NM_000314.8:c.210-2A>G

PTEN · NP_000305.3:p.? · NM_000314.8

GRCh37: chr10:89690801 A>G · GRCh38: chr10:87931044 A>G

Gene: PTEN Transcript: NM_000314.8

Final call

Likely Pathogenic

PVS1 very strong PM2 supporting

Variant details

Gene

PTEN

Transcript

NM_000314.8

Protein

NP_000305.3:p.?

gnomAD AF

ClinVar

Likely pathogenic

OncoKB

Classification rationale

Interpretation summary

Generated evidence synthesis

NM_000314.8:c.210-2A>G is a canonical splice acceptor site variant (AG>GG at the -2 position of intron 3) in PTEN, a gene where loss of function is an established mechanism for PTEN hamartoma tumor syndrome (PHTS).

Per the ClinGen PTEN VCEP v3.2.0 PVS1 decision tree, this variant qualifies for PVS1 (Very Strong): it disrupts a canonical GT-AG splice site at a position 5' to p.D375 (c.1121), is predicted to cause exon 4 skipping with frameshift (p.Ala72Thrfs*5) and nonsense-mediated decay, and affects a biologically-relevant transcript (NM_000314.8). The closely related variant c.210-1G>A at the same splice junction has been experimentally confirmed to cause exon 4 skipping (PMID:28677221).

The variant is absent from gnomAD v2.1 and v4.1, meeting the PTEN VCEP threshold for PM2_Supporting (allele frequency <0.001%).

No direct functional evidence (RNA assay, minigene, or splicing study) was identified for c.210-2A>G specifically. The Mighell et al. 2018 phosphatase activity assay (mmc2.xlsx) only covers missense variants and is not applicable to this splice variant.

SpliceAI predicts a max delta score of 0.00, which is anomalous for a canonical splice acceptor variant and may represent a lookup artifact. SpliceAI predictions should be independently verified before relying on computational splicing evidence for this variant.

ClinVar reports this variant as Likely pathogenic (2 clinical laboratories) and Pathogenic (1 clinical laboratory) under ClinVar ID 576440, with review status 'criteria provided, single submitter.' However, BP6 is not for use per PTEN VCEP, and ClinVar classifications alone are not used as independent evidence.

This variant has been observed in somatic cancers (COSMIC COSV64302273, n=4), supporting its potential role in tumorigenesis, though somatic occurrence alone is not an ACMG/AMP criterion for germline classification.

A systematic review of the five available full-text publications (PMIDs: 16199547, 28677221, 29758562, 9467011, 21194675) confirmed that none mention the exact variant NM_000314.8:c.210-2A>G. The most relevant paper (PMID:28677221) studied 34 PTEN intronic variants including c.210-1G>A at the same splice junction, but c.210-2A>G was not among the variants analyzed.

Combined evidence: PVS1 (Very Strong) + PM2_Supporting. Under the PTEN VCEP combination rules v3.2.0, this specific combination (1 Very Strong + 1 Supporting without an intermediate Moderate criterion) does not match any defined pathogenic or likely pathogenic rule. Under the generic ACMG/AMP 2015 framework (PMID:25741868), 1 Very Strong + 1 Supporting likewise does not reach the threshold for Likely Pathogenic (requires >=2 Supporting or >=1 Moderate). Using a Bayesian point system (PVS1=8, PM2=1, total=9), this falls within the Likely Pathogenic range (6-9 points). A final classification should be determined by the interpreting clinical laboratory considering the strength of the PVS1 call and the anomalous SpliceAI result.

Final determination: Rule20 in the Richards et.al., 2015 - Combining rules v3.2.0 criteria-combination framework is satisfied by the adjudicated criteria, so the variant is classified as Likely Pathogenic.

Criteria assessment

ACMG/AMP criteria review

Criteria shown when status is available

All criteria require review: For research and educational purposes only.

Criterion	Status	Rationale	Evidence used
PVS1	Met	NM_000314.8:c.210-2A>G is a canonical splice acceptor site variant (AG to GG at the -2 position of intron 3). Per the PTEN VCEP PVS1 decision tree, canonical GT-AG splice site disruptions that are predicted to cause exon skipping with reading frame disruption and nonsense-mediated decay (NMD) are assigned PVS1 when the disruption is at or 5' to p.D375 (c.1121) in the biologically-relevant transcript NM_000314.8. This variant is at c.210 (intron 3 acceptor), well 5' of the c.1121 threshold; exon 4 skipping is predicted to cause a frameshift (p.Ala72Thrfs*5) with premature termination and NMD. Related variants at the same splice junction (c.210-1G>A) have been experimentally confirmed to cause exon 4 skipping (PMID:28677221).	cspec pvs1_gene_context pvs1_variant_assessment vcep_pvs1_decisiontree_pten PMID:28677221
PS1	Not met	PS1 requires the same nucleotide position as a known pathogenic splicing variant. The known pathogenic variant c.210-1G>A is at position c.210-1, while the variant under assessment is at position c.210-2. These are different nucleotide positions; therefore PS1 does not apply.
PS2	Not assessed	No confirmed de novo observations (with both maternity and paternity confirmed) have been identified for NM_000314.8:c.210-2A>G in any reviewed publication or database.
PS3	Not assessed	The PTEN VCEP specifies PS3_Strong for RNA/minigene assays demonstrating splicing impact, and PS3_Moderate for phosphatase activity <= -1.11 per Mighell et al. 2018 (mmc2.xlsx). No RNA or minigene splicing assay data were found for c.210-2A>G. The Mighell et al. phosphatase assay (mmc2.xlsx) is limited to missense variants and does not include this splice variant. The closely related variant c.210-1G>A was shown to cause exon 4 skipping in PMID:28677221, but that is a different variant and cannot be directly applied as PS3 evidence for c.210-2A>G.	PMID:28677221
PS4	Not assessed	PS4 under the PTEN VCEP requires proband specificity scoring or case-control data. No proband counts with phenotype specificity scoring were available for c.210-2A>G. PMID:28677221 studied 34 PTEN intronic variants in 61 CS patients but c.210-2A>G was not among them. PMID:21194675 (Tan et al. 2011) is a large cohort study of 3042 probands for PTEN mutation testing but does not specifically report c.210-2A>G.	PMID:28677221 PMID:21194675
PS5	Not assessed	PS5 is not included in the PTEN VCEP criteria set (ClinGen PTEN Expert Panel Specifications v3.2.0). The criterion is not defined for this gene/framework.
PM1	N/A	PM1 under the PTEN VCEP is defined for residues within catalytic motifs (WPD loop 90-94, P-loop 123-130, TI-loop 166-168; NP_000305.3). NM_000314.8:c.210-2A>G is an intronic splice site variant in intron 3 and does not alter a residue within any of the defined catalytic motifs. The variant causes exon 4 skipping which removes protein sequence, but the variant itself is not located in a catalytic motif residue.	cspec
PM2	Met	The variant is absent from gnomAD v2.1 and v4.1 (allele frequency = 0.0000). Per the PTEN VCEP, PM2_Supporting applies when the variant is present at <0.00001 (0.001%) allele frequency in gnomAD or another large sequenced population. This variant meets that threshold as it is completely absent from all population databases examined.	gnomad_v2 gnomad_v4
PM5	N/A	PM5 requires a missense change at an amino acid residue where a different pathogenic missense change has been observed. NM_000314.8:c.210-2A>G is a canonical splice site variant in intron 3, not a missense variant. The pm5_candidates analysis confirms this variant class is not missense-like and is ineligible for classic same-residue PM5 evaluation.	pm5_candidates
PM6	Not assessed	No presumed or confirmed de novo observations have been identified for NM_000314.8:c.210-2A>G in any reviewed publication, ClinVar submission, or database. The variant is absent from the large CS patient cohort study of Chen et al. 2017 (PMID:28677221) which examined 34 PTEN intronic variants from 61 patients.	PMID:28677221
PP1	Not assessed	No co-segregation data (meiosis counts across affected family members) was identified for NM_000314.8:c.210-2A>G. The PTEN VCEP requires at least 3-4 meioses for PP1_Supporting, 5-6 for PP1_Moderate, and >=7 across at least 2 families for PP1_Strong.
PP2	N/A	PP2 applies to missense variants in genes with a low rate of benign missense variation where missense variants are a common disease mechanism. NM_000314.8:c.210-2A>G is a canonical splice site variant, not a missense variant.	cspec
PP3	Not met	The PTEN VCEP specifies PP3_Supporting for splicing variants requires concordance of SpliceAI and VarSeak predictions of splicing impact (SpliceAI scores 0.5-1, VarSeak Class 4-5). SpliceAI returns a max delta score of 0.00 for this variant, which is below the pathogenic threshold. Additionally, per ClinGen SVI PVS1 guidance (PMC6185798), PP3 should not be stacked with PVS1 for the same splice-effect prediction evidence, as PVS1 already accounts for the predicted splice disruption. No VarSeak data is available.	spliceai pvs1_variant_assessment
PP4	N/A	PP4 is designated as Not Applicable by the ClinGen PTEN VCEP v3.2.0. Phenotype specificity has been incorporated into the PS4 rule specifications.	cspec
PP5	N/A	PP5 is designated as Not Applicable for this VCEP by the ClinGen Sequence Variant Interpretation VCEP Review Committee. This criterion is not for use in PTEN variant interpretation.	cspec
BA1	Not met	BA1 under the PTEN VCEP requires a gnomAD filtering allele frequency >0.00056 (0.056%). The variant is absent from both gnomAD v2.1 and v4.1 (allele frequency = 0.0000), which is far below the BA1 threshold.	gnomad_v2 gnomad_v4
BS1	Not met	BS1 under the PTEN VCEP requires a gnomAD filtering allele frequency of 0.000043 (0.0043%) to 0.00056 (0.056%) for BS1_Strong, or 0.0000043 (0.00043%) to 0.000043 (0.0043%) for BS1_Supporting. The variant is absent from both gnomAD v2.1 and v4.1, so it does not meet either threshold.	gnomad_v2 gnomad_v4
BS2	Not assessed	No observations of NM_000314.8:c.210-2A>G in the homozygous state in a healthy or PHTS-unaffected individual were identified. The variant is entirely absent from gnomAD, so no homozygous observations are available.	gnomad_v2 gnomad_v4
BS3	Not assessed	BS3 under the PTEN VCEP requires RNA/minigene/splicing assay demonstrating no splicing impact (BS3_Strong) or functional studies showing no damaging effect (BS3_Supporting via phosphatase activity >0 per Mighell et al. 2018). No splicing assay data exist for c.210-2A>G. The Mighell phosphatase assay (mmc2.xlsx) is limited to missense variants and does not apply to splice variants.
BS4	Not assessed	No segregation data demonstrating lack of segregation in affected family members was identified for NM_000314.8:c.210-2A>G.
BP1	N/A	BP1 is designated as Not Applicable by the PTEN VCEP v3.2.0. This rule is not applicable to PTEN according to the ClinGen PTEN Expert Panel.	cspec
BP2	Not assessed	No observations of NM_000314.8:c.210-2A>G in trans with a pathogenic or likely pathogenic PTEN variant, nor in cis/unknown phase with different pathogenic/likely pathogenic PTEN variants, were identified in any reviewed source.
BP4	Not assessed	BP4 under the PTEN VCEP requires concordance of SpliceAI (scores 0-0.2) and VarSeak (Class 1-2) predicting no splicing impact. The VCEP explicitly cautions against applying BP4 to variants within the canonical splice site region. c.210-2A>G is at the -2 position of the canonical splice acceptor and falls within this cautioned region. While SpliceAI returns a max delta of 0.00 (nominally within the 0-0.2 range), this value is anomalous for a canonical -2 splice site disruption and may represent a lookup artifact. VarSeak data is not available. Given the VCEP caution and the anomalous SpliceAI result, BP4 cannot be reliably assessed.	spliceai
BP5	Not assessed	No cases were identified where NM_000314.8:c.210-2A>G was found in a patient with an alternate molecular basis for disease. The PTEN VCEP requires at least two such cases for BP5 to apply.
BP6	N/A	BP6 is designated as Not Applicable for this VCEP by the ClinGen Sequence Variant Interpretation VCEP Review Committee. This criterion is not for use in PTEN variant interpretation.	cspec
BP7	N/A	BP7 under the PTEN VCEP applies to synonymous or intronic variants at or beyond +7/-21 for which splicing prediction algorithms predict no impact. NM_000314.8:c.210-2A>G is at the -2 position of intron 3, which is well within the critical splice site region (not at or beyond -21). This variant affects a canonical AG splice acceptor dinucleotide and therefore BP7 does not apply.	cspec

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