Nicholas Katsanis, Ph.D.

Transcription

1 Ciliopathies and Oligogenic Phenomena Prof. Center for Human Disease Modeling Duke University 1 Some key questions in human genetics What variants cause disease? What variants are associated with disease modulation? What is the difference? How can we discern context-dependent activity of alleles on phenotype? 2 The rules of pathogenicity 1. Pathogenic variants typically cause a recognizable effect on protein/mrna (strong mutations) Nonsense, deletions haploinsufficiency/dosage Splice truncated proteins or haploinsufficiency Missense loss of function/gain of function Genomic duplication/triploidy dosage Promoter/UTR transcriptional misregulation 3 1

2 Modification of rules for oligogenic traits Mutations can have a subtle effect on protein/mrna function ( weak alleles ) Effect can be deleterious or beneficial Mutations can exhibit non-penetrance Carrier frequency higher in general population 4 Must not be limited by preconceived notions of disease inheritance deduced from pedigree structure 5 Example: motor neuron disease (ALS) Dominant trait in families Mutations in several genes including SOD1 cause ALS In 1994, CNTF null alleles were excluded from causality on the basis of non-segregation/controls In 2002 Giess et al., showed that CNTF is a potential severity/age-of-onset modifier 6 2

3 Age of onset 56 Age of onset 43 CNTF +\ - SOD +\+ CNTF +\ - SOD +\ - Age of onset 55 CNTF +\ - SOD +\+ Badano and Katsanis 7Nature Rev Genet 2002, 3: CNTF +\ - SOD +\+ CNTF +\+ SOD +\+ CNTF +\+ SOD +\ - Asymptomatic 36 CNTF - \ - SOD +\ - Age of onset 25 Models of oligogenicity 1) Direct or indirect complex X X X X Function 8 Badano and Katsanis Nature Rev Genet 2002, 3: ) Pathway rescue model Wild type or B + \ - A B C Effect B - \ - A S C B - \ - ; S + \ - A S C Effect Reduced effect B - \ - ; S - \ - A C S No effect Effect threshold 9 W.T B + \ - B - \ - B - \ - S + \ - B - \ - S - \ - Badano and Katsanis Nature Rev Genet 2002, 3:

4 3) Converging pathway model A D A D A D A D B E XB E B XE XB XE C F C F C F C F Example: Bardet Biedl syndrome and Hirschsprung disease 10 Badano and Katsanis Nature Rev Genet 2002, 3: Moving on from theoretic examples to real time examples 11 Oligogenic architecture: Bardet-Biedl syndrome Retinal dystrophy Post-axial polydactyly Truncal obesity Other major features: Mental retardation Learning difficulties Gonadal malformations Renal malformations 12 An important point to make is the large dissimilarity between patients, both between and within families 4

5 BBS and variable penetrance AR259 - BBS2 AR259 - BBS6 Pentrance Non-penetrance & Expressivity 13 Katsanis et al., Science 2001, 293: BBS and variable expressivity AR768 - BBS1 AR768 - BBS6 14 Badano et al., Hum Mol Genet 2003, 12: No obesity Mild RP No MR Nml devel Nml speech Nml teeth Obese from age 1=90th centile RP/maculopathy MR Dev. delay Speech pathology/delay Crowded teeth Linking BBS to ciliary dysfunction How to make sense of this? - Many rare alleles - Genetic interactions can be difficult to prove statistically This is where biology is critical The planar cell polarity pathway (PCP), also known as the noncanonical pathway or the beta-catenin-independent Wnt-signaling pathway, is necessary for the elaboration structures in a 3D space This was the first hint to suggest the involvement of BBS proteins in complex signal transduction mechanisms 15 5

6 Phenotypes Cilia have been implicated in a number of human genetic disorders Renal, pancreatic, biliary cysts/fibrosis Retinal degeneration Laterality defects Skeletal abnormalities Central / peripheral nervous system defects 16 Nephronophthisis (NPHP) Mild Senior-Loken Syndrome (SLS) Disorders Joubert Syndrome (JBTS) Bardet-Biedl Syndrome (BBS) Meckel-Gruber Syndrome (MKS) Severe Jeune Asphyxiating Thoracic Dystrophy (JATD) Ciliopathies: an expanding group of overlapping clinical entities The challenge: understanding penetrance and expressivity Allelism at a single locus is insufficient to explain phenotypic variability Bardet-Biedl Syndrome Retinal degeneration Skeletal degeneration Pancreatic, biliary cysts/fibrosis Moderate BBS NPH Nephronophthisis Pancreatic, biliary cysts/fibrosis Mild Meckel-Gruber Syndrome Nervous system defects Skeletal degeneration Pancreatic, biliary cysts/fibrosis Severe MKS Zaghloul and Katsanis, 17 Trends Genet. 2010, 26: JBTS NPHP6 SLS Joubert syndrome Pancreatic, biliary cysts/fibrosis Retinal degeneration Nervous system defects Moderate Senior Loken Syndrome Retinal degeneration Pancreatic, biliary cysts/fibrosis Moderate Depletion of ciliary proteins perturbs planar cell polarity/non-canonical Wnt signaling Control Ross et al., Nat Genet 2005, 37: Evaluation of signal output in vivo: a physiologically relevant assay bbs4 MO Gerdes et al., Nat Genet 2007, 39:

7 Hypothesis: Total mutational load in the ciliary proteome determines phenotype Challenge: Deciphering pathogenic potential of missense alleles Major benefit of ciliary biology: Experimentally tractable, physiologically relevant in vivo and in vitro assays 19 Experimental design to assess pathogenicity of human mutations Recessive Dominant Gene suppression injection of MO Injection mutant mrna Zebrafish embryos 1- to 4-cell stage Phenotype Rescue with WT or mutant mrna Hypomorph or loss of function 20 Titrate with WT mrna If rescue: dominant negative If no rescue: gain of function Assays to assess pathogenicity of missense variants In vivo Rescue of morphant phenotype with wild-type vs. mutant human mrna: quantitative analyses 30% epiboly 50% epiboly 65% epiboly 75% epiboly Control mks1 morpholino Rescue 21 Somite trunk length Leitch et al., Nature Genet. 2008, 40: C492W Cell migration 7

8 Deciphering causality of patient mutations Control bbs1 M390R bbs6 T325P Badano et al., Hum Mol Genet 2003, 12: Mapping it all back to humans A B C 23 Zaghloul et al., PNAS : Can we apply these lessons across the ciliary proteome??? 24 8

9 Medical resequencing: A pilot project to probe the total mutational load in ciliopathy patients Gene selection (22 candidates of unknown function): Sanger sequencing (Baylor): Patient selection (94 total): NPHP BBS MKS 25 Phenotypic severity Gherman A, Davis EE, Katsanis N. Nat Genet Sep; 38(9): The ciliary proteome database: an integrated community resource for the genetic and functional dissection of cilia Novel sequence variants detected in 14/22 genes Highest enrichment of novel alleles in TTC21B and unusual patterns in FTM TTC21B has another name IFT139 as a panciliopathy contributor but only if you know how to look for it! IFT139/TTC21B: a significant contributor of pathogenic alleles to ciliopathies 28 individual novel variants unique to patients: 2 frame-shifting 2 nonsense 4 splice junction 20 missense 10 individual novel variants unique to controls: (all missense) 10 individual variants found in cases and controls: (all missense) 9

10 Most IFT139 missense variants are pathogenic Functional testing of missense alleles in an in vivo model: Rescue efficiency of morpholino phenotypes Control Class I Class II Percentage of embryos 100% 80% 60% 40% 20% Normal Class I Class II MO mrna Injection 27/30 IFT139 alleles detected in patients are deleterious in vivo patients harbor pathogenic variants (5.7%) How do pathogenic alleles map back to cases and controls? 29 Mutations in IFT139 cause JATD and NPHP Isolated NPHP Phenotypic spectrum Syndromic/Severe 30 P209L is associated with renal phenotypes in ciliopathies 10

11 An enrichment of pathogenic alleles in ciliopathy cases Similar frequency of novel variants between cases and controls 31 Total pathogenic variant frequency is enriched ~5-fold in N. European cases vs. controls Davis et al., Nat Genet. 2011, 43: Total mutational load in ciliopathies: dissection of a functional ciliary module Gerdes, Davis & Katsanis, Cell. 2009, 137: Medical resequencing of the Intraflagellar Transport complex 20 IFT genes 436 Ciliopathy cases 162 MKS 62 JATD 116 BBS 96 NPHP Severe Mild 336 controls (NHLBI ESP) novel changes 62 nonsense, fs, splice: cases only 138 missense: cases only 136 missense: controls only 38 missense: cases and controls The challenge of 312 coding nonsynonymous variants! Which alleles are responsible for causing disease? Which alleles are responsible for modulating disease? Is there an enrichment in cases versus controls? 33 11

12 Causality under a recessive model...? Anterograde IFT IFT80: JATD Beales et al., Nature Genetics. 2007, 39: IFT88: MKS-like prenatal renal cysts and polydactyly 34 IFT88 M383K: 1. Absent from >5,000 control individuals 2. Present in an additional 6 severe (MKS, JATD) ciliopathy samples McIntyre et al., Nat Med 2012, 18: Mapping alleles back to patients: IFT complex A In vivo scoring of IFT complex A changes Distribution of IFT complex A alleles by disorder Frequency of pathogenic IFT complex A variants IFT complex A alleles/138 total novel missense changes detected in patients: 84% pathogenic Mild Severe IFT complex A load tracks with phenotypic severity Medical resequencing alone is not sufficient to inform locus contribution: Functional assessment is necessary 36 12

13 Context matters!!! 1. Epistasis and trans modification 2. Position of mutations on splice isoforms 3. Local sequence-dependent context Acknowledgments 39 U Michigan, Ann Arbor Edgar Otto Friedhelm Hildebrandt U Pennsylvania, Philadelphia Qi Zhang Qin Liu Eric Pierce U Birmingham, Birmingham Jane Hartley Eamonn Maher U Leeds, Leeds Katarzyna Szymanska Colin Johnson INSERM, Paris Tania Attié-Bitach Sophie Saunier Funding NIH-NICHD, NIDDK, NIMH; MVRF, FFB, EU UCL/ICH, London Philip Beales Peter Scambler ULP, Strasbourg Corinne Stoetzel Hélène Dollfus UCSD, La Jolla Joe Gleeson RWTH, Aachen Carsten Bergmann Baylor, Houston Donna Muzny Richard Gibbs Richard Lewis NISC, Rockville Eric Green 13

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