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Variations in zinc finger protein gene might impact expression of transient receptor potential channel and receptor tyrosine kinase genes contributing to inflammasome dependent inflammatory response and development of clavicular cortical hyperostosis

Background: Unilateral painful swelling of the clavicle might be the first symptom of chronic nonbacterial recurrent multifocal osteomyelitis (CNO/CRMO), but when lacking other inflammatory sites and recurrence, it might also be regarded as clavicular cortical hyperostosis (CCH), separate condition of unknown etiology. To elucidate underlying mechanisms of this disorder we performed various genome, transcriptome and proteome analysis. Methods: Total RNA was isolated from whole blood of 18 new-onset CCH patients and 8 healthy controls, while DNA was extracted from three patients. Targeted exome sequencing was performed using the Nextera Rapid Capture Exome Kit (Illumina). The extracted variants were annotated and filtered using the Variant Studio software (Illumina) and Variant Interpreter (Illumina). DNA microarray gene expression was performed in five CCH and four control patients along with bioinformatical analysis of retrieved data. Carefully selected differentially expressed genes (TRPM2, TRPM3, TRPM7, CASP2, MEFV, STAT3, EIF5A, ERBB2, TLR4, NLRP3, CD24, MYST3) were additionally analyzed by qRT-PCR in all participants. In one patient, the blood cells were processed with cytosine for immunofluorescence microscopy (IF) using TRPM3 and TRPM7 antibody. Results: Exome sequencing identified 428 shared identical variants among affected individuals. Thirty of these variants were not associated with a gene, 121 were in ZNF717 gene and 277 were distributed in 63 other genes. One heterozygous variant in CTBP2 gene, one in HYDIN gene and six in ZNF717 were classified as likely pathogenic. Microarray results and bioinformatical analysis revealed 974 differentially expressed genes, while qRT-PCR analysis showed significantly higher expression of TRPM3 and TRPM7, and lower expression of ERBB2. IF showed high signal of TRPM3 in blood cells of CCH patient. Conclusion: Broad investigation of genetic contribution in CCH patients revealed likely pathogenic variants in ZNF717 gene which encodes a Kruppel- associated box (KRAB) zinc- finger protein from the large group of transcriptional regulators and probably influences observed gene expression patterns. Diligent analysis of those patterns indicated majority of differentially expressed genes were involved in various inflammatory processes, while further investigation confirmed aberrant expression in additional number of patients. Most significant expression divergence was detected in TRPM3 and TPRM7 genes which encodes transient receptor potential channels that are multimodal sensors able to regulate inflammasome and ERBB2 gene which encodes widely expressed cell surface growth factor receptor capable to protect cells during inflammation. Moreover, TRPM3 protein was abundant in blood cells of CCH patient confirming the increased function of this gene. Consequently, our results along with the results of previous studies imply CCH could be a new (auto)inflammatory bone disease entity in which ZNF717 variations could contribute to TRP channel overexpression and abundancy, inflammasome activation and reduced protection during inflammation.

CNO, CRMO, CCH, autoinflammatory disease, WES

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