Irina V. Zorkoltseva

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 (p = 10−4 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.

Genome-Wide Association Study Identifies Novel Loci Associated with Circulating Phospho- and Sphingolipid Concentrations

Phospho- and sphingolipids are crucial cellular and intracellular compounds. These lipids are required for active transport, a number of enzymatic processes, membrane formation, and cell signalling. Disruption of their metabolism leads to several diseases, with diverse neurological, psychiatric, and metabolic consequences. A large number of phospholipid and sphingolipid species can be detected and measured in human plasma. We conducted a meta-analysis of five European family-based genome-wide association studies (N = 4034) on plasma levels of 24 sphingomyelins (SPM), 9 ceramides (CER), 57 phosphatidylcholines (PC), 20 lysophosphatidylcholines (LPC), 27 phosphatidylethanolamines (PE), and 16 PE-based plasmalogens (PLPE), as well as their proportions in each major class. This effort yielded 25 genome-wide significant loci for phospholipids (smallest P-value = 9.88×10−204) and 10 loci for sphingolipids (smallest P-value = 3.10×10−57). After a correction for multiple comparisons (P-value<2.2×10−9), we observed four novel loci significantly associated with phospholipids (PAQR9, AGPAT1, PKD2L1, PDXDC1) and two with sphingolipids (PLD2 and APOE) explaining up to 3.1% of the variance. Further analysis of the top findings with respect to within class molar proportions uncovered three additional loci for phospholipids (PNLIPRP2, PCDH20, and ABDH3) suggesting their involvement in either fatty acid elongation/saturation processes or fatty acid specific turnover mechanisms. Among those, 14 loci (KCNH7, AGPAT1, PNLIPRP2, SYT9, FADS1-2-3, DLG2, APOA1, ELOVL2, CDK17, LIPC, PDXDC1, PLD2, LASS4, and APOE) mapped into the glycerophospholipid and 12 loci (ILKAP, ITGA9, AGPAT1, FADS1-2-3, APOA1, PCDH20, LIPC, PDXDC1, SGPP1, APOE, LASS4, and PLD2) to the sphingolipid pathways. In large meta-analyses, associations between FADS1-2-3 and carotid intima media thickness, AGPAT1 and type 2 diabetes, and APOA1 and coronary artery disease were observed. In conclusion, our study identified nine novel phospho- and sphingolipid loci, substantially increasing our knowledge of the genetic basis for these traits.

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.

Segregation analysis of idiopathic scoliosis: demonstration of a major gene effect.

Segregation analysis using a model with age and gender effects was applied to 101 pedigrees ascertained through a proband with idiopathic scoliosis. The transmission probability model was used to detect major gene effect. When we analyzed the pedigrees where affected status was assigned to persons with a Cobbs angle of more than 5 degrees we did not detect a significant major gene effect. However, when the affected status was assigned to persons with pronounced forms of disease only (a curve of at least 11 degrees) a significant contribution of a major causal gene could be established and inheritance could be described according to a dominant major gene diallele model, assuming incomplete sex and age dependent penetrance of genotypes. According to this model, the pronounced forms of idiopathic scoliosis should never occur in the absence of the mutant allele. This indicates that only the carriers of the mutant allele develop pronounced forms of the disease. At the same time, only a fraction of the carriers of the mutant gene should manifest the disease (30% of males and 50% of females).

Heritability of blood pressure traits and the genetic contribution to blood pressure variance explained by four blood-pressure-related genes.

Objective To study the heritability of four blood pressure traits and the proportion of variance explained by four blood-pressure-related genes. Methods All participants are members of an extended pedigree from a Dutch genetically isolated population. Heritability and genetic correlations of systolic blood pressure, diastolic blood pressure, mean arterial pressure and pulse pressure were assessed using a variance components approach (SOLAR). Polymorphisms of the α-adducin (ADD1), angiotensinogen (AGT), angiotensin II type 1 receptor (AT1R) and G protein β3 (GNB3) genes were typed. Results Heritability estimates were significant for all four blood pressure traits, ranging between 0.24 and 0.37. Genetic correlations between systolic blood pressure, diastolic blood pressure and mean arterial pressure were high (0.93–0.98), and those between pulse pressure and diastolic blood pressure were low (0.05). The ADD1 polymorphism explained 0.3% of the variance of pulse pressure (P = 0.07), and the polymorphism of GNB3 explained 0.4% of the variance of systolic blood pressure (P = 0.02), 0.2% of mean arterial pressure (P = 0.05) and 0.3% of pulse pressure (P = 0.06). Conclusion Genetic factors contribute to a substantial proportion of blood pressure variance. In this study, the effect of polymorphisms of ADD1, AGT, AT1R and GNB3 explained a very small proportion of the heritability of blood pressure traits. As new genes associated with blood pressure are localized in the future, their effect on blood pressure variance should be calculated.

Genetic Architecture of Plasma Adiponectin Overlaps With the Genetics of Metabolic Syndrome–Related Traits

OBJECTIVE Adiponectin, a hormone secreted by adipose tissue, is of particular interest in metabolic syndrome, because it is inversely correlated with obesity and insulin sensitivity. However, it is not known to what extent the genetics of plasma adiponectin and the genetics of obesity and insulin sensitivity are interrelated. We aimed to evaluate the heritability of plasma adiponectin and its genetic correlation with the metabolic syndrome and metabolic syndrome–related traits and the association between these traits and 10 ADIPOQ single nucleotide polymorphisms (SNPs). RESEARCH DESIGN AND METHODS We made use of a family-based population, the Erasmus Rucphen Family study (1,258 women and 967 men). Heritability analysis was performed using a polygenic model. Genetic correlations were estimated using bivariate heritability analyses. Genetic association analysis was performed using a mixed model. RESULTS Plasma adiponectin showed a heritability of 55.1%. Genetic correlations between plasma adiponectin HDL cholesterol and plasma insulin ranged from 15 to 24% but were not significant for fasting glucose, triglycerides, blood pressure, homeostasis model assessment of insulin resistance (HOMA-IR), and C-reactive protein. A significant association with plasma adiponectin was found for ADIPOQ variants rs17300539 and rs182052. A nominally significant association was found with plasma insulin and HOMA-IR and ADIPOQ variant rs17300539 after adjustment for plasma adiponectin. CONCLUSIONS The significant genetic correlation between plasma adiponectin and HDL cholesterol and plasma insulin should be taken into account in the interpretation of genome-wide association studies. Association of ADIPOQ SNPs with plasma adiponectin was replicated, and we showed association between one ADIPOQ SNP and plasma insulin and HOMA-IR.

The apolipoprotein E gene and its age-specific effects on cognitive function

The E4 allele of the apolipoprotein E gene (APOE) is a well-established determinant of Alzheimers disease but its relation to cognitive function is much less understood. We studied the age-specific effects of the APOE*E4 allele on cognitive function and cardiovascular risk factors in 2208 related individuals. APOE*E4 allele was significantly associated with reduced test scores for Adult Verbal Learning Test, particularly on the memory and learning sub domains, in persons older than 50 years of age. The effect of APOE*E4 was independent of the effect of APOE*E4 on vascular risk factors and most pronounced on learning ability. Our findings suggest that APOE*E4 has an effect on cognitive function predominantly in the elderly, independent of vascular risk factors.

Linkage and genome-wide association analysis of obesity-related phenotypes: association of weight with the MGAT1 gene.

As major risk‐factors for diabetes and cardiovascular diseases, the genetic contribution to obesity‐related traits has been of interest for decades. Recently, a limited number of common genetic variants, which have replicated in different populations, have been identified. One approach to increase the statistical power in genetic mapping studies is to focus on populations with increased levels of linkage disequilibrium (LD) and reduced genetic diversity. We have performed joint linkage and genome‐wide association analyses for weight and BMI in 3,448 (linkage) and 3,925 (association) partly overlapping healthy individuals from five European populations. A total of four chromosomal regions (two for weight and two for BMI) showed suggestive linkage (lod >2.69) either in one of the populations or in the joint data. At the genome‐wide level (nominal P < 1.6 × 10−7, Bonferroni‐adjusted P < 0.05) one single‐nucleotide polymorphism (SNP) (rs12517906) (nominal P = 7.3 × 10−8) was associated with weight, whereas none with BMI. The SNP associated with weight is located close to MGAT1. The monoacylglycerol acyltransferase (MGAT) enzyme family is known to be involved in dietary fat absorption. There was no overlap between the linkage regions and the associated SNPs. Our results show that genetic effects influencing weight and BMI are shared across diverse European populations, even though some of these populations have experienced recent population bottlenecks and/or been affected by genetic drift. The analysis enabled us to identify a new candidate gene, MGAT1, associated with weight in women.

Linkage and association analyses of glaucoma related traits in a large pedigree from a Dutch genetically isolated population

Background Despite extensive research on the genetic determinants of glaucoma, the genes identified to date explain only a small proportion of cases in the general population. Methods Genome-wide linkage and association analyses of quantitative traits related to glaucoma were performed: intraocular pressure, size and morphology of the optic disc (individual and combined by method of principal components) and thickness of the retinal nerve fibre layer (RNFL), in a large pedigree from a genetically isolated Dutch population. Results For the size of the optic disc, the study demonstrated a significant linkage signal (logarithm of odds (LOD)=3.6) at the LRP1B region on chromosome 2q21.2-q22.2 and significant association (p=8.95×10−12) with the previously reported CDC7/TGFBR3 locus at 1p22. For parameters describing morphology of the optic disc, the study obtained significant linkage signal (LOD=4.6) at regions SIRPA and RNF24/PANK2 at 20p13 (false discovery rate (FDR) based q value <0.05) and genome-wide significant association (p=2.38×10−9) with a common variant in the RERE gene at 1p36. Suggestive linkage and association signals indicated loci for morphology of the optic disc at 2q31-q33 (IGFBP2 locus) and for RNFL thickness at 3p22.2 (DCLK3 locus) and 14q22-q23 (SIX1 locus). Conclusion This study identified new linkage regions at 20p13 (SIRPA and RNF24/PANK2 loci) and 2q33-q34 (IGFBP2 locus) for parameters describing morphology of the optic disc. The results of the study also suggested common genetic control of these parameters and RNFL thickness by SIX1 and doublecotin family genes. Finally, association signals for the recently reported RERE and LRP1B loci and the well known CDC7, TGFBR3, and ATOH7 loci were replicated.

A Genome-Wide Screen for Depression in Two Independent Dutch Populations

BACKGROUND Depression has a strong genetic component but candidate gene studies conducted to date have not shown consistent associations. METHODS We conducted a genome-wide parametric and nonparametric linkage analysis in a large-scale family-based study including 115 individuals with depression who were identified based on the Hospital Anxiety Depression Scale, Center for Epidemiologic Studies Depression Rating Scale, or use of antidepressive medication. Further, we investigated the most promising chromosomal regions found in the genome-wide linkage analysis with an association analysis in 734 individuals in the family-based study and 2373 individuals in the population-based study. RESULTS Our study demonstrated evidence for significant linkage of depression to chromosome 2p16.1-15 (logarithm of odds [LOD] = 5.13; parametric analysis) and suggestive evidence for linkage in nonparametric analysis to chromosome 5p15.33 (LOD = 2.14), 11q25 (LOD = 2.27), and 19p13.3 (LOD = 2.66). The subsequent association analysis in the family-based study showed region-wide significant association in intron 1 of the OPCML gene on chromosome 11q25 (empirical p value = .04). The association analysis in the population-based study did not show any region-wide significant association, yet showed suggestive association in intron 1 of the APLP2 gene on chromosome 11q25. CONCLUSIONS Our linkage and association studies suggest a locus for depression on chromosomes 2p16.1-15 and 11q25. The linkage to chromosome 11q25 may be, in part, explained by the OPCML or the APLP2 gene. Further, there is evidence for a role of the GNG7 gene (chromosome 19p13.3).