Molecular Biology Reports

, Volume 46, Issue 1, pp 813–822 | Cite as

Identification of porcine CTLA4 gene polymorphism and their association with piglet diarrhea and performance traits

  • Xiaowen Gao
  • Dongchun Guo
  • Mingxing Kou
  • Guiling Xing
  • Andong Zha
  • Xiuqin Yang
  • Xibiao Wang
  • Shengwei Di
  • Jiancheng Cai
  • Buyue NiuEmail author
Original Article


The objective of this study was to evaluate the association between the cytotoxic T-lymphocyte-associated antigen 4 (CTLA4) gene and piglet diarrhea. In this study, the mRNA expression of the CTLA4 gene increased significantly in IPEC-J2 cells after Escherichia coli K88 infection. Single nucleotide polymorphisms (SNPs) located in the 5′ flanking region (SNPs g.107281989C>T) and 3′-untranslated region (3′-UTR; SNPs g.107288753C>A) were identified, and they were in linkage disequilibrium in both Min pigs and the Landrace population. Association analysis showed that Landrace piglets with a TT or AA genotype had a lower diarrhea index, and AA animals had higher average daily gain when compared to CC pigs, respectively (p < 0.05). However, the relationship between SNPs and diarrhea and performance traits in the Min population was not significant. Haplotype analysis indicated that the TC haplotype had the lowest diarrhea index. The 5′ flanking deletion assay suggested that SNP g.107281989C>T was a molecular marker instead of the functional marker. This research demonstrated that genetic variances in the CTLA4 gene had significant effects on Landrace piglet diarrhea resistance.


Pig CTLA4 gene Polymorphism Haplotype Diarrhea 



We thank staff from Lanxi Breeding Farm (Lanxi, Heilongjiang, China) for managing the animals. This study was supported financially by National Natural Science Foundation of China (31301935), Science and Technology Foundation of Education Department, Heilongjiang Province (12541005).

Compliance with ethical standards

Conflict of interest

The authors declare that there is no conflict of interest.

Ethical approval

This study meets the criteria of Institutional Animal Care and approved by the Committee of Northeast Agricultural University.

Informed consent

The owner of the animals was informed and consented in writing.


  1. 1.
    Njau MN, Jacob J (2013) The CD28/B7 pathway: a novel regulator of plasma cell function. Adv Exp Med Biol 785:67–75Google Scholar
  2. 2.
    Alegre ML, Frauwirth KA, Thompson CB (2001) T-cell regulation by CD28 and CTLA-4. Nat Rev Immunol 1(3):220–228Google Scholar
  3. 3.
    Fouad N, Saeed A, Mahedy A (2017) Association of CTLA-4 +49 A/G and CT60 gene polymorphism with Graves’ disease. Egypt J Immunol 24(2):63–70Google Scholar
  4. 4.
    Fang W, Zhang Z, Zhang J, Cai Z, Zeng H, Chen M, Huang J (2015) Association of the CTLA4 gene CT60/rs3087243 single-nucleotide polymorphisms with Graves’ disease. Biomed Rep 3(5):691Google Scholar
  5. 5.
    Ting WH, Chien MN, Lo FS, Wang CH, Huang CY, Lin CL, Lin WS, Chang TY, Yang HW, Chen WF (2016) Association of cytotoxic T-lymphocyte-associated protein 4 (CTLA4) gene polymorphisms with autoimmune thyroid disease in children and adults: case–control study. PLoS ONE 11(4):e0154394Google Scholar
  6. 6.
    Devaraju P, Gulati R, Singh BK, Mithun CB, Negi VS (2016) The CTLA4 +49 A/G (rs231775) polymorphism influences susceptibility to SLE in South Indian Tamils. Tissue Antigens 83(6):418–421Google Scholar
  7. 7.
    Elshazli R, Settin A, Salama A (2015) Cytotoxic T lymphocyte associated antigen-4 (CTLA-4) +49 A>G gene polymorphism in Egyptian cases with rheumatoid arthritis. Gene 558(1):103–107Google Scholar
  8. 8.
    Padma-Malini R, Rathika C, Ramgopal S, Murali V, Dharmarajan P, Pushkala S, Balakrishnan K (2018) Associations of CTLA4 +49 A/G dimorphism and HLA-DRB1*/DQB1* alleles with Type 1 diabetes from South India. Biochem Genet 56:1–17Google Scholar
  9. 9.
    Tavares NA, Santos MM, Moura R, Araújo J, Guimarães RL, Crovella S, Brandão LA (2015) Association of TNF-α, CTLA4, and PTPN22 polymorphisms with type 1 diabetes and other autoimmune diseases in Brazil. Genet Mol Res 14(4):18936–18944Google Scholar
  10. 10.
    Wang J, Liu L, Ma J, Sun F, Zhao Z, Gu M (2014) Common variants on cytotoxic T lymphocyte antigen-4 polymorphisms contributes to type 1 diabetes susceptibility: evidence based on 58 studies. PLoS ONE 9(1):e85982Google Scholar
  11. 11.
    Zhang Y, Zhang J, Deng Y, Tian C, Li X, Huang J, Fan H (2011) Polymorphisms in the cytotoxic T-lymphocyte antigen 4 gene and cancer risk: a meta-analysis. Cancer 117(18):4312–4324Google Scholar
  12. 12.
    Wang Y, Wang X, Zhao R (2015) The association of CTLA-4 A49G polymorphism with colorectal cancer risk in a Chinese Han population. Int J Immunogenet 42(2):93–99Google Scholar
  13. 13.
    Fan C, Zhao X, Xu Z (2015) Associations between the cytotoxic T lymphocyte antigen 4 polymorphisms and risk of bone sarcomas. Tumor Biol 36(1):227–231Google Scholar
  14. 14.
    Rahimifar S, Erfani N, Sarraf Z, Ghaderi A (2010) ctla-4 gene variations may influence cervical cancer susceptibility. Gynecol Oncol 119(1):136–139Google Scholar
  15. 15.
    Jaiswal PK, Singh V, Mittal RD (2014) Cytotoxic T lymphocyte antigen 4 (CTLA4) gene polymorphism with bladder cancer risk in North Indian population. Mol Biol Rep 41(2):799Google Scholar
  16. 16.
    Wang L, Su G, Zhao X, Cai Y, Cai X, Zhang J, Liu J, Wang T, Wang J (2014) Association between the cytotoxic T-lymphocyte antigen 4 +49A/G polymorphism and bladder cancer risk. Tumor Biol 35(2):1139–1142Google Scholar
  17. 17.
    Meijerink E, Fries R, Vögeli P, Masabanda J, Wigger G, Stricker C, Neuenschwander S, Bertschinger HU, Stranzinger G (1997) Two alpha (1,2) fucosyltransferase genes on porcine chromosome 6q11 are closely linked to the blood group inhibitor (S) and Escherichia coli F18 receptor (ECF18R) loci. Mamm Genome 8(10):736–741Google Scholar
  18. 18.
    Kreuzer S, Reissmann M, Brockmann GA (2013) New fast and cost-effective gene test to get the ETEC F18 receptor status in pigs. Vet Microbiol 163(3–4):392–394Google Scholar
  19. 19.
    Zhang B, Ren J, Yan X, Huang X, Ji H, Peng Q, Zhang Z, Huang L (2008) Investigation of the porcine MUC13 gene: isolation, expression, polymorphisms and strong association with susceptibility to enterotoxigenic Escherichia coli F4ab/ac. Anim Genet 39(3):258–266Google Scholar
  20. 20.
    Ren J, Yan X, Ai H, Zhang Z, Huang X, Ouyang J, Yang M, Yang H, Han P, Zeng W, Chen Y, Guo Y, Xiao S, Ding N, Huang L (2012) Susceptibility towards enterotoxigenic Escherichia coli F4ac diarrhea is governed by the MUC13 gene in pigs. PLoS ONE 7(9):e44573Google Scholar
  21. 21.
    Yang QL, Kong JJ, Wang DW, Zhao SG, Gun SB (2013) Swine leukocyte antigen-DQA gene variation and its association with piglet diarrhea in Large White, Landrace and Duroc. Asian–Australas J Anim Sci 26(8):1065–1071Google Scholar
  22. 22.
    Yang QL, Huang XY, Zhao SG, Liu LX, Zhang SW, Huang WZ, Gun SB (2016) Effect of swine leukocyte antigen-DQA gene variation on diarrhea in Large White, Landrace, and Duroc piglets. Anim Genet 47(6):691–697Google Scholar
  23. 23.
    Huang X, Yang Q, Yuan J, Liu L, Sun W, Jiang Y, Zhao S, Zhang S, Huang W, Gun S (2016) Effect of genetic diversity in swine leukocyte antigen-DRA gene on piglet diarrhea. Genes Basel 7(7):36Google Scholar
  24. 24.
    Wang Y, Zhao H, Ma Z, Wang Y, Feng WH (2013) CTLA4 mediated targeting enhances immunogenicity against PRRSV in a DNA prime/killed virus boost strategy. Vet Immunol Immunopathol 154(3–4):121Google Scholar
  25. 25.
    Siepert B, Reinhardt N, Kreuzer S, Bondzio A, Twardziok S, Brockmann G, Nöckler K, Szabó I, Janczyk P, Pieper R (2014) Enterococcus faecium NCIMB 10415 supplementation affects intestinal immune-associated gene expression in post-weaning piglets. Vet Immunol Immunopathol 157(1–2):65–77Google Scholar
  26. 26.
    Kelly D, O’Brien JJ, Mccracken KJ (1990) Effect of creep feeding on the incidence, duration and severity of post-weaning diarrhoea in pigs. Res Vet Sci 49(2):223–228Google Scholar
  27. 27.
    Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(− Delta Delta C(T)) Method. Methods 25(4):402–408Google Scholar
  28. 28.
    Botstein D, White RL, Skolnick M, Davis RW (1980) Construction of a genetic linkage map in man using restriction fragment length polymorphisms. Am J Hum Genet 32(3):314–331Google Scholar
  29. 29.
    Zhou C, Liu Z, Jiang J, Yu Y, Zhang Q (2012) Differential gene expression profiling of porcine epithelial cells infected with three enterotoxigenic Escherichia coli strains. BMC Genomics 13(1):330Google Scholar
  30. 30.
    Xia L, Dai L, Zhu L, Hu W, Yang Q (2017) Proteomic analysis of IPEC-J2 cells in response to coinfection by porcine transmissible gastroenteritis virus and enterotoxigenic Escherichia coli K88. Proteomics Clin Appl 11(11–12):1600137Google Scholar
  31. 31.
    Meijerink E, Neuenschwander S, Fries R, Dinter A, Bertschinger HU, Stranzinger G, Vögeli P (2000) A DNA polymorphism influencing alpha(1,2)fucosyltransferase activity of the pig FUT1 enzyme determines susceptibility of small intestinal epithelium to Escherichia coli F18 adhesion. Immunogenetics 52(1–2):129–136Google Scholar
  32. 32.
    Yang M, Yang B, Yan X, Ouyang J, Zeng W, Ai H, Ren J, Huang L (2012) Nucleotide variability and linkage disequilibrium patterns in the porcine MUC4 gene. BMC Genet 13(1):57Google Scholar
  33. 33.
    Zhou C, Liu Z, Liu Y, Fu W, Ding X, Liu J, Yu Y, Zhang Q (2013) Gene silencing of porcine MUC13 and ITGB5: candidate genes towards Escherichia coli F4ac adhesion. PLoS ONE 8(7):e70303Google Scholar
  34. 34.
    Ji H, Ren J, Yan X, Huang X, Zhang B, Zhang Z, Huang L (2011) The porcine MUC20 gene: molecular characterization and its association with susceptibility to enterotoxigenic Escherichia coli F4ab/ac. Mol Biol Rep 38(3):1593–1601Google Scholar
  35. 35.
    Rampoldi A, Jacobsen MJ, Bertschinger HU, Joller D, Bürgi E, Vögeli P, Andersson L, Archibald AL, Fredholm M, Jørgensen CB, Neuenschwander S (2011) The receptor locus for Escherichia coli F4ab/F4ac in the pig maps distal to the MUC4-LMLN region. Mamm Genome 22(1–2):122–129Google Scholar
  36. 36.
    Fu WX, Liu Y, Lu X, Niu XY, Ding XD, Liu JF, Zhang Q (2012) A genome-wide association study identifies two novel promising candidate genes affecting Escherichia coli F4ab/F4ac susceptibility in swine. PLoS ONE 7(3):e32127Google Scholar
  37. 37.
    Lunney JK, Ho CS, Wysocki M, Smith DM (2009) Molecular genetics of the swine major histocompatibility complex, the SLA complex. Dev Comp Immunol 33(3):362–374Google Scholar
  38. 38.
    Gibson HM, Hedgcock CJ, Aufiero BM, Wilson AJ, Hafner MS, Tsokos GC, Wong HK (2007) Induction of the CTLA-4 gene in human lymphocytes is dependent on NFAT binding the proximal promoter. J Immunol 179(6):3831–3840Google Scholar
  39. 39.
    Lu X, Liu JF, Gong YF, Wang ZP, Liu Y, Zhang Q (2011) Mapping quantitative trait loci for T lymphocyte subpopulations in peripheral blood in swine. BMC Genet 12(1):79–79Google Scholar
  40. 40.
    Galina-Pantoja L, Siggens K, van Schriek MG, Heuven HC (2010) Mapping markers linked to porcine salmonellosis susceptibility. Anim Genet 40(6):795–803Google Scholar
  41. 41.
    Liu G, Kim JJ, Jonas E, Wimmers K, Ponsuksili S, Murani E, Phatsara C, Tholen E, Juengst H, Tesfaye D, Chen JL, Schellander K (2008) Combined line-cross and half-sib QTL analysis in Duroc–Pietrain population. Mamm Genome 19(6):429–438Google Scholar
  42. 42.
    Rückert C, Bennewitz J (2010) Joint QTL analysis of three connected F2-crosses in pigs. Genet Sel Evol 42(1):40Google Scholar

Copyright information

© Springer Nature B.V. 2018

Authors and Affiliations

  1. 1.College of Animal Science and TechnologyNortheast Agricultural UniversityHarbinChina
  2. 2.State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research InstituteChinese Academy of Agricultural Sciences (CAAS)HarbinChina
  3. 3.Lanxi breeding FarmLanxiChina

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