LI Qing-Man, GUO Feng, LING Yi, LI Hui-hui, LIU Fang-fang, LIU Hui, WEN Zhuangfei, SUN Wei-wei, LIU Yi-heng , ZHANG Hai-ying?

1. Hainan Medical University Key Laboratory for Research and Transformation of Tropical Brain Science, Haikou 571199, China

2. Department of Obstetrics and Gynecology, The First Affiliated Hospital of Hainan Medical College, Haikou 570102, China

3. Department of Laboratory, The First Hospital of Nanping City, Fujian Province, Nanping 353006, China

4. Children's Rehabilitation Department, Haikou Hospital of the Maternal and Child Health, Haikou 570102, China

5. Department of Pediatrics, Haikou People’s Hospital, Haikou 570208, China

6. Department of Orthopedics, Haikou Affiliated Hospital of Central South University Xiangya School of Medicine, Haikou 570208, China

Keywords:Congenital heart disease Gene mutation NKX2-5 GATA4 ZIC3

ABSTRACT Objective: Congenital heart disease (CHD) is caused by abnormal cardiac development,which is the most common congenital malformation at home and abroad. NKX2-5, GATA4 and ZIC3 have been shown to be associated with CHD. This experiment explored the relationship between NKX2-5, GATA4 and ZIC3 gene mutations and sporadic CHD in Hainan Province.Methods:To collect 210 sporadic CHD patients in Hainan, the DNA of patients was extracted from blood, and the target gene fragments were amplified. Using high-resolution melting(HRM) and DNA sequencing technology, and we analyzed the sequences of NKX2-5, GATA4 and ZIC3 genes.Results: NKX2-5, GATA4 and ZIC3 genes were sequenced in 210 CHD patients, and seven gene mutations were found, including NKX2-5 heterozygous missense mutation (c.178G>T) and three heterozygous mutations in GATA4 (c.677C>T, c.928A>G,c.1123G>A), three heterozygous mutations in ZIC3 (c.19G>C, c.1255C>G, c.1348C>T),in which NKX2-5 (c.178G>T), GATA4 (c.1123G>A), and ZIC3 (c.1255C>G, c.1348C>T)are new mutation sites. These gene mutations were predicted to be pathogenic mutations by bioinformatics software.Conclusion: Conclusion: Seven gene mutations were found in 210 patients, and it was the first report that the gene mutations of NKX2-5, GATA4 and ZIC3 in Hainan Province associated with the pathogenesis of CHD.?Corresponding author:ZHANG Hai-ying, Professor, Ph.D., E-mail: hyzhang_xjtu2013@aliyun.com; LIU Yi-heng, Chief Physician, E-mail: 1398345154@qq.com.

1. Introduction

Congenital heart disease (CHD) is a disease of the heart structural abnormalities in which lead to cardiac malformations.The pathogenesis of CHD is complex, often caused by mutations in chromosomes or genes that affect heart development, and is sensitive to genetic and environmental perturbations [1, 2]. The highest incidence of CHD was reported in Asia with 9.3 CHD per 1000 live births, followed by Europe (0.82% live births), and the lowest rate reported in North America (0.69% live births) [3, 4].Researchers believe that genetic factors and environmental factors are the main causes of CHD [5].

Both human NKX2-5 and mouse NKX2-5 are belong to the NK homologous gene family and share a conserved domain [6], which plays an important role in the development and differentiation of the heart. NKX2-5 was the first gene to be linked to CHD, and Schott et al. [7] were the first to detect NKX2-5 gene mutations in patients with ventricular septal defect. Studies have found that NKX2-5 is a core regulator transcription factor for cardiac development, NKX2-5 gene mutations can lead to abnormalities in cardiac structure and function [8]. GATA4 is one of the six members of the GATA transcription factor family. Mutations in GATA4 not only lead to abnormal cardiac development [9], but also lead to urogenital defects,hypertension and other diseases [10,11]. The ZIC3 gene encodes a highly conserved zinc finger protein ,in which belongs to the GLI transcription factor superfamily [12]. ZIC3 has multiple functions in morphogenesis, and the regulation of BMP and Wnt signaling pathways by ZIC3 may be critical for normal cardiac development[13].

In this study, we collected 210 non-syndromic sporadic CHD patients and sequenced the NKX2-5, GATA4 and ZIC3 genes of these patients by high-resolution melting (HRM) and DNA sequencing.Research. We analyzed the NKX2-5, GATA4, and ZIC3 gene series in sporadic uncomplicated CHD patients in Hainan, China, and explored the correlation between genotype and phenotype.

2. Materials and methods

2.1 Experimental subjects

From November 2018 to October 2021, we collected 210 sporadic CHD patients in the study. CHD was diagnosed by physical examination and echocardiography by 3 experienced pediatric cardiologists. Patients with syndromic CHD, i.e. syndromic (Charge,DiGeorge, Noonan, Holt-Oram, Marfan, Alagille) and chromosomal disorders were excluded from this study. 210 patients samples collected from from the First Affiliated Hospital of Hainan Medical College, Haikou Maternal and Child Health Hospital, and Haikou People's Hospital (Haikou Hospital Affiliated to Xiangya School of Medicine, Central South University) were collected, 2-5 mL of venous blood was drawn from each patient and filled in tubes containing ethylenediaminetetraacetic acid (EDTA) anticoagulant.The experiments were reviewed by the Ethics Committee of Hainan Medical College (Haikou, China), and informed consent was obtained from all participants or their guardians.

2.2 Main reagents and instruments

Gradient PCR machine (nexus GSX1, Eppendorf AG); agarose gel electrophoresis apparatus; NKX2-5, GATA4 and ZIC3 gene primers(Sangon, Shang Hai); PCR mixture (Invitrogen, U.S.); DNA Marker(TianGen, Guang Zhou).

2.3 Extraction of genomic DNA

DNA was extracted from blood samples according to standard protocols (TIANamp Blood DNA Kit, TIANGEN). DNA quality is required that A260/A280 is controlled between 1.8-2.0.

2.4 PCR amplification, HRM analysis and DNA sequencing

Amplify the target gene fragment (nexus GSX1, Eppendorf AG), screen the mutations of NKX2-5, GATA4 and ZIC3 by HRM technology, and conduct DNA bidirectional sequencing. Variants were initially screened through Exome Variant Server (http://evs.gs.washington.edu/EVS/), and patients who met the mutation diagnosis were confirmed by secondary experiments.

3 Results

3.1 Clinical phenotypes of NKX2-5, GATA4 and ZIC3 mutations

In this study, 210 patients with sporadic CHD were screened for NKX2-5, GATA4 and ZIC3 gene mutations by HRM technology.Sanger sequencing was used to identify potential candidate pathogenic loci, and excluding high-frequency polymorphic loci,potential pathogenic mutation sequences were found. Summary of NKX2-5, GATA4 and ZIC3 gene mutations in sporadic CHD cases(Figure1). The examination results showed that a total of seven variants of NKX2-5, GATA4 and ZIC3 gene were detected in seven CHD patients, and the detection rate was 3%.

3.2 NKX2-5, GATA4 and ZIC3 gene mutations

A nonsense mutation (c.178G>T, p.Glu60X) in NKX2-5 was identified in a patient with ASD and VSD by Sanger sequencing(Figure 2A), which causes structural abnormalities in the heart. We searched the 1000Genomes website (https://www.internationalgenome.org/data), the ClinVar website (https://www.ncbi.nlm.nih.gov/clinvar/) and the gnomAD database (http://gnomad-sg .org/), identified c.178G>T as a new mutation site. Three heterozygous mutations were found in the GATA4 gene, of which c.1123G>A p.Gly375Arg was a novel mutation (Figure 2B), which involved a CCG→CTG mutation at codon 1123 of the GATA4 gene exon. This resulted in the substitution of Gly by Arg at residue 375.This mutation site is located in a highly conserved region of the GATA4 gene (Figure 2C) and alters the domain that binds to other transcription factors. Gene mutation c.677C>T p.Pro226Leu (Figure 2D) was found in a patient with VSD and ASD, and gene mutation c.928A>G p.Met310Val (Figure 2F) was found in a patient with VSD and PDA, we searched The 1000G site and the ClinVar site were analyzed and it was determined that the p.Pro226Leu and p.Met310Val mutations of GATA4 are both known mutations and located in the conserved region (Figure 2E and 2G). Among them,c.677C>T leads to Pro to Leu, and another mutation site c.928A>G leads to Met to Val. We found three novel gene mutations in ZIC3 gene, the first new mutation was detected in a patient with VSD and ASD, ZIC3 gene mutation c.19G>C p.Gly7Arg (Fig 2H), it is located in a conserved sequence (Figure 2I). The second ZIC3 gene nucleotide C was replaced by G at position 1255, c.1255C>G p.Leu419Val (Figure 2J), and Leu was mutated to Val. The third novel mutation, c.1348C>T p.Pro450Ser (Figure 2L), was found in a TOF patient in which C was replaced by T at codon 450 of the ZIC3 gene. c.1255C>G p.Leu419Val and c.1348C>T p.Pro450Ser are located in highly conserved functional domains (Figure 2K and 2M).

3.3 Pathogenicity analysis of NKX2-5, GATA4 and ZIC3 gene mutations

For gene mutations discovered by Sanger sequencing, the experiments used bioinformatics methods (Polyphen2 [http://genetics.bwh.harvard.edu/pph2/], SIFT [http://sift.jcvi.org/] and mutationtaster [https://www.mutationtaster.org/]) to predict the pathogenicity of novel mutation sites in GATA4 and ZIC3 (Table 1). The results showed that using three methods to predict GATA4 p.Pro226Leu, p.Met310Val and p.Gly375Arg all showed that possible pathogenicity. Three mutations in ZIC3 were predicted inconsistently by bioinformatics methods (Table 2), p.Gly7Arg was predicted to be likely pathogenic in polyphen2, and SIFT predicted a high probability of pathogenicity. p.Leu419Val predicted low pathogenicity in polyphen2 and SIFT, however predicted pathogenicity in mutationtaster. p.Pro450Ser is predicted to have no pathogenicity in polyphen2, whereas it is predicted to be highly pathogenic in SIFT.

Tab 1Predicted pathogenicity of NKX2-5, GATA4 and ZIC3 gene mutations

4. Discussion

Genes associated with the occurrence of CHD include NKX2-5,GATA4, ZIC3, TBX1, TBX5, LEFTY, SOX17, and include transforming growth factor beta (TGF β) signal pathway, Shh signal pathway, Notch signal pathway, Wnt signal pathway, and mitogen Activated protein kinase signaling pathway and other related signal transduction pathways [14-24]. We found seven gene mutations in NKX2-5, GATA4, ZIC3, among which five new gene mutations NKX2-5 (c.178G>T), GATA4 (c.1123G>A), ZIC3 (c.19G> C,c.1255C>G, c.1348C>T).

Studies have shown that the NKX2-5 gene plays an important role in the early development of the heart [25]. Some studies have shown that NKX2-5 gene mutation is related to CHD [26]. In 210 CHD patients, a new mutation site c.178G>T p.Glu60X was detected in the NKX2-5 gene, and glutamic acid was mutated to a stop codon,resulting in interruption of protein transcription. The functional domain cannot be transcribed normally, the protein is significantly shorter than the wild-type protein, and there are serious protein structural and functional abnormalities, which may lead to some serious pathological manifestations. By referring to 1000G and ClinVar website, C178G>T is a new mutation site. Among the 210 patients, only 1 patient carried the NKX2-5 mutation site, and the incidence rate was about 0.5%, NKX2-5 mutation detected in 608 CHD patients in the United States at a frequency of 3% [27]. This shows that there is a big difference between in Hainan Province and foreign countries. Larger differences, which may show that the genetic background of NKX2-5 in Hainan Province and other regions is different, resulting in the difference in the pathogenicity of NKX2-5.

GATA4 is considered to be an important transcription factor in the developing heart [28]. Some GATA4 mutations have been shown to cause familial and sporadic CHD. The same mutation may have different clinical manifestations in patients, and the same clinical manifestations may be caused by different mutations [29-31]. We found three different mutations (p.Gly375Arg, p.Pro226Leu,p.Met310Val), these mutations are highly conserved amino acids sequences that may also affect DNA binding or protein-protein interactions of GATA4. Li et al. [32] found that the p.Pro226Leu mutation site is located in the zinc finger domain, and the mutant will reduce the transcriptional activity of GATA4 and reduce the synergistic activation of NKX2-5, thereby affecting the development of cardiac structure. Chen et al [33], found that the p.Gly310Arg mutation site is located in the nuclear localization signal region.The gene mutation leads to changes in protein structure, decreased transcriptional activity, impairs the ability to import into the nucleus,affects transcriptional activation, and ultimately leads to abnormal cardiac structure. GATA4 overexpression induces cardiomyocyte hypertrophy, enhanced sarcomere organization, and a more than 2-fold increase in cell surface area. A number of CHD related GATA4 variants have been reported [34-36]. Although the significance of these mutations is unclear, "silent mutations" have been found to be associated with many genetic diseases.

ZIC3 plays an important role in all aspects of human development.It is involved in the development and regulation of the human heart,bone, gut and urinary system [37]. Mutations in the ZIC3 gene are dominantly inherited, with a pathogenicity rate of approximately 1% [38], resulting in abnormal cardiac development, including atrial isomerism, atrioventricular septal defect, double outlet right ventricle,transposition of the great arteries, and subpulmonary stenosis[39]. Three ZIC3 gene mutation sites were predicted : p.Gly7Arg,p.Leu419Val, p.Pro450Ser. Alessandro De Luca et al. [40] founded a heterozygous mutation at p.Gly17Cys, which affected the nuclear localization function of the protein. The location of p.Gly7Arg and p.Gly17Cys was similar, which may have affected similar functions.The functions of the protein encoded by ZIC3 depend on both its localization in the nucleus and its subsequent ability to activate target genes, and the zinc finger domain plays an important role in these functions. Zhu et al. [41] found that the combination of ZIC3 and GLI3 is an important process in the normal development of the heart.The mutation of ZIC3 p.Lys405Glu will affect the binding ability of ZIC3 and CLI3, thereby affecting the protein function. The position of p.Lys405Glu and p.Leu419Val is close, so it is speculated that ZIC3 p.Leu419Val mutation may also result in inability to bind GLI3 normally, resulting in abnormal cardiac development. p.Ala447Gly is located downstream of the nuclear localization signal of zinc finger structure 2+3 and zinc finger structure ZF4+5, ZIC3 p.Ala447Gly mutation alters subcellular trafficking and prevents downstream transcriptional activation of developmental key genes [42], the position of p.Pro450Ser is close to p.Leu447Val, and it is speculated that it may affect the transcriptional activation of developmental key genes. Collectively, these results suggest that ZIC3 mutations may affect the zinc finger domain, nuclear localization signaling region,and nuclear export signaling leading to the pathogenesis of CHD.

In this study, seven gene mutations in NKX2-5, GATA4 and ZIC3 genes were found in 210 CHD patients that may be associated with congenital sporadic CHD in Hainan Province, China. Detected five new mutation sites NKX2-5 (c.178G>T), GATA4 (c.1123G>A),ZIC3 (c.19G>C, c.1255C>G, c.1348C>T). Only one mutation site was founded in the NKX2-5 gene, and the mutation rate in CHD patients is 0.5%, which is significantly different from that in Western countries. This result suggests that the mutation of NKX2-5 gene may be a risk factor for sporadic CHD patients in Hainan,and NKX2-5 has the value of further study. In conclusion, the results of this study suggest that novel gene mutations of NKX2-5(p.Glu60X), GATA4 (p.Gly375Arg), ZIC3 (p.Gly7Arg, p.Leu419Val,p.Pro450Ser) may play important roles in cardiac function.cause CHD. It is hoped that further verification through animal experiments will be conducted to explore the genetic mechanism of CHD.

Important Notice All authors of this article have read and understood the policy statement of the Journal of Hainan Medical University, and there is no potential conflict of interest in this research.

Contributions FangFang Liu, YiHeng Liu, Yi Ling, ZhuangFei Wen, WeiWei Sun were participated in the collection and interpretation of patient data.Feng Guo and HuiHui Li were involved with acquisition of data.QingMan Li drafted the manuscript with substantial revisions by HaiYing Zhang and Hui Liu. All authors contributed to the editing of the manuscript and agreed on the final content. All authors read and approved the final manuscript.