Abstract
Introduction:
Non-syndromic cleft lip and/or palate (NSCL/P) is a common congenital deformity, influenced by both genetic and environmental factors. The thymocyte selection-associated high-mobility group box-3 (TOX3) gene is suggested to influence NSCL/P development, though its role remains unclear. This study investigates environmental factors associated with NSCL/P and explores the association between TOX3 mutations and these factors.
Materials and Methods:
A cross-sectional retrospective study was conducted amongst 50 Malay patients with NSCL/P who underwent surgery at Hospital Universiti Sains Malaysia (January 2022–January 2023). Participants were selected based on inclusion criteria from electronic medical records. Data on environmental and familial risk factors were obtained using validated pro formas, and TOX3 gene copy number was analysed using quantitative polymerase chain reaction.
Results:
Cleft lip and palate (CL/P) were the most common deformity, accounting for 74% of cases, and 56% of patients were male. Significant risk factors for NSCL/P included consanguineous marriage, family history of clefts, maternal radiation exposure, traditional medicine use, allergies, paternal smoking and infant heart anomalies (P ≤ 0.05). Maternal pregnancy age was inversely correlated with TOX3 expression in CL/P cases (r = −0.387, P = 0.018).
Discussion:
The findings highlight complex gene–environment interactions in NSCL/P aetiology. Genetic predisposition, parental exposures and maternal factors jointly increase risk. Larger studies are needed to confirm these findings and further investigate gene–environment interactions in NSCL/P. The study underscores the role of TOX3 and environmental influences in NSCL/P pathogenesis, supporting targeted prevention and genetic counselling in the Malay population.
Keywords: Cleft lip and palate, environmental risk factors, genetic predisposition, non-syndromic cleft lip and palate, thymocyte selection-associated high-mobility group box-3 gene mutation
INTRODUCTION
Cleft lip with or without cleft palate (CL/P) is amongst the most common congenital craniofacial anomalies, occurring during early pregnancy due to incomplete fusion of facial processes.[1] It is classified into syndromic and non-syndromic types, with non-syndromic cleft lip and/or palate (NSCL/P) representing isolated structural deformities that account for nearly 70% of all CL/P cases.[2] Gender differences exist, as cleft palate is more frequent in females, while CL/P is more common in males.[3] These deformities often cause feeding, speech and hearing difficulties, along with psychosocial and financial burdens. Recognising its global impact, the World Health Organization classified CL/P amongst the major causes of infant morbidity and mortality in 2008.[4] Globally, congenital anomalies account for about 303,000 neonatal deaths annually, with CL/P affecting approximately 1 in 700 live births.[5] The prevalence varies by ethnicity and geography, being highest amongst Asians and Native Americans (1 in 500) and similar in Malaysia (1 in 700).[1,6]
NSCL/P is a multifactorial interaction between genetic and environmental factors.[7] Several genes, including IRF6, MSX1, PVRL1, TGFα, BMP4 and FGF10, have been implicated.[8] Environmental risk factors such as maternal smoking, alcohol use, malnutrition, medication exposure, viral infections and pollutants during early gestation increase the risk.[7] Maternal age below 25 years has been linked to a higher likelihood of giving birth to a child with an orofacial cleft.[9] Genetic linkage studies have identified COL21A1 and TOX3 as novel genes associated with NSCL/P.[10] TOX3, located on chromosome 16q12, encodes a nuclear protein involved in calcium-dependent transcriptional regulation and contains a high-mobility group box domain essential for DNA binding.[11] The mechanism by which TOX3 contributes to CL/P has been unclear. However, it is suggested that TOX3 mutations disrupt fibroblast growth factor signalling, affecting craniofacial fusion during embryogenesis, resulting in abnormal lip and palate fusion and CL/P development.[10]
In the Malay population, there is a higher prevalence of clefts involving the hard and soft palate, compared to Chinese and Indian population,[12] and genetic studies have suggested ethnic-specific genetic influences.[10,13] Environmental contributors such as consanguinity, dietary habits and exposure to local environmental agents may further modify risk.[14] However, the association between TOX3 mutations and environmental factors in NSCL/P has not been studied. This study aims to investigate the association between TOX3 gene mutations and NSCL/P amongst the Malay population and to explore potential gene–environment interactions influencing its occurrence. The null hypothesis posits no significant association between TOX3 mutations and NSCL/P, whereas the alternative hypothesis suggests that TOX3 variations contribute to increased risk in conjunction with environmental factors.
MATERIALS AND METHODS
This cross-sectional retrospective study was conducted from January 2022 to January 2023 at the Reconstructive Sciences Unit, Hospital Universiti Sains Malaysia, to investigate the association between TOX3 gene mutations and environmental risk factors for NSCL/P. Participants were retrospectively identified from hospital records of patients who underwent corrective surgery for cleft lip and/or palate. Eligible subjects included NSCL/P patients aged 3 months to 30 years with no more than one minor anomaly, while those with syndromic clefts, multiple congenital anomalies or complex medical conditions were excluded to ensure a homogeneous group.
A total of 50 participants were included, determined using G*Power software (version 3.1.9.4, Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany) to achieve 80% power with an effect size of 0.5 and a significance level of 0.05. This calculation aimed to provide sufficient power to explore meaningful associations while recognising that a larger sample size would increase the robustness of the findings in future research. Ethical approval was granted by the Universiti Sains Malaysia Human Research Ethics Committee (JEPeM Code: USM/JEPeM/21010010), and informed consent was obtained from all participants or their guardians in accordance with the Declaration of Helsinki. Socio-demographic and environmental information was collected using a validated pro forma questionnaire covering parental health, environmental exposures and family history. Data were gathered through standardised interviews by trained assistants blinded to genetic results. Venous blood samples were obtained under sterile conditions, and genomic DNA was extracted using the QIAamp DNA Mini Kit (Qiagen). DNA concentration and purity were verified using an Infinite® 200 PRO NanoQuant spectrophotometer, with acceptable A260/A280 ratios between 1.7 and 1.9.
The quantitative polymerase chain reaction (PCR) was performed to quantify the copy number of the TOX3 gene using TaqMan copy number assays on the StepOnePlus™ real-time PCR system (Applied Biosystems), with RNase P as a reference. CopyCaller™ Software v2.0 (Applied Biosystems / Life Technologies, Carlsbad, California, USA) was used to calculate copy numbers, accepting results with >95% confidence interval and Z-scores <1.75. Data analysis was conducted using IBM SPSS Statistics version 28. (IBM Corp., Armonk, New York, USA) Descriptive statistics were summarised participant characteristics, while the independent t-tests and one-way analysis of variance were used to compare the groups. Pearson’s correlation was used to assess relationships between TOX3 expression and continuous variables, with significance set at P ≤ 0.05.
RESULTS
Amongst the 50 NSCL/P patients, 74% presented with CL/P, 14% with cleft lip and alveolus (CLA) and 12% with cleft palate only [Figure 1]. Detailed maternal, paternal and infant characteristics are presented in Supplementary Table 1. The frequencies of child arrangement in the family [Figure 2] revealed that firstborn children were most affected – 35.1% in CL/P, 71.4% in CLA and 33.3% in cleft palate-only cases.
Figure 1.
Distribution of non-syndromic cleft lip and palate deformities. NSCL/P: Non-syndromic cleft lip and/or cleft palate
Supplementary Table 1.
Distribution of clefts according to socio-demographic and health characteristics of the study participants
| Variables | n (percentage of group) or mean (SD) | |||
|---|---|---|---|---|
|
| ||||
| CL/P (n=37) | CLA (n=7) | Cleft palate-only (n=6) | Total (n=50) | |
| Maternal characteristics | ||||
| Mean age of marriage (SD), years | 23.38 (3.52) | 22.86 (3.84) | 21.67 (3.77) | 23.10 (3.56) |
| Consanguineous marriage | ||||
| No | 32 (86.5) | 4 (57.1) | 6 (100.0) | 42 (84.0) |
| No | 5 (13.5) | 3 (42.9) | 0 | 8 (16.0) |
| Mothers education | ||||
| Primary | 2 (5.4) | 0 | 2 (4.0) | |
| Secondary | 19 (51.4) | 5 (71.4) | 4 (66.7) | 28 (56.0) |
| Diploma | 8 (21.6) | 1 (14.3) | 1 (16.7) | 10 (20.0) |
| Graduation (bachelor’s) professional degree | 7 (18.9) | 1 (14.3) | 0 | 8 (16.0) |
| Master’s and above | 1 (2.7) | 0 | 1 (16.7) | 2 (4.0) |
| Folic acid intake | ||||
| No | 6 (16.2) | 1 (14.3) | 0 | 7 (14.0) |
| No | 31 (83.8) | 6 (85.7) | 6 (100.0) | 43 (86.0) |
| Exposed to insecticide | ||||
| No | 36 (97.3) | 7 (100.0) | 6 (100.0) | 49 (98.0) |
| No | 1 (2.7) | 0 | 0 | 1 (2.0) |
| Exposed to herbicide | ||||
| No | 32 (86.5) | 6 (85.7) | 6 (100.0) | 44 (88.0) |
| No | 5 (13.5) | 1 (14.3) | 0 | 6 (12.0) |
| Exposed to radiation | ||||
| No | 37 (100.0) | 7 (100.0) | 5 (83.3) | 49 (98.0) |
| No | 0 | 0 | 1 (16.7) | 1 (2.0) |
| Traditional medicine | ||||
| No | 32 (86.5) | 4 (57.1) | 6 (100.0) | 42 (84.0) |
| No | 5 (13.5) | 3 (42.9) | 0 | 8 (16.0) |
| Modern medication | ||||
| No | 34 (91.9) | 6 (85.7) | 4 (66.7) | 44 (88.0) |
| No | 3 (8.1) | 1 (14.3) | 2 (33.3) | 6 (12.0) |
| Smoking | ||||
| No | 37 (100.0) | 7 (100.0) | 6 (100.0) | 50 (100.0) |
| No | 0 | 0 | 0 | 0 |
| Passive smoking | ||||
| No | 23 (62.2) | 3 (42.9) | 4 (66.7) | 30 (60.0) |
| No | 14 (37.8) | 4 (57.1) | 3 (33.3) | 20 (40.0) |
| Alcohol consumption | ||||
| No | 37 (100.0) | 7 (100.0) | 6 (100.0) | 50 (00.0) |
| No | 0 | 0 | 0 | 0 |
| Medical condition | ||||
| No | 27 (73.0) | 6 (85.7) | 2 (33.3) | 35 (70.0) |
| No | 10 (27.0) | 1 (14.3) | 4 (66.7) | 15 (30.0) |
| Diabetes | ||||
| No | 30 (81.1) | 7 (100.0) | 4 (66.7) | 41 (82.0) |
| No | 7 (18.9) | 0 | 2 (33.3) | 9 (18.0) |
| Hypertension | ||||
| No | 35 (94.6) | 6 (85.7) | 6 (100.0) | 47 (94.0) |
| No | 2 (5.4) | 1 (14.3) | 0 | 3 (6.0) |
| Anaemia | ||||
| No | 33 (89.2) | 5 (71.4) | 6 (100.0) | 44 (88.0) |
| No | 4 (10.8) | 2 (38.6) | 0 | 6 (12.0) |
| Fever/infection | ||||
| No | 35 (94.6) | 7 (100.0) | 6 (100.0) | 48 (96.0) |
| No | 2 (5.4) | 0 | 0 | 2 (4.0) |
| Allergy | ||||
| No | 37 (100.0) | 7 (100.0) | 5 (83.3) | 49 (98.0) |
| No | 0 | 0 | 1 (16.7) | 1 (2.0) |
| Paternal characteristics | ||||
| Mean age of marriage (SD), years | 26.95 (4.33) | 26.29 (3.90) | 26.67 (2.87) | 26.82 (4.07) |
| Father education | ||||
| Primary | 6 (16.2) | 1 (14.3) | 0 (0.00) | 7 (14.0) |
| Secondary | 17 (45.9) | 5 (71.4) | 2 (33.3) | 24 (48.0) |
| Diploma | 11 (29.7) | 1 (14.3) | 3 (50.0) | 15 (30.0) |
| Graduation (bachelor’s) professional degree | 3 (8.1) | 0 | 0 | 3 (6.0) |
| Master’s and above | 0 | 0 | 1 (16.7) | 1 (2.0) |
| Exposed to insecticide | ||||
| No | 32 (86.5) | 7 (100.0) | 4 (66.7) | 43 (86.0) |
| No | 5 (13.5) | 0 | 2 (33.3) | 7 (14.0) |
| Exposed to herbicide | ||||
| No | 35 (94.6) | 7 (100.0) | 6 (100.0) | 48 (96.0) |
| No | 2 (5.4) | 0 | 0 | 2 (4.0) |
| Exposed to radiation | ||||
| No | 37 (100.0) | 7 (100.0) | 6 (100.0) | 50 (100.0) |
| No | 0 | 0 | 0 | 0 |
| Traditional medicine | ||||
| No | 36 (97.3) | 7 (100.0) | 6 (100.0) | 49 (98.0) |
| No | 1 (2.7) | 0 | 0 (0.00) | 1 (2.0) |
| Modern medication | ||||
| No | 35 (94.6) | 7 (100.0) | 6 (100.0) | 48 (96.0) |
| No | 2 (5.4) | 0 | 0 | 2 (4.0) |
| Smoking | ||||
| No | 20 (45.1) | 1 (14.3) | 4 (66.7) | 25 (50.0) |
| No | 17 (45.9) | 6 (85.7) | 2 (33.3) | 25 (50.0) |
| Alcohol | ||||
| No | 37 (100.0) | 7 (100.0) | 6 (100.0) | 50 (100.0) |
| No | 0 | 0 | 0 | 0 |
| Medical condition | ||||
| No | 33 (89.2) | 7 (100.0) | 6 (100.0) | 46 (92.0) |
| No | 4 (10.8) | 0 | 0 | 4 (8.0) |
| Diabetes | ||||
| No | 37 (100.0) | 7 (100.0) | 6 (100.0) | 50 (100.0) |
| No | 0 | 0 | 0 | 0 |
| Hypertension | ||||
| No | 33 (89.2) | 7 (100.0) | 6 (100.0) | 46 (92.0) |
| No | 4 (10.8) | 0 | 0 | 4 (8.0) |
| Anaemia | ||||
| No | 37 (100.0) | 7 (100.0) | 6 (100.0) | 50 (100.0) |
| No | 0 | 0 | 0 | 0 |
| Fever/infection | ||||
| No | 37 (100.0) | 7 (100.0) | 6 (100.0) | 50 (100.0) |
| No | 0 | 0 | 0 | 0 |
| Allergy | ||||
| No | 37 (100.0) | 7 (100.0) | 6 (100.0) | 50 (100.0) |
| No | 0 | 0 | 0 | 0 |
| Infant characteristics | ||||
| Gender | ||||
| Male | 20 (54.1) | 6 (85.7) | 2 (33.3) | 28 (56.0) |
| Female | 17 (45.9) | 1 (14.3) | 4 (66.7) | 22 (44.0) |
| Mother’s mean age of pregnancy (SD), years | 30.22 (5.77) | 26.29 (7.25) | 28.0 (8.65) | 29.40 (6.36) |
| Mean birthweight (SD) | 2.89 (0.51) | 2.62 (0.51) | 3.05 (0.38) | 2.87 (0.49) |
| Delivery | ||||
| Normal delivery | 35 (94.6) | 6 (85.7) | 5 (83.3) | 46 (92.0) |
| Preterm delivery | 2 (5.4) | 1 (14.3) | 1 (16.7) | 4 (8.0) |
| Medical disease | ||||
| No | 33 (89.2) | 6 (85.7) | 2 (33.3) | 41 (82.0) |
| No | 4 (10.8) | 1 (14.3) | 4 (66.7) | 9 (18.0) |
| Heart anomaly | ||||
| No | 33 (89.2) | 6 (85.7) | 3 (50.0) | 42 (84.0) |
| No | 4 (10.8) | 1 (14.3) | 3 (50.0) | 8 (16.0) |
| Craniofacial anomaly | ||||
| No | 36 (97.3) | 7 (100.0) | 5 (83.3) | 48 (96.0) |
| No | 1 (2.7) | 0 | 1 (16.7) | 2 (4.0) |
| Extremities anomaly | ||||
| No | 7 (100.0) | 37 (100.0) | 6 (100.0) | 50 (100.0) |
| No | 0 | 0 | 0 | 0 |
| Family history | ||||
| No | 26 (70.3) | 3 (42.9) | 4 (66.7) | 33 (66.0) |
| No | 11 (29.7) | 4 (57.1) | 2 (33.3) | 17 (34.0) |
| First-degree relatives | ||||
| No | 32 (86.5) | 4 (57.1) | 6 (100.0) | 42 (84.0) |
| No | 5 (13.5) | 3 (42.9) | 0 | 8 (16.0) |
| Second-degree relatives | ||||
| No | 29 (78.4) | 5 (71.4) | 4 (66.7) | 38 (76.0) |
| No | 8 (21.6) | 2 (28.6) | 2 (33.3) | 12 (24.0) |
| Positivity side | ||||
| No | 26 (70.3) | 3 (42.9) | 4 (66.7) | 33 (66.0) |
| Yes, father side | 6 (16.2) | 3 (42.9) | 1 (16.7) | 10 (20.0) |
| Yes, mother side | 5 (13.5) | 1 (14.3) | 1 (16.7) | 7 (14.0) |
CL/P: Cleft lip and palate, CLA: Cleft lip and alveolus, SD: Standard deviation
Figure 2.
Frequency of child arrangement in the family amongst all types of cleft deformities
The mean TOX3 gene expression score across all deformities was 2.06, highest in cleft palate-only (2.53), followed by CL/P (2.01) and CLA (1.95) [Table 1].
Table 1.
Distribution of clefts according to mean score of the thymocyte selection-associated high-mobility group box gene amongst infants
| Variable | CL/P (n=37), mean (SD) | CLA (n=7), mean (SD) | Cleft palate-only (n=6), mean (SD) | Total (n=50), mean (SD) |
|---|---|---|---|---|
| TOX3 gene | 2.01 (0.58) | 1.95 (0.62) | 2.53 (1.57) | 2.06 (0.76) |
CL/P: Cleft lip and palate, CLA: Cleft lip and alveolus, TOX3 gene: Thymocyte selection-associated high-mobility group box, SD: Standard deviation
Maternal characteristics showed significant positive correlations between self-reported radiation exposure and CL/P (r = 0.387, P = 0.006) and between allergies and CL/P (r = 0.387, P = 0.006). The radiation exposure in this context was based on the general exposure history provided by participants rather than being quantified through specific measures such as radon levels or X-ray counts. The use of traditional medicine and consanguineous marriage were both correlated with CLA (r = 0.296, P = 0.037), while paternal smoking showed a significant association with CLA (r = 0.288, P = 0.042). Amongst infant factors, heart anomalies were significantly associated with CL/P (r = 0.342, P = 0.015), and family history of first-degree relatives correlated positively with CLA (r = 0.296, P = 0.037) [Table 2 and Supplementary Table 2]. No significant correlations were found between the mother’s pregnancy age, age of marriage for mothers, age of marriage for fathers, birth weight of infants and TOX3 expression with any type of cleft defects [Table 3]. However, a significant negative correlation was noted between the mother’s pregnancy age and the TOX3 expression amongst CL/P cases (r = −0.387, P = 0.018) [Table 4]. TOX3 expression also showed a strong positive correlation with consanguineous marriage in CLA (r = 0.866, P = 0.012) [Table 5].
Table 2.
The correlation between socio-demographic and health characteristics in different types of cleft deformity
| Variables | CL/P (n=37) | CLA (n=7) | Cleft palate-only (n=6) | Total (n=50) | ||||
|---|---|---|---|---|---|---|---|---|
|
|
|
|
|
|||||
| Correlation coefficient | P | Correlation coefficient | P | Correlation coefficient | P | Correlation coefficient | P | |
| Maternal characteristics | ||||||||
| Consanguineous marriage | ||||||||
| No | 0.114 | 0.429 | −0.296* | 0.037 | 0.161 | 0.264 | −0.159 | 0.269 |
| No | −0.114 | 0.429 | 0.296* | 0.037 | −0.161 | 0.264 | 0.159 | 0.269 |
| Folic acid intake | ||||||||
| No | 0.108 | 0.456 | 0.003 | 0.982 | −0.149 | 0.302 | 0.073 | 0.615 |
| No | −0.108 | 0.456 | −0.003 | 0.982 | 0.149 | 0.302 | −0.073 | 0.615 |
| Exposed to radiation | ||||||||
| No | 0.241 | 0.092 | 0.058 | 0.691 | −0.387** | 0.006 | 0.227 | 0.113 |
| No | −0.241 | 0.092 | −0.058 | 0.691 | 0.387** | 0.006 | −0.227 | 0.113 |
| Traditional medicine | ||||||||
| No | 0.114 | 0.429 | −0.296* | 0.037 | 0.161 | 0.264 | −0.217 | 0.131 |
| No | −0.114 | 0.429 | 0.296* | 0.037 | −0.161 | 0.264 | 0.217 | 0.131 |
| Modern medication | ||||||||
| No | 0.202 | 0.159 | −0.028 | 0.845 | −0.242 | 0.090 | 0.064 | 0.657 |
| No | −0.202 | 0.159 | 0.028 | 0.845 | 0.242 | 0.090 | −0.064 | 0.657 |
| Passive smoking | ||||||||
| No | 0.074 | 0.607 | −0.141 | 0.328 | 0.050 | 0.729 | −0.186 | 0.197 |
| No | −0.074 | 0.607 | 0.141 | 0.328 | −0.050 | 0.729 | 0.186 | 0.197 |
| Diabetes | ||||||||
| No | −0.040 | 0.781 | 0.189 | 0.189 | −0.147 | 0.307 | 0.030 | 0.836 |
| No | 0.040 | 0.781 | −0.189 | 0.189 | 0.147 | 0.307 | −0.030 | 0.836 |
| Allergy | ||||||||
| No | 0.241 | 0.092 | 0.058 | 0.691 | −0.387** | 0.006 | 0.227 | 0.113 |
| No | −0.241 | 0.092 | −0.058 | 0.691 | 0.387** | 0.006 | −0.227 | 0.113 |
| Paternal characteristics | ||||||||
| Smoking | ||||||||
| No | 0.137 | 0.344 | −0.288* | 0.042 | 0.123 | 0.394 | −0.147 | 0.308 |
| No | −0.137 | 0.344 | 0.288* | 0.042 | −0.123 | 0.394 | 0.147 | 0.308 |
| Infant characteristics | ||||||||
| Gender | ||||||||
| Male | −0.066 | 0.648 | 0.242 | 0.091 | −0.169 | 0.242 | −0.058 | 0.688 |
| Female | 0.066 | 0.648 | −0.242 | 0.091 | 0.169 | 0.242 | 0.058 | 0.688 |
| Heart anomaly | ||||||||
| No | 0.239 | 0.095 | 0.019 | 0.897 | −0.342* | 0.015 | 0.199 | 0.166 |
| No | −0.239 | 0.095 | −0.019 | 0.897 | 0.342* | 0.015 | −0.199 | 0.166 |
| Family history | ||||||||
| First-degree relatives | ||||||||
| No | 0.114 | 0.429 | −0.296* | 0.037 | 0.161 | 0.264 | 0.016 | 0.914 |
| No | −0.114 | 0.429 | 0.296* | 0.037 | −0.161 | 0.264 | −0.016 | 0.914 |
| Second-degree relatives | ||||||||
| No | 0.094 | 0.516 | −0.043 | 0.766 | −0.081 | 0.577 | 0.284* | 0.046 |
| No | −0.094 | 0.516 | 0.043 | 0.766 | 0.081 | 0.577 | −0.284* | 0.046 |
| Positivity side | ||||||||
| No | 0.152 | 0.292 | −0.197 | 0.170 | 0.005 | 0.971 | 0.292* | 0.039 |
| Yes, father side | −0.160 | 0.268 | 0.231 | 0.107 | −0.031 | 0.832 | −0.069 | 0.636 |
| Yes, mother side | −0.024 | 0.870 | 0.003 | 0.982 | 0.028 | 0.845 | −0.320* | 0.023 |
*Correlation is significant at the level of 0.05 (two-tailed), **Correlation is significant at the level of 0.01 (two-tailed). CL/P: Cleft lip and palate, CLA: Cleft lip and alveolus
Supplementary Table 2.
The correlation between socio-demographic and health characteristics in different types of cleft deformity
| Variables | CL/P (n = 37) | CLA (n = 7) | Cleft palate-only (n = 6) | Total (n = 50) | ||||
|---|---|---|---|---|---|---|---|---|
|
|
|
|
|
|||||
| Correlation coefficient | P | Correlation coefficient | P | Correlation coefficient | P | Correlation coefficient | P | |
| Maternal characteristics | ||||||||
| Consanguineous marriage | ||||||||
| No | 0.114 | 0.429 | −0.296* | 0.037 | 0.161 | 0.264 | −0.159 | 0.269 |
| No | −0.114 | 0.429 | 0.296* | 0.037 | −0.161 | 0.264 | 0.159 | 0.269 |
| Folic acid intake | ||||||||
| No | 0.108 | 0.456 | 0.003 | 0.982 | −0.149 | 0.302 | 0.073 | 0.615 |
| No | −0.108 | 0.456 | −0.003 | 0.982 | 0.149 | 0.302 | −0.073 | 0.615 |
| Exposed to insecticide | ||||||||
| No | −0.085 | 0.559 | 0.058 | 0.691 | 0.053 | 0.716 | −0.222 | 0.122 |
| No | 0.085 | 0.559 | −0.058 | 0.691 | −0.053 | 0.716 | 0.222 | 0.122 |
| Exposed to herbicide | ||||||||
| No | −0.079 | 0.588 | −0.028 | 0.845 | 0.136 | 0.345 | −0.258 | 0.071 |
| No | 0.079 | 0.588 | 0.028 | 0.845 | −0.136 | 0.345 | 0.258 | 0.071 |
| Exposed to radiation | ||||||||
| No | 0.241 | 0.092 | 0.058 | 0.691 | −0.387** | 0.006 | 0.227 | 0.113 |
| No | −0.241 | 0.092 | −0.058 | 0.691 | 0.387** | 0.006 | −0.227 | 0.113 |
| Traditional medicine | ||||||||
| No | 0.114 | 0.429 | −0.296* | 0.037 | 0.161 | 0.264 | −0.217 | 0.131 |
| No | −0.114 | 0.429 | 0.296* | 0.037 | −0.161 | 0.264 | 0.217 | 0.131 |
| Modern medication | ||||||||
| No | 0.202 | 0.159 | −0.028 | 0.845 | −0.242 | 0.090 | 0.064 | 0.657 |
| No | −0.202 | 0.159 | 0.028 | 0.845 | 0.242 | 0.090 | −0.064 | 0.657 |
| Passive smoking | ||||||||
| No | 0.074 | 0.607 | −0.141 | 0.328 | 0.050 | 0.729 | −0.186 | 0.197 |
| No | −0.074 | 0.607 | 0.141 | 0.328 | −0.050 | 0.729 | 0.186 | 0.197 |
| Medical condition | ||||||||
| Diabetes | ||||||||
| No | −0.040 | 0.781 | 0.189 | 0.189 | −0.147 | 0.307 | 0.030 | 0.836 |
| No | 0.040 | 0.781 | −0.189 | 0.189 | 0.147 | 0.307 | −0.030 | 0.836 |
| Hypertension | ||||||||
| No | 0.042 | 0.771 | −0.141 | 0.330 | 0.093 | 0.519 | −0.049 | 0.737 |
| No | −0.042 | 0.771 | 0.141 | 0.330 | −0.093 | 0.519 | 0.049 | 0.737 |
| Anaemia | ||||||||
| No | 0.062 | 0.670 | −0.206 | 0.152 | 0.136 | 0.345 | 0.115 | 0.425 |
| No | −0.062 | 0.670 | 0.206 | 0.152 | −0.136 | 0.345 | −0.115 | 0.425 |
| Fever/infection | ||||||||
| No | −0.121 | 0.403 | 0.082 | 0.570 | 0.075 | 0.603 | 0.052 | 0.722 |
| No | 0.121 | 0.403 | −0.082 | 0.570 | −0.075 | 0.603 | −0.052 | 0.722 |
| Allergy | ||||||||
| No | 0.241 | 0.092 | 0.058 | 0.691 | −0.387** | 0.006 | 0.227 | 0.113 |
| No | −0.241 | 0.092 | −0.058 | 0.691 | 0.387** | 0.006 | −0.227 | 0.113 |
| Paternal characteristics | ||||||||
| Exposed to insecticide | ||||||||
| No | 0.024 | 0.870 | 0.163 | 0.259 | −0.206 | 0.152 | 0.064 | 0.656 |
| No | −0.024 | 0.870 | −0.163 | 0.259 | 0.206 | 0.152 | −0.064 | 0.656 |
| Exposed to herbicide | ||||||||
| No | −0.121 | 0.403 | 0.082 | 0.570 | 0.075 | 0.603 | −0.210 | 0.143 |
| No | 0.121 | 0.403 | −0.082 | 0.570 | −0.075 | 0.603 | 0.210 | 0.143 |
| Exposed to radiation | ||||||||
| No | ||||||||
| No | ||||||||
| Traditional medicine | ||||||||
| No | −0.085 | 0.559 | 0.058 | 0.691 | 0.053 | 0.716 | −0.222 | 0.122 |
| No | 0.085 | 0.559 | −0.058 | 0.691 | −0.053 | 0.716 | 0.222 | 0.122 |
| Modern medication | ||||||||
| No | −0.121 | 0.403 | 0.082 | 0.570 | 0.075 | 0.603 | −0.210 | 0.143 |
| No | 0.121 | 0.403 | −0.082 | 0.570 | −0.075 | 0.603 | 0.210 | 0.143 |
| Smoking | ||||||||
| No | 0.137 | 0.344 | −0.288* | 0.042 | 0.123 | 0.394 | −0.147 | 0.308 |
| No | −0.137 | 0.344 | 0.288* | 0.042 | −0.123 | 0.394 | 0.147 | 0.308 |
| Medical condition | ||||||||
| Diabetes | ||||||||
| No | ||||||||
| No | ||||||||
| Hypertension | ||||||||
| No | −0.175 | 0.225 | 0.119 | 0.411 | 0.109 | 0.452 | −0.226 | 0.114 |
| No | 0.175 | 0.225 | −0.119 | 0.411 | −0.109 | 0.452 | 0.226 | 0.114 |
| Anaemia | ||||||||
| No | ||||||||
| No | ||||||||
| Fever/infection | ||||||||
| No | ||||||||
| No | ||||||||
| Allergy | ||||||||
| No | ||||||||
| No | ||||||||
| Infant characteristics | ||||||||
| Gender | ||||||||
| Male | −0.066 | 0.648 | 0.242 | 0.091 | −0.169 | 0.242 | −0.058 | 0.688 |
| Female | 0.066 | 0.648 | −0.242 | 0.091 | 0.169 | 0.242 | 0.058 | 0.688 |
| Type of delivery | ||||||||
| Normal delivery | 0.161 | 0.263 | −0.093 | 0.518 | −0.118 | 0.415 | 0.074 | 0.607 |
| Preterm delivery | −0.161 | 0.263 | 0.093 | 0.518 | 0.118 | 0.415 | −0.074 | 0.607 |
| Medical disease | ||||||||
| Heart anomaly | ||||||||
| No | 0.239 | 0.095 | 0.019 | 0.897 | −0.342* | 0.015 | 0.199 | 0.166 |
| No | −0.239 | 0.095 | −0.019 | 0.897 | 0.342* | 0.015 | −0.199 | 0.166 |
| Craniofacial anomaly | ||||||||
| No | 0.112 | 0.440 | 0.082 | 0.570 | −0.239 | 0.095 | 0.004 | 0.980 |
| No | −0.112 | 0.440 | −0.082 | 0.570 | 0.239 | 0.095 | −0.004 | 0.980 |
| Family history | ||||||||
| First-degree relatives | ||||||||
| No | 0.114 | 0.429 | −0.296* | 0.037 | 0.161 | 0.264 | 0.016 | 0.914 |
| No | −0.114 | 0.429 | 0.296* | 0.037 | −0.161 | 0.264 | −0.016 | 0.914 |
| Second-degree relatives | ||||||||
| No | 0.094 | 0.516 | −0.043 | 0.766 | −0.081 | 0.577 | 0.284* | 0.046 |
| No | −0.094 | 0.516 | 0.043 | 0.766 | 0.081 | 0.577 | −0.284* | 0.046 |
| Positivity side | ||||||||
| No | 0.152 | 0.292 | −0.197 | 0.170 | 0.005 | 0.971 | 0.292* | 0.039 |
| Yes, father side | −0.160 | 0.268 | 0.231 | 0.107 | −0.031 | 0.832 | −0.069 | 0.636 |
| Yes, mother side | −0.024 | 0.870 | 0.003 | 0.982 | 0.028 | 0.845 | −0.320* | 0.023 |
*Correlation is significant at the level of 0.05 (two-tailed), **Correlation is significant at the level of 0.01 (two-tailed). CL/P: Cleft lip and palate, CLA: Cleft lip and alveolus
Table 3.
The correlation between socio-demographic and health characteristics in different types of cleft deformity
| Variables | CL/P (n=37) | CLA (n=7) | Cleft palate-only (n=6) | Total (n=50) | ||||
|---|---|---|---|---|---|---|---|---|
|
|
|
|
|
|||||
| Correlation coefficient | P | Correlation coefficient | P | Correlation coefficient | P | Correlation coefficient | P | |
| Mother’s pregnancy age | 0.195 | 0.175 | −0.186 | 0.195 | −0.064 | 0.658 | 0.003 | 0.983 |
| Age of marriage for mothers | 0.129 | 0.372 | −0.024 | 0.868 | −0.148 | 0.304 | 0.196 | 0.172 |
| Age of marriage for fathers | 0.092 | 0.843 | 0.044 | 0.760 | −0.086 | 0.553 | 0.093 | 0.521 |
| Birth-weight of infants | 0.017 | 0.904 | −0.172 | 0.232 | 0.160 | 0.266 | −0.191 | 0.184 |
| TOX3 gene | −0.035 | 0.811 | 0.012 | 0.934 | 0.034 | 0.814 | −0.104 | 0.471 |
Correlation is significant at the level of 0.05 (two-tailed), Correlation is significant at the level of 0.01 (two-tailed). CL/P: Cleft lip and palate, CLA: Cleft lip and alveolus, TOX3 gene: Thymocyte selection-associated high-mobility group box
Table 4.
Correlation for the mean score of the thymocyte selection-associated high-mobility group box gene across different types of cleft deformity with various maternal, paternal and infant characteristics
| Variables | Mean score of TOX3 gene | |||||
|---|---|---|---|---|---|---|
|
| ||||||
| CL/P (n=37) | CLA (n=7) | Cleft palate-only (n=6) | ||||
|
|
|
|
||||
| Correlation coefficient | P | Correlation coefficient | P | Correlation coefficient | P | |
| Mother’s pregnancy age | −0.387* | 0.018 | −0.015 | 0.974 | −0.322 | 0.534 |
| Age of marriage for mothers | −0.182 | 0.282 | −0.066 | 0.889 | −0.242 | 0.644 |
| Age of marriage for fathers | −0.078 | 0.645 | −0.133 | 0.776 | −0.248 | 0.635 |
| Birth-weight of infants | 0.063 | 0.710 | 0.134 | 0.775 | −0.200 | 0.705 |
*Correlation is significant at the level of 0.05 (two-tailed), Correlation is significant at the level of 0.01 (two-tailed). CL/P: Cleft lip and palate, CLA: Cleft lip and alveolus, TOX3 gene: Thymocyte selection-associated high-mobility group box
Table 5.
The comparison between the thymocyte selection-associated high-mobility group box gene expression and the mother’s socio-demographic and clinical characteristics
| Variables | Mean score of TOX3 gene | |||
|---|---|---|---|---|
|
| ||||
| CL/P (n=37) | CLA (n=7) | |||
|
|
|
|||
| Correlation coefficient | P | Correlation coefficient | P | |
| Consanguineous marriage | ||||
| No | −0.007 | 0.965 | −0.866* | 0.012 |
| No | 0.007 | 0.965 | 0.866* | 0.012 |
*Correlation is significant at the level of 0.05 (two-tailed), Correlation is significant at the level of 0.01 (two-tailed). CL/P: Cleft lip and palate, CLA: Cleft lip and alveolus, TOX3 gene: Thymocyte selection-associated high-mobility group box
DISCUSSION
The aetiology of NSCL/P is multifactorial, influenced by complex interactions between genetic susceptibility and environmental exposures [Figure 3].[4] The findings highlight key maternal, paternal and infant risk factors, such as consanguineous marriage, family history of clefts, maternal radiation exposure, traditional medicine use, allergy, paternal smoking and infant heart anomalies, each contributing to NSCL/P development. CL/P was the most prevalent deformities (74%), followed by CLA and cleft palate only. This aligns with earlier reports where CL/P was predominant.[15,16] The low frequency of cleft palate-only cases may reflect the hospital-based recruitment bias, as CL/P cases often require multiple surgeries and prolonged follow-up. In addition, data drawn from a single tertiary centre may overrepresent complex cases, limiting generalisability to the wider population.
Figure 3.
Schematic representation of the development and classification of syndromic and non-syndromic cleft lip and/or palate, highlighting the role of genetic (TOX3) and environmental factors and their interaction
A significant finding was the association between consanguineous marriage and CLA, consistent with prior research linking consanguinity with congenital malformations[17] and NSCL/P.[7,18] In this study, consanguinity accounted for 16% of cleft cases with additional anomalies, underscoring its impact. The Malay population shows relatively high rates of consanguineous unions, particularly in rural and less-educated groups.[19,20] Such cultural practices may facilitate the inheritance of recessive alleles contributing to CLA formation, highlighting the need for community-based awareness and genetic counselling initiatives. A notable discovery was the strong positive correlation between consanguineous marriage and TOX3 gene expression amongst CLA patients. This novel finding implies a potential link between genetic inheritance patterns and gene regulation, suggesting that consanguinity might elevate TOX3 gene activity, influencing craniofacial development. Further molecular investigations are needed to clarify this mechanism and its role in orofacial cleft formation.
Family history also emerged as a significant factor. In this study, 34% of patients reported a family history of clefts, consistent with previous studies reporting 22%–35%.[21] A significant correlation between first-degree relatives and CLA reinforces the genetic component in orofacial clefts.[22,23] Familial aggregation suggests that inherited gene variants, including TOX3, may predispose certain lineages, supporting polygenic inheritance models for NSCL/P.[4] The analysis of maternal age revealed no statistically significant association with cleft type, though higher maternal age was linked to CL/P (mean: 30.22 years). This trend parallels previous findings showed no clear relationship between maternal age and cleft type.[23] The weak negative correlation between TOX3 expression and maternal pregnancy age amongst CL/P cases suggests that advanced maternal age may downregulate TOX3 expression, potentially affecting craniofacial morphogenesis. This is the first report of such an association, warranting further genomic and epigenetic investigations, as well as the exploration of potential therapeutic targets for preventing or mitigating the risk of cleft palate.
Overall, TOX3 expression showed a weak inverse correlation with cleft occurrence, implying that lower TOX3 copy numbers may predispose to NSCL/P. Mohamad Shah et al., previously demonstrated a similar trend, identifying TOX3 as a candidate gene linked to NSCL/P in extended Malay families.[10] The lack of statistical significance in this study could stem from a small sample size, population heterogeneity or the gene’s polygenic nature, acting in concert with other loci and environmental triggers. The ethnically mixed background of the Malay population may also introduce allelic variation, potentially obscuring TOX3-specific effects. Larger, multi-ethnic studies could better define its contribution to NSCL/P susceptibility. Parental age at marriage, another debated factor, showed no significant influence on cleft type. Infants with CL/P had the highest mean maternal (23.38 years) and paternal (26.95 years) marriage ages. However, the effects of maternal and paternal age at marriage were not significant for most categories (P > 0.05), consistent with findings by Cheshmi et al.[18] In addition, a meta-analysis reported that elevated cleft risk amongst parents aged >40 years,[24] suggesting that age-related chromosomal alterations or prolonged environmental exposure may elevate risk.[9,25]
Amongst infant anomalies, heart defects were the most prevalent (50%) in cleft palate-only cases and strongly correlated with CL/P, consistent with reports that up to 45% of cleft patients present congenital heart disease.[26,27] Routine echocardiography before surgical correction in cleft patients is therefore recommended. Maternal radiation exposure showed a significant association with cleft palate only (P = 0.006), consistent with a weak correlation observed in prior findings[15] but contrasting with studies’ findings of no link.[28] The binary questionnaire (‘yes/no’) used in this study limited the quantification of exposure type and dose, which may explain inconsistencies. Future studies should incorporate detailed exposure metrics to determine dose–response relationships.
Maternal diabetes showed no significant relationship with cleft risk, echoing findings from other Malaysian and regional studies,[28,29] though some authors report opposite results.[15,30] The absence of association here may reflect the small diabetic subgroup or effective glycaemic control during pregnancy. Tobacco exposure remains a well-documented risk factor for NSCL/P,[31] yet our findings revealed no significant link between maternal smoking and cleft risk, paralleling results reported by many other researchers.[22,28] However, paternal smoking was significantly associated with CLA, aligning with a prior study.[7] It is plausible that exposures such as smoking may exert a teratogenic effect through passive tobacco exposure of the mother or through direct exposure to spermatogenesis, thereby inducing genotoxic effects.[32] This emphasises the role of paternal preconception health in NSCL/P prevention strategies.
Folic acid supplementation and modern medicine were not significantly related to cleft risk, consistent with earlier research.[18,33] However, the lack of specification by drug class prevents definitive interpretation, as certain medications (e.g., anticonvulsants or corticosteroids) are known teratogens.[34] Future research should stratify medication types to delineate specific pharmacologic risks. However, traditional medicine use showed a significant positive correlation with CLA. Traditional and complementary medicine use remains widespread amongst Malays; the National Health and Morbidity Survey 2015 reported that 31.36% had used such remedies,[35] and other studies reported 24.8% usage amongst chronically ill population.[36] Cultural reliance on traditional healing, especially in rural communities, may expose pregnant women to unregulated herbal compounds with teratogenic potential. Supporting this concern, Mbuyi-Musanzayi et al., reported that many pregnant women self-treated with herbal preparations of uncertain safety during early gestation.[37] These findings underscore the urgent need for public education and pharmacovigilance regarding herbal use during pregnancy. Birth order analysis indicated that firstborn children had the highest proportion of cleft deformities. The predominance of firstborns amongst cleft cases mirrors previous observations.[16,18] While biological explanations remain unclear, factors such as limited uterine adaptation, maternal stress or subclinical nutrient deficiencies during the first pregnancy may contribute. Socio-demographic differences, study design and genetic diversity may also influence outcomes across population.
A novel observation in this study was the significant association between maternal allergies and cleft palate-only. Allergic inflammation may disrupt foetal development through immune-mediated pathways or inflammatory cytokines that affect palatal fusion.[38] This potential mechanism warrants exploration, as allergic conditions could represent modifiable maternal risk factors. The study’s strengths include its focus on a specific ethnic group with a unique genetic background, rigorous molecular methods and validated data collection instruments. However, limitations such as small sample size, single-centre recruitment, cross-sectional nature and lack of quantitative exposure measures constrain causal inference. Despite these constraints, the findings contribute valuable preliminary evidence for the role of TOX3 and associated environmental factors in NSCL/P pathogenesis amongst Malays.
Future studies should include larger, multi-ethnic cohorts with matched control groups and longitudinal follow-up to confirm causality. Integrating genomic sequencing and detailed environmental profiling will help clarify TOX3’s role and interactions. Investigating preventive strategies, such as preconception counselling and culturally sensitive awareness campaigns, could reduce the burden of NSCL/P in high-risk population.
CONCLUSIONS
This study identified significant associations between consanguinity, family history, maternal radiation exposure, allergy, traditional medicine use, paternal smoking and infant heart anomalies with the risk of NSCL/P. A significant inverse relationship was observed between maternal pregnancy age and TOX3 expression in CL/P, suggesting that younger maternal age may influence gene regulation. Although overall TOX3 variation did not reach statistical significance, its interactions with environmental exposures point to a multifactorial pathogenesis for NSCL/P in Malays. Public health education programmes geared towards raising awareness of the identified risk factors for the development of NSCL/P would go a long way to prevent the occurrence and reduce the burden. Genetic counselling and early screening for TOX3 mutations may also support prevention efforts.
Declaration of patient consent
The authors certify that they have obtained all appropriate patient consent forms. In the form, the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.
Conflicts of interest
There are no conflicts of interest.
Funding Statement
Nil.
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