Unstable galactose-1-phosphate uridyl transferase: A new variant of galactosemia

454

March, 1971 T h e Journal of P E D I A T R I C S

Unstable galactose-l-phosphate uridyl transferase: A neff rariant of galactosemia The presence of an unstable form of galactose-I-phosphate uridyl transferase in a patient,with clinical galactosemia is described. Galactose-l-phosphate uridyl transferase activity in the red blood cells was approximately 40 per cent of normal and demonstrated storage instability in heparin and isotonic phosphate buffer. Galactose-l-phosphate uridyl transferase from the mother and maternal grandmother demonstrated storage instability and a slower eIectrophoretic mobility than normal. These data suggest the presence of an additional allele at the transferase locus, which may result in a new variant of clinical galactosemia, hereby referred to as the Indiana variant.

Claramma M. Chacko, Ph.D., ~ Joe C. Christian, M.D., Ph.D., and Henry L. Nadler, M.D. C H I C A G O , I L L . , AND I N D I A N A P O L I S , IND.

C L A S S I C G A L A C T O S E M I A is a familial metabolic disorder transmitted as an autosomal recessive defect. The clinical manifestations, which include vomiting, diarrhea, jaundice, failure to thrive, hepatic dysfunction, renal dysfunction, cataracts, and mental retardation, are directly related to From the Department o[ Medical Genetics, Indiana University Medical School, the Department of Pediatrics, Northwestern University Medical School, and Children's Memorial Hospital. Supported by the United States Public Health Service, RR-O0057 and HDO0036, Children's Bureau Project No. 924, Riley Association, and The Chicago Community Trust. Presented to the Society for Pediatric Research, Atlantic City, N. ]., May 2, 1970. r Children's Memorial Hospital, 2300 Children's Plaza, Chicago, Ill. 60614.

Vol. 78, No. 3, pp. 454-460

the inability to metabolize galactose. Restriction of galactose from the diet of an affected infant results in improvement of symptoms and may, in fact, be life saving in the first few months of life?' 2 The red blood cells of patients with classic galactosemia have been shown to be deficient of the enzyme galactose-l-phosphate uridyl transferase (E.C. 2.7.7.12: uridine diphosphoglucose: ~-D-galactose- 1-phosphate uridyl transferase) which will be referred to simply as transferase? Transferase is required for the conversion of galactose-1phosphate to glucose-l-phosphate 4, 5; in its absence galactose-l-phosphate accumulates in red blood cells2 Variants of clinical galactosemia have been described. 7, s Segal and associates7 de-

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lineated a group of Negro patients with typical symptoms of galactosemia in infancy, who were able to oxidize radioactive galactose to C1402. Transferase activity could not be detected in the red blood cells of these patients; however, liver biopsies from 2 of these patients demonstrated transferase with lower specific activity than normal but with similar kinetic parameters. 9 Schapira and Kaplan s have reported an incomplete deficiency of transferase in 2 siblings with clinical galactosemia. Electrophoresis of their transferase demonstrated a slower mobility than normal. In this report evidence for the presence of a new variant of the transferase enzyme is presented. This variant is characterized by storage instability of transferase in hepafin and in phosphate buffer and by an altered electrophoretic mobility. CASE REPORT

The patient, an 18-month-old Caucasian girl, had been seen by the Department of Medical Genetics at the Indiana University Medical School with a tentative diagnosis of galactosemia. The child was the product of a full-term gestation of a gravida 4, para 3, 28-year-old woman. The patient had been placed on Nutramigen at birth because one of her siblings had died at the age of 6 weeks following a stormy course characterized by jaundice, diarrhea, vomiting, hepatomegaly, bilateral nuclear cataracts, and the finding of reducing substances and albumin in the urine. At 4 months of age, she was challenged with cow's milk, to which she responded by excreting large amounts of a reducing substance which was negative for glucose, as shown by the glucose oxidase method. For this reason, she was referred to Riley Hospital for confirmation of the diagnosis of galactosemia. On 2 occasions blood was sent for assay of galactose1-phosphate uridyl transferase; the values of 7.2 and 8.5 units (micromoles of uridine diphosphoglucose consumed per hour per gram of hemoglobin) were obtained in comparison to the normal control value of 25.4 -+ 4.9 units. The patient was again challenged with whole milk and again excreted reducing substances in the urine, which were negative for glucose. The patient was rehospitalized at Riley Hospital for definitive diagnostic tests, including re-

New variant of galactosem{a 4 5 5

peat transferase assay, measurement of galactose1-phosphate in red cells, and a modified galactose tolerance test. The patient's red blood ceils were frozen and shipped to Dr. Helen Berry. Studies demonstrated galactose-l-phosphate levels in red cells compatible with that of a galactosemic patient under dietary control and increased serum galactose and decreased serum glucose levels following oral administration of 80 c.e. of milk. No galactose could be detected in urine after fasting; the specimen taken after ingestion of 80 c.c. of milk revealed 350 mg. of galactose per 100 ml. of urine. These findings were consistent with the diagnosis of galactosemia secondary to transferase deficiency. She has been continued on a galactoserestricted diet; growth and development have been within normal limits. Because of the finding on 2 occasions of transferase activity in the genetics laboratory at Children's Memorial Hospital, blood was obtained from the patient and family members for additional study. MATERIALS

AND METHODS

Blood was collected from the patient, family members, galactosemic subjects, galactosemic heterozygous subjects, and normal control subjects, in heparin and stored at 4 ~ C. until processed. To prepare the hemolysate, the red cells were sedimented by centrifugation, and the plasma and buffy coat were removed. T h e cells were washed 3 times with isotonic saline and any residual bully coat was removed. Hemolysis was achieved by addition of one volume of water, freezing in a dry ice-acetone bath, a n d thawing in a water bath at 37 ~ C. 3 times. Assay of galactose-l-phosphate uridyl transferase. Transferase was assayed either by a modification of the uridine-5-diphosphate glucose ( U D P G ) consumption assay of Mellman and TedescC ~ or by the nicotinamide adenine dinucleotide phosphatelinked assay? 1 The U D P G consumption assay was performed as follows: 0.05 ml. of hemolysate was preincubated at 37 ~ C. for 15 minutes with 0.05 ml. of 0.5M glycylglycine buffer p H 8.7, and 0.05 ml. of 97.5 raM. dithiothreitol. After preincubation, 0.05 ml. of 2.91 raM. U D P G and 0.05 ml. o f 6.9 mM.

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galactose-l-phosphate were added. These test solutions were always run in duplicate. Another set of blank duplicates was also run in which deionized water replaced galaetose1-phosphate. The samples were then incubated at 37 ~ C. for 15 minutes. At the end of the incubation period, 1.75 ml. of 0.15M glycylglycine buffer pH 8.7 was added and the reaction stopped by heating the tubes in a boiling water bath for 5 minutes. After cooling, the tubes were centrifuged and the supernatant was removed. T o determine the amount of UDPG left in the system, 0.5 ml. of the supernatant was incubated with 0.3 ml. of 0.15M glycylglycine buffer pH 8.7 and 0.1 ml. of 0.01M nicotinamide adenine dinucleotide pH 8.0 and 0.1 ml. of 900 units of uridine-5-diphosphate glucose dehydrogenase. Absorbancies were read at 340 and 400 m/~ before and 20 minutes after the addition of uridine-5-diphosphate glucose dehydrogenase. The 400 m? readings were subtracted from the 340 m,a readings to correct for nonspecific absorption. The difference in corrected absorbancies between the average of test samples and the average of blanks represents the amount of U D P G consumed. The results are calculated as micromoles of UDPG consumed per hour per gram of hemoglobin (units). The nicotinamide adenine dinucleotide phosphate-linked assay was performed as follows: 1.0 ml. of reaction mixture contained 0.1 ml. of partially purified enzyme, 50 /~moles of glycylglycine p H 8.7, 1.95 ~moles of D T T , 0.16 /zmoles of MgC12, 0.6 moles of nicotinamide adenine dinucleotide phosphate, 0.1 unit of phosphoglucomutase (rabbit muscle), 0.04 units of glucose-6phosphate dehydrogenase, (type XV--Baker's yeast), 0.007 units of 6-phosphogluconic dehydrogenase (type V--yeast), and 0.6 ~moles of U D P G and 2.0 ~moles of galaerose-l-phosphate. The rate of nicotinamide adenine dinucleotide phosphate reduction was recorded at 340 m~ in a Gilford Model 2000 Spectrophotometer at 37 ~ C. against a blank cuvette from which either UDPG or galactose-l-phosphate was omitted. The reaction was initiated by either the addition

The Journal of Pediatrics March 1971

of enzyme, galactose-l-phosphate, or UDPG. Partial purification of transferase. Partial purification of transferase was accomplished by absorbing 1 volume of dialyzed hemolysate on 1 volume of diethylaminoethylcellulose equilibrated with 0.015M tris-acetate buffer pH 7.4. After being allowed to stand with the diethylaminoethylcellulose at 4 ~ C. for 30 minutes, the slurry was placed on a Buchner funnel and the hemoglobin removed by washing with 0.015M tris-acetate buffer p H 7.4. The transferase was then eluted from the diethylaminoethylcellulose with 1 volume of 0.5M tris-acetate buffer p H 7.4 and frozen until assay. Immediately before assay transferase was concentrated by negative-pressure dialysis in a collodion bag against 0.01M glycylglycine buffer pH 7.5 and diluted in such a way that 0.1 ml. of enzyme in a final volume of 1.0 ml. gave approximately 0.005 to 0.01 optical density units per minute at maximum velocity. Phosphate inhibition. The effects of storage of blood in isotonic phosphate upon transferase activity were studied as follows: Immediately after collection of blood, the red blood cells were placed in 0.10M isotonic sodium phosphate buffer pH 7.4 and stored at 4 ~ C. The cells were washed 3 times in isotonic saline, the hemolysates prepared, and transferase activity determined by the UDPG-consumption method described above. p H Optima studies. The p H optima curve was determined by the UDPG-consumption assay method as described above, substituting 0.05 ml. of 0.5M glycylglycine buffer for pH values between 6.5 and 9.0. Michaelis-Menten constants. The Michaelis-Menten constants of the enzyme for U D P G at 2.0 raM. of galactose-l-phosphate and for galactose-l-phosphate at 0.6 raM. of U D P G were carried out at 37 ~ C. using the nicotinamide adenine dinucleotide phosphate-linked assay with 0.06 to 1.2 raM. of U D P G and 0.2 to 3.0 raM. of galactose-1phosphate. Starch gel electrophoresis. Electrophoresis of transferase in the hemolysates was carried out on a 13 per cent starch gel in 0.01M potassium phosphate buffer p H 7.0, accord-

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New variant of galactosemia

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Number 3

ing to the method described by Mathai and Beutler. 12 Materials. All chemicals used were purchased from Sigma Chemical Company, St. Louis, Mo., with the exception of disthiothreitol which was obtained from California Biochemicals and hydrolyzed starch from Cannaught Medical Research Laboratories, Toronto, Canada. RESULTS

The levels of transferase activity in heparinized red blood cells from normal control subjects, galactosemic patients, and galactosemic heterozygous subjects are shown in Table I. A number of these families have been previously reported by Nadler and associates? 3 Transferase activity from our patient ranged from no detectable activity to 11.0 units. Transferase activity from the mother and maternal grandmother ranged from 16.3 to 18.2 units, while the father's ranged from 10.5 to 14.2 units. The proband's great uncle and his wife had 12.7 and 13.5 units, respectively, while one of the male siblings had 12.4 units of transferase activity. The results of the effect of storage of heparinized blood at 4 ~ C. upon transferase activity were as follows: The proband's transferase activity, initially 11.0 units, fell to undetectable levels within 36 hours, in contrast to the normal control subjects (27.0 units initially) and galactosemic heterozygous subjects (13.0 units initially) in whose blood cells no decrease in transferase activity could be demonstrated during the 72 hours of storage. The father's transferase activity (12.0 units initially) remained stable in contrast to the mother's transferase activity which fell from 17.3 units to 9.8 units during 72 hours of storage. The effect on transferase activity of storage of red Blood cells for 24 hours in phosphate buffer were as follows: Essentially no change could be demonstrated in normal control subjects (25 units to 23 units), while galactosemic heterozygous subjects had a decrease in activity of approximately 20 per cent (12.5 units to 10 units). The father's

Table I. Transferase activity in heparinized red blood cells ~

Subjects Control subjects Obligate heterozygous subjects Galactosemic patients

No. #moles UDPG conof sumed/hr./Gm. patients hemoglobin 3O 33

25.4 + 4.9 12.6 + 6.3

20

1.4 • 1.2

*UDPG consumption method of Mellman and Tedeseo? ~ Mean _+ S.D.

transferase activity felt 17 per cent from 12.0 units to 10.0 units, while the mother's transferase activity decreased from 18.0 units to 9.0 units (50 per cent). The proband's transferase activity could no longer be detected after 24 hours of storage. Incorporation of 0.1M disodium phosphate directly into the preincubation mixture resulted in a 50 per cent reduction in transferase activity in all cases. Transferase activities determined at various pH values from 6.5 to 9.0 in the hemolysates from the proband's parents, normal control subjects, and galactosemic heterozygous subjects revealed a broad curve with an optimum at approximately 8.5 to 8.7. The Michaelis-Menten constants for galactose-l-phosphate at 0.6 mM. of U D P G for normal control subjects, 4.65 x 10-~ M; galactosemic heterozygous subjects, 2.70 x 10-~ M; proband's father, 3.84 x 10-4 M; and mother, 3.67 x 10-4 M, were similar. The Michaelis constant values for U D P G at 2.0 mM. of galactose-l-phosphate were similar in all instances: normal control subjects, 1.03 x 10 -4 M; galactosemie heterozygous subjects, 1.17 x 10-4 M ; proband's father, 9.20 x 10-5 M; and mother, 8.7 x 10-5 M. The recovery of transferase of the proband's hemolysate from diethylaminoethylcellulose was very low, and its yield was further reduced during concentration by negative-pressure dialysis. Hence, Michaelis constant studies could not be attempted at this stage. The results of starch gel electrophoresis of transferase from mother, father, maternal

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The Journal of Pediatrics March 1971

Fig. 1. Starch gel electrophoresis of transferase from the mother (slots 3 and 7), father (slot 6), maternal grandmother (slot 5), control subject (slot 8) and galactosemic heterozygous subject (slot 4), demonstrating decreased mobility in the transferase of mother and maternal grandmother.

grandmother, and normal control subjects are shown in Fig. 1. Transferase from the normal control subjects, galactosemic heterozygous subjects, and the father demonstrates a single fluorescent band at identical positions, whereas the mother and maternal grandmother have a single band with decreased mobility. No transferase band could be detected in hemolysates from galactosemic subjects. The hemolysate of the proband did not reveal any transferase band, possibly due to its inactivation during electrophoresis. DISCUSSION

The finding of transferase activity approximately 35 per cent of normal in a patient with clinical galactosemia is unexpected. The transferase from the patient was highly unstable as demonstrated by the rapid loss of activity upon storage in heparin or in the presence of isotonic phosphate buffer. Transferase activities in the mother and maternal grandmother were approximately 75 per cent of normal and demonstrated instability upon storage in heparin and phosphate buffer. The electrophoretic mobility of the transferase from mother and maternal grandmother was slower than the transferase from normal control subjects or "classic"

galactosemic heterozygous subjects. In contrast, the transferase from the father was approximately 50 per cent of normal and behaved in all studies similar to that from normal control subjects and galactosemie heterozygous subjects. These studies clearly indicate that the patient has a variant form of "classic" clinical galactosemia and that another allele may be added to those previously described at the transferase locus, v, s, 15 The transferase obtained from the mother and maternal grandmother had activity similar to that found in the Duarte variant heterogygote?~ In contrast to the Duarte variant, in which the transferase has a more rapid electrophoretic mobility, ~2 the electrophoretic mobility of their transferase was decreased. The data from the family members suggest that both the gene for classical galactosemia and the gene for the Indiana variant are present in the family (Fig. 2). The electrophoretic mobility, phosphate inhibition, and storage stability of transferase from the great uncle, his wife, and the proband's brother suggested that they were heterozygous for classic galactosemia. The interpretation of data from family members strongly suggests that the patient

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New variant o[ galactosemia

459

T I['1 MALE CARRIER OF TRANSFERASE DEFICIENCY

9 GALACTOSEMIC TRANSFERASE

DEFICIENT HOMOZYGOTE

( ~ FEMALE CARRIER OF INDIANA VARIANT

GALACTOSEMIC, TRANSFERASE/INDIANA DEFICIENT / VARIANT HETEROZYGOTE

1(~ SPONTANEOUS ABORTION DECEASED

9 EXAMINED

Fig, 2. Pedigree of the family as interpreted from data collected in this study.

is a double heterozygote with the "classic" galactosemia gene inherited from the father and the Indiana variant galactosemia gene inherited from her mother. Double heterozygous subjects at the transferase locus involving the classic galactosemic allele and the Duarte allele have previously been reported. 14 These studies of the transferase enzyme suggest that the variant gene has led to the synthesis of a structurally altered protein in which changes in the 3 dimensional configuration of the enzyme m a y have resulted in molecular instability and interference with its function. A similar phenomenon has been evaluated in the case of glucose-6-phosphate dehydrogenase deficiency. 15 I n this instance an unstable enzyme results in markedly shortened half-life with reduction in levels of enzyme activity to as low as 2 to 3 per cent of normal. 16 T h e finding of unstable and therefore functionally deficient enzymes may well be a c o m m o n cause of enzyme deficiency in m a n y of t h e familial metabolic disorders of man. We thank Linda McCrone for excellent tech-

nical assistance and Dr. David Hsia for permitting us to study some of his patients.

REFERENCES 1. Isselbacher, K. J.: Galactosemia, in Stanbury, J, B., Wyngaarden, J. B., and Fredrickson, D. S., editors: The metabolic basis of inherited disease, New York, 1966, McGrawHill Book Company, p. 178. 2. Hsia, D. Y. Y. (editor) : Galactosemia, Springfield, Ill., 1969, Charles C Thomas, Publisher. 3. Isselbacher, K. J., Anderson, E. P., Kurahashi, K., and Kalckar~ H. M.: Congenital galactosemia: A single enzymatic block in galactose metabolism, Science 193: 635, 1956. 4. Leloir, L. F.: Enzymatic transformation of uridine diphosphate glucose into galactose derivative, Arch. Biochem. 33: 186, 1951. 5. Kalckar, H. M., Braganca, B., and MunchPetersen, A.: Uridyl transferases and the formation of uridine diphosphate galactose, Nature 172: 1038, 1953. 6. Schwarz, V., Goldberg, L., Komrower, G. M., and Holzel, A.: Some disturbances of erythrocyte metabolism in galactosemia,'Biochem. J. 62: 34, 1956. 7. Segal, S., Blair, A., and Topper, Y. J.: Oxidation of carbon-14 labled galactose by ;ubjects with clinical galactosemia, Science 136: 150, 1962. 8. Schapira, F., and Kaplan, J. C.: Electrophoretic abnormality of galaCtose-l-phosphate

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I0.

11.

12.

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uridyl transferase in galactosemia, Biochem. Biophys. Res. Commun. 35; 451, 1969. Segal, S., and Rogers, S.: Human liver galactose-l-phosphate uridyl transferase: Activity in the Negro galaetosemic, The Society for Pediatric Research, 40th Annum Meeting, Programs and Abstracts, 1970, p. 50. Mellman, W. J., and Tedesco, T. A.: An improved assay of erythroeyte and leucocyte galactose-l-phosphate uridyl transferase: Stabilization of the enzyme By a thiol protective reagent, J. Lab. Clin. Med. 66: 980, 1965. Beutler, E., and Baluda, M. C.: Biochemical properties of human red cell galactose-1phosphate uridyl transferase (UDPGlucose: a-D-galactose-l-phosphate uridyl transferase E. C. 2. 7. 7. 12) from normal and mutant subjects, J. Lab. Clin. Med. 67: 947, 1966. Mathai, C. K., and Beutler, E.: Electro-

The Journal o[ Pediatrics March 1971

13.

14.

15. 16.

phoretic variation of galactose-l-phosphate uridyl transferase, Science 154: 1179, 1966. Nadler, H. L., Inouye, T., and Hsia, D. Y.-Y.: Clinical galactosemia: A study of 55 cases, in Hsia, D. Y.-Y., editor: Galactosemia, Springfield, Ill., 1967; Charles C Thomas, Publisher, p. 127. Beutler, E., Baluda, M. C., Sturgeon, P., and Day, R. W.: The genetics of galactose-1phosphate uridyl transferase deficiency, J. Lab. Clin. Med. 68: 646, 1966. Harris, H.: Genetieal theory and the inborn errors of metabolism, Brit. Med. J. I: 321, 1970. Piomelli, S., Corash, L. M., Davenport, D. D., Miraglia, J., and Ambrosi, E. L.: In vivo lability of glucose-6-phosphate dehydrogenase in GdA- and Gd Mediterranean deficiency, J. Clln. Invest. 47: 940, 1968.

Erratum. In the article, "Treatment of childhood acute lymphocytic leukemia," by Donald Pinkel, which appeared on pp. 1089-1091 of the December, 1970, issue of the JougxaL, line 12 on p. 1090 should have read: Five-year cure is a traditional measure of success in cancer therapy. Line 20 on p. 1090 should have read: A slightly higher percentage (12/229) was reported for children receiving cyclic or sequential chemotherapy. Line 34 on the same page should have read: In 8 years of experience none of the children with acute lymphocytic leukemia in remission on multiple agent-combination chemotherapy have died of thrombocytopenic hemorrhage. In the second column on p. 1090, line 3 should have read: Administration of daunorubicin (daunomycin, rubidomycin) resulted in complete remission in 4 out of 39 children in one series but in a higher proportion in another.