Hereditary Nephropathy with Focal Segmental Glomerulosclerosis

Joseph P. Gaut, MD, PhD Disclosures

• None

Pathology and Immunology Anatomic and Molecular Pathology Outline

• Background • Clinical Aspects of Hereditary FSGS • Pathologic Aspects of Hereditary FSGS • Genetic Testing • Summary

Pathology and Immunology Anatomic and Molecular Pathology 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

Pathology and Immunology Anatomic and Molecular Pathology • Focal segmental glomerulosclerosis • Diffuse podocyte foot process effacement

Pathology and Immunology Anatomic and Molecular Pathology Focal Segmental Glomerulosclerosis

• A morphologic lesion composed of focal segmental accumulation of extracellular matrix

• 20% of nephrotic syndrome cases in children

• 40% of nephrotic syndrome cases in adults

• Incidence 7/million

• Prevalence of 4%

Pathology and Immunology Anatomic and Molecular Pathology Morphologic Variants of FSGS

D’Agati VD, Kaskel FJ, and Falk RJ. N Engl J Med. 2011; 365(25):2398-2411.

Pathology and Immunology Anatomic and Molecular Pathology A Lesion with Varied Etiologies • Familial/Genetic • Virus-Associated • Drug-Induced • Adaptive • Idiopathic

Pathology and Immunology Anatomic and Molecular Pathology Pathology and Immunology Anatomic and Molecular Pathology Search for and FSGS in PubMed

Current >40 70 Podocin 60 Nephrin Identified 50 Identified 40 30 20 # Publications # Publications 10 0

Year

Pathology and Immunology Anatomic and Molecular Pathology Genes Associated with Hereditary FSGS

Slit Diaphragm Mitochondrial – CoQ10 Biosynthesis •NPHS1 •COQ2 •NPHS2 •COQ6 •PLCE1 •ADCK4 •CD2AP •MTTL1 – mitochondrial DNA •TRPC6 •PDSS2 •ANLN •XPNPEP3 •CRB2 •FAT1 Actin Regulation Rho/Rac/Cdc42 •MAGI2 •KANK1 •KANK2 Actin Cytoskeleton •KANK4 •ACTN4 •ARHGAP24 •INF2 •ARHGDIA •MYO1E •MYH9 Integrin/Laminin •LAMB2 Nuclear •ITGA3 •WT1 •ITGB4 •WDR73 •SMARCAL1 •XPO5 •SCARB2 •NUP93 •NUP107 Other •NUP205 •PTPRO •LMX1B •EMP2 •PAX2 •DGKE •NEIL1 Red = Autosomal Dominant

Pathology and Immunology Anatomic and Molecular Pathology Outline

• Background • Clinical Aspects of Hereditary FSGS • Pathologic Aspects of Hereditary FSGS • Genetic Testing • Summary

Pathology and Immunology Anatomic and Molecular Pathology Clinical Aspects of Hereditary FSGS

• Age of disease onset • Typically younger patients (infants & young children) • Depends on genetic etiology • Steroid resistance • Steroid dependence • Nephrotic syndrome • Positive family history • Syndromic manifestations

Pathology and Immunology Anatomic and Molecular Pathology Age of Onset and Hereditary FSGS

1589 families with SRNS

Onset before 25 years

27 genes evaluated

Sadowski CE et al. J Am Soc Nephrol 2014,26(6):1279-89.

Pathology and Immunology Anatomic and Molecular Pathology Genetic Etiology by Age

Sadowski CE et al. J Am Soc Nephrol 2014,26(6):1279-89.

Pathology and Immunology Anatomic and Molecular Pathology Five Genes Account for Majority of Positive Genetic Diagnoses

Sadowski CE et al. J Am Soc Nephrol 2014,26(6):1279-89.

Pathology and Immunology Anatomic and Molecular Pathology Genetic Etiology by Age

Age of Onset/Steroid Most Common Genes Response Infant, <1 year of age NPHS1, NPHS2, LAMB2, WT1, PLCE1 Age 1-10 years, SRNS NPHS2, LAMB2, WT1, PLCE1 Age 1-10 years, SDNS MAGI2, TENC1, DLC1, CDK20, ITSN1 Age 10-18 or adult w/ +FH INF2, ACTN4, TRPC6, WT1

SRNS – steroid resistant nephrotic syndrome SDNS – steroid dependent nephrotic syndrome

Pathology and Immunology Anatomic and Molecular Pathology • Gene causing mutation identified in 29.5% of families

• Likelihood of genetic etiology decreases with age to ~10% in patients over 12

• Genetic etiology varies with age of disease onset

Pathology and Immunology Anatomic and Molecular Pathology Generalized Features of Inheritance Patterns

Autosomal Recessive Autosomal Dominant • Early onset • Late onset • Loss of function • Gain of function • Infancy/early • Late-onset/slowly childhood progressive • Varied penetrance • Varied disease severity

Pathology and Immunology Anatomic and Molecular Pathology Outline

• Background • Clinical Aspects of Hereditary FSGS • Pathologic Aspects of Hereditary FSGS • Genetic Testing • Summary

Pathology and Immunology Anatomic and Molecular Pathology Pathologic Clues to Hereditary Etiology • Collapsing glomerulopathy • APOL1

• Abnormal mitochondria • COQ2, COQ6, tRNALeu (MELAS)

Hotta O, et al. Int. 2001;59:1236-1243. • Glomerular basement thinning/multilayering • COL4A3, COL4A4, COL4A5

• FSGS with diffuse mesangial sclerosis • WT1, LAMB2, NPHS2, PLCE1, NUP93, NUP205, XPO5

Pathology and Immunology Anatomic and Molecular Pathology Collagen type IV variants and FSGS • COL4A3 and COL4A4 associated with Alport syndrome • Patients may present with TBMD and/or FSGS

Presented with proteinuria and hematuria

Positive family history

FSGS on light microscopy

Thin basement membranes on EM

COL4A3 p.G695R variant

Pathology and Immunology Anatomic and Molecular Pathology • Seven families with FSGS, nephrotic-range proteinuria & hematuria

• One family with thin GBM – same variant as seen in previous case (COL4A3 p.G695R)

• No basement membrane splitting, lamellation, or basket weaving present

• Subset of cases without morphologic clues to diagnosis

Pathology and Immunology Anatomic and Molecular Pathology Pathology is Often Non-Specific in Hereditary FSGS

• Typically NOS pattern

• Diffuse podocyte foot process effacement in non- segmentally sclerotic glomeruli

• Looks identical to primary FSGS

Pathology and Immunology Anatomic and Molecular Pathology Outline

• Background • Clinical Aspects of Hereditary FSGS • Pathologic Aspects of Hereditary FSGS • Genetic Testing • Summary

Pathology and Immunology Anatomic and Molecular Pathology Genetic Testing in FSGS

• Why test? • Aid diagnosis • Identify appropriate treatment • 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

Pathology and Immunology Anatomic and Molecular Pathology Clinical Genomic Testing

Pathology and Immunology Anatomic and Molecular Pathology Single Locus vs. Multiple Gene Testing

Locus C T A T G C T C G • Narrowly targeted specific • Result may trigger testing Analyzeyze Determine additional gene single gene/locus mutation status of testing 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

Pathology and Immunology Anatomic and Molecular Pathology Massively Parallel or ‘Next-Generation’ Sequencing

• Sanger sequencing – 2x read (Bidirectional)

• Next-generation – 100-1000x reads at single position

Pathology and Immunology Anatomic and Molecular Pathology Next-Generation Sequencing

Sanger c. 2000 NGS c. 2016

• 0.01 Gigabase/week • 10,000 Gigabase/week

• $100,000,000/Gigabase • $1/Gigabase

Pathology and Immunology Anatomic and Molecular Pathology Next Generation Sequencing Publications

5000

4000

3000

2000

# Publications # Publications 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

Pathology and Immunology Anatomic and Molecular Pathology Application of NGS Technology

• Patient develops SRNS

• Biopsy reveals FSGS

• A genetic cause is suspected

• Case is referred for genetic sequencing of genes known to be involved in FSGS

Pathology and Immunology Anatomic and Molecular Pathology NPHS1 NPHS2 WT1 Multiple PLCE1 gene testing LAMB2 ACTN4 INF2 . . .

A multitude of genetic variants are identified

The greater number of genes sequenced, the greater number of variants identified

How to determine if a genetic variant is causing disease?

Pathology and Immunology Anatomic and Molecular Pathology Genetic Variant Interpretation

Pathology and Immunology Anatomic and Molecular Pathology Variant Interpretation

• Literature

• Clinical Databases • HGMD, ClinVar

• Locus Specific Databases • Leiden, FH HUS

• Laboratory Specific Databases • EmVClass (Emory)

Pathology and Immunology Anatomic and Molecular Pathology Variant Interpretation

• Frequency Data • dbSNP, 1000 genomes • NHLBI ESP • ExAC Browser

• Effect on • Conservation Data • Grantham scores • In-silico predictions • Protein function • Splicing • Functional Studies

Pathology and Immunology Anatomic and Molecular Pathology Variant Interpretation Classifications

• Pathogenic

• Likely Pathogenic

• Variant of Uncertain Clinical Significance

• Likely Benign

• Benign

Pathology and Immunology Anatomic and Molecular Pathology Clinical Application

• Washington University Experience

• Using standardized variant interpretation criteria

• Patients referred for nephrotic syndrome/FSGS sequencing

• All cases reviewed by a pathologist with subspecialty boards in Molecular Genetics or a clinical laboratory geneticist certified in Clinical Molecular Genetics

• 30 cases sequenced to date as part of routine practice

Pathology and Immunology Anatomic and Molecular Pathology Patient Characteristics

• 16 Male 16 • 14 Female 14 12 • 15 Caucasian 10 • 7 AA 8 • 3 Hispanic 6 • 5 Other Frequency 4 2 0 5 1020304050607080 Age (Years)

Pathology and Immunology Anatomic and Molecular Pathology Washington University Experience

Negative P/LP 17% 10% Diagnostic APOL1 13% Yield = 23%

VUS 60%

Pathology and Immunology Anatomic and Molecular Pathology Differences in Clinical Application

• Clinical population is less well defined compared to a research population with well-characterized families

• Indications for testing vary

• Broader age range tested

• Strict variant interpretations are applied

Pathology and Immunology Anatomic and Molecular Pathology Likely Pathogenic Variants

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

Pathology and Immunology Anatomic and Molecular Pathology Variants of Uncertain Clinical Significance

Pathology and Immunology Anatomic and Molecular Pathology Variant Interpretation Challenges

• Incomplete penetrance

• Polygenic inheritance patterns

• Environmental factors

• Lack of centralized database

• Lack of functional studies

Pathology and Immunology Anatomic and Molecular Pathology Variants of Uncertain Clinical Significance

• Most common classification • Not clinically actionable

• What to do with these variants?

Pathology and Immunology Anatomic and Molecular Pathology Case Revisited

• 2 year old with SRNS

• Pathology – FSGS

• Next generation sequencing performed

Pathology and Immunology Anatomic and Molecular Pathology Genetic Variants Idnentified

• NPHS2 – p.R138Q • NPHS2 – p.R196X

Pathology and Immunology Anatomic and Molecular Pathology Variant Interpretation

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

• PP3 – predicted deleterious effect on protein function Pathogenic

Pathology and Immunology Anatomic and Molecular Pathology Podocin Immunofluorescence

Control Patient

Pathology and Immunology Anatomic and Molecular Pathology Summary • Hereditary FSGS is more common in a younger population

• However, adults with a positive family history may harbor significant genetic variants

• Pathology is helpful in certain cases • COL4 variants, mitochondrial disease, APOL1, DMS

• However, most cases of hereditary FSGS show non-specific findings similar to primary FSGS

• NGS is increasingly utilized, but most commonly yields a diagnosis of VUS

• Need for centralized database, functional studies

Pathology and Immunology Anatomic and Molecular Pathology References

• 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. • Lovric S, et al. Nephrol Dial Transplant. 2016; 31:1802-1813. • 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. • Malone AF, et al. Kidney Int. 2014:86(6):1253-9. • 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. • Mardis ER. Nature. 2011:470:198-203. • Büscher AK, et al. Clin J Am Soc Nephrol. 2016:11:245-253. • 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. • Chen YM, Kikkawa Y, Miner JH. J Am Soc Nephrol. 2011:22(5):849-58. • 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.

Pathology and Immunology Anatomic and Molecular Pathology Thank you

Pathology and Immunology Anatomic and Molecular Pathology