Biallelic mutations in L-dopachrome tautomerase (DCT) cause infantile nystagmus and oculocutaneous albinism.

Infantile nystagmus syndrome (INS) denominates early-onset, involuntary oscillatory eye movements with different etiologies. Nystagmus is also one of the symptoms in oculocutaneus albinism (OCA), a heterogeneous disease mainly caused by defects in melanin synthesis or melanosome biogenesis. Dopachrome tautomerase (DCT, also called TYRP2) together with tyrosinase (TYR) and tyrosin-related protein 1 (TYRP1) is one of the key enzymes in melanin synthesis. Although DCT´s role in pigmentation has been proven in different species, until now only mutations in TYR and TYRP1 have been found in patients with OCA. Detailed ophthalmological and orthoptic investigations identified a consanguineous family with two individuals with isolated infantile nystagmus and one family member with subtle signs of albinism. By whole-exome sequencing and segregation analysis, we identified the missense mutation c.176G > T (p.Gly59Val) in DCT in a homozygous state in all three affected family members. We show that this mutation results in incomplete protein maturation and targeting in vitro compatible with a partial or total loss of function. Subsequent screening of a cohort of patients with OCA (n = 85) and INS (n = 25) revealed two heterozygous truncating mutations, namely c.876C > A (p.Tyr292*) and c.1407G > A (p.Trp469*), in an independent patient with OCA. Taken together, our data suggest that mutations in DCT can cause a phenotypic spectrum ranging from isolated infantile nystagmus to oculocutaneous albinism.

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.

Sequence analysis of tyrosinase gene in ocular and oculocutaneous albinism patients: introducing three novel mutations.

PURPOSE: Albinism is a heterogeneous genetic disorder of melanin synthesis that results in hypopigmented eyes (in patients with ocular albinism) or hair, skin, and eyes (in individuals with oculocutaneous albinism). It is associated with decreased visual acuity, nystagmus, strabismus, and photophobia. The tyrosinase gene is known to be involved in both oculocutaneous albinism and autosomal recessive ocular albinism. In this study, we aimed to screen the mutations in the TYR gene in the nonsyndromic OCA and autosomal recessive ocular albinism patients from Iran. METHODS: The tyrosinase gene was examined in 23 unrelated patients with autosomal recessive ocular albinism or nonsyndromic OCA using DNA sequencing and bioinformatics analysis. RESULTS: TYR gene mutations were identified in 14 (app. 60%) albinism patients. CONCLUSIONS: We found 10 mutations, 3 of which were novel. No mutation was found in our ocular albinism patients, but one of them was heterozygous for the p.R402Q polymorphism.

[Suspected pathogenic mutation identified in two cases with oculocutaneous albinism].

OBJECTIVE: To detect potential mutations in genes related with non-syndromic oculocutaneous albinism I-IV and ocular albinism type I in two couples who had given births to children with albinism. METHODS: All exons of the non-syndromic albinism related genes TYR, OCA2, TYRP-1, MITF, SLC45A2 and GPR143 were subjected to deep sequencing. The results were verified with Sanger sequencing. RESULTS: For the two female carriers, the coding region of the TYR gene was found to harbor a frameshift mutation c.925_926insC, which was also suspected to have been pathogenic. In one of the male partners, a nonsense mutations c.832C>T was found, which was also known to be pathogenic. Another male partner was found to harbor a TYR gene mutation c.346C>T, which was also known to be a pathogenic nonsense mutation. CONCLUSION: The coding region of the TYR gene c.925_926insC (p.Thr309ThrfsX9) probably underlies the OCA1 disease phenotype.

[Prenatal genetic diagnosis for two Chinese families affected with oculocutaneous albinism type â ¡].

OBJECTIVE: To perform genotyping analysis and subsequent prenatal genetic diagnosis for two families affected with oculocutaneous albinism (OCA). METHODS: Direct sequencing of TYR and P genes was performed in two albino probands. Family members were screened for corresponding mutant alleles. Prenatal genetic diagnoses were performed at early pregnancy by chorionic villus sampling (CVS) at mid-pregnancy through amniocentesis. RESULTS: No mutations were detected in the TYR gene in either probands, whereas 4 heterozygous mutations of the P gene were found, namely c.406C>T, c.535A>G, c.808-2A>G and c.2180T>C, among which c.535A>G and c.808-2A>G were novel. In the first round prenatal genetic testing, both fetuses were found to have the same genotypes as the probands. Both families had decided to terminate the pregnancy after genetic counseling. In the second round testing, neither of the fetuses was found to be affected by genotyping. The pregnancies continued and two healthy fetuses were born. CONCLUSION: OCA can be classified by genotyping, with which reliable prenatal diagnosis and feasible genetic counseling may be provided.

African origin of an intragenic deletion of the human P gene in tyrosinase positive oculocutaneous albinism.

Oculocutaneous albinism (OCA) is a genetically heterogeneous hypopigmentation disorder. One of the two major autosomal recessive forms involves the tyrosinase gene (OCA1), while the other form (OCA2) has recently been associated with alterations of the P gene on chromosome 15. OCA2 is about twice as common as OCA1 in African and African-American populations. We now describe an interstitial deletion that removes a single exon of the P gene. In a large family from an inbred population of tri-racial origin, all individuals with OCA2 were found to be homozygous for this allele. Moreover, the same mutant P allele was detected in several unrelated African American individuals with OCA2, but not in Caucasians with OCA2. The detection of the same allele in two unrelated Africans with OCA2 indicates an African origin for this allele.

DeoxyArbutin and its derivatives inhibit tyrosinase activity and melanin synthesis without inducing reactive oxygen species or apoptosis.

Safety is a major concern in developing commercial skin-lightening agents. Here, we report the modulating effects of deoxyArbutin (dA) and its second-generation derivatives - deoxyFuran (dF), 2-fluorodeoxyArbutin (fdA), and thiodeoxyArbutin (tdA) - on tyrosinase, and consequently, on melanization. Results demonstrate that dA and its derivatives inhibit tyrosine hydroxylase and dopa oxidase activity of tyrosinase. The inhibition is dose-dependent, thereby inhibiting melanin synthesis in intact melanocytes, when used at concentrations that retain 95% viability of the treated cells in culture. Herein we demonstrate that dA, and its second-generation derivatives dF, fdA, and tdA, exhibit dose-dependent reductions in melanocyte cell number, primarily due to inhibition of proliferation rather than initiation of apoptosis as exemplified by hydroquinone (HQ), ie, cytostatic as opposed to cytotoxic. Human and murine melanocytes with functional mutations in either tyrosinase or tyrosinase-related protein 1 (Tyrp1) are less sensitive to the cytostatic effects of dA and its derivatives. Minimal amounts of reactive oxygen species (ROS) were generated upon treatment with dA and its derivatives, in contrast to a dramatic amount of ROS induced by HQ. This increase in ROS subsequently induced the expression of the endogenous antioxidant catalase in treated melanocytes. Treatment with exogenous antioxidants provided protection for melanocytes treated with HQ, but not dA and its derivatives, suggesting that HQ exerts more oxidative stress. These studies demonstrate that dA and its derivatives are relatively safe tyrosinase inhibitors for skin lightening or for ameliorating hyperpigmented lesions.

[Early prenatal genetic diagnosis of oculocutaneous albinism type I in seven families].

OBJECTIVE: To evaluate the feasibility of genetic analysis of tyrosinase gene (TYR) in oculocutaneous albinism type I (OCA1). Mutation analysis and prenatal genetic diagnosis of TYR gene for seven pedigrees with OCA1 were performed. METHODS: PCR was used to amplify the exons, exon-intron boundaries and promoter of the TYR gene in the probands and/or their parents. The products were further analyzed by direct sequencing. Prenatal genetic diagnoses were performed by chorionic villus sampling after the genotypes of the probands or their parents were determined. RESULTS: Compound heterozygous mutations were detected in all pedigrees, which included 9 mutations, namely R76Q, c.232insGGG, R116X, R278X, R299H, c.929-930insC, IVS2-11delTT, Q399X and W400L. Among these, R76Q and Q399X were identified for the first time. Seven families have requested prenatal diagnoses. One fetus was detected with double mutations of TYR gene, and the parents have decided to have therapeutic abortion. Two fetuses did not carry the mutations identified in the probands, whilst other four fetuses were carriers of heterozygous mutations. Six families decided to carry on with the pregnancies. And the neonates did not show any symptoms of OCA after birth. CONCLUSION: Direct sequencing of the TYR gene is helpful for genetic counseling, prenatal diagnosis and carriers screening of OCA1.

Albino mice with the point mutation at the tyrosinase locus show high cholesterol diet-induced NASH susceptibility.

Non-alcoholic fatty liver disease (NAFLD) constitutes a metabolic disorder with high worldwide prevalence and increasing incidence. The inflammatory progressive state, non-alcoholic steatohepatitis (NASH), leads to liver fibrosis and carcinogenesis. Here, we evaluated whether tyrosinase mutation underlies NASH pathophysiology. Tyrosinase point-mutated B6 (Cg)-Tyrc-2J/J mice (B6 albino) and C57BL/6J black mice (B6 black) were fed with high cholesterol diet (HCD) for 10 weeks. Normal diet-fed mice served as controls. HCD-fed B6 albino exhibited high NASH susceptibility compared to B6 black, a phenotype not previously reported. Liver injury occurred in approximately 50% of B6 albino from one post HCD feeding, with elevated serum alanine aminotransferase and aspartate aminotransferase levels. NASH was induced following 2 weeks in severe-phenotypic B6 albino (sB6), but B6 black exhibited no symptoms, even after 10 weeks. HCD-fed sB6 albino showed significantly higher mortality rate. Histological analysis of the liver revealed significant inflammatory cell and lipid infiltration and severe fibrosis. Serum lipoprotein analysis revealed significantly higher chylomicron and very low-density lipoprotein levels in sB6 albino. Moreover, significantly higher small intestinal lipid absorption and lower fecal lipid excretion occurred together with elevated intestinal NPC1L1 expression. As the tyrosinase point mutation represents the only genetic difference between B6 albino and B6 black, our work will facilitate the identification of susceptible genetic factors for NASH development and expand the understanding of NASH pathophysiology.

The R402Q tyrosinase variant does not cause autosomal recessive ocular albinism.

Mutations in the gene for tyrosinase, the key enzyme in melanin synthesis, are responsible for oculocutaneous albinism type 1, and more than 100 mutations of this gene have been identified. The c.1205G > A variant of the tyrosinase gene (rs1126809) predicts p.R402Q and expression studies show thermolabile enzyme activity for the variant protein. The Q402 allele has been associated with autosomal recessive ocular albinism when it is in trans with a tyrosinase gene mutation associated with oculocutaneous albinism type 1. We have identified 12 families with oculocutaneous albinism type 1 that exhibit segregation of the c.1205G > A variant with a known pathologic mutation on the homologous chromosome, and demonstrate no genetic association between autosomal recessive oculocutaneous albinism and the Q402 variant. We conclude that the codon 402 variant of the tyrosinase gene is not associated with albinism.

Tyrosinase and ocular diseases: some novel thoughts on the molecular basis of oculocutaneous albinism type 1.

Tyrosinase (TYR) is a multifunctional copper-containing glycoenzyme (approximately 80 kDa), which plays a key role in the rate-limiting steps of the melanin biosynthetic pathway. This membrane-bound protein, possibly evolved by the fusion of two different copper-binding proteins, is mainly expressed in epidermal, ocular and follicular melanocytes. In the melanocytes, TYR functions as an integrated unit with other TYR-related proteins (TYRP1, TYRP2), lysosome-associated membrane protein 1 (LAMP1) and melanocyte-stimulating hormone receptors; thus forming a melanogenic complex. Mutations in the TYR gene (TYR, 11q14-21, MIM 606933) cause oculocutaneous albinism type 1 (OCA1, MIM 203100), a developmental disorder having an autosomal recessive mode of inheritance. In addition, TYR can act as a modifier locus for primary congenital glaucoma (PCG) and it also contributes significantly in the eye developmental process. Expression of TYR during neuroblast division helps in later pathfinding by retinal ganglion cells from retina to the dorsal lateral geniculate nucleus. However, mutation screening of TYR is complicated by the presence of a pseudogene-TYR like segment (TYRL, 11p11.2, MIM 191270), sharing approximately 98% sequence identity with the 3' region of TYR. Thus, in absence of a full-proof strategy, any nucleotide variants identified in the 3' region of TYR could actually be present in TYRL. Interestingly, despite extensive search, the second TYR mutation in 15% of the OCA1 cases remains unidentified. Several possible locations of these "uncharacterized mutations" (UCMs) have been speculated so far. Based on the structure of TYR gene, its sequence context and some experimental evidences, we propose two additional possibilities, which on further investigations might shed light on the molecular basis of UCMs in TYR of OCA1 patients; (i) partial deletion of the exons 4 and 5 region of TYR that is homologous with TYRL and (ii) variations in the polymorphic GA complex repeat located between distal and proximal elements of the human TYR promoter that can modulate the expression of the gene leading to disease pathogenesis.

Membrane-associated human tyrosinase is an enzymatically active monomeric glycoprotein.

Human tyrosinase (hTyr) is a Type 1 membrane bound glycoenzyme that catalyzes the initial and rate-limiting steps of melanin production in the melanosome. Mutations in the Tyr gene are linked to oculocutaneous albinism type 1 (OCA1), an autosomal recessive disorder. Currently, the application of enzyme replacement therapy for a treatment of OCA1 is hampered by the absence of pure hTyr. Here, full-length hTyr (residues 1-529) was overexpressed in Trichoplusia ni larvae infected with a baculovirus, solubilized with detergent and purified using chromatography. Michaelis-Menten kinetics, enzymatic specific activity, and analytical ultracentrifugation were used to compare the hTyr in detergent with the soluble recombinant intra-melanosomal domain, hTyrCtr (residues 19-469). Active hTyr is monomeric in detergent micelles suggesting no stable interactions between protein molecules. Both, hTyr and hTyrCtr, exhibited similar enzymatic activity and ligand affinity in L-DOPA and L-Tyrosine reactions. In addition, expression in larvae is a scalable process that will allow high yield protein production. Thus, larval production of enzymatically active human tyrosinase potentially could be a useful tool in developing a cure for OCA1.

Temperature-sensitive tyrosinase associated with peripheral pigmentation in oculocutaneous albinism.

Several types of autosomal recessive oculocutaneous albinism (OCA) are associated with abnormal tyrosinase function and a generalized reduction in or absence of cutaneous and eye melanin. Each is thought to result from a different mutant allele at the tyrosinase locus, with the mutation producing an enzyme with little or no activity in all involved tissues. In this paper, we report a new type of OCA that results from a tyrosinase allele producing a temperature-sensitive enzyme. The proband had white hair in the warmer areas (scalp and axilla) and progressively darker hair in the cooler areas (extremities) of her body. Melanocyte and melanosome architecture were normal. Quantitative hairbulb tyrosinase (dopa oxidase) assay demonstrated a loss of activity above 35-37 degrees C. Plasma pheomelanin and urine eumelanin intermediates were reduced and correlated with hair melanin content. This is the first temperature-sensitive tyrosinase mutation to be reported in humans and is analogous to the Siamese mutation in the cat and the Himalayan mutation in the mouse.

A tyrosinase gene missense mutation in temperature-sensitive type I oculocutaneous albinism. A human homologue to the Siamese cat and the Himalayan mouse.

Type I oculocutaneous albinism (OCA) is an autosomal recessive disorder in which deficient synthesis of melanin pigment results from abnormal activity of melanocyte tyrosinase. A novel type I OCA phenotype in which hypopigmentation is related to local body temperature is associated with a missense substitution in tyrosinase, codon 422 CGG (Arg)----CAG (Gln). This substitution results in a tyrosinase polypeptide that is temperature-sensitive. This form of type I OCA thus is homologous to the temperature-related forms of albinism seen in the Siamese cat and the Himalayan mouse.

Higher prevalence of OCA1 in an ethnic group of eastern India is due to a founder mutation in the tyrosinase gene.

PURPOSE: Oculocutaneous albinism (OCA) is a group of autosomal recessive disorders characterized by deficient synthesis of melanin pigment and associated with common developmental abnormalities of the eye. It is one of the major causes of childhood blindness in India. The disease is common among an ethnic group (Tili) of Eastern India, which represents about 12.56% of the Bankura district population (approximately 0.4 million) of West Bengal. The purpose of the study was to investigate the molecular lesions causing OCA within this ethnic group for the unequivocal diagnosis of the carriers and attempt to decipher the cause for the high prevalence of OCA. METHODS: Fourteen OCA-affected Tili families consisting a total of 161 individuals, including 26 patients, were recruited for the study. A lack of tyrosinase (TYR) activity among all the patients was ascertained by the tyrosinase hair bulb assay. Mutation screening in the tyrosinase gene (TYR) was done by single strand conformational polymorphism (SSCP) and DNA sequencing. The restriction fragment length polymorphism (RFLP) assay was carried out to determine the frequency of the pathogenic changes among the normal individuals. Haplotype analysis was performed at the TYR locus using a set of informative microsatellite and SNP markers. RESULTS: All the patients were homozygous for a null mutation (c.832C>T, Arg278stop) in TYR exon 2, which might cause a complete loss of enzyme activity. The mutation occurred in the same haplotype background. The frequency of the disease in this ethnic group was estimated to be significantly higher than the world average. CONCLUSIONS: OCA1 in the Tili population is due to the occurrence of a founder mutation in the TYR as indicated by haplotype analysis. Higher prevalence of the mutation in the population group is due to marriage within the same community. The diagnostic RFLP assay can be utilized for genetic counseling and thereby will help to reduce the disease load on the population.

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.

Mutational Analysis of the TYR and OCA2 Genes in Four Chinese Families with Oculocutaneous Albinism.

BACKGROUND: Oculocutaneous albinism (OCA) is an autosomal recessive disorder. The most common type OCA1 and OCA2 are caused by homozygous or compound heterozygous mutations in the tyrosinase gene (TYR) and OCA2 gene, respectively. OBJECTIVE: The purpose of this study was to evaluate the molecular basis of oculocutaneous albinism in four Chinese families. PATIENTS AND METHODS: Four non-consanguineous OCA families were included in the study. The TYR and OCA2 genes of all individuals were amplified by polymerase chain reaction (PCR), sequenced and compared with a reference database. RESULTS: Four patients with a diagnosis of oculocutaneous albinism, presented with milky skin, white or light brown hair and nystagmus. Genetic analyses demonstrated that patient A was compound heterozygous for c.1037-7T.A, c.1037-10_11delTT and c.1114delG mutations in the TYR gene; patient B was heterozygous for c.593C>T and c.1426A>G mutations in the OCA2 gene, patients C and D were compound heterozygous mutations in the TYR gene (c.549_550delGT and c.896G>A, c.832C>T and c.985T>C, respectively). The heterozygous c.549_550delGT and c.1114delG alleles in the TYR gene were two novel mutations. Interestingly, heterozygous members in these pedigrees who carried c.1114delG mutations in the TYR gene or c.1426A>G mutations in the OCA2 gene presented with blond or brown hair and pale skin, but no ocular disorders when they were born; the skin of these patients accumulated pigment over time and with sun exposure. CONCLUSION: This study expands the mutation spectrum of oculocutaneous albinism. It is the first time, to the best of our knowledge, to report that c.549_550delGT and c.1114delG mutations in the TYR gene were associated with OCA. The two mutations (c.1114delG in the TYR gene and c.1426A>G in the OCA2 gene) may be responsible for partial clinical manifestations of OCA.

Detection of 53 novel DNA variations within the tyrosinase gene and accumulation of mutations in 17 patients with albinism.

Oculocutaneous albinism (OCA) in man may be caused by mutations within the tyrosinase gene (TYR) resulting in OCA1. Analysing patients with recessively inherited albinism we found DNA variations in 82 unrelated individuals. 53 out of 78 mutations and polymorphisms revealed by this study are not published previously. The changes include 68 nucleotide substitutions resulting in amino acid changes, stop mutations and polymorphisms as well as four nucleotide insertions and six deletions. Furthermore, we found an accumulation of three to five mutations in 17 patients with OCA1.