Cytidine Deaminase: a New Genetic Polymorphism Demonstrated in Human Granulocytes
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Am J Hum Genet 27:492-497, 1975 Cytidine Deaminase: A New Genetic Polymorphism Demonstrated in Human Granulocytes YAO-SHENG TENG,1 JEANNE E. ANDERSON, AND ELOISE R. GIBLETT Cytidine deaminase (cytidine aminohydrolase; E.C.3.5.4.5.) catalyzes the deamina- tion of cytidine to uridine plus ammonia. It also deaminates deoxycytidine as well as some of the nucleoside analogs, including the antimetabolites cytosine arabinoside and 5-azacytidine, but it does not react with ribonucleotides or deoxyribonucleotides [1]. Cytidine deaminase (CDA) * has been detected in many tissues [1-7]. In human blood, white cells (particularly granulocytes) have high levels of CDA activity, while there is little or no activity in red cells. Immature granulocytes from patients with either myelocytic leukemia or infection have much lower CDA activity levels than mature granulocytes [1]. In mice, but not in man, red cell activity of this enzyme rises steadily and persistently after stimulation of ery- thropoiesis [8]. A molecular weight of 51,000 has been reported for CDA partially purified from human granulocytes [1] as compared with 37,000 for sheep liver CDA [9] and 74,000 for mouse spleen CDA [5]. In this paper, we describe a method for demonstrating the zones of CDA cata- lytic activity after starch gel electrophoresis and report genetic polymorphism of this enzyme in human leukocytes. MATERIALS AND METHODS Preparation of Leukocytes An 8-ml sample of venous blood was collected from human subjects into 2 ml of acid- citrate-dextrose (ACD) formula B or citrate-phosphate-dextrose (CPD) solution. Within 24 hr, concentrates of white cells were prepared from the unrefrigerated blood either by dextran sedimentation or by removal of the top layer of cells after recentrifuging the buffy coat in a hematocrit tube. For dextran sedimentation, the blood was first centrifuged at 400 g at room temperature for 6 min and the plasma removed. The residual cells were then mixed with an equal amount of 3% dextran solution in 0.9% NaCl and permitted to stand for 30 min at room temperature. The top layer was transferred into a 12 ml siliconized centrifuge tube and spun for 6 min at 400 g. The supernatant was discarded, and the reddish cell button was mixed vigorously with 4.5 ml distilled water in a Vortex mixer for 15 sec. After rapid addition of 1.5 ml 0.6 M KCl, the tube was again mixed and then centrifuged at 400 g for 6 min, collecting the white cell pellet at the bottom of Received February 10, 1975; revised March 27, 1975. This work was supported in part by U.S. Public Health Service grant AM 09745 from the National Institutes of Health. 1All authors: Puget Sound Blood Center, Terry at Madison, Seattle Washington 98104. *Abbreviations used in this paper: CDA, cytidine deaminase; CMP, cytidine monophosphate; dCMP, deoxycytidine monophosphate; MTT, 3-(4,5-dimethyl thiazolyl-2)-2,5-diphenyl tetra- zolium bromide; dTTP, deoxythymidine triphosphate. i 1975 by the American Society of Human Genetics. All rights reserved. 492 NEW POLYMORPHISM: CYTIDINE DEAMINASE 493 the tube. For electrophoresis, an extract of the white cells was prepared by adding an equal volume of distilled water, placing the tube at -200 C, rapidly freezing and thawing three times, and centrifuging at 400 g for 10 min. Electrophoresis and Staining The electrophoresis and staining procedures were based on those described by Farron ['10] for detecting the production of ammonia at the sites of activity of another enzyme, arginase. This system requires that starch gel be only minimally buffered, since staining is dependent on the ability of thiol groups in dithiothreitol to reduce a tetrazolium dye nonenzymatically when there is a rise in pH (W. N. Fishbein, cited in [10]). After thor- oughly washing 30 g hydrolyzed starch (Connaught) and 26 g Electrostarch (Hiller) in 500 ml of 0.005 M histidine-HCl buffer, pH 6.7, the starch was left to settle. Most of the buffer was decanted and replaced by an equivalent amount of fresh buffer, pH 6.7. The cathodal electrode buffer was 0.02 M citric acid adjusted to pH 6.2 with 1 N NaOH; for the anode, the buffer pH was adjusted to 6.9. Vertical electrophoresis was performed at 50 mA/20 cm for 19 hr at 40 C. The staining mixture consisted of 15 mg cytidine, 1 ml of 0.3% dithiothreitol, 0.3 ml of 5% aqueous MTT solution, 10 ml distilled water, and 10 ml 2% Jonagar solution (Wilson Diagnostics, Glenwood, Ill.). The Jonagar was dissolved by heating in a flask and then placed in a water bath at 550 C. The cytidine was dissolved in distilled water, placed in the water bath for 1 min, and then mixed with the agar solution. After addition of the dithiothreitol and MTT, the flask contents were mixed rapidly and quickly poured over the sliced starch gel. The gel was incubated at 370 C for 1-2 hr, during which appearance of the dark blue color of reduced MTT signaled the sites of CDA activity. Photographs were taken with a Polaroid camera using a yellow filter. To test substrate specificity, the cytidine in the staining mixture was substituted by equivalent amounts of deoxycytidine, 5-fluorocytidine, cytosine arabinoside, 5-azacytidine, cytosine, CMP, and dCMP. RESULTS Substrate Specificity Zones of enzyme activity were seen when cytidine was present in the staining mixture, and weaker zones of the same mobility appeared when either deoxycytidine, 5-fluorocytidine, cytosine arabinoside, or 5-azacytidine was substituted for cytidine. However, no visible indication of enzyme activity appeared when the substituted substrate was cytosine, CMP, or dCMP. CDA Isozyme Patterns Three different electrophoretic patterns were observed; they are designated CDA 1, CDA 2, and CDA 2-1 in figure 1. Both CDA 1 and CDA 2 consist of single bands, migrating toward the anode about 2 cm and 5 cm, respectively. The CDA 2-1 pattern usually contains four bands, the faintest of which has the mobility of CDA 2, while the other three bands have mobilities intermediate between those of CDA 1 and CDA 2. With heavily concentrated granulocyte extracts, a fifth, fainter band corresponding to that of CDA 1 appears in the CDA 2-1 pattern. Inheritance and Frequency of CDA Phenotypes Since freshly drawn blood is required for preparation of white cells, the members of only four families have so far been tested for their CDA phenotypes. However, 494 TENG ET AL. + 1 2-1 1 2 2 1 FIG. 1.-Photograph of starch gel showing three electrophoretic patterns of cytidine deaminase: CDA 1, CDA 2, and CDA 2-1. as shown in figure 2, the phenotypes of these families are all consistent with autosomal inheritance of two allelic genes, CDA1 and CDA2, with the CDA 1 and CDA 2 phenotypes reflecting homozygosity for their respective alleles and CDA 2-1 representing the heterozygous state. Of 189 Caucasian blood donors living in the Seattle area, 82 had CDA 1, 25 had CDA 2, and 82 had CDA 2-1. From these figures, the CDA' and CDA2 alleles were calculated to have frequencies of-.65 and .35, respectively, and their pheno- type frequencies were found to be in Hardy-Weinberg equilibrium. FIG. 2.-Pedigrees showing four different CDA mating types and their progeny. Open symbol. CDA 1; closed symbol, CDA 2; half-closed, CDA 2-1. NEW POLYMORPHISM: CYTIDINE DEAMINASE 495 DISCUSSION The enzyme detected by the staining technic described in this paper reacted with cytidine, deoxycytidine, 5-fluorocytidine, cytosine arabinoside, and 5-azacy- tidine but not with cytosine, CMP, or dCMP. This specificity corresponds with that of CDA partially purified from normal and leukemic granulocytes [1]. Further support for its identity was obtained when the same electrophoretic patterns were observed by an alternative staining method based on an assay system described by Chabner et al. [11]. Here the ammonia produced at the sites of CDA activity triggers the glutamate dehydrogenase reaction, and the areas of NADH conversion to NAD are seen as defluorescent zones under ultraviolet light. The family and population data clearly indicate the existence of two alleles, CDA1 and CDA2, at an autosomal locus. The high frequency of both CDA alleles makes this genetic system especially useful for performing various kinds of marker studies [12]. There are two major drawbacks to its general use: the necessity of preparing the white cell extracts fairly soon after blood collection and the relative insensitivity of the staining method. Thus far, we have only been able to demon- strate the CDA phenotypes in granulocytes. Preliminary efforts to stain the enzyme in extracts of lymphocytes and fibroblasts have not been successful, suggesting that it may be difficult to assign the CDA locus to its autosome by the use of cell hybridization techniques. The multibanded pattern of the heterozygote is most plausibly explained by assuming that the CDA molecule is a tetramer. Thus, while each of the two homozygote patterns consists of a single homotetramer, the heterozygote pattern consists of five components predominantly represented by the three heterotetramers, with little representation by the CDA 2 homotetramer and even less by the CDA 1 homotetramer. The best studied enzyme with a five-banded pattern is lactate dehydrogenase, which consists of a tetrameric series of five isozymes formed by the products of two separate gene loci. When equal amounts of these two products are mixed in vitro, they combine at random to form tetrads with a binomial distribu- tion (1:4:6:4: 1) of enzyme activity [ 13]. However, in tissue extracts this distribu- tion is not observed, since it would require that both gene products be made in equal amounts, be equally stable, and have equal affinities [14].