Characterization of complex chromosomal rearrangements by targeted capture and next-generation sequencing

Abstract

Translocations are a common class of chromosomal aberrations and can cause disease by physically disrupting genes or altering their regulatory environment. Some translocations, apparently balanced at the microscopic level, include deletions, duplications, insertions, or inversions at the molecular level. Traditionally, chromosomal rearrangements have been investigated with a conventional banded karyotype followed by arduous positional cloning projects. More recently, molecular cytogenetic approaches using fluorescence in situ hybridization (FISH), array comparative genomic hybridization (aCGH), or whole-genome SNP genotyping together with molecular methods such as inverse PCR and quantitative PCR have allowed more precise evaluation of the breakpoints. These methods suffer, however, from being experimentally intensive and time-consuming and of less than single base pair resolution. Here we describe targeted breakpoint capture followed by next-generation sequencing (TBCS) as a new approach to the general problem of determining the precise structural characterization of translocation breakpoints and related chromosomal aberrations. We tested this approach in three patients with complex chromosomal translocations: The first had craniofacial abnormalities and an apparently balanced t(2;3)(p15;q12) translocation; the second has cleidocranial dysplasia (OMIM 119600) associated with a t(2;6)(q22;p12.3) translocation and a breakpoint in RUNX2 on chromosome 6p; and the third has acampomelic campomelic dysplasia (OMIM 114290) associated with a t(5;17)(q23.2;q24) translocation, with a breakpoint upstream of SOX9 on chromosome 17q. Preliminary studies indicated complex rearrangements in patients 1 and 3 with a total of 10 predicted breakpoints in the three patients. By using TBCS, we quickly and precisely defined eight of the 10 breakpoints.

Unbalanced translocations arise from diverse mutational mechanisms including chromothripsis

“…Palindromic AT-rich repeats on Chromosomes 3, 8, 11, 17, and 22 also generate recurrent translocations, the most common of which is the recurrent t(11;22) that causes Emanuel syndrome (Edelmann et al 2001;Kurahashi et al 2003;Gotter et al 2007;Kato et al 2012Kato et al , 2014. Most germline translocations, however, are not recurrent, and sequencing of translocation breakpoints has revealed features of nonhomologous end-joining (NHEJ) and microhomology-mediated break-induced replication (MMBIR) at more than 60 unique translocation junctions (Chen et al 2008;Higgins et al 2008;Sobreira et al 2011;Chiang et al 2012;Robberecht et al 2013). …”

Reproductive outcome of a case with familial balanced translocation t(3;6): implications for genetic counseling

“…The carrier status of balanced translocation is associated with recurrent miscarriage. An apparently balanced translocation may produce a clinical phenotype by gene disruption or altered expression of genes in or around the breakpoint region (Sobreira et al, 2011). Three hypotheses have been proposed to explain such phenotype abnormalities, including a break in a gene, positional effect, and cryptic deletion or duplication (Callier et al, 2007).…”

A Complex Chromosome Rearrangement Involving Four Chromosomes, Nine Breakpoints and a Cryptic 0.6-Mb Deletion in a Boy with Cerebellar Hypoplasia and Defects in Skull Ossification

“…Very few papers have reported the sequencing of complex rearrangement breakpoints [Obenauf et al, 2010;Talkowski et al, 2011]. Recently, Sobreira et al [2011] used targeted breakpoint capture followed by next-generation sequencing in 3 patients with complex chromosomal translocations and defined 8 out of 10 breakpoints, which helped in understanding their mechanism of formation. Chromosomal rearrangements may occur via several mechanisms, which typically reflect the underlying architecture of the genome in the regions surrounding the breakpoints [Gu et al, 2008].…”