Partial hypoxanthine-guanine phosphoribosyl transferase deficiency with full expression of the Lesch-Nyhan syndrome

Summary A patient with the full clinical expression of the classical Lesch-Nyhan syndrome is presented with a residual hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity of 5–10% in erythrocyte lysate and about 30% in fibroblast lysate. The activities of other erythrocyte enzymes of purine metabolism were typical for a classical Lesch-Nyhan patient. The effects of allopurinol therapy on the excretion of urinary purine metabolites were studied by a newly developed isotachophoretic technique.The unusually high residual activity of HGPRT in erythrodytes and fibroblasts of the patient enabled the enzymologic characterization of the mutant enzyme: in fibroblasts the affinities for the substrates hypoxanthine and guanine were normal. However, there was an increased apparent K _m for phosphoribosylpyrophosphate (PRPP), a complete absence of product inhibition by IMP and GMP, and a decreased heat stability. Addition of PRPP did not stabilize the mutant enzyme. In addition to the altered properties of the fibroblast enzyme, the K _m of the erythrocyte enzyme for hypoxanthine was also increased.Immunoprecipitation experiments revealed the presence of an approximately normal amount of material cross-reacting with anti-human HGPRT antiserum. However, it appeared that this cross-reacting material had a decreased stability. When intact erythrocytes were incubated with radiolabeled purine bases, no formation of IMP or GMP could be detected, despite the relatively high residual activity of HGPRT in the hemolysate. The results fit the following hypothesis: as a consequence of a structural mutation affecting the PRPP-site of the enzyme and a decreased heat stability, the activity of the mutant enzyme under in vivo conditions is virtually zero.In the erythrocytes of the patient's mother a normal HGPRT-activity was found. However, the activity in her fibroblasts was lower than normal, while a decreased heat stability and an intermediate behavior towards IMP could be shown.Hair root analysis of several members of the patient's family confirmed the heterozygosity of the mother, whereas no other heterozygotes could be detected. The family anamnesis did not show other cases of Lesch-Nyhan syndrome. These findings were taken as evidence that the patient described in this paper might represent a mutation orginating from the gametes in either of the maternal grandparents.     

Electrophoretic properties of hypoxanthine-guanine phosphoribosyl transferase in erythrocytes of subjects with Lesch-Nyhan syndrome

Hypoxanthine-guanine phosphoribosyl transferase (HGPRT; E.C. 2.4.2.8) has been studied in erythrocytes of patients with Lesch-Nyhan syndrome by polyacrylamide gel electrophoresis. The location of this enzyme in gel was determined by radiochemical assay. Inosine monophosphate (the reaction product of HGPRT with radioactive hypoxanthine and 5-phosphorylribose-1-pyrophosphate) was precipitated in the gel at the site of its formation with lanthanum chloride. The zone containing radioactive inosine monophosphate was located by continuous monitoring of mechanically fractionated gels in a scintillation spectrometer. The sensitivity of this method has permitted the detection of the very low HGPRT activity in the electropherograms of hemolysates of patients with Lesch-Nyhan syndrome. Among six patients, four had a mutant enzyme which migrated 15% faster than the normal; the other two had a mutant enzyme with about 12% faster migration. These mutants were designated HGPRT-LN and HGPRT-LN slow, respectively. These observations indicate that the mutant gene on the X chromosome codes for a protein of altered structure.Aided by U.S. Public Health Service grants Nos. HD 04608 and HD 03015 from the National Institute of Child Health and Human Development and GM 17702 from the National Institute of General Medical Sciences, National Institutes of Health.Presented in part at the 1971 meeting of the Western Society for Pediatric Research, Carmel, California.     

Reversion in expression of hypoxanthine-guanine phosphoribosyl transferase following cell hybridization.

Hybridization of mutant cell lines deficient in hypoxanthine-guanine phosphoribosyl transferase (HGPRT; E.C.: 2.4.2.8) from a variety of established rodent sources with HGPRT plus human cells yielded progeny cells which grew in selective medium containing hypoxanthine, aminopterin and thymidine (HAT). The same result was obtained when the human cell used was an HGPRT minus transformed line derived from a patient with the Lesch-Nyhan syndrome. Electrophoretic analysis indicated that all HAT-resistant progeny clones contained an active HGPRT enzyme which was indistinguishable from the wild type enzyme of the corresponding normal rodent cells. In contrast, no HAT-resistant cells have been obtained when the same HGPRT minus rodent cells were subjected to fusion processes in the absence of human cells or when they fused with similarly derived HGPRT minus mutant cells of other rodents. Reversion in expression of the rodent gene for HGPRT was detected in clones which retained one or more human chromosomes and in clones which contained no detectable human chromosomal material. The observed re-expression of rodent HGPRT in HAT-resistant clones suggests that HGPRT plus as well as HGPRT minus human cells contributed a factor which determined the expression of respective rodent structural genes for HGPRT. In contrast, HGPRT minus rodent cells were unable to induce the synthesis or normal HGPRT in the cells derived from the patient with the Lesch-Nyhan syndrome.     

Azaguanine resistant hamster cell lines not deficient in hypoxanthine-guanine phosphoribosyl transferase

Using a serial selection technique in which Chinese hamster cells were treated first with 8-azaguanine and then subsequently with HAT medium it was found that approximately 15% of azaguanine resistant clones were also resistant to HAT. Several such clones were subcultured and found to be stably resistant to azaguanine, in some cases at a higher level than the usual azaguanine resistant mutants which are HAT sensitive. Measurements of hypoxanthine-guanine phosphoribosyl transferase levels were in some cases lower than the parental line but in three of the clonal lines were higher than the parental strain. The fact that azaguanine resistant lines constitute a biochemically heterogeneous population underscores the importance of careful characterization of mammalian cell culture variants.     

Increased phosphoribosylpyrophosphate synthetase activity in fibroblasts of hypoxanthine-guanine phosphoribosyl transferase deficient patients.

An increased activity of phosphoribosylpyrophosphate synthetase at physiological levels of inorganic phosphate is demonstrated in extracts of skin fibroblast cultures derived from a patient with Lesch-Nyhan syndrome. This eccessive response of the phosphoribosylpyrophosphate synthetase at physiological levels of inorganic phosphate results in increased levels of phosphoribosylpyrophosphate and thus contributes to purine overproduction characteristic of this disorder. The level of enzyme response in skin fibroblast extracts from the carrier mother was between activity of the patient and normals, further suggesting the x-linkage of human phosphoribosylpyrophosphate synthetase.     

Characterization of hypoxanthine-guanine phosphoribosyl transferase in man-mouse somatic cell hybrids by an improved electrophoretic method

The hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity in a group of man-mouse somatic cell hybrids, produced by Sendai virus-mediated cell fusion and HAT selection, has been analyzed by a new electrophoretic technique. Evidence is presented which shows that the hybrid lines derived from fusion of a mouse fibroblast deficient in HGPRT with various human cell strains have an HGPRT activity that is characteristic of the human enzyme, whereas a hybrid line derived from a mouse fibroblast which is deficient in thymidine kinase has an HGPRT activity characteristic of the mouse. This new technique involves electrophoresis of cell extracts on cellulose acetate gel, followed by the localization of the enzyme activity by autoradiography.This research was supported in part by a research grant from the U.S. National Institutes of Health (No. GM-13415).     

Heterogeneity of hypoxanthine guanine phosphoribosyl transferase from human erythrocytes.

Hypoxanthine guanine phosphoribosyl transferase (E.C.: 2.4.2.8) has been purified 4000- to 4500-fold from normal human erythrocytes by three different schemes of protein fractionation. In one scheme, the enzyme was separated by preparative polyacrylamide gel electrophoresis in an LKB Uniphor system and purified by affinity column chromatography employing Sepharose/phosphoribosyl/pyrophosphate. In the second, the enzyme was isolated by isotachophoresis in the presence of Amphiline carrier ampholytes employing a Tris/phosphate/β-alanine ion system. The enzyme was then purified by isotachophoresis in the presence of carrier ampholytes using a Tris/acetate/glycine ion system. The hypoxanthine guanine phosphoribosyl transferase purified by affinity chromatography and isotachophoresis consisted, on immunoelectrophoresis, mainly of one component and had less than 5% impurities. When subjected to analytical polyacrylamide gel electrophoresis, such preparations were resolved into four isoenzymes. In the third scheme, the enzyme was isolated by isoelectric focusing. In this system, the enzyme was also resolved into four isoenzymes. Their isoelectric points were: 5.47, 5.63, 5.74, and 5.84. When subjected to analytical polyacrylamide gel electrophoresis each isoenzyme migrated at a different rate. In sodium dodecyl sulfate gel electrophoresis each isoenzyme yielded one major and one minor band. Protein appearing in the major and minor bands migrated at rates consistent with a molecular size of 33,500 and 26,500, respectively.     

Analysis of human hypoxanthine-guanine phosphoribosyl transferase isozymes by isoelectric focusing in polyacrylamide gel

The method of isoelectric focusing in polyacrylamide gel was used to separate HGPRT isoenzymes in crude hemolysates of human and rat erythrocytes. HGPRT from erythrocytes of a normal human male donor consistently revealed three peaks of activity. Their mean isoelectric points, using pH 5–7 range ampholytes, were, peak I, pI 6.00; peak II, pI 5.8³; and peak III, pI 5.71. Peak III was wide and tailed. It always had a shoulder with a mean pI of 5.62. HGPRT from rat erythrocytes revealed two peaks of activity, corresponding to isoelectric points of 5.90 and 5.80. The method of isoelectric focusing in polyacrylamide gel is presented as a new way of detecting isoenzyme patterns.This study was supported by Grant No. 38-5768 from the Lebanese National Council for Scientific Research and Grant No. 18-5240 from the Medical Research Committee, American University of Beirut.     

Mutations affecting the antigenic properties of hypoxanthine-guanine phosphoribosyl transferase in cultured Chinese hamster cells.

Cells of the mutant Chinese hamster strain RJK10 do not contain either hypoxanthine-guanine phosphoribosyl transferase activity (HGPRT) or protein that cross-reacts immunologically with HGPRT. HGPRT+ revertants have been isolated from RJK10 and those strains produce HGPRT with altered antigenic properties. HGPRT from the revertant cells is less reactive with anti-HGPRT serum than enzyme from the wild-type cells, and enzymes from the two sources are immunoprecipitated independently from mixtures of cell extracts. Thus one or more of the antigenic determinants present on Chinese hamster HGPRT are either missing or present in an altered form on HGPRT from revertants of RJK10. This indicates that RJK10 carries a mutation in the structural gene for HGPRT and that secondary mutations in the gene give rise to the revertants that produce the antigenically altered enzymes.     

Lesch-Nyhan syndrome: Absence of the mutant enzyme in erythrocytes of a heterozygote for both normal and mutant hypoxanthine-guanine phosphoribosyl transferase

Subjects heterozygous for the Lesch-Nyhan syndrome with a deficiency of the X-linked gene for the enzyme hypoxanthine-guanine phosphoribosyl transferase (PRT) would be expected to have two populations of erythrocytes in roughly equal proportions—one type with the normal enzyme and the other type exhibiting the mutant form of the enzyme. In contrast to this prediction, previous studies utilizing an X-linked gene for another enzyme as a marker for the PRT locus have suggested that erythrocytes from heterozygotes consist largely of cells with the normal form of the enzyme. We have recently described a mutant form of hypoxanthine-guanine phosphoribosyl tranferase with altered kinetic properties which allow it to be measured in artificial mixtures with the normal enzyme. The mutant enzyme could not be detected in erythrocyte lysates from a proven heterozygote for both the normal and this mutant form of the enzyme. This provides additional evidence that either inactivation of the X-chromosome in erythropoietic tissue from the heterozygote for PRT deficiency is not random or that random X-chromosome inactivation is followed by selection against erythrocyte precursors with the mutant enzyme.This study was supported in part by USPHS Research Grant No. AM14362, USPHS Training Grant No. AM05620, and a grant (RR-30) from the General Clinical Research Centers Program of the Division of Research Resources, National Institutes of Health.     

Induction of mutations by chemical agents at the hypoxanthine-guanine phosphoribosyl transferase locus in human epithelial teratoma cells

Induction of 6-thioguanine (TG) resistance by chemical mutagens was examined in a line of cells derived from a human epithelial teratocarcinoma cell clone. The cells, designated as P3 cells, have a stable diploid karyotype with 46(XX) chromosomes, including a translocation between chromosomes 15 and 20. Efficient recovery of TG-resistant mutants induced by the direct-acting mutagens: N-methyl-N′-nitro-N-nitrosoguanidine (MNNG); 7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE); and benzo[a]pyrene (B[a]P); activated in a cell-mediated assay, required an expression time of 7 days and a saturation density of 2 × 10⁴ cells/60-mm petri dish. The TG-resistant mutant cells induced by MNNG and BPDE maintained their resistant phenotype 4–6 weeks after isolation. This mutant phenotype was associated with a more than 10-fold reduction in hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity relative to that of the parental P3 cell line, which was shown to catalyze the formation of 4.6 pmoles inosine-5′-monophosphate (IMP)/min/μg protein. Induction of TG resistance was also observed in P3 cells cocultivated in a cell-mediated assay with human breast carcinoma cells, which are capable of polyclinic aromatic hydrocarbon (PAH) metabolism, after treatment with the carcinogenic PAHs: B[a]P, chrysene, 7,12-dimethylbenz[a]anthracene (DMBA), and 3-methylcholanthrene (MCA). The degree of mutant induction in this assay was related to the carcinogenic potency of these PAHs in experimental animals. The most potent mutagen was DMBA, followed in decreasing order by MCA, B[a]P, and chrysene. DMBA, at 0.4 μM, increased the frequency of mutants for TG resistance from 2 for the control to about 200 TG-resistant mutants/10⁶ colony-forming cells (CFC). Benzo[e]pyrene (B[e]P) and pyrene, which are not carcinogenic, were not effective in the assay. None of the PAHs was mutagenic in the P3 cells cultivated in the absence of the PAH-metabolizing cells. These results indicate that the P3 cells can be useful for the study of mutagenesis at the HGPRT locus by direct-acting chemical mutagens, as well as by chemicals activated in a cell-mediated assay.     

Chromosome-mediated transfer of murine alleles for hypoxanthine-guanine phosphoribosyl transferase (HGPRT) and ouabain resistance into human cell lines

Genetic drug-resistance markers were transferred via purified metaphase chromosomes from mouse L cells into the human fibrosarcoma line HT1080 and HeLa S3 cells. Interspecific chromosome-mediated transfer of hypoxanthine-guanine phosphoribosyl transferase (HGPRT; EC 2.4.2.8) from mouse L cells into HGPRT^– HT1080 cells occurred at a frequency of approximately 1×10^–7. The presence of the mouse allele for HGPRT in transferent isolates was confirmed by isoelectric focusing. Transfer of ouabain resistance from mouse L cells to HT1080 and HeLa S3 cells occurred at an average frequency of approximately 4×10^–7. Expression of the mouse trait in transferent isolates was confirmed by their ability to withstand doses of ouabain which would be lethal to spontaneous ouabain-resistant mutants of the human cells but not to mouse L cells. Ouabain-resistant transferents of human cells showed 10⁴- to >10⁵-fold enhanced drug resistance, characteristic of either wild-type or mutant alleles, respectively, from ouabain-resistant donor L cells. Unstable expression of the transferred phenotypes in the absence of selection was seen in some isolates, but expression was lost at slow rates.This work was supported by National Institutes of Health Grant GM30383/21665 to RMB, Core Grants CA14051 to S. E. Luria and CA24538 to E. Mihich, and institutional predoctoral Training Grant GM07287.     

The molecular nature of spontaneous and chemically induced mutations in the hypoxanthine-guanine phosphoribosyl transferase gene in rat liver epithelial cells

The molecular nature of mutations in 6-thioguanine-resistant hypoxanthine/guanine phosphoribosyl transferase (HGPRT)-deficient clones of an adult rat liver (ARL) epithelial cell line mutated by benzo[a]pyrene or aflatoxin B1 was studied. DNA from these clones or spontaneous HGPRT-deficient mutants was subjected to Southern blotting using an HGPRT probe following DNA digestion with the restriction enzymes BamH1, EcoR1, HindIII or XbaI. With either the chemically induced or spontaneous mutants, no difference in restriction fragment pattern was observed between any of the mutants and their wild-type parent. However, differences were found between two lines ARL 6 and ARL 14 and the lines ARL 18, ARL 19 and DNA from Fischer rat hepatocytes. Although the variants did not display loss of HGPRT activity. It is suggested that deletion or loss of a pseudogene sequence could be the basis for the alterations in restriction fragment patterns.     

The dose-response relationship for ethyl methanesulfonate-induced mutations at the hypoxanthine-guanine phosphoribosyl transferase locus in Chinese hamster ovary cells.

The frequency of ethyl methanesulfonate (EMS)-induced mutations to 6-thioguanine resistance in a Chinese hamster ovary cells clone K1-BH4 was studied at many EMS doses including the minimally lethal range (0-100 microng/ml) as well as the exponential killing portion (100-800 microng/ml) of the survival curve. The mutation frequency increases approximately in proportion with increasing EMS concentration at a fixed treatment time. The pooled data for the observed mutation frequency, f(X), as a function of EMS dose X, is adequately described by a linear function f(X)=10(-6)(8.73+3.45 X), where 0 less than or equal to X less than or equal to 800 microng/ml. One interpretation of the linear dose-response is that, as a result of EMS treatment, ethylation of cellular constituents occurs, which is directly responsible for the mutation. Biochemical analyses demonstrate that most of the randomly isolated 6-thioguanine-resistant variants possess a highly reduced or undetectable level of HGPRT activity suggesting that the EMS-induced mutations to 6-thioguanine resistance affect primarily, if not exclusively, the HGPRT locus.     

Dilution of hypoxanthine-guanine phosphoribosyl transferase and other factors affecting the frequency of 6-thioguanine resistance in Chinese hamster lung cells.

Factors influencing the frequency of thioguanine resistant mutations were examined in Chinese hamster lung cells damaged with a carcinogen, N-acetoxy-2-acetyl aminofluorene. Factors such as inoculum density, expression time, and concentration of selective agent were found to have a profound effect on the mutation frequency.Over a range of doses, a longer expression time is required for mutant cells from a more damaged population to reach their maximum frequency. In order to investigate the elements involved in this phenomenon, the increment in the plating efficiency of treated cells as a function of expression time, spontaneous mutation rate per cell per generation, viability of mutant as well as wild type cells, and half life of HGPRTase were evaluated.There was an observed relationship between induced mutation frequency and plating efficiency of treated cells. When treated cells had recovered from effects of the treatment and arrived at the normal level of plating efficiency, they also yielded the maximum frequency of mutations.The estimated mutation rate was 5.5 × 10^?8 per cell per generation. This number is too small to account for the increment in mutation frequency with the increase in the expression time. The mutation frequency of spontaneous origin was 4 × 10^?6 and that of induction of 10^?5 M NA-AAF was 10^?4. Lower growth rates of mutant cells cannot explain this increase in the number of mutants recovered, either.Continuous diminution in the level of HGPRTase, at 35% daily, interpreted as an important factor responsible for the recovery of mutation frequency during expression time, was observed in non-dividing cells. None of a large number of mutants sampled from those isolated had HGRPT activity. This indicates that they are true mutants and are not a result of phenocopy. Only cells completely deficient in HGPRT activity are recovered in TG selection medium. It is suggested, therefore, that this cell line is suitable for mutagenicity testing in the induction of mutation at the HGPRT locus.