chr17-43095924-T-G

Position:

• chr17-43095924-T-G

Variant summary

Our verdict is Likely benign. Variant got -6 ACMG points: 2P and 8B. PVS1_Moderate BP6_Very_Strong

The NM_007294.4(BRCA1):​c.594-2A>C variant causes a splice acceptor change. The variant allele was found at a frequency of 0.0000584 in 1,609,948 control chromosomes in the GnomAD database, with no homozygous occurrence. 3/3 splice prediction tools predicting alterations to normal splicing. Variant has been reported in ClinVar as Benign (★★★).

• Frequency:
  • Genomes: 𝑓 0.000026 (0 hom., cov: 31)
  • Exomes 𝑓: 0.000062 (0 hom.)
• Consequence: BRCA1 NM_007294.4 splice_acceptor
• Scores: Splicing: ADA: 1.000
• Clinical Significance: Benign reviewed by expert panel P:3 U:8 B:7 O:1
• Conservation: PhyloP100: 3.72

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 Likely_benign. Variant got -6 ACMG points.

• PVS1: Splicing +-2 bp (donor or acceptor) variant, product NOT destroyed by NMD, known LOF gene, truncates exone, which is 0.013590844 fraction of the gene. Cryptic splice site detected, with MaxEntScore 6.7, offset of -21, new splice context is: taactagtgtttcttattAGgac. Cryptic site results in inframe change. If cryptic site found is not functional and variant results in exon loss, it results in frameshift change.
• BP6: Variant 17-43095924-T-G is Benign according to our data. Variant chr17-43095924-T-G is described in ClinVar as [Benign]. Clinvar id is 37686.Status of the report is reviewed_by_expert_panel, 3 stars. Variant chr17-43095924-T-G is described in Lovd as [Benign]. Variant chr17-43095924-T-G is described in Lovd as [Pathogenic]. Variant chr17-43095924-T-G is described in Lovd as [Likely_benign].

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	MANE	Protein	UniProt
BRCA1	NM_007294.4	c.594-2A>C		splice_acceptor_variant		ENST00000357654.9	NP_009225.1

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	TSL	MANE	Protein	Appris	UniProt
BRCA1	ENST00000357654.9	c.594-2A>C		splice_acceptor_variant		1	NM_007294.4	ENSP00000350283	P4

Frequencies

GnomAD3 genomes AF: 0.0000263 AC: 4 AN: 152180 Hom.: 0 Cov.: 31

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: 0.0000588

Gnomad OTH AF: 0.00

GnomAD3 exomes AF: 0.0000360 AC: 9 AN: 249876 Hom.: 0 AF XY: 0.0000296 AC XY: 4 AN XY: 135042

Gnomad AFR exome AF: 0.00

Gnomad AMR exome AF: 0.00

Gnomad ASJ exome AF: 0.00

Gnomad EAS exome AF: 0.00

Gnomad SAS exome AF: 0.00

Gnomad FIN exome AF: 0.00

Gnomad NFE exome AF: 0.0000797

Gnomad OTH exome AF: 0.00

GnomAD4 exome AF: 0.0000617 AC: 90 AN: 1457768 Hom.: 0 Cov.: 30 AF XY: 0.0000565 AC XY: 41 AN XY: 725360

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: 0.0000803

Gnomad4 OTH exome AF: 0.0000166

GnomAD4 genome AF: 0.0000263 AC: 4 AN: 152180 Hom.: 0 Cov.: 31 AF XY: 0.0000135 AC XY: 1 AN XY: 74342

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: 0.0000588

Gnomad4 OTH AF: 0.00

Alfa AF: 0.0000553 Hom.: 0

Bravo AF: 0.0000227

TwinsUK AF: 0.000270 AC: 1

ALSPAC AF: 0.000259 AC: 1

ESP6500AA AF: 0.00 AC: 0

ESP6500EA AF: 0.000116 AC: 1

ExAC AF: 0.0000165 AC: 2

ClinVar

Significance: Benign

Submissions summary: Pathogenic:3 Uncertain:8 Benign:7 Other:1

Revision: reviewed by expert panel

Submissions by phenotype

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

Benign, reviewed by expert panel	curation	Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA)	Jun 29, 2017	In our experience this variant always co-occurs in cis with c.641A>G. The haplotype of c.[594-2A>C; 641A>G] has been shown to be not pathogenic from comprehensive clinical studies (PMID:27008870). We recommend that if c.594-2A>C is detected in an individual, presence of c.641A>G should be verified. If co-occurrence is confirmed, the combination of the two variants is classified as not pathogenic/benign (see SCV000282252.1). If c.594-2A>G is detected alone, assume clinical significance uncertain in the absence of further evidence. -
Pathogenic, no assertion criteria provided	clinical testing	Breast Cancer Information Core (BIC) (BRCA1)	May 29, 2002	- -
not provided, no classification provided	clinical testing	Sharing Clinical Reports Project (SCRP)	Mar 15, 2010	- -
Pathogenic, no assertion criteria provided	literature only	Pathway Genomics	Jul 24, 2014	- -

not specified Uncertain:2 Benign:2

Uncertain significance, no assertion criteria provided	research	Research Molecular Genetics Laboratory, Women's College Hospital, University of Toronto	Jan 31, 2014	- -
Uncertain significance, criteria provided, single submitter	clinical testing	Cancer Genetics and Genomics Laboratory, British Columbia Cancer Agency	Apr 18, 2017	- -
Likely benign, criteria provided, single submitter	clinical testing	GeneDx	Jan 02, 2018	This variant is considered likely benign or benign based on one or more of the following criteria: it is a conservative change, it occurs at a poorly conserved position in the protein, it is predicted to be benign by multiple in silico algorithms, and/or has population frequency not consistent with disease. -
Likely benign, criteria provided, single submitter	clinical testing	Women's Health and Genetics/Laboratory Corporation of America, LabCorp	Nov 22, 2023	Variant summary: BRCA1 c.594-2A>C is located in a canonical splice-site and is predicted to affect mRNA splicing resulting in a significantly altered protein due to either exon skipping, shortening, or inclusion of intronic material. Several computational tools predict a significant impact on normal splicing: five predict the variant abolishes a 3 prime acceptor site; one predicts the variant strengthens a cryptic 3 prime acceptor site. In vitro functional studies have shown that the variant in isolation leads to the increased retention of 21 intronic nucleotides immediately upstream exon 10, resulting in a transcript with an in-frame insertion (r.594-21_594-1ins), which is a naturally occurring minor transcript (de la Hoya 2016). However, the variant very frequently, if not always, is found in complex with c.641A>G (located in exon 10), and the two together were shown to cause an out-of-frame exon 10 skipping (likely resulting in NMD), probably driven by the c.641 variant. Notably, though the double mutant allele c.[594-2A>C; 641A>G] did not produce detectable levels of full-length transcripts, it produced normal levels of the delta 9,10 transcripts (a which is a naturally occurring alternative transcript, with an in-frame deletion of exon 9 and 10), predicted to encode a BRCA1 protein with sufficient tumor suppression function to compensate for the lack of full length transcripts (de la Hoya 2016). The variant allele was found at a frequency of 5.1e-05 in 294540 control chromosomes. This frequency is not significantly higher than expected for a pathogenic variant in BRCA1 causing Hereditary Breast And Ovarian Cancer Syndrome (5.1e-05 vs 0.001), allowing no conclusion about variant significance. The c.594-2A>C variant has been reported in the literature in multiple individuals affected with Hereditary Breast and Ovarian Cancer, in most cases as a complex allele in cis with c.641A>G. However, the variant has also been found to co-occur with other pathogenic BRCA mutations in several cases (Rosenthal 2015, Wong-Brown 2016), leaving the pathogenicity of the variant questionable. In addition, a multifactorial analysis considering largescale case-control, segregation and breast cancer pathology information for the complex allele c.[594-2A>C; 641A>G] predicted the complex variant to be not pathogenic (de la Hoya 2016). The following publications have been ascertained in the context of this evaluation (PMID: 22711857, 20104584, 24569164, 19892845, 21702907, 16912212, 22811390, 29254167, 25366421, 25639900, 10408690, 24667779, 16211554, 23239986, 26884819, 27008870). 12 submitters have cited clinical-significance assessments for this variant to ClinVar after 2014. Multiple submitters reported the variant with conflicting assessments (pathogenic n=1, benign/likely benign n=6, VUS n=5), including one expert panel classified it as benign. Based on the evidence outlined above, the variant was classified as likely benign. -

Hereditary cancer-predisposing syndrome Uncertain:2 Benign:1

Likely benign, criteria provided, single submitter	clinical testing	Color Diagnostics, LLC DBA Color Health	Jul 27, 2016	- -
Uncertain significance, criteria provided, single submitter	clinical testing	Ambry Genetics	Apr 10, 2024	The c.594-2A>C intronic variant results from an A to C substitution two nucleotides upstream from coding exon 8 in the BRCA1 gene. This nucleotide position is highly conserved in available vertebrate species. In silico splice site analysis predicts that this alteration will weaken the native splice acceptor site. This alteration is frequently observed as a haplotype BRCA1 c.[594-2A>C; 641A>G] which is classified as benign (de la Hoya M et al Hum Mol Genet. 2016; Mar). The same study evaluated the splice effect of the haplotype and of each alteration independently in a minigene assay. The independent BRCA1 c.594-2A>G variant produced an in-frame transcript that retains 21 nucleotides of intron 7, r.594-21_594-1ins which was also detected at a low level in the control samples (de la Hoya M et al Hum Mol Genet. 2016; Mar). This isoform is also known as Δ10p in the literature. Alterations that disrupt the canonical splice site are expected to cause aberrant splicing, resulting in an abnormal protein or a transcript that is subject to nonsense-mediated mRNA decay. However, as this alteration results in an in-frame insertion that is also present in controls, and since supporting evidence is limited at this time, the clinical significance of this alteration remains unclear. -
Uncertain significance, criteria provided, single submitter	curation	Sema4, Sema4	Jan 14, 2022	- -

Hereditary breast ovarian cancer syndrome Uncertain:1 Benign:2

Likely benign, criteria provided, single submitter	research	CSER _CC_NCGL, University of Washington	May 18, 2016	- -
Uncertain significance, criteria provided, single submitter	clinical testing	Labcorp Genetics (formerly Invitae), Labcorp	Jan 21, 2024	This sequence change affects an acceptor splice site in intron 8 of the BRCA1 gene. Loss-of-function variants in BRCA1 are expected to be pathogenic (PMID: 20104584). However, an in-frame BRCA1 isoform lacking exons 8 and 9 (also known as exons 9 and 10) is highly expressed in blood from unaffected individuals and in normal breast tissue; this isoform may retain protein function and could functionally rescue loss-of-function variants within exons 8-9 (PMID: 24569164). This variant is present in population databases (rs80358033, gnomAD 0.007%). Disruption of this splice site has been observed on the opposite chromosome (in trans) from a pathogenic variant in BRCA1 in at least one individual (PMID: 25639900; Invitae), which suggests that this variant may not be disease-causing. This variant is generally observed on the same chromosome as a second BRCA1 variant, c.641A>G. Comprehensive clinical validation studies have shown that this BRCA1 haplotype c.[594-2A>C; 641A>G] is not disease causing (PMID: 27008870, 25639900). ClinVar contains an entry for this variant (Variation ID: 37686). Experimental studies have shown that this variant is associated with the activation of an in-frame cryptic acceptor splice site 21 nucleotides upstream of the natural splice site in intron 8 (i21d), exon 8 skipping, exon 9 skipping and exons 8-9 skipping (PMID: 27008870, 26913838, 24212087, Invitae). These alternative splicing events are also observed in controls. In summary, the available evidence is currently insufficient to determine the role of this variant in disease. Therefore, it has been classified as a Variant of Uncertain Significance. -
Likely benign, criteria provided, single submitter	clinical testing	University of Washington Department of Laboratory Medicine, University of Washington	Jul 11, 2017	- -

not provided Uncertain:1 Benign:1

Uncertain significance, criteria provided, single submitter	clinical testing	ARUP Laboratories, Molecular Genetics and Genomics, ARUP Laboratories	May 11, 2023	- -
Likely benign, criteria provided, single submitter	clinical testing	Quest Diagnostics Nichols Institute San Juan Capistrano	Jun 20, 2023	- -

Hereditary breast ovarian cancer syndrome;C2676676:Breast-ovarian cancer, familial, susceptibility to, 1 Pathogenic:1

Pathogenic, no assertion criteria provided

research

King Laboratory, University of Washington

Sep 01, 2019

- -

Breast and/or ovarian cancer Uncertain:1

Uncertain significance, criteria provided, single submitter

clinical testing

CHEO Genetics Diagnostic Laboratory, Children's Hospital of Eastern Ontario

May 06, 2020

- -

Malignant tumor of breast Uncertain:1

Uncertain significance, no assertion criteria provided

clinical testing

Department of Pathology and Laboratory Medicine, Sinai Health System

-

The BRCA1 c.594-2A>C variant was identified in the literature in functional, in silico analysis, segregation studies and reviews (Steffensen 2014, Tesoriero 2005, Hoya 2016, Rosenthal 2015). The variant was also identified in dbSNP (ID: rs80358033) as “With Pathogenic, untested allele”; NHLBI GO Exome Sequencing Project in 1 of 8600 European American alleles; Exome Aggregation Consortium database (August 8, 2016) in 2 of 109096 chromosomes (freq. 0.00001833) in the following populations: European (Non-Finnish) in 2 of 59486 chromosomes (freq. 0.00003362), but was not seen in African, East Asian, European (Finnish), Latino, South Asian and Other populations. ClinVar and Clinvitae databases (uncertain significance by Invitae, Ambry Genetics, GeneDx, Genetics Diagnostic Laboratory-CHEO; as likely benign by CSER CC NCGI University of Washington; as pathogenic by University of Washington, Dept of Medicine, Pathway Genetics, BIC and Sharing Clinical Report Projects of Myriad did not provide a classification );Fanconi Anemia Mutation Database-LOVD (3X neutral, and LOVD-3.0 9X); ARUP Laboratories BRCA Mutations Database (5- definitely pathogenic.); GeneInsight COGR database (pathogenic by MESHWCRI and NYG, as unknown significance by CHEO and CVHTHP); the BIC database (15X with clinical importance). The c.594-2A>C variant is predicted to cause abnormal splicing because the nucleotide substitution occurs in the invariant region of the splice consensus sequence. In addition, 5 of 5 in silico or computational prediction software programs (SpliceSiteFinder, MaxEntScan, NNSPLICE, GeneSplicer, HumanSpliceFinder) predict a greater than 10% difference in splicing. A study by Hoya (Hoya 2016) used large-scale genetic and clinical resources from the ENIGMA, CIMBA and BCAC consortia to assess pathogenicity of c.594-2A >C. A recent analysis using family history weighting and co-observation classification modeling indicated that BRCA1 c.594- 2A > C (IVS9-2A > C), previously described to cause exon 10 skipping (a truncating alteration), displays characteristics inconsistent with those of a high risk pathogenic BRCA1 variant. Data indicate that c.594-2A > C is always in cis with c.641A > G. The spliceogenic effect of c.[594-2A > C;641A > G] was characterized using RNA analysis of human samples and splicing minigenes. As expected, c.[594-2A > C; 641A > G] caused exon 10 skipping, albeit not due to c.594-2A > C impairing the acceptor site but rather by c.641A > G modifying exon 10 splicing regulatory element(s). Multiple blood-based RNA assays indicated that the variant allele did not produce detectable levels of full-length transcripts, with a per allele BRCA1 expression profile composed of 70–80% truncating transcripts, and 20–30% of in-frame D 9,10 transcripts predicted to encode a BRCA1 protein with tumor suppression function. The study confirms that BRCA1 c.[594-2A > C;641A > G] should not be considered a high-risk pathogenic variant. Importantly, results from our detailed mRNA analysis suggest that BRCA-associated cancer risk is likely not markedly increased for individuals who carry a truncating variant in BRCA1 exons 9 or 10, or any other BRCA1 allele that permits 20–30% of tumor suppressor function. More generally, the findings highlight the importance of assessing naturally occurring alternative splicing for clinical evaluation of variants in disease-causing genes. A review of the variant by Rosenthal (Rosenthal 2015) detailed the following: The expected classification of BRCA1 c.594-2A >C as pathogenic is based on the predicted impact on mRNA splicing at the intron 9/exon 10 boundary. Splicing analysis algorithms predict that this change disrupts normal mRNA splicing because the consensus splice acceptor sequence at this position is highly conserved. Biochemical studies confirm skipping of exon 10 in transcripts from the BRCA1 c.594-2A >C allele, resulting in the loss of 77 nucleotides and premature truncation o -

Computational scores

Source: dbNSFP v4.3

Name	Calibrated prediction	Score	Prediction
BayesDel_addAF	Uncertain	0.15	D
BayesDel_noAF	Pathogenic	0.16
CADD	Pathogenic	31
DANN	Uncertain	0.98
Eigen	Pathogenic	0.91
Eigen_PC	Pathogenic	0.72
FATHMM_MKL	Uncertain	0.89	D
MutationTaster	Benign	1.0	D;D;D;D;D;D;D;D;D;D;D;D;D;D
GERP RS		4.6
RBP_binding_hub_radar		0.0
RBP_regulation_power_radar		1.1

Splicing

Name	Calibrated prediction	Score	Prediction
dbscSNV1_ADA	Pathogenic	1.0
dbscSNV1_RF	Pathogenic	0.93
SpliceAI score (max)		0.83		Details are displayed if max score is > 0.2
DS_AL_spliceai		0.83		Position offset: -2

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

Publications

LitVar

Other links and lift over

dbSNP: rs80358033; hg19: chr17-41247941;