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.

Characterization of Temperature-Dependent Kinetics of Oculocutaneous Albinism-Causing Mutants of Tyrosinase.

Human tyrosinase (Tyr) is a glycoenzyme that catalyzes the first and rate-limiting step in melanin production, and its gene (TYR) is mutated in many cases of oculocutaneous albinism type 1 (OCA1). The mechanisms by which individual mutations contribute to the diverse pigmentation phenotype in patients with OCA1 have only began to be examined and remain to be delineated. Here, we analyze the temperature-dependent kinetics of wild-type Tyr (WT) and two OCA1B mutant variants (R422Q and P406L) using Michaelis-Menten and Van't Hoff analyses. Recombinant truncated human Tyr proteins (residues 19-469) were produced in the whole insect Trichoplusia Ni larvae. Proteins were purified by a combination of affinity and size-exclusion chromatography. The temperature dependence of diphenol oxidase protein activities and kinetic parameters were measured by dopachrome absorption. Using the same experimental conditions, computational simulations were performed to assess the temperature-dependent association of L-DOPA and Tyr. Our results revealed, for the first time, that the association of L-DOPA with R422Q and P406L followed by dopachrome formation is a complex reaction supported by enthalpy and entropy forces. We show that the WT has a higher turnover number as compared with both R422Q and P406L. Elucidating the kinetics and thermodynamics of mutant variants of Tyr in OCA1B helps to understand the mechanisms by which they lower Tyr catalytic activity and to discover novel therapies for patients.

Billy Idol.

Ruthie Weiss was born with white hair, but her parents did not consider the possibility of there being more to the story until they noticed that she was not visually tracking when she was just a month old. Thus began a long and continuing story of the discovery of Ruthie's albinism, her significant visual impairment, but also her courage and determination to do anything and everything her peers do, if not more. But this story is really about how her parents grew to embrace the impact Ruthie (and importantly Ruthie's disability) had on their lives and the lives of everyone with whom Ruthie interacted. The experience of raising Ruthie ultimately led her parents to think about a world where she might not exist, or at least might not exist with albinism. But it also led them to ponder a future in which children with genetic differences like albinism are gene edited using technologies like CRISPR-Cas9.

Hermansky-Pudlak syndrome and oculocutaneous albinism in Chinese children with pigmentation defects and easy bruising.

BACKGROUND: Determining the etiology of oculocutaneous albinism is important for proper clinical management and to determine prognosis. The purpose of this study was to genotype and phenotype eight adopted Chinese children who presented with oculocutaneous albinism and easy bruisability. RESULTS: The patients were evaluated at a single center; their ages ranged from 3 to 8 years. Whole exome or direct sequencing showed that two of the children had Hermansky-Pudlak syndrome (HPS) type-1 (HPS-1), one had HPS-3, one had HPS-4, and four had non-syndromic oculocutaneous albinism associated with TYR variants (OCA1). Two frameshift variants in HPS1 (c.9delC and c.1477delA), one nonsense in HPS4 (c.416G > A), and one missense variant in TYR (c.1235C > T) were unreported. The child with HPS-4 is the first case with this subtype reported in the Chinese population. Hypopigmentation in patients with HPS was mild compared to that in OCA1 cases, who had severe pigment defects. Bruises, which may be more visible in patients with hypopigmentation, were found in all cases with either HPS or OCA1. Whole mount transmission electron microscopy demonstrated absent platelet dense granules in the HPS cases; up to 1.9 mean dense granules per platelet were found in those with OCA1. Platelet aggregation studies in OCA1 cases were inconclusive. CONCLUSIONS: Clinical manifestations of oculocutaneous albinism and easy bruisability may be observed in children with HPS or OCA1. Establishing definitive diagnoses in children presenting with these phenotypic features is facilitated by genetic testing. Non-syndromic oculocutaneous albinism and various HPS subtypes, including HPS-4, are found in children of Chinese ancestry.

Hermansky-Pudlak Syndrome.

Hermansky-Pudlak syndrome (HPS) is an autosomal recessive disorder that is associated with oculocutaneous albinism, bleeding diatheses, granulomatous colitis, and highly penetrant pulmonary fibrosis in some subtypes, including HPS-1, HPS-2, and HPS-4. HPS pulmonary fibrosis shows many of the clinical, radiologic, and histologic features found in idiopathic pulmonary fibrosis, but occurs at a younger age. Despite knowledge of the underlying genetic defects, there are currently no definitive therapeutic or preventive approaches for HPS pulmonary fibrosis other than lung transplant.

29-year-old man presenting with progressive dyspnea, oculocutaneous albinism, and epistaxis.

A 29-year-old man with a history of oculocutaneous albinism presented to the ED complaining of progressive dyspnea on exertion. One month prior to admission, the patient had begun to experience worsening dyspnea provoked by routine household activities. Additionally, he had developed a nonproductive cough, exacerbated by cold weather. He denied associated chest pain, hemoptysis, fever, chills, or night sweats. He denied any new exposures or sick contacts in the recent past. A review of systems was significant for a history of epistaxis and frequent bruising. Born in Honduras, he had immigrated to the United States approximately 10 years prior to his presentation to our facility. Furthermore, there was no family history of albinism, bleeding disorders, or pulmonary disease.

Comparison of two devices for the treatment of keloid scars with the use of intralesional cryotherapy: An experimental study.

BACKGROUND: Intralesional (IL) cryotherapy is a new technique for the treatment of keloid scars, in which the scar is frozen from inside. Two cryodevices are available, which were recently evaluated. Both devices showed promising results, but differed in clinical outcome. To explain these differences, more understanding of the working mechanism of both devices is required. OBJECTIVE: This experimental study was designed to investigate and compare the thermal behavior of an argon gas- and a liquid nitrogen-based device. Thermal behavior constitutes: (1) minimum tissue temperature (°C), (2) the freezing rate (°C/min). The thermal behavior was measured inside and on the outer surface of the scar. Both devices were tested ex vivo and in vivo. RESULTS: Ex vivo, when determining the maximum freezing capacity, the argon gas device showed a higher end temperature compared to the liquid nitrogen device (argon gas: -120°C, liquid nitrogen: -140°C) and a faster freezing rate (argon gas: -1300°C/min, liquid nitrogen: -145°C/min). In vivo, measured inside the keloid, the argon gas device showed a lower end temperature than the liquid nitrogen device (argon gas: -36.4°C, liquid nitrogen: -8.1°C) and a faster freezing rate (argon gas: -14.7°C/min, liquid nitrogen: -5°C/min). The outer surface of the scar reached temperatures below -20°C with both devices as measured with the thermal camera. CONCLUSION: In conclusion, the argon gas device displayed a lower end temperature and a faster freezing rate in vivo compared to the liquid nitrogen device. Although this resulted in lower recurrence rates for the argon gas device, more hypopigmentation was seen compared to the liquid nitrogen device following treatment. Finally, the low outer surface temperatures measured with both devices, suggest that some hypopigmentation following treatment is inevitable.

Functional characterization of two novel splicing mutations in the OCA2 gene associated with oculocutaneous albinism type II.

Oculocutaneous albinism (OCA) is characterized by hypopigmentation of the skin, hair and eye, and by ophthalmologic abnormalities caused by a deficiency in melanin biosynthesis. OCA type II (OCA2) is one of the four commonly-recognized forms of albinism, and is determined by mutation in the OCA2 gene. In the present study, we investigated the molecular basis of OCA2 in two siblings and one unrelated patient. The mutational screening of the OCA2 gene identified two hitherto-unknown putative splicing mutations. The first one (c.1503+5G>A), identified in an Italian proband and her affected sibling, lies in the consensus sequence of the donor splice site of OCA2 intron 14 (IVS14+5G>A), in compound heterozygosity with a frameshift mutation, c.1450_1451insCTGCCCTGACA, which is predicted to determine the premature termination of the polypeptide chain (p.I484Tfs*19). In-silico prediction of the effect of the IVS14+5G>A mutation on splicing showed a score reduction for the mutant splice site and indicated the possible activation of a newly-created deep-intronic acceptor splice site. The second mutation is a synonymous transition (c.2139G>A, p.K713K) involving the last nucleotide of exon 20. This mutation was found in a young African albino patient in compound heterozygosity with a previously-reported OCA2 missense mutation (p.T404M). In-silico analysis predicted that the mutant c.2139G>A allele would result in the abolition of the splice donor site. The effects on splicing of these two novel mutations were investigated using an in-vitro hybrid-minigene approach that led to the demonstration of the causal role of the two mutations and to the identification of aberrant transcript variants.

Albino mice as an animal model for infantile nystagmus syndrome.

PURPOSE: Individuals with oculocutaneous albinism are predisposed to visual system abnormalities affecting the retina and retinofugal projections, which may lead to reduced visual acuity and Infantile Nystagmus Syndrome (INS). Due to absence of an established mammalian animal model, mechanisms underlying INS remain elusive. In this study, we screened wild-type mice of varying pigmentation for ocular motor abnormalities in order to identify a possible mouse model for INS. METHODS: Three albino mouse strains (CD1, BALB/c, DBA/1), and two normally pigmented strains (129S6, C57BL/6) were screened using infrared oculography. Varying visual stimuli (black or white background, stationary pattern, optokinetic, i.e., horizontally rotating pattern) were displayed to the full (fVF) or anterior visual field (aVF) of the restrained mouse. RESULTS: We found spontaneous nystagmus, specifically jerks and oscillations, in albino mice under all experimental conditions. Median eye velocity was between 0.8 and 3.4 deg/s, depending on the strain. In contrast, the eyes in pigmented mice were nearly stable with a median absolute eye velocity of below 0.4 deg/s. In albino mice, fVF optokinetic stimuli elicited an optokinetic response (OKR) in the correct direction, albeit with superimposed oscillations. However, aVF optokinetic stimuli evoked reversed OKR in these strains, a well known feature of INS. CONCLUSIONS: Based on our results, we endorse the investigated albino mouse strains as new animal models for INS.

Síndrome de Chediak-Higashi / Chediak-Higashi syndrome

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Oculocutaneous albinism.

Oculocutaneous albinism (OCA) is a group of inherited disorders of melanin biosynthesis characterized by a generalized reduction in pigmentation of hair, skin and eyes. The prevalence of all forms of albinism varies considerably worldwide and has been estimated at approximately 1/17,000, suggesting that about 1 in 70 people carry a gene for OCA. The clinical spectrum of OCA ranges, with OCA1A being the most severe type with a complete lack of melanin production throughout life, while the milder forms OCA1B, OCA2, OCA3 and OCA4 show some pigment accumulation over time. Clinical manifestations include various degrees of congenital nystagmus, iris hypopigmentation and translucency, reduced pigmentation of the retinal pigment epithelium, foveal hypoplasia, reduced visual acuity usually (20/60 to 20/400) and refractive errors, color vision impairment and prominent photophobia. Misrouting of the optic nerves is a characteristic finding, resulting in strabismus and reduced stereoscopic vision. The degree of skin and hair hypopigmentation varies with the type of OCA. The incidence of skin cancer may be increased. All four types of OCA are inherited as autosomal recessive disorders. At least four genes are responsible for the different types of the disease (TYR, OCA2, TYRP1 and MATP). Diagnosis is based on clinical findings of hypopigmentation of the skin and hair, in addition to the characteristic ocular symptoms. Due to the clinical overlap between the OCA forms, molecular diagnosis is necessary to establish the gene defect and OCA subtype. Molecular genetic testing of TYR and OCA2 is available on a clinical basis, while, at present, analysis of TYRP1 and MATP is on research basis only. Differential diagnosis includes ocular albinism, Hermansky-Pudlak syndrome, Chediak-Higashi syndrome, Griscelli syndrome, and Waardenburg syndrome type II. Carrier detection and prenatal diagnosis are possible when the disease causing mutations have been identified in the family. Glasses (possibly bifocals) and dark glasses or photocromic lenses may offer sufficient help for reduced visual activity and photophobia. Correction of strabismus and nystagmus is necessary and sunscreens are recommended. Regular skin checks for early detection of skin cancer should be offered. Persons with OCA have normal lifespan, development, intelligence and fertility.

Oculocutaneous albinism.

Oculocutaneous albinism represents a group of inherited skin disorders characterized by a generalized reduction of cutaneous, ocular and pilar pigmentation from the time of birth. Oculocutaneous albinism types 1 and 2 are the most common, but several other types have been described. A defect in the melanin synthesis pathway, resulting in reduced formation of melanin, is responsible for oculocutaneous albinism. Aetiology, clinical manifestations, diagnosis and management are discussed.

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.

Attitudes and beliefs of an albino population toward sun avoidance: advice and services provided by an outreach albino clinic in Tanzania.

OBJECTIVE: To determine an albino population's expectations from an outreach albino clinic, understanding of skin cancer risk, and attitudes toward sun protection behavior. DESIGN: Survey, June 1, 1997, to September 30, 1997. SETTING: Outreach albino clinics in Tanzania. PARTICIPANTS: All albinos 13 years and older and accompanying adults of younger children attending clinics. Unaccompanied children younger than 13 years and those too sick to answer questions were excluded. Ninety-four questionnaires were completed in 5 villages, with a 100% response rate. INTERVENTIONS: Interview-based questionnaire with scoring system for pictures depicting poorly sun-protected albinos. RESULTS: The most common reasons for attending the clinic were health education and skin examination. Thirteen respondents (14%) believed albinism was inherited; it was more common to believe in superstitious causes of albinism than inheritance. Seventy-three respondents (78%) believed skin cancer was preventable, and 60 (63%) believed skin cancer was related to the sun. Seventy-two subjects (77%) thought sunscreen provided protection from the sun; 9 (10%) also applied it at night. Reasons for not wearing sun-protective clothing included fashion, culture, and heat. The hats provided were thought to have too soft a brim, to shrink, and to be ridiculed. Suggestions for additional clinic services centered on education and employment. Albinos who had read the educational booklet had no better understanding of sun avoidance than those who had not (P =.49). CONCLUSIONS: There was a reasonable understanding of risks of skin cancer and sun-avoidance methods. Clinical advice was often not followed for cultural reasons. The hats provided were unsuitable, and there was some confusion about the use of sunscreen. A lack of understanding of the cause of albinism led to many superstitions.

Oculocutaneous albinism types 1 and 3 are ER retention diseases: mutation of tyrosinase or Tyrp1 can affect the processing of both mutant and wild-type proteins.

Various types of oculocutaneous albinism (OCA) are associated with reduced pigmentation in the skin, hair, and eyes that results from mutations in genes involved in melanin synthesis. Immortal mouse melanocyte lines (melan-a, melan-b, and melan-c) provide opportune models with which to investigate the etiology of two different types of OCA (types I and III), which arise from mutations in Tyr and Tyrp1, respectively. We compared intracellular processing, sorting, and degradation of tyrosinase and Tyrp1, and the effects on their catalytic function and melanin synthesis, in these wild-type and mutant melanocytes. A mutation in either Tyr or Tyrp1 increased the time of association of tyrosinase and Tyrp1 with calnexin and Bip, which in turn resulted in the retention of these mutant products in the ER. A mutation in either gene selectively enhanced the duration and efficiency of chaperone interactions (even with the wild-type protein in the mutant melanocytes) and markedly slowed their transport to melanosomes. These results show that OCA1 and OCA3 are (in some cases, at least) ER retention diseases wherein a mutation in one melanogenic protein affects the maturation and stability of the other in the melanogenic pathway.

Hermansky-Pudlak syndrome: models for intracellular vesicle formation.

Hermansky-Pudlak syndrome (HPS) is an autosomal recessive disorder characterized by pigment dilution, nystagmus, decreased visual acuity, a bleeding diathesis, and lysosomal accumulation of ceroid lipofuscin. Electron microscopic evidence demonstrating lack of platelet-dense bodies provides the sine qua non for diagnosing HPS. Ceroid lipofuscinosis is considered to cause several serious complications, including progressive pulmonary fibrosis leading to death in the fourth or fifth decades. Currently, only symptomatic treatment can be offered. Although rare in the general population, HPS occurs in northwest Puerto Rico with a prevalence of 1 in 1800. HPS1, the first gene found to be responsible for HPS, was mapped to chromosome 10q23 and subsequently isolated and sequenced. It consists of 20 exons encoding a 700-amino acid, 79.3-kDa peptide with no homology to any known protein. All 10 HPS1 mutations reported to date, including the 16-bp duplication found in all northwest Puerto Rican patients, result in truncated proteins. The two mutations in the mouse pale ear gene (ep), which is the murine homology of HPS1, cause similarly truncated proteins. The pathologic nature of these truncation mutations may result from unstable mRNA. However, in combination with the absence of any disease-causing missense mutations, it may indicate that the C-terminus of the HPS1 peptide is functionally important. The disorder HPS displays locus heterogeneity, consistent with the existence of 14 mouse strains manifesting both hypopigmentation and a platelet storage pool deficiency. Two mouse models, pearl and mocha, have mutations in the beta3A and delta subunits of the adaptor-3 complex, respectively. This suggests that defective vesicular trafficking, specifically cargo packaging, vesicle formation, vesicle docking, or membrane fusion, may comprise the basic defect in HPS. Studies of the proteins involved in intercompartmental transport for melanosomes, platelet-dense bodies, and lysosomes should lead to a better understanding of the mechanisms of organellogenesis and to more effective therapies for HPS.

Hereditary external ophthalmoplegia synergistic divergence, jaw winking, and oculocutaneous hypopigmentation: a congenital fibrosis syndrome caused by deficient innervation to extraocular muscles.

BACKGROUND: The congenital fibrosis syndrome is a hereditary form of external ophthalmoplegia that is considered to be a primary myopathy. PURPOSE: To document the coexistence of two distinct forms of ocular motor synkinesis in a subgroup of patients with congenital fibrosis syndrome. METHODS: Clinical and intraoperative examination results and extraocular muscle biopsy specimens from four patients with congenital fibrosis syndrome were studied. RESULTS: Three patients displayed a variant of synergistic divergence characterized by simultaneous abduction with intorsion and depression of the synkinetically abducting eye. Three patients had variant of Marcus Gunn jaw winking characterized by elevation of a ptotic eyelid during mouth opening. Three patients had oculocutaneous hypopigmentation. CONCLUSIONS: A subgroup of patients with congenital fibrosis syndrome display two distinct synkinetic ocular movements in conjunction with oculocutaneous hypopigmentation. The patterns of neuronal misdirection implicate a regional innervational disturbance involving cranial nerves III through VI as the underlying cause of diffuse hereditary ophthalmoplegia in these patients.

Molecular basis of congenital hypopigmentary disorders in humans: a review.

Many specific gene products are sequentially made and utilized by the melanocyte as it emigrates from its embryonic origin, migrates into specific target sites, synthesizes melanin(s) within a specialized organelle, transfers pigment granules to neighboring cells, and responds to various exogenous cues. A mutation in many of the respective encoding genes can disrupt this process of melanogenesis and can result in hypopigmentary disorders. Following are examples highlighting this scenario. A subset of neural crest derived cells emigrate from the dorsal surface of the neural tube, become committed to the melanoblast lineage, and are targeted along the dorsal lateral pathway. The specific transcription factors PAX3 and MITF (microphthalmia transcription factor) appear to play a regulatory role in early embryonic development of the pigment system and in associated diseases (the Waardenburg syndromes). During the subsequent development and commitment of the melanoblast, concomitant expression of the receptors for fibroblasts growth factor (FGFR2), endothelin-B (EDNRB), and steel factor (cKIT) also appears essential for the continued survival of migrating melanoblasts. Lack or dysfunction of these receptors result in Apert syndrome, Hirschsprung syndrome and piebaldism, respectively. Once the melanocyte resides in its target tissue, a plethora of melanocyte specific enzymes and structural proteins are coordinately expressed to form the melanosome and to convert tyrosine to melanin within it. Mutations in the genes encoding these proteins results in a family of congenital hypopigmentary diseases called oculocutaneous albinism (OCA). The tyrosinase gene family of proteins (tyrosinase, TRP1, and TRP2) regulate the type of eumelanin synthesized and mutations affecting them result in OCA1, OCA3, and slaty (in the murine system), respectively. The P protein, with 12 transmembrane domains localized to the melanosome, has no assigned function as of yet but is responsible for OCA2 when dysfunctional. There are other genetically based syndromes, phenotypically resembling albinism, in which the synthesis of pigmented melanosomes, as well as specialized organelles of other cell types, is compromised. The Hermansky-Pudlak syndrome (HPS) and the Chediak-Higashi syndrome (CHS) are two such disorders. Eventually, the functional melanocyte must be maintained in the tissue throughout life. In some cases it is lost either normally or prematurely. White hair results in the absence of melanocytes repopulating the germinative hair follicle during subsequent anagen stages. Vitiligo, in contrast, results from the destruction and removal of the melanocyte in the epidermis and mucous membranes.