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.

1. Diagnóstico genético en
enfermedades raras del
metabolismo del hierro: Anemias
raras y Hemocromatosis
26/2/2013 Hospital Vall de Hebrón

2. Mayka Sanchez
CLINICAL GENETIC DIAGNOSTICS:
Advanced Genetic Diagnostic Unit for Rare
Iron Metabolism Disorders
RESEARCH:
Iron & Cancer
Postdocs Ricky Joshi, PhD
Maya Shvarstman, PhD
Technicians Erica Morán, PhD
Jessica Aranda
PhD student Sara Luscieti
Bioinformatician Francisco Fuster

3. Iron is essential for life

4. Iron: a vital biocatalyst that can be toxic
Fenton reaction
Heme proteins
(e.g. hemoglobin,
cytochromes)
e- H 2O 2
Anti-oxidant
defenses
Fe-S cluster
(e.g. proteins of Fe2+ Fe3+ .
mitochondrial
e- transport) OH
Oxidative damage
Di-iron proteins
(e.g. ribonucleotide
reductase)
e-

5. Systemic Iron homeostasis
Heme Hemoglobin
Hepcidin
Ferroportin

6. The importance of Iron balance
Too much… …too little
IRON
DISEASE
Overload Deficiency
Hereditary Hemochromatosis Anemia
Hepatic Cancer

7. Unidad de Diagnóstico Genético Avanzado de Enfermedades
del Metabolismo del Hierrro (UDGAEMH)
Jefe:
Servicios
• Dra. Mayka Sánchez - Hepcidina sérica/plasmática (ELISA)
Técnicos:
• Dr. Erica Morán - Diagnóstico Genético de
• Jessica Aranda
GIF enfermedades raras del metabolismo
Medical Doctors del hierro
network
http://www.imppc.org/resources-and-services/index.html
udgaemh@imppc.org
Acreditación de Calidad:
EMQN EQA en HH-HFE
Designada como unidad EXPERTA por ORPHANET
Acreditación asistencial Generalitat Catalunya

8. Advanced Genetic Diagnostic Unit for Rare Iron Metabolism Disorders
PRICE
CATEGORIES GENE DISEASE OMIM
(euros)
HFE HH1 120 235200
IRON Classical Hemochromatosis HAMP HH2 (juvenile form) 100 613313
OVERLOAD Non-HFE Hemochromatosis
HFE2 HH2 (juvenile form) 100 602390
TFR2 HH3 200 604250
SLC40A1 HH4 160 606069
FTL Hereditary hyperferretinemia with cataracts 50 600886
HYPERFERRITI
Hyperferritinemia FTL Benign Hyperferretinemia 100 600886
NEMIA
FTH Hyperferretinemia with iron overload 100 134770
ALAS2 XLSA 150 300751
STEAP3 Sideroblastic anemia associated to STEAP3 150 609671
ABCB7 XLSA with ataxia 100 301310
Sideroblastic
GLRX5 SA with hepatic iron overload 100 205950
SLC25A38 Non syndromic autosomal recessive CSA 100 205950
RARE IRON- CP Aceruloplasminemia 230 604290
RELATED TF Hypotransferrinemia 250 209300
ANEMIAS Non-sideroblastic Familiar microcytic hypochromic anemia with
SLC11A2 hepatic iron overload
200 206100
TMPRSS6 IRIDA, Iron-refractory iron deficient anemia 200 206200
CDAN1 CDA type I 250 224120
Congenital dyserythropoietic
SEC23B CDA type II 250 224100
anemia
KLF1 Unclassified CDA 150 NA
Precios exentos de IVA si viene de un HOSPITAL

9. Procedimiento de petición de análisis
Consentimiento informado o CONVENIO firmado es OBLIGATORIO para hacer el
estudio.
Muestra: 3 tubos de EDTA (15 ml sangre) + 1 tubo suero sin anticoagulantes
Envío de lunes a miércoles de 9.00 a 17.00h
Contactar por email: udgaemh@imppc.org
http://www.imppc.org/resources-and-services/genetic-diagnostics-metabolism-disorders/es_procedudgaemh.html
http://www.imppc.org/resources-and-services/index.html

10. 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
Hepcidin
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
Ferroportin
-Niveles muy bajos de hepcidina se dan en
HEMOCROMATOSIS HEREDITARIAS
Inhibition of :
-Niveles altos/normales de hepcidina se dan en IRIDA, lo que
Duodenal iron
la diferencia de otras anemias como una anemia ferropenica absorption and
nutricional, anemia debida a enfermedad celíaca, una anemia macrophage iron
debida a talasemias, deficiencia de DMT1, atransferrinemia release
(estas otras anemias presentan niveles bajos de hepcidina).

11. 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)

12. Diagnóstico genético en nuevos
casos de IRIDA
(iron-refractory iron deficiency Anemia)

13. 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. (Du et al., Science, 2008)
(Folgueras et al., Blood 2008)
•Human patients => IRIDA mutations in TMPRSS6 (Finberg KE et al. Nat
Genet. 2008; 40: 569-571)

14. IRIDA (OMIM #206200)
• 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)

15. IRIDA Kindreds. Clinical and Biochemical data
We report 4 IRIDA families (6 patients) with 4 NEW mutations in TMPRSS6
Transferrin
Serum Serum
Patient Consan- RCB Hb MCV sat.
Kindred Ancestry Sex Age 3 6 ferritin iron
number guinity (mm x10 ) (gr/dL) (fl) (%)
(ng/ml) (ug/dL)
IV-1 Spain Yes M 14mo 4,38 6,8 50 5 170 13
A
IV-2 (+) Spain Yes M 12mo 4,88 8,7 56,8 5 90 10
II-1 (+) Venezuela No M 7 years 4,79 10,5 67,7 12 132 36
B
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
LOW LOW LOW LOW
Clinical data reported at diagnosis:
+ Oral iron treatment
¤ Intravenous treatment

16. IRIDA Kindred A . New Nonsense mutation
Lys
Stop
NM_153609.2:
c.[2252_2253insT];[2252_2253insT] Matriptase2 wt (811aa) 752 59
NP_705837.1:p.(Lys752*);(Lys752*) Truncated Matriptase2 (752aa) 752

17. IRIDA Kindred B and C . New Missense mutation
NM_153609.2:c.[861C>A];[861C>A]
NP_705837.1:p.(Thr287Asn);(Thr287Asn)
CONDEL: This variation is predicted to be a deleterious change (score =0.976 ).

18. IRIDA Kindred D. New Missense + frameshift mutation
NM_153609.2:c.[76_81delGGTGA];[=] NM_153609.2:c.[1817T>C];[=]
NP_705837.1:p.(Gly26Trpfs*14);(=) NP_705837.1:p.(Leu606Arg);(=)
Leu606Arg
CONDEL: This variation is predicted to be a deleterious change (score =0.902).

19. New mutations in IRIDA patients
Mutation 1 Mutation 2
(NM_153609.2; (NM_153609.2;
kindred Patient NP_705837.1) NP_705837.1)
Domains
c.2252_2253insT; c.2252_2253insT;
IV-1 Protease/Protease
p.Lys752* p.Lys752*
A
c.2252_2253insT; c.2252_2253insT;
IV-2 Protease/Protease
p.Lys752* p.Lys752*
c.861C>A; c.861C>A;
II-1 CUB I/CUB I
p.Thr287Asn p.Thr287Asn
B
c.861C>A; c.861C>A;
II-2 CUB I/CUB I
p.Thr287Asn p.Thr287Asn
c.861C>A; c.861C>A;
C II-1 CUB I/CUB I
p.Thr287Asn p.Thr287Asn
c.76_81delGGTGA; c.1817 T>C];
D II-1 Transmembrane/Protease
p.Gly26Trpfs*14 p. Leu606Arg

20. 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)

21. Hereditary Hemochromatosis type III
Functional characterization of
Transferrin Receptor 2 Gly792Arg mutation

22. Hereditary Hemochromatosis
•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
Common Rare genetic disease
Type HH 1 HH 3 HJ 2b HJ 2a HH 4
Classic
HFE TFR2 Hemojuvelin Hepcidin Ferroportin
(Chr 6p21.3) (Chr7q22) (Chr1p21) (Chr19q13.1) (Chr2q32)
Onset adult juvenil adult
Heredity Autosomal recessive Autosomal
dominant

23. 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.

24. Case 23 and Case 7. Atypical Hemochromatosis
Proband Case 23: Proband Case 7:
- Woman. 57 years old - Woman. 27 years old
- Diagnosed at 31 years old - Diagnosed at 23 years old
Biochemical and Clinical Symptoms: Biochemical and Clinical Symptoms:
-Ferritin 1700-2200 ng/mL (normal value 200) -Ferritin 3944 ng/mL (Normal value 200)
-Transferrin saturation high -Transferrin saturation 96% (normal value <45%)
- Liver cirrhosis -Serum iron: 236 ug/dL
- Massive deposits of iron (HII: 3,6) -Fibrosis
- Hepatomegaly of 10 cm
- Arthralgia in hands and knees. Treatment:
- Amenorrhea -70 phlebotomies (14 g of iron removed)
Treatment: -EPO
>82 phlebotomies (16.3 g of iron removed)
Relatives: Relatives: Father with hepatopathy, CH, VHC
- Sister with DMI and hypersideremia
- Grandmother dead because of hepatopaty Genetics:
Genetics: -HFE :C282Y -/-; H63D +/+
- HFE : C282Y -/-; H63D -/- - No mutations in HJV and HAMP
- No mutations in HJV and HAMP

25. TFR2 Hemochromatosis. Gly792Arg mutation
CASE 23 CASE 7
G792R
G792R G792R +/-
+/+ +/+
Control Control
Mutated Mutated G
G792R G792R A
Homozygous Heterozygous

26. 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 the
context 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?

27. Functional implication of TFR2 G792R mutation
N-FLAG Cloning and
Site directed mutagenesis
Plasmid
Hs. TFR2 cDNA Protein localization
studies
by immunofluorescence
in HUH7 cells
TFR2-C FLAG
N FLAG-TFR2

28. Permeablized Cells
C-Flag WILDTYPE TFR2 C-FLAG TFR2mut-G792R
Anti-flag ONLY
Anti-E-Cad ONLY
DAPI ONLY
Merged
HUH7

29. Permeablized Cells
N-Flag WILDTYPE TFR2 N-FLAG TFR2mut-G792R
Anti-flag ONLY
Anti-E-Cad ONLY
DAPI ONLY
Merged
HUH7

30. NON-Permeablized Cells
C-Flag WILDTYPE TFR2 C-FLAG TFR2mut-G792R
Anti-flag ONLY
Anti-E-Cad ONLY
DAPI ONLY
Merged
HUH7

31. Bioinformatic studies of TFR2 G792R mutation
rs 80338891: NM_003277: c.[2374 G>A];[2374 G>A]; NP_003218.2: p.Gly792Arg
Structural analysis: TFR2 modelled based on TFR1
Bioinformatic studies indicates:
structure (PDB: 1DE4).
1. Mutation is predicted as DELETERIOUS (SIFT)
1. G792R mutation is in a semi-buried location (relative
/PROBABLY DAMAGING (Polyphen)
accessibility 37.6%), at the inter-phase between
2. G792 is 100% conserved between species
TFR2 monomers.
3. Glycine-Arginine change => large volume and
=> This is consistent with a dimer-disruptive impact of
hydrophobicity change
G792R.
=> Destabilizing effect of G792R mutation on the
structure/function of TFR2 monomer
Collaboration: Dr Xavier de la Cruz and Dr Sergi Lois.

32. 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.

33. Proyecto hiperferritinemia

34. 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)
Research Projects and Contracts
Ramón y Cajal Research Contract
Technician Contrat (Carlos III)
FIS (Health Ministery) – 2009-2012
European Project E-rare –2009-2012
Research Project: Ayuda a la Investigación Ramón Areces 2012-2015.
Proyectos de Investigación Fundamental no Orientada - SAF 2012.
Postdoctoral Fellowship FEBS 2012-2015

35. Gracias por vuestra atención!
Jefe:
Servicios
• Dra. Mayka Sánchez
Técnicos: - Hepcidina sérica/plasmática (ELISA)
• Dr. Erica Morán
• Jessica Aranda - Diagnóstico Genético enfermedades
GIF raras del metabolismo del hierro
Medical Doctors
network
Unidad de Diagnóstico Genético Avanzado de Enfermedades
del Metabolismo del Hierrro (UDGAEMH)
http://www.imppc.org/resources-and-services/index.html
udgaemh@imppc.org
Acreditación de Calidad:
EMQN EQA en HH-HFE
Designada como unidad EXPERTA por ORPHANET
Acreditación asistencial Generalitat Catalunya

37. Gracias por vuestra atención!
Mayka Sanchez
CLINICAL GENETIC DIAGNOSTICS:
Advanced Genetic Diagnostic Unit for Rare Iron Metabolism Disorders
RESEARCH:
Iron & Cancer
Postdocs Ricky Joshi, PhD
Maya Shvarstman, PhD
Technicians Erica Morán, PhD
Jessica Aranda
PhD student Sara Luscieti
Bioinformatician Francisco Fuster

38. Propuesta- Estancias en la UDGAEMH
Estamos interesados en formar a médicos especializados (especialmente
hematólogos) en técnicas de
.
 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/

39. Advanced Genetic Diagnostic Unit for Rare Iron Metabolism Disorders
PRICE
CATEGORIES GENE DISEASE OMIM
(euros)
HFE HH1 120 235200
IRON Classical Hemochromatosis HAMP HH2 (juvenile form) 100 613313
OVERLOAD Non-HFE Hemochromatosis HFE2 HH2 (juvenile form) 100 602390
TFR2 HH3 200 604250
SLC40A1 HH4 160 606069
FTL Hereditary hyperferretinemia with cataracts 50 600886
HYPERFERRIT
Hyperferritinemia FTL Benign Hyperferretinemia 100 600886
INEMIA
FTH Hyperferretinemia with iron overload 100 134770
ALAS2 XLSA 150 300751
STEAP3 Sideroblastic anemia associated to STEAP3 150 609671
Sideroblastic ABCB7 XLSA with ataxia 100 301310
GLRX5 SA with hepatic iron overload 100 205950
SLC25A38 Non syndromic autosomal recessive CSA 100 205950
RARE IRON- CP Aceruloplasminemia 230 604290
RELATED TF Hypotransferrinemia 250 209300
ANEMIAS Non-sideroblastic Familiar microcytic hypochromic anemia
SLC11A2 with hepatic iron overload
200 206100
TMPRSS6 IRIDA, Iron-refractory iron deficient anemia 200 206200
CDAN1 CDA type I 250 224120
Congenital
SEC23B CDA type II 250 224100
dyserythropoietic anemia
KLF1 Unclassified CDA 150 NA
http://www.imppc.org/resources-and-services/index.html
Dra. Mayka Sánchez. udgaemh@imppc.org

40. Gracias por vuestra atención!
Advanced Genetic Diagnostic Unit for Rare Iron Metabolism Disorders
Head:
• Dr. Mayka Sánchez
Technicians:
• Dr. Erica Morán
• Jessica Aranda
GIF
Medical Doctors network
http://www.imppc.org/resources-and-services/index.html
udgaemh@imppc.org
Acreditación de Calidad:
EMQN EQA en HH-HFE
Designada como unidad EXPERTA por ORPHANET
Acreditación asistencial Generalitat Catalunya