Ozren Polašek

Genome-wide association study identifies five loci associated with lung function

Pulmonary function measures are heritable traits that predict morbidity and mortality and define chronic obstructive pulmonary disease (COPD). We tested genome-wide association with forced expiratory volume in 1 s (FEV1) and the ratio of FEV1 to forced vital capacity (FVC) in the SpiroMeta consortium (n = 20,288 individuals of European ancestry). We conducted a meta-analysis of top signals with data from direct genotyping (n ≤ 32,184 additional individuals) and in silico summary association data from the CHARGE Consortium (n = 21,209) and the Health 2000 survey (n ≤ 883). We confirmed the reported locus at 4q31 and identified associations with FEV1 or FEV1/FVC and common variants at five additional loci: 2q35 in TNS1 (P = 1.11 × 10−12), 4q24 in GSTCD (2.18 × 10−23), 5q33 in HTR4 (P = 4.29 × 10−9), 6p21 in AGER (P = 3.07 × 10−15) and 15q23 in THSD4 (P = 7.24 × 10−15). mRNA analyses showed expression of TNS1, GSTCD, AGER, HTR4 and THSD4 in human lung tissue. These associations offer mechanistic insight into pulmonary function regulation and indicate potential targets for interventions to alleviate respiratory disease.

Variability, heritability and environmental determinants of human plasma N-glycome

Plasma glycans were analyzed in 1008 individuals to evaluate variability and heritability, as well as the main environmental determinants that affect glycan structures. By combining HPLC analysis of fluorescently labeled glycans with sialidase digestion, glycans were separated into 33 chromatographic peaks and quantified. A high level of variability was observed with the median ratio of minimal to maximal values of 6.17 and significant age- and gender-specific differences. Heritability estimates for individual glycans varied widely, ranging from very low to very high. Glycome-wide environmental determinants were also detected with statistically significant effects of different variables including diet, smoking and cholesterol levels.

Effects of aging, body mass index, plasma lipid profiles, and smoking on human plasma N-glycans

Protein glycosylation affects nearly all molecular interactions at the cell surface and in the intercellular space. Many of the physiological variations which are part of homeostatic mechanisms influence glycosylation. However, a comprehensive overview of changes in glycosylation caused by aging and common lifestyle parameters is still lacking. After analyzing N-glycans in the plasma of 1914 individuals from the Croatian islands of Vis and Korcula, we performed a comprehensive analysis of the dependence of different glycosylation features (position of fucose, level of galactosylation, sialylation and branching) on aging, smoking, body fat and plasma lipid status. A number of statistically significant associations were observed. Glycosylation changes with aging were especially evident in females, mostly in association with the transition from pre-menopausal to post-menopausal age. Levels of core-fucosylated, non-galactosylated, digalactosylated and disialylated biantennary glycans were shown to be mainly age dependent, but the level of branching and higher levels of galactosylation were found to correlate with lipid status. For the majority of glycans which we analyzed, all examined parameters explained up to 5% of the variance. The only notable exception were non-galactosylated glycans where 20% of the variance was explained mostly by age and blood pressure. In general, only a small fraction of the variability in glycan levels observed in a population was explained by age and other measured parameters, indicating that even in the absence of a genetic template, glycan levels are mostly determined by genetic background and/or specific pathophysiological processes.

Genetic determinants of circulating sphingolipid concentrations in European populations

Sphingolipids have essential roles as structural components of cell membranes and in cell signalling, and disruption of their metabolism causes several diseases, with diverse neurological, psychiatric, and metabolic consequences. Increasingly, variants within a few of the genes that encode enzymes involved in sphingolipid metabolism are being associated with complex disease phenotypes. Direct experimental evidence supports a role of specific sphingolipid species in several common complex chronic disease processes including atherosclerotic plaque formation, myocardial infarction (MI), cardiomyopathy, pancreatic β-cell failure, insulin resistance, and type 2 diabetes mellitus. Therefore, sphingolipids represent novel and important intermediate phenotypes for genetic analysis, yet little is known about the major genetic variants that influence their circulating levels in the general population. We performed a genome-wide association study (GWAS) between 318,237 single-nucleotide polymorphisms (SNPs) and levels of circulating sphingomyelin (SM), dihydrosphingomyelin (Dih-SM), ceramide (Cer), and glucosylceramide (GluCer) single lipid species (33 traits); and 43 matched metabolite ratios measured in 4,400 subjects from five diverse European populations. Associated variants (32) in five genomic regions were identified with genome-wide significant corrected p-values ranging down to 9.08×10−66. The strongest associations were observed in or near 7 genes functionally involved in ceramide biosynthesis and trafficking: SPTLC3, LASS4, SGPP1, ATP10D, and FADS1–3. Variants in 3 loci (ATP10D, FADS3, and SPTLC3) associate with MI in a series of three German MI studies. An additional 70 variants across 23 candidate genes involved in sphingolipid-metabolizing pathways also demonstrate association (pu200a=u200a10−4 or less). Circulating concentrations of several key components in sphingolipid metabolism are thus under strong genetic control, and variants in these loci can be tested for a role in the development of common cardiovascular, metabolic, neurological, and psychiatric diseases.

Genomics Meets Glycomics—The First GWAS Study of Human N-Glycome Identifies HNF1α as a Master Regulator of Plasma Protein Fucosylation

Over half of all proteins are glycosylated, and alterations in glycosylation have been observed in numerous physiological and pathological processes. Attached glycans significantly affect protein function; but, contrary to polypeptides, they are not directly encoded by genes, and the complex processes that regulate their assembly are poorly understood. A novel approach combining genome-wide association and high-throughput glycomics analysis of 2,705 individuals in three population cohorts showed that common variants in the Hepatocyte Nuclear Factor 1α (HNF1α) and fucosyltransferase genes FUT6 and FUT8 influence N-glycan levels in human plasma. We show that HNF1α and its downstream target HNF4α regulate the expression of key fucosyltransferase and fucose biosynthesis genes. Moreover, we show that HNF1α is both necessary and sufficient to drive the expression of these genes in hepatic cells. These results reveal a new role for HNF1α as a master transcriptional regulator of multiple stages in the fucosylation process. This mechanism has implications for the regulation of immunity, embryonic development, and protein folding, as well as for our understanding of the molecular mechanisms underlying cancer, coronary heart disease, and metabolic and inflammatory disorders.

Gaps in policy-relevant information on burden of disease in children: a systematic review

BACKGROUNDnValid information about cause-specific child mortality and morbidity is an essential foundation for national and international health policy. We undertook a systematic review to investigate the geographical dispersion of and time trends in publication for policy-relevant information about childrens health and to assess associations between the availability of reliable data and poverty.nnnMETHODSnWe identified data available on Jan 1, 2001, and published since 1980, for the major causes of morbidity and mortality in young children. Studies with relevant data were assessed against a set of inclusion criteria to identify those likely to provide unbiased estimates of the burden of childhood disease in the community.nnnFINDINGSnOnly 308 information units from more than 17,000 papers identified were regarded as possible unbiased sources for estimates of childhood disease burden. The geographical distribution of these information units revealed a pattern of small well-researched populations surrounded by large areas with little available information. No reliable population-based data were identified from many of the worlds poorest countries, which account for about a third of all deaths of children worldwide. The number of new studies diminished over the last 10 years investigated.nnnINTERPRETATIONnThe number of population-based studies yielding estimates of burden of childhood disease from less developed countries was low. The decreasing trend over time suggests reductions in research investment in this sphere. Data are especially sparse from the worlds least developed countries with the highest child mortality. Guidelines are needed for the conduct of burden-of-disease studies together with an international research policy that gives increased emphasis to global equity and coverage so that knowledge can be generated from all regions of the world.

Common variants in the JAZF1 gene associated with height identified by linkage and genome-wide association analysis

Genes for height have gained interest for decades, but only recently have candidate genes started to be identified. We have performed linkage analysis and genome-wide association for height in approximately 4000 individuals from five European populations. A total of five chromosomal regions showed suggestive linkage and in one of these regions, two SNPs (rs849140 and rs1635852) were associated with height (nominal P = 7.0 x 10(-8) and P = 9.6 x 10(-7), respectively). In total, five SNPs across the genome showed an association with height that reached the threshold of genome-wide significance (nominal P < 1.6 x 10(-7)). The association with height was replicated for two SNPs (rs1635852 and rs849140) using three independent studies (n = 31 077, n=1268 and n = 5746) with overall meta P-values of 9.4 x 10(-10) and 5.3 x 10(-8). These SNPs are located in the JAZF1 gene, which has recently been associated with type II diabetes, prostate and endometrial cancer. JAZF1 is a transcriptional repressor of NR2C2, which results in low IGF1 serum concentrations, perinatal and early postnatal hypoglycemia and growth retardation when knocked out in mice. Both the linkage and association analyses independently identified the JAZF1 region affecting human height. We have demonstrated, through replication in additional independent populations, the consistency of the effect of the JAZF1 SNPs on height. Since this gene also has a key function in the metabolism of growth, JAZF1 represents one of the strongest candidates influencing human height identified so far.

Genome-wide linkage analysis of serum creatinine in three isolated European populations

There is increasing evidence for a role of genetic predisposition in the etiology of kidney disease, but linkage scans have been poorly replicated. Here we performed a genome-wide linkage analysis of serum creatinine on 2859 individuals from isolated villages in South Tyrol (Italy), Rucphen (The Netherlands) and Vis Island (Croatia), populations that have been stable and permanently resident in their region. Linkage of serum creatinine levels to loci on chromosomes 7p14, 9p21, 11p15, 15q15-21, 16p13, and 18p11 was successfully replicated in at least one discovery population or in the pooled analysis. A novel locus was found on chromosome 10p11. Linkage to chromosome 22q13, independent of diabetes and hypertension, was detected over a region containing the non-muscle myosin heavy chain type II isoform A (MYH9) gene (LOD score=3.52). In non-diabetic individuals, serum creatinine was associated with this gene in two of the three populations and in meta-analysis (SNP rs11089788, P-value=0.0089). In populations sharing a homogeneous environment and genetic background, heritability of serum creatinine was higher than in outbred populations, with consequent detection of a larger number of loci than reported before. Our finding of a replicated association of serum creatinine with the MYH9 gene, recently linked to pathological renal conditions in African Americans, suggests that this gene may also influence kidney function in healthy Europeans.

Hypercoagulability in Cushing's syndrome: the role of specific haemostatic and fibrinolytic markers

Objective Hypercoagulability is a commonly described complication in patients with Cushing’s syndrome. Recent clinical studies have indicated various abnormalities of coagulation and fibrinolysis parameters which may be related to that phenomenon. The aim of this study was to investigate the mechanisms underlying the hypercoagulable state in patients with Cushing’s syndrome. Research methods and procedures A wide range of serum markers involved in the processes of blood coagulation and fibrinolysis was measured in a group of 33 patients with Cushing’s syndrome and 31 healthy controls. No participant was taking medication which could influence the result or had known diseases, except hypertension and diabetes, which could affect blood coagulation or fibrinolysis parameters. Results Patients with Cushing’s syndrome had higher levels of clotting factors II (Pxa0=xa00.003), V (Pxa0<xa00.001), VIII (Pxa0<xa00.001), IX (Pxa0<xa00.001), XI (Pxa0<xa00.001) and XII (Pxa0=xa00.019), protein C (Pxa0<xa00.001), protein S (Pxa0<xa00.001), C1-inhibitor (Pxa0<xa00.001) and plasminogen activator inhibitor-1 (PAI-1) (Pxa0=xa00.004). The activity of fibrinolytic markers, plasminogen (Pxa0<xa00.001), antithrombin (Pxa0<xa00.001) and antithrombin antigen (Pxa0=xa00.001) was also increased in the patient group. Conclusion The study has demonstrated hypercoagulability in patients with Cushing’s syndrome manifest as increased prothrombotic activity and compensatory activation of the fibrinolytic system. We propose the introduction of thromboprophylaxis in the preoperative and early postoperative periods, combined with a close follow-up in order to prevent possible thromboembolic events in patients with Cushing’s syndrome.

Estimating human inbreeding coefficients

We have used genealogies and genomic polymorphisms to estimate individual inbreeding coefficients (F) in 50 subjects with an expected range (based on recent genealogies) of F from 0.0 to 0.0625. The estimates were based on two approaches, using genotypes respectively from 410 microsatellite markers (410‐STR panel) and from 10,000 SNPs (10K‐SNP panel). The latter was performed in a sub‐sample of 15 individuals. We concluded that for both marker panels measures of inbreeding based on the excess of homozygosity over Hardy‐Weinberg expectation were not closely correlated with 4‐5 generation genealogical F‐values. For the 10K‐SNP panel we found two alternative measures which correlated more closely with F, based respectively on standard errors and on paired homozygosity of nearby SNPs over distances of 2‐4 cM. We propose an empirical method for estimating standard errors and hence individual F‐values, based on the variation between individual autosomes. This method could provide useful estimates of average F‐values for groups of individuals in population‐based studies of the effects of inbreeding/homozygosity on quantitative traits.