A Han Chinese MO family was investigated in this study (Fig. 1a). The proband (MO1) was a 16-year-old girl suffering from multiple exostoses involving humerus, femur, tibia and fibula (Fig. 1b-c). A group of 200 unrelated healthy subjects, matched for geographical ancestry, were included as controls. Clinical data of each subject was recorded by nurse-administered questionnaires. Each participant underwent careful clinical and computed radiography examination of long bones, truncal and acral joints by two or more experienced orthopedic experts. MO was diagnosed as multiple exostoses, arising from the lateral ends of humerus, ulna, femur, tibiae, fibulae or knee joints. 5 ml EDTA anticoagulated peripheral blood was drawn from 6 family members (Table 1) and from the 200 healthy subjects. Intact chondroma tissues were derived from MO1 and from a patient with extragenetic solitary chondroma (male, aged 3 years) via surgical resection. The collected chondroma tissues were immediately stored in 4% paraformaldehyde solution. This study was approved by the Institutional Review Board (IRB) of Xi'an Jiaotong University. All study subjects or their respective guardians gave their informed written consent by signing a document that had been carefully reviewed by the IRB of Xi'an Jiaotong University.
Genomic DNA was extracted from 5 ml peripheral blood, using E.Z.N.A Blood DNA Midi Kit (Omega Bio-tek, Norcross, USA), according to the manufacturer's protocol. For exome sequencing, 15 microg of extracted DNA was randomly sheared into 250-300 bp fragment libraries by sonication. The purified DNA fragment libraries were captured by NimbleGen 2.1 M capture array (Roche NimbleGen, Madison, USA), followed by 90 bp paired-end sequencing on a Hiseq2000 platform (Illumina, San Diego, USA), according to the manufacturer's protocol.
The raw image files of exome sequencing were processed using Illumina Pipeline software (version 1.7) for base calling. The sequences of each subject were generated as 90 bp pair-end reads. SOAPaligner software (http://soap.genomics.org.cn/index.html) was used to align clean reads to the UCSC human reference genome (version hg19, build 37.1, http://genome.ucsc.edu/). Based on SOAPaligner alignment results, SOAPsnp software (http://soap.genomics.org.cn/index.html) was used for SNP calling. BWA software (http://bio-bwa.sourceforge.net/) was used to identify insertions and deletions (indels) in targeted regions by aligning sequencing reads to the UCSC human reference genome. Low-quality SNP calls and indels were filtered out using the following criteria described previously: 1)consensus quality score >=20; 2) sequencing depth >=4 and <=500; 3) copy number <=2; 4) distance between two adjacent SNPs >=5 bp. Given that common genetic variations were unlikely to be the causal mutations of MO, all identified SNPs and indels were further filtered against the exome data of 30 Chinese Han individuals from the 1000 Genome Project (ftp://www.1000genome.org) and Chinese Han SNP data available in the dbSNP database (http://www.ncbi.nlm.nih.gov/project/SNP/, build 132). SIFT software (version 4.0, http://sift.jcvi.org/) was used to evaluate the impact of amino acid substitution on protein function and synonymous mutations were removed. Missense mutations and frame-shifting indels that were shared by the two affected family members undergoing exome sequencing, were retained as candidate causal mutations for following study.
Sanger sequencing validation was performed in all 6 MO family members (MO1-6) to determine whether the candidate mutations, identified by exome sequencing, co-segregated with MO in the MO family. The population frequencies of the candidate mutations co-segregated with MO were further estimated by Sanger sequencing of 200 unrelated controls with matched geographical ancestry. Sanger sequencing was performed using the standard protocol. Primers used for Sanger sequencing were designed with Primer3 software (http://frodo.wi.mit.edu/).
The paraformaldehyde-fixed chondroma tissues from the MO patient and the extragenetic solitary chondroma patient were rinsed with phosphate buffered saline (PBS), decalcified and embedded in paraffin. Paraffin-embedded chondroma tissues were sectioned (5~8 microm thick), and placed on glass slides. For histochemistry, the tissue slides of MO were dewaxed in xylene, hydrated with graded ethanol, and stained by hematoxylin-eosin (HE), Safranin O (SO) and Toluidine Blue (TB), respectively.
For immunohistochemisty, the dewaxed and hydrated chondroma sections of MO and extragenetic solitary chondroma, were treated with 3% hydrogen peroxide solution for 10 min, rinsed with PBS, and incubated with rabbit polyclonal anti-EXT1 antibody (1:50 working dilution, abcam plc, MA, UK) at 4 C overnight. The chondroma sections were then incubated with secondary antibody (ZHONGSHAN golden bridge biotechnology, China) at 37 C for 15 min, exposed to Streptavidin-Horseradish Peroxidase at 37 C for 15 min, and stained with DAB substrate kit (Vector Laboratories, Burlingame, CA) and Meyer hematoxylin.