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.     

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.     

Identification of a single nucleotide change in a mutant gene for hypoxanthine-guanine phosphoribosyltransferase (HPRTAnn Arbor)

Summary HPRT_{Ann Arbor} is a variant of hypoxanthine (guanine) phosphoribosyl-transferase (HPRT: EC 2.4.2.8), which was identified in two brothers with hyperuricemia and nephrolithiasis. In previous studies, this mutant enzyme was characterized by an increased K_m for both substrates, a normal V_max, a decreased intracellular concentration of enzyme protein, a normal subunit molecular weight and an acidic isoelectric point under native isoelectric focusing conditions. We have cloned a full-length cDNA for HPRT_{Ann Arbor} and determined its complete nucleotide sequence. A single nucleotide change (TG) at nucleotide position 396 has been identified. This transversion predicts an amino acid substitution from isoleucine (ATT) to methionine (ATG) in codon 132, which is located within the putative 5-phosphoribosyl-1-pyrophosphate (PRPP)-binding site of HPRT.     

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.     

Hypoxanthine guanine phosphoribosyltransferase (HPRT) mutations in the Asian population

Mutation of hypoxanthine guanine phosphoribosyltransferase (HPRT) gives rise to Lesch-Nyhan syndrome, which is characterized by hyperuricemia, severe motor disability, and self-injurious behavior, or HPRT-related gout (Kelley-Seegmiller syndrome). The marked heterogeneity of HPRT deficiency is well known, with more than 300 mutations at the HPRT gene locus having been reported (deletions, insertions, duplications, abnormal splicing, and point mutations at different sites of the coding region from exons 1 to 9). We have identified mutations in Asian families with patients manifesting different clinical phenotypes, including rare cases of female subjects, by analyzing all nine exons of the HPRT gene (HPRT1) from genomic DNA and reverse-transcribed mRNA using the polymerase chain reaction technique coupled with direct sequencing. We developed suitable methods to detect the mutations identified from respective families with HPRT deficiency. Then, prenatal genetic diagnoses in HPRT-deficient families were carried out using both mRNA and genomic DNA from chorionic villi or amniotic fluid cells. As shown here in the heterogeneity of HPRT mutations, the spectrum of 70 mutations identified in the Asian population fits the four main conclusions that emerged previously from worldwide analysis.     

Nucleotide sequence analysis of human hypoxanthine phosphoribosyltransferase (HPRT) gene deletions.

We have determined the nucleotide sequences of 10 intragenic human HPRT gene deletion junctions isolated from thioguanine-resistant PSV811 Werner syndrome fibroblasts or from HL60 myeloid leukemia cells. Deletion junctions were located by fine structure blot hybridization mapping and then amplified with flanking oligonucleotide primer pairs for DNA sequence analysis. The junction region sequences from these 10 HPRT mutants contained 13 deletions ranging in size from 57 bp to 19.3 kb. Three DNA inversions of 711, 368, and 20 bp were associated with tandem deletions in two mutants. Each mutant contained the deletion of one or more HPRT exon, thus explaining the thioguanine-resistant cellular phenotype. Deletion junction and donor nucleotide sequence alignments suggest that all of these HPRT gene rearrangements were generated by the nonhomologous recombination of donor DNA duplexes that share little nucleotide sequence identity. This result is surprising, given the potential for homologous recombination between copies of repeated DNA sequences that constitute approximately a third of the human HPRT locus. No difference in deletion structure or complexity was observed between deletions isolated from Werner syndrome or from HL60 mutants. This suggests that the Werner syndrome deletion mutator uses deletion mutagenesis pathway(s) that are similar or identical to those used in other human somatic cells.     

Novel mutations in the human HPRT gene

Inherited mutation of a purine salvage enzyme, hypoxanthine guanine phosphoribosyltransferase (HPRT), gives rise to Lesch-Nyhan Syndrome (LNS) or HPRT-related gout. Here, we report five novel independent mutations in the coding region of the HPRT gene from five unrelated male patients manifesting different clinical phenotypes associated with LNS: exon 2: c.133A > G, p.45R > G; c.35A > C, p.12D > A; c.88delG; exon 7: c.530A > T, p.177D > V; and c.318 + 1G > C: IVS3 + 1G > C splice site mutation.     

Mutations in the hypoxanthine guanine phosphoribosyltransferase gene (HPRT1) in Asian HPRT deficient families

Inherited mutation of hypoxanthine guanine phosphoribosyltransferase, (HPRT) gives rise to Lesch-Nyhan syndrome or HPRT-related gout. We have identified 34 mutations in 28 Japanese, 7 Korean, and 1 Indian families with the patients manifesting different clinical phenotypes, including two rare cases in female subjects, by the analysis of all nine exons of HPRT from the genomic DNA and reverse transcribed mRNA using PCR technique coupled with direct sequencing.     

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.     

Molecular structure and genetic stability of human hypoxanthine phosphoribosyltransferase (HPRT) gene duplications.

We have determined the genetic stability of three independent intragenic human HPRT gene duplications and the structure of each duplication at the nucleotide sequence level. Two of the duplications were isolated as spontaneous mutations from the HL60 human myeloid leukemia cell line, while the third was originally identified in a Lesch-Nyhan patient. All three duplications are genetically unstable and have a reversion rate approximately 100-fold higher than the rate of duplication formation. The molecular structures of these duplications are similar, with direct duplication of HPRT exons 2 and 3 and of 6.8 kb (HL60 duplications) or 13.7 kb (Lesch-Nyhan duplication) of surrounding HPRT sequence. Nucleotide sequence analyses of duplication junctions revealed that the HL60-derived duplications were generated by unequal homologous recombination between clusters of Alu repeats contained in HPRT introns 1 and 3, while the Lesch-Nyhan duplication was generated by the nonhomologous insertion of duplicated HPRT DNA into HPRT intron 1. These results suggest that duplication substrates of different lengths can be generated from the human HPRT exon 2-3 region and can undergo either homologous or nonhomologous recombination with the HPRT locus to form gene duplications.     

Identification of in vivo mutations in exon 5 of the human HPRT gene in a set of pooled T-cell mutants by constant denaturant capillary electrophoresis (CDCE)

Constant denaturant capillary electrophoresis (CDCE), based on co-operative DNA melting equilibria, has the resolving power to separate single nucleotide mutants from wild type sequences. We used this technique to study mutations in a 70-bp isomelting domain of the human HPRT gene, which included the entire exon 5 and its flanking splice donor and acceptor sites. Pooled samples of 6-thioguanine selected T-cell clones from 51 healthy donors representing a total of approximately 1000 individual HPRT mutants were analysed. Slow moving peaks from the heteroduplex part of the CDCE electropherograph were collected and subjected to a second round of PCR and CDCE analysis, followed by DNA sequencing. Five independent mutations were detected. Four were splicing errors; one insertion of CC and two G-->A transitions in the splice donor site of intron 5, and one G-->C transversion in the splice acceptor site of intron 4. The fifth mutation was a missense transversion, T389>G. A reconstruction experiment, in which DNA with known mutation was mixed with wild type DNA, showed the sensitivity of mutation detection to be better than 1:100 under the conditions used in this study. These results demonstrate the high sensitivity of the CDCE-method for mutation screening.     

Molecular analysis of hypoxanthine guanine phosphoribosyltransferase (HPRT) deficiencies: novel mutations and the spectrum of Japanese mutations

Inherited mutation of hypoxanthine guanine phosphoribosyltransferase, (HPRT) gives rise to Lesch-Nyhan syndrome or HPRT-related gout. We have identified a number of HPRT mutations in patients manifesting different clinical phenotypes, by analyzing all nine exons of the HPRT gene (HPRT1) from genomic DNA and reverse transcribed mRNA using the PCR technique coupled with direct sequencing. Recently, we detected two novel mutations: a single nucleotide substitution (430C > T) resulting in a nonsense mutation Q144X, and a deletion of HPRT1 exon 1 expressing no mRNA of HPRT. Furthermore, we summarized the spectrum of 56 Japanese HPRT mutations.     

Identification of two novel mutations in adenine phosphoribosyltransferase gene in patients with 2,8-dihydroxyadenine urolithiasis

Five mutations in the adenine phosphoribosyltransferase (APRT) gene have been described in Japanese patients with APRT deficiency. We investigated the APRT gene from three patients with APRT deficiency and two novel mutations, G133D and V84M, were determined.     

Increased PRPP synthetase activity in cultured rat hepatoma cells containing mutations in the hypoxanthine-guanine phosphoribosyltransferase gene.

Nine independently derived clones of mutagenized rat hepatoma cells selected for resistance to 6-mercaptopurine (6-MP) or 6-thioguanine (6-ThioG) have been isolated. Each has severely reduced catalytic activity of hypoxanthine-guanine phosphoribosyltransferase (HPRT) and seven of them possess significantly increased activities of phosphoribosylpyrophosphate (PRPP) synthetase. The degrees of elevations of PRPP synthetase activities do not correlate with the degrees of deficiencies of HPRT activities. The cells from one of these clones, 1020/12, posses 40% of the normal HPRT catalytic activity and overproduce purines. We have extensively examined the cells from this clone. Immunotration studies of 1020/12 cells indicate that there is a mutation in the structural gene for HPRT. Although they possess increased specific catalytic activities of the enzyme. PRPP synthetase, the catalytic parameters, heat stability, and isoelectric pH of PRPP synthetase from 1020/12 cells are indistinguishable from those of the enzyme from wild-type cells. The cause of purine overproduction by 1020/12 cells appears to be the elevated PRPP synthetase activity, rather than a PRPP "sparing" effect stemming from reduced HPRT activity. Support for this idea is provided by the observation that the complete loss of HPRT activity in a clone derived from 1020/12 cells does not further enhance the levels of PRPP synthetase or purine overproduction. We propose that the elevated levels of PRPP synthetase activity in these HPRT deficient cells result from a mutational event in the structural gene for HPRT, and that this causes the disruption of a previously undescribed regulatory function of this gene on the expression of the PRPP synthetase gene.     

Molecular characterization of two deletion events involving Alu-sequences, one novel base substitution and two tentative hotspot mutations in the hypoxanthine phosphoribosyltransferase (HPRT) gene in five patients with Lesch-Nyhan syndrome

Mutations identified in the hypoxanthine phosphoribosyltransferase (HPRT) gene of patients with Lesch-Nyhan (LN) syndrome are dominated by simple base substitutions. Few hotspot positions have been identified, and only three large genomic rearrangements have been characterized at the molecular level. We have identified one novel mutation, two tentative hot spot mutations, and two deletions by direct sequencing of HPRT cDNA or genomic DNA from fibroblasts or T-lymphocytes from LN patients in five unrelated families. One is a missense mutation caused by a 610C→T transition of the first base of HPRT exon 9. This mutation has not been described previously in an LN patient. A nonsense mutation caused by a 508C→T transition at a CpG site in HPRT exon 7 in the second patient and his younger brother is the fifth mutation of this kind among LN patients. Another tentative hotspot mutation in the third patient, a frame shift caused by a G nucleotide insertion in a monotonous repeat of six Gs in HPRT exon 3, has been reported previously in three other LN patients. The fourth patient had a tandem deletion: a 57-bp deletion in an internally repeated Alu-sequence of intron 1 was separated by 14 bp from a 627-bp deletion that included HPRT exon 2 and was flanked by a 4-bp repeat. This complex mutation is probably caused by a combination of homologous recombination and replication slippage. Another large genomic deletion of 2969 bp in the fifth patient extended from one Alu-sequence in the promoter region to another Alu-sequence of intron 1, deleting the whole of HPRT exon 1. The breakpoints were located within two 39-bp homologous sequences, one of which overlapped with a well-conserved 26-bp Alu-core sequence previously suggested as promoting recombination. These results contribute to the establishment of a molecular spectrum of LN mutations, support previous data indicating possible mutational hotspots, and provide evidence for the involvement of Alu-mediated recombination in HPRT deletion mutagenesis. Received: 21 April 1998 / Accepted: 16 July 1998     

Mutational spectrum of N-hydroxy-N-acetyl-4-aminobiphenyl at exon 3 of the HPRT gene

4-Aminobiphenyl is a human bladder carcinogen present in many environmental sources, including cigarette smoke. It can be metabolized in two steps to the mutagen N-hydroxy-N-acetyl-4-aminobiphenyl (N-OH-AABP). In this study the mutational spectrum of N-OH-AABP-exposed human lymphoblastoid cells (TK6) was determined using HPRT     

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.