scholarly journals Diagnosis of Duchenne/Becker muscular dystrophy and quantitative identification of carrier status by use of entangled solution capillary electrophoresis

1997 ◽  
Vol 43 (5) ◽  
pp. 745-751 ◽  
Author(s):  
Paolo Fortina ◽  
Jing Cheng ◽  
Mann A Shoffner ◽  
Saul Surrey ◽  
Wendy M Hitchcock ◽  
...  
Keyword(s):  
Capillary Electrophoresis ◽  
Muscular Dystrophy ◽  
Genetic Diseases ◽  
Becker Muscular Dystrophy ◽  
Diagnostic Methods ◽  
Carrier Status ◽  
Laser Induced Fluorescence Detection ◽  
General Utility ◽  
Pcr Products ◽  
Quantitative Results

Abstract Use of capillary electrophoresis, a new and useful analytical tool, offers a variety of advantages for nucleic acid analyses, including rapid analysis, automation, high resolution, qualitative and quantitative results, and low consumption of both sample and reagents. We report the first example of the use of entangled solution capillary electrophoresis (ESCE) and laser-induced fluorescence detection (LIF) for separation-based diagnostics in the quantitative analysis of multiplex PCR products for determination of carrier status of Duchenne/Becker muscular dystrophy (DMD/BMD). This ap-proach greatly improved the speed, resolution, and sensitivity of information needed for the diagnosis of DMD/BMD compared with that from conventional diagnostic methods, and is of general utility for diagnosis of genetic diseases.

scholarly journals New approach for diagnostic of Facioscapulohumeral muscular dystrophy based on PCR

2019 ◽  
pp. 3-9
Author(s):  
Н.В. Зернов ◽  
А.А. Гуськова ◽  
М.Ю. Скоблов
Keyword(s):  
Muscular Dystrophy ◽  
Molecular Genetic ◽  
Facioscapulohumeral Muscular Dystrophy ◽  
Diagnostic Methods ◽  
Pulse Field ◽  
Agarose Gel ◽  
New Approach ◽  
Pulse Field Gel Electrophoresis ◽  
Pcr Products ◽  
The Moment

Актуальность. Миодистрофия Ландузи-Дежерина (МЛД) является одной из наиболее часто встречающихся мышечных дистрофий. В 95% случаев заболевание связано с частичной делецией массива повторов D4Z4 на одном из аллелей 4-й хромосомы. Существующие диагностические методики гибридизации по Саузерну и молекулярного комбинга являются ресурсо- и времязатратными. В настоящее время в Российской Федерации молекулярно-генетическая диагностика МЛД не проводится. Цель. Поиск новых подходов к диагностике МЛД для использования в молекулярно-генетических лабораториях. Методы. ДНК выделялась в агарозных блоках и подвергалась обработке эндонуклеазой EcoRI. Полученные фрагменты ДНК разделялись методом пульс-электрофореза в агарозном геле, после этого агарозный гель фрагментировался согласно маркеру молекулярного веса и использовался в качестве матрицы для полимеразной цепной реакции (ПЦР). Принадлежность полученных ПЦР-продуктов к последовательностям повторов D4Z4 4-й хромосомы подтверждалась секвенированием по Сэнгеру. Результаты. Протокол пульс-электрофореза оптимизирован таким образом, что после всех этапов ДНК в агарозном геле пригодна для использования в качестве матрицы для ПЦР. Разработана ПЦР-система специфичной амплификации контрольных ДНК-матриц 4-й хромосомы и подтверждена секвенированием принадлежность получаемых ПЦР-продуктов к последовательности повторов D4Z4 4-й хромосомы. Выводы. Показана возможность использования ДНК в агарозном геле после пульс-электрофореза в качестве матрицы для детекции повторов D4Z4 методом ПЦР. Представленная ПЦР-система специфично амплифицирует последовательности D4Z4 4-й хромосомы. Используя данную ПЦР-систему и геномную ДНК пациента с известной длиной массива повторов D4Z4 проведена успешная диагностика МЛД. Таким образом разработан новый подход к диагностике МЛД для использования в молекулярно-генетических лабораториях. Relevance. Facioscapulohumeral muscular dystrophy (FSHD) is one of the most common muscular dystrophies. In 95% of cases, the disease is associated with partial deletion of the array of the D4Z4 repeats on one of the alleles of the 4th chromosome. The existing diagnostic methods of Southern blotting and molecular combing are quite resource-and time-consuming. At the moment, molecular genetic diagnostic of FSHD is not provided on the territory of the Russian Federation. Aim: to find new approaches for molecular genetic diagnostic of FSHD acceptable for use in standard molecular genetic laboratories Materials and methods: DNA isolated in agarose plugs and treated by the EcoRI restriction enzyme. DNA fragments then were separated by pulse field gel electrophoresis (PFGE) in agarose gel. After PFGE, the agarose gel was fragmented and used as a matrix for PCR. The identity of the obtained PCR products to the sequence of the D4Z4 repeats of the 4th chromosome was confirmed by sequencing by Sanger. Results. The PFGE protocol is optimized in such a way that, after all stages, DNA in agarose gel is suitable for use as a matrix for PCR. We achieve a specific amplification of the control DNA matrices of the 4th chromosome and confirm belonging of the PCR products to the sequence of D4Z4 repeats of the 4th chromosome by the Senger sequencing. Conclusions. This paper shows the possibility of using DNA in agarose gel after PFGE as a matrix for detection of D4Z4 repeats by PCR. The presented PCR system specifically amplify sequence of the 4th chromosome D4Z4 repeats. Using this PCR system and genomic DNA of a patient with a known length of the D4Z4 repeats array, a successful diagnosis of FSHD was performed. Thus, we propose a new approach for FSHD diagnostic, acceptable for use in standard molecular genetic laboratories.

scholarly journals Quantitative Assay of Deletion or Duplication Genotype by Capillary Electrophoresis System: Application in Prader–Willi Syndrome and Duchenne Muscular Dystrophy

2006 ◽  
Vol 52 (12) ◽  
pp. 2203-2210 ◽  
Author(s):  
Chia-Cheng Hung ◽  
Chih-Ping Chen ◽  
Shuan-Pei Lin ◽  
Shu-Chin Chien ◽  
Chien-Nan Lee ◽  
...  
Keyword(s):  
Capillary Electrophoresis ◽  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Quantitative Pcr ◽  
Genetic Diagnosis ◽  
Carrier Status ◽  
Prader Willi Syndrome ◽  
Clinical Genetic ◽  
Genomic Deletions ◽  
Dmd Gene

Abstract Background: Deletions and duplications involving large DNA segments result in underexpression or overexpression, depending on the changes in allele dose, and are known to cause many common disorders. Detection of allele dose variations in the human genome is increasingly important in medical genetic diagnosis. Methods: We used multiplex quantitative PCR coupled with capillary electrophoresis for accurate allele dose determination. In cases of Prader–Willi syndrome (PWS), a total of 24 patients with PWS, as well as 205 control individuals from the general population, were analyzed by use of multiplex quantitative PCR to amplify the FGFR2 gene, the KRIT1 gene, and the SNRPN gene simultaneously. In cases of Duchenne muscular dystrophy (DMD), we optimized the multiplex quantitative PCR to amplify 38 exons to analyze the DMD gene for rapid diagnosis of 12 DMD-affected males, 12 obligate carriers from families, and 50 unaffected female controls. Results: We were able to unambiguously diagnose the deletion genotype in PWS patients and identify all deletion or duplication genotypes and carrier status in DMD-affected cases with 100% sensitivity and specificity. Conclusions: This report describes a novel single assay that can rapidly quantify allele dose to provide accurate clinical genetic diagnosis. This technique offers a valuable alternative for the rapid detection of genomic deletions or duplications and decreases costs because it does not require expensive fluorescent reagents.

Molecular probe protocol for determining carrier status in Duchenne and Becker muscular dystrophies

1990 ◽  
Vol 36 (3) ◽  
pp. 441-445 ◽  
Author(s):  
T W Prior ◽  
K J Friedman ◽  
W E Highsmith ◽  
T R Perry ◽  
L M Silverman
Keyword(s):  
Muscular Dystrophy ◽  
Fragment Length Polymorphism ◽  
Direct Analysis ◽  
Becker Muscular Dystrophy ◽  
Molecular Probe ◽  
Carrier Status ◽  
Deletion Rate ◽  
New Strategy ◽  
Polymerase Chain ◽  
Specific Deletion

Abstract By use of cDNA probes, molecular deletions were identified in 66.6% of 42 patients with Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD). Owing to this high deletion rate, a new strategy for detecting DMD/BMD carriers is feasible in which the polymerase chain reaction is used as an initial screen for detecting the deletions occurring in specific deletion-prone exons. Because the deletions do not occur randomly, specific cDNA probes are utilized first with Southern blot analysis. Identification of a deletion permits direct analysis for DMD carrier status and removes the inherent limitations of the conventional restriction fragment length polymorphism technique. Carrier status is determined by scanning the autoradiographs with a densitometric spectrophotometer or by detection of a junction fragment.

scholarly journals Knowledge of carrier status and barriers to testing among mothers of sons with Duchenne or Becker muscular dystrophy

2016 ◽  
Vol 26 (12) ◽  
pp. 860-864 ◽  
Author(s):  
Lauren Bogue ◽  
Holly Peay ◽  
Ann Martin ◽  
Ann Lucas ◽  
Sindhu Ramchandren
Keyword(s):  
Muscular Dystrophy ◽  
Becker Muscular Dystrophy ◽  
Carrier Status

scholarly journals Analysis of duplications versus deletions in the dystrophin gene in Serbian cohort with dystrophinopathies

2020 ◽  
Vol 77 (4) ◽  
pp. 387-394
Author(s):  
Jasmina Maksic ◽  
Valerija Dobricic ◽  
Lukas Rasulic ◽  
Nela Maksimovic ◽  
Marija Branakovic ◽  
...  
Keyword(s):  
Muscular Dystrophy ◽  
De Novo ◽  
Dystrophin Gene ◽  
Becker Muscular Dystrophy ◽  
Severe Form ◽  
De Novo Mutation ◽  
Carrier Status ◽  
Phenotypic Effect ◽  
Exon 2 ◽  
Large Deletions

Background/Aim. Duchenne muscular dystrophy (DMD) and its allelic form Becker muscular dystrophy (BMD) are X-linked diseases that affect males, characterized by progressive muscle and cardiopulmonary weakness, especially in DMD as a severe form of the disease. They result from mutations in the dystrophin gene, and the most common changes are large intragenic deletions and duplications (80%). One third of patients have de novo mutation and 2/3 of the mothers are estimated as carriers. The aim of the study was to analyze the frequency of duplications versus deletions in the dystrophin gene in patients with dystrophinopathies, as well as to analyze the phenotypic effect of large mutations obtained and to determine the carrier status of female relatives in probands with duplications. Methods. We examined 22 DMD and 35 BMD unrelated patients and 6 female relatives of the probands where duplications were found. We used polymerase chain reaction (PCR) and multiplex ligation-dependent probe amplification (MLPA) methods, according to the protocol, to detect or confirm mutations in probands and female carriers. Results. In probands, there were 34 (59.6%) large deletions (mostly affected exons 44?60) and 6 (10.5%) large duplications in 4 DMD and 2 BMD patients. Also, duplications were found in 3 out of 4 (75%) tested mothers. The distribution of duplications was heterogeneous, affecting N-terminal and central rod domain, and included more exons, except for one DMD patient who had duplication of exon 2. An exception from the Monaco rule was present in 9.5% of DMD and 15.8% of BMD probands, i.e. in 12.5% of DMD/BMD cases. Conclusion. In 57 DMD/BMD probands, we found 59.6% of large deletions and 10.5% of large duplications. The most affected region of the DMD gene was the central rod domain. An exception to Monaco''s rule was present in 12.5% of DMD/BMD cases. Three out of 4 examined proband''s mothers were confirmed as carriers.

Impact of carrier status determination for Duchenne/Becker muscular dystrophy by computer-assisted laser densitometry

1998 ◽  
Vol 75 (2) ◽  
pp. 171-175 ◽  
Author(s):  
Diane J. Allingham-Hawkins ◽  
Leslie K. McGlynn-Steele ◽  
Charlotte A. Brown ◽  
Joanne Sutherland ◽  
Peter N. Ray
Keyword(s):  
Muscular Dystrophy ◽  
Becker Muscular Dystrophy ◽  
Computer Assisted ◽  
Carrier Status ◽  
Laser Densitometry

scholarly journals Simultaneous determination of methylenetetrahydrofolate reductase C677T and factor V G1691A genotypes by mutagenically separated PCR and multiple-injection capillary electrophoresis

1998 ◽  
Vol 44 (2) ◽  
pp. 264-269 ◽  
Author(s):  
Arve Ulvik ◽  
Jicun Ren ◽  
Helga Refsum ◽  
Per Magne Ueland
Keyword(s):  
Capillary Electrophoresis ◽  
Methylenetetrahydrofolate Reductase ◽  
Factor V ◽  
Multiple Injection ◽  
Laser Induced Fluorescence Detection ◽  
Common Genetic Variants ◽  
Pcr Products ◽  
Factor V G1691a ◽  
C677t Mutation ◽  
Factor V Gene

Abstract The C677T mutation of the methylenetetrahydrofolate reductase gene and the G1691A (Leiden) mutation of the factor V gene are established risk factors for thromboembolic disease. We here present an assay for the simultaneous genotyping of these common genetic variants. The assay involves a strategy based on multiplex mutagenically separated PCR performed in a single tube containing six primers. Separation of the resulting four PCR products (197, 207, 233, and 246 bp) was performed by capillary electrophoresis coupled with laser-induced fluorescence detection. The time for the automated electrophoresis was reduced to 2.5 min per sample by performing the capillary electrophoresis analysis in a multiple-injection mode.

Determination of carrier status in Duchenne and Becker muscular dystrophies by quantitative polymerase chain reaction and allele-specific oligonucleotides

1990 ◽  
Vol 36 (12) ◽  
pp. 2113-2117 ◽  
Author(s):  
T W Prior ◽  
A C Papp ◽  
P J Snyder ◽  
W E Highsmith ◽  
K J Friedman ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Muscular Dystrophy ◽  
Gene Dosage ◽  
Quantitative Polymerase Chain Reaction ◽  
Becker Muscular Dystrophy ◽  
Carrier Status ◽  
Chain Reaction ◽  
Allele Specific ◽  
Polymerase Chain

Abstract Detection of carriers of Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD), in the deletion cases, involves calculating gene dosage from Southern blots. We show that the analysis of dosage can also be made from the polymerase chain reaction (PCR) with use of allele-specific oligonucleotides (ASOs). The deletion-prone exons are amplified, transferred to a membrane, and hybridized with ASOs complementary to the exons; the autoradiographic bands are then quantified with a densitometer. After determining the quantitative conditions of the amplification reaction, we were able to identify deletions in a DMD/BMD carrier female. The determination of carrier status via PCR removes several of the technical limitations of Southern analysis and is also cost- and labor-effective.

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.

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