Evidence for X-Linkage of Human Phosphoribosylpyrophosphate Synthetase (Genetic Mapping/Dosage Compensation) RICHARD C

Proc. Natl. Acad. Sci. USA Vol. 75, No. 1, pp. 482-485, January 1978 Medical Sciences Evidence for X-linkage of human phosphoribosylpyrophosphate synthetase (genetic mapping/dosage compensation) RICHARD C. K. YEN, WILLIAM B. ADAMS, CHERI LAZAR, AND MICHAEL A. BECKER* Rheumatology Section, Department of Medicine, San Diego Veterans Administration Hospital; and University of California, San Diego, La Jolla, California 92161 Communicated by J. Edwin Seegmiller, October 31, 1977

ABSTRACT The mode of genetic transmission of human bers of these families (6-8) have not provided evidence for phosphoribosylpyrophosphate synthetase (ribosephosphate random X-chromosome inactivation as predicted by the Lyon pyrophosphokinase; ATP:D-ribose-5-phosphate pyrophospho- transferase; EC 2.7.6.1) was studied in fibroblasts cultured from hypothesis (9). However, in studies of fibroblasts cultured from members of a family with a structurally and electrophoretically the mother of an affected male patient described by Sperling altered phosphoribosylpyrophosphate synthetase that has in- et al. (2), values were found that were intermediate to those creased activity per enzyme molecule. Enzyme activity in fi- shown by cells from the affected son and normal individuals broblast lysates from the daughter of an affected male patient for PRPP synthetase activity, intracellular PRPP content, and was intermediate to the activities in lysates from her father (and purine synthetic rate (10). In addition, fibroblasts from this her affected paternal uncle) and from her mother and other normal individuals. Two bands of enzyme activity corre- woman showed rates of purine synthesis approximating those sponding to normal and mutant phosphoribosylpyrophosphate of her son after growth in a medium designed to select for cells synthetases were found in fibroblast lysates from the daughter with increased enzyme activity, thus providing indirect evi- after cellulose acetate strip electrophoresis. In contrast, only dence of X-linkage of PRPP synthetase (11). mutant enzyme was detectable in lysates derived from the male In the present study, more direct evidence for X-linkage of patients. Fibroblasts cloned from the daughter contained two the structural gene from PRPP synthetase is provided by phenotypically distinct (normal and mutant) populations of cells with respect to phosphoribosylpyrophosphate synthetase ac- demonstration of two distinct clonal populations with respect tivity and electrophoretic mobility. These studies support as- to this enzyme in fibroblasts from a female patient of the pre- signment of the structural gene for human phosphoribosylpy- viously described B. family (7) in which a structural alteration rophosphate synthetase to the X-chromosome. No evidence for in PRPP synthetase leading to increased activity per enzyme the presence of the normal enzyme was found in erythrocyte molecule has been established (5, 12). or lymphocyte lysates or in partially purified erythrocyte en- zyme preparations from the heterozygous daughter, suggesting either nonrandom X-chromosome inactivation in precursors of MATERIALS AND METHODS these cells or selection against hematopoietic cells bearing the normal enzyme after random X-chromosome inactivation. Cells. Fibroblast cultures were established from upper arm skin obtained by punch biopsy and were propagated in mo- 5-Phosphoribosyl-l-pyrophosphate (PRPP) synthetase (ATP: nolayer on 75-cm2 plastic flasks in Eagle's minimum essential D-ribose-5-phosphate pyrophosphotransferase; EC 2.7.6.1) medium supplemented with 2 mM L-glutamine, penicillin (100 catalyzes the reaction between ATP and ribose-5-P to form units/ml), streptomycin (100,ug/ml), and 10% fetal calf serum PRPP and AMP. PRPP is a rate-limiting substrate in purine (13). Clones were derived from fibroblast strains and isolated synthesis de novo as well as an allosteric activator of amido- with cloning rings as described by Ham and Puck (14). phosphoribosyltransferase (EC 2.4.2.14) (1), the first committed Lymphocytes were isolated from freshly drawn blood by enzyme in this pathway. In three families reported to date (2-4), Ficoll-Hypaque gradient centrifugation according to the affected male members with excessive PRPP synthetase activity method of Mendelsohn et al. (15) and incubated overnight at due to apparently different structural alterations in the enzyme 370 in RPMI 1640 medium containing 20% fetal calf serum and show increased intracellular PRPP synthesis, concomitant pu- 2 mM L-glutamine. After centrifugation, contaminating rine overproduction, and gout. Thus, increased PRPP synthetase erythrocytes were removed by resuspending the cell pellet for activity is a prototype for the association of a hereditary disease 10 min at 370 in 17 mM Tris-HCI with 140 mM ammonium state with a structurally aberrant protein of increased rather chloride (pH 7.4). than decreased catalytic activity. Preparation of Cell Lysates. Fibroblasts were harvested by The identification of an excessively active and electropho- treatment with trypsin as described (4), and an aliquot of each retically distinct form of PRPP synthetase (5) provides an op- cell suspension was counted with a Coulter Counter model ZBI. portunity to study the mode of inheritance of the enzyme. Further preparation of lysates was identical for lymphocytes Pedigree data from two of the families thus far reported (6, 7) and fibroblasts. Cells were washed twice in calcium-free Dul- are compatible with either autosomal dominant or X-linked becco's phosphate-buffered saline with 5.5 mM glucose. After transmission of the gene for PRPP synthetase. Father-to-son the final centrifugation, cell pellets were resuspended at a transmission of aberrant forms of the enzyme, a criterion es- density of 4-10 X 106 cells per ml in 8 mM sodium phosphate sential for the exclusion of X-linkage, has not been observed. buffer with 10 mM reduced glutathione/1 mM EDTA (pH 7.4), On the other hand, studies in erythrocytes from female mem- and the cells were lysed by freezing and thawing three times The costs of publication of this article were defrayed in part by the in liquid nitrogen. After centrifugation at 27,000 X g for 40 payment of page charges. This article must therefore be hereby marked "advertisemet" in accordance with 18 U. S. C. §1734 solely to indicate Abbreviation: PRPP, 5-phosphoribosyl 1-pyrophosphate. this fact. * To whom reprint requests should be addressed. 482 Downloaded by guest on September 30, 2021 Medical Sciences: Yen et al. Proc. Natl. Acad. Sci. USA 75 (1978) 483 min, the supernatant layers were dialyzed for 2 hr against the buffer in which the cells were lysed. '-J. 5000 Cellulose Acetate Electrophoresis. A portion of each fi- broblast lysate (3-12 ,tg of protein) was applied to duplicate 400 cellulose acetate strips (Millipore Biomedica) with an applicator, after which electrophoresis was carried out at 40 for 45 min in 0 3000 a Phoroslide electrophoresis apparatus. The electrophoresis buffer was 250 mM tricine with 1 mM sodium phosphate/i mM 22000 magnesium chloride/3% Nonidet/50,M ribose-5-P (pH 8.5) L0. modified from the method of Lebo and Martin (16). After electrophoresis, one strip of each pair was immediately stained 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 at 370 for PRPP synthetase activity as described by Johnson et Relative density al. (17). The control strip was stained in the identical solution FIG. 1. PRPP synthetase activities in normal and B. family fi- from which PRPP was omitted. Comparison of the parallel broblast extracts prepared at various relative cell densities. Cell strips permitted the distinction of bands specific for PRPP numbers corresponding to a relative density of 1.0 varied from strain synthetase activity from those resulting from nonspecific to strain over a range of 8.25 X 105-3.45 X 106 cells per 75-cm2 flask. staining. Each point represents the mean value of three determinations. As previously described (5), electrophoresis of partially pu- Symbols for fibroblast strains are: ,, H.B.; 0, T.B.; A, C.B.; *, Y.B.; rified preparations of human erythrocyte PRPP synthetase was *, normal control; o, C.B. 3; 0, C.B. 2; , C.B. 9. carried out on duplicate Cellogel cellulose acetate strips in a 40 mM barbital/10 mM phosphate buffer solution containing 10 PRPP synthetase activities which were, however, intermediate mM reduced glutathione and 1 mM magnesium chloride (pH to those of her mother and her father or uncle. Although the 8.6). The duplicate gel strips were stained as described above activity of PRPP synthetase in fibroblasts from Y.B. was nearly to permit identification of bands specific for enzyme activi- twice that of the other control fibroblast strain shown in Fig. ty. 1, values for strain Y.B. were within the range of activities Other Methods. PRPP synthetase activity was measured by measured in lysates of fibroblasts cultured from the 10 other a two-step assay (7) in which the PRPP generated from ATP normal individuals (182-697 nmol/hr per mg of protein). and ribose-5-P at 32 mM inorganic phosphate in the first step Electrophoresis Studies. Comparison of purified prepara- was determined by the conversion of [14C]adenine to [14C]AMP tions of normal and B. family erythrocyte PRPP synthetases in the presence of highly purified adenine phosphoribosyl- have established structural and electrophoretic differences in transferase (EC 2.4.2.7) (18). Protein concentration was de- the enzymes (5, 12). PRPP synthetase in lysates of fibroblasts termined by the method of Lowry et al. (19), with bovine serum cultured from these patients also differed from the normal albumin as standard. enzyme in electrophoretic mobility on cellulose acetate strips (Fig. 2). Fibroblast extracts from each of five normal individ- RESULTS uals, including Y.B., showed PRPP synthetase activity in a single Fibroblast Enzyme Activities. Fibroblasts were cultured band with an identical electrophoretic mobility. In contrast, from skin biopsies obtained from B. family members, including the sole band of PRPP synthetase activity demonstrable in patient H.B., his daughter (C.B.), and his brother (T.B.), each lysates of fibroblasts from patients H.B. and T.B. migrated with of whom shows excessive urinary uric acid excretion and diminished electrophoretic mobility. Mixtures of fibroblast erythrocyte PRPP synthetase activity approximately 3-fold lysates from H.B. (or T.B.) and normal individuals showed two higher than the mean activity of normal individuals (7). Fi- enzyme activity-specific bands with mobilities corresponding broblast strains were also developed from 11 clinically normal to those found in the individual lysates. Electrophoresis of fi- individuals, including H.B.'s wife (Y.B.). broblast extracts from patient C.B. produced a pattern of PRPP In order to compare specific activities of PRPP synthetase synthetase activity that was identical to the pattern observed in these fibroblast strains, it was necessary to study the cultures for the mixture of normal and patient H.B. (or T.B.) ex- at comparable stages of cell density (20). Therefore, in these tracts. experiments, internal controls for PRPP synthetase activity at Studies of Fibroblast Clones. Clones were derived from various cell densities were prepared by plating each strain at fibroblasts cultured from patients C.B., H.B., and T.B. and from three different cell densities (4 X 105, 1.3 X 105, and 0.4 X 105 three normal individuals. All of the clones derived from indi- cells per 75-cm2 flask). For each fibroblast strain, cells were viduals other than C.B. (two from T.B., two from H.B., and permitted to grow until the culture plated at the intermediate eight from three normal individuals) showed PRPP synthetase density appeared confluent by microscopic examination. At that activities similar to their respective uncloned parental popu- point, cells from all three flasks were harvested and enumer- lations. Of six putative clones developed from C.B.'s fibroblasts, ated, and lysates were prepared for enzyme assay. On the basis two strains (C.B. 1 and C.B. 2) had PRPP synthetase activities of the cell counts, relative densities were assigned to each of the intermediate to those of normal fibroblasts and those of H.B. flasks, with the flask plated at the highest density given a rela- or T.B. fibroblasts (Fig. 1). Strains C.B. 3 and C.B. 13 had PRPP tive density of 1.0. synthetase activities identical in magnitude to the activity of Fig. 1 shows PRPP synthetase specific activities in dialyzed this enzyme in cloned or uncloned H.B. or T.B. fibroblasts. In fibroblast lysates prepared at different cell densities from the contrast, two additional strains (C.B. 9 and C.B. 16) had enzyme three B. family patients and from normal individuals. A roughly activities indistinguishable from that found in fibroblasts cul- inverse relationship between PRPP synthetase activity and cell tured from C.B.'s mother, Y.B. density was observed for all cell strains. The activities of PRPP The electrophoretic mobility of PRPP synthetase in the pu- synthetase in the fibroblast extracts from patients H.B. and T.B. tative clones derived from patient C.B.'s fibroblasts was also were consistently 3- to 6-fold higher than those in normal cells. examined. Lysates of strains C.B. 1 and C.B. 2 showed two Extracts of fibroblasts from patient C.B. also showed increased PRPP synthetase-specific bands identical in mobility to those Downloaded by guest on September 30, 2021 484 Medical Sciences: Yen et al. Proc. Natl. Acad. Sci. USA 75 (1978)

(-) + (+) Table 1. PRPP synthetase activity in lysates of peripheral blood lymphocytes and erythrocytes from normal individuals and B. family members NORMAL 0D PRPP synthetase activity,* NORMAL 0D nmol/hr per mg protein T. B. Group Lymphocytes Erythrocytes NORMAL Normal + T.B. 0D W.H. 276 (266-286) 77 (74-80) C.B. R.S. 263 (244-281) 72 (69-75) 0D K.A. 242 (224-259) 64 (59-68) C.B. 3 0D C.G. 254(230-278) 83 (79-86) C.B. 9 B. family H.B. 554(545-563) 178 (173-182) T.B. 565 (560-569) 182 (180-185) NORMAL C.B. 536 (500-571) 183 (179-187) NORMAL *Values are the means of two separate determinations of enzyme activity. The range is given in parentheses. T.B. 0i NORMAL j this patient showed only a single band of enzyme activity with + T.B. the increased relative mobility previously described for similar C.B. preparations from patients H.B. and T.B. (5). C.B. 3 0 DISCUSSION The Lyon hypothesis (9) of random X-chromosome inactivation C.B. 9 in female cells during early embryogernesis predicts the exis- tence of two populations of cells in females heterozygous for ORIGIN an X-linked gene: one population that expresses the wild-type FIG. 2. Cellulose acetate strip electrophoresis of normal and B. allele and another that expresses the mutant allele. Demon- family fibroblast PRPP synthetase. PRPP was omitted from the stration of two such phenotypically distinct populations among staining solution for the top strip (e) which, therefore, shows only the cells of heterozygous females has been used in the assign- nonspecific staining. All ofthe other strips shown were developed with- ment of the genes for several biochemical markers, including PRPP in the staining solution (@). Bands seen only after development in the presence of PRPP are PRPP synthetase. (Upper) Stained glucose-6-phosphate dehydrogenase (EC 1.1.1.49) (21), hypo- strips; (Lower) schematic drawing corresponding to the stained xanthine phosphoribosyltransferase (EC 2.4.2.8) (22, 23), and strips. a-galactosidase (EC 3.2.1.22) (24) to the human X chromosome. Similarly, the present studies provide support for the assignment observed in lysates of uncloned C.B. cells. However, strains C.B. of the structural gene for human PRPP synthetase to the X 3 and C.B. 13 showed only the slowly migrating band seen in chromosome. lysates from H.B. or T.B. fibroblasts (Fig. 2). Strains C.B. 9 and A female member of the B. family, affected members of C.B. 16 showed a single band with migration identical to that which have an inherited structurally altered PRPP synthetase of the normal enzyme. These results indicated that strains C.B. with increased activity per enzyme molecule (12), showed in 3 and C.B. 13 and strains C.B. 9 and C.B. 16 were clones with her fibroblast lysates enzyme activities intermediate to those phenotypically distinct populations of cells representing fi- of lysates from normal individuals and her affected father or broblasts with mutant and normal PRPP synthetases, respec- uncle. Furthermore, extracts of fibroblasts from this female tively, and that strains C.B. 1 and C.B. 2 were mixed populations patient contained both normal and mutant PRPP synthetases, containing both types of cells rather than strains of clonal ori- as shown by cellulose acetate strip electrophoresis. Irt contrast, gin. only mutant enzyme was detectable after electrophoresis of Studies in Lymphocytes and Erythrocytes. The activities fibroblast extracts from the affected male patients. Thus, both of PRPP synthetase in lysates of peripheral blood lymphocytes enzyme activities and the migration of the enzyme on elec- isolated from patients H.B. and T.B. were 2-fold greater than trophoresis of extracts of fibroblasts from the B. family were those in identical preparations from normal individuals (Table inconsistent with the previously proposed hypothesis (7) that 1). In contrast to the activities of this enzyme in fibroblasts from the male patients T.B. and H.B. were heterozygotes bearing an patient C.B. (which were intermediate to those of normal in- autosomal mutation with dominant expression. In addition, dividuals and affected males patients), lymphocytes from this these results could not be reconciled with an autosomal recessive patient contained increased PRPP synthetase activity indis- transmission of PRPP synthetase, as was suggested from a study tinguishable in magnitude from the lymphocytes of patients of a suspected primary PRPP synthetase deficiency by Wada H.B. and T.B. Virtual identity in the magnitude of increased et al. (25). PRPP synthetase activity has previously been observed in In order to distinguish between X-linkage and autosomal erythrocyte lysates from these three patients (7), and this codominant inheritance, either of which could account for the finding was confirmed in the present studies (Table 1). In fibroblast activity and electrophoresis findings in the B. family, contrast to the two bands of enzyme activity found after elec- analysis of clonal fibroblast populations derived from the ob- trophoresis of fibroblast lysates from patient C.B., electropho- ligate heterozygote C.B. was undertaken. If hereditary trans- resis of partially purified erythrocyte PRPP synthetase from mission of PRPP synthetase were autosomal codominant, the Downloaded by guest on September 30, 2021 Medical Sciences: Yen et al. Proc. Natl. Acad. Sci. USA 75 (1978) 485 male patients H.B. and T.B. would necessarily expresboth KrQc Foundation, and by Grant AM-18197 (to M.A.B.) and Grant mutant and normal genes. However, in any form of autosomal GM-17702 (to J. E. Seegmiller) from the National Institutes of inheritance, all fibroblasts cultured from patient G.B. would Health. be expected to manifest an identical phenotype. The isolation 1. Holmes, E. W., McDonald, J. A., McCord, J. M., Wyngaarden, from patient C.B.'s fibroblasts of clones with normal (C.B. 9 and J. B. & Kelley, W. N. (1973) J. Biol. Chem. 248, 144-150. C.B. 16) and mutant (C.B. 3 and C.B. 13) phenotypes with re- 2. Sperling, O., Boer, P., Persky-Brosh, S., Kanarek, E. & de Vries, spect to PRPP synthetase activity and electrophoretic mobility A. (1972) Rev. Eur. Etud. Clin. Biol. 17,703-706. is inconsistent with autosomal inheritance of PTPP synthetase 3. Becker, M. A., Meyer, L. J., Wood, A. W. & Seegmiller, J. E. but is entirely compatible with X-linked transmission of this (1973) Science 179, 1123-1126. enzyme. 4. Becker, M. A. (1976) J. Clin. Invest. 57,308-318. Contrary to the in fibroblast of 5. Becker, M. A., Kostel, P. J., Meyer, L. J. & Seegmiller, J. E. (1973) findings extracts, lysates Proc. Natl. Acad. Sci. USA 70,2749-2752. erythrocytes (7) and lymphocytes (Table 1) from patient C.B. 6. de Vries, A. & Sperling, 0. (1973) Urologe A. 12, 153-157. showed activities of PRPP synthetase equal in magnitude to 7. Becker, M. A., Meyer, L. J. & Seegmiller, J. E. (1973) Am. J. Med. those of the affected male patients and partially purified 55,232-242. preparations of erythrocyte PRPP synthetase from all three B. 8. Sperling, O., Eilam, G., Persky-Brosh, S. & de Vries, A. (1972) family patients showed PRPP synthetase activity with the Biochem. Med. 6,310-316. electrophoretic mobility of the mutant enzyme only. Absence 9. Lyon, M. F. (1961) Nature 190,372-373. of expression of the normal allele for PRPP synthetase could 10. Zoref, E., de Vries, A. & Sperling, 0. (1975) J. Clin. Invest. 56, be due either to nonrandom inactivation of X chromosomes 1093-1099. carrying the normal gene for the enzyme in hematopoietic 11. Zoref, E., de Vries, A. & Sperling, 0. (1977) Adv. Exp. Med. Biol. precursor cells of this female or to 76A, 287-292. selection against the popu- 12. Becker, M. A., Kostel, P. J. & Myer, L. J. (1975) J. Biol. Chem. lation of cells with normal PRPP synthetase activity after ran- 250,6822-6830. dom X-chromosome inactivation. Random inactivation re- 13. Rosenbloom, F. M., Henderson, J. F., Caldwell, I. C., Kelley, W. sulting in suppression of X chromosomes bearing only the N. & Seegmiller, J. E. (1968) J. Biol. Chem. 243, 1166-1173. normal allele in hematopoietic precursors of this female het- 14. Ham, R. G. & Puck, T. T. (1960) in Methods in Enzymology, eds. erozygote would seem an unlikely explanation, since in the only Colowick, S. P. & Kaplan, N. 0. (Academic Press, New York), other family reported to date, the obligate heterozygote mother Vol. 5, pp. 90-119. of a patient with excessive PRPP synthetase activity showed 15. Mendelsohn, J., Skinner, S. A. & Kornfeld, S. (1971) J. Clin. In- normal activity of this enzyme in her erythrocytes, despite in- vest. 50, 818-826. creased enzyme activity in her fibroblasts In this 16. Lebo, R. V. & Martin, D. W., Jr. (1977) Adv. Exp. Med. Biol. 76A, (11). latter 570-574. family, nonrandom X-chromosome inactivation or subsequent 17. Johnson, M. G., Rosenzweig, S., Switzer, R. L., Becker, M. A. & selection appears to operate against the mutant allele, thus re- Seegmiller, J. E. (1974) Biochem. Med. 10, 266-275. sembling the situation observed in severe deficiency of hypo- 18. Thomas, C. B., Arnold, W. J. & Kelley, W. N. (1973) J. Biol. xanthine phosphoribosyltransferase (26), in which normal en- Chem. 248, 2529-2535. zyme activity is found in the erythrocytes of obligate heteroz- 19. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. ygous females. Although distinction between nonrandom X- (1951) J. Biot. Chem. 193,265-275. chromosome inactivation and subsequent selection is not 20. Martin, D. W., Jr. & Maler, B. A. (1976) Science 193, 408- presently possible, the observations in erythrocytes and lym- 411. phocytes of patient C.B. appear to represent an example in 21. Davidson, R. G., Nitowsky, H. M. & Childs, B. (1963) Proc. Natl. Acad. Sci. USA 50,481-485. which the product of the mutant allele of an X-linked gene in 22. Rosenbloom, F. M., Kelley, W. N., Henderson, J. F. & Seegmiller, human beings is expressed preferentially (27). J. E. (1967) Lancet ii, 305-306. While an X-linked mode of transmission of human PRPP 23. Migeon, B. R., Der Kaloustian, V. M., Nyhan, W. L., Young, W. synthetase appears to be established by the fibroblast studies J. & Childs, B. (1968) Science 160, 425-427. reported here, confirmation of this finding may be obtained 24. Romeo, G. & Migeon, B. R. (1970) Science 170, 180-181. by the demonstration of synteny with other X-linked bio- 25. Wada, Y., Nishimura, Y., Tanabu, M., Yoshimura, Y., Iinuma, chemical and antigenic markers in appropriate interspecific K., Yoshida, T. & Arakwa, T. (1973) Tohoku J. Exp. Med. 113, somatic cell hybrids, such as those between human and hamster 149-157. cells (28). 26. Nyhan, W. L., Bakay, B., Connor, J. D., Marks, J. F. & Keele, D. K. (1970) Proc. Natl. Acad. Sci. USA 65,214-218. 27. Lyon, M. F. (1970) Phil. Trans. R. Soc. London Ser. B 259, This work was supported in part by the Medical Research Service 41-52. of the Veterans Administration, by a grant to Dr. Seegmiller from the 28. Goss, S. J. & Harris, H. (1975) Nature 255, 680-684. Downloaded by guest on September 30, 2021