Aberrant EXT2 splicing transcripts with premature termination codon.

<p>(A) Screenshot of a CRYP-SKIP output. The input EXT2 sequences included exon 4 (upper) and the flanking intronic sequences (lower). The table on the left lists the summarized values of the predictor variables used in CRYP-SKIP (PESS, putative exonic splicing silencers; NN 5'ss, neural network 5' splice sites; SF2/ASF, the most important SR protein for aberrant splice-site activation, FAS-ESS, ESSs discovered by a fluorescence-activated screen; EIE, exon and intron identity element). PCR-E (shown in light blue) for the mutated sequence is 0.23 in favor of exon skipping. The red vertical mark in the sequence indicates the predicted cryptic donor splice site. The blue vertical mark indicates the predicted acceptor site. (B) BDGP prediction. The red vertical mark indicates the authentic splice sites. The vertical mark indicates the predicted cryptic 5' splice sites with scores >0.90. The blue vertical mark shows the decoy splice site that was confirmed by PCR. (C) Electrophoresis of RT-PCR products derived from the proband, affected individuals, and the controls. Lane 1: DNA marker; Lanes 2, 4, 6: proband and cases; Lanes 3, 5: controls. (D) Direct sequencing of RT-PCR products. Arrow shows the heterozygous insertion of one cryptic splice site 5 bp downstream of the original splice donor site. (E) Clone sequencing of RT-PCR products. The mutant mRNA sequence (c.743+1G>A) has an ATAAG insertion (arrow) compared with the wild-type (WT). (F) Schematic representation of wild-type and aberrant mRNA transcripts. In the wild-type sequence (WT) splicing occurred at the authentic splice sites. In the mutant mRNAs, splicing occurred at the decoy splice site in intron 4 the five additional nucleotides (ATAAG) of intron4 were inserted, which generated the premature termination codon UAA.</p

Mutation analysis and identification of EXT2.

<p>(A) DNA sequences of the EXT2 gene. Arrows indicate the heterozygous G to A transition site in intron 4 of EXT2 from the proband (upper) and one affected individuals (lower). The mutation was not detected in normal family members or in the healthy controls of the same ethnic origin (control, middle). (B) Alignment of EXT2 gene sequences from 43 species. Conservative character analysis indicated that the G residue (shown in red) at the first position of intron 4 was a highly conserved splicing donor site.</p

Decay of mutant EXT2 mRNA and protein.

<p>(A) Levels of EXT2 mRNA by RT-PCR. The mRNA levels were higher in the patients than that in the controls (<i>P</i> = 0.016 in a two-sided Student's t-test). (B) Clone sequencing of wild-type and mutant transcripts. Among the 32 randomly picked clones, 27 (84.4%) were identified as wild-type by direct sequencing, while only five (15.6%) were identified as mutant transcript. (C) Western blots of wild-type and mutant proteins. The band for the predicted truncated protein could not be detected in the HME patients. (D) Comparison of EXT2 protein levels in HME patients and controls. The levels were significantly lower in the patients compared with the controls (<i>P</i> = 0.006). (E) Comparison of EXT1 mRNA expression levels in HME patients and controls. Real-time PCR revealed that EXT1 mRNA was more highly expressed in the patients compared with the controls (<i>P</i> = 0.024). (F) Comparison of EXT1 protein levels in HME patients and controls. The EXT1 levels higher in the patients compared with the controls (<i>P</i> = 0.003). (G) Comparison of HS proteoglycan expression in HME patients and controls. A group of HS proteoglycans around 70 kDa (upper box) were detected in patients and controls; while two HS proteoglycans patterns around 40 kDa and 25–30 kDa (middle and lower boxes) were detected only in the patients.</p

Primers used in mutation screening of the EXT1 and EXT2 genes.

<p>Primers used in mutation screening of the EXT1 and EXT2 genes.</p

Hereditary and clinical features of a Chinese family with HME.

<p>(A) Pedigree of the family with HME. Arrow indicates the proband. Filled circles and squares indicate affected individuals. Open circles and squares indicate normal individuals. Oblique lines indicate the deceased. (B) Postero-anterior positioned radiograph of exostoses (indicated by arrows) in some severely affected individuals. Left image shows the exostosis in juxtaepiphyseal region of right ulna. Middle image shows multiple exostotic lesions in the femur, tibia, and fibular of both lower limbs. Right image depicts excrescences in metaphyseal regions of the femur both sides proximally. (C) Histological lesions of chondrosarcoma in proband under different magnifications (hematoxylin-eosin (HE) stain). One chondrosarcoma site is shown.</p