From the Heritable Disorders Branch/NICHD, National
Institutes of Health, Bethesda, Maryland 20892 and the §
Division of Orthopaedics, The Hospital for Sick Children, Toronto,
Ontario M5G1X8, Canada
Received for publication, December 27, 2000, and in revised form, January 17, 2001
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ABSTRACT |
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We studied four affected individuals from a
family of three generations with Ehlers-Danlos Syndrome II. Type
V collagen transcripts of affected individuals were screened by reverse
transcriptase-polymerase chain reaction. Amplification of the exon
9-28 region of Ehlers-Danlos Syndrome
(EDS)1 is a heterogeneous
group of connective tissue disorders (1). Clinical manifestations in
the milder forms of the disorder occur predominantly in the dermis (loose skin, abnormal scars, easy bruising) and joints
(hyperextensibility), whereas the vascular (aneurysms) and visceral
(ruptures, pneumothoraces) symptoms are found in the severe type IV
form of EDS. A corresponding heterogeneous range of gene defects causes
the broad range of EDS forms. These forms have involved the genes for
type I (EDS VII) or III (EDS IV) collagen or for enzymes involved in
the collagen metabolic pathway (EDS VI and VII) (2). Recently, a
noncollagenous defect in tenascin-X has also been demonstrated in EDS
(3). Furthermore, there must be a number of additional genes in which defective forms cause EDS, because many EDS patients do not have mutations in the genes already described.
In the last 4 years, type V collagen joined the growing list of
matrix molecules associated with Ehlers-Danlos Syndrome. Type V
collagen was a prime candidate for such a role because it is present as
a minor component of the extracellular matrix in tissues in which type
I collagen is the predominant structural molecule, especially skin and
tendon (4). Type V collagen is a fibrillar collagen in which the
central helical region is the same length as that of type I collagen.
It occurs as two forms of heterotrimers or The role of this type V collagen in the matrix appears to involve the
formation of heterotypic fibrils with type I collagen and a direct
effect on regulating the diameter of those fibrils. The formation of
heterotypic fibrils by types I and V collagen was the first
codistribution of different collagen types into fibrils to be
demonstrated (8). Double-labeled immunoelectron microscopy with
colloidal gold-tagged monoclonal antibodies was used to simultaneously
localize types I and V collagen in chick corneal stroma fibers and
demonstrate that type V epitopes were blocked unless fibrils were
partially dissociated. Because fibrils are relatively thin in corneal
stroma and type V is more abundant than in other type I collagen
predominant tissues, a regulatory role for type V was postulated in
limiting the diameter of heterotypic fibrils. This was confirmed by
in vitro studies forming copolymers of types I and V
collagen. Increasing the proportion of type V collagen resulted in a
progressive decrease of fibril diameter (9). The retained N-terminal
portion of type V Both structural defects and null alleles of type V collagen have been
delineated in patients with EDS. Clinically, these patients have had
EDS I or II, the gravis and mitis forms in the older Berlin nosology
(12), now also described collectively as the classical type in the more
recent Villefranche nosology (13). Of the structural defects in type V
collagen, seven were in the In the present study, we have characterized molecularly and
biochemically a type V collagen mutation causing a straightforward null
allele. Steady-state secretion of type V collagen is decreased. Also,
both steady state-secreted collagen and selective salt precipitation of
proband secreted collagen show maintenance of the
Cell Culture--
Dermal fibroblast cultures were established
from skin punch biopsies of individuals III-3, III-10, and IV-5 (Fig.
1). Cells were grown in Dulbecco's modified Eagle's medium, enriched
with 10% calf serum plus 2.0 mM glutamine in the presence
of 5% CO2.
RT-PCR Screening--
Total RNA was isolated from cultured
fibroblasts using Tri Reagent (Molecular Research Center, Inc.,
Cincinnati, Ohio), according to manufacturer protocol (23). Patient and
control total RNA (0.5-1 µg) were reverse-transcribed using murine
leukemia virus reverse transcriptase, oligo(dT), and an RNA PCR core
kit (PerkinElmer Life Sciences) for 1 h at 42 °C. After
cDNA synthesis, regions along both
To detect additional alternatively spliced products, primer E9s or a
sense primer in exon 13, E13s (5'-CCCATGGTCTCAGCCCAGGAGTCCCAG-3', nt
1609-1636) (25)), was paired with an antisense primer I13as (5'-TGAGACAGGGCCCCAGCAGCAGTGCTC-3') annealing to the 3'-end of intron
13.2 After synthesis of cDNA as described above, PCR
was performed at 94 °C for 1 min followed by 35 cycles of
94 °C for 20 s, 68 °C for 15 s, and 72 °C for
40 s.
Subcloning and Sequencing--
RT-PCR of patient fibroblast
total RNA was performed using the primer pair E13s and E28as.
Amplification of cDNA was as follows: 94 °C for 1 min and then 5 cycles of 94 °C for 20 s, 64 °C for 30 s, and 72 °C
for 40 s and 30 cycles of 94 °C for 20 s, 66 °C for
30 s, and 72 °C for 40 s, followed by a final extension at 72 °C for 7 min. PCR amplification of patient fibroblast DNA was performed using a sense primer in intron 13, I13s
(5'-AGGGTGATGGGCTGGGAGCAG-3', nt
Subclones were sequenced using the dideoxy chain termination method
(26) and the Promega fmol DNA cycle sequencing system kit. The
primer for both normal and alternatively spliced cDNA fragments and
the mutant DNA was the pUC reverse primer (Promega, Madison,
Wisconsin). The subclone containing normal DNA was sequenced with the
pUC forward primer (Promega).
Analysis of Leukocyte DNA--
Leukocyte DNA from IV-2, IV-3,
IV-5 and fibroblast DNA from III-3 and III-10 were isolated using the
PureGene DNA Isolation kit (Gentra Systems, Inc., Minneapolis, MN). To
determine the presence of the mutation, DNA was amplified by PCR using
the primer pair I13s and E14as. The 356-bp product was digested with
Aci I (New England Biolabs Inc., Beverly,
Massachusetts), electrophoresed on a 2% agarose gel, and visualized
with ethidium bromide.
RT-PCR of Proband Total RNA from Cycloheximide-Treated
Cells--
Confluent fibroblasts in growth medium were treated with
100 µg/ml cycloheximide for 4 h (27). Total RNA was isolated as described above. RT-PCR used primer pair E9s and E28as and the same
cycling conditions used for screening.
RNA Protection Analysis--
The mutant and normal cDNA
fragment spanning exons 13-28 was re-isolated from the TA
cloning vector (pCR2.1) by digestion with EcoRI (Life
Technologies, Inc.) and subcloned into pGEM3Zf( Protein Analysis--
Fibroblasts from III-3 (P5-10) and the
control cells (GMO 3349, P8-15) were plated at a density of
2.5 × 105 cells/35-mm culture dish and were allowed
to attach overnight at 37 °C. The cells were pre-incubated with
ascorbate for 2 h and then labeled with medium containing 50 µg/ml ascorbate and 325 µCi of
L-[2,3,4,5-3H]proline/well (Amersham
Pharmacia Biotech) for about 16 h. The medium and cell layer
procollagens were harvested separately as described previously (29).
Samples were precipitated with EtOH (half-volume of 95% ethanol) and
then centrifuged at 37,000 × g for 40 min. The pellets
were resuspended in 0.2 M NaCl, 50 mM Tris, pH
7.5. Collagens were prepared by digestion of procollagen with pepsin
(75 µg/ml), electrophoresed on a 5.5% polyacrylamide-2 M
urea-SDS gel, and visualized by autoradiography.
For selective salt precipitation, proband fibroblasts were stimulated
with ascorbate and labeled with
[14C]L-proline (250 mCi/mmol, Amersham
Pharmacia Biotech). Medium was acidified and digested with pepsin (1:10
weight ratio to collagen as measured by the Sircol assay). The
pepsinized sample was concentrated by Centricon 30 and then
successively precipitated with 0.9, 1.2, and 2.0 M NaCl
using a modified procedure from Jimenez et al. (6) and
Rhodes and Miller (30). Samples were electrophoresed on a 5%
polyacrylamide-2 M urea-SDS gel.
Transmission Electron Microscopy of Proband Dermis--
A dermal
punch biopsy was obtained from the upper arm of proband IV-5, and a
control matched for age, gender, and race. The sample was fixed in
2.5% glutaraldehyde and then treated with 1% osmium tetroxide
followed by en bloc staining with 2% uranyl acetate. After
dehydration, the tissue was infiltrated with Spurr's plastic resin.
600-800 Å sections were obtained with an AO Reichert Ultracut
ultramicrotome mounted on copper grids and stained with lead citrate.
The stained grids were examined in a Zeiss EM10 CA transmission
electron microscope, and representative areas were photographed (JFE
Enterprises, College Park, MD).
The Affected Members of an Extended Pedigree Have Symptoms of Mild
EDS II--
Affected individuals were ascertained at two different
medical centers from two branches of a five-generation pedigree and referred independently to the NIH Clinical Center (Fig.
1). Unless otherwise specified, the
molecular and biochemical data were obtained from proband A and her
affected sons.
III-3 is a 58-year-old woman (proband A). Her height is 153 cm (5% for
adult women, 50% for 12-year-old girls), and her span is 160 cm. She
has soft skin on her face and arms. The skin is moderately loose around
the neck and elbows, and moderate bruising was noted. Lacerations have
healed with cigarette paper scars. She reports that her hip and knee
joints were hyperextensible as a child but this has decreased with age.
She carried four pregnancies including a twin pregnancy to term without
premature rupture of membranes or other complications. At age 49, she
had a hysterectomy for uterine prolapse.
IV-5 is the 18-year-old son of III-3. He weighed 10 lb, 1 oz at birth.
He was first evaluated for EDS at age 3.5 years when he was noted to
have thin skin, easy bruising, and a pectus excavatum. At that time,
his height was 97.3 cm (50% for age). The skin around face, neck,
elbows, and knees is moderately hyperextensible. Multiple bruises were
noted on arms and legs. He has several cigarette paper scars. Large
joints are not hyperextensible. Pes planus was noted.
IV-3 is the 24-year-old son of III-3. He weighed 9 lb, 12 oz at birth.
At age 10 years, he had a height of 124 cm (<5% for age, 50% for
7.5-year-old boys). From infancy, he was noted to have moderately loose
skin, easy bruising, and thin scars. He does not have
hyperextensibility of large or small joints.
III-10 is a 46-year-old woman (proband B). Her height is 159 cm (25%
for adult women, 50% for 14-year-old girls). She has moderately loose
skin around the elbows and knees, but facial and neck skin are not
extensible. Her thumbs are hyperextensible, both elbows and knees have
mild recurvatum. Superficial lacerations have healed normally with the
exception of one cigarette paper scar on her leg. There is no evidence
of bruising. Her surviving son reportedly is mildly affected but
declined examination. Two additional pregnancies were complicated by
premature rupture of membranes at 27-28 weeks. The babies died of
complications of prematurity at 14-16 days. No tissues were collected.
RT-PCR of Proband Mutant cDNA Contains a 100-bp Intronic Insertion between Exons
13 and 14--
To define the structure of the abnormal cDNA, the
exon 13-28 region of
To determine whether additional alternative acceptor sites were used,
RT-PCR was performed using an antisense primer complementary to the
3'-end of intron 13 paired with sense primers in exons 9 or 13. Only
the product containing the 100-bp insertion was detected.
The Gene Level Defect Is a Point Mutation in the Exon 14 Splice Acceptor Site--
The region of the
The mutation generates a novel Aci I site. Aci I
digestion of a genomic PCR product containing the mutation was used to
confirm heterozygosity in affected family members (III-3,
III-10, IV-3, and IV-5) and the absence of the mutation in an
unaffected member (IV-2) (Fig. 5).
Use of a Cryptic Splice Acceptor Site Shifts the Collagen Reading
Frame Resulting in the Use of a Premature Termination Codon and a
Decreased Stability of the Mutant Transcript--
The 100-bp intronic
insertion in the
To more accurately quantitate the relative amounts of normal and mutant
Proband Fibroblasts Secrete Reduced Amounts of Type V Collagen with
Normal
The composition of the type V collagen secreted by the proband
fibroblasts was not altered by the haploinsufficiency of Transmissionelectron Microscopy of Proband Dermal Biopsy Reveals
Variation in Size and Shape of Type I Collagen Fibrils--
The
collagen fibrils from a dermal punch biopsy of individual IV-5 were
examined by scanning electron microscopy (Fig.
10) in comparison to the fibrils of a
control matched for age, gender, and race. On the longitudinal section,
a predominantly normal fibril size and arrangement were noted with
occasional wide fibrils (Fig. 10, panel A). On
cross-section, heterogeneity in fibril diameter was noted in the
proband sample as were occasional fibrils with a cauliflower
configuration (Fig. 10, panels B-D). The diameters of 500 fibrils each from proband and control were measured (Fig. 10,
panels B and E). Although the median fibril
diameter is about the same for the proband and control, the proband has
more fibrils with both a larger and smaller diameter.
This report describes a type V collagen splicing defect in a
pedigree with type II of Ehlers-Danlos Syndrome. Because collagen exons
encode an exact multiple of the characteristic
Gly-X-Y helical amino acid sequence (32), splicing
defects that result in the straightforward splicing-out of an exon
would leave the reading frame intact and cause the production of a
structurally abnormal No functional Haploinsufficiency is apparently a general mechanism for a sizable
proportion of EDS I and II cases, of which this is the first case
reported in molecular, biochemical, and ultrastructural detail.
Schwarze et al. (22) examined Nonsense-mediated decay is one type of mRNA surveillance
during which transcripts with premature termination codons are
degraded by a mechanism involving rapid decapping while still
fully adenylated (36). Premature termination codons are known to have a
greater effect on mRNA decay if located early in the transcript
(37), and the termination codon in this pedigree fills that
expectation. However, other elements must also be involved, because
most of the other cases of EDS with haploinsufficiency are located
relatively 3' in the transcript. The sequence context of the
termination codon is also known to be crucial. Downstream
cis-acting elements promote mRNA decay. Specifically,
the presence of one or more ATG codons enhances destabilization,
possibly by promoting translational re-initiation (38). ATG codons are
located 14 and 135 nt downstream of the premature stop codon in the
mutant transcript described here.
Comparison of this null mutation with COL V structural defects provides
new insight on genotype/phenotype relationships of COL V mutations.
Seven structural abnormalities of This distinction between the genotype/phenotype relationships of types
I and V collagen may reside in their different roles in heterotypic
fibrils. For type I collagen null alleles, a diminished quantity of
fibrils does not have a major impact on skeletal tissue and causes mild
osteogenesis imperfecta. In contrast, because of the regulatory role of
type V collagen in fibril formation, both quantitative and structural
defects of type V collagen cause abnormalities of fibril diameter,
which are crucial to the integrity of the skin and tendon. The four
cases of EDS with COL V structural defects for which transmission
electron microscopy of fibrils has been published include
splicing defects of 1(V) yielded normal and larger products from the
proband. Sequencing of cDNA revealed a 100-base pair insertion from
the 3'-end of intron 13 between exons 13 and 14 in one allele. The
genomic defect was identified as an A
2
G
substitution at the exon 14 splice acceptor site. A cryptic acceptor
site
100 nucleotide within intron 13 is used instead of the mutant
splice site. The insertion shifts the reading frame +1 and results in a
stop codon within exon 17. The mutant transcript was much less abundant
than normal allele product in untreated cultured fibroblasts but was
approximately equimolar in cycloheximide-treated cells, suggesting that
the mutation causes nonsense-mediated decay of mRNA. By RNase
protection experiments, the level of mutant transcript was determined
to be 8% that of the normal transcript in untreated proband
fibroblasts. Relative to type I collagen, proband fibroblasts secreted
only 65% of the amount of type V collagen secreted by normal controls.
Selective salt precipitation of proband secreted collagen provided
supportive evidence that the
chain composition of type V collagen
remains
1(V)2
2(V) even in the context of
1(V)
haploinsufficiency. Type V collagen incorporates into type I collagen
fibrils in the extracellular matrix and is thought to regulate
fibril diameter. Transmission electron micrographs of type I collagen
fibrils in a proband dermal biopsy showed greater heterogeneity in
fibril diameter than in a matched control. The proband had a greater
proportion of both larger and smaller fibrils and occasional fibrils
with a cauliflower configuration. Unlike the genotype/phenotype
relationship seen for type I collagen defects and osteogenesis
imperfecta, the null allele in this family appears to cause clinical
features similar to those seen in cases with structural alterations in
type V collagen.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1(V)3
homotrimer. In the dermis, tendon, and bone,
1(V)2
2(V) is the usual trimer composition (5-7).
chains was essential to the regulatory role. This
regulatory model involves the localization of type V trimers at the
periphery of type I fibrils with the helical regions of type V buried
and the N-terminal extensions of type V projecting onto the surface and
limiting fibril growth by steric hindrance (10). This role was
supported by gene targeting experiments of COL5A2 in mice. Homozygous
mice producing structurally abnormal
2(V) lacking the N-telopeptide have reduced tissue strength and fibrils that are heterogeneous in size
(11).
1(V) chain, and three were in the
2(V) chain (14-20). Most cause splicing defects, two cause amino
acid substitutions, and one results from a chromosome translocation.
Transmission electron micrographs of heterotypic fibrils in four
cases are characterized by the irregularity of fibril shape and
variation in diameter. More recently, null alleles of COL5A1 have been
recognized as a cause of a significant proportion of classical EDS (21,
22). Transmission electron micrographs in one patient showed larger and
more irregular fibrils (22).
1(V)2
2(V) chain composition of type V collagen even
in the context of
1(V) haploinsufficiency. Heterotypic dermal
fibrils have greater heterogeneity of diameter than in the control. The
implications of these data for understanding the genotype/phenotype
paradigm in EDS are discussed.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1(V) and
2(V) transcripts
were amplified using Taq polymerase (Life Technologies,
Inc.) (24). PCR reactions contained 2 mM MgCl2,
300 µM each primer, 600 µM dNTP, and 2.5 units of Taq polymerase. The PCR reaction amplifying the
region of
1(V) cDNA from exons 9 to 28 used the forward primer
E9s (5'-GGGAGAACCAGCGATTAT-3', nt 1365-1382) and a reverse primer
E28as (5'-CCTTCAGACCACGGATGC-3', complementary to nt 2396-2413) (25).
Cycling conditions were 2 min at 95 °C and then 35 cycles of
94 °C for 45 s, 58 °C for 30 s, and 72 °C for
45 s, and a 7-min final extension at 72 °C. The 1049-bp
amplification product was digested separately with the restriction
enzymes HhaI and NcoI (Life Technologies, Inc.) electrophoresed on a 2% agarose gel and visualized with ethidium bromide according to manufacturer protocols.
323 to
303),2 and an
antisense primer in exon 14, E14as (5'-ACCCATCGGGCCAGCTGGTCC-3', complementary to nt 1675-1695) (25). PCR conditions were as follows: 94 °C for 1 min and then 35 cycles of 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s, followed by a
final extension at 72°C for 7 min. Products were electrophoresed
on a 1% agarose gel and isolated using QIAquick Gel Extraction Kit.
(QIAGEN, Inc., Valencia, CA). They were then subcloned using the
TA cloning system (Invitrogen, Carlsbad, CA).
) plasmid (Stratagene,
La Jolla, CA). Uniformly labeled antisense riboprobe was synthesized as
described previously (28) using linearized plasmid carrying the mutant
form of exons 13-28, T7 RNA polymerase (Promega), and
[
-32P]CTP. Targets for RNA protection comprised
patient and control fibroblast total RNA (5 µg) or normal and mutant
sense synthetic RNA transcript spanning exons 13-28 (50 ng). T7 or SP6
RNA polymerase (Promega) was used to transcribe normal or mutant sense
target RNA, respectively. Target RNAs were mixed with 2 × 105 cpm of antisense probe, denatured at 85 °C for 5 min, and then hybridized overnight at 50 °C. The hybrids were
digested with RNase A (8 µg/ml Type X, Sigma) as described previously
(28). Digestion products were electrophoresed on 5% polyacrylamide-7 M urea gels.
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Pedigree of family with EDS type II.
IV-5 received attention because of easy bruising in childhood and
III-10 was noted to have abnormal skin during an obstetric admission.
Individuals III-3, III-10, IV-1, IV-3, IV-4, and IV-5 were examined by
J. C. M. Remainder of pedigree was obtained by report.
1(V) RNA Yields both Normal and Larger Size
Products--
The type V collagen mRNAs of a series of patients
with EDS types I and II were screened for structural alterations by
RT-PCR of overlapping regions along both
1(V) and
2(V)
transcripts. Amplification of the region of
1(V) cDNA
encompassing exons 9-28 (17) from patient III-3 yielded two products,
the 1049-bp fragment expected from normal cDNA structure and a
unique minor product ~100 bp larger than the normal fragment
(Fig. 2A). Digestion of the
amplification products with HhaI and NcoI
localized the structural alteration to the region spanning exons 13-23
(Fig. 2, B-C).
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Fig. 2.
Screening of cDNA by RT-PCR amplification
and restriction enzyme digestion. A, analysis of
cDNA by RT-PCR. Selected EDS patients were screened by RT-PCR along
both chains of type V collagen. Amplification of total RNA from patient
III-3 (lane 4) for the exon 9-28 region of 1(V) revealed
a second band approximately 100 bp larger than the expected 1049-bp
fragment from normal cDNA. Lanes 1-3, RT-PCR product
from other EDS patients; lane 4, RT-PCR product from III-3;
lane 5, RT-PCR product from control cell line. B,
digestion of RT-PCR products in panel A with HhaI
and NcoI. Lanes 1 and 3, digestion of
unrelated EDS patients; lane 4, digestion of III-3;
lane 5, control cell line PCR fragment. C, a
schematic presentation of cDNA amplified by RT-PCR and
digested by HhaI and NcoI. Diagram
shows exons, restriction enzyme digestion sites, and sizes of expected
cleavage products.
1(V) cDNA was amplified by RT-PCR,
yielding an 804-bp fragment expected from the normal cDNA and a
fragment ~100 bp larger (data not shown). Both products were
subcloned and sequenced (Fig. 3,
A and B). The 804-bp product contained normal
cDNA sequence for this region (25). The unique larger fragment
contained a 100-bp insertion between exons 13 and 14 derived from the
3'-end of intron 13. Its sequence exactly matches the known intronic sequence (31)2 except for an
A
2
G change within the exon 14 splice acceptor site.
Inspection of intron 13 sequences near the 5'-end of the insertion
reveals a cagt sequence located 100 bp upstream of exon 14, which is
used as the alternative acceptor site.
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Fig. 3.
Sequence of mutant and normal cDNA.
A, the sequence of the cDNA with the 100-bp insertion is
shown. B, a schematic diagram for splicing of
normal and mutant cDNA. Exonic and intronic sequence are shown in
capital and lowercase letters, respectively. The
cagt cryptic acceptor site is underlined, and the bases
involved in the transversion (a g) are in bold.
C, a diagram of
1(V) null allele caused by
100-bp intronic insertion. The insertion causes a shift of +1 in the
reading frame and the occurrence of a premature termination codon (UGA)
downstream in exon 17. The triple helical domain begins in exon
14.
1(V) gene extending
from about 300 bp at the 3'-end of intron 13 into exon 14 was amplified
from the leukocyte DNA of patient III-3, subcloned, and sequenced (Fig.
4). One set of subclones contained normal
intron 13 sequences. A second set contained the same A
2
G transversion in the exon 14 splice acceptor site noted in the
mutant cDNA.
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Fig. 4.
Sequence of normal and mutant genomic DNA
at 1(V) intron 13/exon 14 junction. Sequencing revealed the same A
2
G
change in the exon 14 splice acceptor site as previously noted in the
cDNA sequence. Exonic sequence identified by capital and
lowercase letters indicates intronic sequence. The
arrow indicates the transversion.
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Fig. 5.
Confirmation of mutation status in other
members of pedigree by Aci I digestion of genomic PCR
products. A 356-bp region of 1(V) including the 3'-end of
intron 13 and part of exon 14 was PCR-amplified from DNA of available
family members. The novel Aci I site results in 320 and
356-bp products. Digests were electrophoresed on a 2% agarose gel.
Lane 1, 100-bp ladder; lanes 2-5, family members
II-3, IV-2, IV-3, and IV-5, respectively.
1(V) transcript resulted in a +1 shift of the
collagen reading frame. In the new reading frame, exons 14-16 are
translated into a noncollagenous protein chain, and there is a
premature termination codon (UGA) at the sixth amino acid of
exon 17 (Fig. 3C) (25). We compared the stability of mutant
transcript in proband total RNA isolated from fibroblasts with and
without cycloheximide treatment (27). The exon 9-28 region was
amplified from both RNAs (Fig. 6). In the RT-PCR using RNA from treated cells, the normal and mutant products were approximately equimolar, whereas in the amplification from untreated cells, the mutant product was ~7-fold less abundant than
the normal product. The decreased stability of the mutant transcript
was presumably due to nonsense-mediated decay.
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Fig. 6.
Analysis of mutant transcript stability by
RT-PCR. Total RNA was isolated from fibroblasts of III-3 with and
without cycloheximide treatment. The region of cDNA including exons
9-28 was amplified by RT-PCR. Products were electrophoresed on a 1.2%
agarose gel. Lane 1, 100-bp ladder; lanes 2 and
3, RT-PCR from proband III-3 RNA using RNA from treated and
untreated cells, respectively; lane 4, RT-PCR from control
cell RNA.
1(V) transcript in untreated cells, RNase protection (28) was
performed (Fig. 7). The uniformly labeled
antisense probe for this assay was transcribed from a subclone of
proband mutant cDNA encompassing exons 13-28. Mutant mRNA
would protect a 914-nt probe fragment, whereas normal transcript would
protect fragments of 760 and 54 nt. Protection of the expected probe
fragments was verified using synthetic sense transcripts of the normal
and mutant cDNAs. When the probe was hybridized with proband total RNA, probe protection by mutant transcript appeared to be less than
10% of that protected by the normal transcript. Excision, solubilization, and scintillation counting of the protected fragments corresponding to mutant and normal transcripts demonstrated that the
level of mutant transcript was 8% that of the normal transcript.
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Fig. 7.
Quantitation of normal and mutant
1(V) transcripts by RNase protection.
A, RNase A protection of in vitro transcripts and
total fibroblast RNA. To confirm that this antisense probe would
protect the predicted fragments with equal efficiency, it was
hybridized with synthetic sense transcripts from the same region of
normal and mutant cDNA (lanes N and M).
Hybridization with total RNA (lanes C and ED)
yielded the normal 760-nt product from both proband and control cells.
Only proband RNA protection also contains a minor amount of the full
probe protection product from mutant transcript (arrow).
Lane
,
X HaeIII marker; lane P,
probe. B, diagram of mutant RNA antisense probe and the
expected protection fragments from hybridization with normal and mutant
transcripts. Hybridization of probe to normal RNA does not protect the
intronic insertion present in mutant probe and yields a 760-nt
protected fragment.
Chain Composition--
Procollagen was precipitated from
both the medium and cell layer of fibroblasts from proband III-3 and
control (GMO 3349). Collagens were derived from these samples by pepsin
digestion (Fig. 8A). In the
cell layer sample, the type V
chains appear to have an
approximately equimolar ratio. To normalize the collagen secreted into
the medium, lanes were loaded to achieve equal intensity of
1(I). Longer exposure of the media samples revealed that proband secretion of
1(V) was ~65% that of control cells, supporting the
haploinsufficiency detected at the RNA level. A prominent band was
noted on several preparations migrating between
1(I) and
2(I).
CNBr peptide analysis (Fig. 8B) revealed that in the control
sample this was a mixture of incompletely pepsin-digested
2(I) chain
and some slightly truncated
1(I) chain. For the proband, the band
consisted of an incompletely digested
2(I) chain. The intensity of
this band was variable from preparation to preparation. It had no
apparent significance for type I/type V collagen interactions in EDS.
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Fig. 8.
Collagen synthesis by fibroblasts of proband
III-3 (EDS) and control (C)
cells. A, secreted and cell layer procollagen from
cultured fibroblasts was digested with pepsin and analyzed by
SDS-urea-polyacrylamide gel electrophoresis . B, CNBr
digestion of band migrating between 1(I) and
2(I) in proband and
control samples.
1(V) chain.
The type V collagen appeared to have an
1(V)2
2(V)
composition in the steady-state sample (Fig. 8). In addition, a sample
of secreted collagen was obtained from ascorbate-stimulated proband fibroblasts and subjected to serial selective salt precipitation (Fig.
9). All of the type V collagen appeared
in the 1.2 M NaCl pellet as expected. This sample also had
the expected 2:1 ratio of
1(V) and
2(V) chains.
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Fig. 9.
Fractionation of proband secreted collagen by
sequential salt precipitation. The NaCl concentration at which the
pellet was obtained is indicated below in each lane. Samples
were electrophoresed on a 5% polyacrylamide-2 M urea-SDS
gel.
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Fig. 10.
Transmission electron
micrographs of type I collagen fibrils from a dermal
biopsy of patient IV-5 and matched control sample. A,
longitudinal section of proband collagen fibrils. Occasional wide
fibrils are indicated by small circles. B-D,
cross-sections of proband collagen fibrils. Fibrils with a smaller
diameter than normal are indicated by arrowheads, and
occasional cauliflower configurations are indicated by
arrows. E, cross-section of matched control
collagen fibrils. Magnifications: A, 75,000; B,
48,000; C, 75,000; D, 94,500; and E,
48,000. Bar graph shows the distribution of fibril diameters
in proband and control samples.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
chain. Instead, the A
2
G
defect we delineated in an
1(V) exon 14 acceptor site resulted in
the use of a cryptic acceptor site
100 nt in intron 13. The cryptic
acceptor had the sequence cagt rather than the usual cagg of
1(V)
exon 14 and most other fibrillar collagen splice acceptor sites. In
addition, there were a handful of alternative cagg sites in the 3'-end
of intron 13 in which the position ranged from
57 to
378
nt.2 Careful examination of the mutant transcript by RT-PCR
with an antisense primer at the extreme 3'-end of intron 13 paired with sense primers in exon 9 or 13 revealed no additional alternative splicing products. The exclusive use of the
100 site probably resulted from the presence of a strong adjacent (T/C) run containing 17/19 T or C in the upstream sequence. The cagg site at
64 with 9/14
adjacent T or C nt was the next best candidate and would have been
detected in our assay.
1(V) chain should be produced from this aberrantly
spliced transcript with a 100-nt insertion. First, the 100-nt insertion
shifts the collagen reading frame +1 and causes the occurrence of a
premature stop codon in exon 17. Second, any truncated
chain
translated from the mutant transcript cannot be incorporated into COL
V heterotrimer. Trimer incorporation requires the selection and
alignment region in the C-terminal extension of
chains (33). In the
1(V) chain, exon 14 contains the junction between the N-propeptide
and the helical region (31). Thus, any truncated transcripts will
contain neither
helical sequences nor a C-propeptide and would be
degraded intracellularly. Third, only a minimal amount of even the
nonfunctional truncated chain can be produced because the mutant
transcript is subject to nonsense-mediated decay and is present in the
fibroblasts at only 8% the level of normal transcript. These features
combine to produce a functionally null allele at the transcriptional
level. This is reflected in the quantity of type V collagen secreted by
the fibroblasts of the proband. When the gel loading of secreted
collagen is adjusted to equal intensities of type I collagen (measured
as
1(I) chain), the proband secretes about half as much (65%) type
V collagen as does the control. This confirms that the heterotypic
fibrils of the proband must be formed with a reduced type V/type I
collagen ratio. Selective salt precipitation was done to explore the
question of whether the type V trimer secreted in the context of
1(V) haploinsufficiency still has the same ratio of
1(V) and
2(V) chains. There have been reports demonstrating that the chain
ratio was a function of the tissue extracted. In articular cartilage,
only
1(V) was detectable (30). In placenta (30), synovial membrane,
and skin (34), the two chains were present in a 1:1 ratio. In bone, the
trimer was composed of a 1:1:1 ratio of
1(V),
2(V) and the cartilage
1(XI) chain (7). Furthermore, the recently described
3(V) chain is transcribed in fibroblasts (35). Thus,
haploinsufficiency of
1(V) might alter the molecular configuration
of the type V trimers. In the collagen secreted by our proband, all the
type V collagen forms were precipitated at the expected 1.2 M NaCl, and the
1(V) and
2(V) chains were present in
a 2:1 ratio. This is strongly suggestive that trimer composition is
unchanged, although full proof will await tissue extractions from an
animal model. Because the type V collagen in our proband is secreted in
a reduced amount and with the usual fibroblast
chain composition,
the consequences of this mutant for matrix and for phenotype must result from COL V haploinsufficiency rather than a dominant negative effect.
1(V) RNA from 16 cases of EDS I or II . In seven cases, they were able to identify
either complex splicing abnormalities or small insertions or deletions leading to premature stop codons. Wenstrup et al. (21)
reported the screening of 53 EDS patients for loss of polymorphism
heterozygosity. Twenty-seven patients were heterozygous for at least
one restriction fragment length polymorphism, and eight of them
had only one allele expressed in cDNA. The study of these two sets
of patients suggests that at least one-third of EDS I or II patients
have mutations in
1(V), which results in functional
haploinsufficiency. Furthermore, most of these mutations apparently
result in substantial degradation of mutant transcript by
nonsense-mediated decay.
1(V) and three structural
abnormalities of
2(V) have been reported previously (14-20). Six
abnormalities are single exon-splicing defects, and the others are
amino acid substitutions or more complex insertions and a chromosomal
translocation. The phenotype of these patients with COL V structural
abnormalities covers the range of classical EDS I and/or II. The family
reported here has mild to moderate EDS II. The cases in the screening
series reported by Schwarze et al. (22) and Wenstrup
et al. (21) apparently cover the EDS gravis to mitis range.
Thus, type V haploinsufficiency appears to have a different general
effect on phenotype than does a null allele of type I collagen. For
type I collagen, null alleles of
1(I) are responsible for the
mildest form of osteogenesis imperfecta (type I), whereas structural
defects of type I collagen cause the more severe to lethal types II,
III, and IV of osteogenesis imperfecta. In EDS, the consequences of
haploinsufficiency appear to be similar to those of dominant negative
structural defects.
2(V) exon 27,
1(V) exon 33,
1(V) exon 49, and an
1(V) C1181S substitution (16-19). All show the same
irregularity of fibril shape and variation in diameter. It is
interesting that this is also the fibril pattern seen in this report in
which the COL 5 defect causes haploinsufficiency, because a generalized
increase in fibril diameter might have been predicted. This finding
suggests that the regulation of heterotypic fibril formation in
vivo is not simply based on the ratio of types V and I collagen.
Instead, there is apparently a difference in the distribution of type V
collagen among fibrils with most fibrils being rather normal in size
and shape. In view of the prevalence of null mutations in COL 5 and
these intriguing fibril patterns, a murine model for COL 5 haploinsufficiency could provide important insight to the mechanisms of heteropolymerization.
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ACKNOWLEDGEMENTS |
---|
We thank the members of the family described in this report, especially IV-5 for participation and support. We thank Dr. James B. Sidbury (NICHD, National Institutes of Health) and Judith Benkendorf (Georgetown University Hospital, Washington, D. C.) for separately referring the two branches of the pedigree.
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FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Supported by grants from the Medical Research Council of Canada and the Canadian Arthritis Network.
To whom correspondence should be addressed: Heritable
Disorders Branch/NICHD, National Institutes of Health, Bldg. 10, Rm. 9S241, 9000 Rockville Pike, Bethesda, MD 20892. Tel.: 301-496-6683; Fax: 301-496-0234; E-mail: oidoc@helix.nih.gov.
Published, JBC Papers in Press, January 19, 2001, DOI 10.1074/jbc.M011742200
2 D. S. Greenspan, personal communication.
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ABBREVIATIONS |
---|
The abbreviations used are: EDS, Ehlers-Danlos Syndrome; COL, collagen; RT-PCR, reverse transcriptase-polymerase chain reaction; PCR, polymerase chain reaction; nt, nucleotide; bp, base pair(s).
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