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The age pyramid of Roma populations tips strongly towards the younger age groups and is characterized by a low number of elderly individuals. There is a vast range of environmental factors that influence the age structure of Roma populations. To explore whether a genetic risk for premature mortality also exists in this ethnic minority, 33 single nucleotide polymorphisms (SNPs) in 23 putative longevity genes were investigated in 308 adult Roma living in Croatia, and in Croatian population sample, composed of 314 „Old” (85-101 yrs.) and 97 „Young” (20-35 yrs.) subjects. The cumulative effect of the investigated SNPs, which have previously been related to human longevity, was summarized within Genetic Longevity Score (GLS). After Bonferroni correction the „Old” and „Young” Croatian age groups differ only in the allele frequency in MRE11A locus (rs533984), while the Roma had significantly different allele frequencies from the surrounding majority population in most of the investigated longevity genes loci (in 16 out of the 33 SNPs). However, the Roma’s GLS is equal to those in the „Young” and „Old” Croatian cohorts implying identical chances of surviving to the age of 85 among Roma as to the majority Croatian population, when only genetics is taken into account.

Background/Objectives: Ages of 90.0 and 95.0 are widely considered as thresholds for longevity and extreme longevity, respectively. The goal of this study was to construct unweighted and weighted Genetic Longevity Scores (uGLS, wGLS) and to test their ability to predict a chance of survival beyond these ages. Methods: DNA samples of 314 unrelated elderly individuals (85.0+ years) were genotyped for 42 putative longevity SNPs in 27 genes, and all SNPs that had a p&lt ; 0.20 in univariate analyses were selected for testing in the multivariate logistic regression models. SNPs from the best model for each cut-off age at death were chosen for GLS calculation, with unweighted score representing a sum of longevity-related alleles, and weighted score a sum of each SNP’s value multiplied by its respective beta coefficient from the multivariate model. Results: GLS included nine SNPs (rs7412, rs50871, rs12206094, rs2267723, rs9536314, rs16847897, rs1800629, rs1042522, rs17202060) for the survival age of 90.0, and five SNPs (rs429358, rs12203592, rs4837525, rs6067484, rs1042522) for the age of 95.0. There was no significant sex difference in mean values of any GLS. All four GLSs (uGLS90.0, wGLS90.0, uGLS95.0, wGLS95.0) were positively correlated with age at death (p&lt ; 0.01). ROC curve analysis showed all four scores are predictive for reaching the longevity milestones. With area-under-curve of 0.690, weighted GLS90 was shown to be the most predictive. Conclusion: GLS based on nine longevity SNPs is predictive of survival to 90.0 years, and five SNP-based GLS is predictive of reaching 95.0 years in the elderly Croatian population. References: / Grants: CSF IP-01-2018-2497 (HECUBA).

Introduction: Human longevity is a multifactorial characteristic, influenced by both genetic and environmental factors. This study aimed to explore whether any difference in longevity genes' makeup could be found in two extreme age cohorts originating from the same population. Materials and Methods: 42 SNPs, selected due to their strong and replicated relation to human longevity and their involvement in different metabolic pathways, were genotyped in a Croatian study sample consisting of 411 individuals. Allele and genotype frequencies were compared between 314 individuals aged 85+ and 97 individuals aged 20-35 years. Results: The allele (p = 0.002) and genotype (p = 0.006) frequencies differed only in the rs533984 of the MRE11A gene belonging to the DNA repair pathway, with the longevity allele G being more frequent in the old cohort. A marginal difference is also found for the ApoE rs7412 allele frequency (p = 0.049), with the longevity allele T (determining ε2 isoform) being more frequent in the old cohort. The G allele of rs533984 has been previously confirmed as favourable for surviving to very old age in Danish females. However, this is the first time to our knowledge that the allele and genotype frequencies of rs533984 have been found to differ between old and young cohorts. Conclusions: The differences in allele and genotype distribution between two extreme age groups of the Croatian population open a possibility that the G allele of the MRE11A gene rs533984 locus might contribute to positive age- related selective survival. Funding: Croatian Science Foundation (IP-01-2018-2497).

Activating mutations/deletions in the epidermal growth factor receptor (EGFR) gene are present in 15% of patients diagnosed with non-small cell lung cancer (NSCLC). Exon 19 deletions and codon 858 point mutations of exon 21 are the most common mutations in NSCLC. The presence of point mutations, deletions and insertions within exons 18 - 25 is responsible for a poor response to tyrosine kinase inhibitors. Types 16 and 18 of human papillomavirus (HPV) are considered to be of high risk for cervical cancer development and progression. The aim of this study was to determine the prevalence of HPV infection in NSCLC cells in relation to the presence and frequency of EGFR gene mutations. In this study, 34 NSCLC samples with EGFR gene mutations and 33 NSCLC samples without EGFR gene mutations were analyzed. Furthermore, EGFR gene mutations were divided into classical (ex19del ; L858R) and rare (G719X ; ex20ins ; S768I ; T790M and ex21). Fragments of the E7 gene were amplified by PCR for HPV16 and HPV18 DNA detection. The possible higher prevalence of HPV infection in NSCLC patients with EGFR mutations, compared to NSCLC patients without EGFR mutations, was examined by meta-analysis using data from this and six other studies. More frequent HPV infection was observed in the NSCLC with EGFR gene mutations (HPV16, n=11, p

The CYP2D6 gene is located on chromosome 22q13.1 and consists of nine exons with an open reading frame of 1491 bp coding for a 497-amino acid protein [1]. This gene encodes CYP2D6 enzyme, which is responsible for metabolism of 25% of clinically prescribed drugs, although accounting for around 2% of total CYP content in the liver [2]. Due to difference in pharmacogenetic distribution of CYP2D6, we wanted to determine a phylogeographic distribution of SNP variation in this gene. The 1000 Genomes Project Phase 3 data on 26 worldwide populations with different genetic ancestry were used in this study. 271 polymorphic SNPs were identified and phased into haplotypes (PHASE v2.1). Reconstructed haplotypes were analysed using population genetics softwares in order to explore inter- and intra-population characteristics. In worldwide sample, 433 haplotypes were reconstructed. The highest nucleotide diversity was found in Africa. Highest haplotype diversity was detected in African ancestry populations and lowest in East Asian populations. FST pairwise differences grouped East Asian populations separately from other populations. Principal component analysis showed continental clustering for all but Central and South American populations. Tests of neutrality showed no background selection in any of investigated populations, while mismatch distribution suggests bottleneck effect in East Asian populations. Haplotype distribution showed significant differences between continental population groups. Phylogeographic differences of CYP2D6 gene suggest greater impact of demography than evolutionary forces like selection on SNP variation in this gene. ACKNOWLEDGMENTS The research was funded by Croatian Science Foundation grant (HRZZ-IP- 2014-09-4454) to MPS and DOK-2018-01. REFERENCES [1] S Kimura, M. Umeno, R.C. Skodaj, U.A. Meyert, F.J. Gonzalez, Am. J. Hum. Genet. 45 (1989) 889-904 [2] U. M. Zanger, S. Raimundo, M. Eichelbaum, Naunyn-Schmiedebergs Arch. Pharmacol 369 (2004) 23-37

Aim: High-risk types 16 and 18 of human papillomavirus (HPV) are associated with the development and progression of various cancers. Unlike classical, the presence of rare mutations in epidermal growth factor receptor (EGFR) gene in non-small cell lung cancer (NSCLC) is responsible for incomplete tyrosine kinase inhibitors response. The aim of this study was to determine the prevalence of HPV infection in NSCLC cytological smears in relation to the presence and frequency of EGFR gene mutations. Methods: The two-year retrospective analysis included 67 NSCLC cytological smears obtained from lung cancer patients hospitalized at the Department of Respiratory Diseases Jordanovac, University Hospital Centre Zagreb. Cytological samples were routinely processed to obtain good quality smears with a sufficient number of malignant cells for PCR analysis. The DNA was extracted from NSCLC cytological smears stained by MGG using the Cobas DNA Sample Preparation Kit. Analyzed EGFR gene mutations were divided into classical (ex19del ; L858R) and rare (G719X ; ex20ins ; S768I ; T790M and ex21). The presence of HPV16 and/or HPV18 DNA was detected by amplifying the fragment of the E7 gene. Expected PCR product size was 212 bp for HPV16, and 236 bp for HPV18. Prevalence of HPV infection in NSCLC patients with EGFR mutations compared to NSCLC patients without EGFR mutations was additionally examined by meta- analysis using data from this and six other studies. Results: Among the 67 NSCLC cytological smears, 34 had mutations in the EGFR gene and 33 were free of it. More frequent HPV infection was observed in the NSCLC smears with EGFR gene mutations (HPV16, n=11/34, p

Lifespan is determined by the interaction of genetic, environmental and lifestyle factors. One of the aging theories focuses on immune response, and argues that the immune system loses its effectiveness with age, leading to autoimmunity and a reduced ability to respond to infections. People who can better deal with this problem by activating the anti- inflammatory response live longer. Since interleukin-6 is related to inflammation, its gene is a candidate gene for longevity. Roma is a transnational minority present in many countries of the world, known to have shorter life expectancy than surrounding populations. Objective of this study was to investigate the association between rs1800795 in IL-6-174G/C promoter region and longevity in 315 subjects (171f/144m, age range 18-72 years, 40.35±13.71 yrs), members of socioculturally different and geographically distant Roma groups in Croatia ; two Vlax Roma groups, and one Balkan Roma group. DNA was extracted from peripheral blood using salting-out method and genotypes were determined using the competitive allele specific PCR method (KASP). Results showed that G allele, in previous research associated with longevity, had the lowest allele frequency in the Međimurje Roma (65.2%, p

Introduction: The pharmacogenetic data have been studied extensively due to their clinical importance in the appropriate drug prescription. Aim: to determine allele frequencies of 27 most important ADME polymorphisms (20 of them 1A, 1B or 2A clinical annotation levels of evidence) in the Croatian population and to compare them with the European population. Materials and Methods: The 27 loci from 20 ADME genes were genotyped in the Croatian study sample consisted of 429 participants. Allele frequencies were combined with published data on the Croatian population (weighted frequencies according to sample sizes). The Croatian allele frequencies were compared with the European population average data taken from the gnomAD database (selection criteria: control cases only and non-Finnish). Results: Results showed higher variant allele frequencies in the Croatian population at six loci (rs1045642, rs2279343, rs1799853, rs1057910, rs28371725, rs9923231) and lower at two loci (rs1695, rs1801272). The most significant differences were: 0.4608 vs 0.4880 in ABCB1, 0.0932 vs 0.2430 in CYP2B6*4, 0.1228 vs 0.1470 in CYP2C9*2, 0.3325 vs 0.2980 in GSTP1 and 0.3488 vs 0.4030 in VKORC1 genes, in Europeans and Croatians respectively. Conclusions: Croatian population shows higher variant allele frequencies in several genes responsible for metabolism of anticoagulation drugs (warfarin, acenocumarol and phenprocoumon). This finding indicates higher bleeding and over- anticoagulation risk in Croatians, demanding lower drugs` prescription doses. Also, lower doses of phenytoin and celecoxib, while higher doses of digoxin, fentanyl, bupoprion and efavirenz are more frequently required in Croatians compared with European average. Acknowledgement: Croatian Science Foundation (HRZZ IP-01-2018-2497 and HRZZ-IP-2014-09- 4454).

Od veljače 2020. godine, javno zdravstvene ustanove u svijetu kontinuirano daju i nadopunjavaju preporuke specifičnih i općih mjera zaštite vezane uz smanjenje rizika od zaraze koronavirusom-2 i pojave pandemijske bolesti COVID-19. Dosadašnje spoznaje sugeriraju da se virus prenosi u direktnom kontaktu putem respiratornih kapljica (govorom, kašljanjem i kihanjem) te dodirivanjem inficiranih površina, pa se preporuke kreću u smjeru primjene primjerene respiratorne higijene i higijene ruku, kao i korištenja osobne zaštitne opreme. Počeci zdravstveno-prosvjetiteljskog pristupa iskorjenjivanju bolesti te preventivne medicine sežu unazad stotinjak godina, otkad datira sljedeći citat: „Suzbijanje neznanja, širenje pismenosti i opšteg znanja, od većeg je značaja za unapređenje narodnog zdravlja od mnogih čisto zdravstvenih ustanova.“ Njegov autor je dr. Andrija Štampar, liječnik i specijalist higijene i socijalne medicine, zapamćen kao pionir socijalne pedagogike, zdravstvenog prosvjećivanja naroda. Štampar higijenu smatra znanošću koja treba proučavati okolinu čovjeka s ciljem pronalaska čimbenika štetnih po zdravlje, kako bi se stanovništvo o njima educiralo te mu se pomoglo da usvoji zdrave životne navike. Osim što je idejno oblikovao tada nov zdravstveni sustav, i sam je aktivno djelovao u poboljšanju životnih uvjeta ponajviše seoskog stanovništva pa je tako npr. 1927. u obraćanju stanovnicima sela Mraclin najavio: “… odmah treba preći na posel... u svakoj kući izgradit će se higijenska gnojnica i zahod, izgradit će se higijenski bunari – pumpe sa čistom pitkom vodom, uredit će se nogostupi i odvodni kanali da ne gazite blato, a uvest će se red i čistoća u kuće kako bi smanjili te silne zaraze i bolesti... Sav građevinski materijal, prijevoz materijala i svi stručnjaci bit će besplatni, a vi ćete dati samo radnu snagu.” U izlaganju ću prikazati kakav je pristup i koje metode dr. Štampar koristio u svom radu, te kako je svojom originalnošću i djelotvornošću postao međunarodno prepoznat i tražen kako bi osobno reformirao brojne zdravstvene administracije.

Roma population is a transnational minority present in many countries around the world. They originated in India, and reached Europe around the 11th century following a path through central Asia and present-day Turkey. Some of the Roma settled in the Balkans area, while others kept migrating for centuries to come. It is estimated that today Roma population consists of 15 million people, with 12 million living in Europe. The social structure of Roma groups is deeply influenced by the traditional endogamy which, together with their unique ancestry, shaped Roma genetic structure. It is well known that pharmacological outcome of drug use extensively depends on the patient’s DNA profile, which is largely influenced by the ancestry. Due to poor living conditions, Roma have a higher prevalence of infectious diseases, which is especially pronounced in tuberculosis incidence. It is several times higher than in the general population in southern Europe, and there has been no reduction in the incidence of tuberculosis among the Roma within the last few decades. The first line drug in tuberculosis treatment is isoniazid or isonicotinylhydrazide (INH) which is metabolized by NAT2 gene, one of the most polymorphic drug-metabolizing genes. Due to the importance of NAT2 gene polymorphisms in inter-individual variation in responses to anti-tubercular therapies, we analyzed 439 DNA samples of Roma from Croatia, who come from three culturally and geographically different regions (Baranja, Međimurje, Zagreb). Six SNP loci in NAT2 gene (rs1801279, rs1041983, rs1801280, rs1799929, rs1799930, and rs1208) were genotyped using Kompetitive Allele Specific PCR (KASP) method. Population specific haplotypes were inferred from analyzed SNP loci using Phase ver. 2.1. To determine pharmacogenetic phenotype, haplotypes were translated to the star nomenclature on the basis of the haplotype set translational table from PharmGKB (https://www.pharmgkb.org/). Inter- and intra- population statistical methods were used to analyze differences in phenotype distribution among the three investigated Roma groups. The analyses revealed six distinct haplotypes. When translated to the pharmacogenomic nomenclature, out of the haplotypes present in all three subpopulations, the most frequent was *5B haplotype (42%), followed by *6A haplotype (33.9%), the wild haplotype *4 (21.8%) and *5A haplotype (0.9%). Translating star diplotypes into metabolizing phenotypes revealed that 61% of the investigated subjects were slow and 39% were fast acetylators. Significant difference was found between the three subpopulations after testing the prevalence of metabolizers. Slow metabolizers were more frequently present in Baranja, and Balkan subpopulations. Since slow acetylators have been associated with an increased risk of adverse drug reaction caused by tuberculosis treatment with isoniazid, we can conclude that subpopulations of Baranja and Balkan are at a higher risk when treated by isoniazid. The specific distribution of haplotypes, resulting from socio-cultural isolation, suggests the need for further systematic pharmacogenetic research in this large, transnationally isolated population.

CYP2C8 gene (10q24, composed of 9 exons, spanning 31 kb) encodes a CYP2C8 protein, one of the most important members of the CYP2C subfamily. The CYP2C8 enzyme, which accounts for approximately 7% of CYP content in the liver, oxidizes about 5% of drugs cleared by phase I metabolism, like drugs used in the treatment of diabetes, cancer, malaria (chloroquine), statins, etc. The activity of CYP2C8 varies widely among individuals and there is evidence that genetic variations of CYP2C8 gene might contribute to its variable activity. Some of the most common polymorphisms in the protein coding region are rs11572103 (CYP2C8*2), rs1058930 (CYP2C8*3) and rs72558195 (CYP2C8*4), the variants responsible for reduced enzyme activity. These were analyzed in this study in 440 subjects who belong to three socio-culturally different and geographically distant Roma (Gypsy) groups. Minor allele frequency (MAF) of rs10509681 was the highest in the Međimurje Roma (30.2%) when compared to the Baranja (14.3%) and the Balkan Roma (8.3%) (p

In order to compare two geographically and culturally isolated ethnic groups of Slavic Muslims from Zhupa and Gora regions in Kosovo, we analyzed their quantitative dermatoglyphic traits, compared them with each other and with the majority, Albanian population. The dermatoglyphs were collected from a total of 263 Zhuplyani, 145 Gorani, and 213 Albanians of both sexes. The ANOVA analysis showed more differences between the Albanians and both minority populations, than between Zhupa and Gora regions populations themselves. We also detected selective inertia in Slavic Muslim women. The canonical discriminant analysis grouped the Gora and the Zhupa women together, and at the same time closer to the Gora men than to the Kosovo Plain women. The Gora and Zhupa men were much closer to each other than to the men from Kosovo Plain. To conclude, the Gora and Zhupa populations differ less from each other, than any of them differs from Kosovo Albanians.

The genetic variability of xenobiotic- metabolizing enzymes contributes to individual susceptibility to environmental risk factors (outdoor pollutants, cigarette smoke, diet- related xenobiotics). Glutathione S- transferases (GSTs) are ubiquitous family of multifunctional enzymes which decrease oxidative damage in cells by catalyzing the conjugation of many toxic compounds with glutathione. The GSTP1 is a polymorphic gene whose certain variants, by altering the gene product protein structure, have been investigated as candidate loci involved in the predisposition to pathologic conditions. This study investigated rs1695 (A313G, I105V) and rs1138272 (C341T, A114V) genotypes, alleles and haplotypes in 440 members of three socio- culturally different and geographically distant Roma (Gypsy) groups. The Baranja and the Međimurje Roma groups belong to Vlax (Bayash) Roma who speak Ljimb`d Bayash, while the Balkan Roma group speaks Romani Chib. Haplotypes were inferred using Phase ver. 2.1. Both investigated loci were in Hardy-Weinberg equilibrium in all the three Roma populations. Minor allele frequency (MAF) of rs1138272 was significantly higher in the Baranja (16.4%) and in the Balkan Roma (14.9%) than in the Međimurje Roma (8.8%) (p

Introduction: As during pregnancy a change in body composition and an accretion of water occurs differently in women, body weight gain might not be the best and only measure reflecting changes in fat storage. One useful additional tool for estimating body composition and the amount of body fat, besides weight and BMI, are anthropometric measurements, e.g. body circumferences and skinfold thickness measures. Methods and sample: Anthropometric measurements from 332 pregnant women included in the Croatian Islands’ Birth Cohort Study (CRIBS) were taken in each trimester, during three visits to the gynaecology practices and they included measurements of pregnant woman’s height and weight (self-reported and additionally validated by a measurement at the first visit), middle upper arm and abdomen circumference, and skinfold thicknesses of upper-arm triceps and biceps. All anthropometric measurements were carried out following the standard International Biological Program protocol (1981). The sample was then further divided into three pre-pregnancy BMI and weight gain categories and differences between two groups were tested using One-way ANOVA. Results: All findings of upper arm and abdominal circumferences were significantly (p

The CRoatian Islands Birth Cohort Study (CRIBS) is the first birth cohort study ever conducted in Croatia and Southeastern Europe, designed to prospectively follow a sample of 500 pregnant women and their children up to two years of age. The aim is to assess the prevalence of risk factors for the metabolic syndrome (MetS) and other NCDs in Croatia. This study presents data of 296 mother-child dyads from CRIBS. Maternal age ranged from 19 to 41 years, with the mean age of 29.7 years. On average, women in our study gained 15.3 kg of weight during pregnancy. Their mean pre-pregnancy BMI was 22.8 kg/m2 (15.7 kg/m2 - 45.2 kg/m2). According to their pre-pregnancy BMI, 6.4% of our participants were underweight, 74.4% were normal weight and 19.2% were overweight or obese. Although the majority of our pregnant women was in the normal pre-pregnancy BMI category (74.4%), there were more women aged > 35 in the overweight and obese category. According to our results, the risk of being overweight or obese also increased with the number of pregnancies, which indicates possible accumulation of weight between pregnancies. Additionally, a trend of higher rate of Caesarean section was observed in the overweight/obese category. In conclusion, pregnancy should be planned before 35 years of age, if possible, and women should be encouraged to enter a new pregnancy only when the desired pre-pregnancy body weight is accomplished. This will then also prevent possible adverse pregnancy outcomes, such as Caesarean section.

CYP2D6 enzyme is involved in the metabolism of approximately 25% of the most commonly prescribed drugs metabolized in liver (opioids, beta-blockers, antidepressants, antitumor agent, etc.). CYP2D6 has the largest phenotypical variability among all CYP’s due to the substantial number of polymorphisms that impact its activity. This gene shows population specific variation. Aim was to determine variation within CYP2D6 among three socio- culturally and geographically distinct Croatian Roma groups (Balkan Roma and Vlax Roma from Baranja and Medjimurje) since their specific population history highly influenced their gene pool. NGS method Genotyping-in-Thousands by sequencing was used to sequence whole CYP2D6 gene on 324 Croatian Roma DNA samples. Polymorphic positons were phased using PHASE v2.1.1. Reconstructed haplotypes were translated into star alleles according to P450 Allele Nomenclature Database. Sequencing identified 51 polymorphic positions. Reconstructed haplotypes significantly differed among the three Roma groups (p

Background: The trajectory of blood pressure (BP) in pregnancy is characterized by an early decrease and a late pregnancy increase. Aim: The aim of this study was to define trajectory of systolic (SBP) and diastolic (DBP) blood pressure in 308 pregnant participants in the CRIBS study and to analyse the association of BP with maternal pre-pregnancy BMI and newborns` anthropometry. Methods: Pregnant women included in the CRIBS study had no history of chronic diseases. The BP of CRIBS participants was measured at least once in each trimester. Maternal pre-pregnancy weight was self-reported. Z-scores were calculated for each newborn`s birth weight, length and head circumference using the WHOAnthro software. All deliveries were term births. The analyses were performed using SPSS 10.0 Results: Less than 2% of women had pregnancy- induced hypertension (SBP >140 mmHg and/or DBP >90 mmHg). BP changed through pregnancy: mean systolic BP of 113 mmHg in the 1st and in the 2nd trimester elevated to 116 mmHg in the 3rd trimester (p30 kg/m2) in all three trimesters were significantly higher than in pre-pregnancy underweight, normal weight or overweight women. Positive correlations were detected between maternal SBP in the 2nd trimester and z-scored birth weight, length and head circumference (R2=0.0205, R2=0.0240 and R2=0.0141, respectively) and between DBP in the 2nd trimester and z-scored weight and height (R2=0.0175 and R2=0.0251). No association between gestational age of newborns and maternal BP has been detected. Conclusion: Most of the CRIBS participants had BP within a normal range. The obesity pre-pregnancy was associated with higher BP in pregnancy and the BP in the 2nd trimester of pregnancy correlated with birth size.

The aim of this study was to investigate the relationship between pre-pregnancy body mass index (BMI) and fasting blood glucose level in pregnancy on the anthropometry of newborns. The sample consisted of 171 healthy pregnant women and their newborns from the Croatian Islands' Birth Cohort Study. Peripheral blood of pregnant women was taken in the second trimester and fasting glucose values >5.1 mmol/l were considered elevated. Anthropometric variables (body weight, height and waist circumference) were measured according to the International Biological Program. Pre- pregnancy BMI and fasting glucose levels during pregnancy were significantly positively correlated (p

CILJ: Cjelogenomske asocijacijske studije (GWAS) dosada su identificirale stotine učestalih varijanti gena povezanih s kompleksnim tragovima, međutim te varijante mogu objasniti samo manji dio fenotipske raznolikosti. U posljednje vrijeme sve više pažnje usmjereno je na ulogu epigenetičke varijabilnosti u etiologiji kompleksnih bolesti. Otkrivanje temeljnih genetičkih i epigenetičkih čimbenika koji pridonose povećanom riziku razvoja kardiometaboličkih poremećaja može značajno pridonijeti razvoju novih strategija prevencije i terapije navedenih poremećaja. U ovom istraživanju proveli smo iscrpnu asocijacijsku studiju na razini cijelog epigenoma (EWAS) koristeći longitudinalne uzorke sakupljene u razmaku od 10 godina i integrirali ih s prethodno provedenom GWAS studijom metaboličkih tragova populacije istočnojadranskih otoka Hrvatske. Glavni cilj rada je utvrditi epigenetičke čimbenike koji stoje u podlozi nastanka kardiometaboličkih poremećaja u navedenoj populaciji. METODE: Postavili smo sljedeće hipoteze: (1) stabilne epigenetičke oznake definiraju razvojnu putanju kardiometaboličkih tragova ; (2) dinamične oznake koje se mijenjaju tijekom života ovisno o unutarnjim biološkim procesima i egzogenim stresorima pridonose razvoju kardiometaboličkih poremećaja kasnije u životu ; i (3) te epigenetičke oznake u interakciji sa genetičkom raznolikošću utječu na varijabilnost fenotipa. Za testiranje hipoteza iskoristili smo postojeću bazu od >1, 400 uzoraka koja sadrži fenotipske, okolišne, DNA i GWA podatke prikupljenu na otoku Hvaru 2007.-2008. godine te ponovo uzorkovali >700 osoba iz prethodne studije 10 godina kasnije (2017.). Također smo proveli EWAS koristeći novorazvijeni EPIC 850K BeadChip methylation array na uparenim uzorcima u dvije vremenske točke kako bi procijenili metilacijske uzorke i proveli integrativnu cjelogenomsku i cjeloepigenomsku analizu kako bi istražili njihov zajednički utjecaj na varijabilnost tragova pomoću pristupa sistemske biologije. REZULTATI: Nakon provođenja asocijacijske studije kardiometaboličkih tragova na razini cijelog epigenoma te integrativne cjelogenomske i cjeloepigenomske analize otkriven je relativno malen medijan beta-vrijednosti metilacijskih uzoraka tijekom10 godina. ZAKLJUČAK: Preliminarno istraživanje temeljeno na nasumično odabranih 112 parova (N=224) uzoraka pokazuju da je medijan beta-vrijednosti metilacijskih uzoraka tijekom 10 godina relativno malen što sugerira da su pronađene epigenetičke oznake stabilne značajke kardiometaboličkih tragova. Financirano od strane NIH, projekt broj R56HL128493.

The main goal of this study was to test the association of pre-pregnancy body mass index (BMI) and fasting glucose level in pregnancy on birth size of newborns. The sample consisted of 177 healthy pregnant woman and their newborns from the islands (Hvar and Brač) and the mainland (Split- Dalmatian County) who are participants in the Croatian Islands Birth cohort Study (CRIBS). Anthropometric variables (body weight, height and waist circumference) were measured according to the International Biological Program. Peripheral blood of pregnant women was taken between 22nd and 26th week of gestation, and fasting glucose values >5.1 mmol/L were considered elevated. Statistical analyses were done by SPSS10.0 for Windows. There was a significant positive correlation between pre-pregnancy BMI and fasting glucose levels during pregnancy as well as between pre- pregnancy BMI and birth weight, length, and head circumference of the newborns. Hyperglycemic women gave birth to significantly heavier newborn girls then normoglycemic women (p

CYP2C19 gene is a member of cytochrome P450 family that codes for enzymes involved in the biotransformation of many endogenous and exogenous substances. Roma are a transnational minority population with numerous health problems, often treated with drugs metabolised by CYP2C19 enzyme. Ten CYP2C19 loci were genotyped in 440 Roma from Croatia, seven of which were monomorphic (rs28399504, rs4986893, rs55640102, rs56337013, rs72552267, rs72558186 and rs41291556). Minor allele frequency (MAF) of rs4244285 in the Roma population was 15.5%, similar to the surrounding Croatian population, and MAF of rs12248560 (CYP2C19*17) was 28.3%, higher not only compared to Croatian population, but also to other world populations. MAF of rs3758581 in the Croatian Roma was 18.3%, which is also notably higher than in other populations. Genetic profile of CYP2C19 in Croatian Roma might be the result of reproductive isolation and definitelly should be taken into account in prescribing drugs, to prevent adverse drug reactions.

The Lika Regiment was part of a unique military institution – the Austrian Military Frontier (Militärgrenze, Vojna krajina) – that existed along the entire boundary with the Ottoman Empire from the first quarter of the 16th century until 1881. The original purpose of this frontier establishment was to protect the Habsburgs from the Ottoman attacks. In the course of time, it became a reservoir of cheap and good soldiers governed by military officials and military law, without civil authority. All frontiersmen capable of bearing arms were part of an ever-ready military force who in return enjoyed special privileges as free peasant-soldiers (land fiefs and religious freedom). In order to gain personal and bioanthropological data of all enlisted men from the Lika Regiment born between 1770 and 1830, we have collected and processed materials from the Croatian State Archives in Zagreb and Austrian State Archives in Vienna. In this study, the secular change in height and disease occurence in 530 adult soldiers (17+ year- olds), who served in the Frontier administration, were analyzed in the context of natural hazards and war activities. The analysis showed a significant, non-linear increase in height. A decline in height detected in the 1790-1799 birth cohort correlates with the participation of the Lika Regiment in the Austro- Turkish War (1788-1791) and the War of the First Coalition (1792-1797), plus periods of hunger caused by long-term winters (1791). The health status of these soldiers was exceptional: only 6.4% had some health problems (the most frequent were respiratory tract problems, hernia and tuberculosis), 17.6% of which ended fatally (caused by oedema, typhus, dysentery and tuberculosis). This preliminary analysis will contribute to our knowledge on the life and health of men from the Lika Regiment soldiers.

Background: The genes encoding the cytochrome P450 2C9 enzyme (CYP2C9) and vitamin K-epoxide reductase complex unit 1 (VKORC1) are major determinants of anticoagulant response to warfarin1. Minor allele carriers of both loci respond in additive manner to a lower dose of warfarin, and they are at risk for adverse drug reaction when usual doses are applied. The Roma (Gypsy) are major European transnational minority population of Indian origin and long- lasting reproductive isolation that could result in specific genetic profile implying that the pharmacogenetic information could not just be extrapolated from surrounding populations. Objective: To investigate the separate and joint minor allele frequencies (MAFs) of the two most important pharmacogenetic loci for warfarin dosage - VKORC1 (rs9923231) and CYP2C9*2 (rs1799853) – in the Roma population and to compare them with 20 populations worldwide. Design: Both loci were genotyped in 422 Roma volunteers living in several regions of Croatia. The comparison is made with 20 populations worldwide (1000 Genomes data extended with the published data for the same countries). Results: Locus rs9923231 has wide geographic variability with extremely high MAF among East Asian populations and almost zero in African populations. However, the rs1799853 MAF distribution does not follow the same global distribution since this allele is not present in East Asian populations, resulting with the highest joint minor alleles frequencies in Roma population amounting 10%. They are followed by European (2.5-7.3%) and South American (0.9- 5.4%), while in South Asian (0.3-1.0%), East Asian (0-0.2) and African (0) populations the risk of combined findings of minor alleles at both loci is negligible or not existent. This finding is primarily result of the highest global MAF for rs1799853 in Croatian Roma (21.6%) in combination with high MAF of rs9923231 in this population (46.1%). Conclusions: Both VKORC1 (rs9923231) and CYP2C9*2 (rs1799853) minor alleles contribute to inter-population difference in adequate warfarin dosage. The present investigation points to the specific Roma population as the one with the highest frequency of combined MAFs at both loci. This puts them at high risk of adverse reaction when warfarin is applied without genetic profiling. Acknowledgements: Croatian Science Foundation grant (HRZZ-IP-2014-09-4454) 1 Fung E et al. Semin Thromb Hemostasis 2012 ; 38:893- 904. ADME ; pharmacogenetics ; VKORC1 ; CYP2C9*2 ; Roma ; 1000 genomes populations ; population genetics ; Croatia Roma ; isolation ; population genetics ; ADME ; pharmacogenetics ; VKORC1 ; CYP3A4 ; NAT2 ; 1000 genomes populations ; Croatia

A large transmembrane P-glycoprotein (P-gp) has an important role in the bioavailability of various drugs: chemotherapeutic drugs, antibiotics, immunosupressants, etc. It is a product of ABCB1 gene, a highly polymorphic member of multidrug resistance protein family. Some of the most common polymorphisms in the protein coding region are rs1128503 and rs1045642, and rs3213619 at 5`-UTR end, which were analyzed in this study in 440 subjects who belong to three socio-culturally different and geographically distant Roma (Gypsy) groups. Minor allele frequency (MAF) of rs1128503 was the highest in the Balkan Roma (69.4%) when compared to the Baranja (62.5%) and the Međimurje Roma (54.5%) (p

Woman`s socio-economic status (SES) indicators (age, education, employment status, income), lifestyle in pregnancy (smoking, physical status), pre-pregnancy BMI and health in pregnancy (levels of serum biochemical parameters measured 22nd - 26th week of pregnancy, complications in pregnancy) are known to affect size-at-birth of a newborn. Healthy women with no history of chronic diseases, who conceived naturally and had singleton pregnancies have been participating in the CRoatian Islands` Birth Cohort Study (CRIBS), an ongoing project in the Eastern Adriatic, Croatia. In this preliminary sample (n=316), mothers` SES and health status indicators were tested for the possible association with newborns` (152 females, 146 males) anthropometric measures (weight, length and head circumference) and size according to gestational age. Elevated total, HDL and LDL cholesterol significantly associated with newborns` head circumference, while glucose associated with BMI and weight/length ratio. Women who were before pregnancy overweight or obese gave birth to significantly heavier and longer newborns when compared to pre-pregnancy underweight and normal weight. Women who self- reported intensive level of physical activity during pregnancy gave birth to significantly shorter and lighter children. SES parameters, smoking and complications in pregnancy (IUGR, GDM, preeclampsia, gestational HT, hypothyreosis) showed no association with size at birth.

Background: Proteins involving absorption, distribution, metabolism and excretion (ADME) of many endogenous and exogenous substances play an important role in determining the pharmacokinetics of drugs as well. There is a considerable variation in genes encoding ADME proteins, both within and between populations. Pharmacogenetic data on the isolated populations are rather scarce. Only few data are available on the prevalence of ADME gene variants in the Roma (Gypsy) transnational minority population of Indian origin (1). Objective: We determine the allele frequencies of ADME “core list” markers and compare them with world-wide data in order to elucidate the position of Roma in the global perspective. Design: The sample comprises 439 Croatian Roma from several regions of Croatia with the highest number of Roma minority individuals. Their DNA samples were genotyped for 95 loci from 32 ADME genes using KASP method. Data were analyzed by standard statistical population-genetics methods within the context of ethnically diverse populations from various parts of the world using the 1000 Genomes project and literature data. Results: The analysis placed Croatian Roma among European populations but their proximity to South Asian populations is also evident suggesting that their ADME gene pool is a combination of ancestral (Indian) and more recent (European) layers. Due to their specific genetic history Roma show the outlying position on the global scale in minor allele frequencies of 12 loci: for 10 loci within 9 genes (rs1128503, rs1138272, rs1799853, rs1902023, rs3758581, rs8192709, rs10509681, rs12248560, rs34059508, rs28371725) they have the highest frequency while for two loci in two genes (rs28399433, rs4149117) the lowest frequency of the minor allele. According to the Clinical Pharmacogenetics Implementation Consortium – CIPC guidelines, rs1799853 (CYP2C9) and rs12248560 (CYP2C19) have been clinically annotated as level 1A supporting their high clinical importance. Conclusion: Our results show that Roma population takes specific position within the global ADME genetic landscape and indicate necessity for the assessment of unique genetic profile of Roma in order to achieve the most in the modulation of pharmacotherapy in this population. Acknowledgements: The research was funded by Croatian Science Foundation grant (HRZZ-IP-2014- 09-4454) to MPS. 1. Tomas Ž, Kuhanec A, Škarić-Jurić T, Petranović MZ, Narančić NS, Janićijević B, Peričić M. Pharmacogenomics 2017 ; 18:1575-87.

CYP2C19 gene is a member of cytochrome P450 family that codes for enzyme involved in the biotransformation of 10% of commonly prescribed drugs. Inter-individual variability in CYP2C19- mediated drug metabolism is mostly due to CYP2C19 variations. This study analyses the pharmacogenetic profile of CYP2C19 in three groups of Croatian Roma (440 individuals). Ten CYP2C19 loci were genotyped, seven of which were monomorphic (rs28399504, rs4986893, rs55640102, rs56337013, rs72552267, rs72558186 and rs41291556). Minor allele frequency (MAF) of rs3758581 (defining wild-type allele, CYP2C19*1) was 56.6% in Roma, MAF of rs4244285 (CYP2C19*2) was 15.1% and of rs12248560 (CYP2C19*17) 28.3%. Frequency of the same MAFs in the surrounding majority Croatian population is 61.5%, 14.8% and 23.7%, respectively (Ganoci et al 2017). The more rapid metabolism (vs. wild type) of CYP2C19-targeted drugs is expected in 40% of the Roma (and in as much as 53.1% in the Baranja group). This is a consequence of a high prevalence (33%) of the “rapid metabolizers” (corresponding to *1/*17 genotype) and 7% of “ultrarapid metabolizers” (*17/*17) in the Croatian Roma. The results indicate that genetic profile of CYP2C19 should be taken into account in modulating pharmacotherapy in Roma population.

Health benefits of physical activity during pregnancy include reduced risk of excessive gestational weight gain and conditions such as gestational diabetes, preeclampsia and preterm birth. The ongoing CRoatian Islands Birth Cohort Study (CRIBS) is the first cohort study in the South-Eastern Europe with an aim to assess the prevalence of risk factors (biological, environmental and behavioral) for the Metabolic Syndrome in populations from Dalmatian islands of Hvar and Brač and coastal Split city with its surroundings. At the time of writing, Over 350 pregnant women and 220 of their newborns have been involved in the study. Here we present the preliminary results of testing the association of mothers` self- estimated physical activity during pregnancy with newborns` anthropometric characteristics (birth weight, length and head circumference) using the data from questionnaires and obstetric records of 116 mother – newborn pairs. The difference in weight-at-birth was detected between newborn girls whose mothers were from Low vs. Intensive physical activity categories, as well as from Moderate vs. Intensive physical activity categories. In addition to that, the significant difference in weight-at-birth and height/length-at-birth was detected between newborn boys whose mothers were from Moderate vs. Intensive physical activity categories (p

Introduction: The ADME (absorption, distribution, metabolism and excretion) genes' variation is markedly related to ethnicity and shows distinct geographic patterns. Generally, the knowledge on distribution of ADME genes in isolated populations is limited, particularly in the Roma, transnational minority population of Indian origin. The aim of this study is to determine the allele frequencies ADME “core list” markers and to compare them with world- wide data in order to elucidate the position of Roma in the global ADME genetic landscape. Methods: The 95 loci from 32 ADME genes were genotyped using KASP method in 440 Croatian Roma DNA samples. Data were analyzed using standard statistical population-genetics methods. Results: The analysis of genetic vs. geographic distances placed Croatian Roma among European populations but their proximity to South- Asian populations is also evident. Next, Roma show the outlying position on the global scale in minor allele frequencies of 12 loci: for 9 loci within 8 genes (rs1128503, rs1138272, rs1799853, rs1902023, rs3758581, rs8192709, rs10509681, rs12248560, rs34059508) they have the highest frequency while for three loci in three genes (rs28399433, rs28371725, rs4149117) have the lowest frequencies of the minor allele. Conclusion: The outlying positon in almost 13% of ADME core markers results from the specific genetic history of the Roma population. This finding may be helpful in developing personalized medicines’ protocols in drug therapies for this specific population. The research was funded by Croatian Science Foundation grant (HRZZ-IP-2014-09-4454) to MPS.