Haplotype-based analysis resolves missing heritability in oculocutaneous albinism type 1B.

Oculocutaneous albinism (OCA) is a rare disorder of pigment production. Affected individuals have variably decreased global pigmentation and visual-developmental changes that lead to low vision. OCA is notable for significant missing heritability, particularly among individuals with residual pigmentation. Tyrosinase (TYR) is the rate-limiting enzyme in melanin pigment biosynthesis and mutations that decrease enzyme function are one of the most common causes of OCA. We present the analysis of high-depth short-read TYR sequencing data for a cohort of 352 OCA probands, ∼50% of whom were previously sequenced without yielding a definitive diagnostic result. Our analysis identified 66 TYR single-nucleotide variants (SNVs) and small insertion/deletions (indels), 3 structural variants, and a rare haplotype comprised of two common frequency variants (p.Ser192Tyr and p.Arg402Gln) in cis-orientation, present in 149/352 OCA probands. We further describe a detailed analysis of the disease-causing haplotype, p.[Ser192Tyr; Arg402Gln] ("cis-YQ"). Haplotype analysis suggests that the cis-YQ allele arose by recombination and that multiple cis-YQ haplotypes are segregating in OCA-affected individuals and control populations. The cis-YQ allele is the most common disease-causing allele in our cohort, representing 19.1% (57/298) of TYR pathogenic alleles in individuals with type 1 (TYR-associated) OCA. Finally, among the 66 TYR variants, we found several additional alleles defined by a cis-oriented combination of minor, potentially hypomorph-producing alleles at common variant sites plus a second, rare pathogenic variant. Together, these results suggest that identification of phased variants for the full TYR locus are required for an exhaustive assessment for potentially disease-causing alleles.

Computational Investigation of the pH Dependence of Stability of Melanosome Proteins: Implication for Melanosome formation and Disease.

Intravesicular pH plays a crucial role in melanosome maturation and function. Melanosomal pH changes during maturation from very acidic in the early stages to neutral in late stages. Neutral pH is critical for providing optimal conditions for the rate-limiting, pH-sensitive melanin-synthesizing enzyme tyrosinase (TYR). This dramatic change in pH is thought to result from the activity of several proteins that control melanosomal pH. Here, we computationally investigated the pH-dependent stability of several melanosomal membrane proteins and compared them to the pH dependence of the stability of TYR. We confirmed that the pH optimum of TYR is neutral, and we also found that proteins that are negative regulators of melanosomal pH are predicted to function optimally at neutral pH. In contrast, positive pH regulators were predicted to have an acidic pH optimum. We propose a competitive mechanism among positive and negative regulators that results in pH equilibrium. Our findings are consistent with previous work that demonstrated a correlation between the pH optima of stability and activity, and they are consistent with the expected activity of positive and negative regulators of melanosomal pH. Furthermore, our data suggest that disease-causing variants impact the pH dependence of melanosomal proteins; this is particularly prominent for the OCA2 protein. In conclusion, melanosomal pH appears to affect the activity of multiple melanosomal proteins.

A custom capture sequence approach for oculocutaneous albinism identifies structural variant alleles at the OCA2 locus.

Oculocutaneous albinism (OCA) is a heritable disorder of pigment production that manifests as hypopigmentation and altered eye development. Exon sequencing of known OCA genes is unsuccessful in producing a complete molecular diagnosis for a significant number of affected individuals. We sequenced the DNA of individuals with OCA using short-read custom capture sequencing that targeted coding, intronic, and noncoding regulatory regions of known OCA genes, and genome-wide association study-associated pigmentation loci. We identified an OCA2 complex structural variant (CxSV), defined by a 143 kb inverted segment reintroduced in intron 1, upstream of the native location. The corresponding CxSV junctions were observed in 11/390 probands screened. The 143 kb CxSV presents in one family as a copy number variant duplication for the 143 kb region. In the remaining 10/11 families, the 143 kb CxSV acquired an additional 184 kb deletion across the same region, restoring exons 3-19 of OCA2 to a copy-number neutral state. Allele-associated haplotype analysis found rare SNVs rs374519281 and rs139696407 are linked with the 143 kb CxSV in both OCA2 alleles. For individuals in which customary molecular evaluation does not reveal a biallelic OCA diagnosis, we recommend preliminary screening for these haplotype-associated rare variants, followed by junction-specific validation for the OCA2 143 kb CxSV.

MEK inhibition remodels the active chromatin landscape and induces SOX10 genomic recruitment in BRAF(V600E) mutant melanoma cells.

BACKGROUND: The MAPK/ERK signaling pathway is an essential regulator of numerous cell processes that are crucial for normal development as well as cancer progression. While much is known regarding MAPK/ERK signal conveyance from the cell membrane to the nucleus, the transcriptional and epigenetic mechanisms that govern gene expression downstream of MAPK signaling are not fully elucidated. RESULTS: This study employed an integrated epigenome analysis approach to interrogate the effects of MAPK/ERK pathway inhibition on the global transcriptome, the active chromatin landscape, and protein-DNA interactions in 501mel melanoma cells. Treatment of these cells with the small-molecule MEK inhibitor AZD6244 induces hyperpigmentation, widespread gene expression changes including alteration of genes linked to pigmentation, and extensive epigenomic reprogramming of transcriptionally distinct regulatory regions associated with the active chromatin mark H3K27ac. Regulatory regions with differentially acetylated H3K27ac regions following AZD6244 treatment are enriched in transcription factor binding motifs of ETV/ETS and ATF family members as well as the lineage-determining factors MITF and SOX10. H3K27ac-dense enhancer clusters known as super-enhancers show similar transcription factor motif enrichment, and furthermore, these super-enhancers are associated with genes encoding MITF, SOX10, and ETV/ETS proteins. Along with genome-wide resetting of the active enhancer landscape, MEK inhibition also results in widespread SOX10 recruitment throughout the genome, including increased SOX10 binding density at H3K27ac-marked enhancers. Importantly, these MEK inhibitor-responsive enhancers marked by H3K27ac and occupied by SOX10 are located near melanocyte lineage-specific and pigmentation genes and overlap numerous human SNPs associated with pigmentation and melanoma phenotypes, highlighting the variants located within these regions for prioritization in future studies. CONCLUSIONS: These results reveal the epigenetic reprogramming underlying the re-activation of melanocyte pigmentation and developmental transcriptional programs in 501mel cells in response to MEK inhibition and suggest extensive involvement of a MEK-SOX10 axis in the regulation of these processes. The dynamic chromatin changes identified here provide a rich genomic resource for further analyses of the molecular mechanisms governing the MAPK pathway in pigmentation- and melanocyte-associated diseases.

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

BACKGROUND: Association 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. RESULTS: We 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. CONCLUSIONS: Our meta-analysis of skin pigmentation GWAS in recently admixed populations provides new insights about the genetic architecture of this complex trait.

A curated gene list for expanding the horizons of pigmentation biology.

Over the past century, studies of human pigmentary disorders along with mouse and zebrafish models have shed light on the many cellular functions associated with visible pigment phenotypes. This has led to numerous genes annotated with the ontology term "pigmentation" in independent human, mouse, and zebrafish databases. Comparisons among these datasets revealed that each is individually incomplete in documenting all genes involved in integument-based pigmentation phenotypes. Additionally, each database contained inherent species-specific biases in data annotation, and the term "pigmentation" did not solely reflect integument pigmentation phenotypes. This review presents a comprehensive, cross-species list of 650 genes involved in pigmentation phenotypes that was compiled with extensive manual curation of genes annotated in OMIM, MGI, ZFIN, and GO. The resulting cross-species list of genes both intrinsic and extrinsic to integument pigment cells provides a valuable tool that can be used to expand our knowledge of complex, pigmentation-associated pathways.

Cell-type-specific eQTL of primary melanocytes facilitates identification of melanoma susceptibility genes.

Most expression quantitative trait locus (eQTL) studies to date have been performed in heterogeneous tissues as opposed to specific cell types. To better understand the cell-type-specific regulatory landscape of human melanocytes, which give rise to melanoma but account for <5% of typical human skin biopsies, we performed an eQTL analysis in primary melanocyte cultures from 106 newborn males. We identified 597,335 cis-eQTL SNPs prior to linkage disequilibrium (LD) pruning and 4997 eGenes (FDR < 0.05). Melanocyte eQTLs differed considerably from those identified in the 44 GTEx tissue types, including skin. Over a third of melanocyte eGenes, including key genes in melanin synthesis pathways, were unique to melanocytes compared to those of GTEx skin tissues or TCGA melanomas. The melanocyte data set also identified trans-eQTLs, including those connecting a pigmentation-associated functional SNP with four genes, likely through cis-regulation of IRF4 Melanocyte eQTLs are enriched in cis-regulatory signatures found in melanocytes as well as in melanoma-associated variants identified through genome-wide association studies. Melanocyte eQTLs also colocalized with melanoma GWAS variants in five known loci. Finally, a transcriptome-wide association study using melanocyte eQTLs uncovered four novel susceptibility loci, where imputed expression levels of five genes (ZFP90, HEBP1, MSC, CBWD1, and RP11-383H13.1) were associated with melanoma at genome-wide significant P-values. Our data highlight the utility of lineage-specific eQTL resources for annotating GWAS findings, and present a robust database for genomic research of melanoma risk and melanocyte biology.

The next generation of melanocyte data: Genetic, epigenetic, and transcriptional resource datasets and analysis tools.

The number of melanocyte- and melanoma-derived next generation sequence genome-scale datasets have rapidly expanded over the past several years. This resource guide provides a summary of publicly available sources of melanocyte cell derived whole genome, exome, mRNA and miRNA transcriptome, chromatin accessibility and epigenetic datasets. Also highlighted are bioinformatic resources and tools for visualization and data queries which allow researchers a genome-scale view of the melanocyte.

Identification and functional analysis of SOX10 phosphorylation sites in melanoma.

The transcription factor SOX10 plays an important role in vertebrate neural crest development, including the establishment and maintenance of the melanocyte lineage. SOX10 is also highly expressed in melanoma tumors, and SOX10 expression increases with tumor progression. The suppression of SOX10 in melanoma cells activates TGF-ß signaling and can promote resistance to BRAF and MEK inhibitors. Since resistance to BRAF/MEK inhibitors is seen in the majority of melanoma patients, there is an immediate need to assess the underlying biology that mediates resistance and to identify new targets for combinatorial therapeutic approaches. Previously, we demonstrated that SOX10 protein is required for tumor initiation, maintenance and survival. Here, we present data that support phosphorylation as a mechanism employed by melanoma cells to tightly regulate SOX10 expression. Mass spectrometry identified eight phosphorylation sites contained within SOX10, three of which (S24, S45 and T240) were selected for further analysis based on their location within predicted MAPK/CDK binding motifs. SOX10 mutations were generated at these phosphorylation sites to assess their impact on SOX10 protein function in melanoma cells, including transcriptional activation on target promoters, subcellular localization, and stability. These data further our understanding of SOX10 protein regulation and provide critical information for identification of molecular pathways that modulate SOX10 protein levels in melanoma, with the ultimate goal of discovering novel targets for more effective combinatorial therapeutic approaches for melanoma patients.

Loci associated with skin pigmentation identified in African populations.

Despite the wide range of skin pigmentation in humans, little is known about its genetic basis in global populations. Examining ethnically diverse African genomes, we identify variants in or near SLC24A5, MFSD12, DDB1, TMEM138, OCA2, and HERC2 that are significantly associated with skin pigmentation. Genetic evidence indicates that the light pigmentation variant at SLC24A5 was introduced into East Africa by gene flow from non-Africans. At all other loci, variants associated with dark pigmentation in Africans are identical by descent in South Asian and Australo-Melanesian populations. Functional analyses indicate that MFSD12 encodes a lysosomal protein that affects melanogenesis in zebrafish and mice, and that mutations in melanocyte-specific regulatory regions near DDB1/TMEM138 correlate with expression of ultraviolet response genes under selection in Eurasians.

TFAP2 paralogs regulate melanocyte differentiation in parallel with MITF.

Mutations in the gene encoding transcription factor TFAP2A result in pigmentation anomalies in model organisms and premature hair graying in humans. However, the pleiotropic functions of TFAP2A and its redundantly-acting paralogs have made the precise contribution of TFAP2-type activity to melanocyte differentiation unclear. Defining this contribution may help to explain why TFAP2A expression is reduced in advanced-stage melanoma compared to benign nevi. To identify genes with TFAP2A-dependent expression in melanocytes, we profile zebrafish tissue and mouse melanocytes deficient in Tfap2a, and find that expression of a small subset of genes underlying pigmentation phenotypes is TFAP2A-dependent, including Dct, Mc1r, Mlph, and Pmel. We then conduct TFAP2A ChIP-seq in mouse and human melanocytes and find that a much larger subset of pigmentation genes is associated with active regulatory elements bound by TFAP2A. These elements are also frequently bound by MITF, which is considered the "master regulator" of melanocyte development. For example, the promoter of TRPM1 is bound by both TFAP2A and MITF, and we show that the activity of a minimal TRPM1 promoter is lost upon deletion of the TFAP2A binding sites. However, the expression of Trpm1 is not TFAP2A-dependent, implying that additional TFAP2 paralogs function redundantly to drive melanocyte differentiation, which is consistent with previous results from zebrafish. Paralogs Tfap2a and Tfap2b are both expressed in mouse melanocytes, and we show that mouse embryos with Wnt1-Cre-mediated deletion of Tfap2a and Tfap2b in the neural crest almost completely lack melanocytes but retain neural crest-derived sensory ganglia. These results suggest that TFAP2 paralogs, like MITF, are also necessary for induction of the melanocyte lineage. Finally, we observe a genetic interaction between tfap2a and mitfa in zebrafish, but find that artificially elevating expression of tfap2a does not increase levels of melanin in mitfa hypomorphic or loss-of-function mutants. Collectively, these results show that TFAP2 paralogs, operating alongside lineage-specific transcription factors such as MITF, directly regulate effectors of terminal differentiation in melanocytes. In addition, they suggest that TFAP2A activity, like MITF activity, has the potential to modulate the phenotype of melanoma cells.

Hypoxia-induced HIF1α targets in melanocytes reveal a molecular profile associated with poor melanoma prognosis.

Hypoxia and HIF1α signaling direct tissue-specific gene responses regulating tumor progression, invasion, and metastasis. By integrating HIF1α knockdown and hypoxia-induced gene expression changes, this study identifies a melanocyte-specific, HIF1α-dependent/hypoxia-responsive gene expression signature. Integration of these gene expression changes with HIF1α ChIP-Seq analysis identifies 81 HIF1α direct target genes in melanocytes. The expression levels for 10 of the HIF1α direct targets - GAPDH, PKM, PPAT, DARS, DTWD1, SEH1L, ZNF292, RLF, AGTRAP, and GPC6 - are significantly correlated with reduced time of disease-free status in melanoma by logistic regression (P-value = 0.0013) and ROC curve analysis (AUC = 0.826, P-value < 0.0001). This HIF1α-regulated profile defines a melanocyte-specific response under hypoxia, and demonstrates the role of HIF1α as an invasive cell state gatekeeper in regulating cellular metabolism, chromatin and transcriptional regulation, vascularization, and invasion.

Genomic analysis reveals distinct mechanisms and functional classes of SOX10-regulated genes in melanocytes.

SOX10 is required for melanocyte development and maintenance, and has been linked to melanoma initiation and progression. However, the molecular mechanisms by which SOX10 guides the appropriate gene expression programs necessary to promote the melanocyte lineage are not fully understood. Here we employ genetic and epigenomic analysis approaches to uncover novel genomic targets and previously unappreciated molecular roles of SOX10 in melanocytes. Through global analysis of SOX10-binding sites and epigenetic characteristics of chromatin states, we uncover an extensive catalog of SOX10 targets genome-wide. Our findings reveal that SOX10 predominantly engages 'open' chromatin regions and binds to distal regulatory elements, including novel and previously known melanocyte enhancers. Integrated chromatin occupancy and transcriptome analysis suggest a role for SOX10 in both transcriptional activation and repression to regulate functionally distinct classes of genes. We demonstrate that distinct epigenetic signatures and cis-regulatory sequence motifs predicted to bind putative co-regulatory transcription factors define SOX10-activated and SOX10-repressed target genes. Collectively, these findings uncover a central role of SOX10 as a global regulator of gene expression in the melanocyte lineage by targeting diverse regulatory pathways.

Distinct microRNA expression signatures are associated with melanoma subtypes and are regulated by HIF1A.

The complex genetic changes underlying metastatic melanoma need to be deciphered to develop new and effective therapeutics. Previously, genome-wide microarray analyses of human melanoma identified two reciprocal gene expression programs, including transcripts regulated by either transforming growth factor, beta 1 (TGFß1) pathways, or microphthalmia-associated transcription factor (MITF)/SRY-box containing gene 10 (SOX10) pathways. We extended this knowledge by discovering that melanoma cell lines with these two expression programs exhibit distinctive microRNA (miRNA) expression patterns. We also demonstrated that hypoxia-inducible factor 1 alpha (HIF1A) is increased in TGFß1 pathway-expressing melanoma cells and that HIF1A upregulates miR-210, miR-218, miR-224, and miR-452. Reduced expression of these four miRNAs in TGFß1 pathway-expressing melanoma cells arrests the cell cycle, while their overexpression in mouse melanoma cells increases the expression of the hypoxic response gene Bnip3. Taken together, these data suggest that HIF1A may regulate some of the gene expression and biological behavior of TGFß1 pathway-expressing melanoma cells, in part via alterations in these four miRNAs.

A polymorphism in IRF4 affects human pigmentation through a tyrosinase-dependent MITF/TFAP2A pathway.

Sequence polymorphisms linked to human diseases and phenotypes in genome-wide association studies often affect noncoding regions. A SNP within an intron of the gene encoding Interferon Regulatory Factor 4 (IRF4), a transcription factor with no known role in melanocyte biology, is strongly associated with sensitivity of skin to sun exposure, freckles, blue eyes, and brown hair color. Here, we demonstrate that this SNP lies within an enhancer of IRF4 transcription in melanocytes. The allele associated with this pigmentation phenotype impairs binding of the TFAP2A transcription factor that, together with the melanocyte master regulator MITF, regulates activity of the enhancer. Assays in zebrafish and mice reveal that IRF4 cooperates with MITF to activate expression of Tyrosinase (TYR), an essential enzyme in melanin synthesis. Our findings provide a clear example of a noncoding polymorphism that affects a phenotype by modulating a developmental gene regulatory network.

SOX10 ablation arrests cell cycle, induces senescence, and suppresses melanomagenesis.

The transcription factor SOX10 is essential for survival and proper differentiation of neural crest cell lineages, where it plays an important role in the generation and maintenance of melanocytes. SOX10 is also highly expressed in melanoma tumors, but a role in disease progression has not been established. Here, we report that melanoma tumor cell lines require wild-type SOX10 expression for proliferation and SOX10 haploinsufficiency reduces melanoma initiation in the metabotropic glutamate receptor 1 (Grm1(Tg)) transgenic mouse model. Stable SOX10 knockdown in human melanoma cells arrested cell growth, altered cellular morphology, and induced senescence. Melanoma cells with stable loss of SOX10 were arrested in the G1 phase of the cell cycle, with reduced expression of the melanocyte determining factor microphthalmia-associated transcription factor, elevated expression of p21WAF1 and p27KIP2, hypophosphorylated RB, and reduced levels of its binding partner E2F1. As cell-cycle dysregulation is a core event in neoplastic transformation, the role for SOX10 in maintaining cell-cycle control in melanocytes suggests a rational new direction for targeted treatment or prevention of melanoma.

Integration of ChIP-seq and machine learning reveals enhancers and a predictive regulatory sequence vocabulary in melanocytes.

We take a comprehensive approach to the study of regulatory control of gene expression in melanocytes that proceeds from large-scale enhancer discovery facilitated by ChIP-seq; to rigorous validation in silico, in vitro, and in vivo; and finally to the use of machine learning to elucidate a regulatory vocabulary with genome-wide predictive power. We identify 2489 putative melanocyte enhancer loci in the mouse genome by ChIP-seq for EP300 and H3K4me1. We demonstrate that these putative enhancers are evolutionarily constrained, enriched for sequence motifs predicted to bind key melanocyte transcription factors, located near genes relevant to melanocyte biology, and capable of driving reporter gene expression in melanocytes in culture (86%; 43/50) and in transgenic zebrafish (70%; 7/10). Next, using the sequences of these putative enhancers as a training set for a supervised machine learning algorithm, we develop a vocabulary of 6-mers predictive of melanocyte enhancer function. Lastly, we demonstrate that this vocabulary has genome-wide predictive power in both the mouse and human genomes. This study provides deep insight into the regulation of gene expression in melanocytes and demonstrates a powerful approach to the investigation of regulatory sequences that can be applied to other cell types.

SOX10 directly modulates ERBB3 transcription via an intronic neural crest enhancer.

BACKGROUND: The ERBB3 gene is essential for the proper development of the neural crest (NC) and its derivative populations such as Schwann cells. As with all cell fate decisions, transcriptional regulatory control plays a significant role in the progressive restriction and specification of NC derived lineages during development. However, little is known about the sequences mediating transcriptional regulation of ERBB3 or the factors that bind them. RESULTS: In this study we identified three transcriptional enhancers at the ERBB3 locus and evaluated their regulatory potential in vitro in NC-derived cell types and in vivo in transgenic zebrafish. One enhancer, termed ERBB3_MCS6, which lies within the first intron of ERBB3, directs the highest reporter expression in vitro and also demonstrates epigenetic marks consistent with enhancer activity. We identify a consensus SOX10 binding site within ERBB3_MCS6 and demonstrate, in vitro, its necessity and sufficiency for the activity of this enhancer. Additionally, we demonstrate that transcription from the endogenous Erbb3 locus is dependent on Sox10. Further we demonstrate in vitro that Sox10 physically interacts with that ERBB3_MCS6. Consistent with its in vitro activity, we also show that ERBB3_MCS6 drives reporter expression in NC cells and a subset of its derivative lineages in vivo in zebrafish in a manner consistent with erbb3b expression. We also demonstrate, using morpholino analysis, that Sox10 is necessary for ERBB3_MCS6 expression in vivo in zebrafish. CONCLUSIONS: Taken collectively, our data suggest that ERBB3 may be directly regulated by SOX10, and that this control may in part be facilitated by ERBB3_MCS6.

The pleiotropic mouse phenotype extra-toes spotting is caused by translation initiation factor Eif3c mutations and is associated with disrupted sonic hedgehog signaling.

Polydactyly is a common malformation and can be an isolated anomaly or part of a pleiotropic syndrome. The elucidation of the mutated genes that cause polydactyly provides insight into limb development pathways. The extra-toes spotting (Xs) mouse phenotype manifests anterior polydactyly, predominantly in the forelimbs, with ventral hypopigmenation. The mapping of Xs(J) to chromosome 7 was confirmed, and the interval was narrowed to 322 kb using intersubspecific crosses. Two mutations were identified in eukaryotic translation initiation factor 3 subunit C (Eif3c). An Eif3c c.907C>T mutation (p.Arg303X) was identified in Xs(J), and a c.1702_1758del mutation (p.Leu568_Leu586del) was identified in extra-toes spotting-like (Xsl), an allele of Xs(J). The effect of the Xs(J) mutation on the SHH/GLI3 pathway was analyzed by in situ hybridization analysis, and we show that Xs mouse embryos have ectopic Shh and Ptch1 expression in the anterior limb. In addition, anterior limb buds show aberrant Gli3 processing, consistent with perturbed SHH/GLI3 signaling. Based on the occurrence of Eif3c mutations in 2 Xs lines and haploinsufficiency of the Xs(J) allele, we conclude that the Xs phenotype is caused by a mutation in Eif3c, a component of the translation initiation complex, and that the phenotype is associated with aberrant SHH/GLI3 signaling.