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Replication-coupled Gene Editing in Mammalian cells
van Ravesteyn, T.W.
2019
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van Ravesteyn, T. W. (2019). Replication-coupled Gene Editing in Mammalian cells.
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“DNA neither cares nor knows.
DNA just is. And we dance to its music.”
•
Chapter 5
Functional classification of MMR gene mutations identified in
treatment-related colorectal cancers
Thomas van Ravesteyn
aJohan Hoeksel
aLisanne Rigter
bMonique van Leerdam
bHein te Riele
aaDivision of Tumor Biology and Immunology,
bDepartment of Gastroenterology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands
Based on:
Abstract
Cancer survivors who have been treated with radio- or chemotherapy have
an increased risk for developing second primary malignancies. Molecular
characterization of 54 treatment-related colorectal cancers (t-CRC) that
arose in Hodgkin lymphoma survivors led to the identification of thirteen
tumors with microsatellite instability (MSI). The MSI-phenotype is known
to result from loss of DNA mismatch repair (MMR) activity and several
mechanisms are known to underlie MMR defects. While three MSI t-CRCs
were explained by MLH1 promoter methylation, DNA sequencing of tumor
tissue led to the identification of various somatic MMR gene mutations
in eight MSI t-CRCs. As the majority of the identified subtle variants
haven’t been classified before, we sought to functionally characterize these
by oligonucleotide-directed mutation screening in mouse embryonic
stem cells. Among the twelve variants of uncertain significance tested, we
classified seven variants as deleterious. In combination with analyses for
loss of heterozygosity (LOH) this led to conclusive explanation for the
MSI phenotype of seven out of ten MSI t-CRCs without MLH1 promoter
methylation. We describe the three different combinations of two somatic
hits that caused MMR deficiency and thereby provide insight into the
etiology of t-CRC with a MSI phenotype.
Introduction
Under normal circumstances the DNA mismatch repair (MMR) pathway restores the errors that arise during DNA replication and decreases the rate of spontaneous mutagenesis by 2 to 3 orders of magnitude. The MMR pathway is initiated by binding of heteromeric protein complex MutSα or MutSβ to base-base mismatches or insertion/deletion loops (IDLs). Mismatches and IDLs with one or two extrahelical nucleotides are recognized by MutSα which consist of MutS homologue 2 (MSH2) and MSH6, while MutSβ (MSH2/MSH3) associates with IDLs with up to five extrahelical nucleotides. After binding to the heteroduplex, the exchange of ADP for ATP results in a conformational change that leads to the formation of a DNA sliding clamp. Then a heterodimer consisting of post-meiotic segregation 2 (PMS2) and MutL homologue 1 (MLH1) is recruited and creates nicks into the DNA near the lesion. This provides entry points for 5’-3’ Exonuclease 1 (EXO1) which degrades the nascent DNA strand that contains the falsely incorporated base. DNA polymerases resynthesize the degraded DNA error-free and after ligation by DNA ligase 1 the DNA duplex is restored (4, 5).
Due to the critical role of the MMR pathway in genome maintenance, it is no surprise that a subset of tumors has lost MMR activity. Several diagnostic molecular methods can be used to determine MMR status in tumor material. The characterization of simple nucleotide repeats, named microsatellites, often provides the first indication that MMR is perturbed. Due to the repetitive sequence of these genomic elements, replicative DNA polymerases are more likely to slip and thereby easily incorporate an incorrect number of nucleotides during DNA replication. While in the presence of MMR these alterations are efficiently repaired, the absence of MMR leads to expansion or contraction of microsatellites. Microsatellite instability (MSI) is detected by PCR-based assays that interrogate the length of these markers and thus provides a functional read-out for MMR activity (6–8). In addition to MSI analysis, immuno-histochemical (IHC) staining of tumor material for MSH2, MSH6, MLH1 and PMS2 is often used to determine which MMR gene is affected (9).
In addition to the relevance of MMR for several types of hereditary cancer, also ~15% of sporadic CRCs and ECs show loss of MMR activity (17, 18). One of the mechanisms that causes MMR deficiency involves cytosine methylation of the MLH1 promoter region which results in loss of MLH1 expression (19, 20). Alternatively, somatic aberrations in key MMR genes have been identified to cause MMR deficiency in a small subset of sporadic cancers (21–25). Most recently, a fourth source of MMR-deficient tumors has been identified: CRCs as a second primary malignancy in Hodgkin lymphoma (HL) patients who have been successfully treated with radiotherapy and/or procarbazine (1). Although radio- and chemotherapy are known to be mutagenic and have been associated with an increased risk for developing CRC in treated Hodgkin lymphoma patients (26–29), the mechanism by which radio- and chemotherapy-related CRC (t-CRC) develops is poorly understood.
In this work we used oligonucleotide-directed mutation screening (ODMS) (32, 33) to classify six somatic VUS identified in MSH2, two in MSH6 and four in MLH1. The VUS under study were generated in mouse embryonic stem cells (mESCs) by gene editing with LNA-modified single-stranded oligodeoxyribonucleotides (LMOs) and were subsequently characterized based on resistance to the methylating compound 6-thioguanine (6TG). In combination with LOH analysis, ODMS screening provided conclusive explanation for the MMR defect in five out of six otherwise unexplained MSI t-CRCs.
Methods and Materials
Oligonucleotide-directed mutation screening
For the characterization of VUS identified in MSH2, MSH6 or MLH1 by ODMS we utilized mESCs that were made hemizygous for the gene of interest. Msh2+pur/∆ and Mlh1+pur/∆ cell lines were previously generated by removal of one allele by Cre-lox recombination and the introduction of a non-disruptive puromycin (pur) cassette at the remaining functional allele (33, 34). The Msh6+/- cell line was generated before through insertion of a pur cassette at exon 4 in one of the Msh6 alleles (35).
was analyzed by Sanger sequencing to verify the introduction of the mutation under investigation at the targeted exon/intron. For MLH1 D12G clone D10 we isolated total RNA (High Pure RNA isolation kit, Roche) and used SuperScript II reverse transcriptase (Thermofisher Scientific) to prepare cDNA. PCR and Sanger sequencing was used to characterize Mlh1 mRNA of MLH1 D12G clone D10.
Protein expression analysis by western blot
Western blot was performed as described before (36). In short, whole protein lysates were isolated from 6TGR clones using RIPA buffer with complete protease inhibitor (Roche) and were kept at -80oC. The protein concentration was determined using a BCA protein assay (Pierce) and absorbance was measured at 562 nm on a Tecan infinite M200 plate reader (Tecan). Protein lysates were separated on a 3-8% Tris-Acetate (MSH2/MSH6) or 4-12% Bis-Tris (MLH1) Nupage denaturing gel (Invitrogen). We used rabbit polyclonal primary antibodies against MSH2 (1:500) (37), MSH6 (1:500) (35) and MLH1 (1:1000; SC-581, Santa Cruz Biotechnology) and mouse polyclonal primary antibody against γ-tubulin (1:1000; GTU-88, Sigma-Aldrich). For detection we used goat anti-rabbit and goat anti-mouse and IR dye 800 CW labeled IgG secondary antibodies, blots were scanned on an Odyssey scanner (Li-Cor Biosciences). Protein expression was quantified and normalized against γ-tubulin expression using Image Studio Lite v5.2 (Li-Cor Biosciences).
Results
Functional screening of MMR VUS identified in MSI t-CRCs
planned mutation and its detection in 6TGR colonies by Sanger sequencing indicates that the VUS under study is deleterious to MMR function and thus pathogenic. If variants remain undetected, this indicates their neutral phenotype.
While lack of conservation for MSH6 T213A from human to mice precluded assessment of this variant by ODMS, we were able to design sense and antisense LMOs for each of the remaining twelve VUS in either MSH2, MSH6 or MLH1 (table 2). These LMOs were subsequently used for LMO-directed gene targeting in Msh2+pur/∆, Mlh1+pur/∆ or Msh6+/- mESCs, respectively. To increase the sensitivity of the assay we targeted cells with sense and antisense LMOs in separate screenings. In addition, for optimal targeting efficiencies we supplemented some of the antisense LMOs with a 5’-terminus acridine (Acr) modification (Chapter 2) (39, 40). After 6TG selection we picked clones and isolated their genomic DNA. Since we studied the MSH6 variants in a mESC line that is prone to LOH under pressure of 6TG selection, the presence of both the functional and inactivated Msh6 allele was confirmed by an additional PCR (35). Introduction of the planned mutation was thereafter validated by Sanger sequencing in clones that retained both Msh6 alleles. The presence of a pur-marker at the functional allele of Msh2+pur/∆ and Mlh1+pur/∆ mESCs enabled counter-selection for LOH events in these cell lines and enabled direct genetic analysis of picked clones by Sanger sequencing.
modified cells loss of heterozygosity
6TG selection
Sanger sequencing of 6TGR clones
Msh2+pur/∆ / Mlh1+pur/∆ mESCs MSH2 / MLH1 screening MSH6 screening
LMO transfection
Assess 6TGR clones for loss of heterozygosity by PCR
Msh6+/- mESCs
unmodified cells
Figure 1. Schematic overview of MMR variant classification by ODMS in mESCs. For functional assessment of MSH6, MSH2 and MLH1 gene variants, hemizygous mESCs are transfected with LMOs to introduce the mutation of interest. Thereafter, targeted cells are exposed to 6TG to select for clones that have become MMR-deficient. 6TGR clones are picked, expanded and their gDNA is isolated. Presence of the modification is validated by Sanger sequencing. Since Msh6+/-mESCs are prone to LOH under 6TG selection, in contrast to
Msh2+pur/Δ and Mlh1+pur/Δ mESCs, a PCR approach is used to confirm the presence of both
MSH2 VUS T321I L380S L380Ffs*32 E580Kfs*10 Q645X c.2005+1G>A MSH6 VUS H366Y G1132E MLH1 VUS D12G c.381-1G>C P405S c.1421+6G>A LMO sense antisense sense antisense sense sense sense sense sense antisense sense sense antisense sense antisense sense antisense sense antisense 0/11 0/14 0/10 0/33 28/32 17/24 30/34 36/36 19/24 7/8 8/15 0/18 1/13 0/9 22/24 0/10 0/7 0/9 0/18
Fraction modified clones
Figure 2. ODMS results for unclassified MSH2, MSH6 and MLH1 variants identified in t-CRCs. MMR variants were introduced in hemizygous mESCs using sense and/or antisense LMOs. The fraction of successfully modified clones among total number of analyzed 6TGR clones is indicated schematically and numerically.
Molecular characterization of MLH1 D12G clone D10
C
D
A
B
wild-type MSH6 H366Y (H367Y) wild-type MLH1 c.381-1G>C wild-type MSH2 E580Kfs*10wild-type MSH2 Q645X wild-type MSH2 2005+1G>A
wild-type MSH6 G1132E (G1134E) wild-type MSH2 L380Ffs*32 modified
E
F
G
G G A T T T G C T T C G C D L L R modified modified modified modified modified modified G A A G T T C A A G A T G E V Q D G A A G T T T A A G A T G E V X _ T T A C T G G T A A A A A I T intronic sequence T T A C T G A T A A A A A I T intronic sequence T G A C T G G A C C A A A V T G P N T G A C T G A A C C A A A V T E P N T G T T A A A G A A A T T V K E I T G G T A T C A C G A A A W Y H E T G G T A T T A C G A A AC W Y Y E T G A C T A G A G C A A G intronic sequence A S T G A C T A C A G C A A G intronic sequence A S G G A T T T C T T C G C C D F F A del G T G T T A A G A A A T T G V K K L del A GA
C
B
wild-type MLH1 D12G - clone D10 modified G T C T G G A C G A G A C R L D E T G T C T G G G C G A G A C R L G E T wild-type mutated - R691Q C C A T T C G G A A G C A S I R K Q C C A T T C A G A A G C A S I Q K Q R L A T E K Q Y I L Mlh1 exon16 17 18 19 regular splicing observed splicing c.2072G>A C G A C T G G C C A C T G A G A A G C A G T A T A T A C T Gexon 17 exon 18 del 72 bpD
Figure 4. Molecular characterization of 6TGR clone MLH1 D12G D10. (A) Sanger
Western blot analysis of identified pathogenic MMR variants
After detection of MMR VUS among analyzed 6TGR clones, we assessed MMR protein expression by western blot from two clones for each of the confirmed variants. As MSH2 and MSH6 form a stable heterodimer, their protein stability is heavily dependent on each other. In accordance with their pathogenicity and with the patient data, we observed strongly reduced MSH2 and MSH6 protein levels for all deleterious MSH2 variants (Fig. 5A). For MSH6 variants H367Y and G1134E we also observed strongly reduced protein levels for both of the MSH2/MSH6 heterodimer partners (Fig. 5B). In line with tumor IHC staining, both of the mESC clones modified with intronic variant MLH1 c.381-G>C demonstrated near-complete absence of MLH1 protein expression (Fig. 5C).
Explanation of MSI phenotype in t-CRCs
The obtained ODMS screening results (table 1, MMR variant classification) provided clarification for the observed MSI-phenotype in five out of six unexplained MSI t-CRCs.
t-CRC case A
The combination of MSH2 L380Ffs*32 with LOH of Chr 2 explained the MMR defect in MSI t-CRC case A. Although this tumor also contained a missense mutation in MLH1, P403S, it is unlikely that it contributed to the MSI phenotype. This variant was not detected by our ODMS screening and was also observed in healthy control tissue from the same patient.
t-CRC case B
The identification of MSH6 G1134E as deleterious in combination with previously characterized pathogenic variant MSH6 T1219I explained the loss of MMR-activity in MSI t-CRC case B. Although G1134E led to reduced protein expression in our mESC experiments, T1219I was previously found to be relatively stable in mESCs (32) and may therefore explain the positive staining for all MMR proteins in the tumor sample.
t-CRC case C
ODMS screening and could thereby explain the loss of MSH2 and MSH6 staining in the tumor. Based on our results MSH2 L380S, and MLH1 D12G were classified as non-pathogenic. Unfortunately we consider the ODMS results for MLH1 c.1409+6G>A as inconclusive. Two targeting attempts with sense and antisense LMOs failed to detect any modified 6TGR clone. Although the bases directly next to the splice donor site at MLH1 exon 12 are conserved from man to mice, the lack of intronic conservation at 4 bp distance precludes definitive conclusions concerning its pathogenicity using ODMS in mESCs (Fig. S1). Similarly, lack of conservation precluded assessment of MSH6 T123A by our approach. To classify these variants reliably, alternative methods should be used.
t-CRC case D
Even though MLH1 c.381-1G>C was the only mutation identified in MSI t-CRC case D, its inferred pathogenicity from ODMS screening in combination with LOH of Chr
A
C
B
MSH6 variants MSH2 variants γ-tubulin MLH1 Mlh1 +pur/Δ S44F c.381-1G>C MLH1 variants Msh2 +pur/Δ Msh2 -/-L380Ffs*32 γ-tubulin MSH6 MSH2 E580Kfs*10 Q645X c.2005+1G>A % MSH2 % MSH6 % MSH6 % MSH2 % MLH1 Msh6 +/-Msh6 -/-H366Y G1134E γ-tubulin MSH6 MSH2 100 100 1 2 2 2 0 1 2 2 1 11 10 14 12 9 16 12 10 7 100 100 201 186 158 189 189 100 7 1 1t-CRC case E
For MSI t-CRC case E two MSH2 mutations were identified. While G322D was described before as benign (30) and was detected in healthy control tissue from the same patient, we characterized the second mutation, E580Kfs*10, by ODMS as pathogenic. The presence of this frameshift mutation in combination with LOH of Chr 2 presumably led to loss of the MSH2/MSH6 heterodimer and provides a rationale for MMR-deficiency in this tumor.
Unexplained MSI t-CRC cases
Unfortunately, we were unable to conclusively explain the loss of MMR activity for MSI t-CRC case F. While two mutations were identified, only MSH6 H367Y was found to disrupt MMR activity by our screening method. Although dominant-negative MMR variants have been described in mammalian cells (41–44), such an effect is not to be expected since MSH6 H367Y caused loss of heterodimer stability (Fig. 4B). As results from LOH analysis remained inconclusive this precluded a convincing explanation for the MSI-phenotype in this tumor. Even though DNA sequencing of the tumors covered most of the coding sequences of the MMR genes (MLH1 100%, MSH2 92%, MSH6 97%, PMS2 79%) (45), it cannot be excluded that there are additional disruptive mutations in exonic or otherwise extra-exonic regions that might explain the MSI phenotype. This may also apply to the unexplained MSI t-CRC cases I and J for which only LOH of Chr2 was identified.
Discussion
Although treatment of malignancies with radio- and/or chemotherapy comprising alkylating agents is known to increase cancer risk, the precise mechanism hasn’t been elucidated. Alkylating agents are known to be a major source of genotoxic lesions, including O6-methylguanine (O6-MeG) whose cytotoxicity is intricately connected to the MMR pathway (46). As replicative DNA polymerases preferentially base pair O6 -MeG with thymidine instead of cytosine, mismatches are formed. Although the MMR pathway recognizes and processes these lesions, the O6-MeG persists in the genome. Futile cycles of MMR repair eventually lead to the induction of cellular apoptosis. As MMR-deficient cells are highly resistant to methylating agents, exposure to these compounds provides a growth advantage to cells with pre-existing MMR defects (47). This mechanism might in part explain the increased incidence of MSI-CRC among cancer survivors who have been treated with alkylating chemotherapy. We aimed to gain more insight in the etiology of MSI t-CRCs in Hodgkin lymphoma survivors who received a combination of radiotherapy and procarbazine through molecular analysis of thirteen MSI t-CRCs.
Whereas MLH1 promoter methylation is accountable for loss of MMR activity in up to 80% of sporadic MSI CRCs (20, 48–51), MLH1 promoter methylation was observed for only 3 out of 13 (23%) MSI t-CRCs. The relative low contribution of epigenetic silencing of MLH1 to MSI t-CRC tumorigenesis in this cohort of HL survivors indicates that distinct mechanisms underlie the MMR defect in most of these t-CRCs. Subsequent sequencing of ten t-CRCs without MLH1 promoter methylation led to the identification of seventeen subtle somatic variants in key MMR genes. Subsequent functional analysis of twelve VUS by ODMS enabled us to classify seven somatic MMR variants as deleterious. In combination with LOH analysis, this led to a conclusive explanation for the MSI phenotype of seven out of ten MSI t-CRCs without MLH1 promoter methylation.
t-CRCs demonstrated LOH for Chr 2 only (MSI t-CRC case I and J), one t-CRC had two somatic mutations of which only one in MSH6 was characterized as deleterious (MSI t-CRC case F).
There are several other studies that analyzed the molecular characteristics of sporadic CRC and EC with a MSI phenotype which could not be explained by MLH1 promoter methylation (24, 25, 45, 52, 53). Sourrouille et al. focused on MSI CRC patients with either loss of MLH1 or MSH2 protein expression and reported the presence of double somatic mutations in 3/18 sporadic CRCs. Although LOH analysis was omitted, the MSI-phenotype was explained for 5/18 cases. Interestingly, one patient was diagnosed with LS due to previously unrecognized germline mosaicism (24).
Four other studies included LOH analysis and were thereby able to explain a larger proportion of sporadic MSI-CRCs and –ECs by their own standards (25, 45, 52, 53). Vargas-Parra et al. also sequenced the MSH2 transcript RNA from patient-derived human blood lymphocytes to rule out that the tumor was caused by a germline mutation that affected splicing (53). Most studies used a clear mutation interpretation scheme based on InSIGHT classification, in silico class prediction and the quality of LOH analyses. Tumors with a class 3 (uncertain) MMR VUS in combination with LOH were considered as ‘probably solved’ or ‘solved’ cases by Mensenkamp et al. and Haraldsdottir et al., respectively. However, a class 3 VUS in combination with LOH wasn’t considered as conclusive by Geurts-Giele et al., exemplifying the inconsistency by which sporadic cases of CRC with MSI are currently explained. The variation by which VUS are dealt with in a diagnostic setting emphasizes the need for functional assays that can differentiate pathogenic from neutral variants.
Although we cannot rule out that a somatic or germline mutation has been missed by gene sequencing analysis of tumor material, alternative mechanisms that suppress MMR activity have been described. For example, it has been demonstrated that overexpression of either microRNA(miR)-21 or miR-155 downregulates expression of human MMR genes and can induce a mutator phenotype and MSI (58, 59). Evaluation of miR expression levels in CRC tumors demonstrated an inverse correlation with MSH2 or MLH1 protein levels. Alternatively, in particular MSH2 was found to be destabilized by somatic deletion of one of four genes upstream in the PKCζ pathway, this caused MMR-deficiency and resistance to methylating agents in leukemia cells (60). Interestingly, deletions for one or more of these four PKCζ pathway genes were also found in 14 out of 104 individuals with sporadic colorectal cancer (60). As shown recently, MLH1 protein can be negatively regulated by Casein kinase II (CK2)-dependent phosphorylation at serine 477 of MLH1 (61). Also CK2 was described to be overexpressed in CRC tissue (62).
By studying MMR in detail it was found that the epigenetic histone mark H3K36me3 is essential for the recruitment of human MSH2/MSH6 at mismatches. Therefore loss of the SET domain-containing histone methyl transferase (HMT), SETD2, can result in a MSI-phenotype as well (63). Out of frame deletions at mononucleotide repeats in SETD2 and other SET domain-containing HMTs have been identified in CRC and gastric cancer with a MSI-phenotype (64), but whether this was cause or consequence of the MMR defect remains to be elucidated. Interestingly, potentially deleterious SETD2 variants were recently detected in the germline of Lynch-like syndrome patients (53). Mutations in SETD2 have been identified in lymphoma and leukemia as well and were shown to impair MMR and provide resistance to chemotherapy (65, 66). Finally, based on mouse cell lines and mouse models, multiple reports have demonstrated forms of haplo-insufficiency for MSH2 and MSH6 (37, 67, 68). Hence, it cannot be excluded that under some circumstances the acquisition of a single deleterious mutation results in (partial) loss of MMR activity, which could drive tumorigenesis.
Funding
This work was supported by grant ALW 822.02.01 from the Dutch Organization for Scientific Research.
Acknowledgements
References
1. Hoeijmakers JH (2001) Genome maintenance mechanisms for preventing cancer. Nature 411(6835):366–
74.
2. Garg P, Burgers PMJ (2005) DNA polymerases that propagate the eukaryotic DNA replication fork. Crit
Rev Biochem Mol Biol 40(2):115–28.
3. Kunkel TA, Erie DA (2005) DNA mismatch repair. Annu Rev Biochem 74:681–710.
4. Jiricny J (2006) The multifaceted mismatch-repair system. Nat Rev Mol Cell Biol 7(5):335–46.
5. Aaltonen LA, Peltomäki P, Leach FS, Sistonen P, Pylkkänen L, Mecklin JP, Järvinen H, Powell SM, Jen
J, Hamilton SR (1993) Clues to the pathogenesis of familial colorectal cancer. Science 260(5109):812–6.
6. Thibodeau SN, Bren G, Schaid D (1993) Microsatellite instability in cancer of the proximal colon.
Science 260(5109):816–9.
7. Ionov Y, Peinado MA, Malkhosyan S, Shibata D, Perucho M (1993) Ubiquitous somatic mutations in
simple repeated sequences reveal a new mechanism for colonic carcinogenesis. Nature 363(6429):558–61.
8. Shia J (2008) Immunohistochemistry versus microsatellite instability testing for screening colorectal
cancer patients at risk for hereditary nonpolyposis colorectal cancer syndrome. Part I. The utility of immunohistochemistry. J Mol Diagn 10(4):293–300.
9. Lynch HT, Snyder CL, Shaw TG, Heinen CD, Hitchins MP (2015) Milestones of Lynch syndrome:
1895–2015. Nat Rev Cancer 15(March):181–194.
10. Hemminki A, Peltomäki P, Mecklin JP, Järvinen H, Salovaara R, Nyström-Lahti M, de la Chapelle A, Aaltonen LA (1994) Loss of the wild type MLH1 gene is a feature of hereditary nonpolyposis colorectal cancer. Nat Genet 8(4):405–10.
11. Vasen HF, Wijnen JT, Menko FH, Kleibeuker JH, Taal BG, Griffioen G, Nagengast FM, Meijers-Heijboer EH, Bertario L, Varesco L, Bisgaard ML, Mohr J, Fodde R, Khan PM (1996) Cancer risk in families with hereditary nonpolyposis colorectal cancer diagnosed by mutation analysis. Gastroenterology 110(4):1020–7.
12. Aarnio M, Sankila R, Pukkala E, Salovaara R, Aaltonen LA, de la Chapelle A, Peltomäki P, Mecklin JP, Järvinen HJ (1999) Cancer risk in mutation carriers of DNA-mismatch-repair genes. Int J cancer 81(2):214–8.
13. Hampel H, Frankel WL, Martin E, Arnold M, Khanduja K, Kuebler P, Nakagawa H, Sotamaa K, Prior TW, Westman J, Panescu J, Fix D, Lockman J, Comeras I, de la Chapelle A (2005) Screening for the Lynch syndrome (hereditary nonpolyposis colorectal cancer). N Engl J Med 352(18):1851–60.
14. Hampel H, Frankel W, Panescu J, Lockman J, Sotamaa K, Fix D, Comeras I, La Jeunesse J, Nakagawa H, Westman JA, Prior TW, Clendenning M, Penzone P, Lombardi J, Dunn P, Cohn DE, Copeland , Eaton L, Fowler J, Lewandowski G, Vaccarello L, Bell J, Reid G, De La Chapelle A (2006) Screening for Lynch syndrome (hereditary nonpolyposis colorectal cancer) among endometrial cancer patients. Cancer
Res 66(15):7810–7817.
15. Hampel H, Frankel WL, Martin E, Arnold M, Khanduja K, Kuebler P, Clendenning M, Sotamaa K, Prior T, Westman JA, Panescu J, Fix D, Lockman J, LaJeunesse J, Comeras I, De La Chapelle A (2008) Feasibility of screening for Lynch syndrome among patients with colorectal cancer. J Clin Oncol 26(35):5783–5788.
16. Ward R, Meagher A, Tomlinson I, O’Connor T, Norrie M, Wu R, Hawkins N (2001) Microsatellite instability and the clinicopathological features of sporadic colorectal cancer. Gut 48(6):821–9. 17. Popat S, Hubner R, Houlston RS (2005) Systematic review of microsatellite instability and colorectal
cancer prognosis. J Clin Oncol 23(3):609–18.
18. Kane MF, Loda M, Gaida GM, Lipman J, Mishra R, Goldman H, Jessup JM, Kolodner R (1997) Methylation of the hMLH1 promoter correlates with lack of expression of hMLH1 in sporadic colon tumors and mismatch repair-defective human tumor cell lines. Cancer Res 57(5):808–11.
19. Veigl ML, Kasturi L, Olechnowicz J, Ma AH, Lutterbaugh JD, Periyasamy S, Li GM, Drummond J, Modrich PL, Sedwick WD, Markowitz SD (1998) Biallelic inactivation of hMLH1 by epigenetic gene silencing, a novel mechanism causing human MSI cancers. Proc Natl Acad Sci U S A 95(15):8698–702. 20. Liu B, Nicolaides NC, Markowitz S, Willson JK, Parsons RE, Jen J, Papadopolous N, Peltomäki P, de
Mol Genet 4(11):2065–72.
22. Katabuchi H, van Rees B, Lambers AR, Ronnett BM, Blazes MS, Leach FS, Cho KR, Hedrick L (1995) Mutations in DNA mismatch repair genes are not responsible for microsatellite instability in most sporadic endometrial carcinomas. Cancer Res 55(23):5556–60.
23. Sourrouille I, Coulet F, Lefevre JH, Colas C, Eyries M, Svrcek M, Bardier-Dupas A, Parc Y, Soubrier F (2013) Somatic mosaicism and double somatic hits can lead to MSI colorectal tumors. Fam Cancer 12(1):27–33.
24. Mensenkamp AR, Vogelaar IP, van Zelst-Stams WAG, Goossens M, Ouchene H, Hendriks-Cornelissen SJBB, Kwint MP, Hoogerbrugge N, Nagtegaal ID, Ligtenberg MJLL (2014) Somatic mutations in MLH1 and MSH2 are a frequent cause of mismatch-repair deficiency in Lynch syndrome-like tumors. Gastroenterology 146(3):643–646.e8.
25. Rigter LS, Snaebjornsson P, Rosenberg EH, Atmodimedjo PN, Aleman BM, Ten Hoeve J, Geurts-Giele WR, PALGA group, van Ravesteyn TW, Hoeksel J, Meijer GA, Te Riele H, van Leeuwen FE, Dinjens WN, van Leerdam ME (2018) Double somatic mutations in mismatch repair genes are frequent in colorectal cancer after Hodgkin’s lymphoma treatment. Gut 67(3):447–455.
26. Birdwell SH, Hancock SL, Varghese A, Cox RS, Hoppe RT (1997) Gastrointestinal cancer after treatment of Hodgkin’s disease. Int J Radiat Oncol Biol Phys 37(I):67–73.
27. Hodgson DC, Gilbert ES, Dores GM, Schonfeld SJ, Lynch CF, Storm H, Hall P, Langmark F, Pukkala E, Andersson M, Kaijser M, Joensuu H, Fosså SD, Travis LB (2007) Long-term solid cancer risk among 5-year survivors of Hodgkin’s lymphoma. J Clin Oncol 25(12):1489–1497.
28. Schaapveld M, Aleman BMP, van Eggermond AM, Janus CPM, Krol ADG, van der Maazen RWM, Roesink J, Raemaekers JMM, de Boer JP, Zijlstra JM, van Imhoff GW, Petersen EJ, Poortmans PMP, Beijert M, Lybeert ML, Mulder I, Visser O, Louwman MWJ, Krul IM, Lugtenburg PJ, van Leeuwen FE (2015) Second Cancer Risk Up to 40 Years after Treatment for Hodgkin’s Lymphoma. N Engl J Med 373(26):2499–2511.
29. van Eggermond AM, Schaapveld M, Janus CPM, de Boer JP, Krol AD, Zijlstra JM, van der Maazen RW, Kremer LC, van Leerdam ME, Louwman MWJ, Visser O, De Bruin ML, Aleman BMP, van Leeuwen FE (2017) Infradiaphragmatic irradiation and high procarbazine doses increase colorectal cancer risk in Hodgkin lymphoma survivors. Br J Cancer 117(3):306–314.
30. Thompson BA, Spurdle AB, Plazzer J-P, Greenblatt MS, Akagi K, Al-Mulla F, Bapat B, Bernstein I, Capellá G, den Dunnen JT, du Sart D, Fabre A, Farrell MP, Farrington SM, Frayling IM, Frebourg T, Goldgar DE, Heinen CD, Holinski-Feder E, Kohonen-Corish M, Robinson KL, Leung SY, Martins A, Moller P, Morak M, Nystrom M, Peltomaki P, Pineda M, Qi M, Ramesar R, Rasmussen LJ, Royer-Pokora B, Scott RJ, Sijmons R, Tavtigian SV, Tops CM, Weber T, Wijnen J, Woods MO, Macrae F, Genuardi M (2014) Application of a 5-tiered scheme for standardized classification of 2,360 unique mismatch repair gene variants in the InSiGHT locus-specific database. Nat Genet 46(2):107–115. 31. Houlleberghs H, Goverde A, Dekker M, Lantermans H, Bruno MJ, Hogervorst FBL, van Leerdam
ME, Ruijs M, Spaander MCW, Wagner A, Hofstra RMW, te Riele H Diagnosing Lynch syndrome: identification of pathogenic MLH1 and MSH2 variants in clinical practice. Manuscr Prep.
32. Houlleberghs H, Goverde A, Lusseveld J, Dekker M, Bruno MJ, Menko FH, Mensenkamp AR, Spaander MCW, Wagner A, Hofstra RMW, Te Riele H (2017) Suspected Lynch syndrome associated MSH6 variants: A functional assay to determine their pathogenicity. PLoS Genet 13(5):e1006765. 33. Houlleberghs H, Dekker M, Lantermans H, Kleinendorst R, Dubbink HJ, Hofstra RMW, Verhoef S,
te Riele H (2016) Oligonucleotide-directed mutagenesis screen to identify pathogenic Lynch syndrome-associated MSH2 DNA mismatch repair gene variants. Proc Natl Acad Sci:201520813.
34. Houlleberghs H (2017) Oligonucleotide-directed mutation screening: a functional test to classify mismatch repair gene variants of uncertain significance. Dissertation (Vrije Universiteit Amsterdam). 35. de Wind N, Dekker M, Claij N, Jansen L, van Klink Y, Radman M, Riggins G, van der Valk M, van’t
Wout K, te Riele H (1999) HNPCC-like cancer predisposition in mice through simultaneous loss of Msh3 and Msh6 mismatch-repair protein functions. Nat Genet 23(3):359–62.
36. Wielders EAL, Houlleberghs H, Isik G, te Riele H (2013) Functional analysis in mouse embryonic stem cells reveals wild-type activity for three MSH6 variants found in suspected Lynch syndrome patients.
PLoS One 8(9):e74766.
37. de Wind N, Dekker M, van Rossum A, van der Valk M, te Riele H (1998) Mouse models for hereditary nonpolyposis colorectal cancer. Cancer Res 58(2):248–55.
Hum Mutat 33(12):1617–1625.
39. van Ravesteyn TW, Dekker M, Fish A, Sixma TK, Wolters A, Dekker RJ, Te Riele HPJ (2016) LNA modification of single-stranded DNA oligonucleotides allows subtle gene modification in mismatch-repair-proficient cells. Proc Natl Acad Sci U S A 113(15):4122–7.
40. de Piédoue G, Andrieu-Soler C, Concordet JP, Maurisse R, Sun J-S, Lopez B, Kuzniak I, Leboulch P, Feugeas J-P (2007) Targeted gene correction with 5’ acridine-oligonucleotide conjugates. Oligonucleotides 17(2):258–63.
41. Yang G, Scherer SJ, Shell SS, Yang K, Kim M, Lipkin M, Kucherlapati R, Kolodner RD, Edelmann W (2004) Dominant effects of an Msh6 missense mutation on DNA repair and cancer susceptibility. Cancer
Cell 6(2):139–50.
42. Geng H, Sakato M, DeRocco V, Yamane K, Du C, Erie DA, Hingorani M, Hsieh P (2012) Biochemical analysis of the human mismatch repair proteins hMutSα MSH2(G674A)-MSH6 and
MSH2-MSH6(T1219D). J Biol Chem 287(13):9777–91.
43. Martín-López J V., Barrios Y, Medina-Arana V, Andújar M, Lee S, Gu L, Li GM, Rüschoff J, Salido E, Fishel R (2012) The hMSH2(M688R) lynch syndrome mutation may function as a dominant negative.
Carcinogenesis 33(9):1647–1654.
44. Drost M, Lützen A, van Hees S, Ferreira D, Calléja F, Zonneveld JBM, Nielsen FC, Rasmussen LJ, de Wind N (2013) Genetic screens to identify pathogenic gene variants in the common cancer predisposition Lynch syndrome. Proc Natl Acad Sci U S A 110(23):9403–8.
45. Geurts-Giele WRR, Leenen CHM, Dubbink HJ, Meijssen IC, Post E, Sleddens HFBM, Kuipers EJ, Goverde A, Van Den Ouweland AMW, Van Lier MGF, Steyerberg EW, Van Leerdam ME, Wagner A, Dinjens WNM (2014) Somatic aberrations of mismatch repair genes as a cause of microsatellite-unstable cancers. J Pathol 234(4):548–559.
46. Wyatt MD, Pittman DL (2006) Methylating agents and DNA repair responses: Methylated bases and sources of strand breaks. Chem Res Toxicol 19(12):1580–94.
47. Wojciechowicz K, Cantelli E, Van Gerwen B, Plug M, Van Der Wal A, Delzenne-Goette E, Song J-Y, De Vries S, Dekker M, Te Riele H (2014) Temozolomide Increases the Number of MMR-deficient Intestinal Crypts and Accelerates Tumorigenesis in a Mouse Model of Lynch Syndrome. Gastroenterology. doi:10.1053/j.gastro.2014.07.052.
48. Herman JG, Umar A, Polyak K, Graff JR, Ahuja N, Issa J-PJ, Markowitz S, Willson JK V., Hamilton SR, Kinzler KW, Kane MF, Kolodner RD, Vogelstein B, Kunkel TA, Baylin SB (1998) Incidence and functional consequences of hMLH1 promoter hypermethylation in colorectal carcinoma. Proc Natl Acad
Sci 95(12):6870–6875.
49. Cunningham JM, Christensen ER, Tester DJ, Kim CY, Roche PC, Burgart LJ, Thibodeau SN (1998) Hypermethylation of the hMLH1 promoter in colon cancer with microsatellite instability. Cancer Res 58(15):3455–60.
50. Kuismanen SA, Holmberg MT, Salovaara R, de la Chapell A, Peltomäki P (2000) Genetic and epigenetic modification of MLH1 accounts for a major share of microsatellite-unstable colorectal cancers.
Am J Pathol 156(5):1773–1779.
51. Cunningham JM, Kim CY, Christensen ER, Tester DJ, Parc Y, Burgart LJ, Halling KC, McDonnell SK, Schaid DJ, Walsh Vockley C, Kubly V, Nelson H, Michels V V, Thibodeau SN (2001) The frequency of hereditary defective mismatch repair in a prospective series of unselected colorectal carcinomas. Am J
Hum Genet 69(4):780–90.
52. Haraldsdottir S, Hampel H, Tomsic J, Frankel WL, Pearlman R, De La Chapelle A, Pritchard CC (2014) Colon and endometrial cancers with mismatch repair deficiency can arise from somatic, rather than germline, mutations. Gastroenterology 147(6):1308–1316.e1.
53. Vargas-Parra GM, González-Acosta M, Thompson BA, Gómez C, Fernández A, Dámaso E, Pons T, Morak M, del Valle J, Iglesias S, Velasco À, Solanes A, Sanjuan X, Padilla N, de la Cruz X, Valencia A, Holinski-Feder E, Brunet J, Feliubadaló L, Lázaro C, Navarro M, Pineda M, Capellá G (2017) Elucidating the molecular basis of MSH2-deficient tumors by combined germline and somatic analysis.
Int J Cancer 141(7):1365–1380.
molecules. Science 323(5910):133–8.
55. Clarke J, Wu H-C, Jayasinghe L, Patel A, Reid S, Bayley H (2009) Continuous base identification for single-molecule nanopore DNA sequencing. Nat Nanotechnol 4(4):265–70.
56. de Vree PJP, de Wit E, Yilmaz M, van de Heijning M, Klous P, Verstegen Marjon JAM, Wan Y, Teunissen H, Krijger PHL, Geeven G, Eijk PP, Sie D, Ylstra B, Hulsman LOM, van Dooren MF, van Zutven LJCM, van den Ouweland A, Verbeek S, van Dijk KW, Cornelissen M, Das AT, Berkhout B, Sikkema-Raddatz B, van den Berg E, van der Vlies P, Weening D, den Dunnen JT, Matusiak M, Lamkanfi M, Ligtenberg MJL, ter Brugge P, Jonkers J, Foekens JA, Martens JW, van der Luijt R, van Amstel HKP, van Min M, Splinter E, de Laat Wl. (2014) Targeted sequencing by proximity ligation for comprehensive variant detection and local haplotyping. Nat Biotechnol 32(10):1019–25.
57. Ardui S, Ameur A, Vermeesch JR, Hestand MS (2018) Single molecule real-time (SMRT) sequencing comes of age: applications and utilities for medical diagnostics. Nucleic Acids Res 46(5):2159–2168. 58. Valeri N, Gasparini P, Braconi C, Paone A, Lovat F, Fabbri M, Sumani KM, Alder H, Amadori D,
Patel T, Nuovo GJ, Fishel R, Croce CM (2010) MicroRNA-21 induces resistance to 5-fluorouracil by down-regulating human DNA MutS homolog 2 (hMSH2). Proc Natl Acad Sci U S A 107(49):21098–103. 59. Valeri N, Gasparini P, Fabbri M, Braconi C, Veronese A, Lovat F, Adair B, Vannini I, Fanini F, Bottoni
A, Costinean S, Sandhu SK, Nuovo GJ, Alder H, Gafa R, Calore F, Ferracin M, Lanza G, Volinia S, Negrini M, McIlhatton MA, Amadori D, Fishel R, Croce CM (2010) Modulation of mismatch repair and genomic stability by miR-155. Proc Natl Acad Sci U S A 107(15):6982–7.
60. Diouf B, Cheng Q, Krynetskaia NF, Yang W, Cheok M, Pei D, Fan Y, Cheng C, Krynetskiy EY, Geng H, Chen S, Thierfelder WE, Mullighan CG, Downing JR, Hsieh P, Pui C-H, Relling M V., Evans WE (2011) Somatic deletions of genes regulating MSH2 protein stability cause DNA mismatch repair deficiency and drug resistance in human leukemia cells. Nat Med 17(10):1298–303.
61. Weßbecher IM, Hinrichsen I, Funke S, Oellerich T, Plotz G, Zeuzem S, Grus FH, Biondi RM, Brieger A (2018) DNA mismatch repair activity of MutLα is regulated by CK2-dependent phosphorylation of MLH1 (S477). Mol Carcinog. doi:10.1002/mc.22892.
62. Zou J, Luo H, Zeng Q, Dong Z, Wu D, Liu L (2011) Protein kinase CK2α is overexpressed in colorectal cancer and modulates cell proliferation and invasion via regulating EMT-related genes. J Transl Med 9(1):1–11.
63. Li F, Mao G, Tong D, Huang J, Gu L, Yang W, Li GM (2013) The histone mark H3K36me3 regulates human DNA mismatch repair through its interaction with MutSα. Cell 153(3):590–600.
64. Choi YJ, Oh HR, Choi MR, Gwak M, An CH, Chung YJ, Yoo NJ, Lee SH (2014) Frameshift mutation of a histone methylation-related gene SETD1B and its regional heterogeneity in gastric and colorectal cancers with high microsatellite instability. Hum Pathol 45(8):1674–1681.
65. McKinney M, Moffitt AB, Gaulard P, Travert M, De Leval L, Nicolae A, Raffeld M, Jaffe ES, Pittaluga S, Xi L, Heavican T, Iqbal J, Belhadj K, Delfau-Larue MH, Fataccioli V, Czader MB, Lossos IS, Chapman-Fredricks JR, Richards KL, Fedoriw Y, Ondrejka SL, Hsi ED, Low L, Weisenburger D, Chan WC, Mehta-Shah N, Horwitz S, Bernal-Mizrachi L, Flowers CR, Beaven AW, Parihar M, Baseggio L, Parrens M, Moreau A, Sujobert P, Pilichowska M, Evens AM, Chadburn A, Au-Yeung RKH, Srivastava G, Choi WWL, Goodlad JR, Aurer I, Basic-Kinda S, Gascoyne RD, Davis NS, Li G, Zhang J, Rajagopalan D, Reddy A, Love C, Levy S, Zhuang Y, Datta J, Dunson DB, Davé SS (2017) The Genetic Basis of Hepatosplenic T-cell Lymphoma. Cancer Discov 7(4):369–379.
66. Mar BG, Chu SH, Kahn JD, Krivtsov A V., Koche R, Castellano CA, Kotlier JL, Zon RL, McConkey ME, Chabon J, Chappell R, Grauman P V., Hsieh JJ, Armstrong SA, Ebert BL (2017) SETD2 alterations impair DNA damage recognition and lead to resistance to chemotherapy in leukemia. Blood 130(24):2631–2641.
67. DeWeese TL, Shipman JM, Larrier NA, Buckley NM, Kidd LR, Groopman JD, Cutler RG, te Riele H, Nelson WG (1998) Mouse embryonic stem cells carrying one or two defective Msh2 alleles respond abnormally to oxidative stress inflicted by low-level radiation. Proc Natl Acad Sci U S A 95(20):11915–20. 68. Bouffler SD, Hofland N, Cox R, Fodde R (2000) Evidence for Msh2 haploinsufficiency in mice revealed
