Coronavirus epidemic in Croatia: case fatality decline during summer?

AIM: To describe the SARS-CoV-2 epidemic pattern in Croatia during February-September 2020 and compare the case fatality ratio (CFR) between spring and summer. METHODS: National data were used to calculate the weekly and monthly CFRs, stratified by three age groups: 0-64, 65-79, and 80+ years. We also calculated the standardized mortality ratios (SMR) to offset the differences in age composition. RESULTS: The epidemic consisted of the initial wave, a trough in June, and two conjoined summer waves, yielding 17206 coronavirus disease 2019 cases and 290 deaths. While the number of confirmed cases nearly quadrupled during summer, case fatality estimates decreased; CFR in spring was 4.81 (95% confidence interval 3.91-5.71), compared with 1.24 (1.06-1.42) in summer. The SMR for summer was 0.45 (0.37-0.55), suggesting that the case fatality risk halved compared with spring. Cardiovascular comorbidity was an important risk factor for case fatality (SMR 2.63 [2.20-3.13] during spring and 1.28 [1.02-1.59] during summer). The risk of death in ventilated patients remained unchanged (SMR 0.98 [0.77-1.24]). CONCLUSIONS: The epidemic dynamics suggests summer decline in case fatality, except in ventilated patients. While the effect of comorbidity also decreased, cardiovascular comorbidity remained an important risk factor for death even during summer. A plethora of possible confounders and an ever-changing landscape of SARS-CoV-2 epidemic in Croatia require constant monitoring and evaluation, with an aim to prevent the uncontrolled spread of the virus and a disruption of health care functioning.

Polymorphisms in B3GAT1, SLC9A9 and MGAT5 are associated with variation within the human plasma N-glycome of 3533 European adults.

The majority of human proteins are post-translationally modified by covalent addition of one or more complex oligosaccharides (glycans). Alterations in glycosylation processing are associated with numerous diseases and glycans are attracting increasing attention both as disease biomarkers and as targets for novel therapeutic approaches. Using a recently developed high-throughput high-performance liquid chromatography (HPLC) analysis method, we have reported, in a pilot genome-wide association study of 13 glycan features in 2705 individuals from three European populations, that polymorphisms at three loci (FUT8, FUT6/FUT3 and HNF1A) affect plasma levels of N-glycans. Here, we extended the analysis to 33 directly measured and 13 derived glycosylation traits in 3533 individuals and identified three novel gene association (MGAT5, B3GAT1 and SLC9A9) as well as replicated the previous findings using an additional European cohort. MGAT5 (meta-analysis association P-value = 1.80 × 10(-10) for rs1257220) encodes a glycosyltransferase which is known to synthesize the associated glycans. In contrast, neither B3GAT1 (rs7928758, P = 1.66 × 10(-08)) nor SLC9A9 (rs4839604, P = 3.50 × 10(-13)) had previously been associated functionally with glycosylation of plasma proteins. Given the glucuronyl transferase activity of B3GAT1, we were able to show that glucuronic acid is present on antennae of plasma glycoproteins underlying the corresponding HPLC peak. SLC9A9 encodes a proton pump which affects pH in the endosomal compartment and it was recently reported that changes in Golgi pH can impair protein sialylation, giving a possible mechanism for the observed association.

Can genetics aggravate the health of isolated and remote populations? The case of gout, hyperuricaemia and osteoarthritis in Dalmatia.

INTRODUCTION: The aim of this study was to investigate whether genetics may be considered an additional risk factor for health in isolated and remote populations, compared with their populations of origin. In this study, two remote island population samples from Croatia (from the islands of Vis and the Korcula) were compared with mainland controls from the coastal city of Split. The analyses focused on gout, hyperuricaemia and osteoarthritis, as examples of complex, multifactorial diseases. METHODS: A total of 3006 examinees from all three sites in Dalmatia, Croatia were included in the descriptive part of the study, within a large-scale project of 10,001 Dalmatians. Additionally, a subset of 2428 subjects was genotyped and information on three genomic loci was used in this study. All three loci belong to SLC2A9 gene, considered to have a major role in the regulation of serum uric acid concentration (rs6449213, rs1014290 and rs737267). RESULTS: There was a much a higher prevalence of gout in the isolated populations compared with the mainland sample (3.3% in Vis, 2.2% in Korcula and 1.7% in Split, after age standardization). Furthermore, standardized prevalence of hyperuricaemia (defined as serum uric acid ≥403 mmol/L) was 9.9% in Vis, 5.6% in Korcula and 6.1% in Split. Analysis of the allele frequencies for the three loci of SLC2A9 suggested that in all three instances the prevalence of deleterious genotypes was highest in Vis, followed by Korcula, which had higher or comparable prevalence to the city of Split. Multivariate analysis, adjusted for the main confounder effects indicated that those on the island of Vis, which has the higher degree of isolation, had significantly higher odds ratio for both hyperuricaemia (odds ratio 1.90 95% confidence intervals [1.36-2.64]) and osteoarthritis, but not gout (3.37 [2.14-5.32]). The difference between Split and Korcula included only greater odds for osteoarthritis (1.92 [1.20-3.06]). CONCLUSIONS: Isolated and remote populations that maintain a sufficient level of genetic isolation may suffer not only from consequences of geographic and social isolation, but their population genetic structure may also further contribute to poorer health status and outcomes.

Association of NOS3 tag polymorphisms with hypoxic-ischemic encephalopathy.

AIM: To test the association of NOS3 gene with hypoxic-ischemic encephalopathy (HIE). METHODS: The study included 110 unrelated term or preterm born children (69 boys and 41 girls) with HIE and 128 term and preterm born children (60 boys and 68 girls) without any neurological problems after the second year of life. Children with perinatal HIE fulfilled the diagnostic criteria for perinatal asphyxia. All children were admitted to the Clinical Hospital Split between 1992 and 2008. We analyzed 6 tagging single nucleotide polymorphisms (SNP) within NOS3 gene (rs3918186, rs3918188, rs1800783, rs1808593, rs3918227, rs1799983), in addition to previously confirmed NOS3-associated SNP rs1800779. Genotyping was conducted using real-time polymerase chain reaction (PCR). Association analyses were performed according to allelic and genotypic distribution. RESULTS: Allelic test did not show any SNP association with HIE. SNP rs1808593 showed genotype association (P=0.008) and rs1800783-rs1800779 TG haplotype showed an association with HIE (P<0.001). The study had 80% statistical power to detect (α=0.05) an effect with odds ratio (OR)=2.07 for rs3918186, OR=1.69 for rs3918188, OR=1.70 for rs1800783, OR=1.80 for rs1808593, OR=2.10 for rs3918227, OR=1.68 for rs1800779, and OR=1.76 for rs1799983, assuming an additive model. CONCLUSION: Despite the limited number of HIE patients, we observed genotypic and haplotype associations of NOS3 polymorphisms with HIE.

Common variants in SLC17A3 gene affect intra-personal variation in serum uric acid levels in longitudinal time series.

AIM: To investigate whether intra-personal variation in serum uric acid concentration is influenced by genes that were described to be associated with serum uric acid levels in cross-sectional studies. METHODS: The study included 92 participants from the isolated community of the Croatian island of Vis. For each participant, two uric acid concentration measurements were available, one from 2002 and one from 2003. Changes in uric acid concentration were correlated with a set of 8 genes known to affect it: PDZK1, GCKR, SLC2A9, ABCG2, LRRC16A, SLC17A3, SLC16A9, and SLC22A12. RESULTS: Thirteen participants (14%) had uric acid concentration change greater than 130 micromol/L. Greater variability of uric acid concentration was recorded in women (coefficient of variation 49% vs 12% in men). Two SNPs belonging to SLC17A3 gene (rs9393672 and rs942379) yielded significant association with serum uric acid concentration changes in women. These two single-nucleotide polymorphisms (SNP) explained 0.2%-1.3% of variance for 2002 or 2003 uric acid measurement and 1.1%-1.8% of variance for the average value of these two measurements. CONCLUSIONS: Repeated measurements offer a possibility to enrich the percent of explained variance and contribute to the understanding of the "missing heritability" concept. Although a number of genes have been shown to affect serum uric acid concentration, SLC17A3 seems to have a major role in determination of serum uric acid repeated measurements variation.

Predictive value of 8 genetic loci for serum uric acid concentration.

AIM: To investigate the value of genomic information in prediction of individual serum uric acid concentrations. METHODS: Three population samples were investigated: from isolated Adriatic island communities of Vis (n=980) and Korcula (n=944), and from general population of the city of Split (n=507). Serum uric acid concentration was correlated with the genetic risk score based on 8 previously described genes: PDZK1, GCKR, SLC2A9, ABCG2, LRRC16A, SLC17A1, SLC16A9, and SLC22A12, represented by a total of 16 single-nucleotide polymorphisms (SNP). The data were analyzed using classification and regression tree (CART) and general linear modeling. RESULTS: The most important variables for uric acid prediction with CART were genetic risk score in men and age in women. The percent of variance for any single SNP in predicting serum uric acid concentration varied from 0.0%-2.0%. The use of genetic risk score explained 0.1%-2.5% of uric acid variance in men and 3.9%-4.9% in women. The highest percent of variance was obtained when age, sex, and genetic risk score were used as predictors, with a total of 30.9% of variance in pooled analysis. CONCLUSION: Despite overall low percent of explained variance, uric acid seems to be among the most predictive human quantitative traits based on the currently available SNP information. The use of genetic risk scores is a valuable approach in genetic epidemiology and increases the predictability of human quantitative traits based on genomic information compared with single SNP approach.

Genome-wide association study of anthropometric traits in Korcula Island, Croatia.

AIM: To identify genetic variants underlying six anthropometric traits: body height, body weight, body mass index, brachial circumference, waist circumference, and hip circumference, using a genome-wide association study. METHODS: The study was carried out in the isolated population of the island of Korcula, Croatia, with 898 adult examinees who participated in the larger DNA-based genetic epidemiological study in 2007. Anthropometric measurements followed standard internationally accepted procedures. Examinees were genotyped using HumanHap 370CNV chip by Illumina, with a genome-wide scan containing 316730 single nucleotide polymorphisms (SNP). RESULTS: A total of 11 SNPs were associated with the investigated traits at the level of P<10(-5), with one SNP (rs7792939 in gene zinc finger protein 498, ZNF498) associated with body weight, hip circumference, and brachial circumference (P=3.59-5.73 x 10(-6)), and another one (rs157350 in gene delta-sarcoglycan, SGCD) with both brachial and hip circumference (P=3.70-6.08 x 10(-6). Variants in CRIM1, a gene regulating delivery of bone morphogenetic proteins to the cell surface, and ITGA1, involved in the regulation of mesenchymal stem cell proliferation and cartilage production, were also associated with brachial circumference (P=7.82 and 9.68 x 10(-6), respectively) and represent interesting functional candidates. Other associations involved those between genes SEZ6L2 and MAX and waist circumference, XTP6 and brachial circumference, and AMPA1/GRIA1 and height. CONCLUSION: Although the study was underpowered for the reported associations to reach formal threshold of genome-wide significance under the assumption of independent multiple testing, the consistency of association between the 2 variants and a set of anthropometric traits makes CRIM1 and ITGA1 highly interesting for further replication and functional follow-up. Increased linkage disequilibrium between the used markers in an isolated population makes the formal significance threshold overly stringent, and changed allele frequencies in isolate population may contribute to identifying variants that would not be easily identified in large outbred populations.

Environmental Risk Factors for Type 1 Diabetes Mellitus Development.

Background Although environmental factors induce development of type 1 diabetes mellitus (T1DM) in genetically susceptible individuals, many of those factors have been uncovered. Therefore, the aim of the present study was to analyze associations of T1DM with a wide range of environmental factors. Material and Methods A case-control study was conducted on 249 diabetic and 255 healthy individuals from the Dalmatian region of South Croatia. Data regarding risk factors during pregnancy and early life period of the child were evaluated. Results History of antihypertensive intake (p=0.04) and frequency of stressful life events during pregnancy (p=0.01) were associated with higher risk of T1DM, while hypertension was associated with lower risk of T1DM (p=0.01). Maternal age<25 years at delivery was associated with a higher risk of T1DM (p=0.01).Diabetic patients had a positive family history of T1DM or T2DM (p=0.002) more frequently than controls, while history of infectious diseases was inversely associated with the risk of T1DM (p=0.03). A higher risk of T1DM was significantly associated with earlier introduction of cow's milk (p=0.001), higher number of meals consumed per day (p=0.02), higher frequency of carbohydrate (p=0.001) and meat (p=0.01) consumption and stressful life events during childhood (p=0.02) while earlier introduction of fruit was associated with a lower risk of T1DM (p=0.03) Conclusion This case-control study confirmed associations of a large number of environmental factors with development of T1DM with emphasis on the association of mother's antihypertensive intake during pregnancy, which extends our knowledge about environmental factors related with development of T1DM.

IL12RB2 gene is associated with the age of type 1 diabetes onset in Croatian family Trios.

BACKGROUND: Common complex diseases are influenced by both genetic and environmental factors. Many genetic factors overlap between various autoimmune diseases. The aim of the present study is to determine whether four genetic variants known to be risk variants for several autoimmune diseases could be associated with an increased susceptibility to type 1 diabetes mellitus. METHODS AND FINDINGS: We genotyped four genetic variants (rs2358817, rs1049550, rs6679356, rs9865818) within VTCN1, ANXA11, IL12RB2 and LPP genes respectively, in 265 T1DM family trios in Croatian population. We did not detect association of these polymorphisms with T1DM. However, quantitative transmission disequilibrium test (QTDT, orthogonal model) revealed a significant association between the age of onset of T1DM and IL12RB2 rs6679356 variant. An earlier onset of T1DM was associated with the rs6679356 minor dominant allele C (p = 0.005). The association remained significant even after the Bonferroni correction for multiple testing and permutation. CONCLUSIONS: Variants originally associated with juvenile idiopathic arthritis (VTCN1 gene), sarcoidosis (ANXA11 gene), primary biliary cirrhosis (IL12RB2 gene) and celiac disease (LPP gene) were not associated with type 1 diabetes in our dataset. Nevertheless, association of IL12RB2 rs6679356 polymorphism with the age of T1DM onset suggests that this gene plays a role in defining the time of disease onset.

Genome-wide association study to identify common variants associated with brachial circumference: a meta-analysis of 14 cohorts.

Brachial circumference (BC), also known as upper arm or mid arm circumference, can be used as an indicator of muscle mass and fat tissue, which are distributed differently in men and women. Analysis of anthropometric measures of peripheral fat distribution such as BC could help in understanding the complex pathophysiology behind overweight and obesity. The purpose of this study is to identify genetic variants associated with BC through a large-scale genome-wide association scan (GWAS) meta-analysis. We used fixed-effects meta-analysis to synthesise summary results across 14 GWAS discovery and 4 replication cohorts comprising overall 22,376 individuals (12,031 women and 10,345 men) of European ancestry. Individual analyses were carried out for men, women, and combined across sexes using linear regression and an additive genetic model: adjusted for age and adjusted for age and BMI. We prioritised signals for follow-up in two-stages. We did not detect any signals reaching genome-wide significance. The FTO rs9939609 SNP showed nominal evidence for association (p<0.05) in the age-adjusted strata for men and across both sexes. In this first GWAS meta-analysis for BC to date, we have not identified any genome-wide significant signals and do not observe robust association of previously established obesity loci with BC. Large-scale collaborations will be necessary to achieve higher power to detect loci underlying BC.