The authors state that they have no conflicts of interest.
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.
Paget's disease of bone is a common disorder that can cause considerable morbidity, particularly in older people in whom the disease is more prevalent.(1) The etiology of Paget's disease is incompletely understood, but genetic factors undoubtedly play an important role. Between 15% and 40% of individuals with Paget's disease have at least one affected first-degree relative,(2) and in 25–50% of familial Paget's disease cases, mutations are present in exons 7 or 8 of the sequestosome 1 gene (SQSTM1).(3–5) Paget's disease develops at an earlier age and is more extensive in families with SQSTM1 mutations than in those without mutations.(6)SQSTM1 mutations are thought to be highly penetrant(4,7) and are rare in people without Paget's disease.(3,4,8) Mutations in another gene, VCP, cause the rare syndrome of Paget's disease, dementia, and inclusion body myopathy.(9)
Although genetic factors are unquestionably important in the development of Paget's disease, epidemiologic data from New Zealand, Spain, and Britain has shown significant secular changes. In these countries, the prevalence of Paget's disease and its clinical severity have decreased substantially over the last 30 years.(1,10–12) This change (which has been too rapid to be the consequence of genetic alteration) implies that exposure to some unidentified environmental agent is also important in the etiology of Paget's disease and that exposure to this agent has become less prevalent. We have examined whether adults who have inherited SQSTM1 mutations develop Paget's disease of similar severity and at a similar age to their affected parents. If they are not doing so, this lends further support to the idea that environmental factors are important and that SQSTM1 is a disease-susceptibility gene rather than a disease-causing gene.
MATERIALS AND METHODS
We identified 10 families with a history of familial Paget's disease associated with mutations in SQSTM1. We approached all 82 adult offspring of parents with Paget's disease known to carry a mutation in SQSTM1. The offspring were tested for the presence of the known mutation and had total plasma alkaline phosphatase activity (ALP) measured using a p-nitrophenylphosphate substrate.(12) In those children who had inherited a mutation in SQSTM1, bone scintigraphy with 99Tc-methylene bisphosphonate was performed to detect whether Paget's disease was present. Disease extent was calculated by the scoring method of Coutris et al.(13) Where bone scans suggested Paget's disease, plain radiographs were taken to confirm the findings. Fifty-eight offspring agreed to participate. The study was approved by the Auckland Ethics Committee and was registered with the Australian Clinical Trials Registry (ACTRN012605000294651).
DNA was extracted from blood samples using the Progene DNA purification kit (Gentra Systems). Exon 8 of the SQSTM1 gene was amplified by Paltinum Taq polymerase (Invitrogen) in 35 cycles of PCR at an annealing temperature of 60°C and MgCl2 concentration of 1.5 mM. The forward primer 5′-GGCAGAGTTGAGCAGTGTG-3′ and the reverse primer 5′-CCCGTACAGAGACCTGCAAT-3′ produced 445-bp products that were subsequently purified using the High pure PCR product purification kit (Roche) and sequenced on a 3130XL capillary sequencer (Applied Biosystems).
Comparisons were made between groups for continuous variables using Student's t-test. Kaplan-Meier survival analysis was used to compare the age at diagnosis of Paget's disease between groups using GraphPad Prism version 4.00 for Windows (GraphPad Software, San Diego, CA, USA). Significance level was set at p < 0.05, and all tests were two-tailed. All other statistical analyses were obtained using SPSS for Windows (version 12.0.1; SPSS, Chicago, IL, USA).
The characteristics of Paget's disease at the time of diagnosis in the parents' generation in the 10 families are shown in Table 1. Fifty-eight offspring were tested for the known familial mutation in SQSTM1, and 23 were found to have inherited the mutation. The characteristics of the offspring are shown in Table 2. There were no significant differences in age or serum ALP between offspring who had inherited a mutation and those who had not.
Table Table 1.. Characteristics of Paget's Disease at the Time of Diagnosis in the Parents' Generation
Table Table 2.. Characteristics of Offspring Grouped by Mutation Status
All 23 offspring who inherited mutations in SQSTM1 had bone scintigraphy. Only 4 subjects showed evidence of Paget's disease: the characteristics of these 4 offspring, the 19 offspring who inherited a mutation but had no scintigraphic evidence of Paget's disease, and the 15 affected parents of these 23 offspring are shown in Table 3. The offspring with Paget's disease had lower plasma ALP at diagnosis and less skeletal involvement seen on bone scintigraphy than their parents. Expression of Paget's disease in the offspring was not affected by their sex (p = 0.2) or the sex of their parents (p = 0.8). Scintigraphic evidence of Paget's disease was present in 2 of 5 offspring who inherited a truncating E396X mutation compared with 2 of 18 offspring who inherited a missense mutation (p = 0.19).
Table Table 3.. Characteristics of Paget's Disease in Offspring Who Inherited Mutations of SQSTM1 in Comparison With Their Affected Parents
Figure 1 shows the age of diagnosis of Paget's disease in the 23 offspring who inherited the familial SQSTM1 mutation compared with their 15 affected parents. Fifty-three percent of the parents had a clinical diagnosis of Paget's disease by 45 years of age, whereas in the offspring, 18% had a scintigraphic diagnosis by 45 years and 39% by 54 years of age. There was a 63% reduction in the risk of diagnosis of Paget's at a comparable age in the offspring in comparison with their parents (hazard ratio, 0.37; 95% CI, 0.10–0.89; p = 0.028).
When we restricted the analysis only to the offspring who were older at the time of bone scintigraphy (n = 13) than their affected parent was at the time of diagnosis of Paget's disease (n = 8), the differences in the age of diagnosis of Paget's disease were even more marked (p = 0.0005; Fig. 2). The serum ALP and skeletal involvement on scintigraphy were greater in each parent in comparison with their offspring (p < 0.001, paired t-test).
We found that offspring who inherited a mutation in SQSTM1 from their parents were diagnosed with Paget's disease later in life and had less extensive disease than their parents. Only 4 of 23 offspring had evidence of Paget's disease on bone scintigraphy. In every case, the plasma ALP and amount of skeletal involvement was less in the offspring in comparison with their parent, suggesting less severe and less extensive disease. Kaplan-Meier survival analysis showed that parents were diagnosed at an earlier age in comparison with their offspring and that there was a substantial 63% reduction in the risk of diagnosis of Paget's in offspring compared with parents at comparable ages. In the parents, Paget's disease was diagnosed clinically—on the basis of symptoms and signs and characteristic radiographic and biochemical findings. In contrast, Paget's disease was actively sought by a highly sensitive method (scintigraphy) in their asymptomatic offspring. Because Paget's disease is often present for many years before a clinical diagnosis is made,(14) our study design was biased toward finding no difference. Thus, the size of the effect we found was likely to be an underestimate.
The SQSTM1 gene codes for a scaffold protein (p62) involved in the activation of RANK.(15) Mutations in SQSTM1 were independently identified and associated with Paget's disease by two groups,(3,4) and at least 20 mutations of SQSTM1 have now been reported in association with Paget's disease.(3–5,8,16,17) Such mutations seem to inhibit the binding of ubiquitin to the p62 protein,(18) but how this predisposes to Paget's disease is unknown. Patients with truncating mutations of SQSTM1 have been reported to have earlier onset and more severe disease than those with missense mutations or sporadic Paget's disease.(6,19) Although there were few offspring with Paget's disease in our study, we found that Paget's was more prevalent in those with truncating mutations (40%) than in those with missense mutations (11%), tending to support the view that the phenotype is more severe in the former.
Reports have identified SQSTM1 mutations in 25–50% of familial Paget's disease(3,4,16,20) and 5–15% of sporadic Paget's disease.(3,4,17,20) A number of previous studies have identified carriers of SQSTM1 mutations who do not seem to have Paget's disease based on biochemical screening(3,5,7,8,16,21) or bone scintigraphy.(5,8,16) Many of these people have been <55 years of age. These findings may reflect age-dependent penetrance of SQSTM1. Laurin et al.(4,7) reported the penetrance rate of SQSTM1 mutation carriers was 19% in those <50 years of age, 46% in those 50–59 years of age, and 78% in those >60 years of age. The age range of our cohort of offspring who had inherited a mutation in SQSTM1 was 39–54 years of age. However, when we restricted the analyses only to the offspring who were older at the time of bone scintigraphy than their affected parent was at the time of diagnosis of Paget's disease, the differences in age at diagnosis were more pronounced. Thus, the age-dependent penetrance of SQSTM1 mutations seems to have decreased over time.
A number of different observations suggest that environmental agents may contribute to the pathogenesis of Paget's disease. There is marked geographic variability in the prevalence of Paget's disease, with rates being highest in Britain and countries such as New Zealand with large numbers of people of British descent.(22) In recent decades, the prevalence and severity of Paget's disease has fallen in a number of countries.(3,10,11) Ownership of dogs or cats has been linked to Paget's disease in some(23) but not all studies.(24) Paramyxoviruses have been suggested as causative agents of Paget's disease. Viral-like inclusion bodies have been identified in pagetic osteoclasts, immunochemical studies have shown positive staining for measles virus in pagetic bone, and in-situ hybridization studies have shown measles virus and canine distemper virus in tissue from patients with Paget's disease. However, the role of viruses in Paget's disease remains controversial.(25,26) Our study supports the view that as yet unknown environmental agents contribute to the pathogenesis of Paget's disease.
There are limitations to our study. Only 69% of offspring agreed to take part in the study. Proportionately more women took part in the study, and because men have a higher prevalence of Paget's disease than women,(22) the prevalence of Paget's disease in the offspring may be an underestimate. Our study is small and includes a highly selected number of families, some with severe Paget's disease. The results may not be generalizable to other families with different SQSTM1 mutations and different environmental backgrounds.
In summary, we found that offspring who inherited a mutation in SQSTM1 from their parents were diagnosed with Paget's disease at a later age and had less extensive disease than their parents. Only 17% of offspring had evidence of Paget's disease. Taken together, our results suggest that mutations in SQSTM1confer disease susceptibility but may not be disease-causing, and that as yet unknown environmental agents modify the development of disease. These data support the idea that exposure to these putative environmental agents is diminishing. The adult offspring with SQSTM1 mutations that we studied were relatively young, so further follow-up of the participants with normal bone scintigraphy is required.
This study was funded by the Health Research Council of New Zealand and the Paget's Trust of New Zealand. The authors thank all the families concerned for participating in this study.