Mutations that impair interaction properties of TRIM32 associated with limb-girdle muscular dystrophy 2H.
ABSTRACT TRIM32 belongs to a large family of proteins characterized by a tripartite motif, possibly involved in the ubiquitination process, acting as an E3 ligase. In addition, TRIM32 has six NHL repeats with putative interaction properties. A homozygous mutation at the third NHL repeat (D487N) has been found in patients with limb girdle muscular dystrophy 2H (LGMD2H). This mutation was only identified in the inbred Manitoba Hutterite or their descendants. Interestingly, a mutation in the B-box domain of TRIM32 cosegregates with Bardet-Biedl syndrome type 11 (BBS11). The signs of BBS11 include obesity, pigmentary and retinal malformations, diabetes, polydactyly, and no muscular dystrophy, suggesting an alternative disease mechanism. We aim to ascertain whether D487N is the only pathological LGMD2H allele, limited to Hutterites. We studied the TRIM32 gene in 310 LGMD patients with no mutations at the other known loci. We identified four patients with novel mutated alleles. Two mutations were homozygous and missing in controls. These mutations also clustered at the NHL domain, suggesting that a specific (interaction) property might be abolished in LGMD2H patients. No mutations were found at the B-box region where the BBS11 mutation is found. We tested TRIM32 and its mutants by yeast-two-hybrid assay, developing an interaction test to validate mutations. All LGMD2H mutants, but not the BBS11, lost their ability to self-interact. The interaction of TRIM32 mutants with E2N, a protein involved in the ubiquitination process, was similarly impaired. In conclusion, the mutations here reported may cause muscular dystrophy by affecting the interaction properties of TRIM32.
- SourceAvailable from: PubMed Central[Show abstract] [Hide abstract]
ABSTRACT: Developmental axon branching dramatically increases synaptic capacity and neuronal surface area. Netrin-1 promotes branching and synaptogenesis, but the mechanism by which Netrin-1 stimulates plasma membrane expansion is unknown. We demonstrate that SNARE-mediated exocytosis is a prerequisite for axon branching and identify the E3 ubiquitin ligase TRIM9 as a critical catalytic link between Netrin-1 and exocytic SNARE machinery in murine cortical neurons. TRIM9 ligase activity promotes SNARE-mediated vesicle fusion and axon branching in a Netrin-dependent manner. We identified a direct interaction between TRIM9 and the Netrin-1 receptor DCC as well as a Netrin-1-sensitive interaction between TRIM9 and the SNARE component SNAP25. The interaction with SNAP25 negatively regulates SNARE-mediated exocytosis and axon branching in the absence of Netrin-1. Deletion of TRIM9 elevated exocytosis in vitro and increased axon branching in vitro and in vivo. Our data provide a novel model for the spatial regulation of axon branching by Netrin-1, in which localized plasma membrane expansion occurs via TRIM9-dependent regulation of SNARE-mediated vesicle fusion.The Journal of Cell Biology 04/2014; 205(2):217-32. · 10.82 Impact Factor
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ABSTRACT: Limb-girdle muscular dystrophies (LGMD) are a heterogeneous group of disorders, which has led to certain investigators disputing its rationality. The mutual feature of LGMD is limb-girdle affection. Magnetic resonance imaging (MRI), perioral skin biopsies, blood-based assays, reverse‑protein arrays, proteomic analyses, gene chips and next generation sequencing are the leading diagnostic techniques for LGMD and gene, cell and pharmaceutical treatments are the mainstay therapies for these genetic disorders. Recently, more highlights have been shed on disease biomarkers to follow up disease progression and to monitor therapeutic responsiveness in future trials. In this study, we review LGMD from a variety of aspects, paying specific attention to newly evolving research, with the purpose of bringing this information into the clinical setting to aid the development of novel therapeutic strategies for this hereditary disease. In conclusion, substantial progress in our ability to diagnose and treat LGMD has been made in recent decades, however enhancing our understanding of the detailed pathophysiology of LGMD may enhance our ability to improve disease outcome in subsequent years.Molecular Medicine Reports 03/2014; · 1.17 Impact Factor
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ABSTRACT: Limb-girdle muscular dystrophies (LGMD) are a highly heterogeneous group of muscle disorders, which first affect the voluntary muscles of the hip and shoulder areas. The definition is highly descriptive and less ambiguous by exclusion: non-Xlinked, non-FSH, non-myotonic, non-distal, nonsyndromic, and non-congenital. At present, the genetic classification is becoming too complex, since the acronym LGMD has also been used for a number of other myopathic disorders with overlapping phenotypes. Today, the list of genes to be screened is too large for the gene-by-gene approach and it is well suited for targeted next generation sequencing (NGS) panels that should include any gene that has been so far associated with a clinical picture of LGMD. The present review has the aim of recapitulating the genetic basis of LGMD ordering and of proposing a nomenclature for the orphan forms. This is useful given the pace of new discoveries. Thity-one loci have been identified so far, eight autosomal dominant and 23 autosomal recessive. The dominant forms (LGMD1) are: LGMD1A (myotilin), LGMD1B (lamin A/C), LGMD1C (caveolin 3), LGMD1D (DNAJB6), LGMD1E (desmin), LGMD1F (transportin 3), LGMD1G (HNRPDL), LGMD1H (chr. 3). The autosomal recessive forms (LGMD2) are: LGMD2A (calpain 3), LGMD2B (dysferlin), LGMD2C (γ sarcoglycan), LGMD2D (α sarcoglycan), LGMD2E (β sarcoglycan), LGMD2F (δ sarcoglycan), LGMD2G (telethonin), LGMD2H (TRIM32), LGMD2I (FKRP), LGMD2J (titin), LGMD2K (POMT1), LGMD2L (anoctamin 5), LGMD2M (fukutin), LGMD2N (POMT2), LGMD2O (POMTnG1), LGMD2P (dystroglycan), LGMD2Q (plectin), LGMD2R (desmin), LGMD2S (TRAPPC11), LGMD2T (GMPPB), LGMD2U (ISPD), LGMD2V (Glucosidase, alpha ), LGMD2W (PINCH2).05/2014; 33(1):1-12.
HUMAN MUTATION 0,1^8,2007
Mutations That Impair Interaction Properties
of TRIM32 Associated With Limb-Girdle Muscular
Valentina Saccone,1Michela Palmieri,1Luigia Passamano,2Giulio Piluso,3Germana Meroni,1
Luisa Politano,2,4and Vincenzo Nigro1,3,4?
1Telethon Institute of Genetics and Medicine (TIGEM), Naples, Italy;2Dipartimento di Medicina Sperimentale-Servizio di Cardiomiologia e
Genetica Medica, Seconda Universita ` degli Studi di Napoli, Naples, Italy;3Dipartimento di Patologia Generale, Seconda Universita ` degli Studi di
Napoli, Naples, Italy;4Centro di Eccellenza per le Malattie Cardiovascolari, Seconda Universita ` degli Studi di Napoli, Naples, Italy
Communicated by Christine Van Broeckhoven
TRIM32 belongs to a large family of proteins characterized by a tripartite motif, possibly involved in the
ubiquitination process, acting as an E3 ligase. In addition, TRIM32 has six NHL repeats with putative
interaction properties. A homozygous mutation at the third NHL repeat (D487N) has been found in patients
with limb girdle muscular dystrophy 2H (LGMD2H). This mutation was only identified in the inbred Manitoba
Hutterite or their descendants. Interestingly, a mutation in the B-box domain of TRIM32 cosegregates with
Bardet-Biedl syndrome type 11 (BBS11). The signs of BBS11 include obesity, pigmentary and retinal
malformations, diabetes, polydactyly, and no muscular dystrophy, suggesting an alternative disease mechanism.
We aim to ascertain whether D487N is the only pathological LGMD2H allele, limited to Hutterites. We studied
the TRIM32 gene in 310 LGMD patients with no mutations at the other known loci. We identified four patients
with novel mutated alleles. Two mutations were homozygous and missing in controls. These mutations also
clustered at the NHL domain, suggesting that a specific (interaction) property might be abolished in LGMD2H
patients. No mutations were found at the B-box region where the BBS11 mutation is found. We tested TRIM32
and its mutants by yeast-two-hybrid assay, developing an interaction test to validate mutations. All LGMD2H
mutants, but not the BBS11, lost their ability to self-interact. The interaction of TRIM32 mutants with E2N, a
protein involved in the ubiquitination process, was similarly impaired. In conclusion, the mutations here
reported may cause muscular dystrophy by affecting the interaction properties of TRIM32. Hum Mutat 0, 1–8,
rrrr2007 Wiley-Liss, Inc.
KEY WORDS: TRIM32; LGMD2H; muscular dystrophies; yeast two-hybrid
Limb-girdle muscular dystrophies (LGMDs) include a broad
group of genetically determined progressive muscle disorders
[Angelini, 2004]. By definition, patients should present primary
or predominant symmetrical atrophy of the pelvic and/or shoulder
girdle musculature, and a necrotic regeneration pattern [Emery,
2002; Nigro, 2003]. However, the clinical course is characterized
by a great variability and the term now includes many different
phenotypes ranging from severe forms with onset in the first
decade and rapid progression, to milder forms with later onset and
atypical presentation [Bushby, 1999]. This determines clinical
overlaps with Becker muscular dystrophy, late-onset spinal
muscular atrophy, myotonic dystrophy type 2, Bethlem myopathy,
and Pompe disease. Thus, a clinical examination cannot clinch the
diagnosis [Straub and Bushby, 2006].
LGMD2H (MIM] 254110) is rare, and one of the most
intriguing forms of autosomal recessive limb-girdle muscular
dystrophies. Homozygous p.D487N mutation of the TRIM32 gene
(MIM] 602290) causes LGMD2H in 41 patients from Hutterite
families [Frosk et al., 2002]. Haplotype analyses showed that all
LGMD2H patients share the same allele at 9q33.1 [Weiler et al.,
1998]. This suggests a founder effect arising before the emergence
of the Hutterite religion in central Europe in the 16th century
[Frosk et al., 2002]. LGMD2H onset is usually within the second
or third decade of life, and progression is slow. Most patients
remain ambulatory until the sixth decade of life [Shokeir and
Kobrinsky, 1976; Shokeir and Rozdilsky, 1985].
Published online inWiley InterScience (www.interscience.wiley.com).
The Supplementary Material referred to in this article can be
accessed at http://www.interscience.wiley.com/jpages/1059-7794/
Received 24 May 2007; accepted revised manuscript 29 July 2007.
?Correspondenceto:VincenzoNigro, Prof., Dipartimento di Patolo-
gia Generale, Seconda UniversitaØdegli Studi di Napoli, Naples, Italy.
Grant sponsor: Ricerca di Ateneo; Grant sponsor:Telethon^Unione
Italiana Lotta alla Distro¢a Muscolare (UILDM); Grant number:
GUP04008; Grant sponsor: TIGEM; Grant number: TVNP42TELC;
Grant sponsor: Ministero dell’UniversitaØe della Ricerca; Grant num-
ber: PRIN prot 2004062849_004; Grant sponsor: Ministero della
Salute;Grant number: dlgs.502/92.
rrrr2007 WILEY-LISS, INC.
This same mutation in TRIM32 has recently been found to
cause sarcotubular myopathy (STM) in Hutterite descendants
[Frosk et al., 2005; Schoser et al., 2005], indicating that STM and
LGMD2H are allelic disorders, with STM having a more severe
phenotype. The explanation for this is obscure.
TRIM32 [Fridell et al., 1995] is a ubiquitously expressed protein
that belongs to a family known as TRIM or RBCC [Reymond
et al., 2001]. These genes are characterized by the tripartite motif
(TRIM), which consists of a RING domain, one or two B-box
motifs, and a coiled-coil region (RBCC) [Slack and Ruvkun,
1998]. This motif is usually associated with variable C-terminal
domains [Torok and Etkin, 2001; Meroni and Diez-Roux, 2005].
Members of this family are involved in a broad range of cellular
transcriptional regulation, and ubiquitination and, as a conse-
quence, when altered, they are implicated in many different disease
states. PML [de The et al., 1991; Goddard et al., 1991], RFP
[Hasegawa et al., 1996], and EFP [Ikeda et al., 2000] as TRIM32
[Horn et al., 2004] have been linked to tumor initiation and
progression. MID1 [Dal Zotto et al., 1998; Trockenbacher et al.,
2001], is altered in Opitz syndrome and MUL is associated with
Mulibrey nanism [Avela et al., 2000], two developmental genetic
diseases, while pyrin/marenostrin is implicated in an inflammatory
disease named familial Mediterranean fever [Bernot et al., 1998].
Other studies indicate that TRIM32, consistently with the
presence of the RING domain [Joazeiro and Weissman, 2000], has
E3 ubiquitin ligase activity, binds to the head and neck region of
myosin, and ubiquitinates actin [Kudryashova et al., 2005]. This
suggests that TRIM32 may regulate components of the cytoske-
leton. Recently, in a small consanguineous Israeli Bedouin family,
an SNP microarray genotyping analysis for homozygosity revealed
a new mutation (P130S) in the TRIM32 B-box domain,
cosegregating with Bardet-Biedl syndrome type 11 (BBS11; MIM]
209900). BBS is a pleiotropic disorder characterized by obesity,
pigmentary retinopathy, polydactyly, renal abnormalities, learning
disabilities, and hypogenitalism [Chiang et al., 2006].
In this report we describe the first cases of LGMD2H in a
non-Hutterite population that we linked to the loss of TRIM32
interaction properties [Cao et al., 1998; Cainarca et al., 1999;
Reymond et al., 2001].
The following criteria were used to recruit patients including
limb girdle onset, type of progression, autosomal recessive
inheritance, and dystrophic changes on muscle biopsy.
Blood samples from patients diagnosed with LGMD were taken.
All the possible causative genes were examined by linkage analysis
or mutation detection. About 1,000 DNA samples and 200
biopsies were consecutively collected and stored at the SUN-
Naples Human Mutation Gene Bank (Cardiomyology and Medical
Genetics) and Telethon Institute of Genetics and Medicine. All
samples were analyzed for mutations in the LGMD2A, 2B, 2C, 2D,
2E, 2F, 2G, 2I, 1B, and 1C loci. All exons and intron-flanking
regions were studied.
In about 50% of patients no mutation was found in any of the
LGMD genes. This indicates that we are including a wider
spectrum of muscular phenotype. This can be an advantage to
define alternative phenotypes due to a mutation in a single gene
[Piluso et al., 2005]. Genomic DNA was extracted by phenol/
chloroform to be used for DHPLC analysis. DNA was quantified
and diluted for the amplification by PCR [Underhill et al., 1997].
In the current survey, DNA samples from 310 LGMD patients
and 600 control chromosomes with similar ethnic background were
analyzed by high-throughput DHPLC (HT-DHPLC). Of these
samples, 82% were recruited at the Universities of Naples, Padua,
and Rome. Other patients were from Argentina (one patient;
0.3%), Croatia (six patients; 1.9%), Slovenia (five patients; 1.62%),
Germany (one patient; 0.32%), Greece (two patients; 0.65%), Israel
(six patients; 1.94%), France (five patients; 1.62%), Spain (one
patient; 0.32%), and Turkey (40 patients; 13%).
Polymerase Chain Reaction
The sequence of TRIM32 gene (NM_12210.2) (one coding
exon) and untranslated regions were amplified by PCR from
genomic DNA using seven overlapping fragments with the primers
described in the Supplementary Table S1 (available online at
Primers were compared with results of the web-based program
Primer3 (PRIMER3; primer3_www.cgi, v 0.2; http://frodo.wi.
mit.edu). Each oligonucleotide was also checked by Blastn against
www.ncbi.nlm.nih.gov/BLAST; NCBI, www.ncbi.nlm.nih.gov).
For PCR analysis, 60ng of genomic DNA was amplified with a
DNA Thermocycler System (MWG AG Biotech, Ebersberg,
Germany; MJ Research PTC-100 96 well PCR, Roche, Alameda,
CA). An initial denaturation step at 951C for 7min was set,
followed by 34 cycles (951C for 30s, 60–611C for 1min and 30s,
and 681C for 1min) followed by 951C and a final extension at
681C for 10min. The annealing for the untranslated regions was
The entire TRIM32 gene was analyzed, including the
50untranslated region. We performed comparative mutation
scanning to select amplicons for aberrant DHPLC profiles not
shared by normal controls.
Primers were longer than 25 nucleotides to reduce the allele
preference determined by sequence differences located in the
region of annealing. DHPLC was performed on a WAVE DNA
fragment analysis system (Transgenomic Inc., San Jose, CA)
equipped with a DNASep column (3,500 High Throughput [HT])
employing a UV-C scanner to detect eluted DNA [O’Donovan
et al., 1998].
Based on DHPLC requirements, special buffer formulations and
primer design were used to improve sensitivity and specificity
[Underhill et al., 1997; O’Donovan et al., 1998].
Genomic DNA SequenceAnalysis
Both strands were sequenced using BigDyesTerminator
sequencing chemistry (Applied Biosystems, Foster City, CA). An
ABI3130XL automatic DNA sequencer (Applied Biosystems) was
used to analyze the product of the sequence reaction. We verified
each nucleotide change by direct sequencing of a second amplified
PCR product obtained with different primers. Mutations were
numbered based on protein (GenBank NP_036342) and cDNA
sequence (GenBank NM_012210.2). Nucleotides were numbered
according to international recommendation [den Dunnen and
InVitro Interaction Mating
For functional analysis of TRIM32 novel mutations, we used a
yeast interaction mating assay. The cDNA bait sequences corre-
sponding to the wild-type and mutant TRIM32 (nt: 134–2096 of
2HUMAN MUTATION 0,1^8,2007
Human Mutation DOI 10.1002/humu
NM_012210.2, aa: 1–654) were cloned into pGBKT7 plasmid vector
(Clontech Laboratories Inc., Palo Alto, CA). Prey constructs of
fusion genes encoding TRIM32 single or combined domains, or
combinations of domains (R; R-BB, R-BB-CC, BB-CC-NHL,
CC-NHL, and NHL), were cloned into pGADT7 (Clontech) and
were individually transformed into the Saccharomyces cerevisiae
(S. cerevisiae) Y187 strain.
These lines were mated with the bait lines transformed into
S. cerevisiae AH109 and diploid cells selected on medium lacking
Leu and Trp, Interacting clones were selected on synthetic SD
minimal medium QDO plates (lack of Adenine, Histidine,
Leucine, and Tryptophan) according to the Clontech manufac-
The human p53 and pTD1 proteins were used as positive
controls for the assay. The same system was used to check for
possible interaction between TRIM32 and some Ubiquitin
The cDNA sequences corresponding to TRIM32 (wild-type and
mutated forms) were cloned into pGBKT7 plasmid and trans-
formed in the S. cerevisiae AH109 strain.
The different human Ubiquitin conjugating enzymes cloned
into pGADT7 vector were cotransformed into the Y187 strain.
The positive interaction was observed on selection medium plates,
Selective Dropout lacking Leu, Trp, His, Ade (SD-LWHA), after
incubation at 301C.
To test the correct expression and the amount of TRIM32
mutant proteins in yeast system, we performed a Western Blot
(WB) analysis using anti-myc-tag 9E10 antibody (Abcam plc,
Cambridge, UK) (Supplementary Fig. S1).
The cDNA corresponding to full-length human TRIM32 in
wild-type and mutant forms, were cloned into pcDNA3.1/
HAvector. The cDNA for human E2N (aa.:1-152; cds nt: 367-
825) was cloned into pcS2 Myc-tag vector (Invitrogen Corpora-
tion, Carlsbad, CA).
Expression vectors for Myc-tagged human E2N and HA-tagged
human TRIM32 wild-type and TRIM32 mutants were cotransfected
into COS7 cells. Cells were harvested after 48hr and then
homogenized in lysis buffer (PBS1X, 0.8% NP40, 2mM phenyl-
methylsulfonyl fluoride, 10mg/ml aprotinin, pH 7.8). Cell lysates
were centrifuged at 10,000g for 15 minutes, and the supernatants
preincubated with protein A sepharose (Sigma-Aldrich, St. Louis,
MO) to adsorb nonspecific binding proteins. After centrifugation,
the supernatants were incubated with anti-Myc antibody (9E10) or
anti-HA (Roche Applied Science, Indianapolis, IN).
After incubation with protein A sepharose for 60 minutes,
immunocomplexes were collected by centrifugation. The resin was
washed with an excess amount of lysis buffer followed by elution of
immunoprecipitated proteins with SDS sample buffer.
Transfection was performed using Polyfect (Qiagen GmbH,
Hilden, Germany) according to the manufacturer’s protocol.
COS7 cells were grown on glass coverslip put into 12-well plates
(NUNC A/S, Roskilde, Denmark). They were cultured in
Dulbecco’s modified Eagle’s medium (DMEM) supplemented with
10% (v/v) fetal bovine serum and penicillin-streptomycin (Gibco-
Invitrogen, Carlsbad, CA) and maintained in a 5% CO2incubator
Transfections were carried out using Polyfect reagent with
750ng of pcDNA3MycEGFP (Invitrogen) null vector or with
wild-type TRIM32 or all TRIM32 mutants (D489N TRIM32,
R394H TRIM32, 1559delC TRIM32, D588del TRIM32, and
P130S TRIM32) subcloned in-frame with green fluorescent
protein (GFP). After 36hr, cells were fixed in 4% paraformalde-
hyde/PBS for 10 minutes at room temperature. Coverslips were
mounted with Vectashield mounting medium with DAPI (Vector
Laboratories Inc., Burlingame, CA). Wild-type and mutant
TRIM32 proteins were identified by enhanced GFP (EGPF)
(green) fluorescence. Cells were examined using a Zeiss micro-
scope (Axio Imager A1, Carl Zeiss S.p.A, Milano, Italy) and
analyzed using Axio Vision Rel. 4.5 software. Digital images were
saved and managed by Adobe PhotoShop (Adobe Systems Inc.,
Mountain View, CA).
The effect of the mutations here described on the TRIM32 3-D
structure, was investigated using as a template the crystal structure
of BRAT. This is homologous to TRIM32 (Protein Data Bank
[PDB]; www.rcsb.org/pdb). A model was generated for amino acid
residues 371–643 of NHL. The sequence identity between target
and template for this region was about 40%. The model was
refined using YASARA (Yet Another Scientific Artificial Reality
Application; www.yasara.org), which was shown to increase model
accuracy [Krieger et al., 2004].
We carried out a mutation analysis using DHPLC and
sequencing on a cohort of 310 LGMD patients with different
degrees of severity. All patients were previously examined for the
other LGMD genes. We identified three novel putative mutations
in the TRIM32 gene (p.R394H, p.T520TfsX13, and p.D588del) in
TABLE 1. Summary ofNovel Mutations Detected in theTRIM32 Gene?
?Mutation numbering is based onTRIM32 cDNA (sequence position 1 is theA in the
¢rst ATG codon).The reference sequenceused isGenBankIDNM_012210.2.
FIGURE 1. TRIM32 characteristics. A: The TRIM32 HT2A gene
maps on chr 9q 33.1 (1^3,160bp). Only one exon is coding
(134^2095bp). B: TRIM32 protein is composed of 673 amino
acids (72kDa). Here are shown TRIM32 functional domains:
RING ¢nger domain, B-box type 1 domain, Coiled coil region,
and six NHL repeats.The arrows indicate the mutation location
inTRIM32 protein structure. [Color ¢gure can be viewed in the
onlineissue,whichis available at www.interscience.wiley.com.]
HUMAN MUTATION 0,1^8,20073
Human Mutation DOI 10.1002/humu
four patients (two homozygous and two heterozygous). These
alleles were absent in 600 control chromosomes (Table 1).
Patient 3639 (proband ]1) is a 44-year-old Croatian woman, who
carries a homozygous c.1559delC p.T520TfsX13 variation (Fig. 1;
Supplementary Fig. S2). She complained of a slowly progressive
proximal weakness and muscle wasting, respiratory weakness, and
chronic keratitis; creatine-kinase (CK) levels were in the normal
range. A physical examination in 2001 showed marked atrophy of
the shoulder and abduction of the arms. Facial muscles were also
involved, affecting the ability to close the eyes. Electromyography
(EMG) revealed both neuropathic and myopathic elements. It was
concluded that the disorder combines phenotypic features of LGMD
and facio-scapulo-humeral dystrophy. Her parents had no muscular
involvement. Her brother was affected by Wilson syndrome.
A testing method was devised to detect the c.1559delC
p.T520TfsX13 mutation based on a natural restriction site for
MslI enzyme that is formed by the cytosine deletion. The allele
wild-type was digested once by MslI, the allele of the proband had
two extra fragments (Supplementary Fig. S2).
Patients 3144 and 3996 (probands ]2 and ]3, respectively) are
from Southern Italy and show the same TRIM32 variation,
c.1180G4A p.R394H (Fig. 1; Supplementary Fig. S3).
Proband ]2, heterozygous for this mutation, is a 73-year-old
man presenting with hypertension and type II-diabetes. A chance
occurrence of slightly elevated serum CK values (2.5?), not
associated with muscle symptoms or signs, was revealed at the age
of 64 years and later confirmed. The patient was the last of nine
siblings; five of them died from cardiovascular events.
The last clinical examination showed calf pseudohypertrophy, mild
ankle contractions, and scapular winging, at rest and in upper limb
adduction, especially on the left side. No respiratory involvement was
Proband ]3 is homozygous for c.1180G4A p.R394H TRIM32
allele. Disease onset was in the third decade of life and characterized
by weakness and paresthesia. The patient noted progressive
difficulty in rising from the floor, in climbing stairs, and in walking.
A muscle biopsy, performed at the age of 59 years, showed a
muscular dystrophy with a normal dystrophin staining. The
patient was diagnosed with LGMD. He lost the ability to walk at
the age of 64 years, after a prolonged immobilization for other
Physical examination, showed marked atrophy in both upper
and lower limbs (proximal4distal), with muscle weakness.
Retractions were present at the ankles, knees, and Achilles
tendons. Scapular winging was also observed. Electrocardiography
(ECG) revealed a right bundle branch block. Forced expiratory
vital capacity (FVC) was reduced to 41%.
FIGURE 2. Yeast two-hybrid analysis. The loss of interaction was tested in the yeast two-hybrid assay by cotransformation. A:
Schematic structure of humanfull-lengthTRIM32 cDNA. Numbers indicate nucleotide residue numbers (top, labeled bp) and amino
acid residue numbers (below the nucleotide number, labeled aa). AAA indicates a poly(A1) tail.The structural/functional domains
are shown in gray. Lines below indicate the partialTRIM32 bait constructs that were generated for yeast two-hybrid screens to test
fortheself-interactionproperty. B:Ontheleftistheinteractionmatingoutlineoftheplatepictureontheright.The ¢lledcirclesshow
the growth on selective media (QDO) compared to the positive control represented by the empty circles. Each variation found in
homozygous, compared with p53/pTD1control growth, makesTRIM32 unable to self-interact.TRIM32_P130S remains able to self
interact. NHL domains are responsible for the Coiled-Coil conformation andTRIM self-interaction function. Pictures of the plates
were taken after 5 days of culture at 301C. [Color ¢gure can be viewed in the online issue, which is available at www.interscience.
4HUMAN MUTATION 0,1^8,2007
Human Mutation DOI 10.1002/humu
Patient 3990 (proband ]4) is a 15-year-old boy, heterozygous for
c.1761_1763delGAT p.D588del. Two maternal cousins also
presented high CK levels.
Elevated CK values (4–5?) were found at the age of 8 years
before a minor surgery. The only symptoms of this patient were
muscle cramps after exercise. Physical examination at the age of 11
years showed scapular winging, no pseudohypertrophy of calves,
and normal muscle strength at upper and lower limbs.
This mutation lies at the fifth TRIM32 NHL motif and inserts a
new restriction site for HphI (Fig. 1; Supplementary Fig. S4).
TRIM32 mutants do not mislocalize.
TRIM-32-GFP fusion proteins to investigate whether TRIM32
mutants display a different subcellular localization to wild-type
controls. We transfected COS7 cells by Polyfect reagent, using
750ng of each plasmid.
TRIM32 localizes to discrete cytoplasmic organelles (Golgi,
endoplasmic reticulum [ER]) and in some preparation the
fluorescence is associated with a diffusely stained background
[Reymond et al., 2001].
No TRIM32 mutant proteins showed any significant difference
with the wild-type form. The proteins show a diffusely background,
sometimes accompanied with a spot pattern (Supplementary
LGMD2H mutants lost the ability to self-interact.
novel mutations occur in a protein domain referred to as NHL
domain (beta-propeller) that represents a conserved region,
possibly involved in protein–protein interaction [El-Husseini and
Vincent, 1999]. These mutations could produce a significant
alteration in the structure and function of this domain.
The strong self-association properties found for all of the TRIM
proteins, are attributed to the Coiled Coil (CC) region that is
considered responsible for the formation of higher order
complexes, since TRIM32 Coiled-coil deletion mutants confirm
these properties of the CC region.
To investigate whether the mutant proteins have lost their ability
to self-interact, we utilized the interaction mating technique.
We tested the self-association ability of TRIM32 that carries the
D487N mutation. This mutation abolished self-binding of
TRIM32. The consequence of this single amino acid replacement
was thus similar to the complete deletion of the coiled coil-NHL
domains (Fig. 2B).
We tested all the novel alleles using the same method. The
homozygous mutations p.R394H and p.1559delC cause the
complete loss of TRIM32 homodimerization, while the BBS11
p.D588del, found only in heterozygosity, showed the TRIM32
homodimerization property, albeit with slower growth (Fig. 2B).
This demonstrates that in addition to the CC region, the NHL
domain is important for self-interaction and mutations abolished
E2N is a speci¢c partner of TRIM32 that is lost
TRIM32 is an E3 ubiquitin ligase, due
to the presence of the RING finger domain in its structure,
TRIM32 specifically self-ubiquitinated in the presence of ATP ,
ubiquitin, E1 enzyme, and E2 enzymes [Kudryashova et al., 2005].
We investigated the ability of different ubiquitin conjugating
enzymes to interact with TRIM32 protein.
Results showed that the specific enzyme E2N interacts with the
TRIM32 full-length protein, while E1 and E3 do not (Meroni G.,
Napolitano L., personal communication).
UBE2N gene (Hs.524630; NM_003348.3) encodes a human E2
that is involved in protein degradation, mainly expressed in muscle
The replacement of the arginine with histidine at position
394, the deletion of the cytosine (nt 1559 of TRIM32 coding
sequence) and the substitution of aspartic acid D487 to asparagine
N (pGad-TRIM32_D487N) inhibited the binding to full-length
Aspartic acid deletion in position 588 shows on selective media
a reduced growth, while p.P130S (causative of BBS) displays a
normal growth (Fig. 3).
Structural changes in NHL domains due to several
We created a computer model of six NHL motifs
using the MolIDE program, supported by the BRAT three-
dimensional structure (http://dunbrack.fccc.edu/molide/).
The effect of the novel variations on the DNA-binding capacity
of NHL domain of TRIM32 was investigated using the crystal
structure of BRAT (PDB entry site). A model was constructed for
amino acid residues 371–643 of NHL of TRIM32. The sequence
identity between target and template for this region was 40%.
Subsequently, the model was refined using YASARA, which was
shown to increase model accuracy [Krieger et al., 2004].
We observed a predicted structure with a six-bladed b-propeller.
Each blade is composed of a highly twisted four-stranded
antiparallel b-sheet. When TRIM32 has the mutations p.R394H
or p.D487N, it may take on a different shape, masking the coiled-
coil region. This could prevent the interaction properties (Fig. 4)
[Edwards et al., 2003; Arama et al., 2000]. However, p.D588del
seems to be heavily altered in its structure, albeit the functional
consequence seems to be weaker. There is possibly a difference
between computer prediction and real conformation that could be
solved by direct conformation analyses.
This prediction supports our previous studies that have shown
that mutations in TRIM32 NHL domains abrogate TRIM32 self-
interaction and E2N binding.
To date, LGMD2H has only been described in the Manitoba
Hutterite ancestry. No other haplotype of LGMD2H/STM has
until now been detected and no other mutation has been
associated with these diseases. TRIM32 gene has also been
involved in a completely different disease: a form of Bardet-Biedl
syndrome, BBS11. The allele cosegregating with BBS11 has the
missense mutation p.P130S at B-Box domain of TRIM32.
There are many examples where different mutations in the same
gene can result in different disorders [Capell and Collins, 2006;
Rankin and Ellard, 2006]. To better understand the mechanisms
underlying the development of muscle disease, we genetically
characterized LGMD2H to determine whether D487N allele
identified in Manitoba Hutterites is the only mutation of
The Hutterites are an Anabaptist sect, who lives in colonies on
North America [Nimgaonkar et al., 2000]. The term ‘‘anabaptist’’
comes from the practice of baptizing individuals who had been
baptized previously. They constitute a unique religious and genetic
group. They migrated from Germany to North America in the
1870s. This population shows a high prevalence of autosomal
recessive disorders. This may be due to founder effects.
LGMD forms are also common in this population. Current
estimates exceed 1/400 compared to 1/15,000 of the general
population [Piluso et al., 2005]. There is a second type of LGMD
in the Hutterite population that maps to chromosome 19q31–q33
and is due to homozygosity for the p.L276I mutation in FKRP . This
is also inherited from a common ancestor [Frosk et al., 2005].
HUMAN MUTATION 0,1^8,20075
Human Mutation DOI 10.1002/humu
In 2005, Frosk et al.  demonstrated that LGMD2H
appears to be more frequent in the Schmiedeleut subdivision of
the Hutterites, whereas LGMD2I is more frequent in the
Dariusleut. The clinical heterogeneity for both LGMD2H
and LGMD2I is due to different disease mechanisms. Presumably,
the more severe phenotype was due to a double homozygote
On the basis of our results, we could not exclude a phenotypic
heterogeneity based on additional modifying loci in patients with
p.D487N mutation. These could be due to variations in other
This studywas conducted to assess whether: 1) LGMD2H is aworld-
wide disease or is confined to the Hutterites; 2) there is only a single
specific TRIM32 allele producing a muscular phenotype in a mutation-
specific fashion, as in other genetic disorders (i.e., achondroplasia,
progeria, etc); and 3) to develop a functional test to check alleles.
This is the first report of LGMD2H in a non-Hutterite
p.D588del, and p.T520TfsX13) in our cohort of European
patients. Mutations are all located at NHL repeats and do not
modify subcellular localization of TRIM32. Given the self-
interaction property of TRIM32, we set up a yeast two-hybrid
interaction mating to test this property using TRIM32 mutants.
When we introduced the p.D487N mutation, no self-interaction
was observed. The same result was obtained using mutations
p.R394H and p.T520TfsX13. p.D588del had a weaker interaction
compared with the wild-type protein. p.P130S/BBS11 was
identical to wild-type TRIM32.
The effect of the homozygous mutations was comparable to
the deletion of the entire CC and NHL, suggesting a dramatic
effect of these mutations on protein folding. In contrast, BBS is
a multisystemic disorder and a different mechanism is likely
FIGURE 3. Interaction betweenTRIM32 (wild-type and mutated forms) and E2N. A:Yeast two-hybrid system. Full-length UBE2N
cDNA was cotransformed withTRIM32 cDNAs into S. cerevisiae.The wild-typeTRIM32 bait andTRIM32 p.P130S variation show
the same grade of interaction with E2N.TheTRIM32 variantsTRIM32 pR394H, c.1559delC, and pD487N baits do not interact with
E2N, indicating that theNHL motif inTRIM32 protein is involved in the interactionwith the enzyme.TRIM32 with the aspartic acid
deletioninposition 588 shows only a retarded growth, a reducedinteractionwith E2Nonselectivemedia. B:Co-IP inCOS7 cells to
con¢rmtheinteractionbetweenE2NandTRIM32 (thearrow pointstothemyc-E2N protein).Totallysates (TL) ofcotransfectedcells
were immunoprecipitated with anti-HA antibody.The interaction is revealed by Western blot using anti-myc antibody.The same
experiment was repeated by immunoprecipitatingTL with anti-cmyc antibody and detection by anti-HA antibody.The interaction is
lostbetweentheD487N_TRIM32 andE2Nproteins. [Color ¢gurecanbeviewedintheonlineissue,whichisavailableatwww.inters-
6HUMAN MUTATION 0,1^8,2007
Human Mutation DOI 10.1002/humu
involved, considering that no phenotypic overlap exists between
these two conditions. The difference among TRIM32 alleles was
evident using the yeast two-hybrid direct interaction mating with
TRIM32_D487N and the three novel variations described here.
We demonstrated that mutations impairing self-dimerization of
TRIM32 are associated with muscular phenotypes. Alleles R394H
and T520TfsX13, similar to the p.D487N mutation, prevent
TRIM32 self-interaction. They all occur in the conserved NHL
domains and may hinder coiled coil homodimerization.
TRIM32 is not a muscle-specific protein. We suggest that the
specific damage to the muscle cells could be due to the disruption
of a muscle-specific interaction as a consequence of NHL
mutations. Since TRIM32 has a putative E3 ligase property, we
tested some E2 enzymes for the interaction. E2N, but not E1 and
E3, was able to bind TRIM32 and this binding was abolished in
mutants. The binding was confirmed by co-immunoprecipitation
(co-IP) assay. E2N is a muscle protein and it can confer tissue-
specificity to TRIM32 mutations.
We can hypothesize that these mutations in NHL domains create
a three-dimensional conformational change in the TRIM32 shape
masking the binding. While the two mutations found in
homozygosity are effective in abolishing the property of interaction,
we have some concerns about the significance of the p.D588del
allele since: 1) this allele was not found in homozygosity; 2) the
result of the assay was not clear-cut. Nevertheless, the mutation
was never found in 600 normal chromosomes. A possible
explanation is that we missed the second mutation of TRIM32. It
may be located outside the sequenced part of the gene.
Alternatively, p.D588del is a rare private variant, with no relation-
ship to muscular dystrophy.
The phenotypes of these LGMD2H patients are mild but they share
the characteristics of an irreversible loss of motility after immobiliza-
tion. We hypothesize that TRIM32 could be linked to the process of
atrophic degeneration/regeneration involving massive muscle protein
ubiquitination. When TRIM32 is mutated this process could be
impaired and muscle regeneration could be insufficient.
Since the phenotype of LGMD2H is produced when a partner of
TRIM32 is lost, the observation that there is a great variability in
clinical severity in patients carrying the same mutation suggests
that other factors may interfere in the course of the disease.
We thank Dr. Nina Canki-Klain for patient samples. We thank
Dr. Graciana Diez-Roux for helpful suggestions. We also acknowl-
edge Anna Cuomo for sequencing and Maria Esposito and
Manuela Dionisi at the TIGEM core facility of Mutation
Detection, and Dr. Maria Grazia Esposito at the Cardiomyology
and Medical Genetics for DNA processing. We acknowledge the
SUN-Naples Human Mutation Gene Bank (Cardiomyology and
Medical Genetics), which is a partner of the Eurobiobank network.
This study was supported by grants from Telethon-UILDM
GUP04008 and TIGEM grant TVNP42TELC (to V.N); Ministero
dell’Universita ` e della Ricerca (PRIN prot 2004062849_004)
(to V.N.); and Ministero della Salute (dlgs. 502/92) (to V.N.),
Ricerca di Ateneo (to V.N. and L.Po.).
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Human Mutation DOI 10.1002/humu