Abstract Germline mutations within mismatch repairgenes, such as hMSH2, hMLH1, and hMSH6, have beenshown to be the hallmark of the hereditary nonpolyposiscolorectal cancer (HNPCC) syndrome. The spectrum oftumors associated with mismatch repair gene defects andthe possible relationship between genotype and pheno-type are still unclear. Therefore, the spectrum of tumorsand the possible genotype–phenotype relationship arestill under discussion. Here, we report on a family with anew germline mutation in the hMSH2 gene with a 2-bpdeletion at codons 232 and 233 leading to a frame shiftand a stop at codon 254. Accordingly, immunohisto-chemistry revealed loss of hMSH2 expression in colo-rectal carcinomas of three affected family members. Inthis one family, there was a high penetrance. Interesting-ly, mutational screening of the family revealed a highpenetrance of the mutation affecting four of five testedpeople at risk, with a high mortality rate and a trend to-ward lower age of onset in subsequent generations. Fi-nally, a metachronous breast cancer in one patient turnedout to be a tumor unrelated to microsatellite instabilityphenocopy, i.e., a sporadic tumor unrelated to HNPCCthat expressed the hMSH2 gene and did not show anymicrosatellite instability.

Keywords Hereditary nonpolyposis colorectal cancer (HNPCC) · Genotype–phenotype correlation ·Breast cancer

Introduction

Hereditary nonpolyposis colorectal cancer syndrome(HNPCC) is currently one of the most common forms ofhereditary colorectal cancers and accounts for about2–13% of the total colorectal cancer burden [13, 19].Since there are no single clinical features specific forHNPCC, the diagnosis is based on family history (Ams-terdam or Bethesda criteria) [21] and is confirmed by thedetection of a germline mutation in one of the responsi-ble mismatch repair (MMR) genes. The identification ofthe human homologues of the yeast MMR genes,hMSH2, hMLH1, hMSH6, hPMS1, and hPMS2, offeredthe prospect of genetic screening, leading to an extensivesearch for mutations in HNPCC families [4, 6, 16]. Themajority of the defects have been detected in hMSH2(50%) and hMLH1 (30%) genes, with the other genesbeing only seldom affected [24].

Until now, several hundreds of germline mutationshave been described in hMSH2 and hMLH1 genes. Incontrast to FAP (familiar polyposis), however, the rela-tionship between the type of mutation and the severity ofthe cancer predisposition [15, 17], its penetrance [18],and tumor spectrum is still under debate [11, 12, 13].Here, we describe for the first time a HNPCC familypresenting with some extraordinary, unusual features.

First, germline mutation analysis revealed a new, pre-viously undescribed mutation site in the hMSH2 gene.Second, we could not detect a mutational loss of hMSH2expression in a case of metachronous breast cancer in anaffected family member that may exclude breast cancerfrom the HNPCC tumor spectrum. Finally, a high inci-dence of the mutation was found with a tendency to earlycancer onset in subsequent generations, suggestive of an-ticipation.

Materials and methods

Identification and molecular analysis of the family were done inaccordance with the guidelines of the German cooperative multi-

A. Müller (✉ ) · S. Bolander · H. BeckerDepartment of General Surgery and Human Genetics, University of Göttingen, Robert-Koch-Strasse 40, 37075 Göttingen, Germanye-mail: dramueller@aol.comTel.: +49-551-396132, Fax: +49-551-396106

K. Beyser · J. RüschhoffDepartment of Pathology, Klinikum Kassel, Kassel, Germany

H. ArpsDepartment of Pathology, Klinikum Fulda, Germany

Virchows Arch (2001) 439:191–195DOI 10.1007/s004280100413

O R I G I N A L A RT I C L E

A. Müller · K. Beyser · H. Arps · S. BolanderH. Becker · J. Rüschhoff

Genotype and phenotype of a new 2-bp deletion of hMSH2 at codon 233

Received: 21 September 2000 / Accepted: 5 January 2001 / Published online: 22 March 2001© Springer-Verlag 2001

center study funded by the “Deutsche Krebsgessellschaft” GermanCancer Aid. After taking the family history and obtaining in-formed consent, paraffin-embedded tumor specimens of the threepatients were tested for microsatellite instability (MSI) and MMR.Thereafter, blood was taken for mutational analysis.

Screening for MSI

To carry out the MSI analysis first, DNA was extracted from aparaffin-embedded tumor and corresponding normal tissue. Adja-cent sections were mounted on glass slides and stained with hema-toxylin and eosin for histological examination to ensure that theDNA extracted originated from tumor and normal cells only. Theextracted DNA was then amplified using the polymerase chain re-action (PCR). In accordance with the Bethesda recommendations[3], five microsatellite loci were used to detect MSI for which wecould recently demonstrate the specific diagnostic value [5]. ThePCR products were run on an ABI 310 using fragment analysissoftware. Extra peaks, indicating the presence of MSI in tumorDNA compared with the corresponding normal DNA, were usedfor MSI detection. Shifts at more than one microsatellite locuswere indicative of a tumor with a high rate of MSI.

MMR gene expression

To determine the mismatch protein status, the tumors were sub-jected to immunohistochemical staining using the streptavidin-bi-otin peroxidase complex method with 3,5′ diaminobenzidine asthe chromogen. Briefly, after deparaffinization, the sections werepre-treated by microwaving, and endogenous peroxidase activitywas quenched by incubation with 3% of H2O2 followed by rinsingin phosphate-buffered saline (PBS). Non-specific binding wasblocked by incubation with goat serum for 30 min. PrimaryhMSH2 (clone FE11 0.5 µg/ml; Oncogene Research Products,Cambridge, Mass.), hMLH1 (clone G168–728; 1 µg/ml; Pharmin-gen, San Diego, Calif.), or hMSH6 (AB-1, 0.5°mg/ml OncogeneResearch Products, Cambridge, Mass) antibody was then incubat-ed overnight at 4°C. The next day, the sections were washed withPBS, followed with the incubation of the secondary biotinylatedantibody. After rinsing with PBS, the sections were incubated withstreptavidin-conjugated horseradish peroxidase and then incubatedwith the chromogen 3-amino-5-ethylcarbazole for detection. Acounterstaining was done with hematoxylin.

Mutation analysis

In the autosomal sequencing, we used a DNA primer label withfour different fluorescent dyes. Each fluorescent primer hybridizedto the template DNA and underwent the same synthesis reactionsas in the case of manual sequencing. After elongation of the prim-er, samples containing the four kinds of newly synthesized frag-ments were cooled and subjected to direct laser beam analysis us-ing an ABI sequencer.

Results

Family history

The pedigree disclosed a clear hereditary cancer predis-position with six affected family members spanningover four generations (Fig. 1). There were ten people atrisk, with six being less than 10 years old. Interestingly,a tendency toward lower age of cancer manifestationwas evident throughout the different generations. Theoldest patient died of cancer with unknown primary atthe age of 51 years, and the youngest died at the age of33 years. One affected member is still alive. The genet-ic and molecular analysis was started with this individ-ual. This patient suffered from two synchronous colo-rectal adenocarcinomas located in the colon ascendens(pT3, No, and Mo) and the sigmoid colon (pT1, No,and Mo).

Immunohistology and MSI testing

Tumor specimens of three patients were available frompathological files (provided by H.A.), comprising threecolorectal carcinomas and one breast cancer specimen(of the 48, 49, and 33-year-old patients). All colorectalcancers showed loss of hMSH2 expression (Fig. 2a, b),which was not the case in the breast cancer specimentested in one patient (Fig. 2c). The other two MMRgenes (hMLH1, hMSH6) were highly expressed in allfour tumors. Accordingly, only those carcinomas pre-

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Fig. 1 Hereditary nonpolyposis colorectal cancer (HNPCC) fami-ly with six affected members, of which three were tested for mi-crosatellite instability and mismatch repair gene expression. Muta-tional analysis was done in one patient and five people at risk, ofwhich four exhibited the hMSH2 mutation

Table 1 Microsatellite instability (MSI) results in four carcinomas of three family members

Patient/age Tumor BAT 26 BAT25 APC/D5S346 D2S123 Mfd15/D17S250(years) localization (2p) (4q12) (D5S346) (2p16) (17q11.2)

1/49 Colon + + – + –1/49 Breast – – – – –2/37 Colon + + + + –3/33 Colon – + + – +

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Fig. 2 Mismatch repair gene expression in normal (upper half)and tumor tissue (lower half) in the colonic carcinoma of the pa-tient with metachronous breast cancer. The carcinomatous tissueshows no hMSH2 expression (a) but an intense nuclear stainingwith anti-hMLH1 (b). c Specific positive nuclear staining with an-ti-hMSH2 in the breast cancer specimen

senting a hMSH2 loss displayed instabilities in at leastfour of the five tested microsatellites, indicating theMSI-H type (Fig. 3a).

Mutation analysis

Mutation analysis revealed a new, previously unknown,mutation in the hMSH2 gene which has not yet, to thebest of our knowledge, been reported in the literature.The mutation consists of a 2-bp deletion in a small poly(T)4 run at codons 232 and 233. This mutation led to aframe shift with a stop at codon 254, explaining the lossof hMSH2 expression at the immunohistochemical level(Fig. 3b). Until now, there are no data available regard-ing the penetrance of a mutation in such a family. In ourcase, we found the mutation in four of five asymptoticfamily members. There are five family members current-ly under the age of 10 years, who have not yet been test-ed. At the time of genetic counseling, none of theseshowed clinical signs of having a tumor.

Discussion

Colorectal cancer is one of the common neoplasms inhumans and perhaps the most frequent form of heredi-tary neoplasia. HNPCC is the most common form andaccounts for approximately 2–13% of the colorectal can-cers annually [1, 13]. The syndrome is clinically charac-terized with an early age of onset, a proclivity to theproximal colon, a frequency of mucinous and signet ringcells, and an increase in metachronous and synchronouscolon cancers [7, 10]. These features compose theHNPCC I syndrome. Another subset of HNPCC is desig-nated as type II. Families with type-II HNPCC have allof the features of type I but also harbor extracolonic can-cers [14]. The most common extracolonic tumors are en-dometrial and gastric carcinomas.

The mode of transmission is compatible with autoso-mal dominant inheritance, but the location and character-istics of the putative susceptibility gene are unknown. Astudy by Ponz de Leon et al. [19] demonstrated in a clas-sical segregation analysis of 605 families that cancer riskin families with one affected parent equals that in fami-lies where both parents are healthy. They formed a mod-el of dominant transmission through a major gene withgreatly reduced penetrance in heterozygotes. Herein, weshow a family with a high penetration mutation in thehMSH2 MMR gene that has, to the best of our knowl-edge, not yet been reported. This mutation led to colo-rectal cancers in most affected family members with adouble synchronous cancer in one patient and an unusu-ally high mortality rate, which contrast the recent find-ings of Gryfe et al. [8]. In the parental generation, onlyone member in each family was affected. Therefore, weexpected to find the mutation in the asymptotic familymembers in less than 50% of cases.

To our surprise, we found the germline mutation inthe hMSH2 gene in four of five members tested. Thechildren of the mutation carriers have not been testedyet, but one may expect that they also will have a highrisk of carrying the mutation. All carriers of the germlinemutation are admitted to a pre-screening program and, atpresent, they are asymptomatic. These findings may sug-gest that there are differences in the locus of mutationand the frequency of segregation, resulting in high andlow penetrance mutations. More studies are, however,needed to evaluate the penetration of different mutations.

Interestingly, there was one case of breast cancer inthis family that did not show any MSI (whereas all of thecolorectal cancers showed MSI; Table 1) and no loss ofMMR gene expression [22, 23]. Currently, the inclusionof breast cancer as an integral tumor of the HNPCC syn-

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Fig 3 a Microsatellite analysis with mononucleotide repeat mark-er (BAT 25) shows distinct instability in the colon carcinoma (T1)but not in the metachronous breast tumor (T2). b Heterozygousmutation at codon 232 and codon 233 in the hMSH2 gene. Themutation consists of a 2-bp deletion in a small poly(t)4 run. Lowerhalf wild-type control

drome is controversial. Most studies [2] indicate thatMMR errors are uncommon in human breast cancer and,therefore, breast cancer should not be included as part ofthe HNPCC syndrome. In contrast, a study by Risingeret al. [20] showed in a HNPCC family a case of breastcancer that exhibited widespread MSI and the expressionof the mutant gene. Our findings clearly indicate thatbreast cancer appears not to be a part of the HNPCC tu-mor spectrum, although it should be mentioned that thehistologic tumor type showed some peculiar featureswith an undifferentiated pseudosarcomatous portion be-neath a usual ductal-type cancer.

In summary, we found a new mutation in the hMSH2gene with a high mutation penetration and poor progno-sis in affected patients. Because the breast tumor did notshow the mutation or any MSI, we conclude that the par-ticular case of breast cancer in the patient of this familywas not related to HNPCC.

Acknowledgement We thank R. Fishel (Kimmel Cancer Center,Thomas Jefferson University, Philadelphia, PA) for revising themanuscript. Supported by the Deutsche Krebshilfe (70–2401-Rü 1)n Krebsgesellschaft.

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