We analysed the association of a single nucleotide polymorphism (SNP) in the gene encoding the IL‐12 subunit p40 (IL12B, rs3212227, A>C) with breast cancer. The SNPs allelic and genotypic frequencies were compared between patients (n = 191) and healthy (n = 194) women in a case–control study from Croatia. The major allele (A) was associated with susceptibility to breast cancer (P = 0.003; OR = 1.67; 95% CI: 1.17–2.38). Likewise, the minor allele (C) was significantly correlated with protection (P = 0.003; OR = 0.60; 95% CI: 0.42–0.86). At the genotype level, AA homozygosity was significantly associated with predisposition to disease (P = 0.013; OR = 1.68, 95% CI: 1.09–2.59), whereas the minor allele homozygosity (CC) was correlated with protection to disease (P = 0.020, OR = 0.28, 95% CI: 0.09–0.91). The heterozygous genotype showed no significant correlation with disease. The product of the IL12B gene (IL‐12 p40) can either form a homodimeric cytokine or be part of two pro‐inflammatory (IL‐12 and IL‐23) cytokines. It is presently unclear whether the major allele is associated with higher or lower protein levels of IL‐12 p40 and IL‐12 p70, which are critical in inflammation and adaptive immune responses. However, as the A allele is high producer of IL12B (p40) mRNA, these results might imply that higher levels of IL‐12 p40 (either as homodimers or joined with one or both of the other two subunits) predispose to breast cancer.

Background: Human B cells respond to Toll-like receptor (TLR) 9 stimulation by cytokine production. Results: A rare, novel TLR9 allele fails to activate NF-κB in HEK293 cells. Heterozygous carrier B cells show defective IL-6 and IL-10 production. Conclusion: Heterozygosity for this TLR9 allele modifies CpG oligonucleotide responsiveness. Significance: This is the first analysis of human TLR9 variants in primary cells. Toll-like receptors (TLR) are employed by the innate immune system to detect microbial pathogens based on conserved microbial pathogen molecules. For example, TLR9 is a receptor for CpG-containing microbial DNA, and its activation results in the production of cytokines and type I interferons from human B cells and plasmacytoid dendritic cells, respectively. Both are required for mounting an efficient antibacterial or antiviral immune response. These effects are mimicked by synthetic CpG oligodeoxynucleotides (ODN). Although several hyporesponsive TLR9 variants have been reported, their functional relevance in human primary cells has not been addressed. Here we report a novel TLR9 allele, R892W, which is hyporesponsive to CpG ODN and acts as a dominant-negative in a cellular model system. The R892W variant is characterized by increased MyD88 binding and defective co-localization with CpG ODN. Whereas primary plasmacytoid dendritic cells isolated from a heterozygous R892W carrier responded normally to CpG by interferon-α production, carrier B cells showed impaired IL-6 and IL-10 production. This suggests that heterozygous carriage of a hyporesponsive TLR9 allele is not associated with complete loss of TLR9 function but that TLR9 signals elicited in different cell types are regulated differently in human primary cells.

Breast cancer is the most common malignancy in women, who have over 100-fold higher risk of developing disease than men. Despite the progress in treatment over the past years and relatively good overall survival at present (about 80% have an estimated 5-year survival rate), the disease has still caused over 450,000 deaths worldwide per year in the last decade. Recent novel therapies involve various cytokine treatments that promote anti-cancer-specific adaptive immunity. However, success of therapies might be greatly improved provided all risk factors were known. Cancer has a multi-factorial genetic susceptibility. The immune system can contribute to genetic risk to cancer; first, innate immunity can provide stimulation of cell growth by inflammation, a well-known tumour promotion factor, and second, adaptive immunity can detect and eliminate cancer cells according to the immunosurveillance hypothesis [1]. An increased former capacity or a weakness in the latter ability would increase susceptibility to cancer. Evidence that adaptive immunity has a role in cancer development comes from experiments in various animal models [2–9] and from data showing an increased incidence of cancers in humans with immunodeficiencies [10] or after receiving post-transplantation immunosuppression therapy [11, 12]. In line with this, CD4 T cell and CD1a dendritic cell populations in axillary lymph nodes were highly correlated predictors of disease-free survival in patients with breast cancer [13]. The IL-12 cytokine is important for adaptive immunity with a principal role in the defence against intracellular micro-organisms [14, 15]. It is a 70-kDa heterodimeric protein (IL-12 p70) composed of p35 *Molecular Genetics Laboratory, Department of Oral Biology, Faculty of Dentistry, University of Oslo, Oslo, Norway; School of Medicine, University of Oslo, Oslo, Norway; School of Medicine, University of Zagreb, Zagreb, Croatia; §Department of Medical Oncology, Division of Oncology, University Hospital Center Zagreb, School of Medicine, University of Zagreb, Zagreb, Croatia; –Division of Molecular Medicine, Ru der Bošković Institute, Zagreb, Croatia; and **Clinical Institute for Transfusion Medicine, Universal Hospital Center Rijeka, School of Medicine, University of Rijeka, Rijeka, Croatia

We have sequenced 416 Toll‐like receptor‐2 (TLR2) alleles in 208 subjects in a tuberculosis case–control study in Croatian Caucasian population. We found ten single nucleotide polymorphisms (SNP) among which three were novel (S97S, T138I and L266F). The genotype containing TLR2‐P631H SNP was significantly overrepresented in patients with tuberculosis when compared to contact controls, suggesting a small yet increased risk to disease. The causative agent of tuberculosis is Mycobacterium tuberculosis, which can bind to TLR2 with its lipoprotein coat. The TLR2‐P631H mutant has a dominant negative effect on the wild type TLR2 signalling in transfected HEK293 kidney cells using the NF‐κB‐driven luciferase as a reporter gene with ligands like M. avium extracts, Pam3CysSK4 or FSL‐1 that bind TLR2/TLR1 or TLR2/TLR6 heterodimers, respectively. Studies on internalization from the Regular Madine Darby Canine Kidney cell surface into the early endosomal compartments showed a lower rate of the mutant compared to the wild type. Our data, in combination with a report by others show that the TLR2‐P631H allele could be associated with protection to meningococcal meningitis, suggest that by dominantly inhibiting the response of cells important in the immune response this mutant might confer either protection or susceptibility to meningitis or tuberculosis, respectively.

BACKGROUND Genetic susceptibility to cancer is multifactorial, and it is known that impairment of the immune system could contribute to risk for getting cancer. AIM OF THE STUDY Single-nucleotide polymorphisms (SNPs) of Toll-like receptor (TLR) 2, TLR3, TLR4, and TLR9 genes, which are important for innate immunity, were analyzed for the association with breast cancer. METHODS The SNPs comprised TLR2 (c.597T>C), TLR2 (c.1350T>C), TLR3 (c.1377C>T), TLR4 (c.896A>G), and TLR9 (c.1635A>G). The allelic and genotypic frequencies of these TLR SNPs were compared between patients (n = 130) and controls (n = 101) in a case-control study from Croatia. RESULTS TLR SNPs were not significantly different. From the population genetics viewpoint, we found that a hypomorphic variant of TLR4 (p.Asp299Gly) allele has no specific allelic frequency (8.4%) in the Croatian population (n = 496) compared to other Caucasians (6.5-10%). CONCLUSION These results suggest that polymorphisms in tested TLR genes are not likely to be associated with increased risk for developing breast cancer.

The evolutionary value of landraces is immense, especially when bearing in mind the constant diminishment of crop genetic variability and the development of intensive agriculture. The assessment of morphological and agronomic traits of landraces, together with molecular markers, is certainly a good basis for the efforts toward their protection. Ten maize accessions, originating from the eastern Serbia, were characterized in this study by the use of morphological and molecular analysis. Since the morphological characterization was performed on the plant material grown on two different locations, environmental influence on the development of morphological traits was also addressed. The accessions were significantly differentiated in most of the characteristics that were measured. However, the results have indicated that the plants grown in two experimental plots were also significantly different due to influence of the different environmental conditions. Another part of the study was represented by the field survey conducted in the eastern Serbia, in the Homolje region, in order to determine the presence of the maize landraces and the extent of their utilization in the region. Traditional agricultural practices related to the cultivation of the maize landraces were found to be important for the preservation of the local maize varieties, as well as the cultural diversity related to the cultivation of this crop. However, it was also found that the cultivation of the maize landraces has substantially decreased during the last two decades and, nowadays, it is performed only on small parcels. The preservation of the landraces is a complex task, which should be approached not only through the conservation of the agricultural practices and in situ conservation, but also through proper management and evaluation of existing seed collections. This necessity should be widely recognized as the activity of the national and international interest.

The aim of the study was to determine (1) the frequency and type of mutations in the coding region of the GJB2 gene (sequencing), (2) the frequency of splice site mutation IVS1 + 1G > A in the GJB2 gene (multiplex ligation-dependent probe amplification analysis), (3) possible copy number changes in the GJB2, GJB3, GJB6, and WFS1 genes (multiplex ligation-dependent probe amplification analysis), and (4) the frequency of del(GJB6-D13S1830) in the GJB6 gene in 58 unrelated patients with nonsyndromic hearing loss from Croatia. About 44.8% of our patients presented with mutation in the GJB2 gene. We identified seven sequence variations. Six of them had previously been reported as disease related (35delG, W24X, V37I, L90P, 313del14, and IVS1 + 1G > A), and we report here for the first time one novel variant, -24A > C. We detected the greatest frequency of 35delG allele compared to the other alleles (35.3%). Allelic frequencies of other common mutations accounted for 2.6-0.9% of analyzed chromosomes. Neither GJB6 deletion nor copy number changes in the GJB2, GJB3, GJB6, and WFS1 genes were found. The 35delG/35delG genotype was associated with severe to profound hearing loss in 94% of 35delG homozygotes. High mutation rate (44%) indicates that testing of the GJB2 gene will clarify the genetic cause in almost half of the cases of recessive nonsyndromic hearing loss in Croatia.