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Introduction
Osteoporosis is a systemic skeletal complex disease
characterized by bone loss and decreased bone strength,
leading to increased fracture risk. It is a major health problem
both in Caucasians and in Chinese. Currently, over 200 million people worldwide suffer from this
disease[1].
Bone strength as the ultimate measurement of resistance
to osteoporotic factures (OF) is determined by bone mineral
density (BMD), bone geometry, bone microarchitecture,
and the quality of bone
material[2_4]. Bone geometry can
significantly improve the prediction of osteoporosis or OF
risk[5_7]. Most research have found that women with femoral neck
(FN) fractures have longer hip axis length, larger FN-shaft
angle than controls without
fractures[8,9]. However, Gnudi et
al found no specificity of bone geometric parameters for
spine fracture risk[8]. In addition, bone geometric parameters
as complex traits were under strong genetic
determination[10,11].
It is well known that hip fracture is the most serious and
disabling type of OFs[12]. The ethnic difference of hip
fracture risk is well known[13,14]. Almost all of the studies
consistently reported that the age and sex-adjusted annual rate of
hip fracture was higher in Caucasians than in
Asians[13,14]. The ethnic differentiation of bone geometry may be partially
responsible for the above observations. For example, smaller
bone widths and a shorter hip axis length were detected in
women of African origin compared with Caucasians, and
these observed racial differences could contribute to an
approximately 25% decrease in the risk of hip fracture among
blacks[15]. However, a few studies have focused on the
different bone geometry between Asians and Caucasians, and
whether these ethnic differences of bone geometry come
from the ethnic-specific genetic determination remains
unknown.
Genes encoding the alpha 1 chain of collagen type 1
(COL1A1) and alpha 2-HS-glycoprotein
(AHSG) are 2 important genes, which might influence bone geometry.
COL1A1 is one of most abundant proteins in bone matrix. Mutations
in COL1A1 and collagen type I alpha2
(COL1A2) genes were estimated to be responsible for up to 90% of cases of the
Mendelian disease named osteogenesis imperfecta, which
is associated with a very low bone mass and an increased
fracture risk. A G/T polymorphism affecting the
Sp1 binding site in the first intron of the
COL1A1 gene, also known as rs1800012 or g1546 G>T, was regarded as a predictor of
reduced BMD and increased the risk of fragility fractures,
reported in several studies[16,17]. However, the importance of
the COL1A1 gene on bone phenotypes may not be explained
by the Sp1 polymorphism in Chinese, as the
Sp1 polymorphism was absent in Chinese
population[18]. Another important mutation in the promoter region of
COL1A1 gene, the PCOL2 variance (rs1107946 or g-1997 G>T), was first found
by Garcia-Giralt et al in postmenopausal women of Spanish
origin[19]. Garcia-Giralt et al established a significantly
association between PCOL2 variance and BMD variations at the
lumbar spine and FN[19]. Meanwhile, a strong interaction
between PCOL2 and Sp1 variance was observed in a study
of elderly Caucasian females[20]. In light of foregoing studies,
the PCOL2 polymorphism was selected as a valuable genetic marker to explain the importance of
COL1A1 gene in Chinese.
AHSG as a human plasma glycoprotein has a high level
of serum and mineralized bone[21]. AHSG can bind
Ca2+ with high affinity to prevent apatite formation in the
circulation[22], and may play an important role in bone cell metabolism by
influencing the recruitment of osteoclastic precursors to
bone[23] and modulating bone
resorption[24]. The AHSG phenotypes, characterized by ACG (Thr)/ATG (Met) at amino
acid position 230 at exon 6 and ACC (Thr)/AGC (Ser) at amino
acid position 238 in exon 7, have been proven to contribute
to the genetic influence of lumbar vertebral BMD, femoral
neck BMD[25], and calcaneal broadband ultrasound
attenuation[26]. The SacI polymorphism (rs4918 or g238 C>G)
represents the nucleotide substitution of C_G at amino acid
position 238 in exon 7, which may change the charge of
molecules and generate the relevant mobility on isoelectric
focusing.
To date, only a few studies have focused on the
comparison of bone geometric parameters across different
races[27,28], but no genetic study has simultaneously investigated the
contribution of bone candidate genes on the variation of
bone geometric parameters in both Caucasians and Chinese.
Thus, in the present study, we first compared bone
geometric parameters of FN between the Caucasian and Chinese
population and then tested the effect of the
AHSG and COL1A1 genes on the variation of bone geometric parameters of FN
in both Caucasians and Chinese, our study would
contribute to understand the various genetic determination of bone
geometry between different ethnic populations.
Materials and methods
Subjects The study was supported by Creighton
University (Omaha, USA) and Hu-nan Normal University
(Changsha, China). The Caucasian sample consisted of 605
Caucasian individuals from 157 nuclear families aged from 19
to 87 years. All Caucasian individuals came from the city of
Omaha in the USA. The family size ranged from 3 to 12 with
a mean of 4.2, yielding 372 sibling pairs. The Chinese sample
was composed of 400 nuclear families with a total of 1228
individuals aged from 19 to 80 years. Each nuclear family
was composed of both parents and at least 1 healthy
premenopausal offspring with the average family size of 3.14,
yielding 57 sibling pairs. All the subjects belonged to the
Chinese Han group and were recruited from the city of
Shanghai in China. Before participating in the 2 projects, each
subject signed an informed consent document. Both
populations were collected using the same excluded criterion to
minimize the potential confounding factors on osteoporosis
studies[29].
Phenotyping BMD and bone area of the FN were
measured by dual energy X-ray absorptiometry (DXA). Both
machines were calibrated daily. The short-term precision
in vivo for femoral BMD and bone size was 1.87% and 1.94%,
as well as 0.80% and 0.06% in Caucasians and Chinese,
respectively. In the Hologic systems, the width of the FN
region of interest (ROI) was standardized at 1.5 cm, and W
was the average FN periosteal diameter obtained by
dividing the FN area by 1.5 cm. We used the FN BMD and W to
estimate 5 indices of the FN geometry: (1) cross sectional
area (CSA) __ the area with mineralized bone tissue,
excluding bone marrow space; (2) cortical thickness (CT); (3)
endocortical diameter (ED) __ an estimate of medullary bone
thickness; (4) section modulus (Z) __ a measure of bending
strength, which is consistent with the skeleton load and
implicates in hip fragility; and (5) buckling ratio (BR) __ an
index of bone geometric instability, which indicates the risk
of fracture by buckling[30,31].
Genotyping Genomic DNA was isolated from the
peripheral blood in Caucasians using a commercial isolation kit
(Gentra Systems, Minneapolis, MN, USA) following the
procedure detailed in the kit. In the Chinese, the total genomic
DNA was isolated using the phenol-chloroform extraction
method. The genotyping procedure is the polymerase chain
reaction and restriction fragment length polymorphism
(PCR-RFLP). The PCR reaction mixed was as follows: 0.1_0.3 µg
genomic DNA, 0.2_0.24 mmol/L dNTP (Promega Biotech,
Madison, WI, USA), 1 unit of Taq polymerase (Promega
Biotech, Madison, WI, USA or Sangon Co, Shanghai, China),
respective proportions of MgCl2, oligonucleotide primers and
10×enzyme buffer in a total volume of 25 µL. The PCR was
carried out on either a PE9700 Thermal Cycler (Perkin Elmer
Cetus, Norwalk, CT, USA) or an Omnigene Thermocycler
(Hybaid, Ashford, UK). After the amplification, 8 µL aliquots
of products were digested with the respective restriction
endonucleases for 3_12 h at 37 °C and subjected to
electrophoresis on 2% or 3% agarose gel in 1×TAE buffer
(Tris-Acetate-EDTA) stained with ethidium bromide and detected
under UV light. The information of genotyping the 3
polymorphisms are presented in Table 1.
Statistical analyses We used the quantitative
transmission disequilibrium test program
(QTDT)[32] to test population stratification, total family association, within-family
association, and linkage between the AHSG-SacI,
COL1A1-PCOL2 and Sp1 polymorphisms, and 5 bone geometric
parameters. An orthogonal model in the QTDT analysis decomposed the genotype score into orthogonal between
family (βb) and within family
(βw) components. The βb was
specific to each nuclear family and could be sensitive to the
population structure, but the βw was significant only in the
presence of both linkage disequilibrium (LD) and linkage.
The within-family association only evaluated the
βw, but the total family association evaluated the association at the
population level on all the subjects. Therefore, the total family
association may produce false positive results in the
presence of population stratification/admixture. The QTDT can
also test population stratification by evaluating if the
βb is equal to βw. The above tests were based on a variance
component framework using the maximum likelihood ratio
computed with the QTDT. One thousand permutation tests
using a Monte-Carlo permutation framework were used to
generate the empirical P value to assess the reliability of
within-family association. SPSS (version11, Chicago, IL,
USA) t-tests were used to compare the difference of
unadjusted and adjusted (by age, height, weight, and gender)
bone geometric parameters in both randomly selected
offspring and parent subgroups. All of the phenotypic data
were tested for normality by the Kolmogorov-Smirnov test
implemented in the software Minitab (Minitab Inc, State
College, PA, USA) before the association and linkage tests.
No significant deviation from the normal distribution was
found for the phenotypic data analyzed.
Results
The basic characteristics of the 2 population groups were
summarized in Table 2. Generally, the Chinese have
significant lower CSA, CT, ED, and Z, compared with Caucasians.
The t-test in the offspring showed the significant differences
of all bone geometric phenotypes (except ED) between
Caucasians and Chinese using both unadjusted and adjusted
(by age, height, weight, and gender) data, while in the parent
group, all 5 geometric phenotypes detected significant
differences after being adjusted by age, height, weight, and
gender (P<0.01). The allele frequencies of three
polymorphisms were presented in Table 3. The allele frequency of
the AHSG-SacI polymorphism in the Chinese was lower than
in the Caucasians.
The results of the population stratification, within-family
association, and linkage test were shown in Table 4. The
significant population stratification in Caucasians may have
influenced the results of total family association (data not
shown). In Caucasians, we found significant within-family
association for COL1A1-Sp1 polymorphism with CSA, CT,
ED, BR (P=0.018, 0.002, 0.023, and 0.001, respectively), and
significant linkages were detected with BR
(P=0.039). In Chinese population, the within-family associations between
the COL1A1-PCOL2 polymorphism and CT and BR were
significant (P=0.012 and 0.008, respectively). Although
the AHSG-SacI polymorphism within-family association was
observed with CSA and CT in Caucasians, evidence of
linkage were detected for CT and BR (P=0.042 and 0.014,
respec-tively). However, in Chinese population, neither
association nor linkage results were found for the
AHSG-SacI polymorphism. After one thousand permutation tests,
P<0.009 and P<0.008 were required for an individual test to achieve a
global significance level of 0.05 in Caucasians and Chinese,
respectively. The within-family association
for COL1A1-Spl polymorphism with CSA, CT, ED, and BR were still
significant in Caucasians (P=0.005, <0.001, 0.006, and <0.001,
respectively). In the Chinese, the association for
COL1A1-PCOL2 polymorphism with BR, CSA, CT, and ED was
significant or nominally significant (P=0.002, 0.08, 0.014, and
0.05, respectively) after permutation.
Discussion
Our major findings in this study are as follows: (1) there
is a differential bone geometry between Chinese and
Caucasians; and (2) the COL1A1 gene may be associated
with the variations of the FN bone geometric parameters in
both population groups, and the AHSG gene may be linked
to the variations of the FN bone geometric parameters in
Caucasians, but not in Chinese. These results may provide
some suggestive clues on the ethnic-differential genetic
determination of bone geometry in Caucasians and Chinese,
that is, the 2 ethnic populations may both share some
common and different genes regulating the variation of bone
geometry. This study represents our first efforts in
investigating the importance of the COL1A1 and
AHSG genes on bone geometry in both Caucasians and Chinese.
Our differential bone geometry between the 2 ethnic
populations was partly consistent with another study in
Caucasian and Chinese subjects aged 18_93
years[33], but we failed to detect evidence of significant racial difference on ED in
young female groups. These conflicting results may be the
difference in sampling methods, covariates used for the
adjustment, or some other confounding issue. A review
summarized the ethnic difference in osteoporosis-related
phenotypes between Caucasians and Asians and its potential
underlying genetic determination. The potential genetic
evidence included the different heritability and inheritance mode
of bone phenotypes, the different osteoporosis candidate
genes, and the differential results in related molecular
studies between them[13,14].
The significant within-family association between the
polymorphisms of the COL1A1 gene and bone geometry
suggests that the polymorphisms are likely to be in linkage
disequilibrium with a nearby functional mutation, or the
polymorphisms themselves may have important effects on the
variation of bone geometry in both Caucasians and Chinese.
Simultaneously, the consistent within-family association
across ethnic populations implicates that Caucasians and
Chinese may share the same effects of the
COL1A1 gene on regulating the variation of bone geometry.
The AHSG gene is linked to FN bone geometric
parameters in Caucasians, but not in Chinese. The results were
similar as previous studies, in which within Chinese
population no evidence was found regarding the association
between the AHSG gene and
BMD[34]. Two factors may explain the inconsistent linkage. First, the inconsistent linkage may
be due to the differential linkage power. The power of
linkage analysis is higher in Caucasians than in Chinese. In the
Caucasian sample, the family size ranged from 3 to 12 with a
mean of 4.2, yielding 372 sib pairs, whereas in the Chinese
sample, despite the large number of nuclear families (400),
only 57 sib pairs were informative for the linkage analyses
(since the majority of families had only 1 daughter),
rendering only modest power to detect the true linkage. Second,
the ethnic-specific genetic determination for bone geometry
may be responsible for the inconsistent linkage. A possible
explanation is that there is a functional mutation influencing
bone geometry nearby the AHSG gene on the same
chromosome in Caucasians, but not in Chinese. Interestingly, Xiong
et al recently found linkage evidence for 3q27 with CSA and
CT where the AHSG gene is located in
Caucasians[35].
Our results may support previous findings that the
COL1A1 gene may be a predictor of osteoporosis and OF
risk in Caucasians[16,36]. We found significant within-family
associations between 4 FN geometric parameters (CSA, CT,
ED, and BR) and the COL1A1-Sp1 polymorphism in
Caucasians. Our further analysis found that the ss
genotype groups had higher BR than those with other genotypes.
Therefore, from the present results, we suppose that the
different genotypes of the COL1A1 gene in subjects
regulate the diversity of bone geometry, which will eventually
result in differential OF risk. Further genetic and molecular
researches are needed to confirm this hypothesis.
There were some potential limitations in our study. All 5
FN bone geometric parameters were calculated based on the
DXA-derived BMD and ROI. DXA which only provided a
2-dimensional analysis was used to estimate the
approximation of the 3-dimensional structure of bone, thus the
calculated bone geometric parameters may not be suitable for
reflecting 3-dimensional bone structure features. The mean
values of BMD from the conventional Hologic ROI for FN
(1.5 cm wide) are on average 14% lower than Beck's mean
values of BMD from the narrow neck
region[37]. This difference may result in the lower estimates of CSA, CT, and higher
estimates of ED and BR in our study.
In summary, our results suggest that the
COL1A1 gene may play a role in the FN bone geometric variation in both
Caucasians and Chinese, while the influence of the
AHSG gene may be different in the 2 ethnic groups. The
understanding of ethnic-specific genetic determination for bone
geometry will assist in understanding the pathogenesis of
osteoporosis comprehensively and develop ethnic-specific
approaches for the prevention, diagnosis, and treatment of
osteoporosis.
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