reviewarticle novel implications in molecular diagnosis...

Review ArticleNovel Implications in Molecular Diagnosis of Lynch Syndrome

Raffaella Liccardo1 Marina De Rosa1 Paola Izzo12 and Francesca Duraturo1

1Department of Molecular Medicine and Medical Biotechnology University of Naples ldquoFederico IIrdquo 80131 Naples Italy2CEINGE Biotecnologie Avanzate University of Naples ldquoFederico IIrdquo 80131 Naples Italy

Correspondence should be addressed to Francesca Duraturo duraturodbbmuninait

Received 30 September 2016 Accepted 5 January 2017 Published 29 January 2017

Academic Editor Haruhiko Sugimura

Copyright copy 2017 Raffaella Liccardo et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

About 10 of total colorectal cancers are associated with knownMendelian inheritance as Familial Adenomatous Polyposis (FAP)and Lynch syndrome (LS) In these cancer types the clinical manifestations of disease are due to mutations in high-risk alleles witha penetrance at least of 70 The LS is associated with germline mutations in the DNA mismatch repair (MMR) genes Howeverthe mutation detection analysis of these genes does not always provide informative results for genetic counseling of LS patientsVery often the molecular analysis reveals the presence of variants of unknown significance (VUSs) whose interpretation is not easyand requires the combination of different analytical strategies to get a proper assessment of their pathogenicity In some cases theseVUSs maymake a more substantial overall contribution to cancer risk than the well-assessed severe Mendelian variants Moreoverit could also be possible that the simultaneous presence of these genetic variants in severalMMRgenes that behave as low risk allelesmight contribute in a cooperative manner to increase the risk of hereditary cancer In this paper through a review of the recentliterature we have speculated a novel inheritance model in the Lynch syndrome this could pave the way toward new diagnosticperspectives

1 Introduction

Colorectal cancer (CRC) is a multifactorial disease in whichgenetic and environmental factors are involved FamilialCRC in which one or more first-degree andor second-degree relatives of the index case manifest CRC constitutesapproximately 20 of the total CRC burden [1] High pene-trance mutations confer a predisposition to CRC in the so-called hereditary syndromes responsible for about 2ndash6 ofthe total CRC Low penetrance mutations are found in theremaining part of CRC (about 96) representing a risk factorin both sporadic and familial cases [2 3] CRC syndromesare defined on the basis of clinical pathological and morerecently genetic findings [3] (Table 1)

Accordingly the identification of predisposing genesallows for accurate risk assessment and more precise screen-ing approaches Lynch syndrome (LS) is the most commonhereditary form of CRC with an incidence of 3ndash5 ofall CRCs whereas its primary genetic counterpart namelyFamilial Adenomatous Polyposis (FAP) accounts for lessthan 1 of the total CRC burden [4]

LS and FAP are diseases with autosomal dominant inher-itance caused by germline mutations in the DNA mismatchrepair genes (MMR) or in the Adenomatous Polyposis Colitumor suppressor gene (APC) respectivelyThese syndromesmay also occur in more attenuated forms In FAP syndromeattenuated forms (AFAP) are caused by low penetrancemutations (missense mutations) in the main APC gene orby biallelic loss of the MYH gene (MAP MUTYH-associatedpolyposis with autosomal recessive inheritance) encodinga protein of the Base Excision Repair complex (BER) [5]Variant clinic forms of LS are characterized by the presenceof additional tumors of still unclear etiology in extracoloniclocations Recent studies suggest that an interaction betweenmain genes (MMR) and modifier genes andor environ-mental factors may beat the basis of these tumors Thesevariant syndromes include Muir-Torre syndrome (autosomaldominant) due to MSH2 and MLH1 genes mutations andcharacterized by the presence of cutaneous manifestations(multiple sebaceous adenomas epithelioma and keratoacan-thoma) associated with colorectal and endometrial cancersand Turcot syndrome (autosomal dominant) associated with

HindawiGastroenterology Research and PracticeVolume 2017 Article ID 2595098 12 pageshttpsdoiorg10115520172595098

2 Gastroenterology Research and Practice

APC PMS2 andMLH1 genesmutations and characterized bybrain cancers (glioblastoma and cerebellar medulloblastoma)are associated with colorectal cancer [6 7]

In the process of colorectal carcinogenesis many othergenes are involved such as oncogenes and tumor suppressorgenes that play a key role in the control of cell cycleMutations in these genes are at the basis of rarer inheritedCRC syndromes These are mainly ldquohamartomatous poly-posis syndromesrdquo characterized by the presence of benignadenomas arising from epithelial andor stromal intestinaltissue which increase the risk of developing CRC Thesesyndromes whose characteristics are summarized in Table 1include Peutz-Jeghers syndrome juvenile polyposis Cowdensyndrome and Bannayan-Riley-Ruvalcaba syndrome [8ndash11]

In this review we intend to highlight new insights intothe molecular features of Lynch syndrome in favor of a novelinheritance model in contrast with the classical monogenictransmission this could suggest new genetic and clinicalsurveillance approaches

2 Lynch Syndrome (LS)

21 Genetics Features LS is an autosomal dominant diseasewith recessive phenotype caused by a defect in one of themis-match repair (MMR) genes The main clinical-pathologicalfeatures of the Lynch syndrome are as follows [1ndash11 13]

Autosomal dominant inheritancePenetrance for colorectal cancer (CRC) of 85ndash90Earlier age of onset of CRC (sim45 years ) with respectto general population (69 years)Preferential tumor localization in the right-sidedcolonPresence of multiple synchronous and metachronouscolorectal cancersBetter prognosis than CRCsIncreased risk for extracolonic cancersAccelerated carcinogenesisPoorly differentiated tumors with a marked lympho-cytic peritumoral inflammation recalling features ofthe so-called ldquoCrohnrsquos reactionrdquoMicrosatellite instability

The mismatch repair system was first studied in bacteriain which three proteins MutS MutL and MutH wereidentified In humans at least seven mismatch repair genesare involved in mismatch repair and their names derivefrom their structural homology to the bacterial proteinsthe MutS homologues (MSH) MSH2 on chromosome 2p16MSH3 on chromosome 5q11 and MSH6 on chromosome2p16 the MutL homologues (MLH) MLH1 on chromosome3p21 and MLH3 on chromosome 2p16 the postmeioticsegregation homologues (PMS) PMS1 and PMS2 on chro-mosome 7p22 No MutH homologues have been identifiedin humans [14] MSH2 and MSH6 bind together to forma heteroduplex (MutSa) that predominantly identifies base

pairs mismatched whileMSH2 andMSH3 (MutS120573) combineto identify short insertions or deletions MSH2 is essentialfor both complexes to function while a functional overlapexists between MSH3 and MSH6 MLH1 and PMS2 (MutL120572)or MLH3 (Mutl120574) also bind together to form a heteroduplexthat interacts with MutS120572 or MutS120573 complex stimulatingexcision and resynthesis of the abnormal DNA Similarly toMSH2 also MLH1 is essential for both complexes to repairmismatches Altogether this group of four proteins recruitsexonuclease-1 (EXO1) the proliferating cell nuclear antigen(PCNA) DNA polymerase (Pol120575 or Pol120576) two replicationfactor (RPA and RFC) and a ligase to repair DNA onthe daughter strand at the mismatch point If any of thefour major proteins (MSH2 MLH1 MSH6 or PMS2) isfunctionally inactive mismatches are not repaired [15 16]

22 Microsatellite Instability Consequently a defective DNAMMR system increases the mutation rate and makes thecell vulnerable to mutations in genes controlling cell growth(tumor suppressor genes and oncogenes) resulting in anincreased cancer risk

In case of a defective MMR system mutations occurfrequently in small (usually mononucleotide or dinucleotide)repetitive DNA sequences known as microsatellites InMMR-deficient tumor cells the number of microsatelliterepeat units can deviate from the corresponding normalDNA the number of repeats is usually decreased even thoughit is occasionally found to be increased [17]

Length or size microsatellite variation is known as MSI(microsatellite instability) MSI (formerly referred to as MINor RER replication error) is the molecular hallmark of LSsince approximately 95 of all LS-associated cancers showMSI [13] Althoughmost microsatellite sequences are locatedin noncoding sequences (telomeres and centromeres) manygenes contain repetitive sequences in their coding regionsand some of these genes play key roles in the regulation of cellgrowth Identification of an even growing number of guidegenes and target genes of the mutator phenotype can lead todiscover new complex molecular mechanisms that underliethe process of colorectal tumorigenesis [18]

MSI thereby serves as a reliable phenotypic marker ofMMR deficiency in order to preselect patients eligible forgermline mutation analysis in the MMR genes [19]

However despite the fact that MSI is a reliable markerfor MMR deficiency however until 15 of sporadic CRCsshowed an MSI phenotype This is mainly caused by somatichypermethylation of the MLH1-gene promoter Methylationof the MSH2 promoter has also been reported but it is tobe considered as a heritable somatic methylation because itis caused by a deletion of the last exon of EPCAM that isadjacent to MSH2 on chromosome 2 [18]

Hypermethylation of CpG islands in the MLH1 pro-moter (CIMP phenotype) causes severe inhibition of genetranscription thereby mimicking an inactivating gene muta-tion If both copies of the gene are inactivated (biallelichypermethylation) the MLH1 function is lost This leads tomicrosatellite unstable cancers especially in older patientsTherefore in MLH1-deficient microsatellite unstable (MSI-H) tumors the MLH1 hypermethylation can be assessed to

Gastroenterology Research and Practice 3

Table 1 Hereditary colon cancer syndromes clinical and genetic features (AD autosomal dominant AR autosomal recessive)

Disease(OMIM) Gene Incidence Inheritance Mutation identified

() Penetrance Clinical features

Hereditarynonpolyposiscolorectal cancer(HNPCC)(114500)

MLH1 MSH2MSH6 PMS2MLH3 EPCAM

1 in 400 ADPoint mutation large

rearrangements(60ndash80)

90

Proximal CRCendometrial

carcinoma ovariantumors small bowelcarcinoma urinarytract carcinoma

Classical familialadenomatouspolyposis (FAP)(175100)

APC 1 in 8000 ADPoint mutation large


lt100

100 to gt500adenomatous polyps

of large bowelduodenum stomach

Attenuated FAP(AFAP) (175100) APC lt1 in 8000 AD

Point mutation largerearrangements

(20ndash30)lt100

10 to 100adenomatous polyps


MUTYH-associatedpolyposis (MAP)(608456)

MUTYH lt1 in 10000 ARPoint mutation large


lt100



Muir Torresyndrome(HNPCC)(158320)

MLH1 MSH2 lt1 in 400 ADPoint mutation large


90

CRC endometrialcarcinoma multiplesebaceous adenomas

epitheliomakeratoacanthoma

Turcot syndrome(HNPCC)(276300)

APC PMS2 MLH1 lt1 in 400 ADPoint mutation large


90CRC glioblastoma

cerebellarmedulloblastoma

Peutz-Jegherssyndrome (PJS)(175200)

STK11 (LKB1) 1 in 200000 ADPoint mutation large

rearrangements(90)

95ndash100

lt20 juvenile polyps(PJ) of large bowelduodenum stomach

mucocuta-neousperioral

hyperpigmentationovarian tumorsbreast cancer

Juvenilepolyposissyndrome (JPS)(174900)

SMAD4 BMPR1A 1 in 100000 ADPoint mutation large

rearrangements(60)

90ndash100

5 to 100 JP of largebowel duodenumstomach gastric

cancer

Cowdensyndrome (CS)(158350)

PTEN 1 in 200000 AD Point mutation largerearrangements (80) 90ndash95

Multiple JPlipomasof large bowel

duodenum stomachmucocutaneous

tumors breast cancerendometrial

carcinoma thyroidcancer

Bannayan-Ruvalcaba-Rileysyndrome(BRRS)(153480)

PTEN 1 in 200000 ADPoint mutation large

rearrangements(60)

90ndash95


duodenum stomachmicrocephaly

developmental delayhemangiomatosis


distinguish sporadic CRCs from LS-related cancers More-over recent findings have also identified the BRAF gene asa marker to distinguish LS from sporadic cases of coloncancer [20 21] Indeed an oncogenic BRAF mutation hasbeen described only in one case among several LS tumors[22] Specific activating mutations in the BRAF oncogeneusually the V600Emissense mutation can be detected in 40ndash87 of all sporadic microsatellite unstable tumors [13] Inthis case the survival in the subjects with MSI-H tumors andBRAF mutation is higher than sporadic tumors with BRAFmutation and MSI stable therefore the good prognosis ofMSI-H tumors is not affected by the BRAF genotype [23]Also in another study the MSI-HBRAF mutation grouphad a good prognosis [24] This emphasizes even more theneed to evaluate both BRAF mutation and MSI status inpatients with CRC for an accurate prognosis [23] Theseresults indicate that in cases of MSI BRAF mutations closelycorrelate with MLH1 promoter methylation in sporadic MSICRCs in contrast to LS characterized by germline mutationsin the MMR genes

In 1997 the National Cancer Institute recommended apanel known as the ldquopanel of Bethesdardquo comprising fivemicrosatellites twomononucleotide repeats (BAT25 BAT26)and three dinucleotide repeats (D2S123 D17S250 D5S346)[25] Tumors showing instability at two or more of theserepeats (40 ofmarkers) are defined at high instability (MSI-H) those with instability between 20ndash40 are classifiedas low instability (MSI-L) [26] tumors without alteration(20 or less) are classified as stable (MSS) Subsequently inorder to improve the sensitivity rate and the predictive speci-ficity Bethesda guidelines were revised and other loci wereenclosed in the panel test BAT-25 and BAT-26 besides threeother quasimonomorphic mononucleotide repeats namelyNR21 NR22 and NR24 [27ndash29]

MSI testing is also very important because several piecesof evidence suggested that MSI-H tumors (stage II) areassociated with a favorable prognosis when patients are nottreated with 5-fluorouracil compared to MSI-L and MSSCRC [30 31] These different features are probably relatedto the lymphocytic infiltrate characteristic of MMR-deficienttumors that determines an antitumor immune responsewhich may be abrogated by the immunosuppressive effectsof the chemotherapy [17]

Besides MSI testing analysis of MMR protein expressionby immunohistochemistry (IHC) is routinely performedto identify patients with suspected Lynch syndrome IHCtesting is a specific (100) and sensitive (923) screeningtool to identify MSI-H tumors [13 32] A more detaileddiscussion onMMR IHC is available in a recent review article[33]

23 Clinical Features LS is characterized by a high life-time risk for tumor development especially in the case ofCRC (20ndash70) endometrial cancer (15ndash70) and otherextracolonic tumors (15) These extracolonic malignanciesinclude carcinomas of small intestine stomach pancreasand biliary tract ovarium brain upper urinary tract andskin More recently gastric cancers have been includedin the tumor spectrum of LS [34] The molecular and

clinical-pathological profiles of gastric cancers in LSmutationcarriers have been evaluated and compared with the profilesof sporadic gastric cancers and several differences have beenidentified while there are similarities with canonicalHNPCCspectrum malignancies Stomach can thus be considered asa target tissue in which somatic inactivation (ldquosecond hitrdquo)of MMR genes may occur in carriers of a germline mutation(ldquofirst hitrdquo) [34 35]

24 Clinical Diagnosis of Lynch Syndrome Identificationof MMR gene mutation carriers is critical for improvingcancer surveillance and effectiveness of prevention BeforeMMR genes and their causal role in hereditary CRC cancerwere identified the International Collaborative Group onhereditary nonpolyposis colorectal cancer had established theAmsterdam criteria I in 1990 These criteria were used toidentify families eligible for molecular analysis Subsequentlymodified guidelines (Amsterdam criteria II) were designedto include extracolonic LS-related cancers [1] NeverthelessAmsterdam criteria resulted to be very restrictive and failedto identify a large portion of MMR gene mutation carriersTo overcome this issue Bethesda guidelines which were lessrestrictive and had a sensitivity greater than 90 even with alower specificity (25) were later defined [25]

3 New Insights into the MolecularFeatures of Lynch Syndrome

A long time literature data report that in addition to thepostreplicative repair MMR proteins have developed variousother functions thatmay have relevant roles in carcinogenesis[16] These new roles include the following

(1) DNA damage signalling caused by exogenous car-cinogens (heterocyclic amines oxidative agents andUV radiation) that is achieved through a syner-gistic action between the p53-homologous proteins(p53 p63 and p73) and the MutS120572-MutL120572 complexfurthermore in response to an exogenous damageMLH1 interacts with the protein MRE11 a compo-nent of the ldquoBRCA1 associated surveillance complexrdquo(BASC) and regulates the cell cycle and the apoptoticpathway [36 37]

(2) Prevention of reparative recombination (gene conver-sion) between nonidentical sequences [38]

(3) Promotion of meiotic crossover several studiesin S Cerevisiae and knockout mice have shownthat homologous chromosome recombination duringmeiosis is controlled by MMR proteins in order toavoid mutational events due to deletions insertionsor mismatched bases Among the MMR proteinsMLH1 PMS2 andMLH3 are involved in this processIn fact experimental murine deficiency of one ofthese three proteins is associated with male infertility(defective spermatogenesis) [38 39]

(4) Immunoglobulin diversification based on the ldquoso-matic hypermutationrdquo (SHM) process which is regu-lated by the MutS120572-MutL120572 complex in combination


Mismatch repairin replication

PCNARFC

ATRP53

DNA damagesignaling

MSH2

MLH1MLH1

Regulation ofreparative

recombination(gene conversion)

Promotion of meiotic

crossover and gametogenesis

regulation

MSH2Triplet repeats

expansion

MMR complex

MSH2

MLH1

Modulation of microRNAbiogenesis

MLH1 PMS2PMS2

MSH6

PMS2

MSH3

MSH6

PMS2

MLH3

MSH3

MMR proteins

Immunoglobulindiversification based on the

(SHM) processldquosomatic hypermutationrdquo

AIDPol 120578

Figure 1 New role for MMR proteins

with two other proteins AID (activation-inducedcytidine deaminase) and Pol120583 (DNA Polymeraseldquoerror-pronerdquo) [40] in particular MutS120572 deficiencyis associated with neoplastic transformation of Tlymphocytes [41]

(5) Expansion of repeated triplets (CTG CGG) thatunderlie the pathogenesis of various neurodegenera-tive diseases such as Huntingtonrsquos Disease MyotonicDystrophy and Fragile X Syndrome This mecha-nism is still unknown however experimental evi-dence indicates that although MutS120573 binds theseexpansions the repair is prevented by looping con-formations of these regions [42] Since the tripletexpansion is at the basis of the anticipation of thedisease in the family loss of function of MutS120573 mayhave a protective role against the intergenerationalinstability [43 44]

(6) Modulation ofmicroRNAbiogenesis by interaction ofMMR proteins with the microprocessor complex inparticular MutL120572 specifically binds pri-miRNAs andthe complex DroshaDGCR8 in order to stimulatethe processing of pri-miRNAs to pre-miRNAs in amanner dependent on MutL120572 ATPase activity [45]

These new features indicate that MMR deficiency stronglyaffects cellular resistance to reparative andor apoptotic

response toDNAdamage because impairment of postreplica-tive MMR complexes associated with impairment of compo-nents of other cell systems (Figure 1)

4 Genotype-PhenotypeAssociations in Lynch Syndrome

41 Canonical Features Germline mutations are distributedunevenly along each MMR gene denoting the absenceof mutational hot spots Even the nature of the germlinealterations is varied

Absence of redundant functions for MSH2 and MLH1proteins stresses the importance of these two genes thereforemutations in these genes are associated with aggressive formsof HNPCC characterized by early age of onset typicallyaround 45 years of age high penetrance and high degree ofmicrosatellite instability (MSI-H) [46] The CRC incidenceis similar in subjects with mutations in MLH1 and MSH2(84 and 71 resp) however individuals with alterationsin the MSH2 gene show a higher incidence (48ndash61) ofextracolonic malignancy (endometrial gastric ovarian andkidney cancer) than those carrying mutations in the MLH1gene (11ndash42) [47]

The clinical phenotype is different when minor genesare involved Mutations in MSH6 for example seem tocause a form of ldquoattenuatedrdquo HNPCC characterized by lower


penetrance later age of onset usually around 60 years of ageand MSI-L [48]

Defects in the PMS2 gene are instead associatedwith earlytumor development and microsatellite instability althoughsome features are different with respect to cancers caused bytheMLH1 andMSH2mutations PMS2mutations are associ-atedwith combinedpresence ofmultiple colorectal adenomasand glioblastomas (Turcot syndrome)The specificity of braintumor is probably linked to the accumulation of mutations intarget genes (oncogenes tumor suppressor) more specificallyexpressed in the brain [49] Recently MLH3 variants wereassociated with brain cancer predisposition [50]

In the MSH3 gene missense silent and intronic vari-ations have been mainly identified these mutations areassociated with a severe phenotype in the case they areinherited in combination with each other or associated withvariants in the MSH2 gene [51] In fact MSH3 knockoutmice showed a low susceptibility to cancer development thatcaused late-onset colorectal cancer whereas double mutantMSH3-MSH6 mice showed a very similar phenotype to thatfound in mice lacking MSH2 These results are justified bythe redundant function of the MSH3 and MSH6 genes [52]Moreover MSH3 inactivation is primarily associated withinstability of tetranucleotide repeats (EMAST) that has beenfrequently observed in moderately or poorly differentiatedadenocarcinomas as well as in other cancers including lungkidney ovarian and bladder cancer [14 53]

In recent years numerous studies have found an associa-tion between the development of hematopoietic and intesti-nal tumors in infant age and the presence of homozygousmutations in the MLH1 MSH2 MSH6 and PMS2 genes[54 55] This phenotype was also associated with heterozy-gous mutations in two or more MMR genes suggesting amechanism of compound heterozygosity [56ndash58]

In a subset of LS patients a germline mutation at the31015840 end of the EPCAM (TACSTD1) gene has been identifiedresulting in allelic-specific methylation and transcriptionsilencing of MSH2 which is located upstream of the EPCAMgene The EPCAM gene encodes for the Epithelial CellAdhesion Molecule protein that is involved in cell signallingmigration proliferation and differentiation Accordinglythis mutation may contribute to the development of extra-colonic cancers [59]

42 Noncanonical Features Recently a group of Lynch-likesyndrome patients was described [60] This group mayaccount for as much as 70 of suspected Lynch syndromesubjects Unlike sporadic MSI cancer Lynch-like patients arenearly impossible to differentiate from Lynch patients theyare MSI-positive and cancer tissues express abnormal MMRprotein not only for MLH1 as in sporadic MSI cancers butalso for the other MMR proteins such as MSH2 MSH6 andPMS2 as in Lynch syndrome cancers Lynch-like patientsshow a mean age of onset comparable to LS The onlydifferentiating features between these two syndromes are thelower incidence for CRC and other LS-associated cancers andthe absence of MMR genes germline mutation in Lynch-likesyndrome There are likely three potential reasons for canceronset in Lynch-like patients (a) a genetic process within the

tumors other than germline mutations coupled with secondallele inactivation (b) unknown germline mutations in othergenes than the DNA MMR genes that can drive MSI andor(c) unidentified germline mutations in the DNAMMR genes[12 61]

Mensenkamp et al [62] noted that a considerable numberof MSI-positive tumors lack any known molecular mech-anism for their development Patients were screened forsomaticmutations and for loss of heterozygosity inMLH1 andMSH2 genes This research identified two somatic mutationsin 13 of 25 tumors 8 of whichwereMLH1-deficient and 5wereMSH2-deficient indicating that such acquired mutationsunderlie more than 50 of the MMR-deficient tumors thathave not been found associated with germline mutations orpromoter methylation This is in contrast with LS that isassociated with germline mutations in the MMR genes

Moreover other hereditary factors might play a role intumor development For example deletions affecting genesthat regulate MSH2 degradation were shown to lead toMMRdeficiency and undetectable levels of MSH2 protein More-over cells lacking SETD2 (H3K36 trimethyltransferase SETdomain containing protein 2) displayMSI due to the loss of anepigenetic histonemark that is essential for the recruitment ofthe MSH2-MSH6 complex Whether these mechanisms leadto MSH2-deficient colorectal cancer remains to be clarified[63]

In these cases high-throughput sequencing proceduresplay an important role to identify new constitutive andsomatic mutations in putative genes associated with hered-itary predisposition to cancer [64]

It is also noteworthy that in addition to canonicalinactivation via gene mutation MMR activity can also bemodulated by changes in MMR gene expressionThis type ofalterationmay be the result of mutations occurring in regionsthat are not always routinely analyzed such as the promoterand the 51015840 and 31015840-untraslated regions

Previous studies have defined and characterized the corepromoter regions of hMSH2 (from minus300 to minus17 upstream ofthe start codon) [65] andhMLH1 (fromminus220 tominus39 upstreamof the start codon) [66] subsequent studies have been carriedout to demonstrate that germline mutations in these regionsare involved in LS [67 68]

Regarding mutations in the 31015840UTR of MMR genes a 3-nucleotide (TTC) deletion in the MLH1 31015840UTR was found inleukemia patients [69] This alteration was shown to destroya binding site for miR-422a and there is a downregulationsuggesting a possible role for the miRNA in regulation ofMLH1expression [69]

Therefore cell levels of MMR are likely to be under atight regulation in order to prevent the overproduced proteinwhich may sequester other factors involved in controllingthe mutation rate Potentially adverse consequences of over-produced MLH1 and MSH2 are highlighted by a reportshowing that apoptosis is induced in a human cell linewhen these two genes were expressed under the control ofthe cytomegalovirus (CMV) promoter [70] The dangerousexcess of MMR protein can also be the effect of homod-imerization complex as shown by a study in yeast cellsof Shcherbakova et al [71] showing that the MLH1-MLH1


homodimer replaced the MLH1-PMS1PMS2MLH3 het-erodimer inactivating also the MutS120572 and MutS120573 functionsthus resulting in nonfunctional MMR complex

This concept is also partially extended to other minorMMR genes overexpression of the MSH3 gene in cul-tured mammalian cells selectively inactivates MutS120572 becauseMSH2 is sequestered into a MSH2-MSH3 (MutS120573) complexresulting in reduced MutS120572-dependent repair of base-basemismatches and a strong base substitution mutator pheno-type [72]

Finally severalMSI tumors with unknown cause ofMMRinactivation could display a miRNA down- or upexpressiongenotype that specifically modulate MMR genes [73 74]miRNA expression is in turn regulated by DNA damage [75]miRNAs able to regulate the mismatch repair function aremiR-155 and miR-21 that significantly downregulate the coreMMR proteins MSH2 MSH6 and MLH1 and have beenassociated with a mutator phenotype in particular with MSIinflammatory bowel diseases (IBD) CRCs [76 77]

5 Characterization of the (Variantsof Uncertain Significance) (VUS) inthe MMR Genes

Several mutations identified in the MMR genes are missensesilent or intronic variants The influence of these variantson the development of cancer is often a controversial topictherefore they are classified as ldquoVUSrdquo Variant of UncertainSignificance [78 79]

Several criteria can be applied to assess the possiblepathogenicity of a VUS [57 80] these criteria are as follows(1) de novo appearance (2) segregation with the disease(3) absence in normal individuals (4) change of aminoacid polarity or size (5) occurrence of the amino acidchange in a domain that is evolutionary conserved betweenspecies andor shared between proteins belonging to the sameprotein family (in silico analysis) (6) effects on splicing oron protein function (7) loss of the nonmutated allele due toa large deletion in the tumor DNA (loss of heterozygosity(LOH)) (8) loss of protein expression in the tumor (9)evaluation of MSI in tumor tissues All studies conducted todate show that none of the above criteria including functionalassays is an indicator of pathogenicity if taken alone itis necessary that a combination of strategies be used inorder to lead to a correct assessment of the pathogenicity ofuncertain variants [81] According to these observations aclassification ofMMR sequence variants identified by genetictesting has been proposed based on a 5-class system using amultifactorial likelihood model (Table 2)

Variant-Class 5 includes coding sequence variationresulting in a stop codon (nonsense or frameshift) splicingaberration variants by mRNA assay large genomic deletionsor duplications abrogated mRNAprotein function variantsbased either on laboratory assays on evidence for cosegre-gation with disease and on MSI tumor andor loss of MMRprotein expression

Variant-Class 4 includes IVS+-1 or IVS+-2 mutationsresulting in splicing aberrations variants abrogating

mRNAprotein function based on laboratory assays evi-dence of cosegregation with disease or MSI tumor andorloss of MMR protein expression

Variant-Class 3 includes large genomic duplications mis-sense alterations small in-frame insertionsdeletions silentvariants intronic variants and promoter and regulatoryregion variants for which insufficient molecular evidenceis available and with intermediate clinical effects or lowpenetrance alleles

Variant-Class 2 includes synonymous substitutions andintronic variants with no associated mRNA aberration witha proficient protein expressionfunction and lack of cosegre-gation andor MSS tumor

Variant-Class 1 includes variants reported in control ref-erence groups and excluded as founder pathogenic sequencevariant

According to this classification most of the VUS testedfor theMMRgenes are likely to be pathogenetic and thus theycan be associated with the HNPCC phenotype

For theMLH1 gene 52 out of 73VUS resulted to be patho-genetic (70) similar pathogenicity has been demonstratedfor 25 out of 35 VUS identified in the MSH2 gene (71)(httpswwwinsight-grouporg)

For minor MMR genes the percentage of pathogenicVUSs is reduced due to the milder mutational contributionof these genes to the development of the disease For theMSH6 gene only 1 out of 8 variants studied (13) was foundto have aberrant effects on protein function for the PMS2gene 4 variants were analyzed and all (100) seem to havea causative role in Lynch syndrome for the MLH3 genehowever functional assays have not identified any variantwith certain pathogenetic significance finally for the MSH3gene relevant functional studies have not yet been reported(httpswwwinsight-grouporg)

6 Probability of a (Synergistic Effect) betweenLow Risk Allelic Variants in the MMR Genes

With the advent of high-throughput technologies it isbecoming even more possible to analyze a great number ofpolymorphic variants in large cohorts of cases and controlsof specific cancers such as breast prostate and colorectalcancer thus providing new insights into common mecha-nisms of carcinogenesis In some cases VUSs make a moresubstantial overall contribution to cancer risk than the well-assessed severe Mendelian variants It is also possible that thesimultaneous presence of some polymorphisms and VUSs incancer predisposition genes that behave as low risk allelesmight contribute in a cooperative manner to increase therisk of hereditary cancer [12 64] Therefore current litera-ture data suggest a significant proportion of the inheritedsusceptibility to relatively common human diseases may bedue to the addition of the effects of a series of low frequencyvariants of different genes probably acting in a dominantand independent manner with each of them conferring amoderate but even detectable increase in the relative cancerrisk [50 51 75 81ndash83] Therefore several functional studiesbased on GWAS data related to cancer susceptibility have


Table 2 Proposed classification system for MMR variant interpretation (Colon Cancer Family Registry 2009 InSiGHT Variant InterpretationCommittee 2011)

Class Description Probability of being pathogenic5 Definitely pathogenic gt0994 Likely pathogenic 095ndash0993 Uncertain 005ndash09492 Likely not pathogenic or of little clinical significance 0001ndash00491 Not pathogenic or of no clinical significance lt0001

been performed in an attempt to demonstrate the effectiveassociation and to test the hypothesis of synergistic effectsbetween low risk allelic variants [80 81]

In a recent study on yeast genome it has been shown thatthe minor alleles of the MMR complex cause a weak mutatorphenotype however their interaction causes a more severemutator phenotype [82] In this study 11 polymorphismsand 14 missense variants of uncertain significance previouslyidentified in theMSH2 MLH1 MSH6 and PMS2 genes werestudied by complementation testsThemutator effect of thesevariants was tested singly and in combinationwith each other

In 2011 Kumar et al showed that some variants occurringin domain I of the MSH2 gene in yeast strains (msh2Δ1)behave as weak alleles in the presence of a functional proteinMSH6 as they do not alter the stability of the MutS120572complex However by combining these variants with weakalleles falling in the N-terminal region (NTR) (DNA bindingdomain) of the MSH6 gene a strong mutator phenotype wasfound Moreover the mutator synergistic effect is also foundbetween different systems of DNAdamage response A recentpopulation study by Smith et al [83] has shown that thesimultaneous presence of mutations in the TP53 gene andsingle nucleotide polymorphisms (SNPs) in genes belongingto different repair systems as BER NER MMR and DSBR(Double-Strand Break Repair) complex is associated with anearlier age of onset of breast cancer (lt50 years) Thereforein this case the authors suggest an additive or multiplicativeeffect

The additive effect of low penetrance genes could also bethe cause of atypical Lynch syndromes such as familial CRCtype X [84] With respect to LS the familial CRC type X ismore often located in the distal colon extracolonic cancersare less frequent than in LS and the age of onset is delayedThe sine qua non condition for this diagnosis is the absenceof molecular genetic evidence of LS (MSI IHC or MMRmutations)

7 Scientific Hypothesis and Our Results

Molecular characterization of patients with a clinical diagno-sis of Lynch syndrome currently relies on the identification ofpointmutations and large rearrangements [85 86] byDHPLCandMLPA respectively in themajorMMR genesMLH1 andMSH2

This strategy does not always provide informative resultsfor genetic counseling Indeed many families selected

according to international diagnostic criteria (AmsterdamCriteria and Bethesda Guidelines) do not have a moleculardiagnosis of Lynch syndrome In our experience we haveidentified several patients carrying genetic VUS (missenseintronic and silent variants) not only in main genes MLH1and MSH2 [80] but also in other MMR genes Accordingto international recommendations (Colon cancer FamilyRegistry 2009 InSiGHT Variant Interpretation Commit-tee 2011) we used a multifactorial likelihood model in anattempt to define a pathogenetic role for numerous VUSidentified in MMR genes [50 51 64 85] The segregationanalysis population studies (to exclude the polymorphicnature of the variant) assessment of MSI in tumor tissuesdetection of loss of protein expression in tumor tissues byimmunohistochemical analysis (IHC) in silico analysis by avariety of bioinformatics tools and gene expression studiesare strategies that have to be used to assess an exhaustiveevaluation of the pathogenicity of uncertain variants [85]

In light of literature data indicated that ldquominorrdquo MMRproteins have other functions besides the postreplicativerepair that could be highly relevant in carcinogenesis inour laboratories we have also analyzed the minor MMRgenesMSH6 PMS2MLH3 andMSH3 for germline variantsdetected in patients negative for germline mutations in themajor MMR genes Many of the subjects analyzed in ourseries [50 51 64 85] showed coinheritance of differentgenetic alterations in the minor MMR genes (Table 3) wespeculate a likely additive role of low penetrance alleles in thedisease development in favor of a putative polygenic inheri-tance for Lynch syndrome according to recent literature data[87ndash89]

8 Conclusion

The recent literature data describe theMMRproteins increas-ingly new roles It is now known that MMR proteins notonly have an exclusive role in the repair of DNA mismatchbut are also involved in many other processes relevant incarcinogenesis Therefore some genetic variants may notaffect the repair function but may be responsible for theloss of other important functions related to MMR proteinsIn the light of these new roles of the MMR proteins it isessential to widen the mutations detection in all genes thatare part of MMR complex This will lead to the identificationof numerous VUS in these genes However the study of VUSidentified in MMR genes provides important information


Table 3 Patients carrying variants in several MMR genes MSH6 PMS2 MSH3 and MLH3 lowastpatient 504 showing also the UV in MSH2gene (c984 CgtT) [64]

Patients MSH6 PMS2 MLH3 MSH3 Phenotype

9525ex4

c2633 TgtC(ValgtAla)

ex14c2324 AgtG(AsngtSer)

ex1c2530 CgtT

(ProgtSer) c2533TgtC (SergtPro)

IVS7 -9 TgtC Amsterdam +

013

ex6c665GgtC

(SergtThr) IVS6+16AgtG

ex1c2533 TgtC(SergtPro)

No AmsterdamMSI-H

103ex5

c3261 62insC(Phegtstop)


ex12c1860GgtA(AspgtAsn)

No Amsterdamlater onset MSI-H

423 IVS12-4GgtA

ex1c2530 CgtT(ProgtSer)c2533 TgtC(SergtPro)

Amsterdam + lateronsetMSI-L

015ex5

c3295 97delTT(Ilegtstop)

ex1c666 GgtA (Lys)

c2191 GgtT(ValgtPhe)c2533AgtG(SergtGly)

Amsterdam +MSI-H

210ex4

c2941 AgtG(IlegtVal)

IVS6+16AgtGex13

c2324 TgtC (Phe)

ex1c2530 CgtT(ProgtSer)

IVS6-64 CgtT Amsterdam +MSI not detected

211ex4


IVS12-4 GgtA IVS6-64 CgtT Amsterdam +MSI not detected

416ex11

c1714CgtA(ThrgtLys)

ex 1c2027GgtA(ArggtLys)

IVS6-64 CgtT Amsterdam +MSI-H

504lowast

ex4c693GgtA (Pro)

ex20c2732 TgtG(LeugtTrp)

Amsterdam +MSI-H

on the pathogenicity of the many genetic variants that areidentifying in patients with suspected diagnosis of Lynchsyndrome Very often these variants are causing the diseaseperhaps with a different degree of pathogenicity Sometimesthe simultaneous presence of molecular alterations in severalMMR genes could be causing the onset of tumor All thesereassess the classical model of monogenic transmission infavor of a polygenic inheritance of Lynch syndrome

Therefore these recent findings allow clarifying betterthe genotype-phenotype correlations in Lynch syndromedemonstrating the importance of molecular analysis toimprove the genetic counseling and consequently the clinicalsurveillance

Competing Interests

The authors declare that they have no competing interestsregarding the manuscript

Acknowledgments

This paper is supported by agreement 2010ndash2012 betweenCEINGE and Campania Regional Authority and POR Cam-pania FSE 2007ndash2013

References

[1] H T Lynch K Drescher J Knezetic and S Lanspa ldquoGeneticsbiomarkers hereditary cancer syndromediagnosis heterogene-ity and treatment a reviewrdquo Current Treatment Options inOncology vol 15 no 3 pp 429ndash442 2014

[2] C C Chung and S J Chanock ldquoCurrent status of genome-wideassociation studies in cancerrdquo Human Genetics vol 130 no 1pp 59ndash78 2011

[3] L Valle ldquoGenetic predisposition to colorectal cancer where westand and future perspectivesrdquo World Journal of Gastroenterol-ogy vol 20 no 29 pp 9828ndash9849 2014


[4] M De Rosa R J Dourisboure G Morelli et al ldquoFirst genotypecharacterization of Argentinean FAP patients identification of14 novel APC mutationsrdquo Human Mutation vol 23 no 5 pp523ndash524 2004

[5] M De Rosa M Galatola S Borriello F Duraturo S Masoneand P Izzo ldquoImplication of adenomatous polyposis coli andMUTYH mutations in familial colorectal polyposisrdquo Diseasesof the Colon and Rectum vol 52 no 2 pp 268ndash274 2009

[6] N J Samadder K Jasperson and R W Burt ldquoHereditaryand common familial colorectal cancer evidence for colorectalscreeningrdquo Digestive Diseases and Sciences vol 60 no 3 pp734ndash747 2015

[7] M De Rosa U Pace D Rega et al ldquoGenetics diagnosis andmanagement of colorectal cancer (Review)rdquo Oncology Reportsvol 34 no 3 pp 1087ndash1096 2015

[8] N Carlomagno F Duraturo M Candida et al ldquoMultiplesplenic hamartomas and familial adenomatous polyposis a casereport and review of the literaturerdquo Journal of Medical CaseReports vol 9 no 1 article no 154 2015

[9] M Galatola L Paparo F Duraturo et al ldquoBeta catenin andcytokine pathway dysregulation in patients with manifestationsof the ldquoPTEN hamartoma tumor syndromerdquordquo BMC MedicalGenetics vol 13 article no 28 2012

[10] L Paparo G B Rossi P Delrio et al ldquoDifferential expressionof PTEN gene correlates with phenotypic heterogeneity inthree cases of patients showing clinical manifestations of PTENhamartoma tumour syndromerdquo Hereditary Cancer in ClinicalPractice vol 11 article 8 2013

[11] M Galatola E Miele C Strisciuglio et al ldquoSynergistic effectof interleukin-10-receptor variants in a case of early-onsetulcerative colitisrdquoWorld Journal of Gastroenterology vol 19 no46 pp 8659ndash8670 2013

[12] J M Carethers ldquoDifferentiating lynch-like from lynch syn-dromerdquo Gastroenterology vol 146 no 3 pp 602ndash604 2014

[13] M G F van Lier AWagnerM E van Leerdam et al ldquoA reviewon the molecular diagnostics of Lynch syndrome a central rolefor the pathology laboratoryrdquo Journal of Cellular and MolecularMedicine vol 14 no 1-2 pp 181ndash197 2010

[14] D C Hegan L Narayanan F R Jirik W Edelmann R MLiskay andPMGlazer ldquoDiffering patterns of genetic instabilityin mice deficient in the mismatch repair genes Pms2 Mlh1Msh2Msh3 andMsh6rdquoCarcinogenesis vol 27 no 12 pp 2402ndash2408 2006

[15] S-H Jun TGKim andC Ban ldquoDNAmismatch repair systemclassical and fresh rolesrdquo FEBS Journal vol 273 no 8 pp 1609ndash1619 2006

[16] J Jiricny ldquoThe multifaceted mismatch-repair systemrdquo NatureReviews Molecular Cell Biology vol 7 no 5 pp 335ndash346 2006

[17] F A Sinicrope and D J Sargent ldquoMolecular pathwaysmicrosatellite instability in colorectal cancer prognostic pre-dictive and therapeutic implicationsrdquo Clinical Cancer Researchvol 18 no 6 pp 1506ndash1512 2012

[18] P Alhopuro H Sammalkorpi I Niittymaki et al ldquoCandidatedriver genes in microsatellite-unstable colorectal cancerrdquo Inter-national Journal of Cancer vol 130 no 7 pp 1558ndash1566 2012

[19] A Zaanan K Meunier F Sangar J-F Flejou and F PrazldquoMicrosatellite instability in colorectal cancer from molecularoncogenicmechanisms to clinical implicationsrdquoCellular Oncol-ogy vol 34 no 3 pp 155ndash176 2011

[20] K Imai and H Yamamoto ldquoCarcinogenesis and microsatelliteinstability the interrelationship between genetics and epigenet-icsrdquo Carcinogenesis vol 29 no 4 pp 673ndash680 2008

[21] S G Sharma and M L Gulley ldquoBRAF mutation testingin colorectal cancerrdquo Archives of Pathology and LaboratoryMedicine vol 134 no 8 pp 1225ndash1228 2010

[22] L Wang J M Cunningham J L Winters et al ldquoBRAFmutations in colon cancer are not likely attributable to defectiveDNA mismatch repairrdquo Cancer Research vol 63 no 17 pp5209ndash5212 2003

[23] Y Nakaji E Oki R Nakanishi et al ldquoPrognostic value of BRAFV600E mutation and microsatellite instability in Japanesepatients with sporadic colorectal cancerrdquo Journal of CancerResearch and Clinical Oncology vol 143 no 1 pp 151ndash160 2017

[24] P Lochhead A Kuchiba Y Imamura et al ldquoMicrosatelliteinstability and BRAF mutation testing in colorectal cancerprognosticationrdquo Journal of the National Cancer Institute vol105 no 15 pp 1151ndash1156 2013

[25] C R Boland S N Thibodeau S R Hamilton et al ldquoANational Cancer Institute workshop onmicrosatellite instabilityfor cancer detection and familial predisposition developmentof international criteria for the determination of microsatelliteinstability in colorectal cancerrdquo Cancer Research vol 58 no 22pp 5248ndash5257 1998

[26] E Vilar M E Mork A Cuddy et al ldquoRole of microsatelliteinstability-low as a diagnostic biomarker of Lynch syndrome incolorectal cancerrdquo Cancer Genetics vol 207 no 10-12 pp 495ndash502 2014

[27] R M Xicola X Llor E Pons et al ldquoPerformance of differentmicrosatellite marker panels for detection of mismatch repair-deficient colorectal tumorsrdquo Journal of the National CancerInstitute vol 99 no 3 pp 244ndash252 2007

[28] A Umar C R Boland J P Terdiman et al ldquoRevisedBethesda Guidelines for hereditary nonpolyposis colorectalcancer (Lynch syndrome) andmicrosatellite instabilityrdquo Journalof the National Cancer Institute vol 96 no 4 pp 261ndash268 2004

[29] N Suraweera A Duval M Reperant et al ldquoEvaluation oftumor microsatellite instability using five quasimonomorphicmononucleotide repeats and pentaplex PCRrdquo Gastroenterologyvol 123 no 6 pp 1804ndash1811 2002

[30] D J Sargent S Marsoni G Monges et al ldquoDefective mismatchrepair as a predictive marker for lack of efficacy of fluorouracil-based adjuvant therapy in colon cancerrdquo Journal of ClinicalOncology vol 28 no 20 pp 3219ndash3226 2010

[31] J H Kim and G H Kang ldquoMolecular and prognostic het-erogeneity of microsatellite-unstable colorectal cancerrdquo WorldJournal of Gastroenterology vol 20 no 15 pp 4230ndash4243 2014

[32] H T Lynch P M Lynch S J Lanspa C L Snyder J FLynch and C R Boland ldquoReview of the Lynch syndromehistory molecular genetics screening differential diagnosisand medicolegal ramificationsrdquo Clinical Genetics vol 76 no 1pp 1ndash18 2009

[33] J Shia S Holck G Depetris J K Greenson and D S Klim-stra ldquoLynch syndrome-associated neoplasms a discussion onhistopathology and immunohistochemistryrdquo Familial Cancervol 12 no 2 pp 241ndash260 2013

[34] G Corso D Marrelli and F Roviello ldquoFamilial gastric cancerupdate for practice managementrdquo Familial Cancer vol 10 no2 pp 391ndash396 2011

[35] A Gylling W M Abdel-Rahman M Juhola et al ldquoIs gas-tric cancer part of the tumour spectrum of hereditary non-polyposis colorectal cancer A Molecular Genetic Studyrdquo Gutvol 56 no 7 pp 926ndash933 2007


[36] V OrsquoBrien and R Brown ldquoSignalling cell cycle arrest and celldeath through the MMR Systemrdquo Carcinogenesis vol 27 no 4pp 682ndash692 2006

[37] K Yamane J E Schupp and T J Kinsella ldquoBRCA1 activatesa G2-M cell cycle checkpoint following 6-thioguanine-inducedDNA mismatch damagerdquo Cancer Research vol 67 no 13 pp6286ndash6292 2007

[38] ANicholsonMHendrix S Jinks-Robertson andG F CrouseldquoRegulation of mitotic homeologous recombination in yeastfunctions of mismatch repair and nucleotide excision repairgenesrdquo Genetics vol 154 no 1 pp 133ndash146 2000

[39] G Ji Y Long Y Zhou C Huang A Gu and X WangldquoCommon variants in mismatch repair genes associated withincreased risk of spermDNAdamage andmale infertilityrdquoBMCMedicine vol 10 article 49 2012

[40] S Roa Z Li J U Peled C Zhao W Edelmann and M DScharff ldquoMSH2MSH6 complex promotes error-free repair ofAID-Induced dUG mispairs as well as error-prone hypermu-tation of AT Sitesrdquo PLoS ONE vol 5 no 6 Article ID e111822010

[41] C Jiang M-L Zhao K M Waters and M Diaz ldquoActivation-induced deaminase contributes to the antibody-independentrole of B cells in the development of autoimmunityrdquo Autoim-munity vol 45 no 6 pp 440ndash448 2012

[42] S Tome I Holt W Edelmann et al ldquoATPase domain mutationaffects CTGlowastCAG repeat instability in transgenic micerdquo PLoSGenetics vol 5 no 5 Article ID e1000482 2009

[43] E Dragileva A Hendricks A Teed et al ldquoIntergenerationaland striatal CAG repeat instability in Huntingtonrsquos diseaseknock-in mice involve different DNA repair genesrdquo Neurobiol-ogy of Disease vol 33 no 1 pp 37ndash47 2009

[44] A Seriola C Spits J P Simard et al ldquoHuntingtonrsquos andmyotonic dystrophy hESCs down-regulated trinucleotiderepeat instability and mismatch repair machinery expressionupon differentiationrdquo Human Molecular Genetics vol 20 no 1pp 176ndash185 2011

[45] G Mao X Pan and L Gu ldquoEvidence that a mutation in theMLH1 31015840-untranslated region confers a mutator phenotype andmismatch repair deficiency in patients with relapsed leukemiardquoJournal of Biological Chemistry vol 283 no 6 pp 3211ndash32162008

[46] P Hsieh and K Yamane ldquoDNA mismatch repair molecularmechanism cancer and ageingrdquo Mechanisms of Ageing andDevelopment vol 129 no 7-8 pp 391ndash407 2008

[47] J J Koornstra M J Mourits R H Sijmons A M Leliveld HHollema and J H Kleibeuker ldquoManagement of extracolonictumours in patients with Lynch syndromerdquo The Lancet Oncol-ogy vol 10 no 4 pp 400ndash408 2009

[48] E Lucci-Cordisco V Rovella S Carrara et al ldquoMutations ofthe rsquominorrsquo mismatch repair gene MSH6 in typical and atypicalhereditary nonpolyposis colorectal cancerrdquo Familial Cancervol 1 no 2 pp 93ndash99 2001

[49] E C Chao and S M Lipkin ldquoMolecular models for the tissuespecificity of DNA mismatch repair-deficient carcinogenesisrdquoNucleic Acids Research vol 34 no 3 pp 840ndash852 2006

[50] F Duraturo R Liccardo and P Izzo ldquoCoexistence of MLH3germline variants in colon cancer patients belonging to familieswith Lynch syndrome-associated brain tumorsrdquo Journal ofNeuro-Oncology vol 129 no 3 pp 577ndash578 2016

[51] F Duraturo R Liccardo A Cavallo M D Rosa M Grossoand P Izzo ldquoAssociation of low-risk MSH3 and MSH2 variant

alleles with Lynch syndrome probability of synergistic effectsrdquoInternational Journal of Cancer vol 129 no 7 pp 1643ndash16502011

[52] J Huang S A Kuismanen T Liu et al ldquoMSH6 and MSH3 arerarely involved in genetic predisposition to nonpolypotic coloncancerrdquo Cancer Research vol 61 no 4 pp 1619ndash1623 2001

[53] S-Y Lee H Chung B Devaraj et al ldquoMicrosatellite alter-ations at selected tetranucleotide repeats are associated withmorphologies of colorectal Neoplasiasrdquo Gastroenterology vol139 no 5 pp 1519ndash1525 2010

[54] P Bandipalliam ldquoSyndrome of early onset colon cancershematologic malignancies amp features of neurofibromatosis inHNPCC families with homozygous mismatch repair genemutationsrdquo Familial Cancer vol 4 no 4 pp 323ndash333 2005

[55] J C Herkert R C Niessen M J W Olderode-Berends etal ldquoPaediatric intestinal cancer and polyposis due to bi-allelicPMS2 mutations case series review and follow-up guidelinesrdquoEuropean Journal of Cancer vol 47 no 7 pp 965ndash982 2011

[56] J-W Poley A Wagner M M C P Hoogmans et al ldquoBiallelicgermline mutations of mismatch-repair genes a possible causefor multiple pediatric malignanciesrdquo Cancer vol 109 no 11 pp2349ndash2356 2007

[57] S E Plon D M Eccles D Easton et al ldquoSequence variantclassification and reporting recommendations for improvingthe interpretation of cancer susceptibility genetic test resultsrdquoHuman Mutation vol 29 no 11 pp 1282ndash1291 2008

[58] A Peters H Born R Ettinger P Levonian and K B JedeleldquoCompound heterozygosity for MSH6 mutations in a pediatriclymphoma patientrdquo Journal of Pediatric HematologyOncologyvol 31 no 2 pp 113ndash115 2009

[59] S Y Kang C K Park D K Chang et al ldquoLynch-like syndromecharacterization and comparison with EPCAM deletion carri-ersrdquo International Journal of Cancer vol 136 no 7 pp 1568ndash1578 2015

[60] D D Buchanan C Rosty M Clendenning A B Spurdle andA KWin ldquoClinical problems of colorectal cancer and endome-trial cancer cases with unknown cause of tumor mismatchrepair deficiency (suspected Lynch syndrome)rdquo Application ofClinical Genetics vol 7 pp 183ndash193 2014

[61] C R Boland ldquoThemystery ofmismatch repair deficiency lynchor lynch-likerdquo Gastroenterology vol 144 no 5 pp 868ndash8702013

[62] A R Mensenkamp I P Vogelaar W A G Van Zelst-Stams etal ldquoSomaticmutations inMLH1 andMSH2 are a frequent causeof mismatch-repair deficiency in lynch syndrome-like tumorsrdquoGastroenterology vol 146 no 3 pp 643e8ndash646e8 2014

[63] F Li G Mao D Tong et al ldquoThe histone mark H3K36me3regulates human DNA mismatch repair through its interactionwith MutS120572rdquo Cell vol 153 no 3 pp 590ndash600 2013

[64] F Duraturo R Liccardo A Cavallo M De Rosa and PIzzo Synergistic Effects of Low-Risk Variant Alleles in CancerPredisposition Carcinogenesis edited by Kathryn TonissenInTech Rijeka Croatia 2013

[65] Y Iwahashi E Ito Y Yanagisawa et al ldquoPromoter analysis ofthe human mismatch repair gene hMSH2rdquo Gene vol 213 no1-2 pp 141ndash147 1998

[66] E Ito Y Yanagisawa Y Iwahashi et al ldquoA core promoter anda frequent single-nucleotide polymorphism of the mismatchrepair gene hMLH1rdquo Biochemical and Biophysical ResearchCommunications vol 256 no 3 pp 488ndash494 1999


[67] M Mrkonjic S Raptis R C Green et al ldquoMSH2 minus118TgtCand MSH6 minus159CgtT promoter polymorphisms and the risk ofcolorectal cancerrdquoCarcinogenesis vol 28 no 12 pp 2575ndash25802007

[68] S Raptis M Mrkonjic R C Green et al ldquoMLH1 -93GgtApromoter polymorphism and the risk of microsatellite-unstablecolorectal cancerrdquo Journal of the National Cancer Institute vol99 no 6 pp 463ndash474 2007

[69] G Mao S Lee J Ortega L Gu and G-M Li ldquoModulation ofmicroRNA processing by mismatch repair proteinMutL120572rdquo CellResearch vol 22 no 6 pp 973ndash985 2012

[70] G Zhang E Gibbs Z Kelman M OrsquoDonnell and J HurwitzldquoStudies on the interactions between human replication factorC and human proliferating cell nuclear antigenrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 96 no 5 pp 1869ndash1874 1999

[71] PV ShcherbakovaMCHallM S Lewis et al ldquoInactivation ofDNAmismatch repair by increased expression of yeast MLH1rdquoMolecular and Cellular Biology vol 21 no 3 pp 940ndash951 2001

[72] G Marra I Iaccarino T Lettieri G Roscilli P Delmastroand J Jiricny ldquoMismatch repair deficiency associated withoverexpression of the MSH3 generdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 95 no15 pp 8568ndash8573 1998

[73] D-A Landau and F J Slack ldquoMicroRNAs in mutagenesisgenomic instability andDNA repairrdquo Seminars inOncology vol38 no 6 pp 743ndash751 2011

[74] Y Dong J Yu and S S M Ng ldquoMicroRNA dysregulation asa prognostic biomarker in colorectal cancerrdquo Cancer Manage-ment and Research vol 6 pp 405ndash422 2014

[75] Y Wang and T Taniguchi ldquoMicroRNAs and DNA damageresponse implications for cancer therapyrdquoCell Cycle vol 12 no1 pp 32ndash42 2013

[76] N Valeri P Gasparini C Braconi et al ldquoMicroRNA-21induces resistance to 5-fluorouracil by down-regulating humanDNA MutS homolog 2 (hMSH2)rdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 107 no49 pp 21098ndash21103 2010

[77] M Svrcek N El-Murr K Wanherdrick et al ldquoOverexpressionof microRNAs-155 and 21 targeting mismatch repair proteins ininflammatory bowel diseasesrdquo Carcinogenesis vol 34 no 4 pp828ndash834 2013

[78] F J Couch L J Rasmussen R Hofstra A N A Monteiro MS Greenblatt andN deWind ldquoAssessment of functional effectsof unclassified genetic variantsrdquo Human Mutation vol 29 no11 pp 1314ndash1326 2008

[79] S Syngal E A Fox C Li et al ldquoInterpretation of genetic testresults for hereditary nonpolyposis colorectal cancer implica-tions for clinical predisposition testingrdquo JAMA vol 282 no 3pp 247ndash253 1999

[80] A P Shaik A S Shaik and Y A Al-Sheikh ldquoColorectal cancera review of the genome-wide association studies in the kingdomof Saudi Arabiardquo Saudi Journal of Gastroenterology vol 21 no3 pp 123ndash128 2015

[81] L Le Marchand ldquoGenome-wide association studies and col-orectal cancerrdquo Surgical Oncology Clinics of North America vol18 no 4 pp 663ndash668 2009

[82] D E Goldgar D F Easton G B Byrnes A B SpurdleE S Iversen and M S Greenblatt ldquoGenetic evidence andintegration of various data sources for classifying uncertainvariants into a single modelrdquo Human Mutation vol 29 no 11pp 1265ndash1272 2008

[83] T R Smith W Liu-Mares B O Van Emburgh et al ldquoGeneticpolymorphisms of multiple DNA repair pathways impact age atdiagnosis and TP53 mutations in breast cancerrdquo Carcinogenesisvol 32 no 9 pp 1354ndash1360 2011

[84] N M Lindor K Rabe G M Petersen et al ldquoLower cancerincidence in Amsterdam-I criteria families without mismatchrepair deficiency familial colorectal cancer type Xrdquo JAMA vol293 no 16 pp 1979ndash1985 2005

[85] F Duraturo R Liccardo A Cavallo M De Rosa G B Rossiand P Izzo ldquoMultivariate analysis as amethod for evaluating thepathogenicity of novel genetic MLH1 variants in patients withcolorectal cancer and microsatellite instabilityrdquo InternationalJournal of Molecular Medicine vol 36 no 2 pp 511ndash517 2015

[86] F Duraturo A Cavallo R Liccardo et al ldquoContribution of largegenomic rearrangements in Italian Lynch syndrome patientscharacterization of a novel alu-mediated deletionrdquo BioMedResearch International vol 2013 Article ID 219897 7 pages2013

[87] T A Muranen N Mavaddat S Khan et al ldquoPolygenic riskscore is associated with increased disease risk in 52 Finnishbreast cancer familiesrdquo Breast Cancer Research and Treatmentvol 158 no 3 pp 463ndash469 2016

[88] J Yuan Y Li T Tian et al ldquoRisk prediction for early-onset gas-tric carcinoma a case-control study of polygenic gastric cancerin Han Chinese with hereditary backgroundrdquoOncotarget vol 7no 23 pp 33608ndash33615 2016

[89] B A Talseth-Palmer D C Bauer W Sjursen et al ldquoTargetednext-generation sequencing of 22 mismatch repair genes iden-tifies Lynch syndrome familiesrdquo Cancer Medicine vol 5 no 5pp 929ndash941 2016

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


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Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

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Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


APC PMS2 andMLH1 genesmutations and characterized bybrain cancers (glioblastoma and cerebellar medulloblastoma)are associated with colorectal cancer [6 7]

In the process of colorectal carcinogenesis many othergenes are involved such as oncogenes and tumor suppressorgenes that play a key role in the control of cell cycleMutations in these genes are at the basis of rarer inheritedCRC syndromes These are mainly ldquohamartomatous poly-posis syndromesrdquo characterized by the presence of benignadenomas arising from epithelial andor stromal intestinaltissue which increase the risk of developing CRC Thesesyndromes whose characteristics are summarized in Table 1include Peutz-Jeghers syndrome juvenile polyposis Cowdensyndrome and Bannayan-Riley-Ruvalcaba syndrome [8ndash11]

In this review we intend to highlight new insights intothe molecular features of Lynch syndrome in favor of a novelinheritance model in contrast with the classical monogenictransmission this could suggest new genetic and clinicalsurveillance approaches

2 Lynch Syndrome (LS)

21 Genetics Features LS is an autosomal dominant diseasewith recessive phenotype caused by a defect in one of themis-match repair (MMR) genes The main clinical-pathologicalfeatures of the Lynch syndrome are as follows [1ndash11 13]

Autosomal dominant inheritancePenetrance for colorectal cancer (CRC) of 85ndash90Earlier age of onset of CRC (sim45 years ) with respectto general population (69 years)Preferential tumor localization in the right-sidedcolonPresence of multiple synchronous and metachronouscolorectal cancersBetter prognosis than CRCsIncreased risk for extracolonic cancersAccelerated carcinogenesisPoorly differentiated tumors with a marked lympho-cytic peritumoral inflammation recalling features ofthe so-called ldquoCrohnrsquos reactionrdquoMicrosatellite instability

The mismatch repair system was first studied in bacteriain which three proteins MutS MutL and MutH wereidentified In humans at least seven mismatch repair genesare involved in mismatch repair and their names derivefrom their structural homology to the bacterial proteinsthe MutS homologues (MSH) MSH2 on chromosome 2p16MSH3 on chromosome 5q11 and MSH6 on chromosome2p16 the MutL homologues (MLH) MLH1 on chromosome3p21 and MLH3 on chromosome 2p16 the postmeioticsegregation homologues (PMS) PMS1 and PMS2 on chro-mosome 7p22 No MutH homologues have been identifiedin humans [14] MSH2 and MSH6 bind together to forma heteroduplex (MutSa) that predominantly identifies base

pairs mismatched whileMSH2 andMSH3 (MutS120573) combineto identify short insertions or deletions MSH2 is essentialfor both complexes to function while a functional overlapexists between MSH3 and MSH6 MLH1 and PMS2 (MutL120572)or MLH3 (Mutl120574) also bind together to form a heteroduplexthat interacts with MutS120572 or MutS120573 complex stimulatingexcision and resynthesis of the abnormal DNA Similarly toMSH2 also MLH1 is essential for both complexes to repairmismatches Altogether this group of four proteins recruitsexonuclease-1 (EXO1) the proliferating cell nuclear antigen(PCNA) DNA polymerase (Pol120575 or Pol120576) two replicationfactor (RPA and RFC) and a ligase to repair DNA onthe daughter strand at the mismatch point If any of thefour major proteins (MSH2 MLH1 MSH6 or PMS2) isfunctionally inactive mismatches are not repaired [15 16]

22 Microsatellite Instability Consequently a defective DNAMMR system increases the mutation rate and makes thecell vulnerable to mutations in genes controlling cell growth(tumor suppressor genes and oncogenes) resulting in anincreased cancer risk

In case of a defective MMR system mutations occurfrequently in small (usually mononucleotide or dinucleotide)repetitive DNA sequences known as microsatellites InMMR-deficient tumor cells the number of microsatelliterepeat units can deviate from the corresponding normalDNA the number of repeats is usually decreased even thoughit is occasionally found to be increased [17]

Length or size microsatellite variation is known as MSI(microsatellite instability) MSI (formerly referred to as MINor RER replication error) is the molecular hallmark of LSsince approximately 95 of all LS-associated cancers showMSI [13] Althoughmost microsatellite sequences are locatedin noncoding sequences (telomeres and centromeres) manygenes contain repetitive sequences in their coding regionsand some of these genes play key roles in the regulation of cellgrowth Identification of an even growing number of guidegenes and target genes of the mutator phenotype can lead todiscover new complex molecular mechanisms that underliethe process of colorectal tumorigenesis [18]

MSI thereby serves as a reliable phenotypic marker ofMMR deficiency in order to preselect patients eligible forgermline mutation analysis in the MMR genes [19]

However despite the fact that MSI is a reliable markerfor MMR deficiency however until 15 of sporadic CRCsshowed an MSI phenotype This is mainly caused by somatichypermethylation of the MLH1-gene promoter Methylationof the MSH2 promoter has also been reported but it is tobe considered as a heritable somatic methylation because itis caused by a deletion of the last exon of EPCAM that isadjacent to MSH2 on chromosome 2 [18]

Hypermethylation of CpG islands in the MLH1 pro-moter (CIMP phenotype) causes severe inhibition of genetranscription thereby mimicking an inactivating gene muta-tion If both copies of the gene are inactivated (biallelichypermethylation) the MLH1 function is lost This leads tomicrosatellite unstable cancers especially in older patientsTherefore in MLH1-deficient microsatellite unstable (MSI-H) tumors the MLH1 hypermethylation can be assessed to









90






lt100





(20ndash30)lt100






lt100






90






90CRC glioblastoma




rearrangements(90)

95ndash100






rearrangements(60)

90ndash100


cancer









rearrangements(60)

90ndash95




















PCNARFC

ATRP53

DNA damagesignaling

MSH2

MLH1MLH1





regulation

MSH2Triplet repeats

expansion

MMR complex

MSH2

MLH1


MLH1 PMS2PMS2

MSH6

PMS2

MSH3

MSH6

PMS2

MLH3

MSH3

MMR proteins



AIDPol 120578
























































8 Conclusion





9525ex4



ex1c2530 CgtT



013

ex6c665GgtC



No AmsterdamMSI-H

103ex5





423 IVS12-4GgtA



015ex5


ex1c666 GgtA (Lys)


Amsterdam +MSI-H

210ex4


IVS6+16AgtGex13

c2324 TgtC (Phe)



211ex4



416ex11

c1714CgtA(ThrgtLys)



504lowast

ex4c693GgtA (Pro)


Amsterdam +MSI-H



Competing Interests


Acknowledgments


References



































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























90






lt100





(20ndash30)lt100






lt100






90






90CRC glioblastoma




rearrangements(90)

95ndash100






rearrangements(60)

90ndash100


cancer









rearrangements(60)

90ndash95




















PCNARFC

ATRP53

DNA damagesignaling

MSH2

MLH1MLH1





regulation

MSH2Triplet repeats

expansion

MMR complex

MSH2

MLH1


MLH1 PMS2PMS2

MSH6

PMS2

MSH3

MSH6

PMS2

MLH3

MSH3

MMR proteins



AIDPol 120578
























































8 Conclusion





9525ex4



ex1c2530 CgtT



013

ex6c665GgtC



No AmsterdamMSI-H

103ex5





423 IVS12-4GgtA



015ex5


ex1c666 GgtA (Lys)


Amsterdam +MSI-H

210ex4


IVS6+16AgtGex13

c2324 TgtC (Phe)



211ex4



416ex11

c1714CgtA(ThrgtLys)



504lowast

ex4c693GgtA (Pro)


Amsterdam +MSI-H



Competing Interests


Acknowledgments


References



































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
































PCNARFC

ATRP53

DNA damagesignaling

MSH2

MLH1MLH1





regulation

MSH2Triplet repeats

expansion

MMR complex

MSH2

MLH1


MLH1 PMS2PMS2

MSH6

PMS2

MSH3

MSH6

PMS2

MLH3

MSH3

MMR proteins



AIDPol 120578
























































8 Conclusion





9525ex4



ex1c2530 CgtT



013

ex6c665GgtC



No AmsterdamMSI-H

103ex5





423 IVS12-4GgtA



015ex5


ex1c666 GgtA (Lys)


Amsterdam +MSI-H

210ex4


IVS6+16AgtGex13

c2324 TgtC (Phe)



211ex4



416ex11

c1714CgtA(ThrgtLys)



504lowast

ex4c693GgtA (Pro)


Amsterdam +MSI-H



Competing Interests


Acknowledgments


References



































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





























































8 Conclusion





9525ex4



ex1c2530 CgtT



013

ex6c665GgtC



No AmsterdamMSI-H

103ex5





423 IVS12-4GgtA



015ex5


ex1c666 GgtA (Lys)


Amsterdam +MSI-H

210ex4


IVS6+16AgtGex13

c2324 TgtC (Phe)



211ex4



416ex11

c1714CgtA(ThrgtLys)



504lowast

ex4c693GgtA (Pro)


Amsterdam +MSI-H



Competing Interests


Acknowledgments


References



































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















9525ex4



ex1c2530 CgtT



013

ex6c665GgtC



No AmsterdamMSI-H

103ex5





423 IVS12-4GgtA



015ex5


ex1c666 GgtA (Lys)


Amsterdam +MSI-H

210ex4


IVS6+16AgtGex13

c2324 TgtC (Phe)



211ex4



416ex11

c1714CgtA(ThrgtLys)



504lowast

ex4c693GgtA (Pro)


Amsterdam +MSI-H



Competing Interests


Acknowledgments


References



































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of















































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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