Susceptibility to Paget's disease of bone is influenced by a common polymorphic variant of osteoprotegerin.

To clarify the role of the TNFRSF11B gene encoding osteoprotegerin (OPG), in Paget's disease of bone (PDB) we studied TNFRSF11B polymorphisms in an association study of 690 UK subjects and in a worldwide familial study of 66 kindreds. We found that the G1181 allele of TNFRSF11B, encoding lysine at codon 3 of the OPG protein, predisposes to both sporadic and familial PDB. INTRODUCTION: Paget's disease of bone (PDB) is a common disorder characterized by focal abnormalities of bone turnover. Genetic factors are important in the pathogenesis of PDB, and studies have shown that inactivating mutations of the TNFRSF11B gene, encoding osteoprotegerin (OPG), cause the rare syndrome of juvenile Paget's disease. In this study, we sought to determine whether polymorphisms of the TNFRSF11B gene contribute to the pathogenesis of classical PDB. MATERIALS AND METHODS: We screened for polymorphisms of the TNFRSF11B gene by DNA sequencing of the proximal promoter, coding exons, and intron-exon boundaries in 20 PDB patients and 10 controls. Informative single nucleotide polymorphisms (SNPs), including a G1181C SNP, which predicts a lysine-asparagine substitution at codon 3 of the OPG signal peptide and haplotypes, were related to the presence of PDB in 312 cases compared with 378 controls and to transmission of PDB in 140 affected offspring from 66 kindreds with familial PDB. RESULTS AND CONCLUSIONS: The G1181 allele was significantly over-represented in PDB patients (chi(2) = 5.7, df = 1, p = 0.017, adjusted alpha = 0.024), equivalent to an odds ratio for PDB of 1.55 (95% CI: 1.11-2.16). The distribution of TNFRSF11B haplotypes significantly differed in sporadic PDB cases and controls (chi(2) = 30.2, df = 9, p < 0.001) because of over-representation of haplotypes containing the G1181 allele in cases. The family study showed that the most common haplotype containing the G1181 allele was transmitted more frequently than expected to 140 individuals with familial PDB (chi(2) = 7.35, df = 1, p < 0.01), and the transmission disequilibrium was even more pronounced in a subgroup of 78 familial PDB patients who did not carry mutations of the SQSTM1 gene (chi(2) = 8.44, df = 1, p < 0.005). We conclude that the G1181 allele of TNFRSF11B, encoding lysine at codon 3 of the OPG protein, predisposes to the development of sporadic PDB and familial PDB that is not caused by SQSTM1 mutations.     

Somatic Mutations in SQSTM1 Detected in Affected Tissues From Patients With Sporadic Paget's Disease of Bone

Paget's disease of bone (PDB) is a focal disorder of bone remodeling that leads to overgrowth of affected bone, with rare progression to osteosarcoma. Extensive studies of familial PDB showed that a majority of cases harbor germline mutations in the Sequestosome1 gene (SQSTM1). In contrast, little is known about the mutational status of SQSTM1 in sporadic PDB. We hypothesized that somatic SQSTM1 mutations might occur in the affected tissues of sporadic PDB and pagetic osteosarcoma. We used laser capture microdissection to capture homogeneous populations of cells from the affected bone or tumor of patients with sporadic PDB or pagetic osteosarcoma, respectively. DNA from these samples and appropriate controls was used for sequence analysis and allelic discrimination analysis. Two of five patients with sporadic PDB had SQSTM1^C1215T mutations detected in their affected bone but not in their blood samples, indicating a somatic origin of the mutations. Samples from three of five sporadic pagetic osteosarcoma patients had the SQSTM1^C1215T mutation, whereas the normal adjacent tissue from two of these tumors clearly lacked the mutation, again indicating an occurrence of somatic events. No SQSTM1 mutations were found in primary adolescent osteosarcomas. The discovery of somatic SQSTM1 mutations in sporadic PDB and pagetic osteosarcoma shows a role for SQSTM1 in both sporadic and inherited PDB. The discovery of somatically acquired mutations in both the diseased bone and tumor samples suggests a paradigm shift in our understanding of this disease.     

Intragenic SNP haplotypes associated with 84dup18 mutation in <Emphasis Type="Italic">TNFRSF11A</Emphasis> in four FEO pedigrees suggest three independent origins for this mutation

Familial expansile osteolysis (FEO) is a rare disorder causing bone dysplasia. The clinical features of FEO include early-onset hearing loss, tooth destruction, and progressive lytic expansion within limb bones causing pain, fracture, and deformity. An 18-bp duplication in the first exon of the TNFRSF11A gene encoding RANK has been previously identified in four FEO pedigrees. Despite having the identical mutation, phenotypic variations among affected individuals of the same and different pedigrees were noted. Another 18-bp duplication, one base proximal to the duplication previously reported, was subsequently found in two unrelated FEO patients. Finally, mutations overlapping with the mutations found in the FEO pedigrees have been found in ESH and early-onset PDB pedigrees. An Iranian FEO pedigree that contains six affected individuals dispersed in three generations has previously been introduced; here, the clinical features of the proband are reported in greater detail, and the genetic defect of the pedigree is presented. Direct sequencing of the entire coding region and upstream and downstream noncoding regions of TNFRSF11A in her DNA revealed the same 18-bp duplication mutation as previously found in the four FEO pedigrees. Additionally, eight sequence variations as compared to the TNFRSF11A reference sequence were identified, and a haplotype linked to the mutation based on these variations was defined. Although the mutation in the Iranian and four of the previously described FEO pedigrees was the same, haplotypes based on the intragenic SNPs suggest that the mutations do not share a common descent.     

Identification of sex-specific associations between polymorphisms of the osteoprotegerin gene, TNFRSF11B, and Paget's disease of bone.

We studied the role of TNFRSF11B polymorphisms on the risk to develop Paget's disease of bone in a Belgian study population. We observed no association in men, but a highly significant association was found in women, and this was confirmed in a population from the United Kingdom. INTRODUCTION: Juvenile Paget's disease has been shown to be caused by mutations in TNFRSF11B encoding osteoprotegerin. Although mutations in this gene have never been found in patients with typical Paget's disease of bone (PDB), there are indications that polymorphisms in TNFRSF11B might contribute to the risk of developing PDB. MATERIALS AND METHODS: We recruited a population of 131 Belgian patients with sporadic PDB and 171 Belgian controls. By means of the HapMap, we selected 17 SNPs that, in combination with four multimarker tests, contain most information on common genetic variation in TNFRSF11B. To replicate the findings observed in the Belgian study population, genotyping data of SNPs generated in a UK population were reanalyzed. RESULTS: In our Belgian study population, associations were found for two SNPs (rs11573871, rs1485286) and for one multimarker test involving rs1032129. When subsequently analyzing men and women separately, these associations turned out to be driven by women (56 cases, 78 controls). In addition, three other tagSNPs turned out to be associated in women only. These were rs2073617 (C950T), rs6415470, and rs11573869. Reanalysis of genotyping data from a UK study population indicated that the associations found for C950T and C1181G were also exclusively driven by women (146 cases, 216 controls). Meta-analysis provided evidence for risk increasing effects of the T allele of C950T and the G allele of C1181G in the female population (p = 0.002 and 0.003, respectively). The haplotypes formed by the SNPs associated in the Belgian population were also distributed differentially between female cases and controls. CONCLUSIONS: We showed for the first time that SNPs influencing the risk to develop PDB could be sex-specific. Further research is necessary to identify the causative variants in TNFRSF11B and to elucidate the molecular pathogenic mechanism.     

Two novel mutations at exon 8 of the Sequestosome 1 (SQSTM1) gene in an Italian series of patients affected by Paget's disease of bone (PDB).

PDB is genetically heterogeneous. Mutations of the sequestosome1 gene have been reported in sporadic and familial forms of Paget's in patients of French Canadian and British descent. Mutational analyses in different ethnic groups are needed to accurately investigate hereditary diseases. We describe two novel mutations of sequestosome1 in 62 Italian sporadic patients, confirming the role of the encoded protein in this disorder. INTRODUCTION: Paget's disease of bone (PDB) is a relatively common disease of bone metabolism reported to affect up to 3% of whites over 55 years of age. The disorder is genetically heterogeneous, and at present, there is scientific evidence that at least eight different human chromosomal loci are correlated with its pathogenesis. Mutations of the sequestosome1 (SQSTM1) gene were identified as responsible for most of the sporadic and familial forms of Paget in patients of French Canadian and British descent. Such mutations were located at exon 7 and 8 levels, encoding for the ubiquitin protein-binding domain (UBA) and representing a mutational hot spot area. MATERIALS AND METHODS: To verify the involvement of this gene in Italian subjects affected by PDB, we performed mutational analysis in 62 sporadic PDB cases. RESULTS: We described three different mutations at exon 8 level: P392L, already described in the French Canadian population and families predominantly of British descendent, and two novel mutations consisting of the amino acid substitutions M404V and G425R. No significant differences in the clinical history of PDB have been observed in patients with SQSTM1 mutations in respect to those without. CONCLUSIONS: Even though our findings suggest a minor involvement of the SQSTM1 gene in the pathogenesis of sporadic Italian Paget's cases, the identification of different significant mutations within the SQSTM1 gene in unrelated, but clinically similar individuals, offers extremely convincing evidence for a causal relationship between this gene and PDB. Longitudinal studies are needed to assess the penetrance of genotype/phenotype correlations. Our findings confirm the evidence of a clustered mutation area at this level in this disorder.     

Evaluation of the role of RANK and OPG genes in Paget's disease of bone

Paget's disease of bone (PDB) is one of the most common bone disorders in the western world. PDB is characterized by focal areas of increased osteoclastic bone resorption and bone formation, which leads to the formation of poorly structured bone. These abnormalities of bone turnover and structure predispose affected individuals to various complications including bone pain, deformity, pathological fracture, and an increased risk of osteosarcoma. One of the main mechanisms of osteoclast formation and activation involves the receptor activator of nuclear factor -kappaB (RANK)/RANK ligand (RANKL)/osteoprotegerin (OPG) pathway, where binding of RANKL to RANK results in the differentiation of osteoclast precursors. OPG, on the other hand, acts as an inhibitor of osteoclastogenesis by serving as a decoy receptor for RANKL. Recently, mutations in the RANK gene have been shown to cause familial expansile osteolysis, a rare bone disorder showing great similarity to PDB. We performed mutation analysis in the RANK and OPG genes in 28 PDB patients to investigate whether mutations in these genes could be responsible for PDB. Our data suggest that RANK is not directly involved in PDB in our set of patients, as no mutations in the RANK coding region could be identified and allele frequencies of RANK polymorphisms did not differ in PDB patients as compared with the random population. Also, in the OPG gene, we could not detect PDB-causing mutations. However, of the several polymorphisms identified, one (400 + 4 C/T in intron 2), showed a statistically significant increased frequency for the C allele in PDB patients, suggesting that individuals harboring this allele may be more susceptible for developing PDB.     

Pathogenesis of Paget's disease of bone

Paget's disease of bone is a common condition characterised by increased and disorganised bone turnover which can affect one or several bones throughout the skeleton. These abnormalities disrupt normal bone architecture and lead to various complications such as bone pain osteoarthritis, pathological fracture, bone deformity, deafness, and nerve compression syndromes. Genetic factors play an important role in PDB and mutations or polymorphisms have been identified in four genes that cause classical Paget's disease and related syndromes. These include TNFRSF11A, which encodes RANK, TNFRSF11B which encodes osteoprotegerin, VCP which encodes p97, and SQSTM1 which encodes p62. All of these genes play a role in the RANK-NFkappaB signalling pathway and it is likely that the mutations predispose to PDB by disrupting normal signalling, leading to osteoclast activation. Although Paget's has traditionally be considered a disease of the osteoclast there is evidence that stromal cell function and osteoblast function are also abnormal, which might account for the fact that the disease is associated with increased bone formation as well as resorption. Environmental factors also contribute to Paget's disease. Most research has focused on paramyxovirus infection as a possible environmental trigger but evidence in favour of the involvement of viruses in the disease remains conflicting. Other factors which have been implicated as possible disease triggers include mechanical loading, dietary calcium and environmental toxins. Further work will be required to identify additional genetic variants that predispose to Paget's disease and to determine how the causal mutations and predisposing polymorphisms interact with environmental factors to influence bone cell function and cause the focal bone lesions that are characteristic of the disease.     

Identification of a novel tandem duplication in exon 1 of the TNFRSF11A gene in two unrelated patients with familial expansile osteolysis.

Familial expansile osteolysis (FEO) is a rare autosomal dominant disorder characterized by striking focal expansile osteolytic bone lesions and generalized osteopenia, often accompanied by characteristic early hearing loss and dental disease. The TNFRSF11A gene encodes the receptor activator of nuclear factor-kappaB (RANK), which has been demonstrated to be essential in bone remodeling and osteoclast differentiation. Identical insertional mutations in the first exon of RANK have been identified in all published FEO kindreds. The mutation is an 18 base pair tandem duplication in the sequence coding for the signal peptide of RANK, which causes an increase in NF-kappaB signaling. We report the identification and mutational analysis of two unrelated FEO patients. One had no family history of FEO, but presented with bilateral hearing loss at an early age, deterioration of teeth, and severe pain and swelling in the distal tibia before the age of 20. The second patient had a family history of FEO and exhibited an extensive expansile tibial lesion and lesions in one humerus and a phalanx. She also had early hearing loss and dental disease. Mutational analysis of the TNFRSF11A gene in our patients demonstrated an 18 base pair tandem duplication, one base proximal to the duplications previously reported. This novel mutation results in addition of the same six amino acids to the RANK signal peptide that has been observed previously. Further analysis of the exon 1 sequence demonstrated that it has the ability to form a stable secondary structure that may facilitate the generation of tandem duplications.     

Mutations in Profilin 1 Cause Early-Onset Paget's Disease of Bone With Giant Cell Tumors

Paget's disease of bone (PDB) is a late-onset chronic progressive bone disease characterized by abnormal activation of osteoclasts that results in bone pain, deformities, and fractures. PDB is very rare in Asia. A subset of PDB patients have early onset and can develop malignant giant cell tumors (GCTs) of the bone (PDB/GCTs), which arise within Paget bone lesions; the result is a significantly higher mortality rate. SQSTM1, TNFRSF11A, OPG, VCP, and HNRNPA2B1 have been identified as pathogenic genes of PDB, and ZNF687 is the only confirmed gene to date known to cause PDB/GCT. However, the molecular mechanism underlying PDB/GCT has not been fully elucidated. Here, we investigate an extended Chinese pedigree with eight individuals affected by early-onset and polyostotic PDB, two of whom developed GCTs. We identified a heterozygous 4-bp deletion in the Profilin 1 (PFN1) gene (c.318_321delTGAC) by genetic linkage analysis and exome sequencing for the family. Sanger sequencing revealed another heterozygous 1-bp deletion in PFN1 (c.324_324delG) in a sporadic early-onset PDB/GCT patient, further proving its causative role. Interestingly, a heterozygous missense mutation of PFN1 (c.335 T > C) was identified in another PDB/GCT family, revealing that not only deletion but also missense mutations in PFN1 can cause PDB/GCT. Furthermore, we established a Pfn1-mutated mouse model (C57BL/6J mice) and successfully obtained Pagetic phenotypes in heterozygous mice, verifying loss of function of PFN1 as the cause of PDB/GCT development. In conclusion, our findings reveal mutations in PFN1 as the pathological mechanism in PDB/GCT, and we successfully established Pfn1-mutated mice as a suitable animal model for studying PDB-associated pathological mechanisms. The identification of PFN1 mutations has great diagnostic value for identifying PDB individuals predisposed toward developing GCTs. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).     

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 Paget's 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. © 2010 American Society for Bone and Mineral Research.     

Three novel mutations in SQSTM1 identified in familial Paget's disease of bone.

Mutations in Sequestosome 1 (SQSTM1) have been shown to segregate with familial Paget's disease of bone (PDB). We examined the coding sequence of SQSTM1 in five PDB pedigrees and found three novel mutations clustered around the C-terminal ubiquitin associated domain. Disruptions of the C-terminal domain of SQSTM1 seem to be a leading cause of familial PDB. INTRODUCTION: The characteristic features of Paget's disease of bone (PDB) are caused by focal areas of excessive and uncoordinated bone remodeling. A total of seven genetic loci (PDB1-PDB7) have been reported to be associated with the disease. The gene for Sequestosome 1 (p62; SQSTM1) has been identified as the causative gene for PDB3 in numerous French-Canadian families and families predominantly of British descent. To date, a total of three mutations, all affecting the ubiquitin-associated domain of SQSTM1, have been identified: a single 1215 C to T (P392L) transversion in exon 8, a T insertion in exon 8 (E396X), and a G to A mutation at the splice junction of exon 7 (IVS7 + 1). MATERIALS AND METHODS: DNA was isolated from blood collected from the members of five U.S. PDB pedigrees. Mutation analysis of the coding sequence of the SQSTM1 gene was performed on the proband and other key individuals in the pedigrees. RESULTS: Four of the five families had SQSTM1 mutations. Three of these mutations were novel: a single base deletion in exon 8 at position 1210 (1210delT) resulting in a premature stop codon at amino acid 394, a single C deletion in exon 8 at position 1215 (1215delC) also resulting in a premature stop codon at amino acid 394, and a single 1200 C to T (P387L) transversion in exon 7. CONCLUSION: Noteworthy is the fact that these three SQSTM1 mutations, in addition to the three previously described mutations, are clustered near the C-terminal of the protein. These mutations may be acting in a dominant-negative fashion to disrupt the ubiquitin-binding function, which could result in abnormal activation of the NF-kappaB pathway and the subsequent activation of the osteoclasts. These findings imply that SQSTM1 mutations may play a role in the majority of familial PDB in the United States.     

A nonsynonymous TNFRSF11A variation increases NFκB activity and the severity of Paget's disease

Mutations in the SQSTM1 gene were identified as a common cause of Paget's disease of bone (PDB) but experimental evidence demonstrated that SQSTM1 mutation is not sufficient to induce PDB in vivo. Here, we identified two nonsynonymous single nucleotide polymorphisms (SNPs) (C421T, H141Y and T575C, V192A) in the TNFRSF11A gene, associated with PDB and with the severity of phenotype in a large population of 654 unrelated patients that were previously screened for SQSTM1 gene mutations. The largest effect was found for the T575C variant, yielding an odds ratio of 1.29 (p = 0.003), with the C allele as the risk allele. Moreover, an even more significant p-value (p = 0.0002) was observed in the subgroup of patients with SQSTM1 mutation, with an odds ratio of 1.71. Interestingly, patients with the C allele also showed an increased prevalence of polyostotic disease (68%, 53%, and 51% in patients with CC, CT, and TT genotypes, respectively; p = 0.01), as well as an increased number of affected skeletal sites (2.9, 2.5, and 2.0 in patients with CC, CT, and TT genotypes, respectively, p = 0.008). These differences increased when analyses were restricted to cases with SQSTM1 mutation. In human cell lines, cotrasfection with mutated SQSTM1 and TNFRSF11A(A192) produced a level of activation of NFκB signaling greater than cotrasfection with wild-type SQSTM1 and TNFRSF11A(V192), confirming genetics and clinical evidences. These results provide the first evidence that genetic variation within the OPG/RANK/RANKL system influences the severity of PBD in synergistic action with SQSTM1 gene mutations.     

Genetics of Paget’s Disease of Bone

Paget’s disease of bone (PDB) is a common condition, which is characterised by focal areas of increased and disorganized bone remodeling. Genetic factors play an important role in the disease. In some cases, Paget’s disease is inherited in an autosomal dominant manner and the most common cause for this is a mutation in the SQSTM1 gene. Other familial cases have been linked to the OPTN locus on Chromosome 10p13 and still other variants have been identified by genome wide association studies that lie within or close to genes that play roles in osteoclast differentiation and function. Mutations in TNFRSF11A, TNFRSF11B and VCP have been identified in rare syndromes with PDB-like features. These advances have improved understanding of bone biology and the causes of PDB. The identification of genetic markers for PDB also raises the prospect that genetic profiling could identify patients at high risk of developing complications, permitting enhanced surveillance and early therapeutic intervention.     

Expansile skeletal hyperphosphatasia is caused by a 15-base pair tandem duplication in TNFRSF11A encoding RANK and is allelic to familial expansile osteolysis.

Expansile skeletal hyperphosphatasia (ESH) is a singular disorder characterized in the year 2000 in a mother and daughter with early-onset deafness, premature loss of teeth, progressive hyperostotic widening of long bones causing painful phalanges in the hands, accelerated bone remodeling, and episodic hypercalcemia likely inherited as a highly penetrant, autosomal dominant trait. Absence of large osteolytic lesions with cortical thinning in major long bones, together with bouts of hypercalcemia, indicated that ESH is not a variant of familial expansile osteolysis (FEO). Here, we investigated the molecular basis of ESH after three families with FEO were reported to have an identical 18-base pair tandem duplication (84dup18) in the signal peptide sequence of the TNFRSF11A gene that encodes receptor activator of nuclear factor-kappaB (RANK). We find that ESH is caused by a remarkably similar 15-base pair tandem duplication (84dup15) in TNFRSF11A. Hence, ESH and FEO are allelic diseases and ESH, like FEO, probably reflects increased activity in the skeleton of the RANK target, nuclear factor-kappaB (NF-kappaB).     

Familial Paget's disease of bone: nonlinkage to the PDB1 and PDB2 loci on chromosomes 6p and 18q in a large pedigree.

Paget's disease of bone is a common condition characterized by bone pain, deformity, pathological fracture, and an increased incidence of osteosarcoma. Genetic factors play a role in the pathogenesis of Paget's disease but the molecular basis remains largely unknown. Susceptibility loci for Paget's disease of bone have been mapped to chromosome 6p21.3 (PDB1) and 18q21.1-q22 (PDB2) in different pedigrees. We have identified a large pedigree of over 250 individuals with 49 informative individuals affected with Paget's disease of bone; 31 of whom are available for genotypic analysis. The disease is inherited as an autosomal dominant trait in the pedigree with high penetrance by the sixth decade. Linkage analysis has been performed with markers at PDB1; these data show significant exclusion of linkage with log10 of the odds ratio (LOD) scores < -2 in this region. Linkage analysis of microsatellite markers from the PDB2 region has excluded linkage with this region, with a 30 cM exclusion region (LOD score < -2.0) centered on D18S42. These data confirm the genetic heterogeneity of Paget's disease of bone. Our hypothesis is that a novel susceptibility gene relevant to the pathogenesis of Paget's disease of bone lies elsewhere in the genome in the affected members of this pedigree and will be identified using a microsatellite genomewide scan followed by positional cloning.     

Phenotypic characterization of early onset Paget's disease of bone caused by a 27-bp duplication in the TNFRSF11A gene.

Three different insertion mutations in the TNFRSF11A gene affecting the signal peptide of RANK have been described. An 18-bp duplication at position 84 (84dup18) is associated with the clinical syndrome of familial expansile osteolysis (FEO), whereas a 15-bp duplication at the same site (84dup15) causes the syndrome of expansile skeletal hyperphosphatasia (ESH). Here we report the phenotype of patients harboring a 27-bp duplication at position 75 (75dup27) in RANK. Affected individuals had hearing impairment and tooth loss beginning in the second or third decade. Radiographs of affected bones showed lytic and sclerotic lesions with bony enlargement and deformity. Serum alkaline phosphatase levels were elevated between 2 and 17 times above the normal range. Most patients had pelvic and skull involvement, and all had involvement of the mandible and maxilla. Most patients also had bony enlargement of the small joints of the hands, and one developed hypercalcemia during a period of immobilization. We conclude that the 75dup27 mutation of RANK causes a Paget's disease of bone-like phenotype that is distinct from, but which overlaps with, FEO and ESH. A particularly striking feature was involvement of the mandible and maxilla, but it remains to be seen if this is a specific feature of the 75dup27 mutation until further kindreds with this mutation are reported.     

The Loss of Profilin 1 Causes Early Onset Paget's Disease of Bone

Paget's disease of bone (PDB) is a late-onset disorder frequently caused by mutations in the SQSTM1 gene, leading to hyperactive osteoclasts and resulting in bone pain, deformities, and fractures. However, some more severe forms of PDB—negative for SQSTM1 mutations—have been described, in which the disease degenerates into bone cancers and shows a poor prognosis. Osteosarcoma is the most frequent and aggressive tumor arising in PDB (OS/PDB), with a 5-year survival rate almost nil, but the underlying molecular mechanism is unknown. Here, we investigated an extended pedigree with 11 individuals affected by early onset and polyostotic PDB, mainly interesting the appendicular skeleton. Interestingly, three members also developed secondary osteosarcoma. We performed exome sequencing and identified a 4-bp deletion in the PFN1 gene, resulting in the degradation of the mutant protein. Copy number screening on 218 PDB individuals of our biobank disclosed that four of them (~2%) carry a germline heterozygous deletion of PFN1. The identification of these subjects, who exhibit a particularly severe form of disease, emphasizes the diagnostic value of this genetic screening to identify PDB individuals predisposed to develop osteosarcoma. In fact, we detected allelic imbalance at PFN1 locus also in 8 of 14 (57%) sporadic OS/PDB, further proving its causative role. in vitro experiments also confirmed PFN1 involvement in this form of PDB. Indeed, CRISPR-Cas9-mediated Pfn1 knockout in pre-osteoclasts resulted into enhanced osteoclast differentiation and resorption, with the formation of large osteoclasts never described before in PDB. In addition, Pfn1 lacking pre-osteoblasts lost their differentiation capability and failed to efficiently mineralize bone. Moreover, they acquired features of malignant transformation, including loss of focal adhesions and increased invasion ability. In conclusion, these findings disclose PFN1 haploinsufficiency as the pathological mechanism in OS/PDB. © 2020 American Society for Bone and Mineral Research.     

Guidelines for diagnosis and management of Paget's disease of bone in Japan

We here propose guidelines for the diagnosis and management of Paget's disease of bone (PDB) in Japan. These guidelines provide basic information on the epidemiology, pathophysiology, clinical signs and symptoms, diagnosis, indications for treatment, and available therapy, including orthopedic surgery. PDB is a chronic disorder characterized by focal abnormalities of bone turnover. The characteristic feature of PDB is excessive osteoclastic bone resorption coupled to increased and disorganized bone formation. The most common symptom of PDB is pain in involved bones. The most serious complication of PDB is malignant bone or soft-tissue tumor. PDB is uncommon in Japan; our survey in 2003 found 169 patients with PDB. The prevalence of PDB in Japan is 0.15/100 000; in patients aged 55 years or more, the proportion reaches 0.41/100 000. A careful medical history and physical examination are essential for the diagnosis. The diagnosis of PDB is based on finding the typical features on radiographs. Bone scintigraphy and measurement of serum alkaline phosphatase are sensitive means of screening for PDB. Since PDB is a rare disease in Japan, bone biopsy is quite often used to exclude bone metastases. The only evidence-based indication for treatment of PDB is pain in involved bones. In Japan, etidronate and calcitonin are approved by the Ministry of Health, Labour and Welfare for treating PDB, but currently risedronate is also under development for treating PDB in Japan. Indications for surgical intervention in PDB include unstable fractures, osteoarthritis, malignant soft-tissue tumor, osteosarcoma, and bone deformity.