DOI: https://doi.org/10.21203/rs.3.rs-770260/v1
Background: Hereditary Multiple Osteochondromas(HMO) is a rare genetic musculoskeletal disorder characterized by multiple osteochondromas that form near to the growth plates of many bones. Loss-of-function mutations in EXT1 or EXT2 that encode glycosyltrasferases are the causal mutations for most HMO patients.
Methods: After collecting the family history and clinical information, we used Whole-Exome Sequencing to find the pathogenic mutations in one Chinese Hereditary Multiple Exostoses pedigree. Sanger sequencing and relevant online databases were used to validate the screened variants. Lollipop plots were drew to map the reported mutations from online databases (Multiple Osteochondroma Mutation Database and clinvar)on a linear protein domains by MutationMapper.
Results: A novel heterozygous splicing-site mutation in gene EXT1 (NM_000127:exon5:c.1417+1G>C，chr8:118834703) was found in this pedigree and mutation spectrum of genes EXT1 and EXT2 were demonstrated.
Conclusions: Our results help this pedigree to identify the pathogenic variant and guide the prenatal diagnosis, also expand the mutation spectrum in Hereditary Multiple Osteochondromas.
Hereditary multiple osteochondromas (HMO), also called hereditary multiple exostoses (HME), is a rare genetic disease, inherited in an autosomal dominant manner. Genes EXT1 (8q24.11) and EXT2(11p12) are the causal genes[1, 2], with a penetrance nearly to 100%. Another gene designated EXT3 on chromosome 1 between D19S413 and D19S221 still needs further supported data.[3]
The prevalence of HMO in western countries is estimated around 0.4 to 1 in 50000, peak in Guam 50 in 50000[4]. The major symptoms of the HMO are multiple osteochondromas and related skeletal deformities. These bony exostoses could affect all metaphyses and diaphyses of bones, commonly found at knees, arms, forearm, thigh, pelvis and shoulder. Patients with HME show symptoms at a young age with a palpable mass near long bones, often are diagnosed before 12 years old. The number of the exostoses and functional deformity grow with the time. The exostoses burden per individual is about 31.5 with the mean age of 28.2. Patients with an EXT1 genotype were reported to have significantly greater number of exostoses compared to EXT2 genotype.[5]
Osteochondroma is usually a benign bone mass with a malignant degeneration rates about 0.38-7.0% toward chondrosarcoma.[6, 7] About 3/4 occurred between ages 20–40 and more than half at the pelvis and proximal femur. [7] Surgical excision of the tumor with free margin is suggested and local recurrence is lower than 2% if it was completely resected. Somatic mutation of EXT1 was the molecular basis of chondrosarcoma. Complications of HMO include local compression of adjacent tissues, joints deformity and restricted joint motion, discrepancies of limb-length, shortened stature and osteoarthrosis. Pain and joints dysfunction require surgical treatment to relieve symptoms.
Both EXT1 and EXT2 gene products are glycosyltransferases, which play a important role in the chain elongation step of heparan sulfate synthesis. HS binds with core proteins to produce heparin sulfate proteoglycans(HSPGs), which are abundant in both cell membrane and extracellular matrix and also function with vital signaling proteins ,including bone morphogenetic protein (BMP), to regulating cell function.[8, 9] Animal studies showed that both Ext1+/− and Ext2+/− heterozygous knockout mice developed osteochondromas at a low penetrance.[10, 11] In contrast, mice with stochastic inactivation of ext1 in some chondrocytes demonstrate mulpitle exotoses and bone deformity mimicking human HMO.[12, 13] And also compound heterozygous Ext1+/− Ext2+/− mice and Ext1-deficient mice displayed longbones-associated exotosese. [14, 15] In line with the observation of three-dimensional chondrogenic pellet model, osteochondromas formation arises via a “second hit” that would further lower heparan sulfate level.[16]
Considering the genetic causes and the disease burden of HME, genetic counseling and PGD/PGS ( Preimplantation Genetic Diagnosis/Screening ) is highly suggested. In this study, we collect family history, clinical data, radiological images of a four-generation Chinese HME kindred. Whole-Exome Sequencing was used to identify the pathogenic mutation in this family. Moreover, we review the published literature and two online Multiple Osteochondroma Mutation databases to figure out the hot-spot and mutation sprectrum of EXT1 and EXT2.
Patients
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.
Genetic test
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.[17]
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.
The pedigree of the HME kindred was showed in Figure IA, According to the clinical classification of Mordenti , these 2 patients were moderate type of IB class[20] . Genetic tests found that the proband was with a novel heterozygous splicing-site mutation in gene EXT1 (NM_000127:exon5:c.1417+1G>C，chr8:118834703). This mutation was further validated by Sanger sequencing (Figure IB) and also tested in affected and unaffected family members. Affected members had the same mutation and the unaffected ones were negative. The Radiological image of the proband was demonstrated in Figure (IC-D, CT scan and MRI respectively).
EXT1 and EXT2 are the two causal genes for Multiple Osteochondroma. according to the Multiple Osteochondroma Mutation Database (https://databases.lovd.nl/ , Date last updated August 18, 2021)[20] and Clinivar ( https://www.ncbi.nlm.nih.gov/clinvar) , we found that no hotspot for EXT1 , but for EXT2 the mutation variants were mainly located in the Exostosin region. (Figure2). Of all the 844 variants in EXT1 were reported, in them 714 were unique public pathogenic variants (49% substitutions, 38% deletions, 9% duplications in DNA level),11% were located in splice region and 85% in coding region, and 52% were frameshifts and 28% were stop changes and 16% missense changes in protein level. For gene EXT2 totally 449 public DNA variants were reported, in them 362 variants were pathogenic (65% substitutions, 26% deletions, 6% duplications in DNA level), and 79% located in coding region and 15% were in Splice region. In protein level 51% were frameshifts and 29% stop changes and 15% missense changes.(details in supplements table1-2)
This study indentified a novel splicing-site mutation in EXT1 from a four-generation HME Chinese family. All 2 affected ones which we did genetic tests were found the same site and the unaffected members were not. For both EXT1 and EXT2, the most common mutation type is substitution, followed by deletion and duplication, located mainly on coding region and more than 10% in splicing region. And unlike EXT1, mutation sites of EXT2 were focused on the exostosin region,and rarely in the glycol-transf-64 domain. Consistent with the previous study suggested that mutation hot-spot region of EXT2 were Exon 5. [21]
One limitation of this study is that we did not test all the affected patients, this was due to geographic distance and personal will. Both patients complained about pain and shyness in school. A national wide study in the Netherlands showed that multiple hereditary exostoses damaged both physical and psychological well-being. Pain was the major complain, and about half school attendees experienced problems at school and more than a quarter were unable to join the sport activities.[22] HME impact on patients quality of life similar to osteoarthritis in the mental aspect and women responded even worse in the psychological side. [23]
Another limitation is that the diagnosis was based on the family history, clinical manifestations and radiological results, pathologic diagnosis was lacked because family members were moderate type of IB and did not do the surgical removement. Up to now no consensus on the hereditary multiple osteochondromas (HMO) diagnosis criteria was reported. And clinically
The diagnosis was made when patients with more than one osteochondromas of the juxta-epiphyseal region of long bones was confirmed by radiological images. Additionally these patients were with a positive family history and/or genetic tests indicated positive in gene EXT1 and EXT2. [24] Differential diagnosis was made from Dysplasia Epiphysealis Hemimelica, metachondromatosis (MC), enchondromatosis (Ollier disease and Maffucci syndrome). And the female patient was short-stature, while the other patients were within normal range. Diminished stature was observed in about 37% -53% patients and more severely affected in patients with an EXT1 mutation.[4, 25] The affected patients in this studied showed no sign of malignant degeneration. A international, web-based survey showed that about 2.7% patients underwent malignant transformation at a mean age of 28.6 years. This occurred most commonly on pelvis (8/21)and scapula (4/21) ,followed by proximal femur, rib, skull base, tibia and foot.[6] Whether genetic variants plays a role in the malignant transformation risk is undetermined yet. The molecular basis and characteristic of the HMO patients who had malignant change to chondrosarcoma still need further investigations. A regular doctor visit and yearly physical examination is strongly recommended for the malignant risk.
In conclusion, we found a novel splice-site variant in EXT1 in one Chinese hereditary multiple osteochondromas family. Additionally by digging the online
Multiple Osteochondroma Mutation Database and clinvar, we present the mutation spectrum and lollipop of variants site on protein domains. Multiple Osteochondromas is a chronic skeletal disease with a certain risk of malignant transformation. Medical managements, includes early diagnosis, genetic counseling, regular medical follow-up help to improve the patients’ clinical outcomes. Joint effort of social support and school care are required to help the HMO patients to maintain physical and mental well-being. More cohorts and pathogenesis investigations are needed elucidate the correlation of the genetic basis and clinical manifestations.
HMO Hereditary Multiple Osteochondromas
HME Hereditary Multiple Exostoses
EXT1 Exostosin Glycosyltransferase 1
EXT1 Exostosin Glycosyltransferase 2
Ethics approval and consent to participate: This study was approved by the Ethics Committee of Tongji Hospital and informed written consent was obtained from all the participants and/or their legal guardian(s) for deceased participants. All the methods were carried out in accordance with relevant guidelines and regulations.
Consent for publication: Not Applicable
Availability of Data and Materials: The datasets generated during the current study are available in the following website link
https://databases.lovd.nl/shared/individuals/00382756
Competing interests: The authors declare that they have no competing interests
Funding: This research was supported by grant (Nr1210487149) from the Research Fund of Tongji hospital.
Authors' contributions: WT and YL organized this study and interpreted the patient data. YJL and YHD analysed the data. YHD was a major contributor in writing the manuscript. All authors read and approved the final manuscript.
Acknowledgements: Thanks to Wenjie zhu from Radiology Department Tongji Hospital for helps in CT and MRI images .We show gratitude to the HMO family members who participated in this study.