Systematizing in vivo modeling of pediatric disorders

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1 Systematizing in vivo modeling of pediatric disorders Nicholas Katsanis, Ph.D. Duke University Medical Center Center for Human Disease Modeling Rescindo Therapeutics

4 Task Force for Neonatal Genomics A hybrid structure at Duke Clinical Management & Patient Interaction In vivo/in vitro assay of allele function Genetics/ Genomics Policy Genome-wide sequencing & analysis

5 Our biggest Challenge Our current ability to interpret genetic variation accurately 1. Is the gene relevant to a disease state 2. Is a DNA variant within a gene altering the function of the encoded protein? 3. How do we answer these questions?

6 In vivo assays to model human genetic disease Assays for >750 human disease genes: Nervous system Macro/Microcephaly Optic nerve atrophy Gut motility Carnes et al, PLoS Genet 10:e (2014) Golzio et al, Nature 485: (2012) Cerebellar hypoplasia Peripheral neuropathy Bernier et al, Cell 158: (2014) Margolin et al, N Engl J Med 368: (2013) Gonzaga-Jeugegui et al, Cell Rep2015

49: 373-379 (2012) Lee et al, Mol Biol Cell

7 In vivo assays to model human genetic disease Major organ systems Craniofacial anomalies Vascular integrity Renal atrophy/cysts Chassaing et al, J Med Genet 49: (2012) Lee et al, Mol Biol Cell 23: (2012) Lindstrand et al, Am J Hum Genet 94: (2014) cmlc2:egfp Cardiac malformations Muscular dystrophy R L R L R Hjeij et al, Am J Hum Genet. 93: (2013) L Sarparanta et al, Nat Genet 44: (2012)

8 Systematic Discovery of New Disease Genes Recruit ~50 families/year >10-20 new disease genes/year Phenotypic expansions Complex inheritance CNV dissection Opportunistic Drug Discovery

9 Vignette 1: A novel disease gene in an uncharacterized syndrome DM Proband clinical features Microcephaly Hypotonia Post axial polydactyly bilateral on the feet Severe developmental delay Strabismus Scoliosis Absent clitoris Bilateral vesicoureteral reflux Right complete ureteral duplication Proband: 6 years, 2 months at consent Ethnicity: European American Clinical testing Microarray: NPHP1 deletion (het) Referring clinician (Gallentine) Pediatric Neurology

10 Exome sequencing reveals three candidate genes DM Filter data to identify variants that are: Rare in the general population (<1%) Predicted to alter protein function Inherited from the parents or new from the child Make biological sense DM074 Final candidates Gene Inheritance/segregation Disease association NCAPG2 compound het missense na TTN compound het Cardiomyopathy, Muscular dystrophy GLRA3 de novo missense na

11 A phenotype match Clinical exome sequencing identified NCAPG2 p.t850p mutation in patient 2 Eye anomalies Limb anomalies Brain anomalies Renal/urogenital anomalies Growth defects Spinal anomalies Other anomalies DM074 Strabismus, nystagmus, pigmentary retinopathy, epiblepharon Post axial bilateral polydactyly on feet Microcephaly; dilated cerebral ventricles (prenatally) Small right kidney; kidney cysts (prenatally); vesicoureteral reflux, right complete ureteral duplication Growth failure; hormone treatments recommended Sacral dimple; tethered cord; scoliosis Absent clitoris, craniofacial anomalies DM516 Peter's anomaly, left sided coloboma, glaucoma Clinodactyly; Missing digits on feet Microcephaly History of hydronephrosis Intrauterine growth restriction; failure to thrive Sacral dimple; low lying conus medullaris (tethered cord?) Hearing loss, cardiac defects, neutropenia

12 Suppression of ncapg2 results in microcephaly... Control MO MO+WT MO+p.794 MO+p.609 MO+p.693 MO+p dpf 655 Head size (um) **** **** **** * ** ** **** * p vs MO p vs MO+WT

13 CRISPR-Cas9 editing of ncapg2 results in microcephaly... Control sgrna sgrna+cas9 3 dpf p=< p=< Head size (um) Head size (um) Control sgrna1 sgrna1+cas9

14 Vignette 2: Rare alleles that rescue rare alleles in cis Clinical features Global developmental delay microcephaly feeding issues failure to thrive abnormal muscle tone low immunoglobulins frequent respiratory infections Clinical testing normal female microarray metabolic testing negative extensive genetic testing negative BTG2: Involved in the G1/S transition of the cell cycle NOS2: Nitric oxide synthase 2, inducible TTN: Titin

15 BTG2 is the disease driver Microcephaly Neuronal expr. cell proliferation

16 Conservation of BTG2 141M in multiple species

17 Compensatory Mutations Secondary mutations that alleviate deleterious effect of primary mutations Functional wild-type protein Primary mutation (U) disrupts protein structure/function Compensatory mutation (V) restores structure/function Sayuri & Tetsuro, Frontiers in Microbiology, Vol 2, #267, 2012

18 Finding Potential Compensatory Mutations Human Protein Mutated to -A- in human disease IYKQALIFRLEGNIPESLELFQTCAVLSPQSNDNLKQVARSLFLLGIHKA --V------F----Q------P A A-----K--- --V---Q Q T-----A A-----K--- --V---Q Q---H A----I K--- --V Q Y A K N E---K--- N D N K--- N D N K--- N-----Q N Q D N K Q D N K---

BTG2 has two compensatory mutations Number of

phospho Histone H3 Mo + G6R Mo + G40R Injection * * Mo +

19 BTG2 has two compensatory mutations Number of cells/embryo UI Ctrl Mo Mo + WT Mo + V141M *P<0.01 vs V141M rescue alone Number of cells stained with phospho Histone H3 Mo + G6R Mo + G40R Injection * * Mo + R80K Mo + Q94R Mo + S98R Mo + L128V Mo + A130T Mo + C132Y Mo + L142M Jordan, Frangakis et al, Nature 2015

TFNG Discovery Rate Data from 238 families Inconclusive Genes

12% Probably causal Known gene and known mutation Maybe

alleles; no direct functional assay 16% 72% Known gene and

20 TFNG Discovery Rate Data from 238 families Inconclusive Genes not associated with disease and unable to test functionally 12% Probably causal Known gene and known mutation Maybe causal Novel disease genes harboring candidate pathogenic alleles; no direct functional assay 16% 72% Known gene and novel functional variants Known CNV New gene with strong functional evidence

21 The Big Picture Collaboration and data sharing are key Community engagement is necessary Strong genetics and biochemistry You never know where your winners come from

23 Thank you

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