Characterization of regulatory elements leading to human limb malformations ABSTRACT Limb malformations are the second most common human congenital abnormality with a prevalence of 1 for every 500 births. Although several mutations in genes have been identified that explain syndromic forms (associated with other symptoms) of limb malformations, the characterization of mutations causing non- syndromic/isolated limb malformations has been less successful. A variety of molecular and clinical data suggests that mutations responsible for non-syndromic limb malformations may reside in distant noncoding regulatory sequences such as enhancers (sequences that regulate gene promoters). These data are based on position effects (chromosomal rearrangements that leave the gene intact but remove its regulatory elements) that lead to limb malformations, the observed modular nature of enhancers, and the recent example of a non- syndromic preaxial polydactyly in humans that has been linked to a long distance enhancer of the Sonic Hedgehog (SHH) gene. Long distance regulatory enhancers have traditionally been difficult to identify and very few of them have been characterized for limb regulatory expression thus far. In preliminary studies for this proposal, we have discovered 43 novel human limb enhancers using a mouse enhancer transgenic assay and verified several of them for pectoral fin expression in zebrafish. In order to discover additional human limb enhancers we are using advanced computational tools to dissect the unique sequence signatures in both the novel limb enhancers we discovered and previously reported ones. These signatures allow us to predict novel limb enhancers surrounding known limb-associated genes and throughout the human genome. These predicted limb enhancers will initially be tested in a high-throughput manner in zebrafish for fin expression. Positive fin enhancers will then be reverified in mice for limb expression. All characterized enhancers both in zebrafish and mouse will be available to the biomedical community through a web accessible browser. In addition, we have collected numerous DNA samples of patients with non-syndromic limb malformations and are in the process of collecting numerous more. We will conduct mutation analysis of these DNA samples within limb enhancers, and potential causative nucleotide changes will be tested for their effect on limb formation using the mouse as our model. The identification of causative sequences leading to non-syndromic limb malformations will result in improved patient counseling, the development of molecular testing including prenatal genetic testing, and an increased knowledge about the pathogenesis of human limb malformations and limb development.

Public Health Relevance

Characterization of regulatory elements leading to human limb malformations Mutations in genes leading to limb malformations, the second most common human congenital abnormality with a prevalence of 1 for every 500 births, have been discovered on the majority in a syndromic form (associated with other symptoms). There is a variety of molecular and clinical data to suggest that non- syndromic (isolated) limb malformations can be caused by mutations in distant regulatory noncoding sequences (DNA switches that tell the genes when and where to turn on or off), but only a small number of limb regulatory noncoding sequences have been discovered thus far and only one of these sequences has been linked to human non-syndromic limb malformations. In this proposal we have discovered 43 novel human limb regulatory noncoding sequences and have collected numerous DNA samples from patients with non- syndromic limb malformations enabling us not only to screen these 43 DNA sequences for mutations in non- syndromic patients, but to also identify in a high-throughput manner using advanced computational tools and zebrafish and mouse assays, novel limb regulatory sequences throughout the human genome, which will make for additional limb malformation mutation candidates.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
5R01HD059862-05
Application #
8445255
Study Section
Development - 2 Study Section (DEV2)
Program Officer
Javois, Lorette Claire
Project Start
2009-04-01
Project End
2014-03-31
Budget Start
2013-04-01
Budget End
2014-03-31
Support Year
5
Fiscal Year
2013
Total Cost
$436,784
Indirect Cost
$87,567
Name
University of California San Francisco
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
094878337
City
San Francisco
State
CA
Country
United States
Zip Code
94143
VanderMeer, Julia E; Lozano, Reymundo; Sun, Miao et al. (2014) A novel ZRS mutation leads to preaxial polydactyly type 2 in a heterozygous form and Werner mesomelic syndrome in a homozygous form. Hum Mutat 35:945-8
Erwin, Genevieve D; Oksenberg, Nir; Truty, Rebecca M et al. (2014) Integrating diverse datasets improves developmental enhancer prediction. PLoS Comput Biol 10:e1003677
Birnbaum, Ramon Y; Patwardhan, Rupali P; Kim, Mee J et al. (2014) Systematic dissection of coding exons at single nucleotide resolution supports an additional role in cell-specific transcriptional regulation. PLoS Genet 10:e1004592
VanderMeer, Julia E; Smith, Robin P; Jones, Stacy L et al. (2014) Genome-wide identification of signaling center enhancers in the developing limb. Development 141:4194-8
Kim, Mee J; Oksenberg, Nir; Hoffmann, Thomas J et al. (2014) Functional characterization of SIM1-associated enhancers. Hum Mol Genet 23:1700-8
Camp, J Gray; Frank, Christopher L; Lickwar, Colin R et al. (2014) Microbiota modulate transcription in the intestinal epithelium without remodeling the accessible chromatin landscape. Genome Res 24:1504-16
Oksenberg, N; Haliburton, G D E; Eckalbar, W L et al. (2014) Genome-wide distribution of Auts2 binding localizes with active neurodevelopmental genes. Transl Psychiatry 4:e431
Smith, Robin P; Eckalbar, Walter L; Morrissey, Kari M et al. (2014) Genome-wide discovery of drug-dependent human liver regulatory elements. PLoS Genet 10:e1004648
Zhao, Jingjing; Shi, Hongbo; Ahituv, Nadav (2013) Classification of topological domains based on gene expression and regulation. Genome 56:415-23
Smith, Robin P; Riesenfeld, Samantha J; Holloway, Alisha K et al. (2013) A compact, in vivo screen of all 6-mers reveals drivers of tissue-specific expression and guides synthetic regulatory element design. Genome Biol 14:R72

Showing the most recent 10 out of 26 publications