Ana Miljković

Homozygous loss-of-function variants in European cosmopolitan and isolate populations

Homozygous loss of function (HLOF) variants provide a valuable window on gene function in humans, as well as an inventory of the human genes that are not essential for survival and reproduction. All humans carry at least a few HLOF variants, but the exact number of inactivated genes that can be tolerated is currently unknown—as are the phenotypic effects of losing function for most human genes. Here, we make use of 1432 whole exome sequences from five European populations to expand the catalogue of known human HLOF mutations; after stringent filtering of variants in our dataset, we identify a total of 173 HLOF mutations, 76 (44%) of which have not been observed previously. We find that population isolates are particularly well suited to surveys of novel HLOF genes because individuals in such populations carry extensive runs of homozygosity, which we show are enriched for novel, rare HLOF variants. Further, we make use of extensive phenotypic data to show that most HLOFs, ascertained in population-based samples, appear to have little detectable effect on the phenotype. On the contrary, we document several genes directly implicated in disease that seem to tolerate HLOF variants. Overall HLOF genes are enriched for olfactory receptor function and are expressed in testes more often than expected, consistent with reduced purifying selection and incipient pseudogenisation.

Is experimentally induced pain associated with socioeconomic status? Do poor people hurt more?

Background The association of pain and socioeconomic status is widely reported, yet much less clearly understood. The aim of this study was to investigate the association of experimentally induced pain threshold and tolerance with socioeconomic status. Material/Methods The study sample consisted of 319 adult subjects from the population of the island of Vis, Croatia, which was previously shown to have a high level of social homogeneity. A manual dolorimeter was used to measure mechanical pressure pain threshold (least stimulus intensity) and pain tolerance (maximum tolerance stimulus intensity) on both hands. Pain tolerance interval was defined as the difference between pain tolerance and threshold. Years of schooling and material status were used as socioeconomic estimates. Results Both of the socioeconomic estimates were significantly correlated with pain threshold, tolerance, and tolerance interval (P<0.001). The mixed modeling analysis, controlled for the effects of age, gender, and 4 psychological variables, indicated that education was not a significant predictor in any of the 3 models. However, lower material status was significantly associated with lower pain tolerance (P=0.038) and narrower pain tolerance interval (P=0.032), but not with pain threshold (P=0.506). The overall percentages of explained variance were lower in the tolerance interval model (20.2%) than in pain tolerance (23.1%) and threshold (33.1%), suggesting the increasing share of other confounding variables in pain tolerance and even more so in tolerance interval model. Conclusions These results suggest a significant association between experimentally induced pain tolerance and tolerance interval with material status, suggesting that poor people indeed do hurt more.

Mediterranean diet in the southern Croatia - does it still exist?

Aim To assess the adherence to the Mediterranean diet in the population of Dalmatia in southern Croatia. Methods A cross-sectional study was performed within the 10 001 Dalmatians cohort, encompassing 2768 participants from Korčula and Vis islands and the City of Split, who were recruited during 2011-2014. Using the data obtained from food frequency questionnaire we calculated the Mediterranean Diet Serving Score (MDSS). Multivariate logistic regression was used to identify the characteristics associated with the adherence to the Mediterranean diet, with age, sex, place of residence, education attainment, smoking, and physical activity as covariates. Results The median MDSS score was 11 out of 24 points (interquartile range 8-13), with the highest score recorded on the island of Vis. Participants reported a dietary pattern that had high compliance with the Mediterranean diet guidelines for consumption of cereals (87% met the criteria), potatoes (73%), olive oil (69%), and fish (61%), moderate for consumption of fruit (54%) and vegetables (31%), and low for consumption of nuts (6%). Overall, only 23% of the participants were classified as being adherent to the Mediterranean diet, with a particularly low percentage among younger participants (12%) compared to the older ones (34%). Men were less likely to show good adherence (odds ratio 0.52, 95% confidence interval 0.42-0.65). Conclusion This study revealed rather poor compliance with the current recommendations on the Mediterranean diet composition in the population of Dalmatia. Public health intervention is especially needed in younger age groups and in men, who show the greatest departure from traditional Mediterranean diet and lifestyle.

Neuroticism and pain catastrophizing aggravate response to pain in healthy adults: an experimental study

Background The aim of this study was to investigate the association between neuroticism, pain catastrophizing, and experimentally induced pain threshold and pain tolerance in a healthy adult sample from two regions of the country of Croatia: the island of Korcula and city of Split. Methods A total of 1,322 participants were enrolled from the Island of Korcula (n = 824) and the city of Split (n = 498). Participants completed a self-reported personality measure Eysenck Personality Questionnaire (EPQ) and pain catastrophizing questionnaire Pain Catastrophizing Scale (PCS), followed by a mechanical pain pressure threshold and tolerance test. We have explored the mediating role of catastrophizing in the relationship between neuroticism and pain intensity. Results The results showed that pain catastrophizing partially mediated the relationship between neuroticism and pain intensity, suggesting the importance of pain catastrophizing in increasing vulnerability to pain. The results also indicated gender-related differences, marked by the higher pain threshold and tolerance in men. Conclusions This study adds to the understanding of the complex interplay between personality and pain, by providing a better understanding of such mechanisms in healthy adults.

Nut Consumption and Cardiovascular Risk Factors: A Cross-Sectional Study in a Mediterranean Population

Nuts are often considered beneficial for health, yet few studies have examined determinants of their intake and the associations between nut consumption and various cardiovascular disease risk factors. The aim of this study was to identify factors associated with nut intake in a Mediterranean population, in Croatia, and to investigate the association of nut intake and various cardiovascular risk factors. Methods: Subjects from the Island of Vis, Island of Korčula and the City of Split were included in this cross-sectional study (n = 4416 in total; 4011 without known cardiovascular disease). Survey responses, medical records and clinically relevant measurements were utilized. Multivariate ordinal and logistic regression models were used in the analysis, adjusting for known confounding factors. Results: As low as 5% of all subjects reported daily, and 11% reported weekly, nut consumption. The characteristics associated with more frequent nut intake were female gender (Odds ratio (OR) = 1.39; 95% confidence interval (CI) 1.19–1.62), highest level of education (1.42; 1.15–1.76) and material status (1.58; 1.29–1.93), smoking abstinence (1.21; 1.04–1.42 in never-smokers and 1.22; 1.02–1.46 in ex-smokers), Mediterranean diet adherence (1.87; 1.62–2.15), and absence of central obesity (1.29; 1.09–1.53), absence of diabetes (1.30; 1.02–1.66) and metabolic syndrome (1.17; 1.01–1.36). Subjects who consumed nuts had more favorable waist-to-height (overall p = 0.036) and waist-to-hip ratios (0.033), lesser odds of elevated fibrinogen (p < 0.001 in both weekly and monthly nut consumers) and reduced high-density lipoprotein (HDL) cholesterol (p = 0.026), compared to non-consumers. Conclusions: It appears that frequent nut consumption is an integral part of a healthy lifestyle and better socioeconomic status. A beneficial association of nut intake with cardiovascular risk factors was confirmed in this study.

Heritability Analysis Suggests Comparable Genetic Component of Mechanical Pain Threshold and Tolerance

Genetic basis of pain sensation became one of the hot topics in pain medicine over the past several years. Although numerous studies have suggested the existence of the genetic component of pain threshold, majority of these findings were based on a candidate-gene approach and remained un-replicated, and thus of limited importance (1). Commonly used approach to investigate genetic basis of any trait is to initially estimate heritability, which is defined as the proportion of the total variance of the measured trait attributable to genes. We performed a heritability analysis of the mechanical pain threshold and tolerance in a large-scale pedigree, based on the isolated population of the island of Vis in Croatia, within 10, 001 Dalmatians project (2). A total of 349 subjects were involved in the measurement of mechanical pain threshold and pain tolerance using Algometer Wagner ForceOne. After initial training measurement, subjects were exposed to the actual pain threshold measurement. Three consecutive pain threshold measurements on the dorsal side of the middle phalange of the index finger (on both hands) were performed by a single trained measurer. Additionally, we performed two consecutive measurements of pain tolerance on the same site of both hands. Mixed model analysis was performed using Sequential Oligogenic Linkage Analysis Routines – SOLAR (3), with age and gender as the predictor variables. The results indicated intermediate level of heritability of pain threshold (0.508 for right hand and 0.362 for left hand) and pain tolerance (0.542 for right hand and 0.307 for left hand, notably, the last heritability estimate was not significant ; Table 1). A strong gender and age effects were recorded in all models, with the exception of age in the pain threshold models (Table 1). These results suggest that gene mapping efforts are likely to have similar success both in pain threshold and pain tolerance, despite some previous suggestions that pain threshold is more a physiological function, while tolerance is influenced by stronger modifying effects of different environmental and psychological confounders (4). Furthermore, it should be noted that we detected difference in handedness - the heritability for pain tolerance in left hand was insignificant, with very large standard errors. This could arise from the overall small sample size or it could be pointing to differential mechanisms of pain tolerance in left vs. right, that is dominant, vs. non-dominant hand, which was implied before (5). Regardless of these results, high heritability does not warrant productive gene mapping. Heritability of height was commonly reported as one of the highest ones, generally in the range of 0.92-0.97. However, only creation of a mega-consortium of studies, which consisted of over 180, 000 measured subjects and used genome-wide approach, surpassed previous nearly futile efforts and yielded 180 loci involved in determination of the adult height, and showed a highly polygenic genetic architecture of height (6). If the same is applicable to pain, then we might be expecting a rather complex and lengthy search for genetic architecture of pain. However, the use of hypothesis-free approach, such as genome-wide association study, promises wider and deeper understanding of pain sensation, which is interesting not only for better understanding, but also for the development of new approaches to pain diagnosis and treatment.