Neurofibromatosis type 1-associated tumours: Their somatic mutational spectrum and pathogenesis

  • Sebastian Laycock-van Spyk1,

    Affiliated with

    • Nick Thomas1,

      Affiliated with

      • David N. Cooper1 and

        Affiliated with

        • Meena Upadhyaya1Email author

          Affiliated with

          Human Genomics20115:623

          DOI: 10.1186/1479-7364-5-6-623

          Received: 23 May 2011

          Accepted: 23 May 2011

          Published: 1 October 2011

          Abstract

          Somatic gene mutations constitute key events in the malignant transformation of human cells. Somatic mutation can either actively speed up the growth of tumour cells or relax the growth constraints normally imposed upon them, thereby conferring a selective (proliferative) advantage at the cellular level. Neurofibromatosis type-1 (NF1) affects 1/3,000-4,000 individuals worldwide and is caused by the inactivation of the NF1 tumour suppressor gene, which encodes the protein neurofibromin. Consistent with Knudson's two-hit hypothesis, NF1 patients harbouring a heterozygous germline NF1 mutation develop neurofibromas upon somatic mutation of the second, wild-type, NF1 allele. While the identification of somatic mutations in NF1 patients has always been problematic on account of the extensive cellular heterogeneity manifested by neurofibromas, the classification of NF1 somatic mutations is a prerequisite for understanding the complex molecular mechanisms underlying NF1 tumorigenesis. Here, the known somatic mutational spectrum for the NF1 gene in a range of NF1-associated neoplasms --including peripheral nerve sheath tumours (neurofibromas), malignant peripheral nerve sheath tumours, gastrointestinal stromal tumours, gastric carcinoid, juvenile myelomonocytic leukaemia, glomus tumours, astrocytomas and phaeochromocytomas -- have been collated and analysed.

          Keywords

          NF1 somatic mutations germline mutations pathogenesis tumorigenesis tumour benign malignant

          Introduction

          Neurofibromatosis type 1 (NF1) is a common auto-somal dominantly inherited tumour predisposition syndrome affecting 1/3,000-4,000 individuals worldwide [1, 2]. NF1 manifests a variety of characteristic features that include: hyperpigmentary abnormalities of the skin (café-au-lait macules and inguinal/axillary freckling), iris hamartomas (Lisch nodules) and the growth of benign peripheral nerve sheath tumours (neurofibromas) in the skin. Neurofibromas display many different subtypes and are associated with a variety of different clinical complications. Cutaneous neurofibromas are present in almost all adult NF1 patients [3]. Plexiform neurofibromas (PNFs), a more diffuse type of tumour, are present in 30-50 per cent of NF1 patients, and some 10-15 per cent of these benign tumours are transformed to malignant peripheral nerve sheath tumours (MPNSTs), the main cause of morbidity in NF1 [4]. Other NF1-associated clinical features include: skeletal abnormalities, such as tibial bowing or pseudoarthrosis; skeletal and orbital dysplasia; ostopenia/osteoporosis; aqueduct stenosis; macrocephaly; pectus excavatum; short stature; cardiovascular malformations; learning difficulties; and attention deficit disorder [1, 5].

          Cancer represents the transformation of a cell whose growth is normally tightly controlled into one that is no longer under strict regulation, allowing the cell to multiply uncontrollably and even metastasize. This dramatic alteration in cellular control arises as a consequence of the accumulation of genetic and epigenetic changes: activated oncogenes speed up cell growth through the acquisition of gain-of-function mutations, whereas tumour suppressor genes (TSGs) promote progression by acquiring loss-of-function mutations. TSGs typically encode proteins involved in growth regulation, apoptosis initiation, cellular adhesion and DNA repair. In accordance with Knudson's two-hit hypothesis,[6] both alleles of a TSG must be inactivated for cellular transformation to occur. Typically, a patient will inherit a germline mutation in one TSG allele; a second-hit or somatic mutation then occurs post-fertilisation, thereby inactivating the remaining wild-type allele. Somatic mutation is thus a key event in cancers associated with TSG inactivation. Upon transformation, a cell may acquire many additional somatic mutations elsewhere in the genome, a few of which actively encourage tumour progression, designated as 'driver mutations', while most occur simply because of the increased number of cell replications and are usually of unknown biological consequence and so are designated as 'passenger mutations' [7].

          The NF1 gene encodes neurofibromin, a negative regulator of the Ras/mitogen-activated protein kinase (MAPK) pathway. NF1 is a TSG and, consistent with Knudson's two-hit hypothesis, most patients carry (in all their cells) both a normal and a dysfunctional NF1 gene copy -- the latter harbouring the inherited (germline) mutation. It may be inferred that any tumours that arise will have acquired a second, somatic 'hit' that inactivates the normal NF1 allele, resulting in a complete loss of functional neurofibromin; a double hit (NF1-/-) is critical for NF1 tumorigenesis to occur [8, 9]. The question as to why only a few of these benign tumours eventually go on to become malignant, however, is still puzzling. Consistent with a central role for neurofibromin in cellular function, recent cancer genome sequencing studies have found that somatic NF1 gene mutations occur not only in association with NF1, but also in a number of other common cancers [1016].

          In the context of NF1, few genotype-phenotype correlations are evident. Indeed, marked intrafamilial variation in terms of the clinical phenotype is common [5, 17]. The existence of such families is perhaps an indication of the importance of the second hit, since differences in the type and timing of somatic NF1 mutations may help to explain the variability in patient phenotype [18]. An appreciation of the spectrum of somatic mutations in NF1-associated tumours is therefore essential if we are to understand the molecular pathways involved -- itself a prerequisite for improvements in clinical treatment and the development of new therapeutics. This paper attempts to collate and review the spectrum of somatic NF1 mutations so far reported in NF1-associated tumours, with a view to assessing how they may serve to induce tumour growth and whether or not any genotype-phenotype correlation may be discerned

          The NF1gene: Structure and function

          The NF1 gene spans 283 kilobases (kb) of genomic DNA at 17q11.2 [19] and contains 61 exons [3, 20]. Neurofibromin, the 327 kDa protein encoded by the NF1 gene, is translated from a 12 kb messenger mRNA transcript, and has a number of alternative iso-forms [2124] (reviewed by Upadhyaya [25]). Neurofibromin contains 2,818 amino acids and is expressed at low levels in all cells, with higher levels in the nervous system. It functions as a negative regulator of active Ras, and of the associated Ras/MAPK signalling pathway. Neurofibromin contains a Ras-specific GTPase activating protein (GAP)-related domain which interacts directly with Ras, resulting in a conformational change that greatly stimulates the intrinsic GTPase activity of the Ras protein, thus significantly accelerating the conversion of the active GTP-bound form of Ras into its inactive GDP-bound form and effecting a net decrease in overall mitogenic signalling in the cell. As the Ras/MAPK cascade is critical for the control of cellular growth and differentiation, a lack of functional neurofibromin results in the constitutive activation of this central signalling pathway and in unregulated cell growth [26].

          NF1 tumour biology

          A variety of benign and malignant tumours are associated with NF1 and all involve tumorigenesis of neural crest-derived cells. Several murine models of neurofibromatosis have both successfully recapitulated much of the NF1 human phenotype and shown that NF1 is indeed a classical TSG [27, 28].

          Neurofibromas exhibit extensive cellular heterogeneity, being composed of hyperproliferative Schwann cells (SCs), fibroblasts, mast cells and peri-neural cells. The SCs have been identified as the initiating cell type in neurofibromas and it is only in these cells that the NF1 gene becomes biallelically inactivated [29]. SCs are also the target for various growth factors known to stimulate neurofi-broma formation and growth. What is still not known, however, is the precise cell type within the SC cell lineage in which the somatic mutation occurs, the cell type which subsequently precipitates neurofibroma growth.

          Cutaneous neurofibromas are thought to arise from skin-derived precursor cells (SKPs)[30] and these cells may well be under hormonal control, since most such tumours develop only during puberty [31]. Further, an increase in tumour size and number has also been noted during pregnancy, with some evidence for a postnatal decrease in tumour size [32, 33]. Almost all PNFs appear congenitally and it is thought that they are induced by a somatic NF1 mutation in SC precursors within the embryonic gestational window of 12.5-15.5 days [34]. It may be that this second hit does not render the SC precursor tumorigenic, but instead induces aberrant axonal segregation [35]. The extracellularly expressed transmembranal guidance protein, Sema4F, is strongly downregulated in neurofibromas and it has been suggested that this somehow indirectly promotes SC proliferation by rendering these cells more responsive to environmental signals, possibly through inhibition of axonal re-attachment [36]. In this way, the disruption of normal SC axonal interactions leads to neurofibroma development. An NF1-/+ haploinsufficient cellular environment is also considered necessary, probably because of the growth advantage conferred by the signalling deficiency due to reduced neurofibromin levels. Indeed, Le et al. [30] found that NF1 inactivation is necessary, although not sufficient, for neurofibroma formation, highlighting the importance of the tumour microenvir-onment. There is some evidence to indicate that the haploinsufficiency (NF1-/+) of the other supporting cells (fibroblasts, mast cells and perineurial cells) cooperates in neurofibroma development [37]. Additionally, it has been shown that NF1-/+ haploinsufficient mast cells readily migrate into preneo-plastic nerves, probably in response to Kit ligand, which exhibits four-fold increased levels in nullizygous SCs as compared to normal SCs [38, 39]. The molecular mechanisms underlying both PNF and cutaneous neurofibroma formation are becoming clearer, although the major details are still lacking. It would appear that the key to understanding neurofibroma formation lies in the elucidation of the precise molecular interactions of the haploinsufficient tumour microenvironment within the initial cell type harbouring the biallelically inactivated NF1 gene.

          NF1-associated tumours

          Cutaneous neurofibromas and PNFs

          Neurofibromas are a characteristic feature of NF1 and have a diverse clinical presentation. They are classified as grade 1 tumours by the World Health Organization; they have multiple forms and may affect nerves in any body location. Tumours derived from skin sensory nerves are designated dermal or cutaneous neurofibromas, and usually present as discrete tumours that remain associated with a single nerve ending. Approximately 20-50 per cent of cutaneous neurofibromas exhibit loss of heterozygosity (LOH) at the NF1 locus and the majority of these lesions appear to be due to mitotic recombination [4042]. Tumours associated with larger nerves within the skin may spread within the dermis and appear as a diffuse mass. PNFs are much larger tumours, usually associated with major nerve trunks and nerve plexi. They are generally slow growing, may develop at both internal and external body locations and can often result in major disfigurement. PNFs occur in some 30-50 per cent of patients with NF1 and, although these tumours generally remain benign, some neurological impairment may result from their growth. Approximately 10-15 per cent of PNFs may become malignant.

          While the genetic basis of neurofibroma development is still not fully understood, biallelic NF1 inactivation does seem to be required, as all tumour cells harbour both a constitutional and a somatic NF1 gene mutation [5]. About 70 per cent of PNFs have been reported to display LOH at the NF1 locus;[20] however, there is no obvious correlation between the type or location of germline NF1 mutations in NF1 patients and those of their somatic counterparts arising in their tumours [20].

          Another interesting, although as yet unexplained, observation is that a few patients mildly affected by NF1 who never develop any cutaneous neurofibromas or PNFs have been shown to carry the same germline NF1 mutation (c.2970-2972delAAT) --namely, an in-frame 3-base pair (bp) deletion that leads to the loss of a methionine residue [3].

          MPNSTs

          Cells derived from within some 10-15 per cent of PNFs may eventually undergo malignant transformation into an MPNST. MPNSTs are aggressive and highly invasive soft tissue sarcomas with an annual incidence of 0.16 per cent in NF1 patients, compared with only 0.001 per cent in the normal population,[43] and with a lifetime risk of 8-13 per cent in NF1 individuals [44, 45] (reviewed by Upadhyaya [4]). This form of malignancy represents a major cause of morbidity and mortality in NF1. Malignant transformation usually appears to evolve from within a pre-existing PNF [46]. The distinction between benign PNFs and MPNSTs has been sensitively visualised by non-invasive [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography (FDG-PET) imaging,[47] suggesting a potential role for FDG-PET-based non-invasive imaging in future diagnostic tests. The aberrant molecular pathways that underlie this malignant transformation are still largely unknown, and considerable effort is being directed towards elucidating the molecular defects involved.

          NF1 patients carrying large (usually 1.4-megabase [Mb]) genomic deletions (which remove the entire NF1 gene plus a variable number of flanking genes) have an increased risk of MPNST development in certain patient groups [48, 49]. Indeed, over 90 per cent of MPNSTs have been found to harbour large NF1 somatic deletions [20]. More recently, significantly increased frequencies (relative to the general NF1 population) of PNFs, subcutaneous neurofibromas, spinal neurofibromas and MPNSTs have also been reported in association with molecularly ascertained 1.4 Mb type-1 NF1 deletions [50]. The MPNST-associated deletion breakpoints have been found not to involve the paralogous repetitive sequences that are involved in most germline NF1 deletions [18]. The smallest common region of somatic deletion overlap is, however, restricted to approximately the same ~2.2 Mb interval that contains most of the genes deleted in recurrent constitutional NF1 deletions [51].

          Although it is clear that biallelic NF1 gene inactivation is required for transformation to occur, mutations at the NF1 locus are insufficient to explain the process of tumorigenesis, as most benign neurofibromas also exhibit such biallelic NF1 inactivation. The best evidence for the involvement of other loci relates to the tumour protein 53 gene (TP53), for which several different mutations have been found in MPNSTs that have not been reported in benign neurofibromas [4, 20, 52, 53]. Mice with heterozygous mutations in both their Nf1 and Tp53 genes developed malignancy,[27, 54] an indication, perhaps, that TP53 loss is critical to transformation. The homozygous loss of the cyclin-dependent kinase inhibitor 2A gene (CDKN2A), which encodes p16INK4A and p14ARF, has also been associated with NF1 malignancy [5557]. Another recent report has indicated that phosphatase and tensin homologue deleted on chromosome 10 gene (PTEN) dosage, and/or phosphatidylinositol 3-kinase/AKT8 virus oncogene cellular homologue (PI3K/AKT) pathway activation, may be rate-limiting steps in NF1 malignant transformation [58]. As yet, however, no characteristic gene expression signature has been defined for MPNST development, although several cell-cycle and signalling regulation genes: -- cyclin-dependent kinase inhibitor (CDKN2A); tumour protein 53 (TP53); retinoblastoma 1 (RB1); epidermal growth factor receptor (EGFR); CD44 antigen (CD44); platelet-derived growth factor receptor alpha (PDGFRA); hepatocyte growth factor (HGF); proto-oncogene protein (C-MET) and transcription factor (SOX9) -- are frequently deregulated [4].

          Recent studies of the micro-RNA expression profile of MPNSTs have expanded the pathogenic spectrum associated with this tumour. For example, microRNA-34a (miR-34a) is downregulated in MPNSTs; this microRNA (miRNA) regulates many cell cycle genes and is also upregulated by p53, suggesting that TP53 loss would lead to down-regulation of miR-34a and possibly several other miRNAs. This implies that this could be a critical event in malignant transformation [59]. In similar vein, miR-10b has been reported to be upregulated in SCs from NF1 tumours, while miR-10b inhibition reduced MPNST cell proliferation, migration and invasion [60]. NF1 mRNA is also a specific target for miR-10b,[60] indicating that these miRNAs represent potential therapeutic targets.

          Spinal neurofibromas

          About 40 per cent of NF1 patients present with tumours involving their spinal nerves. This is especially marked in individuals affected with familial spinal neurofibromatosis (FSNF), a variant form of NF1 in which bilateral tumours involving multiple spinal nerve roots are often the only manifestation of NF1 [6163]. Patients with FSNF have been reported to be significantly more likely to harbour missense or splice-site germline mutations compared with patients with classical NF1 [64]. A recent study of the NF1 locus found LOH in eight of 22 spinal tumours analysed, with most (75 per cent) of this LOH being due to mitotic recombination rather than genomic deletions [64].

          Gastrointestinal stromal tumours (GISTs)

          GISTs are the most common mesenchymal tumours of the gastrointestinal tract. Although most GISTs harbour activating somatic mutations of KIT and PDGFRA, the absence of such mutations from NF1-associated GISTs (NF1-GISTs) is probably indicative of a different pathogenetic mechanism. In NF1, the majority (60 per cent) of GISTs develop in the small intestine, whereas sporadic non-NF1 GISTs mainly involve the stomach [65].

          Somatic NF1 mutations have been identified in the interstitial cells of Cajal (ICC) throughout the gastrointestinal tract and in NF1-GISTs lacking KIT or PDGRA mutations [66]. Increased signalling through the Ras/MAPK pathway has also been shown to occur in NF1-GISTS, as opposed to sporadic GISTs. This would seem to indicate that a decrease in neurofibromin level, in the presence of normal c-KIT and PDGFRA levels, leads to tumour formation. It also suggests that NF1 haploinsufficiency is required for ICC hyperplasia, again demonstrating that, although a somatic NF1 mutation is absolutely necessary, it is not sufficient to permit tumorigenesis: additional genetic events required. These observations concur with Knudson's two-hit hypothesis. Somatic inactivation of the NF1 gene through gene deletion; intragenic deletion; and LOH through mitotic recombination have also been described [66, 67].

          Gastric carcinoid

          Gastric carcinoid tumours are associated with multiple endocrine neoplasia, atrophic gastritis and pernicious anaemia but are very rare in NF1 [17]. LOH at the NF1 locus has been demonstrated in a gastric carcinoid tumour derived from an NF1 patient [67].

          Juvenile myelomonocytic leukaemia (JMML)

          Young NF1 patients are at particular risk of developing JMML,[68] a clonal haematopoietic disorder characterised by hypersensitivity (at least in vitro) to granulocyte-macrophage colony-stimulating factor (GM-CSF). Moreover, some 15-20 per cent of JMML patients harbour a somatic NF1 inactivating mutation, even though most exhibit no other NF1 symptoms [69]. Patients may also carry inactivating mutations of other genes, with a recent study identifying that 70-80 per cent of mutations involve genes in the Ras/MAPK pathway, including one tyrosine-protein phosphatase non-receptor type 11 (PTPN11), neuroblastoma RAS viral oncogene homologue (NRAS), and v-Ki-ras2 kirsten rat sarcoma viral oncogene homologue (KRAS) as well as NF1 genes [70]. Additional somatic mutations have also been reported in the casitas B-lineage lym-phoma (CBL) and additional sex combs-like 1 (ASXL1) genes [71]. In most cases, the NF1 gene is lost either via LOH or by compound heterozygous microlesions,[72] which lead to a complete loss of neurofibromin and hyperactive signalling through the Ras/MAPK pathway. LOH may occur through 1.2-1.4 Mb interstitial deletions mediated by low copy number repeat (LCR) elements that flank the NF1 gene [73]. LOH through uniparental interstitial isodisomy (50-52.7 Mb) of chromosome 17 through double mitotic recombination, in an as-yet-unknown initiator cell, has also been reported [72]. The rarity of such events may indicate the existence of a selective advantage, conferred upon the NF1-/- cells, which might explain the propensity of NF1 patients to develop leukaemia [74].

          Astrocytomas (ACs)

          Optic pathway tumours or ACs are found in ~15 per cent of paediatric NF1 patients,[75] with the complete loss of neurofibromin evident in NF1-associated optic gliomas [76]. Approximately 84 per cent of NF1-associated ACs also exhibit LOH in the NF1 region, with many tumours also exhibiting LOH of 17p, suggesting the likely role of TP53 -- or other 17p13-located genes -- in AC formation [77]. As with MPNSTs, biallelic somatic NF1 mutation in ACs is, again, apparently insufficient to induce transformation.

          Phaeochromocytomas (PCs)

          PCs are extremely rare tumours, with only one to six cases observed per million individuals. PCs develop from neural crest-derived chromaffin cells, and the tumour cells produce and release catechol-amines, which cause hypertension and flushing. These are tumours of the adrenal medulla and are primarily associated with mutations of the Ret proto oncogene (RET), von Hippel-Lindau (VHL), succinate dehydrogenase complex, subunit B (SDHB), succinate dehydrogenase complex, subunit C (SDHC), and succinate dehydrogenase complex, subunit D (SDHD) genes, although LOH in the NF1 region, as well as LOH of other loci on both 17q and 17p, have been observed [78, 79].

          Glomus tumours

          Glomus tumours are small ( < 5 mm), benign, but often very painful tumours that develop specifically within the highly innervated glomus body located at the end of each digit. These tumours appear to develop from a-smooth muscle actin-positive cells that have undergone biallelic NF1 inactivation, resulting in increased Ras/MAPK activity [80]. The somatic NF1 mutations often differ between glomus tumours, indicating highly specific tumori-genic events. Brems et al[80]. have suggested that glomus tumours, although rare, should now be recognised as an integral component of the NF1 spectrum of disease.

          The somatic mutational spectrum of NF1-associated tumours

          A review of all published -- and the authors' many unpublished -- somatic NF1 alterations associated with NF1 tumours was undertaken to gain a better appreciation of NF1 tumorigenesis. As of July 2010, at least 577 different somatic NF1 gene changes had been reported in different NF1-associated tumours, with more than half (323/577; 56 per cent) corresponding to LOH in the NF1 gene region, some involving much larger regions of chromosome 17 (Table S1 (Table 4)). The level of LOH detected also differs between cutaneous neurofibromas, PNFs and MPNSTs (40 per cent, 79 per cent and 85 per cent, respectively; Table 1). Table 2 provides the incidence of LOH in the other tumour types, where appropriate evidence has been obtained by multiplex ligation-dependent probe amplification (MLPA), fluorescence in situ hybridisation (FISH) etc; 78 per cent (28/36) of cutaneous neurofibromas, 44 per cent (11/25) of PNFs and 16 per cent (5/31) of MPNSTs display LOH resulting from mitotic recombination. Some 79 per cent (15/19) of the JMML samples that exhibited LOH appear to have lost the entire 17q arm through mitotic recombination, perhaps indicating a significant correlation with this tumour type.
          Table S1

          Summary of germline mutations and loss of heterozygosity (LOH) in NF1-associated tumours

          Patient ID

          Germline mutation

          Type of germline mutation

          LOH

          LOH markers

          Predicted extent of LOH

          Evidence for genomic deletion? MLPA/CGHarrayCGH/FISH

          Probable mechanism

          No. samples with LOH

          Reference

          Dermal neurofibromas

                  

          T190.2

          Exon 2 and 3 deletion

          Two exon deletion

          Yes

          E5 RFLP, I12b,

          IVS27A28.4, J1/J2,

          EVI20, IVS38GT53.0,

          3'NF1, C7CT1/2

          (3'UTR), EW206,

          EW207, D17S798,

          D17S1868

          NF1 and 3' flanking region

          MLPA

          Deletion

          5/23

          1

          T190.6

            

          Yes

          E5 RFLP, I12b,

          IVS27A28.4, J1/J2,

          EVI20, IVS38GT53.0,

          3'NF1, C7CT1/2

          (3'UTR), EW206

          NF1 and 3' flanking region

          MLPA

          Deletion

            

          T190.11

            

          Yes

          E5 RFLP, I12b,

          IVS27A28.4, J1/J2,

          EVI20, IVS38GT53.0,

          3'NF1, C7CT1/2

          (3'UTR)

          NF1 and 3' flanking region

          MLPA

          Deletion

            

          T190.17

            

          Yes

          E5 RFLP, I12b,

          IVS27A28.4, J1/J2

          Intragenic NF1

          MLPA

          Deletion

            

          T190.18ii

            

          Yes

          E5 RFLP

          Intragenic NF1

          MLPA

          Deletion

            

          T206.1

          Ex4b:

          c.499_502delTGTT;

          p.C167GnfsX9

          4 bp

          deletion

          (FS)

           

          LOH

           

          NIA

            

          Unpublished data, Cardiff

          T206.2

             

          LOH

               

          T206.3

             

          LOH

               

          L002_3

          Ex9: c.1246C > T;

          p.Arg416X

          Nonsense

          Yes

          rs29001484, rs4583306,

          NF1 germline mutation,

          rs2055091, rs11869264

          NF1

          Array CGH

          Deletion

          6/28

          2

          L002_5

            

          Yes

          rs29001484, rs4583306,

          NF1 germline mutation,

          rs2055091, rs11869264

          NF1

          Array CGH

          Mitotic recombination

            

          L002_12

            

          Yes

          rs29001484, rs4583306,

          NF1 germline mutation,

          rs2055091, rs11869264

          NF1

          Array CGH

          Mitotic recombination

            

          L002 C

            

          Yes

          NS

           

          NIA

           

          3/38

          3

          T473.1A

          Ex10b: c.1413-

          1414delAG;

          p.Lys471AsnfsX4

          2 bp

          deletion

          (FS)

          Yes

          HHH202, J1J2, IVS27,

          EV120, IVS38

          NF1

          MLPA

          Mitotic recombination

          22/89

          4

          T473.1C

            

          Yes

          HHH202, J1J2, EV120

          NF1

          MLPA

          Mitotic recombination

            

          T473.3

            

          Yes

          J1J2, EV120, IVS38

          NF1

          MLPA

          Mitotic recombination

            

          T473.5

            

          Yes

          HHH202, J1J2, EV120,

          IVS38

          NF1

          MLPA

          Mitotic recombination

            

          T473.7

            

          Yes

          J1J2, EV120

          NF1

          MLPA

          Mitotic recombination

            

          T473.8

            

          Yes

          HHH202, J1J2, EV120,

          IVS38, 3'NF1, EW207,

          D17S949, D17S1822

          NF1 and 3'

          flanking

          region

          MLPA

          Mitotic recombination

            

          T473.10

            

          Yes

          J1J2, EV120, IVS38

          NF1

          MLPA

          Mitotic recombination

            

          T473.14

            

          Yes

          J1J2, EV120, IVS38,

          3'NF1, EW207,

          D17S949, D17S1822

          NF1 and 3'

          flanking

          region

          MLPA

          Mitotic recombination

            

          T473.15

            

          Yes

          J1J2, EV120, IVS38,

          3'NF1

          NF1

          MLPA

          Mitotic recombination

            

          T473.16

            

          Yes

          J1J2, EV120, IVS38,

          3'NF1, EW207,

          D17S949,

          D17S1822

          NF1 and 3'

          flanking

          region

          MLPA

          Mitotic recombination

            

          T473.21

            

          Yes

          J1J2, EV120

          NF1

          MLPA

          Mitotic recombination

            

          T473.35

            

          Yes

          EV120, IVS38

          Intragenic NF1

          MLPA

          Mitotic recombination

            

          T473.30

            

          Yes

          J1J2, EV120, IVS38,

          3'NF1, EW207,

          D17S949

          NF1 and 3'

          flanking

          region

          MLPA

          Mitotic recombination

            

          T473.32

            

          Yes

          J1J2, EV120

          Intragenic NF1

          MLPA

          Mitotic recombination

            

          T473.34

            

          Yes

          EV120, IVS38

          Intragenic NF1

          MLPA

          Mitotic recombination

            

          T225.1

          Ex10b deletion [MLPA]

          Single exon deletion

           

          LOH

           

          NIA

            

          Unpublished data, Cardiff

          T225.3

             

          LOH

               

          T68.2

          Deletion exons 10b-19b

          Partial gene deletion

          Yes

          UT172 - I38 206,207

          Exon 5-3' region

          NIA

            

          Unpublished data, Cardiff

          T68.3

            

          Yes

          UT172 - I38 206,207

          Exon 5-3' region

              

          CLJ1N

          Ex13: c.2041C > T;

          p.Arg681X

          Nonsense

          Yes

          NF1, D17S1800

          NF1 and 3' flanking region

          NIA

           

          32/126

          5, 6, 7

          CLJ2N

            

          Yes

          D17S33, D17S1294,

          NF1, D17S1800,

          D17S798, D17S250,

          D17S787, D17S802

          Majority of 17q

              

          T170.3

          Ex13: c.2041C > T;

          p.Arg681X

          Nonsense

           

          LOH

           

          NIA

            

          Unpublished data, Cardiff

          T170.2

             

          LOH

               

          ABA1N

          Ex13: c.2251 + 2T > C

          Splice site

          Yes

          DS17S1824, D17S841,

          D17S1294, D17S1863,

          NF1, D17S1800,

          D17S1880, D17S798,

          D17S250, D17S787,

          D17S802, D17S784,

          D17S928

          Majority of 17q

          NIA

           

          32/126

          5, 6, 7

          ABA2N

            

          Yes

          DS17S1824, D17S841,

          D17S1294, D17S1863,

          NF1, D17S1800,

          D17S1880, D17S798,

          D17S250, D17S787,

          D17S802, D17S784,

          D17S928

          Majority of 17q

             

          5, 6, 7

          T436

          Ex17: c.2875C > T;

          p.Glu959X

          Nonsense

          Yes

          IVS27, EV120, IVS38

          Intragenic NF1

          MLPA

          Mitotic recombination

          22/89

          4

          T439

            

          Yes

          IVS27, EV120, IVS38

          Intragenic NF1

          MLPA

          Mitotic recombination

            

          T440

            

          Yes

          IVS27, EV120, IVS38

          Intragenic NF1

          MLPA

          Mitotic recombination

            

          T444

            

          Yes

          HHH202, J1J2, IVS27,

          EV120, IVS38, 3'NF1

          NF1 and flanking regions

          MLPA

          Mitotic recombination

            

          T446

            

          Yes

          IVS27, EV120, IVS38

          Intragenic NF1

          MLPA

          Mitotic recombination

            

          T448

            

          Yes

          HHH202, J1J2, IVS27,

          EV120, IVS38, 3'NF1

          NF1 and flanking regions

          MLPA

          Mitotic recombination

            

          T454

            

          Yes

          IVS27, EV120, IVS38

          Intragenic NF1

          MLPA

          Mitotic recombination

            

          EAD1N

          Ex20: c.3419C > G;

          p.Ser1140X

          Nonsense

          Yes

          NS

           

          NIA

           

          32/126

          5, 6, 7

          EAD2N

                   

          CSG3N

          Ex21: c.3525_3526delAA;

          p.Arg1176GlufsX17

          2 bp deletion (FS)

          Yes

          D17S1824, D17S1294,

          NF1, D17S1800,

          D17S1880, D17S798

          NF1 and flanking regions

          NIA

           

          32/126

          5, 6, 7

          CSG38N

            

          Yes

          NF1

          NF1

              

          CSN1N

            

          Yes

          D17S1294, NF1,

          D17S1880, D17S798,

          D17S250, D17S787,

          D17S784, D17S928

          Majority of 17q

              

          CSG1N

            

          Yes

          D17S1294, NF1,

          D17S1800, D17S1880,

          D17S798, D17S250,

          D17S787, D17S802

          Majority of 17q

              

          CSG2N

            

          Yes

          DS17S1824, D17S1294,

          NF1, D17S1800,

          D17S1880, D17S798,

          D17S250, D17S787,

          D17S802

          Majority of 17q

              

          CSG4N

            

          Yes

          DS17S1824, D17S1294,

          NF1, D17S1800,

          D17S1880, D17S798,

          D17S250, D17S787,

          D17S802

          Majority of 17q

              

          CSG5N

            

          Yes

          DS17S1824, D17S1294,

          NF1, D17S1800,

          D17S1880, D17S798,

          D17S250, D17S787,

          D17S802

          Majority of 17q

              

          CSG21N

            

          Yes

          DS17S1824, D17S1294,

          NF1, D17S1800,

          D17S1880, D17S798,

          D17S250, D17S787,

          D17S802

          Majority of 17q

              

          CSG25N

            

          Yes

          D17S1294, NF1,

          D17S1800, D17S1880,

          D17S798, D17S250,

          D17S787, D17S802

          Majority

          of 17q

              

          CSG42N

            

          Yes

          D17S1294, NF1,

          D17S1800, D17S1880,

          D17S798, D17S250,

          D17S787, D17S802

          Majority of 17q

              

          CSG51N

            

          Yes

          DS17S1824, D17S1294,

          NF1, D17S1800,

          D17S1880, D17S798,

          D17S250, D17S787,

          D17S802

          Majority

          of 17q

          FISH

          Mitotic recombination

            

          CSG52N

            

          Yes

          D17S33, DS17S1824,

          D17S1294, NF1,

          D17S1800, D17S1880,

          D17S798, D17S250,

          D17S787, D17S802

          Majority

          of 17q

          NIA

             

          CSG62N

            

          Yes

          NS

               

          T177

          Ex23.1: c.3916 C > T;

          p.Arg1306X

          Nonsense

           

          LOH

           

          NIA

            

          Unpublished data, Cardiff

          T213

             

          LOH

               

          NF44

          UHG_1

          Ex27a: c.4515-2A > T

          Splice site

          Yes

          NF1 germline mutation,

          rs9891455

          NF1

          Array CGH

          Deletion

          6/28

          2

          NF44

          UHG_41

            

          Yes

          rs1018190

          NF1

          Array CGH

          Deletion

            

          T106.1

          Ex37: c.6791dupA;

          p.Tyr2264X

          1 bp

          insertion

          (FS)

          Yes

          IVS38, 3'NF1-1

           

          NIA

            

          Unpublished data, Cardiff

          T106.5

            

          Yes

          I41 - C3

          3' UTR

              

          T106.6

            

          Yes

          J1J2, EVI20, I38, I41,

          C3C7, 206, 207

               

          T210.2

          Ex42: c.7458delC;

          p.Tyr2487IlefsX5

          1 bp

          deletion

          (FS)

             

          30% WG

            

          Unpublished data, Cardiff

          T210.4

               

          30% WG

             

          T210.8

               

          30% WG

             

          T210.4

               

          E8: 30% WG

             

          T210.5

               

          E8: exon duplication

             

          HT1335

          Ex4c: c.7237C > T;

          p.Gln2413X

          Nonsense

             

          E16: del

            

          Unpublished data, Cardiff

          T128.30

          Ex6: c.784C > T;

          p.Arg262Cys

          Missense

          Yes

           

          3' UTR to

          3' region

          NIA

            

          Unpublished data, Cardiff

          T192.4

          Deletion of exons 6-27a [MLPA]

          Multi-exon deletion

           

          LOH: J1J2, EV120,

          HHH202,

              

          Unpublished data, Cardiff

          p062

          Ex7: c.910C > T;

          p.Arg304X

          Nonsense

           

          LOH (6 samples)

           

          Deletion (2 samples)

            

          Unpublished data, Cardiff

          p082

          Ex7: c.910C > T;

          p.Arg304X

          Nonsense

           

          LOH (5 samples)

           

          Deletion(5 samples)

             

          ACF1N

          Ex7: c.910C > T;

          p.Arg304X

          Nonsense

          Yes

          D17S841, D17S1294,

          D17S1863, NF1,

          D17S1880, D17S798,

          D17S250, D17S802,

          D17S784

          Majority of 17q

          FISH

          Mitotic recombination

          32/126

          5, 6, 7

          MIGS1N

          Ex7: c.910C > T;

          p.Arg304X

          Nonsense

          Yes

          D171863, NF1,

          D17S1800, D17S1880

          NF1 and flanking regions

          NIA

           

          32/126

           

          CAG1N

          Ex7: c.979delCinsTT;

          p.Leu327PhefsX3

          Indel (FS)

          Yes

          NF1

          Intragenic NF1

          NIA

           

          32/126

           

          CAG3N

          Ex7: c.979delCinsTT;

          p.Leu327PhefsX3

          Indel (FS)

          Yes

          NS

           

          NIA

           

          32/126

           

          T199

          Ex7: c.983_984delGT;

          p.Cys324X

          2 bp

          deletion

          (FS)

          Yes

          IVS12, J1J2

           

          NIA

            

          Unpublished data, Cardiff

          NF56-2

          Ex9: c.1246C > T;

          p.Arg416X

          Missense

          Yes

          Pin 1, RsaI, AluI, Pin 28,

          530, NF1 3'UTR, Mfd

          15

          NF1 and flanking regions

          NIA

           

          1/6

          8

          T197A

          Ex10a: c.1318C > T

          p.Arg440X

          Nonsense

           

          LOH?

              

          Unpublished data, Cardiff

          CLT1N

          Ex12a:

          c.1754_1757delTAAC;

          p.Thr586SerfsX19

          4 bp deletion (FS)

          Yes

          D17S841, D17S1863,

          NF1, D17S1800,

          D17S1880, D17S787,

          D17S802

          Majority of 17q

            

          32/126

           

          p022

          Ex12a:

          c.1756_1759delACTA;

          p.Thr586ValfsX18

          4 bp deletion (FS)

           

          LOH (9 samples)

           

          Deletion (2 samples)

            

          Unpublished data, Cardiff

          p020

          Ex13: c.2041C > T;

          p.Arg681X

          Nonsense

           

          LOH (2 samples)

           

          Deletion

          (0 samples)

             

          T141.5

          Ex13: c.2233delA;

          p.Ser745AlafsX2

          1 bp

          deletion

          (FS)

          Yes

          202, 12b, IVS27, IVS38,

          3'NF1

              

          Unpublished data, Cardiff

          p103

          Ex15: c.2338A > C;

          p.Thr780Pro

          Missense

           

          LOH (10 samples)

           

          Deletion(3 samples)

            

          Unpublished data, Cardiff

          T22

          Ex17: c.2851-2A > G

          Splice site

          Yes

           

          3'UTR to 3' flanking regions

             

          Unpublished data, Cardiff

          NF253-UHG E

          Ex17: c.2851-2A > G

          Splice site

          Yes

          Not specific

           

          NIA

           

          3/38

          3

          L005 A

          Ex18: c.3113 + 1G > A

          Splice site

                 

          319T1

          Ex19b: c.3208C > T;

          p.Gln1070X

          Nonsense

          Yes

          NF-exon5

          Intragenic NF1

            

          2/15

          9

          p023

          Ex21: c.3525_3526delAA;

          p.Arg1176SerfsX18

          2 bp deletion (FS)

           

          LOH (14 samples)

           

          Deletion (5 samples)

            

          Unpublished data, Cardiff

          p011

          Ex22: c.3826C > T;

          p.Arg1276X

          Nonsense

           

          LOH (5 samples)

           

          Deletion (1 sample)

             

          MASG2N

          Ex22: c.3870 + 1G > T

          Splice site

          Yes

          NF1, D17S1880,

          D17S798, D17S250,

          D17S787

          Majority of 17q

          FISH

          Mitotic recombination

          32/126

          5, 6, 7

          T171

          Ex23.2: c.4084 C > T;

          p.Arg1362X

          Nonsense

           

          LOH

              

          Unpublished data, Cardiff

          p104

          Ex25: c.4309G > T;

          p.Glu1436X

          Nonsense

           

          LOH (3 samples)

           

          Deletion

          (0 samples)

            

          Unpublished data, Cardiff

          p078

          Ex27a: c.4537C > T;

          p.Arg1513X

          Nonsense

           

          LOH (6 samples)

           

          Deletion

          (0 samples)

             

          p084

          Ex27a: c.4572C > G;

          p.Tyr1524X

          Nonsense

           

          LOH (2 samples)

           

          Deletion

          (0 samples)

             

          p102

          Ex29: c.5242C > T;

          p.Arg1748X

          Nonsense

           

          LOH (5 samples)

           

          Deletion

          (2 samples)

             

          p055

          Ex30: c.5710 G > T;

          p.Glu1904X

          Nonsense

           

          LOH (1 sample)

           

          Deletion

          (0 samples)

             

          EMN1N

          Ex30:

          c.5749 + 332A > G

          Splice site

          Yes

          NF1, D17S1800

          NF1 and 3' flanking region

            

          32/126

          5, 6, 7

          p027

          Ex33: c.6226delG;

          p.Ala2076GlnfsX13

          1 bp deletion (FS)

           

          LOH (1 sample)

           

          Deletion (0 samples)

            

          Unpublished data, Cardiff

          p052

          Ex37: c.6791_6792dupA;

          p.Tyr2264X

          1 bp duplication (FS)

           

          LOH (1 sample)

           

          Deletion (1 sample)

             

          T23.6

          Ex41:

          c.7268_7269delCA;

          p.Thr2423SerfsX2

          2 bp deletion (FS)

          Yes

          EVI20, I38, I41, C3

           

          NIA

            

          Unpublished data, Cardiff

          T100

          Ex41: c.7267dupA;

          p.Thr2426X

          1 bp duplication (FS)

          Yes

           

          I38 to 3'UTR

             

          Unpublished data, Cardiff

          T164.1

          Ex41: c.7285 C > T;

          p.Arg2429X

          Nonsense

           

          LOH

              

          Unpublished data, Cardiff

          MAR2N

          NI

          NI

          Yes

          NF1, D17S1800

          NF1 and 3' flanking region

          NIA

           

          32/126

          5, 6, 7

          MOPT2N

          NI

          NI

          Yes

          D17S1824, D17S1294,

          D171863, NF1,

          D17S1800, D17S1880

          NF1 and flanking regions

              

          NGL1N

          NI

          NI

          Yes

          D17S841, D17S1294,

          NF1, D17S1800,

          D17S1880, D17S798,

          D17S250, D17S802,

          D17S784, D17S928

          Majority of 17q

              

          JRR2N

          NI

          NI

          Yes

          D17S1294, D17S1863,

          NF1, D17S1800,

          D17S1880, D17S798,

          D17S250, D17S787

          Majority of 17q

              

          SLC1N

          NI

          NI

          Yes

          D17S33, DS17S1824,

          D17S841, D17S1294,

          NF1, D17S1880,

          D17S798, D17S250,

          D17S784, D17S928

          Majority of 17q

              

          HT1377.1

          NI

          NI

          Yes

               

          Unpublished data, Cardiff

          HT1377.2

          NI

          NI

          Yes

            

          NIA

             

          T109.4

          NI

          NI

          Yes

          I38, I41, 206

          3' region

              

          T167.c

          NI

          NI

          Yes

          IVS27, IVS38, 3'NF1

               

          T192.1

          NI

          NI

          Yes

          202, IVS12, J1J2, IVS27

               

          T197

          NI

          NI

          Yes

          J1J2

               

          T227.2

          NI

          NI

          Yes

          IVS12, J1J2

               

          T230.2

          NI

          NI

          Yes

          202, IVS12, IVS27

               

          T232.2

          NI

          NI

          Yes

          J1J2

               

          T241

          NI

          NI

          Yes

          J1J2

               

          NF253_32

          NI

          NI

          Yes

          rs1018190, rs9891455,

          rs8074061

          NF1

          Array CGH

          Mitotic recombination

          6/28

          2

          T224.1

          NI

          NI

           

          LOH

           

          NIA

            

          Unpublished data, Cardiff

          T162

          NI

          NI

           

          LOH

               

          T172

          NI

          NI

           

          LOH

               

          T179.1

          NI

          NI

           

          LOH

               

          T204.2

          NI

          NI

           

          LOH

               

          T224.2

          NI

          NI

           

          LOH

               

          T173.1

          NI

          NI

           

          LOH

               

          T179.2

          NI

          NI

           

          LOH

               

          T1281.2

          NI

          NI

           

          LOH

               

          T1281.4

          NI

          NI

           

          LOH

               

          T220

          NI

          NI

           

          LOH

               

          T221

          NI

          NI

           

          LOH

               

          T223

          NI

          NI

           

          LOH

               

          T258.1

          NI

          NI

           

          LOH

               

          T258.2

          NI

          NI

           

          LOH

               

          SCs from cutaneous neurofibromas

                  

          T543.2

          Ex4a:

          c.373delGinsATGTGT;

          p.Arg125HisfsX4

          Indel (FS)

          Yes

          J1J2-3'NF1

              

          Unpublished data, Cardiff

          T536A

          Ex40: c.7127_7258del132

          [Exon 40 deletion?]

          132 bp deletion (FS)

          Yes

          J1J2-IVS38

           

          NIA

            

          Unpublished

          data, Cardiff

          T541.2

            

          Yes

          EV120-3'NF1

               

          T541.4

            

          Yes

          EV120-3'NF1

               

          T539

          90 kb Deletion

          Genomic deletion

             

          Duplication:

          Ex19b-25

            

          Unpublished data, Cardiff

          PNFs

                   

          37a

          Ex24: c.4268A > G;

          p.Lys1423Arg

          Missense

          Yes

          HHH202, E5, I12b,

          EVI20,3'NF1-1

          Complete gene deletion

          (1.4 Mb)

          NF1

          (1.4 Mb)

          MLPA

          Genomic deletion

          20/29

          10

          37b

            

          Yes

          IVS27, EVI20, IVS38,

          3'NF1-1 Probable gene

          deletion

          NF1 and 3' flanking

          region

          MLPA

          Genomic deletion

          20/29

          10

          T210.2

          PNF

          Ex42: c.7458delC;

          p.Tyr2486IlefsX15

          1 bp deletion (FS)

          Yes

          LOH detected in only 30% of cells

            

          30% whole gene deletion

           

          Unpublished data, Cardiff

          T261

          PNF

          Ex3: c.288 + 1 delG

          1 bp deletion at a splice site

          LOH IVS38

                

          605-1

          Ex4a: c.289-2A > G

          Splice site

          Yes

          D17S975, IVS27TG24.8,

          IVS27TG28.4,

          D17S1166, D17S1880,

          D17S907, D17S1788,

          D17S1861, D17S1809,

          D17S668, D17S928

          NF1 and flanking regions

          MLPA

          Mitotic recombination

          13/43

          11

          47/T411

          Ex4a:

          c.440_441GC > AA;

          p.Cys147X

          Nonsense

          LOH IVS27,

          IVS38,

          3'NF1-1

          IVS38, 3'NF1-1

          NF1 and 3' flanking

          region

          NIA

           

          20/29

          10

          8/T328

          Ex4b: c.480-2A > G

          Splice site

          LOH:

          IVS27, IVS38

          IVS27, IVS38

          Intragenic NF1

          MLPA

          Mitotic recombination

          20/29

          10

          335-3

          Ex4b: c.528T > A;

          p.Asp176Glu

          Missense

          Yes

          D17S2237,

          IVS27TG24.8,

          D17S1166, D17S1800

          NF1

          MLPA

          Genomic deletion

          13/43

          11

          59

          Ex6: c.752dupA;

          p.Asp241GlufsX7

          Small Insertion (fs)

          Yes

          intron 38 marker 53.0

          Intragenic NF1

          NIA

           

          1/38

          12

          T265.2

          Ex9: c.1186-13delT

          (Pathogenicity?)

          1 bp deletion within a splice site

          LOH ivs27,

          ivs38

                

          374-4

          Ex10a: c.1318C > T;

          p.Arg440X

          Nonsense

          Yes

          IVS27TG24.8,

          IVS27TG28.4,

          D17S1166, D17S1880,

          D17S907, D17S1861

          NF1 and 3' flanking region

          MLPA

          Mitotic

          recombination

          13/43

          11

          14a/

          T412

          Ex13: c.2076C > G;

          p.Tyr692X

          Nonsense

          I12B, 3' NF1

          I12B, 3' NF1-1

          NF1 and 3' flanking region

          MLPA

          Mitotic recombination

          20/29

          10

          T263

          Ex15: c.2326-2A > T

          Splice site

          LOH ivs27

          [rest hom]

                

          22/T394

          Ex16: c.2446C > T;

          p.Arg816X

          Nonsense

          IVS27

          IVS 27, EVI20

          Intragenic NF1

          MLPA

          Mitotic recombination

          20/29

          10

          T437.2

          Ex16: c.2497delT;

          p.Ser833ProfsX7

          1 bp deletion (FS)

          1-6ex

          1,2,3,4a,4b,4c,

          6 deletion

            

          NIA

             

          T212

          Ex16: c.2705deT;

          p.Met902ArgfsX22

          1 bp deletion (FS)

              

          Ex1-Ex 41

          deletion

          [variable ?]

            

          18/T298

          Ex18: c.3113 + 1G > A

          Splice site

          LOH

          :HHH202,

          IVS 27

          IVS 27

          Intragenic

          NF1

          MLPA

          Inconclusive

          20/29

          10

          30/T342

          Ex19a: c.3123G > T;

          p.Met1041Ile

          Missense

          Yes

          Determined by MLPA

          NF1

          (1.4 Mb)

          MLPA

          Genomic

          deletion

          20/29

          10

          5

          Ex20:

          c.345&_3460delCTCA;

          p.Leu1153MetfsX3

          4 bp

          deletion

          (FS)

          Yes

          NF1 gene

          NF1

            

          1/3

          13

          23/

          T373.2

          Ex22: c.3826C > T;

          p.Arg1276X

          Nonsense

          WG deletion

          [mixed cell

          population]

          IVS 27, IVS38

          Intragenic

          NF1

          MLPA

          Inconclusive

          20/29

          10

          452T

          Ex23.2: c.4084C > T;

          p.Arg1362X

          Nonsense

          Yes

          NF-exon5 RFLP, NF-

          (GATN)n intron 26, NF-

          Alu(AAAT)n(i27b), NF-

          EVI2B RFLP(i27b), NF-

          EVI2A RFLP(i27b), NF-

          IVSAC28.4(i27b), NF-

          Evi-20, NF-

          IVS38TG53.0, NF intron

          41 RFLP, D17S57

          (EW206), D17S250,

          D17S1301, D17S384

          NF1 and

          3' flanking

          region

          NIA

           

          4/10

          9

          27/T301

          Ex23.2: c.4095C > A;

          p.Cys1365X

          Nonsense

          Yes

          Determined by MLPA

          Intragenic

          NF1

          MLPA

          Genomic

          deletion

          20/29

          10

          T330

          Ex24: c.4267A > G;

          p.Lys1423Glu

          Missense

          IVS27

            

          NIA

             

          6/T362/

          T395

          Ex24: c.4268A > G;

          p.Lys1423Arg

          Missense

          Yes

          EW206, EW207

          Intragenic

          NF1

          MLPA

          Inconclusive

          20/29

           

          T362

          PNF

          Ex24: c.4268A > G;

          p.Lys1423Arg

          Missense

          LOH:

          HHH202, E5,

          I12b, EVI20,

          3'NF

            

          NIA

             

          T395

          PNF

          Ex24: c.4268A > G;

          p.Lys1423Arg

          Missense

          LOH:IVS27,

          EVI20, IVS38,

          3'NF1-1

                

          317-1

          Ex25: c.4270-2A > G

          Splice site

          Yes

          IVS27TG24.8,

          IVS27TG28.4,

          D17S1166

          NF1

          MLPA

          Genomic

          deletion

          13/43

          11

          T393

          Ex27a: c.4537C > T;

          p.Arg1513X

          Nonsense

              

          Whole gene

          deletion

            

          26/T300

          Ex29:

          c.5227_5229delGTAinsT;

          p.Val1743TyrfsX17

          Indel (FS)

          Yes

          Determined by MLPA

          NF1

          (1.4 Mb)

          MLPA

          Genomic

          deletion

          20/29

          10

          338-2

          Ex29: c.5290delG;

          p.Ala1764LeufsX8

          1 bp

          deletion

          (FS)

          Yes

          D17S783, D17S975,

          IVS27TG28.4,

          D17S1166, D17S1880

          NF1 and

          flanking

          regions

          MLPA

          Genomic

          deletion

          13/43

          11

          952-8

          Ex30: c.5749 + 4delA

          Splice site

          Yes

          D17S975, D17S1880,

          D17S907, D17S1788,

          D17S1861, D17S1809,

          D17S668, D17S928

          NF1 and

          flanking

          regions

          MLPA

          Genomic

          deletion

            

          34/T392

          Ex31:

          c.5750_5754dupGTATT;

          p.Glu1919ValfsX4

          5 bp

          duplication

          (FS)

          Yes

          EVI20, IVS38, 3'NF1-1,

          NF1 and

          3' flanking

          region

          NIA

           

          20/29

          10

          21/T357

          Ex37: c.6791dupA;

          p.Tyr2264X

          1 bp

          duplication

          (FS)

          LOH: EW206

          EW206

          Intragenic

          NF1

          MLPA

          Mitotic

          recombination

            

          T375

          Ex40: c.7237C > T;

          p.Gln2413X

          Nonsense

          16Ex 16

          deletion,

          ex13 & 18

          also lower

            

          NIA

             

          7

          Ex41: c.7285C > T;

          p.Arg2429X

          Nonsense

          Yes

          HHH202, IVS27

          Intragenic

          NF1

          MLPA

          Mitotic

          recombination

            

          15/T407

          Ex46: c.7926dupT;

          p.Lys2643X

          1 bp

          duplication

          (FS)

          LOH 3'NF1-1,

          EW206

          3'NF1-1

          NF1 and

          3' flanking

          region

          MLPA

          Inconclusive

            

          c1 UK/

          T56

          Ex46: c.8035A > T;

          p.Thr2679Ser

          Missense

          LOH IVS27

          IVS27

          Intragenic

          NF1

          NIA

             

          T408

          Segmental NF1 NI

          NI

          LOH: IVS27,

          IVS38,

          3'NF1-1

                

          T377

          Segmental NF1 NI

          NI

          WG deletion

                

          39

          Segmental NF1 NI

          NI

          Yes

          Determined by MLPA

          NF1

          (1.1 Mb)

          MLPA

          Genomic

          deletion

            

          43

          Segmental NF1 NI

          NI

          Yes

          IVS27, IVS38, 3'NF1-1

          NF1 and

          3' flanking

          region

          MLPA

          Mitotic

          recombination

            

          T385.1

          NI

          NI

          Yes

            

          NIA

            

          Unpublished

          data, Cardiff

          T385.2

          NI

          NI

          LOH/del

                

          T316

          NI

          NI

          Yes

          LOH:

          HHH202,

          E5, I12b,

          EVI20,3'NF,

          C71/2, EW206

               

          76, 45-95

          NI

          NI

          Yes

          IVS27AC28.4,

          IVS27TG24.8,

          IVS38GT53

          Intragenic

          NF1

          NIA

           

          8/14

          14

          x1, 47-95

          NI

          NI

          Yes

          IVS27AC28.4, M98509,

          IVS38GT53

          Intragenic

          NF1

              

          x1, 27-97

          NI

          NI

          Yes

          IVS27AC28.4, M98509,

          IVS38GT53

          Intragenic

          NF1

              

          293,

          71-97

          NI

          NI

          Yes

          M98509, IVS27TG24.8,

          IVS38GT53

          Intragenic

          NF1

              

          293,

          124-98

          NI

          NI

          Yes

          M98509, IVS27TG24.8,

          IVS38GT54

          Intragenic

          NF1

              

          290,

          83-97

          NI

          NI

          Yes

          IVS27AC28.4,

          IVS27TG24.8,

          IVS38GT53

          Intragenic

          NF1

              

          290,

          121-98

          NI

          NI

          Yes

          IVS27AC28.4,

          IVS27TG24.8,

          IVS38GT53

          Intragenic

          NF1

              

          292,

          122-98

          NI

          NI

          Yes

          IVS27TG24.8,

          IVS38GT53

          Intragenic

          NF1

              

          PD-T1

          NI

          NI

          Yes

          NF-Alu(AAAT)n(i27b),

          D17S1800

          Intragenic

          NF1

          NIA

           

          4/10

          9

          386T

          NI

          NI

           

          NF-Alu(AAAT)

          n(i27b), NF-

          IVSAC28.4(i27b),

          NF-Evi-20, NF-

          IVS38TG53.0,

          D17S1800

          NF1

              

          454T-V

          NI

          NI

          Yes

          NF-Alu(AAAT)n(i27b),

          NF-EVI2B RFLP(i27b),

          NF-IVSAC28.4(i27b),

          NF-Evi-20, NF intron 41

          RFLP, D17S57

          (EW206), D17S1301

          NF1

              

          NF284-1

          NI

          NI

          Yes

          Exon 28 14bp

          duplication marker

          (specific

          to germline

          lesion found)

          Intragenic

          NF1

            

          1/1

          8

          2654-97

          NI

          NI

          Yes

          Determined by FISH

          Whole

          chromosome

          FISH

          Genomic

          deletion

          1/11

          15

          385

          NI

          NI

          Yes

          D17S975, D17S1880,

          D17S907, D17S1788,

          D17S1861, D17S1809,

          D17S668, D17S928

          NF1 and

          flanking

          regions

          MLPA

          Mitotic

          recombination

          13/43

          11

          389-2

          NI

          NI

          Yes

          D17S975, D17S1307,

          D17S2237,

          IVS27TG28.4,

          D17S1800, D17S1880,

          D17S907, D17S1861,

          D17S1809, D17S668,

          D17S928

          NF1 and

          flanking

          regions

          MLPA

          Mitotic

          recombination

            

          604-4

          NI

          NI

          Yes

          D17S1800, D17S1880,

          D17S907, D17S1861,

          D17S928

          NF1 and

          flanking

          regions

          MLPA

          Mitotic

          recombination

            

          913-5

          NI

          NI

          Yes

          D17S2237,

          IVS27TG24.8,

          D17S1880, D17S1788,

          D17S1861

          NF1 and

          3' flanking

          region

          MLPA

          Genomic

          deletion

            

          612-1

          NI

          NI

          Yes

          D17S1307, D17S2237,

          IVS27TG24.8,

          D17S1166, D17S1800,

          D17S1880

          NF1 and

          flanking

          regions

          MLPA

          Genomic

          deletion

            

          337-5

          NI

          NI

          Yes

          D17S1307, D17S2237,

          IVS27TG24.8,

          D17S1166, D17S1800

          NF1

          MLPA

          Genomic

          deletion

            

          390

          NI

          NI

          Yes

          IVS27TG24.8,

          IVS27TG28.4,

          D17S1166, D17S1800

          NF1

          MLPA

          Genomic

          deletion

            

          49

          NI

          NI

          Yes

          HHH202, E5, I12b,

          EVI20, 3'NF1-1, C71/2,

          EW206

          NF1

          MLPA

          Inconclusive

          20/29

          10

          Spinal neurofibromas

                   

          1

          Ex7: c.899T > C;

          p.Leu300Pro

          Missense

          Yes

          EVI20, IVS38

          Intragenic

          NF1

          MLPA

          Mitotic

          recombination

          8/22

          16

          7

          Ex9: c.1186-13delT

          (Pathogenicity?)

          1 bp

          deletion

          (FS)

          Yes

          IVS27, IVS38

          Intragenic

          NF1

          MLPA

          Mitotic

          recombination

            

          3

          Ex16: c.2410-2A > T

          Splice site

          Yes

          IVS27

          Intragenic

          NF1

          MLPA

          Mitotic

          recombination

            

          11.1

          Ex22: c.3827G > A;

          p.Arg1276Glu

          Missense

          Yes

          IVS38

          Intragenic

          NF1

          MLPA

          Mitotic

          recombination

            

          11.2

            

          Yes

          Deletion of exons

          13 > 16

          Intragenic

          NF1

          MLPA

          Deletion

            

          2

          Ex23.2: c.4066G > A;

          p.Glu1356Lys

          Missense

          Yes

          27, 3'NF1

          NF1

          MLPA

          Mitotic

          recombination

            

          10

          Ex29: c.5242C > T;

          p.Arg1748X

          Nonsense

          Yes

          I12B, Alu1, J1J2 and

          EVI20

          Intragenic

          NF1

          MLPA

          Mitotic

          recombination

            

          MPNSTs

                   

          T196.20

          Deletion exons 2 and 3

          Two exon

          deletion

          Yes

          I12b, IVS27AC28.4,

          EVI20(IVS27B),

          IVS38GT53.0 (IVS38),

          3'-NF1, C7/CT1/2

          (3'-UTR), EW206

          (3extragenic), EW207

          (3'extragenic), D17S798

          NF1 and

          3' flanking

          region

          MLPA

          Mitotic

          recombination

          2/11

          1

          T196.24

            

          Yes

          NF1 exon 5, I12b,

          IVS27AC28.4,

          EVI20(IVS27B),

          IVS38GT53.0 (IVS38),

          3'-NF1, C7/CT1/2

          (3'-UTR), EW206

          (3'extragenic), EW207

          (3'extragenic)

          NF1 and

          3' flanking

          region

          MLPA

          Genomic

          deletion

          2/11

          1

          13

          Deletion exons 2 and 3

          Two exon

          deletion

          Yes

          Ex5, I12b, IVS27, EVI20,

          IVS38,C7CT, EW206,

          EW207,3'NF1

          NF1 and

          3' flanking

          region

          MLPA/CGH

          array

          Genomic

          deletion

          31/34

          17

          7

          Ex4c: c.654 + 1G > T

          Splice site

          Yes

          UT172, HH202, J1/J2,

          EVI20

          NF1

          MLPA/CGH

          array

          Genomic

          deletion

            

          27

          Ex8:

          c.1133_1136delACTG;

          p.Asp378AlafsX7

          4 bp

          deletion

          (FS)

          Yes

          Ex5, J1J2,3'NF1

          NF1 and

          3' flanking

          region

          NIA

             

          9

          Ex11: c.1713G > A;

          p.Trp571X

          Nonsense

          Yes

          D17S182, I12b, J1/J2

          Intragenic

          NF1

              

          10

            

          Yes

          UT172, HH202, J1/J2,

          EVI20, > 2.2Mb

          NF1

          MLPA/CGH

          array

          Genomic

          deletion

            

          23

          Ex12a: c.1318C > T;

          p.Arg440X

          Nonsense

          Yes

          HHH202, EVI20. IVS38

          Intragenic

          NF1

          NIA

             

          14

          Ex12a:

          c.1754_1757delTAAC;

          p.Thr586ValfsX19

          4 bp

          deletion

          (FS)

          Yes

          IVS27, 3'NF1

          NF1 and

          3' flanking

          region

          MLPA/CGH

          array

          Mitotic

          recombination

            

          12

          Ex13: c.2002-14C > G

          Splice site

          Yes

          I12b, IVS27, EVI20,

          IVS38, 3'NF

          NF1 and

          3' flanking

          region

          MLPA/CGH

          array

          Genomic

          deletion

            

          43

          Ex13: c.2041C > T;

          p.Arg681X

          Nonsense

          Yes

          Determined by MLPA

          NF1

          MLPA

          Duplication

          mitotic

          recombination

          6/25

          18

          15

          Ex16: c.2497delT;

          p.Ser833ProfsX7

          1 bp

          deletion

          (FS)

           

          Intragenic Deletion

          (Exons 1-6) MLPA

          Intragenic

          NF1

          MLPA/CGH

          array

          Genomic

          deletion

          31/34

          17

          25

          Ex16: c.2705delT;

          p.Met902ArgfsX22

          1 bp

          deletion

          (FS)

          Yes

          Intragenic deletion

          (exons1-41) MLPA

          Intragenic

          NF1

          MLPA/CGH

          array

          Genomic

          deletion

            

          17

          Ex20:

          c.3457_3460delCTCA;

          p.Leu1153MetfsX3

          4 bp

          deletion

          (FS)

             

          NIA

             

          18

            

          Yes

          3'NF1

          Intragenic

          NF1

          MLPA/CGH

          array

          Genomic

          deletion

            

          459T1

          Ex21: c.3684delC;

          p.Asn1229MetfsX11

          1 bp

          deletion

          (FS)

          Yes

          TP53(INTRON1),

          TP53(INTRON6), NF-

          (GATN)n INTRON26,

          NF-IVSAC28.4(i27b),

          D17S57, D17S250,

          D17S1301, D17S784

          Whole

          chromosome

          NIA

           

          3/5

          9

          8

          Ex22: c.3732delT;

          p.Thr1245Leufsx21

          1 bp

          deletion

          (FS)

          Yes

          Int12, J1J2

          Intragenic

          NF1

            

          31/34

          17

          64

          Ex23.1: c.3368 + 1delG

          1 bp

          deletion at

          a splice site

          Yes

          Determined by MLPA

          NF1

          MLPA

          Genomic

          deletion

          6/25

          18

          56

          Ex25: c.4276C > A;

          p.Gln1426Lys

          Missense

          Yes

          Determined by MLPA

          NF1

          MLPA

          Duplication

          mitotic

          recombination

            

          4

          Ex27a: c.4537C > T;

          p.Arg1513X

          Nonsense

          Yes

          IVS27b

          Intragenic

          NF1

          MLPA/CGH

          array

          Genomic

          deletion

          31/34

          17

          6

          Ex28: c.5003insTG;

          p.Tyr1668LeufsX7

          2 bp

          insertion

          (FS)

          Yes

          I4b, J1J2, EVI20

          NF1

          MLPA/CGH

          array

          Genomic

          deletion

            

          21

          Ex29: c.5234C > G;

          p.Ser1745X

          Nonsense

          Yes

          Partial gene deletion

          Intragenic

          NF1

          MLPA/CGH

          array

          Genomic

          deletion

            

          19

          Ex37: c.6792C > A;

          p.Tyr2264X

          Nonsense

          Yes

          I12b, IVS27, J1J2, EVI20,

          IVS38, C7CT

          NF1

          MLPA/CGH

          array

          Genomic

          deletion

            

          24

          Ex38: c.6961insC;

          p.Leu2321ProfsX5

          1 bp

          duplication

          (FS)

          Yes

          Determined by MLPA

          NF1

          MLPA

          Genomic

          deletion

          6/25

          18

          1

          Ex41:

          c.7268_7269delCA;

          p.Thr2423SerfsX2

          2 bp

          deletion

          (FS)

          Yes

          Intron 41-30

          Intragenic

          NF1

          MLPA/CGH

          array

          Genomic

          deletion

          31/34

          17

          58

          NI

          NI

          Yes

          HHH202, NF1, EW206

          Intragenic

          NF1

           

          Genomic

          deletion

          6/11

          19

          52

          NI

          NI

          Yes

          HHH202, NF1, EW206,

          EW207

          Intragenic

          NF1

           

          Genomic

          deletion

            

          22

          NI

          NI

          Yes

          HHH202

          Intragenic

          NF1

           

          Genomic

          deletion

            

          8

          NI

          NI

          Yes

          EW206, EW207

          Intragenic

          NF1

           

          Genomic

          deletion

            

          2

          NI

          NI

          Yes

          p144D6, pYNZ22.1,

          pYNH37.3, EW503

          NF1 region

          and some

          17p

           

          Genomic

          deletion

          5/6

          20

          3

          NI

          NI

          Yes

          EW503, EW301 (B),

          EW301 (T)

          Intragenic

          NF1

           

          Genomic

          deletion

            

          4

          NI

          NI

          Yes

          p144D6, pYNZ22.1,

          pYNH37.3, EW503,

          EW301 (T), pHHH202,

          EW207 (B), pTHH59

          Whole

          chromosome

           

          Genomic

          deletion

            

          5

          NI

          NI

          Yes

          p144D6, pYNZ22.1,

          pYNH37.3, EW503,

          EW301 (B), EW301 (T),

          pHHH202, EW207

          (B)

          Whole

          chromosome

           

          Genomic

          deletion

            

          10

          NI

          NI

          Yes

          p144D6, pYNZ22.1,

          NF1 region

          and some

          17p

           

          Genomic

          deletion

            

          88-3/14

          NI

          NI

          Yes

          D17S30, TP53, D17S71,

          D17S8, D17S57

          Whole

          chromosome

          G-banded

          chromosome

          17

          duplication

          Genomic

          duplication

          3/9

          21

          88-8

          NI

          NI

          Yes

          D17S30, D17S71

          NF1

          NIA

             

          88-18

          NI

          NI

          Yes

          D17S30, D17S71,

          D17S21, D17S33,

          EVI2B, D17S82

          Whole

          chromosome

              

          1

          NI

          NI

          Yes

          DI7S5, DI7SI, DI7SI37,

          CRYBI, NF1, DI7S146

          NF1 and

          flanking

          regions

          NIA

           

          2/5

          22

          4

          NI

          NI

          Yes

          DI7S34, DI7S5, DI7S146

          NF1 and

          flanking

          regions

              

          1

          NI

          NI

          Yes

           

          NF1

           

          Genomic

          deletion

          1/1

          23

          1

          NI

          NI

          Yes

          NF1 alu, TP53 BHP53

          Whole

          chromosome

          NIA

           

          3/7

          24

          7

          NI

          NI

          Yes

          CRYB1, NF1 alu, TP53

          BHP53

          Whole

          chromosome

              

          8

          NI

          NI

          Yes

          D17S4, D17S74, NF1

          e.31, NF1 alu

          NF1

              

          441T

          NI

          NI

          Yes

          TP53(INTRON6),

          D17S1863,

          D17twbch = S33, NF-

          IVSAC28.4(i27b),

          NF-Evi-20,

          NF-IVS38TG53.0,

          D17S1800, D17S73,

          D17S1301

          Whole

          chromosome

          NIA

           

          3/5

          9

          396T4

          NI

          NI

          Yes

          NF-IVSAC28.4(i27b,

          NF-IVS38TG53.0,

          D17S57, D17S250,

          D17S1301

          NF1 and 3' flanking

          region

              

          2

          NI

          NI

          Yes

          NF1, P16, TP53

          Whole

          chromosome

          NIA

           

          5/8

          13

          5a

          NI

          NI

          Yes

          NF1, P16, TP53

          Whole

          chromosome

              

          5b

          NI

          NI

          Yes

          NF1, P16, TP53

          Whole

          chromosome

              

          6a

          NI

          NI

          Yes

          NF1, P16, TP53

          Whole

          chromosome

              

          6b

          NI

          NI

          Yes

          NF1, P16, TP53

          Whole

          chromosome

              

          2

          NI

          NI

          Yes

          Total gene deletion

          NF1

          MLPA/CGH

          array

          Genomic

          deletion

          31/34

          17

          5

          NI

          NI

          Yes

          Total gene deletion

          NF1

          MLPA/CGH

          array

          Genomic

          deletion

            

          24

          NI

          NI

          Yes

          EVI20, IVS27, IVS38

          Intragenic

          NF1

          MLPA/CGH

          array

          Genomic

          deletion

            

          26

          NI

          NI

          Yes

          NF1 gene deletion

          NF1

          MLPA/CGH

          array

          Genomic

          deletion

            

          48

          NI

          NI

          Yes

          Determined by MLPA

          NF1

          MLPA

          Genomic

          deletion

          6/25

          18

          86

          NI

          NI

          Yes

          Determined by MLPA

          NF1

          MLPA

          Genomic

          deletion

            

          ACs

                   

          T65.1

          Ex24: c.4267A > G;

          p.Lys1423Glu

          Missense

          Yes

          NS

          NF1 3' flanking

          region

          NIA

           

          1/1

          25

          57

          NI

          NI

          Yes

          EW206

          Intragenic

          NF1

          NIA

           

          1/1

          19

          58

          NI

          NI

          Yes

          D17S1849, D17S1863,

          D17S1880

          NF1 and 3'

          flanking

          region

          NIA

           

          2/4

          26

          76

          NI

          NI

          Yes

          D17S1863, D17S1880

          NF1 and 3'

          flanking

          region

              

          182

          NI

          NI

          Yes

          IVS27TG24.8

          Intragenic

          NF1

          NIA

           

          11/12

          27

          185

          NI

          NI

          Yes

          IVS27AC28.4,

          IVS38GT53, D17S804

          NF1 region

          and some

          17p

              

          187

          NI

          NI

          Yes

          IVS27AC28.4,

          IVS38GT53, D17S796

          NF1 region

          and some

          17p

              

          309

          NI

          NI

          Yes

          IVS38GT53, D17S796

          NF1 region

          and some

          17p

              

          330

          NI

          NI

          Yes

          IVS27AC28.4,

          IVS38GT53, D17S520,

          D17S796, D17S804

          NF1 region

          and some

          17p

              

          502

          NI

          NI

          Yes

          IVS27AC28.4, D17S520,

          D17S796

          NF1 region

          and some

          17p

              

          519

          NI

          NI

          Yes

          IVS27TG28.4, M98509,

          IVS27TG24.8,

          IVS38GT53

          Intragenic

          NF1

              

          297

          NI

          NI

          Yes

          IVS27TG28.4, M98509,

          IVS38GT53

          Intragenic

          NF1

              

          609

          NI

          NI

          Yes

          IVS27TG28.4, M98509

          Intragenic

          NF1

              

          20954

          NI

          NI

          Yes

          IVS27TG24.8,

          IVS38GT53

          Intragenic

          NF1

              

          20962

          NI

          NI

          Yes

          IVS27AC28.4, M98509,

          IVS38GT53

          Intragenic

          NF1

              

          1

          NI

          NI

          Yes

          Homozygous

           

          FISH

          Unknown

          3/4

          28

          9

          NI

          NI

          Yes

          Homozygous

           

          FISH

          Genomic

          deletion

            

          10

          NI

          NI

          Yes

          Homozygous

           

          FISH

          Genomic

          deletion

            

          Gastric carcinoid tumours

                   

          1

          Ex37: c.6841C > T;

          p.Gln2281X

          Nonsense

          Yes

          IVS27TG24, D17S250

          Intragenic

          NF1

          NIA

           

          1/1

          29

          GISTs

                   

          1

          Ex27a: c.4537C > T;

          p.Arg1513X

          Nonsense

          Yes

          D17S841, Alu, IVS27GT,

          IVS27CAGT, IVS38,

          3'NF1-1, 3'NF1-2

          NF1 and 3'

          flanking

          region

          MLPA

          Mitotic

          recombination

          1/1

          30

          NF1-3

          Ex45: c.7807delG;

          p.Aal2603LeufsX3

          1 bp

          deletion

          (FS)

          Yes

          Alu, IVS27AC33.1,

          IVS38GT53.0,

          IVS27TG24.8

          Intragenic

          NF1

          Array CGH

          Genomic

          deletion

          1/7

          3

          JMML

                   

          D102

          Ex4b: c.574C > T;

          p.Arg192X

          Nonsense

          Yes

          D17S925, D17S1800,

          D17S1880, D17S855,

          D17S1827, D17S787,

          D17S948, D17S784

          Majority

          of 17q

          SNP array

          Mitotic

          recombination-

          UPD

          4/5

          31

          D115

          Ex13: c.2066delT;

          p.Val689GlyfsX59

          1 bp

          deletion

          (FS)

          Yes

          D17S925, D17S1800,

          D17S1880, D17S855,

          D17S1827, D17S787,

          D17S948, D17S784

          Majority

          of 17q

          SNP array

          Mitotic

          recombination-

          UPD

            

          D003

          Ex22: c.3861_3862delCT;

          p.Cys1288ValfsX21

          2 bp

          deletion

          (FS)

          Yes

          D17S925, D17S1800,

          D17S1880, D17S855,

          D17S1827, D17S787,

          D17S948, D17S784

          Majority

          of 17q

          SNP array

          Mitotic

          recombination-

          UPD

            

          D126

          Ex44: c.7699C > T;

          p.Gln2567X

          Nonsense

          Yes

          D17S925, D17S1800,

          D17S1880, D17S855,

          D17S1827, D17S787,

          D17S948, D17S784

          Majority

          of 17q

          SNP array

          Mitotic

          recombination-

          UPD

            

          1

          NI

          NI

          Yes

          D17S1975, D17S1294,

          UT172, NF1,

          D17S1800, D17S250,

          D17S801, D17S939,

          D17S836, D17S1806,

          D17S1822, D17S1830

          Majority

          of 17q

          FISH

          Mitotic

          recombination-

          interstitial

          isodisomy

          (paternal)

          10/10

          32

          2

          NI

          NI

          Yes

          D17S1975, D17S1294,

          UT172, NF1,

          D17S1800, D17S250,

          D17S801, D17S939,

          D17S836, D17S1806,

          D17S1822, D17S1830

          Majority

          of 17q

          FISH

          Mitotic

          recombination-

          interstitial

          isodisomy

          (paternal)

            

          3

          NI

          NI

          Yes

          D17S1294, UT172,

          NF1, D17S1800,

          D17S250, D17S801,

          D17S939, D17S836,

          D17S1806, D17S1822,

          D17S1830

          Majority

          of 17q

          FISH

          Mitotic

          recombination

          interstitial

          isodisomy

          (paternal)

            

          4

          NI

          NI

          Yes

          D17S1975, D17S1294,

          UT172, NF1,

          D17S1800, D17S250,

          D17S801, D17S939,

          D17S836, D17S1806,

          D17S1822, D17S1830

          Majority

          of 17q

          FISH

          Mitotic

          recombination-

          interstitial

          isodisomy

          (maternal)

            

          5

          NI

          NI

          Yes

          D17S1975, D17S1294,

          UT172, NF1,

          D17S1800, D17S250,

          D17S801, D17S939,

          D17S836, D17S1806,

          D17S1822

          Majority of

          17q

          FISH

          Mitotic

          recombination-

          interstitial

          isodisomy

          (maternal)

            

          6

          NI

          NI

          Yes

          D17S1878, D17S33,

          D17S1975, D17S1294,

          UT172, NF1,

          D17S1800, D17S250,

          D17S801, D17S939,

          D17S836, D17S1806,

          D17S1822, D17S1830

          Majority of

          17q

          FISH

          Mitotic

          recombination-

          interstitial

          isodisomy

          (maternal)

            

          7

          NI

          NI

          Yes

          D17S1294, UT172,

          NF1, D17S1800,

          D17S250, D17S801,

          D17S939, D17S836,

          D17S1806, D17S1822,

          D17S1830, D17S928

          Majority of

          17q

          FISH

          Mitotic

          recombination-

          interstitial

          isodisomy

          (maternal)

            

          8

          NI

          NI

          Yes

          D17S1975, D17S1294,

          UT172, NF1,

          D17S1800, D17S250,

          D17S801, D17S939,

          D17S836, D17S1806,

          D17S1822, D17S1830,

          D17S928

          Majority of

          17q

          FISH

          Mitotic

          recombination-

          interstitial

          isodisomy

          (paternal)

            

          9

          NI

          NI

          Yes

          NF1, D17S1800

          Intragenic

          NF1

          FISH

          Genomic

          deletion

            

          10

          NI

          NI

          Yes

          NF1, D17S1800

          Intragenic

          NF1

          FISH

          Genomic

          deletion

            

          D419

          NI

          NI

          Yes

          D17S925, D17S1841,

          D17S1294, D17S1863,

          D17S1849, D17S1166,

          D17S1800, D17S1880,

          D17S1818, D17S855,

          D17S1827, D17S787,

          D17S948, D17S785,

          D17S784

          Majority of

          17q

          MLPA

          Mitotic

          recombination-

          UPD

          5/10

          33

          D561

          NI

          NI

          Yes

          D17S1294, D17S1863,

          D17S1849, D17S1166,

          D17S1800, D17S1880,

          D17S1818, D17S855,

          D17S1827, D17S787,

          D17S948, D17S785,

          D17S784

          Majority of

          17q

          MLPA

          Mitotic

          recombination-

          UPD

            

          D378

          NI

          NI

          Yes

          D17S1294, D17S1863,

          D17S1849, D17S1166,

          D17S1800, D17S1880,

          D17S1818, D17S855,

          D17S785

          Majority of

          17q

          Array CGH

          Mitotic

          recombination-

          UPD

            

          D341

          NI

          NI

          Yes

          D17S1849, D17S1166,

          D17S1800, D17S1880

          NF1 and

          flanking

          regions

          Array CGH

          Genomic

          deletion

            

          D566

          NI

          NI

          Yes

          D17S1849, D17S1166,

          D17S1800, D17S784

          NF1 and

          flanking

          regions

          Array CGH

          Genomic

          deletion

            

          PCs

                   

          1

          NI

          NI

          Yes

          DI7S34, DI7S137,

          CRYBI, NF1, DI7S4

          Whole

          chromosome

          NIA

           

          7/7

          22

          2

          NI

          NI

          Yes

          CRYBI, DI7S33, NF1,

          DI7S55, DI7S4

          NF1

              

          3

          NI

          NI

          Yes

          DI7S5, DI7S134,

          DI7S58, DI7S33

          Whole

          chromosome

              

          4

          NI

          NI

          Yes

          DI7S33, NF1

          Intragenic

          NF1

              

          5

          NI

          NI

          Yes

          DI7S71, NF1, DI7S226

          Whole

          chromosome

              

          6L

          NI

          NI

          Yes

          DI7S5, NF1, DI7S145,

          DI7S226

          Whole

          chromosome

              

          6R

          NI

          NI

          Yes

          DI7S5, NF1, DI7S145,

          DI7S226

          Whole

          chromosome

              

          1

          NI

          NI

          Yes

          TP53-BAM, TP53 AccII,

          NF1-AE25 (BgIII) SNP,

          THH59-TaqI,

          THH59-PvuII

          Majority of

          17

          NIA

           

          2/7

          34

          1

          NI

          NI

          Yes

          NF1-AE25 (BgIII) SNP,

          THH59-TaqI, THH59-

          PvuII-adrenal corticoid

          tumour

          NF1 and 3'

          flanking

          region

              

          NS

          NI

          NI

          Yes

              

          14/21

          35

          Glomus tumours

                   

          NF1-G2

          Ex42: c.7395_7404del10;

          p.Thr2466SerfsX33

          10 bp

          deletion

          (FS)

          Yes

          Introns 27-38

          Intragenic

          NF1

          Array CGH

          Mitotic

          recombination

          1/7

          36

          CGH, comparative genomic hybridisation; array CGH, high resolution CGH; FS, frame shift; NI, not informative; WG, whole gene; NA, not available; UPD, uniparental disomy; MLPA, multiplex ligation-dependent probe amplification; FISH, fluorescent in situ hybridisation.

          Supplementary Table References

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          2. De Raedt, T., Maertens, O., Chmara, M., Brems, H. et al. (2006), 'Somatic loss of wild type NF1 allele in neurofibromas: Comparison of NF1 microdeletion and non-microdeletion patients', Genes Chromosomes Cancer Vol. 45, pp. 893-904.

          3. Maertens, O., Brems, H., Vandesompele, J., De Raedt, T. et al. (2006), 'Comprehensive NF1 screening on cultured Schwann cells from neurofi-bromas', Hum. Mutat. Vol. 27, pp. 1030-1040.

          4. Thomas, L., Kluwe, L., Chuzhanova, N., Mautner, V. et al. (2010), 'Analysis of NF1 somatic mutations in cutaneous neurofibromas from patients with high tumor burden', Neurogenetics Vol. 11, pp. 391-400.

          5. Serra, E., Puig, S., Otero, D., Gaona, A. et al. (1997), 'Confirmation of a double-hit model for the NF1 gene in benign neurofibromas', Am. J. Hum. Genet. Vol. 61, pp. 512-519.

          6. Serra, E., Ars, E., Ravella, A., Sánchez, A. et al. (2001), 'Somatic NF1 mutational spectrum in benign neurofibromas: mRNA splice defects are common among point mutations', Hum. Genet. Vol. 108, pp. 416-429.

          7. Serra, E., Rosenbaum, T., Nadal, M., Winner, U. et al. (2001), 'Mitotic recombination effects homozygosity for NF1 germline mutations in neu-rofibromas', Nat. Genet. Vol. 28, pp. 294-296.

          8. Eisenbarth, I., Beyer, K., Krone, W. and Assum, G. (2000), 'Toward a survey of somatic mutation of the NF1 gene in benign neurofibromas of patients with neurofibromatosis type 1', Am. J. Hum. Genet. Vol. 66, pp. 393-401.

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          10. Upadhyaya, M., Spurlock, G., Monem, B., Thomas, N. et al. (2008), 'Germline and somatic NF1 gene mutations in plexiform neurofibromas', Hum. Mutat. Vol. 29, pp. E103-E111.

          11. Steinmann, K., Kluwe, L., Friedrich, R., Mautner, V. et al. (2009), 'Mechanisms of loss of heterozygosity in neurofibromatosis type 1-associated plexiform neurofibromas', J. Invest. Dermatol. Vol. 129, pp. 615-621.

          12. Däschner, K., Assum, G., Eisenbarth, I., Krone, W. et al. (1997), 'Clonal origin of tumor cells in a plexiform neurofibroma with LOH in NF1 intron 38 and in dermal neurofibromas without LOH of the NF1 gene', Biochem. Biophys. Res. Commun. Vol. 234, pp. 346-350.

          13. Frahm, S., Mautner, V., Brems, H., Legius, E. et al. (2004), 'Genetic and phenotypic characterization of tumor cells derived from malignant peripheral nerve sheath tumors of neurofibromatosis type 1 patients', Neurobiol. Dis. Vol. 16, pp. 85-91.

          14. Kluwe, L., Friedrich, R. and Mautner, V. (1999), 'Allelic loss of the NF1 gene in NF1-associated plexiform neurofibromas', Cancer Genet. Cytogenet. Vol. 113, pp. 65-69.

          15. De Luca, A., Buccino, A., Gianni, D., Mangino, M. et al. (2003), 'NF1 gene analysis based on DHPLC', Hum. Mutat. Vol. 21, pp. 171-172.

          16. Upadhyaya, M., Spurlock, G., Kluwe, L., Chuzhanova, N. et al. (2009), 'The spectrum of somatic and germline NF1 mutations in NF1 patients with spinal neurofibromas', Neurogenetics Vol. 10, pp. 251-263.

          17. Upadhyaya, M., Kluwe, L., Spurlock, G., Monem, B. et al. (2008), 'Germline and somatic NF1 gene mutation spectrum in NF1-associated malignant peripheral nerve sheath tumors (MPNSTs)', Hum. Mutat. Vol. 29, pp. 74-82.

          18. Bottillo, I., Ahlquist, T., Brekke, H., Danielsen, S. et al. (2009), 'Germline and somatic NF1 mutations in sporadic and NF1-associated malignant peripheral nerve sheath tumours', J. Pathol. Vol. 217, pp. 693-701.

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          20. Menon, A., Anderson, K., Riccardi, V., Chung, R. et al. (1990), 'Chromosome 17p deletions and p53 gene mutations associated with the formation of malignant neurofibrosarcomas in von Recklinghausen neu-rofibromatosis', Proc. Natl. Acad. Sci. USA Vol. 87, pp. 5435-5439.

          21. Glover, T., Stein, C., Legius, E., Andersen, L. et al. (1991), 'Molecular and cytogenetic analysis of tumors in von Recklinghausen neurofibroma-tosis', Genes Chromosomes Cancer Vol. 3, pp. 62-70.

          22. Xu, W., Mulligan, L.M., Ponder, M.A., Liu, L. et al. (1992), 'Loss of NF1 alleles in phaeochromocytomas from patients with type I neurofi-bromatosis', Genes Chromosomes Cancer Vol. 4, pp. 337-342.

          23. Legius, E., Marchuk, D., Collins, F. and Glover, T. (1993), 'Somatic deletion of the neurofibromatosis type 1 gene in a neurofibrosarcoma supports a tumour suppressor gene hypothesis', Nat. Genet. Vol. 3, pp. 122-126.

          24. Lothe, R., Slettan, A., Saeter, G., Brøgger, A. et al. (1995), 'Alterations at chromosome 17 loci in peripheral nerve sheath tumors', J. Neuropathol. Exp. Neurol. Vol. 54, pp. 65-73.

          25. Upadhyaya, M., Han, S., Consoli, C., Majounie, E. et al. (2004), 'Characterization of the somatic mutational spectrum of the neurofibro-matosis type 1 (NF1) gene in neurofibromatosis patients with benign and malignant tumors', Hum. Mutat. Vol. 23, pp. 134-146.

          26. Gutmann, D., Donahoe, J., Brown, T., James, C. et al. (2000), 'Loss of neurofibromatosis 1 (NF1) gene expression in NF1-associated pilocytic astrocytomas', Neuropathol. Appl. Neurobiol. Vol. 26, pp. 361-367.

          27. Kluwe, L., Hagel, C., Tatagiba, M., Thomas, S. et al. (2001), 'Loss of NF1 alleles distinguish sporadic from NF1-associated pilocytic astrocyto-mas', J. Neuropathol. Exp. Neurol. Vol. 60, pp. 917-920.

          28. Gutmann, D., James, C., Poyhonen, M., Louis, D. et al. (2003), 'Molecular analysis of astrocytomas presenting after age 10 in individuals with NF1', Neurology Vol. 61, pp. 1397-1400.

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          Table 1

          Contribution of LOH and NF1 micro-lesions to the somatic NF1 mutational spectrum in different types of NF1-associated tumour

          Tumour type

          LOH

          Point mutations

          Total

          Dermal neurofibroma

          144 (40%)

          211 (60%)

          355

          Plexiform neurofibroma

          67 (79%)

          18 (21%)

          85

          Spinal neurofibroma

          7 (70%)

          3 (30%)

          10

          MPNST

          55 (85%)

          10 (15%)

          65

          Astrocytoma

          18 (100%)

          0 (0%)

          18

          GIST/gastric carcinoid

          3 (38%)

          5 (62%)

          8

          JMML

          18 (95%)

          1* (5%)

          19

          Phaeochromocytoma

          10 (100%)

          0 (0%)

          10

          Glomus tumour

          1 (14%)

          6 (86%)

          7

          Overall

          323 (55%)

          254 (44%)

          577

          * Compound heterozygous NF1 mutations were identified in five of six haemopoietic tumours analysed. As no other normal tissues were available in these five cases, it was not possible to distinguish between the associated germline and somatic NF1 mutations.

          Table 2

          Mechanistic basis of the NF1 gene-associated LOH identified in different NF1-associated tumours

          Tumour type

          Tumour showing mitotic recombination (number & percentile)

          Tumours with genomic deletions (number & percentile)

          Dermal neurofibroma

          28 (76%)

          8 (24%)

          Plexiform neurofibroma

          11 (44%)

          14 (56%)

          Spinal neurofibroma

          7 (88%)

          1 (12%)

          MPNST

          5 (16%)

          26 (84%)

          Astrocytoma

          0 (0%)

          2 (100%)

          GIST/gastric carcinoid

          1 (50%)

          1 (50%)

          JMML

          15 (79%)

          4 (21%)

          Phaeochromocytoma

          0 (0%)

          0 (0%)

          Glomus tumour

          1 (100%)

          0 (0%)

          Tabulated information only given for tumours in which the precise LOH mechanism was identifiable.

          Tumour DNA analysis has also identified 254 somatic NF1 gene lesions, including nonsense, missense, splice site, microdeletion/microinsertions ( < 20 bp), indels (combined insertion-deletion events) and larger ( > 20 bp) deletions/insertions (Tables 3). The consequences of all deletions and insertions for the reading frame were also determined, with five sequence changes being compound heterozygous NF1 mutations found in five haemopoietic tumours; however, with no other tissue available for analysis, it was not possible to differentiate between germline and somatic NF1 point mutations (Table S2 (Table 5)). About 75 per cent (191/254) of the somatic mutations associated with NF1 tumours comprise mutations that are predicted to give rise to truncated proteins. Of these 191 changes, only 18 result from the insertion or duplication of bases; the remaining 173 truncations arise from deletion, nonsense mutation or frameshift events. Splice site mutations form a considerable proportion (39/254; 15.0 per cent) of the mutational spectrum, while missense changes only account for some 9.4 per cent (24/254) of the somatic NF1 mutations.
          Table 3

          The spectrum and percentile distribution of somatic NF1 micro-lesions reported in different NF1-associated tumours

          Tumour type

          Mutation type

           

          Deletion

          Insertion

          Indel

          Nonsense

          Splice site

          Missense

          Truncating

          Total

          Dermal neurofibroma

          82 (39%)

          15 (7%)

          2 (1%)

          59 (28%)

          32 (15%)

          21 (10%)

          158 (75%)

          211

          Plexiform neurofibroma

          6 (33%)

          1 (6%)

          -

          7 (39%)

          2 (11%)

          2 (11%)

          14 (78%)

          18

          Spinal neurofibroma

          -

          -

          -

          -

          2 (66%)

          1 (33%)

          0

          3

          MPNST

          7 (70%)

          1 (10%)

          1 (10%)

          1 (10%)

          -

          -

          10 (100%)

          10

          GIST/gastric carcinoid

          1 (20%)

          -

          -

          3 (60%)

          1 (20%)

          -

          4 (80%)

          5

          JMML*

          *

          *

          *

          1 (100%)

          *

          *

          1 (100%)

          1

          Glomus tumour

          2 (33%)

          1 (17%)

          -

          1 (17%)

          2 (33%)

          -

          4 (67%)

          6

          Overall

          98 (39%)

          18 (7%)

          3 (1%)

          72 (28%)

          39 (15%)

          24 (9%)

          191 (75%)

          254

          * Compound heterozygosity of NF1 mutations in several JMML tumours cases meant it was not possible to distinguish between associated germline and somatic NF1 mutations.

          Table S2

          Summary of germline and somatic point mutations in NF1-associated tumours

          Patient ID

          Germline point mutation

          Type of germline mutation

          Somatic point mutation

          Effect of somatic mutation

          Source

          Dermal neurofibromas

          T196.3

          Ex 2 and 3 deleted

          2 exon deletion

          Ex4c: c.648dup73 p.Leu216

          (through splice site)

          73 bp

          duplication

          (FS)

          1

          T196.12

            

          Ex4c: c.655-1G > A

          Splice site

           

          T196.15

            

          Ex6: c.750delT

          p.Phe250LeufsX30

          1 bp deletion

          (FS)

           

          T196.16

            

          Ex16: c.2534_2557del24

          p.Cys845X

          24 bp deletion

          (In-frame)

           

          T196.7

            

          Ex16: c.2844delA

          p.Gly949AspfX3

          1 bp deletion

          (FS)

           

          T196.4

            

          Ex18: c.3047_c3048delGT

          p.Cys1016SerfsX4

          2 bp deletion

          (FS)

           

          T196.5

            

          Ex27a: c.4537C > T

          p.Arg1513X R

          Nonsense

           

          T196.13

            

          Ex27b: c.4743delG

          p.Asp1582IlefsX21

          1 bp deletion

          (FS)

           

          T196.1

            

          Ex44: c.7721_7722delAA

          p.Lys257Ser4fsX4

          2 bp deletion

          (FS)

           

          T543.1

          Ex4a: c.373delGinsATGTGT

          p.Arg125HisfsX22

          Indel (FS)

          Ex21: c.3568del80

          p.Gly1190HisfsX3

          80 bp deletion

          (FS)

          Unpublished data,

          Cardiff

          T543.3

            

          Ex26: c.4388C > T

          p.Ser1463Phe

          Missense

           

          T128.10

          Ex6: c.784C > T p.Arg262Cys

          Missense

          Ex4b: c.574C > T p.Arg192X

          R

          Nonsense

          Unpublished data,

          Cardiff

          T128.1

            

          Ex8: c.1170delC

          p.Asp390LysfsX6

          1 bp deletion

          (FS)

           

          T128.17

            

          Ex10c: c.1556A > C

          p.Gln519Pro R

          Missense

           

          T128.8

            

          Ex32: c.6055_6056delTC

          p.Ser2019TrpfsX18

          2 bp deletion

          (FS)

           

          NF29a-4

          Ex6: c.801G > A p.Trp267X

          Nonsense

          Ex10a: c.1381C > T

          p.Arg461X

          Nonsense

          37

          NF17-8

            

          Ex10c: c.1528-14_1546del33

          p.Asp510fs (through splice

          site)

          32 bp deletion

          [FS]

           

          NF17-1

            

          Ex10c: c.1641 + 1G > A

          Splice site

           

          NF17-9

            

          Ex18: c.3049C > T

          p.Glu1017X

          Nonsense

           

          NF29a-7

            

          Ex19b: c.3303_3314+7del19

          p.Glu1101 (through splice site)

          19 bp deletion

          [FS]

           

          NF17-15

            

          Ex23.1: c.3916C > T

          p.Arg1306X R

          Nonsense

           

          NF29a-9

            

          Ex27b: c.4756insT

          p.Tyr1586LeufsX14

          1 bp insertion

          (FS)

           

          NF17-18

            

          Ex28: c.5205 + 1G > A

          Splice site

           

          NF17-23

            

          Ex31: c.5772_5775delTTTG

          p.Cys1924TrpfsX4

          4 bp deletion

          (FS)

           

          NF29a-5

            

          Ex40: c.7237_7253del17

          p.Gln2413fsX2

          17 bp deletion

          (FS)

           

          L-002 F

          Ex9: c.1246C > T p.Arg416X

          Nonsense

          Ex3: c.246_247delTC

          p.Glu83SerfsX15

          2 bp deletion

          (FS)

          3

          L-002 A

            

          Ex5: c.655-1G > T

          Splice site

           

          L-002 D

            

          Ex8: c.1105C > T p.Gln369X

          Nonsense

           

          L-002 E

            

          Ex8: c.1153delC

          p.Arg385AlafsX2

          1 bp deletion

          (FS)

           

          L-002 B

            

          Ex22: c.3757_3764del8

          p.Leu1253ThrfsX8

          8 bp deletion

          (FS)

           

          NF282-1

          Ex9: c.1260+1G > A

          Splice site

          Ex23.2: c.4021C > T

          p.Gln1341X

          Nonsense

          8

          NF282-2

            

          Ex23.2: c.4084C > T

          p.Arg1362X

          Nonsense

           

          T473.6

          Ex10b: c.1413_1414delAG

          p.Lys471AsnfsX1

          2 bp deletion

          (FS)

          Ex7: c.890delA

          p.Leu297SerfsX20

          1 bp deletion

          (FS)

          4

          T473.12

            

          Ex12b: c.1884insA p.Tyr628X

          1 bp insertion

          (FS)

           

          T473.11

            

          Ex16: c.2451insG

          p.Ser818ValfsX12

          1 bp insertion

          (FS)

           

          T473.18

            

          Ex22: c.3807insC

          p.Ser1270LeufsX13

          1 bp insertion

          (FS)

           

          T473.20

            

          Ex23.2: c.4087delA

          p.Ser1363ValfsX22

          1 bp deletion

          (FS)

           

          T473.13

            

          Ex31: c.5888A > C

          p.Asn1963Thr

          Missense

           

          T473.33

            

          Ex34: c.6478A > G

          p.Ser2160Gly

          Missense

           

          T473.36

            

          Ex38: c.6859delG

          p.Asp2287ThrfsX18

          1 bp deletion

          (FS)

           

          T473.17

            

          Ex40: c.7128delG

          p.Tyr2377ThrfsX23

          1 bp deletion

          (FS)

           

          T82.3

          Ex12a: c.1754_1757delTAAC

          p.Thr585ValfsX18

          4 bp deletion

          (FS)

          Ex16: c.2445delG

          p.Arg815SerfsX5

          1 bp deletion

          (FS)

          Unpublished data,

          Cardiff

          T82.5

            

          Ex35: c.6621_6625delGTGGA

          p.Gln2207HisfsX11

          5 bp deletion

          (FS)

           

          T77.3

          Ex12a: c.1783G > A

          p.Glu595Lys

          Missense

          Ex16: c.2446C > T

          p.Arg816X R

          Nonsense

          Unpublished data,

          Cardiff

          T77.1

            

          Ex29: c.5242C > T

          p.Arg1748X R

          Nonsense

           

          T77.4

            

          Ex31: c.5839C > T

          p.Arg1947X

          Nonsense

           

          T141.4

          Ex13: c.2233delA

          p.Ser745AlafsX2

          1 bp deletion

          (FS)

          Ex12b: c.1885G > A

          p.Gly629Arg

          Missense

          Unpublished data,

          Cardiff

          T141.13

            

          Ex30: c.5731delT

          p.Ser1911LeufsX9 R

          2 bp deletion

          (FS)

           

          T133

          Ex16: c.2446C > T

          p.Arg816X

          Nonsense

          Ex31: c.5897dupAC

          p.Glu1966HisfsX25

          2 bp

          duplication

          (FS)

          Unpublished data,

          Cardiff

          T137

            

          Ex31: c.5898dupAC

          p.Glu1966HisfsX25

          2 bp

          duplication

          (FS)

           

          T437

          Ex17: c.2875C > T

          p.Gln959X

          Nonsense

          Ex2: c.67A > T p.Ile23Leu

          Missense

          4

          T441

            

          Ex4b: c.586G > T p.Glu196X

          Nonsense

           

          T459

            

          Ex10c: c.1641+2T > G

          Splice site

           

          T433

            

          Ex10c: c.1660C > G

          p.Gln554Glu

          Missense

           

          T469

            

          Ex12a: c.1724delCACA

          p.Ser575X

          4 bp deletion

          (FS)

           

          T468

            

          Ex13: c.2041C > T p.Arg681X

          Nonsense

           

          T472

            

          Ex13: c.2088G > A p.Trp696X

          Nonsense

           

          T463

            

          Ex16: c.2410-3T > G

          Splice site

           

          T451

            

          Ex20: c.3449C > T

          p.Ser1150Leu

          Missense

           

          T456

            

          Ex22: c.3709-2A > G

          Splice site

           

          T450

            

          Ex23.2: c.4084C > T

          p.Arg1362X R

          Nonsense

           

          T442

            

          Ex27b: c.4687_4691del5

          p.Phe1563GlyfsX36

          5 bp deletion

          (FS)

           

          T443

            

          Ex27b: c.4693insG

          p.Ala1565GlyfsX35

          1 bp insertion

          (FS)

           

          T467

            

          Ex29: c.5380C > T

          p.Gln1794X

          Nonsense

           

          T457

            

          Ex34: c.6448A > T

          p.Lys2150X

          Nonsense

           

          T471

            

          Ex38: c.6895delG

          p.Val2299TrpfsX8

          1 bp deletion

          (FS)

           

          T434

            

          Ex44: c.7699C > T

          p.Gln2567X

          Nonsense

           

          T435

            

          Ex44: c.7702C > T

          p.Gln2568X

          Nonsense

           

          T460

            

          Ex46: c.7924delT

          p.Ser2642LeufsX16

          1 bp deletion

          (FS)

           

          CSG6N

          Ex21: c.3525_3526delAA

          p.Arg1176SerfsX18

          2 bp deletion

          (FS)

          Ex4c: c.587-8del6 Splicing

          effect?

          Intronic

          deletion

          6, 7

          CSG13N

            

          Ex9: c.1260 + 1G > A

          Splice site

           

          CSG48N

            

          Ex10c: c.1604A > G

          p.Gln535Arg

          Missense

           

          CSG29N

            

          Ex14: c.2266C > T p.Gln756X

          Nonsense

           

          CSG33N

            

          Ex16: c.2816delA

          p.Asn939IlefsX12

          1 bp deletion

          (FS)

           

          CSG19N

            

          Ex17: c.2928del13

          p.Glu977AsnfsX3

          13 bp deletion

          (FS)

           

          CSG26N

            

          Ex26: c.4514 + 1G > C

          Splice site

           

          CSG44N

            

          Ex31: c.5774delT

          p.Leu1925TrpfsX4

          1 bp deletion

          (FS)

           

          CSG8N

            

          Ex33: c.6292_6322del31

          p.Arg2098PhefsX21

          31 bp deletion

          (FS)

           

          CSG30N

            

          Ex45: c.7908-2A > T

          Splice site

           

          NF482-

          UHG B

          Ex21: c.3525_3526delAA

          p.Arg1176SerfsX18

          2 bp deletion

          (FS)

          Ex4a: c.359_375del17

          p.Phe120X

          17 bp deletion

          (FS)

          3

          NF482-

          UHG C

            

          Ex4c: c.603_621del19

          p.Phe201fsX4

          19 bp deletion

          (FS)

           

          NF482-

          UHG A

            

          Ex8: c.1185 + 1G > A

          Splice site

           

          NF482-

          UHG D

            

          Ex14: c.2252-30_2252-

          6del??insT

          Indel (FS?)

           

          T191.5

          Ex22: c.3721C > T

          p.Arg1241X

          Nonsense

          Ex4b: c.505_524del20

          p.Glu169X

          20 bp deletion

          (FS)

          Unpublished data,

          Cardiff

          T191.9

            

          Ex10b: c.1417delA

          p.Thr473GlnfsX24

          1 bp deletion

          (FS)

           

          T191.1

            

          Ex18: c.2991 + 1 G > A

          Splice site

           

          T191.2

            

          Ex22: c.3721C > T

          p.Arg1241X R

          Nonsense

           

          T175.1

          Ex23.2: c.4084C > T

          p.Arg1362X

          Nonsense

          Ex12a: c.1738insT

          p.Tyr580LeufsX7 R

          1 bp insertion

          (FS)

          Unpublished data,

          Cardiff

          T175.2

            

          Ex31: c.5817C > A

          p.Cys1939X R

          Nonsense

           

          T209.1ii

          Ex28: c.4950C > A

          p.Tyr1650X

          Nonsense

          Ex7: c.1062 + 1G > A R

          Splice site

          Unpublished data,

          Cardiff

          T209.7

            

          Ex10a: c.1318C > T

          p.Arg440X R

          Nonsense

           

          T209.8

            

          Ex15: c. 2326G > A

          p. Ala776Thr R

          Missense?/

          splicing?

           

          T209.5

            

          Ex25: c.4345delA

          p.Ser1449AlafsX12

          1 bp deletion

          (FS)

           

          T209.6

            

          Ex37: c.6790_6806del17

          p.Tyr2264AspfsX8

          17 bp deletion

          (FS)

           

          T506.5

          Ex36: c.6756 + 2T > G

          Splice site

          Ex4b: c.480delG

          p.Arg160SerfsX5

          1 bp deletion

          (FS)

          4

          T506.2

            

          Ex6: c.731_732delAA

          p.Glu244ValfsX5

          2 bp deletion

          (FS)

           

          T506.4

            

          Ex17: c.2987insAC

          p.Val996AspfsX17

          2 bp insertion

          (FS)

           

          T506.8

            

          Ex19b: c.3306insA

          p.Phe1103IlefsX2

          1 bp insertion

          (FS)

           

          T506.1

            

          Ex22: c.3745_3764del20

          p.Ser1249ThrfsX7

          20 bp deletion

          (FS)

           

          T506.9

            

          Ex33: c.6364del114

          p.Glu2122 (through splice site)

          114 bp

          deletion (FS)

           

          T506.6

            

          Ex40: c.7127-3T > G

          Splice site

           

          T106.3

          Ex37: c.6791insA p.Tyr2264Xfs

          1 bp insertion

          (FS)

          Ex13: c.2033delC

          p.Pro678GlnfsX9 R

          1 bp deletion

          (FS)

          Unpublished data,

          Cardiff

          T106.4

            

          Ex26: c.4374_4375delCC

          p.Leu1459X R

          2 bp deletion

          (FS)

           

          T175.1

          Ex37: c.6792C > G

          p.Tyr2264X

          Recurrent

          nonsense

          mutation that

          causes a

          splicing defect

          Ex12a: c.1738insT

          p.Tyr580LeufsX7 R

          1 bp insertion

          (FS)

          Unpublished data,

          Cardiff

          T143.2

            

          Ex19a: c.3124delGTAGinsAT

          p.Val1042IlefsX16

          Indel (FS)

           

          T143.13

            

          Ex30: c.5731delT

          p.Ser1911LeufsX9 R

          1 bp deletion

          (FS)

           

          T175.2A

            

          Ex31: c.5817C > A

          p.Cys1939X R

          Nonsense

           

          T541.3

          Ex40: c.7127_7258del132

          p.Gly2376. Is this a complete

          exon 40 deletion??

          132 bp Inframe deletion

          (FS) Complete

          exon 40

          deletion ??

          Ex12b: c.1888delG

          p.Val630X R

          1 bp deletion

          (FS)

          Unpublished data,

          Cardiff

          T541.1

            

          Ex27b: c.4743insG

          p.Asp1582GlufsX18

          1 bp insertion

          (FS)

           

          T536B

            

          Ex40: c.7169delG

          p.Arg2390LysfsX6

          1 bp deletion

          (FS)

           

          T210.1

          Ex42: c.7458delC

          p.Tyr2487Ilefs

          1 bp deletion

          (FS)

          Ex7: c.1062 + 1G > A R

          Splice site

          Unpublished data, Cardiff

          T210.6

            

          Ex22: c.3870 + 2T > A

          Splice site

           

          T181.3

          E6-27b: Partial deletion of

          gene 90 kb

          Partial gene

          deletion

          Ex3: c.227insG

          p.Glu76GlyfsX30

          1 bp insertion

          (FS)

          Unpublished data,

          Cardiff

          T211.2

            

          Ex7: c.910C > T

          p.Arg304X R

          Nonsense

           

          T211.3

            

          Ex17: c 2855T > A

          p.Leu952X

          Nonsense

           

          T34.1

            

          Ex23.2: c 4108C > T

          p.Gln1370X

          Nonsense

           

          T150.2

            

          Ex34: c.6410delT

          p.Leu2137TyrfsX40

          1 bp deletion

          (FS)

           

          T181.1

            

          Ex34: c.6409_6410delTT

          p.Leu2137ThrfsX19

          2 bp deletion

          (FS)

           

          T198

            

          Ex42: c.7449delT

          p.Ala2484GlnfsX18

          1 bp deletion

          (FS)

           

          C176_3

          NF1 microdeletion

          Genomic

          deletion

          Ex4a: c.479 + 1G > A

          Splice site

          2

          C174

            

          Ex15: c.2326- ?_2409

          Complete exon 15 deletion ?

          Exon

          deletion?

           

          C186

            

          Ex17: c.2990 + 1G > A R

          Splice site

           

          C176_1

            

          Ex28: c.4812C > G

          p.Tyr1604X R

          Nonsense

           

          C176_2

            

          Ex31: c.5927G > A

          p.Trp1976X R

          Nonsense

           

          L-001 D

          NF1 microdeletion

          Genomic

          deletion

          Ex4a: c.396_403del8

          p.Leu134PhefsX21

          8 bp deletion

          (FS)

          3

          L-001 B

            

          Ex19a: c.3189T > A

          p.Cys1063X

          Nonsense

           

          L-001 E

            

          Ex22: c.3774G > A

          p.Trp1258X

          Nonsense

           

          L-001 C

            

          Ex23.2: c.4086_4092del7

          p.Arg1362AlafsX20

          7 bp deletion

          (FS)

           

          L-001 A

            

          Ex28: c.5026_5032del7

          p.Leu1676Alafs10

          17 bp deletion

          (FS)

           

          NF96-1 E

          NF1 microdeletion

          Genomic

          deletion

          Ex13: c.2050C > T

          p.Glu684X

          Nonsense

          3

          NF96-1 B

            

          Ex20: c.3330delT

          p.Phe1110LeufsX2

          1 bp deletion

          (FS)

           

          NF96-1 A

            

          Ex41: c.7394 + 1G > A

          Splice site

           

          NF96-1 C

            

          Ex42: c.7438delG

          p.Glu2480LysfsX22

          1 bp deletion

          (FS)

           

          NF339-

          UHG B

          NF1 microdeletion

           

          Ex3: c.288 + 2T > G

          Splice site

          3

          NF339-

          UHG C

            

          Ex7: c.1007G > A

          p.Trp336X

          Nonsense

           

          NF339-

          UHG D

            

          Ex15: c.2409 + 1G > A

          Splice site

           

          NF339-

          UHG A

            

          Ex27b: c.4697T > A

          p.Leu1566X

          Nonsense

           

          T49.2

          Ex1-42: gene deletion

          E1-42: gene

          deletion

          Ex8: c.1177C > G

          p.His393Asp

          Missense

          Unpublished data,

          Cardiff

          T49.8

            

          Ex8: c.1178A > T

          p.His393Leu

          Missense

           

          T49.1

            

          Ex8: c.1181_1182delTT

          p.Phe394X

          2 bp deletion

          (FS)

           

          T49.5

            

          Ex16: c.2446C > T

          p.Arg816X R

          Nonsense

           

          T49.7

            

          Ex17: c.2953C > T

          p.Gln985X

          Nonsense

           

          T49.3

            

          Ex24: c.4114_4115delGT

          p.Val1372X

          2 bp deletion

          (FS)

           

          T51.3

          Whole gene deletion

          Genomic

          deletion

          Ex7: c.1062 + 1G > A R

          Splice site

          Unpublished data,

          Cardiff

          T51.6

            

          Ex8: c.1179_1180delCT

          p.Phe394LeufsX18

          2 bp deletion

          (FS)

           

          T51.5

            

          Ex11: c.1645_1646delCT

          p.Leu549AlafsX1

          2 bp deletion

          (FS)

           

          T51.4

            

          Ex16: c.2464G > T

          p.Gly822X

          Nonsense

           

          T51.7

            

          Ex41: c.7285C > T

          p.Arg2429X R

          Nonsense

           

          T176.3

          Large deletion

          Genomic

          deletion

          Ex23.2: c.4110 + 1G > C

          Splice site

          Unpublished data,

          Cardiff

          T176.1

            

          Ex28: c.4812C > G

          p.Tyr1604X R

          Nonsense

           

          T176.2

            

          Ex31: c.5928G > A

          p.Trp1976X R

          Nonsense

           

          T217

          Ex1: c.61-1G > C

          Splice site

          Ex12b: c.1900_1907del8

          p.Ile634X

          8 bp deletion

          (FS)

          Unpublished data,

          Cardiff

          T1440

          Ex3: c.264_267delTACA

          p.Thr89Trpfs

          4 bp deletion

          (FS)

          Ex3: c.271G > A p.Glu91Lys

          Missense

          Unpublished data,

          Cardiff

          T183.1

          Ex4a: c.373delGinsATGTGT

          p.Arg125fs

          Indel (FS)

          Ex42: c.7449_7458del10

          p.Leu2483IlefsX15

          10 bp deletion (FS)

          Unpublished data, Cardiff

          T139

          Ex4a: c.434_435delTC

          p.Leu145GlufsX19

          2 bp deletion

          (FS)

          Ex27a: c.4637C > G

          p.Ser1546X

          Nonsense

          Unpublished data,

          Cardiff

          T108.12

          Ex7: c.889-2A > G

          Splice site

          Ex7: c.910C > T p.Arg304X R

          Nonsense

          25

          T199.1

          Ex7: c.983_984delGT

          p.Cys328Xfs

          2 bp deletion

          (FS)

          Ex4b: c.528T > A

          p.Asp176Glu

          Missense

          Unpublished data,

          Cardiff

          T374.5

          Ex10a: c.1318C > T

          p.Arg440X

          Nonsense

          Ex23.1: c.3916C > T

          p.Arg1306X R

          Nonsense

          Unpublished data,

          Cardiff

          T996

          Ex10b: c.1393-32T > C

          Splice site

          Ex6: c.731-11 T > G

          Splice site

          Unpublished data,

          Cardiff

          T227.3

          Ex10b: c.1423insC

          p.Leu475ProfsX9

          1 bp insertion

          (FS)

          Ex15: c.2326-12C > T

          Splice site

          Unpublished data,

          Cardiff

          T161.4

          Ex10b: c.1466A > G

          p.Tyr489Cys

          Missense

          Ex17: c.2990 + 1G > A R

          Splice site

          Unpublished data,

          Cardiff

          T161.3

          Ex10b: c.1466A > G

          p.Tyr489Cys

          Missense

          Ex22: c.3721insC

          p.Arg1241ProfsX7

          1 bp insertion

          (FS)

          Unpublished data,

          Cardiff

          T214

          Ex10b complete exon deletion

          Single exon

          deletion

          Ex22: c.3826C > T

          p.Arg1276X

          Nonsense

          Unpublished data,

          Cardiff

          CLJ8N

          Ex13: c.2041C > T p.Arg681X

          Nonsense

          Ex13: c.2246C > G p.Ser749X

          Nonsense

          6, 7

          T170.1A

          Ex13: c.2041C > T p.Arg681X

          Nonsense

          Ex12a: c.1797G > A

          p.Trp599X

          Nonsense

          Unpublished data, Cardiff

          T1243

          Ex13: c.2197_2214del17

          p.Pro733fs

          17 bp deletion

          (FS)

          Ex36: c.6709C > T

          p.Arg2237X

          Nonsense

          Unpublished data,

          Cardiff

          NF253-

          UHG D

          Ex16: c.2850 + 2A > G

          Splice site

          Ex11: c.1663_1666delTTAG

          p.Leu555IlefsX12

          4 bp deletion

          (FS)

          3

          T193

          Ex17: c.2870delA

          p.Asp957Ilefs

          1 bp deletion

          (FS)

          Ex10a: c.1312G > T

          p.Glu438X

          Nonsense

          Unpublished data,

          Cardiff

          L-004 D

          Ex18: c.3113G > A

          p.Arg1038Lys

          Missense

          Ex27b: c.4729delA

          p.Thr1577LeufsX23

          1 bp deletion

          (FS)

          3

          HT1359.2

          Ex18: c.3113 + 1G > A

          Splice site

          Ex10a: c.1277G > A

          p.Trp426X R

          Nonsense

          Unpublished data,

          Cardiff

          T140.4

          Ex22: c.3732delT

          p.Thr1245LeufsX21

          1 bp deletion

          (FS)

          Ex41: c.7285C > T

          p.Arg2429X R

          Nonsense

          25

          T37.1

          Ex23.2: c.4084C > T

          p.Arg1362X

          Nonsense

          Ex10b: c.1467T > G

          p.Tyr489X

          Nonsense

          Unpublished data,

          Cardiff

          T205.1

          Ex24: c.4196C > A

          p.Ser1399X

          Nonsense

          Ex27a: c.4537C > T

          p.Arg1513X R

          Nonsense

          Unpublished data,

          Cardiff

          T450.3

          Ex27a: c.4537C > T

          p.Arg1513X

          Nonsense

          Ex4b: c.574C > T

          p.Arg192X R

          Nonsense

          Unpublished data,

          Cardiff

          T209.8

          Ex:28: c.4950 C > G

          p.Tyr1650X

          Nonsense

          Ex10a: c.1318 C > T

          p.Arg440X R

          Nonsense

          Unpublished data,

          Cardiff

          NF116-

          UHG A

          Ex28: c.5122insG

          p.Ala1708GlyfsX27

          1 bp insertion

          (FS)

          Ex27a: c.4537C > T

          p.Arg1513X R

          Nonsense

          3

          T1308

          Ex29: c.5546 + 19 T > A

          Splice site

          Ex22: c.3827G > A

          p.Arg1276Gln

          Missense

          Unpublished data,

          Cardiff

          T149.5C

          Ex34:

          c.6512delATGAGAGAinsC

          p.Tyr2171fs

          Indel (FS)

          Ex7: c.988G > A

          p.Ala330Thr

          Missense

          Unpublished data,

          Cardiff

          T89.1

          Ex37: c.6789_6792delTTAC

          p.Asp2264ThrfsX5

          4 bp deletion

          (FS)

          Ex12b: c.1888delG

          p.Val630X R

          1 bp deletion

          (FS)

          25

          T106.1

          Ex37: c.6791insA

          p.Tyr2264XfsX1

          1 bp insertion

          (FS)

          Ex13: c.2033delC

          p.Pro678GlnfsX9 R

          1 bp deletion

          (FS)

          25

          L-004 B

          Ex37: c.6791insA

          p.Tyr2264XfsX1

          1 bp insertion

          (FS)

          Ex23.1: c.3871_3974del103

          Complete exon 23.1 deletion ?

          103 bp

          deletion (FS)

          3

          T1200

          Ex37: c.6791insA

          p.Tyr2264XfsX1

          1 bp insertion

          (FS)

          Ex16: c.2825G > T

          p.Ser942Ile

          Missense

          Unpublished data,

          Cardiff

          CLO1N

          Ex37: c.6792C > A

          p.Tyr2264X

          Nonsense

          mRNA study: Exon 4c skipped

          Splice site?

          6, 7

          T1229

          Ex39: c.7049_7064del16

          p.Cys2350PhefsX19

          16 bp deletion

          (FS)

          Ex13: c.2203T > C

          p.Tyr735His

          Missense

          Unpublished data,

          Cardiff

          T164.1E

          Ex41: c.7285C > T

          p.Arg2429X

          Nonsense

          Ex23.2: c.4084C > T

          p.Arg1362X R

          Nonsense

          Unpublished data,

          Cardiff

          T157.1A

          Ex45: c.7907 + 3A > T

          Splice site

          Ex20: c.3492delC

          p.Ile1165SerfsX2

          1 bp deletion

          (FS)

          Unpublished data,

          Cardiff

          T98.6

          1.5 Mb deletion

          Genomic

          deletion

          Ex34: c.6387A > C

          p.Arg2129Ser

          Missense

          25

          T98

          Complete gene deletion

          Genomic

          deletion

          Ex20: c.3457_3460del4

          p.Leu1153MetfsX3

          4 bp deletion

          (FS)

          Unpublished data,

          Cardiff

          T158.1

          Complete gene deletion

          Genomic

          deletion

          Ex18: c.3058delG

          p.Glu1020LysfsX2 R

          1 bp deletion

          (FS)

          Unpublished data,

          Cardiff

          CCF1N

          Complete gene deletion

          Genomic

          deletion

          mRNA study: exons 12a and

          12b skipped

          Splice site?

          5, 6

          UWA128-

          3

          NI

          NI

          Ex4b: c.543_546delGTAT

          p.Tyr182SerfsX7

          4 bp deletion

          (FS)

          38

          T219.1

          NI

          NI

          Ex9: c.1225_1226delGT

          p.Val409AlafsX18

          2 bp deletion

          (FS)

          Unpublished data,

          Cardiff

          T116

          NI

          NI

          Ex10c: c.1541_1542delAG

          p.Gln514ArgfsX43

          2 bp deletion

          (FS)

          25

          T198.1

          NI

          NI

          Ex10c: c.1555C > T

          p.Gln519X

          Nonsense

          Unpublished data,

          Cardiff

          T128.17

          NI

          NI

          Ex10c: c.1556A > C

          p.Gln519Pro R

          Missense

          25

          T198.2

          NI

          NI

          Ex12a: c.1792A > T

          p.Lys598X

          Nonsense

          Unpublished data,

          Cardiff

          T63.2

          NI

          NI

          Ex13: c.2088delG p.Trp696X

          1 bp deletion

          (FS)

          25

          T146.5

          NI

          NI

          Ex15: c.2326G > A

          p.Ala776Thr R

          Missense/

          splicing?

          Unpublished data,

          Cardiff

          T63.8

          NI

          NI

          Ex15: c.2341_2358del18

          p.His781Ala (in-frame)

          18 bp deletion

          (in-frame)

          25

          T1265.2

          NI

          NI

          Ex17: c.2851-16T > C

          Splice site

          Unpublished data,

          Cardiff

          T233.1

          NI

          NI

          Ex17: c.2879del38 p.Phe960X

          38 bp deletion

          (FS)

           

          T158.2

          NI

          NI

          Ex18: c.3058delG

          p.Glu1020LysfsX2 R

          1 bp deletion

          (FS)

           

          T158.4

          NI

          NI

          Ex18: c.3058delG

          p.Glu1020LysfsX2 R

          1 bp deletion

          (FS)

           

          T192.1

          NI

          NI

          Ex18: c.3113 + 1G > A R

          Splice site

           

          T192.2

          NI

          NI

          Ex18: c.3113 + 1G > A R

          Splice site

           

          NF260-1

          NI

          NI

          Ex22: c.3721C > T

          p.Arg1241X R

          Nonsense

          8

          38

          NI

          NI

          Ex22: c.3727_3728delCT

          p.Leu1243GlyfsX5

          2 bp deletion

          (FS)

          18

          T94

          NI

          NI

          Ex23.2: c.4083insT

          p.Arg1362SerfsX12

          1 bp insertion

          (FS)

          25

          T565

          NI

          NI

          Ex25: c. 4270-2A > G

          Splice site

          Unpublished data,

          Cardiff

          T106.3

          NI

          NI

          Ex26: c.4374_4375delCC

          p.Asp1460X R

          2 bp deletion

          (FS)

          25

          T81.1

          NI

          NI

          Ex27b: c.4662-5C > T

          Splice site

          25

          T1284.5

          NI

          NI

          Ex27b: c.4772 + 5G > A

          Splice site

          Unpublished data,

          Cardiff

          20

          NI

          NI

          Ex33: c.6253_6354 + 5del117

          p.Val2085 (through splice site)

          17 bp deletion

          (FS)

          18

          44

          NI

          NI

          Ex40: c.7127-44_7174del92

          p.Gly2376ValfsX8

          92 bp deletion

          (FS)

          18

          PNFs

          45

          Ex3: c.264_267delTACA

          p.Thr89TrpfsX8

          4 bp deletion

          (FS)

          Ex3: c.271G > A p.Glu91Lys

          Missense

          10

          T399

          Ex3: c.264_267delTACA

          p.Thr89TrpfsX8

          4 bp deletion

          (FS)

          Ex3: c.271G > T p.Glu91X

          Nonsense

          Unpublished data,

          Cardiff

          T7

          Ex4a: c.479 + 1G > A

          Splice site

          Ex16: c.2446C > T

          p.Arg816X R

          Nonsense

          39

          19 UK

          Ex7: c.910C > T p.Arg304X

          Nonsense

          Ex8: c.1177_1178delCA

          p.His393LeufsX16

          2 bp deletion

          (FS)

          Unpublished data,

          Cardiff

          c3 UK

          Ex8: c.1063-2A > G

          Splice site

          Ex7: c.910C > T p.Arg304X R

          Nonsense

           

          14b

          Ex13: c.2076C > G p.Tyr692X

          Nonsense

          Ex4b: c.532_558del27

          p.Glu178 R

          27 bp deletion

          (in-frame)

           

          T318

          Ex13: c.2076C > G p.Tyr692X

          Nonsense

          Ex4b: c.532_558del27

          p.Glu178 R

          27 bp deletion

          (in-frame)

           

          T381.1

          E18: c.3113 + 1G > A

          Splice site

          Ex10a: c.1277G > A

          p.Trp426X R

          Nonsense

           

          T381.2

            

          Ex18: c.3113 + 1G > A R

          Splice site

           

          31

          Ex29: c.5234C > G

          p.Ser1745X

          Nonsense

          Ex9: c.1246C > T p.Arg416X

          Nonsense

           

          c4 UK

          Ex33: c.6289_6290insA

          p.Leu2097fsX2

          1 bp insertion

          (FS)

          Ex27b: c.4706T > G

          p.Leu1569X R

          Nonsense

           

          T155

          Ex33: c.6291insA

          p.Leu2097XfsX9

          1 bp insertion

          (FS)

          Ex27b: c.4706T > G

          p.Leu1569X R

          Nonsense

           

          24

          Complete gene deletion

          Genomic

          deletion

          Ex4b: c.528T > A

          p.Asp176Glu

          Missense

          Unpublished data,

          Cardiff

          T323

          Complete gene deletion

          (1.4 Mb ?)

          Genomic

          deletion

          Ex26: c.4501_4502delCT

          p.Leu1501PhefsX7 R

          2 bp deletion

          (FS)

           

          T369

          Complete gene deletion

          (1.4 Mb ?)

          Genomic

          deletion

          Ex26: c.4501_4502delCT

          p.Leu1501PhefsX7 R

          2 bp deletion

          (FS)

           

          c2 UK

          NI

          NI

          Ex23.2: c.4083insT

          p.Arg1362SerfsX12

          1 bp insertion

          (FS)

          Unpublished data,

          Cardiff

          42

          NI

          NI

          Ex27a: c.4515-2A > G

          Splice site

           

          T329 ?

          NI

          NI

          Ex7: c.952_953delGA

          p.Glu318LysfsX11

          2 bp deletion

          (FS)

           

          Spinal neurofibromas

          1

          Ex7: c.899T > C p.Leu300Pro

          Missense

          Ex24: c.4111-2A > G

          Splice site

          16

          13

          1.4 Mb deletion

          Genomic

          deletion

          Ex21_22 splice site mutation?

          Splice site?

           

          6

          1.4 Mb deletion

          Genomic

          deletion

          Ex27b: c.4690A > G

          p.Lys1564Glu

          Missense

           

          MPNSTs

          53

          Ex4b: c.574C > T p.Arg192X

          Nonsense

          Ex24: c.4203insT p.Glu1402X

          1 bp insertion

          (FS)

          18

          T168

          Ex5: c.663G > A p.Trp221X

          Nonsense

          Ex34: c.6444delA

          p.Val2149SerfsX28

          1 bp deletion

          (FS)

           

          T185

          Ex6: c.773delA

          p.Ser259AlafsX21

          1 bp deletion

          (FS)

          Ex34: c.6410delT

          p.Leu2137TyrfsX41

          1 bp deletion (FS)

           

          37

          Ex16: c.2446C > T p.Arg816X

          Nonsense

          Ex6: c.731-5_741del19

          through a splice site

          19 bp deletion

          (FS)

           

          17

          Ex20: c.3457_3460delCTCA

          p.Leu1153MetfsX4

          2 bp deletion

          (FS)

          Ex31: c.5789delC

          p.Pro1930HisfX6

          1 bp deletion

          (FS)

          17

          20

          1.4 Mb deletion

          Genomic

          deletion

          Ex10c: c.1532delC

          p.Pro511GlnfsX14

          1 bp deletion

          (FS)

          17

          44

          Complete gene deletion

          Genomic

          deletion

          Ex16: c.2446C > T

          p.Arg816X R

          Nonsense

          18

          T184

          Segmental NF NI

          NI

          Ex27a: c.4580_4590del11

          p.Pro1527GlnfsX11 R

          11 bp deletion

          (FS)

          18

          11

          NI

          NI

          Ex27a: c.4580_4590del11

          p.Pro1527GlnfsX11 R

          11 bp deletion

          (FS)

          17

          38

          NI

          NI

          Ex12a: c.1831delCinsTT

          p.Leu611PhefsX3

          Indel (FS)

          18

          GISTs

          NF1-1a

          Ex24: c.4269 + 1G > T

          Splice site

          Ex29: c.5546 + 2T > A

          Splice site

          3

          NF1-1b

            

          Ex29: c.5242C > T

          p.Arg1748X R

          Nonsense

           

          NF1-2a

          Ex37: c.6791insA p.Tyr2264X

          1 bp insertion

          (FS)

          Ex3: c.279T > A

          p.Cys93X

          Nonsense

          3

          NF1-2c

            

          Ex10c: c. del21

          21 bp in-frame

          deletion

           

          NF1-2b

            

          Ex45: c.7846C > T

          p.Arg2616X

          Nonsense

           

          JMML

          D127

          Ex14:

          c.2288_2295dupTGAGGCGC

          /Ex20: c.3366delT

          Compound

          heterozygous

          NF1mutations

          found in

          blood cells

          Ex14:

          c.2288_2295dupTGAGGCGC

          /Ex20: c.3366delT

          Compound

          heterozygous

          NF1mutations

          found in

          blood cells

          31

          CZ051

          Ex12a: c.1748A > G

          p.Lys583Arg/Ex13:

          c.2027delC p.T676TfsX11

           

          Ex12a: c.1748A > G

          p.Lys583Arg/Ex13:

          c.2027delC p.T676TfsX11

            

          D530

          Ex6: c.821T > G p.Leu274Arg

          /Ex34: c.6579 + 1G > C

          With no

          other tissue

          analysed,

          unable to

          differentiate

          germline from

          somatic

          NF1mutations

          Ex6: c.821T > G p.L274R/

          Ex34: c.6579 + 1G > C

          With no

          other tissue

          analysed,

          unable to

          differentiate

          germline from

          somatic

          NF1mutations

          32

          SC049

          Ex3: c.205-2A > G/Ex23.2:

          c.4084C > T p.Arg1362X

           

          Ex3: c.205-2A > G/Ex23.2:

          c.4084C > T p.R1362X

            

          SCO87

          Ex4b: c.482T > G p.Leu161X

          /Ex4b: c.495_498delTGTT

          p.T165TfsX11

           

          Ex4b: c.482T > G p.L161X/

          Ex4b: c.495_498delTGTT

          p.T165TfsX11

            

          D252

          NI

          NI

          Ex29: c.5242C > T

          p.Arg1748X R

          Nonsense

           

          Glomus tumours

          NF1-G8

          Ex4a: c.311T > G p.Leu104X

          Nonsense

          Ex44: c.7727C > A

          p.Ser2576X

          Nonsense

          36

          NF1-G3

          Ex16: c.2546insG

          p.Val850SerfsX15

          1 bp insertion

          (FS)

          Ex29: c.5539_5546dup8

          p.Ser1850ValfsX15

          8 bp

          duplication

          (FS)

           

          NF1-G5

          Ex27a: c.4515-2A > T

          Splice site

          Ex18: c.3113 + 1G > C

          Splice site

           

          NF1-G1

          mRNA study: Exon 29

          partially skipped

          Splice site?

          Ex4a: c.403delC

          p.Arg135GlyfsX30

          1 bp deletion

          (FS)

           

          NF1-

          G10a

          Ex37: c.6789_6792delTTAC

          p.Tyr2264AspfsX5

          4 bp deletion

          (FS)

          Ex2: c.204 + 1G > A

          Splice site

           

          NF1-

          G10b

            

          Ex43: c.7600_7621del22

          p.Lys2534GlyfsX8

          22 bp deletion

          (FS)

           

          ACs

           

          No NF1 somatic mutations

          identified

           

          No NF1 somatic mutations

          identified

            

          Gastric carcinoid tumours

           

          No NF1 somatic mutations

          identified

           

          No NF1 somatic mutations

          identified

            

          PCs

           

          No NF1 somatic mutations

          identified

           

          No NF1 somatic mutations

          identified

            

          FS, frame shift; NI, no information; R, recurrent.

          Any attempt to make direct comparisons between the various tumour types would be unwise at this stage, owing to the paucity of somatic mutation data, especially for the less commonly encountered tumours. Table 3 nevertheless attempts to summarise the available data. The bias inherent in the data is immediately evident, with 211/254 (83 per cent) mutational changes originating from the analysis of cutaneous neurofibroma DNA. Hence, the relative frequencies of the various mutation types in cutaneous neurofibromas are essentially comparable with the germline mutational spectrum, with nonsense mutations, splice site mutations and missense alterations found in cutaneous neurofibromas at frequencies of 28 per cent (59/211), 15 per cent (32/ 211) and 10 per cent (21/211), respectively (Table 3). Table 3 does, however, serve to highlight the high proportion of truncating mutations (191/ 254; ~75 per cent) involved in the somatic inactivation of the NF1 gene in all tumour types, especially cutaneous neurofibromas.

          An additional comparison between the frequency distributions of somatic microlesions and LOH is made in Table 1. There appears to be a marked difference between cutaneous neurofibromas, PNFs and MPNSTs, with 40 per cent, 79 per cent and 85 per cent, respectively, of somatic mutation events represented by LOH. This may be explained in part by the extent of the molecular rearrangements in each tumour type; MPNSTs, for example, would be predicted to exhibit a greater extent of genetic aberration than a benign dermal neurofibroma. The types of analyses performed, however, will have a direct influence on such conclusions, in that either microlesions or LOH may not be screened for in some studies.

          In summary, the more severe MPNSTs show a greater degree of genetic abnormality than other tumour types, with LOH constituting a much more frequent event in these tumours. Further comparison within and between the rarer tumour types would not be valid, however, owing to the relative paucity of mutation data currently available for analysis.

          Mutational mechanisms underlying the known somatic NF1gene lesions

          Somatic inactivation of the NF1 gene may result from different mutational mechanisms and may involve intragenic mutations, LOH and epigenetic modification of the promoter region. Among the 254 somatic NF1 mutations listed in Table S2 (Table 5), 72 nonsense mutations were found, of which 36 involved mutations in just 15 codons in different tumours (codons 192, 304, 426, 440, 816, 1241, 1306, 1362, 1513, 1569, 1604, 1748, 1939, 1976 and 2429), with many previously reported in different tumours or different studies. Ten of these 15 different recurrent nonsense mutations involve C > T or G > A transitions within CpG dinucleotides and are compatible with the endogenous mutational mechanism of methylation-mediated deamination of 5-methylcytosine (5mC). Of these 72 nonsense mutations, 28 have also been reported as germline mutations in NF1 patients (Human Gene Mutation Database [HGMD]),[81] indicating that the same mutational mechanism is operating in both the soma and germline. The importance of this mutational mechanism is evidenced by the finding that 12 of the 15 recurrent somatic nonsense mutations have also been reported independently in the germline (codons 192, 304, 426, 440, 816, 1241, 1306, 1362, 1513, 1569, 1748 and 2429). For the ten of these 15 nonsense mutations that correspond to C > T or G > A transitions within CpG dinucleotides, we may infer that the mutated cytosine must be methylated both in the soma and in the germline, thereby explaining the vulnerability of these sites to methylation-mediated deamination in both cell lineages.

          Among the somatic NF1 mutations listed in Table S2 (Table 5) are 21 different missense mutations. Of these, two (in codons 519 and 776) have been reported more than once in different tumours or different studies, although neither is compatible with methylation-mediated deamination of 5mC. Of the 21 missense mutations, only one (in codon 176) has also been reported in the germline (see HGMD). Since this Asp176Glu mutation has also been reported more than once in NF1-associated tumours, it may well be that this residue is of importance for the function of neurofibromin in both the soma and the germline. Furthermore, this residue is conserved in different species, including Drosophila and Fugu, and has not been identified in 250 unrelated normal individuals.

          Nonsense mutations are not the only type of NF1 mutation to occur recurrently in the soma. Among the somatic NF1 microdeletions listed in Table S2 (Table 5) are five that have been reported more than once in different tumours (c.1888delG, c.2033delC, c.3058delG, c.4374_4375delCC and c.5731delT) with three microdeletions occurring in mononucleotide tracts (G4, C7 and T3, respectively), suggestive of a model of slipped mispairing at the DNA replication fork. Importantly, c.2033delC has also been reported in the germline (see HGMD), indicating that this tetranucleotide stretch is a hotspot for mutation in both the germline and the soma. A microinsertion (c.1733insT, located within a T6 tract) has also been found to occur recurrently in the soma but this has not so far been reported in the germline. The reader interested in a detailed comparative analysis of germline and somatic mutations in human TSGs is referred to Ivanov et al[82].

          NF1gene somatic mutations in non-NF1-associated tumours

          Various studies have identified somatic NF1 gene mutations in non-NF1-associated cancers. Thus, somatic NF1 aberrations have been identified in glioblastoma multiforme (GBM) tumours, lung adenocarcinomas, malignant breast tumours, leukaemia, ovarian serous carcinomas (OSCs) and neuroblastoma [1012, 1416, 83]. Some of the NF1 gene changes are relatively frequent in these tumours and therefore have the potential to represent specific prognostic and diagnostic markers. For example, 23 per cent of sporadic GBM tumours harbour an inactivating NF1 somatic mutation, and this may enable such GBM tumours to differentiate into the mesenchymal molecular subclass [13]. Similarly, in 22 per cent (9/41) of primary OSCs, an NF1 mutation was detected, six of which exhibited biallelic inactivation [12]. Interestingly, all nine of these OSC samples also contained a TP53 mutation, highlighting the likely involvement of this TSG in OSC pathogenesis [12].

          Given the pivotal role that neurofibromin plays in several cell signalling pathways, it is not surprising that its loss will affect distinct molecular subtypes in different cancers. Indeed, the efficacy of any future therapeutic intervention for many tumours will almost certainly hinge upon our ability successfully to identify such molecular subclasses of tumour.

          Prospects for the development of new treatments/therapies

          As the complex picture underlying the molecular nature of NF1 tumorigenesis becomes better defined, the treatment regimens available to patients should greatly improve. Although the future is encouraging, the optimal treatment for NF1 tumours currently rests with their surgical resection, in spite of the high chance of recurrent malignancy. Gottfried and colleagues [84] have suggested that the recruitment of supporting cells around the neurofibroma, coupled with aberrant Remak bundles, could explain how the neurofibroma integrates into the surrounding tissue, and it is this that may lead to the surgical difficulties that often lead to tumour recurrence. Moreover, it has been suggested that surgical interference may even increase the recruitment of surrounding cell types, thereby inadvertently increasing the growth of lesions leading to the formation of new neurofibromas [84]. Surgical biopsy is therefore inherently problematic, and novel therapeutics are urgently required. Clinical and preclinical trials targeting different components of the Ras/MAPK signalling pathway and related growth factor receptors appear to be more promising. It is likely, however, that treatment with multiple drugs may be more effective for NF1 tumours [5].

          Concluding remarks

          Biallelic inactivation of the NF1 gene, resulting in the complete loss of functional neurofibromin, initiates the pathogenic process that eventually results in the formation of nerve sheath tumours. NF1 gene inactivation may occur through relatively subtle lesions that affect just a few DNA bases, or may involve large genomic changes that affect large chromosomal regions, or even the entire chromosome 17. This review demonstrates that NF1-associated tumour types display a considerable degree of variation in terms of the level of LOH detected, with cutaneous neurofibromas, PNFs and MPNSTs. MPNSTs manifest increased levels of deletion-based LOH, whereas cutaneous neurofibromas appear to be associated with a localised deletion of the NF1 gene through mitotic recombination (the situation in PNFs being somewhat intermediate). In MPNSTs, additional mutations at different gene loci are almost certainly involved in the progression of the tumour.

          In terms of the molecular mechanisms of mutagenesis, both methylation-mediated deamination of 5-methylcytosine and slipped mispairing within polynucleotide tracts appear to be responsible for the occurrence of mutation hotspots in both the germline and the soma. For some types of tumour, there is interplay between the soma and the germline, in that the location of the germline mutation can influence the nature, frequency and location of the subsequent somatic mutation [85, 86]. As yet, however, there is no evidence for this phenomenon in the context of NF1 tumorigenesis.

          Although our knowledge of the role of the NF1 gene in tumorigenesis is ever expanding, definitive markers of malignant transformation remain to be discovered. Mouse and other animal models, including zebrafish,[87] have provided new perspectives for research, with various knockout and mutagenesis studies potentiating functional studies. It is already clear that, in order to clarify the role of the NF1 gene in NF1-associated tumours, we must improve our understanding of the significance of the somatic (second-hit) mutations. The brief assessment of the compilation of somatic NF1 mutations in NF1-associated tumour types reported here failed to unearth any specific genotypic correlations. The limited size of the mutation dataset means that reliable conclusions are hard to draw, and that larger and better-defined patient groups will be needed, to allow more reliable comparisons to be made. Additionally, definitive prognostic markers should be identified that permit differentiation between benign neurofibromas that are likely to progress to malignancy and those that are not.

          This review nevertheless emphasises that NF1 is a highly individual condition that exhibits extreme somatic mutational heterogeneity both within and between patients. These are the mutations which are ultimately responsible for the molecular changes that can lead to tumour formation. If we can come to understand how these changes bring about tumorigenesis, we shall be better placed not only with respect to the provision of genetic counselling, but also in terms of exploring new avenues for the development of new drug-based therapies.

          Declarations

          Acknowledgements

          We are grateful to all our NF1 patients and their families for their support. We also thank Laura Thomas and Gill Spurlock for their help with the compilation of mutation data.

          Authors’ Affiliations

          (1)
          Institute of Medical Genetics, School of Medicine, Cardiff University

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