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 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 MicroRNA role in hereditary genetic diseases

2020 ◽  
pp. 5-17
Author(s):  
О.М. Плотникова ◽  
М.Ю. Скоблов
Keyword(s):  
Gene Expression ◽  
Muscular Dystrophy ◽  
Molecular Mechanisms ◽  
Genetic Diseases ◽  
Hirschsprung Disease ◽  
Facioscapulohumeral Muscular Dystrophy ◽  
Diagnostic Methods ◽  
Hereditary Diseases ◽  
Beta Thalassemia ◽  
Genes Expression

На сегодняшний день известно около 7000 наследственных заболеваний. Однако современные методы ДНК диагностики выявляют причину возникновения заболеваний примерно в 40% случаев. Отчасти это обусловлено сложностью и большим разнообразием молекулярных механизмов их патогенеза. МикроРНК являются одним из мощнейших регуляторов экспрессии генов. Однако участие их в патогенезе наследственных заболеваний пока недостаточно изучено из-за сложностей поиска таких нарушений. В данной работе проведён анализ описанных механизмов патогенеза наследственных заболеваний, опосредованных нарушениями регуляции экспрессии генов посредством микроРНК. Такие случаи были выявлены при таких наследственных заболеваниях как муковисцидоз, миодистрофия Дюшенна, бета-талассемия, глаукома, лице-лопаточно-плечевая миодистрофия Ландузи-Дежерина, болезнь Гиршпрунга, синдром Ретта, синдром Туретта, пемфигус (болезнь Хейли-Хейли). To date, about 7,000 hereditary diseases are known. However, modern diagnostic methods reveal the cause of the disease in about 40% of cases. This is partly due to the complexity and wide variety of molecular mechanisms of pathogenesis. MicroRNAs are one of the most powerful genes expression regulators. But their participation in the pathogenesis of hereditary diseases has not yet been studied enough because of the difficulties in finding such disorders. In this work, we collected and analyzed pathogenesis of hereditary diseases mediated by dysregulation of gene expression by microRNA. such cases have been identified for such hereditary diseases as cystic fibrosis, Duchenne muscular dystrophy, beta-thalassemia, glaucoma, facioscapulohumeral muscular dystrophy Landouzy-Dejerine, Hirschsprung disease, Rett syndrome, Tourette syndrome, pemphigus (Hailey-Hailey disease).

scholarly journals Potable water and nosocomial Legionnaires' disease – check water from all rooms in which patient has stayed

1995 ◽  
Vol 114 (2) ◽  
pp. 267-276 ◽  
Author(s):  
T. J. Marrie ◽  
W. Johnson ◽  
S. Tyler ◽  
G. Bezanson ◽  
D. Haldane ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Gel Electrophoresis ◽  
Potable Water ◽  
Pulse Field ◽  
Agarose Gel ◽  
Agarose Gel Electrophoresis ◽  
Pulse Field Gel Electrophoresis ◽  
Potable Water Supply ◽  
Legionnaires Disease ◽  
The Relationship

SummaryWe studied 7 patients with nosocomial Legionnaires' disease to determine the relationship between isolates ofLegionella pneumophilarecovered from potable water and those recovered from patients. Potable water was cultured from all rooms in which patients had stayed prior to the diagnosis of Legionnaires' disease. The 38 isolates ofL. pneumophila(31 environmental, 7 patient) were resolved into 9 distinct patterns by pulse-field gel electrophoresis (PFGE), 3 by plasmid content and 2 each with monoclonal antibodies and conventional agarose gel electrophoresis of small fragments of DNA.Using PFGE it was determined that 4 of the 7 patients were infected withL. pneumophilaidentical to an isolate recovered from the potable water supply in one of the rooms each had occupied prior to the diagnosis of Legionnaires' disease. Patients had resided in a mean of 3·57 rooms before a diagnosis of nosocomial Legionnaires' disease. We conclude that in the setting of contaminated potable water and nosocomial Legionnaires' disease water from all the rooms which the patient has occupied prior to this diagnosis should be cultured. PFGE of large DNA fragments discriminated best among the isolates ofL. pneumophila.

scholarly journals Orofacial Muscle Weakening in Facioscapulohumeral Muscular Dystrophy (FSHD) Patients

Children ◽  
2022 ◽  
Vol 9 (1) ◽  
pp. 96
Author(s):  
Dimitrios Konstantonis ◽  
Kyriaki Kekou ◽  
Petros Papaefthymiou ◽  
Heleni Vastardis ◽  
Nikoleta Konstantoni ◽  
...  
Keyword(s):  
Muscular Dystrophy ◽  
Muscle Weakness ◽  
Fragment Size ◽  
Molecular Genetic ◽  
Muscular Activity ◽  
Facioscapulohumeral Muscular Dystrophy ◽  
Molecular Genetic Analysis ◽  
Family Status ◽  
Facial Growth ◽  
Repeat Array

Background: Facioscapulohumeral muscular dystrophy is the third most commonly found type of muscular dystrophy. The aim of this study was to correlate the D4Z4 repeat array fragment size to the orofacial muscle weakening exhibited in a group of patients with a genetically supported diagnosis of FSHD. Methods: Molecular genetic analysis was performed for 52 patients (27 female and 25 male) from a group that consisted of 36 patients with autosomal dominant pedigrees and 16 patients with either sporadic or unknown family status. The patients were tested with the southern blotting technique, using EcoRI/Avrll double digestion, and fragments were detected by a p13E-11 telomeric probe. Spearman’s correlation was used to compare the fragment size with the degree of muscle weakening found in the forehead, periocular and perioral muscles. Results: A positive non-significant correlation between the DNA fragment size and severity of muscle weakness was found for the forehead (r = 0.27; p = 0187), the periocular (r = 0.24; p = 0.232) and the left and right perioral (r = 0.29; p = 0.122), (r = 0.32; p = 0.085) muscles. Conclusions: Although FSHD patients exhibited a decrease in muscular activity related to the forehead, perioral, and periocular muscles the genotype–phenotype associations confirmed a weak to moderate non-significant correlation between repeat size and the severity of muscle weakness. Orofacial muscle weakening and its association with a D4Z4 contraction alone may not have the significance to serve as a prognostic biomarker, due to the weak to moderate association. Further studies with larger sample sizes are needed to determine the degree of genetic involvement in the facial growth in FSHD patients.

scholarly journals Difficulties in diagnosing amyotrophic lateral sclerosis with lumbosacral debut

2021 ◽  
Vol 9 (3) ◽  
pp. 463-470
Author(s):  
G.A. Leonov ◽  
◽  
A.S. Solomatinа ◽  
A.O. Burshinov ◽  
◽  
...  
Keyword(s):  
Amyotrophic Lateral Sclerosis ◽  
Motor Neuron ◽  
Molecular Genetic ◽  
Genetic Research ◽  
Diagnostic Methods ◽  
Motor Neuron Degeneration ◽  
Laboratory Methods ◽  
The Moment ◽  
The Brain ◽  
Lateral Sclerosis

Description of a clinical case of amyotrophic lateral sclerosis with lumbosacral debut. These data show certain difficulties for neurologists in correct diagnosing ALS or classifying it as a motor neuron disease. The differential diagnosis at admission was carried out with vertebrogenic cervical myelopathy, myasthenia gravis, and neural amyotrophy. During the collection of anamnesis, therapeutic and neurological examinations, evidence of motor neuron degeneration in the cervical-thoracic and lumbosacral parts of the spinal cord was revealed. We used auxiliary diagnostic methods (RCT of the spine and MRI of the brain, clinical and laboratory methods) to exclude other pathologies. The diagnosis of ALS is a verdict for the patient, since there is no effective treatment for the disease at the moment. CONCLUSION: The symptoms of ALS are largely similar to other potentially curable and / or benign diseases of the nervous system, so the patient's examination should be comprehensive, with the possible use of molecular genetic research methods.

Abnormal Processing of the Glycoprotein IIb Transcript due to a Nonsense Mutation in Exon 17 Associated with Glanzmann’s Thrombasthenia

1995 ◽  
Vol 73 (05) ◽  
pp. 756-762 ◽  
Author(s):  
Yoshiaki Tomiyama ◽  
Hirokazu Kashiwagi ◽  
Satoru Kosugi ◽  
Masamichi Shiraga ◽  
Yoshio Kanayama ◽  
...  
Keyword(s):  
Dna Analysis ◽  
Molecular Genetic ◽  
Genetic Defect ◽  
Nucleotide Sequence Analysis ◽  
Type I ◽  
Normal Amount ◽  
Immunoblot Assay ◽  
Glanzmann’S Thrombasthenia ◽  
Glanzmann's Thrombasthenia ◽  
Pcr Products

SummaryWe analyzed the molecular genetic defect responsible for type I Glanzmann’s thrombasthenia in a Japanese patient. In an immunoblot assay using polyclonal anti-GPIIb-IIIa antibodies, some GPIIIa (15% of normal amount) could be detected in the patient’s platelets, whereas GPIIb could not (<2% of normal amount). Nucleotide sequence analysis of platelet GPIIb mRNA-derived polymerase chain reaction (PCR) products revealed that patient’s GPIIb cDNA had a 75-bp deletion in the 3’ boundary of exon 17 resulting in an in-frame deletion of 25 amino acids. DNA analysis and family study revealed that the patient was a compound heterozygote of two GPIIb gene defects. One allele derived from her father was not expressed in platelets, and the other allele derived from her mother had a 9644C → T mutation which was located at the position -3 of the splice donor junction of exon 17 and resulted in a termination codon (TGA). Moreover, quantitative analysis demonstrated that the amount of the abnormal GPIIb transcript in the patient’s platelets was markedly reduced. Thus, the C → T mutation resulting in the abnormal splicing of GPIIb transcript and the reduction in its amount is responsible for Glanzmann’s thrombasthenia.

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