DEVELOPMENT OF A METHODOLOGY FOR ANALYSIS OF HERITABLE REARRANGEMENTS: A CASE STUDY AT THE HUMAN CYP2D6 LOCUS

Authors

• B Alleva
• D Dahiya
• F Pratto
• RD Camerini-Otero

Abstract

In human meiosis, DNA damage in the form of double strand breaks is purposefully generated and are critical for successful cellular division. These DNA breaks cluster in regions of the genome called hotspots. However, certain regions of the genome may be susceptible to improper repair of meiotic breaks which can lead to genetic rearrangements. One major question that has been relatively unstudied in the field is whether these hotspots predispose offspring to heritable genetic rearrangements and disorders. To address this question, we surveyed two lists of genes, “rearrangement-prone” genes and pharmacogenes looking for overlaps with our mapped hotspots. In both surveys, CYP2D6 - a very important pharmacogene known to metabolize ~25% of all clinical drugs, was a top hit. CYP2D6 is highly variable, residing next to two pseudogenes with >90% homology, making genotyping difficult. Therefore, we used high-throughput techniques to bypass these hurdles and uncover previously difficult to detect genetic variation at the CYP2D6 locus. We devised two methodologies to analyze rearrangements in 144 unrelated individuals and 57 parent-offspring trios. First, a long-range, overlapping PCR assay and second, a CRISPR-Cas9 enrichment protocol utilizing long-read sequencing as well as a downstream pipeline to assemble and genotype the CYP2D6 locus. We uncovered 9 different rearrangements including a novel rearrangement. Analysis of rearrangement breakpoints revealed that some rearrangements, including gene fusions, coincided with hotspots suggesting a previously unexamined origin of genetic variation at CYP2D6. Lastly, we discovered a familial trio in which the maternally inherited CYP2D6 had mutational variation likely of meiotic origin.

Scientific Focus Area: Genetics and Genomics

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