Dominantly inherited hyperinsulinism caused by a mutation in the sulfonylurea receptor type 1

ATP-sensitive potassium channels play a major role in linking metabolic signals to the exocytosis of insulin in the pancreatic β cell. These channels consist of two types of protein subunit: the sulfonylurea receptor SUR1 and the inward rectifying potassium channel Kir6.2. Mutations in the genes encoding these proteins are the most common cause of congenital hyperinsulinism (CHI). Since 1973, we have followed up 38 pediatric CHI patients in Finland. We reported previously that a loss-of-function mutation in SUR1 (V187D) is responsible for CHI of the most severe cases. We have now identified a missense mutation, E1506K, within the second nucleotide binding fold of SUR1, found heterozygous in seven related patients with CHI and in their mothers. All patients have a mild form of CHI that usually can be managed by long-term diazoxide treatment. This clinical finding is in agreement with the results of heterologous coexpression studies of recombinant Kir6.2 and SUR1 carrying the E1506K mutation. Mutant K_ATP channels were insensitive to metabolic inhibition, but a partial response to diazoxide was retained. Five of the six mothers, two of whom suffered from hypoglycemia in infancy, have developed gestational or permanent diabetes. Linkage and haplotype analysis supported a dominant pattern of inheritance in a large pedigree. In conclusion, we describe the first dominantly inherited SUR1 mutation that causes CHI in early life and predisposes to later insulin deficiency.

Congenital hyperinsulinism (CHI) is a rare genetic disorder characterized by a dysregulation of insulin secretion that leads to severe hypoglycemia. The severity of the disease varies from a mild form, which responds to treatment with drugs (such as diazoxide) or hormones (like somatostatin), to a severe drug-resistant form, which may necessitate resection of the pancreas. Early diagnosis is important to avoid irreversible brain damage due to prolonged hypoglycemia (1–4).

Mutations in four different genes have been associated with CHI (5–10). Most mutations are associated with the β-cell ATP-sensitive potassium (K_ATP) channel, which plays a major role in the regulation of insulin secretion. This channel is open in the unstimulated β cell, and its activity keeps the resting membrane potential at a hyperpolarized (negative) level. An increase in the extracellular glucose concentration leads to increased β-cell metabolism and thus to a rise in the intracellular ATP concentration and a concomitant fall in intracellular ADP. These nucleotide changes act synergistically to close K_ATP channels, producing depolarization of the β-cell membrane, activation of voltage-gated Ca²⁺ channels, and finally exocytosis of insulin.

The K_ATP channel consists of two types of protein subunit: the sulfonylurea receptor SUR1 and the inward rectifying potassium channel Kir6.2 (11). Mutations in the SUR1 gene are the major known cause of CHI (5). However, a few mutations in Kir6.2 have also been found (6, 7). In most cases, the disease appears to be diffuse, involving all β cells and occurring throughout the pancreas. A focal form has also been described, which is characterized by the somatic loss of maternal alleles and is confined to focal adenomatous lesions of the pancreas (12). All the SUR1 and Kir6.2 mutations described so far have been inherited recessively. However, a gain-of-function mutation in the glucokinase gene (GK) has been shown to cause a dominant form of CHI (8). Furthermore, mutations in the glutamate dehydrogenase gene cause CHI with hyperammonemia (9).

The overall incidence of CHI in Finland is 1:40,000, which is about the same as that reported previously for Northern Europe (13, 14). The clinical phenotype of Finnish patients with CHI is variable. We have previously reported a founder mutation, V187D, in the SUR1 gene, which was detected in 15 Finnish patients with CHI. This causes a severe form of CHI that results from total inactivation of β-cell K_ATP channels (13). Efforts are continuing to identify the genetic defects of the remaining patients with CHI and to correlate the different genotypes and phenotypes. We report here a missense SUR1 mutation that is associated with a mild, diazoxide-responsive form of CHI and, for the first time to our knowledge, shows a dominant mode of inheritance.

Detection of the mutation E1506K. The PCR-SSCP analysis revealed abnormally migrating DNA fragments in seven of the 38 patient samples in exon 37 of SUR1 (Figure 1a). Sequencing of the variant (case 1) led to the detection of a heterozygous GAG→AAG substitution in codon 1506, resulting in an amino acid change from glutamic acid to lysine (E1506K) (Figure 1b). This mutation is located in the second nucleotide binding fold of the SUR1 protein.

Figure 1

Demonstration of the mutation E1506K. (a) SSCP-analysis of the SUR1 exon 37. Results are shown for two individuals carrying the E1506K mutation (Mt) and two controls (Wt). The abnormally migrating DNA fragment is marked with an arrow. The gel was run for 4 hours at 38°C. (b) Sequence analysis reveals a G→A mutation in the genomic sequence resulting in the substitution of glutamic acid for lysine. (c) Detection of the mutation by restriction analysis of PCR-amplified genomic DNA. Mutation E1506K causes the disappearance of a MnlI restriction site, leading to the formation of a new 89-bp digestion product for the mutated allele (arrow). MW, molecular weight marker; Mt, mutant; Wt, wild type.

The presence of this missense mutation results in the disappearance of a MnlI restriction endonuclease site and the formation of a new 89-product that provides a means to confirm and test for its presence (Figure 1c). In all seven cases, the individuals were heterozygous for the mutation and the mutation was maternally inherited. No other mutations in the SUR1, Kir6.2, glucokinase (GCK), or exon 11 and 12 of the glutamate dehydrogenase (GLUD1) genes were found in any of these individuals. The E1506K mutation was not found in 100 normal Finnish chromosomes. We also examined 160 chromosomes of type 2 diabetic patients, and the mutation was not detected in any of them.

Genetic linkage analysis. The large six-generation pedigree with six patients was analyzed for genetic linkage using both nonparametric and parametric approaches. We first tested for the significance of the observation that a haplotype including a rare single nucleotide polymorphism, E1506K, was inherited by all of the affected individuals but by none of those unaffected in the previous generation. This calculation makes no assumptions about the mode of inheritance. The shared haplotype was assumed to be introduced in the pedigree only once in any of the four chromosomes possessed by the founding couple. The likelihood of obtaining the observed haplotype-sharing pattern by chance was 1.2 × 10^–7, strongly supporting the hypothesis that the segregation of CHI in the pedigree was dependent on the segregation of the haplotype.

We then performed two-point linkage analysis with dominant and recessive models of inheritance. All but the last generation of individuals were considered as of unknown phenotype, whereas for the last generation, unaffected and affected individuals were as marked in Figure 2. The linkage of the mutant allele to the phenotype was calculated using a dominant model that assumes an allele frequency of 0.01, full penetrance, and no phenocopies. A maximum LOD score of 6.28 was obtained for θ = 0. A recessive model yielded a maximum LOD score of 4.10 at θ = 0.044, and thus fits the data less well. Using an allele frequency of 0.03, corresponding to the frequency of the conserved haplotype, the figures were LOD 6.00 at θ = 0 and LOD 3.83 at θ = 0.045 for the dominant and recessive models, respectively.

Figure 2

Pedigree and haplotype analysis of a large pedigree carrying the SUR1 mutation E1506K. The order of markers of the haplotype is as follows: D11S1890, D11S921, D11S1888. The haplotypes are presented below each individual. The haplotype 3-4-4 associates with E1506K in all cases.

Clinical findings. The clinical characteristics of the seven pediatric patients who carry the mutation E1506K are presented in Table 1. Five of the patients were large for their gestational age. Five patients presented with hypoglycemia during the first few hours after birth. In the other two cases, the symptoms of hypoglycemia appeared later, at the age of 5 (case 4) and 7 (case 2) months. Hyperinsulinemia during hypoglycemia was used as the major diagnostic criterion for CHI. In case 1, the level of hyperinsulinemia was not documented.

Table 1

Major clinical findings of CHI patients carrying the mutation E1506K

Five patients were treated with diazoxide, and four of them showed a good response (cases 4–7). In case 1, subtotal pancreatectomy was performed at the age of 3.5 years in preference to diazoxide treatment. In one case, the duration of the diazoxide treatment was 5 years (case 4), and the remaining three are still on medication. In the two mildest cases (2, 3), hypoglycemia was treated with extra glucose administration and frequent feeds without the need for medication.

Histological examination was possible only in case 1, who underwent a subtotal pancreatectomy. The resected pancreas was macroscopically unremarkable. After hematoxylin-eosin staining, the histological features were similar in the different parts of the gland: the islets, although slightly irregular in contour, appeared roughly normal, and their number was not increased. In most islets, abnormally large nuclei were observed, corresponding to β cells with an abundant cytoplasm (Figure 3a).

Figure 3

Pancreatic histology of case 1. (a) Hematoxylin-eosin–stained section showing abnormal β cell nuclei (arrows). Objective magnification ×40. (b) Immunoperoxidase staining of insulin reveals irregular islets and small clusters of β cells. Objective magnification ×10.

Immunoperoxidase after anti-insulin antibody stained not only insular but also extrainsular β cells that appeared either in small clusters or scattered within the exocrine tissue (Figure 3b). The labeling intensity of the insulin-containing cells was similar in the different regions of the gland. The distribution of non–β cells also appeared to be normal. The volume density of endocrine tissue measured by point-counting studies of immunostained sections was 1.95% in the CHI pancreas compared with 3.2% in control pancreas; The proportions of β, δ, and α cells were, respectively, 54%, 22%, and 19% versus 53%, 26%, and 19% in normoglycemic controls. The mean radius of 50 selected β-cell nuclei was high compared with the controls (4.61 vs. 3.19 μm), and the index of β-cell nuclear crowding was low (9.94 nuclei per 1,000 μm² of β-cell cytoplasm versus 13.78 nuclei in the controls).

It was possible to extract DNA from three paraffin-embedded tissue blocks originating from different parts of the pancreas. The DNA was partially degraded, probably because the pancreas was resected 23 years earlier. Nevertheless, the primers of the marker D11S1890 amplified a product showing two different alleles (3, 4), which were the same as in the genomic DNA, from all three parts of the pancreas, thus excluding the possibility of somatic loss of heterozygosity in these areas of the pancreas.

At least two of the mothers (cases 1 and 5; generation II), who are sisters, had had symptoms that could be classified as hypoglycemic, during the first 2 years of life. These symptoms always manifested early in the morning and included irritability, convulsions, deviation of the eyes, and even unconsciousness. Unfortunately, no documentation of blood glucose values is available. Their brother had been lethargic and floppy soon after birth, and he died at the age of 2 days. The grandmother of cases 1, 4, and 5 in generation III, and the mothers of cases 4, 6, and 7, had had mild symptoms of hypoglycemia, including trembling and sweating, during fasting. The brother of this grandmother in generation III died at the age of 2 days from unknown causes. The fathers of the seven patients were unrelated, and none of them carried the mutation. The haplotypes inherited paternally were all very different (Figure 2).

All except one of the mothers (the mother of cases 2 and 3, generation II) had impaired glucose tolerance or diabetes during pregnancy. One of them was treated with insulin, and four had pathological values in the OGTT. Two of them have developed diabetes. Furthermore, the grandmother of the affected children (cases 1, 4, and 5) is diabetic. All of these diabetic persons have a tendency to hypoglycemia when treated with sulfonylureas.

To evaluate the β-cell sensitivity to glucose and the tissue sensitivity to insulin, the glucose metabolism of two diabetic mothers was investigated in detail. The results of the studies are shown in Table 2. OGTT values showed diabetic levels according to the World Health Organization criteria (26) in both cases. Both the first-phase and the maximal glucose-stimulated insulin secretory capacity were severely reduced. The M-values during the euglycemic clamp were relatively high compared with those of type 2 diabetic patients evaluated previously in our laboratory.

Table 2

Studies of glucose metabolism in individuals carrying the E1506K mutation (mothers of cases 1 and 4)^A

Recombinant K_ATP channel experiments. We first compared the effect of metabolic inhibition on oocytes co-injected with Kir6.2 and either SUR1 or SUR1-E1506K. Intact oocytes were voltage-clamped using two electrodes, and 3 mM azide was used as a metabolic poison. As shown in Figure 4, oocytes exhibited very small current amplitudes in control solution. Metabolic poisoning induced a large increase in Kir6.2/SUR1-injected oocytes but had little effect on the currents recorded from oocytes injected with Kir6.2/SUR1-E1506K. Subsequent addition of diazoxide in the continued presence of azide, however, activated both Kir6.2/SUR1 and Kir6.2/SUR1-E1506K currents. These currents were blocked by 500 μM tolbutamide, indicating that they flowed through K_ATP channels.

Figure 4

Effects of metabolic inhibition on wild-type and mutant Kir6.2/SUR1 currents. Mean whole-cell current amplitudes recorded at –50 mV first in control solution (black bars), 15 minutes after exposure to 3 mM azide (hatched bars), then in the continued presence of azide plus 340 μM diazoxide (white bars), and, finally, after the further addition of 500 μM tolbutamide (shaded bars). Oocytes were co-injected with mRNA encoding Kir6.2 and either SUR1, SUR1-E1506K, or a 1:1 mixture of SUR1 plus SUR1-E1506K, as indicated. The number of oocytes is given above the error bars.

These results suggest that SUR1-E1506K is capable of forming functional channels with Kir6.2, and that these channels are insensitive to metabolic inhibition but can be opened by diazoxide. This result is consistent with the ability of diazoxide, but not low blood glucose, to inhibit insulin secretion in patients with CHI who were carrying the SUR1-E1506K mutation.

Figure 5a compares the macroscopic currents recorded before and after patch excision from oocytes coexpressing Kir6.2 and either SUR1 or SUR1-E1506K. In all cases, currents recorded in the cell-attached condition were extremely small (< 50 pA), consistent with the results obtained for intact oocytes in control solution. After excision into nucleotide-free intracellular solution, patches excised from oocytes expressing wild-type Kir6.2/SUR1 developed very large currents; this increase in current on patch excision reflects the relief of channel inhibition by ATP present in the oocyte cytoplasm. Smaller, but still substantial, Kir6.2/SUR1-E1506K currents were also observed in excised patches.

Figure 5

(a) Current amplitudes recorded in excised patches. Mean current amplitudes recorded at –100 mV in the cell-attached or inside-out patch configuration from oocytes co-injected with Kir6.2 and either SUR1 or SUR-E1506K as indicated. The number of oocytes is given above the bars. (b) ATP sensitivity of Kir6.2/SUR1-E1506K currents; mean ATP concentration-response relationships for Kir6.2/SUR1-E1506K currents (n = 4). The slope conductance (G) is expressed as a fraction of the mean (G_c) of that obtained in control solution before and after the exposure to ATP: The line is the best fit of the data to Hill equation (IC₅₀ = 9.3 μM and h = 0.96).

We next explored the functional properties of Kir6.2/SUR1-E1506K channels. There was no significant difference in the ATP-sensitivity of Kir6.2/SUR1-E1506K (Figure 5b) and wild-type K_ATP channels. The mean IC₅₀ for ATP inhibition of Kir6.2/SUR1-E1506K currents was 9.3 ± 1.1 μM (n = 4), similar to that measured previously for Kir6.2/SUR1 currents (21 μM; 12 μM) (27, 28). The Hill coefficient was close to unity (h = 0.96 ± 0.1; n = 4), as found before for Kir6.2/SUR1. The values of IC₅₀ and h we observed are also similar to those found for native β cell K_ATP channels (29, 30). Thus, these data indicate that the failure of Kir6.2/SUR1-E1506K channels to respond to metabolic inhibition is not a consequence of an altered ATP sensitivity.

We next examined the effect of MgADP on Kir6.2/SUR1-E1506K channel activity. As shown in Figure 6, a(ii) and b, 100 μM MgADP blocked the channel by approximately 70%. This contrasts with wild-type K_ATP channels, where MgADP stimulates channel activity by approximately 170% (Figure 6b). The lack of MgADP activation may contribute to the failure of metabolic inhibition to stimulate Kir6.2/SUR1-E1506K currents in intact oocytes.

Figure 6

Effects of MgADP, tolbutamide, and diazoxide on Kir6.2/SUR1-E1506K currents. (a) Macroscopic Kir6.2/SUR1-E1506K currents recorded from inside-out patches in response to series of voltage ramps from –110 mV to +100 mV. Tolbutamide (100 μM), ADP (100 μM), diazoxide (340 μM), and ATP (100 μM) were added to the internal solution as indicated by the bars. (b) Mean macroscopic slope conductance recorded in the presence of ATP, ATP plus diazoxide, ADP, or tolbutamide, expressed as percentage of the slope conductance in control solution. The number of oocytes is given above the bars. Inset: Mean macroscopic slope conductance recorded in the presence of 100 μM ATP plus 340 μM diazoxide, expressed as a percentage of the current in the presence of 100 μM ATP for Kir6.2/SUR and Kir6.2/SUR1-E1506K currents. The dashed line indicates the conductance in the presence of 100 μM ATP. The number of oocytes is given above the bars.

The K-channel opener diazoxide is able to reverse, almost completely, the inhibitory effect of 100 μM ATP on wild-type Kir6.2/SUR1 channels (Figure 6b). Diazoxide was far less effective on Kir6.2/SUR1-E1506K channels, although it did increase channel activity to a small extent (Figure 6, a[iii] and b). This can be seen more clearly when the conductance recorded in the presence of ATP and diazoxide is expressed as a percentage of that recorded in ATP alone (Figure 6b, inset). Although the response to diazoxide was small, it is worth emphasizing that because of the very high input resistance of the β cell when the K_ATP channels are closed, even a very small current is capable of producing a large hyperpolarization and thereby inhibiting insulin secretion. The sulfonylurea tolbutamide blocked both wild-type and Kir6.2/SUR1-E1506K currents by a similar amount (Figure 6, a[i] and b).

Unlike most CHI mutations, heterozygosity for E1506K is sufficient to cause the disease. To determine whether this arises from a gene dosage or a dominant negative effect, we examined the effect of coexpressing Kir6.2 with a 1:1 mixture of wild-type and mutant SUR1. Control oocytes were co-injected with Kir6.2, and the same total concentration of either wild-type or mutant SUR1. Figure 4 shows that the amplitude of K_ATP current activated by metabolic inhibition in oocytes expressing Kir6.2 and a 1:1 mixture of wild-type and mutant SUR1 was approximately half that found for oocytes expressing Kir6.2/SUR1 channels. Because the concentration of SUR1 mRNA in the mRNA mixture was also approximately 50% of that in oocytes injected with SUR1 alone, this result indicates that the E1506K mutation does not have a dominant negative effect.

In most cases, CHI is caused by a recessive mutation in the SUR1 gene. The other genes that can cause a similar phenotype include Kir6.2, glucokinase, and glutamate dehydrogenase. Our linkage analyses show unambiguously that CHI in the patients described here depends on the inheritance of the E1506K allele of the SUR1 gene. Nonparametric analysis showed that the observed sharing among patients and nonsharing among healthy sibs in the large pedigree is highly unlikely to be caused by chance (P = 1.2 × 10^–7). Parametric linkage analysis, surprisingly, strongly supported a dominant mode rather than rather than a recessive mode of inheritance.

A dominant rather than a recessive mechanism is also suggested by the inspection of the haplotypes surrounding the SUR1 gene in the patients. For all patients, the other haplotype (not harboring the E1506K variant) was different; specifically, none of the patients was homozygous for the E1506K allele and the surrounding haplotype. If the phenotype was caused by a recessive mechanism, one would expect to see at least some conservation of the haplotypes among the patients, because the patients all come from a small subpopulation and indeed from different branches of a large pedigree. If the inheritance were recessive, the fact that six different haplotypes were observed among the non-E1506K chromosomes would imply that at least six different mutations segregated within this subpopulation, which is unlikely considering the overall rarity of the disease. Furthermore, if one considers the assumption of multiple mutations reasonable, then each of the six mutations must involve noncoding parts of the gene, because no additional coding variants were detected among the patients’ SUR1 alleles. Both coincidences must be so rare that a dominant inheritance of the E1506K allele remains the most likely explanation. There is ample evidence for different mutations within a single gene producing both dominant and recessive disease; the classical example is β-globin and different hemoglobinopathies, some of them dominant, some recessive, depending on the point mutation (31).

Interestingly, all but one of the mothers of E1506K patients had impaired glucose tolerance during pregnancy, including those two who had suffered from hypoglycemia in infancy. Two of them have developed diabetes. Furthermore, the grandmother of cases 1, 4, and 5 has diabetes. The severely blunted first-phase glucose-stimulated insulin secretion reduced maximal glucose-stimulated insulin secretory capacity, and largely normal insulin sensitivity indicated that the mothers of cases 1 and 4 predominantly had the insulin secretion-deficient phenotype of type 2 diabetes.

These observations suggest that after hypoglycemia early in life, the mutation predisposes to the development of insulin deficiency and diabetes. The development of diabetes later in life has also been reported in the dominant form of CHI caused by a gain-of-function glucokinase mutation (8). This is also in agreement with a transgenic animal model of CHI expressing a dominant-negative form of the K_ATP channel subunit Kir6.2 (32). In these mice, hyperinsulinism is evident in the neonatal period but insulin-deficient diabetes develops later, apparently owing to a high frequency of β-cell apoptosis that results in decreased β-cell mass. An increased rate of β-cell apoptosis has also been found in the focal form of CHI (33). Increased β-cell apoptosis is thus a possible explanation for the development of diabetes in our patients.

CHI is associated with β-cell depolarization, increased Ca²⁺ entry, and elevated intracellular Ca²⁺. It is possible that the enhanced energy demand associated with Ca²⁺ extrusion from the β cell, coupled with an elevated [Ca²⁺]_I, is responsible for precipitating β-cell apoptosis. If this is the case, diazoxide therapy, by hyperpolarizing the β cell and limiting Ca²⁺ entry, might delay the onset of apoptosis and progression to diabetes.

All patients carrying the E1506K mutation who were treated with diazoxide responded well. The two mildest cases (2 and 3) needed only a few weeks of intravenous glucose administration and frequent feedings subsequently. One patient (case 1) was treated surgically at the age of 3.5 years. It is possible that this patient, who has since developed diabetes, could have been managed effectively by diazoxide treatment without pancreatectomy.

It is noteworthy that in all six core families, the patients inherited the E1506K allele from their mother (P = 0.0156). This parental skewing could be due to chance, but is also compatible with imprinting, i.e., a functional difference between the maternal and paternal alleles. In this context, it might be of interest to measure the level of SUR1 transcription from maternal and paternal alleles in islet cells.

Previous studies of the focal form of CHI have revealed a loss of maternal alleles resulting in a reduction to homozygosity of paternal SUR1 mutations within the focal lesion (12). To investigate the possibility that a similar mechanism existed in our patients, the resected pancreas of patient 1 was studied (no other patients underwent resection). Although we sought for the loss of heterozygosity in DNA from the resected pancreatic tissue, no loss of alleles was detected. Furthermore, the absence of atrophic resting islets and the observation of large β cells with abnormally large nuclei in different parts of the gland constitute strong arguments in favor of a diffuse form of the disease (34). The volume density of the endocrine cells and their relative proportions are similar in this case to those usually observed in this age group, both in normoglycemic and hypoglycemic infants and children with the diffuse form of CHI (20). The mean β-cell nuclear radius and β-cell nuclear crowding, respectively, 4.61 μm and 9.94 nuclei per 1,000 μm², are characteristic of the diffuse form of the disease and thus allow the exclusion of a focal lesion (21).

The results of the electrophysiological analysis of K_ATP channels containing the CHI mutation SUR1-E1506K are able to account fully for the observed clinical phenotype. The SUR1-E1506K mutation leads to a reduction, but not a complete loss, of K_ATP channels. Consistent with the clinical phenotype, the mutant channels are insensitive to metabolic inhibition but are activated by diazoxide. Our results demonstrate that MgADP is unable to stimulate mutant channel activity. This effect has been reported for other CHI mutations and may partially underlie the inability of metabolic inhibition to stimulate K_ATP channel activity (35). The E1506K mutation lies within the second nucleotide-binding domain (NBD) of SUR1 just distal to the Walker B motif. In other ABC transporters, this motif is involved in binding the Mg²⁺ ion of Mg-nucleotides (36). Mutation of the adjacent aspartate residue (D1505) also abolishes the stimulatory effects of Mg-nucleotides (MgADP, MgATP) and prevents K_ATP channel activation by metabolic inhibition in intact cells (25, 35). It therefore seems plausible that the E1506K CHI mutation abolishes metabolic activation by decreasing Mg²⁺ binding to the Walker motif of SUR1, and thereby impairing Mg-nucleotide interactions.

It is well established that diazoxide activation of β-cell K_ATP channels requires the presence of Mg-nucleotide (MgATP or MgADP) at the intracellular membrane surface (37, 38). Thus, it is not surprising that mutations of NBD1 or NBD2 that abolish the potentiatory effect of MgADP may impair diazoxide activation (25, 39). It is therefore interesting that diazoxide was able to activate Kir6.2/SUR1-E1506K channels, despite the loss of MgADP stimulation. A similar finding has been observed for another CHI mutation (14769R in NBF2) (25) and for a recombinant mutation in the Walker A motif of NBD2 of SUR1 (K11384M) (25). This phenomenon underlies the ability of diazoxide to reduce insulin secretion in patients carrying this mutation.

Our results suggest that the SUR1-E1506K does not exert a dominant negative effect on wild-type SUR1. Instead, the amount of current produced when SUR1 and SUR1-E1506K are expressed in the same oocyte is consistent with the concentration of SUR1 mRNA injected. Our results do not enable us to determine whether or not heteromeric channels containing both wild-type and mutant SUR subunits exist; however, if heteromeric channels are viable, our data suggest the extent to which they are activated by metabolic inhibition is proportional to the number of SUR1 subunits they contain.

All CHI mutations reported to date are inherited in a recessive fashion, which suggests that a 50% reduction in the β-cell K_ATP current is not sufficient to produce a membrane depolarization large enough to cause unregulated insulin secretion. It is therefore somewhat surprising that the E1506K mutation, which produces a 50% reduction in current amplitude when coexpressed with wild-type SUR1 in a 1:1 ratio, shows dominant inheritance. Although it is possible that the level of expression of mutant SURs may differ when heterologously expressed in Xenopus oocytes and in mammalian cells, it does not seem likely that the E1506K mutation would have a dominant negative effect in β cells when it does not in Xenopus oocytes. The reason for the dominant inheritance of the E1506K mutation remains unclear. It is possible, however, that it arises from genetic imprinting, which results in poor expression of the paternally inherited wild-type SUR1 allele.

The consequences of the mutation E1506K and the previously reported Finnish founder mutation V187D differ both in vivo and in vitro. The mutation V187D causes a drug-resistant form of CHI that necessitates pancreatectomy, whereas the E1506K-associated form of CHI responds well to medical treatment with diazoxide. Together, these two mutations explain the genetic basis of about 58% (22/38) Finnish CHI cases. Differentiation of these two forms of the disease is of great importance in clinical decision making. In the case of the mutation E1506K, diazoxide therapy should be the treatment of choice, whereas the patients carrying the V187D mutation will need surgical treatment.

In conclusion, we have found a pedigree in which a dominantly inherited SUR1 mutation causes a diazoxide-responsive form of CHI. This mutation predisposes to the later development of insulin deficiency and diabetes, demonstrating that mild genetic defects in the β-cell K_ATP channels can be a cause of adult-onset diabetes.

We thank P. Kärkkäinen, R. Laitinen, E. Ruotsalainen, and L. Uschanoff for skilled technical assistance; V. Ollikainen for expert help with linkage analysis; and B. Glaser for help with SUR1 promoter sequence analysis. These studies were supported by the Foundation for Paediatric Research (H. Huopio and T. Otonkoski), Yamanouchi Research International, the Wellcome Trust, and the British Diabetic Association (F. Ashcroft). J. Kere is an Investigator of the Center of Excellence for Disease Genetics of the Academy of Finland. T. Otonkoski was the recipient of a Juvenile Diabetes Foundation International Career Development Award. J. Rahier was supported by grant J.4594.99 from the FRSM, Brussels, Belgium.

1. Aynsley-Green, A. Nesidioblastosis of the pancreas in infancy. Dev Med Child Neurol 1981. 23:372-379.
   View this article via: PubMed Google Scholar
2. Permutt, MA, Nestorowicz, A, Glaser, B. Familial hyperinsulinism: an inherited disorder of spontaneous hypoglycemia in neonates and infants. Diabetes Rev 1996. 4:347-355.
3. Meissner, T, Brune, W, Mayatepek, E. Persistent hyperinsulinaemic hypoglycaemia of infancy: therapy, clinical outcome and mutational analysis. Eur J Pediatr 1997. 156:754-757.
   View this article via: PubMed CrossRef Google Scholar
4. Aynsley-Green, A, et al. Practical management of hyperinsulinism in infancy. Arch Dis Child 2000. 82:F98-F107.
   View this article via: CrossRef Google Scholar
5. Thomas, PM, et al. Mutations in the sulfonylurea receptor gene in familial persistent hyperinsulinemic hypoglycemia of infancy. Science 1995. 268:426-429.
   View this article via: PubMed CrossRef Google Scholar
6. Thomas, P, Ye, Y, Lightner, E. Mutation of the pancreatic islet inward rectifier Kir6.2 also leads to familial persistent hyperinsulinemic hypoglycemia of infancy. Hum Mol Genet 1996. 5:1809-1812.
   View this article via: PubMed CrossRef Google Scholar
7. Nestorowicz, A, et al. A nonsense mutation in the inward rectifier potassium channel gene, Kir6.2, is associated with familial hyperinsulinism. Diabetes 1997. 46:1743-1748.
   View this article via: PubMed CrossRef Google Scholar
8. Glaser, B, et al. Familial hyperinsulinism caused by an activating glucokinase mutation. N Engl J Med 1998. 338:226-230.
   View this article via: PubMed CrossRef Google Scholar
9. Stanley, CA, et al. Hyperinsulinism and hyperammonemia in infants with regulatory mutations of the glutamate dehydrogenase gene. N Engl J Med 1998. 338:1352-1357.
   View this article via: PubMed CrossRef Google Scholar
10. Glaser, B, Thornton, P, Otonkoski, T, Junien, C. Genetics of neonatal hyperinsulinism. Arch Dis Child 2000. 82:F79-F86.
    View this article via: CrossRef Google Scholar
11. Aguilar-Bryan, L, Bryan, J. Molecular biology of adenosine triphosphate-sensitive potassium channels. Endocr Rev 1999. 20:101-135.
    View this article via: PubMed CrossRef Google Scholar
12. De Lonlay, P, et al. Somatic deletion of the imprinted 11p15 region in sporadic persistent hyperinsulinemic hypoglycemia of infancy is specific of focal adenomatous hyperplasia and endorses partial pancreatectomy. J Clin Invest 1997. 100:802-807.
    View this article via: JCI PubMed CrossRef Google Scholar
13. Otonkoski, T, et al. A point mutation inactivating the sulfonylurea receptor causes the severe form of persistent hyperinsulinemic hypoglycemia of infancy in Finland. Diabetes 1999. 48:408-415.
    View this article via: PubMed CrossRef Google Scholar
14. Bruining, GJ. Recent advances in hyperinsulinism and pathogenesis of diabetes mellitus. Curr Opin Pediatr 1990. 2:758-765.
    View this article via: CrossRef Google Scholar
15. Inoue, H, et al. Sequence variants in the pancreatic islet beta-cell inwardly rectifying K+ channel Kir6.2 (Bir) gene: identification and lack of role in Caucasian patients with NIDDM. Diabetes 1997. 46:502-507.
    View this article via: PubMed CrossRef Google Scholar
16. Nestorowicz, A, et al. Mutations in the sulonylurea receptor gene are associated with familial hyperinsulinism in Ashkenazi Jews. Hum Mol Genet 1996. 5:1813-1822.
    View this article via: PubMed CrossRef Google Scholar
17. Lehto, M, et al. High frequency of mutations in MODY and mitochondrial genes in Scandinavian patients with familial early-onset diabetes. Diabetologia 1999. 42:1131-1137.
    View this article via: PubMed CrossRef Google Scholar
18. Orita, M, Iwahana, H, Kanazawa, H, Hayashi, K, Sekiya, T. Detection of polymorphisms of human DNA by gel electrophoresis as single-strand conformation polymorphisms. Proc Natl Acad Sci USA 1989. 86:2766-2770.
    View this article via: PubMed CrossRef Google Scholar
19. Lathrop, GM, Lalouel, JM, Julier, C, Ott, J. Strategies for multilocus linkage analysis in humans. Proc Natl Acad Sci USA 1984. 81:3443-3446.
    View this article via: PubMed CrossRef Google Scholar
20. Rahier, J, et al. The basic structural lesion of persistent neonatal hypoglycaemia with hyperinsulinism: deficiency of pancreatic D cells or hyperactivity of B cells? Diabetologia 1984. 26:282-289.
    View this article via: PubMed Google Scholar
21. Sempoux, C, et al. Neonatal hyperinsulinemic hypoglycemia: heterogeneity of the syndrome and keys for differential diagnosis. J Clin Endocrinol Metab 1998. 83:1455-1461.
    View this article via: PubMed CrossRef Google Scholar
22. DeFronzo, RA, Tobin, JD, Andres, R. Glucose clamp technique: a method for quantifying insulin secretion and resistance. Am J Physiol 1979. 237:E214-E223.
    View this article via: PubMed Google Scholar
23. Inagaki, N, et al. Reconstitution of IKATP: an inward rectifier subunit plus the sulfonylurea receptor. Science 1995. 270:1166-1170.
    View this article via: PubMed CrossRef Google Scholar
24. Sakura, H, Ammala, C, Smith, PA, Gribble, FM, Ashcroft, FM. Cloning and functional expression of the cDNA encoding a novel ATP-sensitive potassium channel subunit expressed in pancreatic beta-cells, brain, heart and skeletal muscle. FEBS Lett 1995. 377:338-344.
    View this article via: PubMed CrossRef Google Scholar
25. Gribble, FM, Tucker, SJ, Ashcroft, FM. The essential role of the Walker A motifs of SUR1 in K-ATP channel activation by MgADP and diazoxide. EMBO J 1997. 16:1145-1152.
    View this article via: PubMed CrossRef Google Scholar
26. 1985. Diabetes mellitus. World Health Organization. Technical Report Series, No. 727. Geneva, Switzerland. 10–11.
    View this article via: PubMed Google Scholar
27. Gribble, FM, Tucker, SJ, Seino, S, Ashcroft, FM. Tissue specificity of sulphonylureas: studies on cloned cardiac and beta-cell KKATP channels. Diabetes 1998. 47:1412-1418.
    View this article via: PubMed CrossRef Google Scholar
28. Koster, JC, Sha, Q, Shyng, SL, Nichols, CG. ATP inhibition of KATP channels: control of nucleotide sensitivity by N-terminal domain of the Kir6.2 subunit. J Physiol 1999. 515:19-30.
    View this article via: PubMed CrossRef Google Scholar
29. Ashcroft, FM, Rorsman, P. Electrophysiology of the pancreatic beta-cell. Prog Biophys Mol Biol 1989. 54:87-143.
    View this article via: PubMed CrossRef Google Scholar
30. Ashcroft, FM, Ashcroft, SJ. Properties and functions of ATP-sensitive K-channels. Cell Signal 1990. 2:197-214.
    View this article via: PubMed CrossRef Google Scholar
31. Hemoglobin--beta locus; HBB. http://www.ncbi.nlm.nih.gov/Omim/. Entry no. 141900.
    View this article via: PubMed Google Scholar
32. Miki, T, et al. Abnormalities of pancreatic islets by targeted expression of a dominant-negative KATP channel. Proc Natl Acad Sci USA 1997. 94:11969-11973.
    View this article via: PubMed CrossRef Google Scholar
33. Glaser, B, et al. Hyperinsulinism caused by paternal-specific inheritance of a recessive mutation in the sulfonylurea-receptor gene. Diabetes 1999. 48:1652-1657.
    View this article via: PubMed CrossRef Google Scholar
34. Rahier, J, et al. Partial or near-total pancreatectomy for persistent neonatal hyperinsulinaemic hypoglycaemia: the pathologist’s role. Histopathology 1998. 32:15-19.
    View this article via: PubMed CrossRef Google Scholar
35. Nichols, CG, et al. Adenosine diphosphate as an intracellular regulator of insulin secretion. Science 1996. 272:1785-1787.
    View this article via: PubMed CrossRef Google Scholar
36. Higgins, C. ABC transporters: from microorganism to man. Annu Rev Cell Biol 1992. 8:67-113.
    View this article via: PubMed CrossRef Google Scholar
37. Kozlowski, RJ, Hales, CN, Ashford, MLJ. Dual effects of diazoxide on ATP-K+ currents recorded from insulin secreting cell line. Br J Pharmacol 1989. 97:1039-1050.
    View this article via: PubMed Google Scholar
38. Edwards, G, Weston, AH. The pharmacology of ATP-sensitive potassium channels. Annu Rev Pharmacol Toxicol 1993. 33:597-637.
    View this article via: PubMed CrossRef Google Scholar
39. Shyng, SL, Ferrigni, T, Nichols, CG. Regulation of KATP channel activity by diazoxide and MgADP. J Gen Physiol 1999. 110:643-654.
    View this article via: PubMed CrossRef Google Scholar
40. Vauhkonen, I, et al. Impaired insulin secretion in non-diabetic offspring of probands with latent autoimmune diabetes mellitus in adults. Diabetologia 2000. 43:69-78.
    View this article via: PubMed CrossRef Google Scholar