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 »  Abstract
 »  Introduction
 »  Patients and Methods
 »  Results
 »  Discussion
 »  Acknowledgments
 »  References
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ORIGINAL ARTICLE
Year : 2010  |  Volume : 58  |  Issue : 6  |  Page : 852-856

Gene changes in Duchenne muscular dystrophy: Comparison of multiplex PCR and multiplex ligation-dependent probe amplification techniques


Centre of Medical Genetics, Sir Ganga Ram Hospital, New Delhi, India

Date of Acceptance08-Jul-2010
Date of Web Publication10-Dec-2010

Correspondence Address:
Sudha Kohli
Centre of Medical Genetics, Sir Ganga Ram Hospital, Rajinder Nagar, New Delhi -110 060
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0028-3886.73744

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 » Abstract 

Background: Duchenne muscular dystrophy (DMD) is a common X-linked recessive neuromuscular disorder, affecting 1 in 3,500 live male births. About 65% of cases are caused by deletions; ~5% to 8%, by duplication; and the remaining, by point mutations of the dystrophin gene. The frequency of complex rearrangements (double-deletion and non-contiguous duplications) is reported to be 4%. Aim: In this study, we examined the usefulness of multiplex ligation-dependent probe amplification (MLPA) for screening of deletion and duplication mutations in a group of DMD/ BMD (Becker muscular dystrophy) patients from India. Patients and Methods: We analyzed 180 patients referred from all over India, by both multiplex PCR technique (22 exons) and MLPA (all 79 exons). Results and Conclusion: By multiplex PCR, deletions were detected in 90 (50%) patients. MLPA studies in these cases detected 3 additional deletions, 16 (8.9%) duplications and 2 point mutations. MLPA is useful to verify absence of deletions/ duplications in all 79 exons. This sets the stage to look for point mutations using RNA- or DNA-based tests because of the availability of the drug PTC124. Also, the extent of the deletions and duplications could be more accurately defined by MLPA. The delineation of the precise extent of deletion helps in deciding whether exon-skipping technique would be useful as therapy.


Keywords: Deletions, Duchenne muscular dystrophy, duplications, dystrophin gene, multiplex ligation-dependent probe amplification


How to cite this article:
Kohli S, Saxena R, Thomas E, Singh J, Verma IC. Gene changes in Duchenne muscular dystrophy: Comparison of multiplex PCR and multiplex ligation-dependent probe amplification techniques. Neurol India 2010;58:852-6

How to cite this URL:
Kohli S, Saxena R, Thomas E, Singh J, Verma IC. Gene changes in Duchenne muscular dystrophy: Comparison of multiplex PCR and multiplex ligation-dependent probe amplification techniques. Neurol India [serial online] 2010 [cited 2019 Aug 23];58:852-6. Available from: http://www.neurologyindia.com/text.asp?2010/58/6/852/73744



 » Introduction Top


Duchenne muscular dystrophy (DMD) and its less severe allelic form Becker muscular dystrophy (BMD) are common X-linked recessive neuromuscular disorders, [1],[2] with an estimated worldwide incidence of 1: 4000 male births. [3] The dystrophin gene spans 2.4 Mb of genomic sequence on chromosome Xp21. [4],[5] About 65% of cases are caused by deletions [6],[7] ; ~ 5% to 8%, by duplications [8] ; and the remaining, by point mutations of the dystrophin gene. [9] The frequency of complex rearrangements (double-deletion and non-contiguous duplications) is reported to be 4%. [10]

The genetic test commonly used to detect dystrophin gene mutations is multiplex PCR. Chamberlain et al.[11] developed a kit to test for 9 exons that were the hotspots for deletions. Beggs et al.[12] added another 9 exons to enlarge the number to be screened. Testing for these 18 exons has been the common practice in the laboratories around the world. Using the 18/27 exon test by multiplex PCR, a number of investigators in India have reported the frequency of deletions in the dystrophin gene. [13],[14],[15],[16],[17],[18] Multiplex ligation - dependent probe amplification (MLPA) technique tests for gene changes in all the 79 exons. We used MLPA technique to test for gene changes in dystrophin gene and compared it to the extended Chamberlain-Beggs (C-B) assay.


 » Patients and Methods Top


Patient samples

We studied 180 patients referred from all over India for molecular analysis of the dystrophin gene, by extended Chamberlain-Beggs (C-B) multiplex PCR technique (18 + 4 = 22 exons) as well as by MLPA test for all 79 exons. The sample included 27 patients who had tested negative earlier by the 18/22 exon test and 8 patients that were positive for the deletion but wanted to know the extent of the deletion. Informed consent was taken from all the patients. Genomic DNA was extracted from the leukocytes by a salting-out procedure. [19] Multiplex PCR and MLPA tests were performed on genomic DNA.

Multiplex PCR deletion screening

For multiplex PCR screening, 22 exons 3, 4, 6, 8, 12, 13, 17, 19, 34, 43, 44, 45, 46, 47, 48, 49 50, 51, 52, 53, 60 and the muscle promoter (pm) of the dystrophin gene were tested, using 4 reactions each containing 4 to 5 sets of primers. These sets included 4 exons (34, 46, 49 and 53), [20] in addition to the Chamberlain-Beggs (C-B) protocol, to increase the detection rate as well to define the extent of the deletions. The PCR products were separated on a 3% agarose gel.

Multiplex ligation-dependent probe amplification

Multiplex ligation-dependent probe amplification (MLPA) is a reliable technology for quantitative analysis of the copy-number variation in genes. [21] It has the capability of detecting either large or small genomic deletions or duplications. [22],[23] An MLPA kit that tests for deletions and/ or duplications of all the 79 exons of the dystrophin gene was used (MRC-Holland, Amsterdam, The Netherlands). MLPA was performed according to the published protocol, except that the PCR reaction was in a final volume of 25 μL, and 35 cycles. Samples were tested in duplicate MLPA reactions. The fragments were analyzed on an ABI model capillary sequencer (Applied Biosystems) with the Genescan 500 size standard (Applied Biosystems). Individual peaks corresponding to each exon were identified based on the differences in migration relative to the size standards. The peak area of each fragment was compared to that of a control sample. In the protocol, the relative signal of each probe is determined by dividing each measured peak area (A s) by the sum of all peak areas (Σ As ) of that sample. This relative peak area (A s / Σ As ) is then divided by the relative peak area of the corresponding probe obtained from a control DNA sample (A c / Σ Ac ). For this analysis, Coffalyser software (MRC-Holland) was used. As the DMD gene is located on the X chromosome, a deletion of one or more exons results in total absence of the corresponding MLPA amplification product in male patients. Duplication gives an almost two-fold greater relative peak area in male patients.


 » Results Top


Of 180 DNA samples from both DMD and BMD patients that were analyzed by multiplex PCR, 90 (50%) showed deletions of one or more exons. MLPA analysis of the same 180 samples revealed 93 (51.6%) deletions and 16 (8.9%) duplications [Table 1]. MLPA test detected three additional deletions, viz., exon 18, exons 20-23 and exons 20-22, that were missed by multiplex PCR (because these exons are not included in the multiplex PCR testing protocol).
Table 1: Comparison of deletion/duplication detected by the two techniques (n=180)


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As MLPA test covers all the exons of the DMD gene, it allowed accurate determination of the size of the deletion in all the subjects. Sixteen patients were found to have a deletion larger than that detected by multiplex PCR [Table 2].
Table 2: Comparison of the extent of deletions detected by the two techniques*


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In 2 cases, exon 47 was not deleted by multiplex PCR, but it showed a deletion by MLPA. To explain this discrepancy, we suspected that there would be a polymorphism/ mutation at the probe-binding site of the MLPA. We performed DNA sequencing of exon 47 in the 2 cases and observed a missense (c.6799C>T, p.L2267F) and a nonsense mutation (c.6809T>G, p.L2270X), respectively, in the 2 cases [Figure 1] and [Figure 2]. The mutation c.6809T>G, p.L2270X was located precisely at the ligation site and destroyed the ligation, which resulted in failure of amplification of exon 47 probe in MLPA. The other mutation c.6799C>T, p.L2267F was adjacent to the probe-binding site and may have failed ligation and amplification of the exon 47 probe in MLPA.
Figure 1: DNA sequencing of exon 47 in Case 1

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Figure 2: DNA sequencing of exon 47 in Case 2

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Of 93 cases with deletion tested by MLPA, 28 (30%) carried deletion of a single exon (44, 45, 50, 51, 52, 53). Exon 45 was the most commonly deleted single exon. The commonly involved exons were numbers 45 to 52, at the 3' end of the gene. Of 93 cases, 74 (79.6%) showed deletions in the 3' hot spot region.

MLPA test detected duplications in 16 (8.9%) cases. Eleven of the 16 duplications were less than 10 exons, and 3 cases had large duplications (30, 12 and 18 exons). Duplication of a single exon (number 2) was found in 3 subjects. The region from exon 2 to exon 15 was the hot spot for duplications as 10 out of 16 duplications were in the 5' hot spot region.


 » Discussion Top


Currently full characterization of the mutation in dystrophin gene is recommended for predicting the effect of the mutation on the reading frame of the gene, which is the major determinant of the phenotypic variability. This also determines the eligibility for the mutation-specific treatments currently undergoing trial. [24]

As DMD is an X-linked disorder, deletions can be easily detected in males using PCR technology. However, due to the large numbers of exons (79) in the dystrophin gene, most investigators test only a subset of exons to reduce the cost. [Table 3] lists the frequency of deletions in dystrophin gene reported by investigators from India using multiplex PCR for 18-32 exons. This varied from 63% to 73%.
Table 3: Frequency of deletions in dystrophin gene in India


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The number of deletions detected in our subset of 180 patients were relatively low (93/180; 51.6%). The probable reasons are that the cases referred to us from different centers may have included some with uncertain diagnosis; it also contained 27 cases that had been earlier tested negative for the 18/22 exons deletions. The latter was counterbalanced by inclusion of 8 cases that were positive for the deletion but wanted to define the extent of deletion. If we exclude the 35 cases tested earlier, the remaining 145 cases revealed deletions in 85 (58.6%) cases and duplications in 9 (6.2%) cases. Our own data of over 12 years revealed deletions in 743 (63%) of 1,175 patients (Verma et al., unpublished).

In the present study, MLPA technique detected deletions in 3 additional subjects as compared with multiplex PCR. We were able to determine the full extent of deletions in all the cases. In 16 cases, the deletions observed by MLPA were found to involve more exons than when studied by multiplex PCR.

We detected duplications in 16 (8.9%) of 180 subjects. This is the first report of duplications in patients of DMD from India. Several studies have shown that MLPA is useful in detecting mutation quantitatively, not only for deletions but also for duplications in the dystrophin gene [11],[25],[26],[27],[28],[29] [Table 4].
Table 4: Deletions/duplications in all exons of dystrophin gene reported globally


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White [23] analyzed 102 patients that were negative for deletions and point mutations, and detected duplications in 6% of cases by multiplex amplifiable probe hybridization technique. Similar to our study, duplication frequency was the highest near the 5' end of the gene. We did not detect any noncontiguous duplications, which is unlike what is reported in the literature . [30]

The knowledge of extent of deletion/duplication is critical when predicting the prognosis; as in ~90% of cases, a reading-frame-disrupting mutation will lead to the severe Duchenne phenotype. [31] Mutations that maintain the open reading frame lead to the milder Becker-like phenotype. Moreover, the delineation of the precise extent of the deletion helps in deciding whether exon-skipping technique would be useful as therapy.[32] Antisense-mediated exon-skipping has recently been shown to correct the reading frame and restore dystrophin expression in vitro and in vivo.[33] Exon 51-skipping is being tested in clinical trials. [34]

MLPA is useful to verify absence of deletions/ duplications in all 79 exons. This sets the stage to look for point mutations using RNA- [35] or DNA-based [36] tests. This has become necessary because the drug PTC124 has the ability to bypass stop codon mutations in the gene, thus converting a severe phenotype to a milder one. Currently trials are in progress to examine the usefulness of this drug in a clinical setting. The DMD MLPA test constitutes a simple DNA-based test for deletion/ duplication screening of all 79 exons of the gene. The apparent exon 47 deletion observed in 2 patients caused by point mutations in the probe-binding region, demonstrates that single-exon deletions observed using MLPA should always be confirmed by a second method such as multiplex PCR or sequence analysis. Also, single-exon deletions by m-PCR should be checked with alternate primers for the specific exon. Compared to analyzing limited exons by multiplex PCR, the advantage of MLPA-based screening of all exons is clearly evident in the study.


 » Acknowledgments Top


We thank MRC-Holland, from whom we obtained MLPA kits described in this article; and Lab India for running samples on Applied Biosystems sequencer.

 
 » References Top

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    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]

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