University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Faculty Papers and Publications in Animal Science Animal Science Department 2009 Association and Haplotype Analyses of Positional Candidate Genes in Five Genomic Regions Linked to Scrotal Hernia in Commercial Pig Lines Zhi-Qiang Du Iowa State University Xia Zhao Iowa State University Natascha Vukasinovic Newsham Choice Genetics Fernanda Rodriguez Monsanto Archie C. Clutter Newsham Choice Genetics See next page for additional authors Follow this and additional works at: https://digitalcommons.unl.edu/animalscifacpub Part of the Genetics and Genomics Commons, and the Meat Science Commons Du, Zhi-Qiang; Zhao, Xia; Vukasinovic, Natascha; Rodriguez, Fernanda; Clutter, Archie C.; and Rothschild, Max F., "Association and Haplotype Analyses of Positional Candidate Genes in Five Genomic Regions Linked to Scrotal Hernia in Commercial Pig Lines" (2009). Faculty Papers and Publications in Animal Science. 1033. https://digitalcommons.unl.edu/animalscifacpub/1033 This Article is brought to you for free and open access by the Animal Science Department at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Faculty Papers and Publications in Animal Science by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. Authors Zhi-Qiang Du, Xia Zhao, Natascha Vukasinovic, Fernanda Rodriguez, Archie C. Clutter, and Max F. Rothschild This article is available at DigitalCommons@University of Nebraska - Lincoln: https://digitalcommons.unl.edu/animalscifacpub/ 1033 Association and Haplotype Analyses of Positional Candidate Genes in Five Genomic Regions Linked to Scrotal Hernia in Commercial Pig Lines Zhi-Qiang Du1, Xia Zhao1, Natascha Vukasinovic2, Fernanda Rodriguez3, Archie C. Clutter2, Max F. Rothschild1* 1 Department of Animal Science and Center for Integrated Animal Genomics, Iowa State University, Ames, Iowa, United States of America, 2 Newsham Choice Genetics, West Des Moines, Iowa, United States of America, 3 Monsanto Company, Saint Louis, Missouri, United States of America Abstract Scrotal hernia in pigs is a complex trait likely affected by genetic and environmental factors. A large-scale association analysis of positional and functional candidate genes was conducted in four previously identified genomic regions linked to hernia susceptibility on Sus scrofa chromosomes 2 and 12, as well as the fifth region around 67 cM on chromosome 2, respectively. In total, 151 out of 416 SNPs discovered were genotyped successfully. Using a family-based analysis we found that four regions surrounding ELF5, KIF18A, COL23A1 on chromosome 2, and NPTX1 on chromosome 12, respectively, may contain the genetic variants important for the development of the scrotal hernia in pigs. These findings were replicated in another case-control dataset. The SNPs around the ELF5 region were in high linkage disequilibrium with each other, and a haplotype containing SNPs from ELF5 and CAT was highly significantly associated with hernia development. Extensive re- sequencing work focused on the KIF18A gene did not detect any further SNPs with extensive association signals. These genes may be involved in the estrogen receptor signaling pathway (KIF18A and NPTX1), the epithelial-mesenchymal transition (ELF5) and the collagen metabolism pathway (COL23A1), which are associated with the important molecular characteristics of hernia pathophysiology. Further investigation on the molecular mechanisms of these genes may provide more molecular clues on hernia development in pigs. Citation: Du Z-Q, Zhao X, Vukasinovic N, Rodriguez F, Clutter AC, et al. (2009) Association and Haplotype Analyses of Positional Candidate Genes in Five Genomic Regions Linked to Scrotal Hernia in Commercial Pig Lines. PLoS ONE 4(3): e4837. doi:10.1371/journal.pone.0004837 Editor: Ellen A. A. Nollen, University Medical Center Groningen, Netherlands Received October 31, 2008; Accepted February 6, 2009; Published March 16, 2009 Copyright: ß 2009 Du et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: We appreciate the financial support by Monsanto Choice Genetics and Newsham Choice Genetics, and the additional financial support provided by State of Iowa and Hatch Funds and the support from the College of Agriculture and Life Science in Iowa State University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: mfrothsc@iastate.edu Introduction Our previous genome-wide linkage studies have identified several genomic regions related to the hernia incidence in Most of the inguinal and scrotal hernias (hernia inguinalis and Pietrain-based lines [18,19]. The quantitative trait loci (QTL) scrotalis) are birth defects in human, under the control of genetic regions on Sus scrofa chromosomes (SSC) 2 and 12 have been and environmental factors. However, the molecular mechanism of replicated in seven other independent paternal families derived etiology remains elusive [1–5]. In the pig breeding industry, from three commercial Pietrain-based pig lines [19]. Moreover, in infrequent incidence of inguinal and scrotal hernias can happen the same genomic regions on SSC2 and SSC12, QTL have been for certain pig breeds and lines and genetic factors are believed to drive the hernia development [6–8]. Without clinical examination, identified in another pig breed, Landrace, using affected sib pairs, these two defects cannot be easily distinguished, and most of the which suggests that common genetic origins may be involved even time, this is the case. for different pig populations [20]. We continued to refine the Genome-wide association analyses (GWA) have now been interesting regions by LD analysis, and found three independent frequently applied in the search for human disease susceptibility regions, at approximately 3, 42 and 65 cM on SSC2, and the first loci [9]. This approach could not be realized without the 20 cM region on SSC12, with genes segregating for the risk to advancement of next-generation sequencing and genotyping develop inguinal and scrotal hernias [19]. technologies, as well as the massive efforts spent in the annotation Since the pig whole genome sequencing project is not yet of the structural and functional properties of the human genome completed, we had to use all available sequence information in (HapMap and ENCODE projects) [10,11]. Moreover, attempts to pigs, as well as the comparative information from the human identify the causal genes or gene subnetworks for a disease-linked genome. Taking into consideration the practical concerns of genomic region have employed large-scale candidate gene experimental power in the design of GWA and candidate gene association analyses [12–15], linkage disequilibrium (LD) fine association analyses in human disease studies [9,21,22], and the mapping [16], and more recently, reversely engineered molecular genomic distribution of LD status in pigs [23], we selected 99 posi- network methods [17]. tional candidate genes located in the aforementioned interesting PLoS ONE | www.plosone.org 1 March 2009 | Volume 4 | Issue 3 | e4837 Positional Genes & Pig Hernia regions on SSC2 and SSC12 to conduct a regional large-scale three genes located in or close to region II, the E74-like factor 5 resequencing and association study for the genetic causes of (ELF5), HIPK3 and the kinesin family member 18A (KIF18A), scrotal/inguinal hernia. The possible dysfunction of these genes were highly significantly associated with scrotal hernia (p,0.01) can result in the aberrant collagen metabolism (the most probable (Table 1, Table S2). Even after FDR correction, they were still reason considered for hernia development) [24–27], the smooth significant at p,0.05. The localization of the four SNPs were muscle breakdown [28,29], an altered apoptosis pathway [30], right under the hernia QTL region around 42 cM on SSC2 the sex hormone deregulation [31–33], and the dedifferentiation (Figure 1A), and they were in high LD with each other of fibroblasts derived from the stem cells during epithelial- (Figure 1D). Furthermore, a common haplotype formed by five mesenchymal transition (EMT) [34]. SNPs, ELF5-1, ELF5-3, ELF5-5, ELF5-8 and EHF1, from ELF5 and a neighboring gene EHF, but not HIPK3 and KIF18A, was Results significantly associated with scrotal hernia (p,0.001). In order to further dissect the association from region II on Based on the nucleotide sequence information provided by the SSC2, we added 26 markers surrounding the interesting genes, markers in the three regions associated with scrotal hernia on ELF5 (1 MB region) and KIF18A (6 MB region) (Figure 1E, Table SSC2 at approximately 3, 42 and 65 cM, which existed in S1). At this stage of the genotyping and analyses, the SNP in intron genomic DNA sequences (NCBI accession numbers: BH021488, 3ofKIF18A gave the highest association signal. Furthermore, we DQ648562 and CL352219), we blasted them against the NCBI sequenced 15 exons (exons 2–16), except exon 1 of KIF18A, which nucleotide database, and selected a first set of four important were found in an unordered sequenced BAC clone CH242-13K2 candidate genes distributed in the three specific sub-regions, i.e.