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| ORIGINAL ARTICLE |
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| Year : 2011 | Volume
: 59
| Issue : 6 | Page : 803-809 |
Identification of deletions and duplications in the Duchenne muscular dystrophy gene and female carrier status in western India using combined methods of multiplex polymerase chain reaction and multiplex ligation-dependent probe amplification
Rashna S Dastur1, Munira Y Kachwala1, Satish V Khadilkar2, Madhuri R Hegde3, Pradnya S Gaitonde1
1 Department of Molecular Biology, Institute for Advanced Training and Research in Interdisciplinary Sciences (IATRIS), Therapeutic Drug Monitoring Laboratory, Sion, India
2 Department of Neurology, Medical Research Centre, Bombay Hospital, Mumbai, India
3 Department of Human Genetics, Emory School of Medicine, Atlanta, USA
| Date of Submission | 23-May-2011 |
| Date of Decision | 28-Jun-2011 |
| Date of Acceptance | 05-Aug-2011 |
| Date of Web Publication | 2-Jan-2012 |
Correspondence Address:
Rashna S Dastur
Department of Molecular Biology, Institute for Advanced Training & Reserch in Interdisciplinary Sciences (IATRIS), (TDM-Therapeutic Drug Monitoring Laboratory), Plot 194, Scheme No. 6, Road No. 15, Sion-Koliwada, Sion (East), Mumbai - 400 022
India
 Source of Support: None, Conflict of Interest: None  | Check |
DOI: 10.4103/0028-3886.91355
Background: The technique of multiplex ligation-dependent probe amplification (MLPA) assay is an advanced technique to identify deletions and duplications of all the 79 exons of DMD gene in patients with Duchenne/Becker muscular dystrophy (DMD/BMD) and female carriers. Aim: To use MLPA assay to detect deletions which remained unidentified on multiplex polymerase chain reaction (mPCR) analysis, scanning 32 exons of the "hot spot" region. Besides knowing the deletions and/or duplications, MLPA was also used to determine the carrier status of the females at risk. Materials and Methods: Twenty male patients showing no deletions on mPCR and 10 suspected carrier females were studied by MLPA assay using P-034 and P-035, probe sets (MRC Holland) covering all the 79 exons followed by capillary electrophoresis on sequencing system. Results: On MLPA analysis, nine patients showed deletions of exons other than 32 exons screened by mPCR represented by absence of peak. Value of peak areas were double or more in four patients indicating duplications of exons. Carrier status was confirmed in 50% of females at risk. Conclusion: Combining the two techniques, mPCR followed by MLPA assay, has enabled more accurate detection and extent of deletions and duplications which otherwise would have remained unidentified, thereby increasing the mutation pick up rate. These findings have also allowed prediction of expected phenotype. Determining carrier status has a considerable significance in estimating the risk in future pregnancies and prenatal testing options to limit the birth of affected individuals.
Keywords: Carrier, deletions, Duchenne muscular dystrophy gene, duplications, multiplex ligation-dependent probe amplification
How to cite this article:
Dastur RS, Kachwala MY, Khadilkar SV, Hegde MR, Gaitonde PS. Identification of deletions and duplications in the Duchenne muscular dystrophy gene and female carrier status in western India using combined methods of multiplex polymerase chain reaction and multiplex ligation-dependent probe amplification. Neurol India 2011;59:803-9 |
How to cite this URL:
Dastur RS, Kachwala MY, Khadilkar SV, Hegde MR, Gaitonde PS. Identification of deletions and duplications in the Duchenne muscular dystrophy gene and female carrier status in western India using combined methods of multiplex polymerase chain reaction and multiplex ligation-dependent probe amplification. Neurol India [serial online] 2011 [cited 2022 Mar 14];59:803-9. Available from: https://www.neurologyindia.com/text.asp?2011/59/6/803/91355 |
| » Introduction | |  |
Identifying gene mutations using DNA-based technologies is now the preferred method of diagnosis for most of the inherited diseases. Duchenne muscular dystrophy (DMD) is one of the most common inherited X-linked muscle disorders and affects 1 in 3500 males. [1] Becker's muscular dystrophy (BMD) is a milder allelic form of the disease. DMD/BMD is due to deletions duplications or point mutations in the DMD gene that encodes dystrophin protein. DMD gene is one of the largest genes of the human genome spanning 2.4 MB and consists of 79 exons. [1] It has been reported that 65-70% of the mutations that cause DMD/BMD are due to deletion of one or more exons. More than 98% of deletions are detectable in affected males using multiplex polymerase chain reaction (mPCR). [2],[3] Screening 32 exons of the "hotspot" region offers a rapid and simple method to detect deletions in male patients. [4] Mutations altering the reading frame results in the DMD phenotype and mutations retaining the full-length mRNA cause the milder BMD phenotype; however, exceptions to this rule have been documented. Thus, information on the extent of mutations is critical in predicting progression of the diseases. For patients not showing a deletion in the hotspot region and in patients where the précised extent and delineation of deletions are not detected on mPCR, advanced method of multiplex ligation-dependent probe amplification (MLPA) is used to examine all 79 exons. [5] MLPA is a modification of polymerase chain reaction and permits multiple targets to be amplified with only a single primer pair. [5] This method detects deletions in the remaining non-hotspot regions or duplications, if any. Duplications of exons cannot be seen by mPCR-based assay, due to nonquantitative nature of the technique. [6]
DMD/BMD being an X-linked disorder, in female's deletions is masked by amplification of the normal X-chromosome. PCR approach is not useful in identifying female carriers as mPCR analysis is based on discrimination between presence and absence of PCR product. As there is no effective treatment available for this debilitating disease, identification of carrier females followed by prenatal testing, is essential to prevent the birth of affected male. By using MLPA technique, carrier status of females can ascertained. [7] This method involves simultaneous hybridization and ligation of multiple probes in a single reaction, followed by PCR and analysis by capillary electrophoresis on gene sequencer. [6]
| » Materials and Methods | | |
In the present study, 138 samples of genomic DNA of unrelated DMD/BMD male patients were included over a 1-year period. Of the study samples, 72% showed deletions in the hotspot regions covering 32 exons by mPCR analysis. [2],[3],[4] Of the remaining patients, 20 male patients were further analyzed by MLPA.
All patients referred for 32 exons study had characteristic clinical features of DMD/BMD. The translational reading frame for deletions of 32 exons located in the hotspot regions was predicted by using reading frame checker to distinguish DMD/BMD . [ 8] A total of 30 subjects, 20 male patients [Table 1] and 10 suspected female carriers [Table 2] were included for MLPA analysis. In six ([Table 1]: M3-M8) DMD males confirmed by mPCR, MLPA analysis was carried out to refine the end points. Twelve patients ([Table 1]: M9-M20) with DMD phenotype who showed no deletions on mPCR were subjected to MLPA analysis. Nine females were analyzed for their carrier status [Table 2]. Carrier testing was not offered for minors and was only done in subjects referred by clinicians after obtaining written consent.
MLPA analysis was carried out according to the recommendations of manufacturer (MRC, Holland, Amsterdam, the Netherlands). DNA (20 ng) was denatured and hybridized overnight at 60 °C with SALSA probe mix P034 (DMD exons 1-10, 21-30, 41-50 and 61- 70) and P035 (DMD exons 11-20, 31-40, 51-60 and 71-79). Samples were then treated with ligase 65 for 15 min at 54 °C. The reactions stopped by incubation at 98 °C for 5 mins. Finally, PCR amplification was carried out with specific SALSA FAM PCR primers. Amplification products were run on ABI prism 3100 Genetic Analyzer. The obtained data were analyzed by using Gene Scan 3.7 software.
The peaks obtained after capillary sequencing assigned to specific exons were analyzed. In each sample, for each peak representing individual exon, the relative peak area (RPA) was determined. The mean RPA values in controls were also calculated and considered as 100%, corresponding to the normal value, when no deletion or duplication is present. With each MLPA run, a sample of healthy male and healthy female in case of carrier testing was also included as control.
| » Results | | |
Twenty males showing different ranges of deletion pattern or no deletions on mPCR were taken for MLPA studies.
Deletions
Of the two patients with confirmed deletions of exons 46 to 52 and 48 to 50 respectively on mPCR ([Table 1]: M1 and M2), MLPA analysis showed absence of peaks in the same region as exons deletions in both the patients, thus confirming mPCR results. The deletion regions indicated outframe mutations. Both the patients had elevated serum CPK levels and characteristic clinical features DMD. In six patients [[Table 1]: M3-M8] deletions were detected on mPCR but clear proximal or distal end points were not discernible since all 79 exons are not covered by mPCR. One of the six males, aged 9 years ([Table 1]: M7) showed deletion of exons 17, 19, 20, 21 and 43. In the study of 32 primer set, primers of exon 18 and exons 22 to 42 were not included. Hence, it was not possible to conclude whether the deletions are in two different regions or are continuous. On MLPA, electropherogram revealed absence of peaks of all the corresponding exons spanning from 17 to 43 [Figure 1]a-c. Similarly, other five males, showing incomplete coverage of exon deletions on mPCR were processed for MLPA and showed discrete endpoints enabling to confirm the reading frame of the mutation type [Table 1]. A patient aged 2 years ([Table 1]: M9) with clinical diagnosis of DMD did not show any deletions on mPCR. But MLPA analysis showed absences of peaks corresponding to exons 22 to 42 were clearly seen. | Figure 1: a: Gel picture of DMD male patient showing absence of bands in lane 4,7,10 representing deletions of exons 17, 19, 20, 21 and 43 in dystrophin gene by mPCR
Figure 1: b: MLPA electropherogram of DMD male patient showing absence of peaks in DMD Probe-P034 (MRC- Holland) representing deletions of the exons 21 to 30 and 41 to 43 of the Dystrophin gene
Figure 1: c: MLPA electropherogram of DMD male patient showing absence of peaks in DMD Probe-P035 (MRC- Holland) representing deletions of the exons 17 to 20 and 31 to 40 of the Dystrophin gene
Click here to view |
Duplications
Of the 20 affected males analyzed, 12 ([Table 1]: M9-M20) did not show any deletions on mPCR. On MLPA studies duplications of different exons were identified in four males ([Table 1]: M10-M13). In four patients (M10-M13), relative peak area values corresponding to specific exons were seen to have more than two-fold increase compared to control values [Figure 2]a and b. These duplications were considered reliable as they involved two or more contiguous exons. In nine patients (M14-M20) no deletions /duplications were detected in any of the 79 exons, possibilities of a point mutation in these cases is high or probability of another type of Muscular Dystrophy cannot be ruled out. | Figure 2: a: MLPA electropherogram of DMD male patient showing two-fold increase of peaks in DMD Probe-P034 (MRC- Holland) representing duplications of the exons 44 to 50 and exons 61 to 63 of the Dystrophin gene
Figure 2: b: MLPA electropherogram of DMD male patient showing two-fold increase of peaks in DMD Probe-P035 (MRC- Holland) representing duplications of the exons 51 to 60 of the Dystrophin gene
Click here to view |
Female carrier
Out of 30 samples studied by MLPA, 10 were females ([Table 2]: F1 to F10) with an affected family member and they were analyzed to determine the carrier status.
Carrier diagnosis for known familial mutations
Four females (F1, F2, F4 and F5) were identified to be carriers. In them, the affected relative i.e., the index case was confirmed by identification of the causative mutations. Female F1 aged 40 years had an affected son with exons 45 to 52 deletions detected on mPCR. On MLPA analysis, the relative peak areas were seen to be reduced by more than 50%, corresponding to the same exons i.e. 45 to 52 as reported to be absent in her son [Table 2]. MLPA analysis was also performed to detect the carrier status of a healthy female F4, as her two brothers were affected and showed deletions of exons 49 and 50. On electropherogram of this female F4 revealed 50% drop in peak area values corresponding to exons 49 and 50 as compared to control healthy female values, thereby confirming the carrier status [Figure 3]. | Figure 3: MLPA electropherogram of DMD carrier female showing drop in peak area of the exons 49 and 50 in DMD Probe-P034 (MRC- Holland) of the Dystrophin gene
Click here to view |
Carrier diagnosis for unknown mutations
This group included females for whom affected male was not available for testing and mother with highest prior carrier risk was taken for MLPA analysis.
An asymptomatic female F3, aged 24 years, with two of her deceased brothers affected was referred for carrier testing [Table 2]. MLPA analysis was done on female (F3) along with her mother (F2) suspected to be a carrier. The mother (F2) showed more than 50% drop in RPA values of exons 45 to 50, but RPA values of the daughter (F3) did not show any reduction in any of the values for all 79 exons. Hence, was considered to be normal and not a carrier.
Characterization of a disease manifesting carrier
A symptomatic female (F10) aged 25 years showed limb girdle weakness and high CPK values. The patient had one affected brother who died at the age of 19 years and two maternal uncles who also died from the same disease. Genotype of none of the three affected male of the family was available. The mother (F9) of this patient was asymptomatic and was expected to be a carrier. Deletion of exon 45 in one allele was seen in F10 but the mother (F9) did not show any deletion or duplication in any of the 79 exons [Figure 4]. To rule out assay specific hindrance, MLPA procedure was repeated once again and also sent to other laboratory (Department of Human Genetics, Emory School of Medicine, Atlanta) for re-evaluation. [9] Exon 45 was sequenced to see if the drop in peak area was found to be genuine. Results obtained showed the same deletion of exon 45 only in the daughter F10 with clinical disease. This raises the possibility of the mother F9 having germline mosaisicm since one third of DMD mutations are de novo. | Figure 4: MLPA electropherogram of DMD manifesting carrier female showing drop in peak area of the exon 45 in DMD Probe-P034 (MRC- Holland) of the Dystrophin gene
Click here to view |
| » Discussion | | |
In the present analysis, advantages of MLPA technique for detection of exonic rearrangement in the DMD gene have been established. Comprehensive and comparative data from two different methods mPCR and MLPA indicates that the latter offers a considerable superiority over the established mPCR technique. Multiplex PCR is the method of choice for initial screening as it is a sensitive, robust and is cost effective to assess any of the 32 exons deletions. [3],[10] In contrast, to mPCR, MLPA can be used to identify duplications as well as carrier status in females. [7] Of the DMD patients, 60-75% are due to large deletions, 5-10% are due to duplications and 25- 30% are due to point mutations of the gene. [10],[11] Of the 20 DMD patients with uncertain diagnosis of DMD on mPCR screened with MLPA analysis, duplications of exons in different region of DMD gene were detected in four and one showed deletions of exons [Table 1] not belonging to any of the three primer sets included in mPCR method. In the absence of MLPA evaluation, these patients would have remained unidentified without any genotypic confirmation, an essential step for identifying others in the family including female carriers. [12],[13]
With the introduction of the concept of exon skipping, knowing the exact mutation in a patient is necessary for prognostic evaluation. Studying all the 79 exons for any deletions or duplications is useful in predicting disruption of reading frame which in turn facilitates genotype-phenotype correlation. [14],[15] In this study MLPA analysis allowed confirmation of type of disruption of reading frame in eight patients who showed deletions on mPCR. This helped in establishing a genotype-phenotype correlation and predicting progression of the disease. It has been also reported that mutations in proximal part of the gene have higher probability of becoming familial whereas distal deletions are more sporadic. [15] Four patients in this study showed deletions in the proximal part of the DMD gene.
Studies using MLPA technique from other parts of India have shown similar increase in mutation pick up rate. However, these studies have not included the carrier status of females at risk. [16],[17] Conventional methods of female carrier detection on the basis of pedigree, serum CPK levels, and linkage analysis are not of much relevance in management of the disease. Identification of female carriers for deletion/duplication in the DMD gene is essential to prevent birth of affected children. Investigating females at risk where genotype of an affected male relative is known, MLPA analysis can confirm and exclude the carrier status. Although 30% of the DMD mutations are de novo, the risk of recurrence of the disease in families with a single affected male is due to the carrier status of the mother. [18] In our series, 50% of females at risk were seen to be carriers. One of the females i.e., F10 manifesting the disease showed deletion of exon 45. The possible mechanisms for the symptoms in spite of being a heterozygous include: Nonrandom inactivation of X chromosome or translocation, disrupting the DMD gene. [19],[20] In our study in one of the females, MLPA results did not show any deletions or duplications in spite of her having two affected offsprings (a male and a female). In such a case, the possibility of "germline mosaicsm" is implicated where the mother does not have any mutations in the somatic cells. [19],[20] Such cases exhibiting single exon deletions and germline mosaicism are to be verified by DNA sequencing or microarray technique.
One of the limitations of the MLPA technique is occurrence of false positive results due to small mutations or single nucleotide polymorphism sites coinciding with probe binding regions and ligation site. [21] In our study, seven patients did not show mutations by either of the techniques. One of the possible explanations is occurrence of point mutations in the DMD gene which can be identified only by sequencing. But in Indian scenario, the cost of these technologies restrains further analysis, and cannot be adopted in routine diagnostic protocols. Therefore, confirmatory test by mPCR followed by MLPA analysis (if required) are a relatively simple, reproducible and cost-effective methods of improved genetic diagnosis. The application of these two methods is of utmost relevance to establish the definitive diagnosis, to predict prognosis and prevention the disease.
| » Acknowledgment | | |
We acknowledge the benevolent donation of instruments for Molecular Diagnosis by Lions Club of Babulnath - District 323A1, and Mr. Cavas Fitter in memory of Dhun Cavas Fitter in memory of Dhun Cavas Fitter. Special thanks are also due to Dr. Sasikumar K Menon, Assistant Director, TDM Labs for providing the infrastructural facilities; and Muscular Dystrophy Society; neurologists and pediatric neurologists of Mumbai.
| » References | | |
| 1. | Emery AE. Duchenne and other X linked muscular dystrophies. In: Rimoin DL, Connor JM, Pyeritz, Korf BR, editors. Emery and Rimoin's principles and practice of medical genetics, 4 th ed. London: Harcourt Publishers Limited; 2002a; p. 3266-84. 
|
| 2. | Chamberlain JS, Gibbs RA, Ranier JE, Nga Nguyen PN, Caskey CT. Deletion screening of the duchenne muscular dystrophy, locus via multiplex DNA amplification. Nucl Acid Res 1988;16:11141-56.
|
| 3. | Beggs AH, Koeing M, Boyce FN, Kunkel LM. Detection of 98% of DMD/BMD gene deletions. Hum Genet 1990;86:45-8.
|
| 4. | denDunnen JT. Leiden Muscular Dystrophy pages. Center for Human and Clinical Genetics, Leiden University Medical Center. DNA based diagnostic techniques for DMD/BMD Deletion Detection using multiplex PCR. Available from: http://www.dmd.nl/ [Last updated on 2005a Mar 16].
|
| 5. | Schwartz M, Duno M. Multiplex ligation-dependent probe amplification is superior for detecting deletions/duplications in Duchenne muscular dystrophy. Clin Genet 2005;67:189-91.
|
| 6. | Janssen B, Hartmann C, Scholz V, Jauch A, Zschocke J. MLPA analysis for the detection of deletions, duplications and complex rearrangements in the dystrophin gene: Potential and pitfalls. Neurogenetics 2005;6:29-35.
[PUBMED] [FULLTEXT] |
| 7. | Li H, Ding J, Wang W, Chen Y, Lu W, Shao H, et al. Combining approach with multiplex PCR and MLPA to detect deletion and duplication in DMD patients, carriers, and prenatal diagnosis. Zhonghua Yi Xue Yi Chuan Xue Za Zhi 2009;26:318-22.
[PUBMED] |
| 8. | denDunnen JT. Leiden Muscular Dystrophy pages. Center for Human and Clinical Genetics, Leiden University Medical Center. DMD exonic deletions/duplications reading-frame checker 1.6. Available from: http://www.dmd.nl/. [Last updated on 2005b Mar 16].
|
| 9. | Hegde MR, Chin EL, Mulle JG, Okou DT, Warren ST, Zwick ME. Microarray-based mutation detection in the dystrophin gene. Hum Mutat 2008;29:1091-9.
[PUBMED] [FULLTEXT] |
| 10. | Nadkarni JJ, Dastur RS, Viswanathan V, Gaitonde PS, Khadilkar SV. Duchenne and Becker muscular dystrophies: An Indian update on genetics and rehabilitation. Neurol India 2008;56:248-53.
[PUBMED]  |
| 11. | Koenig M, Hoffman EP, Bertelson CJ, Monaco AP, Feener C, Kunkel LM. Complete cloning of the Duchenne muscular dystrophy (DMD) cDNA and preliminary genomic organization of the DMD gene in normal and affected individuals.Cell 1987;50:509-17.
[PUBMED] [FULLTEXT] |
| 12. | Lalic T, Vossen RH, Coffa J, Schouten JP, Guc-Scekic M, Radivojevic D, et al. Deletions and duplication screening in the DMD gene using MLPA. Eur J Hum Genet 2005;13:1231-4.
[PUBMED] [FULLTEXT] |
| 13. | Marzese DM, Manipel A, Gomez LC, Echeverria MI, Vargas AL, Ferrerio V, et al. Detection of deletion and duplications in the Duchenne Muscular Dystrophy geneby the molecular method of MLPA in the first Argentina affected families. Genet Mol Res 2008;7:223-33.
|
| 14. | Monaco AP, Bertelson CJ, Liechti-Gallati S, Moser H, Kunkel KL. An explanation for the phenotypic differences between patients bearing partial deletions of DMD locus. Genomics 1988;2:90-5.
|
| 15. | Dastur R, Gaitonde P, Khadilkar S, Nadkarni J. Becker muscular dystrophy in Indian patients: Analysis of dystrophin gene deletion patterns. Neurol India 2008;56:374-8.
[PUBMED] |
| 16. | 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.
[PUBMED] |
| 17. | 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.
[PUBMED] |
| 18. | Gatta V, Scarciolla O, Gaspari AR, Palka C, De Angelis MV, Di Muzio A, et al. Identification of deletions and duplications of the DMD gene in affected males and carrier females by multiple ligation probe amplification (MLPA). Hum Genet 2005;117:92-8.
[PUBMED] [FULLTEXT] |
| 19. | Bakker E, Veenema H, Den Dunnen JT, van Broeckhoven C, Grootscholten PM, Bonten EJ, et al. Germinal mosaicism increases the recurrence risk for 'new' Duchenne muscular dystrophy mutations. J Med Genet 1989;26:553-9.
[PUBMED] [FULLTEXT] |
| 20. | Prior TW, Bridgeman SJ. Experience and strategy for the molecular testing of Duchenne muscular dystrophy. J Mol Diagn 2005;7:317-26.
[PUBMED] [FULLTEXT] |
| 21. | Flanigan KM, von Niederhausern A, Dunn DM, Alder J, Mendell JR, Weiss RB. Rapid direct sequence analysis of the dystrophin gene. Am J Hum Genet 2003;72:931-9.
[PUBMED] [FULLTEXT] |
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2]
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