Novel EXT1 and EXT2 mutations identified by DHPLC in Italian patients with multiple osteochondromas

Abstract

We describe the results of an optimised DHPLC-based mutation screening of the EXT1 and EXT2 genes in Italian patients affected by multiple osteochondromas [MO; also referred to as hereditary multiple exostoses (HME) in the literature], using a multistep approach. We first analysed 36 unrelated probands for EXT1 mutations by DHPLC analysis and subsequent direct sequencing of all samples with abnormal elution profile. Negative cases were then screened for EXT2 mutations using the same approach. In patients who tested normal at DHPLC screening, all EXT1 and EXT2 exons and splice-site junctions were directly sequenced. In 7 informative families, we also performed a pre-screening linkage analysis to selectively focus the DHPLC testing on the EXT1 or EXT2 gene. We detected 31 MO-related mutations, of which 23 (74%) were novel. Seven polymorphisms were also found. Twenty-four mutations (77%) were found in EXT1 and 7 (23%) in EXT2. No disease-causing mutations were detected in five of 36 patients, with a mutation frequency of 86%. According with previous studies, most mutations (90%) are loss of function. Neither false positive nor false negative results were obtained. This multistep method can be considered a fast and reliable diagnostic strategy for the detection of EXT1/2 mutations, with excellent sensitivity and specificity.

Mutation Screening of EXT1 and EXT2 by Denaturing High-Performance Liquid Chromatography, Direct Sequencing Analysis, Fluorescence in Situ Hybridization, and a New Multiplex Ligation-Dependent Probe Amplification Probe Set in Patients with Multiple Osteochondromas

“…The observed higher frequency of EXT1 mutations is in agreement with previous mutation studies performed in western populations. 20,21,24,25 For EXT1 we found a frame shift in 11 families, a nonsense mutation in 6 families, and a splice site mutation in 8 families. In seven families a missense mutation was found.…”

“…To facilitate and reduce the cost of this mutation screening, optimization of a DHPLC-based protocol for all EXT1 and EXT2 coding exons has been described. 17,20 After all, most MO patients have point mutations or small deletions or insertions of a few bp in one of both genes and the EXT1 and EXT2 coding regions contain very little polymorphisms, making them very suitable for DHPLC analysis. However, because mutation screening was performed almost exclusively at the sequence level, quantitative (deletions and duplications) and positional (inversions and translocations) changes were not detected by this technique.…”

Genetic models of osteochondroma onset and neoplastic progression: evidence for mechanisms alternative to EXT genes inactivation

“…The 11 coding exons of EXT1 and the 13 of EXT2, along with exonintron junctions, were PCR-amplified using primer pairs and PCR condition previously described (Pedrini et al, 2005). The results of amplification and the presence of right sized PCR reaction products were confirmed by agarose gel electrophoresis.…”

“…The results of amplification and the presence of right sized PCR reaction products were confirmed by agarose gel electrophoresis. DHPLC analysis was carried out using the WAVE DNA Fragment Analysis System 3500HT (Transgenomic, Crewe, UK) equipped with a DNASep column (Transgenomic as already described (Pedrini et al, 2005).…”

“…All identified mutations were compared with those reported in the literature as being disease-causing for MOs (Jennes et al, 2009; MOs Mutation Database: http:// medgen.ua.ac.be/LOVD). The pathogenic effect of novel mutations was confirmed by testing other family members, when available, and by screening at least 200 control chromosomes from 100 unrelated healthy individuals as previously described (Pedrini et al, 2005).…”