Italian Journal of Animal Science ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/tjas20 An association analysis between a polymorphism in the SEC24A gene and lipid traits recorded in Duroc pigs Emilio Mármol-Sánchez, Raquel Quintanilla, María Gracia Luigi-Sierra & Marcel Amills To cite this article: Emilio Mármol-Sánchez, Raquel Quintanilla, María Gracia Luigi-Sierra & Marcel Amills (2021) An association analysis between a polymorphism in the SEC24A gene and lipid traits recorded in Duroc pigs, Italian Journal of Animal Science, 20:1, 1444-1451, DOI: 10.1080/1828051X.2021.1951129 To link to this article: https://doi.org/10.1080/1828051X.2021.1951129 © 2021 The Author(s). Published by Informa View supplementary material UK Limited, trading as Taylor & Francis Group. Published online: 05 Oct 2021. Submit your article to this journal Article views: 440 View related articles View Crossmark data Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=tjas20 ITALIAN JOURNAL OF ANIMAL SCIENCE 2021, VOL. 20, NO. 1, 1444–1451 https://doi.org/10.1080/1828051X.2021.1951129 BRIEF REPORT An association analysis between a polymorphism in the SEC24A gene and lipid traits recorded in Duroc pigs Emilio Marmol-Sancheza, Raquel Quintanillab, Marıa Gracia Luigi-Sierraa and Marcel Amillsa,c a Centre de Recerca en Agrigenomica (CRAG), CSIC-IRTA-UAB-UB, Universitat Autonoma de Barcelona, Bellaterra, Spain; bGenetica i Millora Animal, Intitut de Recerca i Tecnologia Agroalimentaries (IRTA), Caldes de Montbui, Spain; cDepartament de Ciencia Animal i dels Aliments, Universitat Autonoma de Barcelona, Bellaterra, Barcelona, Spain ABSTRACT ARTICLE HISTORY By regulating the expression of low-density lipoprotein receptors (LDLR), the SEC24A protein Received 30 December 2020 modulates the clearance of plasma cholesterol in mice. The whole-genome sequencing of five Revised 21 April 2021 Duroc boars that sired a Duroc purebred experimental population (LIPGEN population, 350 indi- Accepted 25 June 2021 viduals) revealed the segregation of one putative nonsense mutation located at the SEC24A KEYWORDS (rs336401826, c.44G>A) gene. Genotyping of this polymorphic site in the LIPGEN population Nonsense mutation; pig; highlighted the existence of a significant departure from the Hardy Weinberg equilibrium (v2 ¼ lipid traits; skeletal muscle 5.05, p-value ¼ 0.024527). Despite the fact that SEC24A inactivation should affect serum lipid concentrations, we did not observe an association between SEC24A genotype and such traits. A thorough revision of the annotation of the SEC24A rs336401826 SNP revealed that this poly- morphism is probably located in the 5’UTR of the SEC24A gene, rather than at the beginning of the first exon, hence not introducing the predicted premature stop codon that could truncate the SEC24A protein. Given that the annotation of loss-of-function mutations in pigs is far from perfect, careful manual curation of such mutations is recommended before undertaking the ana- lysis of their effects at the experimental level. HIGHLIGHTS  One mutation (c.44 G>A) predicted to disrupt the SEC24A protein segregates in a Duroc pig population.  This polymorphism is not associated with serum lipid levels despite its expected effect on such traits.  Manual curation of the annotation of this putative nonsense polymorphism has revealed that it maps to the 5’UTR of the SEC24A gene. Introduction (Lee et al. 2004). In mammals, there are multiple forms An important fraction of proteins synthesised in the of COPII coat structures specialised in different types endoplasmic reticulum need to enter the secretory of cargos and, in this context, four paralogous SEC24 pathway to reach other intracellular organelles, the genes (SEC24A to SEC24D) have been reported so far cell surface or the extracellular space (Bonifacino and (Zanetti et al. 2011). In mice, the inactivation of the Glick 2004). The transport of these proteins to the SEC24A gene is not lethal but it causes a reduction in Golgi complex, where they can undergo post-transla- plasma cholesterol because the secretion of proprotein tional modifications, is mediated by vesicles coated convertase subtilisin/kexin type 9 (PCSK9), which is a with coat protein complex II (COPII), which are key mediator in the lysosomal degradation of low- assembled through the recruitment of SEC24 and density lipoprotein (LDL) receptors (Lagace 2014), is other proteins (Lee et al. 2004). Indeed, the SEC24 shut off, thus leading to an hepatic upregulation of subunit of the COPII complex contributes to cargo LDL receptors and, in consequence, to an increased selection by recognising specific molecular signals cholesterol clearance (Chen et al. 2013). CONTACT Dr. Marcel Amills marcel.amills@uab.cat Centre for Research in Agricultural Genomics (CRAG), CSIC-IRTA-UAB-UB, Universitat Autonoma de Barcelona, Bellaterra, Spain; Departament de Ciencia Animal i dels Aliments, Universitat Autonoma de Barcelona, Bellaterra, Barcelona, Spain Supplemental data for this article can be accessed here. ß 2021 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ITALIAN JOURNAL OF ANIMAL SCIENCE 1445 In a previous experiment (Marmol-Sanchez et al. calling was performed using the HaplotypeCaller tool 2020, 2021), we sequenced the whole genomes of the according to GATK best practices recommendations. five Duroc boars that sired a purebred population of Finally, SNP variants were retained with SelectVariants 350 Duroc barrows (LIPGEN population), as reported package and quality filtered using the FilterVariants by Gallardo et al. (2008, 2009). The analysis of these tool from GATK v.3.8, according to the following five whole-genome sequences (WGS) revealed the seg- parameters: QD < 2.0, FS > 60.0, MQ < 40.0, regation of a putative nonsense mutation located in MQRankSum < 12.5, ReadPosRankSum < 8.0, SOR the SEC24 Homolog A, COPII Coat Complex > 3.0. Component (SEC24A) gene (our unpublished data). This mutation was recorded at Ensembl repositories Retrieval of the whole-genome sequences from (https://www.ensembl.org/index.html) with the 120 pigs available at public repositories rs336401826 (c.44 G>A, SEC24A) identifier. The goal of the current work was to investigate the segregation of A total of 120 whole-genome sequences from wild this putative nonsense SNP in the LIPGEN population, boars and domestic pigs (Sus scrofa) were retrieved as well as to determine its association with lipid- from the NCBI Sequence Read Archive (SRA) database related traits in order to verify whether SEC24A inacti- (https://www.ncbi.nlm.nih.gov/sra). The 120 selected vation in pigs has the same consequences on fat genome sequences (Supplementary Table 1) were clas- metabolism than those previously reported in mice. sified into Asian domestic pigs (ADM, N ¼ 40), Asian wild boars (AWB, N ¼ 20), European domestic pigs Materials and methods (EDM, N ¼ 40) and European wild boars (EWB, N ¼ 20). Further details about breeds and sample origins can Sequencing the genomes of five Duroc boars be found in Marmol-Sanchez et al. (2020, 2021). All In brief, genomic DNA was purified from blood sam- SRA files from selected porcine individuals were down- ples belonging to the five boars that sired a Duroc loaded from SRA public repositories and converted population of 350 offspring (LIPGEN population), as into fastq format by using the fastq-dump 2.8.2 tool reported by Gallardo et al. (2008, 2009). Subsequently, available in the SRA-toolkit package (ncbi.github.io/ these genomic DNA samples were sequenced at the sra-tools/). Subsequent processing, SNP calling and fil- Centre Nacional d’Analisi Genomica (CNAG, Barcelona, tering were performed analogously to what has been Spain). Paired-end multiplex libraries were prepared reported in the previous section for the 5 whole-gen- according to the instructions of the manufacturer with ome sequenced Duroc sires. the KAPA PE Library Preparation kit (Kapa Biosystems, Wilmington, MA) and processed to generate 100 bp Isolation of genomic DNA and genotyping paired-end reads on a HiSeq2000 Illumina instrument. Base calling and quality control analyses were per- The manipulation of Duroc pigs followed Spanish formed with the Illumina RTA sequence analysis pipe- national guidelines and it was approved by the Ethical line. Quality-checked filtered reads were then Committee of Institut de Recerca i Tecnologia processed and any remaining sequence adapters were Agroalimentaries (IRTA). Genomic DNA was purified trimmed by making use of the Trimmomatic software from blood samples collected from the offspring of (v.0.36) with default parameters (Bolger et al. 2014). the five sequenced Duroc boars by using a standard Trimmed paired-end sequences were aligned against phenol-chloroform extraction protocol (Gallardo et al. the pig Sscrofa11.1 reference genome (Warr et al. 2008, 2009). All Duroc pigs from the LIPGEN popula- 2020) with the BWA-MEM algorithm (Li 2013) and tion were typed with the Porcine SNP60 BeadChip default settings. Sequence alignment map (SAM) for- (Illumina) in a previous experiment (Gonzalez-Prendes matted files were then sorted and transformed into et al. 2017). Moreover, a dedicated TaqMan assay was binary (BAM) formatted files and PCR duplicates were used by the Veterinary Molecular Genetics Service subsequently identified with the MarkDuplicates pack- from the Universitat Auto noma de Barcelona (http:// age from Picard tool (https://broadinstitute.github.io/ svgm.es/es/Home) to genotype the rs336401826 G > A picard/) and removed to perform INDEL realignment SEC24A (N ¼ 320) mutation by employing a with the IndelRealigner package from the Genome QuantStudio 12 K flex Real Time PCR System. The Analysis Toolkit (GATK v.3.8, McKenna et al. 2010). HWChisq module from the HardyWeinberg R package Base quality score recalibration (BQSR) was imple- (https://CRAN.R-project.org/package=HardyWeinberg) mented with BaseRecalibrator from GATK v.3.8. Variant was used to compute a Chi square test of 1446 E. MÁRMOL-SÁNCHEZ ET AL. Table 1. Absolute genotypic frequencies of the rs336401826 (2) backfat thickness (measured between 3rd and 4th (c.44G>A) mutation of the porcine SEC24A gene in a Duroc ribs) and live weight at slaughterhouse for serum lip- pig population (N ¼ 350) and their departure from the Hardy- ids measured at 45 and 190 days of age, and (3) back- Weinberg equilibrium (HWE). fat thickness (measured between 3rd and 4th ribs) for c.44G > A (rs336401826, SEC24A) IMF content; W is the incidence matrix relating pheno- Genotype Observed Expected HWE p-Value types with the corresponding effects; x is the vector of GG 152 160.3 v2 ¼ 5.05 0.024527 GA 149 132.4 the genotypes corresponding to the set of selected AA 19 27.3 polymorphisms; d is the allele substitution effect of the analysed polymorphism; u is a vector of random independence to infer whether genotype frequencies individual genetic effects with a n-dimensional multi- deviate from the Hardy Weinberg equilibrium variate normal distribution MVNn (0, k s  1 K), where (Table 1). s  1 is the variance of the residual errors, k is the ratio between the two variance components and K is Association analyses with lipid traits a known relatedness matrix derived from the SNPs; and e is the vector of residual errors. A false discovery A summary of the lipid-related phenotypes considered rate (FDR) approach (Benjamini and Hochberg 1995) in our experiment is provided in Supplementary Table was used for multiple testing correction. 2. The measurement of lipid traits has been previously described in detail (Gallardo et al. 2008, 2009; Quintanilla et al. 2011). Serum lipid traits were meas- ured after weaning and before starting the fattening Chromosome-wide association analyses period (45 days of age), as well as prior to slaughter Genotyping data for the SEC24A rs336401826 G>A (190 days of age), according to specific experimental nonsense mutation were merged with the genotypes protocols for determining total serum cholesterol of 3684 SNPs mapping to chromosome 2 (SSC2) and (Richmond 1992), low density lipoprotein (LDL, generated with the Porcine SNP60 BeadChip (Illumina, Friedewald et al. 1972), high density lipoprotein (HDL, San Diego, CA) as reported by Gonzalez-Prendes et al. Rafai and Warnick 1994) and triglyceride (Fossati and (2017). The GEMMA software (Zhou and Stephens Prencipe 1982) concentrations. Intramuscular fat (IMF) 2012) was used to perform this association analysis by was measured by Near Infra-red Transmittance (NIT, considering the methods reported previously and Infratec 1625, Tecator Hoganas, Sweden), while muscle chromosome-wide significance was assessed at cholesterol content was assessed following Cayuela et the nominal level (p-value <0.05) as well as after al. (2003). A chromatography analysis of methyl esters correction for multiple testing using the (Mach et al. 2006) was used to determine the fatty Bonferroni approach. acid composition in the C:12 to C:22 range and they were subsequently expressed as a percentage of the IMF for the gluteus medius (GM) and longissimus dorsi (LD) skeletal muscles. Association analyses were per- Multiple sequence alignment of SEC24A formed by applying a univariate linear mixed model transcripts from different species framework with the Genome-Wide Efficient Mixed- We used the Clustal Omega tool of the EMBL-EBI Model Association (GEMMA) software (Zhou and sequence analysis toolkit (Madeira et al. 2019) to gen- Stephens 2012) and the following statistical model: erate a multiple sequence alignment of the annotated y ¼ Wa þ xd þ u þ e coding sequence (CDS) of the 1st exon of the SEC24A where y is the vector of phenotypic observations for transcripts retrieved from eight available porcine every individual; a is a vector containing the intercept assemblies (Sscrofa11.1, USMARC, Bamei, Meishan, plus the included fixed effects, i.e. batch effect with Rongchan, Pietrain, Landrace and Large White), as well four categories (all traits), farm origin effect with three as from the human assembly (GCRh38.p13). Moreover, categories (all traits) and data of extraction with two the MultAlin software (Corpet 1988) was employed to categories within batch (serum lipids traits). The a vec- generate a multiple alignment of the porcine SEC24A tor also contains the regression coefficients of the fol- protein with other homologous SEC24A proteins anno- lowing covariates: (1) IMF content in the LD or GM tated in human (GCRh38.p13), cattle (ARS-UCD1.2), skeletal muscles for LD and GM fatty acid composition, mouse (GRCm38.p6) and dog (CanFam3.1) assemblies. ITALIAN JOURNAL OF ANIMAL SCIENCE 1447 Figure 1. Allelic discrimination plot obtained for the rs336401826 SNP using a TaqMan assay. The x- and y-axes indicate the fluor- escence values of the VIC and FAM dyes, respectively, while the dots are individual sample points. The following pig SEC24A geno- types are depicted in this plot: AA (upper left side, N ¼ 19), GA (middle, N ¼ 149) and GG (down right side, N ¼ 152). Samples that were not successfully genotyped are represented by dots in the lower left side. It can be seen that the three SEC24A geno- types are clearly discernible. Table 2. Nominally significant associations (p-value < 0.05) Results and discussion between fatty acid composition traits and c.44G>A A significant departure from the Hardy Weinberg equi- (rs336401826, SEC24A) genotypes (GG, N ¼ 152; GA, N ¼ 149 librium was found (v2 ¼ 5.05, p-value ¼ 0.024527, and AA, N ¼ 19)a. Phenotypeb dc sed p-Value q-valuee Table 1) for the rs336401826 G>A SEC24A variant, GM (C20:2) 0.0355 0.0129 6.521E-03 2.191E-01 mainly because there was a slight deficit of the two GM (C16:1) 0.1344 0.0504 8.267E-03 2.191E-01 homozygous genotypes. Genotyping errors are one of GM (C14:0) 0.0476 0.0228 3.637E-02 6.006E-01 GM (C16:0) 0.2903 0.1445 4.533E-02 6.006E-01 the most common causes of observing Hardy- a Non-significant association analyses (p-value > 0.05) between lipid traits Weinberg departures, but that does not seem to be and c.44G>A (rs336401826, SEC24A) genotypes are shown in the case because all three AA, GA and GG genotypes Supplementary Table 3. bTrait acronyms are defined in Supplementary Table 2; cd: estimated allele substitution effect; dse: standard error of the are clearly discernible (Figure 1). Another potential substitution effect; eq-value: q-value calculated with the false discovery reason for the Hardy-Weinberg departure observed by rate (FDR) approach (Benjamini and Hochberg 1995). us would be that the frequencies of the SNP might be altered by selection or stochastic factors directly acting concentration due to a specific block in the secretion on it or on a linked nearby mutation. Nevertheless, of PCSK9 (Chen et al. 2013). The rs336401826 the statistical significance of the departure was so (c.44G>A, Trp15) variant is located at codon 15 of a weak that no biological inferences should be made. gene encoding a protein of 1133 amino acid residues, More importantly, no missing genotypes were so the premature stop codon should provoke the full detected, thus evidencing that this putative nonsense inactivation of the protein and marked changes in mutation is not lethal. As previously said, this result blood lipid levels. was anticipated because SEC24A inactivation is Contrary to our expectations, statistical analyses did expected to just reduce plasma cholesterol concentra- not show any significant association between SEC24A tion via up-regulating LDL receptor membrane rs336401826 genotypes (GG, N ¼ 152; GA, N ¼ 149 and 1448 E. MÁRMOL-SÁNCHEZ ET AL. AA, N ¼ 19) and serum cholesterol, LDL, HDL or trigly- average GM C14:0, C16:0, C16:1 and C20:2 contents in ceride concentrations at 45 and 190 days pigs with different rs336401826 genotypes are dis- (Supplementary Table 3). In contrast, significant associ- played in Table 3. ations were found between the nonsense rs336401826 A total of 2951 SNPs mapping to the SSC2 porcine mutation and C14:0, C16:0, C16:1 and C20:2 fatty acid chromosome were successfully analysed in chromo- contents in the GM skeletal muscle (Table 2), although some-wide association analyses, which revealed that only at the nominal level (p-value < 0.05). Data about the rs336401826 SEC24A polymorphism was among the SNPs displaying the most significant associations Table 3. Average C20:2, C16:1, C14:0 and C16:0 fatty acids with GM C16:1 (nominal p-value ¼ 1.577E-03) and GM contents in the gluteus medius (GM) skeletal muscle of pigs C20:2 (nominal p-value ¼ 4.841E-03) phenotypes with GG and AA genotypes for the rs336401826 (c.44G>A) (Figure 2). It should be noted that C14:0, C16:1 and mutation in the porcine SEC24A gene. C20:2 fatty acids are found in very low amounts in the Genotype intramuscular fat of the GM muscle, so it is challeng- GG AA ing to measure them with accuracy. On account of a b c b Phenotype Average % CI Average % CIc this, the results obtained in the current association GM C20:2 0.50 0.49  0.52 0.60 0.55  0.65 GM C16:1 2.90 2.82  2.99 2.59 2.43  2.74 analysis should be interpreted with caution until repli- GM C14:0 1.41 1.37  1.45 1.31 1.20  1.41 cated in other porcine populations. GM C16:0 23.49 23.23  23.74 22.62 22.04  23.19 When we assessed the segregation patterns of the a Trait acronyms are defined in Supplementary Table 2; bAverage fatty SEC24A mutation in the 120 WGS from European and acid content percentage in the gluteus medius (GM) skeletal muscle; cCI: confidence interval for the averaged fatty acid content (a ¼ 0.05). Asian pigs and wild boars, the frequency of the A Figure 2. Manhattan plot depicting the association of the SEC24A rs336401826 variant plus 2950 additional SNPs mapping to SSC2 with C20:2, C16:1, C14:0 and C16:0 fatty acids contents in the gluteus medius (GM) muscle. The blue line represents the chromosome-wide nominal p-value of significance (a ¼ 0.05), while the green line represents the p-value after correction for mul- tiple testing applying the Bonferroni approach. The chromosomal position of the rs336401826 G>A SNP is highlighted in red. ITALIAN JOURNAL OF ANIMAL SCIENCE 1449 Figure 3. Multiple sequence alignment showing the sequence of the protein encoded by the SEC24A gene in different species (human, mouse, cattle, dog and pig). The portion of the pig SEC24A protein that is not homologous to the SEC24A protein sequences from the remaining species is clearly visible, evidencing that the translation start codon of the porcine SEC24A protein sequence is incorrectly annotated in the Sscrofa11.1 assembly. allele was higher than that of the G allele, despite its Yorkshire and Large White pigs carried the G-allele, potential deleterious effect on gene function, and while all the remaining EDM pigs harboured AA this was observed in European and Asian domestic genotypes (Supplementary Table 1). Despite the breeds as well as in European and Asian wild boars high frequency of the A-allele in most porcine (Supplementary Table 1). In the EDM group, the G breeds, the G-allele is the reference allele in the allele was predominantly found in Duroc pigs (N ¼ 7; Sscrofa11.1 porcine assembly (Warr et al. 2020). The 2 GG, 4 GA and 1 AA). Aside the Duroc breed, only most likely reason of this outcome is that this one GA Yucatan pig and three GG Yucatan, assembly was built using PacBio RS II deep long- 1450 E. MÁRMOL-SÁNCHEZ ET AL. read sequencing from one single female pig belong- point, it is difficult to assess how many putative loss- ing to the Duroc breed (Warr et al. 2020), in which of-function mutations are incorrectly annotated in the the G variant is frequent. current pig genome assembly, but caution is advised. In the light of these results, that were not consist- Careful inspection and verification of the annotation ent with a deleterious effect of the nonsense of loss-of-function mutations is recommended before rs336401826 G>A SEC24A mutation, we decided to fur- undertaking the analysis of their effects at the experi- ther investigate the genomic context of this SNP. By mental level. doing so, we determined that the common predicted ATG translation start codon is located 141 nucleotides Acknowledgments downstream from the predicted start codon of SEC24A exon 1 in the Sscrofa11.1 assembly (Supplementary The authors are indebted to Seleccio  Batalle S.A. for provid- Figure 1). Interestingly, in the Rongchang, Large White ing the animal material used in this study. We gratefully acknowledge J. Reixach and J. Soler (IRTA) for their collabor- and Landrace assemblies the SEC24A exon 1 coding ation in the experimental protocols. sequence was correctly annotated, whereas other available porcine assemblies like USMARC, Meishan, Bamei and Pietrain contained a wrong annotation, Disclosure statement analogously to what was found for the Sscrofa11.1 No potential conflict of interest was reported by assembly (Supplementary Figure 1). The alignment of the author(s). the mature SEC24A protein sequences from human, mouse, pigs, cattle and dogs also evidenced that the Funding porcine sequence contains an initial segment that is not present in the remaining species (Figure 3), a This research was partially funded with grants AGL2013- result that once again suggests that the starting 48742-C2-1-R and AGL2013-48742-C2-2-R awarded by the Spanish Ministry of Economy and Competitiveness and grant codon of the porcine SEC24A sequence is incorrectly 2014 SGR 1528 from the Agency for Management of annotated. Altogether, these findings would imply University and Research Grants of the Generalitat de that the rs336401826 G>A SEC24A polymorphism is Catalunya. We also acknowledge the support of the Spanish located within the 5’UTR of the SEC24A transcripts, Ministry of Economy and Competitivity for the Center of rather than at codon 15 of the coding region. Excellence Severo Ochoa 2020-2023 [CEX2019-000902-S] Although the current Sscrofa11.1 assembly (Warr et grant awarded to the Centre for Research in Agricultural Genomics (CRAG, Bellaterra, Spain). Emilio Marmol-Sanchez al. 2020) represents a considerable improvement with was funded with a FPU Ph.D. grant from the Spanish regard to the previous one (Sscrofa10.2, Groenen et al. Ministry of Education [FPU15/01733]. Marıa Gracia Luigi- 2012), there are still annotation problems that need to Sierra was funded with a FPI Ph.D. grant from the Spanish be corrected. The annotation of nonsense mutations Ministry of Economy and Competitivity [BES-2017-079709]. can be particularly error-prone when gene and/or transcript structures are not well known or when the existence of nearby compensatory polymorphisms is Data availability statement ignored. Another important source of error can be pseudogenes, which are expected to contain a much The 3,684 SNPs included in this study were obtained from published porcine SNP60 BeadChip data reported by higher rate of deleterious mutations than that Gonzalez-Prendes et al. (2017). Lipid-related traits and geno- observed in functional genes. As a whole, these find- type data can be accessed at the Figshare public repository ings reinforce the need of manually curating the anno- through the following link: https://figshare.com/projects/ tation of loss-of-function mutations in pigs in order to SEC24A/92765. Whole-genome sequencing data from the 5 be able to predict their functional consequences. 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