3C-PCR: a novel proximity ligation-based approach to phase chromosomal rearrangement breakpoints with distal allelic variants.
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.
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F31HD090780-01 / Eunice Kennedy Shriver National Institute of Child Health and Human Development
DGE1144152 / National Science Foundation
F31 HD090780 / HD / NICHD NIH HHS / United States
P30 CA006516 / CA / NCI NIH HHS / United States
GM061354 / National Institute of General Medical Sciences
P01 GM061354 / GM / NIGMS NIH HHS / United States