Heritability and family-based GWAS analyses of the N-acyl ethanolamine and ceramide lipidome reveal genetic influence over circulating lipids

Certain signalling lipids of the N-acyl ethanolamine (NAE) and ceramide (CER) classes are emerging as novel biomarkers of cardiovascular disease. We sought to establish the heritability of plasma NAEs (including endocannabinoid anandamide) and CERs, and identify common DNA variants influencing the circulating concentrations of the heritable lipid species. Eleven NAEs and thirty CERs were analysed in plasma samples from 999 members of 196 British Caucasian families, using targeted mass spectrometry-based lipidomics (UPLC-MS/MS). Family-based heritability was estimated and GWAS analyses were undertaken. All lipids were significantly heritable over a wide range (h2 = 18%-87%). A missense variant (rs324420) in the gene encoding the enzyme fatty acid amide hydrolase (FAAH), which degrades NAEs, associated at GWAS significance (P<5×10-8) with four NAEs (DHEA, PEA, LEA, VEA). This SNP, previously reported to be associated with addictive behaviour, was associated with an approximately 10% per-allele difference in mean plasma NAE species. Additionally, we have extended the previously described association between rs680379 in the gene encoding the rate limiting step of CER biosynthesis (SPTLC3) and CERs to a wider range of species (e.g. CER[N(24)S(19)] and rs680379 (P =4.82×10-27)). We have shown three novel associations (CD83, SGPP1, FBXO28-DEGS1) influencing plasma CER traits, two of which (SGPP1 and DEGS1) implicate CER species in haematological phenotypes. This first genetic analysis of plasma NAE species, and a wide range of CER mediators, highlights these bioactive lipids as substantially heritable and influenced by SNPs in key metabolic enzymes.

Family-based genome-wide association studies 2 1 9 Linear mixed modelling approaches were used to account for family structure. Family-based genome-wide association analyses were undertaken for each lipid trait found in Table S2. The least significant P-values of the significantly associated SNPs 2 2 4 (P<5x10 -8 ) are depicted as P<X in the manuscript. Significantly associated SNPs were SNPs; expression quantitative trait loci (eQTL) were identified using the GTEx portal undertaken using the GWAS Catalog, and assessment of PheWAS with the UK 2 3 0 Biobank 29 was undertaken using the Gene Atlas Browser (see Web Resources). outcomes are presented in Table S3. As many GWAS associated SNPs were in 2 4 4 linkage disequilibrium, the following SNPs remained in the analysis after the data activity, Figure 1B). Structure-based summations of total abundance and ratios that 2 6 3 have previously been associated in the literature with cardiovascular risk 12 were also 2 6 4 assessed for genetic associations. A list of the species and measurements studied, and 2 6 5 summary statistics are presented in Table S4. Heritability results are depicted in Figure 2 (a detailed list is provided in Table S5). Genome-wide association study of N-acyl ethanolamines 2 7 8 There were conventionally GWAS significant (P<5x10 -8 ) associations between four  Table S6), which catalyses the degradation of NAEs 2 8 3 ( Figure 1A). The leading SNP is a missense variant (rs324420; C385A; P129T) and GTEx confirmed them as liver eQTLs (Table S8) found 20,000 bases downstream of 2 9 9 the gene encoding the third subunit of serine palmitoyltransferase (SPTLC3; Figure   3 0 0 6), which catalyses the rate-limiting step 10 of CER biosynthesis ( Figure 1B). The

0 1
SNPs are associated with differences in the expression of the SPTLC3 gene in the 3 0 2 liver, leading to changes in CER production that are reflected in altered plasma levels. with 19-and 20-carbon sphingosine bases, were found associated with the same SNPs at the SPTLC3 locus (Table S8). A novel association was identified for in Table S7). Association of ceramides and related traits with hematological phenotypes 3 1 5 The Gene Atlas Browser of PheWAS in the UK Biobank study was used to assess the further details in Table S7). All significant SNPs were confirmed eQTLs of DEGS1 in 3 2 3 1 4 whole blood (Table S8). This locus associated with numerous blood cell phenotypes in the UKBiobank data (e.g. rs4653568 and mean platelet (thrombocyte) volume; P=4.77x10 -12 ; Table S8). A further set of SNPs upstream of the gene encoding 3 2 6 sphingosine-1 phosphate phosphatase (SGPP1) were associated with 3 2 7 CER[N(24)S(16)] (e.g. rs7160525, P=5.67x10 -10 ; Figure S7). This enzyme is involved in the recycling of CER[NS] species from sphingosine-1-phosphate and ceramide-1- phosphate (C1P) ( Figure 1B). All signficiant SNPs at this locus were also associated 3 3 0 with blood cell phenotypes, identified in the UK Biobank data (e.g. rs7160525 and 3 3 1 mean platelet (thrombocyte) volume, P=3.28x10 -29 ; Table S8). The significant SNPs traits. between the SPTLC3 locus and the cardiovascular disease endpoints (Table S9). CERs to a wider range of species. Our results indicate that these two genes are the 3 5 4 major loci influencing plasma levels of NAEs and CERs, respectively. In addition, we We show for the first time associations at GWAS significance for NAE species with the SNP (DHEA, LEA, and VEA), though we did not find significant association participants with the missense AA genotype, and those with the wildtype CC 3 7 0 genotype ( Figure S8). resulted in severe neurological side-effects in a Phase I trial, hypothesised due to off-3 9 3 target drug effects 41 . As the functional FAAH SNP rs324420 did not associate with 3 9 4 any adverse phenotypes in the UK Biobank, it is likely that on-target effects of FAAH 3 9 5 inhibitor drugs do not have substantial risks of causing conditions that occurred with 3 9 6 appreciable frequency in UK Biobank.  required to identify the mechanism of the association between genetically determined 4 2 7 plasma ceramide levels and blood cell phenotypes. Association between some CER species and the SPTLC3 SNP rs680379 has been 4 3 8 identified previously through the use of shotgun lipidomics for five CER species for the de novo biosynthesis of CER, this association may have wider implications.

Figure 2: Heritability estimates of N-acyl ethanolamines and ceramides found in human plasma.
This figure depicts the heritability estimated for each lipid species using SNP-based GCTA software (y-axis) and reported pedigree-based QTDT software (x-axis). This data is presented in detail in Table S5.

Figure 4: LocusZoom plot of the association of PEA with FAAH SNP rs324420
The LocusZoom plot depicts the association of N-acyl ethanolamine lipid species PEA with FAAH SNP rs324420 on chromosome 1. The r 2 for each SNP is depicted in colour. The plot was created using the LocusZoom plot tools at http://locuszoom.sph.umich.edu/.

Figure 6: LocusZoom plot of the association of CER[N(24)S(19)] with SPTLC3 SNP rs680379
The LocusZoom plot depicts the association of CER[N(24)S(19)] with FAAH SNP rs680379 on chromosome 1. While there is a group of lead SNPs, this SNP was depicted as it has been identified previously to associate at GWAS with sphingolipid species. The r 2 for each SNP is depicted in colour. The plot was created using the LocusZoom plot tools at http://locuszoom.sph.umich.edu/.