Evaluation of 12 Cleft Candidate Gene Proteins in Human Cleft Lip and Palate Tissue

Objectives
Morphopathogenesis of cleft lip and palate is still an unresolved problem. Defects in certain cleft candidate genes have been associated with orofacial cleft formation. These genes encode proteins which control orofacial tissue growth and formation, but their exact localization in tissue and interrelations have not been well described. The primary aim of this research was to assess and compare the distribution of 12 cleft candidate gene coded proteins in different cleft lip and palate tissue types.

Materials and Methods
Unilateral cleft lip (UCL), bilateral cleft lip (BCL) and cleft palate (CP) mucosal tissue was gathered during cleft plastic surgery. UCL group had 36 patients, BCL – 13 patients, CP – 26 patients. Two control groups were formed from patients without clefts – one with 7 patients who had labial frenectomy and the other with 5 patients from the historical collection of the Institute of Anatomy and Anthropology of Rīga Stradiņš University). Immunohistochemistry was used to identify the following cleft candidate gene proteins: BarH-like Homeobox 1
(BARX1), Distal-less Homeobox 4 (DLX4), Forkhead Box E1 (FOXE1), Homeobox B3 (HOXB3), Muscle Segment Homeobox 2 (MSX2), Paired Box Transcription Factor 7 (PAX7) and 9 (PAX9), Receptor-like Tyrosine Kinase (RYK), Sonic Hedgehog (SHH), SRY-box Transcription Factor 3 (SOX3), Wingless-type MMTV Integration Site Protein 3A (WNT3A) and 9B (WNT9B).

Results
The 12 evaluated proteins were identified within all patient groups and controls with variable distribution. Statistically significant correlations between protein-containing cells were calculated in each group.

Conclusions
WNT9B, WNT3A, SOX3 were the most notable in control tissue, while SHH, PAX7, HOXB3, FOXE1 were found in moderate number and almost no BARX1 and MSX2-containing cells were detected. Increased BARX1, FOXE1, HOXB3, MSX2 was seen in UCL group, while BCL group had reduced SHH and elevated PAX9 and DLX4. UCL, BCL and CP tissue had elevated RYK, MSX2 and reduced WNT9B, WNT3A, SOX3.