Background: Many studies have demonstrated the high efficacy of cell-free nuclear DNA in cancer diagnostics. Compared to nuclear DNA, mitochondrial DNA (mtDNA) exhibits distinct characteristics, including multiple copies per cell and higher mutation frequency. However, the potential applicability of cell-free mtDNA (cf-mtDNA) in plasma and urine remains poorly investigated.Methods: Here, we comprehensively analyzed the fragmentomic and mutational characteristics of cf-mtDNA in urine and plasma samples from controls and cancer patients using next-generation sequencing.Results: Compared to plasma cf-mtDNA, urine cf-mtDNA exhibited increased copy numbers and wider spread in fragment size distributions. Based on 2 independent animal models, urine cf-mtDNA originated predominantly from local shedding and transrenal excretion. Further analysis indicated an enhanced fragmentation of urine cf-mtDNA in renal cell carcinoma (RCC) and colorectal cancer (CRC) patients. Using the mtDNA sequence of peripheral blood mononuclear cells for reference, the mutant fragments were shorter than wild-type fragments in urine cf-mtDNA. Size selection of short urine cf-mtDNA fragments (<150 bp) significantly enhanced the somatic mutation detection. Our data revealed remarkably different base proportions of fragment ends between urine and plasma cf-mtDNA that also were associated with fragment size. Moreover, both RCC and CRC patients exhibited significantly higher T-end and lower A-end proportions in urine cf-mtDNA than controls. By integrating the fragmentomic and mutational features of urine cf-mtDNA, our nomogram model exhibited a robust efficacy for cancer diagnosis.Conclusions: Our proof-of-concept findings revealed aberrant fragmentation and mutation profiles of urine cf-mtDNA in cancer patients that have diagnostic potential.

Phosphodiesterase 2 (PDE2A) modulates the levels of cAMP/cGMP and was recently found to be involved in mitochondria function regulation, closely related to multiple types of tumor progression. This study aimed to estimate the prognostic significance and biological effects of PDE2A on hepatocellular carcinoma (HCC). We comprehensively analyzed the PDE2A mRNA expression in HCC based on The Cancer Genome Atlas (TCGA) database and investigated the effects of PDE2A on the proliferation and metastatic capacity of HCC cells. PDE2A was downregulated in 25 cancer types, including HCC. Lower PDE2A expression was a protective factor in HCC and was negatively associated with serum AFP levels, tumor status, vascular invasion, histologic grade, and pathologic stage of HCC. Moreover, tumors with low PDE2A expression displayed a decreased immune function. Then, the ROC curve was used to assess the diagnostic ability of PDE2A in HCC (AUC = 0.823 in TCGA and AUC = 0.901 in GSE76427). Patients with low PDE2A expression exhibited worse outcomes compared with those with high PDE2A expression. Additionally, GO functional annotations demonstrated the involvement of PDE2A in the ECM organization, systems development, and ERK-related pathways, indicating that PDE2A might regulate HCC growth and metastasis. The in vitro experiments confirmed that overexpression of PDE2A inhibited proliferation, colony formation, migration, and invasion in two HCC cell lines (HLF and SNU-368), while inhibition of PDE2A has the opposite results. The mechanism of PDE2A’s effect on HCC cells is attributed to the change of mitochondrial morphology and ATP content. These data demonstrated that PDE2A closely participated in the regulation of HCC proliferation and metastasis and can be used as a predictive marker candidate and a potential therapeutic target for HCC.

Mitochondrial genome abnormalities can lead to mitochondrial dysfunction, which in turn affects cellular biology and is closely associated with the development of various diseases. The demand for mitochondrial DNA (mtDNA) sequencing has been increasing, and Illumina and MGI are two commonly used sequencing platforms for capture-based mtDNA sequencing. However, there is currently no systematic comparison of mtDNA sequencing performance between these two platforms. To address this gap, we compared the performance of capture-based mtDNA sequencing between Illumina's NovaSeq 6000 and MGI's DNBSEQ-T7 using tissue, peripheral blood mononuclear cell (PBMC), formalin-fixed paraffin-embedded (FFPE) tissue, plasma, and urine samples. Our analysis indicated a high degree of consistency between the two platforms in terms of sequencing quality, GC content, and coverage. In terms of data output, DNBSEQ-T7 showed higher rates of clean data and duplication compared to NovaSeq 6000. Conversely, the amount of mtDNA data obtained by per gigabyte sequencing data was significantly lower in DNBSEQ-T7 compared to NovaSeq 6000. In terms of detection mtDNA copy number, both platforms exhibited good consistency in all sample types. When it comes to detection of mtDNA mutations in tissue, FFPE, and PBMC samples, the two platforms also showed good consistency. However, when detecting mtDNA mutations in plasma and urine samples, significant differenceof themutation number detected was observed between the two platforms. For mtDNA sequencing of plasma and urine samples, a wider range of DNA fragment size distribution was found in NovaSeq 6000 when compared to DNBSEQ-T7. Additionally, two platforms exhibited different characteristics of mtDNA fragment end preference. In summary, the two platforms generally showed good consistency in capture-based mtDNA sequencing. However, it is necessary to consider the data preferences generated by two sequencing platforms when plasma and urine samples were analyzed.

Mitochondria are central eukaryotic organelles in cellular metabolism and ATP production. Mitochondrial DNA (mtDNA) alterations have been implicated in the development of colorectal cancer (CRC). However, there are few reports on the association between mtDNA haplogroups or single nucleotide polymorphisms (SNPs) and the risk of CRC. The mtDNA of 286 Northern Han Chinese CRC patients were sequenced by next-generation sequencing technology. MtDNA data from 811 Han Chinese population controls were collected from two public data sets. Then, logistic regression analysis was used to determine the effect of mtDNA haplogroup or SNP on the risk of CRC. We found that patients with haplogroup M7 exhibited a reduced risk of CRC when compared to patients with other haplogroups (odds ratio [OR] = 0.532, 95% confidence interval [CI] = 0.285–0.937, p = 0.036) or haplogroup B (OR = 0.477, 95% CI = 0.238–0.916, p = 0.030). Furthermore, haplogroup M7 was still associated with the risk of CRC when the validation and combined control cohort were used. In addition, several haplogroup M7 specific SNPs, including 199T>C, 4071C>T and 6455C>T, were significantly associated with the risk of CRC. Our results indicate the risk potential of mtDNA haplogroup M7 and SNPs in CRC in Northern China.