CASE REPORT Familial focal segmental glomerulosclerosis associated with a WT1 gene missense mutation: A case report

Focal s egmental glomerulosclerosis (F SGS) can cause protei nuria and loss o f k idney fun ction, leading to e ndstage renal di s ease (ESRD). Podocyte injury is the ce ntral pathophysiologi cal mechanis m of hereditary FSGS. Numerous mutations in genes e ncoding or affe cting the transcriptional regulation of podocyte cell compar tments have been detected in patients with genetic FSGS. Herein, we report a rare case of familial FSGS with an autosomal dominant WT1 mutation. A 63-year- old man developed pro teinuri a; his reading showed over 1g prote in/day. A pa thological diagn osis of FSG S was made after rena l biops y. H is elder brother an d a 36-year- old son also had ESRD. Heterozygous variant of WT1 (NM_024426.4) c.1373G>A (p.Arg458Gln ) mi s sense was dete cted in the patient a nd his son , by whole-exome sequen cing. Although genetic screening is not a par t of routine practice, it s hould be per for med in such cases to a id a ppropriate tre atment options sel ecting, revealing extra ren al symptoms, and family planning.


Introduction
Focal segmental glomerulosclerosis (FSGS) is characterised by focal scarring of the glomerular capillary tuft and podocyte injury.The prevalence of biopsy-proven FSGS varies according to geographical areas, race, age, accessibility to health care services, and indication for renal biopsy.The causes of FSGS are categorised into primary, secondary, genetic, and undetermined. 1docyte injury is the central pathophysiological mechanism in hereditary FSGS. 2 The WT1 transcription factor gene (WT1) plays a critical role in genitourinary development, and its mutations are involved in several diseases that involve the kidneys and genitals. 3Recent studies have revealed that WT1 deletion is associated with podocytopathy. 4,5Herein, we report a case of familial FSGS with an autosomal dominant WT1 mutation and provide a review of the literature.

Case Report
A 63-year-old man was referred to the nephrology department of CHA Bundang Hospital in Republic of Korea, on February 22, 2018, due to a 10-year history of proteinuria.Additionally, he had a 20-year history of hypertension and a two-month history of gout.Physical examination revealed a maximum blood pressure of 160/100 mmHg and a body mass index of 24.8 kg/m 2 .A urinary dipstick test showed urine protein 2+ and the spot urine protein/creatinine ratio was 1.91 g/g (normal below 0.2 g/g).Serum blood urea nitrogen, creatinine, and albumin levels were 15.7 mg/dL (normal 5-20 mg/dL), 1.0 mg/dL (normal 0.7-1.2mg/dL), and 3.6 g/dL (normal 3.5-5.2g/dL), respectively.An abdominopelvic computed tomography scan showed that both kidneys were normal in size (left, 12.5 cm; right, 12.3 cm).He was asymptomatic, with no extrarenal manifestations.
The kidney biopsy samples revealed the presence of up to 17 glomeruli, of which 8 (47%) showed total sclerosis, and 3 (18%) showed segmental sclerosis.The glomerular basement membrane had a normal thickness with relatively smooth contours and without electron-dense deposits.The podocyte foot processes were widely effaced.The tubules had marked focal atrophy with mononuclear cell infiltration and fibrosis in the interstitium.Hyaline arteriolosclerosis and fibrointimal thickening of small arteries were also observed.Immunofluorescence microscopy showed no immunocomplex or autoantibody deposition.Overall, a pathological diagnosis of moderately advanced FSGS was made (Figure 1 A and B).The patient's deceased older brother had ESRD and a 36year-old son too has end-stage renal failure (ESRD).His son was diagnosed with FSGS at the age of 18 and is currently on chronic haemodialysis (Figure 1 C).Based on family history, genetic analysis on the patient and his family members was performed.This study was approved by the IRB of CHA Bundang Medical Centre and the patient and his son gave informed written consent.Whole-exome sequencing was performed using SureSelect Human All Exon V6 (Agilent, Santa Clara, CA, USA) and sequencing was performed using the NovaSeq platform (Illumina, San Diego, USA). 6The mean depth of coverage was 100× (>10×=99.2%).The pathogenicity of each variant was evaluated according to the recommendations of the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology guidelines. 7The heterozygous variant of WT1 (NM_024426.4)c.1373G>A (p.Arg458Gln) missense, was detected in the patient and his son.This was previously reported to be associated with FSGS and nephrotic syndrome.Moreover, the variant is absent in the gnomAD v2.1.1 dataset and functional studies provide strong evidence that it has a damaging effect on the gene or gene product with in silico tools. 2 Therefore, this variant was classified as pathogenic according to the ACMG guidelines. 7giotensin receptor blockers and calcium channel blockers (Fimasartan 60mg or Amlodipine 5mg) were prescribed to the patient.During follow-up over one year, his serum creatinine was stable at 1.1-1.3mg/dL (normal 0.7-1.2mg/dL), and the urine protein level decreased to 0.362g/g (normal below 0.2g/g).

Discussion
We describe a case of familial FSGS caused by a missense mutation in WT1.Kidney biopsy revealed FSGS with diffuse foot process loss, although the amount of proteinuria and normal serum albumin levels were not consistent with the pathological findings.Genetic testing was performed because of the patient's family history of ESRD; the WT1 mutation, which appeared to be the cause of podocyte abnormalities, was observed in both the patient and his son.Many inherited forms of FSGS have been identified, and most are caused by mutations in podocytes or glomerular basement membrane proteins.WT1 mutations usually present as the autosomal dominant form of hereditary kidney disease, and its penetrance is nearly 90% with various ages of onset.A WT1 mutation is one of the most common causes of familial FSGS and genetic steroidresistant nephrotic syndrome (SRNS), and has been reported to cause 10-20% of nonsyndromic genetic FSGS. 2 The possible genetic causes of FSGS are summarised in   target genes and homodimerisation of gene products, while exons 7-10 encode a DNA-binding domain consisting of four zinc fingers.In the kidneys, WT1 is required for the development and maintenance of podocytes homeostasis; indeed, several podocyte genes, including NPHS1 (nephrin), NPHS2 (podocin), SYNPO (synaptopodin), and Podxl (Podocalyxin), have been identified as WT1 targets.These genes are important components of the slit diaphragm and cytoskeleton of podocytes and are required for cell viability. 51 DNA binding has been suggested to perform both activator and repressor functions, although the exact mechanism remains unknown. 5nterestingly, podocyte-specific genes have multiple WT1 binding sites, which can epigenetically increase the podocytespecific gene expression. 5In addition, WT1 forms a complex along with enhancer proteins to regulate the transcription of target genes. 5In Adriamycin-induced podocyte injury models, it has been proposed that multiple WT1 DNA binding recruits a series of transcription factors and enhancers to induce a repair response at an early stage.foetal WT1:c.1371G>A(pArg458Gln), the missense mutation noted in our case, is presumed to cause modification of the zinc finger portion of the WT1 protein, which affects target DNA binding affinity. 8In the United States, Hall et al first reported the missense mutation (R458Q) in WT1 isoform D in two kindred Northern Europeans with nonsyndromic FSGS. 2 They observed that WT1R458Q overexpression significantly downregulated nephrin and synaptopodin expression, leading to impaired podocyte homeostasis.
Mutations in WT1 cause various pathologies of the genitourinary system. 9he Frasier and Denys-Drash syndromes, associated with WT1 mutation, show abnormal gonadal development and progressive glomerulopathy. 9Wilm's tumour and kidney-urinary dysplasia were observed in 40% and 11% of patients with WT1 mutations, respectively. 10Therefore, additional tests may be needed to rule out extrarenal disease in FSGS patients with suspected WT1 mutation, which may provide appropriate treatment for the patient's extrarenal disease.Interestingly, the age of onset and the severity of the disease differed between the father and son despite the same mutated gene.It is likely to be caused by an autosomal dominant genetic disease that is well expressed as a heterogeneous phenotype.
Genetic FSGS is known to respond poorly to steroids and other immunosuppressants. 11Therefore, if hereditary FSGS is suspected, immunosuppressive treatment is generally stopped and the treatment is focussed on conservative therapies such as Renin-Angiotensin-Aldosterone blockade, optimal blood pressure control, and a low-salt diet.In the present case, the patient maintained kidney function and achieved reduced proteinuria with a Renin-Angiotensin-Aldosterone blocker and antihypertensive treatment.However, in randomised controlled trials, apolipoprotein L1 risk variants have not been reported to affect the response to immunosuppressants in patients with FSGS. 11Further studies on the effects of immunosuppressants in genetic FSGS therapy are needed.

Conclusion
Genetic analysis is not contemplated as routine care for adults with FSGS; however, it may help in selecting treatment options, identifying extrarenal symptoms, and family planning.FSGS patients with positive family history and nonsyndromic glomerulonephritis should be evaluated for genetic causes.Despite not being able to identify all genetic causes, next-generation sequencing (NGS) or target gene panels are useful tools for detecting genetic diseases, including hereditary FSGS.
Patient's consent: Informed consent was taken from the patient prior to manuscript writing.
WT1 is a 50-kb gene with 10 exons on chromosome 11, band p13.WT1 encodes a DNA-binding protein that functions as a transcription factor.Exons 1-6 encode proline-rich regions for the transcriptional regulation of 150 Open Access J Pak Med Assoc

Figure :
Figure: (A) Light microscopy shows focal atrophy and loss of tubules with interstitial infiltration of lymphocytes, hyaline arteriolosclerosis, and intimal fibrosis of the artery (periodic acid-Schiff stain, x200).(B) Electron microscopy of the renal biopsy sample shows diffuse foot process effacement of podocytes without electron-dense deposits (x5,000).(C) Three-generation family pedigree.

Table :
List of genes that cause focal segmental glomerulosclerosis.
Nephrotic syndrome (NS) and FSGS are genetically heterogeneous disorders that represent a spectrum of hereditary renal diseases.The variation of * genes could be more represented as nephrotic syndrome and ** could be more represented as FSGS.OMIM was also added to NS-listing genes: LAMB2, DGKE, ARHGDIA, EMP2 (602334), MAGI2, KANK2, AVIL, and NOS1AP.a AD inheritance pattern in Charcot-Marie-Tooth disease, but an unknown pattern in FSGS.AR, autosomal recessive; AD, autosomal dominant; XLR, X-linked recessive; XL, X-linked; SMu, spontaneous mutation.