Association between polymorphism of TGFA Taq I and cleft Lip and/or palate: a meta-analysis

Facial clefting is one of the most common malformations in humans. Significant differences between populations in the prevalence of cleft lip or palate (CL/P) have been reported, with higher rates found in Asians and American Indians than those observed in Caucasians and Africans. Palate formation is complex, and there are numerous potential untoward possibilities, the most common being delayed shelf horizontalization and inadequate shelf growth [1].

Epidemiologic studies suggest that a number of environmental factors have been examined as risk factors for CL/P, including maternal smoking, exposure to antiepileptic drugs, antiemetic agents and vitamin use during the periconceptual period, maternal metabolic factors, alcohol consumption and exposure to agricultural chemicals [2]. Several studies have suggested that maternal cigarette smoking increased the risk of delivering infants with orofacial clefts [3‐6]. It has previously been shown that maternal periconceptional intake of multivitamins containing folic acid decreased the occurrence of CL/P [6‐8]. However, there is a study showing the different results [9]. A case–control study showed that CL/P was associated with maternal alcohol consumption [10]. However, Christensen and colleagues found that before the pregnancy there were fewer case mothers drinking alcohol than control mothers [11].

The epidemiologic characteristics and risk factors of CL/P are not clear. There is also a strong genetic component to oral clefts. The host susceptibility factors may play an important role in the development of CL/P. Ardinger and colleagues performed the first study to use a case–control design to test candidate genes [12]. They found a significantly statistical association between CL/P and two of 12 markers in five genes, with an intronic Taq1 marker in the transforming growth factor alpha (TGFA) gene showing the strongest association. TGFA encoded by a gene mapped at 2p13, is a secretion protein that binds to the epidermal growth factor receptor (EGFR) and is situated at the palate epithelium during palate closing [13]. TGFA may function as a normal embryonic version of EGF-related growth factor [14]. EGF/TGFA and glucocorticoids are believed to regulate the proliferation and differentiation of palatal epithelial cells both in vitro and in vivo. Moreover, the continued presence of EGF inhibits the fusion process; TGFA is likely to have similar effects. These biological studies suggest that mutations in the TGFA gene might contribute to the development of CL/P, especially for those mutations that affect the timing of the tissue-specific expression of this gene.

The TGFA gene shows a restriction fragment length polymorphism when treated with Taq I restriction enzyme. The mutant allele shows a four-base (TAAT) deletion. In this case, it shows a 178-base pair (bp) C1 allele and a 174-bp C2 allele [15]. TGFA Taq I polymorphism is located at intron 5 and has 602 bp in the 59 direction of the acceptor site of exon 6 [16]. For this polymorphism, C1C1 is wild genotype, C1C2 is heterozygote genotype, and C2C2 is homozygote mutation genotype. In most studies, there are different forms of comparisons such as heterozygote comparison (C1C2 vs. C1C1), homozygote comparison (C2C2 vs C1C1), dominant model (C1C2+C2C2 vs C1C1), recessive model (C2C2 vs C1C2+C1C1) and allelic model (C2 vs C1). Ardinger and colleagues first reported association between the Taq I polymorphisms at the TGFA locus and CL/P susceptibility in a case–control study [12]. This finding has since been replicated in some studies [6, 15, 17‐23]. However, there are still controversies of the effect of TGFA polymorphism on the predisposition of this malformation [24‐35].

The above inconsistent conclusions in the findings of the studies may be attributed to the size of the samples, the ethnic of the sample population and other reasons. In order to contribute to a better understanding of the role of this gene in the occurrence of cleft lip, cleft lip, or cleft lip and palate, we perform an updated meta-analysis on all available case–control studies, combining data together to reach a larger sample size, to get more statistical power to evaluate the association between CL/P susceptibility and TGFA Taq I polymorphism. Understanding the genetic background and etiology of CL/P is essential for both the risk assessment and findings of effective methods for prevention and treatment.

Detailed characteristics of each study are summarized in Table 1. A total of 20 case–control studies including 3824 cases and 7710 controls contributed to the analysis. The subjects in the study were population of Caucasian, African, Hispanic and Asian. There are some studies including more than one ethnic population. There were 14 studies containing Europeans, 4 studies including Hispanics, 3 studies involving Africans and 2 studies comprising Asians. For the Europeans, Hispanics, Africans and Asians, sample sizes ranged from 25 to 1525, 90 to 921, 17 to 69 and 12 to 100, respectively. The total sample size were 2897 cases and 6806 controls for the Europeans, 853 cases and 753 controls for the Hispanics, 41 cases and 72 controls for the Africans and 33 cases and 79 controls for the Asians. The types of controls included population-based, hospital-based and unrelated family members.

NOS results suggested that all of the included studies are high level quality with the score >6.

The individual susceptibility plays an important role in the development of most diseases. Therefore, the studies of genetic susceptibility are helpful for disease prevention, diagnosis, and treatment.

TGFA is a well characterized mammalian growth factor. Previous studies found that although TGFA is expressed in mice during palatogenesis and mice with a null mutation of the TGFA gene have abnormal skin, hair, and eyes, but they do not have CL/P [38, 39]. TGFA is a likely ligand for epidermal growth factor receptor and newborn epidermal growth factor receptor-negative/-negative mice have high incidence of CL/P which may explain the association between TGFA polymorphisms and CL/P [40]. In 1989, it was firstly published that the TGFA Taq I polymorphism contributed to the development CL/P in humans. From then on, a growing number of studies have been done to examine the relationship between TGFA Taq I polymorphism and the risks of CL/P. However, the results are not consistent. In order to better understand the association, we subjected previously published data to meta-analysis to evaluate genetic associations between the TGFA Taq I polymorphism and CL/P susceptibility.

Through this meta-analysis we found that TGFA Taq I polymorphism increased the risk of CL/P. Mitchell adopted a reappraisal and showed that there was a statistically significant association between TGFA and CL/P, However, there was evidence of significant heterogeneity from different studies, regarding an association between genetic variation at the TGFA locus and CL/P remains inconclusive [41]. A meta-analysis of gene-environment interaction showed the suggestive evidence for gene-environment interaction between the infant’s genotype at the Taq1 marker in TGFA and maternal smoking was limited to CP [42]. Meta-review of thyroid cancer gene expression profiling studies identifies important diagnostic biomarkers, including relatively novel or uncharacterized genes such as TGFA [43]. These results of systematic reviews in some extent support the findings in the present meta-analysis.

In order to reduce heterogeneity, we performed the stratified analyses. It is well know that the incidence of most genetic polymorphisms could vary between different ethnic populations and the wide range in TGFA Taq I allele frequencies across different studies suggests the heterogeneity between populations may exist [28]. In the subgroup analysis by ethnicity, we found that TGFA Taq I polymorphism increased the risk of CL/P in Caucasian population, which agreed with Ardinger and colleagues [12], Shiang and colleagues [20], Hwang and colleagues [22], Chenevix-Trench and colleagues [18], Holder and colleagues [17], and disagreed with Lidral and colleagues [27], [28], Stoll and colleagues [25] and Christensen and colleagues [11]. In the subgroup analysis by disease type, we found the ORs of different disease type were statistically significant, which suggested disease type was not mainly result the heterogeneity. In the subgroup analyses of control characteristics, only the ORs of hospital-based control groups were statistically significant. Therefore, subgroup analyses suggested that ethnicity and control characteristics might contribute to the heterogeneity in this meta-analysis. The meta-regression analyses suggested that ethnicity or control characteristics were probably not the major sources of heterogeneity. The heterogeneity might attribute to other factors. The insufficient data are limited to identify the source of heterogeneity only using meta-regression.

Despite trying our best to perform a comprehensive meta-analysis, some limitations exist in our study. Although the results for publication bias in our study were not statistically significant, our analysis used published international studies, which could arose publication bias. Lack of the original data of available studies limited our further evaluation of potential interactions, such as age, gender, family history, environmental factors and lifestyle. There were not significant results in stratified analyses among African and Hispanic population, because there were too few studies after stratifying. Therefore, more studies are needed to provide more evidence on the association between TGFA polymorphism and CL/P in different ethnic populations.

Although CL/P is a complex disease, our study provided evidence showing the important role of TGFA genes polymorphisms in the development of CL/P, genetic factors determining disease susceptibility and severity may facilitate personalized medicine. Further understanding of the interactions between genetic regulatory mechanisms is critical for discovering new therapies for managing human CL/P. Specifically, targeted therapy about the polymorphisms of related genes might be a promising avenue for future CL/P diagnosis and treatment.

In summary, our meta-analysis supports that the TGFA Taq I polymorphism is more likely to contribute to the susceptibility of CL/P, especially in the subgroup of Caucasian population.

TGFA Taq I polymorphism may be associated with the risk of CL/P.