US 2017/0130272 A1 Gianfrancesco (43) Pub

US 20170 130272A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2017/0130272 A1 Gianfrancesco (43) Pub. Date: May 11, 2017

(54) ADAGNOSTIC MARKER FOR PAGETS Publication Classification DISEASE (51) Int. Cl. CI2O I/68 (2006.01) (71) Applicant: Consiglio Nazionale Delle Ricerche, GOIN 33/68 (2006.01) C07K I4/47 (2006.01) Roma (IT) GOIN 33/574 (2006.01) (52) U.S. Cl. (72) Inventor: Fernando Gianfrancesco, Fontegreca, CPC ...... CI2O 1/6886 (2013.01); C12O 1/6883 Caserta (IT) (2013.01); G0IN 33/57407 (2013.01); G0IN 33/6893 (2013.01); C07K 14/47 (2013.01); (73) Assignee: Consiglio Nazionale Delle Ricerche, CI2O 2600/158 (2013.01); C12O 2600/156 Roma (IT) (2013.01); G0IN 2800/50 (2013.01); G0IN 2800/10 (2013.01) (21) Appl. No.: 15/312,501 (57) ABSTRACT The invention relates to an in vitro or in vivo method for (22) PCT Fed: May 21, 2015 diagnosing Paget’s disease or bone tumors associated there with or for determining the predisposition of a subject (86) PCT No.: PCT/B2015/053731 affected by Paget’s disease of bone to develop a bone tumor, the method being based on the detection of a mutation in the S 371 (c)(1), nucleotide sequence of the ZNF687 gene or in the amino (2) Date: Nov. 18, 2016 acid sequence of the protein encoded by the mutated ZNF687 gene. The invention also refers to isolated nucleic (30) Foreign Application Priority Data acids comprising the mutated nucleotide sequence of the ZNF687 gene or a portion thereof and the encoded amino May 21, 2014 (IT) ...... TO2O14AOOO404 acid sequences.

Patent Application Publication May 11, 2017 Sheet 2 of 3 US 2017/0130272 A1

Patent Application Publication May 11, 2017. Sheet 3 of 3 US 2017/0130272 A1

So so 9 Sd n is or C i a. Monocytes Muscle Pre-Osteoblasts GCT/PDB 1 Brain GCT/PDB2 Lung GCT/PDB 3 GCT/PDB 4 Liver GCT/PDB 5 Heart

PanCreas Ovary Kidney

Testis

BOne : .

Blood

Monocytes

Pre-Osteoblasts

Osteoblasts FIG.3 US 2017/O 130272 A1 May 11, 2017

ADAGNOSTIC MARKER FOR PAGETS being Suitable for diagnosing the clinically serious forms of DISEASE the disease, such as for example those with neoplastic 0001. The present invention falls within the field of degeneration of the affected bones. These families do not diagnostic markers. have mutations in the SQSTM1 gene. 0002 More particularly, the present invention relates to a 0012. Therefore, there is a need for a new diagnostic new diagnostic marker for Paget’s disease of bone and bone marker for Paget’s disease of bone which overcomes the tumors associated therewith, Such as for example giant cell drawbacks of the prior art, which particularly allows for tumors (GCT) and osteosarcoma. diagnosing and predicting the risk of developing bone 0003 Paget’s disease of bone is a metabolic disorder tumors associated with Paget’s disease of bone. characterized by focal abnormalities in bone remodeling, 0013 Such a need has now been met by the present which typically causes deformity and increased bone size in inventors who identified a new diagnostic marker for Paget’s one or more areas of the skeleton (monostotic form or disease of bone and/or bone tumors associated therewith, polyostotic form). In some cases, patients affected by Pag particularly osteosarcomas and giant cell tumors, which et's disease of bone—and particularly those affected by its does not show the above-mentioned drawbacks. polyostotic form incur into neoplastic degeneration of the (0014. Such a new marker is the ZNF687 gene, which affected bones (GCT), caused by uncontrolled proliferation encodes for the Zinc finger protein 687, a component of the of the spindle-shaped stromal cells. transcriptional coregulator Z3 complex. 0004. The primary cell abnormality in Paget’s disease of 0015. By whole-exome sequencing and by examining a bone relates to osteoclasts, which show many functional and family including 14 members affected by Paget’s disease of morphological abnormalities. bone, four of which had developed GCT at Paget’s sites, the 0005 Paget’s disease of bone is also characterized by present inventors detected a missense mutation in the specific modifications of the biochemical distribution of ZNF687 gene (c.2810CDG, P937R). The sequencing of 7 non-collagenous proteins in the bone matrix immediately non-related patients affected by GCT as a complication of adjacent to Haversian canals and by bone neoangiogenesis. Paget’s disease of bone has allowed for identifying the same Some of these modifications are very similar to those found mutation in all of the patients, which suggests a founder in bone metastases in cancer patients and in patients affected effect. The same mutation and also a second missense by multiple myeloma. mutation in the same gene (c.725G>T, p. Ser242Ile) were 0006 Epidemiological data point out that Paget’s disease observed in patients with a serious form of Paget’s disease of bone may be considered as a disease that predisposes to of bone. Furthermore, in a non-pagetic patient, affected by the appearance of primary bone neoplasms in Subjects over GCT and with a family history of Paget’s disease of bone 40 years old. The connection between Paget’s disease of (one parent affected) a third mutation was identified (c. 1994 bone and an increased risk of malignant transformation of C>T, P665L) in the ZNF687 gene. the affected bones has been described for the first time by Sir (0016. The ZNF687 gene, which encodes for a member of James Paget in his first paper on osteitis deformans in 1876. the transcriptional regulation Z3 complex, is highly Since then, several literature papers reported a higher inci expressed during osteoblastogenesis and osteoclastogenesis, dence of osteosarcomas and GCT in patients affected by and is dramatically upregulated in tumor tissue derived from Paget’s disease of bone, and neoplastic degeneration of patients affected by giant cell tumor/Paget’s disease of bone. bones affected by Paget’s disease of bone was estimated to ZNF687 knockdown results in a marked decrease in tran occur in 1% of patients, with an increase in the risk as high scription of 2-IIIc receptor of FGF (FGFR2IIIc), revealing a as 30 times over that of the general population (Roodman tight correlation between these two genes. Moreover, by 2001, Gebhart 1998). immunohistochemical analysis of patients affected by giant 0007. In 2002, a few mutations were identified which cell tumor/Paget’s disease of bone, the inventors observed a affect the ubiquitination domain of the protein encoded by strong FGFR2IIIc staining in multinucleated giant cells and the SQSTM1 gene which are detected in 10% and 40% of stromal cells, which supports the role of FGFR2IIIc in the cases of sporadic and familial Paget’s disease of bone, pathogenesis of these tumors. respectively (Laurin et al 2002, Hocking et al 2002). 0017. These and other experimental findings, which will 0008 More recently, other genes (CSF1, OPTN, be described in detail in the experimental section that TNFRSF11A, PML, RIN3, NUP205, TM7SF4) have been follows, allowed the inventors to conclude that the detection associated with the Paget’s disease of bone in patients of mutations in the nucleotide sequence of the ZNF687 gene without the mutation of SQSTM1 (Albagha et al 2010, represents a useful diagnostic tool for Paget’s disease of Gianfrancesco et al 2012, Albagha et al 2011). bone and bone tumors associated therewith, particularly 0009. A familial clustering was also demonstrated in a giant cell tumors and osteosarcomas, as well as a useful few Paget’s pedigrees affected by GCT and less frequently prognostic tool for establishing the predisposition of a by osteosarcoma, particularly in patients of Italian origin subject affected by Paget’s disease of bone of developing a (Rendina et al 2004, Gianfrancesco et al 2013, Wu et al bone tumor associated therewith, particularly giant cell 1991). tumor Or OSteoSarCOma. 0010. The International patent application WO2011/ (0018. The wild-type nucleotide sequence of the ZNF687 128646 describes a method of diagnosis for Paget’s disease gene is illustrated in SEQ ID NO:1. of bone, which comprises testing a sample from a Subject for 0019. One aspect of the present invention is thus an in sequence changes at or within one or more chromosome loci vitro or ex vivo method for diagnosing Paget’s disease of selected from the group consisting of 10p13, 1 pl3.3, 18q21. bone or bone tumor, which comprises the step of determin 8q22.3, 7q33, 14q32.12, 15q24.1, 6p22.3 and Xq24. ing, in a nucleic acid sample of a Subject Suspected of being 0011. However, the currently known diagnostic markers affected by Paget’s disease of bone or bone tumor, the for Paget’s disease of bone have the disadvantage of not presence of at least one mutation in the wild-type nucleotide US 2017/O 130272 A1 May 11, 2017

sequence of the ZNF687 gene as represented by SEQ ID 0031. Such mutated ZNF687 protein is in turn useful for NO:1, the presence of said at least one mutation being the manufacture of specific antibodies, for example mono indicative of Paget’s disease of bone or bone tumor. clonal or polyclonal, chimeric or humanized antibodies, 0020. A further aspect of the present invention is an in which are manufactured by conventional methods, the vitro or ex vivo method for determining the predisposition of accomplishment of which is within the ability of the person a subject affected by Paget’s disease of bone to develop a of ordinary skill in the art. The said antibodies are useful as bone tumor, which comprises the step of determining, in a assay reagents for carrying out immunodiagnostic methods nucleic acid sample of a Subject affected by Paget’s disease within the scope of the present invention, Such as for of bone, the presence of at least one mutation in the example ELISA immunoassay methods. wild-type nucleotide sequence of the ZNF687 gene as 0032. Therefore, another aspect of the present invention represented by SEQ ID NO:1, the presence of said at least is an in vitro or ex vivo method for diagnosing Paget’s one mutation being indicative of a predisposition to develop disease of bone or bone tumor, which comprises the step of a bone tumor. determining, in a biological sample from a Subject Suspected 0021. In a preferred embodiment of both of the methods of being affected by Paget’s disease of bone or bone tumor, mentioned above, the bone tumor is a giant cell tumor or an the presence of at least one ZNF687 protein which is OSteOSarCOma. mutated compared to the wild-type ZNF687 protein 0022. In another preferred embodiment, the mutation is a sequence as represented by SEQ ID NO:8, the presence of missense mutation. said at least one mutated protein being indicative of Paget’s 0023 Examples of missense mutations suitable for use in disease of bone or bone tumor. the methods of the invention are the nucleotide mutation 0033 Still another aspect of the present invention is an in C>G at nucleotide position 2810 of SEQ ID NO:4, the vitro or ex vivo method for determining the predisposition of nucleotide mutation G-T at nucleotide position 725 of SEQ a subject affected by Paget’s disease of bone to develop a ID NO:2 and the nucleotide mutation CDT at nucleotide bone tumor, which comprises the step of determining, in a position 1994 of SEQ ID NO:3. Further mutations particu biological sample from a subject affected by Paget’s disease larly suitable to be detected in the methods of the invention of bone, the presence of at least one ZNF687 protein which are mutations located in the coding region of the gene. is mutated compared to the wild-type ZNF687 protein 0024. The mutations can be detected by any known sequence as represented by SEQ ID NO:8, the presence of method, for example by nucleic acid amplification reaction said at least one mutated protein being indicative of a and detection of the amplification product or by sequencing predisposition to develop a bone tumor. the whole nucleotide sequence of the ZNF687 gene or a 0034. In both of the above-mentioned methods, the portion thereof and comparing it with the wild-type mutated ZNF687 protein compared to the wild-type ZNF687 sequence of ZNF687, used as a known control sequence. protein sequence represented by SEQID NO:8 is preferably 0025. These are per se known methodologies and the selected from SEQ ID NO:5, SEQ ID NO:6 and SEQ ID application thereof to the methods of the invention is well NO:7. within the ability of the person of ordinary skill in the art. 0035. The part that follows, related to Examples and 0026. Another aspect of the invention is an isolated Experimental section, is provided only by way of illustration nucleic acid, preferably a genomic DNA, mRNA or cDNA, and not as a limitation of the scope of the invention as as defined in claim 1. defined in the appended claims. 0027. The isolated nucleic acid of the invention com prises the coding region of the ZNF687 gene and the EXAMPLES mutation is located in said coding region. As previously indicated with reference to the methods of the invention, the 1. Experimental Section mutation is a missense mutation, particularly the nucleotide 0036 Methods mutation C>G at nucleotide position 2810 of SEQID NO:4 0037 Whole-Exome Sequencing Strategy. or the nucleotide mutation G-T at nucleotide position 725 of 0038. The DNA from the four subjects belonging to the SEQID NO:2 or the nucleotide mutation CDT at nucleotide pedigree with GCT/PDB was used for exome sequencing by position 1994 of SEQ ID NO:3. Therefore, preferred iso using the NimbleGen SeqCap EZ Exome TM capture kit lated nucleic acids according to the invention are nucleic (Roche) and the resulting fragments were sequenced by acids with the sequence SEQ ID NO:2 or SEQ ID NO:3 or Illumina Genome Analyzer IIx (Illumina, San Diego, Calif., SEQ ID NO:4. USA) with sequences of 90 bp in length. 58.458.230 0028. Such nucleic acids are suitable for use in the sequences for sample V-4, 55.895.176 sequences for sample manufacture of the corresponding mutated protein, by using V-18, 45.223.865 sequences for sample V-11 and finally recombinant DNA methodologies which are per se known 50.109.914 sequences for sample V-7 were obtained. These and within the ability of the person of ordinary skill in the sequences were aligned with human genomic sequence art GRCh37/hg19 with the software MAQ7 and NextGENe 0029. A further aspect of the present invention is thus an V2.00. A coverage of over 45x for each sample, the sequenc expression vector including a nucleic acid as previously ing of all the exons and the 5' and 3' untranslated regions defined, as well as a host cell transformed with the said were obtained. The analysis for identification of variations Vector. was carried out with the DNA bioinformatics platform 0030 The host cell of the invention is capable of express Nexus. The variants obtained were filtered with those pres ing the mutated ZNF687 protein, for instance the protein ent in the SNP database (dbSNP131). Assuming the syn with the sequence SEQ ID NO:5 or SEQ ID NO:6 or SEQ onymous variants had less chances of being pathogenetic, ID NO:7. the inventors only focused on non-synonymous variants US 2017/O 130272 A1 May 11, 2017

(NS), mutations at splice donor and acceptor sites (SS), and derived from human adult tissues purchased from Stratagene Small coding insertions or deletions (I). with the Cycler DNA Engine Opticon 2 system (Biorad). 0039. Mutational Analysis. One microgram of total RNA was reverse transcribed with 0040. The analysis for validation and segregation of the the RevertAid RT kit (Thermo Scientific). The expressions selected variants was performed by Sanger sequencing. PCR were normalized to the HPRT gene to account for differ amplification and direct sequencing protocols have been ences in the starting material and in the cDNA reaction described previously (Gianfrancesco et al. 2012). Oligo efficiency. An electrophoresis on agarose gel was performed nucleotides used for amplifying and sequencing the ZNF687 to further confirm the specificity of the PCR products. The gene are: oligonucleotides of the ZNF687 transcript (5'-AG GAGTCGTCTTCATCTTCA-3' (SEQ ID NO:27) and 5'-TTCACTGACTTGCCATGCTC-3' (SEQ ID NO:28) exon 2A.: were used to generate a product of 214 bp. The HPRT (SEO ID NO: 9) oligonucleotides (5'-TGGCGTCGTGATTAGTGATG-3' s" - CCTCC TCGTTCCTGTTTTCA-3' (SEQ ID NO:29) and 5'-GCACACAGAGGGCTACAATG (SEQ ID NO: 10) 3' (SEQ ID NO:30) were used to generate a product of 185 5 - CAATGGTGGGGAAGAAACAG-3 bp.

exon. 2B: 0043. Cultures and Primary Cell Lines. (SEQ ID NO: 1) 0044) Cell lines MG63, RAW264.7, U2OS and HEK293 5'-GGACCTGTTTGCTCATTTTG-3' were plated on 60-mm plates using DMEM culture medium (Gibco) supplemented with 10% fetal bovine serum, 2 mM (SEQ ID NO: 12) L-glutamine, 100 U/mL penicillin and 100 mg/mL strepto s' - AACTGTACGCTCACCACCTT-3' mycin (Gibco) until they reached a 70-80% confluence. exon 2 C: 0045 siRNA Transfection (SEQ ID NO: 13) I0046) 1.5x10 cells were plated on 60-mm plates. 24 5 - CGGGAATATCACAAGGACTG-3 hours later, MG63 cells were transfected with a mixture of (SEQ ID NO: 14) small interfering RNAs ZNF687 10 nM (siRNAs) Duplex 5-TATAGGCAGGGAGCAGGTTC-3' Trilencer-27 (Origene) and 26.5 uL of IBAFECT reagent

exon 2D : (Iba Solutions For Life Sciences) in OPTIMEM (1x)+ (SEO ID NO: 15) GlutaMax-I reduced serum media (Gibco, Life Technolo 5'-AATGGTGCCTCGGTGGTGAT-3' gies). Each transfection was performed in triplicate and repeated four times. 48 and 72 hours post-transfection, the (SEQ ID NO: 16) cells were blocked to isolate RNA and proteins. 5 - GAGCATCATGGGGCAGGTTG-3 0047 Protein Extraction and Western Blot Analysis for exon 3 : ZNF687 and FGFR2IIIc (SEQ ID NO: 7) 0048. After 48 and 72 hours of incubation, MG63, s' - CATGCTTTCGCTGCCTGGAG-3' RAW264.7 and U2OS cells were lysed with lysis buffer (5 (SEQ ID NO: 18) ml 100% glycerol, 50 ul tween20, 500 ul Triton 100, 3.5 ml 5 - GAGGGATATAGCAGGGAAGA-3' 5M NaCl, 2.5 ml 1M Tris pH 7.9, 250 ul M MgCl, all in a final volume of 50 ml). The lysed cells were centrifuged at exons 4 - 5: (SEQ ID NO: 19) 14000 g for 5 minutes. Proteins were denatured by boiling s" - CCTTTCTCCACCCTGCTCAT-3 in sample buffer, separated on 8% SDS-PAGE, transferred onto an Immun-Blot PVDF membrane for protein blotting (SEQ ID NO: 2O) (BIO-RAD) and blocked for 1 hour in 5% dried skimmed 5'-ACCCATTCCCCATCCCTCTG-3' milk in TBS-T (10 mM Tris-HCl pH 7.5, 100 mM NaCl, exon 6 : 0.1% (v/v) Tween-20). Antibodies used for protein recog (SEQ ID NO: 21) nition are: anti-ZNF687 polyclonal rabbit antibody s" - CCGTCTTGTCCTCTGCTCTT-3' ab105544 (abcam 1:500), anti-Bek polyclonal rabbit anti (SEQ ID NO: 22) body (c-17): sc-122 (Santacruz 1:100) and anti-B actin 5'-AGGAGAGCAGGGGTAGATGT-3' polyclonal mouse antibody (c4) sc-47778 (Santacruz 1:1000). Goat anti-rabbit antibodies conjugated with peroxi exons 7-8: dase (1:1.000 for ZNF687 protein and 1:2000 for FGFR2 (SEQ ID NO: 23) protein and anti-mouse (diluted 1:15000 in TBST) (Biorad) s' - GCCTGGCTCTGACATCTACC-3' were used as secondary antibodies. (SEQ ID NO: 24) 0049. The Western Blot assay was quantified with the s' - CCTTACCACCACCCCTACTG-3' Image J program. exon 9 : 0050. Osteoclast Formation Assay (SEQ ID NO: 25) 0051 Peripheral blood mononuclear cells were separated s' - GCTGTGCTAGGGCTTTGAGT-3' by Ficol density gradient centrifugation and the cells in (SEQ ID NO: 26) suspension were plated in 24-well plates (3x10). Osteo 5'-AGGCAGCAGTAGGGAAAACC-3' clasts were generated starting from cells cultured for 14-21 days in alpha-minimal essential medium (C.-MEM) 0041 Expression Analysis (SIGMA, Life Science) containing 10% fetal bovine serum 0042. In order to analyze the expression pattern of the (FBS), 2 mM L-glutamine, 100 U/ml penicillin and 100 ZNF687 gene, a real-time PCR was carried out on total RNA mg/ml streptomycin (Gibco) as well as 25 ng/mL macro US 2017/O 130272 A1 May 11, 2017 phage colony stimulating factor (M-CSF) (Peprotech, Rocky The p.P937R mutation segregates with the disease pheno Hill, N.J., USA) and 30 ng/mL soluble human recombinant type in family 2, it being present in patient II-3, but not in sRANK ligand (RANKL) (Peprotech, Rocky Hill, N.J., the healthy sibling II-2. Also the p.S242I mutation segre USA). gates with the disease phenotype, it being present in the two 0052 Results affected siblings (II-1, II-3, II-6) while it has not been 0053 Identification of the Gene Responsible for Giant detected in the non-affected members (II-2, II-4, II-5) (FIGS. Cell Tumor Associated with Paget’s Disease. 2A-2B). This mutation, which determines the substitution of 0054 Recently, the inventors described a family with 14 an isoleucine for a serine in position 242 (p.S242I), is not individuals affected by Paget’s disease, 4 of which with reported in the database “1000 Genomes. Serine in position giant cell tumor (Gianfrancesco et al 2013). A whole-exome 242 is highly conserved in mammals (FIG. 2C) and the sequencing was carried out on the DNA of three patients substitution thereof is predicted as detrimental by the pre affected by PDB (V-4, V-18, V-11), two of which with giant diction programs SIFT. PolyPhen and Align GVGD. In order cell tumor, and of one healthy subject (V-7) (FIG. 1A). By to verify the presence of mutations in the ZNF687 gene also adopting a selection scheme, the variants recorded in the in the GCT not related to the Paget’s disease of bone SNP database (dbSNP131), and then those present in 4 (conventional GCT), the genetic analysis was performed in exomes available at the inventors’ laboratory and derived 44 patients affected by such disease, 38 of which had from 4 subjects affected by diseases that are not related with somatic mutations in the H3F3A gene. This analysis resulted the tested one, were excluded. After such a filtering, the data in the identification of another mutation (c. 1994 CDT, from the three affected individuals were compared, with the P665L) in exon 2 of the ZNF687 gene (FIG. 2D) in a patient exception of variants present in the healthy Subject, identi whose anamnesis had not shown a family history of Paget’s fying 57 variations in 45 candidate genes. Twenty-one of disease of bone. these variants were confirmed by Sanger direct sequencing 0057 The c.2810C-G mutation (p.937R) was assessed in the three patients, yet only one of these showed a by the Taqman assay in two cohorts of Paget’s patients of complete segregation with the disease phenotype in the different ethnic groups. It has been identified in 2 Italian family: c.2810C-G that determines the amino acid substi patients out of 628 and in 2 patients out of 339 of the tution P937R, in exon 6 of the ZNF687 gene located on multi-ethnic cohort (Michou et al 2011). Moreover, this chromosome 1 (1q21.3) in one of the linkage regions mutation was not detected in 564 control individuals of described in the previous work (Gianfrancesco et al 2013). Italian origin and in 269 control individuals of American The mutation is heterozygous in patients and is absent in origin. All the patients bearing the mutation in the ZNF687 non-affected individuals (FIG. 1B). Such a mutation has not gene showed a very serious form of the disease, predicting been detected in a cohort of 564 control individuals from the that these patients are predisposed to development of neo same geographical area and the evolutionary analysis has plastic complications and that the pharmacological treat shown that proline 937 is conserved in all mammals, Sug ment prevents the onset thereof. In fact, all GCT/PDB cases gesting its important biological role (FIG. 1C). To validate show a decreased response to treatment with bisphospho the results, the inventors analyzed the ZNF687 gene in 7 nates and an active persistency of the disease, with ALP patients independently affected by GCT/PDB, 4 of which levels well over the normal value range. Together, these data were from the same localized area of South Italy and three Suggest that the persistency of an active Paget’s disease may were Italian patients or American patients with Italian ances increase the risk of GCT complications, at least in geneti tors (Rendina et al 2006, Gennari et al 2010, Michou et al cally predisposed subjects. 2011). Interestingly, the same mutation c.2810CDG was 0.058 Expression of the ZNF687 gene increases during detected in all GCT/PDB patients, suggesting they share a the differentiation of osteoclasts and osteoblasts. The expres common founder mutation. To validate this hypothesis, the sion profile of the ZNF687 gene was analyzed by using a segregation of rare variants was assessed in genes Surround panel of RNAs extracted from human adult tissues through ing mutation c.2810CDG, detecting heterozygous variations a real-time PCR assay, revealing its expression in all the for SNPs rs149735232 (SV2A), rs138864506 (SETDB1), examined tissues (FIG. 3A). The expression of the gene was rs142842127 (ZNF687), of TA in position 152.185.676 of also analyzed during osteoclastogenesis induced on periph the HRNR gene. This aplotype was not found in any of the eral blood monocytes through treatment with MCSF and 559 tested controls of the same geographical area, Suggest RANKL. Expression of ZNF687 increases significantly dur ing that the mutation is located on a common aplotype which ing the differentiation process (FIG. 3B). This result was identifies all GCT/PDB patients (FIG. 1D). also confirmed during the differentiation process of osteo 0055 Molecular Analysis of the ZNF687 Gene in clasts induced in murine cells. Expression of ZNF687 Patients Affected by Paget’s Disease without Neoplastic mRNA also increases in differentiated osteoblasts compared Degeneration. to their undifferentiated precursors (FIG. 3C). These data 0056. To verify the role of the ZNF687 gene in Paget’s suggest a role for ZNF687 during differentiation of osteo patients without neoplastic degeneration, the whole coding clasts and osteoblasts, showing its involvement in the regu region of the gene, consisting of 9 exons, was analyzed in 22 lation of genes implicated in these two processes. The giant of 37 Paget’s subjects who had tested negative for mutations cell tumor that develops on pagetic bone is the consequence in the SQSTM1 gene. Each of the tested subjects had at least of a modification of both processes. Indeed, in RNA one other family member with the disease, confirming the extracted from the giant cell tumor of pagetic patients, which autosomal dominant inheritance of the disease in these includes the giant cells derived from osteoclasts and the families. This analysis allowed for detecting in the tested stromal cells of osteoblastic origin, the inventors found a subject of family 2 (FIG. 1A) the same mutation c.2810CDG significant increase in ZNF687 expression (FIG. 3D). (P937R) indicated above and a new mutation, c.725G-T Finally, through immunohistochemical techniques, by using (S242I) in exon 2 of the tested subject of family 3 (FIG. 2A). specific antibodies against the ZNF687 protein, a very US 2017/O 130272 A1 May 11, 2017

intense staining was observed in the nucleus of the tumor of the four deoxynucleotides (dATP, dCTP, dTTP, giant cells in GCT/PDB patients compared to PDB patients dGTP), 5x reaction buffer (100 mM Tris, 50 mM (FIG. 3E). Hepes, 12.5 mM Magnesium Sulfate, 50 mM Potas 0059 By Real Time PCR, the expression level of sium Chloride and 50 mM Ammonium Sulfate) and 5 ZNF687 in the peripheral blood of PDB patients without Units of Taq polymerase. mutations in the SQSTM1, PDB patients with mutations in 0068 Amplification Protocol. the SQSTM1 gene and PDB patients with mutations in the 0069. In a final volume of 25 microlitres, 100 ng of ZNF687 gene, was also evaluated. Interestingly, the genomic DNA are combined with 0, 5 microM of each ZNF687 expression levels are significantly higher in PDB oligonucleotide, 5 microlitres of 5x Master mix and H2O. patients with or without a SQSTM1 mutation compared to 0070 The exponential amplification reaction comprises a healthy control subjects (FIG. 3F). Furthermore, a greater first cycle of 3 minutes at 94° C. for the denaturation of the increase (5-folds) was observed in PDB patients with genomic DNA, followed by 40 cycles that comprise: 30 ZNF687 mutations. These results suggest that ZNF687 is seconds at 94° C., 30 seconds at 62° C., 45 seconds at 72° involved in the ethiopathogenesis of Paget’s disease of bone, C.; a final cycle at 72° C. for 10 minutes. The PCR product independently of the patients genetic background. Its can be visualized on a 1.5% agarose gel. expression profile considerably correlates with the clinical 0071 Sequencing seriousness of the disease. 0060 ZNF687 Regulates the Expression of the Receptor 0072 The amplification product is purified with the FGFR2. enzyme exosap and sequenced in a final volume of 20 0061 Recently, a transcriptional coregulating complex microlitres with 2 micromolar oligonucleotide F or R and designated as Z3, formed by proteins ZMYND8, ZNF687 the Dye Terminator Ready Reaction Kit (Applied Biosys and ZNF592, was identified and its interaction with the ERC. tem). The reactions are carried out for 25 cycles that transcription factor was defined (Malovannaya et al 2011). comprise: one step at 95°C. for 10 sec, 55° C. for 5 sec and Bioinformatic analyses allowed for hypothesizing that the 60° C. for 2 min. The sequencing reactions are purified and expression of the FGFR2 gene, which has in its genomic separated on ABI prism 3700 Genetic Analyser (Applied region a binding site for the Z3 complex (recognized in Biosystems). Chip-Seq experiments by ZMYND8 antibody), may be regulated thereby. ZNF687 was silenced by use of a specific 3. Diagnostic Kit According to the siRNA in cell lines MG63 (osteoblast line) and RAW264.7 Invention Assay by the TaqMan Method (osteoclast line). 72 hours later, a decrease in FGFR2IIIc 0073. The diagnostic kit for identifying the causative was observed both at the mRNA and the protein level in both mutation c.2810C-G (P937R) in exon 6 of the ZNF687 gene the cell lines. These results point out that FGFR2IIIc is comprises: regulated by the Z3 complex. As for the ZNF687 gene, an 0.074 a pair of oligonucleotides specific for the expo increase was also observed in the expression of FGFR2 nential amplification reaction of a fragment of the during cell differentiation of osteoclasts and osteoblasts. human ZNF687 gene where the causative mutation is Finally, in sections of tumor tissue of GCT/PDB patients, an located; increase in the expression of the FGFR2 protein, apart from 0075 a forward (F) oligonucleotide probe specific for in mesenchymal stromal cells, was also confirmed in mul the mutated base associated with a reporter fluoro tinucleated giant cells, Supporting its role in the pathogen chrome at the 5' end. esis of this tumor. 0076 Taqman Genotyping Assay 2. Diagnostic Kit According to the 0077. The diagnostic kit for identifying by the TaqMan Invention—Assay by Nucleotide Sequencing method the causative mutation c.2810CDG in exon 6 of the ZNF687 gene comprises: 0062 An example of a diagnostic kit for identifying, by (0078. Two TaqMan(R) MGB probes, of which: nucleotide sequencing, the causative mutation c.2810CDG (P937R) in exon 6 of the ZNF687 gene comprises: 0079 one probe labeled with the fluorochrome VICR) 0063 a pair of oligonucleotides (sense 5'-CCGTCTT for recognition of the Allele 1 sequence (wild type); GTCCTCTGCTCTT-3' (SEQ ID NO:21), anti-sense 0080 one probe labeled with the fluorochrome FAMTM 5'-AGGAGAGCAGGGGTAGATGT-3' (SEQ ID for recognition of Allele 2 (mutated); NO:22) specific for the exponential amplification reac 0081 two sequence-specific sense and anti-sense oli tion of a fragment of the human ZNF687 gene where gonucleotides for amplifying the sequence of interest; the causative mutation is located; I0082 Master Mix TaqMan(R) Universal PCR UP (Ultra 0064 a single oligonucleotide (sense 5'-CCGTCTT Pure) containing: AmpliTaq Gold R DNA polymerase GTCCTCTGCTCTT-3' (SEQ ID NO:21) specific for (1x), the fluorochrome ROXTM as a passive internal the sequencing of the sense Strand of the amplified reference for assuring a better analysis on the real-time fragment; PCR tools, a commercially available reaction buffer 0065 a single oligonucleotide (anti-sense 5'-AGGA and a mixture of the four deoxynucleotides (dATP, GAGCAGGGGTAGATGT-3' (SEQ ID NO:22) spe dCTP, dTTP, dGTP). cific for the sequencing of the anti-sense strand of the I0083 Amplification Protocol amplified fragment; I0084. In a final volume of 25 ul, 1-20 ng of genomic 0.066 a control DNA from a healthy individual not DNA are combined with 12.5 uL of Taq Man Universal PCR bearing the test mutation; Master Mix (2x), 1.25 uL of the mixture of oligonucleotides 0067 a 5x master mix (5 times more concentrated than and sequence-specific probes (20x) and sterile water to the working reaction) comprising: 1 mM of a mixture reach the final volume. US 2017/O 130272 A1 May 11, 2017

0085 Exponential Amplification Reaction 0093 Gianfrancesco F. Rendina D, Di Stefano M, Min I0086 A first cycle of 10 minutes at 95°C. is carried out gione A, Esposito T. Merlotti D, Gallone S. Magliocca S. for activating AmpliTaq Gold(R DNA Taq polymerase, fol Goode A, Formicola D. Morello G, Layfield R. Frattini A, lowed by 40 cycles that comprise: 15 seconds at 92° C. for De Filippo G, Nuti R, Searle M, StraZZullo P. Isaia G, the denaturation of the genomic DNA and 1 minute at 60° C. Mossetti G. Gennari L. A nonsynonymous TNFRSF11A for assuring the annealing of the oligonucleotides and the variation increases NFKB activity and the severity of sequence-specific probes and the Subsequent extension of Paget’s disease. J Bone Miner Res 2012: 27:443-52. the fragment of interest. By using the thermocycler Applied 0094 Rendina D. Mossetti G. Soscia E. Sirignano C, Biosystems 7900HT Fast Real-Time PCR System, amplifi Insabato L. Viceconti R, Ignarra R, Salvatore M, Nunziata cation curves are generated which may then be analyzed V. Giant cell tumor and Paget’s disease of bone in one through the SDS Software v2.2.2 or subsequently for the family: geographic clustering. Clin Orthop Relat Res 7900HT Fast System. 2004; 421:218-24. REFERENCES (0095 Gianfrancesco F, Rendina D, Merlotti D, Esposito T. Amyere M. Formicola D. Muscariello R, De Filippo G, 0087 Roodman G. D. Studies in Paget’s disease and their StraZZullo P. Nuti R, Vikkula M, Gennari L. Giant cell relevance to oncology. Semin Oncol, 2001: 15-21. tumor occurring in familial Paget’s disease of bone: report I0088 Gebhart M, Vandeweyer E. Nemec E. Paget’s of clinical characteristics and linkage analysis of a large disease of bone complicated by Giant Cell Tumor. Clin pedigree. J Bone Miner Res. 2013; 28:341-50. Orthop Relat Res. 1998; 352: 187-93. I0089 Laurin N, Brown J. P. Morissette J, Raymond V. 0096. Wu R K, Trumble T E. Ruwe P A. Familial Recurrent mutation of the gene encoding sequestosome 1 incidence of Paget’s disease and secondary osteogenic (SQSTM1/p62) in Paget disease of bone. Am J Hum sarcoma. A report of three cases from a single family. Clin Genet 2002; 70:1582-88. Orthop Relat Res. 1991; 265:306-9. 0090 Hocking L J, Lucas G. J. Daroszewska A, et al. (0097. Malovannaya A, Lanz R B, Jung SY, Bulynko Y, Domain specific mutations in Sequestosome 1 (SQSTM1) Le NT, Chan DW, Ding C, Shi Y, Yucer N. Krenciute G., cause familial and sporadic Paget’s disease. Hum Mol Kim BJ, Li C, Chen R, Li W. Wang Y. O'Malley B W, Qin Genet 2002: 11:2735-39. J. Analysis of the Human Endogenous Coregulator Com 0091 Albagha O M. Visconti M R. Alonso N. Langston plexome. Cell 2011: 145:787-799. A L, Cundy T. Dargie R, Dunlop M. G. Fraser W D, I0098 Rendina D, Gennari L, De Filippo G, Merlotti D, Hooper MJ. Isaia G. Nicholson G. C. del Pino Montes J. de Campora E. Fazioli F. Scarano G. Nuti R. StraZZullo P. Gonzalez-Sarmiento R, di Stefano M, Tenesa A, Walsh J Mossetti G. Evidence for increased clinical severity of P. Ralston S H. Genome-wide association study identifies familial and sporadic Paget’s disease of bone in Campa variants at CSF1, OPTN and TNFRSF11A as genetic risk nia, southern Italy. J Bone Miner Res 2006: 21:1828-35. factors for Paget’s disease of bone. Nat Genet 2010; 42:520-4. 0099 Gennari L, Gianfrancesco F. Di Stefano M, Ren 0092 Albagha O M, Wani SE, Visconti M R, Alonso N, dina D. Merlotti D, Esposito T. Gallone S, Fusco P. Goodman K. Brandi ML, Cundy T. Chung PY. Dargie R. Rainero I, Fenoglio P. Mancini M. Martini G. Bergui S. Devogelaer J P Falchetti A, Fraser W. D. Gennari L, De Filippo G, Isaia G, StraZZullo P. Nuti R. Mossetti G. Gianfrancesco F. Hooper M J. Van Hul W. Isaia G. SQSTM1 gene analysis and gene-environment interaction Nicholson G C, Nuti R, Papapoulos S. Montes Jael P. in Paget’s disease of bone. J Bone Miner Res 2010; Ratajczak T. Rea SL, Rendina D, Gonzalez-Sarmiento R, 25:1375-84. Di Stefano M, Ward L. C. Walsh J. P. Ralston SH; Genetic 0100 Michou L, Morissette J. Gagnon E. R. Marquis A, Determinants of Paget’s Disease (GDPD) Consortium. Dellabadia M. Brown J P Siris E. S. Novel SQSTM1 Genome-wide association identifies three new suscepti mutations in patients with Paget’s disease of bone in an bility loci for Paget’s disease of bone. Nat Genet 2011; unrelated multiethnic American population. Bone 2011; 43:685-9. 48:456-60.

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS: 3 O

<21 Os SEQ ID NO 1 &211s LENGTH: 371.4 &212s. TYPE: DNA <213> ORGANISM: Homo sapiens <4 OOs SEQUENCE: 1

atgggggata tdaag acccc tigattittgat gacct cottg ctgc ctittga catc cctdac 60

attgatgcga atgaa.gc.cat coattctggg cc agaagaaa atgagggggc tiggaggcc.ca 12O

gggaa.gc.cag aaccaggtgt aggaagtgaa totgaagaca cagcagcagc ct ctgctggg 18O

gatggcc ctg gagttccago Cc aggcCtct gaccatggcc ticcaccgcc agacatttct 24 O

US 2017/O 130272 A1 May 11, 2017 14

- Continued

Val Pro Ala Glin Ala Ser Asp His Gly Lieu Pro Pro Pro Asp Ile Ser 65 70 7s 8O Val Val Ser Val Ile Val Lys Asn Thr Val Cys Pro Glu Glin Ser Glu 85 90 95 Ala Lieu Ala Gly Gly Ser Ala Gly Asp Gly Ala Glin Ala Ala Gly Val 1OO 105 11 O Thr Lys Glu Gly Pro Val Gly Pro His Arg Met Glin Asn Gly Phe Gly 115 12 O 125 Ser Pro Glu Pro Ser Leu Pro Gly Thr Pro His Ser Pro Ala Pro Pro 13 O 135 14 O Ser Gly Gly. Thir Trp Lys Glu Lys Gly Met Glu Gly Lys Thr Pro Leu 145 150 155 160 Asp Leu Phe Ala His Phe Gly Pro Glu Pro Gly Asp His Ser Asp Pro 1.65 17O 17s Lieu Pro Pro Ser Ala Pro Ser Pro Thr Arg Glu Gly Ala Lieu. Thr Pro 18O 185 19 O Pro Pro Phe Pro Ser Ser Phe Glu Lieu Ala Glin Glu Asn Gly Pro Gly 195 2OO 2O5 Met Glin Pro Pro Val Ser Ser Pro Pro Leu Gly Ala Leu Lys Glin Glu 21 O 215 22O Ser Cys Ser Pro His His Pro Glin Val Leu Ala Glin Glin Gly Ser Gly 225 23 O 235 24 O Ser Ile Pro Lys Ala Thr Asp Ile Pro Ala Ser Ala Ser Pro Pro Pro 245 250 255 Val Ala Gly Val Pro Phe Phe Lys Glin Ser Pro Gly His Glin Ser Pro 26 O 265 27 O Lieu Ala Ser Pro Llys Val Pro Val Cys Glin Pro Lieu Lys Glu Glu Asp 27s 28O 285 Asp Asp Glu Gly Pro Val Asp Llys Ser Ser Pro Gly Ser Pro Glin Ser 29 O 295 3 OO Pro Ser Ser Gly Ala Glu Ala Ala Asp Glu Asp Ser Asn Asp Ser Pro 3. OS 310 315 32O Ala Ser Ser Ser Ser Arg Pro Lieu Lys Val Arg Ile Llys Thir Ile Llys 3.25 330 335 Thir Ser Cys Gly Asn Ile Thr Arg Thr Val Thr Glin Val Pro Ser Asp 34 O 345 35. O Pro Asp Pro Pro Ala Pro Lieu Ala Glu Gly Ala Phe Lieu Ala Glu Ala 355 360 365 Ser Leu Lleu Lys Lieu. Ser Pro Ala Thr Pro Thr Ser Glu Gly Pro Llys 37 O 375 38O Val Val Ser Val Glin Lieu. Gly Asp Gly Thr Arg Lieu Lys Gly. Thr Val 385 390 395 4 OO

Leu Pro Wall Ala Thir Ile Glin Asn Ala Ser Thir Ala Met Lieu Met Ala 4 OS 41O 415

Ala Ser Val Ala Arg Lys Ala Val Val Lieu Pro Gly Gly Thr Ala Thr 42O 425 43 O

Ser Pro Llys Met Ile Ala Lys Asn Val Lieu. Gly Lieu Val Pro Glin Ala 435 44 O 445 Lieu Pro Lys Ala Asp Gly Arg Ala Gly Lieu. Gly Thr Gly Gly Glin Lys 450 45.5 460 US 2017/O 130272 A1 May 11, 2017 15

- Continued

Val Asn Gly Ala Ser Val Val Met Val Glin Pro Ser Lys Thr Ala Thr 465 470 47s 48O Gly Pro Ser Thr Gly Gly Gly Thr Val Ile Ser Arg Thr Glin Ser Ser 485 490 495 Lieu Val Glu Ala Phe Asn Lys Ile Lieu. Asn. Ser Lys Asn Lieu. Lieu Pro SOO 505 51O Ala Tyr Arg Pro Asn Lieu. Ser Pro Pro Ala Glu Ala Gly Lieu Ala Lieu 515 52O 525 Pro Pro Thr Gly Tyr Arg Cys Lieu. Glu. Cys Gly Asp Ala Phe Ser Lieu. 53 O 535 54 O Glu Lys Ser Lieu Ala Arg His Tyr Asp Arg Arg Ser Met Arg Ile Glu 5.45 550 555 560 Val Thr Cys Asn His Cys Ala Arg Arg Lieu Val Phe Phe Asn Lys Cys 565 st O sts Ser Lieu. Lieu. Lieu. His Ala Arg Glu. His Lys Asp Llys Gly Lieu Val Met 58O 585 59 O Glin Cys Ser His Lieu Val Met Arg Pro Val Ala Lieu. Asp Gln Met Val 595 6OO 605 Gly Glin Pro Asp Ile Thr Pro Leu Lleu Pro Val Ala Val Pro Pro Val 610 615 62O Ser Gly Pro Lieu Ala Lieu Pro Ala Lieu. Gly Lys Gly Glu Gly Ala Ile 625 630 635 64 O

Thir Ser Ser Ala Ile Thir Thr Wall Ala Ala Glu Ala Pro Wall Lieu Pro 645 650 655 Lieu. Ser Thr Glu Pro Pro Ala Ala Pro Ala Thr Ser Ala Tyr Thr Cys 660 665 67 O Phe Arg Cys Lieu. Glu. Cys Lys Glu Glin Cys Arg Asp Lys Ala Gly Met 675 68O 685 Ala Ala His Phe Glin Gln Leu Gly Pro Pro Ala Pro Gly Ala Thr Ser 69 O. 695 7 OO Asn Val Cys Pro Thr Cys Pro Met Met Leu Pro Asn Arg Cys Ser Phe 7 Os 71O 71s 72O Ser Ala His Glin Arg Met His Lys Asn Arg Pro Pro His Val Cys Pro 72 73 O 73 Glu Cys Gly Gly Asn. Phe Lieu. Glin Ala Asn. Phe Glin Thr His Lieu. Arg 740 74. 7 O Glu Ala Cys Lieu. His Val Ser Arg Arg Val Gly Tyr Arg Cys Pro Ser 7ss 760 765 Cys Ser Val Val Phe Gly Gly Val Asn Ser Ile Llys Ser His Ile Glin 770 775 78O Thir Ser His Cys Glu Val Phe His Lys Cys Pro Ile Cys Pro Met Ala 78s 79 O 79. 8OO

Phe Llys Ser Gly Pro Ser Ala His Ala His Leu Tyr Ser Glin His Pro 805 810 815

Ser Phe Glin Thr Glin Glin Ala Lys Lieu. Ile Tyr Lys Cys Ala Met Cys 82O 825 83 O

Asp Thr Val Phe Thr His Llys Pro Leu Lleu Ser Ser His Phe Asp Glin 835 84 O 845

His Leu Lleu Pro Glin Arg Val Ser Val Phe Lys Cys Pro Ser Cys Pro 850 855 860

Lieu. Lieu. Phe Ala Glin Lys Arg Thr Met Lieu. Glu. His Lieu Lys Asn Thr US 2017/O 130272 A1 May 11, 2017 16

- Continued

His Glin Ser Gly Arg Lieu. Glu Glu Thir Ala Gly Lys Gly Ala Gly Gly 885 890 895 Ala Lieu. Lieu. Thr Pro Llys Thr Glu Pro Glu Glu Lieu Ala Val Ser Glin 9 OO 905 91 O Gly Gly Ala Ala Pro Ala Thr Glu Glu Ser Ser Ser Ser Ser Glu Glu 915 92 O 925 Glu Glu Val Pro Ser Ser Pro Glu Pro Pro Arg Pro Ala Lys Arg Pro 93 O 935 94 O Arg Arg Glu Lieu. Gly Ser Lys Gly Lieu Lys Gly Gly Gly Gly Gly Pro 945 950 955 96.O Gly Gly Trp Thr Cys Gly Lieu. Cys His Ser Trp Phe Pro Glu Arg Asp 965 97O 97. Glu Tyr Val Ala His Met Lys Lys Glu. His Gly Llys Ser Wall Lys Llys 98O 985 99 O Phe Pro Cys Arg Lieu. Cys Glu Arg Ser Phe Cys Ser Ala Pro Ser Lieu. 995 1OOO 1005 Arg Arg His Val Arg Val Asn His Glu Gly Ile Lys Arg Val Tyr O1O 1015 1 O2O Pro Cys Arg Tyr Cys Thr Glu Gly Lys Arg Thr Phe Ser Ser Arg O25 1O3 O 1035 Lieu. Ile Lieu. Glu Lys His Val Glin Val Arg His Gly Lieu Gln Lieu. O4 O 1045 1050 Gly Ala Glin Ser Pro Gly Arg Gly. Thir Thr Lieu Ala Arg Gly Ser O55 O6 O O65 Ser Ala Arg Ala Glin Gly Pro Gly Arg Lys Arg Arg Glin Ser Ser Of O O7 O8O Asp Ser Cys Ser Glu Glu Pro Asp Ser Thr Thr Pro Pro Ala Lys O85 O9 O O95 Ser Pro Arg Gly Gly Pro Gly Ser Gly Gly His Gly Pro Leu Arg OO O5 10 Tyr Arg Ser Ser Ser Ser Thr Glu Glin Ser Leu Met Met Gly Lieu.

Arg Val Glu Asp Gly Ala Glin Glin Cys Lieu. Asp Cys Gly Lieu. Cys

Phe Ala Ser Pro Gly Ser Leu Ser Arg His Arg Phe Ile Ser His

Llys Lys Arg Arg Gly Val Gly Lys Ala Ser Ala Lieu. Gly Lieu. Gly

Asp Gly Glu Glu Glu Ala Pro Pro Ser Arg Ser Asp Pro Asp Gly

Gly Asp Ser Pro Leu Pro Ala Ser Gly Gly Pro Leu. Thr Cys Lys 90 95 2OO Val Cys Gly Llys Ser Cys Asp Ser Pro Lieu. Asn Lieu Lys Thr His 2O5 21 O 215 Phe Arg Thr His Gly Met Ala Phe Ile Arg Ala Arg Glin Gly Ala 22O 225 23 O

Val Gly Asp Asn

<210s, SEQ ID NO 6 US 2017/O 130272 A1 May 11, 2017 17

- Continued

&211s LENGTH: 1237 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 6 Met Gly Asp Met Lys Thr Pro Asp Phe Asp Asp Lieu. Lieu Ala Ala Phe 1. 5 1O 15 Asp Ile Pro Asp Ile Asp Ala Asn. Glu Ala Ile His Ser Gly Pro Glu 2O 25 3O Glu Asin Glu Gly Pro Gly Gly Pro Gly Llys Pro Glu Pro Gly Val Gly 35 4 O 45 Ser Glu Ser Glu Asp Thr Ala Ala Ala Ser Ala Gly Asp Gly Pro Gly SO 55 6 O Val Pro Ala Glin Ala Ser Asp His Gly Lieu Pro Pro Pro Asp Ile Ser 65 70 7s 8O Val Val Ser Val Ile Val Lys Asn Thr Val Cys Pro Glu Glin Ser Glu 85 90 95 Ala Lieu Ala Gly Gly Ser Ala Gly Asp Gly Ala Glin Ala Ala Gly Val 1OO 105 11 O Thr Lys Glu Gly Pro Val Gly Pro His Arg Met Glin Asn Gly Phe Gly 115 12 O 125 Ser Pro Glu Pro Ser Leu Pro Gly Thr Pro His Ser Pro Ala Pro Pro 13 O 135 14 O Ser Gly Gly. Thir Trp Llys Glu Lys Gly Met Glu Gly Llys Thr Pro Lieu 145 150 155 160 Asp Leu Phe Ala His Phe Gly Pro Glu Pro Gly Asp His Ser Asp Pro 1.65 17O 17s Lieu Pro Pro Ser Ala Pro Ser Pro Thr Arg Glu Gly Ala Lieu. Thr Pro 18O 185 19 O Pro Pro Phe Pro Ser Ser Phe Glu Lieu Ala Glin Glu Asn Gly Pro Gly 195 2OO 2O5 Met Glin Pro Pro Val Ser Ser Pro Pro Leu Gly Ala Leu Lys Glin Glu 21 O 215 22O Ser Cys Ser Pro His His Pro Glin Val Leu Ala Glin Glin Gly Ser Gly 225 23 O 235 24 O Ser Ser Pro Lys Ala Thr Asp Ile Pro Ala Ser Ala Ser Pro Pro Pro 245 250 255 Val Ala Gly Val Pro Phe Phe Lys Glin Ser Pro Gly His Glin Ser Pro 26 O 265 27 O Lieu Ala Ser Pro Llys Val Pro Val Cys Glin Pro Lieu Lys Glu Glu Asp 27s 28O 285 Asp Asp Glu Gly Pro Val Asp Llys Ser Ser Pro Gly Ser Pro Glin Ser 29 O 295 3 OO

Pro Ser Ser Gly Ala Glu Ala Ala Asp Glu Asp Ser Asn Asp Ser Pro 3. OS 310 315 32O

Ala Ser Ser Ser Ser Arg Pro Lieu Lys Val Arg Ile Llys Thir Ile Llys 3.25 330 335

Thir Ser Cys Gly Asn Ile Thr Arg Thr Val Thr Glin Val Pro Ser Asp 34 O 345 35. O

Pro Asp Pro Pro Ala Pro Lieu Ala Glu Gly Ala Phe Lieu Ala Glu Ala 355 360 365

Ser Leu Lleu Lys Lieu. Ser Pro Ala Thr Pro Thr Ser Glu Gly Pro Llys US 2017/O 130272 A1 May 11, 2017 18

- Continued

37 O 375 38O Val Val Ser Val Glin Lieu. Gly Asp Gly Thr Arg Lieu Lys Gly. Thr Val 385 390 395 4 OO

Leu Pro Wall Ala Thir Ile Glin Asn Ala Ser Thir Ala Met Lieu Met Ala 4 OS 41O 415 Ala Ser Val Ala Arg Lys Ala Val Val Lieu Pro Gly Gly Thr Ala Thr 42O 425 43 O Ser Pro Llys Met Ile Ala Lys Asn Val Lieu. Gly Lieu Val Pro Glin Ala 435 44 O 445 Lieu Pro Lys Ala Asp Gly Arg Ala Gly Lieu. Gly Thr Gly Gly Glin Lys 450 45.5 460 Val Asn Gly Ala Ser Val Val Met Val Glin Pro Ser Lys Thr Ala Thr 465 470 47s 48O Gly Pro Ser Thr Gly Gly Gly Thr Val Ile Ser Arg Thr Glin Ser Ser 485 490 495 Lieu Val Glu Ala Phe Asn Lys Ile Lieu. Asn. Ser Lys Asn Lieu. Lieu Pro SOO 505 51O Ala Tyr Arg Pro Asn Lieu. Ser Pro Pro Ala Glu Ala Gly Lieu Ala Lieu 515 52O 525 Pro Pro Thr Gly Tyr Arg Cys Lieu. Glu. Cys Gly Asp Ala Phe Ser Lieu. 53 O 535 54 O Glu Lys Ser Lieu Ala Arg His Tyr Asp Arg Arg Ser Met Arg Ile Glu 545 550 555 560 Val Thr Cys Asn His Cys Ala Arg Arg Lieu Val Phe Phe Asn Lys Cys 565 st O sts Ser Lieu. Lieu. Lieu. His Ala Arg Glu. His Lys Asp Llys Gly Lieu Val Met 58O 585 59 O Glin Cys Ser His Lieu Val Met Arg Pro Val Ala Lieu. Asp Gln Met Val 595 6OO 605 Gly Glin Pro Asp Ile Thr Pro Leu Lleu Pro Val Ala Val Pro Pro Val 610 615 62O Ser Gly Pro Lieu Ala Lieu Pro Ala Lieu. Gly Lys Gly Glu Gly Ala Ile 625 630 635 64 O

Thir Ser Ser Ala Ile Thir Thir Wall Ala Ala Glu Ala Pro Wall Leu Pro 645 650 655 Lieu. Ser Thr Glu Pro Pro Ala Ala Leu Ala Thr Ser Ala Tyr Thr Cys 660 665 67 O Phe Arg Cys Lieu. Glu. Cys Lys Glu Glin Cys Arg Asp Lys Ala Gly Met 675 68O 685 Ala Ala His Phe Glin Gln Leu Gly Pro Pro Ala Pro Gly Ala Thr Ser 69 O. 695 7 OO

Asn Val Cys Pro Thr Cys Pro Met Met Leu Pro Asn Arg Cys Ser Phe 7 Os 71O 71s 72O

Ser Ala His Glin Arg Met His Lys Asn Arg Pro Pro His Val Cys Pro 72 73 O 73

Glu Cys Gly Gly Asn. Phe Lieu. Glin Ala Asn. Phe Glin Thr His Lieu. Arg 740 74. 7 O Glu Ala Cys Lieu. His Val Ser Arg Arg Val Gly Tyr Arg Cys Pro Ser 7ss 760 765

Cys Ser Val Val Phe Gly Gly Val Asn Ser Ile Llys Ser His Ile Glin 770 775 78O US 2017/O 130272 A1 May 11, 2017 19

- Continued

Thir Ser His Cys Glu Val Phe His Lys Cys Pro Ile Cys Pro Met Ala 78s 79 O 79. 8OO Phe Llys Ser Gly Pro Ser Ala His Ala His Leu Tyr Ser Glin His Pro 805 810 815 Ser Phe Glin Thr Glin Glin Ala Lys Lieu. Ile Tyr Lys Cys Ala Met Cys 82O 825 83 O Asp Thr Val Phe Thr His Llys Pro Leu Lleu Ser Ser His Phe Asp Glin 835 84 O 845 His Leu Lleu Pro Glin Arg Val Ser Val Phe Lys Cys Pro Ser Cys Pro 850 855 860 Lieu. Lieu. Phe Ala Glin Lys Arg Thr Met Lieu. Glu. His Lieu Lys Asn Thr 865 87O 87s 88O His Glin Ser Gly Arg Lieu. Glu Glu Thir Ala Gly Lys Gly Ala Gly Gly 885 890 895 Ala Lieu. Lieu. Thr Pro Llys Thr Glu Pro Glu Glu Lieu Ala Val Ser Glin 9 OO 905 91 O Gly Gly Ala Ala Pro Ala Thr Glu Glu Ser Ser Ser Ser Ser Glu Glu 915 92 O 925 Glu Glu Val Pro Ser Ser Pro Glu Pro Pro Arg Pro Ala Lys Arg Pro 93 O 935 94 O Arg Arg Glu Lieu. Gly Ser Lys Gly Lieu Lys Gly Gly Gly Gly Gly Pro 945 950 955 96.O Gly Gly Trp Thr Cys Gly Lieu. Cys His Ser Trp Phe Pro Glu Arg Asp 965 97O 97. Glu Tyr Val Ala His Met Lys Lys Glu. His Gly Llys Ser Wall Lys Llys 98O 985 99 O Phe Pro Cys Arg Lieu. Cys Glu Arg Ser Phe Cys Ser Ala Pro Ser Lieu. 995 1OOO 1005 Arg Arg His Val Arg Val Asn His Glu Gly Ile Lys Arg Val Tyr O1O O15 O2O Pro Cys Arg Tyr Cys Thr Glu Gly Lys Arg Thr Phe Ser Ser Arg O25 O3 O O35 Lieu. Ile Lieu. Glu Lys His Val Glin Val Arg His Gly Lieu Gln Lieu. O4 O O45 OSO Gly Ala Glin Ser Pro Gly Arg Gly. Thir Thr Lieu Ala Arg Gly Ser O55 O6 O O65 Ser Ala Arg Ala Glin Gly Pro Gly Arg Lys Arg Arg Glin Ser Ser Of O O7 O8O Asp Ser Cys Ser Glu Glu Pro Asp Ser Thr Thr Pro Pro Ala Lys O85 O9 O O95 Ser Pro Arg Gly Gly Pro Gly Ser Gly Gly His Gly Pro Leu Arg 1 OO 105 11 O

Tyr Arg Ser Ser Ser Ser Thr Glu Glin Ser Leu Met Met Gly Lieu. 115 12 O 125

Arg Val Glu Asp Gly Ala Glin Glin Cys Lieu. Asp Cys Gly Lieu. Cys 13 O 135 14 O Phe Ala Ser Pro Gly Ser Leu Ser Arg His Arg Phe Ile Ser His 145 15 O 155 Llys Lys Arg Arg Gly Val Gly Lys Ala Ser Ala Lieu. Gly Lieu. Gly 16 O 1.65 17 O US 2017/O 130272 A1 May 11, 2017 20

- Continued Asp Gly Glu Glu Glu Ala Pro Pro Ser Arg Ser Asp Pro Asp Gly 1175 118O 1185 Gly Asp Ser Pro Leu Pro Ala Ser Gly Gly Pro Leu. Thr Cys Lys 11.90 11.95 12 OO Val Cys Gly Llys Ser Cys Asp Ser Pro Lieu. Asn Lieu Lys Thr His 12O5 121 O 1215 Phe Arg Thr His Gly Met Ala Phe Ile Arg Ala Arg Glin Gly Ala 122O 1225 123 O Val Gly Asp Asn 1235

<210s, SEQ ID NO 7 &211s LENGTH: 1237 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OO > SEQUENCE: 7 Met Gly Asp Met Lys Thr Pro Asp Phe Asp Asp Lieu. Lieu Ala Ala Phe 1. 5 1O 15 Asp Ile Pro Asp Ile Asp Ala Asn. Glu Ala Ile His Ser Gly Pro Glu 2O 25 3O Glu Asin Glu Gly Pro Gly Gly Pro Gly Llys Pro Glu Pro Gly Val Gly 35 4 O 45 Ser Glu Ser Glu Asp Thr Ala Ala Ala Ser Ala Gly Asp Gly Pro Gly 50 55 60 Val Pro Ala Glin Ala Ser Asp His Gly Lieu Pro Pro Pro Asp Ile Ser 65 70 7s 8O Val Val Ser Val Ile Val Lys Asn Thr Val Cys Pro Glu Glin Ser Glu 85 90 95 Ala Lieu Ala Gly Gly Ser Ala Gly Asp Gly Ala Glin Ala Ala Gly Val 1OO 105 11 O Thr Lys Glu Gly Pro Val Gly Pro His Arg Met Glin Asn Gly Phe Gly 115 12 O 125 Ser Pro Glu Pro Ser Leu Pro Gly Thr Pro His Ser Pro Ala Pro Pro 13 O 135 14 O Ser Gly Gly. Thir Trp Lys Glu Lys Gly Met Glu Gly Lys Thr Pro Leu 145 150 155 160 Asp Leu Phe Ala His Phe Gly Pro Glu Pro Gly Asp His Ser Asp Pro 1.65 17O 17s Lieu Pro Pro Ser Ala Pro Ser Pro Thr Arg Glu Gly Ala Lieu. Thr Pro 18O 185 19 O Pro Pro Phe Pro Ser Ser Phe Glu Lieu Ala Glin Glu Asn Gly Pro Gly 195 2OO 2O5

Met Glin Pro Pro Val Ser Ser Pro Pro Leu Gly Ala Leu Lys Glin Glu 21 O 215 22O

Ser Cys Ser Pro His His Pro Glin Val Leu Ala Glin Glin Gly Ser Gly 225 23 O 235 24 O

Ser Ser Pro Lys Ala Thr Asp Ile Pro Ala Ser Ala Ser Pro Pro Pro 245 250 255

Val Ala Gly Val Pro Phe Phe Lys Glin Ser Pro Gly His Glin Ser Pro 26 O 265 27 O

Lieu Ala Ser Pro Llys Val Pro Val Cys Glin Pro Lieu Lys Glu Glu Asp 27s 28O 285 US 2017/O 130272 A1 May 11, 2017 21

- Continued

Asp Asp Glu Gly Pro Val Asp Llys Ser Ser Pro Gly Ser Pro Glin Ser 29 O 295 3 OO Pro Ser Ser Gly Ala Glu Ala Ala Asp Glu Asp Ser Asn Asp Ser Pro 3. OS 310 315 32O Ala Ser Ser Ser Ser Arg Pro Lieu Lys Val Arg Ile Llys Thir Ile Llys 3.25 330 335 Thir Ser Cys Gly Asn Ile Thr Arg Thr Val Thr Glin Val Pro Ser Asp 34 O 345 35. O Pro Asp Pro Pro Ala Pro Lieu Ala Glu Gly Ala Phe Lieu Ala Glu Ala 355 360 365 Ser Leu Lleu Lys Lieu. Ser Pro Ala Thr Pro Thr Ser Glu Gly Pro Llys 37 O 375 38O Val Val Ser Val Glin Lieu. Gly Asp Gly Thr Arg Lieu Lys Gly. Thr Val 385 390 395 4 OO

Leu Pro Wall Ala Thir Ile Glin Asn Ala Ser Thir Ala Met Lieu Met Ala 4 OS 41O 415 Ala Ser Val Ala Arg Lys Ala Val Val Lieu Pro Gly Gly Thr Ala Thr 42O 425 43 O Ser Pro Llys Met Ile Ala Lys Asn Val Lieu. Gly Lieu Val Pro Glin Ala 435 44 O 445 Lieu Pro Lys Ala Asp Gly Arg Ala Gly Lieu. Gly Thr Gly Gly Glin Lys 450 45.5 460 Val Asn Gly Ala Ser Val Val Met Val Glin Pro Ser Lys Thr Ala Thr 465 470 47s 48O Gly Pro Ser Thr Gly Gly Gly Thr Val Ile Ser Arg Thr Glin Ser Ser 485 490 495 Lieu Val Glu Ala Phe Asn Lys Ile Lieu. Asn. Ser Lys Asn Lieu. Lieu Pro SOO 505 51O Ala Tyr Arg Pro Asn Lieu. Ser Pro Pro Ala Glu Ala Gly Lieu Ala Lieu 515 52O 525 Pro Pro Thr Gly Tyr Arg Cys Lieu. Glu. Cys Gly Asp Ala Phe Ser Lieu. 53 O 535 54 O Glu Lys Ser Lieu Ala Arg His Tyr Asp Arg Arg Ser Met Arg Ile Glu 5.45 550 555 560 Val Thr Cys Asn His Cys Ala Arg Arg Lieu Val Phe Phe Asn Lys Cys 565 st O sts Ser Lieu. Lieu. Lieu. His Ala Arg Glu. His Lys Asp Llys Gly Lieu Val Met 58O 585 59 O Glin Cys Ser His Lieu Val Met Arg Pro Val Ala Lieu. Asp Gln Met Val 595 6OO 605 Gly Glin Pro Asp Ile Thr Pro Leu Lleu Pro Val Ala Val Pro Pro Val 610 615 62O

Ser Gly Pro Lieu Ala Lieu Pro Ala Lieu. Gly Lys Gly Glu Gly Ala Ile 625 630 635 64 O

Thir Ser Ser Ala Ile Thir Thir Wall Ala Ala Glu Ala Pro Wall Leu Pro 645 650 655

Lieu. Ser Thr Glu Pro Pro Ala Ala Pro Ala Thr Ser Ala Tyr Thr Cys 660 665 67 O Phe Arg Cys Lieu. Glu. Cys Lys Glu Glin Cys Arg Asp Lys Ala Gly Met 675 68O 685 US 2017/O 130272 A1 May 11, 2017 22

- Continued Ala Ala His Phe Glin Gln Leu Gly Pro Pro Ala Pro Gly Ala Thr Ser 69 O. 695 7 OO Asn Val Cys Pro Thr Cys Pro Met Met Leu Pro Asn Arg Cys Ser Phe 7 Os 71O 71s 72O Ser Ala His Glin Arg Met His Lys Asn Arg Pro Pro His Val Cys Pro 72 73 O 73 Glu Cys Gly Gly Asn. Phe Lieu. Glin Ala Asn. Phe Glin Thr His Lieu. Arg 740 74. 7 O Glu Ala Cys Lieu. His Val Ser Arg Arg Val Gly Tyr Arg Cys Pro Ser 7ss 760 765 Cys Ser Val Val Phe Gly Gly Val Asn Ser Ile Llys Ser His Ile Glin 770 775 78O Thir Ser His Cys Glu Val Phe His Lys Cys Pro Ile Cys Pro Met Ala 78s 79 O 79. 8OO Phe Llys Ser Gly Pro Ser Ala His Ala His Leu Tyr Ser Glin His Pro 805 810 815 Ser Phe Glin Thr Glin Glin Ala Lys Lieu. Ile Tyr Lys Cys Ala Met Cys 82O 825 83 O Asp Thr Val Phe Thr His Llys Pro Leu Lleu Ser Ser His Phe Asp Glin 835 84 O 845 His Leu Lleu Pro Glin Arg Val Ser Val Phe Lys Cys Pro Ser Cys Pro 850 855 860 Lieu Lieu Phe Ala Gln Lys Arg Thr Met Lieu. Glu. His Lieu Lys Asn Thr 865 87O 87s 88O His Glin Ser Gly Arg Lieu. Glu Glu Thir Ala Gly Lys Gly Ala Gly Gly 885 890 895 Ala Lieu. Lieu. Thr Pro Llys Thr Glu Pro Glu Glu Lieu Ala Val Ser Glin 9 OO 905 91 O Gly Gly Ala Ala Pro Ala Thr Glu Glu Ser Ser Ser Ser Ser Glu Glu 915 92 O 925 Glu Glu Val Pro Ser Ser Pro Glu Pro Pro Arg Pro Ala Lys Arg Pro 93 O 935 94 O Arg Arg Glu Lieu. Gly Ser Lys Gly Lieu Lys Gly Gly Gly Gly Gly Pro 945 950 955 96.O Gly Gly Trp Thr Cys Gly Lieu. Cys His Ser Trp Phe Pro Glu Arg Asp 965 97O 97. Glu Tyr Val Ala His Met Lys Lys Glu. His Gly Llys Ser Wall Lys Llys 98O 985 99 O Phe Pro Cys Arg Lieu. Cys Glu Arg Ser Phe Cys Ser Ala Pro Ser Lieu. 995 1OOO 1005 Arg Arg His Val Arg Val Asn His Glu Gly Ile Lys Arg Val Tyr 1010 O15 1 O2O

Pro Cys Arg Tyr Cys Thr Glu Gly Lys Arg Thr Phe Ser Ser Arg 1025 O3 O 1035

Lieu. Ile Lieu. Glu Lys His Val Glin Val Arg His Gly Lieu Gln Lieu. 104 O O45 1 OSO

Gly Ala Glin Ser Pro Gly Arg Gly. Thir Thr Lieu Ala Arg Gly Ser 105.5 O6 O 1065

Ser Ala Arg Ala Glin Gly Pro Gly Arg Lys Arg Arg Glin Ser Ser 1070 O7 108 O

Asp Ser Cys Ser Glu Glu Pro Asp Ser Thr Thr Pro Pro Ala Lys US 2017/O 130272 A1 May 11, 2017 23

- Continued

Ser Pro Arg Gly Gly Pro Gly Ser Gly Gly His Gly Pro Leu Arg

Tyr Arg Ser Ser Ser Ser Thr Glu Glin Ser Leu Met Met Gly Lieu.

Arg Val Glu Asp Gly Ala Glin Glin Cys Lieu. Asp Cys Gly Lieu. Cys

Phe Ala Ser Pro Gly Ser Leu Ser Arg His Arg Phe Ile Ser His

Llys Lys Arg Arg Gly Val Gly Lys Ala Ser Ala Lieu. Gly Lieu. Gly

Asp Gly Glu Glu Glu Ala Pro Pro Ser Arg Ser Asp Pro Asp Gly

<210 SEQ ID NO 8 &211s LENGTH: 1237 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 8 Met Gly Asp Met Lys Thr Pro Asp Phe Asp Asp Lieu. Lieu Ala Ala Phe 1. 5 1O 15 Asp Ile Pro Asp Ile Asp Ala Asn. Glu Ala Ile His Ser Gly Pro Glu 2O 25 3O Glu Asin Glu Gly Pro Gly Gly Pro Gly Llys Pro Glu Pro Gly Val Gly 35 4 O 45 Ser Glu Ser Glu Asp Thr Ala Ala Ala Ser Ala Gly Asp Gly Pro Gly SO 55 6 O Val Pro Ala Glin Ala Ser Asp His Gly Lieu Pro Pro Pro Asp Ile Ser 65 70 7s 8O Val Val Ser Val Ile Val Lys Asn Thr Val Cys Pro Glu Glin Ser Glu 85 90 95 Ala Lieu Ala Gly Gly Ser Ala Gly Asp Gly Ala Glin Ala Ala Gly Val 1OO 105 11 O Thr Lys Glu Gly Pro Val Gly Pro His Arg Met Glin Asn Gly Phe Gly 115 12 O 125

Ser Pro Glu Pro Ser Leu Pro Gly Thr Pro His Ser Pro Ala Pro Pro 13 O 135 14 O

Ser Gly Gly. Thir Trp Lys Glu Lys Gly Met Glu Gly Lys Thr Pro Leu 145 150 155 160 Asp Leu Phe Ala His Phe Gly Pro Glu Pro Gly Asp His Ser Asp Pro 1.65 17O 17s

Lieu Pro Pro Ser Ala Pro Ser Pro Thr Arg Glu Gly Ala Lieu. Thr Pro 18O 185 19 O US 2017/O 130272 A1 May 11, 2017 24

- Continued

Pro Pro Phe Pro Ser Ser Phe Glu Lieu Ala Glin Glu Asn Gly Pro Gly 195 2OO 2O5 Met Glin Pro Pro Val Ser Ser Pro Pro Leu Gly Ala Leu Lys Glin Glu 21 O 215 22O Ser Cys Ser Pro His His Pro Glin Val Leu Ala Glin Glin Gly Ser Gly 225 23 O 235 24 O Ser Ser Pro Lys Ala Thr Asp Ile Pro Ala Ser Ala Ser Pro Pro Pro 245 250 255 Val Ala Gly Val Pro Phe Phe Lys Glin Ser Pro Gly His Glin Ser Pro 26 O 265 27 O Lieu Ala Ser Pro Llys Val Pro Val Cys Glin Pro Lieu Lys Glu Glu Asp 27s 28O 285 Asp Asp Glu Gly Pro Val Asp Llys Ser Ser Pro Gly Ser Pro Glin Ser 29 O 295 3 OO Pro Ser Ser Gly Ala Glu Ala Ala Asp Glu Asp Ser Asn Asp Ser Pro 3. OS 310 315 32O Ala Ser Ser Ser Ser Arg Pro Lieu Lys Val Arg Ile Llys Thir Ile Llys 3.25 330 335 Thir Ser Cys Gly Asn Ile Thr Arg Thr Val Thr Glin Val Pro Ser Asp 34 O 345 35. O Pro Asp Pro Pro Ala Pro Lieu Ala Glu Gly Ala Phe Lieu Ala Glu Ala 355 360 365 Ser Lieu Lieu Lys Lieu Ser Pro Ala Thr Pro Thr Ser Glu Gly Pro Llys 37 O 375 38O Val Val Ser Val Glin Lieu. Gly Asp Gly Thr Arg Lieu Lys Gly. Thr Val 385 390 395 4 OO

Pro Pro Thr Gly Tyr Arg Cys Lieu. Glu. Cys Gly Asp Ala Phe Ser Lieu. 53 O 535 54 O Glu Lys Ser Lieu Ala Arg His Tyr Asp Arg Arg Ser Met Arg Ile Glu 5.45 550 555 560 Val Thr Cys Asn His Cys Ala Arg Arg Lieu Val Phe Phe Asn Lys Cys 565 st O sts Ser Lieu. Lieu. Lieu. His Ala Arg Glu. His Lys Asp Llys Gly Lieu Val Met 58O 585 59 O

Glin Cys Ser His Lieu Val Met Arg Pro Val Ala Lieu. Asp Gln Met Val US 2017/O 130272 A1 May 11, 2017 25

- Continued

595 6OO 605 Gly Glin Pro Asp Ile Thr Pro Leu Lleu Pro Val Ala Val Pro Pro Val 610 615 62O Ser Gly Pro Lieu Ala Lieu Pro Ala Lieu. Gly Lys Gly Glu Gly Ala Ile 625 630 635 64 O

Thir Ser Ser Ala Ile Thir Thir Wall Ala Ala Glu Ala Pro Wall Leu Pro 645 650 655 Lieu. Ser Thr Glu Pro Pro Ala Ala Pro Ala Thr Ser Ala Tyr Thr Cys 660 665 67 O Phe Arg Cys Lieu. Glu. Cys Lys Glu Glin Cys Arg Asp Lys Ala Gly Met 675 68O 685 Ala Ala His Phe Glin Gln Leu Gly Pro Pro Ala Pro Gly Ala Thr Ser 69 O. 695 7 OO Asn Val Cys Pro Thr Cys Pro Met Met Leu Pro Asn Arg Cys Ser Phe 7 Os 71O 71s 72O Ser Ala His Glin Arg Met His Lys Asn Arg Pro Pro His Val Cys Pro 72 73 O 73 Glu Cys Gly Gly Asn. Phe Lieu. Glin Ala Asn. Phe Glin Thr His Lieu. Arg 740 74. 7 O Glu Ala Cys Lieu. His Val Ser Arg Arg Val Gly Tyr Arg Cys Pro Ser 7ss 760 765 Cys Ser Val Val Phe Gly Gly Val Asn Ser Ile Llys Ser His Ile Glin 770 775 78O Thir Ser His Cys Glu Val Phe His Lys Cys Pro Ile Cys Pro Met Ala 78s 79 O 79. 8OO Phe Llys Ser Gly Pro Ser Ala His Ala His Leu Tyr Ser Glin His Pro 805 810 815 Ser Phe Glin Thr Glin Glin Ala Lys Lieu. Ile Tyr Lys Cys Ala Met Cys 82O 825 83 O Asp Thr Val Phe Thr His Llys Pro Leu Lleu Ser Ser His Phe Asp Glin 835 84 O 845 His Leu Lleu Pro Glin Arg Val Ser Val Phe Lys Cys Pro Ser Cys Pro 850 855 860 Lieu. Lieu. Phe Ala Glin Lys Arg Thr Met Lieu. Glu. His Lieu Lys Asn Thr 865 87O 87s 88O His Glin Ser Gly Arg Lieu. Glu Glu Thir Ala Gly Lys Gly Ala Gly Gly 885 890 895 Ala Lieu. Lieu. Thr Pro Llys Thr Glu Pro Glu Glu Lieu Ala Val Ser Glin 9 OO 905 91 O Gly Gly Ala Ala Pro Ala Thr Glu Glu Ser Ser Ser Ser Ser Glu Glu 915 92 O 925

Glu Glu Val Pro Ser Ser Pro Glu Pro Pro Arg Pro Ala Lys Arg Pro 93 O 935 94 O

Arg Arg Glu Lieu. Gly Ser Lys Gly Lieu Lys Gly Gly Gly Gly Gly Pro 945 950 955 96.O Gly Gly Trp Thr Cys Gly Lieu. Cys His Ser Trp Phe Pro Glu Arg Asp 965 97O 97. Glu Tyr Val Ala His Met Lys Lys Glu. His Gly Llys Ser Wall Lys Llys 98O 985 99 O

Phe Pro Cys Arg Lieu. Cys Glu Arg Ser Phe Cys Ser Ala Pro Ser Lieu. 995 1OOO 1005 US 2017/O 130272 A1 May 11, 2017 26

- Continued

Arg Arg His Val Arg Val Asn His Glu Gly Ile Lys Arg Val Tyr O1O O15 O2O Pro Cys Arg Tyr Cys Thr Glu Gly Lys Arg Thr Phe Ser Ser Arg O25 O3 O O35 Lieu. Ile Lieu. Glu Lys His Val Glin Val Arg His Gly Lieu Gln Lieu. O4 O O45 OSO Gly Ala Glin Ser Pro Gly Arg Gly. Thir Thr Lieu Ala Arg Gly Ser O55 O6 O O65 Ser Ala Arg Ala Glin Gly Pro Gly Arg Lys Arg Arg Glin Ser Ser Of O O7 O8O Asp Ser Cys Ser Glu Glu Pro Asp Ser Thr Thr Pro Pro Ala Lys O85 O9 O O95 Ser Pro Arg Gly Gly Pro Gly Ser Gly Gly His Gly Pro Leu Arg OO O5 10 Tyr Arg Ser Ser Ser Ser Thr Glu Glin Ser Leu Met Met Gly Lieu.

Arg Val Glu Asp Gly Ala Glin Glin Cys Lieu. Asp Cys Gly Lieu. Cys

Phe Ala Ser Pro Gly Ser Leu Ser Arg His Arg Phe Ile Ser His

Llys Lys Arg Arg Gly Val Gly Lys Ala Ser Ala Lieu. Gly Lieu. Gly

Asp Gly Glu Glu Glu Ala Pro Pro Ser Arg Ser Asp Pro Asp Gly

<210s, SEQ ID NO 9 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 9 cctcct cqtt cotgttittca 2O

<210s, SEQ ID NO 10 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer

<4 OOs, SEQUENCE: 10

Caatggtggg gaagaalacag 2O

<210s, SEQ ID NO 11 &211s LENGTH: 2O US 2017/O 130272 A1 May 11, 2017 27

- Continued

&212s. TYPE: DNA <213> ORGANISM: artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 11 ggacctgttt gct cattttg

<210s, SEQ ID NO 12 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 12 aactgtacgc ticaccaccitt

<210s, SEQ ID NO 13 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 13 cgggaat atc acaaggactg

<210s, SEQ ID NO 14 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 14 tataggcagg gag caggttc

<210s, SEQ ID NO 15 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 15 aatggtgc ct cqgtggtgat

<210s, SEQ ID NO 16 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer

<4 OOs, SEQUENCE: 16 gaggat catggggcaggttg

<210s, SEQ ID NO 17 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer US 2017/O 130272 A1 May 11, 2017 28

- Continued

<4 OOs, SEQUENCE: 17 Catgctitt cq Ctgcctggag

<210s, SEQ ID NO 18 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 18 gagggatata gCagggalaga

<210s, SEQ ID NO 19 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 19 cctttctoca ccctgctcat

<210s, SEQ ID NO 2 O &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM; artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 2O acccatt.ccc catcc.ctctg

<210s, SEQ ID NO 21 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 21 ccgt.cttgtc. citctgct citt

<210s, SEQ ID NO 22 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 22 aggaga.gcag ggg tagatgt

<210s, SEQ ID NO 23 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer

<4 OOs, SEQUENCE: 23 gcctggct ct gacatctacc US 2017/O 130272 A1 May 11, 2017 29

- Continued

<210s, SEQ ID NO 24 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 24 ccttaccacc acc cct actg

<210s, SEQ ID NO 25 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 25 gctgtgctag ggctittgagt

<210s, SEQ ID NO 26 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer < 4 OO SEQUENCE: 26 aggcagcagt agggaaaacc

<210s, SEQ ID NO 27 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 27 aggagt cqtc titcatctt.ca

<210s, SEQ ID NO 28 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 28 ttcactgact togc catgctic

<210s, SEQ ID NO 29 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer

<4 OOs, SEQUENCE: 29 tggcgt.cgtg attagtgatg

<210s, SEQ ID NO 3 O &211s LENGTH: 2O US 2017/O 130272 A1 May 11, 2017 30

- Continued

&212s. TYPE: DNA <213> ORGANISM: artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 30 gcacacagag ggctacaatg

1. An isolated nucleic acid which comprises the whole 13. The method according to claim 7, wherein the muta nucleotide sequence of the ZNF687 gene or a portion tion is detected by nucleic acid amplification reaction and thereof, wherein said whole nucleotide sequence of the detection of the amplification product. ZNF687 gene or a portion thereof comprises at least one 14. The method according to claim 7, wherein the muta mutation with respect to the wild-type nucleotide sequence tion is detected by sequencing the whole nucleotide of the ZNF687 gene as represented by SEQ ID NO:1, sequence of the ZNF687 gene or a portion thereof. characterized in that the mutation is selected from the group 15. An isolated mutated ZNF687 protein, consisting of: characterized in that it is encoded by a nucleic acid C>G at nucleotide position 2810 of SEQ ID NO:4, according to claim 1. G>T at nucleotide position 725 of SEQ ID NO:2, and 16. An isolated mutated ZNF687 protein, which consists C>T at nucleotide position 1994 of SEQ ID NO:3. of the amino acid sequence selected from the group con 2. The isolated nucleic acid according to claim 1, com sisting of SEQID NO:5, SEQ ID NO:6 and SEQID NO:7. prising the nucleotide sequence SEQ ID NO:2, SEQ ID 17. An in vitro or ex vivo method for diagnosing Paget’s disease of bone or bone tumor, which comprises the step of NO:3 or SEQ ID NO:4. determining, in a biological sample from a Subject Suspected 3. The isolated nucleic acid according to claim 1, which of being affected by Paget’s disease of bone or bone tumor, is a genomic DNA, an mRNA or a cDNA. the presence of at least one ZNF687 protein which is 4. An oligonucleotide or a probe which comprises from 10 mutated compared to the wild-type ZNF687 protein to 30 consecutive nucleotides of the nucleic acid sequence sequence as represented by SEQ ID NO:8, the presence of according to claim 1 and which includes said mutation. said at least one mutated protein being indicative of Paget’s 5. An expression vector which comprises a nucleic acid disease of bone or bone tumor. sequence according to claim 1. 18. An in vitro or ex vivo method for determining the 6. A host cell which comprises the expression vector predisposition of a subject affected by Paget’s disease of according to claim 5. bone to develop a bone tumor, which comprises the step of 7. An in vitro or ex vivo method for diagnosing Paget’s determining, in a biological sample from a subject affected disease of bone or bone tumor, which comprises the step of by Paget’s disease of bone, the presence of at least one determining, in a nucleic acid sample of a Subject Suspected mutated ZNF687 protein with respect to the wild-type of being affected by Paget’s disease of bone or bone tumor, ZNF687 protein sequence as represented by SEQ ID NO:8, the presence of at least one mutation in the wild-type the presence of said at least one mutated protein being nucleotide sequence of the ZNF687 gene as represented by indicative of a predisposition to develop a bone tumor. SEQ ID NO:1, the presence of said at least one mutation 19. The method according to claim 17, wherein said bone being indicative of Paget’s disease of bone or bone tumor. tumor is a giant cell tumor or an osteosarcoma. 8. An in vitro or ex vivo method for determining the 20. The method according to claim 17, which comprises predisposition of a subject affected by Paget’s disease of contacting said biological sample with an antibody which is bone to develop a bone tumor, which comprises the step of capable of specifically recognizing an isolated mutated determining, in a nucleic acid sample of a Subject affected by ZNF687 protein encoded by a nucleic acid which comprises Paget’s disease of bone, the presence of at least one mutation the whole nucleotide sequence of the ZNF687 gene or a in the wild-type nucleotide sequence of the ZNF687 gene as portion thereof, wherein said whole nucleotide sequence of represented by SEQ ID NO:1, the presence of said at least the ZNF687 gene or a portion thereof comprises at least one one mutation being indicative of a predisposition to develop mutation with respect to the wild-type nucleotide sequence a bone tumor. of the ZNF687 gene as represented by SEQ ID NO:1, 9. The method according to claim 7, wherein said bone characterized in that the mutation is selected from the group tumor is a giant cell tumor or an osteosarcoma. consisting of: 10. The method according to claim 7, wherein said C>G at nucleotide position 2810 of SEQ ID NO:4, mutation is located in the ZNF687 gene coding region. G>T at nucleotide position 725 of SEQ ID NO:2, and 11. The method according to claim 7, wherein said C>T at nucleotide position 1994 of SEQ ID NO:3. mutation is a missense mutation. 21. (canceled) 22. The method according to claim 8, wherein said bone 12. The method according to claim 11, wherein the tumor is a giant cell tumor or an osteosarcoma. missense mutation is selected from the group consisting of 23. The method according to claim 8, wherein said C>G at nucleotide position 2810 of SEQ ID NO:4, mutation is located in the ZNF687 gene coding region. G>T at nucleotide position 725 of SEQ ID NO:2, and 24. The method according to claim 9, wherein said C>T at nucleotide position 1994 of SEQ ID NO:3. mutation is located in the ZNF687 gene coding region. US 2017/O 130272 A1 May 11, 2017 31

25. The method according to claim 9, wherein said mutation is a missense mutation. 26. The method according to claim 8, wherein the muta tion is detected by nucleic acid amplification reaction and detection of the amplification product. 27. The method according to claim 8, wherein the muta tion is detected by sequencing the whole nucleotide sequence of the ZNF687 gene or a portion thereof. 28. The method according to claim 18, wherein said bone tumor is a giant cell tumor or an osteosarcoma. 29. The method according to claim 18, which comprises contacting said biological sample with an antibody which is capable of specifically recognizing an isolated mutated ZNF687 protein encoded by a nucleic acid which comprises the whole nucleotide sequence of the ZNF687 gene or a portion thereof, wherein said whole nucleotide sequence of the ZNF687 gene or a portion thereof comprises at least one mutation with respect to the wild-type nucleotide sequence of the ZNF687 gene as represented by SEQ ID NO:1, characterized in that the mutation is selected from the group consisting of: C>G at nucleotide position 2810 of SEQ ID NO:4, G>T at nucleotide position 725 of SEQ ID NO:2, and C>T at nucleotide position 1994 of SEQ ID NO:3. k k k k k