Functional Analysis of Human MLH1 Variants Using Yeast and in Vitro Mismatch Repair Assays

Research Article

Functional Analysis of Human MLH1 Variants Using Yeast and In vitro Mismatch Repair Assays

Masanobu Takahashi,1 Hideki Shimodaira,1 Corinne Andreutti-Zaugg,2 Richard Iggo,2 Richard D. Kolodner,3 and Chikashi Ishioka1

1Department of Clinical Oncology, Institute of Development, Aging and Cancer, and Tohoku University Hospital, Tohoku University, Sendai, Japan; 2Oncogene Group, Swiss Institute for Experimental Cancer Research, Epalinges, Switzerland; and 3Ludwig Institute for Cancer Research, University of California at San Diego, La Jolla, California

Abstract In the two MMR genes, MLH1 and MSH2, which account for the The functional characterization of nonsynonymous single majority of HNPCC kindred mutations, 31.6% of the MLH1 nucleotide polymorphisms in human mismatch repair mutations and 19.4% of the MSH2 mutations are missense (nonsynonymous single nucleotide polymorphisms) according to (MMR) genes has been critical to evaluate their pathogenicity 4 for hereditary nonpolyposis colorectal cancer. We previously the InSiGHT database. Pathogenic significances of nonsynon- established an assay for detecting loss-of-function mutations ymous single nucleotide polymorphisms are not easily evaluated in the MLH1 gene using a dominant mutator effect of human without a functional assay. Generally, this makes genetic diagnosis MLH1 expressed in Saccharomyces cerevisiae. The purpose of more difficult, especially when phenotype-genotype segregation this study is to extend the functional analyses of nonsynon- analysis is limited because of an ethical issue, the small number of family members, or other reasons. Therefore, functional analysis ymous single nucleotide polymorphisms in the MLH1 gene both in quality and in quantity, and integrate the results to has been needed to interpret the pathogenicity of MLH1 variants evaluate the variants for pathogenic significance. The 101 in genetic diagnoses of HNPCC. MLH1 variants, which covered most of the reported MLH1 Several groups, including ours, have attempted to resolve this nonsynonymous single nucleotide polymorphisms and con- issue by analyzing the functional significance of MLH1 variants by sisted of one 3-bp deletion, 1 nonsense and 99 missense various methods (1–8). We found a dominant mutator effect (DME) variants, were examined for the dominant mutator effect by of wild-type MLH1 on interference in the yeast MMR system and three yeast assays and for the ability of the variant to repair a evaluated the pathogenicity of 20 MLH1 missense variants using heteroduplex DNA with mismatch bases by in vitro MMR this effect for a yeast-based functional assay (1). One of the other assay. There was diversity in the dominant mutator effects strategies was focusing on the well-defined functions of MLH1, and the in vitro MMR activities among the variants. The ATPase activity in the NH2 terminus, and the binding abilities with majority of functionally inactive variants were located around PMS2 in the COOH terminus (2, 4, 7). Another strategy was based the putative ATP-binding pocket of the NH -terminal domain on the assessment of total MMR activity such as in vitro MMR 2 assay, monitoring the MMR rate of cell extracts for heteroduplex or the whole region of the COOH-terminal domain. Integrated functional evaluations contribute to a better prediction of DNA-containing mismatch bases (4, 5), or yeast system measuring the cancer risk in individuals or families carrying MLH1 the replication error rate resulting from the expressed yeast-human variants and provide insights into the function-structure hybrid proteins or equivalent yeast variants (3). An alternative relationships in MLH1. [Cancer Res 2007;67(10):4595–604] approach was to analyze the effects of an MLH1 variant for the expression of MLH1 mRNA and protein and the proliferation rate of cells when an MLH1 variant was introduced into the cells (6). Introduction Recently, MLH1 variants were characterized for the multiple Hereditary nonpolyposis colorectal cancer (HNPCC) is one of functional properties of wild-type MLH1, including protein the most common familial cancer syndromes caused by mainly expression, subcellular localization, MLH1-PMS2 interaction, and germ-line mutations in DNA mismatch repair (MMR) genes, such MMR efficiency (8). However, it still seems to be important to as MLH1, MSH2, MSH6, and PMS2. MMR contributes to genome establish the database on the functional effects of as many variants integrity by correcting replication errors, particularly mismatch as possible by various methods and integrate the accumulated data base pairs or slippages in simple repeat sequences. The MMR for better understanding of MLH1 variants. system is well conserved from Escherichia coli to mammals, and Beyond functional analyses, crystal structure analyses of E. coli the E. coli MMR system, where MutS, MutL, and MutH complexes MutL were used to predict the structural alterations of MLH1 function, has been well analyzed. In mammalian cells, hetero- variants (9, 10). Besides the NH2 terminus, the crystal structure of the dimers of MutS homologues (MSH2-MSH6 and MSH2-MSH3) COOH terminus of E. coli MutL was identified recently (11). recognize replication errors, and the heterodimer of the MutL Consideration of the protein structure permits more comprehensive homologue (MLH1-PMS2) interacts with MutS homologues and analysis of MLH1 and is thought to provide useful information for recruits further repair proteins. interpreting the pathogenesis of MLH1 variants in genetic diagnoses. In this study, we examined 101 MLH1 variants in yeast assays and an in vitro MMR assay, to compare these two assays and to Requests for reprints: Chikashi Ishioka, Department of Clinical Oncology, Institute of Development, Aging and Cancer, and Tohoku University Hospital, develop the functional database for a large number of variations. Tohoku University, 4-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan. Phone: 81-22- 717-8547; Fax: 81-22-717-8548; E-mail: [email protected]. I2007 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-06-3509 4 http://www.insight-group.org/ www.aacrjournals.org 4595 Cancer Res 2007; 67: (10). May 15, 2007

Table 1. Summary of MLH1 variants

Variant Nucleotide Dominant mutator effect In vitro MMR Relative MLH1 SIFT AC+/All Reference change activity (%) expression (%) score families* LacZ GFP ADE2

Missense c E23D 69A>T + + + 25.2 >75 0.00 NI I c b I25F 73A>T + + + 36.4 >75 0.00 NI I c I25T 74T>C + + + 67.2 25–75 0.00 1/1 I P28L 83C>T ÀÀ + 9.2 >75 0.00 1/3 I, S, 8 A29S 85G>T + + + 81.7 >75 0.26 1/1 I, 8 M35R 104T>G ÀÀ À 23.4 25–75 0.00 1/1 I, S c N38D 112A>G À + + 0.0 25–75 0.00 0/1 15 c S44A 130T>G + + + 65.9 >75 1.00 P 16 S44F 131C>T ÀÀ À 23.1 <25 0.00 1/2 I, S c G54E 161G>A ÀÀ + 47.9 >75 0.00 So S c N64S 191A>G ÀÀ + 36.6 >75 0.02 1/1 I, S G67R 199G>A ÀÀ À 5.9 <25 0.00 2/4 I, S, 16, 17 c G67W 199G>T ÀÀ À 7.3 25–75 0.00 1/1 S c I68V 202A>G + + + 79.8 >75 0.02 P 16 I68N 203T>A ÀÀ À 20.8 25–75 0.00 1/1 I. S R69K 206G>A + + + 75.0 >75 0.91 0/1 S C77Y 230G>A À + + 11.2 25–75 0.15 0/1 I, S F80V 238T>G ÀÀ + 23.7 >75 0.07 1/1 S, 8 c T82I 245C>T À + + 27.2 >75 0.00 1/1 18 K84E 250A>G À + + 22.5 >75 0.00 0/1 I, S S93G 277A>G + + + 80.3 >75 0.16 1/2 I, S, 8 c R100P 299G>C À + + 0.0 <25 0.00 NI I E102K 304G>A + + + 44.4 >75 0.00 1/1 I c E102D 306G>T + + + 56.1 >75 0.00 NI I I107R 320T>G ÀÀ À 39.5 25–75 0.00 5/7 I, S, 8, 19 c A111V 332C>T ÀÀ À 25.5 25–75 0.01 2/2 S, 20 T117M 350C>T ÀÀ À 34.8 <25 0.00 12/14 I, S, 15, 21, 22 T117R 350C>G ÀÀ À 25.2 25–75 0.00 1/1 I, S A128P 382G>C ÀÀ À 24.4 25–75 0.01 1/1 I, S D132H 394G>C + + + 63.0 25–75 0.02 0/1 S, 23 c A160V 479C>T + + + 80.9 >75 0.01 NI I c R182G 544A>G + + + 74.3 25–75 0.00 0/1 I, S V185G 554T>G ÀÀ À 8.5 25–75 0.00 2/3 I, S, 8, 15 c S193P 577T>C ÀÀ À 0.0 >75 0.10 1/1 I, S, 24 c E199Q 595G>C + + + 67.7 >75 0.15 0/1 I V213M 637G>A + + + 85.0 >75 0.11 P I, S R217C 649C>T + À + 64.8 >75 0.11 1/2 I, S I219L 655A>C + + + 85.2 >75 0.52 P I I219V 655A>G + + + 60.7 25–75 0.46 P I, S c R226L 677G>T ÀÀ + 39.2 25–75 0.04 2/2 I, S G244D 731G>A ÀÀ À 19.4 >75 0.00 1/1 I, S c G244V 731G>T ÀÀ À 18.8 >75 0.00 So S, 25 c H264R 791A>G + + + 68.7 >75 0.07 1/1 I R265C 793C>T À + + 55.0 25–75 0.00 3/4 I b R265H 794G>A + + + 61.1 >75 0.00 NI I, S c b E268G 803A>G + À + 78.9 25–75 0.05 NI I, S c L272V 814T>G + + + 90.2 >75 0.10 NI I c A281V 842C>T + + + 88.6 25–75 0.27 NI I c K286Q 856A>C + À + 78.6 25–75 0.39 1/1 26 c S295G 883A>G À + + 75.5 25–75 0.46 1/1 I, S c D304V 911A>T ÀÀ + 0.0 >75 0.00 1/1 27 V326A 977T>C + + + 26.9 25–75 0.00 3/5 I, S, 21, 28 H329P 986A>C ÀÀ + 25.7 >75 0.21 2/2 I, S, 8 c V384D 1151T>A + + + 64.8 >75 0.07 P I, S c R389Q 1166G>A + + + 83.6 >75 0.31 NI I

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Table 1. Summary of MLH1 variants (Cont’d)

Variant Nucleotide Dominant mutator effect In vitro MMR Relative MLH1 SIFT AC+/All Reference change activity (%) expression (%) score families* LacZ GFP ADE2

S406N 1217G>A + + + 73.5 >75 0.62 P I, S c T413I 1238C>T + + + 84.0 >75 0.34 NI Unpublished c S420C 1259C>G + + + 70.7 >75 0.13 NI 23 c A441T 1321G>A + + + 71.1 25–75 0.56 1/3 I, S c R474Q 1421G>A + + + 81.1 >75 0.60 0/1 I c D485E 1455T>A + + + 48.3 25–75 0.95 1/1 17 A492T 1474G>A À + + 65.3 >75 0.51 0/1 I, S c P496L 1487C>T + + + 65.7 >75 0.36 NI Unpublished b V506A 1517T>C À + + 67.6 25–75 0.04 1/1 I, S, 18 c E523D 1569G>T + + + 76.6 >75 0.24 So I, 29 Q542L 1625A>T + + + 13.3 >75 0.00 1/1 I, S c L549P 1646T>C ÀÀ À 31.0 25–75 0.00 1/1 I, S c N551T 1652A>C ÀÀ À 78.9 25–75 0.00 1/1 I, S c I565F 1693A>T ÀÀ À 52.5 >75 0.08 NI I, S L574P 1721T>C ÀÀ À 2.9 >75 0.02 1/1 I, S E578G 1733A>G À + + 51.2 25–75 0.12 0/2 I, S L582V 1744C>G + + + 65.6 >75 0.02 1/1 I, S c A586P 1756G>C ÀÀ À 28.0 25–75 0.31 1/1 I, S c L588P 1763T>C + ÀÀ 68.3 >75 0.10 1/1 S c P603R 1808C>G + + + 82.5 >75 0.32 0/1 S c L607H 1820T>A + + + 88.8 >75 0.04 2/2 S, 30 b K618T 1853A>C ÀÀ À 48.7 25–75 0.41 2/4 I, S, 28, 30 c L622H 1865T>A ÀÀ À 69.2 25–75 0.00 1/1 S c P640T 1918C>A ÀÀ À 53.6 >75 0.00 NI Unpublished P648L 1943C>T ÀÀ À 39.2 25–75 0.02 0/1 S c L653R 1958T>G ÀÀ À 12.9 25–75 0.00 0/1 I c P654L 1961C>T ÀÀ À 49.1 25–75 0.00 1/5 I, 8 c I655V 1963A>G + + + 70.6 >75 0.49 NI I c I655T 1964T>C + + + 73.8 >75 0.35 NI 25 R659P 1976G>C ÀÀ À 24.9 <25 0.06 3/3 I, S R659Q 1976G>A À + + 79.7 25–75 0.18 1/1 8 c T662P 1984A>C ÀÀ À 64.0 25–75 0.20 1/1 I, S c E663G 1988A>G + + + 69.7 >75 0.16 1/1 I c E663D 1989G>T À + + 68.5 >75 0.31 1/1 I c L676R 2027T>G ÀÀ À 39.8 25–75 0.00 NI I A681T 2041G>A ÀÀ À 69.8 >75 0.00 3/4 I, S, 8 c R687W 2059C>T ÀÀ À 57.2 25–75 0.02 1/1 S, 20 c Q689R 2066A>G + + + 68.0 25–75 0.52 0/1 S b V716M 2146G>A ÀÀ + 75.1 25–75 0.22 6/11 I, S, 15, 20, 21, 22 H718Y 2152C>T À + + 84.5 25–75 0.00 P I, S L729V 2185C>G + + + 80.3 >75 0.70 NI S c K751R 2252A>G + + + 66.6 >75 0.59 1/2 S, 15 c R755S 2265G>C + + + 7.9 >75 0.00 1/1 18 b K618A 1852-3AA>GC — — + 82.7 25–75 0.44 5/19 I, S, 8, 22 In-frame deletion K618del 1846–48del — — — 38.9 <25 14/19 I, S, 8 Nonsense W714X 2141G>A — — — 0.0 25–75 6/6 I, 17

NOTE: Data on functions in yeast and in vitro MMR assays and on MLH1 protein levels when transiently expressed in HCT116 cells are from this study. SIFT score was calculated by an online program, SIFT, that uses sequence homology to predict whether a substitution affects protein function. If the value is <0.05, the amino acid substitution is predicted to affect protein function. Information on families is from databases or references. Abbreviations: AC, Amsterdam criteria; NI, not informative; So, found as somatic mutation in a patient with sporadic colorectal cancer; P, putative polymorphisms; I, the InSiGHT online database; S, the SWISS-PROT online database. *The number in the left side is the number of families fulfilling the Amsterdam criteria; the number in the right side is the total number of families whose familial information was available from the databases or the various reports. cThe variant has never been evaluated for pathogenicity in functional assays. bThe variant was found also in a family or families carrying the other second mutation in MLH1 or MSH2 gene. The family or families was excluded in this table.

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We also analyzed the distribution of loss-of-function variants on acid substitutions on protein functions (33). Based on the crystal structure of the secondary MLH1 structure or the crystal structure, to predict the E. coli NH2 terminus of MutL (9, 10), the MLH1 structure was predicted the pathogenicity of the MLH1 variants, and to get the structural by a homology modeling method (34) using software ICM-Molsoft (Molsoft basis for defects of MLH1 function. L.L.C.). The secondary structure alignment of the MutL homologues was based on reports (11, 35) and predicted using the PSIPRED server (36). Materials and Methods Results Yeast strains and plasmids. The Saccharomyces cerevisiae haploid strains used in this study were as follows: YPH499 from Stratagene, and DME was detected in the three yeast assays. We previously yCA23 (leu2, his3, trp1, can1, pCYC1-28CDADE2) and yCA25 [leu2, his3, developed a yeast-based MMR assay using a LacZ reporter plasmid D trp1, can1, pCYC1-(CA)15 ADE2]. yCA23 and yCA25 were made by with dinucleotide (GT) repeats (Fig. 1A, left; ref. 1). To establish integrating plasmids pCA2 and pCA3 into the URA3 locus of a haploid simpler methods to detect yeast MMR deficiency, we also derivative of ASZ3 (12). pCA2 and pCA3 were derived from pLS210 (13) developed three distinct assays: a GFP assay using mononucleotide and contain out of frame C28 and (CA)15 repeats at the amino terminus of (C) repeats (Fig. 1A, center) and two ADE2 assays using mono- ADE2 expressed by the CYC1 promoter. Constructs of the low-copy MLH1 nucleotide (C) repeats and dinucleotide (CA) repeats (Fig. 1A, expression plasmid, pCI-ML10, the gap repair vectors, pCI-ML22 and right). In each assay, replication errors of mononucleotide or pCI-ML24, and the reporter LacZ plasmid, pCIZ1, were described previously dinucleotide repeats were observed in yeast colonies transformed (1). The mutation-reporter green fluorescent protein (GFP) plasmid, with wild-type MLH1 due to the DME and expressed reporter pCGFP2A(URA3), was constructed by inserting an enhanced GFP (EGFP) genes, whereas those were not observed in yeast colonies trans- coding sequence with a poly C (C22) tract instead of the b-galactosidase gene, including the poly-GT tract of pCIZ1. The wild-type MLH1 human formed with an empty vector (Fig. 1B). The results of the two ADE2 expression plasmid was constructed by inserting wild-type MLH1 cDNA assays coincided in most of the MLH1 variants and, therefore, into the BamHI site of a pCMV-Neo-Bam (14), and each variant MLH1 the result of the ADE2 assay using mononucleotide (C) repeats was expression plasmid is identical to the wild-type MLH1 expression plasmid adopted as the representative ADE2 assay. The 101 MLH1 variants but contains each mutagenized MLH1 cDNA. were classified into one of the following four categories depending Site-directed mutagenesis. One hundred one MLH1 variants consisted on the results of the LacZ, GFP, and ADE2 assays: (a) DME positive of 99 missense variants, one nonsense variant (W714X), and one 3-bp in- in all three assays (DME3+) contained 43 variants, (b) DME positive frame deletion (K618del), and were constructed by site-directed mutagen- in two assays (DME2+) contained 16 variants, (c) DME positive in esis (Table 1; ref. 1). The information on the majority of MLH1 variants 5 one assay (DME1+) contained 10 variants, and (d) DME negative were based on the InSiGHT and SWISS-PROT databases, whereas others À were based on various publications (15–30). Each of the MLH1 sequences in all three assays (DME ) contained 32 variants. The results of mutated specifically was confirmed by DNA sequencing. These MLH1 the DME phenotypes of all variants are summarized in Table 1. variants included 90 germ-line variants found in HNPCC patients fulfilling In vitro MMR activity of human cell extracts expressing the Amsterdam criteria (AC+) or individuals not fulfilling the Amsterdam MLH1 variants. Although the dominant mutator assays are simple criteria (ACÀ), three somatic variants found in individuals with sporadic and quick methods to detect functional alterations of MLH1 cancer and eight major or putative polymorphisms. variants, DME deficiency may not be directly linked to pathoge- Yeast-based assays. For the LacZ and GFP assays, competent yeast cells nicity on HNPCC onset. To measure the MMR activities of MLH1 YPH499 were transformed with an MLH1 expression plasmid and a reporter variants, we adopted a well-established in vitro MMR assay to A plasmid [pCIZ1 for LacZ assay, pCGFP2 (URA3) for GFP assay], as described analyze the ability of cell extracts to repair DNA substrates with previously (1). In the LacZ assay, the MMR ability of each yeast transformant mismatched bases (4, 5). In the assay, cytoplasmic extracts of could be monitored by the appearance of a blue color on the Xgal plate after 5 days incubation at 37jC (1). In the GFP assay, the MMR ability could be HCT116 cells had no MMR activity (0%) in the in vitro MMR assay monitored by the appearance of green-fluorescent colonies observed under because the cells were defective in MLH1 and PMS2 expressions. fluorescent microscopy after 3 days incubation at 30jC. In the ADE2 assay, The transient expression of wild-type MLH1 and PMS2 in the yCA23 or yCA25 cells were transformed with an MLH1 expression plasmid, cells complemented the defect and the MMR activity (79.7 F 7.8%) and the MMR ability could be monitored by the appearance of a red color on was comparable with that of MMR-proficient HCT116+Ch3 cells a low-adenine plate after 2 or 3 days incubation at 30jC (31). (71.3 F 11.7%). The results indicated that exogenous MLH1 was In vitro MMR assay and Western blot analysis. Human colon cancer able to restore the MMR function of MLH1-deficient cells as also HCT116 cells were cultured in DMEM with 10% fetal bovine serum at described in other reports (4, 5). The MMR activities of the 101 j Â 6 A 37 C. HCT116 cells (3.0 10 ) were transfected with 5 g of each MLH1 MLH1 variants expressed in HCT116 cells were then evaluated. expression plasmid, 5 Ag of a PMS2 expression plasmid, and 1.2 Agof The results indicated that there was diversity (0–90.2%) in the ability pEGFP-C3 (BD Biosciences) using FuGENE6 (Roche). After 24-h incubation, cytoplasmic extracts were prepared and MMR activity in human cell to repair mismatch DNA among the MLH1 variants (Fig. 2A). All of extracts containing heteroduplex DNA was measured in vitro as described the eight putative polymorphisms had >60% of in vitro MMR previously (32). M13mp2 heteroduplex DNA with the T-G mismatch was activity. Therefore, 51 variants that showed 60% or more of used in a MMR reaction with HCT116 cell extracts transfected with each MMR activity were classified into ‘‘MMR+,’’ and 50 showing <60% MLH1 expression plasmid. Each experiment was done twice or more and of MMR activity were classified into ‘‘MMRÀ,’’ in this study. in vitro MMR activity (%) is shown as the mean F SD. The extract was also Protein expression levels among the majority of MLH1 examined in Western blot analyses using an anti-MLH1 antibody (G168-728, variants. In many previous studies on MLH1 protein, non- BD Biosciences), an anti-PMS2 antibody (A16-4, BD Biosciences), and an synonymous single nucleotide polymorphisms have been shown anti-GFP antibody (MMS-118P, Covance). Bands of MLH1 and GFP were to affect protein stability (5, 8, 30, 37). To evaluate the level of quantified by densitometry. expressed MLH1 protein, the 101 variants were transiently Prediction of protein activity and structure. The Sorting Intolerant from Tolerant (SIFT) algorithm was adopted to predict the effects of amino coexpressed with PMS2 in HCT116 colon cancer cells. The two MMR proteins were detected by Western blot analyses and the transfection efficiency was normalized by the cotransfected GFP 5 http://kr.expasy.org/sprot/ level. The representative results of 11 variants for the MLH1 protein

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Figure 1. Functional analysis of MLH1 variants in S. cerevisiae. A, schematic diagram of yeast assays. B, appearance of yeast colonies on indicator media for each functional assay of MLH1 variants. The evaluation of each assay was summarized on the right of the panel.

levels are shown in Fig. 2B. Among these, the protein levels of which 101 variations are plotted by in vitro MMR activities and DME R100P, G67R, T117M, K618del, and R659P variants were lower than showed also correlation between these two assays (Fig. 3B). The those of the wild-type. The level of coexpressed PMS2 was also majority of MMR+ variants showed DME3+ (35 of 51) and MMRÀ reduced in the four variants except for R100P. In S193P and E23D, variants showed DMEÀ,DME1+,orDME2+(42of50;Table1;Fig.3B). PMS2 expressions were reduced although MLH1 expressions Relationship between the function and structure of MLH1. were comparable with wild-type MLH1. Comparing in vitro MMR To elucidate the function-structure relationship of MLH1 variants, activities with MLH1 expression levels, some variants, such as we first compared the in vitro MMR activities with the SIFT score R100P, G67R, T117M, K618del, and R659P, showed MMRÀ with low of the MLH1 variants. SIFT is a program that predicts the effect of MLH1 and/or PMS2 expression levels, whereas other variants, such amino acid substitutions on protein function, based on sequence as E102D, showed MMRÀ despite the retained expression levels of conservation during evolution and the nature of amino acids the two proteins (Fig. 2B). In all, only a slight correlation (R = 0.225, substituted in a gene of interest (33). As shown in Table 1, a P = 0.024) between in vitro MMR activities and MLH1 expression majority of variants sorted as tolerant by SIFT showed MMR+ levels was observed (Fig. 2C), suggesting that the MMR activities of (38 of 48;79%) and those sorted as intolerant showed MMRÀ (38 of MLH1 variants may not solely depend on MLH1 expression levels. 51;75%), suggesting that SIFT had a good accuracy for predicting Correlations between DME phenotype and in vitro MMR functions of variants and there was correlation between amino activities. To examine whether the functions of the DME of MLH1 acids conservations and functions. Second, we focused on the variants correlate with the ability to repair mismatched DNA, we relationships between MLH1 functions and the functional domain. compared the results of yeast assays with the in vitro MMR assay. As shown in Table 1, a large number of MLH1 variants showing The in vitro MMR activities were higher in the DME-positive DME negative in each yeast assay and/or MMRÀ were located variants than in the DME-negative variants in any yeast assay within either of the two functional domains, the NH2-terminal (P < 0.01 by two-sided Mann-Whitney’s U test; Fig. 3A). Moreover, ATPase domain or the COOH-terminal PMS2-interactive domain, in the three yeast assays, more DME-positive results correlated whereas those showing DME+ or MMR+ were distributed with higher in vitro MMR activity (Fig. 3A). The scatter diagram in throughout the whole gene product. Finally, we constructed a www.aacrjournals.org 4599 Cancer Res 2007; 67: (10). May 15, 2007

predicted three-dimensional structural model of the MLH1 NH2- Discussion terminal domain by a homology modeling method to map variants in In this study, we evaluated the functional significance of 101 the predicted structure (Fig. 4A–C). Most of the variants predicted MLH1 variants by yeast-based assay and in vitro assay to provide to be around the ATP-binding pocket were DME negative in at least useful information for understanding of pathogenicity. This À one of the yeast assays or MMR (Fig. 4B). In particular, variants functional characterization of a large number of variants allowed mutated in residues thought to be critical for ATP binding, such as us to compare the property of two assays and to analyze the M35R, N38D, S44F, G67R, I68N, C77R, F80V, T82I, K84E, R100P, and structural basis of functional deficiency. I107R, affected both DME and in vitro MMR activity We showed that the yeast assay distinguished the majority of (Fig. 4C). Unlike the NH2-terminal ATPase domain, sequence identity variations that retained or lost the in vitro MMR activity. These data among the COOH-terminal domains of MutL homologues is not high confirmed the accuracy and usefulness of the yeast assay as a simple enough to construct three-dimensional structure by homology method having an advantage to analyze a large number of variations modeling; however, the detailed analysis of secondary structure without laborious steps. The three kinds of reporter systems predictions and sequence alignments indicates that the structures of identified some variants showing different DME phenotype among the COOH-terminal domains of MutL homologues are similar (Fig. yeast assays (DME1+ or DME2+), although the majority (75 of 101, 4D; refs. 11, 35). Missense variants were classified by the assays and 74.3%) of the MLH1 variants showed consistent DME (DMEÀ or mapped on a COOH-terminal domain alignment of four MutL DME3+). These discrepant variations showing DME1+ or DME2+ homologues, showing that variants in the COOH-terminal domain were supposed to be functionally subtle because these difference were found more frequently in the internal subdomain, especially could be explained by the distinct thresholds of the reporter genes within and around the aC helix, than in the external subdomain and (LacZ, GFP,orADE2), the target nucleotide repeats (mono- or di-), functionally inactive variants were distributed through the whole and/or the location of the repeats (a plasmid or a chromosome). region of the COOH terminus (Fig. 4D). This is supported by our finding that the average values of the Relations between MLH1 functions and clinical features. To in vitro MMR activities depended on the degree of the DME (from investigate the relationship between the functional phenotype in DMEÀ to DME3+; Fig. 3A). Thus, a combination of the three yeast the assays and clinical phenotypes previously reported in the assays has the ability to evaluate functionally subtle variants as well databases or papers, at first, in vitro MMR activities were compared as the in vitro MMR assay. Among variants showing discrepant between AC+ variants and ACÀ variants, considering the total results between the yeast assays and in vitro MMR assay, variants number of families reported for each variant. The AC+ group showing both DME3+ in the yeast assays and MMRÀ in the in vitro showed significantly lower in vitro MMR activities than ACÀ group MMR assay (DME3+/MMRÀ) should be estimated to be pathogenic (P < 0.01; Fig. 5A). Second, the MLH1 protein expression levels in because MMR deficiency should link directly with carcinogenesis by HCT116 cells were compared between AC+ and ACÀ groups. The causing genome instability regardless of the DME in yeast. DMEÀ/ significant difference of the protein level was not shown between MMR+ variants are difficult to be interpreted but possibly defect the two groups (P = 0.68; Fig. 5B). These results suggested that some unknown function in human cells, because the phenotype of there is correlation between MMR defects and a strong family these variants in yeast cells are quite different from that of a wild- history but no correlation between the MLH1 protein instability type. We consider that DME1+ or DME2+ variants can also lose and a strong family history. some function partially for the same reason.

Figure 2. Protein levels of expressed MLH1 and functional assays. A, top, in vitro MMR activities of the 101 MLH1 variants in order of their value. Points, mean MMR activity of wild-type MLH1; bars, SD. Bottom, the corresponding DME status. Gray box, DME positive; black box, DME negative. B, Western blot analyses of 11 representative MLH1 variants coexpressed with PMS2 in HCT116 cells. MLH1 protein levels were normalized by the coexpressed GFP level. The ratios of the variants to the wild-type were shown as the relative MLH1 protein level. The in vitro MMR activities of cell extracts expressing MLH1 variants are also shown below. C, correlation between in vitro MMR activities and the relative protein levels of 101 MLH1 variants. Their relationship was investigated by Pearson’s correlation coefficient test.

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Figure 3. Comparison of the DME of yeast assays with the in vitro MMR activity. A, box-and-whisker plot of in vitro MMR activity in DME-negative and DME-positive MLH1 variants. Horizontal line inside the box, median. Upper and lower limits of the box, 75th and 25th percentiles, respectively. Horizontal bars above and below the box, 90th and 10th percentiles, respectively. n, mean of data points. **, P < 0.01, two-sided Mann-Whitney U test. B, functions of MLH1 variants in an in vitro MMR assay and yeast assays. Wild-type MLH1 and 101 variants are plotted by in vitro MMR activity and DME level. Dotted line, in vitro MMR activity of common polymorphism I219V.

Other functional analyses examining a part of 101 variants agree value is difficult to set up, because we cannot estimate how with our functional evaluation (2–5, 7). For example, 34 MLH1 intermediate or subtle functional defects contribute to the variants were recently analyzed for four distinct functional pathogenesis in HNPCC. However, the information on common properties containing in vitro MMR assay, protein expression, polymorphisms found in the normal populations can be useful to protein localization, and interaction with PMS2, and were evaluated consider the tolerable level predicted to be functionally proficient. for their pathogenicity by integrating all of the results (8). Among The most common polymorphism is I219V, which was reported 20 variants assayed in common with this study, the in vitro MMR from various countries, although the allele frequency is varied from activities were low (<48.7% activity) in their 12 pathogenic variants, 3% to 36% (24, 38, 39). According to our data, I219V retained f60% and high (>60.7%) in their eight nonpathogenic variants. DME of MMR activity and DME3+. The other seven putative poly- phenotype in this study also corresponded with their interpretation morphisms retained also >60% MMR activity, the seven of the eight in 16 of 20 variants. The consistencies in the functionally known showed DME3+ except H718Y showing DME2+. Therefore, we MLH1 variants supported reliability of the functional evaluation for currently propose that both DME3+ and 60% of MMR activity is the the newly analyzed variations in this study. reasonable cutoff value to estimate the variations not associated Our final purpose of analyzing the large number of MLH1 with pathogenicity in HNPCC. Then, 50 MLH1 variations with more variants is to establish a database for understanding the than this MMR activity are categorized into MMR+ ones, and 35 pathogenicity of the gene alterations. One of the major problems DME3+/MMR+ variants are thought to be functionally proficient. in using our data for clinical purpose is that the appropriate cutoff The D132H also retains DME3+/MMR+, which is a polymorphism www.aacrjournals.org 4601 Cancer Res 2007; 67: (10). May 15, 2007

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2007 American Association for Cancer Research. Cancer Research among Israeli populations not associated with HNPCC but the by protein instability, and functional inactivation by structural sporadic colorectal cancer predisposition (23). Thus, we cannot alteration. This suggested that the cutoff values are required in exclude the possibility that even the variations showing DME3+/ both functional level and protein level for overall evaluation. MMR+ might be involved in the sporadic carcinogenesis. Recent studies have indicated that the immunohistochemical Another critical problem is that amino acid substitutions in analysis of MMR proteins is one of the most efficient and sensitive MLH1 affected both protein expression levels and functions. Our screening method to detect abnormalities in MMR proteins data showed that there was just a slight correlation between MLH1 (40, 41). In our data, in vitro MMR activities were impaired without protein levels and in vitro MMR activities (Fig. 2C). Therefore, we reducing MLH1 protein levels in some MLH1 missense variants. predict that there are at least two mechanisms inactivating MLH1 This observation suggested that these pathogenic variants can be function by amino acid substitutions, the shortage of MLH1 protein detected positively by immunohistochemistry, although we cannot

Figure 4. Relationships between putative MLH1 protein structures and functions. A to C, model of the three-dimensional structure of the MLH1 NH2-terminal domain and maps of the MLH1 variants. A, ribbon diagram of the E. coli MutL NH2-terminal domain (left) and an MLH1 NH2-terminal domain simulated by homology modeling (right). B and C, the model of the whole of the MLH1 NH2-terminal domain and the model of the ATPase domain, respectively. Colored balls or bars, amino acid residues examined in this study and the functional information from yeast assays and in vitro MMR assay. Red, pink, and flesh color, DMEÀ, DME1+/2+, and DME 3+ phenotypes, respectively. Green, light green, and yellow, higher (z75% of wild-type), average (50–75%), and (<50% of wild-type) lower in vitro MMR activity, respectively. Blue balls or bars, ADPnP. D, sequence alignment and secondary structure of the COOH-terminal domains of MutL homologues. Arrows and bars, h-sheet and a-helix, respectively. Regular lines, internal subdomain; dotted orange lines, external subdomain. Colored dots, locations of MLH1 variants, representing the functional phenotype, as in (B) and (C).

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dimerization interface (11). The alignment and homology modeling show that L653R, R654L, and R659P resided on the equivalent to the dimmer interface of MutL, which is the putative interface of MLH1 with PMS2. T662P is equivalent site of MutL critical for binding with DNA (Fig. 4D). The homology modeling provides the structural basis of functional deficiency for some variants, although the functionally deficient variations of MLH1 distributed through the whole COOH terminus. The previous studies have shown that MSH6 mutations cause a partial MMR deficiency and related with atypical HNPCC families (42). This suggests the possibility that intermediate functional deficiency is associated with the atypical HNPCC kindred with weak family history or late onset. Based on this hypothesis, one of the methods to validate our functional assay is investigating the relationship between functional evaluations with clinical feature such as family history. The median MMR activity was significantly lower among the AC+ families (38.9%) than the ACÀ families Figure 5. Relationships between the functions and clinical features of MLH1 variants. A, box-and-whisker plot of in vitro MMR activity of MLH1 variants from (65.1%), whereas the median level of relative protein expression families fulfilling the Amsterdam criteria (AC+) or not (ACÀ). B, box-and-whisker has no significant difference between these two groups (Fig. 5). plot of the protein expression levels of the AC+ or ACÀ variants. Line inside the box, median. Upper and lower limits of the box, 75th and 25th percentiles, Therefore, AC status has correlation with our functional data at respectively. Vertical bars above and below the box, 90th and 10th percentiles, least stronger than with protein level. Functions in the assays well respectively. n, mean of data points. **, P < 0.01 by two-sided Mann-Whitney correlated with clinical features, whereas it can be said that the U test. Amsterdam criteria links with functions well and is a very useful clinical diagnostic criteria. Our functional data have consistency directly link the protein amount of transient expression in cell with previous functional assays in a majority of the 45 functionally lines with the endogenous protein level in tumors of the known variants, including some variants evaluated for pathoge- corresponding mutation carrier. Among the 50 variants with nicity by both cosegregation study and functional assay. Precise MMRÀ, 20 (40%) retained >75% of the MLH1 expression level of a family information was described even in the limited number wild-type. We speculate that these 20 variants (E23D, I25F, P28L, among some of the variants newly analyzed in this study. For G54E, N64S, F80V, T82I, K84E, E102D, E102K, S193P, G244V, D304V, example, S193P was detected in three affected members and two H329P, Q542L, I565F, L574P, P640T, and R755S) will be the other members among 12 individuals in an AC+ family (24). This candidate pathogenic variants with difficulty in clear detection variant showed DMEÀ/MMRÀ in our assays and then indicated as loss of protein expression by immunohistochemistry. Among to be pathogenic from both family history and functional assay. them, three variants, P28L, F80V, and P648L, actually have been R687W was found in three affected members but not two healthy shown to retain their protein levels by immunohistochemical members in the AC+ family, suggesting that this variant is likely analyses (8, 37). Functional assays are especially useful for these pathogenic (20). However, our functional data indicated DMEÀ but variants because the abnormality will not be detected by immuno- relatively high MMR activity (57.2%) even below 60% (defined to be histochemistry until subsequent sequence analysis and functional MMRÀ in this study). Yeast assay detected pathogenicity of this assays. variant more clearly than MMR in this case. Although L607H was Several lines of biochemical investigations have shown that the found in one family member with colon cancer but not in two heterodimerization with counterpart proteins such as PMS2 and healthy members in the family, this colon cancer did not show any conformational change by ATP binding are important in the MLH1 microsatellite instability, low staining by immunohistochemistry function (1, 3, 7–10). Mapping 101 MLH1 variants on MLH1 cDNA (29), or functional defect in our assay. Then, pathogenicity of this indicated that the majority of functionally defective MLH1 variants variant is supposed to be in question but functional data correlates (showing DMEÀ, DME1+, or DME2+ and/or MMRÀ) were located with microsatellite instability and immunohistochemical results. within two functional domains, the NH2-terminal ATPase and the Thus, in the several cases in which detailed clinical information is COOH-terminal PMS2-interactive domains. In particular, almost all available, the functional phenotype well correlated with clinical variants involved in the ATP-binding pocket were functionally features for families carrying the corresponding variants. Based on defective (Fig. 4B and C). The homologous structure of the this, it may be expected that the pathogenicity can be well NH2-terminal E. coli MutL provided molecular basis for functional predicted by the functional phenotype in the assay for many of the defects of human MLH1 missense variants (9, 10). P28L, M35R, other variants, especially those showing clear phenotype such as and S44F can disrupt ATP binding and hydrolysis; G67R, I68N, DMEÀ/MMRÀ and DME3+/MMR+. However, we have to accumu- I107R, T117M, and T117R degraded ATP binding pocket; late more data on clinical and functional characteristics and and other variants (A128P, V185G, R226L, G244D, and V326A) evaluate accurate sensitivity and specificity of functional data for can alter or destabilize the overall protein folding. Our functional predicting pathogenicity, to use for practical purposes in clinics, for assay showed that 13 out of these 14 variants were functionally example, to decide whether surveillance of a mutation noncarrier defective and supported the functional prediction based on the should be continued. protein structure. In contrast, some variants probably do not In this study, we analyzed functional significances of 56 MLH1 change the protein structure because the changes are conservative variants that have never been evaluated in any functional assay. (I25F, A29S, S44A, I68V, V213M, and I219L; refs. 9, 10). Recent study Without functional analyses, pathogenicity of missense variants are resolved crystal structure of COOH-terminal MutL and identified estimated usually based on amino acid property, conservation www.aacrjournals.org 4603 Cancer Res 2007; 67: (10). May 15, 2007

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2007 American Association for Cancer Research. Cancer Research among species, or computed prediction like SIFT as a recent way. evaluating cancer risks in individuals or families carrying MLH1 The SIFT has a good accuracy for the functional prediction; variants and may provide clues for better understanding MLH1 however, f20% of variants could fail to be predicted. A similar functions. tendency has been observed in another analysis (8) and in other proteins such as p53 tumor-suppressor protein (data not shown). Acknowledgments Therefore, it is still desirable to carry out biochemical assays to Received 9/21/2006; revised 1/19/2007; accepted 3/8/2007. predict the pathogenicity for variants newly reported if available. Grant support: Ministry of Education, Science, Sports and Culture 17015002 In summary, we examined a large number of MLH1 variants (C. Ishioka) and 16390122 (H. Shimodaira), the Sagawa Foundation for Promotion of using both yeast and in vitro functional assays and characterized Cancer Research (C. Ishioka), the Gonryo Medical Foundation (C. Ishioka), NIH Grant GM50006 (R.D. Kolodner), and the Swiss Cancer League (R. Iggo). the functional alterations of the variants. We confirmed that the The costs of publication of this article were defrayed in part by the payment of page majority of functionally inactive variants were located in the charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. NH2-terminal and COOH-terminal domains, especially around We thank Dr. Thomas A. Kunkel (Laboratory of Molecular Genetics and Laboratory the ATP-binding pocket and the region responsible for hetero- of Structural Biology, National Institute of Environmental Health Sciences, NIH, dimerization with other MutL homologues. The results corre- Department of Health and Human Services, Research Triangle Park, NC) for kindly providing bacteriophage M13mp2, E. coli strains, and human cells; Dr. Alan B. Clark sponded well with observations by the structural analysis of E. coli and Yuka Fujimaki for their technical support for MMR assays; and Michael F. Kane MutL crystals. The study described here should be useful for for sequencing a part of the MLH1 variant expression plasmids.

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2007 American Association for Cancer Research. Functional Analysis of Human MLH1 Variants Using Yeast and In vitro Mismatch Repair Assays

Masanobu Takahashi, Hideki Shimodaira, Corinne Andreutti-Zaugg, et al.

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