In vitro and in silico evidence against a significant effect of the SPINK1 c.194G>A variant on pre-mRNA splicing.

We read with interest the recent publication of Beer and Sahin-Tóth 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>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. It should be noted that the potential effect of c.194G>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. 3 We recently analysed the functional consequences of 24 SPINK1 intronic variants in relation to their associated mRNA splicing phenotypes 5 by means of a fullgene splicing assay in which the fulllength 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. This full-length gene expression system has already proved itself in practice by accurately representing the in vivo situation in the context of the observed splicing patterns of the SPINK1 wild-type gene and two pathogenic splice-site variants, c.87 +1G>A and c.194+2T>C. Naturally, a full-length gene construct corresponds more closely to the in vivo chromosomal context of the studied genes than a minigene construct. We attempted to replicate the abovementioned finding with respect to the SPINK1 c.194G>A variant using our fulllength splicing assay. We first investigated whether c.194G>A would cause aberrant splicing, which was performed as previously described. 5 The c.194G>A variant was introduced into the wild-type fulllength SPINK1 expression vector by directed mutagenesis; reverse transcription-PCR (RT-PCR) analyses of mRNAs from subsequently transfected HEK293T cells revealed a single transcript of similar size to the wild type (figure 1A); sequencing of the RT-PCR products revealed that the variant transcript was correctly spliced as per the wild type. We then performed quantitative RT-PCR analyses of the wild type and c.194G>A variant mRNA expressions as previously described, except that the primers used for amplifying the fulllength target gene transcripts were changed to the primer pair Q1 as described in ref. 6. However, we failed to observe any significant difference between the c.194G>A variant and the wild type in terms of mRNA expression (figure 1B), contrary to the results of the previous authors. The validity of our conclusions was supported by two additional experiments. First, a linear correlation was noted between the mRNA expression level and the amount (0–500 ng) of transfected plasmid harbouring the full-length wild-type SPINK1 genomic sequence (see online supplementary figure S1). This suggested that, under our experimental conditions, we could detect a significant decrease in mRNA expression from HEK293T cells transfected with 500 ng plasmids harbouring a full-length variant SPINK1 genomic sequence. Second, a known SPINK1 variant, c.27delC (p.Ser10fsX5), located in the middle of the exon 1 coding sequence, served as a positive control; it significantly decreased mRNA expression, but this decrease could be reversed by cycloheximide, a known inhibitor of nonsensemediated mRNA decay (see online supplementary figure S2). We further assessed the likely impact of the SPINK1 c.194G>A variant in terms of the disruption of known splice sites and/or creation of new splice sites using the five splicing prediction algorithms viz. SpliceSiteFinderlike, MaxEntScan, NNSPLICE, GeneSplicer and Human Splicing Finder included within the Alamut Visual software suite (V.2.7.1; Interactive Biosoftware, Rouen, France). The c.194G>A variant was predicted to have only slightly reduced scores as compared with the wild-type intron 3 splice donor site (see online supplementary figure S3A). These slightly reduced scores are not