Identification of SQSTM1 mutations in familial Paget's disease in Australian pedigrees

We have conducted a genome-wide scan on a pedigree containing 372 adult members, of whom 49 have PDB. In the present study, we report linkage of a large pedigree to the PDB3 region on chromosome 5q35-qter with a peak multipoint LOD score of 6.77. Sequestosome 1 (SQSTM/p62) has been identified as the causative PDB gene in this region. Six sequestosome 1 mutations have been described to date. Four mutations have been identified in exon 8, 1210delT and 1215delC both resulting in premature stop codon at amino acid 394, 1215C to T (P392L), 1224insT (E396X), one mutation in exon 7, 1200C to T (P387L) and a G to A splice junction mutation at IVS7+1. These mutations cluster in the C terminus of the protein and are predicted to disrupt the ubiquitin binding properties of sequestosome 1. Sequence analysis of the gene encoding sequestosome 1 revealed a single base pair deletion (1215delC) segregating with the majority of affected members in the pedigree. This deletion introduces a stop codon at position 394, resulting in premature termination of the protein (L394X) and loss of the ubiquitin-associated binding domain. Screening of affected members from 10 further PDB families identified the previously reported P392L mutation in one family. No SQSTM1/p62 coding mutations were found in the remaining 9 families or in 113 age-matched controls.     

Novel SQSTM1 mutations in patients with Paget's disease of bone in an unrelated multiethnic American population

More than 20 mutations of the Sequestosome 1 (SQSTM1) gene have been reported in patients of European descent affected by Paget's disease of bone (PDB). In this investigation, a systematic screening for SQSTM1 mutations was conducted in consecutively evaluated unrelated patients with phenotypical PDB living in the New York City area (NY, United States). Seventy unrelated PDB patients with a multiethnic background, mostly of Jewish, Italian American, and Western European ancestries, were recruited. Sequencing of exons 7 and 8 was performed on DNA samples isolated from peripheral blood. Seven patients (10%) had SQSTM1 mutations, of which three had a family history of PDB. Four patients carried the C1215T (P392L) mutation, and three patients carried novel SQSTM1 missense mutations: T1085A (S349T), C1209T (A390V), and T1290A (L417Q) mutations. All PDB patients with SQSTM1 mutations had polyostotic involvement, and the mean number of affected bones was significantly higher in pagetic patient carriers of a SQSTM1 mutation when compared to non-mutated PDB patients (4.0 vs. 2.0, respectively; P = 0.003). Haplotype analysis in patient carriers of the P392L mutation revealed that all P392L mutations were carried by haplotype 2. The SQSTM1 mutation rate in unrelated American patients described in the present study was similar to that reported in European populations.     

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.     

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.     

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.     

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.     

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 of bone in the French population: novel SQSTM1 mutations, functional analysis, and genotype-phenotype correlations.

Mutation screening of the SQSTM1 gene in 94 French patients with PDB revealed two novel point-mutations (A381V and L413F) and two new compound heterozygous genotypes (P392L/A381V and P392L/A390X). Functional analysis showed an increased level of SQSTM1/p62 protein in PDB patients and truncated forms of the protein encoded by the A390X allele. Clinical data indicate that PDB patients with SQSTM1 mutation are younger at PDB diagnosis and have more extensive bone lesions. INTRODUCTION: Paget's disease of bone (PDB) is a common chronic disease of the skeleton, with a strong genetic component. A recurrent mutation (P392L) was first identified on chromosome 5, in the Sequestosome 1 (SQSTM1) gene. Several other mutations of the SQSTM1 gene have been described in PDB patients, affecting the ubiquitin-associated domain (UBA) of the SQSTM1/p62 protein. The objectives of this study were to evaluate the frequency of the SQSTM1 mutations in French PBD patients, to study the expression of the SQSTM1/p62 protein, and to search for genotype-phenotype correlations. MATERIALS AND METHODS: Blood was obtained from 94 unrelated French PDB patients and 100 controls for mutation screening of exons 7 and 8, encoding for the UBA domain of SQSTM1. Epstein-Barr virus (EBV)-immortalized B-cell lymphocytes were established from 13 patients, giving access to functional analysis of the gene and the SQSTM1/p62 expressions using real-time PCR and Western blot. RESULTS: Mutations of the SQSTM1 gene were identified in 12 of the 94 PDB patients (13%). Eight patients carried P392L. Two novel missense mutations were identified: L413F and A381V. This A381V mutation and A390X were found in distinct patients already carriers of P392L. The SQSTM1/p62 protein expression in PDB patients increased when zero, one, or two mutations were present, and SQSTM1 truncated forms were associated with the A390X mutation. The mean age of PDB diagnosis was younger in patients with the SQSTM1 mutation. PDB was more extensive in patients who carried a SQSTM1 mutation. CONCLUSIONS: Mutations of SQSTM1 are present in the French population. PDB patients with and without the SQSTM1 mutation have an increased level of SQSTM1/p62, caused by overproduction of the protein, probably involved in the pathophysiology of PDB. The presence of the SQSTM1 mutation may be a worsening factor for PDB.     

Founder Effect in Different European Countries for the Recurrent P392L SQSTM1 Mutation in Paget’s Disease of Bone

Paget's Disease of Bone (PDB) is one of the most frequent metabolic bone diseases, affecting 1-5% of Western populations older than 55 years. Mutations in the sequestosome1 (SQSTM1) gene cause PDB in about one-third of familial PDB cases and in 2.4-9.3% of nonfamilial PDB cases, with the 1215C-->T (P392L) mutation being the most frequent one. We investigated whether a founder effect of the P392L SQSTM1 mutation was present in Belgian (n = 233), Dutch (n = 82), and Spanish (n = 64) patients without a PDB family history. First, direct sequencing analysis of exon 8 in these three populations showed that the P392L mutation occurred in 17 Belgian patients (7.3%), three Dutch patients without a family history (3.7%), and two Dutch patients with a family history. In the Spanish population, 15.6% of patients (n = 10) had the P392L mutation, including one homozygous mutant. This is by far the highest mutation frequency of all populations investigated so far. Next, we examined the genetic background of 33 mutated chromosomes by analyzing haplotypes. We genotyped four single-nucleotide polymorphisms (SNPs) in exon 6 and the 3'-untranslated region of SQSTM1 (rs4935C/T, rs4797G/A, rs10277T/C, and rs1065154G/T) and used software programs WHAP and PHASE to reconstruct haplotypes. Finally, allele-specific primers allowed us to assign the mutation to one of the two haplotypes from each individual. Sequencing results revealed that all 33 P392L mutations were on the CGTG (H2) haplotype. The chance to obtain this result due to 33 independent mutation events is 3.97 x 10(-14), providing strong evidence for a founder effect of the P392L SQSTM1 mutation in Belgian, Dutch, and Spanish patients with PDB.     

Genetics of Paget's disease of bone

Paget's disease of bone is a chronic bone disease that affects up to 3% of Caucasians older than 55 years. The cause of Paget's disease is unknown but involves genetic factors. Familial cases display an autosomal dominant pattern of inheritance with incomplete penetrance. Genetic heterogeneity has been demonstrated and eight potential susceptibility loci identified. There is sound evidence incriminating Sequestosome 1 (SQSTM1) on the long arm of chromosome 5 (5q35-qter), of which nine mutations have been described in Paget's disease of bone. These mutations are located in exons 7 and 8, which encode a highly conserved ubiquitin-binding domain. The prevalence of SQSTM1 mutations is about 10% in France. Tests for SQSTM1 mutations should be done in patients with Paget's disease of bone, even where the family history is negative. Detection of a mutation allows evaluation of family members to ensure early diagnosis of the disease before complications develop.     

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.     

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.     

Delayed development of Paget's disease in offspring inheriting SQSTM1 mutations.

Familial Paget's disease is associated with mutations in SQSTM1. We compared the age at diagnosis and severity of Paget's disease in parents with SQSTM1 mutations to their offspring who inherited a mutation. At any given age, the offspring were less likely to be diagnosed with Paget's disease and had less severe disease than their parents. INTRODUCTION: Mutations in sequestosome 1 (SQSTM1) occur in 25-50% of cases of familial Paget's disease and are thought to be disease-causing. We sought to determine whether there are differences in age at diagnosis and severity of disease in parents and their offspring who share the same genetic predisposition to Paget's disease. MATERIALS AND METHODS: Eighty-four offspring from 10 families (29 index patients with Paget's disease) with mutations in SQSTM1 were approached, and 58 agreed to participate. The ubiquitin-binding domain region of SQSTM1 was sequenced, and the presence or absence of the known mutation was established. The presence of Paget's disease in offspring who had inherited an SQSTM1 mutation was determined by bone scintigraphy and measurement of serum alkaline phosphatase (ALP). RESULTS: Twenty-three of 58 offspring had inherited a germline mutation in SQSTM1. The mean ALP was 77 U/liter in offspring with mutations and 72 U/liter in those without mutations (p=0.84). Scintiscans from four offspring (mean age, 45 years; mean ALP, 139 U/liter; mean skeletal involvement, 6%) showed evidence of Paget's disease but were normal in the other 19 (mean age, 44 years; mean ALP, 64 U/liter). In comparison, in the 15 parents of the 23 offspring, the mean age of diagnosis was 48 years, the mean ALP was 850 U/liter, and the mean skeletal involvement was 30%. There was a 63% reduction in the risk of being diagnosed with Paget's disease at a comparable age in the offspring compared with the parents (p=0.028). CONCLUSIONS: Only 17% of offspring inheriting an SQSTM1 mutation had evidence of Paget's disease on scintigraphy, and this was diagnosed at a later age and was less extensive than in their affected parents. SQSTM1 thus shows incomplete penetrance. The data are consistent with the hypothesis that an environmental factor is important in the pathogenesis and clinical phenotype of familial Paget's disease and that exposure to this factor may be falling.     

SQSTM1 and Paget’s Disease of Bone

Mutations in the Sequestosome 1 gene (SQSTM1; also known as p62) have recently been identified as the cause of 5q35-linked Pagets disease of bone (PDB). All of the mutations identified to date affect the ubiquitin-associated (UBA) domain of SQSTM1, a region of the protein that binds noncovalently to ubiquitin. In this review we consider the possible functional significance of the SQSTM1-ubiquitin interaction, and consequences of the SQSTM1 UBA domain mutations. Clarification of the in vivo roles of SQSTM1 in bone-cell function will be central to improving our understanding of the molecular pathogenesis of PDB and related conditions.     

SQSTM1 gene analysis and gene‐environment interaction in Paget's disease of bone

Even though SQSTM1 gene mutations have been identified in a consistent number of patients, the etiology of Paget's disease of bone (PDB) remains in part unknown. In this study we analyzed SQSTM1 mutations in 533 of 608 consecutive PDB patients from several regions, including the high‐prevalence area of Campania (also characterized by increased severity of PDB, higher number of familial cases, and peculiar phenotypic characteristics as giant cell tumor). Eleven different mutations (Y383X, P387L, P392L, E396X, M401V, M404V, G411S, D423X, G425E, G425R, and A427D) were observed in 34 of 92 (37%) and 43 of 441 (10%) of familial and sporadic PDB patients, respectively. All five patients with giant cell tumor complicating familial PDB were negative for SQSTM1 mutations. An increased heterogeneity and a different distribution of mutations were observed in southern Italy (showing 9 of the 11 mutations) than in central and northern Italy. Genotype‐phenotype analysis showed only a modest reduction in age at diagnosis in patients with truncating versus missense mutations, whereas the number of affected skeletal sites did not differ significantly. Patients from Campania had the highest prevalence of animal contacts (i.e., working or living on a farm or pet ownership) without any difference between patients with or without mutation. However, when familial cases from Campania were considered, animal contacts were observed in 90% of families without mutations. Interestingly, a progressive age‐related decrease in the prevalence of animal contacts, as well as a parallel increase in the prevalence of SQSTM1 mutations, was observed in most regions except in the subgroup of patients from Campania. Moreover, patients reporting animal contacts showed an increased number of affected sites (2.54 ± 2.0 versus 2.19 ± 1.9, p < .05) over patients without animal contacts. This difference also was evidenced in the subgroup of patients with SQSTM1 mutations (3.84 ± 2.5 versus 2.76 ± 2.2, p < .05). Overall, these data suggest that animal‐related factors may be important in the etiology of PDB and may interact with SQSTM1 mutations in influencing disease severity. © 2010 American Society for Bone and Mineral Research     

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.     

Familial Paget Disease and <Emphasis Type="Italic">SQSTM1</Emphasis> Mutations in New Zealand

Genetic factors play an important role in the pathogenesis of Paget disease of bone (PDB). SQSTM1 is the most important disease-associated gene identified to date. We investigated the relationship of family history, phenotype, and SQSTM1 mutation status in New Zealand (a country with a high prevalence of PDB) in patients with a family history and/or a severe phenotype. We studied 61 unrelated subjects with familial PDB. Family history was subclassified into three groups according to the closeness of the relationship. We also studied a fourth group of 19 unrelated patients defined by early onset and/or severe disease but no family history. The PDB phenotype was defined according to age, alkaline phosphatase activity, and disease extent on scintiscan at the time of diagnosis. Mutations in exon 8 of SQSTM1 were detected by screening of genomic DNA. Four different mutations were identified; the ubiquitous P392L mutation and the truncating mutation E396X accounted for 89% of cases. Overall 26% of patients with familial PBD in New Zealand had disease-associated mutations in the SQSTM1 gene. Mutations were most prevalent (60%) in those with a parent or sibling and at least one other relative affected (P < 0.002). The severity of the phenotype was significantly related to SQSTM1 mutation status but not the strength of the family history (P < 0.005). SQSTM1 mutations were found in 10.5% of patients with early onset and/or severe disease but no family history.