Genetic screens to identify pathogenic gene variants in the common cancer predisposition Lynch syndrome

Drost, Mark; Lützen, Anne; Hees, Sandrine van; Ferreira, Daniel; Calléja, Fabienne M.G.R.; Zonneveld, José B. M.; Nielsen, Finn Cilius; Rasmussen, Lene Juel; Wind, Niels de

doi:10.1073/pnas.1220537110

Cited by 24 publications

(26 citation statements)

References 29 publications

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“…The presence of the planned mutation in the 6TG-resistant cells was confirmed by sequence analysis. Resistance to 6TG has been used previously to select for MMR deficiency in cell cultures that were randomly mutagenized with ethylnitrosourea yielding a catalog of deleterious codon substitutions (29). By using oligo targeting, we can interrogate variants of interest directly.…”

Section: Discussionmentioning

confidence: 99%

Oligonucleotide-directed mutagenesis screen to identify pathogenic Lynch syndrome-associated MSH2 DNA mismatch repair gene variants

Houlleberghs

Dekker

Lantermans

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Single-stranded DNA oligonucleotides can achieve targeted base-pair substitution with modest efficiency but high precision. We show that "oligo targeting" can be used effectively to study missense mutations in DNA mismatch repair (MMR) genes. Inherited inactivating mutations in DNA MMR genes are causative for the cancer predisposition Lynch syndrome (LS). Although overtly deleterious mutations in MMR genes can clearly be ascribed as the cause of LS, the functional implications of missense mutations are often unclear. We developed a genetic screen to determine the pathogenicity of these variants of uncertain significance (VUS), focusing on mutator S homolog 2 (MSH2). VUS were introduced into the endogenous Msh2 gene of mouse embryonic stem cells by oligo targeting. Subsequent selection for MMR-deficient cells using the guanine analog 6-thioguanine allowed the detection of MMR-abrogating VUS. The screen was able to distinguish weak and strong pathogenic variants from polymorphisms and was used to investigate 59 Msh2 VUS. Nineteen of the 59 VUS were identified as pathogenic. Functional assays revealed that 14 of the 19 detected variants fully abrogated MMR activity and that five of the detected variants attenuated MMR activity. Implementation of the screen in clinical practice allows proper counseling of mutation carriers and treatment of their tumors.Lynch syndrome | DNA mismatch repair | MSH2 | variants of uncertain significance | site-directed mutagenesis T he DNA mismatch repair (MMR) system is essential for genome fidelity: It corrects mismatches that may arise during erroneous DNA replication and induces apoptosis if adducts caused by certain DNA-damaging agents cannot be repaired. Newly replicated DNA is scanned for base-base mispairings and loops of unpaired bases by heterodimers composed of MMR proteins mutator S homolog 2 and 6 (MSH2/MSH6) or mutator S homolog 2 and 3 (MSH2/MSH3), respectively. Upon encountering a mismatch, the MSH heterodimers recruit another heterodimer composed of mutator L homolog 1 and postmeiotic segregation increased 2 (MLH1/PMS2) to coordinate downstream repair events (1, 2). Exposure to certain DNA-damaging agents, such as methylating agents and the nucleotide analog 6-thioguanine (6TG), creates lesions in the genome that give rise to mismatches when replicated (3, 4). The DNA MMR system recognizes these mismatches and induces cell death to remove them. Several models propose how DNA MMR activates cell death. One model suggests that DNA MMR recognizes the mismatch and repetitively removes the incorporated nucleotide rather than the lesion itself, creating a cycle of futile repair which ultimately leads to DNA breakage and cell death (3). Another possibility is that MSH2/MSH6 and MLH1/PMS2 bind at the site of the damaged base and act as molecular scaffolds that activate downstream DNA damage-response pathways that result in apoptosis (5, 6). In the absence of a functional DNA MMR system, cells have an increased rate of spontaneous mutagenesis and elevated resistance to DNA-methyla...

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Section: Discussionmentioning

confidence: 99%

Oligonucleotide-directed mutagenesis screen to identify pathogenic Lynch syndrome-associated MSH2 DNA mismatch repair gene variants

Houlleberghs

Dekker

Lantermans

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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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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“…S1). Spontaneous mutant frequencies of the Pole-mutant mES cell lines were determined at the Hypoxanthine Phosphorybosyl Transferase (Hprt) gene as described previously (17). Additional details on these experiments can be found in the Supplementary Methods.…”

Section: Generation Of the Cell-based Modelmentioning

confidence: 99%

Adjuvant Treatment for POLE Proofreading Domain–Mutant Cancers: Sensitivity to Radiotherapy, Chemotherapy, and Nucleoside Analogues

Gool

Rayner

Osse

et al. 2018

Clinical Cancer Research

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Pathogenic proofreading domain mutations are found in many malignancies where they are associated with ultramutation and favorable prognosis. The extent to which this prognosis depends on their sensitivity to adjuvant treatment is unknown, as is the optimal therapy for advanced-staged or recurrent-mutant cancers. We examined the recurrence-free survival of women with -mutant andwild-type endometrial cancers (EC) in the observation arm of the randomized PORTEC-1 endometrial cancer trial ( = 245 patients with stage I endometrial cancer for analysis). Sensitivity to radiotherapy and selected chemotherapeutics was compared between -mutant mouse-derived embryonic stem (mES) cells, generated using CRISPR-Cas9 ( mutations D275A/E275A, and cancer-associated P286R, S297F, V411L) and isogenic wild-type cell lines. In the observation arm of the PORTEC-1 trial ( = 245), women with -mutant endometrial cancers ( = 16) had an improved recurrence-free survival (10-year recurrence-free survival 100% vs. 80.1% for wild-type; HR, 0.143; 95% confidence interval, 0.001-0.996; = 0.049). mutations did not increase sensitivity to radiotherapy nor to chemotherapeutics in mES cells. In contrast,-mutant cells displayed significantly increased sensitivity to cytarabine and fludarabine (IC P286R-mutant vs. wild-type: 0.05 vs. 0.17 μmol/L for cytarabine, 4.62 vs. 11.1 μmol/L for fludarabine; < 0.001 for both comparisons). The favorable prognosis of -mutant cancers cannot be explained by increased sensitivity to currently used adjuvant treatments. These results support studies exploring minimization of adjuvant therapy for early-stage-mutant cancers, including endometrial and colorectal cancers. Conversely, mutations result in hypersensitivity to nucleoside analogues, suggesting the use of these compounds as a potentially effective targeted treatment for advanced-stage-mutant cancers. .

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Genetic screens to identify pathogenic gene variants in the common cancer predisposition Lynch syndrome

Cited by 24 publications

References 29 publications

Oligonucleotide-directed mutagenesis screen to identify pathogenic Lynch syndrome-associated MSH2 DNA mismatch repair gene variants

Oligonucleotide-directed mutagenesis screen to identify pathogenic Lynch syndrome-associated MSH2 DNA mismatch repair gene variants

Massively parallel functional testing ofMSH2missense variants conferring Lynch Syndrome risk

Adjuvant Treatment for POLE Proofreading Domain–Mutant Cancers: Sensitivity to Radiotherapy, Chemotherapy, and Nucleoside Analogues

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