Molecular assay for an intronic variant in NUP93 that causes steroid resistant nephrotic syndrome

Abstract

Advances in molecular genetics have revealed that approximately 30% of cases with steroid resistant nephrotic syndrome (SRNS) are caused by single-gene mutations. More than genes are responsible for SRNS. One such gene is the nucleoporin, 93-KD (NUP93). Thus far, few studies have reported mutations of NUP93 in SRNS. Here, we describe an NUP93 biallelic mutation in a 9-year-old boy with focal segmental glomerular sclerosis (FSGS). Notably, one mutation comprised an intronic variant; we conducted in vivo and in vitro analysis to characterize this variant. We found two heterozygous mutations in NUP93: c.2137-18G>A in intron 19 and a novel nonsense mutation c.727A>T (p.Lys243*) in exon 8. We conducted RNA sequencing and in vitro splicing assays by using minigene construction, combined with protein expression analysis to determine the pathogenicity of the intronic variant. Both RNA sequencing and in vitro splicing assay showed exon 20 skipping by the intronic variant. In protein expression analysis, aberrant subcellular localization with small punctate vesicles in the cytoplasm was observed for the intronic variant. Taken together, we concluded that c.2137-18G>A was linked to pathogenicity due to aberrant splicing. NUP93 variants are quite rare; however, we have shown that even intronic variants in NUP93 can cause SRNS. This study provides a fundamental approach to validate the intronic variant, as well as new insights regarding the clinical spectrum of SRNS caused by rare gene variants.

End‐stage renal disease in a child with focal segmental glomerulosclerosis associated with a homozygous NUP93 variant

“…Also, since the deep introns were not analyzed, there is a possibility of missing deep intronic variants which have been shown to be associated with FSGS. 18 …”

“…Lastly, variants in genes not analyzed in this study could be causative of FSGS in this consanguineous girl. Also, since the deep introns were not analyzed, there is a possibility of missing deep intronic variants which have been shown to be associated with FSGS 18 …”

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).…”

Nuclear pore complexes in development and tissue homeostasis

“…In the past few years, mutations in many genes encoding Nups have been reported to cause diverse kidney diseases, including steroidresistant nephrotic syndrome, Galloway-Mowat syndrome and focal segmental glomerulosclerosis (Bezdícǩa et al, 2018;Braun et al, 2018Braun et al, , 2016Fujita et al, 2018;Hashimoto et al, 2019;Miyake et al, 2015;Park et al, 2017;Rossanti et al, 2019;Rosti et al, 2017;Seeman and Vondrak, 2018;Zhao et al, 2019). The mechanisms through which Nup mutations affect kidney development or function are still poorly understood.…”