Sequence analysis of Mucin 13 gene region associated with piglet diarrhoea in Indian pigs

Mucins are glycoproteins covering the apical surfaces of epithelial cells in gastrointestinal and respiratory tracts which form the first line of defence against pathogens entering these systems (Ringel and Lohr, 2003). They have been thought to play an important role in infectious and inflammatory diseases, cancer and metastasis (Moncada et al., 2003). Mucins have also become target genes in disease resistance research in piglets. Among these mucins, the mucin 13 (MUC13) gene have been proposed to be candidate gene for piglet diarrhoea resistance. Among past few years piglet diarrhoea has emerged as an important disease of piglets (Lalremruata et al., 2015; Pegu et al., 2017) causing mortality and production losses, contributing about 11.5% to 29.5% of total death (Li et al., 2007). The neonatal and post weaning diarrhoea in piglets is caused by E.coli with two main pathotypes involved viz., ETEC (enterotoxigenic E.coli) and EPEC (enteropathogenic E.coli). Due to its’ widespread incidence (56.2% among all types of diarrhoea) researches have focussed in identification of single nucleotide variations in candidate genes that can impart resistance towards ETEC diarrhoea in piglets.
        
The pathogenesis of piglet diarrhoea involves attachment of ETEC fimbriae with their receptors in small intestine of piglet. Expression of these receptors is variable, among within and between breeds and adhesion of receptor with bacterial fimbriae in genetically determined and exhibits Mendelian inheritance (Bijlsma et al., 1982). This fact gives a window to attempt for breeding pigs for resistance towards ETEC mediated diarrhoea using putative candidate genes as markers for indirect selection. However, exact causative mutations responsible for genetic variation in susceptibility to ETEC-F4 are unknown (Schroyen et al., 2012), MUC13 have been proposed to be a putative candidate gene that has been extensively studied for its role in imparting resistance to piglet diarrhoea in piglets. Zhang et al., (2008) found out that the MUC13 is in strong linkage disequilibrium with enterotoxigenic E.coli (ETEC) F4ab/ac receptor locus. Ren et al., (2012) did genome wide mapping using 39,720 informative SNPs and revealed that the most significant markers were proximal to the MUC13 gene in the 2.3-Mb region. Association studies in other diverse outbred pig populations have suggested that MUC13 gene may be most likely to be responsible for resistance / susceptibility towards piglet diarrhoea or it may carry mutations in Linkage Disequilibrium with gene / loci affecting this trait. Owing to its importance in piglet diarrhoea resistance we characterized MUC13 gene (partial sequence) in Indian native pigs using bioinformatic tools.

Total five pairs of primers were used encompassing two major regions of MUC13 gene - intron 6 and exon 7 (partial) – intron 7 – exon 8 (partial) (Table 1). For intron 6 region, the final sequences were submitted to NCBI and accession number were assigned to them as KX686558 (allele 01), KX686559 (allele 02) and KX686560 (allele 03). The annotated sequences of intron 6 region were aligned with that of corresponding sequences of Chinese pig (Sus scrofa,), sheep (Ovis aries), goat (Capra hircus) and cattle (Bos taurus) using Ensembl BLAT server (https://asia.ensembl.org). A heat map was also prepared for pairwise comparison, after sequence comparison (Fig 1). The box 1, 2 and 3 represent the comparison of Indian pig allele 01, 02 and 03 with Chinese pig, sheep, goat and cow. As expected, high similarity was seen between both Indian and Chinese pig, whereas, the sequences of sheep and goat (except in box 2) and cow clustered separately (Fig 1). The overall mean distance between Indian pig allele 01 and other sequences was 1.486, Indian pig allele 02 and other sequences was 1.759 and Indian pig allele 03 and other sequences 6.254. This large mean distance was also expected. 
 

 
For the looking to the function of gene region containing exon 7 (partial), intron 7 (complete) and exon 8 (partial), two PCR primers were used (Table 1) which amplified two regions, 491 bp and 537 bp. The final sequences were submitted to NCBI and accession numbers assigned to them as KX686556 (allele 01) and KX686561 (allele 02). The nucleotide sequences were analysed and compared with that of pig, sheep, goat, and cow using Ensemble BLAT and a heat map was constructed for their comparison using MEGA 7.0 (Fig 2). The box 1 represents the comparison of 01 (KX686556) allele and the box 2 represents the comparison of allele 02 (KX686561) with the related species, respectively (Fig 2). It is evident that sequences from Indian pig and Chinese pig were closely related as with the case in sequences from sheep and goat. This region of MUC13 gene is well defined as compared to the intron 6 region and hence the overall mean distance between Indian pig allele 01and related sequence was 0.149, whereas, mean distance between Indian pig allele 02 was 0.158. The exons 7 and 8 of both the sequence were then translated and joined as per their coordinates to make one single peptide chain. Among the sequences there were 21 sites of disagreement. Histidine, methionine and tryptophan were found in least amount with leucine and cysteine found in maximum amount. The homogeneity test of the sequences showed significant (P<0.05) difference, in terms of evolution by base substitution between Indian pig, Chinese pig and cattle sequences (Fig 3). The overall mean distance between the studied amino acid sequences was 0.23. The sequences were further analysed for secondary structure prediction using phyr2 programme server. The secondary structure showed predominance of β-pleated sheet (Fig 4), as the amino acid chain was small ligand binding prediction could not be performed.  Ren et al., (2012) has reported five SNPs in these regions (one in each segment) on MUC13 gene and they were reported to be associating with resistance to piglet diarrhoea. Sinha et al., (2018a) genotyped these SNPs in Indian native pig population of same region (Bareilly, India) by using PCR-RFLP protocol and found all SNPs to be polymorphic One of these SNP (g.22304A>G) situated in sequence KX686561 was showing a significant effect of genotypes on E.coli F4ab/ac mediated diarrhoea (Sinha et al., 2018a). Earlier, Sinha et al., (2018b) have shown that MUC13 is differentially expressed in jejunum tissue of Indian native pig which is differentially adhesive to diarrhoeagenic E.coli. This is indicative that MUC13 polymorphism may be involved in resistance/ susceptibility to E.coli F4ab/ac mediated diarrhoea (Sinha et al., 2018a, b). 
 

 

 

The current study characterized the regions of MUC13 gene in Indian native pig by sequencing. The only regions reported to be harbouring SNPs that may have significant effect on piglet diarrhoea were studied. Indian native and Chinese pigs showed maximum sequence similarity at nucleotide as well as on amino acid level. As the amino acid sequence was short in length, ligand binding prediction could not be performed. Predicted secondary structure of amino acid chain showed the predominance of beta sheet structure in pig and other species.