Identification and Characterization of an Exonic Duplication in PALB2 in a Man with Synchronous Breast and Prostate Cancer

Abstract

PALB2 (partner and localizer of BRCA2), as indicated by its name, is a BRCA2-interacting protein that plays an important role in homologous recombination (HR) and DNA double-strand break (DSB) repair. While pathogenic variants of PALB2 have been well proven to confer an increased risk of breast cancer, data on its involvement in prostate cancer (PrC) have not been clearly demonstrated. We investigated, using targeted next generation sequencing (NGS), a 59-year-old Caucasian man who developed synchronous breast and prostate cancers. This genetic investigation allowed to identify an intragenic germline heterozygous duplication in PALB2, implicating intronic repetitive sequences spanning exon 11. This variant was confirmed by multiplex ligation probe amplification (MLPA), and genomic breakpoints have been identified and characterized at the nucleotide level (c.3114-811_3202-1756dup) using an approach based on walking PCR, long range PCR, and Sanger sequencing. RT-PCR using mRNA extracted from lymphocytes and followed by Sanger sequencing revealed a tandem duplication r.3114_3201dup; p.(Gly1068Glufs * 14). This duplication results in the synthesis of a truncated, and most-likely, non-functional protein. These findings expand the phenotypic spectrum of PALB2 variants and may improve the yield of genetic diagnoses in this field.

Keywords: PALB2; breast cancer; exon duplication; prostate cancer.

Figure 1

PALB2 Exon 11 duplication testing and breakpoint identification. (A) Schematic representation of the PALB2 normal and mutant alleles, with the duplicated region. The location and orientation of the primers used for specific duplication PCRs are indicated by vertical arrows. (B) PALB2 Exon 11 duplication confirmation by MLPA. (C) Long-range PCR. A specific band of approximately 2.5 kb was detected in the proband’s blood DNA, absent in control samples. (D) Electropherogram showing the breakpoint sequence (forward). Tandem duplication site (TDS) is boxed in orange.

Figure 2

An Alu-mediated duplication mechanism appears to be responsible for exon 11 duplication. (A) Sequence analysis of the LR-PCR product allowed us to identify the duplication conjunctions that are located between c.3114-811 and 3202-1756. The location and orientation of the primers used for specific-duplication PCRs are indicated by vertical arrows. (B) Sequence alignment of the two Alu elements involved in this PALB2 rearrangement showing that these two elements are highly homologous.

Figure 3

PALB2 Exon 11 duplication disrupts normal splicing and leads to frameshift. (A) Agarose gel electrophoresis of the RT-PCR performed with mRNA obtained from the patient and non-carrier controls. An extra band was observed in the patient of about 474 bp, while only the wild-type band was observed in controls (388 bp). (B) Sanger sequencing of the two alleles of RT-PCR product of the sample from proband: the insertion leads to a new stop codon as indicated by the red box. (C) Schematic representation of the two transcripts observed in the proband sample.