MOLECULAR DIAGNOSIS AND MUTATION CHARACTERIZATION IN THALASSEMIAS WANG WEN NATIONAL UNIVERSITY OF SINGAPORE 2004 MOLECULAR DIAGNOSIS AND MUTATION CHARACTERIZATION IN THALASSEMIAS WANG WEN (MBBS, Tianjin Medical University, China) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PAEDIATRICS NATIONAL UNIVERSITY OF SINGAPORE 2004 Acknowledgements First and foremost, I would like to express my deepest gratitude to my supervisor, Associate Professor Samuel S. Chong, who introduced me to the field of thalassemia and many exciting ideas. His endless guidance, warm encouragements, wise counsel, and lots of patience have inspired me throughout the course of my research. I am extremely fortunate and grateful to have benefited from his wisdom and generosity. My sincere appreciations must go to Dr. Denise Goh Li Meng and Dr. Caroline Lee Guat Lay for their helpful suggestions and constructive critiques in the field of nonsense-mediated mRNA decay mechanism and cell culture. I would like to express my appreciation to all the collaborators who have provided samples for assay validation and/or valuable suggestions in the manuscripts. Special thanks must go to Singapore Biomedical Research Council who provided the research fund and department of peadiatrics who provided the opportunity and environment to study. My gratitude has to go to Associate professor and head Quak Seng Hock for his advice on the format and organization of the thesis. I am also grateful to the friends and colleagues in my lab, both past and present, namely, Arnold Tan, Felicia Cheah, Ben Jin, Zhu Haibo, Yang Yayun, Zhou Youyou, Liang Dong, Zeng Sheng and others for their help and advice to my work. It is indeed a great pleasure for me to work with them. Thanks must go to Mr. Wang Baoshuang and Mr. Ren Jianwei who helped me on transfection and western blot. Finally, I would like to thank my husband, Zhenyu, my parents and my sister for their continuous love, encouragement and support. i Table of Contents: Acknowledgements .i Table of Contents: ii Summary .vii List of Tables .ix List of Figures xi Chapter Background and Literature Reviews .1 1.1 Background 1.1.1 Human hemoglobin structure and switching .3 1.1.2 α-Globin genes structure .4 1.1.3 β-Globin genes structure 1.1.4 Genetic classification of thalassemia .6 1.2 α-Thalassemia 1.3 β-Thalassemia 11 1.4 Laboratory diagnosis of thalassemia 14 1.4.1 Non-molecular diagnosis .14 1.4.2 Molecular diagnosis .16 1.5 β-Thalassemia major and nonsense mediated mRNA decay .19 1.5.1 Nonsense mediated mRNA decay (NMD) 21 1.5.2 NMD and β-thalassemia 23 1.6 Objectives 24 Chapter Introduction 26 ii Importance of improved tools for molecular diagnosis of α- and β- 2.1 thalassemia in Southeast Asia 27 2.1.1 Importance of positive controls for the PCR-based diagnosis .27 2.1.2 Importance of multiplex-minisequencing in screening for common α- and β-thalassemia mutations 28 2.1.2.1 Common β-thalassemia mutations in Southeast Asia and India 29 2.1.2.2 Common α-thalassemia non-deletional mutations in Southeast Asia …………………………………………………………………… 29 2.1.3 Importance of screening for anti-3.7 and anti-4.2 α-globin gene triplication 30 2.2 Molecular verification of Hb H disease classification by isoelectric focusing (IEF) …………………………………………………………………………… 33 2.3 Characterization of the relationship between premature termination codon (PTC) and nonsense mediated mRNA decay (NMD) .34 Chapter Materials and Methods 37 Development of improved tools for molecular diagnosis of α- and β- 3.1 thalassemia in Southeast Asia 38 3.1.1 Construction of “reconstituted” positive controls for the 7-deletion multiplex-PCR .38 3.1.1.1 Preparation of seven deletion junction fragments 38 3.1.1.2 Construction of T-vector 39 3.1.1.3 T-A cloning 41 3.1.1.4 Preparation and validation of heterozygous reconstituted positive controls …………………………………………………………………… 44 iii 3.1.2 β-Thalassemia multiplex-minisequencing assay .45 3.1.2.1 DNA samples .45 3.1.2.2 β-Globin gene gap-PCR and ∆619bp mutation detection 49 3.1.2.3 Multiplex-minisequencing .50 3.1.2.4 β-globin gene mutation-detection primers .51 3.1.2.4 Capillary electrophoresis and genotype analysis .52 3.1.3 α-Thalassemia multiplex-minisequencing assay .55 3.1.3.1 DNA samples .55 3.1.3.2 Multiplex-minisequencing .57 3.1.3.3 Capillary electrophoresis and genotype analysis .61 Single-tube multiplex-PCR screen for anti-3.7 and anti-4.2 α-globin 3.1.4 gene triplication/quadruplication .61 3.2 3.1.4.1 DNA samples .61 3.1.4.2 Primer design .61 3.1.4.3 Anti-3.7/4.2 multiplex-PCR .62 3.1.4.4 7-deletion multplex-PCR .63 α-Globin genotyping by PCR and direct sequencing 63 3.2.1 Patient samples .63 3.2.2 Molecular analysis of the α-globin gene cluster 63 3.3 Characterization the relationship between PTC and NMD 66 3.3.1 Plasmid constructs .66 3.3.1.1 Generation of a 3.4 kb β-globin gene fragment with IRE insertion 66 3.3.1.2 Generation of pBS-IREB wild type construct .69 3.3.1.3 Generation of pBS-IREB mutant constructs 72 3.3.1.4 Generation of pIREB-EGFP wild type and mutant constructs 79 iv 3.3.2 Cell culture and transfection 82 3.3.3 RNA extraction and real-time RT-PCR .84 3.3.4 Western blot .88 Chapter Results .90 Improved Tools for Molecular Diagnosis of α- and β-Thalassemia in 4.1 Southeast Asia 91 4.1.1 Successful “reconstituted” positive controls for 7-deletion-multiplex PCR ……………………………………………………………………… 91 4.1.2 β-Thalassemia multiplex-minisequencing assay .91 4.1.3 α-Thalassemia minisequencing assay 97 4.1.4 Single-tube α-globin gene triplication multiplex-PCR 103 4.2 Molecular verification of Hb H disease classification by IEF .106 4.3 Characterization of the relationship between PTC and NMD .107 4.3.1 Western blot analysis of wild type and mutant constructs .108 4.3.2 Nuclear and cytoplasmic levels of PTC-containing transcripts in the global presence of translation 109 4.3.3 Nuclear and cytoplasmic levels of PTC-containing transcripts in the absence of cytoplasmic translation 111 4.3.4 Nuclear and cytoplasmic levels of PTC-containing transcripts in the global absence of translation 112 Chapter Discussion .115 5.1 Advantage of the improved tools for molecular diagnosis of α- and β- thalassemia in Southeast Asia 116 5.1.1 Construction of “reconstituted” positive controls for the 7-deletion multiplex-PCR .116 v 5.1.2 β-Thalassemia multiplex-minisequencing assay .118 5.1.3 α-Thalassemia multiplex-minisequencing assay .122 5.1.4 Single-tube α-globin gene triplication multiplex-PCR 125 5.2 Molecular Verification of Hb H disease Classification by IEF .128 5.3 Characterization of the relationship between PTC and NMD .131 Chapter Conclusion 135 Bibliography .137 Author’s Publications 155 vi Summary Thalassemia, the commonest monogenetic disorder in humans, has historically constituted a serious public heath problem in many parts of the world. In this study, several molecular diagnostic assays for identification of α- and β-thalassemia common mutations in Southeast Asia have been developed and validated. “Reconstituted” genomic DNA samples heterozygous for each of the seven αthalassemia deletions (-α3.7, -α4.2, --SEA, --THAI, -(α)20.5, --MED and –FIL) screened for in the seven-deletion multiplex PCR were generated from the existing patient DNA samples, serving as positive controls to give a more reliable PCR diagnosis results. Additionally, multiplex minisequencing assays were developed to detect the most common Southeast Asia α-and β-thalassemia point mutations together with the most common β-thalassemia deletion – 619bp deletion. Totally the multiplex minisequencing assays can detect common HbA2 mutations (codon0∆1bp, Constant Spring, Paksé, Quong Sze, Suan Dok, codon30∆3bp, codon59) and 16 common HBB mutations (codon41/42, IVSIInt654, IVSI nt5, codon17, -28, -29, codon71/72, codon26, IVSInt1, codon19, initiation codon for translation, codon43, codon27/28, codon8/9, codon35 and ∆619bp deletion). A single-tube multiplex PCR assay was also developed to screen for the presence of α-globin gene triplications (αααanti-3.7 and αααanti-4.2) in apparent β-thalassemia carriers with unexpectedly severe presentation, since extra copies of the α-globin gene may aggravate the severity of βthalassemia. These new molecular diagnostic assays provide a more rapid, efficient, and cost-effective alternative to current thalassemia molecular testing methods in the region. vii In addition, the genotype of 110 Hb H disease samples diagnosed by isoelectric focusing (IEF) from Thailand were characterized by the seven-deletion multiplex PCR and α-globin gene sequencing to evaluate the accuracy of IEF in Hb H disease classification. Several misclassifications of Hb H disease by IEF were found, which highlights the importance of molecular diagnosis in accurate determination of genetic mutations. A reliable protocol to sequence the high GC-rich α-globin genes was also developed. Furthermore, five premature termination codons (PTCs) caused by naturally occurring nonsense or frameshift mutations [codon1 ∆ 1bp GTG→−TG (PTC at codon 3/4 TGA), codon 17 AAG→TAG (PTC at 17 TAG), codon 8/9 +G (PTC at 21/22 TGA), codon 27/28 +C (PTC at 42/43 TGA), and codon 41/42 −TTCT (PTC at 60/61 TGA)] were characterized to study the relationship between the position of PTCs and the cell surveillance mechanism − nonsense-mediated mRNA decay (NMD). The results support the hypothesis that PTCs at or 5’ to codon 17 are resistant to NMD, while PTCs at or 3’ to codon 21/22 are subject to NMD, which is an exception to the rule in the current dogma in NMD. In addition, by regulating translation specifically in the cytoplasm or globally, this study also supports a nucleus-associated NMD mechanism for PTC-containing β-globin gene transcripts. viii 27. Cao A, Galanello R, Rosatelli MC. Genotype-phenotype correlations in betathalassemias. Blood Rev 1994; 8: 1-12. 28. Clarke GM, Higgins TN. 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Clin Chem 2003; 49(10): 1679-82. 5. Sutcharitchan, P*, W Wang*, R Settapiboon, S Amornsiriwat, ASC Tan, and SS Chong. Hemoglobin H disease classification by isoelectric focusing: molecular verification of 110 cases from Thailand. Clin Chem 2005; 51(3): 641-4. *equal contribution first authors 155 [...]... mutations in α-thalassemia involve mutations affecting mRNA splicing, mutations affecting initiation of mRNA tranlation, mutations affecting the poly(A) addition signal, inframe deletions, frameshifts, nonsense mutations and chain termination mutations [9] In α -thalassemias, the defective α-globin chain synthesis cause β-globin chain in excess in RBC The degree of imbalanced globin chain synthesis is a critical... premature termination mutations, and frameshifts or aberrant splicing causing elongated or truncated β-chain variants [26] However, most of the premature termination mutations that cause dominantly inherited β-thalassemia are located in exon 3 or beyond, whereas the termination mutations in exon 1 or 2 are recessively inherited [2] The premature termination mutations that cause dominantly inherited βthalassemia... caused by point mutations β0 -thalassemias are caused by mutations affecting the initiation codon, a splice junction site or mutations producing a nonsense codon or frameshift, whereas β+ -thalassemias are caused by mutations affecting the transcription or mRNA processing [7, 21] Among the rare deletions, only a 619 bp deletion involving the 3’ end of the β-globin gene is common in the Sind and Punjab... the Sind and Punjab populations of India and Pakistan and accounts for 20% of the β-thalassemia alleles in these populations [22, 23] In β -thalassemias, the absence or variable reduction of β-globin chain leads to imbalanced globin chain synthesis and an excess of α-globin chain The excess αchains precipitate in red blood cell precursors and their progeny, leading to ineffective erythropoiesis with large-scale... reduction of α-globin chain form homotetramer β4 (Hb H) Hb H precipitates in red blood cells and can be visualized under microscope [28] 1.4.2 Molecular diagnosis Many DNA analysis techniques have been used in the detection of deletions and point mutations in α- and β-globin genes Since most α -thalassemias are caused by deletions, while β -thalassemias are mostly caused by point mutations, the methods... polymorphism due to point mutations, deletions, and insertions of DNA There are two main varieties of αthalassemia, α+- and α0-thalassemia, depending on the relative output of both α genes per haploid genome In the α+ -thalassemias, one of the linked α-globin genes is inactivated by deletion (−α/αα) or point mutation (ααT/αα) In the α0 -thalassemias, both α-globin genes on one chromosome are inactivated, most... occupying a region of about 70 kilobases [4] The genes are arranged in the order 5’ζ-ψζ-ψα2-ψα1-α2-α1-θ1-3’ (Figure 1-1) The cluster contains 3 functional α-like genes, ζ, α2 and α1 ζ-Globin gene is embryonic α-like gene, expressing only in embryo α2 and α1 genes are adult genes and they are highly homologous, differing only in the sequence within intervening sequence 2 (IVS 2) and in their 3’ non-coding... relative expression level of PTC-containing β-globin gene in cytoplasm and nucleus in the global presence of translation 111 Figure 4-11 The relative expression level of PTC-containing β-globin gene in cytoplasm and nucleus in the absence of cytoplasmic translation 112 Figure 4-12 The relative expression level of PTC containing β-globin gene in cytoplasm and nucleus in the global absence of translation... useful and reliable for confirming the clinical diagnosis, explaining the hematological abnormality, prenatal diagnosis, genetic counseling and population screening for carriers The tests can be subdivided into non -molecular and molecular diagnosis The former is based on hematological changes, such as complete blood count (CBC), Hb H test, HPLC for HbA2 and F quantification, electrophoresis and isoelectric... common α0 -thalassemias in Southeast Asia and Mediterranean region are Southeast Asia deletion (−−SEA) and Med deletion (−−MED), respectively [5] Filipino deletion (−−FIL) and Thai deletion (−−THAI) are predominantly found in the descendants of the Philippines and Thailand [10-16] Although single point mutations or oligonucleotide insertions and deletions are less frequent than deletions in α-thalassemia, . differing only in the sequence within intervening sequence 2 (IVS 2) and in their 3’ non-coding region. Each α-globin genes contains three exons, separating by two introns, or intervening sequences PCR-based diagnosis 27 2.1.2 Importance of multiplex-minisequencing in screening for common α- and β-thalassemia mutations 28 2.1.2.1 Common β-thalassemia mutations in Southeast Asia and India. PTC-containing β-globin gene in cytoplasm and nucleus in the absence of cytoplasmic translation 112 Figure 4-12 The relative expression level of PTC containing β-globin gene in cytoplasm and