APC c.1631T>C, p.(Ile544Thr)

Reasoning

The APC VCEP specifies BS1 at gnomAD Popmax Filtering Allele Frequency at least 0.001% (0.00001). gnomAD v4.1 reports a highest population AF of 0.000304574 (0.03046%) and grpmax FAF of 0.00027828 (0.027828%), each above 0.00001; therefore BS1 is met at strong strength.

Rule

APC VCEP BS1: gnomAD Popmax Filtering Allele Frequency >= 0.001% (0.00001); strong.

Evidence considered

• The governing APC InSiGHT VCEP specifies BS1 at gnomAD Popmax Filtering AF >= 0.00001 (0.001%) and assigns strong strength.
• gnomAD v4.1 reports grpmax FAF 0.00027828 and European (non-Finnish) AF 0.000304574 (0.03046%), both above the VCEP threshold.

Sources

Reasoning

The APC InSiGHT VCEP applies BP1 to APC missense variants except those located in the first 15-amino-acid repeat of the beta-catenin-binding domain, defined as codons 1021-1035. The assessed variant is p.(Ile544Thr) at codon 544, which is outside that exception, so BP1 Supporting applies.

Rule

APC VCEP BP1 Supporting: applicable to APC missense variants except variants in the first 15-amino-acid repeat of the beta-catenin-binding domain, codons 1021-1035.

Evidence considered

• The APC InSiGHT VCEP BP1 rule applies to APC missense variants except those in the first 15-amino-acid repeat of the beta-catenin-binding domain, codons 1021-1035.
• The assessed variant is NP_001120982.1:p.(Ile544Thr), placing it at codon 544 and outside the codon 1021-1035 exception.

Sources

Reasoning

The APC InSiGHT VCEP specifies PS1 for a missense variant producing the same amino-acid change as an established pathogenic or likely pathogenic APC variant. It identifies only p.(Asn1026Ser) and p.(Ser1028Arg) as the current likely pathogenic missense variants; neither has the same p.(Ile544Thr) amino-acid change. The exact variant p.Ile544Thr was listed as a VUS in an APC germline-testing series, not as a pathogenic same-amino-acid comparator.

Rule

APC VCEP PS1: same amino-acid change as a previously established pathogenic or likely pathogenic APC variant; established likely pathogenic missense comparators are p.(Asn1026Ser) and p.(Ser1028Arg).

Evidence considered

• The APC InSiGHT VCEP PS1 rule requires the assessed missense variant to produce the same amino-acid change as an established pathogenic or likely pathogenic APC variant.
• The VCEP states that the only current likely pathogenic APC missense variants are c.3077A>G p.(Asn1026Ser) and c.3084T>A p.(Ser1028Arg); other variants with the same amino-acid changes meet PS1_Moderate.
• The exact variant c.1631T>C p.Ile544Thr was listed among APC variants of uncertain significance in Table 3 of the Mayo Clinic series.

Sources

Reasoning

The APC VCEP defines PM2 supporting as allele frequency <= 0.000003 when allele count is greater than 1, or allele frequency < 0.00001 when allele count is at most 1. gnomAD v4.1 reports allele count 375 and total AF 0.000233606, with grpmax FAF 0.00027828; these exceed the applicable 0.000003 threshold, so PM2 is not met.

Rule

APC VCEP PM2: supporting when AF <= 0.000003 if allele count >1, or AF <0.00001 if allele count <=1.

Evidence considered

• The governing APC InSiGHT VCEP defines PM2 supporting as AF <= 0.000003 when allele count is >1, or AF <0.00001 when allele count is <=1.
• gnomAD v4.1 reports total allele count 375, total AF 0.000233606, and grpmax FAF 0.00027828; because allele count is >1, the applicable threshold is 0.000003 and the observed frequency is above it.

Sources

Reasoning

The variant is missense (NM_001127510.3:c.1631T>C; p.(Ile544Thr)), so PP3 is evaluated through the APC VCEP's in silico splicing path rather than conservation or missense prediction models. The APC specification states that in silico splicing predictors should be used for presumed missense variants and that multiple in silico splicing predictors must support a deleterious effect. SpliceAI reports a maximum delta score of 0.001, with DS_AG 0.000, DS_AL 0.001, DS_DG 0.001, and DS_DL 0.000; these do not support a deleterious splice effect and are below the generic PP3 supporting threshold of 0.2. Therefore PP3 is not met. The REVEL score of 0.422 and BayesDel score of -0.0440841 do not establish PP3 under the governing APC specification, which states not to use computational prediction models for missense conservation/evolution and directs missense assessment to in silico splicing predictors.

Rule

APC VCEP PP3 Supporting: for missense variants, use in silico splicing predictors to reveal possible splicing effects; for non-canonical splicing variants, multiple in silico splicing predictors must support a deleterious effect.

Evidence considered

• The APC VCEP specification assigns PP3 Supporting for presumed missense variants when in silico splicing predictors support a deleterious effect; it also states not to use missense conservation/evolution prediction models.
• SpliceAI maximum delta score is 0.001 (DS_AG 0.000, DS_AL 0.001, DS_DG 0.001, DS_DL 0.000), which is below the generic PP3 supporting threshold of >=0.2 from Jaganathan et al. 2019 (PMID:30661751).
• REVEL is 0.422 and BayesDel is -0.0440841; neither is used to assign PP3 because the governing APC specification directs presumed missense computational assessment to in silico splicing predictors and does not provide a BayesDel cutoff.

Sources

Reasoning

The APC VCEP specifies BA1 when gnomAD Popmax Filtering Allele Frequency is at least 0.1% (0.001). The highest available gnomAD v4.1 population frequency is the European (non-Finnish) AF of 0.000304574 (0.03046%), with grpmax FAF 0.00027828 (0.027828%), both below 0.001; therefore BA1 is not met.

Rule

APC VCEP BA1: gnomAD Popmax Filtering Allele Frequency >= 0.1% (0.001).

Evidence considered

• The governing APC InSiGHT VCEP specifies BA1 at gnomAD Popmax Filtering AF >= 0.001 (0.1%).
• gnomAD v4.1 reports total AF 0.000233606, highest population AF 0.000304574 in European (non-Finnish) individuals, grpmax FAF 0.00027828, and zero homozygotes.

Sources

Reasoning

The APC VCEP requires at least 10 healthy-individual points for BS2 strong or at least 3 points for BS2 supporting, or at least 2 observations in the homozygous state for strong BS2. gnomAD v4.1 reports zero homozygotes and the available variant-specific publication reports the variant in a clinical APC-testing series rather than qualifying healthy individuals; neither VCEP BS2 pathway is satisfied.

Rule

APC VCEP BS2: strong for >=10 healthy-individual points or >=2 homozygous observations; supporting for >=3 healthy-individual points.

Evidence considered

• The APC VCEP defines BS2 supporting as at least 3 healthy-individual points and BS2 strong as at least 10 points or at least 2 homozygous observations; a qualifying healthy individual is specifically defined by the VCEP.
• gnomAD v4.1 reports homozygotes = 0 for 375/1,605,270 alleles, so the homozygous-observation pathway is not met.
• The exact variant was reported once in an APC germline-testing series (PMID:23159591, Table 3), but the paper does not provide qualifying healthy-individual status or homozygous observation for this variant.

Sources

Reasoning

The APC VCEP requires de novo scores derived from phenotype points and parental relationship evidence, with at least 1 de novo score for PS2 at Moderate strength, 2-3.5 for Strong, or at least 4 for Very Strong. The exact variant was reported in one individual in an APC testing series, but the available report provides no maternity or paternity confirmation, parental testing, de novo observation, or phenotype-point information for this individual. Therefore PS2 cannot be assessed.

Rule

≥ 4 de novo scores = Very Strong; 2-3.5 de novo scores = Strong; 1-1.5 de novo score = Moderate. De novo score is curated using APC VCEP Tables 1 and 2.

Evidence considered

• The APC VCEP specifies PS2 as at least 1 de novo score at Moderate strength, 2-3.5 at Strong strength, and at least 4 at Very Strong strength; the de novo score is based on phenotype points and confirmed or unconfirmed parental relationships.
• PMID:23159591 reports NM_001127510.3:c.1631T>C (p.Ile544Thr) in one individual in an APC germline-testing series, but gives no parental testing, confirmed maternity or paternity, or proband phenotype-point data for this variant.

Still missing

• Documented testing of both parents and confirmation of maternity and paternity
• APC VCEP phenotype points for the proband
• A de novo score calculated under APC VCEP Tables 1 and 2

Sources

Reasoning

The APC-specific ClinGen InSiGHT VCEP permits PS3 for this missense variant only when variant-directed RNA or protein functional results meet its specified assay patterns. The available evidence identifies NM_001127510.3:c.1631T>C (p.Ile544Thr) in a germline testing series, but reports no variant-specific functional assay, RNA abnormality, transcriptional activity result, beta-catenin binding result, assay controls, or calibrated quantitative functional measurement. Therefore PS3 cannot be applied at any strength.

Rule

APC VCEP PS3: Very Strong requires an RNA assay showing a premature stop codon or in-frame skipping of exon 13 or 14 with absence of full-length transcript; Strong requires the same finding with less than 10% full-length transcript; Moderate requires specified RNA abnormalities with exon loss/skipping or insertion findings, or other in-frame skipping absent or below 10% full-length transcript; Supporting permits specified RNA transcript abnormalities or increased beta-catenin-regulated transcription activity/decreased beta-catenin binding in the APC beta-catenin-binding domain.

Evidence considered

• The APC InSiGHT VCEP PS3 specification requires defined RNA or protein assay findings, including assay-specific transcript or beta-catenin functional outcomes, for application at calibrated strengths.
• PMID:23159591 lists the exact variant c.1631T>C (p.Ile544Thr) among APC VUSs in a 1,591-case germline testing series, but explicitly provides no variant-specific functional assay, segregation, or phenotype data.
• No available evidence provides assay controls, biological rescue or loss-of-function measurements, RNA transcript quantification, beta-catenin-regulated transcription activity, or beta-catenin binding data for this variant.

Still missing

• A validated variant-directed functional assay with appropriate positive and negative controls is unavailable.
• No quantitative or categorical assay result is available for APC c.1631T>C (p.Ile544Thr).

Sources

Reasoning

The APC InSiGHT VCEP defines PS4 from phenotype points: >=16 for Very Strong, 4-15.5 for Strong, 2-3.5 for Moderate, and 1-1.5 for Supporting, with phenotype points curated using Table 1. PMID:23159591 reports the exact variant NM_001127510.3:c.1631T>C (p.Ile544Thr) in one person among 1,591 APC-testing cases and lists it as a VUS, but provides no variant-specific phenotype details, phenotype-point score, affected-control comparison, odds ratio, relative risk, or confidence interval. Therefore PS4 cannot be assigned from the available evidence.

Rule

PS4: >=16 phenotype points = Very Strong; 4-15.5 = Strong; 2-3.5 = Moderate; 1-1.5 = Supporting, using APC VCEP Table 1.

Evidence considered

• The APC InSiGHT VCEP PS4 rule states: >=16 phenotype points is Very Strong, 4-15.5 is Strong, 2-3.5 is Moderate, and 1-1.5 is Supporting; phenotype points are curated using Table 1.
• The exact variant c.1631T>C p.Ile544Thr was listed in Table 3 of the Mayo Clinic APC germline-testing series as a VUS, observed in 1 of 1,591 consecutive APC-testing cases with reported mutant allele frequency 0.0003; the paper provides no phenotype details for that individual.
• No odds ratio, relative risk, confidence interval, or affected-versus-control enrichment result for this exact variant was identified.

Still missing

• Variant-specific clinical phenotype and APC VCEP Table 1 phenotype-point scoring are unavailable.
• No case-control enrichment statistic, odds ratio, relative risk, or confidence interval is available.

Sources

Reasoning

The APC VCEP PM5 rule requires a different missense variant at the same amino-acid residue that is established as likely pathogenic or pathogenic under APC-specific criteria, with the assessed substitution meeting the required Grantham-distance comparison. The collected PM5 search found zero same-residue candidates at codon 544, but comparator harvesting ended with an HTTP 429 rate-limit error; therefore absence of a qualifying comparator cannot be treated as conclusive. No paper_extracts finding establishes a pathogenic alternate missense at Ile544.

Rule

APC VCEP PM5: a different missense change at the same residue established as pathogenic or likely pathogenic under APC-specific criteria; moderate application additionally requires equal or higher Grantham distance than the reported variant.

Evidence considered

• The APC VCEP PM5 rule requires a different missense variant at the same amino-acid position that is established as pathogenic or likely pathogenic under APC-specific modifications; for moderate PM5, the assessed variant must also have an equal or higher Grantham distance than the reported comparator.
• The PM5 candidate record reports zero same-residue candidates and zero expert-panel or non-expert-majority pathogenic/likely pathogenic candidates, but also records an HTTP 429 rate-limit error during harvesting.
• The exact p.Ile544Thr variant was reported as a VUS in Table 3 of a clinical APC testing series; that report does not establish a pathogenic alternate missense at codon 544.

Still missing

• A verified ClinVar or literature review for pathogenic or likely pathogenic alternate missense variants at APC codon 544 is still required.
• The Grantham-distance comparison cannot be performed without a qualifying codon-544 comparator.

Sources

Reasoning

The APC VCEP requires de novo scores for PM6: 0.5 for Supporting, 1-1.5 for Moderate, and 2-3.5 for Strong. Although the exact variant was listed in one individual, the available evidence does not establish that it was assumed de novo, does not describe parental testing or relationships, and does not provide phenotype points from which a de novo score could be calculated. PM6 therefore cannot be assessed.

Rule

2-3.5 de novo scores = Strong; 1-1.5 de novo scores = Moderate; 0.5 de novo score = Supporting. De novo score is curated using APC VCEP Tables 1 and 2.

Evidence considered

• The APC VCEP specifies PM6 as 0.5 de novo score at Supporting strength, 1-1.5 at Moderate strength, and 2-3.5 at Strong strength, using the APC VCEP de novo-score tables.
• PMID:23159591 lists the exact variant in one individual but reports no parental testing, de novo assessment, relationship confirmation, or phenotype-point data.

Still missing

• Evidence that the variant was absent in tested parents or that de novo status was otherwise assumed
• Parental relationship confirmation status
• APC VCEP phenotype points and resulting de novo score

Sources

Reasoning

The APC VCEP counts only genotype-positive and phenotype-positive affected individuals or obligate carriers with phenotype and assigns PP1 Supporting for 3-4 segregating meioses, Moderate for 5-6 meioses, and Strong for at least 7 meioses in at least 2 families. The available evidence contains no informative relatives, affected family members, pedigree, parental segregation, or meiosis count for this variant. PP1 cannot be assessed.

Rule

Variant segregates in 3-4 meioses in at least 1 family = Supporting; 5-6 meioses in at least 1 family = Moderate; at least 7 meioses in at least 2 families = Strong.

Evidence considered

• The APC VCEP requires phenotype-positive genotype-positive individuals or phenotype-positive obligate carriers and defines PP1 as Supporting for 3-4 meioses, Moderate for 5-6 meioses, and Strong for at least 7 meioses in at least 2 families.
• PMID:23159591 reports the exact variant in one individual without family history, relatives, segregation testing, or informative meioses for the variant.

Still missing

• Pedigree and family identifiers
• Genotypes and phenotype points for affected relatives or obligate carriers
• Count of informative segregating meioses across families

Sources

Reasoning

The APC-specific ClinGen InSiGHT VCEP limits BS3 to validated benign functional evidence: a constitutional RNA assay showing no mRNA abnormality, with biallelic expression or nonsense-mediated-decay inhibition for Strong, or without those controls for Supporting; it also permits Supporting for retained beta-catenin-regulated transcription activity in the APC beta-catenin-binding domain. The available evidence contains no variant-specific RNA or protein assay, control design, or functional result for c.1631T>C (p.Ile544Thr). Therefore BS3 cannot be applied at any strength.

Rule

APC VCEP BS3: Strong requires an RNA assay of a synonymous or intronic variant in a constitutional patient sample showing no mRNA aberration plus biallelic expression and/or nonsense-mediated-decay inhibition; Supporting requires the same RNA finding without those demonstrations, or a protein assay showing beta-catenin-regulated transcription activity comparable to wild type for variants in APC codons 959-2129.

Evidence considered

• The APC InSiGHT VCEP BS3 specification requires a benign RNA or protein assay; Strong RNA evidence additionally requires demonstrated biallelic expression or use of nonsense-mediated-decay inhibition, while Supporting protein evidence requires retained beta-catenin-regulated transcription activity in the specified APC domain.
• PMID:23159591 lists the exact variant c.1631T>C (p.Ile544Thr) but reports no variant-specific functional assay or benign functional result.
• No available evidence supplies the assay controls or measurements needed to establish normal transcript processing or retained APC protein function.

Still missing

• A validated benign functional assay with appropriate controls is unavailable.
• No evidence demonstrates normal mRNA processing, biallelic expression, nonsense-mediated-decay control, retained transcriptional activity, or normal beta-catenin binding for this variant.

Sources

Reasoning

The APC VCEP requires an affected member without the variant to score at least 0.5 phenotype point for BS4 Supporting, or either one such member scoring at least 1 point or two such members each scoring at least 0.5 points for BS4 Strong. No affected relatives lacking the variant, non-segregation result, pedigree, or phenotype-point data are available for this variant. BS4 cannot be assessed.

Rule

Affected member without the variant must score at least 0.5 phenotype point = Supporting; one affected member without the variant scoring at least 1 point, or at least two such members each scoring at least 0.5 points = Strong.

Evidence considered

• The APC VCEP defines BS4 Supporting as an affected member without the variant scoring at least 0.5 phenotype point, and BS4 Strong as one such member scoring at least 1 point or two such members each scoring at least 0.5 points.
• PMID:23159591 provides no affected-relative testing, non-segregation observation, pedigree, or phenotype-point data for the exact variant.

Still missing

• An affected relative or relatives tested for the variant
• Phenotype-point scoring for affected relatives lacking the variant
• Assessment of phenocopy or an alternate pathogenic variant if non-segregation is observed

Sources

Reasoning

The APC VCEP permits BP2 at supporting strength when the variant is observed in trans with a (Likely) Pathogenic APC variant or at least three times with different (Likely) Pathogenic APC variants in unknown phase. The exact APC c.1631T>C (p.Ile544Thr) variant was reported once in an APC germline-testing series, but that report provides no accompanying pathogenic variant, phase, cis/trans determination, or proband-level allelic observation. Therefore the BP2 threshold cannot be established.

Rule

Supporting: observed in trans with a (Likely) Pathogenic APC variant OR observed at least 3 times in unknown phase with different (Likely) Pathogenic APC variants.

Evidence considered

• The APC ClinGen InSiGHT VCEP specifies BP2 supporting when the variant is observed in trans with a (Likely) Pathogenic APC variant or at least three times with different (Likely) Pathogenic APC variants in unknown phase.
• Kerr et al. reported APC c.1631T>C (p.Ile544Thr) in one individual in a 1,591-case APC germline-testing series, but the report gives no variant-specific segregation, pathogenic partner variant, or cis/trans phase information.

Still missing

• No documented pathogenic or likely pathogenic APC variant observed in trans with c.1631T>C.
• No documented cis/trans phase information for the reported individual.
• No evidence of at least three observations with different pathogenic or likely pathogenic APC variants in unknown phase.

Sources

Reasoning

The APC InSiGHT VCEP permits BP5 at Supporting strength only for an alternate genetic basis of a colorectal polyposis phenotype, specifically a qualifying (Likely) Pathogenic variant in another adenomatous polyposis gene. The available evidence reports the exact APC variant in one APC-testing case but does not document a colorectal polyposis phenotype for that individual and does not identify a qualifying alternate pathogenic variant in POLD1, POLE, MUTYH, NTHL1, MSH3, or the specified childhood/adolescent mismatch-repair genes. BP5 therefore cannot be assigned.

Rule

BP5 Supporting: only an alternate genetic basis of a colorectal polyposis phenotype, specifically a qualifying (Likely) Pathogenic variant in another adenomatous polyposis gene.

Evidence considered

• The APC InSiGHT VCEP BP5 rule requires a (Likely) Pathogenic variant in another adenomatous polyposis gene and is only applicable when a colorectal polyposis phenotype is present.
• The available exact-variant literature record identifies the APC variant in one testing case but does not provide the required alternate-gene result or variant-specific colorectal polyposis phenotype.

Still missing

• The clinical phenotype of the individual carrying the exact variant is not documented.
• No qualifying alternate (Likely) Pathogenic variant in another adenomatous polyposis gene is documented.

Sources