CELLULAR METACHROMASIA, A GENETIC MARKER FOR STUDYING THE MUCOPOLYSACCHARIDOSES

241

the rises in the excretions ofH.K. and x.A. are usually much greater than that of H.A. The abnormal metabolism of tryptophan by 13 of the 20 patients who were studied after treatment by mastectomy alone may be a reflection of an increased secretion of oestrogen or, as has been suggested by Hayward (1964), The increased a defective production of androgens. excretions of tryptophan metabolites could not have been a consequence of mastectomy since I have also observed them in untreated patients with carcinoma of the breast

restricted to the skeletal system, seems to be an exception, and this suggests that the abnormal gene is suppressed in skin fibroblasts in this condition. Although these studies have been concerned primarily with the inherited disorders of mucopolysaccharide metabolism, the methodology appears applicable to other genetic disorders, associated with an intracellular accumulation of a normal or abnormal metabolic product. Introduction

THE inherited disorders of mucopolysaccharide (unpublished). metabolism are, by convention, classified into six separate 12 of the 24 patients who had been treated by oophorecsyndromes based on clinical, biochemical, and genetic tomy and mastectomy excreted low amounts of one or more of the three metabolites. These results suggest that studies; in proposing his classification, McKusick (1965) the removal of oestrogen activity brings about a reduction suggested that the heterogeneity existing in this group of in the capacity for conversion of tryptophan to nicotinic mucopolysaccharide disorders could be further elucidated acid. Lojkin (1956) has shown that oophorectomy causes by investigations at the cellular level. We have shown a marked drop in the urinary excretions of Nl-methyl(Danes and Beam 1966a) that under certain conditions the nicotinamide and nicotinic acid in rats, and that this genetic defect can be recognised in cell culture of skin change is reversed by the administration of oestrone and fibroblasts from patients with the Hurler (1919) and progesterone. 8 of the oophorectomised patients had Hunter (1917) syndromes. When the fibroblasts of raised metabolite excretions. This could have resulted patients with these diseases were stained with the metafrom an increase in oestrogen production by the adrenal chromatic dye toluidine-blue 0, the cytoplasm stained red glands, which has been shown to occur in a proportion of indicating the presence of increased cellular mucowomen after oophorectomy (Bulbrook et al. 1958). polysaccharides. Moreover, the degree of cellular metachromasia correlated with the intracellular mucoI thank Dr. G. M. King for allowing me to study his patients. polysaccharide content determined quantitatively (Danes REFERENCES and Bearn 1966b). We have extended these observations Boyland, E., Williams, D. C. (1955) Biochem. J. 60, p. 5. to the other known mucopolysaccharidoses and suggest M. biol. 985. R. Chem. (1956) J. 219, Brown, R., Price, J. Satter, E. J., Wear, J. B. (1960) Acta Un. int. Cancr. 16, 299. that cellular metachromasia can be used as a genetic Wear, J. B. (1955) Proc. Am. Ass. Cancer Res. 2, 7. marker for investigating the mode of inheritance of these Bulbrook, R. D., Greenwood, F. C., Hadfield, G. J., Scowen, E. F. (1958) — —

— —

Br. med. J. ii, 7. L. (1964) Br. J. Surg. 51, 224. Heeley, A. F. (1965) Clin. Sci. 29, 465. Khalafallah, A. S., Abul-Fadl, M. A. M. (1964) Br. J. Cancer, 18, 592. Lojkin, M. E. (1956) J. Nutr. 59, 443. Price, J. M. (1958) Univ. Mich. med. Bull. 24, 461. Brown, R. R., Curreri, A. R., McIver, F. A. (1955) Clin. Res. Proc. 3, 201. Rose, D. P. (1966) Clin. Sci. 31, 265. Walsh, M. P. (1965) Clin. chim. Acta, 11, 263.

Hayward, J.

—

CELLULAR METACHROMASIA, A GENETIC MARKER FOR STUDYING THE MUCOPOLYSACCHARIDOSES B. SHANNON DANES M.D.

Columbia,

Ph.D. Iowa

ASSISTANT PROFESSOR AND ASSOCIATE PHYSICIAN, THE ROCKEFELLER UNIVERSITY, NEW YORK CITY

diseases. Genetic Material and Methods Several families with different genetic mucopolysaccharidoses (see table) have been investigated. Although the patients were diagnosed on clinical and radiological grounds, the urinary excretion of mucopolysaccharides was determined in all patients except those with the Morquio syndrome (Brailsford 1929, Morquio 1929). Specimens of skin were obtained from patients, from certain relatives considered to be carriers by pedigree studies, and from healthy individuals. The method used to establish the cell lines, the preparation of cytological slides, and the subsequent staining of the cells with the metachromatic dye, toluidine-blue 0, have been described in detail elsewhere (Danes and Beam 1966a). Cytological evaluation of all preparations was based on the examination of a hundred fields each containing approximately 100 cells. Since the fibroblast cultures derived from skin of healthy individuals showed essentially no cellular metachromasia (Danes and Beam 1966a), the results have been recorded as either positive or negative.

ALEXANDER G. BEARN M.D. Lond., M.R.C.P., M.R.C.P.E PROFESSOR AND CHAIRMAN, DEPARTMENT OF MEDICINE, UNIVERSITY MEDICAL COLLEGE, AND PHYSICIAN-IN-CHIEF, YORK

From the

CORNELL

THE NEW

HOSPITAL, NEW YORK CITY

Rockefeller University and the Rockefeller University Hospital, New York City

Cell culture of skin fibroblasts seems to offer a means by which inherited disorders of mucopolysaccharide metabolism may be investigated. Since skin fibroblasts are readily available by biopsy and grow well in culture, a cell involved in the metabolic defect can be examined under controlled conditions. Cellular metachromasia, as evidenced by red cytoplasm on staining with toluidine-blue O, seems to be a reliable genetic marker for investigating mucopolysaccharidoses. The Morquio syndrome, in which the abnormalities are Summary

METACHROMASIA OF SKIN FIBROBLASTS GROWN IN CELL CULTURE FROM FAMILIES WITH THE GENETIC MUCOPOLYSACCHARIDOSES

242

(see table). The fibroblast cultures derived from presumed carriers in the same families similarly showed no increase in cellular meta-

Fig. 1—Pedigree of two patients with Hurler’s syndrome (autosomal recessive). The signs (positive and negative) inside the male (square) and female (circle) symbols indicate whether the skin fibroblasts in cell culture showed increased intracellular metachromasia. The small square symbol represents a male stillbirth.

chromasia. Two families with individuals with the Morquio syndrome in association with clinical features of the Hurler syndrome were investigated. In the first family (fig. 3) the affected boy had, in addition to the skeletalde formities associated with the

Morquio syndrome, Results

Skin fibroblasts grown in culture from patients with the Hurler, Hunter, Sanfilippo (Sanfilippo et al. 1963), and Scheie (Scheie et al. 1962) syndromes all contained metachromatic granules (see table). The number of fibroblasts showing metachromatic granules was approximately 60-100°and was relatively constant in replicate cultures derived from different biopsy specimens from the individual. No difference in the size or distribution of metachromatic granules in fibroblasts derived from individuals with the different forms of mucopolysaccharidoses could be detected. In these same families metachromatic granules were seen in cultures of fibroblasts derived from members who seemed clinically normal but who were considered, on the basis of pedigree, to be heterozygous for the abnormal gene (figs. 1 and 2). There seemed to be no quantitative difference in the metachromatic granules in the affected individual and the unaffected carriers. Six families with individuals affected by the Morquio syndrome with involvement of the skeletal system and who showed no clinical evidence of the Hurler syndrome have been examined. The skin fibroblasts from all six affected individuals showed no cellular metachromasia

bilateral corneal clouding, Reilly bodies (Reilly 1941) in the leucocytes of the peripheral blood, and severe mental retardation. In the second family, the affected boy had the typical features of the Morquio syndrome and, in addition, an elongated, shallow sella turcica and X-ray evidence of a widening of the midshaft of the humeri-features

same

Fig. 3-Pedigree of patient with Morquio’s syndrome and some clinical signs of Hurler’s syndrome (autosomal recessive). The signs (positive and negative) inside the male and female symbols indicate whether the skin fibroblasts in cell culture showed increased intracellular metachromasia.

characteristic of the Hurler syndrome. The skin-fibroblast cultures from both affected individuals studied showed cells packed with metachromatic granules. Cultures derived from presumed carriers by pedigree studies showed increased cellular metachromasia

(fig. 3). Discussion

Following the demonstration that the genetic disorders of the Hurler and Hunter syndromes persisted in cell culture of skin fibroblasts (Danes and Beam 1966a), the possibility arose that cell culture might be used to study other mucopolysaccharide

Fig. 2-Pedigree of three patients with Hunter’s syndrome (X-linked recessive). The signs (positive and negative) inside the male and female symbols indicate whether the skin fibroblasts in cell culture showed increased intracellular metachromasia. The small square symbols represent male stillbirths.

disorders. The present results indicate that cellular metachromasia of skin fibroblasts in culture can be used to trace the expression of the abnormal gene for all the mucopolysaccharidoses except the Morquio

syndrome. In the Morquio syndrome, which involves the skeletal system only, the skin

243

BACTERIOLOGICAL FINDINGS IN

This not show cellular metachromasia. observation raises the possibility that Morquio’s syndrome without somatic changes involving other tissues should not be classified as a generalised mucopolysaccharidosis.

fibroblast did

ACUTE OTITIS MEDIA

J. V. DADSWELL M.B.

Patients with the clinical picture of the Morquio syndrome have been reported to excrete an increased amount of keratosulphate in the urine (Pedrini et al. 1962, Maroteux and Lamy 1963). Since keratosulphate is normally found only in cornea, cartilage, and growing bone (Shetlar and Masters 1955, Kaplan and Meyer 1959), it was not altogether surprising to find that the skin fibroblast from an affected individual did not show increased intracellular mucopolysaccharides. If the biopsy specimen from the affected individual or from a known carrier had been obtained from the tissue which synthesises keratosulphate, intracellular metachromasia might have been found.

Lond., M.C.Path.

From the Department of Pathology and Bacteriology, Institute of Laryngology and Otology, London

studies were made on 113 Summary Bacteriological with acute otitis media seen at

patients

the Royal National Throat, Nose, and Ear Hospital, London, between December, 1963, and April, 1964. The organisms most frequently isolated from the ears were &bgr;-hæmolytic streptococci, Staphylococcus pyogenes, Streptococcus pneumoniœ, and Hœmophilus influenzœ. Penicillin is the antibiotic of first choice in treatment, with erythromycin a useful alternative. Introduction

Several investigators (Wiedemann 1954, Robins et al. 1963, Maroteux and Lamy 1965, McKusick et al. 1965) have reported cases of Morquio’s syndrome in which many of the signs and symptoms of Hurler’s syndrome could be found. In the two instances reported here, skin fibroblast cultures from the affected individuals and known carriers showed increased cellular metachromasia (fig. 3, table) and reflected the presence of the abnormal gene. Whereas the six cases of Morquio’s syndrome in which traits were confined to the skeletal system did not show cellular metachromasia. This research was supported by a grant from The National Foundation and supported (in part) by Public Health Service grant

ACUTE otitis media remains a common condition; Fry (1961), from a study of cases in his practice, considered it likely that about a quarter of all children in Great Britain

least one attack. differ as to the incidence of complications. In Reports the series reported by the Medical Research Council (1957), Fry (1961), and Neil et al. (1966) complications were uncommon, but Lowe et al. (1963) found a significant degree of deafness in 25%of their patients six months after an attack. In a preliminary follow-up survey of patients with acute otitis media seen at the Royal National Throat, Nose, and Ear Hospital, London, Shalom and no. Fr-00102 and from the General Clinical Research Centers Sharp (1966) found that, of 36 cases seen some six months Branch of the Division of Research Facilities and Resources. We are after their attack, 2 had serous otitis, and the remainder j most grateful to Dr. R. Archibald, Dr. M. Bryson, Dr. M. Grumbach, showed no evidence of continuing ear trouble. " Glue Dr. G. Jervis, Dr. V. McKusick and Dr. H. Thuline for referring ear " is considered to be more common than formerly and to Miss Dillon for invaluable technical to us, patients Sylvia assistance. (Lancet 1959), possibly because of inadequate antibiotic treatment of otitis or the prophylactic use of antibiotics in for should be addressed to B. S. the D., reprints Requests Rockefeller University, New York, N.Y. 10021, U.S.A. upper-respiratory-tract infections. Adequate antibiotic therapy depends on knowing the REFERENCES nature of the infecting organisms. Studies to determine Brailsford, J. F. (1929) Am. J. Surg. 7, 404. this have given differing results. In this country FriedDanes, B. S., Beam, A. G. (1966a) J. exp. Med. 123, 1. (1966b) ibid. 124, 1181. mann (1957), Morrison (1961), and McNeill (1962) have Hunter, C. (1917) Proc. R. Soc. Med. 10, 104. tended to emphasise the significance of Staphylococcus Hurler, G. (1919) Z. Kinderheilk. 24, 220. Kaplan, D., Meyer, K. (1959) Nature, Lond. 183, 1267. pyogenes in this condition, whereas in reports from the McKusick, V. A. (1965) Circulation, 31, 1. U.S.A. and Scandinavia (Bjuggren and Tunevall 1952, Kaplan, D., Wise, D., Hanley, W. B., Suddarth, S. B., Sevick, M. E., Maumanee, A. E. (1965) Medicine, Baltimore, 44, 445. Lahikainen 1953, Mortimer and Watterson 1956, Gronroos Maroteux, P., Lamy, M. (1963) Presse méd. 71, 2091. et al. 1964) this organism was not found to be prominent Pediat. 312. (1965) J. 67, in the causation of acute otitis media. Morquio, L. (1929) Bull. Soc. Pediat. Paris, 27, 145. Pedrini, V., Lenuzzi, L., Zambotti, V. (1962) Proc. Soc. exp. Biol. Med. The present investigation was undertaken to see whether 110, 847. Reilly, W. A. (1941) Am. J. Dis. Child. 62, 489. any significant change had occurred in the type of causal Robins, M. M., Stevens, H. F., Linker, A. (1963) J. Pediat. 62, 881. organisms, and in this light to review antibiotic therapy. —

suffered

at

—

—

,

—

—

Sanfilippo, S. J., Podosin, R., Langer, L., Good, R. A. (1963) ibid. 63, 837. Scheie, H. G., Hambrick, G. W. Jr., Barnes, L. A. (1962) Am. J. Ophth. 53, 753.

Shetlar, M. R., Masters, Y. F. (1955) Proc. Soc. exp. Biol. Med. 90, Wiedemann, H. R. (1954) Mschr. Kinderheilk. 102, 136.

31.

"... The ballast of factual information, so far from being just about to sink us, is growing daily less. The factual burden of a science varies inversely with its degree of maturity. As a science advances, particular facts are comprehended within, and therefore in a sense annihilated by, general statements of steadily increasing explanatory power and compass-whereupon the facts need no longer be known explicitly, i.e., spelled out and kept in mind. In all sciences we are being progressively relieved of the burden of singular instances, the tyranny of the particular. We need no longer record the fall of every apple." -Sir PETER MEDAwAR, The Art of the Soluble, p. 114. London,1967.

Materials and Methods of a series of 113 patients clinically consists study diagnosed as acute otitis media (with or without mastoiditis) who were seen at the Royal National Throat, Nose, and Ear Hospital over a period of five months (December, 1963, to April, 1964). Their ages ranged from 3 months to 62 years. In 101 cases the disease arose in a previously healthy ear, and in 12 cases the acute episode was superimposed on chronic suppurative otitis media. In all cases either there was a This

discharging

ear or

myringotomy

was

performed.

A swab of the discharge (or fluid from the myringotomy) was obtained and seeded on to the following media: two plates of blood-agar, one plate each of gentian-violet agar, chocolateagar, and MacConkey agar. The swab was then placed in cooked-meat medium. All were incubated aerobically overnight at 370C except one blood-agar plate which was incubated anaerobically. The following day two further blood-agar plates