Microfluidic isolation of extrachromosomal circular DNA through selective digestion of plasmids and linear DNA using immobilized nucleases.

Extrachromosomal circular DNA (eccDNA) refers to small circular DNA molecules that are distinct from chromosomal DNA and play diverse roles in various biological processes. They are also explored as potential biomarkers for disease diagnosis and precision medicine. However, isolating eccDNA from tissues and plasma is challenging due to low abundance and the presence of interfering linear DNA, requiring time-consuming processes and expert handling. Our study addresses this by utilizing a microfluidic chip tailored for eccDNA isolation, leveraging microfluidic principles for enzymatic removal of non-circular DNA. Our approach involves integrating restriction enzymes into the microfluidic chip, enabling selective digestion of mitochondrial and linear DNA fragments while preserving eccDNA integrity. This integration is facilitated by an in situ photo-polymerized emulsion inside microchannels, creating a porous monolithic structure suitable for immobilizing restriction and exonuclease enzymes (restriction enzyme MssI and exonuclease ExoV). Evaluation using control DNA mixtures and plasma samples with artificially introduced eccDNA demonstrated that our microfluidic chips reduce linear DNA by over 99%, performing comparable to conventional off-chip methods but with substantially faster digestion times, allowing for a remarkable 76-fold acceleration in overall sample preparation time. This technological advancement holds great promise for enhancing the isolation and analysis of eccDNA from tissue and plasma and the potential for increasing the speed of other molecular methods with multiple enzymatic steps.

Methods for the purification and detection of single nucleotide KRAS mutations on extrachromosomal circular DNA in human plasma.

BACKGROUNDS: Despite recent advances, many cancers are still detected too late for curative treatment. There is, therefore, a need for the development of new diagnostic methods and biomarkers. One approach may arise from the detection of extrachromosomal circular DNA (eccDNA), which is part of cell-free DNA in human plasma. AIMS: First, we assessed and compared two methods for the purification of eccDNA from plasma. Second, we tested for an easy diagnostic application of eccDNA liquid biopsy-based assays. MATERIALS & METHODS: For the comparison we tested a solid-phase silica purification method and a phenol/chloroform method with salt precipitation. For the diagnostic application of eccDNA we developed and tested a qPCR primer-based SNP detection system, for the detection of two well-established cancer-causing KRAS mutations (G12V and G12R) on circular DNA. This investigation was supported by purifying, sequencing, and analysing clinical plasma samples for eccDNAs containing KRAS mutant alleles in 0.5 mL plasma from 16 pancreatic ductal adenocarcinoma patients and 19 healthy controls. RESULTS: In our method comparison we observed, that following exonuclease treatment a lower eccDNA yield was found for the phenol/chloroform method (15.7%-26.7%) compared with the solid-phase purification approach (47.8%-65.9%). For the diagnostic application of eccDNA tests, the sensitivity of the tested qPCR assay only reached ~10-3 in a background of 105 wild type (wt) KRAS circular entities, which was not improved by general amplification or primer-based inhibition of wt KRAS amplification. Furthermore, we did not detect eccDNA containing KRAS in any of the clinical samples. DISCUSSION: A potential explanation for our inability to detect any KRAS mutations in the clinical samples may be related to the general low abundance of eccDNA in plasma. CONCLUSION: Taken together our results provide a benchmark for eccDNA purification methods while raising the question of what is required for the optimal fast and sensitive detection of SNP mutations on eccDNA with greater sensitivity than primer-based qPCR detection.

Targeted removal of mitochondrial DNA from mouse and human extrachromosomal circular DNA with CRISPR-Cas9.

Extrachromosomal circular DNA (eccDNA) of chromosomal origin is common in eukaryotic cells. Amplification of oncogenes on large eccDNA (ecDNA) can drive biological processes such as tumorigenesis, and identification of eccDNA by sequencing after removal of chromosomal DNA is therefore important for understanding their impact on the expressed phenotype. However, the circular mitochondrial DNA (mtDNA) might challenge the detection of eccDNA because the average somatic cell has hundreds of copies of mtDNA. Here we show that 61.2-99.5% of reads from eccDNA-enriched samples correspond to mtDNA in mouse tissues. We have developed a method to selectively remove mtDNA from total circular DNA by CRISPR/Cas9 guided cleavage of mtDNA with one single-guide RNA (sgRNA) or two sgRNAs followed by exonuclease degradation of the linearized mtDNA. Sequencing revealed that mtDNA reads were 85.9% ± 12.6% removed from eccDNA of 9 investigated mouse tissues. CRISPR/Cas9 cleavage also efficiently removed mtDNA from a human HeLa cell line and colorectal cancer samples. We identified up to 14 times more, and also larger eccDNA in CRISPR/Cas9 treated colorectal cancer samples than in untreated samples. We foresee that the method can be applied to effectively remove mtDNA from any eukaryotic species to obtain higher eccDNA yields.

Telomere length and mitochondrial DNA copy number in multidrug-resistant tuberculosis.

Multidrug resistant tuberculosis (MDR-TB) is a severe disease that requires prolonged chemotherapy and is associated with an increased probability of treatment failure and death. MDR-TB is a state of heightened oxidative stress and inflammation, which could be related to the aging-related processes and immunosenescence. We, therefore, tested the hypothesis that MDR-TB is associated with alterations in aging biomarkers in peripheral blood cells. We investigated 51 MDR-TB patients and 57 healthy individuals and carried out an analysis of covariance to assess the possible impact of different variables on biomarker perturbations. The results showed that MDR-TB patients had significantly reduced telomere length (TL) and increased mitochondrial DNA copy number (mtDNA CN) (P < 0.05) in comparison to the controls, and MDR-TB infection was the main influencing factor. Male sex and extrapulmonary TB strongly influenced mtDNA CN increment, and MDR-TB patients with normal weight had longer telomeres than those who were underweight (P < 0.05). In conclusion, the evidence for shorter telomeres and higher mtDNA CN in the peripheral blood cells of MDR-TB patients was obtained indicating the connection between MDR-TB and aging biomarkers. The observed associations highlight a complicated interplay between MDR-TB and immunosenescence, thus further studies are required to achieve full understanding.

SORLA Expression in Synaptic Plexiform Layers of Mouse Retina.

Sorting protein-related receptor containing LDLR class A repeats (SORLA; also known as LR11) exerts intraneuronal trafficking functions in the central nervous system. Recently, involvement of SORLA in retinogenesis was proposed, but no studies have examined yet in detail the expression pattern of this sorting receptor in the retina. Here, we provide a spatio-temporal characterization of SORL1 mRNA and its translational product SORLA in the postnatal mouse retina. Using stereological analysis, we confirmed previous studies showing that receptor depletion in knockout mice significantly reduces the number of cells in the inner nuclear layer (INL), suggesting that functional SORLA expression is essential for the development of this retinal strata. qPCR and Western blot analyses showed that SORL1/SORLA expression peaks at postnatal day 15, just after eye opening. Interestingly, we found that transcripts are somatically located in several neuronal populations residing in the INL and the ganglion cell layer, whereas SORLA protein is also present in the synaptic plexiform layers. In line with receptor expression in dendritic terminals, we found delayed stratification of the inner plexiform layer in knockout mice, indicating an involvement of SORLA in neuronal connectivity. Altogether, these data suggest a novel role of SORLA in synaptogenesis. Receptor dysfunctions may be implicated in morphological and functional impairments of retinal inner layer formation associated with eye disorders.

Mitochondrial DNA copy number and telomere length in peripheral blood mononuclear cells in comparison with whole blood in three different age groups.

There are more and more studies on telomere length (TL) and mitochondrial DNA (mtDNA), and it has been proven that these factors play a significant role in the aging of the immune system thereby it is important to understand how it varies in different cell types for more accurate conclusions. The aim of this study was to look into dynamics of mtDNA amount in conjunction with TL in peripheral blood mononuclear cells (PBMC) during aging in comparison with whole blood (WB) cells. Overall, 53 samples were divided into three age groups: 20-39 year age group, 40-59 year age group and 60-79 year age group. MtDNA amount was determined by qPCR TaqMan, and TL was measured by Southern blotting of terminal restriction fragments (TRFs). PBMC had much higher mtDNA copy number (CN) amount than WB samples. Furthermore, with age, it increased in PBMC, while in WB mtDNA CN count did not change. TL in the elderly group was shorter in PBMC fraction than in WB cells. It also looked like that in PBMC TL shortened faster than in WB. In conclusions, it appears that during the aging process both mtDNA CN and TL were more stable in WB than in PBMC fraction where changes were more drastically pronounced, but more studies using larger sample cohorts should be performed to confirm this observation.

The prevalence of mitochondrial mutations associated with aminoglycoside-induced deafness in ethnic Latvian population: the appraisal of the evidence.

Aminoglycosides are potent antibiotics which are used to treat severe gram-negative infections, neonatal sepsis, and multidrug-resistant tuberculosis. Ototoxicity is a well-known side effect of aminoglycosides, and a rapid, profound, and irreversible hearing loss can occur in predisposed individuals. MT-RNR1 gene encoding the mitochondrial ribosomal 12S subunit is a hot spot for aminoglycoside-induced hearing loss mutations, however, a variability in the nature and frequency of genetic changes in different populations exists. The objective of this study was to analyze MT-RNR1 gene mutations in a Baltic-speaking Latvian population, and to estimate the prevalence of such genetic changes in the population-specific mitochondrial haplogroups. In the cohort of 191 ethnic non-related Latvians, the presence of two deafness-associated mutations, m.1555A>G and m.827A>G, three potentially pathogenic variations, m.961insC(n), m.961T>G and m.951G>A, and one unknown substitution, m961T>A was detected, and the aggregate frequency of all variants was 7.3%. All genetic changes were detected in samples belonged to the haplogroups H, U, T, and J. The presence of several aminoglycoside ototoxicity-related MT-RNR1 gene mutations in Baltic-speaking Latvian population indicates the necessity to include ototoxicity-related mutation analysis in the future studies in order to determine the feasibility of DNA screening for patients before administration of aminoglycoside therapy.

Mitochondria, its DNA and telomeres in ageing and human population.

In the last decades, studies about ageing have become more essential as our population grows older. The incidence of age-related diseases increases, which pose challenges both for societies and individuals in terms of life quality and economic impact. Understanding ageing and ageing-related processes will help us to slow down or even prevent these diseases and provide opportunities for healthy ageing; additionally, we all want to live longer. Ageing is a consequence of the interaction between processes that occur over time and genetics interacting with various disease states and an individual's lifestyle. There are several hallmarks of ageing that are generally accepted, but neither of the theories appears to be fully satisfactory. The focus of this article is on two theories of ageing: telomere shortening and mitochondrial DNA (mtDNA) alterations and dysfunction. We discuss characteristic molecular features such as mitochondrial haplogroups, telomere length, mtDNA copy number and heteroplasmy, and how all these traits come together in the ageing population. The recent evidence shows the existence of a strong linkage between these two theories suggesting common molecular mechanisms and a complicated telomere-mitochondria interplay during the humans' ageing. However, this relationship is still not completely understood, which is why it needs more attention.

Linkage between mitochondrial genome alterations, telomere length and aging population.

We studied telomere length (TL) and mitochondrial DNA (mtDNA) copy number variations in individuals from Latvian Caucasian population in different age groups. We showed a positive correlation between TL and mtDNA copy number in individuals of up to 90 years of age; however, this correlation was not observed in the 90-100 years age group. While TL shortened with age and mtDNA content decreased with increasing age, in this study it was observed that mtDNA copy number in nonagenarians was slightly higher than in the 60-89 years age group. The presence of heteroplasmy in the mtDNA HVS-I control region did not correlate with TL and mtDNA copy number. TL and mtDNA values also did not differ between mitochondrial haplogroups. In conclusion, while both TL and mtDNA are involved in the aging process and link between these cell components exists, nonagenarians may have differences in senescence-related pathways and systems, which may function as a protective mechanism that allows them to live longer.

Y-Chromosomal Lineages of Latvians in the Context of the Genetic Variation of the Eastern-Baltic Region.

Variations of the nonrecombining Y-chromosomal region were investigated in 159 unrelated Baltic-speaking ethnic Latvians from four different geographic regions, using 28 biallelic markers and 12 short tandem repeats. Eleven different haplogroups (hgs) were detected in a regionally homogeneous Latvian population, among which N1c, R1a, and I1 cover more than 85% of its paternal lineages. When compared its closest geographic neighbors, the composition of the Latvian Y-chromosomal gene pool was found to be very similar to those of Lithuanians and Estonians. Despite the comparable frequency distribution of hg N1c in Latvians and Lithuanians with the Finno-Ugric-speaking populations from the Eastern coast of the Baltic Sea, the observed differences in allelic variances of N1c haplotypes between these two groups are in concordance with the previously stated hypothesis of different dispersal ways of this lineage in the region. More than a third of Latvian paternal lineages belong specifically to a recently defined R1a-M558 hg, indicating an influence from a common source within Eastern Slavic populations on the formation of the present-day Latvian Y-chromosome gene pool.

Comparison of telomere length between population-specific mitochondrial haplogroups among different age groups in a Latvian population.

Population studies have demonstrated that telomere length (TL) displays great diversity among different populations. Previously described controversial findings associated longevity with specific mitochondrial DNA haplogroups (hgs) (e.g., J and U). These observations may be influenced by population diversity, geographic location, and/or specific historic background. The aims of this study were to identify a specific hg which correlates with aging in a Latvian populating and to evaluate the possible association of TL variability with specific mitochondrial hgs. The results show no significant correlation between TL, mitochondrial DNA hgs and longevity. A slight increase in frequency was observed among centenarians of hg H; however, these findings were not statistically significant. TL did not show any statically significant difference, only hg W had slightly longer telomeres among others. An insignificant increase in TL was observed in the 55-89 age group of hg W but in the <90 age group for hg J which also had the longest TL in the 20-45 age group. In conclusion this study indicates that specific mitochondrial DNA hgs do not have a significant, if any, influence on the variation of TL in Latvians.

Point mutations associated with Leber hereditary optic neuropathy in a Latvian population.

PURPOSE: To study mutations associated with Leber hereditary optic neuropathy (LHON) in patients suspected of having this mitochondrial disorder in a Latvian population. Additional aims were to determine the heteroplasmy status of all non-synonymous polymorphisms identified in the current study and to identify the mitochondrial haplogroups of the studied participants because these factors may contribute to the manifestation of LHON. METHODS: Twelve patients, including patients in two families, were enrolled in the current study. LHON was suspected based on the findings of ophthalmologic examinations. In clinically affected individuals, the presence of all previously reported LHON-associated mutations was assessed with sequencing analysis. Additionally, the SURVEYOR endonuclease assay was used to detect heteroplasmy. The mitochondrial haplogroups were identified with restriction analysis and the sequencing of hypervariable segment 1. RESULTS: In one family (mother and son), there was one primary LHON-associated mutation, G11778A. In addition, one rare previously reported LHON-associated polymorphism, A13637G, was detected in two unrelated patients. A non-synonymous polymorphism at T6253C was found in one individual. This mutation was reported in the background of the 3460 mutation among LHON patients in a Chinese population. No non-synonymous point mutations in mitochondrial DNA were found in five of the study participants. CONCLUSIONS: Molecular analysis of 12 patients with suspected LHON confirmed the diagnosis in four patients and allowed the use of appropriate prophylactic measures and treatment. Further investigations and additional studies of different populations are necessary to confirm the role of the non-synonymous polymorphisms A13637G and T6253C in the manifestation of LHON and the associations of these polymorphisms with mitochondrial haplogroups and heteroplasmy.

Dynamics of telomere length in different age groups in a Latvian population.

The shortening of telomeres with ageing is a well-documented observation; however, the reported number of nucleotides in telomeres varies between different laboratories and studies. Such variability is likely caused by ethnic differences between the populations studied. Until now, there were no studies that investigated the variability of telomere length in a senescent Latvian population of the most common mitochondrial haplogroups, defined as H (45%), U (25%), Y chromosomal N1c (40%) and R1a1 (40%). Telomere length was determined in 121 individuals in different age groups, including a control group containing individuals of 20-40 years old and groups of individuals between 60-70 years old, 71-80 years old, 81-90 years old, and above 90 years old. Telomere length was determined using the Southern blot telomeric restriction fragment assay (TRF). Decreased telomere length with ageing was confirmed, but a comparison of centenarians and individuals between 60-90 years of age did not demonstrate a significant difference in telomere length. However, significant variability in telomere length was observed in the control group, indicating probable rapid telomere shortening in some individuals that could lead up to development of health status decline appearing with ageing. Telomere length measured in mononuclear blood cells (MNC) was compared with the telomere length measured in whole peripheral white blood cells (WBC) using TRF. Telomere length in MNC was longer than in WBC for the control group with individuals 20 to 40 years old; in contrast, for the group of individuals aged 65 to 85 years old, measured telomere length was shorter in MNC when compared to WBC.