Galactosaemia in a Brazilian population: high incidence and cost-benefit analysis.

OBJECTIVES: To study the incidence of galactosaemia in the state of São Paulo and the benefit/cost (B/C) ratio of the introduction of neonatal screening for galactosaemia, comparing it with a selective approach. METHODS: An enzymatic-colorimetric assay was used for the screening of total galactose (TG) in a sample of 10% of the births in São Paulo in one year and positive cases were confirmed by the activity of galactose-1-phosphate uridyltransferase (GALT). Detected and referred cases were genotyped using enzyme restriction studies for Q188R, N314D and S135L mutations of the GALT gene. The economic analysis was determined by calculating the B/C ratio and by analysis of sensitivity as a function of the incidence of the disease detected and the variation of the interest rate in the economy. RESULTS: 59 953 newborns were screened for TG, with 3 cases of galactosaemia being identified (0.26% false positives), corresponding to a frequency of 1:19 984 liveborns (95% confidence interval: 1:7494 to 1:59 953). One classical case and one Duarte 2 variant referred to as a selective approach were confirmed. With an incidence of 1:19 984, the B/C ratio was 1.04 for the 11.75% interest rate in effect in Brazil, with values already decapitalized. With a maximum possible incidence of 1:7494, the B/C ratio was 2.79. DISCUSSION: There is an economic advantage in introducing neonatal screening for galactosaemia in the national neonatal screening programme. This advantage could increase with a reduction of the current interest rates in the economy.

Purification and characterization of human erythrocyte uridylyl transferase.

A new method for the purification of human erythrocyte uridylyl transferase (UDPglucose: alpha-D-galactose-1-phosphate uridylyltransferase EC 2.7.7.12) is described. It consists of a hydrophobic purification step associated with hydroxyapatite chromatography and provided for the first time a purification of more than 45 000-fold with a high activity (15 I.U/mg) and a yield of 32%. We show that the enzyme is a dimer and has a molecular weight of 88 000. It can be resolved into three bands by isoelectric focusing with an apparent pI between 5.0 and 5.4. It could be shown by steady-state initial rate measurements that the interconversion of the two substrates of human transferase (Gal-1-P and UDP-glucose) follows ping-pong bi-bi kinetics, with Km values of 0.2 and 0.065 mM, respectively.

Purification of human liver uridylyl transferase and comparison with the erythrocyte enzyme.

Uridylyl transferase (UDP glucose: alpha-D-galactose 1 phosphate uridylyl transferase, EC 2.7.7.12) has been purified 1350-fold from human liver to complete homogeneity. The purification procedure involved ammonium sulfate fractionation, batch treatment, chromatography on DEAE-cellulose, hexylagarose and hydroxylapatite. The specific activity of the homogeneous enzyme was 27 units/mg protein. The liver enzyme was compared to the red cell enzyme previously purified by us. The liver enzyme was similar to the red cell enzyme with respect to subunit molecular weight, kinetic studies and immunological properties. Differences in electrophoretic behaviour were found: the liver transferase has a more basic pI (between 5.5 and 5.8) than that of the erythrocyte enzyme (pI between 5.0 and 5.45). It is very likely that the liver uridylyl transferase and the red blood cell transferase are the same enzymes with post-translational modifications.

Effect of khat on the metabolism of erythrocytes.

The plant khat "Catha Edulis Forsk" is widely distributed among most East African countries, Yemen and many other areas of the world. Administration of khat extract by the intragastric route in rabbits affected the metabolism of erythrocytes. There is a significant decrease in pyruvate kinase and the level of reduced glutathione (P less than 0.001), and a highly significant increase in both glucose-6-phosphate dehydrogenase and glutathione reductase activities (P less than 0.001) in khat-fed rabbits as compared to controls. On the other hand the activity of uridyl transferase as well as the concentration of 2,3-diphosphoglycerate were not significantly changed in experimental khat-fed rabbits (P greater than 0.5).

Problems in the diagnosis of transferase and galactokinase deficient galactosemia.

Galactose in serum and galactose-1-phosphate in erythrocytes were measured in six transferase deficient children to determine if these metabolites could be used in detecting transferase deficient galactosemia. In all six children the galactose levels were normal and the galactose-1-phosphate elevated. The galactose level depends on diet and the rate of metabolism to galactose-1-phosphate and, therefore, should not be used to predict transferase deficient galactosemia. The galactose-1-phosphate level was elevated in all the transferase deficient children because once formed it cannot be metabolized. Measurement of galactose-1-phosphate is difficult and is usually requested to determine whether or not the child is following the galactose restricted diet. In transferase deficient galactosemia, the enzyme hexose-1-phosphate uridylyltransferase is absent. The diagnosis should be determined by measurement of the activity of the enzyme hexose-1-phosphate uridylyltransferase in erythrocytes. In galactokinase deficient galactosemia, the enzyme galactokinase is absent. Galactose levels are elevated but the amount present depends on diet and how soon the blood was collected after the ingestion of galactose containing foods. The diagnosis of galactokinase deficient galactosemia is based on the measurement of the enzyme galactokinase in erythrocytes.

Müllerian duct abnormalities and galactosaemia heterozygosity: report of a family.

Familial aggregates of Müllerian fusion anomalies (MFAs) and of Müllerian aplasia (MA) are rare. I report the case of a girl with MA and 'streak-like' ovaries, whose mother had a MFA. No similar mother-daughter pair appears to have been reported previously. The girl, mother, and maternal grandmother each have low galactose-1-phosphate uridyl transferase activities and are each heterozygous for the 'classic' galactosaemia allele. These findings support previous suggestions that MA may sometimes be related to abnormal galactose metabolism, and further suggest that, in some cases, MFAs may also be related to disordered galactose metabolism.

[Hereditary abnormalities of galactose metabolism: diagnosis and biochemical supervision (author's transl)]. / Anomalies héréditaires du métabolisme du galactose: diagnostic et surveillance biochimiques.

The authors define the main stages of the biochemical study of hereditary abnormalities of galactose metabolism. They review laboratory examinations for detection, enzyme examinations which provide the diagnostic proof, further examinations which permit one to follow the course and efficacy of a galactose-free diet, the demonstration of genetic variants, the technics of antenatal diagnosis and routine neonatal detection.

Identification of galactose-1-phosphate uridyl transferase gene common mutations in dried blood spots.

BACKGROUND: The California newborn screening program uses newborns' dried blood spots (DBS) to screen for more than 45 genetic disorders. Deficiency of galactose-1-phosphate uridyl transferase (GALT) is one of the metabolic genetic disorders screened using newborn DBS. During follow-up tests, common mutations of the GALT gene have been identified using whole blood samples. To avoid the stress of drawing an additional blood sample from newborns who are identified as presumptive positive for galactosemia, we developed a method to test common mutations in the GALT gene using blood spots. METHODS: This method involves DNA extraction from DBS, followed by polymerase chain reaction (PCR), and single nucleotide extension (SNE). SNE products were detected by capillary electrophoresis. RESULTS: In a double-blind study, GALT gene common mutations/variants: IVS2-2A>G, p.S135L, p.T138M, p.Q188R, p.L195P, p.Y209C, p.L218L, p.K285N, and p.N314D were detected in seventy-three DBS which had previously been screened and confirmed as positive in the California Newborn Screening Program. Mutations found using blood spots gave 100% concordance with mutations from previously genotyped whole blood samples. CONCLUSIONS: This blood spot method decreases the genomic test turnaround time of GALT screened positive patients and potentially reduces emotional stress on families required to provide an additional blood draw.

Identification of sequence variation in the galactose-1-phosphate uridyl transferase gene by dHPLC.

Transferase-deficient galactosaemia is an inherited disorder of carbohydrate metabolism, caused by mutation at the galactose-1-phosphate uridyl transferase (GALT) locus. A denaturing high performance liquid chromatography (dHPLC) method was developed for variant scanning of the GALT gene. The method unequivocally identified the Duarte D1, D2, Q188R, and K285N GALT alleles and associated polymorphisms. Length polymorphism in an intronic Alu repeat was characterised and a novel Single Nucleotide Polymorphism (IVS10nt-322g-->t) associated with the D1 allele was identified.

Human red cell galactose-1-phosphate uridylyltransferase (EC 2.7.7.12). Electrophoretically determined polymorphism in Denmark and its use in paternity cases.

The electrophoretically detectable phenotypes of human red cell galactose-1-phosphate uridylyltransferase (GALT) were determined in 2,074 unrelated Danes. The gene frequencies were: GALT1 = 0.9233 and GALT2 = 0.0767. The segregation of phenotypes in 765 mother-child pairs was consistent with autosomal codominant inheritance. One apparent mother-child incompatibility with respect to phenotypes was observed which, however, appeared to be due to the segregation of a silent gene. The results of an investigation of 248 paternity cases are reported, and the application of the GALT polymorphism to paternity cases is discussed.

Biochemical studies of a human low-activity galactose-1-phosphate uridyl transferase variant.

A low activity galactose-1-phosphate uridyl transferase (transferase) variant in a newborn infant has been demonstrated by biochemical studies in erythrocytes and cultured skin fibroblasts. The newborn infant was a galactosaemic suspect identified in a neonatal metabolic screening programme. On breast feeding, he did well without clinical symptoms of galactosaemia during the first 15 days of life. However, substantial amounts of erythrocyte galactose-1-phosphate and urinary galactitol corresponding to the levels in untreated galactosaemic patients, along with mild amino aciduria, were found. The transferase activity, as measured by a sensitive micro kinetic radioisotopic method, was about 7--10% of the normal. On starch gel electrophoresis, the enzyme from the haemolysate had similar mobility as the normal in Tris--glycine buffer, pH 8.8 and phosphate buffer, pH 7.0, but had a slower mobility than that of the normal in the histidine buffer, pH 7.8. The mobility difference was much clearer in a semipurified enzyme preparation. The transferase enzyme in the haemolysate appeared to be more heat labile.

The Chicago variant of clinical galactosemia.

A new variant of clinical galactosemia with two hitherto unidentified alleles on the transferase locus in one family is described. This new clinical variant of transferase has 25% of normal control activity in blood and in skin fibroblasts, and the patient accumulates galactose-1-phosphate in blood on an unrestricted galactose diet. Using starch gel electrophoresis on the hemolysate of the family members, a fast-moving transferase with mobility in between those of the normal control and of the Duarte variant is identified. This new allele is designated as GALTC1 (fast-moving Chicago variant). In addition, a second new allele was documented in this family by studying the instability of the transferase enzyme in hemolysates of family members at 50 degrees C for various time intervals. This new allele is designated as GALTC2 (heat-labile Chicago variant). On the basis of these studies, the transferase genotype of this patient is thought to be a double heterozygote compound, GALTC1/GALTG.

[Clinical and biochemical diagnosis of galactosemia among our cases]. / Kliniczna i biochemiczna diagnostyka galaktozemii na podstawie wlasnych przypadków.

Clinical and biochemical diagnostic studies concerned 17 cases of galactosemia coming from 15 not consauguineous families. Galactosemia was diagnosed between 1-st day and 11-th month of life. Tentative diagnosis based on clinical picture was made in 12 infants, others were detected through family history of galactosemia and/or biochemical newborn screening carried out at the National Research Institute of Mother and Child since 1969. Clinical symptoms of galactosemia occurred in most patients in the first week of life. They were the following (tab. II): hepatomegaly (in 94%), jaundice (81%), splenomegaly (79%), vomitus (62%) and diarrhoea in 56% of patients. Cataract was found in 6 infants (38%). Biochemical diagnosis was based on the results of enzymatic estimation of galactose-1-phosphate uridyl transferase activity in blood, galactose-1-phosphate in red blood cells and galactose in blood and urine. No activity of galactose-1-phosphate uridyl transferase was found in all patients, and the concentration of galactose-1-phosphate was higher than 25 mg/100 ml of red blood cells. High galactose level was observed in blood and urine in all patients with typical clinical course of galactosemia. In 2 patients however without clinical symptoms of the disease only trace amounts of galactose was detected in blood and urine. All these patients were treated with galactose free diet.

Study of five haemogenetic markers (Gc, C3, Bf, Ag, and GALT) in six Indonesian populations and in 12 subgroups of Balinese.

In various ethnic groups of the Indonesian archipelago and of Bali, the polymorphisms of the serum proteins Gc globulin (vitamin D-binding protein), C3 (complement component 3), Bf (complement factor B), Ag x,y (lipoprotein allotypes), and of the red cell enzyme system GALT (galactose-1P-uridyltransferase) were analysed. Among the studied proteins, the Gc system was the most informative one for the anthropologist. Besides considerable differences of frequencies of the common alleles Gc*1F, Gc*1S and Gc*2, a number of rare alleles (1A1, 1A3, 1A8, 1A9, 1A12, 1C2, 1C21, 1C24, and 2C8) and some new ones (1C28, 1C29, 1C30, 2C9) were observed. The presence of Gc*1A1 demonstrates the relationship to the Australo-Melanesian populations, but Mongolian variants (1A3, 1A8, 1A9, 1C2) were also encountered. Within the C3 system a very high frequency of the C3*S allele was observed in all populations. The rare alleles C3*F0.55, C3S1, and C3*S0.5 were observed in some groups. A new allele (C3*F0.35) was detected in a Chinese individual and in a nobleman from Bali. The frequency of the Bf*F allele was rather low in general, and the Bf*S0.7 allele was found in three Indonesian individuals only. The Ag*(x) frequencies were rather high, as it is known for Asiatic populations. Variability among subgroups was not very pronounced. The GALT*2 allele (Duarte variant of the enzyme) was observed very rarely; however, it was present in several populations. Enzyme activities could not be determined, and therefore we cannot tell whether the galactosaemia gene (GALT*0) was present or not.

Galactosaemia: case for neonatal screening illustrated by recent Australian experience.

The varied presentation and clinical features of classical galactosaemia are illustrated by the case histories of seven babies born in Western Australia since January, 1962, and of two babies born in South Australia in whom diagnosis was made as a result of adding galactosaemia to the Guthrie screening programme in October, 1974. All were shown to have a severe deficiency of galactose-1-phosphate uridyl transferase in their red blood cells. We compare our findings with those in 10 galactosaemic babies born in Victoria over a similar period, and show that in both groups these were two main modes of onset: acute and insidious. Jaundice and Escherichia coli infection were prominent in the 13 babies with an acute onset of galactosaemia, while poor weight gain, intermittent vomiting and cataracts were features of the five babies with an insidious onset. An enlarged liver was usually found in both groups. We discuss the various approaches to neonatal screening of galactosaemia in the light of experience in Massachusetts and South Australia. The use of cord blood can be expected to lead to diagnosis before babies with acute onset become ill, while the use of blood collected at five days for the Guthrie test avoids the collection of another routine sample for a relatively rare disorder. The result of red cell transferase assays of parents and siblings of our patients are discussed in relation to their implication for genetic counselling. The relevance of antenatal diagnosis to the prevention of possible intrauterine damage to an affected fetus is pointed out.

[Galactosemia and cow's milk intolerance (author's transl)]. / Galactosemia e intolerancia a proteínas vacunas. Estudio clínico-bioquímico y anatómico.

The clinical, biochemical and anatomical studies of one case of galactosemia with cow's milk intolerance in a one month old infant are reported. Diagnosis was ascertained measuring the enzyme (Gal-1-P-Uridyl-transferase) deficiency with the methods of Anderson, Kalckar and Isselbacher. Overload and enzymatic studies were also carried out in relatives. The interest of this association of galactosemia with cow's milk intolerance, and its clinical and therapeutic implications are pointed out in the discussion.