TSC2 c.5024C>T, p.Pro1675Leu

Reasoning

The variant is NP_000539.2:p.Pro1675Leu. PMID:12111193 explicitly identifies P1675L as a previously reported pathogenic, recurrent disease-causing missense variant, found in two unrelated patients. PMID:12205112 independently reports the exact C5024T/P1675L change in at least 10 unrelated patients and places it at the conserved residue 1675. The available evidence is protein-level and does not indicate that the observation is driven by a splice effect.

Rule

Same amino acid change as a previously established pathogenic variant regardless of nucleotide change.

Evidence considered

• Langkau et al. 2002 (PMID:12111193), Results and Table 1: P1675L was described as previously pathogenic and recurrent in two unrelated patients.
• Kwiatkowski et al. 2002 (PMID:12205112), Materials and Methods/Results: C5024T produces P1675L and had been reported in at least 10 unrelated TSC patients; the residue was described as highly conserved.

Sources

Reasoning

PS3 is met at moderate strength. The exact variant NM_000548.5:c.5024C>T (p.Pro1675Leu) was evaluated in a standardized HEK293T transfection-based functional assay with wild-type TSC2 and the pathogenic p.Arg611Gln control, using at least four independent transfection experiments. Compared with wild-type TSC2, p.Pro1675Leu showed significantly reduced TSC2 signal while TSC1 signal was not significantly reduced, and the T389-phosphorylated S6K/total S6K ratio was significantly increased, indicating impaired inhibition of TORC1 activity. These controls, replication, and biologically relevant TSC1-TSC2/TORC1 readouts support a damaging effect. The strength is moderated because the evidence is from a research-cell assay rather than a validated clinical diagnostic laboratory assay, and a separate cell-cycle study found a wild-type-like G1 response for this mutant under its tested condition.

Rule

Well-established in vitro or in vivo functional studies supportive of a damaging effect on the gene or gene product.

Evidence considered

• Hoogeveen-Westerveld et al. 2013 (PMID:22903760): in a standardized HEK293T transfection-based immunoblot assay, p.P1675L was compared with wild-type TSC2 and pathogenic p.R611Q in at least four independent transfection experiments.
• Hoogeveen-Westerveld et al. 2013 (PMID:22903760), Table 1 Group 3: p.P1675L showed significantly reduced TSC2 signal compared with the wild-type TSC1-TSC2 control, preserved TSC1 signal, and an increased T389-phosphorylated S6K/total S6K ratio, consistent with reduced inhibition of TORC1 activity.
• The assay used variant-relevant TSC1-TSC2 complex and TORC1 pathway readouts, with wild-type and pathogenic controls; this supports a damaging functional effect but does not constitute clinical diagnostic-laboratory validation.
• Gibson et al. 2002 (PMID:12205112): an independent HEK293 cell-cycle assay reported that P1675L increased the G1 fraction similarly to wild-type TSC2 under the tested condition, so this study does not provide an additional unequivocal damaging readout and supports moderating PS3 strength rather than excluding the controlled immunoblot evidence.

Sources

Reasoning

PM2 is met at supporting strength. The variant is absent from gnomAD v2.1, gnomAD v4.1, gnomAD v2.1 non-cancer, and gnomAD v3.1 non-cancer. Thus its observed allele frequency is 0 in the reported datasets and is below the generic PM2 supporting threshold of <=0.0001. No VCEP-specific population source governs this case, so the all-comers datasets remain the default, with the non-cancer searches providing concordant support.

Rule

Absent from controls (or at extremely low frequency if recessive) in Exome Sequencing Project, 1000 Genomes or Exome Aggregation Consortium.

Evidence considered

• gnomAD v2.1 direct query for 16-2137898-C-T: absent.
• gnomAD v4.1 direct query for chr16-2087897-C-T: absent.
• GNOMAD_V2_1_NON_CANCER: absent; the non-cancer result was reported using the v2.1 exome query.
• GNOMAD_V3_1_NON_CANCER: absent; the non-cancer result was reported using the v3.1 genome query.
• Generic PM2 supporting threshold supplied for this case: <=0.0001 (ClinGen SVI recommendation, PMID:25741868).

Sources

Reasoning

PMID:10570911 explicitly reports the matched variant as de novo in patient P28 after examination of the parents' samples. The evidence supports assumed de novo status, while the paper does not document confirmation of maternity and paternity, matching the PM6 definition.

Rule

Assumed de novo, but without confirmation of paternity and maternity.

Evidence considered

• Zhang et al. 1999, patient P28: "P1657L in P28 was confirmed to be a de novo mutation by examination of the parents' samples." The paper does not report independent confirmation of maternity and paternity.

Sources

Reasoning

The variant is a missense substitution, NM_000548.5:c.5024C>T (p.Pro1675Leu), so the generic missense path requires REVEL only; SpliceAI and BayesDel are not used for PP3/BP4 in this situation. REVEL is 0.965, meeting the supplied generic PP3 strong threshold of >=0.932 from the ClinGen SVI REVEL calibration (Pejaver et al. 2022, PMID:36413997).

Rule

Multiple lines of computational evidence support a deleterious effect on the gene or gene product (conservation, evolutionary, splicing impact, etc.). ... PP3 (strong): >= 0.932 — ClinGen SVI REVEL calibration (Pejaver et al. 2022, Am J Hum Genet) (PMID:36413997)

Evidence considered

• Variant consequence is missense: NM_000548.5:c.5024C>T produces NP_000539.2:p.(Pro1675Leu).
• Local REVEL lookup reports a score of 0.965 for the variant; this is above the supplied PP3 strong cutoff of >=0.932.
• The supplied generic calibration identifies the PP3 strong threshold as >=0.932, from the ClinGen SVI REVEL calibration (Pejaver et al. 2022, PMID:36413997).
• SpliceAI max delta 0.008 and BayesDel 0.528466 were not counted because the governing generic path for a missense variant is REVEL only, and no generic ACMG-strength calibration is established for BayesDel.

Sources

Reasoning

PMID:10570911 reports the matched TSC2 variant as de novo after examination of the parents' samples, but the report does not document confirmation of both maternity and paternity. The disease context is TSC, but the defining PS2 requirement is therefore not established.

Rule

De novo (both maternity and paternity confirmed) in a patient with the disease and no family history.

Evidence considered

• Zhang et al. 1999, patient P28: the matched variant was reported as de novo by examination of the parents' samples; the report does not state that maternity and paternity were independently confirmed.

Still missing

• Explicit confirmation of maternity and paternity is not reported.

Sources

Reasoning

The governing rule is: "Reputable source recently reports variant as pathogenic, but the evidence is not available to the laboratory to perform an independent evaluation." ClinVar identifies the exact variant as Pathogenic, but its submission audit reports zero expert-panel submissions and only ordinary laboratory or non-expert assertions; the available exact-variant literature and functional extracts are independently available for evaluation. Under the specified generic PP5 restriction, this does not qualify as PP5.

Rule

Reputable source recently reports variant as pathogenic, but the evidence is not available to the laboratory to perform an independent evaluation.

Evidence considered

• ClinVar exact-match record NM_000548.5(TSC2):c.5024C>T (p.Pro1675Leu) is Pathogenic with review status "criteria provided, multiple submitters, no conflicts" and zero expert-panel submissions in the submission audit.
• The ClinVar record contains ordinary laboratory submissions and non-expert assertions, which are excluded by the specified PP5 rule.
• Exact-variant reports and functional findings are available for independent evaluation, so the evidence is not unavailable to the laboratory.

Sources

Reasoning

BA1 is not met. The variant is absent from gnomAD v2.1 and gnomAD v4.1, with no observed allele frequency or grpmax FAF; therefore it is far below the generic stand-alone threshold of >=0.05. The absence of a high population frequency also avoids the specific concern that a BA1 call would conflict with the established pathogenic ClinVar consensus.

Rule

Allele frequency is above 5% in Exome Sequencing Project, 1000 Genomes or Exome Aggregation Consortium.

Evidence considered

• gnomAD v2.1 direct query for 16-2137898-C-T: absent; no allele count, allele number, point estimate, or grpmax FAF reported.
• gnomAD v4.1 direct query for chr16-2087897-C-T: absent; no allele count, allele number, point estimate, or grpmax FAF reported.
• Generic BA1 default threshold supplied for this case: >=0.05 (ACMG/AMP 2015, PMID:25741868).

Sources

Reasoning

BS1 is not met. The variant is absent from gnomAD v2.1 and gnomAD v4.1, including the available non-cancer searches, so there is no population frequency or grpmax FAF greater than expected for TSC2-related tuberous sclerosis. The generic 0.01 default is not reached, and no maximum-credible-frequency calculation is needed because the observed frequency is zero/not observed rather than elevated.

Rule

Allele frequency is greater than expected for the disorder.

Evidence considered

• gnomAD v2.1 direct query for 16-2137898-C-T: absent; no allele frequency or grpmax FAF reported.
• gnomAD v4.1 direct query for chr16-2087897-C-T: absent; no allele frequency or grpmax FAF reported.
• The non-cancer searches were also absent: GNOMAD_V2_1_NON_CANCER and GNOMAD_V3_1_NON_CANCER; no alternative frequency source was required.
• Generic BS1 default threshold supplied for this case: >=0.01; the governing concept is frequency greater than expected for the disorder (Whiffin et al. 2017, PMID:28518168).

Sources

Reasoning

BS2 is not met. The available population evidence records no observed allele, homozygote, or healthy adult carrier for this variant; absence from gnomAD is not an observation of a healthy adult individual. No separate healthy-adult carrier observation is supplied, so the BS2 requirement is not satisfied.

Rule

Observed in a healthy adult individual for a recessive (homozygous), dominant (heterozygous), or X-linked (hemizygous) disorder, with full penetrance expected at an early age.

Evidence considered

• gnomAD v2.1 and v4.1 report the variant as absent, with no carriers or homozygote count to evaluate.
• No healthy-adult individual carrying this variant is documented in the population evidence supplied for this case.

Sources

Reasoning

BS3 is not met. No well-established functional study demonstrates that p.Pro1675Leu has no damaging effect on TSC2 protein function or splicing. The standardized HEK293T assay instead found significantly reduced TSC2 signal and increased T389-phosphorylated S6K/total S6K ratio relative to wild type. Although an independent HEK293 cell-cycle study reported a P1675L G1 response similar to wild-type TSC2, that single readout does not establish normal overall protein function or normal splicing and does not overcome the damaging results from the controlled TSC1-TSC2/TORC1 assay.

Rule

Well-established in vitro or in vivo functional studies show no damaging effect on protein function or splicing.

Evidence considered

• Hoogeveen-Westerveld et al. 2013 (PMID:22903760), Table 1 Group 3: p.P1675L had significantly reduced TSC2 signal and increased T389-phosphorylated S6K/total S6K ratio compared with wild type, providing evidence against a no-damaging-effect conclusion.
• Gibson et al. 2002 (PMID:12205112): P1675L increased the G1 fraction similarly to wild-type TSC2 in the tested HEK293 cell-cycle assay, but this isolated readout did not demonstrate normal overall TSC2 function or normal splicing.

Sources

Reasoning

BP1 does not apply because the available TSC2 evidence is not consistent with a gene in which disease is caused primarily by truncating variants to the exclusion of established pathogenic missense variation. The exact Pro1675Leu missense variant is reported as pathogenic and recurrent, and functional studies evaluated its protein effect.

Rule

Missense variant in a gene for which primarily truncating variants are known to cause disease.

Evidence considered

• Langkau et al. 2002 (PMID:12111193), Results/Table 1: P1675L was classified as a pathogenic recurrent missense variant.
• Hoogeveen-Westerveld et al. 2013 (PMID:22903760), Group 3/Table 1: p.P1675L was experimentally evaluated and showed reduced TSC2 signal with increased T389-phosphorylated S6K/total S6K ratio.

Sources

Reasoning

The variant is a missense substitution, so the generic BP4 path requires REVEL only; its SpliceAI result cannot substitute for a gray-zone or contrary REVEL result. REVEL is 0.965, which is not <=0.29, the supplied generic BP4 supporting threshold; therefore BP4 is not met. BayesDel is not used because no generic ACMG-strength calibration is established for it.

Rule

Multiple lines of computational evidence suggest no impact on gene or gene product (conservation, evolutionary, splicing impact, etc). ... BP4 (supporting): <= 0.29 — ClinGen SVI REVEL calibration (Pejaver et al. 2022, Am J Hum Genet) (PMID:36413997)

Evidence considered

• Variant consequence is missense: NM_000548.5:c.5024C>T produces NP_000539.2:p.(Pro1675Leu), so BP4 uses the REVEL path only.
• Local REVEL lookup reports a score of 0.965; this does not meet the supplied BP4 supporting cutoff of <=0.29.
• SpliceAI max delta 0.008 was not counted for BP4 because splice prediction is not the applicable path for a missense variant; BayesDel 0.528466 was not used because no generic ACMG-strength calibration is established for BayesDel.
• The supplied generic calibration identifies the BP4 supporting threshold as <=0.29, from the ClinGen SVI REVEL calibration (Pejaver et al. 2022, PMID:36413997).

Sources

Reasoning

The governing rule is: "Reputable source recently reports variant as benign, but the evidence is not available to the laboratory to perform an independent evaluation." The exact ClinVar record is Pathogenic, not benign or likely benign, and reports zero expert-panel submissions; no reputable exact-variant benign or likely-benign assertion is present. Therefore BP6 is not met.

Rule

Reputable source recently reports variant as benign, but the evidence is not available to the laboratory to perform an independent evaluation.

Evidence considered

• ClinVar exact-match record NM_000548.5(TSC2):c.5024C>T (p.Pro1675Leu) reports Pathogenic and zero expert-panel submissions.
• No exact-variant benign or likely-benign assertion from a reputable source was identified in the supplied evidence.

Sources

Reasoning

The governing rule is: "The prevalence of the variant in affected individuals is significantly increased compared to the prevalence in controls. ... Note 1: Relative risk (RR) or odds ratio (OR), as obtained from case-control studies, is >5.0 and the confidence interval around the estimate of RR or OR does not include 1.0." The reviewed exact-variant reports describe affected TSC patients, including two unrelated patients in PMID:12111193, one patient in PMID:10570911, and one patient in PMID:12136241, but provide no unaffected-control counts, matched-ancestry control frequency, OR/RR, or confidence interval. The literature_pass.json packet has no packet.ai_classified_papers entries to verify as PS4. Therefore a case-control comparison and the required OR/RR threshold cannot be assessed; affected-case observations alone do not establish PS4.

Rule

The prevalence of the variant in affected individuals is significantly increased compared to the prevalence in controls. ... Note 1: Relative risk (RR) or odds ratio (OR), as obtained from case-control studies, is >5.0 and the confidence interval around the estimate of RR or OR does not include 1.0.

Evidence considered

• PMID:10570911 reports the exact variant under alternate numbering as P1657L/c.5042C>T in one sporadic TSC patient, confirmed de novo; it reports no control cohort or variant-specific population frequency.
• PMID:12111193 reports P1675L in two unrelated TSC patients and calls it recurrent; the extracted report supplies no unaffected-control comparison or variant-specific control frequency.
• PMID:12136241 reports c.5024C>T/p.1675P>L in patient BHM9001; parent-of-origin data are provided, but no controls or case-control counts are reported.
• PMID:12205112 states that P1675L had been reported in at least 10 unrelated TSC patients, but this functional paper does not provide unaffected-control counts or an OR/RR.
• PMID:22903760 evaluates p.P1675L functionally and does not provide case-control prevalence data.
• gnomAD v2.1 and gnomAD v4.1 report the variant absent, but the available evidence does not provide a matched case frequency and control denominator from which the required OR/RR and confidence interval could be calculated.
• No packet.ai_classified_papers entries were present in literature_pass.json for independent PS4 verification.

Still missing

• A case-control dataset or matched-ancestry control frequency for NM_000548.5:c.5024C>T is required.
• An OR/RR and 95% confidence interval cannot be calculated without case and control counts or frequencies.

Sources

Reasoning

PMID:12205112 places Pro1675 within the TSC2 GAP-related domain and describes the residue as highly conserved. However, the generic PM1 definition requires a critical and well-established functional domain without benign variation, and the available evidence does not establish the relevant domain's benign-variation exclusion. The Cancer Hotspots result was negative, and no VCEP domain table is present.

Rule

Located in a mutational hot spot and/or critical and well-established functional domain (e.g. active site of an enzyme) without benign variation.

Evidence considered

• Kwiatkowski et al. 2002 (PMID:12205112), Figure 1B/functional-analysis discussion: P1675L is within the GAP-related domain and the residue is highly conserved.
• Cancer Hotspots query for TSC2 P1675: no statistically significant hotspot row was found.

Still missing

• A source establishing that the TSC2 GAP-related domain is a critical, well-established functional domain without benign variation.

Sources

Reasoning

The supplied evidence documents the observed P1675Leu change and does not provide a confirmed pathogenic comparator involving a different amino-acid substitution at residue 1675. The PM5 candidate artifact contains zero same-residue candidates, so PM5 cannot be applied or excluded from the available evidence alone.

Rule

Novel missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before.

Evidence considered

• The case's PM5 comparator collection reported zero same-residue candidates; no qualifying different-missense pathogenic comparator was available for adjudication.

Still missing

• A verified pathogenic missense variant at TSC2 residue 1675 with an amino acid change different from Pro1675Leu.

Reasoning

The available variant-specific evidence documents a sporadic/de novo observation and parent-of-origin information, but no affected relatives, informative meioses, or co-segregation results. The multiple unrelated observations are not segregation within a family and cannot establish PP1.

Rule

Co-segregation with disease in multiple affected family members in a gene definitively known to cause the disease. ... May be used as stronger evidence with increasing segregation data.

Evidence considered

• Zhang et al. 1999 evaluated a sporadic patient and parental samples but did not report affected-relative segregation.
• Roberts et al. 2002 reported the variant in patient BHM9001 and identified maternal parent of origin, but no segregation analysis beyond parent of origin was provided.

Still missing

• Family pedigree and genotype-phenotype segregation data are unavailable.

Sources

Reasoning

The available evidence supports TSC2 as a loss-of-function disease gene and documents pathogenic missense variation, including Pro1675Leu, but it does not establish both required PP2 conditions: a low rate of benign missense variation and missense variants as a common disease mechanism. No gene-level benign-missense rate or validated mechanism-frequency assessment is supplied.

Rule

Missense variant in a gene that has a low rate of benign missense variation and where missense variants are a common mechanism of disease.

Evidence considered

• The case evidence describes TSC2 loss-of-function biology and pathogenic missense observations but provides no validated gene-level PP2 assessment or quantitative benign-missense rate.

Still missing

• A gene-level assessment demonstrating low benign missense variation in TSC2.
• A gene-level assessment demonstrating that missense variation is a common disease mechanism in TSC2.

Reasoning

The governing rule is: "Patient’s phenotype or family history is highly specific for a disease with a single genetic etiology." The supplied case evidence identifies the TSC2 variant and literature reports of patients with tuberous sclerosis, but does not provide the evaluated patient's phenotype, family history, or a sufficiently specific clinical presentation. PP4 therefore cannot be assessed from the available case-level information.

Rule

Patient’s phenotype or family history is highly specific for a disease with a single genetic etiology.

Evidence considered

• The case materials identify TSC2-related disease context but do not provide the evaluated patient's phenotype or family history needed for PP4.

Still missing

• The evaluated patient's clinical phenotype and family history are required.
• The phenotype must be assessed for high specificity to a disease with a single genetic etiology.

Reasoning

No affected family members with negative testing for the variant are reported. The available reports provide parental testing or parent-of-origin information, not lack of segregation in affected relatives; therefore BS4 cannot be assessed.

Rule

Lack of segregation in affected members of a family.

Evidence considered

• The available variant-specific reports describe parental testing or maternal parent of origin, but do not report an affected relative who lacks the variant.

Still missing

• Affected-relative genotypes and clinical status are unavailable.

Sources

Reasoning

BP2 requires either observation in trans with a pathogenic variant in a fully penetrant dominant disorder or observation in cis with a pathogenic variant in any inheritance pattern. The available evidence reports the variant in affected individuals and includes parent-of-origin or de novo findings, but does not document a second pathogenic variant, cis/trans phase, or an affected-proband observation establishing either BP2 configuration. Therefore BP2 cannot be assessed from the available evidence.

Rule

BP2: Observed in trans with a pathogenic variant for a fully penetrant dominant gene/disorder or observed in cis with a pathogenic variant in any inheritance pattern.

Evidence considered

• Roberts et al. 2002 (PMID:12136241), Table 6, patient BHM9001: c.5024C>T/p.1675P>L was reported with maternal parent of origin, but no second pathogenic variant or cis/trans phase was reported.
• Zhang et al. 1999 (PMID:10570911), patient P28: the corresponding P1675L-equivalent variant was confirmed de novo by parental testing; de novo status does not establish cis or trans with another pathogenic variant.
• Langkau et al. 2002 (PMID:12111193): P1675L was reported in two unrelated patients as a recurrent mutation, without a variant-specific second pathogenic allele or cis/trans phase result.

Still missing

• A second pathogenic variant and validated cis/trans phase relative to NM_000548.5:c.5024C>T are not documented.
• The available reports do not provide a qualifying BP2 observation in an affected proband.

Sources

Reasoning

The governing rule is: "Variant found in a case with an alternate molecular basis for disease." The available evidence reports the variant in individuals with tuberous sclerosis and provides functional data, but does not establish an alternate molecular diagnosis or another pathogenic molecular basis in a specific case. BP5 cannot be assessed without case-level evidence of an alternate molecular basis.

Rule

Variant found in a case with an alternate molecular basis for disease.

Evidence considered

• The supplied case and literature materials do not document an alternate molecular basis for disease in a specific patient carrying NM_000548.5:c.5024C>T.

Still missing

• A case-level alternate molecular diagnosis or pathogenic variant explaining the disease independently of NM_000548.5:c.5024C>T is required.