Fig. 2

The TNNT1 c.614 + 1delG mutation causes intron 13 inclusion but does not reduce protein abundance. Agarose gels of RT-PCR products derived from type I myofibre-predominant vastus intermedius muscles of wild-type (WT), carrier (CA) and affected (AF) sheep for: a full-length TNNT1 (exon 1–15), b region encoding the protein N-terminus (exons 1–10), and c region encoding the C-terminus (exons 11–14). Expected (normal) product sizes are indicated by black arrows, and unexpected products by red arrows. NT = no template control. Ladder (L) = 100 bp TrackIT (Thermo Fisher, USA). d Schematic of primer sites and amplicons relative to the Ovis aries v3.1 TNNT1 genomic sequence. Amplicons are labelled relative to which panel they appear in. The full-length cDNA is 1001 bp and contains 15 exons. Translation starts in exon 2 and terminates in exon 14. e Sanger sequencing chromatogram of RT-PCR product from affected sheep, aligned to the published ovine TNNT1 cDNA sequence (Accession # KT218690, top sequence). Mismatches are highlighted in red. The causative single nucleotide deletion is indicated by an arrow. Base call quality (Phred quality score) is indicated by blue/grey bars at each position. Only partial intron 13 sequence is shown, up until the new stop codon in intron 13. The total size of intron 13 is 809 bp. Newly-encoded amino acids are indicated above the exon/intron annotations using the single letter amino acid code. Numbers represent amino acid position. f Pairwise comparison of the terminal 14 amino acids from wild-type TNNT1 and the amino acid sequence produced from intron 13 retention. Basic (blue) and acidic (orange) residues are highlighted. Basic residues within this region are known to be important for activation of TNNT1 by Ca²⁺ [31]. g Western blot of TNNT1 (HPA058448) from vastus intermedius muscle samples from 1-year old wild-type, carrier, and affected sheep. Human control is a skeletal muscle sample from quadriceps of a healthy individual. Red arrow indicates expected protein size for all samples (~ 33 kDa). Coomassie-stained myosin gel band (total myosin) was used as a loading control