Tyr Gene Research Articles

A 65 kilobase deletion of the upstream TYR gene region in a family with oculocutaneous albinism type 1

A 65 kilobase deletion of the upstream TYR gene region in a family with oculocutaneous albinism type 1

A missense mutation in the tyrosinase gene explains acromelanism in domesticated canaries.

Acromelanism is a form of albinism observed in several vertebrate species. In mammals, acromelanism is known to be caused by mutations in the tyrosinase gene (TYR) that induce a temperature-sensitive behavior of melanin synthesis, resulting in a characteristic hair color gradient. In birds, several phenotypes consistent with acromelanism have been reported, but their genetic basis remains unknown. This study aimed to identify the genetic basis of an acromelanistic phenotype in domesticated canaries known as pearl and test whether it is caused by the same molecular mechanism described for mammals. To do this, we compared the genomes of pearl and non-pearl canaries and searched for potentially causative genetic mutations. Our results suggest that the pearl phenotype is caused by a mutation in the TYR gene encoding a TYR-P45H missense substitution. Our findings further suggest that reports of acromelanism in other bird species might be explained by TYR mutations.

Common Variants in the TYR Gene with Unclear Pathogenicity as the Cause of Oculocutaneous Albinism in a Cohort of Russian Patients.

Background: oculocutaneous albinism (OCA) is a hereditary impairment of skin, hair, and eye pigmentation. The most common form of albinism is autosomal recessive albinism, caused by mutations in the TYR gene, accounting for approximately 40-50% of all cases of the disease in European populations. Common hypomorphic variants in the TYR gene could lead to a mild form of albinism in a compound heterozygous state with a pathogenic variant. Methods: we examined by allele specific MLPA a cohort consisting of 118 unrelated patients with albinism and 10 parents of these patients. The control cohort consisted of 200 unexamined Russian residents. Results: the patients with albinism were divided into three groups: without pathogenic variants in the TYR gene-70 patients, with one pathogenic variant in the TYR gene-20 patients, and with two pathogenic variants in the TYR gene-28 patients. Among the 20 patients with a single heterozygous variant in the TYR gene, 15 patients had the c.575C>A p.(Ser192Tyr) variant, and 15 had the c.1205G>A p.(Arg402Gln) variant. Both the c.575C>A p.(Ser192Tyr) and c.1205G>A p.(Arg402Gln) variants were identified in 12 patients. In addition to the aforementioned variants, an intronic variant c.1185-6208A>G (rs147546939) was identified in seven patients. Conclusions: the frequencies and the number of alleles c.575A, c.1205A, and c.1185-6208G in different groups of patients and the control group were compared. In this study, we demonstrate that the complex alleles [c.575C>A p.(Ser192Tyr); c.1205G>A p.(Arg402Gln)] and [c.575C>A p.(Ser192Tyr); c.1185-6208A>G; c.1205G>A p.(Arg402Gln)] are associated with oculocutaneous albinism, which is consistent with findings from other researchers.

The co-occurrence of genetic variants in the TYR and OCA2 genes confers susceptibility to albinism

Although rare genetic conditions are mostly caused by DNA sequence alterations that functionally disrupt individual genes, large-scale studies using genome sequencing have started to unmask additional complexity. Understanding how combinations of variants in different genes shape human phenotypes is expected to provide important insights into the clinical and genetic heterogeneity of rare disorders. Here, we use albinism, an archetypal rare condition associated with hypopigmentation, as an exemplar for the study of genetic interactions. We analyse data from the Genomics England 100,000 Genomes Project alongside a cohort of 1120 individuals with albinism, and investigate the effect of dual heterozygosity for the combination of two established albinism-related variants: TYR:c.1205 G > A (p.Arg402Gln) [rs1126809] and OCA2:c.1327 G > A (p.Val443Ile) [rs74653330]. As each of these changes alone is insufficient to cause disease when present in the heterozygous state, we sought evidence of synergistic effects. We show that, when both variants are present, the probability of receiving a diagnosis of albinism is significantly increased (odds ratio 12.8; 95% confidence interval 6.0 – 24.7; p-value 2.1 ×10−8). Further analyses in an independent cohort, the UK Biobank, support this finding and highlight that heterozygosity for the TYR:c.1205 G > A and OCA2:c.1327 G > A variant combination is associated with statistically significant alterations in visual acuity and central retinal thickness (traits that are considered albinism endophenotypes). The approach discussed in this report opens up new avenues for the investigation of oligogenic patterns in apparently Mendelian disorders.

Anti-Melanogenic Potential of Malabar Spinach (Basella alba) in Human Melanoma Cells with Oxidative Stress Suppression and Anti-Inflammatory Activities.

Basella alba has been used in Thai remedies to treat skin disorders, but scientific evidence supporting its efficacy is currently limited. In this study, we investigated the inhibitory effects of B. alba extracts on melanin production using melanoma cells, as well as their impact on oxidative stress and inflammation in keratinocytes. The results demonstrate that B. alba extracts inhibited melanin content and cellular tyrosinase activity in 3-isobutyl-1-methylxanthine (IBMX)-induced melanoma cells by downregulating MITF and the pigmentary genes TYR, TRP-1, and DCT. Interestingly, the MITF regulator gene was inhibited by both the 50% and 95% ethanolic extracts of B. alba with levels of 0.97 ± 0.19 and 0.92 ± 0.09 of the control, respectively, which are comparable to those observed in the arbutin treatment group at 0.84 ± 0.05 of the control. Moreover, after hydrogen peroxide (H2O2) exposure, pretreatment with B. alba reduced lipid peroxidation byproducts and increased the levels of antioxidant-related genes, including SOD-1, GPX-1, and NRF2. Notably, the suppression of the POMC promoter gene in keratinocytes was observed, which may disrupt melanogenesis in melanocytes involving the MC1R signaling pathway. MC1R mRNA expression decreased in the treatments with 50% and 95% ethanolic extracts of B. alba, with relative levels of 0.97 ± 0.18 and 0.90 ± 0.10 of the control, respectively, similar to the arbutin-treated group (0.88 ± 0.25 of control). A significant reduction in nitric oxide was also observed in the B. alba-treated groups, along with a decrease in genes associated with pro-inflammatory cytokines, including IL-1β, IL-6, and COX-2. These findings suggest that B. alba has potential in the prevention of skin-related problems.

Biallelic TYR and TKFC variants in Egyptian patients with OCA1 and new expanded TKFC features

BackgroundOculocutaneous albinism type1 (OCA1) is caused by the TYR gene's homozygous and compound heterozygous variants. TKFC gene variants cause triokinase & FMN cyclase deficiency syndrome with variable multisystemic disorders.ObjectivesTo determine the potential disease-causing variants in two deceased patients presenting atypical OCA1 features by demonstrating three generations for a single family. The two deceased neonates had severe skeletal abnormalities and fatal hypertrophic cardiomyopathy. We also explored the potential mechanisms for the causative relationship between TKFC and multisystem disorders.Patients and methodsDue to the new emerging symptoms that weren’t reported before with the TYR gene, the following methods were performed: Sanger sequencing for the TYR gene, followed by whole exome sequencing, co-segregation, and computational analyses.ResultsExtensive parental consanguinity was found, and consequently an autosomal recessive mode of inheritance was prioritized. Upon performing sequencing and segregation data, the following has been confirmed: positive co-segregation of nonsense homozygous NM_000372.5:c.346C > T p.(Arg116*) variant in TYR gene and multisystem disease-missense homozygous NM_015533.4:c.598G > A p.(Val200Ile) variant in TKFC gene in the two affected index patients who deceased due to hypertrophic cardiomyopathy. Using computational analysis, we found that c.598G > A p.(Val200Ile) pathogenicity has led to the failure of L2-K1 active site closure due to the potential differential fluctuation between valine and isoleucine residues. Subsequently, disruption of endogenous DHA phosphorylation was found. Two potential mechanisms exploring the causative relationship between TKFC gene and multisystem disorders have been suggested.ConclusionsThis study presented a first family with the co-existence of biallelic variants in TYR and TKFC genes associating severe skeletal abnormalities and lethal hypertrophic cardiomyopathy. Neither of these genes would have been pursued in the standard genetic counseling. Such discovery is paving the way for more efficient genetic counseling. Comparing TKFC results with literature data showed that our relevant expanded TKFC variant is the 3rd worldwide.

Visual input regulates melanophore differentiation.

Developmental processes continue in organisms in which sensory systems have reached functional maturity, however, little research has focused on the influence of sensory input on cell and tissue development. Here, we explored the influence of visual system activity on the development of skin melanophores in Xenopus laevis. Melanophore number was measured in X. laevis larvae after the manipulation of visual input through eye removal (enucleation) and/or incubation on a white or black substrate at the time when the visual system becomes functional (stage 40). To determine the developmental process impacted by visual input, migration, proliferation and differentiation of melanophores was assessed. Finally, the role of melatonin in driving melanophore differentiation was explored. Enucleating, or maintaining stage 40 larvae on a black background, results in a pronounced increase in melanophore number in the perioptic region within 24 h. Time lapse analysis revealed that in enucleated larvae new melanophores appear through gradual increase in pigmentation, suggesting unpigmented cells in the perioptic region differentiate into mature melanophores upon reduced visual input. In support, we observed increased expression of melanization genes tyr, tyrp1, and pmel in the perioptic region of enucleated or black background-reared larvae. Conversely, maintaining larvae in full light suppresses melanophore differentiation. Interestingly, an extra-pineal melatonin signal was found to be sufficient and necessary to promote the transition to differentiated melanophores. In this study, we found that at the time when the visual system becomes functional, X. laevis larvae possess a population of undifferentiated melanophores that can respond rapidly to changes in the external light environment by undergoing differentiation. Thus, we propose a novel mechanism of environmental influence where external sensory signals influence cell differentiation in a manner that would favor survival.

Agouti-Signaling Protein and Melanocortin-1-Receptor Mutations Associated with Coat Color Phenotypes in Fallow Deer (Dama dama).

Four dominant coat color phenotypes are found in fallow deer (Dama dama). Brown is the most common. Black, menil, and white occur with varying frequencies. In order to gain insights into the molecular genetic background of these phenotypes, 998 fallow animals (772 brown, 62 black, 126 menil, and 38 white) were examined for mutations in the ASIP, MC1R, TYR, and SLC45A2 genes. In ASIP, two mutations (ASIP-M-E2, located at the boundary from exon 2 to intron 2; and ASIP-M-E3, an InDel of five nucleotides) were found, leading to black fallow deer being either homozygous or heterozygous in combination. There were also two mutations found in MC1R. Whereby the mutation MC1R-M1 (leucine to proline, L48P) homozygous leads to a white coat, while the mutation MC1R-M2 (glycine to aspartic acid, G236D) homozygous is associated with the menil phenotype. When both mutations occur together in a heterozygous character state, it results in a menil coat. Since the mutations in the two genes are only present alternatively, 36 genotypes can be identified that form color clusters to which all animals can be assigned. No mutations were found in the TYR and SLC45A2 genes. Our investigations demonstrate that the four dominant coat colors in fallow deer can be explained by ASIP and MC1R mutations only.

Identifying genetic defects in oculocutaneous albinism patients of West Bengal, Eastern India.

Oculocutaneous albinism (OCA) is a congenital heterogeneous group of autosomal recessive disorders characterized by the absence or loss of melanin in the skin, eyes and hair of the affected individuals. Based on the mutated gene, OCA has been classified into eight sub-types (OCA1-8) with overlapping clinical phenotypes. Mutations in the TYR gene cause OCA1, the most prevalent OCA worldwide including India. Mutations in OCA2 and SLC45A2, both of which regulate melanosomal pH that is critical to TYR activity, cause OCA2 and OCA4 respectively, the other common OCA subtypes in India. In the present study, we have included 54 OCA-affected cases from 41 unrelated families representing 16 different marriage/ethnic groups from 17 districts of West Bengal, India. We pursued a PCR-sequencing based approach followed by bioinformatic analysis to identify mutations in TYR, OCA2 and SLC45A2 genes. Mutations were detected in 27 of the 54 (50%) OCA patients from 18 unrelated families, representing 9 different marriage/ethnic groups from 11 districts of West Bengal. Three TYR variants: NM_000372.4: c.391 A > G, NP_000363.1: p. Lys131Glu; NM_000372.4: c.1037G > T; NP_000363.1: p. Gly346Val, NM_000372.4: c.715 C > T; NP_000363.1:p.Arg239Trp was identified for the first time in Eastern Indian OCA cases. A novel nonsense variant: NM_016180.5: c.389T > A, NP_057264.4: p. Leu130* and a novel synonymous variation NM_016180.5: c.1092 A > G; NP_057264.4: p.364E = were identified in SLC45A2. Additionally, NM_016180.5: c.904A > T; NP_057264.4: p. Thre302Ser was identified for the first time in any Eastern Indian OCA case. We identified 2 previously reported mutations in OCA2. In concordance with previous reports, NM_000372.4: c.832C > T, NP_000363.1: p. (Arg278*) was the commonest TYR mutation. The results of our study enrich the mutational spectrum of the known OCA causing genes in Eastern India, which would facilitate accurate diagnosis, familial screening, carrier detection and containment of the disease load.

P-806 Heritable genome editing in haploid mouse embryonic stem cells

Abstract Study question Can we edit a specific gene by CRISPR-Cas9 in haploid mouse embryonic stem cells? Summary answer We successfully carried out heritable genome editing of a coat pigmentation gene in haploid mouse embryonic stem cells via electroporation. What is known already Embryonic stem cells have been widely used as a substrate to carry out genetic studies due to their availability, low cost and ease of handling. CRISPR-Cas9 has been successful in editing genes in the human model. Gene editing occurred by homology directed repair or non-homologous end joining. The utilization of a haploid genome would confirm the feasibility of non-homologous end joining repair mechanism. Study design, size, duration In the past 3 months, diploid as positive control and experimental haploid embryonic stem cells were independently cultured for genome editing experiments. Electroporation was used as a means to introduce CRISPR-Cas9 into cells. The negative control cohort consisted of embryonic stem cells electroporated without delivering any CRISPR solution. CRISPR-Cas9 was designed to knock out the Tyr gene to create an albino phenotype. Participants/materials, setting, methods Diploid embryonic stem cells were derived from C57BL/6 mice. The haploid counterpart instead was obtained from haploid androgenetic embryos using spermatozoa from Oct4-EGFP mice. CRISPR solution was prepared by combining Tyr gRNA and Cas9 protein. For each experiment, one million cells were suspended in CRISPR solution and an electroporation buffer. Then, electric pulses were applied for electroporation with 4 different parameters. Gene modification at the target site was confirmed using T7E1 cleavage assay. Main results and the role of chance A 584 base pairs region around the CRISPR target site was amplified in all the diploid and haploid mouse embryonic stem cells. After performing T7E1 cleavage assay, editing efficiency was calculated following the manufacturer protocol. Precisely, after testing out different electroporation conditions on diploid mESCs, 1350V/20ms/2 pulses together with the combination of three sgRNAs (exon 1 and exon 2) and Cas9 protein, provided the best genome editing efficiency at 36.8% with a viability of 80%. Interestingly, when delivering only one type of gRNA (exon 2) together with Cas9 protein, we saw an efficiency rising to 77.6%. We then used the same condition to edit the Tyr gene in the genome of haploid androgenetic stem cells and confirmed that also in these cells, we were able to obtain a comparable genome editing efficiency at 68.4%. Limitations, reasons for caution While these experimental observations are limited, it is encouraging that it is possible to target and edit a haploid genome with a single allele. Wider implications of the findings The utilization of a haploid cell culture model paves the way to utilize CRISPR-Cas9 for gamete genome editing. This will provide a consistent and reliable method to correct inheritable genetic conditions on gametes allowing uniform gene editing of all cell progeny. Trial registration number Not applicable

P-805 Sperm and embryo genome editing by CRISPR-Cas9 in a mouse model

Abstract Study question We wonder whether it is possible to edit a specific gene in individual mammalian spermatozoa as well as embryo by using CRISPR-Cas9? Summary answer We were able to successfully edit the coat pigment gene on individual spermatozoa as well as embryos by injecting CRISPR-Cas9 during Piezo-ICSI. What is known already Earlier investigations into embryo genome editing with CRISPR-Cas9 were conducted during the S-phase or zygote stage, encountering obstacles such as mosaicism and loss of heterozygosity. To overcome these challenges, performing genome editing at the gamete level is considered more favorable. While it may be achievable in oocytes, sperm genome editing can be ambitious due to the tightly supercoiled DNA around the protamine cores. In initial attempts, simple permeabilization of the sperm membrane allowed CRISPR-Cas9 penetration into the cell; however, the attempts at genomic editing proved to be unsuccessful. Study design, size, duration In the past several months, oocytes were divided into two groups: half of them were used to perform embryo genome editing, whilst the other half was used to edit exclusively the sperm genome through oocyte-mediated sperm decondensation (OMSD), where a single spermatozoon was injected into an enucleated oocyte and used the ooplasmic machinery for DNA decondensation and editing. In both models, CRISPR-Cas9 was used to knock out Tyr gene to create an albino phenotype. Participants/materials, setting, methods B6D2F1 mice were used to retrieve oocytes and spermatozoa. A cohort of oocytes used for the OSMD approach were enucleated, whilst the others were maintained with an intact nucleus. All oocytes, enucleated or not, were injected with a single spermatozoon with Tyr-sgRNA and Cas9 protein. Embryos were cultured until the 8-cell stage and processed for T7E1 cleavage analysis. Genome editing efficiency was calculated following the manufacturer protocol. Main results and the role of chance Of the 40 oocytes used for the study, 20 intact oocytes were used to perform embryo genome editing in the standard fashion, while 20 were enucleated for OMSD experiments. After undergoing Piezo-ICSI with CRISPR-Cas9 solution, 90.0% (18/20) of intact oocytes fertilized. After 48 hours in culture, diploid embryos reached the 8-cell stage at 88.9% (16/18). The 584-bp region of the target site of extracted DNA was amplified and gene modification of the edited diploid embryo cohort was confirmed in 87.5% (14/16). Compared to the OMSD cohort, enucleated oocytes fertilized at 85.0% (17/20) while cleavage rate was lower at 64.7% (11/17) and sperm genome modification at 81.8% (9/11). Both diploid embryos and haploid androgenetic embryos evidence an editing efficiency of 36.8%. Therefore, we verified that genome editing on embryos and sperm achieved the same editing yield. Limitations, reasons for caution While the experimental observations are still limited, it is important to note that these results in the embryo model may be underestimated by the T7E1 cleavage assay that only recognize heterozygous modification. The technique needs further refinement to optimize targeting and editing efficiency. Wider implications of the findings Sperm genome decondensation through an OMSD approach allows CRISPR-Cas9 to edit the sperm genome in order to correct eventual pathogenic mutations present in the gamete. Moreover, these would be the only method to absolutely ensure uniform generation of resulting embryos. Trial registration number not applicable

Antioxidant, Anti-Inflammation, and Melanogenesis Inhibition of Sang 5 CMU Rice (Oryza sativa) Byproduct for Cosmetic Applications.

Prolonged exposure to environmental oxidative stress can result in visible signs of skin aging such as wrinkles, hyperpigmentation, and thinning of the skin. Oryza sativa variety Sang 5 CMU, an inbred rice cultivar from northern Thailand, contains phenolic and flavonoid compounds in its bran and husk portions that are known for their natural antioxidant properties. In this study, we evaluated the cosmetic properties of crude extracts from rice bran and husk of Sang 5 CMU, focusing on antioxidant, anti-inflammatory, anti-melanogenesis, and collagen-regulating properties. Our findings suggest that both extracts possess antioxidant potential against DPPH, ABTS radicals, and metal ions. Additionally, they could downregulate TBARS levels from 125% to 100% of the control, approximately, while increasing the expression of genes related to the NRF2-mediated antioxidant pathway, such as NRF2 and HO-1, in H2O2-induced human fibroblast cells. Notably, rice bran and husk extracts could increase mRNA levels of HO-1 more greatly than the standard L-ascorbic acid, by about 1.29 and 1.07 times, respectively. Furthermore, the crude extracts exhibited anti-inflammatory activity by suppressing nitric oxide production in both mouse macrophage and human fibroblast cells. Specifically, the bran and husk extracts inhibited the gene expression of the inflammatory cytokine IL-6 in LPS-induced inflammation in fibroblasts. Moreover, both extracts demonstrated potential for inhibiting melanin production and intracellular tyrosinase activity in human melanoma cells by decreasing the expression of the transcription factor MITF and the pigmentary genes TYR, TRP-1, and DCT. They also exhibit collagen-stimulating effects by reducing MMP-2 expression in H2O2-induced fibroblasts from 135% to 80% of the control, approximately, and increasing the gene associated with type I collagen production, COL1A1. Overall, the rice bran and husk extracts of Sang 5 CMU showed promise as effective natural ingredients for cosmetic applications.

Molecular characterization, spatiotemporal expression, and background adaptation regulation of tyrosinase in loach (Misgurnus anguillicaudatus).

The Poyang Lake region is home to large-blackspot loaches (LBL), small-blackspot loaches (SBL), and non-blackspot loaches (NBL), Misgurnus anguillicaudatus. To investigate the impact of tyrosinase on spot development, the complementary DNAs (cDNA) of tyrosinase in M. anguillicaudatus (designated as Matyr) were cloned using the rapid amplification of cDNA ends (RACE)-PCR method. The full-length cDNA for Matyr was 2020 bp, and the open-reading frame comprised 1617 bp, encoding a predicted protein with 538 amino acids. Phylogenetic studies revealed that MaTyr was first grouped with Tyr of Triplophysa tibetana and Leptobotia taeniops, and then Tyr of other cyprinid fish. The quantitative reverse-transcription-PCR results show that Matyr was highly expressed in the muscle, caudal fin, and dorsal skin. The Matyr gene's messenger RNA expression pattern steadily increased from the fertilized ovum period to the somitogenesis period, and from the muscle effect stage to 6 days after fertilization, it considerably increased (p < 0.01). The Matyr hybridization signals with similar location could be found in all developmental stages of three kinds of loaches using whole-mount in situ hybridization (WISH) technology and were the strongest during the organ development period and melanin formation period. Dot hybridization signals in LBLs rapidly spread to the back of the body beginning at the period when the eyes first formed melanin, and their dimensions were larger than those of NBLs during the same time period. The body color of loaches could change reversibly with black/white background adaptation. The α-msh, mitfa, and tyr are mainly expressed in loaches adapted with a black background. Tyr gene could be involved in the development of blackspots and body color polymorphism, and contribute to organ development in the loach.

Discovery of (E)-3-(4-chlorophenyl)-N-(5-hydroxypentyl)acrylamide among N-substituted cinnamamide derivatives as a novel cosmetic ingredient for hyperpigmentation

Hyperpigmentation disorders may result from inappropriate melanin deposition and/or excessive melanin synthesis. They are classified mainly as aesthetic problems, but they can significantly affect human health by decreasing self-esteem. There are available only limited treatment options for hyperpigmentation disorder, among others, cosmetic products applied topically. Depigmenting ingredients were found to be ineffective and characterized by various side effects. As a result, many efforts are made to discover novel, potent, and safe melanogenesis inhibitors for possible use in topical cosmetic depigmenting formulations. Cinnamic acid derivatives constitute a widely tested group for that purpose. This article reports research in the group of N-alkyl cinnamamide derivatives (un)substituted in phenyl ring. Among tested series, (E)-3-(4-chlorophenyl)-N-(5-hydroxypentyl)acrylamide (compound 21) showed the most promising inhibitory properties in mushroom tyrosinase assay (IC50 = 36.98 ± 1.07 µM for monophenolase activity, IC50 = 146.71 ± 16.82 µM for diphenolase activity) and melanin production inhibition in B16F10 mouse melanoma cell line at concentration 6.25 µM resulting probably from decreasing of Tyr, Mitf, Tyrp-1, and Tyrp-2 genes expression. This compound also showed melanin production inhibitory properties in pigmented reconstructed human epidermis when used in 1 % and 2 % solutions in 50 % PEG400. In vitro evaluation of its safety profile showed no cytotoxicity to human keratinocytes HaCaT, human skin fibroblasts BJ, and human primary epidermal melanocytes HEMa, no mutagenicity in the Ames test, no genotoxicity in micronucleus test, no phototoxicity, as well as no skin irritation potential tested in PEG400 solution. This compound was also shown to penetrate across the epidermis to reach the possible site of action. The performed research led to classify (E)-3-(4-chlorophenyl)-N-(5-hydroxypentyl)acrylamide as a novel potential depigmenting cosmetic ingredient.

Whole-exome sequencing combined with in silico protein analysis identifies rare truncating variants in the TYR gene associated with oculocutaneous albinism IA (OCA1A) in an Iranian patient

Oculocutaneous albinism type IA (OCA1A) is an autosomal recessive tyrosinase-negative condition caused by deleterious variants in the TYR gene. Using whole-exome sequencing (WES) followed by Sanger sequencing, we detected rare pathogenic variants (c.286dupA and c.763C > T) within the TYR gene in a 12-year-old Iranian female who met the clinical criteria proposed for OCA. Through comprehensive in silico protein analysis, we elucidated the functional impact of these two variants. c.286dupA induced a frameshift, leading to the loss of the entire copper-binding tyrosinase domain and the only transmembrane melanosome-bound tyrosinase helix. Similarly, c.763C > T introduced a premature stop codon, resulting in the loss of approximately two-thirds of the copper-binding tyrosinase domain and the transmembrane helix. These findings signified a substantial disturbance in the normal function of tyrosinase. Our study presents the first report of co-occurring pathogenic c.286dupA and c.763C > T variants, comprehensively investigating their functional impact using in silico protein tools.

An Efficient Vector-Based CRISPR/Cas9 System in Zebrafish Cell Line.

The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system has been widely applied in animals as an efficient genome editing tool. However, the technique is difficult to implement in fish cell lines partially due to the lack of efficient promoters to drive the expression of both sgRNA and the Cas9 protein within a single vector. In this study, it was indicated that the zebrafish U6 RNA polymerase III (ZFU6) promoter could efficiently induce tyrosinase (tyr) gene editing and lead to loss of retinal pigments when co-injection with Cas9 mRNA in zebrafish embryo. Furthermore, an optimized all-in-one vector for expression of the CRISPR/Cas9 system in the zebrafish fibroblast cell line (PAC2) was constructed by replacing the human U6 promoter with ZFU6 promoter, basing on the lentiCRISPRV2 system that widely applied in mammal cells. This new vector could successfully target the cellular communication network factor 2a (ctgfa) gene and demonstrated its function in the PAC2 cell. Notably, the vector could also be used to edit the endogenous EMX1 gene in the mammal 293T cell line, implying its wide application potential. In conclusion, we established a new gene editing tool for zebrafish cell line, which could be a useful in vitro platform for high-throughput analyzing gene function in fish.

Characterization of the Tyrosinase Gene of Aquarium-Maintained Nyalian Fish (Rasbora sp.)

Rasbora fish is a local Indonesian fish whose population has decreased. In the Province of Bali this fish is called the Nyalian fish and no one has cultivated this fish. Nyalian fish in Bali Province are used as consumption fish and have potential as freshwater ornamental fish. Catching Nyalian fish continuously can cause this fish decrease in population and can cause extinction. Nyalian fish conservation can be done by domestication and adapting fish caught from their natural habitat to aquaculture aquarium, then used in aquaculture activities and ensuring continuity of numbers and avoiding extinction. During the domestication process, the Nyalian fish will experience stress due to changes in their habitat. Stress in fish can be shown by a drastically darker body condition. Parts of the body that blacken or show a darker color are due to the activity of melanin as an active role in it. The Tyrosinase gene is one of the genes responsible for coding pigment patterns. The TYR gene has an important role in instructing the formation of the enzyme Tyrosinase.

Putrescine Upregulates Melanogenesis Through Modulation of MITF Transcription Factor in B16F1 Mouse Melanoma Cells.

Ageing is a biochemical, metabolic and genetic physiological phenomenon. The suppression of melanin biosynthesis, evident in the greying of the hair, is a hallmark of ageing resulting from translation failure, reduced enzyme activity and cellular senescence. Putrescine, the smallest member of the polyamine family and an organic chemical, is present in living mammalian cells and plays a crucial role in regulating skin melanogenesis. Therefore, the purpose of this study is to explore the effect of putrescine on the signalling pathways of melanogenesis in melanoma cells. Melanin production capacity of putrescine was analysed using a tyrosinase activity assay. To assess the cell viability of B16F1 cells exposed to putrescine, a tetrazolium dye MTT assay was performed. The effect of putrescine on melanin synthesis in the presence of H2O2 was evaluated using various in vitro assays in B16F1 cells. The effect of putrescine on melanin production in B16F1 cells was determined using a specific melanin production assay. Gene expression was analysed using real-time polymerase chain reaction (RT-PCR). Furthermore, the effect of putrescine on the expression of proteins related to melanin production in the cells treated with H2O2 was analysed by immunofluorescence and Western blot analysis. Putrescine increased tyrosinase activity and showed no cytotoxicity in B16F1 cells. In addition, putrescine effectively scavenged H2O2, as shown by the reduction of intracellular H2O2 amounts in 2',7'-dichlorofluorescin diacetate analysis, and promoted melanin production in living cells. The stimulation of melanogenesis by putrescine was attributed to the increased expression of Mitf, Tyr, Trp-1 and Trp-2 genes. Immunofluorescence assays revealed that putrescine enhanced the expression of proteins associated with melanogenesis and upregulated TYR, TRP-1 and TRP-2 via the microphthalmia-associated transcription factor (MITF) and increased the expression of methionine sulfoxide reductases A (MSRA) and B (MSRB) in the cells treated with H2O2, effectively promoting melanogenesis. These results suggest that putrescine can be used to stimulate melanin synthesis. This is the first study to investigate the effect of putrescine on the signalling pathways of melanogenesis in B16F1 melanoma cells. The results confirm that putrescine can promote melanogenesis through the expression of TYR, TRP-1 and TRP-2 via the MITF in cells treated with H2O2. Putrescine can be used exclusively as a cosmetic product to prevent premature greying of hair.

Molecular understanding of the therapeutic potential of melanin inhibiting natural products.

With the development of society and the improvement of people's living standards, there is an increasing demand for melanin-inhibiting products that prioritize health, safety, and efficacy. Therefore, the development of natural products that can safely and efficiently inhibit melanin synthesis is of great social significance and has significant market potential. In this paper, by reviewing the literature reported in recent years, we summarized the natural products with inhibition of melanin synthesis effects that have been put into or not yet put into the market, and classified them according to the chemical groups of their compounds or the extraction methods of the natural products. Through the summary analysis, we found that these compounds mainly include terpenoids, phenylpropanoids, flavonoids and so on, while the natural product extracts mainly include methanol extracts, ethanol extracts, and aqueous extracts. Their main inhibition of melanin synthesis mechanisms include: (1) direct inhibition of tyrosinase activity; (2) down-regulation of the α-MSH-MC1R, Wnt, NO, PI3K/Akt and MAPK pathways through the expression of MITF and its downstream genes TYR, TRP-1, and TRP-2; (3) antioxidant; (4) inhibition of melanocyte growth through cytotoxicity; (5) inhibition of melanosome production and transport. This paper provides an in-depth discussion on the research progress of whitening natural products and their market value. The aim is to offer guidance for future research and development of natural skin whitening products.

The molecular landscape of oculocutaneous albinism in India and its therapeutic implications.

Oculocutaneous albinism is an inherited disorder of melanin biosynthesis, characterized by absent or reduced pigmentation of the skin, hair, and eyes. Molecular alterations of genes that cause non-syndromic albinism in Asian Indians are poorly characterized. This information would be useful for developing therapies for this disorder. We analyzed 164 persons with non-syndromic albinism, belonging to unrelated families from all parts of India, for molecular changes in the causative genes. Subjects with white hair, white skin, and red iris had their tyrosinase gene sequenced and were also tested by MLPA for deletions/duplications. Subjects with negative results or with darker skin, golden/brown or darker hair had sequencing of TYR, P, TYRP1, SLC45A2 and GPR143 genes. Pathogenic variants in TYR (OCA1) were observed in 139 (84.7%) patients, in the P gene (OCA2) in 20 (12.2%), in TYRP1 (OCA3) in two (1.2%), in SLC45A2 (OCA 4) in one (0.61%), and in GPR143 (X-linked ocular albinism) in two (1.2%) patients. Of 278 alleles with variants in TYR, 179 (64.3%) alleles had (p.R278*) alteration, suggesting the possibility of therapy with a stop codon readthrough molecule. We report 20 patients with 13 disease associated variants in the P gene and 18 novel pathogenic variants in TYR, P, TYRP1, SLC45A2 and GPR143 genes. The data are compared with those reported from India, Pakistan and rest of the world. The therapeutic options in albinism are briefly described, opening this field for future therapies.