In PROJECT II (Variants from Complementary Genomic Technologies will Identify Candidate Causative CDH Genes) Dr. Charies Lee is completing an extensive platform comparison to evaluate copy number variations (CNV) derived from array CGH platforms, with those from the Affymetrix 6.0 array platform. This work has been done in collaboration between the Lee laboratory, the Sanger Consorslum, and the Sherer laboratory in the University of Toronto, and results are now being readied for publication. We anticipate that this analysis will provide guidance for future interpretations of CNVs from the respective platforms. We will use the high resolution Agilent IM array CGH to study our patients with isolated and syndromic CDH to identify new candidate loci. Parent/patient trios will be analyzed to determine whether an unreported CNV is de novo or inherited. Since birth defects such as CDH are anticipated to be polygenic, we should anticipate that combinations of inherited CNVs which have an increased frequency in patients vs. controls will be strong contributing factors. Affymetrix 6.0 chips will be used to study multiplex CDH families to identify blocks of linkage and for regions of loss of heterozygosity, which, when combined with whole exomic sequencing, can reveal new candidate CDH genes. A bioinformatic algorithm created for this study, CNV connect, will then prioritize all the genes in the CNV loci and those derived from regions loss of heterozygosity to select those that significantly interact with other genes know to contribute to CDH. As the cost of emerging sequencing platforms is progressively reduced, we are planning to undertake whole exomic sequencing on 50 patients with sydromic or complex CDH. Our hypothesis is that variant in common loci will be causative In patients with comorbidities of heart and diaphragm defects. We also hypothesize that similarly, a limited number or even a single variant will be responsible for patients with various syndromic CDH who have a phenotype constellation of CDH with other significant congenital anomalies. We predict that screening this special group's of patients will increase the probability of revealing novel causal variants. For subgenomic sequencing of a larger cohort of patients with isolated Congenital Diaphragmatic Hernia. The Lee and Donahoe laboratories will create the patient specific DNA libraries. The Lee laboratory will then concatamerize on the capture filter all the candidate genes selected by Dr. Pober in Project I and hybridize the patient DNA samples. After elution, next generation sequencing techniques will be employed to sequence all the candidate genes in the entire cohort of 150 patients with isolated CDH. Having the expertise of the Lee laboratory with the consultation of the Seidman laboratory and with his extensive outreach to the Sanger and the Toronto consortia gives added validity to our choice of platforms and the assurity that these technologies will be appropriately selected, used, and interpreted, with the hope of affecting an application to patient care.

National Institute of Health (NIH)
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
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Special Emphasis Panel (ZHD1-DSR-N)
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Massachusetts General Hospital
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Longoni, Mauro; High, Frances A; Qi, Hongjian et al. (2017) Genome-wide enrichment of damaging de novo variants in patients with isolated and complex congenital diaphragmatic hernia. Hum Genet 136:679-691
High, Frances A; Bhayani, Pooja; Wilson, Jay M et al. (2016) De novo frameshift mutation in COUP-TFII (NR2F2) in human congenital diaphragmatic hernia. Am J Med Genet A 170:2457-61
Loscertales, Maria; Nicolaou, Fotini; Jeanne, Marion et al. (2016) Type IV collagen drives alveolar epithelial-endothelial association and the morphogenetic movements of septation. BMC Biol 14:59
Donahoe, Patricia K; Longoni, Mauro; High, Frances A (2016) Polygenic Causes of Congenital Diaphragmatic Hernia Produce Common Lung Pathologies. Am J Pathol 186:2532-43
Sanford, Ethan L; Choy, Kwong W; Donahoe, Patricia K et al. (2016) MiR-449a Affects Epithelial Proliferation during the Pseudoglandular and Canalicular Phases of Avian and Mammal Lung Development. PLoS One 11:e0149425
Longoni, M; Russell, M K; High, F A et al. (2015) Prevalence and penetrance of ZFPM2 mutations and deletions causing congenital diaphragmatic hernia. Clin Genet 87:362-7
Lundby, Alicia; Rossin, Elizabeth J; Steffensen, Annette B et al. (2014) Annotation of loci from genome-wide association studies using tissue-specific quantitative interaction proteomics. Nat Methods 11:868-74
Longoni, Mauro; High, Frances A; Russell, Meaghan K et al. (2014) Molecular pathogenesis of congenital diaphragmatic hernia revealed by exome sequencing, developmental data, and bioinformatics. Proc Natl Acad Sci U S A 111:12450-5
Lage, Kasper (2014) Protein-protein interactions and genetic diseases: The interactome. Biochim Biophys Acta 1842:1971-1980
Russell, Meaghan K; Longoni, Mauro; Wells, Julie et al. (2012) Congenital diaphragmatic hernia candidate genes derived from embryonic transcriptomes. Proc Natl Acad Sci U S A 109:2978-83

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