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Unequal Meiotic Crossover: A Frequent Cause of NF1 Microdeletions

2000; Elsevier BV; Volume: 66; Issue: 6 Linguagem: Inglês

10.1086/302920

1537-6605

C. Correa, Hilde Brems, Conxi Lázaro, Peter Marynen, Eric Legius,

Chromatin Remodeling and Cancer

Neurofibromatosis type 1 is a common autosomal dominant disorder caused by mutations of the NF1 gene on chromosome 17. In only 5%–10% of cases, a microdeletion including the NF1 gene is found. We analyzed a set of polymorphic dinucleotide-repeat markers flanking the microdeletion on chromosome 17 in a group of seven unrelated families with a de novo NF1 microdeletion. Six of seven microdeletions were of maternal origin. The breakpoints of the microdeletions of maternal origin were localized in flanking paralogous sequences, called "NF1-REPs." The single deletion of paternal origin was shorter, and no crossover occurred on the paternal chromosome 17 during transmission. Five of the six cases of maternal origin were informative, and all five showed a crossover, between the flanking markers, after maternal transmission. The observed crossovers flanking the NF1 region suggest that these NF1 microdeletions result from an unequal crossover in maternal meiosis I, mediated by a misalignment of the flanking NF1-REPs. Neurofibromatosis type 1 is a common autosomal dominant disorder caused by mutations of the NF1 gene on chromosome 17. In only 5%–10% of cases, a microdeletion including the NF1 gene is found. We analyzed a set of polymorphic dinucleotide-repeat markers flanking the microdeletion on chromosome 17 in a group of seven unrelated families with a de novo NF1 microdeletion. Six of seven microdeletions were of maternal origin. The breakpoints of the microdeletions of maternal origin were localized in flanking paralogous sequences, called "NF1-REPs." The single deletion of paternal origin was shorter, and no crossover occurred on the paternal chromosome 17 during transmission. Five of the six cases of maternal origin were informative, and all five showed a crossover, between the flanking markers, after maternal transmission. The observed crossovers flanking the NF1 region suggest that these NF1 microdeletions result from an unequal crossover in maternal meiosis I, mediated by a misalignment of the flanking NF1-REPs. Neurofibromatosis type I (NF1 [MIM 162200]) is a common autosomal dominantly inherited disorder with an incidence of ∼1/4,000 individuals (Huson et al. Huson et al., 1989Huson SM Compston DA Harper PS A genetic study of von Recklinghausen neurofibromatosis in south east Wales. II. Guidelines for genetic counselling.J Med Genet. 1989; 26: 712-721Crossref PubMed Scopus (134) Google Scholar). In 60%–70% of the patients, a truncating mutation in the NF1 gene has been found (Heim et al. Heim et al., 1995Heim RA Kam-Morgan LN Binnie CG Corns DD Cayouette MC Farber RA Aylsworth AS et al.Distribution of 13 truncating mutations in the neurofibromatosis 1 gene.Hum Mol Genet. 1995; 4: 975-981Crossref PubMed Scopus (142) Google Scholar). In 5%–10% of the cases, an NF1 gene microdeletion has been described, indicating that this type of rearrangement must be present in ∼1/40,000 to ∼1/80,000 individuals (Clementi et al. Clementi et al., 1996Clementi M Boni S Mammi I Favarato M Tenconi R Clinical application of genetic polymorphism in neurofibromatosis type 1.Ann Genet. 1996; 39: 92-96PubMed Google Scholar; Cnossen et al. Cnossen et al., 1997Cnossen MH van der Est MN Breuning MH van Asperen CJ Breslau-Siderius EJ van der Ploeg AT de Goede-Bolder A et al.Deletions spanning the neurofibromatosis type 1 gene: implications for genotype-phenotype correlations in neurofibromatosis type 1?.Hum Mutat. 1997; 9: 458-464Crossref PubMed Scopus (100) Google Scholar; Rasmussen et al. Rasmussen et al., 1998Rasmussen SA Colman SD Ho VT Abernathy CR Arn PH Weiss L Schwartz C et al.Constitutional and mosaic large NF1 gene deletions in Neurofibromatosis type 1.J Med Genet. 1998; 35: 468-471Crossref PubMed Scopus (70) Google Scholar). So far, 65 patients carrying NF1 microdeletions have been reported in the literature (Kayes et al. Kayes et al., 1994Kayes LM Burke W Riccardi VM Bennett R Ehrlich P Rubenstein A Stephens K Deletions spanning the neurofibromatosis 1 gene: identification and phenotype of five patients.Am J Hum Genet. 1994; 54: 424-436PubMed Google Scholar; Wu et al. Wu et al., 1995Wu BL Austin MA Schneider GH Boles RG Korf BR Deletion of the entire NF1 gene detected by FISH: four deletion patients associated with severe manifestations.Am J Med Genet. 1995; 59: 528-535Crossref PubMed Scopus (81) Google Scholar; Cnossen et al. Cnossen et al., 1997Cnossen MH van der Est MN Breuning MH van Asperen CJ Breslau-Siderius EJ van der Ploeg AT de Goede-Bolder A et al.Deletions spanning the neurofibromatosis type 1 gene: implications for genotype-phenotype correlations in neurofibromatosis type 1?.Hum Mutat. 1997; 9: 458-464Crossref PubMed Scopus (100) Google Scholar; Valero et al. Valero et al., 1997Valero MC Pascual-Castroviejo I Velasco E Moreno F Hernandez-Chico C Identification of de novo deletions at the NF1 gene: no preferential paternal origin and phenotypic analysis of patients.Hum Genet. 1997; 99: 720-726Crossref PubMed Scopus (38) Google Scholar; Upadhyaya et al. Upadhyaya et al., 1998Upadhyaya M Ruggieri M Maynard J Osborn M Hartog C Mudd S Penttinen M et al.Gross deletions of the neurofibromatosis type 1 (NF1) gene are predominantly of maternal origin and commonly associated with a learning disability, dysmorphic features and developmental delay.Hum Genet. 1998; 102: 591-597Crossref PubMed Scopus (137) Google Scholar; López-Correa et al. Lopes et al., 1996Lopes J LeGuern E Gouider R Tardieu S Abbas N Birouk N Gugenheim M et al.Recombination hot spot in a 3.2-kb region of the Charcot-Marie-Tooth type 1A repeat sequences: new tools for molecular diagnosis of hereditary neuropathy with liability to pressure palsies and of Charcot-Marie-Tooth type 1A. French CMT Collaborative Research Group.Am J Hum Genet. 1996; 58: 1223-1230PubMed Google Scholar; Riva et al. Riva et al., 2000Riva P Corrado L Natacci F Castorina P Wu BL Schneider GH Clementi M et al.NF1 microdeletion syndrome: refined FISH characterization of sporadic and familial deletions with locus-specific probes.Am J Hum Genet. 2000; 66: 100-109Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Only a few of those cases are familial (Leppig et al. Leppig et al., 1997Leppig KA Kaplan P Viskochil D Weaver M Ortenberg J Stephens K Familial neurofibromatosis 1 microdeletions: cosegregation with distinct facial phenotype and early onset of cutaneous neurofibromata.Am J Med Genet. 1997; 73: 197-204Crossref PubMed Scopus (91) Google Scholar; Wu et al. Wu et al., 1997Wu BL Schneider GH Korf BR Deletion of the entire NF1 gene causing distinct manifestations in a family.Am J Med Genet. 1997; 69: 98-101Crossref PubMed Scopus (43) Google Scholar). Most patients with NF1 microdeletions are characterized by the presence of a more severe phenotype with a variable facial dysmorphy (28/39 patients with clinical description have coarse face, hypertelorism, ptosis, and/or a Noonan-like face), severe learning disabilities or mild mental retardation, early and excessive development of cutaneous neurofibromas, and, possibly, a higher incidence of malignancies (Kayes et al. Kayes et al., 1994Kayes LM Burke W Riccardi VM Bennett R Ehrlich P Rubenstein A Stephens K Deletions spanning the neurofibromatosis 1 gene: identification and phenotype of five patients.Am J Hum Genet. 1994; 54: 424-436PubMed Google Scholar; Wu et al. Wu et al., 1995Wu BL Austin MA Schneider GH Boles RG Korf BR Deletion of the entire NF1 gene detected by FISH: four deletion patients associated with severe manifestations.Am J Med Genet. 1995; 59: 528-535Crossref PubMed Scopus (81) Google Scholar, Wu et al., 1999Wu R López-Correa C Rutkowski JL Baumbach LL Glover TW Legius E Germline mutations in NF1 patients with malignancies.Genes Chromosomes Cancer. 1999; 26: 376-380Crossref PubMed Scopus (35) Google Scholar; Tonsgard et al. Tonsgard et al., 1997Tonsgard JH Yelavarthi KK Cushner S Short MP Lindgren V Do NF1 gene deletions result in a characteristic phenotype?.Am J Med Genet. 1997; 73: 80-86Crossref PubMed Scopus (101) Google Scholar). In the majority of cases, the microdeletion (17q11.2) is of maternal origin (Lazaro et al. Lazaro et al., 1996Lazaro C Gaona A Ainsworth P Tenconi R Vidaud D Kruyer H Ars E et al.Sex differences in mutational rate and mutational mechanism in the NF1 gene in neurofibromatosis type 1 patients.Hum Genet. 1996; 98: 696-699Crossref PubMed Scopus (80) Google Scholar; Upadhyaya et al. Upadhyaya et al., 1998Upadhyaya M Ruggieri M Maynard J Osborn M Hartog C Mudd S Penttinen M et al.Gross deletions of the neurofibromatosis type 1 (NF1) gene are predominantly of maternal origin and commonly associated with a learning disability, dysmorphic features and developmental delay.Hum Genet. 1998; 102: 591-597Crossref PubMed Scopus (137) Google Scholar). It is thought that the haploinsufficiency of the NF1 gene, in combination with contiguous, genes may be responsible for this particular phenotype (Leppig et al. Leppig et al., 1996Leppig KA Viskochil D Neil S Rubenstein A Johnson VP Zhu XL Brothman AR et al.The detection of contiguous gene deletions at the neurofibromatosis 1 locus with fluorescence in situ hybridization.Cytogenet Cell Genet. 1996; 72: 95-98Crossref PubMed Scopus (44) Google Scholar). Most of the NF1 microdeletions have a size of ∼1.5 Mb, and the deletion breakpoints cluster in a flanking duplicated region (NF1-REPs [López-Correa et al. López-Correa et al., 1999aLópez-Correa C Brems H Lazaro C Estivill X Clementi M Mason S Marynen P et al.Inter-chromosomal homologous recombination could mediate large NF1 gene deletions.Am J Hum Genet Suppl. 1999a; 65: A8Google Scholar; Dorschner et al. Dorschner et al., 2000Dorschner MO Sybert VP Weaver M Pletcher BA Stephens K NF1 microdeletion breakpoints are clustered at flanking repetitive sequences.Hum Mol Genet. 2000; 9: 35-46Crossref PubMed Scopus (156) Google Scholar]). These so-called NF1-REPs are paralogous regions of ∼100 kb (fig. 1) in a direct orientation, with ∼98% of homology (Dorschner et al. Dorschner et al., 2000Dorschner MO Sybert VP Weaver M Pletcher BA Stephens K NF1 microdeletion breakpoints are clustered at flanking repetitive sequences.Hum Mol Genet. 2000; 9: 35-46Crossref PubMed Scopus (156) Google Scholar). We genotyped seven families of patients with a de novo NF1 deletion, to further analyze the molecular mechanisms underlying these deletions. All patients were sporadic cases, and no NF1 manifestations were observed in any of the parents. Each patient fulfilled the NIH criteria for NF1. The patients were evaluated by use of a standard protocol. Clinical features are described in table 1. The presence and the approximate size of the deletion are determined in all patients by use of FISH probes and polymorphic microsatellites located in the NF1-gene region (López-Correa et al. López-Correa et al., 1999bLópez-Correa C Brems H Lazaro C Estivill X Clementi M Mason S Rutkowski JL et al.Molecular studies in 20 submicroscopic neurofibromatosis type 1 gene deletions.Hum Mutat. 1999b; 14: 387-393Crossref PubMed Scopus (53) Google Scholar).Table 1Clinical Features in Patients with an NF1 MicrodeletionFamilyFeature96-396 - 2aPatient with a paternal deletion96-197-198-199-1X173Age (years)514271913237SexFFFFMMFFamilial history−−−−−−−Parental originMPMMMMMCLS+++++++Neurofibromas+−++++−+++−Plexiform neurofibroma−−−+bFacial+cLeg−−Macrocephaly+−+−−−−Facial dysmorphism+−+++++Madelung deformity−−++−−−Scoliosis−−+−−−−Pectus excavatum/carinatum−−+−++−Overgrowth+−++++−Learning difficultiesNA−+++++OtherEpilepsy; unilateral deafnessMPNSTHypertrichosisNote.—Dysmorphic features observed in the reported patients are coarse face, hypertelorism, and ptosis. Note that the oldest patient developed a malignant peripheral nerve-sheath tumor (MPNST) and that two of the patients have a large plexiform neurofibroma. NA = not ascertained. A minus sign (−) denotes presence, and a plus sign (+) denotes presence. No. of plus signs indicates level of severity.a Patient with a paternal deletionb Facialc Leg Open table in a new tab Note.— Dysmorphic features observed in the reported patients are coarse face, hypertelorism, and ptosis. Note that the oldest patient developed a malignant peripheral nerve-sheath tumor (MPNST) and that two of the patients have a large plexiform neurofibroma. NA = not ascertained. A minus sign (−) denotes presence, and a plus sign (+) denotes presence. No. of plus signs indicates level of severity. Peripheral blood samples were obtained from patients with NF1 and from family members, after informed consent was obtained. Grandparental blood samples were collected according to the parental origin of the deletion in six families, and blood from one unaffected sib was collected in one family (96-2). We used six markers, located in the sequences flanking the common deletion region, to elucidate the mechanism underlying the deletion; these markers were D17S842 (AFM240xe5), D17S841 (AFM238vb10), and D17S1863 (AFMc003ze1), mapping centromeric to the deletion region, and 3′NF1-4 (primer sequence and alleles of this marker are available, by request, from the authors), D17S1880 (AFMa072zh9), and D17S798 (AFM179xg11), mapping telomeric to the common deleted region. The localization and genetic distances of the markers are derived from resources at the National Center for Biotechnology Information (NCBI), the Whitehead Institute/MIT Center for Genome Research, Mapping, and the Genome Database linkage maps. Markers localized in the deleted region were IVS 38 (in the NF1 gene), 5′NF1-1 (centromeric to the NF1 gene) and D17S1800 (telomeric to the NF1 gene; fig. 1). The microsatellite markers were analyzed by PCR, as described elsewhere (López-Correa et al. López-Correa et al., 1999bLópez-Correa C Brems H Lazaro C Estivill X Clementi M Mason S Rutkowski JL et al.Molecular studies in 20 submicroscopic neurofibromatosis type 1 gene deletions.Hum Mutat. 1999b; 14: 387-393Crossref PubMed Scopus (53) Google Scholar). The results are represented in Figure 1, Figure 2. All patients had deletions confirmed by FISH and microsatellite analysis. Six patients had microdeletions of maternal origin, as demonstrated by at least two intragenic markers, and their deletion was flanked by the NF1-REPs (fig. 1). One patient (family 96-2) had a shorter deletion of paternal origin, which included only the NF1 gene and the three genes embedded in intron 27b (EVI2A, EVI2B, and OMGP [López-Correa et al. López-Correa et al., 1999bLópez-Correa C Brems H Lazaro C Estivill X Clementi M Mason S Rutkowski JL et al.Molecular studies in 20 submicroscopic neurofibromatosis type 1 gene deletions.Hum Mutat. 1999b; 14: 387-393Crossref PubMed Scopus (53) Google Scholar]). In the group of six patients with deletions of maternal origin, a crossover between flanking centromeric and telomeric markers was observed in five informative cases. In the sixth case, the markers were not informative (X173). Thus, all five informative patients carrying a maternally derived deletion showed a crossover between markers flanking the deletion region. In the patient carrying the smaller deletion of paternal origin, there is no evidence of an interchromosomal recombination in the deletion region on the paternal chromosome. Both sibs inherited the same set of paternal markers flanking the deleted region (fig. 1). The genetic distance between markers D17S841/D17S842 and D17S1880/D17S798 has been estimated as ∼4 cM (NCBI, the Whitehead Institute/MIT Center for Genome Research, and the Genome Database linkage–radiation hybrids maps). The expected number of meiotic recombinations in six individuals would be ∼0.24. The probability of detecting by chance five recombinations in six informative meioses is <5.8×10−7 (binomial distribution; this assumes interference). This finding is highly suggestive of an unequal crossover in maternal meiosis I, at the breakpoint of the deletion and mediated by the NF1-REPs, as being the mechanism leading to the deletion. It has been observed that large DNA deletions resulting in the loss of 1–5 Mb occur with a higher frequency in specific regions of the human genome. The breakpoints of those microdeletions appear to occur at hot-spot regions. Low-copy repeats (REPs, duplicons, or paralogous sequences) have been shown to flank these regions, facilitating the presence of homologous recombination. It has been proposed that pericentromeric regions are particularly prone to this kind of microdeletion. Some of the microdeletion syndromes showing flanking repeats are Prader-Willi/Angelman syndrome on chromosome 15q11.2 (Amos-Landgraf et al. Amos-Landgraf et al., 1999Amos-Landgraf JM Ji Y Gottlieb W Depinet T Wandstrat AE Cassidy SB Driscoll DJ et al.Chromosome breakage in the Prader-Willi and Angelman syndromes involves recombination between large transcribed repeats at proximal and distal breakpoints.Am J Hum Genet. 1999; 65: 370-386Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar), velo-cardio-facial syndrome and DiGeorge syndrome on 22q11 (Edelmann et al. Edelmann et al., 1999Edelmann L Pandita RK Morrow BE Low-copy repeats mediate the common 3-Mb deletion in patients with velo-cardio-facial syndrome.Am J Hum Genet. 1999; 64: 1076-1086Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar), Williams-Beuren syndrome on 7q11.2 (Perez Jurado et al. Perez Jurado et al., 1998Perez Jurado LA Wang YK Peoples R Coloma A Cruces J Francke U A duplicated gene in the breakpoint regions of the 7q11.23 Williams-Beuren syndrome deletion encodes the initiator binding protein TFII-I and BAP-135, a phosphorylation target of BTK.Hum Mol Genet. 1998; 7: 325-334Crossref PubMed Scopus (150) Google Scholar), and hereditary neuropathy with liability to pressure palsies (HNPP [Lopes et al. Lopes et al., 1996Lopes J LeGuern E Gouider R Tardieu S Abbas N Birouk N Gugenheim M et al.Recombination hot spot in a 3.2-kb region of the Charcot-Marie-Tooth type 1A repeat sequences: new tools for molecular diagnosis of hereditary neuropathy with liability to pressure palsies and of Charcot-Marie-Tooth type 1A. French CMT Collaborative Research Group.Am J Hum Genet. 1996; 58: 1223-1230PubMed Google Scholar]) and Smith-Magenis syndrome (SMS; Chen et al. Chen et al., 1997Chen KS Manian P Koeuth T Potocki L Zhao Q Chinault AC Lee CC et al.Homologous recombination of a flanking repeat gene cluster is a mechanism for a common contiguous gene deletion syndrome.Nat Genet. 1997; 17: 154-163Crossref PubMed Scopus (328) Google Scholar) on chromosome 17p11.2. Different reports have been published recently concerning the pathogenesis of these deletions, most specifically regarding the occurrence of inter- versus intrachromosomal rearrangements between the REPs. Concerning the microdeletions located at 15q11.2, 22q11.2, and 7q11.2, no consistent sex-specific occurrence of inter- or intrachromosomal rearrangements have been detected (Carrozzo et al. Carrozzo et al., 1997Carrozzo R Rossi E Christian SL Kittikamron K Livieri C Corrias A Pucci L et al.Inter- and intrachromosomal rearrangements are both involved in the origin of 15q11-q13 deletions in Prader-Willi syndrome.Am J Hum Genet. 1997; 61: 228-231Abstract Full Text PDF PubMed Scopus (54) Google Scholar; Baumer et al. Baumer et al., 1998Baumer A Dutly F Balmer D Riegel M Tukel T Krajewska-Walasek M Schinzel AA High level of unequal meiotic crossovers at the origin of the 22q11.2 and 7q11.23 deletions.Hum Mol Genet. 1998; 7: 887-894Crossref PubMed Scopus (105) Google Scholar; Robinson et al. Robinson et al., 1998Robinson WP Dutly F Nicholls RD Bernasconi F Penaherrera M Michaelis RC Abeliovich D et al.The mechanisms involved in formation of deletions and duplications of 15q11-q13.J Med Genet. 1998; 35: 130-136Crossref PubMed Google Scholar). The molecular rearrangement resulting in the deletion (in the case of HNPP) and duplication (in the case of Charcot-Marie-Tooth disease type A1) of the 1.5-Mb region at 17p11.2 has been proposed to occur by two different and sex-dependent mechanisms. Rearrangements of paternal origin, which are mostly duplications, are generated by interchromosomal recombination, whereas deletions and duplications of maternal origin result from an intrachromosomal rearrangement (Lopes et al. Lopes et al., 1997Lopes J Vandenberghe A Tardieu S Ionasescu V Levy N Wood N Tachi N et al.Sex-dependent rearrangements resulting in CMT1A and HNPP.Nat Genet. 1997; 17: 136-137Crossref PubMed Scopus (51) Google Scholar, Lopes et al., 1998Lopes J Ravise N Vandenberghe A Palau F Ionasescu V Mayer M Levy N et al.Fine mapping of de novo CMT1A and HNPP rearrangements within CMT1A-REPs evidences two distinct sex-dependent mechanisms and candidate sequences involved in recombination.Hum Mol Genet. 1998; 7: 141-148Crossref PubMed Scopus (76) Google Scholar). In contrast, in this study, the maternal NF1 deletions occurred by an interchromosomal recombination in all five informative cases, whereas the paternal deletion occurred by a different mechanism. The observed maternal deletions probably occur by misalignment of the flanking repetitive elements in homologous chromosomes at meiosis I (interchromosomal recombination). The paternal deletion showed a different size and therefore was not mediated by a recombination between the flanking NF1-REPs. This paternal deletion might have originated by a different mechanism. Nevertheless, one patient with a large paternal deletion of ∼1.5 Mb flanked by NF1-REPS has been described elsewhere (Dorschner et al Dorschner et al., 2000Dorschner MO Sybert VP Weaver M Pletcher BA Stephens K NF1 microdeletion breakpoints are clustered at flanking repetitive sequences.Hum Mol Genet. 2000; 9: 35-46Crossref PubMed Scopus (156) Google Scholar). It is not known whether this large paternal microdeletion is mediated by unequal crossover between NF1-REPs, and it would be worthwhile to test this hypothesis. The presence of an interchromosomal recombination theoretically predicts the formation of a duplication product on the other homologous chromosome involved in the unequal crossover. Zygotes carrying such hypothetical duplication are likely to be formed at the same frequency as are zygotes with deletions. So far, no duplications involving the critical region 17q11.2 have been described. Whether the duplication product is viable and gives rise to a recognizable phenotype is still unknown. This reciprocal phenomenon has been described at the 17p11.2 region. Patients with SMS have a common contiguous gene–deletion syndrome in 17p11.2 (Chen et al. Chen et al., 1997Chen KS Manian P Koeuth T Potocki L Zhao Q Chinault AC Lee CC et al.Homologous recombination of a flanking repeat gene cluster is a mechanism for a common contiguous gene deletion syndrome.Nat Genet. 1997; 17: 154-163Crossref PubMed Scopus (328) Google Scholar). Recently, a group of six patients with a mild phenotype has been reported with duplications of the SMS deletion region; those duplications are preferentially of paternal origin and mostly arise from an interchromosomal recombination event. The duplication is probably the reciprocal recombination product of the SMS deletion and represents a paradigm of the homologous recombination mechanism (Potocki et al. Potocki et al., 2000Potocki L Chen KS Park SS Osterholm DE Withers MA Kimonis V Summers AM et al.Molecular mechanism for duplication 17p11.2—the homologous recombination reciprocal of the Smith-Magenis microdeletion.Nat Genet. 2000; 24: 84-87Crossref PubMed Scopus (262) Google Scholar). In our study, six of seven microdeletions were of maternal origin, with apparently the same deletion size. Five of these six arose by an unequal crossover, during meiosis I, as a result of a homologous recombination between NF1-REPs flanking the deletion region. A sex-dependent occurrence of interchromosomal rearrangements in NF1 is hypothesized. A larger series of patients is needed for confirmation of this observation and in order to study the mechanism in the much rarer microdeletions of paternal origin, especially those with the common 1.5-Mb microdeletion region. We thank the patients with NF1 and their families who collaborated in this study. C.L.-C. has a grant from the Vlaamse Liga Tegen Kanker; H.B. is supported by a grant of the Fonds voor Wetenschappelijk Onderzoek Vlaanderen (FWO Vlaanderen) grant G.0238.98; P.M. is research director of the FWO-Vlaanderen; C.L. is supported by Fondo de Investigaciones Sanitarias de la Seguridad Social grant 98-0992 and the Institut Catalá de la Salut; and E.L. is part-time clinical researcher for the FWO Vlaanderen.