6.1 A Comprehensive Functional Annotation of SPINK...
In vitro functional analysis often represents the only practical means to ascertain the pathogenicity of patient-derived sequence variants，particularly when they are individually rare.Various examples have recently been provided in the context of SPINK1 intronic variants.(https://www.daowen.com)
A mini-gene splicing assay is currently the most frequently used method to evaluate the effect of intronic variants on splicing.The SPINK1 c.10lA＞G（p.Asn34Ser）variant and the four intronic variants in linkage disequilibrium with it were previously subjected to such analysis（Kereszturi et al.2009b）.These variants were also analysed in the context of a full gene splicing assay wherein the full-length SPINK1 genomic sequence（approximately 7 kb stretching from the translational initiation codon to the stop codon of the gene）was cloned into the pcDNA3.1/V5-His-TOPO vector（Boulling etal.2012）.This latter expression system is strongly representative of the in vivo situation，as indicated by the observed splicing patterns of the wild-type sequence and two SPINK1 splice site variants，c.87+1G＞A and c.194+2T＞C（Zou et al.2016a）.Employing this assay system，we confirmed previous conclusions with respect to two known pathogenic variants and five presumed non-pathogenic variants，establishing that seven previously reported variants of unknown clinical significance were not of pathological significance（Zou et al.2016a）.We also excluded all ten newly found intronic variants from a pilot resequencing study of the SPINK1 gene from further consideration of their pathological relevance（Zou et al.2016b）.