847 No increase in urinary aminoacids or sugars was detected, except for glycosuria in the oldest woman, a known diabetic.

Discussion A wide range of clinical and biochemical evidence that could be related to cadmium toxicity was found in this small-scale study of the residents of Shipham. Blood-cadmium levels were raised in two-thirds of the residents. This finding is consistent with the estimation of liver-cadmium content carried out by Harvey et a1.15 with in-vivo neutron-activation analysis. In 21 villagers, a mean liver-cadmium concentration nearly five times that in controls. Our findings are less reassuring than their references to industrial exposure would

have

provided some useful guidelines investigation of cadmium toxicity.

cardiovascular disease,18 and replacement with the natural hormone can lower blood-pressure and lipid levels, reduce angina, and normalise the ischaemic

references.

Requests for reprints should be addressed to M. C. REFERENCES

Davies, B. Personal communication. Tucker, A. The Toxic Metals. London, 1972. 3. Kazantzis, G., Flynn, F. V., Spowage, J. S., Trott, D. G. Q. Jl Med. 1963, 1. 2.

32, 165. 4. Glauser, S. C., Bello, C. T., Glauser, E. M. Lancet, 1976, i, 717. 5. Scott, R., Paterson, P. J., Mills, E. A. et al. ibid. 1976, i, 396. 6. Friberg, L., Piscator, M., Nordberg, G., Kjellstrom, T. (editors) Cadmium in the Environment. Cleveland, Ohio, 1974. 7. Evrin, P. E., Wibell, L. Scand. J. clin. Lab. Invest. 1972, 29, 69. 8. Tucker, S. M., Boyd, P. J. R., Thompson, A. E., Price, R. G. Clinica chim.

Acta, 1975, 62, 333. 9. Friberg, L. Acta med. scand. 1950, 238, suppl. 240. 10. Peterson, P., Evrin, P., Berggard, I. J. clin. Invest. 1969, 48, 1189. 11. Wibell, L., Evrin, P., Berggard, I. Nephron, 1973, 10, 320. 12. Kjellström, T., Shiroshi, K., Evrin, P. Environ. Res. 1977, 13, 318. 13. Kjellström, T., Piscator, M. Phadedoc, 1977, 1, 3. 14. Piscator, M., Adamsson, E., Elinder, C. G., Pettersson, B., Steningern, P.

Abstr. 18th Congress of

Occupational Health, Brighton, p. 281. 1975.

T. C., Chettle, D. R., Fremlin, J. H., Al Haddad, I. K., Downey, S. P. M. J. Lancet, 1979, i, 551. 16. Schroeder, H. A. Circulation, 1967, 35, 570. 17. Schroeder, H. A., et al. J. Am. med. Ass. 1966, 195, 81. 18. Poggi, U. L., Argüelles, A. E., Rosner, J., Delaborde, N. P., Cassini, J. H., Volmer, M. C. J. Steroid Biochem. 1976, 7, 229. 19. Jaffe, M. D. Br. Heart J. 1977, 39, 1217.

15.

Harvey,

ANTENATAL DIAGNOSIS OF DUCHENNE MUSCULAR DYSTROPHY A. E. H. EMERY

D. BURT

University Department of Human Genetics, Western General Hospital, Edinburgh V. DUBOWITZ

Department of Pædiatrics and Neonatal Medicine, Hammersmith Hospital, London W12 I. ROCKER

Royal Gwent Hospital, Gwent DIAN DONNAI

R. HARRIS

University Department of Medical Genetics, St. Mary’s Hospital, Manchester P. DONNAI

electrocardiogram. 19 The clinical picture seen in this study, especially in association with a high prevalence of hypertension, evidence of renal damage, and high blood and liver cadmium values, suggests that more detailed studies of the entire population of the village should be carried out as soon as possible. Meanwhile the advice already given about not eating locally grown vegetables and not smoking needs to be emphasised. Although cadmium causes biochemical abnormalities, long-term ingestion is needed before any of the associated illnesses becomes clinically apparent. Industrial exposure is a more serious and .idespread problem. It is hoped that this pilot study will

larger-scale

We thank the residents who took part in the study and Linda McDougal, of Thames Television’s TV Eye programme, who suggested the survey and made it possible. Prof. Lars Friberg of the Karolinska Institute, Fack, Stockholm, kindly arranged blood-cadmium estimations in his laboratory and provided expert advice. We also thank Dr P. O’Gorman, of the Brook Hospital, for zinc and lead analyses, Miss Pat Kind, of St. Thomas’ Hospital, for urinary N.A.G. estimations, and Pharmacia Diagnostics Ltd. for &bgr;-microglobulin

they found

suggest. The serious clinical significance of this high level of exposure to cadmium was shown by the increased prevalence of hypertension and other stigmata of cardiovascular disease, together with evidence of renal tubular damage, of which an increase in P2-microglobulin excretion appears the most constant. In people with additional occupational exposure, as in the 31-year-old nonsmoking man who worked next to a zinc-smelting plant, the clinical and biochemical effects were particularly severe, the blood-cadmium level being fourteen times normal. Cadmium’s nephrotoxicity is generally accepted,3,9 but there is considerable debate about its cardiotoxicity. Administration of cadmium to laboratory animals will consistently produce hypertension.16 Hypertension has also been reported in association with industrial exposure5 and a high water content of the metal .2,17 Bloodcadmium levels have also been found to be higher in untreated hypertensives than in normotensive subjects.4 There are several mechanisms by which cadmium could raise blood-pressure and increase the prevalence of cardiovascular disease. First, though the metal predominantly poisons the tubule,3most chronic renal disorders can contribute to raising the blood-pressure. Secondly, it has been suggested4that cadmium may result in relative sympathetic overactivity by replacing other divalent cations in enzymes which inactivate catecholamines-catechol-o-methyl-transferases and monoamine oxidases. In this way, by acting as an inhibitor of these enzymes, cadmium may elevate the blood-pressure and mood simultaneously, acting as a natural antidepressant. In addition cadmium is known to be toxic to the testis.2 Low testosterone levels have been found in

for

University Department of Obstetrics and St. Mary’s Hospital, Manchester

Gynæcol

As a means of assessing the value of fetal serum-creatine-kinase (S.C.K.) levels in the antenatal diagnosis of Duchenne muscular dystrophy (D.M.D.), fetal muscle from control and at-risk fetuses was studied histologically and the findings were related to fetal S.C.K. levels. Of 7 at-risk fetuses 4 were believed to have normal muscle and all these had normal S.C.K. levels. However, of 3 fetuses with abnormal muscle only 1 had a raised S.C.K. level. At present cau-

Summary

848 tion should be exercised in offering antenatal in D.M.D. on the basis of fetal S.C.K. levels.

diagnosis

TABLE I-DUPLICATE MEASUREMENTS ON FETAL MUSCLE*

Introduction Duchenne muscular dystrophy (D.M.D.) is the comform of muscular dystrophy. It is characterised by progressive muscle wasting and weakness which appears around the age of 3 to 5 years, leads to inability to walk by the age of 12, and causes death in the late teens or early twenties. It is inherited as an X-linked recessive trait affecting males and transmitted by female carriers. There is no effective treatment, and prevention is through genetic counselling. Since the basic biochemical defect is unknown, an indirect test, the fetal serumcreatine-kinase (S.C.K.) level, is being considered as a means of antenatal diagnosis. However, short of allowing an at-risk pregnancy to continue to term and subsequently assessing the status of the newborn male, the only way to determine the value of the test might be to study fetal muscle at termination of pregnancy. There is already evidence that muscle-fibre size, and possibly other indices, are abnormal in at least some at-risk male fetuses.1-4 The aims of the present investigations were three fold-to determine the reproducibility of such quantitative measurements; to compare the opinions of two independent observers on muscle tissue from several control and at-risk male fetuses; and to relate the findings in the muscle tissue to fetal s.c.K. levels.

monest

Methods Material from normal fetuses

was

*All but fetus H were range are in italics.

at

risk for

obtained

at

the time of

neuromuscular disorder. All the mothers of at-risk male fetuses were either proven carriers or had a risk greater than 1 in 5 of being carriers. In each case the sex of the fetus was established before abortion by cytogenetic studies on amniotic-fluid cells obtained at amniocentesis. Gestational ages ranged from 14 to 21 weeks in both groups. Fetal blood-samples were obtained either by fetoscopy or by careful needle aspiration from fetal vessels in the cord at the time of hysterotomy but before delivery of the fetus and placenta. (Blood aspirated from fetal vessels after delivery of the fetus and placenta may have spuriously high S.C.K. levels,5 which may reflect the trauma associated with abortion.) Fetal S.C.K. levels were determined by the method of Rosalki6 and corrected for any contamination by maternal serum and/or amniotic fluid,3 though this was in fact necessary only in a few of the control specimens.

A single biopsy specimen of quadriceps muscle was obtained in each case and cryostat sections were stained with hsmatoxylin and eosin. Fibre size and the proportions of hyaline

eosinophilic fibres were determined by two of us (A.E. and D.B.) by methods previously described.2 Slides were coded and, in some cases where there was adequate material, duplicate slides were made. All duplicates were measured blindwithout knowledge of the source of the material. A series of coded slides were also exchanged and independently assessed by A.E. and V.D. without knowledge of the source of the material or of fetal s.c.K. levels.

Results

between duplicate control and at-risk fetal muscle and there was also complete agreement in the assessment of whether or not a particular specimen was abnormal, abnormality having been defined as two or more measurements outside the normal range (table l). 18 coded slides (5 controls and 13 at-risk) were exchanged and indepenThere

was

good agreement

measurements on

TABLE II-MEASUREMENTS ON AT-RISK FETAL MUSCLE AND FETAL S.C.K.

are

Results outside the normal

and

therapeutic abortion in cases where there was no history of any

*Results outside the normal range

D.M.D.

in italics.

LEVELS*

849 TABLE 111-FETAL S.C.K. LEVELS

(l.V.)

IN MALE FETUSES AT-RISK

FOR D.M.D.

of the study. This work was supported by a research grant from the Muscular Dystrophy Group of Great Britain.

Requests for reprints should be addressed to A.E. REFERENCES

Toop, J., Emery, A. E. H. Clin. Genet. 1974, 5, 230. Emery, A. E. H. Nature, 1977, 266, 472. Mahoney, M. J., Haseltine, F. P., Hobbins, J. C., Banker, B. Q., Caskey, C. T., Golbus, M. S. New Engl. J. Med. 1977, 297, 968. 4. Winn, K. J., Heller, R. H. Clin. Genet. 1978, 13, 335. 5. Larson, P. F., Park, D. C. J. neurol. Sci. 1974, 23, 33. 6. Rosalki, S. B. J. Lab. clin. Med. 1967, 69, 696. 7. Emery, A. E. H., Nelson, M. M., Mayo, O. in Actualitiés de Pathologie Neuro-Musculaire (edited by G. Serratrice); p. 13. Paris, 1971. 8. Pearson, C. M. Brain, 1962, 85, 109. 9. Hudgson, P., Pearce, G. W., Walton, J. N. ibid. 1967, 90, 565. 10. Bradley, W. G., Hudgson, P., Lawson, P. F., Papapetropoulos, T. A., Jenkison, M. J. Neurol. Neurosurg. Psychiat. 1972, 35, 451. 11. Dubowitz, V. Muscle Disorders in Childhood; p. 36. Philadelphia, 1978. 12. Stengel-Rutkowski, L., Scheuerbrandt, G., Beckmann, R., Pongratz, D. Lancet, 1977, 1, 1359. 13. Dubowitz, V., van Iddekinge, B., Rodeck, C. H., Campbell, S., Singer, J. D., Scheuerbrandt, G., Moss, D. W. ibid. 1978, i, 90. 14. Ionasescu, V., Zellweger, H., Cancilla, P. ibid 1978, ii, 1251. 1. 2. 3.

*Value in original publication was misprinted.

dently studied by A.E. and V.D. The assessments were in complete agreement-3 definitely abnormal, 2 possibly abnormal, and the remainder (including all the controls) completely normal. The findings in 7 at-risk fetuses and the fetal S.C.K. levels ("Edinburgh series") are given in table 11. Four specimens assessed as being normal were associated with S.C.K. levels within the appropriate normal range. However, of the 3 muscle specimens judged abnormal, only 1 (B132) was associated with a raised s.c.K. level. Discussion who is at high risk of having a son with may opt for antenatal fetal sexing with selective abortion of any male fetus, and in this way may be guaranteed a daughter who will not be affected. But this is unsatisfactory because at least half the aborted male fetuses will be normal; furthermore, sons (who would A

woman

D.M.D.

have reproduced) are replaced by daughters, a proportion of whom will be carriers, and the effect will therefore be dysgenic.7 A reliable antenatal diagnostic test for this distressing condition is therefore urgently needed. Even in the preclinical stage of the disorder s.c.K. levels may be 20-50 times the upper limit of normal, and muscle is also abnormal: there is increased variation in fibre size and hyalinisation of fibres,8-1O and there may also be foci of degenerating or regenerating fibres. 11 These observations raise the possibility that D.M.D. may be detectable in utero by measurement of fetal s.c.K. with confirmation, perhaps, by examination of fetal muscle. Reported experiences in this regard are summarised in table III. The recent report14 of an apparently normal fetal S.C.K. level and the subsequent birth of a boy who proved to be affected is very disturbing. Unfortunately no details were given as to whether the fetal blood-sample may have been contaminated by amniotic fluid and/or maternal serum which, in our experience, may affect the fetal S.C.K. level by a factor as high as ten. Nevertheless, our finding of 2 fetuses with apparently normal s.c.K. levels despite abnormal muscle likewise suggests that the fetal s.c.K. may not be a reliable antenatal diagnostic test. In any event, caution should at present be exercised in offering antenatal diagnosis of D.M.D. on the basis of fetal s.c.K. levels. not

The A E.

histological findings

will be

reported in

detail later

by V.D.

and

BBe thank Miss G. Dodds and Miss L. Andruzczenko for excellent technical assistance, and Dr E. Blank, Dr S. Duncan, Dr P. Harper and Prof M. Laurence for sending material which formed the basis

A RAPID TECHNIQUE FOR DISTINGUISHING HERPES-SIMPLEX VIRUS TYPE 1 FROM TYPE 2 BY RESTRICTION-ENZYME TECHNOLOGY D. M. LONSDALE Institute

of Virology, Church Street, Glasgow G11 5JR

rapid simplified technique, based on restriction-endonuclease analysis of D.N.A. is it described; distinguishes radioactively labelled herpes-simplex virus type 1 strains from herpes-simplex virus type 2 unambiguously. Large numbers of isolates can be screened in 4-5 days.

Summary

A

Introduction

recognition of two distinct types of herpessimplex virus, a wide variety of characteristics has been used to distinguish the two. Differences between types are demonstrable by plaque morphology,l serology,2,3 virion4 and infected-cell polypeptides,5 D.N.A. density,6 and more recently by the technique of D.N.A. restrictionendonuclease analysis.’ The D.N.A. isolated from herpes-simplex virus type 1 (H.s.v.-l) and herpes-simplex virus type 2 (H.s.v.-2) consists of linear duplex molecules of approximate molecular weight 100 x 106 daltons.6,8 Certain D.N.A. restrictionSINCE the

endonucleases

double-stranded D.N.A. at with D.N.A. palindromic recognition-sequences; any pure species this cleavage produces a set of fragments, the number and electrophoretic profile of which is highly characteristic for that D.N.A.9 The restriction-endonuclease cleavage patterns for H.s.v.-l and H.s.v.-2 D.N.A. are quite distinct for a whole range of these restriction-endonucleases, and where physical maps have been constructed, 10-12 no coincident restriction cleavage sites have so far been found. The simplified technique described here easily allows one worker to type 50 or more isolates unambiguously as H.s.v.-l or H.s.v.-2 in 4-5 days. cleave