LargenumbersofgenevariantswereidentifiedfromgenomicsequencingofCongenitalHeartDisease patients, but lack of functional verification in heart development precludes assigning ?disease gene? status. Genetic control of heart development is conserved from Drosophila to humans, thus investigations in flies can illuminate gene functions in human heart development and disease. We developed a gene validation system in Drosophila to screen large numbers of genes for roles in heart development, and quantitative analysis tools to assess multiple phenotypic parameters. We also developed novel strategies to test patient-derived genetic mutations in flies for in vivo evidence linking specific gene variants to disease. We observed that many histone-modifying genes mutated in disease patientshaverolesinflyheartdevelopment.Weproposestudiesdesignedtoelucidateancestralrolesof histone-modifying genes in heart development and disease, and to generate personalized fly heart diseasemodelsforspecificgenevariants.Usingthehigh-throughputDrosophilagenevalidationscreen for essential roles in heart development, we will test candidate disease genes identified from publicly available datasets and collaborators? unpublished datasets. We will also screen Drosophila genes encodingenzymesforhistonemethylation/demethylationandacetylation/deacetylationforrolesinheart development. Histone modifying genes validated by screening will be phenotyped using multiple quantifiable morphological and functional readouts. We will identify histone modifications that are most important for heart development. Genes will be prioritized based on multiple criteria, and for highest priority genes we will examine the transcriptional profile of heart tissue from flies in which the gene of interestwassilencedincardiaccells.Wewillidentifyconservedtargetsofhistonemodificationeffectsby comparing our results to data from murine models and patient tissue samples. We will generate transgenicandknock-inflymodelstoprovideinvivofunctionalevidenceforinvolvementofhighpriority gene variants in congenital heart disease. In pReplacement, we will express wild type or mutant transgene versions of a given human disease gene in the fly heart while simultaneously silencing the endogenous fly homolog. We will also generate ?knock-in? Drosophila models using CRISPR/Cas9- mediated gene editing. In this pCRISPR approach, the endogenous Drosophila homolog is precisely modifiedtoencodeaproteinwithaminoacidchangesidenticaltothoseencodedinthepatient-derived mutantallele.WewillalsouseDrosophilatomodelpolygenicdiseasebasedonselectedpatients,each ofwhomcarriesmultiplegenevariantsthatincludeonemutanthistone-modifyinggene.

Public Health Relevance

Genomic sequencing of congenital heart disease patients has identified many candidate disease associated genes, but functionally validating genes as disease risk factors requires analysis in an animal model. We developed a Drosophila-based gene validation system to quickly screen large numbers of genes for heart development functions, and discovered essential roles for histone-modifying genes. We propose a comprehensive study of histone modification pathway genes in Drosophila heart development, and establishment and characterization of fly strains bearing specific gene variants found in congenital heart disease patients.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL134940-02
Application #
9529674
Study Section
Cardiovascular Differentiation and Development Study Section (CDD)
Program Officer
Schramm, Charlene A
Project Start
2017-07-20
Project End
2021-06-30
Budget Start
2018-07-01
Budget End
2019-06-30
Support Year
2
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Children's Research Institute
Department
Type
DUNS #
143983562
City
Washington
State
DC
Country
United States
Zip Code
20010
Basu, Madhumita; Zhu, Jun-Yi; LaHaye, Stephanie et al. (2017) Epigenetic mechanisms underlying maternal diabetes-associated risk of congenital heart disease. JCI Insight 2:
Zhu, Jun-Yi; Fu, Yulong; Richman, Adam et al. (2017) Validating Candidate Congenital Heart Disease Genes in Drosophila. Bio Protoc 7: