Search Results (42)

Repetitive DNA represents over 50% of the human genome and is an abundant component of circulating cell-free DNA (cfDNA). We previously showed that cfDNA levels and integrity can predict survival in elderly patients with cardiovascular disease. Here, we aimed to clarify whether a low-pass next-generation sequencing (NGS) approach can characterize the repeat content of cfDNA. Considering the bimodal distribution of cfDNA fragment lengths, we examined the occurrence of repetitive DNA subfamilies separately in dinucleosomal (>250 bp) and mononucleosomal (≤250 bp) cfDNA sequences from 24 patients admitted for heart failure. An increase in the relative abundance of Alu repetitive elements was observed in the longer fraction, while alpha satellites were enriched in the mononucleosomal fraction. The relative abundance of Alu, ALR, and L1HS DNA in the dinucleosomal fraction correlated with different prognostic biomarkers, and Alu DNA was negatively associated with the presence of chronic kidney disease comorbidity. These results, together with the observed inverse correlation between Alu DNA abundance and cfDNA integrity, suggest that the composition of plasma cfDNA could be determined by multiple mechanisms in different physio-pathological conditions. In conclusion, low-pass NGS is an inexpensive method to analyze the cfDNA repeat landscape and identify new cardiovascular disease biomarkers. Full article

Paraspeckles are nuclear membraneless structures composed of a long non-coding RNA, Nuclear-Enriched-Abundant-Transcript-1, and RNA-binding proteins, which associate with numerous mRNAs. It is therefore believed that their cellular function is to sequester in the nucleus their associated proteins and/or target mRNAs. However, little is known about the molecular determinant in mRNA targets that allows their association to paraspeckles, except that inverted repeats of Alu sequences (IRAlu) present in the 3′UTR of mRNAs may allow this association. While in a previous study we established the list of paraspeckle target RNAs in a rat pituitary cell line, we did not find, however, inverted repeated SINEs, the rat equivalent of primate IRAlus in 3′UTR of these RNAs. By developing a candidate gene strategy, we selected a paraspeckle target gene, namely calreticulin mRNA, and we searched for other potential RNA recruitment elements in its 3′UTR, since 3′UTRs usually contain the sequence recognition for nuclear localization. We found a 15-nucleotide sequence surrounded in 5′ by a C-rich sequence, which is present as a tandem repeat in the 3′UTR of this mRNA and which is involved in the nuclear retention by paraspeckles. As shown by mass spectrometry analysis, 6 proteins bound to the 15-nucleotide sequence are paraspeckle proteins and constitute, therefore, bridging proteins between paraspeckles and target mRNAs. Full article

Background. Despite the high transmissibility of SARS-CoV-2, some individuals remain uninfected despite prolonged exposure to a high viral load, suggesting the involvement of an innate resistance mechanism, possibly underpinned by the host’s genetic factors. The angiotensin-converting enzyme-1 (ACE1), ACE2, and C-C Chemokine Receptor 5 (CCR5) polymorphisms have been shown to influence susceptibility to the infection. In this study, the role of ACE1, ACE2, and CCR5 gene polymorphisms in modulating susceptibility to SARS-CoV-2 infection within the context of intimate contact was evaluated. Methods. A cohort of heterosexual couples from Northern Sardinia, characterized by a homogenous genetic background, was recruited during the initial pandemic wave (March–June 2020). In each couple, one partner (index case) tested positive for SARS-CoV-2 by at least two consecutive independent molecular tests (real-time polymerase chain reaction: RT-PCR) on nasopharyngeal swabs. Bed-sharing partners of SARS-CoV-2 positive index cases, resistant and susceptible to the infection, were genotyped for ACE1 287 bp Alu repeat insertion/deletion (I/D) polymorphism, ACE2 G8790A (rs2285666) variant, and a 32-base pair deletion (Δ32) of CCR5. Resistant and susceptible partners to the infection were compared for polymorphisms. Results. Out of 63 couples, 30 partners acquired SARS-CoV-2 infection, while 33 remained uninfected despite intimate exposure. Clinical history was minimal for current or past illnesses. SARS-CoV-2-infected index spouses and partners who acquired the infection developed a mild disease, not requiring hospitalization. The observed distribution of ACE1 I/D and ACE2 G8790A genotypes was consistent with previously reported frequencies in Sardinia and across European populations. None of the study participants carried the CCR5-Δ32 variant. No statistically significant differences (p > 0.05) in the allelic or genotypic frequencies of these polymorphisms were observed between the infected and resistant partners. Conclusions. No differences in the distribution of ACE1, ACE2, and CCR5 polymorphisms between the two groups were detected. These findings suggest that resistance is likely multifactorial, involving a complex interplay of genetic, immunological, and environmental factors. Full article

The double-stranded RNA editing enzyme ADAR1 connects two forms of genetic programming, one based on codons and the other on flipons. ADAR1 recodes codons in pre-mRNA by deaminating adenosine to form inosine, which is translated as guanosine. ADAR1 also plays essential roles in the immune defense against viruses and cancers by recognizing left-handed Z-DNA and Z-RNA (collectively called ZNA). Here, we review various aspects of ADAR1 biology, starting with codons and progressing to flipons. ADAR1 has two major isoforms, with the p110 protein lacking the p150 Zα domain that binds ZNAs with high affinity. The p150 isoform is induced by interferon and targets ALU inverted repeats, a class of endogenous retroelement that promotes their transcription and retrotransposition by incorporating Z-flipons that encode ZNAs and G-flipons that form G-quadruplexes (GQ). Both p150 and p110 include the Zβ domain that is related to Zα but does not bind ZNAs. Here we report strong evidence that Zβ binds the GQ that are formed co-transcriptionally by ALU repeats and within R-loops. By binding GQ, ADAR1 suppresses ALU-mediated alternative splicing, generates most of the reported nonsynonymous edits and promotes R-loop resolution. The recognition of the various alternative nucleic acid conformations by ADAR1 connects genetic programming by flipons with the encoding of information by codons. The findings suggest that incorporating G-flipons into editmers might improve the therapeutic editing efficacy of ADAR1. Full article

Circular RNAs (circRNAs) are a class of unique transcripts characterized by a covalently closed loop structure, which differentiates them from conventional linear RNAs. The formation of circRNAs occurs co-transcriptionally and post-transcriptionally through a distinct type of splicing known as back-splicing, which involves the formation of a head-to-tail splice junction between a 5′ splice donor and an upstream 3′ splice acceptor. This process, along with exon skipping, intron retention, cryptic splice site utilization, and lariat-driven intron processing, results in the generation of three main types of circRNAs (exonic, intronic, and exonic–intronic) and their isoforms. The intricate biogenesis of circRNAs is regulated by the interplay of cis-regulatory elements and trans-acting factors, with intronic Alu repeats and RNA-binding proteins playing pivotal roles, at least in the formation of exonic circRNAs. Various hypotheses regarding pathways of circRNA turnover are forwarded, including endonucleolytic cleavage and exonuclease-mediated degradation; however, similarly to the inconclusive nature of circRNA biogenesis, the process of their degradation and the factors involved remain largely unclear. There is a knowledge gap regarding whether these processes are guided by universal pathways or whether each category of circRNAs requires special tools and particular mechanisms for their life cycles. Understanding these factors is pivotal for fully comprehending the biological significance of circRNAs. This review provides an overview of the various pathways involved in the biogenesis and degradation of different types of circRNAs and explores key factors that have beneficial or adverse effects on the formation and stability of these unique transcripts in higher eukaryotes. Full article

Background/Objective: Large genomic rearrangements of PALB2 gene, particularly deletions and duplications, have been linked to hereditary breast–ovarian cancer. Our research specifically focuses on delineating the intronic breakpoints associated with rearrangements of PALB2 exon 11, which is crucial for understanding the mechanisms underlying these genomic changes in patients with hereditary breast and ovarian syndrome. Methods: By using next-generation sequencing, we identified one duplication and three deletions of PALB2 exon 11, confirmed by Multiplex Ligation-Dependent Probe Amplification analysis. To assess the impact on transcription and potential splicing issues, reverse-transcription PCR was performed on patients’ RNA. For the detailed characterization of intronic breakpoints, the primer walking approach and long-range PCR were implemented, followed by Sanger sequencing. Results: Our analysis revealed a tandem duplication of 5134 base pairs (bp) mediated by AluY repeats located in introns 10 and 11, respectively. Moreover, identical deletions were identified in three unrelated patients, encompassing an approximate 8050 bp region mediated by AluSx elements. Both genomic alterations resulted in a truncated PALB2 protein due to the introduction of a premature stop codon. Conclusions: This study underscores the remarkable instability of intronic regions flanking exon 11 of PALB2 and identifies a previously unreported hotspot involving Alu repeats with very high sequence homology in introns 10 and 11 of the gene. Our findings suggest avenues for further research, such as investigating the prevalence of similar genomic rearrangements in larger cohorts and exploring functional studies to understand how these alterations contribute to hereditary breast cancer pathogenesis. Full article

Background: Global methylation refers to the total methylation in the DNA and can also be inferred from the Line 1 and Alu regions, as these repeats are very abundant in the genome. The main function of DNA methylation is to control gene expression and is associated with both normal and pathological mechanisms. DNA methylation depends on enzymes that generate the methyl radical (e.g., methylenetetrahydrofolate reductase—MTHFR) and attach this radical to the DNA (DNA methyltransferases—DNMT). Genetic variants such as single nucleotide polymorphisms (SNP) in these genes can lead to changes in the activity or expression of MTHFR and DNMT proteins and consequently influence the DNA methylation profile. This review focuses on studies investigating inter-individual variations in the global DNA methylation profile associated with genetic polymorphisms in the MTHFR and DNMT genes. Methods: A narrative review was conducted, taking into account articles published in the last 15 years. Results: It was found that the SNPs rs1801131, rs1801133 and rs1537514 in the MTHFR gene, rs2241531, rs2228611, rs2228612, rs21124724 and the haplotype rs2288349, rs2228611, rs2228612, rs16999593 in the DNMT1 gene, rs2424909, rs998382, rs6058891, rs6058897, rs4911256, rs2889703 and rs1883729 in the DNMT3B were associated with the level of global DNA methylation, including LINE and Alu regions in different contexts. No association was found with polymorphisms in the DNMT3A gene. Conclusions: It is concluded that polymorphisms in the MTHFR and DNMT genes may influence the global DNA methylation profile in health, inflammation, tumours and mental illness. Full article

Tuberous sclerosis complex (TSC) is an autosomal dominant neurodevelopmental disorder and multisystem disease caused by pathogenic DNA alterations in the TSC1 and TSC2 tumor suppressor genes. A molecular genetic diagnosis of TSC confirms the clinical diagnosis, facilitating the implementation of appropriate care and surveillance. TSC1 and TSC2 encode the core components of the TSC1/2 complex (TSC1/2), a negative regulator of the mechanistic target of rapamycin (MTOR) complex 1 (TORC1). Functional analysis of the effects of TSC1 and TSC2 variants on TORC1 activity can help establish variant pathogenicity. We searched for pathogenic alterations to TSC1 and TSC2 in DNA isolated from 116 individuals with a definite clinical diagnosis of TSC. Missense variants and in-frame deletions were functionally assessed. Pathogenic DNA alterations were identified in 106 cases (91%); 18 (17%) in TSC1 and 88 (83%) in TSC2. Of these, 35 were novel. Disruption of TSC1/2 activity was demonstrated for seven TSC2 variants. Molecular diagnostics confirms the clinical diagnosis of TSC in a large proportion of cases. Functional assessment can help establish variant pathogenicity and is a useful adjunct to DNA analysis. Full article

Liquid biopsies are revolutionizing the detection and management of malignant diseases. While repetitive DNA sequences, such as LINE-1 and ALU are established in cell-free DNA (cfDNA) research, their clinical applications remain limited. In this study, we explore human satellite 2 (HSATII), a prevalent repeat DNA sequence in plasma that exhibits increased levels in cancer patients, thereby positioning it as a potential pan-cancer biomarker. We employed targeted sequencing and copy number variation (CNV) analysis using two primer pairs to assess the differential abundance of HSATII sequences in the plasma of breast cancer patients compared to healthy individuals. PCR amplicons of HSATII from 10 patients and 10 control subjects were sequenced, generating 151 bp paired-end reads. By constructing a pooled reference dataset, HSATII copy ratios were estimated in the patients. Our analysis revealed several significant CNVs in HSATII, with certain sequences displaying notable gains and losses across all breast cancer patients, suggesting their potential as biomarkers. However, we observed pronounced fragmentation of cfDNA in cancer, leading to the loss of longer PCR amplicons (>180 bp). While not all observed losses can be attributed to fragmentation artifacts, this phenomenon does introduce complexity in interpreting CNV data. Notably, this research marks the first instance of targeted HSATII sequencing in a liquid biopsy context. Our findings lay the groundwork for developing sequencing-based assays to detect differentially represented HSATII sequences, potentially advancing the field of minimally-invasive cancer screening. Full article

Complex structural chromosome abnormalities such as chromoanagenesis have been reported in acute myeloid leukemia (AML). They are usually not well characterized by conventional genetic methods, and the characterization of chromoanagenesis structural abnormalities from short-read sequencing still presents challenges. Here, we characterized complex structural abnormalities involving chromosomes 2, 3, and 7 in an AML patient using an integrated approach including CRISPR/Cas9-mediated nanopore sequencing, mate pair sequencing (MPseq), and SNP microarray analysis along with cytogenetic methods. SNP microarray analysis revealed chromoanagenesis involving chromosomes 3 and 7, and a pseudotricentric chromosome 7 was revealed by cytogenetic methods. MPseq revealed 138 structural variants (SVs) as putative junctions of complex rearrangements involving chromosomes 2, 3, and 7, which led to 16 novel gene fusions and 33 truncated genes. Thirty CRISPR RNA (crRNA) sequences were designed to map 29 SVs, of which 27 (93.1%) were on-target based on CRISPR/Cas9 crRNA nanopore sequencing. In addition to simple SVs, complex SVs involving over two breakpoints were also revealed. Twenty-one SVs (77.8% of the on-target SVs) were also revealed by MPseq with shared SV breakpoints. Approximately three-quarters of breakpoints were located within genes, especially intronic regions, and one-quarter of breakpoints were intergenic. Alu and LINE repeat elements were frequent among breakpoints. Amplification of the chromosome 7 centromere was also detected by nanopore sequencing. Given the high amplification of the chromosome 7 centromere, extra chromosome 7 centromere sequences (tricentric), and more gains than losses of genomic material, chromoanasynthesis and chromothripsis may be responsible for forming this highly complex structural abnormality. We showed this combination approach’s value in characterizing complex structural abnormalities for clinical and research applications. Characterization of these complex structural chromosome abnormalities not only will help understand the molecular mechanisms responsible for the process of chromoanagenesis, but also may identify specific molecular targets and their impact on therapy and overall survival. Full article

The specific characteristics of k-mer words (2 ≤ k ≤ 11) regarding genomic distribution and evolutionary conservation were recently found. Among them are, in high abundance, words with a tandem repeat structure (repeat unit length of 1 bp to 3 bp). Furthermore, there seems to be a class of extremely short tandem repeats (≤12 bp), so far overlooked, that are non-random-distributed and, therefore, may play a crucial role in the functioning of the genome. In the following article, the positional distributions of these motifs we call super-short tandem repeats (SSTRs) were compared to other functional elements, like genes and retrotransposons. We found length- and sequence-dependent correlations between the local SSTR density and G+C content, and also between the density of SSTRs and genes, as well as correlations with retrotransposon density. In addition to many general interesting relations, we found that SINE Alu has a strong influence on the local SSTR density. Moreover, the observed connection of SSTR patterns to pseudogenes and -exons might imply a special role of SSTRs in gene expression. In summary, our findings support the idea of a special role and the functional relevance of SSTRs in the genome. Full article

Cell responses are usually viewed as transitive events with fixed inputs and outputs that are regulated by feedback loops. In contrast, directed cycles (DCs) have all nodes connected, and the flow is in a single direction. Consequently, DCs can regenerate themselves and implement intransitive logic. DCs are able to couple unrelated chemical reactions to each edge. The output depends upon which node is used as input. DCs can also undergo selection to minimize the loss of thermodynamic entropy while maximizing the gain of information entropy. The intransitive logic underlying DCs enhances their programmability and impacts their evolution. The natural selection of DCs favors the persistence, adaptability, and self-awareness of living organisms and does not depend solely on changes to coding sequences. Rather, the process can be RNA-directed. I use flipons, nucleic acid sequences that change conformation under physiological conditions, as a simple example and then describe more complex DCs. Flipons are often encoded by repeats and greatly increase the Kolmogorov complexity of genomes by adopting alternative structures. Other DCs allow cells to regenerate, recalibrate, reset, repair, and rewrite themselves, going far beyond the capabilities of current computational devices. Unlike Turing machines, cells are not designed to halt but rather to regenerate. Full article

We have developed a new method for promoter sequence classification based on a genetic algorithm and the MAHDS sequence alignment method. We have created four classes of human promoters, combining 17,310 sequences out of the 29,598 present in the EPD database. We searched the human genome for potential promoter sequences (PPSs) using dynamic programming and position weight matrices representing each of the promoter sequence classes. A total of 3,065,317 potential promoter sequences were found. Only 1,241,206 of them were located in unannotated parts of the human genome. Every other PPS found intersected with either true promoters, transposable elements, or interspersed repeats. We found a strong intersection between PPSs and Alu elements as well as transcript start sites. The number of false positive PPSs is estimated to be 3 × 10⁻⁸ per nucleotide, which is several orders of magnitude lower than for any other promoter prediction method. The developed method can be used to search for PPSs in various eukaryotic genomes. Full article

A large body of evidence indicates that environmental agents can induce alterations in DNA methylation (DNAm) profiles. Radiofrequency electromagnetic fields (RF-EMFs) are radiations emitted by everyday devices, which have been classified as “possibly carcinogenic”; however, their biological effects are unclear. As aberrant DNAm of genomic repetitive elements (REs) may promote genomic instability, here, we sought to determine whether exposure to RF-EMFs could affect DNAm of different classes of REs, such as long interspersed nuclear elements-1 (LINE-1), Alu short interspersed nuclear elements and ribosomal repeats. To this purpose, we analysed DNAm profiles of cervical cancer and neuroblastoma cell lines (HeLa, BE(2)C and SH-SY5Y) exposed to 900 MHz GSM-modulated RF-EMF through an Illumina-based targeted deep bisulfite sequencing approach. Our findings showed that radiofrequency exposure did not affect the DNAm of Alu elements in any of the cell lines analysed. Conversely, it influenced DNAm of LINE-1 and ribosomal repeats in terms of both average profiles and organisation of methylated and unmethylated CpG sites, in different ways in each of the three cell lines studied. Full article

Two related tumor suppressor genes, BRCA1 and BRCA2, attract a lot of attention from both fundamental and clinical points of view. Oncogenic hereditary mutations in these genes are firmly linked to the early onset of breast and ovarian cancers. However, the molecular mechanisms that drive extensive mutagenesis in these genes are not known. In this review, we hypothesize that one of the potential mechanisms behind this phenomenon can be mediated by Alu mobile genomic elements. Linking mutations in the BRCA1 and BRCA2 genes to the general mechanisms of genome stability and DNA repair is critical to ensure the rationalized choice of anti-cancer therapy. Accordingly, we review the literature available on the mechanisms of DNA damage repair where these proteins are involved, and how the inactivating mutations in these genes (BRCAness) can be exploited in anti-cancer therapy. We also discuss a hypothesis explaining why breast and ovarian epithelial tissues are preferentially susceptible to mutations in BRCA genes. Finally, we discuss prospective novel therapeutic approaches for treating BRCAness cancers. Full article