INTRINSIC MUTAGENESIS, AN INTERPRETATION OF THE PATHOGENESIS OF XERODERMA PIGMENTOSUM

495 was

discontinued after 20 minutes and the tissues

were

Ringer lactate. The isthmic end of irrigated the polyethylene splint in the graft was then fed into the isthmic stump on the recipient side and passed out through a small incision in the right uterine wall. Anastomosis of the isthmus was completed with 6 nylon sutures through the circular-muscle coat only and tubal peritoneum was oversewn. The splint was now removed. The free edges of the grafted mesentery were sutured to the surrounding peritoneum so that the graft lay in the hollow between the abdominal side-wall and the lumbar muscles. All blood-clot was removed and the abdomen was closed. Chloramphenicol was given for 3 days. 4-6 weeks later the animals were mated. with

warm

resulted from rejection might be small in view of the limited bulk of transplanted tissue. We are grateful to Mr Robert Acland for his expert advice and encouragement with microsurgical techniques, and to Mr Derek Chappell, of the Medical Engineering Department of the Royal Postgraduate Medical School, who helped design Messrs. and make most of the microsurgical instruments. Spingler-Tritt, Chirurgische Nadeln, Jestetten, West Germany, have enthusiastically supported this project and supplied the The diagram was kindly drawn by fine-suture material. Mr Doig Simmonds. This work was supported by the Medical Research Council.

Requests for reprints should be addressed

to

R. M. L. W.

REFERENCES RESULTS

4 rabbits have been subjected to autotransplantation of the fallopian tube. One rabbit has delivered a live litter of nine, 33 days after mating, and another is pregnant. A 3rd rabbit died 36 hours postoperatively because of prolonged anxsthesia. At necropsy the graft appeared viable with patent vessels. In a 4th animal the graft proceeded uneventfully, but the uterine cornu became infarcted because the uterine artery was ligated too far from its anastomosis with the tubal artery. This animal subsequently became xstrous, suggesting that the ovary is viable. DISCUSSION

We believe that these are the first reported pregnancies after transplantation of the fallopian tube and ovary with vascular anastomosis. It is entirely feasible to transplant the tube without the ovary, but we transferred the ovary with the tube for simplicity. Other workers transplanted the entire genital tract in the dog. In 70 experiments they achieved only 1 pregnancy3 In these experiments revascularisation either by wrapping the graft in was obtained or omentum,3s by anastomosis with the great pelvic vessels.1,3 Omental wrapping is unlikely to be useful in human surgery because of resulting tubal adhesions, and anastomosis with the great vessels frequently caused major disturbances to the pelvic vasculature. Moreover, total genital-tract replacement is unlikely to be beneficial to patients because suitable donor tissue is rare and because such grafts are bulky and might cause considerable systemic disturbance if rejected. Tachezy et al.1 transplanted the uterine cornu alone in the rabbit, achieving revascularisation of the graft by burying it in the recipient uterotubal mesentery. 1 pregnancy followed 18 transplants and their poor success-rate is probably due to ensuing disturbance of cornual anatomy and tubal motility and lack of

adequate blood-supply. A microsurgical technique for tubal and vascular anastomosis has the advantage that only the tube needs to be grafted, with minimal disturbance of the surrounding tissues. Cohen8 pointed out that spare human fallopian tubes are readily available, healthy tubes being removed daily at hysterectomy and sterilisation. Homograft rejection will need further assessment, but an encouraging factor is that a transplanted tube need not remain viable after fertilisation and secure implantation of the blastocyst. Once pregnancy was established, any disturbance that

1. 2. 3.

4.

5. 6.

7. 8.

Wingate, M. B., Karasewich, E., Wingate, L., Lauchian, S., Ray, M. Am. J. Obstet. Gynec. 1970, 106, 1171. Scott, J. R., Curtis, J. D. Fertil. Steril. 1972, 23, 217. Barzilai, A., Paldi, E., Gal, D., Hampel, N. Israel J. med. Sci. 1973, 9, 49. Acland, R. D. Br. J. Surg. 1972, 59, 181. Acland, R. D. Br. J. plast. Surg. 1972, 25, 292. O’Leary, J. A., Feldman, M., Gaenssien, D. M. Fertil. Steril. 1969, 20, 757. Tachezy, R., Kučera, E., Macků, F., Studenŷ, B., Trnka, V. Česk. Gynek. 1971. 36, 129. Cohen, B. British Congress of Obstetrics and Gynæcology, 1974.

Hypothesis INTRINSIC MUTAGENESIS, AN INTERPRETATION OF THE PATHOGENESIS OF XERODERMA PIGMENTOSUM F. M. BURNET Basle Institute

*

for Immunology, Basle,

Switzerland

An interpretation is offered of recent work on the autosomal recessive disease xeroderma pigmentosum, based on the concept of intrinsic mutagenesis and its bearing on somatic mutation and ageing in man. It is concluded that observed defects in the physical repair of D.N.A. offer only indirect evidence of what is responsible for the disease and that the real mechanism is an incorporation of informational errors in what is, physiologically, completely repaired D.N.A. The two main manifestations of the disease—multiple skin malignancies and a proportion of patients with serious central-nervous-system anomalies—are both ascribed to somatic mutation resulting from the inheritance of some error-prone enzyme concerned with informational transfer in D.N.A. Possible applications of molecular biological techniques to xeroderma pigmentosum and other clinical conditions are briefly discussed.

Sum ary

FOR many years it has been often remarked in discussions of evolution that if the rate of mutation in a species was below a certain level evolution could not continue, while if it became unduly high the species could not tolerate the excessive burden of deleterious genes. Some level of mutation would be " Present address: School of Microbiology, University of Melbourne, Parkville, Victoria 3052, Australia.

496

optimal for survival and evolutionary advance, and the suggestion has sometimes been raised that mutation-rate might or must be itself controlled by evolution. Over the past two years I have attempted to develop the concept of intrinsic mutagenesis as something under genetic and therefore evolutionary control, and to apply it particularly to the problems of somatic mutation and ageing.l-3 In brief the concept is simply that the existence of error-proneness in the functioning of D.N.A.-polymerases and other key enzymes concerned in the replication and repair of D.N.A. and other informational macromolecules provides a nexus by which the genetic requirements can be expressed. The background of work in bacterial genetics, especially the existence of mutator strains in bacteria and bacterial viruses, is discussed in a book.3 This book has as its central theme an examination of the view that age and age-associated diseases are fundamentally manifestations of accumulating somatic mutation. At the somatic level, it is held, the integrated level of error-proneness in such key enzymes can provide a genetic basis for the characteristic lifespan of mammalian species. More important for the present communication is the question of age-associated diseases, of which malignant disease, autoimmune disease, and degenerative disease of the central nervous system were those taken for major discussion, based in each case on the hypothesis that they are all fundamentally of somatic mutational origin. Other sections of the book are concerned with the possibility that a number of genetic diseases associated with multiple anatomical and functional abnormalities could be related to gross malfunctions of certain of these key enzymes; and there is naturally considerable attention given to the two human conditions known to be associated with experimentally demonstrable defects in D.N.A. repair, progeria and xeroderma pigmentosum (x.p.). Much recent work on x.p. was reviewed at a National Institutes of Health conference.4 This allows a more comprehensive picture of the disease than was previously available to physicians and biologists not au fait with current literature in photobiology. The objective of this paper is to claim, on the basis of that review,4 that the characteristics of x.p. provide almost a categorical proof of the correctness, or at least the usefulness, of the idea of intrinsic mutagenesis as outlined above.

Clinically the disease is a rare autosomal recessive condition involving intense susceptibility to the damaging effect of solar radiation on the skin. A highly significant proportion of cases show congenital and sometimes progressive abnormalities of the central nervous system. The skin changes become evident very early in the form of intense susceptibility of the infants to sunburn and the appearance of copious crops of freckles by the age of As I have discussed elsewhere,5,6 a freckle is more two. readily interpreted as a small self-limiting clone of mutant melanocytes, the mutation being associated with exposure to sunlight, presumably the longer ultraviolet component. Intense freckling with lesions of variable size, shape, and depth of pigmentation of all areas exposed to sunlight is the most consistent feature of the disease. Much more important, however, is the unprecedented liability of these patients to malignant disease of every type that

The essential data from Robbins

et

al.’

A.. B, C, and D are the four complementation groups, and E is the single case with no detectable D.N.A. repair defect. The proportion of the normal uptake of aHTdR is shown as percentage. The four circles and a square is a notional model of the complex gene assumed to be involved, black indicating functional

inactivity

or

anomaly (see text).

tissues exposed to solar ultraviolet (i.e., the of the eye). In order of frequency the tumours observed are basal-cell carcinoma, squamous-cell can occur to

skin and

cornea

carcinoma, acanthoma, malignant melanoma, capillary

angioma, fibroma, are

and

sarcoma.

Premalignant conditions

very common.

Fibroblasts or epidermal cells from skin of such patients have now been extensively investigated in cell culture in regard to their reaction to ultraviolet (u.v.) irradiation and subsequent D.N.A. repair. Most of the relevant information about the cases reviewed at the N.I.H. conference is combined in the figure. In summary, all but one of the cases show a demonstrable reduction in the amount of repair as judged by " unscheduled " uptake of tritiated thymidine in the first 3 hours after heavy u.v. irradiation. The positive examples can be divided into four complementation groups by inducing heterokaryon formation with killed Sendai virus in mixed cell cultures. One typical case (no. 4) gave no evidence of any repair failure, and it should also be noted that positive cultures if followed for more than 24 hours after irradiation in fact took up eventually the same amount of 3HTdR as normal u.v.-irradiated cells. In that sense repair could be said to be complete but variably delayed. In my opinion the findings reviewed by Robbins and his colleagues can be interpreted more widely than they ventured to do, in terms of the concept of intrinsic mutagenesis. The nature of freckles is vital in this consideration. In the N.I.H. review Lutzner refers to my interpretation of the freckles as, a limited clone of mutant melanocytes.6 I can see no other reasonable interpretation. Ordinary observation shows that normal children living in cool temperate climates show highly varied degrees of freckling on exposed skin; many have virtually none. Under the same conditions patients with X.P. show massive freckling from an early age, although from the nature of their condition they are almost certain to have been shielded from direct sunlight to a much greater degree The conclusion is inescapable than normal children. that though exposure to sunlight is necessary for freckling it is of far less importance than some factor intrinsic to the individual and genetically determined. If, as is is a x.p. factor already evident, single genetic anomaly associated with some abnormality of D.N.A. repair, we must assume that other aspects of these children are within the " normal " range. Amongst other things we require to accept the reasonable certainty that in x.p. children and in frecklers and non-frecklers amongst normal chil-

497

dren, exposure to u.v. light and the production of pyrimidine dimers in accessible cells is approximately equivalent. This means that the efficacy of repair without mutation diminishes in the order non-freckler, freckler, x.p.

subject.

It is also of great importance that amongst x.P. cases there is a wide range of effectiveness or rate of physical repair of D.N.A. without any correlated association with symptom intensity. It follows that the rate of somatic mutation as judged by the intensity of Jackling is determined, not by the rate at which physical repair is carried out, but by the proportion of informational errors that have been committed in the course of physiologically complete repair. If a mutant clone is to arise, physiological repair in the initiating mutant and its descendants must be complete or no clone could be produced. The basic deficiency is not in something that can be measured physically by current techniques but in the process by which error is introduced into the final ordering of nucleotide sequence. That error-prone process is indicated by the square blocks in the diagram of the gene complex involved. The four circles stand for four distinguishable variations that are possible within the gene complex which interfere in different ways with the repair process to give physically recognisable effects.

If

that

is correct it greatly earlier contention that mutation is strengthens my not the result of damage by conventional mutagens1 such as u.v. light but wholly the expression of informational error dependent on the structure of the key enzymes concerned in the replicating and repair pro-

interpretation

cesses.

,

.

.

. ,

.

Very similar arguments can be used to support the thesis that malignant disease is, from the evolutionary point of view, an accidental byproduct of the need to have a way by which the average lifespan of a species could be genetically defined.3 It will be remembered that the " non-specific " skin damage seen in a young adult with x.p. shows almost all the features seen in the exposed skin of old persons who have had an outdoor occupation. A high rate of skin carcinoma and melanoma is well known to occur in such people especially where, as in Queensland, the outdoor work in a tropical country is done by lightThe yield of tumours in a skinned Caucasians. single individual with x.p. may be 100 times what is seen in elderly outdoor workers, but the range of tumour types matches closely what would be found in a large series of skin tumours from such a normal population. The x.p. situation is essentially an intensification of the normal one, and it is probably relevant to the normal aetiology of skin cancer. From this onward the is point argument precisely analogous to that used for freckling. The U.v. component of sunlight may be a necessary cause of all the varieties of skin cancer in perhaps 90% of cases, but it clearly requires an additional constitutional factor. In the X.P. patient there is probably something between 104 and 106 times the probability that a given dosage (ergs X time) of u.v. radiation will produce skin cancer as in normal people. Error in repair of u.v. damage to a melanocyte will commonly initiate a freckle, on occasion a malignant melanoma. Similarly, atrophy and benign patches of keratosis in aged normal skin will be occasionally associated with a malignant epithelial tumour. The enormously in-

creased frequency in x.p. must surely indicate error in informational repair associated with some errorprone enzyme. Just as much as a normal cell or such a mildly anomalous one as the freckle melanocyte, the cells of a malignant clone must have physiologically functioning completely repaired D.N.A. The whole pattern points unmistakably toward the somatic mutational origin of human malignant

disease. The final manifestation of x.p. which calls for brief comment is the presence of a substantial proportion of cases of C.N.S. abnormality. The number is far too great to be fortuitous; similar congenital conditions without x.p. are known, but they are all very rare. I have discussed 3 the possibility that this group of what are apparently one-gene anomalies with multiple and varied anatomical and functional expression could represent the action during development of error-prone key enzymes. Clearly u.v. cannot affect the development of the C.N.S. in utero and equally clearly the occurrence in X.P. of such conditions including the de Sanctis-Cacchione syndrome strongly supports the tentative ascription of the group to the same general process. I am not competent to discuss the pathology of genetic congenital anomalies in infants, but it seems likely that an approach along the lines I am suggesting could be rewarding to those interested in understanding their aetiology. As a postscript to this view it should be mentioned that Epstein et al.,while confirming their previous finding8 that fibroblasts from a case of progeria showed greatly diminished ability to repair D.N.A. breaks produced by heavy X irradiation, have found another typical case in which the repair capacity seemed to be unimpaired. The resemblance to case 4 of the x.p. series is unmistakable, and interpretation would follow the same lines. The application of physicochemical methods of assessing the rate of D.N.A. repair has been invaluable in pointing to the importance of D.N.A. damage and repair in human pathology, but concentration on those experimental methods may be misleading as to the mechanism of pathogenesis. If the conception based on intrinsic mutagenesis is to be experimentally established, methods already in use in bacterial and viral genetics will need to be applied to show directly that error-prone key enzymes are present in the cells. Lymphocytes, fibroblasts, or even tumour cells from x.p. patients could well provide ideal material for tests of fidelity of D.N.A. polymerases by any of the available methods which are appropriate or can be adapted to mammalian cells. D.N.A. has essentially the same nature whatever the organism, and the possibility of testing enzymes from mammalian cells, normal and mutant, for their influence on the invitro synthetic systems of the molecular biologists dealing with bacterial and viral material is probably already at hand. One’s impression of the way molecular biology is developing suggests that many other experimental approaches to human pathology in terms of error-prone enzymes concerned with information transfer could be devised.

498

PLACENTAL FREE-FATTY-ACID TRANSFER AND FETAL ADIPOSE-TISSUE DEVELOPMENT: AN EXPLANATION OF FETAL ADIPOSITY IN INFANTS OF DIABETIC MOTHERS ANDREW

J. SZABO

OLGA SZABO

Department of Medicine, New York Medical New York, N.Y. 10029, U.S.A.

College,

A

new hypothesis on adipose-tissue development during intrauterine life is presented. It is proposed that circulating maternal plasma free fatty acids (F.F.A.) are the major precursors of triglyceride fatty acids in the fetal adipose tissue. Maternal F.F.A. is transported across the placenta and is carried in the blood of the fetus to the developing fat cells. After being taken up by the adipocytes, F.F.A. is esterified and is incorporated into the adiposetissue triglyceride pool. Since diabetics have higher plasma-F.F.A. levels than non-diabetic pregnant women, the gradient-dependent diffusion type of transfer of F.F.A. across the placenta is faster in these cases. This results in increased availability in fetal plasma of F.F.A. to be taken up by the adipocytes. It is suggested that glucose is not the major precursor of triglyceride fatty acids—as it was thought to be in the past—but serves as the primary substrate for &agr;-glycerophosphate production. As &agr;-glycerophosphate is necessary for the esterification of F.F.A., hyperglycæmia and hyper-

Sum ary

insulinæmia in fetuses of diabetic mothers may facilitate the esterification process in the fetal adipocytes. This hypothesis assigns a major role to maternal plasma F.F.A. but only a minor role to blood-sugar levels in the pathogenesis of fetal adiposity in infants The hypothesis is therefore of diabetic mothers. compatible with the paradoxical finding that, despite their normal (or near normal) blood-sugar levels, prediabetic women often give birth to obese " diabetictype " babies, while overtly hyperglycæmic diabetic mothers may have normal or even small newborns.

.

available biochemical and physiological observations into a logical sequence of events. Pedersen postulated -and this was subsequently proven by actual observations-that chronic increase in fetal blood-sugar causes hyperplasia of the fetal pancreatic isletsand results in elevated plasma-insulin levels 8 This hyperinsulinaemia was thought to facilitate incorporation of glucose into the fetal adipocytes, and result in quantitatively increased fat synthesis in the fetus. The Pedersen

hypothesis, as summarised above, fails often explain why prediabetic or chemical diabetic mothers deliver " large babies ".9-11 In these women blood-sugar levels are normal or near normal throughout pregnancy. Therefore, if maternal blood-sugar were the major determinant of fetal adiposity, one would expect to find normal-weight babies in this group. - Instead, large babies often precede the diagnosis of diabetes by as much as twenty-five years.*"" In contrast, patients with frank clinical diabetes, and with marked hyperglycaemia throughout their pregnancy, frequently deliver infants of normal or even small size.6,12 According to the glucose/insulin/lipogenesis theory these babies should have been born with increased adipose-tissue mass. In a meticulously designed study by Karlsson and Kjellmer13 mean bloodsugars (three per day from the 30th to 32nd week until delivery in 167 diabetic women) showed no correlation (correlation coefficient 0-09) with the fetal weight. Impaired transport of nutrients to the fetus, due to diabetic vascular disease of the placenta," is frequently offered as an explanation for the paradoxically small babies born to diabetic women. This may be true, but not for glucose transport, since cord bloodsugar correlates well with maternal blood-sugar even in babies born to mothers with long-term diabetes 1; Thus, maternal blood-sugar level cannot be the major factor regulating fetal adipose-tissue triglyceride

to

synthesis. THE HYPOTHESIS

In

attempt to resolve these inconsistencies that be fully explained by the glucose/insulin/lipogenesis theory, we offer the following alternative hypothesis for fetal adipogenesis. Free fatty acids cross the placenta in the human from the mother to the fetus. This transported fatty acid is derived primarily from circulating albumin-bound serum free fatty acids of the mother. To a lesser extent the transported fatty acid may originate from maternal serum triglycerides split to F.F.A. and glycerol by placental lipoprotein-lipase an

cannot

INTRODUCTION

INCREASED adipose-tissue mass of neonates born to diabetic mothers 1,2 is usually ascribed to accelerated triglyceride synthesis by the fetal fat cells. The substrate for this fat synthesis is thought to be the abundantly available glucose that is transported across the placenta from the maternal to the fetal blood. This concept, initially suggested by Jorgen Pedersen,3,4 gained wide acceptance 5,6 because it grouped the

enzymes. SIR MACFARLANE BURNET : REFERENCES 1. 2. 3.

4.

5. 6. 7. 8.

Burnet, F. M. Lancet, 1973, ii, 480. Burnet, F. M. Pathology, 1974, 6, 1. Burnet, F. M. Intrinsic Mutagenesis: a Genetic Approach to Ageing. Lancaster, 1974. Robbins, J. H., Kraemer, K. H., Lutzner, M. A., Festoff, B. W., Coon, H. C. Ann. intern. Med. 1974, 80, 221 (see this for other references on X.P.). Burnet, F. M. Immunological Surveillance. Sydney, 1970. Burnet, F. M. Genes, Dreams and Realities. Lancaster, 1971. Epstein, J., Williams, J. R., Little, T. B. Proc. natn. Acad. Sci. 1973, 70, 977. Epstein, J., Williams, J. R., Little, T. B. Biochem. biophys. Res Commun. (in the press).

As placental F.F.A. transfer has been shown to be primarily gradient-dependent diffusion, conditions resulting in high maternal plasma-F.F.A. levels, such as diabetes mellitus, can result in accelerated transferrate. Thus, it is proposed that from the blood of the diabetic mothers increased quantities of F.F.A. cross the placenta to the fetal side. These transferred circulating free fatty acids are subsequently taken up from the fetal plasma by the adipocytes to be esterified and incorporated into the triglyceride stores of the

fetus.