Human Molecular Genetics, 2000, Vol. 9, No. 1 35-46
© 2000 Oxford University Press
NF1 microdeletion breakpoints are clustered at flanking repetitive sequences
Department of Medicine, University of Washington, 1959 NE Pacific Street, Room I-204, Medical Genetics Box 357720, Seattle, WA 98195, USA and 1Department of Pediatrics, University of Medicine and Dentistry, New Jersey Medical School, Newark, NJ 07103, USA
Received 27 September 1999; Revised and Accepted 3 November 1999.
DDBJ/EMBL/GenBank accession nos AF170177–AF170186.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Neurofibromatosis type 1 patients with a submicroscopic deletion spanning the NF1 tumor suppressor gene are remarkable for an early age at onset of cutaneous neurofibromas, suggesting the deletion of an additional locus that potentiates neurofibromagenesis. Construction of a 3.5 Mb BAC/PAC/YAC contig at chromosome 17q11.2 and analysis of somatic cell hybrids from microdeletion patients showed that 14 of 17 cases had deletions of 1.5 Mb in length. The deletions encompassed the entire 350 kb NF1 gene, three additional genes, one pseudogene and 16 expressed sequence tags (ESTs). In these cases, both proximal and distal breakpoints mapped at chromosomal regions of high identity, termed NF1REPs. These REPs, or clusters of paralogous loci, are 15–100 kb and harbor at least four ESTs and an expressed SH3GL pseudogene. The remaining three patients had at least one breakpoint outside an NF1REP element; one had a smaller deletion thereby narrowing the critical region harboring the putative locus that exacerbates neurofibroma development to 1 Mb. These data show that the likely mechanism of NF1 microdeletion is homologous recombination between NF1REPs on sister chromatids. NF1 microdeletion is the first REP-mediated rearrangement identified that results in loss of a tumor suppressor gene. Therefore, in addition to the germline rearrangements reported here, NF1REP-mediated somatic recombination could be an important mechanism for the loss of heterozygosity at NF1 in tumors of NF1 patients.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Haploinsufficiency for neurofibromin is the likely molecular basis of neurofibromatosis type 1 (NF1), a common autosomal disorder that predisposes to the development of benign and malignant tumors. Genetic, biochemical and proliferative studies of cells from NF1-associated tumors are consistent with a tumor suppressor function for neurofibromin. Tumor suppressor activity is due, at least in part, to a ras-GTPase activating protein (ras-GAP) domain which accelerates the conversion of activated GTP-ras to inactivated GDP-ras (1). Evidence in human and mouse shows that neurofibromin-deficient tumor cells have increased activated ras and dysregulated proliferative properties (2,3), which may be mediated by the ras-dependent mitogen-activated protein kinase signaling pathway (4). Both benign and malignant tumors show homozygous inactivation of NF1 resulting in lack of functional neurofibromin. Although NF1 inactivation in a tumor progenitor cell can occur by numerous mechanisms, the identification of defined intragenic NF1 mutations in primary tumor tissue argues that lack of neurofibromin is central to their development (5–7).
Over 70% of germline mutations of the NF1 gene are intragenic and predict a premature truncation of neurofibromin (8). These mutations are distributed throughout the coding region. They are generally unique for a given patient or family, and are not predictive for any of the diverse clinical manifestations that can develop in this multisystemic disorder. Nearly all NF1 patients develop café-au-lait macules, axillary and inguinal freckling, multiple neurofibromas, and Lisch nodules, which are hamartomas of the iris of the eye. Other significant, but less common, manifestations of the disorder include learning disabilities, optic glioma, bony abnormalities (sphenoid bone dysplasia, pseudoarthrosis, scoliosis), increased risk of specific malignancies, and others (9,10). NF1 has been considered to be primarily a disorder of cells derived from the neural crest, which is supported by recent evidence consistent with neurofibromas arising by clonal proliferation of a neurofibromin-deficient Schwann cell (11).
Previously, we identified five patients that carried a deletion of one entire NF1 allele. These patients were remarkable for an early age (<10 years) at onset of dermal neurofibromas, an increased number or heavy burden of neurofibromas relative to their age, and certain atypical facial features (12,13). The association of an NF1 microdeletion with this phenotype was subsequently confirmed by us and other investigators (14–18). In addition, the identification of families segregating an NF1 microdeletion demonstrated that the rearrangement was co-inherited with the remarkable facial and tumor features (17,19,20).
The molecular basis for precocious neurofibromagenesis in microdeletion patients is unknown. Previously, we estimated the microdeletions at 0.7–2 Mb, which, even accounting for the large 350 kb NF1 gene, implies that many additional genes are deleted (13,14,19). Theoretically, early age at onset of neurofibromagenesis could be attributed to: (i) deletion of the NF1 gene alone; (ii) co-deletion of NF1 and one of the three genes of unknown function that are embedded in an NF1 intron; (iii) co-deletion of NF1 and a novel contiguous gene(s); or (iv) dysregulation of a gene at the deletion breakpoint. We consider it unlikely that neurofibromin haploinsufficiency alone could account for early onset of tumorigenesis. Over 70% of NF1 patients are heterozygous for a mutation that predicts premature truncation of neurofibromin, yet in a population-based study only ~14% of subjects developed dermal neurofibromas before 10 years of age (21,22). However, it is unknown whether neurofibroma development could be ameliorated in any of these patients due to possible residual activity from the mutant NF1 allele. The role of a putative co-deleted locus has been difficult to assess because the number of deletion patients is small and information regarding number and age at onset of neurofibromas and deletion magnitude are not always evaluated or reported. Recently, however, we described 12 unrelated NF1 microdeletion patients with early onset and/or high burden of neurofibromas with deletion breakpoints that clustered in the same centromeric and telomeric locus intervals (K. Maruyama, M. Weaver, K. Leppig, A.S. Aylsworth, M.O. Dorschner, R. Farber, J. Ortenberg, A. Rubenstein, L. Immken, C. Curry and K. Stephens, submitted for publication). Towards mapping and identifying a locus that potentiates neurofibromagenesis in NF1 patients, we constructed a 3.5 Mb physical map of the NF1 region, precisely mapped the deletion, and examined deletion genotype with patient phenotype. We report that the breakpoints in the majority of patients are clustered at flanking genomic segments of paralogous sequence (sequence similarity due to duplication). These results have important implications regarding germline and somatic rearrangements involving NF1.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Construction of a 3.5 Mb contig
Recently, we determined that both the centromeric and telomeric breakpoints in 14 of 15 NF1 patients with submicroscopic deletions were clustered in two distinct marker intervals. Quantitative PCR and the analysis of somatic cell hybrid lines carrying deleted chromosome 17 of each patient mapped the centromeric breakpoints between marker loci D17S2120 and UT172 and the telomeric breakpoints between D17S1800 and D17S1880 (K. Maruyama et al., submitted for publication) (Fig. 1). Although these data were suggestive of breakpoint clustering, the length of each interval was unknown. In addition, the number and unique order of other markers within each of these initial breakpoint intervals was unknown. To refine the location of the deletion breakpoints, we sought to construct a physical map encompassing both breakpoint cluster regions. Initially, chromosome 17 loci reported to map at or near band q11.2 were gleaned from the literature and publicly available electronic databases and screened by PCR against a somatic cell hybrid mapping panel. This placed each locus into one of five possible chromosomal intervals (Fig. 1). Loci that mapped to intervals C and D were used to identify and construct a contig of novel and previously reported bacterial artificial chromosome (BAC) and P1-derived artificial chromosome (PAC) clones. Initial database searches identified five sequenced clones that served as a framework for contig construction. Two BACs, 468F23 and 41C23, were found to harbor AH1 and AN2, respectively, which are end sequences of a previously described NF1 yeast artificial chromosome (YAC) contig (23) (Fig. 2). A 297 kb sequence carrying a large portion of the NF1 gene (GenBank accession no. AC004526), and clones 542B22 and 307A16, were identified from database searches. Together, the three clones 499I20, AC004526 and 41C23 comprise a 476 kb contiguous sequence spanning from intron 1 of the NF1 gene to D17S1800 (Fig. 2). The remainder of the contig was assembled by screening a BAC library with selected loci that mapped in intervals C and D (Figs 1 and 2) and by utilizing newly released chromosome 17 sequences from the Whitehead Institute for Biomedical Research/MIT Center for Genome Research (http://www-genome.wi.mit.edu/ ). The clones comprising the BACs are listed in Table 1.
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The contig consisted of 39 BAC/PAC and two YAC clones (Fig. 2, Table 1). The new marker A16INT linked together the two YACs y947g11 and y815b11 (http://www-genome.wi.mit.edu/ ), creating a YAC contig of the region. In addition, loci prominent for their previous use in genetic mapping and loss of constitutional heterozygosity (LOH) analyses were mapped precisely. UT172, previously estimated to be 1.5 Mb centromeric of NF1 (24), is only ~250 kb distant within BAC 468F23. D17S117 and D17S120 are located ~1 Mb centromeric of NF1; the latter marker actually lies within an intron of the carboxypeptidase D (CPD) gene. D17S798 is located ~1.8 Mb telomeric of NF1.
Fine mapping of the NF1 microdeletion breakpoints
Over 10 loci were placed precisely in each of the breakpoint cluster regions (Fig. 1), thereby facilitating fine mapping of the breakpoints of all 17 microdeletion patients. Fourteen microdeletion patients had proximal breakpoints in the locus interval of SH3GLP2 to CYTOR4 (SHGC-37343) and distal breakpoints in the interval between SH3GLP1 and D17S1880 (Fig. 2). The remaining three deletion cases had at least one novel breakpoint (Fig. 2). Patient UWA113-1 had a novel centromeric breakpoint between FB12A2 and exon 1 of the NF1 gene. Both breakpoints of patient UWA155-1 were novel and located between the intervals defined by SHGC35088–FB12A2 and D17S1656–stSG50857. Patient UWA106-3, who had the largest deletion in our cohort (13), also had two unique breakpoints. The telomeric breakpoint mapped in the interval of D17S73 to FB6F10 and the centromeric breakpoint was mapped previously between D17S1294 and SCL6A4 during construction of a physical contig of the latter gene that encodes the serotonin transporter (25).
The contig provided more precise estimates of the physical lengths of both the region and the patient deletions. Because YACs 947g11 and 815b11, each estimated at 1.7 Mb (http://www-genome.wi.mit.edu/ ), are completely contained within the deletion of UWA106-3, this patient’s deletion is approximated at 3.5 Mb. YAC 947g11 spans from SLC6A4 to A16INT and overlaps 815b11, which extends to just beyond D17S798. The known lengths of sequenced BACs and the average length of 185 kb for non-sequenced BACs derived from the RPC1-11 library were subtracted from the YAC lengths to give the estimated scale in Figure 2. The length of the common NF1 microdeletion was estimated at 1.5 Mb.
NF1 microdeletion breakpoints cluster at repetitive sequences
Fine mapping of the region led to the discovery of two SH3GL expressed pseudogenes, SH3GLP2 and SH3GLP1, that mapped near the breakpoints of the common NF1 deletions (Fig. 2). Because low copy repeats are known to flank deletions/duplications responsible for some contiguous gene syndromes (26), a search for additional multicopy transcripts was initiated. A third expressed pseudogene, SH3GLP3, was reported to map distally at 17q24 (27). BLAST analyses of the SH3GL pseudogenes identified BACs 271K11 and 147L13, which carried SH3GLP2 and SH3GLP3, respectively. The sequence-tagged site (STS)/expressed sequence tag (EST) content of the BAC clones was obtained (http://www-genome.wi.mit.edu/ ) and BLAST analyses identified their locations within each clone. This revealed that two ESTs, WI-12393 and WI-9461, were present in both BACs and located near each respective SH3GL pseudogene. Systematic BLAST analyses of loci reportedly mapping near NF1 in publicly available genome databases revealed that stSG40093 and stSG31654 were not only in BAC 271K11 centromeric to NF1, but were also harbored by BAC 147L13 at chromosome 17q24 (http://www.ncbi.nlm.nih.gov/genemap98 ). Together these analyses identified two clusters of five transcripts for which the order and relative distance between markers was conserved. These clusters of paralogous loci were designated as NF1REP, using the suffixes -P and -D to distinguish the proximal repeat at 17q11.2 from the distal repeat at 17q24 (Figs 2 and 3).
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To determine whether the unsequenced region surrounding SH3GLP1 comprised an additional NF1REP, PCR primers for WI-9461, stSG31654, stSG40093 and WI-12393 were used to amplify these loci from BACs overlapping the SH3GLP1 locus. BAC clones 953C18, 951F11 and 95306 were positive for each of the transcripts; 999E22 was positive for all but the WI-12393 locus. This medially positioned repeat cluster was designated NF1REP-M (Fig. 3). These results demonstrated that chromosome 17 carries at least three clusters of paralogous loci: WI-9461, stSG31654, stSG40093 and WI-12393, each in association with a specific SH3GL pseudogene (Fig. 3). The absence of WI-12393 from BAC 999E22 and preliminary sequence analysis (M. Dorschner, unpublished data) strongly suggests that NF1REP-P and -M are direct repeats of 15–100 kb in length. The repetitive sequences may extend further beyond WI-12393. The breakpoints of the patients carrying the common NF1 microdeletion lie within, or adjacent to, NF1REP regions. The centromeric breakpoints were between SH3GLP2 and CYTOR4, whereas the telomeric breakpoints occurred between SH3GLP1 and K8CEN. Finer mapping of the breakpoints will require the development of REP-specific primers or Southern blot analyses that identify junction fragments. The size and orientation of NF1REP-D is unknown.
Several lines of evidence confirmed that, despite carrying sequences with a high degree of identity, BACs spanning NF1REP-P and -M were localized unambiguously. First, the primers for amplification of SH3GLP1 and SH3GLP2 were locus specific, exploiting base differences in the 5' region of the transcripts (27). BACs 943L10 and 946G8 were the only clones that possessed SH3GLP2, whereas clones 953C18, 951F11, 999E22 and 95306 carried only SH3GLP1. In addition, these BACs harbored the expected unique loci based on our deletion analysis. BAC 943L10 was positive for D17S1863 and CYTOR4, whereas BACs spanning the medial REP contained KIAA0160 or D17S1880.
Mouse orthologs for all of the genes located in interval B (Fig. 1), SLC6A4, CPD, CDK5R1 and the chemokine cluster, have been mapped to the same region of mouse chromosome 11 that carries the NF1 ortholog. It appears that synteny has been conserved between human and mouse for the region from CRYBA1 to at least the chemokine cluster.
NF1 deletion genotype/phenotype and parental origin of deletion
The physical features of the 13 unrelated NF1 microdeletion patients and the four members of family UWA166 are summarized in Table 2. There were no obvious differences detected between the features present in those individuals with the common NF1 deletion and the three with deletions of different lengths. No single feature was present or absent consistently within either group. The location of the putative gene that potentiates neurofibromagenesis was narrowed to an interval of 1 Mb between FB12A2 and SH3GLP1, as defined by the deletion of patient UWA113-1 (Fig. 2). This critical region is known to harbor four genes, two pseudogenes, and seven ESTs (Fig. 2, Table 3).
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A preference for de novo microdeletion of the maternally derived chromosome was observed. Among the eight cases with documented de novo microdeletions, six were derived from the maternal homolog (UWA patients 113-1, 119-1, 147-3, 167-1, 183-1, 184-1) and two from the paternal homolog (UWA106-3, UWA123-3) (13,19, data not shown). Three families inherited NF1 microdeletions. Family UWA166 includes the affected mother UWA166-1 and her three affected children UWA166-2, -3 and -4 (19), patient UWA169-1 inherited NF1 from his affected mother (19,28), and UWA155-1 inherited NF1 from his affected father.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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NF1REP elements
Three NF1REPs were mapped to chromosome 17. NF1REP-P and -M flank the NF1 locus at 17q11.2 and are separated by ~1.5 Mb of DNA. The third, NF1REP-D, is located at 17q24. Each REP is composed of at least five transcripts/ESTs including an SH3GL pseudogene, WI-9461, stSG31654, stSG40093 and WI-12393 (Fig. 3). In a search for proteins containing SH3 (src homology region 3) domains, three functional genes were identified from a fetal brain cDNA library, SH3GL1, SH3GL2 and SH3GL3. These genes map at chromosomes 19p13.3, 9p22 and 15q24, respectively (27), and function in signal transduction, cytoskeleton and aggregation of huntingtin (29–31). In addition, three expressed SH3GL pseudogenes were identified that mapped by FISH to chromosome 17 (Fig. 3) (27). It will be important to characterize the expression of the other paralogous loci, WI-9461, stSG31654, stSG40093 and WI-12393, at each of the NF1REPs. Although these loci were originally isolated as ESTs expressed in multiple tissues (Unigene: www.ncbi.nlm.nih.gov/UniGene/index.html ), it is unclear whether each paralogous locus in NF1REP-P, -M and -D is expressed and whether they represent pseudogenes, functional loci or residual gene fragments.
NF1REP-mediated recombination
We propose that a high degree of homology between NF1REP-P and -M facilitates homologous recombination during meiosis or mitosis resulting in the deletion of intervening sequences. Consistent with this hypothesis, pseudogenes SH3GLP1 and -2 are 97.8% identical, whereas SH3GLP3 shares only 90% identity with either of these (27). Further analysis of the identity between NF1REP-P and -M will require completing the sequence of the NF1REP-M domain; partial sequence analysis shows >98% identity (M. Dorschner, unpublished data). Recombination between the direct repeats NF1REP-P and -M could give rise to NF1 microdeletions by either unequal recombination between sister chromatids or intrachromosomal recombination via a fold-back loop and excision. Distinguishing between these mechanisms will require further analyses to determine whether NF1REP-mediated recombination is associated with a meiotic crossover event. The apparent preference for de novo NF1 microdeletion of the maternally derived chromosomes may provide a clue. Other REP-mediated rearrangements show a sex-dependent mechanism with maternally derived deletions resulting from excision of an intrachromatidal loop (32). Although in other cases, microdeletions mediated by flanking REP domains appear to arise by both mechanisms (33–35). If unequal meiotic recombination between sister chromatids underlies NF1 microdeletion, it would predict the formation of a reciprocal duplication derivative. Whether a 1.5 Mb NF1 duplication product would be stable is unknown; it may quickly undergo recombination and ‘revert’ to a deletion (36). Non-mosaic trisomy 17 has not been reported in a live born, and partial 17p or 17q trisomy is rare and even mosaic cases are uncommon (37), suggesting that many such rearrangements are lethal.
Patterns of REP domains and chromosomal rearrangements
About 10 years ago it became clear that intragenic, or relatively small, deletions, duplications and inversions of the human genome could be mediated by homologous recombination between tandem genes, or other nearby repetitive sequences. Such rearrangements have been well described for the steroid sulfatase, 
-globin, Factor VIII, LDL receptor and other genes (reviewed in refs 36,38). Recently, however, the breakpoints of large contiguous gene deletions and duplications from 1–5 Mb in length were mapped to flanking repetitive sequences of high identity. Such low copy repetitive elements have been designated as REPs, duplicons or paralogous regions (39,40). There is compelling evidence that homologous recombination between REPs is the molecular basis for a number of disorders (Table 4) (reviewed in refs 26,38). The precedence was established for the neuropathies Charcot–Marie–Tooth type 1A (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP). Unequal recombination between flanking REPs during meiosis I results in duplication (CMT1A) or deletion (HNPP) of a 1.5 Mb segment of chromosome 17p11.2 (41,42). The two 24 kb CMT1A-REPs have 98.7% identity with an internal 557 bp recombination hotspot where 21 of 23 breakpoints occurred (43).
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Although the characterization of REPs flanking large contiguous gene rearrangements is in its infancy, variability in REP length, number, complexity and orientation is apparent. REP length varies considerably (Table 4) and may correlate directly with the size of the intervening deletion/duplication. This suggests that recombination between distant REPs may require longer tracts of identity for efficient pairing (26). Although a single CMT1A-REP lies on each side of the CMT1A/HNPP rearrangement, the number of REPs and the apparent preference for recombination between specific REPs can vary considerably. Three Smith-Magenis syndrome (SMS)-REPs are found in the 17p11.2 region, yet nearly all SMS deletions are due to crossover events between the proximal and distal REPs (44). The identification of eight REPs at 22q11 suggests that recombination between specific REP pairs may account for different rearrangements underlying multiple congenital anomaly disorders that map to this chromosomal region, such as velocardiofacial syndrome/DiGeorge syndrome (VCFS/DGS) and cat eye syndrome (CES) (45). REP domains may be very complex repeats that include multiple subrepeats, which can be in tandem or interspersed, inverted or direct in orientation (46–48). REPs may even be dispersed among chromosomes; FISH experiments suggest that copies of the Prader-Willi syndrome/Angelman syndrome (PWS/AS) REP may be at 15q24 and 16p11 (49). The apparent preference for REP domains to occur near the centromere of chromosomes (Table 4) (26) is consistent with reports of a strong bias for these regions to acquire paralogous segments. This phenomenon is referred to as pericentromeric plasticity and presumably accounts for the varied NF1-related fragments that are scattered among the centromeric regions of seven different autosomes (50; reviewed in ref. 39). Homologous recombination events and the resulting chromosomal rearrangements are also dependent on the orientation of the repetitive sequences involved. For example, recombination between direct CMT1A-REPs results in deletion and duplication via unequal crossing-over between chromatids (reviewed in ref. 51), whereas recombination between indirect duplicons can result in either deletions or inversions (52–54).
Unique pathological aspects of NF1REP-mediated recombination
Several aspects of NF1 microdeletions are unique among REP-mediated contiguous gene rearrangements in the human genome. For other disorders, REP-mediated rearrangements commonly account for a large fraction of analyzed cases. For example, >98% of CMT1A cases are caused by a duplication that results in partial trisomy of the 17p11.2 region that includes the PMP22 locus, whereas <2% are due to missense mutations in the PMP22 gene itself (55). In AS, large maternal deletions account for 70% of cases, uniparental disomy and imprinting mutations for an additional 5%, and inactivating mutations in UBE3A for another 5% (56). In marked contrast, only 2–13% of NF1 cases result from NF1 microdeletions (16,18,57,58), whereas >70% result from intragenic mutations that predict premature truncation of neurofibromin (8). Understanding why microdeletion is not the prevalent mutational mechanism may reveal important parameters that affect the efficiency of REP-mediated rearrangements. Perhaps the size and sequence identity of NF1REP-P and -M are comparatively less than those of other genomic disorders, thereby reducing the probability of NF1–REP pairing. Or, polymorphism in the number and orientation of, or identity between, NF1REPs may result in a haplotype that is recombination-prone. A precedent for an inversion polymorphism mediated by flanking repetitive repeats has been established (54).
Our data suggest that NF1 microdeletion may also predispose patients to the development of malignant tumors. This hypothesis is supported by our observation that 2 of the 17 (11%) unrelated microdeletion patients had a neurofibrosarcoma (Table 2, UWA124-3 and UWA155-1). This clearly is greater than the expected occurrence of 1.4–3.5% in NF1 patients (21,59), more so given the young age of the microdeletion patients. In addition, first degree affected relatives of two microdeletion patients died of malignancies (Table 2, UWA155-1 and UWA169-1). Further studies are needed to confirm this hypothesis and to determine whether this effect is mediated by the same putative gene that causes early onset of benign neurofibromas. Two lines of evidence suggest that the increased burden of cutaneous neurofibromas in deletion patients would be an unlikely cause of an apparent increased frequency of malignancy. First, cutaneous neurofibromas do not undergo malignant transformation; in cases where neurofibrosarcomas are associated with a neurofibroma it is either a plexiform neurofibroma or a neurofibroma involving a large nerve or nerve plexus (60). Second, the malignancies of the affected first degree relatives of our patients were central nervous system and fibrosarcoma, not neurofibrosarcoma (Table 2).
NF1REP-P and -M-mediated deletion in early embryogenesis may be an underlying mechanism of somatic mosaicism of NF1. It has been proposed that somatic mosaicism may be common among NF1 patients and could explain, for example, cases of a mildly affected parent with a severely affected child (61,62). Patients with somatic mosaicism for an NF1 deletion have been described (16,57,63–65). Because breakpoints were not mapped in these cases, it is not known whether these deletions involved the entire NF1 gene and/or contiguous genes. The frequency of somatic mosaicism for an NF1 deletion was estimated at 1.5% (16,57). However, this may be underestimated significantly due to the low detection rate of the methods employed.
This is the first report of a REP-mediated rearrangement resulting in the loss of a tumor suppressor gene. Therefore, in addition to the germline rearrangements reported here, NF1REP-mediated somatic recombination could be an important mechanism for the LOH at NF1 in tumors of NF1 patients (5,7,66,67). This hypothesis is consistent with our recent analysis of LOH at NF1 in primary leukemic cells of children affected with NF1 that developed malignant myeloid disorders (K. Stephens, M. Weaver, K. Leppig, K. Maruyama, E.D. Davis, R. Espinosa III, M.H. Freedman, P. Emanuel, L. Side, M.M. LeBeau and K. Shannon, unpublished data). LOH in 2 of 20 tumors arose by an interstitial deletion of a 1–2 Mb segment comparable with the germline deletions described here. Additional informative polymorphisms are needed to determine whether the deletion breakpoints are at NF1REP-P and -M. Other examples of clustered neoplasia-related rearrangements could also result from a REP-mediated recombination mechanism. For example, the interstitial 20q deletion in polycythemia vera and myeloid malignancies (54) and the i(17q)-associated hematologic malignancies (68).
The precocious neurofibromagenesis and severe tumor burden of patients with NF1 microdeletions is consistent with our hypothesis that deletion of a gene or regulatory sequence, in conjunction with neurofibromin haploinsufficiency, potentiates development of neurofibromas. All of the deletion patients showed either childhood onset and/or large numbers of cutaneous neurofibromas (Fig. 2, Table 2), with the exception of UWA166-3 who is only 4 years old. Patient UWA113-1 has the smallest deletion of ~1 Mb, thereby establishing a critical interval between FB12A2 and SH3GLP1 as the location of the putative tumor-promoting gene (Fig. 2). These data excluded the strong candidate gene kinase suppressor of ras (KSR) (69). Currently, the critical region is known to harbor four genes, NF1, OMG, EVI2A and EVI2B, two pseudogenes and seven ESTs (Fig. 2, Table 3). The products of these genes are not strong candidates for potentiating neurofibromagenesis. OMG, EVI2A and EVI2B are genes of unknown function located entirely within intron 27b of the NF1 gene, but they are transcribed from the opposite direction. OMG encodes a glycoprotein, OMgp, which is expressed only in the central nervous system in neurons and oligodendrocytes, and is displayed in central nervous system myelin (70,71). Although growth suppression of NIH3T3 fibroblasts overexpressing OMgp suggests that it plays a role in cell proliferation (72), its lack of expression in the peripheral nervous system makes it a poor candidate. EVI2A and -B genes are more widely expressed and predict a putative transmembrane protein of unknown function (73); it is not known whether they are expressed in Schwann cells, which appear to be the progenitor cells of neurofibromas (11). EVI2A and -B are human orthologs of mouse loci where retroviral integration causes myeloid leukemia. Further investigation, however, revealed that it was inactivation of NF1, not the EVI2 genes, that caused the leukemia (74). The identification of patients deleted for OMG, EVI2A, EVI2B or a segment of NF1 along with flanking sequences would be a direct test of a role for these genes in the early onset of neurofibromas. Assuming exclusion of NF1 and the embedded genes, the critical region is reduced to ~700 kb in length. The seven ESTs that we mapped to this region, and the sequence-ready contig, will provide the basis for identifying and characterizing the putative tumor-modifying gene.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Subjects
Patients described previously include UWA106-3 (12,13); UWA69-3, UWA119-1, UWA123-3 and UWA128-3 (13); UWA166-2 and UWA169-1 (19); UWA147-3, UWA156-1, UWA160-1, UWA167-1, UWA172-1 and UWA176–1 (K. Maruyama et al., submitted for publication). Table 2 includes clinical findings from these reports and more recent clinical evaluations. This study was approved by the Institutional Review Boards of the University of Washington and Children’s Hospital and Regional Medical Center (Seattle, WA). Immortalized cell lines and human/rodent somatic cell hybrid lines carrying a single human chromosome 17 were constructed as described previously (13).
BAC library screening
Marker loci were amplified in the presence of [32P]dCTP as described previously (http://www.sanger.ac.uk ). A cocktail of probes, 1 x 106–107 c.p.m./ml hybridization solution each, was used to screen the RPCI-11 human BAC library, segment 4 (BACPAC Resources, Buffalo, NY; http://bacpac.med.buffalo.edu ) by hybridization. Membranes were prehybridized in 25 ml of hybridization buffer (75) at 65°C for 1 h, hybridized overnight, and washed four to six times at increasing stringency, with a final wash of 0.2x SSC/0.1% SDS for 45 min. Following autoradiography for 1–2 days at –70°C with intensifying screens, positive clone addresses were determined and obtained from BACPAC resources. BAC DNA was isolated from 3 ml overnight cultures using the Qiagen Spin miniprep plasmid kit (Qiagen, Chatsworth, CA) according to the manufacturer’s directions.
STSs, ESTs and generation of new markers
Loci were amplified either as described in the database entry or using a program with an initial denaturation of 94°C for 2 min, followed by 35 cycles of 94°C for 15 s, 59°C for 15 s, and 72°C for 60 s, and a final extension of 8 min. Primers for the amplification of D17S117 and D17S120 were designed from partial sequence analysis of plasmid clones. D17S117 was amplified with primers 5'-AGGATGGACTAGGATTCTTAGTG-3' and 5'-GCTGTCAATCACCAAAGTCGAG-3' for D17S117. D17S210 were amplified with primers 5'-CTCGAAGGTAGGATAGTGACAG-3' and 5'-GATAGTTTGAGCTCAGGAATGTG-3'.
New markers were developed from the ends of BAC clone inserts. DNA was extracted from 300 ml of overnight culture from selected BAC clones using the Qiagen MIDI prep plasmid kit. BAC end termini were sequenced using 0.8–1.0 µg of purified BAC DNA, T7 or SP6 primers, and BigDye terminator chemistry (Applied Biosystems, Foster City, CA). Nucleotide sequences were analyzed with Sequencher 3.0 (Gene Codes, Ann Arbor, MI) and primers were designed (Table 5).
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ACKNOWLEDGEMENTS |
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We thank the NF1 patients and their families for their continued cooperation. This research was supported by the Department of the Army, US Army Medical Research and Material Command grant NF960043 awarded to K.S.
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+ To whom correspondence should be addressed. Tel: +1 206 685 9066; Fax: +1 206 685 4829; Email: mod@u.washington.edu
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