http://informahealthcare.com/hem ISSN: 0363-0269 (print), 1532-432X (electronic) Hemoglobin, 2014; 38(6): 427–430 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/03630269.2014.976793
ORIGINAL ARTICLE
An Alu Element-Mediated 28.5 kb a-Thalassemia Deletion Found in a Chinese Family Jing Yu1*, Jun Xie1*, Liya Luo2, and Zesong Li2 1
Department of Laboratory Medicines, Peking University Shenzhen Hospital, Shenzhen, People’s Republic of China, Shenzhen Key Laboratory of Genitourinary Tumor, Shenzhen Second People’s Hospital, First Affiliated Hospital of Shenzhen University, Shenzhen, People’s Republic of China
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Abstract
Keywords
Over 95.0% of the a-thalassemia (a-thal) cases in southern China are caused by large deletions involving the a-globin gene. Here, we describe the molecular characterization of a novel 28.5 kb deletion that eliminated one of the duplicated a-globin genes in a Chinese family. The deletion breakpoint fragment involved Alu repeat sequences, suggesting a homologous recombination event. Phenotypic analysis on the heterozygous carrier of this deletion revealed that it leads to a very mild phenotype. Because of a 25.0% risk of Hb H (b4) disease in the offspring when in combination with another a0-thal allele, we should not ignore screening the deletion in prenatal diagnosis in order to decrease reproductive risk.
Alu element, a-globin gene, a-thalassemia (a-thal), deletion, homologous recombination
Introduction a-Thalassemia (a-thal) is a common hereditary disorder characterized by deficiency or complete absence of synthesis of a-globin chains. The human a-globin gene cluster is located on the short arm of chromosome 16 and consists of seven genes and pseudogenes arranged in the following linear order: telomere-z- z- a2- a1-a2-a1-y-centromere (1). Most common defects result from large deletions involving one (a/aa, a+-thal) or both (– –/aa, a0-thal) of the a-globin genes, although an increasing number of point mutations have also been described (1,2). Most of the deletional a-thalassemias are the result of homologous or non homologous recombination based upon the presence or absence of homology between parental sequences at the site of recombination (3–5). Deletional a-thalassemias including the Southeast Asia (– –SEA), a3.7 (rightward) and a4.2 (leftward) deletions, are the most common inherited disorders in southern China (6,7). Besides these most common deletions, a large variety of fewer occurring thalassemia deletions have been found in Chinese (8–12) and other populations (13–21). This report characterizes a novel a-thal deletion in a family from Shenzhen in Southern China, which removed 28.5 kb from the a-globin gene cluster, including the a1 and a2 genes; only the a1
*Jing Yu and Jun Xie contributed equally to this study. Address correspondence to Dr. Zesong Li, Shenzhen Key Laboratory of Genitourinary Tumor, Shenzhen Second People’s Hospital, First Affiliated Hospital of Shenzhen University, Shenzhen 518035, Guangdong Province, People’s Republic of China. Tel: +86-755-83669030. Fax: +86-755-8335-6952. E-mail: [email protected]
History Received 25 February 2014 Revised 15 May 2014 Accepted 6 June 2014 Published online 5 November 2014
gene was still present. Thus, this deletion was designated as the a28.5 deletion.
Materials and methods Subjects and hematological analysis The proband, an 18-month-old boy, was referred to our hospital for hematological, biochemical, and DNA analysis because of anemia. Fresh peripheral blood samples of the family members were collected after we obtained approval from the Ethics Committee of the Peking University Shenzhen Hospital, Shenzhen, People’s Republic of China (PRC), and the cooperation of the proband’s family who were from Shenzhen City of Guangdong Province in South China. Routine hematology and red blood cell (RBC) indices were obtained with a Beckman Coulter LH 750 automated cell counter (Beckman Coulter, Miami, FL, USA). The levels of Hb A, Hb A2, Hb H (b4), and Hb F were analyzed on the VARIANT IIÔ high performance liquid chromatography (HPLC) system (Bio-Rad Laboratories, Hercules, CA, USA). Red cell osmotic fragility was tested by the modified one-tube osmotic fragility test (14). Molecular diagnosis Genomic DNA was extracted from peripheral blood leukocytes with the DNeasy Blood and Tissue kit (#69504; Qiagen GmbH, Hilden, Germany). Gap-polymerase chain reaction (gap-PCR) was used to screen for the most common deletional a-thal mutations (– –SEA, a3.7 and a4.2) observed in the Chinese population (6,13). Reverse dot-blot hybridization was carried out to screen the possible presence
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of nondeletional a-thal point mutations and b-thal mutations (6,13). Rare thalassemia deletions with intact a1-globin genes [a16.6 (22), a7.9 (23), a27.6 (8), (a)a5.3 (24) and a2.8 (12)] were screened by gap-PCR with primers reported in the literature and newly-designed primers. Real-time PCR was used to determine gene dosage as described previously with some modification (26). Briefly, 11 pairs of primers were used to amplify specific locations of the a-globin gene cluster spanning a total of 64759 bp (from 103877 to 167961 on chromosome 16 NT_010393.16). Primers were designed and verified using Beacon Designer 7 (Premier Biosoft International, Palo Alto, CA, USA). Data analysis was carried out as described previously (25,26). To characterize the suspected deletion, gap-PCR with forward primer P1 (50 -ATC TAC TTT CTG CTG GGA TTT GCC C30 ) and reverse primer P2 (50 -GGC GGC TCC AGA TTC AGA CTC CT-30 ) was used to amplify across the breakpoints and the PCR product was sequenced directly.
Results Phenotype analysis revealed that the proband and his father presented a typical a-thalassemic trait, with reduced mean corpuscular volume (MCV) (55.7, 68.7 fL, respectively), low percentage of Hb A2 (1.5, 1.9%, respectively), decreased osmotic fragility of the red cells (50.0, 55.0%, respectively). The proband had mild Hb H disease due to the presence of 0.9% Hb H. His mother did not a have microcytic hypochromic anemia phenotype; her nearly normal MCV (81.1 fL), normal Hb A2 value (2.7%) and osmotic fragility of the red cells (70.0%) are shown in Table 1. Single-tube multiplex gap-PCR to screen for the most common deletional a-thal mutations and normal a2-globin gene revealed that the proband was positive for the – –SEA allele, as seen in his father along with the a2-globin allele (Figure 1) that was the only allele seen in the mother. Thus, the proband appears not to have a copy of the a2-globin gene but adult hemoglobin was observed by HPLC suggesting that the a1-globin gene remained intact. The screen for point mutations in a- and b-globin genes appeared normal. The known deletions with intact a1-globin genes [a16.6 (22), a7.9 (23), a27.6 (8), (a)a5.3 (24) and a2.8 (12)] were scanned by gap-PCR with primers reported in the literature and self-designed primers (Supplementary Table),
Figure 2. Schematic overview of the a-globin gene cluster. Genes are indicated as gray boxes. The deletions affecting one or both a-globin genes are indicated as black bars. The downward arrows and numbers indicate the relative position of real-time PCR primers.
Hemoglobin, 2014; 38(6): 427–430
respectively. However, there was no positive result, which suggested an unknown deletion allele (Figure 2). To assess the unknown deletion, gene dosage was determined by comparison of copy number ratios in the proband and in normal controls. The ratios between 0.7 and 1.2 are defined as normal, ratios between 0.4 and 0.6 the presence of one deletion allele, and 0 the presence of two deletion alleles. As shown in Figure 3, the gene copy number from site 4 (NT_010393.16, 138957-138976) to site 11 Table 1. The hematological analysis of the proband and his parents. Parameters Sex-age RBC (1012/L) Hb (g/dL) MCV (fL) MCH (pg) MCHC (g/dL) Hb A (%) Hb A2 (%) Hb F (%) Hb H (%) Osmotic fragility (%) a Genotype
Proband
Mother
Father
M-18 months 6.35 9.5 55.7 15.0 26.8 96.9 1.5 0.7 0.9 50.0 a28.5/– –SEA
F-29 4.40 11.6 81.1 26.0 32.2 97.3 2.7 0.0 – 70.0 a28.5/aa
M-34 6.36 13.5 68.7 21.2 39.0 97.7 1.9 0.4 – 55.0 aa/– –SEA
Figure 1. Molecular analysis of the a-globin gene of the proband’s family by single-tube multiplex gap-PCR for the three most common deletions (– –SEA, a3.7 and a4.2) found in the Chinese population.
A Novel 28.5 kb -Thalassemia Deletion
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DOI: 10.3109/03630269.2014.976793
(NT_010393.16, 167942-167961) in the a-globin gene cluster was reduced. There is a deletion of one allele at sites 4, 5, 9, 10 and 11, and two alleles at sites 6, 7 and 8. The novel deletion, located between sites 3 and 9, owing to the Southeast Asia deletion allele, deletes all regions from site 6 to site 11. The precise breakpoint of this deletion was identified by trying pairs of long-range gap-PCR within the regions uncertain of presence or absence until a specific and reproducible band about 2.4 kb PCR product was obtained by using primers P1 and P2 (Figure 4A). This PCR product was sequenced directly. The sequence analysis showed that the novel mutation removes 28.5 kb from the a-globin gene cluster, including z- z- a2- a1-a2 (NT_010393.16, from 136211-164774), thus it was designated as the a28.5 deletion (Figure 4B). Subsequent analysis showed that there is an Alu homology in the deletional junction fragment. Comparison of the sequence of the a28.5 deletion breakpoint with the flanking wild-type 50 and 30 regions, revealed an Alu homology (136181–136466 at the 50 region flanking the
Figure 3. The scatter plot shows the reduction in copy number at the deleted region of chromosome 16 determined by comparison of copy number ratios in the proband and in normal controls (ratios between 0.7 and 1.2 are defined as normal, ratios between 0.4 and 0.6 indicate the presence of one deletion allele, 0 indicates the presence of two deletion alleles). Figure 4. Identification and characterization of the novel a28.5 allele. (A) The 2401 bp PCR products were generated from both the proband and mother using the P1 and P2 primers, whereas DNA from the father failed to amplify this product. (B) The schematic representation of the arrangement of the novel a28.5 allele. The horizontal arrows mark the Alu sequence. Comparison of the sequence of the a28.5 deletion breakpoint with the wild-type 50 and 30 regions flanking the deletion breakpoint, revealed the Alu homology. The sequences of the parental strands and the recombinant strand are shown.
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deletion breakpoint, and 164758–165034 at the 30 region flanking the deletion breakpoint), which suggests that the novel deletion resulted from an Alu-mediated homologous recombination (Figure 4B).
Discussion It has been reported that there are more than 20 deletions occurring in the human a-globin gene cluster all over the world (27). Of all deletional mutations observed in the Chinese population, three deletion mutations, – –SEA, a3.7 and a4.2, are the most common (6), but rare or novel a-thal mutation have been reported (8) including a compound heterozygote for aaHK/– –SEA that we reported previously (13). Here, we identified a novel large deletion of 28.5 kb in the a-globin gene cluster, removing one a gene in two members of a Chinese family. The new a-globin gene arrangement was given the symbol a28.5 because a 28.5 kb fragment, including the z- z- a2- a1-a2 genes, was removed leaving the a1-globin gene intact (Figure 4B). The breakpoints at the 50 and 30 ends of the deletion were located in an Alu-repetitive sequence that suggested the novel deletion was the result of an Alu element-mediated homologous recombination. Alu sequences, which are present at41.2 million copies, account for more than 10.0% of the human genome (28). It has been reported that the involvement of Alu repeats in recombination events at the human a-globin gene cluster lead to a-thal deletions (3,29). The present case illustrates the importance of molecular genetic diagnosis of a-thal for accurate assessment of reproductive risks. Although the frequency of the a28.5 deletion in the Chinese population is still unknown, it may occur more frequently than expected, especially within communities. When this deletion was found in combination with the most common a-thal deletion allele (– –SEA), it resulted in mild Hb H disease. The existence of rare deletions can pose a diagnostic problem for laboratories in China in which testing is restricted to three common deletion mutations (– –SEA, a3.7 and a4.2). Thus, it is important to characterize
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new and rare mutations so that screening can be comprehensive and allow informed choices for prenatal diagnosis.
15.
Declaration of interest This study was supported in part by grants from the National Natural Science Foundation of China (Grant No. 81272840), and the Young Scientists Fund of the National Natural Science Foundation of China (Grant No. 81201579). The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
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1. Higgs DR, Weatherall DJ. The a thalassaemias. Cell Mol Life Sci. 2009;66(7):1154–1162. 2. Vichinsky EP. a Thalassemia major — new mutations, intrauterine management, and outcomes. Hematology. 2009;2009(1):35–41. 3. Harteveld KL, Losekoot M, Fodde R, et al. The involvement of Alu repeats in recombination events at the a-globin gene cluster: Characterization of two a0-thalassaemia deletion breakpoints. Hum Genet. 1997;99(4):528–534. 4. Rooks H, Bergounioux J, Game L, et al. Heterogeneity of the "gdb-thalassaemias: Characterization of three novel English deletions. Br J Haematol. 2005;128(5):722–729. 5. Higgs DR, Vickers MA, Wilkie AOM, et al. A review of the molecular genetics of the human a-globin gene cluster. Blood. 1989;73(5):1081–1104. 6. Li Z, Li F, Li M, et al. The prevalence and spectrum of thalassemia in Shenzhen, Guangdong Province, People’s Republic of China. Hemoglobin. 2006;30(1):9–14. 7. Li Z, Guo R, Zhang W. Rapid detection of deletional a-thalassemia by an oligonucleotide microarray. Am J Hematol. 2005; 80(4):306–308. 8. Wei XF, Shang X, He DQ, et al. Molecular characterization of a novel 27.6-kb deletion causing a+ thalassemia in a Chinese family. Ann Hematol. 2011;90(1):17–22. 9. Lin M, Wu JR, Huang Y, et al. Clinical and molecular characterization of a rare 2.4 kb deletion causing a+ thalassemia in a Chinese family. Blood Cells Mol Dis. 2012;49(2):83–84. 10. Jia X, Huang R, Lei Z, et al. Detection of a novel large deletion causing a-thalassemia in South China. Exp Mol Pathol. 2013; 95(1):68–73. 11. Long J, Yan S, Lao K, et al. The diagnosis and molecular analysis of a novel 21.9 kb deletion (Qinzhou type deletion) causing a+ thalassemia. Blood Cells Mol Dis. 2014;52(4):225–229. 12. Mo Z, Yu C, Hu Z, et al. A novel deletion of 2.8 kb removing the entire a2-globin gene observed in a Chinese patient with Hb H. Clin Lab. 2012;58(11–12):1309–1312. 13. Li Z, Cai S, Rong K, et al. The first compound heterozygosity for HK aa allele and Southeast Asian deletion allele. Clin Biochem. 2007;40(5–6):407–410. 14. Sessa R, Puzone S, Ammirabile M, et al. Identification and molecular characterization of the – –CAMPANIA deletion, a novel
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a0-thalassemic defect, in two unrelated Italian families (corrected). Am J Hematol. 2010;85(2):143–144. Waye JS, Greenlay B, Eng B. Novel 27.9 kb a0-thalassemia deletion in a Filipino woman. Am J Hematol. 2009;84(3): 197–198. Coelho A, Picanco I, Seuanes F, et al. Novel large deletions in the human a-globin gene cluster: Clarifying the HS-40 long-range regulatory role in the native chromosome environment. Blood Cells Mol Dis. 2010;45(2):147–153. Phylipsen M, Prior JF, Lim E, et al. Thalassemia in Western Australia: 11 novel deletions characterized by multiplex ligationdependent probe amplification. Blood Cells Mol Dis. 2010; 44(3):146–151. Phylipsen M, Vogelaar IP, Schaap RA, et al. A new a0-thalassemia deletion found in a Dutch family (– –(AW)). Blood Cells Mol Dis. 2010;45(2):133–135. Joly P, Lacan P, Labalme A, et al. A novel telomeric (approximately 285 kb) a-thalassemia deletion leading to a phenotypically unusual Hb H disease. Haematologica. 2010;95(5): 850–851. Harteveld CL, Voskamp A, Phylipsen M, et al. Nine unknown rearrangements in 16p13.3 and 11p15.4 causing a- and b-thalassaemia characterised by high resolution multiplex ligationdependent probe amplification. J Med Genet. 2005;42(12): 922–931. Phylipsen M, Traeger-Synodinos J, van der Kraan M, et al. A novel a0-thalassemia deletion in a Greek patient with Hb H disease and b-thalassemia trait. Eur J Haematol. 2012; 88(4):356–362. Rugless MJ, Fisher CA, Old JM, et al. A large deletion in the human a-globin cluster caused by a replication error is associated with an unexpectedly mild phenotype. Hum Mol Genet. 2008; 17(19):3084–3093. Harteveld CL, van Delft P, Wijermans PW, et al. A novel 7.9 kb deletion causing a+-thalassaemia in two independent families of Indian origin. Br J Haematol. 2003;120(2):364–366. Lacerra G, Fioretti G, De Angioletti M, et al. (a)a 5.3: A novel a+-thalassemia deletion with the breakpoints in the a2-globin gene and in close proximity to an Alu family repeat between the a2and a1-globin genes. Blood. 1991;78(10):2740–2746. Fallah MS, Mahdian R, Aleyasin SA, et al. Development of a quantitative real-time PCR assay for detection of unknown a-globin gene deletions. Blood Cells Mol Dis. 2010;45(1):58–64. Yuan JS, Reed A, Chen F, et al. Statistical analysis of real-time PCR data. BMC Bioinformatics. 2006;7:85 (http://www.biomedcentral.com/1421-2105/7/85). Patrinos GP, Giardine B, Riemer C, et al. Improvements in the HbVar database of human hemoglobin variants and thalassemia mutations for population and sequence variation studies. Nucleic Acids Res. 2004;32(Database issue):D537–D541 (http://globin. cse.psu.edu). Lander ES, Linton LM, Birren B, et al. Initial sequencing and analysis of the human genome. Nature. 2001;409(6822): 860–921. Nicholls RD, Fischel-Ghodsian N, Higgs DR. Recombination at the human a-globin gene cluster: Sequence features and topological constraints. Cell. 1987;49(3):369–378.
Supplementary material available online Supplementary Table
ORIGINAL ARTICLE
An Alu Element-Mediated 28.5 kb a-Thalassemia Deletion Found in a Chinese Family Jing Yu1*, Jun Xie1*, Liya Luo2, and Zesong Li2 1
Department of Laboratory Medicines, Peking University Shenzhen Hospital, Shenzhen, People’s Republic of China, Shenzhen Key Laboratory of Genitourinary Tumor, Shenzhen Second People’s Hospital, First Affiliated Hospital of Shenzhen University, Shenzhen, People’s Republic of China
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2
Abstract
Keywords
Over 95.0% of the a-thalassemia (a-thal) cases in southern China are caused by large deletions involving the a-globin gene. Here, we describe the molecular characterization of a novel 28.5 kb deletion that eliminated one of the duplicated a-globin genes in a Chinese family. The deletion breakpoint fragment involved Alu repeat sequences, suggesting a homologous recombination event. Phenotypic analysis on the heterozygous carrier of this deletion revealed that it leads to a very mild phenotype. Because of a 25.0% risk of Hb H (b4) disease in the offspring when in combination with another a0-thal allele, we should not ignore screening the deletion in prenatal diagnosis in order to decrease reproductive risk.
Alu element, a-globin gene, a-thalassemia (a-thal), deletion, homologous recombination
Introduction a-Thalassemia (a-thal) is a common hereditary disorder characterized by deficiency or complete absence of synthesis of a-globin chains. The human a-globin gene cluster is located on the short arm of chromosome 16 and consists of seven genes and pseudogenes arranged in the following linear order: telomere-z- z- a2- a1-a2-a1-y-centromere (1). Most common defects result from large deletions involving one (a/aa, a+-thal) or both (– –/aa, a0-thal) of the a-globin genes, although an increasing number of point mutations have also been described (1,2). Most of the deletional a-thalassemias are the result of homologous or non homologous recombination based upon the presence or absence of homology between parental sequences at the site of recombination (3–5). Deletional a-thalassemias including the Southeast Asia (– –SEA), a3.7 (rightward) and a4.2 (leftward) deletions, are the most common inherited disorders in southern China (6,7). Besides these most common deletions, a large variety of fewer occurring thalassemia deletions have been found in Chinese (8–12) and other populations (13–21). This report characterizes a novel a-thal deletion in a family from Shenzhen in Southern China, which removed 28.5 kb from the a-globin gene cluster, including the a1 and a2 genes; only the a1
*Jing Yu and Jun Xie contributed equally to this study. Address correspondence to Dr. Zesong Li, Shenzhen Key Laboratory of Genitourinary Tumor, Shenzhen Second People’s Hospital, First Affiliated Hospital of Shenzhen University, Shenzhen 518035, Guangdong Province, People’s Republic of China. Tel: +86-755-83669030. Fax: +86-755-8335-6952. E-mail: [email protected]
History Received 25 February 2014 Revised 15 May 2014 Accepted 6 June 2014 Published online 5 November 2014
gene was still present. Thus, this deletion was designated as the a28.5 deletion.
Materials and methods Subjects and hematological analysis The proband, an 18-month-old boy, was referred to our hospital for hematological, biochemical, and DNA analysis because of anemia. Fresh peripheral blood samples of the family members were collected after we obtained approval from the Ethics Committee of the Peking University Shenzhen Hospital, Shenzhen, People’s Republic of China (PRC), and the cooperation of the proband’s family who were from Shenzhen City of Guangdong Province in South China. Routine hematology and red blood cell (RBC) indices were obtained with a Beckman Coulter LH 750 automated cell counter (Beckman Coulter, Miami, FL, USA). The levels of Hb A, Hb A2, Hb H (b4), and Hb F were analyzed on the VARIANT IIÔ high performance liquid chromatography (HPLC) system (Bio-Rad Laboratories, Hercules, CA, USA). Red cell osmotic fragility was tested by the modified one-tube osmotic fragility test (14). Molecular diagnosis Genomic DNA was extracted from peripheral blood leukocytes with the DNeasy Blood and Tissue kit (#69504; Qiagen GmbH, Hilden, Germany). Gap-polymerase chain reaction (gap-PCR) was used to screen for the most common deletional a-thal mutations (– –SEA, a3.7 and a4.2) observed in the Chinese population (6,13). Reverse dot-blot hybridization was carried out to screen the possible presence
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of nondeletional a-thal point mutations and b-thal mutations (6,13). Rare thalassemia deletions with intact a1-globin genes [a16.6 (22), a7.9 (23), a27.6 (8), (a)a5.3 (24) and a2.8 (12)] were screened by gap-PCR with primers reported in the literature and newly-designed primers. Real-time PCR was used to determine gene dosage as described previously with some modification (26). Briefly, 11 pairs of primers were used to amplify specific locations of the a-globin gene cluster spanning a total of 64759 bp (from 103877 to 167961 on chromosome 16 NT_010393.16). Primers were designed and verified using Beacon Designer 7 (Premier Biosoft International, Palo Alto, CA, USA). Data analysis was carried out as described previously (25,26). To characterize the suspected deletion, gap-PCR with forward primer P1 (50 -ATC TAC TTT CTG CTG GGA TTT GCC C30 ) and reverse primer P2 (50 -GGC GGC TCC AGA TTC AGA CTC CT-30 ) was used to amplify across the breakpoints and the PCR product was sequenced directly.
Results Phenotype analysis revealed that the proband and his father presented a typical a-thalassemic trait, with reduced mean corpuscular volume (MCV) (55.7, 68.7 fL, respectively), low percentage of Hb A2 (1.5, 1.9%, respectively), decreased osmotic fragility of the red cells (50.0, 55.0%, respectively). The proband had mild Hb H disease due to the presence of 0.9% Hb H. His mother did not a have microcytic hypochromic anemia phenotype; her nearly normal MCV (81.1 fL), normal Hb A2 value (2.7%) and osmotic fragility of the red cells (70.0%) are shown in Table 1. Single-tube multiplex gap-PCR to screen for the most common deletional a-thal mutations and normal a2-globin gene revealed that the proband was positive for the – –SEA allele, as seen in his father along with the a2-globin allele (Figure 1) that was the only allele seen in the mother. Thus, the proband appears not to have a copy of the a2-globin gene but adult hemoglobin was observed by HPLC suggesting that the a1-globin gene remained intact. The screen for point mutations in a- and b-globin genes appeared normal. The known deletions with intact a1-globin genes [a16.6 (22), a7.9 (23), a27.6 (8), (a)a5.3 (24) and a2.8 (12)] were scanned by gap-PCR with primers reported in the literature and self-designed primers (Supplementary Table),
Figure 2. Schematic overview of the a-globin gene cluster. Genes are indicated as gray boxes. The deletions affecting one or both a-globin genes are indicated as black bars. The downward arrows and numbers indicate the relative position of real-time PCR primers.
Hemoglobin, 2014; 38(6): 427–430
respectively. However, there was no positive result, which suggested an unknown deletion allele (Figure 2). To assess the unknown deletion, gene dosage was determined by comparison of copy number ratios in the proband and in normal controls. The ratios between 0.7 and 1.2 are defined as normal, ratios between 0.4 and 0.6 the presence of one deletion allele, and 0 the presence of two deletion alleles. As shown in Figure 3, the gene copy number from site 4 (NT_010393.16, 138957-138976) to site 11 Table 1. The hematological analysis of the proband and his parents. Parameters Sex-age RBC (1012/L) Hb (g/dL) MCV (fL) MCH (pg) MCHC (g/dL) Hb A (%) Hb A2 (%) Hb F (%) Hb H (%) Osmotic fragility (%) a Genotype
Proband
Mother
Father
M-18 months 6.35 9.5 55.7 15.0 26.8 96.9 1.5 0.7 0.9 50.0 a28.5/– –SEA
F-29 4.40 11.6 81.1 26.0 32.2 97.3 2.7 0.0 – 70.0 a28.5/aa
M-34 6.36 13.5 68.7 21.2 39.0 97.7 1.9 0.4 – 55.0 aa/– –SEA
Figure 1. Molecular analysis of the a-globin gene of the proband’s family by single-tube multiplex gap-PCR for the three most common deletions (– –SEA, a3.7 and a4.2) found in the Chinese population.
A Novel 28.5 kb -Thalassemia Deletion
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DOI: 10.3109/03630269.2014.976793
(NT_010393.16, 167942-167961) in the a-globin gene cluster was reduced. There is a deletion of one allele at sites 4, 5, 9, 10 and 11, and two alleles at sites 6, 7 and 8. The novel deletion, located between sites 3 and 9, owing to the Southeast Asia deletion allele, deletes all regions from site 6 to site 11. The precise breakpoint of this deletion was identified by trying pairs of long-range gap-PCR within the regions uncertain of presence or absence until a specific and reproducible band about 2.4 kb PCR product was obtained by using primers P1 and P2 (Figure 4A). This PCR product was sequenced directly. The sequence analysis showed that the novel mutation removes 28.5 kb from the a-globin gene cluster, including z- z- a2- a1-a2 (NT_010393.16, from 136211-164774), thus it was designated as the a28.5 deletion (Figure 4B). Subsequent analysis showed that there is an Alu homology in the deletional junction fragment. Comparison of the sequence of the a28.5 deletion breakpoint with the flanking wild-type 50 and 30 regions, revealed an Alu homology (136181–136466 at the 50 region flanking the
Figure 3. The scatter plot shows the reduction in copy number at the deleted region of chromosome 16 determined by comparison of copy number ratios in the proband and in normal controls (ratios between 0.7 and 1.2 are defined as normal, ratios between 0.4 and 0.6 indicate the presence of one deletion allele, 0 indicates the presence of two deletion alleles). Figure 4. Identification and characterization of the novel a28.5 allele. (A) The 2401 bp PCR products were generated from both the proband and mother using the P1 and P2 primers, whereas DNA from the father failed to amplify this product. (B) The schematic representation of the arrangement of the novel a28.5 allele. The horizontal arrows mark the Alu sequence. Comparison of the sequence of the a28.5 deletion breakpoint with the wild-type 50 and 30 regions flanking the deletion breakpoint, revealed the Alu homology. The sequences of the parental strands and the recombinant strand are shown.
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deletion breakpoint, and 164758–165034 at the 30 region flanking the deletion breakpoint), which suggests that the novel deletion resulted from an Alu-mediated homologous recombination (Figure 4B).
Discussion It has been reported that there are more than 20 deletions occurring in the human a-globin gene cluster all over the world (27). Of all deletional mutations observed in the Chinese population, three deletion mutations, – –SEA, a3.7 and a4.2, are the most common (6), but rare or novel a-thal mutation have been reported (8) including a compound heterozygote for aaHK/– –SEA that we reported previously (13). Here, we identified a novel large deletion of 28.5 kb in the a-globin gene cluster, removing one a gene in two members of a Chinese family. The new a-globin gene arrangement was given the symbol a28.5 because a 28.5 kb fragment, including the z- z- a2- a1-a2 genes, was removed leaving the a1-globin gene intact (Figure 4B). The breakpoints at the 50 and 30 ends of the deletion were located in an Alu-repetitive sequence that suggested the novel deletion was the result of an Alu element-mediated homologous recombination. Alu sequences, which are present at41.2 million copies, account for more than 10.0% of the human genome (28). It has been reported that the involvement of Alu repeats in recombination events at the human a-globin gene cluster lead to a-thal deletions (3,29). The present case illustrates the importance of molecular genetic diagnosis of a-thal for accurate assessment of reproductive risks. Although the frequency of the a28.5 deletion in the Chinese population is still unknown, it may occur more frequently than expected, especially within communities. When this deletion was found in combination with the most common a-thal deletion allele (– –SEA), it resulted in mild Hb H disease. The existence of rare deletions can pose a diagnostic problem for laboratories in China in which testing is restricted to three common deletion mutations (– –SEA, a3.7 and a4.2). Thus, it is important to characterize
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new and rare mutations so that screening can be comprehensive and allow informed choices for prenatal diagnosis.
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Declaration of interest This study was supported in part by grants from the National Natural Science Foundation of China (Grant No. 81272840), and the Young Scientists Fund of the National Natural Science Foundation of China (Grant No. 81201579). The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
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Hemoglobin Downloaded from informahealthcare.com by University of Connecticut on 04/11/15 For personal use only.
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