Polymorphisms of ATP-binding cassette, sub-family A, member 4 (rs560426 and rs481931) and nonsyndromic cleft lip/palate: A systematic review and meta-analysis CURRENT STATUS: UNDER REVIEW

Background A number of gene loci are closely associated with the incidence of non-syndromic cleft lip/palate (NSCL/P). Herein, this meta-analysis assessed the association between ATP-binding cassette, sub-family A, member 4 (ABCA4) polymorphisms (rs560426 and rs481931) and the risk of NSCL/P by reviewing case-control studies.Methods Four databases including Scopus, Cochrane Library, Web of Science, and PubMed were searched for articles published up to December 2018. The Review Manager 5.3 software was used to calculate the crude odds ratio (OR) and 95% confidence interval (CI).Results Of 82 retrieved studies, 12 were analyzed in this meta-analysis (2,859 NSCL/P patients and 3,792 controls for ABCA4 rs560426 polymorphism and 1,333 NSCL/P patients and 1,884 controls for ABCA4 rs481931 polymorphism). There was no significant association between the polymorphisms and the risk of NSCL/P, with the exception of the allelic model (OR=1.13; 95% CI: 1.01, 1.27; p=0.03), the homozygote model (OR=1.53; 95% CI: 1.01, 2.31; p=0.04), and the recessive model (OR=1.30; 95% CI: 1.03, 1.63; p=0.03) in the Asian ethnicity for rs560426 polymorphism.Conclusion The findings confirmed that G allele and GG genotype of rs560426 polymorphism were significantly associated with the risk of NSCL/P in the Asian population, but not for rs481931polymorphism. Also, there was no association between both polymorphisms (rs560426 and rs481931) with the risk of NSCL/P in the Caucasian and the mixed ethnicities, as well as the source of controls. Therefore, ethnicity may play a significant role in this association.


Introduction
Non-syndromic cleft lip/palate (NSCL/P) is a usual congenital malformation that accounts for more than 70% of facial cleft deformities [1]. Its prevalence ranges from 1/700 to 1/1000, depending on the geographical area and ethnicity [2]. NSCL/P has a multifactorial etiology and therefore both environmental and genetic risk factors can affect its incidence and prevalence [1,3]. Maternal smoking, alcohol consumption, and folic acid and vitamin deficiencies especially during the first trimester of pregnancy have been reported to increase the incidence of NSCL/P [4]. Considering the complex etiology of NSCL/P, studies have shown that interactions of gene-gene and geneenvironment may be related to susceptibility to NSCL/P [3,5,6]. Subsequent genome-wide association studies showed that a number of gene loci are closely associated with the incidence of NSCL/P [7,8].
ATP-binding cassette, sub-family A, member 4 (ABCA4) belongs to the transmembrane protein superfamily [9], which is located on chromosome 1p22.1 [10,11]. The ABCA4 is an ATP-binding cassette transporter that is particularly expressed in the rod and cone photoreceptor cells of the vertebrate retina [9][10][11] and is associated with various ocular diseases [12]. A systematic review reported the significant presence of ocular abnormalities in patients with NSCL/P [13]. The genetic basis of oral clefts is still unclear, and identification of additional risk factors for NSCL/P can greatly help in genetic counseling and may prevent the occurrence or further development of this condition [14]. Therefore, the present meta-analysis aimed to evaluate the association between ABCA4 polymorphisms (rs560426 and rs481931) and the risk of NSCL/P in case-control studies to assess the possible role of these polymorphisms in NSCL/P etiology.

Materials And Methods
The instructions of the PRISMA were applied to design this study [15]. The study was approved by the

Literature search strategy
Four electronic databases including the Web of Science, Cochrane Library, Scopus, and PubMed were searched for articles published until December 2018 without restrictions of language or publication date. The searched terms were: ("ABCA4" or "ATP-binding cassette subfamily A member 4" or "rs560426" or "rs481931") and ("cleft" or "cleft lip" or "cleft palate" or "orofacial cleft" or "oral cleft"). In addition, we checked the references of retrieved articles to find potential articles.

Eligibility criteria
One reviewer (M.S) retrieved the studies from the databases. Then, he excluded duplicate and irrelevant studies. The studies were relevant if they met the following eligibility criteria: (Ӏ) casecontrol design; (ӀӀ) NSCL/P was the outcome of interest; (ӀӀӀ) reporting ABCA4 rs560426 (A > G) and/or rs481931 (C > A) polymorphisms, and (ӀV) having the required data to calculate the odds ratios (ORs) with 95% confidence intervals (CIs). The studies were excluded if they did not have the required data regarding genotype distributions or were animal studies, review articles, letters to the editor reporting previous studies, and family-based studies. The second reviewer (M.M.I) checked the relevant articles based on the eligibility criteria. The differences between tow reviewers were solved by the third reviewer (E.P.-F.L).

Data extraction
Two reviewers (P.L.-J and M.S) independently extracted the data from each study. The information retrieved from the studies involved: the first author's name, the publication year, the ethnic group, the source of controls, the mean age, the number of males in the two groups, the type of disease, the number of patients and controls with each genotype, the genotyping method, and the p-value of Hardy-Weinberg equilibrium in controls. If there was a disagreement between the reviewers, the problem was solved by discussion.

Quality of assessment
One reviewer (M.M.I) distinguished the quality of each included article using the Newcastle-Ottawa Quality Assessment Scale questionnaire (maximum total score = 9 for a case-control study) [16].

Statistical analyses
An analysis was performed by the Review Manager 5.3 using crude OR and 95% CI to show the association between ABCA4 polymorphisms and the risk of NSCL/P in the five genetic models [17].
The Z test was applied to evaluate the pooled OR significance. Heterogeneity across the studies was checked by the Cochrane Q test and I 2 statistic that there was a statistically significant heterogeneity if P <0.1 or I 2 >50%. If there was no significant heterogeneity, the fixed-effects model (Mantel-Haenszel method) was used to estimate the values. Otherwise, we used the random-effects model (DerSimonian and Laird method). The Chi-square test was used to calculate the Hardy-Weinberg equilibrium in the control group of each study. Subgroup analysis was done according to the ethnicity and the source of controls. The Comprehensive Meta-Analysis 2.0 was used to analysis funnel plot using the Egger's and Begg's tests and P<0.05 indicated significant existence of the publication bias.
To evaluate the stability of the results, we used sensitivity analysis.

Study selection
Of 82 retrieved studies, after removing duplicate and irrelevant studies, the full-texts of 21 studies were evaluated (Figure 1). Of 21 studies, nine were excluded with below reasons: three were familybased studies, three did not have the required data, two were letters to the editor reporting previous studies, and one evaluated the mothers of children. Finally, 12 studies were analyzed in this metaanalysis. Table 1 shows some features of the studies included in this meta-analysis that the studies were published from 2011 to 2018. Five studies [6,[18][19][20][21] reported the risk of NSCL/P related to ABCA4 polymorphisms in the Asian ethnicity, four [5,7,14,22] in the mixed ethnicity, and three [23][24][25] in the Caucasian ethnicity. The source of controls in six studies [14,18,19,21,23,24] was hospital-based while six other studies [5][6][7]20,22,25] were population-based. Some studies reported the mean age, the number of males in the two groups, and the type of disease. Of 12 studies, all of them reported ABCA4 rs560426 polymorphism, but only six studies [5,6,21,[23][24][25] reported ABCA4 rs481931 polymorphism. There were different genotyping methods among the studies presented in Table 1.

Study characteristics
The Hardy-Weinberg equilibrium was not seen in controls of two studies [6,19] reporting ABCA4 rs560426 polymorphism while two studies [6,24] reported ABCA4 rs481931 polymorphism. This metaanalysis included 2,859 NSCL/P patients and 3,792 controls reporting ABCA4 rs560426 polymorphism and 1,333 NSCL/P patients and 1,884 controls reporting ABCA4 rs481931 polymorphism. Figure 2 shows the pooled results (combining data from each individual study included) of the risk of NSCL/P related to ABCA4 rs560426 polymorphism based on twelve studies. The heterogeneity in allelic, homozygote, heterozygote, and dominant models was high (I 2 >50%) and therefore randomeffects model was used, but in recessive model, fixed-effects model was used because of low heterogeneity (I 2 <50%).  10)] in the recessive model (GG vs. AA + AG). Based on the genetic models, there was no the risk of NSCL/P related to ABCA4 rs560426 polymorphism.

Pooled analysis
The pooled results of the significant risk of NSCL/P related to ABCA4 rs481931 polymorphism according to six studies is shown in Figure 3. The heterogeneity in all analyses was low and therefore fixed-effects model was used (I 2 <50%

Subgroup analysis
The first subgroup analysis evaluated the pooled analysis based on the ethnicity and the source of controls for the significant risk of NSCL/P related to ABCA4 rs560426 polymorphism ( Table 2)  The second subgroup analysis assessed the risk of NSCL/P related to ABCA4 rs481931 polymorphism according to the ethnicity and the source of controls ( Table 3). There was no association between the significant risk of NSCL/P and rs481931 polymorphism in the Asian, the Caucasian, and the mixed ethnicities, as well as the studies including hospital-based and population-based controls based on the models.

Sensitivity analysis
One study was removed and the cumulative analysis identified that the pooled ORs under all genetic models were stable and trustworthy. As well as, excluding two studies [6,19] that had p-values<0.05 for Hardy-Weinberg equilibrium in controls did not change the overall result regarding the risk of NSCL/P related to ABCA4 rs560426 polymorphism. Excluding two other studies [6,24] did not change the overall result regarding the risk of NSCL/P related to ABCA4 rs481931 polymorphism.

Quality assessment
A mean score of 7.33 was achieved for the quality of all studies ( Table 4). Out of 12 studies, ten studies had score ≥7 and two studies had score < 7. Figure 4 shows the funnel plot of the association between ABCA4 polymorphisms and the risk of NSCL/P using the five genetic models. The Egger's and Begg's tests did not reveal any publication bias except for 4 states: Both tests for A vs. C and AA vs. CC revealed a publication bias and Egger's test for CA vs. CC and CA + AA vs. CC revealed a publication bias (p<0.05).

Discussion
The ABCA4 gene mutations have been associated with a range of autosomal recessive retinal degenerative diseases [24]. NSCL/P is a complex congenital anomaly that shows both clinical and genetic heterogeneity and the genetic basis of NSCL/P have remained unclear [14]. Among the studies reviewed in this meta-analysis, the rs560426 G allele showed 1.36- [21] and 1.34-fold [22] increase in the risk of CL/P compared to the A allele and the rs560426 GG genotype showed 1.74- [19], 3-[20], 1.86- [21], and 1.72-fold [22] increase in the risk of NSCL/P compared to the AA genotype. In contrast, the G allele, GG genotype [23], AG genotype [5,23], A allele [23], AA genotype [24], and CA genotype [23] showed a lower risk of NSCL/P compared to the A allele, AA genotype, AA genotype, C allele, CC genotype, and CC genotype, respectively. This meta-analysis did not exhibit any significant risk of NSCL/P related to ABCA4 polymorphisms (rs560426 and rs481931) except for the Asian ethnicity in which, the G allele and GG genotype identified a significant increase in the risk of NSCL/P compared to A allele and AA genotype, respectively. In addition to some studies mentioned in this meta-analysis, which indicated a link between the ABCA4 gene and the NSCL/P risk, associations of this gene with CL/P in two genome-wide association studies [26,27] and in different populations [28][29][30] highlight the need for further studies on the possible role of ABCA4 gene in the CL/P etiology. Fontoura et al. [23] found significant associations of ABCA4 rs481931 alleles with bilateral and unilateral CL/P. Replication of ABCA4 polymorphisms using independent families from various populations revealed that the Asian families had stronger evidence for ABCA4 compared to the European or American families [19]. The present meta-analysis confirmed this result.
One study [18] suggested that the strong correlation between ABCA4 and CL/P may be due to the impact of nearby genes (such as ARHGAP29), and not the impact of single loci. It is probably that the associated variants in ABCA4 are in close connection disequilibrium with a causal variant included in a neighboring gene, and act as indirect surrogates for a real etiologic variant in NSCL/P patients [23,24,29]. Gene-gene and gene-environment interactions showed that there was an interaction between ABCA4 rs560426 polymorphism with folic acid consumption and V-maf avian musculoaponeurotic fibrosarcoma oncogene homolog B rs11696257 polymorphism [5]. The different rate of the G allele of rs560426 polymorphism in different populations shows that the polymorphism alone is not associated with NSCL/P development, and so, it may not be the causative singlenucleotide polymorphism. Therefore, this apparent incompatibility among populations may identify the complex genetic etiology of NSCL/P [21]. The transmission analysis using case-parent core pedigrees showed that the C allele of rs481931 was significantly over-transmitted from parents to children, indicating that the C allele is associated with the NSCL/P risk. In addition, the C (rs481931)-G (rs560426) haplotype was significantly involved in the occurrence of NSCL/P [6].
The present meta-analysis had several limitations: a small number of studies reviewed, differences in age and sex of patients and genotyping methods, absence of Hardy-Weinberg equilibrium in some studies, a difference of sources of controls and ethnicities evaluated in the studies that created high heterogeneity in some analyses. However, sensitivity analysis identified the stability of the results and also the heterogeneity across the studies was minimal.

Conclusions
The findings of the present meta-analysis confirmed that the G allele and GG genotype of rs560426 polymorphism were significantly associated with the NSCL/P risk in the Asian population. There was no association between both polymorphisms (rs560426 and rs481931) with the risk of NSCL/P in the Caucasian and the mixed ethnicities, as well as the source of controls and even the Asian ethnicity for rs481931polymorphism. Therefore, ethnicity may act as a significant factor in this association.

Consent for publication
Not applicable.

Availability of data and materials
The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.  There was the p-value >0.05 in all analyses and P h indicates P heterogeneity . Abbreviations: OR, odds ratio; CI, confidence interval.   Forest plot of NSCL/P risk related to ABCA4 rs560426 polymorphism using the five genetic models. Abbreviation: NSCL/P, non-syndromic cleft lip with or without a cleft palate.

Funding
20 Figure 3 Forest plot of NSCL/P risk related to ABCA4 rs481931 polymorphism using the five genetic 21 models. Abbreviation: NSCL/P, non-syndromic cleft lip with or without a cleft palate. Figure 4