17-43106453-T-C

Position:

• chr17-43106453-T-C

Variant summary

Our verdict is Pathogenic. Variant got 16 ACMG points: 16P and 0B. PS3 PP4_Strong PVS1

This summary comes from the ClinGen Evidence Repository: The c.212+3A>G variant is an intronic variant occurring in intron 4 of the BRCA1 gene. This variant is present in gnomAD v2.1 (exomes only, non-cancer subset) or gnomAD v3.1 (non-cancer subset) but is below the ENIGMA BRCA1/2 VCEP threshold >0.00002 for BS1_Supporting (PM2_Supporting, BS1, and BA1 are not met). This variant is reported to result in aberrant mRNA splicing. A combination of RT-PCR from patient samples and mini-gene assays demonstrated that the variant impacts splicing by skipping of 22nt from the 3' end of the exon, reported by five studies (PMIDs: 22505045, 11802209, 20215541, 21673748, 24667779). Two of these studies also reported skipping of exon 4 (PMIDs: 20215541, 24667779). Allele-specific expression was reported by one assay on patient-derived material (PMID:22505045), which showed no WT transcript from the variant allele. This is in agreement with two minigene assays (PMIDs: 21673748, 24667779). One study reported no impact on splicing but no results are shown to assess whether the 22nt skipping event (that is seen in controls at low level) was increased (PMID:16619214), and therefore, has been excluded from consideration when assessing the splicing impact of this variant. Appropriate code strength determined by comparison of results to PVS1 decision tree (PVS1 (RNA) met). Reported by one calibrated study to exhibit protein function similar to pathogenic control variants (PMID:30209399) (PS3 met). Multifactorial likelihood ratio analysis using clinically calibrated data produced a combined LR for this variant of 1615.48 (based on Co-occurrence LR=1.298; Family History LR=1245.06), above the threshold for Very strong evidence towards pathogenicity (LR >350) (PP4_Very strong met; PMID:17924331, 31853058).In summary, this variant meets the criteria to be classified as a Pathogenic variant for BRCA1-related cancer predisposition based on the ACMG/AMP criteria applied as specified by the ENIGMA BRCA1/2 VCEP (PVS1 (RNA), PS3, PP4_Very strong). LINK:https://erepo.genome.network/evrepo/ui/classification/CA001412/MONDO:0011450/092

• Frequency:
  • Genomes: 𝑓 0.0000066 (0 hom., cov: 32)
  • Exomes 𝑓: 7.0e-7 (0 hom.)
• Consequence: BRCA1 NM_007294.4 splice_donor_region, intron
• Scores: Splicing: ADA: 0.9986
• Clinical Significance: Pathogenic reviewed by expert panel P:14 U:1 O:2
• Conservation: PhyloP100: 1.29

Genes affected

BRCA1 (HGNC:1100): (BRCA1 DNA repair associated) This gene encodes a 190 kD nuclear phosphoprotein that plays a role in maintaining genomic stability, and it also acts as a tumor suppressor. The BRCA1 gene contains 22 exons spanning about 110 kb of DNA. The encoded protein combines with other tumor suppressors, DNA damage sensors, and signal transducers to form a large multi-subunit protein complex known as the BRCA1-associated genome surveillance complex (BASC). This gene product associates with RNA polymerase II, and through the C-terminal domain, also interacts with histone deacetylase complexes. This protein thus plays a role in transcription, DNA repair of double-stranded breaks, and recombination. Mutations in this gene are responsible for approximately 40% of inherited breast cancers and more than 80% of inherited breast and ovarian cancers. Alternative splicing plays a role in modulating the subcellular localization and physiological function of this gene. Many alternatively spliced transcript variants, some of which are disease-associated mutations, have been described for this gene, but the full-length natures of only some of these variants has been described. A related pseudogene, which is also located on chromosome 17, has been identified. [provided by RefSeq, May 2020]

ACMG classification

Verdict is Pathogenic. Variant got 16 ACMG points.

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	MANE	UniProt
BRCA1	NM_007294.4	c.212+3A>G		splice_donor_region_variant, intron_variant		ENST00000357654.9

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	TSL	MANE	Appris	UniProt
BRCA1	ENST00000357654.9	c.212+3A>G		splice_donor_region_variant, intron_variant		1	NM_007294.4	P4

Frequencies

GnomAD3 genomes AF: 0.00000657 AC: 1 AN: 152220 Hom.: 0 Cov.: 32

Gnomad AFR AF: 0.00

Gnomad AMI AF: 0.00

Gnomad AMR AF: 0.00

Gnomad ASJ AF: 0.00

Gnomad EAS AF: 0.00

Gnomad SAS AF: 0.00

Gnomad FIN AF: 0.00

Gnomad MID AF: 0.00

Gnomad NFE AF: 0.0000147

Gnomad OTH AF: 0.00

GnomAD4 exome AF: 7.03e-7 AC: 1 AN: 1422054 Hom.: 0 Cov.: 27 AF XY: 0.00000141 AC XY: 1 AN XY: 709150

Gnomad4 AFR exome AF: 0.00

Gnomad4 AMR exome AF: 0.00

Gnomad4 ASJ exome AF: 0.00

Gnomad4 EAS exome AF: 0.00

Gnomad4 SAS exome AF: 0.00

Gnomad4 FIN exome AF: 0.00

Gnomad4 NFE exome AF: 9.27e-7

Gnomad4 OTH exome AF: 0.00

GnomAD4 genome AF: 0.00000657 AC: 1 AN: 152220 Hom.: 0 Cov.: 32 AF XY: 0.0000134 AC XY: 1 AN XY: 74358

Gnomad4 AFR AF: 0.00

Gnomad4 AMR AF: 0.00

Gnomad4 ASJ AF: 0.00

Gnomad4 EAS AF: 0.00

Gnomad4 SAS AF: 0.00

Gnomad4 FIN AF: 0.00

Gnomad4 NFE AF: 0.0000147

Gnomad4 OTH AF: 0.00

ClinVar

Significance: Pathogenic

Submissions summary: Pathogenic:14 Uncertain:1 Other:2

Revision: reviewed by expert panel

Submissions by phenotype

Breast-ovarian cancer, familial, susceptibility to, 1 Pathogenic:4 Other:2

Pathogenic, criteria provided, single submitter	clinical testing	Baylor Genetics	Jan 24, 2023	- -
not provided, no classification provided	clinical testing	Breast Cancer Information Core (BIC) (BRCA1)	-	- -
Pathogenic, criteria provided, single submitter	clinical testing	All of Us Research Program, National Institutes of Health	Jan 08, 2024	This variant causes an A to G nucleotide substitution at the +3 position of intron 4 of the BRCA1 gene. Several RNA studies have reported that this variant results in the skipping of exon 4 and/or a partial deletion of exon 4, which are predicted to cause an in-frame deletion in the functionally important RING domain and frameshift, respectively (PMID: 12037674, 21673748, 22505045, 29021971). A functional study has reported that this variant impacts BRCA1 function in a haploid cell proliferation assay (PMID: 30209399). This variant has been reported in multiple individuals and families affected with breast and/or ovarian cancer (PMID: 9150151, 10090482, 15026808, 23199084, 28294317, 29487695). This variant also has been reported as a Belgian founder mutation that was also observed in other populations (PMID: 10595255, 23199084, 29021971). This variant has been identified in 1/31400 chromosomes in the general population by the Genome Aggregation Database (gnomAD). Loss of BRCA1 function is a known mechanism of disease (clinicalgenome.org). Based on the available evidence, this variant is classified as Pathogenic. -
Pathogenic, no assertion criteria provided	clinical testing	Sharing Clinical Reports Project (SCRP)	Feb 17, 2010	- -
not provided, no classification provided	in vitro	Brotman Baty Institute, University of Washington	-	- -
Pathogenic, criteria provided, single submitter	clinical testing	Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA), c/o University of Cambridge	Oct 02, 2015	- -

Hereditary breast ovarian cancer syndrome Pathogenic:3

Pathogenic, criteria provided, single submitter	clinical testing	Laboratory for Molecular Medicine, Mass General Brigham Personalized Medicine	Apr 02, 2020	The c.212+3A>G variant in BRCA1 has been reported in >15 families with hereditary breast and ovarian cancer and is a founder variant in the Belgian population (Claes 1999, Claes 2004, Bhaskaran 2019, Li 2018, Li 2019, Peelen 1997, Breast Information Core Database). This variant was classified as Pathogenic on August 10, 2015 by the ClinGen-approved ENIGMA expert panel (Variation ID 54467). It has also been identified in 0.006% (1/5430) of European chromosomes by gnomAD (http://gnomad.broadinstitute.org). In vitro mini-gene assay and RT-PCR on RNA from blood samples or lymphoblastoid cell lines showed increased in-frame skipping of exon 5 and out-of-frame skipping of the last 22 nucleotides of exon 5. Loss of function of the BRCA1 gene is an established disease mechanism in autosomal dominant hereditary breast and ovarian breast cancer. In summary, this variant meets criteria to be classified as pathogenic for autosomal dominant hereditary breast and ovarian cancer. ACMG/AMP Criteria applied: PS4, PM2, PS3_Moderate, PVS1_Moderate. -
Likely pathogenic, no assertion criteria provided	research	Research Molecular Genetics Laboratory, Women's College Hospital, University of Toronto	Dec 17, 2015	- -
Pathogenic, criteria provided, single submitter	clinical testing	Labcorp Genetics (formerly Invitae), Labcorp	Aug 19, 2023	This sequence change falls in intron 4 of the BRCA1 gene. It does not directly change the encoded amino acid sequence of the BRCA1 protein. RNA analysis indicates that this variant induces altered splicing and may result in an absent or disrupted protein product. This variant is present in population databases (rs80358083, gnomAD 0.007%). This variant has been observed in individual(s) with breast and ovarian cancer (PMID: 9150151, 10090482, 10595255, 15026808, 16619214, 28294317). It has also been observed to segregate with disease in related individuals. For these reasons, this variant has been classified as Pathogenic. Variants that disrupt the consensus splice site are a relatively common cause of aberrant splicing (PMID: 17576681, 9536098). Studies have shown that this variant alters mRNA splicing and is expected to lead to the loss of protein expression (PMID: 21673748, 24667779). Based on a multifactorial likelihood algorithm using genetic, in silico, and/or statistical data, this variant has been determined to have a high probability of being pathogenic (PMID: 21990134). ClinVar contains an entry for this variant (Variation ID: 54467). This variant is also known as IVS5+3A>G. -

not provided Pathogenic:2

Pathogenic, no assertion criteria provided	clinical testing	Joint Genome Diagnostic Labs from Nijmegen and Maastricht, Radboudumc and MUMC+	-	- -
Pathogenic, no assertion criteria provided	clinical testing	Diagnostic Laboratory, Department of Genetics, University Medical Center Groningen	-	- -

not specified Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

ARUP Laboratories, Molecular Genetics and Genomics, ARUP Laboratories

Nov 04, 2016

- -

BRCA1-related cancer predisposition Pathogenic:1

Pathogenic, reviewed by expert panel

curation

ClinGen ENIGMA BRCA1 and BRCA2 Variant Curation Expert Panel, ClinGen

Jun 11, 2024

The c.212+3A>G variant is an intronic variant occurring in intron 4 of the BRCA1 gene. This variant is present in gnomAD v2.1 (exomes only, non-cancer subset) or gnomAD v3.1 (non-cancer subset) but is below the ENIGMA BRCA1/2 VCEP threshold >0.00002 for BS1_Supporting (PM2_Supporting, BS1, and BA1 are not met). This variant is reported to result in aberrant mRNA splicing. A combination of RT-PCR from patient samples and mini-gene assays demonstrated that the variant impacts splicing by skipping of 22nt from the 3' end of the exon, reported by five studies (PMIDs: 22505045, 11802209, 20215541, 21673748, 24667779). Two of these studies also reported skipping of exon 4 (PMIDs: 20215541, 24667779). Allele-specific expression was reported by one assay on patient-derived material (PMID: 22505045), which showed no WT transcript from the variant allele. This is in agreement with two minigene assays (PMIDs: 21673748, 24667779). One study reported no impact on splicing but no results are shown to assess whether the 22nt skipping event (that is seen in controls at low level) was increased (PMID: 16619214), and therefore, has been excluded from consideration when assessing the splicing impact of this variant. Appropriate code strength determined by comparison of results to PVS1 decision tree (PVS1 (RNA) met). Reported by one calibrated study to exhibit protein function similar to pathogenic control variants (PMID: 30209399) (PS3 met). Multifactorial likelihood ratio analysis using clinically calibrated data produced a combined LR for this variant of 1615.48 (based on Co-occurrence LR=1.298; Family History LR=1245.06), above the threshold for Very strong evidence towards pathogenicity (LR >350) (PP4_Very strong met; PMID: 17924331, 31853058). In summary, this variant meets the criteria to be classified as a Pathogenic variant for BRCA1-related cancer predisposition based on the ACMG/AMP criteria applied as specified by the ENIGMA BRCA1/2 VCEP (PVS1 (RNA), PS3, PP4_Very strong). -

Malignant tumor of breast Pathogenic:1

Pathogenic, no assertion criteria provided

clinical testing

Department of Pathology and Laboratory Medicine, Sinai Health System

-

The BRCA1 c.212+3A>G variant was identified in 2 of 1942 proband chromosomes (frequency: 0.001) from individuals or families with breast or ovarian cancer (Chen 2006, Kwong 2018). The variant was also identified in dbSNP (ID: rs80358083) as ""With Pathogenic allele"", ClinVar (classified as pathogenic by five submitters; as likely pathogenic by one submitter and uncertain significance by one submitter) and in LOVD 3.0 (99X as pathogenic). The variant was identified in control databases in 1 of 30976 chromosomes at a frequency of 0.00003 (Genome Aggregation Database Feb 27, 2017). The variant was observed in the European population in 1 of 15010 chromosomes (freq:0.00007), while the variant was not observed in the Other, Latino, European, Ashkenazi Jewish, Finnish, South Asian, African or East Asian populations. The c.212+3A>G variant is located in the 5' splice region but does not affect the invariant +1 and +2 positions. However, positions +3 to +6 are part of the splicing consensus sequence and variants involving these positions sometimes affect splicing. In addition, 3 of 4 in silico or computational prediction software programs (SpliceSiteFinder, MaxEntScan, NNSPLICE, GeneSplicer) predict a greater than 10% difference in splicing. Multiple splicing assay studies using RT-PCR analysis on RNA identify the variant causing in-frame skipping of exon 5 and out-of-frame skipping of the last 22 nucleotides of exon 5 (Houdayer 2012, Steffensen 2014, Thery 2011). In summary, based on the above information, this variant meets our laboratory’s criteria to be classified as pathogenic. -

Familial cancer of breast Pathogenic:1

Pathogenic, no assertion criteria provided

literature only

Center for Precision Medicine, Meizhou People's Hospital

-

- -

Hereditary cancer-predisposing syndrome Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

Ambry Genetics

Jan 24, 2022

The c.212+3A>G intronic pathogenic mutation (also known as IVS5+3A>G) results from an A to G substitution 3 nucleotides after coding exon 3 in the BRCA1 gene. This alteration has been detected in multiple breast and/or ovarian cancer families and is a known Belgian founder mutation, though it has also been reported in German, Dutch, and French families to date (Peelen T. Am. J. Hum. Genet. 1997 May; 60(5):1041-9; Claes K. Br. J. Cancer 2004 Mar; 90(6):1244-51; Claes K. Dis. Markers 1999 Oct; 15(1-3):69-73; Janaviius R. EPMA J 2010 Sep; 1(3):397-412). Although one study utilizing RT-PCR on patient RNA found no effect on normal splicing (Chen X. Hum. Mutat. 2006 May; 27(5):427-35), many other RNA assays show that this alteration results in the use of a cryptic donor site 22 nucleotides upstream from the native site, leading to a frameshift and premature stop codon (Ambry internal data; Meindl A et al. Int J Cancer, 2002 Feb;97:472-80; Houdayer C. Hum. Mutat. 2012 Aug; 33(8):1228-38; Théry JC. Eur. J. Hum. Genet. 2011 Oct; 19(10):1052-8; Steffensen AY. Eur. J. Hum. Genet. 2014 Dec; 22(12):1362-8; Sanz DJ. Clin. Cancer Res. 2010 Mar; 16(6):1957-67). One functional study found that this nucleotide substitution is non-functional in a high-throughput, genome editing, haploid cell survival assay (Findlay GM et al. Nature, 2018 10;562:217-222). Many other alterations impacting the same donor (c.211A>G, c.212G>A, c.212+1G>T, c.212+1G>A, c.212+2T>C) have been shown to have a similar and complete impact on splicing (Vega A et al. Hum Mutat, 2001 Jun;17:520-1; Caleca L et al. PLoS One, 2014 Feb;9:e86924; Colombo M et al. PLoS One, 2013 Feb;8:e57173; Friedman LS et al. Am J Hum Genet, 1995 Dec;57:1284-97; Meindl A et al. Int J Cancer, 2002 Feb;97:472-80; Houdayer C. Hum. Mutat. 2012 Aug; 33(8):1228-38; Sanz DJ. Clin. Cancer Res. 2010 Mar; 16(6):1957-67). Based on the supporting evidence, this alteration is interpreted as a disease-causing mutation. -

Breast and/or ovarian cancer Uncertain:1

Uncertain significance, no assertion criteria provided

clinical testing

Foulkes Cancer Genetics LDI, Lady Davis Institute for Medical Research

Apr 09, 2015

- -

Computational scores

Source: dbNSFP v4.3

Name	Calibrated prediction	Score	Prediction
BayesDel_noAF	Benign	-0.41
CADD	Benign	14
DANN	Benign	0.92
RBP_binding_hub_radar		0.0
RBP_regulation_power_radar		2.9

Splicing

Name	Calibrated prediction	Score	Prediction
dbscSNV1_ADA	Pathogenic	1.0
dbscSNV1_RF	Pathogenic	0.90
SpliceAI score (max)		0.63		Details are displayed if max score is > 0.2
DS_DL_spliceai		0.63		Position offset: 3

Find out detailed SpliceAI scores and Pangolin per-transcript scores at spliceailookup.broadinstitute.org

Publications

LitVar

Other links and lift over

dbSNP: rs80358083; hg19: chr17-41258470;