Supplementary Material for: Detection of a Splice Site Variant in a Patient with Glomerulopathy and Fibronectin Deposits
2017-11-03T10:18:49Z (GMT) by
<b><i>Background/Aims:</i></b> Glomerulopathy with fibronectin deposits (GFND; OMIM: 601894) is a very rare inherited kidney disease caused by pathogenic variants in the <i>FN1</i> gene. Only 9 exonic pathogenic variants in <i>FN1</i>, 9 at the heparin-binding site, and 1 at the integrin-binding site have been reported. No intronic variants in <i>FN1</i> have been detected. <b><i>Methods:</i></b> 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. <b><i>Results:</i></b> RT-PCR using RNA extracted from leukocytes of the proband failed because of the low expression of <i>FN1</i> 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. <b><i>Conclusion:</i></b> 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.