Molecular effect of an OPTN common variant associated to Paget's disease of bone

Paget’s disease of bone (PDB) is a chronic bone disorder and although genetic factors appear to play an important role in its pathogenesis, to date PDB causing mutations were identified only in the Sequestosome 1 (SQSTM1) gene at the PDB3 locus. PDB6 locus, also previously linked to PDB, contains several candidate genes for metabolic bone diseases. We focused our analysis in the most significantly associated variant with PDB, within the Optineurin (OPTN) gene, i.e. the common variant rs1561570. Although it was previously shown to be strongly associated with PDB in several populations, its contribution to PDB pathogenesis remains unclear. In this study we have shown that rs1561570 may contribute to PDB since its T allele results in the loss of a methylation site in patients’ DNA, leading to higher levels of OPTN gene expression and a corresponding increase in protein levels in patients’ osteoclasts. This increase in OPTN expression leads to higher levels of NF-κB translocation into the nucleus and increasing expression of its target genes, which may contribute to the overactivity of osteoclasts observed in PDB. We also reported a tendency for a more severe clinical phenotype in the presence of a haplotype containing the rs1561570 T allele, which appear to be re-enforced with the presence of the SQSTM1/P392L mutation. In conclusion, our work provides novel insight towards understanding the functional effects of this variant, located in OPTN intron 7, and its implication in the contribution to PDB pathogenesis.


Introduction
Paget's disease of bone (PDB) is a metabolic bone disorder that affects between 1% and 3% of Caucasians over the age of 55 [1][2][3]. This disease is characterized by focal abnormal bone remodelling, with increased bone resorption and accelerated, excessive and disorganized new bone formation. The pathophysiology of PDB is currently an area of intensive investigation and both genetic and non-genetic factors have been implicated. Fifteen to 40 percent (15-a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 40%) of affected patients have a first-degree relative with PDB and numerous studies have described large families with PDB exhibiting an autosomal dominant mode of inheritance [3][4][5]. Linkage studies in these families have identified a number of susceptibility loci on chromosomes 6p21 (PDB1) [6], 18q21.   [7], 5q35 (PDB3), 5q31 (PDB4) [5], 2q36 (PDB5) [8], 10p13 (PDB6) [9] and 18q23 (PDB7) [8]. These regions contain many genes that could be plausible candidates based on their known functions. A genome-wide scan in British families with PDB has shown a linkage to the 10p13 (PDB6) locus [9,10] and reanalysis of data from this genome-wide scan confirmed this genetic association [11], namely to the common variant rs1561570 (hg19, chr10:g.13155726 T>C) in the Optineurin (OPTN) gene. Our group has also replicated the strong, statistically significant, genetic association of rs1561570 (p-value = 5.65×10 −7 ) with PDB in the French-Canadian population [12]. OPTN gene has been previously linked to glaucoma [13] and neurodegenerative diseases, like Alzheimer's, Parkinson's or amyotrophic lateral sclerosis [14]. It was also found to be important for several cellular events such as membrane trafficking and maintenance of the Golgi complex, autophagy and protein turnover through the ubiquitin-proteasome pathway [15][16][17][18]. OPTN has been characterized as an autophagy adaptor protein, which regulates selective autophagy of ubiquitin-coated cytosolic proteins, organelles and parasites such as Salmonella enterica. This function depends on the phosphorylation of its LC3-interacting motif by TANK-binding kinase 1 (TBK1) [16,[18][19][20]. OPTN can also mediate protein aggregate removal through a ubiquitin-independent mechanism [21] and, in addition, it seems to be part of the TNFα pathway, contributing to mediate the availability of NF-κB in the cell [22,23]. A possible role for OPTN in bone was recently described by Obaid et al. (2015) who have shown that mice harbouring a loss-of-function mutation in Optn presented an increase in osteoclast activity and in bone turnover, thus indicating that this protein could be acting as a negative regulator of osteoclast differentiation, possibly by inhibiting NF-κB activation [24]. Despite these findings, this animal model did not develop a PDB-like phenotype. Previously, in a different model and in contrast with those results, OPTN was described as being involved in the activation of NF-κB pathway [25]. Indeed, Journo and colleagues (2009) have observed that in human cells, infected with the human T lymphotropic virus type 1, OPTN increased the ubiquitination of Tax1 as well as Tax1-dependent NF-κB signalling, thus promoting NF-κB activation, a result that is in agreement with other studies [26][27][28]. These discrepancies suggest that the pathologies linked to OPTN cannot be associated exclusively to its loss of function, but more likely to its aberrant expression, resulting from either mutant or gain of OPTN function affecting different pathways in distinct biological contexts, and which are not yet fully understood. Therefore, additional work is required to understand the complexity of the events associated to OPTN function, namely using ex-vivo tools such as PDB patients' samples. In the present work the effect in osteoclastogenesis of the most significantly PDB-associated OPTN variant (rs1561570) was assessed using patients' monocytes as study model. These cells are precursors of osteoclasts and can be induced to fuse in order to originate mature osteoclast cells in vitro. The results obtained are expected to contribute to further elucidate the role of OPTN in the pathophysiology of a complex chronic bone disease such as PDB.

Study participants
This study was approved by the CHU de Québec-Université Laval Ethics Committee and all participants have signed a consent form before inclusion in the study. We investigated patients with familial form of PDB (one patient per family, to avoid the biais of relatedness in our genetic association analysis), unrelated PDB patients and healthy controls, all from the French-Canadian population. The intra familial cosegregation of the T allele among affected relatives within familial forms was not performed. All patients and healthy donors studied here were non-carrier of the P392L mutation within the SQSTM1 gene (PDB3 locus). For the PDB osteoclastic phenotype characterization and to assess the potential synergistic effect of rs1561570 with SQSTM1/P392L mutation in osteoclastogenesis, we studied a patient heterozygous for rs1561570 (CT genotype) and also carrier of the P392L mutation in SQSTM1. Phenotype assessment, DNA and RNA extraction methods are detailed in the S1 File.

Bioinformatic analysis
To analyse if rs1561570 was affecting the methylation status at that particular location we used the MethPrimer (http://www.urogene.org/methprimer/), an in silico tool used to predict CpG island by examining the CG dinucleotides and to design specific primers to analyse those CpG islands (S1 Table).

Methylation analysis by bisulfite conversion and sanger sequencing
Sodium bisulfite treatment was performed on 1.5 μg of genomic DNA sample using the Epi-Mark1 Bisulfite Conversion Kit [29] (New England Biolabs Inc., Canada) following the manufacturers' standard protocol. Further details are provided in the S1 File.

Cell culture conditions
The U937 (Human monocytes/lymphoma) and U2OS (Human bone osteosarcoma) cell lines were grown in Dulbecco's modified eagle medium (DMEM), supplemented with 10% fetal bovine serum (FBS), 2mM L-glutamine and 1% penicillin/streptomycin (P/S). The T47D (Human breast carcinoma) cell line was grown in RPMI-1640 medium supplemented with 10% FBS, and 1% P/S. The MG63 (Human bone osteosarcoma) cell line was grown in αMEM supplemented with 10% FBS, and 1% P/S. All cell lines were incubated at 37˚C in a humidified atmosphere containing 5% CO 2 . The medium, FBS, antibiotics and glutamine were obtained from Invitrogen, Portugal. Cell lines were obtained from American Type Culture Collection (ATCC).

Demethylating treatment and immunofluorescence
The localization of NF-κB after treatment with 5-Azacitidine (5-Aza, Sigma-Aldrich, Portugal) for 72h in T47D and U937 cell lines was assessed by immunofluorescence. NF-κB localization was also assessed by immunofluorescence in osteoclasts derived from Peripheral Blood Mononuclear Cells (PBMCs) from healthy donors and PDB patients with all studied genotypes. The immunofluorescence protocol is described in the S1 File.

Preparation of human in vitro-differentiated mature osteoclasts
Human mature osteoclasts were differentiated in vitro using mononuclear cells from blood of healthy controls and PDB patients. PBMCs were obtained by density gradient centrifugation using Ficoll-Hypaque. The cells were resuspended (3x10 6 cells/mL) in OPTI-MEM containing 10% FBS. The cell suspension was added to 6-well plates (9x10 6 cells/well) and to Lab-Tek 8 well-slides (3-6x10 5 cells/well). After 24h, the cells were washed thoroughly and lymphocytefree adherent cells were incubated for three weeks with M-CSF (25 ng/ml, Life technologies, Canada) and RANKL (30 ng/ml, Fitzgerald, Canada).

Osteoclast morphology assessment, TRAP assay and in vitro assessment of bone resorption
Mature osteoclast formation was evaluated by quantification of TRAP-positive multinuclear cells using an acid phosphatase kit (Sigma-Aldrich, Canada), according to the protocols provided by the manufacturer. The bone resorption activity of osteoclasts was tested in vitro, using a previously validated model [30]. The details of the TRAP assay and the in vitro assessment of bone resorption are provided in the S1 File.

Quantitative real-time PCR
In order to test if the most strongly PDB associated OPTN variant had an impact on its gene expression, we performed qPCR as previously described [12]. The details of this analysis are provided in the S1 File.

Statistical analysis
Statistical analysis was performed with GraphPad Prism 5 (GraphPad, La Jolla, CA). Comparisons between two groups were made using a two-tailed Student's t-test. For comparisons between multiple groups, one-way ANOVA followed by Tukey's post-hoc test was used. Differences were considered statistically significant when p<0.05.

Rs1561570 increases in vitro osteoclast differentiation and bone resorption activity
To better assess the effect of rs1561570 on osteoclasts formation, we performed a TRAP assay in osteoclasts derived from the PBMCs of patients carrying the rs1561570 C or the T allele. The presence of a haplotype with at least one T allele for rs1561570 increased significantly osteoclast formation, attested by the mean number of multinucleated cells yielded from PBMCs cultures, when compared to healthy controls (Controls CC 10%, Patients CC 50%, Patients CT/TT 80%, p-value = 0.006) (Fig 1A and 1C). The osteoclasts generated displayed a higher mean number of nuclei than the osteoclasts from healthy controls (Controls CC mean = 3, Patients CC mean = 5, Patients CT/TT mean = 10, p-value = 0.002) (Fig 1B and 1C).
Accordingly, bone resorption was also significantly increased in osteoclast cultures generated from patients with a haplotype carrying at least one T allele for rs1561570, resulting in 3-fold increase in the total bone resorption area when compared to cultures prepared from healthy controls (p-value < 0.001) (Fig 2A and 2B) and 1.4-fold increase when compared to cell cultures prepared from PDB patients carrying two C alleles (p-value = 0.05). We also analysed the bone resorption area from osteoclast cultures of one patient carrying a haplotype with one T allele (CT) in combination with the SQSTM1/P392L mutation. Interestingly, there was an increase of the bone resorption area in the patient with one T allele, suggesting that there might be a synergistic effect of OPTN and SQSTM1 genetic variants in PDB (Fig 2B).

Rs1561570 does not affect autophagy
OPTN has been suggested to be an autophagy adaptor and since impairment in autophagy was reported in PDB, we assessed the autophagy process in patients with a haplotype carrying at least one rs1561570 T allele. By immunofluorescence and western blot analysis we found that (C) The images are representative of the distribution of nuclei number observed in osteoclasts generated from PBMCs derived from healthy controls and PDB patients that were stained for TRAP and counterstained with haematoxylin. (ANOVA, Ã represents a p-value < 0.05, ÃÃ represents a pvalue < 0.01, ÃÃÃ represents a p-value < 0.001, ÃÃÃÃ represents a p-value < 0.0001; non-mutated controls with CC genotype (n = 3), non-mutated patients with CC genotype (n = 5) and patients carrying at least one T allele (n = 3).
https://doi.org/10.1371/journal.pone.0197543.g001 LC3BII expression was impaired, however that effect did not seem to be dependent of the presence of a haplotype with the T allele (S1A and S1B Fig), suggesting that the autophagy impairment may be independent from the rs1561570 genotype.

Rs1561570 causes a change in methylation and increases OPTN expression
Since our previous study has shown a strong association between rs1561570 (located in OPTN intron 7) and PDB [12], we determined the functional effect of rs1561570 in that disease by employing the splice site prediction algorithm, and showed that the allele harbouring a C was likely to create a potential new acceptor site for splicing and disrupt a potential branch point (consensus value for mutant sequence = 69.7%) (S2 Table). To validate this finding, we performed RT-PCR and qPCR using cDNA samples from patients and healthy controls with the three genotypes. Retention of intronic sequences was not observed in the genotypes analysed (results not shown). This result was even more evident in one PDB patient carrying one T allele (CT genotype) plus the SQSTM1/P392L mutation (p-value < 0.001). At least three different wells per patient were analysed. (ANOVA, Ã represents a p-value < 0.05, ÃÃ represents a p-value < 0.01, ÃÃÃ represents a p-value < 0.001, ÃÃÃÃ represents a p-value < 0.0001; non-mutated controls with CC genotype (n = 3), non-mutated patients with CC genotype (n = 5) and patients carrying at least one T allele (n = 3)). https://doi.org/10.1371/journal.pone.0197543.g002 We then studied the predicted impact of rs1561570 in the OPTN methylation status using the Methprimer tool. We found that the allele C was predicted to form a CpG dinucleotide, creating a methylation site, which was not predicted in the presence of the T allele (Fig 3A). After bisulfite treatment, we confirmed that all samples with at least one C allele in their rs1561570 genotype were in fact methylated (Fig 3B). To correlate the methylation site created by the C allele with the OPTN transcriptional levels, we performed qPCR (Fig 3C) and western blot (Fig 3D) analysis using lymphocytes from patients with the three genotypes. Samples carrying a haplotype with the T allele (CT or TT) showed levels of OPTN gene and protein expression higher than samples carrying the CC genotype. This result suggested that the methylated C allele is associated with a decrease in OPTN gene expression levels. The protein expression levels were also measured by western blot analysis in PBMC-derived osteoclasts and the results were in accordance with those obtained by qPCR and western blot analysis in lymphocytes (Fig 3E).

Rs1561570 increases NF-κB translocation into the nucleus
To determine the role of rs1561570 on the osteoclastic phenotype, and since there are previous publications describing OPTN as an important protein in NF-κB pathway [25], we analysed the NF-κB cellular localization by immunofluorescence in PBMC-derived osteoclasts from PDB patients carrying different genotypes of rs1561570 and from healthy controls. Our results showed NF-κB translocation to the nucleus (showed as a ratio between co-localization of NF-κB and DAPI) is increased in patients with genotype CT and TT (Fig 4A). To confirm that the presence of NF-κB in the nucleus was affecting the expression of its target genes, we measured the expression of NF-κB, IL-6, ELK1, TRAP and NFATc1 expression. Our results showed an increased expression of NF-κB target genes related with osteoclastogenesis, such as IL-6, ELK1, TRAP and NFATc1 (Fig 4B)-thus providing additional evidence that an increase of OPTN expression observed in the presence of the T allele in the OPTN methylation status leads to an increase of NF-κB translocation into the nucleus and an increase of its target genes expression.
As a proof of concept, we also analysed the NF-κB cellular localization by immunofluorescence in two different cell lines which carry the rs1561570 C allele-T47D (CT genotype) and U937 (CC genotype)-following a treatment with a demethylating agent, 5-Azacitidine. We were able to confirm that, before the demethylating treatment, NF-κB was localized in the cytoplasm and in the nucleus. However, after OPTN demethylation, NF-κB localization was almost exclusively located in the nucleus (Fig 5A and 5B). To confirm the effect of the demethylating agent we measured OPTN expression and to confirm that the presence of NF-κB in the nucleus was also affecting the expression of its target genes (as in patients carrying the T allele), we measured the expression of NF-κB, IL-6, ELK1, TRAP and NFATc1. Our results showed that there was an increase both in OPTN expression, which was expected after the demethylation treatment, and in the expression of the NF-κB target genes analysed. NF-κB levels were maintained as expected (Fig 5C).
In addition we performed the same procedure using two osteosarcoma cell lines-U2OS (CC genotype) and MG63 (TT genotype) and the results showed that the demethylating agent increased OPTN and NF-κB target genes in U2OS while NF-κB expression was maintained (S2A Fig), suggesting that a change in the methylation status due to the effect of the demethylating agent had an effect in OPTN and in NF-κB target genes expression. In contrast, in the MG63 cell line this treatment did not have any effect in the expression of OPTN or NF-κB target genes (S2B Fig), suggesting that the effect in OPTN expression might be specific for the cells carrying one C allele, thus supporting our hypothesis.

Rs1561570 may have an effect on PDB severity in the presence of SQSTM1 mutations
By comparing the clinical phenotype of patients with PDB carriers of the CC genotype of OPTN rs1561570 with patients carrying one or two T alleles, we observed a tendency, although not significant, for an: i) increase of total serum ALP (sALP) levels, ii) increase of mean number of affected bones, and iii) increase of disease extension measured by Renier's index-related with the number of T alleles in the genotype (S3 Table). These results suggest that rs1561570 could have a dose effect depending on the number of T alleles present in the genotype. In fact, in the presence of the P392L/SQSTM1 mutation and rs1561570 T allele our results showed an increased tendency for i) higher levels of total sALP, ii) higher risk for polyostotic involvement, iii) higher number of affected bones and iv) an increase in the disease extension/Renier's index (S4 Table) with the increase in the number of T alleles. Altogether, these results may suggest a modifier effect of OPTN common variant in the presence of SQSTM1/P392L mutation in patients with PDB, which could lead to an increase in the disease activity and extent, and therefore should be replicated in independent cohorts with higher number of participants.

Discussion
In the present study, we investigated the functional role of the most significantly PDB associated genetic variant of OPTN-rs1561570 (C>T). Previously, we reported that the C allele was more frequent in controls in the French-Canadian population (C allele frequency = 52%, T allele frequency = 48%) while the T allele was more frequent in PDB patients (C allele frequency = 36%, T allele frequency = 64%) [12]. Using in silico tools, we found that the rs1561570 T allele was predicted to cause the loss of a methylation site. With bisulfite treatment, we confirmed that the C allele in that position was always methylated. Furthermore, both qPCR and western blot analysis correlated the presence of the C allele with a decrease of OPTN expression levels. The existence of a correlation between the hypermethylation status of a given gene, resulting in a decrease in gene transcription levels affecting a related disease, has been previously reported in the literature [31][32][33]. Our immunofluorescence analysis also allowed us to confirm that an increase in OPTN levels (due to the demethylation treatment in cells and the presence of at least one T allele in PDB patients-dominant genetic effect) is related to an increase in NF-κB translocation into the nucleus, confirming that a decrease in OPTN methylation (as seen in PDB patients with rs1561570 T allele) increases the translocation of NF-κB into the nucleus, and that is related to an increased expression of NF-κB target genes related with osteoclastogenesis, such as NFATc1 -a master transcription factor of osteoclast differentiation [34], IL6-an important cytokine for osteoclastogenesis [35] and ELK1-a relevant transcription factor in mature osteoclasts [36]. We also showed that the autophagy process is impaired in PDB patients and that it is not dependent on the rs1561570 genetic status. Therefore, we can hypothesize that in healthy individuals, the autophagy process works normally both in the presence of the C allele (when the OPTN expression is controlled at basal levels), and in the presence of the T allele (when the OPTN expression is potentiated due to the loss of a methylation site). In the latter, the efficient autophagy process signals the excessive OPTN for degradation by ubiquitination and recycling of the protein, and thus OPTN expression is reduced to a basal level. In both situations, this will result in normal levels of NF-κB in the nucleus and physiological osteoclastogenesis rate. In PDB patients, where the autophagy process is impaired, in the presence of the rs1561570 C allele, the PDB phenotype might be explained by the presence of other factors, such as other genetic variants or environmental triggers that will lead to an increase in osteoclastogenesis. The effect of rs1561570 T allele in PDB patients may therefore be explained by the increase in OPTN expression associated with a decrease in its degradation due to the autophagy defect present in these patients. Consequently, there is an accumulation of OPTN that promotes NF-κB translocation to the nucleus at a much higher rate than normal, thus also promoting osteoclastogenesis and that in addition to the existence of other genetic variants or environmental triggers potentiate the clinical phenotype of PDB in these patients.
Journo and its co-workers had already described the translocation of NF-κB into the nucleus due to an increase in OPTN expression [25], but upon a virus infection. OPTN is part of a complex, which also includes TAX1 and TAX1BP1, that can activate the IKK complex by ubiquitination, thus leading to the degradation of the NF-κB inhibitor and subsequent release of NF-κB into the nucleus. In the present work, the change in OPTN expression levels caused by one single change in nucleotide may be explained by the alteration of the already reported binding of an enhancer-JUND, a member of the AP1 family that is present in osteoclasts [37][38][39]-in the genomic region containing the T allele, which is lost in the presence of the C allele, as confirmed by the ENCODE project. The ENCODE project shows regions of transcription factor binding, derived from a large collection of ChIP-seq experiments, together with DNA binding motifs identified within these regions by the ENCODE Factor book repository. Indeed, due to the resulting change in DNA conformation in the presence of the T allele, the loss of a JUND binding site around the rs1561570 region of these patients will result in an alteration in OPTN expression levels, promoting an increase in osteoclastogenesis and the development of PDB. Our results are in contrast with data from Obaid et al. (2015) who, based in the description by Zeller and Westra's groups (who have shown that rs1561570 T allele was related to lower levels of OPTN expression [40,41]), analysed the effect of a lack of OPTN in the differentiation of osteoclasts using a mouse knock out for OPTN. Those authors concluded that depletion of OPTN in that model increased the differentiation of osteoclasts (p-value = 0.002), suggesting that OPTN might be a negative regulator of osteoclast differentiation [24]. However, and, unlike in humans, these mice did not develop a PDB-like phenotype [24] , [17]. Interestingly, in the same study, authors also refer that OPTN expression was significantly increased during days 2 to 5 of in vitro osteoclast differentiation using primary bone-marrowderived macrophages, in which they knocked down OPTN by using a small hairpin RNA. In addition, it was found in another set of in vivo works, using either knock-in mice expressing a point mutation in OPTN and abolishing its polyubiquitin binding capacity [42], or mice lacking either the entire C-terminal ubiquitin-binding [43] or the N-terminal TANK-binding kinase 1 (TBK1)-binding domain [44], that mice had retained normal NF-κB responses. These results argued against the fact that a decrease per se in OPTN or in OPTN function could promote osteoclastogenesis by affecting NF-κB signalling pathways.
As referred in a recent review, OPTN was thought to be important for NF-κB signalling because of its strong homology to the NF-κB essential modulator, NEMO-the core element of the inhibitor of NF-κB kinase (IKK) complex -, that is essential for NF-κB activation [18]. Altogether, the lack of a PDB-like phenotype developed in those studies is not in favour of a lack of OPTN expression being the causing factor. As suggested in the same review, it is more likely that the PDB phenotype is the result of an aberrant expression of OPTN, including a possible gain of function [18] which is in agreement with our results. Our study is unique since it analyses, using in vitro and ex vivo techniques, the single effect of rs1561570 in OPTN gene and protein expression in several samples of patients with PDB non carriers of a SQSTM1/P392L mutation, and in different cell types (lymphocytes and osteoclasts derived from patients PBMCs), thus providing, at several levels, clear evidences supporting our hypothesis. Our finding that, in one patient carrying the SQSTM1/P392L mutation, there was a tendency for a more severe clinical phenotype in the presence of a haplotype with the rs1561570 T allele and that osteoclasts from that patient had a higher in vitro bone resorption rate, suggests that there might be a synergistic effect but this should be further investigated with additional patients.
Altogether, the gathered evidence obtained from gene to protein analysis, from in vitro to patient samples, from genetic to morphological cellular features, show that OPTN appears to be important for NF-κB translocation into the nucleus, activation of its target genes and consequent increase in osteoclastogenesis, providing relevant information towards a better understanding on how genetic variants in OPTN can contribute to PDB by acting as modifiers and complementing previous data on the potential functional role of variants in OPTN and how they may contribute to affect the physiological regulatory mechanisms of this gene [45].

Conclusions
In conclusion, we have shown that rs1561570 is responsible for a change in the methylation status of OPTN in patients with PDB and that it has an impact in its expression levels, which may contribute to PDB pathogenesis by affecting NF-κB translocation to the nucleus. Accordingly, to this date, OPTN remains the most likely gene within PDB6 locus to contribute to PDB pathophysiology, possibly in combination with other genetic/environmental factors yet unknown. Values are the mean of at least three independent replicates. (t-test, Ã represents a p-value < 0.05, ÃÃ represents a p-value < 0.01, ÃÃÃ represents a p-value < 0.001, ÃÃÃÃ represents a p-value < 0.0001). (TIF) S1  Table. Comparisons of main clinical characteristics between French-Canadian PDB patients carriers of the rs1561570 TT or TC genotype and the CC genotype carriers, considered as the reference. All 232 patients were non-carrier of the P392L mutation within the SQSTM1 gene (PDB3 locus). (XLSX) S4 Table. Comparisons of main clinical characteristics between French-Canadian PDB patients carriers of the rs1561570 TT or TC genotype and the CC genotype carriers, considered as the reference. All 32 patients were carrier of the P392L mutation within the SQSTM1 gene (PDB3 locus). (XLSX)