Analysis of structural and non-coding variants in patients with limb malformations

• Applicant: Professor Dr. Stefan Mundlos, since 6/2018
• Subject Area: Human Genetics
• Term: from 2008 to 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 60802772

In congenital malformation syndromes generally more than 50% of patients cannot be diagnosed on a molecular level. We hypothesize that a substantial portion of these cases is due to non-coding mutations that interfere with normal gene regulation. With the availability of whole genome sequencing (WGS) this problem can now be approached for the first time. Important additional knowledge to interpret non-coding sequences are the discovery of topologically associated domains (TADs) that separate the genome into bins of regulatory activity and the availability of epigenetic marks that identify enhancers and active promoters. Based on our previous work and our substantial experimental expertise we will exemplarily study a cohort of patients with limb malformations to identify mutations in the non-coding genome. To expand our already existing cohort we will continue to collect patients with limb malformations from our own clinic and from collaborators. Phenotypes will be annotated based on Human Phenotype Ontology terms for the easy identification of similar malformation types. As preparatory work we obtained WGS-data for 30 trios with peromelia (13), oligodactyly (7), mirror image polydactyly (5), and FATCO syndrome (5). All have been tested negative for mutations in the coding genome (exome) and for large CNVs by array-CGH.The data will be first analyzed for sequence variants, then for structural variants using a state of the art bioinformatics pipeline that will also be improved over the course of the project. Variants will be filtered for de novo events as well as rare homozygous and compound heterozygous hits and we will screen for genomic regions with a significant enrichment of such events. We will develop further filtering strategies that take into account new own data and published data mainly from the encode project. By combining RNA-seq from limb buds and results from capture-C covering 470 promoters from important limb genes, as well as genome wide epigenetic marks to identify active and poised enhancers, we are able to identify relevant regulatory regions in the genome that can be used for prioritization. Variants that map in these regions will be considered as good candidates and will be subjected to further analysis. In a previous study we have adapted the CRISPR/Cas9 technology for the efficient and fast generation of mice with structural variations. This technology allows us to produce a mouse with basically any kind of variant within a 10 week period. We will select the most promising variants and generate mice to test their pathogenicity. Detailed studies will be performed on gene expression in the developing limbs using RNA-seq and whole mount in situ hybridization. The phenotype of mutant mice will be studied by µCT analysis, skeletal preparations and histology. Changes of DNA-looping that will be associated with abnormal gene regulation will be investigated using chromosome conformation capture technologies (4C).

• DFG Programme: Research Grants
• Ehemaliger Antragsteller: Professor Dr. Peter Krawitz, until 6/2018