Download Genetic Analysis of Familial Connective Tissue Alterations

Genetic Analysis of Familial Connective Tissue Alterations
Associated With Cervical Artery Dissections Suggests
Locus Heterogeneity
Tina Wiest, PhD; Sonja Hyrenbach, MD; Pinar Bambul, MD; Birgit Erker, MD;
Alessandro Pezzini, MD; Ingrid Hausser, PhD; Marie-Luise Arnold, PhD; Juan José Martin, MD;
Stefan Engelter, MD; Philippe Lyrer, MD; Otto Busse, MD;
Tobias Brandt, MD; Caspar Grond-Ginsbach, PhD
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Background and Purpose—Cervical artery dissections (CAD) can be associated with connective tissue aberrations in skin
biopsies. The analysis of healthy relatives of patients suggested that the connective tissue phenotype is familial with an
autosomal dominant inheritance.
Methods—We performed genetic linkage studies in 3 families of patients with CAD. Connective tissue phenotypes for the
patients and all family members were assessed by electron microscopic study of skin biopsies. A genome-wide linkage
analysis of 1 family (1 patient with 8 healthy relatives) indicated 2 candidate loci. Three genes were subsequently studied by
sequence analysis. Part of the genome was also studied by linkage analysis in 2 further families.
Results—The genome-wide scan in a single family suggested linkage between the hypothetical mutation causing the connective
tissue phenotype and informative genetic markers on chromosome 15q24 (logarithm of the odds score: Z⫽ ⫹2.1). A second
possible candidate locus (Z⫽⫹1.9) was found on chromosome 10q26. Sequence analysis of 3 candidate genes in the
suggestive locus (chondroitin sulfate proteoglycan4 [CSPG4], lysyl oxidase-like1 [LOXL1] and fibroblast growth factor
receptor2 [FGFR2]) did not lead to the identification of a mutation responsible for connective tissue alterations. In 2 additional
smaller families the loci on chromosome 15q24 and 10q26 were excluded by linkage analysis.
Conclusions—Linkage analysis of a large family with CAD-associated connective tissue alterations suggested the presence
of a candidate locus on chromosome 15q2 or on chromosome 10q26. Sequence analysis did not lead to the identification
of a mutated candidate gene in 1 of these loci. The study of 2 additional pedigrees indicated locus heterogeneity for the
connective tissue phenotype of CAD patients. (Stroke. 2006;37:1697-1702.)
Key Words: cerebrovascular disorder 䡲 cervical artery dissection 䡲 genetic linkage analysis
C
ervical artery dissections (CAD) are an important cause
of stroke in young and middle-aged patients.1,2 In few
patients the development of the dissection is thought to be
triggered or caused by mechanical injury, but the pathogenesis of the disease remains unclear in most cases.3
Several risk factors were associated with CAD, as for
instance hereditary connective tissue disorders, in particular
the vascular type of Ehlers-Danlos syndrome (EDS IV),
recent infection, mild hyperhomocysteinemia, ␣-1–antitrypsin deficiency, migraine or fibromuscular dysplasia.4 However, the possible role of these risk factors in the development
of dissections is not self-evident.4
The hypothesis of a structural defect of the arterial wall and
the association with recent infections lead to the study of
several candidate genes in CAD patients by DNA sequence
analysis or by genetic association analysis. Most genes studied
so far were directly involved in the biosynthesis of extracellular matrix components. However, despite many efforts the
candidate gene approach did not yet lead to the identification
of genes that are involved in the pathogenesis of CAD.5 We
therefore aimed at a genome-wide scan by linkage analysis to
search for a candidate locus. However, familial CAD is rare.
The CAD disease phenotype might have a low penetrance,
CAD might occur asymptomatically or unrecognized as such,
and healthy persons without CAD might develop the disease
in the future. We therefore preferred to analyze a subclinical
marker associated with CAD and not the CAD phenotype
itself.
Received January 16, 2006; final revision received April 11, 2006; accepted April 21, 2006.
From the Department of Neurology (T.W., S.H., P.B., M.-L.A., T.B., C.G.-G.), University of Heidelberg, Germany; the Department of Neurology
(B.E., O.B.), City Hospital Minden, Germany; the Department of Neurology (A.P.), University of Brescia, Italy; the Department of Dermatology (I.H.),
University of Heidelberg, Germany; the Department of Neurology (J.J.M.), Sanatorio Allende, Cordoba, Argentina; the Neurological Clinic and Stroke
Unit (S.E., P.L.), University Hospital Basel, Switzerland; and the Department of Neurological Rehabilitation (T.B.), Schmieder-Kliniken Heidelberg,
Germany.
Correspondence to Dr Caspar Grond-Ginsbach, Neurology Department, University of Heidelberg, Im Neuenheimer Feld 400, D-69120 Heidelberg,
Germany. E-mail [email protected]
© 2006 American Heart Association, Inc.
Stroke is available at http://www.strokeaha.org
DOI: 10.1161/01.STR.0000226624.93519.78
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Skin biopsies from most patients with CAD revealed morphological alterations of the dermal connective tissue,6,7 which
suggested the presence of a further unknown risk marker.
Different patterns of connective tissue aberrations were found in
patients with CAD.8 The most common type of connective tissue
aberrations with composite collagen fibrils and fragmentation of
elastic fibers was studied previously in a small pedigree and
found to be familial with an autosomal dominant pattern of
inheritance.9 In this study we present 2 additional and larger
families with inherited connective tissue aberrations of the same
pattern. We performed a genome-wide genetic linkage analysis
to identify a susceptibility locus, responsible for the connective
tissue phenotype and increasing the risk for CAD.
Materials and Methods
Materials
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The index patient of family A had a stroke at the age of 70. Bilateral
CAD resulting in high-grade stenosis of both extracranial internal
carotid arteries was revealed whereas signs of artherosclerotic stenoocclusive disease of the carotid arteries were absent. The index patient
of family B developed a dissection of the right internal carotid artery at
the age of 26. His medical history was presented in more detail
elsewhere.10 Family C was analyzed in a previous exclusion mapping
study.9 Deep-knife skin biopsies were sampled from all 3 index patients
and prepared for histology, electron microscopy and for culturing of
fibroblasts as described.8 All patients showed clear and reproducible
connective tissue alterations of the same (“Ehlers Danlos syndrome-III
like”)8 type. Relatives were informed by the index patients and asked to
participate in the study. The performance of skin biopsies and the
sampling of blood were approved by the local ethical committee
(University of Heidelberg) and required informed consent from each
patient. Eight relatives of patient A, 5 relatives of patient B and 4
relatives of index patient C were phenotyped by an electron microscopic
inspection of a skin biopsy.
EDTA-blood was sampled from all phenotyped family members
and also from their partners. DNA was isolated after SDS-proteinase
K digest, phenol-chloroform extraction and ethanol precipitation following standard procedures.12
Linkage Analysis
Three hundred and eighty-three autosomal microsatellite markers of
the Applied Biosystems MD10 linkage mapping set (mean distance
of the genetic markers 10 cM) were used for an initial genome scan
of family A. Fluorescent amplification products were visualized on
the ABI-310 genetic analyzer and analyzed with the GeneScan
software. Logarithm of the odds (LOD) profiles were calculated in
multipoint analyses for whole chromosomes with the Genehunter
Software (version 2.1).11 The position of the microsatellite markers
were calculated as the mean of male and female cM localization as
indicated in the cedar genetics data bank (http://www.cedar.genetics.soton.ac.uk/summaryml.html). For the estimation of unknown allele frequencies of microsatellite markers we followed the CEPH database
(http://www.cephb.fr/test/cephdb/). Additional microsatellite markers
were selected and analyzed in a second phase of linkage analysis for
chromosome regions with inconclusive results (uninformative genetic markers, LOD-scores ⬎⫺2). In total the segregation of 453
informative microsatellite markers was analyzed in family A.
Moreover, 2 members of the family (a brother of the index patient
as well as his grandson, both displaying a connective tissue phenotype) were analyzed with the aid of Affymetrix 10K single-nucleotide polymorphism (SNP) arrays. Among about ten thousand genotyped markers we found 531 informative SNPs that were suitable for
additional exclusion mapping. Ninety-one of those informative SNPs
were located in regions with low density of informative microsatellite markers and were included in the LOD score calculation.
Family B was studied by linkage analysis for those genomic regions
where we found LOD-scores above ⫺2 in family A. Segregation of 113
microsatellite markers for these genomic regions was analyzed in these
kindred. Family C had previously been analyzed with genetic markers
flanking 32 candidate loci.9 We now analyzed in this family the
segregation of 28 additional CA-repeat markers.
Polymerase Chain Reaction
and Sequencing Analysis
The LOXL1 gene (lysyl oxidase-like 1; GC15P072005; 7 exons) is
located on position 72,005,852 to 72,031,529 of chromosome 15. No
additional degenerate copies were detected by BLAST searching
with single LOXL1 exons as search sequences.
The CSPG4 gene (chondroitin sulfate proteoglycan 4; GC15M073754;
10 exons) is located on position 73,754,019 to 73,792,151 of chrosome 15.
Multiple sequence copies with high similarity to Exons 2 to 8 of the
CSPG4 gene were found during BLAST search. Homologous sequences
are detected on the Y chromosome (AC006328) and in multiple loci of
15q21 (AC019294; AC104758; AC136698; AC135995; AC135735;
AC127482; AC136704; AC044860; AC126605; AC110291; AC005630;
AC010725; AC011295; AC010724; AC012064). In order to design
specific primers for single exons all these different pseudogene sequences were added to the sequence for primer search. Specific primer
sequences were particularly difficult to find for exon 3. For this exon
only few specific primers were found, and nested polymerase chain
reactions were required to generate overlapping fragments not longer
than about 600 bp for direct sequencing analysis.
The FGFR2 (fibroblast growth factor receptor 2 precursor) gene
is located on chromosome 10 (122.473.377 to 123.347.936). The
coding region of FGFR2 was amplified in 4 overlapping fragments
after RT-PCR of RNA from dermal fibroblasts and analyzed by
sequence analysis. The sequence of exons 2 to 5 of the cDNA could
not be analyzed properly in cDNA, because of alternative splicing.
Therefore, exons 2,3,4 and 5 were analyzed in amplified genomic
DNA sequences.
Each sequence region was amplified from family members with
connective tissue alterations as well as relatives without connective
tissue alterations and subsequently studied by sequencing analysis.
Results
Connective Tissue Morphology
Pedigrees of the analyzed families are shown in Figure 1. The
connective tissue morphology of skin biopsies from all family
members was studied by electron microscopy. Mild but significant connective tissue alterations with “flower-like” composite
fibrils in some collagen bundles and with fragmentation of the
elastic fibers (“moth eaten aspect”) were found in the index
patients and in some of their relatives. In all 3 families we found
the same EDS-III like morphology, which is the most common
pattern of morphological aberrations in CAD.22 The connective
tissue alterations of members of family C were presented in
detail before.9 The segregation pattern of the connective tissue
phenotype is in agreement with the assumption of autosomal
dominant inheritance. X-chromosomal markers were not studied, because the observed pattern of inheritance excluded sexlinked transmission.
Genetic Linkage Analysis
DNA from all members of family A was analyzed with 453
informative microsatellite markers and 91 SNPs. The mean
distance between (CA)-repeat markers was 7.6 cM, with a
maximum of 23 cM (between D20S173 and the telomere of
chromosome 20q) and of 26 cM (between D8S505 and
D8S285). For the analysis of the genetic linkage data we
assumed a model of autosomal dominant inheritance with full
penetrance of the connective tissue phenotype, absence of
Wiest et al
Locus Heterogeneity of Cervical Artery Dissections
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Figure 2. Linkage analysis in Family A. Linkage graphs for all
autosomal chromosomes are shown. X-axis: genetic distance
along the chromosomes from pter to qter (in cM); Y-axis: LOD
scores. Arrows indicate regions on chromosomes 10 and 15
with suggestive linkage.
Figure 1. Pedigrees of families (A through C) with CAD patients
(arrows) and relatives. The connective tissue phenotype (filled
symbols) was assessed by electron microscopic analysis of a
skin biopsy. Open symbols indicate relatives with normal connective tissue phenotype. Numbers indicate age (in years) at
which biopsy was taken.
phenocopies, and a mutation prevalence of 0.001 in the normal
population.
LOD scores under ⫺2, virtually excluding the presence of
a hypothetical mutation, were found in 80.7% of the (autosomal) genome of family A (Figure 2). Inconclusive LOD
scores (above ⫺2) were found for the remaining autosomal
region. The maximum LOD score (Z⫽2.1) was detected only
in 1 locus in this family. We initially found perfect cosegregation of 1 allele of marker D15S131 and the connective
tissue phenotype. In a subsequent analysis of 11 other microsatellite markers in close vicinity of D15S131 the candidate locus
with maximum LOD score of 2.1 appeared to include 6 markers
in a genetic region of about 5 cM between D15S131 and
D15S1023 (Figure 3). One further very short region with Z⫽1.9
was detected at chromosome 10 between D10S1757 and
D10S1483. No further suggestive loci were detected in the
genescan of this family.
All regions with LOD scores ⬎⫺2.0 in family A were
subsequently analyzed in family B. The segregation of 113
genetic markers was studied and the results for both pedigrees
were used for the calculation of LOD scores. Both suggestive
loci in family A on chromosomes 10 and 15 were excluded in
family B and very few loci were not excluded (LOD score
⬍⫺2.0) in 2 family analyses. Three loci with slightly positive
LOD score were found in the combined analysis of family A
and B (data not shown). However, all these 3 loci are excluded
by genetic linkage analysis in Family C.
Sequence Analysis of Selected Candidate Genes
Genetic analysis of family A suggested linkage with an assumed
autosomal dominant mutation between genetic markers
D15S131 and D15S1023. Within this region of chromosome 15
we analyzed 2 candidate genes in more detail (LOXL1 and
CSPG4). Because exons 2 to 8 of CSPG4 were found to be
present in many highly homologues copies throughout the
genome, the analysis of these exons turned out to be extraordinary hard. We produced specific sequences of about 75% of
the coding sequence of CSPG4, but we were unable to
generate specific amplification products from parts of exon 3.
The genomic sequences of CSPG4 and LOXL1 in the index
patient were compared with the sequences from his healthy
brother without the connective tissue phenotype and with
published consensus sequences. In CSPG4 we found a
heterozygous G/A substitution at position G5525 in both the
index patient and his brother. The index patient was homozygous and his brother heterozygous for a known SNP in the
LOXL1 gene (Table). The specific amplification of sequences
from ADAMTS7, a third candidate gene within this region,
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Figure 3. Multipoint data for the 2 loci
with suggestive linkage in family A. Most
likely haplotypes were reconstructed on
the basis of the cosegregation of alleles.
Brackets indicate candidate regions in
chromosome 15q and chromosome 10q
with high LOD scores. Boxes delineate
the segregation of haplotypes from the
index patient. All relatives with the connective tissue phenotype share a haplotype for region D15S131–1023. A short
region of chromosome 10 (around marker
D10S209) is common in all subjects with
the connective tissue phenotype, whereas
flanking regions are excluded.
turned out to be too difficult in our hands and we gave up the
initial idea to analyze the coding sequence of this gene completely. In the FGFR2 gene which is located on chromosome
10q26 we found a single silent variant (a G to A substitution at
position 1288). Hence, no sequence variants were found in any
of these genes that are likely to explain the connective tissue
phenotype.
Discussion
Patients with a family history of CAD are rare, and families
with ⬎3 affected members were not described. Hence, a
genetic analysis of CAD on the basis of large families with
affected and unaffected subjects is not possible. However, the
finding of inherited connective tissue alterations in patients as
well as in their healthy relatives enables the genetic analysis
Wiest et al
Locus Heterogeneity of Cervical Artery Dissections
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Genetic Variation in Candidate Genes. Sequence Variation Was Detected in Individuals From Family A With
Connective Tissue Alterations
Gene
Exon Position
Nucleotide Variation
Nucleotide Position
Amino Acid Variation
Amino Acid
SNP-ID
FGFR2
6
1288
G3A
232
V3V
rs 1047100
LOXL1
1
748
G3T
141
R3L
rs 1048661
CSPG4*
10
5525
G3A
1842
R3Q
Not described
Because the LOXL1 G748T and the CSPG4 G5525A SNPs were also found in a sibling with wild-type (normal) connective tissue
phenotype, these nucleotide substitutions are considered as polymorphisms without effect on the pathogenesis of CAD.
*The sequence of part of exon 3 of the CSPG4 gene was not analyzed because we could not generate specific polymerase chain
reaction products for this region of the gene.
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of large families. Not the disease itself is considered as the
mutation phenotype, but the presence of ultrastructural connective tissue alterations. In this genome-wide linkage analysis
study we initiated a candidate locus search for the connective
tissue phenotype.
In family A we identified a suggestive candidate locus
responsible for the connective tissue phenotype. The locus
between D15S131 and D15S1023 on chromosome 15q2 was
the only locus with maximum LOD score (Zmax⫽2.1), attributable to perfect cosegregation of a putative mutation with
highly informative genetic markers. However, this LOD score of
2.1 is below the required standard for the demonstration of
linkage, because the analyzed pedigree does not contain
enough informative meioses. In a second family with the
same connective tissue phenotype, the region between
D15S131 and D15S1023 was excluded as candidate locus for
a disease-causing mutation. All genome regions with inconclusive results (Z⬎⫺2) in family A were studied by genetic
linkage analysis in Family B. Linkage was excluded for the
majority of the genome in this combined genetic analysis of
2 pedigrees. Additional linkage studies in family C finally
resulted in exclusion of the whole genome, which indicated
locus heterogeneity of the connective tissue phenotype.
Locus heterogeneity was also found in linkage studies of
families with familial aorta dissections (with loci on chromosome regions 3p24 –25, 5q13–15 and 11q23–2413–17) and with
intracranial aneurysms (with proposed candidate loci on chromosomes 1p34,18 2p13,19 7q11,20 7q22,21 17cen,22 19q1322,23
and Xp2222).
The connective tissue phenotype in our families has similarities with the findings in EDS-III, a connective tissue
disorder characterized by joint hypermobility. Chromosome
region 15q2 was reported to harbor a candidate gene involved
in a psychiatric disorder associated with joint hypermobility.29,30 Chromosome region 15q2 has a complex sequence
structure with many degenerate long repeats (which, moreover, makes this chromosome region particularly difficult to
study). The original finding of an additional large disease
causing duplication (DUP25)29 in patients with joint hypermobility and a psychiatric disorder was not confirmed by
other researchers.31 The hypothetical unknown gene in this
region, involved in joint hypermobility, is not yet identified.
The suggestive loci on 15q24 and 10q26 that we found in
our linkage studies contains several promising candidate genes.
The CSPG4 gene shows sequence homology with CSPG2,
the versican encoding gene. CSPG2 was repeatedly discussed
as a candidate gene involved in aneurysm formation15,24 and
versican is considered to be a key molecule in vascular
disease.24,25 The LOXL1 gene is highly expressed in large
arteries and involved in elastic fiber deposition.26 Inactivation
of LOX leads in mice to the development of aortic aneurysm
and perinatal death.27 Recently a dramatic down regulation of
LOX gene expression was reported in a patient with spontaneous coronary artery dissection.28 FGFR2 shares sequence
homology with TGFBR2, a gene that was recently shown to
carry mutations in some families with aortic dissections and
aneurysms.32
Possible criticism of the present study might include the
size of family A, which is too small to demonstrate linkage
with certainty and solely enables the detection of suggestive
linkage. However, the occurrence of familial CAD is extremely rare, and the study of the connective tissue phenotype
as a subclinical marker for CAD enabled for the first time a
genome-wide linkage for this disease. A further criticism
might be that we only analyzed the coding sequences of 3
candidate genes. We prefer to perform additional linkage
studies of further large families first. The identification of a
locus with LOD score ⬎3 should have priority over further
sequence analysis of candidate genes.
The identification of the connective tissue phenotype enabled
a genome-wide linkage analysis for the investigation of a genetic
predisposition to cervical artery dissections. We found 2 loci
with suggestive linkage for CAD. The analysis of 3 families
with a similar connective tissue phenotype demonstrated locus
heterogeneity.
Source of Funding
This study was supported in part by the CompetenceNet Stroke of the
German Ministery of Science and Technology (BMBF).
Disclosures
None.
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Stroke. 2006;37:1697-1702; originally published online May 25, 2006;
doi: 10.1161/01.STR.0000226624.93519.78
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