ORIGINAL ARTICLE

Widening the spectrum of deletions and molecular mechanismsunderlying alpha-thalassemia

José Ferrão1 & Marisa Silva1 & Lúcia Gonçalves1 & Susana Gomes1 & Pedro Loureiro1 &

Andreia Coelho1 & Armandina Miranda2 & Filomena Seuanes2 & Ana Batalha Reis3 &

Francisca Pina4 & Raquel Maia5 & Paula Kjöllerström5& Estela Monteiro6,7 &

João F. Lacerda6,8& João Lavinha1,9 & João Gonçalves1,10 & Paula Faustino1,11

Received: 16 March 2017 /Accepted: 1 August 2017# Springer-Verlag GmbH Germany 2017

Abstract Inherited deletions of α-globin genes and/or theirupstream regulatory elements (MCSs) give rise to α-thalasse-mia, an autosomal recessive microcytic hypochromic anemia.In this study, multiplex ligation-dependent probe amplifica-tion performed with commercial and synthetic engineeredprobes, Gap-PCR, and DNA sequencing were used to charac-terize lesions in the sub-telomeric region of the short arm ofchromosome 16, possibly explaining the α-thalassemia/HbHdisease phenotype in ten patients. We have found six differentdeletions, in heterozygosity, ranging from approximately 3.3to 323 kb, two of them not previously described. The deletionsfall into two categories: one includes deletions which totallyremove the α-globin gene cluster, whereas the other includesdeletions removing only the distal regulatory elements andkeeping the α-globin genes structurally intact. An indel wasobserved in one patient involving the loss of the MCS-R2 andthe insertion of 39 bp originated from a complex rearrangement

spanning the deletion breakpoints. Finally, in another case, noα-globin gene cluster deletion was found and the patient re-vealed to be a very unusual case of acquired α-thalassemia-myelodysplastic syndrome. This study further illustrates thediversity of genomic lesions and underlying molecular mecha-nisms leading to α-thalassemia.

Keywords Alpha-thalassemia . AcquiredHbH . ATMDS .

Novel deletions . MLPA

Introduction

Alpha-thalassemias (α-thal) are one of the most common ge-netic recessive disorders worldwide. They involve the impair-ment in the biosynthesis of the α-globin chains of the hemo-globin (Hb) tetramer. Hb is composed of two α-like and two

Electronic supplementary material The online version of this article(https://doi.org/10.1007/s00277-017-3090-y) contains supplementarymaterial, which is available to authorized users.

* Paula Faustinopaula.faustino@insa.min-saude.pt

1 Departamento de Genética Humana, Instituto Nacional de SaúdeDoutor Ricardo Jorge (INSA), Avenida Padre Cruz,1649-016 Lisbon, Portugal

2 Departamento de Promoção da Saúde e Prevenção de Doenças nãoTransmissíveis, INSA, Lisbon, Portugal

3 Serviço de Patologia Clínica, Hospital São Francisco Xavier, CentroHospitalar de Lisboa Ocidental, Lisbon, Portugal

4 Serviço de Hemato-Oncologia, Hospital do Espírito Santo de Évora,Évora, Portugal

5 Unidade de Hematologia, Hospital D. Estefânia, Centro Hospitalarde Lisboa Central, Lisbon, Portugal

6 Faculdade de Medicina, Universidade de Lisboa, Lisbon, Portugal

7 Serviço de Gastroenterologia, Hospital de Santa Maria, CentroHospitalar de Lisboa Norte (CHLN), Lisbon, Portugal

8 Serviço de Hematologia, Hospital de Santa Maria, CHLN,Lisbon, Portugal

9 BioISI, Faculdade de Ciências, Universidade de Lisboa,Lisbon, Portugal

10 ToxOmics, Faculdade de Ciências Médicas, Universidade Nova deLisboa, Lisbon, Portugal

11 ISAMB, Faculdade de Medicina, Universidade de Lisboa,Lisbon, Portugal

Ann HematolDOI 10.1007/s00277-017-3090-y

β-like chains whose genes are arranged in two different clus-ters located on chromosomes 16 and 11, respectively. The α-globin gene cluster is positioned near the telomere of chromo-some 16 (16p13.3) and includes an embryonic ζ-globin geneand two fetal/adult α-globin genes, arranged in the ordertelomere-ζ-α2-α1-centromere, surrounded by widelyexpressed genes [1]. Approximately 25–65 kb upstream ofthe α-globin genes, there are four highly conserved non-coding elements, or multispecies conserved sequences(MCS-R1 to MCS-R4), corresponding to erythroid-specificDNase I hypersensitive sites (HS-48, HS-40, HS-33, HS-10), which are involved in the regulation of the downstreamglobin gene expression. The MCS-R2, or HS-40, has beenshown to be the more important distal regulatory genomicelement for α-globin expression [1].

The two α-globin genes (HBA2 and HBA1) have identicalcoding sequences. The most commonα-thal deletions (-α3.7kb

and -α4.2kb) remove only one α-globin gene and cause, in theheterozygous state, a very mild microcytic hypochromic ane-mia (α+-thal). However, other larger deletions removing bothα-globin genes per allele may be observed, giving rise to amore severe condition (α0-thal). A reduction of approximately75% of the α-globin synthesis (usually corresponding to theloss of three α-globin genes) may lead to moderately severeanemia, associated with the formation of β4 tetramers (in theadult) or γ4 tetramers (in the neonate), resulting in HbH dis-ease or Hb Bart’s disease, respectively; an even higher reduc-tion or complete absence of α-chains results in Hb Bart’shydrops foetalis syndrome [1]. Very rarely, α-thal may occurdue to deletion of the upstream regulatory elements resultingin a severe down regulation of the α-globin gene expression[2–7].

Other unusual basis of α-thal is related with the ATR-16syndrome (OMIM no. 141750). It results from large chromo-somal rearrangements that delete many genes, including theα-globin genes, from the tip of the short arm of chromosome16. ATR-16 is a contiguous gene syndrome where patientspresent α-thal in addition to a variable degree of facialdysmorphism and intellectual disability [8, 9]. Furthermore,another rare syndrome, named ATR-X (OMIM no. 301040),associates α-thal with severe mental retardation and charac-teristic abnormal facial appearance. In this case, the α-globincluster is intact and the syndrome results from a trans-actingmutation in the X-linked ATRX gene. This gene encodes theATRX protein which contains an ATPase/helicase domain andbelongs to the SWI/SNF family of chromatin remodeling pro-teins. Mutations in this gene have been shown to cause diversechanges in the pattern of DNA methylation, which may pro-vide a link between chromatin remodeling, DNAmethylation,and gene expression in developmental processes [10, 11].

While the classic inherited α-thal is common globally, theacquired forms of α-thal are very uncommon. Patients withchronic myeloid disorders such as myelodysplastic syndrome

(MDS; clonal hematopoietic stem cells disorders character-ized by ineffective hematopoiesis and acquired genomic in-stability) may, rarely, develop disorders of hemoglobin syn-thesis, particularly α-thal with high levels of β4 tetramers (HbH inclusions). In these cases, the hematopoietic neoplasiacomplicated with α-thal is termed α-thalassemia-myelodysplastic syndrome (ATMDS; OMIM no. 300448)[12–14]. Most of the reported cases of ATMDS are the resultof acquired somatic point mutations in the ATRX gene [13,15–17]. Another alternative mechanism for acquired α-thal inmyeloid neoplasia is clonal (somatic) deletion of the α-globincluster [12]. However, in several cases of ATMDS, the under-lying molecular defects remain unknown.

Concerning diagnosis, usually, the hematologic phenotypeof microcytic hypochromic anemia is not enough to make thedefinite diagnosis of α-thal, so a molecular procedure has tobe applied, usually as follows: (i) analysis by Gap-PCR (po-lymerase chain reaction amplification using oligo-primersflanking the deletion breakpoints) to detect common deletions,(ii) direct DNA Sanger sequencing for point mutation detec-tion, and (iii) rapid quantitative analysis of gene dosage bymultiplex ligation-dependent probe amplification (MLPA) orfine-tiling array comparative genomic hybridization (aCGH)[6, 18–23].

Herein, we report the results of a molecular analysis, usingMLPAwith commercial plus synthetic probes, Gap-PCR andSanger sequencing, of ten patients with a provisional hemato-logical diagnosis of α-thal or HbH disease.

Materials and methods

Patient’s hematological and biochemical phenotypes

Ten Portuguese patients (eight of them unrelated; Table 1)presenting microcytic hypochromic anemia, normal HbA2

level, absence of iron deficiency, and none of the five morecommon α-thal deletions [-α3.7kb, -α4.2kb, -MED, -SEA,-(α)20.5] were referred to our laboratories to search for pointmutations in the α-globin genes and to scan the 16pter regionfor unknown α-thal causing deletions. Appropriate informedconsent was obtained from all patients studied or of their legalrepresentatives.

Red blood cell indexes were obtained using a BeckmanCoulter LH 750 automated cell counter (Beckman Coulter,Miami, FL, USA). Hemoglobin analysis and HbA2 level mea-surement were performed by automated high performanceliquid chromatography (HPLC; Hb-Gold; Drew ScientificLtd., Barrow-in-Furness, Cumbria, England). Hemoglobincapillary electrophoresis was performed in a Sebia Minicapinstrument (Sebia, Evry, France). HbH inclusion bodies wereobtained by incubating an aliquot of whole blood for 1 h at37 °C with 1% brilliant cresyl blue in buffered saline.

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Screening for point mutations in HBA genes

Genomic DNAs were isolated from peripheral leuko-cytes using the MagNA Pure LC instrument (RocheDiagnostics GmbH, Mannheim, Germany). Screeningfor point mutations in both α-globin genes was per-formed after selective gene PCR amplification usingone common forward primer 5′-GGACTCCCCTGCGGTCCAGG-3′ and the HBA2 3′UTR specific primer 5′-CTCCATTGTTGGCACATTCCGGG-3′ or the HBA1 3′UTR specific primer 5′-CTGCTGTCCACGCCCATGCC-3′. Sanger sequencing was performed using the Big Dyev 1.1 Cycle Sequencing kit (Applied Biosystems) ac-cording to the manufacturer’s instructions. Fluorescentsignals and strand sizes were then discriminated throughcapillary electrophoresis in the automated sequencer3130xl Genetic Analyzer, ABI PRISM (AppliedBiosystems, Foster City, CA, USA), and results wereanalyzed using FinchTV v1.4.0 software (Geospiza,Inc).

Multiplex ligation-dependent probe amplification

MLPA was performed using the commercially availableSALSA MLPA P140B HBA kit (MCR-Holland,Amsterdam, The Netherlands) following the manufac-turer’s instructions and as described in Harteveld et al.

(2005) [21]. In a single MLPA reaction, each patientsample (75 ng of DNA) was tested simultaneously witha positive, a negative, and three normal controls. Theamplified fragments were separated by capillary electro-phoresis according to their size in a 3130xl Genetic Analyzer,ABI PRISM (Applied Biosystems). Peak Scanner v1.0(Applied Biosystems) was used to assess peak quality.Quantitative data were obtained with Coffalyser. Net (MRC-Holland, Amsterdam, The Netherlands) and the peak areaswere used, after standardization, for evaluation of copy numberof specific genomic sequences in each sample. Since some ofthe deletions found remove the entire region of hybridization ofcommercial MLPA probes, synthetic probe usage was manda-tory in order to map those deletions with higher accuracy(Supplementary Table 1).We proceeded according to the onlinemanufacturer’s instructions (http://www.mlpa.com) to designsynthetic probes as described elsewhere [7].

Characterization of an unusual indel complexrearrangement

In order to characterize the breakpoints of the shortest dele-tion found, Gap-PCR was performed using the followingprimers: Fw 5′-GCACAGGGACACAGCTGGACAC-3′and Rv 5′-GATCAGGGAGTGGGGCCAGTGG-3′. The1.1-kb amplified fragment was sequenced as describedabove. Sequence analysis allowed the identification of the

Table 1 Hematological andbiochemical data of thePortuguese patients studied andthe corresponding α-globindeletion found in heterozygosity

Patient Gender/years

Hematological and biochemical parameters α-Globindeletion

Deletionidentity

RBC(1012/L)

Hb(g/dL)

MCV(fL)

MCH(pg)

HbA2(%)

1 F/17 5.47 10.4 63.2 19.0 2.4 Del.1 - -GZ

2 F/39 5.41 11.5 68.0 21.3 – Del.1 - -GZ

3 F/36 5.02 11.4 71.9 22.8 2.3 Del.2a - -VS

4* F/34 4.96 11.0 70.9 22.1 2.5 Del.2a - -VS

5 F/21 5.19 11.4 69.3 22.0 2.3 Del.3a - -CBR

6 F/40 5.2 10.5 65.0 20.1 2.2 Del.4 (αα)b

7* M/10 5.31 10.8 64.7 20.3 2.5 Del.4 (αα)b

8 F/31 5.03 10.7 68.2 21.4 2.3 Del.5 (αα)MM

9 M/6 5.41 11.9 67.4 21.9 – Del.6c (αα)ALT

10 F/61 4.19 7.7 71.6 18.3 1.9 No Del. n.a.

RBC red blood cell count,Hb hemoglobin,MCVmean corpuscular volume,MCHmean corpuscular hemoglobin,n.a. not applicable, patient 4* is the sister of patient 3, patient 7* is the son of patient 6a Novel deletions were nominated according to the patient’s geographical origin: Del.2 = - -VS , where VS standsfor Viseu; Del.3 = - -CBR , where CBR stands for Castelo BrancobDel. 4 resembles one deletion described by Sollaino M.C. et al., 2010 [24]c Del.6 = Indel

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deletion breakpoints and the detection of an insertion se-quencewhichwas studied by in silico analysis.

Genomic nucleotide position and in silico analysis

All genomic nucleotide coordinates were determined ac-cording to the NCBI36/hg18 assembly in the UCSCGenome Browser (https://genome-euro.ucsc.edu/). Insilico analysis for secondary structure prediction wasperformed using Mfold Web Server (http://unafold.rna.albany.edu/?q=mfold/DNA-Folding-Form) [25], andsequence similarity search was done in NCBI BLAST(https://blast.ncbi.nlm.nih.gov/) [26].

Results

Ten Portuguese patients (eight of them unrelated; Table 1)presenting microcytic hypochromic anemia, normal HbA2

level, absence of iron deficiency, and none of the five morecommon α-thal deletions, were referred to our laboratories tosearch for point mutations in the α-globin genes and to scanthe 16pter region for unknown α-thal causing deletions. Oneof the patients (patient 10) also presented high levels of HbH(Fig. 1). Sanger sequencing of the α-globin genes showed nopathogenic single nucleotide variants or micro-deletions/in-sertions. Analysis by MLPA revealed six different deletionsin the α-globin gene cluster (ranging from approximately 3.3to 323 kb); all were found in the heterozygous state (Fig. 2).The three largest deletions (Del. 1, 2, 3) remove the entirecluster, whereas the other three deletions (Del. 4, 5, and 6)remove one or more of the distal regulatory elements keepingthe α-globin genes structurally intact.

Alpha-thalassemia caused by deletions extendingthrough the alpha-globin gene cluster and beyond

In two unrelated patients (Table 1, patients 1 and 2), thedeletion found (Del. 1) removes the entire region covered byMLPA commercial probes except the most centromeric one.Therefore, the α-globin gene cluster is deleted from thesub-telomeric region to a 3′ breakpoint located within a≈ 610 kb region located between TMEM8A and SOX8 genes,as determined by the use of syntheticMLPA probe nos. 52 and53. Thus, this deletion has a minimal length of 323 kb fromchromosome 16 g.43,278 to g.366,331 (Fig. 2).

The second larger deletion (Del. 2) removes the entire re-gion covered by MLPA commercial probes except the first(no. 1) and the last (no. 54). It was found in two Portuguesesisters (Table 1, patients 3 and 4). The use of our syntheticMLPA probe set allowed to conclude that it comprises at least271 kb from chromosome 16 g.95,191 to g.366,331 (Fig. 2).The 5′ breakpoint is located between synthetic probe nos. 9

and 10 within a ≈ 4.8 kb interval and the 3′ breakpoint islocated within a ≈ 610 kb region between the TMEM8A geneand the SOX8 gene as in Del. 1.

Finally, a deletion of at least 125 kb (Del. 3) was found in ayoung woman (Table 1, patient 5). It removes the sub-telomeric region and extends to a region of 1.8 kb betweencommercial probe nos. 44 and 45, downstream of the HBA1gene (Fig. 2).

Alpha-thalassemia caused by deletions removing the distalupstream regulatory elements of the alpha-globin geneskeeping them structurally intact

The three shorter α-thalassemia deletions, observed in fourPortuguese patients, remove one or more of the distal regula-tory regions of the cluster without structurally affecting the α-globin genes. In patients 6 and 7 (Table 1, mother and son,respectively), a deletion (Del. 4) was found segregating withthe α-thalassemia phenotype. It involves the sub-telomericregion, is not less than 88.1 kb long, and its 3′ breakpoint liesin an uncertainty region between synthetic probe no. 22 andcommercial probe no. 23 (Fig. 2). It removes at least threeMCS elements (R1, R2, and R3) located within the NPRL3gene. As MCS-R4 is located within the 3′ breakpoint uncer-tainty region downstream of the NPRL3 gene, it is not knownif it is removed by the deletion. Another deletion, Del. 5, has atleast 90 kb, from the sub-telomeric region down to an uncer-tainty region of the 5.7-kb region between kit probe nos. 23and 24, upstream of the HBZ gene. It removes all the distalupstream regulatory elements.

In this group, the smallest deletion was detected by kitprobe nos. 13 and 14 (Del. 6) in patient 9, within the NPRL3gene, suggesting that only the MCS-R2 (HS-40 site) wasremoved in one of the patient’s alleles. Deletion breakpointcharacterization byGap-PCR followed by sequencing showedthe deletion extends from position chromosome 16 g.103,193to 106,553 removing 3361 bp. In addition, an insertion of39 bp bridging the deletion breakpoints was observed possiblyresulting from a complex rearrangement involving twofragments of DNA from chromosome 3. The foldingprediction of this DNA segment using the Mfold software [25]revealed a probable hairpin loop structure (ΔG = -8.60) (Fig. 3).

Acquired alpha-thalassemia associatedwith myelodysplastic syndrome (ATMDS)

Patient 10 (Table 1) is a 61-year-old Portuguese woman pre-senting an acquired microcytic, hypochromic anemia. In herfamily, there is no history of hematologic disorder or impair-ment of iron homeostasis. Some years earlier, she had normalHb and hematimetric parameters (as shown in SupplementaryTable 2) but, recently, she has developed a myelodysplasticsyndrome. Analysis of her bone marrow aspirate has revealed

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a marked dyserythropoiesis and inversion of the myeloid/erythroid ratio (Supplementary Table 3). Results from Hb iso-electric focusing suggested the presence of HbH that was con-firmed and quantitated (29.7%) by capillary hemoglobin elec-trophoresis (Fig. 1). Supravital staining of peripheral bloodcells demonstrated approximately 50% of HbH-containingcells. Analysis of the α-globin loci by MLPA evidenced nodeletion (Fig. 2; No Del.). The Sanger sequencing of HBA1and HBA2 did not reveal pathogenic gene variants.

Discussion

In this study, MLPA allowed for the detection of copy numbervariation within a region of ≈ 3.8 Mb at the tip of chromosome16p. We used a commercial kit (containing 27 probes hybrid-izing within this region) which proved to be a simple andstraightforward technology suitable for the rapid relative quan-titative analysis of gene dosage and, in this case, for the detec-tion of large deletions associated with α-thal. Further refine-ment of deletion length was achieved by using 27 specific syn-thetically engineered MLPA probes (Supplementary Table 1).Sanger sequencing was used in one case to identify the deletionbreakpoints. Therefore, for α-thal as for other hemoglobinopa-thies, it is of the greatest importance that the results of the

several methodologies used are interpreted as a whole, in orderto correctly determine the molecular basis of the patient’s phe-notype and provide appropriate risk assessment and counseling.

The largest deletion found in unrelated patients 1 and 2(Table 1) removes the entire α-globin gene cluster (-/αα)and consequently accounts for the patients’ erythrocytosis,significant red blood cell microcytosis, and hypochromia, cor-responding to an α0-thal phenotype. This deletion resemblesone previously described, the -GZ deletion [7, 21], which hasthe 3′ breakpoint located between TMEM8A and SOX8 geneswith the latter remaining intact. Although other genes besidesthe α-globin genes are eliminated, our patients appear pheno-typically normal and have a normal intellect which is in ac-cordance with the deletion being smaller than 800 kb [9].Otherwise, it would be expected an α-thal phenotype in asso-ciation with dysmorphic features and intellectual disabilitytypical of the ATR-16 syndrome [9].

The other two larger deletions (Dels. 2, and 3), althoughsmaller than Del. 1, also remove the entire cluster and seem tohave similar pathophysiological consequences. As far as weknow, these two deletions do not resemble any other previ-ously described; thus, they were considered to be novel andwere named according to the patients’ geographical origin:Del.2 = -VS, where VS stands for Viseu; Del.3 = -CBR, whereCBR stands for Castelo Branco (Table 1).

0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300Time in seconds

Capillary electrophoresis of hemoglobins

Z15 Z14 Z13 Z12 Z11 Z10 Z9 Z8 Z7 Z6 Z5 Z4 Z3 Z2 Z1

HbH

HbA2

HbA

Absorban

ceat41

5nm

wavelen

ght

Zones

1

3 4

5

2 6

Fig. 1 Hemoglobin capillaryelectrophoresis (Sebia) from pa-tient 10: peak 1-HbH = 29.7%,peak 5-HbA = 68.0%, and peak 6-HbA2 = 1.0%

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The second category of deletions found in the analyzedgroup of patients is characterized by the removal of one or moreof the distal regulatory regions upstream of the α-globin genecluster. In these cases, despite the in cis α-globin genes beingstructurally intact, they appear to be non-functional or to have avery low expression. Most of the deletions already describedencompassing the MCS regions include the major regulatoryelement, the MCS-R2, together with a variable extent of theflanking DNA. This is also the case of Del. 4 where MCS-R1,MCS-R2, and MCS-R3 located within the NPRL3 gene wereremoved. This deletion resembles the largest rearrangementpreviously described by Sollaino et al., 2010 [24]. Moreover,Del. 5 eliminated the four MCSs elements and seems identicalto one previously described by our group in the Portuguesepopulation, named (αα)MM [3, 7]. On the other hand, here,we report the third Portuguese family (whose propositus is

described in Table 1, patient 9) presenting a very unusual dele-tion of this category removing only the MCS-R2 (≈ 3.3 kb).This deletionwas previously named (αα)ALTwhere ALTstandsfor Alentejo, a region of Southern Portugal [7]. In addition tothe Portuguese cases, an Italian patient was reported presentingalso with this deletion [24] and, recently, another even smallerdeletion (742 bp) also removing only the MCS-R2 was de-scribed in a Chinese family [27].

As far as we know, only three naturally occurringmutants have been reported as resulting from a deletionthat knocks out only the MCS-R2 in both alleles [7, 24,27]. In the Portuguese case, the patient (αα)ALT/(αα)ALT pre-sents a clinically overt HbH disease without requiring bloodtransfusions [7]. Thus, the absence of the regulatory elementdoes not completely abolish the expression of the downstreamα-globin genes (incompatible with life), giving rise to a

a

b

Fig. 2 a Schematic representation of 4 Mb from the sub-telomericregion of chromosome 16p, containing the α-globin gene cluster.MLPA probe hybridization sites are indicated by gray and black arrowsreferring to commercial and synthetic probes, respectively. Each probe isnumbered according to their sequential order of chromosomalhybridization. Probe density may not allow individual numbering andtherefore probe intervals are shown. Black bars represent deleted DNAsequence as determined byMLPA analysis. Thin lines indicate the regionof uncertainty for deletion breakpoints. The oval shape represents thetelomere. MCS-Rs are represented by vertical dark gray bars in or nearby

NPRL3 gene. b MLPA probe ratios (y-axis) were determined bycomparison of their signal quantification in the studied individuals andin normal controls. MLPA probe numbers are displayed in the x-axis.Deleted sequences present a probe amplification ratio around 0.5 whenin heterozygosity and around zero when the target sequence is deleted inboth alleles (exceptions include probes nos. 33 and 36 which hybridize inboth HBA2 and HBA1 and therefore, ratio can vary by 0.25-fold). Probeno. 37 is amplified only in the presence of Hb Constant Spring. *Indicatesbreakpoint location of deletion 3 in the α-globin gene cluster detailedregion

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clinical phenotype less severe than expected [28]. The(αα)ALT deletion may have occurred by a non-homologousrecombinant event as its 3′ breakpoint is located within an Alurepeat, whereas the surrounding region of the 5′ breakpoint isnot located in a repetitive sequence [6]. The in silico analysisof the 39 bp sequence suggests that it may be the result of acomplex molecular mechanism of double-strand break repairby insertion of sequences derived from distant regions of thegenome, termed template sequence insertions [29]. In thiscase, we hypothesize the insertion may have resulted fromtwo fragments of DNA of chromosome 3 (19 nt from positiong.144,280,515 to g.144,280,533 and 18 nt from positiong.157,269,186 to g.157,269,203) with a CC orphan sequencein between. The folding prediction of this sequence revealed ahairpin structure with ΔG = − 8.6 (Fig. 3).

Finally, our patient no. 10 revealed to be a case of ATMDS.Patients with ATMDS are usually recognized when their my-eloid disorder-associated anemia is microcytic and hypochro-mic instead of being normocytic or macrocytic. In our patient,

besides the mentioned microcytic hypochromic anemia, avery high level of HbH (≈ 50% of HbH-containing cells) aswell a considerable amount of HbH in the hemolysate (29.7%)were found, which is in complete agreement with the medianof 30% reported for ATMDS cases [13]. An ATRX somaticmutation is being investigated as a possible cause of theα-thalin this female. This could be clinically relevant because theATRX protein has a conserved DNA methyltransferase do-main. It is thus possible that deregulation of the ATRX genecould lead to a weak response to the DNA hypomethylatingagents used in MDS treatment [30].

In conclusion, this study widens the spectrum of molecularlesions and underlying mechanisms by which α-thal determi-nants are produced as a result of evolutionary dynamics of theα-globin gene cluster. Concerning the inherited cases, despitethe relative rarity of the large deletions in the α-globin cluster,they should be investigated in suspected cases of α-thal (withnegative results for the common α-thal molecular lesions) asthere is a 25% risk of having a child with Bart’s hydrops

a

b

dG = -8.60

T T T T T T T T T T T T TG G G G G G G G G G G G NC C C C C C C C C C C C C C C CA A A A A A AA

Fig. 3 Del. 6 = 3.3 kb deletion + 39 bp insertion (indel). a Sangersequencing electropherogram showing the 39 bp sequence insertionbridging the two deletion breakpoints of Del.6. b The 39 bp sequencemight be the result of a complex rearrangement which has introducedbetween the deletion breakpoints two pieces of DNA from chromosome

3 (19 nt from position g.144,280,515 to g.144,280,533 and 18 nt fromposition g.157,269,186 to g.157,269,203) and two nucleotides (CC) fromunknown origin. DNA folding prediction was obtained by Mfold webserver software [25]

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foetalis or HbH disease if their partner is a carrier of anα0-thalor α+-thal allele, respectively. Moreover, this study empha-sizes the importance of the remote regulatory elements in thelong-range regulation of the α-globin gene expression. On theother hand, the acquired α-thal case in the context of hemato-logic malignancy should be further investigated because if theATRX gene is somatically mutated, it may negatively modu-late the response to MDS treatment with hypomethylatingagents.

Acknowledgements We would like to thank Unidade de Tecnologia eInovação, DGH, INSA, for the technical support.

Author contributions JF, MS, LG, SG, and PL performed the researchlaboratorial work. AC designed the synthetic probes. AM and FS per-formed the hematological characterization of patient 10. PF designed theresearch study, reviewed the study results, performed genotype/phenotype correlations, and wrote the manuscript. JF, MS, and JGreviewed literature/databases and co-wrote the manuscript. ABR, FP,RM, PK, EM, and JFL participated in clinical enrolling/work-up of pa-tients. JL performed a critical revision of the manuscript. All authorsrevised and approved the manuscript final version.

Compliance with ethical standards This study was conducted in ac-cordance with the ethical standards of the institutional research committeeand with the 1964 Helsinki Declaration and its later amendments or com-parable standards.

All persons, or their legal representatives, gave their informed consentprior to their inclusion in the study.

Conflict of interest The authors declare that they have no conflict ofinterest.

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