Identification of pathogenic mutations in 6 Chinese families with multiple exostoses by whole-exome sequencing and multiplex ligation-dependent probe amplification

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Introduction
Hereditary multiple exostoses (HMEs) (MIM #133700, #133701, and %600209), also known as multiple osteochondromas (MOs), is characterized by the presence of multiple exostoses that mainly exist in the metaphysis of long bones. These regions include the humerus, proximal tibia and fibula, and distal femur, with occurrence rates ranging from approximately 70% to 90%. [1,2] These deformities, such as unequal limb length and form, forearm malformation, valgus malformation of the legs and scoliosis, are the main clinical symptoms of patients. [3,4] The penetrance of HME is estimated to be approximately 100%, and the incidence is estimated to be approximately 1 to 2/100,000. [2] HME patients may present mild symptoms or severe symptoms, such as pain and limited mobility, and some patients must undergo multiple surgeries. [5] A mutation in EXT1 (MIM #608177), EXT2 (MIM #608210), or exostosin-3 (EXT3) genes is the cause of HME. The EXT1 and EXT2 are homologous and share an approximately 70% amino acid identity with each other. [6,7] They encode heparin sulfate glycosyl transferases that are composed of 746 and 718 amino acids, respectively, [7][8][9][10] and play an important role in heparan sulfate (HS) synthesis. The synthetic complex is an essential factor in the signal transduction cascade for chondrocyte cell differentiation, ossification, and apoptosis. [11,12] Approximately 70% to 95% of affected individuals have a pathogenic mutation in the EXT1 or EXT2 gene. [13] To date, hundreds of mutations in the EXT1 and EXT2 genes related to HME have been reported. HME has high heritability and teratogenicity. It is necessary to perform genetic diagnosis or prenatal diagnosis, as the offspring of patients have a 50% higher risk of developing the disease. Due to the lack of mutation hotspots and large size of the EXT1 and EXT2 genes, WES and MLPA were performed in this study.

Patients
Six probands of the 6 unrelated Han Chinese families were identified as having HME (Fig. 1). Family histories were collected. Related clinical examinations, such as X-ray imaging, and physical examinations were carried out. For the diagnosis of HME according to the current criteria, [14] at least 2 exostoses must be detected by radiography or palpation in the metaphysis of long bones. Thirtynine blood samples from these families were obtained, which included 21 affected individuals and 18 unaffected individuals. Two hundred unrelated normal individuals were recruited as normal controls. This study was consistent with the tenets of the Declaration of Helsinki and was approved by the ethics committee of the Hunan Jiahui Genetics Hospital (the ethical approval number: 2017011502). Informed consent was obtained from the patient for publication of this case report and accompanying images.

Exome capture and sequencing
Genomic DNA was extracted from peripheral blood cells by the phenol-chloroform extraction method. Exon-containing frag-ments were enriched with the xGen Probes Panel (Integrated DNA Technologies, Coral, IL) and sequenced with the HiSeq 2000 platform (Illumina, San Diego, CA) by the paired-end method. A mean exome coverage of more than 100x was obtained. The sequencing depath was greater than 10x for ninety seven percent of capture regions. Data analysis was performed using the Verita-Trekker and Enliven Bioinformatics analysis pipeline. Variants identified were filtered through the process (see Supplement- Fig. 1, http://links.lww.com/MD/C979). Splicing mutations were checked using Human splicing finder 3.0 (http://umd.be/HSF3/).

Sanger sequencing
Four pair primers (see Supplement- Table 1, http://links.lww.com/ MD/C979) were designed for standard PCR assays using Primer PREMIER Version 5.0 (Premier Biosoft International, Palo Alto, CA). The amplified DNA fragments were directly sequenced by Sanger sequencing (Biosune, http://www.biosune.com). DNAS-TAR software (Madison, WI) was used to analyze the sequencing results by comparison with the reference sequences.

Multiple ligation-dependent probe amplification (MLPA)
The commercial kit #P215-B3 (MRC-Holland, Amsterdam, the Netherlands) was used following the manufacturer's instructions. An ABI 3130 sequencer (Applied Biosystems, Foster City, CA) was used for capillary electrophoresis. Coffalyser.Net software was used for the analysis of MLPA data. The proportion of each peak height was calculated. The normal copy number range (dosage quotient) is between 0.8 and 1.2; a ratio from 0.4 to 0.65 indicates heterozygous deletion, and a ratio from 1.3 to 1.65 indicates heterozygous duplication. Each positive result was repeated.
In family 3 (F3) ( Fig. 1-F3), the proband (III:1), with a height of 160 cm, was a 30-year-old female from Shanxi province. Exostoses occurred around both knees, the right scapula, distal radius, and ulnas, and the right side of the bottom tibia had been progressively worsening since the age of 6. X-rays showed localized bony protrusions around both knee joints, slightly narrowed joint spaces, and multiple bony protrusions in the left distal radius and ulnar (  In family 4 (F4) (Fig. 1-F4), the proband (III:1) was a 6-year-old female from Jiangxi who complained of walking with a valgus gait since the age of 2 years. Exostoses at multiple sites were present, including the right side of the lower tibia and the right bottom of the radial and wrist joint ( Fig. 2: F4A, B). The proband's mother (II:2) and grandfather (I:1, deceased) also exhibited exostoses at multiple sites.
In family 5 (F5) (Fig. 1-F5), the proband (II:3) was a 43-yearold male with a height of 158 cm from Guangxi province. He had discovered exostoses at 3 years of age. He had exostoses at multiple sites and obvious right forearm malformation. The left femoral neck, bilateral femoral heads, bilateral upper end of the tibias, and fibulas had multiple bony protrusions. The pathological examination indicated osteochondroma after surgical resection. His son (III:1) also had exostoses, mostly around the knee joints, with no apparent pain, and they became increasingly obvious during adolescence.
In family 6 (F6) (Fig. 1-F6), the proband (II:4) presented to our clinic with the complaint of pain caused by exostoses around the proximal joints of the extremities since the age of 9. She was 55 years old, had a height of 156 cm, and was from Sichuan province. The abnormal gait had become worse with aging. The exostoses in the upper side of the left femur were obviously increased. The coxa valga was obvious ( Fig. 2: F6A, B). Her daughter (III:1) had similar clinical features. Exostoses appeared at the distal end of the right radius at 4 years of age and obviously increased. The proximal tibias were also affected, and she also had slight pain and an abnormal gait. When the proband came to our clinic, the patient (III:1) was 7 weeks pregnant, and a genetic test and subsequent prenatal diagnosis were performed.

Identification of these mutations
In this study, 6 mutations in EXT1 and EXT2 were detected in 6 unrelated Chinese families with HME and are summarized in detail in Table 1. In F1and F2, no possible pathogenetic variation was detected by WES. MLPA was then used, resulting in the identification of a heterozygous deletion of the EXT1 gene from exon 2 to exon 8 and a heterozygous deletion of the EXT2 gene from exon 2 to exon 4 in the affected individuals of these 2 families, respectively ( Fig. 3-F1, F2). In F3, a c.1197C>G, p. (Tyr399X) heterozygous nonsense mutation in exon 8 of EXT2 was identified in the patients (Fig. 3-F3). In F4, a c.448C>T, p. (Gln150X) transition was observed in exon 1 of the EXT1 gene in the patients (Fig. 3-F4). In F5, a c.1057-2A>T splicing mutation was detected in the exon 3/intron 3 boundary of the EXT1 gene in the proband (II:3) (Fig. 3-F5). In F6, a recurrent small duplication mutation was identified in exon 6 of the EXT1 gene and defined as c.1468dupC (Fig. 3-F6). The prenatal diagnosis was performed after amniocentesis at the 18th week. The same heterozygous duplication mutation was detected in the amniocytes, and the pregnant woman (III:1, F6) terminated the pregnancy. The mutations described above were verified by verification of pedigree. Compared with the affected individuals, the unaffected individuals and 200 unrelated normal individuals did not have the same mutation.

Bioinformatics analysis of mutants
On the basis of the description of UniProtKB-Q16394/Q93063 (http://www.uniprot.org/uniprot/), schematic diagrams of the mutant proteins were drawn, showing the normal and mutated regions of EXT1 and EXT2 (Fig. 3C). These speculative mutants, such as p.

Discussion
HME is a heterogenic monogenic disorder with variable numbers and sizes of exostoses and locations of diseased bones. EXT1 and EXT2 mutations have been detected in approximately 70% to 95% of patients with HME, with EXT1 accounting for 56% to 78% and EXT2 accounting for 21% to 44% of cases. [13] To date (May 5, 2018), in the Multiple Osteochondroma Mutation Database (http://medgen.ua.ac.be/LOVDv.2.0/home.php), the numbers of variations in EXT1 and EXT2 are 436 and 223, respectively. Most of these mutations are detrimental and are predicted to produce truncated proteins. As shown in Fig. 3C, the normal EXT protein is composed of 3 domains, namely, the cytoplasmic domain, transmembrane domain, and luminal domain. In our study, WES was performed first, and when no mutation was found, MLPA was performed to detect the presence of copy variation in the EXT1 and EXT2 genes. In this study, 6 mutations were detected in the EXT1and EXT2 genes, 5 of which are novel mutations. The heterozygous deletion from exon 2 to exon 8 in the EXT1 gene in F1 may result in frameshift mutation from the 322nd amino acid residue and stop gain at 366th, which may result in loss of most of the EXT1 luminal domain. The heterozygous deletion from exon 2 to exon 4 in the EXT2 gene in F2 is speculated to result in the loss of almost entire amino acids. Both of them are large-fragment exon deletions that inevitably change the normal protein structures. The c.448C>T nonsense mutations that were detected in F4 may lead to a truncated protein at the 150th amino acid in EXT1. The c.1197C>G nonsense mutations detected in F3 are similar to those at the site of c.1197C>A, which was reported to be associated with HME in 2009. [13] Both the c.1197C>G and c.1197C>A mutations can change the amino acid codon to a termination codon at the 399th amino acid in EXT2. The Cterminus of the EXT proteins may be the catalytic regions of the enzyme, [14] and nonsense-mediated mRNA decay may also cause haploinsufficiency. These 2 nonsense mutations identified here are speculated to result in the loss of these important catalytic regions and, inevitably, the loss of some or all of the function of these regions. Splice site mutations can induce exon skipping, influence mRNA splicing, or activate cryptic splice sites. Currently, 51 EXT1 splice site mutations have been reported in the Human Gene Mutation Database (HGMD). In our study, a novel c.1057-2A>T heterozygous splicing mutation was detected in the exon 3/intron 3 boundary of the EXT1 gene in F5. These c.1057-2A>C [15] and c.1057-2A>G [16] splicing substitutions that are associated with HME were reported, which indicated the position of c.1057-2 as a key splicing site. We used Human Splicing Finder 3.0 to analyze the splicing mutation. The predicted results showed that the mutation would alter the wild-type (WT) acceptor site and likely affect splicing. Perhaps due to the difficulty of collecting fresh specimens from patients, only a few mutations in EXT1 and EXT2 have been studied at the RNA level. [17,18] The c.1468dupC recurrent frameshift mutation in the EXT1 gene was reported in 2001 [19] and was also identified in F6 in our study. The mutation may cause the replacement of 30 amino acid residues and further produce truncated proteins that truncated at the 519th amino acid residue. These changes in the physical and chemical parameters of mutants, such as the higher instability index, lower aliphatic index, and hydropathicity, indicate the decreased stability and increased degradability of the mutants.
There is a high rate of knee deformity in patients with HME, with approximately one-third of patients developing genu valgum. [20] Bowing deformities of the forearms are even more frequent and are observed in 30% to 60% of patients with HME. [2,21,22] Other skeletal deformities include limb length discrepancy, valgus deformity of the lower extremities, and scoliosis. Obvious forearm deformities and multiple exostoses around the knees were also observed in these probands (V: 2, F1 and III:1, F3). Conspicuous short metacarpals, third brachymetatarsia, and scoliosis were observed in the proband (V: 2, F1). Obvious coxa valga and mild scoliosis were observed in the proband (II: 4, F6). HME can lead to a disproportionate short stature due to the influence of long bone growth plates. We also observed the symptoms of these probands, 4 of whom (III:2, F2;  III:1, F3; II: 3, F5; II: 4, F6) had a height of less than 160 cm. Costal exostoses are mostly asymptomatic, but they can rarely cause intrathoracic complications, including hemothorax and pneumothorax. We did not observe such complications in our patients, except for a patient (V: 2, F1) who had mild symptoms of costal and scapula exostoses. Due to the small sample size, we did not determine the relationship between the genotype and phenotype. However, it is possible that not only the genetic factors but also the living conditions and social environments all contribute to the phenotypic heterogeneity. These mutations in the EXT1 and EXT2 genes may destroy the encoding area structure, impact RNA transcription levels, and subsequently lead to haploinsufficiency. [13,23,24] The encoded mutations may influence hetero-oligomeric complex formation and weaken catalysis in the polymerization of HS. This complex is an essential factor in the signal transduction cascade for regulation of chondrocyte differentiation, ossification, and apoptosis. [12,14] Mutations in the EXT1 and EXT2 genes may lead to structural changes in EXT glycosyltransferases and, subsequently, disturb interactions, lower enzymatic activity and the production of HS, disturb EXT1/EXT2 complex formation, and lead to the occurrence of HME. [13,25] These detected variants in the 6 unrelated Chinese families were not found in ExAC, 1000G, or ESP6500. They also cosegregated with the disease phenotype in these families and were assessed as pathogenic or likely pathogenic variants, according to the standards and guidelines for the interpretation of sequence variants of the American College of Medical Genetics and Genomics (ACMG), which suggests that they may be the genetic pathogenesis of HME. Due to the heterogeneity of HME, as well as lack of mutation hotspots and the large size of the EXT1/EXT2 genes, we effectively applied WES in these patients from 6 Chinese families with HME. Pathogenic mutations were detected in 4 of the patients, including all the point mutations that WES can assess. This could be a routine technique in molecular diagnosis for monogenic disorders when a patient's symptoms are atypical and heterogeneous in the near future.
In conclusion, we identified and evaluated 6 mutations in the EXT1 and EXT2 genes in 6 Chinese families with HME. Among them, 4 and 2 mutations were identified in EXT1 and EXT2, respectively, 5 of which are reported here for the first time. HME exhibits high heritability and teratogenicity; we successfully performed prenatal diagnosis for the pregnant women and prevented the fetuses that may have suffered HME after birth from being born. This study indicates that WES is effective in diagnosing monogenic disorders, such as HME, and extends the mutational spectrums in the EXT1 and EXT2 genes in Chinese patients, which is advantageous for genetic counseling and subsequent prenatal diagnosis.