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

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.

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.

Introduction
Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are X-linked recessive disorders arising due to mutations in the DMD gene. 1 The DMD gene is one of the largest genes in the human genome with a size of more than 2 Mb.This gene spans 79 exons and codes for a 14 kb mRNA that translates a cytoplasmic protein called dystrophin.Due to this large size, mutation detection poses a challenge for routine molecular diagnosis in a clinical setting in many developing countries.Dystrophin protein interacts with other glycoproteins in cell membranes forming the dystrophinglycoprotein complex (DGC) which stabilizes the membranes of muscle fibers. 2 The absolute absence of dystrophin leads to a clinical manifestation of muscle weakness from early childhood in DMD.Subsequent progressive muscle weakness leads to death before the third decade of life due to respiratory or cardiac failure.The presence of partially functional protein results in BMD, a milder phenotype of the disease. 3D is the most frequently inherited muscle disease and reported to affect one in every 3,500 male births. 1 Indonesia is one of the largest countries in the world, with a population of 260 million. 4This suggests that there should be many more DMD cases in Indonesia than the small number of cases that have been reported. 5The true incidence of the disease and the underlying genetic variants are unknown in this region.Lack of awareness of the clinical features associated with the disease and limited use of molecular testing may lead to many undetected cases, resulting in an iceberg phenomenon of under-diagnosis and under-reporting of DMD cases.
Genetic analysis to detect DMD gene mutation is now a gold standard to diagnose DMD/BMD.However, this is not being performed regularly in developing countries like Indonesia.Gene deletions and duplications are reported to be the most common mutations in the DMD gene, encompassing more than 60% of cases. 6Multiplex ligation-dependent probe amplification (MLPA) reaction, a quantitative PCR-based technique, is currently used to routinely amplify all 79 exons of the DMD gene to detect deletions and/or duplications in patients. 7Meanwhile, the identification of point and small mutations for non-deletion or non-duplication cases remains challenging for resource poor labs because of the large number of exons in the DMD gene that requires sequence-by-sequence screening.As such, there is currently a lack of knowledge on the true mutation profile in Indonesian DMD/BMD patients.Future applications of mutation-specific molecular therapies, such as exon skipping, CRISPR-Cas9 mediated correction or stop codon read-through therapy, are currently being investigated for patients carrying mutations such as deletions, duplications, and small mutations. 8Precise mutations analysis is thus necessary to apply the appropriate strategies to patients who are eligible for such treatments. 8hus, the objective of this study was to identify the spectrum of common deletion and duplication mutations in the DMD gene for clinically diagnosed patients in the Indonesian population using the MLPA technique.The study was carried out with a view to informing future therapeutic applications and developing specific approaches for disease management.

Patients
Forty-three male patients from Dr. Sardjito Teaching Hospital and Universitas Gadjah Mada (UGM) Academic Hospital, Yogyakarta, Indonesia were enrolled in this study, from 2017 to 2018.Clinical manifestations of DMD/BMD were found in the patients and supported by the findings of high serum creatine kinase (CK) levels through medical record data.Clinical diagnosis was made by qualified and experienced pediatric neurologists who are also trained molecular geneticists.Up to now, patients have been followed for their clinical management.Genetic analysis was performed based on approval of parents through signed written informed consent.The study protocol was approved by the Medical and Health Research Ethics Committee of the Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada (KE/FK/0890/EC/2018) and National University of Singapore Institutional Review Board (N-19-102E).

Immunohistochemical analysis
Out of 43 subjects, 23 subjects additionally consented to undergo a muscle biopsy procedure.Muscle biopsy in the form of formalin-fixed paraffin-embedded (FFPE) samples were sliced into 3 μm thickness, incubated, deparaffinized, and rehydrated.Antigen retrieval was performed using a Decloaking Chamber NxGen (BioCare Medical, USA) and

REVISED Amendments from Version 2
After receiving feedback from the reviewers, we have made several changes to the manuscript.These changes include addressing issues related to out-of-frame duplication, deletion, and nonsense mutation.We have also added information to the discussion section regarding a 12-nucleotide deletion in exon 65, pediatric cases, and founder mutations.Furthermore, we have corrected the omission of Figure 5 and amended the spelling of "Gower" to "Gowers".Lastly, we have updated reference 3 to ensure accuracy.
Any further responses from the reviewers can be found at the end of the article immunostained with mouse monoclonal Dys-2 antibody (Novocastra, Leica Biosystem, Newcastle, UK, product code NCL-Dys-2, lot number 6062727) to detect dystrophin expression against carboxyterminal domain with dilution 1:25 in phosphate buffer saline (PBS).Diamino-benzidine (DAB) for visualization of positive cells was applied with a semiautomatic intelliPATH FLX (BioCare Medical, USA) according to the manufacturer's instructions.Dys-2 expression was observed under a light microscope by an independent and experienced pathologist by analysing the expression of dystrophin in muscle membrane.Negative expression of Dys-2 is considered as indicative of DMD while faint or focal staining suggests BMD, with normal muscle tissue used as a positive control.

Genomic DNA extraction
Genomic DNA was isolated from 3 mL of EDTA peripheral whole blood samples using Geneaid™ DNA Isolation Kit (Geneaid Biotech Ltd) according to manufacturer's protocol.
PCRs were performed in a volume of 25 μL containing 2 μL of genomic DNA, 12.5 μL of 2Â Go Taq green Master Mix, 1 μL of each primer, and 8.5 μL of nuclease-free water.The PCR cycling conditions of exon 47 were as follows: initial denaturation at 95 °C for two minutes followed by 35 cycles of denaturation at 95 °C for one minute, annealing at 50 °C for one minute, and extension at 72 °C for one minute.Amplification of other exons used the same PCR condition, however, the annealing time of exons 51 and 52, was 50 °C, meanwhile exon 65 was 55 °C.The amplified exons were then sequenced and analyzed for their mutation's status. 9ading frame analysis was performed using online software LOVD exonic deletions/duplications reading-frame checker based on predicted translation of the DMD mRNA arising from the identified deletion/insertion (duplication) of the affected exons.Identified mutations were also compared against previously reported mutations using the UMD-DMD France mutation database (http://www.umd.be/) and the Leiden Muscular Dystrophy pages mutation database accessed on June 6 th , 2021.

Demographic and clinical profile
All 43 subjects recruited for this study were male and showed clinical features of DMD/BMD such as difficulty in walking, muscle weakness, positive Gowers sign at age of onset and increased CK levels in their blood samples.CK levels were elevated, ranging from 1,734 to 40,429 IU/L (mean 9,121.7 IU/L, two patients with unavailable data).The age of onset of the disease ranged from one to nine years old (mean 5.1 years old).Nineteen patients (44.18%) were wheelchair bound, with loss of ambulation starting between nine to 13 years old, with mean age at 11 years old (Table 1).
Out of 43 patients, 35 (81.40%) cases of deletion and duplication could be detected using the MLPA method.Deletions accounted for 69.77% (30 cases), while duplications were found in 11.63% (five cases).Two patients who were initially found to have single exon deletion by MLPA turned out to carry small mutations after further investigation.In the remaining eight patients (18.60%), no deletion nor duplication was detected (Table 1).The identified mutations were screened against two well-known DMD databases, namely UMD-DMD France and Leiden Muscular Dystrophy pages database.Two cases with a novel mutation were identified: dmd19 and dmd21 with duplication spanning exons 2 to 62 (c.(241)_(9433)dup) (Table 1).

Immunohistochemistry results
Out of 23 patients who underwent muscle biopsy, 18 cases (78.26%) showed negative expression of Dys-2 in muscle membrane, suggesting DMD, meanwhile the remaining five cases (21.74%) expressed faint and patchy staining, suggestive of BMD.However, eight discrepant cases were identified.Five faintly staining cases (dmd10, dmd11,   dmd35, dmd39 and dmd54) had out-of-frame mutations, two cases (dmd20 and dmd38) with negative immunostaining were found to carry in-frame mutations, and one case (dmd28) with negative immunostaining carried an out-of-frame mutation but clinically had mild symptoms (Table 2).
Deletion pattern in the DMD gene Initially, 30 deletions (69.77%) cases were discovered using MLPA, consisting of 25 cases with multi-exon deletions and five cases with single exon deletions of exon 47, exon 51, exon 52 and exon 65.Single exon deletion cases were further analysed by PCR.Three cases were confirmed to have single exon deletion of exons 51 or 52.After sequencing, a case with deletion in exon 47 turned out to carry a nonsense mutation (c.6808 del T, p.Leu2271StopCodon) which was discordant with the clinical symptoms of BMD seen in the patient (dmd28).Meanwhile, one case with a deletion in exon 65 (dmd18) turned out to carry a small in-frame deletion of 12 nucleotides (c.9540-9551 del CTGGCTGCTGAA, p. Asn3180-3184Asn).This was clinically discordant with the DMD condition observed in the patient (Table 1).The largest exon deletion in this study spanned exons 3 to 44 (one case).Other large deletions encompassed exons 17-43, exons 7-43, exons 18-47, exons 3-17, exons 48-52, and exons 56-74.The most common type of deletions were deletions of exons 45 to 52, exons 49 to 52, exons 49 to 50, exons 3 to 7 and single deletion of exon 51 (two cases each, respectively) (Table 1).Deletion mostly occurred in the distal hot spot region (exons 45-55) with 20 cases (66.67%) carrying deletions in this region.In eight cases (26.67%) deletions occurred in the proximal hot spot (exons 2-20) region (Figures 1 and 2).Duplications pattern in the DMD gene Out of the 43 DMD cases, duplications occurred in five patients (11.63%): four cases had duplications initiating from exon 2, and one case starting from exon 14.The highest frequency of duplications occurred in exons 14 to 17, found in all five patients.The largest duplication spanned from exons 2 to 62 affecting 40% of these cases (two patients), and the shortest duplication covered exons 14 to 17 in 20% (one patient) (Figures 3 and 4).All duplications (100%) involved the proximal hot spot region.

Association with reading frames
Out of the 30 patients with deletions, only four cases were in-frame, and the remaining 26 cases were out-of-frame (Table 1).One discrepancy was dmd31, with a mutation predicted as in-frame (del 3-44); however, the phenotype was DMD.Meanwhile two cases with single exon deletions detected initially by MLPA turned out to carry a nonsense mutation (case dmd28) and a 12-nucleotide deletion (case dmd18) by sequencing.These mutations were out-frame and in-frame respectively.Both cases were discordant with the reading frame predictions as dmd28 showed a milder phenotype whereas dmd18 showed a severe clinical condition (Table 2).In five patients carrying duplications of the DMD gene, all were out-of-frame with DMD phenotype.
Based on immunohistochemical staining results, three cases (dmd20, dmd28 and dmd38) showed negative staining of dystrophin, however the patients manifested milder phenotype of BMD.Meanwhile five cases (dmd10, dmd11, dmd35, dmd39, and dmd54) showed faint staining of dystrophin (Table 2), even though the genotype-phenotype correlations were consistent with DMD.Among the novel mutations found in this study, two of them (duplication of exon 2 to 62 and deletion of exon 56 to 74) were out-of-frame, while the deletion of exon 7 to 43 was an in-frame mutation (Table 2).Overall, out of 35 patients with deletion and duplication, 32 patients (91.43%) were in accordance with the reading frame rule, meanwhile three (8.57%)patients were discordant (Table 2).Other clinical parameters Out of the 43 patients, 18 patients (41.86%) manifested skeletal deformities, including scoliosis in 14 cases (32.56%), lordosis in two cases (4.65%), ankle deformity in two cases (4.65%), coxae deformity in one case (2.32%), knee deformity in one case (2.32%), and lower leg deformity in one case (2.32%).Some patients had more than one skeletal deformity, such as scoliosis coexisting with ankle deformity.One patient (2.32%) was born with low birth weight, three patients (6.98%) suffered from malnutrition and two patients (2.32%) were reported to have cardiomegaly.Six patients (13.85%) showed early signs of respiratory tract disturbance, such as difficulty in breathing and pneumonia.Some patients reported history of milestone developmental problems, including gross motor skill delays in 14 patients (32.56%), fine motor skill delays in seven patients (16.28%) and speech delays in five patients (11.62%) (Table 1).

Carrier status
Carrier status analysis was performed on mothers and female siblings of patients found to carry deletion or duplication mutations through MLPA analysis.Family members of the patients without deletion or duplication did not undergo this analysis.Twenty-seven available samples from mothers showed that carrier status was confirmed in 14 cases (51.85%).
Five available samples from female siblings of probands showed one case (20%) carrying the mutated DMD gene, meanwhile, the remaining four cases (80%) were negative for the identified mutation in their affected brother.

Molecular therapy applicability
Out of 31 patients whose genotype data was consistent with severe DMD, 46.51% cases are amenable for the application of exon skipping therapy.Therapeutic approaches to skip exons 51, 53, 45, 50, 17, 2, 44, and 8 can be applied in 20 patients (46.51%).The mutation spectrum and molecular therapy applicability for the identified mutations are presented in Tables 3 and 4.

Discussion
In DMD/BMD, the increased permeability of the sarcolemma allows CK from muscle fibers to be released to blood stream, indicating a muscle damage process.The highest levels of CK are commonly observed between two to five years of age and decreases along with disease progression and older age. 10 This pattern is similar to what is observed in the DMD/BMD patients in our study, which also showed high CK levels with mean age of onset at five years of age.
The analysis of fresh frozen muscle specimens is usually a standard procedure for muscle biopsy, however this service is not currently available in Indonesia.Instead, FFPE samples from muscle specimens were used to detect dystrophin expression in sarcolemma membrane.Similar use of such FPPE samples have been previously reported in Thailand, Japan and UK, showing this as a reliable and reproducible technique. 11Our immunohistochemistry (IHC) analysis showed weak staining of dystrophin protein in five DMD patients.Trace-level dystrophin expression in patients with out-offrame DMD mutations has been previously reported in approximately 20% of DMD patients. 12,13The mechanism for this low level of dystrophin has not been fully understood.Possible mechanisms include re-initiation of translation downstream of frameshift mutation and alternative splicing of exon adjacent to deletion boundaries resulting in restoration of open reading frame. 14Indeed, a study by our group previously showed that the formation of splicing silencer by DMD exon 45 deletion junction could explain exon 44 skipping, thereby restoring open reading frame. 15Three other cases with negative dystrophin staining were inconsistent with the BMD genotypes and phenotypes observed.The negative result of dystrophin staining could be due to subjectivity in evaluating the IHC result or insufficient pre-analytical treatment.Moreover, the dystrophin marker used in this study was Dys-2 located in C-terminal, which may not fully capture expression in the rod domain. 16Due to these limitations and the invasive nature of the muscle biopsy procedure, molecular methods of gene analysis from blood draws offer a better option for diagnosis.
The use of the MLPA technique has improved the detection of both single and large intragenic rearrangements because it allows the simultaneous analysis of all 79 exons in the DMD gene, the largest gene in the human genome.Prior to the MLPA technique, approaches such as multiplex PCR using primers that cover sets of commonly deleted exons would yield deletion rates ranging from 40% to 51.2% of DMD/BMD cases as reported in some Asian populations. 17DMD gene analysis in the Indonesian population has been conducted previously using IHC 18 and multiplex PCR 19 methods.However, precise mutations cannot be identified using the IHC method while multiplex PCR is unable to cover all exons in the DMD gene.Our study using MLPA screening showed that deletions were detected in 69.77% cases while duplications were found in 11.63%, providing a molecular diagnosis in 81.4% of total examined cases.This result is comparable to previous MLPA studies of DMD/BMD cases in Asian populations conducted in China 66-95%, 20 India 68%, 21 Vietnam (54%), 22 Malaysia (77%) 23 Japan (70%) 2 and Singapore (75%). 85][26] This detection will contribute to possible application of exon skipping therapy in 60.47% of DMD cases in our patients, similar to some other studies summarized in Table 4.8][29][30] Therapy using eteplirsen (skipping of exon 51) and golodirsen (skipping of exon 53), antisense oligonucleotide drugs approved by the FDA, can be applied in 23.25% of patients while codon read through therapy using compounds such as ataluren, can be applied in one patient.
A critical issue in interpretation of MLPA results is the detection of deletions involving a single DMD exon.In this study, two single exon deletions detected through MLPA turned out to be small point mutations that involve sequences where the probes should ligate.The altered exon sequences inhibited the proper hybridization of the specific probe, thus leading  (exons 51, 53, 45,  50, 17, 2, 44, 8, 55,  63, 18, 19), 2.3% read through del: deletion; dup: duplication; (-): unknown/data not provided.
to the observed deletion of the respective exon during MLPA analysis.Such sequence variations may involve pathogenic small or point mutations, or even a polymorphism that does not disrupt gene function.Therefore, single exon deletions detected by MLPA should always be confirmed with PCR and sequencing as evidenced by our observations in this study. 31Two cases (dmd18 and dmd31) in this study were in-frame mutations, but phenotypically DMD.One possible explanation for the discrepant predicted phenotype in dmd18 is that deletion of exon 65 may disrupt binding to βdystroglycan resulting in a more severe phenotype.However, this requires further investigation.Meanwhile, one case (dmd28) was out-frame in genotype, but phenotypically BMD.Further investigation using mRNA transcripts is needed to reveal the splicing pattern of these cases in order to understand how the discrepancies occurred.Unfortunately, it was not possible to collect additional samples from the patients for further investigations in the current study.Concordance to the reading frame rule in our study was 91.43% (32/35), which is similar to other studies in Asia, including Japan (93%), 1 Singapore (96%), 8 Vietnam (94%), Malaysia (93%) 22 and China (88%). 20 contributing towards the spectrum of mutations in the DMD gene, one novel mutation was observed in two patients, dmd19 and dmd21, involving a duplication of exons 2 to 62 (c.(-182_59) (9187_9246) dup).Duplication frequency is reported to be highest at the 5' end of the gene with exon 2 duplication as the most common single duplication. 1,32This novel mutation in both patients has been deposited into the Leiden Open Variation Database (LOVD) (ID numbers 00383167 and 00383168).
Previous studies on the mutation spectrum of the DMD gene in Japan showed that deletions were found to cluster in a proximal hotspot (exon 2-20) and distal hotspot (exon 45-55), at a frequency of 26% and 65% of all identified deletions, respectively. 1This study showed similar results as deletions occurred more frequently in the distal hot spot region (66.67%), and only 26.67% were in the proximal hot spot region.Duplication mutations appear to be distributed across the DMD gene; however, the proximal hot spot was more frequently duplicated than the distal hotspot, 1 which concurs with our result showing that all duplications (100%) involved the proximal hot spot region.
In this study, the MLPA method was not able to identify deletion nor duplication in eight patients.Small sequence mutations may occur in these cases leading to a negative result in the MLPA since the clinical features were consistent for DMD.Further sequencing analysis using mRNA to detect both point mutation and splicing patterns is needed to reveal the disease-causing mutation.MLPA should be combined with direct Sanger sequencing or whole exome sequencing to obtain higher sensitivity and specificity in the detection of small or point mutations in the DMD gene. 33Additionally, it should be noted that MLPA analysis can generate false positive results by showing an exon deletion when a polymorphism occurs at a primer ligation site. 34Hence, it is always useful to conduct a confirmatory test using another method.
Previously, DMD patients commonly lost their ambulation prior to 12 years of age and could only survive until their late teens. 35However, current treatment such as long-term use of corticosteroids has been reported to prolong ambulation by two to five years or even longer, thereby reducing the need of spinal stabilization surgery, improving cardiopulmonary function, delaying the need for noninvasive nasal ventilation, and increasing survival and the quality of life of patients with DMD. 36Surgical and ventilator support were also reported to increase survival up to the third decade. 37In our study, the mean age of losing ambulatory ability was still below 12 years old and this may be caused by the difficulty of the patients obtaining early and proper clinical management due to challenges in establishing precise diagnosis.This is due to difficulty in getting access to molecular testing.Muscle biopsy can be performed to help with diagnosis using immunohistochemistry analysis, 18 however, not all medical centers have this facility and not all parents agree to this invasive procedure.Due to such limitations in Indonesia, the disease has not been well characterized.Hence, this current study provides evidence of usefulness of deletion and duplication screening for diagnosis in most patients.
Initially, both DMD and BMD patients show skeletal muscle weakness, marked with positive Gowers sign.The natural course of DMD shows that muscular dystrophy will occur progressively followed by deterioration of cardiac and respiratory muscles, leading to early death due to respiratory or heart failure. 35Mutations involving exons 12, 14 to  17, 31 to 42, 45, and 48 to 49 have been reported to enhance cardiac involvement. 38In our study, two patients showed cardiomegaly and only one of them fit with this theory, as this patient had exons 46-50 deletion.Cardiac and respiratory problems were found only in two (2.32%) and six (13.85%) patients, respectively.The small number of patients with cardiac and respiratory problems in this study is perhaps due to the relatively short period of observation.Similar limitations were also encountered in collection of other clinical parameters observed in this study such as musculoskeletal involvement, malnutrition, etc.
Scoliosis is a frequent complication (68-90%) in DMD, meanwhile bone fractures occur in 20-25% of cases and the risk increases along with age and loss of ambulation. 39In our study, cases of skeletal deformity, including scoliosis, were only found in 41.86% of patients, less than previous studies.Some patients were also reported to suffer from malnutrition (6.98%).The weight loss in DMD patients is suspected to be associated with progressive muscle weakness leading to dysphagia and mastication dysfunction. 40Growth delays in individuals with DMD can be observed as short stature 41 or motoric and language delays. 42Even though short stature was not observed in all subjects, some patients (34.88%) were found to develop motoric and language delays.Even though dystrophin expression in the brain is only one-tenth of that observed in muscle, varying degrees of non-progressing cognitive impairment might be exhibited in some patients. 43owever, no mental retardation was observed in this study.
Theoretically, approximately two-thirds of DMD patients inherit their mutations from carrier mothers, meanwhile the remaining one-third are predicted to develop the disease due to spontaneous mutations. 44Our results showed that inherited cases were confirmed in 14 cases (51.85%) out of 27 available samples while 13 cases were due to de novo mutations (48.15%).These results suggest that the rate of de novo mutation in our population is higher than the estimated one-third of all DMD cases.However, there has been one previous study of 150 cases showing a high rate (71%) of de novo mutations as compared to the inherited cases which affected only 49 cases (29%). 45De novo mutations are defined by their presence in one or more progenies and the absence in the mother.The presence of multiple affected offspring from apparently non-carrier parents is caused by germline mosaicism as DMD mutation evolves de novo in the affected patient and the risk of recurrence from germinal mosaicism is estimated to be approximately 4.3%. 46PA has been used to detect carrier status in probands with known deletion or duplication mutations since it is an easy and quick technique. 47Detection 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. 48This study also shows that MLPA analysis can pave the way for future therapeutics in DMD patients by identifying amenable genotypes that can be targeted.
Mutation detection in DMD is essential for patient management especially since advanced therapy such as exon skipping, CRISPR-Cas9 mediated correction or stop codon read-through therapy are mutation specific approaches.Furthermore, molecular diagnosis is also important for genetic counselling to plan future pregnancy.In our study, MLPA was found to be an easy and quick technique to identify deletion and/or duplication mutation and detect copy number of DMD carriers.This study also showed that the majority (81.40%) of DMD gene mutations in Indonesian DMD patients were deletions and duplications.Of these, 60.46% would be amenable to exon skipping therapy.It is important to keep in mind that the data collected in this study was from a single pediatric center.As a result, our study might have excluded milder cases of BMD that only become noticeable in adulthood due to later onset of disease.Further research is necessary to investigate this possibility.Additionally, screening of future patients from more centers in the country can be carried out to add to the existing data in this study.Nonetheless, it should be noted that even when sufficiently large populations of patients are analyzed, the overall genetic composition of dystrophinopathies often yield very similar trends across world populations, as there are no or very few "founder" mutations (because of the severe phenotype and X-linked nature) and most, if not all identified mutations, usually date back to just a few generations.This is the first study showing the feasibility of implementing the MLPA method in detecting DMD gene mutation and revealing the spectrum of common mutations in Indonesia.This is also the first study showing the potential application of exon skipping therapy in the majority of DMD cases in the country.The clinical and molecular characterization of patients in this study provide a better insight of DMD and BMD profiles in the Indonesian population and will shape health policies in patient management.
• Electrophoresis result of single exon deletion of exon 47 and exon 65. directing local health interventions and policies.However, it would be in my opinion also useful to point out that, in sufficiently large populations, the overall genetic composition of dystrophinopathies tends to be very similar across world populations, as there are no or very few "founder" mutations (because of the severe phenotype and X-linked nature) and most, if not all identified mutations, usually date back to just a few generations.
Moreover, I have some comments and suggestions, as listed below: Table 3: Out-of-frame duplications are not amenable to exon skipping.1.
Also skipping of exon 55 for del 56-74 is very theoretical (no dystroglycan binding in the expected protein product).

2.
c.6808delT is not a nonsense mutation, but rather a frameshift deletion of 1 nucleotide; therefore a DMD phenotype is expected.The fact that the patient walks at age 9 (with corticosteroid treatment?)does not exclude a DMD phenotype.This patient should not be treated with ataluren.

3.
How many of the 43 cases are related cases (siblings, cousins, other)?This point is also relevant for the discussion of de novo frequency in the population (the more siblings, the less de novo).

4.
The 12-nucleotide deletion in exon 65 may disrupt binding to β-dystroglycan, a potential explanation for a severe phenotype despite ORF preservation in this patient.

5.
Figure 5 (pedigrees) is not very useful, as X-linked inheritance is expected and always confirmed.
Are the Centers where the data were collected wxclusively pediatric?This may exclude from this population the spectrum of milder BMD which only becomes clinically evident in adult age, and should be considered in the discussion (i.e.underestimation of in-frame mutations).

8.
For reference 3 I would suggest Monaco et al. (1988 1 ), rather than, or in addition to, the current reference.Reviewer Expertise: Muscular dystrophy I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
4. How many of the 43 cases are related cases (siblings, cousins, other)?This point is also relevant for the discussion of de novo frequency in the population (the more siblings, the less de novo).
Response: Thank you for the comment.We checked again and none of related cases was included in this study.
5. The 12-nucleotide deletion in exon 65 may disrupt binding to β-dystroglycan, a potential explanation for a severe phenotype despite ORF preservation in this patient.
Response: Thank you very much for your insight.We have added this to the 4 th paragraph of the discussion section.All changes are highlighted in yellow in the manuscript text.

Figure 5 (pedigrees) is not very useful, as X-linked inheritance is expected and always confirmed.
Response: Thank you for the suggestion, we have removed Figure 5 from the revised manuscript.

"Gowers" instead of "Gower".
Response: Thank you for pointing this out.We have made the revision.
8. Are the Centers where the data were collected exclusively pediatric?This may exclude from this population the spectrum of milder BMD which only becomes clinically evident in adult age and should be considered in the discussion (i.e.underestimation of in-frame mutations).
Response: Thank you very much for this important point.We agree and have added this point in the penultimate paragraph of the discussion section.Monaco et al. (1988 1 ), rather than, or in addition to, the current reference.

For reference 3 I would suggest
Response: Thank you for the suggestion.We have revised reference 3. 10. "However, it would be in my opinion also useful to point out that, in sufficiently large populations, the overall genetic composition of dystrophinopathies tends to be very similar across world populations, as there are no or very few "founder" mutations (because of the severe phenotype and X-linked nature) and most, if not all identified mutations, usually date back to just a few generations".
Response: We have added this suggested comment in the discussion section as "Nonetheless, it should be noted that when sufficiently large populations of patients are analyzed, the overall genetic composition of dystrophinopathies tends to be very similar across world populations, as there are no or very few "founder" mutations (because of the severe phenotype and X-linked nature) and most, if not all identified mutations, usually date back to just a few generations".1.

Competing
Page 4 mentioned that databases were accessed on 6 June 2021.It will be worthwhile to have an updated comparison as almost a year has lapsed.

2.
Table 1 on the column "Age when wheelchair bound", it will be clearer to replace "not yet" with "still ambulant".

3.
Table 2 is redundant as the content would have been explained by Table 1. 4.
Table 3 and 4 may be combined to focus on discrepancy findings (genotype vs phenotype; IHC vs phenotype).

5.
Is the work clearly and accurately presented and does it cite the current literature?Yes therefore a DMD phenotype is expected.The fact that the patient walks at age 9 (with corticosteroid treatment?)does not exclude a DMD phenotype.This patient should not be treated with ataluren.Response: Thank you for pointing out the error.We agree that ataluren will not affect the patient with the frameshift deletion and have revised Table 3. 4. How many of the 43 cases are related cases (siblings, cousins, other)?This point is also relevant for the discussion of de novo frequency in the population (the more siblings, the less de novo).Response: Thank you for the comment.We checked again and none of related cases was included in this study.
5. The 12-nucleotide deletion in exon 65 may disrupt binding to β-dystroglycan, a potential explanation for a severe phenotype despite ORF preservation in this patient.Response: Thank you very much for your insight.We have added this to the 4th paragraph of the discussion section.All changes are highlighted in yellow in the manuscript text.
6. Figure 5 (pedigrees) is not very useful, as X-linked inheritance is expected and always confirmed.Response: Thank you for the suggestion, we have removed Figure 5 from the revised manuscript.
7. "Gowers" instead of "Gower".Response: Thank you for pointing this out.We have made the revision.
8. Are the Centers where the data were collected exclusively pediatric?This may exclude from this population the spectrum of milder BMD which only becomes clinically evident in adult age and should be considered in the discussion (i.e.underestimation of in-frame mutations).Response: Thank you very much for this important point.We agree and have added this point in the penultimate paragraph of the discussion section.9.For reference 3 I would suggest Monaco et al. (19881), rather than, or in addition to, the current reference.Response: Thank you for the suggestion.We have revised reference 3. 10. "However, it would be in my opinion also useful to point out that, in sufficiently large populations, the overall genetic composition of dystrophinopathies tends to be very similar across world populations, as there are no or very few "founder" mutations (because of the severe phenotype and X-linked nature) and most, if not all identified mutations, usually date back to just a few generations".Response: We have added this suggested comment in the discussion section as "Nonetheless, it should be noted that when sufficiently large populations of patients are analyzed, the overall genetic composition of dystrophinopathies tends to be very similar across world populations, as there are no or very few "founder" mutations (because of the severe phenotype and X-linked nature) and most, if not all identified mutations, usually date back to just a few generations".

Figure 2 .
Figure 2. Distribution pattern of exon deletions in Indonesian Duchenne muscular dystrophy/Becker muscular dystrophy patients (n=28).Most exon deletions occurred in rod domain of DMD gene.Out-frame deletions indicated in orange boxes, while in-frame deletions indicated in blue boxes.

Figure 4 .
Figure 4. Distribution pattern of exon duplications in Indonesian Duchenne muscular dystrophy/Becker muscular dystrophy patients (n=5).All exon duplications occurred in the rod domain of the DMD gene and revealed as out-frame mutations (indicated in orange boxes).In-frame duplication was not detected.
A, Bertelson C, Liechti-Gallati S, Moser H, et al.: An explanation for the phenotypic differences between patients bearing partial deletions of the DMD locus.Genomics.1988; 2 (1): 90-95 Publisher Full Text Is the work clearly and accurately presented and does it cite the current literature?Yes Is the study design appropriate and is the work technically sound?YesAre sufficient details of methods and analysis provided to allow replication by others?YesIf applicable, is the statistical analysis and its interpretation appropriate?Yes Are all the source data underlying the results available to ensure full reproducibility?YesAre the conclusions drawn adequately supported by the results?YesCompeting Interests: No competing interests were disclosed.

1 Reviewer
Interests: No competing interests were disclosed.Reviewer Report 25 April 2023 https://doi.org/10.5256/f1000research.143201.r162602© 2023 Sasongko T. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Teguh Haryo Sasongko 1 Graduate School of Medicine, Perdana University-RCSI, Serdang, Malaysia 2 Center for Research Excellence, Perdana University-RCSI, Serdang, Malaysia The authors have sufficiently addressed all concerns.No further comment.Is the work clearly and accurately presented and does it cite the current literature?YesIs the study design appropriate and is the work technically sound?YesAre sufficient details of methods and analysis provided to allow replication by others?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesAre the conclusions drawn adequately supported by the results?YesCompeting Interests: No competing interests were disclosed.ReviewerExpertise: Human Molecular Genetics and Rare Diseases I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.Version Report 22 March 2022 https://doi.org/10.5256/f1000research.77130.r122827© 2022 Sasongko T. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Teguh Haryo Sasongko 1 Graduate School of Medicine, Perdana University-RCSI, Serdang, Malaysia 2 Center for Research Excellence, Perdana University-RCSI, Serdang, Malaysia The authors described their findings on the mutations of DMD gene in Indonesian patients with Duchenne/Becker Muscular Dystrophy.This the first report from Indonesia on the use of MLPA for the molecular diagnosis of DMD, well-written, and with balance clinical, pathological and molecular data.There are a few concerns raised: I believe clinical diagnosis was the basis for patients' inclusion into the study.There are 8 patients without evident deletion or duplication.There should be enough information how these patients were clinically categorized into DMD or BMD or they were simply MD pending molecular confirmation (eg.(a) if the clinical diagnosis/examination was made by qualified neurologist/pediatrician (b) information on dmd22 and dmd26 may be very limited to warrant specific clinical diagnosis of DMD.There was no evident loss of dystrophin in these patients).

Table 4 .
Comparisons of applicability of current molecular therapy reported across different countries (selected examples).

Table 3 .
Cases amenable for molecular therapies.