We investigated the effects of prolactin gene polymorphisms on milk contents in women. The main aim of this work is to determine the genotypes of prolactin and its relationship with some milk chemical contents in women. Genotyping was carried out at Molecular Genetic Laboratory at the College of Agriculture, whereas biochemical assays were performed at the Department of Diseases Analyses at the South Technical University. Blood samples were collected for the prolactin-related gene. DNA was extracted from fifty candidate women. The extra-pituitary prolactin gene promoter 1149 G/T was subjected to XapI restriction enzyme. In this analysis   PRL-Pxa, I products result in three genotypes TT, TG and GG, as well as the population, is under Hardy-Weinberg equilibrium. Our results showed that the highest milk fat yield, milk protein, lactose and sold not fat (SNF) materials percentages were obtained by the genotype GG

AY, Ayied

FA, Ahmed

NM, Awad

American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS)

    21   American Scientific Research Journal for Engineering, Technology,  and Sciences  (ASRJETS) ISSN (Print) 2313-4410, ISSN (Online) 2313-4402 © Global Society of Scientific Research and Researchers  http://asrjetsjournal.org/   An Association of Prolactin Gene Polymorphisms with Some Milk Traits in Women Ahmed FAa, Ayied AYb*, Awad NMc aDepartment of Biology; College of Education for Pure Sciences; University of Basrah/Iraq b,cDepartment of Animal Resources; College of Agriculture; University of Basrah/Iraq aEmail: faizah_noorahmed@yahoo.com  bEmail: asaad.yheia@gmail.com  Abstract We investigated the effects of prolactin gene polymorphisms on milk contents in women. The main aim of this work is to determine the genotypes of prolactin and its relationship with some milk chemical contents in women. Genotyping was carried out at Molecular Genetic Laboratory at the College of Agriculture, whereas biochemical assays were performed at the Department of Diseases Analyses at the South Technical University. Blood samples were collected for the prolactin-related gene. DNA was extracted from fifty candidate women. The extra-pituitary prolactin gene promoter 1149 G/T was subjected to XapI restriction enzyme. In this analysis   PRL-Pxa, I products result in three genotypes TT, TG and GG, as well as the population, is under Hardy-Weinberg equilibrium. Our results showed that the highest milk fat yield, milk protein, lactose and sold not fat (SNF) materials percentages were obtained by the genotype GG.  Keywords: Prolactin gene; Polymorphisms; Women; Milk traits. 1. Introduction  Human prolactin is encoded by a single gene located on chromosome 6 and composed of 5 exons and 5 introns [1] .Transcription of prolactin gene is regulated by two independent promoter regions, in the pituitary gland transcription starts from the promoter of the 1b exon, whereas the second promoter is that of a non-coding exon is active in the extra-pituitary gland [2]. The length of human prolactin cDNA is 914 nucleotide with 618 open reading frame nucleotide, code for a prohormone consists of 227 amino acid [3]. Its peptide signal has 28 amino acids; for this reason, the mature human prolactin composed of 119 amino acids.  ------------------------------------------------------------------------ * Corresponding author.  American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) (2018) Volume   47 , No  1, pp 21-27  22  In mammals, prolactin hormone is best known for regulation of lactation and reproduction; since it binds to mammary receptors and enhances DNA transcription as well as it suppresses sex hormones (LH and FSH) in lactations [4].  The main aim of this work is to determine the genotypes of prolactin and its relationship with some milk chemical contents in women. 2.  Materials and Methods Biochemical assays were measured at the Dairy laboratory at the College of Agriculture, genotyping was analyzed at the laboratory of Genetic Engineering at the College of Agriculture and Department of Diseases Analyses at the South Technical University. 2.1. Milk Samples  Fifty women were studied. 20 ml milk samples were collected from each woman once only. Milk components (fat, protein, lactose, non-fat dried material), Funk Gerber Lacto Flash – Germany.  2.2. DNA Extraction and amplification Blood DNA extraction was carried performed using the Geneaid apparatus with some modifications. For detection of the obtained sample, DNA was electrophoresis in 1% agarose with ethidium bromide [5].  The extra-pituitary PRL-XapI genotypes were analyzed using polymerase chain reaction- RFLP method. A 338 bp fragments of G/T 1149 promoter gene was amplified using the primer forward 5'- AGA ATT GGA GTT CCA GTG CC-3' and reverse 5'- ATC  ACA CTC AAC CAG TTG GC-3' [6].  2.3. Statistical Analysis Data were analyzed using pop gene program [7] to evaluate F- statistics, Fis, and the observed homozygosity and heterozygosity mean for all the three different genotypes, comparisons were calculated using [8]. Additive values, dominance deviation, dominance mean [9]. 3. Results and Discussion 3.1. Amplification Product Amplification product from prolactin gene promoter was performed successfully as observed by others [6] and [10]. As seen from PCR results (Figure1), amplification of PRL primer was 338 bp (single band) using 100 DNA marker was observed in all blood samples. Two alleles and three genotypes were obtained as following: DNA restriction fragments were obtained for PRL-Xap1 polymorphisms; 280 and 56 for GG; 338, 280 and 56 for GT and undigested 338 for TT (Figure 2). American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) (2018) Volume   47 , No  1, pp 21-27  23  The role of genetic polymorphism of G/T PRL-1149 promoter region has been demonstrated in many autoimmune diseases like systemic sclerosis, multiple sclerosis, polymyositis arthritis, psoriatic arthritis and lupus [11, 12, 13, 14].   Figure 1:  Amplification of PCR product of prolactin gene 338 bp.  Figure 2: The polymerase chain reaction product of prolactin gene promoter bp 338 digested with Xap-1 on 2% agarose gel electrophoresis and stained with ethidium bromide. TT genotype of undigested 338 PCR product; GG genotype = 280 and 56 bp. Our results were consistent with findings made by [10] and [6]. Treadwell [10] demonstrated the important role of SNP of G/T 1149 (rs1341239) of the extra-pituitary PRL in a sample of lupus women of African or European ethnicity; and suggested the genotype TT- 1149 may be a risk factor associated with a predisposition of lupus. This study is recommended to be planned with larger population sample and different ethnic background.  A recent study reported the presence of different regulation mechanisms and functional activity of pituitary PRL-1149 polymorphism [6]. PRL-1149 is organ-specific; its expressions in many immune-related organs like the spleen and lymphoid glands [16].  PRL was originally identified as a hormone of pituitary origin, but extra-pituitary tissues also express this protein [16]. Both pituitary and extra-pituitary prolactin coded by the same gene directed by two independent promoters. Extrapituitary PRL expression is a specialized independent single cell such as pituitary transcriptional factor-1 which activates pituitary PRL transcription [17]. Genetic polymorphisms of extra-pituitary promoter have been observed with which contain SNP G/T 1149 (rs 1341239) at the sequence GATA [14]. American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) (2018) Volume   47 , No  1, pp 21-27  24  3.2. Genetic Description Table 1 demonstrates the observed and expected number of genotypes for 50 women enrolled in this study. Genotype GT 27 and 24.03 respectively which is the most frequent among the studied group, followed by GG 17 and 18.48 whereas TT observed and expected numbers were 6 and 7.48 respectively. There were no statistical differences between the group's chi-square (χ2) = 0.78 was not significant. The population was in equilibrium at this restriction site using the Hardy-Weinberg equilibrium. Probability value was (G) 0.79 as observed by [6] who recorded that the number of rheumatoid arthritis (RA) women of genotype GT was higher than both TT and GG genotypes. TT had the least observed arthritis women. Whereas for healthy women; GG and GT were more frequent. TT had the least number among the control group. The expected numbers of the control were higher; chi-square values (χ2) = 0.09 and 1.11 for patients and control respectively. The population was in equilibrium using Hardy-Weinberg equilibrium. Table 1:  Distribution of PRL-1149 G/T Promoter genotypes values Genotypes Observed No. Expected No. χ2 G2 TT 6 7.48 0.78 0.79 GT 27 24.03 GG 17 18.48 Total 50 50  3.3. Genetic Variation Table 2 demonstrates the presence of two alleles; G and T and three genotypes (TT, GT, and GG). G and T allelic frequencies were 0.61 and 0.39 respectively. Genotypes frequencies were 0.12, 0.54 and 0.34 respectively. The prevalence of the heterozygous GT was higher than both TT and GG. Fixation index (Fis) for G and T alleles was ˗0.1349 and 0.1349 respectively, this indicates there no consanguineous marriage as approved by the negative sign. Our findings were in accordance with previously reported   results by [6], who studied PRL-1149 polymorphisms in a sample of rheumatoid arthritis (RA) women. He found that G-allele frequency was higher in patients group compared with the control group, on the contrary, the T allele. The frequency of TT genotype was highest in control group in comparison with the patient's group, whereas the GG and GT genotypes were more abundant in patients than the control group. This difference between allele frequencies can be translated to develop RA among those carrying G allele, but the abundance of T allele seems to be protective. An association between T allele frequency and decreasing susceptibility to RA was observed [18]. The relationship between GG, GT genotypes and the predisposition to RA was noticed [6]. Further confirmation of this result was achieved. The same result was found by [14]; the dominance of GG genotype among the healthy group. GG genotype frequency was higher in RA patients so that could confer a risk for RA in the Iraqi society [6].  The PRL-114 G/T polymorphism among population can be due to ethnic- genetic American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) (2018) Volume   47 , No  1, pp 21-27  25  heterogeneity that might reflect migration history and the impact of natural selection force that shaped genetic variation in a population. [19, 20]. The further interesting finding has been recorded, an association of PRL-114 G/T polymorphism and RA patients; T allele frequency and TT genotypes were higher in RA patients than the control group and vice versa for G allele and GG genotype [6]. Table 2:  Alleles Frequencies, Genotypes frequencies and Fixation Index (Fis) Women total No.  Alleles frequencies Genotypes frequencies        Fis 50 G T TT GT GG T G 0.61 0.39 0.12 0.54 0.34 -0.1349 -0.1349  3.4. Milk chemical Composition Table 3 presents the results of milk chemical composition in women. There is a significant difference between prolactin genotypes for milk chemical composition percentages (fat, protein, ash, humidity, lactose, and non-fat dried materials). The genotype GG had higher milk chemical components were as follows: 3.78% fat, 4.26% protein, 0.26% ash, 5.58% lactose and 10.37% non-fat dried materials that both GT and TT genotypes. TT genotype had higher humidity (93.945) than both GT and GG genotypes.  Prolactin is one of the candidate genes to be studied since it plays a crucial role in the initiation and maintenance of lactation and expression of milk protein genes. Lactation polymorphism is due to genetic background (ethnicity), as well as physiological and environmental factors have a crucial impact on milk traits. Table 3: Milk chemical composition (%) in women ± Standard Deviation Traits Genotypes TT GT GG Fat 0.96±3.78 1.10±2.64 0.93±1.71 Protein 1.14±4.26 2.26±0.84 1.93±0.87 Ash .620±.820 0.97±0.52 0.35±0.28 Moisture 1.28±85.57 0.79±90.90 0.82±93.94 Lactose 0.72±5.58 0.92±3.66 1.24±3.53 Solid not fat 10.37±0.89 6.16±0.99 6.31±0.60 References [1] Owerbach, D, Rutter, WJ, Cooke, NE, Martial, JA, Shows, TB “The prolactin gene is located on chromosome 6 in humans”. Science 212, pp 815–816, 1981.  American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) (2018) Volume   47 , No  1, pp 21-27  26  [2] Berwaer, M, Martial, JA, Davis, JR “Characterization of an up-stream promoter directing extra-pituitary expression of the human prolactin gene”. Mol Endocrinol 8, pp 635–642, 1994. [3] Sinha, Y.N. “Structural variants of prolactin: occurrence and physiological significance”. Endocr Rev 16, pp 354–369, 1995. [4] Nyante, SJ, Faupel-Badger, JM, Sherman, ME, Pfeiffer, RM, Gaudet, MM, Falk, RT, Andaya, et al “Genetic variation in PRL and PRLR, and relationships with serum prolactin levels and breast cancer risk: results from a population based case-control study in Poland”. Breast Cancer Research, 13, R42, pp 1-11, 2011. [5] Williams, J.G.K., Kubelik, A.R, Liak, KJ Rafalski, J and Tingey, SV “DNA polymorphisms amplified by arbitrary primers are useful as enatic marker”. Nucleic Acids Research. 18, pp: 6531-6535, 1990. [6] AL– Azzawie, AF “Extra pituitary prolactin –1149 G/T promoter polymorphism in some rheumatoid arthritis patients”. Al- Kindy College Medical Journal, Vol.11 No. 1, pp 40-44, 2015. [7] Yeh FC Yang RC and Boyle T POPGENE, Version 1.31. “Microsoft Windows-based Freeware for Population Genetics Analysis”. Molecular Biology and Technology Center, University of Alberta, Canada. 1999. [8] SPSS. “Statistical Packages of Social Sciences”. Version 15. USA 2006. [9] Falconer, DS “Introduction to Quantitative Genetics”. 2ed Longman, UK. . 1981. [10] Treadwell, E.L., Wiley, K., Word, B., Melchior, W., Tolleson, W.H., et al “Prolactin and Dehydroepiandrosterone Levels in Women with Systemic Lupus Erythematosus: The Role of the Extrapituitary Prolactin Promoter Polymorphism at −1149G/T”.  Journal of Immunology Research Volume Article ID 435658, 10 p 2015.   [11] Neidhart, M, Gay, RE, Gay, S “Prolactin and prolactin-like polypeptides in rheumatoid arthritis”. Biomed Pharmacotherapy 53, pp 218-222, 1999. [12] Stevens, A, Newton, P, Anderson, NR, Gama, R “Characterization of a prolactin gene polymorphism and its associations with systemic lupus erythematosus”. Arthritis Rheum. 44 (10): pp 2358–2366, 2001. [13] Mellai, M., Miller, A., Shtiller, R., Touby, E “Prolactin and prolactin receptor gene polymorphisms in multiple sclerosis and systemic lupus erythematosus”. Hum. Immunology 64, pp 274–284, 2003. [14] Fojtíková, M, Radim, B, Jiri, VV “Polymorphism of the extra-pituitary prolactin promoter and systemic sclerosis”. Rheumatol. Int. 30 (12), pp 1691–1693, 2010.  American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) (2018) Volume   47 , No  1, pp 21-27  27  [15] Montgomery, DW, Steven, R, Leuis, “A Prolactin production by immune cells”. Lupus 10, pp 665-675, 2001 . [16] Bole-Feysot, C, Goffin, V, Edery, M, Binart, N, and Kelly, PA “Prolactin (PRL) and its receptor: actions, signal transduction pathways and phenotypes observed in PRL receptor knockout mice”. Endocr. Rev, vol.19, no.3, pp 225–268, 1998. [17] Goffin, V., Binart, N., Touraine, P., and P.A. Kelly “Prolactin: the new biology of the old hormone”. Annual Review of Physiology, vol.64, pp 47–67, 2002. [18] Lee, Y.C., Sulli, A., Fasciolo, D. “The PRL –1149 G/T polymorphism and rheumatoid arthritis susceptibility”. Arthritis Rheum, 60 (5), pp 1250–1254, 2009. [19] Kochi, Y., Suzuki, A., Yamada, R., Yamamoto, K. “Ethnogenetic heterogeneity of rheumatoid arthritis-implications for pathogenesis”. Nat. Rev. Rheumatol, 6, pp 290–295, 2010. [20] Guzman-Guzman, I.P., Parra-Rojas, I., Oregón-Romero, E., Ledezma-Lozano, and IY Palafox-Sánchez, CA “The PADI4 haplotypes are associated with anti-CCP levels in rheumatoid arthritis from Western Mexico”. Clin Chem Lab Med 49, S205, Special suppl. 32, 2011.    

An Association of Prolactin Gene Polymorphisms with Some Milk Traits in Women

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An Association of Prolactin Gene Polymorphisms with Some Milk Traits in Women

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