Migration Stimulating Factor (MSF): Its Role in the Tumour Microenvironment.

Migration Stimulating Factor (MSF) is a 70 kDa truncated isoform of fibronectin (FN); its mRNA is generated from the FN gene by an unusual two-stage processing. Unlike full-length FN, MSF is not a matrix molecule but a soluble protein which displays cytokine-like activities not displayed by any other FN isoform due to steric hindrance. There are two isoforms of MSF; these are referred to as MSF+aa and MSF-aa, while the term MSF is used to include both.MSF was first identified as a motogen secreted by foetal and cancer-associated fibroblasts in tissue culture. It is also produced by sprouting (angiogenic) endothelial cells, tumour cells and activated macrophages. Keratinocytes and resting endothelial cells secrete inhibitors of MSF that have been identified as NGAL and IGFBP-7, respectively. MSF+aa and MSF-aa show distinct functionality in that only MSF+aa is inhibited by NGAL.MSF is present in 70-80% of all tumours examined, expressed by the tumour cells as well as by fibroblasts, endothelial cells and macrophages in the tumour microenvironment (TME). High MSF expression is associated with tumour progression and poor prognosis in all tumours examined, including breast carcinomas, non-small cell lung cancer (NSCLC), salivary gland tumours (SGT) and oral squamous cell carcinomas (OSCC). Epithelial and stromal MSF carry independent prognostic value. MSF is also expressed systemically in cancer patients, being detected in serum and produced by fibroblast from distal uninvolved skin. MSF-aa is the main isoform associated with cancer, whereas MSF+aa may be expressed by both normal and malignant tissues.The expression of MSF is not invariant; it may be switched on and off in a reversible manner, which requires precise interactions between soluble factors present in the TME and the extracellular matrix in contact with the cells. MSF expression in fibroblasts may be switched on by a transient exposure to several molecules, including TGFß1 and MSF itself, indicating an auto-inductive capacity.Acting by both paracrine and autocrine mechanisms, MSF stimulates cell migration/invasion, induces angiogenesis and cell differentiation and alters the matrix and cellular composition of the TME. MSF is also a survival factor for sprouting endothelial cells. IGD tri- and tetra-peptides mimic the motogenic and angiogenic activities of MSF, with both molecules inhibiting AKT activity and requiring αvß3 functionality. MSF is active at unprecedently low concentrations in a manner which is target cell specific. Thus, different bioactive motifs and extracellular matrix requirements apply to fibroblasts, endothelial cells and tumour cells. Unlike other motogenic and angiogenic factors, MSF does not affect cell proliferation but it stimulates tumour growth through its angiogenic effect and downstream mechanisms.The epithelial-stromal pattern of expression and range of bioactivities displayed puts MSF in the unique position of potentially promoting tumour progression from both the "seed" and the "soil" perspectives.

[Pregnancy outcomes in women with gestational diabetes: specific subgroups might require increased attention]. / Pacientky s obezitou, hypertenzí a nutností aplikace inzulinu pri diagnóze gestacní diabetes mellitus vyzadují zvýsenou porodnickou péci.

OBJECTIVES: To compare peri-partal parameters between two groups of pregnant women - with and without gestational diabetes mellitus (GDM), to correlate degree of glucose abnormality with incidence of peri-partal morbidity and, finally, to analyse the potential effect of comorbidities (i.e. obesity, hypertension, thyreopathy, polycystic ovary syndrome, trombophylia, anemia, allergy, smoking) on pregnancy outcomes. DESIGN: Epidemiological observational "case-control" study. SETTING: Department of Obstetric and Gynaecology, Faculty Hospital Brno; Department of Internal Medicine, Diabetes Centre, Faculty Hospital Brno; Department of Pathophysiology, Faculty of Medicine, Masaryk University, Brno. METHODS: The study comprised 432 pregnant women (364 with GDM diagnosis, 68 healthy controls) followed during a period 2011-2013. GDM was diagnosed by oral glucose tolerance test in 24-28th week of gestation (by fasting plasma glucose >5,6 mmol/l or >8,8 mmol/l in 60th min or >7,8 mmol/l in 120th min post-75g glucose load). Following peri-partal parameters were studied: ultrasonographic examination before delivery, a date of delivery, length of childbirth, induction, perinatal complications, post-delivery complications, section, abnormity in pH, base excess, Apgar score, birth weight. RESULTS: Subjects with GDM had significantly increased rate of labour induction compared to healthy controls (P = 0.0035, chi-square test). Subgroup of GDM women classified as having a higher risk for adverse perinatal outcomes by a definition of Czech Obstetric and Gynaecology Society had significantly more labour inductions, more sections and instrumental deliveries. New-borns of those mothers had significantly more common worse perinatal outcomes (Apgar score and macrosomia). CONCLUSION: Based on our data risk stratification of GDM subjects according to Czech Obstetric and Gynaecology Society appears relevant and justified.

Genetic and functional analyses of MRAS and HNF1A genes in diabetes and diabetic nephropathy.

Evidence has recently indicated that the MRAS and HNF1A genetic polymorphisms are associated with coronary artery disease. The MRAS and HNF1A genes are located on chromosomes 3q and 12q within the regions where associations with diabetes and diabetic nephropathy occur. We thus performed genetic and functional analyses of these two genes to evaluate their impacts on diabetes and diabetic nephropathy. MRAS and HNF1A genetic polymorphisms were genotyped in 1399 Czech subjects including non-diabetic controls (339), type 1 (243) and type 2 (817) diabetic patients with and without diabetic nephropathy using TaqMan allelic discrimination. Gene expression levels in the kidneys of diabetic Goto-Kakizaki and Wistar rats were detected with real-time RT-PCR. Despite no significance in genetic analysis of diabetic subjects, SNP rs2259816 in the HNF1A gene tended to associate with diabetic nephropathy in type 1 diabetic patients. The hnf1a gene expression was significantly decreased in kidney tissues of Goto-Kakizaki rats compared to Wistar and insulin-treated Goto-Kakizaki rats. There was neither significant association in the MRAS genetic polymorphism with diabetic nephropathy nor variation of mras gene expression in the kidneys of Goto-Kakizaki and Wistar rats. Data from the present study have not proved any significant association of the MRAS and HNF1A genetic polymorphisms with diabetes and diabetic nephropathy in a cohort of Czech population. However, the functional analysis and the trend in genetic analysis suggest that the HNF1A gene may have primary genetic impact on the development of diabetic nephropathy.

Cancer as a metabolic disease and diabetes as a cancer risk?

The prevailing aerobic glycolysis (so called Warburg effect) in cancer cells is according to current understanding the consequence of reprogramming of cellular metabolism during the process of malignant transformation. Metabolic regulation is inseparable component of cell proliferation machinery and has a tight link with activities of oncogenes and suppressor genes. The purpose of metabolic reprogramming of cancer (but also normal intensively proliferating cells) is to incorporate greater fraction of glucose metabolites into newly synthesised macromolecules. Apart from that, aerobic glycolysis confers several other selective advantages to cancer cells. Epidemiological data indicate that type 2 diabetes mellitus is associated with increased incidence of several types of cancer and that cancer mortality can be influenced by certain types of anti-diabetic treatment, however future research is needed to explain whether this relationship might be causal. Deeper knowledge about metabolic properties of rapidly proliferating cells can be exploited for further improvement of anti-cancer, immunosuppressive or anti-inflammatory therapies.

Increased activity of superoxide dismutase in advanced stages of head and neck squamous cell carcinoma with locoregional metastases.

The aim of the study was to investigate relationship between activity of superoxide dismutase (SOD), malondialdehyde (MDA) and tumor necrosis factor alpha (TNFalpha) and between Ala-9Val polymorphism in the gene encoding MnSOD (SOD2) and the initial stage and prognosis of the head and neck squamous cell carcinoma (HNSCC). Prospective study cohort comprised 88 patients who underwent surgical treatment for the diagnosis of HNSCC (53 patients were diagnosed with locoregional metastatic spread (N+) at the time of diagnosis). After the initial surgery subjects were followed for the subsequent period of 26 months during which 14 manifested relapse. Genotypes were detected by the PCR-based methodology. Activity of p-SOD, ery-SOD and TNFalpha were determined by ELISA, and the concentration of MDA by high performance liquid chromatography. Genotype and allele frequencies of the Ala-9Val differed neither between groups defined according to the stage of primary disease (TNM), nor between relapse vs. remission groups after the follow-up (p>0.05). Activity of p-SOD was significantly higher in T3/4 stage compared to T1/2 (p=0.01) and was also higher in N+ compared to N0 patients (p=0.002). Carriers of the Ala/Ala genotype had higher p-SOD activity (p=0.04). There was no significant difference in DFI between SOD2 genotype groups (p>0.05), however, the Ala/Ala group exhibited the shortest median DFI. In conclusion, our results suggest that increased p-SOD at the time of the initial treatment for HNSCC is connected with greater extent and nodal metastatic spread of the initial disease and with an earlier relapse of the disease. Progression of the disease might be further modified by the presence of Ala/Ala genotype of the SOD2. Activity of p-SOD could thus offer diagnostic as well as prognostic value.

Polymorphisms 1704G/T and 2184A/G in the RAGE gene are associated with antioxidant status.

The formation of advanced glycation end products (AGEs) and oxidative stress are supposed to play an important role in the development of diabetic late complications. AGEs can bind to several binding sites including receptor of advanced glycation end products (RAGE). AGE-RAGE interaction results in free radical generation. The aim of the present study was to investigate the impact of previously described polymorphisms in the RAGE gene (G82S, 1704G/T, 2184A/G, and 2245G/A) on the glycoxidation status in non-insulin-dependent diabetes mellitus (NIDDM). A total of 371 unrelated caucasian subjects were enrolled in the study. The NIDDM group consisted of 202 subjects, and the presence of late diabetic complications in 5 particular localizations was expressed as an index (I(compl)). The nondiabetic group included 169 subjects. Glycated hemoglobin (HbA(1c)), glycated stratum corneum proteins (Amadori, AGE), total carotenoids, alpha- and beta -carotene, gamma-tocopherol, lutein, lycopene, and alpha-tocopherol were measured in each subject. Statistically significant differences in allele frequencies between the NIDDM and the nondiabetic groups were observed for the G82S and 2245G/A polymorphisms (P =.047 and .032, respectively). HbA(1c), Amadori, and AGE did not reveal any significant association with any of the polymorphisms analyzed. However, significant differences between subjects bearing "wild-type majority" genotypes 1704GG+2184AA and subjects with "mutated" genotypes were found for total carotenoids (P =.001), alpha-carotene (P =.046), beta-carotene (P =.028), lutein (P =.001), lycopene (P =.006), and alpha-tocopherol (P =.047). I(compl) significantly correlated with the plasma levels of all antioxidants (all P <.01), while no correlation of I(compl) with glycation variables was observed. The newly identified intron polymorphisms in the RAGE gene were proved to be associated with the antioxidant status in NIDDM subjects. The extent of diabetic vascular disease is related to the plasma levels of antioxidants.

Polymorphism Ncol in tumor necrosis factor B is associated with fasting glycemia and lipid parameters in healthy non-obese caucasian subjects.

BACKGROUND: The study was designed to investigate the associations among polymorphisms TNF-B Ncol and TNF-alpha -308G/A, plasma TNF-alpha levels and metabolic and anthropometric parameters related to insulin sensitivity in a set of 113 Caucasian subjects undergoing oral glucose tolerance test (oGTT). METHODS: Genotypes were detected by PCR; BMI, WHR, glycemia during oGTT, fasting immunoreactive insulin, fasting C-peptide, HbA(1c), total cholesterol, triglycerides, HDL, LDL and plasma TNF-alpha levels were measured in each subject. RESULTS: Type 2 diabetes was diagnosed in 10 subjects, impaired glucose tolerance (IGT) in 41, normal glucose tolerance (NGT) in 62. Significant differences among genotypes of the TNF-B Ncol were observed for FPG (P=0.0063), LDL (P=0.0179) and marginally for total cholesterol (P=0.0763) in NGT group. After the classification of NGT subjects into obese and non-obese according to BMI, associations of TNF-B Ncol with FPG, LDL and cholesterol were proved in non-obese subgroup only. TNF-alpha -308G/A polymorphism was not associated with any of the parameters studied. TNF-alpha levels did not revealed difference among NGT, IGT and DM groups or genotype-dependent differences. CONCLUSIONS: Our results indicate significant association of the TNF-B Ncol polymorphism with FPG, LDL and total cholesterol in normoglycemic non-obese Caucasian subjects. This polymorphism could be involved in genetic modulation of glucose and lipid homeostasis and regulation of insulin sensitivity already in healthy state. Disturbances of this regulation could be component of pathogenesis of type 2 diabetes mellitus.

Relations among serum ferritin, C282Y and H63D mutations in the HFE gene and type 2 diabetes mellitus in the Czech population.

Aims of the study were: (i) to determine the prevalence of mutations C282Y and H63D in the HFE gene causing hereditary hemochromatosis in patients with type 2 diabetes mellitus and non-diabetics, (ii) to investigate the relationship among HFE genotypes, serum ferritin and glucose intolerance and (iii) to assess possible association of HFE mutations with the susceptibility to develop late diabetic complications in the Czech population. Two approaches were employed - the case-control study comprising diabetics and non-diabetic controls (n = 326) and the cross-sectional study comprising subjects with a previously unknown defect of glucose tolerance (n = 113, oral glucose tolerance test performed in each subject). Allele frequencies of C282Y and H63D did not differ between diabetic and control groups nor among subjects with normal glucose tolerance (NGT), impaired glucose tolerance (IGT) and diabetes. Ferritin levels significantly differed between diabetic and non-diabetic women (P<1.10 (-3)) and among subjects with NGT, IGT and diabetes (P<0.05). Differences in ferritin levels related to particular genotypes of C282Y and H63D were not detected. Prevalence of diabetes in the first and second quartiles of ferritin distribution differed highly significantly from the prevalence in the third and fourth quartiles in women (P = 0.000037), OR = 3.50 (95% CI, 1.89-6.48). The extent of diabetic late complications did not correlate with ferritin plasma levels.

Polymorphisms in the RAGE gene influence susceptibility to diabetes-associated microvascular dermatoses in NIDDM.

To examine genetic polymorphism in the complete sequence of the Receptor of Advanced Glycation End products (RAGE) gene and its possible associations with diabetes-associated microvascular dermatoses (DAMD). Further, to analyze the distribution of individual genotype combinations on the particular polymorphic loci in the RAGE gene. A part of the RAGE gene spanning a region from -4 to 3334 bp was analyzed on a set of 45 subjects with non-insulin dependent diabetes mellitus (NIDDM) and parallel DAMD by means of PCR with subsequent heteroduplex and single-strand conformation polymorphism (SSCP) analyses. Allele frequencies and genotype combinations of novel common polymorphisms were determined in an associations study comprising four groups of subjects (n=390). Fourteen novel polymorphisms (R77C, V89V, 718G/T, 1704G/T, 1727A1728ins, H305Q, S307C, 2117A/G, 2184A/G, 2245G/A, 2249A/G, 2741G/A, and 3089ACdel) and one described previously (G82S) were identified. Significant association with microvascular dermatoses (MD) irrespective of NIDDM were found for exon mutation 82S (P= .004, after a correction for the number of comparisons P(corr) < .05) and marginally significant for intron variant 1704T (P= .032, P(corr)> .05). Calculated odds ratios for 82S and 1704T were 4.73 (95% CI, 1.51 to 14.77) and 1.73 (95% CI, 0.93 to 3.22), respectively. Certain individual genotype combinations of G82S, 1704G/T, and 2184A/G were significantly associated with the presence of MD (P= .00647) both in diabetic and non-diabetic study populations. The two novel polymorphisms (1704G/T and 2184A/G) together with the G82S were shown to influence the susceptibility to MD independent of diabetes itself.

Polymorphisms in inflammation genes (angiotensinogen, TAP1 and TNF-beta) in psoriasis.

This study focused on the association between plaque psoriasis and polymorphisms of several inflammation genes. Included in the study were 142 Caucasian (Czech) patients with plaque psoriasis and 141 healthy subjects. The genotypes of the polymorphisms in angiotensinogen [M235T ATG, A(-6)G ATG], in transporters associated with antigen processing TAP1 (TAP1*0101, TAP*02011 and TAP1*0301) and in lymphotoxin alpha (TNFbeta) (NcoI in intron 1) were detected by polymerase chain reaction-based methods and restriction enzyme analysis. An increase in B1 (less frequent) allele of NcoI TNFbeta polymorphism was found in psoriatic patients compared to healthy individuals (odds ratio = 1.6, 95% confidence interval 1.13-2.26, P = 0.006). A positive family history of psoriasis was associated with a higher B1 allele frequency in NcoI TNFbeta (P = 0.011). Hardy-Weinberg disequilibrium was found in TAP1 polymorphism A-->G at nucleotide 1207 in psoriatic patients. A case-control difference was found in the allelic concurrence of M235T and A(-6)G ATG polymorphisms. The most frequent population genotypes MMGG, MTAG and TTAA were observed in 92% of patients vs 74% of control subjects (odds ratio 0.29, 95% confidence interval 0.14-0.60, P = 0.0003). A positive history of tonsillitis and/or tonsillectomy was associated with a higher T allele frequency of the M235T ATG polymorphism (P = 0.037) as well as with a higher G allele frequency of the A(-6)G ATG polymorphism (P = 0.022). Polymorphisms in proinflammatory angiotensinogen and TNFbeta genes were associated with plaque psoriasis, a positive family history of psoriasis and with frequent tonsillitis in childhood.

Gene polymorphisms (G82S, 1704G/T, 2184A/G and 2245G/A) of the receptor of advanced glycation end products (RAGE) in plaque psoriasis.

Having in mind the relationships among oxidative stress, psoriasis and common disorders, the association between polymorphisms in the gene encoding the receptor for advanced glycation end products (RAGE) and plaque psoriasis, including patients with a personal history of diabetes mellitus, cardiovascular disorders, cancer and allergy, was investigated. The allele frequencies and genotype distribution combinations of the four polymorphisms in the RAGE gene (6p21.3, G82S, 1704G/T, 2184A/G and 2245A/G) were compared in a case-control study of 272 subjects (130 patients with plaque psoriasis and 142 healthy control subjects of comparable age and sex distribution). The polymerase chain reaction with subsequent restriction analysis was used for detection of genotype variants. There was a significantly higher frequency of the 2184G allele of the 2184A/G RAGE polymorphism in psoriatic patients than in the control subjects (odds ratio 2.18, 95% CI 1.32-3.59, P=0.001). The 2184G allele occurred more often in psoriatic patients with a negative history of cardiovascular diseases (odds ratio 2.38, 95% CI 1.35-4.18, P=0.001, Pcorr=0.004), in those with a negative history of diabetes mellitus (odds ratio 2.05, 95% CI 0.1.22-3.45, P=0.004, Pcorr=0.012) and in those with a negative history of cancer (odds ratio 1.97, 95% CI 1.17-3.31, P=0.007, Pcorr=0.014) compared with the corresponding control subjects. We conclude that the 2184G allele of the RAGE gene is a significant risk factor for plaque psoriasis. The risk is associated with the non-presence of some common, especially cardiovascular, diseases in psoriatic patients.

Distribution of the receptor for advanced glycation end products gene polymorphisms in patients with chronic periodontitis: a preliminary study.

BACKGROUND: Periodontal diseases are viewed today as multifactorial problems initiated and sustained by bacteria but significantly modified by the body's response to bacterial plaque. A recent study suggested that receptor for advanced glycation end products (RAGE) could be involved in the pathophysiology of periodontitis. The aim of this study was to investigate a possible association of 3 common polymorphisms in the RAGE gene with chronic periodontitis. METHODS: We studied 101 Caucasian patients with chronic periodontitis together with 162 orally healthy subjects. Three polymorphisms, one in intron 7 (1704G/T), second in intron 8 (2184A/G), and the third in exon 3 (G82S) of the RAGE gene, were investigated by polymerase chain reaction methods (PCR) with subsequent enzymatic restriction with Bfal, BsmFI, or Alu Il, respectively. RESULTS: A statistically significant difference in allele frequencies between patients and the reference group was found for intron variant 1704G/T (P= 0.02, Pcorr >0.05). There was no significant difference in genotype or allele frequency distributions between groups for intron variant 2184A/G or for the exon variant exchanging amino acid Gly for Ser at position 82 (G82S). CONCLUSIONS: We can speculate that susceptibility to the development of chronic periodontitis could be influenced by the 1704G/T polymorphism of the RAGE gene, independently of diabetes.

[New knowledge on iron metabolism and its disorders]. / Novejsí poznatky o metabolizmu zeleza a jeho poruchách.

Iron is an essential element to all living organisms. It is a component of many proteins with important functions in physiological processes such as oxygen transport, respiration, DNA synthesis, cell cycle regulation and many others. Free iron is highly reactive and its excess can lead to tissue and organ damage. Intestinal absorption of iron is precisely regulated because there is no excretory mechanism for excessive iron. Improved methodology led to the identification of many genes and proteins involved in the iron metabolism and to the understanding of basic processes of iron intake, transport and storage. However, some aspects remain still unclear--primarily the regulation of iron intake according to the body's requirements. Disorders of iron metabolism, both the deficiency and the overload belong to relatively common diseases. Growing understanding of the physiology of the iron metabolism is rapidly reflected in diagnostics, preventive screening and therapy of the iron disorders.

[The Rel/NF-kappa B transcription factors and their biological functions]. / Transkripcní faktory Rel/NF-kappa B a jejich biologické funkce.

The gene family of ubiquitous transcriptional factors Rel/NF-kappa B participates in several critical cellular events ultimately influencing the fate of a cell. In the cytoplasm of all eukaryotic cells, the Rel/NF-kappa B proteins occur bound to their inhibitors, the I kappa B proteins. When stimulated, they become phosphorylated and degraded by 26S proteasome releasing Rel/NF-kappa B. Active Rel/NF-kappa B heterodimers then enter the nucleus, bind to a -kappa B coupling element and start transcription of kappa B-regulated array of genes involved in immune, antiapoptotic and inflammatory events. Part of the pharmacological effects of glucocorticoids, acetylosalicylic acid an ibuprofen may stand on influencing the Rel/NF-kappa B pathway. New compounds with cytotoxic and immunomodulatory properties acting specifically on the cascade of Rel/NF-kappa B signaling system are expected to be synthesized.

[Molecular pathophysiology of late complications diabetes mellitus--hyperglycemia-induced changes]. / Molekulární patofyziologie pozdních komplikací piri diabetes mellitus-- hyperglykémií indukované zmeny.

Late diabetic complications due to vascular and extravascular impairments develop as a consequence of chronic diabetes mellitus. Extent of affection reflects disease duration and therapeutic compensation; however, other modulating factors are involved. Due to growing incidence and permanent shift to younger age diabetes represents serious health problem. T2DM develops in consequence of "dysadaptation" of human genome to rapidly changing environment and life style. Primary prevention of diabetes is rather limited at present, secondary prevention or minimalization of late consequences is practically achievable. Full understanding of pathogenesis and identification of high-risk diabetic subjects will help to upgrade therapeutical options and improve patient's prognosis. This review devoted to late diabetic complications will summarize recent findings about proximal hyperglycaemia-induced alterations leading to common pathogenic action - inhibition of glycolysis on the level of GAPDH due to increased ratio NADH/NAD+, generation of superoxide and intracellular accumulation of dicarbonyls. Activated expression of series of genes leads to tissue remodelation responsible for organ manifestation. Subsequent article will deal with putative genetic susceptibility to their development.

Functional analysis of the common haplotype in the receptor for advanced glycation end-products gene previously identified as a susceptibility factor for diabetic nephropathy.

The Receptor for Advanced Glycation End-products (RAGE) belongs to the family of pattern-recognition receptors and is significantly involved in the molecular mechanisms mediating pro-inflammatory action of hyperglycemia in diabetes. The aim of the current study was to elucidate the possible functional impact of the genetic variability in the AGER gene constituting previously identified risk haplotype for diabetic nephropathy (-429C/-374T/2184G) by testing the haplotype-specific effect on the AGER gene transcriptional activity IN VITRO. Promotor and intron 8 constructs carrying respective substitutions were amplified and cloned into pGL3-Basic reporter vector and subsequently used for transfection of HEK293 cells. Following 48hrs incubation in either normo- (5 mM/L) or hyperglycemic (25 mM/L) culture medium luciferase activity was measured to assess transcriptional efficiency. Risk haplotype was associated with the highest transcriptional activity in hyperglycemia and greatest relative increase of activity between normo- and hyperglycemia conditions (approx. 3-times). We conclude that ascertained functional differences in the regulatory regions of the AGER gene might have significant consequences for the development of hyperglycemia-related pathology in diabetics.

Genetic risk factors for diabetic nephropathy on chromosomes 6p and 7q identified by the set-association approach.

AIMS/HYPOTHESIS: In the present study we investigated potential associations of a set of 45 single nucleotide polymorphisms (SNP) in 20 candidate genes on eight chromosomes with diabetic nephropathy (DN) in type 2 diabetes mellitus. We aimed to compare two methodological approaches suitable for analysing susceptibility to complex traits: single- and multi-locus analyses. MATERIALS AND METHODS: The study comprised a total of 647 subjects in one of three groups: diabetes with or without DN, or no diabetes. Genotypes were detected by PCR-based methodology (PCR only, PCR plus RFLP, or allele-specific PCR). Haplotypes were inferred in silico. Set association (tested using SUMSTAT software) was used for multilocus analysis. RESULTS: After correction for multiple comparisons, only one SNP, in the gene encoding the receptor of advanced glycation end products, AGER 2184A/G (gene symbol formerly known as RAGE) showed a significant association with DN (p = 0.0006) in single-locus analysis. In multi-locus analysis, six SNPs exhibited a significant association with DN: four SNPs on chromosome 6p (AGER 2184A/G, LTA 252A/G, EDN1 8002G/A and AGER -429T/C) and two SNPs on chromosome 7q (NOS3 774C/T and NOS3 E298D), omnibus p = 0.033. Haplotype analysis revealed significant differences between DN and control groups in haplotype frequencies on chromosome 6 (p = 0.0002); however, there were no significant difference in the frequencies of the NOS3 haplotypes on chromosome 7. Logistic regression analysis identified SNPs AGER 2184A/G and NOS3 774C/T, together with diabetes duration and HbA1c, as significant predictors of DN. Testing for interactions between SNPs on chromosomes 6 and 7 did not provide significant evidence for epistatic interaction. CONCLUSIONS/INTERPRETATION: Using the set-association approach we identified significant associations of several SNPs on chromosomes 6 and 7 with DN. The single- and multi-locus analyses represent complementary methods.