Identification of causal genes for nonsyndromic cleft palate via targeted resequencing of candidate genes and functional analysis within the zebrafish model.

• Applicants: Dr. Nina Ishorst; Privatdozentin Dr. Elisabeth Mangold
• Subject Area: Human Genetics
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 542848230

Nonsyndromic cleft palate only (nsCPO) has a prevalence of 1:2000 in the European population and is one of the common congenital malformations. The phenotype is multifactorial with a high estimated heritability of up to 90%. Genome-wide association studies, whose findings are mainly limited to common variants with a low to moderate effect size, could only explain a small proportion of the heritability. Recent literature suggests that rare variants with high effect sizes in genes relevant to facial development may play a significant role in the etiology of nsCPO. To investigate this hypothesis, the applicants performed exome sequencing in nsCPO patient/parent trios and identified rare de novo mutations in potential nsCPO candidate genes. In the proposed project those nsCPO candidate genes will be resequenced in patient/control cohorts using single molecule molecular inversion probes, in order to define candidates for functional characterization in zebrafish via the identification of additional rare mutations. For the functional work, the identified nsCPO candidate genes will be knocked-out and, in a next step, patient-specific mutations will be introduced into the zebrafish genome using CRISPR/Cas9, to investigate a possible phenotypic outcome. These results can be used to infer a correlation between mutations in specific genes and nsCPO. The zebrafish ethmoid plate serves as a model of the human palate in this study. The applicants have extensive expertise in performing and evaluating single molecule molecular inversion probes. In addition, they have access to their in-house multi-ethnic nsCPO patient cohort. Dr. Nina Ishorst already established the methods to knock-out specific candidate genes and to knock-in patient-specific mutations into the zebrafish genome in the lab of the collaboration partner Prof. Benjamin Odermatt. He has extensive experience in zebrafish husbandry and breeding, as well as phenotyping of zebrafish with congenital malformations. The identification of (mutations in) genes specific for palate development will help to understand important signaling pathways and mechanisms of cleft formation and thus contribute significantly to a deeper understanding of the genetic background of nsCPO. In the long term, this knowledge will help in risk prediction and counseling affected families. It might even facilitate the development of individualised prevention strategies.

• DFG Programme: Research Grants
• Co-Investigator: Professor Dr. Benjamin Odermatt