Analysis of dystrophin gene deletions by multiplex PCR in eastern India.
TL;DR: DNA from seventy unrelated patients clinically diagnosed as having DMD/BMD referred from different parts of West Bengal, a few other states and Bangladesh are analyzed using the multiplex polymerase chain reaction (m-PCR) to screen for exon deletions and its distribution within the dystrophin gene.
Abstract: The most common genetic neuromuscular disease of childhood, Duchenne and Becker muscular dystrophy (DMD/BMD) is caused by deletion, duplication or point mutation of the dystrophin gene located at Xp 21.2. In the present study DNA from seventy unrelated patients clinically diagnosed as having DMD/BMD referred from different parts of West Bengal, a few other states and Bangladesh are analyzed using the multiplex polymerase chain reaction (m-PCR) to screen for exon deletions and its distribution within the dystrophin gene. Out of seventy patients forty six (63%) showed large intragenic deletion in the dystrophin gene. About 79% of these deletions are located in the hot spot region i.e., between exon 42 to 53. This is the first report of frequency and distribution of deletion in dystrophin gene in eastern Indian DMD/BMD population.
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Journal Article•
TL;DR: The overall distribution of deletion mutations in the distal 'hot spot' region of the DMD gene was in accordance with DMD/BMD cases investigated elsewhere, and multiplex PCR technology was utilized to demonstrate the frequency of the most commonly found deletions.
Abstract: Introduction: In Saudi Arabia, only limited work has been reported on the
mutation patterns of Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD). This study looks at the spectrum of deletions in the 'hot spot' regions of the DMD gene in Saudi DMD/BMD patients using an enhanced multiplex PCR technique. Material and methods: Twenty-six exons of the DMD gene were analyzed, in eight unrelated DMD/BMD cases aged 4-19 years, using four different multiplexPCR sets. Each multiplex PCR set amplified a total of six or seven exons. Normal controls were included for validation.
Results: Using an optimized multiplex PCR method, 5 out of 8 DMD/BMD patients showed deletions, while the remaining three had no deletions in regions analyzed. Set 1 detected no deletions in any of the patients, whereas each of sets 2, 3 and 4 detected two, four and three deletions respectively. All of these mutations were located in the distal ‘hot spot’ region. No deletions were detected in the proximal ‘hot spot’ region. The normal control samples showed no deletions in any of the 26 exons tested. Conclusions: In this study, multiplex PCR technology was utilized to demonstrate the frequency of the most commonly found deletions in a limited group of Saudi DMD/BMD patients. The overall distribution of deletion mutations in the distal ‘hot spot’ region was in accordance with DMD/BMD cases investigated elsewhere. The study also serves as a good starting point for further investigations into the genetic aspects of the Saudi DMD/BMD population.
Key words: dystrophin, Saudi DMD, BMD, deletions, multiplex PCR, DMD gene.
8 citations
Cites result from "Analysis of dystrophin gene deletio..."
...Comparison with data reported in various countries also show similarities, such as India with 61% [18], Thailand and Greece with 63% each [19-21], Turkey with 59% [22], USA with 65% [10] and Egypt where reported DMD gene mutations vary from 50 to 67% [23]....
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Dissertation•
20 Jul 2013
4 citations
Cites methods from "Analysis of dystrophin gene deletio..."
...The method of choice in India for DMD/BMD diagnosis is multiplex PCR which targets about 18 to 32 exons of the DMD gene to look for whole exon deletions (Banerjee and Verma, 1997; Mallikarjuna Rao et al., 2003; Basak et al., 2006; Dastur et al., 2008)....
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TL;DR: They present clinically with progressive muscle degeneration and weakness, which affects different muscles in different variations, and DMD is the most common form of MDs among children.
Abstract: Muscular dystrophies (MDs) are heterogeneous, genetic muscle disorders.[1-6] Genetic mapping techniques have helped to identify more than 50 diseases, caused by specific gene mutations, which constitute MDs.[1,7,8] Muscle histology has identified changes in the fiber size, the presence of necrotic areas in muscles, and subsequently, the fatty infiltration in MDs.[9-11] MDs are classified according to their phenotypic features, inheritance patterns, age of onset, and the rate of disease progression.[7,10] They present clinically with progressive muscle degeneration and weakness, which affects different muscles in different variations.[1,12-15] DMD is the most common form of MDs among children.[10,16]
3 citations
Cites background from "Analysis of dystrophin gene deletio..."
...For reprints contact: reprints@medknow.com Access this article online Quick Response Code: Website: www.neurologyindia.com DOI: 10.4103/0028‑3886.263203 PMID: xxxx Commentary 718 Neurology India | Volume 67 | Issue 3 | May‑June 2019 study done in South India,[39] and deletion mutations have been more prevalent in Northern, Southern, Western, and Eastern India....
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...718 Neurology India | Volume 67 | Issue 3 | May‐June 2019 study done in South India,[39] and deletion mutations have been more prevalent in Northern, Southern, Western, and Eastern India.[39-41] The prenatal diagnosis can be helpful, so parents can make an informed choice....
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TL;DR: It is concluded that mid-distal region of dystrophin is highly polymorphic in the population of eastern Uttar Pradesh and responsible for pathogenesis of DMD.
Abstract: Introduction: The frequency and distribution of dystrophin gene deletions vary in patients with Duchene/Becker muscular dystrophy (DMD/BMD) Objective: In this study, we aimed to analyze clinical, biochemical, and dystrophin gene deletion pattern, by using multiplex polymerase chain reaction (PCR) in the population of eastern Uttar Pradesh and the adjoining districts of Bihar and Madhya Pradesh Material and Method: After clinical assessment, 225 patients of DMD/BMD were analyzed for deletion in dystrophin gene Clinical features and biochemical parameters were noted For genetic study, all samples were tested for deletion from 25 exons of DMD gene by using multiplex PCR Result: Deletions were detected in 169 (751%) patients of DMD/BMD Deletions were observed in both proximal and mid-distal hot spot regions with maximum deletion localized in the mid-distal hot spot region of the gene The most frequent deletions were observed in exon 50 (149%) and exon 49 (108%) Conclusion: This study concludes that mid-distal region of dystrophin is highly polymorphic in the population of eastern Uttar Pradesh and responsible for pathogenesis of DMD The population of eastern Uttar Pradesh shows similar pattern of deletion in dystrophin gene when compared with other ethnic groups of the Indian population
2 citations
Cites background from "Analysis of dystrophin gene deletio..."
...91% at the proximal hot spot region[17]...
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...In 2006, Basak et al.[17] worked on 70 samples from eastern Indian population, of which 46 patients showed large intragenic deletions in dystrophin gene....
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Journal Article•
TL;DR: This study could be the first to search for the most frequent deletions in the exons of DMD gene in the rural areas of Maharashtra with the help of molecular biology tools.
Abstract: Background: Duchenne Muscular Dystrophy (DMD) is the most common neuromuscular disease of childhood caused by deletion or point mutations in the dystrophin gene. Though the importance of deletion mutations in the dystrophin gene causing DMD have been reported worldwide, no data are available from rural population of Maharashtra. Objectives: This study specifically aimed at the investigation of deletion mutations in the DMD gene from the patients from South-Western Maharashtra. Material & Methods: Fifty patients with clinically diagnosed DMD were analyzed to screen for intragenic deletions in 21 exons within the DMD gene using the multiplex polymerase chain reaction. Results: Amongst the 50 unrelated DMD patients from South-Western Maharashrra we found DMD gene deletions in 47 cases which represent 94 % mutations in DMD patients. Majority of the deletions (85.10%) were located at distal hot spot region that encompasses exons 42-53 and 10.63% of the deletions were located at the proximal hot spot region (exons 2-19). Exons 50, 51, 52 and 53 are most frequently deleted. Conclusion: It is important to note that we could be the first to search for the most frequent deletions in the exons of DMD gene in the rural areas of Maharashtra with the help of molecular biology tools.
2 citations
References
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TL;DR: A rapid, safe and inexpensive method was developed to simplify the deprotein-ization procedure that yielded quantities comparable to those obtained from phenol-chloroform extractions, rendering the entire process of RFLP analysis free of toxic materials.
Abstract: One of the obstacles encountered when extracting DNA from a large number of samples is the cumbersome method of deprotein-izing cell digests with the hazardous organic solvents phenol and isochloroform. Several other non-toxic extraction procedures have been published, but require either extensive dialysis (1) or the use of filters (2). A rapid, safe and inexpensive method was developed to simplify the deprotein-ization procedure. This method involves salting out of the cellular proteins by dehydration and precipitation with a saturated NaCl solution. Buffy coats of nucleated cells obtained from anticoagulated blood (ACD or EDTA) were resuspended in 15 ml polypropylene centrifugation tubes with 3 ml of nuclei lysis buffer (10 mM Tris-HCl t 400 mM NaCl and 2 mM Na 2 EDTA, pH 8.2). The cell lysates were digested overnight at 37°C with 0.2 ml of 10Z SDS and 0.5 ml of a protease K solution (1 mg protease K in 1Z SDS and 2 mM Na2EDTA). After digestion was complete, 1 ml of saturated NaCl (approximately 6M) was added to each tube and shaken vigorously for 15 seconds, followed by centrifugation at 2500 rpm for 15 minutes. The precipitated protein pellet was left at the bottom of the tube and the supernatant containing the DNA was transferred to another 15 ml polypropylene tube. Exactly 2 volumes of room temperature absolute ethanol was added and the tubes inverted several times until the DNA precipitated. The precipitated DNA strands were removed with a plastic spatula or pipette and transferred to a 1.5 ml microcentrifuge tube containing 100-200 pi TE buffer (10 mM Tris-HCl, 0.2 mM Na 2 EDTA, pH 7.5). The DNA was allowed to dissolve 2 hours at 37°C before quantitating. The DNA obtained from this simple technique yielded quantities comparable to those obtained from phenol-chloroform extractions. The 260/280 ratios were consistently 1.8-2.0, demonstrating good deproteinization. Restrictions were performed using a number of different enzymes requiring high, medium or low salt concentrations, all resulting in complete restriction. This procedure has been used in our laboratory on several thousand blood samples for parentage, population and forensic studies. This technique is used with our non-isotopic hybridization procedures (3) rendering the entire process of RFLP analysis free of toxic materials.
19,905 citations
"Analysis of dystrophin gene deletio..." refers methods in this paper
...Deletion screening by multiplex PCR (m-PCR) DNA was isolated from 5-6 ml of EDTA-blood following the standard procedure.([1]) Detection of intragenic deletion by m-PCR was standardized following the methods described earlier([2,3]) and using primer sets purchased from Sigma Chemicals (USA)....
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TL;DR: This procedure utilizes simultaneous genomic DNA amplification of multiple widely separated sequences and should permit deletion scanning at any hemizygous locus and it is demonstrated the application of this multiplex reaction for prenatal and postnatal diagnosis of DMD.
Abstract: The application of recombinant DNA technology to prenatal diagnosis of many recessively inherited X-linked diseases is complicated by a high frequency of heterogeneous, new mutations (1). Partial gene deletions account for more than 50% of Duchenne muscular dystrophy (DMD) lesions, and approximately one-third of all cases result from a new mutation (2-5). We report the isolation and DNA sequence of several deletion prone exons from the human DMD gene. We also describe a rapid method capable of detecting the majority of deletions in the DMD gene. This procedure utilizes simultaneous genomic DNA amplification of multiple widely separated sequences and should permit deletion scanning at any hemizygous locus. We demonstrate the application of this multiplex reaction for prenatal and postnatal diagnosis of DMD.
1,323 citations
"Analysis of dystrophin gene deletio..." refers methods in this paper
...Detection of intragenic deletion by m-PCR was standardized following the methods described earlier([2,3]) and using primer sets purchased from Sigma Chemicals (USA)....
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TL;DR: Using oligonucleotide primer sequences that can be used to amplify eight exons plus the muscle promoter of the dystrophin gene in a single multiplex polymerase chain reaction (PCR) will allow deletion detection and prenatal diagnosis for most DMD/BMD patients in a fraction of the time required for Southern blot analysis.
Abstract: We describe oligonucleotide primer sequences that can be used to amplify eight exons plus the muscle promoter of the dystrophin gene in a single multiplex polymerase chain reaction (PCR). When used in conjunction with an existing primer set, these two multiplex reactions detect about 98% of deletions in patients with Duchenne or Becker muscular dystrophy (DMD, BMD). Furthermore, these primers amplify most of the exons in the deletion prone “hot spot” region around exons 44 to 53, allowing determination of deletion endpoints and prediction of mutational effects on the translational reading frame. Thus, use of these PCR-based assays will allow deletion detection and prenatal diagnosis for most DMD/BMD patients in a fraction of the time required for Southern blot analysis.
642 citations
"Analysis of dystrophin gene deletio..." refers methods in this paper
...Detection of intragenic deletion by m-PCR was standardized following the methods described earlier([2,3]) and using primer sets purchased from Sigma Chemicals (USA)....
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TL;DR: DNA samples from 121 unrelated DMD/BMD patients from North India were analyzed for deletional studies with multiplex PCR and Southern hybridization, and a total of 88 patients showed intragenic deletions in the dystrophin gene.
Abstract: Population-based variations in frequency and distribution of dystrophin gene deletions have been recognized in Duchenne/Becker (DMD/BMD) muscular dystrophy patients. In the present study, DNA samples from 121 unrelated DMD/BMD patients from North India were analyzed for deletional studies with multiplex PCR and Southern hybridization. A total of 88 (73%) patients showed intragenic deletions in the dystrophin gene. The observed proportion of gene deletions is relatively high, particularly compared with that of Asian counterparts. However, the distribution of breakpoints across the gene does not show significant variations.
41 citations
"Analysis of dystrophin gene deletio..." refers background in this paper
...Proportion and patterns of deletions in the dystrophin gene prevalent among the DMD/BMD patients in eastern India detected by multiplex PCR (m-PCR) is reported in the present work....
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...This is the first report of frequency and distribution of deletion in dystrophin gene in eastern Indian DMD/BMD population....
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...E-mail: bjayasri_2000@yahoo.com Neurology India | September 2006 | Vol 54 | Issue 3 310 CMYK311 Basak et al.: Deletion detection in DMD and BMD was available in forty-five families and there was no previous history of the disease in twenty-eight (62...
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...For example, percentage of deletion in the central hot spot region in Chinese is approximately 50% of all deletions,[4] whereas in north India[4] it is around 80%, with which our data conforms....
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...The percentage of cases having gene deletions in eastern India is 65.7%, which is slightly lower than that reported in the north India....
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TL;DR: In the West Midlands region of Britain, Duchenne muscular dystrophy (DMD) is twice as common as expected in Indians, and is less common than expected in Pakistanis.
Abstract: In the West Midlands region of Britain, Duchenne muscular dystrophy (DMD) is twice as common as expected in Indians, and is less common than expected in Pakistanis. Although the numbers are small, they cannot be explained by any bias of ascertainment and are considered to be real. One possible mechanism for the high frequency of DMD in Indians is the presence of repetitive elements in the wild type gene which predispose to mutations.
19 citations
"Analysis of dystrophin gene deletio..." refers background in this paper
...Racial differences in incidence rate of DMD have been suggested.([5]) We have observed a higher incidence of cases in Muslims....
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