scholarly journals Comprehensive Molecular Analysis of DMD Gene Increases the Diagnostic Value of Dystrophinopathies: A Pilot Study in a Southern Italy Cohort of Patients

Diagnostics ◽  
2021 ◽  
Vol 11 (10) ◽  
pp. 1910
Author(s):  
Fatima Domenica Elisa De Palma ◽  
Marcella Nunziato ◽  
Valeria D'Argenio ◽  
Maria Savarese ◽  
Gabriella Esposito ◽  
...  
Keyword(s):  
Molecular Analysis ◽  
Molecular Diagnosis ◽  
Point Mutations ◽  
Diagnostic Procedures ◽  
Becker Muscular Dystrophy ◽  
Diagnostic Value ◽  
Single Step ◽  
Large Deletions ◽  
Human Genes ◽  
Dmd Gene

Duchenne/Becker muscular dystrophy (DMD/BMD) is an X-linked neuromuscular disease due to pathogenic sequence variations in the dystrophin (DMD) gene, one of the largest human genes. More than 70% of DMD gene defects result from genomic rearrangements principally leading to large deletions, while the remaining are small nucleotide variants, including nonsense and missense variants, small insertions/deletions or splicing alterations. Considering the large size of the gene and the wide mutational spectrum, the comprehensive molecular diagnosis of DMD/BMD is complex and may require several laboratory methods, thus increasing the time and costs of the analysis. In an attempt to simplify DMD/BMD molecular diagnosis workflow, we tested an NGS method suitable for the detection of all the different types of genomic variations that may affect the DMD gene. Forty previously analyzed patients were enrolled in this study and re-analyzed using the next generation sequencing (NGS)-based single-step procedure. The NGS results were compared with those from multiplex ligation-dependent probe amplification (MLPA)/multiplex PCR and/or Sanger sequencing. Most of the previously identified deleted/duplicated exons and point mutations were confirmed by NGS and 1 more pathogenic point mutation (a nonsense variant) was identified. Our results show that this NGS-based strategy overcomes limitations of traditionally used methods and is easily transferable to routine diagnostic procedures, thereby increasing the diagnostic power of DMD molecular analysis.

scholarly journals Sequence and Structure Characteristics of 22 Deletion Breakpoints in Intron 44 of the DMD Gene Based on Long-Read Sequencing

2021 ◽  
Vol 12 ◽  
Author(s):  
Chang Geng ◽  
Yuanren Tong ◽  
Siwen Zhang ◽  
Chao Ling ◽  
Xin Wu ◽  
...  
Keyword(s):  
Becker Muscular Dystrophy ◽  
Repetitive Elements ◽  
Loop Formation ◽  
End Joining ◽  
Origin Firing ◽  
Large Deletions ◽  
Long Read ◽  
Non Homologous End Joining ◽  
Dmd Gene ◽  
Palindromic Sequences

Purpose: Exon deletions make up to 80% of mutations in the DMD gene, which cause Duchenne and Becker muscular dystrophy. Exon 45-55 regions were reported as deletion hotspots and intron 44 harbored more than 25% of deletion start points. We aimed to investigate the fine structures of breakpoints in intron 44 to find potential mechanisms of large deletions in intron 44.Methods: Twenty-two dystrophinopathy patients whose deletion started in intron 44 were sequenced using long-read sequencing of a DMD gene capture panel. Sequence homology, palindromic sequences, and polypyrimidine sequences were searched at the breakpoint junctions. RepeatMasker was used to analyze repetitive elements and Mfold was applied to predict secondary DNA structure.Results: With a designed DMD capture panel, 22 samples achieved 2.25 gigabases and 1.28 million reads on average. Average depth was 308× and 99.98% bases were covered at least 1×. The deletion breakpoints in intron 44 were scattered and no breakpoints clustered in any region less than 500 bp. A total of 72.7% of breakpoints located in distal 100 kb of intron 44 and more repetitive elements were found in this region. Microhomologies of 0–1 bp were found in 36.4% (8/22) of patients, which corresponded with non-homologous end-joining. Microhomologies of 2–20 bp were found in 59.1% (13/22) of patients, which corresponded with microhomology-mediated end-joining. Moreover, a 7 bp insertion was found in one patient, which might be evidence of aberrant replication origin firing. Palindromic sequences, polypyrimidine sequences, and small hairpin loops were found near several breakpoint junctions. No evidence of large hairpin loop formation in deletion root sequences was observed.Conclusion: This study was the first to explore possible mechanisms underlying exon deletions starting from intron 44 of the DMD gene based on long-read sequencing. Diverse mechanisms might be associated with deletions in the DMD gene.

scholarly journals Molecular Diagnostics of Duchenne/Becker Muscular Dystrophy Patients by Multiplex Ligation-Dependent Probe Amplification Analysis and Direct Sequencing

2009 ◽  
Vol 12 (2) ◽  
pp. 3-9
Author(s):  
A Todorova ◽  
V Guergueltcheva ◽  
J Genova ◽  
V Mihaylova ◽  
T Todorov ◽  
...  
Keyword(s):  
Muscular Dystrophy ◽  
Molecular Diagnostics ◽  
Direct Sequencing ◽  
Point Mutations ◽  
Direct Analysis ◽  
Index Patient ◽  
Becker Muscular Dystrophy ◽  
Carrier Status ◽  
Dmd Gene ◽  
Dependent Probe

Molecular Diagnostics of Duchenne/Becker Muscular Dystrophy Patients by Multiplex Ligation-Dependent Probe Amplification Analysis and Direct SequencingDuchenne/Becker muscular dystrophy (DMD/BMD), the most common X-linked muscular dystrophy is caused by mutations in the enormously large DMD gene. We screened this gene in 51 unrelated Bulgarian DMD/BMD patients and four families with no living index patient available, by multiplex ligation-dependent probe amplification (MLPA) analysis, which is a powerful tool for detecting deletion/duplication along the DMD gene. This, in combination with direct sequencing, characterized the mutation in all patients, which comprised 42 deletions (82%), six duplications (12%) and three point mutations (6%), and precisely determined all deletion/duplication borders. In all the families with no living index patient available, deletions were detected by direct analysis on the patients' mothers and sisters, proving the value of MLPA for carrier status determination.

scholarly journals Activation-induced Deaminase (AID)-directed Hypermutation in the Immunoglobulin Sμ Region

2002 ◽  
Vol 195 (4) ◽  
pp. 529-534 ◽  
Author(s):  
Hitoshi Nagaoka ◽  
Masamichi Muramatsu ◽  
Namiko Yamamura ◽  
Kazuo Kinoshita ◽  
Tasuku Honjo
Keyword(s):  
B Lymphocytes ◽  
Single Molecule ◽  
Dna Cleavage ◽  
Somatic Hypermutation ◽  
Point Mutations ◽  
Genetic Alterations ◽  
Variable Regions ◽  
Class Switch ◽  
Large Deletions ◽  
Activation Induced Deaminase

Somatic hypermutation (SHM) and class switch recombination (CSR) cause distinct genetic alterations at different regions of immunoglobulin genes in B lymphocytes: point mutations in variable regions and large deletions in S regions, respectively. Yet both depend on activation-induced deaminase (AID), the function of which in the two reactions has been an enigma. Here we report that B cell stimulation which induces CSR but not SHM, leads to AID-dependent accumulation of SHM-like point mutations in the switch μ region, uncoupled with CSR. These findings strongly suggest that AID itself or a single molecule generated by RNA editing function of AID may mediate a common step of SHM and CSR, which is likely to be involved in DNA cleavage.

scholarly journals Mitochondrial DNA Somatic Mutations (Point Mutations and Large Deletions) and Mitochondrial DNA Variants in Human Thyroid Pathology

2002 ◽  
Vol 160 (5) ◽  
pp. 1857-1865 ◽  
Author(s):  
Valdemar Máximo ◽  
Paula Soares ◽  
Jorge Lima ◽  
José Cameselle-Teijeiro ◽  
Manuel Sobrinho-Simões
Keyword(s):  
Mitochondrial Dna ◽  
Somatic Mutations ◽  
Point Mutations ◽  
Human Thyroid ◽  
Thyroid Pathology ◽  
Large Deletions ◽  
Dna Variants ◽  
Mitochondrial Dna Variants

scholarly journals Analysis of Dystrophin Gene Deletions by Multiplex PCR in Moroccan Patients

2002 ◽  
Vol 2 (3) ◽  
pp. 158-160 ◽  
Author(s):  
Aziza Sbiti ◽  
Fatiha El Kerch ◽  
Abdelaziz Sefiani
Keyword(s):  
Hot Spots ◽  
Neuromuscular Disease ◽  
Multiplex Polymerase Chain Reaction ◽  
Dystrophin Gene ◽  
Becker Muscular Dystrophy ◽  
Chain Reaction ◽  
Gene Deletions ◽  
Polymerase Chain ◽  
Dmd Gene ◽  
Moroccan Patients

Duchenne and Becker muscular dystrophy (DMD and BMD) are X-linked diseases resulting from a defect in the dystrophin gene located on Xp21. DMD is the most frequent neuromuscular disease in humans (1/3500 male newborn). Deletions in the dystrophin gene represent 65% of mutations in DMD/BMD patients. We have analyzed DNA from 72 Moroccan patients with DMD/BMD using the multiplex polymerase chain reaction (PCR) to screen for exon deletions within the dystrophin gene, and to estimate the frequency of these abnormalities. We found dystrophin gene deletions in 37 cases. Therefore the frequency in Moroccan DMD/BMD patients is about 51.3%. All deletions were clustered in the two known hot-spots regions, and in 81% of cases deletions were detected in the region from exon 43 to exon 52. These findings are comparable to those reported in other studies. It is important to note that in our population, we can first search for deletions of DMD gene in the most frequently deleted exons determined by this study. This may facilitate the molecular diagnosis of DMD and BMD in our country.

Dystrophinopathy in a Family Due to a Rare Nonsense Mutation Causing Predominant Behavioral Phenotype

2019 ◽  
Vol 18 (04) ◽  
pp. 210-213
Author(s):  
Yohei Harada ◽  
Seth T. Sorensen ◽  
Akilandeswari Aravindhan ◽  
Vikki Stefans ◽  
Aravindhan Veerapandiyan
Keyword(s):  
Intellectual Disability ◽  
Muscular Dystrophy ◽  
Behavioral Problems ◽  
Nonsense Mutation ◽  
Neuromuscular Disorders ◽  
Elevated Serum ◽  
Becker Muscular Dystrophy ◽  
Genetic Changes ◽  
Dmd Gene ◽  
Neurobehavioral Deficits

AbstractDystrophinopathies are a group of X-linked neuromuscular disorders resulting from mutations in DMD gene that encodes dystrophin. The clinical spectrum includes Duchenne muscular dystrophy, Becker muscular dystrophy, X-linked cardiomyopathy, and intellectual disability without involvement of skeletal muscle. Cognitive and behavioral problems are commonly seen among patients with dystrophinopathy. DMD gene is the largest human gene, consisting of 79 exons that produce dystrophin protein. Patients with genetic changes involving shorter dystrophin isoforms such as Dp140 and Dp71 are suggested to have higher rates of intellectual disability, attention-deficit/hyperactivity disorder, and other neuropsychiatric comorbidities. We describe three brothers who presented with prominent neurobehavioral deficits of varying degree, mild proximal weakness, and elevated serum creatine kinase due to a rare nonsense mutation, c.1702C > T; p.Gln568X, in exon 14 of DMD gene. Further studies are needed to better understand the effects of this rare mutation.

scholarly journals Genotype–Phenotype Correlations in Duchenne and Becker Muscular Dystrophy Patients from the Canadian Neuromuscular Disease Registry

2020 ◽  
Vol 10 (4) ◽  
pp. 241
Author(s):  
Kenji Rowel Q. Lim ◽  
Quynh Nguyen ◽  
Toshifumi Yokota
Keyword(s):  
Muscular Dystrophy ◽  
Neuromuscular Disease ◽  
Becker Muscular Dystrophy ◽  
Neuromuscular Disorder ◽  
Test Results ◽  
Disease Registry ◽  
Reading Frame ◽  
Negative Results ◽  
Genetic Test Results ◽  
Dmd Gene

Duchenne muscular dystrophy (DMD) is a fatal neuromuscular disorder generally caused by out-of-frame mutations in the DMD gene. In contrast, in-frame mutations usually give rise to the milder Becker muscular dystrophy (BMD). However, this reading frame rule does not always hold true. Therefore, an understanding of the relationships between genotype and phenotype is important for informing diagnosis and disease management, as well as the development of genetic therapies. Here, we evaluated genotype–phenotype correlations in DMD and BMD patients enrolled in the Canadian Neuromuscular Disease Registry from 2012 to 2019. Data from 342 DMD and 60 BMD patients with genetic test results were analyzed. The majority of patients had deletions (71%), followed by small mutations (17%) and duplications (10%); 2% had negative results. Two deletion hotspots were identified, exons 3–20 and exons 45–55, harboring 86% of deletions. Exceptions to the reading frame rule were found in 13% of patients with deletions. Surprisingly, C-terminal domain mutations were associated with decreased wheelchair use and increased forced vital capacity. Dp116 and Dp71 mutations were also linked with decreased wheelchair use, while Dp140 mutations significantly predicted cardiomyopathy. Finally, we found that 12.3% and 7% of DMD patients in the registry could be treated with FDA-approved exon 51- and 53-skipping therapies, respectively.

scholarly journals Intronic Alternative Polyadenylation in the Middle of the DMD Gene Produces Half-Size N-Terminal Dystrophin with a Potential Implication of ECG Abnormalities of DMD Patients

2020 ◽  
Vol 21 (10) ◽  
pp. 3555
Author(s):  
Abdul Qawee Mahyoob Rani ◽  
Tetsushi Yamamoto ◽  
Tatsuya Kawaguchi ◽  
Kazuhiro Maeta ◽  
Hiroyuki Awano ◽  
...  
Keyword(s):  
Pcr Amplification ◽  
Alternative Polyadenylation ◽  
Coding Region ◽  
Terminal Exon ◽  
Potential Implication ◽  
Human Genes ◽  
Cardiac Muscles ◽  
Rapid Amplification ◽  
Dmd Gene ◽  
Ecg Abnormalities

The DMD gene is one of the largest human genes, being composed of 79 exons, and encodes dystrophin Dp427m which is deficient in Duchenne muscular dystrophy (DMD). In some DMD patient, however, small size dystrophin reacting with antibody to N-terminal but not to C-terminal has been identified. The mechanism to produce N-terminal small size dystrophin remains unknown. Intronic polyadenylation is a mechanism that produces a transcript with a new 3′ terminal exon and a C-terminal truncated protein. In this study, intronic alternative polyadenylation was disclosed to occur in the middle of the DMD gene and produce the half-size N-terminal dystrophin Dp427m, Dpm234. The 3′-rapid amplification of cDNA ends revealed 421 bp sequence in the downstream of DMD exon 41 in U-251 glioblastoma cells. The cloned sequence composing of the 5′ end sequence of intron 41 was decided as the terminal exon, since it encoded poly (A) signal followed by poly (A) stretch. Subsequently, a fragment from DMD exon M1 to intron 41 was obtained by PCR amplification. This product was named Dpm234 after its molecular weight. However, Dpm234 was not PCR amplified in human skeletal and cardiac muscles. Remarkably, Dpm234 was PCR amplified in iPS-derived cardiomyocytes. Accordingly, Western blotting of cardiomyocyte proteins showed a band of 234 kDa reacting with dystrophin antibody to N-terminal, but not C-terminal. Clinically, DMD patients with mutations in the Dpm234 coding region were found to have a significantly higher likelihood of two ECG abnormal findings. Intronic alternative splicing was first revealed in Dp427m to produce small size dystrophin.

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