It might be expected that genetic traits

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1 THE JOURNAL OF JNVESTIOATIVE DERMATOLOOY Copyright 1568 by The Williams & Wilkins Co. Vol. 50, No. 1 Printed in U.S.A. A HUMAN PIGMENTARY DILUTION BASED ON A HERITABLE SUBCELLULAR STRUCTURAL DEFECT THE CHEDIAK-HIGASHI SYNDROME* DOROTHY B. WINDHORST, M.D.,t A. S. ZELICKSON, M.D. AND ROBERT A. GOOD, M.D.t Human pigmentation is recognized to be under the control of multiple genetic factors which are poorly understood. On the basis of current understanding of molecular genetics (1) it is reasonable to expect that Variations in type and amount of pigment may reflect Variations in the function of pigment producing enzymes. In classical albinism, this appears to be the case in that the absence of pigment is associated with a failure of tyrosinase activity (2). Ultrastructural studies of albinism indicate that melanocytes arc present and that the cytoplasmic matrix for melanin deposition, the prcmelanosomc, is essentially normal. It might be expected that genetic traits affecting synthesis of the structural framework for the melanin granule would exist and that such defects would influence the phenotypic expression of pigment. Although hereditary non-albino pigmentary dilutions based on structural differences of melanin granules have been known in lower animals for twenty years (see review by Silvers, (3)), no such anomaly has previously been recognized in humans. We feel that the human hereditary (autosomal recessive) condition known as the Chediak-Higashi syndrome represents an example of just such a condition. We furthermore feel that the correlation of the subccllular pigmcntary defect with other widespread manifestations of the syndrome has profound implications in cell biology. * From the Departments of Pediatrics and Dermatology, University of Minnesota, Minneapolis. t Recipient of a Special Fellowship award of the National Institute of Arthritis and Metabolic Diseases. t American Legion Memorial Heart Research Professor of Pediatrics and Microbiology. Aided in support by the National Foundation, American Cancer Society; Graduate School, University of Minnesota; National Institute of Health (T1-AM , CA-05887, HE-06314, NB-02042). Accepted for publication July 20, MATERIALS AND METHODS A ten-year-old child with a well documented Chediak-Higashi (CH) syndrome (4, 5) was available for study. Her hair color was basically dark brown to black and would probably have been similar to her mother's except for the generalized gray-blue overtone (Fig. 1). She had albinotic fundi as well as nystagmus, and her skin was very pale. Because she bad always tended to sunburn easily, she had not spent much time in the sun. An oval, sbghtly elevated thickening on her back showed a suggestion of brown pigmentation. Clinical impression of a nevus was confirmed on biopsy. Biopsy specimens from normal skin and nevus were fixed in 10% formalin, dehydrated, embedded, sectioned, and stained with Masson's ammoniatcd silver nitrate technique (6). One area of abdominal skin was given a minimal erythema dose of ultraviolet light on 5 days out of 7. Tissue from this area was then processed for the evaluation of tyrosinase activity according to the method of Fitzpatrick et al (7). Scalp hair was plucked, immediately fixed in buffered 1% osmium tctroxidc and embedded in Epon 812. The preparations were sectioned with an LKB ultratome and stained with uranyl acetate. An RCA EMU electron microscope was used to examine the grids. The Aleutian strain of mink have been shown to be an animal model of the Chediak-Higasbi syndrome (8), and the pigmentary modification which the Aleutian gene, if present in the homozygous state, exerts on all other genes has been related to large melanin granules in the hair shaft (9). Aleutian and non-aleutian mink were available for study as a consequence of other work being pursued on this syndrome (10, 11), and specimens of their skin were prepared for examination of the follicular pigment forming area. The possibility that one of the well documented non-albinotic pigmentary dilutions which have been described in mice might be a bomologue of the CH-Aleutian gene was also examined. Mice of the genotypes ln ln, d d, p p, pa pa, and si si were obtained from Jackson Laboratories. Connective tissue spreads and skin biopsies were prepared for examination of mast cells and melanocytes respectively. RESULTS As can be seen (Fig. 2), the compound nevus and normal skin contained granular material which took up the silver stain. The localiza-

2 I B V / FIG. 1. Color photographs of the patient and her mother and of a ranch or "dark" color phase mink with a "blue" or Aleutian mink. The Aleutian mink pigment genotype differs from the dark only at the Aleutian locus. Assuming that the child's normal hair color would have been like her mother's, it can be seen that the degree of pigmentary dilution imposed by the Chediak-Higashi gene is similar to that of the Aleutian state. 10

3 -,rfl THE CHEDIAK-HIGASHI SYNDROME 11 I;' ' Qj,j: I S..s... W B S Fie. 2. Section of lightly pigmented nevus removed, from the affected child's back stained with Masson's silver nitrate technique for melanin: a, illustrates the histology of the compound nevus with little deposition of silver (X 40); b is a higher power view of a nest of nevus cells in the dermis showing the sparse silver deposition in a pattern of irregularly large granules (X 250). V tion of this material was compatible with the expected distribution of melanin-producing cells. The amount was quite reduced from what would normally be expected and the size of the individual silver depositions was unusually large. When IJVL-irradiated skin was frozen, sectioned, and incubated with tyrosine, clear evidence of melanin formation at regular intervals along the basal layer was evident (Fig. 3). The pigment was on grossly different sized granules than in the normal (Fig. 4). In addition there was no evidence of melanin in the keratinizing cells. Examination of melanocytes of the plucked hair with the electron microscope revealed that these cells were depositing melanin on premelanosomal structures which were very large when compared to normal. The melaniring granules ranged in morphologic detail from normal in substructure and size to normal but large. Some were grossly irregular with a suggestion of fusion of separate granules in some areas (Fig. 5). The eye revealed relatively normal (though fewer) granules in the choroid, with grossly deficient melanin granules of the retinal pigmented epithelium. In areas of the latter where melanin granules were present, they were again very large (Fig. 6). Hair follicles of the Aleutian mink produce

4 12 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY tw $t4 tct.i.. / itt' 1 Fio. 3. An unstained section of Chediak-iligashi skin taken from an area irradiated with ultraviolet light, sectioned in the cryostat and incubated with tyrosine. A melanocyte showing large melanin granules is present (X 1000). FIG. 4. Normal caucasian skin prepared in the same way as that illustrated in figure 3. Note the relative size of the melanin granules and the wider dispersion among basal cells. melanin granules which are several times as big as those in mink without the Aleutian trait (Fig.7). No giant granules could be seen in tissue granulocytes in the inbred strains of mice which were examined. The variations in melanin granules were compatible with Moyer's findings (26). D150U5510N The Chediak-Higashi syndrome is a condition in which an unusual pigmentary anomaly is associated with abnormally large granules in the leukoeytes and their precursors (12). These two primary features are evident in infancy, and represent the first observable characteristics of this autosomal recessive trait. As the child grows older he manifests an increased susceptibility to infections and may develop an unusual lymphoma. Most children succumb to one or the other of these complications. Kritzlcr (13) has reviewed the reported cases as has Stegmaicr (14). Both Chediak (15) and Higashi's (16) cases were said to be light-skinned and to have lighter hair than their parents, who were Cuban and Japanese, respectively. Some of the children, however, arc reported to have areas of irregularly increased pigmentation (13). All patients for whom eye findings were given except Saraiva's case (17), were said to show albinotic fundi. There was associated photophobia and nystagmus. Apparently because of the fact that these children arc quite regularly lighter in skin and hair color than their genetic constitution

5 THE CHEDIAK-HIGASHI SYNDROME 13 4 i. 'a* "i; 1St e * I" Cc FIG. 5. Electron microscopic study of a melanocyte in a hair bulb of the child pictured in figure 1. Melanin granules which are well pigmented but larger than usual in relation to the mitochondria are evident. In addition, large but incompletely melanised granules are present, showing lamellar substructure. A centriolar area is also seen (X 46,300).

6 14 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY / F St p S * 0'. C A t ;?!T 'H :j -'-a, 4 '' *,; S -J., 1**. FIG. 6. Unstained sections of eyes illustrating melanin distribution in retinal epithelium and choroid. a, A child who died of Chediak-Higashi syndrome, and b, A normal eye of unknown eye color. Chediak-Higashi patients' eyes are usually blue, although children of dark skinned lineage may have brown eyes. Aleutian mink eyes are usually brown.

7 THE CHEDIAK-HIGASHI SYNDROME 15 A FIG. 7a. Unstained sectien of hair follicles of a non-aleutian mink. b. Unstained section of a hair follicle from an Aleutian mink (both X 1000). warrants, and because of their albinotie fundi, the terms partial and semi-albinism have been applied to describe the pigmentary anomaly. In no instance where the pigment defect is discussed in the literature, however, has a complete absence of pigment been reported. In addition, the association of the C-il anomaly with the abnormality of the leukocytes is a constant finding and is a feature not found in any of the documented variations of albinism currently known in man (2, 14). In classical albinism, the failure of tyrosinase activity is the cause of the failure of melanin deposition. This enzyme failure may be primary, or a consequence of

8 16 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY an inhibitory substance, or possibly a combination of factors (18). There is also the possibility of failure of transport of the substrate, tyrosine (19). Melanocytes are present and are producing melanosomes which appear normal in size and shape (20), but melanin is not deposited thereon. Albinism as present in the human is directly comparable with the albinism which can be found throughout lower forms of life. In the ease of the inbred mouse strains, the genetic locus for albinism has been found to have 5 alleles, all of which modify the major color genes (21). This series of alleles appear to exert increasing degrees of depression of the enzyme tyrosinase (22), so that the highest member of the series permits full expression of other color genes and the lowest member exerts full inhibition of tyrosinase activity. The genetics of the coat colors of mink are well known. In these animals, true albinos are known as well as white strains based on different genetic loei. The Aleutian gene is said to be at a separate locus from that for albino, is autosomal recessive, and when present in its homozygous state, operates to modify the manifestations of all other genes expressing color (23). Animals manifesting the Aleutian trait, regardless of their other color phenotype, demonstrate a bluish tinge to their pelt and are called "blues". The other manifestations include a high susceptibility to infection including the (viral) "Aleutian disease" which is a syndrome of hepatitis and hyperglobulinemia which may eventuate in a monoelonal globulin (? myeloma) disease (24) and death. The Aleutian animals have giant granules in their leukoeytes, as well as giant melanin granules and are thus morphologieally and clinically entirely compatible with the Chediak-Higashi syndrome (8, 11). Lutzner et al (9) have clearly defined the Aleutian trait as having abnormal eytoplasmie granules in any cell which produces membranelimited granules, including secretory granules of the stomach, pancreas, pituitary, and scent glands. Insofar as tissues have become available for ultrastruetural study, giant granules have regularly been found in similarly diverse tissues of the Chediak-Higashi syndrome. The abnormal granules appear to be primarily formed by the cell in which they are found rather than from stores of accumulated material as in Tay-Saehs disease, and the other hereditary lysosomal disease as described by Hers (25). In addition, they coexist in the same cell with normal appearing granules (lysosomes or secretory granules depending on the type of cell). It thus appears that the basis for the pigmentary anomaly of the Chediak-Higashi Syndrome can best be regarded in terms of a structural abnormality of the melanosome, perhaps in the lipo-protein building blocks that are thought to be common to all membranes. That such a basic defect might also be reflected in an overall decrease in tyrosinase activity is reasonable since the tyrosinase activity appears to depend on its arrangement on and relation to the structure of the melanosome (26). However, if the genetics of mice and mink can be applied to man, it should be clear that the genetic locus which exerts this effect is distinct from that which produces the classical and apparently primary tyrosinase defect, albinism. How the evident morphologic defect correlates with the clinical color dilution is a question of some speculative interest. As was mentioned above, some secondary overall decrease in tyrosinase activity may be involved. Equally plausible is the possibility that fewer numbers of melanosomes are produced, or that the aggregation of the melanin on larger granules results in a pseudodeficit in color. It is also possible that the abnormal granules are not easily transferred to keratinizing cells (though this does not seem to be true of the hair), or that the abnormal granule has fewer sites for the deposition of the melanin polymer. Finally, the abnormal granule may be treated as a foreign body by the cell and be broken down by cellular autolytie processes or extruded to be taken up and digested by phagoeytes. Preliminary findings in the nevus have given some support to the latter possibility. It is not unexpected that morphologic variation in the structure of the melanin granule should exist as a function of gene expression. Moyer has demonstrated that such occurs in the mouse pigmentary dilutions (26). That none of the easily available inbred mouse coat color dilutions are homologous to the Chediak-

9 THE CHEDIAK-HIGASHI SYNDEOME 17 Higashi-Aleutian trait is predictable on the basis of Moyer's ultrastuctural studies as well as the shortened life span the trait confers. What is unexpected and extremely thought provoking is the interrelationship which this defect reveals between the melanosomes and granules of many other cell lines. All cells of a given organism carry the same genetic information, though only a part of that information is being used by any given cell as it undergoes differentiation and performs its specialized function. All cells use certain common and very primitive bits of the genetic code, i.e. the information necessary to synthesize the enzymes necessary to maintain cell structure and to perform basic metabolic functions. Thus a genetically determined structural defect of lysosomes would a priori be expected to be present in all cells which produce lysosomes. What is not so evident and what the Chediak-Higashi syndrome suggests is that even in manufacturing their ultimate differentiated and specialized structures such diverse cell lines as neutrophilic leukocytes and melanosomes use common synthetic mechanisms and substances and that the specialized aspects are, so to speak, superimposed on a common primary structure. The question of how a defect of subcellular (? membrane) structure can affect the functioning of the organism so as to produce the extreme susceptibility to disease and malignancy exhibited by these children and by the mink is of prime interest. The eventual understanding of the biochemical defect in this syndrome should provide insight into a basic cell capacity. SUMMARY The pigmentary anomaly of the Chediak- Higashi syndrome appears to be attributable to a failure in control of the size of the melanin granules. This directly relates the color defect to the giant granules seen in the leukocytes, leukocyte precursors, renal tubules, neurons, and other cells of children with Chediak-Higashi disease. The Aleutian strain of mink, which is an animal homologue for the CH trait, also have giant melanin granules. In addition, such mink are known to have giant membrane-bound structures in all ccli lines which produce such cytoplasmic organelles, both lysosomal and secretory in nature. The mink trait is carried at a single genetic locus separate from the locus for albinism and affects the phenotypic expression of all other color genes. On the basis of the pigmentary studies and the analogy of the Chediak-Higashi syndrome to the Aleutian tract in mink, we feel that the Chcdiak-Higashi syndrome represents a human pigmentary dilution based on a structural defect in the melanin granule and that it is only distantly related to classical albinism. REFERENCES 1. Watson, J. D.: Molecular Biology of the Gene. Franklin Press, Fitzpatrick, T. B. and Quevedo, W. C., Jr.: Chapter 14, Albinism. The Metabolic Basis of Inherited Disease. Eds., Stanbury, J., Wyngarden, J. and Fredrickson, D. McGraw- Hill, New York, Second Edition Silvers, W. K.: Genes and the pigment cells of mammals. Science, 134: 368, Page, A. R., Berendes, H., Warner, J. and Good, R. A.: The Chediak-Higashi syndrome. Blood, 20: 330, Windhorst, D. B., Zelickson, A. S. and Good, R. A.: Chediak-Higashi syndrome: Hereditary gigantism of cytoplasmic organelles. Science, 151: 81, Manual of Histologic and Special Staining Techniques: p. 103, Armed Forces Institute of Pathology, Second Edition, McGraw- Hill, Fitzpatrick, T. B., Becker, S. ivy., Jr., Lamer, A. B. and Montgomery, H.: Tyrosinase in human skin: Demonstration of its presence and of its role in human melanin formation. Science, 112: 223, Padgett, G. A., Leader, H. W., Gorham, J. H. and O'Mary, C. C.: The familial occurrence of the Chediak-Higashi syndrome in mink and cattle. Genetics, 49: 505, Lutzner, M. A., Tierney, J. H. and Benditt, E. P.: Giant granules and widespread cytoplasmie inclusions in a genetic syndrome of Aleutian mink. Laboratory Investigation, 14: 2063, Windhorst, D. B.: Studies on a hereditary defect involving lysosomal structure (abstract) Fed. Proc., 25: 358, Windhorst, D. B., White, J. G., Zelickson, A. S., Clawson, C. C., Dent, P. B. Pollara, B. and Good, R. A.: The Chediak-Higashi anomaly and the Aleutian trait in mink: homologous defects of lysosomal structure. N. Y. Acad. Sci. Conference on Leukocyte Chemistry and Morphology. Nov. 3 5, White, J. C.: The Chediak-Higashi syndrome: A possible lysosomal disease. Blood, 28: 143, Kritzler, R. A., Terner, J. Y., Lindenbaum, J., Magidson, J., Williams, H:, Preisig, H. and Phillips, G. B.: Chediak-Higashi syndrome cytologic and serum lipid observations in a case and family. Am. J. Med., 36: 583, Stegmaier, 0. C. and Schneider, L. A.:

10 18 THE JOUENAL OF INVESTIGATIVE DEEMATOLOGY Chediak-Higashi syndrome. Arch. Derm. 91:1, Chediak, M.: Nouvelle anomalie leucocytaire de earactere constitutional et familial. Rev. Hemat., 7: 362, Higashi, 0.: Congenital gigantism of peroxidase granules. Tohoku J. Exp. Med., 59: 315, Saraiva, L. G., Azevedo, M., Correa, J. M., Carvalho, G. and Prospero, J. D.: Anomabus panlenkoeytie granulation. Blood, 14: 1112, Chian, L. T. Y. and Wilgram, George F., Tyrosinase inhibition: Its role in suntanning and in albinism. Science, 155: 198, Kugelman, T P. and Van Scott, E. J.: Tyrosinase activity in melanocytes of human albinos. J. Invest. Dermat., 37: 73, Barnieot, N. A. and Birbeek, M. S. C.: The electron microscopy of human melanocytes. The Biology of Hoir Growth, p Eds. Montagna W. and Ellis, R. A. Academic Press, New York, Billingham, R. E. and Silvers, W. K.: The melanoeytes of mammals. Quart. Rev. Biol., 35: 1, Coleman, D. L.: Effect of gene substitution on the incorporation of tyrosine into the melanin of mouse skin. Arch. Bioehem. Biophys., 96: 562, Shaekleford, R. M.: Genetics of the Ranch Mink. Pilsbury Publishers, New York, Porter, D. D., Dixon, F. J. and Larsen, A. E.: The development of a myeloma hke condition in mink with Aleutian disease. Blood, 25: 736, Hers, H. G.: Inborn lysosomal diseases. Gastroenterology, 48: 625, Moyer, F. H.: Genetic effects on melanosome fine structure and ontogeny in normal and malignant cells. Ann. N. Y. Aead. Se., 100: 584, The eye sections were obtained through the courtesy of Douglas Johnson, M.D. and Robert H. Monahan, M.D., Division of Opthalmology, Department of Surgery, University of Minnesota. They have reported the eye findings in this autopsy previously: Johnson, D. L., Jacobson, L. W., Toyama, R. and Monahan, R. H., Histopathology of the eyes in Chediak-Higashi Syndrome. Arch. Opthal., 75: 84, 1966.