Duchenne muscular dystrophy (DMD) is one of the most common inherited paediatric neuromuscular disorders, affecting 1 in 3500 live male births 1 . It is an X-linked disorder caused by mutations in the DMD gene, one of the largest known human genes covering 2.4 Mb, encoding a 14-kb mRNA and 8 tissue specific Use of multiplex ligation-dependent probe amplification (MLPA) for Duchenne muscular dystrophy (DMD) gene mutation analysis Sakthivel Murugan S.M., Arthi Chandramohan & Bremadesam Raman Lakshmi Sundaram Medical Foundation, Chennai, India Received July 17, 2009 Background & objectives: Duchenne (DMD) and Becker muscular dystrophy (BMD) are X-linked recessive disorders, caused by mutations in the dystrophin gene. Genetic diagnosis of the proband becomes crucial, and forms the base for carrier analysis, genetic counselling, prediction of natural history and prognosis, and eligibility for therapeutic strategies. Traditional multiplex PCR assay is the common method used in India to detect DMD gene deletions, mainly in the hot-spot region. Deletions of exons outside the usual 18 or 21 exons in the hot-spot, duplications and carrier analysis are often left without precise genetic diagnosis and require efficient dosage/quantitative analysis. In this study we evaluated the efficacy of using multiplex PCR (mPCR) of 30 exons followed by multiplex ligation-dependent probe amplification (MLPA), to study deletions and duplications in the DMD gene in patients clinically diagnosed as BMD/ DMD. Methods: Using an algorithm of mPCR and MLPA which was less invasive and cost-effective, we performed retrospective and prospective analysis on 150 male patients. Results: Multiplex PCR could pick up deletions in 103 of the 150 cases. MLPA was able to detect deletions and duplications including nine additional mutations. Further, the borders of the deletions and duplications were more accurately defined by this recent methodology, which enables one to determine the effect of the mutation on the reading frame. In all, including the single exon deletions, MLPA was efficient in accurately confirming mutations in 35 per cent of all cases. Ten novel mutations were identified in this study. Overall, this approach confirmed mutations in 75 per cent of the patients in our study. Interpretations & conclusions: The systematic approach/algorithm used in this study offers the best possible economical mutation analysis in the Indian scenario. Key words Algorithm - Becker muscular dystrophy - Duchenne muscular dystrophy - multiplex ligation-dependent probe amplification (MLPA) promoters and contains 79 exons 2 . Mutations leading to a truncated protein cause the severe phenotype of DMD, whereas mutations retaining the mRNA reading frame cause the more mild phenotype of Becker muscular dystrophy (BMD). Disruption of the translational reading frame seems to be an important Indian J Med Res 132, September 2010, pp 303-311 303
Transcript
Duchenne muscular dystrophy (DMD) is one of the most common inherited paediatric neuromuscular disorders, affecting 1 in 3500 live male births1. It is an X-linked disorder caused by mutations in the DMD gene, one of the largest known human genes covering 2.4 Mb, encoding a 14-kb mRNA and 8 tissue specific
Use of multiplex ligation-dependent probe amplification (MLPA) for Duchenne muscular dystrophy (DMD) gene mutation analysis
Background & objectives: Duchenne (DMD) and Becker muscular dystrophy (BMD) are X-linked recessive disorders, caused by mutations in the dystrophin gene. Genetic diagnosis of the proband becomes crucial, and forms the base for carrier analysis, genetic counselling, prediction of natural history and prognosis, and eligibility for therapeutic strategies. Traditional multiplex PCR assay is the common method used in India to detect DMD gene deletions, mainly in the hot-spot region. Deletions of exons outside the usual 18 or 21 exons in the hot-spot, duplications and carrier analysis are often left without precise genetic diagnosis and require efficient dosage/quantitative analysis. In this study we evaluated the efficacy of using multiplex PCR (mPCR) of 30 exons followed by multiplex ligation-dependent probe amplification (MLPA), to study deletions and duplications in the DMD gene in patients clinically diagnosed as BMD/DMD.Methods: Using an algorithm of mPCR and MLPA which was less invasive and cost-effective, we performed retrospective and prospective analysis on 150 male patients.Results: Multiplex PCR could pick up deletions in 103 of the 150 cases. MLPA was able to detect deletions and duplications including nine additional mutations. Further, the borders of the deletions and duplications were more accurately defined by this recent methodology, which enables one to determine the effect of the mutation on the reading frame. In all, including the single exon deletions, MLPA was efficient in accurately confirming mutations in 35 per cent of all cases. Ten novel mutations were identified in this study. Overall, this approach confirmed mutations in 75 per cent of the patients in our study.Interpretations & conclusions: The systematic approach/algorithm used in this study offers the best possible economical mutation analysis in the Indian scenario.
promoters and contains 79 exons2. Mutations leading to a truncated protein cause the severe phenotype of DMD, whereas mutations retaining the mRNA reading frame cause the more mild phenotype of Becker muscular dystrophy (BMD). Disruption of the translational reading frame seems to be an important
Indian J Med Res 132, September 2010, pp 303-311
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factor in deciding the severity of the disease in DMD, as compared to the extent of deletions or duplications3.
Identification of mutations in probands aids not only in confirming a clinical diagnosis but also allows carrier testing and prenatal diagnosis for family members4. Moreover, the potential therapies being tested for DMD, such as exon skipping and PTC 124 (Ataluren), are absolutely dependant on precise knowledge of the mutation5,6. Further, on a long term perspective the mutation analysis paves a base for phenotypic-genotypic correlation that throws light in understanding the disorder.
The method of choice in India for DMD/BMD diagnosis is multiplex PCR which targets about 18 to 32 exons of the DMD gene to look for whole exon deletions7-11. Multiplex PCR is mostly qualitative or semi-quantitative and serves for the exons in the hot-spot regions12,13. MLPA (multiplex ligation-dependent probe amplification) was developed by Schouten et al14 as a general method to establish copy number of up to 45 nucleic acid sequences in one single reaction. This method has proved a reliable tool for the diagnosis of genetic diseases characterized by large gene deletions and duplications15,16. MLPA has enabled more reliable and faster quantitative detection of the entire dystrophin gene containing 79 exons to study the deletions and duplications17-20.
In this study, we report the usefulness of a systematic approach for deletion and duplication analysis of the DMD gene, using mPCR followed by MLPA in patients suspected to be affected by BMD/DMD.
Material & Methods
Patients: All patients clinically diagnosed with either Duchenne/Becker muscular dystrophy during December 2006 to December 2007 who were referred to Molecular Diagnosic Facility of Dr Rangarajan Memorial Hospital, Chennai, for genetic diagnosis were included in this study. It was mandatory that they brought in the request for genetic analysis, filled in for clinical details by the referring clinician. The clinical details with regard to the most frequently observed symptoms like age of onset, age at loss of ambulation, calf muscle hypertrophy, Gower’s syndrome, including pedigree, were captured in the request form. Of the 150 cases, ranging between 3-36 yr, all being males, 8 were clinically diagnosed as BMD and the remaining as DMD. All samples were tested initially using multiplex PCR. We retrospectively analysed the mPCR results
of 30 exons of these patients and also prospectively analysed samples which did not show any mutation using MLPA. We also included those which showed single exon deletion and those samples where deletion borders as determined by mPCR were not clear (to apply the reading frame rule), for MLPA analysis.
Blood samples with EDTA (3 ml quantities) were collected from patients; DNA was extracted by salting out method21, quantified and stored at -20oC until tested. Informed consent was got from each patient/parent towards genetic analysis and utilization of the results for education and research purpose. The study protocol was approved by the Review Board of Sundaram Medical Foundation.
Multiplex PCR: Multiplex PCR analysis was performed for 30 exons at the central and 5’end hot spot regions4,12,13. The primers for the 30 exons were obtained from www.dmd.nl website. Multiplex PCR was done in 6 sets each consisting of 4-6 exons. The exons tested were 1, 3, 4, 6, 8, 12, 13, 16, 17, 19, 20, 21, 22, 32, 34, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 55 and 60. The reactions were set in a volume of 25 µl, containing 10 pmoles of each primer, 1.5 mM dNTPS, 1U Taq DNA polymerase (Amplitaq Gold), and 250 ng of genomic DNA. The amplification programme for the multiplex was as follows: 93oC for 60 sec, 60oC for 45 sec, 65oC for 60 sec (27 times) followed by final elongation step at 65oC for 10 min. Reaction products were separated on a 2 per cent agarose gel. Multiplex PCR was done for all the samples as the first step to pick out deletions.
Multiplex ligation-dependent probe amplification (MLPA): MLPA analysis was carried out using PO34 and PO35 probes purchased commercially from MRC, Holland (Amsterdam, The Netherlands). The procedures were carried out according to the manufacturer’s recommendations17. Briefly, 100 ng DNA was denatured and hybridized overnight at 60oC with 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). Samples were then treated with Ligase 65 for 15 min at 54oC. The reactions were stopped by incubation at 98oC for 5 min. Finally, PCR amplification was carried out with the specific SALSA FAM PCR primers. Amplification products were run on an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems, USA) with the following modules: capillaries 36 cm, Polymer POP-4, run temperature 60oC, capillary fill volume 184 steps, pre-run voltage 15 kV, pre-run time 180 sec, injection voltage 3.0 kV, injection time 10-30 sec, run voltage 15 kV, data delay
304 INDIAN J MED RES, SEPTEMBER 2010
time 1 sec, run time 1500 sec. The obtained data were analysed by using Genemapper 3.7 Software. Five healthy males and five females without family history of dystrophinopathies were analyzed as controls. They were not age matched controls.
Analysis of MLPA data: The Genemapper results were exported to excel sheet and the data were organized based on product sizes and the peak heights of each probe were used to calculate the dosage quotient using the Andrew’s software (NGRL, UK). The Andrew’s software compares peak heights with internal controls and also with peaks of 5 control samples. This software determines the relative probe signals of each probe by dividing each measured peak area (As) by the sum of all 45 peaks area (ΣAs) of that sample. The relative peak area (As/ΣAs) was then divided by the relative peak area of the corresponding probe obtained from a control DNA sample (Ac/ΣAc). Result are given in terms of normalized ratio and normalized peak heights.
Validation of MLPA method: Methodology and analysis of the MLPA technique was validated using samples obtained from the Molecular Gene Testing and Cytogenetic Laboratories, Centre for Human and Clinical Genetics, Leiden University Medical Centre, Leiden, The Netherlands.
Results
Of the 150 apparently unrelated males, clinically diagnosed as DMD or BMD, tested for DMD gene mutations, pedigree details were available for 134 cases, of which 13 (9.7%) had family history of DMD. Table I shows the spectrum of mutations picked up in our study using the methods of mPCR and MLPA.
Retrospective analysis of DMD gene deletion using mPCR: Multiplex PCR was able to pick up deletions in 103 cases, which accounted for 68.7 per cent of all cases. There were 21 single exon deletions (18.75% of all mutation positive cases). Deletion of exons 44 and 45 accounted for more than 50 per cent of single exon deletions. Most of the deletions were confined to the central hot spot region between exons 44 and 55 (90 cases). The most common of the deletions was that of exons 45-50 in 11 samples. However, deletion of specific exons to assess the framedness was not clear for 23 cases and hence the reading frame rule could not be applied, using the mPCR results. Among the cases for whom the deletion of exons to apply the reading frame rule was clear, 74 showed out-of-frame
deletions and 6 showed in-frame deletions (Table II). After doing MLPA, which screened all the 79 exons in two reactions, specific exons to assess framedness was clear, which resulted in 11 in-frame deletions and 91 out-of-frame deletions. Though only 8 cases were diagnosed as BMD clinically, there were 11 samples with in-frame deletions. These may be exceptions to the reading frame rule, and need to be studied further at the mRNA level and for the protein characteristics. Most of the deletions had their 5’ end between exons 45 and 48 (66 cases) and their 3’end between exons 50 and 55 (60 cases). The commonest breaking point at the 5’ region was intron 44 (34 cases) and that in the 3’ region was intron 51 (28 cases). The most frequently deleted exons were exon 49 and exon 50 (Table I).The fact that our samples were primarily referred by paediatricians could be the reason for low numbers of BMD cases. Samples that showed no deletion, single exon deletion and deletion with borders not clear by mPCR were further tested by MLPA.
Prospective analysis of deletion-negative samples by MLPA: Nine out of 47 deletion-negative cases showed mutations when tested by MLPA. There were 8 duplications and 1 deletion (Table I). Four of the eight duplications were single exon duplications. The single exon deletion (exon 62), which was identified by MLPA, was confirmed by native mPCR to rule out point mutations in the probe ligation site. The commonest duplication was exon 2 duplication, which was seen in 3 cases.
Five of the 8 duplications have their origin at the 3’ region of the gene. Duplications account for 5.3 per cent of all cases suspected to have DMD/BMD. Two of the duplications identified were complex rearrangements involving two separate regions of the DMD gene (Dup Ex 20 & 57 and Dup Ex 45-48 & 53-55). There was also a long duplication spreading from exon 11-40.
Detection of deletion borders by MLPA: Borders of all the 23 cases with end points not clear by mPCR were confirmed by MLPA (Table III). These results become important to check the framedness of the deletion and hence to attempt to understand the genotype-phenotype correlation.
One of the deletions covering exons 10 to 62 (53 of the 79 exons deleted) is a novel deletion where 2/3rd of the exons are deleted spanning almost 1.38 Mb of the gene. One non-contiguous deletion (Del Ex 45-50 & 53-54) was also confirmed by MLPA. Of the samples tested, 17 turned out to be out-of-frame deletions, 5 in-
SAKTHIvEL MURUGAN et al: MLPA FOR DMD GENE MUTATION ANALYSIS 305
Table I. Consolidation of all mutations identified in this study and the methods usedSize of deletion or duplication
Independent cases (N=112)
Exon deleted or duplicated
Fragment deleted or duplicated
IF/OF Method used for identification
Deletions:1 exon (n=21) 7 DEL EX44 c.6291-?_6438+?del OF mPCR & MLPA
6 DEL EX45 c.6439-?_6614+?del OF mPCR & MLPA2 DEL EX51 c.7310-?_7542+?del OF mPCR & MLPA4 DEL EX52 c.7543-?_7660+?del OF mPCR & MLPA1 DEL EX55 c.8028-?_8217+?del OF mPCR & MLPA1 DEL EX62 c.9164-?_9224+?del OF MLPA
2 exons (n=8) 3 DEL EX46-47 c.6615-?_6912+?del OF mPCR 4 DEL EX49-50 c.7099-?_7309+?del OF mPCR 1 DEL EX51-52 c.7310-?_7660+?del IF mPCR
3 exons (n=10) 1 DEL EX45-47 c.6439-?_6912+?del IF mPCR 2 DEL EX46-48 c.6615-?_7098+?del OF mPCR 5 DEL EX48-50 c.6913-?_7309+?del OF mPCR 1 DEL EX50-52 c.7201-?_7660+?del OF mPCR 1 DEL EX53-55 c.7661-?_8217+?del OF mPCR & MLPA
4 exons (n=6) 1 DEL EX46-49 c.6615-?_7200+?del OF mPCR 2 DEL EX48-51 c.6913-?_7542+?del IF mPCR 2 DEL EX49-52 c.7099-?_7660+?del OF mPCR 1 DEL EX51-54 c.7310-?_8027+?del OF mPCR & MLPA
5 exons (n=12) 1 DEL EX3-7 c.94-?_649+?del OF mPCR & MLPA1 DEL EX45-49 c.6439-?_7200+?del IF mPCR8 DEL EX46-50 c.6615-?_7309+?del OF mPCR1 DEL EX48-52 c.6913-?_7660+?del OF mPCR1 DEL EX49-53 c.7099-?_7872+?del IF mPCR & MLPA
6 exons (n=17) 1 DEL EX38-43 c.5326-?_6290+?del OF mPCR & MLPA11 DEL EX45-50 c.6439-?_7309+?del OF mPCR4 DEL EX46-51 c.6615-?_7542+?del OF mPCR1 DEL EX56-61 c.8218-?_9163+?del OF mPCR & MLPA
7 exons (n=3) 3 DEL EX46-52 c.6615-?_7660+?del OF mPCR8 exons (n=9) 9 DEL EX45-52 c.6439-?_7660+?del OF mPCR9 exons (n=3) 1 DEL EX18-26 c.2169-?_3603+?del OF mPCR & MLPA
2 DEL EX45-53 c.6439-?_7872+?del IF mPCR & MLPA10 exons (n=8) 1 DEL EX3-12 c.94-?_1482+?del IF mPCR & MLPA
1 DEL EX8-17 c.650-?_2168+?del OF mPCR & MLPA3 DEL EX45-54 c.6439-?_8027+?del OF mPCR & MLPA3 DEL EX46-55 c.6615-?_8217+?del OF mPCR & MLPA
11 exons (n=2) 1 DEL EX3-13 c.94-?_1602+?del IF mPCR & MLPA1 DEL EX35-45 c.4846-?_6614+?del OF mPCR & MLPA
13 exons (n=1) 1 DEL EX33-45 c.4519-?_6614+?del OF mPCR & MLPA24 exons (n=1) 1 DEL EX20-43 c.2381-?_6290+?del OF mPCR39 exons (n=1) 1 DEL EX3-41 c.94-?_5922+?del IF mPCR & MLPA54 exons (n=1) 1 DEL EX10-62 c.961-?_9224+?del OF mPCR & MLPADouble deletion-8 exons (n=1)
1 DEL EX45-50 &53-54 c.6439-?_7309+?del; c.7661-?_8027+?del
Cannot Assess
mPCR & MLPA
Duplications:Dup 1 exons (n=4) 3 DUP EX2 c.32-?_93+?dup OF MLPA
1 DUP EX44 c.6291-?_6438+?dup OF MLPADup 5 exons (n=1) 1 DUP EX3-7 c.94-?_649+?dup OF MLPADup 30 exons (n=1) 1 DUP EX11-40 c.1150-?_5739+?dup IF MLPADouble duplications (n=2)
frame deletions and 1 non-contiguous deletion. (Table III)Novel mutations in the study: Ten novel mutations (not listed in the www.dmd.nl database) identified in the study are listed in Table Iv. All, except one, were confirmed by MLPA and among these are 3 non-contiguous rearrangements (1 deletion and 2 duplications). MLPA results on single exon deletions: MLPA was performed on 21 samples, which showed single exon deletions by mPCR. Twenty of these 21 samples showed the same deletion by MLPA. One sample which showed exon 60 deletion by mPCR showed exon 56-61 deletion by MLPA (B44 - Table III).
Non-contiguous mutations: In our study 3 non-contiguous mutations were picked up, which included 1 deletion and 2 duplications (Table I). From the clinical data available (not shown here), the severity of the cases was checked based on the age at onset. It was seen that 2 cases, showing exons 45-50 and 53-54 deletion and exons 20 and 57 duplication had an age at onset of 8 yr. The other case having a double duplication of exons 45-48 and 53-55 had an age at onset of 3 ½ yr, suggesting a severe phenotype. Fig. 1 shows a representative picture of MLPA analysis results of the samples showing non-contiguous mutations.
SAKTHIvEL MURUGAN et al: MLPA FOR DMD GENE MUTATION ANALYSIS 307
Table II. Framedness of deletions picked up, before and after MLPAType of Mutation mPCR Results MLPA Results
In this study, we have tested our systematic approach of using mPCR followed by MLPA as a diagnostic tool to precisely detect deletions and duplications in the coding region of the DMD gene and also suggested the over all algorithm towards DMD gene analysis. The above attempt is mainly to ensure that an invasive method like a muscle biopsy need not be the first step to confirm the clinical diagnosis.
Though mPCR is able to offer diagnosis for 68.7 per cent of the patients, precise diagnosis is possible only in approximately 40 per cent of the patients. Following mPCR analysis by MLPA, increases the percentage of patients with a precise diagnosis to 75 per cent. Patients who show no mutation after following this algorithm can then be considered for confirmation by Immunohistochemistry or Western blot on muscle tissue followed by genomic DNA or cDNA sequencing. On an average the cost of mPCR to MLPA is in the ratio of 1:5. The use of MLPA to screen for deletions and duplications in the DMD gene as a starting method for DMD diagnosis has been suggested in recent reports22. But using MLPA as the first screening technique in the Indian scenario will be a costly attempt and our approach of using mPCR as the first step, followed by MLPA will be a prudent & precise way to effectively manage DMD genetic diagnosis.
Multiplex PCR methods allow the detection of approximately 98 per cent of deletions, which accounts for 65 per cent of all mutations12,13. In our study, though mPCR was able to pick up deletions in 68.7 per cent of all the cases, confirmed molecular diagnosis was achieved only in 59 cases (39.3%), due to the fact that precise molecular diagnosis is arrived only if deletions
where exon borders are clear and single exon deletions are confirmed.
Mutations affecting the open reading frame, due to frameshift, generate truncated non-functional dystrophin protein giving rise to severe DMD phenotype. However, mutations not affecting the open reading frame may produce a semi-functional dystrophin protein and usually correlate with mild phenotypes3. In order to assess the reading frame, it is usually necessary to screen the entire gene for determination of the exons involved in deletion or duplication. Though Southern blot analysis was for long the method of choice to establish the exact breakpoints23, since this method was time-consuming and cumbersome, the two recent methods, i.e., Multiplex Amplifiable Probe Hybridization (MAPH)24 and MLPA18-
20, has greatly simplified this analysis, where MLPA has become the method of choice. The exact exon boundaries of the 23 samples that showed unclear borders by mPCR were confirmed by MLPA, the results of which are tabulated in Table III. Table II shows the usefulness of MLPA in confirming the deletion borders and predicting the framedness. We can see that by using MLPA there is an increase in the ability to correctly identify in-frame mutations by 5 per cent and out-of-frame mutations by 17 per cent as compared to mPCR. Though 90 per cent of the cases follow the reading frame rule, 10 per cent show exception to the reading frame rule and have been well documented3.
Single exon deletions picked up by mPCR need to be confirmed using a different set primer pairs, to rule out polymorphisms at the primer annealing site. We have used MLPA for confirming our single exon deletions picked by mPCR, since MLPA probes have exonic ligation sites.
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Table IV. Novel mutations identified in the studyS.No. Sample number Mutation Fragment deleted/duplicated Method1 B80 Exons 20-43 deleted c.2381-?_6290+?del mPCR2 B18 Exons 10-62 deleted c.961-?_9224+?del MLPA3 B110 Exons 45-50 & 53-54 deleted c.6439-?_7309+?del;
MLPA analysis picked up an additional 9 mutations not picked up by mPCR, increasing the pickup percentage by 7 per cent and increases its rate of mutation detection by 19 per cent of all no deletion samples. Most (8 cases) were duplications and only one deletion was picked up outside the hot-spot region. Overall, MLPA was necessary to accurately confirm diagnosis in 35 per cent of all cases. This makes MLPA a very useful and necessary method to be used in the approach towards DMD gene analysis. However, MLPA did not pick up any small insertions/deletions and point mutations in our study, as reported in other studies25,26. Duplications account for approximately
7 per cent of all mutations identified and approximately 5 per cent of all cases, which goes well with published data19,27,28. While White et al28 had described close to 87 per cent of duplications in no deletion/no point mutation cases, we found duplications in 17.4 per cent of no deletion cases. This result goes in line with reports of Wang et al26. There is no specific region, which shows more duplications, and the mutations identified are generally spread out throughout the gene. Only one deletion was picked up by MLPA, which was not tested by mPCR, an exon 62 out-of-frame deletion, which was confirmed by native PCR.
Fig. 1. MLPA results of the three cases showing non-contiguous duplication and deletion.
MURUGAN et al: MLPA FOR DMD GENE MUTATION ANALYSIS 309
In our study we also observed three non-contiguous mutations, one by mPCR, which was further confirmed by MLPA, and two duplications by MLPA. This also shows the importance of screening the whole gene, whilst these non-contiguous mutations could be missed. Reports on non-contiguous deletions exhibit milder phenotypes have been published29,30. Two of our 3 cases (B110 and B108 in Table Iv) were found to be milder phenotypes and one (B181 in Table Iv) showed severe progression taking into account the age of onset of symptoms. Though there are reports on non-contiguous deletions and duplications in literature, there are no reports providing the phenotype of non-contiguous duplications. Further protein studies looking for residual dystrophin might throw light on the pathophysiology of these non-contiguous mutations.
In our study we have also picked up 10 novel mutations not reported in the Leiden database (www.dmd.nl). Most of these (7 cases) are deletions and 3 are non-contiguous rearrangements. The novel mutations are also spread throughout the gene, and the novelty could be attributed only to the extent of the deletion.
In the study reported by Aartsma-Rus et al5, they have used the following deletions (exons 45-50, 45-54, 48-50, 51-55, and 52) to understand and evaluate the feasibility of exon skipping. The study has now
reached the clinical trial stage. From our study we have 23 children who can be eligible participants towards this therapeutic strategy.
Our study has shown that following the systematic approach/algorithm shown in Fig. 2, we were able to detect single and multiple exon deletions and duplication in almost 75 per cent (112 out of 150 samples) of the cases suspected to have DMD/BMD. A study on MLPA analysis of DMD cases from China has reported a pick up rate of 73 per cent25. It is interesting to note that reports by Trimarco A et al31 show 85 per cent pick up rate of mutations (only deletions and duplications) using the log-PCR method.
The unidentified mutations in the study cohort could be point mutations, small insertions or deletions. As per the algorithm suggested, it is only 25 per cent of the cases that require confirmation of dystrophin absence by muscle biopsy followed by cDNA sequencing. This approach helps in; (i) Non-invasive diagnosis in majority of the cases, (ii) replacing muscle biopsy as the first step in diagnosis, and (iii) economical and systematic molecular diagnosis in DMD in close to 75 per cent of the clinically suspected DMD/BMD cases. This systematic approach/algorithm may be used as a precise and cost-effective tool for DMD diagnosis in a developing country like India.
Acknowledgment Authors acknowledge Stitching Porticus Grant, Denmark, for financial support. Authors thank M.S. Swaminathan Research Foundation, Chennai, India, for allowing to use the Genetic Analyser at the centre, also thank all clinicians who referred patients for testing.
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Reprint requests: Dr Bremadesam Raman Lakshmi, Head, Molecular Diagnostics, Counseling, Care & Research Centre Avinashilingam University for Women, Coimbatore 641 043, India e-mail:[email protected]
