Linkage analysis, using multipoint allele-sharing methods, was conducted for four phenotype criteria in 207 extended Icelandic osteoporotic families, containing 1,323 study individuals (see Materials and Methods
). Descriptions of the phenotypes and pedigree sets along with a summary of the linkage results are provided in . We first investigated osteoporosis as it is broadly defined (moderate pedigree set). This is the most generalized osteoporosis phenotype, including individuals with a hip and spine BMD approximately one standard deviation (SD) or more below the average and/or those with osteoporotic fractures and/or those receiving bisphosphonate treatment for osteoporosis. The most prominent peak was on Chromosome 20 at D20S905 (19.90 cM) with an allele-sharing logarithm of the odds (LOD) score of 3.39 (p
value, 4.2 × 10−5
), with four other locations, achieving a LOD score of 1.5 or greater: 16q, two on 18p, and 21q (; ).
Genome-Wide Scan Results and Fine-Mapping on Chromosome 20p12
We next used a definition of a more severe phenotype, with only individuals in the lower 10th percentile of BMD as affected members, but including fracture patients and patients treated for osteoporosis as in the previous run. The most striking feature of this scan was the increase in the peak on Chromosome 20p () that was again at D20S905, but now with a LOD score of 4.93. Two new LOD peaks were observed on Chromosomes 6p and 17p (; ). Compared to the LOD score peaks we had observed when analyzing the moderate pedigree set, for the severe pedigree set both of the peaks on 18p were greatly attenuated, the peak on 16p persisted, and the peak on 21p dropped by about a LOD of 0.5. Two additional analyses were conducted, one considering only hip osteoporosis (hip pedigree set) and the other considering only spine osteoporosis (spine pedigree set). In both cases, the strongest linkage was to Chromosome 20p, but with slightly higher LOD score (3.18) for the hip analysis ().
The results at Chromosome 20p were encouraging, so we decided to genotype 30 additional markers in the region in order to increase the information on identity by descent sharing. At this increased fine-mapping density, the information on sharing was over 95% and the LOD score rose to 5.10 (p
value, 6.3 × 10−7
) at D20S194 (20.35 cM) in the severe phenotype. The LOD score increased for all phenotypes, to 3.99, 3.99, and 3.43 for the moderate, the hip, and the spine pedigree sets, respectively. After applying a Bonferonni adjustment for the four phenotypes tested, the p
value for linkage is 1.6 × 10−6
, which is genome-wide significant according to the dense marker threshold for multipoint genome scans (Lander and Kruglyak 1995
Linkage Disequilibrium Mapping, Association Analysis, and BMP2 Variants
We investigated a 6.6 cM region surrounding our linkage peak that corresponds to a 1.7 Mb segment. This region, which includes up to a drop of 1 in LOD score from the linkage peak on the centromeric side and slightly less on the telomeric side, contains six known genes: BMP2
, LOC51605, C20orf154
, and C20orf42
(B). The BMP2
gene is a strong candidate gene, based on its role in bone formation and osteoblast differentiation (Wozney et al. 1988
; Katagiri et al. 1994
; Fujii et al. 1999
). However, expression analysis showed that four of the genes in the region are expressed in bone marrow or in an osteoblast cell line (BMP2
, and CHGB
). Therefore, the expression data did not help us narrow the list of candidate genes.
The Chromosome 20p12 Linkage Region
To decide which gene in the focused region is most likely to contribute to osteoporosis, we performed a case–control association study by typing many additional polymorphic markers. We used the severe-linkage phenotype as affection status and included all available genotyped patients, including additional familial cases and sporadic cases, in the analysis, 705 in total. BMP2 was our leading candidate, and we genotyped two microsatellites and 15 single nucleotide polymorphisms (SNPs) in the 15 kb region containing BMP2. The other five genes clustered in a 220 kb region. For this region of genes, we genotyped nine microsatellites and 26 SNPs. We genotyped additional markers across the entire region in case there were other unidentified genes or nongene regions associated with the disease. In total in the 1.7 Mb region, we genotyped 41 microsatellites, 99 SNPs that are polymorphic in the Icelandic population (some of the public SNPs are exonic), and 20 other SNPs that we identified by screening all exons and flanking intronic sequences of all six genes or expressed sequence tag (EST) matches. B displays the location of the microsatellites and SNPs used in this analysis relative to the gene locations. Single-marker association and haplotype analyses showed the strongest association to the region of the BMP2 gene (). The BMP2 gene appears to be the only gene within this region, despite our extensive efforts to find additional transcripts (see below). Linkage disequilibrium (LD) analysis across the region, including some SNPs added subsequently, identified about ten blocks of LD, ranging from 10 kb to 250 kb in size. Further blocks of LD are likely to exist in some gaps where no polymorphic markers were found, but these locations tend to be “gene-free.”
Association Analysis in the Focused Region
In order to further understand the haplotype associations and to search for functional variations or SNPs that might capture the risk of osteoporosis, we sequenced the BMP2
gene in 188 patients and 94 controls. The sequenced region covered the 15 kb containing the BMP2
gene, including all exons, introns, and the promoter region, along with another 44 kb of DNA flanking the gene. We genotyped all patients and controls for 63 SNPs from among those identified within the 59 kb region, including SNPs that we had used in this region in the previous analysis. Three of the SNPs change amino acids in BMP2: Ser37Ala, due to a T to G transversion at nucleotide position 116 in exon 2; Ala94Ser, due to a G to T transversion at nucleotide position 287 in exon 2; and Arg189Ser, due to an A to T transversion at nucleotide position 224 in exon 3. The Ala94Ser variant was only found in one family. The Arg189Ser variant is very common and does not show any association to osteoporosis on its own. The Ser37Ala variant, however, changes a conserved amino acid and shows significant association to osteoporosis. It is a relatively rare variant, with allele frequency ranging from 3.0% to 4.9% in patients, depending on the phenotype, versus 0.8% in controls, yielding relative risks (RRs) in the range of 3.8 to 6.3 (); four of the seven phenotypes tested produce p
values smaller than 0.001 after adjusting for the relatedness of the affected (see Materials and Methods
). Given that Ser37Ala is only one of three missense variants identified in BMP2
, we consider this result statistically significant even with adjustment for multiple comparisons.
Association Results for BMP2 SNP Haplotypes and Ser37Ala Missense Variant
Since Ser37Ala is rare and can only account for a small fraction of the affected, we believed there had to be other at-risk variants of BMP2. Through our effort to search for these other variants, we found three SNP haplotypes covering the BMP2 gene that show the strongest association to osteoporosis (; A). The three haplotypes, hapA, hapB, and hapC, defined by a total of six SNPs, are essentially independent of each other (pairwise R2, less than 0.01). The Ser37Ala variant, however, seems to have arisen on the background of hapA so that every chromosome that carries Ser37Ala also carries hapA, but not vice versa. Moreover, it appears that the excess of hapA in the affected can be explained by Ser37Ala; i.e., chromosomes with hapA but not Ser37Ala do not have significant excess in the affected versus controls. The hapB is defined by three SNPs—TSC0271643 (T), P9313 (T), and rs235764 (G)—and spans from within intron 2 of BMP2 and 263 kb upstream of the gene, going across LD blocks (). The upstream SNP is in the region of the most significant single-point microsatellite association. HapC is defined by two SNPs, rs1116867 (A) and D35548 (T), downstream of BMP2 and spans 18 kb (). HapC is entirely contained within one LD block. There is, however, significant LD between some markers across the hapC LD block and the BMP2 LD block. BMP2 is the only known or predicted gene within the segments spanned by the two haplotypes, as previously noted.
To explore the role of Ser37Ala and hapB and hapC in relation to various forms of osteoporosis, we used several other osteoporotic phenotypes in the association analysis, including osteoporotic fractures, whole-body BMD, and pre- and postmenopausal BMD, each analyzed separately (see ). The Ser37Ala variant and hapC are associated with all low BMD phenotypes and with osteoporotic fractures. In fact, the RR for low BMD is higher in premenopausal women than postmenopausal women, indicating that BMP2 may influence the attainment of peak bone mass. HapB, on the other hand, seems more associated with osteoporotic fractures than with BMD phenotypes.
Removing patients who carry the Ser37Ala variant or the disease-associated haplotypes from the linkage analysis, leaving 152 affected in families informative for linkage, the LOD score drops from 5.10 to 3.55. Hence, these variants alone cannot account for the entire linkage signal. Even taking into consideration that there is usually a substantial upward bias in estimation of locus-specific effects from genome scans (Goring et al. 2001
), we believe there are likely to be other variants, probably rare but possibly having very high penetrance, which have yet to be identified. We also cannot rule out that other genes in the region are conferring osteoporosis risk independently or interacting with BMP2
. We have not yet determined the underlying functional variations of disease-associated hapB and hapC. Most importantly, as anonymous haplotypes, proper interpretation of the statistical significance of hapB and hapC has to take multiple comparisons into account. By performing a randomization test (randomizing the patients and controls) for a procedure that considers all haplotypes within the LD block that contains hapC, the smallest adjusted p
value is 0.003 for whole-body BMD (see ), 125 times larger than the unadjusted p
value of 2.4 × 10−5
. Since this adjusted p
value still has not taken into account the relatedness of the patients, the multiple phenotypes tested, and the other LD blocks in the region, we consider hapC at best marginally significant with the Icelandic data alone. HapB goes across LD blocks, and the randomization procedure is performed by considering all possible haplotypes within the region spanned by those blocks, leading to an even larger adjustment factor. As a result, none of the adjusted p
values for hapB are significant (see ). Hence, instead of haplotypes already established to confer increased risk, we considered hapB and hapC only as candidate at-risk haplotypes, identified based on the Icelandic material that required confirmation from a replication study.
Haplotype Confirmation in Danish Cohort
For replication, we used a cohort of Danish postmenopausal women with persistently low BMD and a group of Danish postmenopausal osteoporotic fracture patients. We only typed those SNPs that comprised hapB and hapC as well as the Ser37Ala variant. The results for the Danish study are also shown in . For all six tests, two phenotypes times three variants, the patients have a higher frequency of the variant compared to the controls, and the estimated RRs are overall comparable to those observed in the Icelanders. However, the p values are higher in general due to the smaller sample sizes of the Danish cohort. Nonetheless, hapC gives a p value of 0.0038 for low BMD, which is significant even after adjusting for the six tests performed. The Ser37Ala missense variant is nominally significant for osteoporotic fractures, with an estimated RR of 4.2, and hapB is nominally significant for low BMD, with an estimated RR of 2.1.
Another way of handling the problem of testing multiple variants is to consider them as a group. Define a composite haplotype, hapX, as a chromosome that carries at least one of either the Ser37Ala missense variant, hapB, or hapC. For the Danish cohort, hapX is estimated to have a frequency of 16.3% in the low BMD patients versus 7.3% in controls, giving a one-sided p value of 0.004 with an RR of 2.5. The frequency of hapX in the osteoporotic fracture patients is estimated to be 15.3%, giving a one-sided p value of 0.037 with an RR of 2.2.