Mutation spectrum analysis of <i>DMD </i>gene in Indonesian Duchenne and Becker muscular dystrophy patients

Ery Kus Dwianingsih; Kristy Iskandar; Sunartini Hapsara; Chun Ping Liu; Rusdy Ghazali Malueka; . Gunadi; Masafumi Matsuo; Poh San Lai

doi:10.12688/f1000research.73476.2

Home Browse Mutation spectrum analysis of DMD gene in Indonesian Duchenne and...

ALL Metrics

Views

Downloads

Get PDF

Get XML

Export

▬

✚

Research Article

Revised

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

[version 2; peer review: 1 approved, 1 approved with reservations]

Ery Kus Dwianingsih^1-3, Kristy Iskandar^3-5, Sunartini Hapsara^4,5, [...] Chun Ping Liu⁶, Rusdy Ghazali Malueka^2,3,7, . Gunadi^2,3,8, Masafumi Matsuo⁹, Poh San Lai ⁶

Ery Kus Dwianingsih^1-3, Kristy Iskandar^3-5, [...] Sunartini Hapsara^4,5, Chun Ping Liu⁶, Rusdy Ghazali Malueka^2,3,7, . Gunadi^2,3,8, Masafumi Matsuo⁹, Poh San Lai ⁶

PUBLISHED 07 Feb 2023

Author details Author details

¹ Department of Anatomical Pathology, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada, Yogyakarta, 55281, Indonesia
² Dr. Sardjito General Hospital, Yogyakarta, 55281, Indonesia
³ Genetics Working Group, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada, Yogyakarta, 55281, Indonesia
⁴ Department of Child Health, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada, Yogyakarta, 55281, Indonesia
⁵ Academic Hospital, Universitas Gadjah Mada, Yogyakarta, 55291, Indonesia
⁶ Department of Pediatrics, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, 119228, Singapore
⁷ Department of Neurology, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada, Yogyakarta, 55281, Indonesia
⁸ Pediatric Surgery Division, Department of Surgery, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada, Yogyakarta, 55281, Indonesia
⁹ KNC Department of Nucleic Acid Drug Discovery, Faculty of Rehabilitation, Kobegakuin University, Kobe, 651-2180, Japan

Ery Kus Dwianingsih
Roles: Conceptualization, Investigation, Writing – Original Draft Preparation, Writing – Review & Editing

Kristy Iskandar
Roles: Conceptualization, Investigation, Resources

Sunartini Hapsara
Roles: Conceptualization, Funding Acquisition, Methodology, Supervision

Chun Ping Liu
Roles: Data Curation, Resources, Software

Rusdy Ghazali Malueka
Roles: Formal Analysis, Software, Validation, Visualization, Writing – Review & Editing

. Gunadi
Roles: Funding Acquisition, Software, Validation, Visualization

Masafumi Matsuo
Roles: Conceptualization, Methodology, Supervision

Poh San Lai
Roles: Conceptualization, Funding Acquisition, Methodology, Project Administration, Resources, Supervision, Writing – Original Draft Preparation, Writing – Review & Editing

OPEN PEER REVIEW

REVIEWER STATUS

This article is included in the Genomics and Genetics gateway.

Abstract

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.

Keywords

Duchenne muscular dystrophy, Becker muscular dystrophy, DMD gene, mutation analysis, Indonesia, MLPA

Corresponding author: Poh San Lai

Competing interests: No competing interests were disclosed.

Grant information: This study was supported by the NUS-UGM-Tahir Foundation grant. The funding body had no influence on the study design, data analysis, interpretation of data and writing of the manuscript.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2023 Dwianingsih EK et al. This is an open access article 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.

How to cite: Dwianingsih EK, Iskandar K, Hapsara S et al. Mutation spectrum analysis of DMD gene in Indonesian Duchenne and Becker muscular dystrophy patients [version 2; peer review: 1 approved, 1 approved with reservations]. F1000Research 2023, 11:148 (https://doi.org/10.12688/f1000research.73476.2) First published: 07 Feb 2022, 11:148 (https://doi.org/10.12688/f1000research.73476.1) Latest published: 17 Nov 2023, 11:148 (https://doi.org/10.12688/f1000research.73476.3)

Revised Amendments from Version 1

As the reviewer asked about how the clinical diagnosis of DMD or BMD was made, a statement that the diagnosis was made based on clinical assessment by qualified and experienced pediatric neurologists was added to the Methods section.

In response to reviewer’s suggestion, the term “not yet” for "wheelchair bound" was replaced with “still ambulant” in Table 1. Table 2 has also been deleted while Tables 3 and 4 were combined as recommended. Therefore, all numbering and reference to the Tables in the paper has been updated accordingly throughout the manuscript.

See the authors' detailed response to the review by Luca Bello
See the authors' detailed response to the review by Teguh Haryo Sasongko

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 dystrophin-glycoprotein 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.³

DMD 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.⁴ This suggests that there should be many more DMD cases in Indonesia than the small number of cases that have been reported.⁵ The 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.⁶ Multiplex 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.⁷ Meanwhile, 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.⁸ Precise mutations analysis is thus necessary to apply the appropriate strategies to patients who are eligible for such treatments.⁸ Thus, 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.

Methods

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 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 semi-automatic 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.

Genetic testing

MLPA analysis was conducted to screen all exons of the DMD gene using SALSA MLPA P034 and P035 probe sets (MRC Holland, Netherlands).⁷ The procedure was performed according to the manufacturer’s protocol. Amplified products were separated using the ABI 3130xl Genetic analyzer and data were analyzed by Coffalyser software (MLPA analysis software by MRC-Holland). Patients with single exon deletion were confirmed using conventional PCR. DNA samples from normal healthy individuals were used as reference controls and included in every run. The primer sequences used to amplify the DNA were:

47F 5’-CGTTGTTGCATTTGTCTGTTTCAGTTAC-3’, 47R 5’-GTCTAACCTTTATCCACTGGAGATTTG-3’ (181bp); 51F 5’-GAAATTGGCTCTTTAGCTTGTGTTTC-3’, 51R 5’-GGAGAGTAAAGTGATTGGTGGAAAATC-3’ (388bp); 52F 5’-AATGCAGGATTTGGAACAGAGGCGTCC-3’, 52R 5’-TTCGATCCGTAATGATTGTTCTAGCCTC-3’ (113bp); and 65F 5’-ATTCTCAGAGGAAAAAGGACACTG-3’, 65R 5’-GTCTAACCTTTATCCACTGGAGATTTG-3’ (369bp).

PCRs were performed in a volume of 25 μL containing 2 μL of genomic DNA, 12.5 μL of 2× 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.⁹

Reading 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.

Results

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 Gower 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).

Table 1. Demographic, clinical and genetic characteristic of Duchenne muscular dystrophy (DMD)/Becker muscular dystrophy (BMD) patients.

No	Patient ID	CK level	Age of onset	Age when wheelchair bound	Current age	Clinical features	IHC (dys2)	MLPA	PCR & sequencing validation for single exon deletion	Reading frame prediction	Geno-typing	Pheno-typing
1	dmd1	4906	6	10	11	waddling gait, tiptoe walk, prone to falls, unable to climb stairs, unable to sit without assistance, Gower sign (+), gross and fine motor skill delay, pseudohypertrophy	n/a	del 46–48		Out-frame	DMD	DMD
2	dmd4	4135	5	12	21	waddling gait, tiptoe walk, prone to falls, unable to climb stairs, unable to sit without assistance, Gower sign (+), gross motor skill delay, low birth weight, pseudohypertrophy, scoliosis	negative staining	no del/no dup		n/a	n/a	DMD
3	dmd5	10499	5	10	12	waddling gait, tiptoe walk, prone to falls, Gower sign (+), gross motor skill delay, cardiomegaly, scoliosis, ankle deformity	negative staining	del 53–54		Out-frame	DMD	DMD
4	dmd6	17388	4	not yet	5	unable to climb stairs and sit without assistance, Gower sign (+), cardiomegaly, mild scoliosis	n/a	Del 46–50		Out-frame	DMD	DMD
5	dmd8	2020	4	10	13	difficulty in walking, prone to falls, Gower sign (+), severe obstructive defect of respiratory tract, scoliosis	negative staining	del 17–43		Out-frame	DMD	DMD
6	dmd9	3932	5	10	14	difficulty in walking, prone to falls, Gower sign (+), scoliosis, pneumonia, knee contracture, malnutrition	n/a	del 45–52		Out-frame	DMD	DMD
7	dmd10	11152	5	not yet	7	tiptoe walk, prone to falls, difficulty in standing, Gower sign (+), mild scoliosis	faint staining	del 51	del 51	Out-frame	DMD	DMD
8	dmd11	16550	4	not yet	8	prone to falls, difficulty in walking, Gower sign (+), mild scoliosis, right coxae deformity	faint staining	del 46–51		Out-frame	DMD	DMD
9	dmd14	10846	5	not yet	7	waddling gait, prone to falls, tip toe walk, Gower sign (+), gross and fine motor skill delay, speech delay	negative staining	no del/no dup		n/a	n/a	DMD
10	dmd15	3125	8	not yet	9	Prone to falls, difficulty in walking and climbing, Gower sign (+)	negative staining	del 48–50		Out-frame	DMD	DMD
11	dmd16	8871	4	9	9	difficulty in walking, Gower sign (+), pseudohypertrophy, scoliosis	negative staining	del 45–52		Out-frame	DMD	DMD
12	dmd 17	6239	5	not yet	6	Difficulty in walking, prone to falls, Gower sign (+)	negative staining	del 52	del 52	Out-frame	DMD	DMD
13	dmd18	3255	6	10	13	Lost ambulatory, difficulty in walking, Gower sign (+), gross and fine motor skill delay, speech delay, right lower leg deformity	n/a	del 65	c.9540-9551 del CTGGCTGCTGAA	In-frame	BMD	DMD
14	dmd19	6924	5	not yet	6	difficulty in walking, Gower sign (+), pseudohypertrophy, muscle weakness	negative staining	dup 2–62*		Out-frame	DMD	DMD
15	dmd20	12948	7	not yet	10	Difficulty in walking, prone to falls, waddling gait, Gower sign (+), gross and fine motor skill delay	negative staining	del 7–43		In-frame	BMD	BMD
16	dmd21	28060	6	not yet	7	Difficulty in walking and climbing, prone to falls, waddling gait, Gower sign (+), gross and fine motor skill delay, speech delay, muscle weakness, pneumonia	negative staining	dup 2–62*		Out-frame	DMD	DMD
17	dmd22	no data	5	7	14	Difficulty in walking, Gower sign (+)	n/a	no del/no dup				DMD
18	dmd24	11433	5	Still ambulant	9	Difficulty in walking, Gower sign (+)	n/a	del 47–50		Out-frame	DMD	DMD
19	dmd25	NA	1	Still ambulant	6	Difficulty in walking and climbing, prone to falls, Gower sign (+), gross motor skill delay	n/a	del 49–52		Out-frame	DMD	DMD
20	dmd26	40429	6	Still ambulant	6	Difficulty in walking, Gower sign (+)	n/a	no del/no dup		n/a	n/a	DMD
21	dmd27	8961	6	Still ambulant	6	Difficulty in walking and climbing, tiptoe walking, prone to falls, Gower sign (+), muscle weakness	negative staining	no del/no dup		n/a	n/a	DMD
22	dmd28	4137	9	Still ambulant	9	Difficulty in walking, tiptoe walking, prone to falls, Gower sign (+), muscle weakness, scoliosis	negative staining	del 47	c.6808 del T (stop codon)	Out-frame	DMD	BMD
23	dmd29	8340	5	9	18	Difficulty in walking, tiptoe walking, prone to falls, Gower sign (+), difficulty in breathing, muscle atrophy, upper and lower extremity joint contracture, scoliosis, bedridden	n/a	no del/no dup		n/a	n/a	DMD
24	dmd30	8751	4	8	8	Difficulty in walking, prone to falls, Gower sign (+), gross and fine motor skill delay, inferior flaccid paresis	negative staining	del 51–54		Out-frame	DMD	DMD
25	dmd31	9603	5	10	10	Difficulty in walking, tiptoe walking, Gower sign (+), gross motor skill delay, speech delay, scoliosis	n/a	del 3–44		In-frame	BMD	DMD
26	dmd33	7482	6	Still ambulant	9	Waddling gait, prone to falls, difficulty in walking, Gower sign (+), gross motor skill delay, pseudohypertrophy	n/a	no del/no dup		n/a	n/a	DMD
27	dmd34	6746	2	9	10	Walking difficulty, tiptoe walking, Gower sign (+), muscle weakness, pseudohypertrophy, scoliosis	negative staining	del 49–50		Out-frame	DMD	DMD
28	dmd35	15448	6	Still ambulant	9	Walking difficulty, tiptoe walking, Gower sign (+), muscle weakness	faint staining	dup 14–17		Out-frame	DMD	DMD
29	dmd36	6857	4	Still ambulant	8	Walking difficulty, waddling gait, tip toe walking, prone to falls, Gower sign (+), pseudohypertrophy, lordosis	negative staining	del 18–47		Out-frame	DMD	DMD
30	dmd37	3337	5	Still ambulant	7	Walking difficulty, waddling gait, tip toe walking, gross motor skill delay, speech delay, Gower sign (+), pseudohypertrophy, muscle weakness, malnutrition	negative staining	del 56–74		Out-frame	DMD	DMD
31	dmd38	6607	7	7	8	Walking difficulty, tiptoe walking, Gower sign (+), muscle weakness, pseudohypertrophy, lordosis	negative staining	del 45–49		In-frame	BMD	BMD
32	dmd39	2678	5	10	11	Unable to stand up and walk, muscle weakness	faint staining	del 18–34		Out-frame	DMD	DMD
33	dmd42	1368	5	9	14	Prone to falls, difficulty in walking, ankle contracture, scoliosis	n/a	dup 2–44		Out-frame	DMD	DMD
34	dmd44	12324	5	Still ambulant	9	Difficulty in walking, Gower sign (+)	n/a	del 48–52		Out-frame	DMD	DMD
35	dmd47	13896	4	Still ambulant	6	Difficulty in walking, Gower sign (+)	n/a	del 49–50		Out-frame	DMD	DMD
36	dmd48	7800	2	13	14	Difficulty in walking, Gower sign (+)	n/a	del 3–7		Out-frame	DMD	DMD
37	dmd49	9569	9	Still ambulant	12	Difficulty in walking, Gower sign (+)	n/a	del 3–7		Out-frame	DMD	DMD
38	dmd50	7629	4	9	9	Difficulty in walking and climbing the stairs, easy to fall, Gower sign (+), muscle spasm	n/a	del 5–7		Out-frame	DMD	DMD
39	dmd51	7800	4	10	12	Tiptoe walk, prone to falls, unable to run, Gower sign (+), gross and fine motor skill delay, muscle spasm, Achilles’ contracture	n/a	del 3–17		Out-frame	DMD	DMD
40	dmd53	4865	5	Still ambulant	9	Tiptoe walk, prone to falls, difficulty in climbing stairs, Gower sign (+), muscle weakness, malnutrition	negative staining	del 51	del 51	Out-frame	DMD	DMD
41	dmd54	1734	6	9	11	Difficulty in walking, Gower sign (+), scoliosis	faint staining	dup 2–18		Out-frame	DMD	DMD
42	dmd60	13100	5	Still ambulant	10	Difficulty in walking, Gower sign (+)	n/a	del 49–52		Out-frame	DMD	DMD
43	dmd61	11367	5	Still ambulant	9	Difficulty in walking, prone to falls, Gower sign (+), gross motor skill delay, muscle weakness	n/a	no del/no dup		n/a	n/a	DMD

* Novel mutation.

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).

Table 2. Discrepancies among genotype, phenotype, and immunohistochemistry (IHC) staining.

Subject	Molecular genotype	Clinical phenotype	IHC results (protein)
dmd10	Out-frame (del 51)	DMD	±
dmd11	Out-frame (del 46–51)	DMD	±
dmd18	In-frame (del 65)	DMD*	n/a
dmd20	In-frame (del 7–43)	BMD	-
dmd28	Out-frame (del 47)	BMD*	-
dmd31	In-frame (del 3-44)	DMD*	n/a
dmd35	Out-frame (dup 14–17)	DMD	±
dmd38	In-frame (del 45–49)	BMD	-
dmd39	Out-frame (del 18–34)	DMD	±
dmd54	Out-frame (dup 2–18)	DMD	±

* Genotype-phenotype discrepancy.

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).

Figure 1. Frequencies of deletion of DMD gene exons in Indonesian Duchenne muscular dystrophy/Becker muscular dystrophy patients (n=28).

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.

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.

Figure 3. Frequencies of duplication of DMD gene exons in Indonesian Duchenne muscular dystrophy/Becker muscular dystrophy patients (n=5).

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.

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. Family pedigrees were available in five patients showing x-linked pattern of inheritance (Figure 5).

Figure 5. Five family pedigrees of Duchenne muscular dystrophy patients.

Arrow indicates index cases, square indicates male, circle indicates female, cross-hatching indicates affected individuals, slashed cross indicates deceased individuals, number indicates age (y.o = years old, m.o = months old), black block indicates unknown cause of death.

Molecular therapy applicability

Out of 31 patients whose genotype data was consistent with severe DMD, 60.47% cases are amenable for the application of exon skipping therapy. Therapeutic approaches to skip exons 51, 53, 45, 50, 63, 18, 17, 2, 19, 44, 8 and 55 can be applied in 26 patients (60.47%). One patient with an identified nonsense mutation c.6808 del T can be managed with codon read through therapy. The mutation spectrum and molecular therapy applicability for the identified mutations are presented in Tables 3 and 4.

Table 3. Cases amenable for molecular therapies.

Molecular therapies	Type of mutation	Applicable cases
Molecular therapies	Type of mutation	n	(%)
Skipping of
Exon 51	del48–50, del52, del47–50, del49–50	5	11.63
Exon 53	del45–52, del48–52, del49–52	5	11.63
Exon 45	del46–48, del46–51	2	4.65
Exon 50	del 51	2	4.65
Exon 63	dup 2–62	2	4.65
Exon 18	dup 14–17, del 3–17	2	4.65
Exon 17	del18–47, del18–34	2	4.65
Exon 2	del3–7	2	4.65
Exon 19	dup2–18	1	2.33
Exon 44	del17–43	1	2.33
Exon 8	del5–7	1	2.33
Exon 55	del56–74	1	2.33
Read through	c.6806delT	1	2.33

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

No	Country	Patient number	Del (%)	Dup (%)	Other mutations (%)	No del/No dup (%)	Applicability for current molecular therapies
1	USA (Flanigan et al., 2009)	111	42.9	11	46	(-)	Single exon skipping 59.6%
2	Japan (Takeshima et al., 2011)	442	61	9	29	(-)	single exon skipping 47% (exons 51, 50, 45, 44, 8, 43, 52, 55)
3	India (Polavarapu et al., 2019)	606	81.2	5.4	11.5	1.3	single exon skipping 38.6% (exon 45, 51, 53)
4	Singapore (Tomar et al., 2019)	145	65.5	9.7	21.4	(-)	Single exon skipping 51.6%, multiple exon skipping 32.3%
5	Kuwait (Mohammed et al., 2018)	68	66	5	10	13	Single exon skipping of 16% (exon 51)
6	Eastern European: Poland, Hungary, Lithuania, Romania, Serbia, Croatia, Bosnia, Bulgaria, Ukraine, and Russia (Selvatici et al., 2021)	328	29	11	59	(-)	Single exon skipping 59% (exons 53, 51, 45, 44), 27% for read through
7	Malaysia (Rani et al., 2011)	35	77	6	17	(-)	Single exon skipping 24% (exon 45)
8	Indonesia (this study)	43	65.11	11.63	2.3	23.25	Single exon skipping 60% (exons 51, 53, 45, 50, 17, 2, 44, 8, 55, 63, 18, 19), 2.3% read through

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.¹¹ Our immunohistochemistry (IHC) analysis showed weak staining of dystrophin protein in five DMD patients. Trace-level dystrophin expression in patients with out-of-frame DMD mutations has been previously reported in approximately 20% of DMD patients.¹²^,¹³ The 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.¹⁴ Indeed, 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.¹⁵ Three 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.¹⁶ Due 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.¹⁷ DMD gene analysis in the Indonesian population has been conducted previously using IHC¹⁸ and multiplex PCR¹⁹ 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%).⁸ MLPA is suitable to be performed in developing countries as a screening method since it is simple and fast and allows the diagnosis of DMD in about 85% of cases with the occurrence of 60–70% deletions and 10–15% duplications in the DMD gene found in all DMD patients.¹^,²¹^,²⁴^–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. However, in Kuwait and Malaysia, its applicability is reported to be much lower.¹^,⁸^,²⁷^–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 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.³¹ Two cases (dmd18 and dmd31) in this study were in-frame mutations, but phenotypically DMD. 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

In 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^,³² This 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.¹ This 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.³³ Additionally, 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.³⁴ Hence, 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.³⁵ However, 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.³⁶ Surgical and ventilator support were also reported to increase survival up to the third decade.³⁷ In 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 Gower 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.³⁵ Mutations involving exons 12, 14 to 17, 31 to 42, 45, and 48 to 49 have been reported to enhance cardiac involvement.³⁸ In 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.³⁹ In 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.⁴⁰ Growth delays in individuals with DMD can be observed as short stature⁴¹ or motoric and language delays.⁴² Even 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.⁴³ However, 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.⁴⁴ Our 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%).⁴⁵ De novo mutations are defined by their presence in one or more progenies and the absence in the mother. The presence of multiple affected off-spring 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%.⁴⁶

MLPA has been used to detect carrier status in probands with known deletion or duplication mutations since it is an easy and quick technique.⁴⁷ Detection 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.⁴⁸ This 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.

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.

Data availability

Underlying data

Figshare: Underlying data for ‘Mutation spectrum analysis of DMD gene in Indonesian Duchenne and Becker muscular dystrophy patients’, https://www.doi.org/10.6084/m9.figshare.15172167.⁹

This project contains the following underlying data:

• Demographic, clinical and genetic characteristic of Duchenne muscular dystrophy (DMD)/Becker muscular dystrophy (BMD) patients
• Electrophoresis result of single exon deletion of exon 47, exon 51, and exon 52.
• Electrophoresis result of single exon deletion of exon 47 and exon 65.

Data are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).

Accession numbers^*

Leiden Open Variation Database (LOVD): DMD variant (del 46–48). Accession number DMD_014648, https://databases.lovd.nl/shared/view/DMD?search_VariantOnGenome%2FDBID=%3D%22DMD_014648%22

Leiden Open Variation Database (LOVD): DMD variant (del 53–54). Accession number DMD_105354, https://databases.lovd.nl/shared/view/DMD?search_VariantOnGenome%2FDBID=%3D%22DMD_015354%22

Leiden Open Variation Database (LOVD): DMD variant (del 46–50). Accession number DMD_014650, https://databases.lovd.nl/shared/view/DMD?search_VariantOnGenome%2FDBID=%3D%22DMD_014650%22

Leiden Open Variation Database (LOVD): DMD variant (del 17–43). Accession number DMD_011743, https://databases.lovd.nl/shared/view/DMD?search_VariantOnGenome%2FDBID=%3D%22DMD_011743%22

Leiden Open Variation Database (LOVD): DMD variant (del 45–52). Accession number DMD_014552, https://databases.lovd.nl/shared/view/DMD?search_VariantOnGenome%2FDBID=%3D%22DMD_014552%22

Leiden Open Variation Database (LOVD): DMD variant (del 51). Accession number DMD_015151, https://databases.lovd.nl/shared/view/DMD?search_VariantOnGenome%2FDBID=%3D%22DMD_015151%22

Leiden Open Variation Database (LOVD): DMD variant (del 46–51). Accession number DMD_014651, https://databases.lovd.nl/shared/view/DMD?search_VariantOnGenome%2FDBID=%3D%22DMD_014651%22

Leiden Open Variation Database (LOVD): DMD variant (del 48–50). Accession number DMD_014850, https://databases.lovd.nl/shared/view/DMD?search_VariantOnGenome%2FDBID=%3D%22DMD_014850%22

Leiden Open Variation Database (LOVD): DMD variant (del 52). Accession number DMD_015252, https://databases.lovd.nl/shared/view/DMD?search_VariantOnGenome%2FDBID=%3D%22DMD_015252%22

Leiden Open Variation Database (LOVD): DMD variant (del 65). Accession number DMD_016565, https://databases.lovd.nl/shared/view/DMD?search_VariantOnGenome%2FDBID=%3D%22DMD_016565%22

Leiden Open Variation Database (LOVD): DMD variant (dup 2–62). Accession number DMD_020262, https://databases.lovd.nl/shared/view/DMD?search_VariantOnGenome%2FDBID=%3D%22DMD_020262%22

Leiden Open Variation Database (LOVD): DMD variant (del 7–43). Accession number DMD_010743, https://databases.lovd.nl/shared/view/DMD?search_VariantOnGenome%2FDBID=%3D%22DMD_010743%22

Leiden Open Variation Database (LOVD): DMD variant (del 47–50). Accession number DMD_014750, https://databases.lovd.nl/shared/view/DMD?search_VariantOnGenome%2FDBID=%3D%22DMD_014750%22

Leiden Open Variation Database (LOVD): DMD variant (del 49–52). Accession number DMD_014952, https://databases.lovd.nl/shared/view/DMD?search_VariantOnGenome%2FDBID=%3D%22DMD_014952%22

Leiden Open Variation Database (LOVD): DMD variant (del 47). Accession number DMD_014747, https://databases.lovd.nl/shared/view/DMD?search_VariantOnGenome%2FDBID=%3D%22DMD_014747%22

Leiden Open Variation Database (LOVD): DMD variant (del 51–54). Accession number DMD_015154, https://databases.lovd.nl/shared/view/DMD?search_VariantOnGenome%2FDBID=%3D%22DMD_015154%22

Leiden Open Variation Database (LOVD): DMD variant (del 3–44). Accession number DMD_010344, https://databases.lovd.nl/shared/view/DMD?search_VariantOnGenome%2FDBID=%3D%22DMD_010344%22

Leiden Open Variation Database (LOVD): DMD variant (del 49–50). Accession number DMD_014950, https://databases.lovd.nl/shared/view/DMD?search_VariantOnGenome%2FDBID=%3D%22DMD_014950%22

Leiden Open Variation Database (LOVD): DMD variant (dup 14–17). Accession number DMD_021417, https://databases.lovd.nl/shared/view/DMD?search_VariantOnGenome%2FDBID=%3D%22DMD_021417%22

Leiden Open Variation Database (LOVD): DMD variant (del 18–47). Accession number DMD_011847, https://databases.lovd.nl/shared/view/DMD?search_VariantOnGenome%2FDBID=%3D%22DMD_011847%22

Leiden Open Variation Database (LOVD): DMD variant (del 56–74). Accession number DMD_015674, https://databases.lovd.nl/shared/view/DMD?search_VariantOnGenome%2FDBID=%3D%22DMD_011847%22

Leiden Open Variation Database (LOVD): DMD variant (del 45–49). Accession number DMD_014549, https://databases.lovd.nl/shared/view/DMD?search_VariantOnGenome%2FDBID=%3D%22DMD_014549%22

Leiden Open Variation Database (LOVD): DMD variant (del 18–34). Accession number DMD_011834, https://databases.lovd.nl/shared/view/DMD?search_VariantOnGenome%2FDBID=%3D%22DMD_011834%22

Leiden Open Variation Database (LOVD): DMD variant (dup 2–44). Accession number DMD_020244, https://databases.lovd.nl/shared/view/DMD?search_VariantOnGenome%2FDBID=%3D%22DMD_020244%22

Leiden Open Variation Database (LOVD): DMD variant (del 48–52). Accession number DMD_014852, https://databases.lovd.nl/shared/view/DMD?search_VariantOnGenome%2FDBID=%3D%22DMD_014852%22

Leiden Open Variation Database (LOVD): DMD variant (del 3–7). Accession number DMD_010307, https://databases.lovd.nl/shared/view/DMD?search_VariantOnGenome%2FDBID=%3D%22DMD_010307%22

Leiden Open Variation Database (LOVD): DMD variant (del 5–7). Accession number DMD_010507, https://databases.lovd.nl/shared/view/DMD?search_VariantOnGenome%2FDBID=%3D%22DMD_010507%22

Leiden Open Variation Database (LOVD): DMD variant (del 3–17). Accession number DMD_010317, https://databases.lovd.nl/shared/view/DMD?search_VariantOnGenome%2FDBID=%3D%22DMD_010317%22

Leiden Open Variation Database (LOVD): DMD variant (dup 2–18). Accession number DMD_020218, http://www.umd.be/DMD/4DACTION/WV/368

Consent

Written informed consent for publication of the patients’ details was obtained from the parents of the patients.

Acknowledgements

We would like to thank Klinik Bahasa of Faculty of Medicine, Public Health and Nursing, UGM for assistance in language editing of this manuscript.We also thank Grace Tan from Department of Pediatrics, NUS for assistance with the sequence nomenclatures of the variants. This study was funded by the National University of Singapore (NUHS) - Universitas Gadjah Mada (UGM) Collaboration Seed Fund which was supported by the Tahir Foundation.

References

1. Takeshima Y, Yagi M, Okizuka Y, et al.: Mutation spectrum of the dystrophin gene in 442 Duchenne/Becker muscular dystrophy cases from one Japanese referral center. J. Hum. Genet. 2010; 55(6): 379–388. PubMed Abstract | Publisher Full Text
2. Ervasti JM, Campbell KP: Membrane organization of the dystrophin-glycoprotein complex. Cell. 1991; 66(6): 1121–1131. Publisher Full Text
3. Zatz M, Rapaport D, Vainzof M, et al.: Serum creatine-kinase (CK) and pyruvate-kinase (PK) activities in Duchenne (DMD) as compared with Becker (BMD) muscular dystrophy. J. Neurol. Sci. 1991; 102(2): 190–196. PubMed Abstract | Publisher Full Text
4. Tam CC, O'Driscoll M, Taurel AF, et al.: Geographic variation in dengue seroprevalence and force of infection in the urban paediatric population of Indonesia. PLoS Negl. Trop. Dis. 2018; 12(11): e0006932. PubMed Abstract | Publisher Full Text
5. Wedhanto S, Siregar UP: Duchenne muscular dystrophy. Majalah Kedokteran Indonesia. 2007; 57(9): 312.
6. Okubo M, Goto K, Komaki H, et al.: Comprehensive analysis for genetic diagnosis of dystrophinopathies in Japan. Orphanet J. Rare Dis. 2017; 12(1): 149. PubMed Abstract | Publisher Full Text
7. Schouten JP, McElgunn CJ, Waaijer R, et al.: Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification. Nucleic Acids Res. 2002; 30(12): 57e–557e. PubMed Abstract | Publisher Full Text | Free Full Text
8. Tomar S, Moorthy V, Sethi R, et al.: Mutational spectrum of dystrophinopathies in Singapore: Insights for genetic diagnosis and precision therapy. Am. J. Med. Genet. C Semin. Med. Genet. 2019; 181(2): 230–244.
9. Dwianingsih E, Iskandar K, Hapsara S, et al.: Mutation Spectrum Analysis of DMD gene using MLPA method in Indonesian Duchenne and Becker muscular dystrophy patients. figshare. Dataset.2021. Publisher Full Text
10. Sun SC, Peng YS, He JB: Changes of serum creatine kinase levels in children with Duchenne muscular dystrophy. Zhongguo Dang Dai Er Ke Za Zhi. 2008; 10(1): 35–37. PubMed Abstract
11. Sheriffs IN, Rampling D, Smith VV: Paraffin wax embedded muscle is suitable for the diagnosis of muscular dystrophy. J. Clin. Pathol. 2001; 54(7): 517–520. PubMed Abstract | Publisher Full Text | Free Full Text
12. Arechavala-Gomeza V, Kinali M, Feng L, et al.: Revertant fibres and dystrophin traces in Duchenne muscular dystrophy: implication for clinical trials. Neuromuscul. Disord. 2010; 20(5): 295–301. PubMed Abstract | Publisher Full Text
13. Thanh LT, Nguyen TM, Helliwell TR, et al.: Characterization of revertant muscle fibers in Duchenne muscular dystrophy, using exon-specific monoclonal antibodies against dystrophin. Am. J. Hum. Genet. 1995; 56(3): 725–731. PubMed Abstract
14. Bello L, Pegoraro E: The “Usual Suspects”: Genes for Inflammation, Fibrosis, Regeneration, and Muscle Strength Modify Duchenne Muscular Dystrophy. J. Clin. Med. 2019; 8(5). PubMed Abstract | Publisher Full Text
15. Dwianingsih EK, Malueka RG, Nishida A, et al.: A novel splicing silencer generated by DMD exon 45 deletion junction could explain upstream exon 44 skipping that modifies dystrophinopathy. J. Hum. Genet. 2014; 59(8): 423–429. PubMed Abstract | Publisher Full Text
16. Hoshino S, Ohkoshi N, Watanabe M, et al.: Immunohistochemical staining of dystrophin on formalin-fixed paraffin-embedded sections in Duchenne/Becker muscular dystrophy and manifesting carriers of Duchenne muscular dystrophy. Neuromuscul. Disord. 2000; 10(6): 425–429. PubMed Abstract | Publisher Full Text
17. Lai PS, Takeshima Y, Adachi K, et al.: Comparative study on deletions of the dystrophin gene in three Asian populations. J. Hum. Genet. 2002; 47(10): 0552–0555. PubMed Abstract | Publisher Full Text
18. Dwianingsih EK, Insani MFP, Pratiwi L, et al.: Clinicopathologic and molecular profiles of Duchenne and Becker muscular dystrophy. Paediatr. Indones. 2019; 59(5): 257–264. Publisher Full Text
19. Iskandar K, Dwianingsih EK, Pratiwi L, et al.: The analysis of DMD gene deletions by multiplex PCR in Indonesian DMD/BMD patients: the era of personalized medicine. BMC. Res. Notes. 2019; 12(1): 704. PubMed Abstract | Publisher Full Text
20. Guo R, Zhu G, Zhu H, et al.: DMD mutation spectrum analysis in 613 Chinese patients with dystrophinopathy. J. Hum. Genet. 2015; 60(8): 435–442. PubMed Abstract | Publisher Full Text
21. 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: 303–311. PubMed Abstract
22. Tran VK, Ta VT, Vu DC, et al.: Exon deletion patterns of the dystrophin gene in 82 Vietnamese Duchenne/Becker muscular dystrophy patients. J. Neurogenet. 2013; 27(4): 170–175. PubMed Abstract | Publisher Full Text
23. Rani AQ, Sasongko TH, Sulong S, et al.: Mutation spectrum of dystrophin gene in malaysian patients with Duchenne/Becker muscular dystrophy. J. Neurogenet. 2013; 27(1-2): 11–15. PubMed Abstract | Publisher Full Text
24. Juan-Mateu J, Gonzalez-Quereda L, Rodriguez MJ, et al.: DMD mutations in 576 dystrophinopathy families: a step forward in genotype-phenotype correlations. PLoS One. 2015; 10(8): e0135189. PubMed Abstract | Publisher Full Text
25. Zimowski JG, Massalska D, Holding M, et al.: MLPA based detection of mutations in the dystrophin gene of 180 Polish families with Duchenne/Becker muscular dystrophy. Neurol. Neurochir. Pol. 2014; 48(6): 416–422. PubMed Abstract | Publisher Full Text
26. Ji X, Zhang J, Xu Y, et al.: MLPA application in clinical diagnosis of DMD/BMD in Shanghai. J. Clin. Lab. Anal. 2015; 29(5): 405–411. PubMed Abstract | Publisher Full Text
27. Polavarapu K, Preethish-Kumar V, Sekar D, et al.: Mutation pattern in 606 Duchenne muscular dystrophy children with a comparison between familial and non-familial forms: a study in an Indian large single-center cohort. J. Neurol. 2019; 266(9): 2177–2185. PubMed Abstract | Publisher Full Text
28. Flanigan KM, Dunn DM, von Niederhausern A , et al.: Mutational spectrum of DMD mutations in dystrophinopathy patients: application of modern diagnostic techniques to a large cohort. Hum. Mutat. 2009; 30(12): 1657–1666. PubMed Abstract | Publisher Full Text
29. Mohammed F, Elshafey A, Al-Balool H, et al.: Mutation spectrum analysis of Duchenne/Becker muscular dystrophy in 68 families in Kuwait: The era of personalized medicine. PLoS One. 2018; 13(5): e0197205. PubMed Abstract | Publisher Full Text
30. Selvatici R, Rossi R, Fortunato F, et al.: Ethnicity-related DMD Genotype Landscapes in European and Non-European Countries. Neurol Genet. 2021; 7(1): e536. PubMed Abstract | Publisher Full Text
31. Janssen B, Hartmann C, Scholz V, et al.: MLPA analysis for the detection of deletions, duplications and complex rearrangements in the dystrophin gene: potential and pitfalls. Neurogenetics. 2005; 6(1): 29–35. PubMed Abstract | Publisher Full Text
32. Aartsma-Rus A, Van Deutekom JC, Fokkema IF, et al.: Entries in the Leiden Duchenne muscular dystrophy mutation database: an overview of mutation types and paradoxical cases that confirm the reading-frame rule. Muscle Nerve. 2006; 34(2): 135–144. PubMed Abstract | Publisher Full Text
33. Falzarano MS, Scotton C, Passarelli C, et al.: Duchenne muscular dystrophy: from diagnosis to therapy. Molecules. 2015; 20(10): 18168–18184. PubMed Abstract | Publisher Full Text
34. Niba ETE, Tran VK, Tuan-Phamb LA, et al.: Validation of ambiguous MLPA results by targeted next-generationsequencing discloses a nonsense mutation in the DMD gene. Clin. Chim. Acta. 2014; 436: 155–159. PubMed Abstract | Publisher Full Text
35. Andrews JG, Wahl RA: Duchenne and Becker muscular dystrophy in adolescents: current perspectives. Adolesc. Health Med. Ther. 2018; Volume 9: 53–63. PubMed Abstract | Publisher Full Text
36. Moxley RT 3rd, Pandya S, Ciafaloni E, et al.: Change in natural history of Duchenne muscular dystrophy with long-term corticosteroid treatment: implications for management. J. Child Neurol. 2010; 25(9): 1116–1129. PubMed Abstract | Publisher Full Text
37. Passamano L, Taglia A, Palladino A, et al.: Improvement of survival in Duchenne Muscular Dystrophy: retrospective analysis of 835 patients. Acta Myol. 2012; 31(2): 121–125. PubMed Abstract
38. Yamamoto T, Awano H, Zhang Z, et al.: Cardiac Dysfunction in Duchenne Muscular Dystrophy Is Less Frequent in Patients With Mutations in the Dystrophin Dp116 Coding Region Than in Other Regions. Circ. Genom. Precis. Med. 2018; 11(1): e001782. PubMed Abstract | Publisher Full Text
39. Muntoni F, Bushby K, Manzur AY: Muscular Dystrophy Campaign Funded Workshop on Management of Scoliosis in Duchenne Muscular Dystrophy 24 January 2005, London. UK. Neuromuscul. Disord. 2006; 16(3): 210–219. PubMed Abstract | Publisher Full Text
40. Toussaint M, Davidson Z, Bouvoie V, et al.: Dysphagia in Duchenne muscular dystrophy: practical recommendations to guide management. Disabil. Rehabil. 2016; 38(20): 2052–2062. PubMed Abstract | Publisher Full Text
41. Matsumoto M, Awano H, Lee T, et al.: Patients with Duchenne muscular dystrophy are significantly shorter than those with Becker muscular dystrophy, with the higher incidence of short stature in Dp71 mutated subgroup. Neuromuscul. Disord. 2017; 27(11): 1023–1028. PubMed Abstract | Publisher Full Text
42. Mirski KT, Crawford TO: Motor and cognitive delay in Duchenne muscular dystrophy: implication for early diagnosis. J. Pediatr. 2014; 165(5): 1008–1010. PubMed Abstract | Publisher Full Text
43. Rae MG, O'Malley D: Cognitive dysfunction in Duchenne muscular dystrophy: a possible role for neuromodulatory immune molecules. J. Neurophysiol. 2016; 116(3): 1304–1315. PubMed Abstract | Publisher Full Text
44. Lee T, Takeshima Y, Kusunoki N, et al.: Differences in carrier frequency between mothers of Duchenne and Becker muscular dystrophy patients. J. Hum. Genet. 2014; 59(1): 46–50. PubMed Abstract | Publisher Full Text
45. Sakthivel Murugan SM, Arthi C, Thilothammal N, et al.: Carrier detection in Duchenne muscular dystrophy using molecular methods. Indian J. Med. Res. 2013; 137(6): 1102–1110.
46. Helderman-van den Enden AT, de Jong R , den Dunnen JT , et al.: Recurrence risk due to germ line mosaicism: Duchenne and Becker muscular dystrophy. Clin. Genet. 2009; 75(5): 465–472. PubMed Abstract | Publisher Full Text
47. Verma PK, Dalal A, Mittal B, et al.: Utility of MLPA in mutation analysis and carrier detection for Duchenne muscular dystrophy. Indian J. Hum. Genet. 2012; 18(1): 91–94. PubMed Abstract | Publisher Full Text
48. Bogue L, Peay H, Martin A, et al.: Knowledge of carrier status and barriers to testing among mothers of sons with Duchenne or Becker muscular dystrophy. Neuromuscul. Disord. 2016; 26(12): 860–864. PubMed Abstract | Publisher Full Text

Footnotes

* Representative Accession numbers are shown as some variants have been associated with numerous IDs.

Comments on this article Comments (0)

Version 3

VERSION 3 PUBLISHED 07 Feb 2022

Author details Author details

Ery Kus Dwianingsih
Roles: Conceptualization, Investigation, Writing – Original Draft Preparation, Writing – Review & Editing

Kristy Iskandar
Roles: Conceptualization, Investigation, Resources

Sunartini Hapsara
Roles: Conceptualization, Funding Acquisition, Methodology, Supervision

Chun Ping Liu
Roles: Data Curation, Resources, Software

Rusdy Ghazali Malueka
Roles: Formal Analysis, Software, Validation, Visualization, Writing – Review & Editing

. Gunadi
Roles: Funding Acquisition, Software, Validation, Visualization

Masafumi Matsuo
Roles: Conceptualization, Methodology, Supervision

Poh San Lai
Roles: Conceptualization, Funding Acquisition, Methodology, Project Administration, Resources, Supervision, Writing – Original Draft Preparation, Writing – Review & Editing

Competing interests

No competing interests were disclosed.

Grant information

This study was supported by the NUS-UGM-Tahir Foundation grant. The funding body had no influence on the study design, data analysis, interpretation of data and writing of the manuscript.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Article Versions (3)

version 3

Revised

Published: 17 Nov 2023, 11:148

https://doi.org/10.12688/f1000research.73476.3

version 2

Revised

Published: 07 Feb 2023, 11:148

https://doi.org/10.12688/f1000research.73476.2

version 1

Published: 07 Feb 2022, 11:148

https://doi.org/10.12688/f1000research.73476.1

© 2023 Dwianingsih EK et al. This is an open access article 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.

Download

Export To

metrics

	Views	Downloads
F1000Research	-	-
PubMed Central Data from PMC are received and updated monthly.	-	-

Citations

SEE MORE DETAILS

CITE

how to cite this article

Dwianingsih EK, Iskandar K, Hapsara S et al. Mutation spectrum analysis of DMD gene in Indonesian Duchenne and Becker muscular dystrophy patients [version 2; peer review: 1 approved, 1 approved with reservations]. F1000Research 2023, 11:148 (https://doi.org/10.12688/f1000research.73476.2)

NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article.

track

receive updates on this article

Track an article to receive email alerts on any updates to this article.

Open Peer Review

Version 2

VERSION 2

PUBLISHED 07 Feb 2023

Revised

Views

Reviewer Report 06 Sep 2023

Luca Bello, University of Padova, Padova, Italy

Approved with Reservations

https://doi.org/10.5256/f1000research.143201.r184879

This is a rigorously conducted and well-written study describing the “genetic architecture” of dystrophinopathy in 43 patients from two neuromuscular Centers in an Indonesian city. The literature is quite rich of such papers, showing mutational composition of dystrophinopathy cohorts from different world populations, and these are indeed, as the authors claim, useful for 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.
Also skipping of exon 55 for del 56-74 is very theoretical (no dystroglycan binding in the expected protein product).
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.
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).
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.
Figure 5 (pedigrees) is not very useful, as X-linked inheritance is expected and always confirmed.
“Gowers” instead of “Gower”.
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).
For reference 3 I would suggest Monaco et al. (1988¹), rather than, or in addition to, the current reference.

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?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Yes
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Yes

References

1. Monaco 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

Competing Interests: No competing interests were disclosed.

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.

CITE

Report a concern

Author Response 17 Nov 2023

Poh San Lai, Department of Pediatrics, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, 119228, Singapore

17 Nov 2023

Author Response

Response to reviewer:

1. Table 3: Out-of-frame duplications are not amenable to exon skipping.

Response: Thank you for the comment. We have amended Table 3 to remove ... Continue reading Response to reviewer:

1. Table 3: Out-of-frame duplications are not amenable to exon skipping.

Response: Thank you for the comment. We have amended Table 3 to remove this.

2. Also skipping of exon 55 for del 56-74 is very theoretical (no dystroglycan binding in the expected protein product).

Response: Thank you for the comment. We have amended Table 3 to remove this.

3. 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.

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 4^th 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”.
Response to reviewer:

1. Table 3: Out-of-frame duplications are not amenable to exon skipping.

Response: Thank you for the comment. We have amended Table 3 to remove this.

2. Also skipping of exon 55 for del 56-74 is very theoretical (no dystroglycan binding in the expected protein product).

Response: Thank you for the comment. We have amended Table 3 to remove this.

3. 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.

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 4^th 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”.
Competing Interests: No competing interests were disclosed. Close
Report a concern
Respond or Comment

COMMENTS ON THIS REPORT

Author Response 17 Nov 2023

Poh San Lai, Department of Pediatrics, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, 119228, Singapore

17 Nov 2023

Author Response

Response to reviewer:

1. Table 3: Out-of-frame duplications are not amenable to exon skipping.

Response: Thank you for the comment. We have amended Table 3 to remove ... Continue reading Response to reviewer:

1. Table 3: Out-of-frame duplications are not amenable to exon skipping.

Response: Thank you for the comment. We have amended Table 3 to remove this.

2. Also skipping of exon 55 for del 56-74 is very theoretical (no dystroglycan binding in the expected protein product).

Response: Thank you for the comment. We have amended Table 3 to remove this.

3. 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.

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 4^th 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”.
Response to reviewer:

1. Table 3: Out-of-frame duplications are not amenable to exon skipping.

Response: Thank you for the comment. We have amended Table 3 to remove this.

2. Also skipping of exon 55 for del 56-74 is very theoretical (no dystroglycan binding in the expected protein product).

Response: Thank you for the comment. We have amended Table 3 to remove this.

3. 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.

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 4^th 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”.
Competing Interests: No competing interests were disclosed. Close
Report a concern

Views

Reviewer Report 25 Apr 2023

Teguh Haryo Sasongko, Graduate School of Medicine, Perdana University-RCSI, Serdang, Malaysia; Center for Research Excellence, Perdana University-RCSI, Serdang, Malaysia

Approved

https://doi.org/10.5256/f1000research.143201.r162602

The authors have sufficiently addressed ... Continue reading

CITE

Report a concern

Respond or Comment

Version 1

VERSION 1

PUBLISHED 07 Feb 2022

Views

Reviewer Report 22 Mar 2022

Teguh Haryo Sasongko, Graduate School of Medicine, Perdana University-RCSI, Serdang, Malaysia; Center for Research Excellence, Perdana University-RCSI, Serdang, Malaysia

Approved with Reservations

https://doi.org/10.5256/f1000research.77130.r122827

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, ... Continue reading

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).
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.
Table 1 on the column "Age when wheelchair bound", it will be clearer to replace "not yet" with "still ambulant".
Table 2 is redundant as the content would have been explained by Table 1.
Table 3 and 4 may be combined to focus on discrepancy findings (genotype vs phenotype; IHC vs phenotype).

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?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

No
If applicable, is the statistical analysis and its interpretation appropriate?

Not applicable
Are all the source data underlying the results available to ensure full reproducibility?

No
Are the conclusions drawn adequately supported by the results?

Yes

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Human Molecular Genetics and Rare Diseases

CITE

Report a concern

Author Response 07 Feb 2023

Poh San Lai, Department of Pediatrics, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, 119228, Singapore

07 Feb 2023

Author Response
1. 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
... Continue reading
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).

Response :

a. The diagnosis was made by qualified pediatric neurologists in our hospital, with enough years of experiences and also hold degree of PhD in area of genetic and muscular dystrophy. We have added explanation about this in the method section.

b. DMD/BMD are the most common muscular dystrophy with the patients showing gower signs, difficulty in walking, increase CK level, AST or ALT at clinical level. This clinical screening was recommended by previous study as well (1, 2). Male-young patients with those clinical condition may have high possibility of having DMD gene mutation and enough reason to do screening with either immunohistochemistry (from tissue biopsy) or MLPA (form blood).

Unfortunately for patient DMD22 and DMD26, the muscle biopsy was not performed due to consent problem as it is an invasive procedure. Even though immunohistochemistry result was not available, and genetic analysis of MLPA did not reveal any mutation, we believe the clinical data is enough to include the patients. Further tests need to be done to identify possible small mutations using DNA sequencing. We explained about this in the 7^th paragraph of discussion section (highlighted).

2. 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.

Response: This is an important suggestion. However due to technical difficulties, especially during the pandemic situation, we have been unable to perform a comprehensive database update. Therefore our most current data is from 6 June 2021.

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

Response : Thank you very much for this suggestion. The suggestion has been incorporated in our manuscript.

4. Table 2 is redundant as the content would have been explained by Table 1.

Response : Thank you very much for this suggestion, this table has been removed. Table numbers have been updated accordingly throughout the manuscript.

Table 3 and 4 may be combined to focus on discrepancy findings (genotype vs phenotype; IHC vs phenotype).

Response: Thank you very much for this suggestion, table 3 and 4 have been combined. Table numbers have been updated accordingly throughout the manuscript.
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).

Response :

a. The diagnosis was made by qualified pediatric neurologists in our hospital, with enough years of experiences and also hold degree of PhD in area of genetic and muscular dystrophy. We have added explanation about this in the method section.

b. DMD/BMD are the most common muscular dystrophy with the patients showing gower signs, difficulty in walking, increase CK level, AST or ALT at clinical level. This clinical screening was recommended by previous study as well (1, 2). Male-young patients with those clinical condition may have high possibility of having DMD gene mutation and enough reason to do screening with either immunohistochemistry (from tissue biopsy) or MLPA (form blood).

Unfortunately for patient DMD22 and DMD26, the muscle biopsy was not performed due to consent problem as it is an invasive procedure. Even though immunohistochemistry result was not available, and genetic analysis of MLPA did not reveal any mutation, we believe the clinical data is enough to include the patients. Further tests need to be done to identify possible small mutations using DNA sequencing. We explained about this in the 7^th paragraph of discussion section (highlighted).

2. 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.

Response: This is an important suggestion. However due to technical difficulties, especially during the pandemic situation, we have been unable to perform a comprehensive database update. Therefore our most current data is from 6 June 2021.

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

Response : Thank you very much for this suggestion. The suggestion has been incorporated in our manuscript.

4. Table 2 is redundant as the content would have been explained by Table 1.

Response : Thank you very much for this suggestion, this table has been removed. Table numbers have been updated accordingly throughout the manuscript.

Table 3 and 4 may be combined to focus on discrepancy findings (genotype vs phenotype; IHC vs phenotype).

Response: Thank you very much for this suggestion, table 3 and 4 have been combined. Table numbers have been updated accordingly throughout the manuscript.
Competing Interests: No competing interests were disclosed. Close
Report a concern
Author Response 17 Nov 2023

Poh San Lai, Department of Pediatrics, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, 119228, Singapore

17 Nov 2023

Author Response

Response to reviewer

1.   Table 3: Out-of-frame duplications are not amenable to exon skipping.
Response: Thank you for the comment. We have amended Table 3 to remove this.

... Continue reading Response to reviewer

1.   Table 3: Out-of-frame duplications are not amenable to exon skipping.
Response: Thank you for the comment. We have amended Table 3 to remove this.

2.   Also skipping of exon 55 for del 56-74 is very theoretical (no dystroglycan binding in the expected protein product).
Response: Thank you for the comment. We have amended Table 3 to remove this.

3.   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.
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”.
Response to reviewer

1.   Table 3: Out-of-frame duplications are not amenable to exon skipping.
Response: Thank you for the comment. We have amended Table 3 to remove this.

2.   Also skipping of exon 55 for del 56-74 is very theoretical (no dystroglycan binding in the expected protein product).
Response: Thank you for the comment. We have amended Table 3 to remove this.

3.   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.
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”.
Competing Interests: No competing interests were disclosed. Close
Report a concern
Respond or Comment

COMMENTS ON THIS REPORT

Author Response 07 Feb 2023

Poh San Lai, Department of Pediatrics, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, 119228, Singapore

07 Feb 2023

Author Response
1. 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
... Continue reading
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).

Response :

a. The diagnosis was made by qualified pediatric neurologists in our hospital, with enough years of experiences and also hold degree of PhD in area of genetic and muscular dystrophy. We have added explanation about this in the method section.

b. DMD/BMD are the most common muscular dystrophy with the patients showing gower signs, difficulty in walking, increase CK level, AST or ALT at clinical level. This clinical screening was recommended by previous study as well (1, 2). Male-young patients with those clinical condition may have high possibility of having DMD gene mutation and enough reason to do screening with either immunohistochemistry (from tissue biopsy) or MLPA (form blood).

Unfortunately for patient DMD22 and DMD26, the muscle biopsy was not performed due to consent problem as it is an invasive procedure. Even though immunohistochemistry result was not available, and genetic analysis of MLPA did not reveal any mutation, we believe the clinical data is enough to include the patients. Further tests need to be done to identify possible small mutations using DNA sequencing. We explained about this in the 7^th paragraph of discussion section (highlighted).

2. 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.

Response: This is an important suggestion. However due to technical difficulties, especially during the pandemic situation, we have been unable to perform a comprehensive database update. Therefore our most current data is from 6 June 2021.

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

Response : Thank you very much for this suggestion. The suggestion has been incorporated in our manuscript.

4. Table 2 is redundant as the content would have been explained by Table 1.

Response : Thank you very much for this suggestion, this table has been removed. Table numbers have been updated accordingly throughout the manuscript.

Table 3 and 4 may be combined to focus on discrepancy findings (genotype vs phenotype; IHC vs phenotype).

Response: Thank you very much for this suggestion, table 3 and 4 have been combined. Table numbers have been updated accordingly throughout the manuscript.
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).

Response :

a. The diagnosis was made by qualified pediatric neurologists in our hospital, with enough years of experiences and also hold degree of PhD in area of genetic and muscular dystrophy. We have added explanation about this in the method section.

b. DMD/BMD are the most common muscular dystrophy with the patients showing gower signs, difficulty in walking, increase CK level, AST or ALT at clinical level. This clinical screening was recommended by previous study as well (1, 2). Male-young patients with those clinical condition may have high possibility of having DMD gene mutation and enough reason to do screening with either immunohistochemistry (from tissue biopsy) or MLPA (form blood).

Unfortunately for patient DMD22 and DMD26, the muscle biopsy was not performed due to consent problem as it is an invasive procedure. Even though immunohistochemistry result was not available, and genetic analysis of MLPA did not reveal any mutation, we believe the clinical data is enough to include the patients. Further tests need to be done to identify possible small mutations using DNA sequencing. We explained about this in the 7^th paragraph of discussion section (highlighted).

2. 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.

Response: This is an important suggestion. However due to technical difficulties, especially during the pandemic situation, we have been unable to perform a comprehensive database update. Therefore our most current data is from 6 June 2021.

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

Response : Thank you very much for this suggestion. The suggestion has been incorporated in our manuscript.

4. Table 2 is redundant as the content would have been explained by Table 1.

Response : Thank you very much for this suggestion, this table has been removed. Table numbers have been updated accordingly throughout the manuscript.

Table 3 and 4 may be combined to focus on discrepancy findings (genotype vs phenotype; IHC vs phenotype).

Response: Thank you very much for this suggestion, table 3 and 4 have been combined. Table numbers have been updated accordingly throughout the manuscript.
Competing Interests: No competing interests were disclosed. Close
Report a concern
Author Response 17 Nov 2023

Poh San Lai, Department of Pediatrics, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, 119228, Singapore

17 Nov 2023

Author Response

Response to reviewer

1.   Table 3: Out-of-frame duplications are not amenable to exon skipping.
Response: Thank you for the comment. We have amended Table 3 to remove this.

... Continue reading Response to reviewer

1.   Table 3: Out-of-frame duplications are not amenable to exon skipping.
Response: Thank you for the comment. We have amended Table 3 to remove this.

2.   Also skipping of exon 55 for del 56-74 is very theoretical (no dystroglycan binding in the expected protein product).
Response: Thank you for the comment. We have amended Table 3 to remove this.

3.   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.
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”.
Response to reviewer

1.   Table 3: Out-of-frame duplications are not amenable to exon skipping.
Response: Thank you for the comment. We have amended Table 3 to remove this.

2.   Also skipping of exon 55 for del 56-74 is very theoretical (no dystroglycan binding in the expected protein product).
Response: Thank you for the comment. We have amended Table 3 to remove this.

3.   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.
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”.
Competing Interests: No competing interests were disclosed. Close
Report a concern

Comments on this article Comments (0)

Version 3

VERSION 3 PUBLISHED 07 Feb 2022

Open Peer Review

Reviewer Status

Reviewer Reports

	Invited Reviewers
	1	2
Version 3 (revision) 17 Nov 23		read
Version 2 (revision) 07 Feb 23	read	read
Version 1 07 Feb 22	read

Teguh Haryo Sasongko, Perdana University-RCSI, Serdang, Malaysia; Perdana University-RCSI, Serdang, Malaysia
Luca Bello, University of Padova, Padova, Italy

Comments on this article

All Comments(0)

Add a comment

Browse by related subjects

Back to all reports

Reviewer Report

6 Views

22 Nov 2023 | for Version 3

Luca Bello, University of Padova, Padova, Italy

6 Views Cite this report Responses(0)

Approved

The authors have responded satisfactorily to my comments.

Competing Interests

No competing interests were disclosed.

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.

Respond to this report

Responses (0)

Back to all reports

Reviewer Report

15 Views

06 Sep 2023 | for Version 2

Luca Bello, University of Padova, Padova, Italy

15 Views Cite this report Responses(1)

Approved With Reservations

Table 3: Out-of-frame duplications are not amenable to exon skipping.
Also skipping of exon 55 for del 56-74 is very theoretical (no dystroglycan binding in the expected protein product).
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.
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).
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.
Figure 5 (pedigrees) is not very useful, as X-linked inheritance is expected and always confirmed.
“Gowers” instead of “Gower”.
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).
For reference 3 I would suggest Monaco et al. (1988¹), rather than, or in addition to, the current reference.

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?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Yes
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Yes

References

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Muscular dystrophy

Respond to this report

Responses (1)

Author Response

17 Nov 2023

Poh San Lai, Department of Pediatrics, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, 119228, Singapore

Response to reviewer:

1. Table 3: Out-of-frame duplications are not amenable to exon skipping.

Response: Thank you for the comment. We have amended Table 3 to remove this.

2. Also skipping of exon 55 for del 56-74 is very theoretical (no dystroglycan binding in the expected protein product).

Response: Thank you for the comment. We have amended Table 3 to remove this.

3. 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.

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 4^th 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”.

View more View less

Competing Interests

No competing interests were disclosed.

Back to all reports

Reviewer Report

8 Views

25 Apr 2023 | for Version 2

Teguh Haryo Sasongko, Graduate School of Medicine, Perdana University-RCSI, Serdang, Malaysia; Center for Research Excellence, Perdana University-RCSI, Serdang, Malaysia

8 Views Cite this report Responses(0)

Approved

The authors have sufficiently addressed all concerns. No further comment.

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

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.

Respond to this report

Responses (0)

Back to all reports

Reviewer Report

41 Views

22 Mar 2022 | for Version 1

Teguh Haryo Sasongko, Graduate School of Medicine, Perdana University-RCSI, Serdang, Malaysia; Center for Research Excellence, Perdana University-RCSI, Serdang, Malaysia

41 Views Cite this report Responses(2)

Approved With Reservations

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).
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.
Table 1 on the column "Age when wheelchair bound", it will be clearer to replace "not yet" with "still ambulant".
Table 2 is redundant as the content would have been explained by Table 1.
Table 3 and 4 may be combined to focus on discrepancy findings (genotype vs phenotype; IHC vs phenotype).

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?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

No
If applicable, is the statistical analysis and its interpretation appropriate?

Not applicable
Are all the source data underlying the results available to ensure full reproducibility?

No
Are the conclusions drawn adequately supported by the results?

Yes

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Human Molecular Genetics and Rare Diseases

Respond to this report

Responses (2)

Author Response

07 Feb 2023

Poh San Lai, Department of Pediatrics, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, 119228, Singapore

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).

Response :

a. The diagnosis was made by qualified pediatric neurologists in our hospital, with enough years of experiences and also hold degree of PhD in area of genetic and muscular dystrophy. We have added explanation about this in the method section.

b. DMD/BMD are the most common muscular dystrophy with the patients showing gower signs, difficulty in walking, increase CK level, AST or ALT at clinical level. This clinical screening was recommended by previous study as well (1, 2). Male-young patients with those clinical condition may have high possibility of having DMD gene mutation and enough reason to do screening with either immunohistochemistry (from tissue biopsy) or MLPA (form blood).

Unfortunately for patient DMD22 and DMD26, the muscle biopsy was not performed due to consent problem as it is an invasive procedure. Even though immunohistochemistry result was not available, and genetic analysis of MLPA did not reveal any mutation, we believe the clinical data is enough to include the patients. Further tests need to be done to identify possible small mutations using DNA sequencing. We explained about this in the 7^th paragraph of discussion section (highlighted).

2. 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.

Response: This is an important suggestion. However due to technical difficulties, especially during the pandemic situation, we have been unable to perform a comprehensive database update. Therefore our most current data is from 6 June 2021.

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

Response : Thank you very much for this suggestion. The suggestion has been incorporated in our manuscript.

4. Table 2 is redundant as the content would have been explained by Table 1.

Response : Thank you very much for this suggestion, this table has been removed. Table numbers have been updated accordingly throughout the manuscript.

Table 3 and 4 may be combined to focus on discrepancy findings (genotype vs phenotype; IHC vs phenotype).

Response: Thank you very much for this suggestion, table 3 and 4 have been combined. Table numbers have been updated accordingly throughout the manuscript.

View more View less

Competing Interests

No competing interests were disclosed.

Author Response

17 Nov 2023

Poh San Lai, Department of Pediatrics, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, 119228, Singapore

Response to reviewer

1.   Table 3: Out-of-frame duplications are not amenable to exon skipping.
Response: Thank you for the comment. We have amended Table 3 to remove this.

2.   Also skipping of exon 55 for del 56-74 is very theoretical (no dystroglycan binding in the expected protein product).
Response: Thank you for the comment. We have amended Table 3 to remove this.

3.   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.
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”.

View more View less

Competing Interests

No competing interests were disclosed.

Alongside their report, reviewers assign a status to the article:

Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested

Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.

Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions

[1] 1. Takeshima Y, Yagi M, Okizuka Y, et al.: Mutation spectrum of the dystrophin gene in 442 Duchenne/Becker muscular dystrophy cases from one Japanese referral center. J. Hum. Genet. 2010; 55(6): 379–388. PubMed Abstract | Publisher Full Text

[2] 2. Ervasti JM, Campbell KP: Membrane organization of the dystrophin-glycoprotein complex. Cell. 1991; 66(6): 1121–1131. Publisher Full Text

[3] 3. Zatz M, Rapaport D, Vainzof M, et al.: Serum creatine-kinase (CK) and pyruvate-kinase (PK) activities in Duchenne (DMD) as compared with Becker (BMD) muscular dystrophy. J. Neurol. Sci. 1991; 102(2): 190–196. PubMed Abstract | Publisher Full Text

[4] 4. Tam CC, O'Driscoll M, Taurel AF, et al.: Geographic variation in dengue seroprevalence and force of infection in the urban paediatric population of Indonesia. PLoS Negl. Trop. Dis. 2018; 12(11): e0006932. PubMed Abstract | Publisher Full Text

[5] 5. Wedhanto S, Siregar UP: Duchenne muscular dystrophy. Majalah Kedokteran Indonesia. 2007; 57(9): 312.

[6] 6. Okubo M, Goto K, Komaki H, et al.: Comprehensive analysis for genetic diagnosis of dystrophinopathies in Japan. Orphanet J. Rare Dis. 2017; 12(1): 149. PubMed Abstract | Publisher Full Text

[7] 7. Schouten JP, McElgunn CJ, Waaijer R, et al.: Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification. Nucleic Acids Res. 2002; 30(12): 57e–557e. PubMed Abstract | Publisher Full Text | Free Full Text

[8] 8. Tomar S, Moorthy V, Sethi R, et al.: Mutational spectrum of dystrophinopathies in Singapore: Insights for genetic diagnosis and precision therapy. Am. J. Med. Genet. C Semin. Med. Genet. 2019; 181(2): 230–244.

[9] 9. Dwianingsih E, Iskandar K, Hapsara S, et al.: Mutation Spectrum Analysis of DMD gene using MLPA method in Indonesian Duchenne and Becker muscular dystrophy patients. figshare. Dataset.2021. Publisher Full Text

[10] 10. Sun SC, Peng YS, He JB: Changes of serum creatine kinase levels in children with Duchenne muscular dystrophy. Zhongguo Dang Dai Er Ke Za Zhi. 2008; 10(1): 35–37. PubMed Abstract

[11] 11. Sheriffs IN, Rampling D, Smith VV: Paraffin wax embedded muscle is suitable for the diagnosis of muscular dystrophy. J. Clin. Pathol. 2001; 54(7): 517–520. PubMed Abstract | Publisher Full Text | Free Full Text

[12] 12. Arechavala-Gomeza V, Kinali M, Feng L, et al.: Revertant fibres and dystrophin traces in Duchenne muscular dystrophy: implication for clinical trials. Neuromuscul. Disord. 2010; 20(5): 295–301. PubMed Abstract | Publisher Full Text

[13] 13. Thanh LT, Nguyen TM, Helliwell TR, et al.: Characterization of revertant muscle fibers in Duchenne muscular dystrophy, using exon-specific monoclonal antibodies against dystrophin. Am. J. Hum. Genet. 1995; 56(3): 725–731. PubMed Abstract

[14] 14. Bello L, Pegoraro E: The “Usual Suspects”: Genes for Inflammation, Fibrosis, Regeneration, and Muscle Strength Modify Duchenne Muscular Dystrophy. J. Clin. Med. 2019; 8(5). PubMed Abstract | Publisher Full Text

[15] 15. Dwianingsih EK, Malueka RG, Nishida A, et al.: A novel splicing silencer generated by DMD exon 45 deletion junction could explain upstream exon 44 skipping that modifies dystrophinopathy. J. Hum. Genet. 2014; 59(8): 423–429. PubMed Abstract | Publisher Full Text

[16] 16. Hoshino S, Ohkoshi N, Watanabe M, et al.: Immunohistochemical staining of dystrophin on formalin-fixed paraffin-embedded sections in Duchenne/Becker muscular dystrophy and manifesting carriers of Duchenne muscular dystrophy. Neuromuscul. Disord. 2000; 10(6): 425–429. PubMed Abstract | Publisher Full Text

[17] 17. Lai PS, Takeshima Y, Adachi K, et al.: Comparative study on deletions of the dystrophin gene in three Asian populations. J. Hum. Genet. 2002; 47(10): 0552–0555. PubMed Abstract | Publisher Full Text

[18] 18. Dwianingsih EK, Insani MFP, Pratiwi L, et al.: Clinicopathologic and molecular profiles of Duchenne and Becker muscular dystrophy. Paediatr. Indones. 2019; 59(5): 257–264. Publisher Full Text

[19] 19. Iskandar K, Dwianingsih EK, Pratiwi L, et al.: The analysis of DMD gene deletions by multiplex PCR in Indonesian DMD/BMD patients: the era of personalized medicine. BMC. Res. Notes. 2019; 12(1): 704. PubMed Abstract | Publisher Full Text

[20] 20. Guo R, Zhu G, Zhu H, et al.: DMD mutation spectrum analysis in 613 Chinese patients with dystrophinopathy. J. Hum. Genet. 2015; 60(8): 435–442. PubMed Abstract | Publisher Full Text

[21] 21. 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: 303–311. PubMed Abstract

[22] 22. Tran VK, Ta VT, Vu DC, et al.: Exon deletion patterns of the dystrophin gene in 82 Vietnamese Duchenne/Becker muscular dystrophy patients. J. Neurogenet. 2013; 27(4): 170–175. PubMed Abstract | Publisher Full Text

[23] 23. Rani AQ, Sasongko TH, Sulong S, et al.: Mutation spectrum of dystrophin gene in malaysian patients with Duchenne/Becker muscular dystrophy. J. Neurogenet. 2013; 27(1-2): 11–15. PubMed Abstract | Publisher Full Text

[24] 24. Juan-Mateu J, Gonzalez-Quereda L, Rodriguez MJ, et al.: DMD mutations in 576 dystrophinopathy families: a step forward in genotype-phenotype correlations. PLoS One. 2015; 10(8): e0135189. PubMed Abstract | Publisher Full Text

[25] 25. Zimowski JG, Massalska D, Holding M, et al.: MLPA based detection of mutations in the dystrophin gene of 180 Polish families with Duchenne/Becker muscular dystrophy. Neurol. Neurochir. Pol. 2014; 48(6): 416–422. PubMed Abstract | Publisher Full Text

[26] 26. Ji X, Zhang J, Xu Y, et al.: MLPA application in clinical diagnosis of DMD/BMD in Shanghai. J. Clin. Lab. Anal. 2015; 29(5): 405–411. PubMed Abstract | Publisher Full Text

[27] 27. Polavarapu K, Preethish-Kumar V, Sekar D, et al.: Mutation pattern in 606 Duchenne muscular dystrophy children with a comparison between familial and non-familial forms: a study in an Indian large single-center cohort. J. Neurol. 2019; 266(9): 2177–2185. PubMed Abstract | Publisher Full Text

[28] 28. Flanigan KM, Dunn DM, von Niederhausern A , et al.: Mutational spectrum of DMD mutations in dystrophinopathy patients: application of modern diagnostic techniques to a large cohort. Hum. Mutat. 2009; 30(12): 1657–1666. PubMed Abstract | Publisher Full Text

[29] 29. Mohammed F, Elshafey A, Al-Balool H, et al.: Mutation spectrum analysis of Duchenne/Becker muscular dystrophy in 68 families in Kuwait: The era of personalized medicine. PLoS One. 2018; 13(5): e0197205. PubMed Abstract | Publisher Full Text

[30] 30. Selvatici R, Rossi R, Fortunato F, et al.: Ethnicity-related DMD Genotype Landscapes in European and Non-European Countries. Neurol Genet. 2021; 7(1): e536. PubMed Abstract | Publisher Full Text

[31] 31. Janssen B, Hartmann C, Scholz V, et al.: MLPA analysis for the detection of deletions, duplications and complex rearrangements in the dystrophin gene: potential and pitfalls. Neurogenetics. 2005; 6(1): 29–35. PubMed Abstract | Publisher Full Text

[32] 32. Aartsma-Rus A, Van Deutekom JC, Fokkema IF, et al.: Entries in the Leiden Duchenne muscular dystrophy mutation database: an overview of mutation types and paradoxical cases that confirm the reading-frame rule. Muscle Nerve. 2006; 34(2): 135–144. PubMed Abstract | Publisher Full Text

[33] 33. Falzarano MS, Scotton C, Passarelli C, et al.: Duchenne muscular dystrophy: from diagnosis to therapy. Molecules. 2015; 20(10): 18168–18184. PubMed Abstract | Publisher Full Text

[34] 34. Niba ETE, Tran VK, Tuan-Phamb LA, et al.: Validation of ambiguous MLPA results by targeted next-generationsequencing discloses a nonsense mutation in the DMD gene. Clin. Chim. Acta. 2014; 436: 155–159. PubMed Abstract | Publisher Full Text

[35] 35. Andrews JG, Wahl RA: Duchenne and Becker muscular dystrophy in adolescents: current perspectives. Adolesc. Health Med. Ther. 2018; Volume 9: 53–63. PubMed Abstract | Publisher Full Text

[36] 36. Moxley RT 3rd, Pandya S, Ciafaloni E, et al.: Change in natural history of Duchenne muscular dystrophy with long-term corticosteroid treatment: implications for management. J. Child Neurol. 2010; 25(9): 1116–1129. PubMed Abstract | Publisher Full Text

[37] 37. Passamano L, Taglia A, Palladino A, et al.: Improvement of survival in Duchenne Muscular Dystrophy: retrospective analysis of 835 patients. Acta Myol. 2012; 31(2): 121–125. PubMed Abstract

[38] 38. Yamamoto T, Awano H, Zhang Z, et al.: Cardiac Dysfunction in Duchenne Muscular Dystrophy Is Less Frequent in Patients With Mutations in the Dystrophin Dp116 Coding Region Than in Other Regions. Circ. Genom. Precis. Med. 2018; 11(1): e001782. PubMed Abstract | Publisher Full Text

[39] 39. Muntoni F, Bushby K, Manzur AY: Muscular Dystrophy Campaign Funded Workshop on Management of Scoliosis in Duchenne Muscular Dystrophy 24 January 2005, London. UK. Neuromuscul. Disord. 2006; 16(3): 210–219. PubMed Abstract | Publisher Full Text

[40] 40. Toussaint M, Davidson Z, Bouvoie V, et al.: Dysphagia in Duchenne muscular dystrophy: practical recommendations to guide management. Disabil. Rehabil. 2016; 38(20): 2052–2062. PubMed Abstract | Publisher Full Text

[41] 41. Matsumoto M, Awano H, Lee T, et al.: Patients with Duchenne muscular dystrophy are significantly shorter than those with Becker muscular dystrophy, with the higher incidence of short stature in Dp71 mutated subgroup. Neuromuscul. Disord. 2017; 27(11): 1023–1028. PubMed Abstract | Publisher Full Text

[42] 42. Mirski KT, Crawford TO: Motor and cognitive delay in Duchenne muscular dystrophy: implication for early diagnosis. J. Pediatr. 2014; 165(5): 1008–1010. PubMed Abstract | Publisher Full Text

[43] 43. Rae MG, O'Malley D: Cognitive dysfunction in Duchenne muscular dystrophy: a possible role for neuromodulatory immune molecules. J. Neurophysiol. 2016; 116(3): 1304–1315. PubMed Abstract | Publisher Full Text

[44] 44. Lee T, Takeshima Y, Kusunoki N, et al.: Differences in carrier frequency between mothers of Duchenne and Becker muscular dystrophy patients. J. Hum. Genet. 2014; 59(1): 46–50. PubMed Abstract | Publisher Full Text

[45] 45. Sakthivel Murugan SM, Arthi C, Thilothammal N, et al.: Carrier detection in Duchenne muscular dystrophy using molecular methods. Indian J. Med. Res. 2013; 137(6): 1102–1110.

[46] 46. Helderman-van den Enden AT, de Jong R , den Dunnen JT , et al.: Recurrence risk due to germ line mosaicism: Duchenne and Becker muscular dystrophy. Clin. Genet. 2009; 75(5): 465–472. PubMed Abstract | Publisher Full Text

[47] 47. Verma PK, Dalal A, Mittal B, et al.: Utility of MLPA in mutation analysis and carrier detection for Duchenne muscular dystrophy. Indian J. Hum. Genet. 2012; 18(1): 91–94. PubMed Abstract | Publisher Full Text

[48] 48. Bogue L, Peay H, Martin A, et al.: Knowledge of carrier status and barriers to testing among mothers of sons with Duchenne or Becker muscular dystrophy. Neuromuscul. Disord. 2016; 26(12): 860–864. PubMed Abstract | Publisher Full Text

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

Abstract

Keywords

Revised Amendments from Version 1

Introduction

Methods

Patients

Immunohistochemical analysis

Genomic DNA extraction

Genetic testing

Results

Demographic and clinical profile

Table 1. Demographic, clinical and genetic characteristic of Duchenne muscular dystrophy (DMD)/Becker muscular dystrophy (BMD) patients.

Immunohistochemistry results

Table 2. Discrepancies among genotype, phenotype, and immunohistochemistry (IHC) staining.

Deletion pattern in the DMD gene

Figure 1. Frequencies of deletion of DMD gene exons in Indonesian Duchenne muscular dystrophy/Becker muscular dystrophy patients (n=28).

Figure 2. Distribution pattern of exon deletions in Indonesian Duchenne muscular dystrophy/Becker muscular dystrophy patients (n=28).

Duplications pattern in the DMD gene

Figure 3. Frequencies of duplication of DMD gene exons in Indonesian Duchenne muscular dystrophy/Becker muscular dystrophy patients (n=5).

Figure 4. Distribution pattern of exon duplications in Indonesian Duchenne muscular dystrophy/Becker muscular dystrophy patients (n=5).

Association with reading frames

Other clinical parameters

Carrier status

Figure 5. Five family pedigrees of Duchenne muscular dystrophy patients.

Molecular therapy applicability

Table 3. Cases amenable for molecular therapies.

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

Discussion

Data availability

Underlying data

Accession numbers*

Consent

Acknowledgements

References

Footnotes

Comments on this article Comments (0)

Open Peer Review

Comments on this article Comments (0)

Open Peer Review

Reviewer Status

Reviewer Reports

Comments on this article

Browse by related subjects

Competing Interests Policy

Stay Updated

Accession numbers^*