NF1 c.8161-1_8161delinsCT, p.?

Reasoning

NM_001042492.2:c.8161-1_8161delinsCT alters the canonical -1 acceptor site for NF1 exon 56, and NF1 loss of function is an established germline disease mechanism. The affected exon is represented in the biologically relevant RefSeq transcript and is 217 nucleotides long within an 8,520-nucleotide coding sequence. Under the ClinGen SVI PVS1 framework (PMC6185798), canonical splice variants require assessment of exon skipping and cryptic splice use; skipping this exon would shift the downstream reading frame in the terminal exon, where a resulting premature termination is predicted to escape NMD. Because the affected exon removes less than 10% of the coding sequence and no RNA or functional assay establishes the exact aberrant transcript or demonstrates that the lost C-terminal region is independently critical, the named SVI NMD-escape and <10%-protein-loss downgrade rule supports PVS1 at moderate strength rather than very strong strength. SpliceAI max delta 0.996 supports substantial splice disruption but is not stacked as separate PP3 evidence for this canonical splice assessment.

Rule

ClinGen SVI PVS1 generic framework: canonical splice consequence with predicted NMD escape and less than 10% protein/coding-sequence loss is downgraded to moderate strength when criticality of the retained/lost C-terminal region is not independently established.

Evidence considered

• ClinGen SVI PVS1 recommendations state that a premature termination in the 3'-most exon is generally predicted to escape NMD; when the affected region is not shown to be independently critical, removal of less than 10% of the protein supports PVS1 at moderate strength rather than strong strength (PMID:30192042, PMC6185798).
• VariantValidator maps NM_001042492.2:c.8161-1_8161delinsCT to the canonical acceptor immediately before NF1 exon 56; the transcript annotation places exon 56 at transcript positions 8544-8760 and the terminal exon at 8761-12425, with the coding sequence at positions 384-8903.
• The case gene-level assessment supports NF1 loss of function as a germline disease mechanism and marks the generic PVS1 framework as eligible.
• SpliceAI reports DS_AG 0.702, DS_AL 0.996, DS_DG 0.000, and DS_DL 0.290, with maximum delta 0.996, indicating strong predicted splice disruption.

Still missing

• RNA studies are needed to determine whether exon 56 is skipped, a cryptic splice site is used, or another transcript consequence occurs.
• Functional evidence is unavailable to establish whether the altered C-terminal region is independently critical to NF1 function.

Sources

Reasoning

The retrieved NF1 ClinGen specification contains no structured PM2 rule, so the supplied generic PM2 Supporting threshold of allele frequency <= 0.0001 applies. The variant is absent from gnomAD v2.1 and gnomAD v4.1, and the case evidence also reports absence from GNOMAD_V2_1_NON_CANCER exomes and GNOMAD_V3_1_NON_CANCER genomes. These findings are consistent with an allele frequency of zero in each queried dataset and satisfy PM2 at supporting strength. No governing NF1 rule was available that required a non-cancer or exome-only source instead of the default all-comers datasets.

Rule

PM2 Supporting: variant absent from or at extremely low frequency in an appropriate population dataset, using AF <=0.0001 when no gene-specific rule is available (ClinGen SVI, PMID:25741868).

Evidence considered

• The supplied generic PM2 Supporting threshold is allele frequency <=0.0001 (ClinGen SVI recommendation, PMID:25741868); no retrievable NF1-specific rule overrides it.
• gnomAD v2.1 all-comers reports search_status absent and found false for the variant.
• gnomAD v4.1 all-comers reports search_status absent and found false for the variant.
• The case evidence separately reports absence from GNOMAD_V2_1_NON_CANCER and GNOMAD_V3_1_NON_CANCER; no governing NF1 rule was available that mandated those subset sources.

Still missing

• The NF1 specification was retrieved without structured PM2 rules, so the supplied generic threshold was used.
• The absent records provide no denominators or confidence intervals, only a zero-observation result.

Sources

Reasoning

The variant is a canonical intronic splice-site variant, c.8161-1_8161delinsCT, with no missense consequence; therefore the applicable computational path is SpliceAI only. The NF1 ClinGen specification is present but contains no usable PP3/BP4 rule payload, so the supplied generic calibration applies. SpliceAI reports DS_AG 0.702, DS_AL 0.996, DS_DG 0.000, and DS_DL 0.290; the maximum delta is 0.996. This meets the generic SpliceAI PP3 moderate threshold of >=0.5 from Jaganathan et al. 2019 (PMID:30661751). The SpliceAI result is used only for PP3/BP4 in this group and is not supplemented with REVEL, which was not applicable to this non-missense indel.

Rule

Generic SpliceAI PP3 moderate: maximum delta score >=0.5.

Evidence considered

• The variant is c.8161-1_8161delinsCT at the canonical -1 splice position, so SpliceAI is the applicable computational path.
• SpliceAI reports a maximum delta score of 0.996 (DS_AL), with DS_AG 0.702, DS_DG 0.000, and DS_DL 0.290.
• The NF1 ClinGen specification is available, but its extracted rule payload contains no PP3/BP4-specific assignment; the supplied generic SpliceAI calibration therefore applies.
• The PP3 moderate threshold is >=0.5, from the Jaganathan et al. 2019 SpliceAI calibration (PMID:30661751).

Sources

Reasoning

PP5 is restricted here to an exact-variant ClinVar Expert Panel classification of Pathogenic or Likely pathogenic. ClinVar returned no exact-variant record for NM_001042492.2:c.8161-1_8161delinsCT, and therefore no qualifying Expert Panel assertion is present; ordinary submissions or aggregate labels cannot substitute.

Rule

Apply PP5 only when an exact-variant ClinVar Expert Panel classification is Pathogenic or Likely pathogenic; ordinary laboratory submissions, aggregate labels, and non-Expert Panel assertions do not qualify.

Evidence considered

• ClinVar search for the exact variant returned no result; consequently, no Expert Panel Pathogenic or Likely pathogenic assertion was available.

Sources

Reasoning

The NF1 ClinGen specification is present but provides no retrievable criterion-specific population rule, so the supplied generic BA1 stand-alone threshold of allele frequency >= 0.05 applies. The variant is absent from gnomAD v2.1 and gnomAD v4.1, and the case evidence also reports absence from the available gnomAD v2.1 non-cancer exome and gnomAD v3.1 non-cancer genome entries. No observed allele frequency, ancestry enrichment, or homozygote count can approach the BA1 threshold. BA1 is not met.

Rule

BA1 stand-alone benign: allele frequency >=0.05 in an appropriate population dataset (ACMG/AMP 2015, PMID:25741868); no retrievable NF1-specific override.

Evidence considered

• The retrieved NF1 ClinGen specification has no criterion-specific population rule payload; the supplied generic BA1 stand-alone threshold of allele frequency >=0.05 therefore applies (ACMG/AMP 2015, PMID:25741868).
• gnomAD v2.1 all-comers reports search_status absent and found false for the variant.
• gnomAD v4.1 all-comers reports search_status absent and found false for the variant.
• The case evidence separately reports absence from GNOMAD_V2_1_NON_CANCER and GNOMAD_V3_1_NON_CANCER; no governing NF1 rule was available that required those subsets instead of the default all-comers sources.

Still missing

• The NF1 specification was retrieved without structured population rules, so no NF1-specific BA1 threshold or population-source override could be applied.
• The available gnomAD records contain no AC, AN, ancestry, or homozygote counts because the variant was absent.

Sources

Reasoning

The NF1 ClinGen specification is present but provides no retrievable criterion-specific population rule, so the supplied generic BS1 threshold of allele frequency >= 0.01 applies. The variant is absent from gnomAD v2.1 and gnomAD v4.1, with no observed frequency or ancestry-specific enrichment; it is also reported absent from the available non-cancer subset entries. Therefore the population data do not support an allele frequency greater than expected for NF1 disease, and BS1 is not met.

Rule

BS1 strong benign: allele frequency >=0.01, with per-gene derivation preferred (Whiffin et al. 2017, PMID:28518168); no retrievable NF1-specific override.

Evidence considered

• The supplied generic BS1 threshold is allele frequency >=0.01, with per-gene derivation preferred (Whiffin et al. 2017, PMID:28518168); no retrievable NF1-specific rule overrides it.
• gnomAD v2.1 all-comers reports the variant as absent.
• gnomAD v4.1 all-comers reports the variant as absent.
• The case evidence also reports absence from GNOMAD_V2_1_NON_CANCER and GNOMAD_V3_1_NON_CANCER; no governing NF1 rule was available that mandated these subsets.

Still missing

• The NF1 specification was retrieved without structured population rules, so no NF1-specific BS1 threshold or population-source override could be applied.
• No disease-prevalence or penetrance-calibrated NF1 frequency threshold is present in the available evidence.

Sources

Reasoning

The variant is a canonical intronic splice-site variant, c.8161-1_8161delinsCT, so SpliceAI is the applicable and exclusive computational path; REVEL is not applicable to this non-missense indel. SpliceAI reports a maximum delta score of 0.996, which is above the generic SpliceAI BP4 supporting threshold of <=0.1 from Jaganathan et al. 2019 (PMID:30661751). Therefore the prediction does not support absence of splice impact and BP4 is not met. The NF1 ClinGen specification is present but contains no usable PP3/BP4 rule payload.

Rule

Generic SpliceAI BP4 supporting: maximum delta score <=0.1.

Evidence considered

• The variant is c.8161-1_8161delinsCT at the canonical -1 splice position, so SpliceAI is the applicable computational path.
• SpliceAI reports a maximum delta score of 0.996 (DS_AL), indicating a predicted splice impact rather than a tolerated splice change.
• The BP4 supporting threshold is <=0.1, from the Jaganathan et al. 2019 SpliceAI calibration (PMID:30661751); the observed score is 0.996.
• REVEL was not evaluated because this is not a missense variant, and the NF1 ClinGen specification contains no usable PP3/BP4-specific rule payload.

Sources

Reasoning

BP6 is restricted here to an exact-variant ClinVar Expert Panel classification of Benign or Likely benign. ClinVar returned no exact-variant record for NM_001042492.2:c.8161-1_8161delinsCT, so no qualifying Expert Panel assertion is present; ordinary submissions or aggregate labels cannot substitute.

Rule

Apply BP6 only when an exact-variant ClinVar Expert Panel classification is Benign or Likely benign; ordinary laboratory submissions, aggregate labels, and non-Expert Panel assertions do not qualify.

Evidence considered

• ClinVar search for the exact variant returned no result; consequently, no Expert Panel Benign or Likely benign assertion was available.

Sources

Reasoning

PS2 cannot be assessed because the available case evidence contains no proband-level confirmation of a de novo variant, parental genotypes, maternity/paternity confirmation, or information about the number of tested affected and unaffected relatives. The NF1 ClinGen specification is present but contains no criterion-specific PS2 rule in the retrieved framework payload, so generic ACMG/AMP PS2 evidence requirements apply.

Rule

Generic ACMG/AMP PS2: de novo occurrence in a patient with the disease and no evidence of the variant in either parent; maternity and paternity confirmed for full-strength use.

Evidence considered

• The case evidence identifies NM_001042492.2:c.8161-1_8161delinsCT in NF1 but provides no family segregation or de novo testing data.
• The retrieved NF1 ClinGen framework is incomplete for criterion-specific PS2 instructions, so generic ACMG/AMP requirements were used.

Still missing

• Document the proband's variant result and phenotype.
• Document variant testing in both biological parents.
• Confirm maternity and paternity or otherwise establish biological parentage.
• Provide the number and phenotype of tested relatives, if any.

Sources

Reasoning

The NF1 ClinGen Neurofibromatosis and Schwannomatosis specification was reviewed, but its criterion-specific rule payload is empty and provides no PS3 calibration. The case contains no validated patient RNA study, minigene assay, RT-PCR result, transcript quantification, protein assay, or other functional experiment for NM_001042492.2:c.8161-1_8161delinsCT. SpliceAI predicts splice impact with a maximum delta score of 0.996, but this is computational evidence rather than an experimental assay and cannot establish PS3. Therefore PS3 cannot be assessed.

Evidence considered

• The NF1 ClinGen specification is applicable, but its retrieved criterion-specific rule payload is empty and does not provide a PS3-specific calibration.
• No variant-specific experimental functional evidence was found in the case evidence: there is no patient RNA analysis, minigene assay, RT-PCR, transcript quantification, protein assay, or other validated functional study.
• SpliceAI reports a maximum delta score of 0.996, which predicts splice impact but is computational evidence and does not satisfy the experimental requirement for PS3.

Still missing

• A validated assay directly testing the variant's RNA splicing or NF1 function is absent.
• Assay controls, replicate results, and biologically relevant experimental context are absent.

Sources

Reasoning

PS4 requires disease enrichment in affected individuals or a validated case-control comparison. No variant-specific affected/unaffected cohort counts, odds ratio, or other case-control enrichment result are available for NM_001042492.2:c.8161-1_8161delinsCT, so PS4 cannot be assessed.

Rule

Generic ACMG/AMP PS4 requires significantly increased prevalence of the variant in affected individuals compared with appropriate controls; no applicable NF1 criterion-specific rule text was retrieved from the otherwise applicable VCEP framework.

Evidence considered

• No variant-specific case-control cohort counts, odds ratio, or other validated enrichment statistic were available.

Still missing

• Variant-specific case-control data or an appropriately documented affected-case enrichment analysis are unavailable.

Reasoning

PM6 cannot be assessed because the available case evidence contains no credible de novo observation without parental testing, no proband-level phenotype documentation, and no information establishing whether the parents were tested or whether parentage was confirmed. The NF1 ClinGen specification is present but contains no criterion-specific PM6 rule in the retrieved framework payload, so generic ACMG/AMP PM6 evidence requirements apply.

Rule

Generic ACMG/AMP PM6: assumed de novo occurrence in a patient with the disease, without confirmation of maternity and/or paternity; evidence is weaker than PS2.

Evidence considered

• The case evidence identifies the NF1 variant but provides no parental testing outcome, family history, or proband phenotype suitable for a de novo assessment.
• The retrieved NF1 ClinGen framework is incomplete for criterion-specific PM6 instructions, so generic ACMG/AMP requirements were used.

Still missing

• Document the proband's disease phenotype and variant result.
• Document the evidence that the variant is absent from both parents or that parental testing was unavailable.
• Provide any available maternity/paternity or family-structure information.
• Assess alternative explanations for an apparent de novo finding, including mosaicism.

Sources

Reasoning

PP1 cannot be assessed because the available case evidence contains no affected relatives, unaffected relatives, informative meioses, co-segregation results, or phenotype assessments for family members. The NF1 ClinGen specification is present but contains no criterion-specific PP1 rule in the retrieved framework payload, so generic ACMG/AMP segregation evidence requirements apply.

Rule

Generic ACMG/AMP PP1: co-segregation with disease in multiple affected family members in a gene definitively known to cause the disease; strength depends on the amount and informativeness of segregation evidence.

Evidence considered

• The case evidence contains no family pedigree, relative genotypes, segregation results, or informative meiosis count.
• The retrieved NF1 ClinGen framework is incomplete for criterion-specific PP1 instructions, so generic ACMG/AMP requirements were used.

Still missing

• Provide a pedigree and the phenotype of each informative relative.
• Provide variant genotypes for affected and unaffected relatives.
• Report the number of informative meioses and any non-informative relatives.
• Address phenocopies, reduced penetrance, and age-dependent expression where relevant.

Sources

Reasoning

PP4 requires a highly specific phenotype or family history consistent with the disease. The available case evidence does not provide a patient phenotype, diagnostic criteria, or family-history description that can be evaluated for NF1 specificity, so PP4 cannot be assessed.

Rule

Generic ACMG/AMP PP4 requires a phenotype or family history highly specific for the disease; no applicable NF1 criterion-specific rule text was retrieved from the otherwise applicable VCEP framework.

Evidence considered

• No patient-level phenotype or family-history description suitable for assessing disease specificity was available.

Still missing

• Detailed patient phenotype and family-history information are unavailable.

Reasoning

The retrieved NF1 specification contains no structured BS2 rule, and the available population evidence only establishes that the variant is absent from gnomAD v2.1 and v4.1 and from the reported non-cancer subset entries. Absence does not demonstrate observation in a healthy adult, homozygote, heterozygote, or hemizygote, and the case bundle does not provide NF1-specific penetrance, age-of-onset, or disease-prevalence data needed to assess whether the BS2 full-penetrance premise applies. BS2 cannot be adjudicated from the available evidence.

Rule

BS2 strong benign: observation in a healthy adult for a disorder with full penetrance expected at an early age (ACMG/AMP 2015, PMID:25741868); insufficient observations and disease-model data here.

Evidence considered

• Generic BS2 requires observation in a healthy adult for a disorder in which full penetrance is expected at an early age (ACMG/AMP 2015, PMID:25741868).
• gnomAD v2.1 and gnomAD v4.1 report the variant as absent, so they provide no healthy-carrier or homozygote observation.
• The case evidence reports the variant as absent from the available gnomAD v2.1 non-cancer exome and gnomAD v3.1 non-cancer genome entries as well.

Still missing

• No healthy-adult observation, carrier genotype, homozygote count, or hemizygote observation is available for this variant.
• No NF1-specific penetrance, age-of-onset, or prevalence information is present to determine whether the BS2 full-penetrance premise applies.
• The NF1 specification was retrieved without a structured BS2 rule.

Sources

Reasoning

The NF1 ClinGen Neurofibromatosis and Schwannomatosis specification was reviewed, but its criterion-specific rule payload is empty and provides no BS3 calibration. The case contains no validated assay demonstrating normal splicing, normal NF1 expression, or preserved NF1 function for NM_001042492.2:c.8161-1_8161delinsCT. The available SpliceAI result predicts splice impact with a maximum delta score of 0.996 and does not provide benign functional evidence. Therefore BS3 cannot be assessed.

Evidence considered

• The NF1 ClinGen specification is applicable, but its retrieved criterion-specific rule payload is empty and does not provide a BS3-specific calibration.
• No variant-specific experimental evidence demonstrating normal splicing, normal NF1 expression, or preserved NF1 function was found.
• SpliceAI reports a maximum delta score of 0.996 and is not a benign functional assay; it cannot support BS3.

Still missing

• A validated assay demonstrating normal variant-specific splicing or NF1 function is absent.
• Appropriate positive and negative controls, replicate results, and biologically relevant experimental context are absent.

Sources

Reasoning

BS4 cannot be assessed because the available case evidence contains no affected relatives who were tested, no genotype results showing absence of the variant in affected informative relatives, and no information about phenotype, penetrance, parentage, or the number of informative meioses. The NF1 ClinGen specification is present but contains no criterion-specific BS4 rule in the retrieved framework payload, so generic ACMG/AMP non-segregation evidence requirements apply.

Rule

Generic ACMG/AMP BS4: lack of segregation in affected members of a family; application requires informative family testing and consideration of penetrance, phenocopy, and parentage.

Evidence considered

• The case evidence contains no tested unaffected relatives or family-level non-segregation data.
• The retrieved NF1 ClinGen framework is incomplete for criterion-specific BS4 instructions, so generic ACMG/AMP requirements were used.

Still missing

• Identify affected relatives evaluated for the familial variant.
• Provide their genotypes and clinical phenotypes.
• Provide the pedigree, penetrance context, and number of informative meioses.
• Exclude phenocopy, non-paternity, and reduced or age-dependent penetrance before applying BS4.

Sources

Reasoning

BP2 could be considered for an autosomal dominant disorder if the variant were observed in trans with a pathogenic NF1 variant in an affected individual, but the available case evidence contains no affected-proband genotype, second pathogenic NF1 variant, or phase determination. The criterion cannot be assessed from the available evidence.

Rule

For an autosomal dominant disorder, assess BP2 only when the variant is observed in trans with a pathogenic variant in an affected individual; no such observation or phase evidence is available.

Evidence considered

• The NF1 ClinGen specification identifies neurofibromatosis type 1 as autosomal dominant, making a trans observation with another pathogenic NF1 variant the relevant BP2 scenario.
• The case evidence reports no affected-proband observations, second pathogenic NF1 variant, parental testing, or cis/trans phase information.
• The variant is absent from ClinVar, so no variant-specific clinical submission supplies the missing allelic or phase evidence.

Still missing

• Affected-proband genotype and phenotype documentation are unavailable.
• No second pathogenic or likely pathogenic NF1 variant is documented.
• No parental testing or other phase evidence establishes trans configuration.

Sources

Reasoning

BP5 requires evidence that the affected phenotype is explained by an alternate molecular basis. No documented alternate pathogenic variant, alternate molecular diagnosis, or phenotype attribution resolving the case was provided, so BP5 cannot be assessed.

Rule

Generic ACMG/AMP BP5 requires a pathogenic variant in an alternative gene that explains the patient's phenotype; no applicable NF1 criterion-specific rule text was retrieved from the otherwise applicable VCEP framework.

Evidence considered

• No alternate pathogenic variant, molecular diagnosis, or documented alternate explanation for the phenotype was available.

Still missing

• An evaluated alternate molecular diagnosis or documented alternate cause of the phenotype is unavailable.