The EWS/TEC gene fusion generated by the t(9;22) chromosomal translocation found in extraskeletal myxoid chondrosarcomas encodes a fusion protein containing the amino-terminal domain of the EWS protein fused to the whole coding sequence of the orphan nuclear receptor TEC. We have compared the DNA-binding and transcriptional activation properties of various TEC isoforms and the corresponding EWS/TEC fusion proteins. Band-shift experiments show that the fulllength TEC receptor can e ciently bind the NGFI-B Response Element (NBRE), whereas an isoform lacking the entire carboxyl-terminal domain of the receptor binds much less e ciently the NBRE. Addition of the aminoterminal domain of EWS to either isoforms does not alter signi®cantly their DNA-binding properties to the NBRE. Co-transfection experiments of COS cells and human chondrocytes indicate that whereas TEC moderately activates transcription from a NBRE-containing promoter, the corresponding EWS/TEC fusion protein is a highly potent transcriptional activator of the same promoter, being approximately 270-fold more active than the native receptor. EWS/TEC may thus exert its oncogenic potential in chrondrosarcomas by activating the transcription of target genes involved in cell proliferation.

Quality control is a complex issue for clinical molecular diagnostic applications. In the case of genotyping assays, artifacts such as allele dropout represent a risk of misdiagnosis for amplification-based methods. However, its frequency of occurrence in PCR-based diagnostic assays remains unknown. To maximize the likelihood of detecting allele dropout, our clinical genotyping PCR-based assays are designed with two independent assays for each allele (nonoverlapping primers on each DNA strand). To estimate the incidence of allelic dropout, we took advantage of the capacity of our clinical assays to detect such events. We retrospectively studied their occurrence in the initial PCR assay for 30,769 patient reports for mutations involved in four diseases produced over 8 years. Ninety-three allele dropout events were detected and all were solved before reporting. In addition, 42 cases of artifacts caused by amplification of an allele ultimately confirmed to not be part of the genotype (drop-in events) were detected and solved. These artifacts affected 1:227 genotypes, 94% of which were due to nonreproducible PCR failures rather than sequence variants interfering with the assay, suggesting that careful primer design cannot prevent most of these errors. This provides a quantitative estimate for clinical laboratories to take this phenomenon into account in quality management and to favor assay designs that can detect (and minimize) occurrence of these artifacts in routine clinical use.

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To study the instability of FMR1 triplet repeats in the general population, we screened a prospective sample of 24,449 anonymized mother-offspring pairs and analyzed transmissions of intermediate-size (45-54 triplets) and premutation-size (55-200 triplets) alleles. We screened all mothers for alleles > or = 45 triplets by Southern blot and studied transmission of 545 maternal alleles to their offspring using polymerase chain reaction. Out of 21,411 maternal samples with conclusive results, we identified 250 carriers of at least one intermediate-size allele and 39 carrying a premutation-size allele. Out of a subsample of 430 transmissions of normal-size alleles (< 45 triplets), we observed four (< 1%) unstable transmissions. There were 6/90 intermediate-size unstable alleles (7%) and 11/25 unstable premutation-size alleles (44%). Two mothers transmitted a typical full mutation. The incidence of fragile X syndrome was thus 1/12,225 newborns (upper limit of 95% confidence interval: 1/4638 newborns), but larger in males (1/6209) than females (none detected in over 12,000 newborn females). Intermediate-size alleles were more unstable than normal-size alleles (p = 0.0027), but more stable (about sixfold) than premutation-size alleles (p < 0.0001). Unstable premutation-size alleles harbored the major fragile X haplotype (T50-T42-T62), and this haplotype appeared to be a good predictor of instability in premutations (p = 0.02). Incidence and instability are important to determine the feasibility and cost effectiveness of putative FMR1 screening programs. Carriers of FMR1 alleles of 55+ triplets with no family history of the disease may have a significant risk of expansion to a full mutation in a single generation.