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. 2022 Sep 30:13:918379.
doi: 10.3389/fgene.2022.918379. eCollection 2022.

Identification and characterization of extrachromosomal circular DNA in alcohol induced osteonecrosis of femoral head

Tingting Zhou  1   2 , Shiqiang Ma  1   2 , Yunchao Zhao  1   2 , Donghui Guo  1   2 , Hengjun Wang  1   2 , Mingjie Kuang  2   3 , Xiaoming Li  1   2
Affiliations

Identification and characterization of extrachromosomal circular DNA in alcohol induced osteonecrosis of femoral head

Tingting Zhou et al. Front Genet. .

Abstract

Alcohol-induced osteonecrosis of the femoral head (AIONFH) is a complicated refractory bone disease seen in the clinic. The pathogenesis of AIONFH is still controversial. Extrachromosomal circular DNA (eccDNA) elements have been indicated ubiquitously exist in eukaryotic genomes. However, the characteristics and biological functions of eccDNAs remain unclear in AIONFH. In this study, eccDNAs from AIONFH samples (n = 7) and fracture of femoral neck samples as a control (n = 7) were purified by removing linear DNA and rolling circle amplification. High-throughput sequencing and bioinformatics analysis were performed to study the characterization and biofunction of eccDNAs. We identified more than 600,000 unique eccDNAs. The number of detected eccDNAs in AIONFH was less than that in the control, and eccDNA formation may be related to transcription or other characteristics of coding genes. The eccDNA lengths are mainly distributed between 0.1 kb and 1 kb, with a major peak in 0.358 kb. The bioinformatic analysis showed that 25 significant genes were detected, including MAP3K1, ADCY1, CACNA1S, and MACF1, which contributed to regulating bone formation. GO and KEGG analyses suggested that the related genes derived from exons mainly affected metabolic processes and signal transduction, and bone metabolism-related pathways, such as the MAPK pathway and TGF-β pathway, were enriched. EccDNAs in AIONFH are common and may play an important role in pathogenesis by regulating bone metabolism.

Keywords: alcohol induced osteonecrosis of femoral head; bioinformatics; chromosomal instability; extrachromosomal circular DNA; function of eccDNA.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
EccDNAs number, sizes distribution of AIONFH group and control group. (A). The total amount of eccDNAs detected in each sample. (B). Confidence of identified eccDNAs. (C). Venn diagram showed the consist eccDNAs detected in AIONFH and control samples. (D–F). The length distribution of eccDNAs (<100 kb) in each group.
FIGURE 2
FIGURE 2
EcDNAs number, sizes distribution of AIONFH group and control group. (A). The total amount of ecDNAs detected in each sample. (B). Confidence of identified ecDNAs. (C). Venn diagram showed the consist ecDNAs detected in AIONFH and control samples. (D–F). The length distribution of ecDNAs (100kb–10000 kb) in each group.
FIGURE 3
FIGURE 3
Genomic distribution features of eccDNAs between AIONFH and control. (A–B). The distribution of eccDNAs on chromosomes. (C). The origin region distribution of eccDNA on the genome. (D). the correlation of eccDNA per Mb with coding genes per Mb.
FIGURE 4
FIGURE 4
Genomic distribution of ecDNAs and the correlation with frequency of ecDNAs. (A). The distribution of ecDNA fragment in chromosomes. EcDNA per Mb of AIONFH group and control group: chromosomes (B) and coding genes per Mb (C).
FIGURE 5
FIGURE 5
Bioinformatic analysis of functional attributes connected to eccDNAs changes in gene sets and signaling pathways. (A). Common and specific characteristics of eccDNAs of AIONFH group and control group were showed in Venn diagram. (B). Upregulated and downregulated eccDNAs related genes. (C,D). Volcano plot and hot map show the different genes. (E,F). Top 20 of Gene ontology terms (Biological Processed, Molecular Functions, Cellular Components) enriched in the plot figure. (I). GO biological process classification of genes. (H). Top 20 of KEGG enrichment signal pathways. (J). KEGG function enrichment analysis.
FIGURE 6
FIGURE 6
Bioinformatic analysis of exon region of gene in eccDNAs. (A). Upregulated and downregulated eccDNAs related exon region genes. (B). Volcano plot shows the different genes. (C,D). GO biological process classification of genes and top 20 of GO terms enriched in the plot Figure (E–G). KEGG function enrichment analysis.
FIGURE 7
FIGURE 7
Correlation analysis of eccDNAs (A) and ecDNA (B) quantity between AIONFH group and control group.
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