Imprinting aberrations of SNRPN, ZAC1 and INPP5F genes involved in the pathogenesis of congenital heart disease with extracardiac malformations

Abstract Congenital heart disease (CHD) with extracardiac malformations (EM) is the most common multiple malformation, resulting from the interaction between genetic abnormalities and environmental factors. Most studies have attributed the causes of CHD with EM to chromosomal abnormalities. However, multi‐system dysplasia is usually caused by both genetic mutations and epigenetic dysregulation. The epigenetic mechanisms underlying the pathogenesis of CHD with EM remain unclear. In this study, we investigated the mechanisms of imprinting alterations, including those of the Small nuclear ribonucleoprotein polypeptide N (SNRPN), PLAG1 like zinc finger 1 (ZAC1) and inositol polyphosphate‐5‐phosphatase F (INPP5F) genes, in the pathogenesis of CHD with EM. The methylation levels of SNRPN, ZAC1, and INPP5F genes were analysed by the MassARRAY platform in 24 children with CHD with EM and 20 healthy controls. The expression levels of these genes were detected by real‐time polymerase chain reaction (PCR). The correlation between methylation regulation and gene expression was confirmed using 5‐azacytidine (5‐Aza) treated cells. The methylation levels of SNRPN and ZAC1 genes were significantly increased in CHD with EM, while that of INPP5F was decreased. The methylation alterations of these genes were negatively correlated with expression. Risk analysis showed that abnormal hypermethylation of SNRPN and ZAC1 resulted in 5.545 and 7.438 times higher risks of CHD with EM, respectively, and the abnormal hypomethylation of INPP5F was 8.38 times higher than that of the control group. We concluded that abnormally high methylation levels of SNRPN and ZAC1 and decreased levels of INPP5F imply an increased risk of CHD with EM by altering their gene functions. This study provides evidence of imprinted regulation in the pathogenesis of multiple malformations.


| INTRODUC TI ON
Many types of congenital malformations, such as congenital heart disease (CHD), digestive system malformation, and urinary system malformation, have a greater impact on morbidity and mortality in children than that of single deformity. 1 Studies have shown that 7%-50% of foetuses with CHD are accompanied by extracardiac malformations (EM), especially in patients with a ventricular septal defect (VSD) and right ventricular double outlet (DORV). 2 Malformations in the urinary system, gastrointestinal system, and nervous system are the most common external deformities, while malformations in the respiratory system and skeletal system are relatively rare. [2][3][4][5] EM significantly interrupt the natural history and clinical course of CHD. 1 Due to the mutual influence of CHD with EM, the treatment fee is expensive, but the prognosis is unsatisfactory. Therefore, it is important to understand the pathogenesis of multiple malformations in CHD with EM.
Most studies have attributed the aetiology of syndromes containing CHD with EM to the chromosomal abnormalities and copy number variations (CNVs), such as Down syndrome and Edwards syndrome. However, the aetiology of CHD with EM is not only related to genetic variation, but also epigenetics. Environmental factors are essential for the occurrence of multi-system dysplasia because they can facilitate the generation of epigenetic information related to phenotypic variation and disease, 6 ultimately leading to increased susceptibility to congenital diseases in offspring. 7 Adverse environments such as maternal diseases, nutrition, and even the atmosphere during embryonic development could disturb epigenetic modifications, leading to dysplasia or embryonic lethality. 8 Chamberlain et al 2014 reported that environmental factors during early embryonic development could lead to CHD by influencing the methylation level of heart development-related genes. 9 Previous studies have suggested that smoking factors could change the methylation level of the ERCC1 and ADP-ribose genes in embryos. Additionally, ethanol could lead to the abnormal methylation of PTNP11 or WBSCR1 and WBSCR22 genes, which causes Noonan or Williams syndrome. 10 Zhou et al 2018 reported that arsenic exposure could affect the LINE1 and P16 methylation levels, producing genome-wide methylation abnormalities, as well as CHD and other diseases. 11 However, methylation alterations are effective factors of chromosome structure stability. Mollar et al 2019 reported that methylation modifications occurring during all periods of mitosis could affect kinetochore and chromosome condensation and segregation, which are essential for genome stability. 12 Thus, abnormal methylation modification reduces the integral stability of chromosomes, inducing a 'metastable' status of these genomes that could result in chromosomal deletion and abnormal replication. 13 Imprinted genes comprise elements within the human chromosome that are controlled by epigenetic modifications during early embryonic development. 14 Appropriate imprinting levels of these genes play an essential role in embryonic development, [15][16][17] and genome imprinting substantially affects the development and function of body systems, especially in embryonic stage. 8 Changes in the imprinting level can affect the susceptibility and immunity of certain diseases. 18 Changes in methylation modification can alter the traditional genetic balance of parents, finally affecting the development process of embryos, manifesting as multi-system dysplasia. 19 Presently, the mechanism underlying DNA methylation of imprinted genes in CHD with EM remains unclear. Small nuclear ribonucleoprotein polypeptide N (SNRPN), PLAG1 like zinc finger 1 (ZAC1) and inositol polyphosphate-5-phosphatase F (INPP5F) are imprinted genes that play essential roles in embryogenesis, especially cardiac development.
SNRPN plays a vital role in specific tissues and organ development, including the heart and brain. ZAC1 is an important transcription factor in heart development and influences heart formation. INPP5F is highly expressed in the heart, brain, and other tissues.
In the present study, we selected three imprinted genes, SNRPN, ZAC1, and INPP5F, to study the possible role of DNA methylation modifications in the aetiology of CHD with EM. We focused on children with CHD with EM and analysed the methylation levels of germinal different methylation regions (gDMRs) of the selected three imprinted genes to explore the roles of imprinting establishment on CHD with EM. This study provides a theoretical basis for the pathogenesis of CHD with EM.  Table 1. In general, including nine cases with urinary system diseases (cryptorchidism and), eight cases with digestive system diseases (indirect inguinal hernia, imperforate anus, and abdominal hernia), four cases with nervous system diseases (terminal filament and spina bifida), three cases with motor system diseases (polydactylism, ganglion cysts, and cleft palate) and four cases with other system diseases (hemangioma and sebaceous adenoma). Control samples were confirmed to be disease-free, including 8 females and 12 males with aged of 6m-6y.

| Cell culture
The human colorectal cancer HCT15 cell line, which demonstrates global hypermethylation, was obtained from the American Type Culture Collection . The cells were cultured at 37°C in a humidified 5% CO 2 atmosphere in RPMI 1640 medium (Invitrogen) supplemented with 10% foetal bovine serum (GIBCO). Cells in the exponential growth phase were used for subsequent experiments. For demethylation studies, cultured cells were incubated for 72 h in 0 or 50μmol/l of 5-azacytidine (5-Aza; Sigma-Aldrich), a methylation inhibitor, and the medium was changed daily. DNA with an OD260/OD280 absorbance ratio of 1.8-1.9 was used for subsequent analysis.

| Bisulphite treatment
In total, 500 ng of genomic DNA from each brain tissue sample was subjected to bisulphite treatment using the EZ DNA methylation kit (Zymo Research) according to the manufacturer's instructions.

| Methylation analysis of imprinted genes
The Sequenom MassARRAY platform (CapitalBio) was used to perform quantitative analysis of gene methylation. This system uses matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry combined with RNA base-specific cleavage (MassCLEAVE). The detected pattern is then analysed for its methylation status. Polymerase chain reaction (PCR) primers were designed using Meth primer (http://epide signer.com). For each reverse primer, an additional T7 promoter tag for in vivo transcription was added, as well as a 10-mer tag on the forward primer to adjust for melting temperature differences. One pair of primers was used to amplify the promoter region of each gene. The list of primers is shown in Supplementary S1. Primers were synthesized by Sangon Biotech (Shanghai, China). The spectra methylation ratios were generated by Epityper software version 1.0 (Sequenom). cDNA was stored at -20 °C until required for use in real-time PCR.

| Real-time PCR
Real-time PCR was carried out to compare the mRNA expression levels of SNRPN, ZAC1 and INPP5F relative to that of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The primers were designed using Primer Express® software Version 3.0 (Applied Biosystems). All primers used are shown in Supplementary S1.

Number of cases Age(months) Mean(min-max) Male
Female

| Statistical analyses
The data were stored in the EPI 3.1 Database (EpiData Association) and were analysed using the SPSS 18.0 software package (McGraw-Hill Inc). The methylation level of the imprinted genes was compared between CHD with EM and the control groups by independent sample t test. One-way analysis of variance (ANOVA) was performed to evaluate the differences among different EM systems and CHD subgroups. Odds ratios (ORs) were calculated to evaluate the incidence of CHD with EM correlating with the methylation levels. The data were shown as means and standard deviation. All p-values were two-sided, and P < .05 was considered significant. GraphPad Prism 7 software (GraphPad Software) was used to display the analysis results visually.

| Abnormal methylation modifications of imprinted genes in CHD with EM
Blood samples from 24 cases with CHD with EM and 20 control subjects were obtained for methylation analysis. The characteristics of the subjects are shown in Table 1

| Assessment of the risk of developing CHD with EM
We developed a model to assess the risk of developing CHD with

F I G U R E 2
The methylation level of the ZAC1 gene in the case group is significantly higher than that in the control group. A) Average methylation levels in the case and control groups. The boxes extend from the 25th to 75th percentiles and are divided by a line representing the median of each group. The different methylation levels between the groups of both regions were statistically significant. B) Methylation level of specific CpG sites in the ZAC1 gene. The CpG sites are numbered 1-28 from the 5' end to the 3' end, and the methylation levels of CpG sites numbered 1-23 and 25 in the case group were significantly different than those in the control group. The data were expressed as means ± SD. **P < .01, ***P < .001

F I G U R E 3
Difference between the methylation levels of the INPP5F gene in the case and control groups. A) Average methylation levels in cases with CHD with EM and controls. The methylation level of the case group was lower than that of the control group, and the difference was statistically significant. B) Methylation level of specific CpG sites in the INPP5F gene. The CpG sites are numbered 1-9 from the 5' end to the 3' end in the promoter area of INPP5F. The methylation levels of CpG sites 2-9 in the case group were significant different than those in the case group. The data were expressed as means ± SD. *P < .05, **P < .01, ***P < .001 levels increased the risk of CHD with EM approximately 7-and

| Expression Levels of SNRPN, ZAC1, and INPP5F in the blood samples of cases CHD with EM
To

| SNRPN, ZAC1 and INPP5F transcription negatively correlates with methylation modifications
To explore whether the transcription of SNRPN, ZAC1, and INPP5F was affected by their methylation changes, we established a methylation cell model of HCT15 treated with 5-Aza and examined the methylation and expression levels of SNRPN, ZAC1, and INPP5F.

| D ISCUSS I ON
The pathogenesis of CHD with EM is very complex, and the underlying mechanism remains unclear. 1 In the present study, we demonstrated that the changed methylation modifications of imprinted genes could lead to CHD with the abnormality of different systems.
Our data showed that the methylation levels of SNRPN and ZAC1 increased significantly in CHD with EM, while those of INPP5F decreased significantly. In addition to the changed methylation modifications of these selected imprinted genes, the gene expression levels were negatively altered in CHD with EM. Interestingly, abnormal imprinting increased the risk of CHD with EM.
Imprinting is an epigenetic marking of genomes based on the parental origin, which can generate differential expression of paternal and maternal alleles in certain tissues and developmental phases. These epigenetic marks are set up in the germline and can be maintained and passed down to offspring through mitotic divisions. Changes in methylation modification can alter the traditional genetic balance of parents, finally affecting the development process of embryos and manifesting as multi-system dysplasia, including developmental disorders of the cardiovascular system. 7,8 In this study, first of all, any samples was excluded with abnormal gene duplication/deletion after the analysis of CNVs, which means that epigenetics may contribute more to the pathogenesis of CHD with EM.
On the basis, we selected gDMRs of the imprinted genes SNRPN, ZAC1, and INPP5F to study methylation regulation in CHD with EM.
Our data are the first to hint that not only chromatin mutation but  in pre-mRNA processing and coding through tissue-specific variable splicing events. 23 The gene may play an important role in specific tissues and organ development, especially the heart and brain. 23

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.