Congenital Hyperinsulinism and Glucose Hypersensitivity in Homozygous and Heterozygous Carriers of Kir6.2 (KCNJ11) Mutation V290M Mutation: K_ATP Channel Inactivation Mechanism and Clinical Management

Genomic DNA was isolated from whole blood using the DNeasy Tissue Isolation kit (Qiagen, Valencia, CA). KCNJ11 was amplified by PCR and directly sequenced. The identified V290M mutation was engineered into mouse Kir6.2 cDNA in pCMV6B using the Quikchange site-directed mutagenesis kit (Stratagene, La Jolla, CA) and was confirmed by direct sequencing.

Oral glucose tolerance tests (OGTTs): d-glucose was given orally and blood drawn via peripheral intravenous at baseline and then at hourly intervals. Samples were assayed for serum glucose, insulin, and proinsulin at the Mayo Clinical Laboratory (Rochester, MN). For the mother, the glucose load (Glucola) was 75 g. For the sibling child, the load was 1 g/kg, and the duration of the OGTT was truncated to 3 h due to age. Of note, behavioral changes (e.g., hunger, lethargy) that often followed a meal were reported for the sibling. Given that heterozygous K_ATP channel mutations have been identified in patients with HI (6), the mother requested testing for her and her son. This case report was submitted to the institutional review board at the University of North Carolina and was declared exempt.

Continuous glucose monitoring system (CGMS): Due to parental wishes to decrease the frequency of octreotide injections, medical therapy was adjusted while monitoring under CGMS as an off-label use. A sensor was placed on three separate occasions for Proband 1 (CareLink; Medtronic MiniMed) after application of topical anesthetic. Medtronic (Caremark) provided training to the parents on how to use devices and mark events such as medication and meals, as well as to corroborate hypoglycemic (sensor set at <80) or hyperglycemic (>200) events detected by an external blood glucose meter. Reference ranges were chosen for the alarm settings to avoid hypoglycemia and minimize glycosuria, as well as to avoid excessive finger sticks for the child. Sensors were placed for a maximum of 5 days and corresponded to periods when treated with 1) octreotide alone or 2) octreotide + amlodipine. Of note, the child was tapered off amlodipine while on CGMS for a period of 2 weeks. After monitoring with octreotide alone, amlodipine (0.1 mg/kg divided twice daily) was reintroduced for 5 days prior to CGMS testing. Baseline glucose levels and excursions (high and low) were documented on a continuous basis to evaluate the safety and efficacy of the two treatment regimens.

Fluoro-DOPA (¹⁸F-l-DOPA) positron emission tomography/computed tomography study (4 MBq/Kg of ¹⁸F-l-DOPA administered intravenously 45 min before acquisition) was performed using a hybrid machine (Gemini GXL, Philips Medical Systems). The positron emission tomography scan was performed under general anesthesia and glucose infusion to maintain normoglycemia after 6-h fasting and without stopping medications.

COSm6 cells were cultured in Dulbecco's modified Eagle medium plus 10 mmol/l glucose and supplemented with FCS (10%). Cells were transfected with cDNA using FuGENE 6 Transfection Reagent (Roche Diagnostics, Indianapolis, IN) and then plated on sterile glass coverslips overnight prior to patch-clamp experiments.

Patch-clamp experiments were performed at room temperature on COSm6 cells that fluoresced green under ultraviolet illumination at 3–5 days post-transfection. Membrane patches were voltage-clamped using an Axopatch 1-D amplifier (Axon Instruments, Union City, CA). All currents were measured at a membrane potential of −50 mV. Data were collected using the pClamp8.2 software suite (Axon Instruments, Union City, CA) and Microsoft Excel. Bath and pipette control solutions (K_INT) contained (mM): 150 KCl, 10 HEPES, and 1 EGTA (pH 7.4). Where indicated, ATP was added to the bathing solution as dipotassium salts. Tolbutamide was dissolved in K_INT from a 100 mmol/l stock solution in 100 mmol/l KOH.

COSm6 cells in 12-well plates were incubated for 24 h in culture medium containing ⁸⁶RbCl (1 μCi/ml) 2 days after transfection. Cells were washed twice with Ringer's solution (Basal) (in mM: 118 NaCl, 2.5 CaCl₂, 1.2 KH₂PO₄, 4.7 KCl, 25 NaHCO₃, 1.2 MgSO₄, 10 HEPES; pH 7.4) with or without metabolic inhibition (1 mmol/l 2-deoxy-d-glucose and 2.5 μg/ml oligomycin). At selected time points, solution was removed and replaced with fresh solution; after completion of the assay, cells were lysed with 1% SDS and removed. Collected samples were assayed in a scintillation solution. Raw data are shown as percent ⁸⁶Rb⁺ efflux relative to total content.

The rate constant of K_ATP-specific ⁸⁶Rb⁺ efflux (k₂) was obtained by fitting a single exponential equation:

where apparent rate constant for nonspecific efflux (k₁) was obtained from untransfected cells. Glibenclamide was added to Ringer's solution plus metabolic inhibition from a 1 mmol/l stock solution in DMSO. Results are presented as mean ± SEM. Statistical tests and P values are noted in the figure legends where appropriate.

Mean P_o,zero was estimated from stationary fluctuation analysis of macroscopic currents in isolated membrane patches (10,11). Short (<1 s) recordings of currents were analyzed in zero [ATP] and in 5 mmol/l [ATP] (for estimation of ATP-independent noise). Currents were filtered at 1 kHz and digitized at 3 kHz with 12-bit resolution. Mean patch current (I), and variance (σ) in the absence of ATP were obtained by subtraction of mean current and variance in 5 mmol/l ATP (i.e., assuming all channels fully closed). Single-channel current (i) was assumed to be −3.75 pA at −50 mV, corresponding to wild-type (WT) single-channel conductance of 75 pS (12). P_o,zero was then estimated from the following equation:

The ATP dose–response was quantified by fitting the raw data with a Hill equation:

where I_rel is the current relative to that in the absence of ATP, [ATP] is the ATP concentration, k_1/2 is the half-maximal inhibitory ATP concentration, and H is the Hill coefficient that was fixed at 1.3.

At 48-h post-transfection, cells were washed twice with cold PBS (137 mmol/l NaCl, 2.7 mmol/l KCl, 10 mmol/l Na₂HPO₄, 2 mmol/l KH₂PO₄) and then incubated at 4°C in 300 μl of lysis buffer (150 mmol/l NaCl, 20 mmol/l HEPES, 10 mmol/l EDTA, 1% NP-40, one “Complete Mini” protease inhibitor [Roche Diagnostics, Indianapolis, IN] per 10 ml at pH 7). Lysates were centrifuged for 5 min at 13,000 rpm in 4°C and then transferred to clean microcentrifuge tubes, resolved with SDS-PAGE (7.5% acrylamide), and transferred to polyvinylidene fluoride membranes presoaked in methanol. Filters were blocked overnight in TBS-T (Tris-buffered saline with Tween) buffer (200 mmol/l NaCl, 20 mmol/l Tris-HCl, 0.1% Tween, pH 7.4) plus 5% nonfat dry milk at 4°C. Filters were incubated and rocked for 1 h in a 1:1,000 dilution of anti-SUR1 antibody (affinity-purified from rabbit) in TBS-T plus 5% milk, washed 3 times for 5 min each in TBS-T, then bathed in 1:1,000 dilution of secondary antibody (goat, anti-rabbit IgG, horseradish peroxidase–linked [Pierce]) in TBS-T plus 5% milk. Filters were washed an additional 3 times in TBS-T for 5 min each before appliance of an enhanced chemiluminescence system for detection of horseradish peroxidase (SuperSignal West Pico Chemiluminescent Substrate) and subsequent exposure to autoradiography film (Midwest Scientific, St. Louis, MO).

Figure 1 shows available pedigrees for two families in which the neonatal probands were clinically diagnosed with HI. Proband 1 (female, gestation 38 weeks, birth weight [BW] 3.5 kg, 75–90th centile [13]) was referred by an outside hospital at 2 weeks for evaluation of persistent hypoglycemia (blood glucose <30 mg/dl), despite initiation of dextrose-containing intravenous fluids and diazoxide (15 mg/kg/day). Genotyping (Athena Diagnostics) identified homozygous V290M mutation in KCNJ11 in the proband and heterozygous V290M in each of the unaffected mother (gestation 40 weeks, BW 3.9 kg, 75–90th centile), father (gestation 40 weeks, BW 4.5 kg, 95–97th centile), and male sibling (gestation 35 weeks, BW 3.0 kg, 75–90th centile), but not in an unaffected male sibling (gestation 36 weeks, BW 2.7 kg, 25–50th centile). Additional sequencing revealed no coding mutations in GCK, GLUD1, or ABCC8 genes in the proband. The ancestors of the mother and father came from the same small town in Germany, suggesting the same founder mutations. There is a family history of type 2 diabetes in two maternal great-aunts and a paternal great-grandmother, but no family history of frank hypoglycemia.

Proband 2 was a female born at 38-weeks' gestation (BW 3.4 kg, 75–90th centile) to an Italian family and had the first episode of hypoglycemia at day of life (DOL) 2 (blood glucose 29 mg/dl, treated with glucose infusion). She was referred at 8 weeks to Bambino Gesù Pediatric Hospital for diffuse cyanosis and tremor; plasma glucose was 40 mg/dl with simultaneously elevated insulin of 215 pmol/l and low free fatty acids (220 mmol/l). Diazoxide therapy (15 mg/kg/day) was started and then tapered to 4 mg/kg/day. Genotyping identified heterozygous V290M mutation in KCNJ11 in both the proband and the apparently unaffected father (not available for further testing). Additional sequencing of proband DNA revealed no coding mutations in ABCC8, GCK, or HNF4α genes.

Shortly after birth, proband 1 developed cyanosis, blood glucose <20 mg/dl, and was transferred to neonatal intensive care at an outside hospital. The presence of a heart murmur on DOL 2 prompted an echocardiogram (ECHO) that detected a small patent ductus arteriosus, which resolved spontaneously. Despite frequent breast-feeding, supplemental, and intravenous feeds, hypoglycemic episodes (<30 mg/dl) continued. Diazoxide treatment was initiated at 15 mg/kg/day on DOL 10. Laboratory assessment prior to initiation of diazoxide revealed insulin levels of 56 and 70 pmol/l on two occasions when blood glucose was <40 mg/dl. Pituitary testing at the time of hypoglycemia showed intact counterregulatory responses (not shown). In family 2, both the proband and the father carry the V290M mutation in KCNJ11, yet only the proband suffers from HI. Lack of mutations in other candidate genes suggested paternal uniparental disomy with loss of heterozygosity of the maternal allele, characteristic of focal HI (14). Fluoro-DOPA scanning (Fig. 1 B) provided clear evidence of a focal lesion in the body of the pancreas. Extemporary histological examination performed during surgery revealed focal adenomatous hyperplasia of islet cells, prompting complete excision of the lesion and effectively curing the patient and providing further corroborative evidence of focal HI.

At DOL 16, proband 1 was transferred to the University of North Carolina Children's Hospital. Blood glucose was <40 mg/dl, and the patient was still receiving diazoxide. In addition, the repeat insulin level was 42 pmol/l with blood glucose of 38 mg/dl. She was started on subcutaneous octreotide (4 μg/kg/day given in divided doses every 8 h). Due to persistent hypoglycemia and concomitant tachyphlyaxis, which is commonly found with octreotide (15), dosing was increased steadily to 12 μg/kg/day (divided doses every 6 h). Diazoxide was tapered off and fasting blood glucose was maintained at >60 mg/dl. On DOL 21, ECHO revealed systolic murmur, mild/moderate left ventricular hypertrophy (LVH), and mild hypertension for age (blood pressure 80–90 mmHg [systolic]/30–40 mmHg [diastolic]). Consequently, amlodipine (0.1 mg/kg/day given in divided dosing twice daily orally) was initiated to treat both LVH and associated hypertension. The repeat ECHO at DOL 46 was normal.

Proband 1 is currently 6-years-old with normal A1C (5.6%), impaired fasting glucose-1 (123 ng/ml), Insulin-like growth factor BP3 (4.3 μg/ml), and serum insulin (119 pmol/l) at ambient glucose of 78 mg/dl. Growth is steady between the 25–50 percentile on octreotide (now 8 μg/kg/day divided 4 times daily). Historically, she was allowed to outgrow her amlodipine. Due to the desire to decrease the frequency of injections, reintroduction of amlodipine was evaluated systematically using CGMS (CareLink, Medtronic) (Research Design and Methods).

Given the long-standing debate as to the usefulness of calcium channel antagonists in HI treatment (16,17), CGMS also permitted ascertaining the response to amlodipine, which may inhibit insulin release through direct inhibition of β-cell calcium channels (18) and/or via inhibition of these channels through decreases in cyclic AMP (19). The additive effects of amlodipine plus octreotide therefore would be expected to help to minimize the peak/trough effects of each individual agent. As shown in Fig. 1,C, octreotide alone maintained average blood glucose levels in the desired range, but marked excursions were still present with episodes >300 mg/dl. There was only one episode of blood glucose <60 mg/dl in >3,000 sensor readings (Fig. 1 B). Following the introduction of amlodipine, baseline mean blood glucose rose and stabilized at a higher level than with octreotide alone, and there were no episodes of blood glucose levels <60 mg/dl in the presence of octreotide plus amlodipine. Subsequent introduction of bedtime cornstarch further improved control without dangerous excursions but was not tolerated due to typical side effects of bloating and abdominal discomfort, and it was discontinued.

To examine the mutation effects on K_ATP channel activity, we measured ⁸⁶Rb⁺ efflux across the plasma membrane of COSm6 cells cotransfected with SUR1, and WT Kir6.2, Kir6.2[V290M], or 1:1 mixture of these subunits. Efflux curves were fit with the two-pathway model (Research Design and Methods), untransfected cells providing the efflux rate (k₁) for the non-K_ATP pathway. Homomeric Kir6.2[V290M] (M/M) channels show considerably reduced ⁸⁶Rb efflux rates, with k₂ reduced by ∼80% under metabolically inhibited or diazoxide-stimulated conditions (Fig. 2). Cells expressing heteromeric WT plus V290M (V/M) channels show intermediate efflux rates (k₂, reduced by ∼50%) (Fig. 2 B). These data confirm that the V290M mutation results in reduced K_ATP channel activity in intact cells and predict that insulin hypersecretion will be seen in vivo for heterozygous carriers and more severely so in the homozygous state.

Lysates of COSm6 cells expressing recombinant channels were assayed by immunoblotting (Fig. 3 A). Anti-SUR1 blots show a doublet at ∼170 kDa, corresponding to SUR1 protein, with a similar fraction of complex-glycosylated SUR1 to WT, indicating that the lower activity observed in V290M channels is not due to reduced channel density at the surface of the plasma membrane.

The activity of WT and mutant K_ATP channels was further examined in inside-out membrane patch-clamp experiments (Figs. 3,FIG. 4–5). Upon membrane excision, WT channels typically open to a steady-state level with open probability of 0.3–0.5 (20). In marked contrast, V290M channels open but then exhibit a rapid decay in macroscopic current, typically resulting in much smaller steady-state currents than is observed in WT channels. Following patch excision, V290M channels inactivated with time constant of 2.0 ± 0.5 s (n = 5), while WT channels showed essentially no inactivation following excision (Fig. 3,B). Steady-state ATP sensitivity of V290M channels was similar to WT (Fig. 4,A and C), but current density was considerably lower than WT (Fig. 4,B), reflecting the dramatic inactivation that occurs. Physiologically, the major determinant of channel activation is stimulation by Mg-nucleotides and, as shown in Fig. 4 D, MgADP activation is intact in V290M channels, although inactivation follows the MgADP activation, reducing steady-state currents in MgADP.

Following patch excision, exogenously applied phosphatidylinositol bisphosphate (PIP₂) incorporates into the membrane inner leaflet and typically increases activity of WT channels by two- to threefold as a result of increased open-state stability (21,22) (Fig. 5). Following inactivation to steady-state levels, application of PIP₂ to V290M patches caused much greater relative increase in current (∼50-fold) to 1902.58 ± 560.63 pA (n = 8), similar to the WT patch current on excision. During PIP₂ exposure, inactivation became progressively slower and less complete in response to application and removal of ATP (Fig. 5,B). During this process, the rate and extent of inactivation both decreased in correlation with the estimated open probability (Fig. 6 A) as expected for an inactivation process occurring from the closed state (discussion).

In mouse models of reduced K_ATP, even 50% reduction is sufficient to cause enhanced glucose tolerance and hypersecretion of insulin (23,24). Given that heteromeric V290M/WT channels exhibit ∼50% reduction of K_ATP conductance (Fig. 2) and the anecdotal evidence of hypersensitivity to glucose exhibited by the proband's brother, we pursued OGTT in the mother (Table 1) and the brother (Table 2), both of whom are heterozygous for the V290M mutation. Blood glucose in healthy adults will be below 200 mg/dl at 1 h and below 140 mg/dl at 2 h; in healthy children, it will be ∼100 mg/dl at 1 h (25). Even though neither mother nor brother displays overt HI under-fed conditions, glucose was close to fasting levels in both at 1 h, and even lower at 2 h, suggestive of a “supranormal” response. Proinsulin levels rose during OGTT and, although within the reported ranges for healthy subjects (26), proinsulin-to-insulin ratios at 1 and 2 h were above the normal range for young individuals (26).

We describe two unrelated families with the Kir6.2[V290M] mutation. By recombinant expression of WT or V290M mutant Kir6.2 subunits, we show that the mutation results in partial loss of K_ATP channel activity in the heterozygous case and more severe loss in the homozygous case (Fig. 2). The loss of function results from induction of an inactivation phenomenon (Fig. 3,B), which is similar to that resulting from HI-associated mutations at Kir6.2 residue R301 (27). These mutations resulted in both loss of functional membrane channels and an inactivating phenotype. The V290M mutation seems to reduce channel activity solely through induction of inactivation since surface expression appears normal (Fig. 3,A). Structural analysis suggests that V290 participates in the generation of an inter-subunit salt bridge involving R301 and E290 (Fig. 3,C). We suggest that V290M and R301 mutations induce the same inactivating phenotype by destabilizing this salt bridge, causing the channel to enter a long-lived inactivated state after closure in the absence of ATP. The observed relationship between K_1/2,ATP and open probability is an emergent property of gating schemes in which ATP preferentially stabilizes the closed state of the channel (28), and loss of inactivation paralleling increase in open-state stability with PIP₂ is then predicted by the assumption that inactivation occurs from the unliganded closed state (Fig. 6 B).

In family 1, HI in the neonatal period is clearly associated with the homozygous condition. In the proband of family 2, the mutation presents in a focal lesion with loss of heterozygosity. K_ATP channel activity reduction is graded from the heteromeric to the homomeric expression with severe (>80%) loss in the homomeric case; this is consistent with the clinical HI phenotype of these two probands. In the heterozygous case, reduction of channel activity is ∼50%. Previous extensive studies of animal models of genetic suppression of K_ATP activity predict that such a reduction is sufficient to cause glucose hyperresponsivity with left-shifted glucose-dependence of insulin secretion, but unaltered basal secretion (29,–31). The relevance of this to humans remains unclear, but seems a reasonable explanation for the apparent glucose hyperresponsivity of the two carrier relatives in family 2. In this regard, it is notable that the affected probands in each family and the three unaffected heterozygous carriers in family 1 were at the upper range of normal birthweights and large relative to the unaffected sibling. The anecdotal observation of the mother in family 1 that the 4-year-old heterozygous-carrier child can become hungry and/or lethargic after eating might reflect postprandial hypoglycemia and may warrant caution during meals and/or modification of meal carbohydrate composition for such an individual. There are few published studies focusing on heterozygous carriers of HI mutations. Huopio et al. (32) reported that carriers of the SUR1 V187D mutation had normal glucose tolerance and insulin secretory capacity. However, V187D reduces K_ATP channel activity by affecting trafficking to the surface membrane, and this is readily reversible, even simply by exposure to sulfonylureas (33). It is unclear whether the presence of even a single WT subunit might be sufficient for normal trafficking in the heterozygous case, such that there is minimal effect on K_ATP density in heterozygous carriers. Otherwise, for recessive loss of function HI mutations that do cause reduction in channel density in the heterozygous case, it seems that glucose hyperresponsivity might be a feature, although clearly this requires further study in bigger cohorts.

There are now several reports of patients with HI due to loss-of-function K_ATP mutations “crossing-over” to diabetes in later life, including heterozygous carriers of SUR1 mutations (34,35). Again, this is predicted from mouse models of the loss of K_ATP activity (36,31), which become glucose-intolerant as adults and diabetic on high-fat diets. It is notable that the proinsulin-to-insulin levels in the heterozygous carriers were in the normal range (0.1–0.2) in the fasted state and fell appropriately in the first hour of the OGTT, but then rose to relatively higher levels at 2 and 3 h than has been reported for healthy young subjects, and reminiscent of the elevated ratios seen in type 2 diabetics (26,37). Conceivably, this apparently elevated proinsulin-to-insulin ratio reflects so-called β-cell overwork and might be a harbinger of a tendency to crossover later in life.

Standard treatment options for HI are essentially limited to diazoxide (activating K_ATP and suppressing insulin secretion) or octreotide (long-acting somatostatin analog suppressing glucose actions on the β-cell), glucagon (counterregulator to insulin) or subtotal pancreatectomy (17,38,–40). l-type (dihydropyridine-sensitive, Ca_V1) channel blockers are mechanistically an attractive option to directly modulate insulin secretion. Previous results have been mixed (16,41), but some patients have achieved stable blood glucose levels on nifedipine monotherapy. In the present case, improvement in both LVH and hypertension in addition to hypoglycemia are shown in a neonate on both amlodipine and octreotide. The contribution of amlodipine to blood glucose was assessed more directly using CGMS when the proband became of school-age. The main finding was a rise in basal glucose levels with no values below 70 mg/dl while on the same dose of octreotide in the presence of amlodipine. Given its mechanism of Ca_v1 channel inhibition, this finding is not surprising and supports the results of Aynsley-Green and colleagues (42). In the present case, amlodipine also permitted extension of octreotide dosing from every 6 h to every 8 h, thereby saving 1 injection daily (a significant improvement in quality of life).

A key clinical management question for HI is whether to pursue medical or surgical therapy. Long-term medical therapy, as in this case, presents a less expensive option than subtotal pancreatectomy, which is the procedure that would be indicated by the mutation and mode of inheritance. In addition, excellent glucose control, normal growth and development, and the variable risk of postsurgical diabetes all support this management to date for this child. Moreover, glucose levels normalize and medical therapy can be stopped at some point for many HI children (41,43,–45), whereas patients who undergo major resection of the pancreas will tend to develop diabetes at or around puberty, another consideration when assessing the pros and cons of surgical versus medical management.

We report on HI due to a K_ATP mutation that results in an inactivating channel in two different families. A homozygous-affected child is well controlled with medical therapy without surgery. Clinically unaffected heterozygous carriers show signs of hypersecretion, and we suggest that they may represent an unappreciated cohort with subclinical features.

This work was supported by National Institutes of Health Grant DK69445 (to C.G.N.). F.B. is a member of the Early Onset Diabetes Study Group of the Italian Society of Pediatric Endocrinology and Diabetology.

No potential conflicts of interest relevant to this article were reported.

K.J.L., A.A., and J.C.K. researched data and wrote the manuscript. H.T.K., C.D.-V., A.M., M.P., V.R., J.d.V.d.G., and C.C. researched data. F.B. and C.G.N. wrote the manuscript.

We would like to thank Medtronic for both clinical and technical support during the use of CGMS monitoring for the second subject. Human studies for the second patient and family were carried out under the institutional review board exempt category at the University of North Carolina (K.J.L.).