Clinical Phenotype: Disease Signs & Symptoms

Rational treatment or prevention

Genotype

“Endophenotypes”

Molecular, cellular & whole system

consequences of mutations in

individual genes

Mechanistic information: essential

roles of individual genes; groups of genes clustered

by pathway; systems biology understanding of gene-gene interactions

MAN

MOUSE

Next-gen resequencing to reveal family-specific variants:

Statistical associations: Eg clusters of mutations in particular

pathways

Translation gap

Specific tests for endophenotypes: Cell-based or biomarker assays that can be

applied to human samples

Clinical and commercial translation: targetted development of new

therapeutics/preventions

Preclinical efficacy in mice with pathway defects like humans

Translation Gap: bridged by integrating next-gen sequencing and solid understanding of mechanism from Mendelian mouse mutants

MEDICINE

Fumbling for his recline button, Ted unwittingly instigates a disaster

The genome encodes the buttons and circuits of our immune system - we just need to know how to read the instructions

*

ENU treated C57BL/6 male C57BL/6 female

Random base substitutions in spermatogonial

stem cells

*

G1 progeny: carry ~3000 single nucleotide substitutions spread randomly across paternal chromosomes: ~35 functional variants/G1

G2 progeny: Single nucleotide substitutions segregate

G3 progeny: 12.5% of SNVs brought to homozygosity in each mouse

Systematic screening for recessive Mendelian syndromes in the immune system caused by single nucleotide variants

G0:

C57BL/6 female

Clinical Phenotype: eg susceptibility to infection with M tuberculosis or Poxvirus, allergic dermatitis,

disseminated lymphoid cancer

Preclinical model for treatment or prevention

“Endophenotypes”: eg altered blood cells in flow cytometry, altered antibody response

to immunization

Genotype: mutation mapped to chromosomal interval and identified by DNA sequencing

T Cells

FSC

CD4

CD

3S

SC

CD44

CD

3

CD44

CD

3

Tube 040: lowish CD4s & CD8s & higher %CD44hi in CD8s

IgM

NK1_1

CD

3Ig

DC

D44

B220

CD

3

NK1_1

B Cells

Tube 046: slightly higher IgM

IgE

IgE

IgE

CD

44

IgE

IgG

2aIg

G2a

CD

44

Monocytes

Comment:Mehmet : Sample 37,38,40 and 65 have reduced CD4 and CD8 T cells

Sample 37 and 40 elevated IgE

Chris Goodnow:Stains and gates look great.

I worry that we’re not picking up IgE+ cells in most samples: gating or staining intensity problem?

Tube 040: lowish CD4s & CD8s & higher %CD44hi in CD8s, higher IgE

Tube 046: slightly higher IgM

Tube 037: higher IgE

Plate 14062 Blood FACs bled 064/10 (05.03.10)

I think the technician forgot to scan following mouse:Sample 73 is ENU14Yaa:047:B6:G2 # 2

Mapping and identification of Mendelian mutations in the mouse circa 2009: legacy of the public mouse genome

project

- Mapped over 95 strains with 77 having mutation identified (44 last 3 years)- Many available for researchers to study through the NHMRC Aust. Phenome Bank

Dopey

Mr T-lessMurdoch

Face

Redburst

Lochy

SanRoque

Rockstar

Wavy

Trembles

SocksPharlap

Unmodulated

Blobby

LeukSkywalker

Alberta

Eeyore

Winnie

Tipsy

Nephertiti

Tiny

Zippo

BuffyWillow

PollyWobbles

Plastic

T-wimp

B-blastBthy

Dwarf

Babe Vibes

Nessy

Flipper

Piebald

Senseless

Sweaty

Kenobi

Anarchy

PardonBotero

FatAussie Koy

Malewa

Fatso

Tilcara

6WT33

Anakan

Mozart

Tsavo

Armidilo

Thunder

Captain

Theoden

Jasmine

Xander

Pinky

HipsterT-Bird

Dorian

Lightning

Storm

Pengu

9B6 35 BDown

Jersey

MeioI

9B6 64

Delficio

PIAF

7B6148

Kenny

4AT32

Hoban

Fluoro

B-leg

Joey

LasVegas

8C63

Duane

Bata

Anubis

B52

8B6 27

Mal

Mr Hanky

KrustyPrimurus

11B658

1 2 3 4 5 6 7 8 9 10

11 12 13 14 15 16 17 18 19 X Y

WachinRain

Rose

Collins

ThothSeshat

Example #1: the value of integrating clinical correlations of DNA variants

with understanding of mechanism from the mouse:

Regulating NFkB activity and cell growth in normal, autoimmune and

malignant B cells

Discovering new regulators of B cell growth by forward genetics: phenotypic screening of a library of mice segregating thousands of randomly-induced

single nucleotide substitutions

• Unmodulated detected from B cell screen

• Each dot represent one mouse.

• Dashed lines two SD

• ~ 400 G3 animals

Amount of antigen receptor on B cell surface

Am

ount

of

com

plem

ent

rece

ptor

on

B c

ell s

urfa

ce

Changes in antigen receptor density often indicate

abnormalilities in the BCR signalling pathways

Unmodulated variant breeds true as a simple Mendelian recessive trait

Control

Variant

Mean values for individual offspring from unmodulated affected mouse (un/un) crossed with heterozygous (un/+) carrier

Amount of antigen receptor on B cell surface

Am

ount

of

com

plem

ent

rece

ptor

on

B c

ell s

urfa

ce

Homozygous un/un mice make little antibody upon immunization, because their B cells proliferate poorly to BCR-stimulation

Th1 / IgG2a Th2 / IgG1

Rel

ativ

e am

ount

of

antib

ody

in s

erum

Unmodulated: point mutation in one protein-interaction domain of a MAGUK-family protein, CARD11 (Carma1)

* * * * * * * * *

Jesse Jun

Jun et al 2003. “Identifying the MAGUK protein Carma-1 as a central regulator of humoral immune responses and atopy by genome wide mouse mutagenesis”. Immunity 18:751-762.

Example:MECHANISM STUDY: B cells from mice with inherited CARD11

mutations are selectively crippled for BCR activation of the IKK-NFkB pathway and proliferation

Building up mechanistic information from knockouts, point mutants, knock-downs, etc: essential roles of individual

genes; groups of genes clustered by pathway; …towards a systems-level understanding of how human alleles of different genes might interact

(eg. MYC)

PKCCARD11

Homozygous null mutations in CARD11 disrupt TCR-NFB signalling and cause immunodeficiency, but what about SNVs causing a quantitative decrease?

IKK

IB

NFB

PKC

CD80 (B7.1)

genes for cell growth and division

CD28

* * *

NFAT

Calcineurin

Ca2+

Y YY Y*

Y YY Y** *

*

LckFyn

Zap70

CD4 or

CD8

Y YY Y** *

*

Y YY Y** *

*

Y YY Y** *

*

TCR

MHC

Itk

CD86 (B7.2)

IL-2

IL-2R

JAKSTAT

CARD11(CARMA1)Bcl10MALT1

Ptpn22 (PEP)

Antigen

AireThymic epithelium

CD45

Quantitative decrease in CARD11 signalling preserves T cell activation but diminishes Treg formation, resulting uncontrolled

formation of TH2 cells, hyper-IgE, and mast cell dermatitis

John Altin, Matthew Cook, Jesse Jun

Mouse-to-Man-to-Medicine: translating knowledge of pathways controlling B cell proliferation and resequencing of carefully

phenotyped clinical samples

Robertson MJ et al. 2007. J Clin Oncol 25:1741-6. Phase II study of enzastaurin, a protein kinase C beta inhibitor, in patients with relapsed or refractory diffuse large B-cell lymphoma.

Morschhauser F et al. 2008. Ann Oncol 19:247-253. A phase II study of enzastaurin, a protein kinase C beta inhibitor, in patients with relapsed or refractory mantle cell lymphoma.

Science 2008 319:1676-1679

L298Q unmodulated

Molecular, cellular & whole system

consequences of mutations in

individual genes

Mechanistic information: essential

roles of individual genes; groups of genes clustered

by pathway; systems biology understanding of gene-gene interactions

MAN

MOUSE

Next-gen resequencing to reveal family-specific variants:

Statistical associations: Eg clusters of mutations in particular

pathways

Translation gap

Specific tests for endophenotypes: Cell-based or biomarker assays that can be

applied to human samples

Clinical and commercial translation: targetted development of new

therapeutics/preventions

Preclinical efficacy in mice with pathway defects like humans

Translation Gap: bridged by integrating next-gen sequencing and solid understanding of mechanism from Mendelian mouse mutants

MEDICINE

IMPC GOAL:

A phenotyped mouse mutant for every gene

Genotyped & phenotyped mouse pedigrees: >3 homozygotes/mutation

ES cells with targetted null alleles

ENU-treated male mice with SNVs

Pedigree breeding, genotypic & phenotypic data acquisition & collation

1 deleterious mutation/pedigree ~70 deleterious mutations/pedigree

Chris Goodnow’s group

Adrian Liston

Owen Siggs

Katrina Randall

Jesse Jun

Lina Tze

Anselm Enders

Keisuke Horikawa

Charis Teh

Sally Mapp

Gerard Hoyne

Zuopeng Wu

Aust Phenomics Network

Ed Bertram

Belinda Whittle

The John Curtin School of Medical Research,

The Australian National University, Australia’s Capital City, Canberra

Carola Vinuesa’s Group:

Di Yu

Vicki Athanasopoulos

Michelle Linterman

Diego Silva

Rob Rigby

ACRF, ARC, NHMRC,

The Wellcome Trust,

NIAID-NIH, JDRF, LLRF

Matthew Cook’s Group

Nick Simpson

Oxford & London:

Richard Cornall

Tess Lambe

Facundo Batista

Garvan Institute:

Rob Brink

Fabienne Mackay

Charles Mackay

John Sprent

Stuart Tangye

Tony Basten

Barbara FazekasANU Research

School of Chemistry:Gottfried Otting’s

Group:

Institute for Systems Biology:

Alan Aderem’s group

UCSF:

Art Weiss, Jason Cyster, Lewis Lanier