Cytogenetics Laboratory, Department of Zoology, Institute of Science, Banaras Hindu University, Varanasi, Uttar Pradesh, India.
Department of Cardiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India.
Mutat Res. 2021 Jan-Jun;822:111741. doi: 10.1016/j.mrfmmm.2021.111741. Epub 2021 Mar 1.
CITED2 is a transcription co-activator that interacts with TFAP2 and CBP/ P300 transcription factors to regulate the proliferation and differentiation of the cardiac progenitor cells. It acts upstream to NODAL-PITX2 pathways and regulates the left-right asymmetry. Both human genetic and model organism studies have shown that altered expression of CITED2 causes various forms of congenital heart disease. Therefore, we sought to screen the coding region of CITED2 to identify rare genetic variants and assess their impact on the structure and function of the protein. Here, we have screened 271 non-syndromic, sporadic CHD cases by Sanger's sequencing method and detected a non-synonymous variant (c.301C>T, p.P101S) and two synonymous variants (c.21C>A, p.A7A; c.627C>G, p.P209P). The non-synonymous variant c.301C>T (rs201639244) is a rare variant with a minor allele frequency of 0.00011 in the gnomAD browser and 0.0018 in the present study. in vitro analysis has demonstrated that p.P101S mutation upregulates the expression of downstream target genes Gata4, Mef2c, Nfatc1&2, Nodal, Pitx2, and Tbx5 in P19 cells. Luciferase reporter assay also demonstrates enhanced activation of downstream target promoters. Further, in silico analyses implicate that increased activity of mutant CITED2 is possibly due to phosphorylation of Serine residue by proline-directed kinases. Homology modeling and alignment analysis have also depicted differences in hydrogen bonding and tertiary structures of wild-type versus mutant protein. The impact of synonymous variations on the mRNA structure of CITED2has been analyzed by Mfold and relative codon bias calculations. Mfold results have revealed that both the synonymous variants can alter the mRNA structure and stability. Relative codon usage analysis has suggested that the rate of translation is attenuated due to these variations. Altogether, our results from genetic screening as well as in vitro and in silico studies support a possible role of nonsynonymous and synonymous mutations in CITED2contributing to pathogenesis of CHD.
CITED2 是一种转录共激活因子，可与 TFAP2 和 CBP/P300 转录因子相互作用，调节心脏祖细胞的增殖和分化。它作用于 NODAL-PITX2 途径的上游，调节左右不对称。人类遗传和模式生物研究表明，CITED2 表达的改变导致各种形式的先天性心脏病。因此，我们试图筛选 CITED2 的编码区，以鉴定罕见的遗传变异，并评估它们对蛋白质结构和功能的影响。在这里，我们通过 Sanger 测序法筛选了 271 例非综合征性散发性 CHD 病例，检测到一个非同义变异（c.301C>T，p.P101S）和两个同义变异（c.21C>A，p.A7A；c.627C>G，p.P209P）。非同义变异 c.301C>T（rs201639244）是一种罕见变异，在 gnomAD 浏览器中的等位基因频率为 0.00011，在本研究中的频率为 0.0018。体外分析表明，p.P101S 突变可上调 P19 细胞下游靶基因 Gata4、Mef2c、Nfatc1&2、Nodal、Pitx2 和 Tbx5 的表达。荧光素酶报告基因分析也表明下游靶启动子的激活增强。进一步的，计算生物学分析表明，突变 CITED2 的活性增加可能是由于丝氨酸残基被脯氨酸导向激酶磷酸化所致。同源建模和比对分析还描绘了野生型与突变蛋白之间氢键和三级结构的差异。通过 Mfold 和相对密码子使用偏好计算分析了同义变异对 CITED2 mRNA 结构的影响。Mfold 结果表明，这两个同义变异都可以改变 mRNA 结构和稳定性。相对密码子使用分析表明，由于这些变异，翻译速度降低。总的来说，我们从遗传筛选以及体外和计算生物学研究中得到的结果支持非同义和同义突变在 CITED2 中可能发挥作用，导致 CHD 的发病机制。
