cancer prevention research - characterization of new ......hereditary nonpolyposis colorectal cancer...

Research ArticleSee perspective on p. 1527

Characterization of New Founder Alu-Mediated Rearrangementsin MSH2 Gene Associated with a Lynch Syndrome Phenotype

Lucia P�erez-Cabornero1, Ester Borr�as Flores3, Mar Infante Sanz1, Eladio Velasco Sampedro1,Alberto Acedo Becares1, Enrique Lastra Aras5, Jorge Cuevas Gonz�alez2,Marta Pineda Riu3, Teresa Ram�on y Cajal Asensio4, Gabriel Capell�a Munar3,Cristina Miner Pino1, and Mercedes Dur�an Domínguez1

AbstractIt has been reported that large genomic deletions in the MLH1 and MSH2 genes are a frequent cause of

Lynch syndrome in certain populations. Here, a cohort has been screened and two new founder

rearrangements have been found in the MSH2 gene. These mutations have been characterized by break

point determination, haplotype analysis, and genotype–phenotype correlation. Mutations have been

identified in the MLH1, MSH2, and MSH6 genes in 303 subjects from 160 suspected Lynch syndrome

unrelated families. All subjects were tested using heteroduplex analysis by capillary array electrophoresis.

Multiplex ligation-dependent probe amplification was used to detect rearrangements in mutation-negative

index patients and confirmed by reverse transcriptase PCR. The break point of the deletions was further

characterized by the array comparative genomic hybridization method. Immunohistochemical staining

and microsatellite instability were studied in tumor samples. Hereditary nonpolyposis colorectal cancer–

related phenotypes were evaluated. More than 16% (24 of 160) of the families had pathogenic mutations

(8MLH1, 15MSH2, and 1MSH6). Twelve of these families (50%) are carriers of a novelmutation. Seven of

the 15 positive MSH2 families (47%) are carriers of a rearrangement. The exon 7 deletion and exon 4 to 8

deletion of MSH2 are new founder mutations. The segregation of a common haplotype, a similar

phenotype, and anticipation effects were observed in these families. These findings will greatly simplify

the diagnosis, counseling, and clinical care in suspected Lynch syndrome families and not just in specific

geographic areas, so wide distribution may be explained by migration patterns. Cancer Prev Res; 4(10);

1546–55. �2011 AACR.

Introduction

Hereditary nonpolyposis colorectal cancer (HNPCC)syndrome, or Lynch syndrome (MIM 120435), is probablythe most common form of inherited colorectal cancer(CRC), accounting for 1% to 5% of cases (1). Affectedindividuals have a family history of CRC at an early age,characterized by tumor predominance in the proximalcolon and an association with extracolonic tumors. Germ-line mutations in at least 5 mismatch repair (MMR) genes

(MLH1, MSH2, MSH6, PMS1, and PMS2) have been iden-tified in families fulfilling international criteria for thesyndrome, namely, Amsterdam criteria I or II (2–4), orless stringent criteria referred to as the Bethesda guidelines(5), which lead to tumors characterized by widespreadmicrosatellite instability (MSI).

Identification of inherited predisposition is importantbecause it enables targeted clinical surveillance, whichsignificantly reduces cancer morbidity and mortality inLynch syndrome families (6).

Nearly 90% of the mutations in databases (LeidenOpen Variation Database, LOVD) affect either MLH1(MIM 120436) or MSH2 (MIM 609309; refs. 7, 8). Thevast majority of mutations are nonsense, missense, splic-ing, or frameshift mutations, but a recent report (9)indicates that a substantial percentage of HNPCCs arecaused by gross genomic rearrangements in MMR genealterations undetectable with traditional methods of mu-tation analysis. They account for up to 15% of all path-ogenic mutations in MSH2 and MLH1 (10). Thefrequency of large rearrangements in MSH2 as comparedwith MLH1 depends on the studied population (11).Different prescreening methods have been proposed.

Authors' Affiliations: 1Cancer Genetics Laboratory, IBGM-CSIC, Univer-sity of Valladolid; 2Anatomical Pathology Department, Hospital Comarcalde Medina del Campo, Crta. Peñaranda de Bracamonte, Medina delCampo, Valladolid; 3Hereditary Cancer Program, ICO-IDIBELL, Hospitaletde Llobregat; 4Oncology Department, Hospital de la Santa Creu i Sant Pau,Barcelona; and 5Oncology Department, Hospital General Yag€ue, Burgos,Spain

Corresponding Author: Mercedes Dur�an Domínguez, Cancer GeneticsLaboratory, IBGM-CSIC, University of Valladolid, C/Sanz y For�es 3, 47003Valladolid, Spain. Phone: 34-983184809; Fax: 34-983184800; E-mail:[email protected]

doi: 10.1158/1940-6207.CAPR-11-0227

�2011 American Association for Cancer Research.

CancerPreventionResearch

Cancer Prev Res; 4(10) October 20111546

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Published OnlineFirst July 21, 2011; DOI: 10.1158/1940-6207.CAPR-11-0227

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Here, a combined method of heteroduplex analysis bycapillary array electrophoresis (HA-CAE) and multiplexligation-dependent probe amplification (MLPA) has beenused to screen our HNPCC population (12). Using thisprotocol, numerous different mutations have beendetected and a high proportion has rearrangements, allin the MSH2 gene.Two founder mutations in the MSH2 gene are presented

and characterized here. A similar break point was found inall index cases of the carrier families. These mutations seemto be associated with founder effects, as a common hap-lotype was associated with each; besides, the novel exon 4to 8 deletion seems to be associated with anticipation.Our findings have important implications in the diag-

nosis and management of such families, and these resultswill help to simplify genetic testing for Lynch syndrome.

Patients and Methods

ParticipantsPatients were recruited through the Regional Hereditary

Cancer Prevention Program of Castilla y Le�on (Spain).Informed consent was obtained from 303 subjects belong-ing to 160 unrelated suspected Lynch syndrome families.The control population used was from the National DNABank, a collection of representative DNA samples of theSpanish population. Available DNA, RNA, and tumorblocks were obtained for at least one affected person ineach family.

Mutation analysisGeneral screening of point mutation of the MLH1,

MSH2, and MSH6 genes was carried out using HA-CAE.This method was developed in our laboratory (13), and thevalidation for MMR genes has recently been published(12).Fragments showing an HA-CAE–altered pattern were

sequenced with the BigDye Terminator Sequencing Kitv3.1 (Applied Biosystems) with unlabeled forward andreverse primers on the ABI 3100 DNA Sequencer (4capillaries; Applied Biosystems).

Genomic rearrangement screeningNegative samples by HA-CAE were tested using MLPA.

Genomic rearrangements were evaluated byMLPA (MRC;).MLPA test kits P003 forMLH1/MSH2 and P008 forMSH6/PMS2 were used according to the supplied protocol. Frag-ment analysis of the PCR products was carried out on theABI 3130 Genetic Analyzer and gene dosage calculationand analysis were done using GeneMapper software(Applied Biosystems).

Reverse transcriptase PCRAll the alterations detected by MLPA were confirmed by

reverse transcriptase PCR (RT-PCR). Three microliters oftotal RNA was used to synthesize cDNA with a High Ca-pacity cDNAReverse Transcription kit (AppliedBiosystems)using randomprimers. RT-PCRwas carried outwith specific

primers designed for the coding sequences flanking theputative mutation. To confirm deletions, short ampliconsfrom RT-PCR were sequenced with the following primers:

Exon 4 to 8 deletion: forward in exon 3, 50-gttggagttgg-gtatgtggatt-30; reverse in exon 9, 50-tgttgactgcatcttcttttcc-30

Exon 7 deletion: forward in exon 6, 50-tgctgaataagtgtaa-aacccc-30; reverse in exon 8, 50-ggagaagtcagaacgaagatcag-30.

Immunohistochemical and tumor MSI testingImmunohistochemical (IHC) staining forMLH1,MSH2,

and MSH6 genes in tissue from at least one individual ofthe genomic rearrangement families was analyzed by apathologist in the General Yag€ue Hospital, Burgos, Spain.

Paraffin-embedded tumors from these cases were re-ceived in our laboratory for MSI study. Paired colon tumortissues and normal tissues were microdissected and DNAwas extracted using the DNAeasy Tissue Kit (Qiagen). Afluorescence multiplex PCR to amplify BAT25, BAT26,D2S123, D5S346, and D17S250 was carried out as de-scribed before (14, 15) with slight modifications. Fluores-cently labeled PCR products were detected using the ABI3130 Genetic Analyzer and the GeneScan software. Thetumor was categorized asmicrosatellite stable if none of the5 markers showed instability, as MSI-low if one of the 5markers showed instability, and as MSI-high if 2 or moremarkers showed instability (14).

Break point characterizationAn array comparative genomic hybridization (aCGH)

strategy was used to confirm the deletions identified byMLPA and to identify the location of deletion break points.

One positive sample, from each different family with arearrangement, was tested by a human aCGH 44K speciallydesigned by Nimgenetics for coverage of chromosome 2:47419322-47580004 (NCBI 36).

On the basis of the information obtained from aCGH,primers were designed spanning the putative break pointsfor each case and used in long-range PCR.

Haplotype analysisIndex cases and their relatives were genotyped with 8

microsatellite polymorphic markers surrounding theMSH2 gene and 2 single-nucleotide polymorphisms inthe MSH2 gene. As controls, 96 unrelated DNA samplesfrom the general population of Castilla y Leon (NationalDNA Bank) were also genotyped, and allele frequencieswere estimated.

The 10 markers used were localized in an approximately3.6-Mb region encompassing MSH2 (locus order: cen-D2S123-D2S1248-D2S1247-Clen30-rs3771278-rs2162123-D2S2227-D2S391-Clen27-D2S119-tel) on chromosome 2:47419322-47580004. Fluorescently labeled primers wereused to amplify the microsatellite polymorphic regions.PCR products were analyzed on the Genetic Analyzer3130 using GenMapper 3.7 software (Applied Biosystems).

The 2 intragenic single base substitutions located withinintron 1 and intron 9 ofMSH2 (rs2162123 and rs3771278)

New Founder Rearrangements in MSH2

www.aacrjournals.org Cancer Prev Res; 4(10) October 2011 1547




were screened through high-resolution melting technology(LightCycler 480 Instrument), and samples with alteredcurves were sequenced.

Phenotypic characterizationGenealogic data and phenotype characteristics (gender,

age at onset, cancer history, and characteristic feature oftumors) were evaluated in all rearrangement carrier families.

Statistical methodComparisons between MMR mutation types (point

mutations vs. deletions) in the MSH2 gene were assessedusing the Web resource GraphPad Software.

The variables related to the proband family membersincluded type and number of CRC, endometrial cancer,and/or other Lynch syndrome–related cancers and thecorresponding ages of diagnosis.

The number and type of cancers were treated as acategorical variable. Categorical data were reported as ab-solute values (n) and relative frequencies (%), whereasgroups were compared by analyzing a 2 � 2 contingencytable using Fisher’s exact test. Age was treated as a contin-uous variable and thereafter dichotomized to less than 50or 50 years or more. Continuous data were reported inmean values with their corresponding SD, and groups werecompared using Student’s t test. A 2-sided P < 0.05 wasconsidered statistically significant.

Results

Molecular mutation identificationA total of 24 families with a pathogenic germline mu-

tation were detected in MSH2, MLH1, and MSH6 bycombined HA-CAE–MLPA analysis. Clinicopathologicfeatures, molecular findings of the index patients, andsample numbers are listed in Table 1. Twelve of thesefamilies (50%) are carriers of a novel mutation. Seven ofthem (29.2%) have a rearrangement, all inMSH2. Two newrearrangements encompassing exon 7 and exon 4 to 8deletions were detected in 3 and 4 nonrelated families,respectively.

Identification of the MSH2 recurrent mutationsOne family had previously been detected with exon 7

deletion and 3 families with exon 4 to 8 deletion. (TheMLPA assay, RT-PCR products, and sequencing pattern areshown in ref. 12). New cases of this detection are presentedhere, and the recurrence of these mutations and the found-er effect has been investigated. In total, 4 families (VA17,VA20, VA32, and VA134) were studied for exon 4 to 8deletion and 3 families (VA4, VA169, and VA247) for exon7 deletion.

Table 1 shows that 58 patients (19% of our testedpopulation) were analyzed for a rearrangement, 60% ofwhom are carriers.

A high frequency of MSH2 exon 4 to 8 deletion wasobserved in MSH2 mutation carrier families, whichaccounted for more than 26% (4 of 15).

In total, 33 probands were analyzed, 22 of which arecarriers and 11 are not (double the number of carriers).

IHC and MIS were analyzed in 2 families and bothpresented no staining of the MSH2 protein and MSI-H(see Table 1).

Break point identificationA customized 4 � 44 Agilent platform was used to map

somatic rearrangements (designed by NimGenetics). TheaCGH assay provides a prediction of rearrangementbreak points for the convenient design of primers andsequencing. Eight hundred twenty-two oligonucleotideprobes were used to cover MSH2. A median distancebetween nonoverlapping array probes of 500 bp wasobtained.

The break points predicted from aCGH are shown inFigure 1. For exon 7, aCGH predicts a 0.01 Mb deletion atthe position 47507393-47515906 and presents 71 probeslost (Fig. 1A); for the rearrangement of encoding exons 4 to8, a 0.04 Mb deletion is predicted between positions47492237-47527926, with 284 probes lost (Fig. 1B).

PCR primers were designed in the first probe before andafter the deletion. To confirm common deletions, PCR wascarried out for several index cases of each family with exon7 deletion and exon 4 to 8 deletion. These resulted in adeletion product of 36.7 kb in exon 4 to 8 deletion(g.13272_49953del36681; NG_007110.1.gb) and 9.4 kbin exon 7 deletion (g.28106_37472del9366; NG_0071-10.1.gb), exclusively observed in carriers of the commondeletion and not in deletion-negative controls (see Fig. 1Aand B).

The same mutation was found in exon 7 deletion carrierfamilies and in exon 4 to 8 deletion carrier families. Theidentical break points are positioned within 2 Alu ele-ments (Fig. 1A and B). Thus, the deletion is likely to havearisen through an Alu-mediated recombination. The pres-ence of identical break point sequences in all cases (IDproband in Table 1) is suggestive of a founder mutationbecause a frequently recurring recombination eventwould likely result in at least a few single-nucleotidedifferences.

Haplotype analysisHaplotype analysis was carried out to confirm the com-

mon genetic origin of the deletion rearrangements (Fig. 2).Most of the positive families for the deletion are from asmall area in Castilla y Le�on (in central of Spain), ancestorsof families carrying the exon 4 to 8 deletion came fromValle de las Navas and exon 7 deletion ancestors came fromLerma, both in the province of Burgos.

A shared haplotype was observed cosegregating with themutation (Fig. 2), which was absent in noncarriers of thesefamilies. It provides evidence for a common ancestryamong these families.

Figures 2A and B represent the pedigrees of the 4 Del4_8families and the 3 Del_7 families. The phenotype observedin these families is shown, as are the results of the commonhaplotype segregation.

P�erez-Cabornero et al.

Cancer Prev Res; 4(10) October 2011 Cancer Prevention Research1548




Tab

le1.

Patho

genicmutations

,clinicop

atho

logicfeatures

,an

dmolec

ular

findings

ofthe24

carrierfamilies

Gen

eFa

mily

code

ID proban

d

Gen

der

(male,

female)

Can

cersite

and

ageat

diagno

sis

Mutation

des

igna

tion

Exo

n/intron

Mutation

type

Mutation

status

Loss

ofprotein

express

ion

MSI

status

No.of

carrier

No.of

nonc

arrier

MLH

1VA27

948

3M

Colon

aden

oma,

46c.23

del16

;STO

P11

E-1

Fram

eshift

New

MLH

1/PMS2-

MSI-H

12

VA29

651

8M

Colon

carcinom

a,36

c.77

delA;STO

P35

E-1

Fram

eshift

New

1VA17

030

0M

Colon

carcinom

a,45

c.11

9delT;

p.L40

fsX

E-2

Fram

eshift

MLH

1_00

899

53

VA44

77F

Rec

tum,27

c.30

6þ5G

>AI-3

Splicing

MLH

1_00

175

MLH

1-MSI-H

59

VA67

102

MColon

carcinom

a,59

c.30

6þ5G

>AI-3

Splicing

MLH

1_00

175

34

VA17

530

9M

Colon

carcinom

a,37

c.30

6þ5G

>AI-3

Splicing

MLH

1_00

175

33

VA27

542

9M

Colon

carcinom

a,37

–42

c.18

65T>

A;Le

u622

His

E-16

Misse

nse

pathog

enic

MLH

1_00

643

MLH

1-MSI-H

1

VA2

2M

Tran

sverse

colon,

31c.22

21-222

4de

lCTG

C;in

s30

E-19

Fram

eshift

MLH

1_01

285a

43

MSH2

VA11

719

4F

Uterin

e-co

lon,

47–53

c.22

9_23

0delAG;

p.Ser77

Cys

fsX4

E-2

Fram

eshift

MSH2_

0010

05

3

VA22

438

MColon

-rec

tum,43

–63

c.22

9_23

0delAG;

p.Ser77

Cys

fsX4

E-2

Fram

eshift

MSH2_

0010

0MSH2/MSH6-

MSI-H

76

VA17

86F

End

ometriu

m,60

c.64

6-10

19_1

386þ

2420

del

p.Ile21

6_Gln46

2del

E-4-8

Exo

n4–

8de

letio

nNew

MSH2-

MSI-H

66

VA20

29M

Colon

carcinom

a,31

c.64

6-10

19_1

386þ

2420

del

p.Ile21

6_Gln46

2del

E-4-8

Exo

n4–

8de

letio

nNew

82

VA32

52F

End

ometriu

m,51

c.64

6-10

19_1

386þ

2420

del

p.Ile21

6_Gln46

2del

E-4-8

Exo

n4–

8de

letio

nNew

MSH2/MSH6-

MSI-H

53

VA13

424

6F

End

ometriu

m,42

c.64

6-10

19_1

386þ

2420

del

p.Ile21

6_Gln46

2del

E-4-8

Exo

n4–

8de

letio

nNew

3

VA4

4F

Colon

carcinom

a,39

c.10

77-351

3_12

76þ5

655

p.R35

9RfsX16

E-7

Exo

n7

deletio

nNew

86

VA16

929

9M

Colon

carcinom

a,29

–37

c.10

77-351

3_12

76þ5

655

p.R35

9RfsX16

E-7

Exo

n7

deletio

nNew

MSH2/MSH6-

MSI-H

12

VA24

741

3M

Colon

/prostate/urinary,

47–57

–60

c.10

77-351

3_12

76þ5

655

p.R35

9RfsX16

E-7

Exo

n7

deletio

nNew

44

VA18

832

6F

Colon

carcinom

a,68

c.12

26_1

227d

elAG

p.Gln40

9ArgfsX7

E-7

MSH2_

0032

3MSH2/MSH6-

MSI-H

42

VA25

143

2F

Colon

/end

ometriu

m,

23–35

c.12

16C>T

;p.Arg40

6XE-7

Non

sens

eMSH2_

0031

21

VA6

75M

Colon

-rec

tum,

rectum

-sigma,

47C.166

1G>A

;p.Ser55

4Thr

E-10

Splicing

MSH2_

0099

9aMSH2/MSH6-

MSI-H

1826

(Con

tinue

don

thefollo

wingpag

e)






Ninety-six unrelated samples were also genotyped ascontrols and allele frequencies were estimated; these arealso shown in Figure 2A and B.

Screening of founder rearrangement deletion inMSH2

A PCR test was designed to screen these deletions in first-degree relatives. A routine PCR procedure was optimized.Three primer sequences were used: 1 forward and 2 reverse.The product is a multiplex PCR with the presence of 1 bandin the wild type and 2 bands in deletion carrier samples(Fig. 3). This procedure is faster, cheaper, and easier thanMLPA.

Genotype–phenotype correlationTaking into account the fact that 62.5% of mutations we

have found are in MSH2, largely due to the existence of 2recurrent mutations in this gene that represent approxi-mately half the mutations inMSH2; we have correlated thetype of mutation (punctual or rearrangement) with theoccurrence of extracolonic tumors and the age at diagnosis(Table 2).

The results of Table 2 show no differences between the 2groups in the number of tumors developed (P¼ 1.0000) orthe age at diagnosis, using the threshold of 50 years (P ¼1.0000), unlike the tumor type where the prevalence ofendometrial and urinary system tumors is higher in found-er mutation carriers than in point mutation carriers (P ¼0.47279 and 0.1247).

Ontheotherhand, itmustbeemphasized that themedianage at onset is different in both groups; 46.7 versus 40.67years in CRC males and 50 versus 33 years in CRC females.These data, however, were not statistically significant.

Discussion

Lynch syndrome is a heterogeneous disorder with re-spect to its molecular basis as well as its phenotypicexpression.

A variety of point mutations, as well as large genomicrearrangements, have been reported in LOVD (ref. 16).

Here, 160 families were screened using a combinedmethod previously published by our group (12). Theoverall mutation detection rate in our study is 15% (24of 160 positive families).

We have found 8 MLH1 mutation carrier families (Ta-ble 1), 3 of which have the c.306þ5G>A mutation, whichhas been described as a founder mutation in Spain, and ourgroup has participated in the work (17). Two truncatedmutations in exon 1 atMLH1 are novel, both appearing in aproband with colon cancer before 50 years of age, whereasthe MLH1_01285 mutation has being described by usbefore (18).

Only one family is an MSH6 mutation carrier, this is anovel truncated variant. The carrier shows a late-onsetendometrial cancer with the absence of any staining ofthe MSH6 protein and MSS tumor. Similar results havebeen reported in several studies (19).

Tab

le1.

Patho

genicmutations

,clinicop

atho

logicfeatures

,an

dmolec

ular

findings

ofthe24

carrierfamilies

(Con

t'd)

Gen

eFa

mily

code

ID proban

d

Gen

der

(male,

female)

Can

cersite

and

ageat

diagno

sis

Mutation

des

igna

tion

Exo

n/intron

Mutation

type

Mutation

status

Loss

ofprotein

express

ion

MSI

status

No.of

carrier

No.of

nonc

arrier

VA17

430

5M

Colon

carcinom

a,41

c.22

40_2

241d

elTA

p.Ile7

47ArgfsX2

E-14

Fram

eshift

MSH2_

0113

7MSH2/MSH6-

MSI-H

2

VA19

936

8M

Urin

arytrac

t/co

lon

carcinom

a,50

–52

c.24

70C>T

;p.G

ln82

4XE-15

Non

sens

eMSH2_

0116

3MSH2/MSH6-

MSI-H

32

VA19

143

0F

End

ometriu

m,40

c.26

34G>A

skipping

exon

15E-15

Splicing

MSH2_

0082

32

MSH6

VA14

226

5F

End

ometriu

m,Breas

t,59

–61

c.69

9delT;

STO

P24

5E-4

Fram

eshift

New

MSH6-

41

NOTE

:In

gray

,mutations

notdes

cribed

before.

aMutations

des

cribed

byus

inLO

VD.






The higher rate of mutations in this study appears in theMSH2 gene (nearly double that inMLH1); this is probablybecause of the high number of recurrent mutations in thisgene.Two mutations that affect the splicing are reported here

and which we have described in LOVD. The c.1661G>A

mutation is present in family VA6, which is a largefamily; we have studied 44 members, of which 18 arecarriers (Table 1). This alteration is associated with theabsence of MSH2/MSH6 staining, MSI-H tumor, and aphenotype with colon rectum cancer before the age of 50.The mutation c.2634G>A in exon 15 of MSH2 caused

Figure 1. A, exon 7 deletionrearrangement. a, the break pointspredicted from aCGH. b,rearrangement characterization:PCR amplification, sequencing,and break point sequence.c, break point location andidentification of Alu elements.B, exon 4 to 8 deletionrearrangement. a, the break pointspredicted from aCGH. b,rearrangement characterization:PCR amplification, sequencing,and break point sequence.c, break point location andidentification of Alu elements.

Alu-Y

Intron 6

Deletion Ex7

Alu-Y

Alu-Sg

Alu-Y Alu-Y

Alu-Sg

Alu-Sg

Alu-SgAlu-Sq Alu-SxAlu-SpAlu-Jo

Alu-Sg Alu-Sx

In.8REVIn.6FW

In.3FW In.8REV

860 bp

9.4 kb

36.7 kb

763 bp

Alu-Y/Alu-Sg

Alu-Sq/Alu-Sp

76

6 8

9

987654

3

Intron 7

Intron 3

Deletion Ex 4–8

Alu-Sq

Alu-SpIntron 8

Alu-Sg

Alu-Sq Alu-Sp

860 bp

VA

4

VA

413

VA

299

WT

WT

WT

123

bpM

arke

r

VA

180

VA

307

VA

316

WT

WT

WT

123

bpM

arke

r

VA

86

VA

29

VA

52

WT

WT

WT

123

bpM

arke

r

2: 4738106–4756224, 148 KbaA

B

b

b

a

c

c

–4–2

–10

+1

+2

+4

09NG374

2: 47438106–47586224, 148 Kb 09NG374

–4–2

–10

+1

+2

+4

8






Figure 2. A, a, marker localization and allele frequency in control population of the MSH2 gene in chromosome 2. b–d, representative pedigrees ofexon 7 deletion carrier families. Legend and tumor phenotype are included. B, a, marker localization and allele frequency in control population of theMSH2 gene in chromosome 2. b–e, representative pedigrees of exon 4 to 8 deletion carrier families. Legend and tumor phenotype are included.






endometrial cancer in a mother and her daughter at 43and 40 years of age, respectively.Two recurrent rearrangements have been detected in 7 of

15 MSH2 carrier mutation families (>46%). Genome rear-rangements represent a significant proportion of all path-ogenic mutations in theMMR genes of patients with coloncancer (20).An exon 7 deletion was found in 3 nonrelated families,

the deletion producing a change in the reading frame anda truncated protein. Other rearrangements in this exonhave been described previously (9), but here we exactlycharacterize this mutation in every index case of the 3families and all of them have the identical mutation and ashared haplotype (Fig. 2A). The identification of thebreak points, within the Y and Sg Alu elements, supportsthe hypothesis that the recurrent exon 7 deletion is dueto an Alu repeat–mediated recombination event. These3 families had a high prevalence of CRC, followed byendometrial cancer and prostate tumor, with the majorityof cancers diagnosed before the age of 50 (Fig. 2A). Inrecent years, recurring mutations for various hereditarycancer syndromes have been identified around theworld (21).The most important evidence in our study is a big

deletion that includes 5 exons in the MSH2 gene, an in-frame mutation that produces a shorter protein andappears in 4 nonrelated Amsterdam criteria fulfilledfamilies.The study of the break point in index cases of each of the

4 families and the haplotype analyzed confirms the samemutation in all families (Fig. 2B).We identified that another Alu repeat event that was

involved in the exon 4 to 8 deletion (Fig. 2B).The significant proportion of families in our population

with one of these founder rearrangements (near 50% of the

families) suggests the need to design a simpler, faster, andcheaper method to detect these mutations. Thus, oursample study protocol could be changed and a prescreen-ing of founder mutations can be started.

We have carried out a multiplex PCR (Fig. 3) that ischeaper than the MLPA method.

Table 2 describes the cancer types in affected carriers andthe average age of onset. Most of them developed coloncancer before the age of 45, and the women developedendometrial cancer before the age of 50.

Our data indicate that the anticipation phenomenon isassociated with exon 4 to 8 MSH2 deletion. This is asubjective observation based on evidence from the ageof onset and an exhibition of a more aggressive diseaseseverity (number of cancers/tumors or the stage of thetumor) over successive generations (Fig. 2B).

The anticipation phenomenon has been described inother syndromes (22). The presence of anticipation inHNPCC is more controversial, and some reports providesignificant evidence that MHL1 mutations, in particular,may be associated with anticipation (23). Our data suggestthat MSH2 deletions may well be associated with antici-pation effects. The median age at colon cancer diagnosiswas 44 years in the 4 families (Table 2), but in family VA20,we observed that the difference at onset over 2 generationswas 20 years. In family VA17, the number of cancersincreased over the generations.

Both MSH2 rearrangements observed here occurred be-tween intron 4 and 8. It has been described that this is anAlu-rich region because approximately 84% of Alusequences in MSH2 are located between the promoterand exon 9 and it is possible that these sequences mediatethe recombination observed in this gene (24, 25).

Estimating the age of founder mutations is generally aninexact task (26). It was therefore impossible to estimate

Figure 3. Multiplex PCR ofscreening rearrangements. A,exon 7 deletion, the wild-typesamples show one 586-bp bandand deletion samples show 2bands: 586 bp and an extra bandof 861 bp. B, exon 4 to 8 deletion,the wild-type samples show one1,175-bp band and deletionsamples show 2 bands: 1,175 bpand an extra band of 751 bp.

861 bp861 bp738 bp

615 bp

492 bp

1,175 bp

751 bp

123123 bp 123 bp

Wt: 586 bp Wt: 1,175 bp

Break PointBreak Point

Del: 751 bpDel: 861 bpA B

489 bp 372 bp 68 bp685 bp

Fw15-GGCTCTTCAACTCTGTTGATTAGTT-3 5-CGTAATTAGCTGGGCATGGT-3 5-AGTGAAATGGGAGCCAAAAA-3 5-TCGAGTGGAACTTTAGCCTGTT-3 5-CCGGCCACAATAACAACC-3 5-GGGCAAGTATTAACTCAAACCACAA-3

Fw1Alu-Y Alu-SqRev1 Rev2CTGCAA TGAGCCAC

Rev1 Rev2

Del Wt WtDel Wt Delbp

Del_4–8Del_7

586 bp






whether all our founder carrier families share the samehaplotype. The fraction of haplotype can be inadequate forrecombination events, as we have a small number of carrierfamilies and these show a short genealogy length. Theevidence suggests that the origin of these mutations is arecent event.

Given the high proportion of rearrangements, we haveevaluated the phenotype in MSH2 mutation carriers. Amore severe phenotype (more different tumors, less onset,etc.) in rearrangement carriers was expected (25), butdifferences were not found. We only noticed a less primaryCRC tumor onset in rearrangement carriers than in otherMSH2 mutations carriers.

We detected rearrangements more often in familieswith endometrial or urinary cancer. These data are similarto those published by Geray and colleagues (27). Apossible explanation could be that both rearrangementsoccur in the MSH2 gene and both are founder mutationsin our population.

The differences found are not statistically significant, andthis does not allow us to orient the diagnosis based onmutation type (punctual or rearrangement) in clinicopath-ologically suspect patients of MSH2 mutation.

In conclusion, we have provided genetic evidence thatthe exon 7 deletion and exon 4 to 8 deletion are bothpathogenic foundermutations involved in causingHNPCCin a territory in central Spain. Our data show that largegenomic rearrangements occur in these genes with a highfrequency and emphasize the need to incorporate techni-

ques to routinely detect them. This should facilitate thegenetic diagnosis of Lynch syndrome inourpopulation. Theorigin of theMSH2 founder rearrangement can be linked tospecific geographic areas, and their current distribution iscompatible with the presumed migration pattern in ourcountry. In fact, members of our families have been studiedin other cities of Spain (Madrid, Barcelona, and Seville), andit is for this reason that our results are very important.

Our findings will greatly simplify the diagnosis, counsel-ing, and clinical care in suspected families.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank the National DNA Bank of Salamanca and the ScienceFoundation AECC. We also thank Noemy Mart��nez and Lara Hern�andezfor their excellent technical support and Alan Hynds for his critical readingof the manuscript.

Grant Support

This work has been supported by the University of Valladolid, Valladolid,Spain.

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received May 4, 2011; revised June 16, 2011; accepted July 5, 2011;published OnlineFirst July 21, 2011.

Table 2. Genotype–phenotype correlation in MSH2 mutation carriers

Punctual(truncating)

Rearrangements(founder)

Statisticalcomparison P

Number of families 8 7Number of affected 19 15Males, n (%) 10 (52.6) 6 (40)

n (%) n (%)Number of tumors Several tumors (yes/no) 1.0000

1 tumor 11 (57.9) 8 (53.3)>1 tumor 8 (42.1) 7 (46.7)

Tumor typeColon (CRC) 17 (58.6) 13 (50.0) CRC/EC 0.4727Endometrial cancer (EC) 4 (13.8) 6 (23.1)Urinary system (US) 2 (6.9) 7 (26.9) CRC/USOther cancers 6 (20.7) – 0.1247

Age at diagnosis, y<50 15 (78.9) 11 (73.3%) Age at diagnosis <50 (yes/no) 1.0000>50 4 (21.1) 4 (26.7%)

Age at diagnosis, y Average (SD)a Average (SD)a

Total (males and females) 46.37 � 10.75 40.13 � 10.46 Average age 0.0987CRC (males) 46.71 � 6.24 40.67 � 9.05 Median age 0.1827CRC (females) 50.00 � 23.72 33.00 � 6.89 Median age 0.1657Endometrial (females) 43.00 � 1.41 50.75 � 7.37 Median age 0.2357

NOTE: aStandard deviation.






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2011;4:1546-1555. Published OnlineFirst July 21, 2011.Cancer Prev Res Lucia Pérez-Cabornero, Ester Borrás Flores, Mar Infante Sanz, et al.

Gene Associated with a Lynch Syndrome PhenotypeMSH2Characterization of New Founder Alu-Mediated Rearrangements in

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