Mutations in the hematopoietic transcription factor GATA-1 alter the proliferation/differentiation of hemopoietic progenitors. Mutations in exon 2 interfere with the synthesis of the full-length isoform of GATA-1 and lead to the production of a shortened isoform, GATA-1s. These mutations have been found in patients with Diamond-Blackfan anemia (DBA), a congenital erythroid aplasia typically caused by mutations in genes encoding ribosomal proteins. We sequenced GATA-1 in 23 patients that were negative for mutations in the most frequently mutated DBA genes. One patient showed a c.2T > C mutation in the initiation codon leading to the loss of the full-length GATA-1 isoform

Aspesi, Anna

Carando, Adriana

Dianzani, Irma

Ellis, Steven R

Garelli, Emanuela

Nardi, Margherita

Parrella, Sara

Pavesi, Elisa

Quarello, Paola

Ramenghi, Ugo

Pediatric Blood & Cancer

English

PubMed

PARRELLA, SARA

ASPESI, Anna

PAVESI, Elisa

DIANZANI, Irma

Archivio Istituzionale della Ricerca- Università del Piemonte Orientale

Pediatr Blood Cancer 2014;61:1319–1321BRIEF REPORTLoss of GATA-1 Full Length as a Cause of Diamond–Blackfan Anemia PhenotypeSara Parrella, MSc,1 Anna Aspesi, PhD,1 Paola Quarello, MD, PhD,2 Emanuela Garelli, PhD,3 Elisa Pavesi, PhD,1Adriana Carando, BSc,3MargheritaNardi, MD,4 Steven R. Ellis, PhD,5 Ugo Ramenghi, MD,3 and IrmaDianzani, MD,PhD1*INTRODUCTIONIn mammals, hematopoietic homeostasis is supported by thebalance of the expression of multiple genes specific to each process.The GATA-1 gene is located on the X-chromosome (Xp11.23) andencodes a transcription factor that regulates the development oferythrocytes, megakaryocytic cells, eosinophils, mast cells, anddendritic cells [1]. GATA-1 protein has three functional domains(Fig. 1A): an N-terminal transactivation domain (TD), essential fortranscriptional activation activity, a N-terminal zinc finger (NF),and a C-terminal zinc finger (CF) responsible for the binding toDNA [1,2]. Accumulating evidence supports the notion that defectsinGATA-1 are linked to hematopoietic disorders. Exon 4 mutationshave been identified in families with dyserythropoietic anemia,thrombocytopenia, thalassemia, and erythropoietic porphyria [3].Furthermore, acquired somatic mutations in exon 2, that cause theloss of the N-terminal TD of GATA-1, have been found inindividuals with Down Syndrome (DS) who developed eithertransient myeloproliferative disorder (TMD) or acute megakaryo-blastic leukemia (AMKL) [4]. These mutations prevent thesynthesis of full-length GATA-1 (GATA-1 FL), but allow for thesynthesis of a short isoform of GATA-1 (GATA-1s), that lacks theN-terminal TD [4]. Related germline mutations have also beendescribed. Hollanda et al. [5] reported a c.220G>C mutation inseven-male members of a family all with anemia and trilineagedysplasia. Sankaran et al. [6] identified a mutation in two siblingsaffected by DBA and another mutation in a third male from a cohortof 62 additional DBA patients (Table I). Both mutations occur at thedonor splice site of exon 2 in the GATA-1 gene resulting in exonskipping. Thus only GATA-1s is produced.DBA is a normochromic–macrocytic anemia characterized byimpaired proliferation/maturation of the erythroid precursors in thebone marrow. Approximately, 30% of the patients have aheterogeneous mix of congenital malformations [7]. Sixty percentof patients carry mutations in genes encoding ribosomal proteins(RP). Systematic sequencing of all RP genes in patients with DBAhas allowed the identification of mutations in 11 RP genes [8–10].The remaining 40% of patients have mutations in unknown genes.MATERIALS AND METHODSPatientsAnalysis of the Italian DBARegistry [11] revealed 23 out of 173patients eligible for GATA-1 screening. These patients were allmales and lacked mutations/deletions in the most frequentlymutated DBA genes (RPS10, RPS17, RPS19, RPS24, RPS26,RPL5, RPL11, or RPL35A). Clinical characteristics of this cohortare presented in Supplementary Table I. DBA diagnoses were madeusing the guidelines reported in Vlachos et al. [7] Written informedconsent was provided by all subjects participating in this study.Sanger SequencingThe sixGATA-1 exons were screened for mutations in our cohortusing standard PCR-based Sanger sequencing on genomic DNAextracted from peripheral blood. Coding sequences and exon–intron boundaries were PCR amplified using GoTaq1 Flexi DNAPolymerase (Promega, Madison, WI). PCR products weresequenced in both directions using a Big Dye Terminator v1.1cycle sequencing kit (Applied Biosystems, Foster City, CA).Sequencing data were analyzed using Chromas Lite 2.1.1.RESULTSOne of the 23 patients studied showed a c.2T>C hemizygousmutation in the initiation codon used to synthetize the GATA-1 FL.This mutation was inherited from his mother (Fig. 1B). As aconsequence of this change, GATA-1s is the only isoformMutations in the hematopoietic transcription factor GATA-1 alterthe proliferation/differentiation of hemopoietic progenitors. Muta-tions in exon 2 interferewith the synthesis of the full-length isoformofGATA-1 and lead to the production of a shortened isoform, GATA-1s.These mutations have been found in patients with Diamond–Blackfan anemia (DBA), a congenital erythroid aplasia typicallycaused by mutations in genes encoding ribosomal proteins. WesequencedGATA-1 in 23 patients that were negative for mutations inthe most frequently mutated DBA genes. One patient showed ac.2T>C mutation in the initiation codon leading to the loss of thefull-length GATA-1 isoform. Pediatr Blood Cancer 2014;61:1319–1321. # 2014 Wiley Periodicals, Inc.Key words: anemia; erythropoiesis; Diamond–Blackfan; Gata-1; ribosomal proteinAdditional supporting information may be found in the online versionof this article at the publisher’s web-site.1Department of Health Sciences, University of Eastern Piedmont,Novara, Italy; 2Onco-Hematologic Center, Regina Margherita Child-ren’s Hospital, Turin, Italy; 3Department of Pediatric and Public Health,University of Turin, Turin, Italy; 4Onco-Hematologic Pediatric Center,University Hospital of Pisa, Pisa, Italy; 5Department of Biochemistryand Molecular Biology, University of Louisville, KentuckyGrant sponsor: Istituto Piemontese per la ricerca sulla Anemia diDiamond–Blackfan (to I.D. and U.R.); Grant sponsor: Diamond–Blackfan Anemia Foundation; Grant sponsor: ENERCA;Grant sponsor: Telethon Grant; Grant number: GGP 10181;Grant sponsor: Cariplo; Grant number: 2011-0554; Grant sponsor:PRIN (to I.D.); Grant sponsor: Banca del Piemonte (to U.R.)Conflict of interest: Nothing to declare.Correspondence to: Irma Dianzani, Department of Health Sciences,University of Eastern Piedmont, Via Solaroli, 17, Novara 28100, Italy.E-mail: irma.dianzani@med.unipmn.itReceived 4 October 2013; Accepted 19 December 2013C 2014 Wiley Periodicals, Inc.DOI 10.1002/pbc.24944Published online 22 January 2014 in Wiley Online Library(wileyonlinelibrary.com).expressed. Support for this view comes from a study of DSpatients with TMD [4] where mutations in the initiation codon forGATA-1 FL, that cause the loss of the first methionine, including ac.2T>C mutation, were shown to lead to the exclusive synthesisof GATA-1s.Our is the first study that reports a germline mutation in the firstATG codon of GATA-1 (Fig. 1B).The proband reported here (Supplementary Table I, patientnumber 10) was born at term after an uneventful pregnancy. At theage of 9 months he developed a severe hyporegenerative anemia(Hb 5.5 g/dl, MCV 93 fL, reticulocyte count 40,000/ml) and wasreferred to us based on a suspected diagnosis of DBA. No somaticmalformations were evident. His erythrocyte adenosine deaminaseactivity was slightly increased (1.9U/g Hb, normal values1 0.2U/g Hb). Leukocytes and platelets were in the normalrange. The bone marrow aspirate showed a selective deficiency inerythroid precursors without abnormalities of the other hematopoi-etic lineages. He was treated with a standard dose of prednisone(2mg/kg/day) for 4 weeks which was gradually reduced. Only apartial responsewas observed and his hemoglobin level reached 8 g/dl. Treatment was discontinued after 3 months and his hemoglobinlevel persisted between 8 and 9 g/dl without the need of furthertreatment including red blood cell transfusion.At the age of 4 years he developed a progressive three linearcytopenia. A bone marrow biopsy showed a severe hypocellularmarrow. The karyotype showed mosaicism with a 45XY, -7 clone(65%) and a further 50XXY, þ3, þ8, þ21 clone. On this basis, hewas diagnosed with Myelodysplastic Syndrome (MDS) after whichhe underwent Hematopoietic Stem Cell Transplantation (HSCT)from an unrelated donor. Ten years posttransplant, the patient istransfusion independent and in good health.DISCUSSIONOur study reports a patient with a hyporegenerative macrocyticanemia with a mutation in GATA-1 that interferes with theexpression of GATA-1 FL, leaving GATA-1s as the sole isoformexpressed. Mutations that abolish the expression of GATA-1 FLhave been previously identified in 10 DBA patients from threeindependent families (Table I) [5,6]. These patients showed variousdegrees of anemia, with or without an involvement of otherhematopoietic lineages (Table I). Our patient displayed aprogressive trilinear cytopenia evolving to MDS requiring HSCT.This evolution occurs infrequently in DBA but at an incidencehigher than the general population [12]. Progression to MDS is notrestricted to GATA-1 genotypes since it has also been observed inpatients with mutations in RP genes.DBA is considered the prototype of ribosomopathies, since thediscovery that a mutation in a RP can lead to the impairment of thebiogenesis and function of the ribosomes.GATA-1 is the first and sofar the only non-ribosomal protein gene that when mutated leads toDBA. In contrast to ribosomal proteins, whose essential function inall cells make it difficult to explain such a selective phenotype, arole forGATA-1 as a causative gene in DBA is much more intuitive.Given its role in the transcription of a number of genes required forerythroid development, loss of its transcription activation domain asoccurs in patients with DBA is consistent with the erythroidphenotype.Unresolved is the relationship between DBA caused bymutations in GATA-1 and RPs. It may be possible that RPhaploinsufficiency interferes with the expression of GATA-1 FL.Alternatively, the pathways involving GATA-1 and ribosomeFig. 1. GATA-1 mutation; (A) Schematic representation of GATA-1protein production, Description of normal and alternative translationinitiation sites located at methionine 1 and 84, respectively. The GATA-1 mutation in the first ATG in exon 2 promotes the production of onlyGATA-1 short isoform while in normal condition both isoforms areproduced; (B) Sanger sequencing. Themutation c.2T>C is highlightedin the box.TABLE I. Phenotype Associated to the Mutation of Exon 2 of GATA-1Reference Mutation Patient HematologySankaran et al. [6] c.220G>C Two Brothers Macrocytic anemia with low reticulocyte counts;mild reduction in neutrophils countSankaran et al. [6] c.220delG 1 Anemia without other hematologic abnormalitiesHollanda et al. [5] c.220G>C Seven affected males in a large family Macrocytic anemia and neutropeniaThis paper c.2T>C 1 Anemia and progressive three linear hypoplasiaPediatr Blood Cancer DOI 10.1002/pbc1320 Parrella et al.function may be independent but result in similar phenotypes. Theassociation of somatic GATA-1 mutations with TMD in patientswith DS, raises concerns regarding risks to patients with DBAharboring similarmutations inGATA-1.While the sample size is fartoo small to calculate relative risks for such progression, thetrilineage cytopenia and outgrowth of myelodysplastic clonesobserved in our patient indicate a need for further studies in this areaand the careful surveillance of affected individuals.REFERENCES1. Ferreira R, Ohneda K, Yamamoto M, et al. GATA1 function, a paradigm for transcription factors inhematopoiesis. Mol Cell Biol 2005;25:1215–1227.2. Calligaris R, Bottardi S, Cogoi S, et al. Alternative translation initiation site usage results in twofunctionally distinct forms of the GATA-1 transcription factor. Proc Natl Acad Sci USA 1995;92:11598–11602.3. Ciovacco WA, Raskind WH, Kacena MA. Human phenotypes associated with GATA-1 mutations. Gene2008;427:1–6.4. Kanezaki R, Toki T, Terui K, et al. Down syndrome and GATA1 mutations in transient abnormalmyeloproliferative disorder: Mutation classes correlate with progression to myeloid leukemia. Blood2010;116:4631–4638.5. Hollanda LM, Lima CS, Cunha AF, et al. An inherited mutation leading to production of only the shortisoform of GATA-1 is associated with impaired erythropoiesis. Nat Genet 2006;38:807–812.6. Sankaran VG, Ghazvinian R, Do R, et al. Exome sequencing identifies GATA1 mutations resulting inDiamond-Blackfan anemia. J Clin Invest 2012;122:2439–2443.7. Vlachos A, Ball S, Dahl N, et al. Diagnosing and treating Diamond Blackfan anaemia: Results of aninternational clinical consensus conference. Br J Haematol 2008;142:859–876.8. Boria I, Garelli E, Gazda HT, et al. The ribosomal basis of Diamond-Blackfan Anemia: Mutation anddatabase update. Hum Mutat 2010;31:1269–1279.9. Gazda HT, Preti M, Sheen MR, et al. Frameshift mutation in p53 regulator RPL26 is associated withmultiple physical abnormalities and a specific pre-ribosomal RNA processing defect in Diamond-Blackfan anemia. Hum Mutat 2012;33:1037–1044.10. Landowski M, O’Donohue MF, Buros C, et al. Novel deletion of RPL15 identified by array-comparativegenomic hybridization in Diamond-Blackfan anemia. Hum Genet 2013;132:1265–1274.11. Campagnoli MF, Ramenghi U, ArmiraglioM, et al. RPS19mutations in patients with Diamond-Blackfananemia. Hum Mutat 2008;29:911–920.12. Vlachos A, Farrar JE, Atsidaftos E, et al. Diminutive somatic deletions in the 5q region lead to aphenotype atypical of classical 5q- syndrome. Blood 2013;122:2487–2490.Pediatr Blood Cancer DOI 10.1002/pbcGATA-1 Mutations in DBA 1321

Loss of GATA-1 full length as a cause of Diamond-Blackfan anemia phenotype

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