[CANCER RESEARCH 55. 5677-5680. December 1, 19951 ABSTRACT von Recklinghausen's disease, or type 1 neurofibromatosis, a common familial tumor syndrome, is characterized by the occurrence of multiple benign neoplasms of nerve sheath cells. The disease is caused by germ-line mutations of the NFl gene, which encodes a member of the GTPase activating superfamily of Ras regulatory proteins. We analyzed 5 dinu cleotide repeat loci in DNAs from neurofibromas and matched normal skin from 16 NFl patients. Eight cases (50%) manifested microsateffite alterations. Expansions or compressions of dinucleotide repeats were observed at one locus in four cases and at two loci in one case. Banding patterns compatible with the loss of a microsatellite allele were observed in four cases, including one that also presented microsateffite instability. The surprisingly high frequency of microsatellite alterations suggests that the NFl gene or another gene(s) contributing to the pathogenesis of neurofibromas might be directly or indirectly implicated in the control of genomic integrity. INTRODUCTION von Recklinghausen's neurofibromatosis or NFl3 is one ofthe most common human genetic diseases, affecting approximately 1 in 3500 births (1). The disease is transmitted in a mendelian autosomal dom inant fashion, and predisposes to a wide range of clinico-pathological manifestations, involving primarily tissues deriving from the neural crest. In adults, the most common clinical problems are related to the development of multiple cutaneous and more deeply placed neurofi bromas. These benign, relatively circumscribed lesions, primarily composed of Schwann cells and fibroblasts, slowly but continuously increase in size and number and may eventually lead to severe local or systemic complications (1). NFl has been included among familial tumor syndromes because affected individuals are at an increased risk of developing a variety of neoplasms, particularly neurofibrosarcoma, glioma, pheochromocytoma, and myeloid leukemia (2). NFl is caused by germ-line mutations that inactivate one allele of the tumor-suppressor NFl gene. This gene encodes neurofibromin, a member of the GTPase-activating protein superfamily of ras regula tory proteins (3). Microsatellite instability, characterized by the presence of random shifts in the length of simple repeated sequences, or microsatellites, is a key feature of HNPCC and of a subset of apparently nonhereditary colorectal tumors (4—6).Nerve sheath tumors are not associated with the HNPCC phenotype (7). Microsatellite instability has also been detected in subsets of nonhereditary cancers of the urinary bladder, Received 5/26/95; accepted 10/2/95. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. I This study was supported by an Associazione Italiana per Ia Ricerca sul Cancro grant to R. M-C., by the Consiglio Nazionale delle Ricerche-Applicazioni Cliniche della Ricerca Oncologica Grant 94.01 l61.PF39, and by Ministero dell'Universitá e Ricerca Scientifica e Tecnologica grants (40%) to P. B. 2 To whom requests for reprints should be addressed, at Istituto di Patologia Umana e Medicina Sociale, Università â€oeGabrieleD'Annunzio,― Via dci Vestini 1, 66013 Chieti, Italy. Phone: +39 - (871) - 355-323 or 355-333; Fax: +39 - (871) - 355-322. 3 The abbreviations used are: NFl , type I neurofibromatosis; HNPCC, hereditary non-polyposis colorectal cancer; LOH, loss of heterozygosity. gastrointestinal tract, endometrium, lung, breast, ovary, brain, menin ges, and soft tissues (8—10). Genomic stability is controlled by several mechanisms (11—16).In colorectal tumors, there is evidence that microsatellite instability is caused by functional defects in genes that play a role in mismatch repair (17—21).Intriguingly, there are contradictory data concerning the occurrence of hypersensitivity to chromosomal aberrations and DNA damage in cells from NFl patients (22—27).Chromosomal instability is known to occur in classic hereditary disorders associated with defects in DNA repair (28). In this study, 5 microsatellite loci were analyzed in neurofibroma and normal skin DNAs from 16 NFl patients. Several microsatellite alterations, including mobility shifts, as detected by gain of novel microsatellite alleles missing in the patient's constitutional DNA, and losses of constitutional microsatellite alleles, were observed in neu rofibroma DNAs. Of the 16 individual cases studied, 8 manifested alterations at one or more microsatellite loci. Thus, our results indicate that microsatellite alterations occur at surprisingly high frequency in neurofibromas of NFl patients. PATIENTS AND METHODS Patients. Fourteen unrelated and two related NFl patients, diagnosed ac cording to standard criteria (1), were analyzed. Four of the patients had a family history of disease, 12 were reportedly negative for family history and probably included de novo cases. All 16 patients presented with the typical clinical manifestations of NFl, including cafe-au-lait skin spots, axillary and groin freckling, and multiple cutaneous and s.c. neurofibromas (1). DNA Analysis. Eight-sm sections of formalin-fixed, paraffin-embedded neurofibromas were collected on microscope slides and microdissected with sterile scalpels, referring to hematoxylin and eosin-stained sections to guide the separation of normal skin and of neurofibroma tissue into 1.5-ml polypropilene tubes, containing 1 ml xylene. No attempt was made to dissect out subsets of neurofibroma cells of a single type. The samples were incubated in xylene for 15 mm and pelleted at full speed in a microfuge for I S mm. The xylene was then removed and the pellet was washed twice in absolute ethanol (1 ml). The samples were thoroughly dried under a sterile hood, and 100 pJ ofdigestion buffer, containing 1 MTris-HCI (pH 8.0), 0.5 M EDTA, 0.02% Tween 20, and 100 ,.@g/ml proteinase K were added to each tube. After an incubation of 3 h at 55°C,proteinase K was inactivated at 95°C for 10 mm, and the samples were pelleted at full speed in a microfuge. The supernatant was stored at —20°C until use. One @l of a 1:10 dilution of each sample was used for each PCR reaction. DNA extractions and set-up of PCR reactions were performed in a laboratory distinct from that in which amplified DNAs were manipulated. Two of the microsatellite markers analyzed in this study were those em ployed by Thibodeau et a!. (5) to characterize genomic instability in colorectal cancer. These markers and their respective loci with chromosomal localiza tions were Mid 41 (D17S250) (l7pl2—pl 1.1) and Mfd 27 (D5S107) (5q). The following microsatellite markers containing CA and GA repeats were also analyzed: Mfd 67 (DISIO4) [lq2l—q23;Weber et al., data obtained through GenBank], Mid 39 (D8S87) (chromosome 8; Ref. 29); and DJJS9OS (chro mosome 11; Ref. 30). All of these microsatellites are dinucleotide repeats. Primers and cycling conditions were as described in the relevant references (5, 29, 30). PCRs and electrophoretic separation and autoradiography were as described previously (31). By using the standard PCR protocol, the analysis of 5677 Alterations of Microsatellites in Neurofibromas of von Recklinghausen's Disease1 Laura Ottini, Diana L. Esposito, Antonio Richetta, Marta Carlesimo, Raffaele Palmirotta, Maria Concetta Verl, Pasquale Battista, Luigi Frati, Felice Giacomo Caramia, Stefano Calvieri, Alessandro Cama, and Renato Mariani-Costantini2 Department ofExperimental Medicine IL 0., L F., F. G. C.] and Institute ofDermatology (A. R., M. C., S. C.], University ‘LaSapienza, â€oe 00161 Rome; Istituto Mediterraneo di Neuroscienze, 86077 Pozzilli, Isernia IL F.]; and Institute of Human Pathology and Social Medicine, University â€oeGabrieleD'Annunzio, â€oe 66013 Chieti [D. L E., R. P., M. C. V., P. B.. A. C., R. M-C.J. Italy on May 20, 2017. © 1995 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from