352
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`BRITISH MEDICAL JOURNAL
`
`11 AUGUST 1979
`
`Regular Review
`
`
`
`Mapping haemoglobin genes
`
`D J WEATHERALL
`
`The work for which the Nobel prizes for medicine and physio-
`logy have been given over recent years often seems to the
`clinician completely devoid of any possible practical applica-
`tion. For example, who would have imagined that
`the
`discoveries that led to two recent prizes, the viral reverse
`transcriptases and DNA restriction enzymes, would within a
`few years provide extraordinary insights into the causes of
`some common genetic diseases ? Yet it is these two fundamental
`advances which have opened up the whole field of the detailed
`structural analysis of human genes and which, within the last
`year, have started to yield some remarkable information about
`the molecular basis for some of the inherited diseases of
`haemoglobin production.
`Genetic disorders of haemoglobin synthesis—Many genetic
`disorders result from an abnormality in the structure or rate
`of synthesis of a specific protein or enzyme. The molecular
`structure is determined by the order of nucleotide bases that
`constitute the DNA of the gene (or genes) for the particular
`protein. This genetic information is transferred from the
`nucleus to the cytoplasm of the cell in which the protein is
`synthesised by means of a molecule called messenger RNA,
`which is transcribed from the structural gene—and hence is an
`exact replica of it. The messenger RNA acts as a template for
`protein synthesis as amino-acids are brought to it on specific
`carrier molecules which “find” the right position by interacting
`with triplets of bases (codons). This mechanism—DNA—>
`RNA —>protein——is the central pivot of molecular biology.
`Many human genetic disorders result from point mutations
`in the DNA of a particular gene for a protein or enzyme. A
`single base change in the DNA of the structural gene leads to
`the insertion of an incorrect amino—acid in the protein, and
`this may alter the structure or function of that protein and
`cause disease. Much more commonly, however, genetic
`diseases result from defects in the rate of production of a
`protein or enzyme. In these conditions a protein may not be
`made at all or may be made at a drastically reduced rate.
`Probably the most common and best-studied single-gene
`human disorders due tora reduced rate of protein synthesis are
`the
`inherited disorders of haemoglobin synthesis,
`the
`thalassaemias. These produce major public health problems in
`many parts of the world and are being seen with increasing
`frequency in Britain. Normal adult haemoglobin consists of
`two on-chains and two B—chains ((1252), and there are two major
`forms of thalassaemia, <x—thalassaemia and B-thalassaemia,
`which result
`from defective
`oc— and [3—chain
`synthesis
`respectively.‘ These conditions are genetically heterogeneous,
`and in some forms of ot- or [3-thalassaemia synthesis of ot— or
`{3—chains occurs at a reduced rate, while in others none are
`produced at all. Consequently these disorders provide a model
`for any human genetic disease ‘characterised by a reduced
`
`amount or absence of a gene product. While analysis of
`haemoglobin and its messenger RNAs has given some insight
`into the underlying mechanisms that cause these diseases, a
`complete understanding of their molecular basis can be
`obtained only by direct analysis of the globin genes themselves.
`Analysis of the globin genes—To examine the globin genes
`(or any other genes for that matter) they have to be isolated.
`The first step is to obtain some DNA. Since every cell in the
`body contains an individual’s entire complement of genes
`DNA can be obtained from peripheral blood lymphocytes,
`operation samples such as spleen, skin grown in tissue culture,
`or any other available source. Having obtained a sample of
`DNA, however, we are faced with another
`formidable
`difliculty. There is enough DNA in the cell nucleus to form
`some 10 million genes the size of a globin gene2—so that if the
`globin genes are present in only a few copies we are attempting
`to isolate and examine only one part of DNA in several
`million. Though indirect methods for doing this have been
`available for some years, only recently, with the advent of
`restriction enzyme technology, has this type of problem been
`really amenable to attack.
`found in
`DNA restriction endonucleases are enzymes
`bacteria and capable of slicing DNA at specific base sequences.“
`Presumably bacteria synthesise these enzymes to protect them
`from infecting viruses; they do not chop up their own DNA
`with restriction enzymes because they protect
`the site
`susceptible to attack, probably by methylation of the DNA at
`that site. A whole series of restriction enzymes are now
`available, each cutting DNA at different and highly specific
`sequences. Suppose, therefore, an oc- or B-globin gene is to be
`isolated and its size and structure examined. An individual
`
`globin gene might be contained in about 1600 nucleotide bases
`(1-6 kilobases or kb). A restriction enzyme can be selected to
`cut the DNA on either side of, say, the (3-globin gene so that
`it is present in a conveniently sized piece of DNA, about 4 kb
`long, for example. Of course, breaking up all the DNA in one
`cell with this enzyme will yield many thousands of pieces of
`varying sizes, simply because many “restriction sites” other
`than those near the globin genes will be attacked by the
`enzyme. These pieces may be separated by electrophoresis of
`the mixture in a slab of supporting gel, in which smaller
`pieces migrate faster than the larger ones. Having done this
`we are left with a gel containing a smear of many fragments
`of DNA of diflerent sizes. The next problem is to locate the
`globin genes.
`It is in the isolation of mammalian genes that the viral
`reverse transcriptases come into the story. In 1967 Temin
`predicted that certain tumour viruses must produce an enzyme
`that can synthesise DNA on an RNA template and thus
`reverse the usual flow of genetic information from DNA —>RNA
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`BRITISH MEDICAL JOURNAL
`
`11 AUGUST 1979
`
`to RNA —>DNA. Such enzymes were discovered independently
`by Temin and Baltimore“ 5‘ in 1970, and have now become
`generally available. Hence they can now be used, with globin
`messenger RNA, to produce DNA copies. A sample of reticulo-
`cyte-rich blood is obtained and oc- or (3-globin messenger RNA
`isolated. This is then mixed together with reverse transcriptase
`and all the building blocks required to make DNA (including
`some radioactive bases or phosphate) to produce a radioactively
`labelled copy or complementary DNA (CDNA) which has a
`base composition preciselylcomplementary to the messenger
`RNA from which it was copied. In other words we now have a
`radioactive a- or Q-globin CDNA. How can this be used to find
`the globin genes P DNA is a double-stranded molecule, and
`the backbone of the strands consists of sugars and phosphates.
`The strands are linked by the bases adenine, guanine,
`thymine, and cytosine; and, because of the rules of base
`pairing, cytosine always pairs with guanine and adenine with
`thymine. In fact
`the bases are joined by weak hydrogen
`bonds, so that if DNA is heated the two strands will come
`apart. They come together on cooling. They will pair, however,
`only if there is precise matching of the bases. Thus if the total
`cellular DNA is heated and radioactive cDNA for an oc— or
`
`[5-globin gene is added to the mixture, which is then allowed
`to reanneal, the cDNA will bind (or hybridise) to the total
`cell DNA only if it finds a complementary sequence of DNA
`—in fact, a globin gene. In this way we can use the radioactive
`cDNAs to “look for” the ot- or (3-globin genes.
`Now let us return to our smear of fragments of DNA in the
`gel. Clearly it is not possible to hybridise DNA in the Iniddle
`of a lump of gel, but the DNA fragments can be blotted on to
`an absorbent
`filter and then hybridised with radioactive
`CDNA probes to locate the ot- or (5-globin genes. Bound
`radioactive CDNA can be located by placing an x-ray plate on
`top of the filter after hybridisation to obtain a radioautograph
`of the labelled CDNA. Surprisingly, after all these manoeuvres
`it is possible to obtain very precise pictures of globin genes or
`parts of them on fragments of DNA of different sizes, depend-
`ing on which particular restriction enzymes are used to cut up
`the original DNA.
`The use of restriction enzymes does not stop at producing
`maps of this type, however. Restricted fragments of DNA
`containing globin genes can be inserted into the DNA of
`certain plasmids or bacteriophages—organisms that replicate
`in certain bacteria.“ If the insertion of the plasmid confers a
`selective growth advantage to the bacteria by supplying, for
`example, an enzyme which allows it
`to grow on selective
`media a clone of bacteria can be developed that contain
`replicating plasmid DNA (including the globin genes). These
`bacteria can be isolated for further growth by adding radio-
`active cDNA to the bacterial plates and finding out which
`clones contain the globin genes,
`just as was done for the
`restriction maps. Hence large amounts of pure globin genes
`can be generated in these bacterial factories. These can be
`sequenced to examine their structure or can be used to make
`extremely pure probes for locating globin genes on gels.7 8
`Normal and abnormal human globin gene maps—In the last
`18 months these complex techniques have yielded a vast
`amount of information about the organisation of the human
`globin genes. One surprising finding (which has shaken the
`molecular biologists) is that globin genes, apparently like most
`mammalian genes, are discontinuous.9 In other words, the
`region of DNA that codes for the structure of a globin gene
`is split up into pieces which are separated by one or more
`inserts (or introns), lengths of DNA of up to several hundred
`bases which at the moment have no known function. Apparently
`
`353
`
`the whole gene is transcribed into a long piece of messenger
`RNA and then the inserts are excised and the coding portion
`of the messenger RNA is spliced before being delivered to the
`cytoplasm. Clearly there are many places where this process
`could go wrong.
`A relatively complete map of the a—, B-, and related globin
`genes has now been produced.“’‘12 Using this map, and with a
`whole series of restriction enzymes available to cut the globin
`genes either within or outside the coding sequences, their size
`and organisation can now be studied in a whole variety of
`different genetic disorders of haemoglobin production.
`To make use of the gene maps obtained by restriction
`enzyme analysis of both normal DNA and that obtained from
`patients with genetic disorders of haemoglobin production, we
`need to consider further the heterogeneity of normal human
`haemoglobins.” "In fetal
`life the major haemoglobin is
`haemoglobin F, which has two ac-chains and two -(-chains
`(ot2Y2). The 7 chains are heterogeneous and consist of two
`types of molecules, which differ only at position 136; in one
`type of haemoglobin F position 136 is occupied by glycine
`(G7) and in the other type by alanine (Av). The production of
`Gy— and A7-globin chains is directed by separate gene loci.
`Normal adults have haemoglobin A (c1282) and the minor
`component haemoglobin A2, which consists of oc—chains and
`8—chains (oc282). Most normal adults inherit two on-chain genes
`from each parent, four in all. The Gy-, Ay-, 8-, and [3-globin
`genes lie in a linked cluster on chromosome 11; the anatomy
`of this region has been mapped and is shown in the figure.
`
`
`
`(VA), HPFH 4m_.:j----
`
`G2:6DTHAL ----
`p‘ THAL <—»
`Map of human globin genes. The distances are shown in kilobases (kb).
`The structural genes (97, ‘Y, 3 and B) are shown in boxes interrupted by
`inserts (introns). Deletions which produce hereditary persistence of fetal
`haemoglobin (HPFH), BB thalassaemia, and some forms of B“ thalassaemia
`are indicated by arrows. The broken lines represent uncertainty about
`extent of deletions. Gyfiy HPFH or 8,3 thal, or 57 SB thal are forms of HPFH
`or 3B thalassaemia in which the haemoglobin F contains both 07 and W
`chains or 5y chains only.
`
`Analysis of some of the common forms of thalassaemia by
`restriction endonuclease mapping has begun to disclose a
`variety of dilferent molecular defects, usually involving
`deletions—loss of whole globin genes or parts of them. The
`figure also shows some of the recently discovered deletions of
`genetic material in the GY-Ay-8-[3 gene cluster, which give rise
`to such common clinical disorders as (50 thalassaemia ({3-
`thalassaemia with no (3-chain production); 83-thalassaemia,
`which is a disorder associated with no 8- or (3-chain production;
`and hereditary persistence of fetal haemoglobin, another
`condition in which there is no 8- or (5-chain production but in
`which Y-chain production continues into adult life and makes
`up for the deficit of 6- and 8-chains.”‘“‘ Similarly, some of the
`on-thalassaemias have been shown to be due to deletions of one
`
`or both of the linked on-chain genes, which lie approximately
`2-7 kb apart on chromosome 16.1941 These studies have also
`shown that the ot— and [3-thalassaemias are remarkably hetero-
`geneous at the molecular level, and that in some cases where no
`
`

`
`354
`
`a— or B-chain synthesis occurs the ot— or B—g1obin genes appear
`to be intact. To elucidate these conditions we need to insert a
`
`piece of DNA containing the genes into a bacterial plasmid,
`isolate the particular clone containing the globin gene and
`grow it in large quantities, and then sequence the gene to find
`the precise molecular defect. Several laboratories have already
`succeeded in cloning human S-chain genes, from both normal
`and thalassaemic individuals, and answers to these problems
`should soon be available.
`
`The future—The field of human gene mapping has moved
`incredibly fast over the last few months. Where is all this
`activity leading? Clearly the molecular basis for many of the
`genetic disorders of haemoglobin synthesis will be defined
`over the next year or two. Furthermore, with the increasing
`understanding of how the globin genes are organised we may
`be able to answer some of the questions about how they are
`regulated and switched on and off during development. As
`techniques become available for making appropriate radioactive
`cDNA probes it may be possible to apply the techniques
`developed for studying haemoglobin to the study of other
`human genetic disorders.
`To the physician faced with the management of these
`distressing diseases the most pertinent question is whether all
`this sophisticated knowledge will offer any help in their
`management. The most immediate practical application likely
`to come from this work is the development of easier methods of
`prenatal diagnosis, at least for some of the inherited disorders
`of haemoglobin synthesis. Where these conditions result from
`major gene deletions it should be possible to identify the
`abnormality on relatively small samples of DNA prepared
`from amniotic fluid cells. This approach has already been
`shown to work for certain types of a- and [3-thalassaemia.“
`Where the globin genes are intact, however, and no demon-
`strable abnormality can be found by restriction mapping we
`need other approaches. Almost certainly human DNA will be
`found to be polymorphic in areas other than those coding for
`the structural genes. If such a polymorphism produces a new
`restriction site, and it happens tobe tightly associated with a
`common genetic disorder of haemoglobin synthesis such as
`J3-thalassaemia or sickle—cell anaemia, then restriction mapping
`might offer an approach to the prenatal diagnosis of the
`condition. Such a polymorphism has already been found
`together with the sickle-cell mutation”; but
`though the
`association seemed to be extremely strong when first described
`more recent work suggests that this may not always be the case,
`and that the use of this particular polymorphism will probably
`not give foolproof prenatal diagnosis of sickle-cell anaemia. But
`these are early days, and possibly tighter associations will be
`found between one of the common haemoglobin disorders and
`a related polymorphism in the non-coding parts of the DNA.”
`If so, the prenatal diagnosis of these conditions will be possible
`on the basis of a small amniotic fluid sample rather than the
`current laborious methods requiring fetal blood sampling.
`The ability to isolate, clone, and grow relatively large
`quantities of globin genes raises the possibility of using such
`material for specific gene replacement in patients in whom the
`globin genes are missing or non-functioning. Though the
`technical problems are formidable,
`this approach does not
`seem beyond the realms of feasibility in view of the rapid
`advances. But the difficulties should not be underestimated.
`
`Though globin genes can be introduced into a variety of cells,
`incorporated into the cell’s genome, and even persuaded to
`synthesise some haemoglobin, there is still a long step from
`this remarkable technology to actual “gene therapy.” For
`example, we would probably have to insert the non-coding
`
`BRITISH MEDICAL JOURNAL
`
`11 AUGUST 1979
`
`parts of the gene responsible for regulation as well as those
`that direct
`the structure of the haemoglobin chains, and
`somehow to ensure that the genes are active and under the
`normal regulatory control for protein synthesis. What kind of
`a cell should the globin genes be inserted into? Their place-
`ment into anything later than the stem cell would mean simply
`that the genes went into a terminally maturing population of
`cells that would eventually be lost. Yet how can the globin
`genes be inserted into a stem cell when we do not know what a
`stem cell looks like ? How could the new cell line be encouraged
`to proliferate in preference to the abnormal cell line of the
`patient into whom it was inserted ? Clearly these are formidable
`problems, but it would be a brave man who would say that
`they are insoluble. Progress has been so remarkable in the last
`few years that clinicians should probably be starting to think
`about some of the extremely difficult ethical problems that
`will be presented by the potential for such treatment.
`
`Nuffield Professor of Clinical Medicine,
`University of Oxford
`
`D J WEATHERALL
`
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