Identification of new monogenic disease genes by means of Next Generation Sequencing

• Applicant: Professorin Dr. Kerstin Kutsche, since 9/2018
• Subject Area: Human Genetics
• Term: from 2012 to 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 221465734

Nowadays, whole exome sequencing is the method of choice to find pathogenic sequence variants in patients with a rare hereditary disease. In this projects this approach will be used to identify new disease genes for the Zimmermann-Laband syndrome (ZLS), the Hallermann-Streiff syndrome, a limb duplication syndrome and a familial liver disorder by yet to be performed exome sequencing experiments and for six cases with a rare syndromic disorder by applying a re-engineered bioinformatic analysis pipeline to already existing exome data.The successful identification of new monogenic disease and candidate genes in the first funding period of this project forms the base for further functional studies to understand the pathophysiological consequences of selected genetic disorders. In more detail, structural and functional effects of the ZLS-associated amino acid substitutions in KCNH1 and ATP6V1B2 will be analyzed by cryo-electron microscopy. To determine the composition and the hydrolysis activity of the V-ATPase in fibroblasts of a patient with the recurrent missense mutation p.(Arg485Pro) in ATP6V1B2, encoding the B2 subunit of the V-ATPase, co-immunoprecipitation and ATPase activity assays will be performed. Due to involvement of the V-ATPase in vesicular trafficking processes, we will also use biotinylation assays and immunocytochemistry to analyze the impact of the Arg-to-Pro-substitution in the B2 subunit on trafficking and degradation of the epidermal growth factor receptor. Live cell imaging will be carried out to investigate albumin trafficking in the same cellular system. In our preliminary work, the private missense variant c.44A>G [p.(Gln15Arg)] in STX10, which segregates with short stature, cataract and sensorineural hearing loss in a three-generation family was identified. The encoded protein syntaxin-10 is also implicated in intracellular vesicle transport; therefore biotinylation and hexosaminidase assays will be performed to analyze trafficking of the mannose-6-phosphat receptor, and immunofluorescence analysis will be done to investigate trafficking of the transferrin receptor. Moreover, various molecular biological, biochemical and cell biological techniques will be applied to yet to be discovered putative disease-associated sequence variants to learn about their consequences on the respective protein function.Taken together, this project will make use of a broad methodical spectrum for state-of-the-art human genetic research comprising exome sequencing and functional studies to significantly contribute to genetic and functional characterization of rare syndromic disorders and better understanding of the underlying pathogenesis.

• DFG Programme: Research Grants
• Ehemalige Antragstellerin: Dr. Fanny Kortüm, until 9/2018