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.     

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.     

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.     

HIV-1 TAT-mediated protein transduction of human HPRT into deficient cells

Lesch–Nyhan disease (LND) is a severe and incurable X-linked genetic syndrome caused by the deficiency of hypoxanthine–guanine phosphoribosyltransferase (HPRT), resulting in severe alterations of central nervous system, hyperuricemia and subsequent impaired renal functions. Therapeutic options consist in supportive care and treatments of complications, but the disease remains largely untreatable. Enzyme replacement of the malfunctioning cytosolic protein might represent a possible therapeutic approach for the LND treatment. Protein transduction domains, such as the TAT peptide derived from HIV TAT protein, have been used to transduce macromolecules into cells in vitro and in vivo. The present study was aimed to the generation of TAT peptide fused to human HPRT for cell transduction in enzyme deficient cells. Here we document the construction, expression and delivery of a functional HPRT enzyme into deficient cells by TAT transduction domain and by liposome mediated protein transfer. With this approach we demonstrate the correction of the enzymatic defect in HPRT deficient cells.     

Reaction of antibody to normal human hypoxanthine phosphoribosyltransferase with products of mutant genes.

Immunoglobulin produced in rabbits against normal human red cell hypoxanthine phosphoribosyl transferase (HPRT, EC 2.4.2.8) was used to study cell lysates of individuals with deficient enzyme activity. The reaction of immunoglobulin with HPRT formed partially active insoluble and fully active soluble complexes. The insoluble complexes were separated from soluble complexes and the free enzyme by centrifugation. The soluble complexes and free enzyme were separated by electrophoresis. Hemolysates from 13 patients with the Lesch-Nyhan syndrome who have virtually total deficiency of HPRT activity and 2 patients with hyperuricemia and 2–5% of normal activity were unable to neutralize immunoglobulin and showed no evidence of cross-reacting material (CRM). In contrast, 2 other partially deficient males with 4.5 and 50% of normal actvity, and a partially deficient heterozygous female with 34% of normal activity, were CRM⁺ in this assay. The amount of CRM present in the cells of these 2 males appeared to be disproportionate to their HPRT activity. The heterozygous female contained about 30% of normal CRM which was consistent with the estimated activity provided by her normal cell population. This indicated that her abnormal cells were CRM^?. Absence of CRM in her abnormal cells was consistent with the observed lack of CRM in hemolysates of her hyperuricemic half-brother. These data indicate the presence of considerable heterogeneity in human mutation at the HPRT locus.     

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.     

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.     

The Diagnosis Of HPRT Deficiency in the 21St Century

We have studied 36 patients with HPRT deficiency, 25 with Lesch-Nyhan syndrome and 11 with partial HPRT deficiency (grades 1 to 3). Patients diagnosed with HPRT deficiency have increased 50% since 2000. The most relevant recent advances have been made in molecular diagnosis. Nevertheless, enzyme determinations are still essential for the diagnosis of HPRT deficiency. Therapy for the neurological manifestations of HPRT deficiency has not advanced. Allopurinol remains the drug of choice to diminish uric acid overproduction, but the optimal allopurinol dose must be established in each patient to prevent xanthine or uric acid urolithiasis, a process aided by sequential determination of urinary oxypurines and uric acid.     

The diagnosis of HPRT deficiency in the 21st century

We have studied 36 patients with HPRT deficiency, 25 with Lesch-Nyhan syndrome and 11 with partial HPRT deficiency (grades 1 to 3). Patients diagnosed with HPRT deficiency have increased 50% since 2000. The most relevant recent advances have been made in molecular diagnosis. Nevertheless, enzyme determinations are still essential for the diagnosis of HPRT deficiency. Therapy for the neurological manifestations of HPRT deficiency has not advanced. Allopurinol remains the drug of choice to diminish uric acid overproduction, but the optimal allopurinol dose must be established in each patient to prevent xanthine or uric acid urolithiasis, a process aided by sequential determination of urinary oxypurines and uric acid.     

Hypoxanthine-guanine phosphoribosyltransferase deficiency: analysis of HPRT mutations by direct sequencing and allele-specific amplification

Summary The Lesch-Nyhan syndrome is a severe X chromosome-linked human disease caused by a virtual absence of hypoxanthine-guanine phosphoribosyltransferase (HPRT) activity. A partial deficiency in the activity of this enzyme can result in gouty arthritis. To determine the genetic basis for reduction or loss of enzyme activity, we have amplified and sequenced the coding region of HPRT cDNA from four patients: one with LeschNyhan syndrome (HPRT_Perth) and three with partial deficiencies of HPRT activity, which have been designated HPRT_Urangan, HPRT_Swan and HPRT_Toowong. In all four patients, the only mutation identified was a single base substitution in exons 2 or 3 of the coding region, which in each case predicts a single amino acid substitution in the translated protein. Each base change was confirmed by allele-specific amplification of the patient's genomic DNA. It is interesting to note that the mutation found for HPRT_Perth is identical to that reported for HPRT_Flint. It appears that the two mutations are de novo events.     

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.     

Hypoxanthine-Guanine Phosphoribosyltransferase Deficiency: Biochemical and Molecular Findings in Six Argentine Patients

Hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency is an inborn error of purine metabolism responsible for Lesch-Nyhan Disease (LND) and its partial phenotypes, HPRT-related hyperuricemia with neurologic dysfunction (HRND) and hyperuricemia alone. We report here the recognition of six Argentine patients, two with LND and four with HRND. All patients presented elevated excretion of uric acid, hypoxanthine, and xanthine and decreased HPRT enzyme activities <1 nmol/h/mg Hb. The molecular analysis demonstrated in the two LND patients a novel inherited transition mutation, c.203T >C (L68P), in one subject and a germline transition mutation, c.209G >A (G70E), in the other. In the HRND patients a novel transversion mutation, c.584 A >C (Y195S), was found in three related patients and an inherited transition mutation, c.143G >A (R48H), in the fourth subject.     

Hypoxanthine-guanine phosphoribosyltransferase deficiency: biochemical and molecular findings in six Argentine patients

Hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency is an inborn error of purine metabolism responsible for Lesch-Nyhan Disease (LND) and its partial phenotypes, HPRT-related hyperuricemia with neurologic dysfunction (HRND) and hyperuricemia alone. We report here the recognition of six Argentine patients, two with LND and four with HRND. All patients presented elevated excretion of uric acid, hypoxanthine, and xanthine and decreased HPRT enzyme activities <1 nmol/h/mg Hb. The molecular analysis demonstrated in the two LND patients a novel inherited transition mutation, c.203T >C (L68P), in one subject and a germline transition mutation, c.209G >A (G70E), in the other. In the HRND patients a novel transversion mutation, c.584 A >C (Y195S), was found in three related patients and an inherited transition mutation, c.143G >A (R48H), in the fourth subject.     

The spectrum of inherited mutations causing HPRT deficiency: 75 new cases and a review of 196 previously reported cases

In humans, mutations in the gene encoding the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) are associated with a spectrum of disease that ranges from hyperuricemia alone to hyperuricemia with profound neurological and behavioral dysfunction. Previous attempts to correlate different types or locations of mutations with different elements of the disease phenotype have been limited by the relatively small numbers of available cases. The current article describes the molecular genetic basis for 75 new cases of HPRT deficiency, reviews 196 previously reported cases, and summarizes four main conclusions that may be derived from the entire database of 271 mutations. First, the mutations associated with human disease appear dispersed throughout the hprt gene, with some sites appearing to represent relative mutational hot spots. Second, genotype-phenotype correlations provide no indication that specific disease features associate with specific mutation locations. Third, cases with less severe clinical manifestations typically have mutations that are predicted to permit some degree of residual enzyme function. Fourth, the nature of the mutation provides only a rough guide for predicting phenotypic severity. Though mutation analysis does not provide precise information for predicting disease severity, it continues to provide a valuable tool for genetic counseling in terms of confirmation of diagnoses, for identifying potential carriers, and for prenatal diagnosis.     

Influence of Age and Strain on Striatal Dopamine Loss in a Genetic Mouse Model of Lesch-Nyhan Disease

Abstract : Lesch-Nyhan disease is a neurogenetic disorder caused by deficiency of the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT). Affected individuals exhibit a characteristic pattern of neurological and behavioral features attributable in part to dysfunction of basal ganglia dopamine systems. In the current studies, striatal dopamine loss was investigated in five different HPRT-deficient strains of mice carrying one of two different HPRT gene mutations. Caudoputamen dopamine concentrations were significantly reduced in all five of the strains, with deficits ranging from 50.7 to 61.1%. Mesolimbic dopamine was significantly reduced in only three of the five strains, with a range of 31.6-38.6%. The reduction of caudoputamen dopamine was age dependent, emerging between 4 and 12 weeks of age. Tyrosine hydroxylase and aromatic amino acid decarboxylase, two enzymes responsible for the synthesis of dopamine, were reduced by 22.4-37.3 and 22.2-43.1%, respectively. These results demonstrate that HPRT deficiency is strongly associated with a loss of basal ganglia dopamine. The magnitude of dopamine loss measurable is dependent on the genetic background of the mouse strain used, the basal ganglia sub-region examined, and the age of the animals at assessment.     

Efficacy and Safety of Allopurinol in Patients with the Lesch-Nyhan Syndrome and Partial Hypoxanthine- Phosphoribosyltransferase Deficiency: a Follow-up Study of 18 Spanish Patients

Allopurinol is used widely for the treatment of purine disorders such as gout, but efficacy and safety of allopurinol has not been analyzed systematically in an extensive series of patients with HPRT deficiency. From 1984 to 2004 we have diagnosed 30 patients with HPRT deficiency. Eighteen patients (12 with Lesch-Nyhan syndrome or complete HPRT deficiency, and 6 with partial HPRT deficiency) were treated with allopurinol (mean dose, 6.44 mg/Kg of weight per day) and followed-up for at least 12 months (mean follow-up 7,6 years per patient). Mean age at diagnosis was 7 years (range, 5 months to 35 years). Treatment with allopurinol was associated to a mean reduction of serum urate concentration of 50%, and was normalized in all patients. Mean urinary uric acid excretion was reduced by 75% from baseline values, and uric acid to creatinine ratio was close or under 1.0 in all patients. In contrast, hypoxanthine and xanthine urinary excretion rates increased by a mean of 6 and 10 times, respectively, compared to baseline levels. These modifications were similar in patients with complete or partial HPRT deficiency. In 2 patients xanthine stones were documented despite allopurinol dose adjustments to prevent markedly increased oxypurine excretion rates. Neurological manifestations did not appear to be influenced by allopurinol therapy. Allopurinol is a very efficacy and fairly safety drug for the treatment of uric acid overproduction in patients with complete and partial HPRT deficiency. Allopurinol was associated with xanthine lithiasis.     

Efficacy and safety of allopurinol in patients with the Lesch-Nyhan syndrome and partial hypoxanthine- phosphoribosyltransferase deficiency: a follow-up study of 18 Spanish patients

Allopurinol is used widely for the treatment of purine disorders such as gout, but efficacy and safety of allopurinol has not been analyzed systematically in an extensive series of patients with HPRT deficiency. From 1984 to 2004 we have diagnosed 30 patients with HPRT deficiency. Eighteen patients (12 with Lesch-Nyhan syndrome or complete HPRT deficiency, and 6 with partial HPRT deficiency) were treated with allopurinol (mean dose, 6.44 mg/Kg of weight per day) and followed-up for at least 12 months (mean follow-up 7,6 years per patient). Mean age at diagnosis was 7 years (range, 5 months to 35 years). Treatment with allopurinol was associated to a mean reduction of serum urate concentration of 50%, and was normalized in all patients. Mean urinary uric acid excretion was reduced by 75% from baseline values, and uric acid to creatinine ratio was close or under 1.0 in all patients. In contrast, hypoxanthine and xanthine urinary excretion rates increased by a mean of 6 and 10 times, respectively, compared to baseline levels. These modifications were similar in patients with complete or partial HPRT deficiency. In 2 patients xanthine stones were documented despite allopurinol dose adjustments to prevent markedly increased oxypurine excretion rates. Neurological manifestations did not appear to be influenced by allopurinol therapy. Allopurinol is a very efficacy and fairly safety drug for the treatment of uric acid overproduction in patients with complete and partial HPRT deficiency. Allopurinol was associated with xanthine lithiasis.     

Molecular analysis of mutations in a patient with purine nucleoside phosphorylase deficiency.

Purine nucleoside phosphorylase (PNP) deficiency is an inherited autosomal recessive disorder resulting in severe combined immunodeficiency. The purpose of this study was to determine the molecular defects responsible for PNP deficiency in one such patient. The patient's PNP cDNA was amplified by PCR and sequenced. Point mutations leading to amino acid substitutions were found in both alleles. One point mutation led to a Ser-to-Gly substitution at amino acid 51 and was common to both alleles. In addition, an Asp-to-Gly substitution at amino acid 128 and an Arg-to-Pro substitution at amino acid 234 were found in the maternal and paternal alleles, respectively. In order to prove that these mutations were responsible for the disease state, each of the three mutations was constructed separately by site-directed mutagenesis of the normal PNP cDNA, and each was transiently expressed in COS cells. Lysates from cells transfected with the allele carrying the substitution at amino acid 51 retained both function and immunoreactivity. Lysates from cells transfected with PNP alleles carrying a substitution at either amino acid 128 or amino acid 234 contained immunoreactive material but had no detectable human PNP activity. In summary, molecular analysis of this patient identified point mutations within the PNP gene which are responsible for the enzyme deficiency.     

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.     

Real-time PCR and linkage studies to identify carriers presenting HPRT deleted gene

Lesch-Nyhan syndrome (LNS) is an X-linked genetic disorder resulting in hyperuricemia, choreoathetosis, mental retardation, and self-injurious behavior. It is caused by loss of activity of the ubiquitous enzyme hypoxanthine-guanine-phosphoribosyltransferase (HPRT). The biochemical analysis of residual HPRT activity in patients' red blood cells is the first step in LNS diagnosis, and it precedes molecular study to discover the specific mutation. Unfortunately, biochemical diagnosis of healthy carriers is difficult because HPRT enzymatic activity in blood cells is similar in LNS carriers and in healthy people; genetic tests can help reveal mutations at the genomic or cDNA level, whereas gross deletions involving the first or last exons of HPRT gene are not detectable. Until now, a test based on 6-thioguanine-resistant phenotype of HPRT mutant cells from LNS patients is the only method accepted for the diagnosis of any kind of mutation in carriers. In this work, we introduce a new approach to identify carriers of large deletions in HPRT gene using real-time PCR. Results were validated in a blinded manner with a linkage study and with results obtained in Italian families previously analyzed with selective medium test. Real-time PCR analysis clearly confirmed the results obtained by selective medium; linkage data strengthened real time results, allowing us to follow the allele with the mutated HPRT through the family pedigree. We hope that the real-time PCR approach will provide a useful and reliable method to diagnose LNS carriers of large deletions in HPRT gene.