An historic approach to identifying genetic effects in multifactorial disorders takes advantage of the fact that monozygotic twins are genetically identical, while on average dizygotic twins carry have half of their genes in common. By examining the variance in a continuous trait such as bone mineral density (BMD), geneticists can derive an estimate of the variance attributable to genetic effects, also called the heritability coefficient. Such twin studies indicate that genetic determinants may account for 50 to 85% of BMD variability, depending on age, sex, method of BMD determination, and skeletal site (Ralston,1997). Lumbar spine BMD has the highest heritability, but genetic influences also contribute significantly to BMD variability at the forearm, femoral neck, and Ward's triangle.

An alternative approach is to study single gene disorders caused by severe deletion mutations resulting in osteoporosis. The argument is that milder forms of the disorder may result from partially functional mutations. Such mutations should be observed in individuals bearing the multifactorial trait who have a positive family history or suffer from a more severe or earlier-onset disease than usual. An obvious candidate is osteogenesis imperfecta (OI) or brittle bone disease. The later-onset (or tarda) form (Type I of Sillence) of this condition is characterized by a variable number of fractures but universally accompanied by osteopenia and reduced BMD (Cole & Cohen,1991). In otherwise healthy individuals with early onset primary osteoporosis, the association of pre-menopausal osteoporotic fractures with a positive family history has suggested OI, even without the presence of the distinctive blue sclerae (Paterson et al,1984). In one particular family, the cause was identified as a Gly Cys mutation at position 43 of the COL1A1 gene (Shapiro et al.,1992); in another instance, a Gly Ser mutation at position 661 of the COL1A2 gene was found in a 52 year old woman with blue sclerae but also typical vertebral compression fractures associated with axial osteoporosis (Spotila et al.,1991). While severe or premature osteoporosis may be a common feature of mild OI, COL1A1 mutations in fact account for only a small fraction of osteoporotic disease (Spotila et al.,1994).

Newer molecular methods for identifying genetic loci have been used to assess candidate genes that may act to maintain or regulate bone mineral balance. In 1992, Morrison et al. reported linkage between restriction fragment length polymorphisms (RFLPs) for the vitamin D receptor (VDR) gene and levels of serum osteocalcin, a protein known to be exquisitely sensitive to induction by vitamin D (Cole & Hanley,1991). While this seemed initially of limited consequence, these investigators have more recently described a strong association between VDR polymorphisms and BMD, both in twin pairs and in an adult Australian population (Morrison et al.,1994). On the basis of linkage disequilibrium between BsmI (B/b), Apa1 (A/a), and Taq1 (T/t) restriction sites -- all found in the intron preceding the last exon, their data could be interpreted to indicate that lower BMD at both lumbar and femoral neck regions were caused by a "BAt", as opposed to the "baT", haplogroup (where the lower case letter indicates the presence of the restriction sequence). Subsequent studies looking for similar associations in other populations and for other conditions have yielded variable and sometimes contradictory findings, which have been debated in public and in the literature (Eisman,1995; Peacock,1995). A recent meta-analysis confirms a small but significant effect (Cooper & Umbach,1996) and at least one comprehensive survey of this literature (Gross et al.,1996a) comes to the same conclusions, indicating that: 1) there are substantial geographic and demographic variations in the strength of the association between the BAt haplogroup (B allele) with reduced BMD; 2) there are large populations for which no effect at all is seen; and 3) there are populations for which the b allele is associated with reduced, rather than increased BMD. Still, the evidence for functional differences among the various BbAaTt haplotypes is not strong. It is possible that the association reflects linkage disequilibrium with a different VDR site and there are now promising candidates, both upstream (Gross et al.,1996b). and downstream (Ingles et al.,1997). Moreover, linkage disequilibrium with other physically proximate genes has not been excluded.

Nevertheless, the strength of the evidence supports a significant association and therefore, based on the work of Uitterlinden et al. (1996), we developed an improved haplotyping technique for amplification of a 2229 bp VDR gene fragment bracketing the 3 sites using sequential digestion by all 3 enzymes. The resulting fragment patterns can be interpreted to yield a unique haplogroup (Peltekova et al.,1997). While we find that the haplogroups 1 and 2 (h1=baT and h2=BAt, respectively), described by Morrison et al.(1994) are present in the majority of Caucasians, we found haplogroup 3 (h3=bAT) to be a prevalent third allele in a local Amerindian population (Cole et al.,1996), reflecting their Asian heritage (Tokita et al.,1996 ; Scozzari et al.,1997). We have also positively identified 7 of the 8 possible haplogroups in other sample sets (Peltekova, Cole & Rubin: unpublished observations), indicating a variable degree of linkage disequilibrium in different populations, which may ultimately bear on the variable relationship between genotype to BMD.

Study of specific disease groups with a high rate of osteoporosis has not yet been reported. We chose to examine the hypothesis that hepatic osteodystrophy which characterizes primary biliary cirrhosis (PBC — a progressive cholestatic liver disease characterized by abnormal vitamin D metabolism) could be reflected in decreased relative BMD, as assessed in a cohort of 73 female patients by dual X-ray absorptiometry (Heathcote et al., 1997). Mean age-standardized estimates (z scores) for femoral BMD were significantly reduced, but the relative risk of low BMD (z < -2SD) was elevated for both femoral and spinal sites. BMD correlated with weight (as expected) but did not show significant correlation with clinical parameters except possibly disease duration (p=0.057). In this group homozygotes of haplotype 1 (h1 = baT/baT) had significantly lower BMD than did h1/h2 heterozygotes (baT/BAt), which in turn had lower BMD than h2 homozygotes (BAt/BAt). In contrast to Morrison et al. (1994), we found that the b allele predicted significantly lower corrected BMD at both spinal and lumbar sites.

To ascertain whether this effect was reflected in an unselected population, both BMD and VDR haplotypes were assayed in 702 caucasian women between 18 and 35 years of age who were recruited from the greater metropolitan Toronto area (Rubin et al., in press). We found no significant association between spinal BMD and VDR haplotype, with or without adjustment for height, weight, and age, but we did observe a small, significant association between the BAt (h2) VDR haplotype and higher BMD at the femoral neck, providing us with some assurance that the unexpected association in a much smaller cohort of PBC patients was not an artifact of the disorder itself. In aggregate, these two studies suggest for the first time that risks for secondary osteoporosis (i.e., hepatic osteodystrophy) may be associated with VDR haplotype in much the same way they are for primary postmenopausal osteoporosis.

In addition to VDR, other candidate genes are being assessed. Two single base-pair intron1 sites of the estrogen receptor-alpha (ER1) have been associated with altered BMD (Kobayashi et al.,1996). In a study of peak BMD Willing et al (1998) found significant association of two RFLP's in the first intron of ER1 with femoral neck BMD in a population of 501 Caucasian women. The RFLP's have no known functional consequences for gene expression and therefore any effects on BMD are likely mediated through other functional ER1 variants in linkage disequilibrium. Furthermore, when VDR genotypes are stratified by ER genotype, a significant genotype-genotype interaction was detected - that is, VDR alleles were found to account for significant additional variance in peak BMD (Willing et al., 1998). A similar effect has been seen at the lumbar spine in a postmenopausal Caucasian population, the only other population to date in which this intriguing genotype-genotype interaction has been examined (Gennari et al., 1998).

An Sp1 binding site in the first intron of COLIA1 contains a polymorphism in about 22% of Caucasians which causes reduced binding affinity for the Sp1 protein (Grant et al, 1996). This allele has been shown to associate with post menopausal BMD in Scottish (Grant et al, 1996), English (Grant et al, 1996), Dutch (Uitterlinden et al., 1998), Danish (Langdahl et al., 1998) and French (Roux et al., 1998) Caucasian populations. In the largest study to date, 1778 postmenopausal Dutch women were studied (Uitterlinden et al., 1998). The effects of the Sp1 allele on BMD were found to be dependent on age since menopause, suggesting that this allele may play a role in determining the rate of postmenopausal bone loss. The overall effect on BMD at the spine and femoral neck was small, however, with the Sp1 allele accounting for 2% of the variance in BMD in women aged 75 to 80 years. This is consistent with a polygenic model of inheritance for BMD with many genes playing a role, each with small effects.

More importantly, several studies have shown an effect of the Sp1 allele on fracture risk. Grant et al. (1996) reported a small case-control study documenting a relative risk of vertebral fracture of 3.2 (95% CI 2.0 -14.6) while Uitterlinden et al. (1998) found a relative risk for any fracture type of 1.5 (95% CI 1.1 - 4.4). This finding is especially important since the Sp1 allele is the first genetic marker shown to be associated with significant fracture risk. A Danish case-control study (Langdahl et al., 1998) confirmed and extended these findings, showing that the Sp1 allele conferred an elevated risk for vertebral fracture (OR 11.8; 95% CI 2.6-53) in men and women, a risk that was only partly dependent on BMD. The fracture risk conferred by the Sp1 allele is comparable to the relative risks for vertebral and hip fracture conferred by a 1 SD decrease in vertebral and femoral neck BMD (pooled odds ratio 2.3 to 2.6; Marshall et al., 1996). The effects of the COLIA1 Sp1 allele must be confirmed prospectively and in a wider range of populations before its clinical utility can be judged. With the preliminary data currently available, it appears to be a very promising factor for the future prediction of postmenopausal BMD and fracture risk.

The transforming growth factor beta (TGF-b) superfamily of growth factors comprises more than thirty members grouped in several subfamilies (reviewed in Alevizopoulos and Mermod, 1997). The prototypic member of the TGF-b subfamily is TGF-b1, a multifunctional hormone which plays key roles in cell growth and differentiation, wound healing, inflammation and morphogenesis. In growing bone, TGF-b1-3 isoforms are expressed in defined regions of the growth plate, suggesting a role in control of bone growth. In mature bone, TGF-b1 may be one of the factors which regulates the central interaction of bone turnover - osteoblast-osteoclast coupling. In vitro studies have indicated that estrogen promotes TGF-b1 synthesis by osteoblasts and that both estrogen and TGF-b1 promote osteoclast apoptosis (Hughes et al., 1996). Langdahl et al. (1997) discovered a rare allele of the TGF-b1 gene - a one base pair deletion in intron 4 designated 713-8delC - which was associated with very low bone mass and increased bone specific alkaline phosphatase in osteoporotic women. Yamada et al. (1998) reported the association of a coding polymorphism (designated T29C) which results in the substitution of proline for leucine in the signal peptide of TGF-b1 with lumbar bone density and vertebral fracture frequency in two different Japanese populations of osteoporotics, as compared to normal controls. However, since the case and control groups had different allele frequencies, it is possible that these alleles are associated with survival rather than the osteoporotic phenotype.

Although interactions between genes and environment are difficult to study, they are not intractable. Any functional change in the vitamin D receptor implied by variation in the VDR gene should be accompanied by altered relationships between dietary calcium intake and the internal regulation of calcium metabolism. Recent reports of a strong relationship between VDR polymorphisms and calcium intake (e.g., Kiel et al.,1997) suggest that the association of genotype with BMD is strongest in women with low calcium intake. Similar interactions may occur with vitamin D metabolism, if 25-hydroxyvitamin D levels are low. Certainly, comparisons of VDR genotype correlation with BMD in different geographic locations will require consideration of population genotype variation, intrinsic variations in vitamin D metabolism related to sun exposure, vitamin D fortification policies, and local dietary practices. Undoubtedly, studies of genetic and environmental factors as interacting causes of osteoporosis will be a exciting and fruitful area of future research.

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