Identification of Novel EXT Mutations in Patients with Hereditary Multiple Exostoses Using Whole-Exome Sequencing

Abstract

Objective: To find novel potential gene mutations other than EXT1 and EXT2 mutations, to expand the mutational spectrum of EXT and to explore the correlation between clinical outcome and genotype in patients with hereditary multiple exostoses (HME).

Methods: The study recruited seven families diagnosed with multiple osteochondromas (MO). Family histories and clinical information were collected in detail through comprehensive physical and image examination. Patients with deformities and functional limitations were classified as "severe" and the remaining without functional limitations were classified as "mild," in accordance with previous study. Whole-exome sequencing (WES) was performed on a total of 13 affected individuals, 1 available unaffected relative, and 10 healthy unrelated individuals. Sanger sequencing was used to validate the screened mutations. Finally, the structural change in protein caused by pathogenic mutations was analyzed using information from the relevant database online and we attempted to correlate clinical phenotype with genotype in patients with HME.

Results: Other than EXT1 and EXT2, no novel potential gene mutations were found through WES. We identified nine heterozygous mutations in EXT1 or EXT2. Of these mutations, four have not been reported previously. These are c.996delT in exon 2 of EXT1 (family 1), c.544C > T in exon 3 of EXT2 (family 2), c.1171C > T in exon 7 of EXT2 (family 5), and c.823_- 824delAA in exon 5 of EXT1 (family 7). The other five mutations have already been reported in previous works. It was surprising that we found two mutation sites, in exon 2 and exon 5, respectively, of EXT1 in 1 patient diagnosed with MO, when his father had two mutation sites, in exon 6 and exon 5, respectively, of EXT1 and EXT2 (family 4). In addition, 1 patient showed degeneration, while his father only exhibited slight symptoms (family 7). In our study, among 51 affected patients in seven families, the sex ratio (male vs female) was 58.9% (n = 30) vs 41.2% (n = 21). Male patients seemed to show more severe symptoms compared to females, but because the sample was small, we did not obtain statistically significance results.

Conclusion: Whole-exome sequencing to screen pathogenic gene mutations was applied successfully. Although no third-gene mutation associated with HME was found, a total of nine mutations across EXT1 and EXT2 were identified, four of which are novel. Our results expand the mutational spectrum of EXT and can be used in genetic counseling and prenatal diagnosis for patients with MO.

Keywords: Bone tumor; EXT1; EXT2; Hereditary multiple exostoses; Whole-exome sequencing.

Figure 1

Pedigree of seven families with multiple osteochondroma (MO). Black symbols represent the affected individuals and open symbols represent unaffected individuals. Circles and squares represent females and males, respectively. Arrows identify the probands in each family.

Figure 2

Appearance and radiology of the probands. (A) F1: X‐rays shows multiple exostoses on bilateral distal femur; (B) F2: One obvious bony prominence on his right rib; (C) F3: A huge exostosis on the proximal femur affecting the activation of hip joint; (D–F) (F4–F6, respectively): The X‐ray plain film shows multiple exostoses near the knee requiring surgery; (G) F7: a cauliflower‐like exostosis on the top of the pelvis; (H) F7: The plain radiograph of the distal femur showed extensive osteolytic lesions and obvious calcifications with a soft‐tissue mass.

Figure 3

Mutation analysis. (A) a frameshift mutation, c.996delT in exon 2 of EXT1; (B) a nonsense mutation, c.544C > T in exon 3 in EXT2; (C) a nonsense mutation. c.2053C > T in exon 10 in EXT1; (D) a missense mutation, c.896G > A in exon 5 of EXT2, a point mutation, c.8C > T in exon 2 of EXT2, a deletion, c.1469delT, in exon 6 of EXT1; (E) a nonsense mutation, c.1171C > T in exon 7 of EXT2; (F) a nonsense mutation, c.514C > T in exon 2 of EXT2; (G) a frameshift mutation, c.823_–824delAA in exon 5 in EXT2.