ARTICLE
Auteur(s) : Heather
N Yeowell1, Linda C Walker1, Luitgard M
Neumann2
1Div of Dermatology, Duke Univ Medical Centre,
Durham, NC, USA Fax: (+1) 919-684-3002.
2Institute of Human Genetics, Charité Campus
Virchow-Klinikum, Berlin, Germany
accepté le 28 Juin 2005
Ehlers-Danlos syndrome type VIA, the kyphoscoliosis type (OMIM
no.225400), is a rare autosomal recessive connective tissue
disorder, clinically characterized by soft extensible skin that is
subject to poor scarring and easy bruising, laxity of joints,
severe muscle hypotonia at birth and kyphoscoliosis [1]. The
patients are prone to arterial rupture. The cystic malformations of
the meninges that characterize the patient in this study have not
been previously reported in EDS VIA patients. EDS VIA is confirmed
biochemically by a deficiency of lysyl hydroxylase (LH), also named
procollagen-lysine, 2-oxoglutarate 5-dioxygenase (PLOD), a
posttranslational modifying enzyme in collagen biosynthesis that
hydroxylates specific lysines in helical and non-helical
(telopeptide) collagen. Certain of these hydroxylysines are
precursors for the formation of cross-links that are essential for
the tensile strength of collagen. Therefore any disruption in
collagen lysyl hydroxylation would be predicted to affect the
stability of the extracellular matrix. The deficiency of LH in EDS
VIA patients has been linked to more than 20 mutations in the LH1
gene, the originally-described form of LH [2]. These patients have
been shown to be either homozygous or compound heterozygous for the
mutations in the LH1 gene. With the exception of the most common
mutation of a large duplication of exons 10 to 16 in the LH1 gene
that has been found in approximately one fifth of EDS VI families
studied [3-5], the majority of the other mutations either terminate
or disrupt the reading frame of the protein [2]. A second, rarer
form of EDS VI has been described, EDS VIB (OMIM no. 229200), in
which patients have the clinical phenotype of EDS VI but with
normal levels of LH activity [1, 6]. However, unlike EDS VIA, this
form appears to be genetically heterogeneous [6].The 10-year-old
female patient in this study has, in addition to the typical
characteristics of EDS VIA, cystic malformations of the meninges
that the authors attributed to the connective tissue weakness of
this disorder [7]. A deficiency of LH was indicated by analysis of
urinary cross-links that showed a decrease in the
hydroxylysyl-pyridinoline to lysyl-pyridinoline ratio and supported
a diagnosis of EDS VIA. In the current study we have confirmed this
diagnosis by an assay of LH activity in which we have shown
severely decreased levels of the enzyme in the patient’s skin
fibroblasts.Sequence analysis has identified that the causative
mutation in the LH1 gene predicted to result in the deficiency of
LH activity and the patient’s clinical phenotype is the common
seven exon duplication. The homozygosity of this mutation was
confirmed by its identification in the parents’ DNA, who although
clinically unaffected, are carriers of the duplication. Based on
the mutational analysis of an additional eighteen families with EDS
VIA since the last report [8], we have recalculated the frequency
of the duplicated allele to be 18.3%.
Patient summary
The 10-year-old female patient in this study was the first child of
healthy consanguineous Turkish parents. The three younger brothers
were healthy ( (figure
1) ). Clinical features and photographs of the patient have
been published previously when she was 7 years of age [7]. In
brief, after normal pregnancy and delivery, neonatal muscular
hypotonia as well as congenital kyphoscoliosis and pedes valgi were
noted. An upper brachial plexus palsy developed. Cranial MRI
performed 8 days after birth disclosed an infratentorial subdural
hematoma. At examination at the age of 7 months, muscular hypotonia
was persistent with insufficient head control. At that age she had
undergone a neuromuscular workup including a muscular biopsy
without pathological result. In general, her motor development was
retarded; she began walking at the age of two years. Apart from the
left upper brachial plexus palsy, her neurological status and
intellectual development were normal.
At the age of 7 years the dermatological diagnosis of EDS type
VI was suggested. Analysis of urinary cross-links showed a decrease
in the hydroxylysyl-pyridinoline to lysyl-pyridinoline ratio
indicative of EDS VIA. When seen by us at the age of ten years (
(figure 2A-C) )
she showed a mildly progressive profound kyphoscoliosis which was
treated by external bracing.
Her body height, weight and head circumference were at the
25th percentile. She had a prominent abdomen with an
umbilical hernia. Especially on the forehead ( (figure 2C) ) and both
lower limbs, several hyperpigmented cicatrices with
cigarette-paper-like skin, which had resulted from minor injuries,
were noted. Marked general joint hypermobility without joint
dislocations was present and pedes plani. She presented only mild
skin hyperextensibility, bluish sclerae, teeth abnormalities
(agenesia of all second premolars, malposition of incisive and
canine teeth). The ophthalmologic examination disclosed no
abnormalities. An aortic aneurysm was excluded annually by
echocardiography. In addition to the typical characteristics of EDS
VIA, cystic malformations of the meninges that the authors
attributed to the connective tissue weakness of this disorder [7]
were present.
Materials and methods
Cell culture
Human dermal fibroblasts from the proband (1276) and from a normal
control (842) (GM05659, Coriell Institute for Medical Research,
Camden, NJ), were grown to confluency in Dulbecco’s modified
Eagle’s medium (Invitrogen-Gibco) supplemented with 20%
heat-inactivated calf-serum (Invitrogen-Gibco) as described [9].
Lysyl hydroxylase activity
LH activity in fibroblast extracts was measured with an
L-[4,5-3H] lysine-labeled underhydroxylated procollagen
substrate as described [9]. Prolyl hydroxylase (PH) activity was
measured with a similar underhydroxylated procollagen substrate
labeled with L-[4-3H] proline. The enzyme activities
were quantitated as released 3H2O in a
minimum of 4 assays.
PCR amplification of full length LH1cDNA from patient 1276
cDNA was prepared in a 20 μl reaction with 1 μg total RNA
in ddH2O [10], and amplified using PCR primers under
reaction conditions as described [11]. The PCR products were
electrophoresed on a 1% agarose gel.
PCR amplification over junction of duplication mutation in
genomic DNA
Genomic DNA was isolated from cultured skin fibroblasts using the
Blood & Cell Culture DNA mini kit (Qiagen) according to the
manufacturer’s protocol. PCR was performed on the genomic DNA from
the patient and her parents using primers (JH71 and JH44) based on
introns 9 and 16 to amplify a 960 bp fragment as previously
described [12].
Sequence analysis of duplicated region
Using primers JH71 and JH55 [5], a shorter fragment (~300 bp)
was amplified over the duplication junction and sequenced at the
Duke DNA sequencing facility.
Sequence analysis over polymorphisms: The regions covering the 2
polymorphisms, 318C→T (exon 3) and 1230C→T (exon 12), that are
associated with the duplicated allele, were amplified by PCR from
genomic DNA as described [2].
Results
As described under Patient Summary, the clinical phenotype of the
patient in this study suggested a diagnosis of EDS VI ( (figure 2A-C) ), which is
characterized biochemically by a deficiency of LH. A previous
analysis of urinary cross-links showing a decrease in the
hydroxylysyl-pyridinoline to lysyl-pyridinoline ratio indicated a
deficiency of this enzyme. In the current study, we confirmed the
decreased levels of LH in a tritium release assay that measures
predominantly helical LH activity [6, 11]; severely diminished LH
activity (19 ± 6% of control) was measured in the patient’s skin
fibroblasts. Expression of LH activity in reference to PH activity,
another collagen posttranslational-modifying enzyme whose activity
is unchanged in EDS VIA patients [13], gave a similar decrease.
This reduced level of LH activity confirmed the clinical diagnosis
of EDS VIA.
To identify the mutation(s) in the LH1 gene that causes the
deficiency of enzyme activity and the clinical phenotype of EDS
VIA, we initially amplified full length cDNA for LH1 from the
proband’s dermal fibroblasts by PCR as described [11].
Electrophoresis of the products showed that an abnormally large 3kb
DNA fragment, indicative of the large duplication, was amplified
from the patient’s cDNA in contrast to the normal-sized full length
LH1 cDNA (2.2 kb) amplified from a normal donor (data not
shown).
We then confirmed the presence of the duplication mutation in
genomic DNA by PCR amplification using previously-described primers
(JH71 and JH44) to screen the duplication junction such that a DNA
fragment is only amplified if the duplication is present ( (figure 3) )[8]. We
observed amplification of a 960 bp fragment in genomic DNA
both from the proband and her parents ( (figure 3A) ). The
homozygosity of this mutation was confirmed by its identification
in the DNA from both parents, who although clinically unaffected,
are carriers of the same duplication.
For sequence analysis over the duplication junction, we used
primers (JH71 and JH55) to amplify a shorter (~300 bp) region
over the duplication mutation from the proband’s genomic DNA. The
sequence ( (figure
3B) ) showed that the identical 44 bp Alu sequence was
flanked upstream by sequence from intron 16 and downstream by
intron 9. This confirmed that the duplication had occurred between
identical 44 bp sequences in introns 9 and 16 of the LH1
gene.
As we had previously identified two polymorphic markers in the
LH1 gene that are linked to the duplication mutation, we confirmed
their presence in genomic DNA amplified from both the proband and
her parents [2, 12] (data not shown).
Discussion
One of the clinical hallmarks of EDS VIA, in addition to the lax
joints, fragile hyperextensible skin and generalized connective
tissue weakness that define the Ehlers-Danlos syndromes, is severe
neonatal muscular hypotonia and kyphoscoliosis. Cystic malformation
of the meninges, as described in the current patient, has not been
previously associated with deficiency of lysyl hydroxylase activity
and EDS VIA. In their original clinical description of this patient
[7], the authors suggested that the connective tissue weakness in
EDS may lead to cystic malformation of the meninges. There were no
radiological indications of progression of these spinal lesions
over a 2 year period following the initial examination.
This study describes the identification of a homozygous seven
exon duplication mutation in the LH1 gene in fibroblasts from a
patient with the clinical phenotype of EDS VI. This large
duplication has been shown to be the most common mutation
contributing to the decreased LH activity that results in the
clinical characteristics of EDS VIA [2, 8]. The proband in the
current study was previously suggested to have EDS VIA on the basis
of her clinical characteristics and also her abnormal pyridinoline
cross-linking pattern in urine [7]. We have confirmed this
diagnosis by measurement of severely decreased LH activity in
cultured skin fibroblasts and have identified the causative
mutation in the LH1 gene by mutational analysis. The homozygosity
of the mutation was confirmed in genomic DNA from the parents of
the proband. In other EDS VIA patients who have also been found to
be homozygous for the duplication mutation, there have been no
reports of the occurrence of spinal lesions suggesting, in this
particular patient, the interplay of additional connective tissue
factors influencing formation of the meningeal cysts.
The 8.9 kb duplication occurs via a homologous
recombination of a 44 bp identical sequence, that is located
in a region of Alu repeats in introns 9 and 16, to generate the
duplication of exons 10-16 [8]. The duplication does not disturb
the exon-intron splicing pattern of the LH1 gene; it results in an
abnormally large mRNA (4.2 kb) compared with the normal
3.4 kb mRNA for LH1. Although the duplication does not affect
the catalytic site of the enzyme at the carboxy-terminal of LH1,
the lengthening of the protein is predicted to cause major changes
in the conformation of the protein, which presumably causes the
marked reduction of LH activity in the affected cells. Sequence
analysis of the junction fragment of the duplication [5] has
enabled the selection of specific primers for rapid screening for
this rearrangement in genomic DNA, which we have utilized in the
current study. These primers will only amplify a 960 bp DNA
fragment if the duplication is present and we have observed this
fragment confirming the duplication in both the proband and her
parents. In addition we have amplified and sequenced a shorter
fragment (~300 bp) from genomic DNA to confirm the duplication
junction in the proband. The sequence shows nucleotides from intron
16 and intron 9 flanking the common 44 bp Alu sequence that is
identical in introns 9 and 16 of the LH1 gene.
Earlier reports have shown patients to be both homozygous and
compound heterozygous for this duplication mutation, but in view of
the consanguinuity of the marriage (( figure 1 )), in this study
the homozygosity was not unexpected. We have confirmed that
uniparental isodisomy inheritance (in which both alleles are
inherited from a single parent) is not involved by showing that the
parents, although clinically unaffected, are carriers for the
duplicated alleles. As shown in the pedigree in ( figure 1 ), the proband has
three unaffected brothers, who are all in good health.
Unfortunately, we were unable to obtain their DNA to establish
whether or not they were carriers of the mutation.
A useful tool to examine the possibility of inheritance of a
single mutated ancestral gene is provided by polymorphic markers
that cosegregate with a specific mutation in unrelated patients. As
a result of the comprehensive sequencing to identify mutations in
over 35 patients with EDS VIA, five polymorphic markers have been
identified in the LH1 gene [12]. Two of the polymorphisms, 318C→T
(exon 3) and 1230C→T (exon 12), have been shown to consistently
cosegregate with the large seven exon duplication [12]. This data
indicates that an ancestral single-mutational event for the
duplication may have occurred This observation is supported by
haplotype analysis of 9 EDS VIA families in which association of a
long form of the LH1 allele with the duplication mutation strongly
suggests that the recurrent mutation has originated from a single
ancestral gene [8]. In the current study we identified the presence
of the two duplication-linked polymorphic markers in the LH1 gene
in genomic DNA amplified both from the proband and her parents,
thereby providing further support for both the homozygosity of the
mutation and its ancestral gene linkage.
Over twenty mutations have been identified in the LH1 gene that
are responsible for the LH deficiency in patients with the clinical
phenotype of EDS VIA. With the exception of the large duplication
mutation described in this study, the majority of the other
mutations, which either terminate or disrupt the reading frame of
the protein, result in accelerated degradation of mRNA. The second
most common mutation, after the duplication mutation, is a point
mutation predicted to create a premature termination codon Y511X in
exon 14 of the LH1 gene which has been identified in five unrelated
patients [2, 14]. Two patients are homozygous and three patients
are compound heterozygous for the Y511X mutation, giving an
estimated allelic frequency of approximately 10%.
In an earlier report [8], the frequency of the large duplication
mutation was calculated as 19.1% based on its identification in 13
out of a total 68 gene alleles examined in 35 EDS VIA families.
Together with the identification of 2 duplicated alleles in 30
additional screened alleles [2] and, in our laboratory, the recent
characterization of 4 duplicated alleles from a total of 6 alleles
from 3 patients (including the patient in this study), we have a
current estimate of 19 duplicated alleles out of a total of 104
genetically independent alleles from 53 EDS VIA families. Based on
this, we have recalculated the allelic frequency of this mutation
to be 18.3%. In a general population, screening of 582 alleles has
identified only one positive finding [8], which is not unexpected
considering the rarity of EDS VIA.
In summary, this study describes the identification of the
homozygous large duplication mutation in the LH1 gene that is
causative for the diminished LH activity leading to the clinical
phenotype of EDS VIA in the affected individual. In addition to the
typical characteristics of EDS VIA, the proband had cystic
malformation of the meninges that may have arisen due to the
weakness of connective tissue associated with this disorder [7].
This has not been previously reported in other EDS VIA patients who
are also homozygous for the common duplication and it may suggest
the interplay of other factors associated with the stability of the
extracellular matrix in this patient.
Acknowledgements
This study has been supported in part by NIH Grant AG10215 from the
National Institute on Aging (HNY). We would like to acknowledge the
technical assistance of Kenetta Nunn in this study.
References
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