Biochemical Genetics, Vol. 30, Nos. 7/8, 1992

Adenine Phosphoribosyl Transferase Polymorphism in Baboons John L. VandeBerg 1,2 and Mary Jo Aivaliotis 1

Received 12 Nov. 1991--Final 13 Mar. 1992

Two allelic isozymes of adenine phosphoribosyl transferase (APRT) were detected by starch gel electrophoresis of baboon hemolysates. Extensive family data verified autosomal codominant inheritance. The gene frequencies of five subspecies of baboons differed significantly. The activity of erythrocyte A P R T is sufficiently high to enable the use of this enzyme as a sensitive marker for assessing chimerism in research involving bone marrow transplantation. KEYWORDS: isozyme;genetic marker; primates; paternitytesting;bone marrowtransplantation INTRODUCTION

Adenine phosphoribosyl transferase (APRT; EC 2.4.2.7) is a purine salvage enzyme which catalyzes the reaction of adenine with 5-phosphoribosyl pyrophosphate (PRPP) to yield adenosine-5'-monophosphate and pyrophosphate. It is a dimeric enzyme which is present in all mammalian cell types. Electrophoretic phenotypes in humans have been reported to consist of a common phenotype, APRT-1, and a rare variant, A P R T - X (Mowbray et al., 1972). A P R T deficiency in humans is inherited as an autosomal recessive characteristic (Kamatani et al., 1987). In the absence of APRT, adenine is oxidized to 2,8-dihydroxyadenine, which crystallizes to form urinary stones. As a consequence of our continuing effort to develop genetic markers for baboons in biomedical research applications, this paper presents exten-

This research was supported in part by NIH Grants HL28972,HG00336, and HV53030. 1Department of Genetics, Southwest Foundation for Biomedical Research, P.O. Box 28147, San Antonio, Texas 78228-0147. 2To whom correspondenceshould be addressed. 331 0006-2928/92/0800-0331506.50/0 © 1992 Plenum Publishing Corporation

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sive population and pedigree data on allelic isozymes of APRT in five subspecies of baboons, Papio hamadryas anubis, P. h. cynocephalus, P. h. hamadryas, P. h. papio, and P. h. ursinus (nomenclature per VandeBerg and Cheng, 1986; Williams-Blangero et al., 1990). The APRT gene has been assigned to human chromosome 16q (Tischfield and Ruddle, 1974), Chinese hamster chromosome 3p (Adair et al., 1983), and mouse chromosome 8 (Franche et al., 1977). No chromosomal assignment has been reported for any nonhuman primate species (Lalley et al., 1989). MATERIALS AND M E T H O D S

Unrelated baboons maintained at the Southwest Foundation for Biomedical Research were selected from each subspecies and designated as panel groups. Baboons were assigned to a panel only if the information on their morphological characteristics, pedigrees, and/or capture sites enabled unambiguous assignment to a subspecies [see Williams-Blangero et al. (1990) for additional information]. Washed red blood ceils were stored in an ethylene glycol-citrate solution at -20°C (VandeBerg and Johnston, 1977) or a glycerol solution at -80°C (Cheng and VandeBerg, 1987). Blood clots stored in Tygon tubing segments at -80°C were also used for typing some animals. For routine typing, erythrocytes or clots were lysed in 10 parts of distilled water (v/v). In order to detect low levels of activity in mixing experiments, erythrocytes or clots were lysed in three parts of distilled water. Electrophoresis was conducted in 12% starch gels at 10 V/cm, with cooling, following the method of Harris and Hopkinson (1976). The procedure for localizing APRT was according to Harris and Hopkinson (1976) except that the amount of [14C] adenine in the reaction mixture was reduced to 2 poCi/10 ml. RESULTS AND DISCUSSION

The phenotypic patterns of baboon hemolysates are shown in Fig. 1. The single-banded patterns of the homozygotes are designated APRT A and APRT B, respectively. Heterozygotes exhibit the typical three-banded pattern expected for a dimeric enzyme. The three phenotypes are compared to APRT-1, which is the most common phenotype in humans. The most common baboon phenotype, APRT A, migrated just slightly anodally of human APRT-1. Baboon APRT phenotypes were stable under all three erythrocyte storage conditions. Some of the samples had been at -20°C in ethylene

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333

4-

m

IHUMANI[

Fig. 1. A P R T phenotypes of baboons in lanes 2-4 represent APRT A, APRT AB, and APRT B, respectively, compared to the common human phenotype in lane 1.

1

BABOON 2

3

]

--ORIGIN

4

glycol for as long as 10 years. However, maximal activity of A P R T and other enzymes is maintained by storage of the red blood cells in the form of clots at 80°C (unpublished data). The distributions of A P R T phenotypes and allele frequencies in the panels of unrelated baboons representing each of the five subspecies are shown in Table I. The most common phenotype for all subspecies was A P R T -

Table I. Distribution of A P R T Phenotypes and Gene Frequencies in Panels of Unrelated Baboons APRT phenotype Subspecies

N

A

AB

B

P. h. anubis P. h. cynocephalus P. h. hamadryas P. h. papio P. h. ursinus

121 85 30 81 22

84 81 30 81 22

30 4 0 0 0

7 0 0 0 0

APRT allele frequency

P. h. anubis P. h. cynocephalus P. h. hamadryas P. h. papio P. h. ursinus

A

B

0.818 0.976 1.000 1.000 1.000

0.182 0.024 0.000 0.000 0.000

VandeBerg and Aivaliotis

334 Table II. Family Data for Baboon A P R T

Progeny type b,c Mating type"

N

A

AB

B

A x A A x AB A x B AB x AB AB x B

584 331 40 42 9

583 (583) 167 (165) 0 15 (10.5) 0

U 163 (165) 40 (40) 22 (21) 2 (4.5)

0 1'~ 0 5 (10.5) 7 (4.5)

aReciprocal crosses have been combined because their results did not differ significantly in any instance. bAllele frequencies did not differ significantly between sexes. CExpected n u m b e r s are given in parentheses. dThese progeny were confirmed as cases of mistaken paternity by the use of additional markers (see text).

A. The APRT*B allele was present in both P. h. anubis and P. h. cynocephalus but at a higher frequency in the former subspecies: 0.182 compared to 0.024. No variation was observed among P. h. hamadryas, P. h. papio, and P. h. ursinus, which were all fixed for the APRT*A allele. Family data from 1004 pedigreed progeny and their parents were consistent with autosomal codominant inheritance (Table II). The gene frequencies, determined by direct gene counting, were significantly different among subspecies as determined by a 2 x 5 contingency chi-square test (X2 = 71.38, P < 0.001). The results of testing all pairwise combinations for differences in gene frequencies are shown in Table III. All pairwise combinations of P. h. anubis and other subspecies showed highly significant differences in gene frequencies. No significant differences were found for P. h. cynocephalus in comparison with P. h. hamadryas, P. h. papio, or P. h. ursinus. The baboon APRT polymorphism has proven to be useful for the purpose of pedigree verification (VandeBerg, 1992). In a mixed colony of P. Table III. Chi-Square Values (Above Diagonal) and Associated Probabilities (Below Diagonal) for Differences in Gene Frequencies Between Baboon Subspecies Pairs

P.h. anubis P. h. anubis P. h. cynocephalus P. h. hamadryas P. h. papio P. h. ursinus

-< 0.001 < 0.001 < 0.001 0.002

P.h. cynocephalus 24.31 -0.231 0,050 0.304

P.h. hamadryas

P. h. papio

P.h. ursinus

12.77 1.44 -1 1

33.05 3.58 0 -1

9.46 1.06 0 0 --

APRT Polymorphism in Baboons

335

h. anubis and P. h. cynocephalus, the A P R T alleles had frequencies of 0.84

and 0.16. The probability of detecting an existing pedigree error by the use of this marker alone was calculated to be 0.12. Of 484 triads (sire, dam, progeny) that were members of this colony and typed for APRT, two errors were detected in the assignment of paternity (unpublished data). These pedigree errors were confirmed by the use of other biochemical genetic markers, and the relevant links were broken in the computerized pedigree data base. Six two-allele markers with the same gene frequencies as APRT would be capable of detecting more than 50% of all pedigree errors that existed in this colony [(0.88) 6 = 0.46 would be the proportion of errors not detected]. Another use of the baboon APRT polymorphism has been for quantitative trait linkage analysis. This strategy has been used to determine the chromosomal locations of genes that had been inferred by segregation analysis to influence quantitative traits associated with lipoprotein metabolism. One such analysis provided evidence of linkage between the A P R T locus and a major gene affecting serum levels of apolipoprotein A-I (apo AI) in baboons (Kammerer et al., 1987a, b). A l t h o u g h A P R T itself is not likely to influence lipid metabolism, A P R T and the gene encoding lecithin-cholesterol acyltransferase (LCAT), which is directly involved in lipoprotein metabolism, both map to human chromosome 16q. Consequently, the results of the quantitative trait linkage analysis have prompted an investigation of the baboon L C A T gene in relation to high-density lipoprotein (HDL) phenotypes that contain apo AI. Genetic variation in the L C A T DNA sequence and in LCAT activity levels has been shown to be associated with alterations in H D L phenotypes (Rainwater et al., 1992). The APRT polymorphism also is potentially useful as a sensitive marker to follow engraftment of bone marrow transplants in baboons. In previous in utero bone marrow transplantation experiments (Roodman et aL, 1988; Muirhead et aL, 1990), glucose phosphate isomerase (GPI), which exhibits fixed allelic differences between subspecies, was used as a marker for this purpose. However, the lack of intrasubspecies allelic variation required donors and recipients to be different subspecies. Therefore, the donor and recipient cells were genetically quite different from one another, potentially complicating the experimental outcome (e.g., via immune responses of donor and recipient cells to each other). In order to determine the power of the APRT polymorphism to discriminate donor and recipient cells, we mixed hemolysates from alternative homozygotes at various proportions and subjected the mixtures to electrophoresis. As shown in Fig. 2, a mixture of lysates with phenotypes APRT A and APRT B, at a ratio as high as 99:1, enabled the detection of the minority type. This level of sensitivity is comparable to that for GPI

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VandeBerg and Aivaliotis

ar

A

B m

--ORIGIN 1-1

9:1 19:1 39:1 99:1 199:1

Fig. 2. APRT patterns in mixtures of hemolysates from contrasting homozygotes.

(Roodman et al., 1988). The allele frequency of A P R T * B in P. h. anubis is sufficiently high (0.182). to enable the selection of donor and recipient animals from within this subspecies. We conclude that APRT can be confidently used as a genetic marker in baboons. The phenotypes are easily and unambiguously distinguished from one another, enabling the high level of accuracy required in typing for linkage analysis and pedigree monitoring. In addition, the highly sensitive detection method enables the use of this marker to track the fate of minority cell populations in transplantation experiments. ACKNOWLEDGMENTS

We thank Sarah Williams-Blangero for assisting with data analyses and statistical tests, Bennett Dyke for developing the computer programs for data management, Jim Bridges for entering the data, and Paul Samollow for critically reviewing the manuscript. REFERENCES Adair, G. M., Stallings, R. L., Friend, K., and Siciliano, M. J. (1983). Gene mapping and linkage analysis in the Chinese hamster: Assignment of the genes for the APRT, LDHA, IDH2 and GAA to chromosome 3. Somat. Cell Genet. 9:477. Cheng, M.-L., and VandeBerg, J. L. (1987). Cryopreservation of erythrocytes in small aliquots for isozyme electrophoresis. Biochem. Genet. 25:535. Franche, U., Lalley, P. A., Moss, W., Ivy, J., and Minna, J. D. (1977). Gene mapping in Mus musculus by interspecific cell hybridization: Assignment of the genes for tripeptidase-1 to chromosome 10, dipeptidase-2 to chromosome 18, acid phosphatase-1 to chromosome 12 and adenylate kinase-1 to chromosome 2. Cytogenet. Cell Genet. 19:57. Harris, H., and Hopkinson, D. A. (1976). Handbook of Enzyme Electrophoresis in Human Genetics, North-Holland, Amsterdam.

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Kamatani, N., Terai, C., Kuroshima, S., Nishioka, K., and Mikanagi, K. (1987). Genetic and clinical studies on 19 families with adenine phosphoribosyltransferase deficiencies. Hum. Genet. 75:163. Kammerer, C. M., MacCluer, J. W., VandeBerg, J. L., and Mott, G. E. (1987a). Possible linkage between APRT and a major gene(s) for serum concentrations of lipoproteins and apolipoproteins in baboons. Am. J. Hum. Genet. 41 (Suppl.):A257. Kammerer, C. M., VandeBerg, J. L., Mott, G. E., and Blangero, J. C. (1987b). Preliminary evidence for linkage between adenine phosphoribosyl transferase and serum apolipoprotein A-I in baboons: Results of the sibpair test. Proc. 2nd Int. Congr. Quant. Genet., Abstr. No. 51. Lalley, P. A., Davisson, M. T., Graves, J. A. M., O'Brien, S. J., Womack, J. E., Roderick, T. H., Creau-Goldberg, N., Hillyard, A. L., Doolittle, D. P., and Rogers, J. A. (1989). Report of the committee on comparative mapping. Cytogenet. Cell Genet. 51:503. Mowbray, S., Watson, B., and Harris, H. (1972). A search for electrophoretic variants of human adenine phosphoribosyl transferase. Ann. Hum. Genet. London 36:153. Muirhead, D. Y., Kuehl, T. J., VandeBerg, J. L., Menchaca, E. M., Downs, M. P., and Roodman, G. D. (1990). Mixed lymphocyte culture reactivity of fetal baboons: Application for in utero bone marrow transplantation. Bone Marrow Transplant. 6:263. Rainwater, D. L., Blangero, J., Hixson, J. E., Birnbaum, S., Mott, G. E., and VandeBerg, J. L. (1992). A DNA polymorphism for lecithin:cholesterol acyltransferase (LCAT) is associated with altered LCAT activity and HDL size distributions in baboons. Arterioscler. Thromb. 12:682. Roodman, G. D., VandeBerg, J. L., and Kuehl, T. J. (1988). In utero bone marrow transplantation of fetal baboons with mismatched adult marrow: Initial observations. Bone Marrow Transplant. 3:141. Tischfield, J. A., and Ruddle, F. H. (1974). Assignment of the gene for adenine phosphoribosyltransferase to human chromosome 16 by mouse-human somatic cell hybridization. Proc. Natl. Acad. Sci. USA 71:45. VandeBerg, J. L. (1992). Biochemical markers and restriction fragment length polymorphisms (RFLPs) in baboons: Their power for paternity exclusion. In Martin, R. D., Dixson, A. F., and Wickings, E. J. (eds.), Paternity in Primates: Genetic Tests and Theories, Karger, Basel, p. 18. VandeBerg, J. L., and Cheng, M.-L. (1986). The genetics of baboons in biomedical research. In Else, J. G., and Lee, P. C. (eds.), Primate Evolution, Cambridge University Press, Cambridge, p. 317. VandeBerg, J. L., and Johnston, P. G. (1977). A simple technique for long-term storage of erythrocytes for enzyme electrophoresis. Biochem. Genet. 15:213. Williams-Blangero, S., VandeBerg, J. L., Blangero, J., Konigsberg, L., and Dyke, B. (1990). Genetic differentiation between baboon subspecies: Relevance for biomedical research. Am. Z Primatol. 20:67.