From the Departments of Pathology and
Pediatrics, Vanderbilt University School of Medicine, Nashville,
Tennessee 37232, ** Research Service, Department of Veterans Affairs
Medical Center, Nashville, Tennessee 37212, and the ¶ Department
of Medical Genetics, University of British Columbia,
Vancouver V6T 1Z3, Canada
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ABSTRACT |
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Congenital cutis laxa, a rare syndrome with
marked skin laxity and pulmonary and cardiovascular compromise, is due
to defective elastic fiber formation. In several cases, skin fibroblast
tropoelastin production is markedly reduced yet reversed in
vitro by transforming growth factor- Elastic fibers are the extracellular matrix structures responsible
for the properties of resilience and elastic recoil in all elastic
tissues (1, 2). There are two morphological elements in elastic fibers:
the microfibrillar component and the amorphous component. The
microfibrillar component is made up of 10-12-nm microfibrils that are
composed of at least seven different glycoproteins, including the two
genetically distinct fibrillins, whose genes are the loci for Marfan's
syndrome and congenital contractural arachnodactyly (3, 4). Elastin is
also present in the amorphous component as a cross-linked complex of
hydrophobic proteins synthesized from the single copy, multi-exon
elastin gene (ELN) by extensive alternate usage of several
exons (1, 5-7).
There are several inherited disorders characterized by aberrant elastin
synthesis or degradation. Abnormal elastic fibers are seen in Menkes'
syndrome due to altered copper transport resulting in decreased
activity of lysyl oxidase (8), whereas elastic fibers are prematurely
degraded due to unregulated elastase activity in patients with
In some diseases of elastic tissue, mutations in genes for structural
proteins have been demonstrated. Currently, there are at least three
dominant disorders, Marfan's syndrome (18-20), ectopia lentis (19,
20), and congenital contractual arachnodactyly (19), that are caused by
mutations in fibrillin genes. Most patients with Williams syndrome that
have SVAS are heterozygous for deletions of ELN and
presumably other contiguous genes on chromosome 7q (21, 22). However,
only in patients with SVAS have disruptions or point mutations within
ELN itself been described (15, 16, 23). The connective
tissue features of SVAS and Williams syndrome are consistent with, but
not yet shown to be due to, functional hemizygosity at ELN.
We have previously shown in one cutis laxa cell strain that transcript
instability was the basis of a defect in ELN mRNA
accumulation and tropoelastin production (24). TGF- Clinical Summary--
Patient K.T. was the 3.8-kg full-term male
product of an uncomplicated pregnancy, labor, and delivery to a 23-year
old G2P1 Caucasian mother. The parents denied
consanguinity. Loose skin, stridor, and feeding difficulties were
apparent from birth. Additional clinical findings ascertained during
infancy included moderate subglottic stenosis with floppy airway
structures, redundant mitral and tricuspid valves, mild dilatation of
the proximal aorta and great vessels, and umbilical and inguinal
hernias. He underwent inguinal herniorrhaphies at ages 7 months, 3 years, and 14 years.
At age 17 years, his height and weight are at the 75th percentile. His
physical exam is significant for an aged appearance: smooth, loose skin
lacking elastic recoil; tortuous, pulsatile external carotid arteries;
and a hoarse voice. He complains of fatigue, dyspnea on exertion, and
shortness of breath. His echocardiogram is relatively unremarkable
except for minimal aortic root dilatation; an electrocardiogram reveals
mild right ventricular hypertrophy. Pulmonary function testing shows
reduced expiratory flow, suggestive of fixed or collapsible upper
airway obstruction.
Histologic, ultrastructural, and biochemical analyses of skin and
cultured fibroblasts from the patient have been reported previously
(17, 24). Briefly, dermal collagen fibers appear normal, but elastic
fibers appear fragmented with a paucity of amorphous elastin in the
matrix. Tropoelastin production in cultured fibroblasts from this
patient was the lowest of six cutis laxa patients studied (17), and an
apparent nonspecific increase in type VI collagen production was also
noted (25).
Limited clinical information is available on the second family at this
time. The female proband (WM) was ascertained in 1965 with classical
cutaneous features of cutis laxa at 2 years. This individual gave birth
to an affected son (WS) in 1991, and skin biopsies were cultured from
both individuals in 1993 by Drs. J. Uitto and E. Tan (Jefferson Medical
College). The father and maternal parents were reportedly unaffected,
and the patients have been lost to follow-up.
Tissue Culture--
Skin fibroblasts were grown from skin
biopsies obtained after appropriate consent. Normal human skin
fibroblasts (GM4390) were obtained from the NIGMS Human Genetic Mutant
Cell Repository (Camden, NJ). Cells were grown to confluence in
Dulbecco's modified Eagle's medium (Life Technologies, Inc.)
supplemented with 10% fetal bovine serum (Atlanta Biologicals, Inc.,
Norcross, GA).
cDNA Synthesis--
Confluent cultured cells were washed
twice in phosphate-buffered saline and lysed in 2 ml of 4 M
guanidine isothiocyanate containing 0.1 mM
PCR--
Eleven pairs of overlapping primers were designed to
amplify the complete coding and untranslated regions of ELN
mRNA. Primers were constructed according to the cDNA sequences
for the ELN coding region (GenBankTM accession
number M36860) (27) and 3'-UTR (accession number M17282) (28). Primers
designated F are based on the sense strand sequence, and primers
designated R are based on the complementary strand sequence. The
primers for the elastin coding region were as follows (numbering from
the transcription start site): 96F, 5'-ATCGATCCTGCTGTCCATCCTCCA-3';
419R, 5'-ACACTCCTAAGCCACCAACT-3'; 397F,
5'-AGGAGTTGGTGGCTTAGGAG-3'; 728R, 5'-GCAGTTTCCCTGTGGTGTAG-3'; 709F,
5'-CTGCACCACAGGGAAACTGC-3'; 1064R, 5'-CTCCTGGGACACCAACTACT-3'; 992F,
5'-AAGTATGGAGCTGCTGCAGG-3'; 1410R, 5'-ACTCCGTACTTGGCAGCCTT-3'; 1271F,
5'-GGTGTCGGAGTCGGAGGTAT-3'; 1798R, 5'-AACACCAGCACCAACTCCAA-3'; 1761F,
5'-GTGCTGGTGTTCCTGGACTT-3'; and 2292R, 5'-AGCAGTAGCACCAACGTTGA-3'. The
primers for the elastin 3'-UTR were as follows (numbering from the
3'-UTR): He3'UTR15F, 5'-CTGACTCACGACCTCATCAA-3'; He3'UTR328R, 5'-CAGGAAGATAAGAGCACCAG-3'; He3'UTR302F, 5'-CTACACGCTGGTGCTCTTAT-3'; He3'UTR741R, 5'-GACAGGTCAACCAGGTTGAT-3'; He3'UTR721F,
5'-CATCAACCTGGTTGACCTGT-3'; He3'UTR1092R, 5'-TTCTACTGGGGATACAGCTC-3';
He3'UTR1029F, 5'-TTGTGTCTCGCTGTGATAGA-3'; and He3'UTR1255R,
5'-CCAACAGTTGAAGGCAGATT-3'. The primers for the elastin 5'-UTR were as
follows (numbering from the transcription start site):
PCRs were carried out in a Perkin-Elmer minicycler in 50-µl volumes
using 1-2 µl of reverse transcription products, a 0.2 µM concentration of each primer, a 0.4 mM
concentration of each dNTP, and 2.5 units of Taq polymerase
(Promega, Madison, WI). The reaction cycles consisted of an initial
denaturation at 95 °C for 3 min followed by 95 °C for 1 min,
annealing at 60 °C for 1 min, and extension at 72 °C for 1 min
per cycle for 30 cycles.
Direct DNA Sequencing--
PCR products amplified by different F
and R primer combinations from both cutis laxa and control cell strains
were separated by ethidium bromide-1% agarose gel electrophoresis.
Amplimers of the expected sizes were excised and purified using
Prep-A-Gene DNA purification systems (Bio-Rad). Sequencing was
performed using an ABI PRISMTM 377 DNA Sequencer
(Perkin-Elmer) or manual sequencing using the double-stranded DNA cycle
sequencing system (Life Technologies, Inc.). For mutation confirmation,
three different RT-PCR products were independently sequenced.
Cloning of RT-PCR Products--
RT-PCR products of ~3
kilobases synthesized with primers 96F and He3'UTR741R were separated
by electrophoresis, purified, and cloned into the pNoTA vector using
the PRIME PCR CLONERTM cloning system (5 Prime Restriction Endonuclease Digestion--
The 2012 Heteroduplex Assay--
PCR was performed with primers 1761F and
2292R in the presence of 0.3 µCi of [32P]dCTP for 30 cycles. Equal amounts of PCR products from normal and cutis laxa
fibroblasts were mixed, incubated at 95 °C for 2 min, and gradually
(20-30 min) cooled to 37 °C. After adding 1 µl of loading buffer
to 5 µl of sample, the samples were separated by electrophoresis on a
0.5× Mutation Detection Enhancement gel (FMC Corp. BioProducts,
Rockland, ME).
RNA Stability--
Elastin production and mRNA stability
studies were carried out as described previously (17, 24). RNA was
isolated and purified using the QIAshredderTM and the
RNeasyTM mini kit (QIAGEN Inc., Chatsworth, CA)
according to the manufacturer's instructions.
A Frameshift Mutation of ELNin Cutis Laxa Strain K.T.--
A
single base deletion (2012
Upon sequencing the entire ELN coding region as well as 240 bp of the 5'-UTR and 1.2 kilobases of the 3'-UTR, no other sequence variations could be found between the two cloned patient alleles and
controls. Both alleles contained A at position 1313, the site of an A/G
(Ser422/Gly) polymorphism (29). Several other presumably
less significant base changes were found in the 1.2-kilobase
ELN 3'-UTR of both patient and control strains that differed
from or complemented GenBankTM entry M17282 (28). These
included seven base changes (T174 to A, T196 to
C, C737 to G, A1195 to C, C1216 to
A, T1245 to A, and T1258 to A), four insertions
(C233, C776, C788, and
C861), three deletions (A335, A724,
and A1137), and 15 newly identified residues
(G857, C861, C863,
C876, C924, T925, C926,
G939, C946, C962, C963,
C1018, A1227, A1228, and
C1231). Although these ELN 3'-UTR sequences
differ from the original GenBankTM data, they were
identical in our patient and control fibroblast mRNAs. Further
sequencing of elastin cDNA revealed that exons 22, 23, 26A, and 32 were spliced out of transcripts from both control and K.T. fibroblasts
(Fig. 3), a feature that has been reported previously in normal cells (30).
A Related Mutation in a Second Family Is Alternatively
Spliced--
A second set of fibroblast strains from a kindred with an
affected mother (WM) and son (WS) showed characteristic low
tropoelastin production with high induction of the protein by TGF-
Heterozygosity of the mutation in WM and her son (WS) was established
by direct sequencing of genomic DNA. To confirm the heterozygosity and
to rule out a polymorphism, restriction digests were performed with
Pf1MI (Fig. 5B). 50% of WS and WM DNAs were resistant to enzyme digestion, whereas DNAs from 50 other unrelated individuals were fully susceptible to Pf1MI enzyme digestion
at the exon 30 locus.
TGF- Mutational Consequences--
The two single base deletions in exon
30 each predict a frameshift in the coding region for the elastin
carboxyl terminus. The predicted, truncated protein product would
consist of 667 amino acids (Fig. 7).
Since exon 32 was usually absent, the predicted open reading frame
(ORF) of the mutant transcript would more frequently continue into the
3'-UTR to be translated as a missense structure of 713 amino acids
lacking the distinctive carboxyl terminus of tropoelastin (Fig. 7). In
the less likely event of inclusion of exon 32, the mutation would
create a premature termination codon in exon 32, predicting a
truncated, missense C terminus and a translation product of 667 amino
acid residues. Among the 3'-UTR sequence corrections/additions that
were noted, only the T174 to A substitution would have
functional significance in the translation of the mutant allele since
it would create a novel stop codon (TAA174) in the shifted
ORF. This new stop codon would be 25 amino acids downstream of the
normal translation termination site.
Cutis laxa is a relatively rare connective tissue disease
characterized by genetic heterogeneity and clinical variability (6,
31-33). In all cases, the primary diagnostic feature is loose,
hyperextensible skin with decreased resilience and elasticity, leading
to a premature aged appearance. The skin changes are often accompanied
by extracutaneous manifestations, including pulmonary emphysema,
bladder diverticula, pulmonary artery stenosis, and pyloric stenosis.
Histological examination of the skin in cutis laxa often reveals marked
fragmentation or diminution of elastic fibers (6, 17, 32). This is a
considerably different phenotype than that found in SVAS, a pathology
arising from elastin mutations that lead to functional hemizygosity
(15, 16, 23, 34, 35). Skin fibroblast cultures from many cutis laxa
patients exhibit reduced ELN mRNA levels or tropoelastin
production, but fibroblasts from other affected individuals exhibit
normal levels of elastin production with abnormal elastic fiber
morphology (17, 36). At the time of submission of this manuscript, the
molecular basis for cutis laxa had not been described. However, another group has recently reported autosomal dominant cutis laxa associated with a point deletion in exon 32 (37). This exon was absent from the
cDNAs examined in this study. Unlike the biochemical phenotype
reported here, elastin mRNA is expressed and apparently stable in
this strain, and the data from that report are consistent with a
dominant-negative effect at the level of elastic fiber formation. The
fibroblasts in this study produced insignificant amounts of elastin
mRNA and protein in the absence of TGF- We identified two single base deletions (2012 Analysis of the mutation in WM and WS confirmed ELN as the
cutis laxa locus. Both the similar location in exon 30 and the predicted translational consequences of 2012 The low frequency of polymorphisms in ELN makes analysis of
allelic usage problematic. Transcripts from both normal and mutant alleles were equally unstable, and both transcripts were equally stabilized by TGF- In conclusion, we have found two point deletions in ELN from
individuals with the classical cutaneous phenotype of congenital cutis
laxa. These mutations result in frameshifts that disrupt the sequence
of the conserved carboxyl terminus of tropoelastin and lead to marked
transcript instability. We speculate that variable splicing of the
mutated exon may account for reduced severity and selective tissue
effects. The elastin mutations in SVAS appear to involve functional
hemizygosity that affects the elastin-rich aortic root (14-16).
Complementing these findings, autosomal dominant cutis laxa appears to
arise from dominant-negative effects at the level of both RNA stability
and protein assembly, with the prospect of a novel mechanism for
variable tissue consequences. Identification of additional elastin
mutations in both dominant and recessive forms of cutis laxa will
enable us to understand better the structural biology and regulation of
this important extracellular matrix component.
treatment. We previously
showed that this reversal was due to elastin mRNA stabilization in
one cell strain, and here this behavior was confirmed in skin
fibroblasts from two generations of a second family. cDNA
sequencing and heteroduplex analysis of elastin gene transcripts from
three fibroblast strains in two kindreds now identify two frameshift
mutations (2012
G and 2039
C) in elastin gene exon 30, thus leading
to missense C termini. No other mutations were present in the
ELN cDNA sequences of all three affected individuals.
Transcripts from both alleles in each kindred were unstable and
responsive to transforming growth factor-
. Exons 22, 23, 26A, and 32 were always absent. Since exon 30 underwent alternative splicing in
fibroblasts, we speculate that a differential splicing pattern could
conceivably lead to phenotypic rescue. These two dominant-acting,
apparently de novo mutations in the elastin gene appear to
be responsible for qualitative and quantitative defects in elastin,
resulting in the cutis laxa phenotype.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
1-antitrypsin deficiency (9) and some forms of atrophoderma (10).
Pseudoxanthoma elasticum and Buschke-Ollendorff syndrome are examples
of heritable skin diseases in which increased deposition of cutaneous
or vascular elastin has been demonstrated (11, 12). In contrast,
decreased or aberrant deposition of elastic fibers in certain tissues
is characteristic of Marfan's syndrome (13), supravalvular aortic
stenosis (SVAS)1 (14-16),
and cutis laxa (6, 17).
was able to
increase mRNA stability markedly and to stimulate production of
immunoreactive tropoelastin protein in this cell strain. Since the
metabolic and ultrastructural defect was confined to elastin, we
hypothesized that a structural defect in the ELN transcript
could be responsible for decreased mRNA stability. In this report,
we describe heterozygosity for a frameshift mutation (2012
G) in
ELN in this cutis laxa patient and a similar mutation in two
generations of a second cutis laxa family (2039
C), which we propose
to be responsible for defects in tropoelastin production.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-mercaptoethanol. DNA was sheared by three passages through a
22-gauge needle, and RNA was isolated by extraction in acid
phenol/chloroform (26). Isolated RNA was stored at
70 °C. cDNA
was synthesized using 1-3 µg of total cellular RNA with 200 units of
Moloney murine leukemia virus reverse transcriptase and 0.75 mM gene-specific oligonucleotide primers in a total volume of 20 µl for 60 min at 37 °C.
243F
He-promoter, 5'-GTGTGTGCGTGTGTTGTGTC-3'; and +155R He-intron I,
5'-CTTGAGCGTCTAGTCACCTG-3'. The primers for introns 29 and 30 were as
follows: 212F in intron 29, 5'-GGAGTCTAATGCTCAGCTGT-3'; and 489R in
intron 30, 5'-CACCTTGGCCTACTAGAGTG-3'.
3 Prime,
Inc., Boulder, CO) according to manufacturer's instructions. A total
of 31 white colonies was picked from overnight cultures and propagated
in LB medium for plasmid minipreps. The plasmid DNAs were digested with
BamHI, and positive clones with correctly sized inserts
(2854 bp) were identified by gel electrophoresis.
G deletion in
exon 30 of K.T. created a novel Alw26I restriction site.
RT-PCR products amplified by primers 11 and 12 (476 bp) from K.T.
fibroblasts, a control cell strain, and genomic PCR products amplified
by primers 23 and 24 (277 bp) from the proband, his parents, and 65 unaffected individuals were digested with Alw26I at 37 °C
for 60 min. The 2039
C deletion abolished a PflMI
site in the same PCR product of the mutant allele in WM and WS. DNAs
from an additional 48 individuals were also screened for the same
mutation. The digests were electrophoresed through an 8%
polyacrylamide gel and visualized by ethidium bromide/UV fluorescence.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
G) in exon 30 of ELN was
initially identified by direct sequencing of the RT-PCR products from patient fibroblasts (Fig. 1). This
mutation created a novel Alw26I recognition site
(![N]5GAGAC); heterozygosity for the deletion was
confirmed in the RT-PCR product by the presence of diagnostic 244- and
132-bp bands after digestion of the 476-bp PCR product with
Alw26I (Fig. 2A).
Furthermore, heterozygosity for the mutation was confirmed at the
genomic DNA level in the patient, but the polymorphism was absent in
DNA from both parents (Fig. 2B) and 65 unrelated control
samples (data not shown).
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Fig. 1.
A deletion (2012 G) in ELN in a
cutis laxa patient. RT-PCR products from both patient (K.T.) and
normal fibroblasts were generated by primers 1761F and 2292R (see
"Experimental Procedures") and subjected to automated fluorescent
sequencing. The location of the deletion is indicated by the
arrow.
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Fig. 2.
Confirmation of heterozygosity for the
ELN mutation in strain K.T. A, RT-PCR
products from patient (K.T.) and control (GM4390) fibroblast mRNAs
were digested with Alw26I and separated by 8%
polyacrylamide gel electrophoresis. The mutation creates a novel
Alw26I site (*), thus further cleaving the normal 376-bp
fragment into 244 and 132 bp. B, genomic DNAs from the
patient and his unaffected parents were amplified by PCR with primers
212F and 489R (see "Experimental Procedures"), digested with
Alw26I, and separated by 8% polyacrylamide gel
electrophoresis. The mutation creates a novel Alw26I site
(*), thus further cleaving the normal 120-bp fragment into 97 and 23 bp.
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Fig. 3.
Normal ELN mRNA splicing
pattern in cutis laxa skin fibroblasts. The primary structure of
the coding region was determined by direct cDNA sequencing. Exons
22, 23, 26A, and 32 were spliced out of the ELN mRNA
from both cutis laxa (K.T.) and control fibroblasts.
(Fig. 4). Elastin mRNA only
accumulated in these strains in the presence of TGF-
, and it rapidly
degraded as soon as TGF-
was withdrawn (Fig. 4B).
Heteroduplex analysis revealed a novel band mobility in WM and WS using
primer pairs that spanned the exon 30 region (data not shown). Eight
full-length cDNA clones were derived from WS mRNA and
completely sequenced. Three of the cDNA clones lacked exon 30 (Fig.
5A). Of the five cDNA
clones containing this exon, four showed a deletion of
C2039 (Fig. 5A), which predicts a frameshift
mutation with consequences similar to those of strain K.T.
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Fig. 4.
TGF- reversal of the elastin deficit in WM
and WS is related to transcript instability. A,
fibroblasts from the control strain (GM4390), WS, and WM were analyzed
for tropoelastin (TE) production (9) in the presence and
absence of 10 ng/ml recombinant TGF-
2 (a gift of Genzyme Corp.,
Cambridge, MA). Under basal (5% newborn calf serum) conditions,
tropoelastin production (expressed as molecular equivalents (mol.
eq.)/cell/h ± S.E.) was near the base-line limits of the
enzyme-linked immunosorbent assay in the cutis laxa strains, whereas
production rose to nearly normal levels in the presence of TGF-
.
B, cells pretreated with TGF-
2 for 48 h as described
above were put into fresh medium lacking TGF-
and containing
75 µM 5,6-dichlorobenzimidazole riboside to block
transcription. RNA was isolated immediately, 6, 12, and 24 h
after treatment, and elastin transcripts were quantified in relation to
cyclophilin transcripts by Northern blot hybridization. Consistent with
the production data, elastin mRNA levels were lower in cutis laxa
strains WM and WS and undetectable in the absence of TGF-
(data not
shown). Elastin mRNA levels fell rapidly to base-line levels by
6 h. In control fibroblasts, the t1/2 of
elastin mRNA was >15 h.
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Fig. 5.
Detection of a point deletion in cutis laxa
strains from WM and WS. A, the left panel
shows a segment of the normal sequence within exon 30; the middle
panel (2039 C) illustrates the point deletion (GCCTTA>GCTTA);
and the right panel shows the splice junction between exons
29 and 31. B, genomic DNAs from the two affected strains and
GM4390 were amplified by PCR to yield a 277-bp product. Digestion with
PflMI produced quantitative cleavage of the product of
control cells, whereas only 50% of WM and WS products were
cleaved.
Does Not Change the Ratio of ELN mRNA Expressed from
Two Alleles--
As shown previously for K.T. (24) and above for WM
and WS, TGF-
could in part restore tropoelastin expression in these cutis laxa fibroblasts by stabilizing ELN mRNA. To
examine whether partial restoration of ELN mRNA
stability was functioning through selective expression of
ELN mRNA from the normal allele, RT-PCR products
amplifying the region spanning nucleotides 1671-2292 from
TGF-
-treated and untreated K.T. fibroblasts were analyzed under
semi-quantitative conditions. Amplified products were digested with
Alw26I. The intensity of the 244-bp mutant allele fragment relative to the normal 376-bp fragment did not change after TGF-
exposure (Fig. 6). Similar results were
obtained with RNAs from WS and WM (data not shown). Transcripts of both
alleles had equivalent instability.
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Fig. 6.
Effect of TGF- on the relative abundance
of transcripts from the two ELN alleles in cutis laxa skin
fibroblasts. A 481-bp cDNA region was amplified by RT-PCR
using primers 11 and 12 and mRNAs from TGF-
-treated (K.T./TGF,
lanes marked 1) and duplicate untreated (K.T.,
lanes marked 2 and 3) cells in the
presence of 5 µCi of [32P]dCTP for 20, 27, and 34 cycles. Equal aliquots were digested with Alw26I, separated
by 8% polyacrylamide gel electrophoresis, and visualized by
autoradiography. The sizes of the fragments are indicated. The cDNA
restriction maps are shown in Fig. 2A.
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Fig. 7.
Proposed effect of the ELN
2012 G and 2039
C mutations on translation. The
organization of the domains in the transcript is based on cDNA
sequence analysis of normal and mutant cell strains. In the normal
transcript (top), the open box is the 5'-UTR; the
striped box is the signal peptide; the hatched
boxes are hydrophobic domains; the closed
boxes are cross-link domains; the stippled box
represents cysteine-containing C-terminal exon 36, and the
wavy box is the 3'-UTR. Most domains are encoded by distinct
exons, and exon numbering is shown to emphasize the consensus alternate
splicing pattern present in skin fibroblasts. The positions of the
2012
G and 2039
C deletions are marked by arrows. The
normal and three possible mutant ORFs are illustrated below this.
Normal translation terminates at TGA in exon 36, producing a
tropoelastin molecule of 688 amino acids (aa). Missense
sequence following the frameshift is indicated by hatched
areas in the two mutant ORFs. In the absence of exon 32 (as in
skin fibroblasts), missense translation terminates at a stop codon
(TAA) that is 75 bp 3' to exon 36, producing a defective protein of 713 amino acids. In the presence of exon 32, missense translation
terminates at a premature stop codon (TAG) in exon 32, producing a
defective protein of 667 amino acids. In transcripts lacking exon 30 (rescued ORF), the mutation is skipped, and a 664-residue
tropoelastin is synthesized. kb, kilobases.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
stimulation.
G and 2039
C) in the
coding region of ELN cDNA from two cutis laxa lineages. Analysis of the parental DNA indicated a de novo mutation in
proband K.T., and limited familial information suggests that a de
novo mutation in WM was passed to her son, WS. Both of the
deletions would result in translation of missense protein sequence 3'
to the mutation, and they predict subsequent loss of the functional carboxyl terminus in the tropoelastin molecule by missense and/or premature termination, depending on the more frequent splicing out or
less frequent inclusion of exon 32 (Fig. 7). Thus, different tissues
could produce defective elastin molecules with slightly different
carboxyl termini, depending on the tissue-specific mRNA splicing
pattern. Regardless of the status of exon 32 splicing, each of these
point deletions dictates the loss of two conserved cysteine residues
that create an intramolecular disulfide bridge and that are thought to
be important, together with a basic tetrapeptide tail, for interaction
of tropoelastin with other proteins present in the elastic fibers of
microfibrils (38, 39). Although little or no normal or mutant protein
is expressed in fibroblast cultures, tissues in vivo
(particularly under the influence of TGF-
) might express significant
levels of either defective gene product and lead to a dominant-negative
effect at the level of protein assembly. We may be able to resolve this
issue with C terminus-specific antibodies when tissue becomes available.
G and 2039
C mutations are intriguing, and they may indicate a susceptible site for production of dominant-negative, viable ELN mutations. Exon 30 has
recently been identified, at least in the rat, as a site for a
cis-acting mRNA stability
element.2 More important, our
finding that exon 30 is alternatively spliced may be indicative of a
phenotypic rescue mechanism (40) whereby cells or tissues could escape
the effects of the mutations. The same possibility would apply for the
recent mutation described in exon 32 (37). The fact that all three
affected individuals are not severely incapacitated suggests that
pulmonary and cardiovascular elastic tissues must be relatively spared.
In contrast to the predominant effect of SVAS in the aortic root, both
of the cutis laxa mutations appear to have a largely cutaneous
phenotype. This could be due to tissue-specific or developmentally
regulated differential expression of the two alleles, increased
frequency of alternative splicing of exon 30 (or 32) in non-cutaneous
tissues, or differential consequences of mutant RNA and protein in
different cell types.
2. Both alleles were otherwise identical
throughout the coding region, 223 bp of the 5'-UTR, and 1.2 kilobases
of the 3'-UTR (data not shown). A comparable increase in the amount of
both normal and mutant transcripts by TGF-
stimulation suggests that
at least the proximal regulatory regions of both alleles are intact,
including the response sites for TGF-
(41) and insulin-like growth
factor-1 (42) in the elastin promoter. We have previously shown that
ELN transcription rates are normal in K.T. (24). In
addition, since the point deletion was passed from WM to WS, compound
heterozygosity could only have persisted in WS by donation of an
independent mutation from his paternal ELN allele. These
findings, together with the absence of the deletions in DNAs from both
parents of K.T. and unrelated controls, argue that K.T. and probably WM
are the origins of new dominant mutations that account for the cutis
laxa phenotype. The molecular data predict that if mutant tropoelastin
were synthesized, at least in skin, it would contain defective carboxyl
termini. The quantitative and qualitative defects in tropoelastin
production could readily account for the patients' abnormal
(cutaneous) phenotype. We have not determined the mechanism whereby the
heterozygous mutation appears to exert a dominant effect on
ELN mRNA stability. Nonsense-mediated mRNA decay is
a protective cellular mechanism described for several genes; however,
it acts in cis as a rule (43). Indeed, this mechanism
appears to be operative in some forms of SVAS (16). A dominant,
trans-acting effect might arise if mutant transcripts altered the availability of a rate-limiting factor for elastin mRNA
stability. Elastin mRNA levels drop if secretion is perturbed (44),
and translational or packaging defects may have a feedback effect on
mRNA stability (45). To test these possibilities, current studies
are directed at examining the effects of introducing a mutant
ELN cDNA into elastin-expressing cells.
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ACKNOWLEDGEMENTS |
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We thank J. Rosenbloom and W. Abrams for providing unpublished DNA sequence data for introns 29 and 30. We are extremely grateful to Drs. E. Tan and J. Uitto for providing cell strains and clinical information and to Dr. P. Byers for providing critical contacts.
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FOOTNOTES |
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* This work was supported by Grant AR44431 (formerly Grant GM37387) from the National Institutes of Health and by the Department of Veterans Affairs. This work was reported in preliminary form at a meeting entitled "Elastin and Elastic Tissue" (Maratea, Italy, October 17-20, 1996) and at the 47th annual meeting of the American Society of Human Genetics (Zhang, M.-C., He, L., Yong, S. L., Tiller, G. E., and Davidson, J. M. (1997) Am. J. Hum. Genet. 61, (suppl.) 353 (abstr.).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U77846.
§ Performed this work in partial fulfillment of the doctoral dissertation requirements of Vanderbilt University. Present address: Dermatology Div., Dept. of Medicine, Washington University School of Medicine, St. Louis, MO 63110.
To whom correspondence should be addressed: Dept. of Pathology,
C-3321 MCN, Vanderbilt University School of Medicine, Nashville, TN
37232-2561. Tel.: 615-327-4751 (ext. 5488); Fax: 615-327-5393; E-mail:
jeffrey.m.davidson{at}vanderbilt.edu.
The abbreviations used are:
SVAS, supravalvular
aortic stenosis; TGF-, transforming growth factor-
; UTR, untranslated region; RT-PCR, reverse transcription-polymerase chain
reaction; bp, base pair(s); ORF, open reading frame.
2 M.-C. Zhang and W. C. Parks, submitted for publication.
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