Deletion and duplication screening in the DMD gene using MLPA

Lalic, Tanja; Vossen, Rolf H. A. M.; Coffa, Jordy; Schouten, Jan P.; Guć-Šćekić, Marija; Radivojević, Dušan; Djurisic, M; Breuning, M. H.; White, Stefan J.; Dunnen, Johan T. den

doi:10.1038/sj.ejhg.5201465

Cited by 168 publications

(128 citation statements)

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“…PCR products were analysed on ABI 3130 automated sequencer using Genescan software (Applied Biosystems). The dosage quotient (DQ) was calculated as previously described (Lalic et al,2005).For single exon duplications found with MLPA we always confirmed the data by Real time PCR analysis. MGB-probes (FAM-labelled) and primers designing was performed by Primer Express Software 2.0 (Applied Biosystems) on DMD gene exons 2, 12, 16, 17, 21, 55.…”

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confidence: 60%

Transcriptional behavior of DMD gene duplications in DMD/BMD males

et al. 2009

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DMD gene exons duplications account for up to 5-10 % of Duchenne (DMD) and up to 5-19% of Becker (BMD) muscular dystrophies; as for the more common deletions, the genotype-phenotype correlation and the genetic prognosis are generally based on the "reading frame rule". Nevertheless, the transcriptional profile of duplications, abridging the genomic configuration to the eventual protein effect, has been poorly studied. We describe 26 DMD gene duplications occurring in 33 unrelated patients and detected among a cohort of 194 mutation-positive DMD/BMD patients. We have characterized at the RNA level 16 of them. Four duplications (15%) behave as exception to the reading frame rule. In three BMD cases with out-of-frame mutations, the RNA analysis revealed that exon skipping events occurring in the duplicated region represent the mechanism leading to the frame reestablishment and to the milder phenotype. Differently, in a DMD patient carrying an inframe duplication the RNA behaviour failed to explain the clinical phenotype which is probably related to post-transcriptional-translational mechanisms. We conclude that defining the RNA profile in DMD gene duplications is mandatory both for establishing the genetic prognosis and for approaching therapeutic trials based on hnRNA modulation. reversible, age dependent degeneration (Muntoni et al., 2003).Although dystrophinopathies are inherited as an X-linked condition, about one third of patients have de novo mutations in the DMD gene. The most common pathogenic changes are intragenic deletions, which account for 65% of all mutations, indeed the frequency of duplications may range from 5% to 10% in DMD and from 5 to 19% in BMD patients (Aartsma-Rus et al.,2006;Kesari et al.,2008). The remaining cases (roughly 20%) are thought to be caused by a combination of small mutations (most commonly point mutations resulting in nonsense or frameshift mutations), pure intronic deletions or exonic insertion of repetitive sequences (Muntoni et al, 2003).It has been previously reported that deletions are mostly maternally inherited, whereas duplications preferentially originate in the grandpaternal germline (Hu et al., 1990). This implies that duplications present more frequently as familial cases therefore accounting for a higher risk of recurrence (White et al., 2006).In the past, technical difficulties have hampered the detection of quantitative changes in the huge DMD gene, in particular of duplications, and only in the last years the development of new techniques such as multiplex amplifiable probe hybridization (MAPH) (Armour et al.,2000) and multiplex ligation-dependent probe amplification (MLPA) (Schouten et al., 2002) have greatly simplified it, revealing that duplications underlie the 87% of DMD/BMD cases negative for deletions and point mutations (White et al.,2006).The clinical differences between DMD and BMD patients can be generally explained by the Tony Monaco's hypothesis (Monaco et al.,1988): genomic mutations that disrupt the translational open reading frame lead to a prema...

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confidence: 60%

Transcriptional behavior of DMD gene duplications in DMD/BMD males

et al. 2009

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“…In contrast, in Taiwan, which is geographically near to Japan, MLPA analysis identified a lower incidence of deletions and a higher incidence of duplications (deletion and duplication mutations were found in 36.0 and 24.7% of 89 DMD/ BMD patients, respectively 39 ), compared with other reports that show 60% and 5-10%, respectively. 6,8,40 Although differences in ethnicity should be taken into account, MLPA remains the first line in mutation detection in dystrophinopathy. [6][7][8][9] A total of four deep intron mutations were identified at least 285 bp from the exon/intron boundary; this was possible only because we used detection of novel dystrophin mRNA (Figure 4).…”

Section: Discussionmentioning

confidence: 99%

“…6,8,40 Although differences in ethnicity should be taken into account, MLPA remains the first line in mutation detection in dystrophinopathy. [6][7][8][9] A total of four deep intron mutations were identified at least 285 bp from the exon/intron boundary; this was possible only because we used detection of novel dystrophin mRNA (Figure 4). This analysis provided 1% of detected mutations, and facilitated our perfect mutation detection rate.…”

Section: Discussionmentioning

confidence: 99%

“…[6][7][8][9] However, the identification of small mutations in the dystrophin gene remains challenging, because of the large number of exons and the huge size of the dystrophin gene. By combining quantitative PCR and direct sequencing technologies, the mutation detection rate has risen to 98% of DMD cases (106/108 total cases).…”

Section: Duchenne Muscular Dystrophy (Dmd; Mim (Online Mendelianmentioning

confidence: 99%

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Mutation spectrum of the dystrophin gene in 442 Duchenne/Becker muscular dystrophy cases from one Japanese referral center

et al. 2010

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Recent developments in molecular therapies for Duchenne muscular dystrophy (DMD) demand accurate genetic diagnosis, because therapies are mutation specific. The KUCG (Kobe University Clinical Genetics) database for DMD and Becker muscular dystrophy is a hospital-based database comprising 442 cases. Using a combination of complementary DNA (cDNA) and chromosome analysis in addition to conventional genomic DNA-based method, mutation detection was successfully accomplished in all cases, and the largest mutation database of Japanese dystrophinopathy was established. Among 442 cases, deletions and duplications encompassing one or more exons were identified in 270 (61%) and 38 (9%) cases, respectively. Nucleotide changes leading to nonsense mutations or disrupting a splice site were identified in 69 (16%) or 24 (5%) cases, respectively. Small deletion/insertion mutations were identified in 34 (8%) cases. Remarkably, two retrotransposon insertion events were also identified. Dystrophin cDNA analysis successfully revealed novel transcripts with a pseudoexon created by a single-nucleotide change deep within an intron in four cases. X-chromosome abnormalities were identified in two cases. The reading frame rule was upheld for 93% of deletion and 66% of duplication mutation cases. For the application of molecular therapies, induction of exon skipping was deemed the first priority for dystrophinopathy treatment. At one Japanese referral center, the hospital-based mutation database of the dystrophin gene was for the first time established with the highest levels of quality and patient's number.

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“…3 Most laboratories perform two or more steps to detect mutations in a suspected DMD patient. Multiplex ligationdependent probe amplification (MLPA) or array comparative genome hybridization (aCGH) is used to detect large deletions/duplications; 4,5 if no deletion/duplication is detected, Sanger sequencing of all the DMD exons is performed. 6,7 However, in addition to the high cost and time requirements of the entire process, the results can be inconclusive.…”

Section: Introductionmentioning

confidence: 99%

Targeted next-generation sequencing as a comprehensive test for patients with and female carriers of DMD/BMD: a multi-population diagnostic study

Wei

Dai

et al. 2013

Eur J Hum Genet

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Duchenne and Becker muscular dystrophies (DMD/BMD) are the most commonly inherited neuromuscular disease. However, accurate and convenient molecular diagnosis cannot be achieved easily because of the enormous size of the dystrophin gene and complex causative mutation spectrum. Such traditional methods as multiplex ligation-dependent probe amplification plus Sanger sequencing require multiple steps to fulfill the diagnosis of DMD/BMD. Here, we introduce a new single-step method for the genetic analysis of DMD patients and female carriers in real clinical settings and demonstrate the validation of its accuracy. A total of 89 patients, 18 female carriers and 245 non-DMD patients were evaluated using our targeted NGS approaches. Compared with traditional methods, our new method yielded 99.99% specificity and 98.96% sensitivity for copy number variations detection and 100% accuracy for the identification of single-nucleotide variation mutations. Additionally, this method is able to detect partial deletions/duplications, thus offering precise personal DMD gene information for gene therapy. We detected novel partial deletions of exons in nine samples for which the breakpoints were located within exonic regions. The results proved that our new method is suitable for routine clinical practice, with shorter turnaround time, higher accuracy, and better insight into comprehensive genetic information (detailed breakpoints) for ensuing gene therapy.

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Deletion and duplication screening in the DMD gene using MLPA

Cited by 168 publications

References 24 publications

Transcriptional behavior of DMD gene duplications in DMD/BMD males

Transcriptional behavior of DMD gene duplications in DMD/BMD males

Mutation spectrum of the dystrophin gene in 442 Duchenne/Becker muscular dystrophy cases from one Japanese referral center

Targeted next-generation sequencing as a comprehensive test for patients with and female carriers of DMD/BMD: a multi-population diagnostic study

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