CHARGE syndrome: candidate genes and pathogenesis

Abstract

CHARGE syndrome is a complex malformation syndrome affecting several organ systems like the central nervous system, ear, eye and heart. Heterozygous loss of function mutations in CHD7, a conserved chromatin remodelling enzyme, are responsible to cause about two-thirds of CHARGE syndrome cases. CHD7 is present in large multi-subunit complexes regulating gene transcription in a time and cell type specific manner. In this work, it was shown by co-immunoprecipitation and Duolink proximity ligation assay that CHD7 interacts with the WAR complex members WDR5, ASH2L and RBBP5. Additional direct yeast two-hybrid experiments revealed that CHD7 is most likely associated to this complex via its direct interaction partner CHD8. The WAR complex is one “core” complex of the methyltransferase KMT2D. Mutations in KMT2D are the main cause of Kabuki syndrome, a developmental disorder, showing a remarkable phenotypic overlap to features present in CHARGE syndrome. The results of this work demonstrate a possible link of CHD7 and KMT2D to the same regulatory process of chromatin remodelling and chromatin modification, which might explain the phenotypic overlap of CHARGE syndrome and Kabuki syndrome. It was proposed that CHARGE syndrome belongs to the neurocristopathies. Recent studies demonstrated that CHD7 is required for the formation of multipotent neural crest cells. However, little is known about CHD7 target genes in the process of neural crest cell development. Within this work a genome-wide microarray analysis was performed with embryos (wild-type (Chd7+/+), heterozygous (Chd7Whi/+) and homozygous (Chd7Whi/Whi)) of the Whirligig mouse line which carries a nonsense mutation in the Chd7 gene. 98 genes were identified to be differentially expressed comparing homozygous (Chd7Whi/Whi) to wild-type (Chd7+/+) embryos. In fact, many of these genes are involved in the development of neural crest cells, for example, the specification of neural crest cells, epithelial-to-mesenchymal-transition and guidance of migrating neural crest cells. In this work it was demonstrated that the regulatory effect of Chd7 on Sema3a, a secreted signalling molecule known to have chemorepulsive properties for axons and to guide migrating neural crest cells, is conserved in mouse and Xenopus laevis. Further, a regulatory effect on neural crest cell migration in Xenopus laevis was exposed for Sema3a and Sema3d. In this work three non-synonymous mutations were identified in CHD7 negative CHARGE patients within the SEMA3A gene and the SEMA3D gene, respectively. SEMA3A mutations are known to be involved in the pathogenesis of Kallmann syndrome, a genetic disorder, which represents the mild phenotypic end of CHARGE syndrome. It is assumed that SEMA3A and SEMA3D act as modifiers contributing to a more severe phenotype in CHARGE patients if they are mutated. This would explain the high inter- and intra-familial variability observed in CHARGE syndrome. In general, the results discovered in this work help to better understand the pathogenic mechanism behind CHARGE syndrome.