MLH1 c.1039-6dup, p.?

Reasoning

InSiGHT MLH1 VCEP v2.0 BA1 requires gnomAD v4 Grpmax filtering allele frequency >= 0.001 (0.1%) AND that the variant be excluded as a founder pathogenic variant. In gnomAD v4.1 the joint (exome+genome) grpmax FAF is 0.00117106 (0.117%), which is >= the 0.001 threshold; the highest ancestry-specific AF is African/African American at 0.001426 (78/54,690 alleles). The variant is not a founder pathogenic variant: ClinVar VCV000140797 carries a 9-laboratory consensus of Benign (5) / Likely benign (4) with no expert-panel pathogenic submissions and no founder designation, and the variant is observed across multiple ancestries (NFE 0.0527%, AFR 0.143%, ASJ 0.0288%). A single adult homozygote is present in gnomAD v4 (Ashkenazi Jewish), inconsistent with a fully penetrant dominant Lynch syndrome MLH1 pathogenic allele. Population prevalence/penetrance considerations (Lynch syndrome is a high-penetrance dominant disorder; an MLH1 pathogenic allele at carrier frequency ~0.1% would be incompatible with observed LS prevalence) further support the benign stand-alone call. Note the threshold proximity (grpmax FAF 0.00117 vs 0.001) and the low-complexity/homopolymer (dupA) context flagged in gnomAD-Canada raw data; the filtering-allele-frequency metric is designed to be robust to such artifacts and the allele count (653 total alleles in v4.1, 37 in v2.1) is multi-cohort and too high to be purely artifactual.

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 joint grpmax FAF 0.00117106 (>= 0.001 threshold); total AF 0.000530357 (653/1,231,246 alleles); AFR AF 0.00142622 (78/54,690); 1 homozygote (ASJ)
• gnomAD v2.1 total AF 0.000263237 (37/140,558 alleles), grpmax FAF 0.00082842, 0 homozygotes (cross-version frequency consistency)
• ClinVar VCV000140797: Benign (5 clinical laboratories) and Likely benign (4 clinical laboratories), no expert panel submissions
• InSiGHT MLH1 VCEP v2.0 BA1 rule (cspec 1564688410): gnomAD v4 Grpmax FAF >= 0.001 and variant excluded as founder pathogenic variant

Still missing

• No formal InSiGHT founder-pathogenic-variant exclusion list was present in the evidence bundle; founder exclusion is inferred from the ClinVar 9-laboratory Benign/Likely benign consensus, absence of expert-panel pathogenic submissions, and multi-ancestry distribution.

Sources

Reasoning

c.1039-6dupA is an intronic variant (intron 11, acceptor position -6 relative to exon 12; variant_validator exon 11i; gnomAD VEP most severe consequence splice_region_variant). The governing InSiGHT MLH1 VCEP v2.0 BP4 rule states that for intronic and synonymous variants, SpliceAI predicting no splicing impact with delta score <= 0.1 meets BP4_Supporting (as per Walker et al 2023, PMID 37352859). SpliceAI max delta score = 0.01 (<= 0.1) for this variant -> no predicted splicing impact -> BP4 met at Supporting. The HCI-prior path of BP4 (<0.11) is missense-only and not applicable. No double-counting: this SpliceAI no-impact prediction is counted under BP4 only.

Rule

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 = BP4_Supporting.

Evidence considered

• SpliceAI Lookup (source_registry key 'spliceai'): max delta score 0.01 (DS_AG 0.0, DS_AL 0.01, DS_DG 0.0, DS_DL 0.01) for NM_000249.4:c.1039-6dup.
• InSiGHT MLH1 VCEP v2.0 BP4 rule (source_registry key 'cspec'; CSPEC doc 1564688410): 'For intronic and synonymous variants: SpliceAI predicts no splicing impact with delta score <= 0.1 as per Walker et al 2023.' Walker et al 2023 = PMID 37352859 (Am J Hum Genet 110(7):1046-1067, 2023), per cspec raw_rule_origin.references. Variant is intronic and SpliceAI max delta 0.01 <= 0.1 -> BP4_Supporting met.
• HCI prior BP4 path (<0.11 -> BP4_Supporting) is missense-only per HCI-PRIORS-MLH1.txt index guidance (source_registry key 'vcep_hci_priors_mlh1'); not applicable to this intronic variant.

Still missing

• No RNA/mRNA assay data for this variant; the VCEP BP4 rule is satisfied by SpliceAI delta <= 0.1 alone, so this does not block the decision (mRNA evidence would fall under BS3, outside this group).

Sources

Reasoning

Variant NM_000249.4:c.1039-6dupA (normalized c.1039-6dup) is a single-nucleotide A duplication in intron 11 (variant_validator: start_exon/end_exon '11i'), 6 nucleotides upstream of exon 12, on the MANE Select transcript NM_000249.4. It is not a nonsense/frameshift variant (no PTC; predicted protein consequence p.? on NP_000240.1), not a large single/multi-exon genomic alteration, not at IVS+/-1 or IVS+/-2 (position -6 is non-canonical), not an initiation-codon variant, and not an exon duplication or G>non-G at the last base of an exon. None of the InSiGHT MLH1 VCEP v2.0 PVS1 routes (Very Strong, Strong, or Moderate) is triggered. The only VCEP PVS1 route potentially open to a non-canonical intronic variant is mRNA-assay-confirmed splicing aberration (with no full-length/reference transcript produced and minigene or independent-laboratory confirmation); no RNA/mRNA assay data exist in this case, and the available in silico splicing evidence (SpliceAI max delta = 0.01; DS_AG 0.0, DS_AL 0.01, DS_DG 0.0, DS_DL 0.01) predicts no splicing impact. Population context (gnomAD v4.1 AF 0.053%, 653 alleles including 1 homozygote; grpmax FAF 0.00117) is additionally inconsistent with a highly penetrant null allele, although frequency is not itself a VCEP PVS1 rule. PVS1 therefore not met; no LOF consequence mechanism is established for this variant.

Rule

InSiGHT MLH1 VCEP v2.0 PVS1 — Very Strong: nonsense/frameshift PTC at or before codon 753; large single/multi-exon genomic alteration; IVS+/-1/2 variants with exon skipping/cryptic splice disrupting frame with predicted NMD; mRNA-assay-confirmed splicing aberration (no full-length transcript) leading to PTC or in-frame deletion of a functional domain (minigene/independent-lab confirmation required for non-canonical splice variants); initiation-codon variant. Strong: tandem duplication of >=1 exon causing frameshift; G>non-G at last base of exon (if intron 1-6 bases not GTRRGT); IVS+/-1/2 in-frame skipping in critical region. Moderate: PTC in codons 754-756.

Evidence considered

• cspec
• spliceai
• gnomad_canada
• gnomad_v4
• gnomad_v2

Still missing

• No patient-derived mRNA/cDNA assay (with and without NMD block) or minigene assay for c.1039-6dupA; the VCEP PVS1 mRNA-confirmed splicing-aberration route requires such data for non-canonical splice-region variants.
• No independent-laboratory RNA assay confirmation available for this variant.

Sources

Reasoning

The variant is an intronic duplication (NM_000249.4:c.1039-6dup; predicted protein consequence p.?), so the missense same-amino-acid branch of PS1 cannot apply. The InSiGHT MLH1 VCEP v2.0 splice branch of PS1 requires a confirmed Pathogenic (Strong) or Likely Pathogenic (Moderate) splice variant affecting the same non-canonical splice nucleotide (c.1039-6) with similar or worse SpliceAI prediction; no such comparator variant was identified in any source in the case evidence, and SpliceAI predicts no splicing impact for this variant (max delta score 0.01), well below the VCEP-referenced splice-defect threshold of >=0.2 (Walker et al. 2023, PMID 37352859). PS1 is therefore not met.

Rule

InSiGHT MLH1 VCEP v2.0 PS1 (Pathogenic Strong default): same amino acid change as a previously established Pathogenic missense (not splice) OR variants affecting the same non-canonical splice nucleotide as a confirmed Pathogenic splice variant with similar or worse SpliceAI prediction; PS1_Moderate for same amino acid as Likely Pathogenic missense (with normal RNA result and PM2_supporting) or same non-canonical splice nucleotide as a Likely Pathogenic splice variant with similar or worse SpliceAI prediction.

Evidence considered

• InSiGHT MLH1 VCEP v2.0 PS1 Strong: '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.'
• VariantValidator normalization gives predicted protein consequence NP_000240.1:p.? for c.1039-6dup, so no amino acid change exists for the PS1 same-amino-acid branch.
• SpliceAI lookup reports max delta score 0.01 (DS_AG 0.0, DS_AL 0.01, DS_DG 0.0, DS_DL 0.01) - no predicted splicing impact; the VCEP PP3 rule defines a predicted splice defect as SpliceAI delta >= 0.2 per Walker et al. 2023 (PMID 37352859), so this variant does not approach the 'similar or worse' splicing prediction prong.
• ClinVar VCV000140797 (this exact allele, c.1039-6dup) has 10 submissions classified Benign (5) or Likely benign (4) plus one no-assertion submission, with no expert panel submissions; no pathogenic/likely pathogenic allele at this nucleotide was surfaced in any collected source.

Still missing

• No dedicated query for other variants at the same non-canonical splice nucleotide c.1039-6 (e.g., c.1039-6T>A/G/C substitutions) was present in the case evidence; a ClinVar/InSiGHT same-nucleotide search would formally exclude an unidentified PS1 splice-branch comparator.

Sources

Reasoning

InSiGHT MLH1 VCEP v2.0 PM2_Supporting requires absent/extremely rare allele frequency < 0.00002 (<1 in 50,000 alleles) in the gnomAD v4 dataset. The variant is observed in gnomAD v4.1 at total AF 0.000530357 (653/1,231,246 alleles) — more than 26-fold above the 0.00002 threshold — with grpmax FAF 0.00117106 and highest subpopulation AF 0.00142622 (AFR). The frequency is far too high for the PM2 rarity criterion to be met, so PM2 is not applied. This is consistent with the ClinVar Benign/Likely benign consensus and with the variant being a common, likely benign intronic splice-region variant (SpliceAI max delta 0.01).

Rule

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

Evidence considered

• gnomAD v4.1 total AF 0.000530357 (653/1,231,246 alleles) and grpmax FAF 0.00117106, both far above the PM2 threshold of 0.00002
• InSiGHT MLH1 VCEP v2.0 PM2 rule (cspec 1564688410): AF < 0.00002 (<1 in 50,000 alleles) in gnomAD v4 dataset

Sources

Reasoning

Governing framework is the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel specification for MLH1 v2.0 (cspec, doc 1564688410), in which PM3 is applied via a points system (0.5 points = Supporting; 1-1.5 = Moderate; 2-3.5 = Strong; >=4 = Very Strong) for observations of the variant in trans with a pathogenic/likely pathogenic variant. No such observation exists for NM_000249.4:c.1039-6dupA in any source in this case bundle: (1) a full-bundle search found zero mentions of trans/cis phase, a second MLH1 variant, co-occurrence, proband, or family-history data; (2) ClinVar record 140797 (10 germline submissions: Benign x5, Likely benign x4, no-assertion x1; 2-star 'multiple submitters, no conflicts') has no expert-panel submissions, 0 criterion leads, and 0 usable PMIDs - the two attached PMIDs were rejected in the submission audit, and no submission provides case-level or allelic data; (3) the literature pass contains only guideline/review papers (ACMG standards 25741868, EHTG Lynch guidelines 34043773, EGAPP KRAS 23429431, SEAP/SEOM biomarker 25373533, ACMG/NSGC referral 25394175, NACB tumor markers 19042984, Sherloc 28492532), none of which mention this variant (candidate criteria indexed were PP5/PS4 only); (4) the VCEP pilot-variant workbook for MMR does not contain c.1039-6dupA. Population data were considered and rejected as PM3 evidence: gnomAD v4.1 shows AF 0.000530 (653/1,231,246 alleles) with one homozygote (ASJ) - a single population-database homozygote without proband/phenotype context is not a scorable trans-phase observation under the VCEP rule text available in this bundle, and a 0.05% overall allele frequency (grpmax FAF 0.00117; AFR 0.143%) is far too high for a pathogenic Lynch syndrome allele, consistent with the ClinVar Benign/Likely benign consensus. Therefore no PM3 points can be assigned and PM3 is not met. High confidence in the negative determination based on absence of positive evidence plus countervailing population-frequency data; the only way this could change is if the original clinical case carries family/phase testing data not captured by this pipeline.

Rule

InSiGHT MLH1 VCEP v2.0 PM3 (points-based): Very Strong = >=4 points; Strong = 2-3.5 points; Moderate = 1-1.5 points; Supporting = 0.5 points, for variant observations in trans with a pathogenic/likely pathogenic variant.

Evidence considered

• InSiGHT MLH1 VCEP v2.0 (cspec): PM3 points framework (0.5 points = Supporting; 1-1.5 = Moderate; 2-3.5 = Strong; >=4 = Very Strong) - no scorable observations available
• ClinVar VCV000140797: 10 germline submissions, Benign x5 / Likely benign x4 / no-assertion x1, 2-star review status, 0 expert-panel submissions, 0 criterion leads, 0 usable PMIDs; no trans-phase or co-occurrence data in any submission
• gnomAD v4.1: AF 0.000530 overall (653/1,231,246 alleles), 1 homozygote (ASJ), grpmax FAF 0.00117 - population homozygote without proband phenotype not scored as PM3; high frequency inconsistent with pathogenic allele
• gnomAD v2.1: AF 0.000263 (37/140,558 alleles), 0 homozygotes - no biallelic observations

Still missing

• No observation of c.1039-6dupA in trans with a pathogenic or likely pathogenic MLH1 variant in an affected proband or carrier exists in any consulted source; no phase-confirmation data (parental or offspring testing) are captured anywhere in the case bundle
• ClinVar submissions for VCV000140797 contain no case-level allelic information (0 criterion leads, 0 usable PMIDs); no variant-specific literature was identified

Sources

Reasoning

Splice-impact path (governing InSiGHT MLH1 VCEP v2.0 rule): PP3_Supporting requires a predicted splice defect for non-canonical splicing nucleotides with SpliceAI delta score >= 0.2 (as per Walker et al 2023, PMID 37352859). SpliceAI for NM_000249.4:c.1039-6dup returns max delta score 0.01 (DS_AG 0.0, DS_AL 0.01, DS_DG 0.0, DS_DL 0.01), well below 0.2, so no splice defect is predicted. Missense path (HCI prior >0.68 -> PP3_Supporting; >0.81 -> PP3_Moderate) is not applicable: c.1039-6dupA is an intronic duplication (intron 11, position -6 relative to exon 12), not a missense substitution, and the HCI-PRIORS-MLH1.txt lookup is missense-only (hci_prior found=false). REVEL/BayesDel are not applicable to this intronic variant and no scores were retrieved. PP3 not met.

Rule

Missense variant with HCI prior probability for pathogenicity >0.68 & <=0.81 = PP3_Supporting, >0.81 = PP3_Moderate (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_Supporting.

Evidence considered

• SpliceAI Lookup (source_registry key 'spliceai'; spliceailookup.broadinstitute.org, variant NM_000249.4:c.1039-6dup, hg37, distance 500, mask 0): max delta score 0.01 (DS_AG 0.0, DS_AL 0.01, DS_DG 0.0, DS_DL 0.01).
• InSiGHT MLH1 VCEP v2.0 PP3 rule (source_registry key 'cspec'; CSPEC doc 1564688410): 'Predicted splice defect for non-canonical splicing nucleotides using SpliceAI with delta score >= 0.2 as per Walker et al 2023.' Walker et al 2023 = PMID 37352859 (Walker LC, de la Hoya M, et al., 'Using the ACMG/AMP framework to capture evidence related to predicted and observed impact on splicing: Recommendations from the ClinGen SVI Splicing Subgroup', Am J Hum Genet 110(7):1046-1067, 2023), as listed in cspec raw_rule_origin.references. Observed max delta 0.01 < 0.2 -> splice-impact path of PP3 not met.
• HCI prior path of PP3 (>0.68 -> PP3_Supporting; >0.81 -> PP3_Moderate) applies only to missense substitutions per HCI-PRIORS-MLH1.txt index guidance (source_registry key 'vcep_hci_priors_mlh1'); c.1039-6dupA is intronic, HCI lookup not applicable (hci_prior found=false in prefetch/case_summary).

Sources

Reasoning

InSiGHT MLH1 VCEP v2.0 BS1 requires gnomAD v4 Grpmax FAF >= 0.0001 and < 0.001 (0.01-0.1%). The observed gnomAD v4.1 joint grpmax FAF is 0.00117106, which is ABOVE the upper bound of the BS1 band (0.001), so the BS1 frequency interval is not met; the variant instead satisfies the BA1 stand-alone threshold and BA1 takes precedence. Had gnomAD v2.1 been used (grpmax FAF 0.00082842), the variant would fall inside the BS1 band, but the VCEP rule specifies gnomAD v4, under which BA1 applies.

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 joint grpmax FAF 0.00117106 is not in the BS1 band [0.0001, 0.001)
• InSiGHT MLH1 VCEP v2.0 BS1 rule (cspec 1564688410): gnomAD v4 Grpmax FAF >= 0.0001 and < 0.001 and variant excluded as founder pathogenic variant

Sources

Reasoning

The governing InSiGHT MLH1 VCEP v2.0 BP7 rule applies to 'a synonymous (silent) or intronic variant at or beyond -21/+7 (5'/3' exonic)'. c.1039-6dupA is an intronic duplication located at position -6 relative to exon 12 (intron 11 acceptor side; variant_validator exon 11i), which is within the splice-region window and NOT at or beyond -21/+7. The variant is also not a synonymous variant. The BP7 positional/type criterion is therefore not satisfied. The VCEP note that 'variants may satisfy both BP7 and BP4' does not rescue BP7 here; the SpliceAI no-impact finding is counted under BP4 only.

Rule

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

Evidence considered

• InSiGHT MLH1 VCEP v2.0 BP7 rule (source_registry key 'cspec'; CSPEC doc 1564688410): 'A synonymous (silent) or intronic variant at or beyond -21/+7 (5'/3' exonic). Variants may satisfy both BP7 and BP4.' Variant position c.1039-6 = -6 relative to exon 12 (intron 11), not at or beyond -21 -> criterion not met.
• Variant type/position (prefetch.json variant_validator): NM_000249.4:c.1039-6dupA normalized to NM_000249.4:c.1039-6dup, intronic insertion (protein p.?), variant_exonic_positions 11i/11i; gnomAD VEP most_severe_consequence splice_region_variant.

Sources

Reasoning

No de novo observation is reported for NM_000249.4:c.1039-6dupA in any source in the case bundle. There is no proband case report, no parental testing result, and no maternity/paternity confirmation anywhere in the evidence (source_registry contains only population databases, ClinVar lab submissions, and guideline papers; none report this variant in a proband/family context). The governing InSiGHT MLH1 VCEP v2.0 scores PS2 via a de novo points system (>=4 points Very Strong, 2-3.5 Strong, 1-1.5 Moderate, 0.5 Supporting), but with zero de novo observations there are no points to award. A de novo origin cannot be inferred from ClinVar laboratory classifications (Benign/Likely benign, no criteria detail) per the rule against deriving a criterion from another source's final classification.

Rule

PS2: >=4 de novo points = Very Strong; 2-3.5 = Strong; 1-1.5 = Moderate; 0.5 = Supporting (ClinGen InSiGHT HCRC/PP EP MLH1 v2.0, CSPEC doc 1564688410)

Evidence considered

• InSiGHT MLH1 VCEP v2.0 PS2 rule: de novo points system (0.5-4 points mapping to Supporting through Very Strong); no points without de novo observations

Still missing

• No proband-level de novo observation reported in any source
• No parental testing / maternity-paternity confirmation data
• Proband phenotype consistency data absent (not assessable)

Sources

Reasoning

No variant-specific functional evidence is available for MLH1 c.1039-6dupA. The governing InSiGHT MLH1 v2.0 specification requires calibrated functional assay odds (>2.08 Supporting, >4.3 Moderate, >18.7 Strong), a documented MMR function defect per the VCEP functional assay flowchart, or monoallelic expression with complete loss of expression (<10% of wild-type in cDNA without puromycin); none of these data exist in the case evidence, so PS3 is not assessable.

Rule

InSiGHT MLH1 v2.0 (cspec): PS3_Strong = calibrated functional assays with functional odds for pathogenicity >18.7; PS3_Moderate = calibrated functional assays with functional odds >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 (full-length transcript analysed with and without NMD block); PS3_Supporting = calibrated functional odds for pathogenicity >2.08 and <=4.3

Evidence considered

• InSiGHT MLH1 v2.0 CSPEC (cspec): PS3 calibrated functional odds thresholds reviewed (Supporting >2.08 and <=4.3; Moderate >4.3 and <=18.7, or MMR function defect per functional assay flowchart, or monoallelic expression <10% of wild type; Strong >18.7); no variant-specific assay result maps to any threshold.
• VCEP Functional-assay-SVI-documentation-MMR.xlsx (vcep_functional_assay_svi_documentation_mmr): lists calibrated MMR assays (cell-free MMR repair capacity/CIMRA, chemical selection, hESC CRISPR) with calibration PMIDs (30504929, 31965077, 33357406, 36550560, 36054288, 24362816, 31332305, 30998989); no result recorded for c.1039-6dupA.
• ClinVar (clinvar): 10 submissions (5 Benign, 4 Likely benign per summary; audit usable_for_classification=7) with usable_for_criterion_leads=0, expert_panel_submissions=0, validated_pmids=0; no submission documents functional (PS3/BS3) evidence.
• PMID:25741868: generic ACMG/AMP PS3 definition ('well-established functional studies show a deleterious effect') consulted as fallback framework; the paper does not mention c.1039-6dupA and contains no variant-specific functional data.

Still missing

• No calibrated MMR functional assay result (functional odds for pathogenicity) for c.1039-6dupA
• No MMR function defect data per the VCEP functional assay flowchart for this variant
• No monoallelic expression (cDNA without puromycin) data for this variant
• No RNA splicing/transcript assay (patient cDNA or minigene) data for this variant

Sources

Reasoning

No segregation data exist for this variant. The case bundle contains no pedigrees, no genotyped affected/unaffected relatives, no meioses count, and no paper reporting familial segregation of c.1039-6dupA. The InSiGHT MLH1 VCEP v2.0 requires a combined Bayes Likelihood Ratio from co-segregation in pedigrees (Supporting >2.08, Moderate >4.3-18.7, Strong >18.7 in >=2 families), which cannot be computed without pedigree data. gnomAD allele frequency is population evidence, not segregation evidence, and cannot substitute for PP1.

Rule

PP1: Co-segregation with disease in pedigree(s) with combined Bayes Likelihood Ratio >2.08 (Supporting), >4.3 & <=18.7 (Moderate), >18.7 in >=2 families (Strong) (ClinGen InSiGHT HCRC/PP EP MLH1 v2.0, CSPEC doc 1564688410)

Evidence considered

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

Still missing

• No pedigree or segregation data (no meioses counted, no affected/unaffected relatives genotyped)
• Combined Bayes Likelihood Ratio cannot be computed
• No literature source reports familial segregation of this variant

Sources

Reasoning

PP4 is applicable in the InSiGHT MLH1 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; Moderate: 2 independent tumors; Strong: >=3 independent tumors in >=2 families; an MSI-H tumor with inconsistent protein expression does not meet PP4, and for MLH1 variants MLH1 promoter methylation must be excluded in the tumor). However, the evidence bundle contains no tumor phenotype data for carriers of this variant: no MSI status, no MMR protein IHC results, and no MLH1 promoter methylation results. ClinVar submissions (VCV000140797) carry only generic condition labels ('Carcinoma of colon', 'Hereditary nonpolyposis colorectal neoplasms', 'MLH1-related disorder', 'not provided', 'not specified') with no phenotype detail (usable_for_criterion_leads = 0), and none of the publications in the bundle mention the variant. Evidence is insufficient to evaluate PP4, so it is returned not_assessed.

Rule

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. MSI-H tumor with inconsistent protein expression does not meet PP4. For MLH1 variants, MLH1 promoter methylation is to be excluded in the tumor.

Evidence considered

• CSpec MLH1 v2.0 PP4 thresholds (checked; no qualifying tumor data in bundle)
• ClinVar VCV000140797 submissions (generic condition labels only; usable_for_criterion_leads = 0)
• PMID:34043773 full text screened: no variant mention

Still missing

• No tumor phenotype data for carriers of c.1039-6dupA: MSI status on a standard 5-10 marker panel or tumor genome, MMR protein IHC (MLH1/PMS2 loss consistent with an MLH1 variant location), and MLH1 promoter methylation exclusion

Sources

Reasoning

InSiGHT MLH1 VCEP v2.0 BS2 is a clinical genotype rule, not a population-frequency 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 another LS-spectrum cancer above the median age of onset), with no current or prior evidence of CMMRD manifestations per Aronson et al 2022, and with phase confirmed by testing of parents or offspring. The evidence bundle contains no patient-level genotype data for this variant: no in-trans co-occurrence with a pathogenic MLH1 variant, no CRC/LS cancer age-at-diagnosis observations, and no CMMRD-relevant clinical history (ClinVar submissions carry no such phenotype/phase detail; submission audit shows 0 usable criterion-lead submissions). Therefore BS2 cannot be evaluated and is returned as not_assessed; the required gap is patient-level co-occurrence data.

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 as per Aronson et al 2022 (Refer to 'Table for CMMRD diagnosis.pdf'). Confirmation of phase requires testing of parents or offspring. (InSiGHT MLH1 VCEP v2.0)

Evidence considered

• InSiGHT MLH1 VCEP v2.0 BS2 rule (cspec 1564688410) defines the in-trans co-occurrence + CMMRD-exclusion + phase-confirmation requirements
• ClinVar submission audit (VCV000140797): 0 usable criterion-lead submissions; no co-occurrence/phase observations

Still missing

• No patient-level evidence of in-trans co-occurrence of c.1039-6dupA with a known pathogenic MLH1 variant (phase confirmed by parental/offspring testing) in a CRC patient >45 y or LS-spectrum cancer above median age of onset; no CMMRD clinical assessment data (Aronson et al 2022 criteria) available in the evidence bundle

Sources

Reasoning

BS3 cannot be assessed for this intronic variant: the governing InSiGHT MLH1 v2.0 specification requires demonstration of no associated mRNA aberration (splicing or allelic imbalance) by laboratory assays conducted with nonsense-mediated decay inhibition (Strong), or calibrated functional odds <=0.48 (Supporting) / variant-specific proficient function in protein and mRNA-based lab assays. No RNA-based or calibrated functional assay data exist for c.1039-6dupA; SpliceAI (max delta score 0.01) is an in silico prediction that does not qualify, and ClinVar Benign/Likely benign assertions do not document functional evidence (0 usable criterion leads).

Rule

InSiGHT MLH1 v2.0 (cspec): BS3_Strong = 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 (abnormal transcripts at similar levels in controls considered naturally occurring isoforms); BS3_Supporting = calibrated functional odds >0.05 and <=0.48 OR variant-specific proficient function in protein and mRNA-based lab assays as per the MMR functional assay flowchart

Evidence considered

• InSiGHT MLH1 v2.0 CSPEC (cspec): BS3_Strong for synonymous/intronic variants requires no associated mRNA aberration (splicing or allelic imbalance) as determined by laboratory assays conducted with NMD inhibition; BS3_Supporting requires calibrated functional odds >0.05 and <=0.48 or variant-specific proficient function per the functional assay flowchart.
• SpliceAI (spliceai): max delta score 0.01 (DS_AG 0.0, DS_AL 0.01, DS_DG 0.0, DS_DL 0.01) predicting no significant splice impact; in silico prediction only and does not satisfy the VCEP laboratory-assay requirement for BS3.
• VCEP Functional-assay-SVI-documentation-MMR.xlsx (vcep_functional_assay_svi_documentation_mmr): the cDNA full-length transcript assay (RT-PCR and sequencing; leukocyte culture with active/inhibited NMD via puromycin) is the documented assay framework for intronic variants; no assay was performed or reported for c.1039-6dupA.
• ClinVar (clinvar): Benign/Likely benign submissions exist but carry no documented functional criteria (usable_for_criterion_leads=0, expert_panel_submissions=0); final classifications were not used as functional evidence.
• PMID:25741868: generic BS3 definition ('well-established functional studies show no damaging effect') and mRNA-level assay guidance consulted; no variant-specific study of this variant exists in the retrieved literature.

Still missing

• No RNA-based laboratory assay with NMD inhibition (e.g., cDNA RT-PCR of full-length transcript with and without puromycin from a carrier) demonstrating absence of splicing aberration or allelic imbalance for c.1039-6dupA
• No calibrated functional odds <=0.48 (BS3_Supporting) or <=0.05 (BS3_Strong) for this variant
• No variant-specific proficient-function data in protein and mRNA-based lab assays per the MMR functional assay flowchart

Sources

Reasoning

No evidence of lack of co-segregation is available. The bundle contains no pedigrees showing affected individuals who do not carry the variant or unaffected carriers, so the Bayes Likelihood Ratio for non-segregation required by the InSiGHT MLH1 VCEP v2.0 BS4 rule (Strong <0.05; Supporting >0.05 & <=0.48) cannot be computed. The variant's population allele frequency in gnomAD (grpmax FAF 0.00117, v4.1) is a frequency observation addressed by BA1/BS1/PM2 criteria and is not BS4 segregation evidence.

Rule

BS4: Lack of co-segregation with disease in pedigree(s) with combined Bayes Likelihood Ratio <0.05 (Strong), >0.05 & <=0.48 (Supporting) (ClinGen InSiGHT HCRC/PP EP MLH1 v2.0, CSPEC doc 1564688410)

Evidence considered

• InSiGHT MLH1 VCEP v2.0 BS4 rule: lack of co-segregation Bayes LR thresholds (Strong <0.05; Supporting >0.05 & <=0.48)

Still missing

• No pedigree data demonstrating lack of co-segregation (non-segregation meioses)
• Bayes Likelihood Ratio for BS4 cannot be computed
• No literature source reports non-segregation of this variant

Sources

Reasoning

BP5 is applicable in the InSiGHT MLH1 v2.0 framework (Supporting: 2-3 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 (CRC only)/MLH1 methylation (in LS-spectrum tumor only) with MSI-H/MLH1 loss; Strong: >=4 tumors or >=2 such events). However, no tumor phenotype data exist in the evidence bundle: no MSS status, no MMR IHC results, and no BRAF V600E or MLH1 promoter methylation results for carriers of this variant. The laboratory 'Benign'/'Likely benign' classifications in ClinVar are final assertions, not phenotype evidence, and were not used (a criterion is never inferred from another source's final classification). Evidence is insufficient to evaluate BP5, so it is returned not_assessed.

Rule

BP5 Supporting: 2 or 3 tumors: CRC/Endometrial tumors with MSS and/or no loss of MMR protein expression and/or LS spectrum tumors with loss of MMR protein(s) that is 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

Evidence considered

• CSpec MLH1 v2.0 BP5 thresholds (checked; no qualifying tumor data in bundle)
• ClinVar VCV000140797 submissions (no phenotype detail)
• PMID:34043773 full text screened: no variant mention

Still missing

• No tumor phenotype data for carriers: MSS status, MMR protein IHC, BRAF V600E (CRC) or MLH1 promoter methylation with MSI-H/MLH1 loss

Sources