1 Department of Pediatrics, Haukeland University Hospital, University of Bergen, Norway
2 Center for Medical Genetics and Molecular Medicine, Haukeland University Hospital, University of Bergen, Norway
3 Department of Biochemistry and Biophysics, and Diabetes Research Center, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania
4 Department of Pediatrics, Rambam Medical Center, Haifa, Israel
5 Division of Newborn Medicine, Department of Pediatrics, Gülhane Military Medical Academy, Ankara, Turkey
6 Department of Pathology, the Gade Institute, Haukeland University Hospital, University of Bergen, Norway
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ABSTRACT |
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Neonatal diabetes, insulin-requiring hyperglycemia occurring within the first month of life is often associated with intrauterine growth retardation (IUGR) and can be either transient or permanent (1). Transient neonatal diabetes is associated with abnormalities of chromosome 6, including paternal uniparental disomy and paternal duplications of 6q24, with loss of imprinting (1,2) and increased risk of diabetes later in life. Mutations in the insulin promoter factor-1, a transcription factor implicated in pancreatic development and the regulation of insulin gene expression, result in permanent neonatal diabetes (PNDM) caused by pancreatic agenesis (3). We have recently shown that complete deficiency of the glycolytic enzyme glucokinase is another cause of PNDM (4). Two patients presented with IUGR, permanent insulin requirement from shortly after birth and homozygosity for mutations in the glucokinase gene (GCK). Here we present the results of screening eight cases of PNDM for mutations in glucokinase. Three of these had glucokinase-related PNDM, including a subject who inherited different inactivating mutations from each parent.
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RESEARCH DESIGN AND METHODS |
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Genetic studies.
The exons, flanking introns, and minimal promoter regions of the gene encoding glucokinase were screened for mutations by direct sequencing of the PCR products. In vitro mutagenesis was performed as described in Bjørkhaug et al. (5). The primers 5'-CGTGTCTACGCGCGTTGCGCACATGTGCTCG-3' (F1), 5'-CGAGCACATGTGCGCAACGCGCGTAGACACG-3' (R1), 5'-GCCTTCGGGGACTCCAGCGAGCTGGACGAGTTCC-3' (F2), and 5'-GGAACTCGTCCAGCTCGCTGGAGTCCCCGAAGGC-3' (R2) were used to introduce the appropriate nucleotide changes corresponding to the mutations A378V (F1 and R1) and G264S (F2 and R2).
Kinetic analysis of glucokinase.
Wild-type and mutant forms of human ß-cell glucokinase were generated and expressed as glutathionyl S-transferase (GST) fusion proteins in E. coli; the kinetic properties of the purified proteins were determined as previously described (6). We performed four serial experiments. The wild-type GST-glucokinase preparation was newly made for the current study. Kinetic data may vary as a function of the chemical nature and concentration of the sulfhydryl reagent used in the kinetic analysis. In our studies, 2 mmol/l dithiothreitol was used in the standard kinetic assay. Nonlinear kinetics using the Hill equation were applied. The relative activity index (AI) was calculated as previously described with some modification as AI = (kcat/S0.5nH)(2.5/2.5 + ATPKm)(5nH x 2/5nH + S0.5nH), where kcat is the turnover rate, S0.5 is the concentration of glucose needed to achieve the half-maximal rate of phosphorylation, nH is Hill coefficient for cooperativeness with glucose, and ATPKm is the ATP concentration required for glucokinase activity to be half maximum when glucose is in excess (6). This number indicates the in situ phosphorylation capacity of the enzyme at 5 mmol/l blood glucose. An intracellular ATP concentration of 2.5 mmol/l was assumed. The relative activity index was normalized to a basal blood glucose of 5 mmol/l to account for glucokinase expression.
Mathematical modeling.
A minimal mathematical model was used to assess the impact of the G264S, IVS8 + 2TG, and A378V mutations of glucokinase, in both the homozygous and heterozygous state, on the glucose-stimulated insulin secretion rate (GSIR) (6,16). The modeling was modified to account for adaptation of both alleles in homozygous and heterozygous cases by using the theoretically plausible expression (SnH x 2)/(SnH + S0.5nH), rather than an empirical factor of 0.2 per mmol/l glucose change. S is the glucose level at threshold and "2" indicates that half-maximal induction is achieved at glucose S0.5.
Structural analysis.
A predicted model of the three dimensional structure of wild-type human glucokinase (1 glk; Rasmol Windows version 2.7.2.1) was used to inspect the spatial relations for the previously published homozygous missense mutations causing PNDM (M210K and T228M) (4,9) together with the present missense mutations G264S and A378V.
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RESULTS |
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Family 3.
The male proband (IS2-1) was number three of five siblings (8). His mother was both first and third cousin of the father of the proband in Family 2, and she was also the third cousin once removed of the mother of the proband in Family 2 (Fig. 2). The probands parents were not known to be related. The proband presented with IUGR and was born by vaginal delivery at a gestational age of 38 weeks. His birth weight was 1,870 g (<3rd centile) and his birth length was 44 cm (3rd centile) (Table 1). At age 2 days, he had hyperglycemia (12.0 mmol/l [216 mg/dl]) without ketoacidosis. His psychomotor development has been normal. He was treated with insulin from day 3 of life. At present, he is age 18 years and is treated with insulin daily (0.9 units/kg). His HbA1c is presently 9.4% (reference value for HbA1c in the analytic laboratory was 46.3%). The father died at age 49 years from hepatic failure, but had no history of diabetes. His mother was diagnosed with gestational diabetes during her pregnancies. She is now age 56 years and has developed manifest diabetes (fasting serum glucose 10.1 mmol/l [182 mg/dl]; HbA1c 7.0%). Her diabetes is being treated with diet. The siblings had birth weights of 3,550 (55th centile), 4,300 (96th centile), 2,450 (6th centile), and 3,260 g (45th) centile), and their recent fasting serum glucose levels were 5.6, 5.5, 6.8, and 6.0 mmol/l (101, 99, 122, and 108 mg/dl), respectively.
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Family 1.
In this family, we screened for GCK by direct sequencing and found a novel missense mutation in exon 9 (nucleotide 1,139: GCT to GTT) of GCK, resulting in the substitution of alanine for valine at amino acid residue 378 of the glucokinase protein (designated c.1,139 CT, A378V). Residue 378 is strictly conserved among glucokinase enzymes from man to Drosophila. The proband was homozygous, whereas her parents were heterozygous for the mutation. The mutation was not found in 91 individuals of Norwegian ancestry.
Families 2 and 3.
Because of the known relationship between families 2 and 3 (Fig. 1), we first performed a linkage analysis using four microsatellite markers for the GCK gene (Fig. 2). Because the haplotype pattern suggested a homozygous and a heterozygous GCK mutation in the probands of families 2 and 3, respectively; we therefore sequenced this gene in all available samples. In family 2, we identified a splice site mutation in the second nucleotide of the donor splice site of exon 8, designated IVS8 + 2TG. The proband was homozygous and the parents were heterozygous for this mutation. Family 3 was related to family 2 through the mother of the proband in family 3. The mother had inherited family 2s specific mutation IVS8 + 2T
G. The proband shared this mutation in addition to another GCK mutation in exon 7 (nucleotide 790: -GGC to -AGC), resulting in the substitution of glycine for serine at amino acid residue 264 of the glucokinase protein (designated c.790 G
A, G264S). Residue 264 is strictly conserved from man to Drosophila. Neither IVS8 + 2T
G nor G264S were identified in 91 Norwegian subjects.
Other cases of neonatal diabetes.
We screened five other patients with PNDM (clinical details given in Table 1). Pathogenic mutations in GCK were not identified in any of these patients.
Kinetic analysis of recombinant glucokinase.
We prepared recombinant wild-type, A378V and G264S glucokinase in E. coli and compared the kinetic properties of the purified GST fusion proteins (Table 2) by previously described methods (6,9). The A378V proteins had a relative activity index (IGKB) that was only 0.2% of that of wild-type glucokinase. The kcat of A378V glucokinase was practically the same as that of the wild-type, and the glucose S0.5 was increased 76-fold. ATPKm was increased 27-fold. In contrast, the IGKB of recombinant G264S proteins was near normal (0.86 of wild-type). Hence, G264S glucokinase kcat was nearly equal to that of wild-type, and the glucose S0.5 was only moderately elevated (129% of wild-type). G264S glucokinase ATPKm was 130% of wild-type glucokinase. Thus, A378V severely impairs in vitro glucokinase activity and is the likely cause of PNDM and maturity-onset diabetes of the young (MODY) in family 1. By contrast, G264S has only a modest effect on the enzyme in vitro, if any. We did not test the effect of the IVS8 + 2TG on RNA expression or glucokinase activity, but if intron 8 is not removed there is an in-frame stop codon positioned at nucleotides 479481 of intron 8. This would give rise to a mutant glucokinase protein of 455 amino acids lacking residues 340465 of the normal protein, but with an addition of 160 residues encoded by intron sequences.
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DISCUSSION |
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The clinical picture of our new patients is quite similar to that of the first two cases. The patients had moderate or severe IUGR, birth weights of 1,5501,900 g, and severe hyperglycemia and required subsequent exogenous insulin shortly after birth. This profile fits with the key role played by glucokinase in the regulation of insulin secretion in humans with glucokinase-related diabetes (MODY2) and in mice lacking one or both Gck genes (10,11). That insulin is a potent fetal growth factor is illustrated by our three cases and Gck-/- mice, which are also born growth retarded (11). Our three patients also demonstrate that the fetal growth effect of insulin is most pronounced in the last trimester. Thus, the premature proband of family 1 had a birth weight at the 10th centile, whereas the probands of family 2 and 3 were born at term with birth weights <3rd centile. Although the missense mutation G264S had significant enzyme activity, we believe this mutation is pathogenic as the residue G264 is strictly conserved and there are other members of family 3 at risk with a MODY2 phenotype. Moreover, the compound heterozygous proband of family 3 had severe hyperglycemia at day 2 of life and a birth weight of only 1,870 g, characteristics that are compatible with two defective alleles.
In this regard, it is interesting to compare fetuses with two mutated GCK alleles with those having a GCK mutation on one allele only (12). If a normal fetus is subjected to a diabetic environment (mother heterozygous for a GCK mutation), the combination of intrauterine hyperglycemia and augmented fetal insulin secretion leads to a birth weight increase of 0.5 kg. When the mother and the fetus are both heterozygous, the reduced insulin secretion in the fetus might in theory balance the effect of the maternal hyperglycemia. Hence, the fetus will have a birth weight in the normal range. Should the fetus, but not the mother, be heterozygous for a GCK mutation, the birth weight is lower by
0.5 kg. If the mother is heterozygous and the infant is homozygous, as in our cases, a nonoperating glucokinase renders the infant insensitive to maternal hyperglycemia and severe IUGR may ensue (mean birth weight 1,728 g in the present three and previous two cases) (4).
Which cases of neonatal diabetes should be screened for glucokinase mutations? We would suggest screening primarily diabetic neonates with IUGR of moderate or severe degree who have glucose-intolerant parents. If these criteria are not fulfilled, the absence of glucokinase mutations would not be surprising (Table 1) (1315). It is noteworthy that four of the five cases of glucokinase deficiency had hyperglycemia within the first 2 days of life, illustrating the insulin secretion defect subsequent to the glucokinase deficiency. The proband of family 2 was referred to the hospital in a very severe situation at day 11, suggesting he had hyperglycemia shortly after birth as well.
Is it possible to predict the phenotype of patients with glucokinase mutations from the genotypes? We believe our patients with homozygous or compound heterozygous GCK mutations are important for understanding more of the mechanisms for glucokinase as the glucose sensor. The corresponding residues of the missense mutations M210K, T228M, and A378V are localized either in the cleft leading to or close to the active site of the enzyme (Fig. 4). Thus the effect on the enzyme activity can be assumed to be dramatic and so the phenotype of the patient. In contrast, even though the proband of family 3 had PNDM, his recombinant glucokinase G264S had near normal enzyme activity. Because of its near normalcy, thermal stability tests (16) were also performed (data not shown). Thus, glucokinase G264S was indistinguishable from wild-type and A53S glucokinase (both thermostable under the test conditions) in contrast to the established thermolabile mutant E300K, which was used in parallel as a positive control (16). In this connection, it is puzzling why 10% of the glucokinase mutations identified in diabetic patients are enzymatically normal (16). Why the patients have hyperglycemia at all given their near normal enzyme activities is not yet clear. Clearly, glucokinase may have other roles in the regulation of insulin secretion. There might be other unknown factors interacting with glucokinase that may be involved in the stimulation of insulin release from the granule (17). Alternatively, the mutations could indicate an impaired surface binding site to interacting proteins in this region.
Glucokinase deficiency may be regarded as a recessively inherited inborn error of metabolism, with heterozygous carriers having a mild phenotype (MODY2) (18) and homozygous carriers being associated with PNDM, as a particular severe phenotype.
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ACKNOWLEDGMENTS |
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We would like to thank the members of the families for their participation in this study, and Dr. Graeme I. Bell for comments on the manuscript.
Address correspondence and reprint requests to Pål R. Njølstad, MD, PhD, Department of Pediatrics, University of Bergen, N-5021 Bergen, Norway. E-mail: pal.njolstad{at}uib.no
Received for publication April 7, 2003 and accepted in revised form August 4, 2003
AI, relative activity index; ATPKm, ATP concentration required for glucokinase activity to be half maximum when glucose is in excess; BGPR, ß-cell glucose phosphorylation rate; GSIR, glucose-stimulated insulin release; GST, glutathione S-transferase; IA-2, insulinoma-associated protein 2; IGKB, relative activity index; IUGR, intrauterine growth retardation; IVS, intervening sequence; kcat, turnover rate; MODY, maturity-onset diabetes of the young; nH, Hill coefficient for cooperativeness with glucose; PNDM, permanent neonatal diabetes; S0.5, the concentration of glucose needed to achieve the half-maximal rate of phosphorylation
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REFERENCES |
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