Sideroblastic anemia: molecular analysis of ALAS2 gene in a series of 29 probands and functional

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Sideroblastic anemia: molecular analysis of ALAS2 gene
in a series of 29 probands and functional studies of ten
missense mutations.
Sarah Ducamp, Caroline Kannengiesser, Mohamed Touati, Lo¨ıc Garc¸on,
Agn`es Guerci-Bresler, Jean-Fran¸cois Guichard, Christiane Vermylen, Joaquim
Dochir, H´el`ene Antoine Poirel, Fanny Fouyssac, et al.
To cite this version:
Sarah Ducamp, Caroline Kannengiesser, Mohamed Touati, Lo¨ıc Gar¸con, Agn`es Guerci-Bresler,
et al.. Sideroblastic anemia: molecular analysis of ALAS2 gene in a series of 29 probands and
functional studies of ten missense mutations.. Human Mutation, Wiley, 2011, 32 (6), pp.590.
<10.1002/humu.21455>. <hal-00616698>
HAL Id: hal-00616698
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Human Mutation

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

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Journal: Human Mutation
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Manuscript ID: humu-2010-0359.R1
Wiley - Manuscript type: Research Article
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Date Submitted by the
30-Noev-2010
Author:
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Complete List of Authors: Ducamp, Sarah; INSERM, CRB3, U773; AP-HP, Hôpital Louis
Mourier, Centrre Français des Porphyries
Kannengiesser, Caroline; AP-HP, Hôpital Bichat, genetics; INSERM,
CRB3, U773
Touati, Mohamed; CHU Limoges, Hôpital Dupuytren, Hématologie
clinique R
Garçon, Loïc; AP-HP, Hôpital Saint Antoine, Hématologie Biologique
Guerci-Bresler, Agnès; eCHU de Nancy, Hôpital Brabois, Hématologie
Clinique
Guichard, Jean-François; Hvôpital Sainte Blandine, Médecine
Interne-Pathologie Vasculaire -i Immunologie Clinique
Vermylen, Christiane; Clinique Universitaire Saint-Luc, de Duve
Institute, UCL, Génétique Hémateologique
Dochir, Joaquim; Clinique Universitaire Saint-Luc, de Duve
Institute, UCL, Génétique Hématologique
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Poirel, Hélène; Clinique Universitaire Saint-Luc, de Duve Institute,
UCL, Génétique Hématologique
Fouyssac, Fanny; CHU de Nancy, Hôpital Brabois, Hôpital d'Enfants,
Hématologie Oncologique
Mansuy, Ludovic; CHU de Nancy, Hôpital Brabois, Hôpital d'Enfants,
Hématologie Oncologique
Leroux, Geneviève; AP-HP, Hôpital Avicenne, Hématologie
Tertian, Gérard; AP-HP, Hôpital Bicêtre, Hématologie
Girot, Robert; AP-HP, Hôpital Tenon, Hématologie Biologique
Heimpel, Hermann; Medizinische Universitätsklinik III
Matthes, Thomas; HUG Genève, Hématologie
Talbi, Neila; AP-HP, Hôpital Louis Mourier, Centre Français des
Porphyries
Deybach, Jean-Charles; AP-HP, Hôpital Louis Mourier, Centre
Français des Porphyries
Beaumont, Carole; INSERM, CRB3, U773
Puy, Hervé; INSERM, CRB3, U773; AP-HP, Hôpital Louis Mourier,
Centre Français des Porphyries
John Wiley & Sons, Inc.
Page 1 of 59 Human Mutation
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4 Grandchamp, Bernard; INSERM, CRB3, U773; AP-HP, hôpital
Bichat, genetics
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X-linked Sideroblastic Anemia, ALAS2, heme synthesis,
7 Key Words: protoporphyrin
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Human Mutation Page 2 of 59
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3 Title page
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8 Title
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Sideroblastic anemia: molecular analysis of the ALAS2 gene in a series of 29 probands and
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functional studies of ten missense mutations.
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15 Authors
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17 Sarah Ducamp1,2*, Caroline Kannengiesser1,3*, Mohamed Touati4, Loïc Garçon5, Agnès
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20 Guerci-Bresler6, Jean François Guichard7, Christiane Vermylen8, Joaquim Dochir8, Hélène A.
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22 Poirel8, Fanny Fouyssac9, Ludovic Mansuy9, Geneviève Leroux10, Gérard Tertian11, Robert
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Girot12, Hermann Heimpel13, Thomas Matthes14, Neila Talbi1,2, Jean-Charles Deybach2,
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27 Carole Beaumont1, Hervé Puy1, P2, Bernard Grandchamp1,3
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Affiliation
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1 - INSERM, Centre de Recherche Biomédicale Bichat-Beaujon, U773, Paris, F-75018,
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33 France
34 2 - Assistance Publique des Hôpitaux de Paris, Hôpital Louis Mourier, Centre Français des
35 Porphyries, Colombes, F-92701, France
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3 - Assistance Publique des Hôpitaux de Paris, Hôpital Bichat, Laboratoire de Biochimie
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Hormonale et Génétique, Paris, F-75018, France e
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4 - CHU de Limoges, Hôpital Dupuytren, Hématologie clinique, Limoges, F-87042, France
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40 5 - Assistance Publique des Hôpitaux de Paris, Hiôpital Saint Antoine, Hématologie
41 Biologique, Paris, F-75012, France
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6 - CHU de Nancy, Hôpital Brabois, Hématologie Clinique, Vandoeuvre, F-54511, France
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7 - Hôpital Sainte Blandine, Médecine Interne-Pathologie Vasculaire-immunologie clinique,
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Metz, F-57045, France w
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46 8 - Cliniques universitaires saint-Luc / de Duve Institute / UCL, Génétique Hématologique,
47 Human Molecular Genetics (GEHU), Bruxelles, B-1200 , Belgium
48 9 - CHU de Nancy, Hôpital Brabois - Hôpital d'Enfants, Hématologie Oncologie pédiatrique,
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Vandoeuvre F-54511, France
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10 - Assistance Publique des Hôpitaux de Paris, Hôpital Avicenne, Hématologie, 125 route de
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Stalingrad, Bobigny, F-93009, France
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53 11 - Assistance Publique des Hôpitaux de Paris CHU Bicêtre - Hématologie, Le Kremlin
54 Bicêtre, F-94275, France
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12 - Assistance Publique des Hôpitaux de Paris, Hôpital Tenon, hématologie biologique,
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Paris, F-75970, France
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13 - Medizinische Universitätsklinik III, Ulm, D-89081, Germany
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59 14 - HUG Genève, Service d'Hématologie, 1211 Genève, Switzerland
60 *both these authors participated equally to the work
John Wiley & Sons, Inc.
Page 3 of 59 Human Mutation
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3 Abstract
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X-linked Sideroblastic Anemia (XLSA) is the most common genetic form of sideroblastic
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8 anemia, a heterogeneous group of disorders characterized by iron deposits in the mitochondria
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of erythroid precursors. XLSA is due to mutations in the erythroid-specific 5-aminolevulinate
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synthase (ALAS2) gene. Thirteen different ALAS2 mutations were identified in 16 out of 29
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15 probands with sideroblastic anemia. One third of the patients were females with a highly
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skewed X-chromosome inactivation. The identification of seven novel mutations in the
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20 ALAS2 gene, six missense mutations, and one deletion in the proximal promoter extends the
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22 allelic heterogeneity of XSLA. Most of the missense mutations were predicted to be
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deleterious and ten of them, without any published functional characterization, were
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27 expressed in E. coli. ALAS2 actPivities were assayed in vitro. Five missense mutations resulted
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had decreased activity when assayed in the absence of exogenous pyridoxal phosphate and
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34 increased thermosensitivity. Although most amino-acid substitutions result in a clearly
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decreased enzymatic activity in vitro, a few mutations have a more subtle effect on the protein
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that is only revealed by in vitro tests under specific conditions.
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48 Keys words
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X-linked Sideroblastic Anemia, ALAS2, heme synthesis, protoporphyrin
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Human Mutation Page 4 of 59
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3 Introduction
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8 Congenital sideroblastic anemia (CSA) comprises a group of heterogeneous disorders
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characterized by decreased heme synthesis and mitochondrial iron overload with ringed
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sideroblasts in the bone marrow (For a review, see Camaschella, 2009). The most common
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15 genetic form of CSA, X-linked Sideroblastic Anemia (XLSA, MIM# 300751), results from
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mutations in the specific erythroid gene encoding 5-aminolevulinate synthase (ALAS2, also
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20 known as ALASE, MIM *301300, EC 2.3.1.37) (Fleming, 2002) localized on chromosome
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22 Xp11.21. ALAS is the first enzyme in the heme biosynthesis pathway and catalyzes the
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condensation of glycine and succinyl-coenzyme A into 5-aminolevulinic acid (ALA), the
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27 precursor of the tetrapyrroles (GPibson, et al., 1958; Shemin and Kikuchi, 1958). Pyridoxal 5’-
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29 phosphate (PLP) is the cofactor ofe the enzyme. Most of the 48 reported ALAS2 mutations
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responsible for XLSA are missense mutations localized in exons 4 to 11 (Bergmann, et al.). A
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34 variant in the proximal promoter was first reported as a mutation in an XLSA patient (Bekri,
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et al., 2003), but this variant was subsequently found in unaffected individuals, leading to the
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conclusion that it is a low frequency polymorphism and not a causal mutation (May A., 2005)
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41 Most of these mutations were described at the genomic level without further characterization
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of the mutated protein either in vitro or in vivo. In some patients, reduced ALAS enzymatic
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46 activity has been reported in the bone marrow (Bottomley, et al., 1992; Cotter, et al., 1995;
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48 Cox, et al., 1994; Harigae, et al., 1999a), and in others the mutated cDNA has been expressed
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in E. coli in order to study the activity of mutated protein (Cotter, et al., 1992; Cotter, et al.,
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53 1995; Cotter, et al., 1994; Cox, et al., 1994; Furuyama, et al., 1997; Furuyama, et al., 2006;
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Furuyama, et al., 1998; Harigae, et al., 1999a; Harigae, et al., 1999b; Prades, et al., 1995). In
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addition, in 2005, the crystallographic structure of ALAS2 from Rhodobacter capsulatus (R.
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Page 5 of 59 Human Mutation
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3 capsulatus) was published, making it possible to map the XLSA causing mutations (Astner, et
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al., 2005).
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8 Here we report a series of 29 probands with SA. Thirteen different ALAS2 mutations were
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found in 16 probands. Seven of the 13 mutations had never previously been described,
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including a 48-bp deletion in the proximal promoter region. The functional impact of the 12
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15 remaining missense mutations was assessed in silico using bioinformatic tools. Moreover,
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cDNAs were expressed in E. coli in order to assess the functional consequences of ten amino-
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20 acid substitutions for six novel mutations and four previously reported but so far
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22 uncharacterized mutations. ALAS2 activity, enzyme thermosensitivity and pyridoxine
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responsiveness were studied.
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Human Mutation Page 6 of 59
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3 Materials and Methods
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Patients
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8 We performed genetic analyses in 29 probands (17 males and 12 females) referred to our
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laboratory with a diagnosis of CSA based on the presence of sideroblasts in the bone marrow.
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Patients with syndromic or acquired forms of SA were excluded from this study. These
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15 patients originated from France, other Western European countries (Germany, Switzerland,
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Belgium) and Tunisia. Blood samples for genetic analysis were obtained from the patients or
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20 their parents after they had given signed informed consent in accordance with the
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parameters had been evaluated by standard methods in the respective referring hospitals, and
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27 erythrocyte protoporphyrin wasP measured as previously described (Deacon and Elder, 2001).
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X-inactivation study
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Martin et al., 2007) using the androgen receptor polymorphism as a marker.
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41 Molecular analysis of the ALAS2 gene
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Genomic DNA was extracted from peripheral blood using the QIAamp DNA blood Mini Kit
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46 (Qiagen, CA, USA). Analysis of the ALAS2 gene (Genbank genomic: NG_008983.1,
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48 Genbank mRNA: NM_000032.4, Genbank protein: NP_000023.2) was performed by
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bidirectional direct sequencing. The 11 exons of ALAS2, the proximal promoter (250 bp) and
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53 the exon-intron junctions were amplified by PCR (Supp. Table S1). After purification of PCR
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products (PCR purification kit, Qiagen CA, USA), both strands were sequenced using a Big
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Dye Terminator Cycle Sequencing kit (Applied Biosystems, Life Technologies, Carlsbad,
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60 CA, USA). Sequencing products were purified (Sephadex G50, GE Healthcare, Piscataway,
John Wiley & Sons, Inc.
Page 7 of 59 Human Mutation
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3 NJ, USA), and analyzed using a 3130xl Genetic Analyzer (Applied Biosystems, Life
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Technologies, Carlsbad, CA, USA) and the Seqscape analysis software (v2.6.0) (Applied
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8 Biosystems, Life Technologies, Carlsbad, CA, USA). Identified mutations were confirmed on
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a second sample, when available, or by family study. Numbering of ALAS2 mutations
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followed the international guidelines (www.hgvs.org/mutnomen). For the nucleotide
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sequence of the cDNA; for the amino acid numbering position 1 corresponds to the initiating
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In silico prediction of the functional impact of ALAS2 mutations
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27 The Grantham score (GranthamP, 1974) and six bioinformatic tools were used in silico to
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described (Kannengiesser, et al., 2009). The bioinformatic tools consisted of polyphen
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PANTHER (Brunham, et al., 2005), UMD Predictor (Frederic, et al., 2009) and GVGD
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(Tavtigian, et al., 2006).
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from the number of programs that predicted that the alteration would be deleterious
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