Mapping translocation breakpoints by next-generation sequencing

Abstract

Balanced chromosome rearrangements (BCRs) can cause genetic diseases by disrupting or inactivating specific genes, and the characterization of breakpoints in disease-associated BCRs has been instrumental in the molecular elucidation of a wide variety of genetic disorders. However, mapping chromosome breakpoints using traditional methods, such as in situ hybridization with fluorescent dye-labeled bacterial artificial chromosome clones (BAC-FISH), is rather laborious and time-consuming. In addition, the resolution of BAC-FISH is often insufficient to unequivocally identify the disrupted gene. To overcome these limitations, we have performed shotgun sequencing of flow-sorted derivative chromosomes using "next-generation" (Illumina/Solexa) multiplex sequencing-by-synthesis technology. As shown here for three different disease-associated BCRs, the coverage attained by this platform is sufficient to bridge the breakpoints by PCR amplification, and this procedure allows the determination of their exact nucleotide positions within a few weeks. Its implementation will greatly facilitate large-scale breakpoint mapping and gene finding in patients with disease-associated balanced translocations.

Figure 1.

Ideograms of the derivative chromosomes from the patients. (A) Patient 1, (B) patient 2, (C) patient 3. (Arrows) Breakpoints.

Figure 2.

Solexa sequencing profile of derivative chromosome 9 from patient 1. 1-Mb intervals around the breakpoints on chromosome 7 (A) and 9 (B) are shown. 199,421 and 1,047,649 reads derived from the der (9) were mapped to unique positions on normal chromosomes 7 and 9, respectively. The number of reads was then binned into nonoverlapping 1-kb segments and plotted against the chromosome coordinates. (Arrows) Breakpoints.

Figure 3.

Junction fragment sequences in patients 1 (A), 2 (B), and 3 (C). Normal reference sequences are labeled in italic and normal characters, respectively. Deleted sequences are underlined. (Black box) The 9-bp insertion on the der (12) of patient 3. (Chr) Chromosome, (Der) derivative chromosome.

Figure 4.

PCR products of the two junction fragments from patient 3. (M) Size marker, (C) genomic DNA from a normal individual, (P) genomic DNA from patient 3, (der) derivative chromosome.

Figure 5.

Chromosome breakpoints (arrows) and disrupted genes. (A) Patient 1: Breakpoint on chromosome 7 maps between the 3′ ends of NOM1 and MNX1 (formerly known as HLXB9); breakpoint on chromosome 9 disrupts the 5′ end of a splicing isoform (accession no. AL136545) of TRPM3. (B) Patient 2: Breakpoint on chromosome 4 disrupts EPHA5; no known genes are in the vicinity of the breakpoint on chromosome 5. (C) Patient 3: Breakpoint on chromosome 12 disrupts MAGT4C; no known genes are close to the breakpoint on chromosome 2.