ClinVar Genomic variation as it relates to human health

This variant was observed as part of a predisposition screen in an ostensibly healthy population. A literature search was performed for the gene, cDNA change, and amino acid change (where applicable). Publications were found based on this search. However, the evidence from the literature, in combination with allele frequency data from public databases where available, was not sufficient to rule this variant in or out of causing disease. Therefore, this variant is classified as a variant of unknown significance.

DNA sequence analysis of the MUTYH gene demonstrated a sequence change, c.667A>G, in exon 8 that results in an amino acid change, p.Ile223Val. This sequence change has been described in the gnomAD database with a low population frequency of 0.02% (dbSNP rs200872702). The p.Ile223Val change affects a highly conserved amino acid residue located in a domain of the MUTYH protein that is known to be functional. In-silico pathogenicity prediction tools (SIFT, PolyPhen2, Align GVGD, REVEL) provide contradictory results for the p.Ile223Val substitution. The c.667A>G (p.Ille223Val) sequence change has been reported in the heterozygous state in individuals affected with familial adenomatous polyposis, colorectal adenomas and colon cancer (PMIDs:16287072, 21195604, 20618354, 27705013, 27829682). Functional studies report contradictory results. In vitro studies showed that the protein with this variant had slightly decreased glycosylase activity; however, it had comparable activity in comparison to wild-type protein in a functional complementation assay in E. coli (PMIDs: 20848659, 25820570). Due to these contrasting evidences, the clinical significance of the p.Ile223Val change remains unknown at this time.

In the published literature, this variant has been reported in individuals with classical or atypical adenomatous polyposis, colorectal cancer, prostate cancer, pancreatic cancer, or breast cancer (PMIDs: 12606733 (2003), 16287072 (2006), 17949294 (2007), 21195604 (2011), 23383274 (2013), 27829682 (2016), 27705013 (2016), 27153395 (2016), 29368341 (2018), and 32255556 (2020)). Experimental studies of this variants effects on MUTYH protein function have reported conflicting results (PMIDs: 20848659 (2010) and 25820570 (2015)). Analysis of this variant using bioinformatics tools for the prediction of the effect of amino acid changes on protein structure and function yielded predictions that this variant is disease causing and damaging. Taking into account the available information, we are unable to determine the clinical significance of this variant

The p.Ile223Val variant in MUTYH has been reported in 4 individuals with adenoma tous polyposis as well as in 3 affected relatives from 1 family (Sieber 2003, Ru ssell 2006, Morak 2010, Morak 2011). However, no second MUTYH variant was detect ed in any of the affected individuals. This variant has been identified in 43/12 6744 of European chromosomes by the Genome Aggregation Database (gnomAD, http:// gnomad.broadinstitute.org/; dbSNP rs200872702). In vitro functional studies are conflicting with one study suggesting partial impact on protein function and ano ther study suggesting no impact (Goto 2010, Komine 2015). However, these types o f assays may not accurately represent biological function. Additionally, computa tional prediction tools and conservation analysis do not provide strong support for or against an impact to the protein. In summary, the clinical significance o f the p.Ile223Val variant is uncertain.

Variant summary: MUTYH c.667A>G (p. Ile223Val) results in a conservative amino acid change located in the HhH-GPD domain (IPR003265) encoded protein sequence. Three of five in-silico tools predict a damaging effect of the variant on protein function. The variant allele was found at a frequency of 0.00022 in 252250 control chromosomes, predominantly at a frequency of 0.00037 within the Non-Finnish European subpopulation in the gnomAD database. This frequency is not significantly higher than expected for a pathogenic variant in MUTYH causing MUTYH-associated Polyposis (0.00037 vs 0.0046), allowing no conclusion about variant significance. In a comprehensive literature review spanning 16 years (2003-2019), c.667A>G has been widely reported in the literature in sequencing studies of individuals affected with breast cancer, APC-negative polyposis, attenuated polyposis, as a non-reportable incidental finding in settings of WES, in compound heterozygosity with another MUTYH variant in a colorectal cancer patient with an autosomal dominant family history, with a medium penetrance MUTYH variant in a colorectal cancer patient (e.g., Hilbers_2013, Sieber_2003, Russell_2006, Olschwang_2007, Morak_2010, Morak_2011, Jurgens_2015, Marabelli_2016, Tung_2015, Lorca_2019, Henn_2019, Yurgelun_2017, Maxwell_2016). These report(s) do not provide unequivocal conclusions about association of the variant with MUTYH-associated Polyposis. At-least one co-occurrence with another pathogenic variant has been reported (BRCA2 c.8537_8538del, p. Glu2846Glyfs*22) in a patient with breast cancer, providing supporting evidence for a benign role (Tung_2015). At least two publications report experimental evidence evaluating an impact on protein function. The most pronounced variant effect results in a moderate impairment of adenine DNA glycosylase activity (Goto_2010) which is contrasted by a normal activity in a functional complementation assay in E. coli (Komine_2015). The following publications have been ascertained in the context of this evaluation (PMID: 26377631, 17161978, 17581577, 20848659, 29368341, 23383274, 25569433, 25820570, 31285513, 27705013, 27153395, 20618354, 21195604, 17949294, 20725929, 16287072, 12606733, 25186627, 28135145). Multiple clinical diagnostic laboratories have submitted clinical-significance assessments for this variant to ClinVar after 2014 without evidence for independent evaluation. All laboratories (n = 21) classified the variant as uncertain significance. Some submitters have cited overlapping evidence utilized in the context of this evaluation. Based on the evidence outlined above, the variant was evaluated to retain its classification as uncertain significance.

This missense variant replaces isoleucine with valine at codon 223 of the MUTYH protein. Computational prediction is inconclusive regarding the impact of this variant on protein structure and function (internally defined REVEL score threshold 0.5 < inconclusive < 0.7, PMID: 27666373). Functional studies have shown that this variant causes a mild reduction in MUTYH activity in an enzymatic assay (PMID: 20848659), but could complement the functional deficiency of MutY-null bacteria (PMID: 25820570). This variant has been reported in individuals affected with adenomatous polyposis, colorectal adenomas, colon cancer, breast and/or ovarian cancer, thyroid cancer, or prostate cancer (PMID: 16287072, 12606733, 17949294, 21195604, 20618354, 27705013, 29368341, 30333958, 30680046, 31285513, Oliveira et al. 2021). All reported probands were heterozygous for this variant. This variant has also been identified in 57/282728 chromosomes in the general population by the Genome Aggregation Database (gnomAD) and in a healthy control (PMID: 32980694). The available evidence is insufficient to determine the role of this variant in disease conclusively. Therefore, this variant is classified as a Variant of Uncertain Significance.

Published functional studies are inconclusive: partially reduced glycosylase activity, but mutation rates comparable to wild type (PMID: 20848659, 25820570); Observed in individuals with multiple polyps and/or colorectal cancer, including one who also carried a pathogenic MUTYH variant (phase unknown), and also in individuals with breast or pancreatic cancer (PMID: 16287072, 21195604, 27705013, 27829682, 30680046, 31285513, 31214250, 32255556); In silico analysis indicates that this missense variant does not alter protein structure/function; Also known as Ile195Val or Ile209Val; This variant is associated with the following publications: (PMID: 20848659, 17949294, 19725997, 21235684, 27153395, 30333958, 25820570, 21195604, 16287072, 25569433, 12606733, 23507534, 17161978, 20618354, 23383274, 27829682, 27705013, 29368341, 28492532, 17581577, 31159747, 31214250, 31277343, 30680046, 31285513, 32255556, 20725929, 26377631, 25186627, 28199314, 35264596, 34326862, 35534704, 35980532, 27443514, 32980694)

The MUTYH c.667A>G (p.Ile223Val) missense change has a maximum subpopulation frequency of 0.05% in gnomAD v2.1.1 (https://gnomad.broadinstitute.org/variant/1-45798269-T-C). Both in silico tools and functional assays are not in agreement about the effect of this variant on protein function. A complementation assay evaluating functional deficiency in E.coli demonstrated that glycosylase repair activity was comparable to the wildtype (PMID: 25820570), whereas another study showed a moderate impairment of adenine DNA glycosylase activity (PMID: 20848659). This variant has been reported in individuals with adenomatous polyposis (PMID: 12606733, 16287072, 30680046 ), colorectal cancer (PMID: 21195604, 20618354, 31285513), colorectal adenomas (PMID: 17949294), prostate cancer (PMID: 29368341), breast and/or ovarian cancer (PMID: 30333958), and found to co-occur with a pathogenic BRCA2 mutation (p.Glu2846Glyfs*22) in an individual with breast cancer (PMID: 25186627). This variant has been reported in a 60 year old male with >100 colorectal polyps with colorectal cancer who was compound heterozygous for this variant and the pathogenic p.Tyr179Cys (PM3; PMID: 27705013). This variant is also known as p.Ile195Val and p.Ile209Val in the literature. In summary, this variant meets criteria to be classified as of uncertain significance based on the ACMG/AMP criteria: PM3

This sequence change replaces isoleucine, which is neutral and non-polar, with valine, which is neutral and non-polar, at codon 223 of the MUTYH protein (p.Ile223Val). This variant is present in population databases (rs200872702, gnomAD 0.03%). This missense change has been observed in individual(s) with colorectal polyposis, colon cancer, breast cancer, prostate cancer, and individuals undergoing hereditary cancer predisposition testing (PMID: 12606733, 16287072, 17949294, 20618354, 21195604, 23383274, 27705013, 29368341, 31159747, 31214250, 31285513). ClinVar contains an entry for this variant (Variation ID: 127847). Algorithms developed to predict the effect of missense changes on protein structure and function are either unavailable or do not agree on the potential impact of this missense change (SIFT: "Tolerated"; PolyPhen-2: "Possibly Damaging"; Align-GVGD: "Class C0"). Experimental studies are conflicting or provide insufficient evidence to determine the effect of this variant on MUTYH function (PMID: 20848659, 25820570). 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 missense variant replaces isoleucine with valine at codon 223 of the MUTYH protein. Computational prediction is inconclusive regarding the impact of this variant on protein structure and function (internally defined REVEL score threshold 0.5 < inconclusive < 0.7, PMID: 27666373). Functional studies have shown that this variant causes a mild reduction in MUTYH activity in an enzymatic assay (PMID: 20848659), but could complement the functional deficiency of MutY-null bacteria (PMID: 25820570). This variant has been reported in individuals affected with adenomatous polyposis, colorectal adenomas, colon cancer, breast and/or ovarian cancer, thyroid cancer, or prostate cancer (PMID: 16287072, 12606733, 17949294, 21195604, 20618354, 27705013, 29368341, 30333958, 30680046, 31285513, Oliveira et al. 2021). All reported probands were heterozygous for this variant. This variant has also been identified in 57/282728 chromosomes in the general population by the Genome Aggregation Database (gnomAD) and in a healthy control (PMID: 32980694). The available 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 p.I223V variant (also known as c.667A>G), located in coding exon 8 of the MUTYH gene, results from an A to G substitution at nucleotide position 667. The isoleucine at codon 223 is replaced by valine, an amino acid with highly similar properties. This alteration has been described as heterozygous in individuals with polyposis but without second MUTYH or APC alterations (Sieber OM et al. N Engl J Med. 2003 Feb;348:791-9; Olschwang S et al. Genet. Test. 2007;11:315-20; Lorca V et al. Sci Rep. 2019 07;9:9814; Morak M et al. Clin Genet. 2010 Oct;78:353-63). A 60-year-old Italian patient with polyposis and colorectal cancer was found to have this variant in conjunction with the p.Tyr179Cys MUTYH founder mutation, although phase was not noted (Marabelli M et al. Genet Test Mol Biomarkers. 2016 12;20:777-785). The MUTYH p.I223V alteration has been reported to exhibit slightly lower glycosylation activity (66.9%) compared to wildtype (100%) and has been described as partially defective (Goto M et al. Hum Mutat. 2010 Nov;31:E1861-74; Komine K et al. Hum Mutat. 2015 Jul;36:704-11). Of note, this alteration is also designated as p.I209V (c.625A>G) and p.I195V in published literature. This amino acid position is well conserved in available vertebrate species. In addition, the in silico prediction for this alteration is inconclusive. Since supporting evidence is limited at this time, the clinical significance of this alteration remains unclear.

The MUTYH p.Ile223Val variant was identified in 3 of 878 proband chromosomes (frequency: 0.003) from individuals or families with colorectal adenomas, FAP, or classic polyposis and was not identified in 214 control chromosomes from healthy individuals (Oliver 2003, Olschwang 2007, Russell 2006). The variant was also identified in dbSNP (ID: rs200872702) as "With Uncertain significance allele", ClinVar (classified as uncertain significance by Invitae, Ambry Genetics, GeneDx and nine other submitters), and in UMD-LSDB (1x as unclassified variant). The variant was identified in control databases in 55 of 277080 chromosomes at a frequency of 0.0003 increasing the likelihood this could be a low frequency benign variant (Genome Aggregation Database Feb 27, 2017). The variant was observed in the following populations: African in 1 of 23992 chromosomes (freq: 0.00004), Other in 4 of 6462 chromosomes (freq: 0.0006), Latino in 4 of 34418 chromosomes (freq: 0.0001), European in 43 of 126640 chromosomes (freq: 0.0003), Ashkenazi Jewish in 1 of 10152 chromosomes (freq: 0.0001), South Asian in 2 of 30782 chromosomes (freq: 0.00007), while the variant was not observed in the East Asian, and Finnish populations. To understand the functional consequences of this variant, 47 MUTYH gene variants were generated via site-directed mutagenesis, expressed in MutY - disrupted Escherichia coli, and assessed for their ability to complement the functional deficiency in the E. coli by monitoring spontaneous mutation rates (Komine, 2015). The p.Ile223Val variant retained function but was only partially active in the glycosylase assay. In addition, Morak et al. describe evidence for digenic inheritance in hereditary colorectal cancer with mutations in the base excision repair genes. In a single case, a germline paternal mutation in OGG1, in combination with a maternal MUTYH p.Ile223Val mutation was identified in a female patient with advanced synchronous colon cancer and adenomas at the age of 36 years. This could point towards digenic inheritance for colorectal cancer (CRC) predisposition (Morak 2011), although this remains a single case. The p.Ile223 residue is conserved in mammals and computational analyses (PolyPhen-2, SIFT, AlignGVGD, BLOSUM, MutationTaster) provide inconsistent predictions regarding the impact to the protein; this information is not very predictive of pathogenicity. The variant occurs outside of the splicing consensus sequence and in silico or computational prediction software programs (SpliceSiteFinder, MaxEntScan, NNSPLICE, GeneSplicer) do not predict a difference in splicing. 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.