1. NGS panel - Genetic testing for muscular dystrophy

Muscular dystrophy

August 18, 2017

Clinical features

Muscular dystrophies are inherited neuromuscular diseases characterized by weakness and wasting due to muscle dysfunction. Age of onset, severity, progression and histopathological findings are variable between different subtypes of muscular dystrophies.

Muscular dystrophies include a heterogeneous group of neuromuscular disorders:

Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene located on the X chromosome which encodes protein dystrophin. DMD affects boys in early childhood, causing progression of muscle weakness and resulting gait problems, general motor delay, and speech/learning difficulties. DMD is the most severe form of muscular dystrophy and it is characterized as follows1:

  • Rapid progression of muscle weakness, often with calf hypertrophy
  • Decreased levels of serum CK concentration
  • Onset before age five years
  • Wheelchair dependency before age 13 years
  • Approximately one third of affected shows DMD-associated dilated cardiomyopathy in teenage years.

DMD has a prevalence from 15.9 to 19.5 per 100,000 live births2, 3. A milder form of disease occurs in very few female carriers, where mild form of muscle weakness develops in 2.5-20% of affected female carriers1.

Becker muscular dystrophy (BMD) is another, milder form of muscular dystrophy caused by mutations in DMD. BMD affects older boys and young men, and it occurs in about 1 in 30,000 male births1.

Becker muscular dystrophy is clinically characterized by the following:

  • Progressive symmetric muscle weakness and atrophy with calf hypertrophy
  • In some cases quadriceps femoris weakness is the only sign of disease
  • Activity-induced cramping in some patients
  • Flexion contractures of the elbows
  • Wheelchair dependency after age 16 years
  • Preservation of neck flexor muscle strength differentiates BMD from DMD
  • The most common cause of death in BMD-affected patients is dilated cardiomyopathy and heart failure.

Mutations in the DMD gene cause both DMD and BMD. Deletions of one or more exons of DMD account for approximately 60-70% of pathogenic variants in individuals with DMD and BMD 1, 4. Duplications that lead to in-frame transcripts account for mutations in 5-10% of males with DMD and BMD 4, 5. Single nucleotide variants (single-base changes, small deletions/insertions and splice site changes) account for approximately 25-35% of pathogenic variants in males with DMD and about 10-20% of males with BMD 4, 6.

Emery-Dreifuss muscular dystrophy (EDMD) is another X-linked form of muscular dystrophy that affects young boys, characterized by the following clinical features7:

  • Slowly progressive wasting and weakness, typically of the humero-peroneal/scapulo-peroneal muscles in the early stages
  • Early contractures of the elbow flexors, achilles tendons and neck extensors resulting in limitation of neck flexion, followed by limitation of extension of the spine
  • Cardiac disease with conduction defects and arrhythmias (atrial fibrillation, dilated or hypertrophic cardiomyopathy)
  • Serum CK levels are normal or mildly increased, and muscle histopathology shows nonspecific dystrophic changes.

EDMD is caused by mutations in one of the following genes: EMD (encoding emerin), FHL1 (encoding FHL1), and LMNA, encoding lamin A and C. Mutations in EMD are responsible for ~61%8 of XL-EDMD, mutations in FHL1 for ~10%8 of XL-EDMD, and mutations in LMNA account for ~45% of autosomal dominant EDMD9.

Limb-girdle muscular dystrophy (LGMD) is a large group of childhood to early adulthood-onset diseases, mostly characterized by non-syndromic involvement of skeletal muscles. LGMD affects both men and women and has an estimates of prevalence from 1/14,500 to 1/123,000 10. LGMD includes dystrophies caused by mutations in genes encoding sarcoglycans (SGCA, SGCB, SGCG, SGCD), calpain (CAPN3), dysferilin (DYSF), and other muscle-related genes 10.

Facioscapulohumeral muscular dystrophy (FSH), also known as Landouzy-Dejerine disease, is a late childhood to early adulthood-onset muscular dystrophy that affects both men and women, causing weakness in the muscles of the face, shoulders, and upper arms. FSH occurs in about 4-10/100,000 people11.

Approximately 95% of individuals with FSHD have “contraction” mutations of the D4Z4 macrosatellite locus (within subtelomeric region of chromosome 4q35) 11. The pathologic contraction of the D4Z4 repeat region is associated with an opening of the chromatin structure at the D4Z4 locus. The remaining 5% of FSHD patients do not have contraction of the D4Z4 locus, but were shown to have loss of CpG methylation at all D4Z4 repeat arrays on chromosomes 4 and 10, resulting in FSHD type 2 11.

Myotonic dystrophy type 1 (DM1, Steinert's disease) is an inherited form of muscular dystrophy that affects both men and women, characterized by myotonia and cataracts. Estimates of the prevalence of DM1 range from 1:100,000 in some areas of Japan to 1:10,000 in Iceland, with an overall estimated worldwide prevalence of 1:20,000 12, 13.

The disease has three major subtypes:

  • Mild DM1, characterized by cataract and mild myotonia and normal life span
  • Classic DM1, characterized by myotonia, cataracts, muscle weakness and cardiac conduction abnormalities, and shortened life span
  • Congenital DM1, severe form of disease, characterized by hypotonia and severe generalized weakness at birth, often with respiratory insufficiency and early death; intellectual disability is common.

DM1 should be suspected in newborns who present with one or any of the following:

  • Hypotonia
  • Facial muscle weakness
  • Generalized weakness
  • Positional malformations including club foot
  • Respiratory insufficiency.

Clinical symptoms and the most common signs of myotonic dystrophy type 1 in adult patients include the following:

  • Muscle weakness, starting in distal regions and progressing throughout the body and head
  • Myotonia, sustained muscle contraction, most commonly grip myotonia
  • Posterior subcapsular cataracts, detectable as red/green opacities on slit lamp examination.

DM1 is caused by expansion of a CTG trinucleotide repeat in the non-coding region of DMPK. It is considered that normal alleles have 5-34 CTG repeats, while fully penetrant alleles have more than 50 CTG repeats12.

Myotonic dystrophy type 2 (DM2) is another inherited muscular dystrophy characterized by myotonia (in~90% of affected DM2 patients) and muscle weakness (~82%), and in some patients by cardiac abnormalities, cataracts, insulin-insensitive type 2 diabetes mellitus, and testicular failure14. The prevalence of DM2 appears to differ in various populations, but a higher prevalence is observed in Germany, Poland and Finland 14, 15. Expansion of the CCTG repeat localized within intron 1 of the CNBP (ZNF9) gene causes DM2. The number of CCTG repeats in expanded alleles ranges from approximately 75 to more than 11,000, with a mean of approximately 5000 repeats14.

Oculopharyngeal muscular dystrophy (OPMD) is a rare inherited muscular dystrophy characterized by swallowing difficulties and ptosis. OPMD has an adult age of onset, affecting both males and females, causing weakness in the eye muscles and throat. The estimated prevalence of OPMD is 1/100,000 in France, 1/1000 in the French-Canadian population of Quebec, and 1/600 among Bukhara Jews in Israel15.

OPMD is caused by an expansion of a “GCN” trinucleotide repeat in the first exon of PABPN1 (N represent any of the 4 nucleotides). Normal alleles contain ten GCN trinucleotide repeats, while pathogenic alleles contain 11-17 repeats.

An overview of those genes most commonly associated with muscular dystrophies is listed in the table.


Overview of genes included in the Muscular dystrophy panel

Gene (OMIM)
Chr.locus
Frequency of mutations Clinical features Associated/allelic disorders
ANO5
(608662)
11p14.3
Few families with LGMD2L and MMD3 17 Late onset
Increased serum CK
Muscle weakness Exercise-induced myalgia
GDD (166260); MMD3 (613319); LGMD2L (611307)
CAPN3
(114240)
15q15.1
~40% of all LGMD16
84% for calpainopathy18
Early to adult onset
Proximal limb/trunk muscles atrophy
Scapular winging and scoliosis
Joint contractures
Elevated CK (5-80 times16
LGMD2A (253600)
CAV3
(601253)
3p25.3
>99% for caveolinopathies 19
Rare for HCK, RMD, LGMD, CHM 19
Progressive proximal muscle weakness
Calf hypertrophy
Myalgia, cramps, stiffness after exercise
Muscle hyperirritability
Elevated CK (up to 30 times)19
LGMDIC (607801); LQTS9 (611818); CMH1 (192600); HCK/CPK (123320); MPDT (614321); RMD (606072)
CLCN1
(118425)
7q34
>100% for Myotonia congenita 20 Early onset muscle stiffness
Alleviation of stiffness by brief exercise
Myotonic contractions
Myotonia congenita AD (160800), AR (255700)
COL6A1
(120220)
21q22.3
38% for BTHLM and UCMD 21 Bethlem myopathy:
  • Proximal muscle weakness
  • Variable contractures in finger flexors, elbows, and ankles
Ullrich congenital muscular dystrophy:
  • Congenital weakness and hypotonia
  • Proximal joint contractures
  • Striking hyperlaxity of distal joints
BTHLM1 (158810): UCMD1 (254090)
COL6A2
(120240)
21q22.3
44% for BTHLM and UCMD 21 BTHLM1 (158810): UCMD1 (254090)
COL6A3
(120250)
21q22.3
18% for BTHLM and UCMD 21 BTHLM1 (158810): UCMD1 (254090); DYT27 (616411)
DAG1
(128239)
3p21.31
Few families reported 22 Brain and eye malformations
Profound mental retardation
Congenital muscular dystrophy
LGMD2P (613818); MDDGA9 (616538)
DES
(125660)
2q35
7% for MFM 23
Few cases with LGMD 16
<1% for CMD 24
Mid-adulthood onset of myopathy
Progressive myopathy
Cardiomyopathy signs
LGMD2R (615325); CMD1I (604765); MFM1 (601419); SCPNK (181400)
DMD
(300377)
Xp21.2-p21.1
60%-70% of large deletions1
5%-10% of duplications4
25%-35% of SNVs 5,6
Duchenne muscular dystrophy:
  • Progressive muscle weakness
  • Onset before age 5 years
  • Wheelchair dependency > 13 years
Becker muscular dystrophy:
  • Progressive muscle weakness and atrophy
  • Weakness of quadriceps femoris
  • Activity-induced cramping
  • present, after age 16 years)
  • Wheelchair dependency after 16 years
DMD (310200); BMD (300376); CMD3B (302045)
DNAJB6
(611332)
7q36.3
Few families with LGMD25 Proximal/distal muscle weakness
Facial weakness with bulbar involvement
Respiratory involvement
LGMD1E (603511)
DYSF
(603009)
2p13.2
95% for MMD in Jewish 26
~5% for LGMD in Jewish 10
Childhood or early-adult onset
Calf muscles involvement
Weakness/atrophy of pelvic and shoulder girdle muscles)
LGMD2B (253601); MMD1 (254130); DMAT (606768)
EMD
(300384)
Xq28
~61% of X-linked-EDMD8 Early onset with joint contractures
Slowly progressive muscle weakness initially in a humero-peroneal muscles
EDMD1 (310300)
FHL1
(300163)
Xq26.3
~10% of X-linked EDMD8
FHL1 for for MFM 23
Adulthood onset
Proximal myopathy and atrophy of postural muscles, limited neck flexion, bent spine, respiratory problem, and cardiomyopathy
EDMD6 (300696); RBMX1A (300717); RBMX1B (300718); SPM (300695)
FKRP
(606596)
19q13.32
Several families with LGMD10 and MDDG1 Neonatal hypotonia
Elevated creatine kinase
Muscular dystrophy
Cardiac abnormalities
Brain/eye anomalies
LGMD2I (607155); MDDGA5 (613153); MDDGB5 (606612)
FKTN
(607440)
9q31.2
Several families with LGMD10 and MDDG1 Neonatal hypotonia
Elevated creatine kinase
Muscular dystrophy
Cardiac abnormalities
Brain/eye anomalies
LGMD2M (611588); MDDGB4 (613152); MDDGA4 (253800); CMD1X (611615)
FLNC
(102565)
7q32.1
3% for MFM 23 Slowly progressive weakness and atrophy
Stiffness, cramps
Dyspnea, and dysphagia
Cardiomyopathy is present in 15%-30% 23
MFM5 (609524); CMH26 (617047); MPD4 (614065)
GAA
(606800)
17q25.3
100% for GSD 27 Hypotonia, generalized muscle weakness
Hypertrophic cardiomyopathy
Feeding difficulties, failure to thrive
Respiratory distress, hearing loss
GSD2/Pompe disease (232300)
GMPPB
(615320)
3p21.31
Several families with MDDG1 Onset in infancy
Severe muscle weakness and mental retardation
Microcephaly, cardiac dysfunction, seizures, or cerebellar hypoplasia
MDDGA14 (615350); MDDGB14 (615351); MDDGC14 (615352)
GNE
(603824)
9p13.3
60%-80% of all GNE-related myopathy 28
100% for Middle Eastern Jewish population 28
Onset in late adolescence
Slowly progressive distal muscle weakness
Gait disturbance and foot drop
Nonaka myopathy (605820); Sialuria (269921)
HNRNPDL
(607137)
4q21.22
Few families with LGMD10 Onset is usually in adulthood
Slowly progressive proximal muscle weakness
Develop cataracts before age 50
LGMD1G (609115)
ISPD
(614631)
7p21.2
Several families with LGMD10 and MDDG1 “Walker-Warburg phenotype” (characteristic brain and eye malformations, profound mental retardation, congenital muscular dystrophy) LGMD7c (616052); MDDGA7 (614643)
LAMA2
(156225)
6q22.33
60-100% for LAMA2-CMD 28 Onset at birth
Profound hypotonia with muscle weakness
Contractures of the large joints
Respiratory failure
MDC1A (607855)
LIMS2
(607908)
2q14.3
Several families with LGMD10 Severe and progressive muscle weakness and atrophy in childhood, resulting in loss of independent ambulation. Patients may also develop dilated cardiomyopathy LGMD2W (616827)
LMNA
(150330)
1q22
6% of CDM 24
Several families with LGMD10
45% for EDMD 7
100% for HGPS 29
Onset by 3rd decade
Muscle weakness and cardiac involvement Characteristic facies (receding mandible, narrow nasal bridge and pointed nasal tip)
LGMD1B (159001); CMD (613205); EDMD2 (181350); EDMD3 (616516); CMT2B1 (605888); CDM1A (115200); HGPS (176670)
MYOT
(604103)
5q31.2
9% of MFM 23
Few families with LGMD10
Slowly progressive weakness
Sensory symptoms
Muscle stiffness, aching, or cramps
LGMD1A (159000); MFM3 (609200); Myopathy spheroid bodies (182920)
PLEC
(601282)
8q24.3
8% of EBS 31
6/16 for EGSOG 30
Few families with LGMD10
Early childhood onset
Progressive muscular weakness
Blistering skin changes
LGMD2Q (613723); EBSND (616487); EBSMD (226670); EBSPA (612138); EBSOG (131950)
POMGNT1
(606822)
1p34.1
Few families with LGMD10 and MDDG1 Early infantile onset
Characteristic brain and eye malformations
Profound mental retardation
Progressive muscle atrophy
LGMD3C (613157); MDDGA3 (253280); MDDGB3 (613151); RP76 (617123)
POMGNT2
(614828)
3p22.1
Few families MDDG1 Severe early onset hypotonia
Characteristic brain and eye malformations
Profound mental retardation
Cobblestone Lissencephaly
MDDGA8 (614830)
POMK
(615247)
8p11.21
Few families with LGMD10 Proximal muscular weakness
Delayed motor development
Cognitive impairment
LMGDC12 (616094); MDDGA12 (615249)
POMT1
(607423)
9q34.13
Common for CMD 28
Few families with LGMD 10
Characteristic brain and eye malformations
Profound mental retardation
Cerebellar and retinal malformations
LGMD2K (609308); MDDGB1 (613155); MDDGA1 (236670)
POMT2
(607439)
14q24.3
Few families with LGMD 10 Infantile onset muscular weakness
Less severe brain and eye malformations
Profound mental retardation
LGMD2C (613158); MDDGB2 (613156); MDDGA2 (613150)
SEPN1
(606210)
1p36.11
30%-54% of MDM 32 Hypotonia
Joint laxity
Congenital dislocation of the hip
CFTD (255310); RSMD1 (602771)
SGCA
(600119)
17q21.33
Up to 68% of individuals with childhood onset and ~10% with adult onset 33 Childhood onset (3-15 years)
Proximal muscle weakness
Myoclonic jerk in some patients
Walking difficulty, cramps, exercise intolerance
Calf muscle hypertrophy
Late onset scoliosis
Variable heart involvement
LGMD2D (608099)
SGCB
(600900)
4q12
LGMD2E (604286)
SGCD
(601411)
5q33.2-q33.3
LGMD2F (601287); CMD1L (606685)
SGCE
(604149)
7q21.3
DYT11 (159900)
SGCG
(608896)
13q12.12
LGMD2C (253700)
SMCHD1
(614982)
18p11.32
<5% of FMD 11 Onset before age of 20 years
Weakness in facial muscles, scapular stabilizer, and foot dorsiflexor muscles
FSHD2 (158901); BAMS (603457)
SYNE1
(608441)
6q25.2
Common in French-Canadian population34 Late-onset cerebellar ataxia
Dysarthria
Brisk lower-extremity tendon reflexes
SCA8 (610743); EDMD4 (612998)
SYNE2
(608442)
14q23.2
Rare Proximal upper limb weakness and winged scapulae
Increased serum CK
EDMD4 (612999)
TCAP
(604488)
17q12
1% for DCM 24
3% for LGMD 10
Onset in childhood
Difficulty with walking, running, and climbing
Cardiomyopathy
LGMD2G (601954); CMH25 (607487)
TNPO3
(610032)
7q32.1
Common in Spanish population 35
Several families with LGMD 10
Proximal muscle weakness of lower limbs
Variable onset and presentations
LGMD1F (608423)
TRAPPC11
(614138)
4q35.1
Several families with LGMD 10 Childhood-onset of proximal muscle weakness
Gait abnormalities and scapular winging
LGMD25 10
TRIM32
(602290)
9q33.1
Several families with LGMD 10 Infantile onset
Nonprogressive muscular weakness
Obesity, pigmentary retinopathy, polydactyly, hypogonadism, renal/cardiac abnormalities
LGMD2H (254110); BBS11 (615988)
TTN
(188840)
2q31.2
10%-20% for DCM 24
100% for TMD 36
Severe distal myopathy
Dilated or hypertrophic cardiomyopathy
Early respiratory failure
Tibial muscular dystrophy
LGMD2J (608807); CMD1G (604145); CMH9 (613765); EOMFC (611705); HMERF (603689); TMD (600334)
VCP
(601023)
9p13.3
Majority of IBMPFD 37 Adult-onset proximal and distal muscle weakness
Early-onset Paget disease of the bone
Premature frontotemporal dementia
ALS14 (613954); CMT2Y (616687); IBMPFD1 (167320)

Differential diagnosis

The differential diagnosis of muscular dystrophy-related disorders – depending on the major symptoms in the initial case – includes the following diseases:

  • Emery-Dreifuss Muscular Dystrophy
  • Metabolic Myopathies
  • Spinal Muscular Atrophy
  • Congenital Myopathies
  • Dystrophinopathies.

Testing strategy

CENTOGENE offers an advanced, fast and cost-effective strategy to test large NGS panels and diagnose complex phenotypes based on PCR-free whole genome sequencing and NGS technology. This approach offers an unparalleled advantage by reducing amplification/capture biases and providing sequencing of the entire gene with more uniform coverage.

To confirm/establish the diagnosis, CENTOGENE offers the following testing strategy for muscular dystrophy using NGS Panel Genomic targeted towards this specific phenotype:

Step 1: Whole genome sequencing from a single filter card. The sequencing covers the entire gene (coding region, exon/intron boundaries, intronic and promoter) for all the genes included in the Muscular dystrophy panel. Copy Number Variants analysis derived from NGS data is also included.

Step 2: If no mutation is identified after analysis of the Muscular dystrophy panel, we further recommend continuing the bioinformatics analysis of the data using whole genome sequencing to cover those genes which are either implicated in an overlapping phenotype or could be involved in a similar pathway but are not strongly clinically implicated based on the current information in literature.


Referral reasons

The following individuals are candidates for muscular dystrophy testing:

  • Individuals with a family history of muscular dystrophy and presentation of the most common symptoms
  • Individuals without a positive family history of muscular dystrophy, but with symptoms resembling the disease
  • Individuals with a negative but suspected family history of muscular dystrophy, in order to perform proper genetic counseling.

Test utility

Sequencing, deletion/duplication of the muscular dystrophy panel genes should be performed in all individuals suspected of having muscular dystrophy and suspected phenotypes. In parallel, other genes reported to be related with this clinical phenotype should also be analyzed for the presence of mutations, due to the overlap in many clinical features caused by those particular genes.

Confirmation of a clinical diagnosis through genetic testing can allow for genetic counseling and may direct medical management. Genetic counseling can provide a patient and/or family with the natural history of the muscular dystrophy and related disorders identify at-risk family members, provide disease risks as well as appropriate referral for patient support and/or resources.


More information on CENTOGENE´s Muscular dystrophy panel can be found in our genetic test catalogue.