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Human Molecular Genetics Advance Access originally published online on January 8, 2007
Human Molecular Genetics 2007 16(4):445-452; doi:10.1093/hmg/ddl479
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

The effect of genetic background on the function of Saccharomyces cerevisiae mlh1 alleles that correspond to HNPCC missense mutations

Jennifer J. Wanat{dagger}, Nikhil Singh{dagger} and Eric Alani*

Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA

* To whom correspondence should be addressed at: Department of Molecular Biology and Genetics, Cornell University, 459 Biotechnology Building, Ithaca, NY 14853-2703, USA. Tel: +1 6072544811; Fax: +1 6072556249; Email: eea3{at}cornell.edu

Received September 1, 2006; Revised December 21, 2006; Accepted December 28, 2006


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Germline mutations in the DNA mismatch repair (MMR) gene MLH1 are associated with a large percentage of hereditary non-polyposis colorectal cancers. There are approximately 250 known human mutations in MLH1. Of these, one-third are missense variants that are often difficult to characterize with regards to pathogenicity. We analysed 28 alleles of baker's yeast MLH1 that correspond to non-truncating human mutant alleles listed in online HNPCC databases, 13 of which had not been previously studied in functional assays. Using the highly sensitive lys2::InsE-A14 reversion rate assay, we determined the MMR proficiency conferred by each allele in the S288c strain of Saccharomyces cerevisiae. Seven alleles conferred a null phenotype for MMR and eight others showed significant MMR defects, suggesting that all 15 are likely to be pathogenic in humans. In addition, we observed a strong correlation between these results, limited results from previous functional assays and clinical data. To test whether the potential pathogenicity of certain alleles depends on the genetic background of the host, we examined the mutation rates conferred by the mlh1 alleles in a second yeast strain, SK1, which is ~0.7% divergent from S288c. Many alleles displayed a difference in MMR efficiency between strain backgrounds with decreasing differences as the severity of the MMR defect increased. These findings suggest that genetic background can play an important role in determining the pathogenicity of MMR alleles and may explain cases of atypical colorectal cancer inheritance.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Hereditary non-polyposis colorectal cancer (HNPCC; MIM no. 114500 [OMIM] ) or Lynch syndrome, accounts for 1–6% of total colon cancer cases and segregates in an autosomal dominant fashion within families (1,2, reviewed in 3,4). HNPCC is characterized by an early age of onset, high incidence of cancer in primary relatives and increased susceptibility to a variety of other cancers (5, reviewed in 4).

Germline mutations in the DNA mismatch repair (MMR) genes are associated with a large percentage of HNPCC cases. MMR is a highly conserved process that improves the fidelity of DNA replication approximately 1000-fold by excising DNA polymerase misincorporation errors so that the newly replicated strand is repaired (reviewed in 6). In eukaryotes, MutS{alpha} (MSH2-MSH6) or MutSß (MSH2-MSH3) recognize and bind to DNA mismatches to initiate repair. MutS{alpha} preferentially repairs base–base and single nucleotide loop mispairs, whereas MutSß preferentially repairs insertion/deletion loops up to 17 nucleotides in size. The MutS heterodimers primarily recruit MutL{alpha} (MLH1-PMS1 in baker's yeast, MLH1-PMS2 in humans), which is thought to act as a matchmaker to activate downstream factors that participate in excision and resynthesis steps.

Consistent with a defect in MMR, tumor samples from HNPCC patients display microsatellite instability (7,8). HNPCC is thought to result from the accumulation of mutations in specific tumor suppressor genes that control various cellular processes (reviewed in 9). The genes affected tend to feature short repeat sequences (microsatellites) that suffer frameshift mutations when MMR is compromised, and include the important cell growth inhibition genes TGFß1RII and IGFIIR, the cell cycle regulatory gene E2F4, the proapoptotic gene BAX and the MMR genes MSH2, MSH3 and MSH6 (reviewed in 9).

At present, there are over 500 variants in human MMR genes listed in various databases (http://www.insight-group.org/, 10; http://www.hgmd.org/, 11). Human MMR mutations are primarily found in MLH1 (~50%), MSH2 (~40%) and MSH6 (~10%) (10,12, reviewed in 13). Although the majority of the MLH1 variants are frameshift or nonsense mutations, approximately one-third are missense mutations. Because these mutations do not lead to a truncated protein product, pathogenicity of these variants is often difficult to determine. HNPCC is classified primarily with the Amsterdam I or other less stringent criteria (4,14). The Amsterdam criteria are based on the discovery of colorectal cancer in three or more family members with at least one diagnosis before 50 years of age. In addition, two generations must be affected and two affected persons must be first-degree relatives. However, not all cases of HNPCC satisfying these criteria clearly correspond to mutations in known MMR genes (15–17), and not all mutations in MMR genes observed in colon cancer patients segregate with the disease in Mendelian fashion (17,18, reviewed in 3). Many factors are likely to influence disease penetrance, including environment, epigenetic phenomena and elements in the genetic background of hosts (18–20).

In 2003, two families with colorectal cancer were identified that both featured two separate mutations (msh2, msh6) in MMR genes (18). The msh2 and msh6 mutations appeared to be silent polymorphisms; however, the presence of both mutations in a single individual appeared to be pathogenic. More recently, two msh6 mutations were found to display compound heterozygosity (21). In this case, the patient's parents, who were carriers of one mutation each, were not affected by the disease at 57 and 48 years of age, but the patient displayed an earlier-onset HNPCC phenotype. Interestingly, a missense mutation in codon 127 exon 3 of MSH2 had been described by many authors as a non-pathogenic polymorphism (2). However, when this mutation was associated with an additional mutation in MSH2, known to cause HNPCC on its own, the age of onset was lower than that seen in the pathogenic msh2 mutant alone (22). Together, these reports suggest that pathogenicity is likely to be modulated by genetic background.

HNPCC has a high cure rate if detected early, underscoring the importance of accurate diagnostic tools for this disease (reviewed in 4). It can be difficult to establish or rule out the pathogenicity of a given MMR allele based on clinical data due to the incomplete (80%) lifetime penetrance of HNPCC and the possibility of undetected mutations in other MMR genes. Functional assays, however, can provide important information to define pathogenicity. In this study, we characterized 28 S. cerevisiae mlh1 alleles corresponding to mutant alleles obtained from colorectal cancer patients. We primarily chose alleles that had not previously been characterized in functional assays, and that featured non-truncating point mutations in the hMLH1 protein in conserved residues between yeast and humans. To explore the idea of background-specific penetrance of MMR defects, the S288c-derived MLH1 HNPCC variants were introduced into S288c and SK1 strains of S. cerevisiae that display ~0.7% sequence divergence (20). As shown below, we found that many alleles conferred different phenotypes in the two strain backgrounds. These results suggest that single polymorphisms can display background-dependent defects in MMR and may explain cases of atypical colorectal cancer inheritance.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
mlh1 missense mutations based on HNPCC variants were selected for functional analysis in S288c and SK1 yeast strains because they had not been studied earlier (13 alleles), previous studies yielded conflicting results, or the segregation of the variant with HNPCC in families meeting the Amsterdam criteria was unclear or unknown (Tables 1 and 2; Figs 1 and 2). The mutations analysed in this study mapped primarily to the ATP binding and PMS1/MLH3/PMS2-interaction domains, which were shown previously to be important for MutL homolog function (Fig. 1; 23–26). The mutation rate conferred by each allele was measured in the highly sensitive lys2::InsE-A14 reversion assay (27); wild-type and mlh1{Delta} strains display a four order of magnitude difference in reversion rate (Fig. 2). The data are presented relative to the rate conferred by wild-type S288c MLH1 in the S288c strain background (1X=1.51x10–7).


Figure 4791
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  		Figure 1. Schematic representation of human MLH1 showing the location of mutations with respect to the ATP binding and PMS1/PMS2/MLH3 interaction domains. Amino acid substitutions in the equivalent positions in yeast MLH1 are shown in Table 1. The phenotypes conferred by the yeast mutations in the S288c strain are summarized here.

 


Figure 4792
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  		Figure 2. Characterization of mlh1 alleles in the lys2::InsE-A14 DNA slippage assay. The indicated human alleles are labeled as pathogenic or associated with HNPCC (Y, yes; N, no; I, intermediate; ?, ambiguous). Alleles that have not been examined previously in functional assays are indicated by a ‘U’. Pathogenicity was evaluated in in vitro human MMR assays (12,29), by co-immunoprecipitation, yeast two-hybrid interaction with hPMS2 or hEXO1, and MLH1 function in yeast MMR assays (24,28,30,34–36). Association was determined based on whether an allele segregates with HNPCC in families meeting the Amsterdam criteria (http://www.insight-group.org) except for R265S where the less-stringent Bethesda guidelines are met (33). The yeast alleles based on the human variants (Table 1) were introduced into the S288c and SK1 strain backgrounds and tested for reversion to Lys+. The reversion rate per generation and 95% CI are shown relative to the S288c strain bearing MLH1 (1X=1.51x10–7, n=number of independent cultures examined). As described in the text, introduction of the S288c MLH1 into the SK1 strain background caused an incompatibility between S288c MLH1 and SK1 PMS1. The mlh1-29 allele was shown previously to show a dramatic strain-specific difference in MMR function (32). Group classifications were determined by pair-wise Kruskal–Wallis tests as described in the text and Materials and Methods. Alleles were assigned as NM, non-mutator; LM, low mutator; LI, low intermediate mutator; I, intermediate mutator; M, mutator; HM, high mutator. Group shifts are displayed as values 0–4 to indicate how many group classifications were skipped by that allele between the different strain backgrounds.

 


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  		Table 1. Yeast plasmids bearing human mlh1 variants

 


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  		Table 2. Database information by allele

 
We employed a Kruskal–Wallis test to determine whether the difference in mutation rates between any two strains is significant (Materials and Methods). Individual network diagrams were constructed for the S288c and the SK1 strain backgrounds using pair-wise Kruskal–Wallis tests to place alleles into phenotypic groups. Alleles statistically similar to one another were placed in the same group. Alleles that were not statistically similar were still grouped together if they possessed overlap among the alleles to which they were each independently related. Occasionally, alleles were found to be similar but did not possess significant overlap with their other related partners. In this case, the allele was placed into the class where it had a higher number of related partners and/or was more statistically similar to alleles in that group. MMR proficiency classifications were then assigned in which those alleles falling into the phenotypic group with the lowest median mutation rates were called non-mutators (NM), or wild-type, followed by low mutators (LM), low-intermediate mutators (LI), intermediate mutators (I), mutators (M) and high mutators (HM). The group labels provide a simple way to classify and discuss the six different MMR proficiency groups identified by the Kruskal–Wallis statistical analysis and the subsequent network diagrams.

Unless otherwise stated, alleles will be identified based on their location in yeast MLH1. In the S288c strain background, four alleles conferred non-mutator phenotypes (E676G, Q90G, S415N and K764R), six low mutator (H733Y, M623K, M623A, L272V, A694T and R672Q), three intermediate (R265H, I22T and R265C), eight mutator (L559P, R265S, L666R, C74Y, N61S, P25L, I22F and E20D) and seven high mutator (L627H, P667L, K81E, S190P, R97P, R768W and C74R). Of these, E676G, L272V, I22T, L559P, R265S, L666R, N61S, I22F, E20D, L627H, S190P, R97P and R768W have not been previously tested in functional assays (Fig. 2 and Table 2).

Additional analyses were performed for the yeast alleles that correspond to the K618A and R659Q variants. Whereas the R659Q variant has not been reported to occur independently in HNPCC patients, K618A appears to segregate alone with colorectal cancer in some families that satisfy the Amsterdam criteria (Fig. 2; Tables 1 and 2). Curiously, these variants have been classified as pathogenic in some functional assays but non-pathogenic in others (24,28,29). To determine whether the association of these two mutations caused an MMR defect, we tested each mutation independently and then tested an allele containing both substitutions. Amino acid 618 is not precisely conserved from humans to yeast (residue 623 is an M in yeast). Therefore, we first tested S288c mlh1 with the M623K change, which simulates the normal human allele (30). Because the change did not disrupt MMR (Fig. 2), we made combinations of the analogous K618A (yeast K623A) and R659Q (yeast R672Q) changes. Our results show that the presence of either mutation alone conferred a low mutator phenotype in S288c yeast. The double mutation conferred a similar phenotype.

To determine whether the yeast alleles display a phenotype dependent on genetic background, we introduced the S288c MLH1 derivatives into the SK1 strain, which is ~0.7% sequence divergent from S288c (31). Previously, we found that SK1 PMS1 (kPMS1) is incompatible with S288c MLH1 (cMLH1), such that when S288c and SK1 strains are crossed, progeny harboring cMLH1 and kPMS1 display a small but significant MMR defect (100- to 300-fold increase in mutation rate over wild-type in the lys2::InsE-A14 reversion assay) (20). The molecular incompatibility underlying the cMLH1-kPMS1 effect was due to single amino acid polymorphism differences in MLH1 (D761 in S288c; G761 in SK1) and PMS1 (R818 in S288c; K822 in SK1) with cMLH1-D761-kPMS1-K822 being the incompatible combination (20).

Because of the known incompatibility between cMLH1 and kPMS1, and the fact that all of the alleles were cMLH1-derived, we expected to see higher overall mutation rates in the SK1 background. As shown in Figure 2, baseline mutation rates were higher in the SK1 background for most alleles. The known incompatibility (100- to 300-fold increase in mutation rate above wild-type) (20) is seen by comparing the reversion rates conferred by cMLH1 in the S288c versus the SK1 strain. mlh1-29 was identified as an allele that confers a low mutation rate in the S288c background (9.21-fold) and a completely null phenotype (13 000-fold) in SK1 (32). Given the large range of the assay and the mlh1-29 result, it is clear that despite the known incompatibility, we can easily detect additional strain-specific effects.

To test whether the alleles conferred strain specific phenotypes, the S288c strain set was rank ordered by mean mutation rate (Fig. 2). The SK1 dataset was then analysed by placing each SK1 allele next to its corresponding S288c allele. If the only background-specific difference present in these two yeast strains was the known cMLH1-kPMS1 incompatibility, then the SK1 data should show a rank order pattern similar to that seen for the S288c MMR proficiency groups. As shown in Figure 2, roughly half of the SK1 alleles (e.g. K764R, A694T, R265C, L666R, C74Y) deviated from the S288c rank order. To determine whether the severity of an allele's MMR defect was correlated with the likelihood of genetic background playing a role in its penetrance, we counted the number of categories shifted by that allele between strain backgrounds. For example, the wild-type cMLH1 gene was a non-mutator in the S288c background but an intermediate mutator in the SK1 background, thus showing a shift of three groups (Fig. 2). The E676G, Q90G and S415N alleles also showed this shift, and were thus similar to wild-type MLH1. On the whole, we found that as the mutation rate increased the group shift value decreased. This suggested that there was a threshold above which the genetic background of the carrier no longer affects an allele's pathogenicity. Moreover, it suggested that alleles conferring low mutator to intermediate mutator phenotypes were most likely to show background-specific differences.

Previous functional assays showed that the human R265H and R265C alleles conferred a non-null MMR defect (30). We found that the R265H allele displayed an intermediate mutator phenotype in S288c (219-fold) but a mutator phenotype in SK1 (2450-fold, Fig. 2). Interestingly, the R265C allele conferred a strain-specific defect as an intermediate mutator in S288c (1270-fold) but a high mutator in SK1 (9070-fold, Fig. 2). The allele R265S has not been previously analysed in functional assays (33). We found it to be a mutator in S288c (4190-fold) and a high mutator in SK1 (9340-fold). The large variation in MMR phenotypes among all three alleles along with the differential penetrance associated with the R265C allele suggests that residue 265, which lies within the ATP-binding domain of human MLH1 (Fig. 1), is of particular relevance to our investigation of background-specific effects.

K764R (human K751R) was the only allele that displayed a lower mutation rate in the SK1 background (51.9-fold). Prior to this study, our laboratory identified an Aspartic Acid at residue 761 in yeast cMLH1 to be the major cause of the incompatibility with kPMS1 (20). The close proximity of the K764R mutation to the polymorphism known to cause the incompatibility between cMLH1 and kPMS1 suggests that it acts as a suppressor. In addition, this allele was not shown to be pathogenic in previous functional assays in yeast (34). Taken together, these data suggest the presence of a background-specific difference for K764R that could be a determinant of pathogenicity in humans.

As described above, the Lysine at residue 618 in humans is not conserved in yeast (K618 is M623 in yeast). Interestingly, both of the yeast derivatives, M623K and M623A, and their double mutant combinations, M623K R672Q and M623A R672Q, showed differential penetrance between strain backgrounds, displaying much stronger disruptions of MMR in SK1 (M, 2690- to 3330-fold) than in S288c (LM, 6.52- to 7.55-fold). Although this finding is not directly relevant to human disease since L618/M623 is not a precisely conserved residue, it suggests that nearly silent mutations can cause significant disruptions of MMR function, similar to the yeast mlh1-29 mutation, that are dependent on the genetic background of the host.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
S. cerevisiae provides a simple yet effective system to determine the pathogenicity of HNPCC alleles in different genetic backgrounds. Our analysis of mlh1 alleles that correspond to HNPCC mutations correlated well with data obtained previously in a variety of assays for 13 of the 28 mlh1 mutations examined. These assays included analysis of MLH1 function in in vitro human MMR assays (12,29), analyses of human (35), yeast (34,36), and hybrid yeast–human (30) mlh1 alleles in yeast and biochemical analyses that measured MLH1-PMS2 and MLH1-EXO1 interactions and MLH1 localization (12,24,28,29,34).

In in vitro human MMR assays, the P28L, C77R and K84E mutations (P25L, C74R, K81E in yeast) conferred a defect in MMR (12,29). We found that strains bearing the yeast alleles were mutators or high mutators. These same in vitro assays classified S93G, R659Q, K618A and P654L as MMR proficient (12,29). Similarly, we found in yeast that Q90G (S93G in humans) was a non-mutator (wild-type) and that R672Q (R659Q in humans) and M623A (K618A in humans) were low mutators. Consistent with the prior assays utilizing a yeast system (30), we showed that two alleles, R265H and R265C, conferred intermediate mutator phenotypes. Finally, P667L, classified in humans (P654L) as pathogenic and showing a disruption in protein localization in human cells, conferred a high mutator phenotype in our assay. In contrast, experiments utilizing human cell extracts bearing the MLH1-P654L protein were found to be functional for MMR. This suggests that mutations that disrupt MLH1 localization in humans may be accurately modeled in yeast—an advantage of the yeast system over in vitro MMR assays. Together, the perfect correlation between our results and previous functional characterizations of the 13 aforementioned alleles indicates that the yeast system is a simple and accurate means to classify a large subset of alleles.

Importantly, 13 human alleles (E663G, L272V, I25T, L549P, R265S, L653R, N64S, I25F, E23D, L622H, S193P, R100P and R755W) have not been previously tested in functional assays (Fig. 2 and Table 2). We found that the E663G yeast equivalent conferred a non-mutator phenotype; the L272V equivalent is a low mutator; the I25T equivalent is an intermediate mutator; the L549P, R265S, L653R, N64S, I25F and E23D equivalents are all mutators; and the L622H, S193P, R100P and R755W equivalents are high mutators. This work not only provided functional data for new alleles but also indicates that the yeast system can be effectively used to establish the pathogenicity of alleles for which the clinical data are insufficient (30,34,36).

A limitation of the yeast assay is that pathogenicity can only be easily established for mutations in conserved regions of the MLH1 protein. Also, mutations that deleteriously affect mRNA splicing in humans may not be accurately characterized in yeast. For example, the R672Q mutation, which in humans (R659Q) consistently segregates with colon cancer (along with the K618A mutation), confers a low mutator phenotype in our assay and is likely to be nearly wild-type for MMR function. This mutation is located in a codon that has previously been implicated in proper splicing of the MLH1 transcript, and thus may cause disease by preventing proper splicing of MLH1 (29,37,38).

As shown in Table 2 and Figure 2, the information from major databases frequently disagrees on whether an allele consistently segregates with HNPCC. For example, the A681T allele in humans was classified in functional assays as non-pathogenic (24,28,29,35) but appears to segregate with HNPCC (Fig. 2; http://www.hgmd.org/). L272V and H718Y have not been previously examined in functional assays nor is it known whether these alleles segregate with disease. However, we show that all three corresponding yeast alleles (L272V, A694T and H733Y) display small increases in mutation rate in the S288c background and larger increases in the SK1 background compared to wild-type values, suggesting that genetic background could affect the penetrance of these alleles.

Limited clinical data alone cannot definitively establish or rule out the pathogenicity of a given allele due to the incomplete (80%) lifetime penetrance of HNPCC and the possibility of undetected mutations in other MMR genes. In cases of disagreement, functional assays should play an invaluable role in characterizing a given allele. Our data suggests that L653R, C77Y, P28L, E23D, L622H, P654L, K84E, S193P, R100P, R755W and C77R are likely to be pathogenic in humans, whereas E663G and S406N are unlikely to cause colorectal cancer, despite unclear clinical data for each allele (Table 2 and Fig. 2).

How can we reconcile the fact that some of the alleles found to be functionally wild-type in our assay appear to segregate with HNPCC within families? First, our assay only shows the effect that each allele confers in two different strain backgrounds. It is likely that different results would be seen for different strains of yeast. It is also possible that in these families the mutations are in fact silent polymorphisms, and that colorectal cancer is caused by sporadic mechanisms, such as hypermethylation of the MLH1 promoter (reviewed in 39). It is important to note that the MMR genes are also involved in cellular processes such as genetic recombination and the DNA damage response, raising the possibility that alleles that are functionally wild-type in mutation rate assays may in fact be cancer-causing due to defects in these other processes (reviewed in 40).

In close to half of the families that fulfill the clinical portion of the Amsterdam criteria for HNPCC, a germline mutation in hMLH1 or hMSH2 has not been found (3). Therefore, it is possible that in these patients, polymorphisms may exist in other genes that have not yet been implicated in MMR. These other mutated proteins could interact with the mutated MLH1, causing disease where the mlh1 mutation alone does not. The potential for such background-specific disease manifestations, as demonstrated in this study with yeast alleles, necessitates continued study of the key players in MMR-related diseases.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Strains, plasmids, media
The S288c and SK1 strains used in this study were EAY1366 (MATa leu2, ura3, trp1, his3, lys2::InsE-A14, mlh1{Delta}::KanMX4) and EAY1064 (MATa, ho::hisG, leu2::hisG, ura3, ade2::LK, his4xB, lys2::InsE-A14, mlh1{Delta}::KanMX4), respectively. All mlh1 plasmids are derivatives of pEAA109 (ARSH4, CEN6, LEU2, MLH1; 32). pRS315 (ARSH4, CEN6, LEU2) was described previously (41). All human alleles were previously reported in the following public databases: International Society for Gastrointestinal Hereditary Tumors (InSiGHT) http://www.insight-group.org and the Human Gene Mutation Database (HGMD) http://www.hgmd.org and are listed in Table 2. The human (Genbank accession no. NM_000249) and yeast (Genbank accession no. U07187) proteins were aligned using Vector NTI (Invitrogen Corporation). Mutant mlh1 derivatives of pEAA109 (Table 1) were obtained using the QuickChange XL Site-Directed Mutagenesis protocol (Stratagene, USA). Open reading frames and 75 bp upstream and downstream were sequenced to show that only the desired mutations were created. Plasmids were introduced into EAY1366 and EAY1064 using standard methods (42). Yeast strains were grown in minimal selective media (43).

Lys+ reversion assays
Reversion of the lys2::InsE-A14 allele to Lys+ was measured as described previously (20). In brief, 12–30 independent cultures, from at least three independent transformants per allele, were assayed in each strain background. Single colonies (~2 mM in diameter and capable of growing on lactate as the sole carbon source) grown on minimal Leucine drop-out media were used to inoculate 3 ml cultures of minimal Leucine drop-out media. After 24 h of growth at 30°C, appropriate dilutions of each culture were plated onto minimal Leucine drop-out (permissive) and Leucine, Lysine drop-out (restrictive) minimal plates and incubated for 3 days at 30°C. SK1 strains were sonicated (VirTis VirSonic 100) for 10 s at the first stage in each series of dilutions. The reversion rate µ was determined using the formula µ=f/ln(Nµ), where f is the frequency of revertants and N is the total number of revertants per 1 ml of overnight culture (27). Finally, the median reversion rate of the 12–30 independent cultures tested per allele and its 95% confidence intervals (CIs) are displayed in Figure 2.

Statistical analyses
Pair-wise Kruskal–Wallis tests were performed between all alleles in each strain background using MINITAB (http://www.minitab.com). This is a rank order test (12–30 independent cultures for each strain) that compares median mutation rates between strains. Differences in reversion rate were considered significant when P≤0.05.


   ACKNOWLEDGEMENTS
 
The authors thank members of the Alani laboratory for helpful comments, especially Amy Lyndaker and Jennifer Surtees, and Francoise Vermeylen in the Cornell University Office of Statistical Consulting for advice on data analysis. This study was supported by NIH research grant GM53085. J.J.W. was supported by an NIH training grant and N.S. by an undergraduate research fellowship from the Howard Hughes Medical Institute awarded to Cornell University.

Conflict of Interest statement. None declared.


   FOOTNOTES
 
{dagger} The first two authors contributed equally to this work. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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Y. Fan, W. Wang, M. Zhu, J. Zhou, J. Peng, L. Xu, Z. Hua, X. Gao, and Y. Wang
Analysis of hMLH1 Missense Mutations in East Asian Patients with Suspected Hereditary Nonpolyposis Colorectal Cancer
Clin. Cancer Res., December 15, 2007; 13(24): 7515 - 7521.
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