Journal
of Cutaneous Pathology 1976: 3: 207-216
Giant and "Granular Melanosomes" in Leopard Syndrome: An Ultrastructural Study JAG BitAWAN, M,D,,i DAVID T , PURTII.O, M , D , , I JOHN A, RIORDAN, M,D,,2 VARUN K , SAXENA, M , D , 3 AND LEI; EDELSTEIN, M , D , I , I,"'
Department of Pathologyt, University of Massachusetts Medical School, Department of Pathology', Dermatology:'' and Neurology^', St, Vincent Hospital, Fallon Clinic-', 'Worcester, Massachusetts, Electron microscopy of lentigines was performed to study the pigmentation abnormality in two children with LEOPARD syndrome. Giant melanosomes similar to those seen in cafe-au-Iait spots of neurofibrotnatosis and nevus spilus were found in a lentiginc from one of our cases. Our results show that "spherical granular melanosomes" described in neurofibroniatosis, are lysosomal-like structures associated with the development of complex melanin granules. Our study also demonstrates Ibat immature melanosomes are present in some keratinocytes of LEOPARD syndrome. This finding is in contrast to the prevailing concept that only mature melanosomes are transferred to keratinocytes. The occurrence of individual mehinosotnes of normal size and shape in keratinocytes of skin in whites with LEOPARD syndrome, suggests that neither the size of melanosomes, nor the racial differences are the factors determining the distribution of melanosomes in keratinocytes, (Received for piibtication Jiity 14, 1976)
T h e LEOPARD syndrome is a clinical en- (Jitnbow et al, 1973), in nevus spilus (Kotity (autosotnal dotninant in inheritance), natd et al, 1974) and in nevoid pigmentafirst delineated by Gorlin ct al, in 1969, tion (Eady & Sparrow 1975), In the second Since then, many cases have been reported case we found "spherical granular melanand recently reviewed by Voron et al, osomes" in a lentigine. This type of me(1976), In most publications, the emphasis lanosome has been reported in the hyperhas been on the clinical features of the pigmentcd macular lesions of neurofibrosyndrome. No ultrastructural studies have matosis (Jimbow et al, 1973), Our findings been done in the past to dctcrtnine the na- indicate that these "spherical granular meture of the pigmentary abnormalities. We lanosotnes" are lysosomes or lysosome-like have studied two cases of LEOPARD syn- structures, al times forming complex medrome with both light and electron micro- lanin granules. The present investigation scopy. In one of our cases, giant melan- also focuses attention on the size, shape osomes were seeti in a lentigine. These and distribution of normal melanosomes in structures have been previously described keratitiocytes of two patients with LEOin cafe-au-lait spots of neurofibroniatosis PARD syndiotne.
208
BHAWAN, PURTILO, RIORDAN, SAXENA AND EDELSTEIN
GIANT AND "GRANULAR MELANOSOMES" IN LEOPARD SYNDROME Materials and Methods
Report of cases Case #/. A 13-year-oId white boy of Lebanese descent had bilateral hearing loss at 5 years of age. His clumsiness resulted in frequent injuries. He had large pigmented tnacules in dermatome distribution o n his left flank, abdomen atid thigh. In t h e large pigtnented areas many small lentigines, varying in size from 1 mm to 3 mm in diameter, could be seen (Fig, 1), He had a child-like affect and low normal intelligence. Bilateral 10 to 15 decibel hearing Ioss of a neural type was noted. He had moderate atrophy of his muscles in the forearm and Iower one-third of the legs, Cerebellar function was normal. Deep tend o n reflexes were tnarkedly depressed. He h a d pes cavus. The external gcnitalia were underdeveloped for his age (Fig, 1), The electrocncephalogratn was reported to be mildly abnormal, showing 8-9 per second activity of average voltage in the occipital leads while awake and scattered sharp and spikitig activity during sleep. Nerve conduction studies in his artns and leas (ulnar and pcroneal) revealed slowing of conduction titncs, A 4 mtn skiti punch biopsy was taken frotn a lentigine in the flank, 'We examined both his parents and seven siblings. The father was found to have impaired hearing and pes cavus, but none of the fatnily tnembers was diagnosed as having the syndrome.
209
Case #2, A 12-ycar-oId white girl was seen because of extensive lentigines on the left side of her face, neck, artns and back (Fig, 2), The lentigines varied in size from 0,1 ctn to 0,8 cm in diatneter. They occurred itl dertnatotnal distributioti and were intensely pigtnented. Areas itnmediately surrounding the lentigines were also hyperpigtiicntcd. Neurologic examination was negative atid no hearing loss or other stigmata of the LEOPARD syndrome were found. She was one of five children. Siblings were apparently normal, A punch skin biopsy specitnen was taken from a lentigine of her left arm. She is being followed for the appearance of other features of the syndrome. Light and Electron Microscopic Studies Each skin biopsy specimen was cut into equal halves. One-half was processed for light microscopy and 5 _// sections were cut und stained with hetnatoxylin and eosin. The other half was immediately fixed for 5 h in 3 % glutaraldehyde in 0,1 M cacodylate buffer for electron microscopy. It was then washed in cacodylate buffer (0,1 M), postfixed in ostnium tetroxide, dehydrated in graded ethanols and etnbedded in Epon 812, Thin sections were cut (after choosing an appropriate area from toluidine blue stained 1 /( thick sections) with a diamond knife on ati LKB Ultratome III, The tliit! sections were stained with uranyl acetate and lead citrate atid examined with a Philips EM 301 electron microscope. The sizes of the tnclanosotncs were measured
Fig. 1- A boy with LEOPARD syndrotne (case # 1), Pigmented macules on left side in dermatome distribution. Closer inspection reveals scattered lentigines (arrows). Note the underdeveloped genitalia. Dressing indicates the site of biopsy. Fig. 2. A girl with LEOPARD syndrome (case #2), showing extensive lentigenosis in dermatome distribution. Fig. 3. Reptesentative area of a lentiginc, A marked inctease in lightly stained melanocytes is seen in the lower layers of the epidermis. Possible giant melanosomes are seen in some cells (arrows). Few pigment containing cells (arrowheads) aic present in dermis (D), X400
210
BHAWAN, PURTILO, RIORDAN, SAXENA AND EDELSTEIN
by determining the maxitnutn diatncter of the largest and smallest melanosomes. Results Light Microscopy Increased numbers of tnclanocytcs were seen in the lower epidertnis and to a lesser extent iti the upper detniis. The melanocytes were either distributed singly or in small nests and stained lighter than the adjacent keratinocytes. This latter feature was best seen in the 1 // thick sections of Epon embedded material (Fig, 3), There was increased pigmentation in epidertnis and large pigtnent granules were seen. An occasional melanocytc was present in the upper dermis. Electron Microscopy Electron microscopy confirtned that the cells, staining lightly in thick sections, were indeed melanocytes (Fig, 4), Langcrhans cells were seen with case and occasionally they were seen also in the upper dermis. In addition to these findings, abnortnalities of melanosome size, shape, internal structure and distribution pattern were seen and are described below, A) Ellipsoidal Melanosomes Ellipsoidal melanosotnes resembling normal melanosomes were the conitnonest fortn of granules in both melanocytes and keratinocytes. Great variations in size (from 0,2 ;/tii to 0,6 /
of Cutaneous Pathology 1976: 3: 207-216
Giant and "Granular Melanosomes" in Leopard Syndrome: An Ultrastructural Study JAG BitAWAN, M,D,,i DAVID T , PURTII.O, M , D , , I JOHN A, RIORDAN, M,D,,2 VARUN K , SAXENA, M , D , 3 AND LEI; EDELSTEIN, M , D , I , I,"'
Department of Pathologyt, University of Massachusetts Medical School, Department of Pathology', Dermatology:'' and Neurology^', St, Vincent Hospital, Fallon Clinic-', 'Worcester, Massachusetts, Electron microscopy of lentigines was performed to study the pigmentation abnormality in two children with LEOPARD syndrome. Giant melanosomes similar to those seen in cafe-au-Iait spots of neurofibrotnatosis and nevus spilus were found in a lentiginc from one of our cases. Our results show that "spherical granular melanosomes" described in neurofibroniatosis, are lysosomal-like structures associated with the development of complex melanin granules. Our study also demonstrates Ibat immature melanosomes are present in some keratinocytes of LEOPARD syndrome. This finding is in contrast to the prevailing concept that only mature melanosomes are transferred to keratinocytes. The occurrence of individual mehinosotnes of normal size and shape in keratinocytes of skin in whites with LEOPARD syndrome, suggests that neither the size of melanosomes, nor the racial differences are the factors determining the distribution of melanosomes in keratinocytes, (Received for piibtication Jiity 14, 1976)
T h e LEOPARD syndrome is a clinical en- (Jitnbow et al, 1973), in nevus spilus (Kotity (autosotnal dotninant in inheritance), natd et al, 1974) and in nevoid pigmentafirst delineated by Gorlin ct al, in 1969, tion (Eady & Sparrow 1975), In the second Since then, many cases have been reported case we found "spherical granular melanand recently reviewed by Voron et al, osomes" in a lentigine. This type of me(1976), In most publications, the emphasis lanosome has been reported in the hyperhas been on the clinical features of the pigmentcd macular lesions of neurofibrosyndrome. No ultrastructural studies have matosis (Jimbow et al, 1973), Our findings been done in the past to dctcrtnine the na- indicate that these "spherical granular meture of the pigmentary abnormalities. We lanosotnes" are lysosomes or lysosome-like have studied two cases of LEOPARD syn- structures, al times forming complex medrome with both light and electron micro- lanin granules. The present investigation scopy. In one of our cases, giant melan- also focuses attention on the size, shape osomes were seeti in a lentigine. These and distribution of normal melanosomes in structures have been previously described keratitiocytes of two patients with LEOin cafe-au-lait spots of neurofibroniatosis PARD syndiotne.
208
BHAWAN, PURTILO, RIORDAN, SAXENA AND EDELSTEIN
GIANT AND "GRANULAR MELANOSOMES" IN LEOPARD SYNDROME Materials and Methods
Report of cases Case #/. A 13-year-oId white boy of Lebanese descent had bilateral hearing loss at 5 years of age. His clumsiness resulted in frequent injuries. He had large pigmented tnacules in dermatome distribution o n his left flank, abdomen atid thigh. In t h e large pigtnented areas many small lentigines, varying in size from 1 mm to 3 mm in diameter, could be seen (Fig, 1), He had a child-like affect and low normal intelligence. Bilateral 10 to 15 decibel hearing Ioss of a neural type was noted. He had moderate atrophy of his muscles in the forearm and Iower one-third of the legs, Cerebellar function was normal. Deep tend o n reflexes were tnarkedly depressed. He h a d pes cavus. The external gcnitalia were underdeveloped for his age (Fig, 1), The electrocncephalogratn was reported to be mildly abnormal, showing 8-9 per second activity of average voltage in the occipital leads while awake and scattered sharp and spikitig activity during sleep. Nerve conduction studies in his artns and leas (ulnar and pcroneal) revealed slowing of conduction titncs, A 4 mtn skiti punch biopsy was taken frotn a lentigine in the flank, 'We examined both his parents and seven siblings. The father was found to have impaired hearing and pes cavus, but none of the fatnily tnembers was diagnosed as having the syndrome.
209
Case #2, A 12-ycar-oId white girl was seen because of extensive lentigines on the left side of her face, neck, artns and back (Fig, 2), The lentigines varied in size from 0,1 ctn to 0,8 cm in diatneter. They occurred itl dertnatotnal distributioti and were intensely pigtnented. Areas itnmediately surrounding the lentigines were also hyperpigtiicntcd. Neurologic examination was negative atid no hearing loss or other stigmata of the LEOPARD syndrome were found. She was one of five children. Siblings were apparently normal, A punch skin biopsy specitnen was taken from a lentigine of her left arm. She is being followed for the appearance of other features of the syndrome. Light and Electron Microscopic Studies Each skin biopsy specimen was cut into equal halves. One-half was processed for light microscopy and 5 _// sections were cut und stained with hetnatoxylin and eosin. The other half was immediately fixed for 5 h in 3 % glutaraldehyde in 0,1 M cacodylate buffer for electron microscopy. It was then washed in cacodylate buffer (0,1 M), postfixed in ostnium tetroxide, dehydrated in graded ethanols and etnbedded in Epon 812, Thin sections were cut (after choosing an appropriate area from toluidine blue stained 1 /( thick sections) with a diamond knife on ati LKB Ultratome III, The tliit! sections were stained with uranyl acetate and lead citrate atid examined with a Philips EM 301 electron microscope. The sizes of the tnclanosotncs were measured
Fig. 1- A boy with LEOPARD syndrotne (case # 1), Pigmented macules on left side in dermatome distribution. Closer inspection reveals scattered lentigines (arrows). Note the underdeveloped genitalia. Dressing indicates the site of biopsy. Fig. 2. A girl with LEOPARD syndrome (case #2), showing extensive lentigenosis in dermatome distribution. Fig. 3. Reptesentative area of a lentiginc, A marked inctease in lightly stained melanocytes is seen in the lower layers of the epidermis. Possible giant melanosomes are seen in some cells (arrows). Few pigment containing cells (arrowheads) aic present in dermis (D), X400
210
BHAWAN, PURTILO, RIORDAN, SAXENA AND EDELSTEIN
by determining the maxitnutn diatncter of the largest and smallest melanosomes. Results Light Microscopy Increased numbers of tnclanocytcs were seen in the lower epidertnis and to a lesser extent iti the upper detniis. The melanocytes were either distributed singly or in small nests and stained lighter than the adjacent keratinocytes. This latter feature was best seen in the 1 // thick sections of Epon embedded material (Fig, 3), There was increased pigmentation in epidertnis and large pigtnent granules were seen. An occasional melanocytc was present in the upper dermis. Electron Microscopy Electron microscopy confirtned that the cells, staining lightly in thick sections, were indeed melanocytes (Fig, 4), Langcrhans cells were seen with case and occasionally they were seen also in the upper dermis. In addition to these findings, abnortnalities of melanosome size, shape, internal structure and distribution pattern were seen and are described below, A) Ellipsoidal Melanosomes Ellipsoidal melanosotnes resembling normal melanosomes were the conitnonest fortn of granules in both melanocytes and keratinocytes. Great variations in size (from 0,2 ;/tii to 0,6 /
