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Sucralose is widely used as a non-nutritive sweetener (NNS). However, in order to justify its use as a non-nutritive food additive, sucralose would have to be metabolically neutral. The aim of this study was to examine whether sucralose altered the insulin signaling pathway in an in vitro cell model of Parkinson’s disease (PD)—the dopaminergic differentiated cell line SH-SY5Y. Cells were exposed to sucralose alone and in combination with either insulin or levodopa. Activation of the insulin signaling pathway was assessed by quantifying protein kinase B (AKT) and glycogen synthase kinase 3 (GSK3), as well as the phosphorylated forms of insulin-like growth factor 1 receptor (IGF1-R). Metabolic effects were assayed using MALDI-TOF MS analysis. In the cell viability test, 2 mM sucralose had a negative effect, and levodopa in all combinations had a positive effect. Sucralose treatment alone suppressed GSK3 and IGF1-R phosphorylation in a dose-dependent manner. This treatment also altered the metabolism of fatty acids and amino acids, especially when combined with insulin and levodopa. Suppression of the insulin signaling pathway and sucralose-induced changes in the metabolic profile could underlie a diet-acquired insulin resistance, previously associated with neurodegeneration, or may be an altered response to insulin or levodopa medical therapy.

Context: The role of maternal adrenal steroids is to regulate electrolytes and blood volume of mother during pregnancy. Together with estrogen ( E2) and P4, they regulate fetal access to nutrients, electrolytes and water by regulating maternal volume expansion, perfusion of the uterus and placenta, and substrate availability. Indirectly by controlling it, adrenal steroids can influence mode of delivery. Objective:The hormonal autocrine and paracrine signaling pathway between the mother and the fetal-placental unit could affect the mode of delivery. We investigated the association between the peripartal serum concentration of sex hormone binding protein (SHBG) and 10 selected steroid hormones in pregnant women and the mode of delivery. Patientss and Methods:Serum samples were collected out of 184 women. There were 2 groups of pregnant women whose delivery started with regular contractions: the ones whose pregnancies ended with a vaginal delivery and those who gave birth via urgent C-section. For determining the serum concentrations of SHBG and selected steroid hormones, LC-MS/MS was utilized. Mann Whitney U test was utilized to show difference in serum concentration of SHBG and steroids between these 2 groups. Univariate and multivariate logistic regressions and ROC analysis were utilized to show association between serum concentration of SHBG and selected steroid hormones and mode of delivery. Results:Statistically significant difference was identified between the serum concentrations of corticosterone (p 0.02), cortisol (p 0.04), aldosterone (p 0.007) and dehidroepiandrosterone (DHEAS) (p 0.04) between these two groups. ROC analysis showed that all the examinees whose level of aldosterone is less than 0.916 nmol/L have a 0.27 times greater chance to give birth via C-section (AUC 0.732, 95% CI 0.660-0.796, sensitivity 91.67, specificity 55, 76, p

Objective: The aim of this study was to evaluate the maternal serum concentration of selected endogenous steroid hormones during spontaneous parturition at term and to determinate their association with the need for oxytocin augmentation. PAtients and methods: Blood of 108 healthy pregnant women whose parturition started with the regular spontaneous uterine contractions, with or without a premature rupture of membranes, was drawn at the beginning of the process. LC-MS/MS device was utilized for measurement of the following maternal serum steroid hormones: aldosterone, androstenedione, cortisol, cortisone, corticosterone, dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulphate(DHEA-S), 17-hydroxyprogesterone (17-OHP), progesterone(P4) and testosterone. Mann Whitney U test, Chi-square Test, univariate and multivariate logistic regression and ROC analysis were used for the data analysis. Results: Reference ranges of the selected hormones assessed by the LC-MS/MS in maternal serum of women having spontaneous parturition were established. Statistically significant differences in the serum corticosterone, DHEA and androstenedione between cases requiring oxytocin augmentation and the rest of women having spontaneous parturition were found (p=0.002, p=0.008 and p=0.04 respectively). Concentrations of these steroids were lower in the group of pregnant women who required oxytocin infusion for progression of labor. ROC analysis showed that all the examinees whose DHEA concentration was above 21.6 nmol/L have lower chance to use oxytocin infusion for the labor progression (AUC= 0.649, sensitivity=71.7%, specificity=59.6%, P=0, 006). Conclusion: This study provided reference ranges for the selected maternal serum steroid hormone concentrations at the beginning of parturition. Association of selected steroid hormones with the oxytocin infusion has been established. DHEA is a potential predictor of oxytocin infusion augmentation for successful progression of the parturition.

Premature birth (PTB) is defined as a live birth before 37 weeks of gestation. One of the recognized risk factors for PTB is a maternal and/or fetal genetic predisposition. The aim of the study was to evaluate the roles of four selected genetic variations in fetal and maternal progesterone receptor gene (PGR) and to identify women who may have higher or lower odds for PTB compared to the general population. The study was conducted between November 2017 and January 2021. It was case- control study, which enrolled two groups of pregnant women (109 women who delivered at term and 109 women who had a preterm delivery) and two groups of newborns (109 term and 109 preterm). Venous blood samples were taken from pregnant women and blood from the umbilical cord of the newborns once after obtaining informed consent. Four described genetic variants of PGR (rs1042838, rs1042839, rs10895068, and rs1932836) were analyzed. Statistical analysis has been made. There was statistically significant difference between cases and controls in the distribution of newborns’ allele frequency of minor C allele of the PGR rs1942836 (P=0.03, Fishers exact test) in favor of premature birth. A statistically significant difference between the frequency of the mothers’ minor T allele of rs1042838 (P = 0.005 ; Chi-squared test) and the mothers’ minor T allele of rs1042839 (P = 0.005 ; Chi-squared test) in favor of extremely premature birth has been found. There was a statistically significant difference between the frequency of the newborns’ minor C allele of rs1942836 (P = 0.03 ; Chi-squared Test) and newborns’ heterozygotes CT genotype of rs1942836 (P = 0.03 ; Fishers’ exact Test) when comparing the control group and the early premature group. The present study suggests that patients with selected genetic variants of the progesterone receptor gene (mothers rs1042838 and rs1042839, newborns rs1942836) could have greater odds for premature birth compared to general population.

IgA vasculitis (IgAV) is a desease in which genetic background also plays an important role. Some small studies have indicated the importance of variants in various non-HLA genes in the manifestation of different disease phenotypes. The aim of this research was to investigate single nucleotide polymorphisms (SNPs) of genes HMGB1 and AGER encoding for high mobility group box-1 (HMGB1) and receptor for advanced glycation endproducts (RAGE), both acting as mediators of inflammation, in the susceptibility and clinical features of patients with IgAV. HMGB1 and RAGE gene polymorphisms were genotyped using a real- time polymerase chain reaction. The presence and frequency of polymorphisms in HMGB1 (rs2249825, rs1045411, rs1060348, rs1412125 and rs41369348) and RAGE (rs1800625, rs1800624, rs2070600 and rs3134940) were determined. Clinical data were collected from database of IgAV patients from two Croatian University Centers for pediatric rheumatology. 81 pediatric IgAV patients were included, of whom 45 were boys and 36 girls, as well as 150 age- and sex-matched healthy controls without any history of autoimmune disease. The median (range) age of IgAV patients was 6.25 (4.60-8.20) years, and among them 71.6% had joint involvement, 29.62% had gastrointestinal manifestations, while 27.16% developed nephritis. The purpuric rash which extended from lower extremities to the trunk, upper extremities and face (generalized rash) was present in 43.20% of patients and 27.16% had at least one relapse. Among the analyzed polymorphisms, only in the rs1412125 there was a deviation from the Hardy Weinberger equilibrium. There was no statistically significant association of the analyzed polymorphisms with the IgAV susceptibility, compared to healthy controls. Polymorphism rs2070600 was significantly related with the development of nephritis in IgAV, while rs1412125 was associated with gastrointestinal involvement. The IgAV patients carrying the T allele (rs2070600) of the AGER had significantly increased risk of nephritis development compared with the IgAV patients with homozygous CC genotype in dominant (OR 4.05, CI 1.09-15.03, p = 0.037) and additive genetic models (OR 3.95, CI 1.16- 13.47, p = 0.049). The minor C allele (rs1412125) of the HMGB1 was found to significantly increase the risk of gastrointestinal involvement in overdominant model with an allelic odd ratio of 2.78 (CI 1.04-7.43, p = 0.04). Although neither of analyzed HMGB1 and RAGE polymorphisms was not associated with IgAV susceptibility, our results indicated that these polymorphisms may be involved in the pathogenesis of IgAV with possible effect on different disease phenotypes. SUPPORT: Croatian Science Foundation project IP- 2019-04-8822.

Uvod. U manifestaciji različitih fenotipova IgA vaskulitisa (IgAV) važnu ulogu imaju i varijante gena izvan HLA sustava. Cilj je bio istražiti ulogu polimorfizama pojedinačnih nukleotida gena HMGB1 i AGER koji kodiraju za protein visoke pokretljivosti iz skupine 1 (HMGB1) i receptor za krajnje produkte uznapredovale glikozilacije (RAGE), u predispoziciji i kliničkim značajkama bolesnika s IgAV-om. Ispitanici i metode. Genotipizirani su polimorfizmi gena za HMGB1 i RAGE metodom lančane reakcije polimeraze. Utvrđena je prisutnost i učestalost polimorfizama u HMGB1 (rs2249825, rs1045411, rs1060348, rs1412125 i rs41369348) i RAGE (rs1800625, rs1800624, rs2070600 i rs3134940). Klinički podaci prikupljeni su iz baze podataka bolesnika s IgAV- om iz dva hrvatska tercijarna centra za pedijatrijsku reumatologiju. Rezultati. Istraživanje je obuhvatilo 81 bolesnika s IgAV-om, među kojima je bilo 45 dječaka i 36 djevojčica, te 150 kontrolnih ispitanika koji se po dobi i spolu nisu razlikovali od djece s IgAV- om. Medijan (raspon) dobi bolesnika s IgAV-om iznosio je 6, 25 (4, 60–8, 20) godina, a među njima 71, 6% imalo je zahvaćene zglobove, 29, 62% imalo je gastrointestinalne manifestacije, dok je 27, 16% bolesnika razvilo nefritis. Generalizirani purpurični osip bio je prisutan u 43, 20% bolesnika, a 27, 16% imalo je barem jedan recidiv bolesti. Nije bilo statistički značajne povezanosti analiziranih polimorfizama s predispozicijom za IgAV u usporedbi sa kontrolama. Polimorfizam rs2070600 bio je značajno povezan s razvojem nefritisa u IgAVu dok je rs1412125 bio povezan sa zahvaćanjem probavnog sustava. Bolesnici s IgAV-om koji su imali alel T (rs2070600) u genu AGER imali su značajno veći rizik za razvoj nefritisa u usporedbi s bolesnicima s homozigotnim genotipom CC u dominantnom (OR 4.05, CI 1.09–15.03, p = 0.037) i aditivnom modelu (OR 3, 95, CI 1, 16–13, 47, p = 0, 049). Utvrđeno je da minor alel C (rs1412125) u HMGB1-u značajno povećava rizik od zahvaćanja probavnog sustava u superdominantnom modelu s omjerom izgleda od 2, 78 (CI 1, 04–7, 43, p = 0, 04). Zaključak. Iako niti jedan od analiziranih polimorfizama gena za HMGB1 i RAGE nije bio povezan s predispozicijom za IgAV, naši rezultati pokazali su da ti polimorfizmi mogu biti povezani s različitim fenotipovima IgAV. Potpora: Projekt Hrvatske zaklade za znanost IP- 2019-04-8822

IgA vasculitis (IgAV) or Henoch-Schönlein' s purpura is the most prevalent systemic small vessel vasculitis in childhood. High mobility group box-1 protein (HMBG1) is a pleiotropic cytokine that functions as a pro-inflammatory signal, important for the activation of antigen- presenting cells (APCs) and propagation of inflammation. HMGB1 is implicated in the pathophysiology of a variety of inflammatory diseases. The aim of this study was to investigate the role of single nucleotide polymorphism (SNP)- rs41369348 for HMGB1 gene in the susceptibility and clinical features of patients fulfilling classification criteria for IgAV. DNA was extracted from blood cells of 76 children with IgAV and 150 age-matched healthy controls. Clinical data and laboratory parameters were collected for all IgAV patients. Although there was higher frequency of heterozygous A/delA genotype of this gene polymorphism in IgAV group compared to control group, no genotype difference between those two groups was observed. No statistically significant differences in genotype were disclosed when patients with different IgAV clinical features were compared. In conclusion, in this study polymorphism rs41369348 for HMGB1 was not associated with increased susceptibility to childhood IgAV, nor with its severity or different clinical manifestations.

Premature birth (PTB) is defined as a live birth before 37 weeks of gestation. Besides its association with mortality, there are both acute and chronic morbidities associated with PTB, including long-term sequelae such as cognitive and motor delays. One of the recognized risk factors for PTB is a maternal and/or fetal genetic predispositio. One of main pathways involved in uterine quiescence is progesterone (P4) pathway. A number of single nucleotide polymorphisms (SNPs) have been described in human PGR.The aim of this study was to evaluate the roles of four selected genetic variations in fetal and maternal progesterone receptor gene (PGR) and to identify women who may have higher or lower odds for PTB compared to the general population.

Introduction: The pathogenesis of IgA vasculitis (IgAV) is complex and still insufficiently elucidated. It is a multifactorial disease in the development of which, in addition to numerous environmental factors, the genetic background also plays an important role. Previous genome-wide association study studies have established an association between IgAV susceptibility and the HLA class II genes, although many small studies have indicated the importance of variants in various non-HLA genes in the manifestation of different disease phenotypes. Objectives: The aim of this research was to investigate single nucleotide polymorphisms (SNPs) of genes HMGB1 and AGER encoding for high mobility group box-1 (HMGB1) and receptor for advanced glycation endproducts (RAGE), both acting as mediators of inflammation, in the susceptibility and clinical features of patients with IgAV. Methods: Genomic DNA was extracted from whole blood samples after which the HMGB1 and RAGE gene polymorphisms were genotyped using a real-time polymerase chain reaction. The presence and frequency of polymorphisms in HMGB1 (rs2249825, rs1045411, rs1060348, rs1412125 and rs41369348) and RAGE (rs1800625, rs1800624, rs2070600 and rs3134940) were determined. Clinical data were collected from database with systematic analysis of patients with IgAV in Croatian population from two Croatian University Centers for pediatric rheumatology and nephrology care. Results: The research included 81 pediatric IgAV patients, of whom 45 were boys and 36 girls, as well as 150 age- and sex-matched healthy controls without any history of autoimmune disease. The median (range) age of IgAV patients was 6.25 (4.60-8.20) years, and among them 71.6% had joint involvement, 29.62% had gastrointestinal manifestations, while 27.16% patients developed nephritis. The purpuric rash which extended from lower extremities to the trunk, upper extremities and face (generalized rash) was present in 43.20% of patients and 27.16% had at least one relapse. Among the analyzed polymorphisms, only in the rs1412125 there was a deviation from the Hardy Weinberger equilibrium. There was no statistically significant association of the analyzed polymorphisms with the IgAV susceptibility, compared to healthy controls. However, the two polymorphisms proved to be linked with a well- defined clinical phenotype. Polymorphism rs2070600 was significantly related with the development of nephritis in IgAV, while rs1412125 was associated with gastrointestinal involvement. The IgAV patients carrying the T allele (rs2070600) of the AGER had significantly increased risk of nephritis development compared with the IgAV patients with homozygous CC genotype in dominant (OR 4.05, CI 1.09-15.03, p = 0.037) and additive genetic models (OR 3.95, CI 1.16-13.47, p = 0.049). The minor C allele (rs1412125) of the HMGB1 was found to significantly increase the risk of gastrointestinal involvement in overdominant model with an allelic odd ratio of 2.78 (CI 1.04-7.43, p = 0.04). Conclusion: Although neither of analyzed HMGB1 and RAGE polymorphisms was not associated with IgAV susceptibility, our results indicated that these polymorphisms may be involved in the pathogenesis of IgAV with possible effect on different disease phenotypes. SUPPORT: Croatian Science Foundation project IP- 2019-04-8822

Both genetic (i.e., Catechol-O-methyltransferase Val158Met/COMT-rs4680) and environmental factors (e.g., negative parenting practices including authoritarian and permissive parenting styles) have been implicated as vulnerability factors for future problem behaviors. The present study examined the susceptibility properties of COMT genotype to adverse and favorable parenting styles in relation to childhood problem behaviors. The sample was composed of community children (N = 175 ; 80 boys ; T1age=6.98 ; T2age=9.78) and their parents chosen through a multistage stage random sampling procedure. At T1, children were genotyped for COMT-rs4680, whereas parents rated their parenting styles. DNA was extracted from buccal swabs and genotyped using TaqmanTM SNP genotyping assay on Applied Biosystems™ 7500 Real-Time PCR System. At T2, teachers rated children’s problem behaviors. As in previous studies, we divided the sample into those with the high-activity Val/Val genotype (20.9%) and those with 1 or more Met alleles (79.1%). The results showed no main effects of gender and COMT on teacher-rated problem behaviors. Main effects of all three parenting styles: children with authoritarian and permissive parents were rated as exhibiting more problem behaviors by their teachers, whereas authoritative parenting was related to less conduct problems. There is also a significant interactive effect between authoritarian parenting and COMT rs4680 genotype.

Intellectual disability/mental retardation (ID/MR) is defined as incomplete mental and cognitive development present before the age of 18. There are number of pre-natal and post-natal risk factors that can cause ID/MR but 25%-50% of all have genetic causes. In the general population, the prevalence of ID/MR is about 2%-3%. Use of standard cytogenetic methods analysis of chromosomes GTG banding) and FISH (Fluorescent in Situ Hybridization) reveals only a small number of causes, but when using new molecular genetics techniques (like chromosomal microarray and next generation sequencing), the rate of causes of ID/MR is increased and new candidate genes for ID/MR have been discovered. Establishing a diagnosis of ID/MR is important for the patient and it provides genetic counseling for parents.

Introduction/Background Cervical cancer is worldwide the second most common cancer in women. Immune response appears to be a key determinant in controlling human papilloma virus (HPV) infection. Cell mediated immunity plays a significiant role in progression or regression of neoplastic cervical lesions. Lipid rafts have essential role in T lymphocite activation. Gangliosides play an important role in the formation and stabilization of specific cell lipid membrane domains. This study aims to determine whether ganglioside GM1 expressions in T cell membrane represent risk factor for cervical cancer. Methodology In total, 20 women participated in our study. Peripheral blood samples were obtained from 10 cervical cancer patients and 10 healthy controls at University Hospital for Tumors, Zagreb, Croatia. A 3 mL sample of venous blood was collected from each subject into a test tube containing EDTA as anticoagulant. For T cell subtypes isolation we used Anti CD4 Phycoerythrin antibody and Anti CD8 Peridinin Chlorophyll Protein Complex antibody. Ganglioside GM1 expression in T lymphocytes membranes was analyzed using double labeling antibodies on ganglioside GM1. Results We have not found difference in ganglioside GM1 expression in T lymphocytes membranes between the two groups. Conclusion The present study did not show association of ganglioside GM1 expression in T lymphocytes membranes with cervical cancer development.

The incidence of preterm birth in developed countries is estimated at 7.5% of all births. Incidence of preterm birth in our Clinic is very similar. One of the most daunting complications of preterm birth is cerebral palsy (CP). It is the most frequent cause of child disability. The prevalence of CP is between 2 and 2.5 per 1000 live born babies. Few studies have proven that CP was less frequent in preterm babies who had been exposed to magnesium sulphate antenatal. The aim of this review was to evaluate effects of antepartum intravenous magnesium sulphate given to women in danger of preterm birth as the neuroprotection of a new-born.

Introduction: Breast cancer (BC) is the most common malignant disease in females taking 26 % of all cancer sites. Third generation aromatase inhibitors like anastrozole are becoming more important in treating BC because of their efficacy and better overall safety in the adjuvant treatment. Single nucleotide polymorphism (SNP) in genes encoding drug metabolizing enzymes could have an important role in individual responses to anastrozole therapy including drug efficacy and side effects. Aim: To explore rates of three SNPs (CYP3A4*1B, CYP3A5*3, UGT1A4*2) important in anastrozole metabolism in population of Croatian BC patients, and its possible correlation to anastrozole induced side effects. Materials and methods: 126 BC patients were included in the study of which 82 were postmenopausal patients with ER positive BC treated with anastrozole and 44 were postmenopausal ER positive patients before hormonal adjuvant therapy. DNA for SNPs was genotyped by TaqMan RT-PCR and BMD was measured by DXA. Results: Homozygotes for the wild type A allele of CYP3A5*3 were not detected, moreover mutant G allele homozygotes were predominant with 88%. Wild type homozygotes of CYP3A4*1B were predominant with 94%, and mutant homozygotes were not detected. CYP3A4*1B and CYP3A5*3 SNPs were in 84.3% linkage disequilibrium and 95.1% in group treated with anastrozol and without treatment. Possible association of BMD changes induced by anastrozole therapy with prevalence of the three explored SNPs was not demonstrated. Conclusion: Even though the mutant CYP3A5*3 SNP was predominant, which may result in poor anastrozole metabolism, no significant differences in BMD between the groups were confirmed.

Breast cancer (BC) is the most prevalent cancer in women worlwide. Nowadays, anastrazole is the first choice treatment in postmeopausal women with hormon-dependent BC (HR-BC). Single nucleotide polymorphism (SNP) in genes encoding drug-metabolizing enzymes (DME) could have a critical role in individual responses to anastrozole.

Preterm delivery is defined as delivery occurring before 37 weeks of gestation, and is a major public health problem throughout the world. One of the genetic factors implicated as a factor for the occurrence of preterm birth is genetic polymorphism in progesterone receptor gene (PGR). The aim of this study is to evaluate whether polymorphism in the progesterone receptor gene both in mother and foetus is associated with susceptibility to preterm birth.

Background: Cancer poses a major public health problem worlwide. Breast cancer (BC) is the second most common cancer diagnosed in women in Croatia. Anastrozole has demonstrated a longer disease-free survival and better overall safety in the adjuvant treatment of BC. Single nucleotide polymorphism (SNP) in genes encoding drug metabolizing enzymes could have key role in the individual response to anastrozole therapy and drug safety. Objective: To investigate rates of CYP3A4*1B, CYP3A5*3 and UGT1A4*2 SNPs significant in anastrozole metabolism in BC women in Croatia and its potential correlation to anastrozole induced side effects. Methods: 82 postmenopausal women with estrogen receptor (ER)-positive BC treated with anastrozole and 44 BC controls before therapy were included in the study. DNA for SNPs was genotyped by TaqMan RT-PCR and bone mineral density (BMD) was measured by dual-energy x-ray absorptiometry (DXA). Results: Mutant G allele homozygotes were predominant 88%, and homozygotes for the wild type A allele of CYP3A5*3 were not found. Furthermore wild type homozygotes of CYP3A4*1B were predominant with 94% , and mutant homozygotes were not detected. CYP3A4*1B and CYP3A5*3 SNPs were in 84.3% linkage disequilibrium and 95.1% in group treated with anastrozole and without treatment, respectively. Total hip and femoral neck BMD were lower in the group treated with anastrozole. However, potential correlation of BMD changes induced by anastrozole therapy with prevalence of explored SNPs was not shown. Conclusion: No significant differences in BMD among the groups were confirmed. Although, the mutant CYP3A5*3 SNP was predominant and may result in low anastrozole metabolism.

Incidencija prijevremenog poroda u svijetu je 7.5% od ukupnog broja poroda.Jedna od najtežih posljedica prijevremenog poroda jest cerebralna paraliza u novorođenčadi. Učestalost iste je između 2 i 2.5 na 1000 živorođene djece. Cilj oves tudije je bio ocijeniti efekte te utjecaj terapije magnezijevim sulfatom u trudnica koje su u opasnosti da rode prijevremeno.

The objectives of this study were to validate the direct triplet-primed PCR method (dTP-PCR) for determination of dynamic mutations in the FMR1 gene, and to compare the results of the dTP-PCR method and Southern blot analysis. The number of CGG repeats in the FMR1 gene was determined by the direct triplet-primed PCR method and by melting curve analysis. The cut- off temperature between normal and permutations of the CGG repeats was determined using control samples with a known number of CGG repeats. All patients are classified into four categories based on the DNA melting curve. The clinical performance of the assay was established by 40 previously analyzed samples, yielding results of 100% sensitivity and 90.48% specificity in detection expansions of CGG (>30) repeats in the FMR1 gene. This method is appropriate for the quick determination of allelic changes in the FMR1 gene, screening a population, and identifying mutations or premutation carriers in a population with intellectual disabilities of an unknown cause.

Cilj ove ankete je bio istražiti koji laboratoriji u Hrvatskoj imaju molekularnu dijagnostiku u katalogu svojih pretraga te izdaju nalaze i to među medicinsko-biokemijskim i drugim odgovarajućim laboratorijima. Upitnik je napravljen sa svrhom prikupljanja podataka o laboratorijima kojima će se uputiti detaljna glavna anketa o cjelokupnom dijagnostičkom postupku u praksi. Metode. Upitnik je odrađen uvrštavanjem pitanja u aplikaciju SurveyMonkey (SurveyMonkey Inc. Palo Alto, California, USA). Za kontakte s medicinsko-biokemijskim laboratorijima korišten je registar sudionika HDMBLM-CROQALM (Hrvatski centar za vrednovanje kvalitete u laboratorijskoj medicini) (N=192). Dodatno je pozvano još 15 laboratorija kliničkih instituta, znanstvenih instituta i privatnih poliklinika koji nisu sudionici CROQALM-a zbog spoznaje da se u tim laboratorijima provodi molekularna dijagnostika poligenskih, monogenskih, hematoloških, onkoloških i farmakogenetičkih pretraga te izdaju nalazi za iste. Upitnik je sadržavao 10 pitanja pomoću kojih su prikupljene informacije o podatcima za kontakt, ustanovi i vrsti laboratorija, HDMBLM-podružnici te o tome izvode li samostalno analize molekularne dijagnostike ili se uzorci šalju u suradne institucije. Rezultati. 109 laboratorija je odgovorilo na anketu. 26 laboratorija izdaje nalaze molekularne dijagnostike. 17 laboratorija u kliničkim bolničkim centrima, kliničkim bolnicama i znanstveno-istraživačkim institutima samostalno izvodi analize. 7 znanstveno- istraživačkih laboratorija djelomično, a 2 privatna laboratorija isključivo šalju svoje uzorke u druge institucije. Ako se isključi 11 znanstveno-istraživačkih laboratorija, laboratoriji unutar zdravstvenih ustanova koji izdaju nalaze molekularne dijagnostike su medicinsko-biokemijski (N=8) i laboratoriji u sklopu klinika/odjela za patologiju (N=3), onkologiju (N=2) i transfuzijsku medicinu (N=2). Rezultati molekularne dijagnostike izdaju se pacijentu ili liječniku kao originalan nalaz one institucije koja je napravila samu analizu. Zaključak. Ovim upitnikom utvrdili smo da ćemo surađivati s 24 laboratorija u prikupljanju podataka o molekularnoj dijagnostici u praksi.

Cilj istraživanja bio je ispitati praksu u cjelokupnom laboratorijskom procesu pretraga molekularne dijagnostike u Hrvatskoj. U anketiranje je bilo uključeno 18 od 25 pozvanih laboratorija u kojima se provode pretrage molekularne dijagnostike. Upitnik je upućen putem servisa Surveymonkey, a sadržavao je pitanja o prijeanalitičkoj, analitičkoj i poslijeanalitičkoj fazi te podatke o obrazovanju osoblja koje uvodi nove metode, izrađuje pretrage i potpisuje nalaze. Nove analize uvode visokoobrazovani stručnjaci u području medicine (6/18), medicinske biokemije (12/18), molekularne biologije (7/18), biologije (3/18), kemije (1/18) i biotehnologije (3/18). U izvođenju analiza sudjeluju prvostupnici medicinsko laboratorijske dijagnostike (11/18), zdravstveni tehničari (9/18) i kemijski tehničari (3/18). Nalaze izdaju liječnici (6/18), medicinski biokemičari (13/18), molekularni biolozi (5/18), biolozi (1/18), biotehnolozi (1/18) i prvostupnici medicinsko laboratorijske dijagnostike (1/18). U 7/18 laboratorija procesi su odvojeni u 3 zasebne prostorije, u 8/18 procesi su odvojeni u pre- i post-PCR, dok se u 3/18 laboratorija procesi odvijaju u istom prostoru. Kao mjeru zaštite od kontaminacije 13/18 laboratorija koristi zaštitne PCR kabinete i UV lampe, dok 7/18 koristi i/ili druge načine zaštite. Ispravnost opreme provjerava se godišnjim umjeravanjem (16/19), unutarnjom kontrolom kvalitete (13/18), usporedbom s drugim sustavom (4/18) i pomoću vanjske kontrole kvalitete (2/18). Najzastupljeniji uzorak za analizu je puna krv uz EDTA (15/18), zatim arhivska tkiva uklopljena u parafin (9/18), plazma (6/18), bris bukalne sluznice i solidno tkivo (5/18) te ostale vrste u 4/18 laboratorija. Za izdvajanje DNA najčešće se koristi manualna metoda s komercijalnim kitom na kolonama (12/18) i manualna metoda s komercijalnim kitom (9/18). Anketirani čuvaju uzorke više od godinu dana (DNA (16/16), RNA (6/6) i cDNA (4/6)). Prema ISO 15189 akreditirano je 7/17 laboratorija. Unutarnju kontrolu kvalitete uz svaku seriju uzoraka provodi 17/17 laboratorija, dok u neovisnom programu vanjske procjene kvalitete sudjeluje 14/17 anketiranih. Rezultati istraživanja pokazuju ujednačenost individualnih praksi u cjelokupnome laboratorijskom procesu pretraga molekularne dijagnostike.

Objectives The aim of the study was to establish the association of genetic variants of the ARNTL1 gene with the onset of myocardial infarction. Study design. Case-control study Key words: ARNTL1 gene ; circadian rhythm ; myocardial infarction Participants and Methods The research is conducted on a total of 400 participants. In the first group there were 200 patients with myocardial infarction type 1 hospitalized at the University Hospital Osijek. The second group consisted of 200 healthy controls recruited by a family physician. The goal of the study was to determine the association between single nucleotide polymorphisms of ARNTL1 gene and myocardial infarction. All participants were genotyped for three single nucleotide polymorphisms in the ARNTL1 gene (rs3789327, rs4757144, rs12363415). Results There was no statistically significant difference between polymorphisms of ARNTL1 gene in patients and controls. Research showed that there is no direct link between infarction and single nucleotide polymorphisms in the ARNTL1 gene, but rs3789327 and rs12363415 polymorphisms are associated with cardiovascular risk factors. Diabetes mellitus type 2 was associated with rs12363415 polymorphism, while a history of previous cardiovascular diseases was related to rs3789327 polymorphism. Type 2 diabetes mellitus is a significant risk factor because it has a major role in the development of atherosclerosis and coronary artery disease. Conclusion The results show that there is no association between SNP’s of ARNTL1 gene and myocardial infarction. However, association of SNPs and risk factors for the development of the infarction was found. Based on this, polymorphisms in the ARNTL1 gene has a certain role in the emergence of myocardial infarction.