Experimental Assessment of Splicing Variants Using Expression Minigenes and Comparison with In Silico Predictions

Abstract

Assessment of the functional consequences of variants near splice sites is a major challenge in the diagnostic laboratory. To address this issue, we created expression minigenes (EMGs) to determine the RNA and protein products generated by splice site variants (n = 10) implicated in cystic fibrosis (CF). Experimental results were compared with the splicing predictions of eight in silico tools. EMGs containing the full-length Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) coding sequence and flanking intron sequences generated wild-type transcript and fully processed protein in Human Embryonic Kidney (HEK293) and CF bronchial epithelial (CFBE41o-) cells. Quantification of variant induced aberrant mRNA isoforms was concordant using fragment analysis and pyrosequencing. The splicing patterns of c.1585−1G>A and c.2657+5G>A were comparable to those reported in primary cells from individuals bearing these variants. Bioinformatics predictions were consistent with experimental results for 9/10 variants (MES), 8/10 variants (NNSplice), and 7/10 variants (SSAT and Sroogle). Programs that estimate the consequences of mis-splicing predicted 11/16 (HSF and ASSEDA) and 10/16 (Fsplice and SplicePort) experimentally observed mRNA isoforms. EMGs provide a robust experimental approach for clinical interpretation of splice site variants and refinement of in silico tools.

UGT1A1 Variants c.864+5G>T and c.996+2_996+5del of a Crigler-Najjar Patient Induce Aberrant Splicing in Minigene Assays

“…Thus, c.864+5G>T induced a deletion of 141 nt of exon 1, similar to previously reported variant c.864+1G>C (Gantla et al, 1998), but also a significant proportion of the full-length transcript (29.6%) (Figures 2B-E). Although in some cases splicing does not differ among different cell lines (Sanz et al, 2010;Sharma et al, 2014), these splicing outcomes have been obtained in MCF-7 cells (human breast adenocarcinoma) and should be further confirmed in liver cell lines.…”

Functional assessment of the BMPR2 gene in lymphoblastoid cell lines from Graves’ disease patients

“…After performing the minigene assay for the c.419‐43delT variant to verify its pathogenicity in vitro , we did not find any differences concerning transcript processing between wild‐type and mutant constructions, as this variant is not located in a consensus splicing region . Likewise, we performed functional studies, using lymphoblastoid mRNA from carrier patients and patients without the deletion to confirm the minigene assay results.…”

A synonymous splicing mutation in the SF3B4 gene segregates in a family with highly variable Nager syndrome

“…12 As no patient's RNA was available, to confirm its pathogenicity, we functionally characterized the synonymous variant using a β-globin hybrid minigene assay, which has been effectively used to validate potential splicing variants (Figure 2c). 13 RT-PCR analysis from cells transfected with the mutant construct showed an aberrant splicing compared with controls and yielded two bands (Figure 2d): sequencing of the lower fragment revealed that it corresponds to shorter transcripts owing to the activation of a novel canonical AG/GT 5′ donor splice site within SF3B4 exon 3 and the use of two different AG acceptor sites (the first in position c.536_537, the second three nucleotides downstream) (Figure 2e). The corresponding messenger RNAs (mRNAs) result in the loss of 122 or 125 nucleotides, with the consequent formation of prematurely truncated proteins in both the cases (p.Gly139Glufs*6 or p.Asn140Leufs*4).…”