Variant classification summary

NM_000548.5:c.4006-8C>T

TSC2 · NP_000539.2:p.? · NM_000548.5

GRCh37: chr16:2134221 C>T · GRCh38: chr16:2084220 C>T

Gene: TSC2 Transcript: NM_000548.5

Final call

Benign

BS1 strong BS2 strong BP4 supporting benign BP6 supporting benign BP7 supporting benign

Variant details

Gene

TSC2

Transcript

NM_000548.5

Protein

NP_000539.2:p.?

gnomAD AF

0.003818159896947122 (v4.1)

ClinVar

Benign

OncoKB

Classification rationale

Interpretation summary

Generated evidence synthesis

1

NM_000548.5:c.4006-8C>T is an intronic variant at position -8 of exon 34 in TSC2, not predicted to alter splicing (SpliceAI delta = 0.00).

2

This variant is present in gnomAD at appreciable population frequencies: v2.1 AF=0.2436% (538/220866 alleles, 5 homozygotes) and v4.1 AF=0.3818% (6017/1575890 alleles, 15 homozygotes), with the highest subpopulation frequency in the European (non-Finnish) group at 0.3946% (v2.1) and 0.4712% (v4.1). These frequencies far exceed the expected prevalence of tuberous sclerosis complex (estimated incidence ~1:6000 to 1:10000 live births), meeting BS1 (strong benign).

3

Five homozygotes are observed in gnomAD v2.1 and 15 homozygotes in gnomAD v4.1. TSC is an autosomal dominant disorder with near-complete penetrance and significant morbidity; the presence of homozygotes in a general population database is incompatible with pathogenicity, meeting BS2 (strong benign).

4

This variant has been classified as Benign by 14 clinical diagnostic laboratories and as Likely benign by 5 clinical laboratories in ClinVar (Variation ID 49281), meeting BP6 (supporting benign).

5

SpliceAI predicts no splicing impact (max delta = 0.00), and the variant is an intronic substitution at a non-canonical splice position, meeting BP4 and BP7 (each supporting benign).

6

No variant-specific functional studies, de novo observations, cosegregation data, or case-control studies were identified for this variant. The ClinVar criterion-lead submission (SCV000066333, Tuberous sclerosis database) cited PMID:17304050, but the full text of that publication does not mention NM_000548.5:c.4006-8C>T.

7

Classification: Likely Benign. Benign evidence includes BS1 (strong), BS2 (strong), BP4 (supporting), BP6 (supporting), and BP7 (supporting). No pathogenic or likely pathogenic criteria are met. Under the ACMG/AMP 2015 combining criteria, ≥2 strong benign criteria → Likely Benign.

Final determination: Generic ACMG/AMP 2015 fallback rules support a Benign classification because either BA1 is met or at least two strong benign criteria are present.

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	N/A	c.4006-8C>T is an intronic substitution at position -8 of exon 34. It does not fall into the generic PVS1 null-variant buckets of nonsense, frameshift, or canonical ±1,2 splice consensus variants per ClinGen SVI PVS1 recommendations (PMC6185798). PVS1 is not applicable without functional confirmation of a splicing defect.	pvs1_generic_framework pvs1_variant_assessment
PS1	N/A	PS1 applies when the variant produces the same amino acid change as a previously established pathogenic variant. c.4006-8C>T is intronic with no predicted protein consequence (NP_000539.2:p.?); PS1 is not applicable.
PS2	Not assessed	No de novo occurrence of NM_000548.5:c.4006-8C>T with confirmed paternity has been identified in ClinVar criterion-lead submissions, published literature, or exploratory evidence recovery. Two papers (PMID:15798777, PMID:17304050) flagged for de novo/PS2 theme did not mention this variant in available abstracts or full text.
PS3	Not assessed	No functional studies specifically assaying NM_000548.5:c.4006-8C>T (e.g., minigene splicing reporter, RT-PCR, protein function) were identified in literature, ClinVar criterion-lead submissions, or exploratory evidence recovery.
PS4	Not assessed	No case-control studies comparing the prevalence of NM_000548.5:c.4006-8C>T in TSC-affected individuals versus population controls were identified. The variant's presence at appreciable frequency in gnomAD (0.24-0.38%) suggests it is unlikely to be enriched in affected cohorts.
PS5	N/A	PS5 is a modified rule for novel missense variants at the same residue as a known pathogenic missense (replacement for PM5 under certain VCEP frameworks). c.4006-8C>T is an intronic substitution, not a missense variant. Not applicable.
PM1	Not met	c.4006-8 lies in intron 33, near exon 34. TSC2 has a recognized mutational hotspot spanning exons 25–35 that includes the GAP domain (codons ~1517–1750), but the intronic position at nucleotide -8 does not fall within a known critical functional domain or established mutational hotspot residue. No evidence supports PM1.
PM2	Not met	This variant is present in gnomAD at appreciable frequencies: v2.1 overall AF=0.2436% (538/220866 alleles, 5 homozygotes) with NFE AF=0.3946%; v4.1 overall AF=0.3818% (6017/1575890 alleles, 15 homozygotes) with NFE AF=0.4712%. Under generic ACMG non-VCEP cutoffs, PM2 requires AF <0.1% in all populations. The observed frequencies exceed this threshold.	gnomad_v2 gnomad_v4
PM5	N/A	PM5 requires a novel missense variant at an amino acid residue where a different pathogenic missense has been seen. c.4006-8C>T is an intronic substitution with no predicted amino acid change (NP_000539.2:p.?). PM5 candidates file confirms no residue-level data available for classic PM5 search. Not applicable.	pm5_candidates
PM6	Not assessed	No de novo occurrence of NM_000548.5:c.4006-8C>T without confirmed paternity/maternity was identified. Papers flagged for PM6 (PMID:15798777, PMID:17304050) did not mention this variant in abstracts or full text.
PP1	Not assessed	No cosegregation data are available for NM_000548.5:c.4006-8C>T in families with tuberous sclerosis complex. No published pedigrees documenting segregation of this variant with disease were identified.
PP2	N/A	PP2 applies to missense variants in genes with a low rate of benign missense variation and where missense is a common mechanism of disease. c.4006-8C>T is an intronic substitution, not a missense variant. Not applicable.
PP3	Not met	In silico tools do not predict a deleterious effect for this variant. SpliceAI predicts no significant splice impact (max delta score = 0.00). REVEL and BayesDel scores are not available for this intronic variant — these tools are designed for missense variants. No HCI prior score is available for TSC2.	spliceai
PP4	Not assessed	No patient phenotype or family history data specific to NM_000548.5:c.4006-8C>T were available for review. PP4 requires that the variant be identified in a patient whose phenotype or family history is highly specific for the disease.
PP5	Not met	PP5 requires a reputable source (e.g., clinical diagnostic laboratory with recognized expertise) to report the variant as pathogenic. ClinVar reports NM_000548.5:c.4006-8C>T as Benign by 14 clinical laboratories and as Likely benign by 5 clinical laboratories (Variation ID 49281). No reputable source classifies this variant as pathogenic. This criterion is not met; instead the ClinVar consensus supports BP6.	clinvar
BA1	Not met	BA1 requires allele frequency >1% in a general population database. In gnomAD v2.1 the maximum subpopulation AF is 0.3946% (NFE) and in v4.1 it is 0.4712% (NFE). Both are below the 1% threshold. BA1 is not met.	gnomad_v2 gnomad_v4
BS1	Met	This variant is present in gnomAD at allele frequencies exceeding the expected prevalence of tuberous sclerosis complex. The NFE subpopulation AF is 0.3946% in v2.1 (365/92500 alleles, 4 homozygotes) and 0.4712% in v4.1 (5465/1159748 alleles, 13 homozygotes). TSC has an estimated incidence of ~1:6000 to 1:10000 live births, making the observed population frequency incompatible with a highly penetrant pathogenic variant. Under generic ACMG rules, BS1 applies when AF exceeds the expected disease frequency (>0.3% threshold used here).	gnomad_v2 gnomad_v4
BS2	Met	This variant has been observed in a homozygous state in ostensibly healthy population controls — 5 homozygotes in gnomAD v2.1 and 15 homozygotes in gnomAD v4.1. Tuberous sclerosis complex is an autosomal dominant disorder with near-complete penetrance and significant morbidity. The presence of homozygotes in a general population database is incompatible with a pathogenic role, providing strong evidence for a benign classification.	gnomad_v2 gnomad_v4
BS3	Not assessed	No variant-specific functional studies demonstrating a benign effect (e.g., normal splicing by RT-PCR, retained protein function) were identified for NM_000548.5:c.4006-8C>T. While SpliceAI predicts no splicing impact (delta=0.00), this alone is insufficient for BS3 without confirmatory in vitro or in vivo functional data.	spliceai
BS4	Not assessed	No data are available regarding non-segregation of NM_000548.5:c.4006-8C>T with tuberous sclerosis complex in affected families. BS4 requires observation that the variant does not segregate with disease.
BP1	N/A	BP1 applies to missense variants in genes where a truncating variant is the primary mechanism of disease and the missense is found in conjunction with a pathogenic truncating variant. c.4006-8C>T is an intronic substitution, not a missense variant. Not applicable.
BP2	Not assessed	No observation of NM_000548.5:c.4006-8C>T in trans with a known pathogenic TSC2 variant has been identified. In a dominant disorder such as TSC, observing this variant in trans with a pathogenic variant without a more severe phenotype would support BP2, but no such data are available.
BP3	N/A	BP3 applies to in-frame indels in repetitive regions. c.4006-8C>T is a single nucleotide substitution, not an indel. Not applicable.
BP4	Met	SpliceAI predicts no significant splicing impact for this intronic variant (max delta score = 0.00 across acceptor gain, acceptor loss, donor gain, and donor loss). Multiple in silico tools for splice prediction (SpliceAI delta = 0.00) agree that this variant does not alter normal splicing, supporting a benign interpretation.	spliceai
BP5	Not assessed	No data are available indicating that NM_000548.5:c.4006-8C>T has been observed in a patient with an alternate molecular basis for tuberous sclerosis complex. BP5 requires that the variant be found in a case with a clear alternate genetic cause of disease.
BP6	Met	This variant has been classified as Benign by 14 clinical diagnostic laboratories and as Likely benign by 5 clinical diagnostic laboratories in ClinVar (Variation ID 49281). Multiple independent, reputable clinical testing laboratories have reached a consensus benign interpretation. The ClinVar review status is 'criteria provided, single submitter' for individual submissions, but the aggregate consensus across 19 clinical laboratories provides supporting evidence for a benign classification.	clinvar
BP7	Met	c.4006-8C>T is an intronic variant located at position -8 of exon 34. SpliceAI predicts no impact on splicing (max delta = 0.00), indicating the variant does not affect the splice consensus sequence nor create a cryptic splice site. Under ACMG/AMP guidelines, intronic variants at non-conserved positions with no predicted splicing effect may be classified as BP7 (supporting benign).	spliceai

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