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Early-onset primary osteoarthritis and mild chondrodysplasia.

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674
EARLY-ONSET PRIMARY OSTEOARTHRITIS AND
MILD CHONDRODYSPLASIA
Radiographic and Pathologic Studies with an Analysis of
Cartilage Proteoglycans
PAUL L. KATZENSTEIN, CHARLES J. MALEMUD, MINI N . PATHRIA. JOHN R. CARTER,
ROBERT P. SHEON, and ROLAND W.MOSKOWITZ
Three generations of a nonconsanguineousfamily
with premature onset of primary (idiopathic) osteoarthritis (OA) were studied for clues to the etiopathogenesis of their disorder. Articular symptoms began in their
second and third decades of life and involved multiple
joints, both typical and atypical for primary OA. Radiographs of the majority of involved peripheral joints
showed abnormalities typical of primary OA. Evidence
of chondrodysplasia was found in the spines. Pathologic
examination of femoral heads obtained at total hip
arthroplasty from 3 affected family members showed
moderate to severe OA. Articular cartilage proteoglycans from these specimens were evaluated for aggregatability with hyaluronic acid, levels of chondroitin sulfate
and keratan sulfate, and core protein structure. The
results from each patient’s specimen differed from the
results of the other specimens. We conclude that this
family’s disorder, primary OA associated with a mild
- ~From the Department of Medicine. Division of Rheumatic
Diseases and the Departments of Anatomy, Radiology, and Pathology, Case Western Reserve University and University Hospitals of
Cleveland; and the Medical College of Ohio, Toledo, Ohio.
Supported in part by NIH grants AR-20618 and AG-02205.
Paul L. Katzenstein, MD: Department of Medicine. Case
Western Reserve University (current address: Department of Internal Medicine, Division of Rheumatology and Clinical Immunogenetics, University of Texas Health Science Center, Houston): Charles
J. Malemud, PhD: Departments of Medicine and Anatomy, Case
Western Reserve University; Mini N. Pathria. MD: Department of
Radiology, Case Western Reserve University; John R. Carter, MD:
Department of Pathology, Case Western Reserve University; Robert P. Sheon. MD: Medical College of Ohio; Roland W. Moskowitz.
MD: Department of Medicine. Case Western Reserve University.
Address reprint requests to Charles J. Malemud. PhD,
Department of Medicine, Division of Rheumatic Diseases, Case
Western Reserve University, University Hospitals, 2074 Abington
Road, Cleveland, OH 44106.
Submitted for publication August 29, 1989; accepted in
revised form December 14, 1989.
ArthriHs and Rheumatism, Vol. 33,
No. 5 (May 1990)
chondrodysplasia, was a late-onset overlap form of an
epiphyseal dysplasia, that a defect common to hyaline
articular and physeal cartilage was primary, and that a
single structural proteoglycan abnormality was not
likely to be the underlying cause.
Osteoarthritis (OA) is a common disorder of
synovial joints characterized by degeneration of hyaline articular cartilage, marginal osteophyte formation,
and reactive subchondral bone changes (1). Most
individuals with OA have no identifiable cause for this
disorder. An underlying secondary etiology is suggested, however, when OA develops at an abnormally
young age and involves multiple synovial joint groups
and joints not usually affected in primary (idiopathic)
OA (2). Numerous acquired and inherited disorders
associated with secondary OA, such as trauma, microcrystalline disease, hemochromatosis, and chondrodysplasia, have been described (3). In this report, we
dcscribe 3 generations of a nonconsanguineous family
whose members developed a robust and rapidly progressive, generalized OA beginning at atypically
young ages. Radiographs of affected peripheral joints
showed changes characteristic of primary OA. Radiographic evidence of an underlying mild chondrodysplasia was found in the spine. Gross specimens and
microscopic pathologic specimens of femoral heads
obtained from 3 family members at total hip arthroplasty were evaluated, and newly synthesized and
resident cartilage proteoglycans were analyzed.
PATIENTS AND METHODS
Clinical studies. The family pedigree is shown in
Figure 1. Medical histories, supplemented by inpatient and
675
OA AND CHONDRODYSPLASIA
111
Figure 1. Three-generation pedigree of the family studied. showing
affected individuals (dark symbols). Circles = females; squares =
males; / .= deceased.
outpatient records, were obtained on all family members.
Physical examinations were performed on the surviving first
generation member (1-1). all 7 second generation direct
descendants, and 9 of 19 third generation direct descendants.
Height and lower segment (pubis-to-floor) measurements
were obtained. Upper segment (height minus lower segment)
to lower segment ratios (US:LS) were calculated and com-
pared with values from age-matched normal controls (4). A n
assessment of joint hypermobility was performed (5).
Laboratory studies. The proband (11-9: Figure 1) was
evaluated with a complete blood count (CBC). tests for
Westergren erythrocyte sedimentation rate (ESR). antinuclear antibody, rheumatoid factor. serum electrolytes, liver
function. prothrombin time, serum femtin, serum iron and
percent transferrin saturation, serum thyroxine, serum ceruloplasmin, urinary homogentisic acid, and urinalysis. His 2
symptomatic brothers (11-5 and 11-7) were evaluated with a
CBC, tests for serum electrolytes, liver function. Westergren ESK, HLA-B27 determination, and urinalysis.
Radiologic studies. Standard radiographs were available or were obtained as noted in Table I . All films were read
by a musculoskeletal radiologist and by 2 rheumatologists.
An evaluation for acetabular dysplasia was performed (6).
Pathologic studies. Three affected second generation
members (11-5, 11-7, and 11-9) underwent total hip arthroplasties. Synovial tissue from each was placed in 90% ethanol.
The excised femoral heads were immersed in sterile saline
within 30 minutes of resection. Following removal of portions of articular cartilage for explant culture, multiple
representative blocks of the femoral heads were decalcified
Table 1. Morphometric and radographic findings of the family studied"
~~
~
Family rnembedage
1-1/70
1-2 (deceased]?
11-2/52
11-3151
II-5/49t
II-7/47t$
I 1-9/43t$
11-1 1/40
11- 13/38t
1II-1/30
I I 1-2/25
111-4122
111-5/20
III-6/27
I11-7/26
I1 1-8/24
I II-9/28t
I I I- 10/16t
111-1 1/26
111-12 (deceased)
llI-13/22
I1 I - 14/ 18
111-l5/17
III-I6/IS
I I I- 171I5
III-I~~
~
~~
~
Upper segment:
lower segment
0.846
0.923
1.010
0.805
0.836
0.845
0.955
0.772
-
0.866
0.854
0.918
-
0.854
0.771
0.826
0.799
-
* MCP = metacarpophalangeal; PIP
~
Thoracic
and
lumbar
spine
N
S. OA
N
N
S . OA
S. OA
S. OA
N
S , OA
N
-
N
N
-
N
N
S. O A
S
N
N
N
S
S
S
N
MCP
joints
PIP
joints
DIP
joints
N
N
OA
N
N
N
N
OA
OA
OA
N
OA
OA
OA
OA
N
OA
OA
OA
N
OA
N
N
-
N
-
B
N
N
N
N
N
N
N
N
N
N
N
N
-
N
OA
OA
N
N
N
N
F
N
-
N
-
Hips
Shoulders
Elbows
Wrists
Knees
N
N
-
N
N
N
N
N
N
N
N
-
= proximal interphalangeal; DIP = distal interphalangeal; N = normal; - = measurements not obtained
dysplastic Scheuermann-like changes consisting of irregular endplates. Schmorl's nodes, vertebral body
flattening (platyspondyly). and anterior wedging; OA = osteoarthritis; OD = bilateral osteochondritis dissecans of the capitellurn; F = mild
flattening of the distal fourth MCP joint.
? Family members affected with peripheral OA.
$ These individuals also showed OA of the ankles and OA and flattening of the metatarsal heads.
or radiographs not available; S
=
676
and processed by standard procedures. Microscopic sections were stained with hematoxylin and eosin and with
Masson trichrome. Unstained sections were examined by
polarized microscopy. Microscopic sections from the
proband's tissue were additionally stained with alizarin red,
von Kossa's stain, and alcian blue-periodic acid-Schiff.
Special stains for iron and amyloid deposition were included.
Proteoglycan analysis. Rndiolabeling of cartilage. Resident cartilage was obtained from the 3 affected males in the
second generation (11-5, 11-7. and 11-91. In all cases, contaminating metaplastic cartilage was excluded. Cartilage was
incubated in sulfate-free Dulbecco's modified Eagle's medium (7) containing 10% fetal bovine serum (volume/
volume), antibiotics, Mycostatin and Fungizone. and labeled
with ''SO, (20 pCi/ml) for 20 hours at 37°C. Two of the
samples (11-7 and 11-9) were placed under these conditions
immediately following surgical excision. The third tissue
sample (11-5) was radiolabeled <24 hours after surgery.
Proteoglycan extraction. After 20 hours of incubation. the tissue proteoglycan was extracted by agitation in
4M guanidine hydrochloride (GuHCI). 0.1 M sodium acetate.
pH 5.8, containing protease inhibitors (8) and incubated for
24 hours at 4°C. The culture medium was mixed with an
equal volume of buffered 8M GuHCl and incubated overnight at 4°C. The tissue extract was clarified by centrifugation, and the pellet was analyzed for hexuronic acid (9) to
determine the efficiency of proteoglycan extraction. The
tissue and medium 4M GuHCl extracts were dialyzed separately and exhaustively against double-distilled deionized
water and freeze dried (10). The cloudy suspension that
formed during dialysis was clarified by high-speed centrihgation in a Sorvall model RC-SB centrifuge (Sorvall Centrifuges, Wilmington. DE) at 26,8901: (15,000 revolutions per
minute) for 20 minutes.
CsCl density gradient 14ltracentriJirgation. A highdensity proteoglycan fraction ( A l ) was obtained from the 4M
GuHCl extract by equilibrium centrifugation in CsCl under
associative conditions (10). The precipitate formed by highspeed centrifugation (15.000 rpm precipitate [fraction 15KpI;
see above) was also analyzed by this technique.
Gel Jiltrntion chromatography. The hydrodynamic
profilc of the A l fraction was established by chromatography
on Sepharose CL-2B. A high-density protcoglycan fraction
containing proteoglycan monomer ( A I DI) was generated by
equilibrium centrifugation in CsCl under dissociative conditions, and its hydrodynamic size was established by chromatography on Sepharose CL-2B. The glycosaminoglycan
chain size was estimated after Sepharose CL-6B chromatography (11). The capacity of AID1 to form proteoglycan
aggregates was measured by incubation with umbilical cord
hyaluronic acid and bovine link protein (12). followed by
chromatography on Sepharose CL-2B. A shift in incorporated "SO, from the included volume of the CL-2B column
to fractions 15-19 (encompassing the column void volume) was
used to define the presence of proteoglycan aggregates ( 13).
Trypsin digestion of A I D I . To assess the compositional structure of resident and newly synthesized proteoglycans, AID1 was digested with L-l-tosylamide-2phenylethyl chloromethyl ketone-trypsin (TPCK-trypsin).
Tryptic peptides were chromatographed on DEAE-cellulose
(l4,lS). Two fractions containing >93% of the peptide
KATZENSTEIN ET A L
glycosaminoglycans were obtained-a 0 . 5 M KCI fraction
(DEAE-I) that contained peptides enriched in chondroitin
sulfate, and a 1.OM KCI fraction (DEAE-11) that contained
peptides with which both chondroitin sulfate and keratan
sulfate were associated. Previous studies showed this latter
fraction to be enriched in keratan sulfate (15-17). DEAE-I
and DEAE-II were digested with chondroitinase ABC and
keratanase. or chondroitinase ABC only (l8,19), and the
product was chromatographed on Sepharose CL-6B. Fractions comprising the protein peak (15) were dialyzed and
freeze dried. Protein was electrophoresed on 7.5% polyacrylamide slab gels containing 0.1% sodium dodecyl sulfate
in Tris-glycine buffer (20). The gels were stained with silver
nitrate (21). The I-dimensional peptide map was scanned
using a laser densitometer as previously described (22).
RESULTS
Clinical findings. The proband (11-9) and his
family were evaluated after strikingly similar findings
were noted in the proband and 2 male siblings (11-5 and
11-7). These patients developed limited elbow range of
motion (ROM) in their second and third decades of
life. One (11-5) required radial head resections at ages
16 and 17 for relief of locking and pain. During their
third to fifth decades, these patients developed progressively limited ROM of metacarpophalangeal
(MCP), proximal interphalangeal (PIP), and distal interphalangeal (DIP)joints, shoulders, hips, and knees,
bilaterally. Joint pain remained mild until early in the
fifth decade, when all 3 patients required total hip
arthroplasties for relief of pain and limited ROM. Only
I patient (11-5) reported inflammatory symptoms, consisting of up to 1 hour of morning stiffness in his knees,
hips, and hands. Results of complete physical examinations were notable only for bony enlargement and
limited ROM of MCP, PIP, and DIP (Hebcrden's
nodes) joints and limited ROM of shoulders, elbows,
hips, and knees. Synovitis was absent, and spine
motion was mildly limited. The mean height of these
patients was 167 cm, and their mean US:LS ratio was
0.829 (range 0.805-0.845). The mean height of their 3
unaffected brothers (11-2. 11-3. and 11-1 I ) was 177 cm,
and the mean US:LS ratio was 0.963 (range 0.9231.01). All laboratory test results were normal or negative.
The 3 Unaffected brothers were symptom free
and had normal physical examination results. The sole
second generation female (11-13) reported intermittent
low back discomfort since age 16 and diminished hip
ROM developing during the past several years. Results of her physical examination were notable only for
the mildly limited hip ROM. The father (1-2). who died
677
OA AND CHONDKODYSPLASIA
Figure 2. Anteropostenor pelvic radiographs of family members 11-7 (A) and 11-5 (B). taken at agcs 42 and 46, respectively.
demonstrating severe osteoarthritis (OA) in the absence of dysplasia. In family member 11-7 (A). moderately severe OA is present
bilaterally. In family member 11-5 (B). OA IS more advanced in the left hip 1han in the right hip.
of pneumonia and heart failure at age 75. reportedly
had a syndrome identical to that of his affected male
children. The mother (1-1) was symptom free, and her
physical examination findings were unremarkable. Of
5 affected third generation members, only 1 (111-10)
reported articular symptoms. He noted limited elbow
ROM due to bilateral osteochondritis dissecans of the
capitellum at ages 9 and 10, and intermittent low back
discomfort. Physical examinations of the third generation members produced unremarkable results, except
for the limited elbow ROM.
There was no history of consanguinity, early
death, mental retardation, central or peripheral nervous system disease, ocular disease, internal organ
abnormality, spontaneous fractures. joint contractures, or hypermobility. Four family members (11-5.11-7.
11-9, and 11-1 I ) reported a mild. untested hearing loss
that was attributed to ear infections or occupational
exposure.
Radiologic findings. Radiographs of the father
(1-2) and the 4 affected second generation members
(Figure I ) demonstrated changes consistent with advanced OA, which were largely indistinguishable from
those of primary OA (Figure 2). Articular degeneration
was generalized (Table I ) and involved joints that were
typical and atypical for primary OA. OA occurred at
an unusually young age and involved some clinically
asymptomatic joints. Neither acetabular dysplasia nor
avascular necrosis was present. A radiograph of family
member 111-9. taken at age 28, demonstrated early
changes consistent with OA in the metacarpal heads
(Figure 3).
Chondrodysplasia characterized by irregular
endplates, Schmorl's nodes, vertebral body flattening
(platyspondyly), and anterior wedging was evident in
the spines of 4 second generation individuals and 5
third generation members (Figure 4). Kyphosis beyond 40" was not present except in the unaffected
mother (1-1). Evidence of peripheral joint dysplasia
was largely absent. Of the third generation members,
only 1 (111-10) demonstrated an abnormal epiphyseal
shape, consisting of mild flattening of the fourth
metacarpal distal epiphyses. Bilateral osteochondritis
Figure 3. Radiograph of the right rnetacarpophalangeal joints of
family member 111-9, taken at age 28. showing subchondral cysts
(third metacarpal head) and marginal osteophytes (third and fourth
metacarpal heads) in the absence of dysplasia.
678
KATZENSTEIN ET AL
dissecans was evident in his capitellum, but the contour was otherwise normal.
Radiographs of shoulders, hands, and elbows of
3 affected third generation members (111-16.111-17. and
111-10). taken before skeletal maturity. demonstrated
normal epiphyseal development at these sites. In the
second generation members, epiphyseal contour abnormalities separate from those attributable to OA
were not present.
Pathologic findings. The proband's right femoral
head measured 5 x 4 x 4 cm. The normal curvature
was severely distorted and flattened. particularly in
Figure 5. Hematoxylin and eosin-stained section of right femoral
head articular cartilage taken from the proband (11-9) at age 43.
Degenerative articular cartilage (arrow) and an overlying fibrous
connective tissue pannus (double arrows) are seen (original magnification x 88).
Figure 4. Lateral radiograph of the thoracic spine of family member
11-13, taken at age 38. demonstrating irregular vertebral endplates,
mild platyspondyly. Schmorl's nodes, anterior wedging, marginal
osteophytes. and disc space narrowing.
the anteropostenor plane. Radiographs confirmed this
gross appearance and revealed focal areas of eburnation 4-5 mrn in thickness, cysts up to 2 cm in diameter,
and osteopenia of cancellous bone. Histopathologic
examination showed typical articular cartilage degenerative changes and chondrocyte cytoplasmic vacuoles. A microscopic fibrous connective tissue pannus.
more extensive than usually seen in idiopathic OA and
with pronounced tendinous differentiation, overlay
portions of the articular surface and the eburnated,
weight-bearing bone (Figure 5 ) . This fibrous pannus
was covered by hyperplastic and hypertrophic synovial cells. An exuberant reparative process was evident. There was much reactive new bone and osteoid
and pronounced osteoclastic activity. In addition to
the remaining hyaline cartilage, metaplastic cartilage.
which also had dcgenerative changes, mushroomed up
onto the articular surface. Microscopic foci of osteonecrosis were also seen.
The synovial tissue also showed marked type I
and type I1 synoviocyte hypertrophy, hyperplasia. and
multinucleation. Only scattered chronic inflammatory
cells were seen. Necrotic hyaline cartilage, with a
pronounced foreign body reaction, and necrotic bone
fragments were present. Focal areas of hemorrhage
were seen. Hernosiderin was focally prominent and
found in histiocytes as well as in typc I and I1 synoviocytes in the deepcr layers of the tissue. Most of the
hemosiderin deposition was found in association with
old areas of hemorrhage. Amyloid, calcium, and urate
deposits were absent.
679
OA AND CHONDRODYSPLASIA
r
0‘
C
o
u
v)
400111-5 Al
D
q
p
r
600 Medium Al
0
E
20 25
30
35
Fraction No.
Figure 6. Sepharose CI.2B chromatography of high-density proteoglycan ( A l ) fractions. A l fractions were chromatographed on a
Sepharose CL-7B column (0.4 x I I5 cm). eluted with O.SM sodium
acetate. pH 7. and 0.5-ml fractions were collected. The void volume
(VJ (Kav 0)was determined by the elution of Dextran blue 2000 or
bovine cartilage proteoglycan aggregate. The total column volume
(V,)(KaV 1) was determined with ”SO,. A, A1 fraction from a 4M
guanidine hydrochloride extract of sample 11-9. B, A l from the ISKp
subfraction from sample 11-9. C, A l fraction from sample 11-7. D, A1
fraction from sample LI-5. E. A l fraction from a representative
culture medium.
15
changes typical of OA and a mildly active reparative
process. In sample 11-5, a marked disorganization of
chondrocyte polarity, broad zones of acellular extracellular matrix. and a peripheral fibrous pannus without tendinous differentiation were noted.
Mild type I and type I1 synoviocyte hyperplasia
was noted in sample 11-7, but not in sample 11-5.
Synovial scarring was pronounced, and chronic inflammatory cells were rare. Hemosiderin. amyloid,
calcium, and urate deposits were absent.
Analysis of proteoglycans. Associative CsCl
density gradient ultracentrifugation. The bulk of the
tissue proteoglycan (>70%) was extracted with 4M
GuHCI. as measured by hexuronic acid content. Sixtysix percent, 59.5%. and 70.9% of the newly synthesized proteoglycan in the samples from patients 11-9,
11-7, and 11-5, respectively, reached equilibrium in the
most dense (>1.7 gm CsCl/ml) A l fraction. In contrast, only 16.8% of the newly synthesized proteoglycan secreted into the medium was found in the A l
fraction.
Hexuronic acid measurements of the A l . A2/
A3. and A4 fractions were consistent with the distribution of 3sS04, with the exception that somewhat
more 3SS0,-containing A 1 than hexuronic acidcontaining A1 was found (data not shown).
Hydrodynamic projle of the A1 fraction. In the
3 samples (11-9, 11-7, and 11-3, fractions 15-19 (Kav
04.14) constituted 28.1%. 53.3%, and 16.8% of the A1
fraction, respectively (Figures 6A, C, and D). When
l5Kp A1 subfractions were examined, a distinct void
volume peak was seen (Figure 6B). The A1 fraction of
the medium contained 2 broad peaks of incorporated
3sS0, (Kav0.3 and 0.85) (Figure 6E).
Dissociative CsCl density gradient ultracentrifugation and Sepharose CL-2B chromatography of the
AlDl fraction. The A l D l fraction constituted 89.3%.
97.7%. and 90.9% of the incorporated 35S04in the
samples from patients 11-9.11-7, and 11-5. respectivf ly.
On Sepharose CL-2B, samples 11-9 and 11-7 gave
identical K,, of 0.2, whereas sample 11-5 showed a K,,
of 0.4.
Coniposition analysis of A l D l . The glycosaminoglycan chain size as measured by chromatography of alkaline borohydride-reduced A I D 1 on Sepharose CL-6B was 11.7 x lo3 daltons (Kav0.60). The
Al D1 glycosaminoglycan composition was 34% chondroitin-6-sulfate, and 66% chondroitin-4-sulfate (C4-S), as measured by high performance liquid chromatography of chondroitinase ABC/AC-Il-digested AlDl
(23). No significant increase in disaccharide-4-sulfate
-
The tissues from the proband’s 2 affected siblings were less severely involved in all respects. The
femoral heads measured 5 x 5 x 4.5 cm (11-5) and
5 x 5 x 6 cm (11-7). Both femoral heads showed
680
KATZENSTEIN ET AL
was scen when chondroitinase ABC-digested A 1 D1
was compared with chondroitinase AC-II-digested
AlDl, indicating that the fraction lacked significant
dermatan sulfate content. A positive reaction with
monoclonal antibody 5D4, which is reactive with
keratan sulfate (24). was also obtained (data not
shown). AID1 from samples 11-9 and 11-7 aggregated
efficiently with hyaluronic acid in the presence of
bovine link protein, whereas sample 11-5 did not
aggregate efficiently. The ability of AID1 from sample
11-7 to form proteoglycan aggregates was further substantiated by a strong reaction of reduced and alkylated AID1 with monoclonal antibody 1C6 (24), indicating the presence of the hyaluronic acid-binding
region epitope. Unfortunately, not enough of sample
U-5 was available to test for 1C6 reactivity.
AI DI digestion with TPCK-trypsin and analysis
by sodium dodecyi sulfate-poiyacrylamide gel electrophoresis. The A 1 DI samples showed different tryptic
peptide elution profiles on DEAE-cellulose (Table 2).
The l5Kp AlDl tryptic peptide maps from samples
11-9 and 11-7 were analyzed by densitometric scanning
(Figure 7). Sample 11-9 AID1 showed abundant small
peptides (Figures 7C and D), which were not seen in
sample 11-7 AID1 (Figures 7E and F). In contrast,
sample 11-7 AID1 contained high molecular weight
tryptic peptides (M7.4 kd), which were not seen in the
corresponding fraction from sample 11-9 AID1 (compare Figures 7D and F). Tryptic peptides from sample
11-5 resembled those from sample 11-9. especially in
the content of small tryptic peptides.
B nI 1
n
C
f
.-2
1
I
DISCUSSION
We have describcd a family with a Mendelian
dominant disorder characterized by the precocious
DEAE-cellulose chromatography of L-l-tosylamide2-phenylethyl chloromethyl ketonetrypsin (TPCK-trypsink
digested AIDI*
Table 2.
76 35S0, eluting in pooled DEAE fractions
Sample
DEAE-I (0.5M KCI)
DEAE-11 ( I .OM KCI)
11-9
11-7
11-5
32.9
69.6
47.3
67. I
30.7
46.2
* The AID1 fraction was digested with TPCK-trypsin ( I &sample)
for 6 hours. The DEAE-cellulose column (1.5 x 20 cm) was
equilibrated with 0.01M sodium phosphate bufler, pH 7. Samples
were loaded onto the column and eluted stepwise with O.IM KCI in
sodium phosphate buffer (100 ml). followed by O.5M KCI (100 ml)
and I.OM KCI ( I 0 0 ml). The radioactivity in the pooled fractions was
measured. Less than I% of the "SO, in sample 11-7 and 6.5% of the
"SO, in sample 11-5 was eluted with 0. IM KCI.
Distance mm
Figure 7. Laser densitometric scan of tryptic peptide maps of
high-density proteoglycan monomer fractions from samples 11-7 and
11-9. Sodium dodecyl sulfate-polyacrylamide slab gels were placed
on gel bond film. which was overlaid with cellophane and air dried.
Gels were scanned on an Ultroscan XL laser densitometer (LKB,
Gaithersburg, MD) interfaced with an AT&T PC 6300 computer. The
gel scan XL laser densitometer program (X2400, version 1.20; LKB,
Bromma. Sweden) was used to generate the peaks. and the results
were displayed on an Epson FX-85 printer. Generally, an x-width of 10
units was used. A y-step (ordinate) of 1 unit was used for all gels. A
normalized baseline was generated. The peak widths were variable
depending upon the individual scan. Areas of peaks were calculated by
interpolation. A, Molecular weight markers. B, Protile of chondroitinase ABC and keratanase (1:20) showing the major peak of bovine
serum albumin added to the buffer (66 kd). C, Sample 11-9, DEAE-I. D,
Sample 11-9. DEAE-11. E, Sample 11-7. DEAE-I. F, Sample U-7.
DEAE-11. Low molecular weight tryptic peptides (arrows) and high
molecular weight tryptic peptides (double arrows) are indicated.
OA AND CHONDRODYSPLASIA
onset of generalized OA associated with a mild chondrodysplasia. Six family members (11-5, 11-7. 11-9,
11-13, 111-9. and 111-10) demonstrated the onsct of OA
by clinical history and radiography. at atypically
young ages. A 16-year-old (111-10) and a 28-year-old
(111-9) were the youngest members with radiographic
evidence of OA, which was mild. OA was more severe
and widespread in the older, affected second generation
members. The father (1-2) had early onset of OA by
history, and symptoms and signs identical to those of
his affected children, but radiographs taken prior to
age 50 were not available. Three affected third generation members (111-15, 111-16, and 111-17) had not yet
developed peripheral OA.
Chondrodysplasia was evident in the spines of
10 family members. Spine radiographs of 13 other
family members did not show dysplastic changes.
Morphometric measurements demonstrated this dysplasia to be a short-trunk form. Affected second generation males were a mean of 10 cm shorter than their
3 unaffected brothers, and the US:LS ratios were
smaller and abnormally low.
In contrast, chondrodysplasia was largely absent in peripheral joints. In radiographs of affected
third generation members obtained before skeletal
maturation, epiphyses were normal except for the mild
distal fourth metacarpal flattening in 1 member (III10). Radiographs of affected third generation members
obtained after skeletal maturation did not show any
further epiphyseal contour abnormalities. The early
osteoarthritic changes in the metacarpal heads and
hips of 2 members (111-9 and 111-10) occurred in the
absence of dysplastic changes. Multiple joints of affected first and second generation members demonstrated severe OA and lacked radiographic evidence of
an underlying chondrodysplasia. The flattening seen in
some metacarpal and metatarsal heads of family members in the second generation may have been secondary to OA, or the result of abnormal epiphyseal
formation; the presence of early metacarpal head OA
in the absence of dysplasia in family member 111-9
suggests that the former interpretation is correct.
Thus, in this family, the presence of precocious OA in
otherwise normally shaped epiphyses suggests that
their disorder is due to a primary abnormality of
articular cartilage (25).
The dysplasia in this family is consistent with a
mild, late-onset form of chondrodysplasia. All other
known causes of inherited, generalized OA were eliminated with the available clinical, radiographic, laboratory, and pathologic data. Although Scheuermann's
68 1
disease was the radiographic diagnosis initially assigned to the spinal abnormalities in several affected
family members, the presence of platyspondyly and
the extent of spine involvement make this diagnosis
unlikely.
Chondrodysplasias are a heterogeneous group
of syndromes. that result from disorders of cartilage
growth and development (26.27). They are classified
based on the age at presentation, mode of inheritance,
short limb versus short trunk involvement, predominant long bone segment involved (i.e., epiphysis.
metaphysis, or diaphysis), other specific radiographic
features. and associated nonosseous abnormalities
(28). Spondyloepiphyseal dysplasias (SED) are a subgroup of chondrodysplasias that affect epiphyses only,
and are characterized by platyspondyly, short stature
due to a short trunk, abnormal proximal peripheral
joint epiphyses, and the premature onset of secondary
OA (26,27). Late-onset, mild forms have been described (29,30). Multiple epiphyseal dysplasia (MED)
is similar to SED, but short stature is due to short
extremities, vertebral involvement is less prominent,
and distal peripheral joint epiphyseal involvement is
more severe (26,27.3 I). Late-onset forms of MED
have also been described (32). as well as late-onset,
overlap forms of SED and MED that do not fit easily
into either category (33-36). We believe that the family
described here is best classified as having a late-onset,
overlap form of SED and MED.
Gross and histopathologic evaluations of the
femoral heads disclosed OA of varying severity. One
individual (11-9) had evidence of an unusually extensive fibrous connective tissue pannus and a florid
proliferative synovitis. There were no other atypical
pathologic features. An extensive fibrous pannus has
been previously reported in epiphyseal dysplasias (37)
and in Mseleni disease (38). We speculate that the
varying severities of OA in the 3 femoral heads were
related more to the stage of joint degeneration at the
time of surgery than to the dysplasia directly.
Etiopathologic investigations of the chondrodysplasias have focused on the growth plate (physis)
as the abnormal tissue (39,40). Disordered physeal
architecture, chondrocyte inclusions and degeneration, and extracellular matrix abnormalities have been
noted (39.40). In some cases, the data have been
suggestive of specific pathologic mechanisms (40).
Type 11 collagen abnormalities have been found in
some dysplasias (4143).
Few studies have examined articular cartilage
degeneration in the chondrodysplasias (25) and thc
682
putative etiopathogenic role of proteoglycans (44-46)
in these disorders. In the family described here, the
proteoglycan structural analysis did not indicate a
single common alteration in either newly synthesized
or endogenous core protein. The differences in tryptic
peptide maps of A l D l in the 3 samples suggested that
a proteoglycan core protein variability existed. Although differing proteoglycan peptide maps may also
reflect available tryptic cleavage sites, such maps have
been used to discriminate between large and small
interstitial proteoglycans (47). Thus, a heterogeneous
pattern of proteoglycan alterations, typical of those
found in resident cartilage at the time of total hip
arthroplasty in patients with "idiopathic" OA whom
we have previously described (17.22.48.49). was found
in this family as well.
A void volume peak of fraction A1 on Sepharose CL-2B, typical of the presence of proteoglycan
aggregates, was seen only in the 15Kp subfraction,
presumably due to a concentration of hyaluronic acid
in the latter during dialysis. Other interpretations may
be offered, including the presence of significant
amounts of hyaluronic acid-binding region complexed
to hyaluronic acid, which prevents efficient proteoglycan aggregation (50). The ability of the proteoglycan
monomer fraction (A 1D I ) to form prot eoglycan aggregates in samples 11-7 and 11-9. however, was substantiated by the test tube reaggregation with hyaluronic acid.
The hydrodynamic size of AID1 from samples
11-7 and 11-9 was identical (Kav 0.2) on Sepharose
CL-2B. The Kav, glycosaminoglycan chain size. and
presence of keratan sulfate were typical of proteoglycans from nonarthritic and osteoarthritic femoral head
and knee cartilage (48-52). The C-4-S:C-6-S ratio,
however, was not (48). We have found, typically, that
C-6-S constitutes >60% of the chondroitin sulfate of
osteofemoral cartilage and ostcochondrophytic spurs
(10). In that sense, the features in the cartilage sample
from this affected family member resembled putative
early changes in cartilage composition reported by
others (53).
The smaller size of the A l D l fraction from
sample 11-5 (Kav0.4) was similar to that obtained when
osteoarthritic knee cartilage homogenates were pretreated with p-APMA, an activator of metalloproteinases (54). Combined with the failure of the AlDl
fraction from sample 11-5 to form aggregates with
hyaluronic acid, these results are consistent with modifications in or around the hyaluronic acid-binding
region and chondroitin sulfate attachment domains of
the proteoglycan core protein. Although a smaller,
KATZENSTEIN ET A L
highdensity, newly synthesized proteoglycan (Kav
0.39 on CL-2B) was found in cartilage obtained from a
patient with pseudoachondroplasia ( 4 9 , it did not
differ from the normal cartilage used in that study as a
control. Thus, the reduced AID1 size of sample 11-5
compared with 11-9 and 11-7 may also reflect inherent
proteoglycan polydispersity among cartilage samples.
The disorder in the family described here is
probably due to functionally inadequate articular cartilage ( 5 5 ) . Such a disorder could involve a defect in
integration of individual extracellular matrix components, or a specific change in any one of them. A single
proteoglycan structural alteration does not apparently
underlie the disorder in this family. Linkage of this
disorder to the COL2AI region of the type I1 procollagen gene has been demonstrated (56). An abnormality of type I1 collagen or cartilage proteins would
account for the early onset of articular degeneration
and the abnormal epiphyseal formation seen in the
spine.
ACKNOWLEDGMENTS
We thank Robert Papay for his excellent technical
assistance, and Drs. Joaquin Uy and Stephen Kiechel for
assistance in obtaining the radiographs and pathologic specimens.
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