Users Online: 510
Home Print this page Email this page
Home About us Editorial board Search Browse articles Submit article Ahead of Print Instructions Subscribe Contacts Login 

Previous article Browse articles Next article 
Adv Biomed Res 2014,  3:72

Evaluation of multiplex ligation-dependent probe amplification analysis versus multiplex polymerase chain reaction assays in the detection of dystrophin gene rearrangements in an Iranian population subset

1 Medical Genetics Laboratory, Alzahra University Hospital; Pediatric Inherited Disease Research Center; Pediatric Neurology, Pediatric Neurology Research Center, Isfahan University of Medical Sciences, Isfahan, Iran
2 Medical Genetics Laboratory, Alzahra University Hospital, Isfahan University of Medical Sciences, Isfahan, Iran
3 Medical Genetics Laboratory, Alzahra University Hospital; Department of Genetics and Molecular Biology, Medical School, Isfahan University of Medical Sciences, Isfahan, Iran
4 Pediatric Neurology, Pediatric Neurology Research Center, Isfahan University of Medical Sciences, Isfahan, Iran

Date of Submission15-Sep-2012
Date of Acceptance12-Nov-2012
Date of Web Publication27-Jan-2014

Correspondence Address:
Maryam Sedghi
Medical Genetics Laboratory, Alzahra University Hospital, Isfahan University of Medical Sciences, Isfahan
Login to access the Email id

Source of Support: This study was funded by grant number 189087 from deputy for research, Isfahan University of Medical Sciences, Isfahan, Iran. The authors are grateful to patients with DMD for their participating in this study, Conflict of Interest: None

DOI: 10.4103/2277-9175.125862

Rights and Permissions

Background: The Duchenne muscular dystrophy (DMD) gene is located in the short arm of the X chromosome (Xp21). It spans 2.4 Mb of the human genomic DNA and is composed of 79 exons. Mutations in the Dystrophin gene result in DMD and Becker muscular dystrophy. In this study, the efficiency of multiplex ligation-dependent probe amplification (MLPA) over multiplex polymerase chain reaction (PCR) assays in an Iranian population was investigated.
Materials and Methods: Multiplex PCR assays and MLPA analysis were carried out in 74 patients affected with DMD.
Results: Multiplex PCR detected deletions in 51% of the patients with DMD. MLPA analysis could determine all the deletions detected by the multiplex PCR. Additionally, MLPA was able to identify one more deletion and duplication in patients without detectable mutations by multiplex PCR. Moreover, MLPA precisely determined the exact size of the deletions.
Conclusion: Although MLPA analysis is more sensitive for detection of deletions and duplications in the dystrophin gene, multiplex PCR might be used for the initial analysis of the boys affected with DMD in the Iranian population as it was able to detect 95% of the rearrangements in patients with DMD.

Keywords: Deletion, duchenne muscular dystrophy, duplication, multiplex ligation-dependent probe amplification, multiplex polymerase chain reaction

How to cite this article:
Nouri N, Fazel-Najafabadi E, Salehi M, Hosseinzadeh M, Behnam M, Ghazavi MR, Sedghi M. Evaluation of multiplex ligation-dependent probe amplification analysis versus multiplex polymerase chain reaction assays in the detection of dystrophin gene rearrangements in an Iranian population subset. Adv Biomed Res 2014;3:72

How to cite this URL:
Nouri N, Fazel-Najafabadi E, Salehi M, Hosseinzadeh M, Behnam M, Ghazavi MR, Sedghi M. Evaluation of multiplex ligation-dependent probe amplification analysis versus multiplex polymerase chain reaction assays in the detection of dystrophin gene rearrangements in an Iranian population subset. Adv Biomed Res [serial online] 2014 [cited 2021 Oct 22];3:72. Available from:

  Introduction Top

The Duchenne muscular dystrophy (DMD) gene (Medelian Inheritance in Man MIM 300377) located at Xp21 spans 2.4 Mb and contains 79 exons. [1] The DMD gene is the largest known human gene, and its product is called dystrophin, which is mainly expressed in the skeletal and cardiac muscles, with small amounts expressed in the brain. [2] Dystrophin is comprised of an N-terminal actin-binding domain, 24 spectrin-like repeat units interspersed by four hinge regions, a cysteine-rich domain and a C-terminal domain. It is involved in the linkage between the cytoskeletal actin and the extracellular matrix. [3],[4]

Various mutations in DMD gene result in Duchenne muscular dystrophy and Becker muscular dystrophy (DMD/BMD). [5] DMD (MIM 310200) is the most common X-linked recessive lethal disease affecting one in 3500 newborn males. Affected boys are characterized by progressive dystrophy of the skeletal muscles and are usually wheelchair-bound before the age of 12 years. [6] BMD (MIM 300376) is a milder form of the disease, with an incidence of around one in 12,000 live born males. [7] Patients with BMD have a later age of onset (some as late as 40-50 years of age) and a slower clinical progression. In boys affected by DMD, the prematurely truncated, unstable dystrophins lacking cystein-rich and C-terminal domains are generated. These proteins have completely lost their function as a bridge due to mutations disrupting the open reading frame. In patients with BMD, low levels of full length dystrophin or internally deleted dystrophin resulting from in-frame mutations are detected. [8]

Deletions or duplications are found in about 60-65% and 5-10% of patients with muscular dystrophy, respectively. [9],[10] Although deletions encompass all 79 exons, two deletion hotspots - one including exons 45-55 and the other including exons 2-19 - are recognized. Approximately 30-35% of the cases with BMD/DMD are caused by point mutations (nonsense or splice sites) or small rearrangements. [11]

Multiplex ligation-dependent probe amplification (MLPA) analysis has proven to be a method of choice for the detection of deletions and duplications. [12],[13],[14] Before development of MLPA, detection of deletions and duplications in the DMD gene was mostly carried out by applying Southern blot [15] and multiplex polymerase chain reaction (PCR). Because Southern blotting was a labor-intensive and time-consuming method, it was replaced by the multiplex PCR. [5] The most commonly used multiplex PCR protocols cover 18 exons at the deletion hot spots and detect 90-98% of all deletions. [16],[17],[18] Multiplex PCR is proven to be a method for the rapid detection of deletions in boys affected with DMD; however, it is not efficient for the precise identification of DMD gene duplications in males and heterozygous deletions and duplications in females.

The aim of this study was to prove the efficiency of the MLPA analysis in a subset of Iranian patients. Therefore, we used multiplex PCR assays followed by MLPA analysis in 74 cases with DMD.

  Materials and Methods Top

Patient samples

This study was performed in a total of 74 boys affected with DMD from the central provinces of Iran referred by the neurology specialists to Genetic Lab, Alzahra University Hospital, for genetic analysis of their DMD gene. The diagnosis of DMD was according to the clinical features, electromyography, an elevated serum creatine kinase activity and an X-linked family history. After obtaining informed consent, genomic DNA was extracted from blood leukocytes using a QIAamp DNA mini kit (Qiagen, Hilden, Germany). The exact DNA concentration was determined using a NanoDrop instrument (Thermo 2000c) after dilution to 50 ng/ml.

Multiplex PCR

Two multiplex PCR assays covering exons 1, 3, 4, 6, 8, 12, 13, 17, 19, 43, 44, 45, 46, 47, 48, 50, 51, 52, and 60 in the hot spot regions of the DMD gene were performed in a total volume 50 μl according to the technique described by Chamberleidon and Beggs. [16],[17] Multiplex PCR products were visualized on 2% agarose gel.

MLPA analysis

MLPA analysis was carried out using the SALSA probe mix 034 (DMD exons 1-10, 21-30, 41-50, and 61-70) and 035 (DMD exons 11-20, 31-40, 51-60, and 71-79) according to the manufacturer's instructions. In brief, the ligation reaction was performed using 200 ng of target DNA in the following steps: Denaturation at 98°C for 5 min, hybridization with the SALSA probe mix 034 and 035 at 60°C for 16 h and ligation by Ligase-65 mix at 54°C for 15 min, and then ligase inactivation by incubation at 98°C for 5 min. Finally, multiplex PCR was performed using the specific SALSA FAM PCR primers, dNTPs, PCR buffer and polymerase for 30 cycles (95°C for 30 s, 60°C for 30 s and 72°C for 1 min). The fragments were analyzed on a 3100 capillary sequencer (Applied Biosystems, UK) with a 36-cm capillary array and POP-4 TM polymer (Applied Biosystems, UK) by mixing with 0.2 μl of the GeneScan TM -500 ROX TM size standard (Applied Biosystems, UK) and 10 μl of HiDi Formamide (Applied Biosystems, UK). The results (size and peak area) were analyzed using GeneMarker software.

  Results Top

Multiplex PCR assays indicated that 38 of 74 boys affected with DMD have exon deletions. However, MLPA analysis indicated one more deletion and duplication [Figure 1]. These new mutations identified by MLPA were confirmed by direct sequencing of the relevant exons using primers described earlier. [19] Furthermore, two deletions with different sizes were identified than what was detected by multiplex PCR. In male patients, total absence of MLPA peak areas indicated deletions of one or more exons, and multiplication of MLPA amplification products indicated duplications. Among the 40 identified rearrangements, 36 were located in the central hot spot region, including exons 44-55. Four deletions were identified in the 5' hot spot region.
Figure 1: Multiplex ligation-dependent probe amplification electropherogram. (a) Analysis of individual with deletion of exon 53. (b) Analysis of individual with duplication of exon 51

Click here to view

  Discussion Top

In this study, we performed multiplex PCR assays and MLPA analysis to identify deletions and duplications in the DMD gene. We used multiplex PCR as the first step for identification of deletions in male patients. Then, MLPA analysis was performed to confirm the results obtained by the multiplex PCR.

Because the DMD gene is X-linked, deletions are easily identified by conventional multiplex PCR in male patients. However, this method cannot be used for carrier analysis in women and detection of duplications and uncommon deletions in men with DMD. In addition, multiplex PCR is more difficult to be performed because there are different primer pairs for exons in the hot spot regions, and it is not possible to amplify these exons in a single reaction. However, MLPA can be used to detect rearrangements in all exons of dystrophin gene in both females and males by only two reaction sets. [20],[21] In our study, two multiplex assays covered 19 exons of the DMD gene, and in 51% of the cases deletions were identified. However, because all 79 exons of the DMD gene are included in two separate MLPA kits, MLPA analysis of subjects revealed the exact range of deletion in two patients and two more rearrangements. In two patients, multiplex PCR assays detected deletion of exon 50; however, deletion of exons 49-50 was detected by MLPA analysis. In addition, duplication of exon 51 and deletion of exon 53 were detected by MLPA analysis. Single duplication of exon 51 is not identified in other populations. However; duplication of exons 51-55 was reported in a Taiwanese patient. [22] Exon 51 duplication was identified in an 11-year-old boy whose mother did not show any mutation with MLPA analysis. The identified duplication in the boy might be a de novo mutation, or it could have resulted from maternal germline mosaicism. These findings indicated that MLPA analysis is more sensitive and reliable than conventional multiplex PCR for the detection of exon deletions and duplications.

Although deletions and duplications can occur in any exon of the DMD gene, their frequency is higher in two regions - one located in the 5 region (called minor hot spot) and the other located in the central part (called major hot spot). In the present study, 90% of the rearrangements were located in the central hot spot region between exons 44 and 55. Eight of the 36 deletions located in central hotspot region were deletion of exons 45-51. In another study performed by Khordadpoor-Deilamani et al., [23] 52 of 53 deletions in the dystrophin gene were detected by multiplex PCR. This indicates that 98.11% deletions in dystrophin gene are located in hot spot regions, which is in agreement with our findings.

In the remaining cases without detectable deletion or duplication, after confirmation of the diagnosis by the neurology specialists, DNA sequencing can be used for identification of point mutations, small insertion or deletion that are scattered throughout the dystrophin gene. Alternatively, a dense set of polymorphic and dymorphic markers can be used to track mutation in patients with a family history.

Identification of deletions and duplications in the DMD gene is important for prenatal diagnosis and determination of genotype-phenotype correlation. In recent years, MLPA is recommended to be used as the method of choice for detection of deletions and duplications in the DMD gene. In this study, we detected 95% of the deletions by multiplex PCR, which shows that most of the deletions in our population are located in the hot spot regions. These findings indicate that an initial analysis of patients with DMD using multiplex PCR and then MLPA analysis of patients without detectable mutation is more practical in our population. This approach is described to be a precise and cost-effective tool for DMD diagnosis in developing countries as described by Murugan [24] in the Indian population.

  Acknowledgment Top

This study was funded by grant number 189087 from deputy for research, Isfahan University of Medical Sciences, Isfahan, Iran. The authors are grateful to patients with DMD for their participating in this study.

  References Top

1.Roberts RG, Coffey AJ, Bobrow M, Bentley DR. Determination of the exon structure of the distal portion of the dystrophin gene by vectorette PCR. Genomics 1992;13:942-50.  Back to cited text no. 1
2.Hoffman EP, Brown RH Jr, Kunkel LM. Dystrophin: The protein product of the Duchenne muscular dystrophy locus. Cell 1987;51:919-28.  Back to cited text no. 2
3.Blake DJ, Weir A, Newey SE, Davies KE. Function and genetics of dystrophin and dystrophin-related proteins in muscle. Physiol Rev 2002;82:291-329.  Back to cited text no. 3
4.Ehmsen J, Poon E, Davies K. The dystrophin-associated protein complex. J Cell Sci 2002;115:2801-3.  Back to cited text no. 4
5.Prior TW, Bridgeman SJ. Experience and strategy for the molecular testing of Duchenne muscular dystrophy. J Mol Diagn 2005;7:317-26.  Back to cited text no. 5
6.Emery AE. The muscular dystrophies. Lancet 2002;359:687-95.  Back to cited text no. 6
7.Bushby KM, Thambyayah M, Gardner-Medwin D. Prevalence and incidence of Becker muscular dystrophy. Lancet 1991;337:1022-4.  Back to cited text no. 7
8.Aartsma-Rus A, Van Deutekom JC, Fokkema IF, Van Ommen GJ, Den Dunnen JT. Entries in the Leiden Duchenne muscular dystrophy mutation database: An overview of mutation types and paradoxical cases that confirm the reading-frame rule. Muscle Nerve 2006;34:135-44.  Back to cited text no. 8
9.White SJ, Aartsma-Rus A, Flanigan KM, Weiss RB, Kneppers AL, Lalic T, et al. Duplications in the DMD gene. Hum Mutat 2006;27:938-45.  Back to cited text no. 9
10.Den Dunnen JT, Grootscholten PM, Bakker E, Blonden LA, Ginjaar HB, Wapenaar MC, et al. Topography of the Duchenne muscular dystrophy (DMD) gene: FIGE and cDNA analysis of 194 cases reveals 115 deletions and 13 duplications. Am J Hum Genet 1989;45:835-47.  Back to cited text no. 10
11.Muntoni F, Torelli S, Ferlini A. Dystrophin and mutations: One gene, several proteins, multiple phenotypes. Lancet Neurol 2003;2:731-40.  Back to cited text no. 11
12.Sellner LN, Taylor GR. MLPA and MAPH: New techniques for detection of gene deletions. Hum Mutat 2004;23:413-9.  Back to cited text no. 12
13.Schouten JP, McElgunn CJ, Waaijer R, Zwijnenburg D, Diepvens F, Pals G. Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification. Nucleic Acids Res 2002;30:e57.  Back to cited text no. 13
14.Schwartz M, Dunø M. Multiplex ligation-dependent probe amplification is superior for detecting deletions/duplications in Duchenne muscular dystrophy. Clin Genet 2005;67:189-91.  Back to cited text no. 14
15.Puget N, Stoppa-Lyonnet D, Sinilnikova OM, Pagès S, Lynch HT, Lenoir GM, et al. Screening for germ-line rearrangements and regulatory mutations in BRCA1 led to the identification of four new deletions. Cancer Res 1999;59:455-61.  Back to cited text no. 15
16.Beggs AH, Koenig M, Boyce FM, Kunkel LM. Detection of 98% of DMD/BMD gene deletions by polymerase chain reaction. Hum Genet 1990;86:45-8.  Back to cited text no. 16
17.Chamberlain JS, Gibbs RA, Ranier JE, Nguyen PN, Caskey CT. Deletion screening of the Duchenne muscular dystrophy locus via multiplex DNA amplification. Nucleic Acids Res 1988;16:11141-56.  Back to cited text no. 17
18.Abbs S, Yau SC, Clark S, Mathew CG, Bobrow M. A convenient multiplex PCR system for the detection of dystrophin gene deletions: A comparative analysis with cDNA hybridisation shows mistypings by both methods. J Med Genet 1991;28:304-11.  Back to cited text no. 18
19.Sedlácková J, Vondrácek P, Hermanová M, Zámecník J, Hrubá Z, Haberlová J, et al. Point mutations in Czech DMD/BMD patients and their phenotypic outcome. Neuromuscul Disord 2009;19:749-53.  Back to cited text no. 19
20.Yau SC, Bobrow M, Mathew CG, Abbs SJ. Accurate diagnosis of carriers of deletions and duplications in Duchenne/Becker muscular dystrophy by fluorescent dosage analysis. J Med Genet 1996;33:550-8.  Back to cited text no. 20
21.Abbs S, Bobrow M. Analysis of quantitative PCR for the diagnosis of deletion and duplication carriers in the dystrophin gene. J Med Genet 1992;29:191-6.  Back to cited text no. 21
22.Hwa HL, Chang YY, Chen CH, Kao YS, Jong YJ, Chao MC, et al. Multiplex ligation-dependent probe amplification identification of deletions and duplications of the Duchenne muscular dystrophy gene in Taiwanese subjects. J Formos Med Assoc 2007;106:339-46.  Back to cited text no. 22
23.Khordadpoor-Deilamani F, Akbari MT, Nafissi S, Zamani G. Dystrophin gene mutation analysis in Iranian males and females using multiplex polymerase chain reaction and multiplex ligation-dependent probe amplification methods. Genet Test Mol Biomarkers 2011;15:893-9.  Back to cited text no. 23
24.Murugan S, Chandramohan A, Lakshmi BR. Use of multiplex ligation-dependent probe amplification (MLPA) for Duchenne muscular dystrophy (DMD) gene mutation analysis. Indian J Med Res 2010;132:303-11.  Back to cited text no. 24
[PUBMED]  Medknow Journal  


  [Figure 1]

This article has been cited by
1 Multiplex ligation-dependent probe amplification – a short overview
Valeriu Moldovan,Elena Moldovan
Revista Romana de Medicina de Laborator. 2020; 28(2): 123
[Pubmed] | [DOI]
2 Molecular diagnosis of dystrophinopathies in Morocco and report of six novel mutations
Youssef EL Kadiri,Yassir Selouani,Ilham Ratbi,Jaber Lyahyai,Abdelali Zrhidri,Maryem Sahli,Mouna Ouhenach,Imane Cherkaoui Jaouad,Abdelaziz Sefiani,Aziza Sbiti
Clinica Chimica Acta. 2020; 506: 28
[Pubmed] | [DOI]
3 Predominance of Dystrophinopathy Genotypes in Mexican Male Patients Presenting as Muscular Dystrophy with A Normal Multiplex Polymerase Chain Reaction DMD Gene Result: A Study Including Targeted Next-Generation Sequencing
Miguel Angel Alcántara-Ortigoza,Miriam Erandi Reyna-Fabián,Ariadna González-del Angel,Bernardette Estandia-Ortega,Cesárea Bermúdez-López,Gabriela Marisol Cruz-Miranda,Matilde Ruíz-García
Genes. 2019; 10(11): 856
[Pubmed] | [DOI]
4 Distribution of dystrophin gene deletions in a Chinese population
Yuanyuan Li,Zhuo Liu,Shengrong OuYang,Yanli Zhu,Liwen Wang,Jianxin Wu
Journal of International Medical Research. 2016; 44(1): 99
[Pubmed] | [DOI]


Previous article  Next article
Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
Access Statistics
Email Alert *
Add to My List *
* Registration required (free)

  In this article
Materials and Me...
Article Figures

 Article Access Statistics
    PDF Downloaded296    
    Comments [Add]    
    Cited by others 4    

Recommend this journal