INTRODUCTION

Restriction fragment length polymorphisms (RFLPs) result from DNA base-pair changes that introduce or remove a restriction site, or sequence deletions, additions or rearrangments, that affect the length of DNA between sites. RFLPs promise to be useful in a number of different ways. Their utilisation as ‘genetic signposts’ should eventually permit the saturation of the human genome with evenly-spaced marker loci (White et al, 1985). This new system of markers at the DNA level promises to unify mapping at the cytogenetic and molecular levels, greatly improve the resolution so necessary for accurate gene mapping and linkage studies and give a new perspective on genetic variation. The improved linkage map should provide sufficient markers to localize and diagnose many hitherto undetectable genetic defects and allow the identification of heterozygous carriers.

This article describes a search for RFLPs in the human genome using a random sample of cloned DNA segments. Analysis of the data has permitted a first estimate of heterozygosity in the human genome, an amount large enough to demonstrate the extensive variation which can be exploited in clinical medicine. The clinical applications of recombinant DNA technology to the analysis and diagnosis of human genetic disease are then presented. RFLPs associated either with gene regions or linked DNA segments may permit antenatal diagnosis in cases where it has not proved possible to detect gene defects directly using a cloned gene probe.

INTRODUCTION

The history of the evolution of malaria is not known with any degree of certainty. It is found among all the major groups of terrestrial vertebrates in the Old World, suggesting that the parasites predate the hominids by millions of years. It has been claimed that plasmodial species evolved in tropical Africa or else in South-East Asia. Bruce-Chwatt (1965) pointed out that there were references to malaria dating back to 2000 BC and Hippocrates writing in the 5th Century BC was well aware of malarial fevers. There are also references to malarial fevers in the literature of India, and one writer went so far as to attribute them to mosquitos.

Prior to the 3rd Century BC, the entire population of sub-Saharan Africa consisted of small bands of hunter–gatherers. Such a low population density, coupled with the fact that such people were not tied to settlements near water holes, probably meant that malaria was nowhere hyperendemic. But by about 250 BC the Nok culture made its appearance in what is now northern Nigeria but extending both west and east at the same latitude. This belt was then probably wetter and more thickly wooded than it is today even though it was still to the north of the high forest. It was suitable for cereal growing, and the transition of people from the Stone Age into the Iron Age is thought to have taken place in that region. With farming implements made from iron, the people could cut trees and till the soil more effectively, and expand into the forest areas.

INTRODUCTION

Sir Christopher Wren in 1657 was the first to make intravenous therapy possible, in that he devised an instrument - a needle made from a slender quill fixed to a bladder - by which substances could be injected. As a result, over the next two and a half centuries the sequelae that often followed transfusions came to be recognised, and were documented especially during the Franco-German war when many attempts were made to help the wounded by transfusion under field conditions. But it was the discovery (1875) of serological species specificity, in that mixture of red blood cells of an animal of one species with serum from one of another in vitro leads to agglutination of the red cells, that triggered the discovery of the first blood polymorphism in man. For it was this that prompted Landsteiner to enquire whether differences in agglutination, similar to those in interspecific mixtures, occurred between individuals of the same species.

SYSTEMS

Red cell blood groups

The simplest method of investigation, mixing the serum of one person with the red blood cells of another, led Landsteiner to discover what subsequently became known as the A, B and O blood groups in 1900. The fourth (AB) group was discovered two years later. Then at first there was little advance. In 1910 the Mendelian inheritance of the ABO system was established, and in the same year the A1-A2 subdivisions were discovered by von Dungern and Hirszfeld. That the frequencies differed from one population to another was established by the Hirszfelds' examination of soldiers and prisoners of war of different nationalities.

INTRODUCTION

The contributions made by knowledge of the human blood groups to fundamental genetics and to the nature and pattern of population variation were enormous. Today over 160 red cell antigens are known, and data on the distribution of their frequencies in various populations of the world were collated by Mourant et al (1954, 1976), and brought up to date by Tills et al (1983). But the greatest advances in population genetics in the last few decades came as a result of the development of methods to identify genes governing other variables such as enzymes and proteins involved in fundamental biological functions in the body. Many enzymes and proteins were found to exist in more than one molecular form. Those multiple forms of proteins arising from genetically determined differences in their primary structure are now termed isozymes or isoenzymes (IUB, 1972). The study of the genetic heterogeneity of isozymes was facilitated by the techniques of electrophoresis. Tiselius (1937) developed this technique as moving boundary electrophoresis, but its first direct application to characterise human gene products was by Pauling and his colleagues (1949) who differentiated by zone electrophoresis the product of the mutant hemoglobin gene for sickle cell (HbS) from normal hemoglobin (HbA). This was a crucial discovery, because it meant that the heterozygote and homozygote could be identified directly by an experimental technique, electrophoresis. In the 1950's the technique of zone electrophoresis was perfected by exploration and development of a variety of supporting media such as agarose, cellulose acetate, starch and polyacrylamide.

INTRODUCTION

Human chromosomes exhibit structural variants which occur at considerable frequencies. Although at the Paris Conference on Standardization in Human Cytogenetics (1975) the term “chromosomal heteromorphism” was used for the description of these variants, the term “chromosomal polymorphism” is here used, “chromosomal” to indicate that the variant is at the level of microscopically detectable chromosome structures, “polymorphism” to indicate that it complies with the conventional definition of a genetic polymorphism.

Chromosomal polymorphic variants are constant within a given individual. At least, no consistent differences between the different cell types of one individual are known (though there are special problems with the nucleolar organizer regions, see below). Chromosomal polymorphisms are inherited in a simple Mendelian mode. They can be demonstrated by several techniques, but not all of them by any one technique. They are usually not present in an “all or none” fashion but show different grades, and this makes their evaluation rather difficult in some cases.

The chromosomal polymorphisms were reviewed a few years ago by Verma and Dosik (1980). They are of course also relevant clinically, e.g. as a contributing cause of chromosome abnormalities, as possibly associated with certain diseases, and with tumorgenesis (see, e.g., Atkin, 1977; Kivi & Mikelsaar, 1980; Sutherland, 1983; Glover et al, 1984). In this paper some considerations and new findings on the nature of the chromosomal polymorphisms are presented as well as a brief review of the more recent results of population studies.

TYPES OF POLYMORPHIC REGIONS OF CHROMOSOMES

Table 1 lists different types of chromosomal polymorphisms. The different chromosome bands are placed in one group because of their cytological and molecular similarities.

INTRODUCTION

The HLA system consists of over 100 antigens in six segregant series. Kissmeyer-Nielsen (1968) first identified two closely linked loci, with genes controlling two independent series of antigens, the A and B series, while Sandberg et al (1970) proposed a third locus (C). These are situated close together on chromosome 6 (Fig. 1), but their independence was confirmed by the cross-overs that occur between them (Kissmeyer-Nielsen et al, 1969; Low et al, 1974). Whereas antigens at these loci are recognised by serological methods, the fourth locus (D) was recognised from examination of mixed lymphocyte reactions. When lymphocytes from two individuals are mixed together in appropriate culture conditions, the one stimulates the other to blastoid transformation and proliferation. The extent of the reaction depends upon the degree of histocompatibility between the individuals, and is largely dependent on the D locus. Serological correlates of the MLR were therefore sought, and following the detection on leukaemic cells of B cell alloantigens (Walford et al, 1975), a series of DR (= D-related) antisera were standardised. Extremely close to the DR locus is one controlling a second series (DQ) of antigens (Duquesnoy et al, 1979; De Kretser et al, 1983), while a further locus (DP) governing reactivity in the primed lymphocyte test (secondary MLC) is shown by both cellular and serological techniques (Shaw et al, 1980, 1982; Mawas et al, 1980). The relationship between the specificities defined by MLC and the DR, DP and DQ antigens remains unresolved.

Antigens of the A, B and C series are known as class 1 antigens, and are found on the cells of most body tissues.

INTRODUCTION

All individuals with sickle cell anemia have two β genes that encode a valine residue instead of a glutamic residue in position 6 (Bookchin & Nagel, 1981). Nevertheless the clinical or phenotypic expression of this homozygous genetic abnormality is extremely variable from individual to individual (Labie, 1983; Nagel & Fabry, 1986). Although environmental factors are obviously involved they do not account for most of the variability, and other genetic factors have to be invoked. In the last few years significant progress has been made in this area and the findings from our laboratories and those of others indicate three genetic sources for the variability in the expression of Hb S homozygosity:

SOURCES OF VARIABILITY

Derived from multiple gene origin: sickle cell anemia is a multihaplotype disease

If the Hb S gene had originated only once in history and then expanded in frequency, all sickle cell anemia patients not only would be homozygous for the mutation but would tend to have identical genes and other sequences surrounding the mutated β globin gene. Even if genes or sequences to the left or to the right of the β globin gene could modify the expression of the Hb S gene, a single origin would guarantee that a significant and identical genomic block around this gene would be inherited as an entity by all individuals carrying the Hb S gene. Hence, even in the presence of modifying or epistatic genes located nearby, the variability of phenotypic expression would be minimal unless hot spots for recombination are present.

Following that general misgiving as to our national system of education which, long felt by thoughtful men, found loud and continual expression during the war, Mr Asquith, then Prime Minister, appointed (1916) Committees to consider the position of natural science and of modern languages respectively. After these Committees had reported, a third Committee was set up (1919) to investigate the position of classics in our educational system. The Report of this Committee, recently issued, is a comprehensive document, full of interesting materials, readable and scholarly, as from the character of the Committee might be expected. The history of classical teaching in the several parts of the United Kingdom, its rise and recent decline, are set out in detail, with an abundance of information never before collected. As to the main inference, no mistake is possible. The classical element in British education is disappearing, and will probably soon be gone altogether.

Prefatory Note. The Professorship of Biology was founded in 1908 for a period of five years partly by the generosity of an anonymous benefactor, and partly by the University of Cambridge. The object of the endowment was the promotion of inquiries into the physiology of Heredity and Variation, a study now spoken of as Genetics.

It is now recognised that the progress of such inquiries will chiefly be accomplished by the application of experimental methods, especially those which Mendel's discovery has suggested. The purpose of this inaugural lecture is to describe the outlook over this field of research in a manner intelligible to students of other parts of knowledge.

The opportunity of addressing fellow-students pursuing lines of inquiry other than his own falls seldom to a scientific man. One of these rare opportunities is offered by the constitution of the Professorship to which I have had the honour to be called. That Professorship, though bearing the comprehensive title “of Biology”, is founded with the understanding that the holder shall apply himself to a particular class of physiological problems, the study of which is denoted by the term Genetics. The term is new; and though the problems are among the oldest which have vexed the human mind, the modes by which they may be successfully attacked are also of modern invention.

The alleged Mimicry of Volucellae. 1892. Nature, xlvi, No. 1199, 585, W. B.; ibid.xlvii, No. 1202, 28, Poulton; ibid. No. 1204, 77, W. B.; ibid. No. 1206, 126, Poulton.

The Origin of the Cultivated Cineraria. 1895. Nature, li, No. 1330, 605–607, W. B.; ibid. lii, No. 1331, 3, Thiselton-Dyer; ibid. No. 1332, 29, W. B.; ibid. No. 1333, 54, Weldon and W. Botting-Hemsley; ibid. No. 1334, 78, Dyer and Weldon; ibid. No. 1335, 103, W. B.; ibid. No. 1336, 128, Dyer.

Also notice of the discussion in Gard. Chron. 11 May 1895, p. 588. Also correspondence and MS. notes.

Women's Degrees. Fly sheets. Cambridge University. 1897.

Compulsory Greek. Fly sheets. 1891,1905. Letter to Nature, lxxi, No. 1843, February 1905, 390. Speeches in the Senate House, Cambridge University Reporter, 17 December 1904, pp. 392, 393; ibid. 7 May 1906, pp. 807, 808.

Mendelian Controversy. 1902 and onwards. “Mendel's laws of Alternative (sic) Inheritance in Peas.” W. F. R. Weldon. Biometrika, 1, pt 2, 228–254. “A defence of Mendel's Principles of Heredity.” W. Bateson, with a translation of Mendel's original papers on Hybridisation. Cambridge University Press, 1902. “On the Ambiguity of Mendel's Categories.” W. F. R. Weldon. Biometrika, ii, pt 1, 44–55. “Mr Bateson's Revisions of Mendel's Theory of Heredity.” W. F. R. Weldon. Biometrika, ii, pt 3, 1903.

I visit Canada for the first time in delightful circumstances. After a period of dangerous isolation, intercourse between the centres of scientific development is once more beginning, and I am grateful to the American Association for this splendid opportunity of renewing friendship with my western colleagues in Genetics, and of coming into even a temporary partnership in the great enterprise which they have carried through with such extraordinary success.

In all that relates to the theme which I am about to consider we have been passing through a period of amazing activity and fruitful research. Coming here after a week in close communion with the wonders of Columbia University, I may seem behind the times in asking you to devote an hour to the old topic of evolution. But though that subject is no longer in the forefront of debate, I believe it is never very far from the threshold of our minds, and it was with pleasure that I found it appearing in conspicuous places in several parts of the programme of this meeting.

Standing before the American Association, it is not unfit that I should begin with a personal reminiscence. In 1883 I first came to the United States to study the development of Balanoglossus at the Johns Hopkins summer laboratory, then at Hampton, Va. This creature had lately been found there in an easily accessible place.