Study On Molecular Mechanism Of Osteopetrosis

Background:Osteopetrosis is a rare bone disorder charactered by abnormallyincreased bone density. There are some symptoms of osteopetrosis,including weakened bone resorption, cartilage, calcification andaccumulation of bone tissue which could not be replaced by normal bonetissue and so on. As disease progress of osteopetrosis, significant bonesclerosis, fragility, narrowed bone marrow cavity or even disappearance,anemia and other symptoms are usually found. In the1904, osteopetrosiswas first reported. Till now, a great many of researchers found that therewas a close relationship between abnormal osteoclast and the diseasedevelopment. Over the years, gene diagnosis and treatment ofosteopetrosis were focus by many researchers. So far, dozens ofosteopetrosis-related genes have been found, but effective treatment isstill lacking. In clinical, the genetic diagnosis of osteopetrosis is difficultfor the disease is caused by many genes and the genes sequences arerelative large. Some studies show that whole exome sequencingtechnology may be a new way for osteopetrosis genetic diagnosis, which is and economical and efficientive.Some researchers have indicated that gene expression was not just aone-way flow from the genome, transcriptome, proteome andmetabolome, and they were integrated as a complex regulatory network.Therefore, it may be valuable for the understanding of the pathogenesis oftraditional genetic diseases by our in-depth understanding, application ofthe central dogma, and performing multi-angle studies. Recently, somestudies have shown that some microRNA could control the developmentof some diseases, which indicated microRNA may be some new “drug”for the treatment of human diseases. However, it is necessary tounderstand the differently expressed microRNA and their targets invarious tissues or cells in particular genetic background or disease status,before using microRNA treatment in human diseases. Therefore, thisstudy revealed the disease gene by exome sequencing in a Chineseosteopetrosis family firstly, and then analyzed the differently expressedmicroRNA and protein profiles by microRNA sequencing and iTRAQproteomics respectively in peripheral blood monouclear cells (PBMCs).Finally, the microRNA targets were analyzed by considering the data ofmicroRNA, protein profile and bioinformatics, which may be some newand valuable information for understanding the pathogenesis ofosteopetrosis. Objective:To provide information for prenatal diagnosis and treatment ofosteopetrosis by whole exome sequencing in an osteopetrosis family; tofind osteopetrosis related microRNA and their target gene informationfrom the perspective of microRNA, proteomics, and bioinformatics.Methods:1. Gene mapping and mutation identification of osteopetrosisThe osteopetrosis family with17members was investigated. Thewhole exome of2patients in the osteopetrosis family were captured,amplified and sequenced by high throughput technology. To obtain thecandidate mutation, the sequencing data was analyzed by bioinformaticstechnology, including detection, annotations, filtering of SNP and InDel.Finally, the distribution of candidate mutation in the osteopetrosis familyand normal controls was detected by sanger sequencing.2. Osteopetrosis related microRNA and their target genesanalysisThe PBMCs extracted from6osteopetrosis patients and9controlswere divided into two parts. One of the two parts was used for microRNAsequencing, and the other part was used for differently expressed proteinsanalyze. For microRNA sequencing, the total RNA extracted fromPBMCs were used to microRNA library construction andhigh-throughput sequencing. For the identification of differently expressed proteins, the proteins extracted from PBMCs were labeled byiTRAQ and analyzed by LC-MS/MS. For the identification of microRNAtargets, the data of differently expressed microRNA, proteins, and thepredicted microRNA targets found by StarBase, which includes5softwares, were under consideration. Finally, the expressed microRNAand its target protein were confirmed by RT-qPCR, western blot; andtheir interaction were analyzed in vitro by293T cell culture andluciferase assay.Results:1. Gene mapping and mutation identification of osteopetrosisThe average sequence read depths were83×and81×in targetedexons of the two family patients. The reported mutation CLCN7(Y99C)was found to be a candidate mutation by analyzing the shared geneticvariation in the two family patients. Sanger sequencing found thatCLCN7(Y99C) was detected in6family patients, and could not be foundin other family members and100health controls. The clinical dataindicated that the family members with CLCN7mutation were similar toosteopetrotic patients reported by other researches.2. Osteopetrosis related microRNA and their target gene analysisTotally,38novel microRNA, and123significantly and differentlyexpressed known microRNA were found by microRNA sequencing,including63up-regulated known microRNA, and60down-regulated known microRNA. In iTRAQ Proteomics,173significantly anddifferently expressed proteins were found, including78up-regulatedproteins, and95down-regulated proteins. And totally117pairs ofmicroRNA/target protein were found by association analyze. Theexpression of hsa-miR-320a and Arf1were confirmed by RT-qPCR andwestern blot, and their interaction were confirmed in vitro by293T cellculture and luciferase assay.Conclusion:CLCN7(Y99C) may be the caused mutation of the Chineseosteopetrotic family, and whole exome sequencing is an effective andvaluable tool for the screening of osteopetrosis related genes.There are a large number of significantly different expression ofmicroRNA and proteins in the PBMCs taken from patients withosteopetrosis. MicroRNA may be involved in the pathological process ofosteopetrosis by regulating the expression of their target genes.