The Germline MLH1 K618A Variant and Susceptibility to Lynch Syndrome-Associated Tumors

The Journal of Molecular Diagnostics, Vol. 14, No. 3, May 2012 Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved. DOI: 10.1016/j.jmoldx.2012.01.006

The Germline MLH1 K618A Variant and Susceptibility to Lynch Syndrome-Associated Tumors

Fabiola Medeiros,* Noralane M. Lindor,† Fergus J. Couch,* and W. Edward Highsmith, Jr.* From the Departments of Laboratory Medicine and Pathology* and Medical Genetics,† Mayo Clinic, Rochester, Minnesota

Missense variants discovered during sequencing of cancer susceptibility genes can be problematic for clinical interpretation. MLH1 K618A, which results from a 2-bp alteration (AAG¡GCG) leading to a substitution of lysine to alanine in codon 618, has variously been interpreted as a pathogenic mutation, a variant of unknown significance, and a benign polymorphism. We evaluated the role of MLH1 K618A in predisposition to cancer by genotyping 1512 control subjects to assess its frequency in the general population. We also reviewed the literature concerning MLH1 K618A in families with colorectal cancer. The measured allele frequency of the K618A variant was 0.40%, which is remarkably close to the 0.44% summarized from 2491 control subjects in the literature. K618A was over-represented in families with suspected Lynch syndrome. In 1366 families, the allele frequency was 0.88% (OR ⴝ 2.1, 95% CI ⴝ 1.3 to 3.5; P ⴝ 0.006). In studies of sporadic cancers of the type associated with Lynch syndrome, K618A was overrepresented in 1742 cases (allele frequency of 0.83) (OR ⴝ 2.0, 95% CI ⴝ 1.2 to 3.2; P ⴝ 0.008). We conclude that MLH1 K618A is not a fully penetrant Lynch syndrome mutation, although it is not without effect, appearing to increase the risk of Lynch syndrome-associated tumors approximately twofold. Our systematic assessment approach may be useful for variants in other genes. ( J Mol Diagn 2012, 14:264-273; DOI: 10.1016/j.jmoldx.2012.01.006)

Lynch syndrome, or mismatch repair deficiency syndrome, previously termed hereditary nonpolyposis colorectal cancer (HNPCC), is an autosomal dominant cancer predisposition syndrome caused by mutations in DNA mismatch repair (MMR) genes, most commonly MLH1, MSH2, MSH6, or PMS2, and accounts for approximately 2% to 2.5% of colorectal cancers1–3 and approximately 2% of endometrial cancers.4 MLH1 inactivating mutations lead to an approximately 20-fold increased risk of cancer development and account for 50% of Lynch syndrome

264

cases.5,6 However, several germline missense alterations have been identified in MLH1 whose role in the pathogenesis of colonic epithelial neoplasia is less clear. MLH1 K618A is a prominent example of this problematic category. This variant results from a 2-bp alteration in exon 16, involving nucleotides 1852 and 1853 (AAG¡GCG) and leading to a substitution of lysine to alanine in codon 618. It was first identified in a mutation screening study of Swedish families with suspected HNPCC, performed in 1995.7 Since then, the significance of MLH1 K618A has been debatable, being reported variously in the peer-reviewed literature as a pathogenic mutation, a sequence of unknown significance, and a benign polymorphism. In most instances, this variant has been found incidentally in genetic screening, and no previous study has focused specifically on the significance of MLH1 K618A in hereditary colorectal patients. The K618A variant has been included in several studies evaluating the biochemical function of various missense changes in MLH1. For example, functional studies tabulated at the Mismatch Repair Gene Unclassified Variant Database include eight published studies that report on 28 individual assays for K618A-bearing MLH1 protein. Of these, 6 assays were interpreted as showing pathogenicity, 22 were interpreted as demonstrating that this variant was benign, and 10 were inconclusive (http://www.mmruv.info, last accessed April 20, 2011). Thus, the cancer susceptibility of individuals and families identified with MLH1 K618A is not understood.8 In the present study, we evaluated more precisely the role of MLH1 K618A in predisposition to cancer. We genotyped 1512 control subjects to assess the frequency of this variant in the general population. We also reviewed the experience of the Mayo Clinic Molecular Genetics Laboratory (MGL) in clinical Lynch syndrome testing and identified two cases in which individuals carried both the K618A variant and another, more typical MMR gene mutation. In This work was supported in part by the National Cancer Institute, NIH, under RFA #CA-95-011 via the Mayo Clinic Colon Cancer Family Registry (UO1 CA074800). The content of this manuscript does not necessarily reflect the views or policies of the National Cancer Institute nor any of the collaborating centers in the Cancer Family Registries, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. Accepted for publication January 10, 2012. Address reprint requests to W. Edward Highsmith, Jr., Ph.D., Mayo Clinic, 200 First Street SW, Rochester, MN 55905. E-mail: [email protected].

MLH1 K618A and Lynch Syndrome 265 JMD May 2012, Vol. 14, No. 3

addition, we performed a critical review of literature concerning MLH1 K618A in families with colorectal cancer and summarized the reported frequency of this variant in control subjects and in individuals with colorectal cancer.

Materials and Methods This study was reviewed and approved by the Mayo Clinic Institutional Review Board.

Population Frequency The study sample was 1512 control subjects of European origin who had samples submitted to the Mayo Clinic MGL for cystic fibrosis carrier screening. DNA was isolated from whole blood using a QIAamp 96 DNA blood kit (Qiagen, Valencia, CA). After completion of the ordered service, samples annotated as Caucasian or Northern European ethnicity were selected and strictly anonymized. Briefly, the labels were removed from the sample tubes and the tubes were scrambled. After that, 10% of the samples were discarded and the DNA from the remaining samples was aliquoted into 96-well microtiter trays labeled numerically, starting simply at 1. Although no information other than the ethnicity of the sample collection was retained, the collection was heavily weighted toward women of childbearing age.

Genotyping Methods Analysis of the K618A variant was performed using TaqMan chemistry (ABI Assays-on-Demand; Applied Biosystems, Foster City, CA) at the Mayo Clinic Genotyping Shared Resource. An anonymized sample known to be positive was provided as a positive heterozygous control.

MLH1 Mutation Detection MLH1 whole-gene sequencing and dosage analysis are part of the clinical services offered by the Mayo Clinic MGL for the diagnosis of Lynch syndrome. Under procedures in place since 2005, all 16 exons of the MLH1 gene are amplified in four to five multiplex PCR reactions and are sequenced using ABI BigDye terminators and internal, exon-specific sequencing primers. Data analysis was performed using Mutation Surveyor (SoftGenetics, College Station, PA). For some cases, the ordering physician requested both MLH1 and MSH2 sequencing. In these cases, the MSH2 gene was also sequenced using a similar method. Before 2005, MLH1 (and MSH2) mutation scanning was done by conformation-sensitive gel electrophoresis,9 followed by sequencing with 33P and manual interpretation of the autoradiographs.

In Silico Analysis The protein sequences of MLH1 homologs from 25 species (6 mammals, 4 other vertebrates, 1 echinoderm, 3 insects, 2 protozoan parasites, 1 nematode, 6 fungi, and 2 flowering plants) were aligned with ClustalW2 software (http://www.ebi.ac.uk/clustalw; last accessed May 9, 2011). The online sequence variation analysis packages SIFT (sort intolerant from tolerant; http://blocks.fhcrc.org/sift/ SIFT.html; last accessed May 9, 2011), PolyPhen (polymorphism phenotype; http://www.bork.embl-heidelberg.de/ PolyPhen; last accessed May 9, 2011), Align GVGD (http://agvgd.iarc.fr/agvgd_input.php; last accessed May 9, 2011), and PMut (http://mmb2.pcb.ub.es:8080/Pmut; last accessed May 9, 2011) were used according to the authors’ instructions. The BLOSUM62 matrix was retrieved from the ExPASy website (http://ca.expasy.org; last accessed May 9, 2011).

Statistical Analysis The significance of differences between groups was determined using Fisher’s exact test.

Literature Review All articles mentioning MLH1 K618A listed in the InSIGHT mismatch repair gene variant database8 and the Mismatch Repair Gene Unclassified Variant Database8 were reviewed. A PubMed search yielded several additional references. The review was focused on the following aspects of the scientific articles: i) Reported frequency of MLH1 K618A in colorectal cancer patients with suspected diagnosis of Lynch syndrome; ii) reported frequency of MLH1 K618A in control subjects and in patients with cancers of the type seen in Lynch syndrome (ie, colorectal, gastric, and endometrial cancers) who were not selected on the basis of any high-risk criteria or prior suspicion of having Lynch syndrome (unselected cancers); iii) classification of MLH1 K618A variant as pathogenic mutation, sequence of unknown significance, or benign polymorphism; iv) results for MSI status and MMR protein immunohistochemistry in MLH1 K618A-related colorectal adenocarcinomas when available; and v) findings from functional studies.

Results Genotyping Of a total of 1512 control subjects genotyped, 12 individuals were identified with MLH1 K618A. The resulting allele frequency is 0.40%, with a carrier rate of 1/126.

Microsatellite Instability Testing Before 2008, microsatellite instability (MSI) testing was done using a laboratory-developed panel of 10 short tandem repeat markers, as described by Baudhuin et al.10 This panel contained mono- and dinucleotide markers. Since June 2008, the laboratory has used the MSI Analysis System from Promega (Madison, WI).11

Mayo Clinic MGL Experience Over the past 8 years, out of approximately 600 MLH1 tests, the MGL at the Mayo Clinic has identified 13 cases carrying the K618A variant (Table 1). Because the MGL typically (but not exclusively) performs MLH1 mutation scanning after an individual’s tumor has tested positive

266 Medeiros et al JMD May 2012, Vol. 14, No. 3

Table 1.

Individuals with Colorectal Cancer in Whom Germline MLH1 K618 Was Seen at the Mayo Clinic Molecular Genetics Laboratory

Case no.

MSI

IHC

Other reasons for referral

1 2 3 4 5 6 7 8 9 10 11 12 13

MSI-H MSI-H MSI-L MSI-H Unknown Unknown Unknown Unknown MSS MSS MSS MSS Not done

Loss of MLH1 and PMS2 Normal Loss of MLH1 and PMS2 Loss of PMS2 Unknown Unknown Unknown Unknown Normal Normal Normal Normal Loss of MLH1

Unknown Unknown Unknown Unknown HNPCC, family hx HNPCC, clinical dx HNPCC, clinical dx Unknown Colon cancer, family hx Colon cancer, family hx Colon cancer, family hx Colon cancer, family hx Colon cancer, family hx

Sequencing result MLH1 MLH1 MLH1 MLH1 MLH1 MLH1 MLH1 MLH1 MLH1 MLH1 MLH1 MLH1 MLH1

K618A K618A K618A K618A K618A K618A and MSH2 R487X K618A and MLH1 V506A K618A and MSH2 2204delT K618A K618A K618A K618A K618A

dx, diagnosis; HNPCC, hereditary nonpolyposis colorectal cancer; hx, history; MSI-H, microsatellite instability, high; MSI-L, microsatellite instability, low; MSS, microsatellite stable.

for MSI or loss of MLH1 protein by IHC, or both, this set is biased toward cases with high MSI (MSI-H). Eight of the 13 K618A-positive cases (cases 1 to 8) were tested for clinical purposes. Six of these had MLH1 testing only (cases 1 to 6) and the other two (cases 7 and 8) were tested for MSH2 mutations as well. Cases 1 and 2 were tested by conformation-sensitive gel electrophoresis with follow-up sequencing of positive exons. Of these eight cases, one was referred for MSI-H and absence of MLH1 and PMS2 by IHC, one for MSI-H only, one for low MSI (MSI-L) and absence of MLH1 and PMS2 by IHC, one for MSI-H and absence of PMS2 by IHC, one for a suspected family history of Lynch syndrome, and two for a clinical diagnosis of probable Lynch syndrome; for one case, the reason for testing was not given. Case 6 had a known pathogenic MLH1 mutation, R487X, in addition to the K618A variant. Case 7 had a second MLH1 variant of uncertain significance, V506A, and case 8 had a pathogenic MSH2 mutation, 2204delT. In addition to the clinical laboratory testing, the Mayo Clinic Colon Cancer Family Registry lists five cases with K618A (cases 9 to 13). Colorectal tumors from four of these cases were microsatellite stable (MSS) and had normal IHC; the fifth case exhibited loss of MLH1 by IHC.

In Silico Analysis The lysine (K) at the positions corresponding to the human codon 618 is reasonably well conserved across species, diverging only in the fungi, protozoan parasites, and the worm. Of note, this position is also lysine in the two flowering plants for which a protein sequence was available (Table 2). The results using the online analysis packages were mixed. When the 25 protein sequences described above were loaded, SIFT predicted that all sequence variants were tolerated. PolyPhen predicted that the alanine (A) change was probably damaging; PolyPhen performs its own BLAST search using the input protein identification number. For Swiss Prot ID P40692, the PolyPhen program retrieved 44 sequences, including 23 of the 25 listed above, with additional fungal sequences. The flowering plant sequences were not retrieved. PMut predicted the alanine substitution would be pathological, with a confidence rating

of 6 (on a scale from 0, low confidence, to 9, high confidence). Align GVGD predicted that K618A would be a neutral variant. The BLOSUM62 matrix indicated that lysine to alanine changes had a score of ⫺1, corresponding to a moderately low probability of occurrence. The results of the in silico analyses are summarized in Table 3.

Literature Review Overview A total of 28 articles were identified with studies that had patients or control subjects harboring the MLH1

Table 2. Alignment of MLH1 Homologs Organism Mammals Human (Homo sapiens) Chimpanzee (Pan troglodytes) Dog (Canis familiaris) Opossum (Didelphis marsupialis virginiana) Mouse (Mus musculus) Rat (Rattus norvegicus) Other vertebrates Chicken (Gallus gallus) Frog (Xenopus laevis) Zebrafish (Danio rerio) Pufferfish (Tetraodon nigroviridis) Other animals Sea urchin (Strongylocentrotus purpuratus) Insects Drosophila melanogaster Honeybee (Apis mellifera) Mosquito (Anopheles gambiae) Protozoan parasites, nematodes Plasmodium falciparum Trypanosoma brucei Caenorhabditis elegans Fungi Saccharomyces cerevisiae Ashbya gossypii Neurospora crassa Aspergillus fumigatus Cryptococcus neoformans Ustilago maydis Flowering plants Arabidopsis thaliana Rice (Oryza sativa)

MLH1 homolog . . .KKKAEML. . . . . .KKKAEML. . . . . .KKKAEML. . . . . .KRKAEML. . . . . .KKKAEML. . . . . .KKKAEML. . . . . .KKKTEML. . . . . .KKKTEML. . . . . .KQKAEML. . . . . .KRKSEML. . . . . .KSKADML. . . . . .LKKAPIM. . . . . .LEKADML. . . . . .VSKAPVL. . . . . .YTYNEMY. . . . . .CNWRYML. . . . . .AEHADLL. . . . . .WDMSSML. . . . . .WDMREML. . . . . .VERREML. . . . . .IDRREML. . . . . .RDRQEML. . . . . .LENAEML. . . . . .KEKAEML. . . . . .KEKAEML. . .

MLH1 K618A and Lynch Syndrome 267 JMD May 2012, Vol. 14, No. 3

Table 3.

Online Mutation Classification Tool Analyses of MLH1 K618A Online tool (source)

Interpretation for K618A

Number of aligned sequences

SIFT (http://blocks.fhcrc.org/sift/SIFT.html) PolyPhen (http://www.bork.embl-heidelberg.de/PolyPhen) PMut (http://mmb2.pcb.ub.es:8080/Pmut) AlignGVGD (http://agvgd.iarc.fr/agvgd_input.php) BLOSUM62 (http://ca.expasy.org)

Tolerated Probably damaging Pathological Neutral Low probability of occurrence

25 (user-selected) 44 (not user-selected) 25 (user-selected) 25 (user-selected) Not available

K618A variant: 19 articles concerned the identification of MMR variants in colorectal cancer families with suspected diagnosis of Lynch syndrome and 11 articles evaluated the frequency of MLH1 mutations (including K618A) in cohorts of patients with unselected carcinomas of various sites and normal control subjects. The mutation testing methods varied widely across studies, but most of the studies used direct sequencing or screening techniques followed by direct sequencing. In addition, six functional studies were available. Many published studies used some or all of the online computational tools that we used in the present work (Table 3). Two articles, however, carried the informatics analyses substantially further. MLH1 K618A in Colorectal Cancer Families We identified 19 articles reporting a total of 1545 individuals with suspected diagnosis of Lynch syndrome (Table 4). The studies used different inclusion criteria: either the Amsterdam, modified Amsterdam, or Bethesda criteria.30 –34 MLH1 K618A was encountered in 24 of 1366 subjects, providing an allele frequency of 0.88%. For age at onset, sufficient information was not available to allow comparing K618Apositive cases with all cases. In all 19 articles, the authors attempted to classify the pathogenicity of MLH1 K618A: in 12 articles it was categorized as a mutation, and in 7 articles as a variant of unknown significance. No time-related trend was noted.

Table 4.

Of 24 individuals from colorectal cancer families found to carry MLH1 K618A, 11 were evaluated for tumor MSI status; 3 colonic tumors were MSI-H, 2 were MSI-L, and 6 were MSS. Immunohistochemistry for MLH1 analysis was available for 10 cases; 7 had normal results and 3 had loss of MLH1 expression. Out of all 24 cases, 3 patients were found to carry additional alterations in MMR genes. Two probands (in different families) also carried an MLH1 variant of uncertain significance in addition to K618A: one had V716M and one had V506A. MLH1 K618A in Control Subjects and Unselected Carcinoma Patients Eleven studies investigated the frequency of MLH1 K618A in healthy subjects and in individuals with unselected cancers (Table 5). Of a total of 2869 healthy subjects, the MLH1 K618A alteration was detected in 25 (allele frequency ⫽ 0.44%). Of a total of 1742 individuals with unselected colorectal adenoma or carcinoma, gastric adenocarcinoma, and endometrial adenocarcinoma, the MLH1 K618A alteration was detected in 29 (allele frequency ⫽ 0.83%). Functional Studies Six groups of investigators have reported in vitro MLH1-PMS2 binding assays. Guerrette et al43 and Kondo

MLH1 K618A in Cases with a Strong Family History of Lynch Syndrome

Reference 7

Tannergård et al Mauillon et al12 Weber et al13 Wijnen et al14 Wahlberg et al15 Syngal et al16 Fidalgo et al17 Scott et al18 Salahshor et al19 Müller-Koch et al20 Gille et al21 Caldes et al22 Scartozzi et al23 Ward et al24 Caldes et al25 Same Wolf et al26 Hegde et al27 Belvederesi et al28 Bianchi et al29

Total families (no.)

MLH1 K618A (no.)

39 17 32 125 142 70 20 95 15 254 126 56 37 28 58

1 1 1 1 1 1 1 1 1 1 1 2 1 1 5

46 23 84 99

1 1 1 1

Additional alterations

MLH1 V506A

MLH1 V716M MSH6 IVS5-16C⬎T

MSI

IHC

Classification

NA NA NA NA NA NA NA NA NA NA MSS MSI-H MSI-L MSS 1 MSI-L 4 MSS MSS

NA NA NA NA NA NA NA NA Normal NA NA NA Loss Normal Normal

VUS Mutation Mutation Mutation Mutation VUS Mutation Mutation Mutation Mutation VUS Mutation Mutation VUS VUS

NA

MSI-L NA

Loss Loss

VUS Mutation VUS Mutation

MSI-H, microsatellite instability-high; MSI-L, microsatellite instability-low; MSS, microsatellite stable; NA, not available; VUS, variant of uncertain significance.

268 Medeiros et al JMD May 2012, Vol. 14, No. 3

Table 5.

MLH1 K618A in Control Subjects and Sporadic Neoplasms (Colorectal, Gastric, and Endometrial)

Reference 12

Mauillon et al Farrington et al35 Liu et al36 Samowitz et al37 Hudler et al38 Fearnhead et al39 Pinol et al40 Hampel et al4 Blasi et al41 Bianchi et al29 Barnetson et al42

Healthy individuals (no.)

K618A (no.)

28 26 280 0 100 482 0 140 200 60 1553

0 0 1 0 0 10 0 0 0 0 14

Unselected neoplasms (no.) 0 50 180 130 7 124 91 118 0 110 932

CRC ⬍30 yo CRC CRC MSI-H Gastric carcinoma MSI-H Multiple CRC adenoma CRC, MSI-H/Loss of MLH1 Endometrial carcinoma, MSI-H and MSI-L CRC CRC ⬍55 yo

K618A (no.)

MSI

0 1 3 1 1 4 1 2 0 0 16

NA MSS MSS MSI-H MSI-H NA MSI-H MSI-H NA NA 6 tumors MSS

CRC, colorectal cancer; MSI-H, microsatellite instability, high; MSI-L, microsatellite instability, low; MSS, microsatellite stable; NA, not available; yo, years old.

et al44 described 85% and 95% reductions, respectively, of PMS2 binding to K618A MLH1, compared with wild-type protein. Belvederesi et al28 also described a reduction, but did not provide a quantitative value. In contrast, Raevaara et al,45 Bianchi et al,29 and Kosinski et al46 found normal MLH1-PMS2 in vitro binding activity in their studies. Raevaara et al45 also performed other in vitro MLH1 protein functional studies, including protein expression and stability, subcellular localization, protein-protein interaction, and repair efficiency. MLH1 K618A was found to be normal by all parameters. Wanat et al47 performed a reversion rate assay to determine MMR proficiency in Saccharomyces cerevisiae. There was significant disruption of MMR function, leading the authors to suggest a possible pathogenic potential; however, the authors also note that this finding may be not directly relevant to human disease because this residue is not conserved in yeast. Blasi et al41 investigated the functional role of MLH1 K618A, among other alterations, using the complementation of the phenotype of a MLH1-defective subclone of a human ovarian carcinoma cell line by transfection of constructs encoding altered MLH1 proteins. Measurements of resistance and tolerance to methylating agents, mutation rate at HPRT1, MSI, and steady-state levels of DNA 8-oxoguanine were used to define the MMR status of transfected clones. Transfection with MLH1 K618A was ineffective at reversing the phenotype of the MLH1defective cells analyzed, suggesting a pathogenic role. Perera and Bapat48 transfected the MLH1-deficient colon cell line HCT116 with wild-type or various mutated MLH1 constructs, including MLH1 K618A. These investigators found the expression of MLH1 K618A to be substantially lower than wild-type control expression. They verified their observations using both pulse-chase and cycloheximide protein synthesis inhibition. The half-life of MLH1 K618A was approximately one third that of the wild-type protein (3 to 4 hours versus 10 to 10.5 hours). Notably, they found that the MLH1 K618A protein was able to heterodimerize with PMS2 normally.48 Recently, Perera et al49 introduced a novel method for determining the effect of MLH1 missense mutations on RNA stability. They used a publicly available software package, PeakPicker (http://genomequebec.mcgill.ca/ publications/pastinen), to assess quantitative allele ratios of wild-type and variant sequences in PCR amplified

cDNA. They found that the ratio of K618A to wild-type sequence was greatly reduced (from 0.5 to 0.05) in the lymphocyte RNA from one patient who developed colon cancer at age 34, which suggests that the mutant RNA was unstable; however, they also found that the K618A allele had been passed from the proband’s father, whose allelic ratio was normal (0.5).

In Silico Analysis Chan et al50 used carefully curated multiple species sequence alignments as the platform to evaluate several computational methods for the classification of missense variants in five genes, including MLH1. These authors did not report on specific gene variants, but focused on the predictive values obtained in sets of previously characterized amino acid changes in each of the five genes. They found that when four of four or three of four predictions from SIFT, PolyPhen, Align GVGD, and BLOSUM62 agreed (either benign or deleterious), the overall predictive value was higher than for any of the algorithms alone. Unfortunately, these conditions are not met for K618A (Table 3). In 2005, Stone and Sidow51 described a method for classification of missense mutations that uses both a weighted ortholog alignment and calculation of the change in amino acid properties between wild-type and variant. Because of the correlation between amino acid physicochemical properties, such as polarity and hydropathy, the properties are decorrelated using principal component analysis. Each amino acid then occupies a unique position in a three-dimensional principal component plot and thus can be compared. A noteworthy feature of this multivariate analysis of protein polymorphism (MAPP) algorithm is that it can be optimized for specific genes. Chao et al52 optimized the MAPP method to MMR genes (MAPP-MMR). Using a training set of characterized deleterious and neutral variants and receiver-operator curve analysis, the investigators identified a MAPPMMR score of 4.55 as the optimal threshold for classifying deleterious versus neutral variants. The authors then used this method to calculate a score for 301 MLH1 and MSH2 variants, including MLH1 K618A, for which the score was 5.06.52

MLH1 K618A and Lynch Syndrome 269 JMD May 2012, Vol. 14, No. 3

Table 6.

MLH1 K618A Allele Frequency in Normal Control Subjects, Individuals with Suspected Lynch Syndrome, and Individuals with Sporadic Cancers (Colorectal, Gastric, and Endometrial) Group

Individuals in group (no.)

K618A alleles (no.)

Allele frequency (%)

Normal controls, present study Normal controls, literature† Total, normal controls Lynch syndrome families‡ Sporadic cancers†

1512 2869 4381 1366 1742

12 25 37 24 29

0.40 0.44 0.42 0.88 0.83

P value*

OR (95% CI)

1.00 0.90

0.94 (0.49–1.79) 1.0 (0.62–0.1.7)

0.006 0.008

2.1 (1.3–3.5) 2.0 (1.2–3.2)

*Versus the total number of normal controls. Fisher’s exact test. † References are given in Table 5. ‡ References are given in Table 4. CI, confidence interval; OR, odds ratio.

Discussion Lynch syndrome exhibits substantial genetic heterogeneity, due to mutations in multiple genes, and also allelic heterogeneity, due to a wide variety of mutations in each of the involved genes. The current recommendations for Lynch syndrome testing begin with clinical presentation and family history for colorectal or endometrial cancer. Because ⬎95% of Lynch syndrome colorectal cancer cases exhibit MSI, examination of tumor tissue for MSI and/or IHC evidence of loss of MMR protein expression is highly recommended.53,54 For patients exhibiting MSI-H and/or loss of specific MMR proteins, DNA sequencing of the targeted gene is the next step in the diagnostic algorithm. Even in the face of high allelic heterogeneity, interpretation of MMR gene sequencing is often straightforward, because the majority of mutations are nonsense, frameshifts, or multiexon deletions (all of which are typically assumed to be pathogenic). Nonetheless, a nontrivial fraction of cases reveal missense changes in one or more MMR gene sequences. These are typically more difficult to interpret, because it is often not clear whether a given amino acid change would result in protein dysfunction. This problem is encountered across a great many genetic disorders. Classical methods of determining the effect of a missense mutation on gene product function include segregation analysis within or across families to generate a significant LOD score (log10 of odds score), or performing functional studies of the mutant protein using either cellular or in vitro assays. However, it is seldom possible to gather a large enough number of families to prove pathogenicity using segregation analysis. Furthermore, although measurement of activity of an enzyme may be straightforward, measuring the correct functional parameters for a protein that has multiple interactions and functions may be more problematic. In particular, not all functions or protein interactions disrupted by mutations necessarily influence cancer risk. In addition, evaluation of the effects of mutations on individual functional domains (rather than on full-length proteins) may complicate the interpretation of functional data. Here we report our allele frequency study in a European-origin population and our review of the literature to determine composite allele frequencies in normal control subjects, in individuals with unselected cancers of the type associated with Lynch syndrome, and in individuals with suspected Lynch syndrome based on family history

and MSI and/or IHC testing. The numbers of cases and the allele frequencies for each of these groups is given in Table 6. Significance between groups was tested using Fisher’s exact test. Because the difference between our allele frequency study (allele frequency of 0.40%) did not differ from the results obtained by summing the control cases shown in Table 5 (allele frequency of 0.44%) (P ⫽ 0.864, Fisher’s exact test), the results of these two groups were combined for comparison of the Lynch syndrome and unselected cancer groups. There was a clear over-representation of K618A alleles in the families with suspected Lynch syndrome reported in the literature. In 1366 families reported in the 19 studies reviewed, there were 24 heterozygous carriers of the K618A variant (allele frequency of 0.88%). This is significantly higher than the combined control groups (OR ⫽ 2.1; 95% CI ⫽ 1.3 to 3.5; P ⫽ 0.006). In the studies that reported analysis of sporadic cancers of the type associated with Lynch syndrome (colorectal, gastric, and endometrial cancers), 1742 cases had MLH1 whole-gene sequencing performed, typically because of MSI-H and/or abnormal MMR protein IHC. The K618A allele was over-represented in this group; 29 cases were identified (allele frequency of 0.83%). As for the suspected Lynch syndrome cases, the difference was statistically significant, compared with the control group (OR ⫽ 2.0; 95% CI ⫽ 1.2 to 3.2; P ⫽ 0.008). There was no significant difference in allele frequency of the K618A allele between the individuals with suspected Lynch syndrome and the individuals with unselected Lynch syndrome-associated tumors (P ⫽ 0.89). Barnetson et al42 studied a large number of colorectal cancer patients with an age at onset of ⬍55 years and found that K618A was almost twofold increased in the cases versus population controls; however, the 95% confidence interval was wide (0.93 to 3.95), presumably because, even though they compared 932 cases to 1553 controls, the numbers were too small to detect a significant difference. When pooled with other studies from the literature, however, a sufficient number of cases and controls are available to detect a twofold difference. Based on these observations, wherein germline K618A allele is over-represented in Lynch syndrome cancers but is also not uncommon in the normal population, it appears that this variant is not a classical, high-penetrance mutation, but neither is it completely without effect. We conclude that K618A is a risk factor, but not a highly

270 Medeiros et al JMD May 2012, Vol. 14, No. 3

penetrant mutation, for the development of Lynch syndrome cancers. From the Mayo Clinic MGL series, eight patients were identified as heterozygous carriers of K618A. Because MLH1 sequencing is typically done only after the tumor has been shown to be MSI-H and/or negative for one or more MMR proteins by IHC, this set is heavily biased, and does not cast much light on the issue of whether the K618A variant is pathogenic. Four of these cases (cases 1 to 4) were sent to the MGL from outside facilities; clinical information and age at onset was not available. The other four patients were seen at the Mayo Clinic; two (cases 6 and 8) harbored known pathogenic mutations in addition to the K618A variant (one in MLH1 and one in MSH2). Because no additional family members were available, we were unable to determine whether the mutations in MLH1 were in cis or in trans configuration. Cases of individuals with two pathogenic mutations in one MMR protein or with mutations in more than one MMR protein have been reported (reviewed by Felton et al55). Individuals with biallelic mutation in one MMR gene, including MLH1, characteristically have very early-onset colorectal cancers, hematological malignancies, brain tumors, and a neurofibromatosis-like cutaneous presentation. Neither of our two patients with an additional MLH1 variant had presentations that were unusual for Lynch syndrome. One individual, with an MLH1 genotype of K618A/R487X, developed a mucinous adenocarcinoma of the hepatic flexure as well as a ductal carcinoma of the breast, both at age 41. The other individual, with the K618A variant and a pathogenic 2204delT mutation in MSH2, had a colectomy after developing a carcinoma in situ in the ascending colon at age 54. He was from a sibship of 13, in whom colorectal cancers were diagnosed in three siblings, at ages 35, 42, and 52; breast cancer had been diagnosed at ages 48 and 62. His mother had breast cancer in her 70s and died at age 92. His father had no cancers and died at age 87. Several family members of this individual were tested for both the MLH1 variant and the MSH2 mutation. Two sisters had K618A without the MSH2 mutation. Of these, one had a hysterectomy at age 45 for “dysplasia” (site not specified), and one (as already noted) had breast cancer at age 62. We also included five cases (cases 9 to 13) with K618A from a Mayo Clinic research study in which MLH1 sequencing was done for research purposes and not necessarily because MMR defects have been identified. The Mayo Clinic experience shows that K618A does not occur uniquely in MMR-deficient tumors and that it has little, if any, modulating effect on disease presentation when found in conjunction with a nonsense or frameshift MMR gene mutation. Together, these observations argue against K618A being classified as a pathogenic mutation. The data are consistent with either neutral or modest risk. Several mutation classification tools have been developed and are available online. Among the most widely used are SIFT, PolyPhen, PMut, and Align GVGD. Notably, analysis of the K618A variant with these tools produced mixed results (Table 2). Of the five tools with which K618A was evaluated (the four just listed, and also the BLOSUM62 matrix), three favored a pathogenic interpre-

tation (PolyPhen, PMut, and BLOSUM62) and two favored a benign interpretation (SIFT, Align GVGD). Although these observations may be consistent with the conception of K618A as a low-penetrance or partial-penetrance mutation, more likely they simply illuminate the limitations of current in silico variant classification models. Notably, the MAPP-MMR score for the K618A variant was 5.06, which exceeds the cutoff value of 4.55 for pathogenicity; however, authentic deleterious mutations had a mean value of 16.5, and variants of uncertain significance had a mean value of 10.8. Thus, the value obtained for K618A should be interpreted as equivocal, or consistent with a variant of uncertain significance.52 Functional testing of synthetic or natural gene products has the advantage of not requiring detailed knowledge of family history or characterization of segregation of variants with disease in multiple families. In many cases, assays can be designed to directly assess the effect of specific sequence variants on protein function in vitro. Several groups of investigators have performed a variety of functional studies of mutant MLH1. Six groups have reported the effect of the K618A variant on MLH1-PMS2 binding. The results were mixed, with three studies reporting significantly decreased but not absent PMS2 binding by K618A MLH1, two reporting no difference in PMS2 binding compared with protein with the wild-type sequence, and one reporting ambiguous results. Guerrette et al43 used 35S-labeled MLH1 prepared by a coupled in vitro transcription-translation (IVTT) method to track the binding to a GST-PMS2 fusion protein. 35SMLH1/GST-PMS2 dimers were captured using glutathione-conjugated beads. These authors showed an 85% reduction of K618A MLH1 binding to GST-PMS2. They interpreted the observed loss of MLH1 and PMS2 binding induced by K618A and other missense mutations as consistent with a causative role in the development of Lynch syndrome tumors. Kondo et al44 used a GST-IVTT method (in addition to a yeast two-hybrid system), capturing radiolabeled wild-type and mutant MLH1 proteins with a GST-PMS2 construct. They found that K618A bound approximately 5% of the PMS2 that the wild-type construct did. Belvederesi et al28 used mutant GSTMLH1 constructs prebound to glutathione beads and captured myc-labeled PMS2. In detection of the captured PMS2 by Western blot using anti-myc antisera, PMS2 binding by the K618A construct was decreased, but not eliminated. The authors interpreted these results as ambiguous, and concluded that the pathogenicity of K618A was questionable. Perera and Bapat48 expressed MLH1 constructs in HCT116 cells, incubated cell lysates with anti-MLH1 antisera, captured the complexes with protein A/G beads, and analyzed the relative abundances of MLH1 and PMS2 by Western blotting. Although the amounts of MLH1 and PMS2 were balanced, indicating normal affinity for each other, they noted that the amounts of MLH1 were sharply decreased, compared with controls, and that the decrease in protein expression was due to a decrease in protein stability.48 Kosinski et al46 transfected wild-type or mutant MLH1 along with wild-type PMS2 into HEK293T cells and analyzed the MMR proteins by Western blot after incubation

MLH1 K618A and Lynch Syndrome 271 JMD May 2012, Vol. 14, No. 3

with MLH1 antisera and protein G beads. They found no disruption of PMS2 binding or MLH1 expression. This group also built a detailed molecular model of the interacting domains of MLH1 and PMS2. Although K618 is often described as being in the PMS2 binding domain (eg, Figure 2 in Chao et al 200852), this more detailed model shows that position 618 is not directly involved in PMS2 binding.46 Raevaara et al45 used a somewhat different experimental design. They coexpressed mutant MLH1 and wild-type PMS2 from baculovirus constructs in Sf9 cells. For the interaction studies, lysates of these insect cells were incubated with either anti-MLH1 or anti-PMS2 antibodies. The immune complexes were captured with protein A/G beads, and MLH1 and PMS2 were detected by Western blot. Under this method, the interaction of K618A MLH1 with PMS2 was found to be normal. Bianchi et al29 report the use of a pull-down assay, but the analytical method was not described and the data were not shown. The results of these interaction studies are difficult to interpret. The first three studies (Guerrette et al,43 Kondo et al,44 and Belvederesi et al28) show that the K618A variant has substantially lower ability to bind PMS2, but that it still binds a nontrivial fraction of the available PMS2. The studies by Raevaara et al45 and Kosinski et al,46 however, showed no difference between K618A and wild-type MLH1 constructs with respect to PMS2 binding. The Raevaara group used a method quite different from those of the other four studies, one that may not be able to distinguish between normal and reduced binding. On the other hand, their approach was the only one to use a cellular assay in which the MLH1-PMS2 complex was formed in vivo. In interpreting these results, it is important to keep in mind that none of the described methods (all of which, whether cell-based assays or biochemical assays, are performed in the absence of other members of the DNA MMR complexes, in the absence of DNA, and also in the absence of DNA damage) have been validated for sensitivity and specificity for the prediction of cancer predisposition. In any case, it is clear that the K618A variant does not completely eliminate PMS2 binding. The clinical significance of reduced PMS2 binding is not clear. It may be that this residual binding, with the production of at least some functional MLH1/PMS2 dimer, is consistent with the interpretation of the MLH1 K618A variant not as a classical, fully penetrant mutation but rather as a risk factor for the development of colorectal cancer, as is suggested by the population data. Functional studies of the role of K618A that examined parameters other than PMS2 binding have also yielded discordant results. Raevaara et al45 performed several in vitro studies, including protein expression and stability, subcellular localization, protein-protein interaction, and repair efficiency. They found that K618A MLH1 did not perform differently compared with wild-type protein in any of the assays.45 In contrast, Blasi et al41 found that K618A MLH1 did not complement any of the MMR deficiency phenotypes, tolerance to methylating agents, mutation rate at HPRT1, MSI, and steady-state levels of DNA 8-oxoguanine in an MLH1-defective human cell line. Some have postulated that K618A may be a low-pen-

etrance allele. As already noted, Blasi et al41 found that K618A was nonfunctional in several assays. They also found that it did not segregate with disease in two families, and noted that these observations together are consistent with a low-penetrance Lynch syndrome mutation. Gargiulo et al56 came to the same conclusion after finding K618A in an unaffected branch of a Lynch syndrome family. Our observations confirm these findings and also greatly extend them. In conclusion, we have presented a population frequency study of the germline MLH1 K618A variant and a review of suspected Lynch syndrome cases with this variant seen at the Mayo Clinic. In addition, we have reviewed the literature and summarized the reported allele frequency of this variant in three groups: normal control subjects, individuals from families with suspected Lynch syndrome, and individuals with unselected tumors of the type seen in Lynch syndrome. The allele frequency in our European-origin samples and the frequency abstracted from the literature are remarkably consistent (⬃0.4%). The allele frequency in likely Lynch syndrome families and in individuals with unselected Lynch syndrome type tumors is significantly greater (approximately double), reaching statistical significance in both cases. We have presented data on a Lynch syndrome patient who carries an additional, truncating mutation in the MLH1 gene and showed that this individual does not have the clinical features expected when biallelic MLH1 mutations are present. We have also presented a case in which the proband has mutations in both the MLH1 and MSH2 genes. Such patients do not, however, appear to present with as early and severe symptoms as do patients with biallelic mutations. We used five different publicly available missense analysis tools, with mixed results, and have reviewed the literature concerning functional studies of K618A MLH1 constructs, again with mixed results. Taken together, the data presented here make it clear that the germline MLH1 K618A variant is not a classical, fully penetrant mutation. However, as the increased allele frequency in people with Lynch syndrome associated tumors and the decreased interaction with PMS2 demonstrate, neither is it completely without effect. We conclude that this sequence variant is a risk factor for the development of Lynch syndrome colorectal, endometrial, and gastric tumors. The odds ratio, as calculated from the population data, indicates an approximately twofold increased risk. This will, of course, need to be confirmed in independent association studies with sufficient statistical power to adequately detect such a relatively small effect. Several caveats to our conclusions should be pointed out. First, because the control population genotyped at the Mayo Clinic were not ethnically matched (nor ageand sex-matched) with the cases from the literature, there is the possibility of population stratification and bias. Similarly, the studies that we pooled for analysis were conducted with different populations (Sweden,7 the United States,13 Portugal,17 Spain,22 and Austria26), which may have introduced a bias into our analysis. In addition, it is difficult to weld such different types of analyses into a single, coherent whole. Approaches for achieving this using Bayesian analysis have been proposed and pro-

272 Medeiros et al JMD May 2012, Vol. 14, No. 3

ductively used.57 However, central to such methodology is a numerical result for the different strands of evidence. Here, the only numerical value is the odds ratio from comparing the frequency of K618A in cases and controls. The rest of the information, such as results of the various functional studies, is qualitative in nature. Our findings support the idea that something is wrong with the K618A allele, but do not support classification as a typical, highly penetrant mutation. This study highlights the difficulty in interpreting missense variants that are found by whole-gene sequencing. It further underscores the difficulty in attempting to classify all variants as either mutations or as benign polymorphisms. It is likely that many missense changes will fall into a gray zone (as K618A does), neither causing disease nor being without effect. Furthermore, it is likely that multiple lines of evidence, such as those drawn together here, along with functional assays of known sensitivity and specificity, will be required to uncover genetic risk factors or to identify those variants with modest to moderate protein function-modifying effect. How results of risk or low-penetrance alleles should be communicated to the patient is not clear. Although scientifically noteworthy and valid, such small increases in risk are unlikely to have clinical significance to individual patients or families.

Acknowledgments The authors thank Debora J. Johnson for expert administrative assistance in the preparation of this manuscript.

References 1. 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 VV, Thibodeau SN: The frequency of hereditary defective mismatch repair in a prospective series of unselected colorectal carcinomas [Erratum appeared in Am J Hum Genet 2001, 69:1160]. Am J Hum Genet 2001, 69:780 –790 2. Hampel H, Frankel W, 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: Screening for the Lynch syndrome (hereditary nonpolyposis colorectal cancer). N Engl J Med 2005, 352:1851–1860 3. Salovaara R, Loukola A, Kristo P, KA¨äriäinen H, Ahtola H, Eskelinen M, Härkönen N, Julkunen R, Kangas E, Ojala S, Tulikoura J, Valkamo E, Järvinen H, Mecklin JP, Aaltonen L, de la Chapelle A: Populationbased molecular detection of hereditary nonpolyposis colorectal cancer [Erratum appeared in J Clin Oncol 2000, 18:3456]. J Clin Oncol 2000, 18:2193–2200 4. 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 L, Eaton L, Fowler J, Lewandowski G, Vaccarello L, Bell J, Reid G, DE la Chapelle A: Screening for Lynch syndrome (hereditary nonpolyposis colorectal cancer) among endometrial cancer patients. Cancer Res 2006, 66:7810 –7817 5. Peltomäki P, Vasen H: Mutations associated with HNPCC predisposition—Update of ICG-HNPCC/INSiGHT mutation database. Dis Markers 2004, 20:269 –276 6. Thibodeau SN, French AJ, Cunningham JM, Tester DJ, Burgart LJ, Roche PC, McDonnell SK, Schaid DJ, Vockley CW, Michels VV, Farr GH Jr, O’Connell MJ: Microsatellite instability in colorectal cancer: different mutator phenotypes and the principal involvement of hMLH1. Cancer Res 1998, 58:1713–1718

7. Tannergård P, Lipford JR, Kolodner R, Frödin JE, Nordenskjöld M, Lindblom A: Mutation screening in the hMLH1 gene in Swedish hereditary nonpolyposis colon cancer families. Cancer Res 1995, 55: 6092– 6096 8. Ou J, Niessen RC, Vonk J, Westers H, Hofstra RM, Sijmons RH: A database to support the interpretation of human mismatch repair gene variants. Hum Mutat 2008, 29:1337–1341 9. Ganguly A, Rock M, Prockop D: Conformation-sensitive gel electrophoresis for rapid detection of single-base differences in doublestranded PCR products and DNA fragments: Evidence for solventinduced bends in DNA heteroduplexes [Erratum appeared in Proc Natl Acad Sci USA 1994 May 24;91(11):5217]. Proc Natl Acad Sci USA 1993, 90:10325–10329 10. Baudhuin LM, Burgart LJ, Leontovich O, Thibodeau SN: Use of microsatellite instability and immunohistochemistry testing for the identification of individuals at risk for Lynch syndrome. Fam Cancer 2005, 4:255–265 11. Murphy KM, Zhang S, Geiger T, Hafez MJ, Bacher J, Berg KD, Eshleman JR: Comparison of the microsatellite instability analysis system and the Bethesda panel for the determination of microsatellite instability in colorectal cancers. J Mol Diagn 2006, 8:305–311 12. Mauillon JL, Michel P, Limacher JM, Latouche JB, Dechelotte P, Charbonnier F, Martin C, Moreau V, Metayer J, Paillot B, Frebourt T: Identification of novel germline hMLH1 mutations including a 22 kb Alu-mediated deletion in patients with familial colorectal cancer. Cancer Res 1996, 56:5728 –5733 13. Weber TK, Conlon W, Petrelli NJ, Rodriguez-Bigas M, Keitz B, Pazik J, Farrell C, O’Malley L, Oshalim M, Abdo M, Anderson G, Stoler D, Yandell D: Genomic DNA-based hMSH2 and hMLH1 mutation screening in 32 Eastern United States hereditary nonpolyposis colorectal cancer pedigrees. Cancer Res 1997, 57:3798 –3803 14. Wijnen J, Khan PM, Vasen H, van der Klift H, Mulder A, van LeeuwenCornelisse I, Bakker B, Losekoot M, Møller P, Fodde R: Hereditary nonpolyposis colorectal cancer families not complying with the Amsterdam criteria show extremely low frequency of mismatch-repairgene mutations. Am J Hum Genet 1997, 61:329 –335 15. Wahlberg S, Liu T, Lindblom P, Lindblom A: Various mutation screening techniques in the DNA mismatch repair genes hMSH2 and hMLH1. Genet Test 1999, 3:259 –264 16. Syngal S, Fox EA, Eng C, Kolodner RD, Garber JE: Sensitivity and specificity of clinical criteria for hereditary non-polyposis colorectal cancer associated mutations in MSH2 and MLH1. J Med Genet 2000, 37:641– 645 17. Fidalgo P, Almeida MR, West S, Gaspar C, Maia L, Wignen J, Albuquerque C, Curtis A, Cravo M, Fodde R, Leitao CN, Burn J: Detection of mutations in mismatch repair genes in Portuguese families with hereditary non-polyposis colorectal cancer (HNPCC) by a multimethod approach. Eur J Hum Genet 2000, 8:49 –53 18. Scott RJ, McPhillips M, Meldrum CJ, Fitzgerald PE, Adams K, Spigelman A, du Sart D, Tucker K, Kirk J: Hereditary nonpolyposis colorectal cancer in 95 families: differences and similarities between mutation-positive and mutation-negative kindreds [Erratum appeared in Am J Hum Genet 2001 Feb;68(2):557]. Am J Hum Genet 2001, 68:118 –127 19. Salahshor S, Koelble K, Rubio C, Lindblom A: Microsatellite instability and hMLH1 and hMSH2 expression analysis in familial and sporadic colorectal cancer. Lab Invest 2001, 81:535–541 20. Müller-Koch Y, Kopp R, Lohse P, Baretton G, Stoetzer A, Aust D, Daum J, Kerker B, Gross M, Dietmeier W, Holinski-Feder E: Sixteen rare sequence variants of the hMLH1 and hMSH2 genes found in a cohort of 254 suspected HNPCC (hereditary non-polyposis colorectal cancer) patients: mutations or polymorphisms? Eur J Med Res 2001, 6:473– 482 21. Gille JJ, Hogervorst FB, Pals G, Wignen JT, van Schooten RJ, Dommering CJ, Meijer GA, Craanen ME, Nederlof PM, de Jong D, McElgunn CJ, Schouten JP, Menko FH: Genomic deletions of MSH2 and MLH1 in colorectal cancer families detected by a novel mutation detection approach. Br J Cancer 2002, 87:892– 897 22. Caldes T, Godino J, de la Hoya M, Garcia Carbonero I, Perez Segura P, Eng C, Benito M, Diaz-Rubio E: Prevalence of germline mutations of MLH1 and MSH2 in hereditary nonpolyposis colorectal cancer families from Spain. Int J Cancer 2002, 98:774 –779 23. Scartozzi M, Bianchi F, Rosati S, Galizia E, Antolini A, Loretelli C, Piga A, Bearzi I, Cellerino R, Porfiri E: Mutations of hMLH1 and hMSH2 in patients with suspected hereditary nonpolyposis colorectal cancer: cor-

MLH1 K618A and Lynch Syndrome 273 JMD May 2012, Vol. 14, No. 3

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

relation with microsatellite instability and abnormalities of mismatch repair protein expression. J Clin Oncol 2002, 20:1203–1208 Ward R, Meldrum C, Williams R, Mokany E, Scott R, Turner J, Hawkins N, Burgess B, Groombridge C, Spigelman A: Impact of microsatellite testing and mismatch repair protein expression on the clinical interpretation of genetic testing in hereditary non-polyposis colorectal cancer. J Cancer Res Clin Oncol 2002, 128:403– 411 Caldés T, Godino J, Sanchez A, Corbacho C, de la Hoya M, Lopez Asenjo J, Saez C, Sanz J, Benito M, Ramon y Cajal S, Diaz-Rubio E: Immunohistochemistry and microsatellite instability testing for selecting MLH1, MSH2, and MSH6 mutation carriers in hereditary nonpolyposis colorectal cancer. Oncol Rep 2004, 12:621– 629 Wolf B, Henglmueller S, Janschek E, Ilencikova D, Ludwig-Papst C, Bergmann M, Mannhalter C, Wrba F, Karner-Hanusch J: Spectrum of germ-line MLH1 and MSH2 mutations in Austrian patients with hereditary nonpolyposis colorectal cancer. Wien Klin Wochenschr 2005, 117:269 –277 Hegde M, Blazo M, Chong B, Prior TW, Richards C: Assay validation for identification of hereditary nonpolyposis colon cancer-causing mutations in mismatch repair genes MLH1, MSH2, and MSH6. J Mol Diagn 2005, 7:525–534 Belvederesi L, Bianchi F, Loretelli C, Gagliardini D, Galizia E, Bracci R, Rosati S, Bearzi I, Viel A, Cellerino R, Porfiri E: Assessing the pathogenicity of MLH1 missense mutations in patients with suspected hereditary nonpolyposis colorectal cancer: correlation with clinical, genetic and functional features. Eur J Hum Genet 2006, 14:853– 859 Bianchi F, Galizia E, Bracci R, Belvederesi L, Catalani R, Loretelli C, Giorgetti G, Ferretti C, Bearzi I, Porfiri E, Cellerino R: Effectiveness of the CRCAPRO program in identifying patients suspected for HNPCC. Clin Genet 2007, 71:158 –164 Bellacosa A, Genuardi M, Anti M, Viel A, de Leon P: Hereditary nonpolyposis colorectal cancer: review of clinical, molecular genetics, and counseling aspects. Am J Med Genet 1996, 62:353–364 Benatti P, Sassatelli R, Roncucci L, Pedroni M, Fante R, Di Gregorio C: Tumour spectrum in hereditary non-polyposis colorectal cancer (HNPCC) and in families with “suspected HNPCC”. A populationbased study in northern Italy. Colorectal Cancer Study Group. Int J Cancer 1993, 54:371–377 Rodriguez-Bigas M, Boland C, Hamilton S, Hensom D, Jass J, Khan P, Lunch H, Perucho M, Smyrk T, Sobin L, Srivastava S: A National Cancer Institute Workshop on Hereditary Nonpolyposis Colorectal Cancer Syndrome. J Natl Cancer Inst 1997, 89:1758 –1762 Vasen H, Mecklin J, Meera Khan P, Lynch H: The International Collaborative Group on hereditary nonpolyposis colorectal cancer (ICGHNPCC). Dis Col Rectum 1991, 34:424 – 425 Vasen H, Watson P, Mecklin J, Lynch H: New clinical criteria for hereditary nonpolyposis colorectal cancer (HNPCC, Lynch syndrome) proposed by the International Collaborative Group on HNPCC. Gastroenterology 1999, 116:1453–1456 Farrington SM, Lin-Goerke J, Ling J, Wang Y, Burczak JD, Robbins DJ, Dunlop MG: Systematic analysis of hMSH2 and hMLH1 in young colon cancer patients and controls. Am J Hum Genet 1998, 63:749 –759 Liu T, Tannergård P, Hackman P, Rubio C, Kressner U, Lindmark G, Hellgren D, Lambert B, Lindblom A: Missense mutations in hMLH1 associated with colorectal cancer. Hum Genet 1999, 105:437– 441 Samowitz WS, Curtin K, Lin HH, Robertson MA, Schaffer D, Nichols M, Gruenthal K, Leppert MF, Slattery ML: The colon cancer burden of genetically defined hereditary nonpolyposis colon cancer. Gastroenterology 2001, 121:830 – 838 Hudler P, Vouk K, Liovic M, Repse S, Juvan R, Komel R: Mutations in the hMLH1 gene in Slovenian patients with gastric carcinoma. Clin Genet 2004, 65:405– 411 Fearnhead NS, Wilding JL, Winney B, Tonks S, Bartlett S, Bicknell DC, Tomlinson IP, Mortensen NJ, Bodmer WF: Multiple rare variants in different genes account for multifactorial inherited susceptibility to colorectal adenomas. Proc Natl Acad Sci USA 2004, 101:15992– 15997 Piñol V, Castells A, Andreu M, Castellví-Bel S, Alenda C, Llor X, Xicola RM, Rodríguez-Moranta F, PayÁ A, Jover R, Bessa X; Gastrointestinal Oncology Group of the Spanish Gastroenterological Association: Accuracy of revised Bethesda guidelines, microsatellite instability, and

41.

42.

43.

44.

45.

46.

47.

48.

49.

50.

51.

52.

53.

54.

55. 56.

57.

immunohistochemistry for the identification of patients with hereditary nonpolyposis colorectal cancer. JAMA 2005, 293:1986 –1994 Blasi MF, Ventura I, Aquilina G, Degan P, Bertario L, Bassi C, Radice P, Bignami M: A human cell-based assay to evaluate the effects of alterations in the MLH1 mismatch repair gene. Cancer Res 2006, 66:9036 –9044 Barnetson RA, Cartwright N, van Vliet A, Haq N, Drew K, Farrington S, Williams N, Warner J, Campbell H, Porteous ME, Dunlop MG: Classification of ambiguous mutations in DNA mismatch repair genes identified in a population-based study of colorectal cancer. Hum Mutat 2008, 29:367–374 Guerrette S, Acharya S, Fishel R: The interaction of the human MutL homologues in hereditary nonpolyposis colon cancer. J Biol Chem 1999, 274:6336 – 6341 Kondo E, Suzuki H, Horii A, Fukushige S: A yeast two-hybrid assay provides a simple way to evaluate the vast majority of hMLH1 germline mutations. Cancer Res 2003, 63:3302–3308 Raevaara TE, Korhonen MK, Lohi H, Hampel H, Lynch E, Lönnqvist KE, Holinski-Feder E, Sutter C, McKinnon W, Duraisamy S, Gerdes AM, Peltomäki P, Kohonen-Corish M, Mangold E, MacRae F, Greenblatt M, de la Chapelle A, NyströM M: Functional significance and clinical phenotype of nontruncating mismatch repair variants of MLH1. Gastroenterology 2005, 129:537–549 Kosinski J, Hinrichsen I, Bujnicki JM, Friedhoff P, Plotz G: Identification of Lynch syndrome mutations in the MLH1-PMS2 interface that disturb dimerization and mismatch repair. Hum Mutat 2010, 31:975– 982 Wanat JJ, Singh N, Alani E: The effect of genetic background on the function of Saccharomyces cerevisiae mlh1 alleles that correspond to HNPCC missense mutations. Hum Mol Genet 2007, 16:445– 452 Perera S, Bapat B: The MLH1 variants p.Arg265Cys and p.Lys618Ala affect protein stability while p.Leu749Gln affects heterodimer formation. Hum Mutat 2008, 29:332 Perera S, Li B, Tsitsikotas S, Ramyar L, Pollett A, Semotiuk K, Bapat B: A novel and rapid method of determining the effect of unclassified MLH1 genetic variants on differential allelic expression. J Mol Diagn 2010, 12:757–764 Chan PA, Duraisamy S, Miller PJ, Newell JA, McBride C, Bond JP, Raevaara T, Ollila S, Nyström M, Grimm AJ, Christodoulou J, Oetting WS, Greenblatt MS: Interpreting missense variants: comparing computational methods in human disease genes CDKN2A, MLH1, MSH2, MECP2, and tyrosinase (TYR). Hum Mutat 2007, 28:683– 693 Stone EA, Sidow A: Physicochemical constraint violation by missense substitutions mediates impairment of protein function and disease severity. Genome Res 2005, 15:978 –986 Chao EC, Velasquez JL, Witherspoon MS, Rozek LS, Peel D, Ng P, Gruber SB, Watson P, Rennert G, Anton-Culver H, Lynch H, Lipkin SM: Accurate classification of MLH1/MSH2 missense variants with multivariate analysis of protein polymorphisms-mismatch repair (MAPP-MMR). Hum Mutat 2008, 29:852– 860 Lindor NM, Petersen GM, Hadley DW, Kinney AY, Miesfeldt S, Lu K, Lynch P, Burke W, Press N: Recommendations for the care of individuals with an inherited predisposition to Lynch syndrome: a systematic review. JAMA 2006, 296:1507–1517 Vasen HF, Möslein G, Alonso A, Bernstein I, Bertario L, Blanco I, Burn J, Capella G, Engel C, Frayling I, Friedl W, Hes FJ, Hodgson S, Mecklin JP, Møller P, Nagengast F, Parc Y, Renkonen-Sinisalo L, Sampson JR, Stormorken A, Wijnen J: Guidelines for the clinical management of Lynch syndrome (hereditary non-polyposis cancer). J Med Genet 2007, 44:353–362 Felton KE, Gilchrist DM, Andres SE: Constitutive deficiency in DNA mismatch repair: is it time for Lynch III? Clin Genet 2007, 71:499 –500 Gargiulo S, Torrini M, Ollila S, Nasti S, Pastorino L, Cusano R, Bonelli L, Battistuzzi L, Mastracci L, Bruno W, Savarino V, Sciallero S, Borgonovo G, Nystrom M, Bianchi-Scarrà G, Mareni C, Ghiorzo P: Germline MLH1 and MSH2 mutations in Italian pancreatic cancer patients with suspected Lynch syndrome. Fam Cancer 2009, 8:547–553 Goldgar DE, Easton DF, Byrnes GB, Spurdle AB, Iversen ES, Greenblatt MS; IARC Unclassified Genetic Variants Working Group: Genetic evidence and integration of various data sources for classifying uncertain variants into a single model. Hum Mutat 2008, 29:1265–1272