Susceptibility to DNA damage as a molecular mechanism for non-syndromic cleft lip and palate

Non-syndromic cleft lip/palate (NSCL/P) is a complex, frequent congenital malformation, determined by the interplay between genetic and environmental factors during embryonic development. Previous findings have appointed an aetiological overlap between NSCL/P and cancer, and alterations in similar biological pathways may underpin both conditions. Here, using a combination of transcriptomic profiling and functional approaches, we report that NSCL/P dental pulp stem cells exhibit dysregulation of a co-expressed gene network mainly associated with DNA double-strand break repair and cell cycle control (p = 2.88×10(-2)-5.02×10(-9)). This network included important genes for these cellular processes, such as BRCA1, RAD51, and MSH2, which are predicted to be regulated by transcription factor E2F1. Functional assays support these findings, revealing that NSCL/P cells accumulate DNA double-strand breaks upon exposure to H2O2. Furthermore, we show that E2f1, Brca1 and Rad51 are co-expressed in the developing embryonic orofacial primordia, and may act as a molecular hub playing a role in lip and palate morphogenesis. In conclusion, we show for the first time that cellular defences against DNA damage may take part in determining the susceptibility to NSCL/P. These results are in accordance with the hypothesis of aetiological overlap between this malformation and cancer, and suggest a new pathogenic mechanism for the disease

Expression of <i>Brca1</i>, <i>Rad51</i> and <i>E2f1</i> in the developing facial primordia.

<p>Whole-mount <i>in situ</i> hybridisation (A–F) showing the expression of <i>Brca1</i> (A, D), <i>Rad51</i> (B, E) and <i>E2F1</i> (C, F) in E11.5 (A–C) and E12.5 (D–F) embryos. Expression is indicated by the blue/purple staining. A–C are sagittal views of the developing head whilst A’–C’ are frontal sections through the embryos shown in A–C. A’’–C’’ show high power views through the developing maxillary primordia, lateral and medial nasal processes. E–F are frontal sections through the developing E12.5 palatal shelves. <i>e</i>, eye; <i>l</i>, lateral nasal process; <i>m</i>, medial nasal process; <i>Md</i>, mandibular primordia; <i>Mx</i>, maxillary primordia; <i>ps</i>, palatal shelves.</p

NSCL/P cells exhibit a heterogeneous response to H₂O₂, with distinct patterns of DSB accumulation.

<p>(A) Smaller graph - Comparison of the fraction of γ-H2AX⁺cells between NSCL/P and control cells, at 6 and 24 hours of treatment with H₂O₂. Large graph – Individual quantitation of γ-H2AX⁺ cells, revealing the subgroups (I–III) within the NSCL/P samples. (*) p<0.05. (B) Representative γ-H2AX and PI profiles depicting DSB and cell cycle distribution, for each NSCL/P subgroup and controls. (C) Quantitation of sub-G1 cells, revealing a significant increment in NSCL/P subgroup II, at 24 hours of treatment, as compared to the other subgroups and the controls. (**) F = 6.04; p<0.005. (D) Relative expression of <i>CDC45L</i> following treatment with H₂O₂, revealing a significant decrease in NSCL/P samples at both time points, by comparison to controls. (*) p<0.05.</p

NSCL/P cells exhibit a gene expression profile associated with dysregulation of DNA repair and cell cycle control.

<p>(A) IPA network. DEGs were used to assemble a functional interaction map. Lines with arrowheads represent that one molecule acts on another, while regular lines represent protein interactions. Down-regulated genes are shown as green nodes, whereas genes without differential expression are shown as blank nodes. (B) Top 10 Canonical Pathways significantly enriched among the 228 DEGs (Fisher’s Exact Test, p-value <0.01). (C) ‘Role of BRCA1 in DNA Damage Response’ Canonical Pathway.</p