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