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Human Molecular Genetics, 2003, Vol. 12, No. 18
DOI: 10.1093/hmg/ddg241
2269–2276
Allelic variation in normal human FBN1 expression
in a family with Marfan syndrome: a potential
modifier of phenotype?
Sarah Hutchinson1, Andre Furger1, Dorothy Halliday1, Daniel P. Judge2, Andrew Jefferson3,
Harry C. Dietz2, Helen Firth4 and Penny A. Handford1,*
1
Department of Biochemistry, University of Oxford, Oxford OX1 3QU, UK, 2Department of Medicine, Johns Hopkins
University School of Medicine, Baltimore, Maryland, USA, 3Wellcome Trust Centre for Human Genetics, Roosevelt
Drive, Headington, Oxford OX3 7BN, UK and 4Department of Medical Genetics, Addenbrooke’s Hospital, Cambridge
CB2 2QQ, UK
Received February 28, 2003; Revised and Accepted July 10, 2003
FBN1 mutations cause Marfan syndrome (MFS), an autosomal dominant disorder of connective tissue.
One of the unexplained features of MFS is the pathogenic mechanism that leads to marked inter- and
intra-familial clinical variability, despite complete disease penetrance. An FBN1 deletion patient
[46,XXdel(15)(q15q22.1)] was identified whose fibrillin-1 protein and mRNA levels were significantly higher
than expected for a single FBN1 allele. This suggested that allelic variation in normal FBN1 expression
might occur in MFS families, and have potential clinical implications particularly for those with premature
termination codon (PTC) mutations who usually display low levels of expression from the mutant allele due
to nonsense-mediated decay (NMD). RNA analyses identified a variable reduction in total FBN1 transcript
(78 2.2 to 27.3 2.3%) in three related individuals carrying PTC-causing mutation 932insT, compared with
unaffected control individuals. Both pulse chase analysis of fibrillin-1 biosynthesis and RNase protection
analyses demonstrated that these differences were due to variation in the expression of the normal FBN1
allele and not NMD of mutant RNA. We suggest that differences in normal FBN1 expression could contribute
to the clinical variability seen in this family with MFS, and should be considered as a potential modifier of
phenotype in other cases of MFS.
INTRODUCTION
Marfan syndrome (MFS; OMIM 154700) is one of the major
inherited disorders of connective tissue and results in
prominent skeletal, ocular and cardiovascular manifestations
(1). Over 500 mutations have been identified throughout FBN1
(OMIM 134797) that give rise to MFS and a spectrum of
related ‘fibrillinopathies’ (2). With the exception of a small
number of recurrent mutations, the vast majority of mutations
are unique to families. Premature termination codon (PTC)
mutations resulting from nonsense mutations or insertions/
deletions causing a frameshift constitute 25% of FBN1
mutations so far detected (2). Inter- and intra-familial
variability is a common feature of the disease and genotype–
phenotype relationships have been difficult to deduce.
The affected protein in MFS, fibrillin-1, is a major
component of 10–12 nm connective tissue microfibrils that
are widely distributed in both elastic and non-elastic tissues
(3). Prior to, or during, secretion from the cell profibrillin-1
is proteolytically processed at its N- and C-termini and
assembled in the extracellular matrix to form microfibrils (4–
9). The precise mechanism of normal assembly is poorly
understood and, as a consequence, the pathogenic mechanism(s) that underlie the MFS phenotype remain unclear.
Although direct evidence is lacking, it has been hypothesized
that mutant fibrillin-1 monomers exert a dominant negative
effect impairing the function of normal monomers (10).
Haploinsufficiency has been thought unlikely to be a
pathogenic mechanism in MFS, since PTC patients with
low levels of mutant FBN1 due to nonsense-mediated decay
(NMD) often exhibit milder phenotypes that fall outside the
clinical criteria required for a MFS diagnosis (10). In
addition, in an earlier study a kindred exhibiting isolated
skeletal features was identified which harboured C-terminal
*To whom correspondence should be addressed. Tel: þ44 1865 285347; Fax: þ44 1865 285327; Email: [email protected]
Human Molecular Genetics, Vol. 12, No. 18 # Oxford University Press 2003; all rights reserved
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Human Molecular Genetics, 2003, Vol. 12, No. 18
Table 1. Clinical features of del(15) and MFS patients II.2, II.3, II.12 and III.1
Case
Age
Musculoskeletal
Pectus carinatum
Severe pectus excavatuma
Disproportionate body ratiob
Wrist and thumb signs
Scoliosis of >20
Reduced extension at the elbowsc
Pes planus
Protrusio acetabulaed
Moderate pectus excavatum
Joint hypermobilitye
Highly arched palate with crowding
Typical facial appearance
Cardiovascular
Aortic root dissection
Aortic root dilatationf
Maximum value of the aortic rootg
Mitral value prolapse
Ocular
Ectopia lentish
Myopia
Other
Dural ectasiai
Pneumothorax
Striae
Microcephalyj
Profound learning difficultyj
II.2
47
II.3
48
II.12
40
III.1
24
Del(15)
10
þ
þ
þ
n/a
þ
þ
þ
þ
þ
n/a
þ
þ
þ
þ
þ
þ
þ
n/a
þ
þ
þ
þ
n/a
þ
þ
þ
þ
n/a
þ
þ
þ
þ
70
þ
þ
43
þ
35
þ
þ
>50k
þ
þ
þ
n/a
þ
n/a
þ
þ
þ
þ
n/a
þ
n/a
þ
þ
The clinical features are taken from the Ghent criteria (29).
a
Pectus excavatum requiring surgery.
b
Upper segment /lower segment ratio <0.86 or arm span/height ratio >1.05.
c
<170 .
d
Demonstrated on X-ray.
e
Beighton index >4.
f
The maximum diameter of the aortic root at the sinuses of Valsalva exceeding published body surface area normograms (30).
g
In millimetres.
h
By slit lamp examination.
i
Presence determined using magnetic resonance imaging and a recommended classification (31).
j
Not a feature of MFS.
k
Aortic root replaced.
n/a, Information not available.
mutation R2726W that abolished processing of mutant
profibrillin-1, resulting in its lack of incorporation into
microfibrils (6). It was suggested that this kindred displayed
the phenotype associated with the haploinsufficiency state and
was essentially equivalent to having an FBN1 null allele.
More recently, mouse models with reduced FBN1 expression
have led to the suggestion that disease presentation in MFS is
dependent on a critical threshold of functional microfibrils
(11,12). A defective population or a reduced number of
microfibrils produced by the pathogenic effect of an FBN1
mutation may therefore account for the change in connective
tissue function. Since the level of normal fibrillin-1 protein
has the potential to modulate the pathogenic effect of the
mutant protein, it is possible that allelic variation in normal
FBN1 expression could explain some of the clinical
heterogeneity seen in MFS, particularly in PTC patients
who usually have low levels of mutant transcript due to NMD.
We have studied an isolated patient with an interstitial
deletion of chromosome 15 including the FBN1 locus, together
with three members of an MFS kindred carrying a PTC
mutation 932insT, in order to gain insight into the variability of
normal FBN1 expression. Using both RNA analyses and pulse
chase assays, we assess normal FBN1 transcript and fibrillin-1
protein levels in these individuals and identify significant
differences between them. The potential clinical implications of
these data for MFS pathogenesis are discussed.
RESULTS
Identification of an FBN1 deletion patient [del(15)]
An isolated patient with profound handicap, including a number
of Marfanoid characteristics (Table 1), was investigated for a
suspected chromosomal deletion. Initial cytogenetic analyses
revealed a karyotype of 46,XXdel(15)(q15q22.1) (data not
shown). Fluorescence in situ hybridization (FISH) of patient
interphase cells showed that a single FBN1-specific cosmid
Human Molecular Genetics, 2003, Vol. 12, No. 18
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probe signal (green) was detected in 94 out of 100 interphase
cells in comparison to two red 15q telomere control signals
(Fig. 1). This provided conclusive evidence of a single copy
FBN1 deletion and exclusion of a translocation in this patient.
Subsequent FISH analysis also revealed that the MFAP1 locus,
encoding a microfibril associated protein, was deleted in
this patient. A single MFAP1-specific cosmid probe signal was
detected in 76 out of 100 interphase cells in comparison to two
15q telomere control signals (data not shown).
Pulse chase analysis of fibrillin-1 biosynthesis in del(15)
dermal fibroblasts
Dermal fibroblasts from patient del(15) were established in
order to measure fibrillin-1 protein and RNA levels associated
with expression of a single FBN1 allele. Initially, pulse chase
analyses were performed to investigate profibrillin-1 synthesis.
Comparison of the incorporation of radioactivity by profibrillin1 (P) and internal standard X were used to ascertain levels of
profibrillin-1 synthesis by control and patient fibroblasts as has
previously been described (5,13). Unexpectedly, it was found
that similar amounts of profibrillin-1 were synthesised by both
patient and control fibroblasts at 0 h, despite patient del(15)
having only one FBN1 allele (Fig. 2A). This is in contrast to
profibrillin-1 synthesis by MFS PTC patient fibroblasts which
has previously been shown to be significantly reduced due to
NMD of mutant FBN1 transcript (14,15). These results
demonstrate that patient del(15) fibroblasts express fibrillin-1
protein at a level significantly higher than that expected for a
single FBN1 allele.
Figure 1. Dual-colour fluorescence in situ hybridization of patient del(15) interphase cells using FBN1 specific (green) and 15q telomere (red) control probes
showing the deletion of one FBN1 allele.
Comparative northern blot analysis of patient del(15) and
control fibroblast mRNA verified the pulse chase result
(Fig. 2B). The increased profibrillin-1 protein synthesis
observed in patient del(15) correlated with mRNA levels of
78.4 3.2% from the remaining FBN1 allele, compared with
an unaffected control (100%). Parallel quantitation of mRNA
from an MFS patient control with PTC-causing mutation
5358delT (unpublished data) revealed a reduction in total
fibrillin-1 mRNA expression to 26.8 1.9% of control levels.
Collectively, the del(15) data suggested that differences in
levels of normal FBN1 transcript may occur within MFS
families. If correct, this would be most easily visualized in
MFS patients with PTC mutations where the mutant FBN1
transcript is usually degraded by NMD.
Mutation 932insT was confirmed in individuals II.3, II.12 and
III.1 by direct sequencing of exon 8 of their respective genomic
DNA (data not shown).
Initially RNA extracted from patient dermal fibroblasts was
analysed by northern blot and RNase protection. In each case,
after normalization against GAPDH, fibrillin-1 RNA levels
were expressed relative to an unaffected control (see Materials
and Methods). In both assays, a reduction in fibrillin-1 RNA
was observed for each MFS patient (Fig. 4). However, the
amount of fibrillin-1 RNA from individual III.1 was significantly reduced compared with II.12 and II.3. Comparative
northern blot analysis of patient and control RNA showed that
fibrillin-1 RNA levels from patient II.12 were reduced to
78 2.2% of the control value compared with 27.3 2.3%
for patient III.1 and 26.8 1.9% for an isolated MFS patient
with PTC-causing mutation 5358delT (unpublished data).
Quantitation of RNase protection data demonstrated that the
amount of fibrillin-1 RNA detected from III.1 was 42.4 3.3%
compared to levels of 69.7 4.9% and 64.1 2.5% from
patients II.12 and II.3 respectively.
Analysis of FBN1 RNA in a kindred with PTC
mutation 932insT
RNase protection assays and pulse chase analysis
exclude NMD as a cause of the variable expression
In a previous study, mutation 932insT resulting in a frameshift
and PTC in exon 9 of FBN1, and NMD, was detected in an
MFS kindred (15) (Fig. 3). As in many MFS kindreds, intrafamilial phenotypic variability was observed despite complete
disease penetrance. To investigate levels of FBN1 expression in
this family, three related individuals (II.12, II.3 and III.1) who
differ in their severity of MFS clinical manifestations were
studied (Table 1). Haplotype analysis confirmed that the mutant
allele co-segregated with the MFS phenotype (Fig. 3).
Since it could be argued that the differences in RNA levels
within the MFS 932insT kindred were due to variations in
NMD, further experiments were performed to specifically
analyse RNA and protein derived from the normal FBN1 allele.
An RNase protection probe (MUT) was designed in order
to produce a 447 nucleotide protected fragment when added to
RNA transcribed from the 932insT mutant allele (Fig. 5A). In
contrast, RNA transcribed from the normal allele should mismatch with the probe at the site of the T nucleotide insertion
Northern blot analysis
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Human Molecular Genetics, 2003, Vol. 12, No. 18
Figure 3. Pedigree and FBN1 haplotype segregation of the MFS kindred with
FBN1 mutation 932insT demonstrating co-segregation of the mutant allele (red)
with the MFS phenotype. Extended markers (D15S119, D15S126, D15S1028,
D15S143) were used that occur within 1 Mb of the gene as well as intragenic
markers (MTS1, MTS2, MTS3, MTS4) for complete informativeness (25).
Genotypes that are inferred by pedigree analysis are in brackets. Symbols
corresponding to affected individuals are shaded.
mutant FBN1 transcript from all three individuals was present at
similarly reduced levels (see asterisk, Fig. 5B), therefore
excluding variable rates of NMD in these patients. Pulse chase
analyses verified the RNase protection result, since a significantly increased level of profibrillin-1 synthesis was observed at
0 h in II.12, compared with III.1 (Fig. 5C). The profibrillin-1
band observed in the pulse chase can only have been produced
from the normal allele since the mutant protein is truncated as a
consequence of the PTC mutation. Taken together these data
prove that allelic variation in normal FBN1 expression occurs in
dermal fibroblasts from this family with MFS.
Figure 2. (A) Profibrillin-1 synthesis and secretion by del(15) fibroblasts in comparison to an unaffected control. Cells were pulse-labelled for 30 min with
35
35
L-[ S]methionine/L-[ S]cysteine (0 h) and then chased in the absence of label.
Total cell lysates chased to 2 h are shown. The positions of profibrillin-1 (P) and
protein X are indicated. (B) Northern blot analysis of mRNA extracted from unaffected control fibroblasts (lane 1), an MFS patient control with PTC mutation
5358delT (unpublished data) (lane 2) and del(15) fibroblasts (lane 3).
Hybridization was performed with random primed [a-32P]dCTP-labelled cDNA
probes complementary to fibrillin-1 and GAPDH transcripts. Quantitation values
after normalization using GAPDH mRNA signals are indicated below the figure
expressed as mean SEM from six independent experiments.
and after RNase digestion result in two fragments 293 and 154
nucleotides in length.
RNase protection of fibroblast RNA, using the MUT probe
produced fragments of the expected size (Fig. 5A and B).
Quantitation of these bands (see Materials and Methods)
demonstrated that normal FBN1 allele expression in II.12 and
II.3 (78 2.7 and 71.9 7.2%, respectively) was significantly
greater than that of III.1 (45.5 5.4%; Fig. 5A and B). However
DISCUSSION
In this study we have demonstrated that levels of normal FBN1
transcript are higher than expected for a single allele in isolated
patient del(15) fibroblasts and variable in an MFS kindred with
a previously described PTC mutation. Fibroblast RNA obtained
from affected individuals was analysed by both northern
blotting and allele-specific RNase protection assays and
showed III.1 to have a significantly lower level of normal
transcript than either II.12 or II.3. NMD of mutant transcript
was similar in all three samples. This is the first study to
quantitate normal FBN1 allele expression in clinically affected
patients and complements a recent study highlighting differences in normal allelic expression of six genes in individuals
from the Centre d’Etude du Polymorphisme Humain (CEPH)
genotype database, including FBN1 (16). In this study 2/19
normal individuals showed enhanced expression of FBN1.
Previous attempts to quantify FBN1 transcription in MFS
patients have focused on estimation of mutant FBN1 transcript
levels, particularly in PTC patients where it has been proposed
Human Molecular Genetics, 2003, Vol. 12, No. 18
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Figure 4. (A) Northern blot analysis of dermal fibroblast mRNA extracted from individuals II.12 (lane 1) and III.1 (lane 2) in parallel with a MFS patient with PTC
mutation 5358delT (lane 3) and unaffected control fibroblast mRNA (lane 4). Hybridization was performed using random primed [a-32P]dCTP-labelled cDNA
probes complementary to fibrillin-1 and GAPDH transcripts. Quantitation values after GAPDH normalization are indicated below the figure expressed as
mean SEM from five independent experiments. (B) RNase protection analysis of total RNA extracted from unaffected control cells (lane 1), individual II.12
(lane 2), individual II.3 (lane 3) and individual III.1 (lane 4). Fibrillin-1 (FIB) and GAPDH pBluescript II constructs were used for this experiment (see
Materials and Methods). The resulting protected fragments are indicated above the figure. Lane () shows a digestion control. Lane (þ) shows a fifth of the undigested riboprobes used for the RNase protection. Lane M, size markers indicated in nucleotides. Quantitation values after normalization using protected GAPDH
mRNA are indicated below the figure expressed as mean SEM from four to 10 independent experiments.
that there is a correlation between the level of mutant transcript
and the severity of disease (10,17–19). In these studies the
mutant transcript was expressed as a percentage of normal
transcript, without normalization against a housekeeping gene
such as GAPDH.
The demonstration of allelic variation in FBN1 in normal and
clinically affected individuals suggests a potential role for
normal FBN1 expression in the pathogenesis of MFS. Since
fibrillin-1 is a structural protein of connective tissue, and
transgenic mouse data have demonstrated a critical threshold of
fibrillin-1 microfibrils for tissue function (11,12), it is reasonable to suggest that allelic variation in FBN1 expression could
be a potential modifier of phenotype in MFS, particularly in
PTC families where the population of FBN1 transcripts is
predominantly derived from the normal allele due to NMD. For
example, when expression of a normal FBN1 allele is below a
critical threshold, it is plausible that this could result in more
profound disease. Conversely a higher level of normal FBN1
expression could moderate any pathogenic effect of the mutant
allele resulting in a milder phenotype. A low expressing normal
FBN1 allele could explain the previous observation of two
affected MFS patients with mutations R1541X and R529X that
were shown to have severe disease, despite very low mutant
RNA levels of 2 and 7%, respectively (17,20). Quantitation of
normal FBN1 transcript levels was not performed in these
patients.
The explanation for allelic variation seen in the patient
fibroblasts is unknown. Variation in normal transcript levels is
known to arise from a number of factors including promoter
polymorphisms that affect the binding of transcription factors
and mRNA stability (21–24). In this kindred haplotype analysis
demonstrates that individuals II.12 and II.3 who are moderately
affected, with MFS manifestations in two of the three main
organ systems, and individual III.1 who is more severely
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Human Molecular Genetics, 2003, Vol. 12, No. 18
affected, with manifestations in the skeletal, ocular and
cardiovascular systems, have different normal alleles.
Individual II.2 (Fig. 3, Table 1) who has inherited the identical
FBN1 alleles as II.12 and II.3, exhibits the same system
involvement as II.12 and II.3, although has more severe
cardiovascular manifestations. Unfortunately cells were not
available from II.2 to test RNA and protein levels. It is
interesting to speculate that complete system involvement
(skeletal, cardiovascular, ocular) in this kindred is dependent on
the inheritance of the lower-expressing normal allele as in III.1.
However other modifying factors may also contibute to the
phenotypic spectrum displayed.
Patient del(15), although profoundly affected by the extent of
the chromosomal deletion exhibits a number of Marfanoid
characteristics predominantly affecting the skeletal system
(Table 1). However it is not possible to draw any association
between fibrillin-1 levels in this patient and phenotype
exhibited due to the young age of the patient and the deletion
of the MFAP1 locus which may affect microfibril function,
independently of fibrillin-1.
Establishment of allelic variation in normal FBN1 expression
in fibroblast samples from a single family with MFS does not
by itself prove the influence of the normal allele on intrafamilial
variability. Although an attractive hypothesis, it remains
possible that a different modifier is responsible and it is simply
by chance that, in this family, allelic variation is observed. It is
interesting to note that previous pulse chase studies of PTC
patients have not previously revealed raised levels of
normal fibrillin-1 expression. This may be because the patients
usually studied by this method are index cases with clinically
diagnosed MFS, rather than the more mildly affected family
members, or alternatively it may indicate that allelic variation
in FBN1 is rare and unlikely to be a major factor moderating
phenotype.
Proof will require analysis of larger numbers of MFS
families, particularly those with PTC mutations where allelic
variation can be most easily visualized. However our data on
individuals from a single family and patient del(15) suggest
that, in future, consideration be given to the role of the normal
FBN1 allele in MFS pathogenesis.
MATERIALS AND METHODS
Cell culture and pulse chase analysis of fibrillin-1
Figure 5. (A) RNase protection probe and resulting protected fragments after
RNase digestion using the MUT probe. (B) RNase protection analysis of fibroblast nuclear RNA from two unaffected control individuals (lanes 1 and 2), individual II.12 (lane 3), individual II.3 (lane 4) and individual III.1 (lane 5) using
MUT and GAPDH probes. The GAPDH probe was as used previously
(Fig. 4B). II.12, II.3 and III.1 were overloaded by 1.5, 1.5 and 5, respectively, to enable signal enhancement from the normal allele and detection of the
protected mutant allele. The protected mutant allele (asterisk) can be seen in a
longer exposure of lanes 3–5 on the right. M: size markers indicated in nucleotides. Quantitation values after normalization using protected GAPDH mRNA
are indicated below the figure expressed as mean SEM from six to 10 independent experiments. (C) Pulse chase analysis of individuals II.12 and III.1
with mutation 932insT in comparison to an unaffected control. Cells were
pulse-labelled for 30 min with L-[35S]methionine/L-[35S]cysteine (0 h) and then
chased in the absence of label. Total cell lysates chased to 2 h are shown. The
positions of profibrillin-1 (P) and protein X, used as an internal standard (5,13),
are indicated.
Deletion patient, control and Marfan patient fibroblasts were
established from forearm skin biopsies. The MFS family was
selected for study due to its large size, intra-familial clinical
variability and previously detected PTC mutation (15).
Individual II.2 was unavailable for biopsy. The primary cultures
were maintained in Dulbecco’s modified Eagle medium
supplemented with 10% fetal calf serum, 50 U/ml penicillin,
50 mg/ml streptomycin and 2 mM L-glutamine. For all experiments where only one unaffected control was used it
corresponds to unaffected control 2 in Figure 5B.
Pulse chase analysis of fibrillin-1 from patient and control
fibroblasts was carried out as described previously (13,15) and
profibrillin-1 was confirmed by immunoprecipitation using a prorich region antibody that has been previously characterized (13).
Human Molecular Genetics, 2003, Vol. 12, No. 18
Probe isolation and dual colour FISH
A chromosome 15 cosmid library was constructed at Los Alamos
National laboratory (LANL) and the cosmid library filter obtained
from UK HGMP Resource Centre, Cambridge, UK. The filter
was screened for FBN1 specific cosmids and FBN1 cosmid clone
(LA1537-H4 from the LA15NC01 library) obtained from UK
HGMP Resource Centre, Cambridge, UK. Chromosome preparations were made by treating patient fibroblasts with hypotonic
solution (7.5 mM KCl) for 20 min at 37 C followed by fixing in
3:1 methanol:acetic acid. Slides were prepared following
standard procedures and aged at 20 C.
Probe DNA was biotinylated and ethanol precipitated with
salmon testis DNA (Gibco BRL), E. coli tRNA (Boehringer)
and resuspended in 1 TE. A commercial digoxigenin labelled
chromosome 15q sub-telomeric probe (Appligene Oncor) was
used as a marker for chromosome 15. The cosmid probe and a
50-fold excess of Cot-1 DNA (Gibco BRL) were dried and
resuspended in hybridization solution (50% formamide,
2 SSC, 10% dextran sulfate) to a final concentration of
20 ng/ml. Probe DNA was denatured at 72 C for 5 min and
reannealed at 37 C for 15 min. The 15q sub-telomeric probe
was used according to the manufacturer’s protocol. The slides
were dehydrated in an ethanol series at room temperature and
air-dried, then denatured in 70% formamide at 70 C for 2 min,
quenched in 70% ethanol at 20 C then dehydrated in an
ethanol series.
Probe mix (FBN1 cosmid and 15q probe) was hybridized at
37 C for 20 h followed by washes in 50% formamide at 42 C
for 10 min and 2 SSC at 42 C for 5 min. The biotinylated
probes were detected with Texas red-conjugated streptavidin,
biotinylated anti-streptavidin (Vector Laboratories) and a
final layer of Texas Red-conjugated streptavidin. The digoxigenin probe was detected using mouse anti-digoxigenin
antibody (Roche) and goat anti-mouse Alexa-488 (Molecular
Probes). The slides were counterstained and mounted with
Vectashield (Vector Laboratories) containing 40 ,6-diamidino-2phenylindole (DAPI).
Slides were examined using an Olympus BX-51 epifluorescence microscope coupled to a Sensys CCD camera
(Photometrics). One hundred randomly selected interphase
nuclei were analysed for each hybridisation experiment. Texas
red, Alexa-488 and DAPI fluorescence images were taken
separately then pseudocoloured and merged using the software
package Genus (Applied Imaging International).
2275
were [a-32P]dCTP-labelled by random priming (Roche). The
membrane was hybridized with the fibrillin-1 probe for 16 h at
65 C, washed to a final stringency of 0.2 SSC/0.1% SDS
then autoradiographed. Hybridisation of the membrane to the
GAPDH probe was repeated without stripping and quantitation
carried out by PhosphorImager.
RNase protection
Total RNA was isolated using the hot-phenol method. Volumes
of 450 ml NTE buffer (0.1 M NaCl, 10 mM Tris pH 8, 1 mM
EDTA) and 50 ml 10% sodium dodecyl sulfate were added to
500 ml phenol and heated to 90 C. Cell pellets were added to
the hot phenol mix, phenol–chloroform extracted, and
precipitated. Total RNA pellets were resuspended in 100 ml R
loop buffer (27). Nuclear RNA was extracted by hot phenol
after cytoplasmic/nuclear separation (28).
For total fibrillin-1 analysis fragments of fibrillin-1 cDNA
(nucleotides 1086–1555: FIB) and GAPDH cDNA (nucleotides
84–256: GAPDH) were cloned into pBluescript II (Stratagene).
For II.12, II.3 and III.1 single allele analysis a fragment of
fibrillin-1 cDNA containing the kindred mutation 932insT was
cloned into pBluescript II (nucleotides 639–1086: MUT).
Riboprobes were made from linearized pBluescript II with T7
(GAPDH, MUT) or T3 (FIB) RNA polymerase according to the
manufacturer’s instructions (Promega). Full-length riboprobe
was purified from a denaturing polyacrylamide gel in 300 ml
buffer (0.5 M ammonium acetate, 0.1% SDS, 1 mM EDTA) for
2 h at 37 C. Eluted RNA was ethanol precipitated, resuspended
in water and 50 cps of probe hybridized to total RNA. RNase
protection analysis was performed as described previously (27),
except that the MUT probe experiment (Fig. 5A) was RNasedigested at 37 C.
ACKNOWLEDGEMENTS
The authors thank Dr Sally Rose, Dr Mick Dye, Dr Rob Davies
and Dr Garry Brown for experimental advice and guidance,
Professor Paul Wordsworth for continued support and Dr Lucy
Raymond for taking the del(15) skin biopsy. P.H. and S.H.
thank the British Heart Foundation and MRC for their support.
REFERENCES
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Haplotype analysis of markers D15S119, D15S126, D15S1028,
MTS1, MTS2, MTS3, MTS4, D15S1028 was carried out on
available pedigree DNA as described previously (25).
Northern analysis
Messenger RNA was isolated from primary human fibroblasts
using the Oligotex direct mRNA kit (Qiagen). RNA was
separated on a 0.8% formaldehyde–agarose gel, transferred to
Hybond Nþ (Amersham Pharmacia) and pre-hybridized in
Church buffer (26) at 65 C. Human cDNA probes encoding
fibrillin-1 (nucleotides 1168–1527) and GAPDH (Clontech)
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