Diagnostico funcional y genético de inmunodeficiencias primarias.

Dra. Mónica Martínez-GalloServei d’Immunologia Hospital Universitario Vall d’Hebron, Barcelona

• DefinitionImmunodeficiency is the result of a diverse group of abnormalities of the immune system resulting primarily in an increased incidence of infection.

• Primary ImmunodeficiencyCongenital and hereditary

• Secondary Immunodeficiency Acquired on a transient or permanent basis

Immunodeficiency

B cells Complement

Phagocytes

T cells

PneumococciHaemophilusNeiseeriaMycoplasma

E. coliKlebsiellaSerratiaPseudomonasAspergillus

Herpes spCytomegalovirusListeriaPneumocystis

GiardiaCrytosporidia

Candida

Staphylococcus aureus

Primary immunodeficiency diseases

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

• Defects in recombination of theantigen receptor genes of B & T cells– Recombinant Activating gene 1 (RAG 1)– Recombinant Activating gene 2 (RAG 2)– Artemis deficiency

• Defects in modifiers of geneexpression underlyingmultisystem 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

Lymphocyte phenotypeLymphocyte phenotype

T-B+T-B-

IL2RG (50%)

IL7RA (10%)

JAK3 (10%)

RAG1RAG2DCLRE1C (2%)

ADA/PNP (8%)

Ig Ig T+B- Ig

BTK (50%)

Molecular studies of candidate genesMolecular studies of candidate genes

Clinical suspicion

Functional studiesFunctional studies

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 boySplenomegaly Recurrent episodes of hemolytic anemia compatible with macrophage activation syndrome-14 months- recurrent episodes of hemophagocytosis

Immunodeficiency with autoimmunity

In vitro lymphocyte proliferation assay

0

20

40

60

80

100

120

140

SIN Anti-CD3IL2 +

Anti-CD3 PWM PMA+IONO

x100

0 c.

p.m

.

CONTROL PATIENT

PATIENT

Immunodeficiency with autoimmunity

Mutations in RAG1

Gen: FAS: Normal Gen: RAG2: Normal Gen: SH2D1A Normal

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.Arg973CysGen RAG1Gen RAG1

Mutations in RAG1

Combined Immunodeficiency

- Five years old-girl - Growth retardation and Sepsis - Multiple episodes of fever - Hypogammaglobinemia

0

10

20

30

40

50

60

70

PMA + IONO PHA Anti-CD3 +IL2 No stimulous

x 10

00 c

pm

control

pacient

% Absolute number % Absolute number

T cells CD3+ 58 0,522 x 109/L 55-83 0,7-2,10C3+CD4+ 9 0,081 x 109/L 28-57 0,30-1,40C3+CD8+ 31 0,3 x 109/L 10-39 0,20-0,90

B cell CD19+ 34,00 0,304x 109/L 6-19 0,10-0,50Natural killer cells 8,00 0,007 x 109/L 7-31 0,09-0,60

IgG 275 mg/dl 700-1600 mg/dlIgA <10 mg/dl 40-70 mg/dlIgM 64 mg/dl 40-230 mg/dl

REF VALPATIENT

c.984+1G>AExon 7

N CJH7 JH1JH2JH3JH6 JH5 JH4

FERM Domain SH2 Domain Kinase DomainPseudokinase Domain

c.3208-1G>AExon 24

c.984+1G>AExon 7

N CJH7 JH1JH2JH3JH6 JH5 JH4

FERM Domain SH2 Domain Kinase DomainPseudokinase Domain

c.3208-1G>AExon 24

Mutations in JAK3

c.984+1G>A

Compound mutation

c.984+1G>A

carrierc.3208-1G>A

carrier

c.3208-1G>A

Borte S et al. Blood 2012;119:2552-2555

TRECS and KRECS

NEXT-GENERATION

SEQUENCING

TRIOS STATUSFAMILY MEMBERS OBSERVATIONS

VH45 EXITUS AFFECTED NECROPSY SH2D1A XIAP PRF1VH46 ALIVE MOTHER VH47 ALIVE FATHERVH48 EXITUS AFFECTED NECROPSY FAS SH2D1AVH49 ALIVE MOTHER VH50 ALIVE FATHERVH51 ALIVE AFFECTED BTK NEMOVH52 ALIVE MOTHER VH53 ALIVE FATHERVH54 ALIVE AFFECTED BROTHER EXITUS JAK3 RAG1 RAG2 DCLRE1CVH55 ALIVE MOTHER VH56 ALIVE FATHERVH57 ALIVE AFFECTED AIRE RAG1 RAG2VH58 ALIVE MOTHER VH59 ALIVE FATHERVH60 ALIVE AFFECTED RAG1 RAG2VH61 ALIVE AFFECTED RAG1 RAG2VH62 ALIVE MOTHER VH63 ALIVE FATHER

GENETIC STUDY WITHOUT DEFECT

•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

11 year-old girl previously healthy admitted at the intensive care unit with disseminated varicella-zoster virus (VZV)

GATA-2 deficiency

% Absolute number % Absolute number

CD34+ 0.017

Monocytes 0.00 0 x109 /L 2.5-16 0.2- 1.6

CD19+ 0.20 0.002 x109/L 6-19 0.10-0.50

CD56+ 0 0 x109/L 7-31 0.09-0.60

CD3+ 93 1.024 x109/L 55-83 0.70-2.10

C3+CD4+ 58 0.637x109/L 28-57 0.30-1.40

C3+CD8+ 33 0.365 x109/L 10-39 0.20-0.90IgG 724 mg/dl 700-1600 mg/dlIgA 57 mg/dl 70-40 mg/dlIgM 64 mg/dl 40-230 mg/dl

Infections Disseminated varicella-zoster virusLower respiratory infections

PATIENT REF. VALUE

Under IVIG therapy

PatientControl PatientControl

-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 0,08Monocytes 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 109/L 1,80 0,007 x 109/L 6-19 0,10-0,50Natural 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 0,7-2,10C3+CD4+ 58 0,637 x 109/L 17 0,07 28-57 0,30-1,40C3+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/dlIgA 57 mg/dl ND 40-70 mg/dlIgM 64 mg/dl ND 40-230 mg/dl

REF VALPATIENT 2PATIENT 1

GATA-2 deficiency

•Patient

A C T G T G C C C G A G

His386 STOP

GATA-2 deficiency

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

Gly327 Gln328 Asn329

c.988C>T / p.Arg330X

STOP330

Arg330 Pro331 wild type allele

mutated allele

GATA-2 deficiency

MUTATION c.988C>T

c.1156-1157insAC

Zinc-finger domain 2

DNA double helix

Arg364Ala

Nuclear localization signal domain

c.1156-1157insAC

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 infectionsViralEBV, CMV, PV

•Laboratory findings: CytopeniasTrombocytopenia, aneamia, neutropeniaImpaired or absent NK and T cell cytotoxicity activityHyperferrinaemiaLiver disfunction Lactate dehydrogenase lactic acid Serum transaminases BilirubinHypofibrinogen Intravascular coagulationElevated triglycerides High serum levels of CD25s

Pathophysiology of Haemophagocytic disorders

Sospecha clínica

Ensayo citoxicidad NK

FHLH1 10% Cr9q21-22FHLH2 20-50% PRF1FHLH3 30-40% UNC13D FHLH4 n=10 STX11FHLH5 10% STXBP2

FHLH1 10% Cr9q21-22FHLH2 20-50% PRF1FHLH3 30-40% UNC13D FHLH4 n=10 STX11FHLH5 10% STXBP2

Genetic basis of FHLH:

Presencia de perforina

Ensayo degranulación

UNC13DSTXBP2STX11

PRF1

NK

CD8

PERFORIN

CD3

CD107a

CD56

ProblamenteNo FHLH

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

% m

uert

e ce

lula

rcontrol sano

PACIENTE 1

control patológico

Genetic basis of FHLH:

Sospecha clínica

Ensayo citoxicidad NK

FHLH1 10% Cr9q21-22FHLH2 20-50% PRF1FHLH3 30-40% UNC13D FHLH4 n=10 STX11FHLH5 10% STXBP2

FHLH1 10% Cr9q21-22FHLH2 20-50% PRF1FHLH3 30-40% UNC13D FHLH4 n=10 STX11FHLH5 10% STXBP2

Genetic basis of FHLH:

Presencia de perforina

Ensayo degranulación

UNC13DSTXBP2STX11

PRF1

NK

CD8

PERFORIN

CD3

CD107a

CD56

ProblamenteNo FHLH

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 girlFebrile urinary tract infectionSplenomegaly Progressive liver failure

CASE REPORT

Perforin deficiency FHLH2

0 102 103 104 105

<FITC-A>: PERFORINA

0

102

103

104

105

<A

PC

-A>

: CD

3

42.4 0.22

0.1157.3

0 102 103 104 105

<FITC-A>: PERFORINA

0

102

103

104

105

<A

PC

-A>

: CD

3

42.2 45.1

9.962.79

HEALTHY DONOR

PERFORIN

CD3

0 102 103 104 105

<FITC-A>: PERFORINA

0

102

103

104

105

<A

PC

-A>

: CD

3

34 7.66

29.329.1

Mut PRF1Pro459Leu

ISOTYPECONTROL

Mut PRF1p.Gly477fs X479

100 101 102 103 104

FITC-A

100

101

102

103

104

AP

C-A

76.6 2.6

0.919.9

NK

CD8

PACIENT 1 PACIENT 2

Perforin deficiency

5´… GCC ACA GGG GGG CCC CTG AGG …3´

STOPCODON

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

Patient

Father

Mother

4483delG

MACPF domain

PRF1Cr10

exon1

exon2 exon3

p.Gly477fs X479

4483delG

Rattus norvegicus

Mus musculus

Homo sapiens

New variant

LDFGDVLLATGGPLRLQVWDQDSGRDD

LDFGDVLLATGGPLRLQVWDQDSGRDD

LDFGDVLLATGGPLRLQVWDQDSGRDD

LDFGDVLLATGGPLX

479

481

LDFGDVLLATGGPLRLPVWDQDSGRDDQ481P variant

C2-Calcium-dependent lipid binding domain

24-months old girl, Prolonged symptomatic EBV infectious mononucleosis Pancytopenia

Fever ≥38.5ºC YesSplenomegaly YesCytopenia Anemia (Hgl: 7-8g/dl)

Platelets(36x109/L)Hypofibrinogenia and/or Hypertrigliceridemia HypertrigliceridemiaHaemophagocytosis in bone marrow NoLow or absent NK-cell activity Yes

Diagnostic criteria of HLH

Degranulation defects

10.2 11.6

24.753.6

PERFORIN

CD3

CON

TRO

L

Activacion

CD107a

CD56

PATI

ENT

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

c.2448-11G>A c.753+1G>T c.244811G>Ac.753+1G>T

Healthy P3P3

MOTHERP3

FATHER

+IL-2+ PHA

+IL-2- PHA

CD107a - APC

CD56

-PE

A

c.753+1G>T

c.2448-11G>A

Compound mutation

c.753+1G>T

carrier

c.2448-11G>A

carrier

B

DC

CD107a

Acknowledgements

Hospital Vall d’Hebron Servei d’Immunologia

Laura ViñasDr. Roger ColobranDr. Manuel HernandezDra. Ana Marín-SanchezDr. Ricardo Pujol

Hospital Sant Joan de DéuServei de Pediátria

Dra. Laia AlsinaDra. 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 MartínDra. Concepció Figueras

Servei d’Oncologia i Hematologia PediàtricaDr. Jose Luís Dapena