Abstract
Recent advances in molecular cytogenetics highlight the importance of noncoding structural variation in human disease. Genomic rearrangements can disrupt chromatin architecture, leading to long-range alterations in gene expression. With increasing ability to assess distal gene dysregulation comes new challenges in clinical interpretation of rearrangements. While haplotyping methods to determine compound heterozygosity in a single gene with two pathogenic variants are established, such methods are insufficient for phasing larger distances between a pathogenic variant and a genomic rearrangement breakpoint. Herein, we present an inexpensive and efficient proximity ligation-based method called 3C-PCR for phasing chromosomal rearrangement breakpoints with distal allelic variants. 3C-PCR uses canonical chromosome conformation capture (3C) libraries for targeted distal phasing by implementing a novel nested PCR strategy with primers anchored across the rearrangement breakpoints and subsequent Sanger sequencing. As a proof of concept, 3C-PCR was used to phase a highly variable region 1.3 Mb upstream of a chromosomal rearrangement breakpoint in a balanced translocation. We found that the nested PCR approach amplified the derivative chromosome substrate exclusively and identified the same haplotype by Sanger sequencing reliably. Given its efficacy and versatility, 3C-PCR is ideal for use in phasing chromosomal rearrangement breakpoints with allelic variants located at a genomic distance over a megabase.