Identification of a synonymous variant of NPHP3 causing aberrant splicing and its pathogenicity in a Chinese pedigree

Abstract

Abstract Purpose Nephronophthisis (NPHP) is an autosomal recessive genetic disease. Despite the rapid development of gene detection technology, genetic causes remain unclear in most patients with NPHP. Unidentified pathogenic genes and unrecognized pathogenic variants may contribute to this issue. In this study, we demonstrated the pathogenicity of a synonymous variant of NPHP3 causing aberrant splicing in a Chinese infantile NPHP pedigree. Methods Clinical data and blood samples were acquired. Next-generation sequencing(NGS) and data analysis, evaluation and Sanger sequencing were performed. Nephrocystin 3 expression in kidney tissue was detected by immunofluorescence microscopy. mRNA derived from urine-derived renal epithelial cells (URECs) of the synonymous variant carrier were analyzied. In vitro, minigene splicing report was used to verify the pathogenicity of NPHP3 splicing site mutation. Results Two siblings were born to a couple of nonconsanguineous parents who progressed to end-stage renal disease before 5 years of age. Both siblings had elevated ALT, and their renal pathology showed typical changes of NPHP. Whole-exon sequencing (WES) of the proband’s genomic DNA revealed only a likely pathogenic intron variant of NPHP3, c.2088 + 5G > A. Reanalysis identified a synonymous variant c.2154C > T (p.F718F) of NPHP3, which is predicted to be benign by most tools but may affect splicing by a few and was ignored in the initial bioinformatic analysis. These two variants cosegregate well in this pedigree. The expression of nephrocystin 3 in kidney tissue from the proband was absent, as detected by immunofluorescence. The mRNA isolated from urinary cells of the synonymous variant c.2154C > T carrier showed alternative splicing in NPHP3. The minigene assay showed that c.2088 + 5G > A led to exon 14 skipping in the transcript, while c.2154C > T only led to an increased ratio of exon 15 skipping in the transcript, which indicated that the results of the minigene assay were not always consistent with the results in vivo. Conclusion The synonymous variant compounded with the intron variant of NPHP3 elucidated the previously genetically undefined case of infantile NPHP. These findings suggest that reassessing the pathogenicity of synonymous mutations is necessary, especially when clinical diagnosis is established but pathogenic variants are absent.