Analysis of the impact of DGAT1 p.M435L and p.K232A variants on pre-mRNA splicing in a full-length gene assay

DGAT1 is playing a major role in fat metabolism and triacylglyceride synthesis. Only two DGAT1 loss-of-function variants altering milk production traits in cattle have been reported to date, namely p.M435L and p.K232A. The p.M435L variant is a rare alteration and has been associated with skipping of exon 16 which results in a non-functional truncated protein, and the p.K232A-containing haplotype has been associated with modifications of the splicing rate of several DGAT1 introns. In particular, the direct causality of the p.K232A variant in decreasing the splicing rate of the intron 7 junction was validated using a minigene assay in MAC-T cells. As both these DGAT1 variants were shown to be spliceogenic, we developed a full-length gene assay (FLGA) to re-analyse p.M435L and p.K232A variants in HEK293T and MAC-T cells. Qualitative RT-PCR analysis of cells transfected with the full-length DGAT1 expression construct carrying the p.M435L variant highlighted complete skipping of exon 16. The same analysis performed using the construct carrying the p.K232A variant showed moderate differences compared to the wild-type construct, suggesting a possible effect of this variant on the splicing of intron 7. Finally, quantitative RT-PCR analyses of cells transfected with the p.K232A-carrying construct did not show any significant modification on the splicing rate of introns 1, 2 and 7. In conclusion, the DGAT1 FLGA confirmed the p.M435L impact previously observed in vivo, but invalidated the hypothesis whereby the p.K232A variant strongly decreased the splicing rate of intron 7.

Automated summary

DGAT1 plays a crucial role in fat metabolism and triacylglyceride synthesis. Two DGAT1 loss-of-function variants, namely p.M435L and p.K232A, have been associated with milk production traits in cattle. The p.M435L variant leads to the skipping of exon 16, resulting in a non-functional truncated protein. The p.K232A variant affects the splicing rate of multiple DGAT1 introns, particularly intron 7.