Identification of two new nucleotide mutations (HPRTUtrecht and HPRTMadrid) in exon 3 of the human hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene

Mutations in the X-linked hypoxanthine-guanine phosphoribosyl transferase gene (HPRT) result in deficiencies of HPRT enzyme activity, which may cause either a severe form of gout or Lesch-Nyhan syndrome depending on the residual enzyme activity. Mutations leading to these diseases are heterogeneous and include DNA base substitutions, DNA deletions, DNA base insertions and errors in RNA splicing. Identification of mutations has been performed at the RNA and DNA level. Sequencing genomic DNA of the HPRT gene offers the possibility of direct diagnostic analysis independent on the expression of the mature HPRT mRNA. We describe a Dutch and a Spanish family, in which the Lesch-Nyhan syndrome and a severe partial HPRT-deficient phenotype, respectively, were diagnosed. Direct sequencing of the exons coding for the HPRT gene was performed in both families. Two new exon 3 mutations have been identified. At position 16676, the normally present G was substituted by an A in the Dutch kindred (HPRT_Utrecht), and led to an arginine for glycine change at residue 70. At position 16680, the G was substituted by a T in the Spanish family (HPRT_Madrid); this substitutes a valine for glycine at residue 71. These new mutations are located within one of the clusters of hotspots in exon 3 of the HPRT gene in which HPRT_Yale and HPRT_{New Haven} have previously been identified.     

Human hypoxanthine-guanine phosphoribosyltransferase: a single nucleotide substitution in cDNA clones isolated from a patient with Lesch-Nyhan syndrome (HPRTMidland)

We have determined the molecular basis for hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency in a patient, J.H., with Lesch-Nyhan syndrome. Radioimmunoassay of lysates of erythrocytes or cultured B-lymphoblasts showed that this patient had no detectable HPRT enzyme activity or HPRT protein. HPRT-specific mRNA levels were normal by Northern analysis. We created a cDNA library from mRNA isolated from cultured lymphoblasts derived from this patient. Nucleotide sequencing of full-length HPRT cDNA clones revealed a single nucleotide (nt) substitution: a T-to-A transversion at nt 389. We have designated this variant HPRTMidland. The predicted amino acid (aa) substitution in HPRTMidland is a valine to aspartic acid at aa 130. This substitution is within 2 aa of the amino acid substitution in a previously defined HPRT variant, HPRTAnn Arbor. Both mutations are within a highly conserved sequence in the putative 5-phosphoribosyl-1-pyrophosphate-binding domain. The amino acid substitution in HPRTMidland causes a significant perturbation in the predicted secondary structure of this region. The HPRTMidland mutation affects a different domain of HPRT than the HPRTFlint mutation located at 167 nt away.     

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.     

The incorporation of homologous and heterologous hypoxanthine-guanine phosphoribosyltransferase into mutant cells

Experiments are described leading to partial compensation of a deficiency in the enzyme hypoxanthine-guanine phosphoribosyltransferase in mutant cells by supplying the cells with exogenous purified enzymes. DEAE-dextran is an effective helper agent, whereas poly(L-lysine, lysolecithin and amphotericin B seem to inhibit the entry of the enzymes or their activity. Enzyme preparation from Chinese hamster was found to have different effects in different mutant cell lines. In mutant Chinese hamster cells, the electrophoretic activity pattern remains unchanged for the Chinese hamster enzyme, but changes progressively to faster-moving activity peaks for the human enzyme after several hours. The metabolic effect of the incorporated enzyme is in the range between 3 and 4% of the normal cellular enzyme activity which corresponds to a 10–20 fold increase of hypoxanthine-guanine phosphoribosyltransferase activity in the mutant cells.     

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.     

A conditional mutant deficient in hypoxanthine-guanine phosphoribosyltransferase and xanthine phosphoribosyltransferase validates the purine salvage pathway of Leishmania donovani

Leishmania donovani cannot synthesize purines de novo and express a multiplicity of enzymes that enable them to salvage purines from their hosts. Previous efforts to generate an L. donovani strain deficient in both hypoxanthine-guanine phosphoribosyl-transferase (HGPRT) and xanthine phosphoribosyltransferase (XPRT) using gene replacement approaches were not successful, lending indirect support to the hypothesis that either HGPRT or XPRT is crucial for purine salvage by the parasite. We now report the genetic confirmation of this hypothesis through the construction of a conditional delta hgprt/delta xprt mutant strain that exhibits an absolute requirement for 2'-deoxycoformycin, an inhibitor of the leishmanial adenine aminohydrolase enzyme, and either adenine or adenosine as a source of purine. Unlike wild type parasites, the delta hgprt/delta xprt strain cannot proliferate indefinitely without 2'-deoxycoformycin or with hypoxanthine, guanine, xanthine, guanosine, inosine, or xanthosine as the sole purine nutrient. The delta hgprt/delta xprt mutant infects murine bone marrow-derived macrophages <5% as effectively as wild type parasites and cannot sustain an infection. These data establish genetically that either HGPRT or XPRT is absolutely essential for purine acquisition, parasite viability, and parasite infectivity of mouse macrophages, that all exogenous purines are funneled to hypoxanthine and/or xanthine by L. donovani, and that the purine sources within the macrophage to which the parasites have access are HGPRT or XPRT substrates.     

Mutations which alter splicing in the human hypoxanthine-guanine phosphoribosyltransferase gene.

A large proportion of mutations at the human hprt locus result in aberrant splicing of the hprt mRNA. We have been able to relate the mutation to the splicing abnormality in 30 of these mutants. Mutations at the splice acceptor sites of introns 4, 6 and 7 result in splicing out of the whole of the downstream exons, whereas in introns 1, 7 or 8 a cryptic site in the downstream exon can be used. Mutations in the donor site of introns 1 and 5 result in the utilisation of cryptic sites further downstream, whereas in the other introns, the upstream exons are spliced out. Our most unexpected findings were mutations in the middle of exons 3 and 8 which resulted in splicing out of these exons in part of the mRNA populations. Our results have enabled us to assess current models of mRNA splicing. They emphasize the importance of the polypyrimidine tract in splice acceptor sites, they support the role of the exon as the unit of assembly for splicing, and they are consistent with a model proposing a stem-loop structure for exon 8 in the hprt mRNA.     

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.     

Chinese hamster cells mutant at the hypoxanthine-guanine phosphoribosyltransferase (GPRT) locus. V. Complementation analysis of ts mutants

Four temperature-sensitive HPRT clones were used for hybridological analysis, which led to increase in complementation rate about 5 times. The probability of complementation, in respect of the HPRT locus proved to be rather high: 14 of 45 hybridization-tested mutants had complementation ability (including 3 ts mutants). Analysis of the complementation rate among mutants revealed clear-cut dependence on the selection conditions: clones grown in a medium with 8-azaguanine showed most frequent complementation. The use of mutants with a new phenotype in hybridization analysis revealed four additional complementation groups, three of which are made of temperature-sensitive clones. Biochemical analysis revealed the presence of hybrid forms of the HPRT enzyme in all hybrids tested. This confirms the intragenic character of complementation. At present, the functional map of the HPRT locus is represented by 9 groups, including a group of mutants with no complementation ability.     

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.     

Identification of a single missense mutation in the adenine phosphoribosyltransferase (APRT) gene from five Icelandic patients and a British patient.

We have completely sequenced the adenine phosphoribosyltransferase (APRT) gene from each of six patients--five (I-V) from Iceland and one (VI) from Britain. Cases I and II shared a common ancestor six and seven generations ago, and cases I and V shared a common ancestor seven generations ago, but cases III and IV were unrelated to the above or to each other, over seven generations. Genomic DNA was amplified by PCR, subcloned into M13mp18, and sequenced. Genomic and PCR-amplified DNAs were also analyzed by restriction-enzyme digestion and Southern blotting. The same missense mutation was identified in all six patients. This mutation leads to the replacement of asp (GAC) by val (GTC), at amino acid position 65. The gene sequences from all patients were otherwise identical to our wild-type sequence. The homozygous nature of the mutation was confirmed by sequencing the PCR product directly. All six patients were homozygous for the 1.25-kb TaqI RFLP. The Icelandic patients were also homozygous for the 8-kb SphI RFLP, but the British patient was heterozygous at this site. These studies suggest that a founder effect is likely to be responsible for APRT deficiency in the Icelandic population. The finding of the same mutation in a patient from Britain suggests that this mutation may have originated in mainland Europe.     

Suppressed expression of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) in an irradiation-attenuated Plasmodium berghei XAT strain

Plasmodium berghei XAT (XAT) is a non-reversible, non-lethal type malaria parasite strain derived from the highly virulent lethal P. berghei NK65 (NK65) by X-irradiation. The difference in polypeptide expression between NK65 and XAT was examined in this study. Western blot patterns of the parasite polypeptides showed that a 30-kDa polypeptide was not detected in XAT. In the present paper, we focused the study on the difference in the expression of the 30-kDa polypeptide between XAT and NK65. Although several other significant differences were noted in the spots shown by two-dimensional gel electrophoresis, the 30-kDa polypeptide was isolated by means of preparative 2D-gel electrophoresis followed by HPLC, and N-terminal amino acid sequence of the polypeptide was eventually determined. Complementary DNA clones encoding the 30-kDa polypeptide were isolated and characterized. Full-length cDNA clones from XAT encoded a protein of 231 amino acid residues with a 693-bp open reading frame. The deduced amino acid sequence exhibited 67% identity with that for P. falciparum hypoxanthine-guanine phosphoribosyltransferase (HGPRT; EC 2.4.2.8), suggesting that this protein is P. berghei HGPRT. Northern blot analysis revealed that expression of HGPRT in XAT was only one-eighth of that in NK65. This finding indicates that HGPRT gene expression is markedly suppressed in XAT. The amino acid sequence of HGPRT from NK65 was identical to that from XAT. This finding showed that the amino acid sequence of XAT-HGPRT was not mutated and had not undergone deletion.     

Fine structure mapping of the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene region of the human X chromosome (Xq26).

The Xq26-q27 region of the X chromosome is interesting, as an unusually large number of genes and anonymous RFLP probes have been mapped in this area. A number of studies have used classical linkage analysis in families to map this region. Here, we use mutant human T-lymphocyte clones known to be deleted for all or part of the hypoxanthine-guanine phosphoribosyltransferase (hprt) gene, to order anonymous probes known to map to Xq26. Fifty-seven T-cell clones were studied, including 44 derived from in vivo mutation and 13 from in vitro irradiated T-lymphocyte cultures. Twenty anonymous probes (DXS10, DXS11, DXS19, DXS37, DXS42, DXS51, DXS53, DXS59, DXS79, DXS86, DXS92, DXS99, DXS100d, DXS102, DXS107, DXS144, DXS172, DXS174, DXS177, and DNF1) were tested for codeletion with the hprt gene by Southern blotting methods. Five of these probes (DXS10, DXS53, DXS79, DXS86 and DXS177) showed codeletion with hprt in some mutants. The mutants established the following unambiguous ordering of the probes relative to the hprt gene: DXS53-DXS79-5'hprt3'-DXS86-DXS10-DXS177 . The centromere appears to map proximal to DXS53. These mappings order several closely linked but previously unordered probes. In addition, these studies indicate that rather large deletions of the functionally haploid X chromosome can occur while still retaining T-cell viability.