Arch Dermatol Res (2008) 300:525–529
DOI 10.1007/s00403-008-0884-8

S H O R T CO M MU N I C A T I O N

A novel point mutation in type III collagen gene resulting in exon
24 skipping in a case of vascular type Ehlers-Danlos syndrome
Osamu Okamoto · Tadasuke Ando · Atsushi Watanabe ·Fuminori Sato · Hiromitsu Mimata · Takashi Shimada ·Sakuhei Fujiwara

Received: 23 April 2008 / Revised: 4 August 2008 / Accepted: 22 August 2008 / Published online: 9 September 2008
© Springer-Verlag 2008

Abstract A novel point mutation in a patient with vascular type Ehlers-Danlos syndrome is herein reported. The
patient experienced pneumothorax and multiple arterial
troubles during the past 10 years. The patient’s hand skin
was atrophic, and a paper-like atrophic scar was seen. The
collagen microWbrils from the patient’s skin demonstrated
irregular contour, electron density variegation, and smaller,irregular Wbril diameters. The patient’s Wbroblasts produced
apparently much less type III collagen comparing with control. A skipping of exon 24-originated sequences of thepatient’s mRNA of type III collagen was proved. A single
base pair mutation of thymine to guanine at the second
position of the intron 24 of COL3A1 was Wnally found. Thisis the Wrst report of the mutation of the second position ofexon 24 of COL3A1.
Keywords Ehlers-Danlos syndrome · Type III collagen ·Exon skipping

O. Okamoto (&) · S. Fujiwara
Dermatology, Department of Anatomy,
Biology and Medicine, Faculty of Medicine,
Oita University, 1-1 Idaigaoka, Hasama-machi,
Yufu, Oita 879-5593, Japane-mail: ookamoto@med.oita-u.ac.jp
T. Ando · F. Sato · H. MimataUrology, Department of Tumor Control and Pathogenesis,
Faculty of Medicine, Oita University, 1-1 Idaigaoka,
Hasama-machi, Yufu, Oita 879-5593, JapanA. Watanabe · T. Shimada
Division of Clinical Genetics,
Nippon Medical School Main Hospital,
1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8603, Japan

Introduction
Ehlers-Danlos (EDS) syndrome is a group of diseases demonstrating the abnormalities in the connective tissue, its
central symptoms being hyperelasticity, fragility of theskin, and joint hypermobility [3]. Vascular EDS [2, 16], adisease characterized by hollow organ troubles, i.e., arterialaneurysm, bowel rupture, and pneumothorax, in addition tothe skin symptoms, is one among EDS. The cause of vascular EDS is attributed to the abnormality in the type III collagen [4], which leads to impaired extracellular matrix(ECM) structure formation, and Wnally results in labile hollow organs. The major genetic abnormality of type III collagen is a point mutation and an exon skipping, which
contribute to most of the cases [10]. To date, a number ofmolecular abnormalities of type III collagen is summarized
in the literature, but the information is somewhat limited [9,12]. Thus, among all population of vascular EDS, it is supposed that the genetic and molecular abnormalities are
determined in only a small population of the patients.
Therefore, the accumulation of information about molecular abnormalities would be still important.
In this report, a case of vascular EDS patient is presented. In this patient, the abnormality was found to be askipping of exon 24, which is originated from a one basepair mutation of intron 24.

Case report
A 38-year-old male experienced pneumothorax in 1990,
left renal infarction due to dissecting aneurysm of left renalartery in 1999, right renal infarction due to dissecting aneurysm of right renal artery in 2005 [1], and then pseudoaneurysm of splenic artery, left gastric artery, and intramuscular

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hemorrhage of the left thigh in 2006. The patient did nothave any family history of the arterial trouble. Because of
the repetitive arterial trouble, the patient was transferred toour clinic for examination of vascular EDS.
Upon consultation, the patient’s hand skin was atrophic,
and cutaneous veins were easily visible (Fig. 1a, b). The
patient had hypermobilities in his wrist and Wnger joints(Fig. 1c, d). In the left axillary area, atrophic scars werepresent (Fig. 1e). An angiogram of renal infarction and a
dissecting aneurysm of right renal artery is shown in
Fig. 1f. These clinical features fulWlled three major andminor criteria [2], and the patient was thus clinically diagnosed to have vascular EDS. A skin biopsy was done
from the patient’s forearm. Upon Elastica van Gieson stain,

Fig. 1 Clinical features of thepatient. a and b Appearance ofthe patient’s skin. a Dorsal aspect; b Ventral aspect. Note thatthe patient’s skin is apparently
thin and small cutaneous veinsare clearly visible. c, d Hypermobilities of the patient’s joints.
c Interpharangeal joint of the patient’s thumb can be bent backby nearly 90°. d Thumb sign.
The patient can touch his forearm with his thumb of the sameside. e Paper-like atrophic scaron the left axillary area. Thesescars were formed at the insertion points of tubes for treatmentof pneumothorax in 1990.
f Arterial phase of renoangiogram representing a dissecting
aneurysm in the right renal artery (arrow 1) and a wedgeshaped renal infarction on the renal apex (arrow 2). g Elasticavan Gieson stain of the skinspecimen of the patient. In thepatient, elastic Wbers werestained equally dark as the control. h The same stain of control
specimen

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Arch Dermatol Res (2008) 300:525–529

elastic Wbers of the patient did not show signiWcant abnormality compared with normal ones (Fig. 1g, h).

The patient’s collagen shows abnormal morphology andcomposition
An electron microscopic observation of the patient’s skindemonstrated collagen microWbrils with smaller diameters,irregular contours, and uneven electron densities, comparedwith those of control (Fig. 2a, b). The collagen microWbril
diameter of control was 91.89 § 9.44 nm (mean § SD); incontrast, in the patient, the distribution of the diameters wasbiphasic with the mean § SD of 76.33 § 12.77 nm (Fig. 2c).

Arch Dermatol Res (2008) 300:525–529
Fig. 2 Electron microscopic
view of collagen microWbrils inthe dermis of control individual(a) and from patient of our case(b). White arrows indicate thecollagen microWbrils with irregular contours. Bars indicate
200 nm. c Histograms of collagen microWbril diameter. Emptybars represent that of normal
individual; Wlled bar: that of thepatient of our case. Average
microWbril diameters § SD are
shown on the top of the panel

527

AcontrolCB

patient

control

N= 331, D= 91.89

9.44 nmpatient

N= 302, D= 76.33

12.77 nm9080

Frequency

7060
50403020100512012 012511 511011 011510 51001000-195
5-9900-985
5-8800-875
5-770
0-765
5-660
0-655
5-550
0-545
5-440

microfibril diameter (nm)Analysis using 3H-proline-labeled collagens from Wbroblasts revealed that the position and the amount of 1 (I)and 2 (I) chains from both control and the patient did notdemonstrate any evident diVerence; however, the amount ofboth trimeric and monomeric 1 (III) chains of the patientwere greatly decreased, compared with that of the control(Fig. 3a).

The patient’s COL3A1 showed a novel mutation
Total RNA from cultured Wbroblasts was subjected toreverse-transcriptase PCR (RT-PCR) with primers whichcorrespond to 5⬘ untranslated region and 3⬘ terminal ofCOL3A1 mRNA, respectively. The RT-PCR product waseither directly sequenced, or cloned into a vector, and thenthe sequencing was done [13, 18]. Using the direct
sequencing method, the sequence could not be determinedin progress around the exon 24 of type III collagen. However, it could be Wnally read when the RT-PCR products
were cloned into a vector, namely sequencing based onPCR cloning of the RT-PCR product [13]. Then a genomic PCR using a panel of primers around exon 24 of type
III collagen revealed a single base mutation from T to G at
the second position in the intron 24 (Fig. 3b). The mutation in the second position of exon 24 was the Wrst caseever reported, thus, we identiWed this mutation as a novelone.
Our case fulWlled enough diagnostic criteria for vascular
EDS proposed in 1997 [2] and the diagnosis was conWrmedby the molecular and genomic analysis. Cutis laxa would
be listed as a possible diVerential diagnosis. Our caselacked Xabby, rubber-like skin and no signiWcant morphological abnormality of elastic Wber was observed. Fromthese Wndings, the diagnosis of cutis laxa would be negativein our case.

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Arch Dermatol Res (2008) 300:525–529

Areduction

( )

(+)

(+)

( )

1(III) chain(trimer)
1(III) chain(monomer)1(I) chain
2(I) chain

contr

ol

B

pa

co

tie

nt

ntr

ol

pa
tie
nt

ACCAGGGCCTCCCGTATGTACATTTTTA
orGexon 24control
patient

intron 24

GTATGTGGATGTFig. 3 a Analysis of 3H proline-labeled collagens on SDS-PAGE [17].
Images of pepsin-treated samples are shown. The presence and absence of reduction by 2-mercaptoethanol is indicated on the top of thepanel. The trimeric 1 (III) chain disappears after reduction and it
shifts to the position just above the monomeric 1 (I) chain. b Sequence analysis of PCR product of exon 24-intron 24 junction of thepatient’s genome. Mutated nucleotide is indicated by an arrow in theupper panel, exon 24-intron 24 junction is expressed in the upper bar,and diVerence of genomic sequences at the beginning of intron 24 in
the control and patient’s genomes are contrasted in the middle and bottom bar. The Wrst six sequences of the intron 24 are shown

A mutation in a single allele of COL3A1 can reduce theproduction of normal molecules to one eighth [8]. The Wnalamount of type III collagen in the ECM decreases becauseof the decay of unstable abnormal mRNA, malsecretion orpoor incorporation of remaining seven eighths of abnormal
molecule into the ECM [5–7, 14]. Even if there is no abnormality in the type III collagen molecule, homozygous aswell as heterozygous defect of the collagen leads vascular
EDS phenotypes [11, 14], indicating that merely a decreaseof the amount of type III collagen leads to vascular EDS.
Our case lacked exon 24 (54 bp) of COL3A1, which corresponds to a deletion of 18 amino acids in the second quarterof type III collagen molecule. It is feasible that the Wnalamount of type III collagen incorporated in the collagenmicroWbrils in our case was also less, or varied. Irregular
contours, variable diameter distribution, and variegation ofelectron densities of collagen microWbrils of our case might
reXect this speculation.
As the cause of vascular EDS, point mutations of type III
collagen comprise two-thirds of reported cases, and exon

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skipping follows, which accounts for the rest about one
third of the cases [10]. As the cause of exon skipping,
Schwartze et al. [13] reported that within 42 analyzed casesof EDS with exon skipping, 37 cases had one base mutation
within intron. At the beginning of one intron in pre-mRNA,
acceptor sequences for splicing exist. Mutations within thisregion disrupt the acceptor site, and therefore, splicing in
the exon- intron junction does not occur. Instead, an acceptor site of the preceding intron is spliced [13]; the mutationin our case Wts this model. The mutation of the Wrst base of
the intron sequence is the most common, and then themutations in the second or the Wfth base of the intron arefrequent [13]. Until now, mutation in the fourth base of theintron has not been reported. Concerning exon 24 skipping,
all the known cases had single base mutation of G to A in
the Wrst position of intron and therefore, the mutation fromT to G in the second position of intron 24 in type III collagen is the Wrst case ever reported.
Concerning the formation of impaired ECM in vascular
EDS, information of the relationship between the locationof mutation within type III collagen molecule and the dermal collagen morphology is still limited [15]. Precise analysis based on the accumulation of biochemical as well asmorphological information is awaited.
Acknowlegment The authors thank Mrs. Satoko Sato for an expert
support in histochemical work.

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