DICER1 c.734+6C>T, p.?

Reasoning

The applicable ClinGen DICER1 VCEP specification limits its canonical splice PVS1 rule to intronic positions +/-1 and +/-2. NM_177438.3:c.734+6C>T is a noncanonical intronic variant outside that rule, and SpliceAI predicts no significant splice impact (maximum delta 0.07). Therefore PVS1 is not met; no PVS1 strength tier is assigned.

Rule

ClinGen DICER1 VCEP Version 1.4 PVS1 rule: canonical splice variants at intronic positions +/-1 and +/-2; this +6 variant is outside the specified PVS1 bucket.

Evidence considered

• The DICER1 Version 1.4 VCEP specifies PVS1 for canonical splice variants at intronic positions +/-1 and +/-2, with separate rules for other defined loss-of-function classes.
• The assessed variant is NM_177438.3:c.734+6C>T, a noncanonical intronic change; the case assessment does not establish a truncating consequence or exon-skipping event.
• SpliceAI maximum delta is 0.07, below the supplied 0.1 threshold for a supporting benign splice prediction, and is described as no significant splice impact.
• General ACMG guidance states that splice effects should be confirmed by RNA or protein analysis, but the DICER1 VCEP-specific applicability rule takes precedence here.

Sources

Reasoning

The governing ClinGen DICER1 VCEP v1.4 specifies PM2 at supporting strength when allele frequency is less than 0.000005 across gnomAD, with no more than one allele in any subpopulation and at least 20x coverage. The current gnomAD v4.1 total frequency is 4.6506840e-05 (75/1,612,666 alleles), exceeding the VCEP cutoff by more than ninefold; its highest subpopulation has 67 alleles. The gnomAD v2.1 total frequency is 7.0766400e-05 (20/282,620 alleles), also above the cutoff. The VCEP v1.4 clarification directs use of the most recent and comprehensive gnomAD version and does not require a non-cancer subset. Therefore PM2 is not met.

Rule

DICER1 VCEP v1.4 PM2: allele frequency <0.000005 across gnomAD, no more than one allele in any subpopulation, and at least 20x coverage; supporting strength.

Evidence considered

• DICER1 VCEP v1.4 specifies PM2 supporting when frequency is <0.000005 across gnomAD, with no more than one allele in any subpopulation and at least 20x coverage.
• GnomAD v4.1 reports total frequency 4.6506840e-05 from 75/1,612,666 alleles, with 0 homozygotes; the European (non-Finnish) subpopulation contains 67 alleles and has frequency 5.6826567e-05.
• GnomAD v2.1 reports total frequency 7.0766400e-05 from 20/282,620 alleles, with 0 homozygotes; the European (non-Finnish) subpopulation contains 17 alleles.

Sources

Reasoning

The governing ClinGen DICER1 VCEP v1.4 specifies BA1 as a stand-alone benign criterion when the variant frequency is greater than 0.003 (0.3%) in a gnomAD subpopulation, with more than 2,000 alleles tested and at least 5 alleles present. The highest observed gnomAD v4.1 subpopulation frequency is 5.6826567e-05 in the European (non-Finnish) population (67/1,179,026 alleles), far below 0.003. The gnomAD v4.1 total frequency is 4.6506840e-05 (75/1,612,666 alleles), and gnomAD v2.1 is also far below the threshold. Therefore BA1 is not met.

Rule

DICER1 VCEP v1.4 BA1: frequency >0.003 (0.3%) in a gnomAD subpopulation; subpopulation must have >2,000 alleles tested and at least 5 alleles present.

Evidence considered

• DICER1 VCEP v1.4 specifies BA1 at frequency >0.003 (0.3%) in a gnomAD subpopulation, requiring >2,000 alleles tested and at least 5 alleles present.
• gnomAD v4.1 reports the highest subpopulation frequency as 5.6826567e-05 in European (non-Finnish), based on 67/1,179,026 alleles; this is below 0.003.
• gnomAD v4.1 total frequency is 4.6506840e-05 (75/1,612,666 alleles), and gnomAD v2.1 total frequency is 7.0766400e-05 (20/282,620 alleles).

Sources

Reasoning

The governing ClinGen DICER1 VCEP v1.4 specifies BS1 at frequency greater than 0.0003 (0.03%) in a gnomAD subpopulation, with more than 2,000 alleles tested and at least 5 alleles present. The highest observed gnomAD v4.1 subpopulation frequency is 5.6826567e-05 in European (non-Finnish) (67/1,179,026 alleles), which is below 0.0003. GnomAD v2.1 has a highest subpopulation frequency of 0.000131742, also below the VCEP threshold. Therefore BS1 is not met.

Rule

DICER1 VCEP v1.4 BS1: frequency >0.0003 (0.03%) in a gnomAD subpopulation; subpopulation must have >2,000 alleles tested and at least 5 alleles present.

Evidence considered

• DICER1 VCEP v1.4 specifies BS1 at frequency >0.0003 (0.03%) in a gnomAD subpopulation, requiring >2,000 alleles tested and at least 5 alleles present.
• gnomAD v4.1 reports the highest subpopulation frequency as 5.6826567e-05 in European (non-Finnish), based on 67/1,179,026 alleles; this is below 0.0003.
• GnomAD v2.1 reports a highest subpopulation frequency of 0.000131742 in European (non-Finnish), also below 0.0003.

Sources

Reasoning

The governing DICER1 VCEP permits PS1 for a non-canonical intronic variant at the same nucleotide only when the comparator is asserted pathogenic by the ClinGen DICER1 VCEP and has equal or worse splicing impact, with no splicing difference established by RNA data or concordant MaxEntScan and SpliceAI modeling. The case variant is NM_177438.3:c.734+6C>T, with SpliceAI max delta 0.07, but the available evidence contains no qualifying same-nucleotide pathogenic comparator or comparator splicing result; therefore PS1 cannot be adjudicated.

Rule

For non-canonical intronic splicing variants at the same nucleotide, the variant should have equal or worse splicing impact; the comparator must be asserted pathogenic by the ClinGen DICER1 VCEP, and splicing must be shown equivalent by RNA data or concordant MaxEntScan and SpliceAI modeling.

Evidence considered

• The DICER1 VCEP PS1 rule covers same-amino-acid changes and non-canonical intronic variants at the same nucleotide, requires the comparator to be asserted pathogenic by the ClinGen DICER1 VCEP, and requires no splicing difference based on RNA data or concordant MaxEntScan and SpliceAI modeling.
• The assessed variant is NM_177438.3:c.734+6C>T, and SpliceAI reports a maximum delta score of 0.07.
• No qualifying same-nucleotide pathogenic comparator or comparator splicing result is present in the available case evidence.

Still missing

• A same-nucleotide non-canonical intronic comparator asserted pathogenic by the ClinGen DICER1 VCEP is needed.
• Comparator RNA data or concordant MaxEntScan and SpliceAI splicing assessment is needed.

Sources

Reasoning

The DICER1 VCEP permits PS2 using de novo points, with confirmed maternity and paternity required for a confirmed de novo observation. The available case evidence contains no proband-level de novo observation, parental genotypes, maternity or paternity confirmation, or documented family history sufficient to assign de novo points. Therefore PS2 cannot be assessed.

Rule

DICER1 VCEP v1.4 PS2: apply PS2 from de novo points; Very Strong for >=4 points, Strong for >=2 and <4, Moderate for >=1 and <2, and Supporting for >=0.5 and <1 point. Both maternity and paternity must be confirmed for a confirmed de novo observation.

Evidence considered

• The DICER1 VCEP PS2 rule requires tallying de novo points and states that both maternity and paternity must be confirmed for a confirmed de novo observation.
• No proband-level de novo or parental-testing evidence is documented in the available case materials.

Still missing

• Documented proband phenotype and relevant disease history
• Parental testing results for NM_177438.3:c.734+6C>T
• Confirmation of both maternity and paternity
• Family history and de novo status for the proband

Sources

Reasoning

The DICER1 VCEP Version 1.4 permits PS3 for this intronic variant when an RNA assay demonstrates a damaging splicing outcome or when an in vitro cleavage assay shows failure or severe reduction of either 5p or 3p microRNA production with positive and negative controls. The available case evidence contains only a SpliceAI prediction (maximum delta 0.07), not a variant-specific RNA assay or pre-miRNA cleavage experiment, so PS3 cannot be assigned at any strength.

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); or an 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.

Evidence considered

• The DICER1 VCEP Version 1.4 specifies PS3 Strong or Moderate based on damaging RNA splicing outcomes and PS3 Supporting when an in vitro cleavage assay demonstrates failure or severe reduction of either 5p or 3p microRNA production with positive and negative controls.
• SpliceAI reports a maximum delta score of 0.07 for NM_177438.3:c.734+6C>T; this is a computational prediction and does not establish the functional assay evidence required for PS3.
• No variant-specific RNA splicing result or controlled pre-miRNA cleavage result is present in the reviewed case evidence.

Still missing

• Variant-specific RNA assay with appropriate controls and characterization of the resulting transcript is unavailable.
• Variant-specific in vitro pre-miRNA cleavage assay with positive and negative controls is unavailable.

Sources

Reasoning

The ClinGen DICER1 VCEP specifies PS4 as Strong for "≥4 phenotype points", Moderate for 2–3.5 points, and Supporting for 1–1.5 points. The available case evidence contains no adjudicated PS4 phenotype-point tally, case-control enrichment analysis, or sufficiently documented exact-variant phenotype cohort, so the governing threshold cannot be evaluated. The exact threshold direction is ≥4 phenotype points; because the observed point value is unavailable, no substituted inequality can be established and PS4 remains not assessed.

Rule

DICER1 VCEP Version 1.4: PS4 Strong at ≥4 phenotype points; Moderate at 2–3.5; Supporting at 1–1.5.

Evidence considered

• The DICER1 VCEP Version 1.4 rule states: "≥4 phenotype points" for PS4 Strong, "2 – 3.5 phenotype points" for PS4 Moderate, and "1 – 1.5 phenotype points" for PS4 Supporting.
• No exact-variant case-control enrichment result or adjudicated PS4 phenotype-point tally is present in the reviewed case evidence.

Still missing

• Adjudicated phenotype-point tally or case-control enrichment data for NM_177438.3:c.734+6C>T

Sources

Reasoning

The DICER1 VCEP allows PP1 for phenotype-positive relatives with qualifying specificity and assigns Supporting strength for 3–4 meioses, Moderate for 5–6 meioses, and Strong for at least 7 meioses across at least 2 families. The available case evidence contains no affected relatives, family structure, genotype-phenotype segregation results, or countable meioses, so PP1 cannot be assessed.

Rule

DICER1 VCEP v1.4 PP1: Supporting for 3–4 meioses across at least 1 family; Moderate for 5–6 meioses across at least 1 family; Strong for at least 7 meioses across at least 2 families. Do not apply if BA1 or BS1 is met.

Evidence considered

• The DICER1 VCEP PP1 thresholds are Supporting for 3–4 meioses, Moderate for 5–6 meioses, and Strong for at least 7 meioses across at least 2 families; phenotype-positive relatives must have high-, moderate-, or low-specificity phenotypes.
• No family segregation data or informative meioses are documented in the available case materials.

Still missing

• Pedigree or family structure
• Genotypes and phenotypes of informative relatives
• Number of informative meioses and families
• Phenotype specificity for each reportedly affected relative

Sources

Reasoning

The variant is intronic at c.734+6, so the splice-impact path is applicable. SpliceAI reports a max delta of 0.07, below the generic PP3 supporting threshold of 0.2, but the governing DICER1 specification requires concordance of MaxEntScan and SpliceAI for PP3 and no MaxEntScan result is available. Therefore the required VCEP evidence set is incomplete.

Rule

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

Evidence considered

• The DICER1 VCEP specification defines PP3 for splicing variants using concordance of MaxEntScan and SpliceAI.
• SpliceAI reports max delta 0.07 for NM_177438.3:c.734+6C>T; the generic SpliceAI PP3 supporting cutoff supplied for this review is >=0.2.
• No MaxEntScan result is present in the available case evidence.

Still missing

• MaxEntScan prediction for NM_177438.3:c.734+6C>T is unavailable; the governing DICER1 specification requires concordance with SpliceAI.

Sources

Reasoning

The ClinGen DICER1 VCEP defines PP4 Supporting as a somatic tumor finding of a previously reported DICER1 RNase IIIb hotspot second hit, retention of the germline variant under evaluation, and no additional somatic DICER1 loss-of-function variants. The case evidence provides no tumor sequencing or documented somatic second-hit result, so this rule cannot be assessed.

Rule

DICER1 VCEP Version 1.4 PP4 Supporting: qualifying somatic RNase IIIb hotspot second hit plus retention of the germline variant and no additional somatic DICER1 non-hotspot variants.

Evidence considered

• The DICER1 VCEP PP4 rule requires a previously reported somatic second hit at p.S1344, p.E1705, p.D1709, p.D1713, p.G1809, p.D1810, or p.E1813 and retention of the germline DICER1 variant, with PP4 excluded when additional somatic DICER1 non-hotspot variants are found.
• No qualifying tumor assay or somatic second-hit evidence is documented for this variant.

Still missing

• Tumor sequencing evidence for a qualifying RNase IIIb hotspot second hit, germline-variant retention, and absence of additional somatic DICER1 loss-of-function variants

Sources

Reasoning

The DICER1 VCEP v1.4 permits BS2 at strong strength for 40 or more unrelated females from one source who are tumor-free through age 50, subject to a 40:1 ratio to PS4-eligible probands, or for 2 or more observations of homozygosity in healthy individuals, or one homozygous healthy individual with parental confirmation. It permits supporting strength for 10 or more such tumor-free females with a 10:1 ratio, or 2 or more homozygous observations in individuals lacking clinical information. GnomAD v4.1 and v2.1 report zero homozygotes, which does not provide a positive BS2 observation, and the available case evidence contains no qualifying tumor-free female cohort, healthy homozygote, parental confirmation, or eligible ratio. BS2 is therefore not assessed rather than considered not met.

Rule

DICER1 VCEP v1.4 BS2: strong for 40+ unrelated tumor-free females through age 50 with required 40:1 ratio, or qualifying homozygosity; supporting for 10+ such females with 10:1 ratio, or 2+ homozygous observations without clinical information.

Evidence considered

• DICER1 VCEP v1.4 specifies BS2 strong for 40 or more unrelated tumor-free females through age 50 with a 40:1 ratio to PS4-eligible probands, or 2 or more healthy homozygote observations, or 1 healthy homozygote with parental confirmation.
• The VCEP specifies BS2 supporting for 10 or more unrelated tumor-free females through age 50 with a 10:1 ratio, or 2 or more homozygous observations in individuals lacking clinical information.
• GnomAD v4.1 reports 0 homozygotes and gnomAD v2.1 reports 0 homozygotes; absence of homozygotes is not a qualifying positive BS2 observation.

Still missing

• No qualifying DICER1 BS2 evidence was found: tumor-free female cohort with required age and proband ratio, healthy homozygote observations, or parental confirmation.

Sources

Reasoning

The DICER1 VCEP Version 1.4 permits BS3 Strong when an RNA assay shows no splicing impact more than once, or BS3 Supporting when an in vitro cleavage assay demonstrates production of both 5p and 3p microRNAs from a pre-miRNA with positive and negative controls. The available evidence contains only a SpliceAI prediction (maximum delta 0.07), with no variant-specific RNA assay or controlled cleavage assay, so BS3 cannot be assigned.

Rule

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

Evidence considered

• The DICER1 VCEP Version 1.4 specifies BS3 Strong for intronic or synonymous variants only when no splicing impact is observed by RNA assay more than once.
• The DICER1 VCEP Version 1.4 specifies BS3 Supporting when an in vitro cleavage assay demonstrates production of both 5p and 3p microRNAs from a pre-miRNA with positive and negative controls.
• SpliceAI reports a maximum delta score of 0.07 for NM_177438.3:c.734+6C>T; this computational result is not a functional demonstration of normal splicing or DICER1 cleavage activity.
• No repeated RNA assay or controlled pre-miRNA cleavage assay is present in the reviewed case evidence.

Still missing

• Repeated variant-specific RNA assay showing no splicing impact is unavailable.
• Variant-specific in vitro pre-miRNA cleavage assay showing both 5p and 3p microRNA production with positive and negative controls is unavailable.

Sources

Reasoning

The DICER1 VCEP requires phenotype-positive, genotype-negative first-, second-, or third-degree relatives with a high- or moderate-specificity phenotype for BS4. The available case evidence contains no documented affected relatives, relative genotypes, degree of relationship, or phenotype-specificity information demonstrating non-segregation, so BS4 cannot be assessed.

Rule

DICER1 VCEP v1.4 BS4: Strong when family members with a high- or moderate-specificity phenotype are genotype-negative first-, second-, or third-degree relatives of the proband.

Evidence considered

• The DICER1 VCEP BS4 rule requires phenotype-positive, genotype-negative first-, second-, or third-degree relatives with a high- or moderate-specificity phenotype.
• No genotype-negative affected relatives or other non-segregation evidence are documented in the available case materials.

Still missing

• Affected relatives with qualifying phenotype specificity
• Negative genotype results for the evaluated variant in those relatives
• Degree of relationship to the proband
• Assessment of possible phenocopies or another molecular cause

Sources

Reasoning

The governing ClinGen DICER1 VCEP Version 1.4 rule would support BP2 at Supporting strength for 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 available case evidence contains no affected-proband observation, paired DICER1 variant, phase result, cis/trans determination, or inheritance data for NM_177438.3:c.734+6C>T, so BP2 cannot be assessed.

Rule

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

Evidence considered

• The ClinGen DICER1 VCEP Version 1.4 BP2 rule requires at least one observation in trans with a P/LP DICER1 variant or at least three observations in cis or with phase unknown involving two or more different P/LP DICER1 variants.
• No qualifying affected-proband allelic observation, additional DICER1 variant, phase result, or inheritance information for NM_177438.3:c.734+6C>T is present in the reviewed case evidence.

Still missing

• Affected-proband observations with additional DICER1 variants are not documented.
• Phase information establishing cis or trans with a P/LP DICER1 variant is not documented.
• Inheritance or family-segregation data relevant to the allelic relationship are not documented.

Sources

Reasoning

The variant is intronic at c.734+6, so the splice-impact path is applicable. SpliceAI reports a max delta of 0.07, meeting the supplied generic BP4 supporting threshold of <=0.1, but the governing DICER1 specification requires concordance of MaxEntScan and SpliceAI for BP4 and no MaxEntScan result is available. Therefore the required VCEP evidence set is incomplete.

Rule

DICER1 VCEP BP4 Supporting: for synonymous/intronic/non-coding variants, concordance of MaxEntScan and SpliceAI.

Evidence considered

• The DICER1 VCEP specification defines BP4 for synonymous, intronic, and non-coding variants using concordance of MaxEntScan and SpliceAI.
• SpliceAI reports max delta 0.07 for NM_177438.3:c.734+6C>T; the generic SpliceAI BP4 supporting cutoff supplied for this review is <=0.1.
• No MaxEntScan result is present in the available case evidence.

Still missing

• MaxEntScan prediction for NM_177438.3:c.734+6C>T is unavailable; the governing DICER1 specification requires concordance with SpliceAI.

Sources