peer-reviewedThis work was supported by Enterprise Ireland and by a Teagasc Walsh fellowship to A.F. Keating.Two genetic variants of the bovine β-casein gene (A1 and B) encode a histidine residue at codon 67, resulting in potential liberation of a bioactive peptide, β-casomorphin, upon digestion. An allele-specific PCR (AS-PCR) was evaluated to distinguish between the β-casomorphin-releasing variants (A1 and B) and the non-releasing variants. AS-PCR successfully distinguished β-casein variants in 41 of 42 animals as confirmed by sequence analysis. Overall, while the incidence of the homozygous A1 and B animals (i.e., homozygous for the histidine residue; 21.4%) was lower than that for animals without the histidine residue (30.9% respectively), 69% of animals carried at least one allele for the histidine residue at codon 67.Teagasc Walsh Fellowship ProgrammeEnterprise Irelan

Cairns, M.T.

Keating, A.F.

Ross, R Paul

Smith, T.J.

Irish Journal of Agricultural and Food Research

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99A note on the evaluation of a beta-casein  variant in bovine breeds by allele-specific PCR and relevance to β-casomorphin A.F. Keating1, T.J. Smith2, R.P. Ross3 and M.T. Cairns1†1National Diagnostics Centre, National University of Ireland, Galway, Ireland2National Centre for Biomedical Engineering Science, National University of Ireland, Galway, Ireland3Teagasc, Dairy Products Research Centre, Moorepark, Fermoy, Co. Cork, IrelandTwo genetic variants of the bovine β-casein gene (A1 and B) encode a histidine residue at codon 67, resulting in potential liberation of a bioactive peptide, β-casomorphin, upon digestion. An allele-specific PCR (AS-PCR) was evaluated to distinguish between the β-casomorphin-releasing variants (A1 and B) and the non-releasing variants. AS-PCR successfully distinguished β-casein variants in 41 of 42 animals as confirmed by sequence analysis. Overall, while the incidence of the homozygous A1 and B animals (i.e., homozygous for the histidine residue; 21.4%) was lower than that for animals without the histidine residue (30.9% respectively), 69% of animals carried at least one allele for the histidine residue at codon 67.Keywords: allele-specific PCR; β-casein; bioactive peptidesIrish Journal of Agricultural and Food Research 47: 99–104, 2008†Corresponding author: michael.cairns@nuigalway.ieIntroductionMilk protein has the potential to yield opioid-like peptides following proteolysis. These peptides have been termed “food hormones” and have been reported to elicit their bioactive effects when released from food constituents (Teschemacher, 2003). The major exogenous milk protein-derived opioid peptides are the β-casomorphins, which are liberated upon digestion of cer-tain variants of β-casein, namely the A1 and B variants (Svenberg et al., 1985; Meisel, 1986). The β-casein gene has 12 known genetic variants in the coding sequence of the gene (Farrell et al., 2004). Genetic variants A1, A2, A3 and B are universally distributed in almost all Bos taurus and Bos indicus populations. The base chang-es encoding the amino acid differences between these four variants are located in exon VII that encodes the major part of the mature protein (Bonsing et al., 1988). The β-casein A1 and B variants differ from the A2 variant at position 67 where a histidine replaces a proline. In addition, the B vari-ant differs from the A1 variant in a substitu-tion of arginine for serine at position 122. (Other variants – C, F & G – also have a his-tidine residue at position 67). Importantly, it is the change to histidine at position 67 that has the potential to result in cleav-age occurring upon digestion and a bioac-tive peptide, beta-casomorphin potentially being liberated (Lien et al., 1992; Stewart et al., 1987; Damiani et al., 1992). Consumption of the A1 and B protein variants of bovine β-casein has been impli-cated in the development of diabetes and coronary heart disease (CHD). It has also been shown that β-casein protein from animals with the position 67 base change (A1 or B allele) induced diabetes in NOD mice (Elliott et al., 1988). Elliott et al. (1999) compared the incidence of Type 1 diabetes in 0 to 14 year-old children from 10 different countries with the national consumption of protein from cows’ milk. While total protein consumption was not correlated with diabetes incidence (r = 0.40), the consumption of the β-casein A1 variant was associated with the incidence of diabetes (r = 0.73). There was an even closer relationship between the combined consumption of A1 and B variants and dia-betes (r = 0.98; Elliott et al., 1999). It has been hypothesised that consump-tion of the β-casein A1 and B protein variants are a specific risk factor for the development of CHD based on a study using data measured for dairy breeds in several countries (McLachlan, 2001). Results for France vs. Northern Ireland were particularly striking where disease mortality in middle-aged men was shown to be between three and four times higher in Belfast than in Toulouse, as was their consumption of the β-casein A1 and B protein variants. Cardiovascular disease is one of the biggest killers in Ireland: it accounts for 42% of all deaths – the high-est of any European country and twice the EU average. Ireland has also been reported to have a high incidence of Type 1 diabetes within Europe (Roche et al., 2002). In recent years human consump-tion of β-casein A1 and B protein variants has been brought to the public’s attention, in particular in New Zealand, where A2 milk is marketed by the A2 Corporation as being a lower risk factor for disease development. In this study we investigated the cytosine to adenine base change which results in the amino acid change at position 67 and the potential release of a bioactive peptide. The A1 and B variants (and others with the histi-dine residue at position 67) were therefore combined and referred to as A1 throughout, while other variants are referred to herein as A2. Allele-specific primers were evaluat-ed for their use in assessing the presence of the A1 base change. In addition, a number of genetically unrelated (non-dairy) animals were studied. Materials and MethodsBlood sampling and DNA extractionApproximately 15 ml of blood was col-lected into heparinised tubes from the coccygeal vein of 42 cattle representing 10 breeds. Breeds involved were high genet-ic merit Holstein-Friesian, low genetic merit Holstein-Friesian, Irish-Friesian, Dutch-Friesian, Limousin, Montbeliarde, Charolais, Normande, Norwegian Red and Kerry. With the exception of the Limousin, Charolais and Kerry animals, these sub-100  IRISH JOURNAL OF AGRICULTURAL AND FOOD RESEARCH, VOL. 47, NO. 1, 2008 KEATING ET AL.: BETA CASEIN A1 GENOTYPE INCIDENCE 101jects were part of the herd at Teagasc, Moorepark, Fermoy, Co. Cork. Limousin and Charolais samples were from Teagasc, Grange, Co. Meath, while Kerry samples were obtained from the herd at Muckross House, Killarney, Co. Kerry. DNA extrac-tion was carried out using the Gentra Capture Column™ (Gentra, Minneapolis, MN55441, USA) system for approximate-ly 200 µl of whole blood per animal. Blood was stored at –80 oC and DNA was stored at –20 oC until further use. Primer design and polymerase chain  reaction (PCR)Allele-specific PCR was carried out using a common forward primer (Bwtp3; 5′-GCCCAGATGAGAGAAGTGAGG-3′), and reverse primers with either T or G at the 3' end (5′-GATGTTTTGTGGGAGGC- TGTTAT/G-3′) to amplify an 854 bp frag-ment (Figure 1a). The polymorphisms rep-resented by the A3 variant and the sec-ond polymorphism (codon 122) associated with the B variant were not present in this Figure 1. Comparison of allele-specific PCR results and sequencing validation. (a) Primer design and layout. (b) Sequencing chromatograms representing each of the three genotypes (A1A1, A1A2, A2A2). (c) Three animals (genotype in parenthesis) are shown as examples of typical results. Lane 1: Marker (M). Lane 2, 4, 6: A1-specific primer PCR. Lane 3, 5, 7: A2-specific primer PCR.amplicon and therefore could not inter-fere with the interpretation of the results. Additional primers were used to ampli-fy a 730 bp fragment encompassing the polymorphism for sequence validation (5 ′-GGCCATTGTTAAGGAACTCC-3′;5′-AAGGTGCAGATTTTCAACAT-3′). Primers were synthesised by MWG Biotech (90 Long Acre, Covent Garden, London, WC2E 9RZ). PCR was carried out from a starting template of approximately 200 ng of genomic DNA in a final volume of 50 µl con- taining 1X Taq DNA polymerase buffer (Invi- trogen Ltd, 3 Fountain Drive, Inchinnan Bus- iness Park, Paisley, UK, PA49RF), 1.5 mM MgCl2, 200 µM dNTPs (Promega), 0.3 µM of each primer and 1U Taq polymerase (Invitrogen). After an initial incubation at 95 oC for 2 min, samples were amplified for 30 cycles at 95 oC for 1 min, 58 oC for 1 min and 72 oC for 1 min. PCR product sequenc-ing was carried out by MWG Biotech. The resulting sequences were analysed using the Vector NTI® suite of software (Invitrogen). Results An examination of the electrophoretic analyses for the animals predicted their genotype through the use of AS-PCR (Figure 1), and these results are listed in Table 1 for each of the 42 animals. These PCR products were sequenced and their genotype determined. With the exception of one animal, as indicated, the results matched perfectly. Use of the AS-PCR method distinguished the A1 from A2 base change with a success rate of 97%. Results of the investigation (combining AS-PCR and sequencing data) were as follows: A1A1 – 21.4%, A2A2 – 30.9%, A1A2 – 47.6%. Overall the incidence of A1 allele carri- ers (either one or two copies of A1 allele) was 69%. 102  IRISH JOURNAL OF AGRICULTURAL AND FOOD RESEARCH, VOL. 47, NO. 1, 2008Table 1. Genotypes of animals determined by AS-PCR and sequence analysisDairy breeds Dual purpose breeds Beef breedsAnimal Genotype Animal Genotype Animal Genotype0011 A1A2 0163 A1A1 0292 A2A20026 A1A2 0166 A1A1 191C A1A23048 A1A2 1226 A1A1 0183 A2A29615 A2A2 1267 A1A2 0094 A2A20050 A1A2 1212 A1A1 42L A1A20059 A1A2  0130* A1A2 215W A1A20081 A2A2 1023 A1A2 0069 A2A20876 A1A1 1545 A1A1 0086 A1A20599 A1A2 39 A2A21668 A1A2 40 A2A21270 A2A2 41 A1A21257 A1A2 42 A1A20407 A1A2 43 A2A20287 A1A1 44 A2A20188 A1A1 45 A1A20508 A1A2 46 A2A21535 A1A10191 A2A2* Sample that was incorrectly identified by AS-PCR.  KEATING ET AL.: BETA CASEIN A1 GENOTYPE INCIDENCE 103DiscussionThe incidence of the A1 variant of β-casein has been the focus of much interest in the last decade due to the possibility that a bioactive peptide is liberated from these variants upon enzymatic digestion. While evidence for a clear link between this bioactive peptide and a disease state has not been demonstrated, it is desirable to have some indication of the incidence of this genetic variant in the Irish herd. Neither the base change encoding these variants nor the “wild-type” leads to any recognition site for a restriction enzyme, ruling out the use of restriction fragment length polymorphisms (RFLP) to distin-guish between the variants. Therefore, the use of allele-specific primers, with a single base change (T/G) at the 3' end comple-mentary to the base change of the spe-cific β-casein variant (A1, B primer – T, A2 primer – G) was evaluated. Allele-specific PCR uses two allelic-specific primer sets differing from each other in their 3' ter-minal nucleotide and was first described by Wu et al. (1989). A single band of 854 bp should only result for one or other of the allele-specific (AS) reactions if the template is homozygous or a band should appear with both allele-specific reactions in the case of a heterozygous template. Genotypes were predicted for each ani-mal using the AS-PCR method. A further PCR was used to amplify a 730 bp frag-ment spanning the polymorphic site. The predicted genotypes from AS-PCR was incorrect in the case of one animal. Some further optimisation of the amplification conditions may be appropriate to ensure a 100% success rate. Regardless, we feel that the use of these allele-specific prim-ers successfully distinguished between the genetic variants. When the different bovine breeds were examined and divided into dairy (Holstein Friesian (high or low genetic merit), Irish Friesian, Norwegian Red, Dutch Friesian), dual-purpose (Normande, Montbeliarde, Kerry) and beef (Charolais, Limousin) animals, it was observed that the high-est incidence of the β-casein A1 (or B) allele occurred in the dairy breeds (77%), compared with the dual-purpose (69%). The beef breeds had a lower incidence (50%). This study, however, involved a small number of animals and thus may not be indicative of the overall incidence in the Irish cow herd. Examination of a larger sample of the Irish national dairy herd may therefore be desirable to obtain a more precise estimate of the incidence of the β-casein A1 and B variants, for example using either milk or semen sam-ples. Removal of this genotype from the national herd might prove beneficial for both the health and dairy industries; how-ever the consequences of removal of this milk protein variant should first be care-fully considered. AcknowledgementsThis work was supported by Enterprise Ireland and by a Teagasc Walsh fellowship to A.F. Keating. ReferencesBonsing, J., Ring, J.M., Stewart, A.F. and MacKinlay, A.G. 1988. Complete nucleotide sequence of the bovine β-casein gene. Australian Journal of Biological Sciences 41: 527–537.Damiani, G., Pilla, F., Leone, P. and Caccio, S. 1992. Direct sequencing and bidirectional allele specific polymerase chain reaction of the bovine β-casein B variant. Animal Genetics 23: 561–566.Elliott, R.B., Reddy, S.N., Bibby, N.J. and Kida, K. 1988. Dietary prevention of diabetes in the non-obese diabetic mouse. Diabetologia 31: 62–64.Elliott, R.B., Harris, D.P., Hill, J.P., Bibby, N.J. and Wasmuth, H.E. 1999. Type 1 (insulin-dependent) diabetes mellitus and cow milk: casein variant consumption. Diabetologia 42: 292–296.Farrell, H.M. Jr., Jimenez-Florez, R., Bleck, G.T., Brown, E.M., Butler, J.E., Creamer, L.K., Hicks, C.L., Hollar, C.M., Ng-Kwai-Hang, K.F. and Swaisgood, H.E. 2004. Nomenclature of the pro-teins of cows’ milk – sixth revision. Journal of Dairy Sciences 87: 1641–1674. Lien, S., Alestrom, P., Klungland, H. and Rogne, S. 1992. Detection of multiple β-casein alleles by amplification created restriction sites (ACRS). Animal Genetics 23: 333–338.McLachlan, C.N.S. 2001. β-casein A1, ischaemic heart disease mortality, and other illnesses. Medical Hypotheses 56: 262–272.Meisel, H. 1986. Chemical characterisation and opioid activity of an exorphin isolated from in vivo digests of casein. FEBS Letters 196: 223–227.Roche, E., Menon, A., Gill, D. and Hoey, H.M. 2002. National incidence of Type 1 diabetes in child-hood and adolescence. Irish Medical Journal 95: 115–6, 118. Stewart, A.F., Bonsing, J., Beattie, C.W., Shah, F., Willis, I.M. and Mackinley, A.G. 1987. Complete nucleotide sequences of bovine αs2- and β-casein cDNAs: comparison with related sequences in other species. Molecular Biology and Evolution 4: 231–241.Svenberg, J., de Haas, J., Leimenstoll, G., Paul, F. and Teschemacher, H. 1985. Demonstration of β-casomorphin immunoreactive materials in in vitro digests of bovine milk and in small intestine con-tents after bovine milk ingestion in adult humans. Peptides 6: 825–830.Teschemacher, H. 2003. Opioid receptor ligands der- ived from food proteins. Current Pharmaceutical Design 9: 1331–1344.Wu, D.Y., Ugozzoli. L., Pal, B.K. and Wallace, R.B. 1989. Allele-specific enzymatic amplification of β-globin genomic DNA for diagnosis of sickle cell anaemia. Proceedings of the National Academy of Sciences of the United States of America 86: 2757–2760.Received 12 February 2007104  IRISH JOURNAL OF AGRICULTURAL AND FOOD RESEARCH, VOL. 47, NO. 1, 2008

A note on the evaluation of a beta-casein variant in bovine breeds by allele-specific PCR and relevance to β-casomorphin

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