- Email: [email protected]
Association of polymorphisms of beta-2-microglobulin gene (β2m) with milk IgG1 content in Chinese Holstein dairy cows
Livestock Science 143 (2012) 289–292
Contents lists available at SciVerse ScienceDirect
Livestock Science journal homepage: www.elsevier.com/locate/livsci
Short communication
Association of polymorphisms of beta-2-microglobulin gene (β2m) with milk IgG1 content in Chinese Holstein dairy cows Shengguo Zhao a, 1, Guanglei Liu a, b, 1, Jiaqi Wang a,⁎, Chunlin Zhang a, Dengpan Bu a, Kailang Liu a, Lingyun Zhou a a b
State Key Laboratory of Animal Nutrition, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, P. R. China Shanghai Bright Holstein Co., Ltd., Shanghai 200443, P. R. China
a r t i c l e
i n f o
Article history: Received 5 July 2010 Received in revised form 26 September 2011 Accepted 9 October 2011 Keywords: Beta-2-microglobulin Genotypes Immunoglobulin G1 Milk
a b s t r a c t Beta-2-microglobulin (β2m) is an integral component of the Fc receptor (FcRn) heterodimer which is involved in IgG transfer across mammary epithelial cells. The aim of this study was to estimate the frequency of alleles of β2m and the association of SNPs genotypes as well as haplotypes with milk IgG1 concentration and mass. Blood DNA from 189 Chinese Holstein dairy cows was extracted, sequenced and genotyping was analyzed. Milk IgG1 concentrations from these cows were determined by ELISA and evaluated with respect to their β2m genotypes. The results showed that two single nucleotide polymorphisms (SNPs) and one insertion/deletion (indel) of two base pairs, assorted into four haplotypes, were identified. The genotypes and haplotypes of β2m were found to be correlated (P b 0.05) with milk IgG1 concentration and mass. Dairy cows with homozygous deletion of β2m had the lowest milk IgG1 concentration and mass. These results indicate that β2m genotypes could serve as a marker to identify dairy cows with high IgG1 milk levels. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Immunoglobulin G (IgG) in bovine milk has a multitude of functions including opsonization, complement fixation, prevention of adhesion of pathogenic microbes to endothelial lining, agglutination of bacteria, and neutralization of toxins or viruses. Currently, there are lots of products with enhanced IgG content. More people are consuming this kind of milk products to obtain protective immunity. Consequently milk IgG levels are becoming increasingly important as new IgG enhanced milk products are being developed (Larson et al., 1980; Marnila and Korhonen, 2002).
⁎ Corresponding author at: State Key Laboratory of Animal Nutrition, Ruminant Nutrition Lab, Institute of Animal Science, Chinese Academy of Agricultural Sciences, No. 2, Yuanmingyuan West Road, Haidian District, Beijing, 100193, P.R. China. Tel.: + 86 10 6289 0458; fax: + 86 10 6289 7587. E-mail address: [email protected] (J. Wang). 1 These two authors contribute equally to this work. 1871-1413/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.livsci.2011.10.004
Concentration of IgG1 in milk has been shown to be influenced by a variety of factors, including genetic effects, hormonal regulation, age at calving, parity, nutritional status, premature parturition and stage of lactation (Liu et al., 2009, 2010). In fact IgG1 can be selectively transferred from serum into milk by a Fc receptor (FcRn) mediated mechanism found in mammary gland secretory epithelium (Anderson et al., 2006). The FcRn binds IgG1 at a weak acidic pH on basolateral surface of mammary gland epithelium and following transcytosis, then it releases the IgG1 into milk upon exposure to neutral pH. The FcRn, like MHC class I, is a heterodimer of α-chain (FCGRT) and β2-microglobulin (β2m) (Mayer et al., 2005; Roopenian and Akilesh, 2007). The β2m has been shown to be important for cell surface expression of FcRn and, in absence of β2m, FcRn is retained in endoplasmic reticulum. Furthermore, in absence of β2m, IgG1 binding is decreased compared with that of native FcRn (Kim et al., 2008). The studies have evaluated single nucleotide polymorphisms (SNPs) and haplotypes in bovine FCGRT, and found that they were associated with serum or colostrum IgG
290
S. Zhao et al. / Livestock Science 143 (2012) 289–292
concentration (Laegreid et al., 2002; Zhang et al., 2009). As we know, there is little information about the association between β2m haplotypes and milk IgG concentration. Only β2m haplotypes in exons II and IV in U.S. beef cattle and association with failure of passive transfer in newborn calves have been established (Clawson et al., 2004). Haplotypes of β2m especially of exon IV and its relationship with milk IgG1 content have not been previously researched. In our experiment haplotype structures of β2m exon IV were evaluated to determine their association with milk IgG1 content in Chinese Holstein dairy cows. 2. Materials and methods 2.1. Milk and blood samples collection One hundred eighty-nine Chinese Holstein cows were chosen from a large dairy farm near Beijing. Cows selected to be used in this study had not shown any clinical signs of mastitis (by checking for presence of redness, swelling, hardness and pain in the udder, or presence of clots in milk) for 2 months prior to inclusion in this study. All cows were maintained in a free stall barn and were fed a total mixed ration (TMR) three times daily. The cows with lactation number 1, 2, 3, 4–8 were 64, 45, 47 and 33 respectively. And the cows in 15–49, 50–109, 110–219, >220 days from parturition were 28, 51, 98 and 12 respectively. The average daily milk production of herd was 22.2 ± 6.3 kg. Milk samples were collected from each cow three times (in the morning, at mid-day, and in the evening) in a certain day and were combined in a 4:3:3 ratio by volume to form a composite sample. About 30 mL samples were centrifuged at 5,000g for 15 min at 4 °C to remove the fat component in prior to determining IgG1 concentration. Blood samples were collected into 8 mL Vacutainer tubes (BD Biosciences) from each cow using tail venipuncture procedure. One set of blood samples (5 mL) was used to prepare serum, and the remaining 3 mL was used for DNA extraction. 2.2. Determination of IgG1 concentration in milk and serum Quantitative determination of IgG1 in milk and serum was performed using Bovine IgG1 ELISA Quantitation Kits (Bethyl Laboratories, USA) according to manufacturer's instructions. Dilutions of milk and serum samples were 1:2000 and 1:8000, respectively. Intra-assay and inter-assay coefficients of variation were 5.4% and 3.2%, respectively. Mass of IgG1 for each cow was calculated using a modification of the method of Winger et al. (1995) as follows: IgG1 mass (g) = IgG1 concentration (mg/mL)× milk yield (g)/1.032 (mg/mL), where 1.032 was average specific gravity of milk.
contained 1.5 μL (50 ng/μL) DNA, 1.5 μL of each primers (10 μM), 20.5 μL dd H2O and 25 μL 2 × Taq PCR Mix (Qiagen Science, USA). Thermoprofiles included 94 °C for 5 min; 35 cycles of 94 °C for 30 s, 59.9 °C for 30 s, and 72 °C for 1 min; and a final extension at 72 °C for 10 min.
2.4. Polymorphism detection and genotyping analysis The PCR product was confirmed by agarose gel electrophoresis. The DNA of 475 bp was purified using an Agarose Gel DNA Purification kit (Qiagen Science, USA) and then both strands were sequenced using an ABI 3730XL DNA sequencer (Applied Biosystems, USA). Sequences were compared to GenBank database using BLAST. To identify polymorphisms in 3′-UTR of β2m IV, all sequences were compared using DNAMAN 4.0 software.
2.5. Statistical analysis Goodness of fit to the Hardy–Weinberg equilibrium (HWE) expectation was assessed by the χ 2 test for each locus in the population. Haplotype blocks of each individual and their frequencies were inferred using software PHASE 2.1 (Stephens and Donely, 2003). Genotype and allele frequencies were calculated as percentage of population examined. Association analysis between SNPs genotypes and haplotypes of β2m and IgG1 content and mass was carried out with the GLM procedure using SAS package (SAS Institute, Cary, NC). Data were analyzed with the following linear model. yijkl ¼ μ þ Hi þ Pj þ Mk þ eijkl where yijkl = IgG1 content or mass, μ = herd mean, Hi = lactation number effect, Pj = stage of lactation effect, Mk = genotype effect or haplotype effect, and eijkl = residual error. Lactation number was categorized as 1, 2, 3 and 4–8. Stage of lactation was categorized as 15–49, 50–109, 110–219, >220 days from parturition. The differences between solutions for genotypes and haplotypes were checked using Tukey test.
G
T
AT
deletion
T
C
2.3. DNA extraction and PCR Total genomic DNA was extracted from whole blood samples using the DNA isolation Kit (Qiagen Science, USA) according to manufacturers. The PCR primers (For-5′-GCTACATGTCC ATGTTTGACC-3′ and Rev-5′-TCGGTAGGAAGTTGTTTCAT C-3′) were designed using Oligo 6.0 software based on 3′untranslated region (3′-UTR) of β2m IV (NCBI accession number: NM_173893). The PCR reaction system (50 μL)
SNP1
Indel (2)
SNP2
Fig. 1. Sequence map of polymorphism regions in β2m.
S. Zhao et al. / Livestock Science 143 (2012) 289–292
291
Table 1 Frequencies of genotypes and alleles of each locus in β2m. Loci
Frequencies of genotype, % (number)
SNP1 (G/T)
TT GT GG −−/−− AT/−− AT/AT TT TC CC
Indel(2) (AT/–)
SNP2 (T/C)
1.06 15.87 83.07 3.70 29.10 67.20 90.48 8.99 0.53
(2) (30) (157) (7) (55) (127) (171) (17) (1)
Frequencies of allele, % (number)
Hardy–Weinberg equilibrium χ2 test
Significance P
T G
8.99 (34) 91.01 (344)
0.18
> 0.05
– AT
18.25 (69) 81.75 (309)
0.12
> 0.05
T C
94.97 (359) 5.03 (19)
0.63
> 0.05
3. Results and discussion After genotyping analysis, genetic variations G/T, AT/−−, T/C in the 3′-untranslated region of β2m exon IV were identified and named as SNP1, Indel(2), SNP2 (Fig.1). Three genotypes were also detected in each locus, frequencies of SNPs genotypes and alleles are shown in Table 1. Observed SNPs were in Hardy–Weinberg equilibrium (P > 0.05). Four haplotypes caused by three variations were found, of which H1, H2, H3 and H4 had a frequency of 68.25%, 4.76%, 17.99% and 8.99%, respectively (Table 2). Eight diplotypes were found within the 189 Holstein dairy cows, which were referred to as H1H1, H1H2, H1H3, H1H4, H2H3, H2H4, H3H3 and H4H4. The genotype H1H1 had the highest frequency (44.44%) when compared to other genotypes, and H3H3 (double base-pair deletion) had the frequency of 3.70% (Table 4). Other researchers had identified some other SNPs of β2m.Clawson et al. (2004) identified one and eleven SNPs in β2m exons II and IV in a multi-breed panel of ninety-six beef cattle, respectively. Association of β2m genotype with IgG1 content in milk and serum and mass in milk is summarized in Table 3. For SNP2, genotype TC was found to have the highest (P b 0.05) milk IgG1 mass though have similar IgG1 concentration in serum. Milk IgG1 concentration and mass from homozygous genotype −−/−− of Indel(2) were lower (P b 0.05) than those from genotype of AT/−− and AT/AT. The GLM analysis revealed that haplotypes of β2m were positively associated with milk IgG1 concentration and mass (P b 0.05), but were not associated with serum IgG1 concentration (P > 0.05) (Table 4). The concentration and mass of IgG1 in milk of cows carrying haplotypes H3H3 (double base-pair deletion) were lower (P b 0.05) than in milk of cows with H1H2 and H2H3, but similar (P > 0.05) to concentration and mass of IgG1 of cows with haplotypes H1H1, H1H3, H1H4 and H2H4. Milk IgG is mostly transferred from blood into milk in mammary gland and this is controlled by Fc receptor composed of α-chain encoding by FCGRT and β-chain encoding by β2m (Anderson et al., 2006; Mayer et al., 2005). Zhang et
al. (2009) evaluated haplotypes of bovine FCGRT (encoding α-chain) and their relationship to IgG concentration in bovine colostrum. Four SNPs classified into five haplotypes were identified in forty-nine Holstein–Frisians cows. Haplotype 5 was found to be significantly associated with a high IgG level and haplotype 2 exhibited a similar trend. Laegreid et al. (2002) also identified five SNPs and haplotypes of FCGRT, and indicated that these haplotypes markers were in linkage disequilibrium with genetic risk factors affecting passive transfer of IgG in beef cattle. Adamski et al. (2000) showed that the level of β2m (encoding β-chain) mRNA, not FCGRT, in the mammary gland increased during the switch phase when milk IgG concentration also was increasing. This suggests that expression of Fc receptor in the mammary gland is controlled by expression of β2m (Mayer et al., 2002; Praetor and Hunziker, 2002). In our study, we found that milk IgG1 concentration and mass from dairy cows with homozygous double base-pair deletion were significantly lower than group average. Even though serum IgG1 concentrations from each haplotype had no significant differences, milk IgG1 concentration and mass changed considerably among different haplotypes. Milk IgG1 concentration and mass from haplotype H3H3 were 4.2 and 3.9 times as small as that from haplotype H1H2. Our results indicated that β2m haplotypes are associated with regulation of IgG1 transfer from serum to milk by mammary gland. Therefore, the haplotype of β2m could be considered as a marker to be used for selecting dairy cows that would produce milk with high IgG1 level. Acknowledgments This work was supported by National Natural Science Foundation of China (30871837), Beijing Municipal Natural Science Foundation (6092017) and National International Cooperation Program (2009DFB30530), P. R. China. We are very grateful to Prof. Hans-Martin Seyfert of the Research Institute for the Biology of Farm Animals in Germany and Prof.
Table 2 Frequencies of haplotypes in β2m gene. Haplotype
Type
Frequencies, % (number)
H1 H2 H3 H4
AGAGGTGGG…TTATATGTTA…ATTGTGTGC AGAGGTGGG…TTATATGTTA…ATTGCGTGC AGAGGTGGG…TTAT – GTTA…ATTGTGTGC AGAGTTGGG…TTATATGTTA…ATTGTGTGC
68.25 4.76 17.99 8.99
(129) (9) (34) (17)
292
S. Zhao et al. / Livestock Science 143 (2012) 289–292
Table 3 Least squares means and their standard errors for studied traits as referred to genotypes of β2m gene polymorphism. Loci
Genotype
IgG1 concentration in milk (mg/mL)
IgG1 concentration in serum (mg/mL)
IgG1 mass in milk (g)
SNP1 (G/T)
GT GG TT −−/−− AT/−− AT/AT TT TC CC
0.27 ± 0.10 0.38 ± 0.12 NC 0.15 ± 0.07b 0.35 ± 0.10a 0.38 ± 0.11a 0.33 ± 0.12 0.43 ± 0.14 NC
14.00 ± 6.58 16.00 ± 7.12 NC 19.27 ± 5.53 17.90 ± 7.35 15.06 ± 4.63 15.87 ± 6.70 17.69 ± 6.48 NC
6.60 ± 2.64 8.50 ± 2.75 NC 3.50 ± 1.58b 7.71 ± 2.34a 8.56 ± 3.18a 7.34 ± 3.15b 14.52 ± 3.48a NC
Indel(2) (AT/−−)
SNP2 (T/C)
a,b Least squares means within the same loci column with different superscripts differ significantly (P b 0.05). NC means not calculated (n b 3).
Table 4 Least squares means and their standard errors for studied traits as referred to haplotypes of β2m gene polymorphism. Diplotype
Percentage (%)
IgG1 concentration in milk (mg/mL)
IgG1 concentration in serum (mg/mL)
IgG1 mass in milk (g)
H1H1 H1H2 H1H3 H1H4 H2H3 H3H3 H2H4 H4H4
44.44 7.41 26.46 14.29 1.59 3.70 1.06 1.06
0.36 ± 0.12ab 0.67 ± 0.14a 0.34 ± 0.11ab 0.31 ± 0.12ab 0.51 ± 0.14a 0.16 ± 0.08b NC NC
15.15 ± 8.95 15.03 ± 8.01 17.27 ± 6.38 14.49 ± 8.17 26.73 ± 8.57 19.50 ± 6.45 NC NC
8.09 ± 4.88ab 15.00 ± 6.48a 7.18 ± 4.35ab 7.10 ± 4.19ab 14.55 ± 5.11a 3.80 ± 2.01b NC NC
a,b,c Least squares means in the same column with different superscripts differ significantly (P b 0.05). NC means not calculated (n b 3).
Zhongtang Yu of The Ohio State University in USA for their assistance with the preparation of this manuscript. References Adamski, F.M., King, A.T., Demmer, J., 2000. Expression of the Fc receptor in the mammary gland during lactation in the marsupial Trichosurus vulpecula (brushtail possum). Mol. Immunol. 37, 435–444. Anderson, L.C., Chaudhury, C., Kim, J., Bronson, L.C., Wani, A.M., Mohanty, S., 2006. Perspective—FcRn transports albumin: relevance to immunology and medicine. Trends Immunol. 7, 343–348. Clawson, L.M., Heaton, P.M., Chitko-McKown, G.C., Fox, M.J., Smith, P.L.T., Snelling, M.W., Keele, W.J., Laegreid, W.W., 2004. Beta-2-microglobulin haplotypes in US beef cattle and association with failure of passive transfer in newborn calves. Mamm. Genome 15, 227–236. Kim, J., Bronson, C.L., Wani, M.A., Oberyszyn, T.M., Mohanty, S., Chaudhury, C., Hayton, W.L., Robinson, J.M., Anderson, C.L., 2008. β2-Microglobulin deficient mice catabolize IgG more rapidly than FcRn-α-chain deficient mice. Exp. Biol. Med. 233, 603–609. Laegreid, W.W., Heaton, P.M., Keen, E.J., Grosse, M.W., Chitko-McKown, G.C., Smith, P.L.T., Keele, W.J., Bennett, L.G., Besser, E.T., 2002. Association of bovine neonatal Fc receptor α-chain gene (FCGRT) haplotypes with serum IgG concentration in newborn calves. Mamm. Genome 13, 704–710. Larson, B.L., Heary Jr., H.L., Devery, J.E., 1980. Immunoglobulin production and transport by the mammary gland. J. Dairy Sci. 63 (4), 665–671. Liu, G.L., Wang, J.Q., Bu, D.P., Cheng, J.B., Zhang, C.G., Wei, H.Y., Zhou, L.Y., Zhou, Z.F., Hu, H., Dong, X.L., 2009. Factors affecting the transfer of immunoglobulin G1 into the milk of Holstein cows. Vet. J. 182 (1), 79–85.
Liu, G., Zhang, C., Wang, J., Bu, D., Zhou, L., Zhao, S., Li, S., 2010. Canonical correlation of milk immunoglobulins, lactoferrin concentration and Dairy Herd Improvement data of Chinese Holstein cows. Livest. Sci. 128 (1–3), 197–200. Marnila, P., Korhonen, H., 2002. Immunoglobulins. In: Roginski, H., Fuquay, W.J., Fox, F.P. (Eds.), Encyclopedia of Dairy Sciences. Academic Press, London, UK, pp. 1950–1956. Mayer, B., Zolnai, A., Frenyo, L.V., Jancsik, V., Szentirmay, Z., Hammarstrom, L., Kacskovics, I., 2002. Redistribution of the sheep neonatal Fc receptor in the mammary gland around the time of parturition in ewes and its localization in the small intestine of neonatal lambs. Immunol. 107 (3), 288–296. Mayer, B., Doleschall, M., Bender, B., Bartyik, J., Bosze, Z., Frenyó, L.V., Kacskovics, I., 2005. Expression of the neonatal Fc receptor (FcRn) in the bovine mammary gland. J. Dairy Res. 72, 107–112. Praetor, A., Hunziker, W., 2002. β2-Microglobulin is important for cell surface expression and pH-dependent IgG binding of human FcRn. J. Cell Sci. 115, 2389–2397. Roopenian, C.D., Akilesh, S., 2007. FcRn: the neonatal Fc receptor comes of age. Nat. Rev. Immunol. 7, 715–725. Stephens, M., Donely, P., 2003. A comparison of Bayesian methods for haplotype reconstruction from population genotype data. Am. J. Hum. Genet. 73, 1162–1169. Winger, K.L., Gay, C.C., Besser, E.T., 1995. Immunoglobulin Gl transfer into induced mammary secretions: the effect of dexamethasone. J. Dairy Sci. 78, 1306–1309. Zhang, R., Zhao, Z., Zhao, Y., Kacskovics, I., Eijk, M., Groot, N., Li, N., Hammarstrom, L., 2009. Association of FcRn heavy chain encoding gene (FCGRT) polymorphisms with IgG content in bovine colostrum. Anim. Biotechnol. 20 (4), 242–246.
Contents lists available at SciVerse ScienceDirect
Livestock Science journal homepage: www.elsevier.com/locate/livsci
Short communication
Association of polymorphisms of beta-2-microglobulin gene (β2m) with milk IgG1 content in Chinese Holstein dairy cows Shengguo Zhao a, 1, Guanglei Liu a, b, 1, Jiaqi Wang a,⁎, Chunlin Zhang a, Dengpan Bu a, Kailang Liu a, Lingyun Zhou a a b
State Key Laboratory of Animal Nutrition, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, P. R. China Shanghai Bright Holstein Co., Ltd., Shanghai 200443, P. R. China
a r t i c l e
i n f o
Article history: Received 5 July 2010 Received in revised form 26 September 2011 Accepted 9 October 2011 Keywords: Beta-2-microglobulin Genotypes Immunoglobulin G1 Milk
a b s t r a c t Beta-2-microglobulin (β2m) is an integral component of the Fc receptor (FcRn) heterodimer which is involved in IgG transfer across mammary epithelial cells. The aim of this study was to estimate the frequency of alleles of β2m and the association of SNPs genotypes as well as haplotypes with milk IgG1 concentration and mass. Blood DNA from 189 Chinese Holstein dairy cows was extracted, sequenced and genotyping was analyzed. Milk IgG1 concentrations from these cows were determined by ELISA and evaluated with respect to their β2m genotypes. The results showed that two single nucleotide polymorphisms (SNPs) and one insertion/deletion (indel) of two base pairs, assorted into four haplotypes, were identified. The genotypes and haplotypes of β2m were found to be correlated (P b 0.05) with milk IgG1 concentration and mass. Dairy cows with homozygous deletion of β2m had the lowest milk IgG1 concentration and mass. These results indicate that β2m genotypes could serve as a marker to identify dairy cows with high IgG1 milk levels. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Immunoglobulin G (IgG) in bovine milk has a multitude of functions including opsonization, complement fixation, prevention of adhesion of pathogenic microbes to endothelial lining, agglutination of bacteria, and neutralization of toxins or viruses. Currently, there are lots of products with enhanced IgG content. More people are consuming this kind of milk products to obtain protective immunity. Consequently milk IgG levels are becoming increasingly important as new IgG enhanced milk products are being developed (Larson et al., 1980; Marnila and Korhonen, 2002).
⁎ Corresponding author at: State Key Laboratory of Animal Nutrition, Ruminant Nutrition Lab, Institute of Animal Science, Chinese Academy of Agricultural Sciences, No. 2, Yuanmingyuan West Road, Haidian District, Beijing, 100193, P.R. China. Tel.: + 86 10 6289 0458; fax: + 86 10 6289 7587. E-mail address: [email protected] (J. Wang). 1 These two authors contribute equally to this work. 1871-1413/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.livsci.2011.10.004
Concentration of IgG1 in milk has been shown to be influenced by a variety of factors, including genetic effects, hormonal regulation, age at calving, parity, nutritional status, premature parturition and stage of lactation (Liu et al., 2009, 2010). In fact IgG1 can be selectively transferred from serum into milk by a Fc receptor (FcRn) mediated mechanism found in mammary gland secretory epithelium (Anderson et al., 2006). The FcRn binds IgG1 at a weak acidic pH on basolateral surface of mammary gland epithelium and following transcytosis, then it releases the IgG1 into milk upon exposure to neutral pH. The FcRn, like MHC class I, is a heterodimer of α-chain (FCGRT) and β2-microglobulin (β2m) (Mayer et al., 2005; Roopenian and Akilesh, 2007). The β2m has been shown to be important for cell surface expression of FcRn and, in absence of β2m, FcRn is retained in endoplasmic reticulum. Furthermore, in absence of β2m, IgG1 binding is decreased compared with that of native FcRn (Kim et al., 2008). The studies have evaluated single nucleotide polymorphisms (SNPs) and haplotypes in bovine FCGRT, and found that they were associated with serum or colostrum IgG
290
S. Zhao et al. / Livestock Science 143 (2012) 289–292
concentration (Laegreid et al., 2002; Zhang et al., 2009). As we know, there is little information about the association between β2m haplotypes and milk IgG concentration. Only β2m haplotypes in exons II and IV in U.S. beef cattle and association with failure of passive transfer in newborn calves have been established (Clawson et al., 2004). Haplotypes of β2m especially of exon IV and its relationship with milk IgG1 content have not been previously researched. In our experiment haplotype structures of β2m exon IV were evaluated to determine their association with milk IgG1 content in Chinese Holstein dairy cows. 2. Materials and methods 2.1. Milk and blood samples collection One hundred eighty-nine Chinese Holstein cows were chosen from a large dairy farm near Beijing. Cows selected to be used in this study had not shown any clinical signs of mastitis (by checking for presence of redness, swelling, hardness and pain in the udder, or presence of clots in milk) for 2 months prior to inclusion in this study. All cows were maintained in a free stall barn and were fed a total mixed ration (TMR) three times daily. The cows with lactation number 1, 2, 3, 4–8 were 64, 45, 47 and 33 respectively. And the cows in 15–49, 50–109, 110–219, >220 days from parturition were 28, 51, 98 and 12 respectively. The average daily milk production of herd was 22.2 ± 6.3 kg. Milk samples were collected from each cow three times (in the morning, at mid-day, and in the evening) in a certain day and were combined in a 4:3:3 ratio by volume to form a composite sample. About 30 mL samples were centrifuged at 5,000g for 15 min at 4 °C to remove the fat component in prior to determining IgG1 concentration. Blood samples were collected into 8 mL Vacutainer tubes (BD Biosciences) from each cow using tail venipuncture procedure. One set of blood samples (5 mL) was used to prepare serum, and the remaining 3 mL was used for DNA extraction. 2.2. Determination of IgG1 concentration in milk and serum Quantitative determination of IgG1 in milk and serum was performed using Bovine IgG1 ELISA Quantitation Kits (Bethyl Laboratories, USA) according to manufacturer's instructions. Dilutions of milk and serum samples were 1:2000 and 1:8000, respectively. Intra-assay and inter-assay coefficients of variation were 5.4% and 3.2%, respectively. Mass of IgG1 for each cow was calculated using a modification of the method of Winger et al. (1995) as follows: IgG1 mass (g) = IgG1 concentration (mg/mL)× milk yield (g)/1.032 (mg/mL), where 1.032 was average specific gravity of milk.
contained 1.5 μL (50 ng/μL) DNA, 1.5 μL of each primers (10 μM), 20.5 μL dd H2O and 25 μL 2 × Taq PCR Mix (Qiagen Science, USA). Thermoprofiles included 94 °C for 5 min; 35 cycles of 94 °C for 30 s, 59.9 °C for 30 s, and 72 °C for 1 min; and a final extension at 72 °C for 10 min.
2.4. Polymorphism detection and genotyping analysis The PCR product was confirmed by agarose gel electrophoresis. The DNA of 475 bp was purified using an Agarose Gel DNA Purification kit (Qiagen Science, USA) and then both strands were sequenced using an ABI 3730XL DNA sequencer (Applied Biosystems, USA). Sequences were compared to GenBank database using BLAST. To identify polymorphisms in 3′-UTR of β2m IV, all sequences were compared using DNAMAN 4.0 software.
2.5. Statistical analysis Goodness of fit to the Hardy–Weinberg equilibrium (HWE) expectation was assessed by the χ 2 test for each locus in the population. Haplotype blocks of each individual and their frequencies were inferred using software PHASE 2.1 (Stephens and Donely, 2003). Genotype and allele frequencies were calculated as percentage of population examined. Association analysis between SNPs genotypes and haplotypes of β2m and IgG1 content and mass was carried out with the GLM procedure using SAS package (SAS Institute, Cary, NC). Data were analyzed with the following linear model. yijkl ¼ μ þ Hi þ Pj þ Mk þ eijkl where yijkl = IgG1 content or mass, μ = herd mean, Hi = lactation number effect, Pj = stage of lactation effect, Mk = genotype effect or haplotype effect, and eijkl = residual error. Lactation number was categorized as 1, 2, 3 and 4–8. Stage of lactation was categorized as 15–49, 50–109, 110–219, >220 days from parturition. The differences between solutions for genotypes and haplotypes were checked using Tukey test.
G
T
AT
deletion
T
C
2.3. DNA extraction and PCR Total genomic DNA was extracted from whole blood samples using the DNA isolation Kit (Qiagen Science, USA) according to manufacturers. The PCR primers (For-5′-GCTACATGTCC ATGTTTGACC-3′ and Rev-5′-TCGGTAGGAAGTTGTTTCAT C-3′) were designed using Oligo 6.0 software based on 3′untranslated region (3′-UTR) of β2m IV (NCBI accession number: NM_173893). The PCR reaction system (50 μL)
SNP1
Indel (2)
SNP2
Fig. 1. Sequence map of polymorphism regions in β2m.
S. Zhao et al. / Livestock Science 143 (2012) 289–292
291
Table 1 Frequencies of genotypes and alleles of each locus in β2m. Loci
Frequencies of genotype, % (number)
SNP1 (G/T)
TT GT GG −−/−− AT/−− AT/AT TT TC CC
Indel(2) (AT/–)
SNP2 (T/C)
1.06 15.87 83.07 3.70 29.10 67.20 90.48 8.99 0.53
(2) (30) (157) (7) (55) (127) (171) (17) (1)
Frequencies of allele, % (number)
Hardy–Weinberg equilibrium χ2 test
Significance P
T G
8.99 (34) 91.01 (344)
0.18
> 0.05
– AT
18.25 (69) 81.75 (309)
0.12
> 0.05
T C
94.97 (359) 5.03 (19)
0.63
> 0.05
3. Results and discussion After genotyping analysis, genetic variations G/T, AT/−−, T/C in the 3′-untranslated region of β2m exon IV were identified and named as SNP1, Indel(2), SNP2 (Fig.1). Three genotypes were also detected in each locus, frequencies of SNPs genotypes and alleles are shown in Table 1. Observed SNPs were in Hardy–Weinberg equilibrium (P > 0.05). Four haplotypes caused by three variations were found, of which H1, H2, H3 and H4 had a frequency of 68.25%, 4.76%, 17.99% and 8.99%, respectively (Table 2). Eight diplotypes were found within the 189 Holstein dairy cows, which were referred to as H1H1, H1H2, H1H3, H1H4, H2H3, H2H4, H3H3 and H4H4. The genotype H1H1 had the highest frequency (44.44%) when compared to other genotypes, and H3H3 (double base-pair deletion) had the frequency of 3.70% (Table 4). Other researchers had identified some other SNPs of β2m.Clawson et al. (2004) identified one and eleven SNPs in β2m exons II and IV in a multi-breed panel of ninety-six beef cattle, respectively. Association of β2m genotype with IgG1 content in milk and serum and mass in milk is summarized in Table 3. For SNP2, genotype TC was found to have the highest (P b 0.05) milk IgG1 mass though have similar IgG1 concentration in serum. Milk IgG1 concentration and mass from homozygous genotype −−/−− of Indel(2) were lower (P b 0.05) than those from genotype of AT/−− and AT/AT. The GLM analysis revealed that haplotypes of β2m were positively associated with milk IgG1 concentration and mass (P b 0.05), but were not associated with serum IgG1 concentration (P > 0.05) (Table 4). The concentration and mass of IgG1 in milk of cows carrying haplotypes H3H3 (double base-pair deletion) were lower (P b 0.05) than in milk of cows with H1H2 and H2H3, but similar (P > 0.05) to concentration and mass of IgG1 of cows with haplotypes H1H1, H1H3, H1H4 and H2H4. Milk IgG is mostly transferred from blood into milk in mammary gland and this is controlled by Fc receptor composed of α-chain encoding by FCGRT and β-chain encoding by β2m (Anderson et al., 2006; Mayer et al., 2005). Zhang et
al. (2009) evaluated haplotypes of bovine FCGRT (encoding α-chain) and their relationship to IgG concentration in bovine colostrum. Four SNPs classified into five haplotypes were identified in forty-nine Holstein–Frisians cows. Haplotype 5 was found to be significantly associated with a high IgG level and haplotype 2 exhibited a similar trend. Laegreid et al. (2002) also identified five SNPs and haplotypes of FCGRT, and indicated that these haplotypes markers were in linkage disequilibrium with genetic risk factors affecting passive transfer of IgG in beef cattle. Adamski et al. (2000) showed that the level of β2m (encoding β-chain) mRNA, not FCGRT, in the mammary gland increased during the switch phase when milk IgG concentration also was increasing. This suggests that expression of Fc receptor in the mammary gland is controlled by expression of β2m (Mayer et al., 2002; Praetor and Hunziker, 2002). In our study, we found that milk IgG1 concentration and mass from dairy cows with homozygous double base-pair deletion were significantly lower than group average. Even though serum IgG1 concentrations from each haplotype had no significant differences, milk IgG1 concentration and mass changed considerably among different haplotypes. Milk IgG1 concentration and mass from haplotype H3H3 were 4.2 and 3.9 times as small as that from haplotype H1H2. Our results indicated that β2m haplotypes are associated with regulation of IgG1 transfer from serum to milk by mammary gland. Therefore, the haplotype of β2m could be considered as a marker to be used for selecting dairy cows that would produce milk with high IgG1 level. Acknowledgments This work was supported by National Natural Science Foundation of China (30871837), Beijing Municipal Natural Science Foundation (6092017) and National International Cooperation Program (2009DFB30530), P. R. China. We are very grateful to Prof. Hans-Martin Seyfert of the Research Institute for the Biology of Farm Animals in Germany and Prof.
Table 2 Frequencies of haplotypes in β2m gene. Haplotype
Type
Frequencies, % (number)
H1 H2 H3 H4
AGAGGTGGG…TTATATGTTA…ATTGTGTGC AGAGGTGGG…TTATATGTTA…ATTGCGTGC AGAGGTGGG…TTAT – GTTA…ATTGTGTGC AGAGTTGGG…TTATATGTTA…ATTGTGTGC
68.25 4.76 17.99 8.99
(129) (9) (34) (17)
292
S. Zhao et al. / Livestock Science 143 (2012) 289–292
Table 3 Least squares means and their standard errors for studied traits as referred to genotypes of β2m gene polymorphism. Loci
Genotype
IgG1 concentration in milk (mg/mL)
IgG1 concentration in serum (mg/mL)
IgG1 mass in milk (g)
SNP1 (G/T)
GT GG TT −−/−− AT/−− AT/AT TT TC CC
0.27 ± 0.10 0.38 ± 0.12 NC 0.15 ± 0.07b 0.35 ± 0.10a 0.38 ± 0.11a 0.33 ± 0.12 0.43 ± 0.14 NC
14.00 ± 6.58 16.00 ± 7.12 NC 19.27 ± 5.53 17.90 ± 7.35 15.06 ± 4.63 15.87 ± 6.70 17.69 ± 6.48 NC
6.60 ± 2.64 8.50 ± 2.75 NC 3.50 ± 1.58b 7.71 ± 2.34a 8.56 ± 3.18a 7.34 ± 3.15b 14.52 ± 3.48a NC
Indel(2) (AT/−−)
SNP2 (T/C)
a,b Least squares means within the same loci column with different superscripts differ significantly (P b 0.05). NC means not calculated (n b 3).
Table 4 Least squares means and their standard errors for studied traits as referred to haplotypes of β2m gene polymorphism. Diplotype
Percentage (%)
IgG1 concentration in milk (mg/mL)
IgG1 concentration in serum (mg/mL)
IgG1 mass in milk (g)
H1H1 H1H2 H1H3 H1H4 H2H3 H3H3 H2H4 H4H4
44.44 7.41 26.46 14.29 1.59 3.70 1.06 1.06
0.36 ± 0.12ab 0.67 ± 0.14a 0.34 ± 0.11ab 0.31 ± 0.12ab 0.51 ± 0.14a 0.16 ± 0.08b NC NC
15.15 ± 8.95 15.03 ± 8.01 17.27 ± 6.38 14.49 ± 8.17 26.73 ± 8.57 19.50 ± 6.45 NC NC
8.09 ± 4.88ab 15.00 ± 6.48a 7.18 ± 4.35ab 7.10 ± 4.19ab 14.55 ± 5.11a 3.80 ± 2.01b NC NC
a,b,c Least squares means in the same column with different superscripts differ significantly (P b 0.05). NC means not calculated (n b 3).
Zhongtang Yu of The Ohio State University in USA for their assistance with the preparation of this manuscript. References Adamski, F.M., King, A.T., Demmer, J., 2000. Expression of the Fc receptor in the mammary gland during lactation in the marsupial Trichosurus vulpecula (brushtail possum). Mol. Immunol. 37, 435–444. Anderson, L.C., Chaudhury, C., Kim, J., Bronson, L.C., Wani, A.M., Mohanty, S., 2006. Perspective—FcRn transports albumin: relevance to immunology and medicine. Trends Immunol. 7, 343–348. Clawson, L.M., Heaton, P.M., Chitko-McKown, G.C., Fox, M.J., Smith, P.L.T., Snelling, M.W., Keele, W.J., Laegreid, W.W., 2004. Beta-2-microglobulin haplotypes in US beef cattle and association with failure of passive transfer in newborn calves. Mamm. Genome 15, 227–236. Kim, J., Bronson, C.L., Wani, M.A., Oberyszyn, T.M., Mohanty, S., Chaudhury, C., Hayton, W.L., Robinson, J.M., Anderson, C.L., 2008. β2-Microglobulin deficient mice catabolize IgG more rapidly than FcRn-α-chain deficient mice. Exp. Biol. Med. 233, 603–609. Laegreid, W.W., Heaton, P.M., Keen, E.J., Grosse, M.W., Chitko-McKown, G.C., Smith, P.L.T., Keele, W.J., Bennett, L.G., Besser, E.T., 2002. Association of bovine neonatal Fc receptor α-chain gene (FCGRT) haplotypes with serum IgG concentration in newborn calves. Mamm. Genome 13, 704–710. Larson, B.L., Heary Jr., H.L., Devery, J.E., 1980. Immunoglobulin production and transport by the mammary gland. J. Dairy Sci. 63 (4), 665–671. Liu, G.L., Wang, J.Q., Bu, D.P., Cheng, J.B., Zhang, C.G., Wei, H.Y., Zhou, L.Y., Zhou, Z.F., Hu, H., Dong, X.L., 2009. Factors affecting the transfer of immunoglobulin G1 into the milk of Holstein cows. Vet. J. 182 (1), 79–85.
Liu, G., Zhang, C., Wang, J., Bu, D., Zhou, L., Zhao, S., Li, S., 2010. Canonical correlation of milk immunoglobulins, lactoferrin concentration and Dairy Herd Improvement data of Chinese Holstein cows. Livest. Sci. 128 (1–3), 197–200. Marnila, P., Korhonen, H., 2002. Immunoglobulins. In: Roginski, H., Fuquay, W.J., Fox, F.P. (Eds.), Encyclopedia of Dairy Sciences. Academic Press, London, UK, pp. 1950–1956. Mayer, B., Zolnai, A., Frenyo, L.V., Jancsik, V., Szentirmay, Z., Hammarstrom, L., Kacskovics, I., 2002. Redistribution of the sheep neonatal Fc receptor in the mammary gland around the time of parturition in ewes and its localization in the small intestine of neonatal lambs. Immunol. 107 (3), 288–296. Mayer, B., Doleschall, M., Bender, B., Bartyik, J., Bosze, Z., Frenyó, L.V., Kacskovics, I., 2005. Expression of the neonatal Fc receptor (FcRn) in the bovine mammary gland. J. Dairy Res. 72, 107–112. Praetor, A., Hunziker, W., 2002. β2-Microglobulin is important for cell surface expression and pH-dependent IgG binding of human FcRn. J. Cell Sci. 115, 2389–2397. Roopenian, C.D., Akilesh, S., 2007. FcRn: the neonatal Fc receptor comes of age. Nat. Rev. Immunol. 7, 715–725. Stephens, M., Donely, P., 2003. A comparison of Bayesian methods for haplotype reconstruction from population genotype data. Am. J. Hum. Genet. 73, 1162–1169. Winger, K.L., Gay, C.C., Besser, E.T., 1995. Immunoglobulin Gl transfer into induced mammary secretions: the effect of dexamethasone. J. Dairy Sci. 78, 1306–1309. Zhang, R., Zhao, Z., Zhao, Y., Kacskovics, I., Eijk, M., Groot, N., Li, N., Hammarstrom, L., 2009. Association of FcRn heavy chain encoding gene (FCGRT) polymorphisms with IgG content in bovine colostrum. Anim. Biotechnol. 20 (4), 242–246.