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During the congress, E-Posters will be accessible to all participants on the congress website 24/7, as well as in the E-poster stations in the congress center.
Preparing your E-Poster
Please review the E-Poster format requirements carefully when preparing your E-Poster. Should your E-Poster not meet the mentioned requirements, it may not be displayed as described above.
E-Poster Submission Deadline
Please prepare and upload your E-Poster no later than March 14, 2026 11.59PM CET. After this date, you will no longer be able to prepare and upload your E-poster and it will not be displayed and accessible on the congress website.
Please follow the instructions below to input your abstract title.
Abstract titles should be brief and reflect the content of the abstract.
The NPHS1 gene encodes nephrin, a critical component of the glomerular filtration barrier and a key structural molecule of the slit diaphragm linking adjacent podocytes. Pathogenic NPHS1 variants are major causes of congenital and steroid-resistant nephrotic syndrome (CNS, SRNS). While splice-site variants located at canonical ±1/±2 positions are well established as pathogenic, the functional significance of variants outside these canonical splice sites remains largely uncertain. Such non-canonical variants often remain classified as variants of uncertain significance (VUS) unless their splicing patterns are verified by in vitro or in vivo analyses, making clinical interpretation challenging. This study aimed to elucidate the pathogenicity of non-canonical NPHS1 splice variants using an in vitro splicing minigene assay.
Among 61 splice variants of NPHS1 registered in the Human Gene Mutation Database, 24 were non-canonical splice variants located outside the canonical ±2 positions. Of these, 15 variants previously reported to cause kidney phenotypes and confirmed to exist in compound heterozygous variants were selected. Together with two additional variants identified in our cohort, a total of 17 variants were finally analyzed. For each target exon, intron–exon–intron fragments were cloned into a splicing-reporter vector to generate wild-type (WT) and mutant (MT) constructs. For variants lacking patient-derived DNA, mutations were artificially introduced into WT genomic templates using site-directed mutagenesis. Constructs were transfected into HEK293T cells, and total RNA was extracted, and the splicing pattern was analyzed. Aberrant splicing was defined as (i) WT showing only normal splicing and MT showing only aberrant splicing; (ii) WT showing only normal splicing and MT showing both normal and aberrant splicing; or (iii) WT showing both normal and aberrant splicing and MT showing only aberrant splicing.
Among the 17 variants analyzed, the single-nucleotide substitution sites in gDNA were located in two exonic positions, four at ±3, six at ±5, and one each at −6, +7, −10, −17, and −31. All 17 exhibited aberrant splicing; Exon skipping was observed in six variants and other types of splicing abnormalities in 11. According to the American College of Medical Genetics and Genomics (ACMG) guidelines, all variants had initially been classified as VUS. Based on the in vitro assay results from this study, 15 variants met the PS3 criterion and were reclassified as likely pathogenic.
This study demonstrates that in vitro minigene assays effectively resolve the clinical ambiguity of non-canonical NPHS1 splice variants previously classified as VUS. All variants showed clear splicing abnormalities, and incorporating these results into ACMG interpretation improved the accuracy of pathogenicity assessment. These findings highlight the clinical utility of the minigene assay as a practical tool for improving variant interpretation and diagnosis in NPHS1-related nephrotic syndrome.
