Sharon D. Langley

A prevalent mutation for galactosemia among black Americans

OBJECTIVE To define the mutation causing galactosemia in patients of black American origin who have no galactose-1-phosphate uridyltransferase (GALT) activity in erythrocytes but good clinical outcome. METHODS We discovered a mutation caused by a C-->T transition at base-pair 1158 of the GALT gene that results in a serine-to-leucine substitution at codon 135 (S135L). We developed a method with which to screen populations for its prevalence. We compared galactose-1-phosphate uridyltransferase among erythrocytes, leukocytes, and transformed lymphoblasts, as well as total body oxidation of D-(13C)-galactose to 13CO2 among three genotypes for GALT (S135L/S135L, Q188R/Q188R, and Normal/Normal). RESULTS We found a 48% prevalence of the S135L mutation among 17 black American patients with classic galactosemia and a 1% prevalence in a population of 50 black Americans without galactosemia. The S135L mutation was not found in 84 white patients with G/G galactosemia nor in 87 white control subjects without galactosemia. We found normal whole body oxidation of D-(13C)-galactose by the patient homozygous for S135L and various degrees of enzyme impairment among different tissues. CONCLUSIONS The S135L mutation in the GALT gene is a prevalent cause of galactosemia among black patients. Because GALT activity varies in different tissues of patients homozygous for S135L, they may have a better clinical outcome than patients who are homozygous for Q188R when both are treated from infancy.

A molecular approach to galactosemia

Classical galactosemia (G/G) is caused by the lack of galactose-1-phosphate uridyltransferase (GALT) activity. A more common clinical variant, Duarte/Classical (D/G) produces partial enzymatic impairment. Although neonatal death due to G/G galactosemia has been largely eliminated by populationbased screening and intervention, long-term outcome in some is associated with impaired growth, ovarian failure, dyspraxic speech and neurologic deficits. At least 32 variants in the nucleotide sequence of the GALT gene have been identified and 9 have transferred impaired GALT activity to transformed cells in transfection experiments. We here define the prevalence and biochemical phenotype of two mutations. An A to G transition in exon 6 of the GALT gene converts a predicted glutamine at codon 188 to an arginine (Q188R), and introduces a new HpaII cut site into the gene which enables population screening by polymerase chain reaction. An A to G transition in exon 10 in the GALT gene produces a codon change converting an asparagine to aspartic acid at codon 314 (N314D) and adds an AVA II cut site. We screened a large population for the Q188R and N314D sequence changes to investigate the prevalence of Q188R in G/G galactosemia, the effect of homozygosity for Q188R on outcome, and the prevalence and biochemical phenotype of the N314D sequence change. We found that the Q188R mutation has a prevalence of 62% in a predominately Caucasian population of 107 patients with G/G galactosemia. homozygosity for Q188R was associated with a poor clinical outcome in a subgroup of these patients. The N314D mutation is associated with the Duarte biochemical phenotype with extraordinary concordance.

The 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 ˜24 hours in N314D/N314D cell lines as compared to 50 hours for WT/WT lymphoblasts. Upon exposure to 50°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. Hum Mutat 11:28–38, 1998.

Increased glucose transport by human fibroblasts with a heritable defect in insulin binding.

Insulin and IGF-I binding and their regulation of hexose transport were evaluated in skin fibroblasts cultured from a family (Atl) whose proband had leprechaunism, hypoglycemia, and severe insulin resistance. High affinity insulin binding to proband Atl cells was absent, and partially, but equally, impaired in fibroblasts from his related parents. IGF-I binding to his cultured fibroblasts was within the normal range. Cells from proband Atl had insulin receptor mRNAs similar to control fibroblasts. 3-O-Methyl-D-glucose (OMG) transport by proband Atl was threefold higher than in control fibroblasts (37.7 v 7.6-11 nmol/mL/s) and was insulin-insensitive. Proband Atl fibroblasts had a threefold increase in the Vmax for OMG entry and a concomitant increase in the number of D-glucose-inhibitable cytochalasin B binding sites on their plasma membrane. Similar levels of glucose transporter mRNA were observed in control and proband Atl fibroblasts. These results suggest that fibroblasts from patient Atl have a genetically transmitted mutation in the alpha subunit of their insulin receptor. In the homozygous affected proband, this mutation impairs insulin binding and causes elevated, insulin-insensitive glucose transport. The dysfunction resulting from this mutation is similar to that introduced in Chinese hamster ovary cells by transfection with a truncated alpha subunit.

Glucose transport by cultured human fibroblasts: regulation by phorbol esters and insulin

The regulation of 3-O-methyl-D-glucose (OMG) uptake by insulin and phorbol esters was studied in cultured human skin fibroblasts. Insulin rapidly stimulated OMG uptake through a mechanism independent of new protein synthesis. Maximal insulin effect was reached in 30 min and remained constant up to 12 h. The protein kinase C activators 12-O-tetradecanoyl phorbol 13-acetate (TPA) and phorbol 12,13-dibutyrate (PdBU) promoted an initial rapid stimulation followed by a secondary long-term rise of OMG influx. This latter effect of phorbol esters on OMG influx began after 1 h, reached a maximum in 12-15 h, and was prevented by the simultaneous addition of protein synthesis inhibitors, suggesting that phorbol esters increased the synthesis of new glucose transporters. In accord with this interpretation, phorbol esters, but not insulin, increased mRNA levels for two distinct glucose transporters (GLUT1 and GLUT3) in human fibroblasts. Both the rapid and the long-term effects of phorbol esters on OMG influx were dose-dependent and half-maximal stimulations occurred at 15 nM for both PdBU and TPA. Kinetic analysis of OMG uptake indicated that both effects of phorbol esters were associated with an increase in the Vmax of the transport process, with no significant changes of the Km (4-6 mM). These results suggest that, in human fibroblasts, phorbol esters, unlike insulin, produce a long-term stimulation of OMG uptake, which is dependent upon protein synthesis and is associated with increased levels of GLUT1 and GLUT3 mRNA.

ROLE OF ARGININE 86 OF THE INSULIN RECEPTOR IN INSULIN BINDING AND ACTIVATION OF GLUCOSE TRANSPORT

Mutations in the insulin receptor gene cause the inherited insulin resistant syndrome leprechaunism. Patient Atl-1 with leprechaunism was homozygous for the substitution of Arg-86 with Pro (R86P) in the alpha subunit of the insulin receptor. Fibroblasts homozygous for the mutant receptor had defective insulin binding, but increased glucose transport and receptor kinase activity. The R86P mutation is located in a putative beta turn N-terminal to a proposed insulin binding domain of the receptor [P. DeMeyts, J.L. Gu, R.M. Shymko, B.E. Kaplan, G.I. Bell, J. Whittaker, Mol. Endocrinol. 4 (1990) 409-416]. To get further insight into the mechanism of the paradoxical activation of receptor signalling by the R86P mutation, the codons for proline, alanine, and glycine were substituted in the R86 position of the insulin receptor cDNA by PCR-mediated mutagenesis and stably transfected into Chinese hamster ovary (CHO) cells. Insulin binding increased 10-20 fold in CHO cells transfected with the wild type, the R86A, and the R86G insulin receptor cDNA, but did not increase in cells expressing the R86P mutation. The R86P mutation caused a constitutive activation of insulin receptor phosphorylation in CHO cells, but did not increase basal glucose transport or its sensitivity to insulin stimulation. By contrast, transfection with the wild type and the R86A receptors increased 20-30 fold the sensitivity of glucose transport to stimulation by insulin. The R86G insulin receptor bound insulin normally, but was four times less efficient than the wild type or R86A insulin receptor in increasing the sensitivity for insulin stimulation of glucose transport. These results indicate that position 86 of the insulin receptor alpha subunit is tolerant to substitution by alanine, but not by proline. Substitution with glycine allows insulin binding, but does not activate normally glucose transport, further supporting an essential role of this position in the initiation of insulin receptor signalling of glucose transport.