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Technical Briefs |
1 Gene Sp1 Polymorphism in Premenopausal Women with Primary Osteoporosis: Improved Detection of Sp1 Binding Site Polymorphism in the Collagen Type 1 Gene
1
Biochemistry,
2
Hormonology,
3
Nuclear Medicine,
4
Rheumatology, and
5
Gynaecology, Hospital Clinic, University of Barcelona, 08036 Barcelona, Spain;
Osteoporosis is a common disorder characterized by reduced bone
mass and increased risk of fracture (1). Evidence from
family studies suggests that genetic factors have a major role in the
determination of bone mass (2). Because bone mass and bone
strength are complex phenotypes, their heredity is almost certainly
polygenic. Among the potential candidate genes that might influence
bone mass acquisition, special attention has been directed to the
vitamin D receptor and estrogen receptor genes; results of these
studies, however, have been contradictory (3)(4)(5).
Type I collagen, a protein encoded by the collagen I 
1
(COLIA1) and collagen I 2 (COLIA2)
genes, is the major protein of bone matrix. Genes encoding collagen
type I may be important candidates for the genetic regulation of bone
density because mutations that affect their coding regions have been
associated with osteogenesis imperfecta (6).
A guanine-to-thymidine polymorphism at the first base of a binding site for the transcription factor Sp1 in the first intron of COLIA1 has recently been associated with low bone density and bone fractures in women from various European populations(7)(8)(9)(10)(11). In these studies, the COLIA1 genotypes were determined using a PCR assay with a mismatched primer that introduces a restriction site for the enzyme BalI in polymorphic alleles with the thymidine substitution. The test discriminates two alleles, S and s, which correspond to the presence of guanine and thymidine, respectively. Thus, the absence of the restriction site on both alleles was defined as homozygote SS, the presence of the restriction site was defined as homozygote ss, and the combination of both was defined as heterozygote Ss (7). In this assay, the PCR products containing s alleles are cleaved by BalI, and agarose gel electrophoresis of the fragments reveals a 246-bp band, whereas electrophoresis of uncleaved S alleles reveals a 264-bp band.
To improve the genotyping of S and s alleles, we have modified the method through a change in the reverse primer and in the restriction enzyme, Fnu4HI, which recognizes the sequence GCNGC. By using a polyacrylamide minigel (SE 250 Mighty Small II; Pharmacia), we are able to better resolve the restriction fragments and to genotype subjects more quickly. Moreover, the method has the advantage that the PCR product synthesized has a second restriction site for Fnu4HI located in nucleotide 1437 (GenBank accession no. J03559), which can serve as an internal control for PCR cutting.
Although postmenopausal osteoporosis in women has been well documented, few studies have focused on primary osteoporosis in premenopausal women. In these patients, there are no secondary causes of bone loss, such as menopause or other metabolic disturbances. Therefore, the contribution of genetic factors to the pathogenesis of primary osteoporosis could theoretically be better analyzed.
In this study, we describe a modified method for detecting Sp1 binding site polymorphism in COLIA1 and the distribution of the alleles in women with premenopausal primary osteoporosis.
The patients in this study were 20 premenopausal women (ages 25–52 years; mean age, 40 ± 9 years) with primary osteoporosis who were attending the Rheumatology Service at our Hospital Clinic. The diagnosis of osteoporosis was defined by the presence of one or more atraumatic vertebral crush fractures or by densitometric criteria, i.e., a bone mineral density (BMD) in the lumbar spine less than -2.5 T-score. Lumbar spine BMD (L2–L4) was measured with dual-energy x-ray absorptiometry, using a DPX-L bone mineral analyzer (Lunar Radiation). An automated biochemical profile, complete blood count, and complete hormonal measurements were performed in all patients to exclude an underlying secondary cause of osteoporosis. The results were compared with a control group of 24 healthy women (mean age, 43 ± 7 years; range, 27–52 years) with BMD within the health-related reference interval (lumbar BMD greater than -1 T-score) and no evidence of metabolic bone disorders who had been recruited from the staff of the laboratory. The study was a case-control study and was approved by the ethics committee of the Hospital Clinic.
High-molecular weight DNA was isolated from white blood cells according
to established procedures (12). The intronic polymorphism of
the COLIA1 gene was detected by PCR using the forward primer
(5'-TAACTTCTGGACTATTTGCGGACTTTTTGG-3') described by Grant et al.(7). The reverse primer was changed to
5'-GAAGGTCCAGCCCTCATCCCGCCG-3' to introduce a restriction
site for the Fnu4HI enzyme to detect the G
T substitution.
(The modified nucleotide is underlined.)
Approximately 100 ng of genomic DNA was amplified in 50-µL reactions that included 10 mmol/L Tris-HCl, pH 9, 50 mmol/L KCl, 1.5 mmol/L MgCl2, 0.2 mmol/L dNTPs, 5 µmol/L each primer, and 1.25 U of Taq DNA polymerase (Life Technologies). The PCR reactions were performed in a DNA thermocycler (Omnigene; Hybaid Ltd.) as follows: after a cycle of 94 °C for 4 min, the cycling protocol comprised 40 cycles of 94 °C for 50 s, 60 °C for 10 s, and 72 °C for 30 s. After the last cycle, an extension step of 5 min at 72 °C was performed. The expected size of the specific amplification product was 257 bp. The amplification product (10 µL) was digested with 5 U of Fnu4HI (New England BioLabs) overnight at 37 °C in a total volume of 15 µL; the reaction mixture contained 50 mmol/L potassium acetate, 20 mmol/L Tris acetate, 10 mmol/L magnesium acetate, and 1 mmol/L dithiothreitol. Digestion products were electrophoresed in a 7% polyacrylamide minigel for 20 min and stained with ethidium bromide.
All genotypes were confirmed using the method described by Grant et al.(7).
The 
2 and Fisher exact tests were used to
compare proportions. P <0.05 was considered to be
significant. Odds ratio (with 95% confidence intervals) were
calculated to estimate the relative risk of osteoporosis.
Polyacrylamide gel electrophoresis of PCR-amplified product and
restriction Fnu4HI-digested fragments is shown in Fig. 1
. The S allele was observed as three bands of 180,
54, and 23 bp; and the s allele was observed as two bands of
180 and 77 bp. The assay was performed in 20 min.
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The distribution of the COLIA1 genotypes and alleles in
patients and controls is shown in Table 1
. We observed the same distribution by using the method
described by Grant et al. (7) (data not shown). With this
method, the assay was completed in 50 min. In the control group, the
distribution of genotypes (87.5% SS, 12.5% Ss,
0% ss) and the allelic frequencies (93.7% for the
S allele and 6.2% for the s allele) were
slightly different from those reported for other European populations,
in whom the s allele frequency is 17–23%, which is
slightly higher (7)(9)(10)(11). The reasons for such
differences may be related to both the selection of our healthy
population and the small sample size analyzed. Indeed, these previous
studies (7)(9)(10)(11) included nonselected patients
from larger postmenopausal populations, whereas our healthy controls
consisted of selected premenopausal women with BMD with the reference
interval. Moreover, in the present series, patients with primary
osteoporosis showed higher allelic frequencies for the s
allele (27.5% for the s allele and 72.5% for the
S allele) as well as for the Ss genotype (50%
SS, 45% Ss, 5% ss). The allelic and
genotype distributions showed significant differences between patients
and controls.
|
The odds ratio for osteoporosis in patients with the s allele was 5.68 (95% confidence interval, 1.46–22). Thus, the s allele was present in 27.5% of the women with primary osteoporosis, but only in 6.2% of healthy controls. This overrepresentation of the s allele in patients with primary osteoporosis is consistent with previous reports showing an increased risk of fractures in postmenopausal heterozygote women, suggesting that the COLIA1 Sp1 polymorphism has a potential value as a predictor of osteoporosis.
In conclusion, the introduction of an Fnu4HI restriction site allows us to perform S and s genotyping of the COL1A1 Sp1 polymorphism by PCR-restriction fragment length polymorphism and polyacrylamide minigel electrophoresis. This method is faster, easier, provides better fragment resolution, and is more cost-effective than the method described previously by Grant et al. (7). An additional advantage is that the second Fnu4HI restriction site can serve as an internal control for PCR cutting. Moreover, this study suggests that in premenopausal women with primary osteoporosis, there is a high prevalence of the s allele and the Ss genotype, reinforcing the suggestion that COLIA1 genotype predisposes to osteoporosis.
Footnotes
Services of * address correspondence to this author at: Servicio Bioquimica, Hospital Clinic, C/Villarroel 170, 08036 Barcelona, Spain
fax 34-93-4512203, e-mail lalvarez{at}medicina.ub.es
References
1 gene. Nat Genet 1996;14:203-205.
[Web of Science][Medline]
[Order article via Infotrieve]
I gene to bone density and the risk of osteoporotic fractures in postmenopausal women. N Engl J Med 1998;338:1016-1021.
1 Sp1 polymorphism, bone mass and bone turnover in healthy French premenopausal women: the OFELY study. J Bone Miner Res 1998;13:813-817.
[Web of Science][Medline]
[Order article via Infotrieve]
The following articles in journals at HighWire Press have cited this article:
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  P. Peris, L. Alvarez, J. Oriola, N. Guanabens, A. Monegal, M. J. M. de Osaba, J. Jo, F. Pons, A. M. Ballesta, and J. Munoz-Gomez Collagen type I{alpha}1 gene polymorphism in idiopathic osteoporosis in men Rheumatology, November 1, 2000; 39(11): 1222 - 1225. [Abstract] [Full Text] [PDF]
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