Relevance of CYP2D6 Gene Variants in Population Genetic Differentiation.

A significant portion of the variability in complex features, such as drug response, is likely caused by human genetic diversity. One of the highly polymorphic pharmacogenes is CYP2D6, encoding an enzyme involved in the metabolism of about 25% of commonly prescribed drugs. In a directed search of the 1000 Genomes Phase III variation data, 86 single nucleotide polymorphisms (SNPs) in the CYP2D6 gene were extracted from the genotypes of 2504 individuals from 26 populations, and then used to reconstruct haplotypes. Analyses were performed using Haploview, Phase, and Arlequin softwares. Haplotype and nucleotide diversity were high in all populations, but highest in populations of African ancestry. Pairwise FST showed significant results for eleven SNPs, six of which were characteristic of African populations, while four SNPs were most common in East Asian populations. A principal component analysis of CYP2D6 haplotypes showed that African populations form one cluster, Asian populations form another cluster with East and South Asian populations separated, while European populations form the third cluster. Linkage disequilibrium showed that all African populations have three or more haplotype blocks within the CYP2D6 gene, while other world populations have one, except for Chinese Dai and Punjabi in Pakistan populations, which have two.

Untangling SNP Variations within CYP2D6 Gene in Croatian Roma.

CYP2D6 is a highly polymorphic gene whose variations affect its enzyme activity. To assess whether the specific population history of Roma, characterized by constant migrations and endogamy, influenced the distribution of alleles and thus phenotypes, the CYP2D6 gene was sequenced using NGS (Next Generation Sequencing) method-targeted sequencing in three groups of Croatian Roma (N = 323) and results were compared to European and Asian populations. Identified single nucleotide polymorphisms (SNPs) were used to reconstruct haplotypes, which were translated into the star-allele nomenclature and later into phenotypes. A total of 43 polymorphic SNPs were identified. The three Roma groups differed significantly in the frequency of alleles of polymorphisms 6769 A > G, 6089 G > A, and 5264 A > G (p < 0.01), as well as in the prevalence of the five most represented star alleles: *1, *2, *4, *10, and *41 (p < 0.0001). Croatian Roma differ from the European and Asian populations in the accumulation of globally rare SNPs (6089 G > A, 4589 C > T, 4622 G > C, 7490 T > C). Our results also show that demographic history influences SNP variations in the Roma population. The three socio-culturally different Roma groups studied differ significantly in the distribution of star alleles, which confirms the importance of a separate study of different Roma groups.

Waist to height ratio is the anthropometric index that most appropriately mirrors the lifestyle and psychological risk factors of obesity.

AIM: To determine to what extent the lifestyle and psychological factors influenced the occurrence of obesity in the population of the Mediterranean island of Vis (Croatia); also to relate the examined risk factors to several obesity indicators in order to select the most sensitive one. METHODS: Cross-sectional population study included 960 participants of both sexes (18-93 years) in whom the obesity was estimated using anthropometric indicators. The factor analysis of 55-item Food Frequency Questionnaire (FFQ) was performed as well as the General Health Questionnaire (GHQ12) for the self-assessment of psychological health and wellbeing. FFQ and GHQ12 individual factors' scores, sex, age, physical activity, smoking and socioeconomic status were further used as independent variables in the multiple linear regression analyses with five obesity indicators as dependent variables. RESULTS: The examined risk factors explained the highest proportion of variance of the waist to height ratio (WHtR) in comparison with all other indicators of obesity (body mass index (BMI), waist to hip ratio, sum of skinfolds thicknesses, fat mass percentage). Only the dietary factors showed a significant relation to BMI, while almost all of the tested variables-dietary pattern, psychological structure, smoking habit and physical activity-had a significant association with WHtR. CONCLUSIONS: WHtR is the most useful indicator of obesity as it had a profiled relation with a number of lifestyle factors as well as with psychological health. The present study also revealed the importance of psychological factors for obesity phenotype, particularly the behavioural pattern which could be described as avoidance of dealing with problems.

Characterization of ADME genes variation in Roma and 20 populations worldwide.

The products of the polymorphic ADME genes are involved in Absorption, Distribution, Metabolism, and Excretion of drugs. The pharmacogenetic data have been studied extensively due to their clinical importance in the appropriate drug prescription, but such data from the isolated populations are rather scarce. We analyzed the distribution of 95 polymorphisms in 31 core ADME genes in 20 populations worldwide and in newly genotyped samples from the Roma (Gypsy) population living in Croatia. Global distribution of ADME core gene loci differentiated three major clusters; (1) African, (2) East Asian, and (3) joint European, South Asian and South American cluster. The SLCO1B3 (rs4149117) and CYP3A4 (rs2242480) genes differentiated at the highest level the African group of populations, while NAT2 gene loci (rs1208, rs1801280, and rs1799929) and VKORC1 (rs9923231) differentiated East Asian populations. The VKORC1 rs9923231 was among the investigated loci the one with the largest global minor allele frequency (MAF) range; its MAF ranged from 0.027 in Nigeria to 0.924 in Han Chinese. The distribution of the investigated gene loci positions Roma population within the joined European and South Asian clusters, suggesting that their ADME gene pool is a combination of ancestral (Indian) and more recent (European) surrounding, as it was already implied by other genetic markers. However, when compared to the populations worldwide, the Croatian Roma have extreme MAF values in 10 out of the 95 investigated ADME core gene loci. Among loci which have extraordinary MAFs in Roma population two have strong proof of clinical importance: rs1799853 (CYP2C9) for warfarin dosage, and rs12248560 (CYP2C19) for clopidogrel dosage, efficacy and toxicity. This finding confirms the importance of taking the Roma as well as the other isolated populations`genetic profiles into account in pharmaco-therapeutic practice.

Distinctiveness of the Roma population within CYP2B6 worldwide variation.

AIM: 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. MATERIALS & METHODS: Seven SNP loci (rs12721655, rs2279343, rs28399499, rs34097093, rs3745274, rs7260329 and rs8192709) were genotyped in 439 subjects using Kompetitive Allele Specific PCR (KASP) method. RESULTS: The Croatian Roma took an outlying position in CYP2B6 variation from the worldwide perspective mainly due to their exceptionally high minor allele frequency (MAF) for rs8192709 (12.8%), and lower for rs2279343 (21.1%) compared with south Asian populations. CONCLUSION: 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.

Novel locus for fibrinogen in 3' region of LEPR gene in island population of Vis (Croatia).

Leptin, a possible mediator between energy homeostasis, inflammation and cardiovascular disease (CVD), acts via leptin receptors. We investigated association of single-nucleotide polymorphisms (SNPs) and haplotypes of the leptin receptor gene (LEPR) with several CVD risk factors: body mass index, waist circumference (WC), serum lipids, fibrinogen and C-reactive protein levels. Thirty-one SNPs in and near LEPR gene were analyzed in 986 inhabitants of the island of Vis, Croatia and 29 SNPs in the inland sample (N=499). We assessed linkage disequilibrium (LD), SNP and haplotype associations with the selected phenotypes. rs4291477 significantly associated with fibrinogen (P=0.003) and rs7539471 marginally significantly with high-density lipoprotein (P=0.004), but only in the Vis sample, while rs10493384 marginally significantly associated with triglyceride levels (P=0.006) in the inland sample. SNPs were grouped into eight LD blocks in Vis and in seven blocks in the inland population. Haplotype A-C-A-A-G-A in block 5 in Vis (rs1782754, rs1171269, rs1022981, rs6673324, rs3790426, rs10493380) and haplotype A-A-A-A in block 4 in the inland data (rs1782754, rs1022981, rs6673324, rs1137100) were nominally associated with WC, P=7.085 × 10(-22) (adjusted P=0.0979) and P=5.496 × 10(-144) (adjusted P=0.1062), respectively.

A six decades long follow-up on body size in adolescents from Zagreb, Croatia (1951-2010).

Most studies analysing the influence of socioeconomic deterioration on body size focus on the impact of food shortages and diseases on the growth in early childhood. To evaluate how socioeconomic conditions influence the growth during the adolescence, we tracked the body size of 15-19 year-olds over the last sixty years covering the socialist period (1951-1990), the war (1991-1995) and the transition to capitalistic economy. This study of Zagreb, Croatia, adolescent population provides information on the secular trend in height, weight and Body Mass Index (BMI) and examines their relation with Real Gross Domestic Product. From 1951 to 2010 the girls' height approximately increased by 6.2cm and weight by 6.8kg, while the boys' height increased by 12.2cm and weight by 17.3kg. Prior to 1991 mean BMI in girls was higher than in boys, but from 1991 on, the interrelation between the sexes has been opposite, possibly mirroring the cultural trends that started in mid-1970s and reflecting higher sensitivity of boys to the socioeconomic changes. In conclusion, the secular trend in body size over the investigated period reflects the positive economic trends interrupted by the war. The recent increase in BMI corresponds to the country's economic recovery and indicates the "nutrition transition".

Are the physically active adolescents belonging to the "at risk of overweight" BMI category really fat?

The adolescence is recognized as one of the critical periods for the development of obesity. Children and adolescents who practice sports regularly have higher muscle mass and lower percentage of body fat than their peers who are physically less active. Since body mass index (BMI) is a widely used indicator of overweight/obesity in spite of the fact that it directly measures excess in weight but not in fat, it often misclassifies athletic populations, both children and adults. The specific aim of this study was to evaluate whether BMI adequately assesses fatness in adolescents, especially physically active ones. The analysis was performed on anthropometric data from two surveys (1997 and 2009/2010) of Zagreb secondary school adolescents, 1315 girls and 1034 boys, aged 15-19 years. The group defined as "physically active" consisted of adolescents who practice organized sports (36.2% girls, 44.6% boys), while the "physically inactive" group was made of their peers who practice sport only as a part of physical education in schools. The standardized values, calculated within each sex by survey, were used for comparison of adolescents with different levels of physical activity. Physically active adolescents of both sexes had lower sum of skinfolds mean Z-valutes (Pgirls<0.05, Pboys<0.001); additionally, boys had higher Z-values for body weight (p<0.05) and triceps/subscaputar ratio (indicating peripheral distribution of body fat) (p<0.05) than their less active peers. In order to evaluate whether BMI was adequate indicator for body composition during adolescence, we estimated the concordance of above-median category defined by BMI and the other body fat indicators. The largest discrepancy was found for sum of skinfolds in both sexes and was more pronounced in physically active adolescents. This finding was further confirmed in more extreme BMI category (85th - 95th percentile) which indicated that adolescents categorized as "at risk of overweight" were predominantly characterized by larger lean body mass and not by increased fatness.

Age trends in prevalence of cardiovascular risk factors in Roma minority population of Croatia.

The Roma (Gypsy) are the largest European minority population characterized by poverty, social exclusion as well as by numerous life-style and cultural specificities, which all could have an adverse impact on their cardiovascular health. This study assesses the prevalence of cardiovascular diseases (CVD) risk factors in community-based sample of 430 adult Roma, living in rural area of Croatia, by providing the actual and age-adjusted estimates using the European standard population. The most prominent classical CVD risk phenotypes (blood pressure, obesity, smoking, glucose and lipid profile) were selected, and the standard risk cut-offs were applied. The study has shown that compared to general population of Croatia, the Roma population bears a high CVD risk factors load related to smoking and high glucose level. The CVD risk factors prevalence in Roma also showed important sex and age patterns, the most imposing of which are the findings of higher prevalence of CVD risks in women (especially obesity and triglyceride levels) and the trend of higher body mass index (BMI) level in younger age group (18-34 years) which both stand in contrast to the trends characterizing the general population of Croatia. These findings are complemented by the trend of decreased risk in the oldest age group (65+ years) for all investigated CVD risk factors (with exception of triglycerides level) compared to the 50-64 age group. We conclude that the age and sex CVD risks pattern point to the health transition of this rural Roma population. As we expect the proportion of CVD in the Roma minority of Croatia to increase in the future along with further modernization of their lifestyle, the CVD prevention measures in this population are urgent and should be primarily targeted at women and at the younger segment of this population.

Secular trend of menarche in Zagreb (Croatia) adolescents.

OBJECTIVE: To study the secular trend of menarche in high-school urban adolescent girls (Zagreb, Croatia) over the last two decades, and to evaluate the possible impact of worsening of socio-economic conditions on age at menarche during the Croatian War of Independence (1991-1995). STUDY DESIGN: Three surveys of female adolescents aged 15-19 years: (a) 523 girls interviewed in 1990, (b) 888 girls interviewed in 1997, and (c) 399 girls interviewed in 2010. Self-reported age at menarche (in decimal years) was presented by age groups and year of interview. Differences in mean age at menarche between adolescent age groups as well as between surveys were tested using One-way ANOVA. RESULTS: The mean age at menarche was 12.82 years in 1990, 12.92 years in 1997 and 12.31 years in 2010. It increased by 0.10 years from 1990 to 1997 (p=0.290) and then declined by 0.61 years from 1997 to 2010 (p<0.001). Over the 20-year period, the overall mean age at menarche declined by 0.51 years (p<0.001). CONCLUSION: The age at menarche in Zagreb adolescents continued to decline significantly from 1990 to 2010 (p<0.001), in spite of a statistically insignificant initial increase between 1990 and 1997. When put in broader context, age at menarche mirrored socio-economic changes in the war period.

Angiotensin-converting enzyme deletion allele is beneficial for the longevity of Europeans.

The human angiotensin converting enzyme (ACE) gene is one of the most investigated candidate genes for cardiovascular diseases (CVD), but the understanding of its role among the elderly is vague. Therefore, this study focuses at: (a) testing the association of ACE polymorphism with CVD risk factors among the elderly, and (b) detecting the possible unequal distribution of ACE genotypes between senescent and younger segments of the European populations. The association of ACE I/D polymorphism with CVD health status [hypertension (HT), obesity, dislypidemia] in 301 very old subjects (88.2 ± 5 years; F/M = 221/80) was tested by means of logistic regression analysis. The meta-analysis of D allele frequency in general vs. elderly (80+ years) groups was conducted using all publicly available data for European populations comprising both age cohorts. Multiple multinomial logistic regression revealed that within this elderly sample, age (younger olds, 80-90 years), female sex (OR = 3.13, 95% CI = 1.59-6.19), and elevated triglycerides (OR = 2.53, 95% CI = 1.29-4.95) were positively associated with HT, while ACE polymorphism was not. It was also established that the DD genotype was twice as high in 80+ cohort compared to general population of Croatia (p < 0.00001). This trend was confirmed by the meta-analysis that showed higher D allele frequencies in olds from nine of ten considered European populations (OR = 1.19, 95% CI = 1.08-1.31). The data in elderly cohort do not confirm previously reported role of ACE DD genotype to the development of HT. Moreover, meta-analysis indicated that ACE D allele has some selective advantage that contributes to longevity in majority of European populations.

E2 allele of the apolipoprotein E gene polymorphism is predictive for obesity status in Roma minority population of Croatia.

BACKGROUND AND AIMS: The Roma (Gypsies) are a transnational minority, founder population characterized by unique genetic background modeled by culturally determined endogamy. The present study explores whether the widely found cardiovascular diseases (CVD) risk effects of ACE I/D, APOE (ε2, ε3, ε4), eNOS-VNTR and LEP G2548A polymorphisms can be replicated in this specific population. METHODS AND RESULTS: The community-based study was carried on 208 adult Bayash Roma living in rural settlements of eastern and northern Croatia. Risk effect of four CVD candidate polymorphisms are related to the most prominent classical CVD risk phenotypes: obesity indicators (body mass index and waist circumference), hypertension and hyperlipidemia (triglycerides, HDL and LDL cholesterol). For all of them the standard risk cut-offs were applied. The extent to which the phenotypic status is related to genotype was assessed by logistic regression analysis. The strongest associations were found for ε2 allele of the APOE as a predictor of waist circumference (OR 3.301; 95%CI 1.254-8.688; p = 0.016) as well as for BMI (OR 3.547; 95%CI 1.471-8.557; p = 0.005). It is notable that ε3 allele of APOE gene turned out to be a protective genetic factor determining low lipid levels. CONCLUSION: The strength of the relation and the similarity of the results obtained for both tested indicators of obesity provide firm evidence that APOE plays an important role in obesity development in the Roma population.

Gender-specific growth patterns of transversal body dimensions in Croatian children and youth (2 to 18 years of age).

In a cross-sectional study of growth, 5,260 healthy children of both sexes from Zagreb (Croatia) aged 2 to 18 years were measured. Six transversal body dimensions were studied: biacromial, transverse chest, antero-posterior chest, biiliocristal, bicondylar humerus and bicondylar femur diamters. A significant increase in body diameters has been observed until the age of 14 to 15 in girls and until the age of 16 in boys, showing that girls have a 1 to 2 years shorter period of growth. Compared to boys of the same age, they achieved larger amounts of final transversal bone size throughout the whole growth period. The most pronounced example was the knee diameter that in girls attained 95% of adult size as early as the age of 10. In both genders, the adult size is achieved earlier in widths of the extremities than in those of the trunk. The studied transversal body segments showed different growth dynamics, which is gender-specific. While sexual dimorphism in pelvic and shoulder diameters emerged with pubertal spurt, gender differences in chest and extremities' diameters started early in life. In all ages, boys had larger chest, elbow and knee diameters. In pubertal age boys gained a significantly larger biacromial diameter (from the age of 13 onwards), while girls exceeded them in biiliocristal diameter (from 10 to 14 years). The findings of gender differences were compared to those reported for other European populations and their growth patters were discussed comparing viewpoints.