Genetic analysis of human hypoxanthine-guanine phosphoribosyltransferase deficiency

Hypoxanthine-guanine phosphoribosyltransferase (HPRT; IMP: pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) functions in the purine-metabolic salvage pathway. Two clinical syndromes are associated with a deficiency in HPRT enzyme activity. Virtually complete deficiency leads to the Lesch-Nyhan syndrome, whereas partial deficiency results in hyperuricemia and severe gouty arthritis. Marked heterogeneity in the mutations leading to HPRT deficiency has been found. Mutant enzymes vary with respect to levels of HPRT immunoreactive protein, electrophoretic migration, kinetic properties and amino acid sequence. Analysis of DNA and RNA from patients with HPRT deficiency has revealed point mutations, an internal gene duplication and partial as well as complete gene deletions accounting for the various HPRT mutant enzymes.     

Evaluation of the severity of hypoxanthine-guanine phosphoribosyltransferase deficiency using viable T cells

Peripheral T cells from 3 Lesch-Nyhan patients, 3 normal subjects, and 3 brothers with hypoxanthine-guanine phosphoribosyltransferase (HGPRT) deficiency but without Lesch-Nyhan syndrome (so-called partial deficiency) have been analyzed. Although these brothers contained HGPRT activities neither in the hemolysates nor in the T cell extracts at levels detectable by the regular radioenzyme assay, the enzyme deficiency had not caused any typical neurological symptoms of the Lesch-Nyhan syndrome. Although the T cells from these brothers were at least 10-fold more resistant to 6-thioguanine than normal T cells, they were more than 30-fold less resistant than the T cells from 3 Lesch-Nyhan patients indicating that there is a clear difference in the severity of the enzyme deficiency between the brothers and the Lesch-Nyhan patients. These data indicate that the long-term T cell culture in the medium containing a purine analog whose toxicity depends on a salvaging enzyme is useful for evaluating the severity of the enzyme deficiency in viable cells.     

Mutant chinese hamster cells with a thermosensitive hypoxanthine-guanine phosphoribosyltransferase.

By selecting variants of Chinese hamster cells that were resistant to 6-thioguanine at 39 degrees C, but which would continue to grow in HAT medium at 33 degrees C, we have isolated cell lines with thermosensitive phenotypes. These clones form colonies in HAT medium and incorporate 14-C-hypoxanthine much more efficiently at 33 degrees C than at 39 degrees C. The specific activity of hypoxanthine-guanine phosphoribo-syltransferase is at least 10 times higher in variant cells grown at 33 degrees C than in those grown in 39 degrees C, and the enzymes from the variant clones are inactivated in vitro at 39 degrees C 7-9 times more rapidly than is the enzyme from wild-type cells. The results are consistent with the conclusion that the selected clones have missense mutations in the structural gene for the enzyme.     

Human hypoxanthine-guanine phosphoribosyltransferase deficiency. The molecular defect in a patient with gout (HPRTAshville)

The genetic basis of hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency has been identified by nucleotide sequence analysis of HPRT cDNAs cloned from a patient with gout. A single nucleotide change was identified in two independent clones: an A to G transition at nucleotide 602. Confirmation of a mutation at this site was provided by RNase mapping analysis. The predicted consequence of this transition is an aspartic acid to glycine substitution at amino acid 201. We have designated this variant HPRTAshville. Prior to this report, enzyme activity in HPRTAshville had not been detected by routine assay. Using more sensitive techniques, including an in situ gel assay for HPRT activity, we were able to demonstrate electrophoretic, kinetic, and structural differences between HPRTAshville and normal HPRT. Electrophoretic migration of HPRTAshville has elevated Michaelis constants for 5-phosphoribosyl-1-pyrophosphate and hypoxanthine. Predicted secondary structural alterations may result from the aspartic acid to glycine substitution.     

A review of the molecular basis of hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency

Hypoxanthine-guanine phosphoribosyltransferase (HPRT, EC 2.4.2.8) is a purine salvage enzyme that catalyses the conversion of hypoxanthine and guanine to their respective mononucleotides. Partial deficiency of this enzyme can result in the overproduction of uric acid leading to a severe form of gout, whilst a virtual absence of HPRT activity causes the Lesch-Nyhan syndrome which is characterised by hyperuricaemia, mental retardation, choreoathetosis and compulsive self-mutilation. The HPRT-encoding gene is located on the X chromosome in the region q₂₆–q₂₇ and consists of nine exons and eight introns totalling 57 kb. This gene is transcribed to produce an mRNA of 1.6 kb, which contains a protein encoding region of 654 nucleotides. With the advent of increasingly refined techniques of molecular biology, it has been possible to study the HPRT gene of individuals with a deficiency in HPRT activity to determine the genetic basis of the enzyme deficiency. Many different mutations throughout the coding region have been described, but in the absence of precise information on the three-dimensional structure of the HPRT protein, it remains difficult to determine any consistent correlation between the structure and function of the enzyme.     

Characterization of purine nucleotide metabolism in cultured fibroblasts with deficiency of hypoxanthine-guanine phosphoribosyltransferase and with superactivity of phosphoribosylpyrophosphate synthetase

Cultured fibroblasts with hypoxanthine-guanine phosphoribosyltransferase (HGPRT) deficiency exhibited acceleration of purine synthesis de novo, absence of salvage IMP synthesis from hypoxanthine, but normal total IMP synthesis. Cells with phosphoribosylpyrophosphate synthetase superactivity exhibited acceleration of both de novo and salvage IMP synthesis and increased total IMP synthesis. The study of mutant cells furnished evidence that in normal as well as mutant cells, GMP and AMP are not converted to each other in significant amounts and that these nucleotides are not degraded by nucleotidases. Purine nucleotide degradation in fibroblasts occurs mainly by dephosphorylation of IMP. In HGPRT-containing cells, salvage IMP synthesis from preformed and exogenously supplied hypoxanthine is the main source for IMP production.     

Herpes simplex virus-mediated human hypoxanthine-guanine phosphoribosyltransferase gene transfer into neuronal cells.

The virtually complete deficiency of the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) results in a devastating neurological disease, Lesch-Nyhan syndrome. Transfer of the HPRT gene into fibroblasts and lymphoblasts in vitro and into hematopoietic cells in vivo has been accomplished by other groups with retroviral-derived vectors. It appears to be necessary, however, to transfer the HPRT gene into neuronal cells to correct the neurological dysfunction of this disorder. The neurotropic virus herpes simplex virus type 1 has features that make it suitable for use as a vector to transfer the HPRT gene into neuronal tissue. This report describes the isolation of an HPRT-deficient rat neuroma cell line, designated B103-4C, and the construction of a recombinant herpes simplex virus type 1 that contained human HPRT cDNA. These recombinant viruses were used to infect B103-4C cells. Infected cells expressed HPRT activity which was human in origin.     

Identification of 17 independent mutations responsible for human hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency.

Complete hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency causes the Lesch-Nyhan syndrome, an X-linked, purine metabolism disorder manifested by hyperuricemia, hyperuricaciduria, and neurologic dysfunction. Partial HPRT deficiency causes hyperuricemia and gout. One requirement for understanding the molecular basis of HPRT deficiency is the determination of which amino acids in this salvage enzyme are necessary for structural or catalytic competence. In this study we have used the PCR coupled with direct sequencing to determine the nucleotide and subsequent amino acid changes in 22 subjects representing 17 unrelated kindreds from the United Kingdom. These mutations were confirmed by using either RNase mapping or Southern analyses. In addition, experiments were done to determine enzyme activity and electrophoretic mobility, and predictive paradigms were used to study the impact of these amino acid substitutions on secondary structure.     

Substrate inhibition in a human variant of hypoxanthine-guanine phosphoribosyltransferase

Hypoxanthine-guanine phosphoribosyltransferase from a young man with purine overproduction and decreased purine salvage in fibroblast cultures was found to have low activity at concentrations of purine substrates at which the enzyme from normal individuals showed near maximal activity. The low enzyme activity was not associated with changes in the values of the Km(app) and Vmax(app) for any of the enzyme substrates. However, the enzyme activity was susceptible to substrate inhibition by hypoxanthine and guanine. The values obtained for the true Km, true Vmax, and true Ki for hypoxanthine were 26 +/- 10 microM, 1761 +/- 382 microunits/mg of protein, and 80 +/- 20 microM, respectively. The pattern of the substrate inhibition, as seen on a plot of 1/v versus hypoxanthine concentration, was characteristic of that associated with the formation of a dead-end complex between the inhibitory substrate and an enzyme form with which it normally does not react. The nature of this enzyme form and that of the dead-end complex was determined from double inhibition experiments, which indicated that hypoxanthine interacted with an enzyme-PPi intermediate to form an enzyme-hypoxanthine-PPi dead-end complex. The trapping of the enzyme in this inactive form explains the low activity at high purine base concentrations. Further information as to the nature of the reaction mechanism was obtained from plots of the reciprocal of enzyme activity versus the reciprocal of PP-ribose-P concentration at different fixed hypoxanthine concentrations. A pattern characteristic of uncompetitive substrate inhibition was obtained. This is indicative of an ordered sequential binding of substrates on the enzyme; PP-ribose-P binding before hypoxanthine. Thus, the variant enzyme showed an ordered sequential reaction mechanism, with the inhibitory substrate forming a dead-end complex with an enzyme-PPi intermediate.     

Regulation of purine nucleotide synthesis in human B lymphoblasts with both hypoxanthine-guanine phosphoribosyltransferase deficiency and phosphoribosylpyrophosphate synthetase superactivity.

Human B lymphoblast lines severely deficient in hypoxanthine-guanine phosphoribosyltransferase (HGPRT) were selected for resistance to 6-thioguanine from cloned normal and phosphoribosylpyrophosphate (PP-Rib-P) synthetase-superactive cell lines and were compared with their respective parental cell lines with regard to growth and PP-Rib-P and purine nucleotide metabolism. During blockade of purine synthesis de novo with 6-methylthioinosine or aminopterin, inhibition of growth of all HGPRT-deficient cell lines was refractory to addition of Ade at concentrations which restored substantial growth to parental cell lines. Ade-resistant inhibition of growth of parental lines by 6-methylthioinosine, however, occurred during Ado deaminase inhibition. Insufficient generation of IMP (and ultimately guanylates) to support growth of lymphoblasts lacking HGPRT activity and blocked in purine synthesis de novo best explained these findings, implying that a major route of interconversion of AMP to IMP involves the reaction sequence: AMP----Ado----Ino----Hyp----IMP. PP-Rib-P generation and purine nucleoside triphosphate pools were unchanged by introduction of HGPRT deficiency into normal lymphoblast lines, in agreement with the view that accelerated purine synthesis de novo in this deficiency results from increased availability of PP-Rib-P for the pathway. Cell lines with dual enzyme defects did not differ from PP-Rib-P synthetase-superactive parental lines in rates of PP-Rib-P and purine synthesis despite 5-6-fold increases in PP-Rib-P concentrations, excretion of nearly 50% of newly synthesized purines, and diminished GTP concentrations. Fixed rates of purine synthesis de novo in PP-Rib-P synthetase-superactive cells appeared to reflect saturation of the rate-limiting amidophosphoribosyltransferase reaction for PP-Rib-P. In combination with accelerated purine excretion, increased channeling of newly formed purines into adenylates, and impaired conversion of AMP to IMP, fixed rates of purine synthesis de novo may condition cell lines with defects in HGPRT and PP-Rib-P synthetase to depletion of GTP with consequent growth retardation.     

Adenine phosphoribosyltransferase and hypoxanthine-guanine phosphoribosyltransferase immunoprecipitation reactions in human-mouse and human-hamster cell hybrids.

Male New Zealand White rabbits were immunized with human adenine phosphoribosyltransferase (APRT) and hypoxanthine-guanine phosphoribosyltransferase (HGPRT), which were purified about 2000-fold and 800-fold, respectively, from erythrocytes by DEAE-cellulose chromatography, ammonium sulfate precipitation and preparative polyacrylamide gel electrophoresis. Specific immunoprecipitations of APRT and HGPRT were achieved with the antisera that were obtained and by using polyethylene glycol as a substitute for goat anti-(rabbit) gamma globulin. The activities of the human forms of these enzymes, whether from red blood cells or from cultured cells, were almost completely eliminated under the conditions of immunoprecipitation used. Little or no reduction of APRT and HGPRT activities from mouse and Chinese hamster cells was observed. This discriminatory capacity of the antisera was successfully used for the identification of human APRT and HGPRT in human-mouse and human-hamster cell hybrids using the immunoprecipitation reaction.     

Elevated levels of erythrocyte hypoxanthine phosphoribosyltransferase associated with allelic variation of murine Hprt

Murine stocks with wild-derived hypoxanthine phosphoribosyltransferase (HPRT) A alleles (Hprt a) have erythrocyte HPRT activity levels that are approximately 25-fold (Mus musculus castaneus) and 70-fold (Mus spretus) higher than those of laboratory strains of mice with the common Hprt b allele (Mus musculus: C3H/HeHa or C57B1/6). Since the purified HPRT A and B enzymes have substantially similar maximal specific activities (64 and 46 units/mg of protein, respectively), we infer that these HPRT activity levels closely approximate the relative levels of HPRT protein in these cells. Red blood cells of HPRT A and B mice have similar levels of adenine phosphoribosyltransferase activity (APRT; EC 2.4.2.7) and reticulocyte percentages, which suggests that the elevated levels of HPRT in erythrocytes of HPRT A mice are not secondary consequences of abnormal erythroid cell development. The HPRT activity levels in reticulocytes of HPRT B mice are approximately 35-fold higher than the levels in their erythrocytes and approach the HPRT activity levels in reticulocytes of HPRT A mice. Thus, the marked differences in the levels of HPRT protein in erythrocytes of HPRT A and B mice result from differences in the extent to which the HPRT A and B proteins are retained as reticulocytes mature to erythrocytes. The substantial and preferential loss of HPRT B activity from reticulocytes is paralleled by an equivalent loss of HPRT immunoreactive protein (i.e., CRM) from that cell, and we infer that the HPRT B protein is degraded or extruded as reticulocytes mature to erythrocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Elevated aspartate transcarbamylase and dihydroorotase activities in erythrocytes from patients with hypoxanthine guanine phosphoribosyltransferase deficiency.

Activities of aspartate transcarbamylase and dihydroorotase are increased 6 to 8-fold in erythrocytes from individuals with hypoxanthine guanine phosphoribosyltransferase deficiency. The increased enzyme activities do not appear to be due to enzyme activation.     

Amplification of hypoxanthine-guanine phosphoribosyltransferase genes in chromosome-mediated gene transferents.

Hypoxanthine-guanine phosphoribosyltransferase (HPRT) enzyme activities may be elevated in genetically unstable chromosome-mediated gene transferents selected for transfer of the HPRT gene. Increased levels of HPRT polypeptides in unstable mouse L cell gene transferents were demonstrated by two-dimensional gel electrophoresis and immunoprecipitation. No additional polypeptides were found to be overexpressed. HPRT mRNA levels were elevated 10- to 15-fold in the unstable gene transferent GT427C. Southern blot hybridization experiments showed that overexpression of HPRT correlated with a 5- to 15-fold amplification of HPRT gene sequences in two unstable cell lines. Stabilized gene transferents displayed reduced HPRT copy numbers. The amplification of HPRT gene sequences in the unstable transferent GT427C was associated with the presence of multiple minute chromosome fragments. An average of 9.6 fragments was found per metaphase, but the variation was considerable, ranging from 0 to 53. We conclude that genomic DNA sequences may be amplified in unstable chromosome-mediated gene transferents and that such amplification may be associated with the occurrence of multiple chromosomal fragments.