3 Med Genet 1996;33:636-640

Complex genetic predisposition to cancer in an

extended HNPCC family with an ancestralhMLH1 mutation

P Hutter, A Couturier, R J Scott, P Alday, C Delozier-Blanchet, F Cachat, S EAntonarakis, F Joris, M Gaudin, L D'Amato, J-M Buerstedde

AbstractHereditary non-polyposis colorectal can-cer (HNPCC) is characterised by a geneticpredisposition to develop colorectal can-cer at an early age and, to a lesser degree,cancer of the endometrium, ovaries, uri-nary tract, and organs ofthe gastrointesti-nal tract other than the colon. In themajority offamilies the disease is linked tomutations in one of the two mismatchrepair genes, hMSH2 or hMLH1. We havefound a novel hMLH1 nonsense mutationin a Swiss family with Lynch syndrome,which has been transmitted through atleast nine generations. A different tumourspectrum of neoplasms of the skin, softpalate, breast, duodenum, and pancreaswas observed in three branches of thisfamily, where there was a virtual absenceof colonic tumours. The hMLH1 mutationcould not be detected in members ofthesebranches suggesting that at least a secondgenetic defect predisposing to cancer issegregating in part of the kindred.(JMed Genet 1996;33:636-640)

Key words: HNPCC; Lynch; hMLH1 mutation.

Division de Pathologie,Institut Central desH6pitaux Valaisans, AvGrand-Champsec,1950 Sion, SwitzerlandP HutterA CouturierF CachatF JorisM Gaudin

Dept Forschung,Kantonsspital, Basel,SwitzerlandR J Scott

Division de GenetiqueMedicale, CMU,Geneve, SwitzerlandC Delozier-BlanchetS E AntonarakisL D'Amato

Basel Institut fiirImmunologie, Basel,SwitzerlandP AldayJ-M Buerstedde

Correspondence to:Dr Hutter.

Received 5 February 1996Revised version accepted forpublication 20 March 1996

Hereditary non-polyposis colorectal cancer isprobably the commonest cancer predisposi-tion, accounting for approximately 5% of allcases of colorectal cancers.' 2 This disorder isinherited in an autosomal dominant manner

and has a high level of penetrance. The clinicaldiagnosis relies on the observation of a familialclustering of early onset colorectal cancer.

HNPCC kindreds are commonly defined as

those in which at least three relatives in twosuccessive generations have a histologicallyverified diagnosis of colorectal cancer, one ofwhom should be under 50 years ofage.3Although colorectal carcinoma is the pre-dominant form of disease in HNPCC families,up to 40% of all malignancies in these familiesare extracolonic cancers of epithelial derivedorigin.2 The second most frequently affectedorgan is the endometrium and a higherfrequency of other cancers has also beenreported including the stomach, small intes-tine, upper renal tract, and ovaries. Addition-ally, breast cancer, hepatobiliary cancer, pan-creatic cancer, skin cancers, and sarcomas havebeen reported.4

Recently, heterozygous germline mutationsin any one of the four mismatch repair gene

homologues, hMSH2, hMLHl, hPMS1, andhPMS2, have been shown to be linked toHNPCC.5-9 Germline mutations in hMSH2and hMLHl appear to account for the major-ity of HNPCC families.0-" The products ofthese genes are involved in the process of mis-match repair (MMR) by recognising base-basemismatches and unpaired loops, before excis-ing and replacing them with the correct nucle-otides.'"Evidence suggests that the second wild type

copy of the relevant mismatch repair gene isinactivated in HNPCC tumour cells. 14 Aremarkable feature of tumour DNA isolatedfrom HNPCC patients is its overall genomicinstability, reflecting a mismatch repair defi-ciency. This phenotype is easily detected asshifts in the electrophoretic mobility of micros-atellite DNA sequences containing di- and tri-nucleotide repeats. 15-17 Several studies haveshown that genomic instability is not restrictedto HNPCC, as it is also observed in a substan-tial proportion of the early onset sporadicforms of colorectal cancer.'8 19Here we report a mutation analysis in a large

family showing an unusual tumour spectrum,with typical manifestations of HNPCC in atleast two of its branches but almost exclusivelyextracolonic tumours in three other branches.In the HNPCC subfamilies of the kindred thecausative mutation identified is a G-*A germ-line transition in the hMLH 1 gene, which leadsto the truncation of the mature protein.However, this mutation was not detected inpatients from the other branches of the family,who still developed several types of cancer,suggesting that they might have inherited atleast one different genetic defect stronglypredisposing to cancer.

Material and methodsGENOMIC INSTABILITY IN TUMOURSArchival paraffin embedded tumour tissueswere sectioned and DNA was extracted usingtwo rounds of digestion with proteinase K, fol-lowed by three phenol/chloroform extractionsat 600C. Briefly, tissue sections were depar-affinised with xylene and washed in absoluteethanol. Dried samples were treated with 400gg/ml proteinase K (Boehringer, Mannheim)in 500 jl digestion buffer (10 mmol/l Tris, pH8.0, 1 mmol/l EDTA, 75 mmol/l NaCl, and 1%SDS) at 500C overnight. The pattern of ampli-fication of four microsatellite sequences

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Complex genetic predisposition to cancer in an extended HNPCCfamily

(D6S285, D12S79, D13S225, and GABA55)was compared between DNA from tumour tis-sue and DNA isolated from peripheral bloodcells of the same patient. The DNA was ampli-fied for 30 cycles in 20 gl (5-10 pmol of eachprimer, 50 ,umol/l of each dNTP, and 0.1 unitof Taq polymerase). The upstream primer waslabelled at its 5' end using [y32p] ATP (6000Ci/mmol) and T4 polynucleotide kinase. Fol-lowing PCR, 1-3 p1 of reaction product weredenatured and electrophoresed on denaturing8% polyacrylamide gels (Accugel 19:1 fromNational Diagnostics/ Atlanta). After electro-phoresis, the gels were dried and exposed to xray films for 12-18 hours. The same experi-mental conditions were used to amplify themarkers D3S1277 and D3S161 1, linked to thehMLHl gene.

ANALYSIS OF CDNATotal RNA was extracted from 100 pl of freshblood using Catrimox-14 (Iowa BiotechnologyCorp), and cDNA was generated using ran-

dom hexamers and reverse transcriptase fromGibco/BRL. A two step PCR using nestedprimers was used to amplify the hMLH 1 tran-script in two overlapping fragments. FragmentA contained part of the 5' UTR and codons1-394 and fragment B contained codons 325to the termination codon (756), and part of the3' UTR.

IN VITRO SYNTHESISED PROTEIN ASSAY (IVSP)The above products were used as templates fora nested PCR with a 5' end modified primerthat included signals for transcription by T7polymerase and a eukaryotic consensus se-quence for in vitro coupled transcription-translation (IVSP), in a volume of 25 p1,20 using2 p1 of ['H] leucine (Amersham). The productsof translation were separated on SDS-12.5%polyacrylamide gels (Protogel, NationalDiagnostics/Atlanta), which were then driedand subjected to autoradiography.

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Figure 1 Five generations of the HNPCC kindred under study. Numbers below the symbols are patient identifiers;numbers below the symbols ofpatients preceded with d indicate age of death. Filled symbols refer to patients whose cancerwas verified histologically, shaded symbols to patients whose cancer was determinedfrom family history, and open symbolsto patients with no neoplasm detected. Letters represent the site of the tumour as follows: As, ascending colon; Bd, bile duct;Bo, bone; Br, breast; Ce, caecum; Co, colon; Csu, cancer site unknown; Dn, duodenum; Gli, glioblastoma; Ki, kidney; Ov,ovary; Pa, soft palate; Lu, lung; Si, sigmoid; Sk, skin; St, stomach; Tr, transverse colon. Subjects with a plus were identifiedas carriers of the nonsense mutation at codon 714 of the hMLHI gene, whereas subjects with a minus are not carriers.

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SEQUENCE ANALYSISSequence analysis was carried out either fromproducts of RT-PCR or from products of PCRfrom genomic DNA. When necessary theseproducts were purified using a QIAquick PCRpurification kit (Qiagen, Germany). Sequenc-ing was performed with SequiTherm Polymer-ase (Epicentre Technologies, Madison, WI),using primers labelled at their 5' end with T4polynucleotide kinase, following the conditionsspecified by the manufacturer. Alternatively,genomic sequencing was also performed aspreviously described.2' DNA templates frompeople in whom the mutation reported in thispaper was searched for were sequenced in bothdirections, and results were confirmed bysequencing at least two independent PCRproducts. Sequences of all primers used formicrosatellite amplification, IVSP, and se-quencing are available upon request from PH(e-mail: pierre.hutter@ichv.vsnet.ch).

ResultsThe kindred described here comes from analpine region in southern Switzerland and haspreviously attracted medical attention.22 Newinquiries allowed us to extend the pedigree, asshown in fig 1, and medical records now docu-ment a high incidence of colorectal cancer forthe last six generations.

In search for a possible HNPCC mutation,all exons and exon/intron boundaries of thehMSH2 and hMLH1 genes of patient III.25were sequenced from genomic DNA. Twopolymorphisms were observed, an alreadyreported A to G transition in hMSH2 at nucle-otide + 9 of the donor splice site of intron 10and a novel G to A transition in hMLH1 atnucleotide + 11 of the donor splice site ofintron 13. Apart from these polymorphisms aheterozygous G to A transition was detected inexon 19 of the hMLH1 gene. This single basesubstitution AGTGGAC->AGTAGAC (fig 2)represents a nonsense mutation that changescodon 714W into a termination codon, whichpredicts a loss of the last 42 amino acids of themature protein. Using the in vitro synthesisedprotein assay (IVSP) as a mutation screeningtest, the same mutation was found independ-ently in the germline of a HNPCC patientfrom the same locality, but not known at thattime to be related to the previous patient.Whereas no abnormal transcripts were de-tected in either gene, the IVSP assay fromhMLH1 showed, in addition to the expectedpolypeptide, a slightly smaller product (fig 3),suggesting the presence of a premature termi-nation codon near the COOH-terminal end ofthe encoded protein. Sequence analysis of thelast two exons of the cDNA and of genomicDNA showed the same point mutation as thatfound by genomic sequencing of the hMLH1gene in patient III.25. Further investigation ofthe family history of the above patient allowedidentification of a probable common ancestornine generations ago.

In order to determine whether we were deal-ing with two independent events, allele specificmarkers were used to compare the chromo-somal background of the two patients. For this

purpose we compared the size of two polymor-phic microsatellites, D3S1277 and D3S 161 1,which had been used for the original linkageanalysis of the hMLH1 gene. D3S1277 islocated close to this gene and D3S1611 lieswithin it.8 23 The results indicated that arecombination event had occurred betweenmarker D3S 1277 and the above nucleotide2182 but that the intragenic marker D3S 1611was identical, suggesting that it was inheritedfrom a common ancestor (fig 4). In order toshow that the mutation cosegregated with thedisease, the entire exon 19 was sequenced fromgenomic DNA of 41 other members of the kin-dred (fig 1). The same germline mutation wasfound in all seven patients who had presentedwith an early onset cancer and in four

A C G T

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-G- G -----*A-T

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Figure 2 Sequence analysis of a genomic PCR productfrom patient IV59, containing exon 19 of the hMLH1gene. The G to A transition (heterozygous state) indicatedby an arrow changes codon 714W (TGG) into atermination codon (TAG).

P C1 C2

Figure 3 IVSP assays on RT-PCR products of thehMLH1 genefrom the patient distantly related to thefamily shown in fig 1. In vitro transcription and translationofRT-PCR products were carried out as described inMaterial and methods and the resultant labelled proteinswere separated on an SDS-polyacrylamide gel. Theproducts shown represent the 3' halfgene products obtainedfrom the patient (P) andfrom two non-carriers of themutation (Cl, C2). In addition to a common band, aslightly shorter product (indicated with an arrow) wasdetected in the patient, corresponding to the expectedtruncated protein product. In our experiment the predictedsize of the normal product synthesised is 46.4 kDa, andthat of the truncated product is 41. 7 kDa.

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Complex genetic predisposition to cancer in an extended HNPCCfamily

asymptomatic subjects, of 25years of age (but see Discusspatient), whereas 29 healthyharbour the mutation. As tulHNPCC are expected to exhiinstability (referred to as thtype), we investigated DNA (

paraffin embedded primary clin the patient identified byshows that in this patient threerosatellite sequences investig;to be unstable.As shown in fig 1, a striking

of various cancers is observedof branches A, B, and C of thout typical manifestations of tdrome, apart from patient II.;patients from either branchtavailable, we analysed genoparaffin embedded materialIII.6, III. 18, and IV.3, who ha(onset ovary, stomach, andrespectively. Indeed we were amutation at codon 714 of tifrom genomic DNA extractembedded tissue of the patieniwas identified by the PTT. In Ewas sequenced from subje(IV.22, IV.23, IV.26, IV.31, V.though none of the latterhallmarks of HNPCC. IFhMLH 1 mutation reportedfound in the examined memb

o crsC,>

E

A

Figure 4 Genotype analysis from PCR products generated by amplifipolymorphic markers D3S1611 (A) and D3S1277 (B), situated withhMLHI gene, respectively. Numbers refer to people in fig 1 and P referdistantly related to the family shown in fig 1, whose mutation was detearrows indicate the common alleles shared by mutation carriers for thewhich is not shared (in B) by patient Pfor marker D3S1277. This allfour other mutation carriers in the family (data not shown).

T N T N

Figure Detection ofgenetic instability ofpaired normal (NV) and coDNA from the patient whose mutation was identified by the PTT. Dinipatterns are shown for three loci, D6S285, D12S79, and GABASS, re

locus, tumour DNA exhibits an abnormal pattern, indicating a somatitresult of microsatellite expansion or deletion.

>, 27, 46, and 51ion for the lattersubjects did notmours related to

branches, suggesting that the mutation did notspread to branches A, B, and probably C of thekindred under study.

ibit microsatellite Discussione RER+ pheno- The identification of the molecular defectextracted from a underlying the predisposition to HNPCC inolorectal tumour this family allowed the identification of addi-the PTT. Fig 5 tional branches of the kindred by virtue of thee out of four mic- presence of the identified mutation in patientsated were found living in the same region, and thus to extend

the pedigree further. On the basis of death cer-ly high incidence tificates and the identification of probable car-among members riers segregating with the disease in thisLe pedigree, with- kindred, the MMR mutation could be tracedhe HNPCC syn- back at least nine generations, to the early 18th5. Since no living century.es A or B were Our data have established that the identifiedmic DNA from mutation in the hMLH1 gene is responsible forI from patients the predisposition to HNPCC in several mem-d developed early bers of this family, but that some cancerbreast cancer, patients who are not carriers probably harbour

able to detect the at least one other genetic defect predisposingie hMLH 1 gene to a less restricted range of cancers. The muta-ed from paraffin tion identified in the hMLH1 gene introducest whose mutation a premature stop codon which is expected toaddition, exon 19 lead to a protein lacking the last 42 amino acidscts III.12, IV.1, of its mature form, 19 of which (45.2%) are1, and V.7, even conserved between yeast and man.7 8 In fact,manifested any the last 13 amino acids of yeast and humanmportantly, the proteins are identical. We have confirmed seg-above was not regation of the mutation with the disease in

ers of these three seven patients, whereas people without themutation are all asymptomatic with respect to

CY p an Lynch syndrome. The penetrance of the muta-- tinon appears to be high, since most ascertained

carriers developed a cancer early in life, exceptIII.30 who presented symptoms of a gastric

-~ cancer at the age of 59. Mutation carrier IV.52is cancer free at the age of 51 but has been

B regularly monitored, with ablation of severalintestinal polyps. Although the same mutationhas not been observed in other HNPCC fami-

ication of lies, mutations affecting the same region of thetin and very near the COOH-terminal end of the hMLH1 proteinrs to the patient have been reported in three HNPCC families.cted by the PYT] Thetwo markers, one of The first mutation is a 4 nt deletion beginninglele was also found in at the first position of codon 727, which

produces a frameshift with a new stop codon166 nucleotides downstream, within the 3'

T N untranslated region.8 The second mutation,found by the same authors, is a 4 nt insertionbetween codons 755 and 756, and the thirdmutation is an A to T transversion at + 1 ofthe IVS 18 splice acceptor site.2' AlthoughHNPCC has been known for some time to befrequently associated with an increased risk ofcancer of the uterus, ovary, stomach, and evensmall intestine, skin, and larynx,2 our data indi-cated that the clinical manifestations observedin branches A, B, and C are unlikely to beassociated with the mutation identified in thehMLH1 gene.

Therefore, the presence of at least two muta-tions predisposing to cancer in this kindredillustrates the potential difficulties that can be

lorectal tumour (T) encountered when evaluating cancer riskucleotide repeat among non-carriers of a first mutation incrimi-rspectively. At eachgain of alleles as a nated in cancer predisposition in a small

HNPCC family. Indeed, the additional germ-

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line mutation(s) which has to be postulated toaccount for the high incidence of cancers inbranches A, B, and C may also have beeninherited by some patients from the branchesof the kindred with HNPCC. This would makethe prediction of the cancer risk for non-carriers of the hMLH1 mutation in the wholekindred difficult, as long as additional muta-tions are not identified. As recently suggestedby other workers,"4 one could speculate that thehMLH1 mutation which we identified acts asan allele whose dominant negative or haploin-sufficient effect might lead to a reduced level ofMMR in non-neoplastic cells, thus favouringmutations in other genes involved in thecancers observed in branches A, B, and C. Thiswould be consistent with the observation of anapparent phenomenon of anticipation in thisfamily as well as with the predominance ofmale cancers." Therefore, screening of morepatients for germline mutations in TP53 and inall known MMR genes, starting from thehPMS2 gene whose product is known to forma heterodimeric complex with the proteinencoded by hMLH1"3 25 26 will be performed.In addition, alterations in other genes, such asthe possible target gene encoding receptor II ofthe TGF-P,27 will be searched for in some ofthe primary tumours. The characterisation ofthe hMLH 1 mutation which very strongly pre-disposes to HNPCC in branches D and Fallows presymptomatic diagnosis in the young-est and future members of the family. In addi-tion to the advantage of sparing the anxiety anddiscomfort associated with colonoscopies fornon-carriers of the mutation, enhanced sur-veillance of carriers has recently been shownboth to decrease the incidence of colorectalcancer and to prevent its related deaths.'8

This work was supported by grants from the followinginstitutions to P Hutter: Recherche Suisse Contre le Cancer,Ligue Genevoise Contre le Cancer, Fondation E Boninchi, andFondation pour la Lutte Contre le Cancer. We are grateful toDrs R Berclaz and J-M Cereda for providing patients. We thankA Schonborner and S Fournier for technical help. The BaselInstitute for Immunology is supported by F Hoffmann-LaRoche & Co Ltd.

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