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Gaps between the bedside and the bench: Perspectives from the bench - - PowerPoint PPT Presentation

Mar 20, 2023 144 likes •481 views

Gaps between the bedside and the bench: Perspectives from the bench University of Oregon University of Tbingen Bernardo Blanco-Snchez Antje Bernd Aurlie Clment Eberhart Zrenner Javier Fierro John

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University of Oregon

  • Bernardo Blanco-Sánchez
  • Aurélie Clément
  • Javier Fierro
  • John Postlethwait
  • Jennifer Phillips
  • Alexandra Talafuss
  • Sabrina Toro
  • Phillip Washbourne
  • Jeremy Wegner

University Hospital Cologne

  • Thomas Benzing
  • Hanno Bolz
  • Claudia Dafinger
  • Inge Ebermann
  • Max Liebau
  • Rebecca Ruland
  • Bernhard Schermer
  • Michaela Thoenes

Cologne Center for Genomics

  • Gudrun Nürnberg
  • Peter Nürnberg

McGill University Health Centre Montreal

  • Robert Koenekoop
  • Irma Lopez

Inserm Montpelier

  • Mireille Claustres
  • Anne-Francoise Roux

University of Tübingen

  • Antje Bernd
  • Eberhart Zrenner

Human Genetics Hamburg

  • Ellen Schäfer

VisionForACure.com

Baylor College of Medicine

  • Hugo Bellen
  • Shinya Yamamoto
  • Michael Wangler

Sponsored by the Office of the Director National Institutes of Health, the National Human Genome Research Institute, the National Institute of Child Health & Development, the National Institute on Deafness & Other Communication Disorders, the National Eye Institute, the Usher 1F Collaborative, and the Megan and Vision for a Cure Foundations

Gaps between the bedside and the bench: Perspectives from the bench

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Case study 1: Positive results validate candidate genes Case study 2: Negative results reveal incorrect diagnoses Mind the gaps Undiagnosed Diseases Network

Gaps between the bedside and the bench: Perspectives from the bench

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Case study 1: Positive results validate candidate genes

  • Usher syndrome gene discovery

Case study 2: Negative results reveal incorrect diagnoses Mind the gaps Undiagnosed Diseases Network

Gaps between the bedside and the bench: Perspectives from the bench

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Usher syndrome - the leading cause of deafblindness

  • Prevalence ≈ 1 per 6,000 births in the US

(more common than ALS or Huntington’s Disease)

  • Congenital deafness (~4% of deaf have Usher)

Sensorineural hearing loss Vestibular dysfunction

  • Retinitis pigmentosa

Loss of rod photoreceptors Progressive tunnel vision as cones die

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Type Human Protein: potential function

USH1B MYO7A MyosinV11A: motor activity USH1C USH1C Harmonin: scaffold USH1D CAD23 Cadherin: calcium dependent adhesion USH1E

  • Unknown

USH1F PCDH15 Protocadherin15: adhesion, signaling USH1G USH1G SANS: membrane associated scaffold USH1H

  • Unknown

USH1J CIB2 Calcium and integrin binding protein USH1K

  • Unknown

USH2A USH2A Usherin: Laminin-like transmembrane protein USH2C GPR98 Vlgr1: G-protein coupled receptor, signaling USH2D CIP98 Whirlin: scaffold USH3A CLRN1 Clarin1: 4-pass transmembrane protein USH3B HARS Histidyl-tRNA Synthetase

Multiple Usher genes with multiple functions

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Genetic counseling is important for Usher patients

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Gene discovery is important for Usher patients

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(Hanno Bolz & Inga Ebermann)

Exome sequencing of undiagnosed patients identifies mutations in PDZD7, a gene of unknown function

c.1750-2A>G

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Zebrafish Pdzd7a is localized with other Usher proteins

(Jennifer Phillips)

Pdzd7 + ac-tubulin

Eye Ear

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Stereocilia are defective after pdzd7a knockdown

Control pdzd7a MO

(Bernardo Blanco)

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(Hanno Bolz & Inga Ebermann)

PDZD7 USH2A WT / WT C.C1447QfsX / WT WT / WT C.C1447QfsX / WT WT / WT C.C1447QfsX / WT WT / WT WT / WT p.R56PfsX / WT C.C1447QfsX / C.C1447QfsX WT / WT C.C1447QfsX / C.C1447QfsX C732Flsx / WT A5713FlsX / WT PDZD7 GPR98

PDZD7 mutations are heterozygous in patients with known Usher gene mutations

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Caspase labeling of dying cells

(Ebermann et al., 2010)

PDZD7 -/+ GPR98 -/+

pdzd7a interacts with ush2a & gpr98 in photoreceptor cell death

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PDZD7 binds to USH2A & GRP98 proteins

(Hanno Bolz & Inga Ebermann) V5-USH2A V5-GPR98 V5-PDZD7 36 kD 36 kD 28 kD

FLAG-tag precipitates + anti-V5

HEK293T cells

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PDZD7 forms a quaternary complex

  • f USH 2 proteins

Caspase labeling of dying cells

(Chen et al., 2014)

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Case study 1: Positive results validate candidate genes

  • Usher syndrome gene discovery

PDZD7 causes disease Where are the missing homozygous or compound heterozygous patients?

  • embryonic lethal? (model organism data suggest not)
  • patient pool too small?
  • limited access to patient data?
  • lack of communication (or sharing) among clinicians?

Gaps between the bedside and the bench: Perspectives from the bench

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Case study 1: Positive results validate candidate genes

  • Usher syndrome gene discovery

PDZD7 causes disease Where are the missing homozygous and compound heterozygous patients?

  • embryonic lethal? (model organism data suggest not)
  • patient pool too small?
  • limited access to patient data?
  • lack of communication (or sharing) among clinicians?

Gaps between the bedside and the bench: Perspectives from the bench

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Case study 1: Positive results validate candidate genes

  • Usher syndrome gene discovery

Case study 2: Negative results reveal incorrect diagnoses

  • Joubert syndrome

Mind the gaps Undiagnosed Diseases Network

Gaps between the bedside and the bench: Perspectives from the bench

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Consanguineous family with deafness

(Solaf Elsayed & Hanno Bolz)

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Mapping homozygosity by descent identifies no good candidates

(Solaf Elsayed, Raoul Heller & Hanno Bolz)

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II:3 & II:5 I:1 & I:2

(Solaf Elsayed, Raoul Heller & Hanno Bolz)

Whole exome sequencing for homozygous SNPs identifies mutation in AHI1, a gene responsible for Joubert syndrome Patients Parents

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  • Underdevelopment of the cerebellum and brainstem*
  • Impaired intellectual development, seizures
  • Retinitis pigmentosa
  • Developmental abnormalities
  • Kidney and liver abnormalities

Joubert syndrome - a severe ciliopathy disease

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(Raoul Heller & Hanno Bolz)

Joubert-1 Joubert-2

Homozygous patients have normal CNS MRIs

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Nonsense mutation truncates the protein-protein interaction domain of AHI1

(Solaf Elsayed, Raoul Heller & Hanno Bolz)

Protein-protein interaction domain

cluster of severe disease causing mutations

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cluster of severe disease causing mutations

Targeting upstream in zebrafish gene blocks expression

SPL8

(Jennifer Phillips)

Protein-protein interaction domain

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Upstream targeting produces strong ciliopathy phenotype

(Jennifer Phillips)

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3’ targeting truncates the protein

cluster of severe disease causing mutations

e23i23 SPL8

(Jennifer Phillips)

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Truncated protein has no apparent phenotype

(Jennifer Phillips)

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(Solaf Elsayed, Raoul Heller & Hanno Bolz)

Nonsense AHI1 mutation is not linked to deafness

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Case study 1: Positive results validate candidate genes

  • Usher syndrome gene discovery

Case study 2: Negative results reveal incorrect diagnoses

  • Joubert syndrome

Mind the gaps (perspective from the bench)

  • Barriers to accessing patient data
  • Sociological: clinical vs basic research attitudes
  • Limited access to clinical records: de-identified vs IRB
  • Limited patient data: horde vs share variant & phenotypic data

Undiagnosed Diseases Network

Gaps between the bedside and the bench: Perspectives from the bench

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University of Oregon

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Case study 1: Positive results validate candidate genes

  • Usher syndrome gene discovery

Case study 2: Negative results reveal incorrect diagnoses

  • Joubert syndrome

Mind the gaps (perspective from the bench)

  • Barriers to accessing patient data
  • Sociological: clinical vs basic research attitudes
  • Limited access to clinical records: de-identified vs IRB
  • Limited patient data: horde vs share variant & phenotypic data

Undiagnosed Diseases Network

Gaps between the bedside and the bench: Perspectives from the bench

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University of Oregon

  • Bernardo Blanco-Sánchez
  • Aurélie Clément
  • Javier Fierro
  • John Postlethwait
  • Jennifer Phillips
  • Alexandra Talafuss
  • Sabrina Toro
  • Phillip Washbourne
  • Jeremy Wegner

University Hospital Cologne

  • Thomas Benzing
  • Hanno Bolz
  • Claudia Dafinger
  • Inge Ebermann
  • Max Liebau
  • Rebecca Ruland
  • Bernhard Schermer
  • Michaela Thoenes

Cologne Center for Genomics

  • Gudrun Nürnberg
  • Peter Nürnberg

McGill University Health Centre Montreal

  • Robert Koenekoop
  • Irma Lopez

Inserm Montpelier

  • Mireille Claustres
  • Anne-Francoise Roux

University of Tübingen

  • Antje Bernd
  • Eberhart Zrenner

Human Genetics Hamburg

  • Ellen Schäfer

VisionForACure.com

Baylor College of Medicine

  • Hugo Bellen
  • Shinya Yamamoto
  • Michael Wangler

Sponsored by the Office of the Director National Institutes of Health, the National Human Genome Research Institute, the National Institute of Child Health & Development, the National Institute on Deafness & Other Communication Disorders, the National Eye Institute, the Usher 1F Collaborative, and the Megan and Vision for a Cure Foundations

Gaps between the bedside and the bench: Perspectives from the bench