Alternating hypoglycemia and hyperglycemia in a toddler with a homozygous p.R1419H ABCC8 mutation: an unusual clinical picture

Introduction

Autosomal recessive inheritance of two abnormal ABCC8 or KCNJ11 alleles causes severe forms of hyperinsulinism (HI) (1–4). Encoded by two adjacent genes on chromosome 11p, the sulfonylurea receptor (SUR1) modulates channel activity, whereas the inwardly rectifying potassium channel (Kir6.2) makes up the ion-conducting pore of the hetero-octameric ATP-sensitive potassium channel (K_ATP) (5, 6). The most common outcome of recessively inherited ABCC8 gene mutations is to prevent expression of functional channels, leading to severe HI that presents early in life and is unresponsive to diazoxide. Most patients require subtotal pancreatectomy (7, 8). In some cases, as reported for a splice site mutation of ABCC8, there can be variability in the age and severity of presentation in homozygous individuals (9, 10).

The natural history of patients who do not require pancreatectomy can include remission of the hypoglycemia, with progression to diabetes in early adulthood (11). However, long-term data is limited. These patients have been observed to have both hypoglycemic and hyperglycemic episodes a few years after presentation and pathologic responses to the oral glucose tolerance test have been described after 10 years of age (11). A blunted insulin response to intravenous dextrose was reported, suggesting reduced glucose sensitivity of the SUR-deficient β-cell (12), a condition referred to colloquially as “glucose-blindness”.

In this report, we describe the unusual clinical course and workup of a patient who is homozygous for a p.R1419H mutation in ABCC8, a seemingly severe coding mutation that prevents maturation of K_ATP channels to the cell surface. The patient’s presentation is a rare example of a weak phenotype with relatively late onset, inconsistent with the expected severity of the mutation at the cellular level.

Materials and methods

Results

A previously healthy 16-month-old girl presented with hypoglycemia of 1.2 mmol/L (22 mg/dL) in the context of lethargy during a gastrointestinal infection. Retrospectively, the parents recalled episodes of irritability over the previous 4 months, including episodes of medial eye deviation that could represent hypoglycemic seizures. She had been born at term, weighing 3490 g, after a pregnancy complicated by gestational diabetes, with no documented history of neonatal hypoglycemia. She grew along the 50th percentiles for length and weight. She was still breastfeeding frequently. She had normal motor milestones with mild speech delay.

The patient’s father reported having seizures as an infant in Pakistan. He could not recall whether they were febrile seizures or otherwise associated with intercurrent illness. He had normal intellect and he was diagnosed with diabetes at 28 years of age. His body mass index (BMI) was 22.4 kg/m² (–0.21 standard deviation) at that time. Medical records from the time of diagnosis were unavailable. He was managed by his family physician according to common guidelines with glyburide and metformin, but had not previously been tested genetically. HbA1c at 45 years of age was 8.1% (normal 4.5–6). The patient’s mother had diet-controlled gestational diabetes. Her BMI was 21.6 kg/m² (–0.04 standard deviation). The parents were first cousins through their mothers and second cousins through their fathers, and each of their pairs of parents were also related to each other (Figure 1).

Figure 1

Pedigree chart. Pedigree chart showing the proband (V-18, arrow), her father (IV-3), and mother (IV-11). The family is of Pakistani descent. Her sister is shown in gray because she is asymptomatic and has yet to have genetic testing. Another sister died in an accident. The patient’s mother had two miscarriages.

The patient had recurrent hypoglycemia in hospital and critical samples demonstrated alternating ketotic and non-ketotic hypoglycemia, with detectable insulin levels. An extensive workup was otherwise normal (Tables 1 and 2). Aberrant insulin processing was ruled out as a cause (Supplementary Table 1). Finally, a diagnosis of HI was entertained based on the insulin levels, associated with relatively low β-hydroxybutyrate, as well as a glycemic response to glucagon [increase of 3.4 mmol/L (61 mg/dL)]. Monitoring after a 2 g/kg carbohydrate meal showed post-prandial hyperglycemia, 10.8 mmol/L (193 mg/dL) with relative hypoinsulinemia, 98 pmol/L (16.3 μIU/mL), followed by hypoglycemia, 2.1 mmol/L (38 mg/dL) with relative hyperinsulinemia, 34 pmol/L (5.6 μIU/mL) after 4.5 h (Figure 2), confirming inappropriate regulation of insulin secretion.

Figure 2

The patient’s post-prandial glucose and insulin levels. Monitoring after a 2 g/kg carbohydrate meal showed post-prandial hyperglycemia (10.8 mmol/L, normal <7.8 mmol/L) with relative hypoinsulinemia (98 pmol/L, normal post-prandial insulin 120–300 pmol/L), followed by hypoglycemia (2.1 mmol/L) with relative hyperinsulinemia (insulin 34 pmol/L, normal for hypoglycemia <10 pmol/L).

The patient did not respond to diazoxide and it was stopped at a dose of 12 mg/kg/day due to severe side effects. She also did not respond to boluses of up to 5 μg/kg of octreotide. She was eventually placed on a continuous subcutaneous glucagon infusion (0.7 mg/day) with a significant improvement in blood glucose readings. She had a gastrostomy tube placed with daytime bolus feeds and continuous night-time feeding of Pediasure (Abbott Laboratories, Abbott Park, IL, USA). The glucagon infusion was decreased gradually and stopped altogether after 5 months. She remains on continuous night-time feeding and eats orally frequently during the day. With this regimen, she experiences mild hypoglycemias, as well as postprandial hyperglycemias up to 12.5 mmol/L (225 mg/dL) every 2–3 days.

Microarray studies (Supplementary Table 2) revealed homozygosity for multiple genomic regions, consistent with the family history of consanguinity. The region surrounding KCNJ11 and ABCC8 was identified as the best candidate region for harboring rare variants with major effects on the phenotype. KCNJ11 and ABCC8 mutation analysis was performed at the University of Exeter Medical School, Exeter, UK, and identified a homozygous missense coding mutation, p.R1419H (c.4256G>A) in the ABCC8 gene. Sanger sequencing of the patient and her parents (Supplementary Figure 1A–C) found that the mother was heterozygous and the father homozygous for the same mutation.

Previous functional data in the literature has suggested that the SUR1[R1419H] mutation prevents channels from reaching the cell surface (16). However, the relatively mild phenotypes of the patient and her father contrasted with this “classical” clinical picture of homozygous recessive ABCC8 mutations, which are typically very severe and present early in life (7, 17, 18). Therefore, we tested the effects of this mutation in a reconstituted system, using Rb⁺ efflux assays of CosM6 cells transfected with Kir6.2/KCNJ11 and WT or SUR1[R1419H] to measure functional K_ATP channel expression (Figure 3). Under conditions of metabolic inhibition (1 mM deoxyglucose+100 μM oligomycin), to maximally activate K_ATP channels, cells transfected with the SUR1[R1419H] variant (+Kir6.2) exhibited no Rb⁺ efflux above untransfected cells, consistent with an absence of functional K_ATP channels at the cell surface (Figure 3A). Cells transfected with a 1:1 mixture of WT: SUR1[R1419H] to simulate a heterozygous state exhibited Rb⁺ efflux activity intermediate between the WT-only and R1419H-only SUR1 cells. We examined the effects of SUR1[R1419H] subunits on cell surface maturation of K_ATP using the differential migration of mature and immature glycosylated forms of SUR1 on SDS-PAGE gels (19–21) (Figure 3B and C). Cells transfected with SUR1 alone exhibit a single molecular weight band attributed to an immature form of the protein that fails to reach the plasma membrane. When co-transfected with Kir6.2 channels, a mature glycosylated band is apparent. However, co-transfection of SUR1[R1419H] with WT Kir6.2 channel subunits yields little or none of the mature glycosylated form (not statistically different from SUR1 alone; Figure 3C), consistent with a severe trafficking defect of this channel mutant. We also considered the possibility that the SUR1[R1419H] mutation could increase channel activity and partly counteract the trafficking defects, this might account for the relatively mild phenotype of the patient and her father if small numbers of channels containing the SUR1[R1419H] subunit could reach the cell surface. However, patch clamp recordings showed no significant change in nucleotide sensitivity of K_ATP channels containing the SUR1[R1419H] mutant (Supplementary Figure 2). Overall, the weak Rb⁺ efflux and lack of protein maturation are consistent with trafficking defects that are apparent in many recessively inherited SUR1-mediated hyperinsulinemias (22–24). These data indicate that virtually no functional K_ATP channels are present in the plasma membrane of β-cells of the patient or her father.

Figure 3

K_ATP channels containing SUR1[R1419H] exhibit loss-of-function due to poor trafficking to the cell surface. (A) Rb⁺ efflux assays were performed in CosM6 cells transfected with the indicated plasmids in conditions of metabolic inhibition. K_ATP channels comprising SUR1[R1419H] to recapitulate the homozygous condition exhibit no Rb⁺ efflux above untransfected cells. (B) Cell lysates from CosM6 cells transfected with indicated combinations of Kir6.2, SUR1, and SUR1[R1419H] were separated on SDS-PAGE and probed with an anti-SUR1 antibody (NeuroMab). (C) Spot densitometry was used to determine the relative abundance of mature vs. immature forms of SUR1 in various experimental conditions (n=5 matched experiments). No statistical significance in the mature:immature ratio was observed between cells transfected with SUR1 alone and Kir6.2+SUR1[R1419H].

Discussion

Compared to most reports of homozygous SUR1 mutations, our patient had late onset of symptoms and she exhibited post-prandial hyperglycemia, as well as fasting and rebound hypoglycemia already at presentation. Previous reports have suggested that the R1419H mutation causes a trafficking defect of the K_ATP complex (16), although the only affected patient yet reported was a G70E/R1419H compound heterozygote. That patient had a severe presentation at birth, requiring subtotal pancreatectomy. These authors concluded that the R1419H mutation was consistent with a “channel absent” molecular subclass common to recessive forms of HI (16, 23). Our patient and her father are the first humans to be described with this mutation in a homozygous state, and our in vitro studies confirmed the trafficking defect caused by the R1419H mutation in transfected CosM6 cells.

Mutations that prevent cell surface maturation of K_ATP channels typically lead to a very severe hyperinsulinemic phenotype in homozygotes. The relatively late and unusual clinical presentations of our patient and her father diverge significantly from this prediction. We uncovered one report of extreme phenotypic variability in a family with a homozygous ABCC8 mutation (10), although it should be noted that this report involves a splicing site with unclear consequences on the generation of functional channels. In contrast, the R1419H is a coding mutation that appears to completely suppress expression of functional channels.

We were led to our top candidate genes by the clinical microarray that identified loss-of-heterozygosity (LOH) in multiple chromosomal regions, including around ABCC8 and KCNJ11. We began with a hypothesis that this was an autosomal recessive disease, based on parental consanguinity. We then used microarray analysis as an unbiased tool to create a list of candidate genes. We selected ABCC8 and KCNJ11 as plausible candidate genes based on the fact that there was LOH in the region around these two genes, and on the fact that we suspected an insulin secretion defect on clinical grounds.

Other glucose transporters and other “diabetes genes” for which the patient had LOH included KLF11, SLC2A1, PCSK1, and TCF7L2 (Supplementary Table 2). Identification and validation of potential modifier loci was deemed unfeasible at this stage, because of the small number of affected individuals with the same ABCC8 genotype available for study.

From a phenotypic perspective, the patient exhibited both a poor post-prandial insulin response and fasting hyperinsulinism, illustrating significant dysregulation of insulin secretion. Unfortunately, information on the father’s phenotype over the years was not available, however, the history of unexplained seizures in infancy, and later diabetes in adulthood, can hint to his daughter’s future phenotype. Information regarding insulin dysregulation in patients with recessive forms of SUR1 HI is sparse, mainly because most undergo subtotal pancreatectomy.

Co-existence of hypoglycemias and hyperglycemias has been reported only in long-term follow-up of non-pancreatectomized patients with diffuse SUR1 HI (11). In addition, Grimberg et al. (12) demonstrated the dysregulation of insulin secretion in patients with diffuse SUR1 HI. Lastly, the phenotype of SUR1^–/– knockout mice (25) is surprisingly similar to our patient, as the mice do not have severe hypoglycemia and demonstrate a loss of first-phase and attenuated second-phase glucose-stimulated insulin secretion. Overall, the presence of K_ATP channel-independent pathways (26–28) may enable these patients and animal models to exhibit some sub-optimal glucose tolerance, although it remains unclear what factors underlie the relatively weak phenotype apparent in our patients. Nevertheless, our findings highlight that the inheritance of homozygous loss-of-function mutations of SUR1 does not necessarily translate into an early onset severe hyperinsulinemia. At some level, the fact that this family’s phenotype does not match previously reported phenotypes is surprising, although perhaps it should not be, given the fact that broadening of genotype-phenotype correlations is a frequent outcome for families enrolled in the newer consortia-based genomic sequencing projects (29).

In conclusion, this the first description of a homozygous p.R1419H mutation of the ABCC8 gene. We were led to our candidate gene by looking at relevant regions of homozygosity on microarray. This case is particularly interesting because it diverges from the predicted severe phenotype that typically results from this “channel absent” subclass.