3C-PCR: a novel proximity ligation-based approach to phase chromosomal rearrangement breakpoints with distal allelic variants.
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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. |
Year of Publication | 2018
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Journal | Hum Genet
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Volume | 137
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Issue | 1
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Pages | 55-62
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Date Published | 2018 Jan
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ISSN | 1432-1203
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DOI | 10.1007/s00439-017-1853-0
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PubMed ID | 29196799
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PubMed Central ID | PMC5790632
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Grant list | 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
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