Unusual structures of CCTG repeats and their participation in repeat expansion

AbstractCCTG repeat expansion in intron 1 of the cellular nucleic acid-binding protein (CNBP) gene has been identified to be the genetic cause of myotonic dystrophy type 2 (DM2). Yet the underlying reasons for the genetic instability in CCTG repeats remain elusive. In recent years, CCTG repeats have been found to form various types of unusual secondary structures including mini-dumbbell (MDB), hairpin and dumbbell, revealing that there is a high structural diversity in CCTG repeats intrinsically. Upon strand slippage, the formation of unusual structures in the nascent strand during DNA replication has been proposed to be the culprit of CCTG repeat expansions. On the one hand, the thermodynamic stability, size, and conformational dynamics of these unusual structures affect the propensity of strand slippage. On the other hand, these structural properties determine whether the unusual structure can successfully escape from DNA repair. In this short overview, we first summarize the recent advances in elucidating the solution structures of CCTG repeats. We then discuss the potential pathways by which these unusual structures bring about variable sizes of repeat expansion, high strand slippage propensity and efficient repair escape.

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A Toxic RNA Catalyzes the Cellular Synthesis of Its Own Inhibitor, Shunting It to Endogenous Decay Pathways

10.1101/741926 ◽

2019 ◽

Cited By ~ 1

Author(s):

Raphael I. Benhamou ◽

Alicia J. Angelbello ◽

Eric T. Wang ◽

Matthew D. Disney

Keyword(s):

Small Molecules ◽

Rna Binding ◽

Rna Binding Proteins ◽

Intron Retention ◽

Protein A ◽

List Type ◽

Nucleic Acid Binding ◽

Myotonic Dystrophy Type 2 ◽

Intron 1 ◽

Toxic Rna

SUMMARYMyotonic dystrophy type 2 (DM2) is a genetically defined muscular dystrophy caused by a toxic expanded repeat of r(CCUG) [heretofore (CCUG)exp], harbored in intron 1 of CHC-Type Zinc Finger Nucleic Acid Binding Protein (CNBP) pre-mRNA. This r(CCUG)exp causes DM2 via a gain-of-function mechanism that results in three hallmarks of its pathology: (i) binding to RNA-binding proteins (RBPs) that aggregate into nuclear foci; (ii) sequestration of muscleblind-like-1 (MBNL1) protein, a regulator of alternative pre-mRNA splicing, leading to splicing defects; and (iii) retention of intron 1 in the CNBP mRNA. Here, we find that CNBP intron retention is caused by the r(CCUG)exp-MBNL1 complex and can be rescued by small molecules. We studied two types of small molecules with different modes of action, ones that simply bind and ones that can be synthesized by a r(CCUG)exp-templated reaction in cells, that is the RNA synthesizes its own drug. Indeed, our studies completed in DM2 patient-derived fibroblasts show that the compounds disrupt the r(CCUG)exp-MBNL1 complex, reduce intron retention, subjecting the liberated intronic r(CCUG)exp to native decay pathways, and rescue other DM2-associated cellular defects. Collectively, this study shows that small molecules can affect RNA biology by shunting toxic transcripts towards native decay pathways.HIGHLIGHTSIntron retention in RNA repeat expansions can be due to repeats binding to proteinsSmall molecules that bind RNA repeats and inhibit protein binding can trigger decayA toxic RNA repeat can catalyze the synthesis of its own inhibitor on-siteOn-site drug synthesis most potently affects disease biologyeTOC BLURBThe most common way to target RNA is to use antisense oligonucleotides to target unstructured RNAs for destruction. Here, we show for the first time that small molecules targeting structured, disease-causing RNAs can shunt them towards native decay pathways by affecting their processing.

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A 14-Year Italian Experience in DM2 Genetic Testing: Frequency and Distribution of Normal and Premutated CNBP Alleles

Frontiers in Genetics ◽

10.3389/fgene.2021.668094 ◽

2021 ◽

Vol 12 ◽

Author(s):

Annalisa Botta ◽

Virginia Veronica Visconti ◽

Luana Fontana ◽

Paola Bisceglia ◽

Mario Bengala ◽

...

Keyword(s):

Genetic Testing ◽

Italian Population ◽

Myotonic Dystrophy Type 2 ◽

Italian Experience ◽

Testing Frequency ◽

Intron 1 ◽

Clinical Consequences ◽

Cctg Repeat ◽

Testing And Counseling

Myotonic dystrophy type 2 (DM2) is a multisystemic disorder caused by a (CCTG)n in intron 1 of the CNBP gene. The CCTG repeat tract is part of a complex (TG)v(TCTG)w(CCTG)x(NCTG)y(CCTG)z motif generally interrupted in CNBP healthy range alleles. Here we report our 14-year experience of DM2 postnatal genetic testing in a total of 570 individuals. The DM2 locus has been analyzed by a combination of SR-PCR, TP-PCR, LR-PCR, and Sanger sequencing of CNBP alleles. DM2 molecular diagnosis has been confirmed in 187/570 samples analyzed (32.8%) and is mainly associated with the presence of myotonia in patients. This set of CNBP alleles showed unimodal distribution with 25 different alleles ranging from 108 to 168 bp, in accordance with previous studies on European populations. The most frequent CNBP alleles consisted of 138, 134, 140, and 136 bps with an overall locus heterozygosity of 90%. Sequencing of 103 unexpanded CNBP alleles in DM2-positive patients revealed that (CCTG)5(NCTG)3(CCTG)7 and (CCTG)6(NCTG)3(CCTG)7 are the most common interruption motifs. We also characterized five CNBP premutated alleles with (CCTG)n repetitions from n = 36 to n = 53. However, the molecular and clinical consequences in our cohort of samples are not unequivocal. Data that emerged from this study are representative of the Italian population and are useful tools for National and European centers offering DM2 genetic testing and counseling.

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Characterisation of Non-Pathogenic Premutation-Range Myotonic Dystrophy Type 2 Alleles

Journal of Clinical Medicine ◽

10.3390/jcm10173934 ◽

2021 ◽

Vol 10 (17) ◽

pp. 3934

Author(s):

Jan Radvanszky ◽

Michaela Hyblova ◽

Eva Radvanska ◽

Peter Spalek ◽

Alica Valachova ◽

...

Keyword(s):

Myotonic Dystrophy ◽

Intergenerational Transmission ◽

Grey Zone ◽

Myotonic Dystrophy Type ◽

Nucleic Acid Binding ◽

Pathogenic Role ◽

Myotonic Dystrophy Type 2 ◽

Family Structures ◽

Cctg Repeat

Myotonic dystrophy type 2 (DM2) is caused by expansion of a (CCTG)n repeat in the cellular retroviral nucleic acid-binding protein (CNBP) gene. The sequence of the repeat is most commonly interrupted and is stably inherited in the general population. Although expanded alleles, premutation range and, in rare cases, also non-disease associated alleles containing uninterrupted CCTG tracts have been described, the threshold between these categories is poorly characterised. Here, we describe four families with members reporting neuromuscular complaints, in whom we identified altogether nine ambiguous CNBP alleles containing uninterrupted CCTG repeats in the range between 32 and 42 repeats. While these grey-zone alleles are most likely not pathogenic themselves, since other pathogenic mutations were identified and particular family structures did not support their pathogenic role, they were found to be unstable during intergenerational transmission. On the other hand, there was no observable general microsatellite instability in the genome of the carriers of these alleles. Our results further refine the division of CNBP CCTG repeat alleles into two major groups, i.e., interrupted and uninterrupted alleles. Both interrupted and uninterrupted alleles with up to approximately 30 CCTG repeats were shown to be generally stable during intergenerational transmission, while intergenerational as well as somatic instability seems to gradually increase in uninterrupted alleles with tract length growing above this threshold.

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Ancestral Origin of the First Indian Families with Myotonic Dystrophy Type 2

Journal of Neuromuscular Diseases ◽

10.3233/jnd-210671 ◽

2021 ◽

pp. 1-8

Author(s):

M.J. Damen ◽

M.M.V.A.P. Schijvenaars ◽

A.M. Schimmel Naber ◽

J.M. Groothuismink ◽

M.J.H. Coenen ◽

...

Keyword(s):

Myotonic Dystrophy ◽

Tandem Repeats ◽

Repeat Expansion ◽

European Countries ◽

Myotonic Dystrophy Type ◽

Nucleotide Polymorphisms ◽

Nucleic Acid Binding ◽

Myotonic Dystrophy Type 2 ◽

Ancestral Origin

Background: Myotonic dystrophy type 2 (DM2) is caused by a CCTG repeat expansion in intron 1 of the CCHC-Type Zinc Finger Nucleic Acid Binding Protein (CNBP) gene. Previous studies indicated that this repeat expansion originates from separate founders. Objective: This study was set out to determine whether or not patients with DM2 originating from European and non-European countries carry the previously described European founder haplotypes. Methods: Haplotype analysis was performed in 59 DM2 patients from 29 unrelated families. Twenty-three families were from European descent and 6 families originated from non-European countries (India, Suriname and Morocco). Seven short tandem repeats (CL3N122, CL3N99, CL3N59, CL3N117, CL3N119, CL3N19 and CL3N23) and 4 single nucleotide polymorphisms (SNP) (rs1871922, rs1384313, rs4303883 and CGAP_886192) in and around the CNBP gene were used to construct patients’ haplotypes. These haplotypes were compared to the known DM2 haplotypes to determine the ancestral origin of the CNBP repeat expansion. Results: Of 41 patients, the haplotype could be assigned to the previously described Caucasian haplotypes. Three patients from Morocco and Portugal had a haplotype identical to the earlier reported Moroccan haplotype. Twelve patients from India and Suriname, however, carried a haplotype that seems distinct from the previously reported haplotypes. Three individuals could not be assigned to a specific haplotype. Conclusion: The ancestral origin of DM2 in India might be distinct from the Caucasian families and the solely described Japanese patient. However, we were unable to establish this firmly due to the limited genetic variation in the region surrounding the CNBP gene.

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Marathoning with myotonic dystrophy type 2 (proximal myotonic myopathy) and leukopenia

SAGE Open Medical Case Reports ◽

10.1177/2050313x17703021 ◽

2017 ◽

Vol 5 ◽

pp. 2050313X1770302

Author(s):

Josef Finsterer ◽

Georg Safoschnik ◽

Martina Witsch-Baumgartner

Keyword(s):

Myotonic Dystrophy ◽

Sport Activity ◽

Repeat Expansion ◽

Motor Neuropathy ◽

Myotonic Dystrophy Type ◽

Myotonic Dystrophy Type 2 ◽

Continuous Exercise ◽

Proximal Myotonic Myopathy ◽

Cctg Repeat

Objectives: A mild, slowly progressive course of proximal myotonic myopathy, also known as myotonic dystrophy type 2, over years allowing the patient to continue with extreme sport activity, has been only rarely reported. Methods: Case report. Results: The patient is a 54-year-old female sport teacher who developed myotonia of the distal upper limbs at the age of 32 years. Over the following 22 years, myotonia spreaded to the entire musculature. Myotonia did not prevent her from doing her job and from marathoning and improved with continuous exercise. Additionally, she had developed hypothyroidism, ovarial cysts, incipient cataract, motor neuropathy, hepatopathy, leukopenia, and mild hyper-CK-emia. A heterozygous CCTG-repeat expansion of 500–9500 was found in the CNBP/ZNF9 gene. At the age of 54 years, she was still performing sport, without presenting with myotonia on clinical examination or having developed other typical manifestations of proximal myotonic myopathy. Conclusions: This case shows that proximal myotonic myopathy may take a mild course over at least 22 years, that proximal myotonic myopathy with mild myotonia may allow a patient to continue strenuous sport activity, and that continuous physical activity may contribute to the mild course of the disease.

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Tethering-induced destabilization and ATP-binding for tandem RRM domains of ALS-causing TDP-43 and hnRNPA1

Scientific Reports ◽

10.1038/s41598-020-80524-6 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Mei Dang ◽

Yifan Li ◽

Jianxing Song

Keyword(s):

Conformational Dynamics ◽

Md Simulations ◽

Atp Binding ◽

Rna Recognition Motif ◽

Rna Recognition ◽

Nucleic Acid Binding ◽

General Role ◽

Nmr Characterization ◽

Lateral Sclerosis

AbstractTDP-43 and hnRNPA1 contain tandemly-tethered RNA-recognition-motif (RRM) domains, which not only functionally bind an array of nucleic acids, but also participate in aggregation/fibrillation, a pathological hallmark of various human diseases including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), alzheimer's disease (AD) and Multisystem proteinopathy (MSP). Here, by DSF, NMR and MD simulations we systematically characterized stability, ATP-binding and conformational dynamics of TDP-43 and hnRNPA1 RRM domains in both tethered and isolated forms. The results reveal three key findings: (1) upon tethering TDP-43 RRM domains become dramatically coupled and destabilized with Tm reduced to only 49 °C. (2) ATP specifically binds TDP-43 and hnRNPA1 RRM domains, in which ATP occupies the similar pockets within the conserved nucleic-acid-binding surfaces, with the affinity slightly higher to the tethered than isolated forms. (3) MD simulations indicate that the tethered RRM domains of TDP-43 and hnRNPA1 have higher conformational dynamics than the isolated forms. Two RRM domains become coupled as shown by NMR characterization and analysis of inter-domain correlation motions. The study explains the long-standing puzzle that the tethered TDP-43 RRM1–RRM2 is particularly prone to aggregation/fibrillation, and underscores the general role of ATP in inhibiting aggregation/fibrillation of RRM-containing proteins. The results also rationalize the observation that the risk of aggregation-causing diseases increases with aging.

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The unusual structure of the PiggyMac cysteine-rich domain reveals zinc finger diversity in PiggyBac-related transposases

Mobile DNA ◽

10.1186/s13100-021-00240-4 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Marc Guérineau ◽

Luiza Bessa ◽

Séverine Moriau ◽

Ewen Lescop ◽

François Bontems ◽

...

Keyword(s):

Zinc Finger ◽

Dna Cleavage ◽

Trichoplusia Ni ◽

Catalytic Domain ◽

Structural Diversity ◽

Paramecium Tetraurelia ◽

Lysine Methylation ◽

Unusual Structure ◽

Rich Domain ◽

Cysteine Rich Domain

Abstract Background Transposons are mobile genetic elements that colonize genomes and drive their plasticity in all organisms. DNA transposon-encoded transposases bind to the ends of their cognate transposons and catalyze their movement. In some cases, exaptation of transposon genes has allowed novel cellular functions to emerge. The PiggyMac (Pgm) endonuclease of the ciliate Paramecium tetraurelia is a domesticated transposase from the PiggyBac family. It carries a core catalytic domain typical of PiggyBac-related transposases and a short cysteine-rich domain (CRD), flanked by N- and C-terminal extensions. During sexual processes Pgm catalyzes programmed genome rearrangements (PGR) that eliminate ~ 30% of germline DNA from the somatic genome at each generation. How Pgm recognizes its DNA cleavage sites in chromatin is unclear and the structure-function relationships of its different domains have remained elusive. Results We provide insight into Pgm structure by determining the fold adopted by its CRD, an essential domain required for PGR. Using Nuclear Magnetic Resonance, we show that the Pgm CRD binds two Zn2+ ions and forms an unusual binuclear cross-brace zinc finger, with a circularly permutated treble-clef fold flanked by two flexible arms. The Pgm CRD structure clearly differs from that of several other PiggyBac-related transposases, among which is the well-studied PB transposase from Trichoplusia ni. Instead, the arrangement of cysteines and histidines in the primary sequence of the Pgm CRD resembles that of active transposases from piggyBac-like elements found in other species and of human PiggyBac-derived domesticated transposases. We show that, unlike the PB CRD, the Pgm CRD does not bind DNA. Instead, it interacts weakly with the N-terminus of histone H3, whatever its lysine methylation state. Conclusions The present study points to the structural diversity of the CRD among transposases from the PiggyBac family and their domesticated derivatives, and highlights the diverse interactions this domain may establish with chromatin, from sequence-specific DNA binding to contacts with histone tails. Our data suggest that the Pgm CRD fold, whose unusual arrangement of cysteines and histidines is found in all PiggyBac-related domesticated transposases from Paramecium and Tetrahymena, was already present in the ancestral active transposase that gave rise to ciliate domesticated proteins.

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REscan: inferring repeat expansions and structural variation in paired-end short read sequencing data

Bioinformatics ◽

10.1093/bioinformatics/btaa753 ◽

2020 ◽

Author(s):

Russell Lewis McLaughlin

Keyword(s):

Structural Variation ◽

Sequence Data ◽

Neurological Diseases ◽

Repeat Expansion ◽

Sequencing Data ◽

Short Read ◽

Short Read Sequencing ◽

Repeat Expansions ◽

Paired End Sequencing

Abstract Motivation Repeat expansions are an important class of genetic variation in neurological diseases. However, the identification of novel repeat expansions using conventional sequencing methods is a challenge due to their typical lengths relative to short sequence reads and difficulty in producing accurate and unique alignments for repetitive sequence. However, this latter property can be harnessed in paired-end sequencing data to infer the possible locations of repeat expansions and other structural variation. Results This article presents REscan, a command-line utility that infers repeat expansion loci from paired-end short read sequencing data by reporting the proportion of reads orientated towards a locus that do not have an adequately mapped mate. A high REscan statistic relative to a population of data suggests a repeat expansion locus for experimental follow-up. This approach is validated using genome sequence data for 259 cases of amyotrophic lateral sclerosis, of which 24 are positive for a large repeat expansion in C9orf72, showing that REscan statistics readily discriminate repeat expansion carriers from non-carriers. Availabilityand implementation C source code at https://github.com/rlmcl/rescan (GNU General Public Licence v3).

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Familial adult myoclonic epilepsy (FAME): clinical features, molecular characteristics, pathophysiological aspects and diagnostic work-up

Medizinische Genetik ◽

10.1515/medgen-2021-2100 ◽

2021 ◽

Vol 33 (4) ◽

pp. 311-318

Author(s):

Lorenz Peters ◽

Christel Depienne ◽

Stephan Klebe

Keyword(s):

Clinical Features ◽

Neurological Disorders ◽

Genetic Disorder ◽

Repeat Expansion ◽

Myoclonic Epilepsy ◽

Molecular Characteristics ◽

Autosomal Dominant Disorder ◽

Repeat Expansions ◽

Diagnostic Work ◽

Work Up

Abstract Familial adult myoclonic epilepsy (FAME) is a rare autosomal dominant disorder characterized by myoclonus and seizures. The genetic variant underlying FAME is an intronic repeat expansion composed of two different pentamers: an expanded TTTTA, which is the motif originally present at the locus, and an insertion of TTTCA repeats, which is usually located at the 3′ end and likely corresponds to the pathogenic part of the expansion. This repeat expansion has been identified so far in six genes located on different chromosomes, which remarkably encode proteins with distinct cellular localizations and functions. Although the exact pathophysiological mechanisms remain to be clarified, it is likely that FAME repeat expansions lead to disease independently of the gene where they occur. We herein review the clinical and molecular characteristics of this singular genetic disorder, which interestingly shares clinical features with other more common neurological disorders whose etiology remains mainly unsolved.

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