Purine requirement of cells cultured from humans affected with Lesch-Nyhan syndrome (hypoxanthine-guanine phosphoribosyltransferase deficiency).

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 hypoxanthine, 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 nucleotides. 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, reported 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 nucleotides 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.