1Department of Molecular Genetics, National Institute of Oncology, Budapest, Hungary
2Hereditary Cancers Research Group, Hungarian Academy of Sciences - Semmelweis University, Budapest, Hungary
Copyright © 2022 by the Korean Cancer Association
This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Ethical Statement
The study was approved by the Institutional Ethical Board and the Research and Ethics Committee of the Hungarian Health Science Council (ETT-TUKEB 53720-7/2019/EÜIG) and performed in accordance with the principles of the Declaration of Helsinki. After genetic counseling, written informed consents were obtained from all patients.
Author Contributions
Conceived and designed the analysis: Bozsik A, Papp J, Grolmusz VK, Patócs A, Oláh E, Butz H.
Collected the data: Bozsik A, Papp J, Grolmusz VK, Patócs A, Oláh E, Butz H.
Contributed data or analysis tools: Bozsik A, Papp J, Grolmusz VK, Patócs A, Oláh E, Butz H.
Performed the analysis: Bozsik A.
Wrote the paper: Bozsik A, Papp J, Butz H.
Conflicts of Interest
Conflict of interest relevant to this article was not reported.
Gene | HGVS name | Exon | rs ID | GnomAD frequencya) | Carrier number in our cohort | Splice predictions | |||
---|---|---|---|---|---|---|---|---|---|
ADA | RF | VarSeak | LaBranchoR, RNABP | ||||||
BRCA1 | LRG_292t1:c.4484+4dupA | Ex-14 | NA | NA | 2/3,568 | NA | NA | 5b) | NA |
BRCA1 | LRG_292t1:c.5407-10G>A | Ex-23 | rs273901767 | 4×10−6 | 2/3,568 | 0.999b) | 0.912b) | 5b) | NA |
BRCA1 | LRG_292t1:c.4358-31A>C | Ex-14 | rs764503776 | NA | 1/3,568 | NA | NA | 1 | High, 35%b) |
BRCA2 | LRG_293t1:c.8487G>T | Ex-19 | NA | NA | 4/3,568 | 0.999b) | 0.998b) | 5b) | NA |
BRCA2 | LRG_293t1:c.793G>A | Ex-09 | rs1403242422 | NA | 1/3,568 | 0.067 | 0.398 | 4b) | NA |
ADA, adaptive boosting, ensemble machine learning (scoring interval 0–1); HGVS, Human Genome Variation Society; LaBranchoR, branchpoint position site predictor; RNABP, branchpoint site predictor (probability: 0–100%); NA, not applicable; RF, random Forest, ensemble machine learning (scoring interval 0–1); VarSeak, combined score for splice prediction (scoring interval 1–5).
a) v2.1.1,
b) High scores, that are predictive for aberrant splicing.
Family No. | Variant (HGVS) | Age (at tumor onset) | Tumor type | Histology | No. of family membersa) with HBOC related cancer (see details on the pedigrees) |
---|---|---|---|---|---|
1 | LRG_292t1:c.4484+4dupA | 29 | Breast | Invasive ductal carcinoma; ER+; PR−; HER2−; Ki67: 20% | 1 |
2 | LRG_292t1:c.4484+4dupA | 44 | Ovarian | Ovarian serous papillary carcinoma, high grade; multiplex metastases | 2 |
3 | LRG_292t1:c.4358-31A>C | 49 | Breast | Ductal carcinoma in situ; ER+; PR+; HER2+; Ki67: 10% | 5 |
4 | LRG_292t1:c.5407-10G>A | 48 | Breast | Invasive ductal carcinoma; ER+; PR−; HER2−; Ki67: 30% | None |
5 | LRG_292t1:c.5407-10G>A | 37 | Breast | Invasive ductal carcinoma; ER+; PR+; HER2−; Ki67: 80% | None |
6 | LRG_293t1:c.8487G>T p.(Gln2829His) | 41 | Breast | Invasive ductal carcinoma; ER+; PR−; HER2−; Ki67: 30% | 3 |
7 | LRG_293t1:c.8487G>T p.(Gln2829His) | 36 | Breast | Invasive ductal carcinoma; ER+; PR+; HER2−; Ki67: 15% | 3 |
8 | LRG_293t1:c.8487G>T p.(Gln2829His) | 45 | Breast | Invasive lobular carcinoma; ER+; PR+; HER2−; Ki67: na | 3 |
9 | LRG_293t1:c.8487G>T p.(Gln2829His) | 50 | Breast | Invasive ductal carcinoma; ER+; PR+; HER2−; Ki67: 15% | None |
10 | LRG_293t1:c.793G>A p.(Gly265Arg) | 27 | Breast | Invasive ductal carcinoma; ER−; PR−; HER2−; Ki67: 60% | 1 |
Variant | ACMG class | ACMG criteria | BRCA exchange | ClinVar | ClinVar Review status | Reclassification | ACMG criteria with our evidence | Further supporting evidence |
---|---|---|---|---|---|---|---|---|
BRCA1: c.4484+4dupA | VUS | PM2, PP3, PP4 | No variant listed | No data | NA | LP | PM2, PP3, PP4, PS3a) | |
BRCA1: c.5407-10G>A | VUS | PM2, PP4 | Variant not interpreted | VUS | b) | LP | PM2, PP4, PS3a) | LOH in tumor, Saturation mutagenesis Findlay et al. [23] |
BRCA1: c.4358-31A>C | VUS | PM2, PP4, BP7 | Variant not interpreted | No data | NA | Strong VUS | PM2, PP4, BP7, PS3a) | |
BRCA2: c.8487G>T (p.Gln2829His) | LP | PVS1, PM2, PP3, PP4, BP1 | No variant listed | NA | NA | P | PVS1, PM2, PP3, PP4, BP1, PS3a) | LOH in tumor, Aberrant splicing was formerly detected by Houdayer et al. [24]. |
BRCA2: c.793G>A (p.Gly265Arg) | VUS | PVS1, PM2, PP4, BP1, BP4 | Not interpreted | Conflicting interpretations of pathogenicity: VUS (3) – LB (1) | b) | LB | PM2, PP4, BP1, BP4 |
PVS1: Null variant (nonsense, frameshift, canonical ±1 or 2 splice sites, initiation codon, single or multiexon deletion) in a gene where LOF is a known mechanism of disease; PM2: Variant not found in gnomAD exomes; PP3: Pathogenic computational verdict based on 1 pathogenic prediction from GERP vs. no benign predictions; PP4: Patient’s phenotype or family history is highly specific for a disease with a single genetic etiology; PS3: Well-established in vitro or in vivo functional studies supportive of a damaging effect on the gene or gene product; BP1: Missense variant in a gene for which primarily truncating variants are known to cause disease; BP4: Multiple lines of computational evidence suggest no impact on gene or gene product (conservation, evolutionary, splicing impact, etc.); BP6: Reputable source recently reports variant as benign, but the evidence is not available to the laboratory to perform an independent evaluation; BP7: A synonymous (silent) variant for which splicing prediction algorithms predict no impact on the splice consensus sequence nor the creation of a new splice site and the nucleotide is not highly conserved. ACMG, American College of Medical Genetics and Genomics; LB, likely benign; LOH, loss of heterozygosity; LP, likely pathogenic; NA, not applicable; P, pathogenic; VUS, variant of unknown significance.
a) Supporting pathogenic evidences arising from our tests,
b) Reviewed by expert panel.
Five variants selected for transcript analysis according to in silico predictions
Gene | HGVS name | Exon | rs ID | GnomAD frequency |
Carrier number in our cohort | Splice predictions | |||
---|---|---|---|---|---|---|---|---|---|
ADA | RF | VarSeak | LaBranchoR, RNABP | ||||||
BRCA1 | LRG_292t1:c.4484+4dupA | Ex-14 | NA | NA | 2/3,568 | NA | NA | 5 |
NA |
BRCA1 | LRG_292t1:c.5407-10G>A | Ex-23 | rs273901767 | 4×10−6 | 2/3,568 | 0.999 |
0.912 |
5 |
NA |
BRCA1 | LRG_292t1:c.4358-31A>C | Ex-14 | rs764503776 | NA | 1/3,568 | NA | NA | 1 | High, 35% |
BRCA2 | LRG_293t1:c.8487G>T | Ex-19 | NA | NA | 4/3,568 | 0.999 |
0.998 |
5 |
NA |
BRCA2 | LRG_293t1:c.793G>A | Ex-09 | rs1403242422 | NA | 1/3,568 | 0.067 | 0.398 | 4 |
NA |
ADA, adaptive boosting, ensemble machine learning (scoring interval 0–1); HGVS, Human Genome Variation Society; LaBranchoR, branchpoint position site predictor; RNABP, branchpoint site predictor (probability: 0–100%); NA, not applicable; RF, random Forest, ensemble machine learning (scoring interval 0–1); VarSeak, combined score for splice prediction (scoring interval 1–5).
a)v2.1.1,
b)High scores, that are predictive for aberrant splicing.
Baseline characteristics of index patients from each family
Family No. | Variant (HGVS) | Age (at tumor onset) | Tumor type | Histology | No. of family members |
---|---|---|---|---|---|
1 | LRG_292t1:c.4484+4dupA | 29 | Breast | Invasive ductal carcinoma; ER+; PR−; HER2−; Ki67: 20% | 1 |
2 | LRG_292t1:c.4484+4dupA | 44 | Ovarian | Ovarian serous papillary carcinoma, high grade; multiplex metastases | 2 |
3 | LRG_292t1:c.4358-31A>C | 49 | Breast | Ductal carcinoma in situ; ER+; PR+; HER2+; Ki67: 10% | 5 |
4 | LRG_292t1:c.5407-10G>A | 48 | Breast | Invasive ductal carcinoma; ER+; PR−; HER2−; Ki67: 30% | None |
5 | LRG_292t1:c.5407-10G>A | 37 | Breast | Invasive ductal carcinoma; ER+; PR+; HER2−; Ki67: 80% | None |
6 | LRG_293t1:c.8487G>T p.(Gln2829His) | 41 | Breast | Invasive ductal carcinoma; ER+; PR−; HER2−; Ki67: 30% | 3 |
7 | LRG_293t1:c.8487G>T p.(Gln2829His) | 36 | Breast | Invasive ductal carcinoma; ER+; PR+; HER2−; Ki67: 15% | 3 |
8 | LRG_293t1:c.8487G>T p.(Gln2829His) | 45 | Breast | Invasive lobular carcinoma; ER+; PR+; HER2−; Ki67: na | 3 |
9 | LRG_293t1:c.8487G>T p.(Gln2829His) | 50 | Breast | Invasive ductal carcinoma; ER+; PR+; HER2−; Ki67: 15% | None |
10 | LRG_293t1:c.793G>A p.(Gly265Arg) | 27 | Breast | Invasive ductal carcinoma; ER−; PR−; HER2−; Ki67: 60% | 1 |
Reference sequences: BRCA1: LRG_292t1; BRCA2: LRG_293t1. ER, estrogen receptor; HBOC, hereditary breast and ovarian cancer; HER2, human epidermal growth factor receptor 2; HGVS, Human Genome Variation Society; PR, progesterone receptor.
a)First, second, and third degree.
Variant classification
Variant | ACMG class | ACMG criteria | BRCA exchange | ClinVar | ClinVar Review status | Reclassification | ACMG criteria with our evidence | Further supporting evidence |
---|---|---|---|---|---|---|---|---|
BRCA1: c.4484+4dupA | VUS | PM2, PP3, PP4 | No variant listed | No data | NA | LP | PM2, PP3, PP4, PS3 |
|
BRCA1: c.5407-10G>A | VUS | PM2, PP4 | Variant not interpreted | VUS | LP | PM2, PP4, PS3 |
LOH in tumor, Saturation mutagenesis Findlay et al. [ | |
BRCA1: c.4358-31A>C | VUS | PM2, PP4, BP7 | Variant not interpreted | No data | NA | Strong VUS | PM2, PP4, BP7, PS3 |
|
BRCA2: c.8487G>T (p.Gln2829His) | LP | PVS1, PM2, PP3, PP4, BP1 | No variant listed | NA | NA | P | PVS1, PM2, PP3, PP4, BP1, PS3 |
LOH in tumor, Aberrant splicing was formerly detected by Houdayer et al. [ |
BRCA2: c.793G>A (p.Gly265Arg) | VUS | PVS1, PM2, PP4, BP1, BP4 | Not interpreted | Conflicting interpretations of pathogenicity: VUS (3) – LB (1) | LB | PM2, PP4, BP1, BP4 |
PVS1: Null variant (nonsense, frameshift, canonical ±1 or 2 splice sites, initiation codon, single or multiexon deletion) in a gene where LOF is a known mechanism of disease; PM2: Variant not found in gnomAD exomes; PP3: Pathogenic computational verdict based on 1 pathogenic prediction from GERP vs. no benign predictions; PP4: Patient’s phenotype or family history is highly specific for a disease with a single genetic etiology; PS3: Well-established in vitro or in vivo functional studies supportive of a damaging effect on the gene or gene product; BP1: Missense variant in a gene for which primarily truncating variants are known to cause disease; BP4: Multiple lines of computational evidence suggest no impact on gene or gene product (conservation, evolutionary, splicing impact, etc.); BP6: Reputable source recently reports variant as benign, but the evidence is not available to the laboratory to perform an independent evaluation; BP7: A synonymous (silent) variant for which splicing prediction algorithms predict no impact on the splice consensus sequence nor the creation of a new splice site and the nucleotide is not highly conserved. ACMG, American College of Medical Genetics and Genomics; LB, likely benign; LOH, loss of heterozygosity; LP, likely pathogenic; NA, not applicable; P, pathogenic; VUS, variant of unknown significance.
a)Supporting pathogenic evidences arising from our tests,
b)Reviewed by expert panel.
ADA, adaptive boosting, ensemble machine learning (scoring interval 0–1); HGVS, Human Genome Variation Society; LaBranchoR, branchpoint position site predictor; RNABP, branchpoint site predictor (probability: 0–100%); NA, not applicable; RF, random Forest, ensemble machine learning (scoring interval 0–1); VarSeak, combined score for splice prediction (scoring interval 1–5). v2.1.1, High scores, that are predictive for aberrant splicing.
Reference sequences: First, second, and third degree.
PVS1: Null variant (nonsense, frameshift, canonical ±1 or 2 splice sites, initiation codon, single or multiexon deletion) in a gene where LOF is a known mechanism of disease; PM2: Variant not found in gnomAD exomes; PP3: Pathogenic computational verdict based on 1 pathogenic prediction from GERP vs. no benign predictions; PP4: Patient’s phenotype or family history is highly specific for a disease with a single genetic etiology; PS3: Well-established Supporting pathogenic evidences arising from our tests, Reviewed by expert panel.