(Stroke. 1999;30:1887-1890.)
© 1999 American Heart Association, Inc.
Original Contributions |
Mutations in the COL5A1 Coding Sequence Are Not Common in Patients With Spontaneous Cervical Artery Dissections
From the Departments of Neurology (C.G.-G., R.W., J.H., E.O., T.B., B.W.) and Dermatology (I.H.), University of Heidelberg, Germany; and the Department of Neurology (S.K., O.B.), City Hospital, Minden, Germany.
Correspondence and reprint requests to PD Dr med Brigitte Wildemann, Neurologische Universitätsklinik, Im Neuenheimer Feld 400, 69120 Heidelberg, Germany. E-mail brigitte wildemann{at}med.uni-heidelberg.dewildemann@med.uni-heidelberg.de
 |
Abstract |
|---|
 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
Background and Purpose—The dermal connective tissue of most patients with spontaneous cervical artery dissections (sCAD) contains abnormal collagen fibers. This suggests a predisposing connective tissue defect. The ultrastructural abnormalities in the skin of patients with sCAD have similarity with the morphological alterations in patients with Ehlers-Danlos syndrome type II, a dominant hereditary disorder that has been correlated in some patients to mutations within the genes encoding type V collagen. The aim of this study was to assess the alpha 1 chain of type V collagen (COL5A1) as a candidate gene for sCAD.
Methods—We searched for mutations in the COL5A1 gene in cDNA from cultured fibroblasts of 19 patients with sCAD using single-strand conformational polymorphism analysis and nucleotide sequence analysis of polymerase chain reaction–amplified fragments of the whole COL5A1 coding sequence.
Results—We detected 1 missense mutation leading to a predicted amino acid (192D/N) substitution within the N-terminal propeptide in 2 siblings. All other patients showed regular COL5A1 sequences with some silent polymorphisms.
Conclusions—Mutations in the COL5A1 gene do not appear to be a major factor in the etiology of sCAD.
Key Words: connective tissue disorders • dissection • genetics • mutation
|
Introduction |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
Spontaneous cervical artery dissections (sCAD) are increasingly recognized as a cause of stroke among young and middle-aged patients.1 A predisposing arteriopathy in these patients has been postulated.2 A recent study by Brandt et al3 substantiated the hypothesis of a predisposing defect in the extracellular matrix. These authors found ultrastructural abnormalities in skin biopsies of a majority of patients with sCAD and suggested an association with a connective tissue disorder. Major findings included numerous enlarged and irregular collagen fibrils and pronounced elastic fiber fragmentation. The abnormalities resembled the morphological alterations in patients with Ehlers-Danlos syndrome type II or III (EDS II/III).4 Those changes were not observed in age-matched stroke patients with alternative etiologies of cerebral ischemic infarcts or in healthy control subjects. None of the patients with sCAD had other phenotypic manifestations of a known hereditary connective tissue disorder.
Type V collagen belongs to the family of fibrillar collagens.5 6 7 It is expressed at a low level in many tissues and plays an important role in type I fibrillogenesis by modulating and influencing fibril diameter.8 9 Mutations in COL5A1 and COL5A2 may lead to ultrastructural abnormalities similar to those observed in sCAD patients.10 Furthermore, some patients with EDS I/II carry mutations in the COL5A1or the COL5A2 gene (for reference, see Michalickova et al11 ). The ultrastructural similarities of dermal connective tissue aberrations of EDS II patients and sCAD patients prompted us to start this study and to analyze the full coding sequence of COL5A1 in patients with sCAD.
|
Subjects and Methods |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
We investigated 15 sCAD patients with pronounced electron microscopic alterations in the dermal connective tissue, 4 sCAD patients without ultrastructural abnormalities, and 1 healthy control subject. The mean age of the patients (16 males and 3 females) at the time of dissection was 41.4 years.28–59 One patient suffered from bilateral dissections, and 2 patients subsequently developed a second dissection. Two patients without ultrastructural abnormalities are sibs.
Skin biopsies were taken from the outer side of the upper arm by open deep knife biopsy. Part of the material was processed for electron microscopy according to the method of Hausser and Anton-Lamprecht.4 Fibroblasts from another part of the biopsy were cultured in MEM supplied with 10% FCS. RNA was prepared from cultured skin fibroblasts with RNA-zol (AGS-Heidelberg) and cDNA synthesized with muLV reverse transcriptase and random hexamers (Perkin-Elmer). A mixture of deaza-dGTP and dGTP (3:1) was used for the synthesis of cDNA. DNA was isolated from 3 mL peripheral EDTA blood with DNA-zol (AGS-Heidelberg) or from the midphase of the fibroblast RNA-zol preparations by ethanol precipitation. The whole coding sequence of the COL5A1 cDNA was amplified in the presence of 7% DMSO by polymerase chain reaction (PCR) in 16 overlapping fragments. PCR primers with melting temperatures of approximately 60°C (GC rule) were designed with the aid of the PRIDE program.12 The last primer, located in the 3' untranslated region, was published by Wenstrup et al.13 Amplified DNA fragments were digested with restriction enzymes to produce fragments of 100 to 300 bp for multiplex single-stranded conformational polymorphism analysis (SSCP).14 15 Most fragments were digested twice with 2 different restriction enzymes to generate different patterns of fragments. Following denaturation and subsequent cooling on ice, SSCP fragments were loaded on at least 2 different nondenaturing gel systems (2% or 10% glycerol) and run in a cooled vertical electrophoresis chamber at 15°C overnight. Gels were stained with silver. If single-strand bands were weak or fuzzy or if complementary strands comigrated during electrophoresis, additional electrophoretic conditions were applied by modifying either the run temperature (room temperature or 4°C) or the acrylamide concentration of the gel. PCR products were prepared for dye terminator cycle sequence analysis (Perkin-Elmer) and analyzed at the 310 Abi Prism genetic analyzer following the instructions of the producer.
Genomic DNA was isolated from peripheral blood of several family members of the 2 siblings included in the study as well as from 25 ethnically matched control subjects.
|
Results |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
SSCP indicated the presence of polymorphisms in DNA fragments 0656 to 1157, 1148 to 1512, and 2745 to 3354. In region 0656 to 1157, 2 restriction fragments revealed a polymorphism (Figure
,
panel A). After sequencing of the reamplified PCR products, the
shifts in the SSCP gels were correlated with single-base
heterozygocities at positions 700, 864, 1218, and 3018 of the cDNA
sequence.6 Region 4559 to 5670 of cDNA, encoding the
C-terminal propeptide and part of the triple helix, was
analyzed by direct sequencing in all patients. It contained an
additional, tri-allelic DNA polymorphism at position 4608. The
heterozygocities at positions 864 (22xC/18xT), 1218 (1xC/39xT), 3018
(3xC/37xT), and 4608 (1xA/14xC/25xG) are silent, because substitutions
do not result in changes of the predicted polypeptides. 17 of 20
patients were heterozygous for at least 1 of the polymorphisms in
the COL5A1 coding sequence.
|
The heterozygosity at position 700 in the 2 patients who are
brother and sister (Figure
, panel B) results in a substitution
in the predicted amino acid sequence, with aspartic acid replaced by
asparagine in 1 of both alleles at position 192 of the N-terminal
procollagen amino acid sequence. The TaqI restriction
fragment length polymorphism (RFLP) caused by this mutation was
also studied in genomic DNA. The mother of both patients carries the
mutant allele. It is absent in a healthy brother and the father as
well as in 25 control subjects. A comparison of the predicted
3D-reconstructed polypeptides16 encoded by the
normal and the mutant allele suggests that the amino acid
substitution does not lead to a conformational change of the protein
(data not shown).
|
Discussion |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
The high incidence of ultrastructural dermal connective tissue abnormalities found in patients with sCAD suggests a causal relationship between sCAD and a connective tissue disorder.3 This observation stimulated us to start a search for mutations in a possible candidate gene involved in collagen fibril organization.
Two more groups searched systematically for mutations in patients with sCAD or intracranial aneurysms. Kuivaniemi et al17 analyzed parts of the coding sequence of the a (I) chain of collagen type III in 55 patients. They found no significant mutation. In a recent analysis of the whole COL3A1 coding sequence, mutations were observed neither in patients with intracranial aneurysms nor those with sCAD.18 19
We found 4 silent polymorphisms in the COL5A1 gene. The polymorphism at position 864 that results in an RFLP after digestion with PstI was earlier described as genomic RFLP.20 Seventeen of 20 patients were heterozygous in at least 1 position in the COL5A1 coding sequence. Because these heterozygocities were detected in cDNA, these patients are neither hemizygous for the COL5A1 gene nor do they carry regulatory mutations that result in the transcriptional inactivation of 1 allele (null allele), as described for other collagen genes.21 22 23
The only missense mutation found in this investigation was observed in 2 sibs. It is difficult to prove whether the resulting amino acid substitution has any phenotypic effect. The mother of the patients, who carries the same mutant allele, had a normal ultrasound study of the cervical arteries and did not suffer from symptomatic dissections. Therefore, the mutant condition may not increase the risk of sCAD. However, it is also possible that the mother did not develop sCAD despite an increased risk.
Greenspan et al5 compared the amino acid sequences of the alpha 1 chains of type V collagen in humans and Chinese hamsters. Position 192 of the sequence is not conserved between both species, which suggests that divergence at this position does not disturb an important function. The predicted secondary structures of the proteins encoded by the mutant and the wild-type alleles lead to a similar conclusion.
In summary, the majority of patients with sCAD do not carry mutations in COL5A1. Moreover, we cannot decide whether the only missense mutation detected has any phenotypic effect. Our data therefore suggest that COL5A1 is not a major candidate gene for sCAD.
|
Acknowledgments |
|---|
This study was supported by research grants from the Deutsche Forschungsgemeinschaft (DFG, BR 1858/1-1) and from the Deutsche Schlaganfallhilfe. We thank I. Werner for excellent technical assistance and Prof W. Hacke for excellent working facilities.
Received February 24, 1999; revision received April 8, 1999; accepted June 21, 1999.
|
References |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
1. Guillon B, Levy C, Bousser M-G. Internal carotid artery dissection: an update. J Neurol Sci. 1998;153:146–158.[Medline] [Order article via Infotrieve]
2.
Schievink WI, Wijdicks EFM, Michels VV, Vockley
J, Godfrey, M. Heritable connective tissue disorders in cervical artery
dissections: a prospective study. Neurology. 1998;50:1166–1169.
3. Brandt T, Hausser I, Orberk E, Grau A, Hartschuh W, Anton-Lamprecht I, Hacke W. Ultrastructural connective tissue abnormalities in patients with spontaneous cervico-cerebral artery dissections. Ann Neurol. 1998;44:281–285.[Medline] [Order article via Infotrieve]
4. Hausser I, Anton-Lamprecht I. Differential ultrastructural aberrations of collagen fibrils in Ehlers-Danlos syndrome types I-IV as a means of diagnostics and classification. Hum Genet. 1994;3:394–407.
5.
Greenspan DS, Cheng W, Hoffman GG. The pro-alpha
1(V) collagen chain: complete primary structure, distribution of
expression, and comparison with the pro-alpha 1(XI) collagen chain.
J Biol Chem. 1991;266:24727–24733.
6.
Takahara K, Seto Y, Okazawa K, Okamoto N, Noda
A, Yaoi Y, Kato I. Complete primary structure of human collagen alpha
1(V) chain. J Biol Chem. 1991;266:13124–13129.
7. Takahara K, Hoffman GG, Greenspan DS. Complete structural organization of the human alpha1(V) collagen gene (COL5A1): divergence from the conserved organization of other characterized fibrillar collagen genes. Genomics. 1995;29:588–597.[Medline] [Order article via Infotrieve]
8.
Birk DE, Fitch JM, Babiarz JP, Doane KJ,
Linsenmayer TF. Collagen fibrillogenesis in vitro: interaction of types
I and V collagen regulates fibril diameter. J Cell Sci. 1990;95:649–657.
9. Olsen BR. New insights into the function of collagens from genetic analysis. Curr Opin Cell Biol. 1995;7:720–727.[Medline] [Order article via Infotrieve]
10. Andrikopoulos K, Liu K, Keene DR, Jaenisch R, Ramirez F. Targeted mutation in the COL5A2 gene reveals a regulatory risk for type V collagen during matrix assembly. Nat Genet. 1995;9:31–36.[Medline] [Order article via Infotrieve]
11.
Michalickova K; Susic M; Willing MC, Wenstrup RJ,
Cole WG. Mutations of the alpha2(V) chain of type V collagen impair
matrix assembly and produce Ehlers-Danlos syndrome type I. Hum
Mol Genet. 1998;7:249–255.
12.
Haas S, Vingron M, Poustka A., Wiemann S. Primer
design for large scale sequencing. Nucl Acid Res. 1998;26:3006–3012.
13.
Wenstrup RJ, Langland GT, Willing MC, D'Souza
VN, Cole WG. A splice-junction mutation in the region of COL5A1 that
codes for the carboxyl propeptide of pro a (V) chains results in the
gravis form of Ehlers-Danlos syndrome (type I). Hum Mol
Genet. 1996;5:1733–1736.
14. Grompe M. The rapid detection of unknown mutations in nucleic acids. Nat Genet. 1993;5:111–117.[Medline] [Order article via Infotrieve]
15. Hayashi K. PCR-SSCP: a method for detection of mutations. Genet Anal Tech Appl. 1992;9:73–79.[Medline] [Order article via Infotrieve]
16. Rost B, Sander C. Combining evolutionary information and neural networks to predict protein secondary structure. Proteins. 1994;19:55–72.[Medline] [Order article via Infotrieve]
17.
Kuivaniemi H, Prockop DJ, Wu Y, Madhatheri SL,
Kleinert C, Earley JJ, Jokinen A, Stolle C, Majamaa K, Myllyla VV,
Norrgard O, Schievink WI, Mokri B, Fukawa O, ter Berg JWM, De Paepe A,
Lozano AM, Leblanc R, Ryynanen M, Baxter BT, Shikata H, Ferell RE,
Tromp G. Exclusion of mutations in the gene for type III collagen
(COL3A1) as a common cause of intracranial aneurysms or
cervical artery dissections: results from sequence analysis of
the coding sequences of type III collagen from 55 unrelated patients.
Neurology. 1993;43:2652–2658.
18.
van den Berg JSP, Limburg M, Pals G, Arwert F,
Westerveld A, Hennekam RC, Albrecht KW. Some patients with intracranial
aneurysms have a reduced type III/type I collagen ratio: a
case-control study. Neurology. 1997;49:1546–1551.
19. van den Berg JS, Limburg M, Kappelle LJ, Pals G, Arwert F, Westerveld A. The role of type III collagen in spontaneous cervical arterial dissections. Ann Neurol. 1998;43:494–498.[Medline] [Order article via Infotrieve]
20. Cappa F, Caridi G, Gimelli G, Ghiggeri GM. A new biallelic DNA polymorphism of the human COL5A1 gene. Hum Genet. 1995;95:599–600.[Medline] [Order article via Infotrieve]
21. Thompson JP, Simkevich CP, Holness MA, Kang AH, Raghow R. In vitro methylation of the promoter and enhancer of pro alpha 1(I) collagen gene leads to its transcriptional inactivation. J Biol Chem. 1991 Feb 5;266:2549–2556.
22.
Willing MC, Slayton RL, Pitts SH, Deschenes SP.
Absence of mutations in the promoter of the COL1A1 gene of type I
collagen in patients with osteogenesis imperfecta type I. J
Med Genet. 1995;32:697–700.
23. Prockop DJ, Kivirikko KI. Collagens: molecular biology, diseases, and potentials for therapy. Annu Rev Biochem. 1995;64:403–434.[Medline] [Order article via Infotrieve]
This article has been cited by other articles:
 |
|
 |
|
  C. Grond-Ginsbach, S. Engelter, I. Werner, I. Hausser, U. S. Muller, T. Brandt, and P. Lyrer Alpha-1-antitrypsin deficiency alleles are not associated with cervical artery dissections Neurology, April 13, 2004; 62(7): 1190 - 1192. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Grond-Ginsbach, F. Wigger, M. Morcher, F. von Pein, A. Grau, I. Hausser, and T. Brandt Sequence analysis of the COL5A2 gene in patients with spontaneous cervical artery dissections Neurology, April 9, 2002; 58(7): 1103 - 1105. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Brandt, E. Orberk, R. Weber, I. Werner, O. Busse, B. T. Muller, F. Wigger, A. Grau, C. Grond-Ginsbach, and I. Hausser Pathogenesis of cervical artery dissections: Association with connective tissue abnormalities Neurology, July 10, 2001; 57(1): 24 - 30. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. I. Schievink Spontaneous Dissection of the Carotid and Vertebral Arteries N. Engl. J. Med., March 22, 2001; 344(12): 898 - 906. [Full Text] [PDF]
|
|
|
|
 |
|
 C. Grond-Ginsbach, C. Thomas-Feles, I. Werner, R. Weber, F. Wigger, I. Hausser, and T. Brandt Mutations in the Tropoelastin Gene (ELN) Were Not Found in Patients With Spontaneous Cervical Artery Dissections Stroke, August 1, 2000; 31(8): 1935 - 1938. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||