Paget’s disease of bone (PDB) is a focal metabolic bone disease characterized by excessive and aberrant bone remodeling that results in bone pain, bone deformity and pathologic fracture, followed by secondary osteoarthritis and nerve compression syndromes (1, 2). It is well known that there are pronounced geographical and ethnic differences in the prevalence of PDB (3), with the high prevalence in western countries, especially in European descent. Some studies reported intranational regional differences (4–7) and mainly affects people over 55 years (3, 8, 9). However, the disease is extremely rare in Asia and Africa (1, 2, 10–13). For instance, the incidence rate in Japan is merely 0.00028% (2). So far as we know, the literatures on sporadic PDB in Chinese population are confined to case reports (12–15), no systemic study is available with relatively large sample size up to date. In 2012, we reported our study in thirteen sporadic PDB patients (1). However, the study focused mainly on the mutation detection in SQSTM1.

It is known that genetic background and environmental factors are involved in the pathogenesis of PDB. Although remains doubtful (16), some environmental determinants have been reported to play a role in the pathophysiology of PDB, such as toxins, animal exposure and measles virus infection (17–20), etc. For decades, accumulating evidence have highlighted that genetic factors are important in the pathogenesis of PDB (6, 21–25). Located on human chromosome 5q35, the sequestosome 1 (SQSTM1) gene encodes p62, a scaffold protein with a vital role in both osteoclast differentiation and activity, has been commonly reported among PDB patients (21, 23–25). Additionally, some cases of PDB-like syndromes have been attributed to mutations in VCP, TNFRSF11A, TNFRSF11B, HNRNPA2B1, HNRNPA1, ZNF687 and PFN1, most of which are known to involved in the amyotrophic lateral sclerosis (ALS) and nuclear factor kappa B (NF-kB) signaling pathways (26–32). Recently, several large-scale genome-wide association studies (GWAS) have been conducted to uncover variants at the CSF1, OPTN, TNFRSF11A, PML, RIN3, TM7SF4 and NUP205 loci that may increase susceptibility to PDB (33, 34). For years, we have made great efforts to explore and determine the roles of candidate genes in the development of PDB in Chinese population (26, 27, 31, 35). However, the reported genes could only explain part of the pathogenesis, the pathophysiological mechanism of PDB is needed to be elucidated. Therefore, it is imperative to determine the genetic etiology of PDB and provide guidance for efficient molecular diagnosis. Considering the different genetic background between Chinese and Caucasian population (1, 10–12, 14), the exploration of potential pathogenic genes of PDB in Chinese population also beneficial to explain the pathogenesis of PDB.

In our study, we not only analyzed the clinical characteristics in 50 PDB patients, but also observed the drug efficacy and prognosis by follow-up. Meanwhile, the whole-exome sequencing (WES) was carried out to investigate the underlying gene variants and molecular mechanism of PDB in Chinese population.

The prevalence of PDB is as high as 5% among people above 55 in Western countries (3, 8, 9); however, it is extremely rare in Asians, with only a few cases reported in China (1, 12–15). To gain better insight into the clinical characteristics and reduce the rate of clinical misdiagnosis among Chinese patients, we analyzed the clinical features in 50 Chinese sporadic PDB patients. Moreover, a comprehensive WES analysis was performed to further explore the potential pathogenic genes associated with PDB. To date, this is the largest cohort and most in-depth study in patients with PDB both at the clinical and molecular level in China (1, 26, 27, 31, 46).

As is shown in Table 8 , the clinical manifestations of the 50 Chinese PDB patients were summarized and compared to those of PDB reported in different countries (2, 6, 16, 47–52). It is well known that the majority of patients with PDB are asymptomatic in Western countries (53), yet in our cohort, most patients had clinical manifestations. Unlike individuals in Western countries, Chinese patients presented a higher proportion of polyostotic involvements, as well as remarkable clinical manifestations. The reason is that PDB is extremely rare in China, and most clinicians are not familiar with it. The asymptomatic or slightly symptomatic patients are often easily ignored and hardly to be recognized, which could account at least in part for the greater prevalence of polyostotic and symptomatic forms of PDB in our country. However, it is noteworthy that headache, hearing loss and vision impairment are uncommon in Chinese population (45). Similarly, the mentioned neurological complications were rarely found in Japan (2). Previous studies reported that congestive heart failure occurred in 3% of the PDB patients in the USA (54). However, no such cases were observed in our study. Additionally, it was observed that the incidence of fractures in China was roughly the same as that in other countries. While fibula was the most common site of fractures instead of the femur and vertebra (2, 48).

Of interest, most Chinese patients were early onset, with 66.7% (34/50) of the patients developed symptoms before the age of 55 years, while PDB developed more common in people above 55 years in Caucasian population (51). This may be related to the different genetic backgrounds and need to be further investigated. In addition, the misdiagnosis rate of the disease was as high as 36.0% in Chinese patients and was often misdiagnosed as bone metastasis of malignant tumors and fibrous dysplasia, etc. As a result, 30.0% of the patients underwent bone biopsy, which is unnecessary for diagnosis of PDB in daily clinical practice (36, 45, 55, 56). Likewise, in 2005, Hashimoto et al. demonstrated that bone biopsy was conducted for diagnosis in 55% of the cases in Japan (2). This tendency in application of invasive examination methods also partly reflects the fact that clinicians are unfamiliar with the disease due to the rarity of PDB in Asia, and our main consideration in diagnosis was to exclude possible malignant bone tumors. Therefore, a full understanding of the clinical characteristics and pathogenesis of the disease is key to reducing misdiagnosis and providing timely treatment to patients. It cannot be ignored that the prevalence of the disease is likely to be underestimated in China due to the lack of understanding of the disease by clinicians. In addition, our long-term follow-up results showed that PDB patients responded well to bisphosphonates. Most patients only required zoledronic acid once a lifetime to avoid clinical relapse [defined by recurrence of bone pain (36)]. Therefore, if the patients were correctly diagnosed, the disease would be quickly and effectively controlled, and the symptoms of patients would be greatly alleviated.

Compared with the high proportion (15–26%) of familial aggregation in Western countries (48), sporadic patients are more common in China. Mutations in SQSMT1 had been identified in 40-50% of familial cases and in 9-20% of sporadic individuals in Western countries (23, 24, 57). However, in our study, only 4.0% of the patients carried SQSMT1 mutations, which was much lower than that of in Europe (23–25). Both patients harbored the relatively rare heterozygous identical mutation (M404T) in SQSTM1 instead of the most prevalent P392L PDB-causing mutation in the Caucasian population (21, 23, 24, 58). Taken together, the above data indicated that different genetic backgrounds did exist across various ethnicities, and the M404T mutation in SQSTM1 could be a mutational hotspot in Chinese patients. Whether it represented a founder effect in the Chinese Han nationality needs haplotyping analysis to explore and confirm (6).

A well-known feature of PDB is the increased osteoclast activation. Hence, we have focused primarily on the RANKL-induced NF-κB signaling pathway for a long time. In addition to SQSTM1, our study identified several rare variants (1 in HNRNPA2B1, 1 in NUP205, 2 in WNT16, 3 in RYR3, 2 in RYR1, 2 in CAPN2, and 3 in NUP214) highly related to PDB using in silico tools. Enrichment analysis indicated that the above genes were enriched in neurodegeneration and ALS pathways ( Figure 4 ), which were possibly linked to the pathogenesis of PDB. This is of particular interest because PDB, inclusion body myopathy (IBM), together with frontotemporal dementia (FTD)/ALS lead to an inherited pleiotropic group of inherited disorders, coined the term multisystem proteinopathy (MSP), which is characterized by progressive degeneration of brain, muscle and bone (59, 60). It is widely accepted that MSP is attributed to mutations in VCP, and the acronym IBMPFD is commonly utilized for the above-mentioned complex phenotypes (59, 60). Notably, the syndromes also show genetic overlap with neurodegeneration, and ALS is classified as a neurodegenerative disease. Along with the rapid development of high-speed sequencing technologies, MSP is genetically linked to mutations in the PFN1, HNRNPA1, HNRNPA2B1, SQSTM1 and MATR3 genes (59). Coincidentally, MSP shared genetic overlap with isolated PDB, ALS and other neurodegenerative diseases. In the above diseases, aging is a risk factor (29, 36, 55, 59), no exception for PDB. Till now the relationship between the above genes and diseases remains unclear, further studies should be performed to elucidate the underlying mechanisms related to MSP and isolated PDB.

HNRNPA2B1 encodes a ubiquitously expressed RNA-binding ribonucleoprotein that acts as a binding partner of TDP-43. Accumulating evidence has highlighted those mutations in the HNRNPA2B1 gene are highly associated with MSP, of which PDB is a component (29, 59). In 2017, the heterozygous missense mutation (c.929C>T, p. P310L) in HNRNPA2B1 gene was identified as causative of isolated PDB in a large Chinese pedigree by Xia et al. Consistent with this, we identified another novel heterozygous mutation in the HNRNPA2B1 gene (c.989C>T, p. P330L) in one 32-year-old male with sporadic PDB with a pure bone phenotype. He presented with a severe episode of low back and hip pain for 2 months. The bone scintigraphy indicated increased radionuclide uptake in multiple bone lesions, and X-ray examination showed sclerosis at different levels and ranges in the affected bones, accompanied with an uneven bone density ( Figure 5 ). It is of interest to note that all cases, no matter in Xia’s or our study, presented with relatively early onset (17, 31, 34, 32 years), significantly elevated ALP (1592, 6724, 2042, 2349 U/L) and recurrent fractures (4, 4, 0, 3 times). Taken together, these data indicated that HNRNPA2B1-mutated patients showed a severe symptom, with a remarkable numbers of affected bone sites and increased risks of fractures. It was suggested that PDB patients with severe phenotypes and repeated features, even absence of IBM, FTD, and ALS, should be paid attention to the HNRNPA2B1 gene in molecular diagnosis.

The nuclear pore complex (NPC) is composed of approximately 30 diverse proteins coined nucleoporins (Nups), including nucleoporin 205 and 214, which are encoded by the NUP205 and NUP214 genes, respectively (61). Although the role of the above two genes in bone remains unclear, a large-scale GWAS confirmed the strong signal association of rs4294134 within NUP205 with PDB (33). Of interest, we identified a missense mutation p.Arg615Pro (c.1844G>C) in a 80-year-old woman, who had exhibited relatively severe PDB symptoms with headache and hearing loss for 20 years. GWAS performed by Albagha et al. also demonstrated that the 7q33 locus is susceptible to PDB (33); consistently, our study detected WNT16, which locates within the 7q31 locus and encodes the secreted cysteine-rich glycoproteins that have been implicated in cortical bone homeostasis (62). WNT16 has been previously identified by a GWAS to be associated with BMD, cortical bone thickness and fracture susceptibility (63). Coincidentally, the 29-year-old woman carried the deletion variant p.S38Pfs*15 (c.111delC) in WNT16 suffered from vertebral compression fractures and local cortical destruction both in tibia and fibula, with an extremely high level of ALP (785 U/L). Moreover, the missense variant p.Cys92Phe (c.275G>T) in WNT16 was also found in one 44-year old woman, she presented with progressive temporal bone protrusion and raised ALP level for six years (from 1457 U/L to 2031 U/L). The X-ray examination showed that skull was flocky in appearance without sites involved. Both variant sites were classified to be likely pathogenic mutations by ACMG/AMP. Taken together, the underlying relationship between WNT16 and PDB is worth exploring. Moreover, the recurrent mutant genes in our cohort such as RyR1, RyR3 and CAPN2 are linked to calcium signaling (64, 65). Although the relationship between them and bone metabolism is still unknown, evidence has been reported that Ca²⁺ signaling activates osteoclast precursor differentiation but inhibits resorption in mature osteoclasts (66).

We have considered the possible limitations of our study. As noted above, the sample size of single-center research was still limited due to the rarity of PDB in China, and multiple-center and larger sample size studies are required to provide a more comprehensive characterization of the disease. Second, as a hospital-setting study, the proportion of symptomatic patients may be overestimated compared with the “real-life” characteristics of PDB. In fact, as an authoritative tertiary care level center devoted to bone disease, our findings may underestimate non-skeletal manifestations. Furthermore, it was insufficient to validate the causality between variants and PDB by only computer algorithms and prediction software, and rigorous experimental assays were required before defining a pathogenic variant (32). Also, we may not identify the presence of unknown modifier genes (32). In the future, studies with wider geographic distribution and larger sample capacity are needed to fully clarify our findings, and demonstrate a more comprehensive picture of PDB in Chinese patients.

Overall, due to the rarity of PDB in Asia, our study represents the first comprehensive and largest sample of patients recruited in China to date, both in terms of clinical evaluation and molecular processes. The detection rate of sporadic PDB patients was extremely low, and only 3 of 50 patients carried the known pathogenic gene mutations (including 2 patients with SQSTM1 mutation and 1 patient with HNRNPA2B1 mutation) in our cohort. Besides, we detected other 14 potential pathogenic gene variants, which may have an impact on accurate molecular diagnosis, genetic screening and provide further support for researchers to explore PDB pathogenesis.

The datasets presented in this study can be found in an online repository. The names of the repository and accession number are as follows: National Omics Data Encyclopedia (NODE) [accession: OEP003182]; https://www.biosino.org/node/project/detail/OEP003182.

The studies involving human participants were reviewed and approved by the Ethics Committee of Shanghai Jiao Tong University Affiliated Sixth People’s Hospital. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.

ZZ, GZ and HY contributed to conception and design of the study. XT wrote the first draft of the manuscript. LL wrote sections of the manuscript. XT, LL, GY and ZW collected the samples and organized the database. ZC analysed and interpreted the raw data. ZZ and HY contributed to funding acquisition. All authors contributed to the article and approved the submitted version. XT and LL were co-first authors.

This work was supported by the National Key R&D Program of China (No. 2018YFA0800801), National Natural Science Foundation of China (NSFC) (No. 81770874 and 81974126); the Clinical Science and Technology Innovation Project of Shanghai Shenkang Hospital Development Center (No. SHDC12018120) and Shanghai Key Clinical Center for Metabolic Disease, Shanghai Health Commission Grant (No. 2017ZZ01013).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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