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From symptom to genes: applicability of functional genomic methods in discovering the mechanisms of newly described disease entity

OBJECTIVE: Clavicular cortical hyperostosis (CCH) is a sterile inflammatory bone disorder of unknown etiology clinically characterized by pain and/or swelling of the clavicle. It has been regarded as a variant of chronic nonbacterial/recurrent multifocal osteomyelitis (CNO/CRMO) but due to lack of other inflammatory sites and recurrence it could also be regarded as a separate disease in the spectrum. Therefore, it is of high importance to elucidate the exact mechanisms responsible for the development and progression of the symptoms [1]. METHODS: Total RNA was isolated from whole blood of 18 new- onset, untreated CCH patients and 8 healthy controls. DNA microarray gene expression was performed in 5 CCH and 4 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) where analyzed by qRT-PCR in all participants of the study. In one patient, the blood cells were processed using a cytosine for a immunofluorescence microscopy with TRPM3 and TRPM7 antibody. RESULTS: 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. Immunofluorescence microscopy showed high signal of TRPM3 in blood cells of one patient. CONCLUSIONS: Microarray data analysis revealed that majority of differentially expressed genes in CCH patients are involved in various inflammatory processes, while qRT-PCR analysis confirmed statistically significant expression change of 3 genes. Among them, TRPM3 and TRPM7 are members of transient receptor potential (TRP) gene superfamily, which encodes proteins that act as multimodal sensor cation channels for a wide variety of stimuli, one of which is environmental temperature that in the case of CCH could be elicited by overuse of sterno-clavicular joint (SCJ) [2]. Upon stimulation, TRP channels transduce electrical and/or Ca2+ signals. Dysfunctions in Ca2+ signaling due to altered TRP channel function can have strong effects on a variety of cellular and systemic processes, including the activation and the regulation of the inflammasomes, which are reported to be involved in CRMO pathogenesis [3, 4]. ERBB2, third gene with significant expression change, belongs to a family of genes that encodes for widely expressed cell surface growth factor receptors. Recently it has been shown that ErbB activation promotes protective cellular outcomes during inflammation, hence lower expression of this gene could cause damage due to inflammation [5]. Finally, the results of transcriptome analysis were confirmed on a protein level with a proof-of-concept experiment which indicated high presence of TRPM3 in blood cells of a patient. Based on the results of these and previous studies, we hypothesize that CCH could be an autoinflammatory disease induced by SCJ overuse, TRP channel overexpression, inflammasome activation and reduced protection during inflammation. REFERENCES: 1. Borzutzky A, Stern S, Reiff A, et al. Pediatric chronic nonbacterial osteomyelitis. Pediatrics. 2012 Nov ; 130(5):e1190-1197. 2. Shimizu S, Takahashi N, Mori Y. TRPs as chemosensors (ROS, RNS, RCS, gasotransmitters). Handbook of experimental pharmacology. 2014 ; 223:767-794. 3. Latz E, Xiao TS, Stutz A. Activation and regulation of the inflammasomes. Nature reviews Immunology. 2013 Jun ; 13(6):397-411. 4. Scianaro R, Insalaco A, Bracci Laudiero L, et al. Deregulation of the IL-1beta axis in chronic recurrent multifocal osteomyelitis. Pediatric rheumatology online journal. 2014 ; 12:30. 5. Frey MR, Brent Polk D. ErbB receptors and their growth factor ligands in pediatric intestinal inflammation. Pediatric research. 2014 Jan ; 75(1-2):127-132.

Chronic nonbacterial osteomyelitis; CNO; chronic recurrent multifocal osteomyelitis; CRMO; clavicular cortical hyperostosis; CCH; functional genomics

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