Risk variants in BMP4 promoters for nonsyndromic cleft lip/palate in a Chilean population

Nonsyndromic cleft lip with or without cleft palate (NSCLP [MIM 119530]) is one of the most common human craniofacial birth defects, with both genetic and environmental components involved in its etiology [1]. Its prevalence rate ranges from 1/300 to 1/2500 depending on the ethnic origin of the populations [2]. NSCLP presents a wide variety of primary and secondary medical complications in its rehabilitation. This fact plus medical costs and the emotional burden to patients and their families makes NSCLP a public health problem [3].

The identification of genetic risk factors in NSCLP has been the subject of intensive research in the last decades. In the past few years, the list of NSCLP candidate genes has rapidly increased and their study has been mainly focused in the search for coding mutations [4]. Some reports have estimated that the contribution of certain genes to NSCLP such as MSX1 accounts for 2%, IRF6 12-17% and 6% for the aggregate contribution of FOXE1, GLI2, MSX2, SKI, SATB2 and SPRY2 [5–8]. The increased knowledge that has emerged from all these reports has permitted an improvement in the knowledge of genetic factors involved in NSCLP, especially in families showing a familial aggregation of this disorder [9–11].

An attractive NSCLP candidate gene is bone morphogenetic protein 4 (BMP4, 14q22-23 in humans), a member of the transforming growth factor-beta superfamily. BMP signals regulate many aspects of skeletal development, including cartilage and bone formation during craniofacial and limb development [12]. Gong and Guo have reported that the Bmp4 expression localizes at the site of fusion of the mice facial prominences [13]. The conditional inactivation of Bmp4 in a transgenic mice line results in an isolated cleft lip [14]. The findings of these studies imply that the function of Bmp4 in the ectoderm of the facial processes is to regulate lip fusion.

In humans, few studies on the role of BMP4 in NSCLP have been reported. Lidral and Moreno performed a genome wide scan meta-analysis that showed evidence of linkage between NSCLP and the chromosomal region 14q21-24 [15]. Lin et al. performed an association study in a Chinese population using the non-synonymous single-nucleotide polymorphism (SNP) of BMP4, rs17563 (p.Val152Ala) and described that the C allele carriers showed an increased risk for NSCLP [16]. Recently, Jianyan et al. and Lin et al. have reported an interaction between rs17563 and environmental factors like maternal passive smoking in the expression of NSCLP [17, 18]. The BMP4 coding sequence was analyzed by Suzuki et al. in a sample of patients with subepithelial, microform and overt cleft lip [19]. These authors detected missense and nonsense mutations in 0.7% of these patients which were absent in controls. All these findings support a role for genetic variation of BMP4 in the pathogenesis of NSCLP.

Our group recently reported a mutation screening analysis of BMP4 in a sample of 150 Chilean NSCLP case-parent trios. This analysis considered the coding regions (exons and exon-intron boundaries) and exclude regulatory regions. Due to the absence of causal mutations, we decided to genotype three SNPs (two intronic and one located 5 kb upstream to BMP4) in this same sample of triads. Significant deviations from expected transmissions were observed for haplotypes conformed by rs1957860 and rs762642 [20]. These polymorphisms delimitate a genomic region where a promoter and an enhancer of BMP4 are located [21]. Consequently, in this new study we searched for NSCLP risk variants within the two BMP4 promoters: BMP4.1 (located upstream from exon 1) and BMP4.2 (located upstream from exon 2) (Figure 1) [22] by direct sequencing in a case-control Chilean sample.

The analysis of both BMP4 promoters in cases detected the presence of novel genetic variants. Four of them were found in BMP4.1: c.-5514G > A, c.-5365C > T, c.-5109A > C and c.-5049C > T located at nucleotide 54424454, 54424305, 54424109 and 54423989 respectively (according to genome assembly GRCh37; Figures 1 and 2). The variants c.-5514G > A and c.-5109A > C were present in a heterozygous state in two different cases. The other two variants, c.-5365T and c.-5049T, were found in one male case which inherited them from his healthy mother possibly conforming a haplotype. Among these variants only c.-5109A > C was detected in one of the 336 controls (OR = 2.09; 95% C.I. = 0.13-33.52; p = 0.543) (data not shown). In BMP4.2 only one novel variant was detected corresponding to an eight base pairs deletion (c.-3066_-3059delAACCCGGG; Figures 1 and 3) in 3.9% of cases and in 5.9% of controls. No statistical differences between both group were observed (OR = 0.65; 95% C.I. = 0.33-1.27; p = 0.202; Table 1). Despite its frequency, this variant has not been previously described in genomic databases like Ensembl or Human Mutation Database.

According to Tfsitescan and MatInspector softwares, the change observed in c.-5514 position generates a new site for GATA-1. The variant c.-5365C > T produces a sequence that can be potentially recognized by a RXR heterodimer transcription factor. The change T for C in c.-5049 introduced a previously inexistent site for TCF-1α. Finally, in the case of BMP4.2, this same bioinformatic analyses showed that rs2855530 G allele generated a Sp1 binding motif which is not detected when the C allele is present. Therefore, the bioinformatic analyses of the genetic variants c.-5514G > A, c.-5365C > T and c.-5049C > T within BMP4.1 and the SNP rs2855530 within BMP4.2 showed that they are capable of creating mammalian transcription factor binding motifs.

Regulatory variants are important in the understanding of the phenotypic diversity and the role they play in the susceptibility of complex diseases. However, it is noteworthy that these regulatory variants have not received the same scientific interest in comparison to coding variants [30]. According to the Human Gene Mutation Database http://www.hgmd.cf.ac.uk/ac/index.php approximately 1.6% of single base-pair substitutions described are regulatory mutations. Furthermore, there is abundant evidence indicating that regulatory SNPs (rSNPs) have an impact in the phenotypic diversity and can also affect disease susceptibility interacting with other variants located in their vicinity [31]. All these changes can potentially disrupt the DNA motifs recognized by transcription factors and consequently alter the normal processes of gene activation and/or transcriptional regulation [32]. In this context and taking into account the absent of NSCLP causal mutations in BMP4 coding regions plus the evidence of haplotype association reported by Suazo et al [20], the present report was focused in detecting NSCLP risk variants within both BMP4 promoters. In accordance with our previous report we found NSCLP risk variants within BMP4 promoters. Due to their frequencies it was impossible to establish if these novel variants are in linkage disequilibrium with those SNPs associated with NSCLP described by Suazo et al.

For BMP4.1 three novel substitutions were detected in cases (c.-5514G > A, c.-5365C > T and c.-5049C > T) which can be considered as potential susceptibility variants due to their absence in controls. Moreover, c.-5365T and c.-5049T were found in the same NSCLP case and they were inherited from his healthy mother. Therefore, although that this haplotype can be considered a risk factor it cannot produce the expression of NSCLP by itself and it would need other genetic variants and/or environmental factors absent in this case's mother. In tune with our findings, several mutations have been identified in NSCLP candidate genes mainly in sporadic cases [11]. For this reason these variants can be considered as private mutations from private families. The novel variants described in our study present the same characteristic shared by private mutations from private families.

In BMP4.2 we did not detect novel allelic variants. Regarding polymorphisms, SNP rs2855530 showed an association with NSCLP which presented a sexual dimorphism. Combining the results of association analyses using UNPHASED and PLINK softwares we can conclude that rs2855530 G allele and the G/G+C/G genotype (dominant model) should be considered risk factors but restricted to males due to their higher frequency in male cases than in male controls. On the other hand, the C/C genotype seems to represent a protective factor for male individuals given that its frequency is higher in male controls. Our group has previously reported a sexual or gender dimorphism for NSCLP where an STR allele of MSX1 gene showed significant differences between male cases and male controls [33]. Using animal models, sex-biased gene expression has been reported for gonadal and extragonadal tissues during embryogenesis where the major determinants of these differences are sex hormones [34, 35]. The human adult face displays a sexual dimorphism which seems to be established in the first years of life but could depend on factors expressed in the prenatal life [36]. These evidences and our findings in the present study are closely linked with epidemiological findings showing a higher frequency of NSCLP in males than females.

The bioinformatic analysis of the risk variants predicts that they could create new transcription factor binding motifs which could be involved in NSCLP. The c.-5514A allele of BMP4.1 could introduce a new site for the hematopoietic transcription factor GATA-1 [37]. A different site for this factor has been reported within the human BMP4.1 promoter and it has been demonstrated that it produces a negative effect on BMP4 expression [22]. The c.-5365T allele also generates a consensus sequence for RXR transcription factor which is related with gene expression regulated by retinoic acid (RA). This situation may explain why teratogenic doses of RA induce cleft palate in Rxr-α knockout mice in a lower frequency than in wild-type animals [38]. The BMP4.1 c.-5049T allele introduces a novel site for TCF-1α, a canonical Wnt pathway effector which is expressed in the processes that originate the mice midface [39, 40]. For BMP4.2, the bioinformatic analysis predicted that the rs2855530G allele generates a novel binding site for Sp1, an ubiquitous transcription factor. Sp1 can modulate the gene expression in cellular processes like differentiation, growth and apoptosis, among others [41]. Nevertheless, there is no information about Sp1 inactivation or overexpression related to craniofacial anomalies.

To our knowledge, the present study constitutes the first report detecting novel risk regulatory variants for a NSCLP candidate gene. In this context, three previous studies have associated rSNPs with this birth defect: this is the case of rs642961 located in an IRF6 enhancer, rs28999109 within the PDGF-C promoter, and rs16260 located in the CDH1 promoter [42–44]. The bioinformatic analysis for IRF6 and PDGF-C variants showed that the risk alleles disrupt potential transcription factor motifs. Nevertheless, reporter gene assays demonstrated that significant alterations in gene expression were detected only for the PDGF-C promoter variant [43]. Following the tendency set by these latter articles, functional studies are necessary to confirm our findings.

In summary, we have detected three novel NSCLP potential causal variants in BMP4 promoters which could contribute to approximately 1.2% of this birth defect, as well as a risk SNP allele with a clear sex dependent association (rs2855530). These results are in concordance with our previous report showing the absence of potential causal mutations in the coding sequence of BMP4. The bioinformatic analyses have predicted that all these variants can potentially generate novel transcription factor recognition sites. In future reports will be necessary to confirm the in vivo capability of these variants to alter BMP4 expression using functional and developmental approaches.