Functional Assessment of Human Coding Mutations Affecting Skin Pigmentation Using Zebrafish

Abstract

A major challenge in personalized medicine is the lack of a standard way to define the functional significance of the numerous nonsynonymous, single nucleotide coding variants that are present in each human individual. To begin to address this problem, we have used pigmentation as a model polygenic trait, three common human polymorphisms thought to influence pigmentation, and the zebrafish as a model system. The approach is based on the rescue of embryonic zebrafish mutant phenotypes by “humanized” zebrafish orthologous mRNA. Two hypomorphic polymorphisms, L374F in SLC45A2, and A111T in SLC24A5, have been linked to lighter skin color in Europeans. The phenotypic effect of a second coding polymorphism in SLC45A2, E272K, is unclear. None of these polymorphisms had been tested in the context of a model organism. We have confirmed that zebrafish albino fish are mutant in slc45a2; wild-type slc45a2 mRNA rescued the albino mutant phenotype. Introduction of the L374F polymorphism into albino or the A111T polymorphism into slc24a5 (golden) abolished mRNA rescue of the respective mutant phenotypes, consistent with their known contributions to European skin color. In contrast, the E272K polymorphism had no effect on phenotypic rescue. The experimental conclusion that E272K is unlikely to affect pigmentation is consistent with a lack of correlation between this polymorphism and quantitatively measured skin color in 59 East Asian humans. A survey of mutations causing human oculocutaneous albinism yielded 257 missense mutations, 82% of which are theoretically testable in zebrafish. The developed approach may be extended to other model systems and may potentially contribute to our understanding the functional relationships between DNA sequence variation, human biology, and disease.

The Interplay between Natural Selection and Susceptibility to Melanoma on Allele 374F of SLC45A2 Gene in a South European Population

“…Functional assays for this mutation have not been performed yet, but our results suggest that it could be a cis-acting mutation that may regulate the expression of the gene and contribute to the effect of the SNP L374F. We also acknowledge, however, that this association could be due to the fact that it is in linkage disequilibrium with L374F, whose functional impact has already been described [37]. Further functional work should be done in order to ascertain the significance of this SNP in relation to pigmentation variability.…”

Cyp27c1 Red-Shifts the Spectral Sensitivity of Photoreceptors by Converting Vitamin A1 into A2

“…We first confirmed that the cyp27c1 transcript is significantly increased in TH-treated zebrafish RPE and dorsal bullfrog RPE by quantitative real-time PCR (Figure 2C). We then determined the cellular expression pattern of cyp27c1 , by performing in situ hybridization on eyes from TH-treated albino zebrafish and vehicle controls [26]. The albino strain was used to improve visualization of staining in the RPE, and we confirmed that it undergoes a TH-dependent switch to vitamin A 2 similar to wild-type strains (data not shown).…”

The zebrafish eye—a paradigm for investigating human ocular genetics

“…Knockdown of the zebrafish orthologues tyr (TYR), oca21p (OCA2), tyrp1a and tyrp1b simultaneously (TYRP1), slc45a2 (SLC45A2), slc24a5 (SLC24A5), or c10orf11 (C10ORF11) lead to a reduction or complete absence of melanin in the eye, with varying responses to visual testing immediately after light exposure, phenocopying the varying degrees of hypopigmentation of the eyes of OCA patients and exemplifying the use of zebrafish to model human pathologies of the eye. [65][66][67][68][69] Multiple zebrafish mutants have been identified with visual defects secondary to functional deficits in the RPE. The vps39 mutant carries a mutation in the zebrafish orthologue of VPS39, a gene encoding a component of the vacuole protein sorting (HOPS) membrane-tethering complex, which coordinates vesicle fusion and transport.…”