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Biomedical subjects

R B Gordon

Publications and source records attributed to R B Gordon.

At least 37 records · Page 2Linked to original sources
PubMed1991

The molecular characterisation of HPRT CHERMSIDE and HPRT COORPAROO: two Lesch-Nyhan patients with reduced amounts of mRNA.

A complete deficiency of the purine salvage enzyme, hypoxanthine phosphoribosyltransferase (HPRT; EC 2.4.2.8), in man results in the Lesch-Nyhan (LN) syndrome. Two unrelated patients with the full LN syndrome showed no evidence of a major alteration to the gene encoding HPRT (HPRT) by restriction endonuclease analysis, but exhibited negligible levels of HPRT mRNA on Northern blots. DNA from these patients was characterised further. Amplification, by the polymerase chain reaction (PCR), of individual HPRT-exon fragments from genomic DNA followed by nucleotide (nt) sequence analysis using automated technology, revealed single-base mutations in each patient. One patient has an insertion of a T within exon-2, which places a stop codon in frame, presumably resulting in premature termination of translation of the HPRT mRNA. The other patient has a G----A base substitution at the 5' end of intron-6, at the junction of exon-6 and intron-6. Although dot blot analysis indicated negligible HPRT mRNA in lymphoblast cells from both patients, we were successful in amplifying HPRT cDNA using PCR. Direct nt sequence analysis of the amplified cDNA confirmed the insertion of a T in exon-2 in the one patient and revealed a complete deletion of exon-6 in the other patient, the latter event presumably arising due to aberrant splicing of primary message. Both mutations were also confirmed by hybridisation of amplified genomic DNA with allele-specific oligodeoxyribonucleotide probes. This study illustrates two approaches for analysing DNA mutations at the molecular level and demonstrates the power of PCR technology in the study of genetic diseases.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1991

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

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 Lesch-Nyhan 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 patient's 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.

Alleles↗
PubMed1990

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.

Chromatography, Gel↗
PubMed1990

Identification of a single nucleotide substitution in the coding sequence of in vitro amplified cDNA from a patient with partial HPRT deficiency (HPRTBRISBANE).

The change in genomic DNA responsible for HPRT deficiency has been determined in a patient with urate overproduction and gout. In erythrocyte cell lysates, this patient had approximately 10% of normal HPRT enzyme activity and 26% of immunoidentical HPRT protein. Cultured lymphoblasts derived from this patient were used to extract mRNA. This was reverse transcribed to cDNA, which was then amplified using the polymerase chain reaction. The resulting DNA was cloned and the nucleotide sequence determined. In addition a portion of the sequence was derived from cloned double-stranded cDNA prepared by conventional first and second strand synthesis. A single nucleotide base change (a C----T transition) was detected, which predicts an amino acid substitution of isoleucine for threonine at amino acid 168 of the HPRT protein. The nucleotide substitution creates a BamHI site, confirming a restriction fragment length polymorphism previously reported in this patient.

Adult↗
PubMed1990

Observations on the growth of renal neoplasms.

A retrospective review of the imaging results of 11 patients with 13 solid renal parenchymal neoplasms was performed, allowing analysis of the linear growth rates of these tumors. The study sample included seven pathologically proved renal adenocarcinomas and six lesions that were indicative of a neoplasm radiologically (enhancing parenchymal mass on computed tomographic [CT] scans with documented interval growth), which were followed up for 2-7.8 years. Variable interval tumor growth was demonstrated in every case except one and ranged from 0 to 1.6 cm/y, with an overall mean linear growth rate of approximately 0.5 cm/y. Ten of 11 "small renal neoplasms" (less than or equal to 3.0 cm in diameter) displayed interval growth, with five ultimately measuring greater than 3.0 cm (size range, 3.5-7.0 cm). While the results are preliminary and reflect observations on a very small study sample, it was noted that five of the seven pathologically proved adenocarcinomas appeared homogeneous and well marginated, and all were low-grade, low-stage carcinomas. These grew more slowly and were generally smaller at initial presentation than higher-grade lesions, which demonstrated a more heterogeneous appearance on CT scans.

Carcinoma, Renal Cell↗
PubMed1989

Characterization of genomic DNA, mRNA and enzyme protein in cases of HPRT-deficiency.

1. Immunological quantitation of the HPRT proteins, together with DNA and RNA studies have defined further the heterogeneous nature of HPRT-deficiency in our patients. 2. These studies have dictated possible approaches for further characterisation of the HPRT enzyme in our patients. In the two Lesch-Nyhan patients, both the protein and the usual cDNA approach would appear difficult. 3. A BamHI polymorphism has been detected in Patient A. 4. Sequence data confirmed the creation of this BamHI site by a single C----T transition at position 602 in the coding sequence. 5. Sequencing of other patients is proceeding and use is being made of the Polymerase Chain Reaction (PCR)10 for amplification of specific segments of HPRT coding sequence.

Cell Line, Transformed↗
PubMed1988

Biochemical basis of hypoxanthine-guanine phosphoribosyltransferase deficiency in nine families.

The concentration of hypoxanthine-guanine phosphoribosyltransferase (HPRT) cross-reacting material (CRM) was determined in haemolysates and/or lymphoblast lysates from nine patients with complete or partial deficiency of HPRT activity. Two of the patients had the fully developed Lesch-Nyhan syndrome and although they had undetectable HPRT activity, small amounts of CRM were found. HPRT-specific mRNA was not detected in lymphoblast lysates from one of these patients, while lysates from the other had a much reduced concentration. Samples from three patients with less than 0.1% of normal HPRT activity but with minor or no neurological manifestations were also found to contain small amounts of CRM. The other four patients whose HPRT activities ranged from 3 to 10% of normal were found to have CRM concentrations which varied from 26 to 100% of normal. In one patient with a partial deficiency the Km for 5-phospho-alpha-D-ribosyl-1-pyrophosphate was five times normal.

Humans↗
PubMed1987

Human hypoxanthine-guanine phosphoribosyltransferase. Development of a spectrophotometric assay and its use in detection and characterization of mutant forms.

A simple and rapid spectrophotometric assay for the estimation of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) activity in human tissues is described. It is based on the increase in absorbance at 257.5 nm which occurs when the substrate guanine is converted to its 5'-mononucleotide, GMP. The assay has been developed to measure HGPRT activity in erythrocyte and lymphocyte lysates and in brain homogenates, and has been used in the screening of patients with hyperuricaemia and/or hyperuricosuria for HGPRT deficiency. It has also been used to determine the steady-state kinetic constants of a mutant form of the enzyme. The spectrophotometric assay is compared with the radioactive assay currently used to measure HGPRT activity.

Aged↗
PubMed1987

Synthesis of purines in human lymphoblast cells deficient in methylthioadenosine phosphorylase activity.

Two human lymphoblastic cell lines, deficient in methylthioadenosine phosphorylase (MTAP) activity, were found to have increased rates of de novo purine synthesis. These MTAP- cell lines were K562, an undifferentiated leukemic line and CCRF-CEM, a leukemic line of T-cell origin. Another T-cell line, CCRF-HSB-2 was found to be deficient in activity. However, this line did not demonstrate elevated rates of purine synthesis. Purine metabolism in the above cell cultures was compared with MTAP+ human B-cell lines and two human T-cell lines (MOLT-3 and MOLT-4). In all the MTAP+ cell lines, the rate of de novo purine synthesis was inhibited by the presence of methylthioadenosine in the assay medium (10 microM concentration produced more than 90% inhibition). However, purine synthesis in the MTAP- cells was resistant to inhibition by methylthioadenosine. Adenine in the assay medium inhibited de novo purine synthesis in MTAP+ and MTAP- cells to a similar degree. This inhibition was dose dependent and was elicited by concentrations similar to those of methylthioadenosine. Growth of the cell lines in culture was not affected by either methylthioadenosine or adenine at the concentrations which produced inhibition of purine synthesis. These results suggest that purine synthesis in MTAP+ cells is inhibited by adenine formed from the phosphorolytic cleavage of methylthioadenosine by methylthioadenosine phosphorylase.

Adenine↗
PubMed1987

Purine synthesis de novo in cultured lymphoblast cells derived from patients with gout.

Rates of de novo purine synthesis in lymphoblast cell cultures derived from ten patients with gout were compared with those from control individuals. Since the growth rate of the culture, an assay procedure was developed to account for the variation in lymphoblast growth rates and to permit valid quantitative comparison between purine synthesis in each cell line. Clear differences were demonstrated between the rates of purine synthesis in cells from normal control subjects and those from patients with a deficiency of hypoxanthine-guanine phosphoribosyltransferase activity (HPRT-deficient). Lymphoblasts from the gouty patients showed purine synthesis either within the normal range or intermediate between this and the HPRT-deficient cells. In patients having normal renal function, de novo purine synthesis of lymphoblast cells correlated with the degree of urate production as reflected by the urinary excretion of urate over a 24 h period. Three patients, with demonstrable excessive production of urate in vivo, exhibited increased purine synthesis in lymphoblasts. This increased synthesis did not appear to result from any of the enzyme mutations currently recognized as responsible for abnormal purine metabolism.

Adult↗
PubMed1987

HPRT-deficiency associated with normal PRPP concentration and APRT activity.

Deficiencies of HPRT are usually associated with increased concentrations of PRPP and increased levels of APRT activity in erythrocytes. We report the case of a male with a partial deficiency of HPRT in whom these two parameters were normal. The clinical features of this patient were those associated with severe hyperuricaemia and gout. Studies of intact erythrocytes showed rates of incorporation of [14C]hypoxanthine and of [14C]adenine into purine nucleotides which were almost indistinguishable from normal. However, HPRT activity in erythrocyte lysates was only 9% of normal. In cell extracts of cultured lymphoblasts, the HPRT activity was 20% of control values and the APRT activity was normal. The PRPP concentration and the rate of de novo purine synthesis in cultured lymphoblasts were both intermediate between controls and lymphoblasts from patients with the Lesch-Nyhan syndrome.

Adenine Phosphoribosyltransferase↗
PubMed1987

Pseudoaneurysm with rupture in pancreatic pseudocyst wall as demonstrated by computed tomography.

In chronic pancreatitis, inflammation originating in the pancreas may produce walled-off collections of fluid referred to as pseudocysts. This same histolytic process can also erode into adjacent arteries producing pseudoaneurysms that may hemorrhage secondary to rupture. The most common angiographic finding in patients bleeding secondary to pancreatitis is rupture of a pseudoaneurysm. We present a case of this condition that was first discovered incidentally by computed tomography during evaluation of a right renal carcinoma.

Aneurysm↗
PubMed1987

Hip arthroscopy by the lateral approach.

This report describes a new technique by which arthroscopy of the hip provides complete visualization of the joint space. The arthroscope and operative instruments are inserted by a direct lateral approach over the greater trochanter, with the patient in the lateral decubitus position. The involved leg is held in an abducted and flexed position with traction by pulleys hung overhead. Hip abduction and flexion relaxes the capsule and traction separates the joint so that insertion of the arthroscope is facilitated. Arthroscopic examination of the hip joint was performed in 11 patients. The indications for surgery included diagnosis of unresolved hip pain following failure of conservative treatment, removal of loose bodies, exploration and debridement following fracture and/or dislocation, and evaluation of the arthritic hip prior to definitive surgery.

Acetabulum↗
PubMed1987

Plasma oxipurinol concentrations during allopurinol therapy.

The plasma concentrations of oxipurinol, the chief metabolite of allopurinol, were studied in 66 patients with gout in whom the dose of allopurinol varied between 100 and 400 mg per day. Renal function ranged from normal to moderately impaired. Plasma oxipurinol concentrations correlated directly with both allopurinol dosage and with renal glomerular function as reflected by the plasma creatinine concentration. Plasma oxipurinol concentrations between 30 and 100 mumol/l were generally effective in controlling hyperuricaemia. However, oxipurinol concentrations usually rose above this range if the daily dose of allopurinol exceeded 300 mg in patients with plasma creatinine concentrations of 0.2 mmol/l or more. In patients with normal renal function, a rise of the plasma xanthine concentration to between 6 and 9 mumol/l suggested a satisfactory degree of xanthine oxidase inhibition. These measurements are particularly useful in patients who are still hyperuricaemic despite the usual doses of allopurinol.

Allopurinol↗