Background/Aims: Mutations in KCNJ11, the gene encoding the Kir6.2 subunit of pancreatic and neuronal KATP channels, are associated with a spectrum of neonatal diabetes diseases. Methods: Variant screening was used to identify the cause of neonatal diabetes, and continuous glucose monitoring was used to assess effectiveness of sulfonylurea treatment. Electrophysiological analysis of variant KATP channel function was used to determine molecular basis. Results: We identified a previously uncharacterized KCNJ11 mutation, c.988T>C [p.Tyr330His], in an Italian child diagnosed with sulfonylurea-resistant permanent neonatal diabetes and developmental delay (intermediate DEND). Functional analysis of recombinant KATP channels reveals that this mutation causes a drastic gain-of-function, due to a reduction in ATP inhibition. Further, we demonstrate that the Tyr330His substitution causes a significant decrease in sensitivity to the sulfonylurea, glibenclamide. Conclusions: In this subject, the KCNJ11 (c.988T>C) mutation provoked neonatal diabetes, with mild developmental delay, which was insensitive to correction by sulfonylurea therapy. This is explained by the molecular loss of sulfonylurea sensitivity conferred by the Tyr330His substitution and highlights the need for molecular analysis of such mutations.

Permanent neonatal diabetes mellitus (PNDM) is a rare condition that has been associated with defects in at least 30 different genes [1]. The most prevalent cause of PNDM is an activating mutation in either KCNJ11 or ABCC8 genes, which respectively encode the two subunits (Kir6.2 and SUR1) forming ATP-sensitive potassium (KATP) channels in pancreatic β cells, as well as in the central nervous system (CNS), peripheral nerves, and skeletal muscle [2]. Severe KATP mutations may give rise to developmental delay with (DEND) or without (intermediate DEND) epilepsy in addition to neonatal diabetes [1]. More than 90% of patients with KATP-dependent PNDM respond to oral therapy with sulfonylureas (SU), resulting in long-lasting diabetes control [3, 4]. In addition, SU treatment may improve neurological symptoms [3, 4]. However, patients with DEND may also fail to respond to SU [5]. Importantly, neonatal diabetes with diabetic ketoacidosis (DKA) at onset and/or cerebral edema may impact CNS function, making it difficult to distinguish direct effects of overactive neuronal KATP channels from additional metabolic insults to the brain [6, 7]. Functional assessment of the specific mutation in vitro can provide critical clues about the responsiveness of the mutated channel to SU and shed light on its effect on the CNS [5, 6, 8]. Here, we report a patient with PNDM carrying the heterozygous KCNJ11/ Tyr330His mutation who is unresponsive to SU therapy and consider the molecular underpinnings of this outcome.

Molecular Investigation

Genomic DNA from the affected patient was analyzed on a custom in silico gene panel. Library preparation and clinical exome capture were performed using the Twist Custom Panel Kit (clinical exome: Twist Bioscience, South San Francisco, CA, USA) according to the manufacturer’s protocol and sequenced on the Illumina NovaSeq 6000 platform. The BaseSpace pipeline (Illumina, Inc., San Diego, CA, USA) and TGex software (LifeMap Sciences, Inc., Alameda, CA, USA) were used for variant calling and annotating variants, respectively. Sequencing data were aligned to the hg19 human reference genome. Based on the guidelines of the American College of Medical Genetics and Genomics, a minimum depth coverage of 20× was considered suitable for analysis. Variants were examined for coverage and visualized by the Integrative Genome Viewer (IGV).

Electrophysiology

Mutations were introduced into pcDNA3.1-mouse Kir6.2 (GenBankTM accession no. D50581.1) using site-directed mutagenesis, and specific mutations were confirmed by Sanger sequencing. Cosm6 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 105-units/L penicillin, and 100-mg/L streptomycin. Cosm6 cells were transfected using FuGENE 6 (Promega). Cells were transfected with 0.1-μg GFP cDNA (to identify transfected cells) with 1-μg pECE-hamster SUR1 cDNA (GenBankTM accession no. AAA99201.1) plus either WT or mutant pcDNA3.1-mouse Kir6.2 (0.6 μg), or a mixture of WT and mutant Kir6.2 constructs (0.3 μg:0.3 μg).

Excised, inside-out patch clamp recordings were made 48–72 h post-transfection, at 20–22°C, using a rapid solution exchange Dynaflow Resolve perfusion chip (Cellectricon). Membrane currents were sampled at 3 kHz, filtered at 1 kHz, at a holding potential of -50 mV using an Axopatch 700B amplifier and Digidata 1200 (Molecular Devices). Patch clamp pipettes were made from soda lime glass microhematocrit tubes (Kimble) with tip resistances of 1–2 MΩ when filled with patch solution. The bath and pipette solutions (KINT) contained (in mM): 140 KCl, 10 HEPES, 1 EGTA (pH 7.3 with KOH). Currents were recorded in the presence and absence of nucleotides and drugs as indicated. In Mg2+ nucleotide-containing solutions a 0.5-mM free Mg2+ concentration was maintained by supplementation of MgCl2 as calculated by MAXCHELATOR (https://somapp.ucdmc.ucdavis.edu/pharmacology/bers/maxchelator/index.html), where indicated. KATP currents in nucleotide/drug solutions were normalized to basal current levels in the absence of nucleotides. ATP dose-response curves were fit with a four-parameter Hill equation using the Data Solver Function in Microsoft Excel, where normalized current = Imin + (ImaxImin)/(1 + ([X]/IC50)H), where the current in Kint = Imax = 1, Imin is the normalized minimum current observed in the presence of 10-mM MgATP + 1-μM glibenclamide inhibition which was measured at the end of each experiment and represented the minimal observed KATP current for each recording [X] refers to the concentration of ATP, IC50 is the concentration of half-maximal inhibition, and H denotes the Hill coefficient. Remaining % of ADP-stimulated currents was = ([IGLIBImin]/[IADPImin]) × 100, where IADP was the current in 100-μM MgATP + 500-μM MgADP and IGLIB was the current in 100-μM MgATP + 500-μM MgADP + 1-μM glibenclamide. Data that were tested for statistical significance using unpaired Student’s t tests with Bonferroni correction for multiple comparisons were described in figure legends.

Clinical Features of the Patient

The male proband was delivered at 39+6 weeks gestational age by elective cesarean section because of breech presentation; his birth weight was 2,455 g (small for gestational age). He immediately required cardiopulmonary resuscitation, intubation, and transient mechanical ventilation (Apgar: 2 at 1'; cord arterial pH: 7.0, base excess −16.6 mmol/L) and was promptly transferred to the neonatal intensive care unit because of moderate encephalopathy on Sarnat staging. He underwent neuroprotective whole-body cooling with gradual rewarming after 72 h. During hypothermia he was under parenteral nutrition; frequent capillary blood glucose (BG) monitoring through a point-of-care glucometer showed no abnormal glucose values. After rewarming, capillary BG increased to >250 mg/dL on day 5 of life, in the absence of any stress. Continuous glucose monitoring indicated BG >350 mg/dL with glucosuria, confirming the diagnosis of NDM, without ketonuria or acidosis. Serum insulin (1.8 mIU/L) and C-peptide (0.099 nmol/L) were below the lower limit of the reference range. No dysmorphic features were noted on examination and there was no family history of diabetes. Insulin infusion (0.5–1 U/kg/day) was commenced and carefully titrated using continuous glucose monitoring (Medtronic, Northridge, CA, USA), by tailoring the dose on the trend of BG levels from the previous day. Brain ultrasound revealed unspecific hyperechoic images of periventricular white matter, while brain magnetic resonance imaging performed at day of life 7 displayed minimal bilateral thalamic T1 hyperintensities related to perinatal asphyxia.

Mutation Identification and Sulfonylurea Trial

A spontaneous KCNJ11 variant, c.988T>C [p.Tyr330His] was detected. An identical mutation was cursorily reported in a patient with sulfonylurea-unresponsive DEND [9], and listed in a recent review on KATP variants found in congenital hyperinsulinism and diabetes [1]. Based on the available evidence that includes other variants of KCNJ11 residue 330 [Tyr330Cys, Tyr330Ser, and p.Tyr330Asp [1]] all linked to NDM, either permanent or transient, this missense variant was predicted to be likely pathogenic and the patient was transferred to Bambino Gesù Children’s Hospital for sulfonylurea (glibenclamide) therapeutic trial.

At 48 days of life, oral glibenclamide therapy was started at the initial dose of 0.3 mg/kg/day, along with a continuous intravenous infusion of regular human insulin (0.4 IU/kg/day). Over the following 2 weeks, the glibenclamide dose was progressively increased to 1.5 mg/kg/day, while intravenous insulin infusion was maintained at the same rate, but no substantial changes in BG levels were observed. At 2 months of life the patient was started on subcutaneous insulin therapy with Levemir (4 U/day). Laboratory analysis showed a fructosamine value of 330 μM (reference range 205–285 μM). Abdominal ultrasound showed right inguinal hernia, laparoscopically repaired without any complication. Brain ultrasound revealed no anomalies. The patient was discharged at 3 months of age (weight: 4 kg), with subcutaneous insulin therapy (Levemir 4 U/day), and glibenclamide (1.3 mg/kg/day).

At 4 months of age weight gain was adequate, but BG profile was characterized by marked glycemic variability. After two additional weeks of glibenclamide at ∼1.5 mg/kg/day, serum C-peptide was still undetectable, and daily insulin requirement was unchanged. While no hypoglycemic episode related to sulfonylurea therapy was recorded (Fig. 1), there was also no substantial improvement of glycemic control (time in range [TIR] with sulfonylurea 46%, TIR without sulfonylurea 41%) (Fig. 1a, b). Therefore, glibenclamide treatment was stopped. Pancreatic autoantibodies (GAD, IA2) were confirmed to be negative; fructosamine was at the upper limit of the reference range (283 μM). At 10 months of age, continuous subcutaneous insulin infusion (CSII) with a sensor-augmented insulin pump (MiniMed® 640 G system with SmartGuardTM technology) was initiated. This system allows an automatic stop of insulin delivery based on the prediction of low glucose levels. Glycemic control improved (Fig. 1) and HbA1c levels decreased from 7.1 to 6.4 over 3 months. At the age of 26 months the patient was still unable to crawl. His verbal production was characterized by babbling and 10 clear and contextual words that he failed to combine. Neurodevelopmental outcomes were objectively assessed using Griffith’s Mental Developmental Scales at our follow-up outpatient service, as previously described [10]. An Italian-validated translation of the administration manual was used, as previously described [11]. Mild-to-severe developmental delay without epilepsy (intermediate DEND) was identified, with a global developmental quotient (DQ) of 58 (abnormal below 70). Notably, the worst results were obtained in subscale A (locomotor abilities) with a DQ of 37 rather in other subscales: a DQ of 60 in subscale B (personal-social abilities), a DQ of 62 in subscale C (hearing and speech domain), a DQ of 60 in subscale D (eye and hand coordination), and a DQ of 71 in subscale E (performance and practical reasoning). Unfortunately, infants with neurodevelopmental disabilities could not attend their usual rehabilitation therapies during the first lockdown because of the COVID-19 pandemic. This coincided with the first 2 years of life of the patient, who currently attends psychomotricity and motor physiotherapy sessions. Currently, at the age of 31 months, the patient’s length (82 cm), and weight (10 kg) are both below 3rd percentile for age, according to World Health Organization standards.

Fig. 1.

Plasma glucose levels with insulin/glibenclamide administration 2 weeks CGM profiles (a) during glibenclamide therapy (1.5 mg/kg/day) and Levemir (1 U/kg/day). Glucose average 186 ± 91 mg/dL. TIR (70–180 mg/dL) 46%, time below range (<70 mg/dL) 6%; (b) without glibenclamide. Glucose average 191 ± 91 mg/dL. TIR (70–180 mg/dL) 41%, time below range (<70 mg/dL) 3%; (c) during CSII therapy with Predictive low-glucose suspend (PLGS) system, glucose average 177 ± 79 mg/dL. TIR (70–180 mg/dL) 55%, time below range (<70 mg/dL) 3%.

Fig. 1.

Plasma glucose levels with insulin/glibenclamide administration 2 weeks CGM profiles (a) during glibenclamide therapy (1.5 mg/kg/day) and Levemir (1 U/kg/day). Glucose average 186 ± 91 mg/dL. TIR (70–180 mg/dL) 46%, time below range (<70 mg/dL) 6%; (b) without glibenclamide. Glucose average 191 ± 91 mg/dL. TIR (70–180 mg/dL) 41%, time below range (<70 mg/dL) 3%; (c) during CSII therapy with Predictive low-glucose suspend (PLGS) system, glucose average 177 ± 79 mg/dL. TIR (70–180 mg/dL) 55%, time below range (<70 mg/dL) 3%.

Close modal

Functional Consequences of Tyr330His Substitution

The finding of sulfonylurea insensitivity in this patient is in contrast to the outcome in the vast majority of KATP-dependent PNDM patients [3, 4]. To determine the effect of NDM-associated substitutions at the Kir6.2-Tyr330 position, we co-transfected Cosm6 cells with SUR1 together with wild-type Kir6.2 (Kir6.2-WT), Kir6.2-Tyr330His, or Kir6.2-Tyr330Cys mutant cDNA – the latter was previously identified in patients with PNDM [1]. In inside-out patch clamp recordings, Kir6.2-WT/SUR1 channels were inhibited by ATP with an IC50 of ∼10 μM (at pHi 7.3) whereas in channels comprised of Kir6.2-Tyr330His and Kir6.2-Tyr330Cys ATP sensitivity was decreased ∼380 and ∼70 fold, respectively (Fig. 2a–f). These experiments were performed in the absence of cytoplasmic Mg2+, which isolates the inhibitory effect of ATP-binding to the Kir6.2 subunit [12], showing that this mechanism is impaired by Tyr330 substitution. Significant on-cell currents were observed prior to patch excision in both Tyr330His and Tyr330Cys substituted channels, which is consistent with increased KATP channel activity in normal intact cell conditions (Fig. 2g). When SUR1 was co-transfected with a 1:1 mixture of Kir6.2-WT and Kir6.2-Tyr330His, to mimic the heterozygous context observed in the patient, resultant channels still exhibited striking loss of ATP sensitivity, with IC50 values increased 7-fold (Fig. 2e).

Fig. 2.

ATP sensitivity of recombinant wild-type and mutant KATP channels. Example traces of inside-out voltage clamp experiments of excised patches from Cosm6 cells transfected with SUR1 alongside either Kir6.2-WT (a), a 1:1 mix of Kir6.2-WT and Kir6.2-Tyr330His (b), Kir6.2-Tyr330His (c), or Tyr330Cys (d). Currents were recorded at -50 mV in the presence and absence of ATP as indicated. “On-cell” current levels prior to patch excision are indicated with black arrows and the “o.c.” label, current levels post patch excision in the absence of nucleotides are indicated by the red arrow and “ex.” label. The dashed line represents the zero current level determined in the presence of 10-mM MgATP and 1-μM glibenclamide. e Summary ATP dose-response curves for Kir6.2-WT, a 1:1 mix of Kir6.2-WT and Kir6.2-Tyr330His, Kir6.2-Tyr330His, and Tyr330Cys-containing channels. f IC50 for ATP inhibition. g On-cell currents normalized to the current observed following patch excision. f, g Individual recordings are represented by individual shapes in scatter plots and bars represent mean values ± SEM. *Denotes statistically significance differences determined by unpaired Student’s T test and Bonferroni correction (α = 0.05 divided by number of pairwise comparisons; p< 0.0083 (f); p< 0.0167 (g)). h Representation of Kir6.2 tetramer, to show the location of Tyr330 in Kir6.2.

Fig. 2.

ATP sensitivity of recombinant wild-type and mutant KATP channels. Example traces of inside-out voltage clamp experiments of excised patches from Cosm6 cells transfected with SUR1 alongside either Kir6.2-WT (a), a 1:1 mix of Kir6.2-WT and Kir6.2-Tyr330His (b), Kir6.2-Tyr330His (c), or Tyr330Cys (d). Currents were recorded at -50 mV in the presence and absence of ATP as indicated. “On-cell” current levels prior to patch excision are indicated with black arrows and the “o.c.” label, current levels post patch excision in the absence of nucleotides are indicated by the red arrow and “ex.” label. The dashed line represents the zero current level determined in the presence of 10-mM MgATP and 1-μM glibenclamide. e Summary ATP dose-response curves for Kir6.2-WT, a 1:1 mix of Kir6.2-WT and Kir6.2-Tyr330His, Kir6.2-Tyr330His, and Tyr330Cys-containing channels. f IC50 for ATP inhibition. g On-cell currents normalized to the current observed following patch excision. f, g Individual recordings are represented by individual shapes in scatter plots and bars represent mean values ± SEM. *Denotes statistically significance differences determined by unpaired Student’s T test and Bonferroni correction (α = 0.05 divided by number of pairwise comparisons; p< 0.0083 (f); p< 0.0167 (g)). h Representation of Kir6.2 tetramer, to show the location of Tyr330 in Kir6.2.

Close modal

Residue 330 in Kir6.2 is located in the cytoplasmic C-terminus of the channel subunit, at a solvated intersubunit interface, in close proximity to the ATP-binding site (Fig. 2h) [10, 11]. Conceivably, pH-dependent ionization of the histidine side chain might vary physiologically, and influence ATP sensitivity. To test this, we measured [ATP]-response relationships for Kir6.2-WT/SUR1 and Kir6.2-Tyr330His/SUR1 channels at pHi 6 and pHi 8. As shown in Figure 3a, a subtle change in the rank order of ATP potency across the pH ranges was seen for Kir6.2-Tyr330His/SUR1 channels but the dramatic reduction of ATP sensitivity was present at all pHi. Thus overall mutant channel overactivity and ATP insensitivity appear to be due to loss of the normal Tyr residue, rather than the introduction of a titratable charge, at this location.

Fig. 3.

pH and glibenclamide sensitivity of wild-type and Kir6.2-Tyr330 mutant channels. a Summary ATP dose-response curves from inside-out voltage clamp experiments of excised patches from Cosm6 cells transfected with SUR1 alongside Kir6.2-WT or Kir6.2-Tyr330His at intracellular pH of 7.3, 6.7, or 8.0. Data shown as mean ± SEM from ≥ 4 patches. b Example traces of inside-out voltage clamp experiments of excised patches from Cosm6 cells transfected with SUR1 plus Kir6.2-WT (top) or a 1:1 mix of Kir6.2-WT and Kir6.2-Tyr330His (middle) or Kir6.2-Tyr330His (bottom). Currents were recorded at −50 mV in the presence and absence of ATP, 500-μM ADP and 1-μM glibenclamide as indicated. All nucleotide-containing solutions included 0.5-mM free Mg2+ (see the Methods section). c Summary showing the % of the current observed in the presence of 100-μM ATP and 500-μM ADP which remained upon coadministration of 1-μM glibenclamide. Individual recordings are represented by individual shapes in scatter plot and bars represent mean values ± SEM. *denotes statistically significant differences determined by unpaired Student’s T test and Bonferroni correction (α = 0.05 divided by the number of pairwise comparisons, i.e., p< 0.0167). d Representation of Kir6.2 tetramer associated with a single SUR1 subunit showing the location of Tyr330 at subunit interface, from previously reported cryo-EM structures (PDB 6baa) [10]. Tyr330 (shown as gray spheres) forms part of the ATP-binding site (ATP shown as green/blue/orange/red spheres).

Fig. 3.

pH and glibenclamide sensitivity of wild-type and Kir6.2-Tyr330 mutant channels. a Summary ATP dose-response curves from inside-out voltage clamp experiments of excised patches from Cosm6 cells transfected with SUR1 alongside Kir6.2-WT or Kir6.2-Tyr330His at intracellular pH of 7.3, 6.7, or 8.0. Data shown as mean ± SEM from ≥ 4 patches. b Example traces of inside-out voltage clamp experiments of excised patches from Cosm6 cells transfected with SUR1 plus Kir6.2-WT (top) or a 1:1 mix of Kir6.2-WT and Kir6.2-Tyr330His (middle) or Kir6.2-Tyr330His (bottom). Currents were recorded at −50 mV in the presence and absence of ATP, 500-μM ADP and 1-μM glibenclamide as indicated. All nucleotide-containing solutions included 0.5-mM free Mg2+ (see the Methods section). c Summary showing the % of the current observed in the presence of 100-μM ATP and 500-μM ADP which remained upon coadministration of 1-μM glibenclamide. Individual recordings are represented by individual shapes in scatter plot and bars represent mean values ± SEM. *denotes statistically significant differences determined by unpaired Student’s T test and Bonferroni correction (α = 0.05 divided by the number of pairwise comparisons, i.e., p< 0.0167). d Representation of Kir6.2 tetramer associated with a single SUR1 subunit showing the location of Tyr330 at subunit interface, from previously reported cryo-EM structures (PDB 6baa) [10]. Tyr330 (shown as gray spheres) forms part of the ATP-binding site (ATP shown as green/blue/orange/red spheres).

Close modal

To determine the effect of Tyr330 mutations on sulfonylurea sensitivity, we recorded KATP currents from inside-out patches in the presence of 100-μM ATP/500-μM ADP and the absence or presence of 1-μM glibenclamide (with 0.5-mM free Mg2+). Kir6.2-WT-containing channels were almost completely inhibited by glibenclamide (Fig. 3b, c), as expected [12, 13]. However, there was a significant decrease in the glibenclamide effect on Kir6.2-Tyr330Cys and Kir6.2-Tyr330His-containing channels, with ∼30% and ∼65% remaining currents, respectively (Fig. 3c).

Sulfonylurea Unresponsivity in NDM

Most patients with KCNJ11- (and ABCC8)-associated PNDM respond favorably to SU [1, 3], although very high doses may be required to allow insulin weaning and to maintain optimal metabolic control in patients with the most severe mutations. For instance, we were able to stop insulin in a patient with DEND associated with the KCNJ11-Val59Ala mutation at a glibenclamide dose of 1.75 mg/kg/day [14]. In the present case, however, we saw no improvement of plasma glucose, nor decrease in insulin requirement at a similar glibenclamide dose of 1.5 mg/kg/day maintained for 2 weeks (Fig. 1a, b). Unfortunately, we do not have information on the plasma concentrations of glibenclamide, but a 20-mg oral dose in adults (∼0.3 mg/kg, equivalent to the initial dose used in our patient, was previously found to result in peak plasma concentrations of ∼700 nM [15]. Since the dose of 1.5 mg/kg/day is close to the maximum used in NDM [16], and since toxic effects can be observed at >1-μM plasma concentrations in otherwise normoglycemic individuals [17, 18], further dose escalation was not considered.

PNDM patients with the KCNJ11-Tyr330Cys and KCNJ11-Tyr330Ser variants have previously been reported [19, 20], and the Tyr330Cys variant has been inconsistently linked to a developmental neuronal phenotype: limited clinical data indicate that neurological involvement was present in one sulfonylurea-treated Tyr330Cys patient [21], and in 1of 2-Tyr330Cys patients, both of whom (at the age of 3 and 25) were reported on insulin therapy (0.84 and 0.45 U/kg/day, respectively) [19]. The finding that our patient with the Tyr330His mutation was not responsive to high-dose SU thus seems consistent with the even greater molecular gain-of-function, and more pronounced effect on glibenclamide inhibition, of the histidine substitution compared to the cysteine substitution. Previous retrospective analysis has shown that patients with even very severe molecular gain-of-function can successfully transfer to SU, but this is also dependent on the timing [16, 21, 22]. The earlier transfer is beneficial, and we cannot discount the possibility that time spent prior to sulfonylurea treatment could have been a factor in the outcome for our patient, as we previously found for a 15-year-old sulfonylurea-unresponsive patient with KCNJ11-Gly334Asp, a mutation without obvious effect on sulfonylurea sensitivity [5]. However, SU were introduced relatively early in the current patient (at day of life 48), which suggests that cumulative prior pathology may be an unlikely explanation for SU unresponsivity. Nevertheless, it remains unclear whether the moderate encephalopathy found at birth and the current developmental abnormalities of our patient can be solely attributed to the Tyr330His mutation or whether postpartum complications might also have contributed.

The dramatic loss of ATP sensitivity in the Kir6.2-Tyr330His mutation places it on the severe end of the spectrum of NDM-related molecular dysfunction. Tyr330 is located on the exterior surface of the cytoplasmic C-terminus of Kir6.2 in a loop that also contains NDM-associated mutation F333I, in close proximity to both the ATP-binding site, and the L0-linker region of SUR1 at the interface between subunits (Fig. 3d). The L0-linker has a well-characterized role in regulating both ATP-binding and inhibition, as well as modulating the unliganded open probability of KATP [23-25]. Normal channel function appears strictly dependent on the specific chemistry and/or spatial properties of the tyrosine side chain as substitution to diverse serine, cysteine, and histidine residues all provoke gain-of-function [1, 26].

Glibenclamide acts via binding to the sulfonylurea-receptor in a recently resolved site that is far removed from the location of Tyr330 [10]. The observed decrease in SU sensitivity must therefore arise from an allosteric mechanism, which is consistent with previous reports that open-state stabilizing mutations of Kir6.2 reduce SU sensitivity by decoupling SUR1-Kir6.2 interactions [27].

In conclusion, we report a KCNJ11 mutation that results in a previously uncharacterized Kir6.2-Tyr330His substitution, in a patient with neonatal diabetes. This variant results in very severe gain-of-function and reduced glibenclamide sensitivity of recombinant KATP channels. In addition to sulfonylurea-unresponsive NDM, Kir6.2-Tyr330His causes a spectrum of phenotypic features spanning from relatively moderate CNS functional defects to severe neurologic symptoms, including seizures, i.e., DEND syndrome.

This study is approved by the Ethics Committee of Bambino Gesù Paediatric Hospital, approval # RRC-2018-2365812. Written informed consent was obtained from the parents of the patient for the publication of the details of their medical cases.

S.C. is the editor-in-chief of the journal “Hormone Research in Pediatrics.” The authors declare no other conflicts of interest.

This work was partially supported by R35 Grant HL140024 from the NIH (to C.G.N.) and K99 Grant HL150277 (to C.M.). The funders had no role in the preparation of data or the manuscript.

N.R., D.U.D.R., C.B., E.T., A.P., R.F., A.D., G.V., and S.C. made clinical diagnosis and collected clinical data; M.M. identified the mutation, C.M., J.G., and J.R. performed functional studies; C.M., N.R., D.U.D.R., G.V., F.B., and C.G.N. wrote the paper; F.B., S.C., and C.G.N. conceived the study; and F.B. and C.G.N. critically revised the manuscript.

All data generated or analyzed during this study are included in this article. Further enquiries can be directed to the corresponding author.

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Additional information

Conor McClenaghan, Novella Rapini, and Domenico Umberto De Rose contributed equally.Giovanni Vento, Fabrizio Barbetti, Colin G. Nichols, and Stefano Cianfarani are the senior authors.

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