Characterizing PALB2 intragenic duplication breakpoints in a triple-negative breast cancer case using long-read sequencing

Abstract

Introduction: Accurate identification and characterization of Large Genomic Rearrangements (LGR), especially duplications, are crucial for precise diagnosis and risk assessment. In this report, we characterized an intragenic duplication breakpoint of PALB2 to determine its pathogenicity significance.

Methods: A 52-year-old female with triple-negative breast cancer was diagnosed with a novel PALB2 LGR. An efficient and accurate methodology was applied, combining long-read sequencing and transcript analysis for the rapid characterization of the duplication.

Results: Duplication of exons 5 and 6 of PALB2 was validated by transcript analysis. Long-read sequencing enabled the localization of breakpoints within Alu elements, providing insights into the mechanism of duplication via non-allelic homologous recombination.

Conclusion: Using our combined methodology, we reclassified the PALB2 duplication as a pathogenic variant. This reclassification suggests a possible causative link between this specific genetic alteration and the aggressive phenotype of the patient.

Keywords: LGR; PALB2; breast cancer; long-read sequencing; molecular alteration.

Figure 1

Comprehensive Analysis and Characterization of Tandem Duplications in PALB2. (A) MLPA assay of PALB2. Each dot represents the amplification ratio of a specific probe pair, reflecting the relative amount of the targeted DNA segment compared to three reference samples. Error bars indicate the standard deviation of the measured ratio. The ratio is calculated for each probe and indicates whether there is a deletion (ratio <1), duplication (ratio >1), or no change (ratio ≈1). (B) Sanger sequencing electropherogram of the proband’s cDNA, with black arrows marking the primer positions. The orange shallow points the targeted region. F, forward; R, reverse.

Figure 2

Identification and characterization of the Tandem Duplication Breakpoint in PALB2. (A) IGV visualization of Nanopore Long-Read Sequencing. Purple rectangle depict the genomic length of the insertion. The colored bars, in shades of red and blue, represent sequencing reads that span PALB2 exons 4 to 7. (B) Density estimation plot of insertion length detected by long read sequencing. The discontinuous red line shows the exact insertion length determined by Sanger sequencing. (C) Sanger sequencing electropherogram of the genomic breakpoint region. Black arrows mark the primer positions. The orange shallow points the targeted region. F, forward; R, reverse; I, intron; E, exon.