GeneLink: a database to facilitate genetic studies of complex traits

BACKGROUND: In contrast to gene-mapping studies of simple Mendelian disorders, genetic analyses of complex traits are far more challenging, and high quality data management systems are often critical to the success of these projects. To minimize the difficulties inherent in complex trait studies, we have developed GeneLink, a Web-accessible, password-protected Sybase database. RESULTS: GeneLink is a powerful tool for complex trait mapping, enabling genotypic data to be easily merged with pedigree and extensive phenotypic data. Specifically designed to facilitate large-scale (multi-center) genetic linkage or association studies, GeneLink securely and efficiently handles large amounts of data and provides additional features to facilitate data analysis by existing software packages and quality control. These include the ability to download chromosome-specific data files containing marker data in map order in various formats appropriate for downstream analyses (e.g., GAS and LINKAGE). Furthermore, an unlimited number of phenotypes (either qualitative or quantitative) can be stored and analyzed. Finally, GeneLink generates several quality assurance reports, including genotyping success rates of specified DNA samples or success and heterozygosity rates for specified markers. CONCLUSIONS: GeneLink has already proven an invaluable tool for complex trait mapping studies and is discussed primarily in the context of our large, multi-center study of hereditary prostate cancer (HPC). GeneLink is freely available at

A Large-Scale Zebrafish Gene Knockout Resource for the Genome-Wide Study of Gene Function

With the completion of the zebrafish genome sequencing project, it becomes possible to analyze the function of zebrafish genes in a systematic way. The first step in such an analysis is to inactivate each protein-coding gene by targeted or random mutation. Here we describe a streamlined pipeline using proviral insertions coupled with high-throughput sequencing and mapping technologies to widely mutagenize genes in the zebrafish genome. We also report the first 6144 mutagenized and archived F1’s predicted to carry up to 3776 mutations in annotated genes. Using in vitro fertilization, we have rescued and characterized ~0.5% of the predicted mutations, showing mutation efficacy and a variety of phenotypes relevant to both developmental processes and human genetic diseases. Mutagenized fish lines are being made freely available to the public through the Zebrafish International Resource Center. These fish lines establish an important milestone for zebrafish genetics research and should greatly facilitate systematic functional studies of the vertebrate genome

GeneLink: a database to facilitate genetic studies of complex traits

Abstract Background In contrast to gene-mapping studies of simple Mendelian disorders, genetic analyses of complex traits are far more challenging, and high quality data management systems are often critical to the success of these projects. To minimize the difficulties inherent in complex trait studies, we have developed GeneLink, a Web-accessible, password-protected Sybase database. Results GeneLink is a powerful tool for complex trait mapping, enabling genotypic data to be easily merged with pedigree and extensive phenotypic data. Specifically designed to facilitate large-scale (multi-center) genetic linkage or association studies, GeneLink securely and efficiently handles large amounts of data and provides additional features to facilitate data analysis by existing software packages and quality control. These include the ability to download chromosome-specific data files containing marker data in map order in various formats appropriate for downstream analyses (e.g., GAS and LINKAGE). Furthermore, an unlimited number of phenotypes (either qualitative or quantitative) can be stored and analyzed. Finally, GeneLink generates several quality assurance reports, including genotyping success rates of specified DNA samples or success and heterozygosity rates for specified markers. Conclusions GeneLink has already proven an invaluable tool for complex trait mapping studies and is discussed primarily in the context of our large, multi-center study of hereditary prostate cancer (HPC). GeneLink is freely available at http://research.nhgri.nih.gov/genelink.</p

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 XsJ 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 XsJ, and a c.1702_1758del mutation (p.Leu568_Leu586del) was identified in extra-toes spotting-like (Xsl), an allele of XsJ. The effect of the XsJ 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 XsJ 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.—Gildea, D. E., Luetkemeier, E. S., Bao, X., Loftus, S. K., Mackem, S., Yang, Y., Pavan, W. J., Biesecker, L. G. The pleiotropic mouse phenotype extra-toes spotting is caused by translation initiation factor Eif3c mutations and is associated with disrupted sonic hedgehog signaling

TFAP2 paralogs regulate melanocyte differentiation in parallel with MITF

<div><p>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 <i>TFAP2A</i> 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 <i>Tfap2a</i>, and find that expression of a small subset of genes underlying pigmentation phenotypes is TFAP2A-dependent, including <i>Dct</i>, <i>Mc1r</i>, <i>Mlph</i>, and <i>Pmel</i>. 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 <i>TRPM1</i> is bound by both TFAP2A and MITF, and we show that the activity of a minimal <i>TRPM1</i> promoter is lost upon deletion of the TFAP2A binding sites. However, the expression of <i>Trpm1</i> is not TFAP2A-dependent, implying that additional TFAP2 paralogs function redundantly to drive melanocyte differentiation, which is consistent with previous results from zebrafish. Paralogs <i>Tfap2a</i> and <i>Tfap2b</i> are both expressed in mouse melanocytes, and we show that mouse embryos with <i>Wnt1-Cre</i>-mediated deletion of <i>Tfap2a</i> and <i>Tfap2b</i> 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 <i>tfap2a</i> and <i>mitfa</i> in zebrafish, but find that artificially elevating expression of <i>tfap2a</i> does not increase levels of melanin in <i>mitfa</i> 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.</p></div

TFAP2A peaks are associated with genes involved in pigmentation.

<p>(A-C) Density-based clustering of H3K27ac signal at (A) TFAP2A peaks, (B) MITF peaks, and (C) TFAP2A peaks that overlap MITF peaks in human melanocytes (H3K27ac data from GSM1127072 [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006636#pgen.1006636.ref064" target="_blank">64</a>]), MITF peaks from [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006636#pgen.1006636.ref018" target="_blank">18</a>]). (D) Overlap between genes associated with active TFAP2A peaks and genes associated with active MITF peaks in human melanocytes. (E) Typical enhancers (gray) and super-enhancers (colored) in human melanocytes that overlap neither TFAP2A nor MITF peaks, TFAP2A peaks only, MITF peaks only, or both TFAP2A and MITF peaks. Labels identify melanocyte genes of interest. (F) Diagram of the <i>TRPM1</i> -700 bp promoter element depicting the positions of four TFAP2A binding sites (A1–A4) and the previously reported E-box 1 MITF binding site (E1). (G) Luciferase assays in M21 melanoma cells. Deletion of all four TFAP2A binding sites (ΔAP2) significantly reduced reporter activity compared to the intact <i>TRPM1</i> -700bp element (Student’s t-test, **p = 0.01).</p

TFAP2A binds active enhancers and promoters in mouse melanocytes.

<p>(A) Pie chart showing distribution of mouse TFAP2A peaks with respect to genomic features. TSS, transcription start site; TTS, transcription termination site. (B) Distance from TSS to the nearest TFAP2A peak for genes in three expression categories: highest 1000, median 1000, or lowest 1000. Promoter-proximal TFAP2A peaks are enriched at highly expressed genes (RNA-seq on mouse melan-a cells at GSE87051). (C) Examples of active enhancer signatures defined by H3K4me1 peaks flanking a p300 peak [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006636#pgen.1006636.ref065" target="_blank">65</a>], and partial enhancer signatures, overlapping TFAP2A peaks upstream of the melanocyte differentiation gene <i>Slc45a2</i>. (D) Distance from TSS to the nearest TFAP2A peaks that overlap the active enhancer signature for genes in three expression categories: highest 1000, median 1000, or lowest 1000. (E) Overlap of TFAP2A ChIP-seq peaks in mouse melanocytes with published TFAP2A ChIP-seq peaks in mouse kidney and epididymis cells [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006636#pgen.1006636.ref068" target="_blank">68</a>]. 34% of melanocyte peaks were shared with one or both of the other cell types. (F) MEME-ChIP analysis of unique peaks from each cell type. Melanocyte-unique peaks are significantly enriched for SOX10 and M-Box MITF binding motifs, while kidney-unique and epididymis-unique peaks are not. *All motifs shown are a result of <i>de novo</i> MEME-ChIP enrichment analysis except the M-Box, which we specifically searched using the Analysis of Motif Enrichment (AME) tool [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006636#pgen.1006636.ref109" target="_blank">109</a>].</p

TFAP2A and TFAP2B redundantly regulate murine melanocyte development.

<p>(A-D) Lateral views of E12.0 control (A), <i>Tfap2a</i> SCM (B), <i>Tfap2b</i> SCM (C), or <i>Tfap2a/Tfap2b</i> DCM (D) mouse embryos processed for β-galactosidase (β-gal) staining. β-gal+ cells are a result of recombination of the <i>r26r</i>-allele by the <i>Wnt1-Cre</i> transgene, labeling neural crest cells and derivatives. (E-L) <i>In situ</i> hybridization expression patterns for <i>Pmel</i> (E-H, dorsal views) or <i>Dct</i> (I-L, lateral views) in control (E, I), <i>Tfap2a</i> SCM (F, J), <i>Tfap2b</i> SCM (G, K), or <i>Tfap2a/Tfap2b</i> DCM (H, L) E12.0 mouse embryos. Insets in (A-L) show higher magnification images just above the hindlimb in a similar viewing plane as the low magnification image. (M, N) Lateral views of the trunk processed for ɑ-neurofilament immunoreactivity in an E10.5 control (M) or <i>Tfap2a</i>/<i>Tfap2b</i> DCM (N), revealing developing dorsal root ganglia (a subset labeled with arrowheads). (O, P) Immunofluorescence of cryosections (cross-sectional plane at the level of the hindlimb) of an E12.0 control (O) or <i>Tfap2a</i>/<i>Tfap2b</i> DCM (P), containing a Tomato-reporter (<i>rTMr</i>) allele labeling neural crest cells and derivatives (arrowheads highlight a subset of melanocytes in the ventrolateral pathway). Abbreviations: DCM, double conditional mutant; drg, dorsal root ganglia; hl, hindlimb; nt, neural tube; SCM, single conditional mutant (A = <i>Tfap2a</i> or B = <i>Tfap2b</i>). Scale bars = 250μM, except M, N = 500μM.</p

<i>tfap2a</i> and <i>mitfa</i> display a genetic interaction in zebrafish.

<p>(A-I) Compared to wildtype embryos at 72 hpf (A), melanocytes in <i>mitfa</i>^{<i>z25/z25</i>} mutant embryos (B) are dark but punctate, and melanocytes in <i>mitfa</i>^{<i>w2/z25</i>} trans-heterozygous embryos (C) are pale, punctate, and reduced in number. While heterozygous mutation of <i>tfap2a</i> in the wildtype background (<i>tfap2a</i>^<i>+/-</i>) (D) does not result in a melanocyte phenotype, <i>mitfa</i>^{<i>z25/z25</i>}<i>;tfap2a</i>^<i>+/-</i> mutants (E) and <i>mitfa</i>^{<i>w2/z25</i>}<i>;tfap2a</i>^<i>+/-</i> mutants (F) display a loss of melanocytes in the ventral stripe (brackets). Similarly, the well-characterized phenotype of fewer, paler melanocytes in <i>tfap2a</i>^<i>-/-</i> null mutants (G) is more pronounced in both the <i>mitfa</i>^{<i>z25/z25</i>} mutant background (H) and the <i>mitfa</i>^{<i>w2/z25</i>} mutant background (I). (J, K) Counts of ventral stripe melanocytes (see brackets in A-I) showed no difference between wildtype and <i>tfap2a</i>^<i>+/-</i> embryos (J, n = 10+), while <i>tfap2a</i>^<i>+/-</i> embryos in both <i>mitfa</i> mutant backgrounds had significantly fewer melanocytes in the ventral stripe (K, n = 4–8). One-way ANOVA: *p<0.05, **p<0.01, ****p<0.0001.</p