DICER1 c.3827A>G, p.Asp1276Gly

Reasoning

The variant is NP_803187.1:p.(Asp1276Gly). The collected comparator set contains four candidates but zero same-amino-acid pathogenic ClinGen DICER1 VCEP variants, so the VCEP's same-AA PS1 condition is not satisfied. No PS1 evidence can be derived from the available ClinVar submissions because they are uncertain significance or likely benign rather than pathogenic VCEP assertions.

VCEP rule

For same AA change, must confirm there is no difference in splicing using RNA data or in-silico modeling data (concordance of MaxEntScan and SpliceAI). For non-canonical intronic splicing variants at same nucleotide should have equal or worse splicing impact. ... This rule code can only be used to compare variants asserted as pathogenic by the ClinGen DICER1 VCEP. Likely pathogenic changes do not apply.

Evidence considered

• The variant is NP_803187.1:p.(Asp1276Gly).
• PM5 comparator candidate collection: total candidates seen = 4; same-residue candidates = 0; expert-panel pathogenic/likely pathogenic candidates = 0.
• ClinVar variation 412115 reports uncertain significance submissions and one likely benign submission, with no ClinGen DICER1 VCEP pathogenic assertion.

Sources

Reasoning

The variant is p.Asp1276Gly. The DICER1 VCEP limits PM1 Moderate to the seven specified metal ion-binding residues (p.S1344, p.E1705, p.D1709, p.D1713, p.G1809, p.D1810, p.E1813), and PM1 Supporting to residues p.Y1682-p.S1846 outside those residues. Residue 1276 is neither a listed metal ion-binding residue nor within p.Y1682-p.S1846. vcep_materials.json has no DICER1 domain_tables entry, and the VCEP text names no alternative hotspot route for PM1.

VCEP rule

Putative missense variants at residues affecting metal ion-binding: codons p.S1344, p.E1705, p.D1709, p.D1713, p.G1809, p.D1810, p.E1813 ... Putative missense variants at residues in the RNase IIIb domain (p.Y1682 – p.S1846), besides the metal ion-binding residues (see PM1).

Evidence considered

• DICER1 VCEP PM1 Moderate specifies p.S1344, p.E1705, p.D1709, p.D1713, p.G1809, p.D1810, and p.E1813.
• DICER1 VCEP PM1 Supporting specifies the RNase IIIb interval p.Y1682-p.S1846 excluding the metal ion-binding residues.
• vcep_materials.json domain_tables is empty for DICER1; no additional authoritative domain-table entry is available.

Sources

Reasoning

The DICER1 VCEP PM2 rule requires allele frequency <0.000005 across gnomAD, no more than one allele in any subpopulation, and at least 20x coverage. gnomAD v2.1 has total AF=7.958235181766091e-06 with 2 alleles overall and 2 alleles in European (non-Finnish); gnomAD v4.1 has total AF=4.336594039041737e-06 but 6 alleles in European (non-Finnish). Although the v4.1 total AF is below 0.000005, the subpopulation allele-count requirement is violated in both datasets, so PM2 is not met.

VCEP 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 CSPEC PM2 rule: allele frequency <0.000005 across gnomAD, no more than one allele in any subpopulation, and at least 20x coverage.
• gnomAD v2.1, all-comers exome data: total AF=7.958235181766091e-06, total AC=2, total AN=251312, total homozygotes=0; European (non-Finnish) AC=2 and AF=1.7604394056756568e-05.
• gnomAD v4.1, all-comers data: total AF=4.336594039041737e-06, total AC=7, total AN=1614170, total homozygotes=0; European (non-Finnish) AC=6 and AF=5.08461649280103e-06.
• The available case evidence does not provide a qualifying non-cancer or separate exome-only dataset that changes the governing VCEP v1.4 assessment; the VCEP rule is applied to the available all-comers gnomAD entries.

Sources

Reasoning

The variant is p.Asp1276Gly. The collected PM5 comparator set contains zero same-residue candidates, so no other missense variant at residue 1276 interpreted as pathogenic by the ClinGen DICER1 VCEP is available. Therefore the VCEP PM5 rule cannot be met, regardless of the available SpliceAI result.

VCEP rule

Missense variant under evaluation should have equal or worse Grantham score. Splicing should be ruled out with either RNA data or agreement in splicing predictors (MaxEntScan and SpliceAI) that show no splicing effects. The other variant must be interpreted as pathogenic by the ClinGen DICER1 VCEP. Likely pathogenic changes do not apply. This rule cannot be applied in combination with PM1 or PS1.

Evidence considered

• PM5 comparator candidate collection: total candidates seen = 4; same-residue candidates = 0; expert-panel pathogenic/likely pathogenic candidates = 0; non-expert majority pathogenic/likely pathogenic candidates = 0.
• The variant is p.Asp1276Gly; no collected comparator changes residue 1276.
• SpliceAI max delta score is 0.109, but no qualifying pathogenic same-residue comparator exists.

Sources

Reasoning

The DICER1 VCEP BA1 rule requires frequency >0.003 (0.3%) in a gnomAD subpopulation, with >2,000 alleles tested and at least 5 alleles present. The highest observed subpopulation frequency is 1.7604394056756568e-05 (2/113608 alleles) in gnomAD v2.1 and 1.5997440409534473e-05 (1/62510 alleles) in gnomAD v4.1; both are far below 0.003. Therefore BA1 is not met.

VCEP 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 CSPEC BA1 rule: frequency must be >0.003 (0.3%) in a gnomAD subpopulation, with >2,000 alleles tested and at least 5 alleles present.
• gnomAD v2.1, all-comers exome data: highest subpopulation was European (non-Finnish), AF=1.7604394056756568e-05, AC=2, AN=113608.
• gnomAD v4.1, all-comers data: highest subpopulation was Remaining individuals, AF=1.5997440409534473e-05, AC=1, AN=62510.

Sources

Reasoning

The DICER1 VCEP BS1 rule requires frequency >0.0003 (0.03%) in a gnomAD subpopulation, with >2,000 alleles tested and at least 5 alleles present. The highest observed subpopulation frequency is 1.7604394056756568e-05 in gnomAD v2.1 and 1.5997440409534473e-05 in gnomAD v4.1, both below 0.0003. Therefore BS1 is not met.

VCEP 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 CSPEC BS1 rule: frequency must be >0.0003 (0.03%) in a gnomAD subpopulation, with >2,000 alleles tested and at least 5 alleles present.
• gnomAD v2.1, all-comers exome data: highest subpopulation was European (non-Finnish), AF=1.7604394056756568e-05, AC=2, AN=113608.
• gnomAD v4.1, all-comers data: highest subpopulation was Remaining individuals, AF=1.5997440409534473e-05, AC=1, AN=62510.

Sources

Reasoning

The DICER1 VCEP BS2 rule permits strong evidence for 40 or more unrelated females tumor-free through age 50, 2 or more observations of homozygosity in healthy individuals, or 1 homozygous healthy individual confirmed by parental testing; supporting evidence requires 10 or more such females or 2 homozygous observations in individuals lacking clinical information. The available gnomAD data show zero homozygotes in gnomAD v2.1 and gnomAD v4.1, and no qualifying tumor-free female cohort or additional homozygous observations is documented. Thus no BS2 path is met.

VCEP rule

40+ unrelated females from a single source are tumor-free through age 50 (caveat: ratio of BS2-eligible females to PS4-eligible probands must be ≥ 40:1) OR 2+ observations of homozygosity in healthy individuals OR 1+ observation(s) of homozygosity in a healthy individual with status confirmed by parental testing. ... 10+ unrelated females from a single source are tumor-free through age 50 (caveat: ratio of BS2-eligible females to PS4-eligible probands must be ≥ 10:1) OR 2+ observations of homozygosity in individuals lacking clinical information

Evidence considered

• ClinGen DICER1 VCEP CSPEC BS2 rule includes 40+ unrelated tumor-free females through age 50, 2+ homozygous observations in healthy individuals, or 1 confirmed homozygous healthy individual for strong evidence; supporting paths include 10+ tumor-free females or 2 homozygous observations without clinical information.
• gnomAD v2.1, all-comers exome data: total homozygotes=0.
• gnomAD v4.1, all-comers data: total homozygotes=0.
• No qualifying tumor-free female cohort or qualifying additional homozygous observation is present in the reviewed case evidence.

Sources

Reasoning

The VCEP provides point-based PS2 thresholds, but the available case evidence contains no documented affected proband assessment, parental maternity and paternity confirmation, de novo observation, or de novo-point tally for NM_177438.3:c.3827A>G (p.Asp1276Gly).

VCEP rule

≥4 de novo points; ≥2 but less than 4 de novo points; ≥1 but less than 2 de novo points; ≥0.5 but less than 1 de novo points

Evidence considered

• No proband, parental-testing, or de novo evidence for this variant is documented in the available case-evidence records.
• The VCEP CSPEC lists PS2 as applicable and assigns strength according to the number of de novo points: ≥0.5 but less than 1 for Supporting, ≥1 but less than 2 for Moderate, ≥2 but less than 4 for Strong, and ≥4 for Very Strong.

Still missing

• Documented proband phenotype consistent with DICER1-related tumor predisposition
• Variant testing results for both biological parents establishing maternity and paternity and absence of the variant in both parents
• VCEP de novo-point tally for this proband

Sources

Reasoning

The variant is a missense change, and the available case evidence contains no variant-specific RNA assay demonstrating abnormal splicing and no in vitro pre-miRNA cleavage assay demonstrating failure or severe reduction of 5p or 3p microRNA production. The VCEP's applicable cleavage-assay path requires positive and negative controls; no such assay or controls are documented. Computational splice prediction is not a validated functional assay and does not satisfy the VCEP PS3 rule.

VCEP rule

In vitro cleavage assay shows failure or severely reduced capacity to produce either 5p or 3p microRNAs from a premiRNA (positive and negative controls also performed).

Evidence considered

• Case variant is NM_177438.3:c.3827A>G (NP_803187.1:p.(Asp1276Gly)), classified as missense.
• The available evidence reports only computational splice prediction for this variant (SpliceAI maximum delta score 0.109); no RNA assay or in vitro cleavage assay result is provided.
• The VCEP PS3 supporting path requires an in vitro cleavage assay showing failure or severely reduced capacity to produce either 5p or 3p microRNAs from a premiRNA, with positive and negative controls also performed.

Still missing

• Variant-specific RNA assay results are unavailable.
• Variant-specific in vitro pre-miRNA cleavage results for both 5p and 3p microRNAs are unavailable.
• Positive and negative assay controls are not documented.

Sources

Reasoning

The DICER1 VCEP defines PS4 using phenotype points for unrelated probands, with Strong at ≥4 points, Moderate at 2–3.5 points, and Supporting at 1–1.5 points. The available case evidence contains no proband phenotype-point assignments, no variant-specific case-control counts, and no verified literature observation for NM_177438.3:c.3827A>G; literature_pass.json has an empty ai_classified_papers list, and the reviewed papers do not mention this variant. Therefore PS4 cannot be evaluated from the available evidence. A case-control comparison, if located, would need its counts and an explicitly calculated OR and 95% CI, but none is available here.

VCEP rule

Unrelated probands may contribute up to 1 point each based on phenotype (see Tables 2 & 3 in ruleset) ... Do not apply PS4 if variant meets BA1/BS1 criteria. ... The prevalence of the variant in affected individuals is significantly increased compared to the prevalence in controls. ... 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. ... In instances of very rare variants where case-control studies may not reach statistical significance, the prior observation of the variant in multiple unrelated patients with the same phenotype, and its absence in controls, may be used as moderate level of evidence. ... ≥4 phenotype points ... 2 – 3.5 phenotype points ... 1 – 1.5 phenotype points

Evidence considered

• DICER1 VCEP PS4 rule: unrelated probands may contribute up to 1 point each based on phenotype; the VCEP assigns Strong at ≥4 phenotype points, Moderate at 2–3.5 points, and Supporting at 1–1.5 points.
• literature_pass.json reports ai_classified_papers as an empty list and provides no verified variant-specific PS4 paper evidence.
• PMID:25394175, PMID:25741868, and PMID:28492532 were checked through paper_extracts.json; none mentions DICER1 c.3827A>G or p.(D1276G), and none supplies case-control or phenotype evidence for this variant.

Still missing

• Variant-specific affected-proband phenotype data sufficient to assign DICER1 VCEP phenotype points.
• Any variant-specific case-control counts or matched-ancestry control comparison, with calculated OR and 95% CI.

Sources

Reasoning

The available case evidence contains no affected relatives, variant-positive relatives, phenotype-specific family data, family count, or meiosis count, so the VCEP PP1 thresholds cannot be evaluated.

VCEP rule

≥7 meioses across ≥2 families; 5 – 6 meioses across ≥1 family; 3 – 4 meioses across ≥1 family

Evidence considered

• No segregation data, family pedigree, relative genotypes, relative phenotypes, or meiosis count for this variant is documented in the available case-evidence records.
• The VCEP CSPEC assigns PP1 Supporting to 3–4 meioses across ≥1 family, Moderate to 5–6 meioses across ≥1 family, and Strong to ≥7 meioses across ≥2 families; it also requires phenotype-positive individuals to have high-, moderate-, or low-specificity phenotypes and excludes segregation based only on a single low-specificity phenotype.

Still missing

• Pedigree identifying informative affected relatives and family count
• Genotype and phenotype status for each informative relative
• Count of informative meioses across families
• Confirmation that phenotype-positive individuals meet the VCEP phenotype-specificity requirements

Sources

Reasoning

The VCEP applies PP3 to missense variants through either REVEL ≥ 0.750 or agreement among splicing predictors predicting splicing effects. This variant is missense, and its REVEL score is 0.104, which does not meet the PP3 threshold. SpliceAI is available and has a maximum delta score of 0.109, but no MaxEntScan result is available to establish the required concordant splicing-predictor result for this situation. Because the REVEL path fails and the alternative predictor path cannot be evaluated completely, PP3 is not_assessed rather than not_met.

VCEP rule

For missense variants, REVEL score ≥ 0.750 OR agreement in splicing predictors predict splicing effects. For splicing variants, concordance of MaxEntScan and SpliceAI.

Evidence considered

• VCEP PP3 rule for missense variants: REVEL score ≥ 0.750 OR agreement in splicing predictors predicting splicing effects.
• Local REVEL predictor lookup: NM_177438.3:c.3827A>G, score 0.104; this is below the VCEP PP3 threshold of 0.750.
• SpliceAI Lookup: DS_AG 0.109, DS_AL 0.003, DS_DG 0.083, DS_DL 0.009; maximum delta score 0.109. No MaxEntScan result is present, so concordance of the required splicing predictors cannot be established.

Still missing

• MaxEntScan result for NM_177438.3:c.3827A>G is unavailable; the VCEP's splicing-predictor path cannot be evaluated completely.

Sources

Reasoning

The VCEP's PP4 rule requires tumor testing in a proband carrying the germline variant to show a previously reported somatic DICER1 RNase IIIb hotspot second hit, retention of the germline variant, and no additional somatic DICER1 non-hotspot variants. The available evidence does not document such tumor sequencing, retention, or exclusion of additional non-hotspot variants. The germline missense variant is p.Asp1276Gly, outside the seven hotspot codons named by the VCEP, so the hotspot-specific non-applicability caveat is not triggered; however, the required tumor evidence is missing.

VCEP rule

Somatic tumor testing identifies somatic hotspot second hit and no additional somatic LOF variants. Tumor testing[⁶](#pmid_30311369) of a neoplasm with known DICER1 association in a proband who carries the germline variant under evaluation reveals the following: ... A previously reported somatic second hit of DICER1 in an RNase IIIb-disrupting “hotspot” codon (p.S1344, p.E1705, p.D1709, p.D1713, p.G1809, p.D1810, or p.E1813) AND ... Retention of the germline DICER1 variant under evaluation. PP4 is NOT applicable if: ... The germline variant is a missense variant in one of the seven RNase IIIb “hotspot” codons (see PM1), OR ... Somatic sequencing reveals additional DICER1 non-hotspot variants (could be consistent with sporadic tumorigenesis).

Evidence considered

• The variant is p.Asp1276Gly, whereas the VCEP PP4 hotspot list is p.S1344, p.E1705, p.D1709, p.D1713, p.G1809, p.D1810, or p.E1813; therefore the germline missense hotspot exclusion does not apply.
• The available case evidence contains no tumor sequencing result documenting the required hotspot second hit, germline retention, and absence of additional non-hotspot DICER1 variants.

Still missing

• Tumor sequencing results showing a qualifying somatic RNase IIIb hotspot second hit and retention of the germline variant.
• Evidence that tumor sequencing found no additional somatic DICER1 non-hotspot variants.

Sources

Reasoning

The variant is a missense change, and no variant-specific functional study demonstrates preserved DICER1 activity. In particular, no in vitro cleavage assay documents production of both 5p and 3p microRNAs from a pre-miRNA with positive and negative controls. No validated RNA assay establishes absence of splicing impact. Therefore neither VCEP BS3 path can be evaluated.

VCEP rule

An in vitro cleavage assay must demonstrate the variant produces both 5p and 3p microRNAs from a pre-miRNA (positive and negative controls also performed). An example of an appropriate assay to which criteria could be applied is Wu et al. 2018[⁷](#pmid_28862265).

Evidence considered

• Case variant is NM_177438.3:c.3827A>G (NP_803187.1:p.(Asp1276Gly)), classified as missense.
• The available evidence reports only computational splice prediction for this variant (SpliceAI maximum delta score 0.109); computational prediction is not a functional assay under the VCEP BS3 paths.
• The VCEP BS3 supporting path requires an in vitro cleavage assay demonstrating production of both 5p and 3p microRNAs from a pre-miRNA with positive and negative controls; the VCEP BS3 strong path concerns intronic or synonymous variants with repeated RNA-assay evidence of no splicing impact.

Still missing

• Variant-specific in vitro pre-miRNA cleavage results demonstrating both 5p and 3p microRNA production are unavailable.
• Positive and negative assay controls are not documented.
• Variant-specific RNA assay evidence demonstrating no splicing impact is unavailable.

Sources

Reasoning

The available case evidence does not document any phenotype-positive, genotype-negative first-, second-, or third-degree relative of the proband, so the VCEP BS4 rule cannot be evaluated.

VCEP rule

Family members should be phenotype-positive (must be high- or moderatespecificity phenotype; see phenotype table), genotype-negative 1st, 2nd, or 3rd degree relatives of the proband.

Evidence considered

• No affected-relative phenotype data or relative genotype results are documented in the available case-evidence records.
• The VCEP CSPEC requires phenotype-positive, genotype-negative first-, second-, or third-degree relatives of the proband for BS4 at Strong strength.

Still missing

• Relationship and phenotype status of potentially informative relatives
• Genotype results demonstrating absence of the variant in affected relatives
• Confirmation that relatives have a VCEP high- or moderate-specificity phenotype

Sources

Reasoning

The VCEP BP2 rule requires at least one observation in trans with a pathogenic/likely pathogenic DICER1 variant, or at least three observations in cis or phase unknown with two or more different pathogenic/likely pathogenic DICER1 variants. The available case evidence documents no affected-proband observation, second pathogenic/likely pathogenic DICER1 variant, or cis/trans/unknown phase for NM_177438.3:c.3827A>G (p.Asp1276Gly), so the threshold cannot be evaluated.

VCEP 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

• ClinGen DICER1 and miRNA-Processing Gene Expert Panel Specification v1.4, CSPEC BP2 entry: "≥1 observation in trans with P/LP DICER1 variant or ≥3 observations in cis or phase unknown with 2+ different P/LP DICER1 variants."
• Case evidence for NM_177438.3:c.3827A>G (p.Asp1276Gly) contains no documented affected-proband observation, qualifying second P/LP DICER1 variant, or cis/trans/phase-unknown result.

Still missing

• Documented affected-proband observations involving this variant and another pathogenic/likely pathogenic DICER1 variant.
• Phase information establishing trans, cis, or phase unknown, with the number of qualifying observations.

Sources

Reasoning

The VCEP applies BP4 to missense variants only when both REVEL is < 0.500 and splicing predictors agree that no splicing effects are predicted. This variant is missense; its REVEL score is 0.104 and therefore satisfies the REVEL component. SpliceAI has a maximum delta score of 0.109, but no MaxEntScan result is available to establish the required agreement of the named splicing predictors. Because the complete BP4 rule cannot be evaluated, BP4 is not_assessed.

VCEP 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.

Evidence considered

• VCEP BP4 rule for missense variants: REVEL score < 0.500 and agreement in splicing predictors that no splicing effects are predicted.
• Local REVEL predictor lookup: NM_177438.3:c.3827A>G, score 0.104; this satisfies the VCEP BP4 REVEL threshold.
• SpliceAI Lookup: maximum delta score 0.109, with DS_AG 0.109, DS_AL 0.003, DS_DG 0.083, and DS_DL 0.009. No MaxEntScan result is present, so the required predictor concordance cannot be established.

Still missing

• MaxEntScan result for NM_177438.3:c.3827A>G is unavailable; concordance with SpliceAI cannot be assessed.

Sources