Valosin-Containing Protein (VCP)/p97 Oligomerization.

Valosin-containing protein (VCP), also known as p97, is an evolutionarily conserved AAA+ ATPase essential for cellular homeostasis. Cooperating with different sets of cofactors, VCP is involved in multiple cellular processes through either the ubiquitin-proteasome system (UPS) or the autophagy/lysosomal route. Pathogenic mutations frequently found at the interface between the NTD domain and D1 ATPase domain have been shown to cause malfunction of VCP, leading to degenerative disorders including the inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia (IBMPFD), amyotrophic lateral sclerosis (ALS), and cancers. Therefore, VCP has been considered as a potential therapeutic target for neurodegeneration and cancer. Most of previous studies found VCP predominantly exists and functions as a hexamer, which unfolds and extracts ubiquitinated substrates from protein complexes for degradation. However, recent studies have characterized a new VCP dodecameric state and revealed a controlling mechanism of VCP oligomeric states mediated by the D2 domain nucleotide occupancy. Here, we summarize our recent knowledge on VCP oligomerization, regulation, and potential implications of VCP in cellular function and pathogenic progression.

Randomised trial of genetic testing and targeted intervention to prevent the development and progression of Paget's disease of bone.

INTRODUCTION: Paget's disease of bone (PDB) frequently presents at an advanced stage with irreversible skeletal damage. Clinical outcomes might be improved by earlier diagnosis and prophylactic treatment. METHODS: We randomised 222 individuals at increased risk of PDB because of pathogenic SQSTM1 variants to receive 5 mg zoledronic acid (ZA) or placebo. The primary outcome was new bone lesions assessed by radionuclide bone scan. Secondary outcomes included change in existing lesions, biochemical markers of bone turnover and skeletal events related to PDB. RESULTS: The median duration of follow-up was 84 months (range 0-127) and 180 participants (81%) completed the study. At baseline, 9 (8.1%) of the ZA group had PDB lesions vs 12 (10.8%) of the placebo group. Two of the placebo group developed new lesions versus none in the ZA group (OR 0.41, 95% CI 0.00 to 3.43, p=0.25). Eight of the placebo group had a poor outcome (lesions which were new, unchanged or progressing) compared with none of the ZA group (OR 0.08, 95% CI 0.00 to 0.42, p=0.003). At the study end, 1 participant in the ZA group had lesions compared with 11 in the placebo group. Biochemical markers of bone turnover were significantly reduced in the ZA group. One participant allocated to placebo required rescue therapy with ZA because of symptomatic disease. The number and severity of adverse events did not differ between groups. CONCLUSIONS: Genetic testing for pathogenic SQSTM1 variants coupled with intervention with ZA is well tolerated and has favourable effects on the progression of early PDB. TRIAL REGISTRATION NUMBER: ISRCTN11616770.

Cross-sectional study of patients with VCP multisystem proteinopathy 1 using dual-energy x-ray absorptiometry.

INTRODUCTION/AIMS: VCP multisystem proteinopathy 1 (MSP1), encompassing inclusion body myopathy (IBM), Paget's disease of bone (PDB) and frontotemporal dementia (FTD) (IBMPFD), features progressive muscle weakness, fatty infiltration, and disorganized bone structure in Pagetic bones. The aim of this study is to utilize dual-energy x-ray absorptiometry (DXA) parameters to examine it as a biomarker of muscle and bone disease in MSP1. METHODS: DXA scans were obtained in 28 patients to assess body composition parameters (bone mineral density [BMD], T-score, total fat, and lean mass) across different groups: total VCP disease (n = 19), including myopathy without Paget's ("myopathy"; n = 12) and myopathy with Paget's ("Paget"; n = 7), and unaffected first-degree relatives serving as controls (n = 6). RESULTS: In the VCP disease group, significant declines in left hip BMD and Z-scores were noted versus the control group (p ≤ .03). The VCP disease group showed decreased whole body lean mass % (p = .04), and increased total body fat % (p = .04) compared to controls. Subgroup comparisons indicated osteopenia in 33.3% and osteoporosis in 8.3% of the myopathy group, with 14.3% exhibiting osteopenia in the Paget group. Moreover, the Paget group displayed higher lumbar L1-L4 T-score values than the myopathy group. DISCUSSION: In MSP1, DXA revealed reduced bone and lean mass, and increased fat mass. These DXA insights could aid in monitoring disease progression of muscle loss and secondary osteopenia/osteoporosis in MSP1, providing value both clinically and in clinical research.

Genetic Screening of ZNF687 and PFN1 in a Paget's Disease of Bone Cohort Indicates an Important Role for the Nuclear Localization Signal of ZNF687.

Paget's disease of bone (PDB) is a common, late-onset bone disorder, characterized by focal increases of bone turnover that can result in bone lesions. Heterozygous pathogenic variants in the Sequestosome 1 (SQSTM1) gene are found to be the main genetic cause of PDB. More recently, PFN1 and ZNF687 have been identified as causal genes in patients with a severe, early-onset, polyostotic form of PDB, and an increased likelihood to develop giant cell tumors. In our study, we screened the coding regions of PFN1 and ZNF687 in a Belgian PDB cohort (n = 188). In the PFN1 gene, no variants could be identified, supporting the observation that variants in this gene are extremely rare in PDB. However, we identified 3 non-synonymous coding variants in ZNF687. Interestingly, two of these rare variants (p.Pro937His and p.Arg939Cys) were clustering in the nuclear localization signal of the encoded ZNF687 protein, also harboring the p.Pro937Arg variant, a previously reported disease-causing variant. In conclusion, our findings support the involvement of genetic variation in ZNF687 in the pathogenesis of classical PDB, thereby expanding its mutational spectrum.

Deep brain stimulation in Parkinson disease with valosin-containing protein gene mutation.

BACKGROUND AND PURPOSE: Mutations in the gene encoding valosin-containing protein (VCP) are related to myriad medical conditions, including familial amyotrophic lateral sclerosis, inclusion body myopathy, and frontotemporal dementia. There are several reports of a link between these mutations and early onset Parkinson disease (PD). CASE DESCRIPTION: We report a 53-year-old PD patient with VCP mutation who later developed motor complications, thus receiving subthalamic nucleus deep brain stimulation (DBS) at the age of 56 years. However, myopathy emerged 1.5 years after surgery. CONCLUSIONS: With the phenotype variability of VCP, DBS should be carefully evaluated, considering the possible unfavorable long-term outcomes due to other symptoms of this mutation.

Familial Paget's disease of bone with ocular manifestations and a novel TNFRSF11A duplication variant (72dup27).

INTRODUCTION: Paget's disease of bone (PDB) is a skeletal disorder characterized by disorganized bone remodeling due to abnormal osteoclasts. Tumor necrosis factor receptor superfamily member 11A (TNFRSF11A) gene encodes the receptor activator of nuclear factor kappa B (RANK), which has a critical role in osteoclast function. There are five types of rare PDB and related osteolytic disorders due to TNFRSF11A tandem duplication variants so far, including familial expansile osteolysis (84dup18), expansile skeletal hyperphosphatasia (84dup15), early-onset familial PDB (77dup27), juvenile PDB (87dup15), and panostotic expansile bone disease (90dup12). MATERIALS AND METHODS: We reviewed a Japanese family with PDB, and performed whole-genome sequencing to identify a causative variant. RESULTS: This family had bone symptoms, hyperphosphatasia, hearing loss, tooth loss, and ocular manifestations such as angioid streaks or early-onset glaucoma. We identified a novel duplication variant of TNFRSF11A (72dup27). Angioid streaks were recognized in Juvenile Paget's disease due to loss-of-function variants in the gene TNFRSF11B, and thought to be specific for this disease. However, the novel recognition of angioid streaks in our family raised the possibility of occurrence even in bone disorders due to TNFRSF11A duplication variants and the association of RANKL-RANK signal pathway as the pathogenesis. Glaucoma has conversely not been reported in any case of Paget's disease. It is not certain whether glaucoma is coincidental or specific for PDB with 72dup27. CONCLUSION: Our new findings might suggest a broad spectrum of phenotypes in bone disorders with TNFRSF11A duplication variants.

Clinical, Biochemical, Radiological, and Genetic Analyses of a Patient with VCP Gene Variant-Induced Paget's Disease of Bone.

Paget's disease of bone (PDB) is a rare metabolic bone disorder, which is extremely rare in Asian population. This study aimed to investigate the phenotypes and the pathogenic mutations of woman with early-onset PDB. The clinical features, bone mineral density, x-ray, radionuclide bone scan, and serum levels of alkaline phosphatase (ALP), procollagen type 1 N-terminal propeptide (P1NP), and ß-carboxy-terminal cross-linked telopeptide of type 1 collagen (ß-CTX) were measured in detail. The pathogenic mutations were identified by whole-exon sequencing and confirmed by Sanger sequencing. We also evaluated the effects of intravenous infusion of zoledronic acid on the bones of the patient and summarized the phenotypic characteristics of reported patients with mutation at position 155 of the valosin-containing protein (VCP). The patient only exhibited bone pain as the initial manifestation with vertebral compression fracture and extremely elevated ALP, P1NP, and ß-CTX levels; she had no inclusion body myopathy and frontotemporal dementia. The missense mutation in exon 5 of the VCP gene (p.Arg155His) was identified by whole-exome sequencing and further confirmed by Sanger sequencing. No mutation in candidate genes of PDB, such as SQSTM1, CSF1, TM7SF4, OPTN, PFN1, and TNFRSF11A, were identified in the patient by Sanger sequencing. Rapid relief of bone pain and a marked decline in ALP, P1NP, and ß-CTX levels were observed after zoledronic acid treatment. Previously reported patients with VCP missense mutation at position 155 (R155H) always had myopathy, frontotemporal dementia, and PDB, but the patient in this study exhibited only PDB. This was the first report of R155H mutation-induced early-onset in the VCP gene in Asian population. PDB was the only manifestation having a favorable response to zoledronic acid treatment. We broadened the genetic and clinical phenotype spectra of the VCP mutation.

Pattern of SQSTM1 Gene Variants in a Hungarian Cohort of Paget's Disease of Bone.

Paget's disease of bone (PDB) is characterized by focal or multifocal increase in bone turnover. One of the most well-established candidate genes for susceptibility to PDB is Sequestosome 1 (SQSTM1). Mutations in SQSTM1 have been documented among Western-European, British and American patients with PDB. However, there is no information on SQSTM1 mutation status in PDB patients from the Central- and Eastern-European regions. In this study, we conducted a mutation screening for SQSTM1 gene variants in 82 PDB patients and 100 control participants in Hungary. Mutations of SQSTM1 were detected in 18 PDB patients (21.95%); associations between genotype and clinical characteristics were also analyzed. Altogether, six different exonic alterations, including two types of UTR variants in the SQSTM1 gene, were observed in our PDB patients. Similarly, to previous genetic studies on Paget's disease, our most commonly detected variant was the c.1175C > T (p.Pro392Leu) in nine cases (four in monostotic and five in polyostotic form). We have surveyed the germline SQSTM1 variant distribution among Hungarian patients with PDB. We also highlighted that the pattern of the analyzed disease-associated pathophysiological parameters could partially discriminate PDB patients with normal or mutant SQSTM1 genotype. However, our findings also underline and strengthen that not solely SQSTM1 stands in the background of the complex PDB etiology.

A Panel-Based Sequencing Analysis of Patients with Paget's Disease of Bone Suggests Enrichment of Rare Genetic Variation in regulators of NF-κB Signaling and Supports the Importance of the 7q33 Locus.

Paget's disease of bone (PDB) is a common bone disorder characterized by focal lesions caused by increased bone turnover. Monogenic forms of PDB and PDB-related phenotypes as well as genome-wide association studies strongly support the involvement of genetic variation in components of the NF-κB signaling pathway in the pathogenesis of PDB. In this study, we performed a panel-based mutation screening of 52 genes. Single variant association testing and a series of gene-based association tests were performed. The former revealed a novel association with NFKBIA and further supports an involvement of variation in NR4A1, VCP, TNFRSF11A, and NUP205. The latter indicated a trend for enrichment of rare genetic variation in GAB2 and PRKCI. Both single variant tests and gene-based tests highlighted two genes, NR4A1 and NUP205. In conclusion, our findings support the involvement of genetic variation in modulators of NF-κB signaling in PDB and confirm the association of previously associated genes with the pathogenesis of PDB.

Genetic Determinants of Paget's Disease of Bone.

PURPOSE OF REVIEW: To provide an overview of the role of genes and loci that predispose to Paget's disease of bone and related disorders. RECENT FINDINGS: Studies over the past ten years have seen major advances in knowledge on the role of genetic factors in Paget's disease of bone (PDB). Genome wide association studies have identified six loci that predispose to the disease whereas family based studies have identified a further eight genes that cause PDB. This brings the total number of genes and loci implicated in PDB to fourteen. Emerging evidence has shown that a number of these genes also predispose to multisystem proteinopathy syndromes where PDB is accompanied by neurodegeneration and myopathy due to the accumulation of abnormal protein aggregates, emphasising the importance of defects in autophagy in the pathogenesis of PDB. Genetic factors play a key role in the pathogenesis of PDB and the studies in this area have identified several genes previously not suspected to play a role in bone metabolism. Genetic testing coupled to targeted therapeutic intervention is being explored as a way of halting disease progression and improving outcome before irreversible skeletal damage has occurred.

Structural and Functional Analysis of Disease-Linked p97 ATPase Mutant Complexes.

IBMPFD/ALS is a genetic disorder caused by a single amino acid mutation on the p97 ATPase, promoting ATPase activity and cofactor dysregulation. The disease mechanism underlying p97 ATPase malfunction remains unclear. To understand how the mutation alters the ATPase regulation, we assembled a full-length p97R155H with its p47 cofactor and first visualized their structures using single-particle cryo-EM. More than one-third of the population was the dodecameric form. Nucleotide presence dissociates the dodecamer into two hexamers for its highly elevated function. The N-domains of the p97R155H mutant all show up configurations in ADP- or ATPγS-bound states. Our functional and structural analyses showed that the p47 binding is likely to impact the p97R155H ATPase activities via changing the conformations of arginine fingers. These functional and structural analyses underline the ATPase dysregulation with the miscommunication between the functional modules of the p97R155H.

VCP/p97 increases BMP signaling by accelerating ubiquitin ligase Smurf1 degradation.

The bone morphogenetic protein (BMP)-Smad signaling pathway plays a crucial role in the control of bone homeostasis by regulating osteoblast activity. It is known that the ubiquitin ligase Smad ubiquitination regulatory factor (Smurf)1 is a master negative regulator of BMP signaling, but how its stability and activity are regulated remains poorly understood. Our study showed that valosin-containing protein/p97, the mutations of which lead to rare forms of Paget's disease of bone (PDB)-like syndrome-such as inclusion body myopathy (IBM) associated with Paget's disease of bone and frontotemporal dementia (IBM-PFD)-together with its adaptor nuclear protein localization (NPL)4, specifically interact with Smurf1 and deliver the ubiquitinated Smurf1 for degradation. Depletion of either p97 or NPL4 resulted in the elevation of Smurf1 protein level and decreased BMP signaling accordingly. Mechanically, a typical proline, glutamic acid, serine, and threonine motif specifically existing in Smurf1 is necessary for its recognition and degradation by p97, and this process is dependent on p97 ATPase activity. More importantly, compared with p97 WT, PDB-associated mutation of p97 (mainly A232E) harboring the higher ATPase activity of p97 further promoted Smurf1 degradation, thus increasing BMP signaling activity. Our findings first establish a link between p97 and Smurf1, providing an in-depth understanding of how Smurf1 is regulated, as well as the mechanism of p97-related bone diseases.-Li, H., Cui, Y., Wei, J., Liu, C., Chen, Y., Cui, C.-P., Li, L., Zhang, X., Zhang, L. VCP/p97 increases BMP signaling by accelerating ubiquitin ligase Smurf1 degradation.

Ubiquitin- and ATP-dependent unfoldase activity of P97/VCP•NPLOC4•UFD1L is enhanced by a mutation that causes multisystem proteinopathy.

p97 is a "segregase" that plays a key role in numerous ubiquitin (Ub)-dependent pathways such as ER-associated degradation. It has been hypothesized that p97 extracts proteins from membranes or macromolecular complexes to enable their proteasomal degradation; however, the complex nature of p97 substrates has made it difficult to directly observe the fundamental basis for this activity. To address this issue, we developed a soluble p97 substrate-Ub-GFP modified with K48-linked ubiquitin chains-for in vitro p97 activity assays. We demonstrate that WT p97 can unfold proteins and that this activity is dependent on the p97 adaptor NPLOC4-UFD1L, ATP hydrolysis, and substrate ubiquitination, with branched chains providing maximal stimulation. Furthermore, we show that a p97 mutant that causes inclusion body myopathy, Paget's disease of bone, and frontotemporal dementia in humans unfolds substrate faster, suggesting that excess activity may underlie pathogenesis. This work overcomes a significant barrier in the study of p97 and will allow the future dissection of p97 mechanism at a level of detail previously unattainable.

Ostm1 from Mouse to Human: Insights into Osteoclast Maturation.

The maintenance of bone mass is a dynamic process that requires a strict balance between bone formation and resorption. Bone formation is controlled by osteoblasts, while osteoclasts are responsible for resorption of the bone matrix. The opposite functions of these cell types have to be tightly regulated not only during normal bone development, but also during adult life, to maintain serum calcium homeostasis and sustain bone integrity to prevent bone fractures. Disruption of the control of bone synthesis or resorption can lead to an over accumulation of bone tissue in osteopetrosis or conversely to a net depletion of the bone mass in osteoporosis. Moreover, high levels of bone resorption with focal bone formation can cause Paget's disease. Here, we summarize the steps toward isolation and characterization of the osteopetrosis associated trans-membrane protein 1 (Ostm1) gene and protein, essential for proper osteoclast maturation, and responsible when mutated for the most severe form of osteopetrosis in mice and humans.

A quest for clarity in bone erosion: The role of sequestosome 1 in Paget's disease of bone.

Alterations in the SQSTM1 gene are a putative cause of Paget's disease of bone, yet results are conflicting about how these mutations impact osteoclasts, the cell type believed to be the main pathological contributor. In this issue of JBC, Zach et al. provide important new evidence that the protein encoded by SQSTM1, p62, negatively regulates osteoclastogenesis and demonstrate that aged p62-deficient mice develop bone phenotypes similar to those of Paget's disease. These findings help to clarify the role of this important protein and present new opportunities to interrogate bone biology.

p62/sequestosome 1 deficiency accelerates osteoclastogenesis in vitro and leads to Paget's disease-like bone phenotypes in mice.

The sequestosome 1 gene encodes the p62 protein and is the major genetic risk factor associated with Paget's disease of bone. In 2004, p62 was reported to up-regulate osteoclast differentiation by activating the transcription factors Nfatc1 and NF-κB. Here, we characterized the osteoclastogenic potential of murine p62-/--derived cells compared with WT cells. Our data confirmed previous findings indicating that p62 is induced during murine osteoclast differentiation. Surprisingly, an indispensable role for p62 in in vitro osteoclast differentiation was not reproducible because p62-deficient osteoclasts exhibited robust activation of Nfatc1, NF-κB, and osteoclast marker enzymes. Thus, we concluded that in vitro osteoclast differentiation is not negatively influenced by knocking out p62. On the contrary, our results revealed that p62 deficiency accelerates osteoclastogenesis. Differentiation potential, multinucleation status, and soluble receptor activator of NF-κB ligand (sRANKL) sensitivity were significantly elevated in p62-deficient, murine bone marrow-derived stem cells. Moreover, femur ultrastructures visualized by micro-computed tomography revealed pronounced accumulation of adipocytes and trabecular bone material in distal femora of obese p62-/- mice. Increased tartrate-resistant acid phosphatase activity, along with increased trabecular bone and accumulation of adipocytes, was confirmed in both paraffin-embedded decalcified and methyl methacrylate-embedded nondecalcified bones from p62-/- mice. Of note, Paget's disease-like osteolytic lesions and increased levels of the bone turnover markers CTX-I and PINP were also observed in the p62-/- mice. Our results indicate that p62 predominantly suppresses murine in vitro osteoclast differentiation and highlight previously undetected Paget's disease-like phenotypes in p62-/- mice in vivo.

ZNF687 Mutations in Severe Paget Disease of Bone Associated with Giant Cell Tumor.

Paget disease of bone (PDB) is a skeletal disorder characterized by focal abnormalities of bone remodeling, which result in enlarged and deformed bones in one or more regions of the skeleton. In some cases, the pagetic tissue undergoes neoplastic transformation, resulting in osteosarcoma and, less frequently, in giant cell tumor of bone (GCT). We performed whole-exome sequencing in a large family with 14 PDB-affected members, four of whom developed GCT at multiple pagetic skeletal sites, and we identified the c.2810C>G (p.Pro937Arg) missense mutation in the zinc finger protein 687 gene (ZNF687). The mutation precisely co-segregated with the clinical phenotype in all affected family members. The sequencing of seven unrelated individuals with GCT associated with PDB (GCT/PDB) identified the same mutation in all individuals, unravelling a founder effect. ZNF687 is highly expressed during osteoclastogenesis and osteoblastogenesis and is dramatically upregulated in the tumor tissue of individuals with GCT/PDB. Interestingly, our preliminary findings showed that ZNF687, indicated as a target gene of the NFkB transcription factor by ChIP-seq analysis, is also upregulated in the peripheral blood of PDB-affected individuals with (n = 5) or without (n = 6) mutations in SQSTM1, encouraging additional studies to investigate its potential role as a biomarker of PDB risk.

Rare Inherited forms of Paget's Disease and Related Syndromes.

Several rare inherited disorders have been described that show phenotypic overlap with Paget's disease of bone (PDB) and in which PDB is a component of a multisystem disorder affecting muscle and the central nervous system. These conditions are the subject of this review article. Insertion mutations within exon 1 of the TNFRSF11A gene, encoding the receptor activator of nuclear factor kappa B (RANK), cause severe PDB-like disorders including familial expansile osteolysis, early-onset familial PDB and expansile skeletal hyperphosphatasia. The mutations interfere with normal processing of RANK and cause osteoclast activation through activation of nuclear factor kappa B (NFκB) independent of RANK ligand stimulation. Recessive, loss-of-function mutations in the TNFRSF11B gene, which encodes osteoprotegerin, cause juvenile PDB and here the bone disease is due to unopposed activation of RANK by RANKL. Multisystem proteinopathy is a disorder characterised by myopathy and neurodegeneration in which PDB is often an integral component. It may be caused by mutations in several genes including VCP, HNRNPA1, HNRNPA2B1, SQSTM1, MATR3, and TIA1, some of which are involved in classical PDB. The mechanisms of osteoclast activation in these conditions are less clear but may involve NFκB activation through sequestration of IκB. The evidence base for management of these disorders is somewhat limited due to the fact they are extremely rare. Bisphosphonates have been successfully used to gain control of elevated bone remodelling but as yet, no effective treatment exists for the treatment of the muscle and neurological manifestations of MSP syndromes.

Paget's Disease of Bone.

Paget's disease of bone (PDB) is a chronic and focal bone disorder, characterized by increased osteoclast-mediated bone resorption and a subsequent compensatory increase in bone formation, resulting in a disorganized mosaic of woven and lamellar bone at one or more affected skeletal sites. As a result, bone pain, noticeable deformities, arthritis at adjacent joints, and fractures can occur. In a small proportion of cases neoplastic degeneration in osteosarcoma, or, less frequently, giant cell tumor has been also described at PDB sites. While recent epidemiological evidences clearly indicate a decrease in the prevalence and the severity of PDB, over the past 2 decades there have been consistent advances on the genetic mechanisms of disease. It is now clear that PDB is a genetically heterogeneous disorder, with mutations in at least two different genes (SQSTM1, ZNF687) and more common predisposing variants. As a counterpart to the genetic hypothesis, the focal nature of lesions, the decline in prevalence rates, and the incomplete penetrance of the disease among family members suggest that one or more environmental triggers may play a role in the pathophysiology of PDB. The exact nature of these triggers and how they might interact with the genetic factors are less understood, but recent experimental data from mice models suggest the implication of paramixoviral infections. The clinical management of PDB has also evolved considerably, with the development of potent aminobisphosphonates such as zoledronic acid which, given as a single intravenous infusion, now allows a long-term disease remission in the majority of patients.

Genetic Variation in RIN3 in the Belgian Population Supports Its Involvement in the Pathogenesis of Paget's Disease of Bone and Modifies the Age of Onset.

Paget's disease of bone (PDB) is a common, late-onset bone disorder characterized by focal increase of bone turnover. Mutations in the SQSTM1 gene are found in up to 40% of patients and recent GWAS have led to novel associations with several loci. RIN3, the candidate gene located at the associated 14q32 locus, has recently been studied in a British cohort to elucidate its contribution to the pathogenesis. In this study, we performed a genetic screening of RIN3 in an unrelated cohort to validate these findings and to further explore genetic variation in this gene in the context of PDB. In our screening, we examined the 5' untranslated region (UTR), the exonic regions and the intron-exon boundaries of the gene in a control cohort and a patient cohort. Our findings show clustering of variation similar to the British cohort and support a protective role for common genetic variation (rs117068593, p.R279C) in the proline-rich region and a functionally relevant role for rare genetic variation in the domains that mediate binding and activation of its interaction partner, Rab5. Additive regression models, fitted for the common variants, validated the association of the rs117068593 variant with the disease (OR+/+ 0.315; OR+/- 0.562). In addition, our analyses revealed a potentially modifying effect of this variant on the age of onset of the disease. In conclusion, our findings support the involvement of genetic variation in RIN3 in PDB and suggest a role for RIN3 as a potential modifier of the age of onset of the disease.