TY - JOUR T1 - In vitro and in silico evidence against a significant effect of the <em>SPINK1</em> c.194G&gt;A variant on pre-mRNA splicing JF - Gut JO - Gut SP - 2195 LP - 2196 DO - 10.1136/gutjnl-2017-313948 VL - 66 IS - 12 AU - Hao Wu AU - Arnaud Boulling AU - David N Cooper AU - Zhao-Shen Li AU - Zhuan Liao AU - Jian-Min Chen AU - Claude Férec Y1 - 2017/12/01 UR - http://gut.bmj.com/content/66/12/2195.abstract N2 - We read with interest the recent publication of Beer and Sahin-Tóth1 reporting that exonic variants affecting pre-mRNA splicing contribute to the genetic burden in chronic pancreatitis. One particular variant, affecting the last nucleotide of exon 3 of the SPINK1 gene, c.194G&gt;A, was found to cause an ∼80% reduction in SPINK1 mRNA expression as compared with the wild type in a minigene assay performed in human embryonic kidney 293T (HEK293T) cells. The SPINK1 sequence inserted into the minigene expression vector however comprised only exon 1, exon 2, exon 3, intron 3 and exon 4 of the four-exon gene.1 It should be noted that the potential effect of c.194G&gt;A as a missense mutation (p.Arg65Gln) on protein function has previously been analysed; engineered expression of the full-length mutant coding sequence in Chinese hamster ovary cells and HEK293T cells showed a consistent 50%–60% reduction in protein secretion as compared with the wild type.2 ,3We recently analysed the functional consequences of 24 SPINK1 intronic variants in relation to their associated mRNA splicing phenotypes4 ,5 by means of a full-gene splicing assay in which the full-length 7 kb SPINK1 genomic sequence (including all four exons plus all three introns of the gene) was cloned into the pcDNA3.1/V5-His-TOPO vector.6 This full-length gene expression system has already proved itself in practice by accurately representing the in vivo situation in the context … ER -