Multilokul?rer Morbus Paget bei IBMPFD-Syndrom

Paget's osteodystrophia deformans is a monoostotic or polyostotic disease of the skeletal system with increased bone remodelling, structural modifications and skeletal deformation, typically arranged like a chessboard. The unusual case of a patient is described who had suffered from generalized Paget's disease of the bone for 14?years and also developed progressive myopathy and a behavioural variant frontotemporal dementia. Further cytogenetic diagnostics revealed a point mutation in the valosin-containing protein (VCP, p97) gene on chromosome 9p13-p12 consistent with the finding of inclusion body myopathy with early onset Paget's disease and frontotemporal dementia (IBMPFD syndrome). A causal therapy of this disease is not known. Conservative treatment with bisphosphonate therapy, intensive physiotherapeutic exercise and psychotherapeutic treatment was performed to retard the progression of the disease.     

Identification and characterization of valosin-containing protein (VCP/p97) in untransformed osteoblast-like cells.

A 97-kDa protein called valosin-containing protein (VCP) has been implicated in osteosarcoma metastasis and Paget's disease of bone, two conditions that complicate the course and outcome of orthopaedic surgery. High VCP gene expression is associated with high metastatic potential in osteosarcoma cells, while loss-of-function VCP mutations cause inclusion body myopathy associated with Paget's disease of bone and frontotemporal dementia (IBMPFD). VCP protein expression and regulation have not been examined in normal osteoblasts. The purpose of these studies was to characterize VCP protein expression in control and stressed untransformed osteoblasts. Proteins from confluent MC3T3-E1 mouse osteoblast-like cells were separated by 2D IEF/SDS-PAGE. An abundant spot with a M(r) of 94 kDa and a pI of 5.4 was identified as VCP by MALDI/ToF and peptide mass fingerprint analysis. High constitutive VCP protein expression in subconfluent and confluent resting and mildly physiologically stressed MC3T3-E1 cells was confirmed by Western blotting. When assessed by indirect immunofluorescence in fixed cells or Western blotting of subcellular fractions, VCP was more abundant in the cytoplasm than in the nucleus. Induction of mild physiological stress sufficient to stimulate the ubiquitin-proteasome pathway, which is partially dependent on VCP-mediated targeting of polyubiquitinylated substrates, did not affect steady-state VCP levels or distribution. Thus, VCP is a constitutively abundant protein in untransformed osteoblastic cells under all conditions tested. Such high levels of VCP protein expression in untransformed osteoblastic cells argue against a major causative role for it in metastasis, while the occurrence of Paget's disease in patients with missense VCP mutations supports a major role for VCP in normal osteoblast proliferation and regulation.     

A novel VCP mutation as the cause of atypical IBMPFD in a Chinese family

IntroductionInclusion-body myopathy (IBM) with Paget's disease of bone (PDB) and frontotemporal dementia (FTD), designated as IBMPFD, is a rare, autosomal dominant disorder (MIM 605382). IBMPFD is caused by mutations in the gene that encode valosin-containing protein (VCP). We investigated a Chinese family in which multiple members were diagnosed with PDB and suffered from weakness of the limbs. However, no members of this family were diagnosed with FTD. We made a preliminary diagnosis of PDB, but failed to identify an SQSTM1 mutation in any of the patients. We used whole-exome sequencing to identify the pathogenic gene mutation affecting the Chinese male proband.Materials and methodsAltogether, 254 subjects, including one 56-year-old male proband, four affected, related individuals and additional nine family members from a non-consanguineous Chinese family, and 240 healthy donors were recruited and genomic DNA was extracted. All eight exons and the exon–intron boundaries of the SQSTM1 gene were amplified by polymerase chain reaction (PCR) and directly sequenced in five patients (II13, II4, II5, II8, II9). Using whole-exome sequencing, we identified a novel mutation in VCP as the disease-causing mutation. We confirmed the result by sequencing a 500-bp region of the promoter and the coding region of VCP in all 254 of the participants using Sanger sequencing.ResultsNo mutation in the SQSTM1 gene was identified in the five patients examined using direct Sanger sequencing. However, through whole-exome sequencing we were able to identify a novel missense mutation in exon 3 of the VCP gene (p.Gly97Glu) in the Chinese male proband. This mutation was confirmed using Sanger sequencing. The proband, four affected individuals and three unaffected individuals carried this mutation. We were able to correctly diagnose the patients with atypical IBMPFD. Structural analysis of the p.Gly97Glu mutation in the VCP protein showed that the affected amino‐acid is located in the interface of the protein. This abnormality may therefore interfere with protein function.ConclusionsThis is the first report of a family from China with IBMPFD. A novel VCP mutation was found as the cause of atypical IBMPFD in a Chinese family. Our findings confirm that VCP gene mutations can be a pathogenic cause of IBMPFD.     

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.     

Mutation Screening of the TNFRSF11A Gene Encoding Receptor Activator of NFkB (RANK) in Familial and Sporadic Paget's Disease of Bone and Osteosarcoma

Paget's disease of bone (PDB) is a common disorder characterized by focal areas of increased and disorganized osteoclastic bone resorption, leading to bone pain, deformity, pathological fracture, and an increased risk of osteosarcoma. Genetic factors play an important role in the pathogenesis of Paget's disease. In some families, the disease has been found to be linked to a susceptibility locus on chromosome 18q21-22, which also contains the gene responsible for familial expansile osteolysis (FEO)--a rare bone dysplasia with many similarities to Paget's disease. Insertion mutations of the TNFRSF11A gene encoding Receptor Activator of NF kappa B (RANK) have recently been found to be responsible for FEO and rare cases of early onset familial Paget's disease. Loss of heterozygosity (LOH) affecting the PDB/FEO critical region has also been described in osteosarcomas suggesting that TNFRSF11A might also be involved in the development of osteosarcoma. In order to investigate the possible role of TNFRSF11A in the pathogenesis of Paget's disease and osteosarcoma, we conducted mutation screening of the TNFRSF11A gene in patients with familial and sporadic Paget's disease as well as DNA extracted from Pagetic bone lesions, an osteosarcoma arising in Pagetic bone and six osteosarcoma cell lines. No specific abnormalities of the TNFRSF11A gene were identified in a Pagetic osteosarcoma, the osteosarcoma cell lines, DNA extracted from Pagetic bone lesions, or DNA extracted from peripheral blood in patients with familial or sporadic Paget's disease including several individuals with early onset Paget's disease. These data indicate that TNFRSF11A mutations contribute neither to the vast majority of cases of sporadic or familial PDB, nor to the development of osteosarcoma.     

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.     

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.     

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.     

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.     

Ubiquitin-associated domain mutations of SQSTM1 in Paget's disease of bone: evidence for a founder effect in patients of British descent.

Mutations in the UBA domain of SQSTM1 are a common cause of Paget's disease of bone. Here we show that the most common disease-causing mutation (P392L) is carried on a shared haplotype, consistent with a founder effect and a common ancestral origin. INTRODUCTION: Paget's disease of bone (PDB) is a common condition with a strong genetic component. Mutations affecting the ubiquitin-associated (UBA) domain of sequestosome 1 (SQSTM1) have recently been shown to be an important cause of PDB. The most common mutation results in a proline to leucine amino acid change at codon 392 (P392L), and evidence has been presented to suggest that there may be a recurrent mutation rather than a founder mutation on an ancestral chromosome. Because marked geographical differences exist in the prevalence of PDB, we have investigated the frequency of SQSTM1 mutations in different populations and looked for a founder effect on chromosomes bearing SQSTM1 UBA domain mutations. MATERIALS AND METHODS: We conducted mutation screening of SQSTM1 and performed haplotype analysis using the PHASE software program in 83 kindreds with familial PDB, recruited mainly through clinic referrals in the United Kingdom, Australia, and New Zealand. Similar studies were conducted in 311 individuals with PDB who did not have a family history and 375 age- and sex-matched controls from the United Kingdom. RESULTS: The proportion of patients with familial PDB who had SQSTM1 UBA domain mutations varied somewhat between referral centers from 7.1% (Sydney, Australia) to 50% (Perth, Australia), but the difference between centers was not statistically significant. Haplotype analysis in 311 British patients with PDB who did not have a family history and 375 age- and sex-matched British controls showed that two common haplotypes accounted for about 90% of alleles at the SQSTM1 locus, as defined by common single nucleotide polymorphisms (SNPs) in exon 6 (C916T, G976A) and the 3'UTR (C2503T, T2687G). These were H1 (916T-976A-2503C-2687T) and H2 (916C-976G-2503T-2687G). There was no significant difference in haplotype distribution in PDB cases and controls, but the P392L mutation was found on the H2 haplotype in 25/27 cases (93%), which is significantly more often than expected given the allele frequencies in the normal population (odds ratio, 13.2; 95% CI, 3.1-56.4; p < 0.0001). Similar findings were observed in familial PDB, where 12/13 (92%) of P392L mutations were carried on H2 (odds ratio 17.2; 95% CI, 2.2-138; p = 0.001). CONCLUSIONS: These results provide strong evidence for a founder effect of the SQSTM1 P392L mutation in PDB patients of British descent, irrespective of family history. Our results imply that these individuals share a common ancestor and that the true rate of de novo mutations may be lower than previously suspected.     

Paget’s disease: Epidemiology and pathophysiology

Paget’s disease of bone is a focal disorder of aging bone. The classic late-onset Paget’s disease is often caused by a P392L mutation in the gene SQSTM1, which disturbs signaling pathways in osteoclasts on cell activation. This prevalent mutation is neither necessary nor sufficient to cause Paget’s disease. Its identification, along with the elucidation of other mutations underlying early-onset Paget’s and Paget’s disease seen in association with inclusion body myopathy and frontotemporal dementia, have redefined our understanding of genetic disorders of bone remodeling by emphasizing the importance of environmental determinants in their pathophysiology.     

Evaluation of the Role of the SQSTM1 Gene in Sporadic Belgian Patients with Paget’s Disease

A positional cloning effort in French Canadian families with Pagets disease of bone (PDB) resulted in the identification of a mutation in the sequestosome1 (SQSTM1) gene in a subset of both familial and sporadic PDB cases. This was confirmed in samples of mainly United Kingdom (UK) origin. In this study, we performed both mutation analysis and association studies in order to evaluate the role of this gene in a collection of isolated Belgian PDB patients. A mutation in the SQSTM1 gene was found in only 6 of 111 patients (5.4%). In all cases it involves the P392L mutation, previously shown to be common in both familial and sporadic cases. To perform association studies, we selected 8 single nucleotide polymorphisms (SNPs) and looked for linkage disequilibrium (LD) between these. Haplotype analysis indicated that typing of 3 Tag SNPs (IVS1+633A/C, IVS5–23A/G, and 976A/G) enables us to identify the most common haplotypes. Association studies for the 3 selected SNPs, based on 105 PDB cases without a SQSTM1 mutation and 159 control individuals, did not support a possible influence of natural variants in the SQSTM1 gene either on the pathogenesis of PDB or on the disease severity. In conclusion, our study confirms that the P392L mutation is a recurrent mutation causing PDB in different populations. We were not able to show an association between SQSTM1 polymorphisms and PDB in our population but this clearly needs to be extended to other populations. The presented identification of haplotype Tag SNPs will be of major help for such studies.     

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.     

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.     

Novel UBA domain mutations of SQSTM1 in Paget's disease of bone: genotype phenotype correlation, functional analysis, and structural consequences.

Three novel missense mutations of SQSTM1 were identified in familial PDB, all affecting the UBA domain. Functional and structural analysis showed that disease severity was related to the type of mutation but was unrelated to the polyubiquitin-binding properties of the mutant UBA domain peptides. INTRODUCTION: Mutations affecting the ubiquitin-associated (UBA) domain of Sequestosome 1 (SQSTM1) gene have recently been identified as a common cause of familial Paget's disease of bone (PDB), but the mechanisms responsible are unclear. We identified three novel SQSTM1 mutations in PDB, conducted functional and structural analyses of all PDB-causing mutations, and studied the relationship between genotype and phenotype. MATERIALS AND METHODS: Mutation screening of the SQSTM1 gene was conducted in 70 kindreds with familial PDB. We characterized the effect of the mutations on structure of the UBA domain by protein NMR, studied the effects of the mutant UBA domains on ubiquitin binding, and looked at genotype-phenotype correlations. RESULTS AND CONCLUSIONS: Three novel missense mutations affecting the SQSTM1 UBA domain were identified, including a missense mutation at codon 411 (G411S), a missense mutation at codon 404 (M404V), and a missense mutation at codon 425 (G425R). We also identified a deletion leading to a premature stop codon at 394 (L394X). None of the mutations were found in controls. Structural analysis showed that M404V and G425R involved residues on the hydrophobic surface patch implicated in ubiquitin binding, and consistent with this, the G425R and M404V mutants abolished the ability of mutant UBA domains to bind polyubiquitin chains. In contrast, the G411S and P392L mutants bound polyubiquitin chains normally. Genotype-phenotype analysis showed that patients with truncating mutations had more extensive PDB than those with missense mutations (bones involved = 6.05 +/- 2.71 versus 3.45 +/- 2.46; p < 0.0001). This work confirms the importance of UBA domain mutations of SQSTM1 as a cause of PDB but shows that there is no correlation between the ubiquitin-binding properties of the different mutant UBA domains and disease occurrence or extent. This indicates that the mechanism of action most probably involves an interaction between SQSTM1 and a hitherto unidentified protein that modulates bone turnover.     

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.     

Thirteen Chinese patients with sporadic Paget’s disease of bone: clinical features, SQSTM1 mutation identification, and functional analysis

To increase awareness of the rarity of Paget's disease of bone (PDB) in the Chinese population, we characterized the clinical manifestations and features of 13 Chinese sporadic PDB patients. The clinical features of our Chinese PDB patients show similarities with cases reported in Western countries. The most common lesion sites were the pelvis, femur, and tibia; the next most common lesion sites were the spine and skull. Most patients had a higher serum alkaline phosphatase (ALP) level. Treatment with bisphosphonates was effective. In addition, we screened for PDB-causing mutations and performed a functional analysis in an attempt to elucidate the molecular pathogenesis of PDB. A total of 216 persons, including 13 sporadic PDB patients, three unaffected relatives of 1 patient, and 200 healthy donors, were recruited. All eight exons and exon-intron boundaries of the SQSTM1 gene were amplified by polymerase chain reaction (PCR) and directly sequenced. We identified a 53-year-old man who harbored a heterozygous T-to-C transversion at position 1250 in exon 8 (1250T?>?C), which resulted in a methionine-to-threonine (ATG?>?ACG) substitution at codon 404 (M404T). The M404T mutant SQSTM1 protein exhibited increased NF-κB activation and drove a significantly increased number of osteoclast-like cells (OLCs) that formed in response to RANKL and an increased number of OLC nuclei. This is the first report of an SQSTM1 genetic mutation that contributes to the pathogenesis of PDB in Chinese patients. These results may partially explain the mechanism by which this SQSTM1 mutation contributes to the pathogenesis of sporadic PDB in Chinese patients.     

Genetic Epidemiology of Paget’s Disease of Bone in Italy: sequestosome1/p62 Gene Mutational Test and Haplotype Analysis at 5q35 in a Large Representative Series of Sporadic and Familial Italian Cases of Paget’s Disease of Bone

Families affected by Paget’s disease of bone frequently harbor mutations in the SQSTM1/p62 gene. In this multicentric study we collected 345 sporadic and 12 familial PDB cases throughout Italy, identifying 12 different mutations, 5 of which are newly reported and 3, D335E, A381V, and Y383X, external to the UBA domain. Subjects with truncating mutations, E396X, showed a significantly younger age at clinical diagnosis, while the Y383X subjects had a higher average number of affected skeletal sites. All the mutants exhibited the CGTG-H2 haplotype. In two pairs and one triad of unrelated Italian PDB families from different Italian regions, we detected a common SQSTM1/p62 mutation for each P392L, M404V, and G425R group. Since the CGTG-H2 haplotype frequency was also high in normal subjects, and genetic influence due to migratory fluxes of different ethnic groups exists in the Italian population, to refine the search for a more geographically specific founder effect, we extended the haplotype analysis in these families using polymorphic microsatellite repeat markers, within and flanking the SQSTM1/p62 locus, from chromosome 5q35, other than the exon 6 and 3′UTR polymorphisms. All mutant carriers from two of the three M404V families and from the G425R families exhibited common extended chromosome 5q35 haplotypes, IT01 and IT02, respectively, which may be reflecting influences of past migrations. This may be helpful in estimating the true rate of de novo mutations. We confirm the data on the existence of both a mutational hotspot at the UBA domain of SQSTM1/p62 and a founder effect in the PDB population.     

Sequestosome 1 Mutations in Paget's Disease of Bone in Australia: Prevalence,Genotype/Phenotype Correlation,and a Novel Non‐UBA Domain Mutation (P364S) Associated With Increased NF‐κB Signaling Without Loss of Ubiquitin Binding

Previously reported Sequestosome 1(SQSTM1)/p62 gene mutations associated with Paget's disease of bone (PDB) cluster in, or cause deletion of, the ubiquitin‐associated (UBA) domain. The aims of this study were to examine the prevalence of SQSTM1 mutations in Australian patients, genotype/phenotype correlations and the functional consequences of a novel point mutation (P364S) located upstream of the UBA. Mutation screening of the SQSTM1 gene was conducted on 49 kindreds with PDB. In addition, 194 subjects with apparently sporadic PDB were screened for the common P392L mutation by restriction enzyme digestion. HEK293 cells stably expressing RANK were co‐transfected with expression plasmids for SQSTM1 (wildtype or mutant) or empty vector and a NF‐κB luciferase reporter gene. GST‐SQSTM1 (wildtype and mutant) proteins were used in pull‐down assays to compare monoubiquitin‐binding ability. We identified SQSTM1 mutations in 12 of 49 families screened (24.5%), comprising 9 families with the P392L mutation and 1 family each with the following mutations: K378X, 390X, and a novel P364S mutation in exon 7, upstream of the UBA. The P392L mutation was found in 9 of 194 (4.6%) patients with sporadic disease. Subjects with SQSTM1 mutations had more extensive disease, but not earlier onset, compared with subjects without mutations. In functional studies, the P364S mutation increased NF‐κB activation compared with wildtype SQSTM1 but did not reduce ubiquitin binding. This suggests that increased NF‐κB signaling, but not the impairment of ubiquitin binding, may be essential in the pathogenesis of PDB associated with SQSTM1 mutations.     

Identification of a major locus for Paget's disease on chromosome 10p13 in families of British descent.

Mutations of SQSTM1 are an important cause of PDB, but other genes remain to be discovered. A major susceptibility locus for PDB was identified on chromosome 10p13 by a genome-wide linkage scan in families of British descent, which accounted for the vast majority of cases not caused by SQSTM1 mutations. INTRODUCTION: Paget's disease of bone (PDB) has a strong genetic component, and several susceptibility loci have been identified by genome-wide linkage scans. We previously identified three susceptibility loci for PDB using this approach on chromosomes 5q35, 2q36, and 10p13 in 62 families of mainly British descent, but subsequently, mutations in the SQSTM1 gene were found to be the cause of PDB in 23 families from this cohort. Here we reanalyzed the results of our genome-wide search in families from this cohort who did not have SQSTM1 mutations. MATERIALS AND METHODS: The study population consisted of 210 individuals from 39 families of predominantly British descent with autosomal dominant inheritance of PDB in whom SQSTM1 mutations had been excluded by mutation screening. The average family size was 5.44 +/- 3.98 (SD) individuals (range, 2-24 individuals). Genotyping was performed using standard techniques with 382 microsatellite markers spaced at an average distance of 9.06 cM throughout the autosomes. Multipoint linkage analysis was performed using the GENEHUNTER program under models of homogeneity and heterogeneity. RESULTS: Multipoint parametric linkage analysis under a model of homogeneity and nonparametric linkage analysis under a model of heterogeneity both showed strong evidence of linkage to a single locus on chromosome 10p13 (LOD score, +4.08) close to the marker D10S1653 at 41.43cM. No evidence of linkage was detected at the chromosome 2q36 locus previously identified in this population, and linkage to other candidate loci previously implicated in the pathogenesis of PDB was excluded. CONCLUSIONS: We conclude that there is an important susceptibility gene for PDB on chromosome 10p13 in families of British descent and find no evidence to support the existence of a susceptibility locus on chromosome 2q36 or other previously identified candidate loci for PDB in this population. The gene that lies within the 10p13 locus seems to account for the development of PDB in the vast majority of families of British descent who do not carry SQSTM1 mutations.