rs267607710

Positions:

• chr3-36993668-G-A
• chr3-36993668-G-C

Variant summary

Our verdict is Uncertain significance. Variant got 4 ACMG points: 4P and 0B. PP3_Strong

The NM_000249.4(MLH1):​c.116+5G>A variant causes a splice donor 5th base, intron change involving the alteration of a non-conserved nucleotide. The variant allele was found at a frequency of 0.00000958 in 1,461,686 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 Conflicting classifications of pathogenicity (no stars).

• Frequency:
  • Genomes: not found (cov: 33)
  • Exomes 𝑓: 0.0000096 (0 hom.)
• Consequence: MLH1 NM_000249.4 splice_donor_5th_base, intron
• Scores: Splicing: ADA: 1.000
• Clinical Significance: Conflicting classifications of pathogenicity criteria provided, conflicting classifications P:1 U:10 B:1
• Conservation: PhyloP100: 1.32

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 Uncertain_significance. Variant got 4 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.

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	MANE	Protein	UniProt
MLH1	NM_000249.4	c.116+5G>A		splice_donor_5th_base_variant, intron_variant		ENST00000231790.8	NP_000240.1

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	TSL	MANE	Protein	Appris	UniProt
MLH1	ENST00000231790.8	c.116+5G>A		splice_donor_5th_base_variant, intron_variant		1	NM_000249.4	ENSP00000231790	P1

Frequencies

GnomAD3 genomes Cov.: 33

GnomAD3 exomes AF: 0.0000280 AC: 7 AN: 249680 Hom.: 0 AF XY: 0.0000222 AC XY: 3 AN XY: 135218

Gnomad AFR exome AF: 0.00

Gnomad AMR exome AF: 0.00

Gnomad ASJ exome AF: 0.00

Gnomad EAS exome AF: 0.00

Gnomad SAS exome AF: 0.00

Gnomad FIN exome AF: 0.00

Gnomad NFE exome AF: 0.0000624

Gnomad OTH exome AF: 0.00

GnomAD4 exome AF: 0.00000958 AC: 14 AN: 1461686 Hom.: 0 Cov.: 31 AF XY: 0.00000825 AC XY: 6 AN XY: 727154

Gnomad4 AFR exome AF: 0.0000299

Gnomad4 AMR exome AF: 0.00

Gnomad4 ASJ exome AF: 0.00

Gnomad4 EAS exome AF: 0.00

Gnomad4 SAS exome AF: 0.00

Gnomad4 FIN exome AF: 0.00

Gnomad4 NFE exome AF: 0.0000108

Gnomad4 OTH exome AF: 0.0000166

GnomAD4 genome Cov.: 33

Bravo AF: 0.0000113

EpiCase AF: 0.00

EpiControl AF: 0.0000593

ClinVar

Significance: Conflicting classifications of pathogenicity

Submissions summary: Pathogenic:1 Uncertain:10 Benign:1

Revision: criteria provided, conflicting classifications

Submissions by phenotype

Hereditary cancer-predisposing syndrome Uncertain:3

Uncertain significance, criteria provided, single submitter	curation	Sema4, Sema4	Jan 15, 2022	- -
Uncertain significance, criteria provided, single submitter	clinical testing	Ambry Genetics	Jun 06, 2023	The c.116+5G>A intronic variant results from a G to A substitution 5 nucleotides after coding exon 1 in the MLH1 gene. A different alteration at this nucleotide position (c.116+5G>C) has been classified as pathogenic based on being identified in individuals meeting clinical diagnostic criteria for Lynch syndrome and abnormal splicing was demonstrated by RNA functional studies (Casey G et al, JAMA 2005 Feb; 293(7):799-809; Arnold S et al, Hum. Mutat. 2009 May; 30(5):757-70; Naruse H et al, Fam. Cancer 2009; 8(4):509-17; Thodi G et al, BMC Cancer 2010 Oct; 10:544). This nucleotide position is well conserved in available vertebrate species. In silico splice site analysis predicts that this alteration will weaken the native splice donor site. RNA studies have demonstrated that this alteration results in a splice defect; however, the clinical impact of this abnormal splicing is unknown at this time (Ambry internal data). However, this alteration has been detected in many individuals who do not have a personal or family history that is suggestive of hereditary nonpolyposis colorectal cancer (HNPCC)/Lynch syndrome (Ambry internal data). Since supporting evidence is conflicting at this time, the clinical significance of this alteration remains unclear. -
Uncertain significance, criteria provided, single submitter	clinical testing	Color Diagnostics, LLC DBA Color Health	Mar 03, 2023	This variant causes a G>A nucleotide substitution at the +5 position of intron 1 of the MLH1 gene. Splice site prediction tools suggest that this variant may have a significant impact on RNA splicing. RNA studies have shown that the variant causes abnormal RNA splicing (PMID: 31642931, 32133419). To our knowledge, this variant has not been reported in individuals affected with MLH1-related disorders in the literature. This variant has been identified in 7/249680 chromosomes in the general population by the Genome Aggregation Database (gnomAD). A different variant affecting the same splice donor site (c.116+5G>C) is considered to be disease-causing (ClinVar variation ID: 89658). While the data suggest this nucleotide position is important for normal RNA splicing, the available clinical evidence is insufficient to determine the role of this variant in disease conclusively. Therefore, this variant is classified as a Variant of Uncertain Significance. -

Colorectal cancer, hereditary nonpolyposis, type 2 Uncertain:2 Benign:1

Uncertain significance, criteria provided, single submitter	clinical testing	Counsyl	Apr 25, 2018	- -
Uncertain significance, criteria provided, single submitter	clinical testing	Baylor Genetics	Feb 14, 2024	- -
Likely benign, criteria provided, single submitter	clinical testing	Myriad Genetics, Inc.	Mar 10, 2023	This variant is considered likely benign. This variant is strongly associated with less severe personal and family histories of cancer, typical for individuals without pathogenic variants in this gene [PMID: 25085752]. -

Lynch syndrome Pathogenic:1 Uncertain:1

Likely pathogenic, criteria provided, single submitter	clinical testing	Laboratory for Molecular Medicine, Mass General Brigham Personalized Medicine	Aug 24, 2021	The c.116+5G>A variant in MLH1 has been reported in 1 individual referred for diagnostic genetic testing for hereditary cancer (Karam 2019 PMID: 31642931) and has also been reported by clinical laboratories in ClinVar (Variation ID: 218011). It has also been identified in 0.006% (7/112254) of European chromosomes by gnomAD (http://gnomad.broadinstitute.org). This variant is located in the 5' splice region. Sequencing analysis using patient RNA from blood showed abnormal splicing (Karam 2019 PMID: 31642931). Computational tools also predict a splicing impact. Another variant affecting the same nucleotide (c.116+5G>C) has been reported in several families affected with hereditary non polyposis colorectal cancer and is classified as pathogenic by several clinical labs in ClinVar, including the ClinGen-approved InSiGHT expert panel (Variation ID: 89658). In summary, although additional studies are required to fully establish its clinical significance, this variant meets criteria to be classified as likely pathogenic for autosomal dominant Lynch syndrome. ACMG/AMP criteria applied: PM2_Supporting, PS3_Moderate, PP3, PM5. -
Uncertain significance, criteria provided, single submitter	clinical testing	All of Us Research Program, National Institutes of Health	Dec 01, 2023	This variant causes a G>A nucleotide substitution at the +5 position of intron 1 of the MLH1 gene. Splice site prediction tools suggest that this variant may have a significant impact on RNA splicing. RNA studies have shown that the variant causes abnormal RNA splicing (PMID: 31642931, 32133419). To our knowledge, this variant has not been reported in individuals affected with MLH1-related disorders in the literature. This variant has been identified in 7/249680 chromosomes in the general population by the Genome Aggregation Database (gnomAD). A different variant affecting the same splice donor site (c.116+5G>C) is considered to be disease-causing (ClinVar variation ID: 89658). While the data suggest this nucleotide position is important for normal RNA splicing, the available clinical evidence is insufficient to determine the role of this variant in disease conclusively. Therefore, this variant is classified as a Variant of Uncertain Significance. -

not provided Uncertain:2

Uncertain significance, no assertion criteria provided	clinical testing	Department of Pathology and Laboratory Medicine, Sinai Health System	-	The MLH1 c.116+5G>A variant was not identified in the literature nor was it identified in the following databases: Cosmic, MutDB, Insight Colon Cancer Gene Variant Database, Zhejiang Colon Cancer Database, Mismatch Repair Genes Variant Database, or Insight Hereditary Tumors Database. The variant was identified in dbSNP (ID: rs267607710) as “With Pathogenic, Uncertain significance allele”, ClinVar (1x, uncertain significance), and Clinvitae (1x, uncertain significance). The variant was identified in control databases in 7 of 244674 chromosomes at a frequency of 0.00003 (Genome Aggregation Database Feb 27, 2017). Breakdown of the observations by population include European in 7 of 110346 chromosomes (freq: 0.00006), while the variant was not observed in the African, Other, Latino, Ashkenazi Jewish, East Asian, Finnish, or South Asian populations. The c.116+5G>A variant is located in the 5' splice region but does not affect the invariant +1 and +2 positions. However, positions +3 to +6 are part of the splicing consensus sequence and variants involving these positions sometimes affect splicing. In addition, 4 of 5 in silico or computational prediction software programs (SpliceSiteFinder, MaxEntScan, NNSPLICE, GeneSplicer, HumanSpliceFinder) predict a greater than 10% difference in splicing. Of note, a similar variant, MLH1 c.116+5G>C, is reported on ClinVar as pathogenic by GeneDx and InSiGHT, evidence includes segregation analysis, functional studies and in silico studies. In summary, based on the above information the clinical significance of this variant cannot be determined with certainty at this time. This variant is classified as a variant of uncertain significance. -
Uncertain significance, criteria provided, single submitter	clinical testing	GeneDx	Jun 10, 2021	In silico analysis supports a deleterious effect on splicing; Published functional studies demonstrate abnormal splicing due to inclusion of 227 nucleotides in intron 1 predicted to result in a frameshift (Karam 2019, Landrith 2020); This variant is associated with the following publications: (PMID: 31642931, 32133419, 32719484) -

not specified Uncertain:1

Uncertain significance, no assertion criteria provided

research

Mayo Clinic Laboratories, Mayo Clinic

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- -

Hereditary nonpolyposis colorectal neoplasms Uncertain:1

Uncertain significance, criteria provided, single submitter

clinical testing

Labcorp Genetics (formerly Invitae), Labcorp

Jan 24, 2024

This sequence change falls in intron 1 of the MLH1 gene. It does not directly change the encoded amino acid sequence of the MLH1 protein. It affects a nucleotide within the consensus splice site. This variant is present in population databases (rs267607710, gnomAD 0.006%). This variant has been observed in an individual undergoing hereditary cancer testing (PMID: 31642931). However, it has also been observed in several individuals without clinical features of Lynch syndrome (Invitae). ClinVar contains an entry for this variant (Variation ID: 218011). Variants that disrupt the consensus splice site are a relatively common cause of aberrant splicing (PMID: 17576681, 9536098). Studies have shown that this variant is associated with inconclusive levels of altered splicing (Invitae). This variant disrupts the c.116+5G nucleotide in the MLH1 gene. Other variant(s) that disrupt this nucleotide have been determined to be pathogenic (PMID: 15713769, 19267393, 19685281, 20937110). This suggests that this nucleotide is clinically significant, and that variants that disrupt this position are likely to be disease-causing. In summary, the available evidence is currently insufficient to determine the role of this variant in disease. Therefore, it has been classified as a Variant of Uncertain Significance. -

Computational scores

Source: dbNSFP v4.3

Name	Calibrated prediction	Score	Prediction
BayesDel_noAF	Benign	-0.19
CADD	Benign	22
DANN	Uncertain	0.99

Splicing

Name	Calibrated prediction	Score	Prediction
dbscSNV1_ADA	Pathogenic	1.0
dbscSNV1_RF	Pathogenic	1.0
SpliceAI score (max)		0.60		Details are displayed if max score is > 0.2
DS_DL_spliceai		0.60		Position offset: -5

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

Publications

LitVar

Other links and lift over

dbSNP: rs267607710; hg19: chr3-37035159;