Robust Detection of Somatic Mosaicism and Repeat Interruptions by Long-Read Targeted Sequencing in Myotonic Dystrophy Type 1

Myotonic dystrophy type 1 (DM1) is the most complex and variable trinucleotide repeat disorder caused by an unstable CTG repeat expansion, reaching up to 4000 CTG in the most severe cases. The genetic and clinical variability of DM1 depend on the sex and age of the transmitting parent, but also on the CTG repeat number, presence of repeat interruptions and/or on the degree of somatic instability. Currently, it is difficult to simultaneously and accurately determine these contributing factors in DM1 patients due to the limitations of gold standard methods used in molecular diagnostics and research laboratories. Our study showed the efficiency of the latest PacBio long-read sequencing technology to sequence large CTG trinucleotides, detect multiple and single repeat interruptions and estimate the levels of somatic mosaicism in DM1 patients carrying complex CTG repeat expansions inaccessible to most methods. Using this innovative approach, we revealed the existence of de novo CCG interruptions associated with CTG stabilization/contraction across generations in a new DM1 family. We also demonstrated that our method is suitable to sequence the DM1 locus and measure somatic mosaicism in DM1 families carrying more than 1000 pure CTG repeats. Better characterization of expanded alleles in DM1 patients can significantly improve prognosis and genetic counseling, not only in DM1 but also for other tandem DNA repeat disorders.     

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.     

Epigenetics of Myotonic Dystrophies: A Minireview

Myotonic dystrophy type 1 and 2 (DM1 and DM2) are two multisystemic autosomal dominant disorders with clinical and genetic similarities. The prevailing paradigm for DMs is that they are mediated by an in trans toxic RNA mechanism, triggered by untranslated CTG and CCTG repeat expansions in the DMPK and CNBP genes for DM1 and DM2, respectively. Nevertheless, increasing evidences suggest that epigenetics can also play a role in the pathogenesis of both diseases. In this review, we discuss the available information on epigenetic mechanisms that could contribute to the DMs outcome and progression. Changes in DNA cytosine methylation, chromatin remodeling and expression of regulatory noncoding RNAs are described, with the intent of depicting an epigenetic signature of DMs. Epigenetic biomarkers have a strong potential for clinical application since they could be used as targets for therapeutic interventions avoiding changes in DNA sequences. Moreover, understanding their clinical significance may serve as a diagnostic indicator in genetic counselling in order to improve genotype–phenotype correlations in DM patients.     

Interruptions of the FXN GAA Repeat Tract Delay the Age at Onset of Friedreich’s Ataxia in a Location Dependent Manner

Friedreich’s ataxia (FRDA) is a comparatively rare autosomal recessive neurological disorder primarily caused by the homozygous expansion of a GAA trinucleotide repeat in intron 1 of the FXN gene. The repeat expansion causes gene silencing that results in deficiency of the frataxin protein leading to mitochondrial dysfunction, oxidative stress and cell death. The GAA repeat tract in some cases may be impure with sequence variations called interruptions. It has previously been observed that large interruptions of the GAA repeat tract, determined by abnormal MboII digestion, are very rare. Here we have used triplet repeat primed PCR (TP PCR) assays to identify small interruptions at the 5′ and 3′ ends of the GAA repeat tract through alterations in the electropherogram trace signal. We found that contrary to large interruptions, small interruptions are more common, with 3′ interruptions being most frequent. Based on detection of interruptions by TP PCR assay, the patient cohort (n = 101) was stratified into four groups: 5′ interruption, 3′ interruption, both 5′ and 3′ interruptions or lacking interruption. Those patients with 3′ interruptions were associated with shorter GAA1 repeat tracts and later ages at disease onset. The age at disease onset was modelled by a group-specific exponential decay model. Based on this modelling, a 3′ interruption is predicted to delay disease onset by approximately 9 years relative to those lacking 5′ and 3′ interruptions. This highlights the key role of interruptions at the 3′ end of the GAA repeat tract in modulating the disease phenotype and its impact on prognosis for the patient.     

(Dys)function Follows Form: Nucleic Acid Structure,Repeat Expansion,and Disease Pathology in FMR1 Disorders

Fragile X-related disorders (FXDs), also known as FMR1 disorders, are examples of repeat expansion diseases (REDs), clinical conditions that arise from an increase in the number of repeats in a disease-specific microsatellite. In the case of FXDs, the repeat unit is CGG/CCG and the repeat tract is located in the 5′ UTR of the X-linked FMR1 gene. Expansion can result in neurodegeneration, ovarian dysfunction, or intellectual disability depending on the number of repeats in the expanded allele. A growing body of evidence suggests that the mutational mechanisms responsible for many REDs share several common features. It is also increasingly apparent that in some of these diseases the pathologic consequences of expansion may arise in similar ways. It has long been known that many of the disease-associated repeats form unusual DNA and RNA structures. This review will focus on what is known about these structures, the proteins with which they interact, and how they may be related to the causative mutation and disease pathology in the FMR1 disorders.     

Fluorescence Probe for Detecting CCG Trinucleotide Repeat DNA Expansion and Slip‐Out

Trinucleotide repeat expansion in genomic DNA causes severe neurodegenerative diseases. Fluorescence probes that bind to trinucleotide repeats have potential as diagnostic tools of trinucleotide repeat disorders. Here, we report a novel tricyclic ligand that binds to CCG trinucleotide repeat DNA. The expansion of the aromatic ring system of the 2‐amino‐1,8‐naphthyridine chromophore from the bicyclic to the tricyclic improved the binding ability to the CCG/CCG motif without losing the selectivity and emissive character. The fluorescence sensitively decreased in response to binding to the CCG trinucleotide repeat. The degree of quenching depended on the number of CCG repeats. In addition, the fluorescence detection was applicable to CCG slip‐out DNA.     

Mechanisms of the FMR1 Repeat Instability: How Does the CGG Sequence Expand?

A dynamic mutation in exon 1 of the FMR1 gene causes Fragile X-related Disorders (FXDs), due to the expansion of an unstable CGG repeat sequence. Based on the CGG sequence size, two types of FMR1 alleles are possible: “premutation” (PM, with 56-200 CGGs) and “full mutation” (FM, with >200 triplets). Premutated females are at risk of transmitting a FM allele that, when methylated, epigenetically silences FMR1 and causes Fragile X syndrome (FXS), a very common form of inherited intellectual disability (ID). Expansions events of the CGG sequence are predominant over contractions and are responsible for meiotic and mitotic instability. The CGG repeat usually includes one or more AGG interspersed triplets that influence allele stability and the risk of transmitting FM to children through maternal meiosis. A unique mechanism responsible for repeat instability has not been identified, but several processes are under investigations using cellular and animal models. The formation of unusual secondary DNA structures at the expanded repeats are likely to occur and contribute to the CGG expansion. This review will focus on the current knowledge about CGG repeat instability addressing the CGG sequence expands.     

Fluorescence-Based Binding Characterization of Small Molecule Ligands Targeting CUG RNA Repeats

Pathogenic CUG and CCUG RNA repeats have been associated with myotonic dystrophy type 1 and 2 (DM1 and DM2), respectively. Identifying small molecules that can bind these RNA repeats is of great significance to develop potential therapeutics to treat these neurodegenerative diseases. Some studies have shown that aminoglycosides and their derivatives could work as potential lead compounds targeting these RNA repeats. In this work, sisomicin, previously known to bind HIV-1 TAR, is investigated as a possible ligand for CUG RNA repeats. We designed a novel fluorescence-labeled RNA sequence of r(CUG)₁₀ to mimic cellular RNA repeats and improve the detecting sensitivity. The interaction of sisomicin with CUG RNA repeats is characterized by the change of fluorescent signal, which is initially minimized by covalently incorporating the fluorescein into the RNA bases and later increased upon ligand binding. The results show that sisomicin can bind and stabilize the folded RNA structure. We demonstrate that this new fluorescence-based binding characterization assay is consistent with the classic UV T_m technique, indicating its feasibility for high-throughput screening of ligand-RNA binding interactions and wide applications to measure the thermodynamic parameters in addition to binding constants and kinetics when probing such interactions.     

Disrupting the Molecular Pathway in Myotonic Dystrophy

Myotonic dystrophy is the most common muscular dystrophy in adults. It consists of two forms: type 1 (DM1) and type 2 (DM2). DM1 is associated with a trinucleotide repeat expansion mutation, which is transcribed but not translated into protein. The mutant RNA remains in the nucleus, which leads to a series of downstream abnormalities. DM1 is widely considered to be an RNA-based disorder. Thus, we consider three areas of the RNA pathway that may offer targeting opportunities to disrupt the production, stability, and degradation of the mutant RNA.     

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.     

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.