**3.1. Relationship between G+276T single nucleotide polymorphism of adiponectin gene and markers of insulin resistance in dyslipidemic patients**

In many recent studies, the adiponectin gene has been proposed as a potential candidate gene for insulin resistance but only a few of them have confirmed this relationship. Insulin resistance is considered the key factor in the patogenesis of common disorders, such as atherosclerosis, metabolic syndrome and diabetes mellitus. The genetic backround is likely to be polygenic but the genes involved are mostly unknown.

In our work, we have studied the possible relationship between single nucleotide polymorphism G+276T and IR markers, including lipid and lipoprotein profiles and adiponectin plasma levels in 355 dyslipidemic patients and their first-degree relatives.

#### **3.2. Subjects**

The group consisted of 355 patients attending Lipid Center of 3rd Medical Clinic, Faculty Hospital Olomouc, and their first-degree relatives. Patients had the first examination between January 2004 and January 2006. All patients were examinated by a physician and the family history were collected and medical history with physical examinations were performed. All individuals were tested for secondary hyperlipidemia, especially on the presence of diabetes mellitus, hypothyroidism, hepatic and renal failure and nephrotic syndrome. Violation of the following criteria led to exclusion from the study: hypolipidemic treatment in the previous 6 weeks, the presence of secondary hyperlipidemia, acute infection, acute cardiovascular or cerebrovascular attack within the past 3 months, cardiac disease (NYHA III and IV). Participiants were also divided into three groups. Group G1 included the presence of individuals with clinically manifest atherosclerosis, the group G2 individuals with dyslipidemia defined by Sniderman [25] (apolipoprotein B > 1.2 g/l and/or triglycerides > 1.5 mmol/l) but without clinical signs of the presence of atherosclerosis. Group 3 consisted of healthy individuals with the apolipoprotein B < 1.2 g/l and triglycerides < 1.5 mmol/l. The participants signed informed consent before taking a blood sample for DNA testing. The study was approved by the Ethical Committee of the Faculty of Medicine, Faculty Hospital Olomouc.

#### **3.3. Materials and methods**

684 Lipoproteins – Role in Health and Diseases

11391A and T+45G.

**3.2. Subjects** 

association of SNP T+45G with insulin resistance [22]. The T+276 G polymorphism was associated with higher BMI, lower insulin and adiponectin, but, unlike previous study, in the TT genotype. In discussion the authors analyzed possible causes of these results and conclude the same mentioned above. There is the high probability of the existence of further SNPs or gene mutations, which is in linkage disequilibrium with SNP +276 and which determines its effect. Variations in the adiponectin gene and risk for subsequent type 2

A prospective study focused on the determination of SNPs participation in the development of IR in French population found that variations in the adiponectin gene affects weight gain, body fat distribution and the development and the onset of hyperglycemia, as well as serum adiponectin [24]. At the start of a three-year study, the normoglycemic individuals with no signs of diabetes or impaired glucose tolerance were influenced mainly by two SNPs: G-

An interesting work was published in 2006 in Clinical Chemistry by Hegener et al. [24]; the prospective study monitoring the risk of atherothrombotic disease in individuals with no signs of diabetes. Five SNPs in the gene for adiponectin were investigated in 600 Caucasian men with subsequent aterotrombotic events (myocardial infarction or stroke) and 600 controls. After adjustment for potential risk factors, regression analysis then revealed two variants with a decreased risk of stroke (C-11377G and G-11066A). This study has provided

evidence of links of specific adiponectin gene variants with reduced risk of stroke.

**3.1. Relationship between G+276T single nucleotide polymorphism of** 

to be polygenic but the genes involved are mostly unknown.

**adiponectin gene and markers of insulin resistance in dyslipidemic patients** 

In many recent studies, the adiponectin gene has been proposed as a potential candidate gene for insulin resistance but only a few of them have confirmed this relationship. Insulin resistance is considered the key factor in the patogenesis of common disorders, such as atherosclerosis, metabolic syndrome and diabetes mellitus. The genetic backround is likely

In our work, we have studied the possible relationship between single nucleotide polymorphism G+276T and IR markers, including lipid and lipoprotein profiles and adiponectin plasma levels in 355 dyslipidemic patients and their first-degree relatives.

The group consisted of 355 patients attending Lipid Center of 3rd Medical Clinic, Faculty Hospital Olomouc, and their first-degree relatives. Patients had the first examination between January 2004 and January 2006. All patients were examinated by a physician and the family history were collected and medical history with physical examinations were performed. All individuals were tested for secondary hyperlipidemia, especially on the presence of diabetes mellitus, hypothyroidism, hepatic and renal failure and nephrotic syndrome. Violation of the following criteria led to exclusion from the study: hypolipidemic

diabetes in women has been of interest in the study of Hu et al. [23].

Venous blood for biochemical tests were collected after 12-hour fasting. Total cholesterol, HDL cholesterol and triglycerides were determined enzymatically using an analyzer Modular SWA (Roche, Switzerland), as well as other routine biochemical analyses. LDL cholesterol was calculated using the Friedewald equation for specimens with TG < 4.5 mmol/l (available for 242 subjects). Concentrations of apolipoproteins AI and B were determined by immunoturbidimetric method, as well as C-reactive protein levels, established by highly sensitive method (all Roche, Switzerland). Insulin was determined by IRMA (Immunotech, France). HOMA parameter (homeostatic model) was calculated from the formula: fasting glucose x fasting insulin / 22.5. C-peptide and proinsulin were determined by commercially available kits (Immunotech, France, DRG Instruments GmbH, Germany, respectively). Serum levels of soluble adhesion molecules ICAM 1 and VCAM 1 were analyzed by immunoenzymatic technique (Immunotech, France). Adiponectin determination was performed by the ELISA method (BioVendor, Czech Republic). The following markers of endothelial dysfunction were examined: plasminogen activator inhibitor-1 (PAI-1) and tissue plasminogen activator (tPA), both determined by ELISA methods (Technoclone, Vienna, Austria). Concentrations of adhesion molecules, insulin, proinsulin, C-peptide and adiponectin were measured on samples frozen at - 80 ° C until analysis.

G+276T adiponectin gene SNP was detected by real time polymerase chain reaction with fluorescent hybridization probes (FRET) on the Light Cycler instrument, v.2.0 (Roche), according Fillipi et al [22]. Genotyping was performed after the isolation of DNA from peripheral blood samples using phenol method [26]. DNA isolates were then stored at - 20 °C until analysis. The primer and probe synthesis was made at the in TibMolbiol (Germany). The sequence of oligonucleotides for the detection of SNP +276 G> T were as follows:

Primers:

5'- GGC CTC TTT CAT CAC AGA CC -3'

5'- AGA TGC AGC AAA GCC AAA GT -3'

#### Probes:

#### 5'- AAG CTT TGC TTT CTC CCT GTG TCT A--FL

#### 5'- LCRed640- GCC TTA GTT AAT AAT GAA TGC CTT—PH

Individual genotypes were determined by melting curve analysis after the amplification process. The fluorescence signal was converted and delivered to the graph as the dependency of negative fluorescence change with temperature (y axis) on temperature (x axis). As the result, creation of the characteristic peaks representing the melting temperature of the product and allow to distinguish the genotypes GG, GT and TT was performed. Example of analysis is shown in Figure 2.

#### **3.4. Statistical analysis**

Quantitative data were expressed as a mean ± standard deviation. Parameters with abnormal distribution were logarithmically transformed before statistical analysis. Differences between genotypes in continuous variables were determined by using ANOVA after adjustment for age, gender and waist circumference (SPSS 12.0 statistical package, SPSS Inc., USA). Furthermore, the calculation of frequency of alleles (G and T) and genotypes (GG, GT and TT) in individual groups and subgroups were performed.

**Figure 3.** An example of the melting curve analyses for G+276T polymorphism of adiponectine gene. (melting temperature for T and G alelles: Tm(T) = 54.8 ± 1.5 ºC, Tm(G) = 61.3 ± 1.5 ºC).

#### **3.5. Results**

686 Lipoproteins – Role in Health and Diseases

5'- AAG CTT TGC TTT CTC CCT GTG TCT A--FL

Example of analysis is shown in Figure 2.

**3.4. Statistical analysis** 

5'- LCRed640- GCC TTA GTT AAT AAT GAA TGC CTT—PH

Individual genotypes were determined by melting curve analysis after the amplification process. The fluorescence signal was converted and delivered to the graph as the dependency of negative fluorescence change with temperature (y axis) on temperature (x axis). As the result, creation of the characteristic peaks representing the melting temperature of the product and allow to distinguish the genotypes GG, GT and TT was performed.

Quantitative data were expressed as a mean ± standard deviation. Parameters with abnormal distribution were logarithmically transformed before statistical analysis. Differences between genotypes in continuous variables were determined by using ANOVA after adjustment for age, gender and waist circumference (SPSS 12.0 statistical package, SPSS Inc., USA). Furthermore, the calculation of frequency of alleles (G and T) and

genotypes (GG, GT and TT) in individual groups and subgroups were performed.

**Figure 3.** An example of the melting curve analyses for G+276T polymorphism of adiponectine gene.

(melting temperature for T and G alelles: Tm(T) = 54.8 ± 1.5 ºC, Tm(G) = 61.3 ± 1.5 ºC).

Probes:

In Table 1 the clinical and laboratory characteristics of the groups of dyslipidemic patients divided according to genotypes at position +276 of the gene for adiponectin are shown. Table 2 presents the results of laboratory parameters that differed significantly between each of groups determined by genotype at position +276. The data are adjusted for age, gender and waist circumference. The results show that the GG genotype carriers had significantly higher levels of total cholesterol (GG: 6.54 ± 1.74 mmol/l, GT: 6.18 ± 1.45 mmol/l, TT: 6.25 ± 1.64 mmol/l, p < 0.05) and LDL cholesterol (GG: 4.12 ± 1.49 mmol/l, GT: 3.78 ± 1.31 mmol/l, TT: 3.70 ± 1.34 mmol/l, p < 0.05) than T allele carriers. In heterozygotes, however, the presence of T allele at position +276 was associated with higher concentrations of PAI-1 (GG: 71.50 ± 41.0 μg/l, GT: 81.0 ± 38.7 μg/l, TT: 70.14 ± 44.4 μg/l, p < 0.05). We did not find any significant association with other markers of IR, such as BMI, blood glucose, insulin, or serum adiponectin. Table 3 depicts the frequencies of genotypes and alleles at position +276, Table 4 then presents the distribution of genotypes in groups according to triglyceride levels (cut-off value of TGL = 1.5 mmol/l).


\* GG vs. GT+TT, p < 0.05

**Table 1.** Clinical and laboratory characteristics according to adiponectin genotypes at position +276 (G+276T).


\*only 242 patients included

**Table 2.** Laboratory characteristics according to adiponectin genotypes at position +276 (G+276T) with significant differences between groups (GG vs. GT+TT, after adjustment for sex, age and BMI).


**Table 3.** Genotype and allele frequencies for G+276T polymorphism in dyslipidemic patients.


Chi-square 1.981, p = 0.37

**Table 4.** Genotype frequencies for G+276T polymorphism in dyslipidemic patients according to level of triglycerides (mmol/l).

#### **3.6. Discussion**

Insulin resistance is considered the key factor in the pathogenesis of complex diseases such as atherosclerosis, metabolic syndrome and diabetes mellitus. Genetic background IR is probably multifactorial but the participating genes are largely unknown.

In this study, the relationship of polymorphism G+276T of adiponectin gene and markers of insulin resistance was investigated. We found an association between genotype GT and one marker of IR, PAI-I. However, we found no association with serum adiponectin, insulin, HOMA and BMI. Our work did not confirm the preliminary findings from 2005, where the relationship between the adhesion molecules ICAM 1 and TT genotype was observed [27].

Possible association between SNPs and dyslipidemic phenotypes defined by Sniderman classification, based on serum TGL and apo B, was not seen. We found no linkage (data not specified), even in a situation where the only criterion was TGL alone. The genotype distribution in this case was comparable in both groups.

As shown in Table 1, GG genotype was associated with higher levels of total cholesterol and LDL cholesterol compared with GT and TT genotypes. This was found in our previous study as well [27].

Table 3 displays the fact that distribution of genotypes at position 276 is comparable with those published in previous works [20, 21, 22].

#### **3.7. Conclusions**

688 Lipoproteins – Role in Health and Diseases

\*only 242 patients included

Chi-square 1.981, p = 0.37

triglycerides (mmol/l).

**3.6. Discussion** 

GG GT TT p

PAI-I (μg/l) 71.5 ± 41.0 81.0 ± 38.7 70.14 ± 44.4 < 0.05

**Table 2.** Laboratory characteristics according to adiponectin genotypes at position +276 (G+276T) with significant differences between groups (GG vs. GT+TT, after adjustment for sex, age and BMI).

APM1 G+276T Patients (n = 355)

GG 188 (53 %) GT 144 (41 %) TT 23 (6 %)

G 520 (73 %) T 190 (27 %)

**Table 3.** Genotype and allele frequencies for G+276T polymorphism in dyslipidemic patients.

Genotype GG GT TT TG ≤ 1.5 (n = 225) 119 (53 %) 94 (42 %) 12 (5 %) TG > 1.5 (n = 148) 85 (57 %) 52 (35 %) 11 (8 %)

**Table 4.** Genotype frequencies for G+276T polymorphism in dyslipidemic patients according to level of

Insulin resistance is considered the key factor in the pathogenesis of complex diseases such as atherosclerosis, metabolic syndrome and diabetes mellitus. Genetic background IR is

In this study, the relationship of polymorphism G+276T of adiponectin gene and markers of insulin resistance was investigated. We found an association between genotype GT and one marker of IR, PAI-I. However, we found no association with serum adiponectin, insulin, HOMA and BMI. Our work did not confirm the preliminary findings from 2005, where the relationship between the adhesion molecules ICAM 1 and TT genotype was observed [27].

Possible association between SNPs and dyslipidemic phenotypes defined by Sniderman classification, based on serum TGL and apo B, was not seen. We found no linkage (data not specified), even in a situation where the only criterion was TGL alone. The genotype

probably multifactorial but the participating genes are largely unknown.

distribution in this case was comparable in both groups.

Genotype

Allele

Number 188 144 23 Total cholesterol (mmol/l) 6.54 ± 1.74 6.18 ± 1.45 6.25 ± 1.64 < 0.05 LDL cholesterol (mmol/l)\* 4.12 ± 1.49 3.78 ± 1.31 3.70 ± 1.34 < 0.05

> In summary, our study found only a weak association of adiponectin gene SNP G+276T with IR markers. The relationship of GG genotype and selected quantitative lipid parameters were confirmed, in accordance with several studies. Based on some recent literature we suggest the gene variant G+276T may be marker of one or more haplotypes containing a causal polymorphism determining IR or diabetes mellitus. Differences among populations on the linkage disequilibrium structure may result in association on the disease haplotype with different SNP alleles in different population. More studies will be necessary to perform for evaluation of the influence of G+276T SNP on insulin resistance.
