Toward a clinical diagnostic pipeline for SPINK1 intronic variants

Abstract

BackgroundThe clinical significance of SPINK1 intronic variants in chronic pancreatitis has been previously assessed by various approaches including a cell culture-based full-length gene assay. A close correlation between the results of this assay and in silico splicing prediction was apparent. However, until now, a clinical diagnostic pipeline specifically designed to classify SPINK1 intronic variants accurately and efficiently has been lacking. Herein, we present just such a pipeline and explore its efficacy and potential utility in potentiating the classification of newly described SPINK1 intronic variants.ResultsWe confirm a close correlation between in silico splicing prediction and results from the cell culture-based full-length gene assay in the context of three recently reported pathogenic SPINK1 intronic variants. We then integrated in silico splicing prediction and the full-length gene assay into a stepwise approach and tested its utility in the classification of two novel datasets of SPINK1 intronic variants. The first dataset comprised 16 deep intronic variants identified in 52 genetically unexplained Chinese chronic pancreatitis patients by sequencing the entire intronic sequence of the SPINK1 gene. The second dataset comprised five novel rare proximal intronic variants identified through the routine analysis of the SPINK1 gene in French pancreatitis patients. Employing a minor allele frequency of > 5% as a population frequency filter, 6 of the 16 deep intronic variants were immediately classified as benign. In silico prediction of the remaining ten deep intronic variants and the five rare proximal intronic variants with respect to their likely impact on splice site selection suggested that only one proximal intronic variant, c.194 + 5G > A, was likely to be of functional significance. Employing the cell culture-based full-length gene assay, we functionally analyzed c.194 + 5G > A, together with seven predicted non-functional variants, thereby validating their predicted effects on splicing in all cases.ConclusionsWe demonstrated the accuracy and efficiency of in silico prediction in combination with the cell culture-based full-length gene assay for the classification of SPINK1 intronic variants. Based upon these findings, we propose an operational pipeline for classifying SPINK1 intronic variants in the clinical diagnostic setting.Electronic supplementary materialThe online version of this article (10.1186/s40246-019-0193-7) contains supplementary material, which is available to authorized users.

First estimate of the scale of canonical 5′ splice site GT>GC variants capable of generating wild‐type transcripts

“…The full‐length gene splicing assay preserves better the natural genomic context of the studied variants as compared to the commonly used minigene splicing assay, a point of importance given the highly context‐dependent and combinatorial nature of alternative splicing regulation (Fu & Ares, ). Moreover, the full‐length gene splicing assay can be readily used to evaluate all intronic variants including those located near the first or last exons of the gene (Tang et al, ). Despite these advantages, the full‐length gene assay cannot easily be applied to large‐sized genes owing to the technical difficulties inherent in amplifying and cloning long DNA fragments into the expression vector.…”

5′ splice site GC>GT and GT>GC variants differ markedly in terms of their functionality and pathogenicity

“…First, FABP7 IVS1+2T>C activated a cryptic 5′ splice site GT located 2 bp downstream of the normal one, resulting in the retention of the first 4 bp of the Intron 1 sequence (Lin et al, 2019). Second, SPINK1 c.88‐1G>A resulted in the skipping of the first nucleotide of Exon 3 (Tang et al, 2019).…”

Splicing Outcomes of 5′ Splice Site GT>GC Variants That Generate Wild-Type Transcripts Differ Significantly Between Full-Length and Minigene Splicing Assays

“…Thus, the minigene splicing assay will for the time being remain the mainstream approach for functionally characterizing potential splice-altering variants. However, an inherent drawback of the minigene splicing assay is the lack of the wider genomic sequence context of the gene under study (Zou et al, 2016;Lin et al, 2019Lin et al, , 2020Tang et al, 2019). This could lead to inaccurate results and incorrect conclusions being drawn owing to the complexity of the splicing code (Fu and Ares, 2014;Drexler et al, 2020), as exemplified by the contrasting findings from the study of the SPINK1 c.194G>A variant in a minigene assay (Beer and Sahin-Toth, 2014) and our own FLGSA assay (Wu et al, 2017).…”