In silico prioritization and further functional characterization of SPINK1 intronic variants

BACKGROUND: SPINK1 (serine protease inhibitor, kazal-type, 1), which encodes human pancreatic secretory trypsin inhibitor, is one of the most extensively studied genes underlying chronic pancreatitis. Recently, based upon data from qualitative reverse transcription-PCR (RT-PCR) analyses of transfected HEK293T cells, we concluded that 24 studied SPINK1 intronic variants were not of pathological significance, the sole exceptions being two canonical splice site variants (i.e., c.87 + 1G > A and c.194 + 2T > C). Herein, we employed the splicing prediction tools included within the Alamut software suite to prioritize the ‘non-pathological’ SPINK1 intronic variants for further quantitative RT-PCR analysis. RESULTS: Although our results demonstrated the utility of in silico prediction in classifying and prioritizing intronic variants, we made two observations worth noting. First, we established that most of the prediction tools employed ignored the general rule that GC is a weaker donor splice site than the canonical GT site. This finding is potentially important because for a given disease gene, a GC variant donor splice site may be associated with a milder clinical manifestation. Second, the non-pathological c.194 + 13T > G variant was consistently predicted by different programs to generate a new and viable donor splice site, the prediction scores being comparable to those for the physiological c.194 + 2T donor splice site and even higher than those for the physiological c.87 + 1G donor splice site. We do however provide convincing in vitro evidence that the predicted donor splice site was not entirely spurious. CONCLUSIONS: Our findings, taken together, serve to emphasize the importance of functional analysis in helping to establish or refute the pathogenicity of specific intronic variants. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s40246-017-0103-9) contains supplementary material, which is available to authorized users.