Clin Biochem, Vol. 24, pp. 293-300, 1991 Printed in Canada. All rights reserved.
0009-9120/91 $3.00 + .00 Copyright © 1991 The Canadian Society of Clinical Chemists.
Galactosemia: Screening and Diagnosis ERNEST BEUTLER Scripps Clinic and Research Foundation, Research Institute of Scripps Clinic, Department of Molecular and Experimental Medicine, 10666 North Torrey Pines Road, La Jolla, CA 92037, USA Galactose is normally metabolized to glucose through the coordinated activities of three enzymes: galactokinase, galactose-1phosphate uridyl transferase (GALT), and uridine diphosphoglucose 4-epimerase (epimerase). High concentrations of galactose and their metabolites are toxic to mammals. Hereditary deficiencies of galactokinase and of GALT and perhaps rarely of epimerase cause clinical disorders that can be prevented by early recognition and institution of a galactose-frea diet. The genetics of disorders of galactose metabolisms and the methods used currently for their detection are reviewed. Future prospects in the diagnosis of these disorders are discussed.
by which galactose is metabolized (2). It requires that the galactose be phosphorylated at the 1-position, attached to a pyrimidine, and finally be isomerized to glucose. The three relevant reactions are summarized in Figure 1. Although it was once claimed that an enzyme that oxidizes galactose directly, viz. galactose dehydrogenase, exists in mammals (3,4), the data were found to be due to an artifact (5-7).
KEY WORDS: galactekinase; galactose-l-phosphate uridyl transferase; newborn screening; epimerase; uridine diphosphoglucose epimerase.
Clinical manifestations o f different biochemical abnormalities o f galactose metabolism
Introduction
The inability to metabolize galactose normally is characterized by high blood and urine galactose levels. It was probably first described by GSppert in 1917 (8), and is referred to as galactosemia.
actose (milk sugar), a dissacharide of glucose L and galactose, is the main carbohydrate in the diet of young mammals. Only certain aquatic mammals, such as sea lions, seem to be an exception to this rule (1). Adults, too, ingest galactose, since this sugar is found in glycoproteins and glycolipids. While glucose, the primary energy source for most cells, is metabolized directly after phosphorylation, higher organisms are apparently unable to metabolize galactose without first transforming it into glucose. Without the capability of metabolizing galactose directly, humans must rely on a relatively complex metabolic pathway to convert galactose to glucose before metabolic energy can be extracted from it. THE METABOLISMOF GALACTOSE Understanding the molecular basis of galactosemias had to await unraveling of the pathway
Correspondence: Ernest Beutler, Scripps Clinic and Research Foundation, Research Institute of Scripps Clinic, Department of Molecular and Experimental Medicine, 10666 North Torrey Pines Road, La Jolla, CA 92037, USA. Manuscript received August 28, 1990; accepted December 29, 1990. CLINICAL BIOCHEMISTRY, VOLUME 24, AUGUST 1991
CLASSICAL GALACTOSEMIA(GALACTOSE-1-PHOSPHATE URIDYL TRANSFERASE DEFICIENCY)
Isselbacher and colleagues measured the activities of all three enzymes of galactose metabolism in 10 patients with classical galactosemia. They discovered that the second enzyme in the pathway,
Galactokinase = GaI-1-P + ADP
(1)
Galactose + ATP
(2)
GaI-I-P + UDPG -..
(3)
Epimerase UDPGal -... ~ UDPG NAD
Transferase
"~ Glu-I-P + UDPGal
Figure 1--The pathway by which galactose is converted to glucose. Gal-I-P = galactose-l-phosphate; UDPG = uridine diphosphoglucose; Glu-l-P = glucose-l-phosphate; UDPGal = uridine diphosphogalactose; Transferase -- galactose-l-phosphate uridyl transferase; Epimerase = uridine diphosphoglucose 4-epimerase.
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galactose-l-phosphate uridyl transferase (GALT), was deficient (9). Heterozygotes had about one-half normal activity of GALT in their erythrocytes. Subsequently, many investigators confirmed that this enzyme was lacking in the erythrocytes of galactosemic patients. Infants with the classical form of galactosemia become jaundiced, take feeding poorly and may develop ascites. They often die of sepsis during the first few weeks of life (10). If dietary control is instituted late or compliance with the diet is poor, cataracts and mental retardation develop. The adequate treatment of galactosemia has a dramatic effect on the prognosis of the affected child. Galactosemic children fed a diet containing little or no galactose show satisfactory growth and general health. Intellectual progress may be normal (11) or modestly impaired (12-14). Hypogonadism is common in galactosemic girls as they mature (15,16). Despite the persisting minor stigmata of galactosemia, prompt institution of a galactose-free diet is, quite literally, a matter of life and death. Thus, a rapid and accurate diagnosis deserves a high priority. It is one of few genetically determined disorders that can be treated very effectively once the diagnosis is established. Rarely, patients with galactosemia exhibit normal red cell GALT activity, setting the stage for the discovery of deficiencies of the other two enzymes that are associated with abnormal galactose metabolism. GALACTOKINASE DEFICIENCY
The first defect found in galactosemic children with normal GALT activity was galactokinase deficiency (17). The clinical course of these patients was quite atypical for galactosemia. Although they had not received a galactose-restricted diet, they had none of the stigmata of galactosemia except for cataracts. One parent of a child described earlier also had cataracts (18). This suggested that since galactokinase is the limiting step in galactose metabolism, even a modest reduction of enzyme activity could result in development of cataracts (19). The incidence of the heterozygous state for galactokinase deficiency is more prevalent among patients developing cataracts in the first few years of life than in the general population (20-23). EP1MERASE DEFICIENCY
The other defect, even more rare, is one of U D P G galactose-4-epimerase. In most subjects studied, the defect appears to be limited to circulating blood cells. Activity in liver, cultured skin fibroblasts, and activated lymphocytes have usually been normal (24); however, in one symptom-
294
atic case (see below), the epimerase activity of cultured fibroblasts was deficient. Although affected individuals have elevated red cell galactose1-phosphate levels, they generally do not suffer ill effects and develop normally (24,25). In rare cases, severe clinical symptoms and signs similar to those found in classical galactosemia were associated with epimerase deficiency (26-28); in one of these (27), the diagnosis was later retracted (29).
Genetics and incidence of the galactosemias All three forms of galactosemia are inherited as autosomal recessive disorders. In each case, both parents of severely affected individuals have approximately one-half the normal enzyme activity. CLASSICAL GALACTOSEMIA(GALACTOSE-I-PHOSPHATE URIDYL TRANSFERASEDEFICIENCY)
Before data from large-scale screening programs became available, the most accurate w a y to estimate the expected birth incidence of galactosemia was to ascertain the percentage of the population who were heterozygous for the enzyme deficiencies. However, attempts to do so were confounded by the presence of alleles in the population that lowered enzyme activity, but did not have the capacity to cause galactosemia even in the homozygous state. Most important of these alleles is the Duarte variant. We discovered this polymorphism while attempting to determine the gene frequency for galactosemia (30,31). Family studies revealed the existence of two distinct types of individuals with one-half normal transferase activity. There were those whose parents or children were either normal or had one-half normal enzyme activity. In contrast, in some families both parents and all children of the person expressing one-half normal transferase activity exhibited three-quarters normal enzyme activity (Figure 2). Thus, we deduced that the GALT activity expressed by the Duarte allele is approximately one-half of that expressed by the normal allele. Homozygotes for the Duarte variant have approximately one-half of normal activity and thus mimic carriers for galactosemia. The Duarte variant can be identified by its rapid electrophoretic migration (32-35) and abnormal isoelectric point (36,37) as well as its reduced activity. Even a single Duarte allele is sufficient to prevent the clinical expression of galactosemia; babies born with a galactosemia allele and a Duarte allele have one-quarter of normal enzyme activity. Such children have a reduced capacity to metabolize galactose, with abnormal accumulation of galactose-l-phosphate in their red cells (38,39). However, no ill effects have been found to occur in these individuals (30,31,38,40,41). An infant homozygous for the Duarte variant was believed to
CLINICAL BIOCHEMISTRY, VOLUME 24, AUGUST 1991
GALACTOSEMIA: S C R E E N I N G A N D DIAGNOSIS TYPE G FAMILIES (9)
-eL_ _
10
0
20
: :
30
TYPE 0 FAMILIES(4| • PROPOSITUS
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i
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10 w
0
.
.
.
.
i
20
30
DOUBLY ANOMALOUS FAMILIES (2}
10
20
310
Figure 2--The transferase activity of parents and children of propositi with half-normal (top two panels) and one-quarter normal (bottom panel) transferase activity. The families with half-normal activity could be divided into those in which the gene responsible for transferase deficiency was the gene for galactosemia (type G families) and those in which it was the gene for the Duarte variant (type D families). The distribution of enzyme activity in parents and children confirms the proposed genetics of transferase deficiency, viz. that the individuals in the type D families are homozygous for a mutation that codes for one-half normal transferase activity--the Duarte variant (from Hsia, D.Y.-Y., Galactosemia, 1969. Courtesy of Charles C Thomas, Publisher, Springfield, Illinois).
galactosemia have been estimated to be 0.0021 (44), 0.0024 (53) and 0.004 (54). Screening programs in various populations have shown that the birth frequency of the disease corresponds quite well to these estimates, with an average value 1:62,000 having been reported in studies in various populations (55). The actual reported incidence varies considerably in different populations. In Western Austria, the frequency was 1:13,647, while in Eastern Austria it was 1:43,061 (56). In eight countries in Western Europe, the incidence average was 1:34,000 (57,58). In Switzerland the incidence was 1:57,000 (59) and in New Zealand it was 1:34,000 (60). In Germany, 1:45,282 live births were galactosemic children (61). GALACTOKINASE DEFICIENCY
Estimates of the frequency of galactokinase deficiency are also fraught with difficulty. Among persons of African ancestry, a common allele, the Philadelphia variant that lowers the enzyme activity, has been found (53). Presumably this allele is present albeit at lower levels in non-African populations. The gene frequency estimate for galactokinase deficiency based on heterozygotes is 0.0015, predicting a homozygote frequency of 1:462,000. The birth frequency found in various screening programs ranges from 0:582,000 to 1:168,022 (55). EPIMERASE DEFICIENCY
be symptomatic (42) but was later diagnosed as having the ZZ variant of ~-l-antitrypsin (43). The Duarte allele is very common. We originally estimated its gene frequency to be about 0.0555 (31) and subsequently, in a Greek population, to be 0.0548 (44). Subsequent estimates have been very similar (45,46) but the gene frequency among American blacks (46) and Orientals (46,47) is considerably lower. Thus, some 11% of Caucasian subjects are heterozygous for the Duarte variant. Estimates that are based on electrophoretic mobility only are likely to include heterozygotes for the Los Angeles variant (see below), and are therefore somewhat higher (48). The Los Angeles variant is another m u t a n t allele of GALT. First described by Ng et al. (46), this enzyme has increased activity that is electrophoretically identical to the Duarte variant. The gene frequency of this variant is about 0.025 among Caucasians (45). It is apparently quite rare among Orientals (47). Other rare variants of GALT have also been reported, including the Chicago I and II (49), Rennes (50), Indiana (51) and Berne (52) variants. Only estimates of gene frequency that exclude homozygotes for the Duarte variant provide meaningful data regarding the frequency of true galactosemia alleles in the population. Based on these considerations, the frequency of the alleles for true
CLINICAL BIOCHEMISTRY, VOLUME 24, AUGUST 1991
There are few reliable data regarding the incidence of epimerase deficiency. A screening program in Australia detected 2 infants with this disorder out of 207,000 screened (62). In Japan, screening for epimerase deficiency yielded 2 cases out of 23,049 newborns tested (63). In both studies, all infants detected were clinically normal. Screening n e w b o r n s for galactosemia Because of the high early mortality associated with galactosemia, needless deaths result from limiting diagnostic measures to infants who develop symptoms. In one study, 6 of 10 infants with galactosemia died between the 8th and 18th day of life because diagnosis was delayed to days 7 to 17 (10). Screening newborns for hereditary disorders is best carried out using spots of dried blood. Because such collections are made for phenylketonuria testing, the inclusion of tests for other disorders minimizes the cost of the procedure. Two principal approaches using such samples have been employed to screen for galactosemia. The first measures the activity of GALT itself. The second uses micro-organisms to determine the amount of galactose in the blood sample. We first became interested in screening for galactosemia when Dr. J. Pollard, a chemist at Cal-
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+
Transferase UDPGal + Glu-l-P
,/
Phosphoglucomulase
/
Glu-6-P Jr
Glucose-6-PDehydrogenaseI 6-PGA .-t-
Figure 3--The reactions of the fluorescent screening test for galactosemia. The reaction mixture and hemolysate provide the reagents shown in boxes. When transferase is present, the reactions proceed with the reduction of NADP to NADPH, which is detected by virtue of its fluorescence.
Biochem, told us he developed a method for synthesizing relatively large quantities of galactose-l-phosphate. Thus, current methods for detecting glucose-6-phosphate dehydrogenase (G6PD) deficiency could be adapted to screen for transferase deficiency by linking the transferase reaction, which yielded glucose-l-phosphate, to the G6PD reaction using glucose-6-phosphate as a substrate. The glucose-l-phosphate formed by the transferase could readily be converted to glucose6-phosphate by phosphoglucomutase, an enzyme that is relatively abundant in the erythrocyte. In the 1960's, one of the most convenient methods for screening for G6PD deficiency was the brilliant cresyl blue decolorization test of Motulsky and Campbell-Kraut (64). This test linked the reduction of NADP by glucose-6-phosphate and G6PD to the reduction of brilliant cresyl blue to the reduced leuko-form. For large-scale screening, it was necessary to cover the solution with mineral oil, so we simplified the procedure considerably by using methylene blue as the receptor dye, avoiding auto-oxidation by gassing the solution with CO and stoppering it (65). This method, though useful in screening hundreds (66) and even thousands (67) of subjects at risk, was cumbersome to apply to the millions of infants who required screening to avert needless deaths from galactosemia. Recognizing that the endogenous fluorescence of 296
NADPH could be used as an endpoint of NADP reduction by the linked system made possible a technically simpler technique, the fluorescent spot test (68). This method eliminated the need for anaerobic conditions and a dye and required merely an ultraviolet hand-lamp to appraise the results. The principle of the test is summarized in Figure 3. This test measures the gene product directly, and the results are not dependent upon dietary intake of galactose. The compound heterozygote for galactosemia and the Duarte variant (GALTD/GALT°), an individual with about 1/4 normal enzyme activity, may give false positive results. Moreover, because of its high specificity for GALT, the fluorescent spot test will miss the very rare case of galactokinase deficiency in patients who would also benefit by early restriction of galactose intake. However, since the enzyme is unstable to heating, samples stored at very warm temperatures may give false positive results, causing a high recall rate (61). It has been proposed that this problem may be largely overcome by adding dithiothreitol to the reaction mixture (69). Blood galactose levels are usually measured microbiologically using an auxotrophic microorganism. The E. coli K-12 mutant strain W3805 lacks epimerase and cannot synthesize a normal cell wall unless galactose is present in the medium. The abnormal cell wall formed in the absence of galactose unmasks a receptor for bacteriophage C21, and thus the cell is lysed in galactose deficiency but grows normally in the presence of galactose. When punched out blood spots are placed on a semisolid medium seeded with the microorganism and the phage, the zone of growth around the sample is proportional to the logarithm of its galactose concentration (70). One strength of this approach is that it detects all forms of galactosemia, not just classical galactosemia, the most common form, caused by a deficiency of GALT. Moreover, galactose and galactose-l-phosphate (which accumulates in the red cells of galactosemic subjects) are quite stable, even at warm temperatures. On the other hand, blood galactose levels depend upon dietary intake of galactose. Thus, the galactosemic who is vomiting and not absorbing milk sugar may, theoretically, be missed. The method also appears to be fairly technically demanding (70). For example, agar cannot be used because of its galactose content, and silica gel must be used instead. The bacteria will not grow if inadequately aerated or if repeatedly frozen and thawed. Care must be taken with pH, contamination with other micro-organisms and the quantities of phage and bacteria used. Despite these difficulties, the technique has been applied successfully in different settings (55) and the availability of a test kit may increase its applicability (71). It is unclear to what extent antibiotics may give false positive tests; some antibiotics interfere with a microbiologic test for galactosemia reported earlier (72). CLINICAL BIOCHEMISTRY, VOLUME 24, AUGUST 1991
GALACTOSEMIA: SCREENING AND DIAGNOSIS
In recent years, other methods for the detection of galactose and galactose-l-phosphate in blood spots have been introduced. These depend upon the oxidation of galactose by galactose dehydrogenase with the detection of N A D H by fluorescence (73-75). Although a fluorescent screening test for galactokinase has been published (76), the results obtained are probably invalid because blood glucose can act as a substrate for N A D P H formation in the linked reaction used.
The definitive diagnosis of galactosemia Red blood cells function normally in patients with all types of galactosemia, b u t fortunately they contain the enzymes of galactose metabolism. Thus, they serve as a convenient and powerful tool for the diagnosis of the galactosemias, and the defects underlying all three forms of hereditary galactosemia were originally discovered by examining erythrocytes from affected patients. Whether detected as a result of a screening test on newborns or on the basis of clinical findings, confirmation of the diagnosis of galactosemia requires quantitative assays for the enzymes of galactose metabolism. CLASSICAL GALACTOSEMIA (GALACTOSE-1-PHOSPHATE URIDYL TRANSFERASE DEFICIENCY)
Several different means are available for the measurement of galactose-l-phosphate-uridyl transferase activity in red cells. In this reaction (Figure 1; reaction 2) galactose-l-phosphate is converted to glucose-l-phosphate, while U D P glucose is converted to U D P galactose. The classical method of measuring enzyme activity is to determine the amount of U D P G consumed in a hemolysate incubated with U D P G and galactose-1phosphate. This technique, which designated the U D P G consumption assay, is quite specific and reliable for the diagnosis of galactosemia. However, it is an endpoint assay, and as such it does not directly give true initial enzyme rates. Because the reaction product UDPGal is a competitive inhibitor, the relationship between enzyme added and U D P G consumed is not linear. We developed correction factors which compensate for the non-linearity of the assay (77-79). The U D P G consumption assay loses accuracy when small amounts of enzyme activity are measured. Under these conditions, a large value is subtracted from a slightly larger one in calculating the amount of U D P G consumed. A satisfactory alternative method for estimating GALT depends upon the linkage of glucose1-phosphate formation in the primary reaction to phosphoglucomutase, converting the glucose-lphosphate to glucose-6-phosphate, and then a further linkage to glucose-6-phosphate dehydrogenase, which reduces NADP to NADPH. The rate CLINICAL BIOCHEMISTRY, VOLUME 24, AUGUST 1991
of N A D P H formation can be measured fiuorometrically and is linearly related to GALT activity (80). The same approach has been used widely to develop a spectrophotometric technique (81-83) for transferase assay. In addition to these techniques, radioactive methods have been developed using chromatographic separation of the radioactive substrate galactose-l-phosphate from the radioactive product UDPGal (84-86). This approach, though cumbersome for the measurement of red cell enzyme activity, is valuable when GALT activity of other tissues is to be estimated. In tissues such as cultured amniotic fluid cells, the U D P G consumption assay and the NADP-linked procedures are unreliable because side reactions interfere. GALACTOKINASE DEFICIENCY
Galactokinase activity of red cells is very low and a radioactive assay is more suitable. Our assay is based upon the fact that phosphorylated galactose is strongly negatively charged and binds to the anion exchange paper DE-52. In contrast, galactose is uncharged and readily washed from this medium. Accordingly, hemolysate is incubated with ATP and radioactive galactose, and the mixture is spotted on DEAE paper and vigorously washed (87). After the washed paper is dissolved in scintillation fluid (88), counting provides an accurate measurement of galactokinase activity. EPIMERASE DEFICIENCY
Epimerase may be determined by a two-step procedure: hemolysate or tissue sample is incubated with UDPGal and NAD, a necessary co-factor, the reaction is stopped and the amount of UDPGlucose formed is measured with UDPGlucose dehydrogenase (89). With good instrumentation, it is possible to measure the enzyme by continuously monitoring N A D P H formation in a linked system (90); however, the rate of the reaction is sufficiently slow in normal red cells that the precision of the linked assay is relatively low.
Future prospects Recently, the cDNA for GALT has been cloned and sequenced (91,92), and the molecular biology of galactosemia will undoubtedly increase our understanding of the incidence and population genetics of this group of disorders. Because even classical galactosemia is relatively rare, it is reasonable to assume that many cases arose as new mutations and that the disorder is heterogeneous at a nucleotide level. This assumption is strengthened by the finding that more common genetic disorders occurring at polymorphic frequencies in some populations are heterogeneous. Examples of this finding have been reported in some patients 297
BEUTLER with Gaucher disease (93), Tay-Sachs disease (94), and cystic fibrosis (95,96).
18.
Acknowledgements This work was supported by NIH grant HL25552. This is publication number 6472-MEM from the Research Institute of Scripps Clinic.
19.
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nase in a patient with galactose diabetes. Lancet 1965; 2: 670-1. Monteleone JA, Beutler E, Monteleone PL, Utz CL, Casey. EC. Cataracts, galactosuria and hypergalactosemia due to galactokinase deficiency in a child: studies of a kindred. A m J Med 1971; 50: 403-7. Beutler E, Matsumoto F, Kuhl W, et al. Galactokinase deficiency as a cause of cataracts. N Engl J Med 1973; 288: 1203--6. Harley JD, Mutton P, Irvine S, Gupta JD. Maternal enzymes of galactose metabolism and the "inexplicable" infantile cataract. Lancet 1974; 2: 259--61. Xu GT. The blood galactokinase activity in Chinese with reference to congenital cataract. Chung Hua Yen Ko Tsa Chih 1989; 25: 100-3. Beutler E, Matsumoto F. Galactokinase and cataracts. Lancet 1978; 1:1161 (Letter). Prchal JT, Conrad ME, Skalka HW. Association of presenile cataracts with heterozygosity for galactosemic states and with riboflavin deficiency. Lancet 1978; 1: 12-13. Gitzelmann R, Steinmann B, Mitchell B, Haigis E. Uridine diphosphate galactose 4'-epimerase deficiency. IV. Report of eight cases in three families. Helv Paediatr Acta 1976; 31: 441-52. Ichiba Y, Namba N, Misumi H. Uridine diphosphate galactose 4-epimerase deficiency. A m J Dis Child 1980; 134: 995. Henderson MJ, Holton JB, MacFaul R. Further observations in a case of uridine diphosphate galactose-4-epimerase deficiency with a severe clinical presentation. J Inherited Metab Dis 1983; 6: 17. Garibaldi LR, Canini S, Superti-Furga A, et al. Galactosemia caused by generalized uridine diphosphate galactose-4-epimerase deficiency. J Pediatr 1983; 103: 927-30. Sardharwalla IB, Wraith JE, Bridge C, Fowler B, Roberts SA. A patient with severe type of epimerase deficiency galactosemia. J Inherited Metab Dis 1988; ll(Suppl. 2): 249-51. Garibaldi L, Superti-Furga A, Borrone C. Galactosemia caused by generalized uridine diphosphate galactose-4-epimerase deficiency. J Pediatr 1986; 109: 1074-5. Beutler E, Baluda MC, Sturgeon P, Day R. A new genetic abnormality resulting in galactose-l-phosphate uridyltransferase deficiency. Lancet 1965; 1: 353-4. Beutler E, Baluda MC, Sturgeon P, Day RW. The genetics of galactose-l-phosphate uridyl transferase deficiency. J Lab Clin Med 1966; 68: 646-58. Kuehnl P, Nowicki L, Spielmann W. Untersuchungen zum Polymorphismus der Galaktose-l-phosphaturidyl-Transferase (EC:2.7.7.12) Mittels Agarosegelelektrophorese. Humangenetik 1974; 24: 227-30. Eriksen B, Dissing J. Human red cell galactose-1phosphate uridylyltransferase. H u m Hered 1980; 30: 27-32. Sparkes MC, Crist M, Sparkes RS. Improved technique for electrophoresis of human galactose-l-P uridyl transferase (EC 2.7.7.12). H u m Genet 1977; 40: 93-97. Mathai CK, Beutler E. Electrophoretic variation of galactose-l-phosphate uridyl transferase. Science 1966; 154: 1179-80. Kelley RI, Segal S. Evaluation of reduced activity galactose-l-phosphate uridyl transferase by combined radioisotopic assay and high-resolution isoCLINICAL BIOCHEMISTRY,VOLUME 24, AUGUST 1991
GALACTOSEMIA: SCREENING AND DIAGNOSIS electric focusing. J Lab Clin Med 1989; 114: 152-6. 37. Schapira F, Gregori C, Banroques J, Vidailhet M, Despoisses S, Vigneron C. Isoelectrofocusing of erythrocyte galactose 1 phospho uridyl transferase in a family with both galactosemia and Duarte variants. Hum Genet 1979; 46: 89-96. 38. Schwarz HP, Zuppinger KA, Zimmerman A, Dauwalder H, Scherz R, Bier DM. Galactose intolerance in individuals with double heterozygosity for Duarte variant and galactosemia. J Pediatr 1982; 100: 704-9. 39. Wharton CH, Berry HK, Bofinger MK. Galactose-1phosphate accumulation by a Duarte-transferase deficiency double heterozygote. Clin Genet 1978; 13: 171-5. 40. Kelly S. Significance of the Duarte/classical galactosemia genetic compound. J Pediatr 1979; 94: 93740. 41. Levy HL, Sepe JS, Walton DS, et al. Galactose-1phosphate uridyl transferase deficiency due to Duarte/galactosemia combined variation: Clinical and biochemical studies. J Pediatr 1978; 92: 390-3. 42. Kelly S, Desjardins L, Khera SA. A Duarte variant with clinical signs. J Med Genet 1972; 9: 129-31. 43. Weitkamp LR, Sayre JW, Schwartz RH, Doherty R, Khera SA. Duarte variant with clinical signs has alpha 1-antitrypsin genotype ZZ. J Med Genet 1976; 13: 46--48. 44. Thomakos A, Beutler E, Stamatoyannopoulos G. Variants of galactose-l-phosphate uridyl transferase in the Greek populations. H u m Genet 1977; 36: 335-9. 45. Mulley JC. Polymorphism of human galactose-1phosphate uridyl transferase. Hum Hered 1982; 32: 42-45. 46. Ng WG, Bergren WR, Donnell GN. A new variant of galactose-l-phosphate uridyltransferase in man: the Los Angeles variant. Ann H u m Genet 1973; 37: 1-8. 47. Xu YK, Ng WG. Polymorphism of erythrocyte galactose-l-phosphate uridyltransferase among Chinese. Hum Genet 1983; 63: 280-2. 48. Bissbort S, Koempf J. Population genetics of red cell galactose-l-phosphate-uridyl-transferase (EC: 2.7.7.12). Humangenetik 1972; 17: 79-80. 49. Chacko CM, Wappner RS, Brandt IK, Nadler HL. The Chicago variant of clinical galactosemia. Hum Genet 1977; 37: 261-70. 50. Schapira F, Kaplan J-C. Electrophoretic abnormality of galactose-l-phosphate uridyl transferase in galactosemia. Biochem Biophys Res Commun 1969; 35: 451-5. 51. Chacko CM, Christian JC, Nadler HL. Unstable galactose-l-phosphate uridyl transferase: a new variant of galactosemia. J Pediatr 1971; 78: 454--60. 52. Scherz R, Pflugshaupt R, Buetler R. A new genetic variant of galactose-l-phosphate uridyl transferase. Hum Genet 1976; 35: 51-55. 53. Tedesco TA, Miller KL, Rawnsley BE, Mennuti MT, Spielman RS, Mellman WJ. Human erythrocyte galactokinase and galactose-l-phosphate uridyltransferase: a population survey. A m J H u m Genet 1975; 27: 737-47. 54. Beutler E. Screening for galactosemia. Studies of the gene frequencies for galactosemia and the Duarte variant. Isr J Med Sci 1973; 9: 1323-9. 55. Levy HL, Hammersen G. Newborn screening for galactosemia and other galactose metabolic defects. J Pediatr 1978; 92: 871-7. CLINICAL BIOCHEMISTRY,VOLUME 24, AUGUST 1991
56. Thalhammer O, Scheiber V. Untersuchung fiber die Haufigkeiten angeborener Stoffwechselanomalien in Ost-und West-Osterreich. Humangenetik 1972; 15: 145-9. 57. Schmid-Ri~ter E. Ergebnisse des Europ~iischen Screening-programmes auf heredit~re StoffwechselstSrungen. Monatsschr Kinderheilkd 1973; 121: 205-6. 58. Beckers RG, Wamberg E, Bickel H, et al. Collective results of mass screening for inborn metabolic errors in eight European countries. Acta Paediatr Scand 1973; 62: 413-6. 59. Gitzelmann R. Frfiherfassung von Anomalien im Galaktosestoffwechsel. Monatsschr Kinderheilkd 1976; 124: 654-7. 60. Wilcken B, Raby J, Crotty J, et al. Newborn screening for metabolic disorders in Australia and New Zealand: results for 1983. Med J Aust 1985; 143: 159-60. 61. Mathias D, Bickel H. Follow-up study of 16 years neonatal screening for inborn errors of metabolism in West Germany. Eur J Pediatr 1986; 145: 310-2. 62. Bowling FG, Brown AR. Development of a protocol for newborn screening for disorders of the galactose metabolic pathway. J Inherited Metab Dis 1986; 9: 99-104. 63. Misumi H, Wada H, Kawakami M, et al. Detection of UDP-galactose-4-epimerase deficiency in a galactosemia screening program. Clin Chim Acta 1981; 116: 101-5. 64. Motulsky AG, Campbell-Kraut JM. Population genetics of glucose-6-phosphate dehydrogenase deficiency of the red cell. In: Blumberg BS, ed. Proceedings of the Conference on Genetic Polymorphisms and Geographic Variations in Disease. Pp.
159-80. New York: Grune & Stratton, 1961. 65. Beutler E, Baluda M, Donnell GN. A new method for the detection of galactosemia and its carrier state. J Lab Clin Med 1964; 64: 694-705. 66. Beutler E, Day RW, Baluda M, Polk K. Screening for galactosemia among mentally retarded patients. J Ment Defic Res 1965; 9: 61-68. 67. Beutler E, Irwin HR, Blumenfeld CM, Goldenburg EW, Day RW. Field test of galactosemia screening methods in newborn infants. J A M A 1967; 199: 501-2. 68. Beutler E, Baluda MC. A simple spot screening test for galactosemia. J Lab Clin Med 1966; 68: 137-41. 69. Berry HK, Croft CC. Reagent that restores galactose-l-phosphate uridyltransferase activity in dry blood spots. Clin Chem 1987; 33: 1471. 70. Paigen K, Pacholec F, Levy HL. A new method of screening for inherited disorders of galactose metabolism. J Lab Clin Med 1982; 99: 895-907. 71. Jinks DC, Vollmer D, Orfanos A, Guthrie R. An evaluation of a new test kit to screen for galactosemia. Clin Biochem 1987; 20: 353-7. 72. Clemens P, Voltmer C, Plettner C. Interference by antibiotics with neonatal screening for galactosemia. J Pediatr 1986; I09: 713-4. 73. Greenberg CR, Dilling LA, Thompson R, Ford JD, Seargeant LE, Haworth JC. Newborn screening for galactosemia: a new method used in Manitoba. Pediatrics 1989; 84: 331. 74. Yamaguchi A, Fukushi M, Mizushima Y, et al. Microassay for screening newborns for galactosemia with use of a fluorometric microplate reader. Clin 299
BEUTLER Chem 1989; 35: 1962-4. 75. Orfanos AP, Jinks DC, Guthrie R. Microassay for estimation of galactose and galactose-l-phosphate in dried blood specimens. Clin Biochem 1986; 19: 225-8. 76. Kelly S, Desjardins L, Leikhim E. A fluorescent spot test for the detection of galactokinase deficiency. Clin Chim Acta 1974; 51: 157-61. 77. Beutler E, Baluda MC. Improved method for measuring galactose-l-phosphate uridyl transferase activity of erythrocytes. Clin Chim Acta 1966; 13: 369-79. 78. Beutler E. Galactose-l-phosphate uridyl transferase. In: Yunis JJ, ed. Biochemical methods in red cell genetics. Pp. 289-305. New York: Academic Press, Inc., 1969. 79. Beutler E, Mitchell M. UDPGlu consumption methods. In: Hsia DYY, ed. Galactosemia. Pp. 72-82. Springfield: Charles C Thomas, 1969. 80. Beutler E, Mitchell M. New rapid method for the estimation of red cell galactose-l-phosphate uridyl transferase activity. J Lab Clin Med 1968; 72: 52732. 81. Colombo JP, Moser H, Rosin S, Richterich R, Rossi E. Bestimmung der Galaktose-l-phosphat-uridyltransferase im Capillarblut bei der Galaktos~mie. Klin Wochenschr 1965; 43: 1074-81. 82. Pesce MA, Bodourian SH, Harris RC, Nicholson JF. Enzymatic micromethod for measuring galactose-1phosphate uridylyltransferase activity in human erythrocytes. Clin Chem 1977; 23: 1711-7. 83. Schutgens RBH, Berntssen WJM, Pool L. An improved quantitative assay of galactose-l-phosphate uridyltransferase activity in erythrocytes based on the determination of glucose 1-phosphate generation. Clin Chim Acta 1978; 86: 301--5. 84. Ng WG, Bergren WR, Donnell GN. An improved procedure for the assay of hemolysate galactose-1~4hosphate uridyl transferase activity by the use of C-labeled galactose-l-phosphate. Clin Chim Acta 1967; 15: 489-92. 85. Shin-Biihring Y, Osang M, Ziegler R, Schaub J. A method for galactose-l-phosphate uridyltransferase
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assay and the separation of its isozymes by DEAEcellulose column chromatography. Clin Chim Acta 1976; 70: 371-7. Fensom AH, Benson PF. Assay of galactose-l-phosphate aridyl transferase in cultured Amniotic cells for prenatal diagnosis of galactosaemia. Clin Chim Acta 1975; 62: 189-94. Beutler E, Paniker NV, Trinidad F. The assay of red cell galactokinase. Biochem Med 1971; 5: 325-32. Stocchi V, Dacha M, Bossu M, Fornaini G. Modification of the radioactive method for erythrocyte galactokinase assay. Clin Chim Acta 1978; 89: 371-4. Gitzelmann R. Deficiency of uridine diphosphate galactose-4-epimerase in blood cells of an apparently healthy infant (preliminary communication). Helv Paediatr Acta 1972; 27: 125-30. Beutler E. UDP Glucose-4-epimerase (epimerase). In: Red cell metabolism: a manual of biochemical methods. Pp. 118-9. New York: Grune & Stratton, Inc., 1984. Reichardt JK, Berg P. Cloning and characterization of a cDNA encoding human galactose-l-phosphate uridyl transferase. Mol Biol Med 1988; 5: 107-22. Flach JE, Reichardt JKV, Elsas I_J II. Sequence of a cDNA encoding human galactose-l-phosphate uridyl transferase. Mol Biol Med 1990; 7: 365-9. Zimran A, Sorge J, Gross E, Kubitz M, West C, Beutler E. Prediction of severity of Gaucher's disease by identification of mutations at DNA level. Lancet 1989; 2: 349-52. Myerowitz R, Costigan FC. The major defect in Ashkenazi Jews with Tay-Sachs disease is an insertion in the gene for the a-chain of ~-hexosaminidase. J Biol Chem 1988; 263: 18587-9. White MB, Amos J, Hsu JMC, Gerrard B, Finn P, Dean M. A frame-shift mutation in the cystic fibrosis gene. Nature 1990; 344: 665-7. Lemna WK, Feldman GL, Kerem B, et al. Mutation analysis for heterozygote detection and the prenatal diagnosis of cystic fibrosis. N Engl J Med 1990; 322: 291-6.
CLINICAL BIOCHEMISTRY,VOLUME 24, AUGUST 1991
0009-9120/91 $3.00 + .00 Copyright © 1991 The Canadian Society of Clinical Chemists.
Galactosemia: Screening and Diagnosis ERNEST BEUTLER Scripps Clinic and Research Foundation, Research Institute of Scripps Clinic, Department of Molecular and Experimental Medicine, 10666 North Torrey Pines Road, La Jolla, CA 92037, USA Galactose is normally metabolized to glucose through the coordinated activities of three enzymes: galactokinase, galactose-1phosphate uridyl transferase (GALT), and uridine diphosphoglucose 4-epimerase (epimerase). High concentrations of galactose and their metabolites are toxic to mammals. Hereditary deficiencies of galactokinase and of GALT and perhaps rarely of epimerase cause clinical disorders that can be prevented by early recognition and institution of a galactose-frea diet. The genetics of disorders of galactose metabolisms and the methods used currently for their detection are reviewed. Future prospects in the diagnosis of these disorders are discussed.
by which galactose is metabolized (2). It requires that the galactose be phosphorylated at the 1-position, attached to a pyrimidine, and finally be isomerized to glucose. The three relevant reactions are summarized in Figure 1. Although it was once claimed that an enzyme that oxidizes galactose directly, viz. galactose dehydrogenase, exists in mammals (3,4), the data were found to be due to an artifact (5-7).
KEY WORDS: galactekinase; galactose-l-phosphate uridyl transferase; newborn screening; epimerase; uridine diphosphoglucose epimerase.
Clinical manifestations o f different biochemical abnormalities o f galactose metabolism
Introduction
The inability to metabolize galactose normally is characterized by high blood and urine galactose levels. It was probably first described by GSppert in 1917 (8), and is referred to as galactosemia.
actose (milk sugar), a dissacharide of glucose L and galactose, is the main carbohydrate in the diet of young mammals. Only certain aquatic mammals, such as sea lions, seem to be an exception to this rule (1). Adults, too, ingest galactose, since this sugar is found in glycoproteins and glycolipids. While glucose, the primary energy source for most cells, is metabolized directly after phosphorylation, higher organisms are apparently unable to metabolize galactose without first transforming it into glucose. Without the capability of metabolizing galactose directly, humans must rely on a relatively complex metabolic pathway to convert galactose to glucose before metabolic energy can be extracted from it. THE METABOLISMOF GALACTOSE Understanding the molecular basis of galactosemias had to await unraveling of the pathway
Correspondence: Ernest Beutler, Scripps Clinic and Research Foundation, Research Institute of Scripps Clinic, Department of Molecular and Experimental Medicine, 10666 North Torrey Pines Road, La Jolla, CA 92037, USA. Manuscript received August 28, 1990; accepted December 29, 1990. CLINICAL BIOCHEMISTRY, VOLUME 24, AUGUST 1991
CLASSICAL GALACTOSEMIA(GALACTOSE-1-PHOSPHATE URIDYL TRANSFERASE DEFICIENCY)
Isselbacher and colleagues measured the activities of all three enzymes of galactose metabolism in 10 patients with classical galactosemia. They discovered that the second enzyme in the pathway,
Galactokinase = GaI-1-P + ADP
(1)
Galactose + ATP
(2)
GaI-I-P + UDPG -..
(3)
Epimerase UDPGal -... ~ UDPG NAD
Transferase
"~ Glu-I-P + UDPGal
Figure 1--The pathway by which galactose is converted to glucose. Gal-I-P = galactose-l-phosphate; UDPG = uridine diphosphoglucose; Glu-l-P = glucose-l-phosphate; UDPGal = uridine diphosphogalactose; Transferase -- galactose-l-phosphate uridyl transferase; Epimerase = uridine diphosphoglucose 4-epimerase.
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galactose-l-phosphate uridyl transferase (GALT), was deficient (9). Heterozygotes had about one-half normal activity of GALT in their erythrocytes. Subsequently, many investigators confirmed that this enzyme was lacking in the erythrocytes of galactosemic patients. Infants with the classical form of galactosemia become jaundiced, take feeding poorly and may develop ascites. They often die of sepsis during the first few weeks of life (10). If dietary control is instituted late or compliance with the diet is poor, cataracts and mental retardation develop. The adequate treatment of galactosemia has a dramatic effect on the prognosis of the affected child. Galactosemic children fed a diet containing little or no galactose show satisfactory growth and general health. Intellectual progress may be normal (11) or modestly impaired (12-14). Hypogonadism is common in galactosemic girls as they mature (15,16). Despite the persisting minor stigmata of galactosemia, prompt institution of a galactose-free diet is, quite literally, a matter of life and death. Thus, a rapid and accurate diagnosis deserves a high priority. It is one of few genetically determined disorders that can be treated very effectively once the diagnosis is established. Rarely, patients with galactosemia exhibit normal red cell GALT activity, setting the stage for the discovery of deficiencies of the other two enzymes that are associated with abnormal galactose metabolism. GALACTOKINASE DEFICIENCY
The first defect found in galactosemic children with normal GALT activity was galactokinase deficiency (17). The clinical course of these patients was quite atypical for galactosemia. Although they had not received a galactose-restricted diet, they had none of the stigmata of galactosemia except for cataracts. One parent of a child described earlier also had cataracts (18). This suggested that since galactokinase is the limiting step in galactose metabolism, even a modest reduction of enzyme activity could result in development of cataracts (19). The incidence of the heterozygous state for galactokinase deficiency is more prevalent among patients developing cataracts in the first few years of life than in the general population (20-23). EP1MERASE DEFICIENCY
The other defect, even more rare, is one of U D P G galactose-4-epimerase. In most subjects studied, the defect appears to be limited to circulating blood cells. Activity in liver, cultured skin fibroblasts, and activated lymphocytes have usually been normal (24); however, in one symptom-
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atic case (see below), the epimerase activity of cultured fibroblasts was deficient. Although affected individuals have elevated red cell galactose1-phosphate levels, they generally do not suffer ill effects and develop normally (24,25). In rare cases, severe clinical symptoms and signs similar to those found in classical galactosemia were associated with epimerase deficiency (26-28); in one of these (27), the diagnosis was later retracted (29).
Genetics and incidence of the galactosemias All three forms of galactosemia are inherited as autosomal recessive disorders. In each case, both parents of severely affected individuals have approximately one-half the normal enzyme activity. CLASSICAL GALACTOSEMIA(GALACTOSE-I-PHOSPHATE URIDYL TRANSFERASEDEFICIENCY)
Before data from large-scale screening programs became available, the most accurate w a y to estimate the expected birth incidence of galactosemia was to ascertain the percentage of the population who were heterozygous for the enzyme deficiencies. However, attempts to do so were confounded by the presence of alleles in the population that lowered enzyme activity, but did not have the capacity to cause galactosemia even in the homozygous state. Most important of these alleles is the Duarte variant. We discovered this polymorphism while attempting to determine the gene frequency for galactosemia (30,31). Family studies revealed the existence of two distinct types of individuals with one-half normal transferase activity. There were those whose parents or children were either normal or had one-half normal enzyme activity. In contrast, in some families both parents and all children of the person expressing one-half normal transferase activity exhibited three-quarters normal enzyme activity (Figure 2). Thus, we deduced that the GALT activity expressed by the Duarte allele is approximately one-half of that expressed by the normal allele. Homozygotes for the Duarte variant have approximately one-half of normal activity and thus mimic carriers for galactosemia. The Duarte variant can be identified by its rapid electrophoretic migration (32-35) and abnormal isoelectric point (36,37) as well as its reduced activity. Even a single Duarte allele is sufficient to prevent the clinical expression of galactosemia; babies born with a galactosemia allele and a Duarte allele have one-quarter of normal enzyme activity. Such children have a reduced capacity to metabolize galactose, with abnormal accumulation of galactose-l-phosphate in their red cells (38,39). However, no ill effects have been found to occur in these individuals (30,31,38,40,41). An infant homozygous for the Duarte variant was believed to
CLINICAL BIOCHEMISTRY, VOLUME 24, AUGUST 1991
GALACTOSEMIA: S C R E E N I N G A N D DIAGNOSIS TYPE G FAMILIES (9)
-eL_ _
10
0
20
: :
30
TYPE 0 FAMILIES(4| • PROPOSITUS
__ 0
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10 w
0
.
.
.
.
i
20
30
DOUBLY ANOMALOUS FAMILIES (2}
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20
310
Figure 2--The transferase activity of parents and children of propositi with half-normal (top two panels) and one-quarter normal (bottom panel) transferase activity. The families with half-normal activity could be divided into those in which the gene responsible for transferase deficiency was the gene for galactosemia (type G families) and those in which it was the gene for the Duarte variant (type D families). The distribution of enzyme activity in parents and children confirms the proposed genetics of transferase deficiency, viz. that the individuals in the type D families are homozygous for a mutation that codes for one-half normal transferase activity--the Duarte variant (from Hsia, D.Y.-Y., Galactosemia, 1969. Courtesy of Charles C Thomas, Publisher, Springfield, Illinois).
galactosemia have been estimated to be 0.0021 (44), 0.0024 (53) and 0.004 (54). Screening programs in various populations have shown that the birth frequency of the disease corresponds quite well to these estimates, with an average value 1:62,000 having been reported in studies in various populations (55). The actual reported incidence varies considerably in different populations. In Western Austria, the frequency was 1:13,647, while in Eastern Austria it was 1:43,061 (56). In eight countries in Western Europe, the incidence average was 1:34,000 (57,58). In Switzerland the incidence was 1:57,000 (59) and in New Zealand it was 1:34,000 (60). In Germany, 1:45,282 live births were galactosemic children (61). GALACTOKINASE DEFICIENCY
Estimates of the frequency of galactokinase deficiency are also fraught with difficulty. Among persons of African ancestry, a common allele, the Philadelphia variant that lowers the enzyme activity, has been found (53). Presumably this allele is present albeit at lower levels in non-African populations. The gene frequency estimate for galactokinase deficiency based on heterozygotes is 0.0015, predicting a homozygote frequency of 1:462,000. The birth frequency found in various screening programs ranges from 0:582,000 to 1:168,022 (55). EPIMERASE DEFICIENCY
be symptomatic (42) but was later diagnosed as having the ZZ variant of ~-l-antitrypsin (43). The Duarte allele is very common. We originally estimated its gene frequency to be about 0.0555 (31) and subsequently, in a Greek population, to be 0.0548 (44). Subsequent estimates have been very similar (45,46) but the gene frequency among American blacks (46) and Orientals (46,47) is considerably lower. Thus, some 11% of Caucasian subjects are heterozygous for the Duarte variant. Estimates that are based on electrophoretic mobility only are likely to include heterozygotes for the Los Angeles variant (see below), and are therefore somewhat higher (48). The Los Angeles variant is another m u t a n t allele of GALT. First described by Ng et al. (46), this enzyme has increased activity that is electrophoretically identical to the Duarte variant. The gene frequency of this variant is about 0.025 among Caucasians (45). It is apparently quite rare among Orientals (47). Other rare variants of GALT have also been reported, including the Chicago I and II (49), Rennes (50), Indiana (51) and Berne (52) variants. Only estimates of gene frequency that exclude homozygotes for the Duarte variant provide meaningful data regarding the frequency of true galactosemia alleles in the population. Based on these considerations, the frequency of the alleles for true
CLINICAL BIOCHEMISTRY, VOLUME 24, AUGUST 1991
There are few reliable data regarding the incidence of epimerase deficiency. A screening program in Australia detected 2 infants with this disorder out of 207,000 screened (62). In Japan, screening for epimerase deficiency yielded 2 cases out of 23,049 newborns tested (63). In both studies, all infants detected were clinically normal. Screening n e w b o r n s for galactosemia Because of the high early mortality associated with galactosemia, needless deaths result from limiting diagnostic measures to infants who develop symptoms. In one study, 6 of 10 infants with galactosemia died between the 8th and 18th day of life because diagnosis was delayed to days 7 to 17 (10). Screening newborns for hereditary disorders is best carried out using spots of dried blood. Because such collections are made for phenylketonuria testing, the inclusion of tests for other disorders minimizes the cost of the procedure. Two principal approaches using such samples have been employed to screen for galactosemia. The first measures the activity of GALT itself. The second uses micro-organisms to determine the amount of galactose in the blood sample. We first became interested in screening for galactosemia when Dr. J. Pollard, a chemist at Cal-
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+
Transferase UDPGal + Glu-l-P
,/
Phosphoglucomulase
/
Glu-6-P Jr
Glucose-6-PDehydrogenaseI 6-PGA .-t-
Figure 3--The reactions of the fluorescent screening test for galactosemia. The reaction mixture and hemolysate provide the reagents shown in boxes. When transferase is present, the reactions proceed with the reduction of NADP to NADPH, which is detected by virtue of its fluorescence.
Biochem, told us he developed a method for synthesizing relatively large quantities of galactose-l-phosphate. Thus, current methods for detecting glucose-6-phosphate dehydrogenase (G6PD) deficiency could be adapted to screen for transferase deficiency by linking the transferase reaction, which yielded glucose-l-phosphate, to the G6PD reaction using glucose-6-phosphate as a substrate. The glucose-l-phosphate formed by the transferase could readily be converted to glucose6-phosphate by phosphoglucomutase, an enzyme that is relatively abundant in the erythrocyte. In the 1960's, one of the most convenient methods for screening for G6PD deficiency was the brilliant cresyl blue decolorization test of Motulsky and Campbell-Kraut (64). This test linked the reduction of NADP by glucose-6-phosphate and G6PD to the reduction of brilliant cresyl blue to the reduced leuko-form. For large-scale screening, it was necessary to cover the solution with mineral oil, so we simplified the procedure considerably by using methylene blue as the receptor dye, avoiding auto-oxidation by gassing the solution with CO and stoppering it (65). This method, though useful in screening hundreds (66) and even thousands (67) of subjects at risk, was cumbersome to apply to the millions of infants who required screening to avert needless deaths from galactosemia. Recognizing that the endogenous fluorescence of 296
NADPH could be used as an endpoint of NADP reduction by the linked system made possible a technically simpler technique, the fluorescent spot test (68). This method eliminated the need for anaerobic conditions and a dye and required merely an ultraviolet hand-lamp to appraise the results. The principle of the test is summarized in Figure 3. This test measures the gene product directly, and the results are not dependent upon dietary intake of galactose. The compound heterozygote for galactosemia and the Duarte variant (GALTD/GALT°), an individual with about 1/4 normal enzyme activity, may give false positive results. Moreover, because of its high specificity for GALT, the fluorescent spot test will miss the very rare case of galactokinase deficiency in patients who would also benefit by early restriction of galactose intake. However, since the enzyme is unstable to heating, samples stored at very warm temperatures may give false positive results, causing a high recall rate (61). It has been proposed that this problem may be largely overcome by adding dithiothreitol to the reaction mixture (69). Blood galactose levels are usually measured microbiologically using an auxotrophic microorganism. The E. coli K-12 mutant strain W3805 lacks epimerase and cannot synthesize a normal cell wall unless galactose is present in the medium. The abnormal cell wall formed in the absence of galactose unmasks a receptor for bacteriophage C21, and thus the cell is lysed in galactose deficiency but grows normally in the presence of galactose. When punched out blood spots are placed on a semisolid medium seeded with the microorganism and the phage, the zone of growth around the sample is proportional to the logarithm of its galactose concentration (70). One strength of this approach is that it detects all forms of galactosemia, not just classical galactosemia, the most common form, caused by a deficiency of GALT. Moreover, galactose and galactose-l-phosphate (which accumulates in the red cells of galactosemic subjects) are quite stable, even at warm temperatures. On the other hand, blood galactose levels depend upon dietary intake of galactose. Thus, the galactosemic who is vomiting and not absorbing milk sugar may, theoretically, be missed. The method also appears to be fairly technically demanding (70). For example, agar cannot be used because of its galactose content, and silica gel must be used instead. The bacteria will not grow if inadequately aerated or if repeatedly frozen and thawed. Care must be taken with pH, contamination with other micro-organisms and the quantities of phage and bacteria used. Despite these difficulties, the technique has been applied successfully in different settings (55) and the availability of a test kit may increase its applicability (71). It is unclear to what extent antibiotics may give false positive tests; some antibiotics interfere with a microbiologic test for galactosemia reported earlier (72). CLINICAL BIOCHEMISTRY, VOLUME 24, AUGUST 1991
GALACTOSEMIA: SCREENING AND DIAGNOSIS
In recent years, other methods for the detection of galactose and galactose-l-phosphate in blood spots have been introduced. These depend upon the oxidation of galactose by galactose dehydrogenase with the detection of N A D H by fluorescence (73-75). Although a fluorescent screening test for galactokinase has been published (76), the results obtained are probably invalid because blood glucose can act as a substrate for N A D P H formation in the linked reaction used.
The definitive diagnosis of galactosemia Red blood cells function normally in patients with all types of galactosemia, b u t fortunately they contain the enzymes of galactose metabolism. Thus, they serve as a convenient and powerful tool for the diagnosis of the galactosemias, and the defects underlying all three forms of hereditary galactosemia were originally discovered by examining erythrocytes from affected patients. Whether detected as a result of a screening test on newborns or on the basis of clinical findings, confirmation of the diagnosis of galactosemia requires quantitative assays for the enzymes of galactose metabolism. CLASSICAL GALACTOSEMIA (GALACTOSE-1-PHOSPHATE URIDYL TRANSFERASE DEFICIENCY)
Several different means are available for the measurement of galactose-l-phosphate-uridyl transferase activity in red cells. In this reaction (Figure 1; reaction 2) galactose-l-phosphate is converted to glucose-l-phosphate, while U D P glucose is converted to U D P galactose. The classical method of measuring enzyme activity is to determine the amount of U D P G consumed in a hemolysate incubated with U D P G and galactose-1phosphate. This technique, which designated the U D P G consumption assay, is quite specific and reliable for the diagnosis of galactosemia. However, it is an endpoint assay, and as such it does not directly give true initial enzyme rates. Because the reaction product UDPGal is a competitive inhibitor, the relationship between enzyme added and U D P G consumed is not linear. We developed correction factors which compensate for the non-linearity of the assay (77-79). The U D P G consumption assay loses accuracy when small amounts of enzyme activity are measured. Under these conditions, a large value is subtracted from a slightly larger one in calculating the amount of U D P G consumed. A satisfactory alternative method for estimating GALT depends upon the linkage of glucose1-phosphate formation in the primary reaction to phosphoglucomutase, converting the glucose-lphosphate to glucose-6-phosphate, and then a further linkage to glucose-6-phosphate dehydrogenase, which reduces NADP to NADPH. The rate CLINICAL BIOCHEMISTRY, VOLUME 24, AUGUST 1991
of N A D P H formation can be measured fiuorometrically and is linearly related to GALT activity (80). The same approach has been used widely to develop a spectrophotometric technique (81-83) for transferase assay. In addition to these techniques, radioactive methods have been developed using chromatographic separation of the radioactive substrate galactose-l-phosphate from the radioactive product UDPGal (84-86). This approach, though cumbersome for the measurement of red cell enzyme activity, is valuable when GALT activity of other tissues is to be estimated. In tissues such as cultured amniotic fluid cells, the U D P G consumption assay and the NADP-linked procedures are unreliable because side reactions interfere. GALACTOKINASE DEFICIENCY
Galactokinase activity of red cells is very low and a radioactive assay is more suitable. Our assay is based upon the fact that phosphorylated galactose is strongly negatively charged and binds to the anion exchange paper DE-52. In contrast, galactose is uncharged and readily washed from this medium. Accordingly, hemolysate is incubated with ATP and radioactive galactose, and the mixture is spotted on DEAE paper and vigorously washed (87). After the washed paper is dissolved in scintillation fluid (88), counting provides an accurate measurement of galactokinase activity. EPIMERASE DEFICIENCY
Epimerase may be determined by a two-step procedure: hemolysate or tissue sample is incubated with UDPGal and NAD, a necessary co-factor, the reaction is stopped and the amount of UDPGlucose formed is measured with UDPGlucose dehydrogenase (89). With good instrumentation, it is possible to measure the enzyme by continuously monitoring N A D P H formation in a linked system (90); however, the rate of the reaction is sufficiently slow in normal red cells that the precision of the linked assay is relatively low.
Future prospects Recently, the cDNA for GALT has been cloned and sequenced (91,92), and the molecular biology of galactosemia will undoubtedly increase our understanding of the incidence and population genetics of this group of disorders. Because even classical galactosemia is relatively rare, it is reasonable to assume that many cases arose as new mutations and that the disorder is heterogeneous at a nucleotide level. This assumption is strengthened by the finding that more common genetic disorders occurring at polymorphic frequencies in some populations are heterogeneous. Examples of this finding have been reported in some patients 297
BEUTLER with Gaucher disease (93), Tay-Sachs disease (94), and cystic fibrosis (95,96).
18.
Acknowledgements This work was supported by NIH grant HL25552. This is publication number 6472-MEM from the Research Institute of Scripps Clinic.
19.
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