Evidence Repository - Clinical Genome Resources

The ClinGen Evidence Repository is an FDA-recognized human genetic variant database containing expert-curated assertions regarding variants' pathogenicity and supporting evidence summaries. [Disclaimer]

Variant: NM_024675.4(PALB2):c.2014G>C (p.Glu672Gln)

Curation Version - 1.0
Curation History
JSON LD for Version 1.0

CA151230

126630 (ClinVar)

Gene: PALB2

Condition: hereditary breast cancer

Inheritance Mode: Autosomal dominant inheritance

Link to MONDO

UUID: 033fe989-5072-4c44-957b-9bd48b02178f

Approved on: 2023-04-05

Published on: 2023-04-07

HGVS expressions

NM_024675.4:c.2014G>C

NM_024675.4(PALB2):c.2014G>C (p.Glu672Gln)

NC_000016.10:g.23630140C>G

CM000678.2:g.23630140C>G

NC_000016.9:g.23641461C>G

CM000678.1:g.23641461C>G

NC_000016.8:g.23548962C>G

NG_007406.1:g.16218G>C

ENST00000261584.9:c.2014G>C

ENST00000261584.8:c.2014G>C

ENST00000565038.1:n.87-865G>C

ENST00000568219.5:c.1129G>C

NM_024675.3:c.2014G>C

Benign

BA1 BP1

PM2 BS3 BS1 BP4 PP3

Evidence Links 3

Expert Panel

Hereditary Breast, Ovarian and Pancreatic Cancer VCEP

Criteria Specification Information

Criteria Specification: ClinGen Hereditary Breast, Ovarian and Pancreatic Cancer Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for PALB2 Version 1.0.0

Criteria Specification Approval History

Criteria Specifications for this VCEP

Evidence submitted by expert panel

Hereditary Breast, Ovarian and Pancreatic Cancer VCEP

The c.2014G>C variant in PALB2 is a missense variant predicted to cause a substitution of a glutamic acid by glutamine at amino acid 672 (p.Glu672Gln). This variant has a gnomAD v2.1.1 filtering allele frequency of 0.02846 in non-Finnish European population, which is higher than the HBOP VCEP threshold (>0.001) for BA1, and therefore meets this criterion. This variant is functional in multiple protein assays (PMID: 31636395, PMID: 31757951, PMID: 33964450); however, due to a lack of known positive missense controls with known clinical impact, these assays do not meet the requirements for use by the HBOP VCEP. PALB2, in which the variant was identified, is defined by the HBOP VCEP as a gene for which primarily truncating variants are known to cause disease. In summary, this variant meets the criteria to be classified as benign for autosomal dominant hereditary breast and pancreatic cancer and autosomal recessive FANCN based on the ACMG/AMP criteria applied as specified by the HBOP VCEP. (BA1, BP1)

BA1

The highest population filtering allele frequency in gnomAD v2.1.1 is 0.02846 in non-Finnish European population, which is higher than the ClinGen Hereditary Breast, Ovarian, and Pancreatic Cancer VCEP threshold (>0.001) for BA1, and therefore meets this criterion (BA1).

BP1

PALB2, in which the variant was identified, is defined by the HBOP VCEP as a gene for which primarily truncating variants are known to cause disease (BP1)

PM2

No code specific comments provided, please refer to the summary above or general recommendations provided in the guideline

BS3

Homology-directed repair (HDR) protein assays in mouse mammary tumor cell line B400 (Palb2-/-; Trp53-/-), mouse embryonic stem cell line 129/Ola E14 IB10 (Palb2-/-; Trp53-/), and HEK293T cells showed no significant change in HDR (4.4 fold change in HDR compared to 5.0 fold change in HDR for wild type PALB2 in B400 cells, 83.25% HDR efficiency compared to wild type PALB2 in 129/Ola E14 IB10 cells, 1.1 normalized HR/NHEJ ratio in HEK293T cells) and a PARP inhibition assay in 129/Ola E14 IB10 (Palb2-/-; Trp53-/) cells showed no significant change in cell survival (103.53% survival compared to wild type PALB2) indicating that this variant does not impact protein function (PMID: 31636395, PMID: 31757951, PMID: 33964450); however, due to a lack of positive missense controls with known clinical impact, these assays do not meet the requirements for use by the HBOP VCEP.

Assay: HEK293T cells containing a stably integrated HDR reporter construct (mutant GFP containing ISceI endonuclease target site, T2A linker, out-of-frame mCherry) and depleted of PALB2 by siRNA transfection were transfected with an ISceI endonuclease plasmid (to generate a double stranded DNA break), a GFPdonor plasmid (containing a truncated GFP cDNA capable of correcting the mutated GFP in the reporter construct), and a FLAG-tagged wild type or variant PALB2 cDNA plasmid. HDR is assessed by measuring the ratio of GFP+:mCherry+ cells (representing HR+ events that repaired the mutated GFP and NHEJ+ events which altered the reading frame to restore mCherry, respectively) Material: HEK293T cells containing a stably integrated HDR reporter construct (mutant GFP containing ISceI endonuclease target site, T2A linker, out-of-frame mCherry) Readout: Quantitative; Ratio of GFP+:mCherry+ cells (representing HR+ events and NHRJ+ events, respectively) normalized to wild type PALB2 Controls: wild type PALB2 cDNA, empty vector, 3 LP/P controls, 9 LB/B controls Threshold for normal readout: >0.848 HR/NHEJ ratio Threshold for abnormal readout: <0.689 HR/NHEJ ratio Assay result: approximately 1.1 LMM normalized mean HR/NHEJ Assay result assertion: Normal PubMed:33964450

Assay: In vitro expression of HA-tagged full-length cDNA constructs in cell line, homology directed repair (HDR) fluorescent reporter assay to assess functional capacity of variants. Material: Mouse mammary tumor cell line B400 (Palb2-/-; Trp53-/-) with stably integrated DR-GFP reporter (GFP gene interrupted by I-SceI site, with adjacent native GFP gene). Readout: Quantitative; HDR (measured by GFP+ cells) fold change, normalized & rescaled relative to 1:5 ratio of p.Y551X pathogenic variant control and wild-type PALB2 control, displayed with SEM of independent replicates. Controls: wild type cDNA, 7 LP/P controls, 4 LB/B controls Threshold for normal readout: >2.4 Threshold for abnormal readout: ≤1.7 (34% activity); ≤2.4 (48% activity) for hypomorphic Assay result: 4.4 Assay result assertion: Normal Standard error mean: 0.39 PubMed:31636395

Assay: PALB2 variants introduced by RMCE system (FlpO expression vector + PALB2-RMCE exchange vector) in Palb2KO/Tp53KO mES cells, ~500 neomycin-resistant clones pooled & expanded, then 1x106 cells transfected with I-SceI & mCherry co-expression cell line, HDR assessed by %GFP+ cells among the mCherry+ cells by FACS. Material: Mouse embryonic stem (mES) cell line 129/Ola E14 IB10 (Palb2-/-; Trp53-/-) with stably integrated DR-GFP reporter & RMCE (recombination mediated cassette exchange) system; PALB2 variants made by site mutagenesis of cDNA constructs in RMCE exchange vector. Readout: Quantitative; Relative HR efficiency (mean % of WT GFP+ cells among mCherry+ cells) +/- SEM for 2 replicates. Controls: wild type cDNA, empty vector, 12 LP/P controls, 9 LB/B controls Threshold for normal readout: B/LB ctrls are ≥79.52% of WT Threshold for abnormal readout: <40% of WT HR efficency (HR >60% reduced) Assay result: 83.25% Assay result assertion: Normal Standard deviation: 5.27 Standard error mean: 3.73 PubMed:31757951

BS1

No code specific comments provided, please refer to the summary above or general recommendations provided in the guideline

BP4

The computational splicing predictor SpliceAI gives scores of AL: 0.00/DL: 0.00/AG: 0.00/DG: 0.00, suggesting that the variant has no impact on splicing. However, BP4 was not met since this variant has a putative protein effect.

PP3

No code specific comments provided, please refer to the summary above or general recommendations provided in the guideline

Curation History

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