Variant classification summary

NM_000249.4:c.1515T>C

MLH1 · NP_000240.1:p.(Ser505=) · NM_000249.4

GRCh37: chr3:37070380 T>C · GRCh38: chr3:37028889 T>C

Gene: MLH1 Transcript: NM_000249.4

Final call

Likely Benign

BP4 supporting benign BP7 supporting benign

Variant details

Gene

MLH1

Transcript

NM_000249.4

Protein

NP_000240.1:p.(Ser505=)

gnomAD AF

3.0978089816634492e-06 (v4.1)

ClinVar

Likely benign

OncoKB

Unknown Oncogenic Effect

Classification rationale

Interpretation summary

Generated evidence synthesis

1

NM_000249.4:c.1515T>C is a synonymous variant (p.Ser505=) in MLH1 exon 13, located well beyond splice consensus boundaries at c.1515 (+105 from donor, -43 from acceptor).

2

SpliceAI predicts no splicing impact for this variant (max delta score = 0.00), meeting BP4_supporting under the InSiGHT MLH1 VCEP specification.

3

The variant meets BP7_supporting as a synonymous substitution at or beyond -21/+7 from exonic splice boundaries, where it is unlikely to alter splicing.

4

The variant is present in gnomAD v4.1 at low frequency (grpmax FAF = 2.978e-05; 5/1,614,044 alleles) but does not meet PM2_supporting (threshold <0.00002) or BS1 (threshold ≥0.0001).

5

No functional data, tumor pathology data, co-segregation data, de novo observations, or variant-specific literature were identified for this variant.

6

This variant has been reported in ClinVar as Likely benign by 7 clinical laboratories (ClinVar Variation ID: 413362).

7

Under the InSiGHT MLH1 VCEP v2.0.0 combining rules, two supporting benign criteria (BP4 + BP7) yield a classification of Likely Benign (Rule 19: ≥2 Benign Supporting).

Final determination: Rule19 in the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for MLH1 Version 2.0.0 v2.0.0 criteria-combination framework is satisfied by the adjudicated criteria, so the variant is classified as Likely Benign.

Criteria assessment

ACMG/AMP criteria review

Criteria shown when status is available

All criteria require review: For research and educational purposes only.

Criterion	Status	Rationale	Evidence used
PVS1	N/A	Synonymous variant encoding p.(Ser505=) with no predicted effect on protein sequence. PVS1 is reserved for null variants (nonsense, frameshift, canonical splice, initiation codon) and does not apply to synonymous substitutions under the MLH1 VCEP framework.
PS1	N/A	PS1 requires a different nucleotide change producing the same amino acid change, or the same splice nucleotide as known pathogenic splice variant. This is a synonymous variant with no amino acid change; PS1 is not applicable.
PS2	Not met	No de novo observations have been reported for this variant. No evidence of de novo occurrence was identified in ClinVar submissions, publications, or other sources.
PS3	Not met	No functional data are available for this synonymous variant (p.Ser505=). The variant was not directly tested in any calibrated MMR functional assay. The VCEP functional assay spreadsheet does not include data for this variant, and OncoKB lists Unknown Oncogenic Effect with no associated PMIDs.
PS4	N/A	VCEP InSiGHT MLH1 specification explicitly designates PS4 as Not Applicable for use under this framework.
PS5	N/A	PS5 requires the same amino acid change as an established pathogenic variant from a different nucleotide change. This is a synonymous variant with no amino acid change; PS5 is not applicable.
PM1	N/A	VCEP InSiGHT MLH1 specification explicitly designates PM1 as Not Applicable for use under this framework.
PM2	Not met	VCEP PM2 rule requires gnomAD v4 grpmax filtering allele frequency < 0.00002 (<1 in 50,000 alleles). The observed gnomAD v4 grpmax FAF is 2.978e-05 (0.00002978), which exceeds the 0.00002 threshold. The variant is present in 5/1,614,044 alleles in gnomAD v4.1 and 5/251,472 alleles in gnomAD v2.1. Does not meet the PM2_supporting frequency criterion.	gnomad_v4
PM3	N/A	Recessive disorder trans-configuration criterion; not applicable to the assessment scope for this variant.
PM4	N/A	Protein length change criterion; variant is a synonymous substitution with no effect on protein length.
PM5	N/A	PM5 requires a different missense change at the same amino acid residue as a known pathogenic missense variant. This is a synonymous variant (p.Ser505=) with no amino acid change; PM5 is not applicable.
PM6	N/A	VCEP InSiGHT MLH1 specification explicitly designates PM6 as Not Applicable for use under this framework.
PP1	Not met	No co-segregation data are available for this variant. No pedigrees or family studies were identified in the evidence sources.
PP2	N/A	VCEP InSiGHT MLH1 specification explicitly designates PP2 as Not Applicable for use under this framework.
PP3	Not met	VCEP PP3 requires HCI prior probability >0.68 (missense) or SpliceAI delta ≥ 0.2 for non-canonical splice sites. HCI prior was not found for this variant. SpliceAI max delta score is 0.00, well below the 0.2 threshold. No evidence supports a damaging effect from in silico predictions.	spliceai
PP4	Not met	No tumor data demonstrating MSI-H status or loss of MMR protein expression consistent with the variant location are available for this variant.
PP5	N/A	VCEP InSiGHT MLH1 specification explicitly designates PP5 as Not Applicable for use under this framework.
BA1	Not met	VCEP BA1 requires gnomAD v4 grpmax filtering allele frequency ≥ 0.001 (0.1%). The observed gnomAD v4 grpmax FAF is 2.978e-05, far below the 0.1% threshold. BA1 is not met.	gnomad_v4
BS1	Not met	VCEP BS1 requires gnomAD v4 grpmax filtering allele frequency ≥ 0.0001 and < 0.001 (0.01-0.1%). The observed gnomAD v4 grpmax FAF is 2.978e-05, which is below the lower threshold of 0.0001. BS1 is not met.	gnomad_v4
BS2	Not met	No evidence of co-occurrence in trans with a known pathogenic variant in a patient with CRC after age 45 without clinical manifestations of CMMRD. No phase-confirmed observations available.
BS3	Not met	No functional studies demonstrating a benign effect are available for this variant. The variant has not been evaluated in any calibrated MMR functional assay or other functional system.
BS4	Not met	No lack-of-segregation data are available for this variant. No pedigrees demonstrating non-segregation with disease were identified.
BP1	N/A	VCEP InSiGHT MLH1 specification explicitly designates BP1 as Not Applicable for use under this framework.
BP2	N/A	VCEP InSiGHT MLH1 specification explicitly designates BP2 as Not Applicable for use under this framework.
BP3	N/A	BP3 applies to in-frame deletions and insertions in repetitive regions; variant is a synonymous substitution.
BP4	Met	VCEP BP4 specifies that for synonymous variants, SpliceAI delta score ≤ 0.1 qualifies as BP4_supporting. SpliceAI predicts no splicing impact (max delta = 0.00), consistent with a synonymous variant that does not alter splicing.	spliceai
BP5	Not met	No tumor data demonstrating MSS status or absence of MMR protein expression loss are available for this variant. BP5 requires multiple tumors with MSS and/or no loss of MMR protein expression.
BP6	N/A	VCEP InSiGHT MLH1 specification explicitly designates BP6 as Not Applicable for use under this framework.
BP7	Met	VCEP BP7 applies to synonymous (silent) variants at or beyond -21/+7 from exonic splice boundaries. NM_000249.4:c.1515T>C (p.Ser505=) is a synonymous variant located at position +105 from the exon 13 donor splice site and -43 from the exon 13 acceptor splice site, both well beyond the -21/+7 splice consensus boundaries. This deep exonic synonymous substitution is unlikely to affect splicing.

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