Molecular and Clinical Implications of Variant Repeats in Myotonic Dystrophy Type 1

Myotonic dystrophy type 1 (DM1) is one of the most variable monogenic diseases at phenotypic, genetic, and epigenetic level. The disease is multi-systemic with the age at onset ranging from birth to late age. The underlying mutation is an unstable expansion of CTG repeats in the DMPK gene, varying in size from 50 to >1000 repeats. Generally, large expansions are associated with an earlier age at onset. Additionally, the most severe, congenital DM1 form is typically associated with local DNA methylation. Genetic variability of DM1 mutation is further increased by its structural variations due to presence of other repeats (e.g., CCG, CTC, CAG). These variant repeats or repeat interruptions seem to confer an additional level of epigenetic variability since local DNA methylation is frequently associated with variant CCG repeats independently of the expansion size. The effect of repeat interruptions on DM1 molecular pathogenesis is not investigated enough. Studies on patients indicate their stabilizing effect on DMPK expansions because no congenital cases were described in patients with repeat interruptions, and the age at onset is frequently later than expected. Here, we review the clinical relevance of repeat interruptions in DM1 and genetic and epigenetic characteristics of interrupted DMPK expansions based on patient studies.     

Overview of the Complex Relationship between Epigenetics Markers,CTG Repeat Instability and Symptoms in Myotonic Dystrophy Type 1

Among the trinucleotide repeat disorders, myotonic dystrophy type 1 (DM1) is one of the most complex neuromuscular diseases caused by an unstable CTG repeat expansion in the DMPK gene. DM1 patients exhibit high variability in the dynamics of CTG repeat instability and in the manifestations and progression of the disease. The largest expanded alleles are generally associated with the earliest and most severe clinical form. However, CTG repeat length alone is not sufficient to predict disease severity and progression, suggesting the involvement of other factors. Several data support the role of epigenetic alterations in clinical and genetic variability. By highlighting epigenetic alterations in DM1, this review provides a new avenue on how these changes can serve as biomarkers to predict clinical features and the mutation behavior.     

Genetic and Epigenetic Characterization of a Discordant KMT2A/AFF1-Rearranged Infant Monozygotic Twin Pair

The KMT2A/AFF1 rearrangement is associated with an unfavorable prognosis in infant acute lymphocytic leukemia (ALL). Discordant ALL in monozygotic twins is uncommon and represents an attractive resource to evaluate intrauterine environment–genetic interplay in ALL. Mutational and epigenetic profiles were characterized for a discordant KMT2A/AFF1-rearranged infant monozygotic twin pair and their parents, and they were compared to three independent KMT2A/AFF1-positive ALL infants, in which the DNA methylation and gene expression profiles were investigated. A de novo Q61H NRAS mutation was detected in the affected twin at diagnosis and backtracked in both twins at birth. The KMT2A/AFF1 rearrangement was absent at birth in both twins. Genetic analyses conducted at birth gave more insights into the timing of the mutation hit. We identified correlations between DNA methylation and gene expression changes for 32 genes in the three independent affected versus remitted patients. The strongest correlations were observed for the RAB32, PDK4, CXCL3, RANBP17, and MACROD2 genes. This epigenetic signature could be a putative target for the development of novel epigenetic-based therapies and could help in explaining the molecular mechanisms characterizing ALL infants with KMT2A/AFF1 fusions.     

Epigenetic Distribution of Recombinant Plant Chromosome Fragments in a Human–Arabidopsis Hybrid Cell Line

Methylation systems have been conserved during the divergence of plants and animals, although they are regulated by different pathways and enzymes. However, studies on the interactions of the epigenomes among evolutionarily distant organisms are lacking. To address this, we studied the epigenetic modification and gene expression of plant chromosome fragments (~30 Mb) in a human–Arabidopsis hybrid cell line. The whole-genome bisulfite sequencing results demonstrated that recombinant Arabidopsis DNA could retain its plant CG methylation levels even without functional plant methyltransferases, indicating that plant DNA methylation states can be maintained even in a different genomic background. The differential methylation analysis showed that the Arabidopsis DNA was undermethylated in the centromeric region and repetitive elements. Several Arabidopsis genes were still expressed, whereas the expression patterns were not related to the gene function. We concluded that the plant DNA did not maintain the original plant epigenomic landscapes and was under the control of the human genome. This study showed how two diverging genomes can coexist and provided insights into epigenetic modifications and their impact on the regulation of gene expressions between plant and animal genomes.     

Enhanced anticancer activity of DM1-loaded star-shaped folate-core PLA-TPGS nanoparticles

The efficient delivery of therapeutic drugs into interested cells is a critical challenge to broad application of nonviral vector systems. In this research, emtansine (DM1)-loaded star-shaped folate-core polylactide-d-α-tocopheryl polyethylene glycol 1000 succinate (FA-PLA-TPGS-DM1) copolymer which demonstrated superior anticancer activity in vitro/vivo in comparison with linear FA-PLA-TPGS nanoparticles was applied to be a vector of DM1 for FR⁺ breast cancer therapy. The DM1- or coumarin 6-loaded nanoparticles were fabricated, and then characterized in terms of size, morphology, drug encapsulation efficiency, and in vitro drug release. And the viability of MCF-7/HER2 cells treated with FA-DM1-nanoparticles (NPs) was assessed. Severe combined immunodeficient mice carrying MCF-7/HER2 tumor xenografts were treated in several groups including phosphate-buffered saline control, DM1, DM1-NPs, and FA-DM1-NPs. The antitumor activity was then assessed by survival time and solid tumor volume. All the specimens were prepared for formalin-fixed and paraffin-embedded tissue sections for hematoxylin-eosin staining. The data showed that the FA-DM1-NPs could efficiently deliver DM1 into MCF-7/HER2 cells. The cytotoxicity of DM1 to MCF-7/HER2 cells was significantly increased by FA-DM1-NPs when compared with the control groups. In conclusion, the FA-DM1-NPs offered a considerable potential formulation for FR⁺ tumor-targeting biotherapy.     

Identification and DNA Marker Development for a Wheat-Leymus mollis 2Ns (2D) Disomic Chromosome Substitution

Leymus mollis (2n = 4x = 28, NsNsXmXm), a wild relative of common wheat (Triticum aestivum L.), carries numerous loci which could potentially be used in wheat improvement. In this study, line 17DM48 was isolated from the progeny of a wheat and L. mollis hybrid. This line has 42 chromosomes forming 21 bivalents at meiotic metaphase I. Genomic in situ hybridization (GISH) demonstrated the presence of a pair chromosomes from the Ns genome of L. mollis. This pair substituted for wheat chromosome 2D, as shown by fluorescence in situ hybridization (FISH), DNA marker analysis, and hybridization to wheat 55K SNP array. Therefore, 17DM48 is a wheat-L. mollis 2Ns (2D) disomic substitution line. It shows longer spike and a high level of stripe rust resistance. Using specific-locus amplified fragment sequencing (SLAF-seq), 13 DNA markers were developed to identify and trace chromosome 2Ns of L. mollis in wheat background. This line provides a potential bridge germplasm for genetic improvement of wheat stripe rust resistance.     

Whole Exome Sequencing for a Patient with Rubinstein-Taybi Syndrome Reveals de Novo Variants besides an Overt CREBBP Mutation

Rubinstein-Taybi syndrome (RSTS) is a rare condition with a prevalence of 1 in 125,000–720,000 births and characterized by clinical features that include facial, dental, and limb dysmorphology and growth retardation. Most cases of RSTS occur sporadically and are caused by de novo mutations. Cytogenetic or molecular abnormalities are detected in only 55% of RSTS cases. Previous genetic studies have yielded inconsistent results due to the variety of methods used for genetic analysis. The purpose of this study was to use whole exome sequencing (WES) to evaluate the genetic causes of RSTS in a young girl presenting with an Autism phenotype. We used the Autism diagnostic observation schedule (ADOS) and Autism diagnostic interview revised (ADI-R) to confirm her diagnosis of Autism. In addition, various questionnaires were used to evaluate other psychiatric features. We used WES to analyze the DNA sequences of the patient and her parents and to search for de novo variants. The patient showed all the typical features of Autism, WES revealed a de novo frameshift mutation in CREBBP and de novo sequence variants in TNC and IGFALS genes. Mutations in the CREBBP gene have been extensively reported in RSTS patients, while potential missense mutations in TNC and IGFALS genes have not previously been associated with RSTS. The TNC and IGFALS genes are involved in central nervous system development and growth. It is possible for patients with RSTS to have additional de novo variants that could account for previously unexplained phenotypes.     

Alterations in the Genomic Distribution of 5hmC in In Vivo Aged Human Skin Fibroblasts

5-Hydroxymethylcytosine (5hmC) is a functionally active epigenetic modification. We analyzed whether changes in DNA 5-hydroxymethylation are an element of age-related epigenetic drift. We tested primary fibroblast cultures originating from individuals aged 22–35 years and 74–94 years. Global quantities of methylation-related DNA modifications were estimated by the dot blot and colorimetric methods. Regions of the genome differentially hydroxymethylated with age (DHMRs) were identified by hMeDIP-seq and the MEDIPS and DiffBind algorithms. Global levels of DNA modifications were not associated with age. We identified numerous DHMRs that were enriched within introns and intergenic regions and most commonly associated with the H3K4me1 histone mark, promoter-flanking regions, and CCCTC-binding factor (CTCF) binding sites. However, only seven DHMRs were identified by both algorithms and all of their settings. Among them, hypo-hydroxymethylated DHMR in the intron of Rab Escort Protein 1 (CHM) coexisted with increased expression in old cells, while increased 5-hydroxymethylation in the bodies of Arginine and Serine Rich Protein 1 (RSRP1) and Mitochondrial Poly(A) Polymerase (MTPAP) did not change their expression. These age-related differences were not associated with changes in the expression of any of the ten-eleven translocation (TET) enzymes or their activity. In conclusion, the distribution of 5hmC in DNA of in vivo aged human fibroblasts underwent age-associated modifications. The identified DHMRs are, likely, marker changes.     

Identification of Novel Insulin Resistance Related ceRNA Network in T2DM and Its Potential Editing by CRISPR/Cas9

Background: Type 2 diabetes mellitus is one of the leading causes of morbidity and mortality worldwide and is derived from an accumulation of genetic and epigenetic changes. In this study, we aimed to construct Insilco, a competing endogenous RNA (ceRNA) network linked to the pathogenesis of insulin resistance followed by its experimental validation in patients’, matched control and cell line samples, as well as to evaluate the efficacy of CRISPR/Cas9 as a potential therapeutic strategy to modulate the expression of this deregulated network. By applying bioinformatics tools through a two-step process, we identified and verified a ceRNA network panel of mRNAs, miRNAs and lncRNA related to insulin resistance, Then validated the expression in clinical samples (123 patients and 106 controls) and some of matched cell line samples using real time PCR. Next, two guide RNAs were designed to target the sequence flanking LncRNA/miRNAs interaction by CRISPER/Cas9 in cell culture. Gene editing tool efficacy was assessed by measuring the network downstream proteins GLUT4 and mTOR via immunofluorescence. Results: LncRNA-RP11-773H22.4, together with RET, IGF1R and mTOR mRNAs, showed significant upregulation in T2DM compared with matched controls, while miRNA (i.e., miR-3163 and miR-1) and mRNA (i.e., GLUT4 and AKT2) expression displayed marked downregulation in diabetic samples. CRISPR/Cas9 successfully knocked out LncRNA-RP11-773H22.4, as evidenced by the reversal of the gene expression of the identified network at RNA and protein levels to the normal expression pattern after gene editing. Conclusions: The present study provides the significance of this ceRNA based network and its related target genes panel both in the pathogenesis of insulin resistance and as a therapeutic target for gene editing in T2DM.     

Regulatory T Cells-Related Genes Are under DNA Methylation Influence

Regulatory T cells (Tregs) exert a highly suppressive function in the immune system. Disturbances in their function predispose an individual to autoimmune dysregulation, with a predominance of the pro-inflammatory environment. Besides Foxp3, which is a master regulator of these cells, other genes (e.g., Il2ra, Ctla4, Tnfrsf18, Ikzf2, and Ikzf4) are also involved in Tregs development and function. Multidimensional Tregs suppression is determined by factors that are believed to be crucial in the action of Tregs-related genes. Among them, epigenetic changes, such as DNA methylation, tend to be widely studied over the past few years. DNA methylation acts as a repressive mark, leading to diminished gene expression. Given the role of increased CpG methylation upon Tregs imprinting and functional stability, alterations in the methylation pattern can cause an imbalance in the immune response. Due to the fact that epigenetic changes can be reversible, so-called epigenetic modifiers are broadly used in order to improve Tregs performance. In this review, we place emphasis on the role of DNA methylation of the genes that are key regulators of Tregs function. We also discuss disease settings that have an impact on the methylation status of Tregs and systematize the usefulness of epigenetic drugs as factors able to influence Tregs functions.     

Unravelling Novel Roles of Salivary Exosomes in the Regulation of Human Corneal Stromal Cell Migration and Wound Healing

Salivary exosomes have demonstrated vast therapeutic and diagnostic potential in numerous diseases. This study pioneers previously unexplored roles of SE in the context of corneal wound healing by utilizing primary corneal stromal cells from healthy (HCFs), type I diabetes mellitus (T1DMs), type II DM (T2DMs), and keratoconus (HKCs) subjects. Purified, healthy human SEs carrying tetraspanins CD9+, CD63+, and CD81+ were utilized. Scratch and cell migration assays were performed after 0, 6, 12, 24, and 48 h following SE stimulation (5 and 25 µg/mL). Significantly slower wound closure was observed at 6 and 12 h in HCFs with 5 μg/mL SE and T1DMs with 5 and 25 μg/mL SE. All wounds were closed by 24-hour, post-wounding. HKCs, T1DMs, and T2DMs with 25µg/mL SE exhibited a significant upregulation of cleaved vimentin compared to controls. Thrombospondin 1 was significantly upregulated in HCFs, HKCs, and T2DMs with 25 µg/mL SE. Lastly, HKCs, T1DMs, and T2DMs exhibited a significant downregulation of fibronectin with 25 μg/mL SE. Whether SEs can be utilized to clinical settings in restoring corneal defects is unknown. This is the first-ever study exploring the role of SEs in corneal wound healing. While the sample size was small, results are highly novel and provide a strong foundation for future studies.     

Epigenetics and Helicobacter pylori

Epigenetics regulates gene expression, cell type development during differentiation, and the cell response to environmental stimuli. To survive, bacteria need to evade the host immune response. Bacteria, including Helicobacter pylori (Hp), reach this target epigenetically, altering the chromatin of the host cells, in addition to several more approaches, such as DNA mutation and recombination. This review shows that Hp prevalently silences the genes of the human gastric mucosa by DNA methylation. Epigenetics includes different mechanisms. However, DNA methylation persists after DNA replication and therefore is frequently associated with the inheritance of repressed genes. Chromatin modification can be transmitted to daughter cells leading to heritable changes in gene expression. Aberrant epigenetic alteration of the gastric mucosa DNA remains the principal cause of gastric cancer. Numerous methylated genes have been found in cancer as well as in precancerous lesions of Hp-infected patients. These methylated genes inactivate tumor-suppressor genes. It is time for us to complain about our genetic and epigenetic makeups for our diseases.     

Comparative Efficacy and Safety of P2Y12 Inhibitor Monotherapy and Dual Antiplatelet Therapy in Patients with and without Diabetes Mellitus Undergoing Percutaneous Coronary Intervention

Increasing evidence has shown P2Y12 inhibitor monotherapy is a feasible alternative treatment for patients after percutaneous coronary intervention (PCI) with stent implantation in the modern era. However, patients with diabetes mellitus (DM) have a higher risk of ischemic events and more complex coronary artery disease. The purpose of this study is to evaluate the efficacy and safety of this novel approach among patients with DM and those without DM. We conducted a systematic review and meta-analysis of randomized controlled trials that compared P2Y12 inhibitor monotherapy with 12 months of dual antiplatelet therapy (DAPT) in patients who underwent PCI with stent implantation. PubMed, Embase, Cochrane library database, ClinicalTrials.gov, and three other websites were searched for our data from the earliest report to January 2022. The primary efficacy outcome was major adverse cardiovascular and cerebrovascular events (MACCE): a composite of all-cause mortality, myocardial infarction, stent thrombosis, and stroke. The primary safety outcome was major or minor bleeding events. The secondary endpoint was net adverse clinical events (NACE) which are defined as a composite of major bleeding and adverse cardiac and cerebrovascular events. A total of four randomized controlled trials with 29,136 patients were included in our meta-analysis. The quantitative analysis showed a significant reduction in major or minor bleeding events in patients treated with P2Y12 inhibitor monotherapy compared to standard DAPT (OR: 0.68, 95% CI: 0.46–0.99, p = 0.04) without increasing the risk of MACCE (OR: 0.96, 95% CI: 0.85–1.09, p = 0.50). The number of NACE was significantly lower in the patients treated with P2Y12 inhibitor monotherapy (OR: 0.84, 95% CI: 0.72–0.97, p = 0.019). In DM patients, P2Y12 inhibitor monotherapy was associated with a lower risk of MACCE compared to standard DAPT (OR: 0.85, 95% CI: 0.74–0.98, p = 0.02). Furthermore, P2Y12 inhibitor monotherapy was accompanied by a favorable reduction in major or minor bleeding events (OR: 0.80, 95% CI: 0.64–1.05, p = 0.107). In non-DM patients, P2Y12 inhibitor monotherapy showed a significant reduction in major or minor bleeding events (OR: 0.58, 95% CI: 0.38–0.88, p = 0.01), but without increasing the risk of MACCE (OR: 0.99, 95% CI: 0.82–1.19, p = 0.89). Based on these findings, P2Y12 inhibitor monotherapy could significantly decrease bleeding events without increasing the risk of stent thrombosis or myocardial infarction in the general population. The benefit of reducing bleeding events was much more significant in non-DM patients than in DM patients. Surprisingly, P2Y12 inhibitor monotherapy could lower the risk of MACCE in DM patients. Our study supports that P2Y12 inhibitor monotherapy is a promising alternative choice of medical treatment for patients with DM undergoing PCI with stent implantation in the modern era.     

Altered DNA Methylation Profiles in SF3B1 Mutated CLL Patients

Mutations in splicing factor genes have a severe impact on the survival of cancer patients. Splicing factor 3b subunit 1 (SF3B1) is one of the most frequently mutated genes in chronic lymphocytic leukemia (CLL); patients carrying these mutations have a poor prognosis. Since the splicing machinery and the epigenome are closely interconnected, we investigated whether these alterations may affect the epigenomes of CLL patients. While an overall hypomethylation during CLL carcinogenesis has been observed, the interplay between the epigenetic stage of the originating B cells and SF3B1 mutations, and the subsequent effect of the mutations on methylation alterations in CLL, have not been investigated. We profiled the genome-wide DNA methylation patterns of 27 CLL patients with and without SF3B1 mutations and identified local decreases in methylation levels in SF3B1^mut CLL patients at 67 genomic regions, mostly in proximity to telomeric regions. These differentially methylated regions (DMRs) were enriched in gene bodies of cancer-related signaling genes, e.g., NOTCH1, HTRA3, and BCL9L. In our study, SF3B1 mutations exclusively emerged in two out of three epigenetic stages of the originating B cells. However, not all the DMRs could be associated with the methylation programming of B cells during development, suggesting that mutations in SF3B1 cause additional epigenetic aberrations during carcinogenesis.     

Epigenetic Determinants of CYP1A1 Induction by the Aryl Hydrocarbon Receptor Agonist 3,3',4,4',5-Pentachlorobiphenyl (PCB 126)

Many enzymes involved in xenobiotic metabolism, including cytochrome P450 (CYP) 1A1, are regulated by the aryl hydrocarbon receptor (AhR). 3,3'',4,4'',5-Pentachlorobiphenyl (PCB 126) is a potent ligand for AhR and can thus induce the expression of CYP1A1. Interestingly, we observed that human carcinoma cell lines derived from different types of epithelial cells displayed divergent degrees of CYP1A1 induction after exposure to PCB 126. Since epigenetic mechanisms are known to be involved in cell type-specific gene expression, we sought to assess the epigenetic determinants of CYP1A1 induction in these carcinoma cell lines. In contrast to HepG2 hepatocarcinoma cells, HeLa cervical carcinoma cells showed significantly lower levels of CYP1A1 mRNA expression following PCB 126 exposure. Our results show that the two cell lines maintained differences in the chromatin architecture along the CYP1A1 promoter region. Furthermore, treatment with the epigenetic modifiers, trichostatin A (TSA) and 5-aza-2''-deoxycytidine (5-Aza-dC), significantly increased the expression of CYP1A1 after PCB 126 treatment in HeLa cells. However, we did not observe apparent differences in methylation levels or specific location of CpG DNA methylation between the two cell lines in the analyzed CYP1A1 promoter region. Taken together, our findings suggest that the differences in CYP1A1 expression between HepG2 and HeLa cells are due to differences in the chromatin architecture of the CYP1A1 promoter and thus establish a role of epigenetic regulation in cell-specific CYP1A1 expression.     

Homologous Recombination Deficiencies and Hereditary Tumors

Homologous recombination (HR) is a vital process for repairing DNA double-strand breaks. Germline variants in the HR pathway, comprising at least 10 genes, such as BRCA1, BRCA2, ATM, BARD1, BRIP1, CHEK2, NBS1(NBN), PALB2, RAD51C, and RAD51D, lead to inherited susceptibility to specific types of cancers, including those of the breast, ovaries, prostate, and pancreas. The penetrance of germline pathogenic variants of each gene varies, whereas all their associated protein products are indispensable for maintaining a high-fidelity DNA repair system by HR. The present review summarizes the basic molecular mechanisms and components that collectively play a role in maintaining genomic integrity against DNA double-strand damage and their clinical implications on each type of hereditary tumor.