Combination of conventional multiplex PCR and quantitative real-time PCR detects large rearrangements in the dystrophin gene in 59% of Syrian DMD/BMD patients

Madania, Ammar; Zarzour, Hana; Jarjour, Rami A.; Ghoury, Ifad

doi:10.1016/j.clinbiochem.2010.03.014

Cited by 14 publications

(7 citation statements)

References 17 publications

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“…When compared with our sample, a Turkish sample has also been shown to have a relatively higher rate of deletions within the DMD gene (63.7%) [ 46 ], likely because of admixture with other European ethnicities. Rates of DMD deletions and duplications in some other Middle Eastern populations are more similar to what we found: Egyptian (51.3% deletions) [ 47 ], Iranian (51% deletions) [ 48 ], Moroccan (51% deletions) [ 49 ], and Syrian (49.0% deletions; 9.8% duplications) [ 50 ]. The majority of the reported DMD gene mutations in our Saudi data showed translational reading frame shifts (94.4%), while 5.6% of the mutations did not follow the reading frame rule.…”

Section: Discussionsupporting

confidence: 85%

Molecular characterization of exonic rearrangements and frame shifts in the dystrophin gene in Duchenne muscular dystrophy patients in a Saudi community

Elhawary

Jiffri

Jambi³

et al. 2018

Hum Genomics

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BackgroundIn individuals with Duchenne muscular dystrophy (DMD), exon skipping treatment to restore a wild-type phenotype or correct the frame shift of the mRNA transcript of the dystrophin (DMD) gene are mutation-specific. To explore the molecular characterization of DMD rearrangements and predict the reading frame, we simultaneously screened all 79 DMD gene exons of 45 unrelated male DMD patients using a multiplex ligation-dependent probe amplification (MLPA) assay for deletion/duplication patterns. Multiplex PCR was used to confirm single deletions detected by the MLPA.ResultsThere was an obvious diagnostic delay, with an extremely statistically significant difference between the age at initial symptoms and the age of clinical evaluation of DMD cases (t value, 10.3; 95% confidence interval 5.95–8.80, P < 0.0001); the mean difference between the two groups was 7.4 years. Overall, we identified 147 intragenic rearrangements: 46.3% deletions and 53.7% duplications. Most of the deletions (92.5%) were between exons 44 and 56, with exon 50 being the most frequently involved (19.1%). Eight new rearrangements, including a mixed deletion/duplication and double duplications, were linked to seven cases with DMD. Of all the cases, 17.8% had duplications with no hot spots. In addition, confirmation of the reading frame hypothesis helped account for new DMD rearrangements in this study. We found that 81% of our Saudi patients would potentially benefit from exon skipping, of which 42.9% had a mutation amenable to skipping of exon 51.ConclusionsOur study could generate considerable data on mutational rearrangements that may promote future experimental therapies in Saudi Arabia.Electronic supplementary materialThe online version of this article (10.1186/s40246-018-0152-8) contains supplementary material, which is available to authorized users.

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Section: Discussionsupporting

confidence: 85%

Molecular characterization of exonic rearrangements and frame shifts in the dystrophin gene in Duchenne muscular dystrophy patients in a Saudi community

Elhawary

Jiffri

Jambi³

et al. 2018

Hum Genomics

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“…Therefore, it is difficult to unify the clinical diagnosis methods of DMD/BMD patients. At present, the methods of genetic testing for the DMD gene include PCR amplification, multiplex PCR, Sanger sequencing, real-time PCR, MAPH, MLPA, and NGS [14, 15].…”

Section: Discussionmentioning

confidence: 99%

Genetic analysis of 1051 Chinese families with Duchenne/Becker Muscular Dystrophy

et al. 2019

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Background Duchenne Muscular Dystrophy (DMD) is the most common muscle disease in children, and there are no effective therapies for DMD or Becker Muscular Dystrophy (BMD). Currently, targeted gene therapy treatments have emerged. As a result, genetic diagnosis is the basis of treatment. In addition, genetic and prenatal diagnosis significantly reduces their incidence rates. This study combines the application of multiplex ligation-dependent probe amplification technology (MLPA) and “next-generation” sequencing technology (NGS) as the most economical and efficient method of diagnosis. Therefore, in the diagnosis of DMD/BMD, patients’ MLPA data are first used to detect DMD gene deletions or duplications, and NGS and Sanger sequencing are then applied to exclude MLPA-negative samples. Meanwhile, polymerase chain reaction (PCR) is used to detect single exon deletions to exclude false-positives in MLPA caused by point mutations. Methods In this study, we recruited 1051 proband families of DMD from 2016 to 2018 and had access to information that could identify individual participants during or after data collection. Patients who were diagnosed with DMD were first tested by MLPA. MLPA results with single exon deletions were validated with PCR amplification and Sanger sequencing. The negative results of MLPA were further analysed with NGS and validated by Sanger sequencing. For novel missense mutations, phenotype-genotype correlations were analysed using PolyPhen2 and mutation taster. All methods were performed in accordance with the relevant guidelines and regulations. Results DMD mutations were identified in 1029 families (97.91%, 1029/1051). Large deletions, duplications, and small mutations accounted for 70.41% (740/1051), 8.28% (87/1051), and 19.12% (201/1051) of all cases, respectively. There were 205 small mutation types, 53 of which were novel. The rate of de novo mutations was 39.45% (187/474) and was higher in large duplications (49.53%, 157/317). Among 68 asymptomatic patients (< 3 years old) with unexplained persistent hyperCKaemia upon conventional physical examination, 63 were diagnosed as DMD/BMD according to genetic diagnosis. Conclusion Our results expand the spectrum of DMD mutations, which could contribute to the treatment of DMD/BMD and provide an effective diagnosis method. Thus, the combination of MLPA, NGS and Sanger sequencing is of great significance for family analysis, gene diagnosis and gene therapy.

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“…Many methods are available for detecting deletions and duplications in the DMD gene. Southern blot using cDNA probe , multiplex PCR (mPCR), quantitative mPCR , and high‐resolution melting curve analysis was reportedly used in detecting DMD mutations. The most commonly used method is mPCR that allows for 90∼95% of deletions to be detected in male patients.…”

Section: Introductionmentioning

confidence: 99%

“…Multiplex ligation dependent probe amplification (MLPA) is a technique that had first been reportedly used in DMD analysis in 2004 by Schwartz . Parallel comparison between mPCR and MLPA had been made and reported from many clinic labs, MLPA was reported more superior . Since then MLPA has been increasingly used for deletion/duplication screening of the DMD gene in male patients and female carriers worldwide .…”

Section: Introductionmentioning

confidence: 99%

MLPA Application in Clinical Diagnosis of DMD/BMD in Shanghai

Zhang

et al. 2014

Clinical Laboratory Analysis

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Combination of conventional multiplex PCR and quantitative real-time PCR detects large rearrangements in the dystrophin gene in 59% of Syrian DMD/BMD patients

Cited by 14 publications

References 17 publications

Molecular characterization of exonic rearrangements and frame shifts in the dystrophin gene in Duchenne muscular dystrophy patients in a Saudi community

Molecular characterization of exonic rearrangements and frame shifts in the dystrophin gene in Duchenne muscular dystrophy patients in a Saudi community

Genetic analysis of 1051 Chinese families with Duchenne/Becker Muscular Dystrophy

MLPA Application in Clinical Diagnosis of DMD/BMD in Shanghai

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