完全性肺静脉异位引流潜在致病基因PDX1突变对其基因功能的影响

doi:10.3969/j.issn.1674-8115.2023.10.001

上海交通大学学报（医学版） ›› 2023, Vol. 43 ›› Issue (10): 1219-1226.doi: 10.3969/j.issn.1674-8115.2023.10.001

• 论著 · 基础研究 •

完全性肺静脉异位引流潜在致病基因PDX1突变对其基因功能的影响

冯炜琦(), 张琪, 吴逸卓, 鲁亚南, 于昱()

上海交通大学医学院附属新华医院心血管发育与再生医学研究所，上海 200092

收稿日期:2023-01-16 接受日期:2023-08-28 出版日期:2023-10-28 发布日期:2023-10-28
通讯作者: 于昱 E-mail:fengweiqi1998@sjtu.edu.cn;yuyu@xinhuamed.com.cn
作者简介:冯炜琦（1998—），女，硕士生；电子信箱：fengweiqi1998@sjtu.edu.cn。
基金资助:
国家自然科学基金(81974021);上海市自然科学基金(20ZR1435500)

Effect of potential pathogenic gene PDX1 variants of total anomalous pulmonary venous connection on its gene function

FENG Weiqi(), ZHANG Qi, WU Yizhuo, LU Yanan, YU Yu()

Institute for Developmental and Regenerative Cardiovascular Medicine, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200092, China

Received:2023-01-16 Accepted:2023-08-28 Online:2023-10-28 Published:2023-10-28
Contact: YU Yu E-mail:fengweiqi1998@sjtu.edu.cn;yuyu@xinhuamed.com.cn
Supported by:
National Natural Science Foundation of China(81974021);Natural Science Foundation of Shanghai(20ZR1435500)

摘要/Abstract

摘要：

目的·采用全外显子测序技术筛查完全性肺静脉异位引流（total anomalous pulmonary venous connection，TAPVC）的可能致病基因，并对其功能进行验证。方法·选择2014—2019年在上海交通大学医学院附属新华医院及上海儿童医学中心确诊的100例TAPVC患儿（患儿组）以及120例健康儿童（对照组）为研究对象。收集2组儿童的血液样本，抽提全血基因组DNA并行外显子测序，以筛查TAPVC的潜在致病基因。通过Mutation Taster、SIFT、PolyPhen-2网站筛选致病基因的有害突变位点，并行Sanger测序。构建PDX1野生型（野生组）及突变型（突变组）质粒，转染入HUVEC细胞后，分别采用实时荧光定量PCR（quantitative real-time PCR，qPCR）和蛋白质印迹法检测突变对PDX1的mRNA和蛋白水平的影响。采用STRING数据库进行蛋白与蛋白之间的相互作用分析，并采用qPCR研究PDX1调节的下游基因的表达。结果·在TAPVC患儿中发现致病基因为PDX1，Sanger测序显示该基因存在2个新发突变，即c.C237A （P33T）和c.C237G（P33A）。与野生组相比，2个突变组（CA组、CG组）的PDX1 mRNA水平没有显著变化，但蛋白相对表达量有明显增加，即分别是野生组的2.9倍和3.4倍（P=0.000，P=0.001）。蛋白质相互作用分析的结果显示，PDX1与SOX17相关联；且qPCR结果显示，PDX1过表达可下调HUVEC细胞中SOX17的表达。结论·PDX1的2个新发错义突变可影响其转录后翻译，且PDX1可能是通过调控SOX17来参与TAPVC的发生与发展。

关键词: 完全性肺静脉异位引流, PDX1, 错义突变, SOX17, 人脐静脉内皮细胞

Abstract:

Objective ·To explore the possible pathogenic gene of total anomalous pulmonary venous connection (TAPVC) by whole exon sequencing and verify its function. Methods ·One hundred TAPVC children (case group) and one hundred and twenty healthy children (control group) in Xinhua Hospital and Shanghai Children's Medical Center, Shanghai Jiao Tong University School of Medicine from 2014 to 2019 were included. The blood samples from the two groups of children were collected, and whole blood genomic DNA was extracted for exon sequencing to screen out the potential pathogenic genes of TAPVC. Harmful mutation sites of pathogenic genes were screened through Mutation Taster, SIFT and PolyPhen-2 websites, and then conducted by Sanger sequencing. The wild-type (wild-type group) and mutant (mutant group) plasmids of PDX1 were transfected into HUVEC cells. Quantitative real-time PCR (qPCR) and Western blotting were used to detect the effects of mutations on mRNA and protein levels of PDX1, respectively. The STRING database was used to analyze the interaction between proteins, and qPCR was used to determine the expressions of downstream genes regulated by PDX1. Results ·Pathogenic PDX1 was found in TAPVC children, and Sanger sequencing revealed two novel variants in the gene: c.C237A (P33T) and c. C237G (P33A). Compared with the wild-type group, there was no significant difference in PDX1 mRNA levels in the two mutant groups, but there was a significant increase in relative protein expression of the CA group and CG group, which was 2.9 and 3.4 times higher than the wild-type group, respectively (P=0.000, P=0.001). Protein interaction analysis demonstrated that PDX1 was associated with SOX17. qPCR results showed that overexpression of PDX1 could downregulate the expression of SOX17 in HUVEC. Conclusion ·The two novel PDX1 missense mutations can affect the process of PDX1 post-transcriptional translation, indicating that PDX1 may participate in the occurrence and development of TAPVC by regulating SOX17.

Key words: total anomalous pulmonary venous connection (TAPVC), PDX1, missense mutation, SOX17, human umbilical vein endothelial cell (HUVEC)

中图分类号:

R725.4

冯炜琦, 张琪, 吴逸卓, 鲁亚南, 于昱. 完全性肺静脉异位引流潜在致病基因PDX1突变对其基因功能的影响[J]. 上海交通大学学报（医学版）, 2023, 43(10): 1219-1226.

FENG Weiqi, ZHANG Qi, WU Yizhuo, LU Yanan, YU Yu. Effect of potential pathogenic gene PDX1 variants of total anomalous pulmonary venous connection on its gene function[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2023, 43(10): 1219-1226.

图/表 8

表 1 PDX1 突变质粒的引物序列

Tab 1 Primer sequence for PDX1 mutation plasmids

Primer	Forward sequence (5'→3')	Reverse sequence (5'→3')
C237A mutation	CAGGCACGCAGCGGGGCTGGCGC	CGCGGTCGGGGTGACGCACGGAC
C237G mutation	GCGCCAGCCCCGCTGCGTGCCTG	CGCGGTCGGGGCGACGCACGGAC

表2 qPCR引物序列

Tab 2 Primer sequence for qPCR

Primer	Forward sequence (5'→3')	Reverse sequence (5'→3')
PDX1	TGAGATCCAGTTCGATGTAA	CAAGTAGGTATCAACGGACT
β-actin	GGCCAACCGCGAGAAGATGAC	CAACGATAGGTCCGACACGATAGG

图1 健康儿童及2例TAPVC患儿的 PDX1 测序峰图

Fig 1 Sequence chromatograms of PDX1 missense variants in the healthy child and 2 TAPVC children

表3 2例患儿的 PDX1 的突变位点的特征

Tab 3 Characteristics of mutation sites in PDX1 in the two TAPVC children

Patient ID	Gender	Age/month	Location in age	Base change	Amino change	SIFT	Mutation taster	PolyPhen-2
A03	Male	2	Exon237	C237A	P33T	0 (D)	DC	0.996 (D)
B11	Female	4	Exon237	C237G	P33A	0 (D)	DC	0.997 (D)

图2 PDX1 在不同物种编码的蛋白质序列中的保守性比较

Fig 2 Comparison of the conservation of protein sequences encoded by PDX1 among different species

图3 2个突变对 PDX1 的mRNA和蛋白表达的影响Note：A. Detection of relative expression of PDX1 in HUVECs by qPCR. B. Detection of the expression of PDX1 in HUVECs by Western blotting. C. Quantitative analysis of the expression of PDX1.

Fig 3 Effect of two mutations of PDX1 on its mRNA and protein expression

图 4 PDX1和其他相关蛋白的相互作用网络图Note：PITX2—paired like homeodomain 2; EPHB4—EPH receptor B4; PLXNA2—plexin A2; GJA5—gap junction protein alpha 5; LYVE1—lymphatic vessel endothelial hyaluronan receptor 1; NKX2-5—NK2 homeobox 5; PLCG1—phospholipase C gamma 1; FLT3—fms related receptor tyrosine kinase 3; KDR—kinase insert domain receptor; NOS2—nitric oxide synthase 2; B2M—beta-2-microglobulin; ANGPT1—angiopoietin 1; PTX3—pentraxin 3; TBX3—T-box transcription factor 3; FAS—Fas cell surface death receptor; PDGFRB—platelet derived growth factor receptor beta; HIF1A—hypoxia inducible factor 1 subunit alpha; HCN4—hyperpolarization activated cyclic nucleotide gated potassium channel 4; NRP1—neuropilin 1; ANKRD2—ankyrin repeat domain 2; HMOX1—heme oxygenase 1; PDPN—podoplanin; FGFR2—fibroblast growth factor receptor 2; HAND2—heart and neural crest derivatives expressed 2; BMPR2—bone morphogenetic protein receptor type 2; TNNT2—troponin T2, cardiac type; MYL7—myosin light chain 7; SEMA3D—semaphorin 3D; COL18A1—collagen type ⅩⅧ alpha 1 chain; ACTA2—actin alpha 2, smooth muscle; SHOX2—SHOX homeobox 2; CD34—CD34 molecule; GIT1—GIT ArfGAP 1.

Fig 4 Protein-protein interaction network between PDX1 and other proteins

图5 PDX1 及其突变体的过表达对 SOX17 表达的影响

Fig 5 Effect of overexpression of PDX1 and its mutants on the expression of SOX17

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完全性肺静脉异位引流潜在致病基因PDX1突变对其基因功能的影响

Effect of potential pathogenic gene PDX1 variants of total anomalous pulmonary venous connection on its gene function

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