Identification and characterization of genetic causes of congenital chylothoraces

• Applicants: Privatdozentin Dr. Anja Miriam Boos; Professor Dr. Benjamin Odermatt; Professor Dr. Heiko Reutter
• Subject Area: Human Genetics ; Pediatric and Adolescent Medicine
• Term: since 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 517059954

In congenital chylothoraces (CCT), chyle accumulates in the pleural cavity, the fetal lung is compressed, venous blood flow to the heart is restricted, and fetal hydrops results. Frequently, fetuses die intrauterine or shortly after birth. In surviving newborns, loss of chyle-soluble fluid leads to malnutrition, thrombophilia, and immunodeficiency. About 20 proteins in the VEGFR-3 signaling cascade, have been associated with the expression of congenital lymphatic disorders. Some of these disorders are associated with CCT. The VEGFR-3 downstream signaling cascade involves the RAS/MAPK and PI3K/AKT pathways. Here, CCT also occur in RASopathies associated with Noonan syndrome. Some of the symptoms of these RASopathies can now be treated using inhibitors of the mammalian target of rapamycin (mTOR inhibitors). For the majority of nonsyndromic CCT, neither the genetic causes are known nor targeted drug intervention is possible. Own preliminary candidate gene and exome analyses in patients with non-syndromic CCT identified two likely pathogenic monoallelic variants in the syndromic disease genes FLT4 and GJC2 (Cx47). In two other families, probable pathogenic biallelic variants were found in the PKD1L1 gene. Preliminary studies of PKD1L1 in mouse embryos support this hypothesis. To identify further genetic causes of nonsyndromic CCT, the exomes of 70 affected parent-child trios will be studied in this application. The functional role of the previously identified variants in FLT4, GJC2 (Cx47), and PKD1L1 will be investigated using in vitro lymphangiogenesis assays in human lymph endothelial cells. The effects of the variants on the behavior of lymphangiogenesis will be analyzed in long-term cultures using a flow-through bioreactor. Furthermore, possible new variants identified in the planned exome analyses will be characterized by means of the above-mentioned in vitro studies. To describe the effects of the already identified variants on the formation of CCT in vivo, Morpholino® (MO) knockdown experiments (KD) for the respective zebrafish (zf) orthologs will be performed, or existing knockout (KO) zf lines will be used. The sequence regions in which the identified variants are located are conserved in the zf. Therefore, for in vivo analyses of the identified variants, the respective mRNA sequence and the respective human wild-type mRNA sequence will be injected into double-transgenic zf-KD background lymph reporter lines. The identification and better characterization of novel disease genes and their specific variants will improve counseling of affected families and possibly provide clues for new "drug interventions", analogous to the treatment of RASopathies with mTOR inhibitors.

• DFG Programme: Research Grants