The Spectrum of Hypoxanthine-Guanine Phosphoribosyltransferase (HPRT) Deficiency

Introduction Abbreviations used in this article: APRT, adenine phosphoribosyltransferase, ATP, adenosine triphosphate, DNA, deoxyribonucleic acid, GMP, guanosine monophosphate, HPRT, hypoxanthine-guanine phosphoribosyltransferase, IMP, inosine monophosphate, PCR, polymerase chain reaction, PRPP, phosphoribosylpyrophosphate, RNA, ribonucleic acid The enzyme hypoxanthine-guanine phosphoribosyltransferase (E.C.2.4.2.8., HPRT) plays a crucial role in uric acid synthesis and purine metabolism. This enzyme catalyzes the conversion of hypoxanthine and guanine to inosine monophosphate (IMP) and guanosine monophosphate (GMP), respectively, and uses phosphoribosylpyrophosphate (PRPP) as a cosubstrate and as a source of energy (35). The salvage pathway for purine nucleotide synthesis, catalyzed by HPRT, utilizes only 1 mole of ATP (adenosine triphosphate) per mole of product, as compared to 6 moles utilized by “de novo” purine nucleotide synthesis, and thus saves much energy. In 1964, Lesch and Nyhan (19) described 2 brothers with a clinical syndrome characterized by hyperuricemia, hyperuricosuria, and severe neurologic dysfunction including choreoathetosis, mental retardation, and self-injurious behavior. In 1967, Seegmiller et al (34) reported a complete deficiency of the activity of the enzyme HPRT as the cause of the clinical syndrome. Fortunately, not all patients with HPRT deficiency exhibit the dramatic clinical characteristics of Lesch-Nyhan syndrome. Some patients do not suffer neurologic symptoms and show only uric acid overproduction, gout, and nephrolithiasis (17). In other cases, HPRT deficiency is associated with a lesser degree of neurologic impairment that may eventually prevent an independent life but is without the severity of the neurologic symptoms described by Lesch and Nyhan (26). The prevalence of HPRT deficiency in the population is not accurately known, although an estimate of the minimum frequency of the disorder in Canada is 1 in 380,000 births (5). The HPRT gene is located on the X chromosome in the region q26-q27 (11). Naturally occurring mutations in the HPRT gene are heterogeneous, with most being point mutations or small deletions or insertions, dispersed through all the gene (33). About 30% of the patients have a noninherited mutation (de novo mutation). Most female carriers of HPRT deficiency are healthy, but their somatic cells are mosaics in term of HPRT activity because of random inactivation of the X chromosome (23). A spectrum of clinical consequences of HPRT deficiency has been recognized in small series of patients and in extensive reviews (8,10,17,18,26,29), but the complete spectrum of the neurologic disorder has not been described in a single series of patients examined by the same observers. Since 1984, we have diagnosed 22 patients belonging to 18 different Spanish families with HPRT deficiency. We report here clinical, biochemical, enzymatic, and molecular genetic studies on these patients. Patients and Methods Patients Between 1984 and 1999, we studied, at “La Paz” University Hospital, Madrid, Spain, 22 patients with HPRT deficiency belonging to 18 unrelated Spanish families (Figure 1).Fig. 1: Genealogic trees of 18 Spanish families with HPRT deficiency.Diagnostic criteria HPRT deficiency in the probands was diagnosed on the basis of suggestive clinical symptoms and signs; biochemical results indicative of a severe purine overproduction; and markedly reduced HPRT activity in hemolysates with simultaneously increased adenine phosphoribosyltransferase (APRT) activity. Biochemical determinations: Purine metabolism Purine metabolism was examined after all medications known to influence purine metabolism were discontinued for at least 2 weeks. Colchicine (0.5 mg per day) was given as prophylaxis against acute arthritis to patients who had experienced gout. Patients were placed on a weight maintenance, isocaloric, purinerestricted diet (less than 75 mg per day of purines) with a protein content of 10%–15% for 7 days before the studies. Purine metabolism was examined by determining plasma hypoxanthine, xanthine and uric acid concentrations, and the 24-hour urinary excretion rates of these purines (27). Plasma and urinary hypoxanthine and xanthine were determined by high-performance liquid chromatography (21). Uric acid and creatinine in plasma and urine were measured in a multichannel autoanalyzer (Hitachi 704, Hitachi, Tokyo, Japan). Enzymatic determinations Activities of the enzymes HPRT and APRT were determined in charcoal-treated hemolysates by a high-performance liquid chromatography-based method (30). In order to assess for residual HPRT, the percentage of 14C hypoxanthine incorporated into 14C IMP in intact erythrocyte was also determined (40). In brief, erythrocytes obtained from venous blood were incubated for 40 min at 37 °C in 10 mM HEPES, 125 mM NaCl, 2.6 mM KCl, 5.5 mM glucose, 1 mM CaCl2, 50 mM MgCl2, 18 mM NaH2PO4 and 10 μM [14C] hypoxanthine. At the end of the incubation time, radioactive IMP was separated from hypoxanthine by high-performance liquid chromatography, and radioactivity in the IMP peak was measured. HPRT activity was expressed as the percentage of [14C] hypoxanthine transformed into [14C] IMP. Under these assay conditions, normal values for incorporation of [14C] hypoxanthine into [14C] IMP in erythrocytes were 98%–100%. Molecular analysis Total cellular ribonucleic acid (RNA) was isolated from nucleated blood cells by the guanidium isothiocyanate method (Ultraspec RNA, Biotecx Lab, Inc, Houston, TX). Genomic deoxyribonucleic acid (DNA) was isolated from patients’ samples using the commercial method (Puregene, Gentra Systems, Inc, Minneapolis, MN). Total cellular RNA was reverse transcribed using AMV reverse transcriptase (Promega, USA) and oligo d(T) (15 mer) as primer according to established protocols (31). HPRT transcripts were amplified by polymerase chain reaction (PCR) with HT 3′ and HT 5′ primers as previously described by Davidson et al (6). Both strands of patient cDNAs were sequenced using an automated DNA sequencer (ABI Prism, Model 377). The mutations found in cDNA were verified in genomic DNA. When neither RNA nor HPRT cDNA could not be obtained, genomic DNA was amplified by PCR and the PCR products were sequenced using an automated DNA sequencer according to Gibbs et al (12). Seven families (Z2, H, C2, AnD, C, S2, and Z) were analyzed by use of T-lymphocyte mutant frequency determinations, and cDNA and genomic DNA sequencing as described in Hunter et al (15). The cells from the affected male in family Z did not grow in the presence of 6-thioguanine, while those from the other 6 families did. Results Clinical characteristics Table 1 shows the clinical characteristics of the 22 Spanish patients with HPRT deficiency at presentation to “La Paz” University Hospital. Patients with HPRT deficiency were classified into 2 main syndromes: 1) “classic Lesch-Nyhan syndrome” or “complete HPRT deficiency,” characterized by spasticity, choreoathetosis, mental retardation, and self-injurious behavior. Patients classified in this category were indistinguishable from those described by Lesch and Nyhan (19), although some of them did not show self-injurious behavior; and 2) “partial HPRT deficiency,” also named “Kelley-Seegmiller syndrome” or “HPRT variants.” Patients in this category showed either no neurologic symptoms or mild to severe neurologic manifestations, including different degrees of spasticity and mental retardation. Patients were classified as having “partial HPRT deficiency” if they could perform activities that patients with Lesch-Nyhan syndrome could not (see below).TABLE 1: Clinical characteristics of 22 Spanish patients with HPRT deficiencyFifteen patients, aged 5 months to 31 years, were classified as having Lesch-Nyhan syndrome. At presentation 10 patients showed the typical characteristics of classic Lesch-Nyhan syndrome, including self-injurious behavior. These patients were unable to walk and were extremely dependent on others for daily activities and personal care. Five patients (Table 1, Patients 2, 9, 10, 11, and 14) when first seen did not show the full clinical characteristics described by Lesch and Nyhan. One patient (Figure 1, V Family, subject IV-4;Table 1, Patient 2) belonged to a family with 2 additional living patients who showed the characteristics of Lesch-Nyhan syndrome, including self-mutilation. In contrast to his living uncle and younger brother, this patient who has been followed for 15 years and is now 21 years old, has never showed self-injurious behavior. Another patient (Figure 1, H Family, subject IV-1;Table 1, Patient 11) was diagnosed at age 5 months as having Lesch-Nyhan syndrome because he had 2 uncles with the typical syndrome. This patient has been followed for 6 years and up to now he has never shown self-mutilation. Three patients (Table 1, Patients 9, 10, and 14) suffered psychomotor retardation, spasticity, and choreoathetosis, but did not show self-injurious behavior at presentation. One of the 3 patients (Figure 1, PP Family, subject II-2;Table 1, Patient 9) tended to bite his fingers for about a year, but was educated to prevent this behavior. We have followed this patient for 8 years and have never seen self-inflicted injuries. At age 3 years this patient is able to propel his wheelchair despite severe spasticity. The other 2 patients (Table 1, Patients 10 and 14) have been followed for the last 6 years. They both have been educated to prevent impulsive behavior and have never self-injured. These 3 patients, who initially did not manifest the full clinical syndrome described by Lesch and Nyhan, when growing up did manifest some of the clinical characteristics of the syndrome, including marked psychomotor retardation. They are absolutely unable to stand up or walk (even with much help), and are totally dependent for their personal care. Thus, although these 3 patients did not show self-injurious behavior, they were diagnosed with Lesch-Nyhan syndrome. Seven patients were classified as having “partial HPRT deficiency” or “Kelley-Seegmiller syndrome” when first seen at “La Paz” University Hospital. A review of these patients disclosed an extensive spectrum of clinical abnormalities. At 1 end of the spectrum were 2 patients (Figure 1, F Family, subject II-2;Table 1, Patient 16 and Figure 1, Z1 Family, subject III-1;Table 1, Patient 17) with gout and uric acid overproduction, respectively, as the sole manifestations of their HPRT deficiency. At the other end of the spectrum was Patient 21 (Figure 1, S2 Family, subject III-3;Table 1, Patient 21), who was mentally normal but showed severe dystonia and spasticity, which prevent walking. Nevertheless, he was able to stand up and even initiate some steps with much help. In between, there were 4 patients (Table 1, Patients 18, 19, 20, and 22) with a full spectrum of neurologic symptoms, of variable intensity. None of these partial HPRT-deficient patients showed the mental impairment evidenced by Lesch-Nyhan patients. In addition, all were able to perform certain activities related to personal care that could not be carried out by Lesch-Nyhan patients. In some patients, a precise diagnosis with prognostic implications could not be established when first seen at “La Paz” University Hospital. Four patients (Table 1, Patients 10, 14, 17, 22) with no additional affected family members showed marked heterogeneity with respect to their neurologic symptoms, and, based on clinical grounds and HPRT activity in hemolysates, we could not reliably ascertain whether these patients had Lesch-Nyhan syndrome. This uncertainty prompted investigations to determine whether certain markers could be identified that would help to establish a more accurate prognosis with respect to the neurologic involvement associated with HPRT deficiency. Biochemical studies HPRT-deficient patients showed marked purine overproduction. Mean plasma concentrations of urate, hypoxanthine, and xanthine were significantly increased in patients with Lesch-Nyhan syndrome and in patients with the Kelley-Seegmiller syndrome as compared with control subjects (Table 2). The urinary excretion of uric acid, hypoxanthine, and xanthine, was also markedly elevated in HPRT-deficient patients. However, these biochemical variables were similarly elevated in patients with the Lesch-Nyhan syndrome and the partial HPRT-deficient phenotype.TABLE 2: Purine metabolism in HPRT-deficient patientsEnzymatic studies All patients showed nondetectable or decreased HPRT activity in the hemolysate. HPRT activity ranged from 0 to 10% of that obtained in control subjects (Table 3). Neither patients with the Lesch-Nyhan phenotype, except patient 4 in whom the result was obtained from the literature (14), nor most patients with clinically partial phenotype showed detectable hemolysate HPRT activity. Mean APRT activity in hemolysates was significantly increased as compared with control subjects (see Table 3). No significant correlation was found between APRT activity and the clinical phenotype.TABLE 3: Enzyme activities in hemolysates and in intact erythrocytes of HPRT-deficient patientsTo better HPRT deficiency at the enzyme determinations were in intact erythrocytes and HPRT activity expressed as a percentage of 14C hypoxanthine transformed into 14C IMP. A correlation was found between HPRT activity in intact erythrocytes and neurologic involvement (Figure 2). However, the enzyme activity in intact cells showed values in 2 patients who had different 2: HPRT activity in intact erythrocytes expressed as percentage of 14C hypoxanthine transformed into 14C IMP. Patients were classified in 4 according to their neurologic on others for HPRT and molecular (see Table studies No genetic could be obtained from the members of 5 When we the genetic 1 had (Figure 1, A Family, subject 1, Patient and we could not samples from families living in the Spanish (Figure 1, and and (Figure 1, and F The mutations found in Spanish families are in Table Spanish families we found 3 2 insertions, and 8 point mutations a point and 1 from and 1) were identified through Three mutations HPRT and HPRT were previously identified in unrelated families The phenotype of these previously reported patients with partial HPRT deficiency was to the phenotype in the Spanish patients (Table 1, V Family, Patients 1, 2, and Family, Patient and Family, Patient mutations were identified at “La Paz” University 2, HPRT HPRT HPRT HPRT HPRT HPRT 2, and HPRT 1) and have been reported the Spanish 7 mutations in and all of them to classic Lesch-Nyhan patients. No patient as partial HPRT deficiency a mutation that the protein In the 6 point 1 was a Lesch-Nyhan syndrome and 5 to partial HPRT-deficient characterized in Spanish mutation analysis in this Spanish series diagnosis in at were diagnosed as HPRT-deficient carriers and were (see Figure of the mutations were mutations that in the the of both patients was found to be a Three for This was by DNA analysis of the from a These 3 in 2 female and in 1 male One diagnosis was by biochemical analysis and in 1 HPRT-deficient male (Figure 1, V Family, We have to the spectrum of HPRT deficiency by with 22 patients belonging to 18 different Spanish Patients were at the clinical, biochemical, enzymatic, and molecular genetic and the results were related to the clinical The main results of this are Clinical spectrum of HPRT deficiency This shows that HPRT deficiency may be expressed as different to a spectrum of severity Most patients with HPRT deficiency have been reported as single an extensive review of the literature shows that there is marked heterogeneity with different This has to both and with to the of the different HPRT-deficient Lesch-Nyhan Thus, a clinical of an extensive series of patients from a single examined by the same with some for the of HPRT-deficient clinical is that the clinical of HPRT deficiency was determined by the severity of the neurologic uric acid was of a in all patients. All patients classified in this as having Lesch-Nyhan syndrome did show full on others for their personal could not stand up even when and were to a However, was also that the neurologic disorder could be markedly by a of which was of most Thus, of a phenotype as Lesch-Nyhan syndrome, patients showed marked at least with respect to 3 neurologic spasticity, mental retardation, and self-injurious behavior. shown in this mental and self-injurious behavior may be in patients with a marked molecular Figure 1, Family, subject II-2;Table 1, Patient that would a Lesch-Nyhan may be to the associated with the and to the of these patients, of them to for mentally behavior between 2 and 16 years of and in some is associated or by Some patients did not behavior, as in the of the of a Lesch-Nyhan patient with a and 3 uncles with Lesch-Nyhan syndrome Figure a HPRT deficiency or Kelley-Seegmiller syndrome was diagnosed when no neurologic symptoms were but also when neurologic symptoms were mild or even severe and the patients were able to perform activities in Lesch-Nyhan patients. In this Spanish series 1 patient (Table 1, Patient in the Kelley-Seegmiller syndrome could have been diagnosed as an Lesch-Nyhan at age years he has never and is able to on his We that this patient is better classified as having partial HPRT deficiency with severe neurologic that by no the severity of Lesch-Nyhan patients. enzymatic, and molecular studies Purine to be of a in all of HPRT-deficient patients. In this plasma and urinary uric acid, hypoxanthine, and xanthine concentrations did not between patients with Lesch-Nyhan syndrome and the partial HPRT-deficient HPRT activity in partial ranged from 0 to 10% of control A residual HPRT activity was only in patients with the partial phenotype the of Patient HPRT activity was determined However, a nondetectable HPRT activity did not a Lesch-Nyhan syndrome. Patients with nondetectable HPRT activity in the hemolysate show some degree of HPRT activity in intact and a correlation has been reported between the HPRT activity and the neurologic involvement (26). The results of in patients are with this but HPRT activity in the intact cells showed values in at least 2 patients who had different clinical (see Figure 2). studies have that there may be a correlation between the severity of the molecular and the clinical phenotype Thus, of the mutation in a patient may help to the clinical the Spanish families in whom the molecular could be 7 mutations in and all of them to classic Lesch-Nyhan patients. et al all the known HPRT mutations and 75 cases, different mutations reported in these different HPRT mutations most the protein and have been described in patients with the Lesch-Nyhan the other 6 HPRT mutations the Spanish families did not the protein Five of these 6 mutations were isolated from partial HPRT-deficient patients. In the different mutations by et al single for the of with partial partial are related to that the protein could a minimum of activity. the other mutations that result in are most but not by a complete of activity and to Lesch-Nyhan However, because the of the mutation be used to of HPRT enzyme activity in intact cells may additional the Spanish families studied, only 3 mutations have been described previously in unrelated HPRT-deficient families in these 3 previously reported families the clinical phenotype was to that in the Spanish patients. studies was that mutations in the Spanish HPRT-deficient families are and dispersed all the HPRT This to that there is not a mutation in the Spanish HPRT-deficient the most extensive review of HPRT-deficient patients reported and clinical, biochemical, enzymatic, and molecular studies of this series of 22 HPRT-deficient Spanish patients, we that a with 2 syndrome and Kelley-Seegmiller up to now much and is of We thus a of HPRT deficiency based on the neurologic symptoms of the patients and their for personal care. When the clinical symptoms do not of the HPRT activity and the molecular may be This clinical and and the 3 variables enzymatic, and that have been used to HPRT-deficient patients into complete and partial to this patients with HPRT deficiency could be classified into the 4 (Table of the Spanish HPRT-deficient 1, normal with no neurologic symptoms HPRT deficiency in these patients was by and increased uric acid excretion or gout. These patients were totally independent for daily activities and carried on normal In these patients HPRT activity was than in but both in the hemolysate and in intact in these patients did not the protein The cells of 1 patient in this (Figure 1, Z1 Family, subject did not grow in are 2 other patients cells did not grow in presence of 6-thioguanine, and they both have only and These 2 patients and previously reported patients could be in 2, mild neurologic symptoms This patients with mild neurologic symptoms as some degree of spasticity, or mental retardation. These patients were by their neurologic symptoms, although they were independent for most activities and could HPRT activity was in hemolysate by the but a residual HPRT activity could be in intact found in these patients did not the protein 3 patients with neurologic symptoms severe to an independent life Patients in 3 were mentally normal and could and some personal but a The patient reported by and in diagnosed as HPRT deficiency could have been in this HPRT activity was in the hemolysate by and no residual HPRT activity could be in intact found in these patients did not the protein classic Lesch-Nyhan syndrome (15 with the typical characteristics of classic Lesch-Nyhan syndrome, including mental retardation, choreoathetosis, spasticity, to walk or stand and full for daily activities and personal Some of these patients did not show self-injurious behavior. HPRT activity was in the hemolysate by and no residual HPRT activity could be in intact of the 4 the mutations that affected the protein were only these 4 patients, although some Lesch-Nyhan patients evidenced mutations that did not protein A clinical that we with 4 patients (Table 1, Patients 10, 14, 17, and may when HPRT deficiency is diagnosed in an with no previously affected family members and neurologic symptoms these 4 In of these 4 patients, and molecular studies a that prognostic this of HPRT-deficient patients into 4 may be more in of for and prognosis as compared with the established into and HPRT to be The enzyme hypoxanthine-guanine phosphoribosyltransferase catalyzes the of hypoxanthine and guanine to the purine IMP and GMP, HPRT deficiency is an disorder characterized by uric acid and variable neurologic The complete deficiency of HPRT is of Lesch-Nyhan syndrome by choreoathetosis, spasticity, mental retardation, and self-injurious behavior. In some HPRT-deficient patients the enzyme to be and the neurologic symptoms mild to severe This has prompted the of HPRT deficiency in 2 Lesch-Nyhan syndrome and Kelley-Seegmiller syndrome, which has much A spectrum of clinical consequences of HPRT deficiency has been recognized in small series of patients, but the complete spectrum of the neurologic disorder has not been described in a single series of patients examined by the same observers. We analyzed with 22 patients belonging to 18 different families with HPRT deficiency diagnosed at “La Paz” University in the 16 years. The clinical spectrum of these HPRT-deficient Spanish patients was to the different reported in the in some diagnosed as Lesch-Nyhan The clinical, biochemical, enzymatic, and molecular genetic studies on these 22 patients to a of HPRT deficiency. on the neurologic symptoms, for personal HPRT activity in hemolysate and in intact and protein patients were classified into 4 1 normal with no neurologic symptoms, HPRT activity was detectable in hemolysates and in intact and the mutation did not the protein 2 mild neurologic symptoms that did not prevent independent HPRT activity was detectable in intact and the protein was 3 severe neurologic impairment that an independent no residual HPRT and normal protein 4 (15 clinical characteristics of Lesch-Nyhan syndrome may not show no residual HPRT and in most of 8 patients in whom the mutation could be the mutation affected the protein This of HPRT deficiency into 4 may be more in of for and The of this Spanish series to that HPRT deficiency may be by a spectrum of neurologic the severity of the is associated with mutations some degree of residual enzyme and mutation analysis a for and the 16 years, have to the and care of the patients reported was for this are A V A A A A A and The the for patients to “La Paz” University F A We are to the and the for patient to for to and for their help with the molecular and to for and This is to his We from the University of for his and of the