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Diagnóstico funcional y genético de las inmunodeficiencias primarias
 

Diagnóstico funcional y genético de las inmunodeficiencias primarias

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Seminario de la Dra. Mónica

Seminario de la Dra. Mónica

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  • El termino inmunodeficiencia engloba todo un conjunto de entidades patologicas causadas por alteraciones cuantitativas o cualitativas en un componente o mas del sistema inmunologico. Atendiendo a su causa pueden clasificarse en dos grandes grupos, primarias o secundarias.
  • Figure 1. Identification of CD1c (BDCA-1)+ myeloid dendritic cells in blood by flow cytometric analysis. Analysis of NK cells in a control and the absence in the patient. Molecular study of GATA-2 gene revealed a new mutation (c.1156-1157insAC) leading an early STOP codon (p.Leu386HisfsX2)
  • El resultado será una proteína truncada de 329 aminoácidos (la proteína nativa tiene 480 aminoácidos), El resultado será una proteína truncada de 329 aminoácidos (la proteína nativa tiene 480 aminoácidos),
  • Fourth child of a consanguineous family of Moroccan origin. Previously, two baby boys from the same family had died, the first one from unknown causes at 1 month old, the second one died shortly after birth from multiorgan failure and metabolic acidosis of unknown origin.
  • Guanine Direct sequencing
  • Guanine Direct sequencing

Diagnóstico funcional y genético de las inmunodeficiencias primarias Diagnóstico funcional y genético de las inmunodeficiencias primarias Presentation Transcript

  • Diagnostico funcional y genético de inmunodeficiencias primarias.Dra. Mónica Martínez-GalloServei d’ImmunologiaHospital Universitario Vall d’Hebron, Barcelona
  • • DefinitionImmunodeficiency is the result of a diverse group of abnormalities of theimmune system resulting primarily in an increased incidence of infection.• Primary ImmunodeficiencyCongenital and hereditary• Secondary ImmunodeficiencyAcquired on a transient or permanent basisImmunodeficiency
  • B cellsComplementPhagocytesT cellsPneumococciHaemophilusNeiseeriaMycoplasmaE. coliKlebsiellaSerratiaPseudomonasAspergillusHerpes spCytomegalovirusListeriaPneumocystisGiardiaCrytosporidiaCandidaStaphylococcusaureusPrimary 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 updateFrontiers 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 phosphorylasedeficiencyMutations in several distinct genes SCID
  • Lymphocyte phenotypeLymphocyte phenotypeT-B+T-B-IL2RG (50%)IL7RA (10%)JAK3 (10%)RAG1RAG2DCLRE1C (2%)ADA/PNP (8%)IgIg T+B- IgBTK (50%)Molecular studies of candidate genesMolecular studies of candidate genesClinical suspicionFunctional 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 boySplenomegalyRecurrent episodes of hemolytic anemia compatible withmacrophage activation syndrome-14 months- recurrent episodes of hemophagocytosis
  • Immunodeficiency with autoimmunity
  • In vitro lymphocyte proliferation assay020406080100120140SIN Anti-CD3IL2 +Anti-CD3 PWM PMA+IONOx1000c.p.m.CONTROL PATIENTPATIENTImmunodeficiency with autoimmunity
  • Mutations in RAG1Gen: FAS: NormalGen: RAG2: NormalGen: SH2D1A Normal
  • N CInteraction with RAG2HomeodomainRING+ZnBasicDomainsp.Trp522Cys p.Arg973CysN CInteraction with RAG2HomeodomainRING+ZnBasicDomainsp.Trp522Cys p.Arg973CysGen RAG1Gen RAG1Mutations in RAG1
  • Combined Immunodeficiency- Five years old-girl- Growth retardation and Sepsis- Multiple episodes of fever- Hypogammaglobinemia010203040506070P M A + IO N O P H A A nti-C D 3 +IL2 N o s tim ulo usx1000cpmcontrolpacient% Absolute number % Absolute numberT 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 0,20-0,90B 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 0,09-0,60IgG 275 mg/dl 700-1600 mg/dlIgA <10 mg/dl 40-70 mg/dlIgM 64 mg/dl 40-230 mg/dlREF VALPATIENT
  • c.984+1G>AExon 7N CJH7 JH1JH2JH3JH6 JH5 JH4FERM Domain SH2 Domain Kinase DomainPseudokinase Domainc.3208-1G>AExon 24c.984+1G>AExon 7N CJH7 JH1JH2JH3JH6 JH5 JH4FERM Domain SH2 Domain Kinase DomainPseudokinase Domainc.3208-1G>AExon 24Mutations in JAK3c.984+1G>ACompound mutationc.984+1G>Acarrierc.3208-1G>Acarrierc.3208-1G>A
  • Borte S et al. Blood 2012;119:2552-2555TRECS and KRECS
  • NTRIOS STATUSFAMILYMEMBERS OBSERVATIONSVH45 EXITUS AFFECTED NECROPSY SH2D1A XIAP PRF1VH46 ALIVE MOTHERVH47 ALIVE FATHERVH48 EXITUS AFFECTED NECROPSY FAS SH2D1AVH49 ALIVE MOTHERVH50 ALIVE FATHERVH51 ALIVE AFFECTED BTK NEMOVH52 ALIVE MOTHERVH53 ALIVE FATHERVH54 ALIVE AFFECTED BROTHER EXITUS JAK3 RAG1 RAG2 DCLRE1CVH55 ALIVE MOTHERVH56 ALIVE FATHERVH57 ALIVE AFFECTED AIRE RAG1 RAG2VH58 ALIVE MOTHERVH59 ALIVE FATHERVH60 ALIVE AFFECTED RAG1 RAG2VH61 ALIVE AFFECTED RAG1 RAG2VH62 ALIVE MOTHERVH63 ALIVE FATHERGENETIC 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 novoGATA-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 unitwith disseminated varicella-zoster virus (VZV)GATA-2 deficiencyPatientControl 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 numberNumbers of CD34+ 0,02 0,08Monocytes 0,00 0 x 109/L 0,50 0 x 109/L 2,5-16 0,2-1,6B 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 0,09-0,60T 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 0,20-0,90IgG *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/dlREF VALPATIENT 2PATIENT 1GATA-2 deficiency
  • •PatientA C T G T G C C C G A GHis386 STOPGATA-2 deficiency
  • A C T G T G C C C G A GHis386 STOPA C T G T G C C C G A GHis386 STOPMUTATION c.1156-1157insACGATA-2 deficiency
  • Gly327 Gln328 Asn329c.988C>T / p.Arg330XSTOP330Arg330 Pro331 wild type allelemutated alleleGATA-2 deficiencyMUTATION c.988C>T
  • c.1156-1157insACZinc-fingerdomain 2DNA double helixArg364AlaNuclear localizationsignal domainc.1156-1157insAC
  • Familial Haemophagocytic disordersFHLH is a rare autosomal recessive disorder of immune dysregulation associated withuncontrolled 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: FeverProgressive hepatosplenomegalySkin manifestations non-specificNeurological abnormalitiesOften triggered by infectionsViralEBV, CMV, PV•Laboratory findings: CytopeniasTrombocytopenia, aneamia, neutropeniaImpaired or absent NK and T cell cytotoxicity activityHyperferrinaemiaLiver disfunction Lactate dehydrogenase lactic acidSerum transaminasesBilirubinHypofibrinogen Intravascular coagulationElevated triglyceridesHigh serum levels of CD25s
  • Pathophysiology of Haemophagocytic disorders
  • Sospecha clínicaEnsayo citoxicidad NKFHLH1 10% Cr9q21-22FHLH2 20-50% PRF1FHLH3 30-40% UNC13DFHLH4 n=10 STX11FHLH5 10% STXBP2FHLH1 10% Cr9q21-22FHLH2 20-50% PRF1FHLH3 30-40% UNC13DFHLH4 n=10 STX11FHLH5 10% STXBP2Genetic basis of FHLH:Presencia de perforinaEnsayo degranulaciónUNC13DSTXBP2STX11PRF1NKCD8PERFORINCD3CD107aCD56ProblamenteNo FHLH
  • Sospecha clínicaEnsayo citotoxicidad NK0102030405060708090BASAL 3:1 6:1 12:1 25:1 50:1Ratio Efector: Diana%muertecelularcontrol sanoPACIENTE 1control patológicoGenetic basis of FHLH:
  • Sospecha clínicaEnsayo citoxicidad NKFHLH1 10% Cr9q21-22FHLH2 20-50% PRF1FHLH3 30-40% UNC13DFHLH4 n=10 STX11FHLH5 10% STXBP2FHLH1 10% Cr9q21-22FHLH2 20-50% PRF1FHLH3 30-40% UNC13DFHLH4 n=10 STX11FHLH5 10% STXBP2Genetic basis of FHLH:Presencia de perforinaEnsayo degranulaciónUNC13DSTXBP2STX11PRF1NKCD8PERFORINCD3CD107aCD56ProblamenteNo 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 sample5-weeks-old baby girlFebrile urinary tract infectionSplenomegalyProgressive liver failureCASE REPORTPerforin deficiency FHLH2
  • 0 102103104105<FITC-A>: PERFORINA0102103104105<APC-A>:CD342.4 0.220.1157.30 102103104105<FITC-A>: PERFORINA0102103104105<APC-A>:CD342.2 45.19.962.79HEALTHYDONORPERFORINCD30 102103104105<FITC-A>: PERFORINA0102103104105<APC-A>:CD334 7.6629.329.1Mut PRF1Pro459LeuISOTYPECONTROLMut PRF1p.Gly477fs X479100101102103104FITC-A100101102103104APC-A76.6 2.60.919.9NKCD8PACIENT 1 PACIENT 2Perforin deficiency
  • 5´… GCC ACA GGG GGG CCC CTG AGG …3´STOPCODONA T G G P L RWild typePatientPRF1 exon2 exon34483delG477 4795´… GCC ACA GGG GGC CCC TGA GG …3´A T G G P X
  • PatientFatherMother4483delG
  • MACPF domainPRF1Cr10exon1exon2 exon3p.Gly477fs X4794483delGRattus norvegicusMus musculusHomo sapiensNew variantLDFGDVLLATGGPLRLQVWDQDSGRDDLDFGDVLLATGGPLRLQVWDQDSGRDDLDFGDVLLATGGPLRLQVWDQDSGRDDLDFGDVLLATGGPLX479481LDFGDVLLATGGPLRLPVWDQDSGRDDQ481P variantC2-Calcium-dependent lipidbinding domain
  • 24-months old girl,Prolonged symptomatic EBV infectious mononucleosisPancytopeniaFever ≥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 YesDiagnostic criteria of HLHDegranulation defects
  • 10.2 11.624.753.6PERFORINCD3CONTROLActivacionCD107aCD56PATIENT
  • exó 9 intró9Mutation c.753+1G>TMutation c.753+1G>Twt allelec.2448-11 G>A allelecDNA UNC13DgDNA UNC13DMutation c.2448-11G>AMutation c.2448-11G>ADegranulation defects: Splice donor defects
  • c.2448-11G>A c.753+1G>T c.244811G>Ac.753+1G>TCD107a -APCc.753+1G>Tc.2448-11G>ACompound mutationc.753+1G>Tcarrierc.2448-11G>AcarrierDCCD107a
  • AcknowledgementsHospital Vall d’HebronServei d’ImmunologiaLaura ViñasDr. Roger ColobranDr. Manuel HernandezDra. Ana Marín-SanchezDr. Ricardo PujolHospital Sant Joan de DéuServei de PediátriaDra. Laia AlsinaDra. M. OlayaDra. MA. Martín MateosVall d’Hebron Institut de Recerca (VHIR)Translational Bioinformatics in Neuroscience.Dr. Francisco Javier de la Cruz.Institució Catalana per a la Recercai Estudis Avançats (ICREA)Dr. Sergio LoisUnitat de Malalties Infeccioses i Immunodeficiències PediátriquesDr. Pere SolerDra. Andrea MartínDra. Concepció FiguerasServei d’Oncologia i Hematologia PediàtricaDr. Jose Luís Dapena