3-36993668-G-C

Position:

• chr3-36993668-G-C

Variant summary

Our verdict is Pathogenic. Variant got 14 ACMG points: 14P and 0B. PM2 PP3_Strong PP5_Very_Strong

The NM_000249.4(MLH1):​c.116+5G>C 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 0.000000684 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 Pathogenic (★★★).

• Frequency:
  • Genomes: not found (cov: 33)
  • Exomes 𝑓: 6.8e-7 (0 hom.)
• Consequence: MLH1 NM_000249.4 splice_donor_5th_base, intron
• Scores: Splicing: ADA: 1.000
• Clinical Significance: Pathogenic reviewed by expert panel P:7
• 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 Pathogenic. Variant got 14 ACMG points.

• PM2: Very rare variant in population databases, with high coverage
• 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 3-36993668-G-C is Pathogenic according to our data. Variant chr3-36993668-G-C is described in ClinVar as [Pathogenic]. Clinvar id is 89658.Status of the report is reviewed_by_expert_panel, 3 stars. Variant chr3-36993668-G-C is described in Lovd as [Pathogenic].

Transcripts

RefSeq

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

Frequencies

GnomAD3 genomes Cov.: 33

GnomAD4 exome AF: 6.84e-7 AC: 1 AN: 1461686 Hom.: 0 Cov.: 31 AF XY: 0.00 AC XY: 0 AN XY: 727154

Gnomad4 AFR exome AF: 0.00

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: 8.99e-7

Gnomad4 OTH exome AF: 0.00

GnomAD4 genome Cov.: 33

ClinVar

Significance: Pathogenic

Submissions summary: Pathogenic:7

Revision: reviewed by expert panel

Submissions by phenotype

Hereditary cancer-predisposing syndrome Pathogenic:2

Pathogenic, criteria provided, single submitter	clinical testing	Ambry Genetics	Apr 06, 2023	The c.116+5G>C intronic mutation results from a G to C substitution 5 nucleotides after coding exon 1 in the MLH1 gene. This alteration has been reported in multiple Lynch syndrome families and has segregated with disease in multiple affected family members (Casey G et al, JAMA 2005 Feb; 293(7):799-809; Arnold S et al, Hum. Mutat. 2009 May; 30(5):757-70; Thodi G et al, BMC Cancer 2010; 10():544). In addition, multiple functional analyses have demonstrated that this alteration causes aberrant splicing (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). This variant has been identified in a proband who met Amsterdam I criteria for Lynch syndrome and tumor demonstrated high microsatellite instability with loss of MLH1/PMS2 expression by immunohistochemistry (IHC) (Ambry internal data). In addition, this variant has been identified in probands whose Lynch syndrome-associated tumor demonstrated loss of MLH1/PMS2 expression by IHC (Ambry internal data). 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 abnormal splicing in the set of samples tested (Ambry internal data). This variant is considered to be rare based on population cohorts in the Genome Aggregation Database (gnomAD). Based on the supporting evidence, this alteration is interpreted as a disease-causing mutation. -
Pathogenic, criteria provided, single submitter	clinical testing	Color Diagnostics, LLC DBA Color Health	Jul 23, 2021	This variant causes a G to C nucleotide substitution at the +5 position of intron 1 of the MLH1 gene. Functional RNA studies have shown that this variant causes retention of 227 basepairs of intron, resulting in a frameshift and premature truncation. This variant has been reported in individuals affected with Lynch syndrome or colorectal cancer (PMID: 15713769, 19267393, 20459533, 20937110). This variant has not been identified in the general population by the Genome Aggregation Database (gnomAD). Loss of MLH1 function is a known mechanism of disease (clinicalgenome.org). Based on the available evidence, this variant is classified as Pathogenic. -

Colorectal cancer, hereditary nonpolyposis, type 2 Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

Myriad Genetics, Inc.

Jul 12, 2023

This variant is considered pathogenic. mRNA analysis has demonstrated abnormal mRNA splicing occurs [PMID: 15713769]. -

Carcinoma of colon Pathogenic:1

Likely pathogenic, no assertion criteria provided

clinical testing

Department of Pathology and Laboratory Medicine, Sinai Health System

-

The MLH1 c.116+5G>C variant was identified in 2 of 84 proband chromosomes (frequency: 0.02) from individuals or families with Lynch syndrome (Thodi 2010). The variant was also identified in dbSNP (ID: rs267607710) as "With Pathogenic, other allele", ClinVar (classified as pathogenic by Invitae and InSight; as likely pathogenic by GeneDx), Mismatch Repair Genes Variant Database, and the Insight Hereditary Tumors Database (9x). The variant was not identified in COGR, UMD-LSDB, or Zhejiang University Database. The variant was not identified in the following control databases: the Exome Aggregation Consortium (August 8th 2016), or the Genome Aggregation Database (Feb 27, 2017). The c.116+5G>C 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, 3 of 4 in silico or computational prediction software programs (SpliceSiteFinder, MaxEntScan, NNSPLICE, GeneSplicer) predict a greater than 10% difference in splicing. Several functional splicing studies identify the variant lead to loss of MLH1 protein expression and inclusion of 227nt of intron 1 which causes frameshift and aberrations likely to be considered pathogenic (Arnold 2009, Casey 2005, Naruse 2009, Thompson 2013). In addition, the variant segregate with disease in one family with 5/5 affected carries (Arnold 2009). In summary, based on the above information the clinical significance of this variant cannot be determined with certainty at this time although we would lean towards a more pathogenic role for this variant. This variant is classified as likely pathogenic. -

Lynch syndrome Pathogenic:1

Pathogenic, reviewed by expert panel

research

International Society for Gastrointestinal Hereditary Tumours (InSiGHT)

Sep 05, 2013

Variant causes splicing aberration predicted to produce truncated protein: full inactivation of variant allele. Multifactorial likelihood analysis posterior probability >0.99 -

not provided Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

GeneDx

Dec 01, 2020

Non-canonical splice site variant demonstrated to result in loss-of-function (Casey 2005, Arnold 2009, Naruse 2009); Observed in patients with Lynch-related cancers and tumor studies consistent with pathogenic variants in this gene (Casey 2005, Arnold 2009, Choi 2009, Barrow 2010, Thodi 2010); Not observed in large population cohorts (Lek et al., 2016); Truncating variants in this gene are considered pathogenic by a well-established clinical consortium and/or database; This variant is associated with the following publications: (PMID: 22949379, 20459533, 19698169, 20937110, 15713769, 19685281, 19267393, 32658311) -

Hereditary nonpolyposis colorectal neoplasms Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

Labcorp Genetics (formerly Invitae), Labcorp

Nov 27, 2023

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 not present in population databases (gnomAD no frequency). This variant has been observed in individual(s) with clinical features of Lynch syndrome (PMID: 15713769, 19267393, 19685281, 20937110, 32658311). It has also been observed to segregate with disease in related individuals. ClinVar contains an entry for this variant (Variation ID: 89658). Based on a multifactorial likelihood algorithm using genetic, in silico, and/or statistical data, this variant has been determined to have a high probability of being pathogenic (PMID: 22949379). Variants that disrupt the consensus splice site are a relatively common cause of aberrant splicing (PMID: 17576681, 9536098). Studies have shown that this variant alters mRNA splicing and is expected to lead to the loss of protein expression (PMID: 19685281, 22949379). For these reasons, this variant has been classified as Pathogenic. -

Computational scores

Source: dbNSFP v4.3

Name	Calibrated prediction	Score	Prediction
BayesDel_noAF	Benign	-0.22
CADD	Benign	22
DANN	Uncertain	0.99

Splicing

Name	Calibrated prediction	Score	Prediction
dbscSNV1_ADA	Pathogenic	1.0
dbscSNV1_RF	Pathogenic	0.98
SpliceAI score (max)		0.74		Details are displayed if max score is > 0.2
DS_DL_spliceai		0.74		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;