Homologue-specific chromosome sequencing characterizes translocation junctions and permits allelic assignment

Area of Science:

• Genomics
• Cytogenetics
• Molecular Biology

Background:

• Cytogenetic analysis detects chromosome translocations but struggles with sequence-level breakpoint characterization.
• Flow cytometry enables chromosome sorting, isolating abnormal chromosomes and generating chromosome-specific DNA for detailed analysis.

Purpose of the Study:

• To characterize the sequence-level breakpoints of a derivative chromosome t(9;14) translocation.
• To identify fusion genes resulting from chromosomal rearrangements.
• To evaluate allele-specific chromosome sequencing for comprehensive genomic analysis.

Main Methods:

• Flow cytometry was used to sort derivative chromosome t(9;14) and homologous normal chromosomes 9 and 14.
• Chromosome sequencing was performed on sorted homologue-specific samples.
• Amplicon sequencing targeted genes near the fusion breakpoint to assess variant frequencies and allelic copy number.

Main Results:

• The study identified breakpoint junctions at 9p24.3 and 14q13.1 in the derivative chromosome.
• A novel fusion gene, WASH1-NPAS3, was discovered at the translocation breakpoint.
• Variant frequencies correlated with allelic copy number, validating the sequencing approach.

Conclusions:

• Sequencing of sorted chromosomes allows precise assignment of allelic variants and characterization of abnormal chromosomes.
• Allele-specific chromosome sequencing of homologues is a robust method for distinguishing alleles.
• This technique provides an efficient approach for comprehensive analysis of complex genomic changes, including translocations and fusion gene formation.