Disruption of Type I Collagen Through Genetic Rearrangements or Mutations Is Implicated in the Tumorigenesis of Bizarre Parosteal Osteochondromatous Proliferation (Nora’s Lesion)

doi:10.21203/rs.3.rs-1754250/v1

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Disruption of Type I Collagen Through Genetic Rearrangements or Mutations Is Implicated in the Tumorigenesis of Bizarre Parosteal Osteochondromatous Proliferation (Nora’s Lesion)

https://doi.org/10.21203/rs.3.rs-1754250/v1

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Bizarre parosteal osteochondromatous proliferation (BPOP, or Nora’s lesion) is a benign bone surface lesion which most commonly occurs in the digits of young patients and has a high recurrence rate. Histologically, it is composed of a mixture of disorganized bone, cartilage, and spindle cells in variable proportions and characterized by amorphous “blue bone” mineralization. Recurrent chromosomal abnormalities including t(1;17)(q32-42;q21-23) and inv(7)(q21.1-22q31.3-32) have been reported in BPOP. However, the exact genes involved in the rearrangements remain unknown. In this study, we analyzed 7 BPOP cases affecting the fingers, toe, ulna, and radius of 4 female and 3 male patients, aged 5 to 68 years. RNA sequencing on 5 cases identified genetic fusions between COL1A2 and LINC-PINT, a long non-coding RNA, in 3 cases, and COL1A1::MIR29B2CHG fusion in 1, both validated with fluorescence in situ hybridization (FISH) and reverse transcription-PCR. The remaining fusion-negative case harbored 3 COL1A1 mutations as revealed by whole exome sequencing and confirmed with Sanger sequencing. All these genetic alterations were predicted to cause frameshift and/or truncation of COL1A1/2. The chromosomal locations of COL1A2 (7q21.3), LINC-PINT (7q32.3), COL1A1 (17q21.33), and MIR29B2CHG (1q32.2) were consistent with the breakpoints identified in the previous cytogenetic studies. Subsequent screening of 2 BPOPs by FISH identified one additional case with COL1A1 rearrangement. Our findings are consistent with reported chromosomal abnormalities, and implicate the disruption of type I collagen through COL1A1/2 rearrangements or COL1A1 mutations in the tumorigenesis of BPOP. The prevalence and tumorigenic mechanisms of these COL1A1/2 alterations in BPOP require further investigation.

bizarre parosteal osteochondromatous proliferation

Nora’s lesion

gene fusions

Bizarre parosteal osteochondromatous proliferation (BPOP; Nora’s lesion), first reported by Nora et al in 1983, is a bone surface lesion that occurs more often in the hands and feet of young adult patients.^1–2 The patients usually present with an enlarging painless nodule. Trauma history is reported in a minority of patients. Histologically, BPOP shows disorganized bone, cartilage, and spindle cell components in variable proportions.^3,4 Characteristic matrix with bluish tinctorial quality described as “blue bone” is present in many cases and is considered as the defining feature by some.⁴ The lesions are usually treated with simple surgical excision, but have a high recurrence rate up to 55%.²

Although considered by some earlier reports to be a reactive or reparative process within the same disease spectrum as proliferative periostitis and turret exostosis,⁵ the neoplastic nature of BPOP has been proposed since several cytogenetic studies demonstrated recurrent clonal chromosomal abnormalities (Table 1).^3,6−12 The most frequent chromosomal abnormality reported to date is t(1;17)(q32-42;q21-23) or 1q32 rearrangement alone, found in 7 cases by conventional cytogenetics and/or fluorescence in situ hybridization (FISH).^6–8 The breakpoints on chromosomes 1 and 17 have been narrowed down to within the BAC clones RP11-99A19 and RP11-219F9, respectively.⁶ The other recurrent cytogenetic abnormality reported in BPOP is inv(7)(q21.1-22q31.3-32), described in 4 cases by different authors.^9–12 Currently, there are no deep sequencing data for BPOP reported and the exact genes involved in both chromosomal abnormalities and the prevalence of each genetic event are still unknown. In this study, using RNA sequencing and whole exome sequencing, we identified gene rearrangements of COL1A1/COL1A2 or mutations of COL1A1 in 5 cases of BPOP, suggesting that disruption of type I collagen coding sequences might be implicated in the tumorigenesis of BPOP.

Table 1

Cytogenetic and molecular abnormalities reported in bizarre parosteal osteochondromatous proliferation
Case	Year^reference	Age/Sex	Location	Genetic Alterations /Karyotype	FISH
t(1;17) and associated abnormalities
1	2004⁶	35/M	2nd toe	t(1;17)(q32;q21)	1q32 and 17q21 splits and fusion
2	2004⁶	39/M	3rd digit	--	1q32 and 17q21 splits and fusion
3	2004⁶	40/F	3rd toe	--	1q32 and 17q21 splits and fusion
4	2004⁶	24/F	1st digit	--	1q32 split only
5	2004⁶	44/F	5th digit	Normal karyotype	1q32 and 17q21 splits without fusion
6	2005⁷	39/F	3rd toe	t(1;17)(q42;q23)	--
7	2011⁸	8/F	distal ulna	t(1:17)(q32;q21)	--
inv(7)
8	2009⁹	12/M	metatarsal	inv(7)(q21.1q31.3), inv(6)(p25q15)	--
9	2011¹⁰	59/F	4th digit	inv(7)(q22q32)	--
10	2011¹¹	NA	NA	inv(7)(q22.1q31.3), inv(6)(p25q15)	--
11	2013¹²	36/F	distal radius	inv(7)(q22q32)	--
Others
12	2004³	5/F	radius (diaphysis)	47–48,XX,+1-2r /49,idem,+7	--
13	2004³	7/F	ulna (diaphysis)	Normal karyotype	--
FISH, fluorescence in situ hybridization; --, not done.

Case collection

After the approval of institutional review board, cases diagnosed as Nora’s lesion or bizarre parosteal osteochondromatous proliferation were retrieved from the surgical pathology archives and consultation files of the authors. The H&E slides of cases were reviewed for diagnostic confirmation. Five cases with suitable and available materials were submitted to RNA sequencing. One of them was also submitted to whole exome sequencing (WES). Two additional BPOP cases were screened by fluorescence in situ hybridization (FISH).

RNA sequencing and data analysis

Five cases (cases 1–5) were submitted to RNA sequencing using different platforms, including TruSeq RNA Exome (Illumina, USA; cases 1, 4 & 5), whole transcriptome sequencing (case 3), and TruSight RNA Pan-Cancer Panel (Illumina; case 2), as previously reported.^13–15

Briefly, total RNA was extracted from formalin-fixed and paraffin-embedded (FFPE) or fresh frozen samples using suitable RNeasy kits (Qiagen, USA) following the manufacturer’s instructions. RNA quality and quantity were assessed by a Bioanalyzer 2100 system (Agilent, USA) and Qubit® 2.0 Fluorometer (Life Technologies, USA). For library construction, RNA samples were then subjected to exomic enrichment using TruSeq RNA Exome, target enrichment with the TruSight RNA Pan-Cancer Panel kit, or mRNA capture using magnetic oligo(dT) beads (Illumina). After sequencing by the Illumina NextSeq or HiScanSQ system, reads files (fastq) were mapped to the hg19 reference for fusion gene detection by STAR-Fusion and/or FusionCatcher algorhithms.¹⁶

Whole exome sequencing (WES)

Case

, without promising fusion candidates identified by RNA sequencing, was then submitted for whole exome sequencing to identify other potential driver genetic abnormalities. DNA was extracted from FFPE tissues using QIAamp DNA FFPE Tissue Kit (Qiagen) and ligated to adaptors followed by amplification using the TruSeq Exome Library Prep kit (Illumina). The sample was then sequenced using the NextSeq500 system (Illumina). Quality of the reads file (fastq) was assessed by FastQC and then mapped to human genome hg19 reference. GATK algorithm was used to identify genetic variants/mutations. Variants annotated and filtered were manually checked using Integrative Genomics Viewer¹⁷, with a special focus on genes encoding collagen peptides.

Fluorescence in situ hybridization (FISH)

Custom FISH probes for COL1A1 and COL1A2 were made from BAC clones flanking the genes/regions of interest. The BACs were chosen according to the UCSC genome browser (http://genome.ucsc.edu) and obtained from Invitrogen (USA). BAC clones used were as follows: RP11-939K5, RP11-479I21, RP11-714H24, RP11-93L18, RP11-94C24, and RP11-373E4 for COL1A1; RP11-119H21 and RP11-958G16 for COL1A2. DNA from each BAC was isolated according to the manufacturer’s instructions. The BAC clones were labeled with fluorochromes (fluorescent-labeled dUTPs; Enzo Life Sciences, New York, USA) by nick translation and validated on normal metaphase chromosomes. Four µm-thick FFPE slides were deparaffinized, pretreated, and hybridized with denatured probes. After overnight incubation, the slides were washed, stained with 4’, 6-diamidino-2-phenylindole, mounted with an antifade solution, and then examined under a fluorescence microscope.

Polymerase chain reaction (PCR) and Sanger sequencing

Reverse transcription-PCR or PCR, followed by Sanger sequencing, were performed to validate the gene fusions (cases 1, 3, 4) or mutations (case 5) identified, respectively. The primers for RT-PCR or PCR were designed according to the fusion junction sequences or mutation regions identified (Supplementary Table 1). Depending on the tissue availability of each case, RNA extracted from FFPE (cases 1,4) or fresh frozen tissues (case 3) and DNA extracted from FFPE tissues (case 5) were used.

Clinicopathologic data of the study cases

The patients’ ages ranged from 5 to 68 years old and there were 4 females and 3 males. The lesions involved the fingers (n = 4), toe, ulna, and radius. Case 5 had trauma history 3 years prior to the diagnosis of BPOP, resulting in distal interphalangeal joint deformity. Radiologic studies available in 4 patients showed bone surface lesions typical of BPOP (Fig. 1A & 1B). In addition, case 1 also exhibited features suggesting corticomedullary continuity to the underlying bone (Fig. 1A). Histologically, they showed nodular proliferation of haphazardly arranged cartilage, bone, and spindle cells in variable proportions as well as the presence of blue bone (Fig. 1C-1E). Cases 1 and 2 had more mature lamellar bone with marrow fat towards the stalk of the lesion (Fig. 1C & 1F). There was no significant osteoblastic atypia. Case 5 was an extensively calcified lesion with mixed chondroid and osseous components (Fig. 1G). The chondroid component showed areas of increased cellularity and focal plumper cytomorphology. The irregular calcification pattern typical for blue bone was less prominent in case 5. Case 6 exhibited a multinodular growth pattern, with dermal and subcutaneous soft tissue involvement. Detailed clinicopathologic information was not available for case 3.

COL1A2::LINC - PINT as the resultant genetic event of inv(7) in BPOP

COL1A2 and LINC-PINT gene rearrangements were identified in 3 cases (cases 1–3) with 3 different platforms of RNA sequencing. Two of them harbored reciprocal COL1A2::LINC-PINT and LINC-PINT::COL1A2 fusion transcripts, while only LINC-PINT::COL1A2 sequences could be detected in case 3 (Table 2). The exonic compositions in the 3 cases were diverse, involving exon 6/7, exon 23/24, and exon 50 of COL1A2 and most commonly exons 3 or 4 of LINC-PINT (Table 2; Fig. 2). The fusions were validated by FISH for COL1A2 rearrangements (cases 1 & 2) and RT-PCR (cases 1 & 3) (Fig. 2C & 2D). FISH for COL1A2 rearrangement generally showed small-distance splits of the green and red signals in the lesional cells, consistent with an intrachromosomal inversion. The chromosomal locations of COL1A2 (7q21.3) and LINC-PINT (7q32.3) were in the region of the previously reported inv(7)(q21.1-22;q31.3-32) in BPOP. ^9–12

COL1A1::MIR29B2CHG as the resulting fusion gene of t(1;17) in BPOP

By RNA sequencing, case 4 was found to harbor a COL1A1::MIR29B2CHG fusion instead. No reciprocal fusion transcript was detected. The fusion joined exon 32 of COL1A1 and exon 2 of MIR29B2CHG. FISH for COL1A1 rearrangement and RT-PCR confirmed the gene fusion (Fig. 3A-3D). The chromosomal locations of COL1A1 (17q21.33) and MIR29B2CHG (1q32.2) were consistent with the breakpoints of the previously reported t(1;17)(q32-42;q21-23) in BPOP.^6–8

COL1A1 mutations could serve as an alternative molecular mechanism in BPOP

RNA sequencing identified no promising fusion candidate in case 5. To determine whether genetic mutations could serve as an alternative mechanism by which to alter collagen proteins in BPOP, the DNA of this case was subjected to WES. An analysis focusing on collagen-coding genes found 3 heterozygous mutations in COL1A1 mutations, i.e., c.4263dupC (p.Trp1422fs), c.2664dupC (p.Ser889fs), and c.2217dupA (p.Gly740fs), with allele frequencies of 31%, 26%, and 17%, respectively. The mutations were predicted to result in frameshift and truncation of the coding regions of COL1A1. All three mutations were validated by PCR and Sanger sequencing (Fig. 3E).

FISH screening of additional cases

Two remaining cases without adequate tissue for next-generation sequencing were tested for COL1A1 and COL1A2 rearrangements by FISH. Case 6 showed COL1A1 rearrangement, whereas case 7 was negative for both FISH analyses (data not shown).

The presence of two mutually exclusive recurrent chromosomal rearrangements, t(1;17) and inv(7), in BPOP have been documented by several cytogenetic studies.^9–12 However, the exact genomic breakpoints or fusion genes have not previously been identified. In this study, we identified COL1A1::MIR29B2CHG and COL1A2::LINC-PINT as the fusion genes corresponding to t(1;17) and inv(7), respectively.

In 2004, Nilsson et al. first identified t(1;17)(q32;q21) as the sole cytogenetic abnormality in a case of BPOP.⁶ Further FISH analyses of 4 additional cases showed that all cases had a 1q32 split, while all but one had a 17q21 split, suggesting a recurrent genetic event involving these loci in BPOP (Table 1). Two subsequent reports supported this finding, describing t(1;17)(q42;q23) in a BPOP of the toe and t(1:17)(q32;q21) in a BPOP in the distal ulna.^7,8 The genomic breakpoints were determined by FISH mapping, and approximated to be within BAC clone RP11-99A19 on 1q32 and RP11-219F9 on 17q21.⁶ The 1q32 locus covered by RP11-99A19 encompasses 3 genes: CR1L, CD46 (formerly known as MCP), and MIR29B2CHG. Our data confirmed MIR29B2CHG rearrangement as the critical genetic event in BPOP at the RNA level. As for RP11-219F9 on 17q21, we were not able to find this BAC clone in the current UCSC hg19 and hg38 database to verify its location. In the original study, it was described to be distal to RP11-243D13 on 17q21.32,⁶ compatible with the location of COL1A1 (17q21.33) as identified in our case 4. Although the authors stated that RP11-219F9 covered genes such as NBR2 and NSF, but not COL1A1, we speculate that the sequence alignments back then were imprecise, as the NBR2 and NSF genes lie proximal to RP11-243D13 in the current human genome assembly hg19 and hg38.

The other fusion gene identified in this study, COL1A2::LINC-PINT, matched the cytogenetic abnormality of inv(7)(q21.1-22q31.3-32) in BPOP. Intriguingly, in 2 of the 4 reported BPOPs with inv(7), the lesions showed central continuity with the underlying medullary cavity of bone, an uncommon finding in BPOP.^9,10 Our case 1 with available radiographic information also showed corticomedullary continuity with the underlying bone. Given the limited case number and clinical information available in this study, it remains to be clarified whether this radiographic finding is predictive of the genotype. In the literature, two of the BPOPs reported with inv(7) also had inv(6)(p25q15),^9,11 which was not inferred from our RNA sequencing data.

The pathogenetic mechanisms of these novel fusions remain to be studied. COL1A1 and COL1A2 gene rearrangements are most commonly seen in fibroblastic tumors, such as COL1A1::PDGFB and rarely COL1A2::PDGFB in dermatofibrosarcoma protuberans, COL1A1::USP6 in aneurysmal bone cyst, myositis ossificans, and fibro-osseous pseudotumor of digits, and rare COL1A2::USP6 in nodular fasciitis, as well as occasionally in non-fibroblastic tumors such as COL1A2::PLAG1 fusions in lipoblastoma.^18–24 In these fusions, COL1A1 and COL1A2 often contribute their active promoters, which result in the overexpression of the 3’ fusion partner genes. Indeed, in case 4 the expression of MIR29B2CHG was markedly elevated in comparison with all other mesenchymal tumors subjected to the same RNA exome platform (Supplementary Table 3). Meanwhile, however, LINC-PINT expression approached zero in case 1. Moreover, the expression levels of COL1A1/2 exceeded their fusion partners by at least 3 orders of magnitude in both cases. Collectively, we doubt that the tumorigenic mechanisms mediated by the two novel fusions could be simply or even correctly attributed to the aforementioned “active promoter” model.

Instead, we hypothesize that these gene fusions resulted in the disruption of coding regions of COL1A1 and COL1A2, and hence the production of truncated and dysfunctional type I collagen peptides. For each fusion transcript detected, the collagen proteins are predicted to lose a significant portion from either the N-terminus or C-terminus (Supplementary Table 2). Furthermore, in the fusion transcripts where COL1A2 and COL1A1 were the 5’ fusion partners, stop codons were observed in the 3’ fusion partner genes, not long after the fusion junction (Figs. 2 and 3, and Supplementary Table 2). This suggests a tumorigenic mechanism contributed by the non-coding RNA genes, when serving as the 3’ partner, not much more than providing the stop codons. Importantly, the frameshift mutations of COL1A1 identified in case 5 further supported the possible role of type I collagen disruption in BPOP formation. Although these fusions and mutations are predicted to be heterozygous, the altered collagen products might exert dominant-negative effects that lead to abnormal assembly of collagen fibrils, similar to COL2A1 mutations in a subset of chondrosarcoma.²⁵ In case 5 with COL1A1 mutations, we were unable to determine the germline genotype of the patient because normal DNA material was unavailable; however, since this patient has no known phenotypic association with syndromic diseases, it is most likely that these frameshift mutations are somatic.

Interestingly, rearrangements of another collagen gene, COL12A1, have also been identified in subungual exostosis, another bone surface osteochondromatous lesion bearing some histomorphologic similarity to BPOP. ²⁶ Earlier cytogenetic studies revealed recurrent t(X;6)(q25-26;q15-21) in subungual exostosis.^{3, 27} Using FISH mapping, Storlazzi et al. identified COL12A1 as the breakpoint on chromosome 6, and the breakpoint on chromosome X were found to be COL4A5 in 4 cases and the adjacent IRS4 in 1 case.²⁶ Subsequently, up-regulation of IRS4 was observed in subungual exostosis compared to other chondromatous/osteochondromatous tumors, including BPOP and osteochondroma.²⁸ Together with our findings, it is plausible that BPOP and subungual exostosis are distinct entities with different genetic abnormalities, although both involve alterations in collagen genes.

Both fusion partners, LINC-PINT and MIR29B2CHG, are non-coding RNA genes. LINC-PINT (long intergenic non-protein coding RNA, p53 induced transcript) is a long non-coding RNA functioning as a tumor suppressor relevant in various cancers, including osteosarcoma.^{29, 30} MIR29B2CHG (also known as C1orf132) is the host gene of miR-29b2 and miR-29c, and has been shown to be down-regulated as an alternative longer transcript in triple-negative breast cancer.³¹ We speculate that the gene fusions might also disturb the tumor-suppressing function of these non-coding RNAs. Of potential importance, exon 1 of LINC-PINT contains a highly conserved sequence element essential for its ability to inhibit tumor invasiveness,³² which would be lost in the COL1A2::LINC-PINT fusion. It is noteworthy that the COL1A2::LINC-PINT transcript was not detected in case 3 by RNA sequencing using oligo(dT) selection, implying that the reciprocal transcript LINC-PINT::COL1A2 might be more critical in the tumorigenesis of BPOP. However, it has been known that the 3’ poly-A tails might be very short or even absent in some lncRNAs, thus excluding these lncRNAs from the sequencing libraries with oligo(dT) selection. Whether this could account for the failure to detect the COL1A2::LINC-PINT fusion remains to be determined.

Other gene fusions involving non-coding RNA genes have also been reported, such as MALAT1::GLI1 fusion in plexiform fibromyxoma and gastroblastoma of stomach,^{33, 34} MALAT1::TFEB in renal cell carcinoma,^{35, 36} and MIR143::NOTCH1/2/3 fusion in glomus tumor.³⁷ It has been suggested that these fusions could use the active promoters of the 5’ non-coding RNA genes to upregulate the 3’ oncogenes. Nonetheless, we do not find it quite as plausible that this could apply to the fusions identified in our current study, given the much lower expression levels of these RNA genes than COL1A1/2 and the predicted truncation of the collagen genes, as mentioned above.

In summary, our study revealed that a majority of BPOP cases, including all those subjected to next-generation sequencing, had COL1A1/2 fusions or mutations, which was in keeping with the high prevalence of t(1;17) or inv(7) described in the original paper.⁶ It is possible that a small subset of fusion-negative BPOP might have a cryptic fusion or mutations of either gene, which could elude detection by FISH or even RNA sequencing and therefore might require appropriate next-generation sequencing methods to identify. In addition, the frequently suboptimal tissue quality, probably due to previous decalcification, has prevented us from genotyping a significant subset of cases. The prevalence of these genetic events remains to be further elucidated in a more systematic fashion.

In conclusion, we identified novel COL1A1::MIR29B2CHG and COL1A2::LINC-PINT fusions in BPOP. The locations of these genes were consistent with the previous cytogenetic reports of t(1;17) and inv(7) in BPOP. The current findings further solidify the neoplastic nature of BPOP and shed light on the potential roles of damaged type I collagen in BPOP formation. Further investigations are required to examine the prevalence of these alterations, the contribution of the non-coding RNA fusion partners, other alternative tumorigenic mechanisms in fusion-negative BPOP, and the correlation between genotypes and various clinicopathologic features.

Conflict of interest: None to declare

Funding: This work was partly supported by Shuang Ho Hospital, Taipei Medical University (109TMU-SHH-03 to YCK), Taipei Medical University (TMU108-AE1-B20 to YCK), National Taiwan University Hospital (108-S4295 to JCL), and Ministry of Science and Technology, Taiwan (MOST 109-2326-B-002-010-MY3 to JCL).

Data Availability Statement: The data that support the findings of this study are available from the corresponding author upon reasonable request.

ACKNOWLEDGEMENTS

The authors thank Professor Yoshinao Oda (Kyushu University, Japan) for providing materials, TMU Cancer Translational Core Facility for technical support in molecular testing and analysis, and Mr. Yung-Chuan Chung for technical assistance. This work was partly supported by Shuang Ho Hospital, Taipei Medical University (109TMU-SHH-03 to Y.-C.K.), Taipei Medical University (TMU108-AE1-B20 to Y.-C.K.), National Taiwan University Hospital (108-S4295 to J.-C.L.), and Ministry of Science and Technology, Taiwan (MOST 109-2326-B-002-010-MY3 to J.-C.L.).

CONFLICT OF INTEREST

The authors have disclosed that they have no signiﬁcant relationships with, or ﬁnancial interest

in, any commercial companies pertaining to this article.

AUTHOR CONTRIBUTIONS

Y.-C.K., J.-C.L., A.Y., and K.H.N performed study concept and design; Y.-C.K. and J.-C.L. performed writing of the paper; Y.-C.K., J.-C.L., T.-H.H., K.H.S., and H.I. provided acquisition, analysis and interpretation of data; H.-Y.H., P.C.-H.C., and C.D.M.F. provided material support and review and revision of the paper. All authors read and approved the final paper.

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Tables 2 is available in the Supplementary Files section.

There is NO conflict of interest to disclose.

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Disruption of Type I Collagen Through Genetic Rearrangements or Mutations Is Implicated in the Tumorigenesis of Bizarre Parosteal Osteochondromatous Proliferation (Nora’s Lesion)

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Abstract

Figures

Introduction

Materials And Methods

Case collection

RNA sequencing and data analysis

Whole exome sequencing (WES)

Fluorescence in situ hybridization (FISH)

Polymerase chain reaction (PCR) and Sanger sequencing

Results

Clinicopathologic data of the study cases

FISH screening of additional cases

Discussion

Declarations

References

Tables

Additional Declarations

Supplementary Files

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