Widening the Spectrum of Deletions and Molecular Mechanisms Underlying Alpha-Thalassemia

Ann Hematol DOI 10.1007/s00277-017-3090-y

ORIGINAL ARTICLE

Widening the spectrum of deletions and molecular mechanisms underlying 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 their spanning the deletion breakpoints. Finally, in another case, no upstream regulatory elements (MCSs) give rise to α-thalasse- α-globin gene cluster deletion was found and the patient re- mia, an autosomal recessive microcytic hypochromic anemia. vealed to be a very unusual case of acquired α-thalassemia- In this study, multiplex ligation-dependent probe amplifica- myelodysplastic syndrome. This study further illustrates the tion performed with commercial and synthetic engineered diversity of genomic lesions and underlying molecular mecha- probes, Gap-PCR, and DNA sequencing were used to charac- nisms leading to α-thalassemia. terize lesions in the sub-telomeric region of the short arm of chromosome 16, possibly explaining the α-thalassemia/HbH Keywords Alpha-thalassemia . Acquired HbH . ATMDS . disease phenotype in ten patients. We have found six different Novel deletions . MLPA deletions, in heterozygosity, ranging from approximately 3.3 to 323 kb, two of them not previously described. The deletions fall into two categories: one includes deletions which totally Introduction remove the α-globin gene cluster, whereas the other includes deletions removing only the distal regulatory elements and Alpha-thalassemias (α-thal) are one of the most common ge- keeping the α-globin genes structurally intact. An indel was netic recessive disorders worldwide. They involve the impair- observed in one patient involving the loss of the MCS-R2 and ment in the biosynthesis of the α-globin chains of the hemo- theinsertionof39bporiginatedfromacomplexrearrangement globin (Hb) tetramer. Hb is composed of two α-like and two

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* Paula Faustino 6 Faculdade de Medicina, Universidade de Lisboa, Lisbon, Portugal [email protected] 7 Serviço de Gastroenterologia, Hospital de Santa Maria, Centro 1 Departamento de Genética Humana, Instituto Nacional de Saúde Hospitalar de Lisboa Norte (CHLN), Lisbon, Portugal

Doutor Ricardo Jorge (INSA), Avenida Padre Cruz, 8 1649-016 Lisbon, Portugal Serviço de Hematologia, Hospital de Santa Maria, CHLN, Lisbon, Portugal 2 Departamento de Promoção da Saúde e Prevenção de Doenças não Transmissíveis, INSA, Lisbon, Portugal 9 BioISI, Faculdade de Ciências, Universidade de Lisboa, 3 Serviço de Patologia Clínica, Hospital São Francisco Xavier, Centro Lisbon, Portugal Hospitalar de Lisboa Ocidental, Lisbon, Portugal 10 4 ToxOmics, Faculdade de Ciências Médicas, Universidade Nova de Serviço de Hemato-Oncologia, Hospital do Espírito Santo de Évora, Lisboa, Lisbon, Portugal Évora, Portugal 5 Unidade de Hematologia, Hospital D. Estefânia, Centro Hospitalar 11 ISAMB, Faculdade de Medicina, Universidade de Lisboa, de Lisboa Central, Lisbon, Portugal Lisbon, Portugal Ann Hematol

β-like chains whose genes are arranged in two different clus- (MDS; clonal hematopoietic stem cells disorders character- ters located on chromosomes 16 and 11, respectively. The α- ized by ineffective hematopoiesis and acquired genomic in- globin gene cluster is positioned near the telomere of chromo- stability) may, rarely, develop disorders of hemoglobin syn- some 16 (16p13.3) and includes an embryonic ζ-globin gene thesis, particularly α-thal with high levels of β4 tetramers (Hb and two fetal/adult α-globin genes, arranged in the order H inclusions). In these cases, the hematopoietic neoplasia telomere-ζ-α2-α1-centromere, surrounded by widely complicated with α-thal is termed α-thalassemia- expressed genes [1]. Approximately 25–65 kb upstream of myelodysplastic syndrome (ATMDS; OMIM no. 300448) the α-globin genes, there are four highly conserved non- [12–14]. Most of the reported cases of ATMDS are the result coding elements, or multispecies conserved sequences of acquired somatic point mutations in the ATRX gene [13, (MCS-R1 to MCS-R4), corresponding to erythroid-specific 15–17]. Another alternative mechanism for acquired α-thal in DNase I hypersensitive sites (HS-48, HS-40, HS-33, HS- myeloid neoplasia is clonal (somatic) deletion of the α-globin 10), which are involved in the regulation of the downstream cluster [12]. However, in several cases of ATMDS, the under- globin gene expression. The MCS-R2, or HS-40, has been lying molecular defects remain unknown. shown to be the more important distal regulatory genomic Concerning diagnosis, usually, the hematologic phenotype element for α-globin expression [1]. of microcytic hypochromic anemia is not enough to make the The two α-globin genes (HBA2 and HBA1) have identical definite diagnosis of α-thal, so a molecular procedure has to coding sequences. The most common α-thal deletions (-α3.7kb be applied, usually as follows: (i) analysis by Gap-PCR (po- and -α4.2kb) remove only one α-globin gene and cause, in the lymerase chain reaction amplification using oligo-primers heterozygous state, a very mild microcytic hypochromic ane- flanking the deletion breakpoints) to detect common deletions, mia (α+-thal). However, other larger deletions removing both (ii) direct DNA Sanger sequencing for point mutation detec- α-globin genes per allele may be observed, giving rise to a tion, and (iii) rapid quantitative analysis of gene dosage by more severe condition (α0-thal). A reduction of approximately multiplex ligation-dependent probe amplification (MLPA) or 75% of the α-globin synthesis (usually corresponding to the fine-tiling array comparative genomic hybridization (aCGH) loss of three α-globin genes) may lead to moderately severe [6, 18–23]. anemia, associated with the formation of β4 tetramers (in the Herein, we report the results of a molecular analysis, using adult) or γ4 tetramers (in the neonate), resulting in HbH dis- MLPA with commercial plus synthetic probes, Gap-PCR and ease or Hb Bart’s disease, respectively; an even higher reduc- Sanger sequencing, of ten patients with a provisional hemato- tion or complete absence of α-chains results in Hb Bart’s logical diagnosis of α-thal or HbH disease. hydrops foetalis syndrome [1]. Very rarely, α-thal may occur due to deletion of the upstream regulatory elements resulting in a severe down regulation of the α-globin gene expression Materials and methods [2–7]. Other unusual basis of α-thal is related with the ATR-16 Patient’s hematological and biochemical phenotypes syndrome (OMIM no. 141750). It results from large chromosomal rearrangements that delete many genes, including the Ten Portuguese patients (eight of them unrelated; Table 1)

α-globin genes, from the tip of the short arm of chromosome presenting microcytic hypochromic anemia, normal HbA2 16. ATR-16 is a contiguous gene syndrome where patients level, absence of iron deficiency, and none of the five more present α-thal in addition to a variable degree of facial common α-thal deletions [-α3.7kb,-α4.2kb,-MED,-SEA, dysmorphism and intellectual disability [8, 9]. Furthermore, -(α)20.5] were referred to our laboratories to search for point another rare syndrome, named ATR-X (OMIM no. 301040), mutations in the α-globin genes and to scan the 16pter region associates α-thal with severe mental retardation and charac- for unknown α-thal causing deletions. Appropriate informed teristic abnormal facial appearance. In this case, the α-globin consent was obtained from all patients studied or of their legal cluster is intact and the syndrome results from a trans-acting representatives. mutation in the X-linked ATRX gene. This gene encodes the Red blood cell indexes were obtained using a Beckman ATRX protein which contains an ATPase/helicase domain and Coulter LH 750 automated cell counter (Beckman Coulter, belongs to the SWI/SNF family of chromatin remodeling pro- Miami, FL, USA). Hemoglobin analysis and HbA2 level mea- teins. Mutations in this gene have been shown to cause diverse surement were performed by automated high performance changes in the pattern of DNA methylation, which may pro- liquid chromatography (HPLC; Hb-Gold; Drew Scientific vide a link between chromatin remodeling, DNA methylation, Ltd., Barrow-in-Furness, Cumbria, England). Hemoglobin and gene expression in developmental processes [10, 11]. capillary electrophoresis was performed in a Sebia Minicap While the classic inherited α-thal is common globally, the instrument (Sebia, Evry, France). HbH inclusion bodies were acquired forms of α-thal are very uncommon. Patients with obtained by incubating an aliquot of whole blood for 1 h at chronic myeloid disorders such as myelodysplastic syndrome 37 °C with 1% brilliant cresyl blue in buffered saline. Ann Hematol

Table 1 Hematological and biochemical data of the Patient Gender/ Hematological and biochemical parameters α-Globin Deletion Portuguese patients studied and years deletion identity the corresponding α-globin RBC Hb MCV MCH HbA2 12 deletion found in heterozygosity (10 /L) (g/dL) (fL) (pg) (%)

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, MCV mean corpuscular volume, MCH mean corpuscular hemoglobin, n.a. not applicable, patient 4* is the sister of patient 3, patient 7* is the son of patient 6 a Novel deletions were nominated according to the patient’s geographical origin: Del.2 = - -VS , where VS stands for Viseu; Del.3 = - -CBR , where CBR stands for Castelo Branco b Del. 4 resembles one deletion described by Sollaino M.C. et al., 2010 [24] c Del.6 = Indel

Screening for point mutations in HBA genes (2005) [21]. In a single MLPA reaction, each patient sample (75 ng of DNA) was tested simultaneously with Genomic DNAs were isolated from peripheral leuko- a positive, a negative, and three normal controls. The cytes using the MagNA Pure LC instrument (Roche amplified fragments were separated by capillary electro- Diagnostics GmbH, Mannheim, Germany). Screening phoresis according to their size in a 3130xl Genetic Analyzer, for point mutations in both α-globin genes was per- ABI PRISM (Applied Biosystems). Peak Scanner v1.0 formed after selective gene PCR amplification using (Applied Biosystems) was used to assess peak quality. one common forward primer 5′-GGACTCCCCTGCGG Quantitative data were obtained with Coffalyser. Net (MRC- TCCAGG-3′ and the HBA23′UTR specific primer 5′- Holland, Amsterdam, The Netherlands) and the peak areas CTCCATTGTTGGCACATTCCGGG-3′ or the HBA13′ were used, after standardization, for evaluation of copy number UTR specific primer 5′-CTGCTGTCCACGCCCATGCC- of specific genomic sequences in each sample. Since some of 3′. Sanger sequencing was performed using the Big Dye the deletions found remove the entire region of hybridization of v 1.1 Cycle Sequencing kit (Applied Biosystems) ac- commercial MLPA probes, synthetic probe usage was manda- cording to the manufacturer’s instructions. Fluorescent tory in order to map those deletions with higher accuracy signals and strand sizes were then discriminated through (Supplementary Table 1). We proceeded according to the online capillary electrophoresis in the automated sequencer manufacturer’s instructions (http://www.mlpa.com)todesign 3130xl Genetic Analyzer, ABI PRISM (Applied synthetic probes as described elsewhere [7]. Biosystems, Foster City, CA, USA), and results were analyzed using FinchTV v1.4.0 software (Geospiza, Inc). Characterization of an unusual indel complex rearrangement

Multiplex ligation-dependent probe amplification In order to characterize the breakpoints of the shortest deletion found, Gap-PCR was performed using the following MLPA was performed using the commercially available primers: Fw 5′-GCACAGGGACACAGCTGGACAC-3′ SALSA MLPA P140B HBA kit (MCR-Holland, andRv5′-GATCAGGGAGTGGGGCCAGTGG-3′.The Amsterdam, The Netherlands) following the manufac- 1.1-kb amplified fragment was sequenced as described turer’s instructions and as described in Harteveld et al. above. Sequence analysis allowed the identification of the Ann Hematol deletion breakpoints and the detection of an insertion se- and 10 within a ≈ 4.8 kb interval and the 3′ breakpoint is quence which was studied by in silico analysis. located within a ≈ 610 kb region between the TMEM8A gene and the SOX8 gene as in Del. 1. Genomic nucleotide position and in silico analysis Finally, a deletion of at least 125 kb (Del. 3) was found in a young woman (Table 1, patient 5). It removes the sub- All genomic nucleotide coordinates were determined ac- telomeric region and extends to a region of 1.8 kb between cordingtotheNCBI36/hg18assemblyintheUCSC commercial probe nos. 44 and 45, downstream of the HBA1 Genome Browser (https://genome-euro.ucsc.edu/). In gene (Fig. 2). silico analysis for secondary structure prediction was performed using Mfold Web Server (http://unafold.rna. Alpha-thalassemia caused by deletions removing the distal albany.edu/?q=mfold/DNA-Folding-Form)[25], and upstream regulatory elements of the alpha-globin genes sequence similarity search was done in NCBI BLAST keeping them structurally intact (https://blast.ncbi.nlm.nih.gov/)[26]. The three shorter α-thalassemia deletions, observed in four Portuguese patients, remove one or more of the distal regula- Results tory regions of the cluster without structurally affecting the α- globin genes. In patients 6 and 7 (Table 1, mother and son, Ten Portuguese patients (eight of them unrelated; Table 1) respectively), a deletion (Del. 4) was found segregating with presenting microcytic hypochromic anemia, normal HbA2 the α-thalassemia phenotype. It involves the sub-telomeric level, absence of iron deficiency, and none of the five more region, is not less than 88.1 kb long, and its 3′ breakpoint lies common α-thal deletions, were referred to our laboratories to in an uncertainty region between synthetic probe no. 22 and search for point mutations in the α-globin genes and to scan commercial probe no. 23 (Fig. 2). It removes at least three the 16pter region for unknown α-thal causing deletions. One MCS elements (R1, R2, and R3) located within the NPRL3 of the patients (patient 10) also presented high levels of HbH gene. As MCS-R4 is located within the 3′ breakpoint uncer- (Fig. 1). Sanger sequencing of the α-globin genes showed no tainty region downstream of the NPRL3 gene, it is not known pathogenic single nucleotide variants or micro-deletions/in- if it is removed by the deletion. Another deletion, Del. 5, has at sertions. Analysis by MLPA revealed six different deletions least 90 kb, from the sub-telomeric region down to an uncer- in the α-globin gene cluster (ranging from approximately 3.3 tainty region of the 5.7-kb region between kit probe nos. 23 to 323 kb); all were found in the heterozygous state (Fig. 2). and 24, upstream of the HBZ gene. It removes all the distal The three largest deletions (Del. 1, 2, 3) remove the entire upstream regulatory elements. cluster, whereas the other three deletions (Del. 4, 5, and 6) In this group, the smallest deletion was detected by kit remove one or more of the distal regulatory elements keeping probe nos. 13 and 14 (Del. 6) in patient 9, within the NPRL3 the α-globin genes structurally intact. gene, suggesting that only the MCS-R2 (HS-40 site) was removed in one of the patient’s alleles. Deletion breakpoint Alpha-thalassemia caused by deletions extending characterization by Gap-PCR followed by sequencing showed through the alpha-globin gene cluster and beyond the deletion extends from position chromosome 16 g.103,193 to 106,553 removing 3361 bp. In addition, an insertion of In two unrelated patients (Table 1, patients 1 and 2), the 39 bp bridging the deletion breakpoints was observed possibly deletion found (Del. 1) removes the entire region covered by resulting from a complex rearrangement involving two MLPA commercial probes except the most centromeric one. fragments of DNA from chromosome 3. The folding Therefore, the α-globin gene cluster is deleted from the prediction of this DNA segment using the Mfold software [25] sub-telomeric region to a 3′ breakpoint located within a revealed a probable hairpin loop structure (ΔG = -8.60) (Fig. 3). ≈ 610 kb region located between TMEM8A and SOX8 genes, as determined by the use of synthetic MLPA probe nos. 52 and Acquired alpha-thalassemia associated 53. Thus, this deletion has a minimal length of 323 kb from with myelodysplastic syndrome (ATMDS) chromosome 16 g.43,278 to g.366,331 (Fig. 2). The second larger deletion (Del. 2) removes the entire re- Patient 10 (Table 1) is a 61-year-old Portuguese woman pre- gion covered by MLPA commercial probes except the first senting an acquired microcytic, hypochromic anemia. In her (no. 1) and the last (no. 54). It was found in two Portuguese family, there is no history of hematologic disorder or impair- sisters (Table 1, patients 3 and 4). The use of our synthetic ment of iron homeostasis. Some years earlier, she had normal MLPA probe set allowed to conclude that it comprises at least Hb and hematimetric parameters (as shown in Supplementary 271 kb from chromosome 16 g.95,191 to g.366,331 (Fig. 2). Table 2) but, recently, she has developed a myelodysplastic The 5′ breakpoint is located between synthetic probe nos. 9 syndrome. Analysis of her bone marrow aspirate has revealed Ann Hematol

Fig. 1 Hemoglobin capillary Zones electrophoresis (Sebia) from patient 10: peak 1-HbH = 29.7%, peak 5-HbA = 68.0%, and peak 6- Z15 Z14 Z13 Z12 Z11 Z10 Z9 Z8 Z7 Z6 Z5 Z4 Z3 Z2 Z1 HbA2 = 1.0% HbA 5

HbH 1 Absorbance at415 nm wavelenght

HbA2 2 3 4 6

0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 Time in seconds Capillary electrophoresis of hemoglobins a marked dyserythropoiesis and inversion of the myeloid/ several methodologies used are interpreted as a whole, in order erythroid ratio (Supplementary Table 3). Results from Hb iso- to correctly determine the molecular basis of the patient’sphe- electric focusing suggested the presence of HbH that was con- notype and provide appropriate risk assessment and counseling. firmed and quantitated (29.7%) by capillary hemoglobin elec- The largest deletion found in unrelated patients 1 and 2 trophoresis (Fig. 1). Supravital staining of peripheral blood (Table 1) removes the entire α-globin gene cluster (-/αα) cells demonstrated approximately 50% of HbH-containing and consequently accounts for the patients’ erythrocytosis, cells. Analysis of the α-globin loci by MLPA evidenced no significant red blood cell microcytosis, and hypochromia, cor- deletion (Fig. 2; No Del.). The Sanger sequencing of HBA1 responding to an α0-thal phenotype. This deletion resembles and HBA2 did not reveal pathogenic gene variants. one previously described, the -GZ deletion [7, 21], which has the 3′ breakpoint located between TMEM8A and SOX8 genes with the latter remaining intact. Although other genes besides Discussion the α-globin genes are eliminated, our patients appear pheno- typically normal and have a normal intellect which is in ac- In this study, MLPA allowed for the detection of copy number cordance with the deletion being smaller than 800 kb [9]. variation within a region of ≈ 3.8 Mb at the tip of chromosome Otherwise, it would be expected an α-thal phenotype in asso- 16p. We used a commercial kit (containing 27 probes hybrid- ciation with dysmorphic features and intellectual disability izing within this region) which proved to be a simple and typical of the ATR-16 syndrome [9]. straightforward technology suitable for the rapid relative quan- The other two larger deletions (Dels. 2, and 3), although titative analysis of gene dosage and, in this case, for the detec- smaller than Del. 1, also remove the entire cluster and seem to tion of large deletions associated with α-thal. Further refine- have similar pathophysiological consequences. As far as we ment of deletion length was achieved by using 27 specific syn- know, these two deletions do not resemble any other previ- thetically engineered MLPA probes (Supplementary Table 1). ously described; thus, they were considered to be novel and Sanger sequencing was used in one case to identify the deletion were named according to the patients’ geographical origin: breakpoints. Therefore, for α-thal as for other hemoglobinopa- Del.2 = -VS, where VS stands for Viseu; Del.3 = -CBR, where thies, it is of the greatest importance that the results of the CBR stands for Castelo Branco (Table 1). Ann Hematol

Fig. 2 a Schematic representation of 4 Mb from the sub-telomeric NPRL3 gene. b MLPA probe ratios (y-axis) were determined by region of chromosome 16p, containing the α-globin gene cluster. comparison of their signal quantification in the studied individuals and MLPA probe hybridization sites are indicated by gray and black arrows in normal controls. MLPA probe numbers are displayed in the x-axis. referring to commercial and synthetic probes, respectively. Each probe is Deleted sequences present a probe amplification ratio around 0.5 when numbered according to their sequential order of chromosomal in heterozygosity and around zero when the target sequence is deleted in hybridization. Probe density may not allow individual numbering and both alleles (exceptions include probes nos. 33 and 36 which hybridize in therefore probe intervals are shown. Black bars represent deleted DNA both HBA2 and HBA1 and therefore, ratio can vary by 0.25-fold). Probe sequence as determined by MLPA analysis. Thin lines indicate the region no. 37 is amplified only in the presence of Hb Constant Spring. *Indicates of uncertainty for deletion breakpoints. The oval shape represents the breakpoint location of deletion 3 in the α-globin gene cluster detailed telomere. MCS-Rs are represented by vertical dark gray bars in or nearby region

The second category of deletions found in the analyzed described in Table 1, patient 9) presenting a very unusual dele- group of patients is characterized by the removal of one or more tion of this category removing only the MCS-R2 (≈ 3.3 kb). of the distal regulatory regions upstream of the α-globin gene This deletion was previously named (αα)ALT where ALTstands cluster. In these cases, despite the in cis α-globin genes being for Alentejo, a region of Southern Portugal [7]. In addition to structurally intact, they appear to be non-functional or to have a the Portuguese cases, an Italian patient was reported presenting very low expression. Most of the deletions already described also with this deletion [24] and, recently, another even smaller encompassing the MCS regions include the major regulatory deletion (742 bp) also removing only the MCS-R2 was de- element, the MCS-R2, together with a variable extent of the scribed in a Chinese family [27]. flanking DNA. This is also the case of Del. 4 where MCS-R1, As far as we know, only three naturally occurring MCS-R2, and MCS-R3 located within the NPRL3 gene were mutants have been reported as resulting from a deletion removed. This deletion resembles the largest rearrangement that knocks out only the MCS-R2 in both alleles [7, 24, previously described by Sollaino et al., 2010 [24]. Moreover, 27]. In the Portuguese case, the patient (αα)ALT/(αα)ALT pre- Del. 5 eliminated the four MCSs elements and seems identical sents a clinically overt HbH disease without requiring blood to one previously described by our group in the Portuguese transfusions [7]. Thus, the absence of the regulatory element population, named (αα)MM [3, 7]. On the other hand, here, does not completely abolish the expression of the downstream we report the third Portuguese family (whose propositus is α-globin genes (incompatible with life), giving rise to a Ann Hematol a

TTTGCC GA GGGGGG T AAAA C TT GG CCCCCCCCCCCC TA T G TTT A G TT N A C

dG = -8.60

Fig. 3 Del. 6 = 3.3 kb deletion + 39 bp insertion (indel). a Sanger 3 (19 nt from position g.144,280,515 to g.144,280,533 and 18 nt from sequencing electropherogram showing the 39 bp sequence insertion position g.157,269,186 to g.157,269,203) and two nucleotides (CC) from bridging the two deletion breakpoints of Del.6. b The 39 bp sequence unknown origin. DNA folding prediction was obtained by Mfold web might be the result of a complex rearrangement which has introduced server software [25] between the deletion breakpoints two pieces of DNA from chromosome clinical phenotype less severe than expected [28]. The besides the mentioned microcytic hypochromic anemia, a (αα)ALT deletion may have occurred by a non-homologous very high level of HbH (≈ 50% of HbH-containing cells) as recombinant event as its 3′ breakpoint is located within an Alu well a considerable amount of HbH in the hemolysate (29.7%) repeat, whereas the surrounding region of the 5′ breakpoint is were found, which is in complete agreement with the median not located in a repetitive sequence [6]. The in silico analysis of 30% reported for ATMDS cases [13]. An ATRX somatic of the 39 bp sequence suggests that it may be the result of a mutation is being investigated as a possible cause of the α-thal complex molecular mechanism of double-strand break repair in this female. This could be clinically relevant because the by insertion of sequences derived from distant regions of the ATRX protein has a conserved DNA methyltransferase do- genome, termed template sequence insertions [29]. In this main. It is thus possible that deregulation of the ATRX gene case, we hypothesize the insertion may have resulted from could lead to a weak response to the DNA hypomethylating two fragments of DNA of chromosome 3 (19 nt from position agents used in MDS treatment [30]. g.144,280,515 to g.144,280,533 and 18 nt from position In conclusion, this study widens the spectrum of molecular g.157,269,186 to g.157,269,203) with a CC orphan sequence lesions and underlying mechanisms by which α-thal determi- in between. The folding prediction of this sequence revealed a nants are produced as a result of evolutionary dynamics of the hairpin structure with ΔG=− 8.6 (Fig. 3). α-globin gene cluster. Concerning the inherited cases, despite Finally, our patient no. 10 revealed to be a case of ATMDS. the relative rarity of the large deletions in the α-globin cluster, Patients with ATMDS are usually recognized when their my- they should be investigated in suspected cases of α-thal (with eloid disorder-associated anemia is microcytic and hypochro- negative results for the common α-thal molecular lesions) as mic instead of being normocytic or macrocytic. In our patient, there is a 25% risk of having a child with Bart’s hydrops Ann Hematol foetalis or HbH disease if their partner is a carrier of an α0-thal ligation-dependent probe amplification. Blood Cells Mol Dis – or α+-thal allele, respectively. Moreover, this study empha- 44(3):146 151. doi:10.1016/j.bcmd.2009.12.011 7. Coelho A, Picanço I, Seuanes F, Seixas MT, Faustino P (2010) sizes the importance of the remote regulatory elements in the Novel large deletions in the human α-globin gene cluster: clar- long-range regulation of the α-globin gene expression. On the ifying the HS-40 long-range regulatory role in the native chro- other hand, the acquired α-thal case in the context of hemato- mosome environment. Blood Cells Mol Dis 45(2):147–153. logic malignancy should be further investigated because if the doi:10.1016/j.bcmd.2010.05.010 ATRX gene is somatically mutated, it may negatively modu- 8. Weatherall DJ, Higgs DR, Bunch C, Old JM, Hunt DM, Pressley L, Clegg JB, Bethlenfalvay NC, Sjolin S, Koler RD, late the response to MDS treatment with hypomethylating Magenis E, Francis JL, Bebbington D (1981) Hemoglobin H agents. disease and mental retardation: new syndrome or a remarkable coincidence? N Engl J Med 305(11):607–612. doi:10.1056/ NEJM198109103051103 Acknowledgements We would like to thank Unidade de Tecnologia e 9. Harteveld CL, Kriek M, Bijlsma EK, Erjavec Z, Balak D, Inovação, DGH, INSA, for the technical support. Phylipsen M, Voskamp A, di Capua E, White SJ, Giordano PC (2007) Refinement of the genetic cause of ATR-16. Hum Author contributions JF, MS, LG, SG, and PL performed the research Genet 122(3–4):283–292. doi:10.1007/s00439-007-0399-y laboratorial work. AC designed the synthetic probes. AM and FS per- 10. Gibbons RJ, Suthers GK, Wilkie AO, Buckle VJ, Higgs DR formed the hematological characterization of patient 10. PF designed the (1992) X-linked alpha-thalassaemia/mental retardation (ATR- research study, reviewed the study results, performed genotype/ X) syndrome: localization to Xq12-q21.31 by X inactivation phenotype correlations, and wrote the manuscript. JF, MS, and JG and linkage analysis. Am J Hum Genet 51(5):1136–1149 reviewed literature/databases and co-wrote the manuscript. ABR, FP, 11. Picketts DJ, Higgs DR, Bachoo S, Blake DJ, Quarrell OW, RM, PK, EM, and JFL participated in clinical enrolling/work-up of pa- Gibbons RJ (1996) ATRX encodes a novel member of the tients. JL performed a critical revision of the manuscript. All authors SNF2 family of proteins: mutations point to a common mech- revised and approved the manuscript final version. anism underlying the ATR-X syndrome. Hum Mol Genet 5(12):1899–1907. doi:10.1093/hmg/5.12.1899 Compliance with ethical standards This study was conducted in ac- 12. Steensma DP, Viprakasit V, Hendrick A, Goff DK, Leach J, cordance with the ethical standards of the institutional research committee Gibbons RJ, Higgs DR (2004) Deletion of the α-globin gene and with the 1964 Helsinki Declaration and its later amendments or com- cluster as a acquired α-thalassemia in myelodysplastic syn- parable standards. drome. Blood 103(4):1518–1520. doi:10.1182/blood-2003- All persons, or their legal representatives, gave their informed consent 09-3222 prior to their inclusion in the study. 13. Steensma DP, Gibbons RJ, Higgs DR (2005) Acquired α- thalassemia in association with myelodysplastic syndrome – Conflict of interest The authors declare that they have no conflict of and other hematologic malignancies. Blood 105(2):443 452. interest. doi:10.1182/blood-2004-07-2792 14. Steensma DP, Higgs DR, Fisher CA, Gibbons RJ (2004) Acquired somatic ATRX mutations in myelodysplastic syndrome associated with α thalassaemia (ATMDS) convey a References more severe hematologic phenotype than germline ATRX mutations. Blood 103(6):2019–2026. doi:10.1182/blood-2003-09- 1. Higgs DR (2013) The molecular basis of α-thalassemia. Cold 3360 Spring Harb Perspect Med 3(1):a011718. doi:10.1101/ 15. Nelson ME, Thurmes PJ, Hoyer JD, Steensma DP (2005) A cshperspect.a011718 novel 5’ATRX mutation with splicing consequences in ac- 2. 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