A functional assay–based procedure to classify mismatch repair gene variants in Lynch syndrome

Drost, Mark; Tiersma, Yvonne; Thompson, Bryony A.; Frederiksen, Jane H.; Keijzers, Guido; Glubb, Dylan M.; Kathe, Scott D.; Osinga, Jan; Westers, Helga; Pappas, Lisa M.; Boucher, Kenneth M.; Molenkamp, Siska; Zonneveld, José B. M.; Asperen, Christi J. van; Goldgar, David E.; Wallace, Susan S.; Sijmons, Rolf H.; Spurdle, Amanda B.; Rasmussen, Lene Juel; Greenblatt, Marc S.; Wind, Niels de; Tavtigian, Sean V.

doi:10.1038/s41436-018-0372-2

Cited by 40 publications

(59 citation statements)

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“…We acknowledge that variant classification is extremely important to patient medical management and that biochemical and structural (Drost et al, 2018). For the purposes of understanding LS and MMR variants, we believe that a combination of clinical interpretation, biochemical techniques, and structural methods such as those presented here, can be effectively utilized to study additional VUSs within the MMR genes.…”

Section: Discussionmentioning

confidence: 94%

“…We acknowledge that variant classification is extremely important to patient medical management and that biochemical and structural analyses, while expensive and time consuming, are necessary to aid in the variant interpretation process. Therefore, streamlining assays or new high‐throughput assay development would greatly assist in this process, making classification of variants on a large scale more feasible (Drost et al., ). For the purposes of understanding LS and MMR variants, we believe that a combination of clinical interpretation, biochemical techniques, and structural methods such as those presented here, can be effectively utilized to study additional VUSs within the MMR genes.…”

Section: Discussionmentioning

confidence: 99%

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Biochemical and structural characterization of two variants of uncertain significance in the PMS2 gene

2019

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Lynch syndrome (LS) is an autosomal dominant inherited disorder that is associated with an increased predisposition to certain cancers caused by loss‐of‐function mutations in one of four DNA mismatch repair (MMR) genes (MLH1, MSH2, MSH6, or PMS2). The diagnosis of LS is often challenged by the identification of missense mutations where the functional effects are not known. These are termed variants of uncertain significance (VUSs) and account for 20%–30% of noncoding and missense mutations. VUSs cause ambiguity during clinical diagnosis and hinder implementation of appropriate medical management. In the current study, we focus on the functional and biological consequences of two nonsynonymous VUSs in PMS2. These variants, c.620G>A and c.123_131delGTTAGTAGA, result in the alteration of glycine 207 to glutamate (p.Gly207Glu) and the deletion of amino acid residues 42–44 (p.Leu42_Glu44del), respectively. While the PMS2 p.Gly207Glu variant retains in vitro MMR and ATPase activities, PMS2 p.Leu42_Glu44del appears to lack such capabilities. Structural and biophysical characterization using circular dichroism, small‐angle X‐ray scattering, and X‐ray crystallography of the N‐terminal domain of the PMS2 variants indicate that the p.Gly207Glu variant is properly folded similar to the wild‐type enzyme, whereas p.Leu42_Glu44del is disordered and prone to aggregation.

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Section: Discussionmentioning

confidence: 94%

Section: Discussionmentioning

confidence: 99%

Biochemical and structural characterization of two variants of uncertain significance in the PMS2 gene

2019

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“…We sought to validate these pooled measurements by comparison to traditional, low-throughput functional studies. We collated a set of 184 MSH2 missense variants previously characterized as functionally deleterious or neutral by individual cell-based 15,19,44,[47][48][49][50][51] and/or biochemical assays 52,53 , discarding four variants where multiple studies disagreed and eight predicted to impact splicing, an effect not measured by this approach (SpliceAI 31 score>0.2; Tables S4-5). Our LOF scores agreed with these earlier reports for the great majority of variants covered (158/165, 95.8%; Figure 3A).…”

Section: Pooled Measurements Recapitulate Existing Variant Interpretamentioning

confidence: 99%

Massively parallel functional testing ofMSH2missense variants conferring Lynch Syndrome risk

Jia

Burugula

Chen

et al. 2020

Preprint

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Introduction 3The lack of functional evidence for the majority of missense variants limits their clinical interpretability, 4 and poses a key barrier to the broad utility of carrier screening. In Lynch Syndrome (LS), one of the most 5 highly prevalent cancer syndromes, nearly 90% of clinically observed missense variants are deemed 6 'variants of uncertain significance' (VUS). To systematically resolve their functional status, we performed 7 a massively parallel screen in human cells to identify loss-of-function missense variants in the key DNA 8 mismatch repair factor MSH2. The resulting sequence-function map is substantially complete, covering 9 94% of the 17,746 possible variants, and is highly concordant (≥96%) with existing functional data and 10 expert clinicians' interpretations. The large majority (89%) of missense variants were functionally neutral, 11 perhaps unexpectedly in view of its evolutionary conservation. These data provide ready-to-use 12 functional evidence to resolve the ~1,300 extant missense VUSs in MSH2, and may facilitate the 13 prospective classification of newly discovered variants in the clinic. 14 15 Main text 16 17 Lynch Syndrome (LS, OMIM:120435), the first discovered familial cancer syndrome 1 , confers 18 predisposition to colorectal and endometrial cancers due to the loss of DNA mismatch repair (MMR) and 19 resulting mutagenic burden. Most affected individuals inherit a single loss-of-function variant in one of 20 four MMR factors (MSH2, MLH1, 21 MSH6, PMS2), followed by a somatic 22 'second hit' inactivating the remaining 23 functional copy. Due to high 24 population prevalence (≥1:300) 2,3 , 25 clear genetic etiology, and potential 26 for prevention through intensified 27 surveillance, MMR gene variants are 28 considered clinically actionable 4 . 29 30 Missense changes together comprise 31 20-30% of LS variants 5 , and are 32 particularly challenging to interpret: 33 their functional impacts may range 34 from minimal to profound, most are 35 individually rare, and they exhibit 36 incomplete penetrance 6 . 37 Consequently, the overwhelming 38 majority of LS gene missense variants 39 listed in ClinVar (4,762/5,473, 87.0%) 40 are deemed 'variants of uncertain 41 significance', or VUS 42( Supplementary Fig.1), and cannot 43 be used to guide diagnosis. 45To address these challenges, we 46 performed a deep mutational scan 7 to 47 systematically measure the functional 48 impact of missense variants in the 49 major Lynch Syndrome gene MSH2. 50We established a human cell system 51 to model MSH2 variant function using 52 the mismatch repair proficient 8 cell 53 line HAP1 (Fig. 1a,c). First, to disrupt MMR, we derived clonal MSH2 knockout cells bearing a 19-bp frameshift deletion in MSH2 exon 6, and 55 verified that it lacked detectable MSH2 protein expression ( Supplementary Fig. 2). To restore MMR, we 56 stably reintroduced into cells either wild-type MSH2 cDNA (KO+WT) or a pathogenic founder allele 57 (KO+A636P) on inducible lentiviral constructs. After inducing expression with doxycycl...

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“…An important factor regarding data sets that has emerged recently is how predictors should not be evaluated on variants or proteins that were used to train their prediction models. This circularity could result in predictive values that are artificially inflated [24, 25], and could occur with either likely pathogenic or likely benign variants. We suggest that not enough attention has been assigned to an additional important factor, the likelihood that missense substitution is a major mechanism of pathogenicity for a gene.…”

Section: Discussionmentioning

confidence: 99%

Curated Multiple Sequence Alignment for the Adenomatous Polyposis Coli (APC) Gene and Accuracy of In Silico Pathogenicity Predictions

Karabachev

Martini

Hermel

et al. 2019

Preprint

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22Computational algorithms are often used to assess pathogenicity of Variants of Uncertain 23 Significance (VUS) that are found in disease-associated genes. Most computational methods 24 include analysis of protein multiple sequence alignments (PMSA), assessing interspecies 25 variation. Careful validation of PMSA-based methods has been done for relatively few genes, 26 partially because creation of curated PMSAs is labor-intensive. We assessed how PMSA-based 27 computational tools predict the effects of the missense changes in the APC gene, in which 28 pathogenic variants cause Familial Adenomatous Polyposis. Most Pathogenic or Likely 29 Pathogenic APC variants are protein-truncating changes. However, public databases now 30 contain thousands of variants reported as missense. We created a curated APC PMSA that 31 contained >3 substitutions/site, which is large enough for statistically robust in silico analysis. 32The creation of the PMSA was not easily automated, requiring significant querying and 33 computational analysis of protein and genome sequences. Of 1924 missense APC variants in 34 the NCBI ClinVar database, 1800 (93.5%) are reported as VUS. All but two missense variants 35 listed as P/LP occur at canonical splice or Exonic Splice Enhancer sites. Pathogenicity 36 predictions by five computational tools (Align-GVGD, SIFT, PolyPhen2, MAPP, REVEL) differed 37 widely in their predictions of Pathogenic/Likely Pathogenic (range 17.5-75.0%) and 38 Benign/Likely Benign (range 25.0-82.5%) for APC missense variants in ClinVar. When applied 39 to 21 missense variants reported in ClinVar as Benign, the five methods ranged in accuracy 40 from 76.2-100%. Computational PMSA-based methods can be an excellent classifier for 41 variants of some hereditary cancer genes. However, there may be characteristics of the APC 42 gene and protein that confound the results of in silico algorithms. A systematic study of these 43 features could greatly improve the automation of alignment-based techniques and the use of44 predictive algorithms in hereditary cancer genes. 46Author Summary 47A critical problem in clinical genetics today is interpreting whether a genetic variant is benign or 48 causes disease (pathogenic). Some of the hardest variants to interpret are those that change 49 one amino acid for another in a protein sequence (a "missense variant"). Various computer 50 programs are often used to predict whether mutations in disease-associated genes likely cause 51 disease. Most computer programs involve studying how the gene has changed during 52 evolution, comparing the protein sequences of different species by aligning them with each 53 other. Variants in amino acids that have not tolerated mutation during evolution are usually 54 predicted to be pathogenic, and variants in amino acids that have tolerated variation are usually 55 predicted to be benign. High quality alignments are necessary to make accurate predictions. 56However, creating high quality alignments is difficult, not easily automated, and requires 57 significan...

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A functional assay–based procedure to classify mismatch repair gene variants in Lynch syndrome

Cited by 40 publications

References 32 publications

Biochemical and structural characterization of two variants of uncertain significance in the PMS2 gene

Biochemical and structural characterization of two variants of uncertain significance in the PMS2 gene

Massively parallel functional testing ofMSH2missense variants conferring Lynch Syndrome risk

Curated Multiple Sequence Alignment for the Adenomatous Polyposis Coli (APC) Gene and Accuracy of In Silico Pathogenicity Predictions

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