A total of 44 patients were included in the study. There were 18 males (40.9%) and 26 females (59.1%), with a mean age of 13.307 ± 7.177 years (minimum: 3.5; maximum: 40) and a mean age at diagnosis of 10.773 ± 6.167 years (minimum: 1.5; maximum: 30). The average insulin requirement of patients and the mean level of C-peptide were 0.833 ± 0.358 U/kg/day (minimum: 0.19; maximum: 1.55) and 0.617 ± 0.433 ng/mL (minimum:0.10; maximum: 2.41), respectively. The mean BMI, HbA1c at diagnosis and level of fasting blood glucose were 19.568 ± 4.216 kg/m2 (minimum: 13.80; maximum: 34.50), 9.362 ± 2.705 per cent (minimum: 5.80; maximum: 15) and 273.5 ± 123.941 mg/dL (minimum: 100; maximum: 543), respectively. Presenting complaints included incidental identification of hyperglycemia in 14 patients (31.8%), polyuria and polydipsia in 18 patients (40.9%), ketoacidosis in 7 patients (15.9%), polyuria in 3 patients (6.8%), polydipsia in 2 patients (4.5%). All these cases were negative for ICA, with one of them being positive for IAA and three of them being positive for anti-GAD.
Based on the molecular genetic analysis results, patients were divided into those with a variant classified as possibly pathogenic/pathogenic and others. The clinical and laboratory characteristics belonging to them are given in Tables 2 and 3. Thirteen different genes were analysed in 44 unrelated cases with a pre-diagnosis of MODY. Pathogenic/likely pathogenic variants in 10 (22.7%) cases, variants of uncertain significance (VUS) in 8 (18.2%) cases, likely benign/benign variants in 4 (9%) cases were detected in our study. The distribution of detected pathogenic/likely pathogenic variants were as follows: five variants in the GCK gene, two variants in the HNF1A gene, one variant in the ABCC8 gene, one variant in HNF1B gene and one variant in the CEL gene. VUS were detected in the following numbers: two variants in the HNF1A gene, two variants in the KLF11 gene, two variants in the NEUROD1 gene and one variant in the ABCC8 gene. The distribution of likely benign/benign variants were as follows: two variants in the NEUROD1 gene, one variant in the BLK gene, one variant in the CEL gene (Table 1). Overall, a total of 21 different variants were identified in this study (Table 1). We were able to identify four novel pathogenic/likely pathogenic variants (one in GCK, one in ABCC8, one in HNF1A, one in CEL) and two novel VUS (both in NEUROD1). All variants detected in this study were heterozygous, and the pathogenicity assessments of them are provided in Table 1.
Clinical and molecular analysis results of the variant (+) patients
GCK-MODY (MODY 2)
We detected two pathogenic and three likely pathogenic variants in the GCK gene, all of them being previously described in the literature except for the c.1301G > T (p.Cys434Phe) variant. The corresponding patients are shown in Tables 1 and 2 as P1, P2, P14, P19 and P22, along with the clinical and molecular findings in them.
Fasting blood glucose levels in P1 were between 120 and 180 mg/dL and monitored by diet. In the family history, the fasting blood glucose of the mother of P1 did not exceed 200 mg/dL and she did not use any medication. Through the targeted mutational analysis, it was shown that the mother carries the c.449_451delTCT (p.150delF) variant. The results of the segregation analysis demonstrated that P2 inherited the c.537delG (p.Asn180ThrfsTer25) variant from her father. The father’s fasting blood sugar levels were around 150–160 mg/dL, and he was first diagnosed when he was being examined for other reasons at 25 years old. Currently, he is using oral metformin. Fasting blood glucose levels in P14 were between 100 and 150 mg/dL and monitored by diet. DNA sequence analysis revealed that the patient inherited the variant from his mother. It is worth noting that the patient’s mother and grandmother also have a history of diabetes.
Fasting blood glucose levels in P19 were 90–160 mg/dL and monitored by diet. The same variant (c.1301G > T [p.Cys434Phe]) was found in the patient’s father by Sanger sequencing. In the last routine follow-up visit of P19 who had high fasting sugar for three years, HbA1c was measured as 6.3%, body mass index as 22 kg/m2 and C-peptide as 0.8 ng/mL.
The de novo heterozygous c.790G > A (p.Gly264Ser) variant was detected in P22. The patient was first noticed that he had a high fasting blood glucose level coincidentally. Today, the patient does not use any medication and fasting blood sugar levels vary between 80 and 160 mg/dL. The clinical and molecular findings of the above-mentioned patients were consistent with GCK-MODY.
GCK encodes glucokinase, a 465-residue-long monomeric enzyme that is responsible for glucose phosphorylation in hepatocytes and pancreatic β-cells. Asn180 is located in an α-helical region (helix 5; residues 180–193), while Phe150 is located in a β-stranded region (strand 5; residues 145–150) (Fig. 1A). Although Phe150 lies in the close vicinity of the enzyme’s active site, an analysis of polar interactions occurring between the enzyme and substrate reveals that it does not interact with glucose (Fig. 1B). Similarly, Asn180 itself is not engaged in favorable contacts with glucose. The majority of glucose-binding residues, however, are situated after position 180, meaning that the frameshift variant affecting Asn180 is expected to result in a severely truncated protein (if translated) with no catalytic activity.
ABCC8-MODY (MODY 12)
We detected the c.1943G > A (p.Arg648His) variant as “VUS” in the ABCC8 gene of our 7-year-old male patient, P12, with elevated sugar levels for two years. The allele frequency of this variant is estimated to be 0.000012 in the gnomAD exomes database. The same variant was detected in his mother. In the last routine follow-up visit of P12, fasting blood glucose levels ranged from 100 to 160 mg/dL, insulin requirement was 0.19 U/kg/day, body mass index was 17.7 kg/m2 and C-peptide was 0.87 ng/mL. In the family history, it was noted that the patient’s mother had high blood sugar levels in the past, with the exact value being 124 mg/dL at the time of the follow-up visit. It was also noted that the patient’s grandmother had a history of diabetes, which is being controlled by oral antidiabetics since the age of 30. Clinical and laboratory findings of this family were consistent with MODY.
The heterozygous c.3584C > T (p.Thr1195Ile) variant, which was predicted to be “likely pathogenic”, was detected in the ABCC8 gene of P17. The high levels of fasting sugar in the patient were first noticed 10 years ago, and the patient was on oral antidiabetic agents. He had no insulin requirement, and his fasting blood glucose levels never exceeded 250 mg/dL. The patient’s father had a history of diabetes, and it was noted that he was being treated with insulin. We did not have the opportunity to perform segregation analysis involving the patient’s parents.
ABCC8 codes for the regulatory subunit sulfonylurea receptor 1 (SUR1) which, together with the pore-forming subunit Kir6.2, constitutes the ATP-sensitive potassium (KATP) channels in pancreatic β-cells. Arg648 is located in a cytosolic loop structure between the eleventh transmembrane domain (TM11) and the first nucleotide-binding domain (NBD1) of SUR1 (Fig. 2). Both SUR1 and Kir6.2 contain a tripeptide endoplasmic reticulum retention motif termed RKR. In SUR1, this motif is composed of Arg648-Lys649-Arg650. The occurrence of the RKR motif simultaneously in SUR1 and Kir6.2 ensures that only fully functional β-cell KATP channels are trafficked to the plasma membrane, since each motif is only shielded when the two subunits are properly assembled or folded [5]. Therefore, it is plausible to assume that the Arg648His substitution may lead to dysregulation of intracellular SUR1 trafficking, interfering with the critical role of β-cell KATP channels in the glucose-stimulated insulin secretion pathway.
HNF1A-MODY (MODY 3)
The heterozygous c.505_506delAA (p.Lys169AlafsTer18) variant was observed in P16 in a de novo case of MODY. The presence of high fasting blood glucose levels in the patient was first noticed five years ago by coincidence. Today, the patient’s condition is controlled by low-dose insulin injections, and his fasting blood sugar levels vary between 90 and 200 mg/dL.
The heterozygous c.376 C > T (p.His126Tyr) variant, which was predicted to be “VUS”, was detected as a de novo mutation in P18. The patient’s parents had no previous history of diabetes. This variant is not present in the gnomAD exomes database and has not been reported in the ClinVar archive before. Sequence- or structure-based prediction tools further support the pathogenicity of this variant. The patient has been diagnosed with diabetes for one year, and his blood glucose levels are controlled by oral antidiabetics.
We identified the de novo c.862G > A (p.Gly288Arg) variant in exon 4 of the HNF1A gene. The allele frequency of this variant is calculated to be 0.0000461 in the gnomAD exomes database and has not previously been registered in ClinVar. Sequence- or structure-based prediction tools further support the pathogenicity of this variant and considering familial co-segregation analysis results, we think that this variant may be a disease-causing variant.
We identified the c.476G > A (p.Arg159Gln) variant in exon 2 of the HNF1A gene, which has been classified as pathogenic in ClinVar. In the last follow-up visit of P20, fasting blood glucose values were measured to vary from 100 to 244 mg/dL, insulin requirement to be 0.48 U/kg/day, body mass index to be 23 kg/m2 and C-peptide concentrations to be 0.77 ng/mL. The same variant was detected in his mother and she was diagnosed with diabetes at the age of 25 and it was noticed that she had insulin requirement.
HNF1A encodes hepatocyte nuclear factor1 alpha, a transcription factor that has been shown to regulate the tissue-specific expression of multiple genes including the gene for insulin in pancreatic β-cells and the gene for GLUT2 in glucose-sensing cells [6, 7]. Gly288 appears to be situated in a proline-rich, low-complexity region (residues 287–315) adjacent to the homeobox (HOX) domain of the protein. This region belongs to the C-terminal transactivation domain of the protein, indicating that the Gly288Arg substitution may impair the interactions of HNF1α with coactivators. This, however, remains to be investigated by biochemical and structural approaches. To date, no human HNF1α structures containing the C-terminal transactivation domain have been deposited in the PDB.
HNF1B-MODY (MODY 5)
The heterozygous c.704G > A (p.Arg235Gln) variant was detected as a de novo mutation in P21. The patient was diagnosed with diabetes two years ago, and his blood glucose levels fall in the range of 100–310 mg/dL. In his last follow-up visit, C-peptide was found to be 0.45 ng/mL and HbA1c to be 7.1%. Sequence- or structure-based prediction tools further support the pathogenicity of this variant and considering the segregation analysis results we propose this variant as “likely pathogenic”.
CEL-MODY (MODY 8)
We identified two patients carrying a variant in the CEL gene. P4 was found to bear the c.268A > C variant which was classified as “likely benign” according to the ACMG guidelines. In the last control visit of the patient, who has been followed up with a diagnosis of diabetes for three years, it was noted that fasting blood sugar levels ranged between 100 and 320 mg/dL and postprandial blood glucose levels between 100 and 240 mg/dL, and the patient’s insulin requirement, HbA1c levels, C-peptide concentrations and body mass index were 0.72 U/kg/day, 7.3%, 0.38 ng/mL and 23 kg/m2, respectively. The mother of P4 had no history of diabetes, her fasting blood glucose levels were measured as 110 mg/dL, and it was demonstrated that she also carried the same variant. Because of the low penetrance and variable expression of the CEL gene [8], additional studies are required to better evaluate the pathogenic potential of this variant which has a low allele frequency (0.000112) in the gnomAD exomes database.
CEL codes for bile salt-activated lipase (BAL), a secreted pancreatic enzyme that hydrolyzes a broad spectrum of lipids. Thr90 is located in a conserved 17-residue-long surface loop structure termed the Cys-loop (residues 84–100), which is adjacent to the catalytic site (Fig. 3). The Cys-loop is anchored by a disulfide bridge between Cys84 and Cys100 and forms a cover lid normally responsible for the water–oil interfacial activation of the related triacylglycerol lipases [9, 10]. It could be that simply introducing Pro90 into the Cys-loop or further having a prolyl cis/trans isomerization reaction at position 90 may affect the conformation of the Cys-loop, altering the biological function of BAL. This may eventually lead to pancreatic exocrine dysfunction which could be linked to MODY.
In P5, a heterozygous IVS5-1G > A variant in intron 5 of the CEL gene was detected. We showed that this variant, which has not been reported in the literature before, was also carried by the father of the patient. The effect of the variant on splicing mechanisms was predicted to be “deleterious” by the Human Splicing Finder. Biochemical parameters obtained in the last control visit of P5, who has been followed up with a diagnosis of diabetes for three years, were as follows: fasting blood glucose levels were 122–340 mg/dL, postprandial blood glucose levels were 130–210 mg/dL, C-peptide levels were 0.23 ng/mL and insulin requirement was 1.05 U/kg/day. Autoantibodies that were re-examined at the last examination were negative and BMI was 20 kg/m2. Her father’s blood test revealed fasting blood glucose levels of 128 mg/dL and HbA1c levels of 6.5%. Based on these results, it is plausible to assume that the variant detected in P5 may lead to the MODY phenotype. This, however, needs further studies to be substantiated.
NEUROD1-MODY (MODY 6)
We detected genetic alterations in NEUROD1 in four patients. In the first patient (P6), we identified the heterozygous de novo c.-81C > A variant in the 5' UTR region of the gene. This variant is not present in the gnomAD exomes and genomes databases and has not been reported in ClinVar before. In the last control visit of the patient, who has been followed up with a diagnosis of diabetes for two years, BMI, HbA1c levels and C-peptide concentrations were found to be 27 kg/m2, 8.1%, 1.79 ng/mL, respectively, with negative assay results for autoantibodies. Collectively, these findings suggest that the variant may be a rare polymorphism contributing to type 2 diabetes.
In another patient (P9), the heterozygous de novo c.-71G > A variant in the 5' UTR region of the gene was detected. This variant has an estimated allele frequency of 0.0000637 in the gnomAD genomes database and has not been reported in ClinVar before. The father of the patient had a history of diabetes beginning at about the age of 35 and was on oral antidiabetics. In the last visit of the patient, C-peptide concentrations were 0.2 ng/mL, HbA1c levels were 8.7%, BMI was 19 kg/m2, and autoantibodies were negative except for anti-GAD.
Finally, two patients (P10 and P11) were found to bear the c.590C > A (p.Pro197His) variant in the NEUROD1 gene. The allele frequency of this variant was calculated to be 0.0193 in gnomAD exomes and 0.0129 in gnomAD genomes. The variant has previously been reported as “benign” in Clinvar. According to some in silico tools, such as Mutation Taster, Provean and SIFT, this variant is damaging in nature. By increasing the number of cases available for analysis and having more detailed data regarding the population allele frequencies at this locus, we should be able to draw empirically well-based inferences.
NEUROD1 encodes neurogenic differentiation factor 1, a transcriptional activator that regulates insulin gene expression in pancreatic β-cells through binding to its E-box motif-containing promoter consensus sequence [11]. Pro197 is situated in the protein’s neuronal basic helix-loop-helix (bHLH) transcription factor domain (residues 160–284) which is involved in DNA binding. Therefore, it is tempting to think that Pro197His may lead to dysregulated insulin expression in β-cells, adversely affecting pancreatic cell-fate specification. Further biochemical and structural studies are required to delineate the pathobiology of this substitution. Currently, there are no human NeuroD1 structures deposited in the PDB.
BLK-MODY (MODY 11)
The heterozygous c.211G > A (p.Ala71Thr) variant was identified as de novo mutation in P7. The allele frequency of the variant is estimated to be 0.0116 in gnomAD exomes, while conflicting interpretations of pathogenicity have been reported in ClinVar. Bonnefond et al. stated in their study that the p.Ala71Thr variant, which causes a loss of function of the gene product, is highly unlikely to cause penetrating MODY and may weakly affect the risk of type 2 diabetes, although they also added that this awaits further clarification [12]. The patient has been diagnosed with diabetes mellitus for 2.5 years and tested negative for autoantibodies in the past. The test results from the last follow-up visit revealed that the patient’s fasting blood glucose levels were 90–160 mg/dL, postprandial blood glucose levels were 170–190 mg/dL, C-peptide concentrations were 0.25 ng/mL, BMI was 24 kg/m2 and insulin requirement was 0.57 U/kg/day. Familial co-segregation analysis results and a number of sequence- or structure-based prediction tools support the pathogenicity of c.211G > A (p.Ala71Thr), but additional studies are needed in order to evaluate the causal role of this variant in MODY.
BLK codes for B-lymphocyte kinase, a non-receptor tyrosine kinase that is involved in cellular development, differentiation and signaling. Ala71Thr, which lies in the SH3 domain (residues 58–118) of the protein, has previously been shown to cause MODY by attenuating the biological effects associated with BLK, especially the up-regulation of the transcription factors Pdx1 and Nkx6.1which promote insulin synthesis and secretion in response to glucose in pancreatic β-cells [13]. At present, there are no human BLK structures deposited in the PDB.
KLF11-MODY (MODY 7)
We found a variant (c.1382G > A [p.Arg461Gln]) that we classified as “VUS” in the KLF11 gene of P8. While the pathogenity of this variant has not been clearly elucidated previously, using Mutation Taster and SIFT algorithms we predict that the variant is “damaging”. The variant has an allele frequency of 0.000497 in gnomAD exomes and appears to have been conserved in the evolutionary process. The patient’s father and grandfather had a history of diabetes, and it was noted that the father’s fasting blood glucose levels did not exceed 300 mg/dL and that he did not use insulin at any time. We confirmed that the father is a carrier for the same genetic alteration.
We detected the c.1447C > T (p.Pro483Ser) variant, which we classified as “VUS”, in the KLF11 gene of our 17-year-old female patient (P15) present with elevated sugar levels for two years. The allele frequency of this variant is estimated to be 0.0000637 in the gnomAD exomes database. The last follow-up history of P15 revealed that her fasting blood glucose levels ranged from 100 to 250 mg/dL, insulin requirement was 0.74 U/kg/day, BMI was 21 kg/m2 and C-peptide concentrations were 0.66 ng/mL. Although the patient’s mother and grandmother were known to have a history of diabetes starting after the age of 30, we did not have the opportunity to subject them to molecular genetic analysis.
KLF11 encodes Kruppel-like factor 11, a pancreas-enriched zinc finger transcription factor that regulates the expression of genes related to cellular differentiation and inflammation in early embryonic development. Arg461Gln is situated in the C2H2-type zinc finger domain (residues 454–476) of the protein, suggesting that this substitution may impair the interactions of KLF11 with coactivators, thereby leading to pancreatic β-cell dysfunction. Indeed, KLF11 has previously been reported to activate Pdx1 [14], a transcription factor critical to pancreatic organogenesis and the maintenance of mature β-cell function. The precise mechanism of action of Arg461Gln is yet to be determined experimentally. Currently, there are no human KLF11 structures deposited in the PDB.