Variant Haemoglobins E-Book

Barbara J. Bain

• Herausgeber: John Wiley & Sons
• Kategorie: Fachliteratur
• Sprache: Englisch

Seitenzahl: 169

Veröffentlichungsjahr: 2011

Beschreibung

Variant Haemoglobins - A Guide to Identification is based on the premise that any single diagnostic technique offers only a very provisional identification of a variant haemoglobin. In routine diagnostic practice two techniques are needed as a minimum, with the results being interpreted in the light of the clinical details, blood count, blood film and ethnic origin. This book covers 150 normal and variant haemoglobins that have been studied and carefully documented. Variant Haemoglobins has four introductory chapters followed by an invaluable atlas. The introductory chapters cover * the genetics of haemoglobin synthesis * the principles of tests employed for identification * common haemoglobins of major clinical or diagnostic importance * thalassaemias and related conditions The atlas section comprises 170 full colour pages in which each variant haemoglobin or combination of haemoglobins is illustrated by cellulose acetate electrophoresis at alkaline pH, agarose gel electrophoresis at acid pH, isoelectric focusing and one or more HPLC traces. For ease of reference, the atlas pages are arranged according to the retention time of each haemoglobin on HPLC, this becoming increasingly the primary technique employed in haemoglobin identification. Bringing a mix of necessary scientific expertise and clinical knowledge, each author has more than 30 years experience in the diagnosis of variant haemoglobins. Providing otherwise unavailable information, this unique and practical guide * is illustrated with over 700 high quality colour digital images plus flow charts and line diagrams * covers common and important haemoglobin variants, in addition to many rarer ones * is an essential reference source for diagnosis in the haematology laboratory A remarkably useful book, Variant Haemoglobins will be valuable for haematopathologists, clinical and laboratory haematologists in practice and in training and all laboratory staff involved in haemoglobinopathy diagnosis.

Leseprobe

Contents

Preface

ONE GLOBIN GENES AND HAEMOGLOBIN

Normal haemoglobins and their synthesis

Globin gene structure and function

Nomenclature of haemoglobins

Mutations – what can go wrong?

The proportion of variant haemoglobins

References

Further reading

TWO DIAGNOSTIC PROCEDURES AND PRINCIPLES OF COMMONLY USED TESTS

Clinical and family history

Blood count and blood film

High performance liquid chromatography

Glycated h aemoglobins and HPLC

Cellulose acetate membrane electrophoresis at alkaline pH

Citrate agar gel electrophoresis at acid pH

Agarose gel electrophoresis at acid pH

Isoelectric focusing

Capillary electrophoresis

Sickle solubility test

Haemoglobin H preparation

Tests for instability of haemoglobin

Tests for high and low affinity haemoglobins

Heinz body preparation

Kleihauer test

Tests for haemoglobin M

Investigation of suspected β thalassaemia

Investigation of suspected α thalassaemia

Neonatal screening

DNA analysis

Mass spectrometry

References

THREE COMMON HAEMOGLOBINS OF MAJOR CLINICAL OR DIAGNOSTIC IMPORTANCE

Normal adults and the influence of a cquired disorders on globin chain synthesis

Pregnancy

Acquired conditions affecting globin chain synthesis

Normal and premature neonates

Common and important variant haemoglobins

Haemoglobin C

Haemoglobin E

Haemoglobin D-Punjab (D-L os A ngeles)

Haemoglobin G-Philadelphia

Lepore haemoglobins

Haemoglobin O-Arab

Coexistence of haemoglobin S with other variant haemoglobins and with β thalassaemia

Haemoglobin S plus haemoglobin C

Haemoglobin S plus haemoglobin Lepore

Haemoglobin S plus haemoglobin D-Punjab

Haemoglobin S plus haemoglobin O-Arab

Haemoglobin S plus haemoglobin E

References

FOUR THE THALASSAEMIAS AND RELATED CONDITIONS

Beta thalassaemia heterozygosity

Haemoglobin A2 and β thalassaemia diagnosis

Beta thalassaemia homozygosity and compound heterozygosity

Delta beta thalassaemia

Gamma delta beta thalassaemia

Delta thalassaemia

Hereditary persistence of fetal haemoglobin

Alpha plus thalassaemia

Alpha zero thalassaemia

Haemoglobin H disease

Haemoglobin Bart's hydrops fetalis

References

TECHNICAL NOTES TO AID IN INTERPRETATION OF THE ATLAS PAGES

Atlas pages

Index

This edition first published 2010,© 2010 by Barbara J. Bain, Barbara J. Wild, Adrian D. Stephens, Lorraine A. Phelan.

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Library of Congress Cataloging-in-Publication Data

Variant haemoglobins : a guide to identification / Barbara J. Bain ... [et al.].p. ; cm.Includes bibliographical references and index.ISBN 978-1-4051-6715-41. Hemoglobinopathy–Diagnosis. 2. Hemoglobin polymorphisms – Identification. I. Bain, Barbara J.DNLM: 1. Hemoglobinopathies – diagnosis. 2. Hemoglobins – genetics. WH 190 V299 2010]RC641.7.H35V37 2010616.1'57075–dc22

2010010743

PREFACE

This book is based on our work in the diagnostic haematology laboratories of St Mary's Hospital (Imperial College Healthcare NHS Trust) and King's College Hospital NHS Trust and on the results of a collaboration between one of us (BJW) and Brian Green of Waters Corporation, Micromass UK Ltd in applying electrospray ionization mass spectrometry to this field. Without this development of a procedure for rapidly identifying variants by mass spectrometry, this book would not have been possible. We should like to acknowledge the help of all those with whom we have worked in these laboratories, and in particular that of Alice Gallienne and Elaine Cooper in the Department of Haematological Medicine at King's College Hospital who undertook many of the electrophoretic and chromatographic separations.

Our thanks are due also to Professor Ghulam Mufti for permission to publish material from the King's College Hospital laboratory and to Dr Saad Abdalla and Dr Mark Layton, St Mary's Hospital, for their help, in particular in providing clinical data. Other colleagues have helped with data or specimens from patients with specific conditions.

This guide to identification of variant haemoglobins is based on the premise that any single technique offers only a very provisional identification of a variant haemoglobin. In routine diagnostic practice two techniques are needed as a minimum, with the results being interpreted in the light of the clinical details, blood count, blood film and ethnic origin. Other than for straightforward sickle cell heterozygosity, if a variant haemoglobin is present the use of three techniques, relying on different principles, is recommended. For this guide we have generally been able to show four techniques and virtually all the haemoglobins in the atlas have also had their identity confirmed by electrospray mass spectrometry or, occasionally, by DNA analysis. The exceptions are some examples of common variant haemoglobins such as haemoglobins S, C and E, which have sometimes been identified by traditional techniques. The diagnosis of thalassaemia and related disorders has been confirmed, when necessary, by DNA analysis by the National Haemoglobinopathy Reference Service, Oxford, and we thank Dr John Old and his team for these analyses.

Barbara BainBarbara WildAdrian StephensLorraine Phelan2010

ONE

GLOBIN GENES AND HAEMOGLOBIN

Normal haemoglobins and their synthesis

Haemoglobin is the major protein in the red blood cell. It is a transport protein for oxygen and thus is essential for life. Not all haemoglobin in the human body is the same. During adult life, the major haemoglobin, known as haemoglobin A, comprises about 97% of total haemoglobin. Minor components are haemoglobin A2 and haemoglobin F. During embryonic and fetal life the situation is very different. The embryo has mainly haemoglobins Gower 1, Gower 2 and Portland 1 whereas fetal life is characterized by synthesis of haemoglobin F and increasingly, as gestation proceeds, haemoglobin A.

All normal haemoglobins are composed of two unlike pairs of polypeptide chains known as globin chains, each of which provides a pocket for an iron-containing haem molecule; the globin protects haem from oxidation. It is the different globin chain composition and the interaction between chains that gives the various haemoglobins their differing characteristics. The normal haemoglobins and their constituent chains are summarized in Table 1.1.

Globin chains are encoded by globin genes, which are located in two clusters, one on chromosome 16 and the other on chromosome 11. The α globin cluster is located near the telomere of chromosome 16 and includes a ζ gene and two α genes, in addition to a number of pseudogenes. There is an upstream positive regulatory region designated the locus control region, alpha (LCRA) or HS –40 (since the region is hypersensitive to DNase and is 40 kb upstream of the αglobin cluster). The β cluster is located on chromosome 11 and includes an ε gene, two γ genes, a δ gene and a β gene. It also has an upstream positive regulatory region designated the locus control region, beta (LCRB). These two gene clusters are shown diagrammatically in Figure 1.1.

The synthesis of haemoglobin is complex. Haem is synthesized partly within mitochondria and partly in the cytosol, a total of eight enzymes being required. Its basic structure is that of a porphyrin ring with a Fe+ + (ferrous iron) atom at its centre. Globin chains, like all polypeptides, are synthesized on ribosomes, with α chains being synthesized somewhat in excess of β chains. An α chain is thus able to combine with a β chain that is still attached to its ribosome, to form a dimer, which is then detached. Each globin chain of the dimer incorporates a haem molecule before the dimer associates with another dimer to form a haemoglobin tetramer. The tetrameric structure of haemoglobin is fundamental for its function.

Haemoglobin has a primary structure (the sequence of amino acids), a secondary structure (the alternation of α helixes and non-helical turns), a tertiary structure (the three-dimensional arrangement of the haemoglobin monomer) and a quaternary structure (the relationship of the four haemoglobin monomers to each other in the tetramer). An alteration in the primary structure can affect the secondary, tertiary and quaternary structure of haemoglobin. The tetrameric structure (Figure 1.2) is a major evolutionary improvement on more primitive oxygen-binding proteins. The ability of the monomers to alter their relationship to each other on oxygen binding or dissociation is known as co-operativity. Its effect is that the uptake of oxygen by one monomer facilitates uptake by other monomers, and similarly, release of one oxygen facilitates release of the others. The functional importance of this is that in the oxygen-rich environment of the lungs, oxygen is readily taken up whereas in conditions of relative hypoxia, in peripheral tissues, oxygen is readily given up. It is this co-operativity that is responsible for the normal sigmoid oxygen dissociation curve of haemoglobin (Figure 1.3). Certain abnormal haemoglobins resemble primitive oxygen-binding proteins in that, in hypoxic conditions, they release oxygen less readily than haemoglobin A and the haemoglobin concentration rises to compensate for this; if co-operativity is entirely lost, the haemoglobin oxygen dissociation curve is hyperbolic.

Table 1.1 The normal haemoglobins of man.

Haemoglobin Globin chains Period of life when present Gower 1 ζ2ε2 Embryo Gower 2 α2ε2 Embryo Portland 1 ζ2γ2 Embryo Haemoglobin F α2γ2 Embryo, fetus and neonate; minor component during adult life Haemoglobin A γ2β2 Minor component in fetus, increasing late in gestation and in the neonatal period to become the major haemoglobin during infancy, childhood and adult life Haemoglobin A2α2δ2 Very low levels in infancy; minor component in childhood and adult life

Figure 1.1 Diagram of the α and β globin gene clusters: (a) the β globin gene cluster at 11p15.5 showing the locus control region, beta (LCRB), the ε, Gγ , Aγ ã , δ and β genes and the ψβ pseudogene; (b) the 〈 gene cluster at 16pter-p13.3 showing the locus control region, alpha (LCRA), the ζ, α2 and α1 genes and the pseudogenes, ψζ, ψα2 and ψα 1, and the θ gene (of unknown functionality).

Figure 1.2 Diagram showing the tetrameric structure of haemoglobin A: the α1β1 dimer is at the top and the α2β2 dimer at the bottom; the haem molecules are represented in green.

Although oxygen transport is the major function of haemoglobin it is not the sole function. Haemoglobin also transports CO2 from tissues to lungs and has a buffering capacity, reducing the swings in pH that could otherwise occur. It also has a role in nitric oxide (NO) transport. Haemoglobin can transport nitric oxide to tissues where is causes vasodilation. However, in pathological conditions, binding of NO to haemoglobin is not necessarily beneficial. When there is intravascular haemolysis, as in sickle cell anaemia, free haemoglobin can scavenge nitric oxide leading to undesirable vasoconstriction, which contributes to pulmonary hypertension.

Figure 1.3 Diagram showing the haemoglobin oxygen dissociation curves of haemoglobins A, F and S. Haemoglobin A has a mean P50 (partial pressure at which haemoglobin is 50% oxygenated) of about 26.8 mmHg. Haemoglobin S has a lower affinity than haemoglobin A (P50 about 35.4 mmHg) whereas haemoglobin F has a higher affinity (P50 about 19 mmHg). The partial pressure of oxygen in venous and arterial blood is indicated.

Globin gene structure and function

In order to understand how a globin gene encodes a globin chain it is necessary to know something of the structure and function of genes. Genes are DNA sequences in which a specific nucleotide sequence carries genetic information. Triplets of nucleotides (codons) either encode specific amino acids or, for a minority of sequences, do not encode an amino acid and thus serve as a stop or termination signal. A functioning gene must commence with a promoter sequence to which transcription factors can bind. This sequence is followed by an initiation sequence, which encodes methionine. Genes are composed of exons, which represent the polypeptide encoded, and introns or intervening sequences, which do not. DNA is present as a double strand, i.e. there are two intertwined strands of DNA with complementary sequences. One of these strands, the ‘antisense’ strand serves as a template for RNA synthesis so that the messenger RNA (mRNA) that is ultimately produced carries the same genetic message as the ‘sense’ strand of DNA. In addition to the promoter, which is immediately upstream of the coding sequence of the gene, genes are also influenced by enhancers. These may be located upstream, downstream or even within a gene. In the case of globin genes (and at least three other unrelated genes) there are also upstream sequences that control the transcription of all genes within the cluster, LCRA and LCRB respectively. In addition, there are various genes encoding transactivating factors, mutation of which is a rare cause of thalassaemia; they include ATRX (XH2) (α thalassaemia) and XPD (also known as ERCC2) and GATA1 (β thalassaemia). There are also two loci, at 6q22.3-23.1 and Xp22.2 respectively, that control the number of haemoglobin F-containing cells (F cells). The genetic control of globin chain synthesis is thus highly complex.

The processes involved in globin chain synthesis are shown diagrammatically in Figure 1.4. The term transcription describes the process by which an RNA precursor molecule is synthesized on a DNA template by means of RNA polymerase. Since both introns and exons are represented in this initial (primary) transcript, further processing is necessary. This processing includes removal of the introns (splicing), addition of an upstream 7-methyl guanosine cap (capping) and addition of a downstream polyadenylate tail (polyadenylation). The 7-methyl guanosine cap appears to have a role during translation. Polyadenylation is important for RNA stability. The result of processing is the production of mRNA. The mRNA moves from the nucleus to the cytoplasm where it serves as a template for ribosomal polypeptide synthesis, a process known as translation. The process also requires transport RNA (tRNA) molecules, which transport the designated amino acid to the growing polypeptide chain on a ribosome. Polypeptide chains normally commence with methionine (represented by ATG in the mRNA), which is subsequently removed. Translation stops when a STOP sequence is encountered in the RNA (TAA, TAG or TGA).

A pseudogene is a DNA sequence, which has occurred during the process of evolution, that resembles a gene in structure but does not lead to the synthesis of a protein. The lack of function may be because of a disabling mutation or because of the lack of a critical element for gene expression. Pseudogenes are transcribed but not translated. Occasionally a further mutation converts a pseudogene into a functioning gene. The globin genes include the gene encoding the δ globin chain, which may be seen as being on its way to becoming a pseudogene; alterations in its promoter have led to a low rate of transcription and consequently haemoglobin A2 is quite a low proportion of total haemoglobin.

Figure 1.4 Diagram summarizing the processes of transcription, RNA processing and translation. The DNA molecule with a globin gene is represented in line 1. In the process of transcription, a complementary RNA sequence is synthesized on the DNA template. This creates a messenger RNA (mRNA) precursor molecule, known as heterogeneous nuclear RNA (HnRNA), which must be processed by: (i) the addition of a 7-methyl guanosine cap to the 5′ end of the molecule; (ii) splicing out of the introns; and (iii) polyadenylation of the 3′ end of the molecule. Processing leads to formation of mRNA. Processing is followed by translation, in which there is synthesis of a protein on a ribosome, using the mRNA as a template.

Globin genes are commonly referred to by the same Greek letter as designates the corresponding globin chain. However, they also have ‘official’ names, as assigned by the Human Genome Project (Table 1.2).

Nomenclature of haemoglobins

Early on, the common haemoglobins found were named as haemoglobin A for adult haemoglobin and haemoglobin F for fetal haemoglobin. Haemoglobin A 2, the minor adult haemoglobin first found on starch block electrophoresis in 1955 [1], was so named in 1957 at a meeting of the International Society of Hematology (ISH) [2]. The same group noted that a minor haemoglobin band was often present slightly anodal to haemoglobin A on starch block electrophoresis at alkaline pH [3]; it was named haemoglobin A 3 at the same ISH meeting [2]. subdivided into two peaks that were labelled, in order of their elution, haemoglobin AI and haemoglobin AII [4]; a little later it was found possible to subdivide the haemoglobin A I peak into five smaller peaks, which were called haemoglobins A I a, b, c, d and e in order of their elution [5]. It was later considered that haemoglobin AIe was a storage artefact. Haemoglobin AI a, b and c are all glycated and may increase in diabetes mellitus whereas haemoglobin AId is an ageing peak due to glutathione combining with the cysteine residue at β93 [6], increasing with age of the haemolysate. The haemoglobin previously designated A 3 on electrophoresis was found to be of similar nature to the AIa and AIb peaks seen on cation exchange column chromatography [7] and also on high performance liquid chromatography (HPLC).

Analysis by cation exchange column chromatography showed that haemoglobin A could be

Table 1.2 The globin genes and locus control genes.

It was realized that confusion could be caused by using the designations haemoglobin A2 and haemoglobin AII for different types of haemoglobin and therefore haemoglobin AII of column chromatography was renamed haemoglobin A0. One consequence of the different separations and nomenclatures is that haemoglobin A on electrophoresis is equivalent to the sum of haemoglobin AI and A0 as measured by cation exchange chromatography and by most automated HPLC systems. All variant haemoglobins studied have been shown to have similar adducts to those of haemoglobin A; for instance, haemoglobin S has haemoglobin SI and haemoglobin S0. Haemoglobin F also separates into two peaks, but for a different reason. The main peak is called haemoglobin F0 (it used to be called FII) and the earlier, minor peak on HPLC is called FI. Haemoglobin FI is acetylated and is usually only present in sufficient quantities to be detected in neonatal samples.

Isoelectric focusing will also separate haemoglobin A into haemoglobin A0 and haemoglobin AI and haemoglobin F into haemoglobin F0 and FI