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.

Figure 1. Sequence analysis of the albino gene.

(A and B) Sequence traces of genomic DNA show (A) G→T mutation in slc45a2^nk1 (boxed) and (B) 2aa (6 nt) allele slc45a2 ^Δ422–423 deletion (boxed) (C) Alignment of slc45a2 from various vertebrate species showing disruption of conserved sequences in both the two amino acid deletion (PY red) alb^b4→SJ1 and the nonsense mutation GGA(Gly461)>UGA(stop) (red asterisk) of alb^nk1 zebrafish albino mutants in exon 6. Identical sequences are shown in black.

Figure 2. Effect of human coding polymorphisms on zebrafish mRNA rescue of the albino phenotype.

Lateral views of 48-hpf (A) wild-type zebrafish larva (B) un-injected alb^nk1 zebrafish larva (C-F) alb^nk1 zebrafish larva injected with mRNA (500 pg), coding for indicated variants of zebrafish slc45a2, named according to positions of human variation (human 272 equivalent to zebrafish 303; human 374 equivalent to zebrafish 403). (C) wild-type; (D) E272K mutant; (E) L374F mutant; (F) E272K/L374F double mutant. Note that mRNA rescue in zebrafish does not occur in every cell, which is thought to be due to unequal distribution of the mRNA in the cytoplasm of the originally injected eggs, resulting in unequal distribution of the mRNA among different cells of the embryo. The results shown are typical of a majority of injected embryos in each case; each of embryo in the majority populations contains cells as pigmented as the ones evident in this figure. Scale bar 400, µm.

Figure 3. Dot plot showing no significant correlation of E272K mutation to the Melanin Index in East Asian populations.

Average melanin indices for E/E, E/K and K/K genotypes are 26.4, 26.5, and 25.8, respectively. No significant deviation from Hardy-Weinberg equilibrium was identified for E272K SNP in populations of East Asian ancestry (N = 59). Observed and expected genotype frequencies E/E = 23(23.2); E/K = 28(27.6); K/K = 8(8.2). E and K allele frequencies were 0.63 and 0.37, respectively.

Figure 4. Effect of a human coding polymorphism on zebrafish mRNA rescue of the golden phenotype.

Lateral views of 48-hpf (A and B) wt zebrafish larva (C and D) gol^b1 zebrafish larva (E and F) gol^b1 larva injected with full-length zebrafish slc24a5 (wt) mRNA (500 pg) and (G and H) gol^b1 larva injected with full-length zebrafish slc24a5 mRNA with a single nucleotide change (500 pg), coding for the orthologous human derived A111T allele. Scale bars in (A, C, E, G) 150 µm, (B, D, F, H) 400 µm.

Figure 5. Phylogenetic conservation of amino acid changes associated with OCA.

The Venn diagram illustrates the number of mutations changed across all four OCA genes (TYR, OCA2, TYRP1, and SLC45A2) that can be tested in zebrafish, western clawed frog, and/or medaka on account of wild-type amino acid conservation. Since different mutations can affect different nucleotides in a single codon and different mutations at the same nucleotide can result in different amino acids, the 257 alleles are found in 222 loci.

Figure 6. Flow chart for testing coding mutations based on the HuZOR approach.

Candidate functional coding mutations are first identified from genome sequences. After an orthologue (or potentially a pair of orthologues) is identified, null mutants and/or gene-specific transient functional knockdown embryos are generated with morpholinos or TALENs in zebrafish or other model species. Phenotypes are then scored. Rescue of mutant or functional knockdown phenotypes are then tested by microinjection of wild-type mRNA (and potentially cDNA). If rescue is successful, and if the corresponding amino acid is conserved, zebrafish mRNA containing the orthologous amino acid change is then generated, and ability of the derived mRNA to rescue the mutant phenotype is then tested. Phenotypic rescue by the derived mRNA is evidence in support of the original variant being phenotypically neutral in humans. In contrast, loss of the ability to rescue as a result of an orthologous amino acid change suggests a deleterious effect on protein function. Mutations in regulatory elements, such as a mutation disrupting a phosphorylation site, may potentially have hypomorphic or hypermorphic effects.