Ross B. Gordon

Cystathionine β‐synthase mutations in homocystinuria

The major cause of homocystinuria is mutation of the gene encoding the enzyme cystathionine β‐synthase (CBS). Deficiency of CBS activity results in elevated levels of homocysteine as well as methionine in plasma and urine and decreased levels of cystathionine and cysteine. Ninety‐two different disease‐associated mutations have been identified in the CBS gene in 310 examined homocystinuric alleles in more than a dozen laboratories around the world. Most of these mutations are missense, and the vast majority of these are private mutations. The two most frequently encountered of these mutations are the pyridoxine‐responsive I278T and the pyridoxine‐nonresponsive G307S. Mutations due to deaminations of methylcytosines represent 53% of all point substitutions in the coding region of the CBS gene. Hum Mutat 13:362–375, 1999.

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.

Adenine phosphoribosyltransferase: a simple spectrophotometric assay and the incidence of mutation in the normal population.

The significance of partial deficiency of erythrocyte adenine phosphoribosyltransferase (APRT), reported in a number of subjects with gout, has been investigated by studying its incidence in 700 normal blood donors. Three clearly deficient subjects were found, an incidence not significantly different from that in patients with abnormalities of urate metabolism. A new assay method for APRT is described in which an erythrocyte lysate is incubated with adenine and phosphoribosylpyrophosphate (PRPP) for a given time; both hemoglobin and adenine nucleotide (AMP) are then precipitated with lanthanum phosphate; the change in absorbance of adenine at 260 nm reflects the extent of its conversion to AMP by APRT.

Detection of the carrier state for an X-linked disorder, the Lesch-Nyhan syndrome, by the use of lymphocyte cloning

SummaryUsing a limiting dilution technique, we found that the frequency of thioguanine resistant (TGR) lymphocyte clones was less than 5.0x10-5 in 14 normal individuals, between 9.0x10-3 and 8.9x10-2 in seven heterozygotes for Lesch-Nyhan syndrome, and 0.88 and 0.87 in two hemizygotes. TGR clones from heterozygotes were expanded and had the hemizygote phenotype as evidenced by low hypoxanthine incorporation and severely deficient hypoxanthine-guanine-phosphoribosyl-transferase activity. Enumeration of TGR lymphocyte clones provides a simple technique for detection of heterozygosity for Lesch-Nyan syndrome. A similar approach using lymphocyte cloning may be suitable for detection of the carrier state for other X-linked disorders.

Xanthine-containing calculi during allopurinol therapy.

We report a case of urate overproduction owing to the Lesch-Nyhan syndrome (deficiency of hypoxanthine-guanine phosphoribosyltransferase). Urate crystalluria was controlled by allopurinol therapy but renal calculi developed, which contained a variety of purines, particularly the relatively insoluble xanthine, as well as oxypurinol and hypoxanthine. The potential hazard from the increased amounts of xanthine that are produced during allopurinol therapy for urate overproduction is stressed, as well as the importance of maintaining a high urine flow rate even during such therapy.

Inhibition of Plasmodium falciparum hypoxanthine-guanine phosphoribosyltransferase mRNA by antisense oligodeoxynucleotide sequence.

Comparative biochemical studies between the malaria parasite, Plasmodium falciparum, and its host have revealed differences in a number of metabolic pathways. For example, the parasite has enzymes to carry out de novo pyrimidine synthesis but is not capable of de novo synthesis of purine nucleotides. The parasite must salvage purines from its host to supply the requirement for purine nucleotides. The enzyme required for this salvage is hypoxanthine-guanine phosphoribosyltransferase This enzyme has been suggested as a target for chemotherapy by the design of analogue substrate inhibitors of the parasite enzyme. An alternate strategy, and one which we have been exploring, is the inhibition of expression of the parasite HPRT using antisense oligodeoxynucleotides.

Regulation of purine de novo synthesis in cultured human fibroblasts: The role of P-Ribose-PP

Procedures for assaying the rate of purine de novo synthesis in cultured fibroblast cells have been compared. These were (i) the incorporation of [(14)C]-glycine or [(14)C]formate in alpha-N-formylglycinamide ribonucleotide (an intermediate in the purine synthetic pathway) and (ii) the incorporation of [(14)C]-formate into newly synthesised cellular purines and purines excreted by the cell into the medium. Fibroblast cells, derived from patients with a deficiency of hypoxanthine phosphoribosyltransferase (HPRT-) (EC 2.4.2.8) and increased rates of purine de novo synthesis, were compared with fibroblasts from healthy subjects (HPRT+). Fetal calf serum, which was used to supplement the assay and cell growth medium, was found to contain sufficient quantities of the purine base hypoxanthine to inhibit purine de novo synthesis in HPRT+ cells. This inhibition was the basis of differentiation between HPRT- and HPRT+ cells. In the absence of added purine base, both cell types had similar capacities for purine de novo synthesis. This result contrasts with the increased rates of purine de novo synthesis reported for a number of human HPRT- cells in culture but conforms recent studies made on human HPRT- lymphoblast cells. The intracellular concentration and utilisation of 5-phosphoribosyl-1-pyrophosphate (P-Rib-PP), a substrate and potential controlling factor for purine de novo synthesis, were determined in HPRT- and HPRT+ cells. The rate of utilisation of P-Rib-PP in the salvage of free purine bases was far greater than that in purine de novo synthesis. Although HPRT- cells had a 3-fold increase in P-Rib-PP content, the rate of P-Rib-PP generation was similar to HPRT+ cells. Thus, in fibroblasts, the concentration of P-Rib-PP appears to be critical in the control of de novo purine synthesis and its preferential utilisation in the HPRT reaction limits its availability for purine de novo synthesis. In vivo, HPRT+ cells, in contrast to HPRT- cells, may be operating purine de novo synthesis at a reduced rate because of their ability to reutilise hypoxanthine.

Erythrocyte phosphoribosylpyrophosphate concentrations in heterozygotes for hypoxanthine-guanine phosphoribosyltransferase deficiency

Heterozygotes for the deficiency of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) demonstrate either normal erythrocyte HGPRT activities in families in which the hemizygote shows the Lesch-Nyhan syndrome, or a range of erythrocyte HGPRT activities between 20% of normal and completely normal values in families in which the mutation results in urate overproduction with few or no neurologic signs. Phosphoribosylpyrophosphate (PRPP) concentrations in erythrocytes from heterozygotes for HGPRT deficiency were shown to correlate inversely with HGPRT activities and directly with APRT activities in erythrocyte lysates. Since no evidence of increased synthesis of PRPP was obtained, these elevated PRPP concentrations can best be attributed to reduced utilization of PRPP in the HGPRT-catalyzed reaction. As shown in hemizygotes, the increased PRPP concentrations in these heterozygotes could result in stabilization of the APRT enzyme and elevation of APRT activities.

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

SummaryThe 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 (HPRTPerth) and three with partial deficiencies of HPRT activity, which have been designated HPRTUrangan, HPRTSwan and HPRTToowong. 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 patients genomic DNA. It is interesting to note that the mutation found for HPRTPerth is identical to that reported for HPRTFlint. It appears that the two mutations are de novo events.

Expression of active human hypoxanthine-guanine phosphoribosyltransferase in Escherichia coli and characterisation of the recombinant enzyme

A plasmid, pRG1, has been constructed by incorporating the coding sequence of human hypoxanthine-guanine phosphoribosyltransferase (HPRT) into the expression vector pT7-7. Expression of human HPRT has been achieved in HPRT- Escherichia coli cells transformed with pRG1 and pGP1-2, as shown by: (1) exclusive labelling with [35S]methionine of a polypeptide with the same mobility as purified human HPRT on SDS-PAGE; and (2) measurement of HPRT activity after cell lysis. Although the majority of the recombinant HPRT was present in the particulate fraction after cell lysis and centrifugation, sufficient HPRT activity was present in the supernatant fraction to allow comparison with the HPRT purified from human erythrocytes and the activity in human haemolysates and lymphoblast lysates. Small differences in electrophoretic mobility on native gels were found between HPRT activity from these sources. The Km values of recombinant HPRT for the substrates 5-phospho-alpha-D-ribosyl-1-pyrophosphate and guanine were compared with those of lymphoblast and erythrocyte HPRT.