Liesbeth Van Wesenbeeck

Six novel missense mutations in the LDL receptor-related protein 5 (LRP5) gene in different conditions with an increased bone density.

Bone is a dynamic tissue that is subject to the balanced processes of bone formation and bone resorption. Imbalance can give rise to skeletal pathologies with increased bone density. In recent years, several genes underlying such sclerosing bone disorders have been identified. The LDL receptor-related protein 5 (LRP5) gene has been shown to be involved in both osteoporosis-pseudoglioma syndrome and the high-bone-mass phenotype and turned out to be an important regulator of peak bone mass in vertebrates. We performed mutation analysis of the LRP5 gene in 10 families or isolated patients with different conditions with an increased bone density, including endosteal hyperostosis, Van Buchem disease, autosomal dominant osteosclerosis, and osteopetrosis type I. Direct sequencing of the LRP5 gene revealed 19 sequence variants. Thirteen of these were confirmed as polymorphisms, but six novel missense mutations (D111Y, G171R, A214T, A214V, A242T, and T253I) are most likely disease causing. Like the previously reported mutation (G171V) that causes the high-bone-mass phenotype, all mutations are located in the aminoterminal part of the gene, before the first epidermal growth factor-like domain. These results indicate that, despite the different diagnoses that can be made, conditions with an increased bone density affecting mainly the cortices of the long bones and the skull are often caused by mutations in the LRP5 gene. Functional analysis of the effects of the various mutations will be of interest, to evaluate whether all the mutations give rise to the same pathogenic mechanism.

Involvement of PLEKHM1 in osteoclastic vesicular transport and osteopetrosis in incisors absent rats and humans

This study illustrates that Plekhm1 is an essential protein for bone resorption, as loss-of-function mutations were found to underlie the osteopetrotic phenotype of the incisors absent rat as well as an intermediate type of human osteopetrosis. Electron and confocal microscopic analysis demonstrated that monocytes from a patient homozygous for the mutation differentiated into osteoclasts normally, but when cultured on dentine discs, the osteoclasts failed to form ruffled borders and showed little evidence of bone resorption. The presence of both RUN and pleckstrin homology domains suggests that Plekhm1 may be linked to small GTPase signaling. We found that Plekhm1 colocalized with Rab7 to late endosomal/lysosomal vesicles in HEK293 and osteoclast-like cells, an effect that was dependent on the prenylation of Rab7. In conclusion, we believe PLEKHM1 to be a novel gene implicated in the development of osteopetrosis, with a putative critical function in vesicular transport in the osteoclast.

The Binding Between Sclerostin and LRP5 is Altered by DKK1 and by High-Bone Mass LRP5 Mutations

Low-density lipoprotein receptor–related protein 5 (LRP5), a Wnt coreceptor, plays an important role in bone metabolism as loss-of-function and gain-of-function mutations in LRP5 result in the autosomal recessive osteoporosis-pseudoglioma syndrome and autosomal dominant high–bone mass (HBM) phenotypes, respectively. Prior studies suggested that the presence of HBM-associated LRP5 mutations results in decreased antagonism of LRP5-mediated Wnt signaling. In the present study, we investigated six different HBM-LRP5 mutations and confirm that neither Dickkopf1 (DKK1) nor sclerostin efficiently inhibits HBM-LRP5 signaling. In addition, when coexpressed, DKK1 and sclerostin do not inhibit HBM-LRP5 mutants better than either inhibitor by itself. Also, DKK1 and sclerostin do not simultaneously bind to wild-type LRP5, and DKK1 is able to displace sclerostin from previously formed sclerostin–LRP5 complexes. In conclusion, our results indicate that DKK1 and sclerostin are independent, and not synergistic, regulators of LRP5 signaling and that the function of each is impaired by HBM-LRP5 mutations.

The osteopetrotic mutation toothless (tl) is a loss-of-function frameshift mutation in the rat Csf1 gene: Evidence of a crucial role for CSF-1 in osteoclastogenesis and endochondral ossification

The toothless (tl) mutation in the rat is a naturally occurring, autosomal recessive mutation resulting in a profound deficiency of bone-resorbing osteoclasts and peritoneal macrophages. The failure to resorb bone produces severe, unrelenting osteopetrosis, with a highly sclerotic skeleton, lack of marrow spaces, failure of tooth eruption, and other pathologies. Injections of CSF-1 improve some, but not all, of these. In this report we have used polymorphism mapping, sequencing, and expression studies to identify the genetic lesion in the tl rat. We found a 10-base insertion near the beginning of the open reading of the Csf1 gene that yields a truncated, nonfunctional protein and an early stop codon, thus rendering the tl rat CSF-1null. All mutants were homozygous for the mutation and all carriers were heterozygous. No CSF-1 transcripts were identified in rat mRNA that would avoid the mutation via alternative splicing. The biology and actions of CSF-1 have been elucidated by many studies that use another naturally occurring mutation, the op mouse, in which a single base insertion also disrupts the reading frame. The op mouse has milder osteoclastopenia and osteopetrosis than the tl rat and recovers spontaneously over the first few months of life. Thus, the tl rat provides a second model in which the functions of CSF-1 can be studied. Understanding the similarities and differences in the phenotypes of these two models will be important to advancing our knowledge of the many actions of CSF-1.

Localization of the gene causing autosomal dominant osteopetrosis type I to chromosome 11q12-13.

The osteopetroses are a heterogeneous group of genetic conditions characterized by increased bone density due to impaired bone resorption by osteoclasts. Within the autosomal dominant form of osteopetrosis, the radiological type I (ADOI) is characterized by a generalized osteosclerosis, most pronounced at the cranial vault. The patients are often asymptomatic but some suffer from pain and hearing loss. ADOI is the only type of osteopetrosis not associated with an increased fracture rate. Linkage analysis in two families with ADOI from Danish origin enabled us to assign the disease‐causing gene to chromosome 11q12‐13. A summated maximum lod score of +6.54 was obtained with marker D11S1889 and key recombinants allowed delineation of a candidate region of 6.6 cM between markers D11S1765 and D11S4113. Previously, genes causing other conditions with abnormal bone density have been identified from this chromosomal region. The TCIRG1gene was shown to underly autosomal recessive osteopetrosis (ARO), and, recently, mutations in the LRP5gene were found both in the osteoporosis‐pseudoglioma syndrome and the high bone mass trait. Because both genes map within the candidate region for ADOI, it can not be excluded that ADOI is caused by mutations in either the TCIRG1or the LRP5gene.

A new heterozygous mutation (R714C) of the osteopetrosis gene, pleckstrin homolog domain containing family M (with run domain) member 1 (PLEKHM1), impairs vesicular acidification and increases TRACP secretion in osteoclasts.

We studied phenotypic and cellular aspects in a patient with a heterozygous mutation of the PLEKHM1 gene and obtained some indications regarding the role of the protein in bone cell function. Plekhm1 is involved in osteoclast endosomal vesicle acidification and TRACP exocytosis, contributing to events involved in osteoclast–osteoblast cross‐talk.

Genetic variation in the **TNFRSF11A** gene encoding RANK is associated with susceptibility to Paget's disease of bone

RANK (receptor activator of nuclear factor‐κB), encoded by TNFRSF11A, is a key protein in osteoclastogenesis. TNFRSF11A mutations cause Pagets disease of bone (PDB)–like diseases (ie, familial expansile osteolysis, expansile skeletal hyperphosphatasia, and early‐onset PDB) and an osteoclast‐poor form of osteopetrosis. However, no TNFRSF11A mutations have been found in classic PDB, neither in familial nor in isolated cases. To investigate the possible relationship between TNFRSF11A polymorphisms and sporadic PDB, we conducted an association study including 32 single‐nucleotide polymorphisms (SNPs) in 196 Belgian sporadic PDB patients and 212 control individuals. Thirteen SNPs and 3 multimarker tests (MMTs) turned out to have a p value of between .036 and 3.17 × 10−4, with the major effect coming from females. Moreover, 6 SNPs and 1 MMT withstood the Bonferroni correction (p < .002). Replication studies were performed for 2 nonsynonymous SNPs (rs35211496 and rs1805034) in a Dutch and a British cohort. Interestingly, both SNPs resulted in p values ranging from .013 to 8.38 × 10−5 in both populations. Meta‐analysis over three populations resulted in p = .002 for rs35211496 and p = 1.27 × 10−8 for rs1805034, again mainly coming from the female subgroups. In an attempt to identify the underlying causative SNP, we performed functional studies for the coding SNPs as well as resequencing efforts of a 31‐kb region harboring a risk haplotype within the Belgian females. However, neither approach resulted in significant evidence for the causality of any of the tested genetic variants. Therefore, further studies are needed to identify the real cause of the increased risk to develop PDB shown to be present within TNFRSF11A.

Localization of the Gene Causing the Osteopetrotic Phenotype in the Incisors Absent (Ia) Rat on Chromosome 10q32.1

The incisors absent rat is an osteopetrotic animal model. Segregation analysis in 37 affected animals from an outcross enabled us to assign the disease causing gene to a 4.7‐cM interval on rat chromosome 10q32.1. Further analysis of the genes mapped in this region will provide more insight into the underlying pathogenesis.

A new familial sclerosing bone dysplasia

Osteoscleroses are a heterogeneous group of bone remodeling disorders characterized by an increase in bone density. Here we report on a consanguineous Lebanese family in which two sisters, aged 39 and 36 years, exhibit a severe genu varum, a square‐face appearance, high forehead, slight proptosis of the eyes, symmetric enlargement of the jaw, protruding chin, and short stature. Bone X‐rays showed the presence of hyperostosis of the cranial base and vault with increased density of the orbits, hyperostosis of the bones, thickening of the cortices, diaphyseal modeling defects, cortical thickening of the medullary cavity, mild enlargement of the medullary cavity of the short long bones, short femoral necks, increased width of the ribs, and narrow interpedicular distances of the lower lumbar spine. Osteodensitometry showed values 200% to 300% above values for age. A cervical MRI revealed the presence of a diffuse osteosclerosis with calcification of the posterior vertebral ligament and a narrow canal between C2 and T2. Blood test results were unremarkable. Serum osteocalcin levels were in the normal range, whereas high values of serum C‐telopeptide were noted. A bone biopsy showed only the presence of compact bone and did not allow for histomorphometric analysis. Molecular studies excluded genes known to be involved in sclerosing bone dysplasias as the cause of this condition. In vitro analysis of osteoclast function indicated that contrary to most cases of autosomal recessive osteopetrosis, osteoclasts both formed and resorbed but exhibited a small decrease in resorptive activity compared with osteoclasts generated from normal control individuals. Differential diagnoses are discussed, and the possibility that this may be a novel clinical entity is raised.