Analysis of mRNAs transcribed from the Ragusana Donkey CSN1S2 I and II genes and variability detection

In recent, years donkey's milk (DM) has attracted an increasing interest in human nutrition, since it may represent the best natural substitute of cow’s milk (CM) for children affected by CM protein allergy. The reason lies in a low casein content and a casein to whey protein ratio closer to human milk than to ruminant milk. Recently, the presence of all four casein fractions alfa-s1, beta, alfas2 and k-CN was demonstrated in DM. In particular, concerning the alfas2-CN encoding gene (CSN1S2), two different donkey cDNAs (CSN1S2 I and CSN1S2 II) have been identified. The first (GeneBank FM946022.1) is constituted by 19 exons encoding for a protein (alfas2-I) of 236 amino acids (aa); the second (GeneBank FN298386.2), made of 16 exons, encodes for a peptide (alfas2-II) of 160 aa. Aim of this study was to analyse the transcript profile of donkey CSN1S2 I and CSN1S2 II genes. Milk samples were collected from 8 unrelated donkeys of Ragusana breed. Total RNA was isolated from milk somatic cells. Retrotranscription (RT) was performed using an oligonucleotide dT18, whereas the PCR reaction was accomplished using primers designed using published donkey nucleotide sequences. PCR products were cloned. According to the sequence of randomly chosen clones, the CSN1S2 I gene, beyond a correctly spliced mRNA, transcribes other three minor mRNAs (a) lacking the exon 11, (b) missing the last 35 nucleotides of exon 17, (c) showing the insertion of an extra exon 12 (named 12', intronic insertions of 105 bp) that introduces a premature stop codon. Analysis of RT-PCR products of CSN1S2 II gene evidenced a more remarkable variabilty of the splicing events, mainly regarding the alternative insertion of an extra exon 7 (named 7'), or the alternative skipping of exons 9, 10, 11, 12 or 15. The remaining products are characterized by the combination of deletions described above: e.g. contemporary skipping of 11 and 12, 9 and 15 or 9, 10 and 15. Interestingly, the duplication of the tripeptide KQL at exon 13 previously reported (GeneBank FN298386.2) was not detected in all CSN1S2 II mRNAs sequenced. Therefore, likely it should be considered as an artifact. For CSN1S2 I, seven SNPs were observed, 5 of them responsible for aa changes: p.T73 > A, p.I109 > V, p.I130 > V, p.I146 > T and p.D217 > Y. Similarly, 8 SNPs were observed at CSN1S2 II locus, 6 of which non-synonymous: p.L63 > F, p.H70 > Q, p.D90 > N, p.129A > T, p.H131 > Y and p.F157 > S. The investigation at DNA level will likely clarify the variability found at mRNA level