A missense germline mutation in exon 7 of the MSH2 gene in a HNPCC family from center-italy

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1 DOI /s z ORIGINAL PAPER A missense germline mutation in exon 7 of the MSH2 gene in a HNPCC family from center-italy Francesca Bianchi Æ Eva Galizia Æ Emilio Porfiri Æ Laura Belvederesi Æ Romina Catalani Æ Cristian Loretelli Æ Raffaella Bracci Æ Italo Bearzi Æ Chiara Turchi Æ Alessandra Viel Æ Riccardo Cellerino Received: 26 April 2006 / Accepted: 17 October 2006 Ó Springer Science+Business Media B.V Abstract Introduction Hereditary Non-Polyposis Colorectal Cancer (HNPCC) is an autosomal dominant inherited disease predisposing to the development of colorectal cancers and several other malignancies (endometrium, ovaries, stomach, small bowel, hepatobiliary and urinary tract). HNPCC is caused by germline mutations in any of the MisMatch Repair (MMR) genes. Mutations in MLH1 and MSH2 account for almost 90% of all identified ones. About 15% of mutations identified in MSH2 are missense ones. Patients and methods We studied one family, fulfilling Amsterdam II criteria, referred to our Center for genetic counselling. The proband, and some of her relatives, have been investigated for microsatellite instability (MSI), immunohistochemical MMR protein staining and by direct sequencing and Multiplex Ligation-dependent Probe Amplification (MLPA). Results All patients carried the same novel MSH2 germline missense mutation (R359S) in exon 7, which F. Bianchi E. Galizia L. Belvederesi R. Catalani C. Loretelli R. Bracci R. Cellerino (&) Istituto di Medicina Clinica e Biotecnologie Applicate-Oncologia Medica, Università Politecnica delle Marche, via Tronto, Ancona, Italy r.cellerino@univpm.it F. Bianchi E. Galizia L. Belvederesi R. Catalani C. Loretelli R. Bracci R. Cellerino Centro Regionale di Alta Specializzazione in Genetica Oncologica, Facoltà di Medicina e Chirurgia, Università Politecnica delle Marche, Ancona, Italy E. Porfiri Cancer Research-UK Institute for Cancer Studies, University of Birmingham, Birmingham, UK determines the substitution of an Arginine, which is a basic amino acid, with a polar Serine residue (R359S). The mutation was associated with lack of expression of MSH2 protein and high microsatellite instability in tumour tissues. The same mutation has been detected in one healthy relative. Conclusions The mutation here reported shows a high correlation with phenotype. The mutation is located in an evolutionary conserved domain. Taken together, our findings suggest evidence that the amino acid substitution can be interpreted as pathogenetic. Keywords Missense mutations MSH2 HNPCC Endometrial cancer Colon cancer Abbreviations HNPCC Hereditary Non-Polyposis Colorectal Cancer MMR MisMatch Repair MSI Microsatellite instability IHC Immunohistochemistry R. Bracci Clinica di Oncologia Medica, Università Politecnica delle Marche, Ancona, Italy I. Bearzi Anatomia ed Istologia Patologica, Università Politecnica delle Marche, Ancona, Italy C. Turchi Dipartimento di Neuroscienze-Medicina Legale, Università Politecnica delle Marche, Ancona, Italy A. Viel Oncologia Sperimentale 1, Dipartimento di Ricerca Preclinica ed Epidemiologica, Centro Riferimento Oncologico, IRCCS, Aviano, PN 33081, Italy

2 InSiGHT MLPA Introduction International Society for Gastrointestinal Hereditary Tumours Multiplex Ligation-dependent Probe Amplification Hereditary Non-Polyposis Colorectal Cancer (HNPCC) is an autosomal dominant inherited disease predisposing to the development of colorectal cancers (Lynch syndrome 1) and several other malignancies, such as cancer of endometrium, ovaries, stomach, small bowel, hepatobiliary and urinary tract (Lynch syndrome 2) [1, 2]. Patients affected by HNPCC can be identified on the basis of clinical criteria such as age at onset of neoplasms and characteristics of the family history. Amsterdam criteria were the first guidelines drawn to identify families prone to develop HNPCC [3]. Since Amsterdam criteria were judged too stringent and with inadequate sensitivity, Bethesda guidelines were advised aiming to identify patients who should undergo genetic testing [4, 5]. More recently Bethesda criteria were revised; they are now considered the most accurate clinical criteria for the identification of patients at risk for HNPCC [6, 7]. HNPCC is caused by germline mutations in any of the MisMatch Repair (MMR) genes: MLH1, MSH2, MSH6, MSH3 and PMS2 genes. Mutations in MLH1 and MSH2 account for almost 90% of all identified ones. Failure of the MMR system results in the inability to repair errors that can occur during DNA replication. One of the consequences of this condition is the so called microsatellite instability (MSI), which is the hallmark of HNPCC tumours. Assays for MSI, together with family history, can be used to determine whether a patient could be a carrier of a MMR mutation and if he is worth undergoing genetic tests [8]. Immunohistochemistry (IHC) for the expression of MLH1 and MSH2 proteins in tumour tissue can be used as a complementary screening method, providing information for the presence or absence of the protein tested and, in this way, for the specific gene defect [9]. Microsatellite instability and protein immunostaining are equivalent strategies in terms of cost-effectiveness and, when one of these methods is performed in patients selected according to the revised Bethesda guidelines, they are very useful in identifying patients with HNPCC [7]. In order to diagnose MMR defects the key procedure is the sequencing of the MMR genes looking for mutations. MLH1 and MSH2 mutations are spread in all regions of these genes, without hot spots. Mutations detection strategies must therefore cover the entirety of these genes [10, 11]. Nonsense, splice-site and truncating mutations are considered pathogenetic, whereas missense mutations (which can lead to the substitution of a single amino acid in the proteic sequence) are not a priori diseasecausing and the pathogenetic significance of these amino acid substitutions is often difficult to evaluate. About 30% of mutations identified in MLH1 are missense ones, while MSH2 harbours about 15% of such mutations [1, 12]. Functional impairment of proteins, resulting from these gene alterations, could be the critical factor in deciding on the pathogenicity of a given mutation. Unfortunately, as stated by previous Authors [13], the lack of validation of a functional assay for MSH2 mutations, makes it necessary to use other criteria to predict the pathogenicity of MSH2 missense mutations. Generally accepted criteria are (1) the missense mutation does not occur in general population; (2) the mutation co-segregate with the phenotype in the family; (3) the alteration leads to a non-conservative amino acid change; (4) the codon involved is evolutionary conserved [14]. Here we report a MSH2 missense mutation found in a family fulfilling Amsterdam II criteria. The same mutation was found in another family from the same geographic area and is not reported elsewhere. Patients and methods Family characteristics The proband (D.A.W) was a 70-year-old female (II-1), whose personal and family history fulfilled the Amsterdam II criteria for HNPCC. She was diagnosed with an adenocarcinoma of the right colon when she was 59 and underwent right emicolectomy (pt2n0m0, G2). Subsequently the patient developed an adenocarcinoma of the rectum (pt1n0m0) and a carcinoma of the renal pelvis (pt3n1m0) when she was 70 and was referred to our Center. Detailed patient family history was collected: pedigree was drawn until fourth generation. The recorded data for all relatives included sex, tumour site, age at diagnosis, together with current age or age of death (Figure 1). Patient s family showed a typical HNPCC pedigree (Lynch 2 syndrome). Patient s mother (I-1) had an endometrial cancer when she was 62 and died for an

3 Fig. 1 Family A pedigree: The arrow indicate the proband. The type of tumour, as well as the age of onset, are described. Cancer type: crc, colorectal; urc, urothelial; end, endometrial. Red circles indicate carrier status I II 1 dec 80 y end 62 y urc 80 y 2 dec 65 y IV III y crc 59 y crc 70 y urc 70 y 60 y end 37 y crc 44 y urc 49 y crc 51 y crc 55 y dec 65 y crc 50 y crc 65 y 68 y crc 62 y urothelial cancer at the age of 80; this subject was adopted. Patient s sister (II-2) developed five HNPCC related cancers: endometrial cancer at the age of 37, metachronous colorectal cancers when she was 44, 51 and 55, and ureter cancer at 49. Her brother (II-3) developed two metachronous colorectal cancers when he was 50 and 65 and died at the age of 65. Another proband s brother (II-4) developed a colorectal cancer at the age of 62. Tumour specimens of the proband and of all the relatives mentioned above were available at the Dipartimento di Anatomia Patologica of Ancona and were examined by a unique pathologist (I.B.). Microsatellite instability analysis This analysis was possible for subjects I-1, II-1, II-2, II-3 and II-4. Genomic DNA was isolated from tumour samples and corresponding normal tissues as previously described [15]. Tumour samples were estimated to contain at least 80% neoplastic cells by light microscopy. Tumours were examined for microsatellite instability (MSI) using the 5-markers Bethesda panel (two mononucleotide repeats BAT25 and BAT26 and three dinucleotide repeats D2S, D5S346 and D17S250) recommended by the National Cancer Institute Workshop on MSI for Cancer Detection and Familial Predisposition. Tumours were classified as highly unstable (MSI-H) if at least 40% of the markers showed instability [8]. Oligonucleotide primers were fluorescently labeled (5 -VIC, 5 -NED, 5-6-FAM, 5-6-FAM, 5 -PET), respectively, and the PCR products were evaluated with an ABI Prism 310 Genetic Analyzer based on automated capillary electrophoresis and automated sizing of the alleles by GeneScan 3.7 (Applied Biosystems, Foster City, CA). Expression of MLH1, MSH2 and MSH6 proteins in tumour tissues Tumour slides were available for patients I-1, II-1, II-2, II-3 and II-4. Expression of the MLH1, MSH2 and MSH6 proteins was detected by immunohistochemistry (IHC) on 2-lm sections of formalin-fixed, paraffinembedded tissues, following antigen retrieval as previously described [16]. Primary antibodies used for immunohistochemistry were: anti-mlh1 protein: clone G , PharMingen, San Diego, CA, 1:50 dilution; anti-msh2: clone FE11, Oncogene Research Products, Cambridge, MA, 1:100 dilution; anti-msh6: clone H-141: sc-10798; Santa Cruz Biotechnology, Inc, Santa Cruz, CA 1:250 dilution. Mutation analysis of the MLH1, MSH2 and MSH6 genes Mutation analysis was performed on genomic DNA isolated from peripheral blood lymphocytes according to standard procedures. The 19 MLH1 exons, the 16 MSH2 exons and the 10 exons of MSH6, including flanking intronic regions, were individually amplified and directly sequenced using Applied Biosystems ABI PRISM 310 automated sequencer and the Big Dye terminator cycle sequencing Ready Reaction-Kit (PE Applied Biosystems, Weiterstadt, Germany) as described (primer sequences are available from the corresponding author upon request) [15]. The identification of DNA sequence variants have been confirmed by new PCR and by new sequencing analysis of both DNA strands. Our results were compared with MLH1, MSH2 and MSH6 normal sequence (respectively available in: nucleotide&val=463988;

4 entrez/viewer.fcgi?db=nucleotide&val=432997; Mutation analysis was first performed in patients II-1 (proband) II-2 and II-4 and in four healthy relatives: III- 1, III-2, III-3 and III-4. Informed consent was obtained from each subject. In order to increase the number of studied relatives, we asked to our Ethic Committee if, with the consent of the proband, it was possible to search for the MSH2 missense mutation in two other relatives died of HNPCC related cancers some years before. Ethic Committee and the proband gave their approval. Thus, we performed genetic test on tumour tissues of two other members of the family (subject I-1 and II-3) and we found the same MSH2 missense mutation we described. To exclude that the mutations found in our patients could represent a polymorphysm, each mutation was searched for in the germline of a reference population panel of 100 healthy volunteers and in 110 colorectal cancer not related patients and we compared each mutation with HNPCC Database (the International Society for Gastrointestinal Hereditary Tumours) (InSiGHT) Multiplex Ligation-dependent Probe Amplification (MLPA) analysis Patients studied for MLH1 and MSH2 sequence were also studied with Multiplex Ligation-dependent Probe Amplification (MLPA) analysis. MLPA was performed with 200 ng of normal and tumour DNAs using the MRC-Holland (Amsterdam, Holland) HNPCC probe kit, according to the supplier s protocol. One microliter of the FAM-labelled PCR product was then mixed with 1 (l of fluorescent GeneScan 500 LIZ size standard (Applera) in 15 (l of HiDi Formamide, run on an automatic ABI3100 DNA analyzer, and evaluated with GeneScan sofware (Applera). The electropherograms showed specific peaks corresponding to each exon of MSH2 and MLH1, as well as additional peaks corresponding to control sequences mapping on different chromosomes. A 40 55% decrease of the area of an MSH2 or MLH1 exon peak compared to the wild-type control samples was considered as indicative of a heterozygous deletion of that exon. Results Microsatellite instability analysis Proband (II-1) showed high microsatellite instability (MSI-H): four out the five loci studied (Bethesda panel) resulted altered. Similar results were observed in four affected relatives: patient II-2 showed microsatellite instability in three out of the five markers (MSI-H), patient I-1, II-3 and II-4 exhibited microsatellite instability in two out of the five markers (MSI-H). Expression of MLH1, MSH2 and MSH6 proteins in tumour tissues Study of the proteins expression was conducted in tumour specimens of patients I-1, II-1 (proband), II-2, II-3 and II-4 and, in all cases, normal staining for MLH1 and the lack of expression of MSH2 and MSH6 was observed. Mutation analysis of the MLH1, MSH2 and MSH6 genes The sequence analysis of the entire coding region of MLH1 and MSH6 did not show any mutation. A germline missense mutation was found in exon 7 of MSH2 in patients I-1, II-1, II-2, II-3 and II-4: at nucleotide 1077 an adenine was replaced by a timine (A1077T), leading to the substitution of an Arginine, which is a basic amino acid, with a polar Serine residue (R359S). The mutation here reported is localized in a conserved domain ( SIFT. html polyphen.cgi). This mutation is not reported in ICG-HNPCC/ INSiGHT mutation database ( and was not found in 100 healthy individuals we examined nor in 110 colorectal cancer not related patients. MLPA analysis did not show any evidence of exonic large deletions in all the subject analysed. The same missense MSH2 germline mutation was detected in one of the four healthy relatives analysed: III-4. This subject is a 44-year-old female who was not affected by any malignancy at the time of study, however she is now under investigation for the finding of macrohematuria and high levels of CA125. Interestingly, this genetic alteration was formerly detected and described by our group in a patient (G.M.) who developed colon cancer at the age of 30; he is apparently not related with the family we have here described [15]. However, at that time, GeneScan was not available and microsatellite instability was investigated with other, less reliable, procedures. IHC showed lack of MSH2 protein expression. Recently, we have newly performed MSI analysis by Genescan and we found the presence of MSI-H (60%). G.M. s family shows typical HNPCC features: two other members are

5 Table 1 Results summary (MSI: Microsatellite instability; IHC: Immunohistochemistry; wt: wild type) Subjects Status MSI altered loci (Bethesda panel) IHC MSH2 MLH1 MSH6 MSH2 MUTATION II-1 Alive BAT25, BAT26, D5S346, D17S250 + exon 7 A1077T fi R359S II-2 Alive BAT25, D2S, D17S250 + exon 7 A1077T fi R359S II-4 Alive BAT25, BAT26 + exon 7 A1077T fi R359S II-3 Dead BAT25, BAT26 + exon 7 A1077T fi R359S I-1 Dead BAT25, BAT26 + exon 7 A1077T fi R359S III-1 Alive Healthy Healthy wt III-2 Alive Healthy Healthy wt III-3 Alive Healthy Healthy wt III-4 Alive Healthy Healthy exon 7 A1077T fi R359S affected by gastric cancer. More interestingly, both the families come from the same small area of the Marche Region (The Montefeltro). We were interested in investigating the possible relationship between the two families analysed in this study. The kinship analysis was performed studying mtdna and Y-chromosome polymorphisms. Genetic kinship analysis was performed in three members of the proband family and in G.M. subject. However, the genetic analysis performed only on these four individuals did not show the relationship between the two families. Anyway, the lack of other family members and the fact that subject I-1 of this report was adopted, has hindered the extension of kinship testing and, at present, it is impossible to exclude or confirm any other possible relationship (recent or in the near past) between the two families. (Table 1) Discussion HNPCC represents about 5% of all colorectal cancers and it is the most common hereditary colon cancer syndrome; it can be identified on the basis of age at onset and the characteristics of the family history [17]. Other diagnostic tools have been suggested in order to identify subjects eligible for genetic testing, such as the presence of MSI and the lack of expression of MMR proteins in tumour tissue [18]. MSI-H can be found in more than 90% of HNPCC colorectal tumours while IHC has a sensitivity of about 90% in predicting mutation status of the MLH1, MSH2 or MSH6 genes in colorectal carcinomas [7, 18]. Here we report a MSH2 germline missense mutation caused by the substitution of an adenine with a timine (1077 A fi T) in exon 7 of MSH2 gene. This alteration produces an Arginine to Serine change in position 359 of the primary sequence (R359S). Missense mutations make the interpretation of genotypic data difficult; immunohistochemical analysis of MMR proteins in the tumour can provide clues as to which MMR gene is involved in tumour pathogenesis. Moreover immunostaining may help to solve the status of MSH2 germline variants of uncertain significance because somatic inactivation of MSH2 is a rare event in sporadic microsatellite unstable tumours [7]. The lack of validation of a functional assay for MSH2 mutations, makes it necessary to use the specific criteria previously described [13]. The family we studied fulfils Amsterdam II criteria; all affected family members, in which we could perform genetic tests, carried the same MSH2 mutation (R359S) in exon 7. The mutation here reported is contained in a conserved region and was not found in 60 healthy individuals; moreover it shows a high correlation with phenotype. The A to T transition leads to an amino acid change in which an Arginine, which is a basic amino acid, is substituted by a polar Serine residue. The change is in an evolutionary conserved domain and may indicate the functional relevance of this amino acid for the structure or function of the protein. All family members affected by HNPCC related tumours showed MSI-H and the lack of expression of MSH2 protein in tumour specimens and harboured the same MSH2 germline mutation. Taken together, these findings give strong evidence that this substitution can be interpreted as a disease-causing mutation. The same mutation was previously detected and described [15] in a young patient from the same geographic area. We studied the possible links between the two families, but the insufficient number of relatives and the fact that the mother of the patient here described was adopted, stopped any possibility to further study the existence of links between the two families, and the possible importance of this mutation remains less than speculative. Acknowledgment The authors acknowledge Prof. Tagliabracci A., M.D. for helpful discussion and support.

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