13-48459688-T-C

Variant names:

• chr13-48459688-T-C
• rs769113950
• NM_000321.3:c.1961T>C

Variant summary

Our verdict is Likely pathogenic. The variant received 8 ACMG points: 8P and 0B. PM1 PM2 PM5 PP3_Moderate

The NM_000321.3(RB1):​c.1961T>C​(p.Val654Ala) variant causes a missense, splice region change involving the alteration of a conserved nucleotide. The variant was absent in control chromosomes in GnomAD project. In-silico tool predicts a pathogenic outcome for this variant. 2/3 splice prediction tools predict no significant impact on normal splicing. Variant has been reported in ClinVar as Uncertain significance (★★). Another variant affecting the same amino acid position, but resulting in a different missense (i.e. V654L) has been classified as Likely pathogenic.

• Frequency:
  • Genomes: not found (cov: 30)
  • Exomes 𝑓: 6.8e-7 (0 hom.)
  • Failed GnomAD Quality Control
• Consequence: RB1 NM_000321.3 missense, splice_region
• Scores: Splicing: ADA: 0.8693
• Clinical Significance: Uncertain significance criteria provided, multiple submitters, no conflicts U:2
• Conservation: PhyloP100: 7.67
• Publications: 0 publications found

Genes affected

RB1 (HGNC:9884): (RB transcriptional corepressor 1) The protein encoded by this gene is a negative regulator of the cell cycle and was the first tumor suppressor gene found. The encoded protein also stabilizes constitutive heterochromatin to maintain the overall chromatin structure. The active, hypophosphorylated form of the protein binds transcription factor E2F1. Defects in this gene are a cause of childhood cancer retinoblastoma (RB), bladder cancer, and osteogenic sarcoma. [provided by RefSeq, Jul 2008]

RB1 Gene-Disease associations (from GenCC):

• hereditary retinoblastoma

  Inheritance: AD Classification: DEFINITIVE, STRONG, SUPPORTIVE Submitted by: Labcorp Genetics (formerly Invitae), Orphanet, G2P, Ambry Genetics
• retinoblastoma

  Inheritance: AD Classification: DEFINITIVE Submitted by: ClinGen
• melanoma

  Inheritance: AD Classification: MODERATE Submitted by: Ambry Genetics

ACMG classification

Our verdict: Likely_pathogenic. The variant received 8 ACMG points.

• PM1: In a hotspot region, there are 4 aminoacids with missense pathogenic changes in the window of +-8 aminoacids around while only 3 benign, 24 uncertain in NM_000321.3
• PM2: Very rare variant in population databases, with high coverage
• PM5: Other missense variant is known to change same aminoacid residue: Variant chr13-48456349-G-T is described in ClinVar as Pathogenic/Likely_pathogenic. ClinVar VariationId is 3237045.Status of the report is criteria_provided_multiple_submitters_no_conflicts, 2 stars.
• PP3: MetaRNN computational evidence supports a deleterious effect, 0.866

Variant Effect in Transcripts

ACMG analysis was done for transcript: NM_000321.3. You can select a different transcript below to see updated ACMG assignments.

RefSeq Transcripts

Selected	Gene	Transcript	Tags	HGVSc	HGVSp	Effect	Exon Rank	Protein	UniProt
	RB1	NM_000321.3	MANE Select	c.1961T>C	p.Val654Ala	missense splice_region	Exon 20 of 27	NP_000312.2
	RB1	NM_001407165.1		c.1961T>C	p.Val654Ala	missense splice_region	Exon 20 of 27	NP_001394094.1

Ensembl Transcripts

Selected	Gene	Transcript	Tags	HGVSc	HGVSp	Effect	Exon Rank	Protein	UniProt
	RB1	ENST00000267163.6	TSL:1 MANE Select	c.1961T>C	p.Val654Ala	missense splice_region	Exon 20 of 27	ENSP00000267163.4
	RB1	ENST00000467505.6	TSL:1	n.*1329T>C		splice_region non_coding_transcript_exon	Exon 15 of 22	ENSP00000434702.1
	RB1	ENST00000467505.6	TSL:1	n.*1329T>C		3_prime_UTR	Exon 15 of 22	ENSP00000434702.1

Frequencies

GnomAD3 genomes Cov.: 30

GnomAD2 exomes AF: 0.00000398 AC: 1 AN: 251468 AF XY: 0.00

Gnomad AFR exome AF: 0.00

Gnomad AMR exome AF: 0.00

Gnomad ASJ exome AF: 0.0000992

Gnomad EAS exome AF: 0.00

Gnomad FIN exome AF: 0.00

Gnomad NFE exome AF: 0.00

Gnomad OTH exome AF: 0.00

GnomAD4 exome Data not reliable, filtered out with message: AS_VQSR AF: 6.84e-7 AC: 1 AN: 1461830 Hom.: 0 Cov.: 33 AF XY: 0.00 AC XY: 0 AN XY: 727216

African (AFR) AF: 0.00 AC: 0 AN: 33480

American (AMR) AF: 0.00 AC: 0 AN: 44724

Ashkenazi Jewish (ASJ) AF: 0.0000383 AC: 1 AN: 26132

East Asian (EAS) AF: 0.00 AC: 0 AN: 39694

South Asian (SAS) AF: 0.00 AC: 0 AN: 86258

European-Finnish (FIN) AF: 0.00 AC: 0 AN: 53414

Middle Eastern (MID) AF: 0.00 AC: 0 AN: 5768

European-Non Finnish (NFE) AF: 0.00 AC: 0 AN: 1111970

Other (OTH) AF: 0.00 AC: 0 AN: 60390

GnomAD4 genome Cov.: 30

ExAC AF: 0.00000824 AC: 1

ClinVar

Significance: Uncertain significance

Submissions summary: Uncertain:2

Revision: criteria provided, multiple submitters, no conflicts

Submissions by phenotype

Retinoblastoma Uncertain:1

Jul 19, 2022

Labcorp Genetics (formerly Invitae), Labcorp

Significance: Uncertain significance

Review Status: criteria provided, single submitter

Collection Method: clinical testing

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. Algorithms developed to predict the effect of missense changes on protein structure and function (SIFT, PolyPhen-2, Align-GVGD) all suggest that this variant is likely to be disruptive. ClinVar contains an entry for this variant (Variation ID: 579914). This variant has not been reported in the literature in individuals affected with RB1-related conditions. This variant is present in population databases (rs769113950, gnomAD 0.01%). This sequence change replaces valine, which is neutral and non-polar, with alanine, which is neutral and non-polar, at codon 654 of the RB1 protein (p.Val654Ala).

Hereditary cancer-predisposing syndrome Uncertain:1

Jul 18, 2025

Ambry Genetics

Significance: Uncertain significance

Review Status: criteria provided, single submitter

Collection Method: clinical testing

The p.V654A variant (also known as c.1961T>C) is located in coding exon 20 of the RB1 gene. The valine at codon 654 is replaced by alanine, an amino acid with similar properties. This change occurs in the first base pair of coding exon 20. This amino acid position is well conserved in available vertebrate species. In addition, this alteration is predicted to be deleterious by in silico analysis. Based on the available evidence, the clinical significance of this variant remains unclear.

Computational scores

Source: dbNSFP v4.9

Name	Calibrated prediction	Score	Prediction
AlphaMissense	Pathogenic	0.76
BayesDel_addAF	Pathogenic	0.40	D
BayesDel_noAF	Pathogenic	0.34
CADD	Pathogenic	30
DANN	Uncertain	1.0
DEOGEN2	Pathogenic	0.90	D
Eigen	Pathogenic	0.75
Eigen_PC	Pathogenic	0.74
FATHMM_MKL	Pathogenic	0.99	D
LIST_S2	Uncertain	0.89	D
M_CAP	Uncertain	0.19	D
MetaRNN	Pathogenic	0.87	D
MetaSVM	Pathogenic	0.85	D
MutationAssessor	Uncertain	2.3	M
PhyloP100		7.7
PrimateAI	Uncertain	0.78	T
PROVEAN	Uncertain	-2.4	N
REVEL	Pathogenic	0.88
Sift	Benign	0.089	T
Sift4G	Uncertain	0.020	D
Polyphen		0.97	D
Vest4		0.80
MutPred		0.76		Gain of catalytic residue at Y659 (P = 0.0101)
MVP		0.94
MPC		1.4
ClinPred		0.80	D
GERP RS		5.8
RBP_binding_hub_radar		0.0
RBP_regulation_power_radar		1.7
Varity_R		0.49
gMVP		0.75
Mutation Taster		=22/78	disease causing

Splicing

Name	Calibrated prediction	Score	Prediction
dbscSNV1_ADA	Benign	0.87
dbscSNV1_RF	Pathogenic	0.76
SpliceAI score (max)		0.0		Details are displayed if max score is > 0.2

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

Other links and lift over

dbSNP: rs769113950; hg19: chr13-49033824;