DICER1 c.4206+9del, p.?

Reasoning

BA1 is met under the ClinGen DICER1 VCEP specification: the variant frequency exceeds 0.003 in multiple gnomAD v4.1 subpopulations, including East Asian AF 0.485669 (5524/11374 alleles), and those subpopulations exceed 2,000 tested alleles and contain at least 5 observed alleles.

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

• The DICER1 VCEP v1.4 BA1 rule requires frequency >0.003 in a gnomAD subpopulation, with >2,000 alleles tested and at least 5 alleles present.
• gnomAD v4.1 reports the variant at overall AF 0.352171 (207044/587908 alleles); East Asian AF is 0.485669 (5524/11374 alleles), with 132 homozygotes. Other large subpopulations also substantially exceed the BA1 threshold.

Sources

Reasoning

BS1 is met under the ClinGen DICER1 VCEP specification: the variant frequency exceeds 0.0003 in multiple gnomAD v4.1 subpopulations meeting the required sample-size and allele-count conditions.

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

• The DICER1 VCEP v1.4 BS1 rule requires frequency >0.0003 in a gnomAD subpopulation, with >2,000 alleles tested and at least 5 alleles present.
• gnomAD v4.1 reports AF 0.352171 overall and AF 0.485669 in East Asian individuals (5524/11374 alleles); the qualifying subpopulation observations are far above the BS1 threshold.

Sources

Reasoning

BS2 is met at supporting strength under the ClinGen DICER1 VCEP specification because gnomAD v4.1 contains thousands of homozygous observations, far exceeding the requirement for 2 or more observations of homozygosity in individuals lacking clinical information. No claim is made that these database participants are clinically confirmed healthy.

Rule

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

• The DICER1 VCEP v1.4 BS2 supporting rule allows 2 or more observations of homozygosity in individuals lacking clinical information.
• gnomAD v4.1 reports 7262 homozygotes for the variant overall, including 132 in the East Asian subpopulation and substantial homozygote counts in other large subpopulations.

Sources

Reasoning

NM_177438.3:c.4206+9del is a non-canonical intronic deletion, not a nonsense, frameshift, initiation-codon, exon-deletion, or canonical ±1/2 splice variant. It is therefore outside the DICER1 VCEP PVS1 splice-variant entry conditions. SpliceAI predicts no significant splice impact (maximum delta 0.004), so there is no evidence that this change produces an aberrant transcript, premature termination, or NMD substrate.

Rule

DICER1 VCEP PVS1: apply to canonical splice variants at ±1/2 intronic positions; use the DICER1 PVS1 decision tree for demonstrated aberrant splicing and NMD consequences.

Evidence considered

• The DICER1 VCEP specifies PVS1 for canonical splice variants at intronic ±1/2 positions, with stated exon-specific modifications; c.4206+9 is outside those positions.
• SpliceAI for NM_177438.3:c.4206+9del reports DS_AG 0.004, DS_AL 0.003, DS_DG 0.003, and DS_DL 0.001 (maximum delta 0.004), reported as no significant predicted splice impact.
• The DICER1 PVS1 decision tree assigns NMD-dependent PVS1 strengths only after a loss-of-function consequence, such as frameshift-generating exon skipping or cryptic splicing, has been established.

Still missing

• No RNA study is available; if future transcript data demonstrate a qualifying loss-of-function splice outcome, reassess PVS1 under the DICER1 decision tree.

Sources

Reasoning

PM2 is not met because the variant is common in gnomAD v4.1, with overall AF 0.352171 and multiple subpopulation frequencies above 0.000005. The VCEP PM2 rule therefore fails; the available population data are incompatible with PM2 rarity.

Rule

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

Evidence considered

• The DICER1 VCEP v1.4 PM2 supporting rule requires allele frequency <0.000005 across gnomAD, no more than one allele in any subpopulation, and at least 20x coverage.
• gnomAD v4.1 reports overall AF 0.352171 (207044/587908 alleles), 7262 homozygotes, and East Asian AF 0.485669 (5524/11374 alleles), so the rarity and allele-count requirements are decisively not satisfied.

Sources

Reasoning

This is an intronic deletion with protein consequence p.?; it is not an established in-frame protein indel or stop-loss variant. The DICER1 VCEP PM4 rules require an in-frame indel affecting the RNase IIIb domain for Moderate strength, or an in-frame indel outside that domain and outside specified repeat regions for Supporting strength. Neither condition is met.

Rule

DICER1 VCEP PM4: in-frame indel in RNase IIIb, Moderate; in-frame indel outside RNase IIIb and specified repeat regions, Supporting.

Evidence considered

• Variant normalization gives NM_177438.3:c.4206+9del and NP_803187.1:p.?, indicating no defined protein length change.
• The DICER1 VCEP PM4 rule permits Moderate strength for in-frame indels with a residue in RNase IIIb (p.Tyr1682-p.Ser1846), or Supporting strength for in-frame indels outside RNase IIIb and outside p.Asp606-p.Asp609, p.Glu1418-p.Glu1420, and p.Glu1422-p.Glu1425 repeat regions.
• SpliceAI predicts no significant splice impact for this intronic deletion, providing no predicted basis for an in-frame protein alteration.

Still missing

• No RNA study is available; reassess only if RNA evidence establishes an in-frame protein length alteration.

Sources

Reasoning

DICER1 VCEP PP3 for a splicing variant requires concordance of MaxEntScan and SpliceAI predicting a splicing effect. SpliceAI has a maximum delta score of 0.004 and predicts no significant splice impact; no MaxEntScan result is available. The available prediction therefore does not support PP3.

Rule

DICER1 VCEP PP3: for splicing variants, concordance of MaxEntScan and SpliceAI predicting splicing effects.

Evidence considered

• The governing DICER1 VCEP specification applies PP3_Supporting to splicing variants only with concordance of MaxEntScan and SpliceAI predicting splicing effects.
• SpliceAI for NM_177438.3:c.4206+9del reports DS_AG 0.004, DS_AL 0.003, DS_DG 0.003, DS_DL 0.001 (maximum delta score 0.004) and no significant splice impact.

Still missing

• No MaxEntScan result was supplied; however, the available SpliceAI result predicts no splice impact rather than a deleterious effect.

Sources

Reasoning

The DICER1 VCEP specifies PS2 for confirmed de novo observations, with de novo points tallied to determine strength. The case bundle does not document a proband-level de novo finding, parental genotypes, or confirmation of both maternity and paternity for NM_177438.3:c.4206+9del; therefore no PS2 strength can be assigned.

Rule

De novo points should be tallied using the simplified table for tallying proband points and used to determine the applied strength of PS2. The EP has opted to drop PM6 and exclusively use PS2 for de novo evidence. Strength: Very Strong for >=4 de novo points; Strong for >=2 but <4; Moderate for >=1 but <2; Supporting for >=0.5 but <1.

Evidence considered

• The DICER1 VCEP Version 1.4 rule states that PS2 de novo points should be tallied using the simplified proband-point table and that both maternity and paternity must be confirmed for the de novo evidence definition.
• The case bundle identifies the variant as NM_177438.3:c.4206+9del in DICER1 but provides no case-level parental testing or de novo-point record.

Still missing

• Proband-level variant result and phenotype documentation
• Maternal and paternal testing results for the exact variant
• Confirmation of maternity and paternity
• Any de novo-point details, including the number of qualifying probands

Sources

Reasoning

No variant-specific RNA assay demonstrating an abnormal splicing consequence and no variant-specific in vitro DICER1 pre-miRNA cleavage assay demonstrating failure or severe reduction of 5p or 3p microRNA production were identified in the case evidence bundle. The ClinGen DICER1 VCEP specification requires positive and negative controls for cleavage assays and calibrates splice-assay evidence by the resulting protein consequence; those requirements cannot be evaluated without primary assay data.

Rule

RNA assay shows splicing impact that is out-of-frame, in-frame ≥193 residues, or in-frame with RNase IIIb disruption; PS3_Moderate if PVS1_Strong is applied. RNA assay shows in-frame splicing impact with change in protein length <193 residues and RNase IIIb domain not disrupted. In vitro cleavage assay shows failure or severely reduced capacity to produce either 5p or 3p microRNAs from a pre-miRNA, with positive and negative controls performed.

Evidence considered

• The ClinGen DICER1 and miRNA-Processing Gene Expert Panel Specification version 1.4 defines PS3 as applicable for functional evidence of splicing impact or reduced DICER1 pre-miRNA cleavage, with strength calibrated to the RNA consequence or, for cleavage assays, failure or severe reduction in production of either 5p or 3p microRNA and appropriate positive and negative controls.
• The ACMG/AMP consensus paper states that splice-variant impact requires confirmation by functional RNA or protein analysis; it does not report this variant or provide variant-specific assay results.
• The case bundle contains no variant-specific functional assay result that can be independently assessed for biological relevance, assay validity, controls, reproducibility, or PS3 strength.

Still missing

• Variant-specific RNA assay results, including assay controls, replicate observations, and the resulting transcript/protein consequence, are unavailable.
• Variant-specific pre-miRNA cleavage assay results for both 5p and 3p microRNA production, including positive and negative controls, are unavailable.

Sources

Reasoning

The DICER1 VCEP specification requires phenotype points for PS4 (Supporting: 1–1.5; Moderate: 2–3.5; Strong: ≥4). The bundle contains no phenotype-pointed probands, case-control comparison, or other exact-variant case series from which points can be assigned.

Rule

ClinGen DICER1 and miRNA-Processing Gene Expert Panel Specifications v1.4: PS4 Supporting at 1–1.5 phenotype points, Moderate at 2–3.5 points, and Strong at ≥4 points.

Evidence considered

• DICER1 VCEP PS4 requires phenotype points: Supporting for 1–1.5 points, Moderate for 2–3.5 points, and Strong for at least 4 points.
• No source in the consolidated bundle reports an exact-variant affected-proband count, phenotypes that can be scored under the DICER1 VCEP, or a case-control enrichment calculation.
• gnomAD v4.1 reports this allele at AF 0.352171 (207044/587908 alleles), including 7262 homozygotes; this population observation does not provide PS4 case enrichment.

Still missing

• Phenotype-specific, independent proband observations of NM_177438.3:c.4206+9del with sufficient clinical detail to assign DICER1 VCEP phenotype points, or a valid case-control enrichment analysis.

Sources

Reasoning

The DICER1 VCEP allows PP1 when phenotype-positive relatives co-segregate with the variant and assigns strength by meioses. The case bundle contains no documented affected relatives, familial genotypes, phase information, or countable meioses for this variant, so PP1 cannot be assigned.

Rule

Phenotype-positive individuals should have high, moderate, or low-specificity phenotypes; do not apply if the variant meets BA1/BS1. Strength: Strong for >=7 meioses across >=2 families; Moderate for 5-6 meioses across >=1 family; Supporting for 3-4 meioses across >=1 family.

Evidence considered

• The DICER1 VCEP states that phenotype-positive individuals may support PP1, cautions that a single low-specificity phenotype across multiple individuals is insufficient, and assigns Supporting for 3-4 meioses, Moderate for 5-6 meioses, and Strong for at least 7 meioses across at least 2 families.
• The case bundle provides no familial segregation observations, relative genotypes, or meiosis count for NM_177438.3:c.4206+9del.

Still missing

• Family pedigree and phenotype-specific status of relatives
• Genotypes of phenotype-positive relatives
• Number of informative meioses and family count
• Assessment that reported phenotypes meet the VCEP phenotype-specificity requirements

Sources

Reasoning

The DICER1 VCEP specification defines PP4 through tumor testing: a DICER1-associated neoplasm must retain the germline variant and have a reported somatic RNase IIIb hotspot second hit without additional somatic DICER1 loss-of-function variants. The bundle has no tumor sequencing meeting these requirements.

Rule

ClinGen DICER1 and miRNA-Processing Gene Expert Panel Specifications v1.4: apply PP4 only to the specified tumor-testing pattern; do not apply for a germline missense variant at an RNase IIIb hotspot or if additional somatic DICER1 non-hotspot variants are present.

Evidence considered

• DICER1 VCEP PP4 requires a DICER1-associated neoplasm with retention of the germline variant under assessment plus a previously reported somatic RNase IIIb hotspot at p.S1344, p.E1705, p.D1709, p.D1713, p.G1809, p.D1810, or p.E1813, and no additional somatic DICER1 loss-of-function variants.
• COSMIC reports this exact variant seven times in somatic cancers, but provides neither a qualifying DICER1-associated tumor genotype nor the required paired germline-retention, hotspot-second-hit, and additional-DICER1-variant findings.

Still missing

• Tumor sequencing from a DICER1-associated neoplasm showing retention of NM_177438.3:c.4206+9del and a qualifying somatic RNase IIIb hotspot second hit, with exclusion of additional somatic DICER1 non-hotspot variants.

Sources

Reasoning

No variant-specific RNA assay showing absence of splicing impact, repeated as required by the DICER1 VCEP specification, and no variant-specific in vitro cleavage assay demonstrating production of both 5p and 3p microRNAs with positive and negative controls were identified in the case evidence bundle. Therefore, benign functional evidence cannot be assigned at any strength.

Rule

For intronic or synonymous variants, no splicing impact observed via RNA assay; this should be observed more than once. An in vitro cleavage assay must demonstrate that the variant produces both 5p and 3p microRNAs from a pre-miRNA, with positive and negative controls performed.

Evidence considered

• The ClinGen DICER1 and miRNA-Processing Gene Expert Panel Specification version 1.4 defines BS3_Strong for intronic or synonymous variants with no splicing impact observed by RNA assay more than once, and BS3_Supporting for an in vitro cleavage assay demonstrating production of both 5p and 3p microRNAs from a pre-miRNA with positive and negative controls.
• The ACMG/AMP consensus paper identifies RNA or protein functional analysis as the relevant approach for confirming splice-variant impact, but it does not report this variant or provide benign functional assay data.
• The case bundle contains no variant-specific functional assay result that can establish absence of splicing impact, preserved DICER1 cleavage activity, appropriate controls, or reproducibility.

Still missing

• Variant-specific RNA assay evidence showing no splicing impact is unavailable; the DICER1 VCEP specifies that this should be observed more than once.
• Variant-specific pre-miRNA cleavage assay evidence demonstrating production of both 5p and 3p microRNAs with positive and negative controls is unavailable.

Sources

Reasoning

The DICER1 VCEP allows BS4 for phenotype-positive, genotype-negative first-, second-, or third-degree relatives of the proband. The case bundle contains no documented non-segregating affected relatives or familial genotype results, and therefore does not establish BS4.

Rule

Family members should be phenotype-positive with a high- or moderate-specificity phenotype and genotype-negative; eligible relatives are first-, second-, or third-degree relatives of the proband. Strength: Strong.

Evidence considered

• The DICER1 VCEP BS4 rule requires phenotype-positive, genotype-negative first-, second-, or third-degree relatives and cautions that phenocopies or more than one pathogenic variant in a family can confound apparent lack of segregation.
• The case bundle contains no reported affected genotype-negative relatives or other non-segregation evidence for NM_177438.3:c.4206+9del.

Still missing

• Phenotype-positive relatives with high- or moderate-specificity DICER1-associated phenotypes
• Negative testing for the exact variant in those relatives
• Degree of relationship to the proband
• Evaluation for phenocopies or another pathogenic variant that could explain the phenotype

Sources

Reasoning

The DICER1 VCEP permits BP2 at Supporting strength for at least one observation of the evaluated variant in trans with a P/LP DICER1 variant, or at least three observations in cis or phase unknown with two or more different P/LP DICER1 variants. The case-evidence bundle contains no documented affected-proband observation, second P/LP DICER1 variant, phase determination, parental/offspring testing, or other allelic data establishing any of these thresholds. BP2 cannot be adjudicated from the available evidence.

Rule

Supporting: ≥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 observation in trans with a P/LP DICER1 variant, or at least three observations in cis or phase unknown with two or more different P/LP DICER1 variants.
• The consolidated case-evidence bundle provides variant-level database and literature records but does not document a second DICER1 variant, affected-proband allelic observation, phase, or inheritance testing for NM_177438.3:c.4206+9del.
• The paper-extract records available in this case do not mention NM_177438.3:c.4206+9del and provide no phase or allelic evidence relevant to BP2.

Still missing

• Documented proband or cohort observations of NM_177438.3:c.4206+9del with a second DICER1 variant are absent.
• No pathogenic/likely pathogenic status is documented for a second DICER1 variant in the same individual(s).
• Phase evidence (trans, cis, or explicitly phase unknown) and parental/offspring testing are absent.
• The minimum VCEP threshold of one trans observation or three cis/phase-unknown observations with two or more different P/LP DICER1 variants cannot be evaluated.

Sources

Reasoning

The variant is intronic, and DICER1 VCEP BP4 requires concordance of MaxEntScan and SpliceAI predicting no splicing effects for intronic variants. SpliceAI predicts no significant effect (maximum delta score 0.004), but no MaxEntScan result is available. The VCEP instruction allowing SpliceAI alone outside the MES validation range is limited to prediction of native consensus splice-site impact; this c.4206+9 variant lacks a supplied MES prediction and no applicable VCEP exception permits BP4 from the single SpliceAI result.

Rule

DICER1 VCEP BP4: for synonymous/intronic/non-coding variants, concordance of MaxEntScan and SpliceAI predicting no splicing effects.

Evidence considered

• The governing DICER1 VCEP specification requires MaxEntScan/SpliceAI concordance for BP4 in synonymous, intronic, and non-coding variants.
• SpliceAI for NM_177438.3:c.4206+9del reports a maximum delta score of 0.004 and no significant splice impact; a MaxEntScan result was not supplied.

Still missing

• MaxEntScan prediction for NM_177438.3:c.4206+9del is required to establish the VCEP-required predictor concordance for BP4.

Sources

Reasoning

The variant is at intronic position +9 and thus meets the DICER1 VCEP positional category for BP7 (intronic at or beyond +7). However, the VCEP explicitly requires BP4 before BP7 can be applied. BP4 cannot be assessed because MaxEntScan is unavailable, so BP7 cannot be applied.

Rule

DICER1 VCEP BP7: intronic variant at or beyond +7 to -21; caveat, BP4 must be met.

Evidence considered

• The governing DICER1 VCEP BP7 rule includes intronic variants at or beyond +7 to -21 but requires that BP4 be met.
• SpliceAI predicts no significant splice impact (maximum delta score 0.004), but VCEP BP4 concordance cannot be established without MaxEntScan.

Still missing

• A MaxEntScan result is needed to resolve BP4; BP7 can be considered only if BP4 is met.

Sources