A four-generation Chinese kindred was studied with the permission of the family members, 11 out of 22 members were affected with HME (6 female, 5 male, 5 deceased).The proband III2 , a 53 years old female, visited the rheumatologic department outpatient clinic for multiple joints pain. The palpable mass near the left knee was first noticed at the age 3 and gradually exostoses were affected both knee. She was born of nonconsaguineous Chinese parents with a family history. Physical examinations revealed exostoses in bilateral knee joints and left waist. The patient IV 3 was a 24 years old male with palpable lesions at the bilateral knees and elbow joints. The diagnosis was made by Multi disciplinary team of rheumatology, orthopedic and radiolagic department based on clinical manifestations and radiological images. The other family members’ information was recorded due to statements of the proband and family photos.
We did Whole-Exome Sequencing of the proband III2 , patient IV 3 on Illumina NovaSeq6000 according to the manufacturer’s instruction(Illumina, San Diego, CA, USA).Genomic DNA samples were extracted from peripheral blood using QIAamp RBlood Mini Kit (Qiagen, Hilden, Germany). The quality of genomic DNA was evaluated by agarose gel electrophoresis analysis, and the quantity was measured by
NanoDrop2000 and Qubit3.0. DNA was sheared with M220 Focused-ultrasonicator (Covaris, Woburn, MA, USA). DNA
target regions were captured by hybridizing the genomic DNA
sample library with the xGen RExome Research Panel v1.0 (IDT,
USA). The captured and amplified DNA samples were then sequenced
using Illumina NovaSeq6000 (Illumina, San Diego, CA, USA) with 150 base-paired end reads. The sequence reads were mapped and aligned to Hg37/GRP. Based on Standards and Guidelines for the Interpretation of Sequence Variants recommended by The American College of Medical Genetics and Genomics (ACMG), we analysed the sequencing data.
The following databases were used to filter the variants, the 1000 Genomes Project (http://www.1000genomes.org), Exome Variant Server (http://evs.gs.washington.edu), and Exome Aggregation Consortium (http://exac.broadinstitute.org). Mutation Taster (http://www.mutationtaster.org), PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2 ), and SIFT (Sorting Intolerant from Tolerant) human protein (http://sift.jcvi.org/www/SIFT_enst_submit.html) software
programs were used was used to predict the destructive of amino acid substitution on structure and protein function. Three tools were used to
predict the splice-site variant, namely Human Splicing Finder (http://www.umd.be/HSF3/HSF.shtml), GeneSplicer (http://www.cbcb.umd.edu/software/GeneSplicer/gene_spl), NetGene2(http://www.cbs.dtu.dk/services/NetGene2/). Sanger Sequencing was tested to validate the variants.
To figure out the mutation spectrum of EXT1 and EXT2, we studied the mutations that reported in Multiple Osteochondroma Mutation Database(https://databases.lovd.nl/shared/genes) and Clinivar( https://www.ncbi.nlm.nih.gov/clinvar). The type of mutations was mapped on a linear protein and its function domain (Lollipop plot) by using the online MutationMapper (https://www.cbioportal.org/visualize)[18, 19]. The type of variants was also studied.