Galactose-1-phosphate Uridyltransferase Research Articles

The molecular basis of transferase galactosaemia in South African negroids.

Transferase galactosaemia is an autosomal recessively inherited disorder caused by a deficiency of galactose-1-phosphate uridyltransferase (GALT). Manifestations include jaundice, vomiting, cataracts, mental retardation, speech abnormalities and poor growth. The GALT gene has been mapped and sequenced. The S135L mutation accounts for approximately 48% of galactosaemia alleles in African Americans and has been found to account for about 91% of galactosaemia alleles in negroid South African patients which suggested that the mutation had an African origin. We have calculated the S135L allele frequency (+/- 1SE) in a sample of healthy unrelated negroid South Africans to be 0.0067 (+/- 0.0024). The S135L mutation was also detected in negroid populations from other regions of Africa confirming its African origin.

Classical galactosemia and mutations at the galactose-1-phosphate uridyl transferase (GALT) gene.

Classical galactosemia is caused by a deficiency in activity of the enzyme galactose-1-phosphate uridyl transferase (GALT), which, in turn, is caused by mutations at the GALT gene. The disorder exhibits considerable allelic heterogeneity and, at the end of 1998, more than 150 different base changes were recorded in 24 different populations and ethnic groups in 15 countries worldwide. The mutations most frequently cited are Q188R, K285N, S135L, and N314D. Q188R is the most common mutation in European populations or in those predominantly of European descent. Overall, it accounts for 60-70% of mutant chromosomes, but there are significant differences in its relative frequency in individual populations. Individuals homoallelic for Q188R tend to have a severe phenotype and this is in keeping with the virtually complete loss of enzyme activity observed in in vitro expression systems. Globally, K285N is rarer, but in many European populations it can be found on 25-40% of mutant chromosomes. It is invariably associated with a severe phenotype. S135L is found almost exclusively in African Americans. In vitro expression results are discrepant, but some individuals carrying S135L appear to exhibit GALT activity in some tissues. Duarte 1 (or Los Angeles) and Duarte 2 (or Duarte) variants carry the same amino acid substitution, N314D, even though D1 is associated with increased erythrocyte GALT activity and D2 with reduced activity. N314D is in linkage disequilibrium with other base changes that differ on the D1 and D2 alleles. N314D does not impair GALT activity in in vitro expression systems. However, there are differences in the abundance of GALT protein in lymphoblastoid cells lines from D2 and D1 individuals. It is unclear whether the specific molecular changes that distinguish the D1 and D2 alleles account for the different activities. The considerable genetic heterogeneity documented to date undoubtedly contributes to the phenotypic heterogeneity that is observed in galactosemia. The additional effects of nonallelic variation and other constitutional factors on phenotypic variability remain to be elucidated.

Identification of mutations in the galactose-1-phosphate uridyltransferase (GALT) gene in 16 Turkish patients with galactosemia, including a novel mutation of F294Y

Classical galactosemia caused by deficiency of galactose-1-phosphate uridyltransferase (GALT) is a severe autosomal recessive disorder. We report here molecular analysis of 16 unrelated Turkish galactosemia index cases without GALT activity. Almost 84% of all mutant alleles were identified in this study. The most common molecular defect observed in the Turkish population was Q188R (replacement of glutamine-188 by arginine) (57%). In order to facilitate the determination of unknown mutations in the entire coding region of GALT, we established an approach based on GALT cDNA synthesis and direct sequencing. We have identified one novel candidate galactosemia mutation, a T-to-A transversion at the codon 294 (F294Y) in exon 9 in addition to previously reported three missense (M142K K285N, A320T), one stop codon (E340X), and one silent (L218L) mutations in galactosemia patients which reflect considerable genetic heterogeneity in the Turkish population. © 1999 Wiley-Liss, Inc.

Identification of mutations in the galactose-1-phosphate uridyltransferase (GALT) gene in 16 Turkish patients with galactosemia, including a novel mutation of F294Y Communicated by: R.G.H. Cotton Online Citation: Human Mutation, Mutation in Brief #235 (1999) Online http://journals.wiley.com/1059-7794/pdf/mutation/235.pdf

Classical galactosemia caused by deficiency of galactose-1-phosphate uridyltransferase (GALT) is a severe autosomal recessive disorder. We report here molecular analysis of 16 unrelated Turkish galactosemia index cases without GALT activity. Almost 84% of all mutant alleles were identified in this study. The most common molecular defect observed in the Turkish population was Q188R (replacement of glutamine-188 by arginine) (57%). In order to facilitate the determination of unknown mutations in the entire coding region of GALT, we established an approach based on GALT cDNA synthesis and direct sequencing. We have identified one novel candidate galactosemia mutation, a T-to-A transversion at the codon 294 (F294Y) in exon 9 in addition to previously reported three missense (M142K K285N, A320T), one stop codon (E340X), and one silent (L218L) mutations in galactosemia patients which reflect considerable genetic heterogeneity in the Turkish population. © 1999 Wiley-Liss, Inc.

Elevation of erythrocyte redox potential linked to galactonate biosynthesis: Elimination by Tolrestat

Alternate pathways of galactose metabolism were explored in erythrocytes from normal subjects and patients with galactose-1-phosphate uridylyltransferase (GALT) deficiency incubated with galactose. Micromolar quantities of galactonate accumulated in both normal and mutant cells linearly with time up to 5 hours and with concentrations of galactose up to 25 mmol/L. Galactitol also was found at levels less than one third of the galactonate level, while galactose-1-phosphate concentrations comparable to those of galactonate were found in galactosemic cells. Concomitant with the formation of these galactose metabolites, the erythrocyte redox potential based on measurement of lactate and pyruvate increased fourfold in both cell types. This was due to a 60% to 72% decrease in pyruvate and a 24% to 26% increase in lactate. The oxidation of galactose to galactonate, which is known to generate NADH, is the most likely explanation for the increase in the redox state. The aldose reductase inhibitor (ARI), Tolrestat (Wyeth Ayerst Research, Princeton, NJ), at 70 μmol/L inhibited the formation of both galactonate and galactitol in both cell types without affecting galactose-1-phosphate, and eliminated the increase in the redox potential as indicated by restoration of pyruvate and lactate levels to the levels obtained before exposure of the cells to galactose. A functioning galactonate pathway is a route of galactose disposal in patients with GALT deficiency, but by altering the cellular redox potential, it may also contribute to galactose toxicity.

Characterization of a galactose-1-phosphate uridylyltransferase gene from the marine red alga Gracilaria gracilis.

The metabolism of D-galactose is a major feature of red-algal physiology. We have cloned and sequenced a gene from the red alga Gracilaria gracilis that encodes a key enzyme of D-galactose metabolism, galactose-1-phosphate uridylyltransferase (GALT). This gene, designated GgGALT1, is apparently devoid of introns. A potential TATA box, four potential CAAT boxes, and a repeated sequence occur in the 5'-flanking region. The predicted 369-aa peptide shares significant sequence similarity with GALTs from other organisms (human, 47%; Saccharomyces cerevisiae, 49%; Solanum tuberosum, 49%). Southern-hybridization analysis reveals two related, but apparently not identical, GALT genes in the nuclear genome of G. gracilis. Sequence analysis indicates that the GgGALT1 enzyme lacks a rubredoxin "knuckle" motif, which in bacterial and fungal GALTs is involved in binding zinc. An open reading frame encoding a potential peptidyl tRNA hydrolase occurs 179 bp downstream from the GgGALT1 gene.

Simultaneous occurrence of various mutations and polymorphisms in cis and in trans of the galactose-1-phosphate uridyltransferase gene in a Turkish family with classical galactosemia.

Classical galactosemia, characterized clinically by acute hepatic dysfunction, sepsis, cataract, and failure to thrive, is caused by deficiency of galactose-1-phosphate uridyltransferase (GALT). Galactose restriction normalizes these acute symptoms; however, long-term complications such as intellectual deficits and ovarian failure are conspicuous in the majority of patients. Here we report two Turkish siblings with classical galactosemia. The clinical course of the two children differed markedly: only the older girl suffered from severe acute symptoms during the neonatal period, and she developed greater mental retardation than her younger affected brother. The functional activity of GALT was virtually absent in each affected children. The mother and two healthy siblings exhibited approximately 50% normal GALT activity and the father approximately 25%. Molecular analysis revealed that these two galactosemic siblings were homozygous for a stop codon mutation of E340X in GALT exon 10. Moreover, two additional mutations, a neutral polymorphism L218L and N314D, which are typical for the Duarte-I variant, were found in the same GALT allele. The two healthy siblings and the parents were heterozygous for these combinations of mutations. In addition, the father's second GALT allele revealed three intron mutations at nucleotide position 1105 (G-->C), 1323 (G-->A) and 1391 (G-->A) and the N314D mutation, which correspond to the mutations of Duarte-2 variant. Our findings indicate that in classical galactosemia several distinct mutations can be present in one allele (in cis) of the GALT gene. Therefore it seems to be necessary to examine all introns and exons of the GALT gene in galactosemic patients who do not carry the Q188R mutation or another frequent mutation in the GALT gene.

Cotranscription and Intergenic Splicing of Human Galactose-1-phosphate Uridylyltransferase and Interleukin-11 Receptor α-Chain Genes Generate a Fusion mRNA in Normal Cells: IMPLICATION FOR THE PRODUCTION OF MULTIDOMAIN PROTEINS DURING EVOLUTION

In the past 10 years, much attention has been focused on transcription preinitiation complex formation as a target for regulating gene expression, and other targets such as transcription termination complex assemblage have been less intensively investigated. We established the existence of poly(A) site choice and fusion splicing of two adjacent genes, galactose-1-phosphate uridylyltransferase (GALT) and interleukin-11 receptor alpha-chain (IL-11Ralpha), in normal human cells. This 16-kilobase (kb) transcription unit contains two promoters (the first one is constitutive, and the second one, 8 kb downstream, is highly regulated) and two cleavage/polyadenylation signals separated by 12 kb. The promoter from the GALT gene yields two mRNAs, a 1.4-kb mRNA encoding GALT and a 3-kb fusion mRNA when the first poly(A) site is spliced out and the second poly(A) is used. The 3-kb mRNA codes for a fusion protein of unknown function, containing part of the GALT protein and the entire IL-11Ralpha protein. The GALT promoter/IL-11Ralpha poly(A) transcript results from leaky termination and alternative splicing. This feature of RNA polymerase (pol) II transcription, which contrasts with efficient RNA pol I and pol III termination, may be involved, together with chromosome rearrangements, in the generation of fusion proteins with multiple domains and would have major evolutionary implications in terms of natural processes to generate novel proteins with common motifs. Our results, together with accumulation of genomic informations, will stimulate new considerations and experiments in gene expression studies.

Duarte-1 (Los Angeles) and Duarte-2 (Duarte) variants in Germany: two new mutations in the GALT gene which cause a GALT activity decrease by 40-50% of normal in red cells.

Journal of Inherited Metabolic DiseaseVolume 21, Issue 3 p. 232-235 Article Duarte-1 (Los Angeles) and Duarte-2 (Duarte) variants in Germany: Two new mutations in the GALT gene which cause a GALT activity decrease by 40–50% of normal in red cells Y. S. Shin, Y. S. ShinSearch for more papers by this authorH. G. Koch, H. G. KochSearch for more papers by this authorM. Köhler, M. KöhlerSearch for more papers by this authorG. Hoffmann, G. HoffmannSearch for more papers by this authorA. Patsoura, A. PatsouraSearch for more papers by this authorT. Podskarbi, T. PodskarbiSearch for more papers by this author Y. S. Shin, Y. S. ShinSearch for more papers by this authorH. G. Koch, H. G. KochSearch for more papers by this authorM. Köhler, M. KöhlerSearch for more papers by this authorG. Hoffmann, G. HoffmannSearch for more papers by this authorA. Patsoura, A. PatsouraSearch for more papers by this authorT. Podskarbi, T. PodskarbiSearch for more papers by this author First published: 01 June 1998 https://doi.org/10.1023/A:1005303818858Citations: 11AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume21, Issue3June 1998Pages 232-235 RelatedInformation

Molecular and Biochemical Basis of Galactosemia

Galactosemia is a clinically heterogeneous autosomal recessive inborn error of metabolism caused by deficiency of galactose-1-phosphate uridylyltransferase (GALT). Despite the numerous point mutations identified in the GALT gene, the prevalence of these mutations in different ethnic groups has not been studied. Reports on genotype/phenotype correlation are not consistent due to the small sample sizes studied and the lack of a sensitive enzyme assay. We applied multiplex PCR/ASO dot blot analysis to screen 293 galactosemic patients for 17 known point mutations in exons 5, 6, and 10. Our data demonstrate that only 7 of these mutations were detected in our patients, accounting for 65% of the GALT mutant alleles. Although Q188R is the most common mutation in Caucasian and Hispanic patients, the S135L mutation is most common in African-Americans. Another mutation, F171S, was observed only among African-American patients. An improved, sensitive, and accurate method was used to measure GALT activity in patient's red blood cells. The results indicated that patients homozygous for Q188R have no enzyme activity while those homozygous for S135L had residual enzyme activity. Interestingly, both Q188R/S135L and S135L/F171S compound heterozygotes demonstrated zero enzyme activity. Overall, 85% of Q188R compound heterozygotes also did not have any enzyme activity, whereas the remaining Q188R and the majority of S135L compound heterozygotes expressed variable amounts of GALT activity. We speculate that heterodimeric subunit interaction plays an important role in determining the overall enzymatic activity. Various genotypes thus result in biochemical and clinical heterogeneity among the patients.

Duarte allele impairs biostability of galactose-1-phosphate uridyltransferase in human lymphoblasts.

The Duarte allele (D) is a missense mutation (N314D) that produces a characteristic isoform and partial impairment of galactose-1-phosphate uridyltransferase (GALT) in human erythrocytes, fibroblasts, and transformed lymphoblasts. The position of this amino acid is distant, however, from presumptive catalytic site(s) as deduced from a three-dimensional model of crystallized Escherichia coli galT protein. To evaluate the mechanism(s) involved in the partial impairment of enzymatic activity, we compared the activity, abundance, biological stability, and mRNA of GALT in human lymphoblastoid cell lines cultured from individuals homozygous for wild-type (WT/WT) and Duarte alleles (N314D/N314D). No other nucleotide differences were present in their GALT genes. The apparent Vmax was reduced in N314D/N314D cells to 31 +/- 3.6 compared to WT/WT of 54 +/- 6.5 nmole UDP-galactose formed/g cell protein/hour. Both genotypes had similar apparent KMs for UDP-glucose of 0.142 +/- 0.057 mM and 0.133 +/- 0.056 mM. This reduced Vmax was associated with a reduced abundance of the 86kD GALT dimer as determined by Western blots and densitometry. Using RNase protection assays, this reduced GALT protein in the N314D/N314D cell lines was not associated with reduced abundance of GALT mRNA. Using cycloheximide (3-[2-(3,5-Dimethyl-2-oxocyclohexyl)-2-hydroxyethyl]glutarimide) inhibition of de novo protein synthesis, GALT enzyme activity, and its dimeric protein had a biological T1/2 of approximately 24 hours in N314D/N314D cell lines as compared to 50 hours for WT/WT lymphoblasts. Upon exposure to 50 degrees C for 15 minutes, N314D/ N314D lymphoblasts retained 45% of GALT activity, whereas controls retained 77% activity. Reduced activity and thermal sensitivity caused by the N314D mutation reverted to control values when a lysine was substituted for a glutamic acid at amino acid 203 in cis (E203K). In summary, N314D/N314D lymphoblasts have reduced GALT enzyme capacity, dimeric protein abundance, biological, and thermal stability. We conclude that the substitution of aspartate for asparagine at amino acid 314 in the human GALT protein reduces the biostability of the active enzyme in human lymphoblasts.

Milk consumption, galactose metabolism and ovarian cancer (Australia).

It has been suggested that increased exposure to galactose, due to high consumption of dairy foods or reduced galactose metabolism, is associated with the development of ovarian cancer. We have investigated this in a large case-control study conducted in three Australian states between 1990 and 1993. Approximately 800 histologically-confirmed cases, 800 community controls and 300 controls recruited through breast-screening clinics completed dietary questionnaires. Approximately 100 cases and all breast-screening controls also provided a blood sample for analysis of galactose-1-phosphate-uridyltransferase (GALT). Ovarian cancer risk was positively associated with increasing consumption of whole milk and other full-fat dairy foods, but was not associated with consumption of low-fat dairy foods and was inversely related to consumption of skimmed milk. There was no association between ovarian cancer and GALT except among women with abnormally low GALT who had a non-significant 2.5-fold increased risk of ovarian cancer. These data do not support the hypothesis that galactose plays a major role in the development of ovarian cancer and suggest that reported associations between milk consumption and ovarian cancer are due to the fat content of milk and not to lactose or galactose. An increased risk of ovarian cancer in women with abnormally low levels of GALT cannot, however, be ruled out.

Human UDP-Galactose 4′ Epimerase (GALE) Gene and Identification of Five Missense Mutations in Patients with Epimerase-Deficiency Galactosemia

The galactosemias are a series of three inborn errors of metabolism caused by deficiency of any one of the three human galactose-metabolic enzymes: galactokinase (GALK), galactose-1-phosphate uridyl transferase (GALT), and UDP-galactose 4′ epimerase (GALE). We report here the characterization of the entire coding sequence of theGALEgene and screening for mutations in epimerase-deficient individuals. The humanGALEgene is about 4 kb in size and is divided into 11 exons on chromosome band 1p36. We have identified five mutations in theGALEgene of epimerase-deficient galactosemia patients. The patients were either homozygotes or compound heterozygotes for mutations. These results confirm that epimerase-deficiency galactosemia is the result of missense mutations in theGALEgene and indicate that the disease is characterized by extensive allelic heterogeneity.

Congenital absence of the uterus and vagina (CAUV) is not associated with the N314D allele of the Galactose-1-Phosphate Uridyl Transferase (GALT) gene

Congenital absence of the uterus and vagina (CAUV) is not associated with the N314D allele of the Galactose-1-Phosphate Uridyl Transferase (GALT) gene

Biochemical characterization of the S135L allele of galactose‐1‐phosphate uridylyltransferase associated with galactosaemia

Impairment of the human enzyme galactose-1-phosphate uridylyltransferase (GALT) results in the potentially lethal disorder galactosaemia. The S135L mutation, which accounts for almost 50% of the GALT alleles in galactosaemia patients of African-American descent, has been associated with activities ranging from null to wild-type by different investigators examining cell lysates representing different tissues or model systems. Because of the crude nature of the lysates examined, however, and the absence of quantitative measures concerning GALT abundance in most of those lysates, the available data do not distinguish between differences in GALT enzyme expression/abundance, specific activity, or kinetic constants in these different tissues or systems. In an effort to overcome this uncertainty and investigate the biochemical impact of the S135L substitution on human GALT function under defined conditions, we have overexpressed both wild-type and S135L-mutant GALT sequences in a null-background yeast expression system, and purified both proteins to near homogeneity. Abundance of the wild-type and mutant proteins in crude yeast lysates differed by approximately 2-fold. Kinetic studies of the purified proteins, however, demonstrated that although K(m) values differed by < 2-fold, specific activities differed by 10-fold. Temperature-activity profiles revealed no significant differences, and coprecipitation studies demonstrated that S135L-hGALT subunits remained competent to self-associate in vivo. We conclude that the S135L substitution causes either steric or electrochemical changes sufficiently close to the active site in human GALT to result in partial impairment of the transferase reaction.

Quantitative assessment of whole body galactose metabolism in galactosemic patients.

We employed [1-13C] galactose in isotope kinetic studies to delineate whole body galactose metabolism in vivo in patients with galactose-1-phosphate uridyltransferase (GALT) deficiency. The data in three control and three adult galactosemic subjects, homozygous for the most common GALT gene defect, the Q188R mutation, and with absent RBC GALT activity, revealed an apparent endogenous galactose synthesis rate of 0.53-1.05 mg/kg per hour. Unlike normal children and adults who eliminated 3%-6% and 21%-47% of an intravenous bolus of [1-13C] galactose as 13CO, in expired air in 1 and 5 h respectively, classic galactosemic patients, either Q188R/Q188R or Q188R/unknown, released almost none in 1 h and 3%-6% in 5 h. In contrast, an African-American galactosemic variant patient with a S135L/S135L mutation and no residual RBC GALT activity oxidized [1-13C]galactose to 13CO2 at a rate comparable to control subjects. Individuals homozygous for the Duarte mutation, N314D/N314D and Q188R/ N314D. Q188R/+ and S135L/+ subjects also had normal breath test results. Not surprisingly, the Q188R/Q188R classic galactosemic patient cannot handle an acute galactose load, failing to match a control subject in the rapid conversion of [1-13C]galactose to [13C]glucose and 13CO2. However, classic patients synthesize substantial quantities of galactose de novo and on a lactose-free diet must oxidize comparable amounts of galactose to maintain steady-state levels of galactose and galactose metabolites such as galactose-1-phosphate, galactitol and galactonate. In vivo isotope kinetic analyses may allow us to understand better these aspects of galactose metabolism and, through the use of studies in variant galactosemics, perhaps allow us to begin to unravel the pathophysiology of galactosemia.

Changes in cell surface glycosylation in alpha1,3-galactosyltransferase knockout and alpha1,2-fucosyltransferase transgenic mice.

Inactivation of the alpha1,3-galactosyltransferase (GalT) gene by homologous recombination (knockout [KO] mice) and competition for the enzyme's N-acetyllactosamine substrate by transgenically expressed alpha1,2-fucosyltransferase (H-transferase) are two genetic approaches to elimination of the Gal alpha1,3Gal (alphaGal) epitope, which is the major xenoantigen in pigs against which humans have preformed antibodies. Such genetic manipulations often have unpredictable results. A panel of 19 selected lectins was used to characterize the changes in cell surface glycosylation in GalT KO and H-transferase transgenic mice, compared with nontransgenic littermate controls. GalT KO mice showed complete elimination of the alphaGal epitope, as reported previously. Surprisingly, however, this was associated with only a modest increase in N-acetyllactosamine residues and had little other effect on the pattern of lectin binding. In contrast, the pattern of lectin binding to H-transferase transgenic mouse cells was more profoundly disturbed and indicated, in addition to the expected expression of H substance and suppression of the alphaGal epitope, that there was a marked reduction in alpha2,3-sialylation and exposure of the normally cryptic antigens, sialylated Tn and Forssman antigens. Similar changes in lectin reactivity with porcine aortic endothelial cells were induced by neuraminidase treatment. Lectins were able to bind underlying carbohydrate structures (sialylated Tn and Forssman antigens) that are normally cryptic antigens on H-transferase transgenic mouse spleen and cardiac endothelial cells, probably as a consequence of the reduction in the electronegativity of the cell surface due to reduced sialylation. As humans have preformed anti-Tn and anti-Forssman antibodies, it is possible that these structures may become targets of the xenograft rejection process, including hyperacute rejection.

Black children deficient in galactose 1-phosphate uridyltransferase: Correlation of activity and immunoreactive protein in erythrocytes and leukocytes

A recent study found a high prevalence of a missense mutation (S135L) in the gene for galactose 1-phosphate uridyltransferase (GALT) in black children with galactosemia (J Pediatr 1996;128:89-95). In the present study, GALT activity and GALT protein content were measured in erythrocytes and leukocytes of eight black and seven white galactosemic (GALT-deficient) children, for correlation with the presence of the S135L and Q188R (highly prevalent in white galactosemic children) missense mutations. The S135L mutation was found in 9 of 16 alleles of black children but not in white children; the Q188R mutation was found in 10 of 14 alleles examined in white galactosemic children and in 4 of 16 alleles in black galactosemic children. The GALT activity was near zero in the erythrocytes of white and black galactosemic children (0.26 ± 0.28 vs 0.33 ± 0.25 μmol/hr per gram of hemoglobin, respectively; p = 0.61) (normal 17 to 26 μmol/hr per gram), and no correlation of erythrocyte activity with genotype was observed. The GALT activity was higher in the leukocytes of black galactosemic children compared with white children (5 ± 6 vs 1 ± 2 μmol/hr per gram, respectively) (normal 172 to 374 μmol/hr per gram), but the difference was not statistically significant ( p = 0.11). Analysis by genotype revealed that the two S135L homozygotes had much more leukocyte activity (9 and 17 μmol/hr per gram) than Q188R homozygotes or than all non-S135L allelic genotypes. Compound heterozygotes (S135L/G) had intermediate activity. The GALT protein was not detectable by Western blot in the erythrocytes of either white or black galactosemic children, as determined by antibodies specific for both C- and N-terminal sequences. The GALT protein was undetectable in the leukocytes of white galactosemic children, but leukocytes from black galactosemic children with the S135L mutation contained reduced but readily detectable GALT protein. Erythrocyte galactose 1-phosphate levels were significantly lower in galactosemic children with an S135L mutant allele (1.1 ± 0.2 gm/dl) compared with children who had other mutations (3.1 ± 0.9 mg/dl; p = 0.0001). The correlation of protein content data with activity levels in the blood cells suggests that the S135L missense mutation affects the stability of GALT protein to produce a deficiency state. (J Pediatr 1997;130:972-80)

Altered follicle stimulating hormone isoforms in female galactosaemia patients.

Many women affected with galactosaemia suffer from ovarian dysfunction and have elevated serum levels of follicle stimulating hormone (FSH). We have analysed FSH-glycoprotein isoforms from four galactosaemic and five healthy women. Besides the commonly found FSH species with a median isoelectric point (pI) of 4-5, the sera of the female galactosaemic patients contained qualitatively abnormal FSH isoforms with a pI close to neutral (6.4-7.0). The generally reduced galactosylation in patient samples was confirmed because sera of galactosaemic patients could incorporate 1.7 times more UDP-(14C)galactose than did healthy subjects. Our data indicate that the terminal disaccharides of FSH (a glycoprotein), galactose and sialic acid were partially deficient in three galactosaemic female patients with no galactose-1-phosphate uridyl transferase (GALT) activity in red cells. However, from a female patient with a residual GALT activity (a mild form of galactosaemia), no distinctive deficiency was observed. This again suggest an importance of GALT in retaining a correct FSH structure. Therefore the abundance of neutral FSH isoforms, which was described to have a higher binding affinity to its receptor and no capacity to activate cyclic adenosine mono-phosphate (cAMP), may cause a hormonal dysfunction in classical galactosaemia.

Molecular heterogeneity of classical and Duarte galactosemia: mutation analysis by denaturing gradient gel electrophoresis.

Classical galactosemia is caused by one common missense mutation (Q188R) and by several rare mutations in the galactose-1-phosphate uridyltransferase (GALT) gene. The most common variant of GALT, the Duarte variant, occurs as two types, Duarte-1 (D-1) and Duarte-2 (D-2), both of which carry the sequence change N314D. D-1 increases, whereas D-2 decreases GALT activity. To study the molecular genetics of classical and Duarte galactosemia, we analyzed the GALT mutations in 30 families with classical galactosemia, in 10 families with the D-2 variant and in 3 individuals carrying the D-1 allele by denaturing gradient gel electrophoresis (DGGE). DGGE detected 59 of the 60 classical galactosemia alleles. Q188R accounted for 60%, K285N accounted for 28% of these alleles. Eight novel candidate galactosemia mutations were found. On all D-2 alleles N314D occurred in cis with two intronic sequence changes, on the D-1 alleles in cis with a neutral mutation in exon 7. We conclude that the mutations causing galactosemia are highly heterogeneous and that K285N is a second common galactosemia mutation in our population.