The role of EXT1 gene mutation and its high expression of calcitonin gene-related peptide in the development of multiple exostosis

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Abstract

Objective

Screening and identifying the gene mutation of EXT1, EXT2 and EXT3 associated with multiple exostosis (ME) and the expression in tumor tissues.

Methods

Nine patients with multiple exostosis were collected and genomic DNA was extracted. Polymerase chain reaction (PCR) amplification and direct sequencing techniques were used to screen all exons, 5′ and 3′ ends of the EXT1, EXT2 and EXT3 related causative genes. EXT1, EXT2 and EXT3 gene were screened and quantified by RNA-SEQ and RT-qPCR. The concentration of calcitonin gene-related peptide (CGRP) in peripheral blood of tumor patients and normal controls was detected by ELISA.

Results

Between the two patients with ME, the EXT1 gene was found in one patient to have c.79 T>A mutation, which caused the change of p.M27T, the non polar methionine was replaced by the high frequency mutation of polar threonine, and the rest of patients was found the splicing mutation c.1284 + 8 delAT of the heterozygosity of the EXT1 gene. The serum CGRP concentration of ME patients (623 + 49 pg/ml) was significantly higher than that of normal controls (196 + 68 pg/ml), and EXT1 mutation patients were also higher than non mutation patients.

Section snippets

Object

Nine patients with multiple exostosis treated at the Department of Bone Tumors, First Affiliated Hospital of Fujian Medical University, were diagnosed as systemic multiple osteochondromas by X-ray and CT scan. Histopathological examination was also diagnosed as exostosis (specific typing and staging), including inquiring about family history, and symptoms associated with ME.

Specimen collection and treatment

The redundant paraffin embedded specimens collected from the patients after pathological diagnosis, extracted genomic DNA and reserved at −20 °C after quantification.

Mutation analysis of EXT1/EXT2/EXT3 gene

The 11 exons of the genomic EXT1 gene, 15 exons of the EXT2 gene and 11 exons of the EXT3 gene were amplified respectively. The total PCR reaction volume was 25 μl: 0.2 mmol/l for each of 4 dNTPs, 2.5 μl for 10 × buffer, 10 pmol for each of upstream and downstream primers, 100 ng for DNA template, and Taq DNA Polymerase 2.5u. The

Clinical manifestations and auxiliary examinations

The patient has hips, knees, wrists, ankles or pelvises and other peripheral joints with tumors of different sizes. Typical cases of X-ray (Fig. 1A): multiple osteochondroma of distal femur and upper tibia and fibula. Pathological section (Fig. 1B) shows a continuous fibrous cartilage membrane in the outer layer of tissue, which inner exists a typical cluster of cartilage cells, and near the bone transition zone, the chondrocytes were arranged in a regular manner with a funicular-shape.

Phenotypic diagnosis

Serum

Disscussion

Multiple mutation detection in multiple regions showed that about 80% of the HME gene mutations was micromutation such as nonsense mutation, code shift mutation and splice site mutation, which resulted in the early termination of the corresponding gene translation and partial or complete deletion of the functional genes and eventually resulted in the truncation of the encoded proteins so that the protein functional domain was lost [3]. Partial deletion and insertion mutation of EXT gene is also

Conflict of interest

The authors declare that they have no competing interests.

Acknowledgements

This work was supported by the Joint Fund for Program of Science innovation of Fujian Medical University, China (2017XQ2011).

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