mlh1 and msh2 - hindawi publishing corporation · 2019. 7. 30. · research article novel mutations...

Research ArticleNovel Mutations in MLH1 and MSH2 Genes inMexican Patients with Lynch Syndrome

Jose Miguel Moreno-Ortiz12 Mariacutea de la Luz Ayala-Madrigal1

Jorge Romaacuten Corona-Rivera13 Manuel Centeno-Flores3

Viacutector Maciel-Gutieacuterrez3 Ramoacuten Antonio Franco-Topete3

Juan Armendaacuteriz-Borunda4 Erin Hotchkiss5 Lucia Peacuterez-Carbonell5 Jennifer Rhees5

Clement Richard Boland5 and Melva Gutieacuterrez-Angulo16

1 Instituto de Genetica Humana CUCS Universidad de Guadalajara Sierra Mojada 950 Colonia Independencia44340 Guadalajara JAL Mexico2Trayectoria de Genomica Alimentaria Universidad de la Cienega del Estado de Michoacan de OcampoAvenida Universidad 3000 Colonia Lomas de la Universidad 59103 Sahuayo MICH Mexico3Hospital Civil de Guadalajara ldquoDr Juan I Menchacardquo Salvador de Quevedo y Zubieta 750 44340 Guadalajara JAL Mexico4Departamento de Biologıa Molecular y Genomica CUCS Universidad de Guadalajara Sierra Mojada 950Colonia Independencia 44340 Guadalajara JAL Mexico5GI Cancer Research Laboratory Baylor University Medical Center 3500 Gaston Avenue Suite 250 HoblitzelleDallas TX 75204 USA6Departamento de Clınicas CUAltos Universidad de Guadalajara Carretera a Yahualica Km 7547600 Tepatitlan de Morelos JAL Mexico

Correspondence should be addressed to Melva Gutierrez-Angulo melva73hotmailcom

Received 8 February 2016 Accepted 14 April 2016

Academic Editor Hubert E Blum

Copyright copy 2016 Jose Miguel Moreno-Ortiz et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Background Lynch Syndrome (LS) is characterized by germline mutations in the DNA mismatch repair (MMR) genes MLH1MSH2 MSH6 and PMS2 This syndrome is inherited in an autosomal dominant pattern and is characterized by early onsetcolorectal cancer (CRC) and extracolonic tumors The aim of this study was to identify mutations in MMR genes in threeMexican patients with LS Methods Immunohistochemical analysis was performed as a prescreening method to identify absentprotein expression PCR Denaturing High Performance Liquid Chromatography (dHPLC) and Sanger sequencing complementedthe analysis Results Two samples showed the absence of nuclear staining for MLH1 and one sample showed loss of nuclearstaining for MSH2 The mutations found in MLH1 gene were c2103+1GgtC in intron 18 and compound heterozygous mutantsc1852 1854delAAG (pK618del) and c1852 1853delinsGC (pK618A) in exon 16 In the MSH2 gene we identified mutationc638dupT (pL213fs) in exon 3 Conclusions This is the first report of mutations in MMR genes in Mexican patients with LS andthese appear to be novel

1 Introduction

Lynch Syndrome (LS) previously called hereditary non-polyposis colorectal cancer (HNPCC) was described in theearly twentieth century by A S Warthin and was furthercharacterized in the second half of the twentieth century

by Lynch [1] This syndrome is inherited as an autosomaldominant pattern and is characterized by early onset CRCand other specific extracolonic tumors [2] Most patientswith LS have heterozygous mutations in MLH1 MSH2MSH6 or PMS2 the principal genes in the DNA mismatchrepair (MMR) system [3 4] The proteins encoded by MMR

Hindawi Publishing CorporationGastroenterology Research and PracticeVolume 2016 Article ID 5278024 6 pageshttpdxdoiorg10115520165278024

2 Gastroenterology Research and Practice

genes are involved in the identification and repair of errorsoccurring during S phase and repetitive sequences calledmicrosatellites are particularly vulnerable to mutation inthe absence of DNA MMR activity [4ndash6] The diagnosisof LS is suspected clinically using the Revised BethesdaGuidelines and a definitive diagnosis requires the iden-tification of mutations in one of the LS associated genes[7] A large number of unique mutations are found in LSfamilies and founder mutations are commonly found inrelatively isolated populations [8] The goal of this study wasto identifymutations inMMR genes inMexican patients withLS

2 Material and Methods

21 Patients The study utilized peripheral blood and tumorsamples obtained from three unrelated patients diagnosedclinically with LS at the ldquoDr Juan I Menchacardquo Civil Hospitalof Guadalajara Jalisco Mexico (LS-23 LS-41 and LS-52)The genealogies including age and cancers type associated areshown in Figure 1 Two additional family members affectedwith CRC were analyzed in association with the probandLS-23 (II-2 and II-5) All the patients provided informedconsent The study was performed in accordance with theHelsinki Declaration and considered the ethical aspects ofresearch involving human subjects according to the MexicanGeneral Health LawThis research was approved by the ethicscommittee of Centro Universitario de los Altos Universidadde Guadalajara (CUACINV4942009)

22 Immunohistochemistry Protein expression of MLH1MSH2 MSH6 and PMS2 was analyzed by immunohisto-chemistry (IHC) staining with the DAKO EnVision System-HRP polymer system kit (DakoCytomation Inc CarpinteriaCA) The following antibodies were used for MLH1 clone13271A BD Pharmingen San Diego CA for MSH2 cloneFE11 Oncogene Research Products Boston MA for MSH6clone 44 BD Transduction Laboratories Lexington KYand for PMS2 clone A37 BD Pharmingen One block offormalin-fixed paraffin-embedded tumor tissue was selectedper case IHC was performed as previously reported [9]Briefly 5 120583m thick tissues were cut dewaxed in xylene andrehydrated in graded alcohol concentrations to buffer Theslides were incubated for one hour with the appropriatedilutions of mouse monoclonal antibodies The peroxidasereaction was developed using diaminobenzidine tetrachlo-ride as the chromogen Normal expression of protein wasindicated by the presence of nuclear staining in colon cellsand loss of staining in tumor cells was determined only ifnonneoplastic colonocytes and stromal cells were positivelystained

23 DNA Extraction Genomic DNA was extracted fromperipheral blood samples of patients as previously reported[10] The DNA concentration was measured by spectropho-tometry and the quality was evaluated in 1 agarose gelelectrophoresis

24 dHPLC and DNA Sequencing Specific primers wereused to amplify all 19 exons of MLH1 gene and 16 exons ofMSH2 gene by polymerase chain reaction (PCR) accordingto conditions established by the laboratory The fragmentswere then visualized by gel electrophoresis in a 2 agarosegel stained with ethidium bromide

Denaturing High Performance Liquid Chromatography(dHPLC) was undertaken using the Hitachi WAVE DNAfragment analysis system HSX-3500 (Transgenomic) Analiquot (5 120583L) of the PCR product was directly injected intoa DNA Sep column The samples were analyzed under theoptimum melting temperature determined in the GI CancerResearch Laboratory at Baylor University Medical CenterDallasThe chromatograms of each fragment were comparedwith those of the wild type and fragments containingheteroduplexes and shorter retention times compared to wildtype fragments underwent Sanger sequencing to confirmputative sequence variations

PCR products were purified using the QuickStep 2PCR Purification Kit (EdgeBio Gaithersburg MD) andPERFORMA DTR Gel Filtration Cartridges Kit (EdgeBio)The successive purified products were subjected to cyclesequencing in the forward and reverse directions usinga BigDye Terminator v11 Kit (Applied Biosystems LifeTechnologies) on the automated PRISM 3100-Avant DNASequencer (Applied Biosystems Life Technologies) Thesequences for each fragment were analyzed in the genomebrowser httpgenomeucscedu

25 PCR-RFLP PCR-RFLP was done for a variant localizedin 18 exon-intron boundary of MLH1 using standard PCRconditions in samples of family members of proband LS-23 (II-2 and II-5 Figure 1) The primers were as followsforward 51015840-TTT TGA GGT ATT GAA TTT CTT TGG-31015840 and reverse 51015840-TGA GGT CCT GTC CTA GTC CTG-31015840 A PCR fragment of 191 bp was used for digestion withthe AluI restriction enzyme This cuts the mutant allele andthe fragments generated for heterozygous individuals were191 bp 144 bp and 47 bp

3 Results

MLH1 andMSH2protein expressionwere altered in the tissuesamples evaluated LS-23 and LS-41 showed the absence ofnuclear staining for MLH1 and sample LS-52 showed loss ofnuclear staining for MSH2

All the exons of MLH1 and MSH2 genes were success-fully amplified and screened by dHPLC Samples showingheteroduplex peaks were subjected to DNA sequencing tocharacterize the germlinemutations Patient LS-23 had a sub-stitution at a splice donor site located in the first nucleotidebase of intron 18 ofMLH1 c2103+1GgtC Once we identifiedthe mutation in this patient two additional members withCRC in this family (II-2 and II-5 Figure 1) were screenedby PCR-RFLP as the mutation created a novel restrictionsite (data not shown) The results showed a heterozygousgenotype for the mutation in the analyzed individuals Inpatient LS-41 two different sequence variations were found

Gastroenterology Research and Practice 3

4

3

I

II

III

IV

Colon cancer Colon cancer

Leukemialowastc2103+1GgtC MLH1 gene

45 ylowast

30 ylowast

55 y

10 y 30 y 30 y33 y

35 y 57 ylowast 50 y

(a)

Colon cancer

Leukemia

4

I

II

III

Cervicouterine cancer

Cancer unknown site

52 y 55 y

47 y48 y 7 y45 ylowast

lowastc1852_1854delAAGc1852_1853delinsGC MLH1 gene

(b)

2 2

3

3 2

I

II

III

IV

Isolated cleft palate


38 ylowast

55 y 66 y

33 y

46 y

lowastc638dupT MSH2 gene

(c)

Figure 1 Genealogies from three LS families (a) Family LS-23 (b) Family LS-41 and (c) Family LS-52

in exon 16 of MLH1 One allele had an in-frame deletion ofa codon c1852 1854delAAG (pK618del) and the other dis-played a deletioninsertion of two bases c1852 1853delinsGC(pK618A) which resulted in a lysine to alanine mutationat codon 618 The third sequence variation was duplicationc638dupT in codon 213 in exon 3 of the MSH2 genein patient LS-52 This resulted in the frameshift mutationpL213fs (Figure 2) These data were presented at AmericanSociety of Human Genetics Annual Meeting in 2014 [11]

4 Discussion

The Revised Bethesda Guidelines recommend MSI analysisandor IHC analysis of tumor tissue to select patients fordefinitive DNA sequence analysis [12] Our study identified

three putative LS patients from Mexico initially suspectedbased upon family history and screened by IHC and con-firmed with DNA analyses In the initial screening by IHCwe identified two patients with absent expression of MLH1and one with absent expression ofMSH2

Patient LS-23 had a substitution at a splice donorsite located in the first nucleotide base of intron 18 ofMLH1 c2103+1GgtC This change is located in a canonicalGTAG splice site The phyloP and phastCons values were4281 and 1 respectively suggesting strong evolutionaryconservation [13] This mutation is included in the SNPdatabase as rs267607888 whereas the changes GgtA andGgtT (c2103+1GgtA and c2103+1GgtT) have been reportedin patients with LS in the PubMed database [14ndash20] Vari-ations in this position are classified as likely pathogenic or


T T T T T TC C C C C C CA A A A A A AG G G G G G G G G

105 113 121 129S

(a)

155148141134127T T T T T T TC C CA A A AAG G G G G G G G GN N N N NN N

(b)

337 345 353 361T T TCA A A A A AG G G G N N N N N N N N N N N N NG G

(c)

Figure 2 DNA sequencing results in three LS patients (a) PatientLS-23 with c2103+1GgtC in the MLH1 gene (b) Patient LS-41 withtwo different mutations in the MLH1 gene c1852 1854delAAG(pK618del) and c1852 1853delinsGC (pK618A) (c) Patient LS-52with mutation c638dupT in the MSH2 gene The arrows indicatethe location of the mutations

pathogenic by the InSiGHT Variant Interpretation Commit-tee respectively [21] Mutation GgtC found in this patient(c2103+1GgtC) of MLH1 has apparently been reported onlyin the InSiGHT database [21] The mutational effect onthe MLH1 protein is unclear since mutations in splice sitesequences could promote exon skipping intron retention oractivation of cryptic splice sites [22]

Patient LS-41 had a c1852 1853delinsGC (pK618A)(rs35502531 c1852 1853delAAinsGC) variant in one alleleand a c1852 1854delAAG (pK618del) (rs63751247) sequencevariation in the other allele of MLH1 The K618A variantis classified as nonpathogenic in the InSiGHT database andis not located in the interaction domain with the PMS2protein described between amino acids 531ndash549 and 740ndash756 in MLH1 [23] Thompson et al used multifactoriallikelihood analysis to conclude that c1852 1853delinsGC isnot pathogenic [24] Moreover in a recent study publishedby Abulı et al this variant was found in 14 of 8055 CRCcases and 15 of 10668 controls from seven cohorts Theyconcluded that c1852 1853delinsGC inMLH1 plays no role inthe genetic predisposition to LS [25] In a multifactorial like-lihood analysis byThompson et al in 2013 [24] a pathogeniceffect was found for pK618del which was consistent with aprevious report by Takahashi et al in 2007 [26] Takahashiet al performed functional analyses of different variantsof the MLH1 gene including pK618A and pK618del Theyreported that pK618A has an in vitroMMR activity of 827while that for pK618del was 389 Additionally reducedMLH1 expression (lt25) was observed in association with

the pK618del variant [26] Since most of reports indicatethat c1852 1854delAAG (pK618del) has a pathogenic effectthis should be considered the sequence variant responsiblefor LS in Family LS-41 In a segregation analysis reported byCastillejo et al they concluded that the pK618A polymor-phism should be considered a neutral variant [27] althoughthey do not exclude the variant as a possible susceptibilityfactor in CRC development Moreover the expression assaysdemonstrated that pK618A compromises protein stabilityand decreases the half-life of MLH1 protein by a factor of25ndash35 [28] Perhaps the compound heterozygous variants(c1852 1853delinsGC and c1852 1854delAAG) in MLH1 inpatient LS-41 synergize to predispose to the CRC

Patient LS-52 had a duplication of a single nucleotidec638dupT which resulted in the frameshift mutationpL213fs in the MSH2 gene An in silico analysis of thisduplication using the CCDS1834 sequence (MSH2 gene)and the molecular toolkit program from Colorado StateUniversity (httpwwwvivocolostateedu) [29] indicatedthat the duplication of T generates a protein of 230 aminoacids instead of the wild type protein of 934 amino acids dueto the premature stop in codon 231 Mangold et al analyzed1721 German patients and detected a frameshift mutation inthree patients in this specific region however the alterationin the DNA coding sequence was different c638 639delTG[30] Although frameshift mutations in codon 213 ofexon four of MSH2 gene have been described previously[30 31] the c638dupT alteration has not been reportedin any LS database including the International Society forGastrointestinal Hereditary Tumors Mutation Database(httpinsight-grouporg) [21] the Human Gene MutationDatabase (httpwwwhgmdcfacukacindexphp) [32] ortheMMRGenes Variant Database (httpwwwmedmuncavariants) [33] Therefore this is likely a novelmutation

LS is the most common form of inherited CRC andaccounts for about 3 of all CRC cases [34] In MexicoCRC has a lifetime incidence of 58 according to Globocan2012 [35] The search for MMR genes mutations in CRCpatients is essential for making the diagnosis in LS families[1] The individuals positive for these mutations will havea surveillance according to international guidelines [36] inorder to avoid the cancer development or progression

5 Conclusions

This study reports the c638dupT in MSH2 and thecompound heterozygous alteration c1852 1853delinsGCc1852 1854delAAG in MLH1 as novel findings in LS More-over these findings highlight the importance of identifyingnovel genetic alterations populations that have not beenpreviously studied

Competing Interests

The authors declare that they have no competing interests


Acknowledgments

The authors thank Nurse Elisa Andrade for her technicalassistance at the ldquoDr Juan I Menchacardquo Civil Hospital Thisworkwas supported by aCOECYTJAL-UDGGrant PS-2009-494 Jose Miguel Moreno-Ortiz received a PhD CONACyTFellowship

References

[1] C R Boland andH T Lynch ldquoThe history of Lynch syndromerdquoFamilial Cancer vol 12 no 2 pp 145ndash157 2013

[2] H F A Vasen ldquoClinical description of the Lynch syndrome[hereditary nonpolyposis colorectal cancer (HNPCC)]rdquo Famil-ial Cancer vol 4 no 3 pp 219ndash225 2005

[3] K Lagerstedt Robinson T Liu J Vandrovcova et al ldquoLynchsyndrome (hereditary nonpolyposis colorectal cancer) diagnos-ticsrdquo Journal of the National Cancer Institute vol 99 no 4 pp291ndash299 2007

[4] J V Martın-Lopez and R Fishel ldquoThe mechanism of mismatchrepair and the functional analysis of mismatch repair defects inLynch syndromerdquo Familial Cancer vol 12 no 2 pp 159ndash1682013

[5] E Vilar and S B Gruber ldquoMicrosatellite instability in colorectalcancerthe stable evidencerdquo Nature Reviews Clinical Oncologyvol 7 no 3 pp 153ndash162 2010

[6] C R Boland and A Goel ldquoMicrosatellite instability in colorec-tal cancerrdquoGastroenterology vol 138 no 6 pp 2073ndash2087 2010

[7] R H Sijmons and R M Hofstra ldquoReview clinical aspects ofhereditaryDNAMismatch repair genemutationsrdquoDNARepairvol 38 pp 155ndash162 2016

[8] H T Lynch A de la Chapelle H Hampel et al ldquoAmericanfounder mutation for Lynch syndrome Prevalence estimatesand implicationsrdquo Cancer vol 106 no 2 pp 448ndash452 2006

[9] A Goel T Nagasaka J Spiegel R Meyer W E Lichliterand C R Boland ldquoLow frequency of lynch syndrome amongyoung patients with non-familial colorectal cancerrdquo ClinicalGastroenterology and Hepatology vol 8 no 11 pp 966ndash971e12010

[10] S Gustincich G Manfioletti G del Sal C Schneider andP Carninci ldquoA fast method for high-quality genomic DNAextraction fromwhole human bloodrdquo BioTechniques vol 11 no3 pp 298ndash300 1991

[11] J M Moreno-Ortiz M L Ayala-Madrigal J R Corona-Riveraet al ldquoGermline mutational analysis in Mexican patients withLynch syndromerdquo in Proceedings of the 64th Annual Meeting ofthe American Society of HumanGenetics SanDiego Calif USAOctober 2014

[12] F M Giardiello J I Allen J E Axilbund et al ldquoGuidelineson genetic evaluation and management of lynch syndromea consensus statement by the us multi-society task force oncolorectal cancerrdquoDiseases of the Colon and Rectum vol 57 no8 pp 1025ndash1048 2014

[13] J M Schwarz C Rodelsperger M Schuelke and D SeelowldquoMutationTaster evaluates disease-causing potential ofsequence alterationsrdquo Nature Methods vol 7 no 8 pp575ndash576 2010

[14] J Wijnen P Meera Khan H Vasen et al ldquoMajority of hMLH1mutations responsible for hereditary nonpolyposis colorectalcancer cluster at the exonic region 15-16rdquo American Journal ofHuman Genetics vol 58 no 2 pp 300ndash307 1996

[15] M J W Berends H Hollema Y Wu et al ldquoMLH1 and MSH2protein expression as a pre-screening marker in hereditary andnon-hereditary endometrial hyperplasia and cancerrdquo Interna-tional Journal of Cancer vol 92 no 3 pp 398ndash403 2001

[16] Y Hendriks P Franken JW Dierssen et al ldquoConventional andtissue microarray immunohistochemical expression analysis ofmismatch repair in hereditary colorectal tumorsrdquoTheAmericanJournal of Pathology vol 162 no 2 pp 469ndash477 2003

[17] AWagner A Barrows J TWijnen et al ldquoMolecular analysis ofhereditary nonpolyposis colorectal cancer in the United Stateshigh mutation detection rate among clinically selected familiesand characterization of an American founder genomic deletionof the MSH2 generdquo The American Journal of Human Geneticsvol 72 no 5 pp 1088ndash1100 2003

[18] E Domingo P Laiho M Ollikainen et al ldquoBRAF screeningas a low-cost effective strategy for simplifying HNPCC genetictestingrdquo Journal of Medical Genetics vol 41 no 9 pp 664ndash6682004

[19] L I H Overbeek C M Kets K M Hebeda et al ldquoPatientswith an unexplained microsatellite instable tumour have a lowrisk of familial cancerrdquo British Journal of Cancer vol 96 no 10pp 1605ndash1612 2007

[20] M Bujalkova K Zavodna T Krivulcik et al ldquoMultiplexSNaPshot genotyping for detecting loss of heterozygosity inthe mismatch-repair genes MLH1 and MSH2 in microsatellite-unstable tumorsrdquo Clinical Chemistry vol 54 no 11 pp 1844ndash1854 2008

[21] International Society for Gastrointestinal Hereditary tumours2016 httpinsight-grouporg

[22] R K Singh and T A Cooper ldquoPre-mRNA splicing in diseaseand therapeuticsrdquo Trends in Molecular Medicine vol 18 no 8pp 472ndash482 2012

[23] J Kosinski I Hinrichsen J M Bujnicki P Friedhoff andG Plotz ldquoIdentification of Lynch syndrome mutations in theMLH1-PMS2 interface that disturb dimerization and mismatchrepairrdquo Human Mutation vol 31 no 8 pp 975ndash982 2010

[24] B A Thompson D E Goldgar C Paterson et al ldquoA multi-factorial likelihood model for MMR gene variant classificationincorporating probabilities based on sequence bioinformaticsand tumor characteristics a report from the colon cancer familyregistryrdquo Human Mutation vol 34 no 1 pp 200ndash209 2013

[25] A Abulı L Bujanda J Munoz et al ldquoThe MLH1c1852 1853delinsGC (pK618A) variant in colorectal cancergenetic association study in 18 723 individualsrdquo Public Libraryof Science One vol 9 no 4 Article ID e95022 2014

[26] M Takahashi H Shimodaira C Andreutti-Zaugg R Iggo RD Kolodner and C Ishioka ldquoFunctional analysis of humanMLH1 variants using yeast and in vitro mismatch repair assaysrdquoCancer Research vol 67 no 10 pp 4595ndash4604 2007

[27] A Castillejo C Guarinos A Martınez-Canto et al ldquoEvidencefor classification of c1852 1853AAgtGC in MLH1 as a neutralvariant for Lynch syndromerdquo BMC Medical Genetics vol 12article 12 2011

[28] S Perera and B Bapat ldquoThe MLH1 variants pArg265Cys andpLys618Ala affect protein stability while pLeu749Gln affectsheterodimer formationrdquoHuman mutation vol 29 no 2 p 3322008

[29] The molecular toolkit program of Colorado State UniversityApril 2015 httpwwwvivocolostateedu

[30] E Mangold C Pagenstecher W Friedl et al ldquoSpectrum andfrequencies of mutations in MSH2 and MLH1 identified in


1721 German families suspected of hereditary nonpolyposiscolorectal cancerrdquo International Journal of Cancer vol 116 no5 pp 692ndash702 2005

[31] N Rahner N Friedrichs M Wehner et al ldquoNine novelpathogenic germline mutations in MLH1 MSH2 MSH6 andPMS2 in families with Lynch syndromerdquo Acta Oncologica vol46 no 6 pp 763ndash769 2007

[32] P D Stenson E V Ball M Mort et al ldquoHuman GeneMutationDatabase (HGMD) 2003 updaterdquo Human Mutation vol 21no 6 pp 577ndash581 2003

[33] M O Woods P Williams A Careen et al ldquoA new variantdatabase for mismatch repair genes associated with Lynchsyndromerdquo Human Mutation vol 28 no 7 pp 669ndash673 2007

[34] H T Lynch and A de la Chapelle ldquoHereditary colorectalcancerrdquo The New England Journal of Medicine vol 348 no 10pp 919ndash932 2003

[35] J Ferlay H Shin F Bray D Forman C Mathers and DParkin GLOBOCAN 2012 v10 Cancer Incidence and MortalityWorldwide IARC Cancer Base No 11 International Agency forResearch on Cancer Lyon France 2013 httpglobocaniarcfr

[36] S Syngal R E Brand J M Church F M Giardiello HL Hampel and R W Burt ldquoACG Clinical guideline genetictesting and management of hereditary gastrointestinal cancersyndromesrdquoThe American Journal of Gastroenterology vol 110no 2 pp 223ndash262 2015

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


genes are involved in the identification and repair of errorsoccurring during S phase and repetitive sequences calledmicrosatellites are particularly vulnerable to mutation inthe absence of DNA MMR activity [4ndash6] The diagnosisof LS is suspected clinically using the Revised BethesdaGuidelines and a definitive diagnosis requires the iden-tification of mutations in one of the LS associated genes[7] A large number of unique mutations are found in LSfamilies and founder mutations are commonly found inrelatively isolated populations [8] The goal of this study wasto identifymutations inMMR genes inMexican patients withLS

2 Material and Methods

21 Patients The study utilized peripheral blood and tumorsamples obtained from three unrelated patients diagnosedclinically with LS at the ldquoDr Juan I Menchacardquo Civil Hospitalof Guadalajara Jalisco Mexico (LS-23 LS-41 and LS-52)The genealogies including age and cancers type associated areshown in Figure 1 Two additional family members affectedwith CRC were analyzed in association with the probandLS-23 (II-2 and II-5) All the patients provided informedconsent The study was performed in accordance with theHelsinki Declaration and considered the ethical aspects ofresearch involving human subjects according to the MexicanGeneral Health LawThis research was approved by the ethicscommittee of Centro Universitario de los Altos Universidadde Guadalajara (CUACINV4942009)

22 Immunohistochemistry Protein expression of MLH1MSH2 MSH6 and PMS2 was analyzed by immunohisto-chemistry (IHC) staining with the DAKO EnVision System-HRP polymer system kit (DakoCytomation Inc CarpinteriaCA) The following antibodies were used for MLH1 clone13271A BD Pharmingen San Diego CA for MSH2 cloneFE11 Oncogene Research Products Boston MA for MSH6clone 44 BD Transduction Laboratories Lexington KYand for PMS2 clone A37 BD Pharmingen One block offormalin-fixed paraffin-embedded tumor tissue was selectedper case IHC was performed as previously reported [9]Briefly 5 120583m thick tissues were cut dewaxed in xylene andrehydrated in graded alcohol concentrations to buffer Theslides were incubated for one hour with the appropriatedilutions of mouse monoclonal antibodies The peroxidasereaction was developed using diaminobenzidine tetrachlo-ride as the chromogen Normal expression of protein wasindicated by the presence of nuclear staining in colon cellsand loss of staining in tumor cells was determined only ifnonneoplastic colonocytes and stromal cells were positivelystained

23 DNA Extraction Genomic DNA was extracted fromperipheral blood samples of patients as previously reported[10] The DNA concentration was measured by spectropho-tometry and the quality was evaluated in 1 agarose gelelectrophoresis

24 dHPLC and DNA Sequencing Specific primers wereused to amplify all 19 exons of MLH1 gene and 16 exons ofMSH2 gene by polymerase chain reaction (PCR) accordingto conditions established by the laboratory The fragmentswere then visualized by gel electrophoresis in a 2 agarosegel stained with ethidium bromide

Denaturing High Performance Liquid Chromatography(dHPLC) was undertaken using the Hitachi WAVE DNAfragment analysis system HSX-3500 (Transgenomic) Analiquot (5 120583L) of the PCR product was directly injected intoa DNA Sep column The samples were analyzed under theoptimum melting temperature determined in the GI CancerResearch Laboratory at Baylor University Medical CenterDallasThe chromatograms of each fragment were comparedwith those of the wild type and fragments containingheteroduplexes and shorter retention times compared to wildtype fragments underwent Sanger sequencing to confirmputative sequence variations

PCR products were purified using the QuickStep 2PCR Purification Kit (EdgeBio Gaithersburg MD) andPERFORMA DTR Gel Filtration Cartridges Kit (EdgeBio)The successive purified products were subjected to cyclesequencing in the forward and reverse directions usinga BigDye Terminator v11 Kit (Applied Biosystems LifeTechnologies) on the automated PRISM 3100-Avant DNASequencer (Applied Biosystems Life Technologies) Thesequences for each fragment were analyzed in the genomebrowser httpgenomeucscedu

25 PCR-RFLP PCR-RFLP was done for a variant localizedin 18 exon-intron boundary of MLH1 using standard PCRconditions in samples of family members of proband LS-23 (II-2 and II-5 Figure 1) The primers were as followsforward 51015840-TTT TGA GGT ATT GAA TTT CTT TGG-31015840 and reverse 51015840-TGA GGT CCT GTC CTA GTC CTG-31015840 A PCR fragment of 191 bp was used for digestion withthe AluI restriction enzyme This cuts the mutant allele andthe fragments generated for heterozygous individuals were191 bp 144 bp and 47 bp

3 Results

MLH1 andMSH2protein expressionwere altered in the tissuesamples evaluated LS-23 and LS-41 showed the absence ofnuclear staining for MLH1 and sample LS-52 showed loss ofnuclear staining for MSH2

All the exons of MLH1 and MSH2 genes were success-fully amplified and screened by dHPLC Samples showingheteroduplex peaks were subjected to DNA sequencing tocharacterize the germlinemutations Patient LS-23 had a sub-stitution at a splice donor site located in the first nucleotidebase of intron 18 ofMLH1 c2103+1GgtC Once we identifiedthe mutation in this patient two additional members withCRC in this family (II-2 and II-5 Figure 1) were screenedby PCR-RFLP as the mutation created a novel restrictionsite (data not shown) The results showed a heterozygousgenotype for the mutation in the analyzed individuals Inpatient LS-41 two different sequence variations were found


4

3

I

II

III

IV



45 ylowast

30 ylowast

55 y

10 y 30 y 30 y33 y


(a)

Colon cancer

Leukemia

4

I

II

III


Cancer unknown site

52 y 55 y



(b)

2 2

3

3 2

I

II

III

IV



38 ylowast

55 y 66 y

33 y

46 y


(c)



4 Discussion






105 113 121 129S

(a)


(b)


(c)







5 Conclusions


Competing Interests



Acknowledgments


References












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















4

3

I

II

III

IV



45 ylowast

30 ylowast

55 y

10 y 30 y 30 y33 y


(a)

Colon cancer

Leukemia

4

I

II

III


Cancer unknown site

52 y 55 y



(b)

2 2

3

3 2

I

II

III

IV



38 ylowast

55 y 66 y

33 y

46 y


(c)



4 Discussion






105 113 121 129S

(a)


(b)


(c)







5 Conclusions


Competing Interests



Acknowledgments


References












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















105 113 121 129S

(a)


(b)


(c)







5 Conclusions


Competing Interests



Acknowledgments


References












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

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Acknowledgments


References












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















mlh1 and msh2 - hindawi publishing corporation · 2019. 7. 30. · research article novel mutations...

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