Marina Korolija

Association of PTPN22 C1858T and CTLA-4 A49G polymorphisms with type 1 diabetes in Croatians

In this case-control study the association between the PTPN22 1858T and CTLA-4 49G gene variants and T1D in Croatian population was examined. We found that distribution of PTPN22 C1858T and CTLA-4 A49G genotypes between T1D patient (n=102) and control (n=193) groups differ significantly (p<0.0001 and p=0.012, respectively). Moreover, although the risk alleles of both SNPs are distributed more frequently in patients, the significant difference is observed only for PTPN22 1858T allele (p<0.0001). This is therefore the first evidence that analyzed gene variants contribute to T1D pathogenesis in Croatian population.

Alzheimer’s disease and type 2 diabetes: the association study of polymorphisms in tumor necrosis factor-alpha and apolipoprotein E genes

Type 2 diabetes (T2D) and Alzheimer’s disease (AD) are two progressive disorders with high prevalence worldwide. Polymorphisms in tumor necrosis factor-alpha (TNF-α) and apolipoprotein E (ApoE) genes might be associated with both T2D and AD, representing possible genetic markers for the development of the AD in subjects with T2D. The aim was to determine ApoE and G-308A TNF-α gene polymorphisms in unrelated Croatian Caucasians: 207 patients with sporadic AD, 196 T2D patients and 456 healthy controls. Patients with AD had higher frequency of ApoE4 allele compared to T2D patients and controls. The significant association, observed between ApoE2 allele and T2D, disappeared after the data were adjusted for age and sex. The genotype or allele frequencies of G-308A TNF-α gene polymorphism were similar among the patients with AD, T2D and healthy controls. In conclusion, these results do not support the hypothesis that the A allele of G-308A TNF-α gene polymorphism is associated either with AD or T2D. Our data confirm the association between the ApoE4 allele and AD, and point out the E2 allele of ApoE gene as the possible risk factor for T2D.

Polymorphisms in the IL-18 and IL-12B genes and their association with the clinical outcome in Croatian patients with Type 1 diabetes☆

Genetic variants of IL-18 and IL-12B may be important in immunoregulatory abnormalities, observed in the patients with Type 1 diabetes mellitus (T1DM), that contribute to individual differences in response to a treatment. Therefore, we examined the significance of IL-18-137G/C, IL-18-607C/A, and IL-12B A/C polymorphisms in Croatians (187 patients, 236 controls), not only as factors that contribute to susceptibility to T1DM, but also as determinants of the clinical presentation of disease. The polymorphism screening has been performed using PCR sequence-specific primers (IL-18) or PCR-RFLP (IL-12B) approach. Results were evaluated by GraphPad Prism and Sigma Stat 3.5, Arlequin software and calculator for Hardy-Weinberg equilibrium. The genotype, allele and haplotype distribution were not statistically different between the patients and control subjects. The clinical parameter analysis revealed that patients with minor alleles at each locus, IL-18-137C/-607A, were significantly younger at T1DM onset than carriers of major alleles, IL-18-137G/-607C (20 vs 23.5 years). Moreover, the concomitant presence of minor alleles not only of IL-18 but also of IL-12B, is associated with the risk of disease progression even at younger age. These patients developed diabetes at 16 years of age, what is significantly earlier (p=0.044) compared to 25.5 years of age in patients with common alleles IL-18-137G/-607C/IL-12B A. Furthermore, combined genotype analysis of IL-18 and IL-12B has pointed out that patients with CC/AA/AA genotype have the worst glucose control based on HbA1c (8.7%, range 6.8-13.1%). In conclusion, susceptibility to T1DM in Croatians is not strongly associated with IL-18-137/-607 and IL-12B polymorphisms. These SNPs are associated with the higher risk of earlier disease development and might be implicated in the effectiveness of glycemic control.

Rational Variety Mapping for Contrast-Enhanced Nonlinear Unsupervised Segmentation of Multispectral Images of Unstained Specimen

A methodology is proposed for nonlinear contrast-enhanced unsupervised segmentation of multispectral (color) microscopy images of principally unstained specimens. The methodology exploits spectral diversity and spatial sparseness to find anatomical differences between materials (cells, nuclei, and background) present in the image. It consists of rth-order rational variety mapping (RVM) followed by matrix/tensor factorization. Sparseness constraint implies duality between nonlinear unsupervised segmentation and multiclass pattern assignment problems. Classes not linearly separable in the original input space become separable with high probability in the higher-dimensional mapped space. Hence, RVM mapping has two advantages: it takes implicitly into account nonlinearities present in the image (ie, they are not required to be known) and it increases spectral diversity (ie, contrast) between materials, due to increased dimensionality of the mapped space. This is expected to improve performance of systems for automated classification and analysis of microscopic histopathological images. The methodology was validated using RVM of the second and third orders of the experimental multispectral microscopy images of unstained sciatic nerve fibers (nervus ischiadicus) and of unstained white pulp in the spleen tissue, compared with a manually defined ground truth labeled by two trained pathophysiologists. The methodology can also be useful for additional contrast enhancement of images of stained specimens.

Genetic evaluation of the TNF-α -238G>A and -308G>A promoter polymorphisms in croatian patients with type i diabetes

A case-control study was performed to establish a potential association of two TNF-α gene promoter SNPs (-238G>A and -308G>A) with occurrence of type 1 Diabetes mellitus (T1DM) in Croatian population (174 patients and 193 healthy controls). Genotypes (obtained by polymerase chain reaction-restriction fragment length polymorphism), and the clinical parameters of T1DM patients were statistically evaluated by SPSS 13 and Arlequin software, G*Power 3.0.10 program, and calculator for Hardy-Weinberg equilibrium. The frequency of the risk (A) allele, as well as the distribution of high-expression (GA, AA) genotypes were significantly higher (p < 0.0001) in T1DM patients only at locus -308. The distribution of the -238G/-308A haplotype was also significantly higher in patients compared with controls (27.6% vs 9.6%, p < 0.0001). Gender-dependent analysis revealed that female T1DM -308GA genotype carriers exhibit considerably stronger association with T1DM (odds ratio = 6.37, 95% confidence interval = 3.16-12.85) than male -308GA patients (odds ratio = 2.71, 95% confidence interval = 1.31-5.59). Clinical parameter analysis of T1DM patients revealed significantly decreased level of hemoglobin A(1)c (HbA(1)c) in -238A allele carriers compared with -238G allele carriers (6.55% vs 7.17%, p = 0.022), as well as the tendency of the risk allele carriers at -238 or -308 locus to develop T1DM earlier in life compared with non-risk allele carriers. In conclusion, susceptibility to T1DM in the Croatian population is strongly associated with the TNF-α -308G>A polymorphism, especially in women. In addition, significantly lower HbA(1c) levels found in T1DM -238A allele carriers might indicate better glycemic control in these patients.

Functional and Structural Characterization of FAU Gene/Protein from Marine Sponge Suberites domuncula

Finkel-Biskis-Reilly murine sarcoma virus (FBR-MuSV) ubiquitously expressed (FAU) gene is down-regulated in human prostate, breast and ovarian cancers. Moreover, its dysregulation is associated with poor prognosis in breast cancer. Sponges (Porifera) are animals without tissues which branched off first from the common ancestor of all metazoans. A large majority of genes implicated in human cancers have their homologues in the sponge genome. Our study suggests that FAU gene from the sponge Suberites domuncula reflects characteristics of the FAU gene from the metazoan ancestor, which have changed only slightly during the course of animal evolution. We found pro-apoptotic activity of sponge FAU protein. The same as its human homologue, sponge FAU increases apoptosis in human HEK293T cells. This indicates that the biological functions of FAU, usually associated with “higher” metazoans, particularly in cancer etiology, possess a biochemical background established early in metazoan evolution. The ancestor of all animals possibly possessed FAU protein with the structure and function similar to evolutionarily more recent versions of the protein, even before the appearance of true tissues and the origin of tumors and metastasis. It provides an opportunity to use pre-bilaterian animals as a simpler model for studying complex interactions in human cancerogenesis.

Response to hyperoxia is associated with similar ho-1 gene expression level in lungs of aging CBA mice of both sexes

Beside classical antioxidative enzymes, the response to hyperoxia might be mediated via regulation of other systems, such as heme oxygenase (HO). Ho-1 gene expression is found to be upregulated by hyperoxia in all groups of mice, while HO-1 protein isoform was increased only in 4 months old male mice. In steady-state conditions ho-1 and ho-2 gene expression remained unchanged irrespective of sex or age, which was not the case with protein level of both isoforms. This study suggests that in lungs of CBA mice the response to oxidative stress may be mediated through the interaction of other systems such as heme oxygenase, primarily via upregulation of ho-1 gene expression in both sexes. Contrary to our previous study in liver of hyperoxia treated mice, current results might imply that at conventional oxygen conditions lungs of female mice with the emphasis on aging females, are better prepared for oxidative stress conditions through the increase of HO-activity.

Association of TNF-α and PTPN22 SNPs with the risk and clinical outcome of type 1 diabetes

Type 1 Diabetes mellitus (T1DM) begins with aberrant inflammatory process followed by auto-destruction in genetically susceptible individuals. Therefore, we hypothesized that gain-of-function allelic variants TNF-α-238A, -308A and PTPN22 1858T could be associated not only with T1DM development but also with the clinical outcome in patients of Bosnia and Herzegovina. A total of 402 subjects were enrolled in the association study. SNPs were determined by PCR-RFLP. Data was analyzed by GraphPad Prism and Sigma Stat 3.5 software. Genotypes frequencies at TNF-α-238 and -308 loci were not statistically different between patients and controls. In contrast, distribution of genotypes at the 1858 position of PTPN22 was significantly different, due to higher frequency of gain-of-function gene variants in patients than controls. Moreover, long term glucose regulation (based on HbA1c level) was significantly worse in patients with the risk TNF-α-308A allele than in patients with non-risk (G) allele. However, patients with the risk allele of both genes (TNF-α-308A and PTPN22 1858T) had the worst glycemic control, suggesting that those two work synergistically. In conclusion, in a cohort from Bosnia and Herzegovina TNF-α-308A allele is significantly associated with the worse long-term glucose control, but PTPN22 1858T allele is significantly associated with diabetes development.