A novel pathogenic variant in the glucokinase gene found in two Japanese siblings with maturity-onset diabetes of the young 2

Abstract

Glucokinase is a glycolytic enzyme that catalyzes the phosphorylation of glucose to glucose-6-phospate in the first step of the glycolytic pathway. It also regulates the threshold for insulin secretion from pancreatic beta cells by catalyzing the phosphorylation of glucose and plays an important role as a glucose sensor. Pathogenic variants in the glucokinase gene (GCK) cause non-progressive but persistent mild fasting hyperglycemia, also recognized as maturity-onset diabetes of the young 2 (MODY2). This report presents the case of two Japanese siblings with MODY2, who were initially diagnosed with impaired glucose intolerance at 20 and 17 years of age, and later developed diabetes mellitus. They had no history of obesity, were negative for islet-related autoantibodies and their serum C-peptide level were within the normal range. Diabetic complications were not observed. Next-generation sequencing revealed a novel heterozygous variant in GCK (NM_000162.5: c.1088A>G, p.Asp363Gly) in both siblings. This variant has not been reported previously. In silico functional analyses, using SIFT and MutationTaster, suggested that the variant was damaging. To confirm the functional impact of the mutated GCK, the HiBiT-tagged p.Asp363Gly variant and the wild-type GCK were transiently expressed in HEK293T cells. The cells expressing the variant GCK exhibited 79% less bioluminescence, compared to those expressing the wild-type GCK, suggesting that the pathophysiology of the variant was a result of haploinsufficiency.

GLUCOKINASE is a glycolytic enzyme that serves as a glucose sensor in pancreatic beta cells and hepatic cells [1]. Pathogenic variants in the glucokinase gene, GCK, are associated with early-onset mild fasting hyperglycemia, also known as maturity-onset diabetes of the young 2 (MODY2) [2], which is an autosomal dominant genetic condition. The number of MODY patients in Japan was estimated to be at least 12,000 by the Japan Diabetes Society [3]. In the UK, the minimum prevalence of MODY was estimated to be 108 cases per million [4], while MODY 2 was prevalent in 0.1% of the general population [5]. Precise diagnosis and the classification of diabetes mellitus improve patient’s quality of life by providing precision medicine in those with MODY1, MODY2, and MODY3 [6], however, in the UK, more than 80% of the patients with MODY, including MODY2 were misdiagnosed and had been treated for type 1 or type 2 diabetes, or impaired glucose tolerance (IGT) [4]. We here present a case of siblings with MODY2, who were initially diagnosed with IGT, sharing a novel pathogenic variant p.Asp363Gly in GCK. In silico analysis revealed the variant had deteriorated. The genetic diagnosis was integral to the decision-making process for the treatment, which made life with diabetes less stressful. Moreover, the correct diagnosis enabled patients to understand the prognosis of this disease, which is different from type 2 diabetes, thereby improving their quality of life.

Case Report

The case involved two sisters aged 31 and 36 years. The proband (II-2 in Fig. 1) weighed 2,526 g at birth (3 percentile for 40 weeks of Japanese girls born in 2010, although the precise birth week was unknown). She was diagnosed with IGT at the age of 20 years and had been treated with diet therapy since then. She had not been pointed out for any diabetic complications. At age of 28, she was diagnosed with overt diabetes during her first pregnancy based on HbA1c level of 7.0% (52 mmol/mol), with a body mass index (BMI) 21.9 kg/m². Multiple daily insulin injection therapy was administered during the pregnancy. She gave birth spontaneously at 38 weeks of gestation. Her infant weighed 2,398 g (10 percentile for 38 weeks of Japanese boy in 2010) and did not have any complications. After the delivery, she was treated for type 2 diabetes and therapeutic regimen was switched to oral hypoglycemic agents (OHAs) that included metformin and glimepiride (Fig. 2). When she had been considering the second child, she was referred to our hospital as a suspected case of MODY. The clinical laboratory data including the longitudinal course of HbA1c and fasting plasma glucose levels of the proband were shown in Table 1 and Fig. 2. Her elder sister (Fig. 1 II-1) was diagnosed with type 2 diabetes through a urine test conducted at her junior high school, then she was started to take OHA till 17 years old. After 31 years of age, she was diagnosed with normal to mild fasting hyperglycemia at every annual health check. The latest HbA1c and fasting plasma glucose levels were 6.5% and 104 mg/dL, respectively. She had no history of obesity or diabetic complications. She agreed to undergo the genetic testing by the recommendation of her younger sister (II-2).

Fig. 1

Family pedigree. Males and females are indicated by squares and circles, respectively. The black filling indicates individuals with diabetes mellitus. Numbers are denoted in the upper right side of each individual symbol. The diagonal line indicates a deceased individual. The age at the study is shown below each symbol. The arrow indicates the proband. The proband (II-2) and her elder sister (II-1) possessed a novel GCK variant (NM_000162.5:exon9:c.1088A>G: p.Asp363Gly). Their father died at the age of 61 years due to pancreatic cancer. OHA, oral hypoglycemic agent; n/a, data not available.

Fig. 2

Clinical course of the proband 48 months before the genetic diagnosis. She had been treated with several OHAs after the delivery. Trends of both fasting glucose and HbA1c levels were typical of those observed in patients with MODY2. After the genetic diagnosis, she once stopped to take OHAs, although she has been on insulin treatment according to pre-pregnancy care.

Table 1 Clinical characteristics of the patient

<Hematology>		<Biochemistry>		<Urinalysis>
Hemoglobin	12.4 g/dL	Blood Urea Nitrogen	15.1 mg/dL	Glucose	(–)
Hematocrit	39.5%	Creatinine	0.55 mg/dL	Protein	(–)
Red blood cell	444 × 104/μL	Uric Acid	6.3 mg/dL	Occult blood	(–)
Platelets	27.1 × 104/μL	Aspartate aminotransferase	16 U/L	Ketone	(–)
White blood cell	5,600/μL	Alanine aminotransferase	9 U/L	White blood cell	(–)
		γ-glutamyl Transpeptidase	15 U/L	Albumin	2.9 mg/g·Cr
		HDL cholesterol	83 mg/dL
		LDL cholesterol	114 mg/dL
		Triglyceride	50 mg/dL
		Glucose
		-fasting	110 mg/dL
		-meal after 2 h	148 mg/dL
		serum C-peptide
		-fasting	0.54 ng/mL
		-meal after 2 h	4.01 ng/mL
		Glycated hemoglobin (HbA1c)	6.6%
		GAD-Ab	<5.0 U/mL

After the genetic counseling, written informed consent were obtained from both individuals, and genomic DNA samples were collected from the peripheral blood and subjected to genetic analyses. We analyzed 15 genes reported to cause MODY (HNF4A, GCK, HNF1A, PDX1, HNF1B, NEUROD1, KLF6, CEL, PAX4, INS, BLK, KCNJ11, ABCC8, RFX6, and APPL1) by using the Ion PGM^TM system (Thermo Fisher Scientific, Waltham, MA, USA), covering their coding exons and >10 nucleotides of the 5' and 3' intronic boundaries. The variant calling was performed using Torrent Suite^TM Software version 5 (Thermo Fisher Scientific) on the hg19 human genome assembly. The sequence data achieved an on-average 218.0-fold read depth and covered 96.4% of the targeted region with more than 10-fold read depth. Particularly in the GCK gene, 100% of the exonic and junctional regions were covered more than 20-fold read depth. We have identified the p.Asp363Gly variant in GCK (c.1088A>G, NM_000162.5) in both individuals. The variant was confirmed by Sanger sequencing. The p.Asp363Gly residue was highly conserved across species (Fig. 3) and had not been reported in the literature nor in any database such as the Human Genetic Variation Database (HGVD, https://www.hgvd.genome.med.kyoto-u.ac.jp/), gnomAD (https://gnomad.broadinstitute.org/), and dbSNP150 (http://www.ncbi.nlm.nih.gov/snp/). Online in silico analysis tools had shown that the variant was deleterious by SIFT (score 0.0, https://sift.bii.a-star.edu.sg/), MutationTaster (score 1.0, https://www.mutationtaster.org/) , and CADD (score 26.2, v1.6, http://cadd.gs.washington.edu/), although, benign by PolyPhen-2 (score 0.067, HumDiv, http://genetics.bwh.harvard.edu/pph2/).

Fig. 3

Sanger sequencing chromatogram. The upper panel shows the wild-type sequence (control). The bottom panel shows the GCK variant (arrowed) in the patients. The aspartic acid at this position was highly conserved beyond species as shown in the right panel.

To confirm the functional impact of the p.Asp363Gly GCK, we performed in vitro functional analysis. HEK293T cells were transfected with the variant or wild-type vector expressing glucokinase fused with a carboxyl-terminal HiBiT tag using Lipofectamine 2000 (Thermo Fisher Scientific). Intracellular glucokinase levels were measured via NanoLuc^® luciferase activity using Nano-Glo^® HiBiT Lytic Detection System (Promega Corporation, Madison, WI, USA), 24-h post-transfection. Luminescence was significantly reduced in the cells expressing the p.Asp363Gly GCK compared to those expressing the wild type gene (79% reduction, p < 0.001, Mann-Whitney U test; Fig. 4A). The catalytic activity of the GCK variant was measured via fluorescence spectrometry, using Amplite^TM Fluorimetoric Glucose-6-Phospate Assay Kit (AAT Bioquest, Inc, CA, USA). The luminescence activity of glucose-6-phospate in the HEK293T cells expressing the p.Asp363Gly GCK was significantly decreased compared to those expressing the wild type GCK. This was almost the same as the luminescence activity of the intrinsic glucose-6-phospate observed in the mock-transfected HEK293T cells (p < 0.01), based on the one-way analysis of variance results (Fig. 4B). The results suggested that the pathophysiology of the variant GCK was a result of haploinsufficiency. Including the functional results, the pathogenicity was fulfilled the American College of Medical Genetics and Genomics (ACMG) guidelines 2015 [7], based on the scores of: Pathogenic Strong (PS)3 + Pathogenic Moderate (PM)1 + PM2 + Pathogenic Supporting (PP)1 + PP3.

Fig. 4

(A) HiBiT luminescence assay showing the intracellular glucokinase levels in HEK293T cells. HiBiT-tagged p.Asp363Gly variant and wild-type GCK were transiently expressed in HEK293T cells. Luminescence data from the cells transfected with the mutated plasmid were compared to those transfected with the wild-type plasmid. The data represented as mean ± SD (n = 5). The Mann-Whitney U test was used for statistical comparison. (B) Intracellular glucose-6-phospate concentration in HEK293T cells. The variant (p.Asp363Gly GCK) and wild-type GCK (500 ng each) were transiently expressed in HEK293T cells and cultured in 24-well plate. Intracellular glucose-6-phospate concentrations were calculated by fluorescence spectrophotometry using cell lysate samples in transfected cells. Mock-transfected cells were used to evaluate the intrinsic glucose-6-phospate concentration in the HEK293T cells. The data presented as the mean ± SD (n = 5). One-way ANOVA was used for the statistical comparison.

Discussion

Here, we report the sibling case of MODY2 associated with a novel GCK variant (c.1088A>G, p.Asp363Gly). Both patients had been initially diagnosed as IGT, while the younger sister developed overt diabetes when she was pregnant. Continuous mild fasting hyperglycemia and mild elevation of HbA1c levels were known as the typical phenotypes for MODY2. In addition, the low birthweight of the proband might be associated with the GCK variant, which causes low insulin concentration throughout pregnancy, because the mother had normal glucose tolerance. It is a typical phenotype for the birth weight of GCK pathogenic variant positive babies born to variant negative mothers to be less. Moreover, the relatively low birth weight of the proband’s baby (III-1), who was born with 10 percentile of the birthweight, indicates that he might have inherited the variant, considering the gestational weeks [8].

The in vitro functional study confirmed that the p.Asp363Gly GCK decreased glucokinase levels, as well as its catalytic activity, significantly. Alternative amino acid substitution at the same position, p.Asp363Asn was previously reported as “Conflicting interpretations of pathogenicity” in Clinvar (https://www.ncbi.nlm.nih.gov/clinvar/variation/418228/ accessed on March 3, 2023). However, p.Asp363Asn was predicted only by using in-silico tools, and functional studies have not been published. Therefore, we measured intracellular glucokinase levels using the HiBiT technology, which is a simple method for analyzing the functional property of the p.Asp363Asn GCK, instead of classical enzymatic assays. Previous reports have shown that pathogenic GCK variants caused changes primarily in the kinetic parameters of glucokinase, which reduced its stability and enzymatic capacity, resulting in its oxidation-induced inactivation [9, 10].

In individuals with MODY2, plasma glucose level to secrete insulin is slightly elevated, and typically presents as fasting mild hyperglycemia (99–144 mg/dL), resulting in HbA1c levels ranging 5.7–7.3% for those younger than 40 years old [11]. However, despite persistent mild hyperglycemia after birth, the BMI of individuals with MODY2 was lower, and the prevalence of hypertension and hyperlipidemia was also low, compared to those who were non-diabetic. Strikingly, the prevalence of diabetic macrovascular complications was reportedly lesser in patients with MODY2 than in those who were non-diabetic. This suggested that the deterioration of GCK function was associated with an anti-atherosclerotic effect [12]. In addition, pharmacological treatment does not influence glycemic control in patients with MODY2, therefore, no treatment is required [13]. The findings of previous reports matched those in our case. The precise diagnosis of MODY2 provided a clear prognosis of diabetes and better treatment options, which were quite different from those for type 2 diabetes [6]. Based on the confirmation of the diagnosis of MODY2, we suggested the proband to stop ongoing treatment. However, she was preparing for her second pregnancy and thus, continued the insulin treatment.

Acknowledgments

This work was supported by the Manpei Suzuki Diabetes Research Foundation (N.I.) and Grant-in-Aid (C) from the Ministry of Education, Culture, Sport, Science and Technology (21K06281) to N.I.

Disclosure

Ethics statement

Approval of the research protocol: This research was approved by the Tokyo Women’s Medical University Ethics Committee (215C).

Informed consent: Written informed consent was obtained from all patients.

Approval date of registry and registration number: N/A

Animal studies: N/A

Conflicts of interest

The authors declare no conflict of interest.

References

• 1   Hattersley  AT (1998) Maturity-onset diabetes of the young: clinical heterogeneity explained by genetic heterogeneity. Diabet Med 15: 15–24.
• 2   Hattersley  AT,  Turner  RC,  Permutt  MA,  Patel  P,  Tanizawa  Y, et al. (1992) Linkage of type 2 diabetes to the glucokinase gene. Lancet 339: 1307–1310.
• 3  (2020) Announcement from The Research Committee on the Etiology, Diagnosis and Treatment of Monogenic Diabetes. Research Committee on the Etiology, Diagnosis and Treatment of Monogenic Diabetes, The Japan Diabetes Society. http://www.jds.or.jp/modules/study/index.php?content_id=33 accessed on March 3, 2023 (In Japanese).
• 4   Shields  BM,  Hicks  S,  Shepherd  MH,  Colclough  K,  Hattersley  AT, et al. (2010) Maturity-onset diabetes of the young (MODY): how many cases are we missing? Diabetologia 53: 2504–2508.
• 5   Hattersley  AT,  Patel  KA (2017) Precision diabetes: learning from monogenic diabetes. Diabetologia 60: 769–777.
• 6   Richards  S,  Aziz  N,  Bale  S,  Bick  D,  Das  S, et al. (2015) Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med 17: 405–424.
• 7   Hattersley  AT,  Beards  F,  Ballantyne  B,  Appleton  M,  Harvey  R, et al. (1998) Mutations in the glucokinase gene of the fetus result in reduced birth weight. Nat Genet 19: 268–270.
• 8   Chakera  AJ,  Spyer  G,  Vincent  N,  Ellard  S,  Hattersley  AT, et al. (2014) The 0.1% of the population with glucokinase monogenic diabetes can be recognized by clinical characteristics in pregnancy: the atlantic diabetes in pregnancy cohort. Diabetes Care 37: 1230–1236.
• 9   Sagen  JV,  Odili  S,  Bjørkhaug  L,  Zelent  D,  Buettger  C, et al. (2006) From clinicogenetic studies of maturity-onset diabetes of the young to unraveling complex mechanisms of glucokinase regulation. Diabetes 55: 1713–1722.
• 10   Arden  C,  Trainer  A,  de la Iglesia  N,  Scougall  KT,  Gloyn  AL, et al. (2007) Cell biology assessment of glucokinase mutations V62M and G72R in pancreatic cells: evidence for cellular instability of catalytic activity. Diabetes 56: 1773–1782.
• 11   Steele  AM,  Wensley  KJ,  Ellard  S,  Murphy  R,  Shepherd  M, et al. (2013) Use of HbA1c in the identification of patients with hyperglycaemia caused by a glucokinase mutation: observational case control studies. PLoS One 8: e65326.
• 12   Steele  AM,  Shields  BM,  Wensley  KJ,  Colclough  K,  Ellard  S, et al. (2014) Prevalence of vascular complications among patients with glucokinase mutations and prolonged, mild hyperglycemia. JAMA 311: 279–286.
• 13   Stride  A,  Shields  B,  Gill-Carey  O,  Chakera  AJ,  Colclough  K, et al. (2014) Cross-sectional and longitudinal studies suggest pharmacological treatment used in patients with glucokinase mutations does not alter glycaemia. Diabetologia 57: 54–56.