Hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency: Identification of point mutations in Japanese patients with Lesch-Nyhan syndrome and hereditary gout and their permanent expression in an HPRT-deficient mouse cell line

Two different single nucleotide transitions of hypoxanthine-guanine phosphoribosyltransferase (HPRT) were identified in a Japanese patient with Lesch-Nyhan syndrome (LNS) and a patient with hereditary gout. HPRT enzyme activities in the two patients were severely deficient, but the size and amount of mRNA were normal according to Northern analysis. Entire coding regions of HPRT cDNAs were amplified by PCR and sequenced. A G-to-A substitution at base 208 in exon 3, which predicted glycine 70 to arginine, was detected in the LNS patient (identical mutation with HPRT_Utrecht). A C-to-A substitution at base 73 in exon 2, which predicted proline 25 to threonine, was detected in the gout patient (designated HPRT_Yonago). We transfected normal HPRT cDNA, mutant cDNA with HRPT_Utrecht or mutant cDNA with HPRT_Yonago, respectively, to HPRT-deficient mouse cells and isolated permanent expression cell lines. The HPRT-deficient mouse cells had no detectable HPRT activity and a very low amount of HPRT mRNA. When the HPRT-deficient mouse cells were transfected with normal human cDNA, HPRT enzyme activity increased to 21.8% that of normal mouse cells. The mouse cells transfected with HPRT_Utrecht showed no increase in HPRT activity; however, when the mouse cells were transfected with HPRT_Yonago, the activity increased to 2.4% that of normal activity. The proliferative phenotypes of these cells in HAT medium and in medium containing 6-thioguanine were similar to those of skin fibroblasts from the patients. This series of studies confirmed that each of the two point mutations was responsible for the decreases in HPRT enzyme activity, and the proliferative phenotypes in HAT medium and medium containing 6-thioguanine.     

Hypoxanthine guanine phosphoribosyltransferase deficiency: nucleotide substitution causing Lesch-Nyhan syndrome identified for the first time among Japanese

Summary A previously undescribed nucleotide substitution at codon 51 (CGA to TGA) has been identified using the polymerase chain reaction technique in hypoxanthine guanine phosphoribosyltransferase (HPRT) cDNA; this is the first molecular evidence for a point mutation in a Japanese patient with Lesch-Nyhan syndrome. The present mutation is the 19th nucleotide substitution identified as a germ-line mutation at this locus and the second mutation generating a stop codon. The position of the nucelotide substitution is exactly the same as a previously described mutation HPRT_Toronto, indicating for the first time that nucleotide substitutions at the same position in the sequence of HPRT can generate different mutant alleles, one causing a partial deficiency and the other a complete deficiency. Although the type of nucleotide substitution is different between the two cases, a single base position has twice become the target of a mutation. However, the calculation of the probability of finding substitution mutations at the same base position in the coding region of hprt indicates that there is no evidence for the presence of a hot spot for substitution mutations in the human hprt germ line.     

HPRTSardinia: a new point mutation causing HPRT deficiency without Lesch–Nyhan disease

Hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency always causing hyperuricemia presents various degrees of neurological manifestations, the most severe which is Lesch–Nyhan syndrome. The HPRT gene is situated in the region Xq26-q27.2 and consists of 9 exons. At least 300 different mutations at different sites in the HPRT coding region from exon 1 to exon 9 have been identified. A new mutation in the HPRT gene has been determined in one patient with complete deficiency of erythrocyte activity, with hyperuricemia and gout but without Lesch–Nyhan disease. Analysis of cultured fibroblasts revealed minimal residual HPRT activity mainly when guanine was the substrate. Genomic DNA sequencing demonstrated patient's mother heterozygosity for the mutation and no mutation in her brother. The mutation consists in a C→T transversion at cDNA base 463 (C463T) in exon 6, resulting in proline to serine substitution at codon 155 (P155S). This mutation had not been reported previously and has been designated HPRT_Sardinia. The mutation identified in this patient allows some expression of functional enzyme in nucleated cells such as fibroblasts, indicating that such cell type may add further information to conventional blood analysis. A multicentre survey gathering patients with variant neurological forms could contribute to understand the pathophysiology of the neurobehavioral symptoms of HPRT deficiency.     

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.     

A 13 base pair deletion in exon 1 of HPRTIllinois forms a functional GUG initiation codon

More than 50 mutations in the human hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus have been described, yet only 2 alter the AUG initiation codon. One, variant HPRT₁₁₅₁, results in Lesch-Nyhan syndrome (LNS), and the other, HPRT_Illinois, results in partial HPRT deficiency. Although previously undetectable, we used a sensitive gel assay to demonstrate that HPRT_Illinois is not only active, but has a native Mr indistinguishable from normal. Confirmatory evidence of activity and native Mr is demonstrated following transfection of HPRT cells with expression plasmids containing cDNA sequences representing HPRT_Illinois. These data provide support for the hypothesis that patient RT, or variant HPRT_Illinois, is spared manifestations of the LNS as a result of translation at the newly formed GUG initiation codon.     

HPRTSardinia: a new point mutation causing HPRT deficiency without Lesch-Nyhan disease

Hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency always causing hyperuricemia presents various degrees of neurological manifestations, the most severe which is Lesch-Nyhan syndrome. The HPRT gene is situated in the region Xq26-q27.2 and consists of 9 exons. At least 300 different mutations at different sites in the HPRT coding region from exon 1 to exon 9 have been identified. A new mutation in the HPRT gene has been determined in one patient with complete deficiency of erythrocyte activity, with hyperuricemia and gout but without Lesch-Nyhan disease. Analysis of cultured fibroblasts revealed minimal residual HPRT activity mainly when guanine was the substrate. Genomic DNA sequencing demonstrated patient's mother heterozygosity for the mutation and no mutation in her brother. The mutation consists in a C-->T transversion at cDNA base 463 (C463T) in exon 6, resulting in proline to serine substitution at codon 155 (P155S). This mutation had not been reported previously and has been designated HPRT(Sardinia). The mutation identified in this patient allows some expression of functional enzyme in nucleated cells such as fibroblasts, indicating that such cell type may add further information to conventional blood analysis. A multicentre survey gathering patients with variant neurological forms could contribute to understand the pathophysiology of the neurobehavioral symptoms of HPRT deficiency.     

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 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.     

Clinical and biochemical manifestations and molecular characterization of the mutation HPRT Jerusalem

A novel point mutation (I137T) was identified in the hypoxanthine-guanine phosphoribosyltransferase (HPRT) encoding gene, in a patient with partial deficiency of the enzyme. The mutation, ATT to ACT (substitution of isoleucine to threonine), occurred at codon 137, which is within the region encoding the binding site for 5-phosphoribosyl-1-pyrophosphate (PRPP). The mutation caused decreased affinity for PRPP, manifested clinically as a Lesch-Nyhan variant (excessive purine production and delayed acquisition of language skills). The partial HPRT deficiency could be detected only by measuring HPRT activity in intact fibroblasts (uptake of hypoxanthine into nucleotides).     

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.     

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.     

Clinical and Biochemical Manifestations and Molecular Characterization of the Mutation HPRT Jerusalem

A novel point mutation (I137T) was identified in the hypoxanthine‐guanine phosphoribosyltransferase (HPRT) encoding gene, in a patient with partial deficiency of the enzyme. The mutation, ATT to ACT (substitution of isoleucine to threonine), occurred at codon 137, which is within the region encoding the binding site for 5‐phosphoribosyl‐1‐pyrophosphate (PRPP). The mutation caused decreased affinity for PRPP, manifested clinically as a Lesch–Nyhan variant (excessive purine production and delayed acquisition of language skills). The partial HPRT deficiency could be detected only by measuring HPRT activity in intact fibroblasts (uptake of hypoxanthine into nucleotides).     

A germ line mutation within the coding sequence for the putative 5-phosphoribosyl-1-pyrophosphate binding site of hypoxanthine-guanine phosphoribosyltransferase (HPRT) in a Lesch-Nyhan patient: missense mutations within a functionally important region probably cause disease

Lesch-Nyhan syndrome caused by a complete deficiency of hypoxanthine guanine phosphoribosyltransferase (HPRT) is the result of a heterogeneous group of germ line mutations. Identification of each mutant gene provides valuable information as to the type of mutation that occurs spontaneously. We report here a newly identified HPRT mutation in a Japanese patient with Lesch-Nyhan syndrome. This gene, designated HPRT Tokyo, had a single nucleotide change from G to A, as identified by sequencing cDNA amplified by the polymerase chain reaction. Allele specific oligonucleotide hybridization analysis using amplified genomic DNA showed that the mutant gene was transmitted from the maternal germ line. This mutation would lead to an amino acid substitution of Asp for Gly at the amino acid position 140 located within the putative 5-phosphoribosyl-1-pyrophosphate (PRPP) binding region. Missense mutations in human HPRT deficient patients thus far reported tend to accumulate in this functionally active region. However, a comparison of the data suggested that both missense and synonymous mutations can occur at any coding sequence of the human germ line HPRT gene, but that a limited percentage of all the missense mutations cause disease. The probability that a mutation will cause disease tends to be higher when the missense mutation is within a functionally important sequence.     

Novel mutation in the human HPRT1 gene and the Lesch-Nyhan disease

Lesch-Nyhan disease (LND) is a rare X-linked inherited neurogenetic disorder of purine metabolism in which the enzyme, hypoxanthine-guanine phosphoribosyltransferase (HGprt) is defective. The authors report a novel point mutation that led to HGprt-related neurological dysfunction (HND) in a family in which there was a missense mutation in exon 6 of the coding region of the HPRT1 gene: g.34938G>T, c.403G>T, p.D135Y. Molecular diagnosis is consistent with the genetic heterogeneity of the HPRT1 gene responsible for HGprt deficiency. It allows fast, accurate carrier detection and genetic counseling.     

Mutation in the Human HPRT1 Gene and the Lesch-Nyhan Syndrome

Lesch-Nyhan syndrome (LNS) is a rare X-linked inherited neurogenetic disorder of purine metabolism in which the enzyme, hypoxanthine-guanine phosphoribosyltransferase (HGprt) is defective. The authors report a novel mutation which led to HGprt-related neurological dysfunction (HND) in two brothers from the same family with a missense mutation in exon 6 of the coding region of the HPRT1 gene: c.437T>C, p.L146S. Molecular diagnosis discloses the genetic heterogeneity of the HPRT1 gene responsible for HGprt deficiency. It allows fast, accurate carrier detection and genetic counseling.     

An asymptomatic germline missense base substitution in the hypoxanthine phosphoribosyltransferase (HPRT) gene that reduces the amount of enzyme in humans

A 40-year-old normouricemic (5.5 mg/dl) male showed 46% hemolysate and 37% lymphoblast hypoxanthine phosphoribosyltransferase (HPRT) activities but was otherwise completely free of symptoms. His genomic DNA and cDNA had a missense base substitution (CAT-to-CGT in codon 60) leading to the amino-acid substitution His-to-Arg. Western blot analysis revealed that the amount of HPRT protein in lymphoblasts from this individual was 25%–50% of normal cells, suggesting that the decrease in the amount of enzyme protein was responsible for the partial deficiency. This provides the first clear evidence that a genomic missense mutation at the HPRT locus leads to a decrease in the amount of the enzyme protein but that otherwise it has no evident adverse effects in the hemizygote (asymptomatic mutation). Received: 15 May 1996 / Revised: 22 August 1996     

Unusual Presentation of Kelley-Seegmiller Syndrome

Female carriers of hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency have somatic cell mosaicism of HPRT activity and are healthy. We report a 50-year-old woman without gout or nephrolithiasis. She was never on allopurinol. Normal serum uric acid concentrations, increased plasma hypoxanthine, and xanthine were found. HPRT activity in erythrocytes was surprisingly low: at 8.6 nmol h^?1 mg ^?1 haemoglobin. Mutation analysis revealed a heterozygous HPRT gene mutation, c.215A > G (p.Tyr72Cys). Assessment of X-inactivation ratio has shown that > 75% of the active X-chromosome bears the mutant allele and could explain these unusual, previously undescribed findings.     

Developmental expression of murine HPRT. I. Activities,heat stabilities,and electrophoretic mobilities in adult tissues

Total and specific activity of the enzyme hypoxanthine phosphoribosyltransferase (HPRT) varied widely among six tissues from C3H/f mice; the highest levels of activity were in brain. More striking were thermostability differences in tissue enzymes. Although brain, spleen, and kidney HPRT retained 65% basal activity after 15 min at 85 C, heart, liver, and erythrocyte HPRT retained only 20–30% initial activity. Kidney HPRT behaved as monospecific heat-stable enzyme (K _denaturation=0.022/min, and liver enzyme behaved as monospecific heat-labile enzyme (K _denaturation=0.061/min), while other tissues appeared to contain both forms of the enzyme. Multiple electrophoretic activity bands were present in all tissues; no activity band was restricted to a single tissue. The data presented here are consistent with the hypothesis that the distinct tissue properties of HPRT result from posttranslational modification of the product of a single genetic locus which is expressed in all tissues.This study was supported in part by NIH Grant AM 16722 and by an Institutional Biomedical Grant.     

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.     

Inactivation of the MouseHPRTLocus by a 203-bp Retroposon Insertion and a 55-kb Gene-Targeted Deletion: Establishment of New HPRT-Deficient Mouse Embryonic Stem Cell Lines

To obtain useful hypoxanthine phosphoribosyltransferase (HPRT)-deficient mouse ES cell lines, two different methods were employed: (i) selection of spontaneous 6-TG-resistant mutants and (ii) gene targeting of theHPRTlocus. The first approach resulted in the establishment of E14.1TG3B1, a spontaneous HPRT-deficient cell line with an insertional mutation of 203 bp in the third exon of theHPRTgene. The insert is highly homologous to the B2 mouse repetitive element and has all the expected retroposon characteristics, thus providing an example of gene inactivation by retroposon insertion. This clone exhibited stable 6-TG resistance and high germ-line transmission frequency. Thus E14.1TG3B1 is a useful ES cell line for modifying the mouse genome using theHPRTgene as a selection marker and for transmission at a high frequency into the mouse germ line. The second approach resulted in a 55-kb deletion of the mouseHPRTlocus, demonstrating the feasibility of replacement-targeting vectors to generate large genomic DNA deletions.