We performed a prospective genotype-phenotype study using molecular screening and clinical assessment to compare the severity of disease and the risk of sarcoma in 172 individuals (78 families) with hereditary multiple exostoses. We calculated the severity of disease including stature, number of exostoses, number of surgical procedures that were necessary, deformity and functional parameters and used molecular techniques to identify the genetic mutations in affected individuals. Each arm of the genotype-phenotype study was blind to the outcome of the other. Mutations EXT1 and EXT2 were almost equally common, and were identified in 83% of individuals. Non-parametric statistical tests were used. There was a wide variation in the severity of disease. Children under ten years of age had fewer exostoses, consistent with the known age-related penetrance of this condition. The severity of the disease did not differ significantly with gender and was very variable within any given family. The sites of mutation affected the severity of disease with patients with EXT1 mutations having a significantly worse condition than those with EXT2 mutations in three of five parameters of severity (stature, deformity and functional parameters). A single sarcoma developed in an EXT2 mutation carrier, compared with seven in EXT1 mutation carriers. There was no evidence that sarcomas arose more commonly in families in whom the disease was more severe. The sarcoma risk in EXT1 carriers is similar to the risk of breast cancer in an older population subjected to breast-screening, suggesting that a role for regular screening in patients with hereditary multiple exostoses is justifiable.

EXT1 and EXT2 are two genes responsible for the majority of cases of hereditary multiple exostoses (HME), a dominantly inherited bone disorder. In order to develop an efficient screening strategy for mutations in these genes, we performed two independent blind screens of EXT1 and EXT2 in 34 unrelated patients with HME, using denaturing high-performance liquid chromatography (DHPLC) and fluorescent single-strand conformation polymorphism analysis (F-SSCP). The mutation likely to cause HME was found in 29 (85%) of the 34 probands: in 22 of these (76%), the mutation was in EXT1; seven patients (24%) had EXT2 mutations. Nineteen of these disease mutations have not been previously reported. Of the 42 different amplicon variants identified in total in the cohort, 40 were detected by DHPLC and 39 by F-SSCP. This corresponds to mutation detection efficiencies of 95% and 93% respectively. We have also found that we can confidently distinguish between different sequence variants in the same fragment using F-SSCP but not DHPLC. In light of this, and the similarly high sensitivities of the two techniques, we propose to continue screening with F-SSCP.

We describe here the spectrum and distribution of mutations in the EXT1 and EXT2 genes in the largest reported British Caucasian multiple osteochondromas (MO) population. Furthermore, we report for the first time the screening of the EXT1 and EXT2 promoters, 5'UTRs, and 3'UTRs, and exclude six potential MO candidate genes in individuals without a detectable mutation within the coding region of EXT1 and EXT2. The coding exons of EXT1 and EXT2 were screened in 72 unrelated probands affected with MO. Forty-six different mutations were identified in 56 probands, of which 29 were novel. Mutation in the EXT1 and EXT2 genes each accounted for 50% of the mutations identified. Of the 72 probands, 42 were of British Caucasian descent, which when added to the 41 British Caucasian families previously reported from our total cohort, gave a total of 83 families. This cohort's proportional frequency for EXT1/EXT2 mutation was 53%/47%. We also validated the technique of high-resolution melting analysis in a blind study using 27 unique EXT1 or EXT2 mutations. This technique was found to be sensitive with a detection rate of 100% regarding heterozygote detection for EXT mutation scanning. Furthermore, this technique has a very high throughput and is very cost-effective.