Identifying the causal genetic variants in congenital brain disorders by integrating genome and transcrip-tome sequencing

• Applicant: Professorin Dr. Kerstin Kutsche
• Subject Area: Human Genetics
• Term: since 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 513958071

Rare Mendelian disorders are largely monogenic and comprise over 8,000 distinct entities. Congenital brain disorders, including microcephaly, malformations of the cortex, hindbrain, and corpus callosum, belong to monogenic diseases. Whole-exome sequencing (WES), mainly WES in patient-parents trios, was the main driver in disease gene discovery and led to a comprehensive understanding of the relationship between genes, associated clinical phenotype(s), and molecular mechanisms. Whole-genome sequencing (WGS) is the logical next step in identifying disease-causing variants not detected by WES, such as variants in RNA genes and non-coding regions and structural variants. As a concomitant approach, transcriptome sequencing is useful to search for genes with aberrant expression levels, aberrant splicing, and monoallelic expression and has the power to narrow down the investigation to a subset of candidate genes identified by WGS. To analyze the impact of any potential pathogenic variant, the cellular context in which a disease gene is expressed is important. In most cases, however, tissue specimen affected by the disease are not accessible. Human induced pluripotent stem cells (hiPSCs) that can be established from patient-derived cells and in vitro differentiation of hiPSCs into the cell type of interest are suitable model systems for transcriptome sequencing and studying the cellular-level phenotypes. In this project, we aim to identify the genetic cause in 25 WES-negative individuals with a congenital brain disorder: 7 patients have commissural abnormalities, 13 hindbrain malformations, 3 malformations of cortical development, all with or without microcephaly, and 2 have microcephaly with intrauterine growth retardation. We will first re-analyze existing trio-WES data using an updated bioinformatics workflow to identify a potentially pathogenic variant in a disease or candidate gene in up to 20% of the 25 patients. As a next step, we will perform trio-WGS in 20 genetically unsolved patients to identify a potentially pathogenic variant in a gene or non-coding region in a portion of these patients. A maximum of 5 patients with a candidate non-coding variant or without molecular diagnosis after trio-WGS and their healthy parents will be selected for obtaining primary urinary cells to reprogram them into hiPSCs, differentiate hiPSCs into induced human induced neural stem cells (hiNSCs), and perform transcriptome sequencing in hiNSCs-derived RNA. Integration of WGS and transcriptome data will potentially lead to the discovery or confirmation of the genetic cause in the patients. hiNSCs of one or a maximum of two patient-parent trios in which we have identified the pathogenic variant will be differentiated into human induced cortical neurons (hiNeurons). The morphogenetic phenotype of patient- and parent-derived hiNeurons will be studied to gain insight into the underlying pathophysiology.

• DFG Programme: Research Grants