ClinVar Genomic variation as it relates to human health

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)

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].

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.

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.

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.

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.

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.

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.