Unravelling the role of IRX4 variants in non-syndromic and Down syndrome associated congenital heart disease

The gene IRX4 is a crucial transcription factor for cardiac development, playing a significant role in the formation of the heart. A study analyzed the IRX4 gene in 205 individuals with non-syndromic congenital heart disease (CHD), 24 Down syndrome (DS) cases with CHD, 27 DS cases without CHD, and 150 healthy controls. Researchers discovered two previously unknown variants (p.Ser24Asn and p.Thr217Iso) and one already reported variant (rs2232376) in non-syndromic CHD patients. Importantly, rs2232376 was also identified in DS patients with CHD.

The first and reported variants, both in the N-terminal region, and the second novel variant within the TALE homeodomain, were further investigated. Computational modeling indicated that these variants altered the protein's structure, potentially affecting its ability to bind DNA. Western blotting showed a decreased expression of mutated IRX4 proteins and luciferase reporter assays revealed a decline in activity for the Nanog promoter and HEY2 enhancer, both of which are affected by the mutations.

Moreover, qRT-PCR demonstrated lower mRNA expression levels for downstream targets, including Nfyc, Nppa, and Bmp10.

These findings collectively suggest that the identified IRX4 variants have a pathogenic effect, playing a critical role in regulating various stages of cardiogenesis. The mutations lead to aberrant expression of muteins and downstream target genes, along with compromised promoter activities, all of which contribute to the development of congenital heart disease.