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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., Competing Interests: Declaration of interests The authors declare no competing interests., (Published by Elsevier Inc.)

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., (Published 2021. This article is a U.S. Government work and is in the public domain in the USA.)

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.

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.

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.

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., (Published 2018. This article is a U.S. Government work and is in the public domain in the USA.)

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., (© 2018 Zhang et al.; Published by Cold Spring Harbor Laboratory Press.)

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., (Published 2018. This article is a U.S. Government work and is in the public domain in the USA.)

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-β 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.

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., (Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

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., (Published 2017. This article is a U.S. Government work and is in the public domain in the USA.)

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.

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., (Published by Oxford University Press 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

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 (TGFβ1) 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 TGFβ1 pathway-expressing melanoma cells and that HIF1A upregulates miR-210, miR-218, miR-224, and miR-452. Reduced expression of these four miRNAs in TGFβ1 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 TGFβ1 pathway-expressing melanoma cells, in part via alterations in these four miRNAs., (Published 2014. This article is a U.S. Government work and is in the public domain in the USA.)

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., (Copyright © 2013 Elsevier Inc. All rights reserved.)

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., (©2013 AACR.)

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.

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&#95;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&#95;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&#95;MCS6. Consistent with its in vitro activity, we also show that ERBB3&#95;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&#95;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&#95;MCS6.

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&#95;1758del mutation (p.Leu568&#95;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.

Over 10 years have passed since the first Sox gene was implicated in melanocyte development. Since then, we have discovered that SOX5, SOX9, SOX10 and SOX18 all participate as transcription factors that affect key melanocytic genes in both regulatory and modulatory fashions. Both SOX9 and SOX10 play major roles in the establishment and normal function of the melanocyte; SOX10 has been shown to heavily influence melanocyte development and SOX9 has been implicated in melanogenesis in the adult. Despite these advances, the precise cellular and molecular details of how these SOX proteins are regulated and interact during all stages of the melanocyte life cycle remain unknown. Improper regulation of SOX9 or SOX10 is also associated with cancerous transformation, and thus understanding the normal function of SOX proteins in the melanocyte will be key to revealing how these proteins contribute to melanoma.

Micrognathia, glossoptosis, and cleft palate comprise one of the most common malformation sequences, Robin sequence. It is a component of the TARP syndrome, talipes equinovarus, atrial septal defect, Robin sequence, and persistent left superior vena cava. This disorder is X-linked and severe, with apparently 100% pre- or postnatal lethality in affected males. Here we characterize a second family with TARP syndrome, confirm linkage to Xp11.23-q13.3, perform massively parallel sequencing of X chromosome exons, filter the results via a number of criteria including the linkage region, use a unique algorithm to characterize sequence changes, and show that TARP syndrome is caused by mutations in the RBM10 gene, which encodes RNA binding motif 10. We further show that this previously uncharacterized gene is expressed in midgestation mouse embryos in the branchial arches and limbs, consistent with the human phenotype. We conclude that massively parallel sequencing is useful to characterize large candidate linkage intervals and that it can be used successfully to allow identification of disease-causing gene mutations., (Copyright (c) 2010 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Extensive studies of the biology of the pigment-producing cell (melanocyte) have resulted in a wealth of knowledge regarding the genetics and developmental mechanisms governing skin and hair pigmentation. The ease of identification of altered pigment phenotypes, particularly in mouse coat color mutants, facilitated early use of the pigmentary system in mammalian genetics and development. In addition to the large collection of developmental genetics data, melanocytes are of interest because their malignancy results in melanoma, a highly aggressive and frequently fatal cancer that is increasing in Caucasian populations worldwide. The genetic programs regulating melanocyte development, function, and malignancy are highly complex and only partially understood. Current research in melanocyte development and pigmentation is revealing new genes important in these processes and additional functions for previously known individual components. A detailed understanding of all the components involved in melanocyte development and function, including interactions with neighboring cells and response to environmental stimuli, will be necessary to fully comprehend this complex system. The inherent characteristics of pigmentation biology as well as the resources available to researchers in the pigment cell community make melanocytes an ideal cell type for analysis using systems biology approaches. In this review, the study of melanocyte development and pigmentation is considered as a candidate for systems biology-based analyses.

Utilizing a recently identified Sox10 distal enhancer directing Cre expression, we report S4F:Cre, a transgenic mouse line capable of inducing recombination in oligodendroglia and all examined neural crest derived tissues. Assayed using R26R:LacZ reporter mice expression was detected in neural crest derived tissues including the forming facial skeleton, dorsal root ganglia, sympathetic ganglia, enteric nervous system, aortae, and melanoblasts, consistent with Sox10 expression. LacZ reporter expression was also detected in non-neural crest derived tissues including the oligodendrocytes and the ventral neural tube. This line provides appreciable differences in Cre expression pattern from other transgenic mouse lines that mark neural crest populations, including additional populations defined by the expression of other SoxE proteins. The S4F:Cre transgenic line will thus serve as a powerful tool for lineage tracing, gene function characterization, and genome manipulation in these populations., ((c) 2009 Wiley-Liss, Inc.)

A full understanding of transcriptional regulation requires integration of information obtained from multiple experimental datasets. These include datasets annotating gene expression within the context of an entire organism under normal and genetically perturbed conditions. Here we describe an expression dataset annotating pigment cell-expressed genes of the developing melanocyte and retinal pigmented epithelium lineages. Expression images are annotated and available at http://research.nhgri.nih.gov/manuscripts/Loftus/March2009/. Data are also summarized in a standardized manner using a universal melanoblast scoring scale that accounts for the embryonic location of cells and regional cell density. This approach allowed us to classify 14 pigment genes into four groupings classified by cell lineage expression, temporal-spatial context, and differential alteration in response to altered MITF and SOX10 status. Significant differences in regional populations were also observed across inbred strain backgrounds, highlighting the value of this approach to identify modifier allele influences on melanoblast number and distributions. This analysis revealed novel features of in vivo expression patterns that are not measurable by in vitro-based assays, providing data that in combination with genomic analyses will allow modeling of pigment cell gene expression in development and disease.

Microphthalmia-associated transcription factor (MITF) is involved in melanocyte cell development, pigmentation and neoplasia. To determine whether MITF is somatically mutated in melanoma, we compared the sequence of MITF from primary and metastatic lesions to patient-matched normal DNA. In the 50 metastatic melanoma tumor lines analysed, we discovered four samples that had genomic amplifications of MITF and four that had MITF mutations in the regions encoding the transactivation, DNA binding or basic, helix-loop-helix domains. Sequence analysis for SOX10, a transcription factor, which both acts upstream of MITF and synergizes with MITF, identified an additional three samples with frameshift or nonsense mutations. Microphthalmia-associated transcription factor and SOX10 were found to be mutated in a mutually exclusive fashion, possibly suggesting disruption in a common genetic pathway. Taken together we found that over 20% of the metastatic melanoma cases had alterations in the MITF pathway. We show that the MITF pathway is also altered in primary melanomas: 2/26 demonstrated mutations in MITF and 6/55 demonstrated mutations in SOX10. Our findings suggest that altered MITF function during melanomagenesis can be achieved by MITF amplification, MITF single base substitutions or by mutation of its regulator SOX10.

Expression profile analysis clusters Gpnmb with known pigment genes, Tyrp1, Dct, and Si. During development, Gpnmb is expressed in a pattern similar to Mitf, Dct and Si with expression vastly reduced in Mitf mutant animals. Unlike Dct and Si, Gpnmb remains expressed in a discrete population of caudal melanoblasts in Sox10-deficient embryos. To understand the transcriptional regulation of Gpnmb we performed a whole genome annotation of 2,460,048 consensus MITF binding sites, and cross-referenced this with evolutionarily conserved genomic sequences at the GPNMB locus. One conserved element, GPNMB-MCS3, contained two MITF consensus sites, significantly increased luciferase activity in melanocytes and was sufficient to drive expression in melanoblasts in vivo. Deletion of the 5'-most MITF consensus site dramatically reduced enhancer activity indicating a significant role for this site in Gpnmb transcriptional regulation. Future analysis of the Gpnmb locus will provide insight into the transcriptional regulation of melanocytes, and Gpnmb expression can be used as a marker for analyzing melanocyte development and disease progression.

Sox10 is a dynamically regulated transcription factor gene that is essential for the development of neural crest-derived and oligodendroglial populations. Developmental genes often require multiple regulatory sequences that integrate discrete and overlapping functions to coordinate their expression. To identify Sox10 cis-regulatory elements, we integrated multiple model systems, including cell-based screens and transposon-mediated transgensis in zebrafish, to scrutinize mammalian conserved, noncoding genomic segments at the mouse Sox10 locus. We demonstrate that eight of 11 Sox10 genomic elements direct reporter gene expression in transgenic zebrafish similar to patterns observed in transgenic mice, despite an absence of observable sequence conservation between mice and zebrafish. Multiple segments direct expression in overlapping populations of neural crest derivatives and glial cells, ranging from pan-Sox10 and pan-neural crest regulatory control to the modulation of expression in subpopulations of Sox10-expressing cells, including developing melanocytes and Schwann cells. Several sequences demonstrate overlapping spatial control, yet direct expression in incompletely overlapping developmental intervals. We were able to partially explain neural crest expression patterns by the presence of head to head SoxE family binding sites within two of the elements. Moreover, we were able to use this transcription factor binding site signature to identify the corresponding zebrafish enhancers in the absence of overall sequence homology. We demonstrate the utility of zebrafish transgenesis as a high-fidelity surrogate in the dissection of mammalian gene regulation, especially those with dynamically controlled developmental expression., Competing Interests: The authors have declared that no competing interests exist.

The neural crest (NC) is a population of embryonic stem cells that gives rise to numerous cell types, including the glia and neurons of the peripheral and enteric nervous systems and the melanocytes of the skin and hair. Mutations in genes and genetic pathways regulating NC development lead to a wide spectrum of human developmental disorders collectively called neurocristopathies. To identify molecular pathways regulating NC development and to understand how alterations in these processes lead to disease, we established an N-ethyl-N-nitrosourea (ENU) mutagenesis screen utilizing a mouse model sensitized for NC defects, Sox10(LacZ/+). Out of 71 pedigrees analyzed, we identified and mapped four heritable loci, called modifier of Sox10 expression pattern 1-4 (msp1-4), which show altered NC patterning. In homozygous msp1 embryos, Sox10(LacZ) expression is absent in cranial ganglia, cranial nerves, and the sympathetic chain; however, the development of other Sox10-expressing cells appears unaffected by the mutation. Linkage analysis, sequencing, and complementation testing confirmed that msp1 is a new allele of the receptor tyrosine kinase Erbb3, Erbb3(msp1), that carries a single amino acid substitution in the extracellular region of the protein. The ENU-induced mutation does not alter protein expression, however, it is sufficient to impair ERBB3 signaling such that the embryonic defects observed in msp1 resemble those of Erbb3 null alleles. Biochemical analysis of the mutant protein showed that ERBB3 is expressed on the cell surface, but its ligand-induced phosphorylation is dramatically reduced by the msp1 mutation. These findings highlight the importance of the mutated residue for ERBB3 receptor function and activation. This study underscores the utility of using an ENU mutagenesis to identify genetic pathways regulating NC development and to dissect the roles of discrete protein domains, both of which contribute to a better understanding of gene function in a cellular and developmental setting., Competing Interests: The authors have declared that no competing interests exist.

Haploinsufficiency for the transcription factor SOX10 is associated with the pigmentary deficiencies of Waardenburg syndrome (WS) and is modeled in Sox10 haploinsufficient mice (Sox10(LacZ/+)). As genetic background affects WS severity in both humans and mice, we established an N-ethyl-N-nitrosourea (ENU) mutagenesis screen to identify modifiers that increase the phenotypic severity of Sox10(LacZ/+) mice. Analysis of 230 pedigrees identified three modifiers, named modifier of Sox10 neurocristopathies (Mos1, Mos2 and Mos3). Linkage analysis confirmed their locations on mouse chromosomes 13, 4 and 3, respectively, within regions distinct from previously identified WS loci. Positional candidate analysis of Mos1 identified a truncation mutation in a hedgehog(HH)-signaling mediator, GLI-Kruppel family member 3 (Gli3). Complementation tests using a second allele of Gli3 (Gli3(Xt-J)) confirmed that a null mutation of Gli3 causes the increased hypopigmentation in Sox10(LacZ/+);Gli3(Mos1/)(+) double heterozygotes. Early melanoblast markers (Mitf, Sox10, Dct, and Si) are reduced in Gli3(Mos1/)(Mos1) embryos, indicating that loss of GLI3 signaling disrupts melanoblast specification. In contrast, mice expressing only the GLI3 repressor have normal melanoblast specification, indicating that the full-length GLI3 activator is not required for specification of neural crest to the melanocyte lineage. This study demonstrates the feasibility of sensitized screens to identify disease modifier loci and implicates GLI3 and other HH signaling components as modifiers of human neurocristopathies.

Mutations in the transcription factor Sox10 or Endothelin Receptor B (Ednrb) result in Waardenburg Syndrome Type IV (WS-IV), which presents with deficiencies of neural crest derived melanocytes (hypopigmentation) and enteric ganglia (hypoganglionosis). As Sox10 and Ednrb are expressed in mouse migratory neural crest cells and melanoblasts, we investigated the possibility that SOX10 and EDNRB function through a hierarchical relationship during melanocyte development. However, our results support a distinct rather than hierarchical relationship. First, SOX10 expression continues in Ednrb null melanoblasts, demonstrating that SOX10 expression is not dependent on EDNRB function. Second, Ednrb expression persists in E10.5 Sox10null embryos, demonstrating that Ednrb is not dependent on SOX10 for expression in migratory neural crest cells. Third, over-expression of SOX10 in melanoblasts of mice that harbor null or hypomorphic Ednrb alleles does not rescue hypopigmentation, suggesting that SOX10 overexpression can neither complement a lack of EDNRB function nor increase Ednrb expression. Fourth, mice that are double heterozygous for loss-of-function mutations in Sox10 and Ednrb do not demonstrate synergistically increased hypopigmentation compared to mice that are single heterozygotes for either mutation alone, suggesting a lack of direct genetic interaction between these genes. Our results suggest that SOX10 does not directly activate Ednrb transcription in the melanocyte lineage. Given that SOX10 directly activates Ednrb in the enteric nervous system, our results suggest that SOX10 may differentially activate target genes based on the particular cellular context.

Pancreatic insufficiency (PI) when left untreated results in a state of malnutrition due to an inability to absorb nutrients. Frequently, PI is diagnosed as part of a larger clinical presentation in cystic fibrosis or Shwachman-Diamond syndrome. In this study, a mouse model for isolated exocrine PI was identified in a mouse line generated by a transgene insertion. The trait is inherited in an autosomal recessive pattern, and homozygous animals are growth retarded, have abnormal immunity, and have reduced life span. Mice with the disease locus, named pequeño (pq), exhibit progressive apoptosis of pancreatic acinar cells with severe exocrine acinar cell loss by 8 wk of age, while the islets and ductal tissue persist. The mutation in pq/pq mice results from a random transgene insertion. Molecular characterization of the transgene insertion site by fluorescent in situ hybridization and genomic deletion mapping identified an approximately 210-kb deletion on Chromosome 3, deleting two genes. One of these genes, Serpini2, encodes a protein that is a member of the serpin family of protease inhibitors. Reintroduction of only the Serpini2 gene by bacterial artificial chromosome transgenic complementation corrected the acinar cell defect as well as body weight and immune phenotypes, showing that deletion of Serpini2 causes the pequeño phenotype. Dietary supplementation of pancreatic enzymes also corrected body size, body weight, and immunodeficiency, and increased the life span of Serpini2-deficient mice, despite continued acinar cell loss. To our knowledge, this study describes the first characterized genetic animal model for isolated PI. Genetic complementation of the transgene insertion mutant demonstrates that Serpini2 deficiency directly results in the acinar cell apoptosis, malabsorption, and malnutrition observed in pq/pq mice. The rescue of growth retardation, immunodeficiency, and mortality by either Serpini2 bacterial artificial chromosome transgenic expression or by pancreatic enzyme supplementation demonstrates that these phenotypes are secondary to malnutrition in pq/pq mice., Competing Interests: Competing interests. The authors have declared that no competing interests exist.

Mutation of genes that regulate neural crest-derived melanoblast development and survival can result in reduction and/or loss of mature melanocytes. The reduction in melanocyte number in the skin and hair follicles manifests itself as areas of hypopigmentation, commonly described as white spotting in mice. To date ten genes have been identified which are associated with white-spotting phenotypes in mouse. Seven of these genes are associated with neural crest and melanocyte disorders in humans. This review summarizes the phenotypes associated with mutation of these genes in both mouse and man. We describe our current understanding of how these genes function in development, and explore their complex roles regulating the various stages of melanocyte development.

An in vitro gene complementation approach has been developed to dissect gene function and regulation in neural crest (NC) development and disease. The approach uses the avian RCAS virus to express genes in NC cells derived from transgenic mice expressing the RCAS receptor TVA, under the control of defined promoter elements. Constructs for creating TVA transgenic mice were developed using site-specific recombination GATEWAY (GW), compatible vectors that can also be used to facilitate analysis of genomic fragments for transcriptional regulatory elements. By using these GW vectors to facilitate cloning, transgenic mouse lines were generated that express TVA in SOX10-expressing NC stem cells under the control of the Pax3 promoter. The Pax3-tv-a transgene was bred onto a Sox10-deficient background, and the feasibility of complementing genetic NC defects was demonstrated by infecting the Pax3-tv-a cells with an RCAS-Sox10 expression virus, thereby rescuing melanocyte development of Sox10-deficient NC cells. This system will be useful for assessing genetic hierarchies in NC development. Developmental Dynamics 229:54-62, 2004., (Copyright 2003 Wiley-Liss, Inc.)

Several features of the pigment defect in belted (bt) mutant mice suggest that it occurs as a result of a defect in melanocyte development that is unique from those described for other classical white-spotting mutations. We report here that bt mice carry mutations in Adamts20, a novel member of the ADAMTS family of secreted metalloproteases. Adamts20 shows a highly dynamic pattern of expression in the developing embryo that generally precedes the appearance of melanoblasts in the same region, and is not expressed in the migrating cells themselves. Adamts20 shows remarkable homology with GON-1, an ADAMTS family protease required for distal tip cell migration in C. elegans. Our results suggest that the role of ADAMTS proteases in the regulation of cell migration has been conserved in mammalian development.

Niemann-Pick disease type C (NPC) is a neurodegenerative disorder with major visceral complications, including liver disease that can be fatal before onset of neurodegeneration. We have sought to determine the extent to which visceral disease contributes to neurodegeneration by making transgenic mice in which the wild-type NPC1 protein is expressed primarily in the CNS using the prion promoter. When the transgene was introduced into the npc1(-/-) animals neurodegeneration was prevented, a 'normal' lifespan occurred and the sterility of npc1(-/-) mice was corrected. The rescue did not provide complete neurological correction in the CNS as GM2 and GM3 gangliosides were observed to accumulate in some neurons and glia of transgenic animals. Two of three transgenic lines demonstrated some low-level ectopic expression resulting in correction of visceral phenotypes in liver and spleen. Interestingly, the third transgenic line continued to have moderate histocytosis in liver and spleen, yet had no detectable neurodegeneration. Thus, it is primarily the lack of NPC1 in the CNS and not the secondary effects of the visceral involvement that causes the neurological decline in NPC disease. In addition, the expression levels of NPC1 found in the CNS of transgenic animals were much greater than in normal littermates, demonstrating that overexpression of NPC1 is not harmful and allowing possibilities for genetic therapy interventions that utilize overexpression.

Mutations of genes needed for melanocyte function can result in oculocutaneous albinism. Examination of similarities in human gene expression patterns by using microarray analysis reveals that RAB38, a small GTP binding protein, demonstrates a similar expression profile to melanocytic genes. Comparative genomic analysis localizes human RAB38 to the mouse chocolate (cht) locus. A G146T mutation occurs in the conserved GTP binding domain of RAB38 in cht mice. Rab38(cht)/Rab38(cht) mice exhibit a brown coat similar in color to mice with a mutation in tyrosinase-related protein 1 (Tyrp1), a mouse model for oculocutaneous albinism. The targeting of TYRP1 protein to the melanosome is impaired in Rab38(cht)/Rab38(cht) melanocytes. These observations, and the fact that green fluorescent protein-tagged RAB38 colocalizes with end-stage melanosomes in wild-type melanocytes, suggest that RAB38 plays a role in the sorting of TYRP1. This study demonstrates the utility of expression profile analysis to identify mammalian disease genes.

Avian leukosis type A virus-derived retroviral vectors have been used to introduce genes into cells expressing the corresponding avian receptor tv-a. This includes the use of Replication-Competent Avian sarcoma-leukosis virus (ASLV) long terminal repeat (LTR) with Splice acceptor (RCAS) vectors in the analysis of avian development, human and murine cell cultures, murine cell lineage studies and cancer biology. Previously, cloning of genes into this virus was difficult due to the large size of the vector and sparse cloning sites. To overcome some of the disadvantages of traditional cloning using the RCASBP-Y vector, we have modified the RCASBP-Y to incorporate "Gateway" site-specific recombination cloning of genes into the construct, either with or without HA epitope tags. We have found the repetitive "att" sequences, which are the targets for site-specific recombination, do not impair the production of infectious viral particles or the expression of the gene of interest. This is the first instance of site-specific recombination being used to generate retroviral gene constructs. These viral constructs will allow for the efficient transfer and expression of cDNAs needed for functional genomic analyses.

Regulation of gene expression is a fundamental process by which cells respond to both intracellular and extracellular signals. For a pigment cell, alterations in gene expression regulate the processes of cell migration, lineage restriction, differentiation, type of pigment produced, and progression from a normal pigment cell to that of melanoma. To date, the identification of genes involved in normal pigment cell development has been accomplished by the cloning of individual mutant alleles, a single gene at a time. Current advances in technology have now made it possible to use expression profile analysis to investigate, on a genomic scale, the process of pigment cell development and function. This review compares and contrasts the methods of subtractive suppressive polymerase chain reaction (PCR) and differential display with that of cDNA microarray analysis.

With cDNA microarrays, it is now possible to compare the expression of many genes simultaneously. To maximize the likelihood of finding genes whose expression is altered under the experimental conditions, it would be advantageous to be able to select clones for tissue-appropriate cDNA sets. We have taken advantage of the extensive sequence information in the dbEST expressed sequence tag (EST) database to identify a neural crest-derived melanocyte cDNA set for microarray analysis. Analysis of characterized genes with dbEST identified one library that contained ESTs representing 21 neural crest-expressed genes (library 198). The distribution of the ESTs corresponding to these genes was biased toward being derived from library 198. This is in contrast to the EST distribution profile for a set of control genes, characterized to be more ubiquitously expressed in multiple tissues (P < 1 x 10(-9)). From library 198, a subset of 852 clustered ESTs were selected that have a library distribution profile similar to that of the 21 neural crest-expressed genes. Microarray analysis demonstrated the majority of the neural crest-selected 852 ESTs (Mel1 array) were differentially expressed in melanoma cell lines compared with a non-neural crest kidney epithelial cell line (P < 1 x 10(-8)). This was not observed with an array of 1,238 ESTs that was selected without library origin bias (P = 0.204). This study presents an approach for selecting tissue-appropriate cDNAs that can be used to examine the expression profiles of developmental processes and diseases.

An integrated human-mouse positional candidate approach was used to identify the gene responsible for the phenotypes observed in a mouse model of Niemann-Pick type C (NP-C) disease. The predicted murine NPC1 protein has sequence homology to the putative transmembrane domains of the Hedgehog signaling molecule Patched, to the cholesterol-sensing regions of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and SREBP cleavage-activating protein (SCAP), and to the NPC1 orthologs identified in human, the nematode Caenorhabditis elegans, and the yeast Saccharomyces cerevisiae. The mouse model may provide an important resource for studying the role of NPC1 in cholesterol homeostasis and neurodegeneration and for assessing the efficacy of new drugs for NP-C disease.

Niemann-Pick type C (NP-C) disease, a fatal neurovisceral disorder, is characterized by lysosomal accumulation of low density lipoprotein (LDL)-derived cholesterol. By positional cloning methods, a gene (NPC1) with insertion, deletion, and missense mutations has been identified in NP-C patients. Transfection of NP-C fibroblasts with wild-type NPC1 cDNA resulted in correction of their excessive lysosomal storage of LDL cholesterol, thereby defining the critical role of NPC1 in regulation of intracellular cholesterol trafficking. The 1278-amino acid NPC1 protein has sequence similarity to the morphogen receptor PATCHED and the putative sterol-sensing regions of SREBP cleavage-activating protein (SCAP) and 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase.

Niemann-Pick disease type C (NP-C) is an autosomal recessive lipidosis linked to chromosome 18q11-12, characterized by lysosomal accumulation of unesterified cholesterol and delayed induction of cholesterol-mediated homeostatic responses. This cellular phenotype is identifiable cytologically by filipin staining and biochemically by measurement of low-density lipoprotein-derived cholesterol esterification. The mutant Chinese hamster ovary cell line (CT60), which displays the NP-C cellular phenotype, was used as the recipient for a complementation assay after somatic cell fusions with normal and NP-C murine cells suggested that this Chinese hamster ovary cell line carries an alteration(s) in the hamster homolog(s) of NP-C. To narrow rapidly the candidate interval for NP-C, three overlapping yeast artificial chromosomes (YACs) spanning the 1 centimorgan human NP-C interval were introduced stably into CT60 cells and analyzed for correction of the cellular phenotype. Only YAC 911D5 complemented the NP-C phenotype, as evidenced by cytological and biochemical analyses, whereas no complementation was obtained from the other two YACs within the interval or from a YAC derived from chromosome 7. Fluorescent in situ hybridization indicated that YAC 911D5 was integrated at a single site per CT60 genome. These data substantially narrow the NP-C critical interval and should greatly simplify the identification of the gene responsible in mouse and man. This is the first demonstration of YAC complementation as a valuable adjunct strategy for positional cloning of a human gene.

Treacher Collins syndrome is an autosomal dominant disorder of craniofacial development the features of which include conductive hearing loss and cleft palate. Recently, the Treacher Collins syndrome gene (TCOF1) has been positionally cloned and a series of five mutations within the coding sequence of the gene identified. In the current investigation, seven exons of TCOF1 have been identified which has permitted the identification of additional mutations in the gene. The mutations that have been identified are three distinct deletions and an insertion, which cause a frameshift, and a missense mutation which inactivates a donor splice site with extension of transcription into the intron. To date, all 10 of the mutations which have been reported result in a premature termination codon and are unique to a given family. As these mutations are spread throughout the gene, these observations provide further support for the hypothesis that Treacher Collins syndrome results from haploinsufficiency, although a dominant negative effect cannot, at this stage, be excluded.

Heparan sulfate-N-deacetylase/N-sulfotransferase (HSST) catalyzes both the N-deacetylation and the N-sulfation of heparan sulfate. Previous studies have resulted in the isolation of the human HSST gene from within the Treacher Collins syndrome locus (TCOF1) critical region on 5q. In the present study, the genomic organization of the HSST gene has been elucidated, and the 14 exons identified have been tested for TCOF1-specific mutations. As a result of these studies, mutations within the coding sequence and adjacent splice junctions of HSST can be excluded from a causative role in the pathogenesis of Treacher Collins syndrome.

Treacher Collins syndrome (TCOF1) is a dominant disorder of craniofacial development that has been linked previosuly to a region of chromosome 5q31.3-32. Identification of recombination events in affected individuals has reduced the candidate gene region to a 0.5-Mb area between the loci RPS14 (proximal) and ANX6 [distal]. A transcriptional map of this candidate gene region, generated by analysis of exon amplification clones, has identified the genomic location of four genes, heparan sulfate-N-sulfotransferase-N-deacetylase, glutathione peroxidase, as well as two novel, previously uncharacterized genes. Each of these genes, based on their location, must be considered candidates for TCOF1 locus.

The human CDX1 gene has been isolated from a small intestine cDNA library using a murine Cdx1 cDNA probe. The nucleotide sequence of CDX1 is 81% identical to murine Cdx1 and predicts a 265-amino-acid protein with 85% identity to the mouse protein (98% identity, including conservative amino acid changes). The CDX1 locus has been mapped to a cosmid contig from chromosome 5q31-q33, placing CDX1 approximately 100 kb distal to CSFIR. Expression of CDX1 in adults appears to be limited to the intestine and colon by Northern analysis, suggesting a possible role in the terminal differentiation of the intestine. Further analysis of CDX1 should elucidate the function of caudal-type homeobox genes in human development.