Human Molecular Genetics, 2001, Vol. 10, No. 13 1387-1392
© 2001 Oxford University Press
Recombination hotspot in NF1 microdeletion patients
Center for Human Genetics, Catholic University Leuven, Herestraat 49, B-3000 Leuven, Belgium, 1Department of Medicine, University of Washington, Medical Genetics, Seattle, WA, USA, 2Medical and Molecular Genetics Center-IRO, Hospital Duran i Reynals, Barcelona, Spain, 3Servizio di Genetica Medica, Universita di Padova, Padova, Italy, 4Institute of Medical Genetics, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XN, UK, 5Department of Clinical Genetics, Erasmus University Rotterdam, Rotterdam, The Netherlands, 6Department of Clinical Genetics, Maastricht University, Maastricht, The Netherlands, 7Department of Human Genetics, University of Ulm, Ulm, Germany, 8Departments of Neurology and Pediatrics, University of Pennsylvania, Philadelphia, PA, USA and 9Department of Laboratory Medicine, University of Washington, Seattle, WA, USA
Received February 23, 2001; Revised and Accepted April 18, 2001.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Neurofibromatosis type 1 (NF1) patients that are heterozygous for an NF1 microdeletion are remarkable for an early age at onset and an excessive burden of dermal neurofibromas. Microdeletions are predominantly maternal in origin and arise by unequal crossover between misaligned NF1REP paralogous sequence blocks which flank the NF1 gene. We mapped and sequenced the breakpoints in several patients and designed primers within each paralog to specifically amplify a 3.4 kb deletion junction fragment. This assay amplified a deletion junction fragment from 25 of the 54 unrelated NF1 microdeletion patients screened. Sequence analysis demonstrated that each of the 25 recombination events occurred in a discrete 2 kb recombination hotspot within each of the flanking NF1REPs. Two recombination events were accompanied by apparent gene conversion. A search for recombination-prone motifs revealed a
-like sequence; however, it is unknown whether this element stimulates recombination to occur at the hotspot. The deletion-junction assay will facilitate the prospective identification of patients with NF1 microdeletion at this hotspot for genotype–phenotype correlation studies and diagnostic evaluation. |
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Haploinsufficiency for neurofibromin, the protein product of the NF1 gene, causes the autosomal dominant disorder neurofibromatosis type 1 (NF1) (reviewed in refs 1–3). While the majority of cases are caused by subtle private mutations which predict truncation of neurofibromin (4,5), an estimated 5–22% are heterozygous for a germline deletion spanning the 350 kb NF1 locus (6–9). Early reports that deletion patients were remarkable for facial anomalies and an early age at onset of cutaneous neurofibromas, or for excessive numbers relative to age in cases for which age at onset was unknown (10,11), have been confirmed by the identification of additional patients (7,9,12–16). A few deletion cases without this phenotype have been reported (7,9,16), but because the extent of the deletions was not delineated, it is unclear whether they involved the same loci. These observations led to the hypothesis that the NF1 microdeletion resulted in haploinsufficiency for neurofibromin and for the product of a second contiguous locus, which together potentiated neurofibromagenesis (11,17).
This hypothesis was supported by recent data showing that 80% (n = 17) of microdeletion breakpoints were clustered at paralogous sequences which flank the NF1 gene (17). These paralogs, termed NF1REP-P and -M for proximal and medial, respectively, are 
85 kb in length and in direct orientation. NF1REP-mediated deletion most likely occurs by either interchromosomal recombination between misaligned NF1REP elements or intrachromosomal looping-out (17). The analysis of flanking polymorphic loci in family members of affected individuals with de novo microdeletions revealed that unequal crossing over during maternal meiosis I occurred in five out of six cases (18). This is consistent with earlier findings that 80% of NF1 microdeletions are maternal in origin (6,19).
Other than one expressed pseudogene and four expressed sequence tags (ESTs) (17), nothing is known of putative genes or sequence motifs in the NF1REP elements. Towards elucidating the molecular basis of NF1 microdeletion and the genes involved, we mapped breakpoints, developed a deletion junction PCR assay, and analyzed the sequences of junction fragments. These analyses identified a hotspot for recombination between the NF1REP-P and -M paralogs.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Refinement of NF1 deletion breakpoint intervals
Our previous analyses of somatic cell hybrid lines carrying the deleted chromosome of 15 patients with 1.5 Mb deletions showed that in each case the SH3GLP2 locus in NF1REP-P was retained, while SH3GLP1 in NF1REP-M was lost (17, and patient C12 in this paper) (Fig. 1). Further refinement of the homologous recombination sites required identifying NF1REP-specific nucleotides. The strategy was to use the known NF1REP-P sequence to design primers to amplify sequences from a somatic cell hybrid line carrying a patient’s deleted chromosome 17. Products were analyzed either by direct sequencing or single-stranded conformation polymorphism (SSCP) banding patterns. Results were compared with the sequence or banding pattern of NF1REP-P [AC005562, bacterial artificial chromosome (BAC) 271K11] and the draft sequence of NF1REP-M (AC023278, BAC 640N20; AC021852, BAC 474K4). As summarized in Figure 1, these results identified a common breakpoint interval of
3 kb in seven of the 15 cases in which somatic cell hybrids were analyzed. As predicted, Southern blot analysis of BclI-digested DNA probed with a 200 bp fragment identified a novel deletion junction fragment of 11 kb in a patient, but not in the patient’s healthy parents (Figure 2A). This novel fragment was also detected in three additional unrelated de novo microdeletion patients, but not in their healthy parents (data not shown). Some of the breakpoints of the remaining eight hybrid lines carrying deleted chromosomes appear to cluster at a distinct site, but further sequence analysis is required for precise localization.
|
|
Detection of a deletion-specific junction fragment by PCR
To assay this recombination site in other NF1 microdeletion patients, a deletion junction-specific PCR assay was developed. A forward primer specific for NF1REP-P and a reverse primer specific for NF1REP-M amplified a 3.4 kb junction fragment from DNA of patient C12 and from a somatic cell hybrid line carrying the deleted chromosome of this patient. Specificity of the primers was tested using P1-derived artificial chromosomes (PACs) from the different REPs (NF1REP-M, NF1REP-P, NF1REP-D, PAC with paralogous sequence from chromosome 19p), somatic cell hybrids with a deleted chromosome 17 and control DNA. This assay was then performed on DNA from 54 patients known to carry microdeletions extending beyond the borders of the NF1 gene. The 3.4 kb deletion junction fragment was detected in 25 of 54 patients, but not in DNA from 75 control subjects. Figure 2B shows an example of this deletion junction PCR in patient 98-1 and his healthy parents. These results document the specificity of the assay in detecting only chimeric NF1REP sequences that arose from this specific deletion event. A chimeric NF1REP consisting of NF1REP-M and NF1REP-D could be excluded because this would not result in an NF1 phenotype, but in the deletion of about one-third of the long arm of chromosome 17. This was excluded by molecular analysis of the seven somatic cell hybrids and by cytogenetic analysis [including fluorescence in situ hybridization (FISH)] in the remaining cases.
Sequence analysis of deletion junction fragments
The sequence of the amplified 3.4 kb deletion junction fragment from 25 positive NF1 microdeletion patients was determined by direct cycle sequencing. On the basis of available sequences of NF1REP-P, NF1REP-M and our own sequences of this region, we identified 10 REP-specific nucleotide differences (Fig. 3). Analysis of these nucleotides in the patients revealed that the deletion breakpoints were clustered in a 2 kb region of the junction fragment. Fourteen recombination events occurred in the 670 bp segment, two occurred in a 354 bp segment, and seven in a 967 bp segment (Fig. 3). The parental origin of de novo deletions was predominantly maternal, but paternal deletions also occurred at this hotspot (patients 99-2 and 940174). Apparent gene conversion events were detected in two unrelated patients (Fig. 3B). The deletions of de novo NF1 patients 984412 and 973287 occurred on their maternally-derived chromosomes. The sequence of their mother’s NF1REP-P and -M elements identified polymorphisms that facilitated detection of apparent gene conversion events in the patient’s chimeric REP which maximally spanned 907 and 317 bp.
|
Sequence analysis and structure of NF1REP-P and -M
To investigate the molecular basis of NF1REP-mediated microdeletion, we examined the nucleotide sequence of the REPs for overall sequence identity, GC content, recombination prone motif, and the existence of potentially disrupted transcripts. The structures of NF1REP-P and -M are such that
60 kb of the >85 kb paralogs are identical in their arrangement (M. Dorschner and K. Stephens, unpublished data). These 60 kb segments are 98% identical at the nucleotide level, based on comparisons of the finished sequence of NF1REP-P with the available sequence fragments of the draft sequence of NF1REP-M. Because the sequence quality of NF1REP-M is unknown at this time, the precise degree of nucleotide identity of NF1REP-P and -M may be slightly higher or lower. The nucleotide identity over the entire length of the paralogs appears to be consistent as far as the sequence is available. Pairwise sequence comparisons across the lengths of NF1REP-P and -M did not identify regions with higher or lower nucleotide identities. The average GC content of the NF1REPs is 50%, while the 2 kb hotspot is 12% higher. There is a small region 40–45 kb centromeric to the recombination hotspot with an above average GC content. We have not identified any recombinations in or adjacent to this second region of above average GC content.
A search for recombinogenic motifs and replication-associated sequences in or near the recombination hotspot (Materials and Methods) revealed a -like element in the 670 bp recombination interval (Fig. 3; position 142090 of AC005562). WI-12393 is an EST located within each REP (17). A preliminary examination of the gene that includes this EST suggests that it may be disrupted by the deletions described here. It is unknown, however, whether this transcript represents a functional gene or an expressed pseudogene (data not shown).
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Using an NF1 microdeletion junction-specific PCR assay, we demonstrated that the recombination event between the NF1REP-P and -M paralogs occurred in a 3.4 kb fragment in 46% of cases with deletions that spanned the NF1 gene. Sequence and SSCP analyses of the NF1REPs and junction fragments identified REP-specific polymorphisms that enabled us to narrow the breakpoints to a 1889 bp interval near the telomeric end of the NF1REPs (Fig. 1). Within this segment, recombination events clustered in three intervals (Fig. 3). There was no simple correlation between the recombination site and the parental chromosome which underwent deletion. The majority of de novo microdeletions occurred preferentially on the maternally-derived homolog, consistent with previous data (6,19). Importantly, however, deletion of paternally-derived chromosomes can also occur in this hotspot region (Fig. 3).
Although a number of deletion/duplication disorders are caused by recombination between flanking paralogs (reviewed in ref. 20), this is only the second to be analyzed at the nucleotide level. The breakpoints have been sequenced for duplications and deletions that cause CMT1A and HNPP diseases, respectively. These two different neuropathies are caused by recombination between flanking CMT1A-REP paralogs. The disease phenotype of CMT1A or HNPP depends upon whether the patient carries a duplication or a deletion of the dosage-sensitive PMP22 gene located between the CMT1A-REPs (reviewed in refs 21 and 22). There are striking parallels and differences between NF1 microdeletion and CMT1A/HNPP rearrangements. Each REP-mediated recombination event results in a 1.5 Mb rearrangement, yet the NF1REP is over twice the length of the CMT1A-REP. Both rearrangements show parent-of-origin effects. Eighty percent of NF1 deletions are maternal in origin and are generated primarily by unequal meiotic crossing over between chromosome 17 homologs (6,18,19). CMT1A duplications are paternal in origin (92%) and also arise by unequal meiotic crossing over between chromosome 17 homologs (23). Maternal rearrangements, albeit CMT1A duplication or HNPP deletion, occur by unequal intrachromatid exchange or excision of an intrachromatidal loop, respectively. Both the 85 kb NF1REPs and the 24 kb CMT1A-REPs have discrete recombination hotspots of 2 kb and 557 bp (24), respectively. -like sequences are located in or near both recombination hotspots (Fig. 3) (25). In Escherichia coli, elements stimulate recombination in their general vicinity but whether they can function in a similar manner in humans remains to be verified by experimental data. In addition, a mariner-like transposable element lies 700 bp from the CMT1A hotspot. This element does not express functional transposase, but it may be a target for a transposase expressed from other such elements in the genome (25). Although recombination hotspots have been identified within each REP element, this does not necessarily imply that these are high frequency meiotic recombination sites in the genome. Recently, a sperm analysis showed that unequal recombination between the CMT1A-REPs occurs at an average rate for the male genome (1 cM/Mb) (26). It is possible that the special feature of the recombination hotspot region is the combination of a region of high sequence identity and high GC content. However, until now we were unable to find a recombination in the only other region with an above average GC content. Due to the limited availability of sequence from the medial REP (draft quality, unassembled clones) it is impossible at this time to know if other regions of exact sequence identity exist.
Our findings of a recombination hotspot for NF1 microdeletions and the development of a deletion junction-specific PCR assay have significant implications for research and patient care. NF1 mutations are typically private and scattered throughout the 8.5 kb coding region, making detection difficult (4). Prior to the findings described here, the most prevalent mutation was R1947X, which occurred in 1.5% (n = 255) of patients and is not associated with any particular phenotype (27). The microdeletion hotspot described here probably accounts for 5% of NF1 mutations, based on an estimated microdeletion frequency of 10%. The junction-specific PCR assay will facilitate the identification of the first cohort of NF1 patients with the same mutation. Prospective studies are important to determine whether the deletion is predictive of certain clinical manifestations, such as early age at onset of cutaneous neurofibromas. The majority of NF1 microdeletion patients in the current study were selected by phenotype. To date, available medical records have confirmed that 10 of the 25 patients with deletions at the hotspot showed an early age at onset of cutaneous neurofibromas (<10 years) or an excessive number of tumors relative to their age. A study to assess the phenotype of the remaining patients is in progress.
Although we anticipate that the deletion junction fragment PCR assay may be clinically useful in some cases of NF1, we consider its implementation at this time to be premature. To date, we have screened 75 healthy individuals with the assay conditions as described. We do not know the frequency of false positives nor how it might be affected by minor alterations in assay conditions. It is possible that this recombination is a low frequency event during mitosis of hematopoietic cells in healthy individuals, which could be detected by our robust and sensitive PCR assay. The probability of detecting such false positives may be higher if the deleted cells have a growth advantage. In addition, this assay cannot differentiate a germline NF1 microdeletion patient from one with a somatic mosaic microdeletion. There are documented cases of somatic mosaicism for an NF1 microdeletion, although it is not known whether the recombination events occurred at this hotspot (16,28–30). A priori, the germline patient might be expected to have an early onset and a heavy burden of cutaneous neurofibromas, while the somatic might be expected to have a later onset with fewer neurofibromas or other manifestations. In addition, the risk of a mosaic patient having an affected child may be considerably less than that of a germline NF1 deletion patient. Application of the PCR junction fragment assay to the healthy parents of eight of the 17 de novo microdeletion patients described here was negative. Although none of our de novo deletion patients appear to have a mosaic parent, one such case has been described (16).
It is unclear at this time where the breakpoints of the remaining 54% of NF1 microdeletions occur. Preliminary data suggests that there may be additional recombination hotspots in the NF1REP elements. The development of junction-specific PCR assays for other putative recombination sites will be important for diagnosis, genotype/phenotype analyses, and understanding the molecular basis for recombination-prone sites in the genome.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Subjects and cell lines
Peripheral blood samples were obtained after informed consent from 54 NF1 microdeletion patients and their parents, when available. Previous reports document molecular confirmation of deletion in most of the patients (6,9,12,17). In newly ascertained patients, NF1 microdeletions were confirmed by both analysis of polymorphic markers and FISH, as described previously (12). In all cases the microdeletion was known to extend beyond the borders of the NF1 gene. In addition, rodent/human somatic hybrid cell lines carrying only the deleted chromosome 17 homolog were constructed from a subset of patients (11,17).
Fine mapping of NF1 deletion breakpoints
Breakpoints were mapped in somatic cell hybrids by direct sequencing and/or SSCP of amplified products. For SSCP, 15 µl of loading buffer (0.5% dextran blue, 95% formamide) was added to 15 µl of amplified product, heated at 95°C for 3 min and snap cooled on ice for 1 min. Thirty microlitres was electrophoresed through a 0.5x MDE-gel (FMC BioProducts, Rockland, ME) for 10 h at 4°C, 400 V and visualized by fluoro-imager after Sybr Green staining. Forward (F) and reverse (R) primers for SSCP analysis were: stSG31654 F, 5'-TGTGAGGGGCTCTTTCTATTG-3' and stSG31654 R, 5'-AGAGTGATGTTAGCAGCGCA-3'; stSG40093 F, 5'-TGAAGATGTGGACCTGCTGA-3' and stSG40093 R, 5'-TGTTGCCCAGGCTAGTTTTC-3'; 60T7 F, 5'-ATCCTCCGCTTTTTCTCCTT-3' and 60T7 R, 5'-GTTTTAGGGGAGGCCTGTTC-3' (201 bp); 62T7 F, 5'-TGAGAGGCGGGGTGTATTAG-3' and 62T7 R, 5'-TCCTTCTCCAGCCATGTTTC-3' (187 bp); 58T7 F, 5'-GTATGGGGAGCTGCTTTTCC-3' and 58T7 R, 5'-TTCTGTGAGACCTGGGAAGG-3' (217 bp); 5562-142s F, 5'-TACTCACCCCTAGGCCACAG-3' and 5562-142s R, 5'-ACACACTCAGGGACCAACCT-3' (200 bp); 5562-144s F, 5'-TGGCTCCCTACTGTGTTTCC-3' and 5562-144s R, 5'-TCACACAGCGACTCCTTCAC-3' (186 bp); 5562-145s F, 5'-AAATCCCGGCTTCACAGTTA-3' and 5562-145s R, 5'-GGCTGGTCTCAAACTCTTGG-3' (197bp); and WI-9461 (http://gdbwww.gdb.org/).
A Southern blot of 10 µg of BclI-digested DNA, electrophoresed through 0.6% agarose and transferred to Hybond N+ membrane (Amersham, Buckinghamshire, UK), was probed with a 200 bp PCR product from the breakpoint region (5562-142S). The membrane was washed with 2x SSC and 0.1% SDS at 60°C for 30 min, 0.1x SSC and 0.1% SDS at 70°C for 2x 30 min, and exposed to Hyperfilm MP (Amersham) at –70°C for 72 h.
NF1 deletion junction fragment analysis
The 3.4 kb deletion junction fragment was amplified with primers DCF 5'-TCAACCTCCCAGGCTCCCGAA-3' and DTR 5'-AGCCCCGAGGGAATGAAAAGC-3'. A 25 µl PCR was performed using the Expand Long Template PCR System (Roche Molecular Systems, Indianapolis, IN) with 300 ng DNA, 15 pmoles each primer, 0.35 mM dNTPs, 10x PCR buffer 1, and 2.5 U DNA polymerase. After heating to 94°C for 3 min, samples were subjected to 35 cycles of 94°C for 30 s and 68°C for 2.5 min, with a final extension of 7 min at 68°C. Five microlitres of product was electrophoresed through a 1% agarose gel and visualized by EtBr staining.
Junction fragment products were sequenced by cycle sequencing using either the SequiTherm EXCEL II Long Read (Epicentre, Madison, WI) or the Big-Dye Terminator (Applied Biosystems, Foster City, CA) kits. Extension products were analyzed on either an A.L.F. (Automated Laser Fluorescence sequencer, Pharmacia, Uppsala, Sweden) or an ABI 377 sequencer (Applied Biosystems). Raw nucleotide sequences were analyzed with Sequencher (GeneCodes, Ann Arbor, MI), Clustal W (31), and the vector NTI program (Informax, North Bethesda). The nucleotide sequence of the hotspot region was analyzed for a number of recombination prone motifs including: from E.coli (5'-GCTGGTGG-3'), yeast Ade6-M26 heptamer (5'-ATGACGT-3'), XY32 homopurine-homopyrimidine (5'-AAGGGAGAARGGGTATAGGGRAAGAGGGAA-3'), retroposon LTR (5'-TCATACACCACGCAGGGGTAGAGG-3'), LTR-IS (5'-TGGAAATCCCC-3'), human minisatellite core sequence (5'-GGGCAGGAG-3'), two hypervariable minisatellites (5'-GGAGGTGGGCAGGARG-3') and (5'-AGAGGTGGGCAGGTGG-3'), translin consensus 1 (5'-GCNC[A/T][G/C][G/C][A/T] N(0–2) GCCC[A/T][G/C] [G/C][A/T]-3') and consensus 2 (5'-[C/A]TGCAG N(0–4) GCCC[A/T][G/C][G/C][A/T]-3'), and the binding site for the protein pur (5'-GGNNGAGGGAGARRRR-3'). In addition, we searched for sequences associated with DNA replication including Saccharomyces cerevisiae autonomously replicating sequence (ARS) (5'-WTTTATRTTTW-3'), Schizosaccharomyces pombe ARS consensus (5'-WRTTTATTTAW-3'), consensus scaffold attachment regions (5'-AATAAAYAAA-3', 5'-TTWTWTTWTT-3', 5'-WADAWAYAWW-3' and 5'-TWWTDTTWWW-3'), topoisomerase II binding site (5'-GTNWAYATTNATNNR-3'), and human replication origin consensus (5'-WAWTTDDWWWDHWGWHMAWTT-3'). The recombinogenic and DNA replication-associated motifs were described previously (32 and references therein).
|
ACKNOWLEDGEMENTS |
|---|
We thank the NF1 families who collaborated in this study. We are also grateful to Marleen Willems for establishing the lymphoblastoid cell cultures of patients and the somatic cell hybrid C12. C.L.-C. is supported by a grant by the Vlaamse Liga Tegen Kanker, E.L. is part-time clinical researcher of the Fonds voor Wetenschappelijk Onderzoek Vlaanderen (FWO) and P.M. is research director of the FWO-Vlaanderen. This work is also supported by the National Institute of Health (NIH), grant NS36061 (to J.L.R.), the Fondo de Investigaciones Sanitarias de la Seguridad Social (98-0992) and Institut Catalá de la Salut (to C.L.), the Department of the Army, US Army Medical Material Command grant NF960043 (to K.S.) the Fonds voor Wetenschappelijk Onderzoek Vlaanderen (G.0238.98 to E.L.) and the Catholic University of Leuven (A3255 to E.L.).
|
FOOTNOTES |
|---|
+ To whom correspondence should be addressed. Tel: +32 16 345903; Fax: +32 16 346051; Email: Eric.Legius@med.kuleuven.ac.be
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
1 Friedman, J.M. and Riccardi, V.M. (1999) Clinical and epidemiological features. In Friedman, J.M., Gutmann, D.H., MacCollin, M. and Riccardi, V.M. (eds), Neurofibromatosis. Phenotype, Natural History, and Pathogenesis, 3rd edn. The Johns Hopkins University Press, Baltimore, MD, pp. 29–86.
2 Carey, J.C. and Viskochil, D.H. (1999) Neurofibromatosis type 1: A model condition for the study of the molecular basis of variable expressivity in human disorders. Am. J. Med. Genet., 89, 7–13.[Web of Science][Medline]
3 Huson, S.M. (1994) Neurofibromatosis 1: a clinical and genetic overview. In Huson, S.M. and Hughes, R.A.C. (eds), The Neurofibromatoses: A Pathogenetic and Clinical Overview, 1st edn. Chapman and Hall Medical, London, UK, pp. 160–203.
4 Messiaen, L.M., Callens, T., Mortier, G., Beysen, D., Vandenbroucke, I., Van Roy, N., Speleman, F. and De Paepe, A. (2000) Exhaustive mutation analysis of the NF1 gene allows identification of 95% of mutations and reveals a high frequency of unusual splicing defects. Hum. Mutat., 15, 541–555.[Web of Science][Medline]
5 Ars, E., Serra, E., Garcia, J., Kruyer, H., Gaona, A., Lazaro, C. and Estivill, X. (2000) Mutations affecting mRNA splicing are the most common molecular defects in patients with neurofibromatosis type 1. Hum. Mol. Genet., 9, 237–247.
6 Upadhyaya, M., Ruggieri, M., Maynard, J., Osborn, M., Hartog, C., Mudd, S., Penttinen, M., Cordeiro, I., Ponder, M., Ponder, B.A. et al. (1998) 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., 102, 591–597.[Web of Science][Medline]
7 Valero, M.C., Pascual Castroviejo, I., Velasco, E., Moreno, F. and Hernandez-Chico, C. (1997) Identification of de novo deletions at the NF1 gene: no preferential paternal origin and phenotypic analysis of patients. Hum. Genet., 99, 720–726.[Web of Science][Medline]
8 Rasmussen, S.A., Colman, S.D., Ho, V.T., Abernathy, C.R., Arn, P.H., Weiss, L., Schwartz, C., Saul, R.A. and Wallace, M.R. (1998) Constitutional and mosaic large NF1 gene deletions in neurofibromatosis type 1. J. Med. Genet., 35, 468–471.
9 Cnossen, M.H., van der Est, M.N., Breuning, H., van Asperen, C.J., Breslau-Siderius, E.J., van der Ploeg, A.T., de Goede-Bolder, A., van den Ouweland, A.M.W., Halley, D.J.J. and Niermeijer, M.F. (1997) Deletions spanning the neurofibromatosis type 1 gene: implications for genotype-phenotype correlations in neurofibromatosis type 1? Hum. Mutat., 9, 458–464.[Web of Science][Medline]
10 Kayes, L.M., Riccardi, V.M., Burke, W., Bennett, R.L. and Stephens, K. (1992) Large de novo DNA deletion in a patient with sporadic neurofibromatosis 1, mental retardation, and dysmorphism. J. Med. Genet., 29, 686–690.
11 Kayes, L.M., Burke, W., Riccardi, V.M., Bennett, R., Ehrlich, P., Rubenstein, A. and Stephens, K. (1994) Deletions spanning the neurofibromatosis 1 gene: identification and phenotype of five patients. Am. J. Hum. Genet., 54, 424–436.[Web of Science][Medline]
12 Lopez-Correa, C., Brems, H., Lazaro, C., Estivill, X., Clementi, M., Mason, S., Rutkowski, J.L., Marynen, P. and Legius, E. (1999) Molecular studies in 20 submicroscopic neurofibromatosis type 1 gene deletions. Hum. Mutat., 14, 387–393.[Web of Science][Medline]
13 Wu, B.-L., Austin, M., Schneider, G., Boles, R. and Korf, B. (1995) Deletion of the entire NF1 gene detected by FISH: four deletion patients associated with severe manifestations. Am. J. Med. Genet., 59, 528–535.[Web of Science][Medline]
14 Leppig, K.A., Viskochil, D., Neil, S., Rubenstein, A., Johnson, V.P., Zhu, X.L., Brothman, A.R. and Stephens, K. (1996) The detection of contiguous gene deletions at the neurofibromatosis 1 locus with fluorescence in situ hybridization. Cytogenet. Cell Genet., 72, 95–98.[Web of Science][Medline]
15 Leppig, K., Kaplan, P., Viskochil, D., Weaver, M., Orterberg, J. and Stephens, K. (1997) Familial neurofibromatosis 1 gene deletions: cosegregation with distinctive facial features and early onset of cutaneous neurofibromas. Am. J. Med. Genet., 73, 197–204.[Web of Science][Medline]
16 Tonsgard, J.H., Yelavarthi, K.K., Cushner, S., Short, M.P. and Lindgren, V. (1997) Do NF1 gene deletions result in a characteristic phenotype? Am. J. Med. Genet., 73, 80–86.[Web of Science][Medline]
17 Dorschner, M.O., Sybert, V.P., Weaver, M., Pletcher, B.A. and Stephens, K. (2000) NF1 microdeletion breakpoints are clustered at flanking repetitive sequences. Hum. Mol. Genet., 9, 35–46.
18 Lopez-Correa, C., Brems, H., Lazaro, C., Marynen, P. and Legius, E. (2000) Unequal meiotic crossover: a frequent cause of NF1 microdeletions. Am. J. Hum. Genet., 66, 1969–1974.[Web of Science][Medline]
19 Lazaro, C., Gaona, A., Ainsworth, P., Tenconi, R., Vidaud, D., Kruyer, H., Ars, E., Volpini, V. and Estivill, X. (1996) Sex differences in mutational rate and mutational mechanism in the NF1 gene in neurofibromatosis type 1 patients. Hum. Genet., 98, 696–699.[Web of Science][Medline]
20 Lupski, J.R. (1998) Genomic disorders: structural features of the genome can lead to DNA rearrangements and human disease traits. Trends Genet., 14, 417–422.[Web of Science][Medline]
21 Chance, P.F. (1999) Overview of hereditary neuropathy with liability to pressure palsies. Ann. N. Y. Acad. Sci., 883, 14–21.[Web of Science][Medline]
22 Boerkoel, C.F., Inoue, K., Reiter, L.T., Warner, L.E. and Lupski, J.R. (1999) Molecular mechanisms for CMT1A duplication and HNPP deletion. Ann. N. Y. Acad. Sci., 883, 22–35.[Web of Science][Medline]
23 Lopes, J., Vandenberghe, A., Tardieu, S., Ionasescu, V., Levy, N., Wood, N., Tachi, N., Bouche, P., Latour, P., Brice, A. and LeGuern, E. (1997) Sex-dependent rearrangements resulting in CMT1A and HNPP. Nat. Genet., 17, 136–137.[Web of Science][Medline]
24 Reiter, L.T., Hastings, P.J., Nelis, E., De Jonghe, P., Van Broeckhoven, C. and Lupski, J.R. (1998) Human meiotic recombination products revealed by sequencing a hotspot for homologous strand exchange in multiple HNPP deletion patients. Am. J. Hum. Genet., 62, 1023–1033.[Web of Science][Medline]
25 Kiyosawa, H. and Chance, P.F. (1996) Primate origin of the CMT1A-REP repeat and analysis of a putative transposon-associated recombinational hotspot. Hum. Mol. Genet., 5, 745–753.
26 Han, L.L., Keller, M.P., Navidi, W., Chance, P.F. and Arnheim, N. (2000) Unequal exchange at the Charcot–Marie-tooth disease type 1A recombination hot-spot is not elevated above the genome average rate. Hum. Mol. Genet., 9, 1881–1889.
27 Dublin, S., Riccardi, V.M. and Stephens, K. (1995) Methods for rapid detection of a recurrent nonsense mutation and documentation of phenotypic features in neurofibromatosis type 1 patients. Hum. Mutat., 5, 81–85.[Web of Science][Medline]
28 Ainsworth, P.J., Chakraborty, P.K. and Weksberg, R. (1997) Example of somatic mosaicism in a series of de novo neurofibromatosis type 1 cases due to a maternally derived deletion. Hum. Mutat., 9, 452–457.[Web of Science][Medline]
29 Colman, S.D., Rasmussen, S.A., Ho, V.T., Abernathy, C.R. and Wallace, M.R. (1996) Somatic mosaicism in a patient with neurofibromatosis type 1. Am. J. Hum. Genet., 58, 484–490[Web of Science][Medline]
30 Wu, B.L., Boles, R.G., Yaari, H., Weremowicz, S., Schneider, G.H. and Korf, B.R. (1997) Somatic mosaicism for deletion of the entire NF1 gene identified by FISH. Hum. Genet., 99, 209–213.[Web of Science][Medline]
31 Thompson, J.D., Higgins, D.G. and Gibson, T.J. (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res., 22, 4673–4680.
32 Badge, R.M., Yardley, J., Jeffreys, A.J. and Armour, J.A. (2000) Crossover breakpoint mapping identifies a subtelomeric hotspot for male meiotic recombination. Hum. Mol. Genet., 9, 1239–1244.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  E. Vanneste, C. Melotte, S. Debrock, T. D'Hooghe, H. Brems, J.P. Fryns, E. Legius, and J.R. Vermeesch Preimplantation genetic diagnosis using fluorescent in situ hybridization for cancer predisposition syndromes caused by microdeletions Hum. Reprod., June 1, 2009; 24(6): 1522 - 1528. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H Kehrer-Sawatzki and D N Cooper Mosaicism in sporadic neurofibromatosis type 1: variations on a theme common to other hereditary cancer syndromes? J. Med. Genet., October 1, 2008; 45(10): 622 - 631. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A De Luca, I Bottillo, M C Dasdia, A Morella, V Lanari, L Bernardini, L Divona, S Giustini, L Sinibaldi, A Novelli, et al. Deletions of NF1 gene and exons detected by multiplex ligation-dependent probe amplification J. Med. Genet., December 1, 2007; 44(12): 800 - 808. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. H. Shaikh, R. J. O'Connor, M. E. Pierpont, J. McGrath, A. M. Hacker, M. Nimmakayalu, E. Geiger, B. S. Emanuel, and S. C. Saitta Low copy repeats mediate distal chromosome 22q11.2 deletions: Sequence analysis predicts breakpoint mechanisms Genome Res., April 1, 2007; 17(4): 482 - 491. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Barkan, S. Starinsky, E. Friedman, R. Stein, and Y. Kloog The Ras Inhibitor Farnesylthiosalicylic Acid as a Potential Therapy for Neurofibromatosis Type 1. Clin. Cancer Res., September 15, 2006; 12(18): 5533 - 5542. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K.-W. G. Lam and A. J. Jeffreys Inaugural Article: Processes of copy-number change in human DNA: The dynamics of {alpha}-globin gene deletion PNAS, June 13, 2006; 103(24): 8921 - 8927. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K K Mantripragada, A-C Thuresson, A Piotrowski, T Diaz de Stahl, U Menzel, G Grigelionis, R E Ferner, S Griffiths, L Bolund, V Mautner, et al. Identification of novel deletion breakpoints bordered by segmental duplications in the NF1 locus using high resolution array-CGH J. Med. Genet., January 1, 2006; 43(1): 28 - 38. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R Visser, O Shimokawa, N Harada, N Niikawa, and N Matsumoto Non-hotspot-related breakpoints of common deletions in Sotos syndrome are located within destabilised DNA regions J. Med. Genet., November 1, 2005; 42(11): e66 - e66. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Cruts, R. Rademakers, I. Gijselinck, J. van der Zee, B. Dermaut, T. de Pooter, P. de Rijk, J. Del-Favero, and C. van Broeckhoven Genomic architecture of human 17q21 linked to frontotemporal dementia uncovers a highly homologous family of low-copy repeats in the tau region Hum. Mol. Genet., July 1, 2005; 14(13): 1753 - 1762. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. G. Buckley, C. Jarbo, U. Menzel, T. Mathiesen, C. Scott, S. G. Gregory, C. F. Langford, and J. P. Dumanski Comprehensive DNA Copy Number Profiling of Meningioma Using a Chromosome 1 Tiling Path Microarray Identifies Novel Candidate Tumor Suppressor Loci Cancer Res., April 1, 2005; 65(7): 2653 - 2661. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Kurotaki, P. Stankiewicz, K. Wakui, N. Niikawa, and J. R. Lupski Sotos syndrome common deletion is mediated by directly oriented subunits within inverted Sos-REP low-copy repeats Hum. Mol. Genet., February 15, 2005; 14(4): 535 - 542. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Corfas, M. O. Velardez, C.-P. Ko, N. Ratner, and E. Peles Mechanisms and Roles of Axon-Schwann Cell Interactions J. Neurosci., October 20, 2004; 24(42): 9250 - 9260. [Full Text] [PDF]
|
|
|
|
|
|
C. J. Shaw and J. R. Lupski Implications of human genome architecture for rearrangement-based disorders: the genomic basis of disease Hum. Mol. Genet., April 1, 2004; 13(90001): R57 - 64. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M Venturin, P Guarnieri, F Natacci, M Stabile, R Tenconi, M Clementi, C Hernandez, P Thompson, M Upadhyaya, L Larizza, et al. Mental retardation and cardiovascular malformations in NF1 microdeleted patients point to candidate genes in 17q11.2 J. Med. Genet., January 1, 2004; 41(1): 35 - 41. [Full Text] [PDF]
|
|
|
|
|
|
B Castle, M E Baser, S M Huson, D N Cooper, and M Upadhyaya Evaluation of genotype-phenotype correlations in neurofibromatosis type 1 J. Med. Genet., October 1, 2003; 40(10): e109 - 109. [Full Text] [PDF]
|
|
|
|
|
|
H Kehrer-Sawatzki, S Tinschert, and D E Jenne Heterogeneity of breakpoints in non-LCR-mediated large constitutional deletions of the 17q11.2 NF1 tumour suppressor region J. Med. Genet., October 1, 2003; 40(10): e116 - 116. [Full Text] [PDF]
|
|
|
|
|
|
L. Armengol, M. A. Pujana, J. Cheung, S. W. Scherer, and X. Estivill Enrichment of segmental duplications in regions of breaks of synteny between the human and mouse genomes suggest their involvement in evolutionary rearrangements Hum. Mol. Genet., September 1, 2003; 12(17): 2201 - 2208. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E Petek, D E Jenne, J Smolle, B Binder, W Lasinger, C Windpassinger, K Wagner, P M Kroisel, and H Kehrer-Sawatzki Mitotic recombination mediated by the JJAZF1 (KIAA0160) gene causing somatic mosaicism and a new type of constitutional NF1 microdeletion in two children of a mosaic female with only few manifestations J. Med. Genet., July 1, 2003; 40(7): 520 - 525. [Full Text] [PDF]
|
|
|
|
|
|
R. T. Libby, D. G. Monckton, Y.-H. Fu, R. A. Martinez, J. P. McAbney, R. Lau, D. D. Einum, K. Nichol, C. B. Ware, L. J. Ptacek, et al. Genomic context drives SCA7 CAG repeat instability, while expressed SCA7 cDNAs are intergenerationally and somatically stable in transgenic mice Hum. Mol. Genet., January 1, 2003; 12(1): 41 - 50. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.A. BAILEY and E.E. EICHLER Genome-wide Detection and Analysis of Recent Segmental Duplications within Mammalian Organisms Cold Spring Harb Symp Quant Biol, January 1, 2003; 68(0): 115 - 124. [Abstract] [PDF]
|
|
|
|
|
|
P. STANKIEWICZ, K. INOUE, W. BI, K. WALZ, S.-S. PARK, N. KUROTAKI, C.J. SHAW, P. FONSECA, J. YAN, J.A. LEE, et al. Genomic Disorders: Genome Architecture Results in Susceptibility to DNA Rearrangements Causing Common Human Traits Cold Spring Harb Symp Quant Biol, January 1, 2003; 68(0): 445 - 454. [Abstract] [PDF]
|
|
|
|
|
|
X. Estivill, J. Cheung, M. Angel Pujana, K. Nakabayashi, S. W. Scherer, and L.-C. Tsui Chromosomal regions containing high-density and ambiguously mapped putative single nucleotide polymorphisms (SNPs) correlate with segmental duplications in the human genome Hum. Mol. Genet., August 15, 2002; 11(17): 1987 - 1995. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Viskochil Review Article : Genetics of Neurofibromatosis 1 and the NF1 Gene J Child Neurol, August 1, 2002; 17(8): 562 - 570. [Abstract] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||