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Micromethodology for the Characterization of Hemoglobin Variants

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Micromethodology for the characterization of hemoglobin variants Citation for published version (APA): Plaseska, D. (1994). Micromethodology for the characterization of hemoglobin variants. Rijksuniversiteit Limburg. https://doi.org/10.26481/dis.19940428dp Document status and date: Published: 01/01/1994 DOI: 10.26481/dis.19940428dp Document Version: Publisher's PDF, also known as Version of record Please check the document version of this publication: • A submitted manuscript is the version of the article upon submission and before peer-review. There can be important differences between the submitted version and the official published version of record. People interested in the research are advised to contact the author for the final version of the publication, or visit the DOI to the publisher's website. • The final author version and the galley proof are versions of the publication after peer review. • The final published version features the final layout of the paper including the volume, issue and page numbers. Link to publication General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal. If the publication is distributed under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license above, please follow below link for the End User Agreement: www.umlib.nl/taverne-license Take down policy If you believe that this document breaches copyright please contact us at: [email protected] providing details and we will investigate your claim. Download date: 09 Oct. 2021 MICROMETHODOLOGY FOR THE CHARACTERIZATION OF HEMOGLOBIN VARIANTS :. "id"'vit: .1 -1 ifl PROEFSCHRIFT ter verkrijging van de graad van doctor fn aan de Rijksuniversiteit Limburg te Maastricht, op gezag van de Rector Magnificus, Prof. Dr. H. Philipsen, volgens het besluit van het College van Dekanen, in het openbaar te verdedigen op donderdag, 28 april 1994 om 14.00 uur door Dijana Plaseska geboren te Skopje op 21 mei 1963 Promotores: -- *iHT rKH YOOJOOOHT3MOHO''* Prof. Dr. H.C. Hemker Prof. Dr. T.H.J. Huisman, Medical College of Georgia, USA Beoordelingscommissie: Prof. Dr. P.J. Brombacher (voorzitter) Prof. Dr. L.F. Bernini, Rijksuniversiteit Leiden Prof. Dr. G.D. Efremov, Macedonian Academy of Sciences and Arts Prof. Dr. J.P.M. Geraedts Prof. Dr. W. van der Slik, Rijksuniversiteit Groningen .•?.> n.?.v Q t; qc -'•-»; • : ' •••- ACKNOWLEDGEMENTS I am most grateful to Professor Dr. Titus H.J. Huisman, without whose direction and continuous support the work presented in this dissertation would not have been accomplished. His advice, encouragement, and forbear- ance is deeply appreciated. I would like to express my deepest thanks to Professor Dr. H.C. Hemker for his help in the realization of this dis- sertation. I am glad to be able to record my indebtedness to Professor Dr. Georgi D. Efremov, who has most profoundly influenced my research involvement in the field of hemoglobinopathies. The secretarial, tech- nical, and editorial expertise of Mrs. Marianne F.H. Carver was indispen- sable in the preparation of this manuscript. Finally, I would like to acknowledge the participation of a number of colleagues who have either provided blood samples or have contributed in the experimental part of the work presented here. To all of them I express my deepest gratitude. ,• '"' . JV; .ar. • ."'i•.'.•. i .[".: "sVi "••!'>< ^ !*..**- •-:.'.' ';iK tu-nw I . b?J^ ' ?'?"• ^-"lo v f qtiS .•-.: v.i- :-•;>.. ->»'?' ^f ' •* • f il .'! '' '' '-'u.ii ^."ij iC If- ;:";<*' II;" '( V" To my parents with all my love . .,;, CONTENTS Abbreviations •;...(•"' , , ,,. „>_ -., 1 Introduction and Review of the Literature 1 la Addundum -• n; <. 33 V«i: - . - >J 2 Materials and Methods , J;f,cOo- 51 :3 a Chain Variants Identified by HPLC .J -M371 3a Hb Davenport or o278(EF7)Asn->HisB2 « 73 3b Hb Sun Prairie or a2l30(H13)Ala->ProB2; Sec- b 79 ond Observation in an Indian Adult i: 4 B Chain Variants Identified by HPLC c 85 4a Hb Iowa or a2B2ll9(6H2)Trp->Arg ^ 87 4b Hb Zengcheng or a2B2H4(G16)Leu->Met ' 93 5 B Chain Variants Identified by HPLC and DNA 97 Analyses 5a Hb Yokohama [B31(B13)Leu->Pro] Detected as a 99 De Novo Mutation in a Yugoslavian Boy . ' 5b Hb Volga [B27(B9)Ala->Asp]: Detection of a - De Novo Mutation by Ava II Digestion of PCR- .1 Amplified DNA 6 Y Chain Variants Identified by HPLC 6a Hb F-Jiangsu, the First Y Chain Variant With a Valine->Methionine Substitution: 02^2^^ (H12)Val->Met ^ , „, 6b Hb F-Catalonia or a2^Y2l5(A12)Trp->Arg j r, "119 6c Hb F-Brooklyn or a2^Y266(E10)Lys->Gln ' • !'i25 7 Hb Montreal, a Variant With an Extended B 129 Chain Due to a Deletion and an Insertion at . , , the Same Location Identified by Sequencing of Amplified DNA ' 8 Discussion ft«>r.T,.6*i r>- . 1^*...,.. 9 Summary ^! ie 10 Sanenvatting i;fî ^ * 161 11 Curriculum Vitae '»U'.rj •^^'"••« "* ABBREVIATIONS A£ aminoethylated I ASO allele specific oligonucleotide ^ ««. r-ni*-rs»n* * bp base pair(s) U'nA bovine serum albumin -nbnubi»* CC14 carbontetrachloride i CD(s) codon(s) sto i^ »>«-. <ff^aJsK CF cystic fibrosis "CM- carboxymethyl- ""* --• ' *•' ••..-.. ...--J . =-. ,, CO2 carbondioxide ^ DABITC 4-NN-dimethylaminoazobenzene-4-isothiocyanate fif ï dATP deoxy adenosine triphosphate ^ ddATP dideoxy adenosine triphosphate '' ddCTP dideoxy cytosine triphosphate sHe'V ddGTP dideoxy guanosine triphospate i ddTTP dideoxy thymidine triphosphate GWt.il DEAE- diethyl aminoethyl- > dGTP deoxy guanosine triphosphate DNA deoxyribonucleic acid "!J «3 \i DPG (2,3}-diphosphoglycerate BfTT dithiothreitol , dTTP deoxy thymidine triphosphate 01 * EDTA ethylene diamine tetra acetatedisodium 4 FSC frameshift * Hb(s) hemoglobin(s) HPFH hereditary persistence of fetal Hb HPLC high performance liquid chromatography • HS hypersensitive site(s) ! f i: ! IJJEF isoelectrofocusing • •• - - IVS intervening sequence •rtj ^ dH ,-•> kb kilobase(s) ^> -J;M LCR locus control region MCH mean corpuscular Hb 0." MCHC mean corpuscular Hb concentration MCV mean corpuscular volume j;^ MOPS (3[N-morpholino]propanesulfonic acid) mRNA messenger ribonucleic acid O2 oxygen ^ ;^ nt(s) nucleotide(s) PAGE polyacrylamide gel electrophoresis PCMB p-chloromercuribenzoate PCR polymerase chain reaction S'i PCV packed cell volume PITC phenylisothiocyanate poly A polyadenylation PTC phenylthiocarbamyl CJ PVP polyvynilpyrolidone RBC(s) red blood cell(s) RET(s) retention time(s) SOS sodium dodecilsulphate ss sickle cell anemia ssc sodium saline citrate buffer SSPE sodium saline phosphate EDTA buffer TMAC tetramethyl ammonium chloride TBE Tris borate EDTA buffer TE Tris EDTA buffer TEA Tris EDTA acetate buffer TFA trifluoroacetic acid thai thalassemia Tm melting temperature TMB 5,5'-tetramethylbenzidine UTR(s) untranslated region(s) •»••« ?.JÉ .4T0...Î éf' t'i' •- i .>:.- .••• i.S . ..Vi-;».. V •• .m- £.';!;•:>•-• :••.•- < ' ' " '.' . ":-^ ,. CHAPTER 1 r\- INTRODUCTION ', • '*"•"'"' AND REVIEW OF THE LITERATURE • ^ . - "-, - <: v . : - . , - ., i... î 3 H T Ï' :M ¥ r' S CHAPTER 1 INTRODUCTION Hb has played a historic role in many scientific disciplines such as chemistry, biology, genetics, and medicine. The important functional role of Hb and the relative ease of isolating it in pure form have led to an early characterization, much earlier than that of any other human protein. The amino acid sequences of the globin subunits were among the first to be determined. The three-dimensional structure of Hb has also been determined in detail. The functional properties of the Hb molecule, the factors regulating its activity, and the structural features that underlie its functional behavior, are also well understood. The Hb mole- cule has served as a model for the studies of the structure, and structure- function relationships of proteins in general. The numerous naturally occurring mutations in the globin genes have made Hb a model for studies of the molecular basis of genetic diseases and of gene expression. The mutations in the various globin genes are caused by a variety of mechanisms, including substitutions of one or more nts in the DNA, nt deletions or insertions, and various forms of genetic recombinations. Some of these mutations alter the structure of the globin, thus producing different abnormal gene products or abnormal Hbs, while others decrease the rate of synthesis of the globin chains, leading to the syndromes known as thalassemias. Since 1949, when Hb S was found to be the cause of sickle cell anemia, the number of Hb variants has increased to more than 600. The clinical and hematological consequences have in about one-third of all cases been of sufficient severity to bring the affected individuals to medical atten- tion; this has promoted the detection and identification of some of these abnormalities. In addition, numerous population surveys have been con- ducted in many areas of the world, resulting in the identification of a large number of rare and clinically silent Hb variants. They have also yielded valuable data about some of the more common Hb abnormalities. Much progress has been made in the studies of the Hb variants, mainly because of technological advances that have provided more sensitive methods for their detection, separation, and characterization. Electrophoretic techniques have undoubtedly played the most important role, and these are still the most common methods used for the initial detection of abnormal Hbs. Chromatographic procedures that permit the separation and isolation of mutant Hbs and of individual globin chains have played an important role in the structural analyses of Hb variants. Still, both electropho- retic and classical chromatographic techniques fail to separate many abnor- mal Hbs.
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  • Hemoglobin Variants: Biochemical Properties and Clinical Correlates

    Hemoglobin Variants: Biochemical Properties and Clinical Correlates

    Downloaded from http://perspectivesinmedicine.cshlp.org/ on October 4, 2021 - Published by Cold Spring Harbor Laboratory Press Hemoglobin Variants: Biochemical Properties and Clinical Correlates Christopher S. Thom1,2, Claire F. Dickson3, David A. Gell3, and Mitchell J. Weiss2 1Cell and Molecular Biology Graduate Group, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104 2Hematology Department, Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania 19104 3Menzies Research Institute, University of Tasmania, Hobart, Australia Correspondence: [email protected] Diseases affecting hemoglobin synthesis and function are extremely common worldwide. More than 1000 naturally occurring human hemoglobin variants with single amino acid substitutions throughout the molecule have been discovered, mainly through their clinical and/or laboratory manifestations. These variants alter hemoglobin structure and biochem- ical properties with physiological effects ranging from insignificant to severe. Studies of these mutations in patients and in the laboratory have produced a wealth of information on he- moglobin biochemistry and biology with significant implications for hematology practice. More generally, landmark studies of hemoglobin performed over the past 60 years have established important paradigms for the disciplines of structural biology, genetics, biochem- istry, and medicine. Here we review the major classes of hemoglobin variants, emphasizing general concepts and illustrative examples. lobin gene mutations affecting hemoglobin stitutions, antitermination mutations, and al- G(Hb), the major blood oxygen (O2) carrier, tered posttranslational processing (Table 1). are common, affecting an estimated 7% of the Naturally occurring Hb mutations cause a world’s population (Weatherall and Clegg 2001; range of biochemical abnormalities, some of Kohne 2011). These mutations are broadly sub- which produce clinically significant symptoms.