Sophia Gaynor and Hannah Seberg Will Present Their Research Wednesday, 10/8/2014
NEXT-GENERATION SEQUENCING IN ATTEMPTED SUICIDE
S. Gaynor1, E. Monson1, M. Breen1, K. Novak1, J.B. Potash1, V.L. Willour1
1 University of Iowa, Department of Psychiatry
Suicidal behavior is a complex phenotype with an estimated heritability of 30-50%. While this heritability is partly dependent on the presence of psychiatric disorders, other evidence implicates an independent heritable factor. In order to assess the genetic basis of this independent factor, we are conducting a next-generation targeted sequencing project on 38 candidate genes and two candidate regions in 500 bipolar (BP) subjects that have attempted suicide and 500 BP subjects that have not attempted suicide. The candidate genes and regions were chosen based on hypotheses generated by our lab and evidence from the suicide literature. The target regions for sequencing include all exons of all alternative transcripts, intron-exon boundaries, alternative promoter regions, and any putative regulatory elements identified by ENCODE, including 10kb upstream and downstream of each gene. We currently have completed the sequencing for all of our samples and have data analyzed for 505 of these samples, including 254 BP attempters and 251 BP non-attempters. For these first 505 samples, we found 14,159 unique variant sites following quality control filtering. We performed both individual variant tests and gene burden tests on these variant sites. Our top findings from the individual variant testing include an intergenic region of 2p25 (p=1.20×10-4) and an intronic region of LRRTM4 (p=7.16×10-4). For gene burden testing, our top results based on p-value are DLG3 (p=1.07×10-2) and TMEM132A (p=1.3×10-2). Our top results based on odds ratio are NLGN4X (OR=0.191) and GRIN2B (OR=5.02). We are currently in the process of analyzing the remaining samples, and the addition of these samples will provide more power to identify significant variant or gene associations. The identification of variants associated with suicidal behavior in these candidate genes and regions will help elucidate the biological basis of this complex phenotype.
TFAP2A and MITF work in parallel to activate melanocyte differentiation genes
Hannah Seberg1, Eric Van Otterloo2, Gregory Bonde2, Robert Cornell1,2
1Interdisciplinary Program in Genetics, 2Department of Anatomy and Cell Biology
Transcription factor activator protein 2 alpha (TFAP2A) is widely expressed in the neural crest and multiple neural crest-derived cell types, including melanocytes. Mutations in tfap2a cause pigmentation phenotypes in humans, mice, and zebrafish. However, it is unclear how TFAP2A activity relates to that of lineage-specific Micropthalmia-associated transcription factor (MITF), which directly regulates melanocyte differentiation effectors such as melanin synthesis genes. This issue is complicated by the redundant expression of Tfap2 paralogs. In zebrafish melanocytes, tfap2e is highly expressed along with lower levels of tfap2a and tfap2c. To study the role of multiple paralogs in melanocyte development, we created a tfap2e mutant using zinc finger nucleases. Whereas the number of melanocytes in tfap2a mutants is reduced by about 66%, tfap2e mutants have no discernable phenotype. However, tfap2a/e double mutants display about 50% reduction of melanocytes, suggesting partially redundant functions for tfap2a and tfap2e. We next assessed the genetic interaction between tfap2a and mitfa. Single heterozygous embryos are phenotypically normal, while tfap2a;mitfa double heterozygotes have fewer melanocytes. These data indicate that TFAP2A and MITF interact genetically, but the mechanism of this interaction is unknown. To test the model that TFAP2A and MITF co-activate melanocyte differentiation genes, we identified genes that are likely to be direct targets of TFAP2A. First, we generated a profile of genes that are significantly downregulated in trunks of tfap2a null zebrafish embryos. We then conducted anti-TFAP2A ChIP-seq in human primary melanocytes to create a profile of TFAP2A-bound loci. Genes at the intersection of these profiles include several melanin synthesis genes, such as DCT, PMEL, and OCA2. Many of these genes are also known to be direct targets of MITF. These results provide evidence that TFAP2A and MITF work in parallel to promote melanocyte differentiation, and show that the widely-expressed transcription factor TFAP2A can directly regulate expression of lineage-specific targets.