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Heredity and risk of disease
Environment International Vol. 1, pp. 371-377. Pergamon Press Ltd. 1978. Printed in Great Britain.
Heredity and Risk of Disease
Ranajit Chakraborty, William J. Schull, and Kenneth M. Weiss Center for Demographic and Population Genetics, University of Texas, Health Science Center. Houston Texas. U.S.A. Mathematical modeling of competing risks often uses a single risk function for all individuals within a population. In this paper we use several specific examples to argue that such representations remain approximalions until account is taken of the existence of genetic variability within and between populations, and the impact of this upon liability to disease. The extent of h u m a n genetic variability is also indicated.
Contemporary models of aging or of the risk of specific disabilities such as cancer or coronary artery disease generally assume that the universe of human beings of interest is divisible into one or at most a few populations within which a single risk function applies. Thus, for example, Truett's (67) use of a multiple logistic function to assess the risk of coronary artery disease posits two multivariate frequency distributions, each of which can be represented by a known mathematical function, corresponding to those individuals who will and those who will not develop coronary artery disease. Representations of this nature can and undoubtedly have provided important insights into, the risk of disease or a specific cause of death, and may be a necessary step in the evolution of more biologically meaningful models. We contend, however, that they must remain approximations until such time as account is taken of the existence of genetic variability within and between populations, and the impact of this upon liability to disease. Inherited diseases have been described as the most widespread of all human m a l a d i e s - t h e oldest, and possibly the most burdensome of human ailments (DHEW, 74). Perceptions of what constitutes genetic disease vary ; but these need not trouble us here. Patently, genetic factors can and do make important contributions. directly and indirectly, to a variety of common medical problems such as diabetes and cardiovascular or cerebrovascular disease: and whether the latter are called environmental or genetic diseases is immaterial. Moreover, individuals can and do vary in their responsiveness to drugs, infectious agents, and even diet. We may presume, therefore, that they will differ in their responses to the ever increasing and bewildering array of potential carcinogens and mutagens associated with environmental pollution and changing lifestyles. These differences, many of which are genetic in origin, can
importantly influence the occurrence as well as the course of disease. Possibly some illustrations of genetically determined variability in response to the genotype of another, to drugs, or to elements of our diet will serve to support our contentions (see Table 1).
Genetic variability: interaction with disease I. Hemolytic disease oJ the newborn Almost 40 years ago, Levine and Stetson (39) described a woman whose second pregnancy terminated in a stillbirth and who reacted severely to transfusion with her husband's blood. It was found that she had an antibody in her serum that agglutinated her husband's blood cells and, in fact, agglutinated such cells from about 85°~, of a random sample of U.S. Caucasians. Levine and Stetson conjectured that the hemolytic disease seen in the infant arose as a result of(l) the transplacental isoimmunization of the mother by the fetal red cells that contained an antigen derived from the father and (2) the subsequent passage into the fetal circulation of the immune antibodies that were formed in response to this antigen (see Pickles, 49, for a fuller description). Later studies of their own and others have supported this viewpoint. Hemolytic disease of the newborn, which we now recognize can occur as a result of maternal fetal incompatibility with respect to a number of red-blood-cell antigenic systems of which the most frequent cause is the Rh system, depends upon an interaction between a mother and her fetus, The incidence of hemolytic disease of the newborn due to Rh incompatibility, for example, relates directly to the mating type of the parents with respect to the Rh-locus, and different ethnic groups of the world are at varying degrees of risk depending on the population frequency of the Rh-d gene (see Mourant, 76). 371
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Ranajit Chakraborty, William J. Schull and Kenneth M. Weiss Table 1. Some genetically determined responses to food, drugs, and another's genotype Disorder
"'Causal Agent"
Nature of variation
Hemolytic disease in newborns
Matcrnal genotype
Depending on ethnic variation of d-gene frequency and possible effect of birth order
Sickle cell anemia
Person's own genotype
Ethnic variation of Hb-S gene frequency: association with malaria, case of disease-vector
Lactose intolerance
Food stuff
Ethnic variation in intolerance; identified when exposed to food with lactose added
Thyroid metabolism defects
Iodine dcliciency and overdose
Geographic variation, overdose effect
Isoniazid inactivation
Drug for TB
Genetic variation in the a m o u n t of acetyltransferase in liver cells; racial differences in gene frequency
Suxamethonium sensitivity
Anesthetic
Though rare, genetic variation causes concern in anesthetic uses of the drug
But risk depends upon other factors as well. There exists, or appears to exist, a relationship with birth order (Ceppellini, 52, see also Cavalli-Sforza, 71) and isoimmunization at the Rh locus is known not to be independent of the ABO genotypes of the mother and fetus (for details, see Mollison, 67). Risk is, thus, a function of one's mother's genotype as well as one's own, and events at two different, unlinked loci. (Rh hemolytic disease has been a preventable disorder in the United States since 1968 when Rh immune globulin was licensed for use. This globulin, if administered to Rh negative women with Rh positive fetuses within 72 h of pregnancy termination, will suppress the maternal anti-Rh antibody response.~ 2. Sickh, cell am'mia
A single amino acid substitution in hernoglobin can in the homozygous state result in clinical symptoms of arthralgia, acute attacks of abdominal pain, ulcerations of the lower extremities, and an often fatal anemia. The phenomenon, called sickling, is caused by the formation of large crystal aggregates under conditions of low oxygen tension. The molecular nature of the abnormality, a single amino acid substitution in the Hb-beta chain (glutamic acid for valine at the sixth position), though often lethal in homozygous condition, is maintained in high frequencies in some populations by the presumed advantage of heterozygotes in malaria-infected regions of the world. Resistance to malaria, however, appears substantially more complicated than originally supposed and dependent not only upon the variation in hemoglobin associated with the sickling phenomenon, but also upon variation at the thalassemia loci, the glucose-6-phosphate dehydrogenase locus, and quite possibly the Duffy locus as well. The seriousness of malaria as a health problem can vary too with genetic changes in the mosquito that render it more immune to insecticides and changes in the plasmodium that compromise chemotherapy. Both events have occurred, and malaria is again becoming a
global health problem. Be this as it may, the genotype that may be favored in a malarious environment may be disadvantageous when malaria is not present. We know that individuals heterozygous for the hemoglobin-S gene, who normally appear in good health can under certain circumstances, such as those that attend the hypoxia of altitude, become ill. 3. Lactose intolerance (LIT)
Infant mammals survive on milk, of which disaccharide lactose is a major nutritional constituent. Normally, ingested lactose is cleaved by the enzyme lactase into glucose and galactose, and the latter then metabolized. Occasionally, infants are born who are unable to hydrolyze lactose; they are said to have congenital lactose malabsorption or intolerance, fail to thrive, and generally exhibit a chronic diarrhea which can be life threatening. Much more c o m m o n is a lactose intolerance that develops later. In these cases milk tolerance is usually normal in infancy, but intolerance develops in adolescence or adulthood (Huang, 68; Johnson, 74). The signs and symptoms of the intolerance suggest a direct toxic effect on the intestine--diarrhea, borborygmi, flatulence, and other signs of gastrointestinal distress are common. This form of lactose intolerance, which appears to be recessively inherited in American Negroes and whites (Bayless, 67; Welsh, 68), varies substantially from race to race, and population to population. It is especially common in those peoples whose traditional dietary practices have not included large amounts of milk or milk products in childhood and later years, such as the Amerindians, Eskimos, and many groups of blacks (see Simoons, 73). This has led both individuals and official agencies to be concerned about a suitable policy for using milk and dairy products as dietary supplements in regions where nutrition may be marginal but lactose intolerance common.
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4. Thyroid metabolism The synthesis, storage, secretion, and utilization of the thyroid hormones involve a complex sequence of metabolic events, each of which is probably dependent upon specific enzymatic activity (see Stanbury, 72, for a full explication). Thyroid disease may result from blockage at many steps in this metabolic process. Goiter can and does arise in the presence of ample nutritional iodine. Familial goiter and hypothyroidism may occur when there is failure of the thyroid cell to transport iodide and maintain a favorable concentration gradient inside the follicle lumen, but they may also result from failure to convert inorganic iodide into iodine in the thyroid gland. Other types of familial goiter occur; these involve such disparate bases as diminished or altered thyroglobulin synthesis, impaired response to thyrotropin, and the like. It is reasonable to presume that the sequence of metabolic events listed above may be altered quantitatively without concomitant qualitative changes; that is, through the origin of enzymes that do not block a specific step but diminish or enhance the reaction in question. Even in areas of endemic goiter there occur goiter-free individuals whose nutrition and lifestyles seem no different from those of their goitrous contemporaries. Thus, in the Pedregoso Valley of Chile where 700/0of individuals 10 years of age or older are goitrous, three individuals in ten of similar ages fail to manifest signs of impaired thyroid metabolism (Barzelatto, 68). Goiter in this valley is apparently influenced by two factors, namely, (1) the existence of iodine deficiency and, no less important, (2) the consumption of pinon, the fruit of the araucarian pine. The latter has been shown to sequester iodine and to be goitrogenic in rats. Thus, risk is a function not only of one's genotype, and the availability of iodine, but also of other dietary constituents as well.
toxic, so that the drug is effectively inactivated by acetylation. Shortly after isoniazid began to be used in tuberculosis therapy, it became apparent that the rate at which isoniazid is inactivated by acetylation differs between individuals, though in any one individual the metabolism of the drug remains remarkably constant (Hughes, 54; Bell, 57). It was then shown that people can be divided into two or more or less sharply distinct groups--so-called rapid and slow inactivators--according to the rate at which the inactivation proceeds. The difference in rate has been shown to be genetic in origin; slow inactivators are homozygous for one allele, whereas rapid inactivators are either heterozygous or homozygous for the other allele (Knight, 59; Evans, 60). This difference in isoniazid inactivation is related to a difference in the amount of an enzyme called "acetyltransferase" present in the cells. The existence of these marked individual differences in isoniazid inactivation raises the question of this drug's therapeutic significance for treating tuberculosis. In comparisons of large groups of patients on standardized anti-TB treatment including isoniazid, significant differences have not usually been found between the results of treatments in "rapid" and "slow" inactivators (Evans, 63). But while there may be little or no difference when the drug dosage is optimal, it is likely that if it is suboptimal, for example when the isoniazid is given too infrequently, then differences in response may occur (TB Chemotherapy Center, 70; Ellard, 72). Slow inactivators of isoniazid appear to be somewhat more likely than rapid inactivators to develop a peripheral neuropathy ascribed to the toxic side effects of the drug. This peripheral neuritis, however, is not of much concern now since it can be prevented by the simultaneous administration of pyridoxine. Table 2 gives some idea of the extent of racial variation with respect to the frequencies of the isoniazid inactivation alleles.
5. lsoniazid inactivation 6. Suxamethonium sensitivity Genetically determined differences in the activity of a particular enzyme can result in quite marked differences in the manner in which different individuals metabolize a particular drug, although this may not be associated with any acute clinical consequences. This can be illustrated by the differences that have been found to occur in acetylation of the drug isoniazid, which is widely used in the treatment of tuberculosis. In its acetylated form, isoniazid is much less active therapeutically and is also less
Suxamethonium (succinyl dicholine)was introduced as a muscle relaxant in surgery and electroconvulsion therapy some years ago on the supposition that because of its rapid hydrolysis into active products by serum cholinesterase, its effect should be quite short duration. Occasionally, however [e.g., in about one in every 2000 Europeans (Harris, 75)], a normal dose of the drug causes an extremely prolonged muscular paralysis and apnea
Table 2. Frequency of alleles determining "'rapid" and "'slow" inactivation of isoniazid in different populations (adapted from Harris, 75) Allele Frequency Population U.S. white U.S. black Japanese Ainu Korean Thai Burmese South Indian Alaskan Eskimo Alaskan Indian
Number Tested 305 207 2017 86 65 108 121 850 157 47
"Rapid 0.27 0.27 0.66 0.69 0.67 0.46 0.39 0.19 0.54 0.38
.
.
.
.
Slow'" 0.73 0.73 0.34 0.31 0.33 0.54 0.61 0.81 0.46 0.62
374 lasting for hours instead of the usual period of a few minutes. This rare abnormality is presently ascribed to low levels of serum cholinesterase in these aberrant individuals (Bourne, 52; Evans, 52). This phenomenon is, in fact, genetic; suxamethonium-sensitive individuals with the low levels of serum cholinesterase are homozygous for an abnormal gene which in heterozygotes results in a moderate reduction in enzyme level. Therefore, in populations where the abnormal gene is high in its frequency, administration of this drug would cause frequent problems. Variation in serum cholinesterase has broader implications than the one just cited. It has long been known that potatoes contain a substance, solanine, found principally in the peel and sprouts but present throughout the potato, which can be toxic to humans as well as other animals. Among the symptoms of such poisoning are disturbances in respiration and cardiac activity and significant hemolysis, as well as other evidence of stimulation of the cholinergic system, that is, stimulation of those nerve fibers that cause similar effects to those induced by acetylcholine. The mechanism through which these symptoms come about is seemingly the inhibition of serum and brain cholinesterase. At least two different inherited forms of serum cholinesterase are known; the three phenotypes that arise are differentially inhibited by extracts from the potato (Harris, 62). Numerous Andean populations have consumed potatoes for millenia, literally, and their methods of preparation are known in some instances not to degrade solaine. The implications of this are not clear at present, but obviously warrant exploration. The examples we have chosen are but a 3mall number of many. They have been selected to illustrate tl~e numerous ways in which interactions, gene with environment or gene with gene, can arise. Indeed, so ubiquitous are such interactions that the next level of ~.omplexity to which we turn briefly may seem unnecessary for support of our concerns.
Genetic variability and malignancy Down's syndrome of trisomy-21 has its well-known physical and mental symptoms, but individuals ivith this syndrome are also unusually sensitive to bacterial and viral infections. Because of this susceptibility to infection, few cases survived to adulthood before the advent of antibiotics. Individuals with this syndrome are also prone to leukemia. We recognize individuals who are mosaic with respect to this cytologic abnormality, that is, some of their somatic cells exhibit a supernumerary chromosome 21, but others do not. We do not know, however, whether their risks of infection and leukemia are proportional to the size of the normal cell population. If this is true. then estimation of risk becomes more subtle still. Another line of evidence that warrants note here has recently been provided by Swift (77) in his studies of the risk of malignancy in the parents of individuals with certain rare recessive phenotypes. He conjectures that individuals heterozygous for any one of the genes that in homozygous form give rise to xeroderma pigmentosum, Bloom's syndrome, Fanconi's anemia, or ataxiatelangiectasia have risks of dying from cancer 2 to 12 times higher than those of noncarriers of these gcnes.
Ranajit Chakraborty, William J. Schull and Kenneth M. Weiss In the beginning, the relationship between aryl hydrocarbon hydroxylase inducibility and bronchogenic carcinoma (Kellerman, 73) also seemed to be promising. The initial studies suggested that the inducibility of this enzyme was simply inherited and that "low inducers" were at much smaller risk of bronchogenic cancers when exposed to benzo(c0 pyrene and other polycyclic hydrocarbons in cigarette smoke and polluted air than "high inducers". Subsequent studies have not confirmed this, but the postulated interaction between genetic control of enzyme inducibility and exposure to an environmental agent remains a provocative paradigm.
Extent of human genetic variability So far we have discussed specific cases only. We have seen that there exist genetically determined variability in the liability to certain insect-borne diseases (malaria), as well as in response to dietary constituents (lactose) and pharmacologic agents (isoniazid) that figure large in therapeutic or preventive regimens. But how widespread are these responses ? Is there a limit to genetic variation in man ? If the number of genes is of the order of 500,000 or so, is such a limit measurable? Until relatively recently one could only speculate. However, as our abilities to recognize and measure serological and biochemical variability have grown, speculation has given way to observations that, while not as yet as precise as is desirable, provide important insights. Briefly, the last two decades have seen Karl Landsteiner's (1900) original four red-blood-cell phenotypes grow to no less than 29,952 in 1955: and a decade later in 1965, the immunological distinctions that could be more or less routinely made resulted, in theory, in no less than 2,717,245,440 distinct phenotypes. If the secretor phenomenon (which results in the production of water-soluble forms of the ABH antigens) is included, this number doubles. These distinctions, we emphasize, do not include pathological characteristics of the red blood cell; nor, more importantly, the increasing number of inherited serum proteins or red cell enzymes; nor the histocompatibility antigens, possibly the most variable of all of the so-called genetic markers. The realized variability in these systems is. of course, somewhat less; the genes involved are not ubiquitous, nor in areas where they occur are they all equally frequent. It seems unlikely, however, that the realized variability is less than the "expected" by much more than two orders of magnitude; it is certainly great enough to support the commonplace remark that we have all been uniquely endowed by our creator. A more complete answer to the extent of our genetic variability has begun to emerge through the development of molecular biology. On the theoretical side, Kimura (64) have shown that the number of alleles at a locus that can be maintained in a finite population is fairly large, taking into account the fact that at the molecular level almost an infinite number of alleles may be produced at a locus. (Wright's 1948 paper indicates this possibility even though the structure of the basis of heredity, DNA, was not then known to Wright.) The development of starch gel electrophoresis (Smithies, 55) in combination with a simple staining technique for a specific enzyme activity (Hunter, 57) provided a valuable tool by which genetic heterogeneity in proteins and isozymes could easily be
Heredity and risk of disease
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detected. By 1965, it was already known that natural populations contain a large amount of polymorphism with respect to proteins and enzymes. In a review, Shaw (65) stated, "Enzymes which vary within populations are rather the rule than the exception". An important step in the study of genetic variation in populations was made by Lewontin (66) and Harris (66). The current theory that the primary structure and probably the rate of synthesis of different polypeptides are under genetic control suggests that the genetic variation in a natural population might be measured best by the enzymatic diversity present. Clearly, one cannot currently hope to study all of the enzymes produced by the genome to determine the proportion of genetic loci that show variation. It is possible, however, at least in theory, to study a representative sample of the enzymes of a species and thereby estimate the extent of its genetic variability. But an unbiased estimate will be obtained only if genetic variation is detectable at several loci and the loci are chosen randomly with respect to how variable they are. With the electrophoretic identification of isozymic variation it has become possible to sample genetic loci (by selecting enzymes) in a manner that at least approaches randomness. As might have been expected, even this approach to the sampling of genetic loci falls somewhat short of the ideal. First, only alterations in structural genes are efficiently detected. Detection of variation in the activity of enzymes is poor by electrophoresis and is biased in that we do not know how many regulator genes go undetected. Second, the proteins studied must be soluble, and third, a histochemical staining method for identification of the protein after electrophoresis must be available. These limitations notwithstanding, an increasing body of evidence exists to support the notion that a substantial fraction of genetic loci exhibit variation in p o l y m o r p h i c proportions. Harris (75) has reported findings on 20 arbitrarily chosen enzymes in Europeans and Negroes. Some 27 genetic loci are involved in the structural control of these enzymes. The average heterozygosity for these 20 enzymes, assuming 27 loci, he finds to be 0.054 for Europeans and 0.052 in Negroes. (Excursus: Average heterozygosity is measured in the following manner. Let Pi be the frequency of the ith allele at a locus. The expected
proportion of individuals heterozygous for this gene will be h = 1 - Ep~z, assuming that the population is large and panmictic and that no selection obtains at the locus. The "average heterozygosity" is merely the individual heterozygosities, the H's, summed over all loci under consideration, and divided by the number of loci.) Harris (75) estimates that the average heterozygosity per locus for alleles determining all structural enzyme and protein variants will be three times the values cited above. He argues that approximately one-third of the total variability will be recognizable electrophoretically. This factor, one-third, rests in part on the knowledge that almost two-thirds of all random amino acid substitutions in the hemoglobin molecule will produce no change in the net charge of the molecule and hence will be undetectable electrophoretically. Lewontin (67) suggested another, albeit somewhat similar, approach to the estimation of the average heterozygosity in man. His argument, which utilizes serological data, is based upon the supposition that as more and more bloods are tested, the "detected" heterozygosity will converge on the "true" heterozygosity; somewhat differently stated, data on blood groups will be less and less biased toward polymorphic loci as more and more bloods are studied. As of 1962, Lewontin noted that some 33 blood groups were known; of these, 36.4% were polymorphic and the average heterozygosity was 0.162. Of the next 17 blood groups to be recognized, only one, Dombrock, was polymorphic. If these data and the three blood groups that Lewontin failed to tabulate are considered, then of the 53 systems known in 1968, 13 were polymorphic. The average heterozygosity was correspondingly lower. Nei (74) have recently reexamined these data, but as they and others have noted, the loci associated with the blood groups may not be a random sample of the human genome. To this end, Harris (76) provided an up-to-date summary of our knowledge of enzyme protein variation in man, which is presented in Table 3. Average heterozygosity discussed so far gives some idea of the extent of genetic variability within populations. Recent work on genetic distances also gives estimates of the number of codon or mutational differences per locus between different population groups as obtained from gene frequency data. For example, analyzing data on 35
Table 3. Polymorphism and average heterozygosity as obtained from electrophoretically detectable enzyme variants in European and U.S. white populations (adapted from Harris~ 76) Polymorphic Nonpolymorphic loci (Average loci (Average heterozygosity heterozygosity > 0.02) < 0.02)
Number of loci screened
Average heterozygosity per locus
Oxidoreductases Transferases Hydrolases Lyases lsomerases Ligases
4 8 11 1
20 21 27 9 3
24 29 38 10 3
0.042 0.059 0.087 0.050
Total
24
80
104
0.063 (Weighted average)
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Ranajit Chakraborty, William J. Schull and Kenneth M. Weiss
common b i o c h e m i c a l loci, N e i a n d R o y c h o u d h u r y e s t i m a t e t h a t the net m u t a t i o n a l differerence b e t w e e n the t h r e e m a j o r racial g r o u p s is given by Caucasoid/Negroid Caucasoid/Mongoloid Negroid]Mongoloid
0.023 0.001 0.024
F r o m 21 c o m m o n b l o o d g r o u p loci these are 0.042, 0.034, a n d 0.118, respectively. T h e r e are o t h e r s i m i l a r e s t i m a t e s a v a i l a b l e for p o p u l a t i o n s w i t h i n the s a m e racial g r o u p s at the t r i b a l level [e.g., tribes o f different A m e r i n d i a n p o p u l a t i o n s (see B l a n c o , 75)] o r w i t h i n a single t r i b e at the village level (from d a t a of N e e l et al., see R o t h h a m m e r , 77). N e e d l e s s to say, t h e s e n u m b e r s are s m a l l e r t h a n the v a l u e s above. T h e n u m b e r s we h a v e j u s t cited m i g h t leave o n e w i t h the i m p r e s s i o n t h a t the l i m i t i n g v a r i a b i l i t y is n o t g r e a t e n o u g h to i n v a l i d a t e t h e a s s u m p t i o n of a p p r o x i m a t e u n i f o r m i t y in risk e s t i m a t e s for m a n k i n d as a w h o l e . T h i s is n o t true, h o w e v e r , for t h e v a l u e s j u s t given are averages per locus. T h e d i s t r i b u t i o n of the n u m b e r o f m u t a t i o n a l differences is h i g h l y s k e w e d w i t h a l o n g tail (Nei, 74; C h a k r a b o r t y , 77). So t h e r e are q u i t e a few g e n e t i c loci for w h i c h m u t a t i o n a l differences are m u c h l a r g e r t h a n these a v e r a g e values. S o m e o f t h e s e loci h a v e a s s o c i a t i o n w i t h disease s u s c e p t i b i l i t y as well. C l a r k e ' s (61) s t u d y of the a s s o c i a t i o n of the A B O g r o u p s w i t h d i s e a s e illustrates this aspect. In c o n c l u s i o n , o n e ' s risk of disease as well as o n e ' s s u s c e p t i b i l i t y to e n v i r o n m e n t a l ( m a n - m a d e o r n a t u r a l ) agents depends importantly upon one's genetic b a c k g r o u n d . T h u s , to a s s u m e , as is c o m m o n l y d o n e , t h a t a single f u n c t i o n d e s c r i b e s e q u a l l y well o u r i n d i v i d u a l risks of disease o r d e a t h is to i g n o r e o n e of the m o s t i m p o r t a n t of b i o l o g i c o b s e r v a t i o n s - - m a n ' s inherent g e n e t i c diversity.
A cknowledgement--This research was supported by grants H L- 15614, CA-19311 and GM-19513 from the National Institutes of Health.
References Barzelano J. and Covarrubias E. (1968) Study of endemic goiter in the American Indian, Biomedical Challenges Presented by the American Indian. PAHO Sci. Publ. No. 165, Washington, D.C., 124 132. Bayless T. M. and Rosensweig N. S. (1967) Incidence and implications of lactase deficiency and milk intolerance in white and negro populations, Johns Hopkins Med. J. 121, 54-64. Bell J. C. and Riemensnider D. K. (1957) Use of a serum microbiologic assay technique for estimating patterns of isoniazid metabolit~m, Ann. Reu. Tuberculosis 75, 995. Blanco R. and Chakraborty R. (1975) Genetic distance analysis of twenty-two South American Indian tribes, Hum. Hered. 25, 177 193. Bourne J. G., Collier H. O. J. and Somers G. E. (1952) Succinylcholine [Succinoylcholine): muscle relaxant of short action, Lancet I, 1225. Cavalli-Sforza L. L. and Bodmer W. F. (1971) The Genetics oJHuman Populations, Freeman, San Francisco. Ceppellini R. (1952) In: La Malattia Emolitica del Neonato (R. Ceppellini, S. Nasso, and F. Tecilazich Eds.), Milano. Chakraborty R. (1977) Simulation results with stepwise mutation model and their interpretations, J. Molec. Evol. 9, 313 322. Clarke C. A. (1961) Blood groups and disease, Progress in Medical Genetics (A. G. Steinberg, Ed.) Vol. 1, Grune and Stratton, New York. DHEW (1974)Facts about Genetic Disease, DHEW Publ. No. (NIH) 74-370. Ellard G. A., Abet V. R., Gammon P. T., Mitchison D. A., Lakshminarayan S., Citron K. M., Fox W. and Tall R. 11972) Pharmacology of some slow release preparations of isoniazid of potential use in intermittent treatment of tuberculosis. Lancet I, 340. Evans D. A. P. (1963) Pharmacogenetics, Am. J. Med. 34, 639-662.
Evans D. A. P., Manley K. E. and McKusick V. A. (1960) Genetic control of isoniazid metabolism in man, Br. reed. J. 2, 485~491. Evans F. T., Gray P. W. S., Lehmann H. and Silk E. (1952) Sensitivity to Succinyl-choline in relation to serum cholinesterase, Lancet 1, 1229. Harris H. (1966) Enzyme polymorphisms in man, Proc. R. Soc. Lond. B, 164, 298 310. Harris H. (1975) The Principles of Human Biochemical Genetics, NorthHolland/American Elsevier, Amsterdam. Harris H. and Hopkinson D. L. (1976) Handbook oJ Enzyme Electrophoresis in Human Genetics, North-Holland, Amsterdam. Harris H. and Whittaker M. (1962) Differential inhibition of the serum cholinesterase phenotypes by solanine and solanidine, Ann. Hun. Genet., Lond. 26, 73-76. Huang S. S. and Bayless T. M. (1968) Milk and lactose intolerance in healthy Orientals, Science 160, 83 84. Hughes H. P., Biehl J. P., Jones A. P. and Schmidt L. H. (1954) Metabolism of isoniazid in man as related to the occurrence of peripheral neuritis, Ann. Rev. Tuberculosis 70, 266. Hunter R. L. and Markert C. L. (1957) Histochemical demonstration of enzymes separated by zone electrophoresis in starch gels, Science 125, 1294 1295. Johnson J. D., Kretchmer N. and Simoons F. J. (1974) Lactose malabsorption: Its biology and history, Adv. Pediat. 21, 197 237. Kellerman G., Luyten-Kellerman M. and Shaw C. R. (1973) Genetic variation of aryl hydrocarbon hydroxylase in human lymphocytes, Am. J. hum. Genet. 25, 327-331. Kimura M. and Crow J. F. (1964) The number of alleles that can be maintained in a finite population, Genetics 49, 725 738. Knight R. A., Selin M. J. and Harris H. W. (1959) Genetic factors influencing isoniazid blood levels in humans, Trans. Conf. Chemotherap. Tuberc. (St. Louis) 18, 52 69. Landsteiner K. (1900) Zur kenntnis der antifermentativen, lytischen und agglutinierenden wirkungen des blutserums und der lymphe, Zentbl. Bakt. 27, 357 362. Levine P. and Stetson R. E. (1939) An unusual case of intragroup agglutination, J. Am. reed. Ass. 113, 126-127. Lewontin R. C. (1967) An estimate of average heterozygosity in man, Am. J. hum. Genet. 19, 681 685. Lewontin R. C. and Hubby J. L. (1966) A molecular approach to the study of genic heterozygosity in natural populations. II: Amount of variation and degree of heterozygosity in natural populations of Drosophila pseudoobscura, Genetics 54, 595-609. Mollison P. L. (1967) Blood Transfusion in Clinical Medicine, 4th Ed., Blackwell Scientific Publications, Cambridge, U.K. Mourant A. E., Kopec A. C. and Domaniewska-Sobezak K. (1976). The Distribut ion of the Human Blood Groups and Other Polymorphisms, 2nd Ed., Oxford University Press, London. Nei M. and Roychoudhury A. K. (1974). Genic variation within and between the three major races of man, caucasoids, negroids, and mongoloids, Am. J. hum. Genet. 26, 421~-43. Pickles M. (1949) Haemolytic Disease of the Newborn, 1st Ed., Blackwell Scientific Publications, Oxford. Rothhammer F., Chakraborty R. and Llop E. (1977) A collation of marker gene and dermatoglyphic diversity at various levels of population differentiation, Am. J. Phys. Anthrop. 46, 51-60. Shaw C. R. (1965) Electrophoretic variation in enzymes, Science 149, 936 943. Simoons F. J. (1973) New light on ethnic differences in adult lactose intolerance, Am. J. Dig. Dis. 18, 595 611. Smithies O. (1955) Zone electrophoresis in starch gels: group variations in the serum proteins of normal human adults, Biochem. J. 61,629-641. Stanbury J. B. (1972) Familial goiter, The Metabolic Basis oflnherited Disease (J. B. Stanbury, J. B. Wyngaarden and D. S. Frederickson, Eds.) pp. 223 265. McGraw-Hill, New York. Swift M. (1977) Malignant neoplasms in heterozygous carriers of genes for certain autosomal recession syndromes, Genetics oJHuman Cancers, (J. J. Mulvihill, R. W. Miller and J. F. Fraumeni, Jr., Eds.) pp. 209-216. Raven Press, New York. TB Chemotherapy Center (1970) A controlled comparison of a twiceweekly and three once-weekly regimens in the initial treatment of pulmonary tuberculosis, Bull. WHO, 43, 143. Truen J.. Cornfield, J. and Kannel W. (1967) A multivariate analysis of the risk of coronary heart disease in Framingham, J. chron. Dis. 20, 511--.524. Welsh J. D., Zschiesche O. M., Willits V. L. and Russell L. (1968) Studies of lactose intolerance in families, Archs Intern. Med. (Chicago) 122, 315 321. Wright S. (1948) Genetics of populations, Encyclopaedia Britannica (14thedn.)Vol. 10, pp. 111 112.
Heredity and risk of disease
Discussion Prentice: In Clarke's (61)* study of the association of the ABO groups with diseases, were the control groups matched an age? Chakraborty: As far as I know there is no indication about age matching on the control group. However, this may not be critical since the mean relative indices which were considered to show the association were in terms of relative frequencies of two ABO blood types. For example, for cancer of the stomach a relative mean incidence of A :O was found to be
*Clarke C. A. (1961) Blood groups and disease in Proyress in Medical Geneties, Vol. l (A. G. Steinberg, ed.) Grime and Stratton, New York. t Bodmer W. F. (1974) in Genetic Polymorphisms and Diseases in Man, B. Ramot, ed., 377-392. Academic Press, New York.
377 1.25. This means Ratio of A:O in Stomach Cancer Patients = 1.25. Ratio of A:O in Controls In this case, age matching would be of some consequence only if survival to a certain age is directly or indirectly related to ABO blood types. There is no such evidence to date. Prentice: What diseases have not been associated with specific HLA's? Chakraborty: Thewhole problem of HLA and disease association seems to be quite complex. As a projection of the mouse work on the connection between H-2 and immune response and viral susceptibility, there has been a detailed hunt for the same sort of association in man: namely, between the HLA system and diseases thought to be related to immune response differences. But there are a number of inherent problems, statistical as well as technical, which should be considered before taking such positive evidence of association seriously. Recently, Bodmert presented a detailed account of these sorts of pitfalls of association studies.
Heredity and Risk of Disease
Ranajit Chakraborty, William J. Schull, and Kenneth M. Weiss Center for Demographic and Population Genetics, University of Texas, Health Science Center. Houston Texas. U.S.A. Mathematical modeling of competing risks often uses a single risk function for all individuals within a population. In this paper we use several specific examples to argue that such representations remain approximalions until account is taken of the existence of genetic variability within and between populations, and the impact of this upon liability to disease. The extent of h u m a n genetic variability is also indicated.
Contemporary models of aging or of the risk of specific disabilities such as cancer or coronary artery disease generally assume that the universe of human beings of interest is divisible into one or at most a few populations within which a single risk function applies. Thus, for example, Truett's (67) use of a multiple logistic function to assess the risk of coronary artery disease posits two multivariate frequency distributions, each of which can be represented by a known mathematical function, corresponding to those individuals who will and those who will not develop coronary artery disease. Representations of this nature can and undoubtedly have provided important insights into, the risk of disease or a specific cause of death, and may be a necessary step in the evolution of more biologically meaningful models. We contend, however, that they must remain approximations until such time as account is taken of the existence of genetic variability within and between populations, and the impact of this upon liability to disease. Inherited diseases have been described as the most widespread of all human m a l a d i e s - t h e oldest, and possibly the most burdensome of human ailments (DHEW, 74). Perceptions of what constitutes genetic disease vary ; but these need not trouble us here. Patently, genetic factors can and do make important contributions. directly and indirectly, to a variety of common medical problems such as diabetes and cardiovascular or cerebrovascular disease: and whether the latter are called environmental or genetic diseases is immaterial. Moreover, individuals can and do vary in their responsiveness to drugs, infectious agents, and even diet. We may presume, therefore, that they will differ in their responses to the ever increasing and bewildering array of potential carcinogens and mutagens associated with environmental pollution and changing lifestyles. These differences, many of which are genetic in origin, can
importantly influence the occurrence as well as the course of disease. Possibly some illustrations of genetically determined variability in response to the genotype of another, to drugs, or to elements of our diet will serve to support our contentions (see Table 1).
Genetic variability: interaction with disease I. Hemolytic disease oJ the newborn Almost 40 years ago, Levine and Stetson (39) described a woman whose second pregnancy terminated in a stillbirth and who reacted severely to transfusion with her husband's blood. It was found that she had an antibody in her serum that agglutinated her husband's blood cells and, in fact, agglutinated such cells from about 85°~, of a random sample of U.S. Caucasians. Levine and Stetson conjectured that the hemolytic disease seen in the infant arose as a result of(l) the transplacental isoimmunization of the mother by the fetal red cells that contained an antigen derived from the father and (2) the subsequent passage into the fetal circulation of the immune antibodies that were formed in response to this antigen (see Pickles, 49, for a fuller description). Later studies of their own and others have supported this viewpoint. Hemolytic disease of the newborn, which we now recognize can occur as a result of maternal fetal incompatibility with respect to a number of red-blood-cell antigenic systems of which the most frequent cause is the Rh system, depends upon an interaction between a mother and her fetus, The incidence of hemolytic disease of the newborn due to Rh incompatibility, for example, relates directly to the mating type of the parents with respect to the Rh-locus, and different ethnic groups of the world are at varying degrees of risk depending on the population frequency of the Rh-d gene (see Mourant, 76). 371
372
Ranajit Chakraborty, William J. Schull and Kenneth M. Weiss Table 1. Some genetically determined responses to food, drugs, and another's genotype Disorder
"'Causal Agent"
Nature of variation
Hemolytic disease in newborns
Matcrnal genotype
Depending on ethnic variation of d-gene frequency and possible effect of birth order
Sickle cell anemia
Person's own genotype
Ethnic variation of Hb-S gene frequency: association with malaria, case of disease-vector
Lactose intolerance
Food stuff
Ethnic variation in intolerance; identified when exposed to food with lactose added
Thyroid metabolism defects
Iodine dcliciency and overdose
Geographic variation, overdose effect
Isoniazid inactivation
Drug for TB
Genetic variation in the a m o u n t of acetyltransferase in liver cells; racial differences in gene frequency
Suxamethonium sensitivity
Anesthetic
Though rare, genetic variation causes concern in anesthetic uses of the drug
But risk depends upon other factors as well. There exists, or appears to exist, a relationship with birth order (Ceppellini, 52, see also Cavalli-Sforza, 71) and isoimmunization at the Rh locus is known not to be independent of the ABO genotypes of the mother and fetus (for details, see Mollison, 67). Risk is, thus, a function of one's mother's genotype as well as one's own, and events at two different, unlinked loci. (Rh hemolytic disease has been a preventable disorder in the United States since 1968 when Rh immune globulin was licensed for use. This globulin, if administered to Rh negative women with Rh positive fetuses within 72 h of pregnancy termination, will suppress the maternal anti-Rh antibody response.~ 2. Sickh, cell am'mia
A single amino acid substitution in hernoglobin can in the homozygous state result in clinical symptoms of arthralgia, acute attacks of abdominal pain, ulcerations of the lower extremities, and an often fatal anemia. The phenomenon, called sickling, is caused by the formation of large crystal aggregates under conditions of low oxygen tension. The molecular nature of the abnormality, a single amino acid substitution in the Hb-beta chain (glutamic acid for valine at the sixth position), though often lethal in homozygous condition, is maintained in high frequencies in some populations by the presumed advantage of heterozygotes in malaria-infected regions of the world. Resistance to malaria, however, appears substantially more complicated than originally supposed and dependent not only upon the variation in hemoglobin associated with the sickling phenomenon, but also upon variation at the thalassemia loci, the glucose-6-phosphate dehydrogenase locus, and quite possibly the Duffy locus as well. The seriousness of malaria as a health problem can vary too with genetic changes in the mosquito that render it more immune to insecticides and changes in the plasmodium that compromise chemotherapy. Both events have occurred, and malaria is again becoming a
global health problem. Be this as it may, the genotype that may be favored in a malarious environment may be disadvantageous when malaria is not present. We know that individuals heterozygous for the hemoglobin-S gene, who normally appear in good health can under certain circumstances, such as those that attend the hypoxia of altitude, become ill. 3. Lactose intolerance (LIT)
Infant mammals survive on milk, of which disaccharide lactose is a major nutritional constituent. Normally, ingested lactose is cleaved by the enzyme lactase into glucose and galactose, and the latter then metabolized. Occasionally, infants are born who are unable to hydrolyze lactose; they are said to have congenital lactose malabsorption or intolerance, fail to thrive, and generally exhibit a chronic diarrhea which can be life threatening. Much more c o m m o n is a lactose intolerance that develops later. In these cases milk tolerance is usually normal in infancy, but intolerance develops in adolescence or adulthood (Huang, 68; Johnson, 74). The signs and symptoms of the intolerance suggest a direct toxic effect on the intestine--diarrhea, borborygmi, flatulence, and other signs of gastrointestinal distress are common. This form of lactose intolerance, which appears to be recessively inherited in American Negroes and whites (Bayless, 67; Welsh, 68), varies substantially from race to race, and population to population. It is especially common in those peoples whose traditional dietary practices have not included large amounts of milk or milk products in childhood and later years, such as the Amerindians, Eskimos, and many groups of blacks (see Simoons, 73). This has led both individuals and official agencies to be concerned about a suitable policy for using milk and dairy products as dietary supplements in regions where nutrition may be marginal but lactose intolerance common.
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373
4. Thyroid metabolism The synthesis, storage, secretion, and utilization of the thyroid hormones involve a complex sequence of metabolic events, each of which is probably dependent upon specific enzymatic activity (see Stanbury, 72, for a full explication). Thyroid disease may result from blockage at many steps in this metabolic process. Goiter can and does arise in the presence of ample nutritional iodine. Familial goiter and hypothyroidism may occur when there is failure of the thyroid cell to transport iodide and maintain a favorable concentration gradient inside the follicle lumen, but they may also result from failure to convert inorganic iodide into iodine in the thyroid gland. Other types of familial goiter occur; these involve such disparate bases as diminished or altered thyroglobulin synthesis, impaired response to thyrotropin, and the like. It is reasonable to presume that the sequence of metabolic events listed above may be altered quantitatively without concomitant qualitative changes; that is, through the origin of enzymes that do not block a specific step but diminish or enhance the reaction in question. Even in areas of endemic goiter there occur goiter-free individuals whose nutrition and lifestyles seem no different from those of their goitrous contemporaries. Thus, in the Pedregoso Valley of Chile where 700/0of individuals 10 years of age or older are goitrous, three individuals in ten of similar ages fail to manifest signs of impaired thyroid metabolism (Barzelatto, 68). Goiter in this valley is apparently influenced by two factors, namely, (1) the existence of iodine deficiency and, no less important, (2) the consumption of pinon, the fruit of the araucarian pine. The latter has been shown to sequester iodine and to be goitrogenic in rats. Thus, risk is a function not only of one's genotype, and the availability of iodine, but also of other dietary constituents as well.
toxic, so that the drug is effectively inactivated by acetylation. Shortly after isoniazid began to be used in tuberculosis therapy, it became apparent that the rate at which isoniazid is inactivated by acetylation differs between individuals, though in any one individual the metabolism of the drug remains remarkably constant (Hughes, 54; Bell, 57). It was then shown that people can be divided into two or more or less sharply distinct groups--so-called rapid and slow inactivators--according to the rate at which the inactivation proceeds. The difference in rate has been shown to be genetic in origin; slow inactivators are homozygous for one allele, whereas rapid inactivators are either heterozygous or homozygous for the other allele (Knight, 59; Evans, 60). This difference in isoniazid inactivation is related to a difference in the amount of an enzyme called "acetyltransferase" present in the cells. The existence of these marked individual differences in isoniazid inactivation raises the question of this drug's therapeutic significance for treating tuberculosis. In comparisons of large groups of patients on standardized anti-TB treatment including isoniazid, significant differences have not usually been found between the results of treatments in "rapid" and "slow" inactivators (Evans, 63). But while there may be little or no difference when the drug dosage is optimal, it is likely that if it is suboptimal, for example when the isoniazid is given too infrequently, then differences in response may occur (TB Chemotherapy Center, 70; Ellard, 72). Slow inactivators of isoniazid appear to be somewhat more likely than rapid inactivators to develop a peripheral neuropathy ascribed to the toxic side effects of the drug. This peripheral neuritis, however, is not of much concern now since it can be prevented by the simultaneous administration of pyridoxine. Table 2 gives some idea of the extent of racial variation with respect to the frequencies of the isoniazid inactivation alleles.
5. lsoniazid inactivation 6. Suxamethonium sensitivity Genetically determined differences in the activity of a particular enzyme can result in quite marked differences in the manner in which different individuals metabolize a particular drug, although this may not be associated with any acute clinical consequences. This can be illustrated by the differences that have been found to occur in acetylation of the drug isoniazid, which is widely used in the treatment of tuberculosis. In its acetylated form, isoniazid is much less active therapeutically and is also less
Suxamethonium (succinyl dicholine)was introduced as a muscle relaxant in surgery and electroconvulsion therapy some years ago on the supposition that because of its rapid hydrolysis into active products by serum cholinesterase, its effect should be quite short duration. Occasionally, however [e.g., in about one in every 2000 Europeans (Harris, 75)], a normal dose of the drug causes an extremely prolonged muscular paralysis and apnea
Table 2. Frequency of alleles determining "'rapid" and "'slow" inactivation of isoniazid in different populations (adapted from Harris, 75) Allele Frequency Population U.S. white U.S. black Japanese Ainu Korean Thai Burmese South Indian Alaskan Eskimo Alaskan Indian
Number Tested 305 207 2017 86 65 108 121 850 157 47
"Rapid 0.27 0.27 0.66 0.69 0.67 0.46 0.39 0.19 0.54 0.38
.
.
.
.
Slow'" 0.73 0.73 0.34 0.31 0.33 0.54 0.61 0.81 0.46 0.62
374 lasting for hours instead of the usual period of a few minutes. This rare abnormality is presently ascribed to low levels of serum cholinesterase in these aberrant individuals (Bourne, 52; Evans, 52). This phenomenon is, in fact, genetic; suxamethonium-sensitive individuals with the low levels of serum cholinesterase are homozygous for an abnormal gene which in heterozygotes results in a moderate reduction in enzyme level. Therefore, in populations where the abnormal gene is high in its frequency, administration of this drug would cause frequent problems. Variation in serum cholinesterase has broader implications than the one just cited. It has long been known that potatoes contain a substance, solanine, found principally in the peel and sprouts but present throughout the potato, which can be toxic to humans as well as other animals. Among the symptoms of such poisoning are disturbances in respiration and cardiac activity and significant hemolysis, as well as other evidence of stimulation of the cholinergic system, that is, stimulation of those nerve fibers that cause similar effects to those induced by acetylcholine. The mechanism through which these symptoms come about is seemingly the inhibition of serum and brain cholinesterase. At least two different inherited forms of serum cholinesterase are known; the three phenotypes that arise are differentially inhibited by extracts from the potato (Harris, 62). Numerous Andean populations have consumed potatoes for millenia, literally, and their methods of preparation are known in some instances not to degrade solaine. The implications of this are not clear at present, but obviously warrant exploration. The examples we have chosen are but a 3mall number of many. They have been selected to illustrate tl~e numerous ways in which interactions, gene with environment or gene with gene, can arise. Indeed, so ubiquitous are such interactions that the next level of ~.omplexity to which we turn briefly may seem unnecessary for support of our concerns.
Genetic variability and malignancy Down's syndrome of trisomy-21 has its well-known physical and mental symptoms, but individuals ivith this syndrome are also unusually sensitive to bacterial and viral infections. Because of this susceptibility to infection, few cases survived to adulthood before the advent of antibiotics. Individuals with this syndrome are also prone to leukemia. We recognize individuals who are mosaic with respect to this cytologic abnormality, that is, some of their somatic cells exhibit a supernumerary chromosome 21, but others do not. We do not know, however, whether their risks of infection and leukemia are proportional to the size of the normal cell population. If this is true. then estimation of risk becomes more subtle still. Another line of evidence that warrants note here has recently been provided by Swift (77) in his studies of the risk of malignancy in the parents of individuals with certain rare recessive phenotypes. He conjectures that individuals heterozygous for any one of the genes that in homozygous form give rise to xeroderma pigmentosum, Bloom's syndrome, Fanconi's anemia, or ataxiatelangiectasia have risks of dying from cancer 2 to 12 times higher than those of noncarriers of these gcnes.
Ranajit Chakraborty, William J. Schull and Kenneth M. Weiss In the beginning, the relationship between aryl hydrocarbon hydroxylase inducibility and bronchogenic carcinoma (Kellerman, 73) also seemed to be promising. The initial studies suggested that the inducibility of this enzyme was simply inherited and that "low inducers" were at much smaller risk of bronchogenic cancers when exposed to benzo(c0 pyrene and other polycyclic hydrocarbons in cigarette smoke and polluted air than "high inducers". Subsequent studies have not confirmed this, but the postulated interaction between genetic control of enzyme inducibility and exposure to an environmental agent remains a provocative paradigm.
Extent of human genetic variability So far we have discussed specific cases only. We have seen that there exist genetically determined variability in the liability to certain insect-borne diseases (malaria), as well as in response to dietary constituents (lactose) and pharmacologic agents (isoniazid) that figure large in therapeutic or preventive regimens. But how widespread are these responses ? Is there a limit to genetic variation in man ? If the number of genes is of the order of 500,000 or so, is such a limit measurable? Until relatively recently one could only speculate. However, as our abilities to recognize and measure serological and biochemical variability have grown, speculation has given way to observations that, while not as yet as precise as is desirable, provide important insights. Briefly, the last two decades have seen Karl Landsteiner's (1900) original four red-blood-cell phenotypes grow to no less than 29,952 in 1955: and a decade later in 1965, the immunological distinctions that could be more or less routinely made resulted, in theory, in no less than 2,717,245,440 distinct phenotypes. If the secretor phenomenon (which results in the production of water-soluble forms of the ABH antigens) is included, this number doubles. These distinctions, we emphasize, do not include pathological characteristics of the red blood cell; nor, more importantly, the increasing number of inherited serum proteins or red cell enzymes; nor the histocompatibility antigens, possibly the most variable of all of the so-called genetic markers. The realized variability in these systems is. of course, somewhat less; the genes involved are not ubiquitous, nor in areas where they occur are they all equally frequent. It seems unlikely, however, that the realized variability is less than the "expected" by much more than two orders of magnitude; it is certainly great enough to support the commonplace remark that we have all been uniquely endowed by our creator. A more complete answer to the extent of our genetic variability has begun to emerge through the development of molecular biology. On the theoretical side, Kimura (64) have shown that the number of alleles at a locus that can be maintained in a finite population is fairly large, taking into account the fact that at the molecular level almost an infinite number of alleles may be produced at a locus. (Wright's 1948 paper indicates this possibility even though the structure of the basis of heredity, DNA, was not then known to Wright.) The development of starch gel electrophoresis (Smithies, 55) in combination with a simple staining technique for a specific enzyme activity (Hunter, 57) provided a valuable tool by which genetic heterogeneity in proteins and isozymes could easily be
Heredity and risk of disease
375
detected. By 1965, it was already known that natural populations contain a large amount of polymorphism with respect to proteins and enzymes. In a review, Shaw (65) stated, "Enzymes which vary within populations are rather the rule than the exception". An important step in the study of genetic variation in populations was made by Lewontin (66) and Harris (66). The current theory that the primary structure and probably the rate of synthesis of different polypeptides are under genetic control suggests that the genetic variation in a natural population might be measured best by the enzymatic diversity present. Clearly, one cannot currently hope to study all of the enzymes produced by the genome to determine the proportion of genetic loci that show variation. It is possible, however, at least in theory, to study a representative sample of the enzymes of a species and thereby estimate the extent of its genetic variability. But an unbiased estimate will be obtained only if genetic variation is detectable at several loci and the loci are chosen randomly with respect to how variable they are. With the electrophoretic identification of isozymic variation it has become possible to sample genetic loci (by selecting enzymes) in a manner that at least approaches randomness. As might have been expected, even this approach to the sampling of genetic loci falls somewhat short of the ideal. First, only alterations in structural genes are efficiently detected. Detection of variation in the activity of enzymes is poor by electrophoresis and is biased in that we do not know how many regulator genes go undetected. Second, the proteins studied must be soluble, and third, a histochemical staining method for identification of the protein after electrophoresis must be available. These limitations notwithstanding, an increasing body of evidence exists to support the notion that a substantial fraction of genetic loci exhibit variation in p o l y m o r p h i c proportions. Harris (75) has reported findings on 20 arbitrarily chosen enzymes in Europeans and Negroes. Some 27 genetic loci are involved in the structural control of these enzymes. The average heterozygosity for these 20 enzymes, assuming 27 loci, he finds to be 0.054 for Europeans and 0.052 in Negroes. (Excursus: Average heterozygosity is measured in the following manner. Let Pi be the frequency of the ith allele at a locus. The expected
proportion of individuals heterozygous for this gene will be h = 1 - Ep~z, assuming that the population is large and panmictic and that no selection obtains at the locus. The "average heterozygosity" is merely the individual heterozygosities, the H's, summed over all loci under consideration, and divided by the number of loci.) Harris (75) estimates that the average heterozygosity per locus for alleles determining all structural enzyme and protein variants will be three times the values cited above. He argues that approximately one-third of the total variability will be recognizable electrophoretically. This factor, one-third, rests in part on the knowledge that almost two-thirds of all random amino acid substitutions in the hemoglobin molecule will produce no change in the net charge of the molecule and hence will be undetectable electrophoretically. Lewontin (67) suggested another, albeit somewhat similar, approach to the estimation of the average heterozygosity in man. His argument, which utilizes serological data, is based upon the supposition that as more and more bloods are tested, the "detected" heterozygosity will converge on the "true" heterozygosity; somewhat differently stated, data on blood groups will be less and less biased toward polymorphic loci as more and more bloods are studied. As of 1962, Lewontin noted that some 33 blood groups were known; of these, 36.4% were polymorphic and the average heterozygosity was 0.162. Of the next 17 blood groups to be recognized, only one, Dombrock, was polymorphic. If these data and the three blood groups that Lewontin failed to tabulate are considered, then of the 53 systems known in 1968, 13 were polymorphic. The average heterozygosity was correspondingly lower. Nei (74) have recently reexamined these data, but as they and others have noted, the loci associated with the blood groups may not be a random sample of the human genome. To this end, Harris (76) provided an up-to-date summary of our knowledge of enzyme protein variation in man, which is presented in Table 3. Average heterozygosity discussed so far gives some idea of the extent of genetic variability within populations. Recent work on genetic distances also gives estimates of the number of codon or mutational differences per locus between different population groups as obtained from gene frequency data. For example, analyzing data on 35
Table 3. Polymorphism and average heterozygosity as obtained from electrophoretically detectable enzyme variants in European and U.S. white populations (adapted from Harris~ 76) Polymorphic Nonpolymorphic loci (Average loci (Average heterozygosity heterozygosity > 0.02) < 0.02)
Number of loci screened
Average heterozygosity per locus
Oxidoreductases Transferases Hydrolases Lyases lsomerases Ligases
4 8 11 1
20 21 27 9 3
24 29 38 10 3
0.042 0.059 0.087 0.050
Total
24
80
104
0.063 (Weighted average)
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Ranajit Chakraborty, William J. Schull and Kenneth M. Weiss
common b i o c h e m i c a l loci, N e i a n d R o y c h o u d h u r y e s t i m a t e t h a t the net m u t a t i o n a l differerence b e t w e e n the t h r e e m a j o r racial g r o u p s is given by Caucasoid/Negroid Caucasoid/Mongoloid Negroid]Mongoloid
0.023 0.001 0.024
F r o m 21 c o m m o n b l o o d g r o u p loci these are 0.042, 0.034, a n d 0.118, respectively. T h e r e are o t h e r s i m i l a r e s t i m a t e s a v a i l a b l e for p o p u l a t i o n s w i t h i n the s a m e racial g r o u p s at the t r i b a l level [e.g., tribes o f different A m e r i n d i a n p o p u l a t i o n s (see B l a n c o , 75)] o r w i t h i n a single t r i b e at the village level (from d a t a of N e e l et al., see R o t h h a m m e r , 77). N e e d l e s s to say, t h e s e n u m b e r s are s m a l l e r t h a n the v a l u e s above. T h e n u m b e r s we h a v e j u s t cited m i g h t leave o n e w i t h the i m p r e s s i o n t h a t the l i m i t i n g v a r i a b i l i t y is n o t g r e a t e n o u g h to i n v a l i d a t e t h e a s s u m p t i o n of a p p r o x i m a t e u n i f o r m i t y in risk e s t i m a t e s for m a n k i n d as a w h o l e . T h i s is n o t true, h o w e v e r , for t h e v a l u e s j u s t given are averages per locus. T h e d i s t r i b u t i o n of the n u m b e r o f m u t a t i o n a l differences is h i g h l y s k e w e d w i t h a l o n g tail (Nei, 74; C h a k r a b o r t y , 77). So t h e r e are q u i t e a few g e n e t i c loci for w h i c h m u t a t i o n a l differences are m u c h l a r g e r t h a n these a v e r a g e values. S o m e o f t h e s e loci h a v e a s s o c i a t i o n w i t h disease s u s c e p t i b i l i t y as well. C l a r k e ' s (61) s t u d y of the a s s o c i a t i o n of the A B O g r o u p s w i t h d i s e a s e illustrates this aspect. In c o n c l u s i o n , o n e ' s risk of disease as well as o n e ' s s u s c e p t i b i l i t y to e n v i r o n m e n t a l ( m a n - m a d e o r n a t u r a l ) agents depends importantly upon one's genetic b a c k g r o u n d . T h u s , to a s s u m e , as is c o m m o n l y d o n e , t h a t a single f u n c t i o n d e s c r i b e s e q u a l l y well o u r i n d i v i d u a l risks of disease o r d e a t h is to i g n o r e o n e of the m o s t i m p o r t a n t of b i o l o g i c o b s e r v a t i o n s - - m a n ' s inherent g e n e t i c diversity.
A cknowledgement--This research was supported by grants H L- 15614, CA-19311 and GM-19513 from the National Institutes of Health.
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Heredity and risk of disease
Discussion Prentice: In Clarke's (61)* study of the association of the ABO groups with diseases, were the control groups matched an age? Chakraborty: As far as I know there is no indication about age matching on the control group. However, this may not be critical since the mean relative indices which were considered to show the association were in terms of relative frequencies of two ABO blood types. For example, for cancer of the stomach a relative mean incidence of A :O was found to be
*Clarke C. A. (1961) Blood groups and disease in Proyress in Medical Geneties, Vol. l (A. G. Steinberg, ed.) Grime and Stratton, New York. t Bodmer W. F. (1974) in Genetic Polymorphisms and Diseases in Man, B. Ramot, ed., 377-392. Academic Press, New York.
377 1.25. This means Ratio of A:O in Stomach Cancer Patients = 1.25. Ratio of A:O in Controls In this case, age matching would be of some consequence only if survival to a certain age is directly or indirectly related to ABO blood types. There is no such evidence to date. Prentice: What diseases have not been associated with specific HLA's? Chakraborty: Thewhole problem of HLA and disease association seems to be quite complex. As a projection of the mouse work on the connection between H-2 and immune response and viral susceptibility, there has been a detailed hunt for the same sort of association in man: namely, between the HLA system and diseases thought to be related to immune response differences. But there are a number of inherent problems, statistical as well as technical, which should be considered before taking such positive evidence of association seriously. Recently, Bodmert presented a detailed account of these sorts of pitfalls of association studies.