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Commonality of TRIM32 mutation in causing sarcotubular myopathy and LGMD2H.

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defect in the function of LITAF, as a sorting tool for
protein degradation, may result in accumulation of
misfolded PMP22 and subsequently lead to demyelination and the resulting peripheral nerve pathological features.
In conclusion, molecular genetic analysis allowed the
identification of a compound phenotype in a severe case
of CMT1. The cooccurrence of both PMP22 duplication and a LITAF mutation resulted in a more severe
phenotype. Additional screening for CMT gene mutations can be done for two reasons: either a CMT-like
phenotype exists in the other parent, or the severity of
the phenotype falls outside the known phenotypic variations. In our patient, both reasons are valid. If the diagnosis of CMT1 had not been made in the mother of
the proband, we might not have continued our search
for CMT1 mutations. This underscores that phenotypic
variations of CMT1 disease can be attributed to mutations in other genes. In view of the plethora of genes
involved in CMT disease, many modifier genes for
CMT might exist in the population. Careful neurological and genetic examination of patients and their relatives will probably show similar compound CMT cases.
Commonality of TRIM32
Mutation in Causing
Sarcotubular Myopathy
and LGMD2H
Benedikt G. H. Schoser, MD,1 Patrick Frosk,2
Andrew G. Engel, MD,3 Ursula Klutzny,1
Hanns Lochmüller, MD,1
and Klaus Wrogemann, MD, PhD2
Sarcotubular myopathy (OMIM 268950) is a rare autosomal recessive myopathy first described in two Hutterite
brothers from South Dakota and in two non-Hutterite
brothers from Germany. We report that sarcotubular myopathy (STM) is caused by mutation in TRIM32, the
gene encoding the tripartite motif-containing protein 32.
TRIM32 was found to be the gene mutated in limb girdle
muscular dystrophy type 2H (LGMD2H [OMIM
254110]), a disorder that has been confined to the Hutterite population. The TRIM32 mutation found in the
STM patients is identical to the causative mutation for
LGMD2H (D487N), Haplotype analysis shows that the
disease chromosomes share common ancestry.
Ann Neurol 2005;57:591–595
References
1. Young P, Suter U. The causes of Charcot-Marie-Tooth disease.
Cell Mol Life Sci 2003;60:2547–2560.
2. Dyck PJ, Lambert EH. Lower motor and primary sensory neuron diseases with peroneal muscular atrophy. II. Neurologic,
genetic and electrophysiologic findings in various neuronal degenerations. Arch Neurol 1968;18:619 – 625.
3. Hensels GW, Janssen EA, Hoogendijk JE, et al. Quantitative
measurement of duplicated DNA as a diagnostic test for
Charcot-Marie-Tooth disease type 1a. Clin Chem 1993;39:
1845–1849.
4. Slater HR, Bruno DL, Ren H, et al. Rapid, high throughput
prenatal detection of aneuploidy using a novel quantitative
method (MLPA). J Med Genet 2003;40:907–912.
5. Slater H, Bruno D, Ren H, et al. Improved testing for
CMT1A and HNPP using multiplex ligation-dependent
probe amplification (MLPA) with rapid DNA preparations:
comparison with the interphase FISH method. Hum Mutat
2004;24:164 –171.
6. Street VA, Bennett CL, Goldy JD, et al. Mutation of a putative
protein degradation gene LITAF/SIMPLE in Charcot-MarieTooth disease 1C. Neurology 2003;60:22–26.
7. Bennet CL, Shirk AJ, Huynh HM, et al. SIMPLE mutation in
demyelinating neuropathy and distribution in sciatic nerve. Ann
Neurol 2004;55:713–720.
8. Bonifacino JS, Traub LM. Signals for sorting of transmembraneproteins to endosomes and lysosomes. Annu Rev Biochem
2003;72:395– 447.
9. Pareek S, Notterpek L, Snipes GJ, et al. Neurons promote the
translocation of peripheral myelin protein 22 into myelin.
J Neurosci 1997;17:7754 –7762.
10. Ryan MC, Shooter EM, Notterpek L. Aggresome formation in
neuropathy models based on peripheral myelin protein 22 mutations. Neurobiol Dis 2002;10:109 –118.
In 1973, Jerusalem and coworkers1 described sarcotubular myopathy (STM) as a congenital disorder in two
Hutterite brothers aged 11 and 15 years. Both boys
had mild-to-moderate symmetrical proximal muscle
weakness and wasting, and they experienced minor difficulties on strenuous activity (Cases 1 and 2, respectively, in Jerusalem and colleagues1). The disorder was
thought to be autosomal recessive in nature because the
parents of the two boys were asymptomatic, as well as
consanguineous. STM was distinguished from other
myopathies by its unique structural features.1,2 The
second report of STM was of two brothers aged 33 and
35 years from a small village in southern Germany
From the 1Department of Neurology, Friedrich-Baur Institute,
Ludwig-Maximilians University, Munich, Germany; 2Departments
of Biochemistry and Medical Genetics and Pediatrics and Child
Health, University of Manitoba, Winnipeg, Canada; and 3Department of Neurology, Neuromuscular Research Laboratory, Mayo
Clinic, Rochester, MN.
Received Oct 5, 2004, and in revised form Jan 25, 2005. Accepted
for publication Jan 27, 2005.
Published online Mar 28, 2005, in Wiley InterScience
(www.interscience.wiley.com). DOI: 10.1002/ana.20441
Address correspondence to Dr Schoser, Friedrich-Baur Institute,
Department of Neurology, Ludwig-Maximilians University Munich,
Ziemssenstrasse 1a, 80336 Munich, Germany.
E-mail: bschoser@med.uni-muenchen.de
Schoser et al: STM Is Caused by TRIM32 Mutation
591
(Cases 1 and 2, respectively, in Muller-Felber and colleagues2). The younger brother (UM048) had moderate proximal muscle weakness by 8 years old. By 31
years old, he could walk no further than 10 to 20m,
experienced increasingly severe exercise-induced myalgias, and had winged scapulae, hypertrophied calf muscles, and a positive Gowers’ sign. The older brother
(UM047) had a more benign course with only slight
weakness, mild exercise-induced myalgias, and moderate calf hypertrophy. Muscle biopsy studies demonstrated the same vacuolar changes that Jerusalem and
coworkers1 observed in the two Hutterite brothers, as
well as other myopathic features.
Independently, in 1976, an autosomal recessive muscular dystrophy in Manitoba Hutterites was reported.3
The 11 affected patients had a slowly progressive proximal weakness and wasting with mildly to moderately
increased creatine kinase (CK) levels (57–562U/L; reference range, ⬍40 –50). The facial muscles also were
affected, and it was concluded that the disorder combined phenotypic features of limb girdle muscular
(LGMD) and facioscapulohumeral dystrophies. The
muscle fibers varied in size and contained an increased
number of internal nuclei, and some fibers harbored
small vacuoles. This study was followed up in 1997
using 1 patient from the original publication and 17
more recently ascertained patients.4,5 Facial weakness
was not apparent in any affected individual, but they
did show mild-to-moderate proximal weakness and
wasting and increased CK levels. Linkage analysis demonstrated a novel locus at chromosome 9q31-33, which
was named LGMD2H. Subsequently, 17 additional patients were identified, and the candidate region was extensively refined, leading to the identification of
D487N in TRIM32 as the causative mutation of
LGMD2H.6 Currently, the LGMD2H cohort consists
of 47 D487N homozygotes from both the Schmiedeleut and Lehrerleut subdivisions of the Hutterites. In
addition, another LGMD-causing mutation was identified in a further 21 patients from all three subdivisions of the Hutterites, L276I in FKRP, causing
LGMD2I [OMIM 607155].7 The two disorders are
difficult to distinguish by clinical criteria because of the
extreme phenotypic variability within each patient
group and the common findings of proximal muscle
weakness and wasting and increased serum CK levels.
We hypothesized that STM may be caused by one of
the above two LGMD-causing mutations found in the
Hutterites, because the original STM family was of
Hutterite origin and the clinical presentation of STM
was similar to that of an LGMD.1,2
Materials and Methods
Informed written consent was obtained from patients and
their families. We genotyped the Hutterite (Z01–Z12) and
592
German (UM045–UM048) families for the D487N mutation in TRIM32 and L276I in FKRP using previously published methods.6,7 Genotypes for 10 markers surrounding
the TRIM32 gene were obtained as described previously.6
Haplotypes were constructed manually by minimizing the
number of recombinants and assuming no mutation of
marker alleles. In addition, using standard methods,1,2 we
reevaluated the muscle biopsy findings.
Results
Phenotype of Sarcotubular Myopathy
All four patients were male. The onset of weakness
occurred before reaching 2 years old in Patients 1
(Z06) and 2 (Z04), and at 31 and 4 years old in
Patients 3 (UM048) and 4 (UM047), respectively.
Patients 3 and 4 also reported exercise-induced fatigue and myalgias. Mild facial muscle weakness was
detected in Patients 3 and 4. All patients had proximal limb girdle and neck flexor weakness. In Patients
1 and 2, at 10 and 14 years old, respectively, the
weakness was mild and was accompanied by proportionate decrease in muscle bulk. In Patients 3 and 4,
the proximal weakness was moderate (Patient 3) to
severe (Patient 4), including winged scapulae, hypertrophied calf muscles, and a positive Gowers’ sign accompanied by proximal limb muscle atrophy. In addition, Patients 3 and 4 had Achilles tendon
contractures. The tendon reflexes were hypoactive to
absent in Patient1, normal in Patient 2, and absent in
Patients 3 and 4. In Patients 2 through 4, serum CK
levels were increased up to 5- to 20-fold greater than
the upper limit of normal.
The D487N Mutation Causes the
Sarcotubular Myopathy
All four patients (Z04, Z06, UM047, and UM048)
proved to be homozygous for the D487N mutation,
and none harbored the L276I mutation. Moreover,
haplotyping of the chromosome 9q32 region surrounding the TRIM32 gene indicated not only that the two
families have the same mutation, but also that it was
on a chromosome that shared identity by descent with
the original LGMD2H disease chromosomes (Fig 1).
In all STM patients, segments of many muscle fibers
harbored myriad small abnormal spaces (Fig 2A, B).
Vacuoles were detected in 20 to 30% of the type 2
fibers and less than 4% of the type 1 fibers in frozen
sections.1 Electron microscopy studies showed that the
smallest abnormal spaces arose from focal dilations of
the sarcoplasmic reticulum (Fig 3). Coalescence of the
smaller vacuoles caused larger ones, of which the limiting membranes often degenerated (see Fig 2C). The
membranes limiting the vacuoles showed sarcoplasmic
reticulum–associated ATPase reactivity (see Fig 3),
confirming that the vacuoles arose from the sarcoplasmic reticulum. Regenerating or necrotic fibers were not
© 2005 American Neurological Association
Published by Wiley-Liss, Inc., through Wiley Subscription Services
Fig 1. Chromosome region 9q32 haplotyping of sarcotubular myopathy families with 11-marker haplotype surrounding the
LGMD2H gene, TRIM32. V1 to V4 are variants that Frosk and colleagues6reported; V4 is the causative mutation for LGMD2H
(D487N in TRIM32), and the remaining markers are microsatellites. UM045 to UM048 are from a non-Hutterite family of
German descent reported to have sarcotubular myopathy.2Z01 to Z12 are from a Hutterite family that includes the two brothers
(Z04 and Z06) originally reported to describe sarcotubular myopathy.1 All four affected individuals are homozygous for the D487N
mutation in TRIM32. In addition, haplotypes on which the mutation occurs are nearly identical in both the Hutterite and nonHutterite families, indicating a common origin. Disease-associated haplotypes in black signify identity with the Hutterite LGMD2H
haplotype; regions in gray signify nonidentity. Haplotypes completely in white represent the normal paternal and maternal chromosomes. Alleles in square brackets are inferred. (inset) Marker distances were obtained from the University of California Santa Cruz
(UCSC) human genome browser. Allele frequencies were either reported in Frosk and colleagues6 or were obtained from the Centre
d’Etude du Polymorphisme Humain (CEPH) database (in boldface). NA indicates that frequency was not available; 0 frequency
indicates that the allele was not present in the unaffected individuals typed for the marker.
observed, and inflammatory changes were absent in the
original Hutterite STM patients. However, the two
German STM patients had other myopathic features
that included abnormal variation of muscle fiber diameters, an increased number of internally located nuclei,
and small foci of Z-disk streaming. Immunostains with
antibodies against cytoskeleton proteins including ␣
dystroglycan indicated normal findings.
Discussion
The four STM patients were found to be homozygous
for the D487N mutation in TRIM32. Haplotyping
showed that the chromosomal region surrounding
TRIM32 is identical by descent in all STM and
LGMD2H patients. This is surprising because the German family has no apparent connection with the Hutterite population other than that the Hutterites are
largely Germanic in origin. This raises the possibility
that the D487N mutation arose before the emergence
of the Hutterite religion in central Europe in the 16th
century.
We hypothesize that STM and LGMD2H are the
same disorder. The phenotypic variability is large
among the LGMD2H cohort, from virtually asymptomatic to some older patients who require a wheelchair for ambulation. As such, there is little to differentiate them clinically from the STM patients. Our
initial impressions from examining muscle specimens
of LGMD2H patients is that the vacuolar pathology is
not present at the frequency or severity that it is in the
two families classified as having STM. However, ascertainment of the vacuolar change requires artifact-free
sections, and the minute size of the vacuoles (most
⬍4␮m) may limit their detection in 10␮m-thick sections. To address this hypothesis, we are conducting a
detailed retrospective study of available LGMD2H
muscle specimens and are planning to obtain follow-up
biopsies on some patients.
Schoser et al: STM Is Caused by TRIM32 Mutation
593
Fig 2. Sarcotubular myopathy in the first Hutterite family. (A, B) Resin sections (1 ␮m thick) show myriad small abnormal spaces
in a proportion of the muscle fibers. Note segmental distribution of the abnormal spaces. (C) Electron microscopy shows coalescing
membrane-bound spaces. Some of the limiting membranes are degenerating (asterisk). Bars ⫽ 10 ␮m (A, B); 1 ␮m (C).
A given mutation may lead to different phenotypes
in different families and even within the same family.
For instance, all four different caveolinopathy phenotypes can arise from the same amino acid substitution.8
If our hypothesis is correct, then the D487N mutation
is associated with a spectrum of vacuolar pathology,
594
Annals of Neurology
Vol 57
No 4
April 2005
and STM might prove to be the more histologically
severe variant. Currently, it is unknown whether the
presence or absence of vacuoles has an effect on the
progression of the disease or on patient prognosis.
Many members of the TRIM protein family are involved in human developmental disorders or cancer.9
OMIM and University of California Santa Cruz human genome browser: http://genome.ucsc.edu/
This work was supported by the German Network on Muscular
Dystrophies (MD-NET; 01GM0302, H.L.) funded by the German
Ministry of Education and Research (BMBF), the NIH (National
Institute of Neurological Disorder and Stroke, NS6277, A.G.E.),
the Manitoba Institute of Child Health (K.W.), and the Canadian
Institutes of Health Research (K.W.).
We are indebted to the patients and their families for participating
in our study. We thank I. Rivera, J. Douville, and C. Hirst for
assistance in obtaining genotypes and Drs M. Del Bigio and C.
Greenberg for discussions on phenotype and pathology.
References
Fig 3. Electron cytochemical localization of sarcoplasmic retibulum (SR)—associated ATPase by the method of Tice and
Engel.11 The black reaction product is associated with normal
(asterisk) and dilated components of the SR. Bar ⫽ 0.5 ␮m.
TRIM32 is unique among TRIM proteins in being
linked to cancer without being an oncogenic fusion
protein, and because it causes a myopathy when mutated.10 Initial evidence suggests that TRIM32 is an E3
ubiquitin ligase and may have antiapoptotic activity.10
However, we detected no signs of apoptosis in muscle
specimens of our patients. The D487N mutation occurs in one of six NHL domains of TRIM32, postulated to be critical for the recognition of protein target(s) to be ubiquitinated by this E3 ubiquitinated
ligase. We speculate that the mutation abolishes this
interaction; as a result, the still to be identified target
protein(s) will not be ubiquinated, and hence not degraded by the proteasome machinery, and thus accumulate to greater concentrations in the cell.6
How these observations relate to the pathology of
LGMD2H/STM is unknown. However, our findings
suggest that TRIM32 is likely involved in the generation of the sarcoplasmic reticulum system or in maintaining its structural integrity in skeletal muscle.
1. Jerusalem F, Engel AG, Gomez MR. Sarcotubular myopathy. A
newly recognized, benign, congenital, familial muscle disease.
Neurology 1973;23:897–906.
2. Muller-Felber W, Schlotter B, Topfer M, et al. Phenotypic variability in two brothers with sarcotubular myopathy. J Neurol
1999;246:408 – 411.
3. Shokeir MH, Kobrinsky NL. Autosomal recessive muscular dystrophy in Manitoba Hutterites. Clin Genet 1976;9:197–202.
4. Weiler T, Greenberg CR, Nylen E, et al. Limb girdle muscular
dystrophy in Manitoba Hutterites does not map to any of the
known LGMD loci. Am J Med Genet 1997;72:363–368.
5. Weiler T, Greenberg CR, Zelinski T, et al. A gene for autosomal recessive limb-girdle muscular dystrophy in Manitoba Hutterites maps to chromosome region 9q31–q33: evidence for another limb-girdle muscular dystrophy locus. Am J Hum Genet
1998;63:140 –147.
6. Frosk P, Weiler T, Nylen E, et al. Limb-girdle muscular dystrophy type 2H associated with mutation in TRIM32, a putative E3-ubiquitin-ligase gene. Am J Hum Genet 2002;70:
663– 667.
7. Frosk P, Greenberg CR, Tenese AP, et al. The most common
mutation in FKRP causing limb girdle muscular dystrophy type
2I (LGMD2I) may have occurred only once and is present in
Hutterites and other populations. Hum Mutat 2005;25:38 – 44.
8. Woodman SE, Sotgia F, Galbiati F, et al. Caveolinopathies:
mutations in caveolin-3 cause four distinct autosomal dominant
muscle diseases. Neurology 2004;62:538 –543.
9. Reymond A, Meroni G, Fantozzi A, et al. The tripartite motif
family identifies cell compartments. EMBO J 2001;20:
2140 –2151.
10. Horn EJ, Albor A, Liu Y, et al. RING protein Trim32 associated with skin carcinogenesis has anti-apoptotic and E3ubiquitin ligase properties. Carcinogenesis 2004;25:157–167.
11. Tice LW, Engel AG. Cytochemistry of phosphatases of the sarcoplasmic reticulum. II. In situ localization of the Mg⫹⫹dependent enzyme. J Cell Biol 1966;31:489 – 499.
Electronic Resources
Electronic database information can be obtained via the Internet from the following Web sites: CEPH database: http://
www.cephb.fr/; Online Mendelian Inheritance in Man
(OMIM): http://www.ncbi.nlm.gov/entrez/query.fcgi?db⫽
Schoser et al: STM Is Caused by TRIM32 Mutation
595
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