S0022029921000832jra 368..373

Cluster of differentiation 4 (CD4) is the accessory protein non-covalently bound to the T cell receptor that recognizes an invariant region of MHC class II on antigen presenting cells. Its cytoplasmic tail, physically associated with a protein tyrosine kinase, is important in the activation of helper/inducer T lymphocytes. In Bos taurus, CD4 gene is located on chromosome 5 from which two isoforms are transcribed, with a different number of amino acids due to splicing of exon 7 and variation in the reading frame. The aim of this study was to investigate the sequence of the entire CD4 gene in Simmental sires to evaluate the effects of genomic variants on the indexes of the bulls for milk, fat and protein yields, as well as somatic cell score. The associations among genomic variants and indexes were analysed using the Allele and GLM procedures of SAS 9.4. The analysis indicated that only four of the thirty-one identified SNPs influenced the considered traits. Identified variants insist on coding zones and intronic sequences, where we revealed the presence of sites for transcription factors. To evaluate the existence of haplotypic effects, combinations among the four genomic variants (SNP 3, SNP 8, SNP 11 and SNP 19) were investigated. Six different haplotypic alleles were identified, but only four of them were frequent enough to allow for an evaluation of any haplotypic effect (at least six copies in the examined sample): Hap1, Hap2, Hap3 and Hap6. The analysis of associations between the selected haplotypes in the CD4 gene with milk related indexes showed that bulls with Hap2 (T-A-C-C) had better indexes for milk and protein yields (P < 0.05), whereas the presence of the Hap1 haplotype (A-G-A-T) caused a significant decrease of the index for protein yield (P < 0.05). Frequencies of the two alleles Hap1 and Hap2 (9 and 36% respectively) make them of interest for their possible inclusion in breeding schemes and support the hypothesis of considering this gene as a candidate for the improvement of milkrelated traits in the Simmental breed. Cluster of differentiation 4 (CD4) is a single-pass transmembrane glycoprotein playing an important function in the interaction between T lymphocytes and antigencarrying cells (König and Zhou, 2004). In Bos taurus, CD4 gene is located on chromosome 5 and two isoforms X1 (GenBank Accession Number: XM_024991416) and X2 (XM_024991417) are transcripted. The coded proteins are made of 455 (XP_024847184) and 395 (XP_024847186) amino acids respectively, due to splicing of exon 7 and variation in the reading frame. The influence of some variants in the CD4 genomic sequence is already known in humans for their ability to increase susceptibility to HIV infection (Oyugi et al., 2009) or for their association to the susceptibility and severity of rheumatoid arthritis (Hussein et al., 2012). Also, in the animal field recent researches have highlighted an important role of CD4 in immunity. In particular, Wang et al. (2013) found the CD4 gene to be linked to mastitis resistance in dairy cows. He et al. (2011) highlighted a significant association between SNPs within CD4 gene with mastitis indicators and milk production traits. Usman et al. (2016) showed a significant association between some SNPs of the CD4 gene (between exon 2 and intron 6) and the percentage of fat in milk. Recently, Zeb et al. (2020) showed that four SNPs between exon 2 and exon 4 of the CD4 gene were significantly associated with clinical mastitis incidence and with annual milk yield. Grandoni et al. (2020) identified in two Italian breeds (Friesian and Simmental) and their crosses two new missense mutations in the exon 5: g.103637203 G > T and g.103637191 A > C. The mutations were involved in lack or reduced labeling of anti-CD4 monoclonal antibody clone CC8 detected by flow cytometry assay. In the present study, the entire CD4 gene sequences of Simmental sires were investigated in order to mevaluate the effects of genomic variants on their deregressed multiple across country evaluation (MACE) of estimated breeding values (EBV) indexes for milk, fat and protein yields, and for somatic cell score (SCS). This index allows one to obtain the genetic evaluations of the reproducers, elaborated by the various countries, in a single international evaluation. Using the MACE model, Interbull (International Bull Evaluation Service) evaluates each trait considering the estimates of the genetic correlations between sires in the different countries. This paper documents the presence of a substantial number of polymorphisms in the CD4 gene and their significant associations on milk and protein indexes. Material and methods Data used in this study came from the Italian Simmental Breeders Association (ANAPRI). The Association provided for the complete genomic sequences and the deregressed MACE EBV indexes of 34 Simmental sires of different origins (21 Austrian, 7 Swiss, 4 German and 2 Italian) and born between 1969 and 2006. The sires had an average of 6,316 daughters with a minimum of 95 daughters and a maximum of 42 657. Indexes concerning yields of milk, fat and protein as well as SCS and their genetic basis referred to the years 2013–2015, with an update happening every five weeks. Genetic variants in the CD4 gene In order to investigate the presence of polymorphisms in the coding and regulatory regions of CD4 gene, we used the complementary sequence of the genomic region of chromosome 5 in Dominette’s Bos taurus (GeneBank, Accession Number: NC_037332). This sequence ranges between the nucleotides g.103631000...103660000 (29.000 bp) and it contains both the coding and regulatory regions of the bovine CD4 gene. Genome-wide sequencing of each of the 34 bulls was uploaded to Galaxy server at https://usegalaxy.eu (Version 2.3.4.3). Bowtie 2, tool of the Galaxy software, was used to map reads of the 34 bulls on the region of chromosome 5 including the CD4 gene, after assessing the quality of the raw reads through Galaxy’s FastQC tool. Bioinformatic analysis Bioinformatic analysis was performed using the MATCH programme (Kel et al., 2003) in the TRANSFAC® Professional 10.2 http://www.biobaseinternational.com/ (Matys et al., 2006). A profile search was performed for transcription factor binding sites in the sequenced DNA fragments, focusing on the areas where SNPs had been detected. The ‘vertebrate binding’ and the ‘only high quality’ matrix options were used in the profile selection, with cut-offs for core and matrix similarity set to 0.75 and 0.7 respectively, without any changes in other options. Statistical analysis On each SNP site, the χ test for deviation from Hardy-Weinberg equilibrium, along with expected and observed heterozygosity, polymorphism information content (PIC) and linkage disequilibrium (LD) were calculated using the algorithms provided for by the SAS software 9.4 (ALLELE procedure). The associations between SNPs and traits were analysed using the general linear model (GLM procedure of SAS software 9.4): Yik = μ +Gi + eik where Yik is the phenotype, μ was the overall population mean for the trait, Gi is the fixed effect of the genotype at the SNP (i = AA, AB, BB) and eik is the random error. Significance of the SNP and least-squares means were determined using the Student’s t-test in GLM procedure. The association between haplotypes and traits were analysed by a GLM on each haplotype by inserting the number of copies as a fixed: Yik = μ +Hi + eik, where Yik is the phenotype, μ is the overall population mean for the trait, Hi is the fixed effect of the haplotype [i = 0, individuals with no haplotype; 1, individuals with one copy only (heterozygotes); 2, individuals homozygous for that haplotype] and eik is the random error. Significance of the haplotype and least-squares means were determined using the Student’s t-test in GLM procedure.