Utility of MLPA in mutation analysis and carrier detection for Duchenne muscular dystrophy

Verma, Prashant Kumar; Dalal, Ashwin; Mittal, Balraj; Phadke, Shubha R.

doi:10.4103/0971-6866.96667

Cited by 16 publications

(5 citation statements)

References 20 publications

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“…In our study as represented in Table 2 and Supplementary Table 01, mPCR could provide molecular diagnosis for 55% (131/236) of the patients, of which 76% (100/136) of the patients the exact deletion boundaries accurately detected by mPCR. In line with our findings, studies conducted in South India [ 25 ] and North India [ 26 ] identified (103/150) 68% and (161/217) 74%, respectively, as the mutation detection Percentage by mPCR. Intriguingly in our study 95% (131/138) of the patients with deletion mutations could be diagnosed by mPCR.…”

Section: Discussionsupporting

confidence: 92%

Title-molecular diagnostics of dystrophinopathies in Sri Lanka towards phenotype predictions: an insight from a South Asian resource limited setting

Wijekoon,

Gonawala,

Ratnayake

et al. 2024

Eur J Med Res

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Background The phenotype of Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) patients is determined by the type of DMD gene variation, its location, effect on reading frame, and its size. The primary objective of this investigation was to determine the frequency and distribution of DMD gene variants (deletions/duplications) in Sri Lanka through the utilization of a combined approach involving multiplex polymerase chain reaction (mPCR) followed by Multiplex Ligation Dependent Probe Amplification (MLPA) and compare to the international literature. The current consensus is that MLPA is a labor efficient yet expensive technique for identifying deletions and duplications in the DMD gene. Methodology Genetic analysis was performed in a cohort of 236 clinically suspected pediatric and adult myopathy patients in Sri Lanka, using mPCR and MLPA. A comparative analysis was conducted between our findings and literature data. Results In the entire patient cohort (n = 236), mPCR solely was able to identify deletions in the DMD gene in 131/236 patients (DMD-120, BMD-11). In the same cohort, MLPA confirmed deletions in 149/236 patients [DMD-138, BMD -11]. These findings suggest that mPCR has a detection rate of 95% (131/138) among all patients who received a diagnosis. The distal and proximal deletion hotspots for DMD were exons 45–55 and 6–15. Exon 45–60 identified as a novel in-frame variation hotspot. Exon 45–59 was a hotspot for BMD deletions. Comparisons with the international literature show significant variations observed in deletion and duplication frequencies in DMD gene across different populations. Conclusion DMD gene deletions and duplications are concentrated in exons 45–55 and 2–20 respectively, which match global variation hotspots. Disparities in deletion and duplication frequencies were observed when comparing our data to other Asian and Western populations. Identified a 95% deletion detection rate for mPCR, making it a viable initial molecular diagnostic approach for low-resource countries where MLPA could be used to evaluate negative mPCR cases and cases with ambiguous mutation borders. Our findings may have important implications in the early identification of DMD with limited resources in Sri Lanka and to develop tailored molecular diagnostic algorithms that are regional and population specific and easily implemented in resource limited settings.

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Section: Discussionsupporting

confidence: 92%

Title-molecular diagnostics of dystrophinopathies in Sri Lanka towards phenotype predictions: an insight from a South Asian resource limited setting

Wijekoon,

Gonawala,

Ratnayake

et al. 2024

Eur J Med Res

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“…46 MLPA has been used to detect carrier status in probands with known deletion or duplication mutations since it is an easy and quick technique. 47 Detection of carrier status serves to provide essential information for family counselling regarding future pregnancies, improving quality of life and reducing financial burden for at-risk families. 48 This study also shows that MLPA analysis can pave the way for future therapeutics in DMD patients by identifying amenable genotypes that can be targeted.…”

Section: Discussionmentioning

confidence: 99%

Mutation spectrum analysis of DMD gene in Indonesian Duchenne and Becker muscular dystrophy patients

Dwianingsih,

Iskandar,

Hapsara

et al. 2023

F1000Res

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Background Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are allelic disorders caused by mutations in the DMD gene. The full mutation spectrum of the DMD gene in Indonesian patients is currently unknown. Mutation-specific therapies are currently being developed, such as exon skipping or stop codon read-through therapy. This study was conducted with the aim of identifying the mutation spectrum of the DMD gene in Indonesia to guide future development and application of feasible therapeutic strategies. Methods This study is a cross sectional study that enrolled 43 male patients with a clinical suspicion of DMD or BMD. Multiplex ligation-dependent probe amplification (MLPA) reaction was performed to screen for the common mutations in the DMD gene. Results Out of 43 subjects, deletions accounted for 69.77% (n=30) cases, while duplications were found in 11.63% (n=5) cases. One novel duplication spanning exons 2 to 62 was identified. Deletion mutations clustered around the distal (66.67%) and proximal (26.67%) hot spot regions of the DMD gene while duplication mutations were observed solely at the proximal region. Two false positive cases of single exon deletion detected through MLPA were attributed to sequence mutations affecting primer ligation sites, confirming the need to validate all single exon deletions when using this screening method. Analysis of available maternal DNA samples showed that the rate of de novo mutations (48.15%) appears higher than expected in this population. Out of 31 patients who were classified as DMD based on clinical and genotype characterizations, 60.47% (n=26) of cases were suitable for exon skipping therapy. Conclusion This is the first comprehensive study showing the feasibility of implementing the MLPA method for routine screening of DMD patients in Indonesia. This is also the first study showing the potential applicability of exon skipping therapy in the majority of DMD cases in the country.

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“…Besides, MLPA is an available method which can be used for prenatal diagnosis when the deletions or duplications are not detected by multiplex PCR. [ 23 ] Certainly, sequencing can be used to detect small gene mutations in MLPA-negative individuals. It is essential to perform antenatal diagnosis on the carriers of childbearing age by MLPA primarily.…”

Section: Discussionmentioning

confidence: 99%

Molecular Analysis-Based Genetic Characterization of a Cohort of Patients with Duchenne and Becker Muscular Dystrophy in Eastern China

Zhao

Sun

Shi

et al. 2018

Chinese Medical Journal

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Background:Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are common X-linked recessive neuromuscular disorders caused by mutations in dystrophin gene. Multiplex polymerase chain reaction (multiplex PCR) and multiplex ligation-dependent probe amplification (MLPA) are the most common methods for detecting dystrophin gene mutations. This study aimed to contrast the two methods and discern the genetic characterization of patients with DMD/BMD in Eastern China.Methods:We collected 121 probands, 64 mothers of probands, and 15 fetuses in our study. The dystrophin gene was detected by multiplex PCR primarily in 28 probands, and MLPA was used in multiplex PCR-negative cases subsequently. The dystrophin gene of the remaining 93 probands and 62 female potential carriers was tested by MLPA directly. In fetuses, multiplex PCR and MLPA were performed on 4 fetuses and 10 fetuses, respectively. In addition, sequencing was also performed in 4 probands with negative MLPA.Results:We found that 61.98% of the subjects had genetic mutations including deletions (50.41%) and duplications (11.57%). There were 43.75% of mothers as carriers of the mutation. In 15 fetuses, 2 out of 7 male fetuses were found to be unhealthy and 2 out of 8 female fetuses were found to be carriers. Exons 3–26 and 45–52 have the maximum frequency in mutation regions. In the frequency of exons individually, exon 47 and exon 50 were the most common in deleted regions and exons 5, 6, and 7 were found most frequently in duplicated regions.Conclusions:MLPA has better productivity and sensitivity than multiplex PCR. Prenatal diagnosis should be applied in DMD high-risk fetuses to reduce the disease incidence. Furthermore, it is the responsibility of physicians to inform female carriers the importance of prenatal diagnosis.

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Utility of MLPA in mutation analysis and carrier detection for Duchenne muscular dystrophy

Cited by 16 publications

References 20 publications

Title-molecular diagnostics of dystrophinopathies in Sri Lanka towards phenotype predictions: an insight from a South Asian resource limited setting

Title-molecular diagnostics of dystrophinopathies in Sri Lanka towards phenotype predictions: an insight from a South Asian resource limited setting

Mutation spectrum analysis of DMD gene in Indonesian Duchenne and Becker muscular dystrophy patients

Molecular Analysis-Based Genetic Characterization of a Cohort of Patients with Duchenne and Becker Muscular Dystrophy in Eastern China

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