Prevalence of PALB2 mutations in Australasian multiple-case breast cancer families

Abstract

Introduction: Population-based studies of breast cancer have estimated that some PALB2 mutations confer a breast cancer risk (penetrance) comparable to the average pathogenic mutation in BRCA2. As this risk is of clinical relevance, we sought to identify mono-allelic PALB2 mutations and determine their frequencies in multiple-case breast cancer families attending Familial Cancer Clinics in Australia and New Zealand.

Methods: The youngest affected woman, not known to carry a mutation in BRCA1 or BRCA2, from 747 multiple-case breast cancer families participating in kConFab were selected for PALB2 mutation screening. The coding and flanking intronic regions of PALB2 in DNA extracted from blood were screened using high-resolution melt curve analysis with Sanger sequencing confirmation. Where possible, relatives of women found to carry PALB2 mutations were genotyped for the family-specific mutation, mutant transcripts were characterised and breast tumours arising in mutation carriers were recalled and reviewed. Missense mutations were assessed for potential to disrupt protein function via SIFT, Align GVGD and Polyphen-2.

Results: The mutation screen identified two nonsense mutations (PALB2 c.3113G>A in eight women and PALB2 c.196C>T in one woman), two frameshift mutations (PALB2 c.1947_1948insA and PALB2 c.2982_2983insT each in one woman), 10 missense variants, eight synonymous variants and four variants in intronic regions. Of the four PALB2 mutations identified that were predicted to produce truncated protein products, only PALB2 c.1947_1948insA had not previously been reported. PALB2 c.3113G>A and PALB2 c.196C>T were previously identified in the Australian population whereas PALB2 c.2982_2983insT was previously reported in the UK population. Transcripts derived from three of these mutant PALB2 alleles were vulnerable to nonsense-mediated decay. One missense mutation (PALB2 c.2993G>A) was predicted to disrupt protein function via the three in silico assessment methods applied. The majority of breast cancers arising in carriers that were available for review were high-grade invasive ductal carcinomas.

Conclusions: About 1.5% (95% CI 0.6to 2.4) of Australasian multiple-case breast cancer families attending clinics are segregating protein-truncating mutations in PALB2, most being PALB2 c.3113G>A, p.Trp1038*. Given the prevalence, breast cancer risk, and tumour grade associated with this mutation, consideration of clinical PALB2 testing is warranted.

Figure 1

Characterisation of PALB2 mutation transcripts via RT-PCR. (a) Image of transcripts from PALB2 c.3113G>A visualised on 3% agarose gel. (A) Heteroduplex of the RT-PCR products of the wild-type and the PALB2 r.2997_3113del transcripts. (B) Heteroduplex of the RT-PCR products of the wild-type and PALB2 r.3083_3113del transcripts. (C) RT-PCR product of the wild-type transcript. (D) RT-PCR product of the PALB2 r.3083_3113del transcript. (E) RT-PCR product of the PALB2 r.2997_3113del transcript. FSIs of six wild-type and their corresponding six variant nucleotides at heterozygous positions were recorded for each condition (CT and NCT). Averaged proportions of FSI of the six variant nucleotides to the FSI of the six corresponding wild-type nucleotides for each condition are shown. (b) Image of wild-type transcripts and transcripts from PALB2 c.196C>T visualised on 4% agarose gel. FSIs were recorded for the variant and corresponding wild-type nucleotide at the heterozygous position. (c) Image of transcripts resulting from PALB2 c.1947_1948insA visualised on 2% agarose gel. FSIs were recorded for three variant nucleotides and their corresponding wild-type nucleotides at the heterozygous positions. (d) Image of transcripts from PALB2 c.2982_2983insT visualised on 2% agarose gel. FSIs were recorded for three variant nucleotides and their corresponding wild-type nucleotides at the heterozygous positions. NCT-CT: the difference in FSI proportions in the CT and NCT conditions indicates the semi-quantitative change in relative gene expression levels of the mutant transcripts in each condition. CT, cycloheximide treated; FSI, fluorescence signal intensity; NCT, non-cycloheximide treated; NT, nucleotide; WT, wild-type.

Figure 2

PALB2 protein structure, binding sites of protein partners and transcript alterations due to PALB2 mutations. Schematic diagram of the PALB2 protein showing its predicted functional domains, binding sites of its protein partners and the mutant transcripts resulting from PALB2 mutations identified in this study.