rs63751596

Positions:

• chr3-37040294-G-A
• chr3-37040294-G-C
• chr3-37040294-G-T

Variant summary

Our verdict is Pathogenic. Variant got 18 ACMG points: 18P and 0B. PM1 PM2 PM5 PP3_Strong PP5_Very_Strong

The NM_000249.4(MLH1):​c.1667G>A​(p.Ser556Asn) variant causes a missense, splice region change involving the alteration of a conserved nucleotide. The variant was absent in control chromosomes in GnomAD project. 3/3 splice prediction tools predicting alterations to normal splicing. Variant has been reported in ClinVar as Likely pathogenic (★★). Another variant affecting the same amino acid position, but resulting in a different missense (i.e. S556I) has been classified as Pathogenic.

• Frequency: Genomes: not found (cov: 32)
• Consequence: MLH1 NM_000249.4 missense, splice_region
• Scores: Splicing: ADA: 1.000
• Clinical Significance: Pathogenic/Likely pathogenic criteria provided, multiple submitters, no conflicts P:6 U:1
• Conservation: PhyloP100: 9.74

Genes affected

MLH1 (HGNC:7127): (mutL homolog 1) The protein encoded by this gene can heterodimerize with mismatch repair endonuclease PMS2 to form MutL alpha, part of the DNA mismatch repair system. When MutL alpha is bound by MutS beta and some accessory proteins, the PMS2 subunit of MutL alpha introduces a single-strand break near DNA mismatches, providing an entry point for exonuclease degradation. The encoded protein is also involved in DNA damage signaling and can heterodimerize with DNA mismatch repair protein MLH3 to form MutL gamma, which is involved in meiosis. This gene was identified as a locus frequently mutated in hereditary nonpolyposis colon cancer (HNPCC). [provided by RefSeq, Aug 2017]

ACMG classification

Verdict is Pathogenic. Variant got 18 ACMG points.

• PM1: In a hotspot region, there are 7 aminoacids with missense pathogenic changes in the window of +-8 aminoacids around while only 1 benign, 15 uncertain in NM_000249.4
• PM2: Very rare variant in population databases, with high coverage
• PM5: Other missense variant is known to change same aminoacid residue: Variant chr3-37040294-G-T is described in ClinVar as [Pathogenic]. Clinvar id is 89825.Status of the report is reviewed_by_expert_panel, 3 stars.
• PP3: Splicing scoreres supports a deletorius effect: Scorers claiming Pathogenic: dbscSNV1_ADA, dbscSNV1_RF, max_spliceai. No scorers claiming Uncertain. No scorers claiming Benign.
• PP5: Variant 3-37040294-G-A is Pathogenic according to our data. Variant chr3-37040294-G-A is described in ClinVar as [Likely_pathogenic]. Clinvar id is 449776.Status of the report is criteria_provided_multiple_submitters_no_conflicts, 2 stars. Variant chr3-37040294-G-A is described in Lovd as [Pathogenic].

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	MANE	UniProt
MLH1	NM_000249.4	c.1667G>A	p.Ser556Asn	missense_variant, splice_region_variant	14/19	ENST00000231790.8

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	TSL	MANE	Appris	UniProt
MLH1	ENST00000231790.8	c.1667G>A	p.Ser556Asn	missense_variant, splice_region_variant	14/19	1	NM_000249.4	P1

Frequencies

GnomAD3 genomes Cov.: 32

GnomAD4 exome Cov.: 28

GnomAD4 genome Cov.: 32

ClinVar

Significance: Pathogenic/Likely pathogenic

Submissions summary: Pathogenic:6 Uncertain:1

Revision: criteria provided, multiple submitters, no conflicts

Submissions by phenotype

not provided Pathogenic:2

Likely pathogenic, criteria provided, single submitter	clinical testing	GeneDx	Sep 10, 2019	Not observed in large population cohorts (Lek 2016); Observed in individuals with a personal or family history consistent with pathogenic variants in this gene (Whitworth 2015, Sjursen 2016, Yurgelun 2017); This variant is associated with the following publications: (PMID: 25248401, 27064304, 22949387, 28135145, 30521064, 30720243) -
Likely pathogenic, criteria provided, single submitter	clinical testing	CeGaT Center for Human Genetics Tuebingen	Apr 01, 2023	MLH1: PM1, PM2, PS4:Moderate, PP1 -

Colorectal cancer, hereditary nonpolyposis, type 2 Pathogenic:1

Likely pathogenic, criteria provided, single submitter

clinical testing

Baylor Genetics

Apr 20, 2022

- -

Lynch-like syndrome Pathogenic:1

Likely pathogenic, no assertion criteria provided

clinical testing

Constitutional Genetics Lab, Leon Berard Cancer Center

Jul 01, 2019

- -

Hereditary nonpolyposis colorectal neoplasms Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

Invitae

Dec 29, 2023

This sequence change replaces serine, which is neutral and polar, with asparagine, which is neutral and polar, at codon 556 of the MLH1 protein (p.Ser556Asn). This variant also falls at the last nucleotide of exon 14, which is part of the consensus splice site for this exon. This variant is present in population databases (no rsID available, gnomAD 0.0009%). This missense change has been observed in individuals with colorectal cancer and clinical features of Lynch syndrome (PMID: 27064304, 28135145; Invitae; externalcommunication). It has also been observed to segregate with disease in related individuals. ClinVar contains an entry for this variant (Variation ID: 449776). An algorithm developed to predict the effect of missense changes on protein structure and function (PolyPhen-2) suggests that this variant is likely to be disruptive. Variants that disrupt the consensus splice site are a relatively common cause of aberrant splicing (PMID: 17576681, 9536098). Algorithms developed to predict the effect of sequence changes on RNA splicing suggest that this variant may disrupt the consensus splice site. For these reasons, this variant has been classified as Pathogenic. -

Hereditary cancer-predisposing syndrome Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

Ambry Genetics

Feb 23, 2021

The c.1667G>A pathogenic mutation (also known as p.S556N), located in coding exon 14 of the MLH1 gene, results from a G to A substitution at nucleotide position 1667. The amino acid change results in serine to asparagine at codon 556, an amino acid with highly similar properties. However, this change occurs in the last base pair of coding exon 14, which makes it likely to have some effect on normal mRNA splicing. This alteration has been identified in multiple families fulfilling Amsterdam criteria (Sjursen W et al. Mol Genet Genomic Med. 2016 Mar;4:223-31; Yurgelun MBJ. Clin. Oncol. 2017 Apr;35(10):1086-1095; Jiang W et al. Int J Cancer, 2019 05;144:2161-2168). Another alteration at this same nucleotide position, c.1667G>T, has been shown to result in the use of a cryptic splice site that leads to a frameshift predicted to create a truncated protein (Sharp et al. Hum Mutat. 2004 Sep;24(3):272; Desmet et al. Nucleic Acids Res. 2009 May;37(9):e67). This nucleotide position is highly conserved in available vertebrate species. In silico splice site analysis predicts that this alteration will weaken the native splice donor site. Based on the supporting evidence, this alteration is interpreted as a disease-causing mutation. -

Lynch syndrome 1 Uncertain:1

Uncertain significance, no assertion criteria provided

clinical testing

Ding PR Lab, Sun Yat-sen University Cancer Center

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- -

Computational scores

Source: dbNSFP v4.3

Name	Calibrated prediction	Score	Prediction
AlphaMissense	Pathogenic	0.81
BayesDel_addAF	Pathogenic	0.27	D
BayesDel_noAF	Uncertain	0.020
CADD	Pathogenic	29
DANN	Uncertain	0.98
DEOGEN2	Benign	0.41	T
Eigen	Uncertain	0.55
Eigen_PC	Uncertain	0.43
FATHMM_MKL	Pathogenic	0.99	D
LIST_S2	Benign	0.20	T
M_CAP	Pathogenic	0.39	D
MetaRNN	Uncertain	0.61	D
MetaSVM	Uncertain	0.24	D
MutationTaster	Benign	1.0	D;D;D;D;D;D
PROVEAN	Benign	0.41	N
REVEL	Uncertain	0.43
Sift	Benign	0.27	T
Sift4G	Pathogenic	0.0	D
MutPred		0.41		Loss of helix (P = 0.0076);
MVP		0.94
ClinPred		0.87	D
GERP RS		4.5
RBP_binding_hub_radar		0.0
RBP_regulation_power_radar		1.7

Splicing

Name	Calibrated prediction	Score	Prediction
dbscSNV1_ADA	Pathogenic	1.0
dbscSNV1_RF	Pathogenic	0.99
SpliceAI score (max)		0.86		Details are displayed if max score is > 0.2
DS_DL_spliceai		0.86		Position offset: 0

Find out detailed SpliceAI scores and Pangolin per-transcript scores at spliceailookup.broadinstitute.org

Publications

LitVar

Other links and lift over

dbSNP: rs63751596; hg19: chr3-37081785;