Precision Diagnosis of Maturity-Onset Diabetes of the Young with Next-Generation Sequencing: Findings from the MODY-IST Study in Adult Patients

Abstract

Maturity-onset diabetes of the young (MODY) is a highly heterogeneous group of monogenic and nonautoimmune diseases. Misdiagnosis of MODY is a widespread problem and about 5% of patients with type 2 diabetes mellitus and nearly 10% with type 1 diabetes mellitus may actually have MODY. Using next-generation DNA sequencing (NGS) to facilitate accurate diagnosis of MODY, this study investigated mutations in 13 MODY genes (HNF4A, GCK, HNF1A, PDX1, HNF1B, NEUROD1, KLF11, CEL, PAX4, INS, BLK, ABCC8, and KCNJ11). In addition, we comprehensively investigated the clinical phenotypic effects of the genetic variations identified. Fifty-one adult patients with suspected MODY and 64 healthy controls participated in the study. We identified 7 novel and 10 known missense mutations localized in PDX1, HNF1B, KLF11, CEL, BLK, and ABCC8 genes in 29.4% of the patient sample. Importantly, we report several mutations that were classified as “deleterious” as well as those predicted as “benign.” Notably, the ABCC8 p.R1103Q, ABCC8 p.V421I, CEL I336T, CEL p.N493H, BLK p.L503P, HNF1B p.S362P, and PDX1 p.E69A mutations were identified for the first time as causative variants for MODY. More aggressive clinical features were observed in three patients with double- and triple-heterozygosity of PDX1-KLF11 (p.E69A/p.S182R), CEL-ABCC8-KCNJ11 (p.I336, p.G157R/p.R1103Q/p.A157A), and HNF1B-KLF11 (p.S362P/p.P261L). Interestingly, the clinical effects of the BLK mutations appear to be exacerbated in the presence of obesity. In conclusion, NGS analyses of the adult patients with suspected MODY appear to be informative in a clinical context. These findings warrant further clinical diagnostic research and development in different world populations suffering from diabetes with genetic underpinnings.

Introduction

Maturity-onset diabetes of the young (MODY) accounts for 1–2% of all diabetes cases in the majority of the populations studied (Nkonge et al., 2020; Urakami, 2019). However, misdiagnosis of MODY is a widespread problem and about 5% of patients with type 2 diabetes mellitus (T2DM) and nearly 10% with type 1 diabetes mellitus (T1DM) may actually have MODY (Covantev et al., 2016).

MODY is a monogenic disorder characterized by autosomal dominant inheritance in at least two generations, beta cell dysfunction, and early-onset diabetes in adolescence or young adulthood without evidence of insulin resistance such as obesity and/or acanthosis nigricans (Juszczak and Owen, 2014; Nkonge et al., 2020; Urakami, 2019). MODY represents genetic, metabolic, and clinical heterogeneity, depending on genetic etiology (Gardner and Tai, 2012). Although with some exceptions, MODY patients typically have nonprogressive, mild, stable fasting hyperglycemia and respond to oral diabetes medications such as sulfonylurea (SU) (Urakami, 2019). Patients have measurable C-peptide levels in the presence of hyperglycemia and have normal triglycerides and/or normal or elevated high-density lipoprotein cholesterol (HDL-C) levels (Covantev et al., 2016).

MODY develops with the heterozygous alterations leading to pancreatic beta cell defects such as impaired glucose-stimulated insulin secretion in at least 14 genes (Table 1), most of which are transcription factors, hepatocyte nuclear factor 4α (HNF4A; MODY1), glucokinase (GCK; MODY2), hepatocyte nuclear factor 1α (HNF1A; MODY3), pancreatic and duodenal homeobox 1/insulin promoter factor 1 (PDX1/IPF1; MODY4), hepatocyte nuclear factor 1β (HNF1B; MODY5), neurogenic differentiation 1 (NEUROD1; MODY6), Kruppel-like factor 11 (KLF11; MODY7), carboxyl ester lipase (CEL; MODY8), paired-box-containing gene 4 (PAX4; MODY9), insulin (INS; MODY10), B lymphocyte kinase (BLK; MODY11), ATP-binding cassette transporter subfamily C member 8 (ABCC8; MODY12), potassium channel, inwardly rectifying subfamily J, member 11 (KCNJ11; MODY13), and adaptor protein, phosphotyrosine interaction, PH domain, and leucine zipper containing 1 (APPL1; MODY14) (Nkonge et al., 2020; Prudente et al., 2015).

Table 1.

The Causative Genes for Maturity-Onset Diabetes of the Young Subtypes

Gene	Chr. locus	Definitions and functions	Specific properties
HNF4A (MODY1)	20q13.12	Transcription factor that regulates the pancreatic insulin secretion, glucose transport, and metabolism by interacting with a DNA element in target genes such as serum proteins, enzymes, and tissue-specific transcription factor HNF1A (Covantev et al., 2016; Kim, 2015).	Since HNF4A contains an HNF1A binding site, HNF4A controls the HNF1A transcription with a reverse regulatory cascade (Oliveira et al., 2020). Because of the cross-regulation between HNF1A and HNF4A genes, the clinical features of the MODY1 and MODY3 are similar (Yamagata et al., 1996).
GCK (MODY2)	7p13	Acts as a glucose sensing enzyme and phosphorylates glucose (Covantev et al., 2016; Nkonge et al., 2020; Osbak et al., 2009; Stoffel and Duncan, 1997).	Alterations in the GCK due to heterozygous inactivating mutations are associated with the disorders of glucose regulation, including MODY2 (Estalella et al., 2007). MODY2 is a nonprogressive and complication-free metabolic disease probably due to low levels of free fatty acids and triglycerides, and HbA1c levels, which is just above the upper limit of the normal range (Covantev et al., 2016; Ellard et al., 2008). MODY2 is usually diagnosed incidentally during routine screening or pregnancy (Gardner and Tai, 2012; Kim, 2015).
HNF1A (MODY3)	12q24.31	Transcription factor that regulates the expression of the genes that encode INS, glucose transporter (GLUT) 1 and 2, and sodium/glucose cotransporter 2 (SGLT2) (Nkonge et al., 2020).	Because of the HNF4A-regulated gene expression, MODY1 and MODY3 have a similar clinical phenotype (Kyithar et al., 2011).
PDX1/IPF1 (MODY4)	13q12.2	Homeodomain-containing nuclear transcription factor that acts in pancreas development and insulin gene expression (Kim, 2015; Nkonge et al., 2020).	While heterozygous PDX1 mutations lead to β cell dysfunction and MODY4, homozygous mutations can lead to pancreatic agenesis. Heterozygous carriers of PDX1 mutations have a relatively mild glucose intolerance with a diminished insulin secretion (Deng et al., 2019).
HNF1B (MODY5)	17q12	Homeodomain-containing transcription factor that functions as a homodimer or heterodimer with its homologous partner HNF1A (Chandra et al., 2021; Nkonge et al., 2020). HNF1B plays role in embryonic development, pancreatic cell formation, and β cell transcription factor network (Chandra et al., 2021).	Consistent with its role in the development of the pancreas, kidney, and urogenital tract, pancreatic atrophy and exocrine dysfunction, renal cysts and dysfuctions, and urogenital abnormalities are seen in HNF1B mutation carriers and these patients are diagnosed as renal cysts and diabetes syndrome (Edghill et al., 2008).
NEUROD1 (MODY6)	2q32	Basic helix-loop-helix transcription factor that regulates endocrine pancreatic development and also activates the expression of insulin, ABCC8, GCK, and PAX6 transcription factor genes (Abreu et al., 2020; Nkonge et al., 2020; Zhang et al., 2020).	Mutations cause hyperglycemia. It is also expressed in neuronal cells and explains the neuronal complaints of the patients (Abreu et al., 2020).
KLF11 (MODY7)	2p25	Zinc-finger nuclear transcription factor that controls β cell function by acting as a glucose-inducible regulator of INS and PDX1 gene expression (Nkonge et al., 2020).	KLF11 variants cause increased repression of catalase 1 promoter, suggesting a role in free radical clearance and sensitivity to oxidative stress in β cells (Oliveira et al., 2020). Heterozygous mutations in the KLF11 gene cause β cell dysfunction and impaired insulin secretion (Nkonge et al., 2020).
CEL (MODY8)	9q34	Encodes the bile salt-dependent lipase, (BSDL), which is a major component of the pancreatic juice that is secreted by the pancreas into the digestive tract. CEL provides the digestion of milk, cholesterol, and lipid-soluble vitamins, hydrolysis of esters, and absorption of dietary lipids from the intestine (Oliveira et al., 2020).	CEL-MODY is a progressive disease and MODY8 develops gradually with the occurence of pancreatic exocrine dysfunction and pancreatic lipomatosis from infancy to the 40s (Raeder et al., 2014).
PAX4 (MODY9)	7q32	Zinc-finger nuclear transcription factor that affects the generation, differentiation, development, and survival of pancreatic β cells during fetal development and is required for the regulation of survival and proliferation of β cells in mature pancreas (Delvecchio et al., 2020; Oliveira et al., 2020). PAX4 also represses the promoter activity of insulin and glucagon (Oliveira et al., 2020).	PAX4 mutations inhibit β cell growth and proliferation and lead to impaired glucose-dependent insulin secretion and MODY9, which is associated with ketosis-prone diabetes (Oliveira et al., 2020).
INS (MODY10)	11p15.5	Pancreatic islet hormone secreted by β cells helps to decrease the increased blood glucose levels (Pugliese, 2005).	Lack of or inadequate production of insulin leads to the development of diabetes (Pugliese, 2005). Heterozygous mutations in the INS gene leading to the production of misfolded insulin with reduced biological activity and receptor binding play a role in the development of neonatal diabetes, autoantibody-negative T1DM, and also MODY due to impaired glucose homeostasis and β cell apoptosis (Molven et al., 2008).
BLK (MODY11)	8p23	Member of a nonreceptor tyrosine-kinase SRC family of proto-oncogenes that expressed in B lymphocytes and pancreatic β cells. The BLK protein enhances pancreatic β cell mass by upregulating the PDX1 and NKX6.1, and insulin biosynthesis and secretion (Borowiec et al., 2009).	BLK mutations cause defects in nuclear factor-κB, which is an enhancer of activated β cells and blocks insulin signaling, and lead to insulin resistance (Borowiec et al., 2009). Heterozygous mutations decrease the BLK expression and/or activity, insulin secretion, β cell mass, and lead to diabetes (Borowiec et al., 2009; Nkonge et al., 2020). Patients with BLK mutations have an obese phenotype compared with other MODY patients (Oliveira et al., 2020).
ABCC8 (MODY12)	11p15	Channel protein expressed in the pancreas to form the SUR1 subunit of the ATP-sensitive potassium channel. Opening and closing of this potassium channel regulate glucose-stimulated insulin secretion by coupling blood glucose levels and intracellular ATP concentration to the β cell membrane's electrical activity (Nkonge et al., 2020).	Heterozygous mutations in the KCNJ11 and ABCC8 genes cause defective glucose-stimulated insulin secretion by disrupting the activity of the potassium channel and can result in both hypo- and hyperglycemia by impairing insulin secretion (Nkonge et al., 2020). While the loss-of-function mutations in these genes cause oversecretion of insulin and result in hyperinsulinism of infancy, activating mutations cause the opposite phenotype of diabetes (Flanagan et al., 2009; Nkonge et al., 2020).
KCNJ11 (MODY13)	11p15	The ATP-sensitive potassium (K-ATP) channel, found on the β cell membrane, consists of other four subunits of the inwardly rectifying potassium channel Kir6.2, which is encoded by KCNJ11 (11p15) (Flanagan et al., 2009; Nkonge et al., 2020).	Activating mutations of KCNJ11 alter its interaction with the regulatory SUR1 component and reduce insulin secretion. These mutations cause diabetes by reducing the sensitivity of the K-ATP potassium channel to ATP, and so prevent insulin secretion (Nkonge et al., 2020).

ATP, adenosine triphosphatase; HbA1c, glycosylated hemoglobin A1c; K-ATP, potassium-adenosine triphosphatase; MODY, maturity-onset diabetes of the young; SUR1, sulfonylurea receptor 1; T1DM, type 1 diabetes mellitus.

Genes associated with subtypes MODY1–6 were defined as “causative,” whereas eight genes associated with subtypes MODY7–14 were defined as “probably causative” (Nkonge et al., 2020).

Today, the diagnosis of MODY should be more carefully differentiated because of the increasing prevalence of diabetes, the frequent family history of diabetes among individuals with diabetes, and the younger age at onset of diabetes. In addition to the 14 known responsible genes, as well as the unidentified subtype(s) of the responsible gene defined as MODY X, misdiagnosis due to clinical features overlapping with T1DM and T2DM further increases the complexity of the MODY clinic.

The definitive diagnosis of MODY can only be made by detecting mutations in the genes responsible for MODY using DNA sequencing. More comprehensive genome screening studies are needed in our country to more precisely determine the prevalence and subtypes of MODY. An accurate diagnosis will help to apply appropriate treatment and to identify the risk among family members.

The aim of this study was to identify MODY-specific gene variations in 13 genes (except APPL1) associated with MODY in adult patients with a clinical suspicion of MODY by targeted next-generation DNA sequencing (NGS), and to diagnose MODY subtypes with an integrated analysis by examining genetic, bioinformatic, clinical, and biochemical data together.

Materials and Methods

Characteristics of the study groups

Fifty-one adult patients (aged 23–64 years) with clinical suspicion of MODY and 64 adult healthy controls (aged 24–66 years) were enrolled in this study (so-called MODY-IST-Adult) from Istanbul University, Istanbul Faculty of Medicine, Department of Internal Medicine in Turkey. All patients and controls were subjected to a detailed physical examination and biochemical analysis. The patient group was chosen according to clinical parameters, including age at onset of diabetes between 16 and 40 years, family history of diabetes in a minimum of two generations suggesting an autosomal dominant inheritance in pedigree analysis, preserved endogenous insulin reserve (fasting serum C-peptide ≥0.6 ng/mL); absence of ketoacidosis, no severe microvascular complications plus negative pancreatic autoantibodies (islet cell cytoplasmic, or islet antigen 2, or glutamic acid decarboxylase antibody).

Healthy subjects without any metabolic disease and no family history of diabetes in first-degree relatives were included in the control group during a routine examination.

Written informed consent was obtained from all patients and the study was approved by the Institutional Review Board (Istanbul Faculty of Medicine Ethical Committee: 06.20.2014/922). Clinical parameters (age, sex, body mass index [BMI], medications, chronic diseases, medical interventions, etc.) were recorded using a patient follow-up form. This study was conducted in accordance with the Declaration of Helsinki.

DNA purification and library preparation

Genomic DNA was isolated from peripheral blood samples taken during routine visits using the Epicenter MasterPure™ (Lucigen, WI, USA) DNA purification kit. DNA purity and concentration measurements were performed spectrophotometrically using NanoDrop 2000c (ThermoFisher Sci., MA, USA). Extracted DNA was quantified using the Qubit™ dsDNA HS Assay Kit and the Qubit^® 3.0 Fluorometer (Invitrogen, CA, USA).

Exons and exon/intron boundaries of 13 MODY-related genes, including HNF4A, GCK, HNF1A, PDX1, HNF1B, NEUROD1, KLF11, CEL, PAX4, INS, BLK, KCNJ11, and ABCC8, were amplified through long polymerase chain reaction using Thermal Cycler (C1000; Biorad, USA). Since the APPL1 gene has not yet been associated with MODY at the time of application for project funding, it was not included into the panel. Libraries were constructed using the TruSeq^® Custom Amplicon v1.5 Exome Library Prep kit (Illumina, Inc., San Diego, CA, USA).

Exome sequencing and NGS data analysis

Paired-end sequencing of the libraries was performed on MiSeq 4000 System (Illumina, Inc.). An average sequencing depth of 221 thousand reads per sample was obtained resulting in an average of 90% of targeted bases that covered at least 10 × .

Analysis of NGS data was performed according to the pipeline developed in our previous study (Demirci et al., 2021). Briefly, all samples were analyzed according to the Genome Analysis ToolKit (GATK) best practices pipeline (Van der Auwera et al., 2013). The human genome build37 (GRCh37/hg19) was used as the reference genome (http://genomereference.org). The status, novelty, functional impact, and pathogenicity of variants were assessed, and annotated variants with an overall population frequency of <5% were filtered out and compared between study groups. The Hardy/Weinberg equilibrium (HWE) was tested for each variant, and relative risk (RR) was determined. Visualizations were performed using the R packages cBioPortalData v2.2.8 (Ramos et al., 2020) and forest plot v1.10.1 (https://cran.r-project.org/web/packages/forestplot). The MutationMapper tool of cBioPortal was used to map mutations on proteins and their domains (Gao et al., 2013).

Statistical analyses

Statistical analyses were performed with SPSS v.20.0 (IBM SPSS, Inc., Chicago, IL, USA). Quantitative variables were presented as means and standard deviations, and Student's t-test was used for comparison between two groups. Categorical variables were presented as numbers and percentages, and statistical analyses between the study groups were performed using the chi-square test. p Values <0.05 were considered statistically significant.

Results

Clinical investigation

A comparative analysis of demographic, clinical, and biochemical characteristics of the study groups (Table 2) revealed significant differences in BMI (p = 0.001), fasting blood glucose (FBG) (p = 0.001), glycosylated hemoglobin A1c (HbA1c) (p = 0.001), triglycerides (p = 0.003), HDL-C (p = 0.013), alanine aminotransferase (p = 0.021), high-sensitive C-reactive protein (p = 0.001), thyroid stimulating hormone (p = 0.027), free thyroxine (p = 0.004), white blood cells (p = 0.001), and family history of diabetes (p = 0.001). All parameters, except HDL-C, were higher in the MODY group than control group.

Table 2.

Characteristics of the Study Groups

	Control (n = 64)	MODY (n = 51)	p
Age (year)	42.0 ± 10.8	40.7 ± 11.7	0.550
Age at onset of diabetes mellitus (year)	—	28.7 ± 6.3	—
Gender (female/male) (n)	35/29	37/14	0.058
BMI (kg/m²)	25.4 ± 4.3	29.5 ± 5.5	0.001
FBG (mg/dL)	88.8 ± 9.7	164.0 ± 69.5	0.001
HbA1c (%)	5.5 ± 0.2	7.8 ± 2.0	0.001
(mmol/mol)	37.0 ± 2.7	63.1 ± 24.6
C-peptide (ng/mL)	—	2.67 ± 1.99
Triglycerides (mg/dL)	113.2 ± 68.2	177.0 ± 134.1	0.003
HDL-C (mg/dL)	52.4 ± 14.7	45.5 ± 14.0	0.013
LDL-C (mg/dL)	116.4 ± 35.8	119.0 ± 38.2	0.721
VLDL-C (mg/dL)	23.6 ± 14.6	27.9 ± 11.8	0.109
ALT (U/L)	18.9 ± 10.4	26.1 ± 18.4	0.021
AST (U/L)	17.4 ± 4.3	19.4 ± 8.1	0.166
GGT (U/L)	16.5 ± 4.0	27.0 ± 4.4	0.286
hs-CRP (mg/L)	1.55 ± 0.27	5.67 ± 0.95	0.001
TSH (mIU/L)	1.56 ± 1.09	2.21 ± 1.38	0.027
FT4 (ng/dL)	11.00 ± 6.77	16.08 ± 3.21	0.004
Uric acid (mg/dL)	4.8 ± 1.0	4.3 ± 1.7	0.127
BUN (mg/dL)	12.4 ± 3.6	12.83 ± 5.76	0.648
Creatinine (mg/dL)	2.87 ± 2.09	0.7 ± 0.2	0.325
e-GFR (mL/min./1.73 m²)	101.6 ± 13.0	107.6 ± 25.3	0.208
ACR (mg/g)	—	249.5 ± 166.5
White blood cells ( × 10³ μL)	6.22 ± 1.34	8.74 ± 2.34	0.001
Red blood cells ( × 10⁶ μL)	4.59 ± 0.34	4.53 ± 0.99	0.762
Platelets ( × 10³ μL)	240.29 ± 51.33	271.82 ± 78.68	0.098
Hemoglobin (g/dL)	13.3 ± 1.3	13.0 ± 1.8	0.557
Hematocrit (%)	40.0 ± 4.2	39.2 ± 5.3	0.579
Family history of diabetes (%)	21.6	100.0	0.001
Hypertension (%)	—	42.0

Bold p values indicate statistical significance.

BMI, body mass index; FBG, fasting blood glucose; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein-cholesterol; VLDL-C, very low-density lipoprotein cholesterol; ALT, alanine aminotransferase; AST, aspartate aminotransferase; GGT, gamma glutamyl transferase, hs-CRP, high-sensitive C-reactive protein; TSH, thyroid stimulating hormone; FT4, free thyroxine; ACR, urine albumin-to-creatinine ratio; BUN, blood urea nitrogen; e-GFR, estimated glomerular filtration rate.

The mutation loads of MODY genes

Paired-end sequencing (with an average length of 150 bp) of the exons and exon/intron boundaries (±100 bp) of 13 MODY-related genes resulted in an average sequencing depth of 221 thousand high-quality reads per sample, leading to an average of 90% of targeted bases that covered at least 10 × . Following the GATK best practice pipeline and using the human genome build37 (GRCh37/hg19) as the reference genome, we identified 135 genetic variations in 13 MODY-related genes (Figs. 1 and 2).

FIG. 1.

Mutations identified in MODY genes: ABCC8, BLK, CEL, GCK1, and HNF1A. MODY, maturity-onset diabetes of the young.

FIG. 2.

Mutations identified in MODY genes: HNF1B, HNF4A, INS, KCNJ11, KLF11, NEUROD1, PAX4, PDX1, and PDX1AS1.

Different mutation loads were observed in the MODY genes. The highest number of variations was observed in the ABCC8 gene (n = 30), followed by the HNF1A (n = 17), BLK (n = 15), and CEL (n = 15) genes. Ten variations each were detected in the HNF1B and PDX1 genes, and eight variations each in the KCNJ11 and PAX4 genes. The fewest variations were detected in the genes GCK (n = 6), HNF4A (n = 5), KLF11 (n = 5), NEUROD1 (n = 3), and PDX1-AS1 (n = 3). No variations in the gene INS were detected in the studied population.

Annotated variants in MODY genes

Among the identified variations, 41 variations were MODY-specific, that is, they were detected only in MODY patients compared with the control group. No MODY-specific mutation was detected in the HNF1A and INS genes. MODY-specific variants were analyzed in terms of their genomic features, pathogenicity, functional consequences, and literature ClinVar status (Tables 3 and 4).

Table 4.

Other Mutations Specific to Maturity-Onset Diabetes of the Young-Adult Group

Gene	Location	c.DNA	Alleles, %	Minor allele frequency	Relative risk (95%CI)^a	HWE, p	Literature reference	ClinVar status	RS no. (dbSNP)	Patients	Observed in MODY child group
Synonymous mutations
ABCC8	chr11, 17491706 G>A, exon3, p.Val118 =	c.354C>T	G: 98.0	A = 0.004536	2.27 (1.85–2.78)	0.998	Present study	NR	rs137873871	DAH35	No
ABCC8	chr11, 17491706 G>A, exon3, p.Val118 =	c.354C>T	A: 2.0	A = 0.004536	2.27 (1.85–2.78)	0.998	Present study	NR	rs137873871	DAH35	No
HNF1B	chr17, 36070616 A>G, exon5, p.S367 =	c.1101T>C	G: 96.0	C: 0.016807	2.29 (1.87–2.82)	0.998	Known (Kanca Demirci et al., 2021)	NR	—	DAH19, DAH36, DAH62	Yes
HNF1B	chr17, 36070616 A>G, exon5, p.S367 =	c.1101T>C	C: 4.0	C: 0.016807	2.29 (1.87–2.82)	0.998	Known (Kanca Demirci et al., 2021)	NR	—	DAH19, DAH36, DAH62	Yes
NEUROD1	chr2, 182543177 C>T, exon2, p.L137 =	c.411G>A	C: 98.0	T = 0.000016	2.27 (1.85–2.78)	0.998	Present study	NR	rs910919490	DAH11	No
NEUROD1	chr2, 182543177 C>T, exon2, p.L137 =	c.411G>A	T: 2.0	T = 0.000016	2.27 (1.85–2.78)	0.998	Present study	NR	rs910919490	DAH11	No
KCNJ11	chr11, 17409078C>T, exon1, p.A187 =	c.561G>A	C: 98.0	T = 0.000040	2.27 (1.85–2.78)	0.998	Present study	NR	rs1260020267	DAH44	No
KCNJ11	chr11, 17409078C>T, exon1, p.A187 =	c.561G>A	T: 2.0	T = 0.000040	2.27 (1.85–2.78)	0.998	Present study	NR	rs1260020267	DAH44	No
Intronic variants
HNF4A	chr20, 43035996 T>A, int2	c.225–25T>A	T: 98.0	A = 0.000039	2.27 (1.85–2.78)	0.998	Known (Bennett et al., 2015)	NR	rs200071662	DAH42	No
HNF4A	chr20, 43035996 T>A, int2	c.225–25T>A	A: 2.0	A = 0.000039	2.27 (1.85–2.78)	0.998	Known (Bennett et al., 2015)	NR	rs200071662	DAH42	No
HNF4A	chr20, 43043093 G>A, int4	c.493–54G>A	G: 96.0	A: 0.016807	2.29 (1.87–2.82)	0.985	Present study	NR	—	DAH44; DAH50	Yes
HNF4A	chr20, 43043093 G>A, int4	c.493–54G>A	A: 4.0	A: 0.016807	2.29 (1.87–2.82)	0.985	Present study	NR	—	DAH44; DAH50	Yes
GCK	chr7, 44186292 G>A, int7	c.867–75C>T	G: 98.0	A: 0.008403	2.27 (1.85–2.78)	0.998	Present study	NR	—	DAH62	No
GCK	chr7, 44186292 G>A, int7	c.867–75C>T	A: 2.0	A: 0.008403	2.27 (1.85–2.78)	0.998	Present study	NR	—	DAH62	No
GCK	chr7, 44198754 G>T, int1	c.-35C>A	G: 98.0	T = 0.000040	2.27 (1.85–2.78)	0.998	Present study	NR	rs367774728	DAH60	No
GCK	chr7, 44198754 G>T, int1	c.-35C>A	T: 2.0	T = 0.000040	2.27 (1.85–2.78)	0.998	Present study	NR	rs367774728	DAH60	No
HNF1B	chr17, 36061279 C>T, int6	c.1340–97G>A	C: 96.0	T: 0.016807	2.29 (1.87–2.82)	0.993	Present study	NR	rs77297671	DAH17; DAH23	No
HNF1B	chr17, 36061279 C>T, int6	c.1340–97G>A	T: 4.0	T: 0.016807	2.29 (1.87–2.82)	0.993	Present study	NR	rs77297671	DAH17; DAH23	No
PAX4	chr7, 127251332 G>C, int8	c.890–72C>G	G: 98.0	C: 0.008403	2.27 (1.85–2.78)	0.998	Present study	NR	rs149291099	DAH41	No
PAX4	chr7, 127251332 G>C, int8	c.890–72C>G	C: 2.0	C: 0.008403	2.27 (1.85–2.78)	0.998	Present study	NR	rs149291099	DAH41	No
BLK	chr8, 11414393 C>G, int9,	c.999C>A	C: 98.0	G: 0.008403	2.27 (1.85–2.78)	0.993	Present study	NR	rs749337440	DAH10	Yes
BLK	chr8, 11414393 C>G, int9,	c.999C>A	G: 2.0	G: 0.008403	2.27 (1.85–2.78)	0.993	Present study	NR	rs749337440	DAH10	Yes
BLK	chr8, 11412720 A>G, int7	c.407–121A>G	A: 98.0	G: 0.008403	2.27 (1.85–2.78)	0.998	Present study	NR	rs529387633	DAH35	No
BLK	chr8, 11412720 A>G, int7	c.407–121A>G	G: 2.0	G: 0.008403	2.27 (1.85–2.78)	0.998	Present study	NR	rs529387633	DAH35	No
BLK	chr8, 11418805 C>T, int9	c.1030–6C>T	C: 98.0	T: 0.008403	2.27 (1.85–2.78)	0.998	Present study	NR	rs1318168382	DAH32	No
BLK	chr8, 11418805 C>T, int9	c.1030–6C>T	T: 2.0	T: 0.008403	2.27 (1.85–2.78)	0.998	Present study	NR	rs1318168382	DAH32	No
BLK	chr8, 11418985 G>A, int11	c.1180 + 24G>C	G: 98.0	A: 0.008403	2.27 (1.85–2.78)	0.998	Present study	NR	rs778909298	DAH22	No
BLK	chr8, 11418985 G>A, int11	c.1180 + 24G>C	A: 2.0	A: 0.008403	2.27 (1.85–2.78)	0.998	Present study	NR	rs778909298	DAH22	No
ABCC8	chr11, 17416681 T>A, int36	c.4411 + 38A>T	T: 98.0	A: 0.008403	2.27 (1.85–2.78)	0.998	Present study	NR	—	DAH23	No
ABCC8	chr11, 17416681 T>A, int36	c.4411 + 38A>T	A: 2.0	A: 0.008403	2.27 (1.85–2.78)	0.998	Present study	NR	—	DAH23	No
ABCC8	chr11, 17418689 A>G, int32	c.3988 + 51T>C	A: 98.0	G: 0.008403	2.27 (1.85–2.78)	0.998	Present study	NR	—	DAH54	No
ABCC8	chr11, 17418689 A>G, int32	c.3988 + 51T>C	G: 2.0	G: 0.008403	2.27 (1.85–2.78)	0.998	Present study	NR	—	DAH54	No
ABCC8	chr11, 17449587 A>G, int14	c.2041–98T>C	A: 98.0	G: 0.008403	2.27 (1.85–2.78)	0.998	Present study	NR	rs977603181	DAH28	No
ABCC8	chr11, 17449587 A>G, int14	c.2041–98T>C	G: 2.0	G: 0.008403	2.27 (1.85–2.78)	0.998	Present study	NR	rs977603181	DAH28	No
ABCC8	chr11, 17449784 C>T, int14	c.2040 + 52G>A	C: 98.0	T: 0.008403	2.27 (1.85–2.78)	0.998	Present study	NR	rs1014199318	DAH43	No
ABCC8	chr11, 17449784 C>T, int14	c.2040 + 52G>A	T: 2.0	T: 0.008403	2.27 (1.85–2.78)	0.998	Present study	NR	rs1014199318	DAH43	No
ABCC8	chr11, 17453710 C>A, int11	c.1671 + 41G>A	C: 96.0	A: 0.016807	2.29 (1.87–2.82)	0.993	Present study	NR	—	DAH44; DAH51	No
ABCC8	chr11, 17453710 C>A, int11	c.1671 + 41G>A	A: 4.0	A: 0.016807	2.29 (1.87–2.82)	0.993	Present study	NR	—	DAH44; DAH51	No
ABCC8	chr11, 17464490 G>C, int9	c.1468–61C>G	G: 98.0	C: 0.008403	2.27 (1.85–2.78)	0.998	Present study	NR	rs928776478	DAH46	No
ABCC8	chr11, 17464490 G>C, int9	c.1468–61C>G	C: 2.0	C: 0.008403	2.27 (1.85–2.78)	0.998	Present study	NR	rs928776478	DAH46	No
ABCC8	chr11, 17482272 C>T, int5	c.823–49G>A	C: 96.0	T: 0.016807	2.29 (1.87–2.82)	0.972	Present study	NR	rs146679656	DAH28; DAH44	Yes
ABCC8	chr11, 17482272 C>T, int5	c.823–49G>A	T: 4.0	T: 0.016807	2.29 (1.87–2.82)	0.972	Present study	NR	rs146679656	DAH28; DAH44	Yes
ABCC8	chr11, 17485246 A>G, int3	c.413–95T>C	A: 98.0	G: 0.008403	2.27 (1.85–2.78)	0.998	Present study	NR	—	DAH45	No
ABCC8	chr11, 17485246 A>G, int3	c.413–95T>C	G: 2.0	G: 0.008403	2.27 (1.85–2.78)	0.998	Present study	NR	—	DAH45	No
ABCC8	chr11, 17491831 C>A, int2	c.291–62G>T	C: 98.0	A: 0.008403	2.27 (1.85–2.78)	0.993	Present study	NR	rs11024298	DAH28	Yes
ABCC8	chr11, 17491831 C>A, int2	c.291–62G>T	A: 2.0	A: 0.008403	2.27 (1.85–2.78)	0.993	Present study	NR	rs11024298	DAH28	Yes
UTR variants
HNF4A	chr20, 43058347 C>T, 3′UTR	c.^*42C>T	C: 98.0	T: 0.008403	2.27 (1.85–2.78)	0.998	Present study	NR	rs965250768	DAH46	No
HNF4A	chr20, 43058347 C>T, 3′UTR	c.^*42C>T	T: 2.0	T: 0.008403	2.27 (1.85–2.78)	0.998	Present study	NR	rs965250768	DAH46	No

Table 3.

Missense Mutations Specific to Maturity-Onset Diabetes of the Young-Adult Group

Gene	Location	Observed in MODY child group	c.DNA	a.a change	Pathogenity prediction (SIFT/PolyPhen-2)	Literature status	ClinVar status	RS no. (dbSNP)	Relative risk (95% CI)	HWE, p	Alleles, %	Minor allele frequency (this study)	Patients
ABCC8	chr11,17428190 C>T, exon26	No	c.3308G>A	p.R1103Q	Tolerated/benign	Present study	NR	rs765913448	2.27 (1.85–2.78)	0.998	C: 98.0	T: 0.008403	DAH44
ABCC8	chr11,17428190 C>T, exon26	No	c.3308G>A	p.R1103Q	Tolerated/benign	Present study	NR	rs765913448	2.27 (1.85–2.78)	0.998	T: 2.0	T: 0.008403	DAH44
ABCC8	chr11,17470134 C>T, exon8	No	c.1261G>A	p.V421I	Damaging/possibly damaging	Present study	NR	rs1591846104	2.27 (1.85–2.78)	0.998	C: 98.0	T: 0.008403	DAH12
ABCC8	chr11,17470134 C>T, exon8	No	c.1261G>A	p.V421I	Damaging/possibly damaging	Present study	NR	rs1591846104	2.27 (1.85–2.78)	0.998	T: 2.0	T: 0.008403	DAH12
BLK	chr8, 11421607 T>C, exon 13	No	c.1508T>C	p.L503P	Tolerated/benign	Present study	NR	—	2.27 (1.85–2.78)	0.998	T: 98.0	C: 0.008403	DAH28
BLK	chr8, 11421607 T>C, exon 13	No	c.1508T>C	p.L503P	Tolerated/benign	Present study	NR	—	2.27 (1.85–2.78)	0.998	C: 2.0	C: 0.008403	DAH28
BLK	chr8, 11405576 G>A, exon 4	No	c.211G>A	p.A71T	Damaging/pathogenic	Known (Anik et al., 2015; Borowiec et al., 2009)	MODY11	rs55758736	2.27 (1.85–2.78)	0.998	G: 98.0	A: 0.008403	DAH22
BLK	chr8, 11405576 G>A, exon 4	No	c.211G>A	p.A71T	Damaging/pathogenic	Known (Anik et al., 2015; Borowiec et al., 2009)	MODY11	rs55758736	2.27 (1.85–2.78)	0.998	A: 2.0	A: 0.008403	DAH22
BLK	chr8, 11414273 C>T, exon9	No	c.879C>G	p.H293Q	—/—	Known	MODY11	rs142623841	2.27 (1.85–2.78)	0.998	C: 98.0	T: 0.008403	DAH30
BLK	chr8, 11414273 C>T, exon9	No	c.879C>G	p.H293Q	—/—	Known	MODY11	rs142623841	2.27 (1.85–2.78)	0.998	T: 2.0	T: 0.008403	DAH30
BLK	chr8, 11421448 G>A, exon13	No	c.1349G>A	p.R450H	Tolerated/possibly damaging	Known	MODY11	rs202162624	2.27 (1.85–2.78)	0.998	G: 98.0	A: 0.008403	DAH46
BLK	chr8, 11421448 G>A, exon13	No	c.1349G>A	p.R450H	Tolerated/possibly damaging	Known	MODY11	rs202162624	2.27 (1.85–2.78)	0.998	A: 2.0	A: 0.008403	DAH46
CEL	chr9,135940546 G>A, exon4	No	c.469G>A	p.G157R	Damaging/damaging	Known (Aykut et al., 2018)	NR	rs370344006	2.27 (1.85–2.78)	0.998	G: 98.0	A: 0.008403	DAH44
CEL	chr9,135940546 G>A, exon4	No	c.469G>A	p.G157R	Damaging/damaging	Known (Aykut et al., 2018)	NR	rs370344006	2.27 (1.85–2.78)	0.998	A: 2.0	A: 0.008403	DAH44
CEL	chr9,135944161 T>C, exon8	No	c.1007T>C	p.I336T	Damaging/damaging	Present study	NR	rs368511384	2.27 (1.85–2.78)	0.998	T: 98.0	C: 0.008403	DAH44
CEL	chr9,135944161 T>C, exon8	No	c.1007T>C	p.I336T	Damaging/damaging	Present study	NR	rs368511384	2.27 (1.85–2.78)	0.998	C: 2.0	C: 0.008403	DAH44
CEL	chr9,135946029 A>C, exon10	No	c.1477A>C	p.N493H	Damaging/damaging	Present study	NR	rs748873720	2.27 (1.85–2.78)	0.998	A: 98.0	C: 0.008403	DAH23
CEL	chr9,135946029 A>C, exon10	No	c.1477A>C	p.N493H	Damaging/damaging	Present study	NR	rs748873720	2.27 (1.85–2.78)	0.998	C: 2.0	C: 0.008403	DAH23
CEL	chr9,135946015 T>C, exon 10	No	c.1463T>C	p.I488T	Damaging	Known (Johansson 2014; Sarmadi et al., 2020)	MODY8	rs77696629	2.27 (1.85–2.78)	0.998	T: 98.0	C: 0.008403	DAH45
CEL	chr9,135946015 T>C, exon 10	No	c.1463T>C	p.I488T	Damaging	Known (Johansson 2014; Sarmadi et al., 2020)	MODY8	rs77696629	2.27 (1.85–2.78)	0.998	C: 2.0	C: 0.008403	DAH45
HNF1B	chr17,36070633 A>G, exon5	No	c.1084T>C	p.S362P	—/damaging	Present study	NR	—	2.27 (1.85–2.78)	0.998	A: 98.0	G: 0.008403	DAH62
HNF1B	chr17,36070633 A>G, exon5	No	c.1084T>C	p.S362P	—/damaging	Present study	NR	—	2.27 (1.85–2.78)	0.998	G: 2.0	G: 0.008403	DAH62
HNF1B	chr17,36091625 G>C, exon4	No	c.1006C>G	p.H336D	—/damaging	Known (Alvelos et al., 2015; Karges et al., 2007; Komazec et al., 2019; Ovysyannikova 2020; Thomas et al., 2008	NR	rs138986885	2.27 (1.85–2.78)	0.998	G: 98.0	C: 0.008403	DAH24
HNF1B	chr17,36091625 G>C, exon4	No	c.1006C>G	p.H336D	—/damaging		NR	rs138986885	2.27 (1.85–2.78)	0.998	C: 2.0	C: 0.008403	DAH24
KLF11	chr2, 10188010 C>A, exon3	No	c.546C>A	p.S182R	Tolerated/possibly damaging	Known	MODY7	rs142266428	2.27 (1.85–2.78)	0.998	C: 98.0	A: 0.008403	DAH33
KLF11	chr2, 10188010 C>A, exon3	No	c.546C>A	p.S182R	Tolerated/possibly damaging	Known	MODY7	rs142266428	2.27 (1.85–2.78)	0.998	A: 2.0	A: 0.008403	DAH33
KLF11	chr2, 10188246 C>T, exon3	No	c.782C>T	p.P261L	Tolerated/benign	Known	MODY7	rs148123124	2.27 (1.85–2.78)	0.998	C: 98.0	T: 0.008403	DAH62
KLF11	chr2, 10188246 C>T, exon3	No	c.782C>T	p.P261L	Tolerated/benign	Known	MODY7	rs148123124	2.27 (1.85–2.78)	0.998	T: 2.0	T: 0.008403	DAH62
KLF11	chr2, 10192477 G>A, exon4	Yes	c.1382G>A	p.R461Q	Damaging/damaging	Known (Kanca Demirci et al., 2021)	MODY7	rs199770737	2.27 (1.85–2.78)	0.998	G: 98.0	A: 0.008403	DAH60
KLF11	chr2, 10192477 G>A, exon4	Yes	c.1382G>A	p.R461Q	Damaging/damaging	Known (Kanca Demirci et al., 2021)	MODY7	rs199770737	2.27 (1.85–2.78)	0.998	A: 2.0	A: 0.008403	DAH60
PDX1	chr13,28494372 C>A, exon1	No	c.97C>A	p.P33T	Damaging/damaging	Known (Gragnoli et al., 2005; Hansen et al., 2000; Winter 2003)	MODY4	rs192902098	2.27 (1.85–2.78)	0.998	C: 98.0	A: 0.008403	DAH63
PDX1	chr13,28494372 C>A, exon1	No	c.97C>A	p.P33T	Damaging/damaging		MODY4	rs192902098	2.27 (1.85–2.78)	0.998	A: 2.0	A: 0.008403	DAH63
PDX1	chr13,28494481 A>C, exon1	No	c.206A>C	p.E69A	Damaging/damaging	Present study	NR	—	2.27 (1.85–2.78)	0.998	A: 98.0	C: 0.008403	DAH33
PDX1	chr13,28494481 A>C, exon1	No	c.206A>C	p.E69A	Damaging/damaging	Present study	NR	—	2.27 (1.85–2.78)	0.998	C: 2.0	C: 0.008403	DAH33

CI, confidence interval; HWE, Hardy/Weinberg equilibrium; NR, not reported.

MODY-specific missense mutations were identified in six genes, namely ABCC8 (n = 2), BLK (n = 4), CEL (n = 4), HNF1B (n = 2), KLF11 (n = 3), and PDX1 (n = 2) (Table 3). Among these, seven novel mutations, namely BLK p.L503P, ABCC8 p.R1103Q, ABCC8 p.V421I, CEL I336T, CEL p.N493H, HNF1B p.S362P, and PDX1 p.E69A, were identified for the first time in this study, whereas the remaining nine missense mutations were previously associated with MODY subtypes in ClinVar. The p.G157R and I488T mutations in CEL, the p.H336D mutation in HNF1B, the p.R461Q mutation in KLF11, and the p.P33T mutation in PDX1 have been described in the literature as MODY-associated pathogenic variants.

Moreover, we identified 4 synonymous variants in ABCC8, HNF1B, NEUROD1, and KCJN11; 19 intronic variants in ABCC8 (n = 9), BLK (n = 4), GCK (n = 2), HNF1B (n = 1), HNF4A (n = 2), and PAX4 (n = 1) in addition to a 3′UTR variant in HNF4A (Table 4). The synonymous mutations (i.e., ABCC8 c.334 C>T, NeuroD1 c.411 G>A, and KCNJ11 c. 561 G>A) were also identified for the first time in this study.

Double heterozygosity of PDX1-KLF11 (p.E69A/p.S182R), CEL-ABCC8-KCNJ11 (p.I336, p.G157R/p.R1103Q/A157A), and HNF1B-KLF11 (p.S362P/p.P261L) was observed in three patients (Table 5).

Table 5.

Clinical and Genetic Characteristics of Maturity-Onset Diabetes of the Young Subtypes in the Analyzed 13 Maturity-Onset Diabetes of the Young Genes

	MODY subtypes
	PDX1 (n = 1)	PDX1/KLF11 (n = 1)	HNF1B (n = 1)	HNF1B/KLF11 (n = 1)	NEUROD1 (n = 1)	KLF11 (n = 1)	CEL (n = 2)		BLK (n = 4)				ABCC8 (n = 2)		CEL/ABCC8/KCNJ11 (n = 1)
Patient code	DAH63	DAH33	DAH24	DAH62	DAH11	DAH60	DAH23	DAH45	DAH30	DAH46	DAH28	DAH22	DAH12	DAH35	DAH44
Mutation	P33T	E69A/S182R	H336D	S362P/P261L	L137 =	R461Q	N493H	I488T	H293Q	R450H	L503P	A71T	V421I	V118 =	I336T, G157R/R1103Q/A157A
Double heterozygosity (yes/no)	No	Yes	No	Yes	No	No	No	No	No	No	No	No	No	No	Yes
Sex	Female	Female	Female	Male	Female	Male	Male	Female	Male	Female	Male	Female	Female	Female	Female
Age (years)	26	29	35	41	31	43	39	31	44	55	24	24	36	34	58
Age at diagnosis of diabetes (years)	26	23	25	28	25	25	29	30	37	18	23	22	32	33	33
BMI (kg/m²)	34.8	33.2	23.1 (previous severe obesity)	26.7	26.4	25.6	26.3	32.0	23,7	35.1	19.1	32.5	29.3	30.8	36.6
FBG (mg/dL)	170	153	135	135	103	222	250	101	130	243	121	118	111	147	238
HbA1c (%)	7.2	7.2	6.6	7.6	6.5	9.9	8.1	5.4	6.4	9.4	5.4	—	5.9	6.6	8.6
C-peptide (ng/mL)	2.69	2.56	2.5	3.36	1.25	1.82	2.45	1.97	1.95	2.26	1.69	3.1	3.63	1.99	2.71
Magnesium	—	0.75	0.68	—	0.67	—	—	—	0.9	0.6	—	—	—	—	0.78
ALT (U/L)	10	36	19	49	26	21	24	8	17	22	17	13	21	15	26
AST (U/L)	11	16	16	—	12	20	—	—	16	27	—	18	14	12	18
GGT (U/L)	9	5	11	41	9	11	35	—	19	14	—	—	—	—	27
hs-CRP (mg/L)	2.95	0.67	1.42	2.32	0.56	5.7	0.76	0.60	0.5	4.6	0.48	3.7	4.3	6.6	7.35
ACR (mg/g)	—	—	35	49.5	12	8193	285		2.6	73	—	—	3	1.1	84.6
BUN (mg/dL)	9	9	17	11	7	21	13		13	23	—	9	4	8	21
Creatinine (mg/dL)	0.3	0.7	0.8	0.7	0.8	1.3	0.76	0.6	0.9	1.0	0,78	0.5	0.4	0.5	0.8
e-GFR (mL/min/1.73 m²)	158	117	96	117	98	66	115	2.4	103	63	128	135	134	151	81
Current treatment	Insulin (0.50 IU/kg/day)	Insulin (0.44 IU/kg/day), DPP-4i, metformin	Insulin (1–6 months), now: DPP-4i, metformin	SU, metformin	Metformin	Insulin (6 months+) (0.74 IU/kg/day)	Insulin (6 months+) (0.35 IU/kg/day)	Insulin	SU	Insulin (6 months+) (0.64 IU/kg/day)	No therapy	Insulin (1–6 months) (0.53 IU/kg/day)	Insulin (6 months+) (0.65 IU/kg/day)	Insulin (1–6 months) (0.12 IU/kg/day)	Insulin (6 months+) (0.52 IU/kg/day), DPP-4i, metformin
Hyperlipidemia (yes/no)	Yes	Yes	No	Yes	No	Yes	Yes	Yes	Yes	Yes	No	Yes	No	No	Yes
Hypertension (yes/no)	No	No	No	Yes	No	Yes	No	No	No	Yes	No	No	Yes	No	Yes
Nephropathy (yes/no)	No	No	Yes	Yes	No	Yes	Yes	No	No	Yes	No	No	No	No	Yes
Neuropathy (yes/no)	No	No	Yes	No	No	Yes	Yes	No	Yes	Yes	No	No	No	No	Yes
Retinopathy (yes/no)	No	No	—	No	No	Yes	Yes	No	No	Yes	No	No	No	No	Yes
Thyroid function	Normal	Normal	Normal	Normal	Hypothyroidism	Normal	Normal	Normal	Normal	Normal	Normal	Normal	Normal	Normal	Hyperthyroidism
GDM (yes/no)	Yes	No	Yes	N/A	No	N/A	N/A	Yes	N/A	No	N/A	No	Yes	Yes	No

DPP-4i, dipeptidyl peptidase-4 inhibitor; GDM, gestational diabetes mellitus; N/A, not applicable; SU, sulfonylurea.

Considering that heterozygous MODY mutations in the transactivation, DNA-binding or dimerization domains of proteins, particularly transcription factors, lead to decreased gene expression and activity via haploinsufficiency (Ellard and Colclough, 2006), and nonsense, frameshift, and splicing mutations lead to mRNAs with premature stop codons in the channel- and enzyme-encoding genes (Colclough et al., 2013), we investigated which protein domains the mutations correspond to (Figs. 1 and 2) and whether amino acid substitution affects protein structure and function by estimating the likelihood of pathogenicity of missense mutations. The majority of the missense mutations were predicted to be deleterious or damaging, whereas four mutations, namely KLF11 p.P261L, BLK p.R450H, BLK p.L503P, and ABCC8 p.R1103Q, were classified as tolerated or benign (Table 3).

The RRs changed in the range of 2.27 (1.85–2.78) to 2.29 (1.87–2.82) for all variants, suggesting that MODY risk increased with the presence of variation. In addition, violations of HWE assumptions were observed for most MODY-specific variants (p > 0.05) (Tables 3 and 4).

Discussion

MODY comprises a heterogeneous group of monogenic disorders characterized by early-onset diabetes, pancreatic β cell dysfunction, and autosomal dominant inheritance. Our study of this large series of MODY gene mutations in 51 unrelated patients with a clinical suspicion of MODY once again underscores the substantial genetic heterogeneity of MODY. We identified 41 MODY-specific genetic variations, including 17 missense mutations, 4 synonymous mutations, 19 intronic variants, and one 3′UTR variant, in 13 MODY causative genes.

Seven novel and 10 known missense mutations localized in PDX1, HNF1B, KLF11, CEL, BLK, and ABCC8 genes were detected in 29.4% (n = 15) of MODY patients. Among them, p.E69A and p.P33T mutations in PDX1, p.S362P and p.H336D mutations in HNF1B, p.S182R and p.R461Q mutations in KLF11, p.I336T, p.N493H, p.G157R, and p.I488T mutations in CEL, p.H293Q and p.A71T mutations in BLK, and p.V421I mutation in ABCC8 were predicted to be deleterious or damaging. The missense mutations in KLF11 p.P261L, BLK p.R450H, BLK p.L503P, and ABCC8 p.R1103Q were predicted to be benign.

To the best of our knowledge, ABCC8 p.R1103Q, ABCC8 p.V421I, CEL I336T, CEL p.N493H, HNF1B p.S362P, and PDX1 p.E69A mutations were identified in MODY patients for the first time in this study. Nine of the patients were diagnosed as “MODY X” due to the absence of causative gene mutations in the analyzed MODY genes. Of the remaining patients, 4 were evaluated as T1DM, 22 as T2DM, and 1 as latent autoimmune diabetes in adults. More aggressive clinical features were observed in three patients with double heterozygosity of PDX1-KLF11 (p.E69A/p.S182R), CEL-ABCC8-KCNJ11 (p.I336T, p.G157R/p.R1103Q/p.A157A), and HNF1B-KLF11 (p.S362P/p.P261L) (Supplementary Table S1).

HNF4A mutations associated with the MODY1 subtype were reported less frequently in studies conducted in the Turkish population than in other population studies (Acar et al., 2018; Agladioglu et al., 2016; Anik et al., 2015; Karaoglan and Nacarkahya, 2021; Ozdemir et al., 2018; Ozsu et al., 2018; Yalcintepe et al., 2021).

In the present study, three HNF4A variations (two intronic and one 3′UTR) were detected. One of the intronic variants (c.225–25T>A, rs200071662) was associated with T1DM in a North American cohort (Bennett et al., 2015); however, the other novel intronic variant (c.493–54G>A) and 3′UTR variant (c.*42C>T, rs965250768) were identified in this study and associated with MODY. As in the pediatric/adolescent MODY patients of our previous study (Demirci et al., 2021), we did not find any MODY-specific missense mutation in the HNF4A gene in the adult MODY patients of this study. On the contrary, we detected the HNF4A c.350 C > T p.T117I (rs1800961) missense mutation, which was previously detected in MODY studies in Turkey (Agladioglu et al., 2016; Karaoglan and Nacarkahya, 2021). However, since we also observed the p.T117I mutation in healthy controls, we did not evaluate it as a MODY-specific mutation in this study.

Heterozygous inactivating GCK gene mutations cause MODY2, one of the most common types of MODY. While the nonsense, missense, frameshift, and splice site GCK mutations affect by changing the enzyme's affinity to glucose or its phosphorylating activity, other mutations alter the protein structure or adenosine triphosphatase (ATP) phosphorylation (Doria, 2005). The prevalence of GCK mutations in MODY patients in the Turkish population has been reported to be 25–64% (Agladioglu et al., 2016; Anik et al., 2015; Aykut et al., 2018; Haliloglu et al., 2016; Ucakturk et al., 2017).

In this study, two novel intronic GCK variations, c.-35C>A and c.867–75C>T, were identified, which are localized in intron 1 and intron 7, respectively. However, none of the MODY patients had the missense GCK mutation.

Heterozygous HNF1A gene mutations cause progressive pancreatic beta cell dysfunction and MODY3, the most common subtype of MODY in the European populations (Pearson et al., 2000; Schober et al., 2009; Urbanova et al., 2014). Similarly, many different mutations have been reported in HNF1A, which is one of the most studied genes related to MODY in the Turkish population (Agladioglu et al., 2016; Anik et al., 2015; Demirci et al., 2021; Goksen et al., 2013; Karaca et al., 2017; Karaoglan and Nacarkahya, 2021; Ozdemir et al., 2018).

In fact, many known (p.I27L and p.A98V) and novel (p.I404T, p.T544P, p.T547P, p.F543S, p.S550P, p.L546P, p.S553P, and p.T564P) missense mutations in the HNF1A gene were detected in our study group. However, they were not specific to identify MODY, since they were also observed in the healthy controls. Among these mutations, novel missense p.S553P (n = 2) and p.T564P (n = 1) mutations were detected only in healthy controls.

PDX1/IPF1 mutations are associated with MODY4, T2DM, and gestational diabetes mellitus (GDM) phenotypes (Deng et al., 2019; Gragnoli et al., 2005; Hansen et al., 2000; Mohan et al., 2018). Previous studies conducted in the Turkish population identified missense mutations in PDX1 such as p.G55D (Anik et al., 2015) and p.D76N (Agladioglu et al., 2016) in association with MODY. In the present study, 13 different PDX1 missense mutations were detected in MODY cases, 12 of which were novel (p.Y68S, p.H84P, p.D76A, p.H81P, p.H83P, p.H94P, p.D75A, p.D64A, p.P239Q, p.L73P, p.I65L, and p.E69A) and only p.P33T has been previously presented in ClinVar (Gragnoli et al., 2005; Winter, 2003).

Among these missense mutations, we identified only p.Pro33T and p.E69A, which were predicted to be deleterious by bioinformatic tools, as being specific for MODY. Both mutations are localized in the transactivation domain of the PDX1. These mutations cause downregulation of the PDX1-bound genes, including the transcription factors MNX1 and PDX1, and insulin resistance gene CES1, reducing the differentiation efficiency of pancreatic progenitors and ultimately impairing beta cell differentiation and function (Wang et al., 2019). This study was the first to identify the PDX1 p.E69A missense mutation in MODY patients. Based on the in silico prediction algorithms, SIFT, and PolyPhen-2, the variant, p.E69A (c.A206C), is considered to be pathogenic.

The female patient (DAH33) who carried the PDX1 p.E69A mutation also carried the missense mutation KLF11 p.S182R and under insulin therapy. She was overweight and suffered from polycystic ovary syndrome, hypercholesterolemia, and menstrual irregularity. Her clinical features support a double heterozygosity for MODY4/MODY7. The patient with the PDX1 P33T mutation was a 26-year-old woman (DAH63) receiving insulin therapy. The patient had high FBG, HbA1c, and serum lipid levels, and low liver aminotransferase levels. In this patient, only the PDX1 mutation was present in the genes we examined, and she had obesity and a history of GDM. The PDX1 P33T mutation was first described in a large Italian family study by Gragnoli et al. This mutation has been reported to be associated with MODY4 and GDM phenotypes (Gragnoli et al., 2005). The clinical features of our patient with the p.P33T mutation are consistent with the findings of Gragnoli.

After the first report of HNF1B mutations in the United States by Horikawa et al. (1997), more than 200 mutations were identified, including missense/nonsense, splicing, deletions, and insertions (Tao et al., 2020). In studies screening HNF1B mutations in the Turkish population, six mutations were detected in the HNF1B gene: p.G76C (Anik et al., 2015), p.R235W (Agladioglu et al., 2016), p.R165H (Ozsu et al., 2018), M495K, G464R (Arslan Ates et al., 2021), and p.S367S (Demirci et al., 2021).

In our study group, five of the detected missense mutations of HNF1B gene were novel (p.G356E, p.H368P, p.T376P, p.S7P, and p.S362P) and one of them (p.H336D) was already presented in the dsSNP database. Among these, only p.H336D and p.S362P were MODY-specific mutations. These two mutations have been previously reported in studies conducted in the United States, Serbia, and Germany (Karges et al., 2007; Komazec et al., 2019; Thomas et al., 2008). The p.H336D mutation, in which the histidine located in the activation domain of the Hnf1β protein is replaced by an aspartic acid (Weber et al., 2006), was found in only one patient (DAH24).

A 35-year-old female patient with clinical features of young-onset diabetes (10 years of age), previous severe obesity, GDM, early cataracts, neuropathy, and nephropathy was compatible with the MODY5 phenotype. The male patient with p.S362P mutation (DAH62) also carried the heterozygous KLF11 p.P261L missense mutation. The patient received antidiabetic therapy, and had high FBG, and normal insulin/C-peptide levels, hypercholesterolemia, and hypertension. He had elevated aminotransferases without any other signs of liver dysfunction and a slightly increased urine albumin-to-creatinine ratio (49.5 mg/g). His clinical features support a double heterozygosity for MODY5/MODY7.

In this study, we detected the HNF1B p.S367 synonymous mutation, identified for the first time in our previous study (Demirci et al., 2021), in three patients and two controls. One of the patients with p.S367 (DAH19, 35 years old, female) had mild diabetes mellitus (DM), GDM, obesity, severe insulin resistance, hypertriglyceridemia, a measurable C-peptide level, and was not receiving any antihyperglycemic therapy. The other patient with p.S367 was a 42-year-old woman (DAH36). This patient had thyroid dysfunction, a measurable C-peptide level, and the absence of pancreatic islet autoantibodies.

However, since we observed this synonymous mutation in healthy controls, it was not considered an MODY-specific mutation in this study, and these two patients (DAH19 and DAH36) were diagnosed with T2DM based on their clinical/biochemical features. The other patient carrying p.S367 was a 41-year-old male diagnosed as HNF1B p.S362P/KLF11 P261L double heterozygosity, whose clinical features we described above (DAH62).

Homozygous mutations in NEUROD1 resulted in permanent neonatal diabetes, while heterozygous mutations caused MODY6 (Fajans et al., 2001). So far, there are few NEUROD1 mutations reported to be associated with MODY6 (Zhang et al., 2020). In our country, a few MODY6-related NEUROD1 mutations p.T45A (Yalcin Capan et al., 2020), s.P197H (Agladioglu et al., 2016; Demirci et al., 2021), and p.D202E mutations (Demirci et al., 2021) have been reported.

We detected a novel c.G411A (p.L137) synonymous mutation (located on the bHLH domain of the NeuroD1 protein) in the NEUROD1 gene in a 31-year-old female patient (DAH11). The patient had hypothyroidism and kidney cysts and menstrual irregularity. In this study, NEUROD1 p.T45A and p.P197H missense mutations were commonly detected in both patient and control, and, for that reason, not considered pathogenic. The p.P197H missense mutation in the NEUROD1 gene, which we previously detected in pediatric MODY patients (Demirci et al., 2021), was defined as a single-nucleotide polymorphism (SNP) in this study. This mutation involved in the neuronal helix-loop-helix transcription of the NeuroD1 protein was detected in six patients (DAH3, DAH5, DAH8, DAH10, DAH22, and DAH29) and 10 controls in our study group.

Two of these patients (DAH8 and DAH10) had neuropathy, one of the phenotypic features of MODY6. Five of the patients had no mutations other than p.P197H (DAH3, DAH5, DAH8, DAH10, and DAH29). One patient carrying the p.P197H mutation (DAH22) also carried the BLK mutation (p.A71T). The patient whose clinical phenotype was compatible with BLK-MODY was diagnosed as MODY11.

Mutations in the KLF11 genes that regulate insulin gene expression have been associated with the very rare MODY7 subtype (Neve et al., 2005). There are four KLF11 mutations described in the literature, p.A347S, p.T220M (Neve et al., 2005), p.H418Q (Ushijima et al., 2019), and p.R461Q (Demirci et al., 2021). In the present study, we detected three KLF11 mutations, one known (p.R461Q) (Demirci et al., 2021) and two novel (p.P261L and p.S182R). These KLF11 missense mutations localized to the C4-type zinc finger domains of the Klf11 protein were predicted to be deleterious.

In this study, we identified three missense mutations in the KLF11 gene (p.S182R, p.P261L, and p.R461Q). The male patient with the heterozygous KLF11 p.P261L mutation (DAH62) also carried the heterozygous HNF1B p.S362P missense mutation. The patient (female, 29 years old) who carried the KLF11 p.S182R mutation also carried the missense mutation PDX1 p.E69A (DAH33). The p.R461Q mutation, which was first identified in our previous study of pediatric MODY patients (Demirci et al., 2021), was found in a 43-year-old male patient (DAH60). This patient had high insulin requirements, hypercholesterolemia, neuropathy, nephropathy, and hypertension. The phenotypic effects of this mutation were observed to be more serious than in our previous study. The different effects of the mutation may be due to the longer duration of diabetes in adult patients.

Heterozygous mutations of the CEL gene, which encodes the bile salt-dependent CEL enzyme secreted from the exocrine pancreas, have been associated with MODY8. The phenotype caused by these mutations is defined as diabetes, pancreatic atrophy, and exocrine/endocrine pancreatic dysfunction (Torsvik et al., 2010). In our previous study, we reported one synonymous and one missense mutation in the CEL gene, both benign (p.S712P and p.G150, respectively) (Demirci et al., 2021). In the current study, four deleterious CEL mutations specific to MODY were identified, two known p.G157R (Aykut et al., 2018; Johansson et al., 2011; Raeder et al., 2006; Torsvik et al., 2010) and I488T (Johansen et al., 2014; Sarmadi et al., 2020), and two novel ones (p.I336T and p.N493H).

The patient with deleterious p.I488T mutation was a 31-year-old female (DAH45) and she required insulin treatment, and had dyslipidemia, anemia, and obesity. The I488T mutation is localized in exon 10 of the CEL gene. This mutation was predicted to have a damaging effect on CEL protein by bioinformatic tools. The p.G157R and p.I336T mutations were found in the same patient (DAH44). The female patient who had insulin requirement, uncontrolled diabetes, elevated C-peptide, macrosomia, subclinical hyperthyroidism, retinopathy, neuropathy, stroke, and hypertension was also obese (BMI: 36.6 kg/m²). This patient also heterozygously carried the novel ABCC8 p.R1103Q mutation, which was detected as benign by bioinformatic tools.

The patient carrying the p.N493H mutation was a 39-year-old male (DAH23). The patient with low insulin requirement exhibited the clinical phenotype of moderately uncontrolled diabetes, mild obesity (BMI: 31.2 kg/m²), high C-peptide, hypercholesterolemia, high triglyceride and low HDL-C levels, retinopathy, nephropathy, and neuropathy.

We also observed two missense mutations (T412I and T93S) of the CEL gene with high frequency in both control and patient groups and evaluated them as SNPs. Of these, T412I was localized in the helical domain of the protein and was defined as “benign” in ClinVar. We found this SNP in 35 controls and 29 patients. To our knowledge, the T93S SNP was detected for the first time in this study. The T93S SNP located in the “heparin binding domain” of the protein was detected in eight controls and five patients.

PAX4 mutations are associated with MODY9 and ketosis-prone diabetes, and these rare mutations have been reported in the Asian and Brazilian populations (Abreu et al., 2020; Plengvidhya et al., 2007). In this study, the PAX4 p.H321P mutation, previously reported by Yalcin Capan et al. (2020), was observed in the Turkish population. However, this variation, which was defined as benign by bioinformatic tools, was not MODY-specific as it was common in both MODY patients and healthy controls (MODY: 43.9% vs. controls: 30%).

INS gene mutations are associated with MODY10 (Molven et al., 2008; Piccini et al., 2016). Consistent with the literature on the Turkish population, we did not find any mutations in the INS gene in our study in adult patients with a clinical diagnosis of MODY.

There are rare BLK gene mutations associated with MODY11 in the literature (Bonnefond et al., 2013; Borowiec et al., 2009). In this study, we detected p.H293Q, p.R450H, p.L503P, and p.A71T mutations in the BLK gene. BLK p.L503P mutation was identified in MODY patients for the first time. The p.H293Q (rs142623841) and p.R450H (rs202162624) mutations have been previously presented in ClinVar, however, there are no studies in the literature on their phenotypic effects. BLK p.H293Q damaging mutation was detected in a 44-year-old male patient (DAH30). The patient's clinical findings included mild diabetes, hypercholesterolemia, neuropathy, and erectile dysfunction, and he was nonobese (BMI: 23.7 kg/m²). The potentially damaging p.R450H mutation was found in a 55-year-old obese (BMI: 35.1 kg/m²) female patient (DAH46). The patient with high insulin requirement and uncontrolled diabetes also had dyslipidemia, retinopathy, neuropathy, and nephropathy.

Since we detected p.L503P and p.A71T mutations in our control group as well as MODY patients in our study, we first thought that these mutations were not pathogenic for our society. However, Bonnefond et al. (2013) suggested that the A71T mutation may be mildly “diabetogenic” in the presence of obesity. The 71T mutant allele has been shown to reduce the half-life of the BLK protein (Delgado-Vega et al., 2012). When evaluated together with the report of Bonnefond et al., obesity (BMI: 32.5 kg/m²) and diabetes findings (FBG: 118 mg/dL and C-peptide: 3.1 ng/mL) of a 24-year-old female patient with A71T mutation (DAH22) were found to be compatible with the BLK-MODY phenotype. A71T mutation was also observed in one male (32 years old, BMI: 25.1 kg/m², FBG: 80 mg/dL) and one female (36 years old, BMI: 22.1 kg/m², FBG: 89 mg/dL) in the control group.

Our findings in the patient and control groups confirm that the A71T mutation has a diabetogenic effect in relation to obesity. On the contrary, the BLK p.L503P mutation was detected in one clinically diagnosed patient with MODY (DAH28) and one control subject. A 23-year-old male patient with p.L503P mutation was not obese (BMI: 19.1 kg/m²) and had mild diabetic symptoms (no insulin requirement and mild hyperglycemia). The control case, a 57-year-old female, had normal BMI (26.4 kg/m²) and FBG (99 mg/dL).

When we evaluated the results of all patients with BLK mutation together, we observed that the clinical effects of not only the A71T mutation but also other BLK mutations exacerbated in the presence of obesity. We also observed that hyperglycemic symptoms could be controlled with short-term (1–6 months) insulin therapy in nonobese patients with BLK mutation. In contrast, obese patients had uncontrolled diabetes requiring high-dose insulin (Supplementary Table S1).

Mutations in the ABCC8 and KCNJ11 genes, which encode subunits of the potassium channel (potassium-adenosine triphosphatase) in the beta cell membrane, are associated with MODY12 and MODY13, respectively (Nkonge et al., 2020). These MODY types, which are among the very rare MODY subtypes, are characterized by SU-sensitive diabetes (Bonnefond et al., 2013; Bowman et al., 2012; Urakami, 2019). Clinical manifestations caused by ABCC8 gene mutations have been associated with MODY12, T2DM, GDM (Ovsyannikova et al., 2016), and overweight/obesity (Bowman et al., 2012). Activating mutations of the KCNJ11 gene are associated with neonatal DM, impaired fasting glucose, impaired glucose tolerance, and KCNJ11-MODY. Inactivating KCNJ11 mutations can cause congenital hyperinsulinemic hypoglycemia.

Previous studies showed that missense mutations in ABCC8 and KCNJ11 are very rare in the Turkish population (Arslan Ates et al., 2021; Demirci et al., 2021; Ozdemir et al., 2018; Yalcin Capan et al., 2020).

We identified known p.A1369S, p.V1572, and c.354C>T (p.V118V) and novel p.R1103Q, p.V421I, and p.K1411N mutations in the ABCC8 gene. Among these, the A1369S, V1572I, and K1411N were common in both MODY and control groups. The missense R1103Q and V421I and synonymous c.354C>T (p.V118V) mutations were MODY-specific. The missense mutations (p.R1103Q and p.V421I) presented in ClinVar, however, there are no studies in the literature on their phenotypic effects. Both mutations were located in the transmembrane region of the ABCC8 transporter protein. The p.R1103Q mutation was predicted to be benign, whereas p.V421I was predicted to be damaging.

The female patient with p.R1103Q mutation (DAH44) also carried missense CEL p.I336T (novel), CEL p.G157R (known), and novel synonymous KCNJ11 c.G561A (A157A) mutations. The patient had a phenotype compatible with MODY8/MODY12 double heterozygosity with the clinical features mentioned above in CEL mutations. The clinical features of a 36-year-old female patient (DAH12) carrying the V421I mutation had overweight, mild diabetes, and history of GDM and macrosomic baby. The V118V synonymous mutation was found in a 35-year-old female patient (DAH35). The patient's clinical features (mild obesity, uncontrolled diabetes, nephropathy, retinopathy, GDM, and hypertension) were consistent with the MODY12 phenotype.

To the best of our knowledge, this is the first report that associates these p.R1103Q and p.V421I mutations of the ABCC8 gene with MODY12.

Here, we identified p.L270V (Yalcin Capan et al., 2020) and p.K23E (Chistiakov et al., 2008; Pappa et al., 2011) SNPs in the KCNJ11 gene, which were previously associated with T2DM, in both patient and control groups. In the present study, the KCNJ11 p.S385F mutation, detected in the pediatric/adolescent MODY group in our previous study (Demirci et al., 2021), was observed in only two healthy controls. Therefore, it cannot be considered a pathogenic variant for the adult MODY group. In addition, the KCNJ11 K39E SNP was first observed in this study in both the patient and control groups (in MODY: n = 7; in controls: n = 4).

Conclusions

We have conducted a study in which genetic factors that may be the cause of MODY were evaluated together with the detailed anamnesis and clinical and laboratory parameters of the patients. This study, which analyzes the clinical effects of MODY-specific mutations in detail, draws attention to the importance of evaluating MODY mutations in comparison with a population-based control group. It is noteworthy that the causative MODY variations are different in different populations; however, some common variations are found in our study sample as well as in studies in many other populations.

The findings of our study support the genetic and clinical heterogeneity of MODY in our country, which is a transitional society with many subpopulations, and show the existence of new unique variations that cause MODY in our population. BLK (MODY11) gene mutations were found to be the most common cause of MODY in our study population as recruited from adult index patients and their diabetogenic effects were obesity-dependent. It has been observed that diabetic symptoms are more severe in the presence of obesity in patients with BLK mutation, and require long-term insulin therapy, while hyperglycemic symptoms can be controlled with short-term (1–6 months) insulin support in nonobese patients.

In addition, when we evaluated the MODY patients in our sample, along with their MODY-specific mutations, complications, and comorbidities, we observed that obesity, dyslipidemia, and other comorbid conditions in MODY patients may affect hyperglycemic symptoms and diabetes complications, and thus, treatment approaches and outcomes as in T2DM patients (Supplementary Table S1). A fact that further challenges the MODY in clinical practices is that it is already heterogeneous. Consequently, by revealing the MODY-specific mutations, NGS is currently the most reliable tool to inform the MODY clinic, which is intertwined with the symptoms/complications of other forms of diabetes, and thus, NGS offers an important tool to prevent misdiagnoses.

In addition, it has been observed that having double heterozygous mutations in MODY-related genes is associated with clinical aggressiveness of the disease, which may also contribute to MODY heterogeneity. Our study indicates that some of the MODY cases have defects in unknown MODY genes, referred to as “MODY X.” Therefore, attempts to identify novel causative MODY gene variants using whole-exome sequencing are required in future studies to elucidate the genetic profile of MODY in our population.

Several limitations of the study are noteworthy of emphasis here. First, the current findings of this study only involved a single center, and thus, it may not be appropriate to generalize our results to the entire population. Second, the MODY-associated APLL1 gene (MODY14) was not analyzed.

It is of critical importance to compare the results of NGS-based genetic analysis of MODY patients with healthy individuals from population studies, as well as to evaluate them together with bioinformatic and detailed clinical findings. Thus, it will be possible to predict the clinical course of MODY and to provide the most appropriate treatment to the patients.

In conclusion, NGS analyses of the adult patients with suspected MODY appear to be informative in a clinical context. Nevertheless, these findings warrant further clinical diagnostic research and development in other world populations suffering from diabetes with genetic underpinnings.

Footnotes

Author Disclosure Statement

The authors declare they have no competing financial interests.

Funding Information

This study was funded by the Istanbul University Scientific Research Project Unit (Project no. 44381).

Supplementary Material

Abbreviations Used

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