A mutation in the LDL receptor-related protein 5 gene results in the autosomal dominant high-bone-mass trait

Abstract

Osteoporosis is a complex disease that affects >10 million people in the United States and results in 1.5 million fractures annually. In addition, the high prevalence of osteopenia (low bone mass) in the general population places a large number of people at risk for developing the disease. In an effort to identify genetic factors influencing bone density, we characterized a family that includes individuals who possess exceptionally dense bones but are otherwise phenotypically normal. This high-bone-mass trait (HBM) was originally localized by linkage analysis to chromosome 11q12-13. We refined the interval by extending the pedigree and genotyping additional markers. A systematic search for mutations that segregated with the HBM phenotype uncovered an amino acid change, in a predicted beta-propeller module of the low-density lipoprotein receptor-related protein 5 (LRP5), that results in the HBM phenotype. During analysis of >1,000 individuals, this mutation was observed only in affected individuals from the HBM kindred. By use of in situ hybridization to rat tibia, expression of LRP5 was detected in areas of bone involved in remodeling. Our findings suggest that the HBM mutation confers a unique osteogenic activity in bone remodeling, and this understanding may facilitate the development of novel therapies for the treatment of osteoporosis.

Figure 1

BMD measurements. A plot of the hip versus spine BMD measurements of the family with HBM is shown. Blackened diamonds indicate individuals with the mutation, and shaded squares indicate individuals without the mutation. The criterion used for assigning affection status was a sum >4 of Z scores from hip and spine, as indicated by the diagonal line.

Figure 2

HBM pedigree and haplotypes of the individuals used in the genetic–linkage studies. Blackened symbols represent affected individuals. Symbols containing “N” indicate unaffected individuals. Numbers beneath the symbols show the identification numbers, the Z scores for the spinal and hip BMD, and the alleles for the critical markers on chromosome 11. Question marks (?) denote unknown affection status, genotype, or phase. Untyped genotypes inferred with certainty are included in parentheses. The striped haplotype is shared by all affected individuals; critical crossovers were identified in individuals 44 and 46. Asterisks (*) indicate the 16 additional individuals used to narrow the region. Arrow indicates the proband.

Figure 3

Physical map of the HBM interval. STS markers derived from genes, expressed-sequence tags, microsatellites, random sequences, and BAC end sequences are denoted above the long horizontal line. STSs derived from BAC end sequences are listed with the BAC library address followed by L or R, for the left or right end of the clone, respectively; end sequences derived from BACs from the Genome Systems library are indicated with GS. The two large arrows indicate the location of genetic markers that define the HBM critical region. The horizontal lines below the STSs indicate a minimal tiling path of BAC clones that were sequenced. Open circles indicate that the marker did not amplify from the corresponding BAC. All clones were from the CITB library except for the two preceded by an RPCI-11 prefix. A region that was underrepresented in the library is indicated as a gap. Genes that were analyzed for mutations are shown in their approximate location at the top of the figure. Genes with a prefix of Hs correspond to UniGene entries.

Figure 4

HBM mutation and domain structure of the LRP5 protein. A, Sequence traces illustrating the HBM mutation (arrow) for a core four-member family. Identification numbers are as in figure 2. B, Domain organization of LRP5. The site of the glycine-to-valine change that occurs in the HBM protein is indicated with an arrow. C, Similarity of LRP5 and LDLR. A ribbon representation of LDLR, in which colors are based on homology with the first propeller (and EGF) domains of LRP5, is shown as viewed from the top. Identities are shown in red, and similarities are shown in pink. The location of G516 of the mature LDLR protein (comparable to residue G171 in LRP5) is shown in green. D, Simulation of the LRTP5 G171V mutation in the LDLR YWTD-EGF domain pair. The glycine at amino acid 516 of the mature LDLR protein was replaced with a valine, and the resulting three-dimensional structures were superimposed and viewed from the side. The wild-type structure is shown in blue with G516 in green, and the G516V mutant structure is shown in white with the V516 residue in red.

Figure 5

Northern blot analyses showing the expression of LRP5 in various tissues.

Figure 6

In situ hybridization of the LRP5 gene in bone tissue. A, Localization of LRP5 transcripts in rat endosteum by in situ hybridization with antisense and sense probes; original magnification ×400. Arrow indicates bone-lining cells. B, Localization of LRP5 transcripts in rat metaphysis and growth plate by in situ hybridization with antisense and sense probes; original magnification ×100. Boxes enclose regions that are shown at higher magnification in panel C. C, Localization of LRP5 transcripts in rat proximal metaphysis by in situ hybridization with antisense and sense probes; original magnification ×400.