A distinct human variant of hypoxanthine-guanine phosphoribosyl transferase

March 1978 The Journal o f P E D I A T R I C S

385

A distinct human variant of hypoxanthine-guanine phosphoribosyl transferase A variant form of hypoxanthine-guanine phosphoribosyl transferase has been found in a neurologically normal pediatric patient who presented with hematuria and episodes of oliguria and azotemia. The level of erythrocyte enzyme activity was 3% of normal. Eleetrophoretic mobility was more rapid than normal The Kin for hypoxanthine was approximately ten times normal. ImmunochemicaI analysis indicated that the variant enzyme cross reacted with antibody to normal HPRT. A system is described for the systematic charaeterization of a variant HPRT.

Lawrence Sweetman,* L a Jolla, C a l i f , M e l v i n A, H o e h , H a y w a r d , C a l i f , Bohdan Bakay, Margaret Borden, Phillip Lesh, and William L. Nyhan, L a Jolla, Calif.

H Y P O X A N T H I N E - G U A N I N E phosphoribosyl transferase (E.C.2.4.2.8) catalyzes the conversion of hypoxanthine and guanine to their nucleotides. Evidence for h u m a n variation at the H P R T locus came first from studies of the Lesch-Nyhan syndrome in which a virtual absence of enzyme activity is associated with hyperuricemia and a devastating disorder of the central nervous system. 1,2 Further variation in H P R T has been observed in patients in w h o m the central nervous system has usually been normal. :~-,0 This report describes the properties of the variant enzyme in a boy with a marked deficiency of H P R T activity whose clinical manifestations were limited to those of hyperuricemia and its urinary tract consequences. From the Departments of Pediatrics, University of California San Diego; and the Permanente Medical Group. Supported in part by grants NF 1-377from the National Foundation-March of Dimes, United States Publie Health Service GM 17702from the National Institute of General Medical Sciences, and Public Health Service (DHEW) M C T 000274. *Reprint address: Department of Pediatrics M-O09, University of California San Diego, La Jolla, CA 92093.

0022-3476/78/0392-0385500.50/0

9 1978 Tiae C. V. Mosby Co.

CASE REPORT Patient T.L. was the product of a normal pregnancy and delivery and weighed 4,000 gm at birth. Growth and development were normal: School performance was "fair to average." He presented at 4V2years Of age with gross painless hematuria. Microscopic examination disclosed numerous red blood cells and crystals which appeared to be urate. A diagnosis of acute hemorrhagic cystitis was made, and he was treated with sulfisoxazole. Cultures of the urine were sterile. The hematuria cleared completely within five days. Abbreviations used hypoxanthine-guanine phosphoribosyl transferase HPRT: BUN: blood urea nitrogen APRT: adenine phosphoribosyl transferase 5-phosphoribosyl- 1-pyrophosphate PRPP: IMP: inosinic acid CRM: cross-reacting material sodium fluoride NaF: Michaelis constant K,n: Two weeks later, hematuria recurred along with severe abdominal pain. An intravenous pyelogram showed no visualization after 30 minutes and faint kidney outlines by three hoursl Following negative cystourethrography, he had transient hypertension of 140/100, oliguria, hematuria, and azotemia with a BUN of 78 mg/dl. These problems abated spontaneously. During another bout of hematuria two months later, a second intrave-

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Sweetman et al.

The Journal of Pediatrics March 1978

Table I. Activity of hypoxanthine-guanine phosphoribosyl transferase (HPRT) and adenine phosphoribosyl transferase (APRT) in erythrocytes APRT nmoles A MP/rnin/ml pc

T.L. Controls

950 632 _+ 103

(18)

HPR T (nmoles IMP~rain/ml pc)

Low substrate concentrations T.L. Controls High substrate concentrations T.L. Controls Extrapolations to infinitely high substrate concentrations T.L. Controls

14 1,182 + 140

(20)

63 2,086 _+ 229

(8)

207 2,941

Tile data for the controls represent the means _+ SD. The numbers of control individuals represented are given in parentheses.

Table II. Effects Of sodium flouride on the activity of (hypoxanthine- uanine phosphoribosyl transferase) HPR T (nmoles IMP/min/ml pc) NaF eoncentration

Patient T.L. Control

0

5 mM

9.5 769

8.2 841

l

[

20 mM 1.98

760

The substrate concentrations were 0.01 mM hypoxanthine and 0.25 mM PRPP with 1 /L1 of blood from controls and 50 pl of blood from Patient T.L.

nous pyelogram showed prompt, bilateral excretion and was considered normal. An open renal biopsy was performed; with light and electron microscopy the histology was normal, as were immunofluorescent studies. Following anesthesia, he again developed azotemia as the BUN rose to 80 mg/dl. This episode lasted for three days. Over the next two years he had several minor episodes of hematuria and abdominal pain. At 73/12 years of age, he was admitted to the hospital because of severe abdominal pain, vomiting, hematuria, and azotemia. Many uric acid crystals were noted in the urine. The serum uric acid concentration was 22 mg/ dl. The BUN was 70 and the creatinine 3.7 mg/dl. He was treated with intravenous fluids and there was a prompt resolution of symptoms and change of the BUN and creatinine toward normal within three days. He was then treated with sodium bicarbonat~ 600 mg twice daily. Within three weeks the serum concentration

of uric acid was 7.1 and the BUN 15 mg/dl. The 24-hour urinary uric acid excretion was 430 mg in 900 ml. Following the recognition of the enzyme defect, treatment was started with allopurinol. The dose was gradually increased to 200 mg three times daily to maintain the serum concentration of uric acid between 3 and 6 mg/dl. Sodium bicarbonate administration was discontinued and maintenance of a dilute urine by the oral intake of water encouraged. He has since had only two episodes of hematuria and abdominal pain. At 11 years of age, he is asymptomatic. The mother had a urinary uric acid excretion of 1,000 rag/24 hours and a serum uric acid of 6.0 mg/dl. The father had a history of gout successfully treated with probenecid. METHODS;

Adenine phosphoribosyl transferase was assayed using 10 gl of heparinized blood. 1' The results were expressed as nmoles of adenosine m o n o p h o s p h a t e formed per minute per milliliter of packed cells ( n m o l e s / m i n / m l pc). To assay HPRT, a 10 #1 aliquot of a 1:9 hemolysate of heparinized normal blood or a 10 to 50 /~1 aliquot of heparinized whole blood from Patient T.L. was incubated for 15 minute at 60~ in 1 ml of 60 m M Tris-HC 1, pH 7.4, containing 6 m M MgC12, hypoxanthine-8-14C (1.25 m C i / mmole) and magnesium 5-phosphoribosyl-l-pyrophosphate. 11 H P R T was routinely measured at saturating concentrations of substrate (0.175 m M hypoxanthine and 0.5 m M PRPP) and at subsaturating "screening" concentrations (0.010 m M hypoxanthine and 0.25 m M PRPP) in the search for Kr~ variants. Results were expressed as nmoles of inosinic a c i d / m i n u t e / m l pc. Kinetic properties of the normal and variant H P R T enzymes were determined using 1 and 50 /~1 of blood, respectively. Activity was determined in triplicate with 16 pairs of substrate concentrations ranging from 0.01 to 0.175 m M hypoxanthine and 0.05 to 0.25 m M PRPP. Double reciprocal plots were m a d e for each substrate and reciprocal m a x i m u m velocities replotted against the reciprocal of the alternate Substrate to obtain Km values at saturating concentrations of the alternate substrate and m a x i m u m velocities at infinite concentrations of both substrates. H P R T was separated electrophoretically on polyacrylamide disc gel and assayed radiochemically. 12-~ Sensitivity of the enzyme to inhibition by sodium fluoride was tested 8, ~ in the search for characteristics that would distinguish a variant enzyme. Immunochemical characterization of the enzyme was sought using antibody prepared against purified normal h u m a n H P R T ? 7 The presence of cross-reacting material in erythrocyte lysates was determined by incubation with immunoglobulins followed by the separation of free enzyme from HPRT-antib0dy complexes using disc gel

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Variant of hypoxanthine-guanine phosphoribosyl transferase

387

NORMAL CONTROL ( I pl) 5000

Peak B Peak A

2000 I000

E r 0

L.) ,r

2000

PATIENT

T.L. ( 6 pl)

1000

I

0

2000

I

t

'

I

I

I

I

'~,

,

50

60

7'0

I

I

I

PATIENT R.L. ( 6 pl )

I000

0 ~

'

~ I0

.

~ 20

'

~ 30

~

~ 40

DISTANCE,

?'~'~, ~ - ~ ' ~ ,

80

90

I00

mm

Fig. 1. Electrophoretic and immunochemical analysis of hemolysates from patients with partial deficiency of HPRT activity. The electrophoresis of the enzyme itself is seen in peak A. React/on with immunoprotein results in a soluble complex which forms peak B when the HPRT protein reacts with antibody prepared against purified normal enzyme, as seen in the control and in Patient T.L. Patient R. L. is not related to Patient T.L. electrophoresis. The presence or absence of CRM was also tested by reaction of antibody with a known amount of normal enzyme in the presence and absence of one to four equivalent amounts of enzyme-deficient lysate. Following centrifugation the supernatant solutions were analyzed for HPRT activity. An increase in activity in the presence of the test lysate in the supernatant fluid indicated the presence of CRM. RESULTS The activities of HPRT and APRT in the erythrocytes of the patient are shown in Table I. The activity of HPRT at high concentrations of substrates was 3% of the activity of control hemolysates and at low substrate concentrations 1.2% of control. HPRT activity was stable over time. The means of analyses at 0, 24, and 48 hours were 17, 12 and 14, respectively. The level of activity did not decrease appreciably in assays at 30 minutes up to 14 days after the blood was drawn. The activity of APRT was 50% higher than in controls.

In the control hemolysate there was little effect of NaF on HPRT activity, while in the patient there was a 14% inhibition of activity at 5 mM NaF and an 80% inhibition at 20 mM NaF (Table II). HPRT activity in the erythrocytes of the mother was normal (1,115 nmoles I M P / m i n / ml pc). Kinetic studies of HPRT revealed that in the patient the Km for hypoxanthine was 0.12 mM at infinite concentration of PRPP, while in the control the value was 0.011 mM. The Km values for PRPP at infinite concentration of hypoxanthine were found to be 0.22 mM for the patient and 0.05 mM for the control. The kinetics were of the normal Michaelis-Menten type for both substrates. The electrophoretic pattern for the HPRT of this child was characterized by low activity and a faster rate of anodal migration than normal (peak A in Fig, 1). Migration was similar to that of Patient R,L. who represented an unrelated family with a variant HPRT. ' Hemolysate from Patient T.L. was reacted with immunoglobulin prepared in rabbits against normal human

388

Sweetman et al.

erythrocyte HPRT and then analyzed electrophoretically ~7 (Fig. 1). In the control, the soluble immunoprotein enzyme complex migrates more slowly to form peak B. The electropherogram for Patient R.L. at the bottom of Fig. 1 is obtaiaed with most variant HPRT enzymes. The absence of peak B indicates that there has been no reaction between the protein and the immunoglobulin. The presence of peak A indicates that there is an enzyme protein present which does not react with antibody. In Patient T.L. there was a distinct peak at position B indicating the presence of CRM. Increasing the amount of immunoglobulin increased the size of peak B and decreased that of peak A. With very high amounts of immunoglobulin, both peak A and peak B were titrated out to form larger complexes which did not enter the gel. In the immunoprecipitation assay, the CRM in Patient T.L. was approximately 20% o f normal. Electrophoretic assay following reaction with immunoglobulin with lysates of cultured fibroblasts derived from Patient T.L. confirmed the results obtained with hemolysates. DISCUSSION The clinical phenotype in this patient was unusual. Hematuria and incomplete, transient renal impairment occurred twice following anesthesia and relatively minor procedures. It is now clear that he had hyperuricemia and was excreting increased quantities of urinary uric acid. Presumably, minor procedures induced a catabolic state with breakdown of cells and hence nucleic acids; azotemia, hypertension, and oliguria resulted from the obstruction of tubules by uric acid crystals. There were no stones or sludge in the ureters, either of which can lead to partial or complete obstruction. 1~-~~ The problem was readily managed using parenteral fluid therapy containing alkali. The need for recognition and prompt management deserves emphasis, because renal shutdown, especially if not managed promptly, could be irreversible. Uricosuric drugs are contraindicated in such a patient. The manner of presentation is of interest because it stimulated an investigation for the presence o f nephritis. Once the azotemia has begun to subside in such a patient hyperuricemia may not be impressive. Therefore, quantitative assessment of the urinary excretion of uric acid is essential. The analysis of the variant HPRT in this patient provides a systematic approach to the characterization of the product of the abnormal gene. The initial determination is a quantitative assessment of the level of enzyme activity. We have felt that analysis of the enzyme activity at subsaturating substrate concentrations might be more likely to reveal an unusual variant compared to the measurement of activity at saturating concentrations of

The Journal of Pediatrics March 1978

substrate. For analysis we use both sets of conditions. 1~ Our patient displayed only 1 to 3% of normal erythrocyte HPRT activity. Nevertheless, the phen0type demonstrated none of the neurologic or behavioral features of the Lesch-Nyhan syndrome. Emmerson and Thompson 7 have emphasized the lack of correlation between residual HPRT activity and neurologic symptoms. Kinetic analysis of the enzyme provides further characterization of a variant enzyme. Benke et a121 have described a patient in whom initial analysis of HPRT at saturating concentrations of substrate revealed a normal level of activity. However, the kinetics were abnormal and the Km value for PRPP was about ten times higher than normal. 8 Our patient appeared to be a different extreme in that his quantitative level of enzyme activity was low. The Km for PRPP was about four times normal and the Km for hypoxanthine was approximately ten times the normal value. As in the HPRT described by Benke et al, ~ the HPRT of this patient was more sensitive than normal enzyme to inhibition by fluoride. Electrophoretic analysis indicated a difference in primary structure causing an alteration in charge or molecular radius that led to migration more rapid than normal. Most of the variant enzymes we have studied in this way have migrated more rapidly than normal. 1", 1~, 22 Kinetic analysis along with enzyme activity and electrophoretic behavior should distinguish most variants. Analysis of the enzyme with immunoglobulin for CRM provides another interesting property o f a variant enzyme. It is now generally accepted that patients with the LeschNyhan syndrome do not have CRM, 17' ~8, 54 even though there is clearly an enzyme present as shown by the appearance of a low activity and altered migration on electrophoresis. TM We have also found no CRM in a variant in which there was 5% of normal HPRT activity. ', ~7 The structure of the HPRT protein in this patient must be such that there are available determinants of immunoactivity in common with the normal enzyme. The study provides evidence that among individuals with deficient activity of HPRT there is considerable genetic heterogeneity. REFERENCES

1. Lesch M, and Nyhan WL: A familial disorder of uric acid metabolism and central nervous system function, Am J Med 36:561, 1964. 2. Seegmiller JE, Rosenbloorn FM, and Kelley WN: Enzyme defect associated with a sex-linked human neurological disorder and excessive purine synthesis, Science 155:1682, 1967. 3. Kelley WN, Greene ML, Rosenbloom FM, Henderson JF, and Seegmiller JE: Hypoxanthine-guanine phosphoribosyl transferase deficiency in gout, Ann lntern Med 70:155, 1969.

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Variant of hypoxanthine-guanine phosphoribosyl transferase

4. Kogut MD, Donnell GN, Nyhan WL, and Sweetman L: Disorder of purine metabolism due to partial deficiency of hypoxanthine-guanine phosphoribosyltransferase, Am J Med 48:148, 1970. 5. McDonald JA, and Kelley WN: Lesch-Nyhan syndrome: Altered kinetic properties of mutant enzyme, Science 171:689, 1971. 6. Yfi T, Balis ME, Krenitsky TA, Dancis J, Silvers DN, Elion GB, and Gutman AB: Rarity of x-linked partial hypoxanthine-guanine phosphoribosyltransferase deficiency in a large gouty population, Ann Intern Med 76:255, 1972. 7. Emmerson BT, and Thompson L: The spectrum of hypoxanthine-guanine phosphoribosyltransferase deficiency, Q J Med 42:423, 1973. 8. Benke PJ, Herrick N, and Herbert A: Hypoxanthineguanine phosphoribosyltransferase variant associated with accelerated purine synthesis, J Clin Invest 52:2234, 1973. 9. Fox IH, Dwosh IL, Marchant PJ, Lacroix S, Moore MR, Omura S, and Wyhofsky V: Hypoxanthine-guanine phosphoribosyl-transferase; characterization of a mutant in a patient with gout, J Clin Invest 56:1239, 1975. 10. Sweetman L, Borden M, Lesh, P, Bakay B, and Becker MA.: Diminished affinity for purine substrates as a basis for gout with mild deficiency of hypoxanthine guanine phosphoribosyl transferase, in Mfiller MM, Kaiser E, and Seegmiller JE, editors: Purine metabolism in man II. Regulation of pathways and enzyme defects, New York, 1977, Plenum Publishing Corp., p 319. 11. Bakay B, Telfer MA, and Nyhan WL: Assay of hypoxanthine-guanine and adenine phosphoribosyl transferases. A A simple screening test for the Lesch-Nyhan syndrome and related disorders of purine metabolism, Biochem Med 3:230, 1969. 12. Bakay B: Detection of radioactive components in polyacrylamide gel disc electropherograms by automated fractionation, Anal Biochem 40:429, 1971. 13. Bakay B, and Nyhan WL: Electrophoretic properties of hypoxanthine-guanine phosphoribosyl transferase isoenzymes by disc gel electrophoresis, Biochem Genet 5:81, 1971. 14. Bakay B, and Nyhan WL: Electrophoretic properties of

15.

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18.

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20. 21.

22.

23.

24.

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hypoxanthine-guanine phosphoribosyl transferase in erythrocytes of subjects with Lesch-Nyhan syndrome, Biochem Genet 6:139, 1972. Bakay B, Nyhan WL, Fawcett N, and Kogut MD: Isoenzymes of hypoxanthine-guanine phosphoribosyl transferase in a family with partial deficiency of the enzyme, Biochem Genet 7:73, 1972. Adye JC, and Gots JS: Further studies on genetically altered purine nucleofide pyrophosphorylases of salmonella, Biochim Biophys Acta 118:344, 1966. Bakay B, Becker MA, and Nyhan WL: Reaction of antibody to normal human hypoxanthine phosphoribosyltransferase with products of mutant genes, Arch Biochem Biophys 177:415, 1976. Nyhan WL: Urate nephropathy, in nephrology, Pediatric Edelmann CM Jr, editor: Boston, Little, Brown & Co., (in press). Landgrebe AR, Nyhan WL, and Coleman M: Urinary tract stones resulting from the excretion of oxypurinol, N Engl J Med 292:626, 1975. Howard RS, and Walzak MP: A new cause for uric acid stones in childhood, J Urol 98:639, 1968. Benke PJ, and Herrick N: Azaguanine-resistance as a manifestation of a new form of metabolic overproduction of uric acid, Am J Med 52:547, 1972. Nyhan WL, and Bakay B: Multiple molecular forms of the purine phosphoribosyl transferases, in Markert CL, editor: Isozymes II, physiological function, Proceedings of the First International Conference on Isozymes at Yale University, New Haven, Conn., April 18-20, 1974, New York, 1975, Academic Press, Inc., pp 385-394. Ghangas GA, and Milman G: Radioimmune determination of hypoxanthine phosphofibosyltransferase crossreacting material in erythrocytes of Lesch-Nyhan patients, Proc Natl Acad Sci USA 72:4147, 1975. Upchurch KS, Leyva A, Arnold WJ, Holmes EW, and Kelley WN: Hypoxanthine phosphoribosyltransferase deficiency: Association of reduced catalytic activity with reduced levels of immunologically detectable enzyme protein, Proc Nail Acad Sci USA 72:4142, 1975.