Atiq, Mazen A.

CYP2D6 duplication has important pharmacogenomic implications. Reflex testing with long-range PCR (LR-PCR) can resolve the genotype when a duplication and alleles with differing activity scores are detected. We evaluated whether visual inspection of plots from real-time-PCR-based targeted genotyping with copy number variation (CNV) detection could reliably determine the duplicated CYP2D6 allele. Six reviewers evaluated QuantStudio OpenArray CYP2D6 genotyping results and the TaqMan Genotyper plots for seventy-three well-characterized cases with three copies of CYP2D6 and two different alleles. Reviewers blinded to the final genotype visually assessed the plots to determine the duplicated allele or opt for reflex sequencing. Reviewers achieved 100% accuracy for cases with three CYP2D6 copies that they opted to report. Reviewers did not request reflex sequencing in 49–67 (67–92%) cases (and correctly identified the duplicated allele in each case); all remaining cases (6–24) were marked by at least one reviewer for reflex sequencing. In most cases with three copies of CYP2D6, the duplicated allele can be determined using a combination of targeted genotyping using real-time PCR with CNV detection without need for reflex sequencing. In ambiguous cases and those with >3 copies, LR-PCR and Sanger sequencing may still be necessary for determination of the duplicated allele.

AbstractPrimary aneurysmal bone cyst (ABC) is a benign multiloculated cystic lesion of bone that is defined cytogenetically by USP6 gene rearrangements. Rearrangements involving USP6 are promoter swaps, usually generated by fusion of the noncoding upstream exons of different partner genes with exon 1 or 2 of USP6, thus leading to transcriptional upregulation of full‐length USP6 coding sequence. Testing for USP6 rearrangements is used diagnostically to distinguish it from secondary ABC and other giant cell‐rich primary bone tumors. In this report, we present a case of a 16‐year‐old male with a primary ABC of the left distal femur. USP6 break apart fluorescence in situ hybridization was positive for a rearrangement and conventional chromosome analysis identified a reciprocal X;17 translocation. In order to identify the putative USP6 fusion partner, we performed RNA sequencing and uncovered a novel USP9X‐USP6 promoter swap fusion. This result was confirmed by reverse transcription‐polymerase chain reaction (RT‐PCR) and by mate pair sequencing thus showing the utility of these alternative methodologies in identifying novel fusion candidates. Ubiquitin‐specific protease 9X (USP9X), like USP6, encodes a highly conserved substrate‐specific deubiquitylating enzyme. USP9X is highly expressed in a number of tissue types and acts as both an oncogene and tumor suppressor in several human cancers. We conclude that oncogenic activation of USP6 via USP9X promoter exchange represents a novel driver of primary ABC formation.