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Ported Here, Support the Interpretation That Mutants Are, in Effect, Nucleotide- Deficient

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Ported Here, Support the Interpretation That Mutants Are, in Effect, Nucleotide- Deficient PURINE REQUIREMENT OF CELLS CULTURED FROM HUMANS AFFECTED WITH LESCH-NYHAN SYNDROME (HYPOXANTHINE- GUANINE PHOSPHORIBOSYLTRANSFERASE DEFICIENCY) * BY JEANETTE S. FELIXt AND ROBERT DEMARS GENETICS LABORATORY, UNIVERSITY OF WISCONSIN, MADISON Communicted by R. A. Brink, December 13, 1968 Abstract.-Humans with the Lesch-Nyhan syndrome have an X-chromosomal mutant gene that causes severe neurological and developmental abnormalities. The patients are deficient in hypoxanthine-guanine phosphoribosyltransferase, which converts hypoxanthine to inosinic acid, a major precursor of adenine and guanine nucleotides. Paradoxically, the enzyme defect causes hypernormal de novo synthesis of inosinic acid, which manifests itself as excesses of hypoxan- thine, xanthine, and uric acid. The first step in the de novo pathway is thought to be rate-limiting, due to feedback repression by adenine and guanine nucleo- tides. The derepressed rate of purine production in mutants and their failure to thrive could result from reduction in the amounts of nucleotides derived from inosinic acid to levels that are inadequate for normal feedback control and for nucleic acid synthesis needed in growth. Studies with cultured cells, re- ported here, support the interpretation that mutants are, in effect, nucleotide- deficient. Skin fibroblasts from patients fail to proliferate in media that do not contain supplementary adenine or folic acid, a participant in two stages of purine biosynthesis. The folic acid requirement of mutant cells is at least 50- fold greater than that of normal cells, which can synthesize all the nucleotides needed for growth without exogenous adenine. Both folic acid and adenine supplements are thought to provide mutant cells with the means of making more inosinic acid available for conversion to adenine and guanine nucleotides. It is not clear why the availability of inosinate or its conversion to other nucleo- tides is impaired. Therapy with adenine or folic acid begun at the time of birth may avert development of the disease in mutant males. The relevant gene is X-linked and shows clonal, single-allele-expression: phenotypically normal and phenotypically mutant clones have been derived from females heterozygous for the mutant gene. The phenotypically mutant heterozygous clones have the same requirement for adenine or folic acid as cells from hemizygous mutant males, an indication that the normal allele is repressed in these clones. The adenine-folic acid requirement of mutant cells provides a method of direct, clonal selection for rare, phenotypically normal cells in mutant populations, which is applicable to the single-active-X problem and other in vitro genetic studies. Introduction.-The enzyme hypoxanthine-guanine phosphoribosyltransferase (E.C. 2.4.2.81) carries out the reaction: hypoxanthine (or guanine) + phosphoribosyl pyrophosphate inosinic acid (or guanylic acid, respectively) + pyrophosphate. 536 Downloaded by guest on September 27, 2021 VOL. 62, 1969 GENETICS: FELIX AND DEMARS 53S7 X-linked mutant genes causing apparently complete deficiency of the enzyme evoke the Lesch-Nyhan syndrome in hemizygous human males.2 Cells cul- tured from boys affected with the syndrome can be characterized, as popula- tions, by direct enzyme assay3 or, individually, by the autoradiographic demon- stration of their inability to incorporate radioactive hypoxanthine from their culture medium.4 The locus exhibits clonal, single-allele-expression in diplo-X cells: clones cul- tured from females heterozygous for normal (Jh) and deficiency (jh) alleles are either "normal," having the normal phenotype, or "mutant," having the mutant phenotype, but are not intermediate.6' 6 The presence of "normal" cells in heterozygous "mutant" clones could indicate derepression of the normal allele. The spontaneous derepression rate is less than 104 per cell division,6 but ex- perimentally evoked derepression could yield new information about the Lyon7 or single-active-X8 hypothesis. (See refs. 9-11 for recent reviews.) We describe here conditions for effecting direct selection of rare, phenotypically normal cells in large populations having the mutant phenotype. These experiments began with our observation that skin biopsies from boys af- fected with the syndrome failed to produce cellular outgrowths in standard F4 medium,12 which supported rapid outgrowth from biopsies of unaffected indi- viduals. Our eventual solution to this problem was based on the following rea- soning: de novo purine synthesis occurs at a hypernormal rate in jh cells.'3 This may result from a shortage of adenine and guanine nucleotides, which are known repressors of phosphoribosylpyrophosphate amidotransferase, the first enzyme in the pathway.'4 15 Inosinic acid (IMP) derived from this path- way and other sources is converted to hypoxanthine (plus xanthine and uric acid in the intact human).'6 Lacking hypoxanthine-guanine phosphoribosyl- transferase, jh cells are unable to reconvert this hypoxanthine to IMP and then to nucleotides that are negotiable in the purine economy of the cells. We de- cided that adenine might relieve the apparent nucleotide shortage, since mutant and normal cells have an enzyme, analogous to the transferase, that converts the free base directly to AMP (adenylic acid). This would then be available for restoring feedback repression and conversion to guanine nucleotides. This interpretation does not pinpoint the basis for the nucleotide shortage, but it led to an experimental solution to the cells' difficulties. Addition of adenine to the F4 medium permitted growth of jh cells and "mu- tant" heterozygous clones. The rate depended on adenine concentration and attained maxima comparable to those found in normal cells. We had found empirically that mutant cells grew well in medium F10,17 which also lacks ade- nine but contains higher levels of folic acid than F4 medium. Folic acid par- ticipates in two steps of purine synthesis, and we determined that growth of mutant cells in F10 could be reduced by lowering the concentration of folate. Conversely, elevated levels of folic acid in F4 promoted maximal growth of mutants. Normal (Jh) cells and "normal" heterozygous clones grew well in either medium without supplementary adenine or augmented folic acid. These findings with cultured cells provide new information about the biochemical abnormalities resulting from jh mutations and are laboratory evidence for a Downloaded by guest on September 27, 2021 538 GENETICS: FELIX AND DEMARS PRoc. N. A. S. rational therapy for the disease. A similar suggestion' and attempt' at adenine therapy appeared during the course of our work. The observations presented here also provide the first published method of direct, clonal selec- tion for a biochemically defined marker in diploid human cells-one that can be applied to many sorts of genetic studies in vitro. Materials and Methods.-Skin fibroblasts were cultured2O from three unrelated boys (nos. 252, 253, and 255) with the Lesch-Nyhan syndrome. Four normal strains (nos. 47, 247, 254, and 261) were used as controls. All culture media were supplemented with 15% fetal bovine serum. Stock cultures used for growth experiments were maintained in F4"2 supplemented with 3 X 10-' M adenine, but F10'7 was found to be a generally superior medium for all cells. Clones described in Figure 4 were derived from a proved heterozygous culture (no. 248.1) with the use of previously described Method J1,6 in which only those drops with precisely one microscopically visible cell 3-4 hr after inoculation were used. The clones were stored in liquid N2 after attaining populations of about 106 cells and were thawed for experiments.2' Growth response experiments were performed by inoculating 13-mm diameter glass dishes (Bellco) with about 5 X 101 cells in 0.5 ml of F4 minus adenine. The medium was replaced with 0.5 ml of experimental media 8-18 hr after inoculation and then daily until completion of the experiments. Cell counts were made by trypsinizing each culture with 0.25 ml of 0.25% trypsin (Grand Island Biological) and suspending the cells in 0.9% NaCl. Duplicate O.5-ml aliquots of each suspension were counted with a Coulter counter. Each value presented here is the average of two cul- tures, which agreed to within i+ 10% of their average more than 95% of the time. Results.-A denine requirement: Figure 1 describes the nine-day growth re- sponses of mutant strains as a function of adenine concentration added to F4, beyond the undefined amount contributed by serum (about 10-7 Ml or less). Controls (Fig. 1A) showed no dependence on supplemental adenine, although concentration 2 (10-5 l) sometimes enhanced their growth by 10-15 per cent. Growth of mutants (Fig. 1B) did not occur without adenine supple- ment and attained maximal values at 10-5 M. Concentrations in excess of concentration 4 (10-4 M) were inhibitory to both control and mutant strains. These relations were maintained throughout growth, but only the nine-day counts have been presented for simplicity. Mutant strains occasionally increased without adenine supplement, but never more than 3.5-fold in a nine-day period, where adenine promoted 20-fold or greater increases. Figure 1B also indicates AoA CONTROLS B MUTANTS 24_ 255!A FIG. 1.-Adenine requirement of jh cells. Fibroblasts were given medium ~20 26 F4 supplemented with 15% fetal bovine a:a serum and increasing concentrations of adenine or hypoxanthine. Concentra- tion 0 indicates no added adenine, while other concentrations increased in H,0.16 E CONCENTRATIONREPEAT ~ ~ ~ . \ \_ . threefold steps from 1 (3.3 X 10-6 M) (/1 4 247 \ -i~~~~~~~* 252 to 7 (2.4 X 10-3 M). Cell counts for wi 252 WITH day 0, when experimental media were o NYPOXANTHINE/ added, and for resultant 9-day growth 4-~~~~~~~~~~~- are recorded. 0 1 23 4 56 7 0 1 2 34 567 ADENINE CONCENTRAMiN Downloaded by guest on September 27, 2021 VOL. 62, 1969 GENETICS: FELIX AND DEMARS 539 that jh cells can increase in high concentrations of hy- 24 poxanthine, which they are presumably unable to util- ize.
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