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Full-length gene assays (FLGA) have been routinely used to analyse numerous splicing variants within the human SPINK1 geneA variant on pre-mRNA splicing. Gut 66, 2195–2196 (2017)." href="#ref-CR6" id="ref-link-section-d226774577e452">6,7,8,9,10 and 5’SS GT>GC or GC>GT variants in 43 different genesGT and GT>GC variants differ markedly in terms of their functionality and pathogenicity. Hum. Mutat. 41, 1358–1364 (2020)." href="/articles/s41598-023-36142-z#ref-CR11" id="ref-link-section-d226774577e459">11,GC variants capable of generating wild-type transcripts. Hum. Mutat. 40, 1856–1873 (2019)." href="/articles/s41598-023-36142-z#ref-CR12" id="ref-link-section-d226774577e462"12. In order to assess the spliceogenicity of a variant, the minigene assay is easier to employ than the FLGA and therefore more commonly used. The FLGA although presents two major advantages. First, the analysis of a splicing variant carried by a pre-mRNA transcript which has been generated by an episomal expression vector is more biologically relevant when integrating the whole gene sequence, since the nature and the length of the sequence context is known to impact RNA structures and splicing processes13,14,15. Next, the FLGA allows the assessment of any kind of genetic variants on splicing including deep intronic ones. Therefore, we created a bovine DGAT1 FLGA in order to validate the effect of both p.M435L and p.K232A on splicing in a robust experimental system./p>

Thus, we attempted to replicate the most compelling result from the Fink et al. study which was the decrease in the splicing rate of junction 7 caused by the p.K232A variant4. The possible effects on the splicing of introns 1 and 2 were also tested. In a manner comparable to theirs, we have set up quantitative RT-PCR and we have measured DGAT1 spliced and unspliced transcripts relative expression at each junction, as the average percentage of splicing, in MAC-T cells transfected with pcDNA3.1-DGAT1 constructs. We did not observe any significant difference between (WT) and (K232A) conditions on any splicing parameters related to junction 1, 2 or 7. Conversely, the minigene assay developed by Fink et al. showed a 4-fold increase in terms of spliced transcript relative expression for the p.K232 allele by comparison to the p.A232 allele in the context of the junction 7, what resulted in a dramatic rise of intron 7 splicing ratio. Such a strong discordance may seem a little surprising but it can be explained by differences in terms of methodological choices or technical considerations between both studies. First, and as we have mentioned in the introduction, using a truncated or chimeric genomic sequence of a gene to perform episomal splicing reporter assays is less reliable than using the full gene sequence. A minigene harbouring all exons of a gene but lacking several of its introns as used by Fink et al. may generate strong false positives. This kind of bias has been unmasked by Wu et al., who used FLGA to analyse the human SPINK1 c.194G>A variant previously classified as strongly spliceogenic using minigene assayA variant on pre-mRNA splicing. Gut 66, 2195–2196 (2017)." href="/articles/s41598-023-36142-z#ref-CR6" id="ref-link-section-d226774577e945"6,16. They revealed that this variant actually had no effect on splicing. Subsequently, it is also recognized that cell line-based experiments may suffer from a lack of reproducibility, especially when performed by different experimenters, in different laboratories, and using different cell preparations17. This may have contributed to conflicting results between our study and that of Fink et al./p>T, BTA14:608021C>T), but they were located outside the splice sites and far away from the two studied variants./p>C) and p.K232A (c.694AA>GC) variants were introduced into the pcDNA3.1-DGAT1 (WT) construct by means of the QuikChange II XL Site‐Directed Mutagenesis Kit (Agilent Technologies). Mutagenesis was performed in a 51 μl mixture containing 2.5 U PfuUltra HF DNA polymerase, 1 μl dNTP mix, 5 μl 10× reaction buffer, 3 μl QuikSolution, 20 ng pcDNA3.1-DGAT1, and 125 ng each mutagenesis primer M1 and M2 or alternatively M3 and M4 (Table 1). The PCR program had an initial denaturation at 95 °C for 1 min, followed by 18 cycles of denaturation at 95 °C for 50 s, annealing at 60 °C for 50 s, and extension at 68 °C for 28 min, and a final extension at 68 °C for 7 min. The post-PCR reaction mixture was treated with DpnI at 37 °C for 1 hr then 4 µL of the resulting products were transformed into XL10‐Gold Ultracompetent cells (Agilent Technologies). Transformed cells were spread on LB agar plates with 50 μg/ml ampicillin then several selected colonies were used to prepare miniprep plasmid productions with QIAprep Spin Miniprep Kit (Qiagen). Miniprep plasmids were analysed by Sanger sequencing in order to verify the successful introduction of the desired substitution and one large scale NucleoBond Xtra Midi EF plasmid production was done and quantified for each variant. Constructs harbouring p.M435L and p.K232A variants were named pcDNA3.1-DGAT1 (M435L) and pcDNA3.1-DGAT1 (K232A), respectively./p>A variant on pre-mRNA splicing. Gut 66, 2195–2196 (2017)./p>GT and GT>GC variants differ markedly in terms of their functionality and pathogenicity. Hum. Mutat. 41, 1358–1364 (2020)./p>GC variants capable of generating wild-type transcripts. Hum. Mutat. 40, 1856–1873 (2019)./p>