FSGS is a leading cause of end-stage kidney disease. Genetic factors significantly contribute to the pathogenesis of the diseases. To date, mutations in more than 50 genes have been reported to be involved in FSGS . This number is rising rapidly due to the advancement of sequencing technologies during the last two decades. It is found that mutations involving the slit membrane components (NPHS1, NPHS2, COL4A3/A4/A5, etc.), collagen genes (COL4A3/A4/A5, etc.), podocyte cytoskeleton (ACTN4, INF2, etc.), calcium channel protein (TRPC6, etc.), mitochondrial function (ADCK4, COQ2, COQ6, etc.) and transcription factors (WT1, PAX2, etc.) .
The overall prevalence of FSGS varies in different populations, for example in African Americans it is the most common cause of glomerulonephritis. Its prevalence also varies across different regions of the same country; in China, it varies from 3 to 6 and 14 to 16% of all kidney biopsies in North and South China, respectively . The differences in its prevalence and frequency of familial cases of FSGS proclaim its strong genetic association . In this study, we identified a novel heterozygous missense variant (Chr6: g.43519108A > G, c.T1655C, p.V552A) in XPO5 using WES and Sanger sequencing. This variant was found to be pathogenic in two unrelated Chinese families of adult-onset familial FSGS and FGGS. XPO5 is present on the reverse strand of chromosome 6 and our identified variant (Chr6: g.43519108A > G) corresponds to c.T1655C at complementary DNA level and p.V552A on protein level, respectively.
In our study, we identified a novel heterozygous variant (c.T1655C:p.V552A) in XPO5. The average onset age of our patients was 29.5 years. The proband of family FS-133 was presented with nephrotic range proteinuria (4 g/24 h) and renal insufficiency. Renal biopsy showed FSGS. During follow-up, his proteinuria decreased by more than 50%. His renal function was slightly decreased. The other proband of family CKD-05 had mild proteinuria (0.179 g/24 h) and significantly increased serum creatinine. She was presented with focal glomerulosclerosis (highly suspected FSGS) by renal biopsy. The two probands were both biopsy-proven FSGS (or highly suspected FSGS), but with different clinical features. One of the probands had more proteinuria and slightly decreased renal function, while the other one had less proteinuria but worse renal function. Although the mutation from the same amino site was reported before, there are several features indicating that our identified variant (c.T1655C:p.V552A) exhibited distinct clinical phenotypes that is likely also pathogenetic in our two affected families. Firstly, the identified mutation was not detected in 195 healthy people when Sanger sequencing was performed. Secondly, a good correlation between genotype and phenotype by segregation analysis further strengthens our findings. Thirdly, mutant XPO5 can cause podocyte dysfunction because our patients showed higher levels of proteinuria and glomerulosclerosis. Furthermore, the onset of diseases is different. The average onset age of our patients was 29.5 years while reported cases exhibited proteinuria at a very early age (2 years old). Lastly, our patients exhibited different inheritance pattern with autosomal dominant inheritance pattern, while in the previous study, the inheritance pattern was not determined. In addition, all clinical features suggested that heterozygosity of this variant would still be pathogenic, but just confer the later development of disease onset due to being lower gene expression level. Taking all the data together, we believe that the newly identified variant in these Chinese FSGS patients might represent distinct features with different pathogenesis mechanisms despite mutation from the same amino acid site. We will further explore the potential different mechanisms in future study.
The function of XPO5 has been associated with the SMAD signaling pathway in relation to kidney disease previously. Braun DA et al.  showed XPO5 colocalized in podocytes with synaptopodin, a marker of primary and secondary podocyte foot processes in adult rat glomeruli, in a pattern that has been described for many other products of genes if mutated, could cause SRNS. They described that other proteins like NUP93, importin7 and exportin5 (XPO5) could interact with SMAD proteins. Additionally, they demonstrated that mutations in NUP93 from individuals with SRNS abrogate the interaction with SMAD4 and importin7 by co-IP in HEK293 cells. Similarly, we suggest that our identified variant may affect SMAD pathway. As XPO5 was detected in developing podocytes in adult rat. Hence, any abnormality in XPO5 can cause podocyte injury, which is the major cause of proteinuria and glomerulosclerosis as described by Michio N . Taking all together, genotype–phenotype correlation, conservation analysis, effect on the structure coordination among amino acids and prediction scores of the online tools affirm that our identified mutation is pathogenic.
Other variations at the same amino acid have been reported. Previously, a homozygous XPO5 (p.V552I) variant has been reported in a sporadic case with SRNS and speech development delay without any known inheritance pattern in a 2-year-old Turkish boy. Interestingly, another study  reported the same variant (p.V552I), as somatic mutation in the search of somatic mutation burden in a single case of chondrosarcoma along with other 38 mutations, but they did not mention zygosity. The onset age of the reported SRNS patient was early (2-year-old). The average onset age of our patients was 29.5 years, which is the supporting evidence of variable expressivity related to zygosity status. Hence, homozygous patient (Turkish boy) showed early onset age of the kidney dysfunction while heterozygous patients (our study) showed adult-onset age of the disease. Altogether, the onset age of the disease directly correlates with the status of the zygosity and expressivity. Additionally, a third variant on the same position (p.V552F) is present in some databases without any phenotype association.
In this study, the in silico analysis has also affirmed that the identified variant is pathogenic. In gnomAD database (v3.1.2), the variant is tolerated by SIFT prediction (score: 0.17), which is similar for p.V552I (predicted to be tolerated with a score of 0.16) but other prediction tools like CADD (24.3 vs. 22.9), Fathmm-XF (0.85 vs. 0.0057) and REVEL (0.217 vs. 0.194) showed higher scores for p.V552A than p.V552I (https://gnomad.broadinstitute.org/variant/6-43551371-A-G?dataset=gnomad_r3). They did not functionally characterize p.V552I variant, but they showed that C-terminally GFP-tagged NUP93 precipitates endogenous SMAD4. C-terminally GFP-tagged SMAD4 precipitates endogenous NUP93. Upon co-overexpression, GFP-tagged NUP93 interacts with Myc-tagged SMAD4. Mutations (Lys442Asnfs*14, Gly591Val, Tyr629Cys) identified in individuals with SRNS abrogate SMAD4 interaction. Upon BMP7 stimulation C-terminally GFP-tagged NUP93 interacts with phosphorylated/activated SMAD1/5. C-terminally GFP-tagged NUP93 precipitates endogenous importin7. C-terminally GFP-tagged SMAD4 precipitates endogenous importin7. Upon co-overexpression, GFP-tagged NUP93 interacts with Myc-tagged importin7. Mutations (Lys442Asnfs*14, Gly591Val, Tyr629Cys) identified in individuals with SRNS abrogate importin7 interaction. GFP-tagged SMAD4 precipitates endogenous XPO5. Deletion of the SMAD4 nuclear export signal (NES) (aa142-aa149) abrogates the interaction with XPO5. Altogether, all the proteins have very strong interaction and mutations in XPO5 can cause similar phenotypes as the patients having mutations in NUP93 (K442Nfs*14, G591V, Y629C). So we believe that our identified mutation can be pathogenic based on the higher score of prediction tools. To the best of our knowledge, our study describes the autosomal dominant inheritance pattern in adult-onset of familial FSGS/FGGS and the histopathological changes in association with XPO5 variant for the first time.
The NPCs consist of highly conserved eukaryotic proteins known as NUPs within the nuclear envelope which mediate the nucleocytoplasmic transport of RNAs, ribonucleoprotein (RNP) and other small proteins by exportins and importins . XPO5 plays a role in nucleocytoplasmic shuttling with the interaction of NUPs. Braun et al. have demonstrated that XPO5 was present in developing podocytes along with NUP93 and NUP205 in fetal rat kidney at the capillary loop stage of renal glomerular development . They also described that the defects in NUP93, NUP205 and XPO5 could affect SMAD signaling pathway, proving that SMAD signaling is the relevant pathogenic pathway for early onset SRNS in their patients with homozygous variants . They also demonstrated the colocalization of XPO5 along with synaptopodin in adult rat glomeruli. Synaptopodin is a marker of primary and secondary podocyte foot processes and has been defined as a marker for some other products of genes in association with SRNS, if mutated [23,24,25]. Interestingly, all the patients having variants in the genes colocalizing with synaptopodin had homozygous variants and an early onset age of the disease [23,24,25].
FSGS can present with massive proteinuria with or without impaired renal function . It is a leading cause of end-stage kidney disease. It accounts for about 20% of nephrotic syndrome in children. The renal biopsy of family FS-133’s proband showed FSGS and global sclerosis which were in accordance with his clinical features such as nephrotic range proteinuria and renal insufficiency. The renal biopsy of proband from CKD-05 family showed focal glomerulosclerosis (highly expected FSGS) with increased serum creatinine level.
Till date, few variants in exportins (only in EXPO1 and EXPO5) have been reported and described in association with chronic lymphocytic leukemia, Wilms’ tumor, autism, multiple myeloma and minimal change nephrotic syndrome (MCNS) with SRNS, respectively [7, 9, 10, 26]. Moreover, Neggers JE et al. have demonstrated that a heterozygous variant confers a similar defect in Expo1 cargo as a homozygous substitution in human cell lines . To the best of our knowledge, four variants have been reported in XPO5, of which only one homozygous variant has been identified in a boy with kidney disease and the other three variants have been found in patients with Wilms tumor, autism, and multiple myeloma. A homozygous variant in XPO5 (c.G1654A, p.V552I) in a 2-year-old Turkish boy has been reported in association with SRNS and speech development delay, while renal biopsy showed MCNS . Here, we implicate NPC-associated protein, XPO5, in the pathogenesis of FSGS in two Chinese families with different findings. The Turkish boy had a homozygous variant, while our patients have heterozygous. Furthermore, the Turkish boy exhibited MCNS at an early age (2 years), while our patients were adults and affected with focal glomerulosclerosis by renal biopsy. The Turkish boy was also primary SRNS and showed a response to CsA while our patient (the proband of family FS-133) showed only partial remission of proteinuria after immunosuppressant therapy including CTX . Thus, our findings confirm that the identified variant was causing adult-onset biopsy-proven FSGS for the first time.
In this study, we performed direct sequencing of 40 adult-onset Chinese FSGS families, 77 sporadic FSGS cases and 157 CKD families (non-FSGS) and identified a novel heterozygous variant (c.T1655C:p.V552A) in XPO5. All the patients were carrying variants, while the normal individuals were wild type. Our findings showed that the identified variant in XPO5 is causing adult-onset FSGS and its rate is low in Chinese familial FSGS (2.5%, 1/40) and in CKD (0.64%, 1/157).
Interestingly, the variant rate of NUPs was relatively lower in the Chinese population based on previous studies [28,29,30,31,32]. In contrast to prior studies related to NUPs variants, the XPO5 variants discovered in families recruited in this study had heterozygous variant. The in silico analysis of the identified heterozygous variant in this study affirms that it is pathogenic and might cause the adult-onset FSGS/FGGS in our patients. Furthermore, we suggest the functional analysis of identified variant in vivo by using different model organisms. This study has two limitations, firstly the unavailability of validation at protein levels and secondly, the absence of the in vivo and in vitro functional evidence for the pathogenesis of the identified variant.
In summary, this is the first study that demonstrates a novel heterozygous variant of XPO5 causing adult-onset of FSGS in two Chinese families segregating in an autosomal dominant pattern. Thereby, we describe a novel XPO5 protein to a new pathogenicity for FSGS. Additionally, the identification of the new inheritance pattern and the difference in onset age for XPO5 can help us understand the recurrence risk within the families and the genetic counseling of such complicated impending nephropathies. This study will be helpful for nephrologists using genetic analysis to make the early diagnosis, disease management, future treatment and genetic counseling. Lastly, we implicate that SMAD signaling pathway has a high potential for new approaches toward FSGS therapy.