This article is mentioned in:

Introduction

Down syndrome (DS) or Down's syndrome, also known as trisomy 21, is an autosomal abnormality induced by an extra copy of chromosome 21 and is the most common birth defect among children worldwide (1,2). Children with DS usually have severe mental retardation (3) and delayed development (4), and are prone to gastrointestinal malformations (5). In children, almost 50% of DS cases are accompanied with congenital heart disease (6), and the risk of developing acute leukemia is 20 times higher than that of the normal population globally (7). In addition, patients with DS generally have shorter life expectancy (8,9).

DS is the most common cause of mental retardation and malformation in newborns. During meiosis, chromosome 21 in the egg does not separate, therefore, an extra copy of chromosome 21 is produced (10). When the sperm and the egg fuse, the embryo has 47 chromosomes, with three copies of chromosome 21 (11). An extra chromosome 21 leads to the overexpression of its genes, causing nerve dysfunction in vivo, and affecting the normal growth and development of children (12). At present, prenatal diagnosis is the optimal approach in preventing DS, however, there are no effective drugs for treatment of the disease. Thus, it is important to investigate the molecular mechanisms of DS.

Previous studies have suggested that the elevated gene expression of human chromosome 21 (HSA21) is responsible for specific aspects of the DS phenotype. Arron et al (13) showed that certain characteristics of the DS phenotype can be associated with the increased expressions of two HSA21 genes, namely those encoding the transcriptional activator, regulator of calcineurin 1 (DSCR1-RCAN1), and the protein kinase, dual-specificity tyrosine phosphorylation-regulated kinase (DYRK)1A. The overexpression of a number of HSA21 genes, including DYRK1a, synaptogenin 1 and single-minded homolog 2, results in learning and memory defects in mouse models, suggesting that trisomy of these genes may contribute to learning disability in patients with DS (14,15).

The abnormal copy number of chromosome 21 is the primary genetic characteristics of DS. Therefore, the present study applied a variety of bioinformatics tools to determine the genetic fragments in chromosome 21. The methylated sites in bisulfite-sequencing (seq) data were detected, differentially methylated regions between DS and control samples were determined, and the adjacent genes of differential DNA methylation regions were identified. Subsequently, the functions of the abnormal demethylated genes were predicted using Gene Ontology (GO) enrichment analyses. The differentially expressed genes (DEGs) between the DS and control samples were screened. Furthermore, the interactions/associations between the proteins encoded by selected genes were determined, and a protein-protein interaction (PPI) network was constructed. The present study aimed to identify the key genes involved in DS, and may be able to establish the theoretical foundation for the targeted therapy of DS.

Materials and methods

Data sources

The bisulfite-seq data GSE42144 deposited by Jin et al (16) was downloaded from the Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) of the National Center for Biotechnology Information, which was based on the platform of the Illumina Genome Analyzer IIx (Illumina, Inc., San Diego, CA, USA). GSE42144 included three placental samples (GSM1032059, GSM1032060 and GSM1032061) from patients with DS and three normal control samples (GSM1032070, GSM1032071 and GSM1032072). According to the quality control results of RNA-seq data, two RNA-seq data from patients with DS (GSM1033476 and GSM1033478) and five RNA-seq data from normal control samples (GSM1033470, GSM1033471, GSM1033472, GSM1033473 and GSM1033474) were also used in the present study.

Alignment of bisulfite-seq data and detection of DNA methylation

For all bisulfite-seq data, Bismark (www.bioinformatics.bbsrc.ac.uk/projects/bismark/) (17) and Bowtie 2 (bowtie-bio.sourceforge.net/bowtie2/index.shtml) (18) software were applied to perform read alignment, analyze methylated DNA signaling and output cytosine methylation sites in the genome. All parameters were set at default values.

Differential DNA methylation and corresponding adjacent gene analysis

The BiSeq tool (19) was used to determine differentially methylated regions between the placenta samples of patients with DS and normal control samples. The false discovery rate of each significant differentially methylated CpG cluster was ≤0.1. The methylated CpG clusters with a length of <100 bp were merged and defined as a differentially methylated DNA region. In addition, the length of each differentially methylated DNA region was required to be ≥50 bp. If the distance between the center of the differentially methylated DNA region and the transcription start site of a specific gene ranged between 3,000 and 500 bp, the differentially methylated DNA region was considered to have the potential to affect the gene, and this gene was defined as an adjacent gene of the differentially methylated DNA region.

Alignment of RNA-seq data and calculation of gene expression

Tophat (4) software was used to perform read alignment, with the University of California Santa Cruz (genome.ucsc.edu) hg19 genome sequences as a reference. For read alignment, up to two base mismatches were permitted in one read. Only the reads which mapped to specific genome locations were retained for further analysis. The other parameters were set to the defaults. On combining with the Refseq gene annotations, the transcripts were assembled and gene expression values were calculated using Cufflinks and Cuffdiff tools (5). The calculated gene expression values were based on the fragments per kilobase of transcript per million fragments mapped method (20).

Analysis of DEGs

The paired t-test (21) was used to identify DEGs between the DS and control samples. P<0.01 and |log2fold-change|≥2 were used as the cut-off criteria.

Function annotation of the adjacent genes

The Database for Annotation, Visualization, and Integrated Discovery (22) was used to perform GO enrichment analysis for the adjacent genes of the differentially methylated DNA regions. The GO terms were classified into biological process, molecular function and cellular component categories. P<0.05 was used as the cut-off criterion.

PPI network construction

The interaction associations of the proteins encoded by selected genes were determined using the Search Tool for the Retrieval of Interacting Genes (STRING) database (23). All parameters were set to defaults. A PPI network was then constructed using Cytoscape (24).

Results

Identification of differential DNA methylation regions in DS

Based on the Bisulfite-seq data, a total of 74 CpG regions had significant differential DNA methylation between the DS and normal samples, including 68 demethylated regions, accounting for 92%, and six regions with higher levels of methylation, compared with those of the normal samples.

For a single chromosome, the majority of the abnormal DNA methylation regions were detected in chromosomes 7 and 17, showing a total of seven aberrantly methylated DNA regions. In chromosome 21, five abnormal demethylated DNA regions were found (Fig. 1).

Figure 1. Distribution of differentially methylated DNA regions in each chromosome. Blue indicates hypomethylated DNA regions and red indicates hypermethylated DNA regions. DS, Down syndrome; chr, chomosome.

Identification of adjacent genes of the differentially methylated DNA

Compared with the control samples, a total of 43 adjacent (protein-coding) genes were identified in the DS samples with demethylated promoter regions and one adjacent gene, chromosome 19 open reading frame 80, which is known to be located in chromosome 19, was identified with upregulated methylation in its promoter region (Table I).

Table I. Number of adjacent genes with differentially methylated DNA regions in Down syndrome samples.

Table I.

Number of adjacent genes with differentially methylated DNA regions in Down syndrome samples.

Methylation	Protein-coding genes (n)	Noncoding genes (n)
Hypomethylated	43	0
Hypermethylated	1	0

In the autosomal chromosomes, there were six DS-associated genes with demethylated promoter regions in chromosome 17. The number of abnormal genes in other chromosomes ranged between one and three. No genes were found to be affected by abnormal DNA demethylation in the sex chromosomes.

The distributions of the genes on chromosomes are listed in Table II. Among these, only Runt-related transcription factor 1 (RUNX1) was found to be located on chromosome 21, and the demethylation of the promoter region of this gene was significant (Table II).

Table II. Distribution of adjacent genes with demethylated regions in DS sample chromosomes.

Table II.

Distribution of adjacent genes with demethylated regions in DS sample chromosomes.

Chromosome	n	Gene
1	2	LOC730144, EIF1
2	3	ATG4B, LRRFIP1, STRADB
3	1	EXOG
4	1	FRYL
5	3	BOD1, B4GALT7, BRD9
6	2	MAP3K4, ID4
7	4	PAXIP1, POLM, FAM131B, AGAP3
8	2	ATAD2, REEP4
9	2	BARX1, KIAA1539
10	2	OTUD1, GPRIN2
11	1	SLC1A2
12	1	ALDH2
13	3	HMGB1, HMGB1L10, FARP1
14	1	RTN1
15	2	TYRO3, PCSK6
16	1	FA2H
17	6	UTP18, LOC730144, C17ORF56, EIF1, TIMP2, MRM1
18	1	ABHD3
20	2	CBFA2T2, C20ORF20
21	1	RUNX1
22	3	HMGB1, OSBP2, HMGB1L10

Functional enrichment of abnormal demethylated genes

The present study subsequently analyzed the functions of the 43 abnormally demethylated genes. Combined with GO functional annotation, the five genes, high mobility group box (HMGB)1, HMGB1L10, inhibitor of DNA binding 4 (ID4), leucine-rich repeat flightless-interacting protein 1 and core-binding factor, Runt domain, α subunit 2; translocated to, 2 (CBFA2T2) were found to be involved in the biological process of negative regulation of transcription, whereas the three genes, BARX homeobox 1, DNA polymerase mu and RUNX1, were associated with immune system development. In addition, the present study found that solute carrier family 1 member 2, ID4 and tissue inhibitor of matrix metalloproteinase 2 were predominantly involved in forebrain development and regulation of neurogenesis. The HMGB1, HMGB1L10, ID4, RUNX1 and CBFA2T2 genes possessed the capabilities of transcription factor binding, according to the molecular function terms (Table III).

Table III. Functional annotation of the adjacent genes with demethylated promoter regions in Down syndrome samples.

Table III.

Functional annotation of the adjacent genes with demethylated promoter regions in Down syndrome samples.

Category	GO term	Genes (n)	Gene
BP	GO:0016481~negative regulation of transcription	5	HMGB1, HMGB1L10, ID4, LRRFIP1, CBFA2T2
BP	GO:0002520~immune system development	3	BARX1, POLM, RUNX1
BP	GO:0032147~activation of protein kinase activity	2	MAP3K4, STRADB
BP	GO:0030855~epithelial cell differentiation	2	BARX1, CBFA2T2
BP	GO:0030900~forebrain development	2	SLC1A2, ID4
BP	GO:0050767~regulation of neurogenesis	2	ID4, TIMP2
CC	GO:0005694~chromosome	4	HMGB1, BOD1, BARX1, HMGB1L10
CC	GO:0019898~extrinsic to membrane	3	OSBP2, PCSK6, FARP1
MF	GO:0008134~transcription factor binding	5	HMGB1, HMGB1L10, ID4, RUNX1, CBFA2T2
MF	GO:0016564~transcription repressor activity	3	ID4, LRRFIP1, CBFA2T2
MF	GO:0030528~transcription regulator activity	6	BARX1, ATAD2, ID4, LRRFIP1, RUNX1, CBFA2T2

[i] GO, Gene Ontology; BP, biological process; CC, cellular component; MF, molecular function.

Functional enrichment analysis showed that ID4 was not only involved in neuronal differentiation, but also functioned in transcriptional suppression. The demethylation of its promoter region led to the increased expression level of ID4 (Table III).

Analysis of DEGs

Combined with the RNA-seq data, the present study analyzed transcriptome differences between the DS samples and normal samples, and identified a total of 584 DEGs, including RUNX1, which were upregulated (Table IV).

Table IV. Number of differentially expressed genes in Down syndrome samples, compared with control samples.

Table IV.

Number of differentially expressed genes in Down syndrome samples, compared with control samples.

Expression	Genes (n)	Transcription factors (n)
Upregulated	584	24
Downregulated	0	0

Based on the detection of tissue-specific gene database, 208 of the 584 DEGs (36%) were found to have specific expression in brain tissue. By contrast, 52 DEGs in the DS samples were expressed specifically in neutrophils, the pituitary, peripheral nervous system, stomach and T-cells, which was substantially lower than the number of brain tissue-specific genes (Fig. 2).

Figure 2. Distribution of tissue-specific differentially expressed genes in Down syndrome samples.

Finally, with the addition of transcription factor data, the present study identified 24 DEGs with transcriptional regulatory function, of which the eight transcription factors, zinc finger protein 43, early growth response (EGR)3, nuclear receptor subfamily 4, group A, member 2 (NR4A2), nuclear receptor subfamily 3, group C, member 2 (NR3C2), LIM homeobox 2, gastrulation brain homeobox 2, pentraxin-related gene, rapidly induced by interleukin-1β and nuclear factor I/A, were brain-tissue specific (Table V).

Table V. Differentially expressed genes with transcriptional regulatory function and brain tissue specificity.

Table V.

Differentially expressed genes with transcriptional regulatory function and brain tissue specificity.

Transcription factor	Description	Brain-specific
ZNF43	Zinc finger protein 43	Yes
EGR3	Early growth response 3	Yes
EGR2	Early growth response 2	No
ZFY	Zinc finger protein, Y-linked	No
MITF	Microphthalmia-associated transcription factor	No
NR4A2	Nuclear receptor subfamily 4, group A, member 2	Yes
NR3C2	Nuclear receptor subfamily 3, group C, member 2	Yes
FOXN1	Forkhead box N1	No
HOXB13	Homeobox B13	No
VAX2	Ventral anterior homeobox 2	No
HNF4G	Hepatocyte nuclear factor 4, γ	No
SIX4	SIX homeobox 4	No
FOXP1	Forkhead box P1	No
HESX1	HESX homeobox 1	No
ERCC8	Excision repair cross-complementing rodent repair deficiency, complementation group 8	No
HOXA1	Homeobox A1	No
HOXB5	Homeobox B5	No
LHX2	LIM homeobox 2	Yes
PAX8	Paired box 8	No
GBX2	Gastrulation brain homeobox 2	Yes
LHX6	LIM homeobox 6	No
PTX3	Pentraxin-related gene, rapidly induced by IL-1β	Yes
NFIA	Nuclear factor I/A	Yes
NFIB	Nuclear factor I/B	No

Association between differential methylation and dysregulation

In order to examine the potential link between abnormal methylation and dysregulation in DS samples, the present study integrated their data combined with protein-protein interactions the STRING database, and found only one PPI network (Fig. 3). The network contained five genes, including NR4A2, EGR2, EGR3, RUNX1 and hepatocyte nuclear factor 4, γ (HNF4G). There were several interactions, including RUNX1-NR4A2, NR4A2-EGR2 and NR4A2-EGR3, in the PPI network.

Figure 3. Motif analyses between the genes with demethylated DNA in promoter regions and DEGs in DS samples. Red nodes represent significantly upregulated genes and the orange node represents a moderately upregulated gene. The V-node represents the gene in which the promoter region was significantly demethylated, and circle nodes indicate the genes without significantly altered methylation. The blue lines indicate protein-protein interactions. RUNX1, Runt-related transcription factor 1; NR4A2 nuclear receptor subfamily 4 group A member 2; EGR, early growth response; HNF4G hepatocyte nuclear factor 4, γ.

Discussion

As a genetic disease in which an individual has 47 chromosomes instead of the usual 46 (25), DS affects ~1/730 live births and occurs in all populations equally (26). In the present study, bioinformatics tools were used to determine the genetic fragments associated with DS. A total of 74 CpG regions had significant differential DNA methylation between the DS and normal samples. There were five abnormal DNA demethylated regions in chromosome 21. A total of 43 adjacent genes with demethylation in promoter regions and one adjacent gene with upregulated methylation in promoter regions were identified in the DS samples. In addition, 584 upregulated genes were identified, including 24 genes with transcriptional regulatory function. Only NR4A2, EGR2, EGR3, RUNX1 and HNF4G were involved in the PPI network.

In the present study, upregulated RUNX1 was located on chromosome 21, and the demethylation of the promoter region of this gene was significant. Functional enrichment analysis showed that RUNX1 was associated with immune system development and possessed the capabilities of transcription factor binding. It is reported that the expression of RUNX1 in megakaryoblasts in children with DS and acute megakaryocytic leukemia is lower, compared with cases of acute megakaryocytic leukemia without DS (27,28). The risk of developing dementia of Alzheimer's disease in individuals with DS is higher, compared with that of the general population, and a variant within RUNX1 is closely linked with dementia of Alzheimer's disease in DS (29). A previous study reported that RUNX1 and NR4A2 can coordinately regulate the differentiation of T cells (30,31). The transcription factor, NURR1, which is also known as NR4A2, is important in the functional maintenance, development and survival of midbrain dopaminergic neurons (32). As with DS, Parkinson's disease is also a disorder of the central nervous system, and decreased expression levels of NURR1 may contribute to the identification of Parkinson's disease and other neurlogical disorders (33). The transcription factors, EGR2 and EGR3, are members of the Egr family, which is involved in regulating the peripheral immune response, and EGR2 may serve as a potential target in neuroinflammation therapy for its host defense role in the central nervous system immune response (34). According to the results of the present study, transcription factors EGR3 and NR4A2 were identified as brain-tissue specific. In the PPI network, several interactions were identified, including RUNX1-NR4A2, NR4A2-EGR2 and NR4A2-EGR3, indicating that RUNX1 and NR4A2 may be involved in DS by coordinately regulating EGR2 and EGR3.

As a member of the ID family, ID4 inhibits the differentiation or the DNA binding of basic helix-loop-helix transcription factors, regulating genes, which are important in neuronal differentiation (35). A previous study demonstrated that ID1, ID2, ID3 and ID4 are promising primary targets for methyl-CpG binding protein 2-regulated neuronal maturation, which may be responsible for the development of Rett syndrome, a neurodevelopmental disorder (36). In the present study, functional enrichment analysis indicated that ID4 possessed the capabilities of transcription factor binding, and that ID4 was involved in neuronal differentiation and transcriptional suppression. Therefore, it was hypothesized that the upregulated expression level of ID4 may be associated with the symptoms of severe mental retardation and stunting of the nervous system, which are observed in patients with DS.

In conclusion, the present study performed integrated bioinformatics analyses of DNA methylation and RNA-seq data to identify genes, which may be correlated with DS. A total of 43 adjacent genes with demethylation of promoter regions and one adjacent gene with upregulated methylation of its promoter region were identified in the DS samples. In addition, 584 upregulated genes were identified, which included 24 genes with transcriptional regulatory function. RUNX1, NR4A2, EGR2, EGR3 and ID4 may be correlated with DS. However, their mechanisms of action in DS remain to be fully elucidated and further experimental validation is required.

References