Human phenotypes associated with
germline IKZF1 mutations
David Boutboul
Département d’Immunologie Clinique, Hôpital Saint Louis U1163, Institut Imagine
IKZF1
• = IKAROS = LyF1 = ZNF1A1 (1994)
• 7p12.2
• 8 exons (first exon = non coding)
• Hematopoietic transcription factor
• Major form = Ik-1
ZF1 ZF2 ZF3 ZF4 ZF5 ZF6
DNA-BINDING DOMAIN DIMERIZATION DOMAIN
Ikaros localizes at pericentromic
heterochromatin
NIH3T3
Ikaros is mainly involved in lymphoid
development
Splenic B-cell Anti-CD40+ IL4
T-cell Anti-CD3 Positive regulation of T-cell differentiation
(DNTT, CD8, CD4)
Control of cellular proliferation (ex: MYC)
Positive regulation of B-cell differentiation (IGLL1, RAG ½)
Ikaros is tightly regulated
ISOFORMS
Myeloid Lymphoid
2/3 4 5 6 7 8
IK-1
IK-2
IK-3
IK-5
IK-6
IK-7
Exons
DNA Binding Domain
ZF1 ZF2 ZF3 ZF4 ZF5 ZF6
Dimerization domain
IK-H 3B
IK-8 IK-4
1 Only one or no ZF in
the DNA B Domain Dominant-negative
effect
Insertion 60 nt
IK-X
The Ikaros family
Helios
Eos
Pegasus?
Ikaros
Aiolos
Post translational modifications
Phosphorylation
Sumoylation
H 0 3 6 12 24 48 72 96
D14 PHA-derived T-cell blasts
Anti-CD3/CD28
Mouse models of Ikaros inactivation
NULL MUTATION
« Normal myeloid development »
Severe T-cell lymphopenia with oligoclonal repertoire
Absent B, NK and plasmacytoid dendritic cells
Fraction of Ikzf1
null/+mice develops T-cell malignancy
DOMINANT-NEGATIVE MUTANT
Myeloid defect leading to lethality of the homozygous mutant (anemia)
Absent B cells and abnormal peripheral T cells
Secondary occurrence of thymic lymphoid proliferation (100%)
in the heterozygous mutant
Human phenotypes associated with IKZF1
somatic variants
IKZF1/B-ALL
Δ IKZF1
75% BCR-ABL+ BCP-ALL 15% pediatric BCP-ALL 30-50% adult BCP-ALL Poor outcome
Haploinsufficiency vs Dominant-negative effect?
Olsson et Johansson, BJH 2015
Human phenotypes associated with
IKZF1 germline variants
Somatic variants Germline variants
Mouse models
IKZF1 p.Y210C heterozygous mutation
Germline?
Early pancytopenia with reduced-cellularity bone marrow
Absent peripheral B lymphocytes
Normal Ig levels
Absent memory T cells/ Proliferation defect upon PHA and CD3
Death at D40 following BMT
ZF1 ZF2 ZF3 ZF4 ZF5 ZF6
DNA-binding domain Dimerization domain
Y210C
T cells
NIH3T3
Heterozygous DNA-binding domain mutations CVID phenotype
Incomplete penetrance No clinical T-cell defect
2 patients (on 29) developed B-ALL (at 3 and 5 years of age)
Severe (progressive) B-cell lymphopenia, with absent memory B-cells
Normal memory T-cell number
Normal proliferation upon anti-CD3 stimulation Normal protein expression (WB)
No overt myeloid defect
Haplo-insufficiency
Our story
2 m.o (P1)
Bacterial lung infection
Severe viral illnesses (SRV, adenovirus) PCP pneumonia (17 m.o)
Cryptosporidiosis with cholangitis
Died from cholangitis complications following BMT (2 y.o)
6 m.o (P2)
PCP pneumonia (2 episodes) Localized onychomycosis
Molluscum contagiosum Recurrent mouth ulcers
Well under IVIG and cotrimoxazole (13 y.o)
Patient 1 Patient 2 Peripheral blood
lymphocyte counts
2300/mm3 (16 m.o) 5200/mm3 (7 y.o)
CD3+ 2200/mm3 (96%) 4628/mm3 (89%)
CD4+ T cells 43% 61%
Naive CD4+ 92% 97%
CD8+ T cells 38% 20%
Naive CD8+ 85% 55%
Central memory CD8+ 1% 5%
Effector memory CD8+ 4% 19%
TEMRA cell 11% 21%
CD19+CD20+ B cells 0,3% 9%
Naïve B cells 98%
NK cells 1% 2%
IgG (mg/dL) 35 329
IgA (mg/dL) undetectable <58
IgM (mg/dL) undetectable <42
Functional B-cell studies N.D. Normal B-cell proliferation (3H) and IgE production upon CD40L/IL4 stimulation
T-cell proliferation defect
Shown on CD8+ T cells/PBMC/ Acquired at Day6 Same picture on CD4+ T cells
Ctrl
P1
P2
Anti-CD3
1μg/mL Anti-CD3
10μg/mL
Beads Anti CD3/28
Anti-CD3 0.05 μg/mL + IL-2 500 UI/mL
Unstimulated Stimulated
Cell Trace Violet
Absent proliferation to antigens Normal proliferation to PHA
T-cell activation defect
Shown as CD25 expression on CD8 T cells/PBMC Same picture on CD4+ T cells
Beads Anti-CD3/28 Unstimulated Anti-CD3
1μg/mL Anti-CD3
10μg/mL Unstimulated
Anti-CD3 0.05 μg/mL + IL-2 500 UI/mL
Ctrl
P1
P2
CD25
Whole Blood WES (04/2016)
Patient 1 Patient 2
Common SNVs 1332 967
Intronic or silent 591 438
Compound heterozygous 64 45
Remaining heterozygous 419 384
Genes in common 1
IK-1 isoform
ZF1 ZF2ZF3 ZF4 ZF5 ZF6
DNA-binding domain Dimerization domain
P2 mutation
c.476A>C, p.N159T CADD score=26,7 P1 mutation
c.476A>G, p.N159S CADD score=24
Hypothesis : Compound heterozygous mutation (recessive model)
Heterozygous (de novo or incomplete penetrance) mutation (dominant model)
Excluded Benignity No expression or fuction in the immune system
IKZF1
IKZF1 ex.1 ex.8
ZF1ZF2ZF3 ZF4 ZF5 ZF6
DNA-binding domain Dimerization domain
Family G c.476A>C, p.N159T
CADD score=26,7 Family A-F
c.476A>G, p.N159S CADD score=24
c.476A>G (p.N159S) Family C
WT/WT
WT/Mut WT/WT
C1
c.476A>G (p.N159S) Family D
WT/Mut
D1
WT/WT WT/WT
WT/WT
c.476A>C (p.N159T) Family G
WT/Mut
G1
WT/WT WT/WT WT/WT
c.476A>G (p.N159S) Family A
WT/WT
WT/Mut WT/WT
A1
c.476A>G (p.N159S) Family B
WT/Mut WT/WT WT/WT
B1
Family E c.476A>G (p.N159S)
WT/Mut
E1 Family F
c.476A>G (p.N159S)
WT/Mut
F1
WT/WT WT/WT
WT/WT WT/WT
E1
N159 mutations are de novo mutations impacting a highly conserved amino acid
Patient Mutation Origin
Age at first sympto
ms
Infections
Bacterial Others
Extra- hematopoietic manifestations
Outcome (age in
years) Pneumocystis
pneumonia (age) Viral
A1 p.N159S Caucasian
(Europe) 2 m.o yes (17 m.o) Severe SRV bronchiolitis Klebsiella sp. lung infection
Cryptosporidial
cholangitis No HSCT
Died (2)
B1 p.N159S Asian
(Japan) 2 m.o yes (2, 3 and 17 y.o) No
Lung abscess, recurrent sinopulmonary
infections, bronchiectasis
Pulmonary Mycobacterium avium
complex infection
Pulmonary
aspergillosis No Alive (17)
C1 p.N159S Caucasian
(US) 1.5 y.o yes (2 y.o)
Severe SRV bronchiolitis Adenoviral illness requiring
cidofovir
Recurrent otitis media
and pneumonia No
Recurrent mouth
ulcers HSCT, alive and well
D1 p.N159S Caucasian
(US) 2 m.o yes (2 y.o) Usual chickenpox
Lung abscess, recurrent sinopulmonary
infections, bronchiectasis
No No Alive and well
E1 p.N159S Caucasian
(US) 1 y.o yes (1y.o) Recurrent HSV (oral,
genital), usual chickenpox
Streptococcus pneumoniae meningitis;
Pseudomonas sp.pneumonia; skin
abscesses
Recurrent thrush;
Candida parapsilosis
fungemia
DVT/PE;
pancreatitis Alive (28)
F1 p.N159S Asian
(Japan) 9 m.o yes (20 m.o) No
Severe pneumonia, otitis media, chronic
sinusitis
Recurrent oral
candidiasis T-ALL
HSCT Alive and well
(19) G1 p.N159T Caucasian
(Europe) 6 m.o yes (6 m.o, 5 y.o) Usual chickenpox,
molluscum contagiosum No Localized
onychomycosis
Recurrent mouth ulcers
Alive and well (12)
y.o, year old. m.o, month old, SRV syncitial respiratory virus, HSCT, hematopoietic stem cell transplantation, T-ALL T-cell acute lymphoblastic leukemia
N159 patients display an early-onset CID
A1 (16 m.o) B1 (17 y.o) C1 (4 m.o) D1 (16 m.o) E1 (26 y.o) F1 (9 m.o/4
y.o) G1 (7 y.o)
B cells
CD19+ (cells.mm-3) 0 32 7 0 7 15/26 468
CD19+ (%) 0 0.5 0.1 0 1.1 6/1.3 9
CD21+CD27+ / CD19+
(memory) (%) 0.7 n.d 2
Immunoglobulin levels (mg. dL-1)
IgG (1 / 2 / 3 / 4) 0.35 0.18 0 0.65 0.4 0.21 3.29
IgM 0 0.13 0 <0.03 0.29 0.09 0
IgA 0 0.05 0 <0.05 0.12 <0.05 0
N159 patients show severe B-cell lymphopenia with low or absent Ig
N159 mutations are associated with a singular phenotype
N159 mutations are associated with multiple functional T-cell defects
PBL
anti-CD3/CD28 24h stimulation
CID phenotype
Cryptosporidiosis in one patient
Incomplete proliferation defect upon anti-CD3
Null proliferation to antigens
Associated monocyte/mDC defect?
No monocytopenia but...
DC and monocyte defects associated with N159 mutations
LPS 24h-stimulation
low doses anti-CD3 stimulation
Other myeloid defects?
A1 (16 m.o) B1 (17 y.o) C1 (4 m.o) D1 (16 m.o) E1 (26 y.o) F1 (9 m.o/4
y.o) G1 (7 y.o)
Leukocytes (cells..mm-3) 4600 7700 7820 11500 1030 6100/2600 4700
Hemoglobin (g/dL)/
Mean Corpuscular Volume
12.6/78 11.2/91 12.7/93.2 9.9/87.6 10.0/86.1 10.5/80 12.3/77
Neutrophils (cells.mm-3) 1900 704 720 280 200/900 5650/494 1300-1400
Monocytes (cells..mm-3) 260 360 640 5080 40/80 150/1170 376
Dendritic cells
pDC (% total peripheral
blood leukocytes) n.d 0.02 n.d n.d n.d n.d 0.07
Total mDC (% total peripheral blood leucocytes)
n.d 0.09 n.d n.d n.d n.d 0.017
Why are N159 mutants associated with a more
severe phenotype?
Ikaros N159 expression is similar to control
T cells HEK/293T cells
Dominant-negative effect of the N159 mutants on PC-HC
localization
NIH3T3 cells Ik/He/Aio -
**** p<10-4
N159 amino-acid substitutions maintain the ability to bind DNA in silico
Is there an impact on other lymphoid partners?
Control
R143W
N159T 0
2 4 6 8
% of Helios positive cells
Naive CD4+ T cells
Control
R143W
N159T 0
5 10 15
% of Helios positive cells
Central memory CD4+ T cells
Control
R143W
N159T 0
10 20 30
% of Helios positive cells
Effector memory CD4+ T cells
Control
R143W
N159T 0
10 20 30 40
% of Helios positive cells
Naive CD8+ T cells
Control
R143W
N159T 0
10 20 30 40 50
% of Helios positive cells
Central memory CD8+ T cells
Control
R143W
N159T 0
20 40 60 80
% of Helios positive cells
Effector memory CD8+ T cells
Control
R143W
N159T 0
20 40 60 80
% of Helios positive cells
Effector memory RA+ CD8+ T cells
Is there an impact on other lymphoid partners?
IKZF3/Aiolos
HK, SDR
Genotype-phenotype correlation?
Haplo-insufficiency Negative dominance
IKAROS heterozygous germline LOF mutations
CVID-like phenotype
Incomplete penetrance
Progressive loss of B-cells
Autoimmune complications
ITP/IgA vasculitis/SLE
Pure B-cell disease?
CID-like phenotype
Early-onset
100% PJ Pneumonia
Mostly de novo
Lymphoid defect
Absent B-cells
T-cell memory defect
Proliferation and activation defect
Defect in IL-2 signaling
Myeloid defect
Low mDC
Central cytopenias
Erythroid abnormalities?
Increased ALL risk
2
1
Ikaros dominant negative mutations share similarities
with other PIDs
Combined immune deficiency
T-cell memory defect
IL2 signaling defect
Myeloid defect
Central cytopenias/AA
mDC defect
Increased ALL
risk
GATA2
deficiency
B-NK- Mono-DC- BM failure
AD IRF8
deficiency
B+NK+
mDC1-
NfKB LOF
Thank you!
Institut Imagine Akihiro Hoshino Zoé Van de Wyngaert Sylvain Latour
Capucine Picard Sarah Winter Emmanuel Martin Christelle Lenoir Mathieu Furasso Sébastien Fournier Norbert Minet Debora Jorgé Steicy So(b)rino Claire Soudais Camille Bachelet Romain Lévy Benoît Roch Pratick Revy
Jean-Pierre de Villartay Frédéric Rieux-Laucat Alain Fischer
Plateforme d’imagerie UFR Necker
Hôpital Necker Patients and families
Despina Moshous Ludovic Lhermitte
Vahid Asnafi GRAALL Felipe Suarez Olivier Hermine
Hôpital de la Conception Catherine Farnarier
Vincent Barlogis Frédéric Vély
NIH
Hyesun Kuehn Sergio Rosenzweig
Tala Shahin
Münich/Istanbul Fabian Hauck
Gül Ozdemir
Tokyo/Nagoya Hirokazu Kanegane
Seiji Kojima
Ikaros, Daedalus and Naucrate
• 8 cas mutés sur 197 LAL-T (4%) (dont 3/7 ETP-ALL)
• 1 mutation germinale* (sur 3 prélèvements testés)
essential splicing ho R184W
IKZF1/T-ALL?
Targeted sequencing, GRAALL, L. LHERMITTE, V. ASNAFI, in progress
ZF1 ZF2 ZF3 ZF4 ZF5 ZF6
DNA-binding domain Dimerization domain
N114Efs*
N159S L273Y
R274Ifs S105L
missense frameshift
*germline
dominant negative mutation haplo-insufficiency mutation unknown mechanism
S364W(phospho-sérine, LOF, Ukun PNAS 2012)
Patients carrying Dimerization Domain (DD) mutants
Autoimmune cytopenias/Hypogammaglobulinemia/Burkitt lymphoma/T-ALL Monomères vs multimères d’IKZF1?
Interactions avec les autres membres?
Modifications post traductionnelles?
Kuehn et al, Blood 2020
Two new families…
Diagnosis =13 y.o
Evans syndrome (ITP+AIHA) Celiac disease (15 y.o)
AutoAb positivity (ANA/smooth muscle+) Rituximab responsive
IKZF1 ex.1 ex.8
ZF1 ZF4 ZF5 ZF6
Heterozygous c.548 G>A, p.R183H mutation CADD score =33 (MSC=7.4)
Not seen in gNOMAD De novo in P1
ZF2ZF3
Diagnosis=41 y.o
Pseudotumor cerebri/radiotherapy Rosai-Dorfman disease (47 y.o)/Steroid Non-typed diabetes
ANA/Smooth muscle + 1/160
Diagnosis =16 y.o
Diffuse nodal lymphoid hyperplasia Evans syndrome (ITP+AIHA)
Type 1 diabetes
Autoimmune hepatitis
AutoAb positivity (ANA, ACAN, anti-gliadine, anti- TPO)
Rituximab responsive
P1 P2
Immunological features of PBMCs from patients with IKZF1
R183Hmutations
Same aspects are observed in the mother and the brother of P2
Ctrl1 P1
CD4+
CD8+
Normal number of T, B and NK cells High CD45RA MFI on CD4+ and CD8+ T cells
Decreased TCM and TEM CD8+ T cells Normal recent thymic emigrants Normal Treg numbers (CD4+FoxP3+CD25+)
Normal naive/memory B cell repartition Increased transitional B cells
Increased CD21low B cells
Normal or increased gammaglobulins (P2) Increased pDC in P2
Mild eosinophilia in P2
Ctrl2 P2
CCR7
CD45RA
Functional T-cell defects in patients with IKZF1
R183Hmutations
102 103 104 105
Ctrl1
102 103 104 105 102 103 104 105
Ctrl2 P1
NS
Anti-CD3 0.1 μg/mL
1 μg/mL
10 μg/mL
Anti-CD3/CD28 beads
CTV CD4+T cells
CD25
anti-CD3 1 μg/mL
+ lenalidomide 10 mmol
Ctrl1
P1
Ctrl3 P2
CD4+PHA-derived T cells-blasts
Why is R183H mutant associated with a singular clinical and biological
phenotype ?
Overexpression in HEK/293 T cells pcDNA3.1
IK1
KU80
Ctrl P1
24h- CD3/CD28 stimulated
T cells Overexpression
in NIH3T3 pcDNA3.1
100% IK1-R183H
50% IK1-WT + 50%IK1-R183H 100% IK1-WT
R183H mutant behaves as a gain-of-function mutant
haplo R162Q WT R183H WT + Ab R183H + Ab
IB : IKZF1 EMSA 293T cells IKBs4 seq Same results for gSat8 seq
EV WT R183H R162Q
0 50 100 150
Relative Hes1 Luc activity
pCMV6-DDK
*
*
0.0062
0.0093
*0.0057
*0.0092
* Paired t test (N=3)
H. Kuehn T. Shahin Luciferase assay
pGL3-Hes1 293T cells
I. Callebaut
IKZF1 ex.1 ex.8
ZF1 ZF2ZF3 ZF4 ZF5 ZF6
DNA-binding domain
Dimerization domain Family G
c.476A>C, p.N159T CADD score=26,7
Family A-F c.476A>G, p.N159S
CADD score=24
R162L R162Q R162W H167R C147R R143W*
R184Q R184W*
H191R
R183H (n=4, FRL) CADD score =33
Y210C
S384L
CADD score =24 T398M* (Evans, FRL)
Genotype-phenotype correlation
IKZF1 GERMLINE MUTATIONS ASSOCIATED PHENOTYPES
IKZF1 LOF mutations
CID-like phenotype Early-onset 100% PJ Pneumonia
Mostly de novo Lymphoid defect
Absent B-cells T-cell memory defect
Proliferation and activation defect Defect in IL-2 signaling
Myeloid defect Low mDC Central cytopenias Erythroid abnormalities?
Haplo-insufficiency Negative dominance
CVID-like phenotype Incomplete penetrance Progressive loss of B-cells Autoimmune complications
ITP/IgA vasculitis/SLE
Pure B-cell disease?
Increased ALL risk 2
1
Immune dysregulation phenotype POLYCLONAL LYMPHOPROLIFERATION
ROSAI DISEASE EVANS SYNDROME
PGA-II No infection
Incomplete penetrance T-cell hyperproliferation Increased Tr and CD21low B
cells
Myeloid expansion?
Ongoing studies T /B/monocytes RNAseq
T Reg function LRBA/CTLA4 expression
New mutants IKZF1 GOF mutations
