Spondylocheiro dysplastic form of the Ehlers-Danlos syndrome--an autosomal-recessive entity caused by mutations in the zinc transporter gene SLC39A13.

Abstract

We present clinical, radiological, biochemical, and genetic findings on six patients from two consanguineous families that show EDS-like features and radiological findings of a mild skeletal dysplasia. The EDS-like findings comprise hyperelastic, thin, and bruisable skin, hypermobility of the small joints with a tendency to contractures, protuberant eyes with bluish sclerae, hands with finely wrinkled palms, atrophy of the thenar muscles, and tapering fingers. The skeletal dysplasia comprises platyspondyly with moderate short stature, osteopenia, and widened metaphyses. Patients have an increased ratio of total urinary pyridinolines, lysyl pyridinoline/hydroxylysyl pyridinoline (LP/HP), of approximately 1 as opposed to approximately 6 in EDS VI or approximately 0.2 in controls. Lysyl and prolyl residues of collagens were underhydroxylated despite normal lysyl hydroxylase and prolyl 4-hydroxylase activities; underhydroxylation was a generalized process as shown by mass spectrometry of the alpha1(I)- and alpha2(I)-chain-derived peptides of collagen type I and involved at least collagen types I and II. A genome-wide SNP scan and sequence analyses identified in all patients a homozygous c.483_491 del9 SLC39A13 mutation that encodes for a membrane-bound zinc transporter SLC39A13. We hypothesize that an increased Zn(2+) content inside the endoplasmic reticulum competes with Fe(2+), a cofactor that is necessary for hydroxylation of lysyl and prolyl residues, and thus explains the biochemical findings. These data suggest an entity that we have designated "spondylocheiro dysplastic form of EDS (SCD-EDS)" to indicate a generalized skeletal dysplasia involving mainly the spine (spondylo) and striking clinical abnormalities of the hands (cheiro) in addition to the EDS-like features.

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ARTICLE
Spondylocheiro Dysplastic Form of the Ehlers-Danlos
Syndrome—An Autosomal-Recessive Entity Caused
by Mutations in the Zinc Transporter Gene SLC39A13
Cecilia Giunta,1Nursel H. Elc ¸ioglu,2Beate Albrecht,3Georg Eich,4Ce ´line Chambaz,1
Andreas R. Janecke,5Heather Yeowell,6MaryAnn Weis,7David R. Eyre,7
Marius Kraenzlin,8and Beat Steinmann1,*
We presentclinical,radiological,biochemical, andgeneticfindingson sixpatientsfrom twoconsanguineous familiesthat show EDS-like
features and radiological findings of a mild skeletal dysplasia. The EDS-like findings comprise hyperelastic, thin, and bruisable skin,
hypermobility of the small joints with a tendency to contractures, protuberant eyes with bluish sclerae, hands with finely wrinkled
palms, atrophy of the thenar muscles, and tapering fingers. The skeletal dysplasia comprises platyspondyly with moderate short stature,
osteopenia, and widened metaphyses. Patients have an increased ratio of total urinary pyridinolines, lysyl pyridinoline/hydroxylysyl
pyridinoline (LP/HP), of ~1 as opposed to ~6 in EDS VI or ~0.2 in controls. Lysyl and prolyl residues of collagens were underhydroxylated
despite normal lysyl hydroxylase and prolyl 4-hydroxylase activities; underhydroxylation was a generalized process as shown by mass
spectrometry of thea1(I)- and a2(I)-chain-derivedpeptidesof collagentypeI andinvolvedatleast collagentypesI andII.A genome-wide
SNP scan and sequence analyses identified in all patients a homozygous c.483_491 del9 SLC39A13 mutation that encodes for a mem-
brane-bound zinc transporter SLC39A13. We hypothesize that an increased Zn2þcontent inside the endoplasmic reticulum competes
with Fe2þ, a cofactor that is necessary for hydroxylation of lysyl and prolyl residues, and thus explains the biochemical findings. These
data suggest an entity that we have designated ‘‘spondylocheiro dysplastic form of EDS (SCD-EDS)’’ to indicate a generalized skeletal
dysplasia involving mainly the spine (spondylo) and striking clinical abnormalities of the hands (cheiro) in addition to the EDS-like
features.
Introduction
The Ehlers-Danlos syndrome (EDS) is a heterogeneous
group of heritable disorders of connective tissue character-
ized by articular hypermobility, skin hyperelasticity, and
tissue fragility. It affects skin, ligaments, joints, blood ves-
sels, and internal organs. The natural history and mode
of inheritance differ between the six major types.1,2
Among them, the kyphoscoliotic type of the Ehlers-
Danlos syndrome (EDS VI [MIM 225400]) is characterized
at birth by severe muscular hypotonia (requiring often
invasive neuromuscular workup), kyphoscoliosis that is
progressive, severe joint hypermobility and luxations, and
marked skin hyperelasticity. In addition, there is fragility
of the skin with abnormal scarring, osteopenia without
a tendency to fractures, often a Marfanoid habitus and
microcornea, and occasionally rupture of the arteries and
the eye globe. The disorder is caused by a deficiency of the
enzyme collagen lysyl hydroxylase (LH1; EC 1.14.11.4;
procollagen-lysine,2-oxoglutarate 5-dioxygenase), which
normally hydroxylates lysyl residues in -Xaa-Lys-Gly-
sequences of the helical region of the collagen a chains
to -Xaa-Hyl-Gly-. In bone and other tissue collagens, two
Hyl residues, one in each of two amino telopeptides or
two carboxyl telopeptides, together with one Hyl or one
Lys residue ofthe triple helix form the trivalentpyridinium
crosslinks hydroxylysyl pyridinoline (HP) and lysyl pyridi-
noline (LP), respectively. In addition, some helical Hyl resi-
dues of the a chains serve as attachment sites for carbo-
hydrate units (either galactose or glucosyl-galactose). As
a consequence, lysyl hydroxylase (LH1) deficiency results
in underhydroxylation of lysyl residues, fewer glycosylated
hydroxylysyl residues in collagens, and an abnormal cross-
link formation with consequent mechanical instability of
the affected tissues, as seen in these patients. As a result of
underhydroxylation and underglycosylation, the collagen
a chains produced by fibroblasts in culture display a faster
electrophoretic mobility on SDS-PAGE gels. Furthermore,
theenzymedeficiencygivesrisetoanabnormalurinaryex-
cretionpatternofLPandHPcrosslinks. Theratioofurinary
total LP to HP in patients with EDS VI is high (5.99 5 0.99;
range4.30–8.10;n¼17)ascomparedwithnormalcontrols
(0.1950.02;0.12–0.25;n¼179)3andisdiagnosticforEDS
VI.2,4,5The diagnosis of EDS VI may then be confirmed by
measuring the activity of the enzyme in cultured skin
fibroblasts6and/or directly by mutation analysis of PLOD1
(MIM 153454), which encodes the enzyme.7
Here, we report on a novel clinical entity overlapping
with but distinguishable from EDS VI that is caused by
a mutation in the zinc transporter gene SLC39A13 (MIM
1Division of Metabolism and Molecular Pediatrics, University Children’s Hospital, CH-8032 Zurich, Switzerland;2Marmara University Hospital, T-34660
Istanbul, Turkey;3Institut fu ¨r Humangenetik, Universita ¨tsklinikum, Essen, Universita ¨t Duisberg-Essen, D-45122 Essen, The Netherlands;4Pediatric Radi-
ology, Kantonsspital, CH-5000 Aarau, Switzerland;5Division of Clinical Genetics, Innsbruck Medical University, A-6020 Innsbruck, Austria;6Division of
Dermatology, Duke University Medical Center, Durham, NC 27710, USA;7Department of Orthopaedics and Sports Medicine, University of Washington,
Seattle, WA 98195, USA;8Division of Endocrinology and Diabetes, University Hospital, CH-4031 Basel, Switzerland
*Correspondence: beat.steinmann@kispi.uzh.ch, beat.steinmann@access.uzh.ch
DOI 10.1016/j.ajhg.2008.05.001. ª2008 by The American Society of Human Genetics. All rights reserved.
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608735). The clinical features of 6 patients from 2 con-
sanguineous families with Ehlers-Danlos syndrome-like
features, short stature, finger contractures, distinct radio-
logical features, elevated ratios of LP to HP, underhydroxy-
lated collagens in culture despite higher than normal and
normal in vitro activities of lysyl hydroxylase and prolyl
4-hydroxylase, respectively, are described.
Subjects and Methods
The anthropometric, salient clinical and radiological features, and
results of urinary pyridinolines of the six patients and the other
members of the two families are summarized in Tables 1–4 and
Figures 1–3. Routine blood and urine analyses were normal or not
contributory.ThekaryotypesinP1/IandP3/IIwerenormal.Family
members participating in the study gave their informed consent.
Family I
The pedigree of family I is shown in Figure 1A. The proband (P1/I)
was referred to one of us (BA) for evaluation at the age of 10 years
because of short stature, walking pain, and a waddling gait.
P1/I was born spontaneously after an uneventful pregnancy at
term. Birth weight was 2500 g (<3rdcentile), length was 50 cm
(10th–25thcentile), and head circumference was not recorded. At
birth, protruding eyes and reddish eye lids were noted. There
was no muscular hypotonia. Psychomotor development was
normal. Deciduous and permanent teeth erupted late.
Figure 1.
Family I and family II are shown in (A) and (B), respectively. Affected individuals are depicted by black symbols. Genotypes for 39 micro-
satellite-marker loci spanning the linkage interval on chromosome 11p11.2-p13 are shown. The haplotypes are displayed by vertical bars.
The disease-associated haplotype is denoted by a red box around the vertical bars. Recombinations in both families define a candidate
region flanked by markers D11S1779 and D11S4191.
Pedigrees and Haplotype Analysis
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At the age of 10 years, he was short (height 122 cm, 2 cm < 3rd
centile; weight 26 kg, 10thcentile; and head circumference 52 cm,
10th–25thcentile). He had down-slanting palpebral fissures, pro-
truding eyes, slightlybluish sclerae,and a normal corneadiameter.
All the deciduous teeth were present, but one permanent tooth of
the lower jaw was missing. His skin was thin and translucent with
an easily visible venous pattern; the scars over the knees, sheens,
and elbows had a cigarette-paper-like appearance. The hands
looked prematurely aged. The fingers were slender and tapering,
the palms had a thin, finely wrinkled and tight skin, and the
thenar muscles were atrophic so that the thumbs could not
be adducted.
Follow-up examinations at ages 11.5, 13, and 14.5 years (Tables
1 and 2) showed that the standing height remained less than third
centile. He had pain in the knees and occasionally in the hips.
Chest and spine deformities and Marfanoid features were not pres-
ent. With time his skin manifestations became more apparent
with easy bruisability and slow wound healing resulting in thin
cigarette-paper-like scars. For radiological findings, see below,
Table 3, and Figures 3B and 3D.
A brother, P2/I, was born spontaneously at term after an un-
eventful pregnancy. Birth weight was 2490 g (<3rdcentile), length
52 cm (50thcentile), and head circumference 34 cm (10th–25th
centile). No abnormalities were noted after birth. He walked inde-
pendently at the age of 1 year. Clinical examination at the age of
2 years showed body measurements within the normal range
(standing height 82 cm, weight 10 kg, head circumference
50 cm). He had down-slanting palpebral fissures and large, slightly
protruding eyes with blue sclerae. His skin was normal. The hands
showed tapering of the fingers and a fine texture of the skin of the
palms. He had hypodontia with missing deciduous and perma-
nent teeth of the lower jaw.
Follow-up examinations at ages 5, 6.5, and 8.5 years (Tables 1
and 2) showed that the standing height followed 10thcentile.
The skin of the palms was more wrinkled, and there was atrophy
of the thenar. His skin was thin and translucent, with atrophic
scars. He had occasional pain in both knees. For radiological find-
ings, see below, Table 3, and Figures 3A and 3E.
Theconsanguineousfamily originatesfrom North-WesternIraq.
The parents were healthy, second cousins (Figure 1A). Both par-
ents (F1/I and M1/I) and a daughter (S1/I) had normal skin, hands,
joints, sclerae, and eyes and no spine or chest deformities.
Family II
Thepedigreeof familyII is shownin Figure1B. Theproband, P3/II,
was referred to one of us (NE) for evaluation at age 7.5 years
because of short stature and finger contractures.
P3/II was born after an uncomplicated pregnancy at term with
a birth weight of 2750 g (3rdcentile). Perinatal history was un-
eventful, and psychomotor development was normal. Her family
realized at age 1 year that her ‘‘wrists were weak’’ and ‘‘that she
could not hold any objects with her hands and fingers.’’ At
7.5 years, she had a proportionally small stature with a standing
height 109 cm (5 cm < 3rdcentile), weight 15.6 kg (3 kg < 3rdcen-
tile), head 47 cm, arm span 99 cm, lower segment 52.5 cm, and
upper/lower segment ratio of 1.08. There was hypotelorism, and
the eyes were mildly protuberant with blue sclerae and a normal
cornea diameter. There was a high palate, delayed teeth eruption,
minimal umbilical hernia, and hypoplastic mammillae. Her skin
was soft, velvety, and easily bruisable with scars. There was joint
laxity especially of the small joints and elbows. There were atro-
phies of the thenar and hypothenar on both hands, and the right
thumb was dislocated. On the hands there were some mild con-
tracture-like deformities. The palms were wrinkled without real
dermatoglyphics but with many thin lines. Flexion contractures
were present on the first, third, and fifth fingers. There were bilat-
eral pes planus and mild crura vara.
Follow-up at 10.1 years showed that the anthropometric mea-
sures (Table 1) remained similar. Her hands while shaking felt
Figure 2.
Changes
(A) P3/II at age 10.1 years. The thumb is
hypermobile and can easily be adducted
to the forearm; the fingers are tapering
and display flexion contractures.
(B) P3/II at age 10.1 years. The palm is ex-
cessively wrinkled, the thenar is hypotro-
phic, the fingers are tapering, and the end-
phalange of the thumb contracted.
(C) P6/III at age 12.5 years. The palms are
wrinkled, the thenar and hypothenar mus-
cles are hypotrophic, the fingers are taper-
ing, and there is partial syndactyly of
fingers 2 and 3, and 3 and 4.
(D) P6/III at age 12.5 years. The dorsal
aspect of the hands show tapering fingers,
broadened regions over the interphalan-
geal joints with abundant skin, and flexion
contractures, especially of the fifth fingers.
Hands with Characteristic
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like ‘‘bags filled with little bones’’; there was thumb instability on
both sides, and the thumbs could easily touch the radius by flex-
ion (Figure 2A). She was able to dislocate and reduce some phalan-
geal joints of other fingers. The fingers were long, spider-like, not
straight, and tapering; there was also a dynamic swan neck defor-
mity particularly of the fifth fingers and less so of the first and
third fingers. Ligamentuous laxity was observed by pulling on
the distal phalanges, giving a telescoping appearance. Thenar
and hypothenar atrophy was obvious (Figure 2B), and to
strengthen the grip, she used a stress ball. Her lower legs looked
atrophic, and severe bilateral pes planovalgus needed support;
swan-neck deformities were present on small toes. She had a phys-
iotherapeutic program with weight bearing, swimming, and bicy-
cling. The large joints (arms, hips, and knees) had a normal range
of motion; the elbows were broadened. The skin was smooth,
easily bruisable, slow healing, and somewhat hyperelastic, and
the palms were wrinkled with abnormal dermatoglyphics (Fig-
ure 2B); the skin was particularly thin over the dorsal side of hands
and feet. For radiological findings, see Table 3 and Figure 3F.
An affected sister of the proband, P4/II, was born at term with
feet presentation and a weight of 3000 g; her perinatal history
was uneventful and neuromotor development was normal.
At age 4.5 years, the eyes were mildly protuberant with blue
sclerae and astigmatism. There was a bifid uvula, malocclusion
of teeth, and bilaterally broadened elbows. The skin was smooth,
velvety, and translucent particularly on legs and feet with easily
visible veins and only a few atrophic scars and ecchymoses. Both
hypermobile thumbs reached easily the radius. The palms were
wrinkled, the thenar and hypothenar looked atrophic, and her
thumb could easily be subluxated and replaced. There were bilat-
eral severe pes planus and positional genua valga, and when the
subject walked, it looked like ‘‘that there were not enough bones
to hold the feet strong.’’ Arms, hips, and lower extremities had
a full range of motion; she presented with bilateral genua recur-
vata and bilateral medial and lateral instability of knees, instability
of ankles, bilateral flat feet that required physiotherapy and supra-
malleolar orthesis. Her skin was always fragile and easily bruisable
with slow wound healing and few atrophic scars.
An affected cousin, P5/III, was admitted at age 28 years because
of short stature and weakness of his fingers and toes.
P5/III was born at term with a weight of 3000 g and a length of
50 cm. He presented with finger contractures and bilateral club
foot at birth, and these were treated by conservative means until
the age of 7 years. His psychomotor development was normal.
He had always complained about his short stature and weakness
of his finger and toe tips; his fine motor skills of finger (and toe)
tips were never as good as that of his peers but did not restrict
his own routine day care. His fingers (particularly the fifth) were
getting thinner and tapering with time on both sides. He had sev-
eral dislocations of his fingers after moderate trauma. His mother
Figure 3.
Thoracic and Lumbar Spines, the Hands,
the Pelvis, and the Knees
(A) P2/I at age 3.5 years. Note mild to
moderate flattening, osteopenia, and ir-
regular endplates of the vertebral bodies.
(B) P1/I at age 11.5 years. There are simi-
lar findings to (A) with flattening, irregular
endplates and osteopenia of the vertebral
bodies.
(C) P5/III at age 28 years. Again, platy-
spondyly, osteopenia, and irregular end-
plates of the vertebral bodies with depres-
sions resembling notochordal remnants are
seen.
(D) P1/I at age 11.5 years. Note the mildly
short metacarpals and phalanges with wid-
ening of the ends and relative narrowing of
the diaphyses. The epiphyses of the short
tubular bones are flat. Osteopenia is not
present.
(E) P2/I hand at age 3.5 years. Skeletal
features of this younger child are similar
but less pronounced as compared to (A);
alterations of shape and flat epiphyses of
the short tubular bones are noted.
(F) Pelvis of P3/II at age 10 years. Small
ilia, mild flattening of the proximal epiph-
yses, and short and wide femoral necks are
seen.
(G) Knee of P5/III at age 28 years. The
knees show normal-sized epiphyses and
a normal metaphyseal contour on the
long tubular bones, but the intercondylar
fossa of the distal femur is shallow in this
adult patient.
Radiographs of the Low
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described his skin as fragile, bruisable with atrophic scars after
mild trauma during childhood but this improved with time.
At age 28 years, he had a short stature with a short trunk (Tables
1 and 2). The palpebral fissures were slightly down-slanting, the
eyes were mildly protuberant with white sclerae—unlike in child-
hood when they were bluish—and there was moderate hyperopia
(þ3.25 Dpt). Theskin was velvety, smooth,rather thinwith mildly
translucentappearance,andhyperelastic, withexcessiveskinfolds
over the broadened phalangeal joints. Both hands had muscular
weakness with atrophy of the thenar, the hypothenar, and the
interosseal muscles that made dorsiflexion difficult; there were
no sensory deficits. The fingers were tapering with flexion contrac-
tures of the right third to fifth fingers and left fourth and fifth fin-
gers that restricted the small-joint mobility in these fingers, and he
could not bend his thumb. There was no hypermobility of elbows
or genua recurvata, and flexion of the trunk was limited. Varicose
veins over the dorsum of the feet were apparent. For radiological
findings, see below, Table 3, and Figures 3C and 3G.
P6/III is the affected sister of P5/III and complained about the
abnormalities of her skin and hands. She was born at term and
had a normal perinatal history and a normal psychomotor devel-
opment. Her skin had been always hyperelastic, thin, very bruis-
able, and fragile with slow wound healing. According to her
mother, her fingers wereless contractedat birthcomparedtothose
of her affected brother (P5/III), and although they improved with
exercise, she lost some skills in her right hand during the years,
such as buttoning and unbuttoning her clothes and carrying a
cup.
At age 12.5 years, she was moderately short (Table 1). The teeth
were malocclusive with irregular borders. The skin was velvety,
smooth, thin, and translucent, with cigarette-paper-like atrophic
scars; and over the hands and feet, the skin looked rather old
with enlarged, easily visible vessels and was atrophic over the fin-
ger and toe tips. The skin was loose over hands and finger joints,
and the palms were wrinkled (Figures 2C and 2D). The metaphy-
seal regions of elbow, wrist, and proximal interphalangeal joints
were broadened. The fingers were tapering, with flexion contrac-
tures particularly on her left metacarpophalangeal and interpha-
langeal joints and swan-neck deformities, and the fascia and/or
tendon were rather shortened between the thumb and the second,
third, and fourth fingers, and there was some atrophy of the inter-
osseous, thenar, and hypothenar muscles. There were bilateral flat
feet. The eyes were moderately myopic, and the sclerae were
bluish. For radiological findings, see below and Table 3.
The parents were consanguineous (Figure 1B), originating from
the Southeastern part of Turkey, and had normal eyes and sclerae,
no chest or spine deformities, and normal hands, joints, and skin.
There was moderate familial microcephaly of M2/II, S2/II, S3/II,
and P4/II (Table 1).
Radiological Findings
The radiological findings are summarized in Table 3. X-ray exam-
inations of the hands, knees, and spine were performed in all pa-
tients and some healthy first-degree relatives (F2/II; S3/II; and M3/
III). The pelvis was radiographed in all patients but P2/I, the chest
was radiographed in P3/II and P4/II, and the skull was radio-
graphed in P3/II and P6/III (Table 3).
All patients had mild to moderate flattening of vertebral bodies
(platyspondyly). In addition, mild to moderate osteopenia was
present in all but P5/III. Irregular endplates of the vertebral bodies
were shown in P1/I and P5/III (Figures 3B and 3C, respectively);
in P2/I, platyspondyly was already present at age 3.5 years
(Figure 3A). The only adult patient of the series had endplate
depressions resembling notochordal remnants (Figure 3C).
The hands were abnormal in all patients. Mildto moderate alter-
ation of the shape of metacarpals and phalanges consisting of wid-
eningoftheendsandrelativenarrowingofthediaphyseswaspres-
ent in all patients to a mild or moderate degree. The epiphyses of
the short tubular bones, however, were flat in all patients (Figures
3D and 3E) except in P3/II. This latter feature distinguishes the
present patients from progressive pseudorheumatoid arthropathy
Table 1.Anthropometric Synopsis of the Members of the Two Families at Last Investigation
Individuals (Age in
Years); Sex Height, cm (Centile)a
Sitting Height,
cm (Centile)c
Arm Span, cm Weight, kg (Centile)a
Head Circumference,
cm (Centile)d
P1/I (14.5); m
P2/I (8.5); m
F1/I (38); m
M1/I (31); f
S1/I (12); f
P3/II (10.1); f
P4/II (4.5); f
F2/II (43); m
M2/II (36); f
S2/II (13); m
S3/II (5.5); f
P5/III (28); m
P6/III (12.5); f
F3/III (56); m
M3/III (48); f
S4/III (20.5); f
S5 (29); f
146 (8 cm < P3)
123 (P10)
172 (P25)
158 (P25)
149 (P25)
121 (5 cm < P3)
95 (P3)b
183 (P90)
155 (P25)
151 (P25)
113 (P75)b
152 (14 cm < P3)
144 (P3)
na (‘‘normal’’)
157 (P25)
162 (P50)
na (‘‘normal’’)
76 (P3)
70 (P25)
101 (P97)
92 (P90-97)
80 (P75)
64 (4 cm < P3)
54 (1 cm < P3)
92 (P25-50)
82 (P3)
77 (P10-25)
60 (P25)
79 (8 cm < P3)
73 (P3)
na (‘‘normal’’)
82 (P3)
81 (P3)
na
140
119
na
na
na
108
86
175
150
143
105
156
135
na (‘‘normal’’)
155
153
na
51 (P10-25)
25 (P25)
93 (>P97)
73 (P97)
44 (P25-50)
20 (3 kg < P3)
15 (P25)b
87 (P90-97)
51 (P10)
37 (P10)
17 (P10)b
63 (P10)
40 (P10)
na (‘‘normal’’)
na (‘‘normal’’)
55 (P50)
na (‘‘normal’’)
56 (P75)
53 (P50)
56 (P50)
56 (P75)
54 (P75)
49 (P2)
48 (P2)b
57 (P75)
52 (P2)
51 (P2)
47 (P2)b
57 (P50)
55 (P75)
na (‘‘normal’’)
58 (P98)
55 (P50)
na (‘‘normal’’)
m, male; f, female; and na, not available; characteristics of the patients are given in bold. ‘‘normal’’ is as judged by the family.
aTurkish centiles.37
bTurkish centiles below age 6 years.38
cSwiss centiles.39
dHead.40
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of childhood (MIM 208230), which otherwise bears similar radio-
graphic features to our patients.
The ileum was small in all patients (P2/I was not examined). An
abnormality of the proximal femurs consisting of mildly flat prox-
imal femoral epiphyses and short and wide femoral necks was
present in most patients (Figure 3F). The knees, however, showed
normal-sized epiphyses and a normal metaphyseal contour of the
long tubular bones. The intercondylarfossa of the distal femur was
shallow in P1/I and P5/III (Figure 3G).
The chest was examined in P3/II and P4/II, both with normal
findings. Theskull (Towne’s projection) of P6/III showed innumer-
able, small Wormian bones of the lambdoid suture (not shown),
whereas the skull of P3/II was normal.
X-rays of the examined hands, knees, and spine were normal in
the first-degree relatives (F2/II, S3/II, and M3/III; not shown) who
were clinically not affected and who had normal ratios of urinary
crosslinks (Table 4).
Analysis of Pyridinium Crosslinks by HPLC
Spot urine samples and isolated peptide fractions from urine
collections were acid hydrolyzed and analyzed by reverse phase
Table 2. Salient Clinical Findings in the Six Patients at Last Investigation
P1/I P2/I P3/IIP4/IIP5/IIIP6/III
Skin
hyperelastic
velvety, smooth
thin, translucent
bruisable
scars atrophic, cigarette paper-like
scars hypertrophic
(þ)
–
þ
þ
þ
þ
(þ)
–
þ
–
þ
nr
(þ)
þ
þ
þ
þ
–
(þ)
þ
(þ)
þ
þ
–
þ
þ
þ
þ (infancy)
–
þ (knee, ankle)
þ
þ
þ
þ
þ
–
Hands
palms wrinkled
fingers tapering
thenar hypotrophy
þ
þ
þ
þ
þ
þ
+
+
+
þ
(þ)
þ
(þ)
þ
þ
+
+
+
Feet
flat feet
þþþþþ
+
Joints
hypermobility
hypermobility of major joints
hypermobility of minor joints (and
subsequent contractures)
history of subluxations
broadened metaphyseal regions (elbow,
wrist, interphalanges)
wrist sign
thumb sign
nr
–
(þ)
nr
–
–
þ
–
+ (contractures)
þ
þ
þ
(þ)
–
þ (contractures) + (contractures)
(þ)
–
–
–
–
–
þ
þ
þ
þ
þ
þ
–
+
–
–
–
–
–
–
–
–
–
–
–
–
Skeletal
Marfanoid habitus
chest deformity
spine deformity
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
Face and Eyes
down-slanting palpebral fissures
sclerae bluish
protuberant
myopia
cornea diameter (mm)
þ
þ
þ
þ
nr
þ
þ
þ
–
11
hypothelorism
þ
(þ)
–
11
–
þ
(þ)
astigmatism
11
(þ)
– (þ as a child)
(þ)
hyperopia
nr
–
(þ)
(þ)
þ
nr
Oral
cleft palate/ bifid uvula
teeth (hypodontia), small
retrognathia
–
–
–
–
þ
–
high palate
small, malocclusion small, malocclusion caries tendency
––
bifid uvula––
irregular ends of teeth
––
Cardiac Signs
heart murmur (MVP, AI)––––––
The following symbols and abbreviations are used: nr, not recorded; þ, present; (þ), mildly present; and –, absent; findings marked by symbols printed in
bold are depicted in Figure 2.
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1295

Page 7

HPLC in heptafluorobutyric acid as previously reported.5,8The
average intra-assay and inter-assay coefficients of variation for LP
and HP were <8% and <6%, respectively.3
Collagen Peptides Isolation from Urine
Crosslinked telopeptides were isolated from urine with immuno-
affinity columns with mAb 1H11, specific to crosslinked N-
telopeptides of type I collagen (NTx) resulting from bone resorp-
tion, or with mAb 2B4, which binds to crosslinked C-telopeptides
oftypeIIcollagen(CTx-II)resultingfromgrowth-cartilagedegrada-
tion.AntibodywascoupledtoCNBr-activatedSepharose4B(Amer-
sham Biosciences). For NTx isolation, the 1H11-column was equil-
ibrated in 50 mM HEPES, pH 7.2, loaded with urine diluted with
equilibrating buffer, and washed with 0.125 M NaCl, 0.025 mM
HEPES, pH 7.2, and bound peptides were eluted with 1 M NaCl
and 0.025 mM CAPS (N-cyclohexyl-3-aminopropanesulfonic
acid), pH 10.4. For CTx-II isolation, the 2B4-column was equili-
brated and loaded similarly, and the bound peptides were eluted
with 0.1 M NaCl and 0.1 M glycine, pH 2.5.8
Dermal Fibroblast Cultures
Cells strains were established from skin biopsies from patients P1/I
and P2/I and their father (F1/I) and mother (M1/I), as well as their
unaffected sister (S1/I), and from patient P3/II and her mother
(M3/II), by routine procedures. Cells were maintained under stan-
dard conditions, and radiolabeled collagen samples were prepared
by digestion with pepsin, precipitated with ethanol, separated on
a 5% SDS-polyacrylamide gel, and visualized by fluorography.9
Amino Acid Composition of Dermis
Skin biopsies were hydrolyzed with 6 N HCl in nitrogen at 110?C
for 24 hr as described.10
Stability of Collagens
Thermal stability of triple helical collagens was assessed by the
resistance to proteases at increasing temperatures.11
Collagen Purification and Mass Spectrometry
Cultures of exponentially growing fibroblasts from patients
and controls were supplemented with 10% fetal-calf serum and
50 mg/ml ascorbic acid; the culture medium was serially collected
3timesa week andreplacedby freshlyprepared supplemented me-
dia;andthecollectedmediumwasstoredat?20?Candpooledover
a 2 week period. The combined medium was then acidified to 3%
acetic acid and digested with pepsin (50 mg/ml) at 4?C for 24 hr.
Collagens were precipitated with 1.0 M NaCl and run on SDS-
PAGE either directly or after digesting a portion with CNBr in
70% formic acid. The a chains or individual CNBr peptides were
cut from the gel and subjected to in-gel trypsin digestion. Electro-
spray MS was performed on the tryptic peptides with an LCQ
Deca XP ion-trap mass spectrometer equipped with in-line liquid
chromatography (LC) (ThermoFinnigan) that used a C8 capillary
column (300 mm 3 150 mm; Grace Vydac 208MS5.315) eluted at
4.5 ml per min. The LC mobile gradient was made from buffer A
(0.1% formic acid plus 2% buffer B in MilliQ water) and buffer B
(0.1% formic acid in 3:1 acetonitrile:n-propanol v/v). The sample
stream was introduced into the mass spectrometer with an electro-
spray ionization source (ESI) under atmospheric pressure. The
spray voltage was 3 kV and the inlet temperature 160?C.12
Lysyl Hydroxylase and Prolyl 4-Hydroxylase
Enzyme activities were measured in extracts of fibroblasts from
two patients and a control (GM05659, Coriell Institute for Medical
Research) as described.6In brief, cells were grown to confluency in
Dulbecco’s modified Eagle’s medium (Invitrogen-GIBCO) supple-
mented with 10% heat-inactivated fetal-calf serum (Sigma). Lysyl
hydroxylase activity was measured with an L-[4,5-3H] lysine-
labeled underhydroxylated procollagen substrate, and prolyl
hydroxylase was determined with a similar underhydroxylated
procollagen substrate labeled with L-[4-3H] proline. The enzyme
activities were quantitated as released
four independent assays.
3H2O in a minimum of
DNA and RNA Extraction
Genomic DNA was isolated from peripheral blood leukocytes or
cultured fibroblasts with standard techniques. Total RNA was iso-
lated from cultured dermal fibroblasts (23 T75 flasks) with an
RNeasyMini Kit(QIAGEN),in accordance withthemanufacturer’s
protocols. Prior to RNA extraction, one of the two T75 fibroblast
flasks was incubated for 6 hr with 1000 mg/ml cycloheximide in or-
der for prevention of nonsense-mediated mRNA decay. Transcript
Table 3. Salient Radiological Findings in the Six Patients
CodeP1/I P2/I P3/IIP4/II P5/III P6/III
Hand
CAa
SAa
Broad ends of
short tubular bones
Flat epiphyses
Short metacarpals
and phalanges
Osteopenia
11.5 3.5
11.5 3.0
+
9.9
8.9
þ
5.3
5.0
þ
28
mature 14
þ
12.5
+
þ
+
+
+
–
–
–
þ
–
na
þ
þ
–
–––
þ
––
Knee
Shallow
intercondylar
fossa
þ
–––+–
Spine
Platyspondyly
Osteopenia
Irregular
endplates
+
+
+
+
+
+
þ
þ
–
þ
þ
–
+
–
+
þ
þ
–
Pelvis
Small, broad
ileum
Flat capital
femoral
epiphyses
Broad and short
Femoral necks
þ
ne+
þþþ
þ
ne+
þ
–
þ
þ
ne+––
þ
Additional Regions
Chest
Skull
normal normal
normalWormian
bones
DXA total t score
?5.8
þ1.1
?3.8
aCA, chronological age; SA, skeletal age;41þ, present; (þ) mildly present;
–,absent;ne,notexamined;na,notapplicable;findingsmarkedbysymbols
printed in bold are depicted in Figure 3.
1296
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Page 8

quantification was performed by real-time quantitative PCR with
the ABI Prism 7700 sequence detection system (Applied Biosys-
tems) as previously described.7
Genome-wide Microsatellite-Marker Analysis
A genome-wide scan was performed on all individuals of family II
with 218 highly polymorphic microsatellite markers distributed
with an average spacing of 20 cM (ABI Prism Linkage Mapping
Set 2.5, Applied Biosystems). Microsatellite analysis was carried
out by standard semiautomated methods with an ABI 3100 Ge-
netic Analyzer (Applied Biosystems). Parametric multipoint link-
age analysis was performed by GENEHUNTER v2.1r5. Haplotypes
were reconstructed with GENEHUNTER v2.1r5.
Genome-wide SNP Scan and Linkage Analysis
A genome-wide scan with SNP-arrays (Affymetrix GeneChip Hu-
man Mapping 10K array Xba 142 2.0) was performed on all indi-
viduals of family II in accordance with the manufacturer’s instruc-
tions. The array interrogates 10,204 SNPs with a mean intermarker
distance of 258 kb, equivalent to 0.36 cM. Genotypes were called
by the GeneChip DNA Analysis Software (GDAS v2.0, Affymetrix).
We assumed a fully penetrant autosomal-recessive trait locus with
a disease-allele frequency of 0.0001 and a marker-allele frequency
estimated from our sample. Multipoint parametric linkage analy-
sis and haplotyping were carried out with GENEHUNTER v2.1r5
and ALLEGRO v. 2.0,13respectively, through stepwise use of a slid-
ing window with sets of 100 and 50 SNPs. Data handling was
performed by EasyLinkage Plus v.5.05.14Themicrosatellite-marker
content of the linked ~26.22 cM genomic region (deCode genetic
map) between rs1350960 and rs870333 on chromosome 11p11.2-
q13 (~36 Mb) was examined with the NCBI Map Viewer (build
36.1).Genotypingof39highlypolymorphicmicrosatellitemarkers
spaced at intervals of ~0.2–0.5 cM was carried out by PCR and frag-
ment analysis of amplified products with ABI Prism 310 and ABI
3100GeneticAnalyzers(AppliedBiosystems).Multipointparamet-
ric linkage analysis and haplotyping were carried out with GENE-
HUNTER v2.1r5 and ALLEGRO v.2.0,13under EasyLinkage Plus
v.5.05. Haplotypes were displayed with HaploPainter v.029.5.15
Sequencing and Mutation Analysis
of Candidate Genes
Positional candidate genes including SLC39A13 (NM_152264)
withinthe~6.72cMhomozygouscandidateregion(58.73–64.96cM
according to the deCode genetic map, ~16.9 Mb in size between
Table 4.
Individuals
Ratios of Total Urinary Pyridinolines (LP/HP) in Affected and Unaffected Family Members, Controls, and EDS VI
Subjects Age (y)Single Values (LP/HP)N MeanRange1 SD Reference
P1/I 11.5–14.5 0.76/0.67 (26.4.01); 0.73 (7.2.02); 0.91;
0.74 (10.3.04)
0.64/0.60 (26.4.01); 0.74 (7.2.02); 0.83;
0.77 (10.3.04); 0.82 (19.4.06)
0.89 (13.11.03); 1.07 (13.2.04); 1.04
(26.10.04); 0.93 (19.4.06); 0.92 (27.6.06)
0.79 (13.11.03); 0.98 (13.2.04); 0.91
(26.10.04); 0.99 (19.4.06); 0.90 (27.6.06)
0.79 (26.10.04); 0.73 (19.4.06)
1.25 (26.10.04); 1.13 (19.4.06)
0.26 (9.7.01); 0.22 (17.10.02)
0.34 (9.7.01); 0.30 (17.10.02); 0.35
(16.4.06)
0.31 (19.4.06)
0.324 (27.6.06)
n.a.
0.39 (19.4.06)
0.27 (17.10.02)
0.27 (19.4.06)
0.20 (27.6.06)
0.19/0.19 (19.4.06)
4 0.76 0.67–0.91
P2/I 3.5–8.55 0.74 0.60–0.83
P3/II 7.5–10.15 0.97 0.89–1.07
P4/II 1.9–4.55 0.91 0.79–0.99
P5/III
P6/III
F1/I
M1/I
26.8–28.3
11.0–12.5
38
31
2
2
2
3
0.76
1.19
0.24
0.33 0.30–0.35
F2/II
M2/II
F3/III
M3/III
S1/I
S2/II
S3/II
S4/III
EDS VI homozygotes
EDS VI obligate
heterozygotes
Control children
Control children
Control children
Control children
Control children
Control children
Control children
Control men
Control women
43
36
36
48
10
13
5.5
3.5
10 (1–28)
38 (26–54)
1
1
—
1
1
1
1
1
17
7
0.31
0.32
—
0.39
0.28
0.27
0.20
0.19
5.97
0.33
4.30–8.10
0.30–0.37
0.99
0.035
5
(B.S., unpublished data)
0–14
0–1
>1–3
>3–5
>5–8
>8–11
>11–14
56 (28–69)
41 (26–52)
83
15
14
15
12
14
13
44
52
0.19
0.17
0.19
0.19
0.21
0.18
0.19
0.20
0.20
0.12–0.25
0.12–0.21
0.15–0.25
0.13–0.25
0.15–0.25
0.12–0.24
0.16–0.23
0.15–0.23
0.12–0.22
0.030
0.03
0.04
0.04
0.03
0.03
0.02
0.02
0.02
3
3
3
Note that biological replicates are indicated by the dates of urinary collection in parentheses; analytical duplicates are indicated by slashes; values in bold
were obtained by an independent method,8all others are obtained as described.5The rows in italics refer to the data of patients and parents with EDS VI
and highlight the difference to those of the patients described in the text. ‘‘n.a.’’ is short for not available.
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Page 9

D11S1779 and D11S4191) were prioritized for sequencing analy-
ses on the basis of the known or putative functions of the encoded
proteins, as well as on the basis of their expression profiling. Mu-
tation screening of the candidate genes was performed by fluores-
cent bidirectional sequencing of genomic as well as cDNA-ampli-
fied PCR products on an ABI 3100 automated sequence
detection system (Applied Biosystems). PCR conditions and
primer sequences are available from the authors upon request.
Conservation Analysis
Multiple sequence alignment of the SLC39A13 (ZIP13) protein
was performed with ClustalW and BOXSHADE programs, with
sequences from human (Q96H72), orangutan (Q5R6I6), mouse
(Q8BZH0), rat (Q2M1K6), bovine (A5D7H1), chicken (Q5ZI20),
and zebrafish (Q8AW42).
Results
Because the clinical findings in patient 1 (P1/I) did not es-
tablish a diagnosis, a skin biopsy was taken for electron-
microscopy study and fibroblast culture. Elastin was nor-
mal in amount and structure, and the collagen fibrils
were normal in shape and diameter; specifically, no large
fibrils with an irregular contour or round fibrils with an ab-
normally wide range of diameters were seen as in the clas-
sical form of EDS (EDS I/II [MIMs 130000 and 130010]).16
The collagens produced by his cultured skin fibroblasts
showed on SDS-PAGE a slightly increased electrophoretic
mobility of the a(I)-chains of collagen type I, seen also
for P2/I, P3/II, and P5/III studied later. However, the faster
migration of the collagen chains was not consistently ob-
served (data not shown; see also the legend to Figure 4)
and was less apparent than on SDS-PAGE of EDS VI colla-
gen. In EDS VI, in addition to the a1(I)- and a2(I)-chains
of collagen type I, the a1(V)-, the a2(V)-, and the a3(V)-
chains of collagen type V and the crosslinked chains b11
and b12 of collagen type I display a faster electrophoretic
mobility.2Thus, we concluded that lysyl residues in colla-
gens were underhydroxylated in these patients, albeit less
than in individuals with EDS VI.
Previously, we have shown that underhydroxylation of
collagen is reflected in the urine by the excretion of an ab-
normal LP/HP pattern and that a markedly increased LP/
HP ratio (5.97 5 0.99; range 4.30–8.10; n ¼ 17) was diag-
nostic for EDS VI.2,4,5,8Therefore, we measured the ratio
of total urinary pyridinolines in the present patients and
their family members. The ratio LP/HP was elevated in
all six patients (mean 0.89 5 0.18; range 0.67–1.25), re-
mained so over an observation period of up to 5 years,
and was independent of age (Table 4). The ratio was clearly
lower in these patients than in individuals with EDS VI but
markedly higher than in controls of all ages (mean 0.19 5
0.02; range 0.12–0.25; n ¼ 179),3and there was no overlap
of the ratios between the individuals with EDS VI, the pres-
ent affected subjects, and the controls (Table 4). It is of
interest that in seven obligate heterozygotes for EDS VI,
the LP/HP ratio was indistinguishable (0.33 5 0.03;
0.30–0.37) from those of five obligate heterozygotes of
the present subjects (0.32 5 0.05; 0.24–0.39) despite the
fact that homozygotes for EDS VI had an LP/HP ratio far
higher (~6) than that of the affected subjects with a LP/
HP ratio of ~1.
The total amount of pyridinolines, i.e., lysyl pyridino-
line plus hydroxylysyl pyridinoline expressed per creati-
nine was normal as compared to age-matched controls.
This means that the formation of LP was increased at the
expense of synthesized HP, as observed also in patients
with EDS VI.4
Analysis of the immunopurified crosslinked N-telopep-
tide of collagen type I and of the crosslinked C-telopeptide
of collagen type II from the urines of P1/I and P2/I revealed
that both collagen types were underhydroxylated at the
triple helical crosslinking sites. The underhydroxylation
of collagen lysyl residues ranged between that of the con-
trols and that of the subjects with EDS VI (Table 5).
As a next step, we measured hydroxylation of lysyl and
prolyl residues in collagens produced in vivo and in cul-
ture. Acid hydrolysis of a skin biopsy of P5/III showed
that lysyl residues were underhydroxylated, i.e., the Hyl-
to-Lys ratio was 0.09 (5 controls: 0.14 5 0.02) and the
Hyl-to-Hyp ratio was 0.05 (controls: 0.10 5 0.02); for com-
parison, the Hyl-to-Lys and Hyl-to-Hyp ratios in five
patients with EDS VI were 0.008 5 0.006 and 0.004 5
0.002, respectively, and thus were more reduced than in
P5/III.
Because hydroxylation of collagen prolyl residues con-
fers stability to the triple helical molecules,17we explored
the possibility of lowered stability to proteases at increas-
ing temperatures.11Thermal stability of collagens pro-
duced by fibroblasts from P1/I and P3/II was normal for
collagentypes I, III, and V.Obviously,the general underhy-
droxylation of these collagens is moderate (see below) and
not sufficient to lower their melting temperatures.
Mass-spectral analysis of a tryptic peptide from the a1(I)-
chain of collagen type I (Figure 4A), composed of amino
acid residues 658–684, confirmed that lysyl residues in col-
lagen samples produced by fibroblasts of P1/I and P3/II
were consistently underhydroxylated (Figure 4C). Specifi-
cally, hydroxylation of lysyl residue 684 in the peptide
was 12% and 27% in P1/I and P3/II as opposed to 41%
and 36% in the two controls. Similarly, in a tryptic peptide
of a CNBr peptide from the a2(I)-chain of collagen type I
(Figure 4B), composed of amino acid residues 193–219
(GEVGLP*GLSGPVGPP*GNP*GANGLTGAK*),
ation of lysyl residue 219 was 46%, 48%, 74%, and 83%
in P1/I, P3/II, and two controls, respectively (Figure 4D).
Inspection of the mass-spectral results from the same in-
gel digests in the controls and the subjects showed, in
addition, partial but significant underhydroxylation at
the single 3-Hyp site in the a1(I)-chain and underhydrox-
ylation at a nearby 4-Hyp residue (Figure 5). Specifically, 3-
hydroxylation of prolyl residue 986 was 88%, 93%, 100%,
and 100%, and 4-hydroxylation of prolyl residue 981
was 67%, 78%, 93%, and 92% in P1/I, P3/II, and the two
hydroxyl-
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Page 10

controls, respectively. These results indicate that lysyl
underhydroxylation and prolyl 4-underhydroxylation oc-
cur along the whole molecule and are not confined to spe-
cific residues, and the single site of prolyl 3-hydroxylation
is also underoccupied compared with control collagen.
The in vitro activities of the enzymes lysyl hydroxylase
and prolyl 4-hydroxylase were measured in fibroblasts of
P1/I and P3/II in four independent assays. Lysyl hydroxy-
lase activities were normal to higher-than-normal range,
whereas prolyl 4-hydroxylase activities were within the
normal range (Table 6). Because the in vitro assay was
done under optimal substrate and cosubstrate concentra-
tions, a Kmvariant for the binding of substrates and cosub-
strates to the putative mutant enzyme was considered, and
such a variant might reduce the enzyme activity in vivo
under suboptimal conditions. Therefore, we explored the
possibility of PLOD1 as a candidate gene. However, analy-
ses of linkage, amounts of transcripts, and sequencing ex-
cluded it as a candidate; similarly, PLOD3 (MIM 603066)
and PLOD2 (MIM 601865) were excluded.
Figure 4.
(A) SDS-PAGE resolution of collagen a1(I)- and a2(I)-chains of collagen type I from pepsin-treated culture medium. The a1(I) chains
were excised from the gel and subjected to in-gel digestion with trypsin. Lanes 1–4 refer to samples obtained by fibroblasts from
P1/I, P3/II, and controls 1 and 2. Please note that the electrophoretic mobility of the a(I) chains from the patients’ samples is barely
different from that of the controls.
(B) SDS-PAGE resolution of CNBr-peptides prepared from collagen type I from culture medium. Peptide a2(I)CB4 was excised from the gel
and subjected to in-gel digestion by trypsin. Lanes 1, 2, and 3 were from patients P1/I, P3/II, and a control, respectively.
(C) Mass-spectral scans across the regions of the LCMS profiles from the a1(I) chains of one patient and a control that capture the post-
translational variants of the tryptic peptide. The lowest panel shows an MS/MS spectrum from the 11492þprecursor ion, which establishes
the identity of the peptide sequence including placement of hydroxyl groups on the three prolyl residues and the single lysyl residue.
Similar MS/MS spectra showed that the 11412þion lacked the hydroxyl group (16 mass units) of the lysyl residue 684 in the two patients.
(D) Mass-spectral scans across regions of the LCMS profiles from peptide a2(I)CB4 of one patient and a control that capture posttrans-
lational variants of the tryptic peptide shown. The lowest panel shows the MS/MS spectrum from the 12182þprecursor ion, which estab-
lishes the identity of the peptide including the placement of hydroxyl groups on three prolines and the single lysine. Similar MS/MS spec-
tra showed that the 12102þion lacked the hydroxyl (16 mass units) on Lys219 in the two patients.
Decreased Lysyl Hydroxylation of the Collagen a1(I) and a2(I) Chains Revealed by TMS
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The discrepancy of reduced hydroxylation of collagens
produced in vivo or formed in intact cells and the normal
lysyl hydroxylase and prolyl hydroxylase activities mea-
sured in cell homogenates in vitro prompted us to explore
the hypothesis of in vivo reduced enzyme activities due to
impaired cellular uptake of vitamin C, a cofactor of both
enzymes. We hypothesized that SLC23A2 (MIM 603791)
encoding the sodium-dependent vitamin C transporter 2
(SVCT2)18,19would be defective in these patients. How-
ever, linkage and mutation analysis in the two families
ruled out SVCT2 as a candidate. We likewise excluded
genes involved in prolyl hydroxylation of collagen,
P4HA1 (MIM 176710), P4HA2 (MIM 600608), P4HB
(MIM 176790), P4HA3 (MIM 608987), and LEPRE1 (MIM
610339) by linkage analysis with microsatellite markers
within and/or flanking the genes.
A 20 cM low-density genome scan was then performed
on family II. A LOD score of 1.6 was obtained for a 20ptel
chromosomal region. However, the only candidate gene
SOX12 (MIM 601947) within the region was excluded by
mutation analysis in F1/I, M2/II, and P3/II.
A genome-wide SNP scan with the Affymetrix Gene-
Chip10 K SNPs array identified a region on chromosome
11p11.2-q13 flanked by the recombinant SNPs rs1350960
and rs870333 in family II defining a critical interval of
~36 Mb. Fine-mapping with 39 highly polymorphic micro-
satellite markers within this region, and including all
membersoffamilyI,reducedthecriticalregiontoa16.9Mb
interval between markers D11S1779 and D11S4191,
with a combined maximal LOD score of 5.3 (Figure 6).
Within this region, several genes were regarded as relevant
functional candidates because of their expression in con-
nective tissues; however, sequencing at the genomic as
well as at the cDNA level in M2/II, P3/II, M3/III, and P5/III
detectedonlyknownpolymorphicvariantsin26candidate
genes except SLC39A13 (NM_152264.2), in which we de-
tected a 9 bp in-frame deletion in exon 4, c.483_491 del9
(p.F162_164 del). All six patients were homozygous, and
all parents as well as siblings S1/I, S2/II, and S4/III were
Table 5.
Lysyl Residues in Bone Collagen Type I and Cartilage Collagen
Type II
Site-Specific Underhydroxylation of Crosslinking
LP/HP Molar Ratio
Total UrineNTx-I Peptides CTx-II Peptides
Control
P1/I
P2/I
EDS VI*
0.18
0.91
0.83
5.26
0.40
3.00
3.57
20.0
<0.04
0.30
0.13
0.50
The NTx-I LP/HP ratio is a surrogate index of the aggregate ratio of Lys/Hyl
at K930 in a1(I) plus K933 in a2(I) chains of bone collagen type I, and the
CTx-II LP/HP ratio is a surrogate index of the Lys/Hyl ratio at K87 in a1(II)
chains of growth-plate cartilages. Underhydroxylation was assessed
by HPLC analysis of the LP/HP pyridinoline ratio in urinary crosslinked
peptides.
* The patient with EDS VI was studied previously.8
Figure
4-Hydroxylation Revealed by TMS
Mass spectra are shown for the tryptic peptide containing Pro986,
the site of 3-Hyp formation in the a1(I) chain from one patient
and one control fibroblast cultures run on SDS-PAGE (shown in
Figure 4A).
(A) Mass-spectral scans encompassing all variants of the tryptic
peptide detected in the LC profiles (~28 min elution time). Four
posttranslational variants are revealed in the patient’s collagen.
(B) MS/MS spectra of the four peptide variants differing by 16 mass
units identify the placement of hydroxyl groups. Underhydroxyla-
tion at the 3-Hyp position and the two 4-Hyp positions can be
quantified from the identified structures and their relative abun-
dance as shown in Figure 4.
5. DecreasedProlyl 3-Hydroxylationand Prolyl
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heterozygous for this mutation, whereas S3/II was homo-
zygous for the wild-type allele (Figure 7A). The mutation
creates a Tail restriction-endonuclease recognition site
(ACGTD) that was used for assessment of its frequency in
182 control individuals (51 with the same ethnic back-
ground); restrictiondigestionof364chromosomesshowed
no mutation.
Discussion
The work-up of the initial proband with Ehlers-Danlos
syndrome-like features and short stature revealed a pattern
of urinary pyridinolines that was abnormal but less pro-
nounced than in subjects with the kyphoscoliotic form
of the Ehlers-Danlos syndrome (EDS VI). The subsequent
study of five additional cases showed that they all had
moderate underhydroxylation of collagen lysyl and prolyl
residues despite normal lysyl hydroxylase and prolyl 4-
hydroxylase activities in cell extracts and a clinical pheno-
type clearly distinct from EDS VI. These findings and
a search of the literature indicated, to the best of our
knowledge, that they all had a hitherto unrecognized clin-
ical entity, which we named spondylocheiro dysplastic
form of EDS (SCD-EDS) (see below).
SCD-EDS is inherited as an autosomal-recessive trait
caused by mutations in SLC39A13 on chromosome
11p11.2. Clinically, it is characterized by the following fea-
tures: (1) postnatal growth retardation, growth parallel be-
low or along the lower centiles, moderate short stature; (2)
hyperelastic, velvety, thin skin with an easily visible ve-
nous pattern and bruisability that leads to atrophic scars;
(3) slender, tapering fingers, wrinkled palms, and consider-
able thenar (and hypothenar) atrophy; (4) hypermobility
ofthesmalljoints,especiallythefingers,whichresultslater
in contractures; (5) broadened metaphyseal regions of the
elbows, wrists, knees, and interphalangeal joints; (6) protu-
berant eyes with bluish sclerae and normal cornea diame-
ter; (7) primary platyspondyly, osteopenia of the axial skel-
eton, widening of the ends with relative narrowing of the
diaphysesandflatepiphysesofmetacarpalsandphalanges,
small ileum, mildly flat proximal femoral epiphyses, and
short and wide femoral necks; and (8) a ratio of lysyl pyri-
dinoline to hydroxylysyl pyridinoline (LP/HP) of ~1.0.
There are clear differences and similarities between sub-
jects with EDS VI and SCD-EDS. The phenotype of EDS VI
differs from that of SCD-EDS bysevere muscular hypotonia
from birth, progressive kyphoscoliosis, severe hypermobil-
ity of small and large joints, absence of contractures of fin-
gers, and normal height (in the absence of severe kypho-
scoliosis), often by a Marfanoid habitus and microcornea,
occasionally by ruptures of the eye globe and of large ar-
teries, by the absence of platyspondyly,5,20–24and by the
high LP/HP ratio. On the other hand, individuals with
SCD-EDS do have platyspondyly, wrinkled palms, tapering
fingers, enlarged metaphyseal regions over the elbows, the
wrists, and the knees, and the interphalangeal joints that
are not observed in individuals with EDS VI. SCD-EDS
shares common features with EDS VI such as smooth,
Table 6.
Activities in Patients’ Fibroblasts
Lysyl Hydroxylase and Prolyl 4-Hydroxylase
Lysyl Hydroxylasea
Prolyl Hydroxylasea
P1/I
P3/II
142 5 27; 105–166
126 5 16; 109–147
103 5 10; 95–115
120 5 22; 86–133
aActivities of four independent assays are expressed as percentages of
control values and given as mean 5 SD and range.
Figure 6.
The parametric multipoint LOD score anal-
ysis for 39 microsatellite markers spanning
the linkage interval
11p11.2-q13 in families I and II is shown.
Because of space constraints, only 31
markers are shown. Genetic distances, in
cM, based on the deCODE map and the
LOD scores are shown on the x axis and
on the y axis, respectively.
Results of Linkage Analysis
on chromosome
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1301

Page 13

thin, hyperelastic, and fragile skin with atrophic scars,
hypermobile small joints, bluish sclerae, and osteopenia.
Platyspondyly is observed in SCD-EDS but not in EDS VI
despite the complete lack of LH1 activity in the latter con-
dition. Hydroxylysine content in bone collagen of the
original EDS VI patients,25however, was 40%–100% of
the normal content, whereas in skin it was 5%–10%. The
difference in lysyl hydroxylation of collagen in bone and
skin is explained by the different activity ratios of lysyl hy-
droxylase to prolyl hydroxylase measured in bone cells and
skin fibroblasts from these patients; their bone cells and fi-
broblasts had activity ratios of 46%–48% and 8% ofnormal
activity ratios, respectively (Krane et al., 1980, Calcif. Tis-
sue Int. 31, 57, abstract). These authors speculated on the
presence of different isoenzymes with variable tissue ex-
pression and different substrate specificities that have
been characterized recently.26In SCD-EDS, the disturbed
zinc homeostasis (see below) is expected to inhibit all
iron-dependent collagen hydroxylases, including those
in bone and growth-plate cartilages, and so may have
more severe consequences on the vertebrae than isolated
LH1 deficiency in EDS VI. Thus, the platyspondyly in
SCD-EDS may be explained by the combined underhy-
droxylation of lysyl and prolyl residues in bone and carti-
lage, perhaps in combination with the normal muscular
tone that thus may lead to platyspondyly; note that severe
muscular hypotonia per se in genetic disorders similar to
EDS VI and in acquired conditions result in tall vertebral
bodies.
The features observed in SCD-EDS, its disease locus, and/
or its autosomal-recessive mode of inheritance distinguish
it from skeletal dysplasias such as ‘‘SEMD with joint laxity
(SEMD-JL) Beighton type’’ (MIM 271640), ‘‘spondyloepi-
metaphyseal dysplasia with multiple dislocations’’ (MIM
603546), and ‘‘spondylometaphyseal dysplasia with cone-
rod dystrophy’’ (MIM 608940).27
The genetic defect in the two families with SCD-EDS is a
homozygous 9 bp deletion in exon 4 of SLC39A13 (Fig-
ure 7A), which encodes for the zinc transporter SLC39A13
(ZIP13) that is widely expressed in most tissues. This pro-
tein is a member of the LIV-1 subfamily of ZIP zinc Trans-
porters (LZT),28a highly conserved group of eight trans-
membrane domain proteins known to transport zinc and/
or other metal ions from the extracellular space or from
theorganellarlumenintothecytoplasm.29Theyaremainly
situatedontheplasmamembrane,butatleastonemember,
SLC39A7 (HKE4 or ZIP7 [MIM 601416]), is located on
the ER30and on the Golgi31membranes and transport
zinc into the cytosol.30Protein sequence alignments have
shown that human SLC39A13 and SLC39A7 are phyloge-
neticallycloselyrelatedZIPtransporters32thatlackahighly
conserved CPALLY motif, which immediately precedes the
first transmembrane domain28and characterizes all other
LZTfamily membersthat arelocalized onthe plasmamem-
brane. Thus, in analogy to SLC39A7, the zinc transporter
SLC39A13 may also reside on intracellular membranes.32
Indeed, our prediction analysis for subcellular localization
of SLC39A13, based on its amino acid sequence with the
online PSLDoc server, resulted in a 55% likelihood that
SLC39A13 resides on the membrane of intracellular organ-
elles, whereas the probability of it being localized on the
plasma membrane of the cell was only 17%; for compari-
son, the probability of SLC39A7 to be located on these
membranes is 60% and 17%, respectively. Although the
exact localization of ZIP13 is not known so far, our
biochemical findings (see below) strongly suggest it is on
the ER membrane.
The mutation found in our patients causes a deletion of
amino acid residues 162–164 (Phe-Leu-Ala) within the
transmembrane domain III, which is highly conserved
(Figure 7B). We suspect that the deletion disturbs the
proper folding of the protein and thus impairs 3D confor-
mation and function of the transporter.
Synthesis and posttranslational modifications of the
nascent a chains of the various collagen types occur in the
ER. Among the different modifications, hydroxylation of
Figure 7.
Comparative Analysis of the ZIP13
Protein
(A) Electropherograms indicating the wild-
type (S3/II), the homozygous (P3/II), and
the heterozygous (M2/II) mutation se-
quences. The 9 bp deletion in exon 4 of
SLC39A13 (c.483_491 del9; p.F162_164
del) is boxed.
(B) ClustalW alignment and BOXSHADE
analysis of the protein sequence of the
transmembrane domain
around the mutation (the deleted amino
acids FLA are boxed). The protein sequence
is highly conserved across vertebrates,
from primates through to birds and primi-
tive fish.
Mutation Identification and
IIIof ZIP13
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Page 14

collagen lysyl and prolyl residues are due to highly con-
served enzymatic processes that involve Fe2þ, a-ketogluta-
rate, O2, ascorbate as cofactors, cosubstrates, and reducing
agent.17We suspect that an increased concentration of
Zn2þin the ER competes with Fe2þfor binding to lysyl hy-
droxylase, prolyl 4-hydroxylase, and prolyl 3-hydroxylase,
thus impairing hydroxylation of lysyl and prolyl residues.
Indeed, Zn2þwas found to be an effective competitive in-
hibitor with respect to Fe2þfor prolyl 4-hydroxylase33
and for lysyl hydroxylase.34Proper hydroxylation of colla-
gen lysyl residues is important for functional crosslinks as
demonstrated by its deficiency in EDS VI, whereas 4-hy-
droxylation of collagen prolyl residues is important for
the stability of the triple helix17as shown in experimental
scurvy.35
As a consequence of the defect, collagen hydroxylysyl
residues (Hyl) are formed to a lesser extent in SCD-EDS
than in controls as reflected by the abnormally high
urinary LP/HP ratio and the lower Hyl contents of dermal
collagens and of collagens formed by cultured fibroblasts.
Underhydroxylation of collagen lysyl and prolyl residues
observed in these individuals occurs along the entire mol-
ecule, is not confined to specific residues (although prefer-
ential effects dependent on sequence context are possible),
and affects at least collagen types I and II (Table 5). The
underhydroxylationof lysyl residues
although less pronounced than in EDS VI—suggests a rele-
vant impairment of lysyl hydroxylase activity in SCD-EDS.
However, its activity measured in fibroblast homogenates
of SCD-EDS subjects is in the normal to higher-than-
normal range. This is consistent with our hypothesis of
Zn2þinduced inhibition of this and related hydroxylases.
Homozygous EDS VI patients with a complete lack of
LH1 still have ~15%–20% of residual lysyl hydroxylase
activity in cell extracts, an outcome has been attributed
to the presence of its isoform LH3, and this results in a uri-
nary LP/HP ratio of ~6. In obligate heterozygotes for EDS
VI, the estimated residual activity of 60%–65% (50% plus
15%–20%)2leads to an LP/HP ratio of ~0.3, which is clearly
higher than in controls (~0.2) but similar to that in obli-
gate heterozygotes for SCD-EDS (Table 4). By extrapolating
these results, we expect the lysyl hydroxylase activity
in vivo to be considerably reduced in SCD-EDS. Moreover,
itispossiblethattheslightbutsignificantprolyl3-underhy-
droxylation contributes to the phenotype. In osteogenesis,
imperfecta type VII (OI type VII [MIM 610682]) caused by a
homozygous mutation in CRTAP12(MIM 605497) hydrox-
ylation of prolyl residue 986 in the a1(I)-chain from cul-
tured skin fibroblasts and from a bone biopsy was ~70%
of the control, compared with 90% in the patients’ sam-
ples. It is unclear whether the deficiency in 3-Hyp in OI
(moderate in OI type VII and nearly complete in OI type
II [MIM 166210] and OI type III [MIM 259420]) is simply
a marker or has pathological effects on matrix collagen.
If the latter is true, it could contribute to the osteopenia
in SCD-EDS. However, the pathogenesis may be more com-
plex and may involve zinc as an intracellular second mes-
inSCD-EDS—
senger36and as an essential component of many proteins;
thus, a disturbance of intracellular zinc homeostasis could
affect many processes inside and outside the cell and add
to the phenotype. Nevertheless, the common biochemical
effect of mutations in PLOD1 and SLC39A13 on lysyl hy-
droxylation and the abnormal collagen crosslinks strongly
implies that a similar defect in collagen structure is a key
element of both phenotypes. Collagen assembly is com-
plex, requiring for example collagen type V as an essential
template for collagen type I deposition, so consequences of
underhydroxylation in more collagen gene products than
simply types I and II will need to be examined to under-
stand fully the pathogenic mechanism. The observed
underhydroxylation of collagen types I and II may explain
the clinical findings in skin, joints, and the skeleton of our
patients. It is an open question whether underhydroxyla-
tion of other collagen types is involved and whether this
may be clinically relevant and responsible for subtle clini-
cal features, i.e., as in the vascular form of EDS (EDS IV
[MIM 130050], due to mutations in collagen type III), the
classical form of EDS (EDS I/II, due to mutations in colla-
gen type V), Bethlem myopathy ([MIM 158810] due to
mutations in collagen type VI), epidermolysis bullosa dys-
trophica ([MIM 131750] due to mutations in collagen type
VII), and various chondrodysplasias (due to mutations
other than in collagen II such as in collagen types IX and
XI) and as in other collagen disorders.
We have chosen the name ‘‘spondylocheiro dysplastic
form of EDS (SCD-EDS)’’ to indicate a generalized skeletal
dysplasia involving mainly the spine (spondylo) and strik-
ing clinical abnormalities of the hands (cheiro) in addition
to the common EDS-like features.
As a diagnostic tool for SCD-EDS, the measurement of
the urinary LP/HP ratio appears to be specific, reliable,
and rapid; however, more subjects are needed for estimat-
ing the biochemical range of the LP/HP ratio as well as the
clinical spectrum. The prevalence of this entity is not
known. The common mutation in the two families may
be explained by a founder effect in this geographic region.
Indeed, an identical disease haplotype is indicated by the
finding of identical alleles at the two SLC39A13-flanking
satellite marker loci in our families. On the other hand,
the disorder may have been underreported so far. With
this publication, we hope to attract more cases of this
entity in order to identify the spectrum of disease-causing
mutations and the resulting clinical and biochemical
variability.
Acknowledgments
We are grateful to all patients and their family members for partici-
pating in this study. Our thanks go to Michael Raghunath (Mun-
ster) for sending material of the index patient; to Ingrid Hauser
(Heidelberg) for the ultrastructural assessment of the skin biopsy;
to Jo ¨rg Schaper (Dusseldorf) for initial radiological considerations;
to Angelika Schwarze (Zurich), Udo Redweik (Zurich), and Chris-
tine Plu ¨ss (Basel) for expert technical assistance; to Konrad Oexle
The American Journal of Human Genetics 82, 1290–1305, June 2008
1303

Page 15

(Zurich), Ann Randolph (Zurich), and Frank Neuheiser (Zurich) for
their help in the initial candidate linkage, genome-wide mapping
andmutationanalyses,respectively; to LindaWalker(Durham) for
the enzyme assays; to Albert Schinzel (Zurich) for karyotyping;
and to Andres Giedion, Patricie Paesold-Burda, and Matthias
Baumgartner (Zurich) for useful discussions. This work was sup-
ported by grants from the Swiss National Science Foundation
(grant Nr.3200B0-109370/1), the Wolfermann-Na ¨geli Stiftung to
B.S., and NIH (AR 37318, AR 36794) to D.R.E.
Received: March 31, 2008
Revised: April 29, 2008
Accepted: May 2, 2008
Published online: May 29, 2008
Web Resources
The URLs for data presented herein are as follows:
BOXSHADE, http://www.ch.embnet.org/software/BOX_form.html
ClustalW, http://www.ebi.ac.uk/Tools/clustalw2/index.html
EasyLinkage,http://www.uni-wuerzburg.de/nephrologie/molecular_
genetics/molecular_genetics.htm
Entrez Gene, http://www.ncbi.nlm.nih.gov/sites/entrez
ExPASy, http://www.expasy.ch/sprot/
NCBI Map Viewer, http://www.ncbi.nlm.nih.gov/ mapview/
Online Mendelian Inheritance in Man (OMIM), http://www.ncbi.
nlm.nih.gov/Omim
PSLDoc, protein subcellular localization prediction based on
modified document classification methods, www.bio-cluster.
iis.sinica.edu.tw/~bioapp/PSLDoc
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