Genome-wide association study reveals a QTL and strong candidate genes for umbilical hernia in pigs on SSC14

Genome-wide association study reveals a QTL and strong candidate genes for umbilical hernia in... Background: Umbilical hernia is one of the most prevalent congenital defect in pigs, causing economic losses and substantial animal welfare problems. Identification and implementation of genomic regions controlling umbilical hernia in breeding is of great interest to reduce incidences of hernia in commercial pig production. The aim of this study was to identify such regions and possibly identify causative variation affecting umbilical hernia in pigs. A case/control material consisting of 739 Norwegian Landrace pigs was collected and applied in a GWAS study with a genome-wide distributed panel of 60 K SNPs. Additionally candidate genes were sequenced to detect additional polymorphisms that were used for single SNP and haplotype association analyses in 453 of the pigs. Results: The GWAS in this report detected a highly significant region affecting umbilical hernia around 50 Mb on SSC14 (P < 0.0001) explaining up to 8.6% of the phenotypic variance of the trait. The region is rather broad and includes 62 significant SNPs in high linkage disequilibrium with each other. Targeted sequencing of candidate genes within the region revealed polymorphisms within the Leukemia inhibitory factor (LIF) and Oncostatin M (OSM) that were significantly associated with umbilical hernia (P < 0.001). Conclusions: A highly significant QTL for umbilical hernia in Norwegian Landrace pigs was detected around 50 Mb on SSC14. Resequencing of candidate genes within the region revealed SNPs within LIF and OSM highly associated with the trait. However, because of extended LD within the region, studies in other populations and functional studies are needed to determine whether these variants are causal or not. Still without this knowledge, SNPs within the region can be used as genetic markers to reduce incidences of umbilical hernia in Norwegian Landrace pigs. Keywords: Genome-wide association study, GWAS, Umbilical, Hernia, Pigs, LIF, OSM Background a hernia is the potential entrapment of intestines Hernias are of the most common congenital defects in through this opening. Physical injury, nutrition, excessive pigs which often leads to poor animal welfare and severe pressure, muscular weakness and heredity have been of- economic losses for pig producers. The most common fered as causes of hernia [1]. Umbilical hernias often ap- types of hernias in pigs are umbilical and inguinal/scro- pears in pigs at 9 to 14 weeks of age, with incidences tal hernia. Umbilical hernia is diagnosed by the protru- reported to range from 0.4 to 1.2% [1, 2]. The prevalence sion of abdominal contents beneath the skin at the navel of scrotal/inguinal hernias is shown to be approximately (umbilicus). It is generally accepted that weakened sup- in the same range, between 0.5 and 1.5%, and with an portive muscles around the umbilical stump or navel estimated heritability of around 0.3 in different breeds area of the animal causes the umbilical opening not to [3, 4]. Several studies have explored the genomics of close properly and intestines protrude through the intes- scrotal/inguinal hernia [4–7], but very few have devoted tinal wall to form the “ball-like” structure. The threat of efforts to decipher the genetic architecture of umbilical hernia. This is mainly because umbilical hernia is relatively rare and sometimes complicated to diagnose properly. * Correspondence: eli.grindflek@norsvin.no It is generally accepted that genetics influence con- Norsvin SA, Storhamargata 44, 2317 Hamar, Norway genital umbilical hernias, but mode of inheritance and Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Grindflek et al. BMC Genomics (2018) 19:412 Page 2 of 9 genetic architecture are more or less undiscovered, and (approximately smaller or larger than baseball size, re- h estimates of umbilical hernia are reported to be very spectively). Based on this, 165 hernias were classified as low in pigs (0.06–0.08) [1, 8]. The frequencies and heri- “small” and 267 were classified as “large”. Additionally, tabilities of umbilical hernia also differs between species. the time of first observation was recorded, together with In cattle, for example, both frequencies (0.1–15%) and records on whether the piglet previously had a history of heritability (h = 0.4) are reported to be much higher [9, navel inflammation or not. The size-score of the umbil- 10] than in pigs. A few studies have tried to decipher the ical hernias was considered not to be valuable as a co- genetic structure of the umbilical hernia. Ding et al. [7] variate in the analysis. The first observation of hernias observed significant linkage between markers and umbil- ranged between 2 and 24 weeks, with the average of ical hernia in pigs on 12 different chromosomes, while 14 weeks. Only 2 piglets were previously observed to have Ron et al. [11] detected a locus on chromosome 8 linked an inflammation, and this was therefore not accounted for with umbilical hernia in cattle. Recently, Long et al. [12] in the statistical analysis. All animals were cared for ac- published a study suggesting a copy number variation cording to laws and internationally recognized guidelines (CNV) on SSC14 to be involved in development of um- and regulations controlling experiments with live animals bilical hernia in pig. Also in humans some studies have in Norway (The Animal Protection Act of December 20th, been done on structural variations like deletions and 1974 (revision: FOR-2010-08-06-117), and the Animal duplications, suggesting that such variations also could Protection Ordinance Concerning Experiments with Ani- play a role in occurrence of umbilical hernia [13–15]. mals of January 15th, 1996), according to the rules given Additionally, two studies in other species suggest that by Norwegian Animal Research Authority. umbilical hernia is associated with the function of KIP2 cyclin-dependent kinase inhibitory protein p57 ,a DNA extraction regulator of cell proliferation [16, 17]. DNA was extracted from porcine blood, leukocytes or Studies on genetic control of umbilical hernia so far is semen using the MagAttract DNA Blood Midi M48 not clear, although indicating that the disorder is com- protocol on the Bio-Robot M48 (Qiagen, Hilden, plex and affected by multiple causative genes and vari- Germany). The concentration and quality of samples ants. Therefore, the aim of this study was to collect a were measured on a Nanodrop, ND-1000 spectropho- proper case/control material and identify genomic regions tometer (NanoDrop Technologies, DE, USA), and on a affecting the frequency of umbilical hernia in pigs, using 1420 Victor plate reader (Turku, Finland) using Pico- GWAS on a high resolution SNP panel followed up by Green fluorescence (Molecular Probes, OR, USA), and candidate gene approaches. thereafter normalized to 50 ng/ul in 96-well plates. Methods Genotyping, sequencing and quality control Animals and phenotypes Genome wide association study (GWAS) The overall frequency of umbilical hernia for Landrace The genotyping was performed at CIGENE (www.cigen- in Norwegian nucleus herds was found to be only 0.55%, e.no), Norwegian University of Life Sciences, Norway. and the heritability (h ) was also estimated to be very Genotyping for the GWAS was performed using the low (0.065). Due to the low incidence of umbilical hernia iScan (Illumina, San Diego, CA, USA) platform with the in Norwegian Landrace, samples had to be collected PorcineSNP60 array according to manufacturer’s in- over time-periods of altogether 4 years. To generate suf- structions. Image intensity, data processing, clustering ficient number of case/control samples we collected data and genotype calling were performed using the genotyp- and samples from all the nucleus herds in Norsvin ing module in the Genome Studio software (Illumina, (Hamar, Norway). Blood samples were collected from a San Diego, CA, USA). Altogether, including the hernia total of 369 purebred Norwegian Landrace piglets dis- pigs, their full-sibs and their parents, 739 Norwegian playing umbilical hernia, along with blood from 202 Landrace were genotyped for 64,232 SNPs. The SNP phenotypically unaffected fullsibs within all affected lit- markers passing the quality control had call rate above ters. The affected pigs were distributed on 259 litters, 0.9, minor allele frequency (MAF) above 0.01, propor- with each litter containing 1 to 6 affected piglets from tion of genotyped above 0.25, and proportion of geno- 110 sires. Altogether, 168 parents were genotyped. Sam- type errors below 0.025. The average call rate across ples were obtained from 35 pig breeding farms, where samples was 0.997, and no samples were excluded from several sires were used on different farms. The farmers analysis due to unacceptable call rates from the genotyp- reported the cases and thereafter the diagnostic proce- ing. Animals with pedigree errors (altogether 17 animals) dures were performed by a Norsvin-breeding technician. were removed from the study. After quality control, 554 The umbilical hernias were subjectively classified into animals were available for the case/control, in addition two categories by approximate size “small” or “large” to 168 parents. The most frequent failing category is Grindflek et al. BMC Genomics (2018) 19:412 Page 3 of 9 non-informative markers (12,259 SNPs). Beagle v. 3.3.1 highest MAF were selected, and additional five SNPs lo- was used to impute sporadically missing genotypes in cated in other genes were removed due to low MAF the final genotype file [18]. In total 49,049 high quality (MAF < 0.01). Therefore, 31 SNPs were used for the final SNP markers, were positioned in the porcine genome as- statistical analyses (Table 2). Candidate genes with more sembly Build 10.2 (Sanger Institute). than one SNP were included for haplotype analysis. Haplotypes and LD blocks were constructed using the Candidate gene study software Haploview v.4.2 [28]. Candidate genes were chosen based on GWAS results in Structural models of the OSM protein were generated this study and putative role in development of umbilical by using the SWISS-MODEL workspace [29], using hernia from literature [6, 16, 17, 19–25]. In total, eight amino acid sequence input files for the OSM translated positional and/or biological candidate genes in seven dif- from DNA sequence with the two different alleles of the ferent regions were selected for sequencing: Leukemia missense single point mutation. QMEAN [30] were used inhibitory factor (LIF), Oncostatin-M (OSM), Macro- to evaluate the quality of the model in 6 different terms: phage migration inhibitory factor (MIF), cyclin-depen- (1) C_beta interaction energy, (2) all-atom pairwise en- dent kinase inhibitory protein p57KPI2 (CDKN1C), ergy, (3) solvation energy, (4) torsion angle energy, (5) Pyrroline-5-carboxylate reductase 1 (PYCR1), Versican solvent accessibility agreement and (6) total (VCAN), Matrix metallopeptidase-13 (MMP13), and QMEAN-score. Furthermore, GROMOS empirical force Vimentin (VIM). Names, positions from www.ensem- field energy [31] was estimated for each amino acid of bl.org and references are listed in Table 1. the protein chain. SNP discovery was performed by PCR resequencing of genomic DNA and cDNA from Norwegian Landrace Statistical analyses with umbilical hernia and healthy siblings. Primers were Genome wide association study (GWAS) designed using Primer3 [26]. The programmes Phrap, The association analyses were carried out for the 49,049 Phred and PolyPhred (v.4.06) were used to identify puta- SNPs mapped on the 18 porcine autosomes using pig tive SNPs from the PCR resequencing chromatograms, genome assembly Build10.2. The minor allele frequen- and the Consed programme was used to visually confirm cies of the selected SNPs were uniformly distributed be- the putative SNPs [27]. For the SNP detection, eight her- tween 0.01 and 0.5. Genome-wide association analysis nia pigs and eight healthy pigs were used for sequencing. was performed with the GenABEL package (version 1.6–5) No SNPs were detected in the positional candidates MIF in R [32] using the structured association approach and CDKN1C, but 50 SNPs were obtained in six other (“qtscore”) for binary traits [33], which is in principle a genes. Primer assays for use in the Sequenom MassAR- Cochran-Armitage test. Umbilical hernia was the binomial RAY system were designed using MassARRAY Assay trait and gender was added as a covariate, although signifi- Design Software, with multiplexes between 12 and 19. cant differences between gender was not observed (p =0.7). Genotyping was done according to the manufacturer’s Gender-factor have not either previously been reported in instructions in the IPLEX protocol. The MassARRAY pigs, but in cattle, however, umbilical hernia have been Typer software was used for the automated genotype foundtobemorefrequentlyoccurringinfemale compared calling (Sequenom, San Diego, USA). Altogether, 46 to males [10]. SNPs in six different candidate genes were successfully The p-values corrected for genomic control (GC) of a genotyped for 463 animals; 201 pigs with umbilical her- 1-df test were accepted to represent proof of genome-wide nia, 135 healthy siblings and 127 parents. Due to high association at p < 0.001 (−log10(p) = 3). Collection of sam- SNP density in VCAN (16 SNPs) the six SNPs with ples was done during intensive periods of altogether four Table 1 Eight positional and/or biological candidate genes were used for resequencing Gene name Position References Leukemia inhibitory factor (LIF) 14:50263722–50,266,807 [19] Oncostatin M (OSM) 14:50281701–50,286,188 [20, 21] Macrophage migration inhibitory factor (MIF) 14:53282552–53,283,339 [22] Cyclin-dependent kinase inhibitory protein p57KPI2 (CDKN1C) Unplaced [16, 17] Pyrroline-5-carboxylate reductase 1 (PYCR1) Unplaced [6] Versican (VCAN) 2:93783042–93,895,497 [23] Matrix metallopeptidase 13 (MMP13) 9:37493636–37,504,552 [24] Vimentin (VIM) 10:48227005–48,235,670 [25] Positions are in chromosome:basepairs Grindflek et al. BMC Genomics (2018) 19:412 Page 4 of 9 Table 2 SNPs within candidate genes used for association analysis a b c d Gene Name SNP name Location Alleles MAF P %var Versican VCAN_1 Exon 8 A/G 0.45 n.s. 0.2 Versican VCAN_2 Exon 8 G/A 0.45 n.s. 0.4 Versican VCAN_3 Exon 8 T/G 0.45 n.s. 0.4 Versican VCAN_4 Exon 8 C/T 0.45 n.s. 0.2 Versican VCAN_5 Exon 8 C/A 0.38 n.s. 0.4 Versican VCAN_6 Exon 8 C/T 0.38 n.s. 0.4 Leukemia inhibitory factor LIF_1 Intron 1 A/G 0.47 0.001 8.6 Matrix metallopeptidase 13 MMP13_1 Intron 7 G/A 0.33 n.s. 0.08 Matrix metallopeptidase 13 MMP13_2 Intron 7 T/G 0.33 n.s. 0.05 Matrix metallopeptidase 13 MMP13_3 Exon 8 A/G 0.33 n.s. 0.1 Matrix metallopeptidase 13 MMP13_4 Intron 8 C/T 0.33 n.s. 0.03 Matrix metallopeptidase 13 MMP13_5 Intron 8 T/C 0.32 n.s. 0.01 Matrix metallopeptidase 13 MMP13_6 Intron 8 G/T 0.33 n.s. 0.3 Oncostatin M OSM_1 Exon 3 C/T 0.47 0.001 7.7 Oncostatin M OSM_2 Exon 3 T/C 0.47 0.001 8.6 Oncostatin M OSM_3 Exon 3 T/C 0.47 0.001 8.6 Oncostatin M OSM_4 Exon 3 G/T 0.47 0.0008 8.6 Oncostatin M OSM_5 Exon 3 A/G 0.47 0.001 8.6 Oncostatin M OSM_6 Intron 3 C/T 0.47 0.001 7.7 Oncostatin M OSM_7 Intron 3 T/C 0.47 0.001 7.7 Oncostatin M OSM_8 Intron 3 A/G 0.47 0.001 7.7 Pyrroline-5-carboxylate reductase 1 PYCR1_1 5’ UTR A/T 0.11 n.s. 2.5 Pyrroline-5-carboxylate reductase 1 PYCR1_2 5’ UTR C/T 0.13 n.s. 1.0 Pyrroline-5-carboxylate reductase 1 PYCR1_3 Intron 1 C/T 0.03 n.s. 1.0 Vimentin VIM_1 Intron 5 C/T 0.21 n.s. 0.2 Vimentin VIM_2 Intron 5 A/G 0.36 n.s. 0.3 Vimentin VIM_3 Intron 5 C/T 0.17 n.s. 0.01 Vimentin VIM_4 Intron 5 A/G 0.19 n.s. 0.6 Vimentin VIM_5 Intron 5 C/T 0.15 n.s. 0.00 Vimentin VIM_6 Intron 5 C/A 0.34 n.s. 0.3 Vimentin VIM_7 Intron 5 C/A 0.11 n.s. 0.7 major/minor allele, where risk allele is indicated in bold minor allele frequency (MAF) p-value (p > 0.001 is n.s) the percentage of explained phenotypic variance (%var) years, but it was seven years between the first and final col- Y ¼ μ þ snp=haplotype þ sex þ id þ e lection of samples. Therefore, a population stratification analysis (“strata”) was performed to find whether the popu- where Y is the binomial trait “case” or “control”. The lation during some generations of selection was grouped SNP, coded as 0, 1, or 2 for homozygote allele 1, hetero- into several genetic populations or not. However, only one zygote, homozygote allele 2, respectively, was fitted as a population was defined and correction for stratification fixed effect. In case of the haplotype associations, each was not added in the final analysis. haplotype allele combination was assigned its own level and fitted as a fixed effect. Sex was fitted as a fixed effect Candidate gene study whereas animal id was treated as a random effect, using For the single SNP-marker and haplotype association a pedigree based relationship matrix to account for analysis the ASReml v.2.00 [34] was used to conduct population structure. The F-statistics was calculated for association analysis using the following model: each SNP or haplotype and the significance level was set Grindflek et al. BMC Genomics (2018) 19:412 Page 5 of 9 at a corresponding p-value of 0.001. The genetic vari- Table 3 The most significant 60 k SNPs for umbilical hernia a b c ance explained by a SNP was calculated from the esti- Marker name SSC Position MAF P-value %var mated genotype effects and the observed genotype INRA0043998 14 50,652,198 0.48 7.43e-05 8.1 frequencies. It was expressed as a percentage of the total ASGA0063261 14 50,672,335 0.48 7.43e-05 8.1 phenotypic variance obtained from the model without ASGA0063262 14 50,738,530 0.48 7.43e-05 8.1 the genotype effect. ALGA0077446 14 50,761,608 0.48 7.43e-05 8.1 ASGA0063267 14 50,776,775 0.48 7.43e-05 8.1 Results Genome-wide association study (GWAS) ALGA0077450 14 50,792,395 0.48 7.43e-05 8.1 Assuming a corrected significance level of P < 0.001, 129 MARC0024032 14 50,824,208 0.48 7.43e-05 8.1 SNP markers reached the significant level. Almost all of INRA0044003 14 50,847,851 0.48 7.43e-05 8.1 them, 126 SNPs, were located on SSC14 between posi- MARC0112737 14 50,917,403 0.48 7.43e-05 8.1 tions 47.16 Mb and 58.91 Mb. The other three single ALGA0077457 14 50,938,144 0.48 7.43e-05 8.1 SNP associations were obtained on SSC1 around ASGA0063274 14 50,964,903 0.48 7.43e-05 8.1 102 Mb at the Porcine Build10.2 genome sequence (http://www.ensembl.org), on SSC8 around 88 Mb, H3GA0040130 14 50,992,818 0.50 7.43e-05 7.9 and on SSC17 around 68 Mb, respectively. The Man- ASGA0063217 14 49,288,281 0.49 9.21e-05 8.6 hattan plot of all chromosomes is shown in Fig. 1. a minor allele frequency (MAF) With a more strict significance level of P < 0.0001, 62 the genomic control corrected p-value the percentage of explained phenotypic variance (%var) SNP markers were significant and all of them were located between 48.04–50.99 Mb at SSC14. The most significant SNPs (p <7 ×10–5) are listed in Table 3. Leukemia inhibitory factor (LIF) was the two genes lo- Figure 2 shows the association results for SSC14 with cated within the SSC14 QTL. For OSM, all the eight sin- characterized genes and LD within the QTL region gle SNP markers, and the haplotype combining all the (P < 0.0001). eight SNPs, were significantly associated with umbilical hernia (P < 0.001) and found to explain up to 8.6% of the Candidate genes for umbilical hernia phenotypic variance (Tables 2 and 4). As shown in Fig. 2, Six candidate genes were included in the candidate gene all eight SNPs within OSM are in complete LD with each association study. Four of them were based on biological other. Only two different haplotypes were obtained in knowledge and previously known associations to umbil- the data set, with frequencies of 0.53 and 0.46 for haplo- ical hernia, and two of them were chosen based on the type 1 and 2, respectively. The haplotype frequencies for QTL position obtained on SSC14 and biological func- the affected/un-affected animals were 0.72/0.28 for the tion. The Oncostatin-M precursor gene (OSM) and 11, 0.54/0.46 for the 12 and 0.55/0.45 for the 22, Fig. 1 Manhattan plot of genome-wide association results. The x axis represents the genome in physical order whereas the y axis shows –log10 p-value for all SNPs. The line corresponds to a significance level of P < 0.001 Grindflek et al. BMC Genomics (2018) 19:412 Page 6 of 9 Fig. 2 Association results for SSC14. a A QTL region of 62 SNPs on SSC 14 reached a significance level of P < 0.00015 (solid line) and they were localized between 48.04 and 50.99 Mb. b Map of characterized genes in the QTL region and their orientation, based on information available from Ensembl, where candidate genes are indicated in red. c An LD plot was constructed using Haploview for the QTL region. R was used as a measurement of LD, where a darker color represents a higher R value. The red triangle covers the SNPs in the candidate genes and they are all in complete LD respectively. Constructing haplotypes within a block be- had umbilical hernia, for the heterozygote 2 animals 53% tween 48 and 51 Mb, covering both OSM and LIF (see had hernia, and for the homozygote 12 51% had hernia, Fig. 2), we got two major haplotypes (0.54 and 0.33) and which may suggest a dominant effect of the haplotype, with remaining haplotypes having a frequency of 0.07 or with the haplotype 1 as the risk allele. The OSM_1 SNP less. For the homozygote haplotype 1, 80% of the animals located in exon 3 (Table 2) is non-synonymous causing an Table 4 Results from the haplotype analyses a b Gene name #haplotypes P-value % Var Versican (VCAN) 7 0.03 0.1 Matrix metallopeptidase 13 (MMP13) 5 0.04 0.02 Oncostatin M (OSM) 2 0.0012 6.0 Pyrroline-5-carboxylate reductase 1 (PYCR1) 6 0.03 0.07 Vimentin (VIM) 11 0.13 0.1 haplotypes were constructed within genes based on SNPs in Table 2 and are presented with the number of haplotypes with a frequency > 2% the percentage of explained phenotypic variance Grindflek et al. BMC Genomics (2018) 19:412 Page 7 of 9 amino acid shift from Serine to Glycine. This seems to the extracellular matrix [37]. Diaphragmatic, inguinal, cause only minor changes in the protein structure ob- and umbilical hernia are all associated with connective tained by SWISS-MODEL [29]. However, the GROMOS tissue weakness, and previously extracellular matrix pro- empirical force field energy was shown to be positive (un- teins such as collagens, fibronectin, elastin and matrix favorable environment) with Serine and slightly negative metalloproteinases have been suggested to be involved (favorable environment) when changed to Glycine (results [38, 39]. Moreover, a candidate gene study for scrotal not shown). Only one polymorphism was obtained by se- hernia in pigs obtained a highly significant association quencing for the other positional candidate gene (LIF)on between PYCR1 and hernia [6]. Several matrix metallo- SCC14. This marker was also significantly associated with proteinase genes are suggested to be associated with umbilical hernia (p < 0.001) and found to be in perfect LD membrane weakening and rupture. One of them, MMP13 with OSM (Fig. 2). None of the polymorphisms detected on SSC9, is shown to obtain an important role in wound in the four candidate genes outside the QTL-region on healing by coordinating cellular activities important in the SSC14 (VCAN, MMP13, PYCR1 and VIM) obtained sig- growth and maturation of granulation tissue, including nificant associations with umbilical hernia (Table 2). myofibroblast function, inflammation, angiogenesis, and proteolysis [24]. MMP13 was for example shown to affect Discussion the structural integrity and mechanical stability of the In this study, a genome wide association analysis was connective tissue in both indirect and direct scrotal her- conducted in the Norwegian Landrace population, to- nias [38]. The VCAN is shown to display high adhesive gether with studies of positional and biological candidate ability to endothelial cells and facilitated tube-like struc- genes for umbilical hernia. ture formation, and is previously suggested as a candidate The most convincing result from the GWAS was to be involved in development of umbilical hernia [23]. In around 50 Mb on SSC14. Three candidate genes in this this study the VCAN gene, located on SSC2, was chosen region were investigated in more detail; OSM, LIF and as a biological candidate gene. Finally, vimentin (VIM), MIF (Macrophage migration inhibitory factor). OSM is lo- which is located on SSC10, was added as a biological can- cated at SSC14 base pair position 50,281,701–50,286,188, didate gene due to its function of being a marker of imma- LIF is located at SSC14 base pair position 50,263,722– ture smooth muscle cells and being involved in collagen 50,266,807 and MIF at position 53,282,553–53,283,313. structure [25], as well as having suggested involvement in The three genes were sequenced at the genomic level in inflammatory/immune response [40]. eight hernia pigs and eight healthy pigs, but SNPs were Since there are variability in level of expression inci- only detected in the OSM and LIF genes. OSM and LIF dences of umbilical hernia, we are aware that defining have similar gene structure and functions, both are pleio- the trait as a case or control trait is one of the limita- tropic cytokines that belong to the interleukin-6 (IL-6) tions in this study. However, no other way of recording family, and they can interact with each other [35]. These the trait have successfully been applied, in either this cytokines play a crucial role in diverse biological events study or others. Statistical packages suitable for analyz- like growth promotion and cell differentiation, as well as ing binary traits were therefore performed for GWAS embryogenesis and inflammatory responses to injury and study as well as for the single SNP/haplotype association infection [19–21]. LIF is shown to be the inducer of the study. Limited number of genomic studies have investi- acute phase protein synthesis affecting the cell recruit- gated associations between genes/genetic markers and ment into the area of damage or inflammation (reviewed umbilical hernia. A genome wide scan in White Duroc by [19]). MIF is also involved in immune responses by and Erhualian, using 194 microsatellites and two differ- regulation of cytokine secretion and the expression of re- ent statistical methods, obtained significant QTL regions ceptors that are involved in innate immunity [22], but no for umbilical hernia in altogether 11 chromosomes [7]. variations within the MIF gene were detected in the pigs The most promising loci were revealed on SSC7 and resequences in this study. Even though inflammation at SSC10. None of the SNPs from our GWAS or candidate the umbilicus at weaning was not previously found to be study are located within or close to the significant associated with hernia development [1], our results may chromosomal regions in Ding et al. [7]. The putative suggest the involvement of immunological factors in the QTLs detected on SSC14 in Ding et al. [7] are located development of umbilical hernia in pigs. more telomeric (~ 140 and 149 Mb). Also, in a study by PYCR1 was added as a biological candidate gene due Liao et al. [41] two suggestive loci were found on two to its importance in conversion of glutamate to proline other locations, SSC2 and SSC17. In cattle, Ron et al. and suggested role in cell growth regulation [36]. Proline [11] suggested that a umbilical hernia allele found in the is essential for the stabilization and a major component study was dominant or codominant with partial pene- of collagen, which accounts for three-quarters of the dry trance. The locus detected was located on the centro- weight of skin and is the most prevalent component of meric end of the bovine chromosome 8, comparative to Grindflek et al. BMC Genomics (2018) 19:412 Page 8 of 9 SSC14 position 14–15 Mb. In our study no significant and candidate gene studies and contributed in writing the paper. All authors have read and approved the final manuscript. SNP associations were obtained in, or close to, this region. The discrepancy between studies are probably due to sev- Ethics approval and consent to participate eral reasons. Low heritability indicate that it is many genes All animals were raised in Norwegian nucleus herds, private owned, and were cared for according to laws and internationally recognized guidelines involved and that the trait is highly influenced by environ- and regulations controlling experiments with live animals in Norway (The mental factors. Different causative alleles and a variety of Animal Protection Act of December 20th, 1974, and the Animal Protection allele frequencies in different breeds would highly affect Ordinance Concerning Experiments with Animals of January 15th, 1996). Protocol for sampling and recording were approved by the Norwegian which QTLs that are detectable in different breeds. Animal Research Authority (reference no. ID2570). Phenotypes in this study are used according to “Nucleus herd agreement” between each of the farmers and Norsvin. Regulation about sampling and veterinary care are Conclusions following the “The regulation of veterinary and other animal health A highly significant QTL for umbilical hernia in Norwegian personnel” from the Norwegian Ministry of Agriculture and Food (§34; LOV- Landrace pigs was detected between 48 and 51 Mb on 2001-06-15-75-§34). SSC14 (P < 0.00015) explaining up to 8.6% of the pheno- Competing interests typic variance for umbilical hernia. Resequencing of candi- The authors declare that they have no competing interests. date genes provided SNPs and haplotypes for association analyses and SNPs within positional candidate genes LIF Publisher’sNote and OSM in the SSC14. Due to the large degree of LD in Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. the region, future functional studies are needed to assign the causal variant(s) and further studies should be con- Author details 1 2 ducted to investigate the effect of this QTL region in other Norsvin SA, Storhamargata 44, 2317 Hamar, Norway. Department of Animal and Aquacultural Sciences, Centre for Integrative Genetics (CIGENE), pig breeds. SNPs within the QTL region can be used as Norwegian University of Life Sciences, PO Box 5003, Ås, Norway. genetic markers to reduce incidences of umbilical hernia in Norwegian Landrace pigs. Received: 2 August 2017 Accepted: 22 May 2018 Abbreviations References CDKN1C: Cyclin-dependent kinase inhibitory protein p57KPI2; LIF: Leukemia 1. Searcy-Bernal R, Gardner IA, Hird DW. Effects of and factors associated with inhibitory factor; MIF: Macrophage migration inhibitory factor; MMP13: Matrix umbilical hernias in a swine herd. J Am Vet Med Assoc. 1994;204(10):1660–4. metallopeptidase-13; OSM: Oncostatin M; PYCR1: Pyrroline-5-carboxylate 2. Petersen HH, Nielsen EO, Hassing AG, Ersboll AK, Nielsen JP. Prevalence of reductase 1; VCAN: Versican; VIM: Vimentin clinical signs of disease in Danish finisher pigs. Vet Rec. 2008;162(12):377–82. 3. Vogt DW, Ellersieck MR. Heritability of susceptibility to scrotal herniation in Acknowledgements swine. Am J Vet Res. 1990;51(9):1501–3. We would like to thank the breeding technicians in Norsvin for collecting 4. Sevillano CA, Lopes MS, Harlizius B, Hanenberg EH, Knol EF, Bastiaansen JW. blood samples and recording data, the nucleus herds for providing Genome-wide association study using deregressed breeding values for information and allowing us to collect samples, BioBank AS for doing most cryptorchidism and scrotal/inguinal hernia in two pig lines. Genetics, of the DNA-extraction, Hanne Hamland for helping out with genotyping and selection, evolution : GSE. 2015;47:18. organization of data, Dr. Ina Andersen-Ranberg for discussions and for 5. Grindflek E, Moe M, Taubert H, Simianer H, Lien S, Moen T. Genome-wide adding general information about hernia status in Norwegian nucleus herds, linkage analysis of inguinal hernia in pigs using affected sib pairs. BMC and Dr. Thomas Moen and Dr. Harald Grove for helping out with pro- Genet. 2006;7:25. grammes for data handling. We are also grateful to Dr. Anneleen Stinckens 6. 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A unique loop structure in oncostatin M determines binding affinity toward oncostatin M receptor and leukemia inhibitory factor receptor. J Biol Chem. 2012;287(39):32848–59. 36. De Ingeniis J, Ratnikov B, Richardson AD, Scott DA, Aza-Blanc P, De SK, Kazanov M, Pellecchia M, Ze R, Osterman AL, et al. Functional specialization in proline biosynthesis of melanoma. PLoS One. 2012;7(9):e45190. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png BMC Genomics Springer Journals

Genome-wide association study reveals a QTL and strong candidate genes for umbilical hernia in pigs on SSC14

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Life Sciences; Life Sciences, general; Microarrays; Proteomics; Animal Genetics and Genomics; Microbial Genetics and Genomics; Plant Genetics and Genomics
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Abstract

Background: Umbilical hernia is one of the most prevalent congenital defect in pigs, causing economic losses and substantial animal welfare problems. Identification and implementation of genomic regions controlling umbilical hernia in breeding is of great interest to reduce incidences of hernia in commercial pig production. The aim of this study was to identify such regions and possibly identify causative variation affecting umbilical hernia in pigs. A case/control material consisting of 739 Norwegian Landrace pigs was collected and applied in a GWAS study with a genome-wide distributed panel of 60 K SNPs. Additionally candidate genes were sequenced to detect additional polymorphisms that were used for single SNP and haplotype association analyses in 453 of the pigs. Results: The GWAS in this report detected a highly significant region affecting umbilical hernia around 50 Mb on SSC14 (P < 0.0001) explaining up to 8.6% of the phenotypic variance of the trait. The region is rather broad and includes 62 significant SNPs in high linkage disequilibrium with each other. Targeted sequencing of candidate genes within the region revealed polymorphisms within the Leukemia inhibitory factor (LIF) and Oncostatin M (OSM) that were significantly associated with umbilical hernia (P < 0.001). Conclusions: A highly significant QTL for umbilical hernia in Norwegian Landrace pigs was detected around 50 Mb on SSC14. Resequencing of candidate genes within the region revealed SNPs within LIF and OSM highly associated with the trait. However, because of extended LD within the region, studies in other populations and functional studies are needed to determine whether these variants are causal or not. Still without this knowledge, SNPs within the region can be used as genetic markers to reduce incidences of umbilical hernia in Norwegian Landrace pigs. Keywords: Genome-wide association study, GWAS, Umbilical, Hernia, Pigs, LIF, OSM Background a hernia is the potential entrapment of intestines Hernias are of the most common congenital defects in through this opening. Physical injury, nutrition, excessive pigs which often leads to poor animal welfare and severe pressure, muscular weakness and heredity have been of- economic losses for pig producers. The most common fered as causes of hernia [1]. Umbilical hernias often ap- types of hernias in pigs are umbilical and inguinal/scro- pears in pigs at 9 to 14 weeks of age, with incidences tal hernia. Umbilical hernia is diagnosed by the protru- reported to range from 0.4 to 1.2% [1, 2]. The prevalence sion of abdominal contents beneath the skin at the navel of scrotal/inguinal hernias is shown to be approximately (umbilicus). It is generally accepted that weakened sup- in the same range, between 0.5 and 1.5%, and with an portive muscles around the umbilical stump or navel estimated heritability of around 0.3 in different breeds area of the animal causes the umbilical opening not to [3, 4]. Several studies have explored the genomics of close properly and intestines protrude through the intes- scrotal/inguinal hernia [4–7], but very few have devoted tinal wall to form the “ball-like” structure. The threat of efforts to decipher the genetic architecture of umbilical hernia. This is mainly because umbilical hernia is relatively rare and sometimes complicated to diagnose properly. * Correspondence: eli.grindflek@norsvin.no It is generally accepted that genetics influence con- Norsvin SA, Storhamargata 44, 2317 Hamar, Norway genital umbilical hernias, but mode of inheritance and Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Grindflek et al. BMC Genomics (2018) 19:412 Page 2 of 9 genetic architecture are more or less undiscovered, and (approximately smaller or larger than baseball size, re- h estimates of umbilical hernia are reported to be very spectively). Based on this, 165 hernias were classified as low in pigs (0.06–0.08) [1, 8]. The frequencies and heri- “small” and 267 were classified as “large”. Additionally, tabilities of umbilical hernia also differs between species. the time of first observation was recorded, together with In cattle, for example, both frequencies (0.1–15%) and records on whether the piglet previously had a history of heritability (h = 0.4) are reported to be much higher [9, navel inflammation or not. The size-score of the umbil- 10] than in pigs. A few studies have tried to decipher the ical hernias was considered not to be valuable as a co- genetic structure of the umbilical hernia. Ding et al. [7] variate in the analysis. The first observation of hernias observed significant linkage between markers and umbil- ranged between 2 and 24 weeks, with the average of ical hernia in pigs on 12 different chromosomes, while 14 weeks. Only 2 piglets were previously observed to have Ron et al. [11] detected a locus on chromosome 8 linked an inflammation, and this was therefore not accounted for with umbilical hernia in cattle. Recently, Long et al. [12] in the statistical analysis. All animals were cared for ac- published a study suggesting a copy number variation cording to laws and internationally recognized guidelines (CNV) on SSC14 to be involved in development of um- and regulations controlling experiments with live animals bilical hernia in pig. Also in humans some studies have in Norway (The Animal Protection Act of December 20th, been done on structural variations like deletions and 1974 (revision: FOR-2010-08-06-117), and the Animal duplications, suggesting that such variations also could Protection Ordinance Concerning Experiments with Ani- play a role in occurrence of umbilical hernia [13–15]. mals of January 15th, 1996), according to the rules given Additionally, two studies in other species suggest that by Norwegian Animal Research Authority. umbilical hernia is associated with the function of KIP2 cyclin-dependent kinase inhibitory protein p57 ,a DNA extraction regulator of cell proliferation [16, 17]. DNA was extracted from porcine blood, leukocytes or Studies on genetic control of umbilical hernia so far is semen using the MagAttract DNA Blood Midi M48 not clear, although indicating that the disorder is com- protocol on the Bio-Robot M48 (Qiagen, Hilden, plex and affected by multiple causative genes and vari- Germany). The concentration and quality of samples ants. Therefore, the aim of this study was to collect a were measured on a Nanodrop, ND-1000 spectropho- proper case/control material and identify genomic regions tometer (NanoDrop Technologies, DE, USA), and on a affecting the frequency of umbilical hernia in pigs, using 1420 Victor plate reader (Turku, Finland) using Pico- GWAS on a high resolution SNP panel followed up by Green fluorescence (Molecular Probes, OR, USA), and candidate gene approaches. thereafter normalized to 50 ng/ul in 96-well plates. Methods Genotyping, sequencing and quality control Animals and phenotypes Genome wide association study (GWAS) The overall frequency of umbilical hernia for Landrace The genotyping was performed at CIGENE (www.cigen- in Norwegian nucleus herds was found to be only 0.55%, e.no), Norwegian University of Life Sciences, Norway. and the heritability (h ) was also estimated to be very Genotyping for the GWAS was performed using the low (0.065). Due to the low incidence of umbilical hernia iScan (Illumina, San Diego, CA, USA) platform with the in Norwegian Landrace, samples had to be collected PorcineSNP60 array according to manufacturer’s in- over time-periods of altogether 4 years. To generate suf- structions. Image intensity, data processing, clustering ficient number of case/control samples we collected data and genotype calling were performed using the genotyp- and samples from all the nucleus herds in Norsvin ing module in the Genome Studio software (Illumina, (Hamar, Norway). Blood samples were collected from a San Diego, CA, USA). Altogether, including the hernia total of 369 purebred Norwegian Landrace piglets dis- pigs, their full-sibs and their parents, 739 Norwegian playing umbilical hernia, along with blood from 202 Landrace were genotyped for 64,232 SNPs. The SNP phenotypically unaffected fullsibs within all affected lit- markers passing the quality control had call rate above ters. The affected pigs were distributed on 259 litters, 0.9, minor allele frequency (MAF) above 0.01, propor- with each litter containing 1 to 6 affected piglets from tion of genotyped above 0.25, and proportion of geno- 110 sires. Altogether, 168 parents were genotyped. Sam- type errors below 0.025. The average call rate across ples were obtained from 35 pig breeding farms, where samples was 0.997, and no samples were excluded from several sires were used on different farms. The farmers analysis due to unacceptable call rates from the genotyp- reported the cases and thereafter the diagnostic proce- ing. Animals with pedigree errors (altogether 17 animals) dures were performed by a Norsvin-breeding technician. were removed from the study. After quality control, 554 The umbilical hernias were subjectively classified into animals were available for the case/control, in addition two categories by approximate size “small” or “large” to 168 parents. The most frequent failing category is Grindflek et al. BMC Genomics (2018) 19:412 Page 3 of 9 non-informative markers (12,259 SNPs). Beagle v. 3.3.1 highest MAF were selected, and additional five SNPs lo- was used to impute sporadically missing genotypes in cated in other genes were removed due to low MAF the final genotype file [18]. In total 49,049 high quality (MAF < 0.01). Therefore, 31 SNPs were used for the final SNP markers, were positioned in the porcine genome as- statistical analyses (Table 2). Candidate genes with more sembly Build 10.2 (Sanger Institute). than one SNP were included for haplotype analysis. Haplotypes and LD blocks were constructed using the Candidate gene study software Haploview v.4.2 [28]. Candidate genes were chosen based on GWAS results in Structural models of the OSM protein were generated this study and putative role in development of umbilical by using the SWISS-MODEL workspace [29], using hernia from literature [6, 16, 17, 19–25]. In total, eight amino acid sequence input files for the OSM translated positional and/or biological candidate genes in seven dif- from DNA sequence with the two different alleles of the ferent regions were selected for sequencing: Leukemia missense single point mutation. QMEAN [30] were used inhibitory factor (LIF), Oncostatin-M (OSM), Macro- to evaluate the quality of the model in 6 different terms: phage migration inhibitory factor (MIF), cyclin-depen- (1) C_beta interaction energy, (2) all-atom pairwise en- dent kinase inhibitory protein p57KPI2 (CDKN1C), ergy, (3) solvation energy, (4) torsion angle energy, (5) Pyrroline-5-carboxylate reductase 1 (PYCR1), Versican solvent accessibility agreement and (6) total (VCAN), Matrix metallopeptidase-13 (MMP13), and QMEAN-score. Furthermore, GROMOS empirical force Vimentin (VIM). Names, positions from www.ensem- field energy [31] was estimated for each amino acid of bl.org and references are listed in Table 1. the protein chain. SNP discovery was performed by PCR resequencing of genomic DNA and cDNA from Norwegian Landrace Statistical analyses with umbilical hernia and healthy siblings. Primers were Genome wide association study (GWAS) designed using Primer3 [26]. The programmes Phrap, The association analyses were carried out for the 49,049 Phred and PolyPhred (v.4.06) were used to identify puta- SNPs mapped on the 18 porcine autosomes using pig tive SNPs from the PCR resequencing chromatograms, genome assembly Build10.2. The minor allele frequen- and the Consed programme was used to visually confirm cies of the selected SNPs were uniformly distributed be- the putative SNPs [27]. For the SNP detection, eight her- tween 0.01 and 0.5. Genome-wide association analysis nia pigs and eight healthy pigs were used for sequencing. was performed with the GenABEL package (version 1.6–5) No SNPs were detected in the positional candidates MIF in R [32] using the structured association approach and CDKN1C, but 50 SNPs were obtained in six other (“qtscore”) for binary traits [33], which is in principle a genes. Primer assays for use in the Sequenom MassAR- Cochran-Armitage test. Umbilical hernia was the binomial RAY system were designed using MassARRAY Assay trait and gender was added as a covariate, although signifi- Design Software, with multiplexes between 12 and 19. cant differences between gender was not observed (p =0.7). Genotyping was done according to the manufacturer’s Gender-factor have not either previously been reported in instructions in the IPLEX protocol. The MassARRAY pigs, but in cattle, however, umbilical hernia have been Typer software was used for the automated genotype foundtobemorefrequentlyoccurringinfemale compared calling (Sequenom, San Diego, USA). Altogether, 46 to males [10]. SNPs in six different candidate genes were successfully The p-values corrected for genomic control (GC) of a genotyped for 463 animals; 201 pigs with umbilical her- 1-df test were accepted to represent proof of genome-wide nia, 135 healthy siblings and 127 parents. Due to high association at p < 0.001 (−log10(p) = 3). Collection of sam- SNP density in VCAN (16 SNPs) the six SNPs with ples was done during intensive periods of altogether four Table 1 Eight positional and/or biological candidate genes were used for resequencing Gene name Position References Leukemia inhibitory factor (LIF) 14:50263722–50,266,807 [19] Oncostatin M (OSM) 14:50281701–50,286,188 [20, 21] Macrophage migration inhibitory factor (MIF) 14:53282552–53,283,339 [22] Cyclin-dependent kinase inhibitory protein p57KPI2 (CDKN1C) Unplaced [16, 17] Pyrroline-5-carboxylate reductase 1 (PYCR1) Unplaced [6] Versican (VCAN) 2:93783042–93,895,497 [23] Matrix metallopeptidase 13 (MMP13) 9:37493636–37,504,552 [24] Vimentin (VIM) 10:48227005–48,235,670 [25] Positions are in chromosome:basepairs Grindflek et al. BMC Genomics (2018) 19:412 Page 4 of 9 Table 2 SNPs within candidate genes used for association analysis a b c d Gene Name SNP name Location Alleles MAF P %var Versican VCAN_1 Exon 8 A/G 0.45 n.s. 0.2 Versican VCAN_2 Exon 8 G/A 0.45 n.s. 0.4 Versican VCAN_3 Exon 8 T/G 0.45 n.s. 0.4 Versican VCAN_4 Exon 8 C/T 0.45 n.s. 0.2 Versican VCAN_5 Exon 8 C/A 0.38 n.s. 0.4 Versican VCAN_6 Exon 8 C/T 0.38 n.s. 0.4 Leukemia inhibitory factor LIF_1 Intron 1 A/G 0.47 0.001 8.6 Matrix metallopeptidase 13 MMP13_1 Intron 7 G/A 0.33 n.s. 0.08 Matrix metallopeptidase 13 MMP13_2 Intron 7 T/G 0.33 n.s. 0.05 Matrix metallopeptidase 13 MMP13_3 Exon 8 A/G 0.33 n.s. 0.1 Matrix metallopeptidase 13 MMP13_4 Intron 8 C/T 0.33 n.s. 0.03 Matrix metallopeptidase 13 MMP13_5 Intron 8 T/C 0.32 n.s. 0.01 Matrix metallopeptidase 13 MMP13_6 Intron 8 G/T 0.33 n.s. 0.3 Oncostatin M OSM_1 Exon 3 C/T 0.47 0.001 7.7 Oncostatin M OSM_2 Exon 3 T/C 0.47 0.001 8.6 Oncostatin M OSM_3 Exon 3 T/C 0.47 0.001 8.6 Oncostatin M OSM_4 Exon 3 G/T 0.47 0.0008 8.6 Oncostatin M OSM_5 Exon 3 A/G 0.47 0.001 8.6 Oncostatin M OSM_6 Intron 3 C/T 0.47 0.001 7.7 Oncostatin M OSM_7 Intron 3 T/C 0.47 0.001 7.7 Oncostatin M OSM_8 Intron 3 A/G 0.47 0.001 7.7 Pyrroline-5-carboxylate reductase 1 PYCR1_1 5’ UTR A/T 0.11 n.s. 2.5 Pyrroline-5-carboxylate reductase 1 PYCR1_2 5’ UTR C/T 0.13 n.s. 1.0 Pyrroline-5-carboxylate reductase 1 PYCR1_3 Intron 1 C/T 0.03 n.s. 1.0 Vimentin VIM_1 Intron 5 C/T 0.21 n.s. 0.2 Vimentin VIM_2 Intron 5 A/G 0.36 n.s. 0.3 Vimentin VIM_3 Intron 5 C/T 0.17 n.s. 0.01 Vimentin VIM_4 Intron 5 A/G 0.19 n.s. 0.6 Vimentin VIM_5 Intron 5 C/T 0.15 n.s. 0.00 Vimentin VIM_6 Intron 5 C/A 0.34 n.s. 0.3 Vimentin VIM_7 Intron 5 C/A 0.11 n.s. 0.7 major/minor allele, where risk allele is indicated in bold minor allele frequency (MAF) p-value (p > 0.001 is n.s) the percentage of explained phenotypic variance (%var) years, but it was seven years between the first and final col- Y ¼ μ þ snp=haplotype þ sex þ id þ e lection of samples. Therefore, a population stratification analysis (“strata”) was performed to find whether the popu- where Y is the binomial trait “case” or “control”. The lation during some generations of selection was grouped SNP, coded as 0, 1, or 2 for homozygote allele 1, hetero- into several genetic populations or not. However, only one zygote, homozygote allele 2, respectively, was fitted as a population was defined and correction for stratification fixed effect. In case of the haplotype associations, each was not added in the final analysis. haplotype allele combination was assigned its own level and fitted as a fixed effect. Sex was fitted as a fixed effect Candidate gene study whereas animal id was treated as a random effect, using For the single SNP-marker and haplotype association a pedigree based relationship matrix to account for analysis the ASReml v.2.00 [34] was used to conduct population structure. The F-statistics was calculated for association analysis using the following model: each SNP or haplotype and the significance level was set Grindflek et al. BMC Genomics (2018) 19:412 Page 5 of 9 at a corresponding p-value of 0.001. The genetic vari- Table 3 The most significant 60 k SNPs for umbilical hernia a b c ance explained by a SNP was calculated from the esti- Marker name SSC Position MAF P-value %var mated genotype effects and the observed genotype INRA0043998 14 50,652,198 0.48 7.43e-05 8.1 frequencies. It was expressed as a percentage of the total ASGA0063261 14 50,672,335 0.48 7.43e-05 8.1 phenotypic variance obtained from the model without ASGA0063262 14 50,738,530 0.48 7.43e-05 8.1 the genotype effect. ALGA0077446 14 50,761,608 0.48 7.43e-05 8.1 ASGA0063267 14 50,776,775 0.48 7.43e-05 8.1 Results Genome-wide association study (GWAS) ALGA0077450 14 50,792,395 0.48 7.43e-05 8.1 Assuming a corrected significance level of P < 0.001, 129 MARC0024032 14 50,824,208 0.48 7.43e-05 8.1 SNP markers reached the significant level. Almost all of INRA0044003 14 50,847,851 0.48 7.43e-05 8.1 them, 126 SNPs, were located on SSC14 between posi- MARC0112737 14 50,917,403 0.48 7.43e-05 8.1 tions 47.16 Mb and 58.91 Mb. The other three single ALGA0077457 14 50,938,144 0.48 7.43e-05 8.1 SNP associations were obtained on SSC1 around ASGA0063274 14 50,964,903 0.48 7.43e-05 8.1 102 Mb at the Porcine Build10.2 genome sequence (http://www.ensembl.org), on SSC8 around 88 Mb, H3GA0040130 14 50,992,818 0.50 7.43e-05 7.9 and on SSC17 around 68 Mb, respectively. The Man- ASGA0063217 14 49,288,281 0.49 9.21e-05 8.6 hattan plot of all chromosomes is shown in Fig. 1. a minor allele frequency (MAF) With a more strict significance level of P < 0.0001, 62 the genomic control corrected p-value the percentage of explained phenotypic variance (%var) SNP markers were significant and all of them were located between 48.04–50.99 Mb at SSC14. The most significant SNPs (p <7 ×10–5) are listed in Table 3. Leukemia inhibitory factor (LIF) was the two genes lo- Figure 2 shows the association results for SSC14 with cated within the SSC14 QTL. For OSM, all the eight sin- characterized genes and LD within the QTL region gle SNP markers, and the haplotype combining all the (P < 0.0001). eight SNPs, were significantly associated with umbilical hernia (P < 0.001) and found to explain up to 8.6% of the Candidate genes for umbilical hernia phenotypic variance (Tables 2 and 4). As shown in Fig. 2, Six candidate genes were included in the candidate gene all eight SNPs within OSM are in complete LD with each association study. Four of them were based on biological other. Only two different haplotypes were obtained in knowledge and previously known associations to umbil- the data set, with frequencies of 0.53 and 0.46 for haplo- ical hernia, and two of them were chosen based on the type 1 and 2, respectively. The haplotype frequencies for QTL position obtained on SSC14 and biological func- the affected/un-affected animals were 0.72/0.28 for the tion. The Oncostatin-M precursor gene (OSM) and 11, 0.54/0.46 for the 12 and 0.55/0.45 for the 22, Fig. 1 Manhattan plot of genome-wide association results. The x axis represents the genome in physical order whereas the y axis shows –log10 p-value for all SNPs. The line corresponds to a significance level of P < 0.001 Grindflek et al. BMC Genomics (2018) 19:412 Page 6 of 9 Fig. 2 Association results for SSC14. a A QTL region of 62 SNPs on SSC 14 reached a significance level of P < 0.00015 (solid line) and they were localized between 48.04 and 50.99 Mb. b Map of characterized genes in the QTL region and their orientation, based on information available from Ensembl, where candidate genes are indicated in red. c An LD plot was constructed using Haploview for the QTL region. R was used as a measurement of LD, where a darker color represents a higher R value. The red triangle covers the SNPs in the candidate genes and they are all in complete LD respectively. Constructing haplotypes within a block be- had umbilical hernia, for the heterozygote 2 animals 53% tween 48 and 51 Mb, covering both OSM and LIF (see had hernia, and for the homozygote 12 51% had hernia, Fig. 2), we got two major haplotypes (0.54 and 0.33) and which may suggest a dominant effect of the haplotype, with remaining haplotypes having a frequency of 0.07 or with the haplotype 1 as the risk allele. The OSM_1 SNP less. For the homozygote haplotype 1, 80% of the animals located in exon 3 (Table 2) is non-synonymous causing an Table 4 Results from the haplotype analyses a b Gene name #haplotypes P-value % Var Versican (VCAN) 7 0.03 0.1 Matrix metallopeptidase 13 (MMP13) 5 0.04 0.02 Oncostatin M (OSM) 2 0.0012 6.0 Pyrroline-5-carboxylate reductase 1 (PYCR1) 6 0.03 0.07 Vimentin (VIM) 11 0.13 0.1 haplotypes were constructed within genes based on SNPs in Table 2 and are presented with the number of haplotypes with a frequency > 2% the percentage of explained phenotypic variance Grindflek et al. BMC Genomics (2018) 19:412 Page 7 of 9 amino acid shift from Serine to Glycine. This seems to the extracellular matrix [37]. Diaphragmatic, inguinal, cause only minor changes in the protein structure ob- and umbilical hernia are all associated with connective tained by SWISS-MODEL [29]. However, the GROMOS tissue weakness, and previously extracellular matrix pro- empirical force field energy was shown to be positive (un- teins such as collagens, fibronectin, elastin and matrix favorable environment) with Serine and slightly negative metalloproteinases have been suggested to be involved (favorable environment) when changed to Glycine (results [38, 39]. Moreover, a candidate gene study for scrotal not shown). Only one polymorphism was obtained by se- hernia in pigs obtained a highly significant association quencing for the other positional candidate gene (LIF)on between PYCR1 and hernia [6]. Several matrix metallo- SCC14. This marker was also significantly associated with proteinase genes are suggested to be associated with umbilical hernia (p < 0.001) and found to be in perfect LD membrane weakening and rupture. One of them, MMP13 with OSM (Fig. 2). None of the polymorphisms detected on SSC9, is shown to obtain an important role in wound in the four candidate genes outside the QTL-region on healing by coordinating cellular activities important in the SSC14 (VCAN, MMP13, PYCR1 and VIM) obtained sig- growth and maturation of granulation tissue, including nificant associations with umbilical hernia (Table 2). myofibroblast function, inflammation, angiogenesis, and proteolysis [24]. MMP13 was for example shown to affect Discussion the structural integrity and mechanical stability of the In this study, a genome wide association analysis was connective tissue in both indirect and direct scrotal her- conducted in the Norwegian Landrace population, to- nias [38]. The VCAN is shown to display high adhesive gether with studies of positional and biological candidate ability to endothelial cells and facilitated tube-like struc- genes for umbilical hernia. ture formation, and is previously suggested as a candidate The most convincing result from the GWAS was to be involved in development of umbilical hernia [23]. In around 50 Mb on SSC14. Three candidate genes in this this study the VCAN gene, located on SSC2, was chosen region were investigated in more detail; OSM, LIF and as a biological candidate gene. Finally, vimentin (VIM), MIF (Macrophage migration inhibitory factor). OSM is lo- which is located on SSC10, was added as a biological can- cated at SSC14 base pair position 50,281,701–50,286,188, didate gene due to its function of being a marker of imma- LIF is located at SSC14 base pair position 50,263,722– ture smooth muscle cells and being involved in collagen 50,266,807 and MIF at position 53,282,553–53,283,313. structure [25], as well as having suggested involvement in The three genes were sequenced at the genomic level in inflammatory/immune response [40]. eight hernia pigs and eight healthy pigs, but SNPs were Since there are variability in level of expression inci- only detected in the OSM and LIF genes. OSM and LIF dences of umbilical hernia, we are aware that defining have similar gene structure and functions, both are pleio- the trait as a case or control trait is one of the limita- tropic cytokines that belong to the interleukin-6 (IL-6) tions in this study. However, no other way of recording family, and they can interact with each other [35]. These the trait have successfully been applied, in either this cytokines play a crucial role in diverse biological events study or others. Statistical packages suitable for analyz- like growth promotion and cell differentiation, as well as ing binary traits were therefore performed for GWAS embryogenesis and inflammatory responses to injury and study as well as for the single SNP/haplotype association infection [19–21]. LIF is shown to be the inducer of the study. Limited number of genomic studies have investi- acute phase protein synthesis affecting the cell recruit- gated associations between genes/genetic markers and ment into the area of damage or inflammation (reviewed umbilical hernia. A genome wide scan in White Duroc by [19]). MIF is also involved in immune responses by and Erhualian, using 194 microsatellites and two differ- regulation of cytokine secretion and the expression of re- ent statistical methods, obtained significant QTL regions ceptors that are involved in innate immunity [22], but no for umbilical hernia in altogether 11 chromosomes [7]. variations within the MIF gene were detected in the pigs The most promising loci were revealed on SSC7 and resequences in this study. Even though inflammation at SSC10. None of the SNPs from our GWAS or candidate the umbilicus at weaning was not previously found to be study are located within or close to the significant associated with hernia development [1], our results may chromosomal regions in Ding et al. [7]. The putative suggest the involvement of immunological factors in the QTLs detected on SSC14 in Ding et al. [7] are located development of umbilical hernia in pigs. more telomeric (~ 140 and 149 Mb). Also, in a study by PYCR1 was added as a biological candidate gene due Liao et al. [41] two suggestive loci were found on two to its importance in conversion of glutamate to proline other locations, SSC2 and SSC17. In cattle, Ron et al. and suggested role in cell growth regulation [36]. Proline [11] suggested that a umbilical hernia allele found in the is essential for the stabilization and a major component study was dominant or codominant with partial pene- of collagen, which accounts for three-quarters of the dry trance. The locus detected was located on the centro- weight of skin and is the most prevalent component of meric end of the bovine chromosome 8, comparative to Grindflek et al. BMC Genomics (2018) 19:412 Page 8 of 9 SSC14 position 14–15 Mb. In our study no significant and candidate gene studies and contributed in writing the paper. All authors have read and approved the final manuscript. SNP associations were obtained in, or close to, this region. The discrepancy between studies are probably due to sev- Ethics approval and consent to participate eral reasons. Low heritability indicate that it is many genes All animals were raised in Norwegian nucleus herds, private owned, and were cared for according to laws and internationally recognized guidelines involved and that the trait is highly influenced by environ- and regulations controlling experiments with live animals in Norway (The mental factors. Different causative alleles and a variety of Animal Protection Act of December 20th, 1974, and the Animal Protection allele frequencies in different breeds would highly affect Ordinance Concerning Experiments with Animals of January 15th, 1996). Protocol for sampling and recording were approved by the Norwegian which QTLs that are detectable in different breeds. Animal Research Authority (reference no. ID2570). Phenotypes in this study are used according to “Nucleus herd agreement” between each of the farmers and Norsvin. Regulation about sampling and veterinary care are Conclusions following the “The regulation of veterinary and other animal health A highly significant QTL for umbilical hernia in Norwegian personnel” from the Norwegian Ministry of Agriculture and Food (§34; LOV- Landrace pigs was detected between 48 and 51 Mb on 2001-06-15-75-§34). SSC14 (P < 0.00015) explaining up to 8.6% of the pheno- Competing interests typic variance for umbilical hernia. Resequencing of candi- The authors declare that they have no competing interests. date genes provided SNPs and haplotypes for association analyses and SNPs within positional candidate genes LIF Publisher’sNote and OSM in the SSC14. Due to the large degree of LD in Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. the region, future functional studies are needed to assign the causal variant(s) and further studies should be con- Author details 1 2 ducted to investigate the effect of this QTL region in other Norsvin SA, Storhamargata 44, 2317 Hamar, Norway. Department of Animal and Aquacultural Sciences, Centre for Integrative Genetics (CIGENE), pig breeds. SNPs within the QTL region can be used as Norwegian University of Life Sciences, PO Box 5003, Ås, Norway. genetic markers to reduce incidences of umbilical hernia in Norwegian Landrace pigs. Received: 2 August 2017 Accepted: 22 May 2018 Abbreviations References CDKN1C: Cyclin-dependent kinase inhibitory protein p57KPI2; LIF: Leukemia 1. Searcy-Bernal R, Gardner IA, Hird DW. Effects of and factors associated with inhibitory factor; MIF: Macrophage migration inhibitory factor; MMP13: Matrix umbilical hernias in a swine herd. J Am Vet Med Assoc. 1994;204(10):1660–4. metallopeptidase-13; OSM: Oncostatin M; PYCR1: Pyrroline-5-carboxylate 2. Petersen HH, Nielsen EO, Hassing AG, Ersboll AK, Nielsen JP. Prevalence of reductase 1; VCAN: Versican; VIM: Vimentin clinical signs of disease in Danish finisher pigs. Vet Rec. 2008;162(12):377–82. 3. Vogt DW, Ellersieck MR. Heritability of susceptibility to scrotal herniation in Acknowledgements swine. Am J Vet Res. 1990;51(9):1501–3. We would like to thank the breeding technicians in Norsvin for collecting 4. Sevillano CA, Lopes MS, Harlizius B, Hanenberg EH, Knol EF, Bastiaansen JW. blood samples and recording data, the nucleus herds for providing Genome-wide association study using deregressed breeding values for information and allowing us to collect samples, BioBank AS for doing most cryptorchidism and scrotal/inguinal hernia in two pig lines. Genetics, of the DNA-extraction, Hanne Hamland for helping out with genotyping and selection, evolution : GSE. 2015;47:18. organization of data, Dr. Ina Andersen-Ranberg for discussions and for 5. Grindflek E, Moe M, Taubert H, Simianer H, Lien S, Moen T. Genome-wide adding general information about hernia status in Norwegian nucleus herds, linkage analysis of inguinal hernia in pigs using affected sib pairs. BMC and Dr. Thomas Moen and Dr. Harald Grove for helping out with pro- Genet. 2006;7:25. grammes for data handling. We are also grateful to Dr. Anneleen Stinckens 6. 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BMC GenomicsSpringer Journals

Published: May 29, 2018

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