Thomas D. Palella

Cyclosporine-Induced Hyperuricemia and Gout

To evaluate the frequency and the pathogenesis of hyperuricemia and gout during cyclosporine therapy, we studied renal-transplant recipients who were treated with either cyclosporine and prednisone (n = 129) or azathioprine and prednisone (n = 168). Among the patients with stable allograft function and serum creatinine concentrations below 265 mumol per liter, hyperuricemia was more common in the cyclosporine group than in the azathioprine group (84 percent vs. 30 percent; P = 0.0001). Gout developed in nine patients (7 percent) in the cyclosporine group, but no episodes occurred in the azathioprine group. Serum urate levels became elevated in 90 percent of the patients in the cyclosporine group who were treated with diuretics, as compared with 60 percent of those not treated with diuretics (P = 0.001); in the azathioprine group, the corresponding values were 47 percent and 15 percent (P = 0.0001). Serum urate levels did not correlate with trough blood cyclosporine levels in a selected subgroup (n = 40) of patients from the cyclosporine group, who were studied from 4 to 96 weeks after transplantation. Detailed studies of urate metabolism in six cyclosporine-treated patients revealed normal turnover rates for urate and decreases in creatinine and urate clearance, as compared with seven control subjects. We conclude that hyperuricemia is a common complication of cyclosporine therapy and is caused by decreased renal urate clearance. Gouty arthritis is the cause of considerable morbidity among renal-transplant recipients who receive cyclosporine.

Expression of human HPRT mRNA in brains of mice infected with a recombinant herpes simplex virus-1 vector

Complete deficiency of the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) results in a devastating neurological disease, the Lesch-Nyhan syndrome. This disorder has been identified as a candidate for initial attempts at somatic cell gene therapy. We have previously reported the construction of a recombinant herpes simplex virus type 1 (HSV-1) vector containing human hprt cDNA sequences under the regulatory control of the viral thymidine kinase gene (tk) [Palella et al., Mol. Cell. Biol. 8 (1988) 457-460]. Infection of HPRT- cultured rat neuronal cells with these vectors resulted in transient expression of human hprt. In this paper, we report the expression of human hprt mRNA transcripts in the brains of mice infected in vivo with this vector by direct intracranial inoculation. Human hprt transcripts were distinguished from endogenous mouse transcripts by RNase A mapping using riboprobes transcribed from human hprt cDNA. These initial studies demonstrate the transfer and transcription of a human gene in brain cells by direct in vivo infection with recombinant HSV-1 vectors.

Herpes simplex virus-mediated human hypoxanthine-guanine phosphoribosyltransferase gene transfer into neuronal cells.

The virtually complete deficiency of the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) results in a devastating neurological disease, Lesch-Nyhan syndrome. Transfer of the HPRT gene into fibroblasts and lymphoblasts in vitro and into hematopoietic cells in vivo has been accomplished by other groups with retroviral-derived vectors. It appears to be necessary, however, to transfer the HPRT gene into neuronal cells to correct the neurological dysfunction of this disorder. The neurotropic virus herpes simplex virus type 1 has features that make it suitable for use as a vector to transfer the HPRT gene into neuronal tissue. This report describes the isolation of an HPRT-deficient rat neuroma cell line, designated B103-4C, and the construction of a recombinant herpes simplex virus type 1 that contained human HPRT cDNA. These recombinant viruses were used to infect B103-4C cells. Infected cells expressed HPRT activity which was human in origin.

Molecular basis of hypoxanthine-guanine phosphoribosyltransferase deficiency in ten subjects determined by direct sequencing of amplified transcripts.

Hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency is an inborn error of purine metabolism. Mutant HPRT gene sequences from patients deficient in enzyme activity have previously been characterized by cDNA cloning or amino acid sequencing techniques. The presence of HPRT-specific mRNA in nearly all deficient subjects, as well as the small size of the HPRT mRNA (1,400 bp), make the polymerase chain reaction (PCR) an alternative for the identification of mutations at this locus. In this report we use the PCR to identify previously undetermined mutations in HPRT mRNA from B lymphoblasts derived from 10 deficient individuals. Six of these variants contain single point mutations, three contain deletions, and one contains a single nucleotide insertion. Several of these mutations map near previously identified HPRT variants, and are located in evolutionarily conserved regions of the molecule.

A molecular survey of hypoxanthine-guanine phosphoribosyltransferase deficiency in man.

We characterized 24 unrelated patients with a deficiency of the enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) in an attempt to better understand the nature and spectrum of mutations that underlie this prototype-inherited disease. Lymphoblast cell lines derived from each patient were analyzed at multiple molecular levels including the structure and function of the residual HPRT enzyme, messenger RNA (mRNA), and gene. Our studies demonstrate the following: (a) at least 16 of the 24 patients represent unique and independent mutations at the HPRT structural gene; (b) the majority of cell lines have normal quantities of mRNA but undetectable quantities of enzyme; (c) 33% of patients retain significant quantities of structurally altered, functionally abnormal, HPRT enzyme variants; and (d) a minority of patients are void of both enzyme and mRNA, possibly representing examples of aberrations in gene expression. Our studies provide direct evidence for marked genetic heterogeneity in this disorder and illustrate the kinds of mutations and mutational consequences that underlie inherited disease in humans.

Cellular immune deficiency with autoimmune hemolytic anemia in purine nucleoside phosphorylase deficiency

Immunologic and metabolic abnormalities were studied in a five year old boy with 0.07 per cent of normal erythrocyte purine nucleoside phosphorylase activity. The clinical course is characterized by severe autoimmune hemolytic anemia, a transient neurologic disorder with tremor and ataxia, and minor infectious illnesses. There is severe lymphopenia with decreased absolute numbers of T and B lymphocytes. Mitogen-stimulated blastogenesis is reduced, but response to allogeneic lymphocytes is normal. A monoclonal IgG protein is present. There is hypouricemia, elevated plasma inosine level, hypouricosuria and an increase in the urinary concentration of inosine and guanosine. The pattern of heterozygote distribution in the patients family is compatible with an autosomal recessive trait in which heterozygotes are identifiable. In addition, the unusual laboratory and clinical manifestations of this patient illustrate the heterogeneity of the clinical syndrome associated with purine nucleoside phosphorylase deficiency.

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

SummaryHPRTAnn 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 Km for both substrates, a normal Vmax, 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 HPRTAnn Arbor and determined its complete nucleotide sequence. A single nucleotide change (T→G) 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.

Genetic basis of hypoxanthine guanine phosphoribosyltransferase deficiency in a patient with the Lesch-Nyhan syndrome (HPRTFlint)

The molecular basis for complete hypoxanthine guanine phosphoribosyltransferase (HPRT) deficiency has been determined in a patient with Lesch-Nyhan syndrome. A B-lymphoblastoid cell line derived from this patient expresses normal amounts of HPRT mRNA yet no detectable immunoreactive protein as determined by radioimmunoassay. These findings suggest either a decreased rate of translation or accelerated degradation due to enhanced proteolytic susceptibility. cDNAs synthesized from this patients RNA have a single nucleotide (nt) substitution, a C----A transversion at nt 222. RNase A cleavage analysis confirms the presence of a mutation at this position within mRNA isolated from lymphoblasts from patient A.C. This transversion predicts a phenylalanine to leucine replacement at amino acid position 73 in the translated protein. We have designated this mutant HPRTFlint. The mutation in HPRTFlint disrupts a strongly conserved region among PRTases from Escherichia coli, rodents and man, suggesting an important role for this region for the normal function of HPRT. Since it is unlikely that this amino acid substitution alters the translational rate, we hypothesize that disruption of the secondary structure within this region renders HPRTFlint more susceptible to proteolysis.

Identification of a single nucleotide change in the hypoxanthine-guanine phosphoribosyltransferase gene (HPRTYale) responsible for Lesch-Nyhan syndrome.

Complete deficiency of hypoxanthine-guanine phosphoribosyltransferase (HPRT) causes the Lesch-Nyhan syndrome. Previous characterization of a mutant form of HPRT, HPRTYale, from a subject with the Lesch-Nyhan syndrome revealed normal mRNA and protein concentrations, no residual catalytic activity, and cathodal migration upon PAGE. We have cloned and sequenced HPRTYale cDNA. The nucleotide sequence of full-length HPRTYale cDNA revealed a single nucleotide substitution compared with normal HPRT cDNA: G----C at nucleotide position 211. This transversion predicts substitution of arginine for glycine at amino acid position 71, explaining the cathodal migration of HPRTYale. Chou-Fasman secondary structure analysis predicts a change in the probability of beta-turn formation in the region containing the mutation. Inclusion of the bulky arginine side chain in place of glycine probably disrupts protein folding as well. Cloning mutant forms of cDNA allows identification of specific mutations, provides insight into mutational mechanisms, and facilitates structure-function analysis of mutant proteins.

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.