In our cohort the number of patients studied is 36, corresponding to 72 alleles for each gene – these are distributed in 20 patients from the first group (40 alleles) and 16 from the second one (32 alleles). For some genetic variants the number is less due to unsuccessful analysis. Figure 1 and 2 show the results from the genotyping of 12 genetic variants at risk genes in diabetic patients with and without CVD, respectively.
1.1 Results from the genotyping of factors for congenital thrombophilia – Table 2
Results from the genotyping of Factor V Leiden and R2
Altogether for all patients a frequency of 5,5% was found for Factor V Leiden mutation (Table 2) – it is two-fold increase than the population frequency in the world (1,9%) and in Europe (2,9%). According to 1000 Genomes database the frequency of the heterozygotes is 2% and in our cohort it was 11%. We found also a higher than population frequency for FV H1299R (R2) – 9,7% in comparison to frequency of 5,7% in the world and 6% in Europe. No connection between both FV mutations and cardiovascular complications has been established.
Table 2. Allelic and genotypic frequencies of factors for congenital thrombophilia
Allele/Genotype
|
DM with CVD
|
DM without CVD
|
All
|
FV (Leiden)
FV G/A
FV A/A
FV (R2)
FV H/R
FV R/R
|
1/40 (2.5%)
1/20 (5%)
0
3/34 (8.8%)
3/17 (17.6%)
0
|
3/32 (9.4%)
3/16 (18.8%)
0
3/28 (10.7%)
3/14 (21.4%)
0
|
4/72 (5.5%)
4/36 (11%)
0
6/62 (9.7%)
6/31 (19.4%)
0
|
Prothrombin 20210A
Prothrombin G/A
Prothrombin A/A
|
1/40 (2.5%)
1/20 (5%)
0
|
0
0
0
|
1/72 (1.4%)
1/36 (2.8%)
0
|
PAI-1 4G
PAI-1 4G/5G
PAI-1 4G/4G
|
26/40 (65%)
12/20 (60%)
7/20 (35%)
|
15/30 (50%)
7/15 (46.7%)
4/15 (26.7%)
|
41/70 (58.6%)
19/35 (54.3%)
11/35 (31.4%)
|
Factor XIII 34L
Factor XIII V/L
Factor XIII L/L
|
3/40 (7.5%)
3/20 (15%)
0
|
5/32 (15.6%)
5/16 (31.2%)
0
|
8/72 (11.1%)
8/36 (22.2%)
0
|
β-Fibrinogen -455 A
β-Fibrinogen -455 G/A
β-Fibrinogen -455 A/A
|
11/40 (27.5%)
11/20 (55%),
p<0.03
0
|
5/32 (15.6%)
3/16 (18.7%)
1/16 (6.2%)
|
16/72 (22.2%)
14/36 (38.9%)
1/36 (2.7%)
|
HPA-1b
HPA 1a/1b
HPA 1b/1b
|
3/40 (7.5%)
3/20 (15%)
0
|
6/32 (18.7%)
6/16 (37.4%)
0
|
9/72 (12.5%)
9/36 (25%)
0
|
Results from the genotyping of ProthrombinG20210A
We found a frequency of 1,4% and it is comparable to the world population frequency of 0,8% and to that in Europe – 1,1% (Table 2). The mutation was found only in DM patients with CVD not reaching statistical significance.
Results from the genotyping of PAI –14G/5G
We found higher frequency of the pathogenic allele 4G – 58,6% (Table 2) compared to 26,9% world population frequency according to Ensemble genome database. The frequency of the homozygotes 4G/4G was 31,4% in comparison to 20,9% in the world and 29,4% in Europe.
Results from the genotyping of Factor XIII V34L
In our cohort we found lower frequency of 11,1% for the minor allele (Table 2) compared to 21,9% world population frequency and 25,2% in Europe. It is important to note that in the group with CVD the frequency is even lower – 7,5%, which suggests a protective role of this genetic variant.
Results from the genotyping of β-Fibrinogen-455 G/A
The overall allelic frequency of the pathological allele in our group was 22,2% which is higher than the world population frequency – 16,9%, and close to that in Europe – 20,3%. According to 1000 Genomes database the population frequency of the heterozygotes is 22% and we found it 38,9% (Table 2). It increases significantly in the group with CVD compared to the one without CVD – 55% versus 18,7% - figure 3
Results from the genotyping of HPA1(GPIIIa L33P)
We found allelic frequency of 12,5% (Table 2) which is comparable to the world population frequency – 12,1%; and that in Europe – 15,2%.
In order to conclude about the factors contributing to congenital thrombophilia we found higher frequencies for most of them than in the world population frequency but not reaching statistical significance (Figure 4A). The highest frequency was found for PAI-1 variant in patients with DM. The frequency of Factor XIII polymorphism was lower than that in the world which is in accordance to the suggested protective role of the polymorphism. When comparing the frequencies in the groups with and without CVD only the variants of PAI-1 and Fibrinogen show higher frequency in the group with CVD – figure 4B.
1.2 Results from the genotyping of MTHFRC677T and A1298C
The allelic frequency of MTHFR 677T we found was 25% (Table 3) and is a little lower than that of world population – 31%, and that in Europe – 32%. The allelic frequency of MTHFR 1298C in our study was 38,9% and it is higher than that in world – 29%, and in Europe – 32%.
Table 3. Allelic and genotypic frequencies of MTHFR C677T и MTHFR A1298C
Allele/Genotype
|
DM with CVD
|
DM without CVD
|
All
|
MTHFR 677T
|
9/40 (22.5%)
|
9/32 (28.1%)
|
18/72 (25%)
|
MTHFRC/T
|
5/20 (25%)
|
5/16 (31.2%)
|
10/36 (27.8%)
|
MTHFRT/T
|
2/20 (10%)
|
2/16 (12.5%)
|
4/36 (11.1%)
|
MTHFR 1298C
|
19/40 (47.5%)
|
9/32 (28.1%)
|
28/72 (38.9%)
|
MTHFRA/C
|
11/20 (55%)
|
5/16 (31.2%)
|
16/36 (44.4%)
|
MTHFRC/C
|
4/20 (20%)
|
2/16 (12.5%)
|
6/36 (16.7%)
|
1.3 Results from the genotyping of ACEI/D
We found a frequency of the homozygotes for the pathologic allele of 36,1% (Table 4) which is higher than population frequency in Europe – 25%.
Table 4. Allelic and genotypic frequencies of ACE I/D
Allele/Genotype
|
DM with CVD
|
DM without CVD
|
All
|
ACEDel
|
22/40 (55%)
|
21/32 (65.6%)
|
43/72 (59.7%)
|
ACEI/D
|
10/20 (50%)
|
7/16 (43.7%)
|
17/36 (47.2%)
|
ACED/D
|
6/20 (30%)
|
7/16 (43.7%)
|
13/36 (36.1%)
|
1.4 Results from the genotyping of ApoBR3500Q and ApoE
The ApoB mutation was not found in any of the patients and its world population frequency is 1:5000.
The frequency of the risk ApoE allele E4 we found was 13,9% (Table 5) and is comparable to the world population frequency – 13,8% and that in Europe – 16,1%
Table 5. Allelic and genotypic frequencies of Apo E2/E3/E4
Allele/Genotype
|
DM with CVD
|
DM without CVD
|
All
|
ApoE3/E4
|
2/20 (10%)
|
3/16 (18.7%)
|
5/36 (13.9%)
|
ApoE2/E4
|
1/20 (5%)
|
0
|
1/36 (2.8%)
|
ApoE3/E3
|
17/20 (85%)
|
13/16 (81.3%)
|
30/36 (83.3%)
|