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Dec 20, 2008 - Abstract Hereditary non-polyposis colorectal cancer is a cancer predisposition syndrome known to be caused by heterozygous germline ...
Familial Cancer (2009) 8:187–194 DOI 10.1007/s10689-008-9227-3

Homozygosity of MSH2 c.1906G?C germline mutation is associated with childhood colon cancer, astrocytoma and signs of Neurofibromatosis type I Helen Toledano Æ Yael Goldberg Æ Inbal Kedar-Barnes Æ Hagit Baris Æ Rinnat M. Porat Æ Chen Shochat Æ Dani Bercovich Æ Eli Pikarsky Æ Israela Lerer Æ Isaac Yaniv Æ Dvorah Abeliovich Æ Tamar Peretz

Published online: 20 December 2008 Ó Springer Science+Business Media B.V. 2008

Abstract Hereditary non-polyposis colorectal cancer is a cancer predisposition syndrome known to be caused by heterozygous germline mutations in DNA mismatch repair genes (MMR) most commonly hMLH1, hMSH2, hMSH6. Heterozygous mutations in one of these genes confer an increased risk, mainly for colon and endometrial cancer. Recently, several publications identified that biallelic mutations in the MMR genes are associated with a more severe phenotype, including childhood malignancies and

H. Toledano and Y. Goldberg contributed equally to the paper. H. Toledano  I. Yaniv Schneider Children’s Medical Center of Israel and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel Y. Goldberg (&)  T. Peretz Sharett Institute of Oncology, Hadassah-Hebrew University Medical Center, P.O. Box 12000, 91120 Jerusalem, Israel e-mail: [email protected] I. Kedar-Barnes  H. Baris The Raphael Recanati Genetic Institute Rabin Medical Center, Beilinson Hospital, Petah Tikva, Israel R. M. Porat  E. Pikarsky Department of Pathology, Hadassah-Hebrew University Medical Center, Jerusalem, Israel C. Shochat  D. Bercovich The Human Molecular Genetics & Pharmacogenetics lab, Migal - Galilee Bio-Technology Center, Kiryat-Shmona, Israel I. Lerer  D. Abeliovich Department of Human Genetics, Hadassah-Hebrew University Medical Center, Jerusalem, Israel D. Bercovich Tel Hai Academic College, Tel Hai, Israel

signs of neurofibromatosis type I (NF1). We report on a nonconsanguineous Ashkenazi Jewish family with two affected siblings with features of NF1, colon cancer and astrocytoma at age 13 and 14. Their mother developed endometrial cancer at age 54. Their father had leukoplakia of the vocal cords with a family history of pancreatic cancer. Molecular and pathology studies were done on the tumor tissue and on genomic DNA of family members. Tumor testing demonstrated a high degree of microsatellite instability (MSI analysis), expression of MLH1 and absence of expression of both MSH2 and MSH6 proteins. A biallelic c.1906G [ C (p.A636P) mutation in the hMSH2 gene was detected in the blood of one affected child. Parental genetic testing showed that each parent was heterozygote for the mutation. The c.1906G [ C mutation is a founder mutation in the Ashkenazi Jewish population. To our knowledge this is the first report of homozygosity for this founder mutation. Keywords HNPCC, NF1  Ashkenazi  Bi-allelic  MMR  MSI  MSH2 Abbreviations AC Amsterdam criteria CCS Childhood cancer syndrome CRC Colorectal cancer DHPLC Denaturing high performance liquid chromatography HNPCC Hereditary non-polyposis colorectal cancer IHC Immunohistochemistry MLPA Multiplex ligase dependent probe amplification MMR Mismatch repair MMRMismatch repair deficiency D MSI Microsatellite instability NF1 Neurofibromatosis type 1

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Introduction Hereditary non-polyposis colorectal cancer (HNPCC) is an autosomal dominant inherited condition known to be associated with a predisposition to several malignancies, mainly colorectal cancer (CRC) and endometrial cancer. Other malignancies such as ovarian, gastric, small intestine, pancreas, bile duct, urinary tract and brain tumors have also been associated with HNPCC [1, 2]. Typically, affected individuals with HNPCC present with cancer in the fourth or fifth decade but in some families the presentation may be in the third decade or later in the sixth decade [2]. Germline and somatic mutations in the MMR genes have been implicated in HNPCC and in 15–20% of sporadic CRC [1, 3]. In HNPCC, defined by the presence of a MMR gene mutation, mutations in hMLH1 and hMSH2 account for approximately 90% of cases, hMSH6 for 10% and hPMS2 accounts for less than 5% [4]. Biallelic mutations in the MMR genes lead to a distinctive syndrome, characterized by hematological malignancies, tumors of brain and bowel early in childhood, often associated with signs of neurofibromatosis type 1 (NF1). This syndrome has been referred to as childhood cancer syndrome (CCS) [5] and mismatch repair deficiency syndrome (MMR-D) [6]. In 1999, Wang et al. were the first to describe the association between compound heterozygous mutation in MLH1 gene, early onset extracolonic cancers and signs of NF1 [7]. Menko et al. described a child with multiple cafe´-au-lait spots (CLS), oligodendroglioma and rectal cancer who was homozygous for a mutation in hMSH6 gene [8]. Kru¨ger et al. [9] reported on six children from two consanguineous families with a homozygous PMS2 mutation, who suffered from glioblastoma, colorectal cancer, lymphoma and other HNPCCassociated tumors at early ages. Kru¨ger et al. also reviewed reports of 43 individuals with biallelic MMR germline mutations in 23 different families. Brain tumors occurred in most families, followed by hematological malignancies and intestinal tumors [9]. All affected individuals with CCS had clinical features characteristic of NF1: either CLS, or CLS and axillary freckling. The median age of tumor onset was 9 years, ranging from 1 to 24 years. Mouse knockouts for all MMR genes have been generated [10–12]. When comparing the tumor spectra of these homozygous mouse models to the spectrum of humans with biallelic mutations it becomes clear that they are similar in terms of frequent lymphoma development and relatively low abundance of gastrointestinal tumors. A difference is that brain tumors are very rare and neurofibromas and CLS are absent in mouse MMR knockouts. In Msh2 deficient mice there is an increasing rate of somatic mutations in genes associated with tumorigenesis [13]. Knockout mutants of the three major MMR genes in

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zebrafish mimic distinct features of the human disease and are complementary to mouse models. They develop predominantly, neurofibromas/malignant peripheral nerve sheath tumors at low frequencies [14]. Several mutations in HNPCC have been described as founder mutations in certain populations; two founder mutations in the MLH1 gene account for 63% of all deleterious mutations identified in HNPCC families in the Finnish population [15]. The MSH2 c.1906G [ C mutation was initially described by Yuan et al. in an Ashkenazi Jewish family that fulfilled the Amsterdam criteria for the clinical diagnosis of HNPCC [16]. This finding was confirmed by Foulkes et al. [17]. The mutation results in a substitution of alanine to proline at codon 636 (A636P) in the MSH2 protein, associated with an alteration in MSH2 Crystal structure. Foulkes et al. [17] found that all CRC tumors, from carriers of the mutation, were MSI-H and were negative for the expression of both MSH2 and MSH6 proteins in all tumors examined by IHC studies. This finding could be explained by the fact that MSH2 and MSH6 form a functional complex-MutSa, as a result MSH2 loss often causes concurrent loss of MSH6. The MSH2 c.1906G [ C mutation was examined in a population-based series of Ashkenazi Jews with colorectal cancer and shown to be rare (0.44%), yet highly penetrant; In a combined consecutive series from Israel, New York and Toronto, the A636P mutation was found in 8 of 1,345 (0.59%) Ashkenazi Jewish CRC cases versus 0 of 1,588 healthy Ashkenazi Jewish controls [17]. It has been reported that 20–30% of Ashkenazi Jewish families that fulfill Amsterdam criteria carry this mutation [18, 19]. Hereby, we describe a family with two siblings who died from cancer in childhood. One was affected with features of NF1 and astrocytoma and the other sibling with features of NF1, astrocytoma and CRC. Genetic studies for HNPCC done on one sibling found that he was homozygous for a c.1906G [ C (A636P) mutation in the MSH2 gene. Both parents were found to be heterozygous for the mutation. None of them come from a family that complies with the Amsterdam Criteria, or fulfills the Bethesda guidelines.

Materials and methods Patient data The proband, a 14-year-old boy, presented with a twomonth history of fatigue, mild weight loss, falls, intermittent melena and fresh blood per rectum. A blood count revealed hemoglobin of 7.7 g/dl with microcytic indices. Past history was remarkable for the fact that since childhood he had been noted to have multiple CLS on the skin.

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He was presumed to have NF1 and had been seen intermittently in an NF clinic. Brain MRI studies 2 years earlier reportedly showed changes in the temporal region consistent with the diagnosis of NF1. On examination at the pediatric day center he appeared pale, thin and unwell. He had torticollis and ataxia. A family history of a brother who had died of a malignant brain tumor was reported. The clinical examination and his family history prompted an immediate MRI of the brain which revealed two separate lesions. One was in the temporal area—this had been present on the MRI from 2 years previously but was considerably enlarged and the other was a large lesion in the pons with an appearance of a diffuse brainstem glioma that had not been evident on the previous MRI (Fig. 1a). He underwent resection of the temporal lesion only and pathology revealed a fibrillary astrocytoma (grade II). He then had a gastrointestinal workup for the anemia, which included an upper GI endoscopy and a barium study of the small bowel that were reported as normal. However, colonoscopy revealed a large mass in the rectum 3 cm from the anal sphincter and there were 20–30 polyps throughout the large bowel of different sizes and biopsies were taken. Pathology revealed the polyps to be tubulo-villous adenomata and the rectal mass was a well differentiated adenocarcinoma. Tumor markers CEA and CA19-9 were normal. MRI demonstrated involvement of the peri-rectal fat and PET-CT showed uptake in the mass and several of the polyps but not in the lymph nodes and there was no evidence of distant metastases. In view of the average 1 year life expectancy following diagnosis of a brain stem glioma there was debate regarding the management of the rectal tumor. However, longer than average survival has been described for gliomas in some genetic conditions such as Turcot syndrome and after discussion with the family it was decided to start

aggressive management of his rectal tumor. Since immediate surgery for the rectal mass would have involved permanent loss of sphincter function, initial chemo-radiation with the hope of shrinking the tumor and performing sphincter-sparing surgery at a later date was attempted. At the same time, he began suffering from increasing rightsided weakness and falls and MRI showed increased size of the brain stem lesion so that he received radiation therapy simultaneously to the brain and rectum. Imaging studies performed 6 weeks after completion of radiation showed an improvement in the brain lesion but increase in size of the rectal mass, making sphincter sparing surgery impossible. Since colonic polyposis is a premalignant condition and some of his polyps were already large and positive on the PET scan he underwent panproctocolectomy with permanent ileostomy. Pathology confirmed adenocarcinoma of the rectum without lymph node involvement and approximately 100 polyps in the large bowel. Post-operatively he received adjuvant chemotherapy for the rectal tumor with the FOLFOX regimen but had early relapse at the rectal stump 2 months post-operatively. He received second line therapy with FOLFIRI and Avastin but by 11 months post diagnosis he was suffering from intractable rectal pain as well as neurological deterioration and he died at 1 year following the diagnosis. At the time of his diagnosis with two concurrent tumors and the death of their previous son, increasing family anxiety prompted referral to genetic counseling. Physical examination by the geneticist revealed: head circumference 51 cm (\2%), multiple CLS scattered over the body of which 21 were between 15 and 55 mm in diameter and multiple smaller spots. There was inguinal and axillary freckling. Asymmetrical facies (right [ left) was evident. Neurological examination was normal except for mild imbalance exhibited on tandem walk test only. Ophthalmological evaluation excluded Lisch nodules but bilateral retinal

Fig. 1 MRI of brain tumor. a MR T2 weighted sagittal image of the brain at diagnosis. Hyperintensity of the pons and medulla is seen, compatible with a brain stem tumor. b, c MR T1 weighted fat suppressed axial image of the lower pelvis post Gadolinium injection.

b At diagnosis: irregular thickening of the rectal wall is seen, indicative of tumoral involvement. 10 months post diagnosis. c Local recurrence is evident: a large necrotic mass is seen between the sacrum and the bladder

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hyperpigmentation raised the possibility of congenital hypertrophy of the retinal pigment epithelium (CHRPE). Targeted physical examination of his family members included paternal head circumference of 54.4 cm (25%), maternal head circumference of 52 cm (2%). No CLS were observed on his father’s and mother’s dermal examination. His healthy siblings had one CLS each.

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MSI analysis was performed by a fluorescence-based PCR method as described [21]. PCR products were analyzed on ABI Sequencer (3100) using GeneScan and Genotyper software (PE Applied Biosystems). Immunohistochemistry analysis

The proband was the youngest of four siblings: two apparently healthy siblings and another brother who was diagnosed with anaplastic astrocytoma and NF1 features and died at the age of 13. During the proband’s treatment his father received radiation for severe, progressive laryngeal leukoplakia and his mother was diagnosed age 54 with endometrial carcinoma and underwent a hysterectomy. A paternal grandfather had been diagnosed with pancreatic cancer at age 61 and died soon after. All living first-degree relatives had a colonoscopy that was reported as normal (Fig. 2).

Five micro meter paraffin sections were de-waxed and hydrated through graded ethanols, antigen retrieval was done either with 20 mM citrate buffer pH 6.0 (hMSH2) or with Borg Decloaker (Biocare Medical) pH 9.5 (hMLH1 and hMSH6) in a pressure cooker (Biocare Medical). Slides were incubated with the indicated primary antibody, diluted 1:50 in CAS-Block (Zymed) for overnight at 48C, washed with Optimax (biogenex), incubated with MACH 3 Mouse HRP Polymer (Biocare Medical) and developed with DAB. Normal cells showing strong nuclear staining for the MMR proteins were used as an internal positive control. Mouse monoclonal antibodies used were: hMLH1: clone G168-15; hMSH2: clone FE11; hMSH6: clone BC/ 44 from Biocare Medical.

Tumor testing

DHPLC homozygous mutations screening

Tumor tissue was obtained from archived paraffin blocks. Normal and pathological tissue of the colon were differentially marked on the slide. DNA extraction from the paraffin embedded tissues was performed as described [20]. DNA was extracted from peripheral blood lymphocytes using the QIAGEN DNA Isolation kit (QIAGEN, Germany).

Mutation testing of the MMR genes was performed using a combination of DHPLC and semiquantitative fluorescent multiplex–PCR analysis. To identify homozygous mutations, 10 ll PCR product of wild-type DNA, and 10 ll PCR product of sample DNA were mixed 1:1 and denatured at 95°C. This enabled detection of homozygous

Family history

Fig. 2 An abbreviated pedigree of the reported family: Squares males; circles females; diagonal bars deceased; black minisquares cancer; MSH2/ MSH2—homozygous for the c.1906C [ G mutation; w/w—wild-type for mutation; w/MSH2 heterozygous for mutation

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Homozygosity of MSH2 c.1906G?C Ashkenazi mutation

mutations by formation of a heteroduplex [22]. All exonic fragments of each gene, including intron junctions, were amplified individually. PCR and DHPLC (WAVE, Transgenomic Inc., Omaha, NE) were preformed as described previously [18, 23]. Testing for the Ashkenazi Jewish mutation The c.1906G [ C was tested by allele specific amplification as described in [18]. PCR conditions: 94°C—20 min; 35 cycles of 94°C—30 s; 61°C—1 min; 72°C—1 min; final extension 20 min—72°C. PCR products run on 3% NuSieve-Agarose gel (FMC-CAMBREX) in TBE buffer, stained with ethidium bromide and visualized under UV illumination.

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proband, 14-year-old boy and from the mother. Immunostaining of the adenocarcinoma of the rectum from the 14-year-old patient shows nuclear staining for MLH1 both in the tumor and in the normal cells (Fig. 3a) indicating the retained protein, whereas the adenocarcinoma cells show loss of nuclear staining for MSH2 and MSH6 (Fig. 3b, c). Interestingly, the normal cells were also devoid of nuclear staining for MSH2. The mother’s endometrial adenocarcinoma showed a similar pattern of loss of MSH2 and MSH6 staining, with retained MLH1 staining as demonstrated in Fig. 3d–i. MSI (child, mother)

Tumor analysis

both tumors showed high degree of MSI in DNA extracted from tumor tissue (Fig. 4) and showed microsatellite stability in DNA extracted from peripheral leukocytes, as shown in Fig. 4. As can be seen, no difference in the pattern of stability was seen between the heterozygote and the homozygote samples.

IHC

Molecular analysis

Immunostaining with antibodies against MLH1, MSH2 and MSH6 was performed on tumor samples derived from the

Based on the results obtained from tumor testing, we tested DNA from peripheral leukocytes. Genetic studies on the

Fig. 3 Immunostaining with antibodies against MLH1, MSH2 and MSH6: Positive staining with antibodies against MSH2 and MSH6 is represented by the presence of nuclear brown staining. Thick arrows indicate tumor cells. Arrow heads indicate internal positive control cells, either stromal or epithelial. Sections of moderately differentiated

adenocarcinoma of the rectum (a–c) show retained nuclear staining for MLH1 (a) and loss of MSH2 and MSH6 staining (b, c). Endometrial tissue with positive MLH1 staining in normal (d) and tumor cells (e) but negative MSH2 and MSH6 staining in tumor nuclei (g, i)

Results

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Fig. 4 DHPLC and direct sequencing of the homozygote son and heterozygote mother. Screening for mutations in the MSH2 gene by the DHPLC and direct sequencing of PCR fragments with abnormal chromatograms in comparison to wild-type demonstrates the existence of a homozygous mutation (A636P). a, b The homozygous

mutation (son) was detected by mixing the patient PCR products with a wild-type PCR (1:1) before denaturation and reaniling (b). c Heterozygous DNA (mother) was detected by regular screening of PCR products after denaturation and reaniling

proband revealed a homozygous c.1906G [ C germline mutation in the hMSH2 gene (Fig. 5). Both parents were found to be carriers of a heterozygous mutation. The proband’s sister did not carry the mutation and a living brother was found to be a healthy carrier for the mutation (Fig. 2).

the Ashkenazi c.1906G [ C founder mutation in the MSH2 gene. Both had features of NF1, one died from Astrocytoma, and the other had colon cancer and Astrocytoma at age 13 and 14, respectively. Their mother developed endometrial cancer at age 54. Their father had leukoplakia of the vocal cords with a family history of pancreatic cancer. Neither of the parental families fulfilled the Amsterdam Criteria. In contrast to the well-known phenotype caused by heterozygous MMR gene mutations, little is known about the phenotype of the very rare biallelic MMR mutation carriers. Until recently, less than 100 carriers with biallelic

Discussion We report two siblings from a non-consanguinous Ashkenazi family who present with CCS due to biallelic inheritance of Fig. 5 MSI analysis by the BAT25 marker in peripheral blood and tumor tissue from heterozygote mother and homozygous son. The pattern of stability in the blood and the pattern of instability in tumor is similar

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MMR germline mutations have been reported (reviewed in [9]). Only two families with mutations in the MSH2 gene were reported [24, 25]. To our knowledge there have been no previous reports of homozygosity for the Ashkenazi c.1906G [ C founder mutation in the MSH2 gene. The clinical phenotype of the affected brothers raised the diagnosis of CCS. However, given the clinical presentation of a brain tumor and multiple colonic polyps ([100) suggesting Turcot syndrome, a blood sample of the proband was sent for sequencing of the APC gene. Sequencing of exon 1–14 and 3,000 bases from the 50 of exon 15 did not reveal any mutations (data not shown). Though the proband presented with features of NF1, the lack of suggestive family history and some features that did not support this diagnosis (small head circumference, no neurofibromas, and presence of multiple colonic polyps) did not support this diagnosis. The age of onset of malignancies, the presence of the features of NF1 in both siblings and their absence in both parents, supported the diagnosis of CCS, but the absence of a suggestive family history did not corroborate the diagnosis. However, this is in line with previous reports of biallelic MMR gene mutation carriers; all had features of NF1, mostly multiple CLS, but seldom fulfilled the NIH diagnostic criteria for NF1. Interestingly, an absence of significant family history has been reported in cases of CCS; Kru¨ger et al. have already reported that family history does not seem to be a good predictor for identifying CCS, while the early onset of hematological malignancies, brain or intestinal tumors together with signs of NF1 identified CCS in 80% (20 out of 25) of families with biallelic MMR gene germline mutation carriers [9]. It has been suggested that the NF1 gene is an early target in embryogenesis of carriers of biallelic MMR gene mutations. Puisieux reports that the NF1 gene appears to be a preferential mutational target [26]. This is further supported by Wang et al. [27], showing a higher rate of mutations in the NF1 gene in highly unstable human cell lines and tumors, and in Mlh1 knockout mice compared to MMR-sufficient tissue. Still, the link between these two syndromes or the common features is yet to be clarified. Patients carrying a homozygous deletion of an MMR gene have a different spectrum of tumors than that of heterozygotes; specifically, these patients have a higher occurrence of nervous system tumors. While we cannot definitely explain this different phenotype we would like to suggest two possible explanations that should be further explored: 1. The window of opportunity hypothesis—it is possible that some tumors can only develop if the initiating mutation occurs up to a certain time point in life. If this is the case, than it is clear why homozygotes will be more susceptible to such tumors. 2. The non-cell autonomous effect hypothesis—it has been shown that mast cells lacking one allele of NF1 play an important role in the

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pathogenesis of plexiform neurofibromas in patients with NF1 in a non-cell autonomous way through the secretion of factors that facilitate the growth of tumor cells. It is possible that in homozygotes for MMR genes the microenvironmental deficiency plays a similar role in the tumors unique for this syndrome. We identified MSI-H in the tumor tissues from the proband and a stable pattern in leukocyte DNA. Stable pattern in non-neoplastic cells is in line with other reports [28, 29] and with some knockout models of the MMR genes. However, some others report MSI in non tumoral cells [7] confirming the constitutional defect in DNA MMR. It has been suggested that the different results may be explained by the lack of clonal populations in the peripheral blood sample, or from the relative advantage of stable cells in the normal tissue. A characteristic phenomenon of HNPCC tumors is loss of nuclear expression of MMR proteins detectable by immunohistochemical procedures [30, 31]. Indeed, IHC for MSH2 expression performed in both examined tumors, (namely the proband’s colorectal tumor and the mother’s endometrial adenocarcinoma), exhibited clear loss of MSH2 expression (Fig. 3). Our results, that tumor cells harboring the missense A636P mutation show absence of MSH2 protein by immunohistochemical analysis, are in agreement with the data reported by Foulks et al. [17]. While the correlation between the absence of MSH2 staining and the pathogenicity of the missense mutation is notable, the fact that a single amino acid substitution in MSH2 protein results in both distortion of the antigenic site and MMR function loss may indicate that the mutated protein is unstable. Testing recombinant MSH2-A636P protein for MMR efficiency by in vitro assay showed a complete loss of function [32]. A636 is a non-conserved residue adjacent to a conserved region near the ATP-DNA binding region. Therefore, another suggested explanation for the functional loss is that an inflexible adenosine to proline substitution may cause steric hindrance which not only changes the antibody recognition site, but also alters an otherwise conserved region in the protein and possibly, interferes with its function. The MSH2 c.1906G [ C mutation was shown to be relatively rare among unselected Ashkenazi CRC patients, yet highly penetrant [33]; surprisingly, none of the parental families in our pedigree fulfilled the AC, and hardly comply with the Bethesda guidelines. Of note, we and others have previously reported few Ashkenazi families with this mutation that also did not even comply with the Bethesda guidleines [18]. The incidence of the c.1906G [ C founder mutation is estimated to be very low in the Ashkenazi population. Accordingly the chance of such an event to occur is very rare. But, it is important that children, who have multiple

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tumors and CLS, will be tested for the possibility of biallelic MMR mutations [34]. One should be aware of the occurrence of this syndrome also among non-consanguineous families, given the presence of a founder mutation. Acknowledgments This work was supported, in part, by the Israeli Cancer Association, and by the Levinace Friedl foundation.

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