Disclosure of Relevant Hereditary Nephropathy with Focal Financial Relationships Segmental Glomerulosclerosis USCAP requires that all planners (Education Committee) in a position to Joseph P. Gaut, MD, PhD influence or control the content of CME disclose any relevant financial relationship WITH COMMERCIAL INTERESTS which they or their spouse/partner have, or have had, within the past 12 months, which relates to the content of this educational activity and creates a conflict of interest.
Disclosures Outline
• No financial disclosures • Background • Clinical Aspects of Hereditary FSGS • Pathologic Aspects of Hereditary FSGS • Genetic Testing • Summary
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A 2 year old presents with proteinuria
• Caucasian boy transferred from an outside hospital with severe nephrotic syndrome and pneumonia
• No extra-renal syndromic manifestations
• Focal segmental glomerulosclerosis • Diffuse podocyte foot process effacement
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1 Focal Segmental Glomerulosclerosis Morphologic Variants of FSGS
• A morphologic lesion composed of focal segmental • Columbia Classification accumulation of extracellular matrix • NOS • 20% of nephrotic syndrome cases in children • Perihilar • Cellular • 40% of nephrotic syndrome cases in adults • Tip • Collapsing • Incidence 7/million
• Prevalence of 4%
D’Agati VD, Kaskel FJ, and Falk RJ. N Engl J Med. 2011; 365(25):2398‐2411.
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A Lesion with Varied Etiologies • Familial/Genetic • Virus-Associated • Drug-Induced • Adaptive • Idiopathic
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Genes Associated with Hereditary FSGS
Slit Diaphragm Mitochondrial – CoQ10 Biosynthesis •NPHS1 •COQ2 Search for Gene and FSGS in PubMed •NPHS2 •COQ6 •PLCE1 •ADCK4 •CD2AP •MTTL1 – mitochondrial DNA •TRPC6 •PDSS2 Current •ANLN •XPNPEP3 >40 genes •CRB2 70 •FAT1 Actin Regulation Rho/Rac/Cdc42 Podocin •MAGI2 •KANK1 60 •KANK2 Nephrin Identified Actin Cytoskeleton •KANK4 50 Identified •ACTN4 •ARHGAP24 40 •INF2 •ARHGDIA •MYO1E 30 •MYH9 Integrin/Laminin •LAMB2 Publications 20 Nuclear •ITGA3 # •WT1 •ITGB4 10 •WDR73 •SMARCAL1 Lysosome 0 •XPO5 •SCARB2 •NUP93 •NUP107 Other •NUP205 •PTPRO Year •LMX1B •EMP2 •PAX2 •DGKE •NEIL1 Red = Autosomal Dominant
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2 Outline Clinical Aspects of Hereditary FSGS
• Background • Age of disease onset • Clinical Aspects of Hereditary FSGS • Typically younger patients (infants & young children) • Pathologic Aspects of Hereditary FSGS • Depends on genetic etiology • Genetic Testing • Steroid resistance • Summary • Steroid dependence • Nephrotic syndrome • Positive family history • Syndromic manifestations
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Five Genes Account for Majority of Age of Onset and Hereditary FSGS Positive Genetic Diagnoses
• 1589 families with SRNS 2% 2% 7% NPHS1 • Onset before 25 years 3% NPHS2 4% 34% • 27 genes evaluated WT1 7% PLCE1 • < 1 year old ‐ 60% with molecular diagnosis LAMB2 SMARCAL1 INF2 • >12 years old ‐ ~10% molecular diagnosis 16% TRPC6 Other • Genetic diagnosis identified in 29.5% of families 25%
Sadowski CE et al. J Am Soc Nephrol 2014,26(6):1279‐89. Sadowski CE et al. J Am Soc Nephrol 2014,26(6):1279-89.
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Genetic Etiology by Age Generalized Features of Inheritance Patterns
Age of Onset/Steroid Most Common Genes Autosomal Recessive Autosomal Dominant Response <1 year of age NPHS1, NPHS2, LAMB2, WT1, • Early onset • Late onset PLCE1 • Loss of function • Gain of function Age 1-10 years, SRNS NPHS2, LAMB2, WT1, PLCE1 • Infancy/early • Late-onset/slowly Age 1-10 years, SDNS MAGI2, TENC1, DLC1, CDK20, ITSN1 childhood progressive Age 10-18 or adult w/ +FH INF2, ACTN4, TRPC6, WT1 • Varied penetrance • Varied disease SRNS – steroid resistant nephrotic syndrome severity SDNS – steroid dependent nephrotic syndrome
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3 Pathologic Clues to Hereditary Outline Etiology
• Background • Collapsing glomerulopathy • APOL1 • Clinical Aspects of Hereditary FSGS • Pathologic Aspects of Hereditary FSGS • Glomerular basement • Genetic Testing thinning/multilayering • Summary • COL4A3, COL4A4, COL4A5
• FSGS with diffuse mesangial sclerosis • WT1, LAMB2, NPHS2, PLCE1, NUP93, NUP205, XPO5
• Abnormal mitochondria • COQ2, COQ6, tRNALeu (MELAS)
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Collagen type IV variants and FSGS • COL4A3 and COL4A4 associated with Alport syndrome • Patients may present with TBMD and/or FSGS
• Seven families with FSGS, nephrotic-range proteinuria & Presented with proteinuria and hematuria hematuria
Positive family history • One family with thin GBM – same variant as seen in previous case (COL4A3 p.G695R) FSGS on light microscopy • No basement membrane splitting, lamellation, or basket weaving Thin basement membranes on EM present COL4A3 p.G695R variant • Subset of cases without morphologic clues to diagnosis
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Pathology is Often Non-Specific in Hereditary FSGS Outline
• Typically NOS pattern • Background • Clinical Aspects of Hereditary FSGS • Diffuse podocyte foot process effacement in non- • Pathologic Aspects of Hereditary FSGS segmentally sclerotic glomeruli • Genetic Testing • Summary • Looks identical to primary FSGS
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4 Clinical Genomic Testing Genetic Testing in FSGS
• Why test? • Aid diagnosis • Identify appropriate treatment • Discontinue steroids • Continue steroid e.g. PLCE1 • CoQ10 supplements • Start calcineurin inhibitors – TRPC6
• Determine risk of disease recurrence post-transplantation • Assess risk of kidney donors • Prenatal diagnosis • Family screening
Brown EJ, Pollak MR, and Barua M. Kidney Int. 2014;85:1030‐1038
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Massively Parallel or ‘Next-Generation’ Sequencing Single Locus vs. Multiple Gene Testing • Sanger sequencing – 2x read (Bidirectional)
Locus C T A T G C T C G • Narrowly targeted specific • Result may trigger testing Analyze Determine additional gene single gene/locus mutation status of testing • Next-generation – 100-1000x reads at single position limited region
NPHS1 NPHS2 • Cost effective Multiple WT1 PLCE1 • Efficient/time‐saving gene testing LAMB2 • Yields unexpected ACTN4 INF2 findings Analyze multiple Determine mutation status relevant genes of all relevant genes simultaneously
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Next-Generation Sequencing Next Generation Sequencing Publications
Sanger c. 2000 NGS c. 2016 5000 4000
• 0.01 Gigabase/week • 10,000 Gigabase/week 3000 2000 Publications
• $100,000,000/Gigabase • $1/Gigabase # 1000 0 2005 2007 2009 2011 2013 2015 2017 Year
• Next-generation sequencing citations in PubMed • Total = 15,715 • 4,655 in 2016 • 1 in 2005
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5 Application of NGS Technology NPHS1 • Patient develops SRNS NPHS2 WT1 Multiple PLCE1 gene testing LAMB2 • Biopsy reveals FSGS ACTN4 INF2 . • A genetic cause is suspected . .
• Case is referred for genetic sequencing of genes known to A multitude of genetic variants are identified be involved in FSGS The greater number of genes sequenced, the greater number of variants identified
How to determine if a genetic variant is causing disease?
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Variant Interpretation Genetic Variant Interpretation • Literature
• Clinical Databases • HGMD, ClinVar
• Locus Specific Databases • Leiden, FH HUS
• Laboratory Specific Databases • EmVClass (Emory)
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Variant Interpretation Variant Interpretation Classifications • Frequency Data • dbSNP, 1000 genomes • Pathogenic • NHLBI ESP • ExAC Browser • Likely Pathogenic
• Effect on Protein • Conservation Data • Variant of Uncertain Clinical Significance • Grantham scores • In-silico predictions • Likely Benign • Protein function • Splicing • Functional Studies • Benign
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6 Clinical Application Patient Characteristics
• Washington University Experience • 16 Male 16 • 14 Female • Using standardized variant interpretation criteria 14 12 • 15 Caucasian • Patients referred for nephrotic syndrome/FSGS sequencing 10 • 7 AA 8 • 3 Hispanic • All cases reviewed by a pathologist with subspecialty 6 • 5 Other Frequency boards in Molecular Genetics or a clinical laboratory 4 geneticist certified in Clinical Molecular Genetics 2 0 • 30 cases sequenced to date as part of routine practice 5 1020304050607080 Age (Years)
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Washington University Experience Differences in Clinical Application
• Clinical population is less well defined compared to a research population with well-characterized families Negative P/LP 17% 10% • Indications for testing vary Diagnostic APOL1 13% Yield = 23% • Broader age range tested
• Strict variant interpretations are applied
VUS 60%
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Likely Pathogenic Variants Variants of Uncertain Clinical Significance
Age Gene 32 INF2 71 INF2 75 COL4A3
Typically worked up in setting of transplantation
All have a pathologic diagnosis of FSGS
All have a positive family history
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7 Variant Interpretation Challenges Variants of Uncertain Clinical Significance
• Incomplete penetrance • Most common classification • Not clinically actionable • Polygenic inheritance patterns
• Environmental factors • What to do with these variants?
• Lack of centralized database
• Lack of functional studies
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Case Revisited Genetic Variants Idnentified
• 2 year old with SRNS • NPHS2 – p.R138Q • NPHS2 – p.R196X • Pathology – FSGS
• Next generation sequencing performed
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Variant Interpretation Podocin Immunofluorescence
Variant MAF (overall) GERP SIFT
NPHS2 nonsense G-A p.Arg196X 0 4.79 n/a
NPHS2 missense C-T p.Arg138Gln 0.000676 5.82 0
• PVS1 – nonsense/truncating mutation
• PS1 – previously described pathogenic variant
• PM2 – low frequency relative to controls Control Patient • PP3 – predicted deleterious effect on protein function Pathogenic
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8 Summary References • Hereditary FSGS is more common in a younger population • Sadowski CE et al. J Am Soc Nephrol 2014; 26(6):1279-89. • D’Agati VD, Kaskel FJ, and Falk RJ. N Engl J Med. 2011; 365(25):2398-2411. • However, adults with a positive family history may harbor • Lovric S, et al. Nephrol Dial Transplant. 2016; 31:1802-1813. significant genetic variants • Pollak MR. Adv Chronic Kidney Dis. 2014;21(5):422-5. • Brown EJ, Pollak MR, and Barua M. Kidney Int. 2014;85:1030-1038 • Chatterjee R, et al. PLoS One. 2013:8(10):e76360. • Pathology is helpful in certain cases • Malone AF, et al. Kidney Int. 2014:86(6):1253-9. • COL4 variants, mitochondrial disease, APOL1, DMS • Vivante A and Hildebrandt F. Nat Rev Nephrol. 2016:12:133-146. • Laurin LP, et al. Nephrol Dial Transplant. 2014:29:2062-2069. • Fogo AB. Nat Rev Nephrol. 2015:11:76-87. • However, most cases of hereditary FSGS show non-specific • Mardis ER. Nature. 2011:470:198-203. • Büscher AK, et al. Clin J Am Soc Nephrol. 2016:11:245-253. findings similar to primary FSGS • Kestilä M, et al. Mol Cell. 1998:1:575-582. • Boute N, et al. Nature Gen. 2000:24:349-354. • Giglio S, et al. J Am Soc Nephrol. 2015:26:230-236. • NGS is increasingly utilized, but most commonly yields a • Chen YM, Kikkawa Y, Miner JH. J Am Soc Nephrol. 2011:22(5):849-58. diagnosis of VUS • Hildebrandt F. Lancet. 2010:375:1287-1295. • MacArthur DG, et al. Nature. 2014:508:469-476. • Liapis H and Gaut JP. Pediatr Nephrol. 2013:28(8):1207-19. • Need for centralized database, functional studies
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Thank you
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