Tyrosinase (TYR) gene sequencing and literature review reveals recurrent mutations and multiple population founder gene mutations as causative of oculocutaneous albinism (OCA) in Pakistani families.

PURPOSE: To investigate eight previously unreported Pakistani families with genetically undefined OCA for mutations in TYR. METHODS: Sanger sequencing of TYR has been performed in eight families with OCA phenotype. Mutation analysis was performed to establish the pathogenic role of novel mutation. Bioinformatics analysis was performed to predict the structural and functional impacts on protein due to the mutation. RESULTS: In this study, we identified six likely pathogenic variants of TYR (c.272 G>A, c.308 G>A, c.346C>T, c.715 C>T, c.832 C>T and c.1255 G>A), including one novel variant (c.308 G>A; p.Cys103Tyr), segregating as appropriate in each family. Cys103 lies in the highly conserved region of the tyrosinase enzyme, and p.Cys103Tyr is predicted to disturb enzymatic function via alteration of the configurational orientation of TYR leading to a more rigid polypeptide structure. We have also reviewed the mutation spectrum of TYR in Pakistani ethnicity. Published data on OCA families proposed that ~40% have been associated with genetic variations in the TYR gene. The mutations reported in this study have now been described with varying frequencies in Pakistani families, including very rare/unique mutations. CONCLUSION: A literature review of TYR gene mutations in Pakistani populations, combined with our genetic data, identified a number of gene mutations likely to represent regional ancestral founder mutations of relevance to Pakistani populations, in addition to sporadic and recurrent 'hotspot' mutations present repeatedly in other regions worldwide.

Oral levodopa rescues retinal morphology and visual function in a murine model of human albinism.

Albinism is a group of disorders characterized by pigment deficiency and abnormal retinal development. Despite being a common cause for visual impairment worldwide, there is a paucity of treatments and patients typically suffer lifelong visual disability. Residual plasticity of the developing retina in young children with albinism has been demonstrated, suggesting a post-natal window for therapeutic rescue. L-3, 4 dihydroxyphenylalanine (L-DOPA), a key signalling molecule which is essential for normal retinal development, is known to be deficient in albinism. In this study, we demonstrate for the first time that post-natal L-DOPA supplementation can rescue retinal development, morphology and visual function in a murine model of human albinism, but only if administered from birth or 15 days post-natal age.

A Japanese Family With Autosomal Dominant Oculocutaneous Albinism Type 4.

Purpose: We report the clinical characteristics of a Japanese family with autosomal dominant oculocutaneous albinism and a SLC45A2 gene mutation. Methods: A total of 16 members of a Japanese family with general hypopigmentation and foveal hypoplasia underwent detailed clinical examinations. We evaluated the severity of foveal hypoplasia using spectral-domain optical coherence tomography (SD-OCT) and graded it according to the criteria of Thomas et al. DNA was extracted from 17 family members and used for genome-wide single nucleotide polymorphism genotyping and linkage analysis. Mutational search was performed for the SLC45A2 gene responsible for oculocutaneous albinism type 4 (OCA4). Results: All 16 patients exhibited hypopigmentation of their hair and/or iris. They showed foveal hypoplasia, including 3 patients with grade 1 foveal hypoplasia, 7 with grade 2, and 6 with grade 3. No patient had grade 4 foveal hypoplasia. Optical coherence tomography showed macular ganglion cell complex thinning in the temporal area, and a slight reduction of visual field sensitivity in the centrotemporal area. A maximum multipoint parametric logarithm of the odds (LOD) score of approximately 2.00 to 3.56 was obtained on chromosome 5, spanning approximately 7.2 Mb between rs13187570 and rs395967 that included the SLC45A2 gene. All affected members showed a novel heterozygous variant, c.208T>C (p.Y70H), in the SLC45A2 gene, which supported a diagnosis of OCA4. Conclusions: The present study reports a very rare family with autosomal dominant OCA4 whose diagnosis was confirmed by a mutational analysis. Most family members exhibited mild general hypopigmentation and low-grade foveal hypoplasia.

Oculocutaneous albinism type 1: link between mutations, tyrosinase conformational stability, and enzymatic activity.

Oculocutaneous albinism type 1 (OCA1) is an autosomal recessive disorder caused by mutations in the tyrosinase gene. Two subtypes of OCA1 have been described: severe OCA1A with complete absence of tyrosinase activity and less severe OCA1B with residual tyrosinase activity. Here, we characterize the recombinant human tyrosinase intramelanosomal domain and mutant variants, which mimic genetic changes in both subtypes of OCA1 patients. Proteins were prepared using site-directed mutagenesis, expressed in insect larvae, purified by chromatography, and characterized by enzymatic activities, tryptophan fluorescence, and Gibbs free energy changes. The OCA1A mutants showed very low protein expression and protein yield and are enzymatically inactive. Mutants mimicking OCA1B were biochemically similar to the wild type, but exhibited lower specific activities and protein stabilities. The results are consistent with clinical data, which indicates that OCA1A mutations inactivate tyrosinase and result in severe phenotype, while OCA1B mutations partially inactivate tyrosinase and result in OCA1B albinism.

A histopathological study of melanocytic and pigmented skin lesions in patients with albinism.

BACKGROUND: Oculocutaneous albinism (OCA) is a group of genetic disorders characterized by diminished pigmentation of the skin, hair and eyes. Individuals with OCA are at increased risk to develop sun-induced skin malignancies. The incidence of malignant melanoma in OCA individuals is, however, very low. The aim of this study was to document pigmented and melanocytic skin lesions occurring in patients with OCA. METHODS: A prospective study was performed. Sixteen patients with OCA presenting at the Oncology and Dermatology Departments at Universitas Academic Hospital Annex in Bloemfontein, South Africa, were included. Selected clinically pigmented and/or melanocytic lesions were biopsied and studied by light microscopy. RESULTS: Twenty-four punch biopsies were taken. Ten dendritic freckles and 10 melanocytic nevi were confirmed histologically. The nevi, which occurred in eight patients, were found on sun-protected skin. All the freckles occurred on sun-exposed skin. Twelve patients had current or previous skin malignancies. No melanomas were present in the study population. Other skin lesions ranged from solar keratoses to squamous cell carcinomas. CONCLUSION: The majority of pigmented lesions were dendritic freckles that occurred on sun-exposed skin. None of the patients had a current or previous diagnosis of malignant melanoma.

An intracellular anion channel critical for pigmentation.

Intracellular ion channels are essential regulators of organellar and cellular function, yet the molecular identity and physiological role of many of these channels remains elusive. In particular, no ion channel has been characterized in melanosomes, organelles that produce and store the major mammalian pigment melanin. Defects in melanosome function cause albinism, characterized by vision and pigmentation deficits, impaired retinal development, and increased susceptibility to skin and eye cancers. The most common form of albinism is caused by mutations in oculocutaneous albinism II (OCA2), a melanosome-specific transmembrane protein with unknown function. Here we used direct patch-clamp of skin and eye melanosomes to identify a novel chloride-selective anion conductance mediated by OCA2 and required for melanin production. Expression of OCA2 increases organelle pH, suggesting that the chloride channel might regulate melanin synthesis by modulating melanosome pH. Thus, a melanosomal anion channel that requires OCA2 is essential for skin and eye pigmentation.

Computational screening of disease-associated mutations in OCA2 gene.

Oculocutaneous albinism type 2 (OCA2), caused by mutations of OCA2 gene, is an autosomal recessive disorder characterized by reduced biosynthesis of melanin pigment in the skin, hair, and eyes. The OCA2 gene encodes instructions for making a protein called the P protein. This protein plays a crucial role in melanosome biogenesis, and controls the eumelanin content in melanocytes in part via the processing and trafficking of tyrosinase which is the rate-limiting enzyme in melanin synthesis. In this study we analyzed the pathogenic effect of 95 non-synonymous single nucleotide polymorphisms reported in OCA2 gene using computational methods. We found R305W mutation as most deleterious and disease associated using SIFT, PolyPhen, PANTHER, PhD-SNP, Pmut, and MutPred tools. To understand the atomic arrangement in 3D space, the native and mutant (R305W) structures were modeled. Molecular dynamics simulation was conducted to observe the structural significance of computationally prioritized disease-associated mutation (R305W). Root-mean-square deviation, root-mean-square fluctuation, radius of gyration, solvent accessibility surface area, hydrogen bond (NH bond), trace of covariance matrix, eigenvector projection analysis, and density analysis results showed prominent loss of stability and rise in mutant flexibility values in 3D space. This study presents a well designed computational methodology to examine the albinism-associated SNPs.

MITF mutations associated with pigment deficiency syndromes and melanoma have different effects on protein function.

The basic-helix-loop-helix-leucine zipper (bHLHZip) protein MITF (microphthalmia-associated transcription factor) is a master regulator of melanocyte development. Mutations in the MITF have been found in patients with the dominantly inherited hypopigmentation and deafness syndromes Waardenburg syndrome type 2A (WS2A) and Tietz syndrome (TS). Additionally, both somatic and germline mutations have been found in MITF in melanoma patients. Here, we characterize the DNA-binding and transcription activation properties of 24 MITF mutations found in WS2A, TS and melanoma patients. We show that most of the WS2A and TS mutations fail to bind DNA and activate expression from melanocyte-specific promoters. Some of the mutations, especially R203K and S298P, exhibit normal activity and may represent neutral variants. Mutations found in melanomas showed normal DNA-binding and minor variations in transcription activation properties; some showed increased potential to form colonies. Our results provide molecular insights into how mutations in a single gene can lead to such different phenotypes.

The etiology and molecular genetics of human pigmentation disorders.

Pigmentation, defined as the placement of pigment in skin, hair, and eyes for coloration, is distinctive because the location, amount, and type of pigmentation provides a visual manifestation of genetic heterogeneity in pathways regulating the pigment-producing cells, melanocytes. The scope of this genetic heterogeneity in humans ranges from normal to pathological pigmentation phenotypes. Clinically, normal human pigmentation encompasses a variety of skin and hair color as well as punctate pigmentation such as melanocytic nevi (moles) or ephelides (freckles), while abnormal human pigmentation exhibits markedly reduced or increased pigment levels, known as hypopigmentation and hyperpigmentation, respectively. Elucidation of the molecular genetics underlying pigmentation has revealed genes important for melanocyte development and function. Furthermore, many pigmentation disorders show additional defects in cells other than melanocytes, and identification of the genetic insults in these disorders has revealed pleiotropic genes, where a single gene is required for various functions in different cell types. Thus, unravelling the genetics of easily visualized pigmentation disorders has identified molecular similarities between melanocytes and less visible cell types/tissues, arising from a common developmental origin and/or shared genetic regulatory pathways. Herein we discuss notable human pigmentation disorders and their associated genetic alterations, focusing on the fact that the developmental genetics of pigmentation abnormalities are instructive for understanding normal pathways governing development and function of melanocytes.

Functional interactions between OCA2 and the protein complexes BLOC-1, BLOC-2, and AP-3 inferred from epistatic analyses of mouse coat pigmentation.

The biogenesis of melanosomes is a multistage process that requires the function of cell-type-specific and ubiquitously expressed proteins. OCA2, the product of the gene defective in oculocutaneous albinism type 2, is a melanosomal membrane protein with restricted expression pattern and a potential role in the trafficking of other proteins to melanosomes. The ubiquitous protein complexes AP-3, BLOC-1, and BLOC-2, which contain as subunits the products of genes defective in various types of Hermansky-Pudlak syndrome, have been likewise implicated in trafficking to melanosomes. We have tested for genetic interactions between mutant alleles causing deficiency in OCA2 (pink-eyed dilution unstable), AP-3 (pearl), BLOC-1 (pallid), and BLOC-2 (cocoa) in C57BL/6J mice. The pallid allele was epistatic to pink-eyed dilution, and the latter behaved as a semi-dominant phenotypic enhancer of cocoa and, to a lesser extent, of pearl. These observations suggest functional links between OCA2 and these three protein complexes involved in melanosome biogenesis.

Held under arrest: Many mature albino RPE cells display polyploidal features consistent with abnormal cell cycle retention.

A population of peripheral retinal pigment epithelium (RPE) cells in mature pigmented rats are retained in the cell cycle and divide, as determined by Ki67 and BrdU labeling. Their cell cycle rate is approximately 5 days. Ten times as many Ki67 positive cells are found in albinos compared with pigmented animals, but it is not known if they actually divide or only express cell cycle markers. In spite of the increased number of cells expressing cell cycle markers, we show here using BrdU, that levels of cell division in albino RPE are similar to those in pigmented animals and have a similar cell cycle rate. Hence, cell cycle activity does not progress through to cell division in the majority of albino RPE cells. Peripheral RPE cells in albinos are different from those in pigmented animals. Many have very large distorted or highly fragmented nuclei. These data along with patterns of Ki67 and BrdU labeling are consistent with such cells being retained abnormally in the cell cycle, replicating their DNA, but not able to progress through to full cell division. Hence, there are two populations of RPE cells in albinos, those undergoing normal cell division and those that appear to be arrested in the cell cycle. These abnormalities are present from early postnatal stages.

AAV-mediated tyrosinase gene transfer restores melanogenesis and retinal function in a model of oculo-cutaneous albinism type I (OCA1).

Oculo-cutaneous albinism type 1 (OCA1) is characterized by congenital hypopigmentation and is due to mutations in the TYROSINASE gene (TYR). In this study, we have characterized the morpho-functional consequences of the lack of tyrosinase activity in the spontaneous null mouse model of OCA1 (Tyr(c-2j)). Here, we show that adult Tyr(c-2j) mice have several retinal functional anomalies associated with photoreceptor loss. To test whether these anomalies are reversible upon TYR complementation, we performed intraocular administration of an adeno-associated virus (AAV)-based vector, encoding the human TYR gene, in adult Tyr(c-2j) mice. This resulted in melanosome biogenesis and ex novo synthesis of melanin in both neuroectodermally derived retinal pigment epithelium (RPE) and in neural crest-derived choroid and iris melanocytes. Ocular melanin accumulation prevented progressive photoreceptor degeneration and resulted in restoration of retinal function. Our results reveal novel properties of pigment cells and show that the developmental anomalies of albino mice are associated with defects occurring in postnatal life, adding novel insights on OCA1 disease pathogenesis. In addition, we provide proof-of-principle of an effective gene-based strategy relevant for future application in albino patients.

Oculocutaneous albinism type 4: six novel mutations in the membrane-associated transporter protein gene and their phenotypes.

Oculocutaneous albinism type 4 (OCA4) is an autosomal recessive hypopigmentary disorder caused by mutations in the Membrane-Associated Transporter Protein gene (SLC45A2). The SLC45A2 protein is a 530-amino-acid polypeptide that contains 12 putative transmembrane domains, and appears to be a transporter that mediates melanin synthesis. Eighteen pathological mutations have been reported so far. In this study, six novel mutations, p.Y49C (c.146A > G), p.G89R (c.265G > A), p.C229Y (c.686G > A), p.T437A (c.1309A > G), p.T440A (c.1318A > G) and p.G473D (c.1418G > A) were found in eight Japanese patients with various clinical phenotypes. The phenotypes of OCA4 were as various as the other types of OCA and probably depended on the mutation sites in the SLC45A2 gene.

Heterologous expression of tyrosinase recapitulates the misprocessing and mistrafficking in oculocutaneous albinism type 2: effects of altering intracellular pH and pink-eyed dilution gene expression.

The processing and trafficking of tyrosinase, a melanosomal protein essential for pigmentation, was investigated in a human epithelial 293 cell line that stably expresses the protein. The effects of the pink-eyed dilution (p) gene product, in which mutations result in oculocutaneous albinism type 2 (OCA2), on the processing and trafficking of tyrosinase in this cell line were studied. The majority of tyrosinase was retained in the endoplasmic reticulum-Golgi intermediate compartment and the early Golgi compartment in the 293 cells expressing the protein. Coexpression of p could partially correct the mistrafficking of tyrosinase in 293 cells. Tyrosinase was targeted to the late endosomal and lysosomal compartments after treatment of the cells with compounds that correct the tyrosinase mistrafficking in albino melanocytes, most likely through altering intracellular pH, while the substrate tyrosine had no effect on the processing of tyrosinase. Remarkably, this heterologous expression system recapitulates the defective processing and mistrafficking of tyrosinase observed in OCA2 albino melanocytes and certain amelanotic melanoma cells. Coexpression of other melanosomal proteins in this heterologous system may further aid our understanding of the details of normal and pathologic processing of melanosomal proteins.

Identification of compounds that bind mitochondrial F1F0 ATPase by screening a triazine library for correction of albinism.

A triazine-based combinatorial library of small molecules was screened in albino murine melanocytes to identify compounds that induce pigmentation. Six compounds (of 1536 screened) produced at least 3-fold increases in pigmentation. Immunohistochemical studies demonstrated that the compounds conferred correct routing of the mistrafficked enzyme tyrosinase, which is critical to normal melanogenesis. Affinity matrices of the immobilized compounds allowed the cellular target to be identified as the mitochondrial F1F0-ATP synthase. Oligomycin and aurovertin B, small molecules known to inhibit the mitochondrial ATP synthase, were shown to compete with the triazine-based compounds for their cellular target in albino melanocytes and confer similar effects on pigmentation and tyrosinase rerouting. This is the first demonstration of the mitochondrial ATP synthase as a potential therapeutic target for restoring pigmentation in albino melanocytes.

Anterior chamber optical coherence tomography study of human natural accommodation in a 19-year-old albino.

Anterior segment optical coherence tomography is a new method to explore the anterior chamber. The target can be focused and defocused with positive or negative lenses to reproduce the conditions of natural accommodation. We studied accommodation in an albino patient because the absence of pigment allows the infrared beam to penetrate the iris and explore the modifications of the ciliary body and the crystalline lens during natural accommodation in a human subject.

The etiology of oculocutaneous albinism (OCA) type II: the pink protein modulates the processing and transport of tyrosinase.

Oculocutaneous albinism (OCA) is caused by reduced or deficient melanin pigmentation in the skin, hair, and eyes. OCA has different phenotypes resulting from mutations in distinct pigmentation genes involved in melanogenesis. OCA type 2 (OCA2), the most common form of OCA, is an autosomal recessive disorder caused by mutations in the P gene, the function(s) of which is controversial. In order to elucidate the mechanism(s) involved in OCA2, our group used several antibodies specific for various melanosomal proteins (tyrosinase, Tyrp1, Dct, Pmel17 and HMB45), including a specific set of polyclonal antibodies against the p protein. We used confocal immunohistochemistry to compare the processing and distribution of those melanosomal proteins in wild type (melan-a) and in p mutant (melan-p1) melanocytes. Our results indicate that the melanin content of melan-p1 melanocytes was less than 50% that of wild type melan-a melanocytes. In contrast, the tyrosinase activities were similar in extracts of wild type and p mutant melanocytes. Confocal microscopy studies and pulse-chase analyses showed altered processing and sorting of tyrosinase, which is released from melan-p1 cells to the medium. Processing and sorting of Tyrp1 was also altered to some extent. However, Dct and Pmel17 expression and subcellular localization were similar in melan-a and in melan-p1 melanocytes. In melan-a cells, the p protein showed mainly a perinuclear pattern with some staining in the cytoplasm where some co-localization with HMB45 antibody was observed. These findings suggest that the p protein plays a major role in modulating the intracellular transport of tyrosinase and a minor role for Tyrp1, but is not critically involved in the transport of Dct and Pmel17. This study provides a basis to understand the relationship of the p protein with tyrosinase function and melanin synthesis, and also provides a rational approach to unveil the consequences of P gene mutations in the pathogenesis of OCA2.

Mislocalization of melanosomal proteins in melanocytes from mice with oculocutaneous albinism type 2.

More than 10% of admissions worldwide to institutions for the visually impaired are due to some form of albinism. The most common form, oculocutaneous albinism type 2, results from mutations at the p locus. The function of the p gene is yet to be determined. It has been shown that melanocytes from p -null mice exhibit an abnormal melanosomal ultrastructure in addition to alterations in activity and localization of tyrosinase, a critical melanogenic enzyme. In light of these observations, we examined tyrosinase trafficking in p -null vs wildtype mouse melanocytes in order to explore p function. Electron microscopy of wildtype melan-a and p -null melan-p1 cells demonstrated accumulation of tyrosinase in 50 nm vesicles throughout the cell in the absence of p, an observation corroborated by an increase in tyrosinase activity in vesicle-enriched fractions from melan-p1 compared to melan-a cells. Misrouting in the absence of p was not limited to tyrosinase; a second melanosomal protein, tyrosinase-related protein 1, also trafficked incorrectly. In melan-p1, mislocalization led to secretion of tyrosinase into the medium. Adding tyrosine to the medium was found to partially correct tyrosinase trafficking and to reduce secretion; the cysteine protease inhibitor E64 also reduced secretion. We propose that p is required by melanocytes for transport of melanosomal proteins. In its absence, tyrosinase accumulates in vesicles and, in cultured melanocytes, is proteolysed and secreted.

The Hermansky-Pudlak syndrome (HPS) protein is part of a high molecular weight complex involved in biogenesis of early melanosomes.

Hermansky-Pudlak syndrome (HPS) is a rare autosomal recessive disorder in which oculocutaneous albinism, bleeding tendency and a ceroid-lipofuscin lysosomal storage disease result from defects of multiple cytoplasmic organelles: melanosomes, platelet dense granules and lysosomes. The HPS polypeptide, a 700 amino acid protein which is unrelated to any known proteins, is likely to be involved in the biogenesis of these different organelles. Here, we show that HPS is a non-glycosylated, non-membrane protein which is a component of two distinct high molecular weight complexes. In non-melanotic cells the HPS protein is contained almost entirely in an approximately 200 kDa complex that is widely distributed throughout the cytosol. In melanotic cells the HPS protein is partitioned between this cytosolic complex and a >500 kDa complex that appears to consist of the approximately 200 kDa complex in association with membranous components. Subcellular fractionation, immunofluorescence and immunoelectron microscopy studies indicate that the membrane-associated HPS complex of melanotic cells is associated with tubulovesicular structures, small non-coated vesicles, and nascent and early-stage melanosomes. These findings suggest that the HPS complex is involved in the biogenesis of early melanosomes.