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Etiology of limb girdle muscular dystrophy 1D1E determined by laser capture microdissection proteomics.

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Etiology of Limb Girdle
Muscular Dystrophy 1D/1E
Determined by Laser Capture
Microdissection Proteomics
Steven A. Greenberg, MD,1
Mohammad Salajegheh, MD,1
Daniel P. Judge, MD,2 Matthew W. Feldman, MS,2
Ralph W. Kuncl, PhD, MD,3 Zachary Waldon, BS,4
Hanno Steen, PhD,4 and
Kathryn R. Wagner, MD, PhD5
Limb girdle muscular dystrophy 1D/1E (OMIM nomenclature LGMD1D, Human Gene Nomenclature Committee
LGMD1E), a skeletal and cardiac myopathy, has previously been linked to chromosome 6q23. We used laser
capture microdissection to isolate cytoplasmic inclusions
from skeletal muscle from a patient with LGMD1D/1E,
performed mass spectrometry–based proteomics on
these minute inclusions, and identified through bioinformatics desmin as their major constituent. Sequencing in
this patient and family members identified the genetic
basis of the previously reported 6q23 linked LGMD1D/
1E to be due to an intron splice donor site mutation
(IVS3þ3A>G) of the desmin gene located on chromosome 2q35.
ANN NEUROL 2012;71:141–145
he limb girdle muscular dystrophies (LGMDs) are a
diverse group of inherited disorders with variably
affected skeletal and cardiac muscle. Classification schemes
include 7 dominant and 14 recessive disorders labeled as
LGMDs, and there are many other muscle disorders that
could be categorized as LGMDs (eg, the myofibrillar myopathies). Traditional diagnostic approaches to LGMD
include phenotypic characterization, pattern of inheritance,
pathological evaluation of muscle biopsy specimens through
histochemistry, immunohistochemistry, electron microscopy,
and targeted genetic testing.
Limb girdle muscular dystrophy type 1D/1E
(LGMD1D/1E), also known as dilated cardiomyopathy
type 1F (CMD1F) (named LGMD1D in the Online
Mendelian Inheritance in Man database [OMIM]
#602067; named LGMD1E in the Human Gene Nomenclature Committee database [HGNC] #2106),1 is an
autosomal dominant skeletal and cardiac myopathy with
cardiac conduction defects. Genome-wide linkage analysis
of a single family identified a chromosome 6q23 locus
for LGMD1D/1E in 1997. However, no other families
have since been reported with a skeletal or cardiac myopathy linked to this locus. Here, we report the use of an
alternative pathological approach to identification of the
genetic basis for LGMD1D/1E in this family, through a
method involving laser capture microdissection and mass
spectrometry-based proteomics. This approach identified
the genetic basis of LGMD1D/1E to be a mutation in
desmin located on chromosome 2q35.
Subjects and Methods
Proband and Pedigree
The proband developed exertional dyspnea and limb weakness at
the age of 25 years. Over the ensuing 15 years, he developed a
progressive cardiomyopathy with first-degree atrioventricular
block, right bundle branch block, and nonsustained ventricular
tachycardia. A pacemaker–defibrillator was implanted, and subsequent progression of heart failure led to cardiac transplantation.
Proximal limb weakness, greater in the legs than the arms, was
progressive. Evaluation at age 40 years showed serum creatine
kinase to be mildly elevated (150–350U/l). Family history
suggested an autosomal dominant pattern of inheritance.
Genetic Analysis
DNA sequencing was performed in a combination of clinical
and research laboratories. After informed consent, blood was
obtained from participating family members, and DNA was
extracted using standard methods (DNAEasy Kit; Qiagen, Valencia, CA). Primers were designed to amplify each exon and
the exon–intron boundaries of genes for which Clinical Laboratory Improvement Amendments–certified laboratory testing was
not available. Bidirectional dideoxy sequencing was performed.
From the 1Department of Neurology, Division of Neuromuscular Disease,
Brigham and Women’s Hospital and Children’s Hospital Informatics
Program, Children’s Hospital Boston, Harvard Medical School, Boston,
MA; 2Center for Inherited Heart Disease, Division of Cardiology, Johns
Hopkins University, Baltimore, MD; 3Departments of Neurology and Brain
and Cognitive Sciences, University of Rochester School of Medicine and
Dentistry, Rochester, NY; 4Proteomics Center at Children’s Hospital
Boston, Harvard Medical School, Boston, MA; 5Center for Genetic Muscle
Disorders, Kennedy Krieger Institute and Departments of Neurology and
Neuroscience, Johns Hopkins School of Medicine, Baltimore, MD.
Address correspondence to SA Greenberg, Brigham and Women’s
Hospital Department of Neurology, Division of Neuromuscular Disease
and Children’s Hospital Boston, Harvard Medical School, 75 Francis
Street, Boston, MA 02115. E-mail:
Received Jun 6, 2011, and in revised form Sep 27, 2011. Accepted for
publication Oct 7, 2011.
View this article online at DOI: 10.1002/ana.
C 2011 American Neurological Association
of Neurology
Laser Capture Microdissection and Proteomic
Laser capture microdissection was used to isolate cytoplasmic
inclusions, and mass spectrometry and bioinformatics were used
to identify their protein contents. Methods have been previously
described in detail.2–5 Briefly, a Veritas microdissection system
(Arcturus Engineering, Mountain View, Calif) and CapSure HS
LCM caps (Arcturus #LCM0214) were used to capture cytoplasmic inclusions from hematoxylin and eosin–stained 7mm muscle
sections, and separately to capture unaffected myofibers. Captured material was frozen (overnight at 80 C), and proteins
were solubilized (with 30ml extraction buffer consisting of
100mM NH4HCO3, 1% sodium dodecyl sulfate, 8M urea, and
10mM dithiothreitol incubated for 2 hours at 37 C), purified
(centrifugation at 5,000 g for 5 minutes, cleared from urea
and sodium dodecyl sulfate [SDS] using a C12 filter system),
dried in an Ependorf (Hamburg, Germany) Vacufuge, and stored
at 80 C. Purified protein was reconstituted in 1 LDS loading buffer (Invitrogen, Carlsbad, CA), loaded, and run in a 10%
Bis/Tris gel to the end of the stacking layer.
All mass spectrometric protein analysis was performed at
the Proteomics Center at Children’s Hospital Boston, using a
previously described approach.4,5 Bands of interest were excised
from the Coomassie-stained SDS–polyacrylamide gel electrophoresis gel, washed with 100mM ammonium bicarbonate and
acetonitrile, reduced with 10mM dithiothreitol at 56 C for 45
minutes, and alkylated for 30 minutes at room temperature, in
the dark, with 55mM iodoacetamide. Samples were digested
with sequencing grade trypsin (Promega, Madison, WI) at a
concentration of 12.5ng/ll in 100mM ammonium bicarbonate
at 377deg;C overnight. Peptides were extracted with 100mM
ammonium bicarbonate and acetonitrile, and then dried in a
SpeedVac. Samples were resuspended in 5% acetonitrile and
5% formic acid before direct injection into the liquid chromatography (LC)–mass spectrometry (MS) system comprising a
nanoLC AS-2 autosampler, a nanoLC ultra1D HPLC pump
(both Eksigent, Dublin, CA), and an FT-LTQ Ultra mass spectrometer (Thermo Scientific, San Jose, CA). The LC system for
the mass spectrometer featured a reversed-phase column packed
in-house into PicoTip Emitters (New Objective, Woburn, MA)
using Magic C18 (3 lm, 200 Å; Michrom Bioresources,
Auburn, CA) packing material. The peptides were eluted with a
30-minute linear gradient, and data were acquired in a data-dependent fashion, that is, the 6 most abundant species were
selected for fragmentation by collision-induced dissociation.
The raw files were converted into .mgf files using scripts written in-house.6 For each fragment ion spectrum, only the 200
most intense fragment ions were exported into the .mgf file.
The mass spectrometric data were searched against an IPIHuman (v3.56) database using the protein identification software Mascot (v2.2.04; Matrix Sciences, London, UK). Search
criteria included tryptic cleavage specificity, 1 missed cleavage,
carbamidomethyl as a fixed modification (C), and Gln->pyroGlu (N-term of Q), deamidation (NQ), and oxidation (M) as
variable modifications. Mass tolerances of 10ppm and 0.8Da
were used for precursor and fragment ions, respectively. Only
peptides with a Mascot score above 25 and proteins with 2 or
more peptide assignments were accepted.
Pathology and Genetic Evaluation
Endomyocardial biopsy specimens showed fibrosis and
myocyte hypertrophy. Skeletal muscle biopsy frozen sections
revealed abundant densely staining cytoplasmic inclusions,
which were rounded in the center of fibers and frequently
wedge-shaped if subsarcolemmal. Histochemically, cytoplasmic inclusions were densely eosinophilic with hematoxylin
and eosin, dark green on modified Gomori trichrome,
completely adenosine triphosphatase (ATPase) negative,
strongly positive (diastase-resistant) by the periodic acidSchiff (PAS) reaction, and acid phosphatase negative, and
excluded oxidative activity, yet were surrounded by an
intense rim of mitochondrial enzymes (Fig 1). Menadionelinked nitro blue tetrazolium (M-NBT) staining was positive in occasional cytoplasmic inclusions (see Fig 1D), qualifying them as reducing bodies. By electron microscopy, the
cytoplasmic inclusions were composed of fibrils and granulofilamentous, osmiophilic material in a trabecular pattern
within large perinuclear or subsarcolemmal collections, with
interspersed abundant glycogen but excluding both recognizable myofibrils and mitochondria (except those mitochondria clumped on the outside rim of the inclusion).
The dense dappled material was sometimes continuous
with, and had the same density as, Z-band (see Fig 1E).
These ultrastructural features account for the histochemical
appearance as eosinophilic, ATPase-negative, nicotinamide
adenine dinucleotide–negative, PAS-positive proteinaceous
inclusions. Variation in myofiber size, multiple internal
nuclei, mild endomysial fibrosis, rare red-rimmed vacuoles,
and isolated degenerating fibers were also seen.
Further phenotypic investigation of the extended family
excluded the previously reported 6q23 locus as the sole cause
of disease due to the absence of cosegregation of this locus
with the phenotype in unequivocally affected family members. Genetic analysis was performed using a panel of clinical
tests in the proband’s affected sister. No mutations were identified in CAV3, FKRP, LMNA, or MYOT; dysferlin was present in blood by Western blot. Because of positive M-NBT
staining of some of the cytoplasmic inclusions similar to that
of reducing body myopathy due to FHL1 mutations,7 additional genetic testing was performed on FHL1 and the closely
related FHL2 and FHL3; no mutations were identified.
Laser Capture Microdissection/Proteomics/
Genetic Identification of a Desmin Mutation
Laser capture microdissection and proteomic studies
identified desmin as the major constituent of the
Volume 71, No. 1
Greenberg et al: Etiology of LGMD1E
FIGURE 1: Skeletal muscle pathology. (A, B) Hematoxylin and eosin–stained muscle with cytoplasmic inclusions (white arrowheads). (C) Modified Gomori trichrome–stained cytoplasmic inclusions. (D) Menadione-linked nitro blue tetrazolium–stained frozen muscle sections. (E, F) Electron microscopy shows subsarcolemmal myofibrillar material.
cytoplasmic inclusions (Fig 2). Five peptide sequences
unique to desmin were identified in the cytoplasmic inclusions, whereas no desmin peptides were identified in unaffected myofibers. Sequencing of family members disclosed
a previously reported disease-causative mutation in the
desmin gene þ3 position of the splice donor site of intron
3 (IVS3þ3A>G) that cosegregated with disease (Fig 3).8
We have identified the genetic basis of LGMD1D/1E as
a mutation in the gene for desmin. Desmin myopathies
January 2012
are a diverse group of disorders representing a subcategory of myofibrillar myopathies, and should be considered in patients with M-NBT–staining reducing bodies.
Skeletal and cardiac myopathy with cardiac conduction
defects has previously been reported in desmin mutations
disrupting the intron 3 donor splice site.9 Genetic linkage studies depend critically on phenotype characterization, and subsequent refinements of pedigree details can
alter locus assignment, as in this family, where age-dependent penetrance changed the phenotypic status of
critical family members. As this is the only family
of Neurology
FIGURE 2: Laser capture microdissection and mass spectrometric identification of desmin in cytoplasmic inclusions. (A) Frozen
section stained with Histogen stain to provide sufficient contrast to visualize cytoplasmic inclusions (black arrowhead) using an
Arcturus Veritas laser capture microdissection microscope. (B) Magnified view of a myofiber containing cytoplasmic inclusions.
(C) View of the myofiber seen in B, removed from the muscle section after laser microdissection. (D) Mass spectrometric protein analysis of the 5 most abundant proteins present in laser-dissected unaffected myofibers, compared with myofibers containing cytoplasmic inclusions, identifies desmin only in those myofibers with cytoplasmic inclusions. Five different desmin
sequences were identified by mass spectrometry. (E) Spectrum corresponding to 1 of these sequences (FASEASGYQDNIAR)
with mascot score of 84 and an expect value of 2.6e-07.
reported as having a 6q23 chromosomal locus, and they
have now been characterized as having 2q35-located desmin mutations, the current OMIM assignments
LGMD1D and CMD1F, and current HGNC assignment
LGMD1E should be appropriately revised.
This study highlights the utility of laser capture
microdissection for isolating pathological lesions from
muscle tissue and their subsequent analysis by proteomic
methods for clinical diagnosis. This approach has previously been used to identify the genetic basis of reducing
body myopathy,7 deficiency of titin in dermatomyositis
atrophic myofibers,2 and previously characterized myonuclear tau in inclusion body myositis as neurofilament H
protein.3 Although technologically challenging, this
Volume 71, No. 1
Greenberg et al: Etiology of LGMD1E
FIGURE 3: Pedigree. (A) Originally reported pedigree demonstrating autosomal dominant inheritance affecting at least 4 generations. (B) Portion of the family involved in this report. (C) Revised pedigree for this portion of the family with corrected
phenotypes. Males are represented by squares and females by circles. An arrow designates the proband. Affected individuals
are represented in black. The presence (1) or absence (2) of DES mutation IVS313A>G is also shown.
method combines traditional methods of neuromuscular
microscopic pathology with state-of-the-art protein identification techniques, and is a valuable addition to neuromuscular genetic diagnosis.
Salajegheh M, Kong SW, Pinkus JL, et al. Interferon-stimulated
gene 15 (ISG15) conjugates proteins in dermatomyositis muscle
with perifascicular atrophy. Ann Neurol 2010;67:53–63.
Salajegheh M, Pinkus JL, Nazareno R, et al. Nature of ‘‘Tau’’ immunoreactivity in normal myonuclei and inclusion body myositis. Muscle Nerve 2009;40:520–528.
Parker KC, Walsh RJ, Salajegheh M, et al. Characterization of
human skeletal muscle biopsy samples using shotgun proteomics.
J Proteome Res 2009;8:3265–3277.
Parker KC, Kong SW, Walsh RJ, et al. Fast-twitch sarcomeric and
glycolytic enzyme protein loss in inclusion body myositis. Muscle
Nerve 2009;39:739–753.
Renard BY, Kirchner M, Monigatti F, et al. When less can yield
more—computational preprocessing of MS/MS spectra for peptide
identification. Proteomics 2009;9:4978–4984.
Schessl J, Zou Y, McGrath MJ, et al. Proteomic identification of
FHL1 as the protein mutated in human reducing body myopathy.
J Clin Invest 2008;118:904–912.
Park KY, Dalakas MC, Goebel HH, et al. Desmin splice variants
causing cardiac and skeletal myopathy. J Med Genet 2000;37:
Arbustini E, Pasotti M, Pilotto A, et al. Desmin accumulation restrictive cardiomyopathy and atrioventricular block associated with desmin gene defects. Eur J Heart Fail 2006;8:477–483.
Funded in part by NIH, NINDS 5R21NS057225 to S.A.G.
We thank L.C. Warsing and M. Lehar for technical
Potential Conflicts of Interest
R.W.K.: consultancy, Ardea Biosciences.
Messina DN, Speer MC, Pericak-Vance MA, McNally EM. Linkage
of familial dilated cardiomyopathy with conduction defect and muscular dystrophy to chromosome 6q23. Am J Hum Genet 1997;61:
January 2012
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etiology, limba, girdle, proteomic, 1d1e, microdissection, determiners, capture, muscular, laser, dystrophy
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