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COL6A1 genomic deletions in Bethlem myopathy and Ullrich muscular dystrophy.

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This work was supported entirely by a grant from The Wellcome
Trust (ref 061140, M.H.).
We are very grateful to the patients who participated in this study
and to Drs P. Nachev and P. Sumner for their comments.
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COL6A1 Genomic Deletions
in Bethlem Myopathy and
Ullrich Muscular Dystrophy
Guglielmina Pepe, MD,1 Laura Lucarini, PhD,1,2
Rui-Zhu Zhang, MD,2 Te-Cheng Pan, PhD,2
Betti Giusti, PhD,1 Susana Quijano-Roy, MD, PhD,3,4
Corine Gartioux, MA,3 Katharine M. D. Bushby, MD,5
Pascale Guicheney, PhD,3 and Mon-Li Chu, PhD2
We have identified highly similar heterozygous COL6A1
genomic deletions, spanning from intron 8 to exon 13 or
intron 13, in two patients with Ullrich congenital muscular dystrophy and the milder Bethlem myopathy. The
5ⴕ breakpoints of both deletions are located within a
minisatellite in intron 8. The mutations cause in-frame
deletions of 66 and 84 amino acids in the amino terminus of the triple-helical domain, leading to intracellular
accumulation of mutant polypeptides and reduced extracellular collagen VI microfibrils. Our studies identify a
deletion-prone region in COL6A1 and suggest that similar mutations can lead to congenital muscle disorders of
different clinical severity.
Ann Neurol 2006;59:190 –195
Mutations in collagen VI genes, COL6A1, COL6A2,
and COL6A3, cause Bethlem myopathy (BM) and Ullrich congenital muscular dystrophy (UCMD).1 Collagen VI is a ubiquitous connective tissue component
comprising three subunits, the ␣1(VI), ␣2(VI), and
␣3(VI) collagen chains, which are folded into a monomer through their central triple-helical domains.2– 4
The monomers undergo intracellular assembly into
dimers and tetramers, which then are secreted extracel-
From the 1Department of Medical and Surgical Critical Care and
Center for the Study of Molecular and Clinical Level of Chronic,
Degenerative, and Neoplastic Diseases to Develop Novel Therapies,
University of Florence, Florence, Italy; 2Department of Dermatology and Cutaneous Biology, Jefferson Institute of Molecular Medicine, Thomas Jefferson University, Philadelphia, PA; 3Institut National de la Santé et de la Recherche Médicale U582, Institut de
Myologie, IFR 14, Groupe Hospitalier Pitié-Salpêtrière; Université
Pierre et Marie Curie, Paris; 4Unité de Neurophysiologie Hôpital
D’Enfants Armand Trousseau, Paris et Unité de Neurologie Pédiatrique, Service de Pédiatrie, Hôpital Raymond Poincaré, Garches,
France; and 5Institute of Human Genetics, University of Newcastle
upon Tyne, Newcastle upon Tyne, United Kingdom.
Received Jul 22, 2005, and in revised form Sep 9. Accepted for
publication Sep 14, 2005.
Published online Nov 8 2005 in Wiley InterScience
( DOI: 10.1002/ana.20705
Address correspondence to Dr Chu, Department of Dermatology
and Cutaneous Biology, Thomas Jefferson University, 233 South
10th Street, Philadelphia, PA 19107.
© 2005 American Neurological Association
Published by Wiley-Liss, Inc., through Wiley Subscription Services
lularly and assembled into microfibrils.5,6 BM, caused
by dominant mutations, is a relatively mild, childhoodonset condition characterized by muscle weakness and
joint contractures.1 UCMD, resulting from either recessive or dominant mutations,7–11 is a severe disorder
presenting with muscle wasting, joint contractures, and
distal hyperlaxity.1 The first dominant mutation reported in a severe UCMD patient involves a COL6A1
genomic deletion of exons 9 and 10 with the 5⬘ breakpoint located immediately downstream of a minisatellite in intron 8.10 Here, we report two larger COL6A1
gene deletions, in which the 5⬘ breakpoints are again
located in intron 8 within the minisatellite, in two patients afflicted with BM and moderate UCMD.
Patients and Methods
Patient A, a white female with French nonconsanguineous
parents, presented with bilateral talus feet deformities and
hip dislocation at birth and axial hypotonia during the first
year of life. She walked at 13 months. At age 5 years, she
showed diffuse amyotrophy, hyperlaxity, and mild weakness
on examination. Creatine kinase levels were elevated at 5 and
8 years (437 and 269U/L, respectively; upper normal limit,
135U/L). At 22 years of age, clinical examination showed
kyphoscoliosis, mild diffuse amyotrophy, hyperlaxity of the
wrists and the distal phalanges of the fingers, and mild generalized muscle weakness sparing the face. She was able to
walk 500m without aid and could help herself up from a
chair with one hand. Mild to moderate joint contractures
were observed in the proximal phalanges of the fingers, hip
abductors, and in the Achilles tendons. Her skin was thin
and showed marked striae, especially in the lower lumbar
regions, buttocks, and upper thighs, but no hypertrophic
scars were observed. Electrodiagnostic studies showed myopathic changes on electromyogram at 5 years and normal
motor and sensory nerve conduction velocities at 5 and 19
years (see Patient 14 in Quijano-Roy and colleagues12). Brain
magnetic resonance imaging was normal. A muscle biopsy
demonstrated a dystrophic pattern at 5 years of age. Laminin
␣2 chain immunolabeling was normal on skin at 19 years of
age. The diagnosis of UCMD was made based on the predominant distal laxity.
Patient B was a 32-year-old British man, with nonconsanguineous parents. His mother had a history of joint tightness
and difficulty walking since early adulthood. He had no contractures at birth and no torticollis. He walked at the age of
2 years and always had a problem with toe-walking, for
which he twice had Achilles tendon release procedures performed, with subsequent recurrence of the problem. He suffered from a relatively nonprogressive muscle weakness, such
that he was unable to walk further than a couple of miles
and needed to use a rail to climb stairs. On examination at
the age of 26 years, he had a rigid spine and prominent contractures of the shoulders, elbows, wrists, finger flexors, hips,
knees, and Achilles tendons. He had proximal and distal
weakness and wasting in the upper limbs and predominantly
Fig 1. COL6A1 mRNA deletions in Patients A and B. (A) cDNA and amino acid sequences of the mutant COL6A1 mRNAs showing a deletion of exons 9 to 13 in Patient A and a deletion of exons 9 to 14 in Patient B. Arrowheads mark exon borders. Nucleotide and amino acid positions are indicated (COL6A1 mRNA reference sequence, NM_001848). (B) Schematic diagram of normal
and mutant ␣1(VI) collagen chains containing in-frame deletions in the triple-helical domain. The normal ␣1(VI) collagen chain consists of a triple-helical domain (TH) of 336 amino acids flanked by N- and C-terminal globular domains made up of von Willebrand
factor–like motifs (N1, C1, C2). The TH domain contains a unique cysteine residue at position 345 (depicted as “C”), which is important for dimer assembly. Patients A and B contain in-frame deletions of different sizes at the N terminus of the triple-helical domain and the cysteine residue is absent in Patient B. The amino acid positions of the deletions are shown in parentheses.
Pepe et al: COL6A1 Genomic Deletions
Fig 2. COL6A1 gene deletions in Patients A and B. (A) Genomic DNA sequences of the deletion junctions in Patients A and B and
their alignment with the normal COL6A1 genomic sequence (NT_011515). Exon sequences are in capital letters. Arrows mark the
breakpoints of the deletions and the nucleotide positions are indicated. Note that 6bp of inserted sequences is present at the deletion
junction of Patient A and that the 3⬘ breakpoint in Patient B is located at ⫺16 of intron 13, which removes the branch site of the
splice acceptor. (B) Schematic diagram of the multiexon deletions in COL6A1, showing the region containing exons 7 to 15 (filled
boxes). Exon 8 is the first exon coding for the triple-helical domain, and exons 9 to 15 each encode discrete numbers of Gly-Xaa-Yaa
repeats. A minisatellite (open box, VNTR) is present at the 5⬘ portion of intron 8. The gene deletions in Patients A and B and in
Patient UC1 reported elsewhere10 begin at different positions within or immediately downstream of the minisatellite.
Fig 3. Consequences of gene deletions in fibroblasts from Patients A and B. (A) Fibroblasts grown in the presence of 50␮g/ml
L-ascorbic acid for 4 days after confluence were fixed in 100% methanol at ⫺20 C for 10 minutes and immunostained with an
antibody specific for the ␣1(VI) collagen chain. The antibody labeling was detected with Cy3-conjungated goat anti–rabbit IgG.
(B) Western blot analysis of cell lysates and culture media from fibroblasts of a control, Patient A, and Patient B with an antibody
specific for the ␣1(VI) collagen chain. Ten micrograms of total protein from the cell lysates and 100␮l of culture medium precipitated with 0.9ml of 100% ethanol were analyzed. (C) Immunoprecipitation of cell lysates and culture media from fibroblasts of a
control individual, Patient A, and Patient B with an antibody specific for the ␣3(VI) collagen chain. Fibroblasts were labeled with
[35S]cysteine overnight. Note that some fibronectin (FN) is also precipitated from the culture media. Samples for Western analyses
and immunoprecipitation were reduced with 25mM DTT and separated on 3 to 8% polyacrylamide gels. The mutant ␣1(VI) collagen chains are depicted as ␣1(VI)m.
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Figure 3
Pepe et al: COL6A1 Genomic Deletions
distal wasting in the lower limbs. He had prominent scars.
Mutations in emerin and lamin A/C were excluded; there
was no muscle biopsy performed. BM was diagnosed based
on the prominent contractures.
The genetic and clinical studies were performed in accordance with institutional review board–approved protocols
and informed consent.
Mutational and Biochemical Analyses
Mutation detection was performed by reverse transcription
polymerase chain reaction (RT-PCR) amplification of the
three collagen VI mRNAs and direct DNA sequencing as detailed.10,13 Genomic DNA regions between introns 3 and 18
of COL6A1 were PCR-amplified using primers 5⬘ATGAGACCCACAGGCGTTATT-3⬘ and 5⬘-CACGTCCACAGCTACATGG-3⬘and the Expand Long Template PCR system (Roche, Indianapolis, IN). Analyses of collagen VI protein
production in fibroblasts by immunostaining, immunoprecipitation, and Western blotting were performed as described.10
Mutational Analysis
We detected COL6A1 mRNA deletions of 198bp
(c.805_1002del, NM_001848) corresponding to exons 9 to 13 in Patient A and 252bp (c.805_1056del)
encoded by exons 9 to 14 in Patient B (Fig 1A). The
resulting mutant chains contain in-frame deletions of
66 and 84 amino acids, respectively, at the
N-terminal region of the triple-helical domain (see
Fig 1B). At the genomic level, Patient A carried a
heterozygous gene deletion of 2.49kb spanning from
IVS8⫹248 to exon13⫹16, whereas Patient B harbored a heterozygous gene deletion of 2.35kb between IVS8⫹790 and IVS13⫺16 (Fig 2A). The gene
deletion in Patient B removed the branch point of the
splice acceptor site in intron 13. Hence, although the
intron 13/exon 14 junction sequence remained intact,
exon 14 was skipped in the mature mRNA. The
5⬘ breakpoints of both deletions occurred within a
minisatellite located at the 5⬘ portion of intron 8 (see
Fig 2B). The gene deletion in Patient A was not
found in her parents and sister and thus is de novo.
The deletion in Patient B was found in his 2-year-old
daughter, who is asymptomatic thus far. The genomic
DNA of his mother was not available for analysis.
Consequences of the Mutations
Immunostaining with an antibody against the ␣1(VI)
collagen chain14 showed that fibroblasts from both patients deposited few collagen VI microfibrils extracellularly (Fig 3A). Western blot analysis showed that the
shortened ␣1(VI) collagen chains from both patients
were preferentially accumulated inside the cells in comparison with their normal counterparts, and substantial
amounts of the shortened chains were secreted into the
media (see Fig 3B). Using an antibody specific for the
␣3(VI) collagen chain,15 both normal and shortened
Annals of Neurology
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␣1(VI) collagen chains could be coimmunoprecipitated
with the other two chains from cell lysates of these two
patients, demonstrating that both mutant chains could
be assembled into monomers (see Fig 3C). In contrast,
only small amounts of the mutant ␣1(VI) collagen
chains could be coimmunoprecipitated in culture media from both fibroblasts, indicating that the mutant
monomers were not efficiently secreted or were unstable after secretion (see Fig 3C).
The 5⬘ deletion breakpoints of both patients are located in a minisatellite in intron 8 of Col6A1, which
comprises tandem repeats of the sequence ccagatggagggga(t/c)ggcggg(a)gt. There are 32 repeats in the
NCBI COL6A1 reference sequence (NT_011515).
Minisatellites constitute chromosomal fragile sites and
have been associated with recurrent translocation
breakpoints.16 In both patients, the DNA regions involved in deletion formation do not show extensive sequence identity, suggesting that the deletion occurred
through a mechanism of nonhomologous recombination.17 In Patient A, a 6bp insertion was present in the
deletion junction (see Fig 2A), consistent with a
Ku-mediated nonhomologous end joining of DNA termini.18 A similar deletion previously reported in a severe UCMD patient starts immediately downstream of
the minisatellite in intron 8 and ends in intron 10 (see
Fig 2B, Patient UC1).10 Together, these findings indicate the presence of a deletion-prone region involving
the minisatellite in intron 8 of COL6A1.
Characterization of fibroblasts from both patients
did not show significant differences in the consequences of the two mutations, even though the deletion removed a cysteine important for collagen VI
dimer assembly in the BM patient but not in the
UCMD patient. Coimmunoprecipitation studies show
that monomers consisting of mutant chains with large
triple-helical deletions are unstable and inefficiently secreted, thereby preventing a strong dominant effect
(see Fig 3C). In contrast, the mutant monomers from
the patient with a smaller heterozygous deletion of exons 9 and 10 are efficiently secreted and can act dominantly on microfibillar assembly.10 This provides an
explanation for the milder phenotypes of the two patients in this study as compared with the severe
UCMD patient with a smaller deletion reported previously.10
In comparing the clinical features of Patients A and
B, the main difference observed, other than the severity
of illness, is the presence or absence of marked joint
laxity in the initial stages of the disease. Although Patient A displays the characteristic feature of UCMD,
her subsequent clinical course, with the maintenance of
independent mobility at the age of 22 years, is in keeping with the Bethlem myopathy phenotype, despite
displaying relatively few contractures. Given that the
biosynthetic consequences of the mutations in fibroblasts are similar, the differences in the clinical presentations of these two patients may not be directly related
to the extent of genomic deletions but instead may involve other modifying factors such as a protein that
could serve a similar function as collagen VI. In this
regard, recent studies have suggested molecular heterogeneity in BM and UCMD.11,13,19
In conclusion, our studies demonstrate that the presence of a minisatellite in COL6A1 intron 8 predisposes
that area of the gene to multiexon deletions in collagen
VI–related muscular dystrophies. The prevalence of
multiexon deletions in BM and UCMD may be underestimated because this type of mutation is not detectable by genomic DNA analysis of individual exons
and may not be detectable by short-range RT-PCR
analysis. This finding underscores the necessity of longdistance RT-PCR and protein analyses in the accurate
molecular diagnosis of collagen VI–related muscle disorders.
This work was supported by the NIH (National Institute of Arthritis and Muscoskeletal and Skin Diseases, AR38912, M.-L.C.), the
European Community Grant (QLG1-CT-1999-00870, G.P.), the
Institut National de la Santé et de la Recherche Médicale, (P.G.),
Association Française contre les Myopathies, (P.G.), the Institut des
Maladies Rares, (P.G.), and the Muscular Dystrophy Campaign for
Centre Grant support (K.M.D.B.).
We thank the patients and their families for their participation of
this study and Dr F. Renault, who counseled Patient A.
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Pepe et al: COL6A1 Genomic Deletions
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myopathy, ullrich, muscular, deletion, genomics, col6a1, dystrophy, bethlem
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