Darko Grujii 1 Ljiljana Mirkov 2 Kristina Virijevi

Darko Grujičić 1*, Ljiljana Mirkov 2, Kristina Virijević 1, Nikola Pivljaković 1, Dragoslav Marinković 3, Olivera Milošević-Djordjević 1, 4 1 University of Kragujevac, Faculty of Science, Department of Biology and Ecology, Kragujevac, Serbia 2 Health Care Center, Kragujevac, Serbia 3 Serbian Academy of Sciences and Arts, Belgrade, Serbia 4 University of Kragujevac, Serbia, Faculty of Medical Sciences, Department of Genetics, Kragujevac, Serbia *Corresponding author, E-mail: darko@kg. ac. rs Homozygous-recessive characteristics (HRCs) and biochemical parameters as biomarkers of cardiovascular disease risk

Abstract Ø Cardiovascular diseases (CVD) are a leading cause of death globally. The aim of our study was to analyse homozygousrecessive characteristics (HRCs) and some biochemical parameters such as concentration of glucose, cholesterol, triglyceride in blood in the group of CVD patients and in the control sample, as well as to evaluate whethere is a predisposition to the occurrence of CVD, when these biomarkers are concerned. This study analysed presence, distribution, and individual variability of 20 selected genetically controlled morphophysiological traits among 90 CVD patients (45 males and 45 females), average 62. 80 ± 10. 20 and 90 healthy controls (45 males and 45 females), average 50. 39 ± 9. 17. Our results showed a significant difference in the individual variation of HRCs between the patients and controls (χ²=315. 34; p < 0. 0001). Statistically significant differences were found in the frequency of 9 out of 20 analyzed HRCs. The average value of HRCs in group patients was significantly higher, comparing to the control sample (6. 37 ± 1. 71 vs. 4. 97 ± 1. 68; p < 0. 001), while variability decreases compared to the control sample (VCVD= 0. 27%; VC= 0. 34%). There is not a statistical difference in the HRC between males and females in analyzed samples. In the group of CVD patients, the frequency of HRCs for males was 6. 56 ± 1. 69, while for females, it was 6. 18 ± 1. 74. In controls the frequency of HRCs for males was 5. 02 ± 1. 74, and for females it was 4. 91 ± 1. 64. This study showed difference in the type of distribution HRCs; in the CVD patients’ the most were with 5 to 8/20, while in control sample were with 3 to 6/20 recessive traits. Regarding biochemical parameters, our results showed a significantly higher concentration of glucose and triglycerides in the blood the CVD patients compared to control sample (t=3. 14, p < 0. 001; t=4. 20; p < 0. 0001). The distribution of ABO blood type frequencies between tested samples showed that in both samples the most present is A blood type, but significantly more present in the sample of CVD patients (51. 1% vs. 37. 2%). The results of multiple linear regression analysis of tested variables (gender, smoking, glucose, cholesterol, triglyceride, HRCs) showed that only HRCs and triglyceride concentration have a significant impact on the onset of the CVDs. Our conclusion is that the increased degree of recessive homozygosity and decreased variability homozygous-recessive characteristics of CVD patients versus controls, regardless of the gender, indicates a potential genetic predisposition for CVD, including biochemical parameters such as elevated concentration triglycerides can significantly contribute to the development of this predisposition.

Ø Cardiovascular diseases (CVD) are a leading cause of death globally. Ø The aim of our study was to analyse homozygous-recessive characteristics (HRCs) and some biochemical parameters such as concentration of glucose, cholesterol, triglyceride in blood in the group of CVD patients and in the control sample, as well as to evaluate whethere is a predisposition to the occurrence of CVD, when these biomarkers are concerned. Ø This study analysed presence, distribution, and individual variability of 20 selected genetically controlled morphophysiological traits among 90 CVD patients (45 males and 45 females), average 62. 80 ± 10. 20 and 90 healthy controls (45 males and 45 females), average 50. 39 ± 9. 17.

Table 1. Frequency of HRCs among control (C) and cardiovascular (CVD) patients (m-men; f-female) No. C sample; N = 90 f Total (%) χ² Homozygous-recessive characteristics m 1. Blond hair 8 4 12 (13. 33%) 7 13 20 (22. 22%) 5. 33 2. 3. 4. 5 6. 7. 8. 9. 10. Continuous frontal hairline Straight hair Double hair whorl Blue eyes Attached ear lobe Ear without Darwinian notch Inability to Longitudinally Tongue Roll Guttural “r” Daltonisms 12 37 13 15 11 11 15 0 1 22 28 10 9 17 18 12 2 0 34 (37. 78%) 65 (72. 22%) 23 (25. 56%) 24 (26. 67%) 28 (31. 11%) 29 (32. 22%) 27 (30. 00%) 2 (2. 22%) 1 (1. 11%) 18 37 16 17 14 17 25 8 3 16 29 22 14 8 13 29 5 0 34 (37. 78%) 66 (73. 33%) 38 (42. 22%) 31 (34. 44%) 22 (24. 44%) 30 (33. 33%) 54 (60. 00%) 13 (14. 44%) 3 (3. 33%) 0. 00 0. 02 9. 78 2. 04 1. 29 0. 03 27. 00 60. 5 4. 00 11. 12. 13. 14. Right thumb over left thumb (hand clasping) Top joint of the thumb >45 o Left-handedness Digital index: finger longer (men) i. e shorter (women) than the ring finger Mid-phalangeal hair absence Soft hair Retraction of anterior teeth Retracted chin Proximal thumb hyperextensibility Inability to transversally Tongue Roll 23 12 4 16 19 15 0 10 42 (46. 67%) 27 (30. 00%) 4 (4. 44%) 26 (28. 89%) 25 18 1 12 23 12 2 22 48 (53. 33%) 30 (33. 33%) 3 (3. 33%) 34 (35. 56%) 0. 86 0. 33 0. 25 2. 46 16 17 2 7 5 1 7 29 2 11 5 1 23 (25. 56%) 46 (51. 11%) 4 (4. 44%) 18 (20. 00%) 10 (11. 11%) 2 (2. 22%) 12 29 8 14 3 11 18 31 6 14 2 9 30 (33. 33%) 2. 13 60 (66. 67%) 4. 26 14 (15. 56%) 25. 00 28 (31. 11%) 5. 56 5 (5. 56%) 2. 50 20 (22. 22%) 162. 00 χ²=315. 34; df=19; р <0. 001 15. 16. 17. 18. 19. 20. m CVD sample; N = 90 f Total (%)

Table 2. Average values of HRCs in the group of patients with CVD and the control Cm No. of persons 45 HRCs index (Х ± SD) 5. 02± 1. 74 Variability (V) (%) 0. 35 Cf 45 4. 91± 1. 64 0. 33 C Total 90 4. 97± 1. 68 0. 34 CVDm 45 6. 56± 1. 69 0. 26 CVDf 45 6. 18± 1. 74 0. 28 CVD Total 90 6. 37± 1. 71 0. 27 Student’s t- test Р t HRC Cм/Cf =0. 31; df=88 > 0. 05 t HRC CVDм/CVDf=1. 05; df=88 > 0. 05 t HRC C/CVD = 5. 54 ; df= 178 < 0. 001

Table 3. Distribution of HRCs in the group of patients with CVD and in control sample Number of HRCs person m 1. 1 / / / 2. 1 1 2 / 1 1 3. 7 10 17 1 2 3 4. 9 9 18 4 6 10 5. 11 7 18 8 6 14 6. 6 12 18 9 8 17 7. 5 4 9 9 11 20 8. 5 / 5 10 8 18 9. / 2 2 1 3 4 10. / / / 3 m- male; f- female Control sample N = 90 f Total m f CVD patients N = 90 Total

Figure 1. Distribution of HRCs in the group of patients with CVD and control sample 1 -10/20

Table 4. Average values of biochemical parameters among CVD patients and controls Glucose (Х ± SD) Cholesterol (Х ± SD) Triglycerides (Х ± SD) Cm 5. 36± 1. 00 5. 12± 0. 95 1. 24± 0. 36 Cf 5. 54± 0. 95 5. 45± 0. 98 6. 12± 1. 83 5. 66± 0. 84 5. 88± 1. 43** 5. 48± 1. 06 5. 29± 1. 01 5. 26± 1. 62 5. 65± 1. 27 5. 45± 1. 46 1. 25± 0. 68 1. 22± 0. 55 1. 64± 0. 77 1. 66± 0. 86 1. 65± 0. 81*** Cm+f CVDm CVDf CVDm+f t. CVD/C glucose =3. 14, p < 0. 01; t. CVD/C triglycerides =4. 20; p < 0. 0001

Table 5. Frequencies of ABO blood types and Rh among CVD patients and controls A B AB O Rh+ Rh- Cm 17 8 4 14 39 4 Cf 15 11 6 11 36 7 C Total 32 (37. 2%) 19 (22. 1%) 10 (11. 6%) 25 (29. 1%) 75 (87. 2%) 11 (12. 8%) CVDm 25 5 5 10 42 3 CVDf 20 4 2 17 39 5 45 (51. 1%) 9 (10. 0%) 7 (8. 0%) 17 (31%) 81 (91. 0%) 8 (9. 0%) CVD Total

Table 6. The results of multiple linear regression analysis of tested variable Coefficientsa Unstandardized Coefficients Model 1 B (Constant) sex Std. Error 2. 429. 034 . 329. 078 . 010 . 049 HRC -. 105 Glucose Standardize d Coefficients Beta t Sig. 95. 0% Confidence Interval for B Lower Upper Bound . 034 7. 385. 432 . 000. 667 1. 778 -. 121 3. 079. 189 . 016 . 199 . 842 -. 088 . 108 . 021 -. 384 -4. 969 . 000 -. 147 -. 063 -. 075 . 040 -. 147 -1. 862 . 065 -. 154 . 005 . 015 . 034 . 447 . 656 -. 053 . 083 -. 135 . 061 -. 191 -2. 222 . 028 -. 255 -. 015 ABO . 025 . 030 . 064 . 837 . 404 -. 034 . 084 Rh . 076 . 123 . 048 . 621 . 536 -. 167 . 320 smoking Cholesterol Triglycerides a. Dependent Variable: sample . 038

Conclusions Based our results we demonstrated that: 1. Increased degree of recessive homozygosity was observed for tested homozygousrecessive characteristics in CVD patients compared with healthy persons, regardless of gender. 2. There is a reduced variability of the examined HRCs in CVD patients compared to healthy persons, regardless of gender. These findings indicate a potential genetic predisposition to CVD. 3. Of all tested biochemical parameters (glucose, cholesterol, triglycerides), only elevated concentration triglycerides can significantly contribute to the development of this predisposition.