Clinical and molecular findings in osteoporosis-pseudoglioma syndrome

Abstract

Mutations in the low-density lipoprotein receptor-related protein 5 gene (LRP5) cause autosomal recessive osteoporosis-pseudoglioma syndrome (OPPG). We sequenced the coding exons of LRP5 in 37 probands suspected of having OPPG on the basis of the co-occurrence of severe congenital or childhood-onset visual impairment with bone fragility or osteoporosis recognized by young adulthood. We found two putative mutant alleles in 26 probands, only one mutant allele in 4 probands, and no mutant alleles in 7 probands. Looking for digenic inheritance, we sequenced the genes encoding the functionally related receptor LRP6, an LRP5 coreceptor FZD4, and an LRP5 ligand, NDP, in the four probands with one mutant allele, and, looking for locus heterogeneity, we sequenced FZD4 and NDP in the seven probands with no mutations, but we found no additional mutations. When we compared clinical features between probands with and without LRP5 mutations, we found no difference in the severity of skeletal disease, prevalence of cognitive impairment, or family history of consanguinity. However, four of the seven probands without detectable mutations had eye pathology that differed from pathology previously described for OPPG. Since many LRP5 mutations are missense changes, to differentiate between a disease-causing mutation and a benign variant, we measured the ability of wild-type and mutant LRP5 to transduce Wnt and Norrin signal ex vivo. Each of the seven OPPG mutations tested, had reduced signal transduction compared with wild-type mutations. These results indicate that early bilateral vitreoretinal eye pathology coupled with skeletal fragility is a strong predictor of LRP5 mutation and that mutations in LRP5 cause OPPG by impairing Wnt and Norrin signal transduction.

Figure 1

Disease-associated missense mutations in LRP5. A, Schematic depiction of LRP5 protein and the sites of missense amino acid substitutions that have been associated with OPPG. Homozygous mutations in patients with OPPG are noted in bold above the protein. Heterozygous mutations in patients with OPPG are shown below the protein. Two missense mutations that occur in cis on a single allele are marked with an asterisk (*). Missense mutations that are associated with autosomal dominant FEVR are shown in italics, and mutations associated with autosomal recessive FEVR are in bold italics. All underlined mutations were tested ex vivo. B, Single-letter amino acid ClustalW alignments of residues surrounding the 20 OPPG-associated missense mutations for LRP5, LRP6, and arrow. The amino acid residues altered by the missense mutations are shaded in the species in which the residue is evolutionarily conserved. The seven putative OPPG-causing mutations that were tested ex vivo are shown in the upper half of the panel.

Figure 2

Trafficking and posttranslational modification of OPPG-LRP5 mutants in 293T cells. A, WT and OPPG-causing LRP5 constructs that express a myc-tagged, truncated polypeptide lacking the transmembrane and cytoplasmic domains (LRP5N-myc) were transiently transfected into 293T cells. Western-blot analyses were performed to detect the recombinant protein in the conditioned medium (“CM”) and cell lysate (“Lysate”) with use of an anti-myc antibody. Note the relative efficiencies of different mutants to transit the cell and be secreted into the conditioned medium compared with the WT protein. T390K is present in the cell lysate but is not secreted into the conditioned medium, whereas G520V is secreted into the conditioned medium at rates comparable to WT. Equal loading of cell lysate and conditioned medium in each lane is demonstrated by immunodetection of cell lysate with an anti-tubulin antibody, D-10-HRP (Santa Cruz Biotechnology), and Coomassie staining of conditioned medium, respectively. B, Western-blot analysis of conditioned medium (“CM”) and cell lysate (“LY”) from 293T cells expressing WT or G520V LRP5N-myc protein. Note that, when the conditioned medium was digested with N-glycosidase (“DG”), the molecular weight of the secreted protein was decreased. Other OPPG-associated mutant proteins were tested in the same assay and gave similar results (data not shown).

Figure 3

OPPG-causing missense mutations impair Wnt and Norrin signaling. Fold-induction of luciferase activity in 293T cells expressing WT or OPPG-causing missense mutants are expressed in combination with Wnt1-v5 (A), Wnt10b (B), and Norrin and Fzd4 (C). Thirty hours after transfection, firefly luciferase activity was measured and normalized to Renilla luciferase activity. Note that four mutants have no signal transduction and that three (G404R, D434N, and G610R) have markedly reduced signal transduction (white bars); WT-LRP5 signal transduction was not inhibited when coexpressed with an OPPG mutant (gray bars), which indicates that the OPPG mutants do not exert a dominant negative effect (A and B). C, OPPG mutants have reduced ability to transduce Norrin signal. D, Western blot of cell lysates from transfected 293T cells used in the Wnt1-v5 signal transduction assay shown in panel A, demonstrating comparable expression of WT and OPPG-causing LRP5 receptors. Lysates were separated by SDS-PAGE and were immunodetected with an anti-LRP5/LRP6 antibody, 3801–100 (Biovision). An anti-tubulin antibody was used to demonstrate that comparable amounts of cell lysates were loaded.

Figure 4

FEVR-causing missense mutations variably affect Wnt and Norrin signaling. Fold-induction of luciferase activity in 293T cells expressing WT or FEVR-causing missense mutants in combination with Norrin and Fzd4 (A) or Wnt1-v5 (B). Mutants associated with autosomal recessive FEVR are underlined. Note that the Y1168H mutant could not transduce Norrin or Wnt signal, and mutants T173M and E1367K could transduce signal from both ligands (white bars). Coexpression of WT and FEVR-causing mutant receptors did not interfere with the WT receptor’s ability to transduce Norrin or Wnt-v5 signal (gray bars), which indicates that these mutations do not have dominant negative effects.