The New Compound Heterozygous Mutation of NUP Nephropathy: Report of Two Cases and Literature Review


 Backgrounds: NUP nephropathy is identified as a rare monogenic cause of steroid-resistant nephrotic syndrome recently. To explore the relationship between NUP mutation and renal disorders, we provide two cases and a literature review of the genotypical and phenotypical features in patients with NUP nephropathy.Results: We reported two patients with newly diagnosed NUP nephropathy who carried a compound heterozygous mutations in NUP107 and NUP93 gene respectively. Both patients were diagnosed steroid-resistant nephrotic syndrome and progressed to end-stage renal disease in childhood. While the mutation c.1537+1G>A in NUP93 gene was previously described, the mutations c.460A>G and c.1085C>T in NUP107 gene and c.1472A>T in NUP93 gene were novel. We also summarized the phenotypic and genetic spectrum of NUP nephropathy in eighty-six reported patients who carried 50 different mutations in 6 NUP genes (NUP107, NUP93, NUP205, NUP85, NUP133, NUP160). The majority of them were Asians (66/86, 76.7%). The mutation c.2492A>C and c.1079-1083del in NUP107 had been identified as the founder mutations in East Asian[1-3], while c.1772G>T and c.1886A>G in NUP93 might be the founder mutations in Western Europe and Turkish respectively. Nephrotic syndrome was the most common renal manifestation (68/86, 79.1%). Although the renal prognosis was poor that 80.8% (59/73) of them developed end-stage renal disease within the first two decades, the outcome of renal transplantation in NUP nephropathy is better than patients with other steroid-resistant nephrotic syndrome. Focal segmental glomerulosclerosis was the most prevalent renal biopsy pathologic type (56/65, 86.1%). Various extra-renal manifestations were found in 44.8% (26/58) of patients. Neurological involvement was the most common extra-renal presentation (22/26, 84.6%), including microcephaly (13/22, 59.1%), intellectual disability (12/22, 54.5%), and global developmental delay (10/22, 45.5%). Diverse abnormalities of the facial appearance (8/26, 30.8%), short stature (5/26, 19.2%),and contain convergent strabismus (4/26, 15.4%) had also been reported. There are significant differences in extra-renal manifestations between different genomics. Conclusions: The renal manifestation of NUP nephropathy is highly consistent that most patients suffered early-onset SRNS with FSGS. More than half of the patients had extra-renal symptom concomitantly. Asians showed potential susceptibility to NUP nephropathy. Despite the limited reports, some genotype-phenotype correlations have been gradually revealed.


Background
Nuclear pore complexes (NPCs) are the largest protein assemblies in eukaryotic cells that embedded in nuclear membrane ubiquitously. It plays important role in gene expression, protein synthesis and macromolecular transmembrane process. Relatively stable sub-units that are composed of nucleoporins make up NPCs supramoleculares. Despite there are distinct categories of nucleoporins among different species, the structure of NPCs is conservatively evoluted [4].
There are more than 30 nucleoporins (NUPs) discovered in human NPCs. Recent studies revealed that mutations of nucleoporin (NUP) genes can affect the binding and interaction between different NPCs sub-units,which may alter the structure or function of NPCs leading a range of diseases [5]. Since the rst case of nephrotic syndrome (NS) caused by NUP107 mutation reported in 2015 [1], dozens of cases who were conjectured to be NUP nephropathy have been reported worldwide. However, the role of NUP gene mutations in nephropathy is still unclear [6]. In this report, the clinical characteristics of two Chinese cases with steroid-resistant nephrotic syndrome (SRNS) who discovered NUP93 and NUP107 gene biallelic mutations respectively were summarized. In addition, to explore the relationship between NUP mutation and renal disorders, we provide a literature review of the genotypical and phenotypical features in patients with NUP nephropathy reported to date.

Methods
Two children with SRNS that progressed to end-stage renal disease (ESRD) were presented. Gene detection approval and Written informed consents were obtained from the patients' parents.

Exome sequencing
Genomic DNA was isolated from peripheral blood leukocytes. The puri ed DNA fragments were ampli ed with PCR reactions after ligation of adaptor. Exome sequencing was performed after SeqCap EZ Choice XL Library (Roche NimbleGen) targeted capturing on Illumina HiSeq 2000. Sequencing data were analyzed by NextGene Software (V2.3.4). Nucleotide positions refer to GRCh37/hg19 (http://grch37.ensembl.org). Genome Aggregation Database (gnomAD, http://gnomad.broadinstitute.org/) was used to determine the prevalence of identi ed variants in coding regions. To con rm the variants, Sanger sequencing was performed in patients and their parents.

Patient 1
This patient was a 3-year-7-month-old girl who is the rst child of a non-consanguineous family without family history of renal disease. She developed seizures when she was 3 months old. Although multiple anti-epileptic drugs were used, seizures was not well controlled. She presented with global developmental delay and detected proteinuria by regular physical examinations at the age of 2 years. One year later, she was admitted to the emergency department for edema and oliguria. The patient was diagnosed NS because of macroalbuminuria (3 + protein on urine dipstick), hypoalbuminemia (serum albumin 13.6g/L) and hypercholesterolemia (serum cholesterol 20.42mmol/L). Prednisone with daily dose of 2mg/kg/d was given orally for four weeks, but no response was observed. Due to steroid-resistant, prednisone was gradually tapered off after 3 months of mycophenolate and 1.5 months of tacrolimus were used sequentially. Despite those treatments, the patient's symptoms and signs were not improved. Her renal function deterioriated with serum creatinine increased to 398 µmol/L at the age of 3 years 5 months and hemodialysis was initiated due to hyperkalemia and anuria. Whole-exome sequencing of the proband showed a compound heterozygous NUP107 missense mutation (c.460A > G and c.1085C > T) inherited from her unaffected father and mother respectively. Neither of these mutations have been reported previously. The allele frequency of this variant in the general population is very low. The variant sequence was highly conserved across different species (see Fig. 1.). The patient is currently waiting for kidney transplantation.

Patient 2
A 1-year-2-months old girl was admitted to local hospital with severe bilateral lower extremity edema. Laboratory test revealed severe proteinuria (urine protein/creatinine ratio > 2) and hypoalbuminemia (< 2.5 g/dL). Value of serum creatinine was not recorded initially and normal renal function was selfreported. Prednisone 2mg/kg for 4 weeks was prescribed, but her proteinuria persistented. Renal biopsy was performed at the age of 1 year and 4 months and focal segmental glomerulosclerosis (FSGS) was found. Cyclosporine and tacrolimus were used successively but stopped because of ineffectiveness. Five months since the onset of symptoms, her serum creatinine level elevated to 344µmol/L gradually and commenced peritoneal dialysis. Targeted exome sequencing revealed NUP93 compound heterozygous mutations (c.1537 + 1G > A/c.1472A > T). Sanger sequencing con rmed that the novel mutation c.1472A > T was inherited from her unaffected father while the reported mutation c.1537 + 1G > A [7,8] was inherited from her unaffected mother while c.1472A > T was inherited from her father. Corticosteroids and immunosuppressive agents were then progressively tapered off in two months. During the waiting period for kidney transplantation, her urine output decreased gradually. Cardiac ultrasound showed progressively enlarged left heart, mild mitral valve insu ciency and left ventricular ejection fraction decreased (43%). However, no clinical signs of cardiac insu ciency was observed. She underwent allograft renal transplantation when she was 1-year-9-month old. After kidney transplantation, she had transient proteinuria for about one month. Her renal function became normal 3 days post-operation and maintained stable until now. During follow-up, echocardiogram returned to normal 5 months later.

Literature review
Since the discovery of the linkage between NUP107 gene mutation to SRNS syndrome in 2015, renal involvement resulting from NPCs mutation has been observed in six NUP genes (NUP107 [OMIM #616730] [1][2][3][9][10][11][12][13] [9,19,20]). We studied the correlations between NUP gene mutations and clinical manifestations by reviewing the literatures published on PubMed Database (http://www.pubmed.com) up to January 2021. The retrieval strategy is provided in Table S1. Seventy-four articles were retrieved initially and 21 articles were included nally after manual screen. We summarized the clinical manifestations by using standardized vocabulary provided in Human Phenotype Ontology (https://hpo.jax.org/).
c.303G > A Exon 4 hom (11) p.M101I NS (6) P (3) FSGS (4),  was poor that 80.8% (59/73) patients developed end-stage renal disease within the rst two decades, and among them 72.9% (43/59) occurred even in the rst decade. Fortunately, compared with other SRNS patients, the outcome of renal transplantation in NUP nephropathy is excellent that only one case suffered relapse of NS post-transplantation. Sixty-ve patients performed renal biopsy and 86.1% (56/65) of patients showed FSGS. Electron microscopy revealed partial effacement of foot processes without electron dense deposition. Various extra-renal manifestations were found in 44.8% (26/58) of patients while 32.6% (28/86) of them did not mention relevant information. Neurological involvement was the most common extra-renal presentation (22/26, 84.6%) (see Table 3). For those who harboured NUP107 gene mutations, some were presented with microcephaly, impaired intellectual development and steroidresistant nephrotic syndrome, which this triad is similar to Galloway-Mowat Syndrome (OMIM #616730) but lack of structural abnormality of brain. Abnormalities of the facial appearance including sloping forehead, narrow forehead, smooth philtrum, micrognathia, cleft lip and palate were seen in patients with NUP107 or NUP133 mutations. Short stature (5/26, 19.2%), contain convergent strabismus (4/26, 15.4%), and muscle hypotonia (4/26, 15.4%) had also been reported. Table 3 Comparison between NPC associated renal diseases (including our cases).   Discussion SRNS account fors 30% of the primary nephrotic syndrome in children, it is challenging due to that 30% − 50% of them gradually progress to ESRD within 10 years. This progress may be more rapid in patients with genetic background [11]. NUP nephropathy is identi ed as a rare monogenic cause of SRNS recently.
Due to the limited cases reported previously, the association between clinical and genetic features remain unclear. Hence, the diagnosis of NUP nephropathy are mainly based on genetics analysis. In this study, we presented the clinical and genetic data of two Chinese children with early-onset SRNS. Three novel mutations in NUP genes (two in NUP107 and one in NUP93) were detected in these patients, thus expanding the variant spectrum of NUP nephropathy and raising concern about carrier screening and genetic counselling for their family members in the future. Furthermore, we summarized the clinical, pathologic and genetic features of 86 reported cases to explore the correlation between phenotype and genotype in NUP nephropathy.
NUP nephropathy was initially reported in 9 patients with NUP107 gene biallelic mutations by Miyake et al. [1]. The results from the in vitro assays showed that the p. D831A substitution weak the binding between NUP107 and NUP133. Although mutations in NUP nephropathy varied among the reported cases, founder mutations in some regions emerged gradually. The mutation c.2492A > C and c.1079-1083del in NUP107 had been identi ed as the founder mutations in East Asian [1][2][3], while c.1772G > T and c.1886A > G in NUP93 might be the founder mutations in Western Europe and Turkish respectively [8,16]. Including our cases, East Asian race accounting for more than half of the reported cases with NUP nephropathy. A multicenter study in Korean revealed that NUP107 gene is one of the top 5 causative genes of SRNS or FSGS in pediatric patients [2]. However, as a novel genetic etiology of early-onset renal disease, it has not been given adequate attention at present. Current researches on genetic spectrum of renal disease in children did not include [21][22][23][24] or only include part of [11] NUP genes mutation detection. For the potential susceptibility of NUP nephropathy in Asians, NUP gene detection should be considered in gene panel of renal disease in Asia.
Both renal clinical and pathologic manifestations of NUP nephropathy are highly consistent that most patients suffered SRNS with FSGS in their early life.
Apoptotic cells in the glomeruli and renal tubules cells were mentioned in some cases, which it might be the typical change of NUP nephropathy [1]. Small amount of immune complex deposition was observed in some cases but massive deposition of immune complex was reported in only one patient with IgA 4 + by immuno uorescence [19]. Two patients underwent repeated renal biopsy. One patient was found to have increased IgA deposition [19] and the other had pathological transformation from minimal change disease (MCD) to FSGS after 2 years [1], suggesting that performing renal biopsy at different time points may give different impressions of the pathological types. According to the limited reports, patients with NUP nephropathy were unresponsive to either steroids or immunosuppressants. Thus, it is recommended to perform genetic examination early in patients with SRNS or FSGS to avoid unnecessary long-term administration to steroids or immunosuppressants.
Rapidly progressive renal insu ciency, which it may be a result of treatment failure, is not uncommon and can increase the therapeutic di culty for children with NUP nephropathy, especially in young children. Yet not all reported cases had ESRD. A girl presented SRNS at the age of 10 years and renal function remainded normal until the last follow-up at the age of 24 years, dissimilar from her sister who carried the same biallelic heterozygous mutation in NUP107 had SRNS at the age of 11 years but progressed to ESRD one year later [1]. The pathophysiology of renal function deterioration in NUP nephropathy has not yet been studied, thus further researches are required to clarify the interacting mechanisms. In progressed patients, the effect of renal transplantation were better than those without NUP gene mutation [3,11]. Only 1 case suffered plasma exchange dependent recurrent NS after renal transplantation. Fortunately, his proteinuria disappeared after comprehensive treatment therapy including rituximab and PE [25].
Extra-renal manifestations have made the comprehensive management of children with NUP nephropathy even more challenging. This study concluded that the involvement of nervous system is common, but there are signi cant differences between different genomics. All of the patients who carried c.303G > A mutations in NUP107 had microcephaly, and most of them with concomitant global developmental delay or intellectual disability [9,12,13]. However, no extrarenal involvement was reported in patients with mutations of c.2492A > C and c.1079-1083del in NUP107, which they are considered as the founder mutations in East Asia [1,11]. The differences mentioned above exhibit a potential correlation between phenotype and genotype. Besides, nearly one-third of the cases did not provide relevant information about the extra-renal involvement. Due to the high expression of NUP in vivo, systemic symptoms deserve further attention from physicians during disease course.

Conclusion
Timely genetic testing may guide early diagnosis of NUP nephropathy to avoid prolonged steroid or immunosuppressant exposure in patients with SRNS. In Asian population, NUP genes detection should be routine considered in children with SRNS or FSGS. Extra-renal involvement in patients with NUP nephropathy is not uncommon, which it should be given more attention. Although patients with NUP nephropathy often progress to ESRD, their long-term prognosis after renal transplantation are favorable.

Declarations
Ethics approval and consent to participate Ethics approval was not required for this study protocol since it was a retrospective analysis of observational data and does not use any tissue collected from human subjects.

Consent for publication
All authors read and approved the nal manuscript.