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

Abstract

Multiple osteochondromas (MO) is an autosomaldominant skeletal disorder characterized by the formation of multiple cartilage-capped protuberances. MO is genetically heterogeneous and is associated with mutations in the EXT1 and EXT2 genes. In this study we describe extensive mutation screening in a set of 63 patients with clinical and radiographical diagnosis of MO. Denaturing high-performance liquid chromatography analysis revealed mutations in 43 patients. Additional deletion analysis by fluorescence in situ hybridization and a newly developed multiplex ligation-dependent probe amplification probe set identified one patient with an intragenic EXT1 translocation , three patients with a partial EXT1 deletion , and one patient with a partial EXT2 deletion. Thirty-six patients harbored an EXT1 mutation (57%) , and 12 had an EXT2 mutation (19%). We show that our optimized denaturing high-performance liquid chromatography/sequencing/multiplex ligation-dependent probe amplification protocol represents a reliable and highly sensitive diagnostic strategy for mutation screening in MO patients. Clinical analysis showed no clear genotype-phenotype correlation in our cohort of MO patients.

Exon-level array CGH in a large clinical cohort demonstrates increased sensitivity of diagnostic testing for Mendelian disorders

“…The positive rate of 9.3% for CREBBP intragenic deletions in 75 patients with Rubinstein-Taybi syndrome and of 7.3% for EXT1 or EXT2 deletions in 55 patients with multiple exostoses falls in the range reported by others. 10 Finally, consistent with previous reports, 1 out of 13 patients …”

Clinical and molecular studies of EXT1/EXT2 in Bulgaria

“…In our patients with EXT1 and EXT2 mutations the tibia, femur and humerus were the most frequently affected bones, which explains the common complications and deformations seen in these bones by Jennes et al 2008. Our patients with EXT2 mutations showed a smaller number of affected bones.…”

“…The exon numbering is according to Clines et al 1997. MLPA analysis was performed with Salsa® MLPA® kit P215 (Jennes et al 2008). FISH analysis was performed with EXT1 cosmid probes 46F10 and 90D8 (Ahn et al 1995) to confirm large deletions.…”

Tiling resolution array-CGH shows that somatic mosaic deletion of the EXT gene is causative in EXT gene mutation negative multiple osteochondromas patients

“…Mutation screening of the EXT1 or EXT2 genes using standard sequencing for all exons including exon-intron junction points and MLPA testing using exon dedicated probe set for each exon coding regions were normal as reported earlier (Jennes et al, 2008;Wuyts et al, 2005). These mutation-negative patient samples were tested using the custom made EXT-tiling-array.…”

“…The complexity of osteochondroma formation, however, is not yet completely clarified by the bi-allelic inactivation as mosaic distribution of cells with retained chromosome 8 regions (presumably normal cells) was observed in the cartilage cap of both human osteochondromas and animal model systems (de Andrea et al, 2010;Jones et al, 2010;Bovee et al, 2010) The majority of the hereditary cases (70-75%) are caused by point mutations resulting in truncated proteins (Wuyts and Van Hul, 2000;Signori et al, 2007;Lonie et al, 2006;Pedrini et al, 2005;Jennes et al, 2009). Deletions involving single or multiple exons were found in about 10 % of all hereditary cases using Multiplex Ligation-dependent Probe Amplification (MLPA) (Jennes et al, 2008;Fredman et al, 2004;Vink et al, 2005). However, in about 10-15 % of the MO cases, genomic alterations can not be detected implying the potential role of other alterations such as inversions, translocations or somatic mosaicism (Vink et al, 2005).…”