Hereditary podocytopathies in adults: the next generation

Transcription

1 Hereditary podocytopathies in adults: the next generation Olivia Boyer Néphrologie pédiatrique Institut Imagine Hôpital Necker Enfants Malades, Université Paris Descartes Paris, France

2 Hereditary podocytopathies Podocyte cytoskeleton Glomerular filtration barrier Hereditary podocytopathies Congenital NS Steroid-resistant NS Idiopathic FSGS Foot process effacement NS Proteinuria Machucca et al. HMG 2009 Faul, Trends Cell Biol 2007 Welsh, Nat Rev Nephrol 2012

3 The study of familial glomerulopathies led to unravel the physiology of the glomerular filtration barrier Nephrin Rodewald and Karnovsky, 1974 Tryggvason K, JASN 1999 Endlich K, Curr Opin Nephrol Hypertens 2001

4 Growing number of genes involved in podocytopathies Adapted from Machuca et al. HMG 2009 Cytoskeleton Cdc42 Slit Diaphragm Rac1 Cdc42 Arhgap24

5 Growing number of genes involved in podocytopathies 1991 WT1 NPHS1, NPHS2, SMARCAL1 LAMB2, TRPC6 PLCE1, CD2AP, MYH9, INF2 PTPRO, ITGA3, NXF5, LMX1B ACTN4, CD151 PDSS2 COQ2 SCARB2 ARHGAP24, ARHGDIA ITGB4 COQ6 TTC21B MYO1E Positional Cloning, homozygosity mapping in large/consanguineous families 18 Mb Slide courtesy of C. Antignac

6 Input from positonal cloning Analyses the cotransmission of a morbid trait (NS/Pu) with microsatellite markers spred along the whole genome (linkage analyses, homozygosity mapping) location in the genome Sequencing of candidate genes at the locus mutation Direct identification of genes involved in hereditary diseases, which pathophysiology is unknown Requires several informative families or large consanguineous familles Limited by incomplete penetrance

7 NPHS2 mutations (podocin) are the 1st cause of autosomal recessive steroid resistant nephrotic syndrome AR-SRNS NS 4 years, ESKD 10 years > 110 mutations - 42% familial cases % sporadic cases R138Q (32%): retained in the endoplasmic reticulum Age at Dx 2 years < 10 years for mutants located at the plasma membrane Congenital NS % of cases (ESKD 6 years) Expression in podocytes Slit diaphragm protein Late onset NS/Pu R229Q + 1 pathogenic mutation 19% of adult NS (Pu 25 years, ESKD 32 years) WT Podocine R138Q Calnexin (RE) Homodimer Boute et al., Nat Genet 2000 Bouchireb, Boyer et al., Hum Mut 2014 Roselli et al., Traffic 2004

8 Adult patients carry the NPHS2 polymorphism p.r229q + 1 pathogenic mutation Series of 105 cases / 96 families No adult patient with 2 pathogenic mutations 18 patients with 1 pathogenic mutation + p.r229q p.r229q: non neutral polymorphism (~ 3%) reduced interaction with nephrin Age at Dx: 24.9 years ( years) Age at ESKD: 32 years ( years) 1 st step of genetic analyses in adults: search for p. R229Q variant Tsukagushi, JCI 2002 Machuca, Hummel et al., KI 2009

9 NPHS2 mutations and incomplete penetrance 3% of parents should be affected? o x R229Q ; R138Q x R138Q ; wt x x R138Q ; R138Q 6/129 parents of affected children are asymptomatic carriers of the association [p.r229q];[mutation] Tory et al, Nat Genet 2014

10 The effect of the p.r229q NPHS2 mutation depends on the 2 nd mutation Pathogenic dimers [p.r229q];[cterminal mutation] Non pathogenic dimers [p.r229q];[nterminal mutation] R229Q + A284V R229Q + WT R229Q + R138Q Podocin retained in the cell Patients are affected Podocin at the plasma membrane Patients are unaffected Patients with [p.r229q];[n-terminal mutation] are unaffected Genetic counselling: A couple carrying an NPHS2 mutation in exons 1 6 in one member and R229Q in the other are not at risk of having an affected child o R229Q ; wt o x x R229Q ; R138Q R138Q ; wt Tory et al, Nat Genet 2014

11 TRPC6 mutations are responsible for 5% of AD FSGS TRPC6 TRPC6 (HSF-AD) Calcium channel Identified in a large family from New Zealand with late onset Pu and ESKD in 60% of patients Mutations in 9/179 families (5%) Incomplete penetrance Intracellular Ca influx Winn et al., Kidney Int 1999 Winn et al., Science 2005 Reiser et al., 2005

12 ACTN4 mutations are responsible for 4% of AD FSGS ACTN4 ( -actinin 4) Actin-bundling protein ACTN4 Missense mutations in 3 large pedigrees with AD FSGS 5/141 familial FSGS (4%) Proteinuria with FSGS (2 nd decade), progressive CKD lateonset ESKD in some patients (~50 yrs) Incomplete penetrance Kaplan et al., Nat Genet 2000 Weins et al., JASN 2005 Weins et al., PNAS 2007 Increased affinity for actin

13 Mutations in INF2 are the main causes of AD FSGS Exon 2 p.a13t p.l42p p.l76p * Exon 4 p.r177h p.e184k p.e184q p.s186p p.y193h p.l198r* p.n202d p.a203d p.r214c p.r214h p.r218w p.r218q* p.e220k* * * * * # Exon 6 p.l245p % * Brown et al., Nat Genet 2010 # Lee, Ped Nephrol 2010 Boyer et al., JASN 2011 Gbadegesin et al., KI 2011 % Sanchez-Ares et al., KI 2012 Reference Brown 2010 Barua 2012 # tested families # mutated families % mutations Age at onset Pu Age at ESKD % yrs yrs Lee % 7-30 yrs 14 yrs Boyer % 5-44 yrs yrs Gbadegesin % yrs yrs TOTAL % 5-72 yrs yrs Interindividual variability & incomplete penetrance 3/405 = 0.8% of sporadic cases Bown et al., Nat Genet 10 Boyer et al. JASN 11

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15 Mutations in the INF2 DID alter the localisation of the protein and actin polymerization Enhances actin polymerization Promotes actin depolymerization and filament sievering Role in intracellular protein transport in association with Cdc42 and MAL MYELIN And Lymphocyte protein Madrid, Dev Cell 2010 Andres-Delgado, Blood 2010 Brown et al., Nat Genet 2009

16 Input from candidate gene approaches Amyotrophy Steppage gait Pes cavus CMT + FSGS 13 reported cases Age Pu 1-28 yrs Nephrotic syndrome 3/11 ESKD after yrs Prevalence: 1/2500 Reduced deep tendon reflexes Common genetics? INF2 partners Cdc42 and MAL are crucial for myelination INF2 = good candidate? Lemieux et al., Can Med Assoc J 1967

17 Mutations in INF2 are the main causes of FSGS associated with Charcot Marie Tooth disease Cohort of 16 unrelated families with FSGS-CMT : mutations in 75% cases No mutation in 150 patients with CMT and no renal involvement CMT : 13 years (5-28 years), 4 with deafness Proteinuria : 18 years (10-21 years) ESKD: 21 years (12-47 years) Dominant inheritance Mutants alter the INF2-mediated polymerization & depolymerization INF2 & MAL colocalize and interact in Schwann cells Boyer et al., N Engl J Med 2011

18 Other podocyte cytoskeleton genes Plasma membrane Ciclosporin + GTP GEF GDP INF2 Rho-GDP Rho-GTP Formin WASP α-actinin 4 FAK vinculin talin pax ILK Myosine actin 1E Rho-GDP GDI Pi GAP ARHGAP24 (Rho-GAP24) Actin rearrangement dys ARHGDIA collagen IV laminin Faul, Trends Cell Biol 2007 Machuca et al. HMG 2009

19 ARHGAP24 mutations are responsible for autosomal dominant FSGS Cdc42 Rho-GAP24 ARHGAP24 Rac1 EXPERIMENTAL BACKGROUND Rho-GAP24 inhibits Rac1 and lamellipodia formation during podocyte differentiation RhoA ESKD 29 yrs sequencing of 310 patients with FSGS loss-of-function mutation in the GAP domain 1 family (0.3%) with ESKD in childhood or adulthood FSGS 20 yrs CKD 32 yrs ESKD 12 yrs Q156R mutant fails to inhibit Rac1 Akilesh S, et al., JCI 2011

20 Input from high throughput sequencing XXXXX variants Exome sequencing Filters Genomic DNA Exome capture Sequencing Candidate genes Functional studies Disease-causing variant

21 Exome sequencing in a large family of AD FSGS 22 yrs: Pu K Bx: FSGS 59 yrs: GFR 61 ml/min/1.73m2 II:1 III:1 het 36 yrs: Pu 58 yrs: ESKD Cause? RTx II:2 III:2 II:3 het III:3 het I:1 I:2 II:4 III:4 No mutation Family F. III:5 6 yrs: SRNS K Bx: MC Variable Pu Lost to F/U 37 yrs: NS, FSGS, CKD 54 yrs: ESKD II:5 II:6 II:7 No mutation IV:1 het III:6 het 40 yrs: ESKD Cause? II:8 II:9 26 yrs: gravidic NS K Bx: FSGS GFR 120 ml/min/1.73m2 7 yrs: microalbuminuria GFR 115 ml/min/1.73m2 10 affected 4 generations 5 with DNA No extra-renal involvement Linkage analyses: Large region of interest 118 Mb on chromosomes 1,2,3,4,6,9,11,15,17,19 No known genes

22 Exome sequencing in a large family of AD FSGS 3845 variants Family F. I:1 I:2 II:1 II:2 II:3 het II:4 II:5 II:6 II:7 No mutation II:8 II:9 III:1 het III:2 III:3 het III:4 No mutation III:5 III:6 het Filters Number of variants Common to the 5 pts 3845 IV:1 het Within the regions of interest non exonic regions synonymous variants 20 - polymorphisms dbsnp 13 LMX1B Mutation p.r246q segregating in the family - polymorphisms 1000 genomes 11 - polymorphisms EVS 8 - Polymorphisms Necker 2 Boyer O et al., JASN 2013

23 R246 LMX1B mutations lead to FSGS without Nail Patella syndrome Nail-Patella syndrome Developmental defects of dorsal limb structures, kidney and eye, with nail dysplasia, patellar abnormalities, elbow dysplasia, iliac horns, nephropathy (2-65%) and glaucoma Patients Nail Patella No extra-renal disorder recorded in the patients No typical EM lesions of the glomerular basement membrane in the biopsy sample Patients Dreyer et al., Nat Genet 1998 Bongers and Gubler, Ped Nephrol 2002 Patients Nail Patella

24 Mutations in the homeodomain-containing transcription factor LMX1B Screening of 73 additional AD-FSGS families R246Q in one R246P in another R246Q Marini et al, Genet Med, 2010 The 2 mutations involve the same residue on LMX1B predicted to strengthen the interaction between LMX1B and DNA Boyer O et al., JASN 2013

25 Another contribution of exome sequencing 2 families late onset glomerular Pu 3 affected sibs with DNA in each family 1) Linkage analysis homozygosity region on chromosome 2 (LOD score = 2.4) 2) Exome sequencing Only 1 common mutated gene TTC21B p.p209l homozygous mutation segregating with disease 3) TTC21B screening in other families with similar phenotype 7/39 patients SRNS with a similar phenotype and p.p209l Homozygous mutation No mutation in 280 controls Huynh Cong E et al., JASN in press

26 TTC21B p.p209l mutation is a cause of nephronophthisis Anterograde IFT-B Kinesine Axoneme Nephronophthisis : a renal ciliopathy Autosomal recessive Due to mutations in ciliary genes Tubulo-interstitial nephritis without proteinuria ± extra-renal features Primary cilium IFT139 Retrograde IFT-A Dyneine 2 Transition zone Basal body Interstitial fibrosis, tubular basement membrane thickening Cortico-medullary cysts Mechano-chemical sensor Cell polarity Homozygous p.p209l mutation in 5 patients with isolated nephronophthisis TTC21B: 1 st ciliary gene involved in podocytopathies

27 Maturation of podocytes leads to cilia loss and IFT139 relocalization Cilia No cilia Primary cilium in fetal rat kidney Primary cilium and IFT139 expression during kidney development Fetal rat podocyte Adult rat podocyte K. Ichimura et al., 2010 Fetal Podocytes Cilia IFT139 Basal body Adult podocytes No cilia IFT139 Along the microtubule network Huynh Cong E et al., JASN in press

28 IFT139 knock-down leads to primary cilium and cytoskeleton defects partally rescued by the p.p209l hypomorphic variant Abnormal backwheel shape cells Shortened cilia Huynh Cong E et al., JASN in press

29 Patients with the p.p209l TTC21B homozygous mutation (IFT139) have a primary tubular and glomerular nephropathy Patients with FSGS also have Patients with nephronophthisis also tubulo-interstitial lesions have FSGS lesions and glomerular reminiscent of nephronophthisis proteinuria (1-7g/d) Origin Portugal and North Africa ESKD: 22 years 1 st ciliary gene leading to a glomerular disease Opens a new chapter on primary tubulo-glomerular nephropathies Huynh Cong E et al., JASN in press

30 Genetic heterogeneity in SRNS/FSGS More than 20 genes have been identified in non syndromic or syndromic forms of hereditary FSGS/SRNS. Some genes are mutated in very few families. Direct sequencing of genes long & expensive Gene (exons) # of identifed mutations # of families tested PTPRO (26) MYO1E (28) ARHGAP24 (8) 1 (+/-2) 310 ARHGDIA (19) Technical and ethical issues in interpreting WES data Need to develop new diagnostic tools EuRenomics (coordinateur: F. Schaefer, Heidelberg) Podocyte kit (30 genes)

31 Thanks Institut Imagine - Necker Géraldine Mollet Olivier Gribouval Evelyne Huynh-Cong Albane Bizet Kalman Tory Stéphanie Woerner Marie-Josèphe Tête Sophie Saunier Rémi Salomon Marie-Claire Gubler Corinne Antignac Inserm U702 - Tenon Emmanuelle Plaisier Pierre Ronco Equipe accueil Limoges Heidelberg Franz Schaefer Madrid Miguel Alonso Clinicians