MLH1 c.1558+4C>T, p.?

Reasoning

InSiGHT MLH1 VCEP v2.0 PM2 (Supporting) is met when the allele frequency is absent/extremely rare, < 0.00002 (<1 in 50,000 alleles) in gnomAD v4. The observed gnomAD v4.1 total AF is 2.478e-06 (4/1,613,954 alleles), and the joint grpmax filtering AF is 4.43e-06; both are below the 2e-05 threshold, with zero homozygotes. The African/African American subpopulation AF (2.667e-05; 2/74,988 alleles) marginally exceeds the threshold but rests on only 2 alleles and is handled by gnomAD's filtering AF, which remains well below the threshold; this is a threshold artifact of small counts and does not change the assessment. gnomAD v2.1 (AF 7.958e-06; 2/251,334) and absence in gnomAD-Canada v1.0 are consistent. PM2_Supporting met.

Rule

Absent/extremely rare allele frequency < 0.00002 (<1 in 50,000 alleles) in gnomAD v4 dataset (InSiGHT MLH1 VCEP v2.0, PM2_Supporting)

Evidence considered

• gnomAD v4.1 (gnomad_v4): total AF 2.478e-06 (4/1,613,954 alleles; <1 in 50,000), joint grpmax FAF 4.43e-06, homozygotes 0; AFR subpopulation AF 2.667e-05 (2/74,988)
• gnomAD v2.1 (gnomad_v2): total AF 7.958e-06 (2/251,334 alleles), homozygotes 0
• gnomAD-Canada v1.0 (gnomad_canada): variant absent
• InSiGHT MLH1 VCEP v2.0 (cspec) PM2 rule: absent/extremely rare allele frequency < 0.00002 (<1 in 50,000 alleles) in gnomAD v4 dataset, strength Supporting

Sources

Reasoning

VCEP BP4 rule for intronic variants: SpliceAI predicts no splicing impact with delta score <= 0.1 (InSiGHT MLH1 VCEP v2.0, 'as per Walker et al 2023'). SpliceAI max delta score for c.1558+4C>T is 0.00, which is <= 0.1, so BP4 is met at Supporting strength. The alternate HCI-prior path (<0.11) is missense-only and does not apply to this intronic variant. The SpliceAI prediction is counted once (for BP4); it is not double-counted across PP3/BP4/BP7.

Rule

BP4_Supporting: Missense variant with HCI-prior probability of pathogenicity <0.11 as per hci-priors.hci.utah.edu/PRIORS; OR For intronic and synonymous variants: SpliceAI predicts no splicing impact with delta score <= 0.1 as per Walker et al 2023. (InSiGHT MLH1 VCEP v2.0)

Evidence considered

• SpliceAI (SpliceAI Lookup, Broad Institute) for NM_000249.4:c.1558+4C>T: max_delta_score = 0.00; DS_AG = 0.00, DS_AL = 0.00, DS_DG = 0.00, DS_DL = 0.00.
• InSiGHT MLH1 VCEP v2.0 (cspec doc 1564688410) BP4 rule: 'For intronic and synonymous variants: SpliceAI predicts no splicing impact with delta score <= 0.1 as per Walker et al 2023' — threshold 0.1 named by the governing VCEP specification; max delta 0.00 <= 0.1, BP4 Supporting met.

Sources

Reasoning

Governing framework is the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel (VCEP) specification for MLH1 v2.0 (CSPEC doc 1564688410, framework_mode=vcep). PVS1 is Applicable in this VCEP. Variant NM_000249.4:c.1558+4C>T is an intronic substitution at +4 of intron 13 (VariantValidator exon annotation '13i'), i.e. a non-canonical splice-site variant with no predicted protein consequence (p.?). It is not a nonsense or frameshift variant (PTC at/before codon 753 or at codons 754-756), not an initiation-codon variant, not a large genomic alteration, and not an IVS+/-1 or +/-2 splice variant; the VCEP PVS1_Strong 'G>non-G at last base of exon' rule does not apply because the variant is intronic at +4, not the last exonic base. SpliceAI reports max delta score 0.00 (no predicted splice impact), and no patient-derived mRNA/minigene assay data is present in the case, so the VCEP PVS1 rule for mRNA-assay-confirmed splicing aberration (which for non-canonical splice-site variants requires minigene or independent-laboratory RNA confirmation and evidence the variant allele produces no full-length/reference transcript) cannot be satisfied. No PVS1 category applies; the criterion is not met.

Rule

InSiGHT MLH1 VCEP v2.0 PVS1: PVS1_VeryStrong for nonsense/frameshift PTC at/before codon 753; large genomic alterations of single/multi-exon size; IVS+/-1 or +/-2 variants with out-of-frame exon skipping or cryptic splice use predicted to undergo NMD; variants with mRNA assays from patient constitutional samples showing the variant allele produces no full-length transcript and leads to a PTC or in-frame deletion disrupting a functional domain (minigene or independent-lab RNA confirmation required for non-canonical splice-site variants); initiation-codon variants. PVS1_Strong for G>non-G at last base of exon if first 6 bases of the intron are not GTRRGT; IVS+/-1 or +/-2 with in-frame skipping of a critical region; tandem duplication of >=1 exon causing frameshift. PVS1_Moderate for PTC at codons 754-756.

Evidence considered

• cspec: MLH1 VCEP v2.0 PVS1 rules (Very Strong: nonsense/frameshift PTC at or before codon 753, large genomic alterations, IVS+/-1 or +/-2 with NMD-predicted out-of-frame skipping, mRNA-assay-confirmed splicing aberration with no full-length transcript, initiation codon; Strong: tandem exon duplication frameshift, G>non-G at last base of exon if first 6 intronic bases not GTRRGT, IVS+/-1 or +/-2 in-frame skipping of critical region; Moderate: PTC at codons 754-756) - variant is intronic +4 (non-canonical) and no category applies
• spliceai: max delta score 0.00 (DS_AG 0.0, DS_AL 0.0, DS_DG 0.0, DS_DL 0.0) - no predicted splice impact
• pvs1_variant_assessment: consequence_class 'unknown', canonical_splice_consensus false, variant_bucket 'other', apply_generic_pvs1_framework false - variant is not in the generic PVS1 null-variant buckets

Still missing

• No patient-derived mRNA/minigene assay data is available; the VCEP PVS1 mRNA-aberration rule for non-canonical splice-site variants is untestable without RNA studies (and requires confirmation in a minigene assay or an additional RNA assay from an independent laboratory).
• VCEP PVS1 decision-tree PDF (PVS1_DecisionTree_MMR.pdf) is present in the gene VCEP folder but was not summarized in vcep_materials.json; PVS1 assessment relies on the CSPEC ruleset text.

Sources

Reasoning

Variant is NM_000249.4:c.1558+4C>T (MLH1), an intronic variant at the +4 position of intron 13 (a non-canonical splice nucleotide) with no predicted protein consequence (NP_000240.1:p.?). Under the governing InSiGHT MLH1 VCEP v2.0 specification, PS1_Strong requires either (a) a predicted missense substitution encoding the same amino acid change as a different nucleotide change previously established by this VCEP as Pathogenic, or (b) a variant affecting the same non-canonical splice nucleotide as a confirmed Pathogenic splice variant with similar or worse splicing in silico prediction using SpliceAI; PS1_Moderate applies the same-nucleotide logic against a Likely Pathogenic splice variant. Scenario (a) is inapplicable because this variant produces no amino acid change. For scenario (b), no variant at c.1558+4 classified by this VCEP as Pathogenic or Likely Pathogenic is present anywhere in the case evidence (ClinVar lists only this exact variant, VUS x3 and Likely Benign x3, no expert panel submissions; the PM5 comparator collection contains 0 candidates), and independently the SpliceAI max delta score for c.1558+4C>T is 0.00, so the required 'similar or worse splicing in silico prediction' cannot be satisfied. PS1 is therefore not met.

Rule

A predicted missense substitution that encodes the same amino acid change with a different underlying nucleotide change previously established by this VCEP as Pathogenic (not a predicted or confirmed splice defect). OR Variants affecting the same non-canonical splice nucleotide as a confirmed Pathogenic splice variant with similar or worse splicing in silico prediction using SpliceAI. [PS1_Strong; PS1_Moderate analogous with Likely Pathogenic splice variant]

Evidence considered

• cspec: InSiGHT MLH1 VCEP v2.0 PS1 rule text — PS1_Strong requires a predicted missense substitution encoding the same amino acid change via a different nucleotide previously established as Pathogenic by this VCEP, OR a variant affecting the same non-canonical splice nucleotide as a confirmed Pathogenic splice variant with similar or worse SpliceAI prediction; PS1_Moderate applies the same non-canonical splice nucleotide logic against a Likely Pathogenic splice variant
• spliceai: SpliceAI max delta score = 0.00 for NM_000249.4:c.1558+4C>T (all DS_AG/AL/DG/DL = 0.0), i.e., no splicing impact predicted, which cannot satisfy the 'similar or worse splicing in silico prediction' requirement of the VCEP PS1 splice-nucleotide scenario
• pm5_candidates: PM5 comparator collection found 0 total candidates and 0 same-residue candidates; variant consequence class is 'unknown' (not missense), so no same-residue/same-change comparator exists
• clinvar: ClinVar variation 187689 contains only submissions for c.1558+4C>T itself (Uncertain significance x3, Likely benign x3, single-submitter, no expert panel); no Pathogenic/Likely Pathogenic variant at the same splice nucleotide was identified

Still missing

• No dedicated same-nucleotide comparator search (e.g., InSiGHT/LOVD variants at c.1558+4, such as c.1558+4C>G or c.1558+4C>A, with VCEP classifications) was present in the evidence bundle; the not_met conclusion rests on absence of such comparators in ClinVar and the PM5 candidate collection plus the SpliceAI max delta of 0.00, which independently fails the 'similar or worse' SpliceAI requirement.

Sources

Reasoning

Variant NM_000249.4:c.1558+4C>T is an intronic variant at donor position +4 (non-canonical splice nucleotide), so the VCEP PP3 splice path applies: SpliceAI delta score >= 0.2 is required for PP3_Supporting (InSiGHT MLH1 VCEP v2.0, 'as per Walker et al 2023'). SpliceAI max delta score = 0.00 (DS_AG 0.00, DS_AL 0.00, DS_DG 0.00, DS_DL 0.00), which is well below the 0.2 threshold, so no splice defect is predicted and PP3 is not met. The alternate HCI-prior path is missense-only and does not apply: c.1558+4C>T is intronic and is absent from the HCI prior lookup table (variant_not_in_hci_table). REVEL and BayesDel are not available for this variant (found=false) and are missense predictors not applicable to an intronic variant; BayesDel additionally has no verified published threshold available to this pipeline.

Rule

PP3_Supporting: Missense variant with HCI prior probability for pathogenicity >0.68 & <=0.81 as per hci-priors.hci.utah.edu/PRIORS; OR Predicted splice defect for non-canonical splicing nucleotides using SpliceAI with delta score >= 0.2 as per Walker et al 2023. PP3_Moderate: HCI prior >0.81. (InSiGHT MLH1 VCEP v2.0)

Evidence considered

• SpliceAI (SpliceAI Lookup, Broad Institute) for NM_000249.4:c.1558+4C>T: max_delta_score = 0.00; DS_AG = 0.00, DS_AL = 0.00, DS_DG = 0.00, DS_DL = 0.00.
• InSiGHT MLH1 VCEP v2.0 (cspec doc 1564688410) PP3 rule: 'Predicted splice defect for non-canonical splicing nucleotides using SpliceAI with delta score >= 0.2 as per Walker et al 2023' — threshold 0.2 named by the governing VCEP specification; 0.00 < 0.2, not met.
• HCI prior lookup (vcep_hci_priors_mlh1 / HCI-PRIORS-MLH1.txt): c.1558+4C>T not present (variant_not_in_hci_table); missense-only table, PP3 HCI-prior path (>0.81 Moderate; >0.68-0.81 Supporting) not applicable to this intronic variant.

Still missing

• No spliceogenic in-silico prediction above threshold; direct RNA/minigene splicing assay would be needed to further resolve splice impact, but that is outside this group's computational-evidence scope.

Sources

Reasoning

InSiGHT MLH1 VCEP v2.0 BA1 requires gnomAD v4 Grpmax filtering allele frequency >= 0.001 (0.1%) with the variant excluded as a founder pathogenic variant. The observed gnomAD v4.1 joint grpmax FAF is 4.43e-06 (total AF 2.478e-06; 4/1,613,954 alleles), approximately 226-fold below the 0.001 threshold. The highest population-specific AF (African/African American, 2.667e-05; 2/74,988 alleles) is also far below the threshold. gnomAD v2.1 total AF is likewise only 7.96e-06. The founder-variant exclusion clause is not triggered because the allele frequency does not approach the BA1 threshold. BA1 not met.

Rule

GnomAD v4 Grpmax filtering allele frequency >= 0.001 (0.1%) and variant is excluded as founder pathogenic variant (InSiGHT MLH1 VCEP v2.0)

Evidence considered

• gnomAD v4.1 (gnomad_v4): total AF 2.478e-06 (4/1,613,954 alleles), exome 3/1,461,808, genome 1/152,146, joint grpmax FAF 4.43e-06, AFR AF 2.667e-05 (2/74,988), homozygotes 0
• gnomAD v2.1 (gnomad_v2): total AF 7.958e-06 (2/251,334 alleles), homozygotes 0
• InSiGHT MLH1 VCEP v2.0 (cspec) BA1 rule: GnomAD v4 Grpmax filtering allele frequency >= 0.001 (0.1%) and variant excluded as founder pathogenic variant

Sources

Reasoning

InSiGHT MLH1 VCEP v2.0 BS1 requires gnomAD v4 Grpmax filtering allele frequency >= 0.0001 and < 0.001 (0.01-0.1%) with the variant excluded as a founder pathogenic variant. The observed gnomAD v4.1 joint grpmax FAF is 4.43e-06, below the 0.0001 lower bound of the BS1 window; the highest population-specific AF (African/African American, 2.667e-05; 2/74,988 alleles) is also below 0.0001. gnomAD v2.1 AF (7.96e-06) is likewise below. The allele frequency is far too low to trigger BS1. BS1 not met.

Rule

GnomAD v4 Grpmax filtering allele frequency >= 0.0001 and < 0.001 (0.01-0.1%) and variant is excluded as founder pathogenic variant (InSiGHT MLH1 VCEP v2.0)

Evidence considered

• gnomAD v4.1 (gnomad_v4): total AF 2.478e-06 (4/1,613,954 alleles), joint grpmax FAF 4.43e-06, AFR AF 2.667e-05 (2/74,988), homozygotes 0
• gnomAD v2.1 (gnomad_v2): total AF 7.958e-06 (2/251,334 alleles), AFR AF 6.152e-05 (1/16,254), homozygotes 0
• InSiGHT MLH1 VCEP v2.0 (cspec) BS1 rule: GnomAD v4 Grpmax filtering allele frequency >= 0.0001 and < 0.001 (0.01-0.1%) and variant excluded as founder pathogenic variant

Sources

Reasoning

Under the governing InSiGHT MLH1 VCEP v2.0, BS2 is not a population-frequency criterion: it requires co-occurrence in trans with a known pathogenic MLH1 sequence variant in a patient with colorectal cancer after age 45 (or another Lynch-spectrum cancer above the median age of onset), without CMMRD manifestations, with phase confirmed by parental/offspring testing. No such case-level in-trans co-occurrence data is present anywhere in this case bundle (no co-occurrence, phase, or pedigree observations). Population databases show zero homozygotes in gnomAD v2.1 and v4.1, and the variant is absent from gnomAD-Canada v1.0, so no homozygous-observation or healthy-adult evidence supports BS2 either. BS2 not met on available evidence.

Rule

Co-occurrence in trans with a known pathogenic sequence variant in the same gene in a patient with colorectal cancer after age 45 (or other LS cancer above the median age of onset for that cancer in LS), and who has no previous or current evidence of clinical manifestations of CMMRD; confirmation of phase requires testing of parents or offspring (InSiGHT MLH1 VCEP v2.0)

Evidence considered

• InSiGHT MLH1 VCEP v2.0 (cspec) BS2 rule: in-trans co-occurrence with a known pathogenic sequence variant in the same gene in a patient with CRC after age 45 (or other LS cancer above median age of onset) without CMMRD manifestations; phase confirmation required
• No in-trans co-occurrence, phase, or pedigree observations present in the case bundle (evidence.json, case_summary.json, ClinVar submissions)
• gnomAD v4.1 and v2.1: homozygote count 0 in all populations; gnomAD-Canada v1.0: variant absent

Still missing

• Case-level in-trans co-occurrence with a known pathogenic MLH1 variant (with phase confirmation and CRC after age 45 or other LS cancer above median age of onset, and no CMMRD features) was not available in this case bundle; such data would be required to consider BS2 met under the InSiGHT MLH1 VCEP v2.0 definition

Sources

Reasoning

VCEP BP7 requires 'a synonymous (silent) or intronic variant at or beyond -21/+7 (5'/3' exonic)'. c.1558+4C>T is intronic at donor position +4, i.e. only 4 nucleotides into the intron on the 3' side of exon 13; +4 is not 'at or beyond +7', so the positional requirement is not satisfied and the variant is not synonymous. BP7 is therefore not met. (The VCEP notes variants may satisfy both BP7 and BP4, but BP4 is met here via the SpliceAI rule, not via BP7.)

Rule

BP7_Supporting: A synonymous (silent) or intronic variant at or beyond -21/+7 (5'/3' exonic). Variants may satisfy both BP7 and BP4. (InSiGHT MLH1 VCEP v2.0)

Evidence considered

• InSiGHT MLH1 VCEP v2.0 (cspec doc 1564688410) BP7 rule: 'A synonymous (silent) or intronic variant at or beyond -21/+7 (5'/3' exonic). Variants may satisfy both BP7 and BP4.' — c.1558+4C>T is at +4, not at or beyond +7, and is not synonymous; BP7 not met.
• VariantValidator/transcript annotation (prefetch): NM_000249.4:c.1558+4C>T is intronic (exon 13i, donor +4), protein p.? — no synonymous (exonic) change.

Sources

Reasoning

Governing framework is the ClinGen InSiGHT VCEP MLH1 v2.0 specification, which adjudicates de novo evidence via a PS2 points system (0.5 points = Supporting; 1-1.5 = Moderate; 2-3.5 = Strong; >=4 = Very Strong). No de novo evidence is present in this case: no parental testing results (neither parent genotyped or both confirmed negative), no reported de novo occurrence of c.1558+4C>T in the proband, and no sibling/other-family-member testing that could contribute de novo points. ClinVar VCV000187689 (6 submissions: 3 VUS, 3 LB; 1-star conflicting) contains no parental or de novo fields, and none of the 9 papers in the literature pass report this variant. With zero de novo points derivable from available sources, PS2 cannot be applied and remains not assessed pending parental testing data.

Rule

PS2 de novo points (InSiGHT VCEP MLH1 v2.0): >=4 points = Very Strong; 2-3.5 points = Strong; 1-1.5 points = Moderate; 0.5 points = Supporting.

Evidence considered

• CSPEC InSiGHT VCEP MLH1 v2.0 PS2 rule (de novo points: 0.5 Supporting / 1-1.5 Moderate / 2-3.5 Strong / >=4 Very Strong); no proband or parental testing data in the case bundle to award any de novo points
• ClinVar VCV000187689 checked for parental/de novo observations in all 6 submissions; none present

Still missing

• No parental testing results available (maternity/paternity confirmation and both parents' genotypes needed to establish a de novo event)
• No de novo observation of c.1558+4C>T reported in the proband by any source
• No sibling or other family member testing to support de novo points under the VCEP PS2 points system

Sources

Reasoning

PS3 is applicable under the InSiGHT MLH1 v2.0 VCEP specification (source_registry key: cspec) and requires variant-specific functional evidence: calibrated functional assays with functional odds for pathogenicity > 2.08 (Supporting), > 4.3 and <= 18.7 OR MMR function defect following the functional assay flowchart OR monoallelic expression with complete loss of expression (<10% of wild-type in cDNA without puromycin) (Moderate), or > 18.7 (Strong). No variant-specific functional data exist for MLH1 c.1558+4C>T in this case: no calibrated assay result (e.g., the VCEP-approved MLH1 hESC CRISPR repair assay or cell-free CIMRA documented in vcep_functional_assay_svi_documentation_mmr), no MMR function defect result per the flowchart, and no monoallelic/allele-specific expression data. The VCEP functional assay documentation describes methodology and calibration thresholds only and lists no per-variant result for this variant; the EP pilot-variant workbook contains no entry for c.1558+4C>T (it lists the distinct variant c.1558+1G>A, which was not used as evidence). All five available publication full texts were screened and none mention the variant. A ClinVar submission audit signal (SCV000684753) states functional studies have not been reported for this variant. Evidence is therefore insufficient; PS3 is not assessed pending functional assay or mRNA/minigene assay data.

Rule

Calibrated functional assays with functional odds for pathogenicity > 18.7 (Strong); >4.3 and <= 18.7 OR MMR function defect following functional assay flowchart OR variants with monoallelic expression: complete loss of expression (<10% of wild-type in cDNA without puromycin) of the variant allele (Moderate); calibrated functional odds for pathogenicity >2.08 and <= 4.3 (Supporting).

Evidence considered

• cspec: InSiGHT MLH1 v2.0 PS3 rule tiers (functional odds >2.08 Supporting; >4.3 and <=18.7 or MMR defect per flowchart or monoallelic expression <10% of wild-type Moderate; >18.7 Strong) - no variant-specific assay result is available to apply any tier.
• vcep_functional_assay_svi_documentation_mmr: VCEP-approved calibrated assays with documented controls and thresholds (e.g., MLH1 hESC CRISPR assay with OddsPath-functional normal/abnormal readout thresholds); document contains methodology/calibration only, no per-variant result for c.1558+4C>T.
• vcep_vcep_pilot_variants_mmr: InSiGHT EP pilot variant workbook contains no entry for c.1558+4C>T; the listed MLH1 c.1558+1G>A is a different variant and was not used as evidence.
• clinvar: Variation 187689 has 6 submissions (3 VUS, 3 Likely benign) with no validated functional-study PMIDs; submission audit flagged only that functional studies have not been reported (signal from SCV000684753, not validated); submissions' classifications were not used as evidence.

Still missing

• No calibrated functional assay result for c.1558+4C>T (e.g., OddsPath-functional MLH1 repair assay or cell-free CIMRA per VCEP functional assay SVI documentation).
• No MMR function defect assessment per the VCEP functional assay flowchart for this variant.
• No monoallelic expression / allele-specific expression data (cDNA without puromycin, <10% of wild-type threshold) for this variant.
• No publication reporting functional studies of c.1558+4C>T was identified (all available full texts screened, none mention the variant).

Sources

Reasoning

Governing framework is the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis EP MLH1 v2.0 specification (cspec, precedence rank 1), which makes PM3 Applicable as a points-based criterion: co-occurrence with a known pathogenic/likely pathogenic (P/LP) sequence variant in the same gene in a patient with clinical features consistent with CMMRD per Aronson et al 2022 (Table for CMMRD diagnosis); for MLH1 variants the variant must also meet PM2_Supporting (gnomAD v4 AF < 0.00002). Points: P/LP in trans = 1.0; P/LP phase unknown = 0.5; >=4 points = Very Strong, 2-3.5 = Strong, 1-1.5 = Moderate, 0.5 = Supporting. The case bundle contains no proband-level data on which to award points: ClinVar VCV000187689 (6 clinical laboratory submissions: 3 VUS, 3 Likely benign; 1 somatic-origin; no expert panel submissions, no criterion-lead submissions) reports no co-occurrence with a second MLH1 variant and no phase information; gnomAD v2.1 (2/251,334 alleles) and v4.1 (4/1,613,954 alleles) show zero homozygotes, i.e., no biallelic individuals; no locus-specific (InSiGHT/LOVD) records, case reports, or publications mentioning this variant are present in the bundle; SpliceAI predicts no splice impact (max delta 0.00). No observation of a P/LP MLH1 variant in trans or of unknown phase with c.1558+4C>T in a CMMRD-consistent patient could be identified, so 0 PM3 points can be assigned. Because the bundle lacks any carrier/proband genotype or phase data, this is an absence-of-evidence situation rather than a confirmed exclusion, so the criterion is recorded as not assessed rather than met; the population-frequency gate (AF 2.48e-6, grpmax FAF 4.43e-6, both < 0.00002) would be satisfied were co-occurrence data to appear.

Rule

Co-occurrence with a known pathogenic/likely pathogenic sequence variant in the same gene in a patient with clinical features consistent with CMMRD as per Aronson et al 2022 (Table for CMMRD diagnosis). For MLH1 variants the variant must also meet PM2_Supporting. Points: P/LP in trans = 1.0; P/LP phase unknown = 0.5; sum across cases: >=4 points = Very Strong, 2-3.5 = Strong, 1-1.5 = Moderate, 0.5 = Supporting (InSiGHT MLH1 VCEP v2.0).

Evidence considered

• InSiGHT MLH1 VCEP v2.0 PM3 rule text (points system, CMMRD requirement, PM2_Supporting gate for MLH1)
• ClinVar VCV000187689 submission audit: 6 submissions (3 VUS, 3 Likely benign), 0 expert panel submissions, 0 criterion-lead submissions, no co-occurrence or phase data in any submission
• gnomAD v4.1: 4/1,613,954 alleles, 0 homozygotes, total AF 2.47839e-06, grpmax FAF 4.43e-06
• gnomAD v2.1: 2/251,334 alleles, 0 homozygotes, total AF 7.95754e-06

Still missing

• No reported co-occurrence of c.1558+4C>T with a second pathogenic/likely pathogenic MLH1 variant in any proband (no InSiGHT/LOVD or case-report data in the bundle)
• No phase information (trans vs cis vs unknown) available for any carrier
• No CMMRD phenotype/clinical data (Aronson et al 2022 criteria) for any reported carrier
• Proband-level genotype data absent from this variant-centric case bundle

Sources

Reasoning

The governing InSiGHT VCEP MLH1 v2.0 PP1 rule requires co-segregation with disease in pedigree(s) quantified as a combined Bayes Likelihood Ratio (BLR >2.08 and <=4.3 = Supporting; >4.3 and <=18.7 = Moderate; >18.7 in >=2 families = Strong), with BLR computed per the cosegregation method referenced in CSPEC footnote f (Belman et al., Genet Med 2020, PMID 32773770; COOL v3 recommended tool). No segregation data exists in this case: no pedigrees, no affected-carrier/meioses counts, no combined BLR are reported by ClinVar submissions, the literature pass (which indexes no paper for PP1), or any other bundle source. PP1 is therefore not assessed pending segregation data.

Rule

PP1 co-segregation (InSiGHT VCEP MLH1 v2.0): combined Bayes Likelihood Ratio >18.7 in >=2 families = Strong; >4.3 and <=18.7 = Moderate; >2.08 and <=4.3 = Supporting.

Evidence considered

• CSPEC InSiGHT VCEP MLH1 v2.0 PP1 rule (combined Bayes LR >2.08-4.3 Supporting / >4.3-18.7 Moderate / >18.7 in >=2 families Strong; method per Belman et al. 2020 PMID 32773770 as cited in CSPEC footnote f)
• ClinVar VCV000187689 checked for segregation data in all 6 submissions; none present

Still missing

• No pedigree data: no families reported with variant-positive affected members to count meioses
• No combined Bayes Likelihood Ratio computable from available sources
• VCEP PP1_Strong additionally requires >=2 families with combined BLR >18.7; none available

Sources

Reasoning

PP4 is applicable in the InSiGHT MLH1 VCEP v2.0 framework (Supporting: 1 CRC/Endometrial MSI-H tumor using a standard panel of 5-10 markers or tumor genome and/or loss of MMR protein expression consistent with the variant location; for MLH1 variants, MLH1 promoter methylation must be excluded in the tumor; Moderate: 2 tumors; Strong: >=3 independent tumors in >=2 families). However, the consolidated evidence bundle contains no tumor phenotype data whatsoever for carriers of NM_000249.4:c.1558+4C>T: no MSI status, no MMR IHC results, no MLH1 promoter methylation testing, and no proband phenotype descriptions. ClinVar submissions provide classifications only without phenotype/tumor detail. No retrieved publication mentions this variant, so no tumor-level evidence can be drawn from the literature. Evidence is insufficient to count any MSI-H tumor or protein-expression finding, therefore PP4 cannot be adjudicated.

Rule

InSiGHT MLH1 VCEP v2.0 PP4: Supporting = 1 CRC/Endometrial MSI-H tumor using a standard panel of 5-10 markers or tumor genome and/or loss of MMR protein expression consistent with the variant location (MLH1 promoter methylation excluded for MLH1 variants); Moderate = 2 independent tumors; Strong = >=3 independent tumors in >=2 families.

Evidence considered

• InSiGHT MLH1 VCEP v2.0 (cspec) PP4 rules: Supporting = 1 CRC/Endometrial MSI-H tumor using a standard panel of 5-10 markers or tumor genome and/or loss of MMR protein expression consistent with the variant location; MLH1 promoter methylation must be excluded for MLH1 variants.
• ClinVar VCV000187689: no tumor phenotype (MSI/IHC/methylation) reported in any of the 6 submissions.

Still missing

• No tumor data available for any carrier: MSI status (standard 5-10 marker panel or tumor genome), MMR IHC (MLH1/PMS2 loss consistent with MLH1), and MLH1 promoter methylation exclusion are all absent.
• No proband phenotype or family-history detail is present in ClinVar submissions or retrieved publications.

Sources

Reasoning

BS3 is applicable under the InSiGHT MLH1 v2.0 VCEP specification (cspec). For this intronic variant (c.1558+4C>T, 4 bp into intron 12 at the donor splice region), BS3 Strong requires either calibrated functional assays with functional odds for pathogenicity <= 0.05 or 'synonymous substitutions and intronic variants with no associated mRNA aberration (either splicing or allelic imbalance) as determined by laboratory assays conducted with nonsense-mediated decay inhibition'; BS3 Supporting requires calibrated functional odds >0.05 and <=0.48 or variant-specific proficient function in protein and mRNA-based lab assays per the MMR functional assay flowchart. No such laboratory evidence exists for this variant: no mRNA assay with NMD inhibition (cDNA +/-(puromycin) full-length transcript analysis), no allelic imbalance study, no calibrated functional odds, and no proficient-function protein/mRNA assay. SpliceAI predicts no significant splicing impact (max delta score 0.00), but this is an in silico prediction and the VCEP BS3 rule explicitly requires laboratory assays, so it cannot meet BS3. ClinVar contains 3 Likely benign submissions, but none provide validated functional or mRNA assay evidence (no validated PMIDs, no criterion leads) and final classifications from other sources were not used as evidence. Evidence is therefore insufficient; BS3 is not assessed pending variant-specific mRNA (with NMD block) or calibrated functional assay data.

Rule

Calibrated functional assays with functional odds for Pathogenicity <= 0.05 OR synonymous substitutions and intronic variants with no associated mRNA aberration (either splicing or allelic imbalance) as determined by laboratory assays conducted with nonsense-mediated decay inhibition (Strong); calibrated functional assays with functional odds for Pathogenicity >0.05 and <= 0.48 OR variant-specific proficient function in protein and mRNA-based lab assays as per MMR functional assay flowchart (Supporting).

Evidence considered

• cspec: InSiGHT MLH1 v2.0 BS3 rule - calibrated functional odds <= 0.05 OR (for synonymous/intronic variants) no associated mRNA aberration as determined by laboratory assays with NMD inhibition (Strong); functional odds >0.05 and <=0.48 OR variant-specific proficient function in protein and mRNA-based lab assays per flowchart (Supporting).
• vcep_functional_assay_svi_documentation_mmr: documents the approved mRNA/splicing assay (cDNA analysis of full-length transcripts with active/inhibited NMD using puromycin; allelic loss <=10% abnormal, biallelic 50+/-10% normal) and calibrated assay thresholds; no result for c.1558+4C>T.
• spliceai: max delta score 0.00 (DS_AG/AL/DG/DL all 0.0); in silico prediction only - consulted to confirm no predicted splice impact, but does not satisfy the VCEP BS3 laboratory-assay requirement.
• vcep_vcep_pilot_variants_mmr: no entry for c.1558+4C>T in the EP pilot list (functional codes for this variant not assigned by the EP in this dataset).
• clinvar: Variation 187689 - 3 Likely benign submissions lack validated functional/mRNA assay evidence (no validated PMIDs, usable_for_criterion_leads = false); classifications were not used as BS3 evidence.

Still missing

• No mRNA assay conducted with nonsense-mediated decay inhibition (cDNA +/- puromycin, full-length transcript analysis) demonstrating absence of splicing aberration or allelic imbalance for this intronic variant, as required by the VCEP BS3 Strong rule.
• No calibrated functional assay result with functional odds for pathogenicity <= 0.48 for this variant.
• No variant-specific proficient function in protein and mRNA-based lab assays per the MMR functional assay flowchart.
• SpliceAI max delta score 0.00 is in silico only and does not substitute for the laboratory assays required by the VCEP BS3 rule.

Sources

Reasoning

The governing InSiGHT VCEP MLH1 v2.0 BS4 rule requires demonstrated lack of co-segregation quantified as a combined Bayes Likelihood Ratio (BLR >0.05 and <=0.48 = Supporting; <0.05 = Strong). No non-segregation evidence exists in this case: no affected non-carriers or unaffected carriers reported, no pedigrees, and no combined BLR derivable from ClinVar submissions (which contain no segregation fields) or from any literature source in the bundle (no paper reports this variant). Absence of segregation data is not evidence of non-segregation, so BS4 is not assessed rather than not met.

Rule

BS4 lack of co-segregation (InSiGHT VCEP MLH1 v2.0): combined Bayes Likelihood Ratio <0.05 = Strong; >0.05 and <=0.48 = Supporting.

Evidence considered

• CSPEC InSiGHT VCEP MLH1 v2.0 BS4 rule (combined Bayes LR <0.05 Strong / >0.05 and <=0.48 Supporting; method per Belman et al. 2020 PMID 32773770 as cited in CSPEC footnote f)
• ClinVar VCV000187689 checked for non-segregation data in all 6 submissions; none present

Still missing

• No non-segregation observations (affected non-carriers or unaffected carriers of c.1558+4C>T) reported
• No pedigree data or combined Bayes Likelihood Ratio computable from available sources
• VCEP BS4 thresholds (<0.05 Strong; >0.05 and <=0.48 Supporting) cannot be evaluated without segregation testing data

Sources

Reasoning

BP5 is applicable in the InSiGHT MLH1 VCEP v2.0 framework (Benign Supporting: 2 or 3 CRC/Endometrial tumors with MSS and/or no loss of MMR protein expression and/or LS spectrum tumors with loss of MMR protein(s) inconsistent with the gene demonstrating genetic variation, OR 1 BRAF V600E (colon only)/MLH1 methylation (in LS spectrum tumor only) with MSI-H/MLH1 loss; Strong: >=4 such tumors or >=2 BRAF V600E/MLH1 methylation events). The criterion requires positive tumor evidence of an alternate molecular basis for disease in carriers. The evidence bundle contains no tumor molecular data for any carrier of NM_000249.4:c.1558+4C>T: no MSI status, no MMR IHC, no BRAF V600E testing, no MLH1 promoter methylation data, and no case reports in the retrieved literature (variant absent from all 5 full-text papers). With no tumor data, an alternate molecular basis cannot be confirmed or refuted, so BP5 cannot be adjudicated.

Rule

InSiGHT MLH1 VCEP v2.0 BP5 (Benign Supporting): 2 or 3 CRC/Endometrial tumors with MSS and/or no loss of MMR protein expression and/or LS spectrum tumors with loss of MMR protein(s) inconsistent with the variant gene, OR 1 BRAF V600E (colon only)/MLH1 methylation (in LS spectrum tumor only) with MSI-H/MLH1 loss.

Evidence considered

• InSiGHT MLH1 VCEP v2.0 (cspec) BP5 rules: Supporting = 2 or 3 tumors with MSS and/or no loss of MMR protein expression and/or LS spectrum tumors with inconsistent MMR protein loss, OR 1 BRAF V600E (colon)/MLH1 methylation (LS spectrum tumor) with MSI-H/MLH1 loss.
• ClinVar VCV000187689: no tumor phenotype or alternate-basis evidence attached to any of the 6 submissions.

Still missing

• No tumor molecular data for any carrier: MSI status, MMR IHC, BRAF V600E (colon tumors), and MLH1 promoter methylation results are all absent from the evidence bundle and the retrieved literature.
• No documented alternate molecular basis (e.g., biallelic/somatic MMR-independent tumor etiology) for any case carrying this variant.

Sources