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Rare α0-thalassemia deletions detected by MLPA in five unrelated Brazilian patients

Abstract

Alpha-thalassemias are among the most common genetic diseases in the world. They are characterized by hypochromic and microcytic anemia and great clinical variability, ranging from a practically asymptomatic phenotype to severe anemia, which can lead to intrauterine or early neonatal death. Deletions affecting the α-globin genes, located on chromosome 16p13.3, are the main causes of α-thalassemia. Multiplex ligation-dependent probe amplification (MLPA) can be used to detect rearrangements that cause α-thalassemia, particularly large deletions involving the whole α cluster and/or deletions in the HS-40 region. Here, MLPA was used to investigate the molecular basis of α-thalassemia in five unrelated patients, three of whom had Hb H disease. In addition to the -α3.7 deletion identified in the patients with Hb H disease, four different α0 deletions removing 15 to 225 kb DNA segments were found: two of them remove both the α genes, one affects only the regulatory element (HS-40) region, and another one extends over the entire α cluster and the HS-40 region. These results illustrate the diversity of α-thalassemia deletions in the Brazilian population and highlight the importance of molecular investigation in cases that present with microcytosis and hypochromia without iron deficiency and normal or reduced Hb A2 levels..

Keywords:
α-Thalassemia; Hb H disease; multiplex ligation-dependent probe amplification; MLPA; Brazilian population

Thalassemias are among the most frequently found genetic diseases in populations. They are caused by mutations that affect the globin genes, reducing or preventing synthesis of one or more globin chains. The α-thalassemias, characterized by reduced α-globin synthesis, are generally caused by deletions that partially or completely remove one (-α) or both (--) α genes in the haploid genome or, more rarely, the α-globin major regulatory element (HS-40). They are classified as either α+, when there is partial synthesis of α chains, or α0, when there is no production of these chains (Bunn and Forget, 1986Bunn HF and Forget BG (1986) Hemoglobin: Molecular, Genetic and Clinical Aspects. W.B. Saunders Company, Philadelphia, 690 p.; Harteveld and Higgs, 2010Harteveld CL and Higgs DR (2010) Alpha-thalassaemia. Orphanet J Rare Dis 5:13.; Galanello and Cao, 2011Galanello R and Cao A (2011) Gene test review. Alpha-thalassemia. Genet Med 13:83-88.).

Individuals that are heterozygous for α+-thalassemia (-α/αα) have minimal or no hematological changes, while individuals homozygous for α+-thalassemia (-α/-α) and heterozygous for α0-thalassemia (--/αα) show moderate microcytosis and hypochromia. The presence of only one functional α gene (--/-α) results in chronic, moderate or severe hemolytic anemia, jaundice and hepatosplenomegaly, a condition known as Hb H disease. Homozygosity for α0-thalassemia (--/--) leads to Hb Bart's hydrops fetalis syndrome with severe tissue hypoxia. Without medical intervention it is incompatible with life and leads to intrauterine or early neonatal death (Bunn and Forget, 1986Bunn HF and Forget BG (1986) Hemoglobin: Molecular, Genetic and Clinical Aspects. W.B. Saunders Company, Philadelphia, 690 p.; Harteveld and Higgs, 2010Harteveld CL and Higgs DR (2010) Alpha-thalassaemia. Orphanet J Rare Dis 5:13.; Galanello and Cao, 2011Galanello R and Cao A (2011) Gene test review. Alpha-thalassemia. Genet Med 13:83-88.).

α-Thalassemia is estimated to affect around 5% of the population worldwide, with the -α3.7 deletion being the most common alteration (Piel and Weatherall, 2014Piel FB and Weatherall DJ (2014). The α-thalassemias. N Engl J Med 371:1908-1916.). In Brazil, its prevalence is high (Sonati et al., 1991Sonati MF, Farah SB, Ramalho AS and Costa FF (1991) High prevalence of alpha-thalassemia in a Black population of Brazil. Hemoglobin 15:309-311.; Couto et al., 2003Couto FD, Albuquerque ABL, Adorno V, Neto JPM, Abbehusen LF, Oliveira JLB, Reis MG and Gonçalves MS (2003) α-Thalassemia 2, 3.7 kb deletion and hemoglobin AC heterozygosity in pregnancy: A molecular and hematological analysis. Clin Lab Haem 25:29-34.; Adorno et al., 2005Adorno EV, Couto FD, Neto JPM, Menezes JF, Rêgo M, Reis MG and Gonçalves MS (2005) Hemoglobinopathies in newborns from Salvador, Bahia, Northeast Brazil. Cad Saude Publica 21:292-298.; Wagner et al., 2010Wagner SC, de Castro SM, Gonzalez TP, Santin AP, Filippon L, Zaleski CF, Azevedo LA, Amorin B, Callegari-Jacques SM and Hutz MH(2010) Prevalence of common α-thalassemia determinants in south Brazil: Importance for the diagnosis of microcytic anemia. Genet Mol Biol 33:641-645.; Cardoso et al., 2012Cardoso GL, Takanashi SYL and Guerreiro JF (2012) Inherited hemoglobin disorders in an Afro-Amazonian community: Saracura. Genet Mol Biol 35:553-556.; De Medeiros Alcoforado et al., 2012De Medeiros Alcoforado GH, Bezerra CM, Lemos TMAM, Oliveira DM, Kimura EM, Costa FF, Sonati MF and Medeiros TMD (2012) Prevalence of α-thalassemia 3.7 kb deletion in the adult population of Rio Grande do Norte, Brazil. Genet Mol Biol 35:594-598.). However, Hb H disease, which is found primarily in Southeast Asia, the Middle East and the Mediterranean, has only rarely been reported in Brazil, where most cases are the result of an interaction of the -α3.7 deletion with the --MED, -(α)20.5, or --SEA deletions (Sonati et al., 1992Sonati MF, Kimura EM, Grotto HZ, Tavella MH and Costa FF (1992) HbH disease associated with the (--MED) deletion in a Brazilian Black woman. Acta Haematol 87:145-147.; Wenning et al., 2000Wenning MR, Kimura EM, Costa FF, Saad STO, Gervasio S, Jorge SB, Borges E, Silva NM and Sonati MF (2000) α-Globin genes: Thalassemic and structural alterations in a Brazilian population. Braz J Med Biol Res 33:1041-1045., 2002Wenning MRSC, Harteveld CL, Giordano PC, Kimura EM, Saad STO, Costa FF and Sonati MF (2002) Hemoglobin H disease resulting from the association of the -α3.7 rightward deletion and the (αα)MM deletion in a Brazilian patient. Eur J Haematol 69:179-181., 2009Wenning MR, Mello MP, Andrade TG, Lanaro C, Albuquerque DM, Saad ST, Costa FF and Sonati MF (2009) PIP4KIIA and beta-globin: Transcripts differentially expressed in reticulocytes and associated with high levels of Hb H in two siblings with Hb H disease. Eur J Haematol 83:490-493.; Kimura et al., 2009Kimura EM, Oliveira DM, Fertrin K, Pinheiro VR, Jorge SEDC, Costa FF and Sonati MF (2009) Hb H disease resulting from the association of an αº-thalassemia allele [-(α)20.5] with an unstable α-globin variant [Hb Icaria]: First report on the occurrence in Brazil. Genet Mol Biol 32:712-715.). Combinations of the -α3.7 deletion with new or rare α0 deletions started to be detected in the Brazilian population more recently, suggesting that the prevalence of Hb H disease may be underestimated (Suemasu et al., 2011Suemasu CN, Kimura EM, Oliveira DM, Bezerra MAC, Araújo AS, Costa FF and Sonati MF (2011) Characterization of alpha thalassemic genotypes by multiplex ligation-dependent probe amplification in the Brazilian population. Braz J Med Biol Res 44:16-22.).

The deletions that most commonly cause α-thalassemia in populations [-α3.7, -α4.2, -(α)20.5, --MED, --SEA, --FIL, --THAI] are easily detected by multiplex gap PCR (Chong et al., 2000Chong SS, Boehm CD, Higgs DR and Cutting GR (2000) Single tube multiplex-PCR screen for common deletional determinants of alpha-thalassemia. Blood 95:360-362.), a technique that can only be used to screen known deletions. Multiplex ligation-dependent probe amplification (MLPA) is a sensitive technique that allows relative quantification of target regions in the genome and can be used to detect gene deletions and duplications and estimate their lengths (Schouten et al., 2002Schouten JP, McElgunn CJ, Waaijer R, Zwijnenburg D, Diepvens F and Pals G (2002) Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification. Nucleic Acids Res 30:e57.; Harteveld et al., 2005Harteveld CL, Voskamp A, Phylipsen M, Akkermans N, den Dunnen JT, White SJ and Giordano PC (2005) Nine unknown rearrangements in 16p13.3 and 11p15.4 causing alpha- and beta-thalassaemia characterised by high resolution multiplex ligation-dependent probe amplification. J Med Genet 42:922-931.; Stuppia et al., 2012Stuppia L, Antonucci I, Palka G and Gatta V (2012) Use of the MLPA assay in the molecular diagnosis of gene copy number alterations in human genetic diseases. Int J MolSci 13:3245-3276.). MLPA was used here to investigate the molecular basis of α-thalassemia in five unrelated patients, three of whom had Hb H disease.

This study was approved by the Research Ethics Committee at the School of Medical Sciences, Unicamp (CEP/FCM/Unicamp) under reference number 918/2007 dated February 18, 2007. All patients or responsibles gave their written consent.

Five unrelated patients (P) with suspected thalassemia were referred to our laboratory for investigation. Three of them had Hb H disease, while the other two presented with microcytosis, hypochromia and normal Hb A2 levels without iron deficiency. Familial analysis could only be carried out for four patients (Tables 1 and 2).

Table 1
Hematological and molecular data of Patients P1 - P3 and their families, and P4.
Table 2
Hematological and molecular data of Patient P5 and her family.

A Sysmex hematology analyzer (Sysmex XE2100, Sysmex, Kobe, Japan) was used for cell counts and hematological indices; cation-exchange high-performance liquid chromatography (HPLC) (Variant, Bio-Rad Laboratories, Inc., Hercules, CA, USA) and electrophoresis on cellulose acetate in neutral and alkaline pHs were used in the hemoglobin analysis. Hb H inclusion bodies were observed in the three patients with Hb H disease after whole blood was incubated with brilliant cresyl blue (Dacie and Lewis,1995Dacie JV and Lewis SM (1995) Practical Haematology. 8th edition. Churchill Livingstone, Edinburgh, 609 p.).

Genomic DNA was extracted from peripheral blood leukocytes using a commercial kit (QIAamp DNA Blood Mini Kit, Qiagen® GmbH, Hilden, Germany), and multiplex-gap-PCR was used to screen for the seven most common α-thalassemia deletions [-α3.7, -α4.2, -(α)20.5, --MED, --SEA, --FIL, --THAI] (Chong et al., 2000Chong SS, Boehm CD, Higgs DR and Cutting GR (2000) Single tube multiplex-PCR screen for common deletional determinants of alpha-thalassemia. Blood 95:360-362.). A search for the most prevalent non-deletional mutations (αHphIα, αNcoIα, ααNcoI, αTSaudiα) was carried out after selective amplification of the α genes, followed by analysis with the respective restriction enzymes, or, in the case of TSaudi, a specific nested PCR (Kattamis et al., 1996Kattamis AC, Carnaschella C, Sivera P, Surrey S and Fortina P (1996) Human α-thalassemia syndromes: Detection of molecular defects. Am J Hematol 53:81-91.). The -α3.7 deletion was only detected in the three patients with Hb H disease. Hematological and molecular data for the five patients are shown in Table 1.

To identify the α0 deletions, MLPA was performed with the SALSA MLPA P140 C1 HBA kit (MRC-Holland, Amsterdam, The Netherlands), which allowed to examine the region extending from the telomeric region of chromosome 16p to the DECR2 gene (approximately 360 kb of DNA). Comparative analysis of the fragments was performed using the Coffalyser. Net software to evaluate possible changes in the number of copies of the α locus in the samples.

In the three cases with Hb H disease (P1, P4 and P5), -α3.7 was present in combination with the α0 deletion, while the two other cases were heterozygous for α0-thalassemia. P1 and P3 had the same pattern of deletions in MLPA, including probes 318 to 283 and the region between them, with a deletion extending from ψζ (the zeta pseudogene) to the downstream region of the α1 gene. P2 had a deletion restricted to the HS-40 region that involved probes 236 to 364 and left the genes in the α cluster structurally intact, while P4 had a deletion between probes 292 and 400 that also extended from the ψζ gene to the downstream region of the α1 gene. In P5, the deletion detected affected a larger fragment, extending from the telomere to part of the RGS11 gene and involving probes 463 to 472. The five deletions are schematically shown in Figure 1.

Figure 1
Schematic representation of chromosome 16p13.3. The oval represents the telomeric region, the arrows the locations of the probes and the boxes the genes. The black bars correspond to the deleted fragments, the dotted lines denote the first and last deleted probes delimiting the segments containing the breakpoints (adapted from MRC-Holland, provider of the MLPA kit).

Familial studies revealed the α0 allele in P1's father and the -α3.7 deletion in his mother and brother. P2 inherited the deletion in the HS-40 region from her mother, but no deletions were detected in her father. P3 has a daughter with the same molecular defect. P4's family was not available for familial analysis. P5's mother had the -α3.7 deletion, while the α0 allele was inherited from his father; of the patient's six siblings, one had the -α3.7 deletion, two sisters had the α0 deletion and two brothers also had Hb H disease. Only one member of this family of nine (one of the patient's brothers) did not have any deletions. Figure 2 shows the pedigrees of the four families studied.

Figure 2
Pedigrees of the families studied. (A) P1 has Hb H disease (-α3.7 and α0 alleles), while his mother and brother are heterozygous for the -α3.7 deletion and his father for the α0 deletion. (B) P2 and her mother and brother are heterozygous for the α0 deletion, while her father has normal α-genotype (no deletion). (C) P3 and her daughter are heterozygous for the α0 deletion. (D) P5 and two of her brothers have Hb H disease (-α3.7 and α0 alleles); her mother and two sisters are heterozygous for the -α3.7 deletion, while her father and one of her brothers are heterozygous for the α0 deletion. Only one of the siblings does not have any deletions.

Multiplex-gap-PCR is the most widely used method to screen for α-thalassemia and can identify the deletions that most frequently affect populations worldwide. However, there are cases of patients with reduced MCV and MCH values, normal or reduced Hb A2 and Hb F and normal iron status in whom a diagnosis could not be reached. In these patients it is important to investigate rare or new deletions that affect the α genes and/or their regulatory elements. The combination of two α0 deletions results in Hb Bart's hydrops fetalis syndrome, while the association of these with more common deletions, such as the -α3.7 deletion, causes Hb H disease, a moderate or severe type of hemolytic anemia that may require blood transfusions and/or splenectomy. Even for heterozygotes, the correct diagnosis is extremely important, as microcytosis and hypochromia are frequently interpreted as indications of iron deficiency (Borges et al., 2001Borges E, Wenning MRSC, Kimura EM, Gervásio SA, Costa FF and Sonati MF (2001) High prevalence of alpha-thalassemia among individuals with microcytosis and hypochromia without anemia. Braz J Med Biol Res 34:759-762.) and incorrectly treated.

Patients P1 and P3 appear to have a 15 kb deletion (positions 162735-177934 according to the USCS Genome Browser, March 2006), which is similar to that described as --GB by Harteveld et al. (2005)Harteveld CL, Voskamp A, Phylipsen M, Akkermans N, den Dunnen JT, White SJ and Giordano PC (2005) Nine unknown rearrangements in 16p13.3 and 11p15.4 causing alpha- and beta-thalassaemia characterised by high resolution multiplex ligation-dependent probe amplification. J Med Genet 42:922-931. in a Dutch individual of mixed ethnic backgrounds, and found again, by Phylipsen et al. (2010)Phylipsen M, Prior JF, Lim E, Lingam N, Vogelaar IP, Giordano PC, Finlayson J and Harteveld CL (2010) Thalassemia in Western Australia: 11 novel deletions characterized by multiplex ligation-dependent probe amplification. Blood Cells Mol Dis 44:146-151., in three unrelated individuals of Arabic, Indian and unknown origin. Patient P2 has a deletion of approximately 97 kb that removes the α-MRE (positions 46407-143677 according to the USCS Genome Browser, March 2006); this is similar to the (αα)MM deletion reported by Romão et al. (1991)Romão L, Osório-Almeida L, Higgs DR, Lavinha J and Liebhaber SA (1991) α-Thalassemia resulting from deletion of regulatory sequences far upstream of the α-globin structural genes. Blood 78:1589-1595. in a child from the Azores, and by Wenning et al. (2002)Wenning MRSC, Harteveld CL, Giordano PC, Kimura EM, Saad STO, Costa FF and Sonati MF (2002) Hemoglobin H disease resulting from the association of the -α3.7 rightward deletion and the (αα)MM deletion in a Brazilian patient. Eur J Haematol 69:179-181. in a Brazilian family. In P4, the deletion of about 22 kb (positions 159487-181215 of the USCS Genome Browser, March 2006) appears to be the same as that described by Phylipsen et al. (2010)Phylipsen M, Prior JF, Lim E, Lingam N, Vogelaar IP, Giordano PC, Finlayson J and Harteveld CL (2010) Thalassemia in Western Australia: 11 novel deletions characterized by multiplex ligation-dependent probe amplification. Blood Cells Mol Dis 44:146-151. in two unrelated individuals of Indian and unknown origin. P5 has a deletion of approximately 225 kb (positions 46407-271806 according to the USCS Genome Browser, March 2006) that affects the entire α cluster and the α-MRE, and is of similar length to that described by Suemasu et al. (2011)Suemasu CN, Kimura EM, Oliveira DM, Bezerra MAC, Araújo AS, Costa FF and Sonati MF (2011) Characterization of alpha thalassemic genotypes by multiplex ligation-dependent probe amplification in the Brazilian population. Braz J Med Biol Res 44:16-22. in a Brazilian patient. It is possible that the deletions found in the patients studied here are the same as those previously described, and that have they been introduced to Brazil by immigration. However, as their breakpoints have not yet been accurately identified, we cannot rule out the possibility that one or even all of these are new deletions.

With the development of new techniques and technologies, an increasing number of novel and rare deletions compromising both alpha genes have been detected, suggesting that their frequencies (and heterogeneity) may be underestimated in populations (Gilad et al., 2014Gilad O, Dgany O, Noy-Lotan S, Krasnov T, Elitzur S, Pissard S, Kventsel I, Yacobovich J and Tamary H (2014) Characterization of two unique α-globin gene cluster deletions causing α-thalassemia in Israeli Arabs. Hemoglobin 38:319-324.; Brieghel et al., 2015Brieghel C, Birgens H, Frederiksen H, Hertz JM, Steenhof M and Petersen J (2015) Novel 31.2 kb deletion in a Palestinian family with α-thalassemia. Hemoglobin 39:346-349.; de-la-Cruz-Salcedo et al., 2016de-la-Cruz-Salcedo EI, Ibarra B, Rizo-de-la-Torre LC, Sánchez-López JY, González-Mercado A, Harteveld CL and Perea-Diaz FJ (2016) Molecular analysis of complex cases of alpha- and beta-thalassemia in Mexican mestizo patients with microcytosis and hypochromia reveals two novel alpha(0)-thalassemia deletions --(Mex1) and --(Mex2). Int J Lab Hematol 38:535-542.; Hu et al., 2016Hu L, Shang X, Yi S, Cai R, Li Z, Liu C, Liang Y, Cai D, Zhang F and Xu X (2016) Two novel copy number variations involving the α-globin gene cluster on chromosome 16 cause thalassemia in two Chinese families. Mol Genet Genomics 291:1443-1450.; Mota et al., 2017Mota NO, Kimura EM, Ferreira RD, Albuquerque DM, Ribeiro DM, Santos MN, Costa FF and Sonati MF (2017) A novel αº-thalassemia deletion in a Brazilian child with Hb H disease [--(BRAZ)]. Int J Lab Hematol 39:e106-e109.; Wang et al., 2017Wang Y, Liu C, Zhang L, Du L, Zhou W, Huang S, Liu L and Yin A (2017) Identification of a novel 44.6-kb deletion causing α0-thalassemia in southern China. Int J Lab Hematol 39:e94-e97.; Wu et al., 2017Wu MY, He Y, Yan JM and Li DZ (2017) A novel selective deletion of the major α-globin regulatory element (MCS-R2) causing α-thalassemia. Br J Haematol 176:984-986.). Our findings highlight the importance of using MLPA in the characterization of rare deletions, allowing the molecular basis of α-thalassemia to be elucidated when conventional methods fail. In addition to allowing correct diagnosis and treatment of carriers, characterization of these changes is important in genetic counseling, as it allows couples to understand the risk of having an affected child and to make an informed reproductive decision. Furthermore, knowledge of the size of these deletions and the genes and regulatory elements affected by them can greatly help to elucidate the genetic recombination mechanisms in the affected regions and the functions of the α-globin genes, about which little is yet known to date.

Acknowledgments

This study received financial support from the State of São Paulo Research Foundation-FAPESP (grant number 2014/00984-3; fellowship no. 2015/21184-8) and the National Council for Scientific and Technological Development (CNPq). N.O. Mota was the recipient of a fellowship from the Agency for the Support and Evaluation of Graduate Education (CAPES) of the Brazilian Ministry of Education.

References

  • Adorno EV, Couto FD, Neto JPM, Menezes JF, Rêgo M, Reis MG and Gonçalves MS (2005) Hemoglobinopathies in newborns from Salvador, Bahia, Northeast Brazil. Cad Saude Publica 21:292-298.
  • Borges E, Wenning MRSC, Kimura EM, Gervásio SA, Costa FF and Sonati MF (2001) High prevalence of alpha-thalassemia among individuals with microcytosis and hypochromia without anemia. Braz J Med Biol Res 34:759-762.
  • Brieghel C, Birgens H, Frederiksen H, Hertz JM, Steenhof M and Petersen J (2015) Novel 31.2 kb deletion in a Palestinian family with α-thalassemia. Hemoglobin 39:346-349.
  • Bunn HF and Forget BG (1986) Hemoglobin: Molecular, Genetic and Clinical Aspects. W.B. Saunders Company, Philadelphia, 690 p.
  • Cardoso GL, Takanashi SYL and Guerreiro JF (2012) Inherited hemoglobin disorders in an Afro-Amazonian community: Saracura. Genet Mol Biol 35:553-556.
  • Chong SS, Boehm CD, Higgs DR and Cutting GR (2000) Single tube multiplex-PCR screen for common deletional determinants of alpha-thalassemia. Blood 95:360-362.
  • Couto FD, Albuquerque ABL, Adorno V, Neto JPM, Abbehusen LF, Oliveira JLB, Reis MG and Gonçalves MS (2003) α-Thalassemia 2, 3.7 kb deletion and hemoglobin AC heterozygosity in pregnancy: A molecular and hematological analysis. Clin Lab Haem 25:29-34.
  • Dacie JV and Lewis SM (1995) Practical Haematology. 8th edition. Churchill Livingstone, Edinburgh, 609 p.
  • de-la-Cruz-Salcedo EI, Ibarra B, Rizo-de-la-Torre LC, Sánchez-López JY, González-Mercado A, Harteveld CL and Perea-Diaz FJ (2016) Molecular analysis of complex cases of alpha- and beta-thalassemia in Mexican mestizo patients with microcytosis and hypochromia reveals two novel alpha(0)-thalassemia deletions --(Mex1) and --(Mex2). Int J Lab Hematol 38:535-542.
  • De Medeiros Alcoforado GH, Bezerra CM, Lemos TMAM, Oliveira DM, Kimura EM, Costa FF, Sonati MF and Medeiros TMD (2012) Prevalence of α-thalassemia 3.7 kb deletion in the adult population of Rio Grande do Norte, Brazil. Genet Mol Biol 35:594-598.
  • Galanello R and Cao A (2011) Gene test review. Alpha-thalassemia. Genet Med 13:83-88.
  • Gilad O, Dgany O, Noy-Lotan S, Krasnov T, Elitzur S, Pissard S, Kventsel I, Yacobovich J and Tamary H (2014) Characterization of two unique α-globin gene cluster deletions causing α-thalassemia in Israeli Arabs. Hemoglobin 38:319-324.
  • Harteveld CL and Higgs DR (2010) Alpha-thalassaemia. Orphanet J Rare Dis 5:13.
  • Harteveld CL, Voskamp A, Phylipsen M, Akkermans N, den Dunnen JT, White SJ and Giordano PC (2005) Nine unknown rearrangements in 16p13.3 and 11p15.4 causing alpha- and beta-thalassaemia characterised by high resolution multiplex ligation-dependent probe amplification. J Med Genet 42:922-931.
  • Hu L, Shang X, Yi S, Cai R, Li Z, Liu C, Liang Y, Cai D, Zhang F and Xu X (2016) Two novel copy number variations involving the α-globin gene cluster on chromosome 16 cause thalassemia in two Chinese families. Mol Genet Genomics 291:1443-1450.
  • Kattamis AC, Carnaschella C, Sivera P, Surrey S and Fortina P (1996) Human α-thalassemia syndromes: Detection of molecular defects. Am J Hematol 53:81-91.
  • Kimura EM, Oliveira DM, Fertrin K, Pinheiro VR, Jorge SEDC, Costa FF and Sonati MF (2009) Hb H disease resulting from the association of an αº-thalassemia allele [-(α)20.5] with an unstable α-globin variant [Hb Icaria]: First report on the occurrence in Brazil. Genet Mol Biol 32:712-715.
  • Mota NO, Kimura EM, Ferreira RD, Albuquerque DM, Ribeiro DM, Santos MN, Costa FF and Sonati MF (2017) A novel αº-thalassemia deletion in a Brazilian child with Hb H disease [--(BRAZ)]. Int J Lab Hematol 39:e106-e109.
  • Phylipsen M, Prior JF, Lim E, Lingam N, Vogelaar IP, Giordano PC, Finlayson J and Harteveld CL (2010) Thalassemia in Western Australia: 11 novel deletions characterized by multiplex ligation-dependent probe amplification. Blood Cells Mol Dis 44:146-151.
  • Piel FB and Weatherall DJ (2014). The α-thalassemias. N Engl J Med 371:1908-1916.
  • Romão L, Osório-Almeida L, Higgs DR, Lavinha J and Liebhaber SA (1991) α-Thalassemia resulting from deletion of regulatory sequences far upstream of the α-globin structural genes. Blood 78:1589-1595.
  • Schouten JP, McElgunn CJ, Waaijer R, Zwijnenburg D, Diepvens F and Pals G (2002) Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification. Nucleic Acids Res 30:e57.
  • Sonati MF, Farah SB, Ramalho AS and Costa FF (1991) High prevalence of alpha-thalassemia in a Black population of Brazil. Hemoglobin 15:309-311.
  • Sonati MF, Kimura EM, Grotto HZ, Tavella MH and Costa FF (1992) HbH disease associated with the (--MED) deletion in a Brazilian Black woman. Acta Haematol 87:145-147.
  • Stuppia L, Antonucci I, Palka G and Gatta V (2012) Use of the MLPA assay in the molecular diagnosis of gene copy number alterations in human genetic diseases. Int J MolSci 13:3245-3276.
  • Suemasu CN, Kimura EM, Oliveira DM, Bezerra MAC, Araújo AS, Costa FF and Sonati MF (2011) Characterization of alpha thalassemic genotypes by multiplex ligation-dependent probe amplification in the Brazilian population. Braz J Med Biol Res 44:16-22.
  • Wagner SC, de Castro SM, Gonzalez TP, Santin AP, Filippon L, Zaleski CF, Azevedo LA, Amorin B, Callegari-Jacques SM and Hutz MH(2010) Prevalence of common α-thalassemia determinants in south Brazil: Importance for the diagnosis of microcytic anemia. Genet Mol Biol 33:641-645.
  • Wang Y, Liu C, Zhang L, Du L, Zhou W, Huang S, Liu L and Yin A (2017) Identification of a novel 44.6-kb deletion causing α0-thalassemia in southern China. Int J Lab Hematol 39:e94-e97.
  • Wenning MR, Kimura EM, Costa FF, Saad STO, Gervasio S, Jorge SB, Borges E, Silva NM and Sonati MF (2000) α-Globin genes: Thalassemic and structural alterations in a Brazilian population. Braz J Med Biol Res 33:1041-1045.
  • Wenning MRSC, Harteveld CL, Giordano PC, Kimura EM, Saad STO, Costa FF and Sonati MF (2002) Hemoglobin H disease resulting from the association of the -α3.7 rightward deletion and the (αα)MM deletion in a Brazilian patient. Eur J Haematol 69:179-181.
  • Wenning MR, Mello MP, Andrade TG, Lanaro C, Albuquerque DM, Saad ST, Costa FF and Sonati MF (2009) PIP4KIIA and beta-globin: Transcripts differentially expressed in reticulocytes and associated with high levels of Hb H in two siblings with Hb H disease. Eur J Haematol 83:490-493.
  • Wu MY, He Y, Yan JM and Li DZ (2017) A novel selective deletion of the major α-globin regulatory element (MCS-R2) causing α-thalassemia. Br J Haematol 176:984-986.

Internet Resources

  • University of California Santa Cruz (UCSC) Genome Browser, http://genome.ucsc.edu (accessed March 2006).
    » http://genome.ucsc.edu
  • Associate Editor: Angela M. Vianna-Morgante

Publication Dates

  • Publication in this collection
    02 Oct 2017
  • Date of issue
    Oct-Dec 2017

History

  • Received
    16 Dec 2016
  • Accepted
    23 July 2017
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