Download Evidence for a role of the genomic region of the gene encoding for

ARTHRITIS & RHEUMATISM
Vol. 52, No. 5, May 2005, pp 1437–1442
DOI 10.1002/art.21020
© 2005, American College of Rheumatology
Evidence for a Role of the Genomic Region of the
Gene Encoding for the ␣1 Chain of
Type IX Collagen (COL9A1) in Hip Osteoarthritis
A Population-Based Study
B. Z. Alizadeh,1 O. T. Njajou,1 C. Bijkerk,1 I. Meulenbelt,2 S. C. De Wildt,2 A. Hofman,1
H. A. P. Pols,1 P. E. Slagboom,2 and C. M. van Duijn1
Results. Affected sibpairs with radiographic hip
OA shared alleles identical by descent at markers 8B2
and 12B1 significantly more often than expected
(mean ⴞ SD 0.66 ⴞ 0.07 and 0.65 ⴞ 0.08, respectively;
P < 0.05). No excess sharing for radiographic OA was
observed at other joint sites. When comparing the
frequency of marker 8B2 and 12B1 alleles in subjects
with radiographic OA and controls, the frequency of
8B2 alleles in subjects with radiographic OA differed
significantly(P ⴝ 0.01) from that in controls.
Conclusion. Our data suggest that susceptibility
for hip OA is conferred within or close to the COL9A1
gene in linkage disequilibrium with the COL9A1 5098B2 marker.
Objective. Type IX collagen proteoglycan is an
important protein in collagen networks and has been
implicated in hip osteoarthritis (OA). We studied 2
COL9A1 markers (509-8B2 and 509-12B1) in relation to
radiographic OA, within the framework of the Rotterdam Study, a population-based study of 7,983 subjects
ages 55 years and older.
Methods. We used 2 different designs, as follows:
1) a linkage study of 83 probands with multiple joints
affected with radiographic OA and their 221 siblings,
yielding 445 sibpairs who participated in the study, and
2) an association study in a series of 71 patients with
radiographic hip OA and 269 controls without radiographic OA. All subjects were characterized for the 2
COL9A1 markers, 509-8B2 and 509-12B1. The mean test
was used to assess the proportion of alleles shared in
concordantly affected and unaffected sibpairs. The chisquare test was used to compare the allele distributions
in patients and controls.
Osteoarthritis (OA) is a complex disorder that is
common worldwide (1) and is the leading cause of
disability and pain among the elderly (2). Results of
family-based and candidate gene studies demonstrated a
clear genetic component, particularly for early-onset
primary OA (3,4). One of the main pathologic characteristics of OA is degradation of hyaline cartilage. The
collagen fibril network is one of the main components of
hyaline cartilage, and this network maintains the integrity of hyaline cartilage and prevents its degradation (5).
Type IX collagen links type II collagen–containing
fibrils to the rest of the cartilage matrix and thus plays a
role in cartilage integrity (6). Type IX collagen is
composed of 3 genetically distinct ␣ polypeptide chains,
i.e., ␣1(IX), ␣2(IX), and ␣3(IX), which are encoded by
COL9A1, COL9A2, and COL9A3, respectively (6).
A deficiency of polypeptide chain ␣1(IX) has
been shown to lead to functional abnormalities in type
Supported by the Erasmus Medical Center, Erasmus University, Rotterdam, The Netherlands Organization for Scientific Research, The Netherlands Organization for Health Research and Development, the Research Institute for Diseases in the Elderly, the
Ministry of Education, Culture and Science, the Ministry of Health,
Welfare and Sports, the European Commission, and the Municipality
of Rotterdam.
1
B. Z. Alizadeh, MD, DSc, O. T. Njajou, PhD, C. Bijkerk,
PhD, A. Hofman, MD, PhD, H. A. P. Pols, MD, PhD, C. M. van Duijn,
PhD: Erasmus Medical Center, Rotterdam, The Netherlands; 2I.
Meulenbelt, PhD, S. C. De Wildt, MD, P. E. Slagboom, PhD: Leiden
University Medical Center, Leiden, The Netherlands.
Address correspondence and reprint requests to C. M. van
Duijn, PhD, Department of Epidemiology and Biostatistics, Erasmus
Medical Center, PO Box 1738, 3000 DR Rotterdam, The Netherlands.
E-mail: [email protected].
Submitted for publication August 19, 2004; accepted in
revised form February 1, 2005.
1437
1438
ALIZADEH ET AL
Figure 1. Flow chart showing participation of probands and siblings in the linkage
study and cases and controls in the association study. ROA ⫽ radiographic
osteoarthritis.
IX collagen fibrils, which lead to instability of hyaline
cartilage (7). This observation suggests that mutations in
the COL9A1 gene that lead to a nonfunctional ␣1(IX)
polypeptide may be implicated in OA. Some evidence
supports this view. OA develops in transgenic mice that
express a nonfunctional protein as well as in knockout
mice (8), which suggests that COL9A1 may be a candidate gene for OA in humans. Some evidence suggests
that COL9A1 is involved in hip OA (9,10). Studies of
affected sibpairs demonstrated linkage of COL9A1 to
severe forms of hip OA in women (9,10). However,
association studies failed to show any relationship. Another question that remains to be answered is whether
COL9A1 is involved in joints other than the hip.
In the present study, we investigated 2 polymorphisms in the COL9A1 gene (509-8B2 and 12B1) in
relation to radiographic OA at different joint sites, in 2
independent studies: a sibpair study including 445 sibpairs with radiographic hip, knee, or hand OA or spinal
disk degeneration, and an association study of 71 patients with radiographic hip OA and 269 controls without radiographic OA.
PATIENTS AND METHODS
Study population. The present study was conducted
within the framework of the Rotterdam Study, a populationbased cohort study of chronic diseases in 7,983 subjects ages 55
years and older (11). The medical ethics committee of the
Erasmus Medical Center approved the study, and written
informed consent was obtained from all participants. Baseline
examinations took place between 1990 and 1993 by means of a
structured interview in which a standardized questionnaire was
used. Figure 1 presents a flow chart of participation of the
study population. From the total cohort of subjects ages 55 to
65 years (n ⫽ 2,593), a random cohort including 944 noninstitutionalized persons was drawn, and subjects were scored for
radiographic OA of the hip, knee, or hand and for disk
degeneration of the spine.
Radiographic examination. Radiographs of the hip,
knee, and hand joints of participants of the Rotterdam Study
and their siblings were scored for the presence of radiographic
OA. Radiographs of the spine were evaluated for the presence
of disk degeneration using the standard Kellgren/Lawrence
(K/L) grading system (12). A diagnosis of radiographic OA was
considered for any joint with a K/L score of ⱖ2. Two independent readers scored all radiographs. After assessment of each
set of ⬃150 radiographs, the scores of the 2 readers were
evaluated. Whenever the scores differed by ⱖ2 points, or when
the score given by one reader was 1 and the score given by the
other reader was 2, a consensus was agreed upon. All radiographs were scored before genotyping, with the readers
blinded to clinical data.
Linkage study. For the linkage analysis, probands were
derived from the random cohort. Persons who had radiographic OA at ⱖ2 joint sites of the 4 joint groups (i.e., hips,
knees, hands, or spine) were selected as probands. Individuals
with both radiographic hand OA and disk degeneration of the
spine, which was the most common combination observed, also
had to have Heberden’s nodes in order to be included as
probands. This criterion was applied to maximize the proba-
THE COL9A1 GENE AND OSTEOARTHRITIS
1439
Table 1. Characteristics of the study population*
Linkage study
Association study
Characteristic
Probands
Siblings
Cases
Controls
Number
Age, mean ⫾ SD years
Women, %
BMI, mean ⫾ SD kg/m2
BMD, mean ⫾ SD cg/cm2
83
60.90 ⫾ 2.71†
69.22
27.36 ⫾ 4.23
0.91 ⫾ 0.13†
221
65.80 ⫾ 8.02
50.25
26.71 ⫾ 4.01
0.86 ⫾ 0.14
71
60.76 ⫾ 2.43‡
41.66
26.45 ⫾ 3.46
0.90 ⫾ 0.13
269
59.71 ⫾ 2.84
49.07
25.65 ⫾ 3.26
0.87 ⫾ 0.13
* BMI ⫽ body mass index; BMD ⫽ bone mineral density.
† P ⬍ 0.05 versus siblings.
‡ P ⱕ 0.05 versus controls.
bility of a subject having a genetic form of radiographic OA. A
total of 221 probands (response rate 88%) were willing to
contribute to the study, yielding 708 live-borne siblings (Figure
1). Four hundred fifty siblings of 101 probands were not
eligible for the study due to sibling death, refusal, emigration,
disease, or nonresponse. In total, 258 siblings and 120 probands derived from 120 pedigrees were included in the study
(Figure 1). The siblings were examined at the research center,
using the same protocol and methods that were used to
examine participants in the random cohort.
Association study. Within the random cohort, 72 persons with radiographic hip OA were genotyped. The 269
persons who did not have radiographic OA of the hip, knee, or
hand joints were selected as controls (Figure 1).
Genotyping for COL9A1 markers 509-8B2 and 12B1.
Participants were genotyped for COL9A1 509-8B2 and 12B1
short tandem repeat polymorphisms (STRPs) according to the
protocol described by Warman and colleagues (13). In the
sibpair study, genotyping was successful for 85 probands and
241 siblings. In the association study, genotyping was successful
for 71 cases and all controls, except for 8B2 in 1 control subject
(Figure 1).
Statistical analysis. Linkage study. Familial relationships between siblings were confirmed using genealogic data.
Six half-sibs were excluded from the analysis (Figure 1).
Mendelian inconsistency in pedigrees was checked using the
MARKERINFO module. Given the sibling genotypes in nuclear families, this module reconstructs sibling genotype sets
and thereafter the parental genotypes. Pedigrees with Mendelian inconsistency are identified whenever 1 or 2 alleles of the
studied markers in any sibling do not match with the family
genotype sets. Two probands and 14 full siblings who belonged
to 4 pedigrees and had Mendelian inconsistencies in 1 or both
of the 2 markers were excluded from the analysis (Figure 1).
The remaining 83 probands and 221 siblings (belonging to 100
pedigrees) yielded a total of 445 sibpairs.
Sibpairs were classified as follows: concordantly affected (both siblings had radiographic OA), concordantly
unaffected (neither sibling had radiographic OA), and discordant (1 sibling was affected, and the other sibling was unaffected at the studied joint site). We used the mean test, which
is a powerful test for additive inheritance, to compare the
average proportion of alleles shared identical by descent (IBD)
with the expected value of 0.5 (14). On average, sibpairs share
half of alleles of a given locus IBD. Concordantly affected
sibpairs should share ⬎50% of alleles IBD at COL9A1 if this
locus is linked to radiographic OA. The analysis was adjusted
for age and sex, which are the 2 major determinants of OA.
Sibpair data were analyzed using the program Statistical
Analysis for Genetic Epidemiology, version 4.4 (New Orleans:
Louisiana State University Medical Center).
Association study. Allele and genotype frequencies for
the 8B2 and 12B1 markers were estimated by counting alleles
and estimating the sample proportion. Allele and genotype
proportions were tested for Hardy-Weinberg equilibrium. The
chi-square test was used to compare allele frequencies between
subjects with radiographic OA and controls.
RESULTS
Tables 1 and 2 show the characteristics of the
study population. Compared with probands, the mean
age of siblings was significantly higher (P ⬍ 0.001), and
the mean bone mineral density was significantly lower
(P ⬍ 0.05). In the final analysis, each pedigree contributed, on average, 4.5 (range 1–36) sibpairs to the linkage
study (Table 2). Among the probands, 33% had radiographic hip OA, 78% had radiographic knee OA, 78%
had radiographic OA of the hand joints, and 64% had
spinal disk degeneration. Among the siblings, 7% had
radiographic hip OA, 19% had radiographic knee OA,
75% had radiographic OA of the hand joints, and 79%
Table 2.
Frequency of families by the number of sibpairs
No. of
sibpairs
No. of
families
No. of
sibpairs contributed
1
3
6
10
11
15
21
28
36
54
20
12
4
1
3
3
1
2
54
60
72
40
11
45
63
28
72
1440
ALIZADEH ET AL
Table 3. Frequency of COL9A1 509-8B2 and 509-12B1 alleles shared IBD according to site of radiographic OA*
COL9A1 marker/sibpair
phenotype
509-8B2
Concordantly
Concordantly
509-12B1
Concordantly
Concordantly
Joint site with radiographic OA
Hip
Knee
Hand
Spine
affected
unaffected
0.66 ⫾ 0.07† (11)
0.50 ⫾ 0.02 (327)
0.49 ⫾ 0.05 (41)
0.51 ⫾ 0.02 (205)
0.51 ⫾ 0.02 (251)
0.52 ⫾ 0.06 (26)
0.52 ⫾ 0.02 (243)
0.54 ⫾ 0.06 (39)
affected
unaffected
0.65 ⫾ 0.08† (11)
0.49 ⫾ 0.02 (327)
0.50 ⫾ 0.05 (30)
0.49 ⫾ 0.02 (212)
0.50 ⫾ 0.02 (251)
0.52 ⫾ 0.07 (26)
0.50 ⫾ 0.02 (243)
0.50 ⫾ 0.06 (39)
* Values are the mean ⫾ SD (n). Data for discordant sibpairs are not presented.
† P ⬍ 0.05, indicating a significant increase in the proportion of alleles shared identical by descent (IBD), based on the expected
value of 0.5.
had spinal disk degeneration. In the association study,
no significant differences in sex, body mass index, or
bone mineral density between patients with radiographic
hip OA and controls were observed. Patients were
slightly (1 year) older than controls (P ⫽ 0.05). The
allele and genotype proportions were in HardyWeinberg equilibrium.
Table 3 shows the results of the linkage analysis
in affected and unaffected sibpairs. Affected sibpairs
with radiographic hip OA (n ⫽ 11) had significantly
increased allele sharing (P ⬍ 0.05) in the COL9A1 8B2
(mean ⫾ SD 0.66 ⫾ 0.07) and 12B1 (0.65 ⫾ 0.08)
markers shared IBD. The 11 sibpairs with radiographic
hip OA belonged to 9 families consisting of a total of 19
siblings (1 family contributed 3 affected siblings).
Among the sibpairs with radiographic OA at the hip
Table 4. Frequency of COL9A1 509-8B2 and 12B1 alleles according
to the presence of radiographic hip OA*
Radiographic hip OA
COL9A1 marker 509-8B2
Allele 5
Allele 6
Allele 7
Allele 8
Allele 9
Other†
COL9A1 marker 509-12B1
Allele 2
Allele 4
Allele 5
Allele 6
Allele 7
Allele 8
Other†
Present
Absent
50 (0.35)
27 (0.19)
14 (0.10)
10 (0.07)
20 (0.14)
21 (0.15)
231 (0.43)
140 (0.26)
29 (0.05)
22 (0.04)
62 (0.12)
52 (0.10)
14 (0.10)
50 (0.35)
27 (0.19)
15 (0.11)
5 (0.03)
23 (0.16)
8 (0.06)
96 (0.18)
172 (0.32)
67 (0.12)
44 (0.08)
33 (0.06)
92 (0.17)
34 (0.06)
P
0.01
joints, 3 pairs were homozygous for the COL9A1 8B2
allele 5/allele 6 genotype (i.e., both siblings had the 5/6
genotype), 2 pairs were homozygous for 5/5, and 1 pair
was homozygous for 4/6. The remaining sibling pairs
were heterozygous for 8B2 (i.e., 2 pairs had a 5/5 and 5/6
genotype set, 1 pair had 5/2 and 5/6, 1 pair had 5/4 and
9/4, and 1 pair had 5/6 and 9/6). When considering the
12B1 marker, 2 sibpairs were homozygous for the 4/6
genotype, 1 pair for the 4/8 genotype, and 1 pair for the
4/4 genotype. The rest of the sibpairs were heterozygous
for 12B1 (i.e., 2 sibpairs had 4/4 and 4/8 genotype sets, 2
pairs had 4/6 and 5/6, 1 pair had 4/8 and 8/8, 1 pair had
4/4 and 4/6, and 1 pair had the 3/6 and 3/5 genotype set).
No significant differences for the other joints were
observed. The numbers of alleles shared in affected and
unaffected sibpairs were extremely similar, suggesting
that there is no evidence for a role of COL9A1 in
radiographic OA of other joints.
The frequency of the 8B2 or 12B1 alleles was not
significantly different between controls without radiographic OA and the total population. Table 4 shows the
frequency of the 8B2 and 12B1 alleles according to the
presence of radiographic hip OA. The frequency of 8B2
alleles differed significantly (P ⫽ 0.01) between subjects
with radiographic hip OA and controls without radiographic OA. The frequency of 12B1 alleles was not
significantly different between subjects with radiographic hip OA and controls without radiographic OA.
0.10
* Values are the number (frequency). OA ⫽ osteoarthritis.
† Alleles with a frequency of ⬍0.05.
DISCUSSION
In this population-based study, we observed that
affected sibpairs with radiographic hip OA shared a
significantly higher number of alleles IBD at 2 markers
at COL9A1 (the 8B2 and 12B1 STRPs). Furthermore, in
the association study, we observed that the 8B2 marker
was significantly associated with radiographic hip OA.
THE COL9A1 GENE AND OSTEOARTHRITIS
The positive linkage of the COL9A1 locus in our
sibpairs confirmed earlier findings of linkage in female
sibpairs with hip OA (9,10), although we could not
stratify for sex because the numbers were too low for a
meaningful statistical analysis. Despite the fact that in
our study the number of sibpairs was small, the excess of
sharing was statistically significant. Also, in our association study, we observed a significant relationship between the COL9A1 8B2 marker and radiographic hip
OA. The relevance of our finding is not completely clear,
because the significance was marginal, various alleles
together contribute to the association, and no association with the nearby 12B1 marker was observed. One
previous association study on the relationship between
the COL9A1 markers 8B2 and 12B1 and radiographic
OA has been reported. In that study, no association of
8B2 or 12B1 with severe hip OA was observed in 146
women selected from families with OA (9).
Two important points should be considered when
interpreting the difference between our findings and
those of Loughlin and colleagues (9). First, in contrast to
a linkage study, the relationship in an association study
can be easily missed because the marker used in the 2
studies is not very powerful for association analysis due
to a large number of rare alleles. The genetic information content of a marker depends on the heterozygosity
index, a function of marker allele frequencies, as well as
the location of the marker on genome map and the
functional effect of the marker variants. In the present
study, the polymorphic nature of the studied markers
resulted in multiple strata of cases and controls, thus
demolishing the power of the association study. Second,
although we hypothesize that the COL9A1 locus contributes to OA susceptibility, it is likely that the 8B2
marker is not causally related to radiographic OA.
Marker 8B2 is located in COL9A1 intron 4, which
resides 17.7 kb downstream of the start of a 65-kg
haplotype block within COL9A1. This haplotype block is
encompassed between intron 1 (⫺501) and intron 34
(⫹32) (9). Thus, marker 8B2 may be in strong linkage
disequilibrium with other COL9A1 mutations. Marker
12B1 is located 14.3 kb upstream of exon 1 and resides
outside the COL9A1 haplotype block. Furthermore,
COL9A1 mapped to a region where other fibrilassociated collagen with interrupted triple helix
(FACIT)–like collagens (e.g., COL19A1) (15) have been
also mapped. Although the association of marker 8B2
with hip OA might be explained by linkage disequilibrium with adjacent loci, which suggests that an OA
susceptibility locus may map near to the COL9A1 locus,
several experimental studies support the role of the
1441
COL9A1 locus in OA (7,8). Those studies (7,8) showed
that early-onset OA developed in COL9A1–knockout
mice.
Taken together with earlier findings, our results
suggest that OA susceptibility may map within or near
the COL9A1 gene, with 509-8B2 being simply a marker
for this. Our sibpair data showed no evidence for a role
of COL9A1 in other forms of OA. Further studies are
necessary to identify the underlying mutation in
COL9A1 or within a nearby OA susceptibility locus.
ACKNOWLEDGMENTS
We greatly appreciate the contributions to the Rotterdam Study from the general practitioners and pharmacists of
the Ommoord district.
REFERENCES
1. Lawrence RC, Helmick CG, Arnett FC, Deyo RA, Felson DT,
Giannini EH, et al. Estimates of the prevalence of arthritis and
selected musculoskeletal disorders in the United States. Arthritis
Rheum 1998;41:778–99.
2. Odding E, Valkenburg HA, Algra D, Vandenouweland FA,
Grobbee DE, Hofman A. Associations of radiological osteoarthritis of the hip and knee with locomotor disability in the Rotterdam
Study. Ann Rheum Dis 1998;57:203–8.
3. MacGregor AJ, Antoniades L, Matson M, Andrew T, Spector TD.
The genetic contribution to radiographic hip osteoarthritis in
women: results of a classic twin study. Arthritis Rheum 2000;43:
2410–6.
4. Felson DT, Couropmitree NN, Chaisson CE, Hannan MT, Zhang
Y, McAlindon TE, et al. Evidence for a Mendelian gene in a
segregation analysis of generalized radiographic osteoarthritis: the
Framingham Study. Arthritis Rheum 1998;41:1064–71.
5. Mendler M, Eich-Bender SG, Vaughan L, Winterhalter KH,
Bruckner P. Cartilage contains mixed fibrils of collagen types II,
IX, and XI. J Cell Biol 1989;108:191–7.
6. Diab M, Wu JJ, Eyre DR. Collagen type IX from human cartilage:
a structural profile of intermolecular cross-linking sites. Biochem J
1996;314:327–32.
7. Hagg R, Hedbom E, Mollers U, Aszodi A, Fassler R, Bruckner P.
Absence of the ␣1(IX) chain leads to a functional knock-out of the
entire collagen IX protein in mice. J Biol Chem 1997;272:20650–4.
8. Fassler R, Schnegelsberg PN, Dausman J, Shinya T, Muragaki Y,
McCarthy MT, et al. Mice lacking ␣1 (IX) collagen develop
noninflammatory degenerative joint disease. Proc Natl Acad Sci
U S A 1994;91:5070–4.
9. Loughlin J, Mustafa Z, Dowling B, Southam L, Marcelline L,
Raina SS, et al. Finer linkage mapping of a primary hip osteoarthritis susceptibility locus on chromosome 6. Eur J Hum Genet
2002;10:562–8.
10. Mustafa Z, Chapman K, Irven C, Carr AJ, Clipsham K, Chitnavis
J, et al. Linkage analysis of candidate genes as susceptibility loci
for osteoarthritis-suggestive linkage of COL9A1 to female hip
osteoarthritis. Rheumatology (Oxford) 2000;39:299–306.
11. Hofman A, Grobbee DE, de Jong PT, van den Ouweland FA.
Determinants of disease and disability in the elderly: the Rotterdam Elderly Study. Eur J Epidemiol 1991;7:403–22.
1442
12. Kellgren JH, Lawrence JS, editors. The epidemiology of chronic
rheumatism, atlas of standard radiographs. Oxford: Blackwell
Scientific; 1963.
13. Warman ML, Tiller GE, Polumbo PA, Seldin MF, Rochelle JM,
Knoll JH, et al. Physical and linkage mapping of the human and
murine genes for the ␣1 chain of type IX collagen (COL9A1).
Genomics 1993;17:694–8.
ALIZADEH ET AL
14. Guo X, Elston RC. Two-stage global search designs for linkage
analysis I: use of the mean statistic for affected sib pairs. Genet
Epidemiol 2000;18:97–110.
15. Yoshioka H, Zhang H, Ramirez F, Mattei MG, Moradi-Ameli M,
van der Rest M, et al. Synteny between the loci for a novel
FACIT-like collagen locus (D6S228E) and ␣1 (IX) collagen
(COL9A1) on 6q12-q14 in humans. Genomics 1992;13:884–6.