Analysis of CSN2 variants in Friesian Holstein cows and their association with milk protein allergy and production traits

Mohammad Ilham Dhiaurridho, Firmansyah Tristadika Prakosa, Firna Fauziatul Karimah, Salsabilla Ramadhana, Ine Febriantama, Thoriq Aldri Bramastya, Ahmad Pramono, Muhammad Cahyadi


Objective: The objective of this study was to analyze CSN2 variants in Indonesian Friesian Holstein (FH) cow and their association with milk protein allergy and production traits.

Methods: Genomic DNA was extracted from bloods of twelve Indonesian FH cow. Exon 7 of the CSN2 was amplified using novel primer pair to produce 683 bp amplicon. The primers were 5’-ACCCCAATTTCTTAACCAAACCA-3’ as a forward primer and 5’-CATCAGAAGTTAAACAGCACAGT-3’ as a reverse primer. The PCR products were analyzed to determine the nucleotide sequence of CSN2 using Bioedit version 7.2.5. Moreover, Hardy-Weinberg equilibrium (HWE) was calculated and one-way analysis of variance (ANOVA) was conducted to associate between CSN2 variants and milk production traits.

Results: Two polymorphisms, c.350A>C and c.516G>C, were identified in the CSN2 exon 7. Base substitution from adenine (A) to cytosine (C) of c.350A>C changed amino acid codon from histidine (CAU) to proline (CCU), and base substitution guanine (G) to cytosine (C) of c.516G>C changed amino acid codon from arginine (AGG) to serine (AGC). The CC genotype frequency for c.350A>C SNP was 33% and they were able to produce A2 CSN2 variant which is favorable for preventing lactose intolerance. In addition, there were no association between c.350 A>C and c.516 G>C SNP of the CSN2 with milk production traits.

Conclusions: In conclusion, A1 and A2 variants of CSN2 were identified in Indonesian FH population and they did not associate with milk production in Indonesian FH.



CSN2; Friesian Holstein; lactose intolerance; A2 milk; milk production

Full Text:



Haug, A., A. T. Høstmark, and O. M. Harstad. 2007. Bovine milk in human nutrition – a review. Lipids Health Dis. 6:1-6. Doi: 10.1186/1476-511X-6-25

Heyman, M. B., Committee on Nutrition. 2006. Lactose intolerance in infants, children and adolescents. Pediatrics. 118:1279-1286. Doi: 10.1542/peds.2006-1721

Sitepu, G. A. S., E. R. R. Putri, and Inayah. 2020. Isolasi enzim laktase untuk mengurangi kadar laktosa susu bagi penderita intoleransi laktosa. Prosiding Industrial Research Workshop and National Seminar (IRWNS). p. 720-724. Doi: 10.35313/irwns.v11i1.2106

Pal, S., K. Woodford, S. Kukuljan, and S. Ho. 2015. Milk intolerance, beta-casein and lactose. Nutrients. 7:7285-7297. Doi: 10.3 390/nu7095339

Chia, J. S. J., J. L. McRae, S. Kukuljan, K. Woodford, R. B. Elliot, B. Swinburn, and K. M. Dwyer. 2017. A1 beta-casein milk protein and other environmental predisposing factors for type 1 diabetes. Nutr. Diabetes. 7:1-7. Doi: 10.103 8/nutd.2017.16

Ng-Kwai-Hang, K. F. and F. Grosclaude. 2003. Genetic polymorphism of milk proteins. In: Fox, P. F., and P. L. H. McSweeney, editors, Advanced dairy chemistry-1 Proteins. Springer, Boston, MA. p. 739-816.

Truswell, A. S. 2005. The A2 milk case: A critical review. Eur. J. Clin. Nutr. 59(5):623-631. Doi: 10.1038/sj.ejcn.1602104

Lambers, T. T., S. Broeren, J. Heck, M. Bragt, and T. Huppertz. 2021. Processing affects beta-casomorphin peptide formation during simulated gastrointestinal digestion in both A1 and A2 milk. Int. Dairy J. 121:1-6. Doi: 10.1016/j.idairyj.2021.105099

Teagan, S. E., K. L. Dawson, J. L. Keenan, and A. S. Day. 2021. A simple method to generate β-Casomorphin-7 by in vitro digestion of casein from bovine milk. J. Funct. Foods. 85:1-8. Doi: 10.1016/j.jff.2021. 104631

Thiruvengadam, M., B. Venkidasamy, P. Thirupathi, I. Chung, and U. Subramanian. 2021. β-Casomorphin: A complete health perspective. Food Chem. 337:127765. Doi: 10.1016/j.foodchem.2020.127765

Deng, Y., B. Misselwitz, N. Dai, and M. Fox. 2015. Lactose intolerance in adults: biological mechanism and dietary management. Nutrients. 7:8020-8035. Doi: 10.3390/nu7095380

Taylor, S. K. Dwyer, K. Woodford, and N. Kost. 2017. Systematic review of the gastrointestinal effects of A1 compared with A2 β-casein. Adv. Nutr. 8:739-748. Doi: 10.3945/an.116.013953

Miluchova, M., A. Trakovická, and M. Gabor. 2009. Analysis of polymorphism of beta casein of Slovak Pinzgau cattle by PCR-RFLP for alleles A1 and A2. Sci. Papers Anim. Sci. and Biotechnologies. 42:288-292.

Citek, J., L. Hanusova, L. Liskovcova, E. Samkova, O. Hanus, L. Hasonova, Z. Krizova, and L. Vecerek. 2019. Polymorphisms in CSN3, CSN2 and LGB genes and their relation to milk production dairy cattle in in the Czech Republic. Acta Univ. Agric. et Silvic. 67:19-24. Doi: 10.11118/actaun201967010019

Grada, A. and K. Weinbrecht. 2013. Next-generation sequencing: Methodology and application. J. Invest. Dermatol. 133:1-4. Doi: 10.1038/jid.2013.248

Rangel, A. H. N., L. G. Zaros, T. C. Lima, L. H. F. Borba, L. P. Novaes, L. F. M. Mota, and M. S. Silva. 2017. Polymorphism in the beta casein gene and analysis of milk characteristicsin Gir and Guzerá dairy cattle. Genet. Mol. Res. 16:1-9. Doi: 10.4238/gmr16029592

Sebastiani, C., A. Chiara, C. Marcella, T. Martina, C. Giulia, F. Stefano, and B. Massimo. 2020. Frequencies evaluation of β-casein gene polymorphisms in dairy cows reared in Central Italy. Anim. 10:252. Doi: 10.3390/ani10020252

Montgomery, G. W., and J. Sise. 1990. Extraction of DNA from sheep white blood cells. New Zealand J. Agric. Res. 33:437-441. Doi: 10.1080/00288233.1990.10428440

Nei, M., and S. Kumar. 2000. Moleculer evaluation and phylogenetic. Oxford University Press Inc., New York, USA.

Bruce, W. 1981. Basic population genetics. Columbia University Press, New York, USA.

Sambrook, J. and D. W. Russel. 1989. Molecular cloning: a laboratory manual. Cold-Spring Harbor Laboratory Press, New York, USA.

Vougiouklaki, D., D. Antonopoulos, S. Allexeli, and D. Houhoula. 2020. Identification of polymorphisms of gene CSN2 of b-casein in Greek Cow breeds (holstein) by restriction fragment length polymorphism. J. Agric. Sci. 12:32-39. Doi: 10.5539/jas.v12n11p32

Handoyo, D. and A. Rudiretna. 2000. Prinsip umum dan pelaksanaan polymerase chain reaction (PCR) [General principles and implementation of polymerase chain reaction]. Unitas. 9:17-29.

Huang, W., F. Peñagaricano, K. R. Ahmad, J. A. Lucey, K. A. Weigel, and H. Khatib. 2012. Association between milk protein gene variants and protein composition traits in dairy cattle. Int. J. Dairy Sci. 95:440-449. Doi: 10.3168/jds.2011-4757

Jianqin, S., X. Leiming, X. Lu, G. W. Yelland, J. Ni, and A. J. Clarke. 2016. Effects of milk containing only A2 beta casein versus milk containing both A1 and A2 beta casein proteins on gastrointestinal physiology, symptoms of discomfort, and cognitive behavior of people with self-reported intolerance to traditional cows’ milk. J. Nutr. 15:1-16. Doi: 10.1186/s1293 7-016-0147-z

Duifhuis-Rivera, T., C. Lemus-Flores, M. A. Ayala-Valdovinos, D. R. Sánchez-Chiprés, J. Galindo-García, K. Mejía-Martínez, and E. González-Covarrubias. 2014. Polymorphisms in beta and kappa casein are not associated with milk production in two highly technified populations of Holstein cattle in México. J. Anim. Plant. Sci. 24:1316-1321.


  • There are currently no refbacks.