Angelika C Roehl

Julia Vogt

Tanja Mussotter

Antje N Zickler

Helene Spoeri

Josef Hoegel

Nadia A Chuzhanova

Katharina Wimmer

Lan Kluwe

Victor-Felix Mautner

Helene Spöri

Josef Högel

David N Cooper

Hildegard Kehrer-Sawatzki
email: hildegard.kehrer-sawatzki@uni-ulm.de

John Wiley

Inc Sons

Intrachromosomal mitotic non-allelic homologous recombination is the major molecular mechanism underlying type-2 NF1 deletions

Keywords: mitotic non-allelic homologous recombination, Neurofibromatosis type 1, genomic disorders, non-B DNA, parental and chromosomal origin, recombination hotspot, microdeletions Neurofibromatosis type 1 (NF1), mitotic non-allelic homologous recombination

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Introduction

Genomic disorders result from microdeletions or microduplications of several hundred kilobases whose formation is mediated by the local DNA sequence architecture, in particular region-specific low-copy repeats (LCRs) or segmental duplications [Shaw and Lupski, 2004;[START_REF] Lupski | Genomic disorders ten years on[END_REF][START_REF] Mefford | Duplication hotspots, rare genomic disorders, and common disease[END_REF]Zhang et al., 2009a;[START_REF] Carvalho | Evolution in Health and Medicine Sackler Colloquium: Genomic disorders: a window into human gene and genome evolution[END_REF].

Recurrent microdeletions and their reciprocal microduplications are caused predominantly by non-allelic homologous recombination (NAHR) [START_REF] Gu | Mechanisms for human genomic rearrangements[END_REF][START_REF] Stankiewicz | Structural variation in the human genome and its role in disease[END_REF]. The molecular mechanisms underlying NAHR are assumed to be similar to those of allelic homologous recombination (AHR), but, instead of an allelic homologous template, NAHR employs a similar yet non-allelic template to repair the initiating double strand break (DSB) [START_REF] Steele | Allelic and ectopic interactions in recombination-defective yeast strains[END_REF][START_REF] Hurles | Recombination hotspots in nonallelic homologous recombination[END_REF][START_REF] Sasaki | Genome destabilization by homologous recombination in the germ line[END_REF]. Homologous recombination (HR) is in general a very precise repair mechanism for DNA lesions such as DSBs [START_REF] Mao | DNA repair by nonhomologous end joining and homologous recombination during cell cycle in human cells[END_REF]. Thus, no 'scars' of the DSB-repair process, such as small insertions or deletions, are apparent at the breakpoint junctions repaired by HR, as they often are at the breakpoints of DNA lesions repaired by non-homologous end-joining (NHEJ) [START_REF] Lieber | The mechanism of double-dtrand DNA break repair by the nonhomologous DNA end-joining pathway[END_REF].

Chromosomal regions harbouring multiple duplicated sequences, such as the NF1 gene region, are inherently prone to recurrent NAHR-mediated rearrangements. By contrast, nonrecurrent rearrangements tend to be mediated by NHEJ or by replication-based mechanisms such as 'fork stalling and template switching' (FoSteS) [START_REF] Lee | A DNA replication mechanism for generating nonrecurrent rearrangements associated with genomic disorders[END_REF][START_REF] Vissers | Rare pathogenic microdeletions and tandem duplications are microhomology-mediated and stimulated by local genomic architecture[END_REF]Zhang et al., 2009b]. Although the breakpoint regions of non-recurrent chromosomal rearrangements do not exhibit extensive sequence similarity, they often occur close to LCRs [Shaw et al., 2004;[START_REF] Lupski | Genomic disorders: molecular mechanisms for rearrangements and conveyed phenotypes[END_REF][START_REF] Bauters | Nonrecurrent MECP2 duplications mediated by genomic architecture-driven DNA breaks and break-induced replication repair[END_REF][START_REF] Zhang | Identification of uncommon recurrent Potocki-Lupski syndrome-associated duplications and the distribution of rearrangement types and mechanisms in PTLS[END_REF]. This suggests that the unusual genome architecture associated with LCRs may serve to Large deletions in the NF1 gene region at 17q11.2 (MIM# 613113) belong to the abovementioned group of genomic disorders [START_REF] Jenne | Complete physical map and gene content of the human NF1 tumor suppressor region in human and mouse[END_REF][START_REF] Forbes | Genomic context of paralogous recombination hotspots mediating recurrent NF1 region microdeletion[END_REF]. Two common types of recurrent NF1 deletion have been identified which differ in terms of their size and breakpoint location: type-1 NF1 deletions span 1.4-Mb and are mediated by NAHR between LCRs termed NF1-REP A and NF1-REP C [START_REF] Dorschner | NF1 microdeletion breakpoints are clustered at flanking repetitive sequences[END_REF][START_REF] Jenne | Molecular characterization and gene content of breakpoint boundaries in patients with neurofibromatosis type 1 with 17q11.2 microdeletions[END_REF]Lopez-Correa et al., 2001]. The majority of type-1 NF1 deletions are maternally inherited germ-line deletions [Upadhyaya et al., 1998;[START_REF] López-Correa | Unequal meiotic crossover: a frequent cause of NF1 microdeletions[END_REF][START_REF] Steinmann | Copy number variations in the NF1 gene region are infrequent and do not predispose to recurrent type-1 deletions[END_REF] whose breakpoints are located within two hotspot regions of meiotic recombination termed PRS1 and PRS2 [START_REF] López-Correa | Recombination hotspot in NF1 microdeletion patients[END_REF][START_REF] Forbes | Genomic context of paralogous recombination hotspots mediating recurrent NF1 region microdeletion[END_REF]. By contrast, type-2 NF1 deletions encompass only 1.2-Mb and their breakpoints are located within the SUZ12 gene (MIM# 606245) and its pseudogene SUZ12P which immediately flank the NF1-REPs (Fig. 1). In individuals with type-2 NF1 deletions, somatic mosaicism with normal cells is frequently observed, indicating an early postzygotic (mitotic) origin for these deletions [START_REF] Petek | 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[END_REF][START_REF] Kehrer-Sawatzki | High frequency of mosaicism among patients with neurofibromatosis type 1 (NF1) with microdeletions caused by somatic recombination of the JJAZ1 gene[END_REF][START_REF] Steinmann | Type 2 NF1 deletions are highly unusual by virtue of the absence of nonallelic homologous recombination hotspots and an apparent preference for female mitotic recombination[END_REF]. Whereas an estimated 70% of all NF1 deletions are type-1, only 10-20% are type-2 which are therefore much less frequently encountered than the germline type-1 NF1 deletions [START_REF] Kehrer-Sawatzki | High frequency of mosaicism among patients with neurofibromatosis type 1 (NF1) with microdeletions caused by somatic recombination of the JJAZ1 gene[END_REF].

Although NAHR is a major cause of genome instability in humans, the underlying molecular mechanisms are not well understood. Different types of NAHR have been observed that are distinguishable by virtue of the chromosomal origin of the resulting rearrangements, in other words whether they have arisen via inter-or intra-chromosomal NAHR [reviewed by [START_REF] Gu | Mechanisms for human genomic rearrangements[END_REF]. The chromosomal origins of the various deletions or duplications known to be responsible for genomic disorders have been generally ascertained by microsatellite marker analysis [Thomas et al., 2006 and references therein]. Although type-1 NF1 deletions have previously been shown to arise by interchromosomal NAHR during maternal meiosis I [START_REF] López-Correa | Unequal meiotic crossover: a frequent cause of NF1 microdeletions[END_REF], much less is known about the chromosomal origin and underlying generative mechanism of type-2 NF1 deletions. Until now, only four type-2 deletions had been analysed in terms of their chromosomal origin [START_REF] Steinmann | Type 2 NF1 deletions are highly unusual by virtue of the absence of nonallelic homologous recombination hotspots and an apparent preference for female mitotic recombination[END_REF]. In this study, we have used microsatellite markers and SNP arrays to characterize 14 additional type-2 NF1 deletions in order to acquire insight into the molecular mechanism(s) underlying these unusual somatic deletions. Our study has demonstrated that SNP array analysis represents a valuable tool not only to characterize genomic rearrangements such as microdeletions and microduplications, but also to determine their chromosomal origin. In addition, comparative analysis of type-1 and type-2 deletion breakpoint junctions revealed substantial differences in terms of the precise locations and DNA sequence characteristics of the microdeletion breakpoints that appear to be suggestive of basic mechanistic differences between NAHR events depending upon whether they have occurred during meiosis or during early postzygotic/mitotic cell divisions.

Patients, Materials and Methods

Patients and deletion breakpoint identification

In this study, we have investigated the mechanisms underlying 18 type-2 NF1 deletions. The precise breakpoints of 13 of the 18 type-2 deletions studied here have been previously described [START_REF] Kehrer-Sawatzki | High frequency of mosaicism among patients with neurofibromatosis type 1 (NF1) with microdeletions caused by somatic recombination of the JJAZ1 gene[END_REF][START_REF] Steinmann | Type 2 NF1 deletions are highly unusual by virtue of the absence of nonallelic homologous recombination hotspots and an apparent preference for female mitotic recombination[END_REF]. The breakpoints of 5 newly identified type-2 NF1 deletions were characterized in this study employing the methods summarized in Supp. Table S1. The sequences of the PCR primers used to identify the breakpoints are available from the authors upon request. The clinical features of these 5 newly identified patients with type-2 NF1 deletions are summarized in Supp. Table S2.

Search for the reciprocal duplication

In order to search for duplications reciprocal to the somatic deletions observed in patients with type-2 NF1 deletions mediated by NAHR, we performed duplication-specific F o r P e e r R e v i e w 7 breakpoint-spanning PCRs with forward primers designed to bind specifically to the SUZ12 sequence, and a reverse primer specific for the SUZ12P sequence according to the assay principle described by Turner et al. [2008] (Supp. Table S3). PCR reactions with paralogspecific primers were performed using genomic DNA isolated from peripheral blood of the patients and Platinum Taq polymerase (Invitrogen).

Analysis of somatic mosaicism

Mosaicism for cells with and without the deletion was investigated in the 5 newly identified type-2 NF1 deletions characterized in this study. To this end, FISH analysis was performed on primary blood lymphocytes cultivated for 72h with phytohaemagglutinin. In each case, at least 200 blood cells were evaluated by interphase FISH. FISH analysis was not performed on whole blood cell populations without cultivation. If buccal cells were available for FISH analysis, ~100 interphase nuclei were evaluated. Mosaicism was also investigated by microsatellite marker analysis of DNA isolated from peripheral blood samples and buccal swabs as described [START_REF] Kehrer-Sawatzki | High frequency of mosaicism among patients with neurofibromatosis type 1 (NF1) with microdeletions caused by somatic recombination of the JJAZ1 gene[END_REF]. The results of the mosaicism analysis in all 18 patients with type-2 NF1 deletions so far characterized by ourselves are summarized in (Supp. Table S4).

In order to investigate potential mosaicism for the type-2 deletion in peripheral blood samples taken from the parents of patients 2358 and 2429, deletion-specific breakpointspanning PCR was performed. The primers used for these assays bind to sites of paralogous sequence variants (PSVs) between SUZ12 and SUZ12P. Using such specific primer combinations, only the deletion-specific breakpoint-spanning fragments are amplified by PCR and not sequences that derive from SUZ12 or SUZ12P on the wild-type chromosome 17. The primer sequences used to PCR amplify across the deletion breakpoints of patients 2358 and 2429 are available on request.

Microsatellite marker analysis

Polymorphic microsatellite markers located on chromosome 17 were investigated to determine the chromosomal origin of the deletions, viz. whether the deletions originated via intrachromosomal or interchromosomal recombination. Four of the eighteen deletions (those of patients IL-39, KCD, HC and 697) were previously analysed by this method [START_REF] Steinmann | Type 2 NF1 deletions are highly unusual by virtue of the absence of nonallelic homologous recombination hotspots and an apparent preference for female mitotic recombination[END_REF], whereas 14 patients were investigated for the first time here (Supp. Fig. S1). The parental origin of each chromosome 17 carrying a deletion was also determined. To this end, DNA from all patients was investigated together with DNA from their parents and, if available, their siblings. Genomic DNA was extracted either from venous blood, from buccal swabs or from saliva as indicated in Supp. Fig. S1. Microsatellite markers were also genotyped using DNA extracted from human/mouse somatic cell hybrids containing either the deletion-bearing or the wild-type chromosome 17 of the patient (Supp. Fig. S1). Primer sequences used to PCR-amplify microsatellite markers are available from the authors on request.

SNP array analysis

Sixteen of the 18 patients harbouring somatic type-2 NF1 deletions were genotyped by means of the Human SNP arrays 6.0 (Affymetrix, St. Clara, CA) using DNA extracted from peripheral blood lymphocytes from the patients as previously described [START_REF] Roehl | Extended runs of homozygosity at 17q11.2: an association with type-2 NF1 deletions?[END_REF]Supp. Table S5].

Bioinformatic and statistical analysis

Sequences spanning the breakpoint (recombination) regions of the 16 type-2 NF1 deletions mediated by NAHR were screened for the presence of 109 different DNA sequence motifs (and their complements) of length ≥5 nucleotides (nt) which have been reported to be associated with DNA breakage, repair and recombination [START_REF] Cullen | High-resolution patterns of meiotic recombination across the human major histocompatibility complex[END_REF] , 2003;[START_REF] Ball | Microdeletions and microinsertions causing human genetic disease: common mechanisms of mutagenesis and the role of local DNA sequence complexity[END_REF][START_REF] Myers | A common sequence motif associated with recombination hot spots and genome instability in humans[END_REF][START_REF] Chuzhanova | Gene conversion causing human inherited disease: evidence for involvement of non-B-DNAforming sequences and recombination-promoting motifs in DNA breakage and repair[END_REF][START_REF] Makridakis | Genomic analysis of cancer tissue reveals that somatic mutations commonly occur in a specific motif[END_REF]. A similar analysis was performed in our previous study [START_REF] Steinmann | Type 2 NF1 deletions are highly unusual by virtue of the absence of nonallelic homologous recombination hotspots and an apparent preference for female mitotic recombination[END_REF] but this time the number of recombination-associated motifs investigated was increased from 37 to 109, since in the meantime many more recombination-associated motifs have been identified. In addition, both the actual recombination regions and 100 nt segments flanking them on either side, were analysed by complexity analysis [START_REF] Gusev | On the complexity measures of genetic sequences[END_REF] in order to identify direct, inverted and symmetric repeats ≥ 6 nt separated by no more than 20 bp. Such repeats are capable of non-B DNA formation, in particular slipped, cruciform and triplex structures [START_REF] Wells | Non-B DNA conformations, mutagenesis and disease[END_REF].

For each of the above searches, the statistical significance of our findings was assessed by comparison with 1000 control datasets using z-score statistics [as described in Chuzhanova et al., 2009]. Two regions of chromosome 17, flanking the NF1 gene but not including any of the known recombination hotspots, were used to generate matching control datasets: [coordinates 26,109,477 to 26,359,000 (centromeric to the NF1 gene, between but not including either SUZ12P or the NF1-REPs A and B) and 26,889,356 to 27,203,003 (telomeric to the NF1 gene up to, but not including either SUZ12 or NF1-REP C); human genome assembly hg18 (NCBI build 36)].

The GC-content of the deletion breakpoint regions was determined using RepeatMasker of each overlapping dinucleotide according to the method described by [START_REF] Breslauer | Predicting DNA duplex stability from the base sequence[END_REF].

Results

Deletion breakpoint localization

We initiated this study by identifying the breakpoints of five previously uncharacterized type-2 NF1 deletions. Utilizing paralogous sequence variants (PSVs) to differentiate between the SUZ12 gene and its pseudogene, the breakpoint regions were identified with the highest possible resolution and assigned to recombination regions (RRs) of between 64 bp and 225 bp (Table 1;patients 2358patients , 585, 1956patients , 2429, UC172), UC172). These RRs represent segments of absolute sequence identity between SUZ12 and SUZ12P and must contain the deletion breakpoints themselves. NAHR is the mechanism underlying all five newly characterized type-2 NF1 deletions since the breakpoints were identified at homologous sites between SUZ12 and SUZ12P. The breakpoint data from these five newly characterized type-2 deletions were then combined with the breakpoint data from 13 previously reported cases [START_REF] Steinmann | Type 2 NF1 deletions are highly unusual by virtue of the absence of nonallelic homologous recombination hotspots and an apparent preference for female mitotic recombination[END_REF] in order to make a general assessment of the molecular mechanism underlying type-2 NF1 deletions.

Recombinational mechanism underlying type-2 NF1 deletions

Non-allelic homologous recombination (NAHR) was responsible for the type-2 deletions in 16 of the 18 NF1 patients analysed (Table 1). In only two of the 18 patients studied (patients HC and 928) were the deletion breakpoints not located at homologous sites within SUZ12 and SUZ12P [START_REF] Steinmann | Type 2 NF1 deletions are highly unusual by virtue of the absence of nonallelic homologous recombination hotspots and an apparent preference for female mitotic recombination[END_REF].

Somatic mosaicism

Deleted: was deemed to be Deleted: most likely Deleted: , since the deletion breakpoints were found to be located at homologous sites within the SUZ12 gene and its pseudogene SUZ12P. Indeed, NAHR appears to be the major mechanism responsible for type-2 NF1 deletions ( 11 Somatic mosaicism of cells with and without the type-2 NF1 deletion was noted in 3 of the 5 newly identified patients with type-2 deletions. Taken together with the results of our previous analysis on mosaicism in patients with type-2 NF1 deletions [START_REF] Steinmann | Type 2 NF1 deletions are highly unusual by virtue of the absence of nonallelic homologous recombination hotspots and an apparent preference for female mitotic recombination[END_REF], 16 of the 18 patients studied exhibited somatic mosaicism, indicative of an early postzygotic/mitotic origin for the deletions (Supp. Table S4).

In two patients (2429 and 2358), neither FISH performed on primary blood samples, nor microsatellite marker analysis using DNA from buccal swabs, provided any hint as to the presence of normal cells lacking the NF1 deletion. In addition, neither breakpoint-spanning PCR with primers designed to amplify across the deletion breakpoints, nor FISH analysis of peripheral blood samples, revealed any evidence of low-level mosaicism for the respective deletions in the parents of these patients. The 16 year-old male patient 2429 presented with a severe clinical phenotype characterized by multiple cutaneous and subcutaneous neurofibromas, an MPNST and dysmorphic facial features (Supp. Table S2). The severe manifestations of NF1 exhibited by patient 2429 are consistent with the conclusion that he possesses a de novo germline type-2 NF1 deletion.

Female patient 2358 was 10 years old at the time of investigation and had multiple café-aulait spots, mild facial dysmorphism, mild developmental delay and a plexiform neurofibroma.

Although it is very likely that patient 2358 possesses a de novo germline type-2 NF1 deletion, we were unable to confirm this postulate by skin biopsy analysis since it was declined by the parents of patient 2358.

Chromosomal origin of the type-2 NF1 deletions mediated by NAHR

NAHR causing 16 of the 18 type-2 NF1 deletions under investigation is initiated by the misalignment of the SUZ12 and SUZ12P sequences which exhibit 96.2% similarity over their shared 45 kb lengths. In principle, NAHR could occur within a chromatid, between sisterchromatids or between chromosomes. at different stages during the cell cycle. Thus, 12 intrachromosomal NAHR during G1-phase, which has been found to occur in mammalian cells [START_REF] Stanfield | Cloning and characterization of small circular DNA from Chinese hamster ovary cells[END_REF], would be expected to give rise to a deletion plus an excised circular DNA (Fig. 2A). By contrast, intrachromosomal NAHR post-replication may occur either within one chromatid (intrachromatid NAHR) or between sister chromatids (interchromatid NAHR) (Fig. 2B). Finally, interchromosomal NAHR may occur between non sister-chromatids either in G1-phase (Fig. 3A) or post-replication (Fig. 3B). Depending upon the stage in the cell cycle, the chromosomal consequences of NAHR will be very different.

When NAHR occurs between chromosomes, the deletion-causing NAHR event would be expected to give rise to sequence homozygosity distal to the deletion (Fig. 3B).

Interchromosomal (allelic) homologous recombination does indeed occur in somatic cells and leads to loss of heterozygosity (LOH) which is a common cause of tumorigenesis [START_REF] Hagstrom | Mitotic recombination map of 13cen-13q14 derived from an investigation of loss of heterozygosity in retinoblastomas[END_REF][START_REF] Howarth | A mitotic recombination map proximal to the APC locus on chromosome 5q and assessment of influences on colorectal cancer risk[END_REF] and has been well documented in NF1-associated tumours [START_REF] Garcia-Linares | Dissecting loss of heterozygosity (LOH) in neurofibromatosis type 1-associated neurofibromas: importance of copy neutral LOH[END_REF][START_REF] De Raedt | Conservation of hotspots for recombination in low-copy repeats associated with the NF1 microdeletion[END_REF][START_REF] Upadhyaya | The spectrum of somatic and germline NF1 mutations in NF1 patients with spinal neurofibromas[END_REF]Steinmann et al., 2009;[START_REF] Garcia-Linares | Dissecting loss of heterozygosity (LOH) in neurofibromatosis type 1-associated neurofibromas: importance of copy neutral LOH[END_REF]. In order to investigate the presence of extended regions of homozygosity encompassing the majority of 17q distal to the NF1 type-2 deletions, which would be indicative of the deletions having resulted from interchromosomal NAHR, we evaluated SNP array results for the 16 patients in whom it could be established that the somatic deletions had been mediated by NAHR. The use of SNP arrays to detect somatic loss of heterozygosity was used here for the first time to ascertain the chromosomal origin of type-2 NF1 deletions.

In our previous study, SNP arrays were used to analyse runs of homozygosity (ROHs) in the NF1 gene and its immediate flanking regions [START_REF] Roehl | Extended runs of homozygosity at 17q11.2: an association with type-2 NF1 deletions?[END_REF]. However, in the context of the present study, these SNP array data acquired new utility by helping to determine the presence or absence of an extended region of homozygosity encompassing the majority of chromosome 17q distal to the type-2 NF1 deletions. The presence of homozygosity extending over many Mb of 17q would have suggested that interchromosomal NAHR had caused these S6, Fig. 4). It is most unlikely that these ROHs had originated via interchromosomal recombination events mediating the NF1 deletions since similarly sized ROHs (between 200 kb and 600 kb) were also observed in 41 of 60 CEU individuals evaluated as controls [START_REF] Roehl | Extended runs of homozygosity at 17q11.2: an association with type-2 NF1 deletions?[END_REF]Supp. Table S7]. Since the prevalence of ROHs ranging in size from 200 to 600 kb turned out not to be significantly different in patients with type-2 NF1 deletions as compared with CEU controls (p = 0.37; two-tailed Fisher's Exact test), we concluded that the 16 type-2 NF1 deletions were not generated via interchromosomal NAHR because this mechanism would have given rise to extended regions of homozygosity on 17q. Instead, the 16 deletions were considered to have been caused by intrachromosomal NAHR. To confirm this initial assessment, we performed microsatellite marker analysis using DNA from the patients and their family members as well as DNA from human/mouse somatic cell hybrids containing either the wild-type or the deletion-containing chromosome 17 from the respective patients (Supp. Fig. S1). This analysis indicated that 17 of the 18 type-2 NF1 deletions originated from intrachromosomal recombination. Only in one of the 18 patients, patient 2429, was the type-2 deletion probably caused by interchromosomal NAHR during paternal meiosis since this was the most likely mechanism to account for the observed haplotypes (Supp. Fig. S1-N, Table 2). FISH analysis of primary blood lymphocytes failed to yield any evidence for somatic mosaicism for the deletion in either patient 2429 (Supp. Table S4) or his parents. Deletion breakpoint-specific PCR assays did not detect the deletion in peripheral blood samples of the parents of patient 2429. We therefore concluded that this patient possesses a de novo germline type-2 NF1 deletion. In 13 of the 18 type-2 NF1 deletions investigated, we were able to establish unequivocally whether the deletion had occurred on the paternal or maternal chromosome 17. Four deletions were found to have occurred on the paternal chromosome whereas 9 deletions had occurred on the maternal chromosome (Table 2). Hence, there was no significant preference for the deletions to occur on either the maternal or paternal chromosome (p = 0.267; two-sided exact version of the goodness-of-fit χ 2 -test).

As schematically depicted in Figure 2B, intrachromosomal NAHR between sisterchromatids should in principle also yield cells harbouring duplications reciprocal to the deletions. In order to search for these reciprocal duplications, we performed breakpointspanning PCR with forward primers designed to bind specifically to the SUZ12 sequence, and a reverse primer specific for the SUZ12P sequence according to the assay principle described by Turner et al. [2008] (Supp. Table S3). However, no such reciprocal duplications were detected in DNA isolated from the primary blood samples of any of the patients with type-2 NF1 deletions.

Analysis of a possible clustering of type-2 NF1 deletion breakpoints

Meiotic recombination hotspots have been identified that are confined to genomic regions of between ~500 bp and ~4 kb [START_REF] Reiter | Human meiotic recombination products revealed by sequencing a hotspot for homologous strand exchange in multiple HNPP deletion patients[END_REF]Lopez-Correa et al., 2001;[START_REF] Kauppi | Where the crossovers are: recombination distributions in mammals[END_REF][START_REF] Paigen | Mammalian recombination hot spots: properties, control and evolution[END_REF]. Much less is however known about the existence of mitotic recombination hotspots in general. Using our combined dataset of a total of 16 type-2 deletions of postzygotic origin, we did not identify any significant clustering of breakpoints in the patients tested (Kolmogorov-Smirnov-test, p = 0.205). Despite the general absence of breakpoint clusters, the deletion breakpoints in patients WB and UC172 were nevertheless found to occur within the same 177 bp interval (Fig. 1). The breakpoint regions of the 16 deletions mediated by NAHR were assigned by reference to the paralogous sequence variants (PSVs) which serve to distinguish SUZ12 and SUZ12P.

Structural features of the recombination regions which could have promoted NAHR

These breakpoint regions, also termed recombination regions (RRs), span between 46bp and 225 bp (as summarized in Table 1) and represent segments of absolute sequence identity between SUZ12 and SUZ12P. Since the RRs must contain the deletion breakpoints, they were screened for the presence of known recombination-promoting motifs potentially involved in DNA breakage and repair. However, the only recombination-associated motif found to be overrepresented within the RRs as compared to a control dataset was the χ-like element (CCWCCWGC) and its complementary motif (p < 0.05). Although this motif was found to be overrepresented as compared with the control dataset, it was only observed in 2 of the 16 RRs examined (Supp. Table 8).

In addition to the above motif search, the RRs and extended RRs (which included additional 100 bp segments flanking the RRs on either side) were screened for the presence of repeats capable of mediating non-B DNA structure formation. Inverted repeats were found to be overrepresented within the RRs (p < 0.001) whereas both direct and inverted repeats were overrepresented within the extended RRs (p < 0.001) as compared to control datasets. Taken together, the RRs of 14 of the 16 deletions (88%) caused by NAHR were found to harbour short repeats capable of forming non-B DNA structures (Supp. Figure S2).

Prevalence of Alu elements within the recombination regions

The breakpoints of structural variants in the human genome, such as deletions and insertions mediated by NAHR, have been found to be disproportionately associated with SINE/Alu elements [START_REF] Witherspoon | Alu repeats increase local recombination rates[END_REF][START_REF] Lam | Nucleotide-resolution analysis of structural variants using BreakSeq and a breakpoint library[END_REF]. Consistent with these findings, Alu elements were found to be significantly overrepresented at the type-2 deletion breakpoints as compared with the Alu frequency within the 45 kb segment of the SUZ12 gene homologous to S9). Indeed, 10 of the 16 type-2 NF1 deletions mediated by NAHR actually overlapped with SINE/Alu elements.

DNA stability and GC content

In previous studies, breakpoint regions of structural variants generated by meiotic NAHR events have been found to be (i) biased towards GC-rich regions and (ii) to manifest higher DNA helix stability and lower DNA flexibility as compared with rearrangements caused by non-homologous end joining (NHEJ) [Lopes-Correa et al., 2001;[START_REF] Visser | Identification of a 3.0-kb major recombination hotspot in patients with Sotos syndrome who carry a common 1.9-Mb microdeletion[END_REF][START_REF] Lam | Nucleotide-resolution analysis of structural variants using BreakSeq and a breakpoint library[END_REF]. In order to permit comparison of the physical features of the breakpoints respectively associated with meiotic and mitotic NAHR events within the NF1 gene region, DNA stability and GC-content were determined for both the mitotic type-2 deletion breakpoint regions characterized here, and the PRS1 and PRS2 hotspot regions of meiotic recombination which contain the type-1 NF1 deletion breakpoints. Both the DNA stability and GC-content of the breakpoint regions of type-2 NF1 deletions were found to be significantly lower than in the PRS1 and PRS2 hotspots (p < 0.0001, one-sample t-test; Supp. Tables S10,S11).

Discussion

In this study, we have employed a combination of SNP array and microsatellite marker analysis to determine the chromosomal origin of type-2 NF1 microdeletions and to ascertain the underlying generational mechanism(s). Our analysis revealed that 17 of the 18 type-2 NF1 deletions were generated by an intrachromosomal mechanism. Further, 16 of the 18 type-2 NF1 deletions resulted from intrachromosomal NAHR, whereas the remaining two deletions exhibited breakpoints at non-homologous locations within SUZ12 and SUZ12P (Tables 1 and2). The postzygotic origin of 16 of the 17 type-2 deletions caused by intrachromosomal recombination was evidenced by the presence of mosaicism with normal cells without the S4). Only one of the 18 type-2 NF1 deletions under investigation, the deletion in patient 2429, was mediated by interchromosomal NAHR. In our view, it is unlikely to be a mere coincidence that this was also a documented case of de novo germline type-2 NF1 deletion. Taken together, our findings indicate that type-2 NF1 deletions of early postzygotic origin arise predominantly by intrachromosomal NAHR (Fig. 2). However, the occasionally encountered germline type-2 NF1 deletion mediated by interchromosomal NAHR serves to demonstrate that meiotic recombination is feasible between SUZ12 and SUZ12P even although it is infrequent.

Whereas most type-2 NF1 deletions are of postzygotic origin, the vast majority of type-1 NF1 deletions with breakpoints located within the NF1-REPs A and C are mediated by interchromosomal NAHR during maternal meiosis [START_REF] López-Correa | Unequal meiotic crossover: a frequent cause of NF1 microdeletions[END_REF][START_REF] Steinmann | Copy number variations in the NF1 gene region are infrequent and do not predispose to recurrent type-1 deletions[END_REF]. This mirrors the origin of the germline microdeletions underlying Williams-Beuren syndrome, DiGeorge syndrome and Angelman syndrome which are also predominantly generated by interchromosomal NAHR during either maternal or paternal meiosis [Thomas et al., 2006 and references therein]. The predominance of interchromosomal NAHR events during meiosis may be related to the unique pairing of homologous chromosomes during synapsis, which differs quite radically from any chromosome pairing during the postzygotic cell cycle. Interchromosomal recombination during meiosis may be facilitated by the close synapsis between homologous chromosomes during the prophase of meiosis I, which in mammals lasts for many hours [START_REF] Adler | Comparison of the duration of spermatogenesis between male rodents and humans[END_REF]. Synapsis is fully established at the zygotene stage of meiotic prophase I and involves the intimate association of homologues in the synaptonemal complex (SC) [reviewed in [START_REF] Roeder | Meiotic chromosomes: it takes two to tango[END_REF][START_REF] Gerton | Homologous chromosome interactions in meiosis: diversity amidst conservation[END_REF][START_REF] Ding | From meiosis to postmeiotic events: alignment and recognition of homologous chromosomes in meiosis[END_REF]. The SC appears to facilitate recombination between non-sister chromatids, as evidenced by the observation that yeast mutants which lack the ability to form SCs display a ~10-fold lower recombination rate [START_REF] Rockmill | Meiosis in asynaptic yeast[END_REF][START_REF] Xu | Meiotic cells monitor the status of the interhomolog recombination complex[END_REF]. The pairing of homologous chromosomes in human somatic cells has been visualized by the Deleted: (most likely during paternal meiosis) Deleted: , Deleted: whether allelic or non-allelic, Deleted: However, at least in yeast, chromosome synapsis is not an absolute requirement for recombination to occur [Sym and Roeder, 1994;Storlazzi et al., 1996;[START_REF] Deleted | S. pombe linear elements: the modest cousins of synaptonemal complexes[END_REF][START_REF] Ding | From meiosis to postmeiotic events: alignment and recognition of homologous chromosomes in meiosis[END_REF]. ¶ F o r P e e r R e v i e w 18 analysis of chromosome territories in interphase nuclei, although both inter-chromosome and tissue-specific differences in pairing frequency exist [START_REF] Zeitz | A probabilistic model for the arrangement of a subset of human chromosome territories in WI38 human fibroblasts[END_REF]. However, the pairing of homologous chromosomes during mammalian mitosis is probably different in nature and much less stable than the inter-homologue pairing within the meiotic SC [reviewed by [START_REF] Meaburn | Cell biology: chromosome territories[END_REF]. Despite these surmised differences in chromosome pairing, observations made in yeast mitotic cells have suggested that meiotic and mitotic recombination are likely to share mechanistic similarities. In yeast, it has been shown that homologous recombination is mediated by joint molecule intermediates whose strand composition and size are identical to those of the canonical double Holliday junctions (DHJ) structures observed in yeast meiosis [START_REF] Bzymek | Double Holliday junctions are intermediates of DNA break repair[END_REF]. DHJs form preferentially between yeast sister chromatids during mitotic DSB repair whereas in meiosis a preference for interhomologue recombination has been observed [START_REF] Bzymek | Double Holliday junctions are intermediates of DNA break repair[END_REF]. These observations are analogous to the situation we observe in humans in the context of NF1 deletions: intrachromosomal NAHR is the major mechanism causing type-2 NF1 deletions of postzygotic origin, whereas interchromosomal NAHR during maternal meiosis is responsible for the germline type-1 NF1 deletions.

It is unclear at what point, during the cell cycle, intrachromosomal NAHR gives rise to type-2 NF1 deletions. Most mitotic recombination events are likely to occur during G1-, S-or G2-phase rather than mitosis proper [START_REF] Lafave | Mitotic recombination: why? when? how? where?[END_REF][START_REF] Moynahan | Mitotic homologous recombination maintains genomic stability and suppresses tumorigenesis[END_REF]. The methods adopted in our study (SNP array and microsatellite marker analysis) did not allow us to ascertain whether the intrachromosomal NAHR events occurred in cells during G1-phase or post-replication. The outcomes of intrachromosomal NAHR in the different phases of the cell cycle can be quite diverse. If intrachromosomal NAHR were to have occurred between sister-chromatids (post-replication), then cells bearing the reciprocal duplication might also have arisen in addition to those cells harbouring the type-2 deletion (Fig. 2B). We therefore sought evidence for the presence of the duplication reciprocal to the 19 type-2 deletions by performing FISH on interphase blood cells and PCR on peripheral blood lymphocytes using primers specifically designed to PCR amplify across the predicted duplication breakpoints. In principle, this latter method should have been capable of detecting even small numbers of cells harbouring the duplication. This notwithstanding, we failed to detect the presence of any such duplication in the peripheral blood lymphocytes of patients in whom intrachromosomal NAHR had been established. It is possible that the putative duplication is not represented in the DNA isolated from peripheral blood cells. Even although no evidence for the presence of duplications was forthcoming in this study, we cannot exclude the possibility that post-replication recombination events caused the type-2 NF1 deletions (Fig. 2B). In yeast mitotic cells, homologous recombination is largely confined to the S-and G2-phases. During these time periods, the sister chromatid is available to mediate DBS repair [START_REF] Sjögren | Sister chromatid cohesion is required for postreplicative double-strand break repair in Saccharomyces cerevisiae[END_REF][START_REF] Krogh | Recombination proteins in yeast[END_REF][START_REF] Moynahan | Mitotic homologous recombination maintains genomic stability and suppresses tumorigenesis[END_REF]. Thus, we may also infer that type-2 NF1 deletions are likely to have been generated by intrachromosomal NAHR between sister chromatids rather than within one chromatid.

Analogous to this situation, intrachromosomal NAHR between sister chromatids has been shown to be the major mechanism underlying duplications at the α-globin locus in human blood cells [START_REF] Lam | Processes of de novo duplication of human alpha-globin genes[END_REF].

Allelic and non-allelic meiotic recombination events occur preferentially at specific hotspots, which are usually confined to regions of ~500 bp up to ~3-4 kb [START_REF] Reiter | Human meiotic recombination products revealed by sequencing a hotspot for homologous strand exchange in multiple HNPP deletion patients[END_REF]Lopez-Correa et al., 2001;[START_REF] Kauppi | Where the crossovers are: recombination distributions in mammals[END_REF][START_REF] Lupski | Hotspots of homologous recombination in the human genome: not all homologous sequences are equal[END_REF][START_REF] Kurotaki | Sotos syndrome common deletion is mediated by directly oriented subunits within inverted SoS-REP low-copy repeats[END_REF][START_REF] Myers | A fine-scale map of recombination rates and hotspots across the human genome[END_REF][START_REF] Visser | Identification of a 3.0-kb major recombination hotspot in patients with Sotos syndrome who carry a common 1.9-Mb microdeletion[END_REF][START_REF] De Raedt | Conservation of hotspots for recombination in low-copy repeats associated with the NF1 microdeletion[END_REF][START_REF] Lindsay | A chromosomal rearrangement hotspot can be identified from population genetic variation and is coincident with a hotspot for allelic recombination[END_REF]Torres-Juan et al., 2007;Turner et al., 2008;[START_REF] Wu | Anatomy of mouse recombination hot spots[END_REF][START_REF] Zhang | Identification of uncommon recurrent Potocki-Lupski syndrome-associated duplications and the distribution of rearrangement types and mechanisms in PTLS[END_REF][START_REF] Paigen | Mammalian recombination hot spots: properties, control and evolution[END_REF].

Importantly, some NAHR hotspots have been shown to operate exclusively during meiosis and not during mitosis [Turner et al., 2008]. In the NF1 context, ~80% of all meiotic type-1 deletions display breakpoints located within the PRS1 and PRS2 hotspots, which span 2.9 kb and 3.5 kb, respectively [START_REF] Forbes | Genomic context of paralogous recombination hotspots mediating recurrent NF1 region microdeletion[END_REF][START_REF] De Raedt | Conservation of hotspots for recombination in low-copy repeats associated with the NF1 microdeletion[END_REF]. No such hotspot of deletions are not randomly distributed. Indeed, we noted a disproportionate number of short sequences within the recombination regions (RRs) that are capable of forming non-B DNA.

These repeats may well have contributed to the formation of DSBs [START_REF] Wells | Non-B DNA conformations, mutagenesis and disease[END_REF] that then triggered the NAHR events which ultimately gave rise to the type-2 NF1 deletions. There may also have been some synergy between these repeats and the Alu elements noted at the breakpoints of 10 of the 16 intrachromosomal NAHR-mediated type-2 deletions. Alu elements are well known recombinogenic sequences [START_REF] Witherspoon | Alu repeats increase local recombination rates[END_REF][START_REF] Konkel | A mobile threat to genome stability: The impact of non-LTR retrotransposons upon the human genome[END_REF] and hence probably also contributed to local DSB formation and subsequent NAHR underlying the type-2 deletions.

A degenerate 13 bp sequence motif has recently been found to be enriched within recombination hotspots [START_REF] Myers | A common sequence motif associated with recombination hot spots and genome instability in humans[END_REF]. Although this motif is present within the PRS2 hotspot in the NF1-REPs, it was absent from the breakpoint regions of the type-2 NF1 deletions (Supp. Table S8). However, while primary sequence determinants appear to be necessary, they are insufficient to activate recombination [START_REF] Arnheim | Mammalian meiotic recombination hot spots[END_REF][START_REF] Jeffreys | The rise and fall of a human recombination hot spot[END_REF][START_REF] Myers | Drive against hotspot motifs in primates implicates the PRDM9 gene in meiotic recombination[END_REF]. Thus, in addition to the presence of specific sequence motifs, other factors such as histone modification [START_REF] Buard | Distinct histone modifications define initiation and repair of meiotic recombination in the mouse[END_REF], the binding of specific proteins e.g. Prdm9 [Mets and Myers, 2009;[START_REF] Parvanov | Prdm9 controls activation of mammalian recombination hotspots[END_REF][START_REF] Baudat | PRDM9 is a major determinant of meiotic recombination hotspots in humans and mice[END_REF] and chromatin structure [START_REF] Berchowitz | A positive but complex association between meiotic double-strand break hotspots and open chromatin in Saccharomyces cerevisiae[END_REF] are likely to influence both the location and activity of meiotic recombination hotspots [Székvölgyi and Nicolas, 2009;[START_REF] Paigen | Mammalian recombination hot spots: properties, control and evolution[END_REF]. We have noted marked differences between type-1 and type-2 NF1 deletion breakpoints in terms of their physical properties. Thus, both GC-content and DNA duplex stability were considerably higher within the meiotic PRS1 and PRS2 NAHR hotspots causing type-1 NF1 deletions than within the mitotic type-2 NF1 deletion breakpoints (Supp.

Tables S10,S11 ). Hence, we conclude that in addition to differences in chromosomal origin, there are probably substantial differences between mitotic and meiotic NAHR events in the NF1 gene region with respect to the presence or absence of recombination hotspots as well as GC-content and DNA stability in the respective breakpoint regions. Perhaps significantly, Spo11, which binds to DSBs and initiates meiotic NAHR, is not expressed in mammalian mitotic cells [START_REF] Shannon | Differential gene expression of mammalian SPO11/TOP6A homologs during meiosis[END_REF][START_REF] Nogués | Baseline expression profile of meioticspecific genes in healthy fertile males[END_REF]. It remains to be seen whether these differences between meiotic and mitotic NAHR are specific to the NF1 gene region or whether they may instead reflect a more general difference in the mechanism(s) underlying germline and somatic gross chromosomal rearrangements. [START_REF] Kehrer-Sawatzki | High frequency of mosaicism among patients with neurofibromatosis type 1 (NF1) with microdeletions caused by somatic recombination of the JJAZ1 gene[END_REF] c as determined in this study : the deletion was not caused by NAHR and hence recombination regions were not observed. Since the breakpoints of the deletions were located in regions that are not homologous between SUZ12 and SUZ12P, the breakpoints could be determined precisely. Nucleotide numbering is according to hg18, NCBI 36.

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1 Supplementary Figure S1-A: Analysis of microsatellite markers on chromosome 17 using DNA extracted from blood samples taken from patient 811-1 and his family members. Patient 811-1 has a constitutional type-2 NF1 deletion. He is the son of patient 811-M (his mother) who is mosaic for the deletion. Some 93% of cells in the blood of patient 811-M were found to exhibit the deletion as determined by FISH [START_REF] Steinmann | Type 2 NF1 deletions are highly unusual by virtue of the absence of nonallelic homologous recombination hotspots and an apparent preference for female mitotic recombination[END_REF]. The horizontal rectangular box includes those markers located within the deletion region. Marker alleles in parentheses displayed reduced signal intensity, indicative of somatic mosaicism involving both normal cells and cells bearing the deletion.

--: reduced signal intensity of markers which would have been indicative of somatic mosaicism but could not be formally assessed as a consequence of homozygosity.

--*: a second allele was not visible, although heterozygosity was inferred from the grandpaternal haplotype. del: deletion of the allele. Nucleotide numbering is according to hg18, NCBI 36.

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2 Supplementary Figure S1-B: Analysis of microsatellite markers on chromosome 17 using DNA extracted from peripheral blood taken from patient KCD and hybrid cell lines containing only one chromosome 17 from this patient. Hybrid cell line #3 contains the chromosome 17 with the type-2 NF1 deletion whereas hybrid cell line #25 contains the normal chromosome 17 which was subject to the deletion in a number of cells. Hybrid cell line #101 contains the wild-type chromosome 17 of the patient. All three somatic cell hybrids were obtained by fusion of skin fibroblasts from patient KCD with mouse cell line B82. In skin fibroblasts from patient KCD, 51% of cells carried the deletion whilst 49% of cells were normal. In peripheral blood, the deletion was detected in 92% of cells as determined by FISH [START_REF] Kehrer-Sawatzki | High frequency of mosaicism among patients with neurofibromatosis type 1 (NF1) with microdeletions caused by somatic recombination of the JJAZ1 gene[END_REF][START_REF] Steinmann | Type 2 NF1 deletions are highly unusual by virtue of the absence of nonallelic homologous recombination hotspots and an apparent preference for female mitotic recombination[END_REF]. Marker alleles in parentheses displayed reduced signal intensity, indicative of somatic mosaicism involving both normal cells and cells bearing the deletion. The horizontal rectangular box includes those markers located within the deletion region. del: deletion of the allele. Nucleotide numbering is according to hg18, NCBI 36.

D17S1873

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4 Supplementary Figure S1-D: Analysis of microsatellite markers on chromosome 17 using DNA extracted from blood taken from patient 736 and hybrid cell lines #1 and #26 containing either the deletion-bearing or the normal chromosome 17 from the patient, respectively. Hybrid cell line #15 contains the wild-type chromosome 17 which was subject to the deletion in a number of cells. Patient 736 displays mosaicism for the type-2 NF1 deletion. In peripheral blood, the deletion was detected in 94% of cells while 59% of her buccal smear cells had the deletion as determined by FISH [START_REF] Kehrer-Sawatzki | High frequency of mosaicism among patients with neurofibromatosis type 1 (NF1) with microdeletions caused by somatic recombination of the JJAZ1 gene[END_REF][START_REF] Steinmann | Type 2 NF1 deletions are highly unusual by virtue of the absence of nonallelic homologous recombination hotspots and an apparent preference for female mitotic recombination[END_REF]

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5 Supplementary Figure S1-E: Analysis of microsatellite markers on chromosome 17 using DNA extracted from blood taken from patient 1630 and hybrid cell line #36 containing the wild-type chromosome 17 of the patient. Patient 1630 displays mosaicism for the type-2 NF1 deletion. In peripheral blood, the deletion was detected in 92% of cells as determined by FISH [START_REF] Steinmann | Type 2 NF1 deletions are highly unusual by virtue of the absence of nonallelic homologous recombination hotspots and an apparent preference for female mitotic recombination[END_REF]. The horizontal rectangular box includes those markers located within the deletion region. Marker alleles in parentheses displayed reduced signal intensity, indicative of somatic mosaicism involving normal cells and cells bearing the deletion.

--: reduced signal intensity of markers which would have been indicative of somatic mosaicism but could not be formally assessed as a consequence of homozygosity.

--*: a second allele was not visible, although heterozygosity was inferred form the paternal haplotype.

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7 Supplementary Figure S1-G: Analysis of microsatellite markers on chromosome 17 using DNA extracted from blood samples taken from patient 585 and her parents. Patient 585 has a mosaic type-2 NF1 deletion. The horizontal rectangular box includes those markers located within the deletion region. n.d.: not determined. Marker alleles in parentheses displayed reduced signal intensity, indicative of somatic mosaicism involving both normal cells and cells bearing the deletion.

--: reduced signal intensity of markers which would have been indicative of somatic mosaicism but could not be formally assessed as a consequence of homozygosity.

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8 Supplementary Figure S1-H: Analysis of microsatellite markers on chromosome 17 using DNA from blood samples taken from patient 488. The proportion of cells harbouring the deletion was 98% in peripheral blood and 56% of buccal smear cells, as determined by FISH [START_REF] Kehrer-Sawatzki | High frequency of mosaicism among patients with neurofibromatosis type 1 (NF1) with microdeletions caused by somatic recombination of the JJAZ1 gene[END_REF][START_REF] Steinmann | Type 2 NF1 deletions are highly unusual by virtue of the absence of nonallelic homologous recombination hotspots and an apparent preference for female mitotic recombination[END_REF]

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9 Supplementary Figure S1-I: Analysis of microsatellite markers on chromosome 17 using DNA from patient 1502 and her family members as well as DNA extracted from somatic cell hybrid cells (hybrid #Kl.1-5) containing only the deletion-bearing chromosome 17 from patient 1502. The proportion of cells harbouring the deletion was 97% in peripheral blood and 70% in buccal smear cells, as determined by FISH [START_REF] Steinmann | Type 2 NF1 deletions are highly unusual by virtue of the absence of nonallelic homologous recombination hotspots and an apparent preference for female mitotic recombination[END_REF]

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11 Supplementary Figure S1-K: Analysis of microsatellite markers on chromosome 17 using DNA extracted from blood from patient IL39-III2 and his family members. Patient IL39-III2 has a constitutional type-2 NF1 deletion inherited from his mother (patient IL39) who is mosaic for a type-2 deletion as determined by FISH [START_REF] Petek | 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[END_REF]. The deletion was observed in 70% of peripheral blood lymphocytes and 15% of fibroblasts from patient IL39. DNAs extracted from somatic cell hybrids containing only the chromosome 17 with the deletion of patient IL39-III2 (hybrid cell line #15) and from hybrids containing the wild-type chromosome from patient IL39-III2 (#17) were also analysed. The horizontal rectangular box includes those markers located within the deletion region. del: deletion of the allele.

--: reduced signal intensity of markers which would have been indicative of somatic mosaicism but could not be formally assessed as a consequence of homozygosity.

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13 Supplementary Figure S1-M: Analysis of microsatellite markers on chromosome 17 using DNA from blood samples from patient 928 and her father. DNA samples extracted from somatic cell hybrids carrying only the deletion-bearing chromosome 17 (hybrid #Kl.1) or the wild-type chromosome (hybrid #Kl.3) from patient 928 were also analysed. The deletion was observed in 80% of neurofibroma cells and 55% of buccal smear cells in patient 928 as determined by FISH [START_REF] Kehrer-Sawatzki | High frequency of mosaicism among patients with neurofibromatosis type 1 (NF1) with microdeletions caused by somatic recombination of the JJAZ1 gene[END_REF][START_REF] Steinmann | Type 2 NF1 deletions are highly unusual by virtue of the absence of nonallelic homologous recombination hotspots and an apparent preference for female mitotic recombination[END_REF]

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14 Supplementary Figure S1-N: Analysis of microsatellite markers on chromosome 17 using DNA from blood samples from patient 2429 and his family members as well as DNA extracted from hybrid cell line #Kl.1-5 containing only the chromosome 17 with the deletion from patient 2429. In the brother of patient 2429, a crossover between markers D17S1833 and D17S1788 has to be assumed on the paternal chromosome whilst a crossover between markers D17S1861 and D17S1809 on the maternal chromosome has to be assumed to explain the observed haplotypes.

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15 Supplementary Figure S1-O: Analysis of microsatellite markers on chromosome 17 using DNA from blood samples from patient SB and her family members as well as DNA extracted from somatic cell hybrids #S10 and hybrid #B9 containing respectively only the wildtype chromosome 17 and only the deletion-bearing chromosome 17 from patient SB. The horizontal rectangular box includes those markers located within the deletion region. Patient SB has a germline type-2 NF1 deletion inherited from her mother (patient WB) who exhibited somatic mosaicism for the deletion. The deletion was observed in 94% of blood cells from patient WB, as determined by FISH [START_REF] Kehrer-Sawatzki | High frequency of mosaicism among patients with neurofibromatosis type 1 (NF1) with microdeletions caused by somatic recombination of the JJAZ1 gene[END_REF][START_REF] Steinmann | Type 2 NF1 deletions are highly unusual by virtue of the absence of nonallelic homologous recombination hotspots and an apparent preference for female mitotic recombination[END_REF]

Hybrid #S10

Supplementary Figure S1-P: Analysis of microsatellite markers on chromosome 17 using DNA from blood samples from patient UC172 and her family members as well as DNA extracted from somatic cell hybrids #1 and #2 containing only the wild-type chromosome 17 and only the deletion-bearing chromosome 17 from patient UC172, respectively. The deletion was observed in 86% of blood cells from this patient as determined by FISH (this study). The horizontal rectangular box includes those markers located within the deletion region. Marker alleles in parentheses displayed reduced signal intensity, indicative of somatic mosaicism involving normal cells and cells bearing the deletion.

--: reduced signal intensity of markers which would have been indicative of somatic mosaicism but could not be formally assessed as a consequence of homozygosity. del: deletion of the allele. S1-Q: Analysis of microsatellite markers on chromosome 17 using DNA from blood samples from patient 938 and her family members as well as DNA extracted from a somatic cell hybrid containing only the chromosome 17 bearing the deletion of patient 938. The deletion was observed in 91% of peripheral blood lymphocytes and 80% of buccal smear cells in patient 938 as determined by FISH [START_REF] Kehrer-Sawatzki | High frequency of mosaicism among patients with neurofibromatosis type 1 (NF1) with microdeletions caused by somatic recombination of the JJAZ1 gene[END_REF][START_REF] Steinmann | Type 2 NF1 deletions are highly unusual by virtue of the absence of nonallelic homologous recombination hotspots and an apparent preference for female mitotic recombination[END_REF]. Marker alleles in parentheses were visible as peaks with strongly reduced signal intensity, indicative of somatic mosaicism involving normal cells and cells bearing the deletion. The horizontal rectangular box includes those markers located within the deletion region.

--: reduced signal intensity of markers which would have been indicative of somatic mosaicism but could not be formally assessed as a consequence of homozygosity. --*: a second allele was not visible, although heterozygosity was inferred form the maternal haplotype. del: deletion of the allele. Nucleotide numbering is according to hg18, NCBI 36. 26,093,151-26,093,196 (27,297,456-27,297,501) CAAAATGCAAAAATTAGCCAGGCGTGGTGGTGCACACCTGTAGTCCCAGC 27,297,297,551 TACTCGGGAGGCTGAGGCAGGAGAATTGCCTGAACCCGGGAGGTGGAGGT 27,297,297,601 Patient KCD GCTTGGGTGGTGTTTGTTCATTCATTAACTACCTTTAGAGAGAATGCAGA 26,095,095,260 ACTAAAAATATTAGATTCTTAAAAAAATAGAGTTGGAAAATTTGTCACAG 26,095,095,310 TGCATTTTTCTTTTCCTTCTAGTTTTAAATCACAGCTTACGAATTTAGAT 26,095,095,419 AGGATACACACTTAATGATGCATCATTTATGCATCTCATTGTTTTCCCAT (27,299,299,721) TGTTCTGTT GACTGGTAAAAGACTTCCTTTTTTTCAGTTTGGCTTCCTTCTATTTTAAG [START_REF] Lam | Processes of de novo duplication of human alpha-globin genes[END_REF]299,299,771 TTTGGCTTTGGAATTAAGTGATTTTTTTACTTGGATTATAAAATGATATT 27,299,299,821 Patient 697 CCATGCCTGGCCTTATTTTTATTTTTTGAGACAAGGTCTCGCTTTGTCAC 26,100,100,262 CGAGGCTGGAGTGTAGTGGCATAATGATAGGTCACTGATGGATTGAACTC 26,100,100,312 GTGGACTAAAGGGATCCTCCTGCCTCAGCCTCCTGAGTAGCTGGGACTAC 26,100,100,381 AGGCATGTACTGTCACACC (27,304,304,699) TCACTAATTTTTTGAATTTTTGTAGAGTCAGGGTCTCATCATCTTGCCCA [START_REF] Lam | Processes of de novo duplication of human alpha-globin genes[END_REF]304,304,749 GGCCAGTTTTGAACTCCTATCCTCAAGTGATCCTCCCACCTCGACATCCT 27,304,304,799 Patient 736 AGCCTCCCAAAGCGCTGGGATTATAGCCGTGAGCCACTGCCCCCAGCCGG 26,104,104,540 TTTTTGTATTTTTAGCAGAGACGGAGTTTCACCATGTTGGCCAGGCTGGT 26,104,104,590 CTCGAATTCTTGACCTCAAGTTATCCACCAGCCTCAGCCTCCCAAAGTGC 26,104,104,678 TGGATTACAGGTGTGAGCCACTGCACCTGGCCCTGGAT (27,308,308,982) TACAGGTGTGAGCCACTGCACCTGGCCCTGGCTCACCTTTTGTCCTTTTT [START_REF] Lam | Processes of de novo duplication of human alpha-globin genes[END_REF]308,308,982 TTTTTTTTTTTTTTTTTTTTGAGACAGAGTCTTGCTCTGTCGCCCAGGCT 27,309,309,082 Patient 1630 TTATCTTATGAAAAAAATTTGTTGGGTCATTTTCTGTAAGACACTGCTAG 26,108,108,772 GTGCTAGGGACACTAATAAAAAGATGTATATGGAAAAATAGTGACTAAAC 26,108,108,822 AGTGGTAAATAAGAGTGCTTGTAAAGGGATAAAGTGGTCATTTAGCTCTT 26,108,108,934 GATTGATTGGGTTTAGGGTCTTTGTAGAAGAGGGAGAGAGTATTTTGCTG (27,313,313,271) GATACTTTATTA TTTTCTGCAAGCCTGACCAGGCACAGTGGCTCATGCCTATAATCGCAGCA [START_REF] Lam | Processes of de novo duplication of human alpha-globin genes[END_REF]313,313,321 CTTGGGGAGGCTGAGGTGGGATGATCACTTGCACCCAGGAGTTCATTACC 27,313,313 GTTATGAATGTCTCCTGATCAACAGCTGTATTCTGTTTTGCAGTTTAAAG 26,109,109,864 GTACTTCAAAAGCTTATTGGTGCTTCTGTTTGTTTTATCCCATCCTTATA 26,109,109,914 GTTTTTTTCTATTGTTAATAGATGTTTGTGAAAATAGAAGGAGCACTAGT 26,109,109,978 TCTAGTTCATGCTT (27,316,316,619) GCTTTTGCTTTTCCATAGCTTCTAAAACTAACAGTGAAAATGATATATTG [START_REF] Lam | Processes of de novo duplication of human alpha-globin genes[END_REF]316,316,669 AAACAGTAAATTATAGATCTATACCAGATTTGTAATTGTGTATTTCATTG 27,316,316,719 Patient 585 ATTGGTGCTTCTGTTTGTTTTATCCCATCCTTATAGTTTTTTTCTATTGT 26,109,109,929 TAATAGATGTTTGTGAAAATAGAAGGAGCACTAGTTCTAGTTCATGCTTT 26,109,109,979 CTTTTGCTTTTCCATAGCTTCTAAAACTAACAGTGAAAATGATATATTGA 26,109,110,109 AACAGTAAATTATAGATCTATACCAGATTTGTAATTGTGTATTTCATTGC (27,316,316,750) TATTTGAAGTATAAAGTAATGGTTCTGTAT CATTTATTGGGAAGGTTGAATTTCCATAGCTTCAAAATATTCAAATTTTT 27,316,316,800 TTGCATGGATTTAGCTACGCTTTTCAAAATTTCCTTTTGATGAAGTCATT 27,316,316,850 Patient 488 TTGGAAGGCTGAGGCGGGTGTATCATGAGGCCAGGAGTTCAGGACCAGCC 26,111,111,498 TGGCCAATATGGTGAAACCCCATCTCTACTAAAAATATAAACATTAGCCG 26,111,111,548 GGCGTGGTGGTGGGTGCCTGTAGTCCCAGTTACTTGGGAGACTTAGGCAG 26,111,111,605 AAGAATC (27,318,318,245) ACTTGAACCTGAGAGTCGGAGGTTGCAATGAGCTGAGATTGCGTTACTGC [START_REF] Lam | Processes of de novo duplication of human alpha-globin genes[END_REF]318,318,295 ACTCCAGCCTGGGCGACAGAGCGAGACTCCATCTCAAAAAAAAAAAAAAA 27,318,318,345 Patient 1502 TGAGAGTCAGAGGTTGCAGTGAGCTGAGATTGTGTTACTGCACTGCAGCC 26,111,111,664 TGGGCGACAGAGCGAGACTCCATCTCAGAAAAAAAAAGGGGGGGAAAATT 26,111,111,714 ACTTATCAGCTTATCCATCATAAGTCTGTGTATATGGCATATATTTTTAT 26,111,111,905 TATGCAATGGAATAAAACCATTATTAGAAACATGCCAGGTTGGTTGTCTT (27,318,318,545) GGTATCGTTTAGTAAGAAACAAAGATTGAAAATGAGTCCTGGTGGGCCGG GCACGGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGC TGAGGCGGGTGGATCACAAGGTCAGGAGATCGAGACTATCCTGGCTAACA [START_REF] Lam | Processes of de novo duplication of human alpha-globin genes[END_REF]318,318,595 TGGTGAAACCCCGTCTCTACTAAAATACAAAAAAACTAGCTGGGTGTCGT 27,318,318,645 Patient 1956 GCTTGGGCAACACAGACCCCATCTCTACAAAAATTAGCCAGGTATGGTGG 26,112,112,314 GTATGTCCTGTAGTTCTAGGTACTTGGAAGTCCAAGATGGCAGGATGGCA 26,112,112,364 TAAGCTCAGGAATTCAAGGTTACAGTTACCTATGATTGCACAACTCTACT 26,112,112,510 CCAGGCTGGGCAACAGAGTGAGACTCTGTCTCCAAAAAATCCCAAATATT (27,318,319,061) AGACTGGGCATGGTGGCTCACAGCTATAATGTCAACACCTTGGGAG GCTGAGATGGAAGAATAGCTTGAGGCTTGCCTGGGCAACATAGGGAGAAC [START_REF] Lam | Processes of de novo duplication of human alpha-globin genes[END_REF]319,319,111 CTGTCTGTAAGAAGTAAAAAAAGATTAACTGGGCACAGTGGCACATGCCT 27,319,319,161 Patient IL39 GTTGCCCAATGCCTTAGAACAAAAATTTTTTTTTGTATGTTCATGGATTC 26,115,115,455 ATCTATTATAATAGTTCTGGATGTTATTGAAGCTATTTGATGAATTATTT 26,115,115,505 ATTAAATATTCAGTTATGAACTGTTAATACCTTTGGGACTTTAAAAA 26,115,115,552 (27,322,322,108) CAAGTTATGGAGGACTACTCTAGAACCTTAATTTGTAAAGCCTGTGTTAA [START_REF] Lam | Processes of de novo duplication of human alpha-globin genes[END_REF]322,322,158 TTTACATAGAGAATATAGACTATGGTATTCAAAATTAACACCCCTAAATT 27,322,322,208 Patient 1104 ATTTATTAAATATTCAGTTATGAACTGTTAATACCTTTGGGACTTTAAAA 26,115,115,551 AAAAGTTATGGAGGACTACTCTAGAACCTTAATTTGTAAAGCCTTTGTTA 26,115,115,601 GTTAATTTACATAGAGAATATAGACTATGGTATTCAAAATTAACACCCCT 26,115,115,677 AAATTTTTTGTAAGCCAAGATATTCT (27,322,322,229) AGATAGTAAATAATATCTTGATTTTTGTTATCCCATTTAAACTGTAGAGA [START_REF] Lam | Processes of de novo duplication of human alpha-globin genes[END_REF]322,322,279 AATTCTGGGGAAAGCATCTAAAAATAGCTCTGATAGCTCTGTCTCTACGT 27,322,322 TACCATGCCCAGCTAATTTTTGTGTTTTTAGAGAGATGAGGTTTCACCAT 26,122,122,896 GTTGGCCAGGCTGGTCTGGAGCTCCTGACCTCACATCATCCACCTGCCTA 26,122,122,946 AGCCTCCCAAAGTGCTGGGATTACAGGTGAGCCACCGTGCCCGGCCAGTG 26,122,123,171 TCTTTTTAAAGTCAGAAATTTTATTGTGGATCGAAGATTTAAATGGAAAA (27,329,329,817) CAATAGAACCATAAATGTACTAGAAAATAGTGGTCATGTCTGTGGGGGAA ACAATTAGAATGAGGCGCATTGGACTTCATCGATGCTGTTAATTGTTCTA TTTTAGGCAGCATGGGGTATGTTCT ATTTTCTTTTTTAATCACTGTACCTGACACATATGTTTACACATTTCTTG [START_REF] Lam | Processes of de novo duplication of human alpha-globin genes[END_REF]329,329,867 TAAGAAAAAGATAATGCTTTTATTTTAAAGAAAAAGTGCCAAGAAGAAAC 27,329,329,917 Patients WB and UC172 TCAGGCTGGTCTTGAACTCCCGACCTCAGGTGATCCTCCCACCTCAGCCT 26,125,125,179 CCCGAAGTGTGGGGATTACGGGCATGAGCCACTGCGTCGGGCCAACCTTC 26,125,125,229 TCTTACCTCTGCTCTGAAGTTGTGATTTCTTCAAGGAGTCCTGATTCCTT 26,125,125,406 ATAGAAGAATAGTATTTTTAAGCCAAAATCTGCATGTTAGATGTGTAAAA (27,331,332,070) ATTGTTTTTAAAATCTCACAGAGGGCTGAGTGCAGTGGCTCACACTTGTA ATCTTAGTACTTTGGAAGACCAAGGTG AGAGGATCTCTTGAGCCCAGGAGTCTGAGGCTGTAGTGAGCTATGATTGT 27,332,332,120 GTCACTGCACTCTAGCCTGGGTGACACAGCGACTGTCAAAAAAAAAAACC 27,332,332,170 Patient 938 GCTATGGACTACTTAGAAGGTTGAGCACATTATAGTTATGAACTCCCATT 26,127,127,595 TTTGATTGATGTTTTCTTCCCCAAATGCTAATTCATGTTGGAAGTAGAGG 26,127,127,645 CCTTTGTTTTTTATACTTTAAAAAACACAAGTAAATGATCTAGTCAGAGC 26,127,127,756 ATTTAACGGAAGGTATCATTCCTTTTTTTTTTTTTTTTTTGAGGGGGAGT (27,334,334,446) CTTGCTCTGT T GCCCAGGCTGGAGTGCAGTGGCATGGTCTCGGCTCACTGCAACCTCCACC [START_REF] Lam | Processes of de novo duplication of human alpha-globin genes[END_REF]334,334,496 TCCCGGGTTCAAGCGATTCTCCTGTCTCAGCCTCCCAAGTAGCTGTGATT 27,334,334

D17S1873

                UC172 d f (6) yes     86                
a: mosaicism was investigated by microsatellite marker analysis and putative heterozygosity of markers located within the deletion interval using DNA isolated from peripheral blood samples (primary blood cells) and not from lymphoblastoid cell lines. b: as determined in [START_REF] Steinmann | Type 2 NF1 deletions are highly unusual by virtue of the absence of nonallelic homologous recombination hotspots and an apparent preference for female mitotic recombination[END_REF] c: as determined in [START_REF] Kehrer-Sawatzki | High frequency of mosaicism among patients with neurofibromatosis type 1 (NF1) with microdeletions caused by somatic recombination of the JJAZ1 gene[END_REF] The DNA stability of the RRs of the type-2 NF1 deletions was significantly lower than in the PRS1 (∆G°: 2.008) and PRS2 (∆G°: 2.152) hotspot regions of type-1 NF1 deletions (P < 0.0001; one-sample t-test). CI: confidence interval assigned under the assumption of a normal distribution. The average ∆G° of the complete SUZ12 gene is 1.871, of the SUZ12P pseudogene 1.877, and of the NF1-REP A 1.976.

(

  http://www.repeatmasker.org/cgi-bin/WEBRepeatMasker) and a program provided by EnCor Biotechnology Inc. (http://www.encorbio.com/protocols/Nuc-MW.htm).The potential clustering of type-2 deletion breakpoints mediated by NAHR was assessed by means of the Kolmogorov-Smirnov-test, comparing the distribution function F N (X) of the breakpoint localizations within the SUZ12 gene identified in the patients with a was determined by calculating the average dissociation free energy (∆G°)

  , evaluation of the SNP array data for the complete set of SNPs on chromosome 17 served to exclude the occurrence of extended regions of homozygosity distal to the NF1 deletions. Only short ROHs (between 202 kb and 568 kb in length) immediately flanking the type-2 NF1 deletions in a telomeric direction were noted (Supp. Table

  0.0001; exact goodness-of-fit χ 2 test) (Supp. Table

Deleted:

  for the first time Deleted: not only Deleted: but also Deleted: unequal recombination between the highly homologous SUZ12 and SUZ12P repeats.

Deleted:

  involving the NF1 gene region Deleted: As indicated in Figure 2, t

  Deleted: it may well be that the Deleted: responsible for Deleted: occur post-replication Deleted: Once again, we base this assumption upon observations made i Deleted: in which Deleted: for which cohesion between sister chromatids appears to be essential confined to a few kilobases and occurring in the majority of patients, is evident with type-2 NF1 deletions. This notwithstanding, the breakpoints of type-2 NF1

  Deleted: of type-2 deletions Deleted: , CCNCCNTNNCCNC, Deleted: both allelic and non-allelic homologous

Figure 1 :

 1 Figure 1: Map of the NF1 gene region at 17q11.2 showing the positions of the breakpoints identified in the 18 patients with type-2 NF1 deletions. (A) The relative positions of the NF1 gene, the SUZ12 gene and its pseudogene (SUZ12P) are specifically indicated. The other genes located in this region are represented by black bars. (B) The positions of the type-2 deletion breakpoints arising from NAHR identified in most patients (indicated by vertical red lines) were located at homologous sites within SUZ12 and SUZ12P. By contrast, the type-2 deletion breakpoints in patients HC and 928 (indicated by vertical blue lines) were not located at homologous sites within SUZ12 and SUZ12P. The exons within the SUZ12 and SUZ12P sequences are denoted by numbered vertical bars. The circles represent the juxtaposed breakpoints of patients WB and UC172 which occurred within identical 174 bp intervals.

Figure 2 :Figure 3 :

 23 Figure 2: Intrachromosomal NAHR between the SUZ12 gene (red rectangle) and the highly homologous SUZ12P sequence (blue rectangle) is accompanied by the maintenance of heterozygosity distal to the rearrangements. (A) If intrachromosomal NAHR occurs during G1-phase, this yields, after replication and mitosis, cells bearing the deletion on one chromosome. The reciprocal acentric fragment is lost during subsequent cell divisions. Type-2 NF1 deletions occur during the embryonic development of affected patients and hence, in addition to cells harbouring the deletion, normal cells lacking the deletion are invariably also present in the soma of the respective patients (not shown in this schema). (B) If intrachromosomal NAHR occurs post-replication, this could either be within one chromatid or between sister-chromatids. Only NAHR between sister-chromatids yields cells with the reciprocal duplication.

Figure 4 :

 4 Figure 4: Map of the NF1 gene region (and the genes located therein) in relation to the relative positions of runs of homozygosity (ROHs; indicated as blue bars) in a size range of 202 kb to 568 kb immediately flanking the type-2 NF1 deletions in a telomeric direction. Such ROHs were noted in 13 patients. The type-2 NF1 deletions are depicted as grey bars.

Table 1

 1

	) since

Table 1 : Localization and extent of the type-2 deletion breakpoint regions (recombination regions) identified in 18 NF1 patients Patient Deletion breakpoint regions in SUZ12P Deletion breakpoint regions in SUZ12

 1

	Length of the	Breakpoint
	recombination	localization within
	region (bp)	SUZ12P/SUZ12

a as determined in

[START_REF] Steinmann | Type 2 NF1 deletions are highly unusual by virtue of the absence of nonallelic homologous recombination hotspots and an apparent preference for female mitotic recombination[END_REF]

b as determined in

Table 2 : Mechanisms underlying the 18 type-2 NF1 deletions and parental origin of the chromosomes 17 harbouring the deletions

 2

	Patient	Type of	Mechanism as	Mechanism as	Parental origin of the
		recombination	determined by	determined by	chromosome 17 harbouring
		causing the	SNP arrays	microsatellite	the type 2 NF1 deletion
		deletions		marker analysis b
	811-M	NAHR	intrachromosomal	intrachromosomal	paternal
	KCD	NAHR	intrachromosomal	intrachromosomal	n.d.
	697	NAHR	intrachromosomal	intrachromosomal	maternal
	736	NAHR	intrachromosomal	intrachromosomal	n.d.
	1630	NAHR	intrachromosomal	intrachromosomal	paternal
	2358	NAHR	n.d.	intrachromosomal	maternal
	585	NAHR	intrachromosomal	intrachromosomal	maternal
	488	NAHR	intrachromosomal	intrachromosomal	paternal
	1502	NAHR	intrachromosomal	intrachromosomal	maternal
	1956	NAHR	intrachromosomal	intrachromosomal	n.d.
	IL39	NAHR	intrachromosomal	intrachromosomal	maternal
	1104	NAHR	intrachromosomal	intrachromosomal	n.d.
	928 a	NHEJ	intrachromosomal	intrachromosomal	maternal
	2429	NAHR	n.d.	interchromosomal	paternal
	WB	NAHR	intrachromosomal	intrachromosomal	n.d.
	938	NAHR	intrachromosomal	intrachromosomal	maternal
	HC a	NHEJ	n.d.	intrachromosomal	maternal
	UC172	NAHR	intrachromosomal	intrachromosomal	maternal
	n.d.: not determined
	a: The deletions of patients 928 and HC have breakpoints in apparently non-homologous
	regions between SUZ12 and SUZ12P.
	NHEJ: non-homologous end joining; NAHR: non-allelic homologous recombination

  . Marker alleles in parentheses displayed reduced signal intensity, indicative of somatic mosaicism involving both normal cells and cells bearing the deletion. The horizontal rectangular box includes those markers located within the deletion region. del: deletion of the allele. --: reduced signal intensity of markers which would have been indicative of somatic mosaicism but could not be formally assessed as a consequence of homozygosity.

				Human Mutation		Page 50 of 91
	Position of marker	Marker	Patient 736	Hybrid	Hybrid	Hybrid
	on chromosome 17			Blood	#15	#1	#26
	24,481,596 -24,481,738	D17S1873	146	140	140	140	146
	24,885,942 -24,886,214	D17S1841	274	272	272	272	274
	25,124,113 -25,124,371	D17S975	258	254	254	254	258
	26,497,479 -26,497,687	D17S1307	210	(204)	204	del	210
	26,641,851 -26,642,254	D17S2237	400	(396)	396	del	400
	26,664,798 -26,665,027	IVs27TG24.8	280	--	280	del	280
	26,668,420 -26,668,626	IVs27AC28.4	205	(207)	207	del	205
	26,673,142 -26,673,342	D17S1166	202	(198)	198	del	202
	26,960,942 -26,961,211	D17S1800	270	(276)	276	del	270
	28,038,006 -28,038,197	D17S1880	192	174	174	174	192
	30,890,423 -30,890,752	D17S907	324	286	286	286	324
	31,148,163 -31,148,318	D17S1833	154	154	154	154	154
	33,160,167 -33,160,320	D17S1788	156	160	160	160	156
	Nucleotide numbering is according to hg18, NCBI 36.
			John Wiley & Sons, Inc.

479 -26,497,687 26,594,230 -26,594,438 26,641,851 -26,642,254 26,664,798 -26,665,027 26,668,420 -26,668,626 26,673,142 -26,673,342 26,960,942 -26,961,211 28,038,006 -28,038,197 29,161,843 -29,162,110 30,890,423 -30,890,752 31,148,163 -31,148,318 33,160,167 -33,160,320

	24,481,596 -24,481,738 24,885,942 -24,886,214 25,124,113 -25,124,371 on chromosome 17 26,497,Position of marker	D17S1873 D17S1841 D17S975 D17S1307 Marker	Patient 1630 Blood 124 272 254 --134 268 258 210	Hybrid #36 134 268 258 210	130 264 262 200 Father of 124 272 254 210 patient 1630 Saliva	134 268 258 210 Mother of patient 1630 Saliva 130 274 254 210
		D17S1849	207	--	207	207	207	207	207
		D17S2237	404	(396)	404	396	400	404	400
		IVs27TG24.8	272	--	272	272	280	272	272
		IVs27AC28.4	207	--	207	207	207	207	213
		D17S1166	194	--*	194	192	198	194	194
		D17S1800	270	(278)	270	278	278	270	270
		D17S1880	188	174	188	174	174	188	192
		D17S1850	266	270	266	270	266	266	270
		D17S907	336	304	336	304	286	336	304
		D17S1833	154	154	154	154	156	154	154
		D17S1788	156	156	156	156	156	156	156
	Nucleotide numbering is according to hg18, NCBI 36.

Page 51 of 91 John Wiley & Sons, Inc. Human Mutation F o r P e e r R e v i e w 6 Supplementary Figure S1-F:

  Analysis of microsatellite markers on chromosome 17 using DNA from blood samples and buccal mucosa taken from patient 2358 and family members. The horizontal rectangular box includes those markers located within the deletion region. del: deletion of the allele; n.d.: not determined.

		Patient 2358			Father of		Brother of
		Blood			patient 2358		patient 2358
						Buccal smear		Buccal smear
	22,336	D17S783	241	n.d.	n.d.	247	251	247	251
		D17S1873	142	n.d.	n.d.	140	126	140	126
		D17S1841	272	n.d.	n.d.	272	272	272	272
		D17S975	254	n.d.	n.d.	258	258	258	258
		D17S1307	del	210	del	210	210	210	210
		D17S1849	del	207	del	207	207	207	207
		D17S2237	del	400	del	400	400	400	400
		IVs27TG24.8	del	272	del	272	282	272	282
		IVs27AC28.4	del	205	del	205	205	205	205
		D17S1166	del	194	del	194	202	194	202
		D17S1800	del	270	del	270	266	270	266
		D17S1880	192	n.d.	n.d.	192	192	192	192
		D17S1850	268	n.d.	n.d.	270	270	270	270
		D17S1293	260	n.d.	n.d.	264	268	264	268
		D17S907	286	n.d.	n.d.	340	286	340	286
		D17S1833	156	n.d.	n.d.	154	156	154	156
		D17S1788	156	n.d.	n.d.	156	166	156	166
	Nucleotide numbering is according to hg18, NCBI 36.

,241 -22,336,481 24,481,596 -24,481,738 24,885,942 -24,886,214 25,124,113 -25,124,371 26,497,479 -26,497,687 26,594,230 -26,594,438 26,641,851 -26,642,254 26,664,798 -26,665,027 26,668,420 -26,668,626 26,673,142 -26,673,342 26,960,942 -26,961,211 28,038,006 -28,038,197 29,161,843 -29,162,110 29,584,279 -29,584,562 30,890,423 -30,890,752 31,148,163 -31,148,318 33,160,167 -33,160,320 Position of marker on chromosome 17

	Marker	Patient
	247	241	241
	140	120	142
	272	264	272
	258	254	254
	210	210	214
	207	209	207
	400	396	400
	272	280	272
	205	211	207
	194	194	194
	270	268	268
	192	190	192
	270	270	268
	264	280	260
	340	286	286
	154	156	156
	156	166	156

2358 Buccal smear Mother of patient 2358 Buccal smear Page 52 of 91 John Wiley & Sons, Inc. Human Mutation

  . The horizontal rectangular box includes those markers located within the deletion region. del: deletion of the allele. Nucleotide numbering is according to hg18, NCBI 36.

	D17S783	247	247	247	247	241	247	241
	D17S1873	118	130	118	130	138	118	124
	D17S1841	264	264	264	264	272	264	272
	D17S975	254	254	254	254	254	254	258
	D17S1532	172	169	172	169	169	172	169
	D17S1307	210	del	210	210	210	210	206
	D17S1849	207	del	207	207	207	207	207
	D17S2237	400	del	400	400	400	400	400
	D17S2162	258	del	258	258	258	258	258
	IVs27TG24.8	272	del	272	272	272	272	272
	IVs27AC28.4	207	del	207	207	207	207	213
	D17S1166	194	del	194	192	194	194	194
	D17S1800	282	del	282	266	276	282	270
	D17S1880	174	190	174	190	192	174	192
	D17S1850	266	268	266	268	270	266	270
	D17S1293	264	272	264	272	288	264	264
	D17S907	286	286	286	286	316	286	286
	D17S1833	154	154	154	154	154	154	154
	D17S1788	166	166	166	166	154	166	160
	D17S1809	143	145	143	145	143	143	141

,596 -24,481,738 24,885,942 -24,886,214 25,124,113 -25,124,371 26,497,479 -26,497,687 26,594,230 -26,594,438 26,641,851 -26,642,254 26,664,798 -26,665,027 26,668,420 -26,668,626 26,673,142 -26,673,342 26,960,942 -26,961,211 28,038,006 -28,038,197 29,161,843 -29,162,110 30,890,423 -30,890,752 31,148,163 -31,148,318 33,160,167 -33,160,320 Position of marker on chromosome 17

  . Marker alleles in parentheses displayed reduced signal intensity indicative of somatic mosaicism involving both normal cells and cells bearing the deletion. The horizontal rectangular box includes those markers located within the deletion region. del: deletion of the allele; n.d.: not determined.

	Marker						Hybrid	Hybrid
							#FV-2-1	#FV-4
	Marker	Patient 1502 Blood	Patient 1502 Buccal smear	Hybrid #Kl.1-5 124 272	Father of patient 1502 Saliva 124 138 patient 1502 Mother of Saliva 272 264	Daughter of patient 1502 Blood
	D17S1873 D17S1841 24,481130 274	124 272	n.d. n.d.	n.d. n.d.	124 272 258 del	130 274 258 210	136 264	254 210	124 272	134 272	130 274	136 270
	D17S975	254	254	n.d.	n.d.	254 del	254 404	254	404	254	254	254	266
	D17S1307	202	del	n.d.	n.d.	del del	202 280	210	272	206	210	202	204
	D17S1849	225	del	n.d.	n.d.	del del	225 209	233	207	191	207	225	209
	D17S2237	396	del	n.d.	n.d.	del del	396 202	396	194	400	400	396	404
	IVs27TG24.8 IVs27AC28.4 D17S1166 D17S1800 D17S1880 D17S1850 D17S907	280 207 198 278 190 266 286	del del del del 190 268 324	n.d. 207 198 n.d. n.d. n.d. n.d.	n.d. (205) (202) n.d. n.d. n.d. n.d.	del del del del 190 268 324 del 184 276 316 154 156	280 207 198 278 190 266 286 266 184 276 316 154 156	280 207 202 272 194 266 316	266 182 260 286 154 156	280 205 202 276 190 268 324	280 205 200 276 194 268 316	280 207 198 278 190 266 286	272 207 192 278 186 286 316
	D17S1833	154	164	n.d.	n.d.	164	154	154		164	154	154	154
	D17S1788	156	156	n.d.	n.d.	156	156	159		156	159	156	160
	Nucleotide numbering is according to hg18, NCBI 36.

142 -26,673,342 26,960,942 -26,961,211 28,038,006 -28,038,197 30,890,423 -30,890,752 31,148,163 -31,148,318 33,160,167 -33,160,320 40,162,539 -40,162,644 Position of marker on chromosome 17

			Human Mutation			Page 58 of 91
	D17S1873 D17S1841 D17S975 D17S1849 D17S1166 D17S1800 D17S1880 D17S907 D17S1833 D17S1788 D17S1861 Marker 26,673,Marker 24,481,596 -24,481,738 24,885,942 -24,886,214 25,124,113 -25,124,371 26,594,230 -26,594,438	152 267 263 del del del 196 314 158 155 95 Patient 120 267 255 235 195 276 178 334 158 155 93 IL39-III2 Blood	Hybrid #17 of IL39-III2 120 267 255 235 195 276 178 334 158 155 93	152 267 263 ------196 314 158 155 95 Patient IL39 (mother of IL39-III2) Blood 136 265 259 211 195 Hybrid Hybrid #15 of IL39-III2 152 267 263 del #Kl.2 241 124 272 254 169 202 241 124 272 258 169 210 del 205 207 276 del 280 274 186 196 198 194 330 314 270 276 146 158 174 190 159 155 328 286 95 95 154 154	120 267 255 235 195 276 178 334 158 155 93 Father of patient IL39-III2 Blood 140 275 255 211 203 268 194 290 160 155 97	152 267 263 211 195 276 196 314 158 155 95 Grandmother 144 275 259 237 303 268 196 318 156 153 95 of patient IL39-III2 Blood
				158	154
	Nucleotide numbering is according to hg18, NCBI 36.
			John Wiley & Sons, Inc.

084 -26,614,348 26,641,851 -26,642,254 26,664,798 -26,665,027 26,668,420 -26,668,626 26,673,142 -26,673,342 26,960,942 -26,961,211 28,038,006 -28,038,197 29,161,843 -29,162,110 30,890,423 -30,890,752 31,148,163 -31,148,318 33,160,167 -33,160,320 40,162,539 -40,162,644 60,127,912 -60,128,058

  . The horizontal rectangular box includes those markers located within the deletion region. del: deletion of the allele; n.d.: not determined. Nucleotide numbering is according to hg18, NCBI 36.

	24,481,596 -24,481,738 24,885,942 -24,886,214 25,124,113 -25,124,371 26,497,479 -26,497,687 26,614,Position of marker on chromosome 17	D17S1873 D17S1841 D17S975 D17S1307 NF1PCR3 Marker	Patient 928 Blood 138 274 258 del del 130 264 254 210 266	Hybrid #Kl. 3 130 264 254 210 n.d.	138 274 258 del del Hybrid #Kl.1	130 264 254 210 266 Father of 144 264 254 210 262 Blood patient 928
		D17S2237	404	del	404	del	404	404
		IVs27TG24.8	272	del	272	del	272	274
		IVs27ac28.4	207	del	207	del	207	207
		D17S1166	192	del	192	del	192	194
		D17S1800	276	del	276	del	276	278
		D17S1880	182	184	182	184	182	182
		D17S1850	270	270	270	270	270	270
		D17S907	323	286	323	286	323	286
		D17S1833	154	154	154	154	154	156
		D17S1788	166	166	166	166	166	154
		D17S1861	102	102	102	102	102	102
		D17S1809	145	141	145	141	145	145

24,481,596 -24,481,738 24,885,942 -24,886,214 25,124,113 -25,124,371 26,497,479 -26,497,687 26,641,851 -26,642,254 26,664,798 -26,665,027 26,668,420 -26,668,626 26,673,142 -26,673,342 26,960,942 -26,961,211 28,038,006 -28,038,197 30,890,423 -30,890,752 31,148,163 -31,148,318 33,160,167 -33,160,320 40,612,539 -40,162,644 60,127,912 -60,128,058 Position of marker on chromosome 17

  The horizontal rectangular box includes those markers located within the deletion region. del: deletion of the allele; n.d.: not determined.

	Marker	Patient 2429	Patient 2429	Hybrid	Father of	Mother of	Brother of
			Blood	Buccal smear	#Kl.1-5	patient 2429	patient 2429	patient 2429
								Blood	Blood	Blood
	D17S1873		134	130	n.d.	n.d.	134	134	140	130	126	140	126
	D17S1841		278	272	n.d.	n.d.	278	278	272	272	272	n.d.	n.d.
	D17S975		258	254	n.d.	n.d.	258	258	258	254	258	258	258
	D17S1307		del	206	n.d.	n.d.	del	210	210	206	210	210	210
	D17S2237		del	400	n.d.	n.d.	del	400	404	400	396	404	396
	IVs27TG24.8	del	274	del	274	del	272	280	274	280	280	280
	IVs27AC28.4	del	207	del	207	del	203	205	207	209	205	209
	D17S1166		del	194	del	194	del	192	202	194	202	202	202
	D17S1800		del	276	n.d.	n.d.	del	278	276	276	272	276	272
	D17S1880		192	174	n.d.	n.d.	192	174	192	174	192	192	192
	D17S907		328	316	n.d.	n.d.	328	300	328	316	312	328	312
	D17S1833		154	156	n.d.	n.d.	154	156	154	156	154	154	154
	D17S1788		166	160	n.d.	n.d.	166	160	166	160	156	160	156
	D17S1861		108	96	n.d.	n.d.	108	106	108	96	104	106	104
	D17S1809		142	138	n.d.	n.d.	142	140	142	138	144	140	138
	Nucleotide numbering is according to hg18, NCBI 36.

851 -26,642,254 26,664,798 -26,665,027 26,668,420 -26,668,626 26,673,142 -26,673,342 28,038,006 -28,038,197 29,584,279 -29,584,562 30,890,423 -30,890,752 31,148,163 -31,148,318 33,160,167 -33,160,320 40,162,539 -40,162,644 Position of marker on chromosome 17

  ; del: deletion of the allele. Nucleotide numbering is according to hg18, NCBI 36.

	Marker	Patient SB		Hybrid	Patient WB	Sister of
		Blood		#B9	(Mother of patient SB) Blood	patient SB Blood
	D17S1873 D17S1841 D17S975 D17S1307 D17S1849 D17S2237 26,641,130 24,481,596 -24,481,738 24,885,942 -24,886,214 25,124,113 -25,124,371 26,497,479 -26,497,687 26,594,230 -26,594,438 266 264 214 209 400	134 278 260 del del del	130 266 264 214 209 400	134 278 260 del del del	134 278 260 del del del	126 272 260 206 207 400	126 272 260 206 207 400	130 266 264 214 209 400
	IVs27TG24.8	272	del	272	del	del	272	272	272
	IVs27AC28.4	209	del	209	del	del	213	213	209
	D17S1166	194	del	194	del	del	194	194	194
	D17S1880	174	182	174	182	182	182	182	174
	D17S1293	284	276	284	276	276	284	284	284
	D17S907	324	324	324	324	324	320	320	324
	D17S1833	154	154	154	154	154	154	154	154
	D17S1788	160	156	160	156	156	156	156	160
	D17S1861	102	102	102	102	102	92	92	102

230 -26,594,438 26,641,851 -26,642,254 26,664,798 -26,665,027 26,668,420 -26,668,626 26,673,142 -26,673,342 26,960,942 -26,961,211 28,038,006 -28,038,197 30,890,423 -30,890,752 31,148,163 -31,148,318 33,160,167 -33,160,320 60,127,912 -60,128,058 Position of marker on chromosome 17

  Analysis of microsatellite markers on chromosome 17 using DNA from blood samples from patient HC and his parents. DNAs derived from somatic cell hybrids either carrying the chromosome 17 with the deletion (hybrid #Kl.1) or bearing the wild-type chromosome 17 of patient HC (hybrid # Kl.6), were also analysed. The horizontal rectangular box includes those markers located within the deletion region. Marker alleles in parentheses displayed reduced signal intensity indicative of somatic mosaicism involving both normal cells and cells bearing the deletion.

	Page 63 of 91	Human Mutation
	D17S2093 D17S1841 D17S975 D17S1532 D17S1849 D17S2237 IVs27TG24.8 IVs27AC28.4 D17S1166 D17S1800 D17S1880 D17S907 D17S1833 D17S1788 D17S1809 Marker Patient HC 118 160 264 258 169 207 400 272 213 194 276 196 286 154 166 145 Patient 938 Blood Blood 140 160 272 254 169 (229) ----* (207) (202) --* 190 286 156 166 149 26,594,144 140 160 272 254 169 229 400 274 207 202 268 190 286 156 166 149 Mother of patient 938 Hybrid del Hybrid #Kl.1 Blood 24,481,596 -24,481,738 24,693,853 -24,694,013 24,885,942 -24,886,214 25,124,113 -25,124,371 25,708,122 -25,708,291 160 272 272 169 207 404 280 207 194 270 190 312 154 156 145 140 160 272 254 169 del del del del del del 190 286 156 166 149 D17S783 241 239 22,336,241 -22,336,481 D17S1873 136 144 24,481,596 -24,481,738 D17S1841 D17S975 D17S1532 NF1PCR3 D17S2237 IVs27AC28.4 D17S1800 D17S1880 D17S907 D17S1833 D17S1788 270 254 163 266 404 213 268 188 314 162 154 272 258 163 --* --* --* --* 174 286 156 164 24,885,942 -24,886,214 25,124,113 -25,124,371 25,708,122 -25,708,291 F o r P e e r R e v i e w D17S1849 207 --* 26,594,	Father of Father of patient 938 Blood patient HC 118 160 264 258 169 207 400 272 213 194 276 196 286 154 166 145 Blood 130 241 241 160 136 130 272 258 169 233 404 280 213 202 280 192 316 154 166 147 270 254 163 266 404 213 268 188 314 162 154 272 258 169 262 400 209 280 188 324 154 154 207 233	Mother of patient HC Blood 239 144 272 258 163 262 404 207 276 174 286 156 164 207
	Supplementary Figure S1-R: --: reduced signal intensity of markers which would have been indicative of somatic mosaicism but could not be formally assessed as a
	consequence of homozygosity.
	--*: a second allele was not visible, although heterozygosity was inferred form the maternal haplotype.
	del: deletion of the allele. n.d.: not determined.
	Nucleotide numbering is according to hg18, NCBI 36.
		John Wiley & Sons, Inc.		18

230 -26,594,438 26,614,048 -26,614,348 26,641,851 -26,642,254 26,668,420 -26,668,626 26,960,942 -26,961,211 28,038,006 -28,038,197 30,890,423 -30,890,752 31,148,163 -31,148,318 33,160,167 -33,160,320

  Non-B DNA-forming repeats in the breakpoint-spanning regions, also termed recombination regions (RRs; grey), of the 16 type-2 NF1 deletions mediated by NAHR. Sequences flanking the RRs in a proximal direction are marked in yellow. Sequences flanking the RRs in a telomeric direction are marked in blue. Paralogous sequence variants are highlighted in bold. Direct repeats are shown in pink or red letters. Inverted repeats are highlighted in green. Symmetric repeats are underlined. Inverted repeats capable of non-B DNA structure formation were found to be overrepresented within the RRs (p<0.001) whereas both direct and inverted repeats were overrepresented within the extended RRs (p<0.001). Extended RRs also encompass 100 bp in centromeric and telomeric directions, marked in yellow and blue, respectively. Nucleotide numbering is according to hg18, NCBI 36.

	Position of marker on chromosome 17 Supplementary Figure S2: Patient 811-M Marker	n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. Patient HC n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. Buccal smear	239 144 272 258 163 del del del	Hybrid #Kl.6 241 136 270 254 163 207 266 404	239 130 264 262 173 207 274 404
	del AGTTGAATACAATAAATAGCTCTTTTTTGGCCAGGCATGGTGGCTCACAC 26,093,051-26,093,100 213 (207) CTTTAATCCTAGCACTTTGGGAGACAGGGGTGGATAGATCACCTGAGGTC 26,093,101-26,093,150	213	207
	r o F AGGAGTTCAAGACCAGCCTGACCAACATGGTGAAACCCTGTCTCTA.	268 n.d. 314 n.d. n.d.	(276) n.d. 286 n.d. n.d.	del 174 286 156 164	268 188 314 162 154	280 174 324 154 154
	P
	e
	e r
	R
	e
	v i e
		w
				20

Table S2 :

 S2 Clinical features of the five newly identified type-2 NF1 deletion patients Multiple CALM, axillary and inguinal freckling, hypotony, hypertelorism, plexiform neurofibroma on the right calf, no dermal neurofibromas, no spinal or intra-abdominal tumours as determined by MRI, T2 hyperintensities in the cerebellum, mild developmental delay, attends primary school.More than 6 CALM, axillary freckling, Lisch nodules, scoliosis, no neurofibromas, attends primary school but is behind normal educational level for age, no significant developmental delay or dysmorphic facial features.Multiple CALM, axillary and inguinal freckling, Lisch nodules, height 182cm; head circumference 60cm, one MPNST, congenital heart disease, >1000 subcutaneous neurofibromas, ~ 500 cutaneous neurofibromas, plexiform neurofibromas on face and neck, corpus callosum aplasia, IQ 84, learning problems, hypertelorism, saddle nose, dysmorphic facial features, large hands and feet, hyperflexibility of joints, funnel chest, broad neck.

	Supplementary Patient Sex (age	Mosaicism detected	Clinical features Proportion (%) of cells harbouring the deletion as
		in years)	by marker analysis a			determined by FISH analysis of
					of
				blood	buccal cells	blood		buccal	skin fibroblast	neuro-	urine
								cells	cultures	fibroma
	2358 811-M b	f (10) f (35)		yes	   	93		   		   	   	   
	KCD c	f (34)		Multiple CALM, axillary and inguinal freckling, plexiform neurofibroma yes     92     51        
	585 697 b	f (7) f (11)		on the neck, no intellectual disabilities or psychomotor developmental delay. no     97 59            
	736 b	f (68)		yes	   	94		59		   	   	   
	1956 1630 b 2358 d 585 d	m (9) f (15) f (10) f (7)	F o r F o r F o r yes     F no no o r yes yes	92 100    		           		           	           	           
	P P P     P e e 98 r e e r e yes 97 e Multiple CALM, axillary and inguinal freckling, no dermal or no 56         no 70         subcutaneous neurofibromas, no intellectual disabilities or psychomotor     ,546     developmental delay. CALM: Cafe-au-lait macules 2429 m (16) 488 b f (33) UC172 f (6) 1502 b f (26) e r 1956 d m (9) yes     92                 e r IL39 c f (60) yes     70     15        
	R R MPNST: malignant peripheral nerve sheath tumour R R 1104 b f (36) yes     84	8		   	   	15
	928 b	f (39)		no	   	e e e e 100 55		   	80	   
	2429 d WB c	m (16) f (65)		no no	no    	100 94	v i e v i e v i e     v i e    	       	       	       
	938 a HC a	f (35) m (9)		yes no	    yes	91 100		80	w w w     w	   	   
										24

Table S7 :

 S7 Positions of runs of homozygosity (ROHs) ≥ 200 kb located distal to the type-2 NF1 deletion interval in 60 CEU individuals as determined by the analysis of phased haplotypes downloaded from HapMap (HapMap Data Rel 24/phaseII Nov08, on NCBI B36, assembly dbSNPb126)[START_REF] Roehl | Extended runs of homozygosity at 17q11.2: an association with type-2 NF1 deletions?[END_REF]

	Supplementary CEU individuals Supplementary	Position a of the ROH on chromosome 17	Length (bp)
			start		end
	NA06985		27,352,718		27,593,261	240,544
	NA06993		27,352,718		27,580,395	227,678
	NA06994		27,605,972		27,904,495	298,524
	NA07000		27,352,718		27,605,972	253,255
	NA07022		27,420,000		27,651,777	231,778
	NA07055		27,651,777		27,853,777	202,001
	NA07056		27,651,777		27,883,082	231,306
	NA07345 NA11831 NA11832	F o r F o r 27,352,718 27,352,718 27,630,414 27,352,718 27,352,718 F o r F o r		27,605,972 27,580,395 27,909,678 27,636,926 27,580,395	253,255 227,678 279,265 284,209 227,678
	NA11839 NA11840		P e P e 27,352,718 27,651,777 P P 27,352,718 e e		27,593,261 27,883,082 27,605,972	240,544 231,306 253,255
	NA11881 NA11882		e r e r 27,352,718 27,633,000 27,352,718 e r e r	27,636,926 27,853,777 27,580,395	284,209 220,778 227,678
	NA11992 NA11995 NA12003 NA12004 NA12005 NA12006 NA12043 NA12044 NA12056		R e v i e w R e v i e w 27,352,718 27,580,395 27,651,777 27,883,082 R R 27,352,718 27,573,486 27,352,718 27,593,261 e e 27,352,718 27,593,261 27,352,718 27,593,261 27,352,718 27,610,212 v i e v i e 27,352,718 27,580,395 27,352,718 27,593,261 w w	227,678 231,306 220,769 240,544 240,544 240,544 257,495 227,678 240,544
			27,605,972		27,883,082	277,111
	NA12057		27,651,777		27,883,082	231,306
			27,352,718		27,605,972	253,255
	NA12145		27,651,777		27,855,741	203,965
	NA12146		27,352,718		27,593,261	240,544
	NA12154		27,352,718		27,580,395	227,678
	NA12155 d: as determined in this study NA12156 : not determined NA12236	27,352,718 27,633,000 27,352,718	]	27,883,082 27,844,289 27,636,926	530,365 211,290 284,209
	NA12249		27,352,718		27,897,975	545,258
	NA12264		27,352,718		27,593,261	240,544
	NA12716		27,352,718		27,855,741	503,024
						40 42

Table S8 :

 S8 Motifs identified in the recombination regions (RRs) of 16 type-2 deletions caused by NAHR between SUZ12 and SUZ12P Y=C/T, W=A/T, S=G/C, M=A/C, K=G/T, N= any base. The complementary sequence was also investigated, but the corresponding sequence motifs are not listed. * Sequences significantly overrepresented at the 5% level in RRs.

		Human Mutation	Page 88 of 91
	Supplementary
	Motif	Consensus sequence a	Number of motifs (and
			their complements)
			identified within the RRs
	Vaccinia topoisomerase I consensus	YCCTT	7
	cleavage site
	Immunoglobulin heavy chain class	GAGCT, TGGGG,	13
	switch repeat	GGGCT,GGGGT, TGAGC
	Chi and chi-like elements	CCWCCWGC	2*
	Human minisatellite consensus sequence GCWGGWGG	2*
	Short polypurine/ polypyrimidine tracts	R 5 /Y 5	16
	Long polypurine/ polypyrimidine tracts Murine parvovirus recombination hotspot CTWTTY R 10 /Y 10 Deletion hotspot consensus sequence TGRRKM DNA polymerase arrest site WGGAG DNA polymerase β frameshift hotspot ACCCWR Hamster deletion hotspot sequence TGGAG F o r F o r	1/2 9 13 7 3 2
	Hamster and human APRT deletion hotspot P P Super hotspot motifs c e TTCTTC CCAAR, CCCAG AGCTG CCACCA e e r e r	3 13 6 4 4
		R R
		e e
		v i e v i e
		w w
	John Wiley & Sons, Inc.	44

a : R=A/G,

Table S10 :

 S10 DNA stability, measured as ∆G° values, within the recombination regions (RR) of 14 patients with mosaic type-2 NF1 deletions mediated by NAHR

	Patient	∆G° (kcal) of 50 bp in	∆G° (kcal) within the RR	∆G° of a 3 kb sequence including
		the centre of the RR	(bp)	and flanking the RR
	811-M	1.94	1.94 (45)	1.78
	KCD	1.75	1.75 (108)	1.79
	697	1.96	1.95 (68)	1.94
	736	2.04	2.03 (87)	1.94
	1630	1.77	1.79 (111)	1.87
	585	1.6	1.66 (129)	1.86
	488	1.97	1.96 (56)	1.88
	1502	1.82	1.81 (190)	1.87
	1956	1.87	1.89 (145)	1.89
	IL39	1.61	1.61 (46)	1.87
	1104	1.63	1.63 (75)	1.86
	WB	1.75	1.77 (174)	1.8
	UC172	1.75	1.77 (174)	1.8
	938	1.68	1.7 (110)	1.79
	Mean	1.8	1.8	1.85
				[95% CI: 1.82-1.88]

John Wiley & Sons, Inc.Human Mutation

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Acknowledgement

This work was supported by grants from the DFG [KE 724/7-1; KE 724/9-1 (H.K.S.) and MA 2299/7-1 (V.F.M.)] and from the Deutsche Krebshilfe (#108793, H.K.S., V.F.M.).

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Human Mutation

F o r P e e r R e v i e w

10 Supplementary Figure S1-J: Analysis of microsatellite markers on chromosome 17 using DNA extracted from a blood sample from patient 1956 and hybrid cell lines containing only one chromosome 17 from the patient. Hybrid cell line #FIII-1 contains only the chromosome 17 harbouring the type-2 NF1 deletion; hybrid cell line #FV-2-1 contains the wild-type chromosome 17 which was subject to the deletion in a number of cells. Hybrid cell line #FV-4 contains the wild-type chromosome 17 of the patient. In blood lymphocytes of patient 1956, 92% of the cells harboured the deletion as determined by FISH (in the present study). The horizontal rectangular box includes those markers located within the deletion region. Marker alleles in parentheses displayed reduced signal intensity, indicative of somatic mosaicism involving both normal cells and cells bearing the deletion.

--: reduced signal intensity of markers which would have been indicative of somatic mosaicism but could not be formally assessed as a consequence of homozygosity. del: deletion of the allele. Nucleotide numbering is according to hg18, NCBI 36. 12 Supplementary Figure S1-L: Analysis of microsatellite markers on chromosome 17 using DNA from blood samples from patient 1104 as well as DNA extracted from somatic cell hybrids bearing the two wild-type chromosomes 17 of the patient. The deletion was observed in 84% of peripheral blood lymphocytes, 15% of urine cells and 8% of buccal smear cells from this patient [START_REF] Steinmann | Type 2 NF1 deletions are highly unusual by virtue of the absence of nonallelic homologous recombination hotspots and an apparent preference for female mitotic recombination[END_REF]. Marker alleles in parentheses displayed reduced signal intensity, indicative of somatic mosaicism involving both normal cells and cells bearing the deletion. The horizontal rectangular box includes those markers located within the deletion region. Nucleotide numbering is according to hg18, NCBI 36. Sequence analysis of PCR products amplified from somatic cell hybrids containing only the chromosome 17 with the deletion WB b

D17S783

Sequence analysis of PCR products amplified from somatic cell hybrids containing only the chromosome 17 with the deletion 938 a

Sequence analysis of PCR products amplified from somatic cell hybrids containing only the chromosome 17 with the deletion HC a

Sequence analysis of PCR products amplified from somatic cell hybrids containing only the chromosome 17 with the deletion UC172 c

Sequence analysis of PCR products amplified from somatic cell hybrids containing only the chromosome 17 with the deletion a breakpoints were identified as previously described [START_REF] Steinmann | Type 2 NF1 deletions are highly unusual by virtue of the absence of nonallelic homologous recombination hotspots and an apparent preference for female mitotic recombination[END_REF] b breakpoints as determined in [START_REF] Kehrer-Sawatzki | High frequency of mosaicism among patients with neurofibromatosis type 1 (NF1) with microdeletions caused by somatic recombination of the JJAZ1 gene[END_REF]. c breakpoints were identified in the present study.

Supplementary Table S3 To determine whether Alu elements were overrepresented in the breakpoint regions of the 16 type-2 NF1 deletions mediated by NAHR, the number of Alu sequences in the breakpoint regions was ascertained by Repeatmasker analysis (http://www.repeatmasker.org/cgibin/WEBRepeatMasker). Ten Alu sequences were identified in the 16 breakpoint regions with a mean length of 114 bp. The SUZ12 gene and SUZ12P pseudogene are highly homologous over 45 kb as determined by BLAT analysis (http://genome.ucsc.edu/). Within this 45 kb sequence, 71 Alu elements were identified. Accordingly, in 1825 bp of SUZ12 (the total length of the breakpoint regions), 2.9 Alu elements would be expected. However, since we observed 10 Alu elements in the breakpoint regions, it was concluded that Alu elements were significantly overrepresented at the type-2 deletion breakpoints (p < 0.0001; exact exact goodness-of-fit χ 2 test).

Supplementary Table S11: GC-content of the recombination regions (RRs) of the 14 type-2 NF1 deletions mediated by NAHR during the mitotic cell cycle Patient GC-content of the RRs within the SUZ12 gene (%) GC-content of 3 kb including and flanking the RRs within the SUZ12 gene (%) 811-M 50.0 34.8