SLC45A2 Research Articles

SLC24A5 plays fundamental roles in regulating melanophore development in Cyprinidae fish

The mechanism of color pattern formation in animals is complicated. Oujiang color common carp (Cyprinus carpio var. color) is an excellent fish model for studying the molecular mechanism of pigmentation. Solute carrier family 24 member 5 (SLC24A5) was identified as a key gene in regulating melanophore development and melanin distribution in organisms. In this study, functional experiments were conducted on the SLC24A5 gene in Oujiang color common carp. The full-length of SLC24A5 mRNA was cloned and obtained. In Oujiang color common carp, qRT-PCR and whole-mount in situ hybridization indicated that SLC24A5 was highly expressed in tissues in the presence of melanin and initiated its expression along with the presence of melanin during embryo development. The SLC24A5 knocked-out via CRISPR/cas9 caused an obvious loss of melanophore in larva and adult zebrafish. In addition, the homozygous SLC24A5−/− zebrafish mutant phenotype was rescued by the over expression of the SLC24A5 from Oujiang color common carp. Results suggested that SLC24A5 is essential for regulating melanophore development and that the functional and structures of SLC24A5 are conserved in Cyprinidae fish.

Genome Editing Using the CRISPR-Cas9 System to Generate a Solid-Red Germline of Nile Tilapia (Oreochromis niloticus).

In recent years, there has been increasing demand for red tilapia, which are commercial strains of hybrids of different tilapiine species or red variants of highly inbred Nile tilapia. However, red tilapia phenotypes are genetically unstable and affected by environmental factors, resulting in nonuniform coloration with black or dark-red color blotches that reduce their market value. Solute carrier family 45 member 2 (SLC45A2) is a membrane transporter that mediates melanin biosynthesis and is evolutionarily conserved from fish to humans. In the present study, we describe the generation of a stable and heritable red tilapia phenotype by inducing loss-of-function mutations in the slc45a2 gene. For this purpose, we identified the slc45a2 gene in Nile tilapia and designed highly specific guide RNAs (gRNA) for its genomic sequence. Multiplex microinjection of slc45a2-specific ribonucleoproteins to Nile tilapia zygotes induced up to 97-99% albinism, including loss of melanin in the eye. Next-generation sequencing of the injected zygotes demonstrated that all injected fish carried mutant alleles with variable mutagenesis efficiencies. Sanger sequencing of the genomic target region in the slc45a2 gene from fin clips, sperm, and F1 offspring of a highly mutant male identified various genomic indels and germline transmission of the sperm-identified indels. Overall, this work demonstrates the generation of somatic and germline slc45a2 mutant alleles, which leads to complete albinism in Nile tilapia.

Antiretroviral Drug Transporters and Metabolic Enzymes in Circulating Monocytes and Monocyte-Derived Macrophages of ART-Treated People Living With HIV and HIV-Uninfected Individuals.

Membrane-associated drug transport proteins and drug metabolic enzymes could regulate intracellular antiretroviral (ARV) drug concentrations in HIV-1 target cells such as myeloid cells. We investigated the expression of these transporters and enzymes in monocyte subsets and monocyte-derived macrophages (MDMs) isolated from peripheral blood mononuclear cells (PBMCs) of HIV-uninfected individuals (HIV-negative) and people living with HIV receiving viral suppressive antiretroviral therapy (ART; HIV+ART) and examined plasma and intracellular ARV concentrations. Monocytes were isolated from PBMCs of 12 HIV-negative and 12 HIV+ART donors and differentiated into MDMs. The mRNA and protein expression of drug transporters and metabolic enzymes were analyzed by quantitative real-time polymerase chain reaction and flow cytometry, respectively. ARV drug concentrations were quantified in plasma, PBMCs, monocytes, and MDMs by LC-MS/MS. The mRNA expression of relevant ARV transporters or metabolic enzymes, ABCB1/P-gp, ABCG2/BCRP, ABCC1/MRP1, ABCC4/MRP4, SLC22A1/OCT1, SLC29A2/ENT2, CYP2B6, CYP2D6, and UGT1A1, was demonstrated in monocytes and MDMs of 2 to 4 HIV-negative donors. P-gp, BCRP, and MRP1 proteins were differentially expressed in classical, intermediate, and nonclassical monocytes and MDMs of both HIV+ART and HIV-negative donors. Intracellular concentrations of ARVs known to be substrates of these transporters and metabolic enzymes were detected in monocytes of HIV+ART donors but were undetectable in MDMs. In this study, we demonstrated the expression of drug transporters and metabolic enzymes in monocytes and MDMs of HIV-negative and HIV+ART individuals, which could potentially limit intracellular concentrations of ARVs and contribute to residual HIV replication. Further work is needed to assess the role of these transporters in the penetration of ARVs in tissue macrophages.

Evident hypopigmentation without other ocular deficits in Dutch patients with oculocutaneous albinism type 4

To describe the phenotype of Dutch patients with oculocutaneous albinism type 4 (OCA4), we collected data on pigmentation (skin, hair, and eyes), visual acuity (VA), nystagmus, foveal hypoplasia, chiasmal misrouting, and molecular analyses of nine Dutch OCA4 patients from the Bartiméus Diagnostic Center for complex visual disorders. All patients had severely reduced pigmentation of skin, hair, and eyes with iris transillumination over 360 degrees. Three unrelated OCA4 patients had normal VA, no nystagmus, no foveal hypoplasia, and no misrouting of the visual pathways. Six patients had poor visual acuity (0.6 to 1.0 logMAR), nystagmus, severe foveal hypoplasia and misrouting. We found two novel variants in the SLC45A2 gene, c.310C > T; (p.Pro104Ser), and c.1368 + 3_1368 + 9del; (p.?). OCA4 patients of this Dutch cohort all had hypopigmentation of skin, hair, and iris translucency. However, patients were either severely affected with regard to visual acuity, foveal hypoplasia, and misrouting, or visually not affected at all. We describe for the first time OCA4 patients with an evident lack of pigmentation, but normal visual acuity, normal foveal development and absence of misrouting. This implies that absence of melanin does not invariably lead to foveal hypoplasia and abnormal routing of the visual pathways.

Rapid Generation of Pigment Free, Immobile Zebrafish Embryos and Larvae in Any Genetic Background Using CRISPR-Cas9 dgRNPs.

The ability to carry out high-resolution, high-magnification optical imaging of living animals is one of the most attractive features of the zebrafish as a model organism. However, increasing amounts of pigmentation as development proceeds and difficulties in maintaining sustained immobilization of healthy, living animals remain challenges for live imaging. Chemical treatments can be used to suppress pigment formation and movement, but these treatments can lead to developmental defects. Genetic mutants can also be used to eliminate pigment formation and immobilize animals, but maintaining these mutants in lines carrying other combinations of transgenes and mutants is difficult and laborious. In this study, we show that CRISPR duplex guide ribonucleoproteins (dgRNPs) targeting the slc45a2 (albino) and chrna1 (nic1) genes can be used to efficiently suppress pigment formation in and immobilize F0 injected animals. CRISPR dgRNPs can be used to generate pigment-free, immobile zebrafish embryos and larvae in any transgenic and/or mutant-carrying background, greatly facilitating high-resolution imaging and analysis of the many transgenic and mutant lines available in the zebrafish.

Genetic loci associated with skin pigmentation in African Americans and their effects on vitamin D deficiency.

A recent genome-wide association study (GWAS) in African descent populations identified novel loci associated with skin pigmentation. However, how genomic variations affect skin pigmentation and how these skin pigmentation gene variants affect serum 25(OH) vitamin D variation has not been explored in African Americans (AAs). In order to further understand genetic factors that affect human skin pigmentation and serum 25(OH)D variation, we performed a GWAS for skin pigmentation with 395 AAs and a replication study with 681 AAs. Then, we tested if the identified variants are associated with serum 25(OH) D concentrations in a subset of AAs (n = 591). Skin pigmentation, Melanin Index (M-Index), was measured using a narrow-band reflectometer. Multiple regression analysis was performed to identify variants associated with M-Index and to assess their role in serum 25(OH)D variation adjusting for population stratification and relevant confounding variables. A variant near the SLC24A5 gene (rs2675345) showed the strongest signal of association with M-Index (P = 4.0 x 10−30 in the pooled dataset). Variants in SLC24A5, SLC45A2 and OCA2 together account for a large proportion of skin pigmentation variance (11%). The effects of these variants on M-Index was modified by sex (P for interaction = 0.009). However, West African Ancestry (WAA) also accounts for a large proportion of M-Index variance (23%). M-Index also varies among AAs with high WAA and high Genetic Score calculated from top variants associated with M-Index, suggesting that other unknown genomic factors related to WAA are likely contributing to skin pigmentation variation. M-Index was not associated with serum 25(OH)D concentrations, but the Genetic Score was significantly associated with vitamin D deficiency (serum 25(OH)D levels less than 12 ng/mL) (OR, 1.30; 95% CI, 1.04–1.64). The findings support the hypothesis suggesting that skin pigmentation evolved responding to increased demand for subcutaneous vitamin D synthesis in high latitude environments.

Chemical Imaging of Retinal Pigment Epithelium in Frozen Sections of Zebrafish Larvae Using ToF-SIMS.

Variants of the SLC24A5 gene, which encodes a putative potassium-dependent sodium-calcium exchanger (NCKX5) that most likely resides in the melanosome or its precursor, affect pigmentation in both humans and zebrafish (Danio rerio). This finding suggests that genetic variations influencing human skin pigmentation alter melanosome biogenesis via ionic changes. Gaining an understanding of how changes in the ionic environment of organelles impact melanosome morphogenesis and pigmentation will require a spatially resolved way to characterize the chemical environment of melanosomes in pigmented tissue such as retinal pigment epithelium (RPE). The imaging mass spectrometry technique most suited for this type of cell and tissue analysis is time-of-flight secondary ion mass spectrometry (ToF-SIMS) because it is able to detect many biochemical species with high sensitivity and with submicron spatial resolution. Here, we describe chemical imaging of the RPE in frozen-hydrated sections of larval zebrafish using cryo-ToF-SIMS. To facilitate the data interpretation, positive and negative polarity ToF-SIMS image data were transformed into a single hyperspectral data set and analyzed using principal component analysis. The combination of a novel protocol and the use of multivariate data analysis allowed us to discover new marker ions that are attributable to leucodopachrome, a metabolite specific to the biosynthesis of eumelanin. The described methodology may be adapted for the investigation of other classes of molecules in frozen tissues from zebrafish and other organisms.

Identification and Computational Analysis of Novel TYR and SLC45A2 Gene Mutations in Pakistani Families With Identical Non-syndromic Oculocutaneous Albinism.

Non-syndromic oculocutaneous albinism (nsOCA) is an inherited disorder of melanin biosynthesis with autosomal recessive mode of inheritance, presenting either hypopigmented or depigmented skin, hair, and eyes. It is genetically heterogeneous with seven loci (OCA1–OCA7) reported to date. In the present study, we have reported three consanguineous families (A, B, C) presenting identical nsOCA phenotypes. Sanger sequencing revealed a novel [NM_000372.5: c.826 T > C, p.(Cys276Arg)] and a recurrent variant [NM_000372.5: c.832C > T, p.(Arg278∗)] in tyrosinase (TYR) in families A and B, respectively. Microsatellite marker-based homozygosity mapping linked family C to OCA4. Sequence analysis identified a novel insertion variant (NM_016180.5: c.1331_1332insA) in the SLC45A2. Further, in silico mutagenesis and dynamic simulation approaches revealed that a novel Cys276Arg variant abolished the cysteine bridge and might contribute toward decreased stability of the TYR protein. Our study expands the mutation spectrum of the TYR and SLC45A2 genes and emphasizes that molecular investigations are essential for accurate disease diagnosis.

Spectrum of pathological genetic variants among 405 Chinese pedigrees affected with oculocutaneous albinism

To determine the spectrum of pathological genetic variants among 405 Chinese pedigrees affected with oculocutaneous albinism (OCA). A total of 405 OCA patients were collected. High-throughput sequencing (The panel included TYR, OCA2, TYRP1 and SLC45A2 genes), Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA) were used to analyze the genetic variants and patterns of each subtype. The overall detection rate of genetic variants was 79.9% (647/810), and the variants included missense variants (57.3%, 371/647), frameshift variants (22.9%, 148/647), nonsense variants (13.9%, 90/647), splicing variants (5.6%, 36/647), and microdeletions (0.3%, 2/647). Thirty-six novel variants were detected. Of the 405 patients, 306 have carried 2 variant alleles (75.6%, 306/405), 35 carried 1 variant alleles (8.6%, 35/405), while no variant was detected in 64 patients. Among the 306 genetically diagnosed OCA patients, OCA1 was the most common form (74.5%, 228/306), compared with OCA2 (15.0%, 46/306), OCA3 (0.7%, 2/306) and OCA4 (9.8%, 30/306), respectively. One patient was found to harbor homozygous c.1262-4_c.1262-3insTAGA variant of the TYRP1 gene. Another patient was found to carry compound heterozygous variants of c.1214C>A (p.T405N) and c.1338delinsCG(p.V447Gfs*19) of the TYRP1 gene. High-throughput sequencing in combination with Sanger sequencing and MLPA can effectively detect genetic variants associated with OCA. Above finding has expanded variant spectrum of OCA, which can facilitate genetic and prenatal diagnosis of this disease in China.

Non-syndromic Oculocutaneous Albinism: Novel Genetic Variants and Clinical Follow Up of a Brazilian Pediatric Cohort.

Oculocutaneous albinism (OCA) is a genetic disorder characterized by skin, hair, and eye hypopigmentation due to a reduction or absence of melanin. Clinical manifestations include vision problems and a high susceptibility to skin cancer. In its non-syndromic form, OCA is associated with six genes and one chromosomal region. Because OCA subtypes are not always clinically distinguishable, molecular analysis has become an important tool for classifying types of OCA, which facilitates genetic counseling and can guide the development of new therapies. We studied eight Brazilian individuals aged 1.5–18 years old with clinical diagnosis of OCA. Assessment of ophthalmologic characteristics showed results consistent with albinism, including reduced visual acuity, nystagmus, and loss of stereoscopic vision. We also observed the appearance of the strabismus and changes in static refraction over a 2-year period. Dermatologic evaluation showed that no participants had preneoplastic skin lesions, despite half of the participants reporting insufficient knowledge about skin care in albinism. Whole-exome and Sanger sequencing revealed eight different mutations: six in the TYR gene and two in the SLC45A2 gene, of which one was novel and two were described in a population study but were not previously associated with the OCA phenotype. We performed two ophthalmological evaluations, 2 years apart; and one dermatological evaluation. To the best of our knowledge, this is the first study to perform clinical follow-up and genetic analysis of a Brazilian cohort with albinism. Here, we report three new OCA causing mutations.

Generation of albino via SLC45a2 gene targeting by CRISPR/Cas9 in the marine medaka Oryzias melastigma

To produce albinism in the marine medaka Oryzias melastigma, we disrupted the solute carrier family 45 (SLC45a2) gene by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 with a single guide RNA (sgRNA). Selected sgRNAs were able to target a SLC45a2 gene as confirmed by genotyping and heteroduplex mobility assay (HMA). Of the survived embryos after injection, 54.2% and 60.0% embryos exhibited albinism phenotype by sgRNA1 and sgRNA2, respectively. Deep sequencing at the on-target sites showed different insertion and deletion (indel) mutation profiles near the DNA cleavage sites, indicating high efficacy of producing SLC45a2 knock-out mutants by this method. Moreover, HMA at the potential off-target sites revealed that off-target activity would be induced at a low rate, or not induced at all. This albino marine medaka will be a good model for marine molecular ecotoxicology in establishment of diverse in vivo endpoints, and the application of this efficient gene targeting method in the marine medaka would be useful tool for mechanistic approaches.

Identification of a novel missense variant in SLC45A2 associated with dilute snowdrop phenotype in Gypsy horses

SUMMARY SLC45A2 mutations are responsible for several dilution phenotypes in horses, including cream, pearl, and sunshine. We sequenced the SLC45A2 gene in a horse that possessed a diluted coat color but tested negative for any known dilution genotypes. We identified a novel homozygous missense mutation in ECA21: SLC45A2:c.305G>A; p.(Arg102Gln) which we have named snowdrop (Csno). The snowdrop dilution is autosomal recessive and dilutes both red and black pigment in the homozygous state, creating a phenotype similar to homozygous cream. We also identified this mutation in the heterozygous state in several relatives, including the sire and dam of the affected horse, where it has no visible effect on phenotype. Based on these data, the snowdrop variant produces a recessive dilution similar to cream, and its discovery confirms that multiple SLC45A2 alleles cause dilute phenotypes in horses.

Indel locations are determined by template polarity in highly efficient in vivo CRISPR/Cas9-mediated HDR in Atlantic salmon

Precise gene editing such as CRISPR/Cas9-mediated homology directed repair (HDR) can increase our understanding of gene function and improve traits of importance for aquaculture. This fine-tuned technology has not been developed for farmed fish including Atlantic salmon. We performed knock-in (KI) of a FLAG element in the slc45a2 gene in salmon using sense (S), anti-sense (AS) and double-stranded (ds) oligodeoxynucleotide (ODN) templates with short (24/48/84 bp) homology arms. We show in vivo ODN integration in almost all the gene edited animals, and demonstrate perfect HDR rates up to 27% in individual F0 embryos, much higher than reported previously in any fish. HDR efficiency was dependent on template concentration, but not homology arm length. Analysis of imperfect HDR variants suggest that repair occurs by synthesis-dependent strand annealing (SDSA), as we show for the first time in any species that indel location is dependent on template polarity. Correct ODN polarity can be used to avoid 5′-indels interrupting the reading frame of an inserted sequence and be of importance for HDR template design in general.

Whole exome sequencing identifies a novel pathogenic variation [p.(Gly194valfs*7)] in SLC45A2 in the homozygous state in multiple members of a family with oculocutaneous albinism in southern India.

Deleterious mutations within the SLC45A2 gene, encoding membrane-associated transporter protein (MATP), are responsible for type 4 oculocutaneous albinism. The cytogenetic location of SLC45A2 is 5p13.2 and it comprises seven exons located over around 40kb. Its encoded protein, MATP, is 530 amino acids long and has 12 putative transmembrane domains. MATP is synthesized within melanocytes. It is in these cells that melanogenesis takes place and the melanin is contained within specialized organelles called melanosomes. Previous studies have shown that when MATP expression was reduced using small interfering RNA in MNT-1 melanoma cells, pH was lowered within melanosomes, they became poorly melanized and tyrosinase activity within melanocyteswas also reduced. This type of albinism produces a broad spectrum of phenotypes, ranging from complete absence of melanin to brown hair and brown irides. In the current study, blood was collected from a family in which four members had oculocutaneous albinism, showing a complete absence of melanin in skin, hair and eyes. Screening of the TYR gene using the extracted DNA showed no mutation and therefore whole exome sequencing analysis was performed. A novel deletion mutation c.579delG [p.(Gly194Valfs*7)] in the SLC45A2 gene, predicted to be pathogenic and to result in both frameshift and premature termination of the MATP chain, was identified. These data add to the information pertaining to the mutation spectrum of OCA4.

Urinary extracellular vesicles: Origin, role as intercellular messengers and biomarkers; efficient sorting and potential treatment options

Urinary extracellular vesicles (uEVs) are a heterogenous group of vesicles consisting mainly of microvesicles and exosomes that originate predominantly (99.96%) from kidney, the urinary tract epithelium and the male reproductive tract. Secreted EVs contain molecular cargo from parental cells and provide an attractive source for biomarkers, a potential readout of physiological and pathophysiological mechanisms, and events associated with the urinary system. uEVs are readily enriched and isolated from urine samples and we review 6 standard methods that allow for downstream analysis of the uEV cargo. Although the use of uEVs as a surrogate readout for physiological changes in tissue protein levels is widespread, the protein abundance in uEVs is affected significantly by mechanisms that regulate protein sorting and secretion in uEVs. Data suggest that baseline kidney tissue and uEV levels of apical membrane-associated electrolyte transport proteins are not directly related in human patients. Recent evidence indicates that EVs may contribute to physiological and pathophysiological intercellular signalling and EVs confer protection against renal ischemia-reperfusion injury. The therapeutic use of EVs as information carriers has mainly been explored in vitro and a major hurdle lies in the translation of the in vitro findings into an in vivo setting. Thus, the EV research field is moving from a technical focus to a more physiological focus, allowing for a deeper understanding of human physiology, development of diagnostic tools and potential treatment strategies for precision medicine.

Meta-analysis of GWA studies provides new insights on the genetic architecture of skin pigmentation in recently admixed populations

BackgroundAssociation studies in recently admixed populations are extremely useful to identify the genetic architecture of pigmentation, due to their high genotypic and phenotypic variation. However, to date only four Genome-Wide Association Studies (GWAS) have been carried out in these populations.ResultsWe present a GWAS of skin pigmentation in an admixed sample from Cuba (N = 762). Additionally, we conducted a meta-analysis including the Cuban sample, and admixed samples from Cape Verde, Puerto Rico and African-Americans from San Francisco. This meta-analysis is one of the largest efforts so far to characterize the genetic basis of skin pigmentation in admixed populations (N = 2,104). We identified five genome-wide significant regions in the meta-analysis, and explored if the markers observed in these regions are associated with the expression of relevant pigmentary genes in human melanocyte cultures. In three of the regions identified in the meta-analysis (SLC24A5, SLC45A2, and GRM5/TYR), the association seems to be driven by non-synonymous variants (rs1426654, rs16891982, and rs1042602, respectively). The rs16891982 polymorphism is strongly associated with the expression of the SLC45A2 gene. In the GRM5/TYR region, in addition to the rs1042602 non-synonymous SNP located on the TYR gene, variants located in the nearby GRM5 gene have an independent effect on pigmentation, possibly through regulation of gene expression of the TYR gene. We also replicated an association recently described near the MFSD12 gene on chromosome 19 (lead variant rs112332856). Additionally, our analyses support the presence of multiple signals in the OCA2/HERC2/APBA2 region on chromosome 15. A clear causal candidate is the HERC2 intronic variant rs12913832, which has a profound influence on OCA2 expression. This variant has pleiotropic effects on eye, hair, and skin pigmentation. However, conditional and haplotype-based analyses indicate the presence of other variants with independent effects on melanin levels in OCA2 and APBA2. Finally, a follow-up of genome-wide signals identified in a recent GWAS for tanning response indicates that there is a substantial overlap in the genetic factors influencing skin pigmentation and tanning response.ConclusionsOur meta-analysis of skin pigmentation GWAS in recently admixed populations provides new insights about the genetic architecture of this complex trait.

Oculocutaneous Albinism with Squamous Cell Carcinoma, Bowen's Disease and Actinic Keratosis: A Case Report

Oculocutaneous Albinism with Squamous Cell Carcinoma, Bowen's Disease and Actinic Keratosis: A Case Report

A candidate gene approach identifies variants in SLC45A2 that explain dilute phenotypes, pearl and sunshine, in compound heterozygote horses

Variations in the SLC45A2 gene are responsible for the dilution phenotypes cream and pearl in domestic horses. Cream dilution is inherited in an incomplete dominant manner, diluting only red in the heterozygous state but both red and black pigments when two alleles are present. The pearl dilution is recessive and dilutes only the red and black pigment in the homozygous state or when paired with a cream allele. Horses that inherit one copy of pearl (Cprl ) and one copy of the dominant cream allele (CC r ) display a dilution phenotype similar to that of homozygous cream, suggesting that pearl is the result of a different variation in the same gene responsible for cream. We sequenced SLC45A2 in two 'false double dilute' horses that appeared phenotypically homozygous cream but tested as possessing only a single CC r allele. We also sequenced one known pearl carrier to screen for putative causal variants. The missense variant ECA21:SLC45A2:c.985G>A; p.Ala329Thr (Cprl ) was present in one false double dilute and the pearl carrier and was also genotyped in an additional 126 horses for statistical evaluation. The genotype matched the expected phenotype in all horses (P-value=6.5×10-41 ) and is identical to a pearl variant found previously. The second false double dilute horse and one non-dilute offspring genotyped as heterozygous for a novel missense variant ECA21:SLC45A2:c.568G>A (p.Gly190Arg), the proposed Csun variant (for the name of the horse). This variant produces a recessive dilution similar to pearl and indicates that multiple alleles of SLC45A2 result in dilution phenotypes in the domestic horse.

In silico screening of deleterious single nucleotide polymorphisms (SNPs) and molecular dynamics simulation of disease associated mutations in gene responsible for oculocutaneous albinism type 6 (OCA 6) disorder

Solute carrier family 24 member 5 (SLC24A5) is a gene that is associated with oculocutaneous albinism type 6 (OCA6) disorder and is involved in skin and hair pigmentation. It is involved in the maturation of melanosomes and melanin synthesis. SLC24A5 gene is located in the chromosomal position of 15q21.1. The present study involves the use of computational techniques in order to obtain a detailed picture of the most probable mutations that are associated with SLC24A5. From the observed result it was found that the mutation S145F is most deleterious and disease associated is predicted using several bioinformatics tools. The 3-D structures of native and mutant (S145F) were modeled in order to understand protein functionality using ab initio Robetta server. The modeled structure validation was done with ERRAT, Verify-3D, Procheck and RAMPAGE Ramachandran plot analysis. The most validated structure undergoes molecular dynamics simulations (MDS) study to understand the structural and functional behaviour of the native and mutant proteins. The MDS result showed the more flexibility in the native SLC24A5 structure. Due to mutation in the SLC24A5 protein structure it became more rigid and might disturb the conformational changes and glycosylation function of protein structure and might play role in inducing the OCA6. This study provides a significant insight into the underlying molecular mechanism involved in albinism associated with OCA6. It further helps scientists to develop a drug therapy against OCA 6 disease.Communicated by Ramaswamy H. Sarma

A tRNA-based multiplex sgRNA expression system in zebrafish and its application to generation of transgenic albino fish

The CRISPR/Cas9 system can be introduced into zebrafish as transgenes. Namely, expression of single-guide RNA (sgRNA) and controlled expression of Cas9 in transgenic zebrafish enables the study of gene functions in specific cell types. This transgenic CRISPR/Cas9 approach would be more useful if multiple sgRNAs could be expressed simultaneously since we could knock-out a gene more efficiently or disrupt multiple genes simultaneously. Here we describe a novel system to express multiple sgRNAs efficiently in zebrafish, that relies on the endogenous tRNA processing machinery. We cloned nine endogenous zebrafish tRNA genes, fused them to sgRNAs, and demonstrated that an active sgRNA can be produced from a precursor transcript containing either of these tRNAs. To show a proof of principle, we constructed transgenic fish expressing Cas9 under the control of the ubiquitin promoter and a single transcript containing three distinct sgRNAs, that targeted the slc45a2 (albino) gene, fused to tRNAs under the control of the U6 promoter. We found that the Tg(ubb:SpCas9,u6c:3xslc45a2-sgRNA) harbored mutations in all of the target sites in the albino gene and showed nearly complete albino phenotypes, which were amenable to imaging experiments. Thus, the tRNA-based multiplex sgRNA expression system should facilitate gene knock-out studies in transgenic zebrafish.