clinical studies cancer research mlh1 founder mutations...
TRANSCRIPT
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1 Founder Mutations with Moderate Penetrance in
R
nish Lynch Syndrome Families
Borràs1, Marta Pineda1, Ignacio Blanco8, Ethan M. Jewett10, Fei Wang12, Àlex Teulé2, Trinidad Caldés15,l Urioste16,17, Cristina Martínez-Bouzas18, Joan Brunet9, Judith Balmaña3, Asunción Torres19,a Ramón y Cajal4, Judit Sanz5, Lucía Pérez-Cabornero20, Sergi Castellví-Bel6, Ángel Alonso21,
Lanas22, Sara González1, Víctor Moreno7, Stephen B. Gruber13,14, Noah A. Rosenberg10,11, ar Mukherjee12, Conxi Lázaro1, and Gabriel Capellá1ractThe
SpanisfunctihistorRNA atainmc.1865from cc.306+causes(age-cc.1865ogenicconfir
sed riskisk has b
s' Affiliatiod'Oncologil Genètic en, Hospitaleebron; 4Seau; 5Instituenterologial Clínic; 7Unlogia i Univena, Spain;d'Oncologima de Conal Josep Tne and BioLife ScienMedicine,
Health; 13Deity of Michity of Michratorio de
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variants c.306+5G>A and c.1865T>A (p.Leu622His) of the DNA repair gene MLH1 occur frequently inh Lynch syndrome families. To understand their ancestral history and clinical effect, we performedonal assays and a penetrance analysis and studied their genetic and geographic origins. Detailed familyies were taken from 29 carrier families. Functional analysis included in silico and in vitro assays at thend protein levels. Penetrance was calculated using a modified segregation analysis adjusted for ascer-ent. Founder effects were evaluated by haplotype analysis. The identified MLH1 c.306+5G>A andT>A (p.Leu622His) variants are absent in control populations and segregate with the disease. Tumorsarriers of both variants show microsatellite instability and loss of expression of the MLH1 protein. The5G>A variant is a pathogenic mutation affecting mRNA processing. The c.1865T>A (p.Leu622His) variantdefects in MLH1 expression and stability. For both mutations, the estimated penetrance is moderate
umulative colorectal cancer risk by age 70 of 20.1% and 14.1% for c.306+5G>A and of 6.8% and 7.3% forT>A in men and women carriers, respectively) in the lower range of variability estimated for other path-Spanish MLH1 mutations. A common haplotype was associated with each of the identified mutations,
ming their founder origin. The ages of c.306+5G>A and c.1865T>A mutations were estimated to be 53 tod 12 to 22 generations, respectively. Our results confirm the pathogenicity, moderate penetrance, and
122 anfounder origin of the MLH1 c.306+5G>A and c.1865T>A mutations. These findings have important implicationsfor genetic counseling and molecular diagnosis of Lynch syndrome. Cancer Res; 70(19); 7379–91. ©2010 AACR.
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ch syndrome (MIM 120435) is an autosomal-dominantion caused by germline mutations in mismatch repair) genes MLH1, MSH2, MSH6, and PMS2 (1). It is char-zed by early-onset colorectal cancer (CRC) and an
of other cancers (1). At age 70, cumulativeeen estimated to be 27% to 66% for men
genetihave o
ns: 1Laboratori de Recerca Translacional, Instituta, IDIBELL, Hospitalet de Llobregat; 2Programa deCàncer, Institut Català d'Oncologia, Hospital Duran it de Llobregat; 3Servei d'Oncologia Mèdica, Hospitalrvei d'Oncologia Mèdica, Hospital de la Santa Creu it d'Oncologia Corachan-Centre Mèdic; 6Servei de, Institut de Malalties Digestives i Metabòliques,itat de Bioinformàtica i Bioestadística, Institut Catalàrsitat de Barcelona, IDIBELL, Hospitalet de Llobregat,8Programa de Consell Genètic en Càncer, Instituta, Hospital Germans Trias i Pujol, Badalona, Spain;sell Genètic en Càncer, Institut Català d'Oncologia,rueta, Girona, Spain; 10Center for Computationalinformatics and 11Department of Human Genetics,ces Institute, University of Michigan; 12Department ofUniversity of Michigan Medical School and School ofpartments of Internal Medicine and Human Genetics,igan Medical School; 14Department of Epidemiology,igan School of Public Health, Ann Arbor, Michigan;Oncología Molecular, Hospital Clínico San Carlos;
16DepInvestiEnfermMolecuGenètiBiologGenéticde EnfZarago
Note:Resear
E. Borr
CorresL'HospFax: 34
doi: 10
©2010
ls.org
2% to 47% for women; risk for endometrial canceraries between 14% and 40% (2–5).eral founder mutations have been described in MMR(6). In MLH1, founder mutations were identified in sev-opulations (6–8), where they can explain a substantialn of Lynch syndrome occurrences (9, 10), facilitating
c diagnosis. In Spain, founder mutations in MMR genesnly been identified in the MSH2 gene (11, 12).ar tamento de Genét ica Humana, Centro Nac ional degaciones Oncológicas; 17Centro de Investigación en Red deedades Raras, Madrid, Spain; 18Laboratorio de Genéticalar, Hospital de Cruces, Bizkaia, Spain; 19Unitat de Consellc, Hospital Universitari Sant Joan, Reus, Spain; 20Instituto deía y Genética Molecular, Valladolid, Spain; 21Servicio dea, Hospital Virgen del Camino, Pamplona, Spain; and 22Servicioermedades Digestivas Hospital Universitario, Universidad deza, Zaragoza, Spain
Supplementary data for this article are available at Cancerch Online (http://cancerres.aacrjournals.org/).
às and M. Pineda contributed equally to this work.
ponding Author: Gabriel Capellá, Gran Via 199-203, 08907italet de Llobregat, Barcelona, Spain. Phone: 34-932607952;-942607466; E-mail: [email protected].
.1158/0008-5472.CAN-10-0570
American Association for Cancer Research.
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R founder mutations reported thus far are heteroge-in function and frequency (6). Most of them encodeted proteins and are readily categorized as patho-mutations. However, founder MMR variants with un-pathogenicity have also been identified (11, 13, 14).
vestigate the functional effect of MMR variants, nu-s assays at the RNA and protein level have been de-ed (15, 16). The classification of a variant as ae-causing mutation requires integration of data fromnt sources, including its frequency in control popu-s, its cosegregation with the disease in families, itspathologic features, and its properties in functionals (17).have identified and characterized two frequently oc-g MLH1 variants in the Spanish population. To addresslinical significance, we examined their penetrance and
med functional studies. The founder effect hypothesis TableanalysSupple
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rials and Methods
ts and samplestal of 57 families were included in this study. Twenty-amilies were carriers of c.306+5G>A or c.1865T>Avariants (17 and 12 families, respectively) and wereed through Spanish genetic counseling and molecularstic laboratories. Twenty-eight families were carrierser pathogenic MLH1 germline mutations assessed attalan Institute of Oncology (ICO). Detailed family his-from at least three generations and geographic originsbtained. Clinical data collection included tumor loca-ge at diagnosis, microsatellite instability (MSI) testing,munohistochemistry of MMR proteins in tumors.
logic investigations based on interviews failed to iden-lationships between individuals from different families.udy protocol was approved by the Human ResearchCommittees of participating centers, and informedt was obtained from all subjects evaluated.omic DNA was extracted from whole blood using theene DNA kit (Qiagen) or from formalin-fixed, paraffin-ded tissues using the QIAamp DNA Mini kit (Qiagen).amples from 325 cases and 309 controls in a hospital-CRC case-control study conducted to assess gene-nment interactions in relation to CRC risk (18) were used.
ning for the c.306+5G>A and c.1865T>Amutations and loss of heterozygosity analysisening for the c.306+5G>A and c.1865T>A MLH1 muta-was performed by conformation-sensitive capillaryophoresis (CSCE) in DNA samples from the samease-control study (Supplementary Table S1; conditionsble on request; ref. 18). DNAs with patterns differinghose of controls were amplified using nonfluorescentr and sequenced with BigDye Terminator v.3.1 Cyclencing kit (Applied Biosystems). The methods for anal-
loss of heterozygosity (LOH) of the variant c.1865T>AH1 in tumors are included in Supplementary Methods.sess efied u
r Res; 70(19) October 1, 2010
utational methodsfollowing computational methods were used to predictications in splice-sites or exonic splicing enhancer sites:port (19), NNSplice (20), Rescue_ESE (21), and ESEfinderhe Polyphen (23) algorithm was used to predict thegenicity of p.Leu622His variant using default settings.lustalw program was used to align the amino acidnce of MLH1 in 13 phylogenetically diverse species.
of the c.306+5G>A and c.1865T>A MLH1tions on mRNA processing and stabilityal RNA was extracted from peripheral blood lympho-using Trizol (Invitrogen), and cDNA was synthesizedrandom primers (Invitrogen) and SuperScript II reverseriptase (Invitrogen). Specific primers were used to am-he appropriate MLH1 coding region (SupplementaryS1). The methods for allele-specific expression (ASE)is of the variant c.1865T>A of MLH1 are descibed inmentary Methods.
irected mutagenesis and constructioneu622His-MLH1pcDNA3.1+ vector containing wild-type (WT) MLH1(Genbank accession no. NM_000249.2) was kindlyed by Dr. R. Kolodner (Ludwig Institute for Cancerrch, UC San Diego School of Medicine, La Jolla, USA).utant p.Leu622His-MLH1 cDNA was constructed usinguikChange Site-Directed Mutagenesis kit (Stratagene)rimer 5′-AAGAAGAAGGCTGAGATGC(A)TGCAGAC-TCTCTTTG-3′ (sense strand; mutagenesis site is in pa-ses). Sequencing was used to verify the presence of theion. The cDNA insert between XhoI and BamHI sitesbcloned into WT MLH1-pcDNA3.
ulture and transfectionT116 [deficient for endogenous MLH1 (24)] and 293ere obtained from the American Type Culture Collec-esuscitated from stocks frozen at low passage within 6s of purchase, and cultured as described (25, 26). Cellere routinely tested by Mycoplasma presence and ver-y morphology, growth curve analysis, and expression ofns (e.g., MLH1 and PMS2). HCT116 cells were trans-with pGFP (green fluorescent protein) and WT
-pcDNA3.1 or p.Leu622His-MLH1-pcDNA3.1 vector us-pofectamine 2000 (Invitrogen) and Plus (Invitrogen).fection efficiency was measured by cytometry 24 hoursransfection.
and PMS2 protein expression andity experimentstein extraction from peripheral blood lymphocytes and16 and 293 cells was performed as described elsewhereLH1 and PMS2 expression levels were examined by
AGE, followed by Western blotting analysis with anti-and anti-PMS2 antibodies (clones G168-15 and A16-4;osciences). β-Tubulin antibody (Sigma) was used to as-
qual loading in all lanes. Band intensities were quanti-sing Quantity One software (Bio-Rad), and expressionCancer Research
of MLThe dethe nofectedcells.by trede nov
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age athe ag70 yeathe caat agesparseEC, fediagnoMMRavailabage wage of(deadTo c
al likeand gethe enmatiotion obias. Aratio (MMRhazardbackgin thethe po(30). Twith texp[g(for thgðtÞ¼k = 1,assumobtainTo
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H1 and PMS2 was normalized to β-tubulin expression.crease of protein expression was calculated by dividingrmalized protein expression in p.Leu622His-trans-cells by the expression in WT MLH1–transfected
The stability of WT and variant MLH1 was assessedating cells with cycloheximide, a global inhibitor ofo protein synthesis, as described (25).
ation of age-specific cumulative riskused information on the occurrence of CRC and EC ines of MMR mutation-positive index cases to estimatend gender-specific incidences of CRC and EC (females)R mutation carriers by maximum likelihood using
ied segregation analysis (2). The method was imple-d in MENDEL (v3.3.5; refs. 28, 29).tives were assumed to be followed from 20 years ofnd to be censored at the age of CRC diagnosis, ate of death, at the age of last follow-up, or at agers, whichever occurred earliest. We did not ignoreses beyond age 70 but assigned them the same risk70 to avoid the larger variances observed when onlydata are available. In estimating the risk of CRC andmale relatives were censored at age of CRC or ECsis, whichever was diagnosed first. Information onmutation status in relatives was included wheneverle. For individuals with missing age information, theas imputed based on relationship with the proband,the proband, and deceased status at last follow-upor alive).orrect for ascertainment, we maximized the condition-lihood of observing the phenotypes (CRC and/or EC)notypes (c.306+5G>A or c.1865T>A MLH1 variants) oftire pedigree given the phenotypic and genotypic infor-n of the index case (proband) and phenotypic informa-f the pedigree to account for multiplex ascertainmentge-specific cumulative risks (penetrance) and hazardHR) estimates of CRC and EC risks in families withgene variants were calculated assuming a proportionals model, with λ(t) = λ0(t)exp[g(t)], where λ0(t) is theround incidence. They were compared with the risksgeneral population, which were assumed to followpulation incidence from the Spanish Cancer Registrieshe age-specific relative risks in carriers comparedhe population rates are modeled through the functiont)]. We estimated the age-specific log HR parameterse two age intervals <50 and ≥50, assuming thatPn
k¼1
exp½�k�; a piecewise constant HR in the kth age band2. In all analysis, cancer incidences in noncarriers wereed to follow the population cohort–specific rates ased through the Spanish Cancer Registries.construct confidence intervals (CI) for the log(HR)ates, we assumed that the maximum likelihoodtes of the parameters were asymptotically normallyuted with covariance matrix given by the inverse ofsher information matrix. Cumulative risk (e.g., pene-) and its 95% CI were calculated from the cumulative
nce Λ(t) given by �ðtÞ ¼Pnk¼1iktk exp½�k�, where ik is the
ation incidence obtained from the Spanish Cancerthe unalogy
acrjournals.org
ries, tk is the length of the kth age interval, and βk isg(RR) in the kth age interval k = 1, 2. The cumulativegiven by F(t) = 1 − exp[−Λ(t)] and a 95% CI for F(t) isp½��ðtÞ � 1:96
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiV arð�ðtÞÞp �, where
ðtÞÞ ¼Xn
k¼1
i2kt2kV arð�kÞ exp½2�k�
þ 2Xn
j < k;k¼1
ikijtktj½V arð�kÞV arð�jÞ�1=2 expð�kþ �jÞcorrð�k; �jÞ:
type tests and CIs were then used based on the pointte and the estimated SEs (28, 29).analysis accounted for both genotyped and ungeno-relatives. Missing genotype information was handledluding the allele frequency as a parameter in the like-and then maximizing the marginal likelihood of thetype and genotype data of the entire pedigree, sum-over all possible configurations of the unobservedpe matrix, given the observed genotypes. Cumulativeand HR for CRC and EC were estimated separatelyales and females; risk of EC was estimated onlyales.
type analysislotype analysis was performed using three MLH1-nucleotide polymorphisms and seven microsatelliters (Supplementary Table S1; conditions available on re-). One hundred and twenty-two DNA samples fromers of the studied families and 50 control individualsmly selected from the CRC case-control study (18) wereed, including individuals that come from the areas ofof the founders. To deduce the mutation-associatedype, intrafamilial segregation analysis was performedthe assumption that the number of crossovers
en adjacent markers was minimal. The frequency ofe haplotypes in the control population was estimatedPHASE2 (default settings; ref. 31).
ation of founder mutation ageused a modification of the method of Schroeder andgues (32) to estimate the time to the most recenton ancestor (TMRCA) separately for the c.306+5G>A1865T>A alleles. This method uses a count of the num-recombination events that have occurred on copiesancestral mutant haplotype, together with an estimaterecombination map length of the haplotype, to estimatetal time length of the genealogy of sampled copies of at allele in units of generations. In our application of theach (32), to estimate TMRCA from the genealogy length,ied on a multiplier c(n), a ratio of TMRCA to tree lengthas estimated from coalescent simulations with sampleThe TMRCA estimate was then taken as an estimate ofe of the mutation, and CIs were obtained by considering
certainty in converting the length estimate for the gene-into an estimate of TMRCA. Because analysis of familyCancer Res; 70(19) October 1, 2010 7381
Table
Family
A. c.30A1
A2
A3
A4
A5
A6
A7A8
A9A10A11
A12
A13
A14
A15
A16A17
B. c.18B1
B2
B3
B4
B5
Borràs et al.
Cancer R7382
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9) October 1, 2010
affected
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carriers
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+ + R
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ancer Re
Referral center Criteriamet
Affectedcarrier
Gender Tumortype
Age ofonset
MSI IHC CRClocation
CRC stage:TNM/DukesMLH1 MSH2 MSH6
6+5G>A
families ICO, B arcelona AC A1.1 F CRC 38 + − + + R T4N 0M0A1.2
F CRC 41 R T2N 0M0 A1.3 M CRC 69 + − + + R T3N 0M0ICO, B
arcelona BC A2.1 F CRC 28 + − + + Rc T2N 0M0 A2.2 M CRC 51 + − + + R T3N 0M0ICO, B
arcelona BC A3.1 M CRC 38 A3.8 M CRC 52 R T2N 0M0 A3.1 0 M CRC 52GC
70 ICO, B arcelona AC A4.1 F CRC 38 + NV + + R T3N 1MxA4.2
F CRC 60 + NV + + R T4N xMx ICO, B arcelona BC A5.1 F CRC 50 + − + + R T2N xM0DC
68 CRC 80 Rc T3N 0M0ICO, B
arcelona BC A6.1 F CRC 38 + NV + NV L T4N 1Mx A6.2 F CRC 49 L T2N 0MxICO, B
arcelona BC A7.1 F CRC 52 + − + + R T1N 0M0 HCSC , Madrid/ BC A8.1 F BC* 48HUL
B, Zaragoza 3CRC 50 + NV + + R,R,L B1,B 1,B2 HCSC , Madrid AC A9.1 F CRC 20 + − L T3N 2Mx CNIO, Madrid BC A10.1 F CRC 30 + R B 1 H. Cru ces, Bizkaia BC A11.1 M CRC 59 R T2N 0M0A11.2
M CRC 48 L T3N 0M0 H. Cru ces, Bizkaia BC A12.1 F EC 51TC*
65 ICO, B arcelona AC A13.1 F EC2CRC
4242 − + + R,R T3N M0,T3N
0M0 ICO, B arcelona AC A14.2 M CRC 45 LCRC
49 IBGM, Valladolid AC A15.1 F CRC 27 + − + + Rc T4N 0M0A15.2
F CRC 50 R2
Valladolid ACic, ACA15.8
M CRC 39 R T3N 0MxH. ClinBar
celona/HVCA17.1A17.1
M
CRC 59 + −−+
+ RPamplona1 M CRC 35 + + R C
65T>A
families ICO, B arcelona BC B1.1 M CRC 27 + − + + R T3N 0M0CRC
39 L T1N 0M0 H. St J oan, Reus AC B2.1 M 2CRC 56 R,L T1N 0M0H. St P
au, AC B2.2B3.1M
CRC 24 + − + NV L T3N 0M0 F CRC 43 + − + NV R T3N 0M0Bar
celona B3.2 F CRC 28 L T3M 1N0 ICO, B arcelona AC B4.1 F CRC 33 + − + NV R T3N 2MxB4.2
M CRC 42 R T3N 0M0 OC* 58ICO, Girona AC B5.1 M CRC 42 + − + + R T3N0M0
search
historthe mwas pepast, swas inrials a
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pectedtion oc.306+codingin carridenti(28 paicancidenti9% ofcollabof Tabfamilie
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gatedand wly. Thememb21 una
Pathoc.186
Table
Family ie
sDM
B6 1
B7B8B9B10B11B12
NOTc.18AbbOncPauH. CBC,canprot*Tumor type not included in Lynch syndrome tumor spectrum.
Spanish MLH1 Founder Mutations
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y revealed no relationships among sampled families inost recent three to five generations, our age estimationrformed from a reference point four generations in theo that at the end of the estimation process, the estimatecreased by four generations. See Supplementary Mate-nd Methods for further details.
lts
ification of frequently occurring germlinevariants
tational screening of MMR genes performed in sus-Lynch syndrome patients at ICO led to the identifica-f two frequently occurring germline MLH1 variants:5G>A and c.1865T>A. No other alterations in MLH1sequence or at exon-intron boundaries were detectediers. Of the 83 families with germline MMR alterationsfied at ICO, 45 were carriers of an MLH1 alterationthogenic mutations and 17 variants of unknown signif-e). The c.306+5G>A and c.1865T>A variants werefied in nine and four families, constituting 20% andall MLH1 carrier families, respectively. An extendedorative study with other referral centers (see legend
le 1) enabled the identification of 16 additional carriers. In all, we identified 17 families with c.306+5G>A andForpredic
acrjournals.org
h c.1865T>A MLH1 variants (Table 1). Neither of theseriants was detected by CSCE in a panel of paired CRCand controls, reducing the probability of being poly-isms or CRC risk alleles.
ial clinical featuresst of the included families fulfilled the modified Am-m criteria (Table 1). The majority of tumors diagnosedriers belonged to the Lynch syndrome spectrum. Then age at diagnosis was 49.5 years (range, 20–80) for5G>A carriers and 45.5 years (range, 24–79) forT>A carriers. CRC and EC tested were microsatellitele (MSI+), and informative cases showed loss of ex-on of the MLH1 protein (Table 1).h c.306+5G>A and c.1865T>A MLH1 variants cosegre-with cancer in 29 and 21 affected members, respectively,ere absent in 37 and 18 unaffected members, respective-c.306+5G>A variant was also identified in 21 unaffecteders (median age, 34.0; range, 22–63), and c.1865T>A inffecteds (median age, 29.0; range, 18–41).
genicity assessment of the c.306+5G>A and5T>A MLH1 variants
1. Cli
nical features of affected carriers (Cont'd )the c.30ted the
6+5Gcreat
>A variant, splicing prion of a new donor si
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rral center Critermeta Affectcarri
edr
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r Tumotyper Age oonset
f MSI
LH1
IHC
MSH2
MSH6CRClocatio
nCRCTNM/
tage:ukes
HCSC
, Madrid AC B6. F CRC 38 + − + + R D B6. 2 F EC 53 + − + +CRC
65 L B B6. 3 F EC 55 + − + + B6. 4 M CRC 32 R T3N 2MxHCSC
, Madrid BC B7. 1 M CRC 59 + − + + IOC, B arcelona BC B8. 2 F CRC 43 − + + HVH, Barcelona BC B9. 1 M CRC 55 + − + + R B ICO, B arcelona AC B10. 1 F EC 54 + NV + + CNIO, Madrid AC B11. 1 M CRC 56 + R A CNIO, Madrid AC B12. 5 F EC 38BC*
79 B12.7 F CRC 48 + R AB12.8 F 2CRC 51 R AE: A. Clinical features of affected carriers of variant c.306+5G>A of MLH1. B. Clinical features of affected carriers of variant65T>A of MLH1.reviations: ICO, Institut Català d'Oncologia; HCSC, Hospital Clínico San Carlos; CNIO, Centro Nacional de Investigacionesológicas; H. Cruces, Hospital de Cruces; HVH, Hospital Vall d'Hebron; H. St Joan, Hospital Universitari Sant Joan; H. St, Hospital de la Santa Creu i Sant Pau; IDOC, Institut d'Oncologia Corachan; IBGM, Instituto de Biología y Genética Molecular;linic, Hospital Clinic; HVC, Hospital Virgen del Camino; HULB, Hospital Universatario Lozano Blesa; AC, Amsterdam criteria;Bethesda criteria; M, male; F, female; BC, breast cancer; CRC, colorectal cancer; DC, duodenal cancer; EC, endometrialcer; GC, gastric cancer; OC, otorhinolaryngological cancer; TC, thyroid cancer; IHC, immunohistochemical analysis of MMReins in tumor tissue; NV, not evaluable result; R, right; L, left; Rc, rectum.
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with losuppomutation via a mechanism in which MLH1 protein expressionis decreased.
1. Characterization of the c.306+5G>A MLH1 variant. A, schematic overview of MLH1 exons 2 to 4 with a representation of the normal and aberrantipts caused by the c.306+5G>A mutation. Black arrows represent primers used for RT-PCR amplification. B, acrylamide gel showing RT-PCR
t sequ
Figure 2. Expression and stability of the p.Leu622His variant of MLH1 inHCT116 cells. A, time course of expression of WT MLH1 andp.Leu622His (L622H) variants. Top and middle, cell lysates probed withanti-PMS2 and anti-MLH1 antibodies; bottom, to verify equal proteinloading, cell lysates were probed with variant anti-tubulin antibody.Untransfected HCT116 and 293 cells were used as controls. Onerepresentative experiment among three is shown. The ranges of decreasein MLH1 expression at 24, 48, and 72 h were 35% to 55%, 42% to75%, and 81% to 100%, respectively. The ranges of decrease in PMS2expression at 24, 48, and 72 h were 57% to 89%, 48% to 55%, and43% to 64%, respectively. B, stability of WT MLH1 and p.Leu622Hisvariant as assessed by cycloheximide (CHX) treatment. “+” and “−”represent treatment with cycloheximide or DMSO as vehicle,respectively. The decrease in expression at 1, 4, 6, and 9 h after
e transcription-PCR (RT-PCR) analysis on RNA fromocytes of carriers confirmed the generation of thisnt mRNA transcript, r.303_307delugagg, expected tote a truncated protein. This transcript was associatedn increased amount of the r.208_306del alternativetutional transcript, corresponding to the in-frameng of MLH1 exon 3 (Fig. 1). Although the variant5G>A is pathogenic at the RNA level, neither abnor-ands nor differences in MLH1 protein expression wereed in lymphocytes from c.306+5G>A carriers, as as-by Western blotting (data not shown).MLH1 c.1865T>A variant is predicted to generate these change p.Leu622His, which was classified as path-by the Polyphen algorithm and MAPP-MMR andredictions (33, 34). The affected amino acid residuely conserved and is located at the interaction domainutL (the homologue of MLH1 in Escherichia coli;). Spliceport and NNSplice did not predict any effectNA processing, whereas ESEfinder and Rescue_ESEted the creation of a new potential binding site forroteins. In lymphocytes of c.1865T>A carriers, differ-neither at the mRNA level [as assessed by RT-PCRnot shown) and allele-specific expression analysislementary Fig. S1)] nor at the protein expression levelnot shown) were observed. The transient transfectionp.Leu622His variant into the HCT116 cell line (24),lacks endogenous MLH1, resulted in diminishedexpression when compared with WT MLH1–transfectedFig. 2A). MLH1 heterodimerizes with PMS2, stabilizingression (25). As expected, the diminished p.Leu622Hisssion was associated with lower PMS2 expressionred with WT MLH1–transfected cells (Fig. 2A). Aftereximide treatment, MLH1 expression was lower in both22His-transfected cells and WT MLH1–transfected cells.er, the effect was greater in p.Leu622His-transfectedindicating that p.Leu622His protein was less stableT MLH1 (Fig. 2B). Five of the six tumors analyzed
ts obtained from c.306+5G>A carriers and a noncarrier. On the right, direc
c.1865T>A carriers lost the MLH1 WT allele (Supple-ry Fig. S1), suggesting a growth advantage associated
cycloheand 47represe
r Res; 70(19) October 1, 2010
ss of the WT protein. Together, these lines of evidencert the notion that the variant c.1865T>A is a pathogenic
encing of the RT-PCR products is shown.
ximide treatment was 39%, 64%, 55%, and 61% in WT MLH1%, 97%, 99%, and 100% in p.Leu622His, respectively. Onentative experiment among three is shown.
Cancer Research
CumupenetA to
trancewere fcumulSpanisc.306+exceedCRC cCRC i14.1%is 7.7for femage inhigherdeclinrisk foage 706.3 (95Alth
riers60 yeatinuesfor maHR for1.1–18genera0–10.0In c
estimafor me10.5 (9
femalewith Hc.306+withinotherpenetr
HaploAnc
cameancestfrom tgeograthe twa singby hapThe
dualstion fhaploD3S12has ac.1865cy of tthe dic.1865To
mateon theAt thely rec
Table 2. Characteristics of the study population by mutation type
+ 5 r
No. pr 2Total n 7No. fe 4No. m 1No. ind 2No. ind 8No. fir 6No. su 0No. te 2Percen 0Media 0Media 3Percen 7PercPercPerc
NOTE: Blood- and nonblood-related relatives are included.
Spanish MLH1 Founder Mutations
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lative risks and HRs derived from therance analysistal of 1,936 individuals were included in the pene-analysis, 57 of which were probands and 388 of whichirst-degree relatives of probands (Table 2). Age-specificative risks of CRC by decade compared with risks inh Cancer Registries (30) are shown in Table 3A. For5G>A carriers, the risk of CRC by age 50 significantlys the cumulative risk in the general population. Risk ofontinues to increase, and by age 70, lifetime risk fors estimated at 20.1% (95% CI, 1.4–35.9%) for men andfor women (95% CI, 1.2–25.2%). The overall HR for CRC(95% CI, 2.9–20.6) for males and 9.0 (95% CI, 3.6–22.6)ales (Table 3B). The age-specific HR estimates in thetervals 20 to 49 and 50 to 69 clearly indicate muchrelative risk compared with the general population,
ing in the age range from 50 to 69 years. Cumulativer EC also exceeds that of the general population atyears, equaling 7.2% (95% CI, 0–16.9%) with HR of% CI, 1.4–27.9; Table 3).ough the cumulative risk of CRC for c.1865T>A car-exceeds the risk in the general population by agers, it is lower than for c.306+5G>A. Risk of CRC con-to increase and by age 70 is 6.8% (95% CI, 0–15.8%)les and 7.3% (95% CI, 0–16.5%) for females. The overallCRC is 2.4 (95% CI, 06–10.2) for males and 4.5 (95% CI,.0) for females. Risk for EC tends to exceed that of thel population at age 70 years, equaling 3.4% (95% CI,%) with HR of 2.9 (95% CI, 0.4–23.6; Table 3).arriers of other Spanish MLH1 pathogenic mutations,ted lifetime risk for CRC is 26.3% (95% CI, 7.8–41.1%)
n and 10.7% for women (95% CI, 0–16.8%), with HR of5% CI, 5.0–21.8) for males and 5.4 (95% CI, 2.0–14.9) forin famtype w
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s, and cumulative risk for EC is 6.5% (95% CI, 0–13.1%)R of 5.7 (95% CI, 1.9–17.0; Table 3). Therefore, both5G>A and c.1865T>A mutations show a penetrancethe range of variability estimated in families with
MLH1 mutations, with a nonsignificant trend to lowerance for the c.1865T>A mutation.
type analysis and estimation of mutation ageestors of families carrying the c.306+5G>A mutationfrom the Ebro river valley in northern Spain, whereasors of families with the c.1865T>A mutation originatedhe region of Jaén in southern Spain (Fig. 3). Commonphic origins of carrier families suggested that each ofo mutations could have occurred as a unique event inle founder individual. This hypothesis was confirmedlotype analysis (Table 4).existence of a clear shared haplotype in carrier indivi-permitted estimation of mutation age. In this estima-or the c.306+5T>A mutation, we considered diseasetypes in the region extending from D3S2369 to98 (D3S1612 to D3S1298 for c.1865T>A; Table 4), whichrecombination map length of 0.9796 cM (2.2578 cM forT>A). In the control population, the estimated frequen-he identified minimum common haplotype, excludingsease mutation, was 0.069 for c.306+5T>A and 0.048 forT>A (Supplementary Table S2).estimate the age of the mutation, we relied on an esti-of the number of recombination events that occurredancestral disease haplotype for each disease mutation.5′ end of the common haplotype of c.306+5G>A, a like-ombination event at marker D3S2369 was detected
ily A10as lost; at the 3′ endin 6 of the 17
Ca
(D3S1298), tfamilies, lik
ncer Res; 70
c.306
5G>A c.186 T>A Othe MLH1 mutationsobands/pedigrees 1
7 1 28 o. individuals 51 4 44 975 males 25 7 20 444 ales 24 0 22 486 ividuals with missing gender information 1 7 2 45 ividuals with missing age information 22 6 20 442st-degree relatives 8
9 10 193 bjects genotyped 8 8 6 174 sted mutation-positive subjects 5 0 4 88 tage of mutation-positive subjects among subjects genotyped 5 6.8 7 .0 50.5 n age at diagnosis of CRC in men (range) 52 (3 8–80) 43 (2 –65) 41 (25–80) n age at diagnosis of CRC in women (range) 50 (2 0–80) 48 (2 –87) 44 (18–77) tage of CRC diagnosed before age 50 among affected subjects 3 8.3 5 .1 73.7 tage of CRC or EC affected subjects among carriers 5 6.0 4 .6 64.8 en 7entage of CRC or EC affected subjects among noncarriers 2.6 0 0entage of CRC or EC affected subjects among nontested individuals 10.1 12.1 7.5
he common haplo-ely in two separate
(19) October 1, 2010 7385
Tablec.1 6
A
Age (y)
% Cumulativerisk population carriers (95% CI)
ulativepulation carriers (95% CI)
20
304050
6070
20304050
60 0.55 3. .27 14.70
B
Age (y)
CRC (males) CRC (females)
20– 9
50– 9
Ove l
20– 9
50– 9
Ove
NOTcorrcum(95%Abb
Borràs et al.
Cance7386
8
4
6
ra
4
6
ra
Eeu
rev
r R
3. Age-specific cumulative risk and HR estimates for CRC and EC in carriers of c.306+5G>A,5T>A, and other pathogenic MLH1 mutations
iation: NA,
es; 70(19) O
not available.
ctober 1, 2010
C
RC (males) CR C (females)% Cumulativ
e risk MLH1 % Cumrisk po% Cumulativ
e risk MLH1Cancer R
c.306+
5G>A c.186 5T>A OthermutaMLH1tions
c.306+
5G>A c.1865 T>A OthermutatMLH1ions
0 0
0 0 0 0 0 00
.01 0.0 8 0.046 0.12 0.4 0.
10 063 0
.01 0.0
9 0.054 0.24 0.07 0.3
53
00
.06 0..28 2.1 4 0.6 8 2. 90 0.06 0.
.24 2.1
4 1.0 7 1. 3 (0.04– 4.20) (0–146 2.4
.64) (0.78–
0 10.
4.96)
00 0
(0.17–
4.06) (0–2.29 3.1
54) (0–2
8 3.8
.59)
5
1.00 7. .72 6.2.87 20.13 6.80 26.32 1.67 14.05 7.26 10.74(1.42–
35.95) (0–15.80) (7.85–41.07) (1.20–2 5.23) (0–16.50) (0–16.80)EC (fem
ales) CRC and EC (females)0 0
0 0 0 0 0 0 0 0 013 0.0
06 0.
011 0
.01 0.1 2 0.097 0.3
4 0.05 0.466
00
.02 0..13 0.8 2 0.3 8 0. 74 0.08 0.
.37 4.4
1 1.6 3 2.1 0 (0–2 .02) (0–142 1.6
.16) (0–10 3..54)1 1
(1.25–
7.47) (0–3.41 5.550) (0.43–0 7.0
3.74)7
1.17 7.16(0–16.88)
3.37(0–10.05)
6.5(0–13.12)
2.84 29.58(9.34–45.29)
11.9(0–24
8.24)
16.2(3.32–2
45.67)
HR
(95% CI)c.306+
5G>A c.186 5T>A OthermutaMLH1tions
c.306+
5G>A c.1865 T>A OthermutatMLH1ions
14.
8973.23)4.1(0.18–
095.72)
32.(12.57–
4283.57)
13.3
660.94)9.5(1.33–6
68.44)
12.6(3.49–4
35.62)
(3.03–5.
3719.72)1.8(0.325–
910.95)
3.(0.88–
2111.70)
(2.93–6.8
824.28)1(NA
)2.3(0.48–1
01.05)
(1.47– (1.95–l HR 7.72 2.42 10.48 8.99 4.4
(2.89– 20.59) (0.57–10.23) (5.04–21.81) (3.58–2 2.56)8(1.11–18.01)
5.42(1.97–14.93)
EC (fem
ales) CRC and EC (females)20.
00144.19)5.2(0.15–1
285.08)
16.(3.19–
0280.42)
22.5
267.70)9.1(1.62–5
31.61)
20.3(7.84–5
02.27)
(2.77–3.
6724.51)2.1(0.15–
732.45)
2.(0.55–
9815.92)
(7.49–8.4
122.78)1.7(0.16–1
38.49)
1.9(0.54–
97.34)
(0.55– (3.10–ll HR 6.32(1.43–27.94)
2.92(0.36–23.57)
5.71(1.91–17.02)
12.17(5.92–25.01)
4.43(1.37–14.36)
5.73(2.59–12.71)
: A. Age-specific cumulative risk of CRC and EC for male and female carriers of MLH1 mutations compared withsponding values for the population incidences as reported in the Spanish Cancer Registries (95% CIs are provided forlative risk at ages 50 and 70). B. Age-specific and overall HR for CRC and EC for male and female carriers ofMLH1 mutationsWald CI is provided for the HR parameters).
esearch
recomwe coessaryc.1865curredof theFro
we esL ¼ k=and Lfrom(see S5 × 10tion sestimacEbro LEing 25Simila0.25694 = 15tion)generato 22
Discu
WeMLH1amongthe firpopulaThe
iant cwith oand c.in exppared
of muWesteThe
scribe(39, 40clusivwas dcotranactivitincontransfsubstaity, whcounttationWT almutattrafficOnc
shownfromSpanisdata rfoundetranctrancefact tMLH1to theuse ofonly onote, pilies alationStoffeare obgeograsyndromentathe expolyge
Figure 3. Map of Spain showing locations where Lynch syndromefamilies with the c. 306+5G>A mutation (white circles) and the c.1865T>Amutation (black circles) originated. For six families (four c.306+5G>Aand two c.1865T>A), the original location is not known. The Ebro valleyis highlighted in dark gray. The insert shows a larger-scale map of theJaén pr
Spanish MLH1 Founder Mutations
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bination events (Table 4A). Therefore, for c.306+5T>A,unted a minimum of three recombination events nec-to explain all disease haplotypes in the region. ForT>A, we counted a minimum of one event, which oc-at the 3′ end (D3S1298) and which explained the losscommon haplotype by 5 of the 12 families (Table 4B).m the map lengths (r) and recombination counts (k),timated genealogy lengths (L) using the formular. We obtained LEbro ¼ 3=0:009796 ¼ 306 generations
Jaén=1/0.022578=44 generations. To estimate TMRCAthese lengths, we required an estimate of the ratio cupplementary Materials and Methods). By simulating7 trees under a coalescent model of constant popula-ize with n = 17 lineages representing 17 families, weted cEbro ¼ 0:2324, which gives an estimate of HEbro ¼bro ¼ 3=0:009796 ¼ 75 generations (1,879 years assum-years per generation) for the c.306+5G>A mutation.rly, using n = 12 lineages, we estimated cJaén =, producing an age estimate of H Jaén ¼ cJaénLJaén +generations (384 years assuming 25 years per genera-for c.1865T>A. The 95% CI for HEbro is 53 to 122
ovince.
tions, and the corresponding interval for H Jaén is 12 Fouin tradin MLAshkesyndroreportidentifrequeof Lyn+5G>A45 famnonfowith mfoundin the
generations (Supplementary Table S2).
ssion
have identified and characterized two frequentvariants, c.306+5G>A and c.1865T>A (p.Leu622His),Spanish Lynch syndrome families. They represent
st founder MLH1 mutations identified in the Spanishtion.predicted effect of the previously reported MLH1 var-.306+5G>A (36) on splicing was confirmed, consistentbservations on the nearby mutations c.306+2dupT306+4A>G (37, 38). At the protein level, no differences
ression were observed in mutation carriers when com-with controls, presumably because of a lack of stabilityOurthe vi
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tated MLH1 proteins or due to the limitations of thern blotting assay.c.1865T>A variant (p.Leu622His) was previously de-
d as a germline MLH1mutation in two Spanish families). Prior functional assays for p.Leu622His were incon-e to assess its pathogenicity: In yeast-based assays, iteficient in MMR activity (33, 41), whereas HCT116 cellssfected with MLH1 and PMS2 retained partial MMRy (69.2%). The effect on protein expression levels wasclusive (33). In vitro expression studies in MLH1-ected HCT116 cells indicated that this mutation mayntially reduce MLH1 expression by affecting its stabil-ich in turn leads to a reduction in the expression of itserpart PMS2. A pathogenic role for the p.Leu622His mu-was further supported by the frequent loss of theMLH1lele observed in tumors (42). An additional effect of theion on the capacity to bind PMS2 or on the intracellularking of MLH1/PMS2 could not be ruled out.e the functional effect of the founder mutations was, we explored whether penetrance estimates differedother MLH1 mutations. The estimated penetrance ofh MLH1 mutations was moderate and consistent witheported for Dutch families (5). The penetrance ofer mutations was within the range of variability of pen-e observed for Spanish MLH1 mutations. The pene-for c.1865T>A missense mutation may be lower, a
hat may be attributed to the observed decrease inexpression. The penetrance estimates are sensitivetype of ascertainment correction, and elevate withan unconditional likelihood or likelihood conditionedn the proband, a phenomenon also noted in (2). Ofenetrance estimates for Spanish Lynch syndrome fam-re lower than those reported in North American popu-s (2, 3). The fact that we have used the same method asl and colleagues (2) and that similar cumulative risksserved in the United States and Spain suggests thatphic differences in cancer risk might exist in Lynchme carriers. Several factors including lifestyle, environ-l factors, mutational spectrum, or, as recently reported,istence of distinct weak alleles capable of producingnic effects (43) may account for this observation.nder mutations in MMR genes have an important effectitional “founder populations”: Four different mutationsH1 and MSH2 genes in Finland, Newfoundland, and innazi Jews account for between 25% and 50% of all Lynchme cases in these populations (9, 10, 13, 44). Before our, two founder mutations in the MSH2 gene had beenfied in Spanish Lynch syndrome families, although theirncies were low in our population (11, 12). In the cohortch syndrome families from ICO, the identified c.306and c.1865T>A mutations account for 28.8% of theilies carrying MLH1 alterations (13 founders and 32unders) and represent 17.6% (13 of 74) of all familiesutations in MMR genes. In addition, the c.306+5G>A
er accounts for up to 25% ofMLH1mutations identifiedEbro basin area (data not shown).
detection of founder mutations in MLH1 supportsew that they can occur not only in groups commonly
Cancer Res; 70(19) October 1, 2010 7387
Table 4. Haplotypes associated with the c.306+5G>A (A) and c.1865T>A (B) mutations in carrier families
A
Marker (Mb onchromosome 3)
D3S1609(29.915)
D3S1612(34.565)
D3S2369(36.472)
c.306+5G>A(37.018)
rs4234259(37.024)
rs179997(37.029)
D3S1611(37.044)
rs9876116(37.059)
D3S3623(37.419)
D3S1298(38.024)
D3S3564(42.394)
FamiliesA1 254 106 103 Yes G A 256 G 224 199 209A2 250//254 106 103 Yes G A 256 G 224 199 213A3 250 96 103 Yes G A 256 G 224 205 213A4 254 92 103 Yes G A 256 G 224 205 201/211A5 254/256 92 103 Yes A/G A 250/256 A/G 224 205 201/213A6 250 104 103 Yes G A 256 G 224 205 201/213A7 250 96/104 103 Yes G A 256 G 224 201 211A8 250/254 96/106 103 Yes G A 256 G 218/224 199/201 203/209A9 254 96 103 Yes A/G A 256/260 A/G 216/224 197/199 213/215A10 250/256 96/104 101 Yes G A/G 256 G 224 199 203/215A11 250 96 103 Yes G A 256 G 224 199 209/213A12 250 96/104 103 Yes G A/G 256 G 216/224 195/199 201/215A13 250 96 103 Yes G A/G 256 G 220/224 199/209 209/213A14 252 96 101/103 Yes G A/G 256 G 224 205 211A15 250//254 96 103 Yes G A 256 G 224 199 213A16 250//254 96 103 Yes G A 256 G 224 199 213A17 254 96 103 Yes A/G A 256 G 224 199 209//213
Max 1.55 Mb
Min 0.40 Mb
Shared alleles 103 Yes G A 256 G 224 199Control frequencies (%) 67 0 44 66 44 44 12 30
(Continued on the following page)
Borràs
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Table 4. Haplotypes associated with the c.306+5G>A (A) and c.1865T>A (B) mutations in carrier families (Cont'd)
B
Marker (Mb onchromosome 3)
D3S1609(29.915)
D3S1612(34.565)
D3S2369(36.472)
rs4234259(37.024)
rs1799977(37.029)
D3S1611(37.044)
rs9876116(37.059)
c.1865T>A(37.064)
D3S3623(37.419)
D3S1298(38.024)
D3S3564(42.394)
FamiliesB1 256 104 101 G G 256 G Yes 222 205 207B2 256 104 101 G G 256 G Yes 222 205 207B3 252//256 92/104 101 G G 256 G Yes 222 205 201//207B4 256 104 101 G G 256 G Yes 222 205 201B5 250 104 101 G G 256 G Yes 222 197 203B6 250 104 101 G G 256 G Yes 222 205 207B7 250/256 104 101/103 A/G A/G 256 G Yes 222 205 211B8 250/256 104 101/103 G A/G 256 G Yes 222 205 201/207B9 256 104 101 G G 256 G Yes 222 197 203B10 250/254 92/104 101 A/G A/G 256/260 A/G Yes 214/222 197/199 201/203B11 250/256 96/104 101 G G 256 G Yes 222 197/199 203/213B12 250//256 104 101 G G 256 G Yes 222 197 209
Max 3.46 Mb
Min 0.95 Mb
Shared alleles 104 101 G G 256 G Yes 222 205Control frequencies (%) 32 31 44 34 44 44 0 22 10
NOTE: The “/” symbol indicates that the phase of the disease haplotype cannot be established. The “//” symbol indicates that recombination has likely occurred within a family.Intragenic MLH1 markers are shown in a box. Dark gray shading indicates nonrecombinant haplotypes. The sizes of the minimum and maximum conserved haplotypes areshown at the bottom. Inferred haplotypes of subjects in the fifth generation in each family are in bold. The frequencies of disease-associated alleles among a panel of controlDNAs are shown at the bottom.
Spanish
MLH
1Found
erMutatio
ns
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the Ebthe mufact thtain racenturin theyearsthan cmounbandsquentthe peer mutheir cterns.severabinatiwhichlengthage. Hsensibgeogra
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Borràs et al.
Cance7390
as founder populations but also in geographicallyed subsets of larger populations. These two founderions were not detected in a prior study of 1,222 inci-ases of CRC in Spain (EPICOLON; ref. 45). This studyied 11 germline MMR mutations, 4 in MLH1. The lackection of founder mutations may be due to the lower of identified mutations or the relatively low cover-the areas of origin.ilies carrying the c.306+5G>A mutation cluster withinro river basin, in northern Spain, and we estimate thattation arose ∼1,879 years ago. Taking into account theat the river valley is geographically isolated by moun-nges, and the Ebro river was navigable until the 19thy, we hypothesize that the mutation arose somewherevalley and was distributed along the river over the
(Fig. 3). The c.1865T>A mutation is younger (384 years).306+5G>A, and its distribution is restricted to thetainous province of Jaén. It is noteworthy that pro-have been identified in Madrid and Barcelona, fre-destinations of internal migratory movements duringriod 1960 to 1970. Thus, the origin of the MLH1 found-tations can be linked to specific geographic areas, andurrent distribution can be explained by migration pat-Allele age estimation is generally imprecise (46) due tol factors, including uncertainty in the count of recom-on events on ancestral haplotypes, the accuracy withthe recombination count predicts the true genealogy, and the accuracy with which TMRCA reflects alleleowever, the relatively recent values obtained are
le for rare mutations restricted to relatively isolated Recestrom-Lahti M, Kristo P, Nicolaides NC, et al. Founding mutationsAlu-mediated recombination in hereditary colon cancer. Nat Med5;1:1203–6.
10. MoAghe
11. Memutal26
12. Mepafam
13. FoMScoGe
14. CeGrandro
15. SpPreclin
16. Codevar
17. GoGrsoHu
18. Mo
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high prevalence and moderate penetrance of the5G>A and c.1865T>A variants have implications forular and clinical approaches to founder mutations insettings. In specific populations such as the Spanishation, initial screening for founder mutations increasesficiency of testing of molecular diagnostic labs. Evenimportantly, careful clinical characterization of founderions can lead to mutation-specific counseling andved clinical care.
osure of Potential Conflicts of Interest
otential conflicts of interest were disclosed.
owledgments
hank the staff at the Genetic Counseling Units who have participatedstudy and Michael DeGiorgio for helpful discussions about then age estimates.
Support
ació La Caixa grant BM 04-107-0; Fundació Gastroenterologia Dr.co Vilardell grant F05-01; Ministerio de Educación y Ciencia grants4-07579-04, SAF 06-6084, and SAF09-7319; Spanish Networks RTICCCD06/0020/1050, 1051, 0021, and 0028; Acción en Cáncer (Instituto dearlos III); Fundació Roses Contra el Càncer; Fondo de Investigacióna (CP 03-0070); and NIH grant CA81488.costs of publication of this article were defrayed in part by the paymentcharges. This article must therefore be hereby marked advertisement innce with 18 U.S.C. Section 1734 solely to indicate this fact.
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