Muscle without a Matrix: A Biological Love Story Gone Wrong

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Transcript Muscle without a Matrix: A Biological Love Story Gone Wrong

Muscle without a Matrix:

A Biological Love Story Gone Wrong

Corey Cannon, MS3 Russell Romano-Kelly, MS3 Corbin Shawn, MS3 Presentation given by 3rd year medical students at Pediatric Neurology Grand Rounds, Valentines Day (2/14/2014)

Chief Complaint:

Increased laxity and muscle weakness


5 year old former term baby who has been followed at Shriner’s Neuromuscular clinic for increased laxity and muscle weakness.

Initial visit in November 2011 (age 3) for muscle weakness. • Parents report hypotonia since birth, but no subsequent feeding, no swallowing difficulties and never requiring a ventilator.

• Hypotonia persistently manifested as difficulty getting up from the floor, unsteady with frequent falls and weakness.

HPI - Follow up visit May 2012

• Saw genetics for significant joint laxity and concern for Ehlers Danlos Syndrome, which genetics did not feel was significant. No testing was sent. • Family concern about upper extremities weakness due to difficulty with using steering wheel on toy car. • Muscle biopsy planned Example of great motor activity

PMH  Developmental Hx • • • – Sat 7 months, didn’t walk until 18 months, frequent falls. No regression and has been improving with time. Normal cognitive and language development.

  Medical Hx - Congenital hypotonia. Delayed motor milestones. Surgical Hx - Muscle Biopsy 4/17/2013  Meds: None  Family Hx – Younger brother healthy, but older sibling born at 7.5 mo G.A who died at 8 days of life likely from respiratory issues. Negative for any similar problems. No consanguinity.  Social Hx – Parents are from Mexico.

Physical Exam

Vitals: Height: 113cm (80%) , Weight: 23kg (90%) , HOC: 53cm (~75%) General: Awake, alert, oriented. Has prominent forehead . No dysmorphic features.

CV: RRR, no murmurs Resp: Breathing comfortably on room air.

Abdomen: no hepatosplenomegaly Derm: small erythematous papules over upper arms, triceps area, and mildly on forearms. No neurocutaneous stigmata.

Neurological Exam

• Mental Status: pleasant and interactive, follows commands Language: normal speech and cognition. Cranial nerves: intact Sensation: intact to light touch. Motor: Tone: significant hypotonia throughout , + axillary slippage and joint laxity, especially with flexion at • the wrist , + hyperextensible finger extension and at knees. + mild contractures at bilateral elbows.

Power: diffuse muscle weakness 4/5 throughout, but neck flexor 2/5. + significant head lag when pulled from the lying position.

Reflexes: DTRs 1+ throughout . No clonus or Babinski. Gait/Station: + hyperlordotic and + waddling gait . Other: Mild scapular winging. + Gowers maneuver.

Differential Diagnosis

 Limb-girdle Muscular Dystrophy  Ehlers- Danlos Syndrome  Emery-Dreifuss Muscular Dystrophy  Central Core disease and Fiber type Disproportion  Collagen VI Congenital Myopathies

Work - Up

Labs (11/2011): Aldolase mildly elevated. ALT/AST normal. Total CK normal. EMG/NCS (3/2012): normal. Muscle biopsy (4/2013): evidence of muscular dystrophy with multiple lobulated fibers.

SMN1 gene (4/2013): normal.

Follow up visit 6/14/2013

Over last few months, he seems a little stronger and his falls are less frequent. He still had significant laxity and muscle weakness.

Molecular tests for collagen 6 mutations were performed.

Overall, we think this is…

Collagen 6 Muscular Dystrophy!


 Most abundant protein in the human body  Main component of connective tissue in humans  tendons, ligaments and skin  Produced by fibroblast cells  Basic structural unit is the triple helix  At least 16 different subtypes of collagen, 80-90% in humans is type I, II, and III




Major Collagen Molecules

Representative tissues

Skin, tendon, bone, ligaments, dentin, interstitial tissues Cartilage, vitreous humor Skin, muscle, blood vessels All basal laminaes Skin, tendon, bone, ligaments, dentin, interstitial tissues, fetal tissues Most interstitial tissues Cartilage, vitreous humor;

Commonly Associated Diseases

Osteogenesis Imperfecta, Ehlers- Danlos Syndrome Ehlers – Danlos Syndrome Alport Syndrome Ehlers – Danlos Syndrome Collagen VI Myopathies

Discoverers of the Collagen VI Myopathies

 Ullrich Congenital Muscular Dystrophy  Named after Otto Ullrich (1894-1957), German pediatrician and published first paper about the disorder in 1930 paper in the German literature  Bethlem Myopathy  Named after Jaap Bethlem (1924-) who first described Bethlem myopathy in paper coauthored by George van Wijngaarden published by


journal in 1976


A Spectrum of Disease

Severe Ullrich CMD Typical Ullrich CMD Intermediate Collagen VI Myopathy Bethlem Myopathy


Presentation of UCMD

   may initially show reduced fetal movement     Hypotonia Weakness Hyperlaxity of distal joints Joint contractures of elbows, knees, spine, neck Clubfoot (rare) Dysphagia with transient feeding difficulties

Presentation of UCMD (continued)

 Propensity for abnormal (atrophic, keloid) scars  Prominent keratosis pilaris of extensor surfaces  In severe cases may not gain the ability to walk, but majority walk by 2 years of age  Loss of ability usually by adolescence  Eventual respiratory insufficiency  Cranial and heart musculature is preserved

Presentation of Bethlem Myopathy

 Similar symptoms to UCMD but milder with wide variability  May first be diagnosed in adulthood but signs may be present in infancy   Hypotonia, torticollis, foot deformities Congenital contractures usually resolve by age 2  Patients rarely fully symptomatic before 5 years of age  May have weakness in proximal distribution without contractions or prominent contractures without weakness

Early Symptoms of Bethlem Myopathy

Presentation of Bethlem Myopathy (continued)

 Typical contractures of the Achilles tendon and elbows around the beginning of adolescence  Progress to affect long finger flexors, shoulders and spine  Bethlem Sign  Eventual walking difficulties  Increased risk of restrictive lung disease and subsequent respiratory insufficiency


A Spectrum of Disease


Natural History

    Ullrich Congenital Muscular Dystrophy    Hyperlaxity, hypertonia, joint contractures may be present at birth mean onset of disease by 12 months Muscle weakness is progressive Disability aggravated by significant contractures in large joints Loss of ability to walk usually by early teenage years Respiratory insufficiency usually occurs before loss of ability to walk and manifests first as nocturnal hypoxemia  Deterioration imminent, but not necessarily associated with age or severity at onset  Bethlem Myopathy   Joint contractures may be present at birth but may resolve by age 2 Patients experience progressive deterioration and eventual loss of ability to ambulate in 4 th or 5 th decade of life  Significant decrease in muscle strength reported also around 4 th or 5 th decade of life


 Detection of mutations by microarray and sequencing in collagen VI gene  Disease caused by mutation in α-chain peptides α1 (encoded by COL6A1), α2 (COL6A2) or α3 (COL6A3)  Diagnosis typically depends on clinical features  Muscle biopsy may be useful adjunct showing myopathic or dystrophic changes with collagen VI immunolabelling normal in BM but moderately to severely reduced in UCMD  Prenatal diagnosis only considered for UCMD (not BM) in rare case studies



models knock-out mouse  Exhibit little weakness with mild neuromuscular disorder  Increased apoptosis of myocytes  Prevented with cyclosporin to inactivate cyclophilin D (CyD), resulting in improvement of muscular function  Impairment of mitochondrial autophagy


 Cell anchorage is an important factor in the prevention of apoptosis  Collagen VI-deficient cell cultures show decreased adhesion to extracellular matrix




Collagen VI-related myopathy

b Figure 2 | Immunohistochemical identification of collagen  VI in the muscle. Images Laminin γ-1 (green) showing dual immunohistochemical labeling for collagen  VI (red) and the basement marker laminin subunit γ-1 (green). a | Note the colocalization of collagen VI and laminin γ-1 in the basement membrane in a healthy individual, which results in a yellow color. b | In a patient with collagen  VI-related myopathy, a gap is evident between the collagen  VI staining and the basement membrane staining. This patient has a dominant-negative mutation, so that altered collagen  VI can be excreted into the matrix but is then not able to function or interact properly.

pericellular distribution around cells without basement membranes, such as tendon fibroblasts.

41 Three major collagen  VI genes have been identified —








lie in a head-to-head arrangement on chromo some 21, whereas


is on chromosome 2.

42 Three additional genes have been identified by comparative analysis as being related to


; 43,44 in mice all three genes,






, are expressed, whereas in humans


is interrupted by a translocation and is no longer fully functional. The tissue distribution of the collagen α5(VI) chain and collagen α6(VI) chain proteins is more limited than that of the original three chains.

45 The peptides encoded by




consist of short α-chain domains flanked by N-terminal and C-terminal globular domains of about equal size.

46 The product of


contains a similar-sized α-chain domain; however, its N-terminal globular domain is much larger compared with




, and is alternatively spliced. The C-terminal domain of the


transcript is proteolytically processed once secreted into the extracellular matrix as part of the assembled collagen  VI (Figure 4).

47 Collagen VI undergoes an extensive assembly process in the cell before being secreted into the extra cellular matrix.

42,48 A basic understandi ng of this assembly process is important to comprehend the consequences of the various disease-causing mutations. The assembly begins with the formation of the basic monomer, which is composed of one of each of the three α-chain subunits encoded by






. Similarly to other collagens, the three α-chains first associate at their C1-terminal globular domains and come to lie adjacent to each other. Hydrogen bonding then links the three α-chains in a zipper-like fashion from the C-terminal to the N-terminal end, resulting in a triple-helical structure (Figure  4).

The next assembly step involves the formation of an antiparallel dimer, stabilized by disulfide bridging between crucial cysteines in the N-terminal ends of the triple-helical domain and in the globular domain.

49–51 Two such dimers then associate in a staggered parallel orientation to form a tetramer, which is again stabilized by disulfide bridging between cysteine residues in the triple-helix regions.

49,50,52 These tetramers are secreted into the extracellular space, where they associate in an end-to-end fashion to form characteristic ‘beads on a string ’ collagen VI microfibrillar structures, which have a periodicity of 100 –105 nm and a diameter of 4.5 nm (Figure  4).


Physiological roles of collagen  VI

In the extracellular matrix, collagen  VI interacts with a large number of matrix molecules (Box  3). The identity of the molecular partner (or partners) mediating the interaction of collagen  VI in the muscle basement mem brane is not yet known. One possible candidate would be collagen type  IV, 40 the most important collagenous component of basement membranes. Since collagen  VI binds to biglycan, 55 which interacts with the sarcoglycan and dystroglycan complex, 56,57 collagen  VI might be indi rectly linked to muscle cell surface receptors via biglycan and the dystrophin-associated protein complex.

Possible functions of collagen  VI pertaining to various cell types have been suggested. These functions include the promotion of adhesion, 58–60 proliferation, 61 migra tion 62,63 and survival.

64,65 Collagen VI also has crucial roles in the regulation and differentiation of adipo cytes and of normal and malignant mammary ductal cells.

63,64,66 Judging by the clinical features seen in patients with collagen  VI-related myopathies, the tissues where collagen  VI has the most important roles include muscle, tendon and skin, although abnormalities in collagen  VI might also have important, albeit more-subtle, effects in other tissues. In tendons, collagen  VI is expressed by the resident tendon fibroblasts, around which it assumes a close pericellular distribution.

41 In muscle, the cell of origin for collagen  VI is the interstitial fibroblast.

67,68 The collagen  VI-related myopathies are quite unique, there fore, in that they are non-cell-autonomous disorders of  muscle.

A mouse model of collagen  VI deficiency has been generated by knockout of the collagen  VI


locus, resulting in a complete absence of collagen  VI expres sion in muscle.

69 In contrast to the human disease, these mice only show a mild neuromuscular disorder without much overt weakness.

69 Muscles from these animals also show an increased incidence of apoptosis, which corre lates with facilitated breakdown of the potential across the mitochondrial permeability pore when primed with oligomycin.

65 This effect was preventable by inactiva tion of cyclophilin D using ciclosporin, its derivative Debio0025, 70–72 or genetic inactivation of cyclophilin D.

73 These interventions also resulted in decreased apopto sis and improved physiological functioning of isolated muscles from the collagen  VI-knockout animals.

65,72 Subsequent work using the same model suggests that impairments in autophagocytic flux (including impaired mitochondrial autophagy) also occur in the muscle cells of collagen  VI-deficient mice, and possibly exacerbate the accumulation of defective mitochondria.

74 384 | JULY 2011 | VOLUME 7 nrneurol © 2011 Macmillan Publishers Limited. All rights reserved


 Ullrich CMD  Classically AR, though AD patterns of inheritance exist (usually

de novo

mutations)  AR forms result in complete absence of collagen VI in the extracellular matrix due to nonsense mutations, splice-site mutations, and intragenic deletions  AD/sporadic forms result from in-frame skipping of exons in the N terminus of the α-chain domains


 Bethlem CMD  AD predominate, but AR exist  Exon-14 skipping mutations of C terminus of α-1 chain most common  Result in disrupted formation of the monomers from the three peptide subunits, thus decrease tetramer formation  25% of patients have no known mutation in the COL6 genes

Treatment and Management

 Prior to the introduction of respiratory management, collagen VI myopathies were typically survivable to the teens  Sleep studies often needed for nocturnal hypoxemia  Can be managed for years with noninvasive bilevel positive airway pressure ventilation  Scoliosis can be managed with a trunk orthosis, such as a Garchois brace  Regular stretching, standing, splinting, and serial casting for contractures

Future directions

 Most promising target is to halt apoptosis in myocytes  Inhibition of cyclophilin D with ciclosporin or DEBIO-025 (alisporivir)  Small study of 5 patients showed stabilized mitochondrial function and decreased apoptotic nuclei via biopsy after 4 weeks of therapy with ciclosporin, though no strength testing was performed  More research is required to elucidate exact mechanism responsible for myocytes becoming susceptible to apoptosis when the extracellular matrix is deficient of collagen VI

Case Update

 Most recent visit 1/10/2014 - Still not able to stand alone, has to hold on to objects/handles in order to pull himself up from chair. Recently began using braces. Denies trouble swallowing or chewing or respiratory distress.

 Results for Collagen 6 testing done on 11/27/2013 showed mutation in the collagen 6A1 gene. Two heterozygous mutations were noted.   P.GLY 287GLU which was predicted to be pathogenic P.ALA112THR, which clinical relevance is not yet known.



Collagen: The Fibrous Proteins of the Matrix. Molecular Cell Biology. 4th edition. Lodish H, Berk A, Zipursky SL, et al. New York: W.H Freeman. 2000 2.

Bethlem J, Wijngaarden GK. Benign Myopathy, With Autosomal Dominant Inheritance. Brain. (1976) 99: 91-100. 3.


Lampe AK, Bushby KM. Collagen VI related muscle disorders. J Med Genet 2005.

Bönnemann CG. The collagen VI-related myopathies: muscle meets its matrix. Nat. Rev. Neurol. 7, 379 –390 (2011) 5.

Nagappa M, Atchayaram N, Narayanappa G. A large series of immunohistochemically confirmed cases of congenital muscular dystrophy seen over a period of one decade. Neurol India 2013;61:481-7 6.

Jobisis GJ, Boers JM, Barth PG, de Visser M. Bethlem myopathy: a slowly progressive congenital muscular dystrophy with contractures. Brain. (1999) 122 (4): 649-655.doi: 10.1093/brain/122.4.649

References (continued)


Nadeau, A. et al. Natural history of Ullrich congenital muscular dystrophy. Neurology 73, 25 –31 (2009). 8.

Wang, C. H.

et al.

Consensus statement on standard of care for congenital muscular dystrophies.

J. Child. Neurol.

25, 1559 –1581 (2010). 9.

Orrenius S, Zhivotovsky B, Nicotera P. Regulation of cell death: the calcium-apoptosis link. Nature Reviews Molecular Biology 2003 Jul, 4, 552-565.


Jaalouk DE, Lammerding J. Mechanotransduction done awry. Nat Rev Mol Cell Biol. 2009 Jan;10(1):63-73.