The role of longevity-related genetic variant interactions as predictors of survival after 85 years of age.

Genome-wide association studies and candidate gene studies have identified several genetic variants that might play a role in achieving longevity. This study investigates interactions between pairs of those single nucleotide polymorphisms (SNPs) and their effect on survival above the age of 85 in a sample of 327 Croatian individuals. Although none of the SNPs individually showed a significant effect on survival in this sample, 14 of the 359 interactions tested (between SNPs not in LD) reached the level of nominal significance (p<0.05), showing a potential effect on late-life survival. Notably, SH2B3 rs3184504 interacted with different SNPs near TERC, TP53 rs1042522 with different SNPs located near the CDKN2B gene, and CDKN2B rs1333049 with different SNPs in FOXO3, as well as with LINC02227 rs2149954. The other interaction pairs with a possible effect on survival were FOXO3 rs2802292 and ERCC2 rs50871, IL6 rs1800795 and GHRHR rs2267723, LINC02227 rs2149954 and PARK7 rs225119, as well as PARK7 rs225119 and PTPN1 rs6067484. These interactions remained significant when tested together with a set of health-related variables that also had a significant effect on survival above 85 years. In conclusion, our results confirm the central role of genetic regulation of insulin signalling and cell cycle control in longevity.

Genes Involved in DNA Damage Cell Pathways and Health of the Oldest-Old (85+).

Some sources report a connection of cellular senescence with chronic pathological conditions; however, the association between particular cellular processes and general health is rarely examined. This study aims to test the relationship of general health with DNA damage pathways that play a crucial role in senescence. The association of ten selected SNPs with subjective and objective general health and functional ability indicators has been tested in 314 oldest-old people from Croatia. Multivariate logistic regression was employed to simultaneously test the impact of variables potentially influencing targeted health and functional ability variables. The best model, explaining 37.1% of the variance, has six independent significant predictors of functional ability scores: rs16847897 in TERC, rs533984 in MRE11A, and rs4977756 in CDKN2B, chronic disease count, Mini-Mental State Examination scores, and age at surveying. In conclusion, the examined ten loci involved in DNA damage repair pathways showed a more significant association with self-rated health and functional ability than with the number of disease or prescribed medicaments. The more frequent, longevity-related homozygote (GG) in rs16847897 was associated with all three aspects of self-assessments-health, mobility, and independence-indicating that this TERC locus might have a true impact on the overall vitality of the oldest-old persons.

Infection with human cytomegalovirus, Epstein-Barr virus, and high-risk types 16 and 18 of human papillomavirus in EGFR-mutated lung adenocarcinoma.

AIM: To assess the frequency of human cytomegalovirus (HCMV), Epstein-Barr virus (EBV), and high-risk types of human papillomavirus (HPV16 and HPV18) infections in lung adenocarcinoma samples. METHODS: Lung adenocarcinoma cytological smears and their DNA isolates were obtained from patients hospitalized at the Department for Lung Diseases Jordanovac, Zagreb, in 2016 and 2017. Overall, 67 lung adenocarcinoma samples were examined: 34 with epidermal growth factor receptor gene (EGFR) mutations and 33 without EGFR mutations. The EGFR mutation status and virus presence were assessed with a polymerase chain reaction, and random samples were additionally tested for EBV with Sanger sequencing. HCMV, EBV, HPV16, and HPV18 infections were evaluated in relation to EGFR mutation, smoking status, and sex. A meta-analysis of available data about HPV infection in non-small cell lung cancer was performed. RESULTS: More frequent HCMV, EBV, HPV16, and HPV18 infections were observed in lung adenocarcinoma samples with EGFR mutations than in samples without these mutations. Coinfection of the investigated viruses was observed only in lung adenocarcinoma samples with mutated EGFR. In the group with EGFR mutations, smoking was significantly associated with HPV16 infection. The meta-analysis showed that non-small cell lung cancer patients with EGFR mutations had a higher odds of HPV infection. CONCLUSION: HCMV, EBV, and high-risk HPV infections are more frequent in EGFR-mutated lung adenocarcinomas, which indicates a possible viral impact on the etiology of this lung cancer subtype.

Genetic scores for predicting longevity in the Croatian oldest-old population.

Longevity is a hallmark of successful ageing and a complex trait with a significant genetic component. In this study, 43 single nucleotide polymorphisms (SNPs) were chosen from the literature and genotyped in a Croatian oldest-old sample (85+ years, sample size (N) = 314), in order to determine whether any of these SNPs have a significant effect on reaching the age thresholds for longevity (90+ years, N = 212) and extreme longevity (95+ years, N = 84). The best models were selected for both survival ages using multivariate logistic regression. In the model for reaching age 90, nine SNPs explained 20% of variance for survival to that age, while the 95-year model included five SNPs accounting for 9.3% of variance. The two SNPs that showed the most significant association (p ≤ 0.01) with longevity were TERC rs16847897 and GHRHR rs2267723. Unweighted and weighted Genetic Longevity Scores (uGLS and wGLS) were calculated and their predictive power was tested. All four scores showed significant correlation with age at death (p ≤ 0.01). They also passed the ROC curve test with at least 50% predictive ability, but wGLS90 stood out as the most accurate score, with a 69% chance of accurately predicting survival to the age of 90.

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.

From Croatian Roma to 1000 Genomes: The Story of the CYP2D6 Gene Promoter and Enhancer SNPs.

The CYP2D6 gene encodes an enzyme responsible for the metabolism of ~20% of clinically prescribed drugs. In this study, 18 SNPs from the enhancer and promoter regions of CYP2D6 in 323 Roma from Croatia were genotyped, to find out whether the demographic history of Roma affected the distribution of the studied SNPs and their linkage disequilibrium (LD) values, with the major SNPs defining the CYP2D6 star alleles. No differences were found between the three Roma groups in allele and genotype frequencies. The distribution of LD values of Roma was compared with LD values of European and Asian populations. Regulatory CYP2D6 SNPs (rs5758550, rs28624811, rs1080985 and rs1080983) showed similar distribution and the highest LDs with rs16947 from the gene-coding region in all populations. In the promoter region, a complete LD between rs1080989 and rs28588594, and between rs1080983 and rs28624811, was found in Croatian Roma and investigated populations from 1000 genomes. A high LD was also found between rs1080985 from the promoter and rs5758550 from the enhancer region. SNP rs28735595 from the gene promoter region had the highest LD, with two gene region SNPs, rs1058164 and rs1135840. To conclude, the Croatian Roma population shows an LD pattern of the CYP2D6 gene region similar to the 1000 Genomes European and Asian populations.

Testing the Institute of Medicine (IOM) recommendations on maternal reproductive health and associated neonatal characteristics in a transitional, Mediterranean population.

BACKGROUND: High pre-pregnancy body mass index (BMI) and excessive gestational weight gain (GWG) are significant risk factors for maternal and neonatal health. AIM: To assess pre-pregnancy BMI and GWG during pregnancy and their association with different maternal and neonatal characteristics in the transitional Mediterranean population from the Eastern Adriatic islands. SUBJECTS AND METHODS: Two hundred and sixty-two mother-child dyads from the CRoatian Islands' Birth Cohort Study (CRIBS) were included in the study. Chi-square test, ANOVA, and regression analysis were used to test the association between selected characteristics. RESULTS: In total, 22% of women entered pregnancy as overweight/obese and 46.6% had excessive GWG. Pre-pregnancy overweight and obesity were significantly associated with elevated triglycerides uric acid levels, and decreased HDL cholesterol in pregnancy. Excessive GWG was associated with elevated fibrinogen and lipoprotein A levels. Women with high pre-pregnancy BMI and GWG values were more likely to give birth to babies that were large for gestational age (LGA), additionally confirmed in the multiple logistic regression model. CONCLUSION: High maternal pre-pregnancy BMI and excessive GWG were both significantly associated with deviated biochemical parameters and neonatal size. More careful monitoring of maternal nutritional status can lead to better pre- and perinatal maternal healthcare.

Pharmacogenetic distinction of the Croatian population from the European average.

AIM: To compare the Croatian and European population in terms of allele frequencies of clinically relevant polymorphisms in drug absorption, distribution, metabolism, and excretion (ADME) genes. METHODS: In 429 Croatian participants, we genotyped 27 loci in 20 ADME genes. The obtained frequencies were merged with the published frequencies for the Croatian population by sample size weighting. The study sample obtained in this way was compared with the average data for the European population from the gnomAD database. RESULTS: Variant allele frequencies in the Croatian population were higher in three and lower in two polymorphisms (Benjamini-Hochberg-corrected P values: 0.0027 for CYP2B6*4 rs2279343, CYP2C9*2 rs1799853, and VKORC1 rs9923231; 0.0297 for GSTP1 rs1695; 0.0455 for CYP2A6 rs1801272) compared with the European population. The most marked difference was observed for CYP2B6*4 (9.3% in Europe vs 24.3% in Croatia). The most clinically relevant findings were higher variant allele frequencies in two polymorphisms related to lower warfarin requirements: VKORC1*2 (34.9% in Europe vs 40.1% in Croatia) and CYP2C9*2 (12.3% in Europe vs 14.7% in Croatia). This indicates that three-quarters of Croatian people have at least one variant allele at these loci. Variants in genes GSTP1 and CYP2A6 were significantly less frequently observed in Croatia. CONCLUSIONS: Croatian population has a higher bleeding and over-anticoagulation risk, which is why we recommend the prescription of lower doses of anticoagulation drugs such as warfarin and acenocoumarol. Lower phenytoin, and higher bupropion and efavirenz doses are also recommended in the Croatian population.

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.

CVD Risk Factors in the Ukrainian Roma and Meta-Analysis of Their Prevalence in Roma Populations Worldwide.

The Roma population suffers from severe poverty, social exclusion, and some of the worst health conditions in the industrialized world. Herein, we report on cardiovascular disease (CVD) risk factors in the Ukrainian Roma and present a meta-analysis of the prevalence of CVD risk factors in 16 Roma populations worldwide. The meta-analyses of CVD risk factors in Roma (n = 16,552) vs. non-Roma majority population of the same country (n = 127,874) included publicly available data. Ukrainian field survey included 339 adults of both sexes and outcomes of interest were hypertension, body mass index (BMI), smoking, education, and employment status. Furthermore, 35.7% of the Ukrainian Roma were hypertensive, 69.3% unemployed, and 48.4% never went to school. Ukrainian Roma women were more likely to be underweight and more prone to be hypertensive, with odds of hypertension increasing with age, BMI, and positive smoking status. Meta-analyses showed that, in comparison with non-Roma worldwide, the Roma bear significantly higher risk factor loads related to smoking (OR = 2.850), diabetes (OR = 1.433), abdominal obesity (OR = 1.276), and metabolic syndrome (OR = 1.975), with lower loads for hypertension (OR = 0.607) and BMI ≥ 25 kg/m2 (OR = 0.872). To conclude, the CVD risk factors which are more common in Roma than in the majority population may reflect their poor health-related behaviors and inadequate access to health education.

Three Patterns of Inheritance of Quantitative Dermatoglyphic Traits: Kosovo Albanian Twin Study.

Dermatoglyphs are epidermal ridge configurations on the fingers, palms and soles that are formed during fetal development, and therefore only the intrauterine environment can have any influence on their formation. This study aims at investigating the genetic and environmental contribution in determining quantitative dermatoglyphic traits in 32 monozygotic (MZ) and 35 dizygotic (DZ) same-sex twins from the Albanian population of Kosovo. All genetic analyses were run in the statistical program Mx. After assumptions testing, based on the pattern of MZ-DZ correlations, univariate models were fitted to the data in order to estimate additive genetic (A), common (C) and individual (E) environmental influences for all variables. The exception was the atd-angle for which a model with nonadditive genetic (D) influences was tested, since DZ correlations were less than half of MZ correlations. Goodness of fit of the full ACE or ADE model was compared to the saturated model. The fit of nested models (AE, CE, DE or E) was compared to the full models (ACE or ADE). Our results indicate that additive genetic component strongly contributes to individual differences in finger ridge counts (49-81%), and weakly (0-50%) on the formation of the palmar ridge counts between the palmar triradii a, b, c, and d. The specific pattern found for the atd-angle implies the impact of a nonadditive genetic component, possibly the effect of a major gene. Further, more powered studies are needed to confirm this pattern, especially for resolving the issue of the huge difference in MZ and DZ twin similarity for the atd-angle palmar trait.

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.

Role of MYC in B Cell Lymphomagenesis.

B cell lymphomas mainly arise from different developmental stages of B cells in germinal centers of secondary lymphoid tissue. There are a number of signaling pathways that affect the initiation and development of B cell lymphomagenesis. The functions of several key proteins that represent branching points of signaling networks are changed because of their aberrant expression, degradation, and/or accumulation, and those events determine the fate of the affected B cells. One of the most influential transcription factors, commonly associated with unfavorable prognosis for patients with B cell lymphoma, is nuclear phosphoprotein MYC. During B cell lymphomagenesis, oncogenic MYC variant is deregulated through various mechanisms, such as gene translocation, gene amplification, and epigenetic deregulation of its expression. Owing to alterations of downstream signaling cascades, MYC-overexpressing neoplastic B cells proliferate rapidly, avoid apoptosis, and become unresponsive to most conventional treatments. This review will summarize the roles of MYC in B cell development and oncogenesis, as well as its significance for current B cell lymphoma classification. We compared communication networks within transformed B cells in different lymphomas affected by overexpressed MYC and conducted a meta-analysis concerning the association of MYC with tumor prognosis in different patient populations.

The prognostic and predictive value of excision repair cross-complementation group 1 (ERCC1) protein in 1288 patients with head and neck squamous cell carcinoma treated with platinum-based therapy: a meta-analysis.

Excision repair cross-complementation group 1 (ERCC1) protein has been extensively investigated as a prognostic and predictive factor for platinum-based treatment in head and neck squamous cell carcinoma (HNSCC) but with inconsistent results. We performed the present meta-analysis to better elucidate this issue in advanced HNSCC. A literature search was conducted using the PubMed and Web of Science databases. The inclusion criteria were head and neck cancer patients with platinum-based treatment and evaluation of the correlation between ERCC1 expression and clinical outcomes [objective response rate (ORR), progression-free survival (PFS), and overall survival (OS), both unadjusted and adjusted estimates]. In high vs. low pooled analyses, high ERCC1 expression was associated with unfavorable OS [hazard ratio (HR) = 1.95, 95 % confidence interval (CI) 1.18-3.21, p = 0.009], PFS (HR = 2.39, 95 % CI 1.74-3.28, p = 0.000) and ORR (odds ratio = 0.48, 95 % CI 0.23-0.98, p = 0.044). In the subgroup analysis of adjusted OS estimates, ERCC1 was a predictor of shorter survival in Asians (HR = 3.13, 95 % CI 2.09-4.70, p = 0.000) and Caucasians (HR = 2.02, 95 % CI 1.32-3.07, p = 0.001) but of longer survival in South Americans (HR = 0.17, 95 % CI 0.07-0.40, p = 0.000). Immunohistochemistry proved to be of predictive value irrespective of used antibody (p = 0.009). In the stratified analysis according to the tumor site, ERCC1 expression was associated with OS in nasopharyngeal cancer (HR = 2.72, 95 % CI 1.79-4.13, p = 0.000). ERCC1 has a potential to become predictive and prognostic factor enabling treatment tailoring in HNSCC patients.

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".

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.

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.