Association of Vitamin D receptor polymorphisms with osteoporosis in Mexican postmenopausal women
Lisker, Ruben
KEY WORDS: OSTEOPOROSIS, VITAMIN D RECEPTOR POLYMORPHISMS, MEXICAN POST-MENOPAUSAL WOMEN, BSMI AND FOKI RESTRICTION ENZYME POLYMORPHISMS
Abstract
It has been reported that Vitamin D receptor polymorphisms are associated with osteoporosis, particularly those demonstrated by the BsmI and FokI restriction enzymes. Herein we report the results of a case-control study performed in postmenopausal Mexican women. We studied 65 osteoporotic women ( or = -1.5 SD BMD of young normal females. Restriction enzymes BsmI and FokI were used to identify polymorphisms. Odds ratios and their 95% confidence intervals were calculated, and analysis was performed controlling for age as a covariate. The BsmI genotypes revealed a higher frequency of the bb genotype in cases than in controls, contradicting much of the literature that suggests this genotype protects females against osteoporosis. Regarding the FokI genotypes, we were unable to confirm that the FF genotype has a protective effect against osteoporosis. The inconsistencies found in the literature and the results obtained in the present work suggest to us that other genetic and nongenetic factors are involved in the occurrence of osteoporosis, confounding the results of the possible association of osteoporosis and VDR polymorphisms.
Morrison et al. (1994) reported a strong association between a Vitamin D receptor (VDR) BsmI polymorphism and bone mineral density (BMD) measured at the femoral neck and lumbar spine. Absence of the BsmI restriction site identifies the B allele and its presence the b allele. Of the three genotypes, BB was associated with a lower BMD and bb with a higher one. In a meta-analysis of 16 publications, Cooper and Umback (1996) found evidence suggestive of a moderate effect of VDR polymorphisms on BMD at the hip, spine, and radius with approximately a 2% decrease in BB individuals compared to bb ones. They suggested that the heterogeneous results are due to ethnic, age, and calcium-intake differences, as well as stochastic variability due to different sample sizes. Other studies not included in that meta-analysis have shown inconsistent results (Zmuda et al. 2000), including that of a lower BMD associated with genotype bb rather than BB (Houston et al. 1996).
Another VDR polymorphism (Gross et al. 1996), identified with restriction endonuclease FokI and due to a C[arrow right]T transition, creates a new restriction site three codons proximal to the usual downstream start site. The presence of the FokI site, designated f, starts translation from the first ATG codon, while the allele lacking this site, designated F, initiates translation three codons up. Of the three possible genotypes, the homozygous FF individuals had a BMD in the lumbar spine 12.8% higher than that of ff individuals. Subsequent reports, however, have been inconsistent (Zmuda et al. 2000).
Herein we report the results of a case-control study, performed in Mexico, searching for the association of osteoporosis in postmenopausal women with the VDR Bsml and FokI restriction enzymes polymorphisms.
Materials and Methods
Cases and controls were recruited from the Department of Nephrology and Mineral Metabolism of our Institute, where they were selecting postmenopausal women for a phase III double-blind therapeutic trial. The Institute’s ethics committee approved the project, and participants signed a letter of informed consent prior to giving the blood sample needed for the study. We excluded individuals who had diseases capable of influencing calcium and phosphorus metabolism, such as hyperparathyroidism, renal failure, and chronic liver disease. We selected as cases 65 women who, at the femoral neck and/or the lumbar spine, had a bone mineral density or = -1.5 SD. Only women with a normal body mass index (between 19 and 25) were included.
Bone mineral density was measured with a Norland XR, 26 Densitometer. Between 5 and 7 mL of peripheral blood was collected from each individual and used for both the VDR polymorphisms identification and the study of blood groups systems ABO, Rh, MN, and Duffy.
VDR Mutation Analysis. DNA extractions were performed according to the usual procedure (Miller et al. 1988). Two regions of the VDR gene were amplified for the analysis of different mutations, one consisting of 800 base pairs (bp) corresponding to exon 9 for the Bsml site, and the other of 265 bp in exon 2, the FokI site. The primers used and assay conditions followed those recommended by Morrison et al. (1994) and Gross et al. (1996).
Statistical Analysis. Genotype and status associations were evaluated through chi-square tests. Odds ratios (OR) and their 95% confidence intervals (CI) were calculated taking the bb and ff genotypes as the reference groups. Alpha value was set at p = 0.06, according to Bonferroni’s correction. In order to better appraise the associations, both gene systems were dichotomized according to the following scheme: BB+Bb versus bb, and FF+Ff versus ff. Age differences between cases and controls were tested through an independent sample t test. Odds ratios were adjusted for age effects with standard procedures.
According to a power analysis, the study of 65 cases and 57 controls allowed (assuming [alpha] = 0.05 and [beta] = 0.20) detection of population ORs equal to or less than 0.35 (i.e., a risk reduction of more than 65%) for an exposure prevalence of 65% (B,b system), and ORs equal to or less than 0.30 for exposure prevalence of 85% (F,f system).
Results
The mean age and SD (years) were 65.2 and + or -6.8 in the cases, compared to 56.5 and + or -6.0 in the controls. Cases were significantly older than controls, p
Table 1 shows the genotypes for the BsmI and FokI polymorphisms in cases and controls. The first one showed a highly significant difference between them (p
Crude and age-adjusted odds ratios (Table 1) for the Bb system showed substantially lower values than unity, ranging from 0.20 to 0.08, suggesting a “protective” effect of the B allele. The age adjustment further supported this association. When genotypes BB and Bb were added, the OR was significantly below unity, 0.11 in the crude analysis and 0.09 in the age-adjusted one.
Table 1 also shows the results obtained with the FokI enzyme. There were no statistical differences between patients and controls (p
There were no significant differences in terms of age-adjusted BMD across the studied genotypes either in cases or controls. Blood group results showed no significant differences between cases and controls.
Discussion
The similarity of blood group results between cases and controls indicates that ethnically they are comparable and similar to other Mexican Mestizo groups from Mexico City (Lisker et al. 1995).
The fact that our cases were not in Hardy-Weinberg equilibrium for the BsmI polymorphism may not be unusual if that polymorphism has some etiologic relation to osteoporosis. It is disturbing however, that the control group showed similar results, although at a lower level (p
Much fewer data are available for the FokI polymorphism. Except for one study in blacks (Harris et al. 1997) that gave a figure of 0.194 for the f allele, other published data show gene frequencies around 0.400, regardless of ethnic origin. Our findings of 0.373 and 0.395 for cases and controls, respectively, are close to this figure.
To correct for age differences between cases and controls we adjusted the ORs for age effects by multiple logistic regression analysis for the BsmI and FokI polymorphisms (Table 1). Regarding the BsmI genotypes, the frequency of the homozygote bb was significantly higher in cases than in controls (p
According to our results (Table 1) the bb genotype, far from having a protective effect for osteoporosis, seems to represent a risk factor. This is not due to a lack of statistical power, but because it pointed towards the opposite side of the OR continuum. In the only study performed in Mexico (Jaramillo et al. 1999), no difference was found in femur and spine BMD in relation to BsmI genotypes. Gong et al. (1999) analyzed 75 full papers and summaries published on this subject up to January 1997, concluding that several VDR alleles, including B, were associated with lower hip and spine bone mass more often than the expected 5% rate under the null hypothesis. They believe that BMD is associated with BsmI VDR polymorphisms, and that some other genetic and nongenetic factors (calcium intake, weight, age at nursing, among others) interfere with the identification of this association. We agree with this assessment.
Acknowledgments This study was supported by the Consejo Nacional de Ciencia y Tecnologia (CONACYT), Mexico, under grant 0138P-M9506.
Received 3 September 2002; revision received 24 February 2003.
Literature Cited
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RUBEN LISKER,1* MARIA A. LOPEZ,1 SALOMON JASQUI,2 SERGIO PONCE DE LEON ROSALES,3 RICARDO CORREA-ROTTER,2 SERGIO SANCHEZ,4 AND OSVALDO M. MUTCHINICK1*
1 Department of Genetics, Instituto Nacional de Ciencias Medicas y Nutricion Salvador Zubiran, Tlalpan 14000, D.P. Mexico.
2 Department of Nephrology and Mineral Metabolism, Instituto Nacional de Ciencias Medicas y Nutricion Salvador Zubiran, Tlalpan 14000, D.F. Mexico.
3 Department of Clinical Epidemiology, Instituto Nacional de Cieneias Medicas y Nutricion Salvador Zubiran, Tlalpan 14000, D.F. Mexico.
4 Blood Bank, Instituto Nacional de Ciencias Medicas y Nutricion Salvador Zubiran, Tlalpan 14000, D.F. Mexico.
* Contributed equally.
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