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ARHGDIA: a Novel Gene Implicated in Nephrotic Syndrome

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ARHGDIA: a Novel Gene Implicated in Nephrotic Syndrome Developmental defects J Med Genet: first published as 10.1136/jmedgenet-2012-101442 on 22 February 2013. Downloaded from ORIGINAL ARTICLE ARHGDIA: a novel gene implicated in nephrotic syndrome Indra Rani Gupta,1,2,3 Cindy Baldwin,4 David Auguste,4,5 Kevin C H Ha,3,6 Jasmine El Andalousi,2 Somayyeh Fahiminiya,3,6 Martin Bitzan,1,2 Chantal Bernard,7 Mohammad Reza Akbari,8,9 Steven A Narod,8,9 David S Rosenblatt,1,3,4 Open Access 3,6 2,4,5 Scan to access more Jacek Majewski, Tomoko Takano free content ▸ Additional material is ABSTRACT the barrier into the urine. The glomerular filtration published online only. To view Background Congenital nephrotic syndrome arises barrier consists of fenestrated endothelial cells, the please visit the journal online fi (http://dx.doi.org/10.1136/ from a defect in the glomerular ltration barrier that acellular glomerular basement membrane that is jmedgenet-2012-101442). permits the unrestricted passage of protein across the about 300 nm thick, and specialised epithelial cells barrier, resulting in proteinuria, hypoalbuminaemia, and called podocytes. The majority of children with For numbered affiliations see end of article. severe oedema. While most cases are due to mutations congenital nephrotic syndrome have a monogenic in one of five genes, in up to 15% of cases, a genetic basis for their disease due to mutations in genes Correspondence to cause is not identified. We investigated two sisters with that affect the structure and function of the actin Dr Indra Rani Gupta, a presumed recessive form of congenital nephrotic cytoskeleton within podocytes.1 In a large survey, Department of Pediatrics, ∼ Division of Nephrology, syndrome. 85% of cases of congenital nephrotic syndrome Montreal Children’s Hospital Methods and results Whole exome sequencing were attributed to mutations in NPHS1 (nephrin), and McGill University, 2300, identified five genes with diallelic mutations that were NPHS2 (podocin), LAMB2 (laminin β2), or WT1 rue Tupper—E222, Montreal, shared by the sisters, and Sanger sequencing revealed (Wilms tumour suppressor 1), while the remaining Quebec, Canada H3H 1P3; that ARHGDIA that encodes Rho GDP (guanosine 15% were genetically uncharacterised.1 Subsequent [email protected]; α α PLCE1 Dr Tomoko Takano, Division of diphosphate) dissociation inhibitor (RhoGDI , OMIM to this report, mutations in (phospholipase Nephrology, McGill University 601925) was the most likely candidate. Mice with C ɛ) were also identified in cases of congenital Health Centre, 3775 University targeted inactivation of ARHGDIA are known to develop nephrotic syndrome, more frequently in the pres- Street, Room 236, Montreal, severe proteinuria and nephrotic syndrome, therefore this ence of a renal biopsy showing diffuse mesangial Quebec, Canada H3H 1P3; 23 [email protected] gene was pursued in functional studies. The sisters sclerosis. Establishing a genetic diagnosis has harbour a homozygous in-frame deletion that is important implications for the treatment of con- CB, DA, KCH, JEA and SF predicted to remove a highly conserved aspartic acid genital nephrotic syndrome; most of the heritable contributed equally. residue within the interface where the protein, RhoGDIα, forms do not respond to immunosuppressive interacts with the Rho family of small GTPases therapy, leaving bilateral nephrectomy followed by http://jmg.bmj.com/ Received 22 November 2012 Revised 24 January 2013 (c.553_555del(p.Asp185del)). Rho-GTPases are critical dialysis and transplantation as the only therapeutic Accepted 31 January 2013 regulators of the actin cytoskeleton and when bound to option.4 Published Online First RhoGDIα, they are sequestered in an inactive, cytosolic We report two sisters with congenital nephrotic 22 February 2013 pool. In the mouse kidney, RhoGDIα was highly syndrome who were born to consanguineous expressed in podocytes, a critical cell within the parents of Pakistani origin. Since no coding muta- glomerular filtration barrier. When transfected in tions were identified in the five aforementioned α HEK293T cells, the mutant RhoGDI was unable to bind genes, we hypothesised that the girls had a novel on September 24, 2021 by guest. Protected copyright. to the Rho-GTPases, RhoA, Rac1, and Cdc42, unlike the recessive form of congenital nephrotic syndrome. wild-type construct. When RhoGDIα was knocked down Using whole exome sequencing, we discovered that in podocytes, RhoA, Rac1, and Cdc42 were both girls have an in-frame deletion in ARHGDIA hyperactivated and podocyte motility was impaired. The that encodes the protein known as Rho GDP proband’s fibroblasts demonstrated mislocalisation of (guanosine diphosphate) dissociation inhibitor α RhoGDIα to the nucleus, hyperactivation of the three (RhoGDIα, OMIM 601925). Rho-GTPases, and impaired cell motility, suggesting that the in-frame deletion leads to a loss of function. CASE HISTORY Conclusions Mutations in ARHGDIA need to be The proband presented at 3 weeks of age with gen- considered in the aetiology of heritable forms of eralised oedema and was found to have severe nephrotic syndrome. hypoalbuminaemia and proteinuria and was diag- nosed with congenital nephrotic syndrome. The family history revealed that the parents were INTRODUCTION Pakistani in origin and consanguineous. A renal Congenital nephrotic syndrome is a rare kidney dis- biopsy was performed at 1 month of age and this order characterised by the presence of massive pro- showed severe glomerular changes consistent with fi – To cite: Gupta IR, teinuria, hypoalbuminaemia, and generalised diffuse mesangial sclerosis ( gure 1A D). The Baldwin C, Auguste D, et al. oedema in the first 3 months of life. It arises from a patient rapidly deteriorated and developed end J Med Genet 2013;50: defect in the glomerular filtration barrier that stage renal failure at 3 months of age that was – 330 338. permits the unrestricted passage of protein across treated by haemodialysis. She received a cadaveric 330 Gupta IR, et al. J Med Genet 2013;50:330–338. doi:10.1136/jmedgenet-2012-101442 Developmental defects J Med Genet: first published as 10.1136/jmedgenet-2012-101442 on 22 February 2013. Downloaded from Figure 1 Kidney biopsy of the proband at the age of 1 month. Kidney tissue was processed and stained with periodic acid Schiff (PAS) (A) and PAS methenamine (PASM) silver (B, D) for light microscopy or for electron microscopy (C). The patient’s glomeruli are notably abnormal; they are small for her age and show hypercellularity, increased extracellular matrix, and contracted/collapsed glomerular tufts that are surrounded by cuboidal/ immature podocytes (A, arrows) or by vacuolated/hypertrophic podocytes (B, arrows). These changes are consistent with diffuse mesangial sclerosis. A normal glomerulus from an age matched infant is shown for comparison (D). Electron micrograph images from the patient’s kidney biopsy (C) show diffuse foot process effacement (arrow), thinning of the glomerular basement membrane (arrowhead), and swollen endothelial cells (Endo). This figure is only reproduced in colour in the online version. renal transplant at the age of 2 years, but the graft never func- The bioinformatics analysis of exome sequencing data was tioned due to venous thrombosis in the transplant, and so the carried out as previously described.56Briefly, high quality patient remains on dialysis. The second child was diagnosed trimmed reads were aligned to the human reference genome with congenital nephrotic syndrome on day 16 of life when her (hg19 assembly) by Burrows-Wheeler Aligner (V.0.5.9),7 produ- laboratory studies revealed severe hypoalbuminaemia with pro- cing a coverage at >5× read depth for 86% and 82% of teinuria (figure 2A). The mother requested no further treatment exomes in patient 1 and patient 2, respectively. for this baby who subsequently died at the age of 2 months. Variants were determined using SAMtools V.0.1.7, mpileup and varFilter.8 For each called position, a minimum of two http://jmg.bmj.com/ METHODS variant reads and >20% single nucleotide variants or >15% Ethics approval indels (small insertions or deletions) variant reads were consid- Sequencing analyses, human fibroblast cultures and experiments ered. Common polymorphisms and systematic false positives fi were approved by the Research Ethics Board of the Montreal were removed by ltering the variants against our in-house Children’s Hospital/McGill University Health Center. Parental exome database, containing more than 500 individuals. Functional annotation of the remaining variants, those previ- written informed consent was obtained for the outlined studies. on September 24, 2021 by guest. Protected copyright. ously seen in less than five individuals in our in-house exome database, was carried out using ANNOVAR,9 which cross- Whole exome sequencing analysis references variants against public databases (dbSNP132 and the μ A total of 3 g of genomic DNA from each affected sister was 1000 Genomes project) and annotates them according to the used for whole exome sequencing. The DNA of patient 1, the type of mutations (intronic, exonic, untranslated region (UTR), proband, was subjected to the Agilent Sure-Select Human All etc) and the score of SIFT, PolyPhen-2 and PHASTCONS Exon V.2 Kit (Santa Clara, California, USA) which targets tools.10 11 Novel variants had an allele frequency <0.5% in the fi approximately 44 Mb of the human exome de ned by the 1000 Genomes dataset and were predicted to be non- National Center for Biotechnology Information (NCBI) consen- synonymous (ie,
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