INGID04-10-2012

Development in genetics and PID

PIDs

• How many PIDs ? A need to identify them, to understand pathophysiology

• Diagnosis, treatment, prevention

• Most PIDs are inherited monogenic diseases

Genes are made of ADN, contain informations to make proteins

based on

the genetic code which relies on an alphabet consisting in 4

« letters » :A,T,C,G and « words made of 3 letters » (triplet of

nucleotides)

There are approximately 25 000 genes (a small 3% of our genome)

From chromosomes to genes

From gene to protein

ADN

Gene

Transcription

Splicing : trimming of introns

Translation

Mutation

Mutations result from mistakes during DNA

replication,

these events are frequent :

nucleotide[s] substitution, deletion,

inversion

the new DNA will thus differ from the original

copy

Most are neutral, some are deleterious

Frequency of de novo

mutations

A. Kong et al, Nature 2012

from which 10 % are deleteriousmean = 6/newborn !

Ataxia telangiectasiaMost SCID..,Most HLH,LAD,…..

Inheritance (1)

Hyper IgE syndrome (STAT3)Di George syndromeNeutropenia..

Inheritance(2)

X-linked SCIDXLA (Bruton’s disease)CGD (2/3)Wiskott Aldrich syndome..

Inheritance (3)

Genetics and PID

> 180 genes

• Candidate genes  ≥ 1984

• Gene mapping (segregation of polymorphic markers) ≥

1984

• Positional cloning ≥ 1985

• Whole exome sequencing ≥ 2010

• Whole genome sequencing 2011…

From mutations to diseases

-> Correlation between « phenotypes » and « genotypes »

-> Modifier genes

-> Somatic mutations

Step 1 : DNA bank generation

Fragmentation

GenomicDNA

Ligation of adaptor

Adaptors : same primers used in PCR for amplification

Genomic bank

Next generation sequencing3 steps

->1 image / base added

Principle of sequencing :

*Sequencing by synthesis*dye-terminator reversible*small fragments

Support =FlowCellStep 2- Clonal amplificationStep 2- Clonal amplification

Step 3- SequencingStep 3- Sequencing

Bank

NGS sequencing example (Illumina)

Genetics and PID

Diagnosis

Prognosis

Treatement

Screening

Genetic counseling

Diagnosis

Prognosis

Treatement

Screening

Genetic counseling

Phagocytic cell disorders

XL-CGD CYBB gene mutations

Kuhns et al. NEJM 2010

B cell PIDs

TNF-homologous extracellular domain

Transmembranedomain

22 33 44 5511

1261

non sense mutationmissense mutation

insertion, frameshift

deletion, frameshift

deletion, in frame

splice-site mutation

gross deletion

insertion, in frame

X-linked CD40L-deficiency

CD40L on activated T cells

Control Patient

T-cell PIDs

N1

C770

CCAT DNA-B linker SH2 TA2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 22 2320 21

Q644delV463del

R382W/Q/L

F384L/S

T389IT412S

R423Q

H437Y/P

S465AN472D

S611NS614G

V637L/M

V638G P639A

N647DE652K

Y657C

I665NS668Y

V713M/L

K642E

V343F/L

R335W H332Y

K340N

Intron11A>G

Intron12G>T

R432MN466K

Q469H

K531E K591

T620A F621V

T622IS636F/Y

Q644P

K340N / T341del E690_P699del

T708N/SK709E

F710C

T714I

Y705

Hyper IgE syndrome (A.Dom.)-STAT3 mutations

AT: Amino-terminal Domain CC: Coiled-coil Domain TA: Transactivation Domain

Genetics and PID

> 180 genes

• Candidate genes  ≥ 1984

• Gene mapping (segregation of polymorphic markers) ≥

1984

• Positional cloning ≥ 1985

• Whole exome sequencing ≥ 2010

• Whole genome sequencing 2011…

From mutations to diseases

-> Correlation between « phenotypes » and « genotypes »

-> Modifier genes

-> Somatic mutations

PerforinNonsenseMissense

Mutations Exon 1 Exon 2 Exon 3

100101.1.010

25

50

75

100

Sp

ecifi

c lysis

(%

)

Effector/target

P1P2

control

HLH resulting from perforin defect in FHL2Genotype/Phenotype correlation

0

3

6

9

12

15

18

21

0,1-3 1-6 7-12

13-23 24-60 >61Age of onset (months)

non sense mutationsmissense mutations with complete loss of activitymissense mutations retaining partial activity

from I. Voskoboinik et al.,2006

Pati

en

t n

um

ber

Defective cytotoxic activity

Genetics and PID

> 180 genes

• Candidate genes  ≥ 1984

• Gene mapping (segregation of polymorphic markers) ≥

1984

• Positional cloning ≥ 1985

• Whole exome sequencing ≥ 2010

• Whole genome sequencing 2011…

From mutations to diseases

-> Correlation between « phenotypes » and « genotypes »

-> Modifier genes

-> Somatic mutations

Autoimmune LymphoProliferative Syndrome

Onset < 5 years (0-18y)

Splenomegaly (hepatomegaly)

Adenopathy

Lymphoproliferation(benign)

Immunologicaldisorders

ALPS

Ct

TCR

CD4+CD8

30%

0,4%«double negative»T cells (Tcr CD4 - CD8-)

hyper Ig (G,A)

autoimmunity (2/3 patients)

Inherited dominant Fas mutations (with partial clinical penetrance)

Age at presentation0

2

4

6

8

1 01 01 52 0yrs

(ALPS, Canale-Smith Syndrome)

Mutations of FAS causative of ALPS

1836

D.D.

S227fs (X 229)

T254K

G256K

7

Ex 8 (P201fs X 204)

I243RI243T

K280fs (X 321)S214fs (X 224)

W265fs(X 296)W265C

8

D253fs (X 263)R234X, R234QR234P

K177fs (X194)

dup 20bp fs307X350

1

1 2 3 4 5SP

E63X

TM6

S6 fs (X12)

L143fs(X170)

Ex 4 (H95fs X133)

Ex 6

V123fs (X 170)

W173X L268P

V233LV233F

C88G

E100G

G237SG237DG237D

Extra Cellular Domain (ECD) Intra Cellular Domain (ICD)

Selective advantage conferred to lymphocytes by somatic mutations of FAS

Genetics and PID

• Diagnosis

• Prognosis

• Treatement

• Screening

• Genetic counselling

• Diagnosis

• Prognosis

• Treatement

• Screening

• Genetic counselling

Therapy of PID as based on the study of disease mechanisms

• Protein

• Non sense mutations, suppression of premature termination

• Gain of glycosylation mutations

• Neutralization of cytokines

• By pass of cellular defects

• Cell therapy stem cells, thymus

• Gene therapy addition, replacement, inhibition

Principle of gene therapy

Adapted from M. Kay et al, 2001

The vector: to induce integration of the transgene in the genome of the target cell

Vector

target cell

Vectoruncoating

ChromosomalDNA

Integrated therapeuticexpression cassette

Therapeutic mRNAand protein

Retrovirus

SCID and gene therapy

Ex vivo gene therapy of SCID-X1

amphotropic MFGB2 vector

patients with no HLA matched donor,reinjection of transduced CD34 + cells, no chemotherapy

patients with no HLA matched donor,reinjection of transduced CD34 + cells, no chemotherapy

Outcome of gene therapy trials in 20 patients with SCIDX1(Paris-London combined data)

85% (disease-free survival)

0

20

40

60

80

100su

rviv

al (%

)

0 2 4 6 8 10 12

Years

90% (overall survival)

normal quality of life, school attendance, normal growth and development free of therapy (n=10), IG substitution (n=8)

(median 10.3 y., 5.7 to 13.5 y.)

14

Gene therapy of SCID - Data summary

• SCID X1 2 trials (Paris, London)

n=20 alive 18 (median 10.3 y., 5.6 to 13.5 y.)

SAE 5 (alive, cured 4)

• ADA 3 trials (Milan, London, NIH/L.A.)

n=36 all alive, 25 off ERT (median 5.0 y., 1.0 to 11.5

y.)

SAE 0SAE: serious adverse eventERT: enzyme replacement therapy

SAE: serious adverse eventERT: enzyme replacement therapy

New trial

U5U5 Y

R RQMP ΔSD PREProm. IL2RG

EF1(S)RSV

New retroviral SIN vector

New trial, 9 patients enrolledMulticenter trial

(3-18 months follow-up)

New trial, 9 patients enrolledMulticenter trial

(3-18 months follow-up)

enhancer

SIN-MLV- pSRS11.EFS.IL2RG.pre

SCID-X1 Gene Therapy 2Kinetics of T lymphocytes recovery/transgene expression

months after months after c-gene therapyc-gene therapy

0

750

1500

2250

3000

3750

4500

0 2 4 6 8 10 12 14 16 18

CD

3+

/µl

CD

3+

/µl

P1

P3

0

200

400

600

800

1000

0 2 4 6 8 10 12 14 16 18

P1

P2P3

CD

31

+C

D45

RA

+/C

D4

/µl

CD

31

+C

D45

RA

+/C

D4/µ

l

CD132 c expression on

CD3

Vector copy number in T cells 1.0 to 1.2

Gene therapy hematopoietic stem cells

T lymphocyte

NK lymphocyte

B lymphocyte

Myeloidprogenitor

cell

Lymphoid progenitor

cell

erythrocyte

granulocyte

dendritic cell

macrophage

osteoclast

platelets

Stem cells

DiseasesDiseases

primary immunodeficiencies

hemoglobinopathies

primary immunodeficiencies

metabolic diseases

osteopetrosis

SCIDSCID

Wiskott Aldrichsyndrome

Wiskott Aldrichsyndrome

Gene therapy hematopoietic stem cells

T lymphocyte

NK lymphocyte

B lymphocyte

Myeloidprogenitor

cell

Lymphoid progenitor

cell

erythrocyte

granulocyte

dendritic cell

macrophage

osteoclast

platelets

Stem cells

DiseasesDiseases

primary immunodeficiencies

hemoglobinopathies

primary immunodeficiencies

metabolic diseases

osteopetrosis

SCIDSCID

Wiskott Aldrichsyndrome

Wiskott Aldrichsyndrome

CGD,LAD

HLH,IPEX

Genetics and PID

• Diagnosis

• Prognosis

• Treatement

• Screening

• Genetic counseling

• Diagnosis

• Prognosis

• Treatement

• Screening

• Genetic counseling

An example of a prenatal genetic diagnosis of an autosomal recessive disease (SCID)

Affectedfoetus

Genetics and PID

• Diagnosis

• Prognosis

• Treatement

• Screening

• Genetic counselling

• Diagnosis

• Prognosis

• Treatement

• Screening

• Genetic counselling

Fan HC et al, Nature

2012

Fetal genes in mother’s blood

Conclusion

• Genetic diagnosis will enter more and more frequent

daily practice of PID medicine

• Monogenic will turn to more complex (genome influence)

• Therapeutic development expected

• Preimplantatory and Prenatal diagnosis made easier

• (Pre)neonatal screening

• Ethical issues

Biotinylated probes

Capture « Whole Exome » 50 Mb of exomes

Design of capture phases

Liquid phase hybridization

NGS bank

Avidine beads

Exomic bank generation

Exomes bank

SEQUENCINGSEQUENCING

Sequences captures

by magnet

Filtering steps (artefacts, known variants,…)

~30 000 to 50 000 variants /person

Exome sequence analysis