The Association of TOX3 Copy Number Variation with Gene Expression and Susceptibility to Nonsyndromic Cleft Lip And/or Palate in a Malay Cohort

Nonsyndromic cleft lip and/or palate (NSCL/P) is a common congenital malformation with genetic influences. While Thymocyte selection-associated high mobility group box 3 (TOX3) is involved in other developmental processes, its role in NSCL/P remained unexplored. This study investigated the association between TOX3 copy number, expression, and NSCL/P in 64 Malay NSCL/P cases and 64 normal controls. Samples from patients undergoing cleft repair surgery and eligible volunteers for the control group were quantified via quantitative polymerase chain reactions (qPCR). A higher mean of TOX3 copy number was found in cases (2.195 +/- 0.689) compared to controls (1.962 +/- 0.558; p < 0.05). Similarly, a higher TOX3 expression was observed in cases (0.014 [IQR 0.024]) compared to controls (0.006 [IQR 0.019]; p < 0.001). Unadjusted analyses showed higher TOX3 copy number (OR = 1.850; p < 0.05) and its expression associated with NSCL/P. However, these associations were nullified after adjusting for sex and age (p > 0.05). Instead, male sex emerged as a significant independent predictor for NSCL/P (adjusted OR = 4.03; p < 0.001). Besides, an inverse, weak correlation was observed between TOX3 copy number and expression in NSCL/P patients (rho = -0.285; p < 0.05) indicating the potential role of epigenetics in this condition. While male sex strongly contributed to the NSCL/P condition, our results suggest that TOX3 is not an independent genetic risk factor for NSCL/P in this population. These results highlight sex as a primary demographic risk factor and underscore the importance of considering demographic context in genetic association studies.