PIGA GERMLINE MUTATIONS CAUSE DECREASED HEPCIDIN EXPRESSION AND A NOVEL SUBTYPE OF HEREDITARY HEMOCHROMATOSIS
Author(s): ,
Oriana Marques
Affiliations:
Department of Pediatric Oncology, Hematology, Immunology and Pulmonology,Universitätsklinikum Heidelberg,Heidelberg,Germany
,
Lena Muckenthaler
Affiliations:
Department of Pediatric Oncology, Hematology, Immunology and Pulmonology,Universitätsklinikum Heidelberg,Heidelberg,Germany
,
Silvia Colucci
Affiliations:
Department of Pediatric Oncology, Hematology, Immunology and Pulmonology,Universitätsklinikum Heidelberg,Heidelberg,Germany
,
Joachim Kunz
Affiliations:
Department of Pediatric Oncology, Hematology, Immunology and Pulmonology,Universitätsklinikum Heidelberg,Heidelberg,Germany
,
Thomas Bast
Affiliations:
Pediatric Epilepsy Centre,Diaconia Kork,Kehl-Kork,Germany
,
Piotr Fabrowski
Affiliations:
Cancer Drug Development Group,DKFZ,Heidelberg,Germany
,
Sandro Altamura
Affiliations:
Department of Pediatric Oncology, Hematology, Immunology and Pulmonology,Universitätsklinikum Heidelberg,Heidelberg,Germany
,
Britta Höchsmann
Affiliations:
Department of Transfusion Medicine,Universitätsklinikum Ulm,Ulm,Germany
,
Monika Langlotz
Affiliations:
Flow Cytometry & FACS Core Facility, Centre of Molecular Biology,University of Heidelberg,Heidelberg,Germany
,
Nicole Hofmeister-Mielke
Affiliations:
Laboratory for Hematological Diagnostics,Universitätsklinikum Heidelberg,Heidelberg,Germany
,
Andreas Kulozik
Affiliations:
Department of Pediatric Oncology, Hematology, Immunology and Pulmonology,Universitätsklinikum Heidelberg,Heidelberg,Germany
Martina Muckenthaler
Affiliations:
Department of Pediatric Oncology, Hematology, Immunology and Pulmonology,Universitätsklinikum Heidelberg,Heidelberg,Germany
EHA Library. Marques O. 06/09/21; 324508; S100
Oriana Marques
Oriana Marques
Contributions
Abstract
This abstract is embargoed until Friday, June 11, 09:00 CEST

Presentation during EHA2021: The Presidential Symposium will be held on Friday, June 11, 2021 (16:00 - 17:30 CEST) and will be accessible for on-demand viewing until August 15, 2021 on the Virtual Congress platform.

Abstract: S100

Type: Presidential Symposium

Session title: Presidential Symposium

Background

Hereditary Hemochromatosis (HH) is a common genetic disease of systemic iron overload caused by decreased activity of the hepcidin/ferroportin regulatory axis. The most common form of HH is caused by homozygosity for the HFE p.C282Y mutation, while rarer, juvenile subtypes can be caused by mutations in transferrin receptor 2, hemojuvelin, hepcidin or the hepcidin receptor ferroportin. Some patients with inherited hemochromatosis-like phenotypes do not carry mutations in these genes known to be involved in iron homeostasis.


We have recently diagnosed iron overload in 3 patients with epileptic encephalopathy. The clinical picture included normal red blood cell parameters, increased serum transferrin saturation, elevated liver iron and decreased serum hepcidin levels. Exome sequencing excluded mutations in known HH-associated genes and identified novel germline mutations in the phosphatidylinositol glycan class A (PIGA) gene. Somatic and germline pathogenic PIGA mutations are observed in patients with Paroxysmal nocturnal haemoglobinuria (PNH) and PIGA deficiency, respectively. PIGA is a protein involved in the first step of GPI anchor biosynthesis, which is important for the dynamics and cell membrane attachment of proteins.  

Aims

GPI-anchored proteins involved in iron metabolism include HJV and ceruloplasmin. We therefore hypothesized that the iron overload observed in our patients was caused by failure in GPI-anchorage of these proteins. Impaired HJV surface expression would impair iron sensing and result in low hepcidin expression and increased iron uptake while altered ceruloplasmin levels may contribute to enhanced cellular iron retention.

Methods

We performed CRISPR/Cas12a-mediated knockout of PIGA in the hepatocytic cell line Hep3B. The expression of key players in iron homeostasis was analysed in WT and PIGA KO clones by gene and protein expression analysis. Rescue experiments were performed in PIGA KO clones by simultaneously overexpressing HJV and PIGA from constructs that either coded for PIGA WT protein or carried the mutations identified in our patients.

Results

PIGA mRNA transcript and protein were inactivated in the PIGA KO clones in comparison with clones that underwent the same CRISPR/Cas12a-mediated procedure but in which PIGA was not ablated (PIGA WT). Protein analysis detected decreased/absent levels of GPI-anchored proteins, such as CD59, Ceruloplasmin and HJV. We show that WT cells respond with increased hepcidin mRNA expression when HJV is overexpressed, while PIGA KO cells fail to do so. Simultaneous overexpression of HJV and a PIGA WT construct rescued hepcidin expression in PIGA KO cells to similar levels like WT but hepcidin remained significantly lower when the PIGA mutants L344P, R77Q, L110del and R412* were overexpressed.

Conclusion

We have discovered that GPI-anchoring of HJV is crucial for its function in regulating hepcidin. The results demonstrate that PIGA mutations are responsible for a novel form of juvenile hemochromatosis.

Keyword(s): Hemochromatosis, Hepcidin, Iron metabolism

This abstract is embargoed until Friday, June 11, 09:00 CEST

Presentation during EHA2021: The Presidential Symposium will be held on Friday, June 11, 2021 (16:00 - 17:30 CEST) and will be accessible for on-demand viewing until August 15, 2021 on the Virtual Congress platform.

Abstract: S100

Type: Presidential Symposium

Session title: Presidential Symposium

Background

Hereditary Hemochromatosis (HH) is a common genetic disease of systemic iron overload caused by decreased activity of the hepcidin/ferroportin regulatory axis. The most common form of HH is caused by homozygosity for the HFE p.C282Y mutation, while rarer, juvenile subtypes can be caused by mutations in transferrin receptor 2, hemojuvelin, hepcidin or the hepcidin receptor ferroportin. Some patients with inherited hemochromatosis-like phenotypes do not carry mutations in these genes known to be involved in iron homeostasis.


We have recently diagnosed iron overload in 3 patients with epileptic encephalopathy. The clinical picture included normal red blood cell parameters, increased serum transferrin saturation, elevated liver iron and decreased serum hepcidin levels. Exome sequencing excluded mutations in known HH-associated genes and identified novel germline mutations in the phosphatidylinositol glycan class A (PIGA) gene. Somatic and germline pathogenic PIGA mutations are observed in patients with Paroxysmal nocturnal haemoglobinuria (PNH) and PIGA deficiency, respectively. PIGA is a protein involved in the first step of GPI anchor biosynthesis, which is important for the dynamics and cell membrane attachment of proteins.  

Aims

GPI-anchored proteins involved in iron metabolism include HJV and ceruloplasmin. We therefore hypothesized that the iron overload observed in our patients was caused by failure in GPI-anchorage of these proteins. Impaired HJV surface expression would impair iron sensing and result in low hepcidin expression and increased iron uptake while altered ceruloplasmin levels may contribute to enhanced cellular iron retention.

Methods

We performed CRISPR/Cas12a-mediated knockout of PIGA in the hepatocytic cell line Hep3B. The expression of key players in iron homeostasis was analysed in WT and PIGA KO clones by gene and protein expression analysis. Rescue experiments were performed in PIGA KO clones by simultaneously overexpressing HJV and PIGA from constructs that either coded for PIGA WT protein or carried the mutations identified in our patients.

Results

PIGA mRNA transcript and protein were inactivated in the PIGA KO clones in comparison with clones that underwent the same CRISPR/Cas12a-mediated procedure but in which PIGA was not ablated (PIGA WT). Protein analysis detected decreased/absent levels of GPI-anchored proteins, such as CD59, Ceruloplasmin and HJV. We show that WT cells respond with increased hepcidin mRNA expression when HJV is overexpressed, while PIGA KO cells fail to do so. Simultaneous overexpression of HJV and a PIGA WT construct rescued hepcidin expression in PIGA KO cells to similar levels like WT but hepcidin remained significantly lower when the PIGA mutants L344P, R77Q, L110del and R412* were overexpressed.

Conclusion

We have discovered that GPI-anchoring of HJV is crucial for its function in regulating hepcidin. The results demonstrate that PIGA mutations are responsible for a novel form of juvenile hemochromatosis.

Keyword(s): Hemochromatosis, Hepcidin, Iron metabolism

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