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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.

Aim To investigate the influence of specific intrapopula - tion genetic structures on interpopulation relationships. Special focus was the influence of island population isola - tion on the substructuring of the Croatian population, and the influence of regional population groups on the sub - structuring of Southeast European populations. Methods Autosomal short tandem repeat (STR) loci were analyzed by using four forensic parameters: matching probability (PM), power of discrimination (PD), power of exclusion (PE), and polymorphic information content (PIC) on a sample of 2877 unrelated participants of both sexes. A sample set comprising 590 participants was analyzed for the first time, and 2287 participants were included from previous studies. The analysis was performed with Power - Stats v. 1.2. Results The analysis of forensic parameters for all nine loci in the Croatian subpopulations showed the largest devia - tions in the populations of the islands of Korčula and Hvar. The smallest deviations were found in the mainland popu - lation. As for Southeast European populations, the largest deviations were found in the population of North Mace - donia, followed by Romania, Albanians from Kosovo, and Montenegro, while the smallest deviations were found in the population of Hungary. Conclusion The comparison of forensic parameters be - tween different subpopulations of Croatia and Southeast Europe indicates that the isolation of individual Croatian subpopulations and rare alleles in their gene pool affect the values of forensic parameters. Specific features of (sub) populations should be taken into account for appropriate sampling of the total population when creating a DNA da - tabase of STR markers.

Background/Objectives: Human longevity is influenced both by genetic and non-genetic factors, where genetic variability accounts for 25% of human life expectancy variation (1). We aimed to elucidate SNPs that are significantly related to longevity as defined by cut-off age of 90.0 in a sample of elderly persons of European origin. Methods: 42 SNPs – selected due to strong or repeatedly found association with human longevity in other studies – were genotyped in 314 individuals aged 85.0+ from Croatia. Univariate and multivariate logistic regressions were performed with genotypic data coded as: 2 = longevity allele homozygotes ; 1 = heterozygotes ; 0 = non-longevity allele homozygotes. Results: 16 SNPs that reached inclusion criteria (p

The goal was to perform a meta-analysis of synthesized data and investigate the influence of specific intrapopulation genetic structures on interpopulation relationships. Special focus was the influence of island population isolation on the substructuring of the Croatian population, and the influence of regional population groups on the substructuring of Southeast Europe. A long-term goal is to develop a model of appropriate sampling of the total population when creating a database of genetic STR markers that would properly reflect all the characteristics of included subpopulations. Autosomal STR loci were analyzed using four forensic parameters (match probability, power of discrimination, power of exclusion and the degree of polymorphism) on a sample of 2877 unrelated participants of both sexes. The analysis was performed using the statistical package PowerStats v1.2. The comparison of forensic parameters between different subpopulations of Croatia and Southeast Europe indicates that the isolation of individual Croatian subpopulations and certain rare alleles in their gene pool affect the values of forensic parameters. Specific features of (sub)populations should be taken into account for appropriate sampling of the total population when creating a DNA database of STR markers.

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).

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

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).

Romi su transnacionalna manjinska populacija indijskog porijekla koja nastanjuje većinu europskih zemalja i broji oko 12 milijuna pripadnika. Primjer su populacije utemeljitelja koja je ostala u višestoljetnoj socio-kulturnoj izolaciji, što je ostavilo tragove u njihovoj zalihi gena. Geni ADME, odgovorni za apsorpciju, distribuciju, metabolizam i izlučivanje lijekova, pokazuju značajnu varijabilnost među populacijama. Unatoč dokazanoj funkcionalnoj ulozi, malo se zna o njihovoj distribuciji u izoliranim populacijama. Među gene ADME spadaju i članovi genske porodice NAT koji kodiraju arilaminske N-acetiltransferaze, kod ljudi zastupljeni s dva funkcionalna gena ; genima NAT1 i NAT2. S obzirom na važnost ova dva gena u metabolizmu lijekova i ksenobiotioka, cilj ovog rada je bio utvrditi utjecaj specifične genetske povijesti romskih populacija na raspodjelu alela i haplotipova u genima NAT1 i NAT2. Na uzorcima DNA 439 pripadnika romske populacije iz triju geografski udaljenih regija (Baranja, Međimurje i Zagreb), određeno je 7 polimorfizama jednog nukleotida (SNP) gena NAT1 (rs4986988, rs4986989, rs56379106, rs4987076, rs4986990, rs5030839, rs56172717) te 6 SNP-lokusa gena NAT2 (rs1801279, rs1041983, rs1801280, rs1799929, rs1799930, rs1208). U svrhu određivanja intra- i interpopulacijskih razlika, korištenjem softvera Phase (ver. 2.1.) rekonstruirani su hapolotipovi koji su potom analizirani statističkim metodama populacijske genetike. Radi utvrđivanja farmakogenetičkog fenotipa, haplotipovi su prevedeni u službenu farmakogenetičku zvjezdastu (star) nomenklaturu. Analizom je utvrđena statistički značajna razlika u razdiobi genotipova lokusa gena NAT2 između triju geografski udaljenih romskih populacija u Hrvatskoj. Prisutnost najučestalijih haplotipova utvrđena je u svim istraživanim populacijama, dok se kod gena NAT2 javio jedinstven haploptip u populaciji Roma iz Baranje. Genetske udaljenosti između triju istraživanih populacija pokazale su veće vrijednosti kod gena NAT2, nego li kod gena NAT1. Prijevod haplotipova u zvjezdastu nomenklaturu omogućio je određivanje farmakogenetsog fenotipa Roma: 61% ispitanika su spori, a 39% brzi acetilatori lijekova. Spori acetilatori su povezani s povećanim rizikom od ADR-a (engl. Adverse Drug Reaction – štetna reakcija na lijek) izazvanog liječenjem tuberkuloze (TBC) izonijazidom. Specifična raspodjela haplotipova, koja je nastala kao posljedica socio-kulturne izolacije, upućuje na potrebu za daljnjim farmakogenetskim istraživanjima kako bi se u što većoj mjeri izbjegao potencijalni rizik od ADR-a i očuvalo zdravlje.

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

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

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

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.

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

The european wildcat Felis silvestris silvestris inhabits wide areas in Europe and is considered a subspecies genetically close to the domestic cat Felis silvestris catus. The two subspecies have different habitat preference, but as human activity causes undisturbed areas to disappear the two subspecies come increasingly into contact, allowing for hybridization and the disappearence of the genetically pure wildcat populations. While hybridization has been confirmed throughout Europe, the genetic structure of wildcats in Croatia has not been previosly studied. With relatively conserved forest habitats, Croatia might contain a wildcat population largely unaffected by hybridization. If present, such population is important in maintaining of the wildcat genetic structure for both conservation of the existing wildcat populations and reintroduction of the genetically pure wildcats into areas from which they have disappeared. Muscle, blood and oral mucosa samples have been obtained from both wildcats and domestic cats. After DNA extraction, microsatellite markers have been amplified by high fidelity DNA polymerase using multiplex touchdown PCR and analysed by capilary electrophoresis. Bayesian clustering using Structure 2.3.4 clearly separated the wildcat, domestic and hybrid cat populations and the genetic variability of each population has been analyzed using Arlequin 3.5.2.2. and CLUMPAK software. The results show low incidence of hybrids, and the existance of subpopulations in both wildcats and domestic cats. These results indicate the need for further wildcat habitat preservation, and might be used as a basis in tracking, monitoring and conservation of the wildcat population in 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

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.

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.

Aim was to determine variation of CYP2B6 gene within the genetically specific Croatian Roma (Gypsy) population originating from India and to examine it in the worldwide perspective. Seven SNP loci (rs12721655, rs2279343, rs28399499, rs34097093, rs3745274, rs7260329, rs8192709) were genotyped in 439 subjects using KASP method. The Croatian Roma took an outlying position in CYP2B6 variation from the worldwide perspective mainly due to their exceptionally high MAF for rs8192709 (12.8%), and lower MAF for rs2279343 (21.1%) compared to south Asian populations. This study provides the first data of several CYP2B6 polymorphisms in Roma population and indicates the need for systematic investigation of the most important pharmacogenes' variants in this large, transnationally isolated population worldwide.

ADME genes exhibit significant variation among the human populations due to the past demographic and evolutionary events. 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 variation of CYP2B6 among Croatian Roma. It is one of the most polymorphic ADME genes in humans and plays role in several drug metabolizing pathways (e.g. efavirenz and nevirapine, bupropion). Within the broadly designed project, we investigated the variation of a large panel of ADME genes among the Roma minority populations of Croatia. Genotyping was done using KASP method. Here we present CYP2B6 gene variation, which was detected by genotyping seven SNP loci (rs12721655, rs2279343, rs28399499, rs34097093, rs3745274, rs7260329, rs8192709) in 440 DNA samples belonging to the three socio-culturally and geographically distinct Croatian Roma groups. Results: Three of the investigated loci (rs12721655, rs28399499, rs34097093) were monomorphic in all samples. MAFs of polymorphic loci (rs2279343, rs3745274, rs7260329, rs8192709) ranged 17-30%, 12-26%, 24-45%, 5- 17%, respectively, with significant differences among the investigated groups. Moreover, MAF of locus rs8192709 is in two of the three Roma populations four times higher than the average global frequency. Significant LD values between different pairs of loci were detected in all three investigated populations indicating recent founder effect. Conclusion: Our results suggest that each investigated Roma population has unique CYP2B6 genetic profile which should be considered in the modulation of pharmacotherapy due to its possible clinical importance.

Introduction: Arylamine N-acetyltransferase 2 (NAT2) plays an important role in metabolism of therapeutic drugs and exogenous chemicals present in the diet and the environment. In addition, numerous association studies confirmed a link between NAT2 acetylator phenotypes and cancer risk. However, these studies were predominantly conducted in populations of European origin. Roma population is a transnational minority population of Indian origin for which data on variability of NAT2 gene is very limited, presenting a problem in regard to information on drug efficacy and safety. As many Roma people live from collecting secondary raw material, they are exposed to exogenous possibly cancerogenic chemicals, which can increase cancer risk in genetically susceptible individuals. The aim of this study was to determine genetic variability of NAT2 in Croatian Roma population.Methods: NAT2 gene polymorphisms (rs1041983, rs1801280, rs1799929, rs1799930, rs1208, rs1799931, rs1801279 and rs1805158) were genotyped in a sample of 440 individuals from three Roma groups as a part of genotyping project aimed at determining variability of ADME and several other selected genes involved in drug metabolism. Genotyping was performed using KASP method. Results: Loci rs1801279 and rs1805158 were monomorphic in all three Roma groups. Allele distribution of 3 polymorphic loci, rs1801280, rs1799929 and rs1208, differed significantly among the investigated groups. In addition, MAFs in Croatian Roma are notably different from the average global frequency. LD between investigated loci is very high in all three Roma groups.Conclusion: Results of this study confirm expected specific genetic profile of NAT2 in Croatian Roma population which results from their high level of isolation. This genetic specificities should be taken into account in pharmacotherapy. In addition, very high LD level suggest a possibility of using tag SNPs in genotyping analysis of these loci in Roma populations.

Background: Evidence from studies conducted in community dwelling older adults indicate the beneficial effect of resilience on health and longevity but this relationship has not been adequately examined in the institutional settings. Resilience connotes the ability to adapt positively to adversity and can be well preserved at the very end of the lifespan. Exploring the resilience in institutionalized elderly, especially in those of very advanced age, can help to increase our understanding of how institutions can facilitate successful aging. The purpose of this study was to investigate the resilience among the oldest-old residents of long-term care institutions and to determine its association with age, sociodemographic factors and health status. Methods: Data were collected from the interviews with 330 persons aged 85-101 years in 13 retirement homes of Zagreb (Croatia) using cross-sectional design. The instruments used in the study included resilience scale, questions about demographic and socioeconomic characteristics (age, sex, marital status, education, pension status) and health status (chronic conditions, Activities-of-Daily- Living, Mini-Mental-State-Examination, Mini- Nutritional-Assessment, stress, parental longevity). Resilience was analyzed as a dichotomous variable (higher versus lower) using logistic regression. Independent variables in the models comprised all the above mentioned sociodemographic and health characteristics. Results: A total of 42% of subjects had higher resilience level. Age showed no association with the resilience without adjusting for any confounders. After controlling for sociodemographic and health characteristics, nonagenarians were in fact more resilient than the octogenarians (odds ratio - OR:1.91 ; 95% confidence interval – CI:1.02-3.55). There was no association with sex, education nor pension status but still having a spouse had strong positive effect (OR:3.48 ; CI:1.33-9.07). Among potential health confounders, only the good nutritional status showed association with higher resilience (OR:2.20 ; CI:1.26-3.83). Additionally, coping with stressful life events had a significant positive effect on resilience (OR:2.27 ; CI:1.05-4.91). Conclusion/Implications: The findings indicate that resilience does not decline in very advanced age. The study has quantified the role of sociodemographic and health factors associated with resilience among the oldest-old Croatian institutionalized persons. Policies and programs to promote resilience would have long-term and positive effects on the well- being for senior persons and their families.

Objective: The role of adiponectin in hypertension (HT) is still a matter of debate. Obtained conflicting results could be mostly explained with diversity of subjects included in different studies. Our aim was to analyze association of adiponectin and its gene polymorphisms with blood pressure (BP) in a group of normotensive (NT) and untreated HT subjects with normal kidney function. Design and method: Analyses was conducted in 192 subjects (68 m ; age 44 (IQR 31–53)). Those with diabetes, treated HT and chronic kidney disease (eGFR < 60 ml/min) were excluded. BP was measured using Omron M6 device following ESH/ESC guidelines. Adiponectin concentration was determined by ELISA. Genotyping was done using PCR-FRET. Genetic association analyses were done with Arlequin and haplotype analysis with Unphased. Results: There were no differences in plasma adiponectin values (mg/L) between HT and NT (9.75 ; iqr:7.44–17.88 vs.11.35 ; iqr:7.43–12.63 ; P = 0.17) and no associations with systolic or diastolic BP (P > 0.05). Ninety-three persons had genotype −11377C/C, 77 had −11377C/G and 18 −11377G/G ; minor allele frequency (MAF) 30%, with no difference between NT and HT. Polymorphism −11377C > G was not associated with HT in the dominant, codominant, overdominant, recessive or additive model (all p > 0.05). Polymorphism −11391G > A (164 were dominant homozygots and 22 were heterozygots ; MAF 12%, with no difference between NT and HT) also was not associated with HT (OR 2.12 [0.73–6.16]). Haplotype analysis established three haplotypes, C-G (freq. 64%), G-G (30%), C-A (6%) which showed no association with HT well. Conclusions: In our group of NT and untreated HT with normal kidney function adiponectin was not associated with BP even after adjustment for other risk factors. Even more, we did not find an association of two common adiponectin gene polymorphisms with HT. Adiponectin-BP relationship is complex and differs between specific populations. Our conclusions should not be extrapolated to subjects with other characteristics. Studies on larger number are needed.

Institut za antropologiju proveo je 2012. godine multidisciplinarno antropološko i epidemiološko istraživanje populacije Roma koja živi na području grada Zagreba, a koje je uključilo 173 osobe (79 muškaraca, 94 žene), dobi 39, 9+12, 7 godina (18-72). Uz niz biometrijskih mjerenja, provedena je opsežna anketa koja je uz teme o radu, školovanju, broju djece, stanovanju, diskriminaciji i zdravlju uključivala i prehrambeni upitnik koji je temeljem prethodnih istraživanja prilagođen ovoj populaciji. Pitanja vezana uz prehranu uključila su: učestalost konzumacije alkoholnih pića (vino, pivo, žestoka pića), kave i vode, pojedinih prehrambenih namirnica (mliječnih proizvoda, mesa ili ribe, voća ili povrća, grahorica ili jaja) te pitanja o broju i dnevnom rasporedu obroka. Cilj ovog rada bio je ustanoviti glavne karakteristike prehrambenih navika ove ranjive populacije. U opisu obroka vidljiva je visoka preferencija mesa i mesnih prerađevina uz niski udio svih ostalih namirnica. Analiza odgovora o vrsti hrane u obroku pokazala je izrazitu zastupljenost „mesa i krumpira“ ili hamburgera/ćevapa/pečenog mesa kao sadržaja glavnih obroka u danu. Međutim, najistaknutiji nalaz jest da Romi grada Zagreba jedu prosječno 2, 7 obroka dnevno (1-7) pri čemu postoji značajna povezanost između broja obroka i sata prvog i drugog obroka ; naime vidljivo je odgađanje sata prvog obroka ukoliko je broj obroka u danu manji. Pokazalo se da 75% osoba jede od jednog do tri obroka dnevno od toga 41, 4% jede manje od tri obroka na dan (28, 6% muškaraca i čak 52, 2% žena). Prva skupina, a to su osobe koje su jele 1-2, 5 obroka dnevno značajno se kod oba spola razlikovala od skupina koje su dnevno jele 3 i više obroka. Tako su žene prve skupne prvi obrok su imale u prosjeku u 11, 5h, a muškarci u 12, 0h, što je bilo značajno kasnije od vremena prvog obroka drugih skupina koji je padao u vrijeme oko 10h (9, 9 muškarci, 9, 8 žene). Drugi obrok je kod oba spola prve skupine padao u 17h, što je značajno kasnije od vremena drugog obroka ostalih skupina koje je padao između 13, 5 i 14, 5 sati. Muškarci koji jedu manje od 3 obroka dnevno imaju značajno viši prosječan indeks tjelesne mase (31, 1) u odnosu na one koji jedu 3 i više obroka dnevno (28, 3). Također, kod muškaraca (ali ne i kod žena) bilo je značajno više pretilih osoba među onima koji jedu manje od tri obroka dnevno. Muškarci iste skupine su i stariji od onih koji jedu 3 i više obroka dnevno (48, 6 vs. 36, 5 g). Žene koje jedu manje od 3 obroka dnevno, imaju više vrijednosti glukoze natašte (6, 3 vs 5, 2 mmol/L) kao i kožnih nabora tricepsa i potkoljenice te su starije od onih koje jedu 3 i više obroka dnevno (41, 6 vs 36, 3 g). Također, kod žena je u skupini koja jede manje od 3 obroka dnevno češći povišeni ukupni kolesterol te ih ima više koje imaju subskapularni kožni nabor veći od 18mm. Rezultati provedenog istraživanja ukazuju na zabrinjavajuću prehrambenu praksu u populaciji Roma grada Zagreba koja se sastoje od malog broja obroka u danu, što ima vidljive posljedice na antropometrijske i biokemijske pokazatelje zdravstvenog statusa u ovoj populaciji.

The ADME genes exhibit significant variation among the human populations due to the past demographic and evolutionary events. Genetic distinctiveness is especially pronounced in isolated populations where the exchange of genes with other populations is minimal and where the increased frequency of otherwise rare or private alleles emerges. The example of such population are the Roma, the transnational minority population of Indian origin with centuries long sociocultural isolation which left traces in their gene pool showing considerable differences in comparison to other populations. Therefore, we investigated the variation of a large panel of ADME genes among several Roma minority populations residing in Croatia. Here we present the results of CYP2B6 gene variation which was detected by genotyping five SNP loci (rs12721655, rs2279343, rs28399499, rs34097093, rs3745274, rs7260329, rs8192709) in the three socioculturally and geographically distinct Roma populations living in northern, central and eastern regions of Croatia. Two of the investigated loci (rs28399499, rs34097093) were monomorphic in all samples, while locus rs12721655 was polymorphic only in Roma population from the northern Croatian region of Medjimurje. Its MAF was 21.5% that is considerably high since the global MAF is

Self-perceived health (SPH) is a powerful indicator of the general health status of older people that has been widely investigated in community dwelling populations. Its determinants have rarely been studied in institutional settings. The objective of this study was to identify the main factors that have an impact on SPH among the oldest old residents of old people's homes. In the extensive anthropological study, face-to-face interviews were carried out with 330 individuals aged 85-101 years in 13 public and private old peoples' homes in Zagreb (Croatia) and its surroundings. SRH was assessed by a single-item health measure with five options. It was analyzed as a dichotomous variable (fair/poor versus excellent/very good/good) using logistic regression. Independent variables in the models included sociodemographic and health characteristics. A total of 51% of subjects rated their health as excellent or very good. There was no association with sex or age nor with marital status, education or pension status. The adjusted odds ratio (95% confidence interval) for worse health perception was 1.36 (1.19- 1.55) for each additional chronic condition, 1.42 (1.01-1.86) for each additional assistive device, and 1.67 (1.05-2.71) when comparing functionally dependent residents and those functionally independent. Feeling of loneliness and poor social relations were also strongly associated with worse health perception. Bad nutritional status and cognitive impairment showed significant independent effects. This study found a positive association between subjective and objective health in the oldest age. Chronic conditions, functional status and social interaction were the main determinants of self-perceived health among Croatian institutionalized elderly persons. Thus, the study revealed that, with the exception of socioeconomic factors, the same determinants of health perception in the community are also relevant in institutions among persons of very advanced age.

BACKGROUND: Roma (Gypsy) are transnational European disempowered minority population, and the increasing prevalence of obesity and diabetes suggests that they are undergoing the epidemiological and nutritional transition. The genetic architecture of this population is also specific, characterized by their remote origin (India) and substantial endogamy. Concluding, they are socioeconomically and genetically very different from the surrounding majority European populations. AIM: To explore the association of serum leptin and leptin (LEP) and leptin receptor (LEPR) genes with anthropometric and biochemical nutritional status biomarkers in Croatian Roma population. METHODS: The associations of leptin and 12 polymorphic sites in LEP (9) and LEPR (3) genes with anthropometric and biochemical indicators of nutritional status have been analyzed in 206 men and 208 women of Roma origin. The associations have been tested using ANOVA and chi2 test (for categorical variables constructed using standard cut-offs). RESULTS: The results showed that leptin concentrations as well as leptin system genes are associated with investigated indicators of nutritional status in Roma. The consistent impact of leptin genes on total cholesterol/HDL and LDL/HDL ratios in women is a prominent finding of this study. This relation (quantitative and qualitative variables) was significant in 6 out of 12 SNPs in women, while none of 12 SNPs showed significance in men. Several anthropometric indicators of obesity also showed significant association with LEP and LEPR loci (upper arm circumference, waist-hip ratio, triceps-subscapular skinfold ratios). Interestingly, serum leptin showed no relation to investigated LEP and LEPR loci. CONCLUSION: This study confirmed the important role of leptin, which showed significant association with almost all anthropometric indicators of obesity as well as the relation with several indicators of lipid and glycemic status. The consistently strong association of cholesterol ratio with LEP and LEPR polymorphisms in Roma women is an unexpected and outstanding finding of this study.

Our recent extensive research of the Roma (Gypsy) population in Croatia pointed to a significant load of risk factors for the different diseases which accompany economic transition, indicating a rising prevalence of cardiovascular diseases (CVD) in the future. This may lead to the increase of appropriate drugs consumption in this population. The active transport of numerous medications for CVD treatment is mediated by OATP1B1 protein encoded by SLCO1B1 gene. This gene has numerous variants which can cause adverse drug response and some of them significantly differentiate among populations. The Roma are transnational minority whose gene pool results from their Indian ancestry, subsequent admixture with surrounding populations and their socio-cultural isolation. Their reactions to different drugs are poorly documented. Therefore we analyzed 8 SNP loci within SLCO1B1 gene in 434 Roma individuals from three socio-culturally different Roma groups (Balkan Roma, Vlax Roma from Baranja and Medjimurje). All but one locus, rs4149056, were monomorphic. The polymorphic locus shows significantly different genotype distributions among the investigated populations (p

Razlog i cilj istraživanja: Kronološka dob mjeri broj godina života dok je biološka dob mjera kvalitete funkcioniranja organizma te predstavlja jednu općenitu smjernicu koja ukazuje treba li kod pojedinca snažnije preventivno i terapijski intervenirati kako bi se usporio proces starenja i razvoj kroničnih bolesti te tako održalo sadašnje zdravstveno i funkcionalno stanje i odgodio nastup nesamostalnosti. Cilj istraživanja je analizom antropoloških i biomedicinskih obilježja osoba duboke starosti identificirati najrelevantnije biomarkere zdravstvenog statusa koji bi mogli biti od koristi u svakodnevnoj antropološko- gerontološkoj i gerijatrijskoj praksi. Sve analizirane parametre karakterizira njihov značaj za više organskih sustava te su stoga oni potencijalno dobri biomarkeri ukupnog zdravstvenog i funkcionalnog stanja. Metode: Uzorak čine 325 institucionalizirane osobe duboke starosti (85-101 godine života) s područja grada Zagreba. Provedena je faktorska analiza širokog spektra varijabli povezanih sa zdravljem koja je uključivala neinvazivnu zdravstvenu biometriju (antropometrija, mjerenje krvnog tlaka, ultrazvučna denzitometrija), osnovne biokemijske pretrage seruma (lipidni, glikemijski i proteinski status) te anketno utvrđene karakteristike zdravstvenog statusa. Rezultati: Prvi faktor je kod oba spola definiran s dobi ispitanika nasuprot antropometrijski utvrđenom prehrambenom statusu, na čelu s tjelesnom težinom. Drugi faktor je kod oba spola definiran koštanim statusom (ultrazvučno utvrđena gustoća petne kosti), no on je kod muškaraca kontrastiran s nekoliko varijabli koji upućuju na lošiji zdravstveni status (veći broj kroničnih bolesti, veći broj korištenih lijekova) dok je kod žena kontrastiran s biokemijskim varijablama koje definiraju lipidni status (trigliceridi, omjer LDL/HDL kolesterol, ukupni kolesterol) i proteinski status (globulini, ukupni proteini). Zaključak: U osoba duboke starosti (85+ godina) kod oba spola prisutna je negativna povezanost između dobi i tjelesne mase (što su osobe starije to imaju manju tjelesnu težinu, indeks tjelesne mase, opseg nadlaktice, debljinu kožnih nabora). Kod muškaraca, ali ne i kod žena, gustoća koštanog tkiva istaknula se kao dobar pokazatelj ukupnog zdravstvenog statusa. Provedena preliminarna analiza ukazuje na postojanje spolno specifičnih antropometrijskih i biokemijskih indikatora zdravstvenog statusa. S obzirom na velik značaj koji koštano-mišićni sustav ima za pokretnost i samostalnost starijih osoba te time na njihovu neovisnost o tuđoj pomoći, ovi rezultati daju novi prilog jer ukazuju na direktnu vezu između parametra koštanog statusa i ukupnog zdravstvenog statusa.

The Institute for Anthropological Research has undertaken multidisciplinary anthropological, molecular-genetic and epidemiological investigations of Roma populations in Croatia in order to gain results which will improve living and health conditions of Roma in Croatia and contribute to better inclusion of Roma communities in society. In order to collect research data we carried out several field studies in Roma settlements of three socio-culturally different Roma groups (two groups of Bayash/Vlax Roma, speaking two distinct dialects of archaic Romanian language – the Baranja group speaks mostly the Muntean dialect, the Međimurje group uses Ardelean, and Balkan Roma Romani chib). Since genetic history is an important part of Roma population identity, one of our main goals was to determine genetic structure of Roma populations in Croatia by analyzing different DNA markers (mtDNA, autosomal-STR, Y-STR and X-STR loci). Due to their different inheritance pattern the analyses of ancestrally informative markers (Y-chromosome and mtDNA) elucidated full genetic structure of Roma populations in Croatia. The analyses of Y chromosome and mtDNA confirmed the Indian origin of all Roma populations in Croatia and their subsequent on route isolation. The population of Bayash Roma harbored unique maternal lineages which implies long isolation period among different Roma groups. Analyses of autosomal STR loci showed significantly lower gene diversity in Roma populations in comparison with geographically proximal majority population and low gene flow among Roma populations and majority population, while the analyses of X chromosome STR loci showed LD values characteristic for isolates. All three investigated population groups show common Indian origin and admixture with majority population. Nevertheless, intra and interpopulation analyses suggest significant differences among investigated populations in terms of different degree of endogamy, accompanied by the genetic drift, and different sources of admixture and migration routes. Since the endogamy shapes the genetic structure of Roma populations in Croatia we have extended our genetic investigation on the genes which are responsible for absorption, distribution, metabolism and excretion of drugs (ADME genes) and significantly differentiate among populations. The general objective of our on-going project is to determine the variations of ADME genes in the Roma minority population of Croatia. Our assessment of Roma’ unique genetic profile will contribute to the medical practice through the modulation of pharmacotherapy in Roma population and highlight the population uniqueness in ADME genes profile, which will, hopefully, encourage similar studies of this minority population estimated to around 15 million people.

Contemporary populations’ genomic diversity reflects past demographic and evolutionary events. Genetic distinctiveness is especially pronounced in isolated populations where the exchange of genes with other populations is minimal. The Roma, transnational minority population of Indian origin is an example of a founder population with centuries’ long sociocultural isolation which left traces in their gene pool showing considerable differences compared with other populations. The general objective of this study is to determine the variations mtDNA lineages in tree socio-culturally different Roma populations from Croatia, in order to gain insight into their temporal and spatial ancestral differences. Therefore we analyzed mtDNA of 160 Balkan Roma, 200 of Vlax Roma from Baranja and 150 Vlax Roma from Međimurje. All three populations show the same major haplogroups M and H, suggesting their common Indian origin and substantial admixture with surrounding population, respectively. Nevertheless intra and interpopulation haplotype analyses suggest significant differences among investigated populations in terms of different degree of endogamy, accompanied by genetic drift, and different sources of admixture and migration routs.

Roma are an example of genetically isolated population, which preserved its uniqueness until present day due to their socio-cultural characteristics. Genetically isolated populations have been recognized as an appropriate model for investigations of various genetic diseases. The Roma population was, so far, investigated for numerous monogenetic disorders but not for complex diseases in spite of their high prevalence among Roma. Association studies aimed at finding disease mutations are dependent on the extent of linkage disequilibrium (LD) levels in a given population. The main goal of this investigation is to determinate the level of LD and isolation and inbreeding among three socio-culturally different Roma populations in Croatia. We genotyped 7 X-chromosome microsatellite markers located in the region previously studied for LD estimation in genetic isolates. PCR fragments were analysed on ABI Prism 310 DNA analyzer. Genescan v3.1 software was used for processing the results, and tandem software was used for rounding allele sizes to valid integers. Various software were used to measure pairwise and multiallelic LD values. Results indicate elevated levels of LD in investigated Roma populations, and the comparison with other isolated populations showed similarity of Međimurje sample with highly isolated and small but stable populations, while Baranja and Zagreb sample exhibited characteristics similar to populations more open to gene flow and therefore less isolated than Međimurje sample. Levels of LD in Croatian Roma populations suggest a possibility for their use in the identification of genes underlying complex diseases by means of LD association studies.

AIM: To explore the interaction of two leptin system genes and nutritional status biomarkers in Croatian Roma population as an example of the socio-cultural and genetic isolate. METHODS: The associations of 12 polymorphic sites in leptin (LEP) and leptin receptor (LEPR) genes with 20 anthropometric and 25 biochemical indicators (from serum or plasma) of nutritional status have been analyzed in total sample (413 participants) as well as in sex- and region-specific subsamples. The urban and rural subsamples have been matched for age and sex structure. The potential associations have been tested using ANOVA (for original measurements) and chi2 test (for categorical variables constructed using standard cut-offs) while the structure of the relationships has been studied using principal component analysis. RESULTS: The study has shown significant sex and regional differences in the impact of leptin system genes on nutritional biomarkers. Overall, the genetic influences on morphological traits have been more pronounced in men where the LEPR polymorphisms had a major role. The most significant finding is was the association of LEPR1019 polymorphism with body mass index and subscapular skinfold in men. On the other side, LEP loci have shown association with lipid profile and this effect was more pronounced in women. CONCLUSION: Two most important genes of the leptin system - LEP and LEPR - have diverse roles in their impact on the biomarkers of nutritional status: the former being more related to biochemical profile and the later being associated with the quantity and distribution of body fat.

The Roma (Gypsy) are the transnational European minority population characterized by poverty, social exclusion as well as by numerous life-style and cultural specificities. It has been hypothesised recently that they are experiencing a transition from traditional to a more sedentary lifestyle with an excess of caloric intake. Such lifestyle changes could have important public health consequences since they are expected to be accompanied with epidemiological transition ; from contagious to common complex morbidities as the major health problem in this population. To explore the evidence of this transition this study focuses on obesity. It aims to compare the anthropometric indicators of obesity as well as the obesity and overweight prevalences (estimated using body mass index, BMI) in urban vs. rural adult Roma living in Croatia (413 participants in total). The two groups - urban (living in Zagreb, the capital of Croatia) and rural Roma (living in Baranja and Međimurje counties) - were matched for their age (40.6 yrs ; p=0.997) and sex structure (46.9% vs. 52.5% men ; p = 0.256). The study has shown that urban Roma have significantly higher prevalence of obesity (BMI 30+) compared to their rural counterparts (36.7% vs. 24.0%, p = 0.005). The overweight and obesity prevalence (BMI 25+) was also higher in urban than in rural Roma (66.8% vs. 57.1%, p = 0.043). Men and women were not different (according to t-test) in their average BMI (27.5 ; +5.3 in men and 26.8 ; +6.2 in women, p=0.222). Urban-rural difference was more pronounced in males: all investigated anthropometrical variables related to obesity showed to be consistently higher in urban men (triceps and subscapular skinfolds, waist, hip and upper arm circumferences) while only two of them (triceps and subscapular skinfolds) showed similar regional differences in women. The study has shown that the Roma population bears a high obesity risk, which is especially present in urban environment and in men. High prevalence of obesity in Roma population, which was recently observed in some other countries as well, indicates health transition of this ethnic group and the necessity of adequate preventive measures.

Romi su transnacionalna manjina indijskog porijekla. Danas su sastavljeni od brojnih grupa različitih socio-kulturnih značajki, a kod svih je izražen učinak utemeljitelja prilikom osnivanja. Zbog stroge endogamije, romske grupe podložne su utjecajima genskog pomaka, što za posljedicu ima postojanje brojnih razlika u njihovoj genskoj zalihi. Kako bismo uočili te razlike, proučavali smo pripadnike različitih grupa koje postoje u Hrvatskoj, a pričaju različitim jezicima (Romani-chib i ljimba d’ bjaš). Analizirali smo njihovu genetičku strukturu koristeći autosomalne (STR) i uniparentalne (mtDNA i kromosom Y) markere, na temelju kojih smo odredili intra- i inter-populacijske parametre. Rezultati su ukazali da su romske grupe prisutne u Hrvatskoj bile izložene različitim stupnjevima miješanja s okolnim populacijama i genskog pomaka, a razlike nastale između grupa uslijed toga ukazuju na važnost socio-kulturnih značajki u oblikovanju genetičke strukture u populacijama utemeljitelja.

Genotypic and phenotypic differences between populations can be created by adaptation to different living conditions such as climate. Recently it was found that the frequencies of some genetic variants involved in metabolic pathways are correlated with climate zones. Since the Roma are a population which descends from area belonging to different climate zone then they presently live the goal of this study was to determinate how it is reflected in their gene pool. Since the leptin system is recognized as important regulator of metabolism we analyzed 29 SNPs associated with leptin system in Croatian Roma population. Allelic frequencies of these loci are compared between Romani and other worldwide populations. We found out that allele frequency distribution in worldwide populations follows isolation by distance model and 6 investigated loci show significant correlation with latitude as a proxy for climate conditions. Investigated loci in Roma gene pool reflect process of admixture with Asian and European populations.

Unatoč svojoj raznolikosti, sve današnje romske grupe dijele zajedničko indijsko porijeklo a time i brojne privatne mutacije, od kojih mnoge uzrokuju rijetke monogenske bolesti. U ovom istraživanju promatran je utjecaj učinka utemeljitelja na genetičko opterećenje određenih romskih grupa, s 4 monogenske bolesti uzrokovane mutacijama svojstvenim isključivo romskim populacijama. Proučavane su dvije populacije Roma Bajaša prisutne na području Hrvatske, koje prema migracijskoj podjeli spadaju u vlaške Rome. Istraživane bolesti javljaju se različitom učestalošću u pojedinim romskim skupinama, ovisno o njihovim migracijskim obrascima i socio-kulturnim obilježjima. Nasljedna senzo-motorna neuropatija – tip Lom (HMSNL) javlja se isključivo u osoba romskog podrijetla, a učestalost mutiranog alela iznosti od 2% do 16%, ovisno o pojedinoj skupini. Kod pripadnika vlaške migracijske kategorije prosječna učestalost je oko 11%. Alel koji uzrokuje GM1 gangliozidozu (GM1) javlja se s niskim učestalostima (oko 2%) u različitim romskim grupama, a visoku učestalost (10%) doseže kod Rudara, izrazito endogamne skupine vlaških Roma naseljenoj u Bugarskoj. Mišićna distrofija zdjeličnog pojasa – tip 2C (LGMD2C) do sada je otkrivena u bugarskim skupinama balkanskih Roma i zapadno-europskim Romima, a učestalost mutiranog alela varira od 2% do 6%. Kod vlaških Roma do sada nije zabilježena. Sindrom kongenitalnog katarakta, facijalnog dismorfizma i neuropatije (CCFDN) do danas je dijagnosticiran u otprilike 150 Roma koji pripadaju različitim migracijskim kategorijama, a svi pacijenti su homozigoti za istu mutaciju. Kod vlaških Roma učestalost nosioca iznosi 2, 64%. U ovom istraživanju analizirano je 426 uzoraka, metodom PCR-RFLP, a pronađene mutacije potvrđene su sekvenciranjem. U populaciji Baranje pronađeni su nosioci za nasljednu motornu i senzornu neuropatiju – tip Lom, čija učestalost iznosi 1, 5%. Za ostale 3 ispitivane bolesti (GM1 gangliozidoza, sindrom kongenitalnog katarakta, facijalnog dismorfizma i neuropatije i mišićna distrofija zdjeličnog pojasa – tip 2C) nosioci nisu pronađeni. Iz rezultata je vidljivo da je genetičko opterećenje u ispitivanim populacijama hrvatskih Roma Bajaša niže u odnosu na ostale skupine vlaških Roma. Ovakavi rezultati mogu biti posljedica smanjenog toka gena između vlaških romskih skupina, te izrazite endogamije unutar skupina tijekom ropstva u Rumunjskoj, što je moglo dovesti do izostanka mutiranih alela u skupinama vlaških Roma u Hrvatskoj, te povećane učestalosti u drugim vlaškim skupinama. Osim toga, prisutnost mutiranog alela u populaciji Roma iz Baranje može biti i posljedica nedavnog toka gena iz romskih populacija u kojima je ova mutacija prisutna s većom učestalošću.