A combined analytical approach reveals novel EXT1/2 gene mutations in a large cohort of Italian multiple osteochondromas patients.

Multiple osteochondromas (MO), also known as hereditary multiple exostoses (HME), is one of the most common hereditary musculoskeletal diseases in Caucasians (1/50,000) with wide clinical variability and genetic heterogeneity. Two genes have thus far been identified as causing the disease, namely EXT1 and EXT2. Various methods to detect mutations in the EXT genes have been used. Here a cohort of 100 MO patients belonging to unrelated Italian families have been analyzed by single-strand conformation polymorphism (SSCP) analysis or by denaturing high performance liquid chromatography (DHPLC). However, neither of these techniques can detect deletions or duplications of entire exons. Families that were negative at SSCP/DHPLC analysis underwent two-color multiple ligation-dependent probe amplification (MLPA) analysis. By these complementary techniques mutation detection was significantly improved and 26 novel mutations have been revealed as well as 18 previously described mutations to give a total of 44 different mutations. Thus we can conclude that combining MLPA with DHPLC in point-mutations negative MO families, the detection of mutations in EXT genes can significantly improve the identification of both point-mutations and mid-size rearrangements. More important, we were able to characterize all those patients who were negative at the first PCR-based method screening.