Seminario de la Dra. Mónica

1. Diagnostico funcional y genético de inmunodeficiencias primarias.
Dra. Mónica Martínez-Gallo
Servei d’Immunologia
Hospital Universitario Vall d’Hebron, Barcelona

2. • Definition
Immunodeficiency is the result of a diverse group of abnormalities of the
immune system resulting primarily in an increased incidence of infection.
• Primary Immunodeficiency
Congenital and hereditary
• Secondary Immunodeficiency
Acquired on a transient or permanent basis
Immunodeficiency

3. B cells
Complement
Phagocytes
T cells
Pneumococci
Haemophilus
Neiseeria
Mycoplasma
E. coli
Klebsiella
Serratia
Pseudomonas
Aspergillus
Herpes sp
Cytomegalovirus
Listeria
Pneumocystis
Giardia
Crytosporidia
Candida
Staphylococcus
aureus
Primary immunodeficiency diseases

4. 1. Combined immunodeficiencies.
2. Well-defined syndromes with immunodeficiency.
3. Predominantly antibody deficiencies.
4. Diseases of immune dysregulation.
5. Congenital defects of phagocyte number, function, or both.
6. Defects in innate immunity.
7. Autoinflammatory disorders.
8. Complement deficiencies.
Primary immunodeficiency diseases: classification 2011 update
Frontiers in Immunology 2011.V2-54

5. • Defects in recombination of the
antigen receptor genes of B & T cells
– Recombinant Activating gene 1 (RAG 1)
– Recombinant Activating gene 2 (RAG 2)
– Artemis deficiency
• Defects in modifiers of gene
expression underlying
multisystem disorders:
– Cartilage Hair Hypoplasia
– SCID with alopecia and nail dystrophy
• Defects in cytokine receptors & signaling
– γc deficiency, X-linked SCID
– JAK3 deficiency
– IL-7 receptor α chain deficiency
– CD 45 deficiency
– CD 3 δ chain deficiency
• Defects in the purine pathway enzymes
– ADA deficiency
– Purine nucleoside phosphorylase
deficiency
Mutations in several distinct genes SCID

6. Lymphocyte phenotypeLymphocyte phenotype
T-B+T-B-
IL2RG (50%)
IL7RA (10%)
JAK3 (10%)
RAG1
RAG2
DCLRE1C (2%)
ADA/PNP (8%)
IgIg T+B- Ig
BTK (50%)
Molecular studies of candidate genesMolecular studies of candidate genes
Clinical suspicion
Functional studiesFunctional studies

7. Immunodeficiency with autoimmunity
•Histiocytosis: skin biopsy ruled it out
•Cutaneous T-cell lymphoma (Sezary)
•ALPS: No biomarkers. No mutations in TNFRSF6 (FAS) or KRAS
•Hemophagocytic lymphohistiocytosis (HLH)
•Leaky-SCID with Omenn or GVHD (maternal or post-transfusional)
-9 month-old boy
Splenomegaly
Recurrent episodes of hemolytic anemia compatible with
macrophage activation syndrome
-14 months- recurrent episodes of hemophagocytosis

8. Immunodeficiency with autoimmunity

9. In vitro lymphocyte proliferation assay
0
20
40
60
80
100
120
140
SIN Anti-CD3
IL2 +
Anti-CD3 PWM PMA+IONO
x1000c.p.m.
CONTROL PATIENT
PATIENT
Immunodeficiency with autoimmunity

10. Mutations in RAG1
Gen: FAS: Normal
Gen: RAG2: Normal
Gen: SH2D1A Normal

11. N C
Interaction with RAG2Homeodomain
RING+Zn
Basic
Domains
p.Trp522Cys p.Arg973Cys
N C
Interaction with RAG2Homeodomain
RING+Zn
Basic
Domains
p.Trp522Cys p.Arg973Cys
Gen RAG1Gen RAG1
Mutations in RAG1

12. Combined Immunodeficiency
- Five years old-girl
- Growth retardation and Sepsis
- Multiple episodes of fever
- Hypogammaglobinemia
0
10
20
30
40
50
60
70
P M A + IO N O P H A A nti-C D 3 +IL2 N o s tim ulo us
x1000cpm
control
pacient
% Absolute number % Absolute number
T cells CD3+ 58 0,522 x 109
/L 55-83 0,7-2,10
C3+CD4+ 9 0,081 x 109
/L 28-57 0,30-1,40
C3+CD8+ 31 0,3 x 109
/L 10-39 0,20-0,90
B cell CD19+ 34,00 0,304x 109
/L 6-19 0,10-0,50
Natural killer cells 8,00 0,007 x 109
/L 7-31 0,09-0,60
IgG 275 mg/dl 700-1600 mg/dl
IgA <10 mg/dl 40-70 mg/dl
IgM 64 mg/dl 40-230 mg/dl
REF VALPATIENT

13. c.984+1G>A
Exon 7
N CJH7 JH1JH2JH3JH6 JH5 JH4
FERM Domain SH2 Domain Kinase DomainPseudokinase Domain
c.3208-1G>A
Exon 24
c.984+1G>A
Exon 7
N CJH7 JH1JH2JH3JH6 JH5 JH4
FERM Domain SH2 Domain Kinase DomainPseudokinase Domain
c.3208-1G>A
Exon 24
Mutations in JAK3
c.984+1G>A
Compound mutation
c.984+1G>A
carrier
c.3208-1G>A
carrier
c.3208-1G>A

14. Borte S et al. Blood 2012;119:2552-2555
TRECS and KRECS

15. N
TRIOS STATUS
FAMILY
MEMBERS OBSERVATIONS
VH45 EXITUS AFFECTED NECROPSY SH2D1A XIAP PRF1
VH46 ALIVE MOTHER
VH47 ALIVE FATHER
VH48 EXITUS AFFECTED NECROPSY FAS SH2D1A
VH49 ALIVE MOTHER
VH50 ALIVE FATHER
VH51 ALIVE AFFECTED BTK NEMO
VH52 ALIVE MOTHER
VH53 ALIVE FATHER
VH54 ALIVE AFFECTED BROTHER EXITUS JAK3 RAG1 RAG2 DCLRE1C
VH55 ALIVE MOTHER
VH56 ALIVE FATHER
VH57 ALIVE AFFECTED AIRE RAG1 RAG2
VH58 ALIVE MOTHER
VH59 ALIVE FATHER
VH60 ALIVE AFFECTED RAG1 RAG2
VH61 ALIVE AFFECTED RAG1 RAG2
VH62 ALIVE MOTHER
VH63 ALIVE FATHER
GENETIC STUDY WITHOUT DEFECT

16. •Myelodysplastic syndrome (MDS)/acute myeloid leukemia (AML)
•Mono-MAC syndrome/DCML deficiency
-predisposition to nontubercular mycobacterial infection
-Deficiency in monocytes, dendritic cells, B and NK lymphocyte
-autosomal dominant or de novo
GATA-2 deficiency
(Hsu et al. Blood 2011) (Dickinson et al. Blood 2011)
Gene GATA-2

17. 11 year-old girl previously healthy admitted at the intensive care unit
with disseminated varicella-zoster virus (VZV)
GATA-2 deficiency
PatientControl PatientControl

18. -20 year-old man
-Lymphoedema and lymph nodes agenesia since the age of 3-years old.
-Multiple and recurrent episodes of erysipelas
-Pancytopenia+ granulomatous lung and bone marrow lesions.
-Responding to empiric therapy for Mycobacteria.
% Absolute number % Absolute number % Absolute number
Numbers of CD34+ 0,02 0,08
Monocytes 0,00 0 x 109
/L 0,50 0 x 109
/L 2,5-16 0,2-1,6
B cell CD19+ 0,00 0,002 x 10
9
/L 1,80 0,007 x 10
9
/L 6-19 0,10-0,50
Natural killer cells 0,06 0 x 109
/L 0,50 0,002 x 109
/L 7-31 0,09-0,60
T cells CD3+ 93 1,024 x 109
/L 94 0,38 55-83 0,7-2,10
C3+CD4+ 58 0,637 x 109
/L 17 0,07 28-57 0,30-1,40
C3+CD8+ 33 0,365 x 109
/L 36 0,14 10-39 0,20-0,90
IgG *724 mg/dl * Under IVIG therapy ND 700-1600 mg/dl
IgA 57 mg/dl ND 40-70 mg/dl
IgM 64 mg/dl ND 40-230 mg/dl
REF VALPATIENT 2PATIENT 1
GATA-2 deficiency

19. •Patient
A C T G T G C C C G A G
His386 STOP
GATA-2 deficiency

20. A C T G T G C C C G A G
His386 STOP
A C T G T G C C C G A G
His386 STOP
MUTATION c.1156-1157insAC
GATA-2 deficiency

21. Gly327 Gln328 Asn329
c.988C>T / p.Arg330X
STOP330
Arg330 Pro331 wild type allele
mutated allele
GATA-2 deficiency
MUTATION c.988C>T

22. c.1156-1157insAC
Zinc-finger
domain 2
DNA double helix
Arg364Ala
Nuclear localization
signal domain
c.1156-1157insAC

23. Familial Haemophagocytic disorders
FHLH is a rare autosomal recessive disorder of immune dysregulation associated with
uncontrolled T cell and macrophage activation and hypercytokinaemia.
Fatal unless a HSCT is performed.
•70-80% Symptoms of HLH in the first year of life
•Most common symptoms: Fever
Progressive hepatosplenomegaly
Skin manifestations non-specific
Neurological abnormalities
Often triggered by infectionsViralEBV, CMV, PV
•Laboratory findings: CytopeniasTrombocytopenia, aneamia, neutropenia
Impaired or absent NK and T cell cytotoxicity activity
Hyperferrinaemia
Liver disfunction Lactate dehydrogenase lactic acid
Serum transaminases
Bilirubin
Hypofibrinogen Intravascular coagulation
Elevated triglycerides
High serum levels of CD25s

24. Pathophysiology of Haemophagocytic disorders

26. Sospecha clínica
Ensayo citoxicidad NK
FHLH1 10% Cr9q21-22
FHLH2 20-50% PRF1
FHLH3 30-40% UNC13D
FHLH4 n=10 STX11
FHLH5 10% STXBP2
FHLH1 10% Cr9q21-22
FHLH2 20-50% PRF1
FHLH3 30-40% UNC13D
FHLH4 n=10 STX11
FHLH5 10% STXBP2
Genetic basis of FHLH:
Presencia de perforina
Ensayo degranulación
UNC13D
STXBP2
STX11
PRF1
NK
CD8
PERFORIN
CD3
CD107a
CD56
Problamente
No FHLH

27. Sospecha clínicaEnsayo citotoxicidad NK
0
10
20
30
40
50
60
70
80
90
BASAL 3:1 6:1 12:1 25:1 50:1
Ratio Efector: Diana
%muertecelular
control sano
PACIENTE 1
control patológico
Genetic basis of FHLH:

28. Sospecha clínica
Ensayo citoxicidad NK
FHLH1 10% Cr9q21-22
FHLH2 20-50% PRF1
FHLH3 30-40% UNC13D
FHLH4 n=10 STX11
FHLH5 10% STXBP2
FHLH1 10% Cr9q21-22
FHLH2 20-50% PRF1
FHLH3 30-40% UNC13D
FHLH4 n=10 STX11
FHLH5 10% STXBP2
Genetic basis of FHLH:
Presencia de perforina
Ensayo degranulación
UNC13D
STXBP2
STX11
PRF1
NK
CD8
PERFORIN
CD3
CD107a
CD56
Problamente
No FHLH

29. At admission:
-Pancytopenia: Anemia (Hb: 7-8g/dl)
Neutropenia (0.5x109
/L)
Trombocytopenia (13.9x109
/L)
-Low plasma fibrinogen: 1.90 g/L
-High plasma ferritin: 51336 ng/ml
-High plasma triglycerides: 152 mg/dL
-LDH: 4339 UI/ml
-Haemophagocytosis was detected in a bone marrow aspiration sample
5-weeks-old baby girl
Febrile urinary tract infection
Splenomegaly
Progressive liver failure
CASE REPORT
Perforin deficiency FHLH2

30. 0 10
2
10
3
10
4
10
5
<FITC-A>: PERFORINA
0
10
2
10
3
104
10
5
<APC-A>:CD3
42.4 0.22
0.1157.3
0 10
2
10
3
10
4
10
5
<FITC-A>: PERFORINA
0
10
2
10
3
104
10
5
<APC-A>:CD3
42.2 45.1
9.962.79
HEALTHY
DONOR
PERFORIN
CD3
0 10
2
10
3
10
4
10
5
<FITC-A>: PERFORINA
0
10
2
10
3
10
4
10
5
<APC-A>:CD3
34 7.66
29.329.1
Mut PRF1
Pro459Leu
ISOTYPE
CONTROL
Mut PRF1
p.Gly477fs X479
100
101
102
103
104
FITC-A
100
101
102
103
104
APC-A
76.6 2.6
0.919.9
NK
CD8
PACIENT 1 PACIENT 2
Perforin deficiency

31. 5´… GCC ACA GGG GGG CCC CTG AGG …3´
STOP
CODON
A T G G P L R
Wild type
Patient
PRF1 exon2 exon3
4483delG
477 479
5´… GCC ACA GGG GGC CCC TGA GG …3´
A T G G P X

32. Patient
Father
Mother
4483delG

33. MACPF domain
PRF1
Cr10
exon1
exon2 exon3
p.Gly477fs X479
4483delG
Rattus norvegicus
Mus musculus
Homo sapiens
New variant
LDFGDVLLATGGPLRLQVWDQDSGRDD
LDFGDVLLATGGPLRLQVWDQDSGRDD
LDFGDVLLATGGPLRLQVWDQDSGRDD
LDFGDVLLATGGPLX
4
7
9
4
8
1
LDFGDVLLATGGPLRLPVWDQDSGRDDQ481P variant
C2-Calcium-dependent lipid
binding domain

34. 24-months old girl,
Prolonged symptomatic EBV infectious mononucleosis
Pancytopenia
Fever ≥38.5ºC Yes
Splenomegaly Yes
Cytopenia Anemia (Hgl: 7-8g/dl)
Platelets(36x109/L)
Hypofibrinogenia and/or Hypertrigliceridemia Hypertrigliceridemia
Haemophagocytosis in bone marrow No
Low or absent NK-cell activity Yes
Diagnostic criteria of HLH
Degranulation defects

35. 10.2 11.6
24.753.6
PERFORIN
CD3
CONTROL
Activacion
CD107a
CD56
PATIENT

36. exó 9 intró
9
Mutation c.753+1G>TMutation c.753+1G>T
wt allele
c.2448-11 G>A allele
cDNA UNC13DgDNA UNC13D
Mutation c.2448-11G>AMutation c.2448-11G>A
Degranulation defects: Splice donor defects

37. c.2448-11G>A c.753+1G>T c.244811G>A
c.753+1G>T
CD107a -APC
c.753+1G>T
c.2448-11G>A
Compound mutation
c.753+1G>T
carrier
c.2448-11G>A
carrier
DC
CD107a

38. Acknowledgements
Hospital Vall d’Hebron
Servei d’Immunologia
Laura Viñas
Dr. Roger Colobran
Dr. Manuel Hernandez
Dra. Ana Marín-Sanchez
Dr. Ricardo Pujol
Hospital Sant Joan de Déu
Servei de Pediátria
Dra. Laia Alsina
Dra. M. Olaya
Dra. MA. Martín Mateos
Vall d’Hebron Institut de Recerca (VHIR)
Translational Bioinformatics in Neuroscience.
Dr. Francisco Javier de la Cruz.
Institució Catalana per a la Recerca
i Estudis Avançats (ICREA)
Dr. Sergio Lois
Unitat de Malalties Infeccioses i Immunodeficiències Pediátriques
Dr. Pere Soler
Dra. Andrea Martín
Dra. Concepció Figueras
Servei d’Oncologia i Hematologia Pediàtrica
Dr. Jose Luís Dapena