J Med Genet: first published as 10.1136/jmg.13.2.91 on 1 April 1976. Downloaded from Journal of Medical Genetics (1976). 13, 91-95. Use of phytohaemagglutinin stimulated lymphocytes to study effects of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) deficiency on polynucleotide and protein synthesis in the Lesch-Nyhan syndrome R. 0. McKERAN AND R. W. E. WATTS From the Division of Inherited Metabolic Diseases, Medical Research Council, Clinical Research Centre, Harrow, Middlesex Summary. The incorporation of ['4C]thymidine and [14C]uridine into the nucleoprotein, and [14C]phenylalanine into the protein by phytohaemagglutinin (PHA) stimulated lymphocytes from a patient with the Lesch-Nyhan syndrome [hypoxanthine-guanine phosphoribosyl transferase (EC 2.4.2.8. HGPRT) de- ficiency] and controls, was studied over 72 hours of incubation, with and without azaserme to block de novo purine biosynthesis. No difference was observed between the values obtained for Lesch-Nyhan and control lymphocytes, when PHA- stimulated without added azaserine. The percentage reduction in the incorporation ofprecursors into nucleoprotein andprotein after PHA stimulation in thepresence of azaserme was more obvious in the lymphocytes ofthe patient with the Lesch-Nyhan syndrome than in the controls after the shorter incubation periods at the lower rates http://jmg.bmj.com/ of synthesis. Blocking the de novo purine biosynthetic pathway, in control PHA stimulated lymphocytes, inhibited transformation, whereas loss ofthe purine salvage enzyme HGPRT did not have this effect. These results are compatible with the view that the brain and bone-marrow damage that occur in the Lesch-Nyhan syndrome are the result of lack of HGPRT in tissues with little de novo purine biosynthetic capability. Other tissues with both purine biosynthetic and salvage pathways are less vulnerable to the enzyme defect. on September 26, 2021 by guest. Protected copyright. Some possible mechanisms by which HGPRT deficiency could act are discussed. We suggest that inability to increase the supply ofguanylic acid (GMP) in response to a mitotic stimulus may mediate the effect of HGPRT deficiency. The Lesch-Nyhan syndrome is a rare sex-linked Purine ribonucleotides are formed either by de recessive neurological disorder associated with the novo synthesis from small molecular weight pre- virtually complete absence of the purine salvage cursors or by direct reaction of a purine base with enzyme hypoxanthine-guanine phosphoribosyl- phosphoribosylpyrophosphate (PRPP*), catalysed transferase (EC 2.4.2.8; HGPRT; Table I). by the purine phosphoribosyltransferase enzymes Occasional cases of severe gout and/or uric acid (Fig. 1). Some tissues (e.g. fibroblasts, Rosen- urolithiasis, sometimes with minor neurological bloom et al, 1968) have both pathways, others (e.g. abnormalities, are associated with partial HGPRT brain and bone-marrow) lack, or have very little deficiency (Kelley et al, 1967, 1969). purine de novo biosynthetic activity (Howard, Received 9 June 1975. * a-D-ribofuranose-l-pyrophosphate-5 phosphate. 91 J Med Genet: first published as 10.1136/jmg.13.2.91 on 1 April 1976. Downloaded from 92 McKeran and Watts TABLE I nucleotides (GMP and AMP). This need is MAIN CLINICAL AND BIOCHEMICAL FEATURES normally met by purine synthesis de novo and by the OF THE LESCH-NYHAN SYNDROME HGPRT catalysed purine salvage pathway. Thus, 1: Delayed motor development, apparent by 3 to 4 months in the presence of azaserine the lymphocyte becomes 2: Choreoathetosis by 1 year a model of a brain cell or a bone-marrow stem cell 3: Compulsive self-mutilation at 18 months to 2 years 4: Spasticity at about 1 year from the point of view of the metabolic pathway 5: Aggressive behaviour 6: Mental retardation which it has available for purine ribonucleotide 7: Small stature 8: Brain weight less than normal with neuronal loss, astrocytic synthesis. hypoplasia, foci of necrosis, and focal perivascular demyelina- tion, principally affecting the cerebral cortex, cerebellum, and Methods mid-brain nuclei 9: Megaloblastic anaemia Cell separation. Blood was withdrawn under 10: Hyperuricaemia and hyperuricaciduria 11: Virtually complete deficiency of hypoxanthine-guanine phos- sterile conditions after an ovemight fast from a patient phoribosyltransferase (EC 2.4.2.8 HGPRT). with complete absence of HGPRT and controls of comparable age and sex. Lymphocytes were separated from heparinized blood (0.1 ml, 1:1000 heparin, no preservative added) by the ficol-triosil method (Boyum, Kerson, and Appel, 1970; Lajtha and Vane, 1968). 1958). Fibroblasts derived from patients with the Growth of phytohaemagglutinin stimulated Lesch-Nyhan syndrome show no impairment of lymphocytes from patient with Lesch-Nyhan growth in vitro, whereas granulocyte/macrophage syndrome and controls. The pellet of cells was progenitor cells cultured from bone-marrow fail to resuspended to a final concentration of 1 x 106 lympho- cytes/ml in Dulbecco's modification of Eagle's medium, grow normally and form fewer colonies, which are containing benzylpenicillin (200 units/ml), streptomycin also smaller than colonies from control subjects base (0.2 mg/ml), and pooled human serum gelatin (McKeran et al, 1974). This suggests that similar (final concentration 10% v/v). The cell suspension restricted growth in brain development, as a result (200 ,ul, 200 000 lymphocytes) was added to the wells of of lack of HGPRT in a tissue with little purine de microtitre plates. Aliquots (10 ,ul) of [2-14C]thymidine novo biosynthetic ability, might be a factor in the (8.3 nCi, 0.134 pmol), [2-14C]uridine (50 nCi, pathogenesis of brain damage in the Lesch-Nyhan 0.826 pmol), and L[U-14C]phenylalanine (30 nCi, syndrome (McKeran et al, 1974). 0.057 pmol) were added to separate wells at 0, 24, and These propositions have been further studied in 48 hours, with incubation for 24 hours at 37°C in 50o CO2 and air. Experiments were performed in triplicate phytohaemagglutinin (PHA) stimulated lym- on three occasions with and without PHA stimulation phocytes by blocking purine de novo biosynthesis (10lO added at zero time) [each vial contained reagent http://jmg.bmj.com/ with azaserine. Blast transformation in lympho- grade PHA suspended in 25 ml Dulbecco's modified cytes involves a rapid burst of polynucleotide Eagle's medium with benzylpenicillin (200 units/ml) and synthesis with an acute demand for purine mono- streptomycin base (0.2 mg/ml)]. hosphoribosyl pyrophosphate (PRPP) on September 26, 2021 by guest. Protected copyright. Purine .Fc Biosynthesis miimyl qlyci noidomFeA De novo L Azoserine IFr Ti inhibition Fc)rmyl Qlycinomidine DNA -rilbonucreotide RNA Cyclic GMP .Inioinnic ocid < Adenylicacid Cyclic AMP G uanine Adenine ATP FIG. 1. A simplified diagram to show the major features of the purine biosynthetic de novo and salvage pathways in the production of purine ribonucleotides for DNA, RNA and protein synthesis. J Med Genet: first published as 10.1136/jmg.13.2.91 on 1 April 1976. Downloaded from Use ofphytohaemagglutinin stimulated lymphocytes 93 Plates were centrifuged for 10 minutes at 250 x g5V and [14C]uridine into the nucleoprotein, and 4°C. The supernatant was discarded, the individual [14C]phenylalanine into the protein of PHA cell pellets washed once with NaCl (200 1l, 0.154 M), stimulated lymphocytes from a patient with the trichloracetic acid (200 ,lI, 1 M) and finally with methanol Lesch-Nyhan syndrome and control patients is (200 u1l). Each cell pellet was dissolved in NaOH shown in Fig. 2. No reduction was detected (200 ,ul, 1 M) and aliquots (100 I-L) counted in 10 ml of a scintillator solution having the following composition: between the values obtained for the Lesch-Nyhan 2,5-diphenyloxazole (PPO) 6 g, p-bis (2-5-phenyloxa- patient and controls with PHA stimulation. zolyl) benzene (POPOP) 0.12 g, Triton ( x 100) 500 ml, The percentage reduction in the incorporation of methanol 100 ml, xylene 1 litre. The efficiency of [14C]thymidine, and [14C]uridine into the nucleo- counting was 71 %. protein, and [14C]phenylalanine into the protein of lymphocytes from a patient with Lesch-Nyhan Growth of phytohaemagglutinin stimulated syndrome and controls with PHA in the presence of lymphocytes from a Lesch-Nyhan patient and 0.3 mmol azaserine is shown in Fig. 3. Azaserine controls in presence of azaserine. The experiment progressively inhibited the incorporation of pre- described above was repeated on two occasions, with duplicate wells containing 0.3 mmol azaserine to inhibit cursors into lymphocyte nucleoprotein and protein. the conversion of a-N-formylglycinamide ribonucleotide This was more obvious in the lymphocytes from the (FGAR), to a-N-formylglycinamidine ribonucleotide patient with Lesch-Nyhan syndrome during the and thus block the 4th reaction on the pathway of purine biosynthesis de novo. 100- a 90 T Results 80- 70 The incorporation of [14C]thymidine, and 60o 50 2000- 40- 30- 500- 1000- ioo 80 500-~ Soo 0 E 670-0- _- http://jmg.bmj.com/ 30000- b 60- 50- 2 5 000- 0 u 0 20000- x 15 000- -v 10000- 5000- 0 on September 26, 2021 by guest. Protected copyright. 6U90_0 j 0 803- 700 -c 50- 600- 40- 500- 400- 300- 30 200- 20 100 10. s 1 O_ 0 12 24 36 48 60 72 00 12 24 36 48 bb Hours Hours FIG. 2. The incorporation of (a) [14C]thymidine, (b) [14C]uridine FIG. 3. The percentage reduction in the incorporation of (a) and (c) [14C]phenylalanine into the nucleoprotein and protein of [14C]thymidine, (b) [14C]uridine, and (c) [14C]phenylalanine into the T nucleoprotein and protein of Lesch-Nyhan and control PHA. Lesch-Nyhan and control PHA stimulated lymphocytes. 0 Mean stimulated lymphocytes in the presence of 0.3 mmol azaserine and extreme range of 6 observations on blood in the same patient 0 Mean and extreme range of 4 observations on blood in the same T with the Lesch-Nyhan syndrome.