Variant classification summary

NM_000314.8:c.253+5G>T

PTEN · NP_000305.3:p.? · NM_000314.8

GRCh37: chr10:89690851 G>T · GRCh38: chr10:87931094 G>T

Gene: PTEN Transcript: NM_000314.8

Final call

VUS

PM2 supporting

Variant details

Gene

PTEN

Transcript

NM_000314.8

Protein

NP_000305.3:p.?

gnomAD AF

ClinVar

Pathogenic

OncoKB

Classification rationale

Interpretation summary

Generated evidence synthesis

1

NM_000314.8:c.253+5G>T is an intronic variant at the +5 position of the PTEN donor splice site. This variant is absent from gnomAD v2.1 and v4.1 population databases (PM2_Supporting).

2

SpliceAI strongly predicts a deleterious splicing effect (max delta score 0.91; acceptor loss 0.91, donor loss 0.85), consistent with disruption of normal splicing at the exon 4-intron 4 junction.

3

The PTEN VCEP PVS1 decision tree applies to canonical GT-AG ±1,2 splice site disruptions. c.253+5G>T at the +5 position does not fall within the canonical splice site branch of the decision tree, and PVS1 is not met under the VCEP framework.

4

This variant is reported in ClinVar (VariationID 427617) as Pathogenic by four clinical laboratories and Likely pathogenic by one, with review status 'criteria provided, single submitter.' No expert panel review is available.

5

The variant has been observed in somatic cancers (COSMIC COSV64298366, n=2), consistent with a role in tumorigenesis, though somatic observations are not directly applicable to germline ACMG/AMP criteria.

6

Key functional evidence may exist in PMID:28677221 (Chen et al. 2017), which characterized cryptic splicing in 34 germline PTEN intronic variants from Cowden syndrome patients, but full-text confirmation that c.253+5G>T was specifically studied is unavailable. Until splicing assay data are confirmed, PS3 cannot be applied.

7

Multiple criteria require additional evidence to be fully assessed: PS3 (RNA/mini-gene splicing assay), PS4 (proband specificity scores), PP3 (VarSeak concordance with SpliceAI), PS1 (comparison to other pathogenic variants at c.253+5), and PP1/BS4 (segregation data).

Final determination: No criteria-combination rule matched the adjudicated criteria in the Richards et.al., 2015 - Combining rules v3.2.0 framework, so the variant remains a Variant of Uncertain Significance pending human review.

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	Not met	The PTEN VCEP PVS1 decision tree covers canonical GT-AG ±1,2 splice site disruptions that lead to exon skipping or cryptic splice site usage with NMD prediction. c.253+5G>T is at the +5 position of the donor splice site, which is outside the canonical ±1,2 positions evaluated by the decision tree. The variant does not fit any branch of the PTEN-specific PVS1 decision tree (nonsense, frameshift, canonical splice sites, deletions, duplications, or initiation codon).	cspec vcep_pvs1_decisiontree_pten pvs1_variant_assessment
PS1	Not assessed	The VCEP PS1 rule allows application when a different variant at the same nucleotide position is a known pathogenic splicing variant with equal or greater predicted impact. Data on other variants at the c.253+5 position (e.g., c.253+5G>A or c.253+5G>C) and their SpliceAI predictions were not available for comparison.	cspec
PS2	Not assessed	No de novo observations for this variant were identified in the literature or ClinVar submissions. De novo data are required to apply PS2 under the VCEP framework.	cspec clinvar
PS3	Not assessed	The VCEP PS3_Strong rule can be applied when an RNA, mini-gene, or other assay demonstrates impact on splicing. PMID:28677221 (Chen et al. 2017) is a study of cryptic splicing in 34 germline PTEN intronic variants from Cowden syndrome patients and likely contains relevant functional data, but the full text is unavailable for confirmation that c.253+5G>T was specifically studied. The mmc2.xlsx phosphatase activity dataset applies only to missense variants and is not relevant to this intronic variant.	cspec clinvar
PS4	Not assessed	The VCEP PS4 rule requires probands with specificity scores. ClinVar reports this variant as Pathogenic by 4 clinical laboratories and Likely pathogenic by 1, with review status 'criteria provided, single submitter.' However, no proband counts with phenotype specificity scores are available for formal PS4 assessment. The variant is absent from gnomAD, consistent with rarity in the general population.	clinvar gnomad_v2 gnomad_v4
PS5	N/A	PP5 is declared 'Not Applicable for this VCEP' by the ClinGen PTEN Expert Panel v3.2.0. PS5 as the stronger form of PP5 is similarly not applicable under this VCEP framework.	cspec
PM1	N/A	The VCEP PM1 rule is limited to residues within the PTEN catalytic motifs (NP_000305.3 residues 90-94, 123-130, 166-168). c.253+5G>T is an intronic variant at the +5 position of intron 4 and does not alter a catalytic motif residue.	cspec
PM2	Met	This variant is absent from gnomAD v2.1 (exomes) and gnomAD v4.1 (exomes), meeting the PTEN VCEP PM2_Supporting threshold of <0.001% (0.00001) allele frequency.	gnomad_v2 gnomad_v4 cspec
PM5	N/A	PM5 requires a missense change at an amino acid residue where a different pathogenic missense change has been observed. c.253+5G>T is an intronic (splice site) variant, not a missense variant, and the variant does not encode an amino acid change (NP_000305.3:p.?).	cspec pm5_candidates
PM6	Not assessed	No de novo observations for this variant were identified. The VCEP PM6 rule requires one or more de novo observations depending on confirmation status and strength level.	cspec
PP1	Not assessed	No co-segregation data are available for this variant. The VCEP PP1 rule requires co-segregation with disease in multiple affected family members with specific meioses counts for each strength level.	cspec
PP2	N/A	PP2 is specific to missense variants in genes with a low rate of benign missense variation where missense variants are a common disease mechanism. c.253+5G>T is an intronic splice site variant, not a missense variant.	cspec
PP3	Not assessed	SpliceAI predicts a deleterious splicing impact for this variant (max delta score 0.91; acceptor loss delta 0.91, donor loss delta 0.85), well above the >0.2 threshold. However, the VCEP PP3 rule for splicing variants requires concordance of both SpliceAI and VarSeak. VarSeak data are not available, so the required concordance cannot be confirmed.	spliceai cspec
PP4	N/A	The PTEN VCEP v3.2.0 declares PP4 'Not Applicable.' Phenotype specificity has been incorporated into the rule specifications for PS4 Use 2.	cspec
PP5	N/A	The PTEN VCEP v3.2.0 declares PP5 'Not Applicable for this VCEP.' This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee.	cspec
BA1	Not met	This variant is absent from gnomAD v2.1 and v4.1. The VCEP BA1 threshold is a gnomAD filtering allele frequency >0.00056 (0.056%). An absent variant does not meet this threshold.	gnomad_v2 gnomad_v4 cspec
BS1	Not met	This variant is absent from gnomAD v2.1 and v4.1. The VCEP BS1 threshold ranges from 0.0000043 (0.00043%, Supporting) to 0.00056 (0.056%, Strong). An absent variant does not meet either threshold.	gnomad_v2 gnomad_v4 cspec
BS2	Not met	This variant is absent from gnomAD v2.1 and v4.1 with no homozygous observations. The VCEP BS2 rule requires observation in the homozygous state in a healthy or PHTS-unaffected individual. No such observations are available.	gnomad_v2 gnomad_v4 cspec
BS3	Not met	The VCEP BS3_Strong rule requires an RNA, mini-gene, or other splicing assay demonstrating no splicing impact. No such evidence is available for this variant. The mmc2.xlsx phosphatase activity dataset (Mighell et al. 2018, PMID:29706350) applies only to missense variants and does not provide evidence for this intronic variant. In the absence of experimental data showing no splicing impact, BS3 cannot be applied.	cspec vcep_mmc2
BS4	Not assessed	No segregation data are available for this variant. The VCEP BS4 rule requires lack of segregation in affected members of one family (Supporting) or two or more families (Strong).	cspec
BP1	N/A	The PTEN VCEP v3.2.0 declares BP1 'Not Applicable.'	cspec
BP2	Not assessed	No co-occurrence or phase data are available for this variant. The VCEP BP2 rule requires observation in trans with a pathogenic or likely pathogenic PTEN variant, or at least three observations in cis/phase unknown with different pathogenic/likely pathogenic PTEN variants.	cspec
BP4	Not met	The VCEP BP4 rule for splicing variants requires SpliceAI scores in the 0-0.2 range (concordant with VarSeak Class 1-2) indicating no splicing impact. SpliceAI predicts a deleterious splicing impact for this variant with a max delta score of 0.91 (acceptor loss 0.91), well above the benign threshold. BP4 requires prediction of no impact, which is contradicted by the available in silico evidence.	spliceai cspec
BP5	Not assessed	The VCEP BP5 rule requires the variant to be found in a case with an alternate molecular basis for disease (at least two such cases, with the other gene/disorder being highly penetrant and no phenotypic overlap with PTEN). No such cases have been identified for this variant.	cspec
BP6	N/A	The PTEN VCEP v3.2.0 declares BP6 'Not Applicable for this VCEP.' This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee.	cspec
BP7	N/A	The VCEP BP7 rule applies to intronic variants 'at or beyond +7/-21.' c.253+5G>T is at the +5 position of the donor splice site, which is within the splice consensus region (+3 to +6) and does not meet the positional requirement of 'at or beyond +7.' Additionally, SpliceAI predicts splicing impact (delta 0.91), which would contradict the BP7 requirement that splicing prediction algorithms predict no impact.	spliceai cspec

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