2.1. Population Characteristics: The characteristics of participants are demonstrated in Table 1. Initially, 20,002 participants from TCGS families were included in the study. The cohort is female-dominated (54.4%), with a mean age of 46.3 ± 20.2 years. Concerning the glycemic status, 3,043 participants (15.2%) were classified as diabetic, 5,835 (29.1%) were classified as pre-diabetics, and 11,124 (55.6%) were classified as non-diabetics. Analysis of diabetic cases phenotypically showed that females accounted for 56% of all diabetic participants. The average age of the diabetic participants was 65.4\(\pm\)14.7 years. Participants with diabetes showed a mean BMI of 29.8\(\pm\)5.3 kg/\({\text{m}}^{2}\), which is higher in comparison with 25.9\(\pm\)5.0 kg/\({\text{m}}^{2}\), for normoglycemic participants.
2.2. MODY-Causing Variants Replication: Using ClinVar database results for "Maturity onset diabetes of the young" to identify candidate variants on 16 MODY genes, 3040 variants were extracted. Among these variants, 710 were available in the TCGS gVCF database. After filtering variants with MAF < 0.0015%, mean sequencing depth > 20×, and clinical significance of pathogenic/likely pathogenic (P/LP), six variants on six different MODY genes were retained. Table 2 depicts the features of detected variants. Forty-five participants from 24 families carried the aforementioned six variants. Table 3 demonstrates the MODY carrier’s laboratory data, diabetes family history, and more detailed features.
2.3. Novel MODY-Causing Variants Finding: We filtered out all the available variants on the 16 MODY genes in TCGS data which brings 647,705 variants. Based on MAF < 0.001%, mean sequencing depth > 20×, and a CADD Phred score > 20, 116 variants remained. After evaluating the disease penetrance, complete disease penetrance (100%) was observed in three variations in three MODY-related genes including KLF11-MODY7, PAX4-MODY9, and HNF1B-MODY5 (Table 2). None of these variants are reported as MODY-causing mutations in the ClinVar databases. These three potentially novel MODY variants were detected in 10 individuals from 5 families and all of the detected novel mutations were missense (Table 3). Notably, almost all potentially novel mutations were detected in cases classified as DM based on their phenotype data, except one KLF11-MODY7 carrier which was a normoglycemic girl who was only 12 years old until the last phase of the study.
2.4. Familial Pattern: Regarding the clinical characteristics and disease transmission in MODY carriers’ pedigree, the result for each subtype of MODY is mentioned below. For some subtypes of the disease, we could not find any marker in our population (subtypes 4,6,11–14). The rest will be explained in detail:
2.4.1. MODY 1: There was one HNF4A variant found, that had previously been classified as a pathogenic variant by ClinVar database. Additionally, this particular variant has been listed as an AD variant in Online Mendelian Inheritance in Man (OMIM) (125850). One proband was the carrier of this well-known missense mutation (p.Val130Ile). A 33-year-old Iranian woman was diagnosed with diabetes at the age of 22 with a maternal family history of the disease. She had not been treated with any glucose-lowering agents and has a normal BMI of 19.05 kg/\({\text{m}}^{2}\). Her older brother, who had a normal BMI, was also diagnosed with diabetes at the age of 28. Despite the lack of information regarding her father's glycemic status, her mother and two maternal aunts had young-onset diabetes and normal BMIs. Additionally, her mother had dyslipidemia (especially low HDL), hypertension, and diabetic retinopathy, all of which are suggestive of the microvascular complications linked to HNF4A-MODY1 [3]. The traits of this pedigree are shown in Fig. 1, suggesting a chance of maternal transmission of the variation. Indeed, the variant's AD transmission pattern and 100% disease penetrance indicate that it has a causative impact on MODY presentation in our population.
Table 1
Demographic and clinical characteristics of Tehran Cardiometabolic Genetic Study (TCGS) cohort.
|
All
|
Diabetics
|
Pre-Diabetics
|
Non-Diabetics
|
Number of subjects (n and %)
|
20002
|
3043 (15.2)
|
5835 (29.1)
|
11124 (55.6)
|
Age (mean ± SD)
|
46.3 ± 20.2
|
65.4 ± 14.7
|
51.5 ± 19.1
|
38.4 ± 17.6
|
Female %
|
10883(54.4)
|
1707(56.0)
|
2888 (49.4)
|
6288 (56.5)
|
BMI (kg/\({\text{m}}^{2}\))
|
27.4 ± 5.3
|
29.8 ± 5.3
|
28.4 ± 5.1
|
25.9 ± 5.0
|
Laboratory Data (mean ± SD)
|
|
|
|
|
FPG (mg/dl)
|
98.4 ± 28.2
|
137 ± 51.4
|
95.9 ± 9.3
|
87.0 ± 6.3
|
OGTT (mg/dl)
|
118 ± 44.1
|
211 ± 79.9
|
125 ± 31.5
|
98.3 ± 19.2
|
Triglyceride (mmol/l)
|
134 ± 86.4
|
166 ± 121
|
145 ± 82.6
|
115 ± 68.0
|
Cholesterol (mmol/l)
|
182 ± 39.9
|
180 ± 44.5
|
189 ± 39.9
|
178 ± 37.4
|
HDL cholesterol (mmol/l)
|
47.5 ± 11.1
|
45.2 ± 10.8
|
46.6 ± 11.0
|
49.0 ± 11.1
|
LDL cholesterol (mmol/l)
|
98.6 ± 33.8
|
93.2 ± 37.6
|
104 ± 33.9
|
96.2 ± 31.7
|
Blood Pressure (mean ± SD)
|
|
|
|
|
SBP (mmHg)
|
114 ± 18.1
|
126 ± 19.2
|
117 ± 17.9
|
108 ± 14.9
|
DBP (mmHg)
|
75.4 ± 10.6
|
77.9 ± 10.9
|
77.1 ± 10.4
|
73.3 ± 10.4
|
DM Onset Glycemic Status †
|
|
|
|
|
FPG (mg/dl)
|
|
147.6 ± 56.5
|
|
|
OGTT (mg/dl)
DM treatment (n and %)
|
|
246.5 ± 83.5
|
|
|
Insulin
|
|
298 (9.70)
|
|
|
Sulphnylurea
|
|
1207 (39.6)
|
|
|
Metformin
|
|
1379 (45.3)
|
|
|
AGIs
|
|
17 (0.50)
|
|
|
Other
|
|
7 (0.00)
|
|
|
BMI, body mass index. FPG, fasting plasma glucose. OGTT, oral glucose tolerance test. HDL, high-density lipoprotein. LDL, low-density lipoprotein. SBP, systolic blood pressure. DBP, diastolic blood pressure. DM, diabetes mellitus. † The reported laboratory data pertain to each diabetic participant’s onset of diabetes. AGIs, Alpha-glucosidase inhibitors. |
Table 2
Information of MODY variants in the Tehran Cardiometabolic Genetic Study (TCGS) cohort.
MODY Subtype†
|
chr:position:ref:alt
|
rs
|
Protein Change
|
MAF
|
Penetrance (%)
|
Diagnosis (n)
|
CADD phred
|
Consequence
|
ClinVar-Clinical significance
|
SIFT
|
Polyphen-2
|
HNF4A-MODY1
|
chr20:44413696:G:A
|
rs377476335
|
p.Val130Ile
|
0.000328
|
100
|
DM
|
22.2
|
Missense
|
P/LP
|
D
|
PD
|
GCK-MODY2
|
chr7:44149764:C:G
|
rs772754004
|
p.Ile225Met
|
0.000711
|
61.5
|
DM (3)
Pre DM (5)
N.D (4)
N.A (1)
|
6.324
|
Missense
|
P
|
D
|
PD
|
HNF1A-MODY3
|
chr12:120994313:-:C
|
rs766191969
|
p.Pro289fs
|
0.000355
|
100
|
DM
|
|
Frameshift
|
P
|
-
|
-
|
HNF1B-MODY5
|
chr17:37739468:C:T
|
rs764561297
|
p.Tyr172=
|
0.001422
|
12.5
|
DM (1)
Pre DM (5)
N.D (2)
|
9.416
|
Synonymous
|
P
|
-
|
-
|
chr17:37731615:C:T ‡
|
rs780035561
|
p.Ser342Phe
|
0.000355
|
100
|
DM
|
20.7
|
Missense
|
US
|
D
|
Benign
|
KLF11-MODY7
|
chr2:10046160:T:C ‡
|
rs1661191198
|
p.Met18Thr
|
0.000711
|
100
|
DM
|
23.3
|
Missense
|
NR
|
T
|
Benign
|
CEL-MODY8
|
chr9:133071270:C:CC
|
rs193922638
|
p.Val593fs
|
0.000423
|
0
|
Pre DM
|
17.15
|
Frameshift
|
LP
|
-
|
-
|
PAX4-MODY9
|
chr7:127615015:G:C ‡
|
rs759379107
|
p.Ser75Arg
|
0.000711
|
100
|
DM
|
25
|
Missense
|
NR
|
D
|
PD
|
INS-MODY10
|
chr11:2160956:C:T
|
rs121908278
|
p.Arg6Cys
|
0.001422
|
38
|
DM (8)
Pre DM (8)
N.D (3)
N.A (2)
|
21.2
|
Missense
|
P
|
D
|
Unknown
|
† Identified variants including 1) previously reported pathogenic (P) or likely pathogenic (LP) MODY-causing mutations in ClinVar databases, and 2) novel MODY variants in TCGS cohort data. ‡ Demonstrates three novel MODY variants. all novel identified variants showed complete disease penetrance (100%) and mutation carriers were classified as diabetics based on phenotype data. Chr:Pos, Chromosome:Position of identified variant based on genome build CRCh38. Ref, reference allele. Alt, Alternative allele. MAF, Minor Allele Frequency. Penetrance is shown as the percentage of mutation carriers with diabetes. Diagnosis is based on phenotype data. N, Number of subjects. DM, Diabetes Mellitus. Pre DM, Pre-diabetes. N.D, Non-Diabetics. N.A, Not Available glycemic data. CADD, Combined Annotation-Dependent Depletion Phred score. US, a variant of Uncertain Significance. D, Deleterious. T, Tolerated. PD, is Probably Damaging. NR, Not Reported. |
Table 3
Clinical characterization and laboratory data of index MODY carriers
|
Previously-Reported Variants
|
Novel Variants
|
MODY Subtype
|
MODY 1
|
MODY 2
|
MODY 3
|
MODY 5
|
MODY 8
|
MODY 10
|
MODY 5
|
MODY 7
|
MODY 9
|
Case n/ Family n†
|
1 / 1
|
13 / 7
|
1 / 1
|
8 / 3
|
1 / 1
|
21 / 11
|
4 / 1
|
5 / 3
|
1 / 1
|
Age, y
|
33
|
48.0 ± 21.8*
|
24
|
47.8 ± 15.7
|
43
|
49.7 ± 15.8
|
58.7 ± 17.7
|
54.8 ± 33.9
|
44
|
Female, n (%)
|
1 (100)
|
5 (38.4)
|
1 (100)
|
6 (75)
|
1 (100)
|
12 (57.1)
|
2 (50)
|
3 (60)
|
0 (0)
|
BMI (kg/\({\mathbf{m}}^{2}\))
|
22.2
|
26.5 ± 3.3
|
24.7
|
25.8 ± 4.9
|
32.2
|
27.6 ± 4.8
|
30.1 ± 4.4
|
29.4 ± 10.8
|
31.8
|
Underweight %
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
20
|
-
|
Normal %
|
100
|
33.3
|
100
|
40
|
-
|
46.6
|
-
|
-
|
-
|
Overweight %
|
-
|
44.4
|
-
|
40
|
-
|
33.3
|
50
|
40
|
-
|
Obese %
|
-
|
22.2
|
-
|
20
|
100
|
20.0
|
50
|
40
|
100
|
Glycemic Status, n (%)
|
|
|
|
|
|
|
|
|
|
Diabetic
|
1 (100)
|
2 (16.6)
|
1 (100)
|
1 (12.5)
|
-
|
3 (15.7)
|
4 (100)
|
4 (80.0)
|
1 (100)
|
Pre-Diabetic
|
-
|
6 (50.0)
|
-
|
5 (62.5)
|
1 (100)
|
8 (42.1)
|
-
|
-
|
-
|
Non-Diabetic
|
-
|
4 (33.3)
|
-
|
2 (25.0)
|
-
|
8 (42.1)
|
-
|
1 (20.0)
|
-
|
missing
|
-
|
1
|
-
|
-
|
-
|
2
|
-
|
-
|
-
|
Laboratory Data
|
|
|
|
|
|
|
|
|
|
FPG (mg/dl)
|
130
|
120 ± 65.3
|
171
|
95 ± 13.7
|
101
|
103 ± 44.0
|
152 ± 41.3
|
109 ± 30.5
|
109
|
OGTT (mg/dl)
|
256
|
174 ± 135
|
260
|
155 ± 83.3
|
138
|
128 ± 38.5
|
231 ± 138
|
275 ± 65.4
|
225
|
Triglyceride (mmol/l)
|
109
|
156 ± 100
|
158
|
95.5 ± 43.1
|
200
|
158 ± 82.5
|
179 ± 34.1
|
114 ± 38.3
|
92
|
Cholesterol (mmol/l)
|
196
|
186 ± 50.2
|
150
|
196 ± 61.8
|
239
|
194 ± 38.6
|
180 ± 52.5
|
193 ± 33
|
129
|
HDL cholesterol (mmol/l)
|
43
|
42.4 ± 8.8
|
46
|
55.3 ± 5.6
|
54
|
46.2 ± 10.9
|
33 ± 8.2
|
42.4 ± 8.1
|
37
|
LDL cholesterol (mmol/l)
|
124
|
103 ± 40
|
60
|
111 ± 52.5
|
142
|
107 ± 35.4
|
101 ± 48.3
|
119 ± 29.2
|
58
|
Serum creatinine (µmmol/l)
|
1.1
|
1 ± 0.1
|
0.8
|
0.9 ± 0.09
|
0.8
|
1.13 ± 0.1
|
1.25 ± 0.5
|
1.04 ± 0.1
|
1.4
|
Blood Pressure
|
|
|
|
|
|
|
|
|
|
SBP
|
68
|
74.2 ± 6
|
75
|
74.7 ± 7.4
|
89
|
72.9 ± 9.8
|
90.5 ± 10.1
|
70 ± 7.3
|
81
|
DBP
|
94
|
118 ± 22.5
|
100
|
122 ± 19.5
|
131
|
111 ± 17.1
|
154 ± 13.1
|
110 ± 20.6
|
120
|
Family History of Diabetes ‡
|
|
|
|
|
|
|
|
|
|
First-degree relative
|
1 (100)
|
-
|
1 (100)
|
-
|
-
|
2 (66.6)
|
4 (100)
|
2 (50)
|
-
|
Second-degree relative
|
1 (100)
|
-
|
1 (100)
|
-
|
-
|
-
|
4 (100)
|
-
|
-
|
DM Onset Glycemic Status ‡§
|
|
|
|
|
|
|
|
|
|
FPG (mg/dl)
|
101
|
241 ± 89.0
|
128
|
130
|
-
|
182 ± 104
|
169 ± 38.2
|
134 ± 18.1
|
109
|
OGTT (mg/dl)
|
228
|
401 ± 174
|
232
|
256
|
-
|
217 ± 8.7
|
249 ± 42.4
|
347 ± 9.8
|
225
|
Treatment (n and %) ‡
|
|
|
|
|
|
|
|
|
|
Insulin
|
-
|
-
|
-
|
-
|
-
|
-
|
1 (25)
|
-
|
-
|
Sulphnylurea
|
-
|
-
|
-
|
-
|
-
|
1 (33.3)
|
3 (75)
|
2 (50)
|
-
|
Metformin
|
-
|
1 (50)
|
-
|
1 (100)
|
-
|
-
|
2 (50)
|
1 (25)
|
-
|
† Case n/Family n: number of MODY carriers/number of carrier’s families. The reports for MODY 1-3-8-9 don’t contain mean and standard deviation due to only one proband. N, Number of subjects. BMI, Body Mass Index. FPG, Fasting Plasma Glucose. OGTT, Oral Glucose Tolerance Test. HDL, High-Density Lipoprotein. LDL, Low-Density Lipoprotein. SBP, Systolic Blood Pressure. DBP, Diastolic Blood Pressure. DM, Diabetes Mellitus. ‡ The reported data are only among the carriers with diabetic phenotype. § The reported laboratory data pertain to each diabetic participant’s onset of diabetes. * Mean ± SD. |
2.4.2. MODY 3: One HNF1A variant was found that had previously been reported as a pathogenic variant by ClinVar. Additionally, this particular variant was previously identified as a MODY-causing AD variation in OMIM (600496). One proband had this well-known frameshift mutation (insertion p.Pro289fs). A 24-year-old Iranian woman with a strong diabetes familial history was diagnosed with diabetes at age 13. She wasn't receiving any glucose-lowering agents with poor glycemic control (FPG = 171 mg/dL). Her BMI was 22.73 kg/\({\text{m}}^{2}\), which is considered normal. Despite her father being normoglycemic, her mother, her maternal grandmother, and three of her maternal uncles all developed diabetes before the age of 18 and required insulin treatment. All the maternal uncles were normal weight or slightly overweight. Furthermore, dyslipidemia was found in each of the aforementioned maternal family members. Additionally, the eldest uncle's history of angioplasty and the other uncle's history of diabetic retinopathy are indicative of the microvascular complications associated with HNF1A-MODY3 [3]. Figure 2 demonstrates the characteristics of the family pedigree. The AD pattern of transmission and complete disease penetrance (100%) of this variant shows that it has also a causative impact on MODY presentation in our population.
2.4.3. MODY 5: Between the two HNF1B variations, one was previously described in ClinVar and the other one was a potentially novel HNF1B-MODY5 variant. Regarding the previously-reported variant, as the disease penetrance of this variant was 12.5% and none of the carriers showed the special clinical manifestations for Renal Cysts and Diabetes Syndrome (RCAD) related to MODY5, it appears that this mutation in our community has no clinical significance. A novel variant in HNF1B was also found, which was reported with unspecified disease conditions in ClinVar database [3, 32]. Four patients from a single family had this missense variation. A 76-year-old woman with diabetes who had been enrolled in the cohort trial was the first carrier. This mutation was present in each of her two children, who were both diagnosed with diabetes. Her grandson, a 34-year-old male with diabetes who was first diagnosed at age 30, was the final carrier. The population's response to this variation revealed complete penetrance. Although AD transmission and complete disease penetrance were observed, as most carriers were overweight or obese with none of the symptoms of RCAD, more investigations are needed to confirm the causative impact of this variant. The family pedigree is available in Fig. 2.
2.4.4. MODY 7: A novel KLF11 variant that has not previously been reported in ClinVar was found. Five probands from three families were identified to have this missense variant. Except for one person, a 12-year-old boy, all of these carriers were diabetics, demonstrating complete disease penetrance of this variant in our community. Examining the variation transmission, however, could not confirm the AD inheritance for the diabetic phenotype.
2.4.5. MODY 9: Unreported in the ClinVar database, one novel variation of PAX4 was found. The variation was discovered in one candidate. Diabetes was discovered in the sole carrier, giving this variation complete penetrance. The carrier's 8-year-old child was the only family member in our cohort whose glycemic status was not available, therefore the variant transmission could not be reached.
2.4.6. Since the other MODY subtype carriers (GCK-MODY2, CEL-MODY8, INS-MODY10) were not diabetic or didn’t show any AD inheritance for the phenotype in their families, it appears that these variants haven’t causative impact on MODY manifestation in our population.
2.5. MODY Genetic Spectrum in Iran: Overall, there were 55 MODY variant carriers from 29 families in the TCGS cohort study. The mean age was 49.3 ± 23 years, 31 (56.3%) carriers were female. 17 (32.6%) were diabetic, 20 (38.4%) were pre-diabetic, 15 (28.4%) were non-diabetics and the glycemic status of 3 participants was unavailable.
2.6. Protein Structure Modeling and IDPRs: We have pinpointed the locations of the three most probable MODY markers on the chromosomes. Figures 1–2 highlight the genomic locations, transcripts and pathogenicity of these markers within the respective genes, providing a comprehensive overview of their spatial distribution on the chromosomes. Thanks to the Swiss Model web server, the 3D structure of the proteins encoded by the MODY genes has been modeled. Figure 1 displays the HNF4A-MODY1's 3D structure. However, due to the poor quality and coverage of the models of both HNF1A-MODY3 and HNF1B-MODY5, we assessed their Intrinsically Disordered Protein Regions (IDPRs). Also, it shows the results of the IUpred webserver. These results are in agreement with their 3D models' quality control parameters.