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Adenosine deaminase and nucleoside phosphorylase activity in patients with immunodeficiency syndromes
CLINICAL
IMMUNOLOGY
AND
13, 156-160
IMMUNOPATHOLOGY
(1979)
Adenosine Deaminase and Nucleoside Phosphorylase Activity in Patients with lmmunodeficiency Syndromes SHI-HAN
HANS D. OCHS,’ C. RONALD ELOISE R. GIBLETT
CHEN,*
*Department of Pediatrics Washington School
SCOTT,
and the Center for Inherited Diseases, of Medicine, and the Pager Sound Blood Seattle. Washington 98195
AND
University Center.
oj
Received November 9, 1978 Red cell adenosine deaminase (ADA) and nucleoside phosphorylase (NP) activities were measured in 62 patients with various immunodeticiency syndromes and in 67 adult and 37 infant controls. NP activity was found to be within the normal range (1206 + 144 UigHb in adults, 1266 ? 270 UigHb 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 UigHb (with 95% confidence interval of 22.5-58.1 UigHb) for normal adult controls (n = 67) and 35 UigHb (95% confidence interval 21.6-60.8 UigHb) 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 live 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.
INTRODUCTION
Adenosine deaminase (ADA EC 3.5.4.4.) deficiency as a cause of combined immune deficiency (CID) was first described by Giblett and associates in 1972 (1). Shortly thereafter, a second enzyme involved in the purine salvage pathway, purine nucleoside phosphorylase (NP EC 2.4.2.1.) was also demonstrated to be deficient in some children who had severe T-cell deficiency (2, 3). Because of this association between the purine salvage pathway enzymes and immune function, we have determined the activities of ADA and NP in red blood cells from 62 patients with a variety of immune deficiency syndromes. MATERIALS
AND METHODS
A total of 62 patients with various immunodeficiency syndromes were studied. They were classified according to the recommendations of the WHO Committee (4). Included were 17 patients with CID who had severe to moderate impairment of B- and T-cell function, 7 with X-linked infantile agammaglobulinemia (X-LA), 6 with ataxia telangiectasia (AT), 4 with Wiskott-Aldrich syndrome (WAS), 17 I Dr. Ochs is an investigator of the Howard Hughes Medical Institute. 156
0090-1229n9/060156-05$4ll.O0/0 Copyright All rights
0 1979 by Academic Press. Inc. of reproduction in any form reserved.
ADENOSINE
DEAMINASE
157
with variable immunodeficiency syndromes (VID), and 11 with other immunodeficiency syndromes, including 4 patients with NP deficiency (2, 5, 6), 1 with immunodeficiency and thymoma, 2 with thymic hypoplasia, and 2 with selective IgA deficiency. Blood specimens from these patients, 67 normal adult controls and 37 infant controls (aged 1-12 months), were collected into acid citrate dextrose solution. Fractions of lymphocytes, granulocytes, and red cells were prepared using Ficoll -Hypaque gradient centrifugation (7). Red cells were fractionated into young and old populations by a method described by Murphy (8). Hemolysates and extracts of white cells and cultured skin fibroblasts were prepared as described previously (9). Enzyme activities of ADA and NP were measured as described (10, 11) and one unit (U) of enzyme activity was defined as pmol/hr of the substrate converted at 25°C. Specific activities were expressed as U/gHb for red cells, U/lo9 cells for white cells, and U/g protein for cultured skin fibroblasts. RESULTS Red cell NP activities were 1206 t 144 U/gHb for the normal adult controls and 1266 2 270 U/gHb for the infant controls (1 - 12 months). The NP activities of all but four patients were within the normal range. All of these four patients had defective T-cell immunity and normal B-cell function. Two had no detectable NP activity, while the other two (brothers) had approximately 0.2% of the normal mean. The clinical and biochemical findings of these patients have been reported elsewhere (2, 5, 6). Red cell ADA activities were 36 U/gHb (geometric mean, 22.5-58.1 UigHb 95% confidence interval) for the normal adult controls and 35 U/gHb (geometric mean, 21.6-60.8 U/gHb 95% confidence interval) for the infant controls. The red cell ADA activities of the patients were distributed over a wide range and varied with the immunological classification (Fig. 1). In those patients with X-LA, AT, and WAS, the red cell ADA activity was within the normal range. Six of the 17 patients with CID had undetectable or very low ADA activities in their red cells, and 7 of the remaining patients in this group had enzyme levels ranging from 75-135 U/gHb, mean 99.7 U/gHb, significantly greater than 2 SD above the normal mean. Of the 17 patients classified as having VID, 2 identical twin sisters had markedly elevated red cell ADA activities (185 and 212 U/gHb), amounting to five to six times the mean of the control red cells. Their red cell enzyme activities and those of other family members are indicated in the pedigree shown in Fig. 2. In contrast to the twins, other members of this family had normal red cell ADA activity. ADA activity of fractionated red cells was studied in one of the twins. The top layer consisted of young cells and showed more enzyme activity (250 U/gHb) than either the mixed unseparated cells (193 U/gHb), or the older red cells of the bottom layer (190 U/gHb). The ADA activity of each fraction was five to six times greater than in the corresponding normal red cell fractions: 41 U/gHb for the top layer, 36 and 35 U/gHb for the mixed and the bottom layers of normal control. In contrast to the greatly increased ADA activity in the red cells, the enzyme activity in granulocytes, nonstimulated peripheral blood lymphocytes, and cultured skin tibroblasts was not significantly different from normal controls (Table 1). The ADA activity of granulocytes and lymphocytes from the mother of the twins was normal.
158
CHEN ET
AL.
220-
200-
180-
160-
140-
.
4 P
I20-
‘0 3 5
I . .
IOO-
. .
80-
.
.
60-
.
:
l
:
l
,
l .:
.
1 3 !
4020.
0’
:
:
; :
I
contrch
X-LA
’ AT
’ WAS ’
CID ’ VI0
bfharlm’
CN=67)
FIG. 1. Distribution of red cell ADA activities in 62 patients with various immunodeticiency dromes. For controls, values indicate geometric mean and 95% confidence interval.
syn-
DISCUSSION Of the 17 patients with CID, deficiency of ADA was found in 6, but in 7 others the enzyme was increased at least 2 SD above the normal mean. In addition, a pair of twins with variable immunodeficiency had red cell ADA levels five to six times greater than normal. These twin sisters were tested on five occasions during a 4-year period and the values were always elevated, implying that genetic factor(s) may be operating in the expression of the ADA allele. Electrophoretic and kinetic
1
7
349
32 9
111 11
& 36 5
39 5
212 3 185 6
FIG. 2. Pedigree of the family with elevated red cell ADA activity. The enzyme activity (U/gHb) is indicated for each family member. Red cell ADA activity in 67 normal controls is 36 f 7 UigHb.
ADENOSINE
DEAMINASE
TABLE
1
ADA SPECIFIC ACUVIIXES ShrN FIBROBLASIX
IN RED CELLS, LYMPHOCYTES, FROM Two PATIENTS WITH ELEVATED .~ND NORMAL COKTROLS”
Red cells WgHb) Adult controls
36
[671"
(range 21-58.2)
Lymphocytes (U/lOy cells) 42.8
159
[31
(range 22.5-60.1)
GRANULOCYTES.
ADA ACTIVIV,
Granulocytes (U/lOy cells) 28.2
[31
(range 12-40)
Patient 1
212 (5)’ (range 172 - 246)
85.2
(2)
25.2
(2)
Patient 2
185 (3 (range 161-221)
46.8
(2)
34.8
(2)
22.2
(1)
Mother
32.9
(2)
ND
AND CULWREII THEIR MOTHER.
Cultured skin fibroblasts Wig protein) 15 WI (range 6.4-34.0)
ND” 11
(2) ND
(’ The values are the means of the determinations b Number of individuals tested. c Number of independent determinations. ” Not determined.
properties of the ADA prepared from red cells of one of these twins were not different from the normal enzyme, suggesting that the structural gene for ADA is not altered. Thus, the increase in ADA activity appears to represent an overproduction of the normal enzyme, apparently confined to the red cell. Autosomal dominant inheritance for a much greater increase in red cell ADA activity (60- to 70-fold of normal) has been demonstrated by Valentine et al. in a patient with mild hemolytic anemia and normal ADA levels in other tissues (12). Autosomal dominant inheritance of increased ADA activity in red cells and peripheral blood lymphocytes was also suggested in another family in which two siblings died of severe CID and leukopenia (13). The mode of inheritance in the family we studied (Fig. 2) is not clear because of the small pedigree and incomplete sampling. However, if the increased activity of ADA found in our twin patients was determined by a single autosomal dominant allele, we would have had a reasonable chance (16: 1, in view of four additional family members) of finding another family member with increased activity. This and other studies have demonstrated that abnormalities of red cell ADA activity occur in various unrelated syndromes- extremely elevated in one form of hemolytic anemia (12), significantly elevated in some patients with CID and VID, and absent in some patients with severe CID (1, 14). Such variation in ADA activity in different clinical disorders may implicate a defect of purine metabolism within the nucleated red cell precursors or an abnormality in a biochemical mechanism regulating ADA production. Sicilian0 er al. (15) have recently demonstrated the activation of the ADA gene in a human chorionic cell line by forming hybrid clones with a mouse cell line. They interpreted their findings as evidence for genetic regulation of ADA expression. Although we anticipated the absence of ADA in some patients with CID, the finding of significant elevations of ADA in the red cells of some patients with CID
160
CHEN
ET
AL.
and VID was unexpected. It is possible that these patients have an unrecognized enzyme deficiency which is responsible for their abnormal immune status and the increased ADA synthesis in red blood cells. ACKNOWLEDGMENTS This work was supported in part by USPHS Grants AI 12617, AI 07073, GM 15253, and HL 17265, and a grant from the National Foundation-March of Dimes (6-7). A portion of this work was conducted through the Clinical Research Center Facility of the University of Washington with the support of National Institutes of Health Grant RR-37.
REFERENCES 1. Giblett, E. R., Anderson, .I. E., Cohen, F., Pollara, B., and Meuwissen, H. J., Lancer. 2, 1067. 1972. 2. Giblett, E. R., Ammann, A. J.. Wara, D. W.. Sandman, R., and Diamond, L. K.. Lancer 1, 1010, 1975. 3. Stoop, J. W., Zegers, B. J. M., Hendrickx, G. F. M., Siegenbeek van Heukelom, L. H., Staal, G. E. J., de Bree, P. K.. Wadman, S. K., and Ballieux, R. E., N. Engl. J. Med. 296, 651, 1977. 4. Fudenberg, H. H., Good, R. A., Hitzig, W., Kunkel, H. G., Roitt, I. M., Rosen, F. S., Rowe, D. S., Seligmann, M., and Soothill, J. R., N. Engl. J. Med. 283, 656, 1970. 5. Biggar, W. D., Giblett. E. R., Ozere, R. L.. and Grover. B. D.. J. Pediat. 92, 354, 1978. 6. Rich, K., Arnold. W., FOX, I.. and Palella, T., Pediar. Res. 12, 485, 1978. 7. Boyum, A., Tissue Aniigens 4, 269. 1974. 8. Murphy, J. R., J. Lab. Clin. Med. 82, 334, 1973. 9. Chen, S. H., Scott, C. R., and Swedberg, K. R., Amer. J. Human Genet. 27, 46. 1975. 10. Hopkinson, C. A., Cook, P. J. L., and Harris, H., Ann. Human Genet. 32, 361, 1%9. 11. Kalckar, H. M., J. Biol. Chem. 167, 461, 1947. 12. Valentine, W. N., Paglia, D. E.. Tartaglia, A. P., and Glisanzo, F., Science 195, 783, 1977. 13. Ownby, D. R., Pizzo, S., Blackman, L., Gall, S. A., and Buckley, R. H.,J. Pediat. 89,382,1976. 14. Meuwissen, H. J., Pickering, R. J., Moore, E. C., Pollara, B., In “Combined Immunodeficiency Disease and Adenosine Deaminase Deficiency-A Molecular Defect” (H. J. Meuwissen, R. J. Pickering, E. B. Hook, B. Pollara, I. H. Porter, and S. Kelly, Eds.), p. 73, Academic Press, New York, 1975. 15. Siciliano, M. J., Bordelan. M. R., and Kohler. P. O., Proc. Nai. Acad. Sci. U.S.A. 75,936, 1978.
IMMUNOLOGY
AND
13, 156-160
IMMUNOPATHOLOGY
(1979)
Adenosine Deaminase and Nucleoside Phosphorylase Activity in Patients with lmmunodeficiency Syndromes SHI-HAN
HANS D. OCHS,’ C. RONALD ELOISE R. GIBLETT
CHEN,*
*Department of Pediatrics Washington School
SCOTT,
and the Center for Inherited Diseases, of Medicine, and the Pager Sound Blood Seattle. Washington 98195
AND
University Center.
oj
Received November 9, 1978 Red cell adenosine deaminase (ADA) and nucleoside phosphorylase (NP) activities were measured in 62 patients with various immunodeticiency syndromes and in 67 adult and 37 infant controls. NP activity was found to be within the normal range (1206 + 144 UigHb in adults, 1266 ? 270 UigHb 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 UigHb (with 95% confidence interval of 22.5-58.1 UigHb) for normal adult controls (n = 67) and 35 UigHb (95% confidence interval 21.6-60.8 UigHb) 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 live 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.
INTRODUCTION
Adenosine deaminase (ADA EC 3.5.4.4.) deficiency as a cause of combined immune deficiency (CID) was first described by Giblett and associates in 1972 (1). Shortly thereafter, a second enzyme involved in the purine salvage pathway, purine nucleoside phosphorylase (NP EC 2.4.2.1.) was also demonstrated to be deficient in some children who had severe T-cell deficiency (2, 3). Because of this association between the purine salvage pathway enzymes and immune function, we have determined the activities of ADA and NP in red blood cells from 62 patients with a variety of immune deficiency syndromes. MATERIALS
AND METHODS
A total of 62 patients with various immunodeficiency syndromes were studied. They were classified according to the recommendations of the WHO Committee (4). Included were 17 patients with CID who had severe to moderate impairment of B- and T-cell function, 7 with X-linked infantile agammaglobulinemia (X-LA), 6 with ataxia telangiectasia (AT), 4 with Wiskott-Aldrich syndrome (WAS), 17 I Dr. Ochs is an investigator of the Howard Hughes Medical Institute. 156
0090-1229n9/060156-05$4ll.O0/0 Copyright All rights
0 1979 by Academic Press. Inc. of reproduction in any form reserved.
ADENOSINE
DEAMINASE
157
with variable immunodeficiency syndromes (VID), and 11 with other immunodeficiency syndromes, including 4 patients with NP deficiency (2, 5, 6), 1 with immunodeficiency and thymoma, 2 with thymic hypoplasia, and 2 with selective IgA deficiency. Blood specimens from these patients, 67 normal adult controls and 37 infant controls (aged 1-12 months), were collected into acid citrate dextrose solution. Fractions of lymphocytes, granulocytes, and red cells were prepared using Ficoll -Hypaque gradient centrifugation (7). Red cells were fractionated into young and old populations by a method described by Murphy (8). Hemolysates and extracts of white cells and cultured skin fibroblasts were prepared as described previously (9). Enzyme activities of ADA and NP were measured as described (10, 11) and one unit (U) of enzyme activity was defined as pmol/hr of the substrate converted at 25°C. Specific activities were expressed as U/gHb for red cells, U/lo9 cells for white cells, and U/g protein for cultured skin fibroblasts. RESULTS Red cell NP activities were 1206 t 144 U/gHb for the normal adult controls and 1266 2 270 U/gHb for the infant controls (1 - 12 months). The NP activities of all but four patients were within the normal range. All of these four patients had defective T-cell immunity and normal B-cell function. Two had no detectable NP activity, while the other two (brothers) had approximately 0.2% of the normal mean. The clinical and biochemical findings of these patients have been reported elsewhere (2, 5, 6). Red cell ADA activities were 36 U/gHb (geometric mean, 22.5-58.1 UigHb 95% confidence interval) for the normal adult controls and 35 U/gHb (geometric mean, 21.6-60.8 U/gHb 95% confidence interval) for the infant controls. The red cell ADA activities of the patients were distributed over a wide range and varied with the immunological classification (Fig. 1). In those patients with X-LA, AT, and WAS, the red cell ADA activity was within the normal range. Six of the 17 patients with CID had undetectable or very low ADA activities in their red cells, and 7 of the remaining patients in this group had enzyme levels ranging from 75-135 U/gHb, mean 99.7 U/gHb, significantly greater than 2 SD above the normal mean. Of the 17 patients classified as having VID, 2 identical twin sisters had markedly elevated red cell ADA activities (185 and 212 U/gHb), amounting to five to six times the mean of the control red cells. Their red cell enzyme activities and those of other family members are indicated in the pedigree shown in Fig. 2. In contrast to the twins, other members of this family had normal red cell ADA activity. ADA activity of fractionated red cells was studied in one of the twins. The top layer consisted of young cells and showed more enzyme activity (250 U/gHb) than either the mixed unseparated cells (193 U/gHb), or the older red cells of the bottom layer (190 U/gHb). The ADA activity of each fraction was five to six times greater than in the corresponding normal red cell fractions: 41 U/gHb for the top layer, 36 and 35 U/gHb for the mixed and the bottom layers of normal control. In contrast to the greatly increased ADA activity in the red cells, the enzyme activity in granulocytes, nonstimulated peripheral blood lymphocytes, and cultured skin tibroblasts was not significantly different from normal controls (Table 1). The ADA activity of granulocytes and lymphocytes from the mother of the twins was normal.
158
CHEN ET
AL.
220-
200-
180-
160-
140-
.
4 P
I20-
‘0 3 5
I . .
IOO-
. .
80-
.
.
60-
.
:
l
:
l
,
l .:
.
1 3 !
4020.
0’
:
:
; :
I
contrch
X-LA
’ AT
’ WAS ’
CID ’ VI0
bfharlm’
CN=67)
FIG. 1. Distribution of red cell ADA activities in 62 patients with various immunodeticiency dromes. For controls, values indicate geometric mean and 95% confidence interval.
syn-
DISCUSSION Of the 17 patients with CID, deficiency of ADA was found in 6, but in 7 others the enzyme was increased at least 2 SD above the normal mean. In addition, a pair of twins with variable immunodeficiency had red cell ADA levels five to six times greater than normal. These twin sisters were tested on five occasions during a 4-year period and the values were always elevated, implying that genetic factor(s) may be operating in the expression of the ADA allele. Electrophoretic and kinetic
1
7
349
32 9
111 11
& 36 5
39 5
212 3 185 6
FIG. 2. Pedigree of the family with elevated red cell ADA activity. The enzyme activity (U/gHb) is indicated for each family member. Red cell ADA activity in 67 normal controls is 36 f 7 UigHb.
ADENOSINE
DEAMINASE
TABLE
1
ADA SPECIFIC ACUVIIXES ShrN FIBROBLASIX
IN RED CELLS, LYMPHOCYTES, FROM Two PATIENTS WITH ELEVATED .~ND NORMAL COKTROLS”
Red cells WgHb) Adult controls
36
[671"
(range 21-58.2)
Lymphocytes (U/lOy cells) 42.8
159
[31
(range 22.5-60.1)
GRANULOCYTES.
ADA ACTIVIV,
Granulocytes (U/lOy cells) 28.2
[31
(range 12-40)
Patient 1
212 (5)’ (range 172 - 246)
85.2
(2)
25.2
(2)
Patient 2
185 (3 (range 161-221)
46.8
(2)
34.8
(2)
22.2
(1)
Mother
32.9
(2)
ND
AND CULWREII THEIR MOTHER.
Cultured skin fibroblasts Wig protein) 15 WI (range 6.4-34.0)
ND” 11
(2) ND
(’ The values are the means of the determinations b Number of individuals tested. c Number of independent determinations. ” Not determined.
properties of the ADA prepared from red cells of one of these twins were not different from the normal enzyme, suggesting that the structural gene for ADA is not altered. Thus, the increase in ADA activity appears to represent an overproduction of the normal enzyme, apparently confined to the red cell. Autosomal dominant inheritance for a much greater increase in red cell ADA activity (60- to 70-fold of normal) has been demonstrated by Valentine et al. in a patient with mild hemolytic anemia and normal ADA levels in other tissues (12). Autosomal dominant inheritance of increased ADA activity in red cells and peripheral blood lymphocytes was also suggested in another family in which two siblings died of severe CID and leukopenia (13). The mode of inheritance in the family we studied (Fig. 2) is not clear because of the small pedigree and incomplete sampling. However, if the increased activity of ADA found in our twin patients was determined by a single autosomal dominant allele, we would have had a reasonable chance (16: 1, in view of four additional family members) of finding another family member with increased activity. This and other studies have demonstrated that abnormalities of red cell ADA activity occur in various unrelated syndromes- extremely elevated in one form of hemolytic anemia (12), significantly elevated in some patients with CID and VID, and absent in some patients with severe CID (1, 14). Such variation in ADA activity in different clinical disorders may implicate a defect of purine metabolism within the nucleated red cell precursors or an abnormality in a biochemical mechanism regulating ADA production. Sicilian0 er al. (15) have recently demonstrated the activation of the ADA gene in a human chorionic cell line by forming hybrid clones with a mouse cell line. They interpreted their findings as evidence for genetic regulation of ADA expression. Although we anticipated the absence of ADA in some patients with CID, the finding of significant elevations of ADA in the red cells of some patients with CID
160
CHEN
ET
AL.
and VID was unexpected. It is possible that these patients have an unrecognized enzyme deficiency which is responsible for their abnormal immune status and the increased ADA synthesis in red blood cells. ACKNOWLEDGMENTS This work was supported in part by USPHS Grants AI 12617, AI 07073, GM 15253, and HL 17265, and a grant from the National Foundation-March of Dimes (6-7). A portion of this work was conducted through the Clinical Research Center Facility of the University of Washington with the support of National Institutes of Health Grant RR-37.
REFERENCES 1. Giblett, E. R., Anderson, .I. E., Cohen, F., Pollara, B., and Meuwissen, H. J., Lancer. 2, 1067. 1972. 2. Giblett, E. R., Ammann, A. J.. Wara, D. W.. Sandman, R., and Diamond, L. K.. Lancer 1, 1010, 1975. 3. Stoop, J. W., Zegers, B. J. M., Hendrickx, G. F. M., Siegenbeek van Heukelom, L. H., Staal, G. E. J., de Bree, P. K.. Wadman, S. K., and Ballieux, R. E., N. Engl. J. Med. 296, 651, 1977. 4. Fudenberg, H. H., Good, R. A., Hitzig, W., Kunkel, H. G., Roitt, I. M., Rosen, F. S., Rowe, D. S., Seligmann, M., and Soothill, J. R., N. Engl. J. Med. 283, 656, 1970. 5. Biggar, W. D., Giblett. E. R., Ozere, R. L.. and Grover. B. D.. J. Pediat. 92, 354, 1978. 6. Rich, K., Arnold. W., FOX, I.. and Palella, T., Pediar. Res. 12, 485, 1978. 7. Boyum, A., Tissue Aniigens 4, 269. 1974. 8. Murphy, J. R., J. Lab. Clin. Med. 82, 334, 1973. 9. Chen, S. H., Scott, C. R., and Swedberg, K. R., Amer. J. Human Genet. 27, 46. 1975. 10. Hopkinson, C. A., Cook, P. J. L., and Harris, H., Ann. Human Genet. 32, 361, 1%9. 11. Kalckar, H. M., J. Biol. Chem. 167, 461, 1947. 12. Valentine, W. N., Paglia, D. E.. Tartaglia, A. P., and Glisanzo, F., Science 195, 783, 1977. 13. Ownby, D. R., Pizzo, S., Blackman, L., Gall, S. A., and Buckley, R. H.,J. Pediat. 89,382,1976. 14. Meuwissen, H. J., Pickering, R. J., Moore, E. C., Pollara, B., In “Combined Immunodeficiency Disease and Adenosine Deaminase Deficiency-A Molecular Defect” (H. J. Meuwissen, R. J. Pickering, E. B. Hook, B. Pollara, I. H. Porter, and S. Kelly, Eds.), p. 73, Academic Press, New York, 1975. 15. Siciliano, M. J., Bordelan. M. R., and Kohler. P. O., Proc. Nai. Acad. Sci. U.S.A. 75,936, 1978.