow Babies and Children's Hospital, 2101 Adelbert Rd., Cleveland, OH 44106.

312

Adenosine deaminase deficiency accounts for approximately 20% to 25% of cases of autosomal recessive severe combined immunodeficiency. In the majority of patients, the onset of clinical disease occurs within 1 to 6 months of age, with failure to thrive, diarrhea, persistent oral and perianal candidiasis, and recurrent viral, bacterial, and

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ADA AXP dAXP EHNA PBMC PEG-ADA SAHase SCID

A D A deficiency with late onset o f recurrent infections

Adenosine deaminase Total adenine ribonucleotides Total adenine deoxyribonucleotides Erythro-9-(2-hydroxyl-3-nonyl)adenine Peripheral blood mononuclear cells Polyethylene glycol modified ADA S-adenosylhomocysteine hydrolase Severe combined immunodeficiency

other opportunistic infections. 1 In 10% to 15% of patients, the onset of disease may occur later than 6 months. 1'2 Rarely, patients with clinical onset of disease after the second year of life have been recognized. 3,4 W e describe a 5-year-old girl with _<1% of normal A D A activity in erythrocytes, peripheral blood mononuclear cells, and T cell line, who demonstrates the variability of clinical presentations associated with A D A deficiency. The patient has had an excellent early clinical response to enzyme replacement therapy with polyethylene glycol-modified bovine A D A . 5 CASE REPORT This five-year-old girl was seen in our allergy-immunology clinic for evaluation of recurrent infections. Her family history was unremarkable. She had had only minor, self-limited upper respiratory tract infections until age 3 years, but then had recurrent episodes of fever and sinusitis, chronic bronchitis, and pneumonia at 4 and 5 years of age. At the age of 41/2years, she had pneumococcal bacteremia with septic arthritis of the left hip. Two blood cell counts performed 1 month before her episode of septic arthritis revealed leukocyte counts of 2000 and 5000 cells/ram 3, respectively, with lymphocyte counts of 640 and 1777 cells/mm 3. All infections responded well to antibiotic treatment, hut new infections started soon after discontinuation of therapy. Physical examination revealed a well-developed girl (weight and height in the 50th percentile for age) without skin rashes or skeletal abnormalities. Tonsils were absent, and only small bilateral (0.5 • 0.5 cm) submandibular lymph nodes were palpable. Routine laboratory tests performed over an 8-month period when the patient was between 5 and 6 years of age revealed leukocyte counts ranging from 3700 to 18,400 cells/mm 3 with persistent lymphopenia, (126 to 925 cells/mm3) and eosinophilia (805 to 4732 cells/mm3). Blood chemistry values and urinalysis results were normal. Chest roentgenograms did not demonstrate a thymus shadow or bone abnormalities. Lateral neck roentgenograms revealed an absence of adenoidal tissue. Immunologic evaluation revealed reduced numbers of T lymphocytes and severely reduced blastogenic responses to mitogens. Levels of IgG (844 mg/dl), IgM (79 mg/dl), and IgA (111 mg/dl) were normal. IgE levels were elevated (2000 and 3000 U/ml on two determinations). IgG 1, 3, and 4 subclass levels, determined on two occasions by an immunoradiometric assay (Specialty Laboratories Inc., Los Angeles, Calif.), were normal (IgGl: 629 mg/dl and 537 mg/dl;

31 3

Table I. Erythrocyte enzyme activities in patient and parents ( n m o l / h r / m g protein) Enzyme

Patient

Mother

Father

Control

ADA

0

12.8

12.4

21.1

SAHase PNP

0.12 2862

4.9 1743

3.2 2100

3.4 1691

PNP. Purine nucleoside phosphorylase.

IgG3:31 and 16 mg/dl; IgG4:72 and 44 mg/dl). IgG2 was 79 and 37 mg/dl, respectively (normal 60 to 400 mg/dl), on two determinations. The following specific antibody titers were present in protective levels: anti-measles antibodies (1:64), antidiphtheria toxoid antibodies (0.118 U/ml), and anti-tetanus toxoid antibody (0.67 U/ml). Anti-B isohemagglutinin titer was 1:8. The preimmunization and postimmunization anti-hemophilus polysaccharide antibody levels were 0.07 #g/ml and 0.06 #g/ml, respectively. No antibodies against pneumococcal types 3, 7, 9, and 14 polysaccharide were detected. C3 and C4 levels were normal: 117 mg/dl and 71 mg/dl, respectively. At age 5Y2 years, intravenous therapy with immunoglobulin at a dose of 400 mg IgG/kg every 4 weeks was begun. During the following 6 months, a marked improvement was noted, with reduction in the frequency and severity of the episodes of cough, wheezing, and fever, but monthly antibiotic treatments were still required. Further studies were conducted to identify a casue of the patient's deficient cellular immunity and to evaluate therapy with PEG-ADA, which was initiated at 6 years of age after ADA deficiency was diagnosed.

METHODS T cell subsets were enumerated with the use of monoclonal antibodies for immunofluorescent labeling and flow cytometry on P B M C isolated by Ficoll-Hypaque gradients. The following monoclonal antibodies (Ortho Pharmaceutical Corp., Raritan, N.J.) were used: O K T l l and OKT3 (CD2 and CD3, T lymphocytes), O K T 4 (CD4, helper-inducer T cells), O K T 8 (CD8, suppressor-cytotoxic T cells), O K T 6 (CD1, common thymocytes), and O K I a (activated T lymphocytes, B lymphocytes, and some monocytes). The B lymphocytes were identified with the F(ab')2 fragment of anti-human immunoglobulin antibody (Kallestad Diagnostics, Austin, Tex.). Natural killer cells were identified with the monoclonal antibody anti-Leu-lla (CD16) (Becton Dickinson Immunocytometry Systems, Mountain View, Calif.). Proliferative responses of lymphocytes to mitogens were determined by measurement of [aH]-thymidine incorporation by P B M C , incubated for 3 days with optimal concent~ration of phytohemagglutinin, concanavalin A, and pokeweed nitrogen. Enzyme studies were performed independently at Wash-

3 14

Levy et al.

The Journal of Pediatrics August 1988

Table II. Metabolic response to treatment with PEG-ADA Erythrocyte nucleotldes (/~mol/ml RBC)

Weeks of therapy

PEG-ADA dose (U/kg)

Plasma ADA (/~mol/hr/ml)

AXP

dAXP

%dAXP

0 1 2 3 6 10 15 32

0 5 10 15 15 15 15 15

0.081 3.997 10.194 11.948 21.535 16.687 16.591 16.534

1.029 1.117 1.051 1.125 0.931 0.923 1.164 1.420

0.204 0.088 0.044 0.028 0.007 0.005 0.007 0.005

16.5 7.3 4.1 2.4 0.7 0.6 0.6 0.3

Normal values (#mol/ml erythrocytes):AXP = 1.465 + 0.38; dAXP = <0.001.

ington University and Duke University. At Washington University, ADA, S-adenosylhomocysteine hydrolase, and purine nucleoside phosphorylase were assayed as reported previously:: The ADA, SAHase, and purine nucleoside phosphorylase activities in erythrocytes, PBMC, and an interleukin-2-dependent CD4 (20%) and CD8 (80%) positive T cell line were determined at Duke University by methods used previously:'7'8 The levels of total adenine ribonucleotides and deoxyribonucleotides in erythrocytes were determined as described previously: Treatment was given 5 with PEG-ADA kindly provided by Enzon, Inc. (South Plainfield, N.J.) Plasma A D A activity and erythrocyte A X P and dAXP levels were determined after 24 hours, 48 hours, and 3, 5, and 7 days after the first three weekly injections, and on the seventh day after each subsequent injection. For these determinations, plasma and pelleted erythrocytes obtained from heparinized blood were frozen and shipped on dry ice to Duke University. Lymphocyte subpopulation enumeration, blastogenic responses to mitogens, complete blood cell counts, blood chemistry studies, and urinalysis were performed every 4 weeks. RESULTS The patient's A D A activity was markedly decreased, the activity of purine nucleoside phosphorylase was elevated. A decreased SAHase activity, caused by inactivation of this enzyme because of the accumulation of 2'-deoxyadenosine, 7,8 was also present. Approximately 50% of the normal level of A D A activity was present in the erythrocytes of the patient's parents (Table I). The results from the studies at Washington University were independently confirmed by assays at Duke University Medical Center, where erythrocyte A D A activity was found to be 0.6% of normal (0.33 n m o l / h r / m g cell protein; n o r m a l = 56.7---20.5 n m o l / h r / m g cell protein) and SAHase activity was 5.2% of normal (0,22 nmol/hr/mg;

normal -- 4.2 _+ 1.9 n m o l / h r / m g protein). Approximately 1% of normal ADA activity was present in the patient's PBMC: 11.6 nmol/hr/mg protein, in comparison with 1055 _+ 256 nmol/hr/mg protein in control PBMC. The activity of A D A in cultured interleukin-2-dependent T cells was 7.1 nmol/hr/mg protein. The residual low level of A D A activity in the patient's PBMC extracts was not inhibited by 5 #mol EHNA, which inhibited over 98% of A D A activity of control cells. The EHNA-resistant A D A activity in cells and plasma of ADA-deficient children with SCID has been shown to be due to a distinct adenosine amino hydrolase activity that is not the product of the A D A gene. 9-H Weekly intramuscular injections of PEG-ADA were begun when the patient was 6 years of age, while intravenous immunoglobulin therapy was continued. During the first 3 weeks of treatment, the dose of P E G - A D A was increased from 5 units/kg to a final dose of 15 units/kg. At this dose, trough (preinjection) levels of plasma ADA were maintained at approximately one and one-half times the normal level of total erythrocyte A D A activity5 (Table II). Before treatment, dAXP represented 16.5% of the erythrocyte adenine nucleotide pool; with treatment, dAXP decreased to <0.6% (Tab!e II). In addition, the level of erythrocyte SAHase activity increased almost 20-fold, to a maximum value of 3.78 n m o l / h r / m g protein. This nearly complete correction of the erythrocyte biochemical abnormalities caused by deoxyadenosine is very similar to the response of the first two patients treated with PEGADA) Before treatment there was a marked decrease in the number of total lymphocytes, total T ceils (CD2, CD3 positive cells), and T-helper cells (CD4 cells). These values increased into the normal range by 2 months of treatment and persisted in this range after 6 months of treatment (Table III). Lymphocyte blastogenic responses to mitogens were

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ADA deficiency with late onset o f recurrent infections

31 5

T a b l e III. Lymphocyte subpopulation analysis before and after P E G - A D A treatment During treatment Before treatment

1 mo

Marker

%

(range)* No, (cells/ram 3)

Total Lymphocytes Count CD2 CD3 CD4 CD8 SIg OKIa CD16 CD1

--

126-925

57-88 63? 21-32 23-40 9-34 30-48 6-8 47

176-920 244 74-220 53-418 30-105 70-148 19-63 15

CD4/CD8 ratio

No. (cells/ mm 3)

%

2 mo No. (cells/ mm 3)

%

1040

59 38 26 15 28 32 26

0.53-1.39

614 395 270 156 291 233 270

6 mo No. (cells/ mm 3)

%

2035

52 50 27 30 21 30 20

1058 1018 488 611 427 611 407

Normal range ( m e a n _+ 2 SD) No. (cells/ mm 3)

%

3540

80 56 39 22 8 12 11

2832 1982 1381 779 283 425 389

ND

ND

ND

1.73

0.80

1.77

1125-11373

75-95 60-88 39-68 12-34 5-17 5-17 0-18 0-4

844-10806 675-10010 439-7735 135-3868 56-1934 56-1934 0-860 0-4555

1.3-4.6

ND. Not done. *Results Of tests performed over a period of 8 months when the patient was between 5 and 6 years of age. ~'Only one test performed.

T a b l e IV. Peripheral blood lymphocyte proliferative responses to mitogens (mean counts per minute for triplicate cultures)

Before treatment:

During treatment ( c p m )

Stimulator

(range)"

I mo

2 mo

6 mo

Normal values (cpm)

Phytohemagglutinin Concanavalin A Pokeweed mit0gen

3,406-9,339 1,562-7,026 2,754-4,578

57,765 8,811 6,370

188,136 65,677 9,450

168,186 42,290 18,187

30,000-180,000 40,000-155,000 20,000-95,000

*Results of three tests performed over 8 months when the patient was between 5 and 6 years of age.

markedly decreased on three occasions when tested before treatment, but they increased into the normal range by 2 months after treatment and remained normal or nearly normal over 6 months of treatment (Table IV). T r e a t m e n t with P E G - A D A has been well tolerated, with no signs of toxic effects or allergic reactions. During the 6 months since treatment was begun, the patient has had only one (self-limited) upper respiratory tract infection. N o further antibiotic treatment has been required. DISCUSSION Despite in vitro evidence for significant cellular immunodeficiency detected at 5 years of age, our patient did not have typical clinical features such as failure to thrive, chronic diarrhea, Candida infections, Pneumocystis carinii pneumonia, or severe or life-threatening viral infections in the first months of life, as seen in 80% to 90% of patients with A D A deficiency? In 10% to 15% of the cases,

apparent clinical Onset may occur later, but both humoral and cellular immunity become severely defective and death usually occurs before 3 years of age. a,~z W e are aware of only two other ADA-deficient patients with late onset of disease. Giblett et al? described a 31h-year-old girl with clinical onset of disease after 2 years of age, although she had lymphopenia and eosinophilia as early as 2 weeks of age; she had persistently normal immunoglobulin values with specific deficiency of isoagglutinins and antibodies to diphtheria. A m m a n n et al? described an 8-year-old girl with pneumonia followed by recurrent upper respiratory tract infections at the age of 3 years; she had residual A D A activity in her P B M C (1.9% control), which might explain the late onset of symptoms and mild clinical course. Immunocompetent and asy.mptOmatic individuals with absent A D A activity in theie erythrocytes, but with 2% to 70% o f normal A D A activity in their P B M C , have also been described? 3~5 In contrast, the patient we describe has

3 16

Levy et al.

The Journal of Pediatrics August 1988

approximately 1% of normal ADA activity in her lymphocytes, which is not above values found in typical ADAdeficient patients with SCID and early onset of disease. ~,4,13Her late onset of symptoms of immunodeficiency and benign clinical course may be due to the presence of an unstable enzyme that rapidly loses activity in vitro. This possibility is raised by the finding of a somewhat lower level of deoxyadenosine nucleotides in her erythrocytes than has been found in the erythrocytes of other ADAdeficient children with classic SCID: 17% dAXP in comparison with 40% to 60% in typical ADA-deficient patients?' 16, 17

the biochemical abnormalities responsible for causing the immune system defect in ADA deficiency? Immunologic recovery in our patient was somewhat more rapid than observed in the first two patients, perhaps because of the less severe immune defect in this patient. This encouraging response to PEG-ADA therapy emphasizes the importance of recognizing the heterogeneous clinical presentation of ADA deficiency.

Our patient's history of chronic and recurrent sinopulmonary infections, and one major infection with Streptococcus pneumoniae starting at 3 years of age, suggested the possibility of hypogammaglobulinemia or IgG2 subclass deficiency. The suspicion of a functional selective IgG2 subclass deficiency in our patient was confirmed by the lack of antibody response to Haemophilus influenzae vaccine. Her good clinical response t o treatment with immunoglobulin lends support to the clinical relevance of her specific antibody deficiency~ It is possible that other children with selective antibody deficiency to polysaccharides with or without IgG2 subclass deficiency may also have abnormal cellular immunity caused by ADA deficiency. Our patient also had persistent eosinophilia and elevated IgE levels, abnormalities that previously had been described only in patients with other cellular immunodeficiencies, such as DiGeorge syndrome, X-linked SCID, Nezelof syndrome, Wiskott-Aldrich syndrome, and the hyper-IgE syndrome. 18-2~ Bone marrow transplantation is the preferred treatment for immunodeficiency caused by ADA deficiency. However, histocompatible donors are frequently not available, and engraftment has been difficult to achieve in some ADA-deficient patients treated with T cell-depleted marrow from haploidentical donors. 22In our patient the milder disease did not warrant the potential risk of ablative preconditioning,, which would have been required to carry out haploidentical marrow transplantation. We elected, therefore, to treat her with enzyme replacement, which has now been simplified with the introduction of PEGADA. 5,23 The attachment of polyethylene glycol greatly prolongs the circulating life and reduces the immunogenicity of bovine ADA, 24 permitting long-term administration of purified ADA by weekly intramuscular injection? PEG-ADA therapy is more convenient and better tolerated than partial exchange transfusion, and it avoids the risks of iron overload, virus transmission, and sensitization to erythrocyte antigens. In addition, because higher levels of circulating ADA activity can be achieved, PEG-ADA is more effective than erythrocyte transfusion in eliminating

1. Hirschhorn R. Genetic deficiencies of adenosine deaminase and purine nucleosidephosphorylase: overview,genetic heterogeneity and therapy. Birth Defects 1983;19:73-81. 2. Polmar SH. Metabolic aspects of immunodeficiencydisease. Semin Hematol 1980;17:30-43. 3. Giblett ER, Anderson JE, Cohen F, et al. Adenosinedeaminase deficiency in two patients with severely impaired cellular immunity. Lancet 1972;2:1067-9. 4. Ammann AJ, Cowan MJ, Martin DW, et al. Dipyridamole and intravenous deoxycytidine therapy in a patient with adenosine deaminase deficiency. Birth Defects 1983;19:11720. 51 Hershfield MS, Buckley RH, Greenberg ML, et al. Treatment of adenosine deaminase deficiency with polyethylene glycol-modified adenosine deaminase. N Engl J Med 1987; 316:589-96. 6. Fernandez-Mejia C, Debatisse M, Buttin G. Adenosineresistant Chinese hamster fibroblast variants with hyperactive adenosine-deaminase: an analysis of the protection against exogenous adenosine afforded by increased activity of the deamination pathway. J Cell Physiol 1984;120:321-8. 7. Hershfield MS. Apparent suicide inactivation of human lymphoblast S-adenosylhomocysteinehydrolase by 2'-deoxyadenosineand adenine arabinoside: a basis for a direct toxic effect of an analog of adenosine. J Biol Chem 1979;254: 22-5. 8. Hershfield MS, Kredich NM, Ownby DR, et al. In vivo inactivation of erythrocyte S-adenosylhomocysteine hydrolase by 2'-deoxyadenosine in an adenosine deaminase deficient patient. J Clin Invest 1979;63:807-11. 9. Daddona PE, Kelley WN. Characteristics of an aminohydrolase distinct from adenosine deaminase in cultured human lymphoblasts. Biochem Biophys Acta 1981;658:280-90. 10. Schrader WP, Pollara B, Meuwissen H. Characterization of the residual adenosine deaminating activity in the spleen of a patient with combined immunodeficiencydisease and adenosine deaminase deficiency. Proc Natl Acad Sci USA 1978; 75:446-50. 11. Ratech H, Hirschhorn R. Serum adenosine deaminase in normats and in a patient with adenosine deaminase deficient severe combined immunodeficiency. Clin Chim Acta 1981; 115:341-7. 12. Giblett ER. ADA and PNP deficiencies: how it all began. Ann NY Aead Sci 1985;451:1-8. 13. Hirschhorn R, Roegner V, Jenkins T, et al. Erythrocyte adenosine deaminase deficiency without immunodeficiency: evidence for an unstable mutant enzyme. J Clin Invest 1979;64:1130-9.

We thank Dr. Lawrence V. Hofmann for the referral and continued primary care of this patient. REFERENCES

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ADA deficiency with late onset o f recurrent infections

14. Jenkins T. Red blood cell adenosine deaminase deficiencyin a "healthy" !Kung individual [Letter]. Lancet 1973;2:736. 15. Perignon JL, Hamet M, Cartier P, et al. Complete adenosine deaminase (ADA) deficiencywithout immunodeficiencyand with primary hyperoxalemia in a 12-year-old boy. J Clin Chem Biochem 1979;17:406. 16. Hershfield MS, Kurtzburg J, Alyar VN, et al. Abnormalities in S-adenosylhomocysteinehydrolysis. ATP catabolism and lymphoid differentiation in adenosine deaminase deficiency. Ann NY Acad Sci 1985;451:78-86. 17. Kredieh NM, Hershfield MS. Immunodeficiency diseases caused by adenosine deaminase deficiencyand purine nucleoside phosphorylase deficiency. In: Scriver CR, Beaudet AL, Sly WS, Valle D, eds. The metabolic basis of inherited disease, 6th ed. New York: McGraw-Hill (in press). 18. Polmar SH, Waldmann TA, Terry WD. IgE in immunodeficiency. Am J Pathol 1972;69:499-512. 19. Kikkawa Y, Kamimura K, Hamajima T, et al. Thymic almyphoplasia with hyper IgE globulinemia. Pediatrics 1973; 51:690-6.

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20. Buckley RH, Fiseus SA. Serum IgD and IgE concentrations in immunodefieiencydiseases. J Clin Invest 1975;55:157-65. 21. Donabedian H, Gallin JI. The hyperimmunoglobulinE recurrent infection (Job's) syndrome: a review of the NIH experience and the literature. Medicine 1983;62:195-208. 22. Buckley DH, Schiff SE, Sampson HA, et al. Developmentof immunity in human severe primary T-cell deficiencyfollowing haploidentical bone marrow stem cell transplantation. J lmmunol 1986;136:2398-407. 23. Polmar SH, Stern RC, Schwartz AL, et al. Enzyme replacement therapy for adenosine deaminase deficiencyand severe combined immunodeficiency.N Engl J Med 1976;295:133743. 24. Davis S, Abuchowski A, Park YK, Davis FF. Alteration of the circulating life and antigenic properties of bovine adenosine deaminase in mice by attachment of polyethyleneglycol. Clin Exp Immunol 1981;46:64%52.

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