ORAN 2010 new Genetic syndrome

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New genetic developmentsin nephrotic syndromeA. Bensman*, C. Antignac*** Hôpital d’enfants Armand Trousseau** Hôpital Enfants MaladesPARIS

Idiopathic nephrotic syndrome (NS) : 

Idiopathic nephrotic syndrome (NS) Steroid-sensitive NS T-cell disorder Circulating permeability factor Steroid-resistant NS Ciclo-sensibility Recurrence after transplantation Structural anomalies of the glomerular filtration barrier Focal Segmental Glomerulosclerosis Diffuse Mesangial Sclerosis

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Idiopathic nephrotic syndrome Unexpected efficacy of Rituximab (antiCD20) in multirelapsing minimal change nephrotic syndrome B cell disorder

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Georges Deschênes

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Hereditary forms of non syndromic NS Autosomal recessive inheritance Congenital NS of the Finnish type (NPHS1 nephrin - 19q13) Steroid-resistant NS (NPHS2 podocin-1q25-31) NS with Diffuse Mesangial Sclerosis (PLCE1) Pierson sd (LAMB2 - 3p14-p22) - ocular anomalies Autosomal dominant inheritance WT1 - male pseudohermaphroditism FSGS-1 locus: chrom 19q13 (ACTN4) FSGS-2 locus: chrom 11q21-q22 (TRPC6) Associated with mitochondrial cytopathy Usually associated with extra-renal symptoms, but proteinuria/NS might be the only symptom for months (Goldenberg et al., Pediatric Nephrol, 2005) Coenzyme Q deficiency (ubiquinone) - mitochondria proliferation by EM - possible response to treatment by ubiquinone supplementation

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Pierson syndrome Diffuse mesangial sclerosis and ocular anomalies (mainly microcoria) ± psychomotor delay, neuro-muscular disease Mutations in LAMB2 (32 exons) encoding laminin b2 expressed in GBM and in intraocular muscles (Zenker et al., 2004) Recently, LAMB2 mutations in: - early onset NS without ocular or neurological symptoms - childhood onset NS with ocular abnormalities

WT1 mutations : 

WT1 mutations Denys-Drash syndrome (Wilms’ tumor, male pseudohermaphroditism and diffuse mesangial sclerosis) Mutations in exons 8 and 9 Frasier syndrome (FSGS and male pseudohermaphroditism) Mutations in the donor splice site of intron 9 WT1 exons 8 and 9 mutations found in 6/115 (5%) patients with sporadic SRNS without external genital abnomalies (all phenotypically females) (Ruf., Kid Int 2004) WT1 mutations can be detected both in DMS and in FSGS

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Hereditary NephroticSyndromes From Kerjaschki et al. 2001 NPHS1: (nephrin) Cong NS ACTN4: AD - FSGS WT1 Frasier and Drash sd Cd2ap Neph1 Fat NS in mouse LMX1B In Nail-Patella sd Smarcal 1 in Schimke disease LAMB2: Pierson sd NPHS2 (podocin) SRNS Normal glomerular filtration barrier TRPC6: AD - FSGS PLCE1 SCARB2 (Limp2) in AMRF

Nephrin gene mutations in congenital NS of the Finish type : 

Nephrin gene mutations in congenital NS of the Finish type Over 70 mutations, >50% missense mutations In 16/21, defective intracellular nephrin transport, probably caused by nephrin misfolding (Liu et al., 2001) Nephrin mutations present >80% of severe NS observed in the first days of life, without extra-renal symptoms * Ruotsalainen et al., (1999) Extracellular domain Ig-like

NPHS1 (nephrin) mutations in patients with non congenital nephrotic syndrome : 

NPHS1 (nephrin) mutations in patients with non congenital nephrotic syndrome NPHS1 mutation screening in 142 unrelated patients with SRNS without NPHS2 mutations 11 patients compound heterozygous for NPHS1 mutations with mean age of onset of NS: 3 years (range 6 months - 8 years) and of ESRD (5 patients): 13.6 years (range 6 - 25) Histology at presentation: FSGS in 3 cases, minimal changes in 7 cases 14 mutations (12 novel): 6 nonsense and frameshift, 2 splice site and 6 missense 11% of patients with SRNS < 5 years present NPHS1 mutations

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Autosomal recessive inheritance Early onset in childhood Rapid progression to ESRD No recurrence after transplantation FSGS Podocyte protein Hairpin-like structure Located at the cytoplamic face of the slit diaphragm Interacts with nephrin Familial steroid-resistant nephrotic syndrome NPHS2 encoding podocin

Clinical Features of SRNS : 

Clinical Features of SRNS Mean±SEM * p<0.001 Clinical Features of patients with SRNS and NPHS2 mutations Mean±SEM * p<0.001

NPHS2 mutations in late-onset FSGS (>18yrs)Necker experience : 

NPHS2 mutations in late-onset FSGS (>18yrs)Necker experience Screening of 105 cases from 96 families (18 families from South America) resistant to immunosuppressive therapy without recurrence after transplantation (72 sporadic) No patient with 2 pathogenic mutations 18 patients (15 families - 9 from South America) with 1 pathogenic mutation (9 A284V ) + R229Q R229Q: non neutral polymorphism associated with a decreased binding to nephrin (Tsukagushi et al., 2002) Rate of mutation South America: 9/18 Others: 6/78 (7.6%) Mean age at onset : 24.9 years (18.5 - 39 years) Mean age of ESRD: 32 years (27.7 - 38.5 years) Frequence of the A284V in the patients from Spanish origin R229Q variant to be screened in first intention in adult patients

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Néphrin NPHS2 mutations responsible of CNF (Ruf et al, 2004 ; Hinkes et al, 2007) NPHS1 mutations responsible of SRNS ? Congenital NS of the Finish type NPHS1 gene mutations Mostly missense mutations leading to the retention of the mutant protein the ER Steroid-resistant NS (SRNS) NPHS2 gene mutations R138Q: most frequent mutation Podocin

PLCE1 mutations (Hinkes et al., 2006) : 

PLCE1 mutations (Hinkes et al., 2006) Identified using a combination of homozygosity mapping and cDNA microarrays from rat glomeruli Phospholipase C Hydrolyses membrane phospholipids inositol triphosphate (IP3) diacylglycerol Localization in the podocyte Interaction between PLC1 and IQGAP1 (which interacts with nephrin) Clinical symptoms 12 pts with truncating mutations Proteinuria by 4 yrs (median: 0.8 yrs) ESRD by 5 yrs (median: 0.9 yrs) Diffuse mesangial sclerosis 2 patients (1 family) with missense mutations Onset at 2 and 8.8 years FSGS

PLCE1 mutations: Necker experience : 

PLCE1 mutations: Necker experience Detection of PLCE1 mutations in 9/22 children with DMS PLCE1 mutations in 5/67 familial cases with isolated SRNS and FSGS without mutations in NPHS1 or NPHS2 Either truncating (3) or missense (2) mutations Onset between 10 months and 4 yrs ESRD by 6.5 yrs PLCE1 is involved in early onset NS with DMS or FSGS. It is difficult to draw phenotype-genotype correlation.

Diffuse Mesangial Sclerosis : 

Diffuse Mesangial Sclerosis Drash syndrome (WT1) Pierson syndrome (LAMB2) PLCE1 gene mutations Galloway-Mowat syndrome Early-onset NS Rapid evolution to ESRD FSGS and DMS can be found in patients with WT1, PLCE1 and LAMB2 mutations and might represent the extreme phenotypes of the same physiopathological mechanisms

Oligogenic inheritance ? Protective effect arising from other members of the phopholipase C family in the unaffected individuals ? : 

Oligogenic inheritance ? Protective effect arising from other members of the phopholipase C family in the unaffected individuals ? Phenotypic variability of PLCE1 mutations 3 unaffected individuals from 3 unrelated families with DMS present with PLCE1 mutations in the homozygous state Two patients seemed to respond to steroid- or cyclosporin therapy (Hinkes et al., 2006) No obvious renal phenotype in the mouse model

MYH9 as a major-effect risk gene for FSGS among African Americans : 

MYH9 as a major-effect risk gene for FSGS among African Americans MYH9 mutations in Epstein syndrome (glomerulopathy with deafness and macrothrombocytopeny) and Fetchner syndrome (with leukocyte inclusions) Encodes the nonmuscle myosin heavy chain IIA, expressed in the podocyte. MYH9 variants (exons 14 through 23) strongly associated to idiopathic and HIV-associated FSGS (P=4 X 10-23) in a cohort of 852 African Americans; also associated to hypertensive ESRD but not to type 2 diabetes ESRD (Kopp et al, Nature Genet, September 2008) Same results obtained in an additional cohort of African Americans with ESRD (Kao et al., Nature Genet, September 2008) Variants in genes involved in rare Mendelian disorders can be associated to susceptibility to more frequent diseases with complex inheritance

Function of the slit diaphragm proteins : 

Function of the slit diaphragm proteins Podocin, nephrin, CD2AP, TRPC6 and NEPH proteins associate in lipid rafts Nephrin and CD2AP are linked to the actin cytoskeleton Nephrin is phosphorylated and, in turn, activates downstream phosphorylation pathways Podocin enhances nephrin-induced signaling Role of CD2AP in endocytosis The whole complex is anchored to the cytoskeleton and is involved not only in podocyte and slit diaphragm architecture but also in cell signaling

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Response to steroïd or cyclosporine therapy PLCEA mutation Hinkes et al, Nat Genet 2006, 38 : 1397405 WT1 Alport Syndrome Non immulogical mechanisms

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Response to steroïd or cyclosporine therapy Stabilization of the actin cytoskeleton in kidney podocytes (Peter Mundel, nature medicine 2008, 14 : 931-938)

Practical approach : 

Practical approach Necessary to perform genetic testing in congenital NS and in SRNS starting in childhood Search for any extra-renal symptom (especially genital anomaly, eye or brain disorder…) Electron microscopy for looking for abnormal mitochondria Molecular screening

Conclusions : 

Conclusions Crucial role of the podocyte in the formation and maintenance of the glomerular filtration barrier Genetic heterogeneity Mutations in NPHS2 (podocin) and NPHS1 (nephrin) found in both congenital and SRNS A284V, hot-spot of mutations in patients from Spanish origin? Focal Segmental Glomerulosclerosis vs Diffuse Mesangial Sclerosis Extreme phenotypes of the same physiopathological mechanisms Interest of the molecular screening Genetic counseling Help for the treatment Some mutations might be amenable to treatment by chemical chaperones

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Department of Pediatric Nephrology (Trousseau Hospital) - Tim Ulinski - Béatrice Letavernier - Olivier Dunand Acknowledgments

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Inserm U574 - Necker Eduardo Machuca Fabien Nevo Olivier Gribouval Ernie Esquivel Aurélie Philippe Steffi Weber Emeline Verna Vincent Morinière Marie-Claire Gubler Department of Pediatric Nephrology Marie-Josèphe Tête Patrick Niaudet All the clinicians who referred the patients Acknowledgments