DOI: 10.5061/dryad.rn8pk0p6k
title: A new mechanism for a familiar mutation – bovine DGAT1 K232A modulates gene expression through multi-junction exon splice enhancement

description: The DGAT1 gene encodes an enzyme responsible for catalysing the terminal
 reaction in mammary triglyceride synthesis, and underpins a well-known
 pleiotropic quantitative trait locus (QTL) with a large influence on milk
 composition phenotypes. Since first described over 15 years ago, a
 protein-coding variant K232A has been assumed as the causative variant
 underlying these effects, following in-vitro studies that demonstrated
 differing levels of triglyceride synthesis between the two protein
 isoforms. In the current study, we used a large RNAseq dataset to
 re-examine the underlying mechanisms of this large milk production QTL,
 and hereby report novel expression-based functions of the chr14
 g.1802265AA&gt;GC variant that encodes the DGAT1 K232A substitution.
 Using expression QTL (eQTL) mapping, we demonstrate a highly-significant
 mammary eQTL for DGAT1, where the K232A mutation appears as one of the top
 associated variants for this effect. By conducting in vitro expression and
 splicing experiments in bovine mammary cell culture, we further show
 modulation of splicing efficiency by this mutation, likely through
 disruption of an exon splice enhancer as a consequence of the allele
 encoding the 232A variant. Although the relative contributions of the
 enzymatic and transcription-based mechanisms now attributed to K232A
 remain unclear, these results suggest that transcriptional impacts
 contribute to the diversity of lactation effects observed at this locus.
description: Genotypes Animals were genotyped
 with the Illumina BovineHD BeadChip by GeneSeek. A subset of 115 SNPs in
 the 1 Mbp interval centred on <em>DGAT1 </em>K232A
 (chr14:1302265–2302265) were assessed for this study.
 RNAseq-derived genotypes for K232A (chr14:1802265G&gt;A SNP)
 were also included in the analyses, as this variant is the first base of
 the <i>DGAT1</i> K232A MNP. All genotypes
 were recoded using PLINK to 0, 1 or 2 to represent the number of
 alternative alleles for each marker (i.e. 0, 1, and 2 to represent the
 homozygous reference, heterozygous, and homozygous alternative genotypes,
 respectively). In addition to these genotypes, 3,128
 imputed whole-genome sequence (WGS) derived variants in the 1 Mbp interval
 of interest were used. These were imputed with Beagle v4, using a
 reference population of 556 animals (not included in this
 dataset). RNA Seq splicing
 phenotypes RNA sequence data were mapped to the
 UMD3.1 genome using Tophat2, locating an average of 88.9 million
 read-pairs per sample. The splicing efficiency
 phenotypes for each individual RefSeq <i>DGAT1 </i>junction
 were calculated as the ratio of exonic reads to intronic reads
 corresponding to the junction (of spliced and unspliced reads,
 respectively). Reads were considered exonic if they bridged the splicing
 junction i.e. mapped to the 3′ end of the preceding exon and the 5′ end of
 the following exon. Reads were considered intronic or unspliced if they
 mapped to the 3′ end of the preceding exon and through the intron-exon
 boundary into the intron. 
 Association analyses Association analyses
 for splicing efficiency were also conducted using ASReml-R to fit
 pedigree-based mixed models. This analysis fitted 115 BovineHD SNPs plus
 K232A genotypes derived from the RNAseq data, with phenotypes defined as
 described above.