Hypopigmentation in Angelman syndrome

Chromosome region 15q is thought to contain one or more genes that are important for melanin pigment synthesis in the hair, skin, and eyes. Hypopigmentation has been identified in the Prader-Willi (PWS) and Angelman (AS) syndromes. We have examined 6 individuals with AS to further characterize the pigment pattern in this condition. The age of the 5 girls and one boy ranged from 2.4 to 7.0 years. None has obvious albinism. Hair color ranged from light blond to brown. Skin was type I in 3 and type II in 3. Eye changes included nystagmus in 2, strabismus in 4, and reduced retinal pigment in 5. The mean hairbulb tyrosinase activity was 0.37 ± 0.44 pmol/hb/120 min for the individuals with AS, with a range of 0.00 to 1.13 (normal brown control 1.49 ± 0.79, normal blond control 1.50 ± 0.85). Electron microscopic examination of hairbulb melanocytes showed normal melansome and melanocyte architecture and number, but reduced melanin formation, with many stage II and III premelanosomes but few stage IV fully melanized melanosomes. Hypopigmentation characterized by light skin, reduced retinal pigment, low hairbulb tyrosinase activity, and incomplete melanization of melanosomes is part of the phenotype of AS, and is similar to that found in PWS. © 1993 Wiley-Liss, Inc.     

Copper deficiency and pigmentation in the rat: morphofunctional aspects.

The effects of a low copper diet on pigmentation, pigment cell and melanosome morphology have been investigated in ACI/T male rats. After a three months treatment the fur and skin pigmentation is reduced as compared to the controls. The melanocytes of the treated rats show the phenotype of active pigment cells while some melanosomes are abnormally differentiated: both lamellar and granular organelles are present in the same pigment cell and mosaic age melanosomes appear. The abnormal melanosome structure expressed by the treated-rat melanocytes is also evident in vitro. After incubation with deoxycholate the melanosomes from the low-copper diet treated rats are much more altered than those from the control rats. The phenotype of the rats starved for copper seems to mimic as regards pigmentation the phenotype of the mouse Mo (mottled) mutation that is an experimental model of the Menkes' kinky hair syndrome. In conclusion copper deficiency seems to affect both the morphology and function of the pigment cells.     

Immunoprecipitation of melanogenic enzyme autoantigens with vitiligo sera: evidence for cross-reactive autoantibodies to tyrosinase and tyrosinase-related protein-2 (TRP-2)

In the present study we describe the detection of TRP-2 antibodies in vitiligo patients using in vitro³⁵S-labelled human TRP-2 in a radioimmunoassay. Of 53 vitiligo sera examined in the assay, three (5.9%) were found to be positive for TRP-2 antibodies. In contrast, 20 control sera, sera from 10 patients with Hashimoto's thyroiditis and sera from 10 patients with Graves' disease were all negative. All three patients positive for TRP-2 antibodies (mean age 54 years, age range 50–63 years) had had vitiligo of the symmetrical type for more than 1 year and all of them also had an associated autoimmune disorder: Graves' disease in one and autoimmune hypothyroidism in two. In addition, antibodies to the melanogenic enzyme tyrosinase were present in their serum. To examine any immunological cross-reactivity between TRP-2 and tyrosinase, the three vitiligo sera positive for TRP-2 antibodies were preabsorbed with COS-7 cell extract containing either expressed TRP-2 or tyrosinase, and subsequently used in the radioimmunoassay. These absorption studies indicated that preincubation with both proteins inhibited the immunoreactivity of the positive sera in the immunoassay using in vitro translated ³⁵S-TRP-2. This antibody cross-reactivity suggests the humoral response to the two melanogenic enzymes in these patients may not be entirely independent.     

Molecular basis of albinism: mutations and polymorphisms of pigmentation genes associated with albinism

Albinism, caused by a deficiency of melanin pigment in the skin, hair, and eye (oculocutaneous albinism [OCA]), or primarily in the eye (ocular albinism [OA]), results from mutations in genes involved in the biosynthesis of melanin pigment. The lack of melanin pigment in the developing eye leads to fovea hypoplasia and abnormal routing of the optic nerves. These changes are responsible for the nystagmus, strabismus, and reduced visual acuity common to all types of albinism. Mutations in six genes have been reported to be responsible for different types of oculocutaneous and ocular albinism, including the tyrosinase gene (TYR) and OCA1 (MIM# 203100), the OCA2 gene and OCA2 (MIM# 203200), the tyrosinase-related protein-1 gene (TYRP1) and OCA3 (MIM# 203290), the HPS gene and Hermansky-Pudlak syndrome (MIM# 203300), the CHS gene (CHS1), and Chediak-Higashi syndrome (MIM# 214500), and the X-linked ocular albinism gene and OA1 (MIM#300500). The function of only two of the gene products is known tyrosinase and tyrosinase-related protein-1 both of which are enzymes in the melanin biosynthetic pathway. Continued mutational analysis coupled with function/structure studies should aid our understanding of the function of the remaining genes and their role in albinism. Mutation and polymorphism data on these genes are available from the International Albinism Center Albinism Database web site (http://www.cbc.umn.edu/tad).     

Micro-anatomy related antigen expression in melanocytic lesions

The in situ expression of antigens associated with melanosomes (gp-100), pigmentation (PAA), tyrosinase (TRP-1), melanoma (MAA-1/MAA-2), and HLA-DR was investigated immunohistochemically in frozen archival specimens of common acquired melanocytic naevi, in dysplastic melanocytic naevi, and in lymph node metastases of melanoma. Expression of these antigens was also studied in established cultured normal human melanocytes, naevus-derived melanocytes and melanoma cell lines of varying metastatic potential, by immunohistochemistry and flow cytometry. Compared with normal melanocytes, melanocytic naevi exhibited increased expression of gp-100, PAA, and TRP-1 in the lesional cells at or very near the dermo-epidermal junction, but with diminishing expression towards the intra-dermal base of the lesions. In contrast, expression of MAA-1 and MAA-2 was observed in melanocytes throughout the dermal part of the naevi. Melanocytes located at the basal layer of the epidermis were positive only for gp-100, PAA, and TRP-1 antigens. Dysplastic melanocytic naevi showed staining of gp-100, PAA, TRP-1, HLA-DR, MAA-1, and MAA-2 of junctional lesional melanocytes, but less intense than that of common acquired naevi. These antigens were not detectable in the dermal part of the dysplastic naevi. Expression of these antigens in lymph node metastases of melanoma was either positive or negative. Similar results regarding antigen expression were observed in all cultured melanocytic cells, both by immunohistochemistry and by flow cytometry. The present data suggest that analysis of these antigens may contribute to the discrimination of common acquired melanocytic naevi from their dysplastic counterparts. Furthermore, variations in the levels of expression in naevi may be consistently related to the micro-anatomy of the lesions, indicating that the micro-environment may have an influence on the expression levels of these antigens in different lesional melanocytes.     

Neural differentiation in the OTT-6050 mouse teratoma: Enzymatic and immunofluorescence characterization of a tumor fraction showing melanogenesis in neuroepithelial cells

Summary A pigmented tumor fraction, designated IB-9, obtained following cellular dissociation and elutriation procedures applied to the solid transplants of the OTT-6050 mouse teratoma cell line, was characterized enzymatically and by immunofluorescence for the presence of tyrosinase and tyrosine hydroxylase (TH). Enzymatic assays of the pigmented tumors were compared with those obtained on non-pigmented teratoma-derived tumors, on pigmented tumors obtained from the mouse melanoma B16 line as a control for tyrosinase activity, and on whole brains of adult 129/J mice as a control for TH activity.All the teratoma-derived tumors, including the IB-9 fraction, showed a predominance of TH over tyrosinase activity. The levels of TH activity appeared independent of the presence or the extent of melanin pigment. All pigmented teratoma-derived tumors showed low levels of tyrosinase activity.On the basis of the enzymatic assays, the IB-9 tumors were divided into two groups: group I, which showed low enzyme activity, almost certainly entirely tyrosinase; and group II, in which the enzyme activity appeared largely due to TH, with presumably a very low background of tyrosinase activity. Immunofluorescence demonstrated the localization of TH activity to non-pigmented cells of the IB-9 fraction, whereas the pigmented cells showed absence of TH activity.These findings, taken in conjunction with the presence by electron microscopy of premelanosomes and melanosomes, indicate that pigment formation associated with melanosomal differentiation in the neural cells of IB-9 with the histologic patterns of primitive CNS neuroepithelium results from tyrosinase activity only and is therefore unrelated to the metabolic pathways involved in catecholamine synthesis and degradation. It is suggested that, at this stage of differentiation and in this system, the expression of catecholamine synthesis via tyrosine hydroxylase in neuroepithelial cells, and of melanin pigment via tyrosinase, are probably mutually exclusive.Supported by Research Grant CA 11689 of the National Cancer Institute; MH 23861 of the National Institute of Mental Health; and Neuropathology Training Grant NS 5 T32 NS 7111 of the National Institute of Neurological and Communicative Diseases and Stroke, USPHS     

Mammalian tyrosinase-related protein-1 is recognized by autoantibodies from vitiliginous Smyth chickens. An avian model for human vitiligo.

The Smyth line (SL) chicken is an animal model for the human acquired depigmentary disorder vitiligo. Affected birds from this line express a postnatal loss of melanocytes in feather and ocular tissues. This vitiligo-like depigmentation is considered to be a disorder with two interacting components: melanocyte dysfunctions and autoimmune reactions. Previously, SL chicks were shown to express high levels of circulating autoantibodies that bind to chicken melanocyte proteins with molecular masses between 65 and 80 kd. Three mammalian melanocyte proteins known to have isoforms in this molecular mass range are tyrosinase, tyrosinase-related protein (TRP)-1 and TRP-2. Of these, only tyrosinase is reported to be expressed in chicken melanocytes. The results presented in this study indicate that, of these three candidate proteins, TRP-1 is the primary antigen recognized by the SL autoantibodies. SL autoantibodies recognize a chicken melanocyte protein that is different from that of tyrosinase or the candidate chicken TRP-2. In addition, several types of experiments incriminate TRP-1 as the primary mammalian melanocyte antigen recognized by SL autoantibodies. We further verified that chicken melanocytes expressed messages for TRP-1 by finding positive signals on Northern blots of chicken melanocyte RNA probed with mammalian TRP-1 cDNA fragments. Therefore, we conclude from these results that the SL autoantibodies primarily recognize TRP-1 in mammalian melanocytes and suggest that chicken melanocytes express a homologue of TRP-1 (the human gp75 and the murine brown/b locus protein).     

Morphological variations of pigment granules in eyes of the rhesus monkey

Melanin granules of rhesus monkey eyes develop in four stages. Early in stage I, they are small, spherical or elongated, membrane-limited vesicles containing parallel arrays of membranes oriented along the long axis and having regular periodicity. Melanization of these membranes, which is indicated by increased thickness and electron opacity, leads to the formation of stages II and III granules. In stage IV, their internal structure is completely obliterated by electron-dense melanin. During this process of development, melanosomes are transformed from thin, long vesicles into large ovoid bodies. By 60 days of gestation, melanosomes have developed in the pigmented epithelium of the iris, ciliary body, and retina, those in the retina being more mature and fully pigmented than the others. Stromal pigment cells of the uveal tract develop later than those of the pigmented epithelium. Pigment first appears in the iris at 140 days, then in the ciliary body, and lastly (150 days) in the choroid, where only well-developed melanosomes are found. Melanosomes of the stromal pigment cells as well as those of the pigmented epithelium may have their origin in the iris. Early melanosomes (stage II) are present almost exclusively in the iridial cells whereas mature forms are found in the ciliary and retinal cells.     

Choroidal melanocytes - a model for studying the aging process in nonreplicative differentiated cells.

Choroidal melanocytes differentiate and cease to divide early in postnatal life. They have characteristic organelles, the melanosomes, which are formed from tyrosine in a reaction catalyzed by tyrosinase. As animals become old, enzyme activity declines and the melanosomes show changes that are quantitatively and qualitatively correlated to the animal's increasing age.     

Assignments of the tyrosinase related protein-1 and -2 genes to human chromosome bands 9p23 and 13q32.1 by in situ hybridization

To determine the precise chromosomal localization of tyrosine related protein-1 and -2 (TRP-1 and TRP-2) genes by fluorescence in situ hybridization, we used DNAs isolated from human bacterial artificial chromosome clones. They contain genomic sequences with approximately 120 kb inserts for TRP-1 and TRP-2. The TRP-1 and TRP-2 genes were assigned to human chromosome bands 9p23 and 13q32.1, respectively. These results confirmed the previously mapped location for the TRP-1 gene and more precisely located the TRP-2 gene, which had previously been mapped to chromosome 13q31-q32.     

Blue nevus and melanosis of the prostate. Electron-microscopic and immunohistochemical studies

Two cases of blue nevus and one case of melanosis of the prostate were studied with ultrastructural and immunohistochemical methods. All patients complained of urinary obstruction, and the clinical impression in all was benign prostatic hyperplasia. Melanin was present in the stroma of the prostate in all cases. In one, pigment was also demonstrated both in benign and malignant epithelial cells. Electron microscopically, melanosomes in different stages were present in the two white patients, but only mature stage IV melanosomes were demonstrated in the black patient. The melanin in epithelial cells consisted only of mature melanosomes. Immunohistochemically, the stromal cells that contained melanin stained with S-100 protein. The evidence suggests that the pigmented cells in the prostatic stroma are melanocytes and the melanin in the glandular epithelium is a result of the transfer of pigment from the stromal melanocytes.     

Oa1 knock-out: new insights on the pathogenesis of ocular albinism type 1

Ocular albinism type I (OA1) is an X-linked disorder characterized by severe reduction of visual acuity, strabismus, photophobia and nystagmus. Ophthalmologic examination reveals hypopigmentation of the retina, foveal hypoplasia and iris translucency. Microscopic examination of both retinal pigment epithelium (RPE) and skin melanocytes shows the presence of large pigment granules called giant melanosomes or macromelanosomes. In this study, we have generated and characterized Oa1-deficient mice by gene targeting (KO). The KO males are viable, fertile and phenotypically indistinguishable from the wild-type littermates. Ophthalmologic examination shows hypopigmentation of the ocular fundus in mutant animals compared with wild-type. Analysis of the retinofugal pathway reveals a reduction in the size of the uncrossed pathway, demonstrating a misrouting of the optic fibres at the chiasm, as observed in OA1 patients. Microscopic examination of the RPE shows the presence of giant melanosomes comparable with those described in OA1 patients. Ultrastructural analysis of the RPE cells, suggests that the giant melanosomes may form by abnormal growth of single melanosomes, rather than the fusion of several, shedding light on the pathogenesis of ocular albinism.     

Progressive cytologic changes during the development of delayed feather amelanosis and associated choroidal defects in the DAM chicken line. A vitiligo model.

Newly hatched Gallus domesticus chicks of the delayed amelanotic (DAM) line have phenotypically normal down pigmentation. Functioning pigment cells are present in the down plumage, choroid, and retinal pigment epithelium. However, histologic and ultrastructural studies reveal that after hatching regenerating feather melanocytes synthesize melanosomes with abnormal, irregularly shaped surfaces and pigmented extensions. Eventually retraction of melanocytic dendrites and clumping of pigment occurs concomitantly with intracellular compartmentalization of the abnormal melanosomes. Melanocyte degeneration is accompanied by the appearance of mononuclear leukocytes (MNLs) in the pulp of the regenerating feathers. Concurrently, melanocytes cease to migrate into the regenerating feather epithelium, and the result is amelanosis. Changes in choroidal melanocytes are first evident as swelling of cell bodies and associated dendrites. Ultrastructurally, the choroidal melanocytes demonstrate increased cytoplasmic material, melanosomal irregularities, retraction of dendrites, melanosome compartmentalization, and eventual necrosis. Concurrently, MNLs arrive and remove the pigment from the choroid. The authors conclude that a basic melanocyte defect precedes the arrival of immunocytes in the delayed cutaneous and choroidal amelanosis in the genetic DAM vitiligo model of the chicken.     

The predominant defect in dilute melanocytes is in melanosome distribution and not cell shape,supporting a role for myosin V in melanosome transport

Mice with mutations at the dilute locus, which encodes the heavy chain of a type V unconventional myosin, exhibit a reduction in coat colour intensity. This defect is thought to be caused by the absence in dilute melanocytes of the extensive dendritic arbor through which these cells normally deliver pigment-laden melanosomes to keratinocytes. The data on which this conclusion has been based can also be explained, however, by a defect in the outward transport of melanosomes within melanocytes of normal shape. To resolve this question, we compared the shape and pigment distribution within melanocytes present in primary cultures prepared from the epidermis of C57BL/6J pups that were either wild type (D/D) at dilute or homozygous for the dilute null allele d120J. These same comparisons were also performed on melanocytes in situ, where antibodies to the membrane tyrosine kinase receptor cKIT were used to visualize melanocy te cell shape independent of pigment distribution. Wild type melanocytes were found to be dendritic and to have melanosomes distributed throughout their dendrites both in vitro and in situ. Mutant melanocytes were also found to be dendritic in both cases, but their melanosomes were highly concentrated in the cell body and largely excluded from dendrites. We conclude, therefore, that the predominant defect in dilute melanocytes is in melanosome distribution, not cell shape. These results argue that the myosin V isoform encoded by the dilute locus functions in dendritic extensions to move melanosomes from their site of formation within the cell body to their site of intercellular transfer at dendritic tips. This conclusion is consistent with our recent demonstration by immunolocalization that the dilute myosin V isoform associates with melanosomes in mouse melanocytes This revised version was published online in July 2006 with corrections to the Cover Date.     

Melanosomes and MHC class II antigen-processing compartments

Melanosomes are specialized intracellular compartments within melanocytes and retinal pigment epithelial cells that function in the synthesis, storage, and secretion of melanins, which are the major pigments made by mammals. The mechanisms that regulate the formation of melanosomes, and the pathways by which constituent proteins are targeted to them, are related to those involved in the biogenesis of major histocompatibility complex (MHC) class II antigen-processing compartments. Consequently, diseases that affect pigmentation may also affect antigen presentation to T cells. Moreover, many of the tissue-specific proteins that localize to melanosomes and participate in melanin formation double as tumor-associated antigens that are targets for T cells in patients with melanoma. Our studies on melanosome biogenesis are providing new ways of thinking about antigen-processing compartments and the mechanisms regulating presentation of tumor-associated antigens.