The Somatic Mutation Paradigm in Congenital Malformations: Hirschsprung Disease as a Model.

Patients with Hirschsprung disease (HSCR) do not always receive a genetic diagnosis after routine screening in clinical practice. One of the reasons for this could be that the causal mutation is not present in the cell types that are usually tested-whole blood, dermal fibroblasts or saliva-but is only in the affected tissue. Such mutations are called somatic, and can occur in a given cell at any stage of development after conception. They will then be present in all subsequent daughter cells. Here, we investigated the presence of somatic mutations in HSCR patients. For this, whole-exome sequencing and copy number analysis were performed in DNA isolated from purified enteric neural crest cells (ENCCs) and blood or fibroblasts of the same patient. Variants identified were subsequently validated by Sanger sequencing. Several somatic variants were identified in all patients, but causative mutations for HSCR were not specifically identified in the ENCCs of these patients. Larger copy number variants were also not found to be specific to ENCCs. Therefore, we believe that somatic mutations are unlikely to be identified, if causative for HSCR. Here, we postulate various modes of development following the occurrence of a somatic mutation, to describe the challenges in detecting such mutations, and hypothesize how somatic mutations may contribute to 'missing heritability' in developmental defects.

Cellular mechanisms underlying Pax3-related neural tube defects and their prevention by folic acid.

Neural tube defects (NTDs), including spina bifida and anencephaly, are among the most common birth defects worldwide, but their underlying genetic and cellular causes are not well understood. Some NTDs are preventable by supplemental folic acid. However, despite widespread use of folic acid supplements and implementation of food fortification in many countries, the protective mechanism is unclear. Pax3 mutant (splotch; Sp2H ) mice provide a model in which NTDs are preventable by folic acid and exacerbated by maternal folate deficiency. Here, we found that cell proliferation was diminished in the dorsal neuroepithelium of mutant embryos, corresponding to the region of abolished Pax3 function. This was accompanied by premature neuronal differentiation in the prospective midbrain. Contrary to previous reports, we did not find evidence that increased apoptosis could underlie failed neural tube closure in Pax3 mutant embryos, nor that inhibition of apoptosis could prevent NTDs. These findings suggest that Pax3 functions to maintain the neuroepithelium in a proliferative, undifferentiated state, allowing neurulation to proceed. NTDs in Pax3 mutants were not associated with abnormal abundance of specific folates and were not prevented by formate, a one-carbon donor to folate metabolism. Supplemental folic acid restored proliferation in the cranial neuroepithelium. This effect was mediated by enhanced progression of the cell cycle from S to G2 phase, specifically in the Pax3 mutant dorsal neuroepithelium. We propose that the cell-cycle-promoting effect of folic acid compensates for the loss of Pax3 and thereby prevents cranial NTDs.