Genomic organization and evolution of the NF1 microdeletion region

Genomics 84 (2004) 346 – 360 www.elsevier.com/locate/ygeno

Genomic organization and evolution of the NF1 microdeletion region

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Thomas De Raedt, Hilde Brems, Catalina Lopez-Correa, Joris Robert Vermeesch, Peter Marynen, and Eric Legius * Center of Human Genetics, KULeuven, Herestraat 49, 3000 Louvain, Belgium Received 12 December 2003; accepted 26 March 2004 Available online 10 May 2004

Abstract Five to 10% of neurofibromatosis type 1 (NF1) individuals have a microdeletion (1.5 Mb) encompassing the entire NF1 region and neighboring genes. Microdeletion patients have a distinct phenotype with a more severe tumor burden. Most of the microdeletion breakpoints cluster in flanking paralogous regions (NF1REPs). We describe the complete genomic region covering the NF1 microdeletion and an extensive analysis of the genomic and transcriptional organization of the NF1REPs. The flanking NF1REPs have a total length of about 75 kb and are composed of several fragments. One of these fragments originated from chromosome 19 and contains a hot spot for microdeletion breakpoints. The analysis of the genomic organization of the NF1 microdeletion region and of the NF1REPs in particular is important for understanding the mechanism by which NF1 microdeletions are formed. This analysis will also help to identify loci potentially involved in the pathogenesis of the increased tumor load and malignancy risk observed in NF1 microdeletion patients. D 2004 Elsevier Inc. All rights reserved. Keywords: Neurofibromatosis 1; NF1 gene; Gene deletion; Low-copy repeats; Microdeletion

Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder with an incidence of about 1/2500 and a prevalence of 1/4000 [1]. NF1 is a tumor suppressor gene located at 17q11.2 [2]. The main features of the NF1 phenotype are neurofibromas, cafe´-au-lait spots, axillary freckling, Lisch noduli, and learning disabilities. Most of the individuals with NF1 have a small mutation in the NF1 gene (point mutation, small deletion, insertion, or duplication). Five to 10% of NF1 individuals have a microdeletion [3– 5]. This microdeletion encompasses the NF1 gene and its surrounding regions. Individuals with an NF1 microdeletion frequently show a typical phenotype with more neurofibromas at an earlier age, a lower average IQ, facial dysmorphies, and an increased risk for the development of malignant peripheral nerve sheath tumor (MPNST) [6– 11]. The microdeletion region has a length of 1.5 Mb and is flanked by large paralogous sequences called NF1REPs [12,13]. Most NF1 microdeletions are formed during an interchromosomal recombination in meio$ Sequence data from this article have been deposited with the DDBJ/EMBL/GenBank Data Libraries under the accession numbers listed in Table 1. * Corresponding author. Fax: +32-16-346051. E-mail address: [email protected] (E. Legius).

0888-7543/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ygeno.2004.03.006

sis I between misaligned flanking paralogous sequences [14]. Forty-five percent of the microdeletion breakpoints are located in a 2-kb recombination hot spot located in these flanking paralogous sequences [15]. Up to 5% of the human genome is composed of segmental duplications. Their length ranges from 1 to 200 kb with a high sequence identity. It is thought that segmental duplications evolved during recent hominoid evolution (35 million years ago (Mya)) [16,17]. Segmental duplications can be divided further into two classes, interchromosomal and intrachromosomal duplications. Intrachromosomal segmental duplications are restricted to one chromosome or chromosome band. Interchromosomal duplications are located on nonhomologous chromosomes; many of them seem to be present in pericentromeric and subtelomeric regions [16]. In recent years several disorders in which deletions or duplications are mediated by segmental duplications have been described (Williams – Beuren syndrome (7q11.23), Charcot – Marie –Tooth 1A and hereditary neuropathy with liability to pressure palsies (17p11.2), Smith– Magenis syndrome (17p11.2 –p12), Prader – Willi and Angelman syndromes (15q11 – q13), velocardiofacial syndrome (22q11), and neurofibromatosis type 1 (17q11.2)) [18]. In this article we report on the specific genomic organization of the NF1

T. De Raedt et al. / Genomics 84 (2004) 346–360 Fig. 1. The physical map of the NF1 microdeletion region. All identified genes and pseudogenes are indicated with green and red arrows, respectively, and are mentioned in Table 1. Vertical lines above the baseline represent the exons of the genes. STS, EST, and polymorphic markers are indicated under the baseline. Polymorphic markers 3VNF1-1, 3VNF1-2, and 5VNF1-1 were described by Lopez-Correa et al. [22]. The NF1 microdeletion breakpoint hot spot is indicated with a vertical black arrow. NF1REPs a, b, and c are indicated with boxes. The green box represents sequence homologous to the KIAA0563-related gene, the red box and barred red box represent the chromosome 19 derived sequence. The yellow box corresponds to sequence homologous to SMURF2. The blue box represents sequence homologous to JJAZ1. The sequence centromeric to NF1REPa and telomeric to NF1REPc is paralogous to other both intra- and interchromosomal regions (dotted line). These regions were excluded from being part of the NF1REPs because they are not duplicated in the NF1 microdeletion region. The band indicated in gray corresponds to the region with the highest sequence identity between NF1REPs a and c (98.6%). At the top a plot of the GC percentage is given. The NF1 microdeletion breakpoint hot spot corresponds to a peak in GC%. The sequenced BACs and the assembled PAC contig spanning the region are shown at the bottom.

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Table 1 Overview of PAC end ESTs and STSs present in the NF1 microdeletion

, , , , ,

, , , , , , , , ,

,

, ,

, , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , ,

, , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , ,

, , , , , , , , , , , , , , , ,

, , , , , , , , , , , , , , , ,

, , , , , , , , , , , ,

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349

Table 1 (continued)

+, identical sequence mapped in BAC; 0, paralogous copy present in BAC, but identical sequence is mapped to other (paralogous) region. Position 1 of the contig is the first base of BAC AC006050. STSs indicated with * are not mapped to the NF1 microdeletion region. The colors correspond to the different subfragments of the NF1REPs.

microdeletion region and on the structure and origin of the segmental duplications flanking the NF1 microdeletion. The construction of the genomic map of the NF1 microdeletion region was not straightforward due to the presence of these segmental duplications and the absence of correct mapping data in the electronic databases. This map confirms the recently published map of Jenne et al. [19]. This confirmation is important, as a contradictory genomic organization of the NF1 region was published [20].

Results Genomic organization: a PAC contig spanning the NF1 microdeletion region We used a hybrid cell line (C12) containing the common NF1 microdeletion to map 17q11.2 STSs and ESTs in the

deletion region. To map the same markers centromeric or telomeric to NF1 we used a hybrid cell line containing a derivative chromosome 22 from a reciprocal translocation t(17;22) containing part of the long arm of chromosome 17 from NF1 intron 31 to the telomere [21]. A chromosome walk was initiated in both centromeric and telomeric directions starting from NF1. We screened the human PAC libraries RPCI-1, -3, and -5 using ESTs and STSs known to be included in the deletion region. Newly isolated clones were analyzed by fluorescence in situ hybridization (FISH) on metaphase chromosome spreads from NF1 deletion patients. End clones of deleted PACs were then used for additional screening of the PAC library and further chromosome walking. BACs mapping to the region were identified with BLAST searches of the databases. These experiments resulted in the assembly of a set of overlapping clones encompassing the genomic region that is commonly deleted in NF1 microdeletion patients (Fig. 1).

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This physical map of the NF1 microdeletion region contains a total of 90 PACs and about 180 PCR markers with a density of about 1 marker every 8 – 10 kb. Table 1 gives an overview of sequenced PAC ends, known ESTs, and STSs present in the NF1 microdeletion region. Their positions in the BACs and the complete contig are indicated. In our mapping effort we noticed that several STS/EST markers were duplicated in the NF1 microdeletion region. These observations were confirmed by FISH analysis of PACs located in these regions (Fig. 2). The PACs showed crosshybridization to several other genomic regions, both intraand interchromosomal locations (chromosome 19). These results are indicative of the presence of segmental duplications. These segmental duplications or low-copy repeats

(LCR) are called NF1REPs and flank the microdeletion region [12]. In this report the flanking NF1REPs are called NF1REPa and NF1REPc. We found an additional internal NF1REP, called NF1REPb. The NF1 microdeletion breakpoints were mapped to the flanking NF1REPs a and c [12,15]. Database and PCR analysis allowed us to identify a total of 17 genes in the microdeletion region. Table 2 gives an overview of the expressed sequences/genes located in the NF1 microdeletion region. It is remarkable that the genomic region flanking the NF1 gene on the telomeric side contains very few genes. For a long time the genomic organization of the NF1 microdeletion region remained incomplete. With some recent submissions of BAC sequences to the databases we were able to close the last gaps in the genomic sequence of the region. With the completion of the genomic sequence in the region the length of the NF1 microdeletion is calculated to be 1.41 Mb. We also calculated the GC% of the entire NF1 microdeletion region. The NF1 microdeletion breakpoint hot spot, indicated with an arrow on Fig. 1, coincides with a peak in GC%. Several regions with different average levels of GC% can be identified. At the 3V end of the NF1 gene a sharp rise in average GC content is visible. This transition point coincides with a switch in the extent of linkage disequilibrium [23]. Fig. 1 gives an overview of the microdeletion region with the GC% plot, the PAC and BAC map, and the STS markers. The presence of the genes and the NF1REPs is also indicated. Structural organization of the NF1REPs

Fig. 2. FISH results in human, chimpanzee, gorilla, orangutan, and rhesus macaque. BAC RP11-9B11 located on human 17q21.2, in red, is used as a control to identify the human chromosome 17 ortholog in the ape metaphase spreads. The arrow indicates the NF1 region (17q11.2). With probe PAC 1040B18 (green) multiple green signals are observed in human, chimpanzee, gorilla, and orangutan. In rhesus macaque only one strong signal in the 17q21 orthologous region is consistently observed. In about 50% weak hybridization signals are observed in the 17q11 and 17p regions. In all apes hybridization is seen in the 19p13 orthologous region. With probe 896B22 mapped to human chromosome region 19p13.12 hybridization can be seen in all apes on the chromosome 19 ortholog. A signal is also observed in the 17q11 orthologous region of chimpanzee and gorilla, but not in orangutan and rhesus macaque.

Genes located in the NF1REPs of 17q11.2 One of the genes present in the flanking NF1REPs is the KIAA0563-related gene. This gene has 12 exons (3017 bp, cDNA) and contains EST WI-12393. The presence of the KIAA0563-related gene sequences in the NF1 microdeletion region was analyzed with PCR and Southern blot using the described PAC contig as template (data not shown). These results together with BLAST sequence analysis of BAC clones (AC005562, AC027793, AC127025) allowed us to identify the current genomic organization of the KIAA0563related gene (Fig. 3). NF1REPa is located centromeric of the NF1 gene and contains 7 of the 12 exons of the KIAA0563related gene; one of these, exon 8, is present twice. Exons 8 to 12 are in the same orientation. The duplicated copy of exon 8 is inverted (exon 8r) and exons 4 and 5 are also in opposite orientation and located telomeric of exon 12 (exons 4r and 5r). NF1REPb is located between NF1REPa and the NF1 gene and contains 6 exons of the gene related to KIAA0563. Exons 6 to 11 are missing; the distance between exon 5 and exon 12 is 5 kb. NF1REPc is located telomeric of the NF1 gene. It contains 12 exons and an inverted duplication of exons 2 to 5 telomeric to exon 12 (exons 2r, 3r, 4r, and 5r). The hot spot for NF1 microdeletion breakpoints identified so far [15] is located between exon 12 and

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Table 2 List of genes in the NF1 microdeletion region

The genes represented in color are located in the NF1REPs, the specific color used is dependent on the presence of that gene in a certain subfragment of the REP and is the same color as used in Fig. 1. When available Unigene cluster, Locus ID, and GenBank numbers of the genes are given.

the rearranged exon 5r. In NF1REPs a and c the gene related to KIAA0563 is in direct orientation, in NF1REPb it is in inverse orientation compared to the two other copies. Only NF1REPc contains 12 exons in the same orientation and only the NF1REPc copy of the KIAA0563-related gene is expressed. The NF1REPc copy has conserved intron –exon boundaries and has 100% sequence identity with the sequence we obtained from RT-PCR experiments in fibroblast and Schwann cell cDNA (data not shown). LOC147172 is a gene with unknown function and has 9 exons and a total length of 1384 bp. In NF1REPa 9 exons are present and only exons 3 to 6 are present in NF1REPb. Sequence comparison of LOC147172 with the expressed sequence database showed that exons 4, 5, and 6 have a very high sequence identity with SMURF2 (91.5%); exons 7, 8, and 9 of LOC147172 correspond to exons 9, 10, and 11 of the KIAA0563-related gene (sequence identity of 98.4%). Sequencing of a PCR-amplified fragment of LOC147172 from fibroblast cDNA showed that the expressed sequence has 100% sequence identity with the sequence in NF1REPa. This transcript has an open reading frame of 1259 bp. LOC342662: SMURF2 has been mapped to the 17q22 – q23 region (19 exons, 2916-bp cDNA). Sequence corresponding to exons of SMURF2 are present in NF1REPs a and b. SMURF2 exons 1, 4, 5, and 6 are present in NF1REPa (91.5% sequence identity, 488 bp); exons 1, 3, 4, 5, and 6 are present in NF1REPb (91.6% sequence

identity). The copy in NF1REPa is part of the LOC147172 gene (see LOC147172). The largest ORF in NF1REPb is only 336 bp. The EST databases contain several ESTs with 100% sequence identity to the partial copy of SMURF2 in NF1REPb. The ESTs transcribed from NF1REPb correspond only to exons 3, 4, and 5 of SMURF2 and are grouped under the locus name LOC342662. It remains unclear though if this transcribed copy of NF1REPb has any function. LOC147817 is present in both NF1REPs a and c. A transcribed copy of this sequence is located on 19p13.12 and has two exons (2262-bp cDNA). The chromosome 19 locus has a sequence identity of about 96% with the copies in NF1REPs a and c. Neither the copy of 19p13.12 nor the copies in NF1REPs a and c have large ORFs (294 bp for 19p13.2, 270 bp for NF1REPs a and c). Only the chromosome 19 copy is represented in the EST database, and it is only represented once (BU615889). It is not clear if this is functionally important. Two copies of JJAZ1, also known as KIAA0160, are present in the microdeletion region but are not part of the NF1REPs. We will discuss these genes here because they are also present in a duplicated region flanking the NF1 gene (represented in blue in Fig. 1). One copy is localized telomeric of NF1REPa and the other centromeric of NF1REPc. JJAZ1 has 16 exons and a total length of 4441 bp of cDNA. The copy flanking NF1REPa contains exons 1

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to 9 and is probably a pseudogene. The copy centromeric of NF1REPc contains 16 exons and has 100% sequence identity to the cDNA sequence present in the databases. The intron – exon boundaries are conserved. Recently a microdeletion breakpoint in both copies of the JJAZ1 gene has been demonstrated in an NF1 person mosaic for a microdeletion [24]. It has also been shown that the functional copy of JJAZ1 is sometimes disrupted in endometrial stromal sarcomas [25]. Genomic structure of NF1REPs a, b, and c (NF1 microdeletion region) NF1REPs a, b, and c are not exact duplicates. During database analysis and homology searches it became evident that the NF1REPs of the microdeletion region are composed of several fragments in different combinations, orientation, and order. The total length of NF1REPa is 72 kb and the length of NF1REPc is 73 kb. The middle REP (NF1REPb) has a length of about 39 kb. Only 25.8 kb of these are also present in NF1REPs a and c. Some sequences centromeric to NF1REPa and telomeric to NF1REPc are also paralogous to other both intra- and interchromosomal regions (indicated with dotted line in Fig. 1 at the level of the NF1REPs). These regions were excluded as being part of the NF1REPs because they are not duplicated in the NF1 microdeletion region. NF1REPb has three interruptions (5.7, 6.3, and 2.2 kb, respectively). The first interruption in NF1REPb contains a copy of exon 3 of SMURF2, the second is located between exon 6 of SMURF2 and exon 12 of the KIAA0563related gene, the third interruption is located in exon 1 of the KIAA0563-related gene (see Fig. 3). The sequence identities of the different NF1REP subfragments are shown in Table 3. The KIAA0563-related gene is represented in green, fragments of it are present in all three NF1REPs located in the microdeletion region. The KIAA0563-related gene is in direct orientation in the flanking NF1REPs (a and c) and in inverse orientation in NF1REPb (Fig. 3). Only the copy in NF1REPc is expressed and the fragments in NF1REPa are part of LOC147172. The duplicated and inverted exons of the KIAA0563-related gene are also represented in green. The fragment represented in red is present only in NF1REPs a and c and not in NF1REPb (Figs. 1 and 3). FISH experiments and database analysis

(comparison of AC005562 (NF1REPa) with AC011509 (chromosome 19)) showed that the red and the barred red fragments were also present on 19p13.12. The fragment represented by the barred red block is not further duplicated on chromosome 17 and is not considered a part of the NF1REPs. The only chromosome 19 homologous sequences (red) present in NF1REPc are located between exon 12 and the rearranged exon 5r of the KIAA0563-related gene. The microdeletion breakpoint hot spot described by Lopez-Correa et al. [15] is located in the chromosome 19-derived sequences. This hot spot is located in a region with high GC% content compared to the surrounding regions (Fig. 1) and it also contains a CpG island. The fragments represented in yellow are present only in NF1REPs a and b and are in direct orientation. The fragments represented in blue (Fig. 1) are located on the telomeric side of NF1REPa and on the centromeric side of NF1REPc and have a lower sequence identity (94%) then the NF1REPs (Table 3). The JJAZ1 gene/pseudogenes are located in this ‘‘blue’’ fragment. These duplicated blue fragments are not considered subfragments of the NF1REPs. Other related segmental duplications Using FISH and database searches we identified seven additional NF1REP-related paralogous sequences containing fragments of the KIAA0563-related gene. All of the paralogs containing fragments of the KIAA0563-related gene identified so far are located on chromosome 17. Three are present in the NF1 microdeletion region (NF1REPs a, b, and c), 6 at 17q21 (NF1REPs d, e, f, g, h, and i), and 1 at 17q24 (NF1REPj). The accession numbers of BACs containing these paralogs are given in Table 4. Fig. 4 gives a schematic overview of the comparison between the different paralogs. They can be classified as intrachromosomal segmental duplications. The different paralogous sequences have high sequence identities, ranging from 89 to 99.5%. Fig. 5A represents the phylogenetic analysis of NF1REPs a, c, d, e, and j based on sequence comparison using a common genomic sequence between exons 8 and 11 of the gene related to KIAA0563. The analysis of NF1REPs b, c, d, e, and f

Fig. 3. Part of the genomic and transcriptional organization of (a) NF1REPa, (b) NF1REPb, (c) NF1REPc, and (d) SMURF2 is shown. A represents the genomic structure, B the transcripts from that particular genomic sequence. Sequences identical or homologous to SMURF2 are represented in yellow, sequences identical or homologous to the KIAA0563-related gene in green. The red fragment is derived from a sequence on chromosome 19 (not drawn to scale (31.3 kb)). The 2.7-kb NF1 microdeletion breakpoint hot spot [15] is indicated by a hatched box in the red fragment. The vertical black lines represent exons. The number indicated above an exon corresponds to the exon number in the transcribed copy of the gene (the KIAA0563-related gene in green, SMURF2 in yellow). The scale is indicated at the bottom. (a) NF1REPa contains 4 exons of SMURF2 (exons 1, 4, 5, and 6) and copies of 7 different exons of the KIAA0563-related gene (4, 5, 8, 9, 10, 11, and 12). Exons 4 and 5 are inverted and transposed (indicated with r), exon 8 is internally duplicated and inverted (8r). A transcript from NF1REPa named LOC147172 has 9 exons, some of which are homologous to SMURF2 and the KIAA0563-related gene. The exons indicated with an asterisk are not found in the SMURF2 transcript from 17q22 – q23 and may represent cryptic exons. (b) NF1REPb contains 5 exons (exons 1, 3, 4, 5, and 6) of SMURF2 and 6 of the KIAA0563-related gene (exons 1, 2, 3, 4, 5, and 12). Exon 1 of the KIAA0563-related gene is interrupted (2.2 kb between 1b and 1a). Exon 3 of SMURF2 is located in one of the interruptions of NF1REPb (5.7 kb) compared with NF1REPa. A transcript from NF1REPb named LOC342662 has 3 exons corresponding to exons 3, 4, and 5 of SMURF2. (c) NF1REPc contains the KIAA0563-related gene (12 exons), which is transcribed. NF1REPc contains no sequence homology to SMURF2. (d) SMURF2 is located in 17q22 – q23. It contains 19 exons. The genomic distance between exon 1 and exon 2 of SMURF2 (17q22 – q23) is about 55 kb (not drawn to scale).

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Table 3 Sequence identity (%) of the subfragments of NF1REPs a, b, and c NF1REPc

NF1REPb

NF1REPa Red Green Yellow

98.5% 98.6% -

97.5% 97.1%

NF1REPb Green

97.2%

-

represented by the red and red barred blocks in Fig. 1 and 56% is sequence unique for this human chromosome 19 locus. In human, chimpanzee, and gorilla strong signals were observed on both the orthologous chromosome regions 17q11 and 19p.13. A signal was observed only on the orthologous chromosome region 19p13 and not on 17 in orangutan and rhesus macaque (Fig. 2).

Discussion (Fig. 5B) is based on sequence comparison using a genomic sequence between exons 2 and 5. The two regions used in this phylogenetic analysis are shown in Fig. 4. Only sequences in which the considered region was present were used in this analysis. Most of the paralogs from chromosome band 17q21 have an insertion in intron 5 of the KIAA0563-related gene (black-green barred box on Fig. 4). NF1REPd has the largest insertion (15 kb). The other paralogs from 17q21 have a shorter insertion derived from the same 15-kb sequence. Presence of NF1REP sequence in nonhuman primates To detect the presence of orthologous NF1REPs in nonhuman primates we performed FISH analysis on metaphase spreads of cells from chimpanzee, gorilla, orangutan, and rhesus macaque. The FISH results are shown in Fig. 2 and all results are summarized in Table 5. PAC 1040B18 is located in NF1REPc (Fig. 1) and was used as FISH probe. This PAC contains part of the sequence paralogous to chromosome 19 (represented in red on Fig. 1). Thirty-one percent of PAC 1040B18 sequence is paralogous to chromosome 19, 30% contains the rest of NF1REPc represented in green on Fig. 1, 29% consists of unique chromosome 17 sequence, and 10% is paralogous to the Smith – Magenis syndrome REPs (SMS-REPs). PAC 1040B18 hybridized strongly to the 17q11.2 region (NF1 region) and to a lesser extent to 17q21 and 17q24 in human. Sometimes a weak signal was observed in 17p11.2. PAC 1040B18 also hybridized to three different loci on the ortholog of human chromosome 17 in chimpanzee, gorilla, and orangutan (Fig. 2). Only a single strong signal could be detected consistently on the chromosome 17 ortholog of the rhesus macaque at the orthologous position 17q21 (at the position of control probe 9B11 in red). In addition weak signals could be observed at two other positions in 13 of 25 chromosome 17 orthologs analyzed in rhesus macaque (Fig. 2). We also consistently observed a signal on the ortholog of chromosome 19 in the higher apes (gorilla, chimpanzee) and in human (Fig. 2). In only some metaphases of rhesus macaque and orangutan, probe 1040B18 shows weak hybridization to the chromosome 19 ortholog. In addition we performed FISH analysis with PAC 896B22 (located on human chromosome 19). Forty-four percent of probe 896B22 consists of sequence paralogous to the sequence

The present map shows that most genes present in the NF1 microdeletion region are located centromeric of NF1. Our mapping effort revealed the presence of three related segmental duplications in the NF1 microdeletion region (NF1REPs a, b, and c) (Fig. 1). The flanking NF1REPs are large (72 and 73 kb) and composed of several fragments. We did not consider the JJAZ1-containing sequences (blue fragments in Fig. 1) as parts of NF1REPs a and c. They are both separated from NF1REPs a and c by 55 kb of sequence that is unique to the 17q11.2 region. Moreover the sequences indicated in blue have a lower sequence identity, indicating that the duplication occurred probably independent of the NF1REPs. The NF1 microdeletion breakpoint hot spot described in Lopez-Correa et al. [15] is located in the chromosome 19-derived sequence in the flanking NF1REPs (a and c). The hot spot is located between exon 12 and the rearranged exon 5r (Fig. 3) of the KIAA0563related gene on both flanking NF1REPs. Forty-five percent of all NF1 microdeletions with breakpoints in the NF1REPs are localized in this 2-kb hot spot [15]. Meiotic recombination hot spots are usually small (1– 2 kb) and are often associated with high GC% content, CpG islands, and the presence of poly(A) and poly(T) stretches [26,27]. The NF1 microdeletion breakpoint hot spot region has a high GC content and contains remnants of a promoter region and a CpG island, all in concordance with the presence of a meiotic recombination hot spot [15]. It would be interesting to check if this breakpoint hot spot is also a hot spot for natural recombination. Several smaller deletions have been reported, and some of them show breakpoints in JJAZ1 and its pseudogene, Table 4 GenBank accession numbers of sequences corresponding to the different NF1REPs Chromosome band

NF1REP

Accession number(s)

17q11.2

NF1REPa NF1REPb NF1REPc NF1REPd NF1REPe NF1REPf NF1REPg NF1REPh NF1REPi NF1REPj

AC005562, AC027793 AC127025, AC046170 AC005670, AC091132 AC005829, AC091178 AC037487 AC005332

17q21

17q24

AC127024 AC003041 AC068152 AC090419

T. De Raedt et al. / Genomics 84 (2004) 346–360 Fig. 4. Overview of the different regions present in the NF1REPs so far identified on chromosome 17. Most NF1REPs contain exons 1 to 5 of the gene related to KIAA0563. The barred lines on green background represent an insertion of about 15 kb between exons 5 and 6 of the gene related to KIAA0563. The sequence used for the phylogenetic analysis of the different NF1REPs is indicated in gray. The first gray band represents the sequence from exon 2 to 5 and the second from exon 8 to 11.

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Fig. 5. (A) Phylogenetic analysis of NF1REPs a, c, d, e, f, and j based on sequence comparison from genomic sequence between exons 8 and 11 of the gene related to KIAA0563. (B) Phylogenetic analysis of NF1REPs b, c, d, e, f, g, h, i, and j based on sequence comparison from genomic sequence between exons 2 and 5. Only NF1REPs for which the analyzed sequence was present in one fragment were taken into account. The values at the bottom are the values calculated in the distance matrix.

located in the sequence indicated in blue in Fig. 1 [24]. None of these breakpoints have been localized at the nucleotide level so far. It is interesting to note that the breakpoint in the smaller NF1 microdeletions mapping to JJAZ1 and its pseudogene occurred in patients with somatic mosaicism and represent a different mechanism (intrachromosomal recombination during mitosis). Although FISH and molecular analysis indicated that the deletion breakpoint in smaller deletions is localized in the NF1REPb region (internal NF1REP) [19], the exact location of the breakpoint has not been identified yet at the nucleotide level. Therefore it remains unclear to what extent NF1REPb is involved in any of the other smaller deletions.

Several genes/pseudogenes are located in the NF1REPs of 17q11.2. Three of these are transcribed and may be functional genes (KIAA0563-related gene, LOC147172, and LOC342662). The transcribed copy of the KIAA0563related gene is located in NF1REPc. The transcribed copy of LOC147172 is located in NF1REPa and the transcribed copy of LOC342662 is localized in NF1REPb. LOC147172 is composed of partial duplications of SMURF2 (17q22 – q23) and the KIAA0563-related gene (17q11.2). LOC147172 can be considered an example of new gene formation due to exon shuffling. This kind of new gene formation in paralogous sequences has been observed before in the REPs of CMT1A [28].

T. De Raedt et al. / Genomics 84 (2004) 346–360 Table 5 Summary of FISH data PAC 1040B18

Human

Chimpanzee

Gorilla

Orangutan

Rhesus macaque

PAC 896B22

Chr. 17

Chr. 19

17q11 17q21 17q24 17q11 17q21 17q24 17q11 17q21 17q24 17p 17q11 17q21 17p (weak in 13/25) 17q11(weak in 13/25) 17q21 (strong)

19p13

17q11

Chr. 17

19p13

Chr. 19

19p13

17q11

19p13

19p13

17q11

19p13

19p13

No signal

19p13

19p13 (weak)

No signal

19p13

The common NF1 microdeletion breakpoint is located between exon 12 and the rearranged exon 5r of the KIAA0563-related gene. Therefore the transcribed copy of the KIAA0563-related gene (NF1REPc) is deleted in at least 45% of the NF1 microdeletion individuals (Fig. 3). In these individuals LOC147172 is not deleted. Both the JJAZ1 gene and its pseudogene are in direct orientation located on the internal sides of NF1REPs a and c (sequence represented in blue on Fig. 1). Both the pseudogene and the transcribed copy are deleted in 45% of the NF1 microdeletion cases. The NF1REPs are composed of several subfragments, with different sequence identity, orientation, and order. This heterogeneity was observed before in the flanking paralogs of the Williams – Beuren syndrome region (7q11.2) [29]. The complex organization is evidence of a long history of insertions, deletions, and inversions. A total of 10 NF1REP-related segmental duplications were identified. The NF1REP-related segmental duplications on chromosome 17 are located in three chromosome bands (17q11.2, 17q21, and 17q24). Therefore they can all be classified as intrachromosomal segmental duplications. The genomic organization of the NF1 microdeletion region presented here confirms and details the map published by Jenne et al. [19]. In the Jenne et al. paper EST WI-6742 was inadvertently labeled MGC11316, which is an alias for KIAA1821 and RAB11-FIB4. In fact the WI-6742 transcript is different from RAB11-FIB4 and is named HSA272196. The data presented here, in Jenne et al. [19], and in Kehrer-Sawatzki et al. [30] are not compatible with a duplication of NF1 in this region as suggested by others [20]. Sequence comparison represented in Fig. 5 shows that paralogs located within one chromosome band have a higher sequence identity then paralogs located in different chromosome bands. Two explanations can be given for this observation: (1) The NF1REPs were duplicated to a

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certain chromosome band in one time period and duplicated within this chromosome band in a more recent period. (2) There is an interaction between the NF1REPs located close to each other. Gene conversion events between paralogs of the same chromosome band would decrease their differentiation. Gene conversion as a mechanism to keep two sequences from diverging has been observed in the male-specific region of the Y chromosome [31] and in the REPs of CMT1A [32]. We analyzed the presence of the NF1REP orthologs in chimpanzee, gorilla, orangutan, and rhesus macaque by FISH analysis. The separation from the human lineage was 5.5 Mya for chimpanzee, 6.7 Mya for gorilla, 8.2 Mya for orangutan, and 25 Mya for the rhesus macaque. Our FISH analysis demonstrated that the NF1REPs were present and duplicated in many of the nonhuman apes. Hybridization signals at multiple regions were present on the chromosome 17 ortholog in chimpanzee, gorilla, and orangutan. In rhesus macaque the results were different. With the same PAC 1040B18 (NF1REPc) a strong signal at the region orthologous to human 17q21 was observed. In only about 50% of the chromosomes 17 analyzed were very weak signals observed at two other loci. The loci of these two weak signals are orthologous to two loci that show strong hybridization in orangutan. One of these loci coincides with the NF1 region (arrow in Fig. 2), the other locus is located on the short arm of the orthologous chromosome 17. In the other apes, however (chimpanzee, gorilla, and orangutan), all signals were strong, with the signal in the NF1 region the strongest of the three. This is not the case in rhesus macaque. The weak 17q11.2 signal could be explained by cross-hybridization with a SMS-REP fragment in probe 1040B18 (10% of probe) or the 17q11.2 unique sequence (29% of probe). The presence of an SMSREP fragment could also potentially explain the weaker signal in 17p of rhesus macaque. A weak signal in this region is also sometimes observed in human with PAC 1040B18. Park et al. [35] showed that in human the SMSREP is also duplicated along chromosome 17, with hybridization signals for example in 17q11.2. The SMS-REPs are already duplicated in rhesus. Alternatively the data presented here are also compatible with the possibility that the NF1REP orthologs in rhesus macaque were already present at three regions on chromosome 17. The strong signal in the 17q21 orthologous position might be caused by the presence of multiple copies of the NF1REP ortholog. In human we observed seven NF1REP-related copies in this region. On the basis of the sequence divergence between the different segmental duplications one can calculate that the oldest duplication of NF1REP-containing material (indicated in green in Fig. 4) on chromosome 17 took place about 22.1 Mya (89% sequence identity), if a mutation frequency of 1% in 2.1 Mya [33] is assumed. This is very close to the estimated date of the separation of rhesus macaque from the human lineage (25 Mya); therefore the presence of multiple signals on the chromosome

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17 ortholog in rhesus macaque is also compatible with a duplication of the NF1REPs. With probe 1040B18 (NF1REPc) cross-hybridization is observed with the chromosome 19 orthologs. This is to be expected as 31% of PAC 1040B18 is homologous to chromosome 19 sequence. We further investigated the history of the fragment in the NF1REPs that is paralogous with chromosome 19 (red and red barred fragment on Fig. 1) with probe 896B22 (chromosome 19-derived probe). Close to half of probe 896B22 consists of sequence that is paralogous between chromosome 17 and 19 in human. In species in which the chromosome 19 sequence was already duplicated to chromosome 17 one would expect to see a strong signal on both chromosomes 19 and 17. This is observed in human, chimpanzee, and gorilla but not in orangutan and rhesus macaque. The last two have FISH signals only on chromosome 19, indicating that in orangutan and rhesus macaque this sequence is located on chromosome 19 only. The duplication of this chromosome 19 region to the chromosome 17 locus must have occurred after the separation of orangutan from the human lineage (8.2 Mya). The sequence identity between the chromosome 19-derived sequence of NF1REPs a and c on one hand and the sequence on chromosome 19 on the other hand is 96%. It can be estimated from the sequence divergence that the duplication must have occurred about 8.4 Mya. This is very close to the separation of orangutan from the human lineage (8.2 Mya) and is compatible with the FISH data. The FISH data show that initially the NF1REPs were duplicated along chromosome 17 (at least 8.2 Mya, before the separation of orangutan); in a later stage a sequence from chromosome 19 was duplicated and inserted into the NF1REP present in the 17q11.2 region (between 6.7 Mya, the separation of gorilla, and 8.2 Mya, the separation of orangutan). This was probably followed by the duplication of the NF1REPs in the 17q11.2 region because only the NF1REPs of 17q11.2 (NF1 region) contain fragments paralogous to chromosome 19. It is interesting to note that the data presented here are very similar to data observed in other pericentromeric deletion/duplication syndromes. For example the Smith – Magenis and Charcot – Marie – Tooth genomic region (17p11.2) presents a very similar patchwork of duplicated sequences [34]. Similar to the NF1REPs, several SMSREPs are also observed on chromosome 17, even in 17q11.2. They also originated in recent hominoid evolution (after the separation of the New World monkeys from the premonkeys) [35]. All these observations indicate that many segmental duplication events took place in recent hominoid evolution. These duplication events gave rise to a complex genomic architecture predisposing the human genome to both meiotic and mitotic deletion/duplication events at specific locations. The identification of the complete genomic organization and gene content of the NF1 microdeletion region

was necessary to determine the number and type of genes deleted in the NF1 microdeletion patients. It also sheds some light on the mechanism and origin of the deletion and the evolutionary history of the LCRs in that region. An interesting type of experiment to unravel further the mechanism of microdeletion formation would be to analyze the rate of meiotic recombination in the deletion breakpoint hot spots of the NF1REPs. We previously showed that the formation of REP-mediated NF1 microdeletions is associated with a meiotic crossover between misaligned flanking REPs [14,15]. We hypothesize that the NF1 microdeletion breakpoint hot spot might also be a hot spot for normal meiotic recombination. This would explain why the majority of the microdeletion breakpoints are located in a small but distinct (recombining) region of the NF1REPs. It is possible that segmental duplications predispose to the formation of microdeletions only if hot spots for recombination are present in these segmental duplications. Comparing the genotype and phenotype of NF1 microdeletion patients with different deletion sizes and breakpoints might help in identifying genes or mechanisms contributing to the specific NF1 microdeletion phenotype, such as the higher tumor load and elevated risk for malignancy [11].

Materials and methods Cell lines In our mapping efforts two hybrid cell lines were used. One hybrid cell line contains the common NF1 microdeletion (C12), the other hybrid a derivative chromosome 22 from a reciprocal translocation t(17;22) containing the long arm of chromosome 17 from NF1 intron 31 to the telomere [21]. Cell lines from gorilla (Gorilla gorilla) were established in our center. The cell lines from rhesus macaque (PMK, ECACC 98020308) (Macaca mulatta) and orangutan (EB185) (Pongo pygmaeus) were obtained from the European Collection of Cell Cultures and the chimpanzee cell line (AG 0693987) (Pan troglodytes) was obtained from Coriell Cell Repository. Fluorescence in situ hybridization PAC, BAC, P1, and cosmids were labeled by nicktranslation with biotin-16 – dUTP or digoxigenin-11 –dUTP. Chromosome spreads from human (Homo sapiens), chimpanzee (Pan troglodytes), gorilla (G. gorilla), orangutan (Po. pygmaeus), and rhesus macaque (M. mulatta) were prepared using standard cytogenetic techniques. The protocol used for FISH was essentially the same as described in Vermeesch et al. [36]. In addition FISH on metaphase spreads of orangutan and rhesus macaque was performed

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using low-stringency washing conditions. Following hybridization overnight at 37 jC, the slides were washed 5 min in 2 SSC/25% formamide solution at 42 jC, 5 min in 2 SSC solution at 42 jC, and 3 min in 4 SSC, 0.05% Tween 20, pH 7.0. Subsequently, biotin and digoxigenin detection was performed as described [36]. P1 clone P1-9 (f65 kb including NF1 exons 2– 11) was used as probe for the NF1 gene [37]. For the analysis of the origin of the chromosome 19-derived fragment we used PAC 896B22, located on chromosome 19. To probe the NF1REPs we used PAC 1040B18, located in NF1REPc. The control probe for chromosome 17 was BAC 9B11; this probe hybridizes to human chromosome band 17q21.1 and contains the STAT3 gene.

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up table only. Other BLAST-related programs used were ‘‘MEGABLAST’’ and ‘‘BLAST 2 sequences’’. The repeat content of a given sequence was determined by the program REPEATMASKER (http://ftp. genome.washington.edu/cgi-bin/RepeatMasker). For the phylogenetic analysis we initially compared the different sequences with CLUSTALW (http://www2.ebi.ac.uk/ clustalw/). A tree was generated from this aligned sequence using the TreeTop-Phylogenetic Tree Program (http://www.genebee.msu.su/services/phtree_reduced.html). The evolution of the GC% in a sequence was analyzed with the program Vector NTI (7). The prediction of functional domains in the gene related to KIAA0563 was done with the SMART program (http://www.smart. embl-heidelberg.de/).

PAC contig construction High-density membranes of the genomic RPCI-1, -3, and -5 human PAC library from Pieter de Jong (Roswell Park Cancer Institute, Buffalo, NY, USA) (http://bacpac. med.buffalo.edu/) were hybridized with 32P-labeled STSs and ESTs. Positive clones were isolated and DNA was prepared using standard methods. The identified PACs were initially confirmed by PCR using the probe from the original screening. The marker content and relative positions of the different PACs were established by PCR analysis. Different STSs and ESTs located in the region have been tested in these clones to clarify their chromosomal positions in the region and build the PAC contig. To connect the individual PACs STSs were developed from the ends of the clones by vectorette PCR [38]. The ends of the PACs were sequenced and new STSs were generated. Single-strand confirmation polymorphism and sequencing were used to distinguish between STSs located in the paralogous sequences [39]. We also mapped all STSs using somatic cell hybrids containing only chromosome 17 with a microdeletion or a derivative chromosome 22 containing part of chromosome 17 from intron 31 of NF1 until the telomere of the long arm of chromosome 17. This allowed us to map every STS in or out of the microdeletion region and centromeric or telomeric of NF1. Bioinformatics tools used for analysis The BLASTn program (http://www.ncbi.nlm.nih.gov/ BLAST/) at the National Center for Biotechnology Information was used to screen the public database for sequences related to the NF1REPs. The search for new paralogous sequences was done against the ‘‘nr’’ database and the ‘‘htgs’’ database. The nr database contains all nonredundant GenBank, EMBL, DDBJ, and PDB sequences, the htgs database contains all unfinished highthroughput genomic sequences. To avoid problems with repetitive elements in the sequence we used the following filters: low complexity, human repeats, masked for look-

Acknowledgments We thank Prof. Dr. David Viskochil (Department of Pediatrics, University of Utah, Salt Lake City, UT, USA) for providing the NF1-specific clone P1-9 and Dr. Hildegard Kehrer-Sawatzki (Department of Human genetics, University of Ulm, Ulm, Germany) for the hybrid cell line containing the derivative chromosome 22 from a reciprocal t(17;22) translocation [21] and Reinhilde Toelen for technical assistance with the FISH experiments. T.D. is supported by the Vlaams Instituut voor de Bevordering van Wetenschappelijk-Technologisch Onderzoek in de Industrie. E.L. is a part-time clinical researcher of the Fonds voor Wetenschappelijk Onderzoek Vlaanderen (FWO). This work is also supported by the FWO (Grant G.0096.02 to E.L.) and an Interuniversity Attraction Poles grant from the Federal Office for Scientific, Technical, and Cultural Affairs, Belgium (2002 – 2006; P5/25).

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