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Diabetic ketoacidosis (DKA) is the leading cause of mortality in a type 1 diabetic mellitus (T1DM) in pediatric population. The prevalence of the DKA in the developed world ranges from 15% to 61% and in Croatia it is 33 to 36%. In the past few decades in Europe there have been no significant changes in the prevalence of DKA in patients with newly T1DM. In the previous preventive program known as the “Parma campaign”, which was conducted between 1991 and 1998 and reduced the prevalence of DKA from the initial 78% to the cumulative prevalence during the campaign of 12.5%. The aim of the prevention program was to reduce the prevalence of DKA in the Southern part of Croatia in children and adolescents with newly diagnosed T1DM.

Hashimoto’s thyroiditis (HT) is the most common autoimmune thyroid disorder defined as a chronic inflammation of the thyroid gland. It is characterized by the infiltration of lymphocytes and progressive destruction of thyroid tissue. Another distinguished feature of HT is the presence of elevated thyroid autoantibodies against thyroid peroxidase (TPOAb) and thyroglobulin (TgAb). The aim of this study was to explore the genetic background of HT-associated traits, namely thyroid volume and thyroid antibodies, in HT patients by genome-wide association analysis (GWAS). The study included 430 HT patients recruited at the University Hospital Split (Croatia). Diagnosis of HT patients was based on clinical findings and laboratory parameters according to ETA recommendations and guidelines for Management of Subclinical Hypothyroidism. Participants were genotyped using the Illumina Infinium HumanCoreExome genotyping platform and imputed using the “1000 Genomes” reference panel and IMPUTE2 software. Association analysis of the resulting 8, 592, 875 genetic markers was performed using GEMMA software. Three analyzed traits were adjusted for covariates under standard linear regression model: thyroid volume was adjusted for gender, age, BSA, TSH, and levothyroxine therapy status whereas TPOAb and TgAb levels were adjusted for gender. Derived residuals were further included in the association analysis as a new phenotype for each trait under the linear mixed model, which accounts for population stratification and relatedness. We identified two genome-wide significant associations of intronic variants inside the AATF and CNDP1 genes (p=1.36×10-8, p=3.08×10-8, respectively) with thyroid volume. An additional genetic variant near the MIR8054 gene was suggestively associated with thyroid volume (p=2×10-7). No SNP reached genome-wide significance for thyroid antibodies ; however, we identified two suggestive associations of genetic variants near the FAM13A and TRIM61 genes with TPOAb levels (p=6.30×10-8, p=4.89×10-7, respectively) and two suggestive associations of genetic variants near GTF3AP6 and inside the CA10 gene with TgAb levels (p=2.27×10-7, p=6.48 ×10-7, respectively). Our most interesting signal resides within AATF (apoptosis antagonizing transcription factor), which is involved in the regulation of gene transcription and cell proliferation. Overexpression of this gene interferes with MAP3K12-induced apoptosis, whereas, inhibition of the same MAP3K12 activity is driven by MBIP, a gene that has already been associated with thyroid cancer and TSH level, giving further evidence of the biological relevance of this pathway in thyroid function. The second hit resides inside CNDP1 (carnosine dipeptidase 1), which is a human carnosinase mainly expressed in the central nervous system. In conclusion, our study identified novel, biologically interesting genes associated with thyroid volume and several suggested genes associated with thyroid antibodies in HT patients. These findings represent a good basis for further exploration of underlying genetics of HT determinants.

Type 1 diabetes mellitus (T1DM) is characterized by a chronic progressive inflammatory autoimmune reaction that causes the selective elimination of pancreatic β cells. Proinflammatory cytokines, interleukin-18 (IL-18) and tumor necrosis factor alpha (TNF-α ), both have been implicated in the pathogenesis of T1DM. Additionally IL-18 plays a role in regulation of TNF-α production. Therefore we tested an association of two single nucleotide polymorphisms (SNP) in promoter regions of these genes with the susceptibility to T1DM in the Dalmation population (South Croatia). 134 T1DM patients and 132 control subjects were tested for a G-137C change in a promoter region of IL-18 gene by a sequence specific PCR. TNF-α G-308A promoter variant was genotyped in a larger group of T1DM patients (206) and controls (144) by standard PCR followed with NcoI endonuclease restriction. Data were analysed using chi-square test. A distribution of IL-18 G-137C and TNF-α G-308A promoter variants was equal between the T1DM patients and the controls (p=0.9448 and p=0.0655, respectively). The present study provided no evidence of association of IL-18 and TNF-α promoter variants with susceptibility to T1DM in Dalmatian population. However as genotype frequencies for TNF-α G-308A polymorphism were close to the limit of statistical significance (p=0.0655) further analysis should be conducted.

Vitamin D and its receptor gene (VDR) have immunomodulatory properties and influence insulin secretion. Recent data indicate VDR polymorphisms to be associated with type 1 diabetes mellitus (T1DM) and therefore were studied FokI polymorphism in exon 2. 134 individuals with T1DM and 132 control subjects from the Dalmatia population of South Croatia were examined. Fok I genotyping was performed by PCR amplification followed by FokI endonuclease restriction. Data were analysed using the chi-square test. On the basis of our previous research, haplotype analysis of VDR polymorphisms BsmI, ApaI and TaqI and their association with susceptibility to T1DM have been investigated. Estimates of haplotypes were made using statistical program EH+. FokI polymorphism showed unequal distribution (p=0.049) between T1DM cases and controls indicating ff genotype to be associated with development of T1DM and Ff genotype to be a protective one. BsmI, ApaI and TaqI polymorphisms were found to be in strong linkage disequilibrium (LD) (p=0.0001). Bat haplotype was observed 11 times in control and not once in T1DM individuals (p=0.0007) and therefore revealed it's possible protective role. No significant LD between FokI and any other polymorphisms was detected but, nevertheless, bf haplotype was significantly more frequent in T1DM patients (p=0.0168) while the aF haplotype was observed more times in controls (p=0.0413). Our findings indicate that VDR FokI polymorphism and several VDR allele variants are associated with susceptibility to T1DM in the Dalmatian population.

Vitamin D and interleukin-1 have been suggested to play a role in the pathogenesis of type 1 diabetes mellitus (T1DM). Therefore, we have studied the influence of vitamin D receptor (VDR) and interleukin-1 receptor (IL1-R1) gene polymorphisms on susceptibility to T1DM in the Dalmatian population of South Croatia. We genotyped 134 children with T1DM and 132 control subjects, except for the FokI polymorphism where we extended control group for additional 102 subjects. VDR gene polymorphism FokI displayed unequal distribution (p=0.0049) between T1DM and control group, with the "ff" genotype occurring more frequently in the T1DM individuals while VDR gene polymorphism Tru9I did not differ in frequency between studied groups. All tested polymorphisms of IL1-R1 gene, PstI, HinfI and AluI (promoter region) and PstI-e (exon 1B region) did not display difference among cases and controls. Haplotype analysis of VDR gene (FokI, BsmI, ApaI, TaqI, Tru9I) and of IL1-R1 gene (PstI, HinfI, AluI, PstI-e) found VDR FbATu (p=0.0388) and IL1-R1 phap' (p=0, 0419) haplotypes more frequently in T1DM patients while BatU haplotype occured more times in the control group (p=0, 0064). Our findings indicate that VDR FokI polymorphism and several VDR and IL1-R1 haplotypes are associated with susceptibility to T1DM in the Dalmatian population.

The polygenetic susceptibility to type 1 diabetes (T1DM) is well established and recent studies have demonstrated linkage of a vitamin D receptor gene (VDR), interleukin-1 receptor type 1 gene (IL1R1) and Neuro D/BETA2, a transcription factor of the insulin gene, to disease. We studied 134 individuals with T1DM and 132 control subjects. The genotyping was performed using PCR and BsmI, ApaI and TaqI restriction enzymes for VDR polymorphism, PstI, HinfI and AluI for IL1R1 polymorphism and MwoI for NeuroD/BETA2 polymorphism. Data were analysed using the c2 test. VDR gene polymorphisms are associated with an increased risk of T1DM in Dalmatian population caused with differences in Taq I genotype distribution between T1DM and control subjects. The genotype combination, which conferred strongest susceptibility to T1DM, was BBAAtt (P=0.002). Interestingly, the BAt haplotype was found to be a risk factor in a German population, the only European population tested thus far. All tested polymorphisms of IL1R1 gene and NeuroD/BETA2 gene did not display difference among case and control subjects. Our data implement involvement of vitamin D in pathogenesis of T1DM.