Introduction of newborn screening for galactosemia to Arkansas.
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Chromosome Aberrations↗
Biomedical subjects
J B Gibson
Publications and source records attributed to J B Gibson.
Retrotransposon insertion induces an isozyme of sn-glycerol-3-phosphate dehydrogenase in Drosophila melanogaster.
The insertion of the blood retrotransposon into the untranslated region of exon 7 of the sn-glycerol-3-phosphate dehydrogenase-encoding gene (Gpdh) in Drosophila melanogaster induces a GPDH isozyme-GPDH-4-and alters the pattern of expression of the three normal isozymes-GPDH-1 to GPDH-3. The process of transcript terminus formation inside the retrotransposon insertion reduces the level of the Gpdh transcript that contains exon 8 and increases the level of the transcript that contains exons 1-7. The induced GPDH-4 isozyme is a translation product of the three transcripts that contain fragments of the blood retrotransposon. The mechanism of mutagenesis by the blood insertion is postulated to involve the pause or termination of transcription within the blood sequence, which in turn is caused by the interference of a DNA-binding protein with the RNA polymerase. Thus, we show the formation of a new functional GPDH protein by the insertion of a transposable element and discuss the evolutionary significance of this phenomenon.
Alleles↗
Endogenous synthesis of galactose in normal men and patients with hereditary galactosaemia.
Despite restricted ingestion of lactose, patients with galactose-1-phosphate uridyltransferase deficiency have raised concentrations of galactose metabolites in blood and urine. Endogenous production of galactose may underlie this phenomenon. Using isotopically labelled galactose in a continuous intravenous infusion, we employed the steady-state flux method to calculate endogenous galactose production rate in three normal men and three patients with classic galactosaemia. We found that galactosaemic patients and normal subjects synthesise gram quantities of galactose per day. The rate of synthesis ranged from 0.53-1.05 mg/kg per h. Endogenous production of galactose may be an important factor in the pathogenesis of the complications of the brain and ovary, and could explain the persistent elevation of galactose metabolites in patients despite dietary restriction of galactose.
Adult↗
Effect of glucose and galactose loading in normal subjects on red and white blood cell uridine diphosphate sugars.
In order to test the assumption that red blood cell (RBC) uridine diphosphate galactose content is regulated in part by dietary galactose and to help comprehend factors influencing RBC and white blood cell (WBC) uridine diphosphate hexose concentrations in vivo, oral loading studies were performed with 50 g of galactose or 75 g of glucose in normal adults. While elevations of blood glucose did not influence RBC or WBC UDPhexose levels, increased blood galactose concentrations caused transient increases in both RBC and WBC UDPgalactose. In both RBC and WBC, the UDPgalactose-4-epimerase was rate limiting. In comparison to RBC, WBC had larger changes in UDPgalactose levels, synthesized additional UDPglucose through the action of UDPglucose pyrophosphorylase and began to restore the equilibrium ratio between UDPglucose and UDPgalactose concentrations while the plasma galactose level was still increasing. Thus, galactose ingestion alters the steady-state levels of UDPhexoses in circulating cells. The modulation of UDPglucose and UDPgalactose concentrations, occurring after the presentation of galactose to cells, as a result of the combined actions of GALT and UDPgalactose-4-epimerase, may be important in determining rates of synthesis of complex glycoconjugates.
Administration, Oral↗
In vivo oxidation of [13C]galactose in patients with galactose-1-phosphate uridyltransferase deficiency.
We developed an intravenous and oral [13C]galactose breath test for the in vivo study of galactose metabolism. Following an intravenous bolus of 7 mg/kg of [1-13C]galactose in the fasting state, normal children and adults eliminated 3-6% and 21-47% of the bolus as 13CO2 in expired air collected over 1 and 5 h, respectively. Comparable fractional elimination was seen when the dose was given orally. Patients with galactosemia who have barely detectable or absent galactose-1-phosphate uridyltransferase (GALT) activity in erythrocytes and are homoallelic for the Q188R gene mutation, when given a 7 mg/kg intravenous bolus had barely detectable 13CO2 in air samples in the first hour, but eventually eliminated as much as 3.6% of the dose in 5 h. A galactosemia/Duarte (Q188R/N314D) compound heterozygote and a homozygous Duarte subject, as well as a subject with one normal allele and one Q188R allele, showed normal in vivo oxidation. An assessment of whole body galactose metabolism can be made with this procedure. Further use of this in vivo modality in patients with different genetic backgrounds should increase our understanding of genotype-phenotype relationships in hereditary galactosemia.
Adolescent↗
Gonadal function in galactosemics and in galactose-intoxicated animals.
Gonadal dysfunction, specifically hypergonadotropic hypogonadism, in female galactosemics is an almost universal finding. Minimal transferase activity may modulate ovarian function and the time of menopause. In contrast, male galactosemics have a relatively low risk of gonadal dysfunction. Animal models have variously suggested prenatal and postnatal insults which may play a role in the gonadal pathology. Several candidate toxic states may be involved. Current dietary restrictions are inadequate to prevent ovarian failure. Until such time as the pathophysiology is better understood, therapy will remain palliative and supportive. Exogenous estrogen and progesterone have roles in assisting pubertal changes and in prevention of the sequelae of a post-menopausal state.
Animals↗
Comparison of erythrocyte uridine sugar nucleotide levels in normals, classic galactosemics, and patients with other metabolic disorders.
By limiting galactosylation mechanisms, a cellular deficiency of the uridine sugar nucleotide, UDPgalactose, has been implicated as a cause of the long-term complications seen in patients with classic galactosemia despite dietary treatment. As a result, great interest has been generated in the accurate assessment of UDPgalactose, as well as UDPglucose, from which UDPgalactose may be derived by the function of a ubiquitous, active UDPgalactose-4-epimerase. Since several series of values for the concentration of these compounds in red blood cells (RBCs) of galactosemics have been flawed by the use of methods subsequently shown to be unsuitable, we have quantified erythrocyte UDPgalactose and UDPglucose levels by an accurate high-performance liquid chromatography (HPLC) assay in 116 normals, 76 galactosemics, and 39 patients with other metabolic disorders. These large groups have permitted the evaluation of age, diet, and genotype as influential factors in the steady-state RBC levels of the sugar nucleotides. The data show that age is an important determinant of RBC levels, with children younger than 10 years having higher values than individuals older than 10 years. Mean UDPgalactose levels in galactosemic children younger than 10 years and those older than 10 years were 30% and 18% lower, respectively, than levels in comparable normals. Although the mean differences were highly significant, there was considerable overlap of individual values. There was no difference in UDPglucose levels between galactosemics and normals.(ABSTRACT TRUNCATED AT 250 WORDS)
Adolescent↗
Molecular analysis of a Drosophila melanogaster sn-glycerol-3-phosphate dehydrogenase allozyme variant that has cold labile activity.
A rare naturally occurring allele, GpdhACb62, at the sn-glycerol-3-phosphate dehydrogenase locus in Drosophila melanogaster, encodes an enzyme with an electrophoretic mobility that is more cathodal than that produced by the common slow electrophoretic allele. After electrophoresis and staining of extracts of single adult flies there is a single band of activity corresponding in position to GPDH-1, but, using highly concentrated extracts, a faint band corresponding to GPDH-3 is observed. In GpdhACb62 homozygotes there is about 26% of the normal level of activity in adults, and less than 6% in third instar larvae. The reduction in activity is significantly greater than the decrease in GPDH immunologically cross-reacting material (CRM). Northern analyses, and rapid amplification of the cDNA ends (RACE) of the 3' regions of the transcripts, show that the levels and structures of the poly(A)+RNAs are similar in homozygotes for GpdhACb62 and for a normal activity allele GpdhAC8. Hybridization to oligonucleotide probes specific for the GPDH-1 and GPDH-3 transcripts was of a similar intensity in GpdhACb62 and GpdhAC8 adult flies. In third instar larvae the main transcript is for GPDH-3 and again the hybridization signals were similar in each line. The activity of the enzyme produced by GpdhACb62 was unstable both at 50 degrees C and at 0 degrees C. The activity lost at 0 degrees C was recovered by incubation at 20 degrees C. The complete GpdhACb62 gene, and the partial Gpdh tandem duplication 3' to this gene, were cloned and sequenced. Comparisons with two normal activity GpdhF genes revealed 31 unique changes in the first copy of GpdhACb62. In exon 4, a T to G substitution changes cysteine to glycine and may disrupt a disulphide bond and be responsible for the distinctive properties of GPDH-ACb62.
Alleles↗
Molecular heterogeneity of naturally occurring sn-glycerol-3-phosphate dehydrogenase low-activity variants in Drosophila melanogaster.
Northern analyses of two low-activity sn-glycerol-3-phosphate dehydrogenase (Gpdh) alleles extracted from natural populations of Drosophila melanogaster showed that one of them, GpdhACyg22, produced wild-type levels of a normal sized (1.7-kb) mRNA but the other, GpdhAMB5, had very low levels of a 1.7-kb mRNA together with low levels of a transcript 200 bp larger. The two variant genes were cloned and sequenced. Compared with normal activity alleles, there were two nucleotide differences in the DNA sequence of GpdhACyg22 which were in first-codon positions and would be expected to give rise to Asn-13-->Tyr and Arg-272-->Cys substitutions. The second of these changes is most likely to account for the altered properties of the enzyme. In contrast, none of the nucleotide differences in GpdhAMB5 would give rise to amino acid substitutions, but a 76-bp deletion in the 5' region removed the normal TATA box and there was a 20-bp insertion in the same region. One of the two transcripts was derived from the use of a substitute TATA box sequence in the insertion, but the 1.9-kb transcript had heterogeneous 5' ends that were not associated with substitute TATA box sequences. The two transcripts either are produced at a lower rate or are less stable than the normal mRNA.
Alleles↗
Uridine diphosphate hexoses in leukocytes and fibroblasts of classic galactosemics and patients with other metabolic diseases.
To examine uridine diphosphate hexose (UDPhexose) content of cells that have more complete metabolic patterns than erythrocytes, which have been commonly used in the study of galactosemia, the concentrations of uridine diphosphate galactose (UDPgalactose) and uridine diphosphate glucose (UDPglucose) were determined in white blood cells (WBC) and fibroblasts cultured from skin biopsies. Leukocyte UDPgalactose and UDPglucose values were determined in 60 normal individuals, 14 classic galactosemics, and 18 patients with other metabolic diseases on protein-restricted and low-lactose diets. There was no difference in the average concentration of these compounds between any of these groups. There was no relationship between age and WBC UDPhexose content or correlation of WBC and erythrocyte UDPhexose levels in the same blood specimens. WBC from galactosemic individuals differ from their red blood cells because the former do not show the low average UDPgalactose levels and abnormal UDPglucose to UDPgalactose ratio previously reported for erythrocytes from galactose-1-phosphate uridyltransferase-deficient individuals. Fibroblast cell lines from 10 normal and 10 galactosemic individuals, cultured and grown to confluence in glucose medium, also showed no difference in nucleotide sugar concentrations. Thus far, of the cell types easily available, red blood cells appear to be unique in showing an abnormality in nucleotide sugar metabolism. The fact that galactosemic fibroblasts demonstrate no abnormality in the concentration of these compounds suggests that the defective galactosylation that has been observed in galactosemic fibroblasts is not due to unavailability of UDPgalactose.
Adolescent↗
Defective P element insertions affect the expression of sn-glycerol-3-phosphate dehydrogenase alleles in natural populations of Drosophila melanogaster.
A distinctive and geographically widespread category of low-activity variant at the Gpdh locus in Drosophila melanogaster is shown to have defective P elements inserted between the TATA box and the transcription start site. In four examples the insertion was a single 1.15 kilobase (kb) KP element, whereas in another variant there were two KP elements in tandem. A sixth example contained a 0.61 kb defective P element. The target site for all of the insertions is GTGCAAAC. There was no sequence variation either between the insertions or in comparison with two other KP elements previously described from natural populations. The insertions cause a reduction in GPDH mRNA, and are the most likely cause of the low GPDH activity.
Alleles↗
Uridine diphosphoglucose content of human erythrocytes: assessment by conversion to uridine diphosphoglucuronate.
To settle the ongoing controversy regarding differential uridine diphosphoglucose (UDPG) and uridine diphosphogalactose (UDPGal) content of erythrocytes, which may be important in evaluating the metabolic abnormality in patients with galactosemia, we derived a combined enzymatic-high-performance liquid chromatography (HPLC) assay. Uridine diphosphoglucuronate (UDPGA), the unique product of UDPG dehydrogenase activity, was separated and quantified by HPLC in extracts of human erythrocytes. The quantity of UDPGA produced in cell filtrates incubated with the enzyme corresponds to the amount of UDPG directly determined by HPLC. The amount of UDPGA produced was independent of the enzyme purity or activity used. On the other hand, the amounts of UDPG estimated by fluorometric measurement of the production of reduced nicotinamide adenine dinucleotide varied with the enzyme purity and activity. The combined enzymatic-HPLC method confirms the direct determinations of UDPG content of normal erythrocytes. The results indicate that, under appropriate conditions, the fluorometric-based assay will give accurate estimates of UDPG, but the direct HPLC method yields consistent and correct UDPG and UDPGal determinations.
Chromatography, High Pressure Liquid↗
Concentration of white blood cell UDPgalactose and UDPglucose determined by high performance liquid chromatography.
We have applied a HPLC method to separate and quantitate UDPgalactose (UDPGal) and UDPglucose (UDPGlu) in human white blood cells (WBCs). Trichloroacetic acid-treated, protein-free filtrates were chromatographed on an anion-exchange column (Dionex CarboPac PA-1) using a gradient of 20-40% potassium phosphate buffer (pH 4.5). Recoveries of UDPGal and UDPGlu ranged from 93 to 106%, and the method was linear over a wide range of WBC protein concentrations. Volumes of blood as low as 2.5 ml (2.2 mg WBC protein) could be used to achieve quantitative recovery of the sugar nucleotides. The mean values and standard deviations of UDPGal and UDPGlu in 33 normal individuals ranging in age from 1 day to 65 years were 12.4 +/- 4.2 and 31.5 +/- 9.3 mumol/100 g protein, respectively. No statistical differences in UDPGal and UDPGlu values were observed between children and adults. No correlation was established between the concentrations of UDPGal and UDPGlu and either total WBC count or the number of lymphocytes obtained from Coulter counter analysis. There was no relationship between the concentrations of UDPGal and UDPGlu in WBCs and RBCs which were prepared from the same blood specimen.
Adolescent↗
Restriction site variation, gene duplication, and the activity of sn-glycerol-3-phosphate dehydrogenase in Drosophila melanogaster.
Restriction site variation in a 25-kb region including the sn-glycerol-3-phosphate dehydrogenase (Gpdh) locus has been assessed in 29 single female D. melanogaster lines from the Cardwell (Australia, QLD) population. The Gpdh locus was duplicated in about one-third of the lines, although the duplication was incomplete and lacked exons 1 and 2. There was no restriction site variation in the duplicated region. Three insertions were found in the gene region but only one affected GPDH activity. The lines with the duplication had higher levels of GPDH activity and protein amount than did nonduplicated lines. This effect was also observed in lines extracted from two other Australian populations. The duplication is shown to have a similar structure in each population investigated and is also present in populations from China and Africa. It is suggested that the effect of the duplication on GPDH activity, which might be due to structural factors affecting transcription at the Gpdh locus, could account for the worldwide distribution of the duplication.
Animals↗
The alcohol dehydrogenase polymorphism in natural populations of Drosophila melanogaster: restriction map variation in the region of the Adh locus in populations from two hemispheres.
Restriction endonuclease variation in the 12 kb region surrounding the Adh locus was measured in seven Australian and six Chinese populations of Drosophila melanogaster. There is a higher level of nucleotide-substitution variation in the Australian populations than in the Chinese, which is possibly a reflection of their origins. None of the restriction site polymorphisms, nor any of the insertions, showed a significant association with latitude. A 0.2 kb deletion varied with latitude in the Chinese populations. In accordance with previous studies, a majority of the insertions were located in a region 1.5-3.5 kb 3' from the Adh coding region, and a majority of the deletions were at a site 3 kb 5' to the Adh coding region. Two of the insertions shared homologies with known mobile elements. Overall, the data suggest that restriction endonuclease variation in the Adh region is not related to the cline in Adhs frequencies.
Alcohol Dehydrogenase↗
The alcohol dehydrogenase polymorphism in natural populations of Drosophila melanogaster: ADH activity variation restriction site polymorphism and the Adh cline.
Alcohol dehydrogenase activity has been measured in 186 iso-second chromosome lines--104 from seven Australian populations and 82 from six Chinese populations. Restriction endonuclease variation in the Adh gene region in these lines has previously been described (Jiang & Gibson, 1991). The mean ADH activity of AdhF and AdhS lines was significantly higher in the Chinese samples than in the Australian samples. In each population on both continents the mean activity of the AdhF lines is significantly higher than that of the AdhS lines. Six lines homozygous for a thermostability variant, AdhFChD (detected in four of the Chinese populations), had intermediate levels of ADH activity and protein amount. In a subset of the lines with the highest and lowest levels of ADH, there was a correlation of 0.69 between ADH activity and ADH CRM. None of the restriction site variants was consistently associated with the amount of ADH activity. Associations between BamHI (-7.2), the Adh polymorphism and ADH activity suggest that there are modifiers of ADH 5' to the gene. The deletion (0.2) at position -2.8 on the restriction map (Jiang & Gibson, 1991) was associated with increased levels of ADH activity in AdhS lines from China. Two unique insertions in the gene region were associated with low activity in AdhF lines and a null activity allele had a deletion removing most of exon 2. A single line with a duplication of a part of the Adh coding region and of the 5' regulatory section had relatively high ADH activity. Considering all the data, the main factor affecting ADH activity levels in populations is the frequency of AdhF.
Alcohol Dehydrogenase↗
Molecular relationships between alcohol dehydrogenase null-activity alleles from natural populations of Drosophila melanogaster.
Alcohol dehydrogenase null-activity alleles extracted from a number of natural populations of Drosophila melanogaster in Tasmania were shown to be molecularly similar by probing, with an oligonucleotide specific to an inserted region in intron 2 of the gene, genomic DNA amplified by the polymerase chain reaction. This insertion had previously been shown to be the cause of the loss of activity in one of the null alleles whose DNA sequence was known. Three Adh null alleles from mainland populations did not contain the insertion. Two of these null alleles, extracted from the Coffs Harbour population in different years, were cloned, and their DNA sequences showed that they were identical and that both had a 438-bp deletion which removed most of exon 2. The third null allele, identified in a sample of flies from Chateau Tahbilk, was shown by 4-bp restriction-endonuclease mapping to contain a 320-bp insertion in intron 1, although this may not be the cause of the loss of activity. The data show that at least three different Adh null alleles have been found in Australian populations and that at least two have been maintained as heterozygotes over a period of years.
Alcohol Dehydrogenase↗
Low activity sn-glycerol-3-phosphate dehydrogenase variants in natural populations of Drosophila melanogaster.
Eleven Australian natural populations of Drosophila melanogaster were screened by electrophoresis for evidence of null and/or low activity variants at the Gdph locus. Of 5018 alleles investigated, 57 had markedly lower GDPH activity, as judged by their heterozygous phenotypes, than control alleles. GPDH assays of 13 of these variant alleles showed that whilst they were heterogeneous in their properties and included electrophoretic variants and two categories of low-activity variant, there were no true null alleles. The most common low-activity variants exhibited dominance for GPDH activity in heterozygotes with normal alleles, and were shown to share this property with an allele previously isolated from a London (UK) population (Langley et al., 1981).
Alleles↗