Abstract
BCOR is an epigenetic regulator altered by various mechanisms including BCOR-internal tandem duplication (BCOR-ITD) in a wide range of cancers. Six different BCOR-ITD in the 3’-part of the coding sequence of exon 15 have been reported ranging from 89 to 114 bp in length. BCOR-ITD is a common genetic alteration found in clear cell sarcoma of the kidney and primitive myxoid mesenchymal tumor of infancy (PMMTI) and it characterizes a new type of central nervous system tumor: “CNS tumor with BCOR-ITD”. It can also be detected in undifferentiated round cell sarcoma (URCS) and in high-grade endometrial stromal sarcoma (HGESS). Therefore, it is of utmost importance to search for this genetic alteration in these cancers with the most frequent technique being RNA-sequencing. Here, we developed a new droplet PCR assay (dPCR) to detect the six sequences characterizing BCOR-ITD. To achieve this goal, we used a single colored probe to detect both the duplicated region and the normal sequence that acts as a reference. We first generated seven synthetic DNA sequences: ITD0 (the normal sequence) and ITD1 to ITD6 (the duplicated sequences described in the literature) and then we set up the optima dPCR conditions. We validated our assay on 19 samples from a representative panel of human tumors (9 HGNET-BCOR, 5 URCS, 3 HGESS, and 2 PMMTI) in which BCOR-ITD status was known using at least one other method including RNA sequencing, RT-PCR or DNA-methylation profiling for CNS tumors. Our results showed that our technique was 100% sensitive and specific. DPCR detected BCOR-ITD in 13/19 of the cases; in the remaining 6 cases additional RNA-sequencing revealed BCOR gene fusions. To conclude, in the era of histomolecular classification of human tumors, our modified dPCR assay is of particular interest to detect BCOR-ITD since it is a robust and less expensive test that can be applied to a broad spectrum of cancers that share this alteration.
Highlights
BCL-6 transcriptional corepressor (BCOR) gene is located at Xp11.4 and comprises 16 exons encoding a ubiquitously expressed transcriptional repressor [1, 2]
Figure 2A1 shows that the signal height of the positive droplet cluster is strictly different from the negative droplet cluster as seen for the negative PCR control (NTC)
The positive control (C+) has three droplet clusters that differ according to their fluorescence levels, with the lowest signal (1,000< >3,000 AU) for negative droplets, a “normal sequence” cluster (3,000< >5,000 AU) of the same intensity level as ITD0p and a third cluster of higher intensity (>5,000 AU) corresponding to a characteristic “BCOR-ITD” signal
Summary
BCL-6 transcriptional corepressor (BCOR) gene is located at Xp11.4 and comprises 16 exons encoding a ubiquitously expressed transcriptional repressor [1, 2]. The principal isoform, encoded by 14 exons, gives rise to a protein of 1,755 amino acids. BCOR protein contains two main functional binding domains. The BCL-6 binding domain allows binding to the POZ domain of BCL-6 and increases its function as a repressor of transcription [2]. The other domain is the polycomb-group RING finger homolog (PCGF) ubiquitin-like fold discriminator (PUFD), a domain binding to some of the PCGF proteins forming repressive complexes involved in epigenetic histone modification. BCOR is part of one of the six currently described non-canonical variants of the polycomb repressive complex 1, the PRC1.1 [3]
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