DICER1 c.735-23A>G, p.?

Reasoning

NM_177438.3:c.735-23A>G sits at intronic position -23, outside the DICER1 VCEP's MaxEntScan-validated acceptor window (intron -20 through first 3 nt of exon), so per the VCEP's own PP3/BP4 instructions SpliceAI scores alone should be used for this variant. SpliceAI predicts no splicing effect (DS_AG=0.001, DS_AL/DG/DL=0.0, max delta score=0.001), consistent with the evidence_sentence 'SpliceAI predicts no significant splice impact for this variant (max delta score = 0.00).' This satisfies the BP4 rule for intronic variants (concordance of splicing predictors showing no effect, applied via the VCEP-mandated SpliceAI-only path for this out-of-MES-range position).

Rule

For synonymous/intronic/non-coding variants, concordance of MaxEntScan and SpliceAI (SpliceAI alone applied here per VCEP instructions since variant is outside the MES-validated window)

Evidence considered

• SpliceAI Lookup result for NM_177438.3:c.735-23A>G: DS_AG=0.001, DS_AL=0.0, DS_DG=0.0, DS_DL=0.0, max delta score=0.001.
• DICER1 VCEP cspec BP4 rule: 'For missense variants, REVEL score < 0.500 and agreement in splicing predictors that no splicing effects are predicted. For synonymous/intronic/non-coding variants concordance of MaxEntScan and SpliceAI.'
• DICER1 VCEP cspec PP3/BP4 instructionsToUse: SpliceAI scores alone govern for variants outside the MES-validated acceptor range of intron -20 through exon+3; c.735-23 lies at position -23, outside this range.

Sources

Reasoning

BP4 is met for this variant (SpliceAI predicts no splicing impact), satisfying the DICER1 VCEP's BP7 caveat that 'Variant must meet BP4 to apply BP7'. The variant is intronic at position c.735-23, which is beyond -21 from the acceptor site, meeting the BP7 rule clause 'Intronic variant at or beyond +7 to -21 positions' directly without needing to invoke the conservation-based alternate clause.

Rule

Intronic variant at or beyond +7 to -21 positions, with BP4 prerequisite met

Evidence considered

• DICER1 VCEP cspec BP7 rule: 'Silent variant OR Intronic variant at or beyond +7 to -21 positions OR Other intronic or non-coding variant if the variant is the reference nucleotide in >=1 primate and/or >=4 mammalian species. Caveat: Variant must meet BP4 to apply BP7.'
• Variant position c.735-23A>G is at intron position -23, beyond the -21 threshold specified in the BP7 rule.
• BP4 determination (this group): SpliceAI max delta score 0.001, no splicing effect predicted, satisfying the BP4 prerequisite for BP7.

Sources

Reasoning

The governing DICER1 VCEP allows PM2 at Supporting only when allele frequency is <0.000005 across gnomAD, with no more than one allele in any subpopulation and at least 20x coverage. gnomAD v4.1, the most recent and comprehensive dataset specified for use by the VCEP, reports overall AF 1.3798595697203617e-05 (21/1,521,894 alleles), 19 alleles in the European non-Finnish subpopulation, and maximum subpopulation AF 1.7406440382941687e-05. These observations exceed both the frequency and per-subpopulation allele-count requirements; therefore PM2 is not met.

Rule

Allele frequency <0.000005 across gnomAD with no more than one allele in any subpopulation and at least 20x coverage.

Evidence considered

• ClinGen DICER1 VCEP v1.4 specifies PM2 Supporting as allele frequency <0.000005 across gnomAD, with no more than one allele in any subpopulation and at least 20x coverage; it also instructs users generally to use the most recent or most comprehensive gnomAD version.
• gnomAD v4.1 reports overall AF 1.3798595697203617e-05 (21/1,521,894 alleles), maximum subpopulation AF 1.7406440382941687e-05 in Admixed American individuals, and 19 alleles in the European non-Finnish subpopulation; homozygotes = 0.

Still missing

• The bundle does not provide a per-variant 20x coverage confirmation, although the PM2 frequency and allele-count requirements are already not met in gnomAD v4.1.

Sources

Reasoning

NM_177438.3:c.735-23A>G is a deep intronic variant (position -23, acceptor side), outside the DICER1 VCEP's MaxEntScan validation range (intron -20 through first 3 nt of exon for acceptor sites). Per VCEP instructions, SpliceAI alone governs for variants outside this MES-validated window. SpliceAI predicts no splicing effect (max delta score 0.001, all four delta scores <=0.001), which is far below any threshold indicating a splicing effect, so the PP3 splicing-impact path does not apply. The variant is not missense (it is intronic/non-coding), so the REVEL/BayesDel missense sub-path is not applicable, and no verified published BayesDel threshold is available to this pipeline regardless. No basis exists to apply PP3.

Rule

For splicing variants, concordance of MaxEntScan and SpliceAI (MES alone excluded outside MES-validated acceptor window; SpliceAI governs)

Evidence considered

• SpliceAI Lookup result for NM_177438.3:c.735-23A>G: DS_AG=0.001, DS_AL=0.0, DS_DG=0.0, DS_DL=0.0, max delta score=0.001, evidence_sentence 'SpliceAI predicts no significant splice impact for this variant (max delta score = 0.00).'
• DICER1 VCEP cspec PP3 instructionsToUse: 'All variants should be assessed by MaxEntScan (MES) and SpliceAI for predicting de novo and cryptic splice sites. However, for predicting impact to consensus splice sites, SpliceAI scores alone should be considered for variants outside the MES validation threshold... (MES validation threshold = ... intronic position -20 through first 3 nucleotides of exon (acceptor sites))' - this variant at position -23 falls outside that window.
• DICER1 VCEP cspec PP3 rule: 'For missense variants, REVEL score >= 0.750 OR agreement in splicing predictors predict splicing effects. For splicing variants, concordance of MaxEntScan and SpliceAI.' - not met since SpliceAI shows no effect and this is not a missense variant.
• prefetch.json revel: score null, found false; bayesdel: score null, found false, so no missense in-silico evidence is available regardless.

Sources

Reasoning

The governing DICER1 VCEP specification requires a frequency >0.003 in a gnomAD subpopulation, with >2,000 alleles tested and at least 5 alleles present. The highest reported gnomAD v4.1 subpopulation frequency is 1.7406440382941687e-05 in Admixed American individuals (1/57,450 alleles); this is far below the BA1 threshold.

Rule

Frequency >0.003 (0.3%) in gnomAD subpopulations. Subpopulations must have >2,000 alleles tested and a minimum of 5 alleles present.

Evidence considered

• ClinGen DICER1 VCEP v1.4 specifies BA1 as frequency >0.003 in a gnomAD subpopulation, with >2,000 alleles tested and at least 5 alleles present.
• gnomAD v4.1 reports the highest subpopulation frequency as 1.7406440382941687e-05 (1/57,450 alleles) in the Admixed American population; the overall frequency is 1.3798595697203617e-05 (21/1,521,894 alleles).

Sources

Reasoning

The governing DICER1 VCEP specification requires a frequency >0.0003 in a gnomAD subpopulation, with >2,000 alleles tested and at least 5 alleles present. The highest reported gnomAD v4.1 subpopulation frequency is 1.7406440382941687e-05 in Admixed American individuals (1/57,450 alleles), which is below the BS1 threshold.

Rule

Frequency >0.0003 (0.03%) in gnomAD subpopulations. Subpopulations must have >2,000 alleles tested and a minimum of 5 alleles present.

Evidence considered

• ClinGen DICER1 VCEP v1.4 specifies BS1 as frequency >0.0003 in a gnomAD subpopulation, with >2,000 alleles tested and at least 5 alleles present.
• gnomAD v4.1 reports the highest subpopulation frequency as 1.7406440382941687e-05 (1/57,450 alleles) in the Admixed American population; this is below 0.0003.

Sources

Reasoning

The DICER1 VCEP permits BS2 Strong for 2 or more observations of homozygosity in healthy individuals, or one homozygous healthy individual with parental confirmation, and permits BS2 Supporting for 2 or more homozygous observations in individuals lacking clinical information. gnomAD v4.1 reports zero homozygotes, and the case bundle contains no qualifying healthy-individual observations or parental testing.

Rule

Strong: 40+ unrelated females from a single source are tumor-free through age 50, or 2+ observations of homozygosity in healthy individuals, or 1+ observation of homozygosity in a healthy individual with status confirmed by parental testing. Supporting: 10+ unrelated females from a single source are tumor-free through age 50, or 2+ observations of homozygosity in individuals lacking clinical information.

Evidence considered

• ClinGen DICER1 VCEP v1.4 requires qualifying homozygous observations in healthy individuals or the specified healthy cohort criteria for BS2.
• gnomAD v4.1 reports 0 homozygotes for the variant; no individual-level healthy homozygote observations, clinical status, or parental confirmation are provided in the case evidence.

Sources

Reasoning

The ClinGen DICER1 VCEP Version 1.4 permits PS2 based on de novo points, with both maternity and paternity confirmation required for the standard de novo evidence model. The case-evidence bundle contains no proband-level de novo observation, parental genotypes, maternity or paternity confirmation, phenotype-linked case report, or de novo point tally for NM_177438.3:c.735-23A>G. Therefore PS2 cannot be assessed.

Rule

De novo points should be tallied using the simplified table for tallying proband points; PS2 is Very Strong for >=4 points, Strong for >=2 and <4 points, Moderate for >=1 and <2 points, and Supporting for >=0.5 and <1 point. The VCEP drops PM6 and uses PS2 instead for de novo evidence.

Evidence considered

• The governing ClinGen DICER1 VCEP Version 1.4 specifies that de novo points determine PS2 strength and that both maternity and paternity should be confirmed.
• The consolidated case-evidence bundle provides no case-specific proband, parental-testing, maternity, paternity, or de novo segregation data for this variant.

Still missing

• Proband phenotype and disease-specific clinical context
• Parental testing results for the variant
• Documented maternity and paternity confirmation
• Independent de novo point tally and source-level case documentation

Sources

Reasoning

No RNA splicing assay or in vitro pre-miRNA cleavage assay data for NM_177438.3:c.735-23A>G were found in the case evidence bundle. The DICER1 VCEP specification (v1.4) requires either an RNA assay demonstrating splicing impact (out-of-frame, in-frame >=193 residues, or RNase IIIb disruption) or an in vitro cleavage assay showing failure/reduced production of 5p or 3p microRNAs (with positive and negative controls) to apply PS3. Literature triage returned only a general ACMG/AMP guidelines paper (PMID:25741868), with no variant-specific functional study identified. SpliceAI computational prediction (max delta score 0.00, no significant splice impact predicted) is not equivalent to functional assay evidence and cannot substitute for PS3.

Still missing

• No RNA-based splicing assay (e.g., minigene, patient RNA/cDNA analysis) for this variant identified in the evidence bundle or literature triage.
• No in vitro pre-miRNA cleavage assay data for this variant identified.
• Literature search returned only 1 PMID (general ACMG/AMP guidelines), no variant-specific functional publication.

Reasoning

The ClinGen DICER1 VCEP framework makes PS4 applicable and requires phenotype-point evidence from unrelated probands, with case-control enrichment preferred where available. The case bundle contains no exact-variant case series, unrelated-proband phenotype observations, case-control counts, or odds ratio/confidence interval, so PS4 cannot be assigned.

Rule

Unrelated probands may contribute up to 1 point each based on phenotype; do not apply PS4 if BA1/BS1 is met or if a qualifying alternative molecular cause or sporadic tumor profile explains the phenotype. Strength: Supporting = 1-1.5 phenotype points; Moderate = 2-3.5; Strong = >=4.

Evidence considered

• The DICER1 VCEP Version 1.4 PS4 rule requires unrelated-proband phenotype points: Supporting for 1-1.5 points, Moderate for 2-3.5 points, and Strong for at least 4 points.
• The available case bundle provides no exact-variant proband phenotype observations or case-control enrichment statistics for NM_177438.3:c.735-23A>G.

Still missing

• Exact-variant clinical case data identifying unrelated affected probands and their DICER1 phenotype points are absent.
• No case-control enrichment analysis, odds ratio/relative risk, or confidence interval is available.
• No evidence is available to exclude probands with an alternative high-penetrance molecular cause or a tumor profile that would make the event sporadic under the VCEP caveats.

Sources

Reasoning

The ClinGen DICER1 VCEP Version 1.4 applies PP1 according to the number of phenotype-positive meioses: Supporting for 3-4 meioses, Moderate for 5-6 meioses, and Strong for at least 7 meioses across at least 2 families. The case-evidence bundle contains no affected relatives, phenotype descriptions, variant-positive relatives, family structure, or meiosis count. PP1 therefore cannot be assessed.

Rule

Phenotype-positive individuals may have high-, moderate-, or low-specificity phenotypes; PP1 is Supporting for 3-4 meioses across at least 1 family, Moderate for 5-6 meioses across at least 1 family, and Strong for at least 7 meioses across at least 2 families.

Evidence considered

• The governing ClinGen DICER1 VCEP Version 1.4 requires phenotype-positive relatives and assigns PP1 strength by informative meioses: 3-4 Supporting, 5-6 Moderate, and >=7 across >=2 families Strong.
• The consolidated case-evidence bundle contains no family segregation data or informative meiosis count for this variant.

Still missing

• Family pedigree and relationship degree
• Phenotype status and specificity for each relative
• Genotypes of affected relatives
• Number of informative meioses and number of families

Sources

Reasoning

The DICER1 VCEP specifies PP4 for a proband with the germline variant whose DICER1-associated neoplasm has a somatic RNase IIIb hotspot second hit, retains the germline variant, and has no additional somatic DICER1 loss-of-function variants. No tumor-testing or proband phenotype result for this exact variant is present in the case bundle.

Rule

Apply Supporting PP4 only when tumor testing in a proband carrying the germline variant identifies a previously reported somatic RNase IIIb hotspot second hit and retains the germline variant, without additional somatic DICER1 loss-of-function variants; PP4 is not applicable for a germline missense hotspot variant or qualifying additional somatic non-hotspot variants.

Evidence considered

• The DICER1 VCEP Version 1.4 PP4 rule requires a DICER1-associated neoplasm with a somatic second hit at p.S1344, p.E1705, p.D1709, p.D1713, p.G1809, p.D1810, or p.E1813 and retention of the germline variant, with no additional somatic DICER1 non-hotspot variants.
• The case bundle contains no tumor sequencing or other exact-variant clinical evidence establishing those PP4 requirements.

Still missing

• No exact-variant proband or tumor report is available.
• Somatic hotspot second-hit status, germline-variant retention, and exclusion of additional somatic DICER1 loss-of-function variants are unknown.

Sources

Reasoning

No RNA splicing assay data demonstrating absence of splicing impact for NM_177438.3:c.735-23A>G were found in the case evidence bundle. The DICER1 VCEP specification (v1.4) requires an RNA assay (observed more than once) showing no splicing impact for intronic variants to apply BS3_Strong, or an in vitro cleavage assay showing normal 5p/3p microRNA production for BS3_Supporting. Neither type of functional evidence for this specific variant was found. SpliceAI in silico prediction of no significant splice impact (max delta score 0.00) is relevant to PP3/BP4/BP7 computational criteria, not to BS3, which per this VCEP specification requires wet-lab RNA or cleavage assay data.

Still missing

• No RNA-based splicing assay demonstrating lack of splicing impact for this variant identified in the evidence bundle or literature triage.
• No in vitro pre-miRNA cleavage assay data (with controls) for this variant identified.
• Literature search did not surface a variant-specific functional study to evaluate.

Reasoning

The ClinGen DICER1 VCEP Version 1.4 requires phenotype-positive, genotype-negative first-, second-, or third-degree relatives for BS4. The case-evidence bundle contains no affected relatives, relative genotypes, pedigree relationships, or phenotype-specific family data. There is therefore insufficient evidence to assess lack of segregation, and BS4 cannot be applied.

Rule

Strong: family members should be phenotype-positive with a high- or moderate-specificity phenotype and genotype-negative for the variant, as first-, second-, or third-degree relatives of the proband.

Evidence considered

• The governing ClinGen DICER1 VCEP Version 1.4 defines BS4 as phenotype-positive, genotype-negative first-, second-, or third-degree relatives and cautions that phenocopies or another pathogenic variant can mimic lack of segregation.
• The consolidated case-evidence bundle contains no family members with the required phenotype and genotype data.

Still missing

• Phenotype-positive relatives with high- or moderate-specificity DICER1-associated phenotypes
• Variant testing results for those relatives
• Pedigree relationship and family context
• Evaluation for phenocopies or another pathogenic variant that could explain the phenotype

Sources

Reasoning

The DICER1 VCEP framework permits BP2 at Supporting strength when there is at least one observation in trans with a pathogenic or likely pathogenic DICER1 variant, or at least three observations in cis or with phase unknown involving two or more different pathogenic or likely pathogenic DICER1 variants. The case-evidence bundle contains no affected-proband or family-level observations, no second DICER1 variant, and no phase, parental, or inheritance data for this variant. The threshold cannot be evaluated, so BP2 is not assessed rather than called not met.

Rule

≥1 observation in trans with P/LP DICER1 variant or ≥3 observations in cis or phase unknown with 2+ different P/LP DICER1 variants.

Evidence considered

• The DICER1 VCEP BP2 rule requires at least one trans observation with a P/LP DICER1 variant, or at least three cis or phase-unknown observations involving at least two different P/LP DICER1 variants.
• The consolidated case-evidence bundle provides no report of phase, cis/trans status, parental testing, offspring testing, affected probands with another DICER1 variant, or a documented second DICER1 variant for this case.

Still missing

• Affected-proband or family-level observations involving NM_177438.3:c.735-23A>G are not available.
• No co-occurring pathogenic or likely pathogenic DICER1 variant is documented for assessment of cis or trans phase.
• No parental testing, offspring testing, or other phase-resolving data are available.

Sources