Optimization of Real-Time PCR-melting for detection of the Cholesterol-deficiency mutation in Holstein Friesian cattle

• Andrea Branda-Sica Instituto Nacional de Investigación Agropecuaria (INIA), INIA Las Brujas, Unidad de Biotecnología. Canelones, Uruguay
• Paula Nicolini Universidad de la República, Centro Universitario de Tacuarembó, Instituto Superior de la Carne, Área Biología Molecular. Tacuarembó, Uruguay
• Rody Artigas Universidad de la República, Facultad de Veterinaria, Unidad Académica de Genética y Mejora Animal. Montevideo, Uruguay
• Maria Teresa Federici Instituto Nacional de Investigación Agropecuaria (INIA), INIA Las Brujas, Unidad de Biotecnología. Canelones, Uruguay
• Silvia Llambi Universidad de la República, Facultad de Veterinaria, Unidad Académica de Genética y Mejora Animal. Montevideo, Uruguay. https://orcid.org/0000-0003-2594-9338

DOI: https://doi.org/10.52973/rcfcv-e32137

Keywords: Cholesterol deficiency, Holstein Friesian, real-time PCR-melting

Abstract

The purpose of this study was to optimize a real-time PCR-melting analysis for reliable and economical detection of the 7.5 Kb mutant insert of the BoERVK bovine transposable element in exon 5 of the Apolipoprotein B (APOB) gene, which causes cholesterol deficiency — CD — (OMIA 001965-9913). This technique was also used to perform a preliminary molecular screening to detect this mutation in a DNA sample of Holstein Friesian cows (HFc) of six commercial dairy farms from different regions of Uruguay. By amplifying the 170 and 146 bp PCR products, two genotypes were clearly identified: homozygote (wild type wt/wt) and heterozygote (carrier of the CD mutation: MUT/wt). The homozygous wt/wt genotype was detected in the representative sample of 103 HFc. It is concluded that Real-Time PCR-melting analysis is a fast, easily interpretable, low cost, and highly accurate technique for detecting this mutation, which can be implemented in genetic selection programs to prevent the spread of the disease in HFc

References

Ansevin, A.T.; Vizard, D.L.: Brown, B.W.; McConathy, J. High-resolution thermal denaturation of DNA. I. Theoretical and practical considerations for the resolution of thermal subtransitions. Biopolym. 15: 153-174. 1976.

Briano-Rodríguez, C.; Romero, A.; Llambí, S.; Branda-Sica, A.; Federici, M.; Giannitti, F.; Caffarena, D.; Schild, C.; Casaux, M.L.; Dutra, F. Lethal and semi-lethal mutations in Holstein calves in Uruguay. [Mutações letais e semi-letais em bezerros da raça Holandesa no Uruguai.] Anim. Prod. Cien. Rural. 51(7): e20200734. 2021. https://doi.org/h2gt.

Cole, J.B.; Null, D.J.; VanRaden, P.M. Phenotypic and genetic effects of recessive haplotypes on yield, longevity, and fertility. J. Dairy Sci. 99(9): 7274-7288. 2016.

Charlier, C. The Role of Mobile Genetic Elements in the Bovine Genome. Plant and Animal Genome Conference XXIV, San Diego, CA, January 9-13, USA, Abstract W636. 2016. On Line: https://bit.ly/3HSHy7f. 15/03/2022.

Charlier, C.; Georges, M.; Harland, C.; Coppieters, W. Detecting cholesterol deficiency mutation in cattle. European Patent Application N° EP 3 181697 A1. European Patent Office. 19pp. 2017. On Line: https://bit.ly/3OqZCHl. 15/03/2022.

Chien, A.; Edgar, D.B.; Trela, J.M. Deoxyribonucleic acid polymerase from the extreme thermophile Thermus aquaticus. J. Bacteriol. 127(3): 1550-1557. 1976.

Green, M.R.; Sambrook, J. Agarose Gel Electrophoresis. Chapter 2. In: Green, M.R.; Sambrook, J. (Eds.) Molecular Cloning: A Laboratory Manual. 4th. Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. Pp 94-99. 2012.

Gross, J.J.; Schwinn, A.C.; Schmitz-Hsu, F.; Barenco, A.; Neuenschwander, T.F.; Drögemüller, C.; Bruckmaier, R.M. The APOB loss-of-function mutation of Holstein dairy cattle does not cause a deficiency of cholesterol but decreases the capacity for cholesterol transport in circulation. J. Dairy Sci. 102: 10564-10572. 2019. https://doi.org/h2gv.

Gross, J.J.; Schwinn, A.C.; Schmitz-Hsu, F.; Menzi, F.; Drögemüller, C.; Albrecht, C.; Bruckmaier, R.M. Rapid Communication: Cholesterol deficiency-associated APOB mutation impacts lipid metabolism in Holstein calves and breeding bulls. J. Anim. Sci. 94: 1761-1766. 2016. https://doi.org/f8nndm.

Gundry, C.N.; Vandersteen, J.G.; Reed, G.H. Pryor, R.J.; Chen, J.; Wittwer, C.T. Amplicon melting analysis with labeled primers: a closed-tube method for differentiating homozygotes and heterozygotes. Clin. Chemistry. 49: 396-406. 2003.

Hall, T.A. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl. Acids. Symp. Ser. 41: 95-98. 1999.

Kamiñski, S.; Ruoeæ, A. Cholesterol Deficiency–new genetic defect transmitted to Polish Holstein-Friesian cattle. Polish J. Vet. Sci. 19(4): 885–887. 2016.

Kipp, S.; Segelke, D.; Schierenbeck, S.; Reinhardt, F.; Reents, R.; Wurmser, C.; Pausch, H.; Fries, R.; Thaller, G.; Tetens, J.; Pott, J.; Piechotta, M.; Grünberg, W. A new Holstein haplotype affecting calf survival. Interbull Annual Meet. 49: 49-53. 2015.

Kipp, S.; Segelke, D.; Schierenbeck, S.; Reinhardt, F.; Reents, R.; Wurmser, C.; Pausch, H.; Fries, R.; Thaller, G.; Tetens, J.; Pott, J.; Haas, D.; Raddatz, B.B.; Hewicker-Trautwein, M.; Proios, I.; Schmicke, M.; Grünberg, W. Identification of a haplotype associated with cholesterol deficiency and increased juvenile mortality in Holstein cattle. J. Dairy Sci. 99: 8915-8931. 2016. https://doi.org/h2gz.

Kumar, A.; Gupta, I.D.; Kumar, S.; Vineeth, M.R; Kumar, D.; Mohan, G.; Jayakumar, S.; Kumar-Niranjan, S. First report of colesterol deficiency associated APOB mutation causing calf mortality in Indian Holstein Fresian population. Indian J. Anim. Sci. 91(2): 148-150. 2021.

Li, Y.; Fang, L.; Liu, L.; Zhang, S.; Ma, Z.; Sun, D. The cholesterol-deficiency associated mutation in APOB segregates at low frequency in Chinese Holstein cattle. Can. J. Anim. Sci. 99(2): 332-335. 2018.

Menzi, F.; Besuchet-Schmutz, N.; Fragnière, M.; Hofstetter, S.; Jagannathan, V.; Mock, T.; Raemy, A.; Studer, E.; Mehinagic, K.; Regenscheit, N.; Meylan, M.; Schmitz-Hsu, F.; Drögemüller, C. A Transposable element insertion in APOB causes cholesterol deficiency in Holstein cattle. Anim. Genet. 47(2): 253-257. 2016. doi: https://doi.org/f8fjm5.

Miller, S.A.; Dykes, D.D.; Polesky, H.F. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 12:319-325. 1988.

ONLINE MENDELIAN INHERITANCE IN ANIMALS (OMIA). Faculty of Veterinary Science, University of Sydney, 2011. On line: https://bit.ly/39GVPHk/. 28/03/2022.

Pozovnikova, M.V.; Gladyr, E.A.; Romanenkova, O.S.; Vasileva, O.K.; Leibova, V.B.; Tyshchenko, V.I.; Dementeva, N.V. Screening of haplotype for cholesterol deficiency genetic defect in the Russian Holstein cattle population. Pol. J. Vet. Sci. 23(2): 313-315. 2020. https://doi.org/h2g5.

Saleem, S.; Heuer, C.; Sun, C.; Kendall, D.; Moreno, J.; Vishwanath, R. Technical Note: The role of circulating low-density lipoprotein levels as a phenotypic marker for Holstein cholesterol deficiency in dairy cattle. J. Dairy Sci. 99(7): 5545-5550. 2016. https://doi.org/f8s6hv.

Schütz, E.; Wehrhahn, C.; Wanjek, M.; Bortfeld, R.; Wemheuer, W.E.; Beck, J.; Brenig, B. The Holstein Friesian Lethal Haplotype 5 (HH5) Results from a Complete Deletion of TFB1M and Cholesterol Deficiency (CDH) from an ERV-(LTR) Insertion into the Coding Region of APOB. PLoS One 11(6): e0154602. 2016. https://doi.org/h2hj.

VanRaden, P.; Null, D. Holstein haplotype for cholesterol deficiency (HCD). 2015. On line: https://bit.ly/3xG2fyv. 29/03/2022.

White, H; Potts, G. Mutation scanning by high resolution melt analysis. Evaluation of Rotor-Gene 6000 (Corbett Life Science), HR-1 and 384 well LightScanner (Idaho Technology). 2006. National Genetics Reference Laboratory. Wessex. On Line: https://bit.ly/3y4QLWw. 29/03/2022.