Genetic and Metabolic Disease in Pediatrics: Butterworths International Medical Reviews

UK

1

Genetic causes of deviant metabolism. What do we know? What can we do?

Charles R. Scriver and June K. Lloyd

Publisher Summary

This chapter discusses the genetic causes of deviant metabolism. Disrupted homeostasis underlies any disease and has proved to be a useful paradigm to interpret the cause, pathogenesis, and manifestations of even that classic genetic but nonmetabolic disease, Down’s syndrome. To appreciate the origins of dishomeostasis in a disease, it is necessary to consider the process of displacement. Homeostasis has metrical parameters; their statistics are the central tendency (mean), the dispersion (standard deviation), and the character of dispersion (skewness and kurtosis). Deviation of a value beyond the normal boundaries of a biological parameter can reflect only two events (mutation or experience); one or other acts predominantly or, as is most often the case, they act together. Displacement, therefore, reflects either an extrinsic experience overwhelming the process or an intrinsic event subverting it or a combination of the two. Health of modern man living in a developed society has resulted largely from ascendency over causes of the first type. When mortality and morbidity were most often the manifestations of extrinsic causes, it was sufficient to control these causes; as a result, collective health and longevity improved.

Claude Bernard (1878) said: ‘Constancy of the milieu interieur is the condition of a free and independent existence’. This idea reflected a new awareness about life on earth. There was form: the awareness that living things on earth have certain limits to form which are their own, reflecting evolutionary processes and physical laws that are not miraculous or supernatural. There were signs: manifestations of specific forms and functions and ways to describe them. And there were mechanisms: explanations of how things work. Homeostasis, the term devised (Cannon, 1932) to fix the idea of adaptive constancy of biological functions in living systems, was perceived as a product of mechanisms serving the integrity of form.

Knowledge of heredity preceded knowledge of its mechanism. The origin of continuity in form, and in the mechanisms that sustain living systems, was mysterious until the discovery of DNA (Schrödinger, 1944). Now that we understand the ‘phenotype paradigm’ (Figure 1.1) and the processes of replication, transcription and translation of information encoded in DNA (Crick, 1970), we perceive that the gene is a relative ‘constant’; it determines species and the persistence of their form (phenotype) in the individual descendants. But life is a process whereby genotype and thus phenotype is undergoing variation, and in the resultant interactions with experience, organisms can fail or succeed. The consequences of variation are measured in biological terms; either individuals reproduce adequately or they do not. Their adaptation and their success reflects homeostasis in the individual members of the species. Humans prefer not to measure their own success merely in biological terms; we see the consequences of selection extended to encompass behaviour and culture (Skinner, 1981). Nevertheless, adaption in any context implies measures of normality. Deviant values indicate dishomeostasis, the cost of which is disadaption or, as we usually call it, disease. We tend to think about obvious physiological and metabolic parameters when we invoke homeostasis. But the idea in its broadest terms, that disrupted homeostasis underlies any disease, has proved to be a useful paradigm to interpret the cause, pathogenesis and manifestations of even that classic genetic but non-metabolic disease, Down’s syndrome (Shapiro, 1983).

Figure 1.1 The ‘central dogma’ of biology (Crick, 1970) is also the ‘phenotype paradigm’. The diagram indicates pathways of information transfer: DNA→DNA (replication); DNA→RNA (transcription); RNA→protein (translation). Solid arrows indicate conventional transfers; interrupted arrows represent special (e.g. reverse transcription) or unidentified transfers. The vectorial relations (direction of arrow) imply that mutation in DNA influences phenotype (protein) but that perturbations in phenotype (e.g. disrupted homeostasis by extrinsic events) cannot alter genotype

The medical model of disease is both universal and simple (WHO, 1980). Manifestations of disease (impairment, disability or handicap, either permanent or temporary) reflect a process (pathogenesis) which has a cause. Medicine is largely concerned with the biological and social manifestations of disease and with process.

The biological consequences (manifestations) of disease can be categorized. They affect longevity, reproductive capability, development (somatic, intellectual and behavioural), and viability, and it is even possible to measure systematically the consequences of a genetic disease in these simple terms (Costa, Scriver and Childs, 1985). For example, Tay-Sachs disease, which impairs postnatal development and kills the individual before reproductive life has begun, is clearly catastrophic; on the other hand, a disorder with primary cosmetic significance and little biological cost such as the Treacher Collins-Franceschetti’s syndrome, can influence social interaction and exert costs in access to schooling and work. Accordingly, behavioural costs which may put an individual on the social ‘side-line’ are a consequence of disease that will concern physicians. Whilst the social consequences of genetic and metabolic diseases are very important (Raine, 1977), they are not the principal focus of this volume, which concentrates on the biological manifestations of some genetic diseases, and offers some explanations for them.

Manifestations are the stuff of medical practice – and curricula. But the processes behind their appearance are of equal importance since they will determine treatment. Mechanisms of disease involve the processes of disadaptation which are often not as well understood as the manifestations. For example, we apparently know almost all there is to know about the manifestations of phenylketonuria; we still do not understand the mechanism(s) of its major manifestation – the mental retardation. The cause of disease must be our ultimate concern. To know the cause enhances prediction and treatment and may even enable prevention.

To appreciate the origins of dishomeostasis in a disease it is necessary to consider the process of displacement. Homeostasis has metrical parameters (Figure 1.2); their statistics are the central tendency (mean), the dispersion (standard deviation), and the character of dispersion (skewness and kurtosis). Deviation of a value beyond the ‘normal’ boundaries of a biological parameter can reflect only two events (mutation or experience); one or other acts predominantly or, as is most often the case, they act together. Displacement therefore reflects either an extrinsic experience overwhelming the process or an intrinsic event subverting it, or a combination of the two.

Figure 1.2 Frequency distributions of a metrical phenotype. Left: the quantitative parameter (abscissa) has both central tendency and dispersion; they are described by conventional statistics: mean value (central tendency); and standard deviation, skewness, and kurtosis (dispersions). If genotype ‘proposes’ the phenotype, and gene products which have been selected as adaptive for homeostasis are centripetal in their effects on the dispersion of values, then experiences (various environmental forces) can be said to ‘dispose’ the normal phenotype with centrifugal effects. Right (top): dispersion of phenotypic values can be altered by a deviant experience (Ev) which overwhelms homeostasis. (Bottom): dispersion altered by mutation (Gv) which modifies the gene products maintaining homeostasis; the modified phenotype may then be skewed (quasi-continuous variation) or bimodal (discontinuous variation). Inborn errors, in their fully expressed forms, tend to confer discontinuous variation

Health of modern man living in a developed society has resulted largely from ascendency over causes of the first type. When mortality and morbidity were most often the manifestations of extrinsic causes it was sufficient to control these causes; as a result collective health and longevity improved. This is the story of public health, the improvement in environment, the discovery of vaccines and immunization procedures, and the awareness of vitamins and other nutritional requirements. Even so, disease and premature death have not disappeared. If a ‘specific disease’ continues to occur, albeit at a lower frequency (and incidence) in the population, we must ask: ‘Why is this so’? The eternal question in medicine – ‘Why does this person have this disease at this time’? – is still and always will be pertinent (King, 1982). It must follow that if the disease persists, yet its causes due to environmental events are less, then causes of intrinsic origin must now be relatively more important. That is to say heritability of the phenotype (or disease, or dishomeostasis) has increased. That is why the content of paediatric disease in modern referral hospitals has such a high genetic component (Hall et al., 1978) and why we must be interested in the genetic causes of disease.

DNA is the ultimate constant in living systems. Of course, the message in the nucleotide code is not constant throughout all living species, and that fact explains differences among species and individuals. Mutations at specific loci are sudden, one-time-only events in lineages; but once they occur, they will persist if they are not genetic lethals. The mutations will descend in their lineages, and they will spread in the population. The ubiquity of mutations, their allelic heterogeneity, and the phenomenon of genetic polymorphism are now widely appreciated (Vogel and Motulsky, 1979). Of major concern, in this book, is the phenotypic significance of the genetic diversity which is characteristic of man.

ILLUSTRATIONS OF THEMES

This book reflects the organizational intentions of the editors; the authors provide the substance to fulfil those intentions. To know an address is one thing; to get there and back is another. Pembrey describes the journey in Chapter 2. If the central dogma or sequence paradigm (DNA→mRNA→protein) is the basis of biology, then methods that reveal the links between DNA and phenotype are of great interest to physicians. The recent development of molecular genetics is the technical domain which permits us to move ‘backward’ from disease phenotype to discover and read the gene; it is also the technique that will help us to move ‘forward’ from mutation in DNA to discover phenotypes (proteins) presently unidentified but known to be associated with disease. For example, the aberrant cellular mechanisms of Duchenne muscular dystrophy, Huntington’s disease, and cystic fibrosis may soon be discovered by identifying the relevant DNA sequences and interpreting their products. Molecular genetics can now predict causes of certain diseases and, with suitable options, their manifestations can be prevented.

Next, Sparkes describes an important development in modern human genetics: the assignment of specific genes to particular places (loci) on chromosomes. Vesalius gave us modern human anatomy and every physician has had to learn it; the emerging map of human gene loci constitutes a ‘neoVesalian anatomy’ of importance equal to its namesake of earlier times. There is even now an emerging genetic morbid anatomy (McKusick, 1983), specifying the chromosomal loci, and DNA sequences, at which particular diseases find their causes in mutation. These developments represent a new form in medicine whose signs may be daunting to the uninitiated. The purpose of Sparkes’ chapter is to familiarize the signs. Their relevance to manifestations of disease needs no further defence.

Genes specify polypeptide products; the latter are the true primary phenotype. Form determines the function of polypeptides. Deviation from the normal form initiates the process of disease. Two chapters in the book deal with new ideas about form; their authors are as responsible as anyone for a refined understanding about the process of disease. In Chapter 4, Byers and Bonadio nominally discuss osteogenesis imperfecta – a ‘classical’ disease with a long and interesting history – but the actual focus of their chapter is a major protein – collagen, its form, and its synthesis. They make it clear that the aberrant collagen of osteogenesis imperfecta reflects mutation at specific gene loci now mapped to chromosomes 7 and 17. Tremendous genetic heterogeneity underlies osteogenesis imperfecta and it is fairly recent knowledge that this Mendelian phenotype is in fact a group of diseases; molecular genetics in the past 3–4 years has revealed that there are a multitude of variant osteogenesis imperfecta phenotypes. Osteogenesis imperfecta will undoubtedly be a model of disease as fascinating as the thalassaemias. The new knowledge about collagen has practical as well as intrinsic value. Genetic counselling, at present based on a state of ‘informed uncertainty’, will improve when it is feasible to read the gene(s) and predict the associated phenotype.

An important new dimension in knowledge about cellular forms came with the discovery of pre (or pro) polypeptides. Phenotype is specified by one gene, and modified by the product of another to yield the next or final form of the first. Cytosolic processing and transport of mitochondrial ornithine transcarbamylase is a familiar example; Sly and Sundaram discuss others in Chapter 5. The particular mechanisms for routing proteins through the endoplasmic reticulum to the Golgi apparatus and thence to the lysosome were discovered only recently. I-cell disease, an inborn error of lysosomal function, focused interest on the possibility that aberrant processing could explain this and perhaps other lysosomal diseases. From the studies described here, we can now characterize our understanding of form at three levels. First, the existence of cellular pathways for secretion (e.g. of collagen); and endocytosis (e.g. of LDL cholesterol). Second, informational organization of protein sorting (e.g. leader sequences in collagen and mannose-6-phosphate on oligosaccharide chains of lysosomal proteins). Third, the chemical machinery for labelling and translocation of proteins (e.g. phosphorylases and clathrin coats). As in the case of osteogenesis imperfecta, we see that Mendelian phenotypes pointed the way to genes and their products which, in the area described by Sly and Sundaram, involve critical aspects of protein traffic in human cells.

Four separate chapters by Robinson, Brusilow, Smith, and Thompson take up a traditional theme: the inborn error of metabolism. Garrod (1909) coined the term and by it we believe he intended to formalize his much more important theme of human biochemical individuality (Garrod, 1902). There is danger in the succès d’estime of the inborn errors. First, their rarity as specific entities lured physicians away from perceiving their importance as markers of loci involved in universal homeostatic networks. Second, because most patients with so-called inborn errors are homozygotes (or genetic compounds) and their manifestations are usually dramatic, we often fail to ask whether the heterozygotes might not also suffer some consequence of genotype after some delay during their lifetime. Third, we tend to forget that heterozygotes are much more prevalent in the population relative to affected homozygotes. Accordingly, there are practical implications in the four ‘inborn error’ chapters besides the obvious ones about classification, diagnosis, and treatment of diseases.

There are other messages here, often subtle, always interesting. For instance, D-glucose was selected as the principal fuel for eukaryotic evolution, apparently because of its chemical properties (Bunn and Higgins, 1981); it spends vastly less time in the alkyl form relative to the ring structure and therefore is unlikely, under normal homeostasis, to glycosylate proteins with disadaptive consequences for the organism. Evolution of eukaryotes took place in an environment rich in phosphorus and oxygen. Therefore, it is not surprising that energy metabolism is grounded in glucose oxidation and oxidative phosphorylation. It follows that any perturbation of these highly conserved processes will have a profound impact on cellular homeostasis. Lactic acidosis is a marker of several forms of such perturbation. The occurrence of genetic forms of lactic acidosis should come as no surprise to modern paediatricians; nor should the genetic heterogeneity of the phenotype surprise us, considering the complexity of the pathways.

Metabolic interlocks are part of homeostasis. Pyruvate, lactate, and ammonia metabolism are interlocked through argininosuccinate synthesis. A portion of the aspartate used for the synthesis of argininosuccinate (ASA) derives from pyruvate carboxylation. The most severe forms of pyruvate carboxylase deficiency impair aspartate homeostasis sufficiently to block ASA synthesis and cause hyperammonaemia. An analogous relationship has been seen in the neonatal form of argininosuccinicaciduria. Occasional patients have acidosis and lactic acidaemia (S. Krawawych, personal communication, 1984), this abnormality is corrected by citrate infusion. Depletion of a tricarboxylic acid intermediate, namely fumarate, because it is locked in ASA, is a putative mechanism to explain additional metabolic dishomeostasis in this disease.

The human life form is inconceivable without a mechanism to excrete ammonium ion, a by-product of many reactions and an obligatory product of our diet. Inborn errors of the urea cycle are yet further examples of our inability to tolerate dishomeostasis in critical metabolic events. Brusilow spells out this message in clear terms. Two further messages come across in his chapter: first we are not yet very skilled in relaxing the consequences of mutations that impair urea synthesis, even though we know so much about them; second, some heterozygotes (for example, females with X-linked ornithine transcarbamylase deficiency), although free of major symptoms in the normal environment, may suffer subtle impairments in their intelligence. These facts imply that we have a way to go in our search for better ways to prevent these consequences, which relate equally to many other ‘inborn errors’.

Phenylketonuria is one of the ‘good news’ stories in medical genetics. Smith makes that clear. Yet it is also evident that not all forms of phenylalanine dishomeostasis are likely to be well-treated. The exquisite homeostasis of tetrahydrobiopterin-dependent hydroxylations is so demanding that it may never be possible to correct completely the consequences of its imbalance in Mendelian phenotypes with impaired tetrahydrobiopterin synthesis or regeneration. Moreoever, prenatal treatment is probably necessary in these disorders if normal brain development in the third trimester is to be attained. On the other hand, there is clear evidence that an important cause of the mental retardation associated with disorders of phenylalanine hydroxylation has been largely prevented in those societies practising newborn screening and early treatment of typical phenylketonuria. Here then is a useful prototype for genetic screening with the goal of predicting risk and offering early medical intervention.

Paediatricians are arbiters of some antecedents of adult-onset disease, in particular when genotype is a significant determinant of phenotype. Thompson introduces this theme in his chapter on the hyperlipidaemias. Although paediatricians may never, in a lifetime of practice, see a child with homozygous, compound, or polygenic hyperlipidaemia, they will frequently encounter families in which members with genetically determined hyperlipidaemia occur and where a young parent of the patient may already have manifestations of lipid dishomeostasis. The fascinating new knowledge about low-density-lipoprotein receptor-deficient hypercholesterolaemia indicates that it may be possible, with new modes of treatment, to restore metabolic homeostasis and prevent disease in some forms of hyperlipidaemias. Accordingly, a carefully tuned ear to the family history may yield the clue upon which paediatricians can act to prevent premature disease in other members of the family.

The ‘inborn errors’ which perturb metabolic homeostasis at any point in time can have deviant morphogenesis and development as an associated consequence. The final chapters in this book illustrate this concept.

Stacey and Levy describe, in separate chapters, what might be called metabolic ‘teratogenesis’. They discuss the effects on fetal development of metabolic dishomeostasis caused by variant maternal genotypes. Mammals commit maternal metabolic reserves to intrauterine development and, in the human, a critical phase of our big-brained development occurs in utero (Martin, 1981). Some have suggested that interaction between certain heterozygous maternal genotypes and homozygous mutant fetal genotypes will produce fetal metabolic dishomeostasis but studies of these relationships as they affect phenylalanine and tyrosine metabolism, for example, reveal no apparent risk to the fetus (Scriver et al., 1980). Nonetheless, tight maternal metabolic homeostasis in utero is still essential. Modern studies of maternal diabetes (Freinkel, 1980) show that improvements in maternal blood glucose homeostasis during conception, embryogenesis and fetal development are accompanied by a reduced frequency of congenital malformation and improved fetal viability. Stacey expands these ideas to consider how variant genotypes expressed in the pregnant woman, the fetus itself, or the placenta, might modify fetal development.

As management of genetic disease in the present generation proceeds we have reason to ask: ‘What will happen in the next generation after treatment of genetic disease if the first yields well-adapted fertile females?’ The answer is glimpsed in maternal phenylketonuria; unless there are societal and medical procedures to deal with the problem (Cartier et al., 1982), the deviant metabolic process in the mother has the potential for harm to the fetus. Here, indeed, is a challenge for the paediatrician, whose responsibilities now extend into family medicine and to the obstetrical circumstances of his grown-up female patient.

In the last chapter of the book, Pinsky and Kaufman describe the ‘androgen pathway’, its known Mendelian variants, and their effects on sexual differentiation. Something as important as sexual dimorphism could not be expected to be a simple process, and it is indeed invested with many gene loci and complex interactions between their products. The studies described here are exquisite models to aid our understanding of the heirarchical layered process of development and the essential homeostasis required to maintain the heirarchy. Furthermore, we know now, for example, that androgen responsiveness of pulmonary mesenchymal cells determines alveolar development and this may be a factor in the pathogenesis of hyaline membrane disease.

To return to the major question: ‘Why does this patient have this disease at this time?’ the chapters in this volume, in different ways, address cause, process, and manifestations of disease; they explain the why of disease. The theme common to all chapters is genetic cause. When cause is genetic, the physician can predict and sometimes prevent recurrence in families and, in some cases, for example phenylketonuria, occurrence in populations. It is a new style of medicine; some call it ‘genetic medicine’ (Scriver et al., 1978), or an ‘evolutionary view of disease in man’ (Scriver, 1984). Access to tools of molecular genetics anticipates a revolution in diagnosis of cause and prevention of disease; we stand on its threshold.

There will follow many new challenges, among them moral and social issues that must be faced. Understanding the genetic cause and metabolic consequences cannot always be expected to lead to effective treatment. Within the examples selected for this book we can see that there is no or little likelihood of reversing the processes in osteogenesis imperfecta or the urea cycle disorders. Paediatricians in their enthusiasm for genetic medicine, have to accept that much of treatment will always be inadequate, that there will never be a treatment for some disorders, and that prenatal diagnosis is likely to be the best we can offer. This is an option for life, and individuals and society have to make the choices. Our role is to advise them of the options and to indicate routes of access to them, even if we ourselves are unable to enact the options. In doing that we answer Kant’s questions: ‘What can I know? On the basis of that knowledge, what ought I do?’

References

BERNARD, C. Les Phenomenes de la Vie; 1. Librairie J-B Baillière et Fils, Paris, 1878:879.

BUNN, H. F., HIGGINS, P. J. Reaction of monosaccharides with proteins: possible evolutionary significance. Science. 1981; 213:222–224.

CANNON, W. J.The Wisdom of the Body. New York: W. W. Morton, 1932.

CARTIER, L., CLOW, C. L., LIPPMAN-HAND, A., MORISETTE, J., SCRIVER, C. R. Prevention of mental retardation in offspring of hyperphenylalaninemic mothers. American Journal of Public Health. 1982; 72:1386–1390.

COSTA, T. M., SCRIVER, C. R., CHILDS, B. The effect of Mendelian disease on human health. A measurement. American Journal of Medical Genetics. 1985. [(in press)].

CRICK, F. H.C. Central dogma of molecular biology. Nature. 1970; 227:561–563.

FREINKEL, N. The Banting Lecture, 1980. Of pregnancy and progeny. Diabetes. 1980; 29:1023–1035.

GARROD, A. E. The incidence of alkaptonuria. A study in chemical individuality. Lancet. 1902; 2:1616–1620.

GARROD, A. E.Inborn errors of metabolism: The Croonian Lectures delivered before the Royal College of Physicians of London in June 1908. London: Frowde, Hodder and Stoughton, 1909.

HALL, J. G., POWERS, E. K., MCILVAINE, R. T., EAN, V. H. The frequency and financial burden of genetic disease in a pediatric hospital. American Journal of Medical Genetics. 1978; 1:417–436.

KING, L. S. Medical Thinking. A Historical Preface. Princeton: Princeton University Press, 1982; 187–203.

MARTIN, R. D. Relative brain size and basal metabolic rate in terrestrial vertebrates. Nature. 1981; 293:57–60.

MCKUSICK, V. A. Mendelian Inheritance in Man, 6th edn. Baltimore: Johns Hopkins Press, 1983. [Catalogs of autosomal dominant, autosomal recessive and X-linked phenotypes].

RAINE, D. N.Medico-social Management of Inherited Metabolic Disease. Lancaster: MTP Press, 1977.

SCHRÖDINGER, E.What is Life? Cambridge: Cambridge University Press, 1944.

SCRIVER, C. R. An evolutionary view of disease in man. Proceedings of the Royal Society of London. 1984; 220:273–298.

SCRIVER, C. R., COLE, D. E.C., HOUGHTON, S. A., LEVY, H. L., GRENIER, A., LABERGE, C. Cord-blood tyrosine levels in the full-term phenylketonuric fetus and the ‘justification hypothesis’. Proceedings of the National Academy of Sciences USA. 1980; 77:6175–6178.

SCRIVER, C. R., LABERGE, C., CLOW, C. L., FRASER, F. C. Genetics and medicine: an evolving relationship. Science. 1978; 200:946–952.

SHAPIRO, B. L. Down syndrome – a disruption of homeostasis. American Journal of Medical Genetics. 1983; 14:241–269.

SKINNER, B. F. Selection by consequences. Science. 1981; 213:501–504.

VOGEL, F., MOTULSKY, A. G.Human Genetics. Problems and Approaches. New York: Springer-Verlag, 1979.

WORLD HEALTH ORGANIZATION (1980) International Classification of Impairments, Disabilities and Handicaps. Geneva: WHO

2

Genes

Marcus Pembrey

Publisher Summary

This chapter discusses the new techniques for analysis of DNA that can be used to detect specific mutations, or to at least track the transmission of the chromosome region that carries the mutant gene within the family. Some areas of medical genetics have moved rapidly from being largely academic to having, in addition, great practical importance in the management of a family at risk of further affected children. One such area is genetic linkage. Another area is the matter of genetic heterogeneity within what appears to be a single disease. A crucial distinction in deciding on the strategy used for genetic prediction is whether the mutations affect the same gene locus. Thus, genetic heterogeneity is primarily divided into allelic heterogeneity where a single gene locus is involved and nonallelic heterogeneity where separate gene loci—often on different chromosomes—are involved.

Introduction

The structure and function of genes

The genetic code and translation

Coding sequences and the organization of genes

Regulation of gene transcription

RNA processing

Variation in the way genes determine the structure of functional proteins

Mutations

Amino acid substitutions

Point mutations that effect termination of translation

Defective splicing or processing of RNA

Gene deletions

Mutations effecting transcription

Genetic heterogeneity in inherited disease

Direct analysis of DNA in genetic prediction

Outline of the technical approach

Restriction fragment length polymorphism

Gene-specific and chromosome region specific DNA probes

Gene detection

Gene tracking

References

INTRODUCTION

It is now more than 100 years since Mendel’s death, and the word ‘gene’ has long been used by biologists, physicians and the informed public, yet direct analysis of human genes and the way they are arranged has only been possible in the last seven years or so. It is not surprising, therefore, that although most physicians have some idea of the different patterns of Mendelian inheritance, autosomal dominant, autosomal recessive and X-linked, they may have rather limited information on what genes actually are and the mechanism by which they dictate the structure of proteins.

Contemplation of how the activity of all the genes is orchestrated during embryological and fetal life to allow the development of a healthy newborn, let alone the continued short-term regulation of gene activity in metabolic homeostasis, is an awe-inspiring business. So awe inspiring, in fact, that it tends to inhibit the casual visitor to the land of molecular biology from even attempting to extract those principles that are relevant to his or her clinical practice. Some of the difficulties, of course, stem from our fragmentary knowledge of what actually goes on and, contrary to what one might imagine, it is an area where more detail can make it easier, not harder, to understand. There is, however, a simplification and that is that the study of genetic defects that show simple Mendelian inheritance, as most of the inborn errors of metabolism do, allows one to concentrate on a single gene locus. Admittedly the activity of other genes may modify the outcome, but for practical purposes the differences between those who have the disease and those who do not almost certainly resides in differences in the nucleotide sequence of the particular gene involved. Partial inherited disorders, such as many of the congenital malformations or diabetes mellitus, for example, present great difficulties because the genetic influence can vary from one case to another. Indeed the whole subject of multifactorial inheritance appears to be full of paradoxes if one tries to consider the genetic component separately from the environment in which the genes are operating.

This review will confine itself to the simpler task of single genes and the mutations in them that cause so-called monogenic disorders. This still means a variety of different mechanisms, for not only are different proteins encoded in the genome in a variety of ways but there are a number of different types of mutation that can occur. I will also confine myself to ‘constitutional’ mutations and not consider somatic mutations such as appear to underlie malignant transformation, for example.

Another purpose of this review is to outline the way in which the new techniques for analysis of DNA can be used to detect specific mutations, or if this is not practical to at least track the transmission of the chromosome region that carries the mutant gene within the family. Some areas of medical genetics have moved rapidly from being largely academic to having, in addition, great practical importance in the management of a family at risk of further affected children. One such area is genetic linkage discussed in the next chapter. Another is the matter of genetic heterogeneity within what appears to be a single disease, and here it will be argued that a crucial distinction in deciding on the strategy used for genetic prediction is whether the mutations affect the same gene locus. Thus genetic heterogeneity is primarily divided into allelic heterogeneity, where a single gene locus is involved, and non-allelic heterogeneity where separate gene loci, often on different chromosomes, are involved.

In discussing the different types of mutation that can occur I will draw heavily on the haemoglobinopathies, because much of Nature’s known repertoire can be illustrated within this single group of disorders affecting the adult haemoglobin molecule. Only time will tell whether the mutations that disrupt the genes encoding key enzymes in man’s metabolic pathways will turn out to be ‘more of the same’ or whether entirely novel disruptive mechanisms to the synthesis of normal proteins will be discovered. To date there seem to be two main consequences of gene mutations: the synthesis of an abnormal protein that may have an altered function, or the reduced or complete absence of synthesis of a particular protein. There are other possibilities, such as the primary excess synthesis of a gene product, the production of a normal protein at the wrong time during development or in an inappropriate tissue, but clinically significant examples of these are wanting at the present time.

THE STRUCTURE AND FUNCTION OF GENES

The genetic code and translation

A working definition of a gene will emerge during this section but, in essence, it is that part of the DNA double helix within the chromosome that codes for a single RNA molecule that in turn usually dictates the synthesis of a polypeptide at a ribosome in the cell cytoplasm. It will be recalled that DNA consists of two strands, each with a sugar–phosphate backbone and a linear array of any combination of the four nucleotide bases, adenine (A), guanine (G), cytosine (C) and thymine (T). The two DNA strands are held together by the complementary nature of the nucleotide base sequences; adenine only pairs with thymine, and guanine with cytosine (Watson and Crick, 1953). When transcription takes place one of the two DNA strands acts as a template for the formation of an RNA molecule that will have a complementary base sequence. The only difference is that RNA has uracil (U) instead of thymine. The genetic code consists of a series of codons or base triplets, and as there are four different bases there are 64 combinations of three. Every codon except three codes for one of the 20 amino acids: UAA, UAG and UGA code for the termination of translation of the messenger RNA (mRNA) into a polypeptide chain (Crick et al., 1961; Watson, 1965). AUG is the codon for methionine, but also has the role of initiating translation of mRNA (Darnbrough et al., 1973; Schreier and Staehelin, 1973).

Once in the cytoplasm the mRNA associates with one or more ribosomes which allow amino acids to be assembled into polypeptide chains in accordance with genetic code of that particular mRNA. Translation is achieved with the support of other intermediary molecules called transfer RNA (tRNA) (Rich and Kim, 1978). These molecules bind an amino acid at one end, while the other end is capable of recognizing the mRNA codon for that particular amino acid (Schimmel and Söll, 1979). Initiation of translation is a complex process in which the mRNA binds to a ribosome together with the initiation tRNA, the whole process being facilitated by the temporary involvement of proteins called initiation factors (Hunt, 1980). Defects in the translation process itself (as opposed to the faithful translation of wrong messages) have not as yet been shown to be a feature of monogenic disease. It has been known for a long time that even the mRNA molecule in the cytoplasm is considerably longer than the sequence from initiation codon to termination codon corresponding to the polypeptide chain and, as will be seen inFigure 2.1, the unprocessed product of transcription of a gene, the primary transcript, is longer still.

Figure 2.1 Schematic representation of the β-globin gene and the way it is transcribed into nuclear RNA which is then processed by addition of a CAP structure at the 5′ end and a polyadenyl tail at the 3′ end, and the removal of intervening sequences (IVS I and IVS II), before passing into the cytoplasm as messenger RNA. The position of codons 1 (corresponding to the first amino acid of the β chain), 30, 31, 104, 105 and 146, plus the initiation and termination codons, are