Yong Loo Lin School of Medicine

NGS 6882A
BIOLOGY OF DISEASE:
Duchenne Muscular Dystrophy

Assoc Professor Lai Poh San

Department of Paediatrics
YLL School of Medicine
National University of Singapore
National University Hospital
Email : poh_san_lai@nuhs.edu.sg

Copyright ©, 2020
 Bridging Biology to Disease

Providing an understanding of
human disease from a bench to
bedside perspective

Lecture 1 : Basic biology and clinical

pathology underlying human disease
(A/P Reshma Taneja)

Lecture 2 : Clinical perspectives of the

disease (A/P Lai Poh San)
 In this lecture, we will ..........
1. Review the clinical symptoms of Duchenne muscular dystrophy

2. Differentiate between Duchenne and Becker muscular dystrophies

3. Understand the genetics underlying this X-linked disease

4. Review approaches available for diagnosis & carrier screening

5. Learn about interpretation of DNA results and some challenges in

molecular diagnosis and genetic counselling

6. Highlight potential molecular therapies for this disorder

1. DISEASE
 DEFINITION OF MUSCULAR DYSTROPHIES
• Group of diseases with degeneration of muscle cells
• At least nine major forms of MDs
• Muscular dystrophy = Muscle wasting (disease)
Duchenne muscular dystrophy
Becker muscular dystrophy (allelic form of disease)

Dr. Guillaume Benjamin Amand Duchenne Dr. Peter Emil Becker

Desribed first case Becker and Kiener (1955)
Reported on the areas of muscles affected A new form of the disorder?
Arch. Gen. Med. 1868 vol 11:8) “Arch Psychiatr Nervenkr 193:427–448
(From Dr Craig McDonald)
Cardinal clinical features
TYPICAL PRESENTING SYMPTOMS

• Late walking, difficulties in climbing stairs

• Frequent falling
• Tendency to walk on toes
• Tight achilles tendon
• Gower’s sign
• Calf pseudo-hypertrophy
• Progressive, proximal weakness
• Lumbar lordosis, scoliosis
• May have developmental speech delays
A key clinical sign

What is Gower’s sign?

Gower’s maneuver – describes typical movement associated

with weakness of the proximal muscles, namely those of the
lower limb. This sign is typical of DMD patient.

The patient has to use both his hands and arms to walk up his
own body from a squatting position due to lack of hip and thigh
muscle strength.
 Sir William Richard Gowers’, 1879
Typical sign: Proximal weakness (lower limb muscles), use hands &
arms to walk up own body from prone or squatting position into upright
position instead of relying on their legs. “Gowers sign or manoeuvre”
What is the difference between DMD and BMD at the clinical level?

Duchenne muscular dystrophy (DMD) Becker muscular dystrophy (BMD)

• Incidence: 1 in 3,500 male births
• Early childhood, delayed milestones
• Skeletal muscle involvement
• Wheel-chair bound by about 12 years’
• Cardiomyopathy, respiratory arrest
• Life expectancy : second/third decade
• Incidence: 1 in 18,000 male births
• Later-onset
• Similar but less severe symptoms
• Remain ambulatory until later
• Slower progression
• Life expectancy longer (mid-40s to 60s)

 DMD and BMD are allelic disorders

 Caused by mutations in same gene
 But different mutations can lead to either DMD or BMD
 What are the family concerns in this situation?

Mother with Reproductive Options

a DMD son
1. No children
2. Adopt a Child
3. Play Genetic Roulette
Termination/Acceptance
DMD patient

What is causing the disease in my family?

What is pattern of inheritance (isolated or sporadic cases)?
Will my next child be affected?
What is the prognosis? (allelic or milder forms of similar disorders)
2. GENETICS
 Gene responsible for DMD is X-linked
2.3 Mb gene (79 exons)
14 kb transcript → 424 kDal protein

Female carriers can transmit

Affects males only

But females
can be
carriers

https://sites.google.com/site/hannahcgenetics/medical
Disease: DMD/BMD Gene: DMD gene Protein: Dystrophin

 Mutations in same gene can cause either DMD or BMD (two phenotypes)

 Types of mutations : deletions/duplications or point mutations

 Frame-shift mutations
Severe phenotype (DMD): out-of-frame or truncating mutations
Milder phenotype (BMD): in-frame or missense mutations

 Protein in absent in DMD but reduced or defective in BMD

 What are frame-shift mutations?
A frameshift mutation is a mutation caused by insertions or
deletions of a number of nucleotides in a DNA sequence that is
not divisible by three
CGA UUA GGG UAC AAA CGA
Arg Leu Gly Tyr Lys Arg

Original reading frame

THE FAT CAT AND THE OLD RAT RAN

In-frame mutation (deletion of 3 nucleotides)

THE CAT AND THE OLD RAT RAN Missing 3 alphabets

Out-of-frame mutation (deletion of 2 nucleotides)

THE FA AT AND THE OLD RAT RAN Missing 2 alphabets

THE FAA TAN DTH EOL DRA TRA N

 Difference between
Normal splicing of exons
DMD & BMD
at the molecular level
Deletion mutation

DMD : out-of-frame mutations

BMD : in-frame mutations

Duplication mutation
Different
clinical outcomes
due to type
of mutations!
Point mutation

Le Rumeur 2015
Bos J Basic Med Sci 15(3): 14-20
3. DIAGNOSIS
- Different techniques and approaches -
 DIAGNOSING DMD/BMD

• (1) Clinical Presentations

• (2) Biochemical tests (serum creatine kinase)
• (3) Muscle Biopsies
• (4) Molecular diagnosis

Thinking point:
What are some of the limitations
with each of the above approaches?
(1) Clinical Presentations
http://drustapbio.wikia.com/wiki/Duchenne_Muscular_Dystrophy
(2) Biochemical assay
(serum creatine kinase)
 Serum creatine kinase (CK) as a biochemical marker

- Patients have ↑CK levels

10 to 50 fold increase

- Female carriers elevated levels

- Used for carrier screening

Bunch et al 2015
- Limitations:
• CK catalyzes the reversible ** reference values?
phosphorylation of creatine to ** fluctuating levels?
phosphocreatine and of ADP to ATP, to ** also ↑ in muscle injury
generate cellular ATP
or other diseases (other MDs
• Muscle ↑ level of CK
• Cell damage → CK leaks into serum
myocardial infarction, etc.)
(3) Muscle Biopsy Analysis
Staining for dystrophin protein

Normal Patient
 (4) Molecular diagnosis

What are the two types of common mutations in this disease?

A. Screening for gross deletions
or duplications
The molecular detection method needs to take into
consideration the mutation profile of the disease

Types of common mutations in DMD/BMD

DMD BMD
60-70% gross deletions 80% gross deletions
10% duplications 5% duplications
15-30% point mutations 10-15% small point mutations

Deletion/duplication – dosage analysis

Point mutations – sequence analysis
(nonsense, splice site mutations, missense, indels, etc.)

GENE
Deletions and Duplications in DMD gene
Involve whole or multi-exons
Clustered around two hot spot regions (5’ and 3’)

Lupez-Hernandez et al (2015)
Int J Med Sci 16: 5334-5346
 Multiplex PCR

• Amplifying and analyzing of > 1 exon at a time

• Single reaction/tube (multiple exons amplified)
• Target hot-spots (commonly mutated exons)
 DNA analysis : Detection deletions
(Quick & easy!)

exons Control Patient Patient Control exons

19 48
3 44
8 51
43
45
50
53
13 47
1
42
6 60
4 52
5’ 3’

Multiplex PCR for common hot-spots

Limitation: Deletion Screening using hot-spot
regions may not offer diagnosis for ALL patients!
87.5%

75.0%

63.0% 65.0%
60.2%
51.2% 50.0%

40.0%
32.4%

Singapore Japan Vietnam Thailand Brazil Cyprus N America Canada UK &

Europe
Comparative Dystrophin Gene Deletion
Frequencies in Populations – may be differences Lai et al, 2002
among different populations J Hum Genet 47: 552-555
From multiplex PCR for selected hot-spot exons to analysis to cover all exons

LOG-PCR analysis for all 79 exons

DMD gene – 79 exons
Known mutations - common
4 multiplex rxns (cover 57.5 kb)
All exons (except 78 & 79)
Evenly spaced products (211 bp – 1,742 bp)

Primer design – constant spacing

log Xn = log X (n-1) + 0.047 (X=amplicon size)
28-32 mers
GC-rich 3’ ends (13 C or Gs with at least 3 C/G end)

Multiplex PCR chemistry –

polyols & co-solvents, trehalose, etc.
Cycling conditions -

High resolution electrophoresis required

Limitation: Technical expertise to optimize assay

Trimarco et al (2008)
 MLPA (multiplex ligation probe amplication) analysis
Lali et al 2005; Schwartz et al 2005; Todorova et al 2008
Currently most common method for molecular diagnosis
• Dosage-based analysis for ALL exons
• Can detect both deletions and duplications
• Can detect carriers
• Interpretation: absence of peaks, half or double peaks areas

• Convenient kit-based assays

• Commercially available:
MLPA Holland
 MLPA is based on dosage analysis

Detecting deletions

Detecting carriers
B. Screening for point mutations
DNA Diagnosis: detecting point mutations
1. Pre-screening to narrow location (exon)
and/or
2. Direct sequencing
Remember:
DMD is largest gene in human genome
Spans 2.3 Mb with 79 exons

• All 79 exons : Screen first by methods like heteroduplex analysis, SSCP

(Single-strand Conformation Polymorphism) analysis, DHPLC (Denaturing
High-performance Liquid Chromatography) analysis, High Resolution Melt
(HRM) Analysis, etc.
• Cheap, quick, high throughput and easy to interpret
• Then, identified abnormal fragment is sequenced
High resolution melt (HRM) analysis
Quick analysis of all 79 exons of DMD through artificial heterozygote screening
Analysis of individual exons performed in quick and cheap screening
Differentiate between wild type (normal) from mutant (homozygote) and carriers (heterozygote)

• Melting of dsDNA to ssDNA

• Captured “live”
• Real-time high resolution
 DNA sequencing (precise direct diagnosis)
Amino Acid Type of
Exon Nucleotide change
change mutation

4 c.196A>T p.Lys66X Nonsense

8 c.653T>G p.Val218Gly Missense

8 c.831G>T p.Gln277His Missense

c.IVS5-1G>A (c.358-
6 1G>A) Splicing

7 c.565C>T p.Gln189X Nonsense

Identification of 8 c.745C>T p.Gln249X Nonsense

nucleotide changes 10 c.976G>T p.Glu326X Nonsense

68 c.9955T>C p.Cys3319Arg Missense

Blood : DNA
Muscle biopsy : mRNA/CDNA
• Sanger Sequencing
• Nex-gen Sequencing
Spectrum of mutations in DMD/BMD patients

Types of mutations among

patients with identified mutations

Distribution among type

of point mutations
4. CARRIER SCREENING

& PRENATAL DIAGNOSIS

PRENATAL DIAGNOSIS FOR DMD

• Mutation already identified

in patient (son)
• Prenatal sample from
mother
• Chorionic villi or amniotic
cells sampling
• Molecular analysis carried
out on extracted DNA
 What is a Pedigree Diagram in Genetics?
A pedigree is a chart diagram of a family tree showing genetic relationships of family members. The diagram
reflects inheritance of a trait or disease though several generations. Such diagrams are used by clinical
geneticists and genetic counsellors to aid in explaining information of disease risk for various genetic conditions.

Standard symbols and lines are used to represent the family members and their relationships.

Proband = index case

https://teaching.ncl.ac.uk/bms/wiki/index.php/Pedigree
Case 1
 CASE STUDY 1

Father Mother

Fetus
Patient ?
11 weeks
Is fetus affected?
Gene deletion identified
(Gene promoter, Pm to exon 8)
F : Father Promoter
Patient has gene
M : Mother deletions from
MK + - F M S P CV Promoter to exon 8
S : Sister
P : Patient
CV : prenatal DNA

Exon 3 Exon 4
MK : marker
+ : control + - F M S P CV M +- F M S P CV
- : control

Exon 6 Exon 8
+ - F M S P CV M +- F M S P CV

Prenatal Results : ? Is the fetus normal or affected?

Case 2
CASE STUDY 2

12 weeks
?

Is fetus affected?
Deletion of two exons
(exons 48-49)
Prenatal diagnosis by MLPA analysis of all 79 coding exons

Control

Look at exons 48-49

Case 2
(fetus DNA)

Is fetus affected?
 CARRIER SCREENING

• Not all females in affected family are carriers

• Can be detected via molecular (DNA) tests
• Can be direct or indirect
Direct : for known mutation
Indirect : unidentified mutation
• Carry heterozygote mutation (on one X-chromosome)
or detect the affected haplotype/chromosome using
indirect markers linked to the X-chromsome
 LINKAGE ANALYSIS
• Linkage analysis is useful for unidentified
2 5 Locus A (2, 5)
causative mutation
• Can detect carriers or affected (proband or fetus)
• Uses probes linked to mutation or disease gene 9 16 Locus B (9, 16)
• Probes detect genotype at each locus or
haplotype (for each chromosome).
Genotype: 2,5 or 9,16
Haplotype: 2,9 and 5,16

Locus A For X-linked diseases, males

have one chromosome, so
only one haplotype observed

http://www.biology-pages.info/R/RFLPs.html
Identifying carriers : 3 Case Studies
Case 1
 Genetic implications (Counselling)
• Patient (75)
• Mother (77) is definite carrier
• Younger brother (78) of patient is also affected and he
presented with symptoms later at 6 years
• Future prenatal diagnosis possible Questions:
• Female carriers can be identified (future siblings) • Why is mother a definite carrier?
Dys I 4 3 4 Dys I • Why does father only have one
Dys III 4 2 4 Dys III
87-1/B
87-8/T
2
2
1
1
2
1
87-1/B
87-8/T
chromosome/haplotype?
87-15/B
87-15/X
1
1
2
1
1
1
87-15/B
87-15/X • Which is the affected X-
I24/P 2 2 1 I24/P
I38/T
Str 44
1
8
1
13
2
8
I38/T
Str 44
chromosome (haplotype)?
Str 45
E48/M
8
2 76 77
7
2
11
1
Str 45
E48/M • Is brother (78) affected?
Str 49 2 3 1 Str 49
Str 50
J66
1
2 CK = 412
3
3
5
2
Str 50
J66
• Why is brother (78) not shaded?
Bstr H1 4 CK = 365 (10/2/95) 4 4 Bstr H1
BStr62 2 2 2 BStr62
3 Dys I
Dys I 3
2 Dys III
Dys III 2
1 87-1/B
87-1/B 1
1 87-8/T
87-8/T 1
2 87-15/B
87-15/B 2
1 87-15/X
87-15/X 1
I24/P
I38/T
2
1
75 78
2
1
I24/P
I38/T
13 Str 44
Str 44
Str 45
13
7
7 Str 45 one X chromosome
2 E48/M
E48/M 2
CK = 30,654 U/L 3 Str 49
Str 49 3
Str 50 3 CK = 6,220 (10/2/95)
Patient
3
3
Str 50
J66
two X chromosomes
J66 3
4 Bstr H1
Bstr H1 4
2 BStr62
BStr62 2
Case 2
 Questions:
GENETIC RECOMBINATION 5
4
4
4
DYSI
DYSIII
• Which is the affected
2 2 87-1/BstNI

chromosome?
2 1 87-8/TaqI
2 1 87-15/BamHI
2 1 87-15/XmnI
2
2
1
2
I24/PstI
I38/TaqI
• Why is III.4 not an
I 2
1 1
5
1
3
E48/MseI
STR50
affected male?
CK=128U/L 2 2 J66
• How many definite
4 4 4 4 5 4 5 4 5 4 4 4 female carriers in the
4 4 4
family?
4 4 4 4 4 4 4 4 4
2 1 2 1 2 1 2 1 2 1 2 2
1 2 1 2 2 2 2 2 2 2 1 2
II 2
2
1 2 2
2
1
1
3 4 5 2
2
2
2
6 2
2
2
2
7 2
2
2
2
2
2
1
1
8 9 2
2
• Are II.8 and III.6
1
2
2
2
1
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
1
2
carriers?
2 2 1 2 1 2 1 2 1 2 1 2
3 3 3 3 5 3 5 3 5 3 5 1
1 2 2 2 2 2 2 2 2 2 2 2

4 4 4 5 4 5 4 4 4
III 4 1 2 4 2 4 3 4 4 5 4 4 6 4 4
2 2 2 2 1 2 2 2 2
1 1 2 2 2 2 1 1 2
1 1 2 2 2 2 1 1 2
1 1 1 2 2 2 1 1 2
1 1 1 2 2 2 1 2 1
2 2 2 2 2 2 2 2 2
1 1 2 1 2 1 1 1 2
3 3 2 5 3 5 3 5 1
2 2 2 2 2 2 2 2 2

In this pedigree, II.2 was shown to be a carrier by linkage analysis while carrier
risk was excluded in II.5, II.6, II.7, III.3, III.5. Intragenic recombination was
found in II.8 between markers 87-15/XmnI and I24/PstI. Thus, her carrier risk
status and that for her daughter’s (III.6) cannot be clarified (ie. unknown).
Case 3
 Linkage analysis using X-markers
Haplotypes (maternal and paternal)

DYSI 1 3 4
DYSIII 2 2 4
87-1/BstNI 2 2 2
87-8/TaqI 2 1 1
87-15/BamHI 1 1 2

I 87-15/XmnI
I24/PstI
1
1
1
1
2
1
I38/TaqI 2 1 2
E48/MseI 2 2 2

1
3 1
2
STR50 3
J66 1 3 1

Which is the affected X-chromosome (haplotype)?

Are the females carriers?

4 4 3 4 4
4 4 2 4 4
2 2 2 2 2
1 2 1 2 1

II 2
1
1
1
1
1
1
1
1
1
1
1
2
1
1
2 2 1 2 2
2 2 2 2 2
1
1 I.1 I.2 3
2
3
3 I.3 3
2
1
1
5. THERAPY
Current Clinical Management For DMD
• There is no “cure" for DMD
Current clinical management of DMD
supportive, providing temporary relief of signs and
symptoms & management of pulmonary, cardiac, psychosocial issues, etc.
Multidisciplinary care
 Physiotherapists, orthopaedics, respiratory physicians,
cardiologists, medical social workers/psychologists, neurologists
 Aim at restoring muscle strength & motor function
 Steroid treatment – start between 4 to 6 years old THERAPY?
 Exercise (physiotherapy)
 Posture & pain management (non-ambulatory stage)
 Supportive treament for scoliosis & weak bones (calcium & Vit D
supplementation, bisphosphonates, etc.
 Pumonary and cardiology surveillance
 Alternative supplements: carnitine, Coenzyme Q10, creatine, etc.
Genetic counselling, carrier screening, prenatal & preimplantation diagnosis
 APPROACHES FOR THERAPY

Pharmacological Cell-based Molecular therapy

- Growth factors - Myoblast - Utrophin upregulation
(IGF-1, myostatin, transfer
- Gene delivery (DNA)
TNF-, etc.) - Satellite cells (transgene, mingene, etc)
- Metabolites
- Progenitors - Gene correction (RNA)
(creatine, taurine, Stem cells: (AONs (antisense) mediated-
resveratrol,
CD133+ splicing, translational
coenzyme Q10)
- Hematopoietic read-through)
- Clinically used drugs
Muscle-derived - Genome editing (DNA)
(Sodium
Pericyte (zinc finger nucleases,
4-phenylbutyrate,
Mesoangioblast meganucleases, CRISPR/Cas9)
Simvastatin)
Antisense Therapy
Antisense Therapy (causes exon skipping)
• Exon 50 is a (single) deletion hotspot and causes DMD
• Induction of Exon 51 skipping leading to deletion of two exons
• Deletion of both Exons 50 and 51 can restore reading frame
• This converts severe DMD to milder BMD

DNA

mRNA
Patient DMD (del exon 50)

AON Antisense oligonucleotide:

Interferes with splicing of exons
causing an exon to be skipped
DNA

mRNA
Patient treated with AO BMD (del exons 50-51)
 • 100 mg/2 mL (50 mg/mL) in single-dose vial
• 500 mg/10 mL (50 mg/mL) in single-dose vial

o FDA approval in 2016

o Applicable to 13% of patients
o Targets skipping of exon 51
o Actual cost ~ $750,000 to $1.5m/year
o 30 mg/kg/week infusion
o Sales of $300 million (2018)
Gene Editing Therapy
GENE EDITING : CRISPR/Cas9 targeting in DMD
delete/insert/replace sequences Double stranded breaks (DSBs) in DNA
repaired endogenously by either
homology-directed repair (HDR) or
nonhomologous end-joining (NHEJ)

Four platfoms for Targeted repair DSBs:

• zinc finger nucleases (ZFNs)
transcription activator-like effector
• (TALE)-nucleases (TALENs)
• meganucleases
• CRISPR/Cas system

CRISPR - Clustered regularly

interspaced short palindromic
repeats
Cas9 – CRISPR associated
nuclease

Ousterout et al 2015 (Nature Comm. 6:6244)

 In Vitro studies
2015

- iPSCs derived from patient (del exon 44)

Hongmei Lisa Li, Naoko Fujimoto, Noriko Sasakawa, Saya Shirai, Tokiko
Ohkame, Tetsushi Sakuma, Michihiro Tanaka, Naoki Amano, Akira Watanabe,
Hidetoshi Sakurai, Takashi Yamamoto, Shinya Yamanaka, Akitsu Hotta
Precise Correction of the Dystrophin Gene in Duchenne Muscular Dystrophy
Patient Induced Pluripotent Stem Cells by TALEN and CRISPR-Cas9
- Three correction methods tested
- Knock-in method was most effective
- Corrected iPSCs differentiated back to muscle cells
- Immunofluoresence staining studies
- Minimal off-target effects
- TALEN and CRISPR equally effective

Stem Cell Reports Volume 4, Issue 1, 2015, 143–154

http://dx.doi.org/10.1016/j.stemcr.2014.10.013
In Vivo studies
2016 Long et al 2016, Science 6271: 400-403.

Restoration of 4-15%
- mdx mice (exon 23)
- AAV-9 to deliver
CRSIP/Cas9 components
- Permanent change
- Blood-brain barrier?
- Off-target sites? Why is CRISP/Cas9
approach subject to such effects?
in Vitro and in vivo studies
2017 First use of alternative
Cas9 endocuclease
called Cpf1 from
Prevotella & Francisella 1
Generates sticky 5’ overhangs
Able to target AT rich-regions
Sci. Adv. 2017; 3: e1602814
12 April 2017
CRISPR-Cpf1 correction of muscular dystrophy mutations
in human cardiomyocytes and mice (Zhang et al 2017).
- Streptococcus pyogenes cas9 (SpCas9) most widely used Cas9
endonuclease
- Limitations : G-rich PAM, thus cannot do genome editing of AT-rich
regions
- Large size is disadvantage for packaging & delivery for AAV vectors
- Staphylococcus aureus cas9 (SpCas9) smaller but PAM sequence is
longer & complex
- Thus, a smaller CRISPR enzyme will enable higher cutting efficiency
- New RNA-guide endonuclease called Cpf1
- Prefers T-rich PAM at 5’ end of spacer
- Splice acceptor site is T-rich and offers ideal PAM sequence for
genome editing by Cpf1
DMD iPSC-derived cardiomyocytes express dystrophin
(red colour) after Cpf1-mediated genome editing by reframing
 SUMMARY
1. DMD is an incurable disorder
2. Diagnosis is important for clinical management
3. Symptoms may resemble other types of muscle disorders
4. Supportive care for patients and therapeutics to alleviate symptoms
5. Prenatal diagnosis options, carrier identification
6. Challenges in interpreting DNA results
7. Holistic view : Disease has impact on family, diagnosis may have social implications
8. Multi-disciplinary understanding (physiology, biology, anatomy, genetics, etc.) provide
better tools to manage, diagnose & treat disease
9. Critical thinking in applying appropriate techniques from bench for clinical applications

Providing an understanding of
human disease from a bench to
bedside perspective
USEFUL GENERAL REFERENCES FOR YOUR OWN INTEREST
About the Disease
• Chamberlain (1991). Curr Opin Genet Dev 1:11-14
• Muntoni (2003). Lancet Neurol. 2:731-40
About Molecular Diagnosis
• Luce et al (2014). Muscle Nerve 49: 249-256
• Laing et al (2011). Clin Biochem Rev 32:129-34
About Clinical Management
• Manzur et al (2008). Arch Dis Child 2008;93: 986-990
• Bushby et al (2010). Lancet Neurol. 9(1):77-93
About Therapy
• Benedetti et al (2013). FEBS J. 280, 4263–4280
• Mah et al (2016). Neuropsychiatric Disease & Treatment 12: 1795-1807
• Aartsma-Rus & Krieg (2017). Nucleic Acids Therapeutics 27: 1-3
• Li et al (2015). Stem Cells Report 4: 143-154
• Ousterout et al (2015). Nature Comm. 6:6244
• Long et al (2016). Science 6271: 400-403
• Zhang et al (2020). Science Advances 6: 6812
• Mollanoori et al (2020). Genes & Diseases. https://doi.org/10.1016/j.gendis.2019.12.007
Overview on DMD in Singapore
• Tomar et al (2019). Am J Med Genetics (C)181: 230-244