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 beta-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 x 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

Automated workflow-based exploitation of pathway databases provides new insights into genetic associations of metabolite profiles

Background: Genome-wide association studies (GWAS) have identified many common single nucleotide polymorphisms (SNPs) that associate with clinical phenotypes, but these SNPs usually explain just a small part of the heritability and have relatively modest effect sizes. In contrast, SNPs that associate with metabolite levels generally explain a higher percentage of the genetic variation and demonstrate larger effect sizes. Still, the discovery of SNPs associated with metabolite levels is challenging since testing all metabolites measured in typical metabolomics studies with all SNPs comes with a severe multiple testing penalty. We have developed an automated workflow approach that utilizes prior knowledge of biochemical pathways present in databases like KEGG and BioCyc to generate a smaller SNP set relevant to the metabolite. This paper explores the opportunities and challenges in the analysis of GWAS of metabolomic phenotypes and provides novel insights into the genetic basis of metabolic variation through the re-analysis of published GWAS datasets. Results: Re-analysis of the published GWAS dataset from Illig et al. (Nature Genetics, 2010) using a pathway-based workflow (http://www.myexperiment.org/packs/319.html), confirmed previously identified hits and identified a new locus of human metabolic individuality, associating Aldehyde dehydrogenase family1 L1 (ALDH1L1) with serine/glycine ratios in blood. Replication in an independent GWAS dataset of phospholipids (Demirkan et al., PLoS Genetics, 2012) identified two novel loci supported by additional literature evidence: GPAM (Glycerol-3 phosphate acyltransferase) and CBS (Cystathionine beta-synthase). In addition, the workflow approach provided novel insight into the affected pathways and relevance of some of these gene-metabolite pairs in disease development and progression. Conclusions: We demonstrate the utility of automated exploitation of background knowledge present in pathway databases for the analysis of GWAS datasets of metabolomic phenotypes. We report novel loci and potential biochemical mechanisms that contribute to our understanding of the genetic basis of metabolic variation and its relationship to disease development and progression

Precision engineering for PRRSV resistance in pigs: Macrophages from genome edited pigs lacking CD163 SRCR5 domain are fully resistant to both PRRSV genotypes while maintaining biological function

Porcine Reproductive and Respiratory Syndrome (PRRS) is a panzootic infectious disease of pigs, causing major economic losses to the world-wide pig industry. PRRS manifests differently in pigs of all ages but primarily causes late-term abortions and stillbirths in sows and respiratory disease in piglets. The causative agent of the disease is the positive-strand RNA PRRS virus (PRRSV). PRRSV has a narrow host cell tropism, limited to cells of the monocyte/macrophage lineage. CD163 has been described as a fusion receptor for PRRSV, whereby the scavenger receptor cysteine-rich domain 5 (SRCR5) region was shown to be an interaction site for the virus in vitro. CD163 is expressed at high levels on the surface of macrophages, particularly in the respiratory system. Here we describe the application of CRISPR/Cas9 to pig zygotes, resulting in the generation of pigs with a deletion of Exon 7 of the CD163 gene, encoding SRCR5. Deletion of SRCR5 showed no adverse effects in pigs maintained under standard husbandry conditions with normal growth rates and complete blood counts observed. Pulmonary alveolar macrophages (PAMs) and peripheral blood monocytes (PBMCs) were isolated from the animals and assessed in vitro. Both PAMs and macrophages obtained from PBMCs by CSF1 stimulation (PMMs) show the characteristic differentiation and cell surface marker expression of macrophages of the respective origin. Expression and correct folding of the SRCR5 deletion CD163 on the surface of macrophages and biological activity of the protein as hemoglobin-haptoglobin scavenger was confirmed. Challenge of both PAMs and PMMs with PRRSV genotype 1, subtypes 1, 2, and 3 and PMMs with PRRSV genotype 2 showed complete resistance to viral infections assessed by replication. Confocal microscopy revealed the absence of replication structures in the SRCR5 CD163 deletion macrophages, indicating an inhibition of infection prior to gene expression, i.e. at entry/fusion or unpacking stages

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

HIF-1BETA determines ABCA1 expression under hypoxia in human macrophages-A potential target for cardiovascular protection

Background: ATP-binding cassette transporter A1 (ABCA1) is a plasmamembrane protein that is primarily involved in high-density lipoprotein(HDL) production in liver cells and in the elimination of intracellularcholesterol in peripheral tissues. Functional deficiency of this protein leadsto low levels of circulating HDL, a clinical condition called Tangier’s disease. In peripheral tissues impaired ABCA1 function may lead to localcholesterol accumulation. Aim: Atherosclerotic lesion associated lipid-loaded macrophages expressABCA1 as a primary reverse cholesterol transporter. These macrophagesare exposed to regions local hypoxia inside the lesions, which may have amajor impact on ABCA1 mediated reverse cholesterol transport. We studiedthe effect of hypoxia on ABCA1 regulation and cholesterol efflux in humanmacrophages. Methods: Primary human monocyte derived macrophages and hepato-cytes as well as monocytic and hepatocytic cell lines were cultivated invitro in order to analyze the binding of the hypoxia-inducible factor 1(HIF-1) complex to the ABCA1 promoter and to determine the ABCA1response to HIF-1 signaling in normoxic and hypoxic conditions. To evaluate the functional effect of hypoxia on ABCA1 mediated lipid effluxtotal cholesterol was measured from the culture media in hypoxic conditions and after transduction of the cells with constitutively active HIF-1alpha adenovirus construct. The results were confirmed in human suben-dothelial fat depositions and pre-eclamptic placentas. Results: We found that HIF-1 specifically binds to the HIF-1 responseelement of the ABCA1 promoter, and the HIF-1 complex increasesABCA1 promoter activity along with ABCA1 expression. Primary humanmacrophages exposed to hypoxia or expressing constitutively active HIF-1alpha respond with a potent change in ABCA1 expression, which showsa strong correlation with HIF-1beta expression (r 0.95–0.91). Moreover,ABCA1-mediated cholesterol efflux was also found to be regulated byHIF-1beta under hypoxia. In vivo, macrophages prepared from humanatherosclerotic lesions ABCA1 levels show a strong correlation with HIF-1beta expression. This in vivo regulatory mechanism was confirmed inhuman pre-eclamptic placentas, a clinical condition with severe localhypoxia. Conclusions: These results demonstrate that HIF-1beta availability deter-mines ABCA1 expression and cholesterol efflux in macrophages underhypoxia. The individually observed differences in HIF-1beta expression,therefore, may contribute to the interpersonal variability of atheroscleroticlesion progression. HIF-1beta as a potential drug target may open novelpathways for cardiovascular protection therapies

Accompanying antibodies during immunization tored blood cell antigens

Background: Alloantibodies in some patients are accompanied by au-toantibodies. These autoantibodies may react with all red blood cells (RBC),or they may show a partial blood group antigen specificity that is usuallyspecific for the patient’s own antigens. However, discrimination betweenauto- and alloantibodies may be obscure in some cases. Aim: We describe a patient with alloimmunization and accompanyingantibodies that showed characteristics of both allo- and autoantibodies. Methods: Immunohematologic standard methods were used for serologic-and genotyping, antibody differentiation, adsorption and elution. Patient and Results: A 36-year old female, blood group A, ccD.EE, K-,Jk(a-), Fy(a), with liver cell carcinoma was repeatedly transfused because ofsurgical procedures. This patient developed antibodies against C, e, Jk(a)and subsequently Fy(b). In addition, accompanying autoantibodies withoutblood group specificity could be demonstrated by adsorption and elutionusing RBC of the blood group ccD.EE Jk(a-) Fy(b-). The autoantibodies werereactive with RBC of all blood groups. Apart from autoantibodies, additionally anti-C could be adsorbed to and eluted from the ccD.EE Jk(a-)Fy(b-) RBC. Anti-G could be excluded with D-positive RBC. Summary: This case illustrates the potential autoreactivity of newlyformed alloantibodies, a behavior that has sometimes been calledpseudoalloreactive autoantibodies or mimicking type antibodies thatresemble a Matuhasi-Ogata phenomenon. This case also illustrates thecomplexity of immunization that may not only include accompanyingautoantibodies, but also accompanying alloantibodies

Analyzing time-dependent microarray data using independent component analysis derived expression modes from human macrophages infected with F. tularensis holartica.

The analysis of large-scale gene expression profiles is still a demanding and extensive task. Modern machine learning and data mining techniques developed in linear algebra, like Independent Component Analysis (ICA), become increasingly popular as appropriate tools for analyzing microarray data. We applied ICA to analyze kinetic gene expression profiles of human monocyte derived macrophages (MDM) from three different donors infected with Francisella tularensis holartica and compared them to more classical methods like hierarchical clustering. Results were compared using a pathway analysis too[, based on the Gene Ontology and the MeSH database. We could show that both methods lead to time-dependent gene regulatory patterns which fit well to known TNF alpha induced immune responses. In comparison, the nonexclusive attribute of ICA results in a more detailed view and a higher resolution in time dependent behavior of the immune response genes. Additionally, we identified NF kappa B as one of the main regulatory genes during response to F. tularensis infection