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

udgaemh@imppc.org

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: udgaemh@imppc.org

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/index.htmludgaemh@imppc.org

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 msanchez@imppc.org

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. udgaemh@imppc.org

Advanced Genetic Diagnostic Unit for Rare Iron Metabolism Disorders

http://www.imppc.org/resources-and-services/index.html

udgaemh@imppc.org

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!