4-Pyridone-3-carboxamide-1-ß-D-ribonucleoside triphosphate (4PyTP), a novel NAD metabolite accumulating in erythrocytes of uremic children: a biomarker for a toxic NAD analogue in other tissues?

We have identified a novel nucleotide, 4-pyridone 3/5-carboxamide ribonucleoside triphosphate (4PyTP), which accumulates in human erythrocytes during renal failure. Using plasma and erythrocyte extracts obtained from children with chronic renal failure we show that the concentration of 4PyTP is increased, as well as other soluble NAD(+) metabolites (nicotinamide, N(1)-methylnicotinamide and 4Py-riboside) and the major nicotinamide metabolite N(1)-methyl-2-pyridone-5-carboxamide (2PY), with increasing degrees of renal failure. We noted that 2PY concentration was highest in the plasma of haemodialysis patients, while 4PyTP was highest in erythrocytes of children undergoing peritoneal dialysis: its concentration correlated closely with 4Py-riboside, an authentic precursor of 4PyTP, in the plasma. In the dialysis patients, GTP concentration was elevated: similar accumulation was noted previously, as a paradoxical effect in erythrocytes during treatment with immunosuppressants such as ribavirin and mycophenolate mofetil, which deplete GTP through inhibition of IMP dehydrogenase in nucleated cells such as lymphocytes. We predict that 4Py-riboside and 4Py-nucleotides bind to this enzyme and alter its activity. The enzymes that regenerate NAD(+) from nicotinamide riboside also convert the drugs tiazofurin and benzamide riboside into NAD(+) analogues that inhibit IMP dehydrogenase more effectively than the related ribosides: we therefore propose that the accumulation of 4PyTP in erythrocytes during renal failure is a marker for the accumulation of a related toxic NAD(+) analogue that inhibits IMP dehydrogenase in other cells.

A population study of Lesch-Nyhan disease in the UK.

AIM: The aims of this study were to identify all people with Lesch-Nyhan disease (LND) born in the UK between 1988 and 2008, and to obtain a clinical profile including age at diagnosis, genetic background, family history, neurological signs, and medications. METHOD: Potential participants were contacted through the British Paediatric Neurology Surveillance Unit. Questionnaires were sent to each child's paediatric neurologist or primary consultant. Two purine laboratories provided metabolic information. RESULTS: Twenty-three live males with LND in the 0- to 20-year age band and eight live males over the age of 20 years were identified. Thirty-one live people with LND were identified in the UK in 2008, giving a prevalence of 1 in 2 million people. Over the 20 years of study, there was a mean incidence rate of 0.18 per 100 000 live births, range 0 to 0.5. INTERPRETATION: To our knowledge, this study is the first to provide details of the prevalence and incidence of LND in the UK. The data highlight that clinical profiles, at the time of diagnosis, and management of the disease are variable. There is the need for ongoing monitoring of allopurinol dosage and metabolic screening.

Synthesis of genomic and subgenomic RNA in mosquito cells infected with two Sindbis virus nsP4 mutants: influence of intracellular nucleoside triphosphate concentrations.

Cells infected with Sindbis virus (SV) make two positive-strand RNAs, a genomic-length RNA (G) RNA and a subgenomic (SG) RNA. In cells infected with SVstd, and in general in cells infected with wt alphaviruses, more SG RNA is made than G RNA. How the balance between synthesis of G RNA and SG RNA is regulated is not known. SVpzf and SVcpc are nsP4 mutants of SV which, in mosquito cells, make more G RNA than SG RNA. When low concentrations of pyrazofurin (inhibits the synthesis of UTP and CTP) were added to SVpzf-infected cells, the yield of virus was increased, and the ratio of SG/G RNA was changed from <1 to >1. These effects were reversed by uridine. In SVcpc-infected cells, but not in SVstd-infected cells, synthesis of viral RNA was inhibited by the addition of either uridine or cytidine, and viral yields were lowered. Our findings suggest that the activities of the viral RNA-synthesizing complexes in cells infected with SVpzf or SVcpc, in contrast to those in SVstd-infected cells, are sensitive to high concentrations of UTP or CTP. Using a cell-free system that synthesizes both SG and G RNA, we measured viral RNA synthesis as a function of the UTP/CTP concentrations. The results indicated that the presence of the SVpzf mutations in nsP4 and the SG promoter produced a pattern quite different from that seen with the SVstd nsP4 and SG promoter. As the UTP/CTP concentrations were increased, the SVpzf system, in contrast to the SVstd system, made more G RNA than SG RNA, reflecting the situation in cells infected with SVpzf.

A novel nucleotide found in human erythrocytes, 4-pyridone-3-carboxamide-1-beta-D-ribonucleoside triphosphate.

We report the identification of a hitherto unknown nucleotide that is present in micromolar concentrations in the erythrocytes of healthy subjects and accumulates at levels comparable with the ATP concentration in erythrocytes of patients with chronic renal failure. The unknown nucleotide was isolated and identified by liquid chromatography with UV and tandem mass detection, (1)H nuclear magnetic resonance and infrared spectroscopy as 4-pyridone-3-carboxamide-1-beta-D-ribonucleoside triphosphate (4PYTP), a structure indicating association with metabolism of the oxidized nicotinamide compounds. Subsequently, we demonstrated formation of 4PYTP in intact human erythrocytes during incubation with the chemically synthesized nucleoside precursor 4-pyridone-3-carboxamide-1-beta-D-ribonucleoside (4PYR). We noted preferential accumulation of monophosphate of 4PYR (4PYMP) over 4PYTP as well as a decrease in erythrocyte ATP concentration during incubation with 4PYR. Both the 4PYR phosphorylation and ATP depletion were blocked by an inhibitor of adenosine kinase. Plasma concentration of 4PYR was detectable but very low (0.013 +/- 0.006 microm) in contrast with the high daily urine excretion of this compound (26.7 +/- 18.2 micromol/24 h) in healthy subjects, indicating much greater renal clearance than other nicotinamide metabolites, nucleosides, or creatinine. We also noted a 40-fold increase in 4PYR plasma concentration in patients with chronic renal failure (0.563 +/- 0.321 microm). We suggest that 4PYTP formation in the erythrocytes is a hitherto unknown process aimed at sequestering potentially toxic 4PYR in a form that could be safely transported and subsequently released and excreted during passage of erythrocytes through the kidney.

An unusual patient with kidney stones composed of 1-methyluric acid.

An unusual case with kidney stones composed mainly of 1-methyluric acid is described. The patient, a Caucasian male of Celtic descent, reportedly drank at least eight cups of coffee per day and had a long history of rheumatoid arthritis, gouty attacks and renal colics--the latter attributed to nephrocalcinosis and analgesic nephropathy. He was treated with allopurinol. At 54 years, a bilateral nephrolithotomy was performed. Stone samples were analysed by thermogravimetry and infrared spectroscopy and reported to be 12-25% calcium oxalate, the remainder being organic uric acid-like material. Analysis of the extracts by HPLC confirmed that the organic material contained 67% of 1-methyluric acid and 33% of uric acid. Possible mechanisms leading to the precipitation of 1-methyluric acid from urine are discussed. We conclude that the high caffeine intake resulted in extremely elevated urinary concentrations of 1-methyluric acid favouring the formation of 1-methyluric acid stones.

Aprt/Opn double knockout mice: osteopontin is a modifier of kidney stone disease severity.

BACKGROUND: Osteopontin (OPN) is reported to have two distinct functions in kidney disease: Promotion of inflammation at sites of tissue injury, and inhibition of calcium oxalate monohydrate stone formation. However, many of the studies supporting these functions were carried out in animal models of acute renal injury or in cultured cells; thus, the role of OPN in chronic renal disease is not well defined. We examined the role of OPN in adenine phosphoribosyltransferase (Aprt) knockout mice, in which inflammation and formation of 2,8-dihydroxyadenine (DHA) kidney stones are prominent features, by generating Aprt/Opn double knockout mice. METHODS: We characterized the phenotypes of six- and 12-week-old Aprt-/- Opn-/-, Aprt-/- Opn+/+, Aprt+/+ Opn-/-, and Aprt+/+ Opn+/+ male and female mice using biochemical, histologic, immunohistochemical, and in situ hybridization techniques. RESULTS: At 6 weeks of age, there was no difference in phenotype between double knockout and Aprt knockout mice. At 12 weeks, there was increased adenine and DHA excretion, renal crystal deposition, and inflammation in double knockout versus Aprt knockout male mice. Double knockout and Aprt knockout female mice at 12 weeks had less pathology than their male counterparts, but kidneys from double knockout females showed more inflammation compared with Aprt knockout females; both genotypes had similar levels of DHA crystal deposition. CONCLUSION: We conclude that (1) OPN is a major inhibitor of DHA crystal deposition and inflammation in male mice; and (2) OPN is a major modifier of the inflammatory response but not of crystal deposition in female mice. Thus, separate mechanisms appear responsible for the tissue changes seen in DKO males versus females.

Pyrimidine pathways in health and disease.

Genetic defects involving enzymes essential for pyrimidine nucleotide metabolism have provided new insights into the vital physiological functions of these molecules in addition to nucleic acid synthesis. Such aberrations disrupt the haematological, nervous or mitochondrial systems and can cause adverse reactions to analogue therapy. Regulation of pyrimidine pathways is also known to be disrupted in malignancies. Nine genetic defects have now been identified but only one is currently treatable. Diagnosis is aided by the accumulation of specific metabolites. Recently, progress has been made in understanding the molecular mechanisms underlying inborn errors of pyrimidine metabolism, together with the key clinical issues and the implications for the future development of novel drugs and therapeutic strategies.

Hereditary hyperuricemia and renal disease.

Hyperuricemia and gout have long been known to run in families. As well as an apparently multifactorial genetic component to classic gout itself, 2 rather unusual sex-linked single-gene disorders of purine biosynthesis or recycling have been defined: deficiency of the enzyme hypoxanthine-guaninephosphoribosyl transferase (HPRT), and overactivity of PPriboseP synthase. Both result in overproduction of urate, hyperuricemia, and secondary overexcretion that may lead to acute or chronic renal damage. Familial juvenile hyperuricemic nephropathy (FJHN) and autosomal-dominant medullary cystic kidney disease (ADMCKD) are more common but less well-defined hyperuricemic conditions resulting from a decrease in the fractional excretion of filtered urate, with normal urate production. Although having features in common, ADMCKD is distinguished in particular by the presence of medullary cysts. One major group of both disorders is associated with mutations in the gene for uromodulin, but this accounts for only about one third of cases, and genetic heterogeneity is present. Whether the genes involved in these latter disorders contribute to the polygenic hyperuricemia and urate underexcretion of classic gout remains unexplored.

A mutant of Sindbis virus which is able to replicate in cells with reduced CTP makes a replicase/transcriptase with a decreased Km for CTP.

We reported earlier the isolation and characterization of a Sindbis virus mutant, SV(PZF), that can grow in mosquito cells treated with pyrazofurin (PZF), a compound that interferes with pyrimidine biosynthesis (Y. H. Lin, P. Yadav, R. Ravatn, and V. Stollar, Virology 272:61-71, 2000; Y. H. Lin, H. A. Simmonds, and V. Stollar, Virology 292:78-86, 2002). Three amino acid changes in nsP4, the viral RNA polymerase, were required to produce this phenotype. We now describe a mutant of Sindbis virus, SVCPC, that is resistant to cyclopentenylcytosine (CPC), a compound that interferes only with the synthesis of CTP. Thus, in contrast to SVPZF, which was selected for its ability to grow in mosquito cells with low levels of UTP and CTP, SVCPC was selected for its ability to grow in cells in which only the level of CTP was reduced. Although SV(PZF) was cross-resistant to CPC, SVCPC was not resistant to PZF. Only one amino acid change in nsP4, Leu 585 to Phe, was required for the CPC resistance phenotype. The viral replicase/transcriptase generated in SVCPC-infected mosquito cells had a lower Km for CTP (but not for UTP) than did the enzyme made in SVSTD-infected mosquito cells. SV(PZF) and SVCPC represent the first examples of viral mutants selected for the ability to grow in cells with low levels of ribonucleoside triphosphates (rNTPs). Further study of these mutants and determination of the structure of nsP4 should demonstrate how alterations in an RNA-dependent RNA polymerase permit it to function in cells with abnormally low levels of rNTPs.

Mycophenolate mofetil treatment following renal transplantation decreases GTP concentrations in mononuclear leucocytes.

MMF (mycophenolate mofetil) has been proven to provide an effective immunosuppression by non-competitive selective reversible inhibition of IMPDH (inosine monophosphate dehydrogenase), the enzyme playing a crucial role in GTP biosynthesis. However, the exact metabolic changes induced by inhibition of IMPDH in target cells of the immune system have been the subject of recent debate. The aim of the present study was to evaluate whether MMF treatment produced sustained changes in the guanosine nucleotide pool of MNLs (mononuclear leucocytes) in vivo. Sixty-two renal failure patients were divided into three groups: chronic renal failure patients undergoing haemodialysis (CRF-HD; n=20) and two groups of patients after renal transplantation, the first on AZA (azathioprine; TN-AZA; n=23) and the second treated with MMF (TN-MMF; n=19). In addition, MNLs from 25 healthy subjects were analysed as controls. Anion-exchange HPLC was used to quantify purine and pyrimidine nucleotides in MNLs. We report a significant decrease in GTP and the total MNL guanine nucleotide pool in the TN-MMF group (P<0.05) compared with control, CRF-HD and TN-AZA groups, although no significant differences were found between any of the other groups. Adenine nucleotide concentrations in MNLs were decreased in the TN-AZA group, but not in the TN-MMF group compared with the CRF-HD group and controls. There were no differences in CTP concentrations, but UTP concentrations were decreased in the CRF-HD, TN-AZA and TN-MMF groups compared with controls. MMF caused a significant and sustained decrease in the guanine nucleotide pool in MNLs from renal transplant recipients. This decrease contrasts with the elevation in GTP reported in erythrocytes of MMF-treated patients.

Mycophenolate mofetil, an inhibitor of inosine monophosphate dehydrogenase, causes a paradoxical elevation of GTP in erythrocytes of renal transplant patients.

The immunosuppressant MMF (mycophenolate mofetil) has increasingly replaced AZA (azathioprine) in renal transplantation. MMF is a prodrug of MPA (mycophenolic acid), which inhibits lymphocyte IMPDH (inosine monophosphate dehydrogenase), thereby drastically decreasing GTP concentrations essential to lymphocyte proliferation in vitro and in vivo. Erythrocyte GTP concentrations are commonly elevated in severe renal disease, but normalize following successful engraftment. Consequently, elevated GTP in renal transplant recipients might signal impending loss of immunosuppression and graft failure. In the present study, we compared erythrocyte nucleotides and plasma metabolites in two groups of 25 patients after renal transplantation, both receiving prednisolone and cyclosporin A, but one group receiving MMF and the other AZA. No patients had recent allograft biopsy evidence of rejection. Erythrocyte GTP concentrations at MMF commencement were 50.4+/-23.4 micromol/l. An increase occurred during the first 3 months after transplant when MMF was used de novo, stabilizing at 146.7+/-62.9 micromol/l after 4 months. This was significantly higher (P=2.5 x 10(-6)) than erythrocyte GTP (40.4+/-15.9 micromol/l) in the AZA group, which was essentially unchanged from values immediately after successful transplantation. The effect of MMF on erythrocyte GTP levels was reversible, since GTP levels fell when MMF therapy was terminated. The results demonstrate paradoxically high GTP concentrations in erythrocytes of renal transplant patients receiving MMF. MPA may stabilize reticulocyte IMPDH, allowing the protein to persist during erythropoiesis. This behaviour is in marked contrast with the decrease in GTP levels seen in white blood cells of patients on chronic MMF therapy.

Origin and characteristics of an unusual pyridine nucleotide accumulating in erythrocytes: positive correlation with degree of renal failure.

BACKGROUND: Our aim was to identify an unusual nucleotide accumulating, with precursors, in erythrocytes in uraemia and to establish its relationship to the candidate uraemic toxin, N1-methyl-2-pyridone-5-carboxamide (M2Py). METHODS: This nucleotide, plasma creatinine and M2Py were measured in four categories of patients: mild chronic renal failure (CRF), end-stage renal failure (ESRF), haemodialysis (HD) and continuous ambulatory peritoneal dialysis (CAPD); also in healthy controls and patients after successful kidney transplantation (Post-Tx). RESULTS: The nucleotide was identified using liquid chromatography and mass spectrometry as 2-pyridone-5-carboxamide ribonucleoside triphosphate (2PyTP). In the patient groups, mean 2PyTP concentrations are lowest in CRF (21.8 micromol/l), increasing with degree of renal failure through ESRF (55.1 micromol/l) and HD patients (70.9 micromol/l) and being highest in CAPD patients (216.7 micromol/l). Following successful transplantation, 2PyTP concentrations fall to the control range. Plasma concentrations of M2Py correlate with 2PyTP in the erythrocytes; both are undetectable in patients lacking molybdenum cofactor, needed to oxidise N-methylnicotinamide. CONCLUSION: Our evidence indicates that M2Py accumulates in parallel with the nucleotide 2PyTP in renal failure: either may be a uraemic toxin, since both increase with the degree of renal failure. Elevated concentrations of 2PyTP in CAPD patients reflect a longer half-life for erythrocytes than in HD patients.

Atypical familial juvenile hyperuricemic nephropathy associated with a hepatocyte nuclear factor-1beta gene mutation.

BACKGROUND: Familial juvenile hyperuricemic nephropathy (FJHN) is a dominantly inherited condition characterized by young-onset hyperuricemia, gout, and renal disease. The etiologic genes are unknown, although a locus on chromosome 16 has been identified in some kindreds. Mutations in the gene encoding hepatocyte nuclear factor (HNF)-1beta have been associated with dominant inheritance of a variety of disorders of renal development, particularly renal cystic disease and early onset diabetes; hyperuricemia has been reported in some kindreds. METHODS: To assess a possible role for the HNF-1beta gene in some FJHN kindreds we sequenced the HNF-1beta gene in subjects from three unrelated FJHN families with atypical features of renal cysts or abnormalities of renal development. We also compared serum urate levels in subjects with HNF-1beta mutations with populations of controls, type 2 diabetic subjects, and subjects with mild chronic renal failure without HNF-1beta mutations. RESULTS: A splice-site mutation in intron 2, designated IVS2+1G>T, showed complete co-segregation with FJHN in one family with diabetes. Serum urate levels were significantly higher in the HNF-1beta subjects compared with the normal control subjects (384 micromol/L vs. 264 micromol/L, P = 0.002) and the type 2 diabetic subjects (397 micromol/L vs. 271 micromol/L, P = 0.01). Comparison of serum urate levels in the HNF-1beta subjects with gender-matched subjects with renal impairment of other causes did not reach significance (402 micromol/L vs. 352 micromol/L, P = 0.2). CONCLUSION: Hyperuricemia and young-onset gout are consistent features of the phenotype associated with HNF-1beta mutations, but the mechanism is uncertain. Families with HNF-1beta mutations may fit diagnostic criteria for FJHN. Identification of HNF-1beta patients by recognizing the features of diabetes and disorders of renal development is important in resolving the genetic heterogeneity in FJHN.

Genetic basis of inosine triphosphate pyrophosphohydrolase deficiency.

Inosine triphosphate pyrophosphohydrolase (ITPase) deficiency is a common inherited condition characterized by the abnormal accumulation of inosine triphosphate (ITP) in erythrocytes. The genetic basis and pathological consequences of ITPase deficiency are unknown. We have characterized the genomic structure of the ITPA gene, showing that it has eight exons. Five single nucleotide polymorphisms were identified, three silent (138G-->A, 561G-->A, 708G-->A) and two associated with ITPase deficiency (94C-->A, IVS2+21A-->C). Homozygotes for the 94C-->A missense mutation (Pro32 to Thr) had zero erythrocyte ITPase activity, whereas 94C-->A heterozygotes averaged 22.5% of the control mean, a level of activity consistent with impaired subunit association of a dimeric enzyme. ITPase activity of IVS2+21A-->C homozygotes averaged 60% of the control mean. In order to explore further the relationship between mutations and enzyme activity, we examined the association between genotype and ITPase activity in 100 healthy controls. Ten subjects were heterozygous for 94C-->A (allele frequency: 0.06), 24 were heterozygotes for IVS2+21A-->C (allele frequency: 0.13) and two were compound heterozygous for these mutations. The activities of IVS2+21A-->C heterozygotes and 94C-->A/IVS2+21A-->C compound heterozygotes were 60% and 10%, respectively, of the normal control mean, suggesting that the intron mutation affects enzyme activity. In all cases when ITPase activity was below the normal range, one or both mutations were found. The ITPA genotype did not correspond to any identifiable red cell phenotype. A possible relationship between ITPase deficiency and increased drug toxicity of purine analogue drugs is proposed.

Severe pyridine nucleotide depletion in fibroblasts from Lesch-Nyhan patients.

The relationship between a complete deficiency of the purine enzyme hypoxanthine-guanine phosphoribosyltransferase and the neurobehavioural abnormalities in Lesch-Nyhan disease remains an enigma. In vitro studies using lymphoblasts or fibroblasts have evaluated purine and pyrimidine metabolism with conflicting results. This study focused on pyridine nucleotide metabolism in control and Lesch-Nyhan fibroblasts using radiolabelled salvage precursors to couple the extent of uptake with endocellular nucleotide concentrations. The novel finding, highlighted by specific culture conditions, was a marked NAD depletion in Lesch-Nyhan fibroblasts. ATP and GTP were also 50% of the control, as reported in lymphoblasts. A 6-fold greater incorporation of [(14)C]nicotinic acid into nicotinic acid- adenine dinucleotide by Lesch-Nyhan fibroblasts, with no unmetabolized substrate (20% in controls), supported disturbed pyridine metabolism, NAD depletion being related to utilization by poly(ADP-ribose) polymerase in DNA repair. Although pyrimidine nucleotide concentrations were similar to controls, Lesch-Nyhan cells showed reduced [(14)C]cytidine/uridine salvage into UDP sugars. Incorporation of [(14)C]uridine into CTP by both was minimal, with more than 50% [(14)C]cytidine metabolized to UTP, indicating that fibroblasts, unlike lymphoblasts, lack active CTP synthetase, but possess cytidine deaminase. Restricted culture conditions may be neccesary to mimic the situation in human brain cells at an early developmental stage. Cell type may be equally important. NAD plus ATP depletion in developing brain could restrict DNA repair, leading to neuronal damage/loss by apoptosis, and, with GTP depletion, affect neurotransmitter synthesis and basal ganglia dopaminergic neuronal systems. Thus aberrant pyridine nucleotide metabolism could play a vital role in the pathophysiology of Lesch-Nyhan disease.

Accumulation of plasma N-methyl-2-pyridone-5-carboxamide in patients with chronic renal failure.

Intracellular catabolism of NAD in mammalian cells occurs mainly via reaction catalyzed by poly(ADP-ribose) polymerase (PARP) with the release of nicotinamide, which is then metabolized predominantly to N-methyl-2-pyridone-5-carboxamide (2PY). PARP could be activated by binding to broken DNA and is known to be involved in DNA repair mechanisms, cell stress response and regulation of apoptosis. 2PY may accumulate under disease conditions resulting in accelerated DNA damage and retention of catabolic products. Our hypothesis was that chronic renal failure would lead to elevation of 2PY and potentially to inhibition of PARP and related physiological mechanisms. In the present study we: (a) compared plasma 2PY concentration in healthy subjects and in patients with chronic renal failure (CRF); (b) evaluated the relationship between plasma 2PY concentration and the severity of CRF; (c) evaluated the effect of hemodialysis treatment and kidney transplantation on 2PY concentration. We found that the plasma 2PY concentration in healthy subjects is 0.83+/-0.18 microM but it could increase up to 40 microM in patients with CRF. A significant correlation was found in CRF between plasma 2PY and creatinine concentration. A single hemodialysis treatment was associated with significant reduction of plasma 2PY concentration after the hemodialysis, but it increased rapidly 48 h after the end of treatment. Successful kidney transplantation was associated with return of 2PY concentration to the normal range. In conclusion, our results indicated significant production of 2PY in humans. In healthy subjects 2PY is cleared from the plasma by excretion in the urine. Altered excretion by the kidney leads to increase in plasma concentration of 2PY. It is possible that 2PY may play a significant role in the development of uremic toxemia, especially as an inhibitor of poly(ADP-ribose)polymerase.

Restriction of a Sindbis virus mutant in BHK cells and relief of the restriction by the addition of adenosine.

SV(PZF) is a mutant of Sindbis virus (SV) which we selected on the basis of its ability to replicate in mosquito cells treated with pyrazofurin (PZF), a drug which inhibits pyrimidine nucleotide biosynthesis (Lin et al., 2000, Virology 272, 61-71). Three mutations, A6627U, A7543U, and C7593A, were identified in the nsP4 (the viral RNA polymerase) coding region, which were required for the PZF-resistant phenotype. We report here that SV(PZF) has a second phenotype. Its replication in BHK cells is severely restricted; yields of SV(PZF) from BHK cells are 100- to 1000-fold lower than the yields of standard SV (SV(STD)). However, addition of adenosine to the SV(PZF)-infected cultures completely relieves this restriction and results in yields comparable to those observed with SV(STD). Adenosine has no effect on the yield of SV(STD) from BHK cells. Synthesis of the viral structural proteins is markedly depressed in SV(PZF)-infected BHK cells, as is synthesis of the viral subgenomic (SG) RNA from which these proteins are translated. In contrast, normal amounts of genomic RNA are made. Experiments with mutagenized viruses indicated that the SV(PZF) mutation, C7593A, by itself, was sufficient to produce the restriction phenotype. However, this mutation not only changes Pro 609 of nsP4 to Thr, it also changes the nucleotide at the minus sign5 position of the SG promoter. To evaluate the relative contributions of the change in nsP4 and the change in the SG promoter to the restriction phenotype, we made use of double SG viruses, in which nsP4 and the promoter for the SG RNA which encodes the structural proteins can be changed independent of each other. Our results indicated that both the change in nsP4 and the change in the SG promoter were required to produce the full restriction phenotype. We suggest that the changes in nsP4 and the SG promoter destabilize the RNA initiation complex assembled at the SG promoter and that since ATP is the initiating nucleotide in the SG RNA transcript, the increased level of ATP resulting from the addition of adenosine is able to compensate for this destabilization and restore the synthesis of SG RNA to normal levels.

Partial hypoxanthine-Guanine phosphoribosyltransferase deficiency as the unsuspected cause of renal disease spanning three generations: a cautionary tale.

Hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency is an X-linked defect of purine metabolism. Clinical manifestations are usually related to the degree of enzyme deficiency: complete HPRT deficiency (Lesch-Nyhan syndrome) presenting with severe neurologic or renal symptoms, or partial HPRT deficiency (Kelley-Seegmiller syndrome) manifesting as a gout-urolithiasis syndrome. A 3-generation kindred is described in which the recognition of partial HPRT deficiency in 2 adolescent male siblings presenting with uric acid lithiasis led to the diagnosis in 2 maternal uncles already in renal failure of unknown cause. This report highlights the importance of clinical awareness leading to early diagnosis, appropriate diagnostic methodology, and therapy of a treatable inherited disorder of purine metabolism for the prevention of renal failure.