Molecular screening of bovine β-casein (CSN2) A2 variant in Indonesian Holstein dairy cattle as attempt to produce digestive friendly milk

Cow’s milk-intolerance is a digestive problem on people who not able to digest milk. This problem may relate to the variant of (3-casein (CSN2), especially A1, suggested due to (3-casomorphins (BCM-7) formation during enzymatic digestion, for that selecting cattle free BCM-7 become a concern to produce digestive friendly milk. This study aimed to differentiate A1 and A2 allele variant of CSN2 gene in selected population of Indonesian Holstein cattle. In total 70 cows DNA were collected, and fragment of CSN2 exon 7 which contain Single Nucleotide Polymorphism (SNP) rs43703011 and rs43703013 were amplified. Variant analysis was done by mutation site analysis using PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) with Mspl restriction enzyme and DNA sequence for confirmation. Result shows 2 allele variants of (3-casein that are B type, representing A1 family variant, and A2 in mutation site rs43703013C>G. We found A2 allele in the studied population is superior in frequency than A1 (0.916 vs. 0.084). Of that, 8.6% cattle were heterozygotes that is BA2 and 91.4% were homozygotes A2A2. Cattle which carry A1 allele variant should be excluded from dairy cattle breeding program for further milk production free of BCM-7.


Introduction
Cow milk intolerance is a gastrointestinal problem which commonly occur in people who are not able to digest cow's milk or dairy products. It is a complex problem of importance to public and individual health. One of the elements in milk intolerance is lactose malabsorption or intolerance [1]. Additionally, in some cases, the milk intolerance is also accompanied with other potential health risk problem [2]. Previous review shows that milk intolerance may associated with BCM-7 during digestion in human  [1]. BCM-7 is a polypeptide known as the result of A1 digestion but not A2 β-casein which affects gastrointestinal motility and inflammation [3]. In here milk with A2 protein, believed to be relatively friendly to be consume compared to the A1 milk. β-casein or CSN2 is one of main milk protein which has 13 variants, the most common is A1 and A2 variant protein [4]. Milk with A1 protein produce 4 times more of BCM-7 compared to A2 milk [5]. Earlier study reporting that BCM-7 has potential to be associated with health risk problem such as the risk of diabetes type-1, hearth diseases, arteriosklerosis, infant dead, autism, schizophrenia, and others [6,7]. The A1 β -casein milk led to significantly higher stool consistency values compared with the A2 β-casein milk. There was also a positive association between abdominal pain and stool consistency on A1 diet, but not A2 diet. This shows the differences in gastrointestinal responses in adult humans who consuming milk containing β-casein. It appears that BCM-7 may function as an immunosuppressant and impair tolerance to dietary antigens in the gut immune system [8]. In vitro studies indicate that BCM-7 can be produced from A1 and B milk during typical digestive processes; however, BCM-7 is not a product of A2 digestion.
The β-casein variant A1, A2, and B are commonly found in European dairy cattle breed and received much attention due to their influence on milk technology properties and on human health [9]. The two major variant A1 and A2 can be considered as β-casein "families", which include at least 10 subvariants. Those within the A1 family are B, C, D, F and G, while the A2 family are A3, E, H1, H2 and I [5]. The different of sub-variants within A1 family still unproved to have any effect on the release of BCM-7. However, there is a solid evidence that the B variant resulted in a particularly high release of BCM-7 compared to A1 variant [10]. Considering the food safety reason to human health, this study has objective to differentiate dairy cattle which carry different CSN2 allele variant namely A1 and A2 using a molecular approach. Further, the result could be applied in breeding dairy cattle free of BCM-7 to produce friendly digestive milk for human consumption.

Sample collection dan DNA extraction
In total 70 cows of Indonesian Holstein dairy cattle were randomly sampled from two locations. The location is in Baturaden (-7.303969142727606, 109.24800625409786) and Boyolali (7.580411827901281, 110.58255720101133) Indonesia. Those two locations were known to have large population of Indonesian Friesian Holstein dairy cattle.
Fresh blood for genomic DNA (gDNA) source were collected through coccygeal vein using 21G × 1.5 venoject needle attached to collection tube with EDTA contained. The procedure during blood collection was supervised by a veterinary medicine for animal ethically approval. Samples then stored in 5°C and transported to laboratory for long-term storage until DNA extraction. The gDNA were extracted using Wizard® Genomic DNA Purification Kit (Promega, USA) according to the protocol supplied by the company. Following extraction, gDNA then stored in -20°C for genotyping process.

β-casein genotyping
In this study, we differentiate β-casein allele variant of B and A2 on SNP site at g.8267C>G. According to Ensembl database (http://asia.ensembl.org/index.html), this site of mutation is recorded as rs43703013 (ENSBTAT00000003409.6: c.516G>C). Polymorphisms of CSN2 were detected by employing PCR-RLFP method then confirmed with DNA sequencing analysis. Gene fragment of CSN2 at exon 7 was amplified using primer listed in Table 1, position of amplicon as well as the mutation site was illustrated in Figure 1.
In every PCR reaction a positive control using GAPDH gene, and negative control by replacing the gDNA to ddH2O were performed to ensure both reactions were working and no contamination available. We use PCR reactions which consist of 1 µl gDNA sample, 1 µl each for forward and reverse primer (IDT, Singapore), 5 µl GoTaq® Master Mixes (Promega, USA), and 12 µl sterile ddH2O. All the PCR reactions were set at 35 cycles and visualized in electrophoresis reaction using 2% agarose gel. Resulted PCR product then digested using MspI restriction enzyme (Thermo Scientific, USA) which cut the DNA at restriction site 5'…C^CGG…3'. In total 5µl of PCR product were incubated at 37°C for 40 minutes along with 5 unit of MspI. Next, digestion results then visualized in 2% agarose gel. β-casein allele types were determined by observing the resulted DNA band, A2 allele is indicated by digested PCR product into 208 and 25 bp. Meanwhile B allele was not digested. Part of PCR product simultaneously sent for sequencing using Sanger Methods. Resulted sequence then analyzed for amplified target confirmation.

Similarity and sequencing analysis.
The DNA sequencing in this study was interpreted using Unipro Ugene v. 39 software [13]. Subsequently, the result was confirmed by BLASTN on the NCBI website (https://blast.ncbi.nlm.nih.gov/Blast.cgi) [14].

Allele and genotype frequency.
Allele and genotype frequency were analyzed using formula from Nei and Kumar [15], as described in formulas (1) and (2). Where: is allele frequency i, is the sum of genotype ii individual, is the sum of heterozygote genotype ij, is total individual.

=
(2) is genotype frequency ii, is the sum of homozygote genotype ii, is total individual. Later, the genotype distribution in population was determined by Hardy-Weinberg Equilibrium (HWE) and statistically analyzed by chi-square test (χ 2 ) [15].

Results and discussion
Part of exon 7 of CSN2 gene was successfully amplified in this study as shown in Figure 2. The PCR reaction was able to amplify 233 bp of targeted DNA by primers designed in the earlier study [11]. Control positive (+) using GAPDH confirming that reaction was running well, and there is no contamination occurred as confirmed no DNA band in control negative (-) reaction. Additionally, the sequencing result of PCR product samples confirming the amplicon is part of CSN2 exon 7 with similarity 98.97% (Table 2).   Following amplification, genotyping was done by restriction enzyme digestion. As the result, 2 types of alleles namely B and A2 variant were revealed. In genotype variation, we found 2 genotypes that are BA2, shown by two DNA bands, and A2A2 which only single band (Figure 3). Furthermore, in our studied population, we didn't find any cattle carry BB genotype. The sequence analysis to confirm that result is presented in chromatogram (Figure 4), shows the SNP different in the mutation site from G (allele B) to C (allele A2). Analysis of allele show that allele A2 was higher in frequency compared to  (Table 3). HWE analysis shows no significant (P>0.05) different between genotype, which means the studied population is normally distributed.  Commonly CSN2 A1 variant has only found in European dairy cattle (Bos taurus) origin [9]. However, the variation of CSN2 allele now can be found in Bos indicus or other different dairy cattle worldwide. Purebred Asian and African cattle produce milk containing only the A2 beta-casein type, although some cattle produce A1 beta-casein as a consequence of crossbred ancestry. The A1 and A2 variant was reported in all dairy cattle both in Bos taurus and Bos indicus. For example the I, C, and B variant was found in Bos taurus and Bos indicus [16,17], while the E variant was reported only in Bos taurus [9]. Interestingly CSN2 B variant was reported to be existed in Bos javanicus which known to be Indonesian native cattle [18]. Notably, the mutations of CSN2 have been spreading and inherited since long time ago as part of evolutionary process of cattle domestication.
The result of higher frequency of A2 allele in this study compared to B allele, is in alignment with the previous report [19]. It is reported that A2 allele variant of CSN2 in Holstein cattle population studied in China, Rumania, and Italy have higher frequency compared to allele A1, B, I, A3, and C. Though the area of those studied populations are different but seem there are the same trend to produce digestive friendly milk is being a concern. This can be assumed due to the growing evidence of the negative effect of BCM-7 to human health risk factors.
CSN2 B is the variant of CSN2 which resulting highest BCM-7 compared to other variant during digestion [1,20]. In here, B variant particularly high release of BCM-7 compared to A1 variant [10]. It is explained that BCM-7 associated with lactose intolerance by inhibiting the lactase enzyme activity then affecting intestinal condition, thus lead to the problem of digestive system [1].
The growing demand of milk product and its safety, a concern to produce safer and friendly milk product, then manifested in the dairy cattle selection program. As we know, Friesian Holstein dairy cattle is the majority breed reared in Indonesia. This breed is selected and imported from either New Zealand or Australia to establish the herd population [21]. Since a lot of claims of unfavorable effect of BCM-7 to human health, New Zealand and Australia started to select and sold milk containing A2