Alu Repetitive Elements Research Articles

Abstract A007: DNA methylation analysis comparing high-grade serous ovarian cancer to the normal ovary with adjustment for cell type variation identifies tumor-specific methylation alterations

Abstract A007: DNA methylation analysis comparing high-grade serous ovarian cancer to the normal ovary with adjustment for cell type variation identifies tumor-specific methylation alterations

LDLR gene rearrangements in Czech FH patients likely arise from one mutational event

BackgroundLarge deletions and duplications within the low-density lipoprotein receptor (LDLR) gene make up approximately 10% of LDLR pathogenic variants found in Czech patients with familial hypercholesterolemia. The goal of this study was to test the hypothesis that all probands with each rearrangement share identical breakpoints inherited from a common ancestor and to determine the role of Alu repetitive elements in the generation of these rearrangements.MethodsThe breakpoint sequence was determined by PCR amplification and Sanger sequencing. To confirm the breakpoint position, an NGS analysis was performed. Haplotype analysis of common LDLR variants was performed using PCR and Sanger sequencing.ResultsThe breakpoints of 8 rearrangements within the LDLR gene were analysed, including the four most common LDLR rearrangements in the Czech population (number of probands ranging from 8 to 28), and four less common rearrangements (1–4 probands). Probands with a specific rearrangement shared identical breakpoint positions and haplotypes associated with the rearrangement, suggesting a shared origin from a common ancestor. All breakpoints except for one were located inside an Alu element. In 6 out of 8 breakpoints, there was high homology (≥ 70%) between the two Alu repeats in which the break occurred.ConclusionsThe most common rearrangements of the LDLR gene in the Czech population likely arose from one mutational event. Alu elements likely played a role in the generation of the majority of rearrangements inside the LDLR gene.

Firefighting, per- and polyfluoroalkyl substances, and DNA methylation of genes associated with prostate cancer risk.

Prostate cancer is the leading incident cancer among men in the United States. Firefighters are diagnosed with this disease at a rate 1.21 times higher than the average population. This increased risk may result from occupational exposures to many toxicants, including per- and polyfluoroalkyl substances (PFAS). This study assessed the association between firefighting as an occupation in general or PFAS serum levels, with DNA methylation. Only genomic regions previously linked to prostate cancer risk were selected for analysis: GSTP1, Alu repetitive elements, and the 8q24 chromosomal region. There were 444 male firefighters included in this study, with some analyses being conducted on fewer participants due to missingness. Statistical models were used to test associations between exposures and DNA methylation at CpG sites in the selected genomic regions. Exposure variables included proxies of cumulative firefighting exposures (incumbent versus academy status and years of firefighting experience) and biomarkers of PFAS exposures (serum concentrations of 9 PFAS). Proxies of cumulative exposures were associated with DNA methylation at 15 CpG sites and one region located within FAM83A (q-value <0.1). SbPFOA was associated with 19 CpG sites (q < 0.1), but due to low detection rates, this PFAS was modeled as detected versus not detected in serum. Overall, there is evidence that firefighting experience is associated with differential DNA methylation in prostate cancer risk loci, but this study did not find evidence that these differences are due to PFAS exposures specifically.

Targeted long-read sequencing identifies and characterizes structural variants in cases of inherited platelet disorders

BackgroundGenetic diagnosis of inherited platelet disorders (IPDs) is mainly performed by high-throughput sequencing (HTS). These short-read–based sequencing methods sometimes fail to characterize the genetics of the disease. ObjectivesTo evaluate nanopore long-read DNA sequencing for characterization of structural variants (SVs) in patients with IPDs. MethodsFour patients with a clinical and laboratory diagnosis of Glanzmann thrombasthenia (GT) (P1 and P2) and Hermansky–Pudlak syndrome (HPS) (P3 and P4) in whom HTS missed the underlying molecular cause were included. DNA was analyzed by both standard HTS and nanopore sequencing on a MinION device (Oxford Nanopore Technologies) after enrichment of DNA spanning regions covering GT and HPS genes. ResultsIn patients with GT, HTS identified only 1 heterozygous ITGB3 splice variant c.2301+1G>C in P2. In patients with HPS, a homozygous deletion in HPS5 was suspected in P3, and 2 heterozygous HPS3 variants, c.2464C>T (p.Arg822∗) and a deletion affecting 2 exons, were reported in P4. Nanopore sequencing revealed a complex SV affecting exons 2 to 6 in ITGB3 (deletion-inversion-duplication) in homozygosity in P1 and compound heterozygosity with the splice variant in P2. In the 2 patients with HPS, nanopore defined the length of the SVs, which were characterized at nucleotide resolution. This allowed the identification of repetitive Alu elements at the breakpoints and the design of specific polymerase chain reactions for family screening. ConclusionThe nanopore technology overcomes the limitations of standard short-read sequencing techniques in SV characterization. Using nanopore, we characterized novel defects in ITGB3, HPS5, and HPS3, highlighting the utility of long-read sequencing as an additional diagnostic tool in IPDs.

Impact of genetic structural variants in factor XI deficiency: identification, accurate characterization, and inferred mechanism by long-read sequencing

BackgroundCongenital factor XI (FXI) deficiency is a probably underestimated coagulopathy that confers antithrombotic protection. Characterization of genetic defects in F11 is mainly focused on the identification of single-nucleotide variants and small insertion/deletions because they represent up to 99% of the alterations accounting for factor deficiency, with only 3 gross gene defects of structural variants (SVs) having been described. ObjectivesTo identify and characterize the SVs affecting F11. MethodsThe study was performed in 93 unrelated subjects with FXI deficiency recruited in Spanish hospitals over a period of 25 years (1997-2022). F11 was analyzed by next-generation sequencing, multiplex ligand probe amplification, and long-read sequencing. ResultsOur study identified 30 different genetic variants. Interestingly, we found 3 SVs, all heterozygous: a complex duplication affecting exons 8 and 9, a tandem duplication of exon 14, and a large deletion affecting the whole gene. Nucleotide resolution obtained by long-read sequencing revealed Alu repetitive elements involved in all breakpoints. The large deletion was probably generated de novo in the paternal allele during gametogenesis, and despite affecting 30 additional genes, no syndromic features were described. ConclusionSVs may account for a high proportion of F11 genetic defects implicated in the molecular pathology of congenital FXI deficiency. These SVs, likely caused by a nonallelic homologous recombination involving repetitive elements, are heterogeneous in both type and length and may be de novo. These data support the inclusion of methods to detect SVs in this disorder, with long-read–based methods being the most appropriate because they detect all SVs and achieve adequate nucleotide resolution.

Estimation of ALU Repetitive Elements in Plasma as a Cost-Effective Liquid Biopsy Tool for Disease Prognosis in Breast Cancer

Liquid biopsy is widely recognized as an efficient diagnostic method in oncology for disease detection and monitoring. Though the examination of circulating tumor cells (CTC) is mostly implemented for the assessment of genomic aberrations, the need of complex methodologies for their detection has impeded its acceptance in low-resource settings. We evaluated cell-free DNA (cfDNA) as a liquid biopsy tool and investigated its utility in breast cancer patients. Total cell-free DNA was extracted from the plasma of breast cancer patients (n = 167) with a median follow-up of more than 5 years, at various stages of the disease. Quantitative PCR was performed to estimate the copy numbers of two fractions of ALU repetitive elements (ALU 115 and ALU 247), and DNA integrity (DI) was calculated as the ratio of ALU 247/115. Mutations in TP53 and PIK3CA in the cfDNA were estimated by next-gen sequencing (NGS) in a subset of samples. Associations of the levels of both the ALU fragments with various clinico-pathological factors and disease-free survival at various stages were examined. Nomogram models were constructed with clinical variables and ALU 247 levels to predict disease-free survival and the best performing model was evaluated by decision curve analysis. DI and ALU 247 levels were significantly lower (p < 0.0001) in the post-operative plasma when compared to their pre-surgery levels. DI and ALU 247 were found to be significantly higher in patients with metastasis (p < 0.05). Patients with higher levels of ALU 247 in their post-operative plasma had significant poor disease-free survival (p = 0.005). Higher levels of ALU 247 in the circulation also correlated with low tumor-infiltrating lymphocytes (TIL) within their primary tumors in the ER-negative breast cancer subtype (p = 0.01). Cox proportional hazard analysis confirmed ALU 247 as an independent variable of disease-free survival both in univariate and multivariate analysis [HR 1.3 (95% CI 1.047 to 1.613, p = 0.017)]. The nomogram model showed that the addition of ALU 247 with other variables significantly improved (C-index 0.823) the predictive ability of the model. Our results confirm the utility of cfDNA as an evolving liquid biopsy tool for molecular analysis. Evaluation of larger fragments of cfDNA estimated through ALU 247 can provide vital information concurrent with the pathological process of disease evolution in breast cancer and warrants expansion to other cancer types.

DNA methylation status of interspersed repetitive sequences in patients with migraine.

To analyse the methylation status of the Long Interspersed Nuclear Element-1 (LINE-1) and Short Interspersed Nuclear Element Alu (Alu) of peripheral blood mononuclear cells (PBMCs) from patients with migraine compared with healthy control subjects. This case-control study recruited patients with migraine without aura and age-matched healthy control subjects. PBMCs were purified from peripheral blood samples. Methylation levels and patterns of LINE-1 and Alu sequences were evaluated using combined bisulfite restriction analysis-interspersed repetitive sequences polymerase chain reaction. A total of 84 patients with migraine and 82 age-matched healthy controls were enrolled in the study. High levels of unmethylated cytosines in both the LINE-1 and Alu repetitive elements were observed in the migraine group compared with the control subjects. In addition, a significant difference was detected in the methylation level of LINE-1 between TT and CC genotype groups of the methylenetetrahydrofolate reductase gene. These results suggest that analysis of epigenetic biomarkers in PBMCs may help to identify patients at a higher risk of migraine development.

Multiplexing Cas9 activity with multi-target CRISPR.

We present multi-target CRISPR, an approach whereby a degenerate gRNA directs Cas9 to simultaneously target a large number of epigenetically diverse genomic locations. This multiplexing of CRISPR is enabled by targeting the highly abundant Alu repetitive elements. Combined with different next-generation sequencing readouts, multi-target CRISPR offered new insights into Cas9 activity and the ensuing cellular response. First, by interrogating the same protospacer in different genomic contexts, multi-target CRISPR showed that Cas9 binding is more efficient at open chromatin regions, and Cas9 cleavage is enhanced near transcribed regions. We then combined multi-target CRISPR with ATAC-Seq to unveil an increase in chromatin accessibility around Cas9 DSBs, which spreads over a window of ∼1 kb centered at the cut site. We attribute this chromatin opening to nucleosomes being evicted from the damaged region. Notably, the extent of chromatin opening is independent of the chromatin context in which the lesions occurs, suggesting that nucleosome eviction is a robust part of the DNA damage response. By combining multi-target CRISPR with photo-activatable gRNAs, we determined the characteristic time-scales of DSB-induced chromatin relaxation and found that nucleosome removal occurs within ∼30 min. Last, we combined multi-target CRISPR with a photo-deactivatable gRNA to measure chromatin dynamics after DSB repair completion. Once repair is completed, chromatin accessibility levels returned to their original pre-cleavage state within 1 h, revealing a quick repositioning of nucleosomes around the repaired cut site. Based on these measurements, we propose a model in which chromatin undergoes fast and transient opening in response to Cas9 breaks to facilitate recruitment of repair factors. In conclusion, multi-target CRISPR enables high-throughput studies of Cas9 activity and DNA repair in cellulo, revealing new insights on the interplay between Cas9 activity and chromatin.

Traffic-Related Air Pollution and Ground-Level Ozone Associated Global DNA Hypomethylation and Bulky DNA Adduct Formation.

Studies have indicated that air pollution, including surface-level ozone (O3), can significantly influence the risk of chronic diseases. To better understand the carcinogenic mechanisms of air pollutants and identify predictive disease biomarkers, we examined the association between traffic-related pollutants with DNA methylation alterations and bulky DNA adducts, two biomarkers of carcinogen exposure and cancer risk, in the peripheral blood of 140 volunteers-95 traffic police officers, and 45 unexposed subjects. The DNA methylation and adduct measurements were performed by bisulfite-PCR and pyrosequencing and 32P-postlabeling assay. Airborne levels of benzo(a)pyrene [B(a)P], carbon monoxide, and tropospheric O3 were determined by personal exposure biomonitoring or by fixed monitoring stations. Overall, air pollution exposure was associated with a significant reduction (1.41 units) in global DNA methylation (95% C.I. -2.65-0.04, p = 0.026). The decrement in ALU repetitive elements was greatest in the policemen working downtown (95% C.I. -3.23--0.49, p = 0.008). The DNA adducts were found to be significantly increased (0.45 units) in the municipal officers with respect to unexposed subjects (95% C.I. 0.02-0.88, p = 0.039), mainly in those who were controlling traffic in downtown areas (95% C.I. 0.39-1.29, p < 0.001). Regression models indicated an increment of ALU methylation at higher B(a)P concentrations (95% C.I. 0.03-0.60, p = 0.032). Moreover, statistical models showed a decrement in ALU methylation and an increment of DNA damage only above the cut-off value of 30 µg/m3 O3. A significant increment of 0.73 units of IL-6 gene methylation was also found in smokers with respect to non-smokers. Our results highlighted the role of air pollution on epigenetic alterations and genotoxic effects, especially above the target value of 30 µg/m3 surface-level O3, supporting the necessity for developing public health strategies aimed to reduce traffic-related air pollution molecular alterations.

Circulating cell-free endometrial DNA level is unaltered during menstruation and in endometriosis.

Is circulating cell-free DNA (cirDNA) from the endometrium elevated during menstruation and in endometriosis? Endometrial cirDNA does not increase during menstruation and is not elevated in endometriosis. Changes in cirDNA associated with common benign conditions are a potential source of false positives in cancer diagnostic applications, but also present an opportunity for biomarker development for diseases such as endometriosis. Elevated cirDNA has been reported in endometriosis patients compared to healthy community controls, but no difference in total or endometrial cirDNA has been found between patients with endometriosis and patients with other gynaecological conditions. Likewise, menstruation is a potential driver of changes in cirDNA levels and tissue profile, but total and endothelial cirDNA do not increase during menstruation. For endometriosis comparisons, 59 participants with surgically confirmed endometriosis and 27 laparoscopic patients without endometriosis (hospital controls) were prospectively recruited, while 25 healthy community participants (healthy controls) were recruited in a university setting. Total and endometrial cirDNA and cirDNA fragmentation were measured across the three groups. For menstrual comparisons, 36 matched non-menstruating and menstruating samples were collected from healthy women recruited within a university setting, and the endometrial cirDNA was compared between the two groups. cirDNA was extracted from venous blood plasma then quantitated by quantitative PCR of ALU repetitive element (115 bp) and TP53 gene sequence (105 bp) for total concentration. cirDNA derived from the endometrium was quantitated by methylation-specific droplet digital PCR of a FAM101A region (69 bp) after bisulfite conversion of the DNA. A cirDNA size fragmentation ratio was obtained by quantifying a long segment of ALU repetitive element (247 bp) and expressing the amount relative to the 115 bp ALU target. No differences in cirDNA level were found in any comparison populations in this study. Mean total cirDNA was unchanged between healthy controls (ALU-115-3.31 ng/ml; TP53-2.73 ng/ml), hospital controls (ALU-115-3.47 ng/ml; TP53-2.83 ng/ml) and endometriosis patients (ALU-115-3.35 ng/ml; TP53-2.66 ng/ml). Likewise, endometrial cirDNA was unchanged between healthy controls (18.3 copies/ml), hospital controls (20.6 copies/ml) and endometriosis patients (22 copies/ml). Endometrial cirDNA did not change during menstruation (non-menstruating: 38 copies/ml; menstruating: 33 copies/ml). Irrespective of endometriosis diagnosis, blood from patients undergoing laparoscopy (hospital controls: 0.77; endometriosis patients: 0.79), had a significantly higher cirDNA size ratio than community-recruited healthy controls (0.64), indicating increased abundance of long cirDNA fragments. It was not possible to completely match the age, BMI and parity between the three cohorts investigated, however of these, only age has been shown to influence circulating DNA levels and not within the age range of our cohort. Blood from community-recruited healthy women and women undergoing laparoscopy was collected via antecubital vein venepuncture (processed within 3 h) and with either peripheral cannula or venepuncture (processed within 6 h), respectively, which could potentially impact the size distribution of circulating DNA fragments. For the collection of non-menstruating phase blood samples, we did not differentiate between follicular phase, ovulation and luteal phase. Thus, only the mensturating samples were collected at a consistent phase, and any fluctuations in cirDNA that occur at the other phases may have obscured small changes during menstruation. There is no evidence that cirDNA has potential as a diagnostic biomarker for endometriosis. Endometriosis, representing a common benign gynaecological condition, and menstruation, representing a normal physiological occurrence in women, should not affect methylation-based diagnostics in other disease areas, including oncology. N.L.Y.: Australian Government Research Training Program (RTP) Stipend through The University of New South Wales, Translational Cancer Research Network PhD Scholarship Top-Up Award via the Cancer Institute NSW, Beth Yarrow Memorial Award in Medical Science, UNSW Completion Scholarship; C.E.H.: Gynaecological Oncology Fund of the Royal Hospital for Women; K.W.: Ovarian Cancer Research Foundation and CAMILLA AND MARC. C.E.F.: UNSW Women's Wellbeing Academy and the Australian Human Rights Institute. We declare the following competing interest: K.W. holds stock in Guardant Health, Exact Sciences and Epigenomics AG. No other authors have competing interests. N/A.

Association of Sat-a and Alu methylation status with HCV-induced chronic liver disease and hepatocellular carcinoma

BackgroundThe combination of epigenetic and genetic abnormalities contributes together to the development of liver cancer. The methylation status of the repetitive elements (REs) in DNA has been investigated in a variety of human illnesses. However, the methylation patterns of Sat-α and Alu REs in chronic liver disease (CLD) and hepatocellular carcinoma (HCC) caused by hepatitis C virus (HCV) have never been studied before. MethodologyIn this study, 3 groups of participants including 50 patients having HCV-induced CLD, 50 patients having HCV-induced HCC, and 46 healthy subjects were subjected to measurement of Sat-α and Alu methylation using the quantitative MethyLight assay. ResultsSat-α and Alu methylation percentages decreased significantly in both CLD and HCC, compared to control. Also, a significant Sat-α hypomethylation was detected in HCC, compared to CLD. In addition, Sat-α and Alu methylation showed a significant decline as lesion size grew. However, only Sat-α hypomethylation was significantly increased in association with portal vein thrombosis and the MELD score. Sat-α methylation percentage had the highest sensitivity and specificity for diagnosing HCC (100% and 84.4%) followed by α-fetoprotein (80% and 84.4%) and Alu methylation (66% and 61.5%). Furthermore, there was a strong positive correlation between Sat-α and Alu methylation. ConclusionsMeasuring Sat-α and Alu methylation provides us with a new tool for early detecting HCV-induced CLD and hepatocarcinogenesis. Sat-α has the potential to be utilized as an independent predictive parameter for HCC development and progression because of its ability to distinguish between CLD and HCC with their different MELD scores.

Self or Non-Self? It Is also a Matter of RNA Recognition and Editing by ADAR1

Simple SummaryA fundamental feature of innate immune cells is to detect the presence of non-self, such as potentially harmful nucleic acids, by germline-encoded specialized receptors called pattern recognition receptors (PRRs). ADAR1 is one key enzyme avoiding aberrant type I interferon (IFN-I) production and immune cell activation by the conversion of adenosine to inosine (A-to-I) in double-stranded RNA (dsRNA) structures that arise in self mRNA containing specific repetitive elements. This review intends to give an up-to-date and detailed overview of the ADAR1-mediated ability to modulate the immune response in autoimmune diseases and cancer progression.A-to-I editing is a post-transcriptional mechanism affecting coding and non-coding dsRNAs, catalyzed by the adenosine deaminases acting on the RNA (ADAR) family of enzymes. A-to-I modifications of endogenous dsRNA (mainly derived from Alu repetitive elements) prevent their recognition by cellular dsRNA sensors, thus avoiding the induction of antiviral signaling and uncontrolled IFN-I production. This process, mediated by ADAR1 activity, ensures the activation of an innate immune response against foreign (non-self) but not self nucleic acids. As a consequence, ADAR1 mutations or its de-regulated activity promote the development of autoimmune diseases and strongly impact cell growth, also leading to cancer. Moreover, the excessive inflammation promoted by Adar1 ablation also impacts T and B cell maturation, as well as the development of dendritic cell subsets, revealing a new role of ADAR1 in the homeostasis of the immune system.

Multi-target CRISPR for high-throughput studies of double-strand break repair and genetic barcoding at endogenous sites

CRISPR has revolutionized biology by enabling highly versatile and precise genome editing. In the canonical application of CRISPR, a guide RNA (gRNA) is rationally designed to direct the nuclease to an individual genomic locus. However, for many other applications of CRISPR, it is desirable to target a large number of genomic locations at once. Here, we demonstrate efficient multiplexing of CRISPR using multi-target gRNAs (mgRNAs). By targeting mutations of the Alu repetitive element dispersed throughout the genome, mgRNAs introduce a user-defined number of on-target double-strand breaks (DSBs).

Angiotensin-Converting Enzyme Receptor Genotype and its Activity Level as Potential Predictors of the Severity COVID-19 among Iraqi Patients

Objectives: The purpose of this study is to determine whether the angiotensin-converting enzyme activity and various biomarkers are used to investigate the severity of Covid-19 and to study the genetic variation occurs in angiotensin-converting enzyme-2 (ACE-2) receptor in severe Covid-19-related genes in the Iraqi population of Kerbala Province. Methods: This case-control study was conducted on 176 subjects who survived hospitalization and diagnosed by physician. Various biomarkers including ferritin, C-reactive protein, lactate dehydrogenase, alanine aminotransferase, aspartate aminotransferase, angiotensin-converting enzyme-2 activity levels were determined. Accordingly, they were divided into three groups: 59 of them were infected with severe Covid-19, 54 of them were infected with moderate Covid-19 and 63 of them were checked and obtained as apparently healthy control. Severe and moderate patients were collected from Al-Hayat tertiary center at Al-Hussein Medical City, Kerbala Health Directorates, Kerbala – Iraq during Oct., 2020- July, 2021 with matched age ranged between (23-88) years. Blood samples of apparently healthy and Covid-19 samples were subjected to genomic DNA extraction within 24-48 hours of aspiration. The genomic DNA extracted was subjected to electrophoresis through 1.5% of agarose gels which was detected by staining with the fluorescent dye ethidium bromide and then visualized by illumination with UV light to confirm the presence and integrity of the extracted DNA. Results: Genotyping of ACE-2 (I/D) polymorphism (rs4646994), which has a high prevalence, was performed by polymerase chain reaction assay. The amplification of an Alu repetitive element in an intron of the ACE-2 has shown three potential genotypes of I/I and D/D as homozygous, and I/D as heterozygous. Individuals with normal homozygous (DD) revealed band of (190 bp), while individuals with normal (II) revealed band of (490 bp), and the individuals with heterozygous (ID) revealed two bands (190, 490 bp) respectively. Every severe Covid-19 group carried (DD) allele genotype, moderate group carried (ID and II) alleles and finally the control group carried (DD, ID, II) alleles genotype. Conclusion: In the ACE-2 polymorphism, the D/D genotype allele is implicated as a risk factor for severe Covid-19 patients, in Iraqi population.

Lack of relationship between Alu repetitive elements in angiotensin converting enzyme and the severity of diabetic retinopathy.

BackgroundAngiotensin-converting enzyme (ACE) stimulates angiogenesis that leads to the development of diabetic retinopathy (DR). Alu repetitive elements in ACE gene increase the expression of this enzyme. We investigated the frequency of Alu repetitive elements, insertion/deletion (I/D) polymorphism, in angiotensin-converting enzyme among diabetic retinopathy patients and whether this polymorphism is associated with the severity of retinopathy in Jordanians with type 2 diabetes.MethodsA total of 277 subjects participated in this case/ control study (100 diabetic patients without DR, 82 diabetic patients with DR, and 95 healthy control). Blood samples were withdrawn, followed by DNA extraction. Alu repetitive elements were examined by polymerase chain reaction followed by gel electrophoresis.ResultsThe genotype and allele frequencies among diabetic patients, were close to healthy controls (genotypes, II 44.4 vs. 44.7%, ID 44.4 vs. 42.6%, DD 12.2 vs. 12.8%, P = 0.402 and 0.677 respectively, alleles, I 65.6 vs. 66%, D 34.4 vs. 34%, P=0.863). Complicated diabetics with retinopathy showed similar genotype and allele frequency to those without complications. The severity of diabetic retinopathy in affected individuals was not correlated with I/D polymorphism (P=0.862).ConclusionsWe conclude that the presence of Alu repetitive elements did not increase the development or progression risk to retinopathy in Jordanian type 2 diabetic patients. No association between I or D alleles with the severity of DR was detected.

Relationship of angiotensin converting enzyme (I/D) polymorphism (rs4646994) and coronary heart disease among a male Iraqi population with type 2 diabetes mellitus.

Insertion deletion (I/D) polymorphism (rs4646994) in the angiotensin-converting enzyme (ACE) has a substantial effect on coronary heart disease (CHD). The amplification of an Alu repetitive element in an intron of the ACE has shown three potential genotypes of I/I and D/D as homozygous, and I/D as heterozygous. The objective of this study was to investigate the association between the ACE gene polymorphism and CHD among male Iraqi patients with and without type2 diabetes mellitus (T2DM). A case-control study of total 217 male subjects participated in this study, divided into three groups; Group 1 including 86 CHD patients with T2DM, group 2 including 78 CHD patients without T2DM, and group 3 including 53 age and sex-matched healthy individuals (as a control group). Genotyping of ACE (I/D) gene was performed using polymerase chain reaction (PCR) technique. The II allele was significantly more frequent in CHD patients without T2DM compared to the control population, but not from those patients with T2DM (p < 0.05). Nonetheless, the ID allele was significantly more frequent in each of CHD with T2DM and control populations compared to the CHD without T2DM. The DD allele was significantly more frequent in CHD patients with T2DM compared to each of CHD patients without T2DM and control populations (p < 0.05). We conclude that the D/D and I/D genotypes are implicated as risk factors for development of CHD with T2DM, but not CHD without T2DM among the male Iraqi population. However, larger sample sizes are needed to monitor the CHD patients and to validate this study.

Circulating cell-free DNA variables as marker of ovarian cancer patients: A pilot study.

Minimal invasive blood-based molecular markers are evaluated as promising biomarkers in malignant diseases these days. In this pilot study, we investigated the potential of cell-free DNA (cfDNA) concentration and cell-free DNA Integrity (cfDI) as blood-based diagnostic markers for ovarian cancer patients in a retrospective study cohort. cfDNA concentration and cfDI were determined in the plasma of 37 ovarian cancer patients and 28 healthy controls, by measuring ALU and LINE1 repetitive DNA elements using quantitative real-time PCR. A high correlation was observed between the results of ALU and LINE1. The correlated co-efficiency between the values of cfDNA concentration and cfDI was 0.86 and 0.71. As for the results between cases and controls, no or just borderline significant difference was observed in cfDI after age adjustment (P= 0.40 for ALU and P= 0.05 for LINE1) while cfDNA concentration showed a significant difference between ovarian cancer patients and healthy controls groups (P= 0.03 for ALU and P= 3.00 E-03 for LINE1). cfDNA concentration of ALU and LINE1 had an AUC of 0.81 (0.70-0.91). ALU and LINE1 cfDI reached an AUC of 0.60 (95% CI: 0.46-0.73). The combination of these markers reached the best diagnostic power with an AUC of 0.84. cfDNA variables might be potentially diagnostic biomarkers in ovarian cancer, in combination with additional molecular markers. However, further studies are needed to confirm the diagnostic ability of cfDNA variables (cfDNA concentration and cfDI).

Adenosine-to-inosine RNA editing in the immune system: friend or foe?

Our body expresses sensors to detect pathogens through the recognition of expressed molecules, including nucleic acids, lipids, and proteins, while immune tolerance prevents an overreaction with self and the development of autoimmune disease. Adenosine (A)-to-inosine (I) RNA editing, catalyzed by adenosine deaminases acting on RNA (ADARs), is a post-transcriptional modification that can potentially occur at over 100 million sites in the human genome, mainly in Alu repetitive elements that preferentially form a double-stranded RNA (dsRNA) structure. A-to-I conversion within dsRNA, which may induce a structural change, is required to escape from the host immune system, given that endogenous dsRNAs transcribed from Alu repetitive elements are potentially recognized by melanoma differentiation-associated protein 5 (MDA5) as non-self. Of note, loss-of-function mutations in the ADAR1 gene cause Aicardi-Goutières syndrome, a congenital autoimmune disease characterized by encephalopathy and a type I interferon (IFN) signature. However, the loss of ADAR1 in cancer cells with an IFN signature induces lethality via the activation of protein kinase R in addition to MDA5. This makes cells more sensitive to immunotherapy, highlighting the opposing immune status of autoimmune diseases (overreaction) and cancer (tolerance). In this review, we provide an overview of insights into two opposing aspects of RNA editing that functions as a modulator of the immune system in autoimmune diseases and cancer.

TDP-43 regulates transcription at protein-coding genes and Alu retrotransposons

The 43-kDa transactive response DNA-binding protein (TDP-43) is an example of an RNA-binding protein that regulates RNA metabolism at multiple levels from transcription and splicing to translation. Its role in post-transcriptional RNA processing has been a primary focus of recent research, but its role in regulating transcription has been studied for only a few human genes. We characterized the effects of TDP-43 on transcription genome-wide and found that TDP-43 broadly affects transcription of protein-coding and noncoding RNA genes. Among protein-coding genes, the effects of TDP-43 were greatest for genes <30 thousand base pairs in length. Surprisingly, we found that the loss of TDP-43 resulted in increased evidence for transcription activity near repetitive Alu elements found within expressed genes. The highest densities of affected Alu elements were found in the shorter genes, whose transcription was most affected by TDP-43. Thus, in addition to its role in post-transcriptional RNA processing, TDP-43 plays a critical role in maintaining the transcriptional stability of protein-coding genes and transposable DNA elements.

Discovering human transcription factor physical interactions with genetic variants, novel DNA motifs, and repetitive elements using enhanced yeast one-hybrid assays.

Identifying transcription factor (TF) binding to noncoding variants, uncharacterized DNA motifs, and repetitive genomic elements has been technically and computationally challenging. Current experimental methods, such as chromatin immunoprecipitation, generally test one TF at a time, and computational motif algorithms often lead to false-positive and -negative predictions. To address these limitations, we developed an experimental approach based on enhanced yeast one-hybrid assays. The first variation of this approach interrogates the binding of >1000 human TFs to repetitive DNA elements, while the second evaluates TF binding to single nucleotide variants, short insertions and deletions (indels), and novel DNA motifs. Using this approach, we detected the binding of 75 TFs, including several nuclear hormone receptors and ETS factors, to the highly repetitive Alu elements. Further, we identified cancer-associated changes in TF binding, including gain of interactions involving ETS TFs and loss of interactions involving KLF TFs to different mutations in the TERT promoter, and gain of a MYB interaction with an 18-bp indel in the TAL1 superenhancer. Additionally, we identified TFs that bind to three uncharacterized DNA motifs identified in DNase footprinting assays. We anticipate that these enhanced yeast one-hybrid approaches will expand our capabilities to study genetic variation and undercharacterized genomic regions.