diagnóstico genético en enfermedades raras del metabolismo del hierro: anemias raras y...
DESCRIPTION
Seminario dirigido por la Dra. Mayka Sánchez, Principal Investigator. Iron and Cancer Group Head of the Advanced Genetic Diagnostic Unit of Rare Iron Disorders Institute of Predictive and Personalized Medicine of Cancer (IMPPC). Consulta el vídeo de la presentación después de la última diapositiva.TRANSCRIPT
26/2/2013 Hospital Vall de Hebrón
Diagnóstico genético en enfermedades raras del
metabolismo del hierro: Anemias raras y Hemocromatosis
CLINICAL GENETIC DIAGNOSTICS:
Advanced Genetic Diagnostic Unit for Rare Iron Metabolism Disorders
RESEARCH:
Iron & Cancer
Mayka Sanchez
Postdocs Ricky Joshi, PhDMaya
Shvarstman, PhD Technicians Erica Morán, PhD
Jessica ArandaPhD student Sara LuscietiBioinformatician Francisco Fuster
Iron is essential for life
e-
Fe3+Fe2+
e-
Fe-S cluster(e.g. proteins of mitochondriale- transport)
Heme proteins(e.g. hemoglobin,cytochromes)
Di-iron proteins(e.g. ribonucleotidereductase)
H2O2
Anti-oxidantdefenses
Oxidative damage
Iron: a vital biocatalyst that can be toxic
Fenton reaction
OH.
Systemic Iron homeostasis
Heme Hemoglobin
Hepcidin
Ferroportin
The importance of Iron balance
Too much… …too little
Deficiency
Anemia
Overload
Hereditary Hemochromatosis Hepatic Cancer
IRON
DISEASE
Unidad de Diagnóstico Genético Avanzado de Enfermedades del Metabolismo del Hierrro (UDGAEMH)
http://www.imppc.org/resources-and-services/index.html
Jefe: • Dra. Mayka
SánchezTécnicos:• Dr. Erica Morán• Jessica ArandaGIFMedical Doctors network
Acreditación de Calidad:EMQN EQA en HH-HFEDesignada como unidad EXPERTA por ORPHANETAcreditación asistencial Generalitat Catalunya
Servicios
- Hepcidina sérica/plasmática (ELISA)
- Diagnóstico Genético de
enfermedades raras del
metabolismo del hierro
CATEGORIES GENE DISEASE PRICE (euros) OMIM
IRON OVERLOAD
Classical HemochromatosisNon-HFE Hemochromatosis
HFE HH1 120 235200
HAMP HH2 (juvenile form) 100 613313
HFE2 HH2 (juvenile form) 100 602390
TFR2 HH3 200 604250SLC40A1 HH4 160 606069
HYPERFERRITINEMIA Hyperferritinemia
FTL Hereditary hyperferretinemia with cataracts 50 600886FTL Benign Hyperferretinemia 100 600886FTH Hyperferretinemia with iron overload 100 134770
RARE IRON-RELATED
ANEMIAS
Sideroblastic
ALAS2 XLSA 150 300751STEAP3 Sideroblastic anemia associated to STEAP3 150 609671ABCB7 XLSA with ataxia 100 301310
GLRX5 SA with hepatic iron overload 100 205950
SLC25A38 Non syndromic autosomal recessive CSA 100 205950
Non-sideroblastic
CP Aceruloplasminemia 230 604290TF Hypotransferrinemia 250 209300
SLC11A2 Familiar microcytic hypochromic anemia with hepatic iron overload 200 206100
TMPRSS6 IRIDA, Iron-refractory iron deficient anemia 200 206200
Congenital dyserythropoietic anemia
CDAN1 CDA type I 250 224120
SEC23B CDA type II 250 224100
KLF1 Unclassified CDA 150 NA
Advanced Genetic Diagnostic Unit for Rare Iron Metabolism Disorders
Precios exentos de IVA si viene de un HOSPITAL
Procedimiento de petición de análisis
http://www.imppc.org/resources-and-services/genetic-diagnostics-metabolism-disorders/es_procedudgaemh.html
http://www.imppc.org/resources-and-services/index.html
Consentimiento informado o CONVENIO firmado es OBLIGATORIO para hacer el estudio. Muestra: 3 tubos de EDTA (15 ml sangre) + 1 tubo suero sin anticoagulantesEnvío de lunes a miércoles de 9.00 a 17.00h Contactar por email: [email protected]
Advanced Genetic Diagnostic Unit for Rare Iron Metabolism Disorders
- Puesta a punto de medida de la HEPCIDINA
SERICA/PLASMATICA por ELISA
- La hepcidina es la hormona que controla el metabolismo del
hierro, mediante el bloqueo de la absorción de hierro por el
intestino y el bloqueo de la liebración de hierro de los
macrófagos. La hepcidina ejerce su función sobre la
ferroportina
- Niveles muy bajos de hepcidina se dan en
HEMOCROMATOSIS HEREDITARIAS
- Niveles altos/normales de hepcidina se dan en IRIDA, lo
que la diferencia de otras anemias como una anemia
ferropenica nutricional, anemia debida a enfermedad celíaca,
una anemia debida a talasemias, deficiencia de DMT1,
atransferrinemia (estas otras anemias presentan niveles
bajos de hepcidina).
Hepcidin
Ferroportin
Inhibition of :Duodenal iron absorption and
macrophage iron release
Patient’s recruitment (2010-2013)
- 13 Hyperferritinemias with cataracts => Seq. IRE FTL: 7 known Mutations, 2 new. - Poster número PO-382. Erica Morán et al. Congreso de Nacional Hematología 2010. - Chapter of book 2010. The IRP/IRE regulatory network. ISBN 979-953-307-162-5, InTech- 2 NEW MUTATIONS Publication in OJRD Luscieti et al., 2013. IF: 5.074 (2011 JCR).
- 2 hyperferretinemias without cataracts => Seq. FTL / FTH, No mutations.- 26 Hemochromatosis => Seq. genes HFE, TFR2, HAMP, HJV, SLC40A1. - 1 case C282Y HFE. - 4 cases TFR2, molecular studies ongoing, 3 NEW MUTATIONS. Publication in preparation- 3 cases SLC40A1 (FPN), 2 NEW MUTATIONS
- 10 Sideroblástic Anemias (CSA) => Seq. genes ALAS2, SLC25A38, ABCB7, GLRX5, STEAP3. -2 ALAS2 (known mutation R452C, P520L+possible new splicing mutation).
- 2 SLC25A38 NEW MUTATIONS. Publication Kannengiesser C*, Sanchez M*, Sweeney M* et al., . Haematologica. 2011 ).
- 19 non-sideroblastic Anemias => Seq. genes TF, CP, TMPRSS6, DMT1.- 4 families TF (5 NEW MUTATIONS). Publication submitted BJH, Publication in preparation - 4 families IRIDA (6 patients) 4 NEW MUTATIONS. Publication in preparation - IRIDA Review Publication Haematologica De Falco L*, Sánchez M*, et al. 2013- 1 new entity case NGS5 ongoing.
- 15 CDAI/II => Seq. genes SEC23B, CDAN1, KLF1 - SEC23B: 4 NEW MUTATIONS (2 missense, 1 splicing, 1 insertion), 2 known mutations - CDAN1: 1 NEW missense MUTATION
Total Cases 85, 101 affected patients, 155 studied persons (affected and non-affected)
Diagnóstico genético en nuevos casos de IRIDA
(iron-refractory iron deficiency Anemia)
Gene TMPRSS6, Protein MATRIPTASE-2
• Mask mouse. ENU mutagenesis mice. Inappropriate high hepcidin levels. Positional cloning: TMPRSS6 gene (membrane-bound serine protease), splicing error. (Dr. Beutler´s lab. Science 2008).
• TMPRSS6 KO mouse. Dr. Lopez-Otin´s lab. Matriptase-2 is an essential regulator of iron homeostasis. Severe iron deficiency anemia.
(Folgueras et al., Blood 2008) • Human patients => IRIDA mutations in TMPRSS6 (Finberg KE et al. Nat
Genet. 2008; 40: 569-571)
(Du et al., Science, 2008)
• Anemia microcítica e hipocroma sin respuesta a hierro oral y
respuesta parcial al hierro intravenoso
• Infancia / adolescencia
• Autosómico recesivo con baja prevalencia <1 / 1,000,000
(Orphanet)
• Primer caso descrito en humanos en el 2008 (Finberg et al. 2008)
• 44 pacientes reportados en 29 familias (14 publicaciones)
• Mutaciones gen TMPRSS6, proteina Matriptase-2, serin proteasa
membrane-bound. Regulador negativo de la hepcidina
• Niveles de hepcidina inapropiadamente normales o altos
(ELISA en la UDGAEMH)
IRIDA (OMIM #206200)
IRIDA Kindreds. Clinical and Biochemical data
Kindred Patient number Ancestry Consan-
guinity Sex Age RCB(mm3 x106)
Hb (gr/dL)
MCV(fl)
Transferrin sat.(%)
Serum ferritin (ng/ml)
Serum iron
(ug/dL)
AIV-1 Spain Yes M 14mo 4,38 6,8 50 5 170 13
IV-2 (+) Spain Yes M 12mo 4,88 8,7 56,8 5 90 10
BII-1 (+) Venezuela No M 7 years 4,79 10,5 67,7 12 132 36
II-2 (+¤) Venezuela No F 1 year 5,29 10,3 61 6 367 26
C II-1 (+¤) Colombia No M 4 years 4,34 9,7 69 6 30 15
D II-1 (+¤) Spain No M 5 years 4,95 9,7 74 4 40 13
Clinical data reported at diagnosis:+ Oral iron treatment¤ Intravenous treatment
We report 4 IRIDA families (6 patients) with 4 NEW mutations in TMPRSS6
LOW LOWLOW LOW
Matriptase2 wt (811aa)
Truncated Matriptase2 (752aa)
Lys
Stop
752
752 59
IRIDA Kindred A . New Nonsense mutation
NM_153609.2:c.[2252_2253insT];[2252_2253insT]NP_705837.1:p.(Lys752*);(Lys752*)
IRIDA Kindred B and C . New Missense mutation
CONDEL: This variation is predicted to be a deleterious change (score =0.976 ).
NM_153609.2:c.[861C>A];[861C>A]NP_705837.1:p.(Thr287Asn);(Thr287Asn)
NM_153609.2:c.[76_81delGGTGA];[=] NP_705837.1:p.(Gly26Trpfs*14);(=)
NM_153609.2:c.[1817T>C];[=] NP_705837.1:p.(Leu606Arg);(=)
IRIDA Kindred D. New Missense + frameshift mutation
Leu606Arg
CONDEL: This variation is predicted to be a deleterious change (score =0.902).
New mutations in IRIDA patients
kindred Patient Mutation 1
(NM_153609.2;NP_705837.1)
Mutation 2(NM_153609.2;NP_705837.1) Domains
A
IV-1 c.2252_2253insT; p.Lys752*
c.2252_2253insT; p.Lys752* Protease/Protease
IV-2 c.2252_2253insT; p.Lys752*
c.2252_2253insT; p.Lys752* Protease/Protease
B
II-1 c.861C>A;p.Thr287Asn
c.861C>A;p.Thr287Asn CUB I/CUB I
II-2 c.861C>A;p.Thr287Asn
c.861C>A;p.Thr287Asn CUB I/CUB I
C II-1 c.861C>A;p.Thr287Asn
c.861C>A;p.Thr287Asn CUB I/CUB I
D II-1 c.76_81delGGTGA;p.Gly26Trpfs*14
c.1817 T>C]; p. Leu606Arg Transmembrane/Protease
Conclusiones
• Es altamente sugestivo que las variaciones encontradas en estas familias sean causantes de su clínica
• Nuestros resultados extienden el patrón de mutaciones en el gen TMPRSS6 asociadas a IRIDA, confirmando la importancia de los análisis genéticos para el diagnóstico de la enfermedad
• Es importante conocer los niveles de hepcidina (UDGAEMH)
Hereditary Hemochromatosis type IIIFunctional characterization of
Transferrin Receptor 2 Gly792Arg mutation
• Iron-overload genetic disease. Increased dietary iron absorption. • Hepatic iron accumulation. Hepatic cancer.• High sFerritin (>1000 ng/ml) + High Transferrin saturation (>80%)• Cirrosis, Hepatic cancer, Cardiomyopathy, Diabetes, Arthritis, Impotence,
Hyperpigmentation, Hypogonadotropic hypogonadism
HFE HepcidinHemojuvelinTFR2
adult juvenil
(Chr 6p21.3) (Chr19q13.1)(Chr1p21)(Chr7q22)
Ferroportin
(Chr2q32)
Onset adult
Heredity Autosomal recessive Autosomal dominant
Type HH 1 HH 3 HJ 2b HJ 2a HH 4 Classic
Rare genetic diseaseCommon
Hereditary Hemochromatosis
Type III Hereditary Hemochromatosis – TFR2
• Type III HH was first described in two Sicilian families by Clara Camaschella and collaborators in 2000 (Nat. Genetics)
• TFR2 is a homologue of TFR1, type II transmembrene membrane protein expressed mostly in the liver and CD71+ early erythroids.
• At least 50 families and 69 patients have been described with mutations in TFR2.
Case 23 and Case 7. Atypical Hemochromatosis
Proband Case 23:- Woman. 57 years old- Diagnosed at 31 years old
Biochemical and Clinical Symptoms:- Ferritin 1700-2200 ng/mL (normal value 200)- Transferrin saturation high- Liver cirrhosis- Massive deposits of iron (HII: 3,6)- Hepatomegaly of 10 cm- Arthralgia in hands and knees. - AmenorrheaTreatment:>82 phlebotomies (16.3 g of iron removed) Relatives: - Sister with DMI and hypersideremia- Grandmother dead because of hepatopatyGenetics:- HFE : C282Y -/-; H63D -/- - No mutations in HJV and HAMP
Proband Case 7:- Woman. 27 years old- Diagnosed at 23 years old
Biochemical and Clinical Symptoms:- Ferritin 3944 ng/mL (Normal value 200)- Transferrin saturation 96% (normal value <45%)- Serum iron: 236 ug/dL- Fibrosis
Treatment:- 70 phlebotomies (14 g of iron removed) - EPO
Relatives: Father with hepatopathy, CH, VHC
Genetics:- HFE :C282Y -/-; H63D +/+ - No mutations in HJV and HAMP
TFR2 Hemochromatosis. Gly792Arg mutation
MutatedG792R
Homozygous
Control
G792R+/+
G792R+/+
CASE 23 CASE 7
GA
MutatedG792R
Heterozygous
Control
G792R+/-
TFR2 Gly792Arg mutation
VARIANT FOUND IN TRF2 GENE rs 80338891: NM_003277: c.[2374 G>A]+[2374 G>A]; NP_003218.2: p.Gly792Arg;
G792R mutation was described in heterozygosity in a patient that carriers 2 additional mutations (Lee and Barton, 2006)
“The functional effect of TFR2 G792R is unknown, and in thecontext of the present patient, it is irrelevant” (because this patient carriers two other mutations R445Q and R396X in compound heterozygosity)
First time G792R mutation is described in homozygosity (Case 23).
What is the functional consequences of the G792R mutation?
Plasmid Hs. TFR2 cDNA
N-FLAG Cloning and Site directed mutagenesis
Protein localization studies
by immunofluorescencein HUH7 cells
Functional implication of TFR2 G792R mutation
TFR2-C FLAGN FLAG-TFR2
DAPI ONLY
Anti-flag ONLY
Merged
Anti-E-Cad ONLY
Permeablized CellsC-Flag WILDTYPE TFR2 C-FLAG TFR2mut-G792R
HUH7
Permeablized Cells
DAPI ONLY
Anti-flag ONLY
Merged
Anti-E-Cad ONLY
HUH7
N-Flag WILDTYPE TFR2 N-FLAG TFR2mut-G792R
DAPI ONLY
Anti-flag ONLY
Merged
Anti-E-Cad ONLY
NON-Permeablized Cells
HUH7
C-Flag WILDTYPE TFR2 C-FLAG TFR2mut-G792R
rs 80338891: NM_003277: c.[2374 G>A];[2374 G>A]; NP_003218.2: p.Gly792Arg
Bioinformatic studies indicates:1. Mutation is predicted as DELETERIOUS
(SIFT) /PROBABLY DAMAGING (Polyphen)2. G792 is 100% conserved between species3. Glycine-Arginine change => large volume and
hydrophobicity change => Destabilizing effect of G792R mutation on the
structure/function of TFR2 monomer
Bioinformatic studies of TFR2 G792R mutation
Structural analysis: TFR2 modelled based on TFR1 structure (PDB: 1DE4).1. G792R mutation is in a semi-buried location (relative
accessibility 37.6%), at the inter-phase between TFR2 monomers.
=> This is consistent with a dimer-disruptive impact of G792R.
Collaboration: Dr Xavier de la Cruz and Dr Sergi Lois.
Conclusions
• Genetic analysis of TFR2 revealed a previously described but uncharacterized change Gly792Arg in 2 Spanish families. A putative splicing variation is also present in the proband of Case 7 in heterozygous state.
• First time this variation has been described in homozygosity (Case 23).
• Co-IF analysis revealed that the TFR2 Gly792Arg mutated protein is unable to reach the cell membrane where its function in iron metabolism takes place.
• Bioinformatic and computational analysis on TFR2 Gly792Arg mutation suggest that the mutation may destabilize both the monomer and the dimmer of TFR2, hence affecting its functionality.
Proyecto hiperferritinemia
Research Projects and ContractsRamón y Cajal Research ContractTechnician Contrat (Carlos III)FIS (Health Ministery) – 2009-2012European Project E-rare –2009-2012Research Project: Ayuda a la Investigación Ramón Areces 2012-2015. Proyectos de Investigación Fundamental no Orientada - SAF 2012. Postdoctoral Fellowship FEBS 2012-2015
Agradecimientos• Iron and cancer group. Mayka Sanchez’s lab (IMPPC)
• Dr. Ricky Joshi• Francisco Fuster
• Advanced Genetic Diagnostic Unit for Rare Iron Metabolism Disorders Mayka Sanchez’s lab (IMPPC)
• Dr. Erica Morán• Jessica Aranda
• Dra. Bienvenida Argiles, Hospital de la Fe (Valencia, Spain)• Dra. Cristina Sanz, Hospital Clínic de Barcelona• Dr. Xavier de la Cruz and Dr. Sergio Lois (Vall de Hebrón, Spain)• Grupo Ibérico de Ferropatología (GIF)
Unidad de Diagnóstico Genético Avanzado de Enfermedades del Metabolismo del Hierrro (UDGAEMH)
http://www.imppc.org/resources-and-services/[email protected]
Jefe: • Dra. Mayka
SánchezTécnicos:• Dr. Erica Morán• Jessica ArandaGIFMedical Doctors network
Acreditación de Calidad:EMQN EQA en HH-HFEDesignada como unidad EXPERTA por ORPHANETAcreditación asistencial Generalitat Catalunya
Servicios
- Hepcidina sérica/plasmática (ELISA)
- Diagnóstico Genético enfermedades raras del metabolismo del hierro
Gracias por vuestra atención!
CLINICAL GENETIC DIAGNOSTICS:Advanced Genetic Diagnostic Unit for Rare Iron Metabolism Disorders
RESEARCH:Iron & Cancer
Mayka Sanchez
Postdocs Ricky Joshi, PhD Maya Shvarstman,
PhD Technicians Erica Morán, PhD
Jessica ArandaPhD student Sara LuscietiBioinformatician Francisco Fuster
Gracias por vuestra atención!
Propuesta- Estancias en la UDGAEMH
Estamos interesados en formar a médicos especializados (especialmente hematólogos) en técnicas de Genética molecular aplicadas a enfermedades raras del metabolismo del hierro.
Oportunidad de colaborar y realizar estudios genéticos y funcionales en pacientes de nuestra unidad.
Objetivo: aprender a trabajar en un laboratorio de diagnóstico genético, hacer investigación y publicar los datos en revistas internacionales.
Financiación: nula ! (Así está la cosa... de mal…)
Posibilidad de pedir contrato Rio Hortega y otros contratos dependiendo del Cv del candidato.
Interesados contactar con:Dra. Mayka Sanchez [email protected]
Para más detalles sobre nosotros ver:http://www.imppc.org/research-activities/cancer-and-iron/http://www.imppc.org/resources-and-services/genetic-diagnostics-metabolism-disorders/
CATEGORIES GENE DISEASE PRICE (euros) OMIM
IRON OVERLOAD
Classical HemochromatosisNon-HFE Hemochromatosis
HFE HH1 120 235200
HAMP HH2 (juvenile form) 100 613313
HFE2 HH2 (juvenile form) 100 602390TFR2 HH3 200 604250
SLC40A1 HH4 160 606069
HYPERFERRITINEMIA Hyperferritinemia
FTL Hereditary hyperferretinemia with cataracts 50 600886
FTL Benign Hyperferretinemia 100 600886FTH Hyperferretinemia with iron overload 100 134770
RARE IRON-RELATED
ANEMIAS
Sideroblastic
ALAS2 XLSA 150 300751
STEAP3 Sideroblastic anemia associated to STEAP3 150 609671
ABCB7 XLSA with ataxia 100 301310GLRX5 SA with hepatic iron overload 100 205950
SLC25A38 Non syndromic autosomal recessive CSA 100 205950
Non-sideroblastic
CP Aceruloplasminemia 230 604290TF Hypotransferrinemia 250 209300
SLC11A2 Familiar microcytic hypochromic anemia with hepatic iron overload 200 206100
TMPRSS6 IRIDA, Iron-refractory iron deficient anemia 200 206200
Congenital dyserythropoietic anemia
CDAN1 CDA type I 250 224120SEC23B CDA type II 250 224100
KLF1 Unclassified CDA 150 NA
Advanced Genetic Diagnostic Unit for Rare Iron Metabolism Disorders
http://www.imppc.org/resources-and-services/index.htmlDra. Mayka Sánchez. [email protected]
Advanced Genetic Diagnostic Unit for Rare Iron Metabolism Disorders
http://www.imppc.org/resources-and-services/index.html
Head: • Dr. Mayka SánchezTechnicians:• Dr. Erica Morán• Jessica ArandaGIFMedical Doctors network
Acreditación de Calidad:EMQN EQA en HH-HFEDesignada como unidad EXPERTA por ORPHANETAcreditación asistencial Generalitat Catalunya
Gracias por vuestra atención!