The future role of molecular and cell biology in medical practice in the tropical countries

David Weatherall

Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, UK Downloaded from https://academic.oup.com/bmb/article/54/2/489/285007 by guest on 27 September 2021

Molecular and cell biology have a great deal to offer tropical medicine in the future. As well as helping to understand the population genetics and dynamics of both infectious and non-infectious diseases, they promise to provide a new generation of diagnostic and therapeutic agents, and to play a major role in the development of new vaccines and other approaches to the control of disease in tropical communities.

Over the last 20 years there has been a gradual shift in the emphasis of basic biomedical research from the study of disease in patients and their organs to its definition at the level of molecules and cells. This new trend has been underpinned by a remarkable new technology which has made it possible to isolate and sequence genes, study their function and transfer them across the species barrier. In the short time during which this field has evolved, a great deal has been discovered about human pathology at the molecular level. Many monogenic diseases have been characterised, much has been learnt about the molecular and cell biology of cancer, and a start has been made in defining the different genes that comprise the complex interactions between nature and nurture that underlie many of the major killers of Western society. Enough is known already to suggest that this knowledge will have major implications for the development of more precise diagnostic and therapeutic agents in the future. There is little doubt that tropical medicine will benefit from this new technology, particularly as the demography of disease changes in emerging countries. The World Health Organization have predicted that by the year 2020 there will be a major shift in the pattern of disease1. As social conditions and standards of hygiene and nutrition improve, there

Correspondence to: will be a gradual decline in infant and childhood mortality due to Professor Sir David infectious disease. On the other hand, it is predicted that there will be a Weatherall, Institute of steady increase in diseases of 'Westernisation', including heart disease, Molecular Medicine. diabetes, other forms of vascular disease, and the major psychoses. It is University of Oxford, John Radcliffe Hospital, already apparent from the epidemic of insulin-resistant diabetes that is Oxford OX2 9DS, UK affecting many of these countries, that populations respond in quite

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different ways to the introduction of high energy diets2. Though it is likely that both environmental and genetic factors are involved, there is increasing evidence that inheritance may play an important role in these different responses. It is important, therefore, that medical practice in the tropics prepares itself for the remarkable possibilities that the rapidly moving sciences of

molecular and cell biology will have to offer it in the future. This has Downloaded from https://academic.oup.com/bmb/article/54/2/489/285007 by guest on 27 September 2021 particular implications for medical education; specialists in the field will have to be able to communicate with those working in the basic sciences so that their technology can be adapted most effectively for the benefit of the health of communities in the tropical world.

Technical advances

The application of molecular and cell biology to the study of human disease, or molecular medicine as it is rather optimistically called, has developed on the back of the technical advances of molecular biology3'4. One of the first was the discovery of how to isolate DNA and to cut it up into pieces of different sizes using restriction endonucleases, that is enzymes isolated from various bacteria that will slice DNA at predictable sequences of nucleotide bases. An early and quite seminal advance in the application of this approach to human pathology was called Southern blotting after the name of its inventor, Edwin Southern. In this technique restriction enzyme digests of DNA are separated into different sized fragments by electrophoresis in gels and then simply blotted onto nitrocellulose filters on which they can be immobilised. By constructing radioactive probes to hunt for particular genes on these filters it was possible to analyse potential disease loci for major deletions or re-arrangements of the particular genes involved. / It soon became possible to take mixtures of restricted DNA and to insert the different fragments into bacterial plasmids or other 'foreign' DNA vectors. This was the beginning of the era of recombinant DNA technology. The inserted DNA could be grown in bacteria and, hence, it became possible to construct libraries containing most of the human genome from which it was possible to isolate any gene of interest. Methods were soon developed for rapid sequencing of DNA and hence it became feasible to define the precise mutations in many single gene disorders. And by carrying out genetic linkage studies using highly variable regions of DNA as markers to pinpoint genes for diseases of unknown cause, and to deduce the function of their products from their sequence, a technique called 'reverse genetics', it became possible to characterise the molecular pathology of many common monogenic disorders of unknown cause.

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The next important step was to take isolated human genes and persuade them to function, either in cultured cells or in laboratory animals. This made it possible to learn about the major regulatory regions that are involved in ensuring that genes are expressed in the correct tissues at the right stages of development and at an appropriate level. Furthermore, as methods for sequencing genes became more

efficient and were automated it became clear that it would be possible Downloaded from https://academic.oup.com/bmb/article/54/2/489/285007 by guest on 27 September 2021 to determine the complete sequence of the genome of any organism. Currently, this has already been achieved in the case of some bacteria and varieties of yeast, and the Human Genome Project, that is the determination of the complete sequence of the DNA of a human being, is well on course for completion early in the next millennium. Although the full benefits of this field for the improvement of health may not be evident for many years, and particularly until new developments in biomathematics and computer technology help us to understand how all our genes are orchestrated to subserve the complex metabolic functions of intact cells, organs, and whole organisms, there is no doubt that along the way there will be a steady accumulation of information that will make a major impact on tropical medicine. It is beyond the scope of this brief review to describe all these possibilities and hence I shall simply summarise a few that are already well advanced and try to predict some of the more important possibilities in the future.

Molecular genetics in the tropics

Single gene disorders

Although many diseases are inherited in a simple Mendelian pattern, and are seen in every part of the world, most of them occur at a very low frequency which probably reflects the mutation rate. However, there are a few groups of genetic disorders which occur much more commonly and which will pose an important public health problem in the future. There is increasing evidence that they have reached their high frequency in many tropical countries by natural selection, reflecting heterozygote advantage against different forms of malaria. It is probably through this mechanism that the inherited disorders of haemoglobin have become the commonest human monogenic diseases5 Human adult haemoglobin consists of two pairs of a chains and two pairs of P chains (

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amino acid substitutions or other structural alterations in the a or P globin chains. The second and more common disorders are those due to a reduced rate of synthesis of the a or P globin chains, the a and P thalassaemias. Studies at the molecular level have led to a broad understanding of the structure and regulation of the globin genes and of the molecular pathology of both the structural variants and the 5

thalassaemias . Downloaded from https://academic.oup.com/bmb/article/54/2/489/285007 by guest on 27 September 2021 Although several hundred structural haemoglobin variants have been described only three, haemoglobins S, C and E, reach very high frequencies in some tropical countries. The homozygous state for the sickle cell gene, sickle cell anaemia, is a major cause of childhood morbidity and mortality in sub-Saharan Africa, the Mediterranean region, the Middle East and in parts of India5. It also occurs frequently in countries with large African immigrant populations. Although much remains to be learnt about the mechanisms of sickling and the reasons for the remarkable clinical heterogeneity of sickle cell anaemia, considerable progress has been made towards its better management and prevention from research at the molecular and cellular level6. Haemoglobin E reaches very high frequencies throughout Bangladesh, Burma, and in many countries in southeast Asia. Although its homozygous state is characterised by a mild hypochromic anaemia, because it is synthesised at a reduced rate, when inherited together with P thalassaemia it often produces a crippling thalassaemic disorder. This condition, haemoglobin E/p thalassaemia, is, globally, the commonest serious inherited disorder of haemoglobin7. The P thalassaemias, which result from over 150 different mutations of the P globin gene, occur commonly throughout the Mediterranean region, in parts of Africa, throughout the Middle East and the Indian sub-continent, and in many regions of southeast Asia5. Together with haemoglobin E/p thalassaemia, they constitute an increasing public health problem now that childhood mortality rates due to malnutrition and infection are falling and affected children are living long enough to require treatment7. The a thalassaemias are also very heterogeneous at the molecular level, and the severe forms, or a° thalassaemias, which occur most frequently in the Mediterranean region and southeast Asia, are associated with death in utero. The milder forms, the a+ thalassaemias, which occur frequently in many parts of sub-Saharan Africa, throughout the Mediterranean region, the Middle East and southeast Asia, are associated with mild hypochromic anaemias. Through an understanding of the molecular pathology of these conditions, better forms of treatment have been developed and it has been possible to establish prenatal diagnosis programmes which have led to a marked reduction in their frequency in many parts of the Mediterranean region8. However, their control by carrier detection and

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prenatal diagnosis is not yet established in many countries, and in some may not be acceptable for religious and other ethical reasons. Thus the better management of these diseases offers a major challenge to the field of molecular and cell biology, and for medical practice in the tropics, for the next millennium. Downloaded from https://academic.oup.com/bmb/article/54/2/489/285007 by guest on 27 September 2021 The haemoglobin disorders and malaria

Our new-found ability to identify different forms of thalassaemia at the molecular level has recently re-opened up the question of whether these diseases have reached their high frequencies because of heterozygote protection against severe forms of malaria. While this was known to be the case for sickle cell disease9, until very recently there was little evidence that the same mechanism underlies the particularly high frequency of the thalassaemias. Recent work has shown, however, that the milder forms of a thalassaemia offer a highly significant level of protection against Plasmodium falciparum malaria, though, remarkably, affected babies during the few years of life seem to be more prone to infection, by both P. vivax and P. falciparum10'11. This quite unexpected observation, which highlights the value of direct DNA analysis for genotyping large populations, offers a novel conceptual framework for understanding how a* thalassaemia might protect against malaria11. It appears that homozygotes are more susceptible to malaria, but only at a time of development when the disease rarely kills. Thus, it is possible that this may provide them with an immunising dose of malaria which offers later protection. Interestingly, in areas in which both types of malaria occur, the earliest infections in life are usually due to P. vivax. Furthermore, there is some evidence that there may be cross- immunisation between the two common forms of malaria12. Thus the fact that young babies with a thalassaemia are more susceptible to P. vivax early in life may explain their later resistance to P. falciparum.

Other genetic polymorphisms and malaria

It is now clear that many other red cell disorders and polymorphisms have been shaped by malaria. Again, it is research at the molecular level which has enabled these issues to be clarified. Glucose-6-phosphate dehydrogenase (G6PD) deficiency results from many different mutations at the G6PD locus. Recent studies suggest that both female heterozygotes and male hemizygotes have a reduced risk of malaria, in the range of 50%13. Protection is also mediated by different red cell surface antigens; the molecular basis for the absence of the Duffy

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antigen, observed many years ago to be associated with resistance to P. vivax malaria, has now been identified as a substitution in the binding site for the GATA1 erythroid transcription factor at position -156 to the promoter14. By gene mapping analyses, the Melanesian form of ovalocytosis, which is quite difficult to identify with certainty, has been shown to provide complete protection against cerebral malaria (S.J.

Allen, personal communication). Downloaded from https://academic.oup.com/bmb/article/54/2/489/285007 by guest on 27 September 2021 Genetic variability due to selection by malaria is not confined to genes that are expressed in red cells15. Certain polymorphisms of the HLA-DR system are associated with protertion against both cerebral malaria and severe malarial anaemia16. While earlier studies of associations of this type were bedevilled by the possibility that the histocompatibility antigens were simply acting as informative markers for other genes that might be genuine immune response determinants, analyses at the molecular level has shown quite clearly that these genes and their products are, in themselves, immune response agents through determinant selection. For example, studies of the HLA-B53 association with malaria have shown how this might be mediated through the identification of HLA-B53 cytotoxic lymphocytes that identify a particular parasite epitope17. Another important malaria-related polymorphism that has been discovered recently involves the gene encoding tumour necrosis factor-a (TNFa). Gambian children who are homozygous for a single base change at position -308 of the promoter of the TNFa gene have a significantly increased likelihood of dying from cerebral malaria18. In vitro reporter gene studies suggest that this polymorphism can increase levels of TNF expression, an observation that is compatible with evidence that excessive TNF production is a major factor in the pathogenesis of cerebral malaria. Another potentially important functional polymorphism related to malarial infection which has been unearthed recently involves a member of the immunoglobulin supergene family called ICAM-1. This protein has a wide range of immunological functions and it has also been found to have an important role in the adherence of P. falciparum-iniected red cells in cerebral malaria. It has been found that, in parts of Africa, there is a high prevalence of a polymorphism of this protein which appears to be a predisposing factor for cerebral malaria19. It is possible that this may have been selected for the role that it may play in other forms of infection but that it is disadvantageous in the case of those due to P. falciparutn. Now that it is possible to study genetic disease and polymorphisms in large populations at the DNA level, a remarkable picture of their pattern of distribution is evolving. It is clear, for example, that in the case of a and B thalassaemia, every population has its own particular mutations. The sickle cell mutation occurs in Africa, the Mediterranean and India, but not further east; haemoglobin E is found all over southeast Asia,

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Burma and Bangladesh, but not further west. Melanesian ovalocytosis is confined to Melanesia and adjacent regions. Different HLA-DR polymorphisms appear to be protective in particular populations. It is likely that these observations reflect the relatively recent appearance of malaria as the principal agent that has maintained these polymorphisms, a notion that is strengthened by the molecular analysis of the

relationship between globin gene polymorphisms and their associated Downloaded from https://academic.oup.com/bmb/article/54/2/489/285007 by guest on 27 September 2021 restriction fragment length polymorphism haplotypes9. Malaria must have been a particularly powerful selective agent to have recruited so many deleterious and other traits in such a short evolutionary time.

Genetic susceptibility to infections other than malaria

Although it has been suspected for a long time that individual responses to infection may have a strong genetic basis, it is only with the advent of the molecular era that it has been possible to investigate this important possibility20*21. Until very recently studies in this field utilised the 'candidate gene' approach, that is research workers made an educated guess about what type of gene might be involved in host-defence mechanisms and then proceeded to test whether particular polymorphisms are associated with different degrees of susceptibility. One of the first candidates was the HLA-DR gene family; early studies using serotyping have now been strengthened by an analysis of polymorphisms of this system at the DNA level. Convincing HLA-DR associations have been found with a variety of parasitic illnesses including leishmaniasis, onchocerciasis and filariasis20. Strong associations have also been demonstrated with either chronic carriage or rates of viral clearance in patients infected with hepatitis B virus22-23. The candidate gene approach has also been valuable for studying genetic variability in susceptibility to bacterial infection. As well as malaria, TNF polymorphisms have been found to modify the course of meningococcal meningitis, lepromatous leprosy and trachoma24"26. Increased susceptibility to bacterial infections has also been related to several polymorphisms of the gene for the mannose-binding protein (MBP)27. It has long been known that the ability to secrete the soluble form of ABO blood group antigens into saliva and other body fluids, that is the secretor status, may be associated with variable susceptibility to bacterial infection. Recently, the molecular basis for the non-secretor phenotype has been found to be a nonsense allele at the gene locus involved, Sec218. Preliminary studies suggest that this polymorphism occurs widely as the basis for the non-secretor phenotype and, hence, it should now be relatively easy to relate secretor status to varying susceptibility to infection by studies at the DNA level.

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Important progress is also being made towards a better understanding of individual susceptibility to viral disease using the candidate gene approach. The relationship between HLA-DR polymorphisms and the rates of clearance of hepatitis B virus has already been mentioned. The puzzling observation that some patients who are regularly exposed to the human immunodeficiency virus (HIV) do not become infected has 29

recently been explained, at least in some cases, at the molecular level . Downloaded from https://academic.oup.com/bmb/article/54/2/489/285007 by guest on 27 September 2021 It turns out that CD4-positive T-cells of such individuals are highly resistant in vivo to the entry of primary macrophage-tropic virus but are regularly infected with transformed T-cell adapted viruses. These individuals are homozygous for a defect in the Ckr-5 gene, which encodes for the co-receptor for primary HTV isolates. The molecular defect in this case appears to be a defective Crk-5 allele containing an internal 32 basepair deletion. Quite recently, a different approach has been used to search for genes that may modulate response to infection. In this case, linkage analyses have been carried out using DNA polymorphisms to pinpoint genes that govern the intensity of infection by Schistosoma mansoni. Hitherto such 'blind' genome searches have been extremely difficult, but, with the availability of a linkage map of most of the human genome, this approach offers an extremely valuable way of finding disease-susceptibility genes. In this case, a gene that modifies the degree of infection by S. mansoni was found to lie on chromosome 5q31-q33, a region that encodes IL4, IL5 and several other immunological mediators30. There is no doubt that this powerful approach will be extremely valuable for identifying other genes that modify susceptibility to infection.

Studies of the genomes of infectious organisms

The development of techniques for rapidly sequencing DNA, and then- automation, are making it possible to sequence the entire genomes of micro-organisms in a relatively short time. A full list of bacteria and other agents that are being analysed in this way has been published recently31.It is already clear that a knowledge of the primary DNA sequence of an infectious organism can yield valuable information, particularly the identification of different virulence genes. These studies can be combined with assays for function. For example, it is possible to define different gene transcripts at particular phases of infection. The methodology for this type of analysis is developing rapidly; using microchip technology it is feasible to study the output of literally thousands of genes at different phases of infection. This provides a valuable profile of which genes are likely to be of particular functional importance with respect to virulence. This information has major

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implications for strategies directed at the development of new anti- microbial, anti-viral and anti-parasitic agents. Analysis of the genomes of pathogenic bacteria have other important implications. These organisms face a particularly daunting task because infections occur within a very short time during which they encounter a wide variety of host polymorphisms, a range of immune mechanisms, and environments which may already be modified by the administration of anti-microbial drugs. It is now becoming clear that bacteria, and Downloaded from https://academic.oup.com/bmb/article/54/2/489/285007 by guest on 27 September 2021 probably other organisms, have a remarkable repertoire of different genetic systems with which to tackle these problems32. One mechanism of this kind, that is of considerable current interest, is the generation of mutator alleles, genes or gene families which can lead to an increase in mutation rate. Such alleles may predispose a wide variety of different genes to potentially beneficial mutations, and may hitchhike because the mutator allele and the selected alleles may be linked, especially in asexual clonal populations. It is now clear that viruses, bacteria and parasites may all have subsets of genes that are excessively prone to mutation through a variety of molecular mechanisms. These hypermutable genes encode surface molecules, such as adhesins and invasins, which are intimately involved in interactions with host molecules. Hence, populations of micro-organisms can use these combinatorial systems rapidly to generate phenotypic variation which can influence antigenicity, motility, chemotaxis, attachment, and a variety of other qualities which may alter their virulence. The potential molecular mechanisms for these remarkable adaptive changes, which are not yet fully understood, have been summarised recently32. Clearly, these genes offer another potential target for novel chemotherapeutic agents.

Diseases due to 'Westernisation' As mentioned earlier, there is a remarkable variation between different populations in their response to the diets and lifestyles of more affluent Western societies. For example, there is currently an increasingly serious world epidemic of type 2 diabetes, with or without associated obesity. In some countries that have been exposed to high energy diets for the first time, up to 70% of the population is affected. In the Pima Indians, this form of diabetes is associated with gross obesity and an extremely high frequency of gallstones, a constellation of disorders which has been called 'the new world syndrome'. Although there is currently much debate about the role of nature and nurture in the generation of the high frequency of diabetes it seems very likely that genetic factors play an important role; this form of diabetes shows an extremely high degree of heritability in twin studies2.

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This remarkable susceptibility to diabetes as a response to high energy diets may reflect what has been called a 'thrifty genotype'. The idea is that, during the early dissemination of Homo sapiens, populations that had to undergo long journeys and periods of dietetic deprivation may have undergone intense selection for a variety of different genes involved in more effective energy storage; it is just this genetic make-up that now

makes their distant progeny more likely to develop diabetes. Since there Downloaded from https://academic.oup.com/bmb/article/54/2/489/285007 by guest on 27 September 2021 are probably many ways in which this type of susceptibility might be mediated, different genes may be involved in different populations. It will be extremely productive, therefore, to try to identify them by DNA- based linkage studies. Because this form of diabetes is an important risk factor for vascular disease, a better understanding of its pathogenesis is of particular importance. Diabetes is not the only common disease of Westernisation that is being found at different frequencies in the emerging countries. There are remarkable differences in the frequency of coronary artery disease and hypertension which may also have a genetic basis. Similarly, it is becoming clear that childhood respiratory disorders, particularly asthma, also vary in frequency in a way which may reflect genetic as well as environmental factors. These common non-infectious diseases will play an increasingly important role in determining the pattern of disease in the tropical world. Since susceptibility to them may reflect the action of completely different genes to those that are involved in Western societies, this aspect of medicine in the tropics will be increasingly important in the future. The tools of molecular genetics, particularly linkage analysis using DNA polymorphisms to find the particular genes involved, will provide an extremely valuable approach to this important problem (see also Forrester, Cooper and Weatherall in this issue).

Diagnosis and therapy

The recombinant DNA era offers a wide variety of new possibilities for the more accurate diagnosis and definitive treatment of many of the current diseases of tropical climates as well as for the non-infectious disorders that may gradually take their place.

Diagnostics

DNA-based technology offers a wide variety of new diagnostic approaches to infectious disease3-4. The development of the polymerase chain reaction (PCR), which makes it possible rapidly to amplify any

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chosen piece of DNA from the genome of a micro-organism, has provided a valuable new approach to the diagnosis of bacterial, viral and parasitic illnesses. As the genes that are involved in the development of resistance to antibiotics and other therapeutic agents are defined, this approach is also providing valuable information about the emergence of resistant strains of organisms in populations. PCR-based technology is also being used for both carrier detection and prenatal diagnosis of important genetic diseases in the tropics, particularly the Downloaded from https://academic.oup.com/bmb/article/54/2/489/285007 by guest on 27 September 2021 haemoglobinopathies. DNA can be obtained by chorion villus sampling during the first trimester of pregnancy; the increasing use of this approach in prenatal diagnosis programmes has already resulted in a marked reduction in the number of cases of fJ thalassaemia in many Mediterranean populations8. Currently, plans are being developed to apply this technology more widely in the rural populations of southeast Asia. For example, in Thailand it is hoped that it will be possible to reduce the number of births of children with serious forms of thalassaemia by approximately 30% over the next 20 years. Similarly, as more is learnt about the genes involved in susceptibility to diseases, such as type 2 diabetes, it should be possible to define individuals at particularly high risk for developing these conditions in response to changes in diet and lifestyle.

Prevention and treatment of tropical diseases

Recombinant DNA technology offers many different new approaches to the development of vaccines33. A variety of methods are being explored. One of the earliest successes was the development of a vaccine against hepatitis B. In this case, a gene for an appropriate antigen was cloned in yeast to produce a safe and effective vaccine. The advantage of this technique is that the genetically engineered yeast cell contains information for only a particular viral gene, and hence there is no possibility that a complete virus or any other infectious agent could arise during the production of the vaccine. In other cases, live recombinant vectors are being used. More recently there has been considerable interest in DNA immunisation. In this case, inoculation with plasmid DNA vectors that encode immunogenic proteins appears to induce both humoral and cell-mediated immune responses, which quite often seem to provide protective immunity. Although this approach is still in its infancy, and its overall efficacy and safety remain to be determined, in the long-term it may offer a safer and cheaper alternative to more conventional vaccines. As more is learnt about the virulence genes of microorganisms, and about_the__gattern of gene expression during infections, it should be

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possible to define specific targets for the development of new forms of chemotherapy. In effect, therapeutics will move from traditional medicinal chemistry to the era of designer drugs. There is also much current interest in defining the different molecular mechanisms for drug resistance. A variety of members of a ubiquitous family of membrane transporter proteins have been identified as being major players in the development of resistance to a wide variety of chemotherapeutic agents. As more is learnt about how drug resistance is mediated, through Downloaded from https://academic.oup.com/bmb/article/54/2/489/285007 by guest on 27 September 2021 changes in the genes that encode for these proteins, it should be possible to design drugs to by-pass these resistance pathways.

Postscript

It has only been possible to outline a few of the applications of molecular and cell biology to medicine in the tropics. But enough has been achieved already to make it clear that this field has enormous possibilities for improving the health of the populations of the emerging countries. Currently, this is a complex research field, the fruits of which are expensive. International health agencies and the governments and pharmaceutical industries of richer countries will have to recognise its importance for world health in the future, and ensure that its applications for the diseases of the emerging countries are not neglected.

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