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Mar 7, 2010
Dear Editors
On behalf of my co-authors, I am submitting the manuscript entitled
“Linkage of three polymorphisms on chromosome 20p12 to ossification
of the posterior longitudinal ligament of spine and its severity in Han
Chinese patients” for possible publication in your journal.
We certify that we have participated sufficiently in the work to take
public responsibility for the appropriateness of the experimental design
and method, and the collection, analysis, and interpretation of the data.
We have reviewed the final version of the manuscript and approve it for
publication. To the best of our knowledge and belief, this manuscript has
not been published in whole or in part nor is it being considered for
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Best Regards
Yours Sincerely,
Liang Yan
Linkage of three polymorphisms on chromosome 20p12 to
ossification of the posterior longitudinal ligament of spine and its
severity in Han Chinese patients
YAN Liang1, ZHAO Wei-guang1, LI Jin-jun2, YANG Hui3, WANG Hao1and LIN Xin1
1Department
of Orthopaedics, Beijing Tiantan Hospital, Capital Medical University (CMU),
Beijing 100050, China
2Department of Orthopaedics, Beijing Friendship Hospital, Capital Medical University (CMU),
Beijing, 100050, China
3Beijing Institute for Neuroscience, Capital Medical University (CMU), Beijing Center of Neural
Regeneration and Repair, Key Laboratory for Neurodegenerative Disease of the Ministry of
Education, Beijing 100069, China
First Author: YAN Liang
Beijing Tiantan Hospital
Capital Medical University (CMU)
Beijing 100050, China
E-mail: [email protected]
Corresponding Author: LIN Xin, MD, PhD
Beijing Tiantan Hospital
Capital Medical University (CMU)
Beijing 100050, China
Tel: +86 10 67098437
E-mail address: [email protected]
Co-Corresponding Author:  WANG Hao, MD, PhD
Beijing Tiantan Hospital
Capital Medical University (CMU)
Beijing 100050, China
Tel: +86 10 67098437
E-mail address: [email protected]
Acknowledgments
The authors thank the DNA donors for making this study possible.
This research was supported by grants from the National Nature Science Foundation of China (No.
30872599), and the Beijing Nature Science Foundation of China (No. 7092028).
Keywords ossification of the posterior longitudinal ligament; single
nucleotide polymorphisms; susceptibility; polymerase chain reaction
Background Ossification of the posterior longitudinal ligament (OPLL)
is characterized by replacement of ligamentous tissue with new ectopic
bone formation, and has a strong genetic background. Because of the
abnormal bone metabolic features and the strong genetic component,
osteoporosis is a related disorder with OPLL. Three polymorphisms on
chromosome 20p12 were identified associated with the risk of
osteoporosis and osteoporotic fracture. The rs996544 (C/T) “TT” and
rs965291 (G/A) “AA” genotypes conferred higher risks for vertebral and
hip fractures. The osteoporosis haplotype is defined by two
polymorphisms, rs1116867 (A) and D35548 (T). However, it remains
unknown whether these three polymorphisms predispose to an increased
frequency and severity of OPLL in Han Chinese patients.
Methods A total of 420 OPLL patients and 506 age- and sex-matched
controls were studied. Three single nucleotide polymorphisms (SNPs),
rs996544 (C/T), rs965291 (G/A) and rs1116867 (A/G), were analyzed by
direct sequencing. Associations between these SNPs with the occurrence
and extent of OPLL were statistically evaluated.
Results There was no significant association between the rs996544 (C/T)
polymorphism and the prevalence of OPLL. The rs1116867 (A/G)
polymorphism “AG” genotype was associated with the occurrence of
OPLL. The rs1116867 (A/G) polymorphism “G” allele was associated
with the occurrence of OPLL, but not with the extent of OPLL. The
rs965291 (G/A) polymorphism in female patients was statistically
different between cases and controls (P<0.05). The rs965291 (G/A)
polymorphism “A” allele was associated with the occurrence of OPLL in
female patients. For the rs965291 (G/A) polymorphism, patients with the
“A” allele (genotype, “AG” or “AA”) showed a significantly greater
number of ossified cervical vertebrae than those without the “A” allele
(genotype, “GG” P<0.05), particularly in female patients.
Conclusions The rs1116867 (A/G) and rs965291 (G/A) polymorphisms
on chromosome 20p12 are associated with the occurrence and the extent
of OPLL, at least in Han Chinese subjects. Our data should advance our
understanding of the molecular etiology of OPLL and may guide
approaches to prevent the onset of OPLL.
Ossification of the posterior longitudinal ligament (OPLL) is
pathological ectopic ossification of this ligament at the cervical and
thoracic spine, causing myeloradiculopathy as a result of chronic pressure
on the spinal cord and nerve roots [1, 2]. OPLL of the spine was first
reported in Japan and has even been called “a Japanese disease,” because
many more cases have been reported in Japanese and other Asian
populations than in non-Asiatic populations. The prevalence of OPLL in
Japan is 1.9–4.3% of the general population aged >30 years old [3, 4].
Among Chinese individuals, the prevalence of OPLL ranges from 0.44 to
8.92%, with a mean prevalence of 3.08% [5]. Many clinical studies
conducted in Japan have suggested that OPLL is a multifactorial disease
in which complex genetic and environmental factors interact [1, 6–9].
Studies have shown that OPLL is prevalent with a high concordance in
twins and families, and is associated with human leukocyte antigen
haplotypes [10, 11]. Because genetic factors appear to play a crucial role
in OPLL, molecular genetic studies are important to better understand the
molecular etiologies of OPLL and will lead to the development of new
therapeutic approaches. On the other hand, osteoporosis is a bone
metabolic disorder that has been extensively investigated [12-15].
Osteoporosis is a common disease that is characterized by a reduction in
bone mass, microarchitectural deterioration of bone tissue and an
increased risk of fracture [16-18]. Recently, a compound osteoporosis
phenotype was reported to be linked to chromosome 20p12. Furthermore,
three single nucleotide polymorphisms (SNPs) of rs996544 (C/T),
rs965291 (G/A) and rs1116867 (A/G) were found to be associated with
osteoporosis and increase the risk of osteoporosis and osteoporotic
fracture [19-21]. Because abnormal bone metabolic features and strong
genetic components of OPLL and osteoporosis have been reported, but
with limited information about the extent of the overlap, OPLL could be
widespread among individuals with osteoporosis.
The purpose of this study was to investigate the associations between
three SNPs [rs996544 (C/T), rs965291 (G/A), rs1116867 (A/G)] with
susceptibility to OPLL of the spine and its severity in Han Chinese
patients. To our knowledge, we are the first to report an association
between these three SNPs and OPLL.
METHODS
Disease criteria and subjects
This study was approved by the ethical committee at Beijing Tiantan
Hospital Capital Medical University. Informed consent was obtained
from all participants in this study.
Overall, 420 patients with OPLL and 506 age- and sex-matched
healthy control subjects without OPLL participated in this study. All the
participants are from third grade class A hospitals in Beijing and live in
the Beijing region. The characteristics of the OPLL patients and controls
are shown in Table 1. The diagnosis of OPLL was based on radiologic
findings including radiographs, computed tomography (CT) and magnetic
resonance imaging (MRI) of the cervical spine according to the criteria
reported by Tsuyama [22]. The severity of OPLL was determined based
on the number of ossified cervical vertebrae on lateral radiograph films
and CT images. Patients were also stratified according to the extent of
ossification. The patients with ankylosing spondylitis and metabolic
diseases
associated
rickets/osteomalacia,
with
OPLL,
osteoporosis,
such
diffuse
as
hypophosphate
idiopathic
skeletal
hyperostosis (DISH) and hyperparathyroidism were excluded according
to radiographic and biochemical examinations. Women who had taken
drugs such as estrogen, progesterone, glucocorticoids, bisphosphonates,
alfacalcidol and calcitriol were also excluded.
Genomic DNA analysis
SNPs for genotyping were obtained from the two public databases:
NCBI dbSNP (http://www.ncbi.nlm.nib.gov/SNP/) and Ensembl Genome
(http://www.ensembl.org/index.html). Venous blood (5 ml) was collected
in tubes containing EDTA (50 mmol/l of disodium salt). The blood
samples were stored at –20°C until use for genomic DNA extraction and
SNP genotyping. Genomic DNA was isolated using Wizard Genomic
DNA Purification Kits (Promega Corporation, USA) and polymerase
chain reaction (PCR) was performed with a standard protocol using sense
and antisense primers for each factor (Table 2). Reactions were
performed in a total volume of 50 μl containing 0.5 μg of genomic DNA;
1 μl of each primer (20 μM); 8 μl of dCTP, dTTP, dGTP and dATP
mixture (each 2.5 μM); 0.5 μl TAKARA LA Taq DNA polymerase
(TakaRa Biotechnology Dalian Co., Ltd); 5 μl MgCl2 (25 mM); 5 μl 10×
LA PCR Buffer Ⅱ (Mg2+ Free); and double distilled H2O (ddH2O) to 50
μl. The PCR products (including the three SNPs) were analyzed by direct
sequencing using BigDye Terminator cycle sequencing on an ABI
3730XL POP7 DNA sequencing analysis 5.2 (Applied Biosystems).
Statistical analysis
The two groups were compared using Student unpaired t test.
Hardy-Weinberg equilibrium and the genotypic and allelic distribution
were evaluated using χ2 tests. The nonparametric Mann-Whitney U test
was used to compare the number of ossified cervical vertebrae between
the two groups. Analysis of variance was used to compare group means.
A P value less than 0.05 was considered statistically significant.
RESULTS
There were no significant differences in age, height, body weight, or
personal history between the case and control subjects. Within the OPLL
cases, there were significantly more men than women compared with the
control group. The distributions of genotype and allele types of the three
SNPs among OPLL cases and controls are shown in Table 3. The
genotype distributions in the case and control subjects were in
Hardy-Weinberg equilibrium, which suggests that the study population
was genetically homogenous, and that selection bias was avoided.
The number of subjects in the total study population with the
rs996544 (C/T) “CC,” “CT” and “TT” genotypes was 779, 147 and 0,
respectively, with frequencies of 84, 16 and 0%, respectively. The number
of subjects with the rs965291 (G/A) “AA,” “AG” and “GG” genotypes
was 67, 320 and 539, with frequencies of 7, 35 and 58%, respectively.
The number of subjects with the rs1116867 (A/G) “AA,” “AG,” “GG”
genotypes was 228, 464 and 234, with frequencies of 24, 50 and 26%,
respectively.
Comparing the distributions of the genotype of these three SNPs,
there was a statistical difference among the rs1116867 (A/G) “AA,”
“AG” and “GG” genotypes (P<0.05) (Table 3). The “AG” genotype was
associated with the occurrence of OPLL (Figure 1). However, no
association was found between male and female cases in terms of the
rs1116867 (A/G) genotypes (data was not shown). Furthermore, there
was no statistical difference among the rs965291 (G/A) “AA,” “AG,” and
“GG” genotypes (P=0.898). In addition, the rs965291 (G/A) in the male
(P=0.779) and female (P=0.742) cases was not significantly associated
with controls. The rs965291 (G/A) polymorphism in females was
statistically different between cases and controls (P<0.05) (Figure 2),
whereas the difference in males was not significant (P=0.099) (Table 4).
These results suggest that the genetic background of this polymorphism
affects the initiation of OPLL more strongly in women than in men. There
was no statistical difference among the rs996544 (C/T) “CC,” “CT” and
“TT” genotypes (P=0.548).
Comparing the allelic distributions of the three SNPs, there was a
significant association between the rs1116867 (A/G) polymorphism and
the occurrence of OPLL in the cervical spine. In addition, the “G” allele
was significantly more frequent in OPLL cases than in control subjects
(P<0.05). Among females with OPLL, the rs965291 (G/A) “A” allele was
significantly associated with the occurrence of OPLL (P<0.05) (Table 5).
To study the contribution of these polymorphisms to the severity or
promotion of OPLL, we investigated the association between the three
SNPs with the number of ossified cervical vertebrae in cases with OPLL.
Regarding the rs996544 (C/T) polymorphism, patients with the “T” allele
(genotype, “CT” or “TT”) showed no increase in the number of ossified
cervical vertebrae than those without the “T” allele (genotype, “CC”
P=0.362). Similarly, for the rs1116867 (A/G) polymorphism, patients
with the “G” allele (genotype, “AG” or “GG”) showed no increase in the
number of ossified cervical vertebrae than those without the “G” allele
(genotype, “AA” P=0.423). However, for the rs965291 (G/A)
polymorphism, patients with the “A” allele (genotype, “AG” or “AA”)
had a significantly greater number of ossified cervical vertebrae
compared with patients without the “A” allele (genotype, “GG” P<0.05).
Female patients with the “A” allele (genotype, “AG” or “AA”) had a
greater number of ossified vertebrae than those without the “A” allele
(genotype, “GG” P<0.05). This finding was not observed among males
(Figure 3). The present results indicate that the “A” allele promotes
ectopic ossification in the cervical spine among patients with OPLL,
particularly among females.
DISCUSSION
OPLL is characterized by replacement of ligamentous tissue by
ectopic new bone formation in the spine and often causes narrowing of
the spinal canal. OPLL is a common disorder among elderly populations
and is the leading cause of spinal myelopathy in Asian populations.
Although various systemic and local factors such as abnormal
carbohydrate or calcium metabolism, aging and hormonal disturbances
have been suggested as causes of OPLL, the etiology of OPLL is not fully
understood. However, the possible involvement of genetic factors has
been proposed based on the high incidence of OPLL in families and
among twins [22, 23]. According to a study of probands and their
relatives within the second degree of consanguinity, OPLL was present in
23% of relatives, exhibiting autosomal dominant inheritance. The rarity
of the disease among white populations of Europe and the United States,
despite the relatively high prevalence among Japanese populations, also
suggests a genetic predisposition. Therefore, the development of OPLL
may be affected by an interaction between genetic and environmental
factors. Previous studies have shown that restriction fragment length
polymorphisms of the estrogen receptor and interleukin-1 (IL-1) genes,
and
SNPs
of
pyrophosphatase
the
transforming
(NPPS),
leptin
growth
receptor,
factor-β,
COL11A2
nucleotide
and
bone
morphogenetic protein-2 (BMP-2) genes are associated with the
development of OPLL [24-28]. Our research group has also previously
shown that the Ser37Ala (T/G) and exon 3 (–726) T/C polymorphisms
are associated with the occurrence of OPLL, but not with extensive OPLL
in the cervical spine. Meanwhile, the Ser87Ser (A/G) polymorphism “G”
allele was reported to promote the extent of OPLL [29, 30].
In the present study, one of the three SNPs on chromosome 20p12,
the rs965291 (G/A) polymorphism (20:6491074), is located upstream of
the BMP-2 gene, and span 25 kb. The population genotypes and allele
frequencies of rs965291 (G/A) supplied by the NCBI dbSNP database
showed that these were the only “AG” or “GG” genotypes in Han
Chinese people. However, the current study also identified 43 subjects
with
the
“AA”
genotype.
The
rs996544
(C/T)
polymorphism
(20:6796219) is located downstream of the BMP-2 gene and spans 20 kb.
Similarly, the rs1116867 (A/G) polymorphism (20:6778164) is located
downstream of the BMP-2 gene and spans 18 kb. Previous studies have
shown that these three polymorphisms on chromosome 20p12 were
associated with the risk of osteoporosis and osteoporotic fracture [19, 20].
The rs996544 (C/T) “TT” genotype and rs965291 (G/A) “AA” genotype
were associated with higher risk of vertebral and hip fractures. The
osteoporosis haplotype is defined by two polymorphisms, rs1116867 (A)
and D35548 (T). BMP-2 was suggested as a candidate gene involved in
the predisposition to OPLL; these three SNPs are located near the BMP-2
gene and may affect the initiation or severity of OPLL by regulating the
expression of BMP-2 gene.
Our study showed that the rs1116867 (A/G) polymorphism is
associated with the occurrence of OPLL, but not with more extensive
OPLL in the cervical spine. Furthermore, the “AG” genotype is
associated with the occurrence of OPLL. The results indicate that the “G”
allele is a risk factor for genetic susceptibility to OPLL, but is not
associated with the extent of heterotopic ossification in the cervical spine.
The rs965291 (G/A) polymorphism in female patients is associated with
the occurrence of OPLL and that the genetic background of this
polymorphism may affect the initiation of OPLL more strongly in women
than in men.
The present results show that the rs965291 (G/A) polymorphism is
associated with the severity or promotion of OPLL, but not with its rate
of occurrence in the cervical spine, while the rs965291 (G/A) “A” allele
promotes the extent of OPLL. Patients not carrying the rs965291 (G/A)
“G” allele had a greater number of ossified vertebrae than those carrying
the “G” allele. Furthermore, the analysis adjusted for sex showed that the
female patients with the rs965291 (G/A) “A” allele (genotype, “AG” or
“AA”) had a greater number of ossified vertebrae than those without the
“A” allele (genotype, “GG”). These results indicate that the “A” allele
promotes the extent of OPLL, while the “G” allele restricts ectopic
ossification in the cervical spine in OPLL patients, particularly in female
patients.
The functional impact of the three SNPs neighboring the BMP-2 gene
is uncertain. However, because the three SNPs are located upstream or
downstream of the BMP-2 gene, they may yield alternative transcript or
result in gene products with abnormal function, or induce overexpression
of BMP-2 protein in the posterior longitudinal ligament matrix.
Overexpression of the BMP-2 protein can augment the signaling activity
of endogenously produced BMPs. Furthermore, the posterior longitudinal
ligament matrix shows an imbalance between the expression of BMP-2
agonists and the potential existence of antagonists. Thus, the BMP-2
protein may be refractory to inhibitors such as noggin. Further
augmentation of the BMP-2 signal via the SMADs and p38 mitogen
activated protein kinase signaling pathways may promote ectopic
ossification in the posterior longitudinal ligament of the cervical spine
[31-35].
In conclusion, the present results demonstrate that the rs1116867
(A/G) “AG” genotype is associated with the occurrence of OPLL. The
rs1116867 (A/G) “G” allele is associated with the occurrence of OPLL,
but not with the extent of OPLL. The rs965291 (G/A) “A” allele
promotes the extent of OPLL. Among female patients with OPLL, the
rs965291 (G/A) “A” allele is not only associated with the occurrence of
OPLL, but is also associated with the extent of OPLL, at least in Han
Chinese subjects. Our data should advance our understanding of the
molecular etiology of OPLL and may guide approaches to prevent the
onset of OPLL.
Acknowledgments
The authors thank the DNA donors for making this study possible. This
research was supported by grants from the National Nature Science
Foundation of China (No. 30872599) and the Beijing Nature Science
Foundation of China (No. 7092028).
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Table 1. Characteristics of OPLL cases and healthy control subjects
ALL
Number
Age (years)
Body weight
(kg)
Height (cm)
Personal
history of:
Smoking
Alcohol use
Food
preference
Type of OPLL
Continuous
Mixed
Segmental
localized
Male
Female
OPLL
Controls
OPLL
Controls
OPLL
Controls
420
32-77
(55.2±9.9)
51-84
(59.7±6.4)
150-178
(163.1±6.6)
506
31-79
(54.8±7.6)
50-85
(59.1±8.0)
152-180
(164.1±9.8)
232
32-77
(56.9±10.2)
63-84
(60.1±9.4)
165-178
(168.6±6.3)
288
31-79
(55.7±5.7)
61-85
(61.3±6.3)
167-180
(169.3±3.6)
188
34-75
(52.7±9.0)
51-71
(57.7±9.6)
150-170
(158.6±3.5)
218
32-78
(51.6±7.2)
50-72
(56.9±7.8)
152-169
(159.0±4.4)
167
68
28
221
94
47
151
52
16
204
81
22
16
16
12
17
13
25
155
88
139
38
78
52
81
21
77
36
58
17
Age, body weight and height are expressed as means ± SD
Table 2. Primers for each SNP
SNP
rs965291
rs99654
rs1116867
Sense
CAGTCTATGAACATGGGATA
CAGACACCGGGAAACCAT
CTGATCTTTGAATTGGGTAA
Antisense
Product
size
(bp)
Annealing
temperature
(°C)
TAAGTTGGGCAAAGTGAA
CAGACCGACAGCAGCAAC
AGTGGAGGCAGAGTGGAA
500
505
486
54
60
55
Table 3. Genotypic and allelic distributions of the three SNPs in OPLL
cases and healthy control subjects
SNPs
Genotype, n (%)
OPLL (n=420)
Control (n=506)
Allele n (%)
OPLL (n=420)
Control (n=506)
rs996544
CC
350
(83)
429
(85)
P=0.548
C
770
(92)
935
(92)
P=0.566
CT
70
(17)
77
(15)
rs965291
TT
0
(0)
0
(0)
T
70
(8)
77
(8)
AA
32
(8)
35
(7)
P=0.898
A
207
(25)
247
(24)
P=0.907
AG
143
(34)
177
(35)
rs1116867
GG
245
(58)
294
(58)
G
633
(75)
765
(76)
AA
80
(19)
148
(29)
P=0.002
A
387
(46)
533
(53)
P=0.005
AG
227
(54)
237
(47)
GG
113
(27)
121
(24)
G
453
(54)
479
(47)
Table 4. Genotypic distributions of rs965291 (G/A) classified by sex in
OPLL cases and healthy control subjects
SNP
Genotype
OPLL cases (n=420)
Male (n=232)
Female (n=188)
Control subjects (n=506)
Male (n=288)
Female (n=218)
rs965291 (G/A)
AA
32
19
13
35
19
16
AG
143
83
60
177
81
96
GG
245
130
115
294
188
106
Note: Differences in genotypic and allelic distribution classified by sex between OPLL cases and
controls subjects were analyzed using χ2-tests
Male of OPLL and control, P=0.779
Female of OPLL and control, P=0.742
Male of OPLL and male of control, P=0.099
Female of OPLL and female of control, P=0.033
Table 5. Allelic distributions of rs965291 (G/A) in female OPLL cases
and female control subjects
SNP
Allele n (%)
OPLL cases
Controls
rs965291 (G/A)
A
86(23)
128(29)
G
290(77)
308(71)
P=0.036
Figure 1. Two-dimensional computed tomography (2D-CT) and sequencing results of a
62-year-old man with OPLL. A) The 2D-CT scan revealed ossification of the posterior
longitudinal ligament in the cervical spine (C4–6, Continuous type). B) Direct sequencing of the
PCR products showed an AG heterozygotic mutation in the rs1116867 (A/G) polymorphism.
Green line: A allele; black line: G allele; blue line: C allele; red line: T allele.
Number of ossified vertebrae
Figure 2. Two-dimensional computed tomography (2D-CT) and sequencing results of a
59-year-old woman with OPLL. A) The 2D-CT scan revealed ossification of the posterior
longitudinal ligament in the cervical spine (C2-7, Mixed type). B) Direct sequencing of the PCR
products showed an AG heterozygotic mutation in the rs965291 (G/A) polymorphism. Green line:
A allele; black line: G allele; blue line: C allele; red line: T allele.
10
*
8
*
6
N.S.
A allele(+)
A allele(-)
4
2
0
All
Male
rs965291(G/A)
Female
Figure 3. The number of ossified vertebrae of OPLL patients in subjects classified by sex and
rs965291 (G/A) genotype. Data are expressed as means ± SEM. The difference in the number of
ossified vertebrae between carriers (closed bar) and non-carriers (open bar) of each allele was
statistically analyzed. *: P<0.05; N.S.: not significant.