Dataset Information

3C-PCR: a novel proximity ligation-based approach to phase chromosomal rearrangement breakpoints with distal allelic variants.

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.

SUBMITTER: Schilit SLP

PROVIDER: S-EPMC5790632 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

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3C-PCR: a novel proximity ligation-based approach to phase chromosomal rearrangement breakpoints with distal allelic variants.

Schilit Samantha L P SLP Morton Cynthia C CC

Human genetics 20171201 1

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 ar ...[more]

PMID: 29196799

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Project description:BACKGROUND: Genomes undergo large structural changes that alter their organisation. The chromosomal regions affected by these rearrangements are called breakpoints, while those which have not been rearranged are called synteny blocks. We developed a method to precisely delimit rearrangement breakpoints on a genome by comparison with the genome of a related species. Contrary to current methods which search for synteny blocks and simply return what remains in the genome as breakpoints, we propose to go further and to investigate the breakpoints themselves in order to refine them. RESULTS: Given some reliable and non overlapping synteny blocks, the core of the method consists in refining the regions that are not contained in them. By aligning each breakpoint sequence against its specific orthologous sequences in the other species, we can look for weak similarities inside the breakpoint, thus extending the synteny blocks and narrowing the breakpoints. The identification of the narrowed breakpoints relies on a segmentation algorithm and is statistically assessed. Since this method requires as input synteny blocks with some properties which, though they appear natural, are not verified by current methods for detecting such blocks, we further give a formal definition and provide an algorithm to compute them. The whole method is applied to delimit breakpoints on the human genome when compared to the mouse and dog genomes. Among the 355 human-mouse and 240 human-dog breakpoints, 168 and 146 respectively span less than 50 Kb. We compared the resulting breakpoints with some publicly available ones and show that we achieve a better resolution. Furthermore, we suggest that breakpoints are rarely reduced to a point, and instead consist in often large regions that can be distinguished from the sequences around in terms of segmental duplications, similarity with related species, and transposable elements. CONCLUSION: Our method leads to smaller breakpoints than already published ones and allows for a better description of their internal structure. In the majority of cases, our refined regions of breakpoint exhibit specific biological properties (no similarity, presence of segmental duplications and of transposable elements). We hope that this new result may provide some insight into the mechanism and evolutionary properties of chromosomal rearrangements.

Dataset Information

3C-PCR: a novel proximity ligation-based approach to phase chromosomal rearrangement breakpoints with distal allelic variants.

Publications

3C-PCR: a novel proximity ligation-based approach to phase chromosomal rearrangement breakpoints with distal allelic variants.

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