Markedly Reduced Activity of Lysyl Oxidase in Skin and Aorta from a Patient with Menkes' Disease Showing Unusually Severe Connective Tissue Manifestations1
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003 1-3998/90/2802-0 137$02.00/0 PEDIATRIC RESEARCH Vol. 28. No. 2. 1990 Copyright 0 1990 International Pediatric Research Foundation. Inc. Prin~edin C! S..4 Markedly Reduced Activity of Lysyl Oxidase in Skin and Aorta from a Patient with Menkes' Disease Showing Unusually Severe Connective Tissue Manifestations1 PETER M. ROYCE AND BEAT STEINMANN ABSTRACT. In Menkes' disease, a severe disturbance of (15-17). the concept has arisen of a functional intracellular copper handling appears to render copper unavailable for copper deficiency in which. despite even elevated levels of intra- copper-requiring processes. We have measured the activity cellular copper, copper is unavailable to copper-dependent en- of lysyl oxidase, the copper-dependent enzyme that initi- zymes (2. 4. 14. 18). ates the cross-linking of collagen and elastin, in extracts Most features of the disorder are explicable on the basis of of skin and aorta obtained at autopsy from a patient with impaired function of copper-dependent enzymes (4, 14, 18). unusually marked connective tissue manifestations, and Thus, the connective tissue abnormalities were logically proposed found it to be only 6-12% of normal, thus suggesting a as being principally the result of deficient crosslink formation of basis for these alterations. (Pediatr Res 28: 137-141,1990) collagen and elastin consequent upon decreased activity of lysyl oxidase (1 3, 19), the copper-dependent enzyme that functions to Abbreviations initiate the process through the oxidative deamination of the 6- NH2 group of lysyl or hydroxylysyl residues within the nonhelical EMEM, Eagle's minimum essential medium with Earle's telopeptides of the collagen a-chains, or of specific lysyl residues salts, containing 10% (vol/vol) FCS in elastin, to form the aldehydic precursors of the cross-links (20-22). The activity of lysyl oxidase in cultures of fibroblasts derived from a number of patients with Menkes' disease has previously been reported to be reduced quite considerably (19, 23). We Menkes' disease (MIM 30940 in ref. I) is an X-linked. reces- recently had the opportunity to study a patient with unusually sively inherited disorder in which, although the precise nature of severe connective tissue manifestations, and report here the first the basic defect remains unknown, disturbed copper handling determinations of tissue lysyl oxidase activity in the disorder. In appears to render copper unavailable to copper-requiring proc- extracts of both skin and aorta obtained at autopsy, lysyl oxidase esses (2-4). It has an estimated frequency of 1 in 50 000 to 100 activity was drastically reduced to 6-14% of control values. Quite 000 live births (4) and. although a mild form of the disorder does surprisingly, despite this, the solubility of collagen in the two appear to exist (5-7). and despite the longer survival of a small tissues appeared to be no greater than normal. This finding is. proportion of cases both with and without copper therapy (4, 8- nevertheless, in accord with ultrastructural evidence indicative 1 1). death usually occurs before 3 y of age, most frequently from of a far greater disruption of elastin than collagen (24, and respiratory infections such as pneumonia (3, 4). The disorder is Baccarani-Contri M, Pasquali-Ronchetti I, Royce PM, Stein- characterized principally by growth retardation, progressive cer- mann B, unpublished observations). ebral degeneration, distinctive facial features, hypopigmentation ofskin and hair, a tendency to recurrent episodes of hypothermia, CASE REPORT and abnormal hair (3. 4). There are also connective tissue alter- ations, which include bone changes reminiscent of those seen in A detailed case report has already appeared (25). the essential scurvy, Wormian bones in the skull, and arterial abnormalities features of which are as follows. The patient was the 2nd child that may lead to thrombosis and rupture, although these features of young, healthy, unrelated parents, who had previously had a of the disorder are not usually the most prominent (3.4). clinically normal daughter. He was delivered normally at 37.5 Serum levels of copper and ceruloplasmin are greatly below wk, weighed 2.94 kg, and was 51 cm in length. His skin was normal, and although this was initially regarded simply as the noted to be dry, with little s.c. fat, and lax over the whole body, result of intestinal malabsorption of copper (12, 13), Menkes' particularly the hands and feet. An episode of hypothermia on disease has proved to be extremely recalcitrant to parenteral the 2nd day of life necessitated a 24-h spell in an incubator. On copper therapy (4, 14). After the demonstration of a copper the 6th day of life, it was noticed that both humeri and the right accumulation phenotype in cultured fibroblasts from patients clavicle and second rib were fractured. Radiography of the right arm showed diminished bone density. and numerous Wormian Received Novemhcr 29. 1989: accepted March 9. 1990. Correspondence: Dr. P. M. Royce, Ston\vechselahteilung. Kinderspital. Stein- bones were apparent in the skull. At about 10 wk of age. he wiesstrasse 75, CH-8032 Zurich, Switzerland. became apathetic and took his feed poorly, and shortly afterwards Supported hy Grants 3.905.083 and 3.905.086 from the Swiss National Science suffered convulsions, at first focal. and later generalized. He was Found:~tion. taken into hospital, where he remained until his death at approx- I Presented in part at the 19th Annual Meeting of the SWISSSocieties for Experimental Biology. Lausanne. Switzerland [Experientia 1987, 43:667(abstr)] imately 11 mo of age. Serum copper was undetectable. and and the Annual Meeting of thc European Society for Pediatric Research. Padova. ceruloplasmin virtually undetectable. Cranial computerized to- Italy [Pediatr Res 1987. 22:237(ahstr)]. both in 1987. mography showed progressive cerebral atrophy and subdural 138 ROYCE AND STEINMANN I hematomas. Scalp hairs were sparse and twisted. Additional standing the extracts for 24 h, they were dialyzed exhaustively indications of generalized connective tissue involvement were against 0.5 M acetic acid, lyophilized, and subjected to SDS- bladder diverticula and diffuse arterial anomalies consisting of PAGE, as previously described (31). an intimal hyperplasia associated with a duplication and frag- Further samples of skin and aorta were cut finely with a scalpel, mentation of the internal elastic lamina, particularly in the case homogenized in 1 M NaCI/O.O5 M Tris HCI, pH 7.4 at 4°C with of the aorta and coronary arteries. The cerebral arteries were a Polytron homogenizer (3 times for 15 s), then extracted se- markedly tortuous. quentially with this solution, 0.5 M acetic acid, and 8 M urea, for 24-h periods (23). The extracts were dialyzed exhaustively against 0.5 M acetic acid, then both they and the insoluble MATERIALS AND METHODS residue were lyophilized and hydrolyzed in 6 M HCI at 110°C Tisstie~and cc4ls. Skin and aorta obtained at autopsy of the for 24 h. Hydroxyproline in the hydrolysates was determined by patient and controls were stored at -70°C until used. Fibroblast automated amino acid analysis. cultures were established from skin biopsies of the patient and each of his parents by Prof. G. Pescia, Centre Hospitalier Univ- RESULTS ersitaire Vaudois, Lausanne, Switzerland. Cultures were also I established from skin biopsies of control subjects, and grown in Copper incorporation studies with cliltured Jihrohlasts. The EMEM. "Contrast" fibroblast lines from two patients with combination of the patient's clinical symptoms and the virtual Menkes' disease were purchased, one each from the Human undetectability of serum copper and ceruloplasmin when inves- Genetic Mutant Cell Repository, Camden, NJ (cell line GM tigated at about 6 mo of age was suggestive of Menkes' disease. 1981) and the American Type Culture Collection, Rockville, The diagnosis was firmly established by the results of h4C~uptake MD (cell line CRL 1230). studies on cultured fibroblasts (32). kindly performed by Dr. T. Determination c?f copper content uf tissues. Samples of skin Tonnesen of the John F. Kennedy Institute, Glostrup, Denmark. and aorta were dried to constant weight and dissolved in concen- which show (Table 1) that over a 20-h period, the copper uptake trated HNO, (Aristar grade; BDH Chemicals, Poole, England) of fibroblasts from the patient falls in the middle of the range for (26). Copper in the solutions was measured using a Perkin-Elmer Menkes' patients. The results also show that although the father model 5000 atomic absorption spectrophotometer equipped with is normal, the mother is quite clearly heterozygous for the an HGA 500 programmable graphite furnace (Perkin-Elmer, disorder. Nonvalk. CT). Copper content oftissues. The copper content af both skin and Lysyl osidase assay. Before extraction of lysyl oxidase, S.C. fat aorta obtained from the patient at autopsy was increased, in the was scraped away from skin and aorta was cleaned of adherent case of the former to about 1.5 times normal, and in that of the tissue. Tissues were homogenized as previously described (27), latter by -2-fold (Table 2). except that only a single extraction with first PBS and then 4 M L.vs.vl oxidase activi1.v in fibroblast czrltures. It has been well urea/0.05 M Tris HCI, pH 7.7 at 4°C was performed. established that in skin fibroblast cultures 90% or more of the Fibroblasts were grown in 75 cm' flasks in EMEM until they total lysyl oxidase activity is present in the medium (23, 33, and had just reached confluent density. The cell layers were washed Royce PM, unpublished results). Consequently, lysyl oxidase twice with serum-free EMEM and then incubated in 5 mL of activity was determined only in serum-free medium in which this medium supplemented with ascorbic acid (50 pg/mL) for 24 fibroblasts had been cultured.