KCNJ11 readthrough variant in a patient with congenital hyperinsulinism

Abstract

KCNJ11 is one of the major causative genes for congenital hyperinsulinism (CHI) characterized by neonatal and infantile hypoglycemia. Although one readthrough KCNJ11 variant has been identified in a patient with CHI, the pathogenicity of the substitution has yet to be confirmed. Here, we identified two heterozygous nucleotide substitutions separated by one nucleotide (c.[1170C>T;1172G>T], alternative description, c.1170_1172delinsTTT) in one allele of KCNJ11 in a neonate with CHI and his father with mild CHI-compatible features. The c.1170C>T and c.1172G>T variants were assumed to be silent p.(Ser390Ser) and readthrough p.(Ter391LeuextTer93) substitutions, respectively. These results suggest that a mutant KCNJ11 protein containing 93 additional amino acids at the C-terminus is likely to exert dominant-negative effects on the wildtype protein and that monoallelic KCNJ11 readthrough variants constitute a rare etiology of CHI.

Highlights

● We report a family with congenital hyperinsulinism with two KCNJ11 substitutions..

● Mutant Kir6.2 with 93 extra C-terminal amino acids likely to be dominant-negative.

● Monoallelic KCNJ11 readthrough variants can cause congenital hyperinsulinism.

Introduction

Congenital hyperinsulinism (CHI) is a rare endocrinopathy characterized by neonatal and infantile hypoglycemia (1, 2). CHI is commonly caused by loss-of-function variants in KCNJ11, which encodes Kir6.2, and ABCC8, which encodes sulfonylurea receptor 1 (SUR1). Kir6.2 and SUR1 jointly form the ATP-sensitive potassium (K_ATP) channel in pancreatic β-cells (2, 3). The loss of K_ATP channel activity leads to persistent membrane depolarization and inappropriate insulin release (3). The Human Gene Mutation Database (HGMD, https://www.hgmd.cf.ac.uk/ac/index.php) includes 156 KCNJ11 variants associated with autosomal dominant or recessive CHI. It is believed that recessive KCNJ11 variants disrupt the expression or membrane trafficking of the protein, while dominant variants encode mutant proteins with no or impaired channel-gating activity (4). While a heterozygous readthrough substitution (c.1171T>C, p.Ter391ArgextTer93) of KCNJ11 has been identified in one patient with CHI (5), the pathogenicity of such variants remains to be determined.

Case Report

We report a boy with CHI who carried a hitherto unreported readthrough variant of KCNJ11. He was born at 40 wk of gestation with a normal body weight [3,384 g, +0.53 standard deviation (SD)] and length (52.2 cm, +1.53 SD). He was delivered through an emergency cesarean section due to fetal tachycardia and maternal fever. He showed hypoglycemia shortly after birth and underwent continuous glucose infusion. Laboratory examinations at 8 d of age demonstrated hypoglycemia (37 mg/dL) with a relatively high serum insulin level (11.3 μU/mL) and a relatively low capillary beta-hydroxybutyrate level (0.4 mmol/L). Abdominal ultrasonography showed no abnormalities. He was clinically diagnosed with CHI and treated with diazoxide from 21 to 40 d of age. Since he often showed hypoglycemia, despite being treated with a high dose of diazoxide (15 mg/kg/d), he was subjected to continuous subcutaneous octreotide injection from 36 d of age. From 43 d of age, he had euglycemia under octreotide injection and enteral formula. At eight months of age, he had a large body weight (11.5 kg, +3.28 SD) and length (79.5 cm, +3.75 SD). His developmental milestones were normal. He underwent no imaging studies to localize the pancreatic lesion. None of his family members except for the paternal great-grandfather had diabetes (Fig. 1A). Allegedly, his father was born at term with a large body weight of 4,100 g (> +2 SD) and required frequent feeding during the neonatal period. In addition, his paternal aunt weighed 4,500 g at birth (> +3 SD). No family members besides his father and paternal aunt were born heavy-for-date or required frequent feeding during the neonatal period.

Fig. 1.

Clinical and molecular findings of the patient. (A) Family pedigree. The black square indicates the proband (P), and the gray square and circle indicate his family members with mild features of congenital hyperinsulinism. The asterisks depict individuals for whom sequence analysis was performed. “Wildtype” and “Variants” indicate a normal allele and an allele containing the two KCNJ11 variants, respectively. (B) Sanger sequencing of KCNJ11. Two heterozygous variants in KCNJ11 (c.[1170C>T;1172G>T], alternative description, c.1170_1172delinsTTT) were identified in the patient and his father. The c.1170C>T and 1172G>T variants are predicted to be silent p.(Ser390Ser) and readthrough p.(Ter391LeuextTer93) substitutions, respectively. (C) The structure of mRNA and protein of KCNJ11/KCNJ11. The termination codons are indicated by arrowheads. Domains of wildtype Kir6.2 are shown as arrows. The orange and blue boxes of the mRNA depict untranslated and coding regions, respectively. The striped blue box indicates 279 nucleotides added to the coding region of the mutant mRNA. Thus, the mutant protein contains 93 additional amino acids at the C-terminus.

Genetic Analysis

Genetic tests were performed after obtaining written consent from the parents of the patient. This study was approved by the Ethics Committee of the National Center for Child Health and Development. Genomic DNA samples were obtained from the blood of the patient and his parents. Targeted next-generation sequencing of ABCC8, AIRE, FOXP3, GATA6, GCK, GLUD1, HADH, HNF1A, HNF1B, HNF4A, INS, INSR, KCNJ11, KLF11, NEUROD1, PDX1, and WFS1 was performed for the patient (Kazusa DNA Research Institute, Chiba, Japan). The results showed two heterozygous nucleotide substitutions separated by one nucleotide (c.[1170C>T;1172G>T], alternative description, c.1170_1172delinsTTT) in KCNJ11 (NM_000525.4). Sanger sequencing confirmed the presence of these variants in the patient and his father (Fig. 1B). No rare variants were identified in the other genes tested. The c.1170C>T variant was a synonymous substitution p.(Ser390Ser), while the c.1172G>T variant was predicted to cause readthrough and add 93 amino acids to the C-terminus of the protein p.(Ter391LeuextTer93) (Fig. 1C). The c.1170C>T and c.1172G>T variants (and the c.1170_1172delinsTTT variant) were not found in Japanese Multi Omics Reference Panel 54KJPN (https://jmorp.megabank.tohoku.ac.jp/), ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/), or HGMD. The allele frequencies of the c.1170C>T and c.1172G>T variants in the Genome Aggregation Database (https://gnomad.broadinstitute.org/) were 1/1,612,056 and 0, respectively. The UCSC genome browser (https://genome.ucsc.edu/) showed that c.1170C and c.1172G are highly conserved. Mutation taster (https://www.genecascade.org/MutationTaster2021/#transcript) and CADD (https://cadd.gs.washington.edu/) scored the c.1170C>T variant as benign, and the c.1172G>T variant as deleterious and benign, respectively. According to the ACMG/AMP guidelines (https://www.broadinstitute.org/videos/variant-classification-using-acmgamp-interpreting-sequence-guidelines), the two variants were assessed as variants of uncertain significance (PM2 + PP4 + BP7 and PM2 + PM4 + PP4, respectively).

Discussion

We identified two nucleotide substitutions in one KCNJ11 allele in a boy with CHI. The clinical features of the patient were comparable with those of previously reported patients with paternally inherited heterozygous KCNJ11 variants (5). His father with the same variants, was born large for gestational age and required frequent feeding during the neonatal period, which was possibly indicative of mild CHI (6). The difference in the clinical severities between our patient and his father can be explained by the broad phenotypic variation of CHI caused by dominant KCNJ11 variants (7, 8). The phenotypic variations may reflect specific genetic or epigenetic factors, such as somatic loss of the maternal chromosome 11p15.5 region, or environmental factors. However, we could not investigate these factors in our patient, because he did not undergo pancreatectomy or imaging examinations.

The c.1172G>T variant (or the c.1170_1172delinsTTT variant) was predicted to add 93 amino acids to the C-terminus of Kir6.2 and thereby, alter the structure of the C-terminus cytoplasmic domain (Fig. 1C). Most known KCNJ11 variants associated with autosomal dominant CHI resided in the cytoplasmic domains (2,3,4, 8,9,10) and are assumed to create mutant proteins that undergo normal membrane trafficking, but retain low or no channel-gating activity (4). In vitro assays have suggested that these mutant proteins exert dominant-negative effects on wildtype Kir6.2 (3, 4, 9, 10). In addition, a previously reported patient with paternally inherited KCNJ11 c.1171T>C p.(Ter391ArgextTer93) presented with persistent CHI despite the removal of a histologically confirmed focal lesion. The patient was managed with diazoxide and regular feeds after partial pancreatectomy (5). Hence, the c.1172G>T variant (or the c.1170_1172delinsTTT variant) of our patient is likely to be a further example of a dominant KCNJ11 variant. Alternatively, the mutant mRNA may be degraded through mRNA surveillance mechanisms, such as no-go decay or nonsense-mediated mRNA decay (11). However, this is unlikely, because KCNJ11 belongs to single-exon genes, which usually escape nonsense-mediated decay (12). Moreover, previous studies have suggested that amorphic variants of KCNJ11 typically cause CHI in an autosomal recessive manner (13).

In conclusion, the results of this study indicate that monoallelic readthrough variants of KCNJ11 constitute a rare etiology of CHI. It is likely that a mutant Kir6.2 containing 93 additional amino acids at the C-terminus exerts dominant-negative effects on the wildtype protein.

Conflict of interests

The authors declare no conflict of interest.