Detection of a Splice Site Variant in a Patient with Glomerulopathy and Fibronectin Deposits

Abstract

Background/Aims: Glomerulopathy with fibronectin deposits (GFND; OMIM: 601894) is a very rare inherited kidney disease caused by pathogenic variants in the FN1 gene. Only 9 exonic pathogenic variants in FN1, 9 at the heparin-binding site, and 1 at the integrin-binding site have been reported. No intronic variants in FN1 have been detected. Methods: We found a pathogenic intronic variant in intron 36 (c.5888–2A>G) located at the heparin-binding site. To determine whether this mutation influences splicing processes, we conducted RT-PCR analysis and an in vitro splicing assay using minigene construction. Results: RT-PCR using RNA extracted from leukocytes of the proband failed because of the low expression of FN1 mRNA in leukocytes. We conducted in vitro functional splicing analysis using minigenes and found that c.5888–2A>G caused a 12 bp deletion at exon 37 by the activation of a novel splicing acceptor site within exon 37. We were able to detect the same abnormal transcript in mRNA extracted from the patient’s urinary sediment and confirmed the pathogenicity of c.5888–2A>G by both RT-PCR using the patient sample and an in vitro splicing assay. Conclusion: Intronic variants can cause GFND. Minigene analysis is useful for determining the pathogenicity of the intronic variants and could be used for all inherited kidney diseases.

The Contribution of COL4A5 Splicing Variants to the Pathogenesis of X-Linked Alport Syndrome

“…Variants can be introduced to the minigene by using site directed mutagenesis allowing transcript analysis even if patient sample is not available (33,34). We have analyzed several novel intronic variants in various inherited kidney diseases using this assay (35)(36)(37)(38)(39)(40). As for intronic variants of COL4A5, several studies report using the minigene assay including our studies (11,15,41).…”

Usefulness of functional splicing analysis to confirm precise disease pathogenesis in Diamond-Blackfan anemia caused by intronic variants in RPS19

“…In recent years, in vitro functional splicing analyses using minigene constructs have been used as an alternative approach to assess the pathogenicity of splicing variants in various inherited diseases. [25][26][27][28][29][30][31] To date, few reports of splicing analysis using a minigene assay have been published in the field of inherited hematological disease. [32][33][34][35][36][37][38][39][40][41] The only report that describes using the minigene assay in cases of congenital bone marrow failure is a functional splicing analysis of familial DBA caused by the RPS7 variant.…”