In vitro disease modeling of oculocutaneous albinism type 1 and 2 using human induced pluripotent stem cell-derived retinal pigment epithelium.

Oculocutaneous albinism (OCA) encompasses a set of autosomal recessive genetic conditions that affect pigmentation in the eye, skin, and hair. OCA patients display reduced best-corrected visual acuity, reduced to absent ocular pigmentation, abnormalities in fovea development, and/or abnormal decussation of optic nerve fibers. It has been hypothesized that improving eye pigmentation could prevent or rescue some of the vision defects. The goal of the present study was to develop an in vitro model for studying pigmentation defects in human retinal pigment epithelium (RPE). We developed a "disease in a dish" model for OCA1A and OCA2 types using induced pluripotent stem cells to generate RPE. The RPE is a monolayer of cells that are pigmented, polarized, and polygonal in shape, located between the neural retina and choroid, with an important role in vision. Here we show that RPE tissue derived in vitro from OCA patients recapitulates the pigmentation defects seen in albinism, while retaining the apical-basal polarity and normal polygonal morphology of the constituent RPE cells.

The Phenotypic and Mutational Spectrum of the FHONDA Syndrome and Oculocutaneous Albinism: Similarities and Differences.

Purpose: The purpose of this study was to further expand the mutational spectrum of the Foveal Hypoplasia, Optic Nerve Decussation defect, and Anterior segment abnormalities (FHONDA syndrome), to describe the phenotypic spectrum, and to compare it to albinism. Subjects and Methods: We retrospectively collected molecular, ophthalmic, and electrophysiological data of 28 patients molecularly confirmed with FHONDA from the Netherlands (9), Israel (13), France (2), and the United States of America (4). We compared the data to that of 133 Dutch patients with the 3 most common types of albinism in the Netherlands: oculocutaneous albinism type 1 (49), type 2 (41), and ocular albinism (43). Results: Patients with FHONDA had a total of 15 different mutations in SLC38A8, of which 6 were novel. Excluding missing data, all patients had moderate to severe visual impairment (median visual acuity [VA] = 0.7 logMAR, interquartile range [IQR] = 0.6-0.8), nystagmus (28/28), and grade 4 foveal hypoplasia (17/17). Misrouting was present in all nine tested patients. None of the patients had any signs of hypopigmentation of skin and hair. VA in albinism was better (median = 0.5 logMAR, IQR = 0.3-0.7, P 0.006) and the phenotypes were more variable: 14 of 132 without nystagmus, foveal hypoplasia grades 1 to 4, and misrouting absent in 16 of 74. Conclusions: Compared to albinism, the FHONDA syndrome appears to have a more narrow phenotypic spectrum, consisting of nonprogressive moderately to severely reduced VA, nystagmus, severe foveal hypoplasia, and misrouting. The co-occurrence of nystagmus, foveal hypoplasia, and misrouting in the absence of hypopigmentation implies that these abnormalities are not caused by lack of melanin, which has important implications for understanding the pathogenesis of these features.

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 case report: Co-occurrence of Wilson disease and oculocutaneous albinism in a Chinese patient.

RATIONALE: Both Wilson disease (WD) and Oculocutaneous Albinism (OCA) are rare autosomal recessive disorders that are caused by mutations on chromosome 13 and chromosome 11, respectively. Here, we report on a patient with coexisting WD and OCA, initially presenting episodes of tremors. PATIENT CONCERNS: WD is a disorder of copper metabolism. The main sites of copper accumulation are the liver and the brain, resulting in hepatic symptoms. OCA is a disorder of melanin biosynthesis, characterized by a generalized reduction in pigmentation of the eyes (oculo-), skin (-cutaneous), and hair. DIAGNOSIS: The diagnosis of WD was confirmed by neurological symptoms, metabolism tests, and MRI scans. Interestingly, the patient also had very light skin color, blond hair and eyebrows, and dark brown eyelashes and irises. Because the association of dermatologic signs in WD has rarely been reported, OCA was highly suspected based on these clinical findings. Genetic analysis was subsequently conducted, and the results revealed the p. (Arg778Leu) mutation in 1 allele and the p. (Asn1270Ser) mutation in the other allele of the ATP7B gene, confirming the diagnosis of WD; the p. (D456fs) mutation in 1 allele and the p. (R299H) mutation in the other allele of the TYR gene, confirming the diagnosis of OCA. The family history was positive for WD with a 14-year-old younger brother also being diagnosed with it. Her parents are negative for OCA and WD. INTERVENTIONS: Sodium dimercaptopropanesulfonate (DMPS) was given during hospitalization. D-penicillamine and zinc sulfate treatment was initiated after discharge for long-term control. OUTCOMES: Postural and intention tremor disappeared, and other symptoms and signs markedly improved after treatment. LESSONS: In this study, we reported on the first case of a child who simultaneously presented WD and OCA, bringing up the possibility of a presumable link between these 2 rare diseases.

Functional validation of the albinism-associated tyrosinase T373K SNP by CRISPR/Cas9-mediated homology-directed repair (HDR) in rabbits.

BACKGROUND: Oculocutaneous albinism (OCA) is a group of autosomal recessive disorders characterized by reduced melanin that are caused by mutations in the gene encoding tyrosinase (TYR), which is the rate-limiting enzyme in the production of the pigment melanin. Many studies or meta-analyses have suggested an association between the TYR T373K SNP and OCA1, but there is limited biochemical and genetic evidence to support this association. METHODS: We overexpressed TYR-WT and TYR-T373K mutants on HK293T cells and tested the changes of melanin production and tyrosinase activity. Then we generated TYR-K373T knock-in (KI) rabbits by microinjection of ssDNA and synthesized RNAs targeting C1118A using CRISPR/Cas9-HDR to observe the formation of melanin. FINDINGS: We demonstrated that the T373K mutation in TYR can reduce tyrosinase activity, leading to an absence of melanin synthesis at the cell-level. The gene-edited TYR-K373T rabbits exhibited rescued melanin production in hair follicles and irises, as inferred from the evident decrease in pigmentation in TYR-T373K rabbits, thus providing functional validation of the albinism-associated T373K SNP at the animal level. INTERPRETATION: Our study provides the first animal-level functional validation of the albinism-associated TYR K373T SNP in rabbits, and these results will facilitate gene therapy of OCA1 in pre-clinical settings in the future. FUND: The National Key Research and Development Program of China Stem Cell and Translational Research, the Strategic Priority Research Program of the Chinese Academy of Sciences, the Guangdong Province Science and Technology Plan Project, and the Program for JLU Science and Technology Innovative Research Team.

Behavioural changes controlled by catecholaminergic systems explain recurrent loss of pigmentation in cavefish.

Multiple cave populations of the teleost Astyanax mexicanus have repeatedly reduced or lost eye and body pigmentation during adaptation to dark caves. Albinism, the complete absence of melanin pigmentation, is controlled by loss-of-function mutations in the oca2 gene. The mutation is accompanied by an increase in the melanin synthesis precursor l-tyrosine, which is also a precursor for catecholamine synthesis. In this study, we show a relationship between pigmentation loss, enhanced catecholamine synthesis and responsiveness to anaesthesia, determined as a proxy for catecholamine-related behaviours. We demonstrate that anaesthesia resistance (AR) is enhanced in multiple depigmented and albino cavefish (CF), inversely proportional to the degree of pigmentation loss, controlled by the oca2 gene, and can be modulated by experimental manipulations of l-tyrosine or the catecholamine norepinephrine (NE). Moreover, NE is increased in the brains of multiple albino and depigmented CF relative to surface fish. The results provide new insights into the evolution of pigment modification because NE controls a suite of adaptive behaviours similar to AR that may represent a target of natural selection. Thus, understanding the relationship between loss of pigmentation and AR may provide insight into the role of natural selection in the evolution of albinism via a melanin-catecholamine trade-off.

Computational analysis of histidine mutations on the structural stability of human tyrosinases leading to albinism insurgence.

Misfolding and structural alteration in proteins lead to serious malfunctions and cause various diseases in humans. Mutations at the active binding site in tyrosinase impair structural stability and cause lethal albinism by abolishing copper binding. To evaluate the histidine mutational effect, all mutated structures were built using homology modelling. The protein sequence was retrieved from the UniProt database, and 3D models of original and mutated human tyrosinase sequences were predicted by changing the residual positions within the target sequence separately. Structural and mutational analyses were performed to interpret the significance of mutated residues (N180, R202, Q202, R211, Y363, R367, Y367 and D390) at the active binding site of tyrosinases. CSpritz analysis depicted that 23.25% residues actively participate in the instability of tyrosinase. The accuracy of predicted models was confirmed through online servers ProSA-web, ERRAT score and VERIFY 3D values. The theoretical pI and GRAVY generated results also showed the accuracy of the predicted models. The CCA negative correlation results depicted that the replacement of mutated residues at His within the active binding site disturbs the structural stability of tyrosinases. The predicted CCA scores of Tyr367 (-0.079) and Q/R202 (0.032) revealed that both mutations have more potential to disturb the structural stability. MD simulation analyses of all predicted models justified that Gln202, Arg202, Tyr367 and D390 replacement made the protein structures more susceptible to destabilization. Mutational results showed that the replacement of His with Q/R202 and Y/R363 has a lethal effect and may cause melanin associated diseases such as OCA1. Taken together, our computational analysis depicts that the mutated residues such as Q/R202 and Y/R363 actively participate in instability and misfolding of tyrosinases, which may govern OCA1 through disturbing the melanin biosynthetic pathway.

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.

Cytoplasmic Control of Sense-Antisense mRNA Pairs.

Transcriptome analyses have revealed that convergent gene transcription can produce many 3'-overlapping mRNAs in diverse organisms. Few studies have examined the fate of 3'-complementary mRNAs in double-stranded RNA-dependent nuclear phenomena, and nothing is known about the cytoplasmic destiny of 3'-overlapping messengers or their impact on gene expression. Here, we demonstrate that the complementary tails of 3'-overlapping mRNAs can interact in the cytoplasm and promote post-transcriptional regulatory events including no-go decay (NGD) in Saccharomyces cerevisiae. Genome-wide experiments confirm that these messenger-interacting mRNAs (mimRNAs) form RNA duplexes in wild-type cells and thus have potential roles in modulating the mRNA levels of their convergent gene pattern under different growth conditions. We show that the post-transcriptional fate of hundreds of mimRNAs is controlled by Xrn1, revealing the extent to which this conserved 5'-3' cytoplasmic exoribonuclease plays an unexpected but key role in the post-transcriptional control of convergent gene expression.

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.

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.

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.

Characterization of a bystander effect induced by the endocrine-disrupting chemical 6-propyl-2-thiouracil in zebrafish embryos.

This study was conducted to evaluate possible bystander effects induced by the model chemical 6-propyl-2-thiouracil (PTU) on melanin synthesis. Zebrafish (Danio rerio) embryos were treated with PTU by either microinjection exposure, via waterborne exposure or indirectly through bystander exposure. Melanin content, related mRNA and protein expression were examined at the end of exposure (36 h post-fertilization). Direct exposure to PTU decreased the melanin content, up-regulated mRNA expressions of oculocutaneous albinism type 2 (OCA2), tyrosinase (TYR), dopachrometautomerase (DCT), tyrosinase-related protein 1 (TYRP1) and silver (SILV), and increased the protein expressions of TYR and SILV. Bystander exposure also up-regulated mRNA and protein expressions of TYR and SILV but increased melanin contents. Correlation analysis demonstrated that mRNA expressions of OCA2, TYR, DCT, TYRP1, SILV and protein expressions of TYR and SILV in bystander exposure groups were positively correlated with corresponding expressions in microinjection exposure groups. The results might have environmental implications and highlight the need to consider the bystander effects when assessing potential risks of endocrine-disrupting chemicals.

Change in visual acuity in albinism in the early school years.

PURPOSE: To determine whether binocular best-corrected visual acuity (B-BCVA) improves in the early school years in patients with albinism and whether this is related to type of albinism, ocular pigment, or appearance of the macula. METHODS: Patients with albinism seen between 5.5 and 9 years (Visit A) and 9.5 and 14 years of age (Visit B), with visits separated by at least 2.5 years, were included. Type of albinism, B-BCVA, glasses wear, iris pigment and macular transparency grade, and presence or absence of an annular reflex and melanin in the macula were recorded. RESULTS: Mean B-BCVA was 20/84 at Visit A and 20/61 at Visit B (P < .001). B-BCVA improved in 80%. Improvement in B-BCVA and glasses wear, iris grade, macular grade, macular melanin, and annular reflex were weakly correlated. However, a moderate correlation was found between measured B-BCVA and iris grade at Visit A (r = 0.485, P < .001) and Visit B (r = 0.467, P < .001), and the presence of macular melanin at Visit A (r = 0.436, P < .001) and Visit B (r = 0.482, P < .001). CONCLUSIONS: B-BCVA often improves in albinism in the early school years and this observation should be included in counseling. The etiology is unknown but may be related to change in nystagmus, use of precise null point, developmental maturation, and/or some of the ocular characteristics evaluated in this study.

Screening of TYR, OCA2, GPR143, and MC1R in patients with congenital nystagmus, macular hypoplasia, and fundus hypopigmentation indicating albinism.

BACKGROUND: A broad spectrum of pigmentation of the skin and hair is found among patients diagnosed with ocular albinism (OA) and oculocutaneous albinism (OCA). Even though complexion is variable, three ocular features, i.e., hypopigmentation of the fundus, hypoplasia of the macula, and nystagmus, are classical pathological findings in these patients. We screened 172 index patients with a clinical diagnosis of OA or OCA based on the classical findings, to evaluate the frequency of sequence variants in tyrosinase (TYR), P-gene, P-protein (OCA2), and the G-protein-coupled receptor 143 gene, OA1 (GPR143). In addition, we investigated the association of sequence variants in the melanocortin receptor 1 gene (MC1R) and OCA2. METHODS: Pigmentation of the hair, skin, iris, and fundus were included in the evaluation of OCA and OA. Male OA patients showing X-linked inheritance were screened for GPR143. Females showing OA without family history were regarded as representing autosomal recessive OA (OA3). Direct sequencing was applied to PCR products showing aberrant single-strand conformation polymorphism-banding patterns. RESULTS: Fifty-seven male index patients were screened for OA. We identified 16 potentially pathogenic sequence variations in GPR143 (10 novel) in 22 males. In TYR, we identified 23 (7 novel), and in OCA2 28 (11 novel) possibly pathogenic variants. Variants on both alleles were identified in TYR or OCA2 in 29/79 OCA patients and 14/71 OA patients. Sequence changes in TYR were identified almost exclusively in OCA patients, while sequence changes in OCA2 occurred in OCA and OA patients. MC1R sequencing was performed in 47 patients carrying mutations in OCA2 and revealed MC1R mutations in 42 of them. CONCLUSIONS: TYR gene mutations have a more severe effect on pigmentation than mutations in OCA2 and the GPR143 gene. Nevertheless, mutations in these genes affect the development of visual function either directly or by interaction with other genes like MC1R, which can be deduced from a frequent association of MC1R variants with p.R305W or p.R419Q in OCA2.

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.