aa-utp SMAD plays a pivotal role in the

SMAD4 plays a pivotal role in the pathological progression of vascular disorder (Zhang et al., 2016). SMAD4, a member of Smad family, is a central mediator in the canonical TGF-β signaling. It regulates biological processes important for the pathogenesis of TAAD, such as SMCs migration and proliferation, extracellular matrix degradation (Mao et al., 2012). SMAD4-deficient SMCs can trigger aortic wall aa-utp by producing chemokines and recruiting macrophages (Zhang et al., 2016). Besides, SMAD4 also play essential roles in cardiogenesis, blood vessel remodeling, maturation and integrity (Jiao et al., 2003; Lan et al., 2007; Qi et al., 2007; Song et al., 2007). The TGF-β signaling network consists of a complex of ligands, receptors, and transcriptional coregulators that have important effects in vascular remodeling and matrix degradation (Jones et al., 2009). Mutations of members (TGFBR1/2, FBN1) in this network are causative for disorders hallmarked by aortic aneurysm, e.g. Marfan syndrome (MFS) or Loeys–Dietz syndrome (LDS) (Loeys et al., 2005, 2006). Mutations in a TGF-β ligand-TGF-β2 gene have been recognized as a cause of TAAD with MFS (Boileau et al., 2012). SMAD4 encodes the only co-smad in TGF-β signaling (Mao et al., 2012). The mutations of SMAD4 gene are also important in progression of aortopathy. In Heald\'s retrospective study, the authors described a high prevalence (38%) of aortopathy in patients with juvenile polyposis (JPS) and hereditary hemorrhagic telangiectasia (HHT) is attributed to SMAD4 mutations (Heald et al., 2015). Gallione and Andrabi also reported that SMAD4 mutations could cause a combined JPS & HHT syndrome and vascular disorders (for example, aortic root dilation, multiple arteriovenous malformation) (Andrabi et al., 2011; Gallione et al., 2004). Thus, it is interesting to explore the association between variants of SMAD4 and the pathological progression of TAAD, which may shed light on the role of SMAD4 in pathogenesis of TAAD and provide a maker for disease diagnosis. To investigate the genetic effect of SMAD4 on TAAD, five tagging SNPs were initially genotyped in 202 subjects and 400 healthy controls. The potential function of the significant SNP in the screening dataset was further analyzed by bioinformatic software. A series of experiments was conducted to investigate the potential molecular mechanism of the significant SNP.
Materials and Methods
Results
Discussion The TGF-β superfamily is composed of various secreted polypeptides which regulate cell proliferation, differentiation and tissue morphogenesis in many organisms (Xie et al., 2014). SMAD4 has been recently identified as the unique co-smad which mediates the responses to TGF-β and related factors. Multiple studies in animal models showed that the deficiency of SMAD4 contributed to the development of thoracic aortic aneurysm or other vascular disorder (Lan et al., 2007; Mao et al., 2012; Zhang et al., 2016). In the present study, we investigated the genetic effects of SMAD4 on the pathogenesis of TAAD. Five tagger SNPs were genotyped in 202 TAAD patients and 400 healthy controls. There was a significant association between individuals carrying at least one T allele of rs12455792 and an increased risk of TAAD. In functional analysis, we found that the variant reduced SMAD4 expression, thus promote proteoglycans degradation and SMCs apoptosis, which were important pathophysiological processes in TAAD (Fig. 5). rs12455792 is located in the 5′UTR of SMAD4. This locus is predicted as a transcription factors binding site, with proximal transcriptional regulatory potential. While allele C changes to T, both the number of potential binding factors and affinity scores are decreased. Sequence logo graph indicated a high conservation of C allele in Sox6 binding site (Supplemental Fig. 2). These analyses determined the significance of this locus in transcriptional regulation. In view of the evidences shown in Fig. 1, rs12455792 C→T change reduced the transcriptional activity, altered the RNA folding structure, and decreased SMAD4 expression. Except for the reduced affinity of transcription factor and -650 region DNA, the C to T substitution also changed RNA folding structure greatly. RNA hairpin structure might act like a hinge that open up DNA and bind the kinase domain. However, the variant of C to T might attenuated the function of RNA hairpin structure, thus down-regulated gene expression. To the best of our knowledge, rs12455792 is a functional SNP involved in the pathogenesis of TAAD. Although it has been reported in cancer metastasis (Li et al., 2012), the present study is the first to demonstrate potential function of this SNP.