Investigating the Role of NADSYN1 and NAD+ Synthesis in Experimental Models of VACTERL Association
Abstract
VACTERL association is diagnosed in infants with multiple congenital malformations in Vertebral, Anal, Cardiac, Tracheal, Esophageal, Renal and Limb (VACTERL) tissues. Recently, mutations in KYNU, HAAO or NADSYN1 genes of the nicotinamide adenine dinucleotide (NAD+) synthesis pathway have been identified in patients with VACTERL association. Similar defects were observed in offspring of mice undersupplied with NAD+ biosynthesis precursors, tryptophan, or vitamin B3. VACTERL phenotypes have also been recapitulated in Kynu and Haao knockout mice and rescued by vitamin B3 supplementation. These findings strongly support the concept that NAD+ synthesis, previously considered a universal requirement in all cells, is especially necessary for the development of several, primarily mesodermal, organ systems. We hypothesize that nadsyn1.L is differentially regulated in early development with higher expression or activity in mesodermal derivatives. To test this, we first examined the expression of nadsyn1.L in early Xenopus laevis frog embryos. nadsyn1.L transcripts were detectable by in situ hybridization in the hatching gland and developing pronephric kidney in Xenopus. Given kidney defects are a key feature of VACTERL association, we are using CRISPR/Cas9 gene editing to test whether mutations in nadsyn1.L result in developmental defects in the kidney. Preliminary evidence suggests that nadsyn1.L mutations in Xenopus embryos cause diminished kidney pronephros complexity. These experiments will provide groundwork for understanding how compromised NAD+ synthesis results in the specific developmental defects that lead to VACTERL association.