rs63751665

chr3-37001053-G-Achr3-37001053-G-Cchr3-37001053-G-T

Variant summary

Our verdict is Likely pathogenic. Variant got 6 ACMG points: 6P and 0B. PP3_Moderate PP5_Strong

The NM_000249.4(MLH1):c.306G>A(p.Glu102=) variant causes a splice region, synonymous change. The variant allele was found at a frequency of 0.00000379 in 1,582,496 control chromosomes in the GnomAD database, with no homozygous occurrence. In-silico tool predicts a benign outcome for this variant. 3/3 splice prediction tools predicting alterations to normal splicing. Variant has been reported in ClinVar as Uncertain significance (★★★).

• Frequency:
  • Genomes: 𝑓 0.0000066 (0 hom., cov: 32)
  • Exomes 𝑓: 0.0000035 (0 hom.)
• Consequence: MLH1 NM_000249.4 splice_region, synonymous
• Scores: Splicing: ADA: 0.9998
• Clinical Significance: Uncertain significance reviewed by expert panel P:4 U:2
• Conservation: PhyloP100: 6.33

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 Likely_pathogenic. Variant got 6 ACMG points.

• 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 3-37001053-G-A is Pathogenic according to our data. Variant chr3-37001053-G-A is described in ClinVar as [Uncertain_significance]. Clinvar id is 232603.Status of the report is reviewed_by_expert_panel, 3 stars. We mark this variant Likely_pathogenic, oryginal submissions are: {Pathogenic=2, Uncertain_significance=2, Likely_pathogenic=2}. Variant chr3-37001053-G-A is described in Lovd as [Likely_pathogenic].

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	MANE	UniProt
MLH1	NM_000249.4	c.306G>A	p.Glu102=	splice_region_variant, synonymous_variant	3/19	ENST00000231790.8

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	TSL	MANE	Appris	UniProt
MLH1	ENST00000231790.8	c.306G>A	p.Glu102=	splice_region_variant, synonymous_variant	3/19	1	NM_000249.4	P1

Frequencies

GnomAD3 genomes AF: 0.00000657 AC: 1 AN: 152092 Hom.: 0 Cov.: 32

Gnomad AFR AF: 0.0000242

Gnomad AMI AF: 0.00

Gnomad AMR AF: 0.00

Gnomad ASJ AF: 0.00

Gnomad EAS AF: 0.00

Gnomad SAS AF: 0.00

Gnomad FIN AF: 0.00

Gnomad MID AF: 0.00

Gnomad NFE AF: 0.00

Gnomad OTH AF: 0.00

GnomAD3 exomes AF: 0.0000159 AC: 4 AN: 251334 Hom.: 0 AF XY: 0.0000221 AC XY: 3 AN XY: 135846

Gnomad AFR exome AF: 0.0000615

Gnomad AMR exome AF: 0.00

Gnomad ASJ exome AF: 0.00

Gnomad EAS exome AF: 0.00

Gnomad SAS exome AF: 0.0000980

Gnomad FIN exome AF: 0.00

Gnomad NFE exome AF: 0.00

Gnomad OTH exome AF: 0.00

GnomAD4 exome AF: 0.00000350 AC: 5 AN: 1430404 Hom.: 0 Cov.: 26 AF XY: 0.00000561 AC XY: 4 AN XY: 713570

Gnomad4 AFR exome AF: 0.0000305

Gnomad4 AMR exome AF: 0.00

Gnomad4 ASJ exome AF: 0.00

Gnomad4 EAS exome AF: 0.00

Gnomad4 SAS exome AF: 0.0000467

Gnomad4 FIN exome AF: 0.00

Gnomad4 NFE exome AF: 0.00

Gnomad4 OTH exome AF: 0.00

GnomAD4 genome AF: 0.00000657 AC: 1 AN: 152092 Hom.: 0 Cov.: 32 AF XY: 0.00 AC XY: 0 AN XY: 74286

Gnomad4 AFR AF: 0.0000242

Gnomad4 AMR AF: 0.00

Gnomad4 ASJ AF: 0.00

Gnomad4 EAS AF: 0.00

Gnomad4 SAS AF: 0.00

Gnomad4 FIN AF: 0.00

Gnomad4 NFE AF: 0.00

Gnomad4 OTH AF: 0.00

ClinVar

Significance: Uncertain significance

Submissions summary: Pathogenic:4 Uncertain:2

Revision: reviewed by expert panel

Submissions by phenotype

Lynch syndrome Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

All of Us Research Program, National Institutes of Health

Oct 02, 2023

This synonymous variant does not change the encoded amino acid at codon 102 of the MLH1 protein, but it causes a G to A substitution at the last nucleotide of exon 3 of the MLH1 gene. Splice site prediction tools suggest that this variant may have a significant impact on RNA splicing by impairing the intron 3 splice donor site. RNA studies have shown that this variant results in exon 3 skipping resulting in an in-frame deletion within the ATPase domain, which is important for the mismatch repair activity (ClinVar: SCV001414684.3). Another variant at this position (c.306G>T) has also been observed to cause exon 3 skipping (PMID: 22736432). The MLH1 c.306G>A, p.Glu102= variant has been reported in individuals affected with Lynch syndrome tumors displaying loss of MLH1 on immunohistochemistry (ClinVar: SCV001414684.3, SCV000276776.7, SCV000618506.2), breast cancer (PMID: 32039725), and homozygous in a patient with constitutional mismatch repair deficiency syndrome (ClinVar: SCV000276776.7). Other variants at the same nucleotide position are classified as Pathogenic in ClinVar (Variation ID: 90150, 90151). This variant has been identified in 4/251334 chromosomes in the general population by the Genome Aggregation Database (gnomAD). Based on the available evidence, this variant is classified as Pathogenic. -

not provided Pathogenic:1

Likely pathogenic, criteria provided, single submitter

clinical testing

GeneDx

Jun 05, 2023

Alters the last nucleotide of the exon and is predicted to destroy the splice donor site and result in aberrant splicing, although in the absence of functional evidence the actual effect of this sequence change is unknown; Observed in individuals with colon cancer displaying loss of MLH1 on immunohistochemistry (IHC) studies at this laboratory and in an individual with breast cancer in published literature (da Costa e Silva Carvalho et al., 2020); Not observed at significant frequency in large population cohorts (gnomAD); Deletions involving coding exons of this gene are a known mechanism of disease (HGMD); This variant is associated with the following publications: (PMID: 32039725) -

Hereditary nonpolyposis colorectal neoplasms Pathogenic:1

Likely pathogenic, criteria provided, single submitter

clinical testing

Invitae

Jul 16, 2023

This sequence change affects codon 102 of the MLH1 mRNA. It is a 'silent' change, meaning that it does not change the encoded amino acid sequence of the MLH1 protein. RNA analysis indicates that this variant induces altered splicing and likely results in a shortened protein product. This variant is present in population databases (rs63751665, gnomAD 0.01%). This variant has been observed in individual(s) with clinical features of Lynch syndrome (Invitae). ClinVar contains an entry for this variant (Variation ID: 232603). Variants that disrupt the consensus splice site are a relatively common cause of aberrant splicing (PMID: 17576681, 9536098). Studies have shown that this variant results in skipping of 3, but is expected to preserve the integrity of the reading-frame (Invitae). This variant disrupts the ATP binding and hydrolysis domain of the MLH1 protein, which is important for the mismatch repair activity (PMID: 22753075, 21404117). While functional studies have not been performed to directly test the effect of this variant on MLH1 protein function, this suggests that disruption of this region of the protein is causative of disease. This variant disrupts the c.306G nucleotide in the MLH1 gene. Other variant(s) that disrupt this nucleotide have been determined to be pathogenic (PMID: 9536098, 17510385, 17576681, 21520333, 22736432, 23729658, 26659639, 26681312). This suggests that this nucleotide is clinically significant, and that variants that disrupt this position are likely to be disease-causing. In summary, the currently available evidence indicates that the variant is pathogenic, but additional data are needed to prove that conclusively. Therefore, this variant has been classified as Likely Pathogenic. -

Hereditary cancer-predisposing syndrome Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

Ambry Genetics

Jan 29, 2023

The c.306G>A pathogenic mutation (also known as p.E102E), located in coding exon 3 of the MLH1 gene, results from a G to A substitution at nucleotide position 306. This nucleotide substitution does not change the codon at position 102. However, this change occurs in the last base pair of coding exon 3, which makes it likely to have some effect on normal mRNA splicing. This alteration has been observed in the homozygous state in a patient with clinical features of constitutional mismatch repair deficiency (CMMRD) and in a second patient without consistent clinical features of CMMRD (Ambry internal data, interlaboratory correspondence). This alteration has been observed in the heterozygous state in a proband whose Lynch-syndrome associated tumor demonstrated loss of both MLH1/PMS2 expression on immunohistochemistry (IHC) (Ambry internal data). In a cohort of hereditary breast and ovarian cancer patients (N=95), this alteration was identified once in an individual with invasive ductal carcinoma diagnosed at the age of 40 (da Costa E Silva Carvalho S et al. BMC Med Genomics, 2020 02;13:21). Another alteration at the same position, c.306G>T, has been reported to result in abnormal splicing and has been detected in multiple individuals with family histories meeting Amsterdam criteria and/or loss of PMS2 expression on IHC with high microsatellite instability in their Lynch syndrome-associated tumors (Ambry internal data). In silico splice site analysis predicts that this alteration will weaken the native splice donor site. RNA studies have demonstrated this alteration results in abnormal splicing in the set of samples tested (Ambry internal data). Based on the total supporting evidence, this alteration is interpreted as a disease-causing mutation. However, in the homozygous state, this alteration may be associated with a variable phenotype with respect to CMMRD. Clinical correlation is advised. -

not specified Uncertain:1

Uncertain significance, criteria provided, single submitter

clinical testing

Women's Health and Genetics/Laboratory Corporation of America, LabCorp

Aug 02, 2021

Variant summary: MLH1 c.306G>A (p.Glu102Glu) alters a conserved nucleotide located close to a canonical splice site and therefore could affect mRNA splicing, leading to a significantly altered protein sequence. Several computational tools predict a significant impact on normal splicing: Three predict the variant weakens a 5 prime donor site. However, these predictions have yet to be confirmed by functional studies. The variant allele was found at a frequency of 1.6e-05 in 251334 control chromosomes. The available data on variant occurrences in the general population are insufficient to allow any conclusion about variant significance. c.306G>A has been reported in the literature in one individual affected with Breast Cancer (Carvalho_2020). This report does not provide unequivocal conclusions about association of the variant with Hereditary Nonpolyposis Colorectal Cancer. To our knowledge, no experimental evidence demonstrating an impact on protein function has been reported. Four clinical diagnostic laboratories have submitted clinical-significance assessments for this variant to ClinVar after 2014 without evidence for independent evaluation. Multiple laboratories reported the variant with conflicting assessments (pathogenic/likely pathogenic n=2, VUS n=2). Another variant affecting same nucleotide, c. 306G>T, has been reported to associate with colorectal cancer (HGMD database). Based on the evidence outlined above, the variant was classified as uncertain significance. -

Lynch syndrome 1 Uncertain:1

Uncertain significance, reviewed by expert panel

curation

International Society for Gastrointestinal Hereditary Tumours (InSiGHT)

Jun 13, 2018

Criteria changed for variants in last base of exon therefore downgrade classification -

Computational scores

Source: dbNSFP v4.3

Name	Calibrated prediction	Score	Prediction
BayesDel_noAF	Benign	-0.19
Cadd	Benign	21
Dann	Benign	0.96
RBP_binding_hub_radar		0.0
RBP_regulation_power_radar		2.7

Splicing

Name	Calibrated prediction	Score	Prediction
dbscSNV1_ADA	Pathogenic	1.0
dbscSNV1_RF	Pathogenic	0.90
SpliceAI score (max)		0.88		Details are displayed if max score is > 0.2
DS_DL_spliceai		0.88		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: rs63751665; hg19: chr3-37042544;