Publications by authors named "Sarah Ducamp"

17 Publications

  • Page 1 of 1

A mutation in the iron-responsive element of is a modifier of disease severity in a patient suffering from associated erythropoietic protoporphyria.

Haematologica 2021 07 1;106(7):2030-2033. Epub 2021 Jul 1.

Universitat Internacional de Catalunya (UIC), Department of Basic Sciences, Iron metabolism: Regulation and Diseases. Sant Cugat del Vallès, Barcelona; BloodGenetics S.L. Diagnostics in Inherited Blood Diseases. Esplugues de Llobregat.

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http://dx.doi.org/10.3324/haematol.2020.272450DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8252951PMC
July 2021

XPO1 regulates erythroid differentiation and is a new target for the treatment of β-thalassemia.

Haematologica 2020 09 1;105(9):2240-2249. Epub 2020 Sep 1.

INSERM UMR 1163, CNRS ERL 8254, Laboratory of Cellular and Molecular Mechanisms of Hematological Disorders and Therapeutical Implications, Paris, France; Imagine Institute, Université Paris Descartes, Sorbonne Paris-Cité et Assistance Publique-Hôpitaux de Paris, Hôpital Necker, Paris, France; Laboratory of Excellence GRex, Paris, France; Service d'Hématologie, Faculté de Médecine Paris Descartes, Sorbonne Paris-Cité et Assistance Publique-Hôpitaux de Paris Hôpital Necker, Paris, France.

β-thalassemia major (β-TM) is an inherited hemoglobinopathy caused by a quantitative defect in the synthesis of β-globin chains of hemoglobin, leading to the accumulation of free a-globin chains that aggregate and cause ineffective erythropoiesis. We have previously demonstrated that terminal erythroid maturation requires a transient activation of caspase-3 and that the chaperone Heat Shock Protein 70 (HSP70) accumulates in the nucleus to protect GATA-1 transcription factor from caspase-3 cleavage. This nuclear accumulation of HSP70 is inhibited in human β-TM erythroblasts due to HSP70 sequestration in the cytoplasm by free a-globin chains, resulting in maturation arrest and apoptosis. Likewise, terminal maturation can be restored by transduction of a nuclear-targeted HSP70 mutant. Here we demonstrate that in normal erythroid progenitors, HSP70 localization is regulated by the exportin-1 (XPO1), and that treatment of β-thalassemic erythroblasts with an XPO1 inhibitor increased the amount of nuclear HSP70, rescued GATA-1 expression and improved terminal differentiation, thus representing a new therapeutic option to ameliorate ineffective erythropoiesis of β-TM.
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http://dx.doi.org/10.3324/haematol.2018.210054DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7556489PMC
September 2020

Evidence in the UK Biobank for the underdiagnosis of erythropoietic protoporphyria.

Genet Med 2021 01 2;23(1):140-148. Epub 2020 Sep 2.

Deparment of Medicine, Division of Pulmonary and Critical Care Medicine, Massachusetts General Hospital, Boston, MA, USA.

Purpose: Erythropoietic protoporphyria (EPP), characterized by painful cutaneous photosensitivity, results from pathogenic variants in ferrochelatase (FECH). For 96% of patients, EPP results from coinheriting a rare pathogenic variant in trans of a common hypomorphic variant c.315-48T>C (minor allele frequency 0.05). The estimated prevalence of EPP derived from the number of diagnosed individuals in Europe is 0.00092%, but this may be conservative due to underdiagnosis. No study has estimated EPP prevalence using large genetic data sets.

Methods: Disease-associated FECH variants were identified in the UK Biobank, a data set of 500,953 individuals including 49,960 exome sequences. EPP prevalence was then estimated. The association of FECH variants with EPP-related traits was assessed.

Results: Analysis of pathogenic FECH variants in the UK Biobank provides evidence that EPP prevalence is 0.0059% (95% confidence interval [CI]: 0.0042-0.0076%), 1.7-3.0 times more common than previously thought in the UK. In homozygotes for the common c.315-48T>C FECH variant, there was a novel decrement in both erythrocyte mean corpuscular volume (MCV) and hemoglobin.

Conclusion: The prevalence of EPP has been underestimated secondary to underdiagnosis. The common c.315-48T>C allele is associated with both MCV and hemoglobin, an association that could be important both for those with and without EPP.
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http://dx.doi.org/10.1038/s41436-020-00951-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7796935PMC
January 2021

p53 activation during ribosome biogenesis regulates normal erythroid differentiation.

Blood 2021 01;137(1):89-102

Université de Paris, Institut Cochin, Unité Mixte de Recherche (UMR) 8104, Centre National de la Recherche Scientifique (CNRS), INSERM U1016, Paris, France.

The role of ribosome biogenesis in erythroid development is supported by the recognition of erythroid defects in ribosomopathies in both Diamond-Blackfan anemia and 5q- syndrome. Whether ribosome biogenesis exerts a regulatory function on normal erythroid development is still unknown. In the present study, a detailed characterization of ribosome biogenesis dynamics during human and murine erythropoiesis showed that ribosome biogenesis is abruptly interrupted by the decline in ribosomal DNA transcription and the collapse of ribosomal protein neosynthesis. Its premature arrest by the RNA Pol I inhibitor CX-5461 targeted the proliferation of immature erythroblasts. p53 was activated spontaneously or in response to CX-5461, concomitant to ribosome biogenesis arrest, and drove a transcriptional program in which genes involved in cell cycle-arrested, negative regulation of apoptosis, and DNA damage response were upregulated. RNA Pol I transcriptional stress resulted in nucleolar disruption and activation of the ATR-CHK1-p53 pathway. Our results imply that the timing of ribosome biogenesis extinction and p53 activation is crucial for erythroid development. In ribosomopathies in which ribosome availability is altered by unbalanced production of ribosomal proteins, the threshold downregulation of ribosome biogenesis could be prematurely reached and, together with pathological p53 activation, prevents a normal expansion of erythroid progenitors.
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http://dx.doi.org/10.1182/blood.2019003439DOI Listing
January 2021

Mutations in the iron-sulfur cluster biogenesis protein HSCB cause congenital sideroblastic anemia.

J Clin Invest 2020 10;130(10):5245-5256

Department of Pathology, Boston Children's Hospital, Boston, Massachusetts, USA.

The congenital sideroblastic anemias (CSAs) can be caused by primary defects in mitochondrial iron-sulfur (Fe-S) cluster biogenesis. HSCB (heat shock cognate B), which encodes a mitochondrial cochaperone, also known as HSC20 (heat shock cognate protein 20), is the partner of mitochondrial heat shock protein A9 (HSPA9). Together with glutaredoxin 5 (GLRX5), HSCB and HSPA9 facilitate the transfer of nascent 2-iron, 2-sulfur clusters to recipient mitochondrial proteins. Mutations in both HSPA9 and GLRX5 have previously been associated with CSA. Therefore, we hypothesized that mutations in HSCB could also cause CSA. We screened patients with genetically undefined CSA and identified a frameshift mutation and a rare promoter variant in HSCB in a female patient with non-syndromic CSA. We found that HSCB expression was decreased in patient-derived fibroblasts and K562 erythroleukemia cells engineered to have the patient-specific promoter variant. Furthermore, gene knockdown and deletion experiments performed in K562 cells, zebrafish, and mice demonstrate that loss of HSCB results in impaired Fe-S cluster biogenesis, a defect in RBC hemoglobinization, and the development of siderocytes and more broadly perturbs hematopoiesis in vivo. These results further affirm the involvement of Fe-S cluster biogenesis in erythropoiesis and hematopoiesis and define HSCB as a CSA gene.
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http://dx.doi.org/10.1172/JCI135479DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7524500PMC
October 2020

The molecular genetics of sideroblastic anemia.

Blood 2019 01 6;133(1):59-69. Epub 2018 Nov 6.

Department of Pathology, Boston Children's Hospital, Boston, MA.

The sideroblastic anemias (SAs) are a group of inherited and acquired bone marrow disorders defined by pathological iron accumulation in the mitochondria of erythroid precursors. Like most hematological diseases, the molecular genetic basis of the SAs has ridden the wave of technology advancement. Within the last 30 years, with the advent of positional cloning, the human genome project, solid-state genotyping technologies, and next-generation sequencing have evolved to the point where more than two-thirds of congenital SA cases, and an even greater proportion of cases of acquired clonal disease, can be attributed to mutations in a specific gene or genes. This review focuses on an analysis of the genetics of these diseases and how understanding these defects may contribute to the design and implementation of rational therapies.
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http://dx.doi.org/10.1182/blood-2018-08-815951DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6318428PMC
January 2019

Finely-tuned regulation of AMP-activated protein kinase is crucial for human adult erythropoiesis.

Haematologica 2019 05 11;104(5):907-918. Epub 2018 Oct 11.

Institut Cochin, INSERM U1016

AMP-activated protein kinase (AMPK) is a heterotrimeric complex containing α, β, and γ subunits involved in maintaining integrity and survival of murine red blood cells. Indeed, α , and mice develop hemolytic anemia and the plasma membrane of their red blood cells shows elasticity defects. The membrane composition evolves continuously along erythropoiesis and during red blood cell maturation; defects due to the absence of Ampk could be initiated during erythropoiesis. We, therefore, studied the role of AMPK during human erythropoiesis. Our data show that AMPK activation had two distinct phases in primary erythroblasts. The phosphorylation of AMPK (Thr172) and its target acetyl CoA carboxylase (Ser79) was elevated in immature erythroblasts (glycophorin A), then decreased conjointly with erythroid differentiation. In erythroblasts, knockdown of the α1 catalytic subunit by short hairpin RNA led to a decrease in cell proliferation and alterations in the expression of membrane proteins (band 3 and glycophorin A) associated with an increase in phosphorylation of adducin (Ser726). AMPK activation in mature erythroblasts (glycophorin A), achieved through the use of direct activators (GSK621 and compound 991), induced cell cycle arrest in the S phase, the induction of autophagy and caspase-dependent apoptosis, whereas no such effects were observed in similarly treated immature erythroblasts. Thus, our work suggests that AMPK activation during the final stages of erythropoiesis is deleterious. As the use of direct AMPK activators is being considered as a treatment in several pathologies (diabetes, acute myeloid leukemia), this observation is pivotal. Our data highlighted the importance of the finely-tuned regulation of AMPK during human erythropoiesis.
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http://dx.doi.org/10.3324/haematol.2018.191403DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6518903PMC
May 2019

Dyserythropoiesis evaluated by the RED score and hepcidin:ferritin ratio predicts response to erythropoietin in lower-risk myelodysplastic syndromes.

Haematologica 2019 03 4;104(3):497-504. Epub 2018 Oct 4.

Department of Hematology, CHU Grenoble-Alpes, Grenoble.

Erythropoiesis-stimulating agents are generally the first line of treatment of anemia in patients with lower-risk myelodysplastic syndrome. We prospectively investigated the predictive value of somatic mutations, and biomarkers of ineffective erythropoiesis including the flow cytometry RED score, serum growth-differentiation factor-15, and hepcidin levels. Inclusion criteria were no prior treatment with erythropoiesis-stimulating agents, low- or intermediate-1-risk myelodysplastic syndrome according to the International Prognostic Scoring System, and a hemoglobin level <10 g/dL. Patients could be red blood cell transfusion-dependent or not and were given epoetin zeta 40 000 IU/week. Serum erythropoietin level, iron parameters, hepcidin, flow cytometry Ogata and RED scores, and growth-differentiation factor-15 levels were determined at baseline, and molecular analysis by next-generation sequencing was also conducted. Erythroid response (defined according to the International Working Group 2006 criteria) was assessed at week 12. Seventy patients, with a median age of 78 years, were included in the study. There were 22 patients with refractory cytopenia with multilineage dysplasia, 19 with refractory cytopenia with unilineage dysplasia, 14 with refractory anemia with ring sideroblasts, four with refractory anemia with excess blasts-1, six with chronic myelomonocytic leukemia, two with del5q-and three with unclassifiable myelodysplastic syndrome. According to the revised International Prognostic Scoring System, 13 had very low risk, 47 had low risk, nine intermediate risk and one had high-risk disease. Twenty patients were transfusion dependent. Forty-eight percent had an erythroid response and the median duration of the response was 26 months. At baseline, non-responders had significantly higher RED scores and lower hepcidin:ferritin ratios. In multivariate analysis, only a RED score >4 (=0.05) and a hepcidin:ferritin ratio <9 (=0.02) were statistically significantly associated with worse erythroid response. The median response duration was shorter in patients with growth-differentiation factor-15 >2000 pg/mL and a hepcidin:ferritin ratio <9 (=0.0008 and =0.01, respectively). In multivariate analysis, both variables were associated with shorter response duration. Erythroid response to epoetin zeta was similar to that obtained with other erythropoiesis-stimulating agents and was correlated with higher baseline hepcidin:ferritin ratio and lower RED score. .
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http://dx.doi.org/10.3324/haematol.2018.203158DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6395339PMC
March 2019

Mutation in human elevates levels of aminolevulinate synthase and protoporphyrin IX to promote erythropoietic protoporphyria.

Proc Natl Acad Sci U S A 2017 09 5;114(38):E8045-E8052. Epub 2017 Sep 5.

Division of Hematology, Brigham & Women's Hospital, Harvard Medical School, Boston, MA 02115;

Loss-of-function mutations in genes for heme biosynthetic enzymes can give rise to congenital porphyrias, eight forms of which have been described. The genetic penetrance of the porphyrias is clinically variable, underscoring the role of additional causative, contributing, and modifier genes. We previously discovered that the mitochondrial AAA+ unfoldase ClpX promotes heme biosynthesis by activation of δ-aminolevulinate synthase (ALAS), which catalyzes the first step of heme synthesis. CLPX has also been reported to mediate heme-induced turnover of ALAS. Here we report a dominant mutation in the ATPase active site of human CLPX, p.Gly298Asp, that results in pathological accumulation of the heme biosynthesis intermediate protoporphyrin IX (PPIX). Amassing of PPIX in erythroid cells promotes erythropoietic protoporphyria (EPP) in the affected family. The mutation in inactivates its ATPase activity, resulting in coassembly of mutant and WT protomers to form an enzyme with reduced activity. The presence of low-activity CLPX increases the posttranslational stability of ALAS, causing increased ALAS protein and ALA levels, leading to abnormal accumulation of PPIX. Our results thus identify an additional molecular mechanism underlying the development of EPP and further our understanding of the multiple mechanisms by which CLPX controls heme metabolism.
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http://dx.doi.org/10.1073/pnas.1700632114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5617249PMC
September 2017

Comprehensive Proteomic Analysis of Human Erythropoiesis.

Cell Rep 2016 08 21;16(5):1470-1484. Epub 2016 Jul 21.

INSERM U1016, Institut Cochin, 75014 Paris, France; Centre National de la Recherche Scientifique (CNRS), UMR8104, 75014 Paris, France; Université Paris Descartes, Sorbonne Paris Cité, 75014 Paris, France; Laboratory of Excellence GReX, 75015 Paris, France; Plateforme de Protéomique de l'Université Paris Descartes (3P5), 75014 Paris, France; Ligue Nationale Contre le Cancer, Equipe Labellisée, 75014 Paris, France. Electronic address:

Mass spectrometry-based proteomics now enables the absolute quantification of thousands of proteins in individual cell types. We used this technology to analyze the dynamic proteome changes occurring during human erythropoiesis. We quantified the absolute expression of 6,130 proteins during erythroid differentiation from late burst-forming units-erythroid (BFU-Es) to orthochromatic erythroblasts. A modest correlation between mRNA and protein expression was observed. We identified several proteins with unexpected expression patterns in erythroid cells, highlighting a breakpoint in the erythroid differentiation process at the basophilic stage. We also quantified the distribution of proteins between reticulocytes and pyrenocytes after enucleation. These analyses identified proteins that are actively sorted either with the reticulocyte or the pyrenocyte. Our study provides the absolute quantification of protein expression during a complex cellular differentiation process in humans, and it establishes a framework for future studies of disordered erythropoiesis.
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http://dx.doi.org/10.1016/j.celrep.2016.06.085DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5274717PMC
August 2016

Human Erythroid 5-Aminolevulinate Synthase Mutations Associated with X-Linked Protoporphyria Disrupt the Conformational Equilibrium and Enhance Product Release.

Biochemistry 2015 Sep 2;54(36):5617-31. Epub 2015 Sep 2.

Department of Molecular Medicine, Morsani College of Medicine, University of South Florida , Tampa, Florida 33612, United States.

Regulation of 5-aminolevulinate synthase (ALAS) is at the origin of balanced heme production in mammals. Mutations in the C-terminal region of human erythroid-specific ALAS (hALAS2) are associated with X-linked protoporphyria (XLPP), a disease characterized by extreme photosensitivity, with elevated blood concentrations of free protoporphyrin IX and zinc protoporphyrin. To investigate the molecular basis for this disease, recombinant hALAS2 and variants of the enzyme harboring the gain-of-function XLPP mutations were constructed, purified, and analyzed kinetically, spectroscopically, and thermodynamically. Enhanced activities of the XLPP variants resulted from increases in the rate at which the product 5-aminolevulinate (ALA) was released from the enzyme. Circular dichroism spectroscopy revealed that the XLPP mutations altered the microenvironment of the pyridoxal 5'-phosphate cofactor, which underwent further and specific alterations upon succinyl-CoA binding. Transient kinetic analyses of the variant-catalyzed reactions and protein fluorescence quenching upon binding of ALA to the XLPP variants demonstrated that the protein conformational transition step associated with product release was predominantly affected. Of relevance is the fact that XLPP could also be modeled in cell culture. We propose that (1) the XLPP mutations destabilize the succinyl-CoA-induced hALAS2 closed conformation and thus accelerate ALA release, (2) the extended C-terminus of wild-type mammalian ALAS2 provides a regulatory role that allows for allosteric modulation of activity, thereby controlling the rate of erythroid heme biosynthesis, and (3) this control is disrupted in XLPP, resulting in porphyrin accumulation.
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http://dx.doi.org/10.1021/acs.biochem.5b00407DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4573335PMC
September 2015

Antisense oligonucleotide-based therapy in human erythropoietic protoporphyria.

Am J Hum Genet 2014 Apr 27;94(4):611-7. Epub 2014 Mar 27.

Institut National de la Santé et de la Recherche Médicale, U1149, Centre de Recherches sur l'Inflammation, F-75018 Paris, France; Assistance Publique-Hôpitaux de Paris, Centre Français des Porphyries, Hôpital Louis Mourier, 178 Rue des Renouillers, F-92701 Colombes, France; Université de Versailles Saint Quentin en Yvelines, F-78035 Versailles, France; Assistance Publique-Hôpitaux de Paris, Laboratoire de Biochimie Hormonale et Génétique, Hôpital Ambroise Paré, F-92100 Boulogne Billancourt, France; Laboratory of Excellence GR-Ex, 75000 Paris, France.

In 90% of people with erythropoietic protoporphyria (EPP), the disease results from the inheritance of a common hypomorphic FECH allele, encoding ferrochelatase, in trans to a private deleterious FECH mutation. The activity of the resulting FECH enzyme falls below the critical threshold of 35%, leading to the accumulation of free protoporphyrin IX (PPIX) in bone marrow erythroblasts and in red cells. The mechanism of low expression involves a biallelic polymorphism (c.315-48T>C) localized in intron 3. The 315-48C allele increases usage of the 3' cryptic splice site between exons 3 and 4, resulting in the transcription of an unstable mRNA with a premature stop codon, reducing the abundance of wild-type FECH mRNA, and finally reducing FECH activity. Through a candidate-sequence approach and an antisense-oligonucleotide-tiling method, we identified a sequence that, when targeted by an antisense oligonucleotide (ASO-V1), prevented usage of the cryptic splice site. In lymphoblastoid cell lines derived from symptomatic EPP subjects, transfection of ASO-V1 reduced the usage of the cryptic splice site and efficiently redirected the splicing of intron 3 toward the physiological acceptor site, thereby increasing the amount of functional FECH mRNA. Moreover, the administration of ASO-V1 into developing human erythroblasts from an overtly EPP subject markedly increased the production of WT FECH mRNA and reduced the accumulation of PPIX to a level similar to that measured in asymptomatic EPP subjects. Thus, EPP is a paradigmatic Mendelian disease in which the in vivo correction of a common single splicing defect would improve the condition of most affected individuals.
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http://dx.doi.org/10.1016/j.ajhg.2014.02.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3980518PMC
April 2014

Molecular and functional analysis of the C-terminal region of human erythroid-specific 5-aminolevulinic synthase associated with X-linked dominant protoporphyria (XLDPP).

Hum Mol Genet 2013 Apr 20;22(7):1280-8. Epub 2012 Dec 20.

AP-HP, Centre Franc¸ais des Porphyries, Hoˆ pital Louis Mourier, 178 rue des Renouillers, 92701 Colombes Cedex,France.

Frameshift mutations in the last coding exon of the 5-aminolevulinate synthase (ALAS) 2 gene were described to activate the enzyme causing increased levels of zinc- and metal-free protoporphyrin in patients with X-linked dominant protoporphyria (XLDPP). Only two such so-called gain-of-function mutations have been reported since the description of XLDPP in 2008. In this study of four newly identified XLDPP families, we identified two novel ALAS2 gene mutations, a nonsense p.Q548X and a frameshift c.1651-1677del26bp, along with a known mutation (delAGTG) found in two unrelated families. Of relevance, a de novo somatic and germinal mosaicism was present in a delAGTG family. Such a phenomenon may explain the high proportion of this mutation in XLDPP worldwide. Enhancements of over 3- and 14-fold in the catalytic rate and specificity constant of purified recombinant XLDPP variants in relation to those of wild-type ALAS2 confirmed the gain of function ascribed to these enzymes. The fact that both p.Q548X and c.1651-1677del26bp are located in close proximity and upstream from the two previously described mutations led us to propose the presence of a large gain-of-function domain within the C-terminus of ALAS2. To test this hypothesis, we generated four additional nonsense mutants (p.A539X, p.G544X, p.G576X and p.V583X) surrounding the human XLDPP mutations and defined an ALAS2 gain-of-function domain with a minimal size of 33 amino acids. The identification of this gain-of-function domain provides important information on the enzymatic activity of ALAS2, which was proposed to be constitutively inhibited, either directly or indirectly, through its own C-terminus.
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http://dx.doi.org/10.1093/hmg/dds531DOI Listing
April 2013

ALAS2 acts as a modifier gene in patients with congenital erythropoietic porphyria.

Blood 2011 Aug 7;118(6):1443-51. Epub 2011 Jun 7.

Biochemistry and Molecular Genetics Unit, Hospital Clinic, IDIBAPS, University of Barcelona, Barcelona, Spain.

Mutations in the uroporphyrinogen III synthase (UROS) gene cause congenital erythropoietic porphyria (CEP), an autosomal-recessive inborn error of erythroid heme biosynthesis. Clinical features of CEP include dermatologic and hematologic abnormalities of variable severity. The discovery of a new type of erythroid porphyria, X-linked dominant protoporphyria (XLDPP), which results from increased activity of 5-aminolevulinate synthase 2 (ALAS2), the rate-controlling enzyme of erythroid heme synthesis, led us to hypothesize that the CEP phenotype may be modulated by sequence variations in the ALAS2 gene. We genotyped ALAS2 in 4 unrelated CEP patients exhibiting the same C73R/P248Q UROS genotype. The most severe of the CEP patients, a young girl, proved to be heterozygous for a novel ALAS2 mutation: c.1757 A > T in exon 11. This mutation is predicted to affect the highly conserved and penultimate C-terminal amino acid of ALAS2 (Y586). The rate of 5-aminolevulinate release from Y586F was significantly increased over that of wild-type ALAS2. The contribution of the ALAS2 gain-of-function mutation to the CEP phenotype underscores the importance of modifier genes underlying CEP. We propose that ALAS2 gene mutations should be considered not only as causative of X-linked sideroblastic anemia (XLSA) and XLDPP but may also modulate gene function in other erythropoietic disorders.
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http://dx.doi.org/10.1182/blood-2011-03-342873DOI Listing
August 2011

Sideroblastic anemia: molecular analysis of the ALAS2 gene in a series of 29 probands and functional studies of 10 missense mutations.

Hum Mutat 2011 Jun 24;32(6):590-7. Epub 2011 Feb 24.

INSERM, Centre de Recherche Biomédicale Bichat-Beaujon, Paris, France.

X-linked Sideroblastic Anemia (XLSA) is the most common genetic form of sideroblastic anemia, a heterogeneous group of disorders characterized by iron deposits in the mitochondria of erythroid precursors. XLSA is due to mutations in the erythroid-specific 5-aminolevulinate synthase (ALAS2) gene. Thirteen different ALAS2 mutations were identified in 16 out of 29 probands with sideroblastic anemia. One third of the patients were females with a highly skewed X-chromosome inactivation. The identification of seven novel mutations in the ALAS2 gene, six missense mutations, and one deletion in the proximal promoter extends the allelic heterogeneity of XSLA. Most of the missense mutations were predicted to be deleterious, and 10 of them, without any published functional characterization, were expressed in Escherichia coli. ALAS2 activities were assayed in vitro. Five missense mutations resulted in decreased enzymatic activity under standard conditions, and two other mutated proteins had decreased activity when assayed in the absence of exogenous pyridoxal phosphate and increased thermosensitivity. Although most amino acid substitutions result in a clearly decreased enzymatic activity in vitro, a few mutations have a more subtle effect on the protein that is only revealed by in vitro tests under specific conditions.
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http://dx.doi.org/10.1002/humu.21455DOI Listing
June 2011

C-terminal deletions in the ALAS2 gene lead to gain of function and cause X-linked dominant protoporphyria without anemia or iron overload.

Am J Hum Genet 2008 Sep 4;83(3):408-14. Epub 2008 Sep 4.

Department of Medical Biochemistry and Immunology, University Hospital of Wales and, School of Medicine, Cardiff University, Cardiff CF14 4XN, UK.

All reported mutations in ALAS2, which encodes the rate-regulating enzyme of erythroid heme biosynthesis, cause X-linked sideroblastic anemia. We describe eight families with ALAS2 deletions, either c.1706-1709 delAGTG (p.E569GfsX24) or c.1699-1700 delAT (p.M567EfsX2), resulting in frameshifts that lead to replacement or deletion of the 19-20 C-terminal residues of the enzyme. Prokaryotic expression studies show that both mutations markedly increase ALAS2 activity. These gain-of-function mutations cause a previously unrecognized form of porphyria, X-linked dominant protoporphyria, characterized biochemically by a high proportion of zinc-protoporphyrin in erythrocytes, in which a mismatch between protoporphyrin production and the heme requirement of differentiating erythroid cells leads to overproduction of protoporphyrin in amounts sufficient to cause photosensitivity and liver disease.
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http://dx.doi.org/10.1016/j.ajhg.2008.08.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2556430PMC
September 2008
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