IMPROVED MUTATION ANALYSIS OF THE NF1 GENE

Abstract: The present invention relates to a method for mutation analysis of the neurofibromatosis 1 (NF1) gene of a patient comprising the steps of extracting DNA from peripheral blood lymphocytes of said patient, establishing an EBV transformed B-lymphoblastoid cell line using lymphocytes from said patient, treatment of said EBV transformed B-lymphoblastoid cell line culture with puromycin, immediate extracting RNA of cultures of said cell line, amplifying said RNA using suitable primers, and, obtaining peptide fragments by means of <i>in vitro</i> transcription/translation of said amplified fragments. This new approach allows a much faster, sensitive and reliable analysis of any NF1 mutation that is normally difficult to perform due to unstable and wrong-spliced transcripts. Using this technique, mutations within the NF1 gene can be detected in 83% of all cases, which is significantly higher compared to previously described analysis techniques (71%), and do not result in false positives which are expectable from other setups. This invention also relates to the identification of new hotspots and specific NF1 mutations. This invention comprises diagnostic kits for the detection of described specific mutations and hotspot domains, compounds correcting the structure of specific mutated NF1 proteins and <i>in vitro</i> and <i>in vivo</i> systems that can be used to screen for these therapeutic compounds.

Description: Improved mutation analysis of the NF1 gene The present invention relates to the field of improved methods for genetic diagnosis of Neurofibromatosis type 1 (NF1 ). More particularly, the present invention relates to an optimized mutation analysis of the NF1 gene by a faster and reliable protein truncation analysis leading to identification of at least 83% of mutations in familial as well as sporadic NF1 patients fulfilling the N.I.H. diagnostic criteria. The current technology allows to define the mutation profile of the NF1 gene. Neurofibromatosis type 1 is one of the most common autosomal dominant disorders, affecting about 1 :3500 individuals in all ethnic groups. The main characteristics are cutaneous and subcutaneous neurofibromas, cafe-au-lait (CAL) skin spots, iris Lisch nodules and freckling (Huson et al., 1994). Other features found in only a minority of patients include scoliosis, macrocephaly, pseudarthrosis, short stature, malignancies and learning disabilities. One of the most feared complications of NF1 is malignancy. The disorder shares some features common to heritable cancer syndromes due to mutations in a tumor suppressor gene. These are: i) a tissue-restricted occurrence of primary cancers in neural crest and myeloid lineage cells; ii) a decreased age of onset of malignancies compared to the general population; iii) the occurrence of multiple primary tumors in some patients. The NF1 gene has been mapped to 17q1 1.2 and was positional cloned (Cawthon et al., 1990; Viskochil et al., 1990; Wallace et al., 1990). The NF1 gene is approximately 350 kb in size, contains 60 exons and codes for an ubiquitously expressed 11 - to 13-kb transcript with an open reading frame ceding for 2818 amino acids (Marchuk et al. 1991). The central portion of the coding sequence exhibits homoiogy to the GTPase activating proteins (GAPs) and the protein can down regulate the Ras pathway through this GAP-related domain (reviewed by Kim and Tamanoi, 1998). The mutation rate in the NF1 gene is one of the highest known for human genes (reviewed by Huson and Hughes, 1994) with approximately 50% of all NF1 patients presenting as sporadic cases expected to carry de novo germline mutations. For these patients, only identification of the pathogenic germline mutation allows for presymptomatic/prenatal testing in offspring. Mutation is located in one of both alleles resulting in the synthesis of a mutated form in addition to the WT protein. A decrease in the WT NF1 protein content results in a diseased state of the cell. Since NF1 is a dominant disorder, individuals that are heterozygous for a NF1 mutation may still express NF1 at 50% or reduced levels. In tumors, a second hit (loss of Heterozygosity or point mutation) in the remaining allele further reduces the neurofibromin content in the cells. Despite the high frequency of this disorder in all populations, relatively few mutations have been identified at the molecular level. Mutational analysis in NF1 patients has proven to be laborious and is hampered by the large size of the gene, the large number of exons, the presence of several pseudogenes (Cumming et al., 1993; Hulsebos et al., 1996) and the wide variety of mutations (i.e. nonsense, frameshift and missense mutations, small insertions or deletions, large deletions encompassing the total gene, translocations, etc). Many techniques have been applied for the study of NF1 mutations with detection rates varying from 10 to 65% (Abernathy et al, 1997, Fahsold et al., 2000, Ars et al., 2000). A limited number of mutational „hotspots" have been reported: R1947X (C5839T) in exon 31 , the 4-bp region between nucleotides 6789 and 6792 in exon 37, both implicated in about 2% of the NF1 patients (reviewed by Upadhyaya and Cooper, 1998). Also exon 4b is considered to contain mutational hotspots by others (Toliat et al., 2000). Recently we found that another mutational hotspot resides in exon 10b and harbors a missense mutation associated with aberrant splicing (Messiaen et al., 1999). So far, no studies attempted to delineate the mutational spectrum in the NF1 gene by extensive analysis using a combined cascade of complementary techniques. A high mutation detection rate is especially important if genetic testing is requested for the offspring of sporadic patients in which only identification of the pathogenic mutation can allow for prenatal/predictive testing. Moreover, once a technology will be available that is able to find the mutation in the NF1 gene with high sensitivity, this will enable to help with the diagnosis of patients that do not fulfill to the N.I.H. diagnostic criteria yet. The protein truncation test (PTT) is a form of mutation detection first described in 1993 by Roest et al. The PTT allows to analyze the total coding region of a gene by in vitro transcription and translation of RT-PCR fragments and will specifically detect mutations that result in a truncated protein due to the occurrence of a premature stop codon or due to f.i. an in frame skipping of exons or segments of exons. The protein truncation test (PTT) based on in vitro protein synthesis, was first applied by Heim et al. (1995) to the whole NF1 coding sequence in 21 unrelated patients and 14 mutations were identified at the cDNA level (66% detection rate). However, the authors were able to identify the mutation at the genomic level in only 11 of the patients, what brings us to a detection level at the genomic level of only 11/21 (52%). No reasons for this failure were discussed in the paper. Park et al. (1998) studied 14 unrelated patients and 10 mutations were disclosed (71 %). Here too, one of the alterations seen in the cDNA could not be resolved at the genomic DNA level, what brings us to a- detection level of 9/14 (64%). Whereas the experiments of Heim et al. (1995) started from either blood cells or lymphoblastoid cell lines, Park et al. (1998) exclusively used peripheral blood. In order to be able to implement high-throughput screening using PTT, major improvements of the procedures are required (Den Dunnen and Van Ommen, 1999). Especially, laboratory testing for NF1 mutations is difficult. A protein truncation test is commercially available, but its sensitivity, specificity and predictive value have not been established (Rasmussen and Friedman, 2000). The aim of the present invention is thus to provide an optimized mutation analysis method for the NF1 gene which is faster and more reliable than presently known protein truncation analysis systems. This optimized system can be applied to develop a kit that can be used for fast genetic NF1 diagnosis. In addition, the invention also relates to the use of this optimized method to characterize new hotspot domains and specific mutations allowing the definition of the mutation profile of the NF1 gene. These aims have been met by the following embodiments. The present invention relates more particularly to a method for mutation analysis of the NF1 gene of a patient comprising the steps of (see figure 1 and 2): a) isolating peripheral blood lymphocytes of said patient, b) establishing an EBV transformed B-lymphoblastoid cell line with said peripheral blood lymphocytes of said patient, or short-term culturing of the blood lymphocytes by phytohaemaglutinin (PHA) stimulation c) treatment of the EBV transformed B-lymphoblastoid cell line or the short term cultures with a protein synthesis inhibitor, d) immediate extraction of RNA of cultures of said EBV transformed B-lymphoblastoid cell line, e) amplifying said RNA using suitable primers generating cDNA fragments covering the whole NF1 gene coding region (RT-PCR), and f) obtaining peptide fragments by means of in vitro transcription/translation of said amplified fragments of step e). According to a further embodiment, the present invention also relates to a method as described above wherein f) is followed by at least on of the following steps (see figure 1): g) separation of said peptide fragments; h) cycle sequencing of the cDNA fragments that resulted in a truncated peptide in the PTT assay. Cycle sequencing of cDNA prepared from EBV transformed B-lymphoblastoid cell lines that were treated with and without a protein synthesis inhibitor are compared. This allows, as explained below, to detect more easily the aberrant fragments and allows to give information on the stability of the mutant transcript in the affected cells, i) evaluation of the cycle sequencing chromatograms with respect to putative splice sites using "Splice Site Prediction using Neural Networks" (http://www- hgc.lbl.gov/projects/splice.html, Messiaen et al. (2000)) if internal cDNA deletions are seen to allow to determine the plausible site of the genomic alteration. j) Identification of the genomic mutation by cycle sequencing of the exons of interest. Genetic analysis creates the possibility of finding gene mutations at an early stage in the disease development so symptoms can be reduced or even stopped in its early phase, if treatment becomes available. The present invention provides a method for the genetic analysis of the neurofibromatosis type 1 (NF1) gene. The large size of the gene and relatively insensitive techniques have made detection of causative mutation difficult especially for this NF1 gene. As NF1 is one of the most common autosomal dominant disorders with a high mutation rate, it is of prime interest to medicine that an efficient and reliable method is available to diagnose this disease. Screening for NF1 mutations, particularly in neonates and young children who are often a/oligosymptomatic, is thus one of the major applications of the present invention. Since the NF1 gene is ubiquitously expressed, test samples of the subject can be obtained from a variety of tissues or blood. An NF1 test can also be included in panels of prenatal tests since NF1 DNA, RNA or protein can also be assessed in amniotic fluid and chorion villi. Further description of the invention will illustrate the technique starting from blood cells, nevertheless, explained principles can also be applied when starting from other cells. Analysis can be performed on blood sent from any location such as private doctor practices or hospitals. Peripheral blood lymphocytes can be cultured for a short time, using phytohaemaglutinin stimulation. Alternatively, EBV transformed cell lines also allow repetitive analyses in a controlled environment. Preferably, the present invention provides a method as defined above wherein said protein synthesis inhibitor might be chosen from a group comprising puromycin, actinomycinD, cycloheximide or a possible analogue thereof. All of these prevent nonsense mediated decay of the mutant transcript. Mutations can be missed starting from a culture without puromycin treatment, due to the instability of the mutant transcript and the "premature termination codon induced" mRNA decay. Puromycin is preferred in the proposed method. Puromycin is a tRNA analogue causing chain termination and blocks nonsense-mediated decay in cell lines as was demonstrated to be the case for the gene (Andreutti-Zaugg et al., 1997). The effect of puromycin on the stability of the NF1 mutant transcript was not experimentally investigated before. The present inventors proved for the first time that the addition of puromycin to cell cultures could also improve the analysis of mutant transcripts of the NF1 gene thereby increasing the efficiency of NF1 genetic diagnosis. Until now, for the NF1 mutation analysis no indication was available when RNA isolation should be started. The inventors point out that production of newly made NF1 messenger RNA in specific conditions is of prime importance. Indeed, according to present invention it is essential that RNA is extracted immediately from the cultures of said EBV transformed B- lymphoblastoid cell lines or short-term cultures of PHA stimulated blood lymphocytes once they are retrieved from the incubator. As shown by the inventors, incubation of cell cultures at room temperature will influence the splicing of the NF1 messenger creating alternatively spliced products which may influence the interpretation of the NF1 analysis. The inventors showed that a crucial parameter in the successful application of the PTT to find the disease causing mutation is the quality of the RNA that is used to start the procedure. It was noticed that starting from RNA extracted from peripheral blood cells, kept for a while at room temperature, very often "spurious" bands were present after RT-PCR as well as on the PTT SDS-PAGE. It is obvious for a skilled person in the art that this would also account for possible artifacts if one incubates said EBV transformed B-lymphoblastoid cell lines at room temperature. Therefore it is also suggested by the inventors that RNA should be immediately extracted from the cell lines once they are removed from the incubator. The incubator creates an optimized environment for cell growth with stable CO 2 pressure and temperature (37°C). Consequently, if cells are immediately analyzed after removal, epigenetic factors will not have the time to influence the activation of cryptic splice sites leading to the occurrence of these deleted transcripts. The inventors showed that RNA extracted from "aged" blood samples leads to increased skipping of exons and hence mimics - in the absence of a genomic alternation - the presence of a mutation. This results in a wrong interpretation of the genomic background of the patient, which can not be allowed in medical diagnosis. Although it was already observed that infidelity of the splicing process could occur in specific gene transcripts of TSG101 and FH/T (Gayther et al., 1997) when RNA was isolated from "aged" blood, it seemed that this infidelity is gene specific and not a generalized phenomenon. Indeed this does not occur in other transcripts of other tumor suppressor genes such as BRCA1, BRCA2, BRUSH1, hMSH2, IGF2 receptor, PGDβ and RB (Gayther et al, 1997). Therefore it was not evident to find this phenomenon for the NF1 gene. According to a preferred embodiment, the present invention also relates to a method as defined above wherein RNA is immediately extracted from immediately isolated peripheral blood lymphocytes of said patient for further analysis of the mutations present in the DNA of said patient (figure 1 ). Also in this case epigenetic factors would not have the time to influence the activation of cryptic sites. The term 'immediately' implies not longer than 2 hours after blood collection and preferably as soon as possible. Often RNA can not be extracted immediately after prelevation of the blood, which is often the case in clinical practice when samples are sent from abroad, lymphocytes are revived by short term (48- 168 hours) culture using phytohaemagglutinin (PHA) stimulation at 37°C. Short-term culture moreover allows to obtain a much larger cell population for the extraction of the RNA. Preferably, the methods according to the present invention involve a reverse transcriptase (RT) step followed by an amplification step, which is a polymerase chain reaction (PCR) (figure 1 ). This step allows the amplification of gene fragments covering the whole coding domain of the NF1 gene, which makes the analysis of a gene consisting of multiple exons more feasible. Preferably, the present invention provides a method as defined above wherein said RNA extracted in step d) is total RNA. Isolation of total RNA is less expensive compared to the isolation of mRNA and will still result in the amplification of specific gene products when amplification conditions and primers are chosen appropriate as described by the method. Amplification products can be used to verify the corresponding DNA sequence or used to produce corresponding proteins (figures 1 and 2). The said PCR products can be used in an in vitro translation system, so NF1 -peptide fragments can be made. In addition, the present invention preferably provides methods as defined above wherein step f) is followed by a separation of said peptide fragments. This separation can be done by any technique known in the art such as SDS PAGE (one or two dimensional). In previous experiments, as described by Heim et al. (1995) and Park et al. (1998), isotopic 35 S-Methionine is incorporated in the peptides so separated peptides can be easily visualized using radiography. Contrarily to the prior art, the inventors changed the label to 3 H-Leucine. Changing the label increases the sensitivity of the mutation analysis significantly. This increase can be explained by the fact that the NF1 protein is rich in Leucine residues compared to the number of Methionines present in this protein and allows therefore a higher incorporation of specific label thereby increasing the detection efficiency. Alternatively to SDS-PAGE, mass spectrometry or Malditoff can be used to analyse the peptide population obtained. The present invention also provides a method that is as sensitive that it is possible to identify the NF1 mutation in sporadic patients presenting as somatic mosaic. More precisely, the sensitivity of the test allows to detect the NF1 mutation if present in at least 10% of the cells that are under investigation (figure 17). Up to now, somatic mosaicism could only be detected via FISH analysis and only for patients carrying large deletions. Other studies failed to identify the mutation with equal efficiency in sporadic patients (Ars et al. (1995): 51 % detection rate in sporadic patients; Fahsold et al. (2000): 53% detection rate without specification between sporadic versus familial; our data: 83% detection in sporadic cases by PTT alone). So, by increasing the detection efficiency we are able to detect this aberration even when a point mutation is present. This is of uttermost importance as for sporadic patients only the identification of the pathogenic mutation allows for presymptomatic/prenatal diagnosis in future generations. The present invention also provides a method as defined above wherein in case a truncated peptide is observed by means of protein separation, the amplified cDNA fragment obtained in step e) is analyzed by cycle sequencing allowing the characterization of the effect of the mutation at the mRNA level. It is not excluded that this amplified cDNA fragment can be analyzed without any hint given by such an in vitro PTT system. Moreover, comparison of the cDNA analysis from cells treated with and without puromycin allows to give information on the stability of the mutant mRNA in the affected cells. As stable mRNA may result in the production of a truncated neurofibromin, this information may point to novel putative functional domains in neurofibromin. Preferably said analysis may be performed by means of cycle sequencing of a suitable fragment by means of suitable primers. From the length of the peptide fragment, it is mostly known which primers will be suitable. Primers for amplification of each of these fragments are known or can be readily developed (see also table 2 and 3 or any given table). Primers that are used for fragment amplification can also be applied to sequence respective amplified fragment. In this latter case, primers are labeled as known by a person skilled in the art. Preferably such methods according to the present invention will employ as primers of step e) primers as represented in Figure 2 or in any of the tables or in the Examples or figure legends. Prefered methods according to the present invention employ non-isotopically labeled primers. Said label is chosen from a group comprising fluorescein, biotin, Cy5, FAM6, TAMRA, ROX. The generated cDNA fragments may be further analyzed by means of ALF-sequencer (Pharmacia), ABI-370 (Perkin Elmer) or any other sensitive semi- or automatic sequencing system. Currently, no lab in the world performs routine NF1 mutation diagnostic assays for large numbers of patients. One of the reasons for this is the inability to find high sensitivity and high specificity methodology for routine diagnostic testing of NF1 gene mutations. It is therefore highly desirable to have an improved diagnostic methodfor the presence or absence of NF1 mutation. By the present optimized Protein Truncation Test >83% of the germline mutations in NF1 patients fulfilling the N.I.H. diagnostic criteria can be identified. The spectrum of mutations that can be detected by PTT is limited to nonsense mutations, frameshift mutations, splice mutations, deletions not encompassing the region flanked by the used primers, all leading to a premature termination codon. Additional methods are needed to detect missense mutations, small (less than 80 nucleotides) in frame deletions and/or insertions, large deletions and cytogenetic abnormalities such as translocations. The method of the invention relates to a hierarchical system for effective molecular diagnosis of NF1 disease-associated mutations. The spectrum of mutations reveals the high incidence of unusual splice mutations. Many of these mutations will be missed using genomic scanning techniques as many splicing mutations are caused by intronic mutations outside the canonical splice donor/acceptor sequences. Moreover, some mutations called "silent" at the genomic level, create a novel splice donor or acceptor site and are proven to be pathogenic by the RNA-based mutation detection methods. In the hierarchical system, the second level of analysis for patients that score negative with the optimized PTT system includes methods to detect missense mutations and/or small in frame insertions and/or deletions. These analyses can be performed by means of heteroduplex analysis (HA) and/or single stranded conformation polymorphism (SSCP) analysis and/or denaturing gradient gelelectrophoresis (DGGE) and/or conformation sensitive gelelectrophoresis (CSGE) and/ or immediate cycle sequencing (with or without subcloning). Said HA or single stranded confirmation analysis is performed to detect aberrant migrating PCR fragments which are then further analyzed by cycle sequencing. A preferred combined approach for the characterization of an NF1 germline mutation according to the present invention involves a protein truncation test from EBV transformed cell lines as detailed above and in the examples and figure legends followed by direct cDNA and gDNA sequencing, heteroduplex analysis followed by direct gDNA sequencing, Southern blot analysis using probes GE2-FF13-FF1 -FB5D-AE25-P5-B3A as described in Marchuk et al, 1991 (These clones were a kind gift of Francis Collins), FISH (fluorescence in situ hybridization) analysis using intragenic cosmid or PAC clones and cytogenetic analysis. Preferably, the present invention provides a method for mutation analysis of the NF1 gene of a patient as defined above wherein said primers are located flanking exon 4b, 7, 10a-10c, 13, 23.2, 27a, 29, 37 or 39 of the NF1 gene respectively, as represented on Figure 7 and 8. We have found that the mutation spectrum of NF1 and the identification of mutational hotspots as defined before was biased by the limitations of the technology that was used as well as by the fact that the total coding region of the NF1 gene was not screened in older studies. We describe as examples thereof several hotspot domains for mutations in the NF1 gene which were not identified before: i.e. exon 7, exon 10a-10b-10c, exon 13 (2033insC), exon 23.2 (R1362X), exon 27a (R1513X), exon 29 (R1849X), exon 39 (2266delNF). Assay kits for screening and diagnosis of mutations within these specific novel hotspots in accordance with the principles of the present invention are also provided. Focusing on these domains will improve the speed in which mutations can be diagnosed. The present invention also provides a method for detecting besides previously published mutations also the following novel specific frame shifts, nonsense or splice mutations (see table 1 ): K33K (99del105), C93Y (278G>A), C187Y (560G>A), R192X (574C>T), 603-604insT (idem), Q209X (625C>T), 819-821 delCCT (idem), 889- 454del474nt(888del174), 987-988insA (idem), 1261 -19G>A (1260insTTTG I I I I I CTCTAGTC), W425X (1275G>A), R461X (1381C>T), Y489C (1465del62), 1466insC (idem), 1527+5G>A (1392del135), E524X (1570G>T), 1605insA (idem), S536X (1607C>A), 1642-3C>G (1641 del80), 2305insT (idem), 2585insA (idem), 2836insT (idem), 2850+2del6 (2617del233), 2851 -6del4 (2850del140), Q959X (2875C>T), Q963X (2887C>T), 2990+3A>C (2850del140), Y1044X (3132C>A), 3193delC (idem), V1093M (3277G>A/ 3274 del40), 3108-3C>G (3314del182), E1123X (3367G>T), 3457delCTCA (idem), Q1174X (3520C>T), 3704delA (idem), 3708+1 G>C (3496del212), 4026delG (idem), 4299delC (idem), Q1494X (4480C>T), 4515-2A>T (4515-14ins14/4515- 17ins17), 4773-2A>T (4772del433/ 4772del293), 5033delG (idem), 5117delT (idem), R1849 (502del341/5205del544), S1755X (5264C>G), S1765X (5215del90/5294C>A), 5567delT (idem), 5798delC (idem), Q1966X (5896C>T), 6577delGAGgta (6364del215), R2237X (6709C>T), 6858G>C (6756del102), 7127-12T>A (7126del132/7127-1 OinslO), 7268delCA (idem), K2401X (7201 A>T), R2429X (7285C>T), 7884-7885delGT (idem), 8016delA (idem). Nomenclature for the mutations found for the NF1 protein is as recommended by Antonarakis (1998). Effect of the mutation at the mRNA level is between parentheses. All mutations were verified to be present at the genomic DNA level. The novel balanced translocation t(14;17)(q32;q1 1.2) interrupted the NF1 gene: PAC928b9 was found on the der(17) and PAC1002g3 was found on der(14). The present invention points to a number of regions in the NF1 gene that can be skipped "in frame" by specific mutations in the genomic DNA and result in the production of a stable mRNA. Also a number of missense mutations were identified. As both types of mutations may lead to the production of a truncated/altered neurofibromin, these mutations may point to novel functional domains of neurofibromin. So far only the central GAP-related domain has been well characterized (GRD in figures 7 and 8). A region involved in cAMP-mediated signaling exists in Drosophila and probably in humans as well but its location in the NF1 gene is not yet defined. Neither has the region that mediates the association of neurofibromin to the microtubules been defined. Careful mutation analysis like the study presented here may point to the regions involved in these and other functions of the NF1 gene. Specifically the following regions may be important and are first described now: In frame skipping of the last 105 nt of exon 2 (mediated by K33K), C83Y (exon 3), C93Y (exon 4b), 274delL (exon 6), In frame skipping of exon 10b (mediated f.i. by 1527+5G>A), In frame skipping of both exon 11 and 12a (mediated f.i. by IVS12a+1 G>T), L847P (exon 16), In frame skipping of the first 90 nt of exon 29 (mediated by f.i. S1765X), In frame skipping of exon 37 (mediated f.i. by 6792C>A, 6792C>G, K2286N), in frame skipping of exon 40 (mediated f.i. by IVS39-12T>A).The established EBV-cell lines harboring these specific mutations are valuable tools to perform further protein research to define additional functional regions onto the NF1 protein. E.g. specific transcripts can be isolated from these EBV cell lines and used to setup two-hybrid screenings. The present invention also relates to a diagnostic kit for mutation analysis of the NF1 gene of a patient comprising primers specifically amplifying the gene domains containing the novel specific mutations or the novel mutation hotspot regions as mentioned above. The present invention also relates to a diagnostic kit for mutation analysis of the NF1 gene of a patient comprising probes specifically detecting the gene domains containing novel mutation hotspots regions or specific mutations as mentioned above. The term "nucleic acid" refers to a single stranded or double stranded nucleic acid sequence present in a biological sample, said nucleic acid may consist of deoxyribonucleotides or ribonucleotides or may be amplified cDNA or amplified genomic DNA. The term "probe" refers to single stranded oligonucleotides and may consist of deoxyribonucleotides or ribonucleotides, nucleotide analogues or modified nucleotides, or may be amplified cDNA or amplified genomic DNA. The probes used in the process of the invention can be produced by any method known in the art, such as cloning of recombinant plasmids containing inserts including the corresponding nucleotide sequences, if need be, by cleaving the latter out from the cloned plasmids upon using the appropriate nucleases and recovering them (e.g., by fractionation according to molecular weight). The probes can also be synthesized chemically, for instance, by the conventional phopho-triester method. The probes of the invention can optionally be labelled using any conventional label. This may include the use of labelled nucleotides incorporated during the polymerase step of the amplification or by any other method known to the person skilled in the art. The term "primer" refers to a single stranded nucleotide sequence capable of acting as a point of initiation for synthesis of a primer extension product which is complementary to the nucleic acid strand to be copied. The length and the sequence of the primer must be such that they allow to prime the synthesis of the extension products. Preferably the primer is about 5-50 nucleotides. Specific length and sequence will depend on the complexity or the required DNA or RNA targets, as well as on the conditions of primer use such as temperature and ionic strength. The present invention also advantageously provides nucleic acid sequences of at least approximately 15 contiguous nucleotides of the NF1 gene or mutant versions thereof, preferably from 15 to 50 nucleotides. These sequences may, advantageously be used as probes to specifically hybridize to sequences of the invention as defined above or primers to initiate specific amplification or replication of sequences of the invention as defined above, or the like. They may also be used in diagnostic kits or the like for detecting the presence of a nucleic acid according to the invention. These tests generally comprise contacting the probe with the sample under hybridizing conditions and detecting the presence of any duplex or triplex formation between the probe and any nucleic acid in the sample. Identification of specific mutations in the NF1 gene also has therapeutic implications. A method for identifying a compound correcting the defective structure of the mutated NF1 protein is one of the examples. A mutated NF1 protein can result from a specific mutation of the NF1 coding region as described above. Modulation of NF1 function can be accomplished by the use of therapeutic agents or drugs. These can be designed to interact with different aspects of the NF1 protein structure or function; a drug can correct its defective structure or increase its affinity for a substrate or cofactor. Efficacy of a drug or agent can be identified by a screening program in which nodulation is monitored in vitro using cell systems in which a defective NF1 protein is expressed. Alternatively, drugs can be designed to modulate NF1 activity from knowledge of the structure correlation of the NF1 protein and from knowledge of the specific defect in the various NF1 mutant proteins (Capsey et al., 1988). This invention also relates to model systems comprising an NF1 gene mutation, as defined above, which can be used to screen for therapeutic agents. In both in vitro and in vivo models mutant NF1 proteins are expressed and used to screen for correction of the mutant NF1 activity. In the in vitro tests both purified NF1 protein or cell lines expressing the mutant NF1 protein can be used; in the in vivo models, transgenic animals expressing the mutant NF1 protein can be employed. Transgenic mice carrying a mutation in one of the NF1 genes show clear pathological symptoms. Vogel et al. (1999) described that cis- Nf1 +I' :p53 1'1' mice exhibit a significant incidence of soft tissue sarcomas. The presence of the heterozygous NF1 mutation accelerates tumorigenesis and alters the tumor spectrum in the content of the p53 ~ ' ~ background. In addition, chimeric mice composed in part of NfV 1' cells carrying homozygous NF1 alterations do develop neurofibromas (Cichowski et al., 1999). Consequently, both mouse models provide the means to test therapeutic strategies. The following examples and Figure legends merely serve to illustrate the invention and are by no way to be understood as limiting the present invention. Figures and table legends: Tables Table 1 : Mutations detected by analysis of the whole coding region of the NF1 gene by PTT, HA, cytogenetic and FISH analysis. Table 2: NF1 cDNA sequencing primers Table 3: Primers for amplification of all exons of the NF1 gene for HA. Table 4: HA-PCR primers and conditions. Table 5: Mutations detected by analysis of the whole coding region of the NF1 gene by PTT and HA by analysing 67 unrelated NF1 patients. Table 6: Comparison of the sensitivity to detect NF1 mutations by direct cDNA cycle sequencing starting from Pmin and Pplus EBV cultures as measured by the ratio between mutant/wild-type peak height on sequencing chromatograms. Table 7: Consensus values (CV) and Splice Site Scores (SSS) of splice sites (ss) involved in splicing mutations in the NF1 gene Figures Figure 1: Overview of the optimized NF1 genetic mutation analysis. Peripheral blood lymphocytes are taken from the patients and DNA is extracted from the lymphocytes. Concomitant an EBV-transformed lymphoblastoid cell line is initiated or a short term culture of PHA stimulated lymphocytes is started. Once the culture is well established, the culture is split and 1 culture (P+ culture; wherein puromycin is added) is incubated with 200 μg/ml puromycin for 16 hours, the second culture (P- culture; wherein no puromycin is added) is further incubated in the RPMI 1640 culture medium'. In order to get a good signal to noise ratio for the cycle sequencing of cDNA, total RNA from the P+ culture is extracted. cDNA is prepared using random hexamers and the coding region is amplified in 5 overlapping fragments using a modified forward primer containing a T7 promotor sequence, the KOZAK sequence and a methionine start codon in frame with the sequence to be analyzed. Afterwards in vitro transcription/translation peptide fragments are separated by SDS-PAGE. If a truncated peptide is present, the RT-PCR fragment leading to this truncated peptide is analyzed by cycle sequencing. Once the cDNA sequence pattern has been interpreted we continue the analysis at the genomic level by cycle sequencing of the DNA extracted directly from the lymphocytes covering the whole coding domain of the NF1 gene. Hence, it can be excluded that the mutation that is identified was introduced due to the EBV transformation itself. Figure 2: Schematic overview of the total coding region of the NF1 gene (60 exons drawn to scale) and position of the 5 overlapping RT-PCR fragments used for in vitro transcription/translation. The position of the 5 overlapping RT-PCR fragments is denoted. Exon numbers are indicated. Overlap between the neighboring fragments is indicated in amino acids (AA). Small vertical bars indicate the position of the sequencing primers that are used to perform direct cycle sequencing of the RT-PCR fragments. Underneath are pictures from the SDS-PAGE showing the normal peptides obtained after in vitro transcription/translation of the 5 fragments. On every gel a protein marker ( 14 C Methylated proteins CFA626, Amersham) is loaded. 69 kDa denotes the size of the largest peptide band from the protein marker. Smaller fragments of the protein marker are 46 kDa, 30 kDa and 14.3 kDa in size. GRD denotes the Gap-related domain, this is a domain with catalytic GTPase-stimulating activity (Kim and Tamanoi,1998). The GRD spans the amino acids 1 172-1538 of the protein. Figure 3: Influence of puromycin on the analysis of NF1 exon 20. RNA was extracted from an EBV transformed lymphoblastoid cell culture with and without incubation with 200 μg/ml puromycin for 15 hours. A. PTT from Pplυs and Pmin EBV cultures from NF1 patient NF-039. Lane 1: protein markers (sizes in kDa). Lanes 2, 3, Pplus and Pmin cultures, with and without puromycin treatment, the presence of a truncated peptide was discerned by PTT analysis. The intensity of the truncated peptide compared to the wild type peptide was greatly enhanced after puromycin treatment. B. Starting from the cultures that were not treated with puromycin, direct cycle sequencing of the cDNA exon 20 without subcloning was unable to identify unambiguously he presence of the nonsense mutation in exon 20, due to the nonsense-mediated mRNA decay. C. Starting from the puromycin treated cultures, direct cycle sequencing of the cDNA without subcloning gave unambiguous results leading to the clearcut identification of a mutation in Exon 20: 3367GtoT, E1123X. Figure 4: Influence of puromycin on the analysis of NF1 exon 10c. A. PTT from Pmin and Pplus EBV cultures from a normal control and NF1 patient NF- 033. Lane 1: protein markers (sizes in kDa). Lanes 2, 4, Pplus cultures. Lanes 3, 5, Pmin cultures. Lanes 2 and 3: normal control showing only wild-type (WT) NF1. Lanes 4 and 5: patient NF-033 showing truncated peptide due to the presence of NF1 stop codon mutation at amino acid 524. B. cDNA sequence chromatograms of patient NF-033 of Pmin (upper panel) and Pplus (lower panel) EBV cultures. Arrow, heterozygous peak: GAA>TAA at amino acid 524. Another example illustrating the PTT results obtained starting from RNA extracted from EBV transformed lymphoblastoid cell cultures immediately after removal from the incubator and treated with and without puromycin. Both with and without puromycin treatment, the presence of a truncated peptide was discerned. The intensity of the truncated peptide compared to the wild type peptide was greatly enhanced after puromycin treatment. Sequencing results are only shown for the fragments leading to the peptides shown in lanes 4 and 5. Here too, starting from the puromycin treated cultures, direct cycle sequencing of the cDNA without subcloning gave unambiguous results leading to the clearcut identification of a mutation in Exon 10c: 1570GtoT, E524X. The mutation would again have been missed if only direct cycle sequencing would be performed starting from the culture without puromycin treatment, due to the instability of the mutant transcript and the "premature termination codon induced" mRNA decay. Due to this decay the mutant messenger is only present in small quantities and relevant seuence information is lost as it does not peak above the background/noise peaks. Figure 5: Automated Laser Fluorescence (ALF) based fragment analysis of NF1 exon 7-skipping that is present in "aged" blood and in EBV cell lines carrying a specific nonsense mutation in exon 7, but that is not present in fresh blood nor in EBV cell lines not carrying a NF1 mutation in exon 7. Illustration of the importance to extract the RNA immediately after prelevation for unprocessed blood or after removal from the incubator for cell cultures. Total RNA was extracted from Pplus and Pmin EBV cultures and from peripheral blood lymphocytes either processed immediately ("fresh"), or kept at room temperature and processed 48 hours after prelevation ("aged") and semi-quantitative RT-PCR analysis of E7 skipping using a 5'-fluorescein labelled forward primer in E6 (5 1 - TTGACTTGGTGGTGGTTT-3') and a reverse primer in E8 (5'- TTGAGAATGGCTTACTTGGA-3'). Lane 1, fresh peripheral blood lymphocytes. Lane 2, "aged" peripheral blood lymphocytes. Lane 3 and 5, Pmin EBV culture from patient NF-027 (A) and NF-064 (B) both carrying the mutation R304X. Lane 4 and 6, Pplus EBV culture from patient NF-027 (A) and NF-064 (B both carrying the mutation R304X. Lane 7 and 8, Pmin (lane 7) and Pplus {lane 8) EBV culture from patient NF-019 carrying the mutation R2264X. Fragment analysis of RT-PCR products using a 5' fluorescein labeled forward primer located in exon 6 and a reverse primer located in exon 8. 20 cycles of amplification were performed, i.e. the exponential phase of the PCR well before a plateau is reached. Hence the experiments are semi-quantitative. RT-PCR fragments were separated on a 5% denaturing polyacrylamide gel on an ALF automated DNA sequencer (Pharmacia). Lengths of the fragments were evaluated with Fragment Manager software using internal and external markers (M). The quantity of each transcript was determined as the area under the curve, which was also sized by means of Fragment Manager software (Pharmacia) and normalized against the sum of all the fragments obtained for a particular sample. This is expressed as skip/total. The analysis showed that -9% of the transcripts in "aged' blood (48 hours at room temperature) did not contain the exon 7 (exon 7 skipping) (lane 2). Patient A and B, both carrying an identical nonsense mutation in the exon 7 showed a slightly higher level of exon 7 skipping in their transcripts (lane 3 to 6). Exon 7 skipping was not present (at least not in amounts that are detectable with the technology used) in fresh blood samples in which RNA was extracted immediately after prelevation lane 1 ), nor in EBV cell lines from patients that had no mutation in NF1 exon 7 (lanes 7 and 8) (patient C). P+ denotes with puromycin treatment, P- denotes without puromycin treatment. M denotes internal size markers. Figure 6: ALF based fragment analysis of NF1 exon 37-skipping that is present in "aged" blood and in EBV cell lines carrying a specific nonsense mutation in exon 37, but that is not present in fresh blood nor in EBV cell lines carrying a NF1 mutation in another exon. Another illustration of the importance to extract the RNA immediately after prelevation for unprocessed blood or after removal from the incubator for cell cultures. Fragment analysis of RT-PCR products using a 5' fluorescein labeled forward primer located in exon 36 and a reverse primer located in exon 38. 20 cycles of amplification were performed. RT-PCR fragments were separated on a 5% denaturing polyacrylamide gel on an ALF automated DNA sequencer (Pharmacia). Lengths of the fragments were evaluated with Fragment Manager software using internal and external markers (M). The quantity of each transcript was determined as the area under the curve, which was also sized by means of Fragment Manager software (Pharmacia) and normalised against the sum of all the fragments obtained for a particular sample. This is expressed as skip/total. The analysis showed that -14% of the transcripts in aged blood (48 hours at room temperature) did not contain the exon 37 (exon 37 skipping). P+ denotes with puromycin treatment, P- denotes without puromycin treatment. M: denotes internal size markers. Patient C, carrying a nonsense mutation in the exon 37 showed a significantly higher level of exon 37 skipping in his transcripts. Exon 37 skipping was almost absent (-2%) in fresh blood samples (lane 1) and not detectable in EBV cell lines from patients that had a mutation in another exon of NF1 (lanes 5 and 6). Figure 7: Distribution of the mutations identified by the protein truncation test (PTT) of the total coding region of the NF1 gene by analyzing 105 patients. The figure gives a schematic representation of the total coding region of the NF1 gene drawn to scale. In 85 of the 105 patients a mutation in the NF1 gene could be visualised using PTT (see Table 1 ). Exon numbers are indicated as described by Viskochil D. (1998). A mutational hotspot is defined by the occurrence of at least 2 independently arisen mutations in 2 unrelated persons at the same nucleotide. Examples are R304X (exon 7), R440X (exon 10a), R461X (exon 10a), Y489C (exon 10b), 2033-2034insC (exon 13), R1362X (exon 23.2), R1513X (exon 27a), R1849Q (exon 29), 2366delNF (exon 39). Also the finding of two different mutations at the same spot (e.g. 1465-1466insC and 1466A>G in exon 10b) is in itself indicative of a mutational hotspot (Cooper et al., 1998) Moreover, if the number of mutations identified in the number of patients analysed is weighted against the size of the exons, a number of mutation dense regions stand out: i.e. exon 7, 10a-10b-10c and exon 37. Exon 31 was previously described to contain a mutational hotspot, i.e. R1947X (Upadhyaya and Cooper, 1998). We only found this mutation once so far, but is was found already by several research groups and hence it is a recurrent mutation. The denomination of this mutation as a mutational hotspot may however be due to a bias, as till recently only limited portions of the gene were investigated by research groups and preferentially, groups searched in those regions where mutations were reported by others before. Figure 8: Distribution of all mutations identified so far by us using the PTT for the total coding region of the NF1 gene in 105 patients, and heteroduplex analysis, FISH analysis, Southern blot analysis and cytogenetic analysis in patients in which no mutation was identified by PTT. The heteroduplex analysis has not yet been completed for all exons in all patients that were negative in the PTT assay. So far, 6 interesting missense mutations and/or in frame deletions were disclosed: C93Y (exon 3), C187Y (exon 4b), 274delL (exon 6), L847P (exon 16), 991delM (exon 17), 2366delNF (exon 39). Their distribution is given on top of the bar representing the total coding region of the NF1 gene. Besides, a deletion of the total gene was found in 4 patients and 1 translocation t(14;17)(q32;q11.2). All mutations identified so far by analysing all together 105 patients are summarized in Table 1. Figure 9A and 9B: Figure 9A: Overview of the "mutation rich" regions in the NF1 gene. In our study exons 7, 10a, 10b, 10c and 37 stand up as particularly mutation-rich regions. In order to define the percentage of the coding region of a certain exon, we made the ratio between the number of nucleotides of a given exon and the number of nucleotides of the total coding region (i.e. 8457 nt). In the denominator the total coding region from ATG to TGA was taken, with the exception of the alternatively spliced exons 9br, 23a and 48a, in which no mutations have ever been found. Figure 9B: Overview of the recurrent mutations found in this study. Between parentheses is denoted: the exon that is prone to the recurrent mutation, number of patients found in this specific study with the specific mutation, whether the patients are sporadic (S) or familial (F). If 2 apparently unrelated patients with an identical mutation are found and patients are familial cases, haplotype analysis was performed to confirm that both patients are indeed unrelated and hence that the mutations arose independently. Figure 10. Illustration of the power of the current methodology detecting 2 different cryptic splice acceptors in IVS26, activated by mutation IVS26-2A>T. Genomic DNA, cDNA and subcloned cDNA sequence chromatograms of 4515-2A>T (IVS26-2A>T) in patient NF-044. A. genomic DNA sequencing chromatogram showing presence of mutation 4515-2A>T. B. starting from puromycin treated EBV cell lines, direct cycle sequencing chromatograms of the exon 26 & 27 region showed that besides normal transcipts containing exon 27 derived from the Wild Type allele, also 2 mutant transcript populations were present: a fraction contain an insertion of the last 14 nt of IVS26 (mutant transcripts) and another fraction contain an insertion of the last 17 nt of IVS26 (mutant transcripts). Mutant transcripts are formed due to use of two different cryptic splice acceptors in IVS26 (see Table 9). C. cDNA sequencing chromatograms of the 2 mutant transcript populations after subcloning formed in patient NF-044. A fraction contain an insertion of the last 14 nt of IVS26 (left panel) and another fraction contain an insertion of the last 17 nt of IVS26 (right panel). Figure 11. Illustration of the power of the current methodology detecting 2 different misspliced transcripts that are formed due to presence of the mutation IVS39-12T>A. PTT, cDNA and genomic DNA sequence chromatograms of IVS39-12T>A in patient NF-005. A. PTT results. Lanes 1 and 2. peptides synthesized in vitro from normal control EBV cultures. Lane 3: peptides synthesized in vitro from a Pmin EBV culture of patient NF-005. Arrowheads indicate presence of 2 different truncated peptides: one derived from transcripts in which exon 40 is skipped, another derived from transcripts formed by use of a novel splice acceptor in I VS 39 leading to insertion of the last 10 nt of I VS39. B. cycle sequencing of the mutant cloned cDNA transcripts. Upper panel, transcripts with an insertion of the last 10 nucleotides of IVS39 due to use of the novel splice acceptor (gtttgtttgtttgtttgttag/ttttttgtag) created by 7127-12T>A. Transcripts lead to a truncated peptide of 209 amino acids after in vitro translation. Lower panel, transcripts with E40 skipping resulting in a peptide shortened by only 44 amino acids after in vitro translation. C. cycle sequencing of the splice acceptor site genomic region of E40. Arrow, heterozygous peak showing the 7127-12T>A mutation. Figure 12: genomic and cDNA analysis of a patient with the mutation Y489C or 1466A>G in exon 10b resulting in the creation of a novel splice door site. Exon 10b of the NF1 gene: 1466A to G (Y489C) is a "missense mutation" masquerading a splicing mutation. The mutation creates a novel splice donor site that succesfully competes with the normal unaltered splice donor leading to skipping of the last 62 nt of exon 10b. A. Cycle sequencing without subcloning of the genomic region of exon 10b in patient NF-017 showing transition of A to G at nt 1466 resulting in the formation of a splice donor site CT/gtaag; B. Cycle sequencing of the mutant cloned cDNA allele in patient NF-017. The last 62- bp of exon 10b are skipped and immediately followed by exon 10c, resulting in the formation of a stop codon at amino acid 489; C. Schematic diagram of the genomic region surrounding exon 10b. Shaded boxes represent exons, normal and novel splice donor sequences are denoted. By the transition of A to G at nt 1466 a novel splice donor is formed. Figure 13: genomic and cDNA analysis of a patient showing mutation V1093M or 3277G>A in exon 19b resulting in the formation of a novel splice donor site that is used by the splicing machinery. Exon 19b of the NF1 gene: 3277GtoA (V1093M) is a "missense mutation" masquerading a splicing mutation. The mutation creates a novel splice donor site that competes with the normal unaltered splice donor leading to skipping of the last 40 nt of exon 19b. Novel information is obtained on splice preferences in the NF1 gene using Splice Site Prediction using Neural Networks (http://www- hgc.lbl.gov/projects/splice.html). Evaluation of the' sequences using this in silico prediction shows that wild type exon 19b contains a very weak splice donor and can be inactivated even by creation of another weak splice donor upstream. A. Cycle sequencing without subcloning of the genomic region of exon 19b in patient NF-063 showing substitution of G to A at nt 3277 resulting in the formation of a weak splice donor site TG/gtatg; see Table 9 B. Direct cycle sequencing of the mutant cDNA alleles in patient NF-063. The last 40- bp of exon 19b are skipped and immediately followed by exon 20; C. Schematic diagram of the genomic region surrounding exon 19b. Shaded boxes represent exons, normal and novel splice donor sequences are denoted. By the substitution of G to A at nt 3277 a novel splice donor is formed. Figure 14: 5294C>A (S1765X) nonsense mutation. Exon 29 of the NF1 gene: 5294CtoA (S1765X is a "nonsense mutation" masquerading a splicing mutation. The mutation creates a novel weak splice acceptor site that competes with the normal unaltered splice acceptor leading to skipping of the first 90 nt of exon 29. Novel information is obtained on splice preferences in the NF1 gene using Splice Site Prediction using Neural Networks (http://www-hgc.lbl.gov/projects/splice.html). Exon 29 contains a strong splice acceptor that however already can be inactivated even by creation of a weak splice acceptor downstream. A. Cycle sequencing without subcloning of the genomic region of exon 29 in patient NF-009 showing substitution of C to A at nt 5294 resulting in the formation of a novel splice acceptor site; see Table 9 B. Direct cycle sequencing of the mutant cDNA alleles in patient NF-009. The first 90- bp of exon 29 are; C. Schematic diagram of the genomic region surrounding exon 29. Shaded boxes represent exons, normal and novel splice acceptor sequences are denoted. By the substitution of C to A at nt 5294 a novel splice donor is formed. Figure 15: effect of nonsense mutations on splicing. PTT, cDNA and gDNA sequencing results of patient NF-027 with mutation R304X and PTT results of patient NF-003 and NF-019 with mutationY2264X. A. PTT results using primers encompassing NF1 exons 28-38. Lane 1 & 2 normal and truncated peptides formed in patients NF-003 (lane 1 ) and in patient NF-019 (lane 2). We identified the first nonsense mutations causing skipping of an exon containing these specific nonsense mutations (6792C>A and 6792C>G; published in Messiaen et al., 1997). Hoffmeyer et al. (1998) subsequently claimed that other nonsense codons in the NF1 gene are disparate splice effectors. We show using our detailed mutation analysis technology that only few nonsense mutations in the NF1 gene are inducing exon skipping, i.e; the formerly described 6792C>A and 6792C>G. We disagree with Hoffmeyer et al that the predominant effect of R304X is skipping of exon 7, see Figure 15 B and C. Our experiments showed that the predominant effect of presence of R304X is formation of a stopcodon, as clearly evidenced on PTT assay and by cycle sequencing of the cDNA. B. PTT results using primers encompasing NF1 exons 1 -12a. Analysis of 2 patients with a truncated peptide using the abovementioned primers starting from EBV transformed lymphoblastoid cell cultures not treated with puromycin and immediate RNA extraction after removal from the incubator. Lane 2, 3, 4 are normal controls and show presence of the normally observed bands not further discussed or relevant in this figure. Lane 5 shows presence of a truncated peptide in another patient carrying a mutation in the region between exons 28-38. Lane 6 shows presence of a truncated peptide of approximately 33 kDa in patient NF-027. Lane 1 : protein marker. C. cycle sequencing without subcloning of (a) gDNA of patient NF-027 showing presence of the 910C>T substitution changing R304 into a stopcodon, (b) cDNA of patient NF-027 Pmin EBV culture. Arrow, presence of the TGA allele in a minor fraction of the transcripts (unequal expression), (c) cDNA of patient NF-027 Pplus EBV culture. Arrow, presence of the TGA allele present in equal amounts due to the inhibition of the nonsense mediated mRNA decay. 33 kDa is the size expected in the PTT reaction when the nonsense codon is retained and the optimized PTT could correctly identify the major effect of R304X on transcription. Figure 16: Use of the combined cascade of testing allows making distinction between (even very rare) polymorphism and bona fide pathological mutations. The evaluation of the pathological effect of missense mutations is very difficult in the absence of knowledge of all functional domains in a protein. Often missense mutations are reported as bona fide mutations although firm data underscoring these conclusions are missing (Lambert et al., 2000). We identified a missense mutation in exon 45 of the NF1 gene, R2616Q: this mutation was not found in 300 normal control chromosomes, the amino acid Arginine is conserved in Drosophila, mouse, rat and Fugu and change of arginine for glutamine is predicted to cause a dramatic change in the polypeptide chain . Still, the missense mutation did not segregate with the disorder in the family that was studied. By PTT of the total coding region the real pathogenic lesion was identified, i.e. R304X in exon 7, illustrating the strength of the technology. Efficient and correct molecular mutation analysis is extremely important, as the most immediate result of the current findings is the ability to provide presymptomatic and/or prenatal diagnosis. Figure 17: Somatic mosaicism for R2429X in a sporadic NF1 patient NF-075. Patient NF-075 is a male patient born in 1989 and has 2 small CAL spots (<5mm), subcutaneous neurofibromas supraclavicular and a plexiform neurofibroma surrounding the R atrium and septum and invading the pericard, and multiple internal neurofibromas in the mediastinum, freckling in left axilla and 2 isch noduli in left eye. A: schematic representation of fragment 5, small bars denote the presence of sequencing primers to study this fragment, asterisk denotes the position of the stopcodon found in patient NF-075. B: panel 1 : PTT analysis from fragment 5 and separation of the peptides on a 15%SDS- PAGE and 20hrs exposure of autoradiograms in 2 normal control cell lines treated with puromycin (lane 2, 3) , in patient NF-055 EBV cell lines treated with (lane 4) and without (lane 5) puromycin. This patient NF-055 carries the germline mutation R2429X. In patient NF-075 a weak truncated band at exactly the same position as the truncated peptide previously detected in another patient NF-055 was revealed. Only with an optimal signal to noise ratio it is possible to discern such a faint truncated band. PTT, starting from RNA extracted immediately after taking the EBV cell line from the incubator, and using the very sensitive 3 H-Leucine incorporation, can effectively pinpoint the region of interest for further molecular study. panel 2: PTT analysis from fragment 5 and separation of the peptides on a 10%SDS-PAGE and 20hrs exposure of autoradiograms in cell lines from patient NF-055 (lanes 1 and 2) and patient NF-075 (lanes 2 and 4), treated with puromycin (lane 1 and 3) and without puromycin (lanes 2 and 4) panel 3: same gel as in panel 2 but with a longer exposure time (60hrs instead of 20hrs). Using the longer exposure time the truncated peptide in patient NF-075 can be more readily seen. C. (1 ). genomic DNA direct cycle sequencing chromatograms of NF-055 reveals presence of mutation R2429X in his blood lymphocytes. Equal quantity of mutant versus wild type allele is present, as can be expected for a germline mutation present on 1 Λ/F7-copy in all cells; (2) genomic DNA direct cycle sequencing chromatograms of a normal control person; (3) Genomic DNA direct cycle sequencing of NF-075 reveals at that sequence the presence of a small signal that might indicate presence of a T nucleotide at position 7285 in a fraction of the cells. Cycle sequencing in itself is not sensitive enough to give any pathological significance to such a signal; (4) Further analysis of the genomic DNA of patient NF-075 by subcloning revealed presence of mutation R2429X in a fraction of his blood cells. This is the first sporadic patient that could be identified to be a "somatic mosaic" for a nonsense mutation in the NF1 gene. Fragment analysis (not shown) showed that the mutation is present in <10% of the blood cells. Examples Example 1. Patients From all patients EBV transformed B-lymphoblastoid cell lines and/or short-term phytohaemaglutinin stimulated blood lymphocyte culutes were established to allow for extensive RNA and DNA based studies. DNA was extracted directly from the lymphocytes as well. Patients were ascertained in the Center of Medical Genetics of Ghent, Brussels and Liege (Belgium). Before the analysis was initiated a detailed clinical evaluation was performed and the clinical features of all patients were documented using the "NNFF International NF1 Mutation analysis Consortium" form. Only patients that fulfilled the diagnostic criteria as proposed by the NIH Consensus Statement in 1988 (Stumpf et al.) and updated in 1997 (Gutmann et al.) were admitted to the study. The study was approved by the ethics committee of the University Hospital Ghent (Belgium).. Example 2. DNA Isolation, RNA Isolation and cDNA Synthesis. EBV-transformed cell lines were grown in RPMI 1640. Prior to RNA isolation, the EBV transformed cell culture was split. In order to prevent nonsense mediated mRNA decay, one subculture was maintained in the presence of puromycin [16 hours, 200 μg/ml puromycin (Sigma, p7255), further called Pplus culture ], while in the other subculture no puromycin was added (further called Pmin culture). RNA was extracted from both types of cultures for all patients. Total cellular RNA was extracted with TRIzol LS Reagent (Gibco BRL, 10296-010) using the manufacturer's instructions. cDNA was synthesized with 2-3 μg total RNA using random hexamers (Amersham Pharmacia Biotech) and 200 U Superscript II Reverse transcriptase (Gibco BRL). Example 3. Protein truncation test Primers used for the protein truncation test (PTT) assay have been described by Heim et al. (1995). PTT was performed using an optimized protocol (Claes et al., 1998). The sensitivity of the technique was further enhanced using puromycin treated EBV- transformed cell cultures and/or phytohaemaglutinin stimulated blood lymphocyte cultures and RNA extraction immediately after removal from the incubator. We analyzed all protein samples on a 10% and 15% SDS-PAGE gel in order to maximize the detection of the very large and small abnormal peptide fragments. Example 4. Improved PTT Results. As the vast majority of mutations reported so far are predicted to generate a premature stop codon, it was chosen to start with the protein truncation test (PTT) in the first step of a combined mutation approach consisting of a cascade of techniques applied in the mutation analysis of the NF1 gene. However, the efficiency to detect truncating mutations by PTT depends on the stability as well as on the purity of the mutant mRNA under investigation. As nonsense-mediated mRNA decay has been documented in mutant NF1 alleles (Hoffmeyer et al, 1995) it was decided to also develop an optimized PTT for the NF1 gene using puromycin-treated EBV cell lines. Puromycin is a tRNA analogue causing chain termination and blocks nonsense-mediated decay in cell lines as was demonstrated to be the case for the MSH2 gene (Andreutti-Zaugg et al., 1997). The effect of puromycin on the stability of the NF1 mutant transcripts was not investigated before. 67 EBV cell lines from NF1 patients were established. Established cell lines were grown until a T25 culture flask contained approximately 100 clusters with a diameter of 0.2-0.3 mm/cm 2 and were then divided in two separate wells (10cm 2 ): one well was treated for 15 hours with puromycin (200 μg/ml), the other well was further incubated in the RPMI-1640 tissue culture medium. Total RNA was extracted from both cultures and further analyzed by the protein truncation test. Although we did not miss a truncated peptide using RNA from the cultures without puromycin treatment - mainly due to the fact that a PTT system based on isotopic incorporation of 3 H-Leucine is very sensitive - the direct cycle sequencing of all fragments leading to a truncating peptide was surprisingly greatly facilitated starting from cDNA prepared from the puromycin treated cultures (Figure 3 , 4 and 15). By the optimized PTT analysis, the germline mutation at the cDNA and gDNA level in 83% of the patients studied was succesfully identified. Another crucial parameter in the successful application of the PTT to find the disease causing mutation is the quality ol the RNA that is used to start the procedure. It was noticed that starting from RNA extracted from peripheral blood cells, very often "spurious" bands were present after RT-PCR as well as on the PTT SDS-PAGEs. The origin of these spurious bands was further explored by comparing the transcripts obtained when RNA is extracted from the peripheral blood lymphocytes directly after prelevation or after 48 hours incubation at room temperature. The rationale for these experiments were twofold: i) in the natural situation the blood sample often stays for some time in the doctor's room and is transported hereafter to the lab. The time needed for this transport depends on the place from where the sample is referred, but can take more than 24 hours; ii) it has been reported that some tumor suppressor genes such as TSG101 and FHIT often show deletions at the cDNA level for which no mutations in the DNA can be found. Especially in tumor tissues and in "aged" (means that the blood sample was kept at room temperature for 60 hours before RNA was extracted) blood samples it was seen that both genes exhibit infidelity of the splicing process. Noteworthy, the authors observed that the breakpoints of the deleted transcripts coincide with cryptic splice donor or acceptor sites or with the skipping of entire exons. The splicing infidelity was observed to be gene specific and did not occur in other gene transcripts that were analyzed, i.e. BRCA1, BRCA2, hMSH2, IGF2, RB amongst others. Epigenetic factors may influence the activation of these cryptic sites leading to the occurrence of these deleted transcripts. Therefore, semi-quantitative experiments on the ratio of transcripts skipping of exons 7 and 37 compared to transcripts that do contain exons 7 and 37 were carried out. RNA was extracted from different sources, including fresh blood, "aged" blood at room temperature for 48 hours, EBV cell lines from normal control persons, EBV cell lines from patients harboring specific mutations in exon 7 and 37 leading to increased skipping of the respective exons (Figures 5 and 6). These experiments clearly demonstrated that RNA extracted from "aged" blood samples leads to increased skipping of these exons and hence mimics - in the absence of a genomic alteration - the presence of a mutation. The NF1 gene has to be considered to be a gene that is prone to alterations in the RNA processing in response to epigenetic factors. As all missense mutations, small in frame insertions / deletions as well as large deletions and chromosomal rearrangements necessarily escape detection by the PTT analysis, all patients in which no mutation was identified in the first step were further analysed with a second battery of analyses. This second step included: DNA heteroduplex analysis of all 60 exons, FISH analysis using 3 intragenic cosmid/PAC clones, Southern blot analysis using 5 intragenic probes and finally karyotyping. The combined approach surprisingly led to identification of the germline mutation in 64 out of 67 patients (>95%, Table 5). By PTT alone the germline mutation (after cDNA and genomic sequencing) could be identified in 56 out of the 67 patients analysed (83%). This is the highest detection rate reported so far. This study indicates that 29% of the germline mutations in the NF1 gene are associated with aberrant splicing, a frequency that is much higher than that reported in surveys of other human genetic disorders (Krawczak et al., 1992 and Ruttledge et al., 1996), but is reminiscent of the situation found in the ATM gene (Teraoka et al., 1999). Given the fact that splicing errors as the cause for Neurofibromatosis type 1 are particularly frequent in the NF1 gene it is of paramount importance to avoid the occurrence of spliced transcripts that arise due to epigenetic factors such as incubation at room temperature of the blood lymphocytes outside their natural habitat (the bloodstream). This can be obtained by working with EBV lymphoblastoid cell lines and extraction immediately after cultures are withdrawn from the incubator. Example 6: Exon 10b of the NF1 gene represents a mutational hotspot and harbors a recurrent missense mutation Y489C associated with aberrant splicing With the current technology several novel mutational hotspots were identified. One such a mutational hotspot resides in exon 10b. This region harbors a missense mutation that masquerades a splicing mutation. Material and methods NF1 patients For all patients, the diagnosis of NF1 was based upon the presence of two or more of the diagnostic criteria proposed by the NIH Consensus Statement in 1988 (Stumpf et al., 1988) and updated in 1997 (Gutmann et al., 1997). The study was approved by the Institutional Ethical Committees and informed consent was obtained from the patients studied. Patients were recruited randomly without bias as they were seen for medical follow up and genetic advise. Patients were recruited as part of a general mutation study. 37 patients were contributed by the Dpt of Medical Genetics of Gent and by the Service de Genetique, Hopital Erasme Brussels. Nucleic acid extraction DNA and RNA samples were obtained from 37 unrelated NF1 patients by extraction from EBV- transformed lymphoblastoid cell lines. Total cellular RNA and genomic DNA was isolated as described (Messiaen et al., 1997). cDNA analysis and in vitro transcription/translation analysis First strand cDNA was synthesised by random priming (Messiaen et al., 1997) and cDNA was amplified using 5 primer pairs for amplification of the total coding region (10). 4 μl PCR product was used in an optimized in vitro transcription/translation reaction as described (Messiaen et al., 1997; Claes et al., 1998). An identical truncated peptide fragment of 55 kDa was observed in 2 out of 37 patients by in vitro transcription/translation of the fragment spanning exons 1 to 12a and the corresponding cDNA was analysed by cycle sequencing with and without subcloning using 0.15 μM fluorescein isothiocyanate (FITC) labeled primers, designated by nucleotide positions: 5'-CTTCGGAATTCTGCCTCT-3' (400-418), 5'-CTGATATGGCTGAATGTG-3' (719-736), 5'- GCCTGTGTCAAACTGTGT-3' (967-984) and 5'- CACACCCAGCAATACGAA -3' (1367-1384) and the Thermo Sequenase fluorescent labelled primer cycle sequencing kit (Amersham). Samples were loaded on a 6% LongRanger gel (FMC) containing 7M urea and analysed on an ALF automated DNA sequencer. In order to check for the presence of the missense mutation Y489C in a fraction of the cDNA, RT-PCR fragments were cloned using the pCR-TOPO cloning kit (Invitrogen) and 90 individual clones were further analysed by cycle sequencing. Genomic DNA analysis Exon 10b was amplified using the primer pair as described (Purandare et al., 1994) and PCR products were further analysed by cycle sequencing without subcloning (Messiaen et al., 1997). Mutations are reported according to the recommendations of the Nomenclature Working Group (Antonarakis, 1998), with the start site of translation denoted as nucleotide 1 both for cDNA and genomic alterations. Results The total coding region of the NF1 gene was analysed by the protein truncation test in 37 unrelated NF1 patients from which an EBV lymphoblastoid cell line was available (Heim et al., 1995). In 2 patients an identical shortened fragment of approximately 55 kDa was discerned in the region encompassing the exons 1 to 12a. In both patients in vitro transcription/translation for the other regions only showed normal sized fragments. By electrophoresis of the RT-PCR fragments from patient 1 two discrete bands were discerned on a 1.5% agarose gel, i.e. a normal sized band of 1868-bp and a band that was approximately 60-bp smaller. In patient 2 however only a normal sized band was seen, indicating that the truncated protein of identical size was formed in a different way in this patient. cDNA sequencing in this region indeed revealed a different mutation in both patients. In patient 2, an insertion of C at nt 1465-1466 in exon 10b was found, immediately resulting in the creation of a stop codon at this site. In patient 1 , a deletion/skipping of the last 62 nucleotides of exon 10b was observed in RNA from both lymphocytes and the EBV- lymphoblastoid cells (Fig.12B). Here too, the immediate result is formation of a stop codon at this site, explaining the identical picture seen by protein truncation analysis. Further analysis of exon 10b at the genomic level confirmed the presence of an insertion 1465insC in patient 2. In patient 1 however, a missense mutation was identified: A1466G, changing the codon for Tyr to Cys (Y489C) (Fig. 12A). Both parents of this sporadic patient did not carry this missense mutation. This missense mutation masquerades a splicing defect: indeed substitution of A to G at position 1466 of the genomic DNA creates a new splice donor site (CT/GTAAG) (Fig. 12C). Analysis of the normal and mutant sequence using the program for splice site prediction by neural network (http://www- hqc.lbl.gov/proiects/splice.html) showed a 0.86 score for the normal exon 10b donor site (GCTTTGT/gtaagtat) and a higher 0.97 score for the new donor site created by the missense mutation Y489C (AGAAGCT/gtaagtat). RT-PCR fragments from an EBV lymphoblastoid cell line of patient 1 were cloned and 90 individual clones were further analysed by cycle sequencing in order to check for the presence of the missense mutation in a fraction of the cDNA. In 50 cDNA clones showing a normal sized band of 1868-bp on a 1.5% agarose gel, only the wild type sequence was found and in none of them the missense mutation was present. In 40 clones containing a slightly smaller insert (approximately 60-bp) as evidenced by agarose gel electrophoresis, the smaller size was due to the skipping of the last 62 nucleotides of exon 10b along with intron 10b in the mRNA (Fig. 12B). This indicates that the major outcome of the mutation Y489C at the cDNA level is skipping of the last 62 nucleotides of exon 10b. Although Y489C and 1465- 1466insC are different mutations at the genomic DNA level, both result in the formation of a premature stop codon at exactly the same position well before the GAP domain of neurofibromin. As the finding of two different mutations at the same spot (i.e. 1465- 1466insC and 1466A>G) is in itself indicative of a mutational hotspot (Cooper et al., 1998) these findings prompted us to analyze exon 10b in a larger patient population. We now identified the mutation Y489C in 4 unrelated NF1 patients on a total number of investigated proven NF1 patients of 105, allowing to estimate prevalence of this mutation in the NF1 population to be about 4%. Example 7: Exhaustive mutation analysis of the NF1 gene allows to identify 95% of mutations and reveals a high frequency of unusual splicing defects. Patients, Materials and Methods Patient samples In this prospective study, 67 unrelated index patients seen at the Departments of Medical Genetics of Ghent University Hospital, Universite de Liege, Vrije Universiteit Brussel and Universite Libre de Bruxelles for clinical follow-up and genetic counseling were included. Clinical features of all patients were documented using the "NNFF International NF1 Mutation analysis Consortium" form. Only patients fulfilling the diagnostic criteria as proposed by the NIH Consensus Statement in 1988 (Stumpf et al., 1988) and updated in 1997 (Gutmann et al., 1997) were included. The ethics committee of the Ghent University Hospital approved the study. From all patients EBV transformed B-lymphoblastoid cell lines were established. 38 patients presented as de novo cases and 29 patients were familial. The familial or sporadic nature of the mutation was verified by analysis of family members. All mutations were verified on a second independent sample. The mutations were absent on 100 unrelated normal chromosomes. RNA Isolation and cDNA Synthesis Prior to RNA isolation, the EBV transformed cell culture was split. In order to prevent nonsense mediated mRNA decay, one subculture was maintained in the presence of puromycin [16 hours, 200 μg/ml puromycin (Sigma, p7255), further called Pplus culture ], while in the other subculture no puromycin was added (further called Pmin culture). RNA was extracted from both types of cultures for all patients. Total cellular RNA was extracted with TRIzol LS Reagent (Gibco BRL, 10296-010) using the manufacturer's instructions. cDNA was synthesized with 2-3 μg total RNA using random hexamers (Amersham Pharmacia Biotech) and 200 U Superscript II Reverse transcriptase (Gibco BRL). RT-PCR and PTT Primers used for the amplification of the total NF1 cDNA in 5 overlapping fragments (F1- F5) were as previously described (Heim et al., 1995). 3-5 μl PCR product, 20 μM amino acid mix minus Leucine (Promega, L4610) and 1.6 μl 3 H Leucine (specific activity 1 mCi/mmol; Amersham) were added to the TNT™ Coupled Reticulocyte Lysate System (Promega). Reactions were performed as described (Claes et al., 1998). Samples were subjected to electrophoresis in a 10% and 15% SDS-polyacrylamide gel (Protean II Bio- rad, 20 x 24 cm gels) and run for 16 h at 30 mA (10% gels) and 40 mA (15% gels). 14 C methylated protein (Amersham Pharmacia Biotech CFA626) was used as a protein-weight marker. Synthesized polypeptides were visualized by autoradiography after 20 and 60 h exposure to X-ray film. Semi-quantitative analysis of splicing variants not associated with mutations cDNA was subjected to 20 cycles of PCR using the following primer pairs: 5'-FITC-TTGACTTGGTGGATGGTTT-3' (cDNA 749-777) and 5'- TTGAGAATGGCTTACTTGGA-3' (cDNA 1096-1077) for analysis of exon 7 (E7) skipping; 5'-FITC- GGGCAGATAAAGCAGATAAT-3'(cDNA 6721-6740) and 5'- CCGGATTGCCATAAATAC-3' (cDNA 7029-7012) for analysis of E37 skipping. Semi-quantitative analysis of the transcripts was performed on a 5% denaturing acrylamide gel on an ALF automated DNA sequencer (Amersham Pharmacia Biotech) as described (Lambert et al., 1998). The nature of shortened transcripts was verified after subcloning by direct cycle sequencing as described (Messiaen et al., 1997). Splice site scores The sequence environment of all splice mutations was analyzed using Splice Site Prediction by Neural Network (SSPNN) and a Splice Site Score (SSS) was obtained (URL adress: http://www.fruitflv.org/seq tools/splice.html). For all 5' and 3' splice sites (ss) the consensus values (CV) were calculated as developed by Shapiro and Senepathy (1987). cDNA sequencing Autoradiograms from PTT gels allowed to predict the most plausible position of the mutations causing the specific truncated peptides and RT-PCR fragments were cycle sequenced in the corresponding regions with the Thermo Sequenase™ fluorescent labeled primer cycle sequencing kit (Amersham Pharmacia Biotech) using 5'-fluorescein or 5'-Cy5 labeled sequencing primers distributed along the coding sequence (sequences available upon request). Heteroduplex Analysis (HA) Exons were amplified from genomic DNA. For some exons PCR primers were developed using OLIGO V5 software (Table 6). For other exons PCR primers were as described (Purandare et al., 1994; Hoffmeyer et al., 1998, Maynard et al., 1997; Abemathy et al., 1997; Cawthon et al., 1990; Li et al., 1995). Exons 1 and 49 were not yet studied. For the larger exons 16, 21 , 28, 29, 31 , 33, 35, 37 and 38 the sensitivity of the HA was improved by digestion with a specific RE in order to obtain fragments with an optimal size between 200-300 nt. After amplification, fragments were denatured at 98°C for 5' and allowed to reanneal at 68°C for 1 hour. 2-4 μl of the PCR product was mixed with 8 μl loading buffer (25% bromophenolblue, 25% xyleencyanol, 30% glycerol) and loaded on a 1 X MDE gel (FMC, Rockland, Maine) containing 10% glycerol. After electrophoresis, gels were stained with EtBr (0.5 μg/l) and evaluated under a transilluminator. Aberrant fragments were further analyzed by cycle sequencing using the forward amplification primer or a nested primer for sequencing. Cytogenetic analysis and fluorescence in situ hybridisation (FISH) Patients in whom no mutation was found by PTT and HA were analyzed by cytogenetic analysis and FISH. Cytogenetic analysis was performed on PHA stimulated G-banded metaphases according to standard procedures. To detect submicroscopic deletions dual color FISH was performed according to Van Roy et al (1994) using PAC clones 22 (926B9; 5' NF1) and 13 (1002G3; 3' NF1) described by Correa et al (1999). Southern Blot analysis 6 μg of genomic DNA was digested with 30 U of EcoR\ and Bgl\\ (both Gibco BRL) for 6 hours at 37°C. Digested DNA was electrophoresed in 0.8% agarose and transferred to positively charged Nylon membranes (Hybond N + Amersham). Hybridisation was carried out using standard procedures (Sambrook et al., 1989) using cDNA probes GE2, FF13, FB5D, P5 and B3A as described (Marchuk et al., 1991 ). Results Mutation detection rate using the combined approach and mutational spectrum We identified a bona fide pathogenic mutation in 64 of 67 unrelated NF1 patients (95.5%) (Table 5), including all 29 familial cases and 35 of 38 sporadic patients. By the optimized PTT starting from puromycin-treated EBV-lymphoblastoid cell lines, the mutation was completely characterised both at the cDNA and gDNA level in 56 patients (56/67 patients; 83.5%): 25 were nonsense (25/67; 37%), 12 frameshift (12/67; 18%: 5 insertions and 7 deletions of one or a few basepairs) and 19 in-frame or out-of-frame splice mutations (19/67; 28%). Further investigation of the remaining patients by HA resulted in the identification of 6 missense mutations and/or deletions of single amino acids (9%): C93Y, C187Y, L847P, 2970-2972delAAT or 991delM and 7096-7101 delAACTTTor 2266delNF. 18 of the 44 (41%) single base-pair substitutions were due to a C>T or G>A transition at CpG dinucleotides, known to be prone to mutation if methylated. A deletion of the entire NF1 gene was found by FISH analysis as evidenced by the absence of the 5' (926B9) and 3' (1002G3) PAC clone. Cytogenetic and FISH analysis showed a balanced translocation in a large NF1 family: t(14;17)(q32;q11.2), interrupting the NF1 gene as PAC 926B9 was found on the der (17) and PAC 1002G3 on the der (14) (data not shown). 32 of the mutations identified (including the translocation) are novel compared to the most recent data (International NF1 Mutation Analysis Consortium before, March 1999; URL address: http://www.nf.org/nf1 ene/nf 1 qene.home.html). Ars et al, 2000, Fahsold et al., 2000 and most recent overview of published data by Upadhyaya and Cooper, 1998). Translation inhibition facilitates detection of Premature Termination Codons (PTCs) by PTT and direct cycle sequencing Nonsense mediated mRNA decay compromises most RNA-based mutation detection methods, but can be circumvented using puromycin (Andreutti-Zaugg et al., 1997). Starting from RNA extracted from Pmin EBV cultures, PTT detects truncated peptides even if mutant transcripts are highly unstable. However, direct cycle sequencing of cDNA fragments using fluorescent dyes is severely impaired by the nonsense mediated decay and the signal-to-noise ratio is far better starting from Pplus EBV cultures. Representative results are shown in Figure 4B. We compared the sensitivity of PTT and direct cycle sequencing in 13 EBV lymphoblastoid cell cultures treated with and without puromycin (6, Figure 3, 4 , 15 and 17). By PTT all truncated peptides from both types of cultures were discerned after 60h exposure of autoradiograms. However only in 7 of the Pmin EBV cultures the mutant transcript was unambiguously identified by direct cDNA sequencing. In the remaining 6 cultures, the expression of the mutant transcripts was highly reduced compared to the normal transcripts with the ratio of mutant to wild-type peak height in sequencing chromatograms being <0.35. Direct cDNA cycle sequencing can not reveal unambiguously the pathogenic mutation in these cases. In contrast, in all Pplus EBV cultures the mutation was identified and the ratio between mutant and wild-type peak height in sequencing chromatograms varied between 0.8 and 1.00. Distribution of the mutations, mutational hot spots and recurrent mutations Mutations seem to be equally distributed along the gene. However, some exons may have a higher mutation density (Figure 9A). In 15% of the patients studied, a mutation was found within the exons 10a-10b-10c, although this region comprises only 4.5% of the coding region. In this region 3 recurrent mutations (R440X, R461X and Y489C) were found. In E37, comprising only 1.2% of the coding region, a mutation was found in 5.9% of the patients. Ten recurrent mutations were identified in 20 unrelated patients and together account for 30% of the mutations found in this study (5): R304X, R440X, R461X, Y489C, 2033-2034insC, R1362X, R1513X, R1849Q, Y2264X and 7096-7101 delAACTTT (Figure 9B). As NF1 haplotypes were different in both families carrying the mutation R1849Q, recurrence can not be due to identity by descent. R304X, R440X, R461 X, R1362X, R1513X, R1849Q are C>T or G>A substitutions at CpG dinucleotides, which may explain their recurrence. There is no clue to explain the recurrence of 1466A>G (Y489C) looking at the sequences surrounding this mutational hotspot. We found this mutation in 5/232 unrelated patients (Messiaen et al., 1999). The recurrence of 2033insC may be caused by slippage of the polymerase in a stretch of 7 cytosines. Mutation Y2264X (C6792A and C6792G) resides in a sequence environment containing direct AC-repeats as well as palindromic sequences. The recurrence of 7096delAACTTT may be caused by slipped mispairing between two AACTTT tandem repeat sequences. Missense mutations in the NF1 gene and their pathogenicity We identified 6 genuine missense mutations or deletions of single amino acids, i.e. C93Y, C187Y, L847P, 2970-2972delAAT and 7096-7101 delAACTTT. These were absent on 300 normal control chromosomes, conserved during evolution in Rat (D45201 ), Mouse (L10370), Fugu (AF064564) and Drosophila (L26501 ), segregated with the disorder in 5 familial cases or verified to be de novo in 1 sporadic case. If the total coding region would be studied uniquely at the genomic level 4 more mutations could erroneously be considered to be missense mutations: Y489C (2X), V1093M (1X) and R2616Q (1X). Y489C was documented as a splice mutation (Messiaen et al., 1999). V1093M acts similarly as a splice mutation by creating a novel splice donor in the middle of E19b. R2616Q, found in a familial patient NF-027, was not found in 300 control normal chromosomes, is predicted to cause a dramatic change in the polypeptide chain and is conserved in Rat, Mouse, Fugu and Drosophila (Figure 16). However, this alteration did not segregate with the disorder within the family (Figure 16). The index patient was compound heterozygous for R2616Q and R304X, the latter identified by PTT. R304X is the genuine pathogenic mutation in this family as her healthy daughter inherited the R2616Q allele and the affected daughter the R304X mutation. This finding underscores the importance of the analysis of the total coding region for truncating mutations before firm conclusions can be made on the pathogenicity of missense mutations. Mutations resulting in splicing defects Splicing errors were detected in 19/67 (28%) patients (Table 5 and 7). Only 4 splice mutations were at the canonical GT splice donor or AG splice acceptor, i.e. IVS12a+1 G>T, 6577delGAGgta, IVS26-2A>T and IVS27b-2A>T. 6 mutations were at less conserved positions of the 5' or 3' splice site (ss): I VS16+3delaaagtg, R1849Q, K2286N, IVS16-6delcttt, IVS19b-3C>G and IVS39-12T>A. One nonsense and 2 missense mutations create a novel 5' or 3' ss and are splice mutations, i.e. S1765X, Y489C and V1093M. Y2264X (C6792A and C6792G) result in E37 skipping and R304X results partially in E7 skipping besides retention of the nonsense codon (Figure 5). Both mutations do not alter the existing normal ss nor create novel ones and may exert their effect by altered interaction between an exonic splice enhancer and mRNA splicing factors (Messiaen et al., 1997, Hoffmeyer et al., 1998). The remaining 25 nonsense mutations were not associated with splicing defects and hence nonsense mediated exon skipping is rather exceptional in NF1. Consensus values (CVs) according to Shapiro and Senepathy (1987) and splice site scores (SSSs) according to Splice Site Prediction by Neural Networks (SSPNN) were calculated for all splice sites involved in splicing mutations (Table 7). Simple skipping of an exon due to a mutation at the 5' or 3' consensus ss of that exon was observed in only 4 cases, i.e. 6577delGAGgta, K2286N, I VS16-6delcttt and IVS19b- 3C>G. Only K2286N results in a transcript that, if translated, would leave the reading frame intact. Remaining splice mutations had complex effects. IVSl2a+1G>T leads to skipping of both E11 and 12a. Noteworthy, CV and SSS for the normal 5' and 3' ss of E11 and for the 3' ss of E12a are very weak which may explain the concerted skipping of both exons if a mutation affects the 5' ss of E12a. R1849Q (5547G>A) results in transcripts lacking E29 (ex29del) and transcripts lacking both E29+30 (ex29/30del) in equal amounts. The same transcripts were found in a patient with mutation IVS29+1 G>C (Osborn et al., 1999). This region is involved in tissue-specific alternative splicing with ex29del expressed only in human brain tissues and ex29/30del detectable at low levels in all tissues (Park et al., 1998). Constitutive increase of the ex29del expression in non-brain tissues seems to be the pathogenic lesion causing NF1 in this family. Seven mutations induced - beside exon skipping in some cases - the activation of a cryptic ss or the use of a novel created ss, i.e. IVS16+3delaaagtg, Y489C, V1093M, IVS26- 2A>T (Figure 10), IVS39-12T>A (Figure 11 ) and S1765X.For all mutations affecting the 5' ss of NF1 exons, the CV and SSS of the mutated ss (M) were lower than the wild type ss (N) sequences but differences between the SSSs were more pronounced. Y489C (Figure 12) and V1093M (Figure 13) both create a novel 5' ss with an almost identical CV compared to the wild type 5' ss. Prediction of ss strength by SSPNN gives slightly better results (Table 7). The natural 5' ss of E19b is unusual for primates (AA/gtaaat, rare nucleotides underlined), which may explain why it is inactivated even by the weak donor ss created by V1093M. Mutations at the 3' ss had a lower (3/5) or almost identical (2/5) CV and SSS compared to the wild type 3' ss. Both the CVs or SSSs fail to predict whether the mutation will lead to exon skipping and/or activation of a cryptic 3' ss. IVS16-6delcttt and IVS39-12T>A both disrupt a tandem repeat, cttt and gttt respectively. For both mutations, the CV and SSS of the mutant sequence are identical compared to the wild type 3' ss, yet missplicing occurs and the outcome of both mutations differs. IVS16- 6delcttt leads to "simple" E17 skipping, although a strong cryptic 3' ss resides 57 nt upstream (SSS 0.96) and -if activated- could result in the in frame insertion of 29 amino acids. For IVS39-12T>A the mutant 3' sequence (gtttgttagttttttgtag/ggtacag) still has a high SSS (0.99) yet apparently gets inactivated and a novel created 3' ss at IVS39-12 (gtttgtttgtttgtttgttag/ttttttgtaggg) is used partially leading to a transcript that retains the last 10 nucleotides of IVS39 .forming a peptide of 209 amino acids after in vitro translation. The mutation further causes skipping of E40 leading to a peptide shortened by only 44 amino acids. Both truncated peptides were discerned by PTT (Figure 1 1 ) illustrating the power of this technique to detect multiple mutant transcripts. The novel 3' ss created by S1765X has a lower CV and SSS compared to the wild type ss, yet in frame skipping of the first 90 nucleotides of E29 is observed (Figure 14). An intranuclear scanning mechanism capable of recognizing nonsense codons as proposed by Dietz and Kendzior (1994) and primarily concerned with the maintenance of an open reading frame may mediate this outcome. R304X and splicing R304X was shown by Hoffmeyer et al (1997) to result in in-frame E7 skipping without retention of the nonsense codon in the mutant transcripts. We studied 3 patients with mutation R304X from 2 unrelated families. Starting from RNA extracted from Pplus and Pmin EBV cultures, PTT clearly showed a truncated peptide of approximately 33 kDa, the size expected when the nonsense codon is retained in all patients (Figure 15). Cycle sequencing of cDNA from the Pmin cultures showed unequal expression of the mutant transcript containing the nonsense codon (Figure 15). Semi-quantitative RT-PCR analysis of the transcripts showed that E7 skipping was present in a minor fraction of the transcripts (6-13%; Figure 5). Puromycin treatment did not alter the ratio of the E7-skipped versus full- length transcript (Figure 5). In EBV lymphoblastoid cell lines, Ex7del transcripts were undetectable in normal control persons (data not shown) and in patients with the mutation Y2264X (Figure 5). Our results indicate that at least in EBV cultures E7 skipping is not the major outcome of mutation R304X. We cannot exclude whether or not cell type dependent splicing differences may underly the apparent discrepancies between both studies. Variant splicing due to environmental factors and not associated with mutations. In specific tumor suppressor genes such as TSG101 and FHIT, some transcripts with internal deletions are not necessarily associated with a genomic mutation and can be found in the RNA from normal tissues as well, especially in lymphocytes not processed immediately after prelevation ("aged" blood) (Gayter et al., 1997). Initially PTT was developed starting from blood samples, but often spurious background bands were visible on autoradiograms, urging us to develop the technology starting from EBV transformed cell lines. Some blood samples are inevitably delayed in transit from the hospital to the laboratory and the background bands may be caused by misspliced NF1 transcripts in "aged" blood cells that lead to the formation of truncated peptides in the PTT. We analysed blood samples from 4 unrelated normal control persons for 2 regions in the NF1 transcript: the region where E7-skipping was observed to a low extent in patients with the mutation R304X and the region where equal expression of transcripts with E37-skipping was observed in patients with the mutation Y2264X (Messiaen et al., 1997). Semi-quantitative analysis showed that skipping of E7 and 37 was undetectable in all control samples processed immediately after prelevation of the blood. In all 4 "aged" blood samples however, a fraction of the transcripts showed skipping of E7 and E37. Typically the ratio of misspliced to full-length transcripts for E7 and E37 ranged between 0.09 and 0.14. Analyses starting from RNA that was not extracted immediately after prelevation of the blood samples can result in the occurrence of shorter transcripts in RT-PCR. This "noise" may in some cases obscure the real "signal" that is formed by the bona fide mutation. Discussion In this study 67 unrelated typical NF1 patients were analysed using a cascade of complemenatry techniques and the mutation was identified in 64 patients (>95%). Hence a sensitive molecular diagnostic test for NF1 can be achieved if classical NF1 patients are studied with multiple complementary and optimized techniques. By PTT we identified mutations in 56/67 patients (83%). Missense mutations or small in frame insertions/deletions were found by HA in 6/67 patients (9%). 18 out of 44 single base pair substitutions were due to a C>T or G>A transition at CpG dinucleotides. By cytogenetic and/or FISH analysis 1 deletion of the entire gene and 1 balanced translocation t(14;17)(q32;q11.2) interrupting the NF1 gene was found. The mutations were evenly distributed along the NF1 coding sequence. However, exons 10a-10c and E37 seem to be more mutation-rich as would be expected if mutations were distributed at random. We did not find E4b to be a remarkably mutation-rich region, as indicated by Fahsold et al (in press). This may be due to our smaller patient cohort. Alternatively, some of the recurrent E4b mutations reported by Fahsold et al may be identical by descent as no data on sporadic versus familial status or haplotypes are available. If, for both studies, we consider the number of E4b mutations versus the number of patients that were studied instead of versus the number of mutations that were found, a similar pick-up rate for E4b is obtained (16 out of 521 patients by Fahsold et al and 2 out of 67 patients in this study; both -3%). Ten mutations were recurrent in our study and each account for -2.9% of the germline mutations. The high number of recurrent mutations was unexpected. Our results suggest that the exons 7, 10a-10c, 13, 23.2, 27a, 29, 37, 39 harbour recurrent mutations and that these exons, together with exons 4b, 22 and 31 reported by others to contain recurrent mutations (Fahsold et al., in press and Upadhyaya and Cooper, 1998), should be implemented with priority in NF1 mutation analysis. A mutation was identified in 36 out of 39 sporadic patients (92%). This study shows for the first time that also in sporadic NF1 patients the pathogenic mutation can be identified with high efficiency. The most immediate result of this effort is the ability to provide presymptomatic/prenatal testing in the offspring of sporadic patients. Moreover, a sensitive test can help to diagnose young children presenting with NF1 -related symptoms, but not (yet) fulfilling the N.I.H. diagnostic criteria. The presence of somatic mosaicism in conditions with a high new mutation rate as in NF1 has been predicted (Hall, 1988). Comparisons between mutation detection rates after analyzing the total coding region in sporadic versus familial NF1 patients were not published so far. In our study the gDNA direct sequencing chromatograms of 2 patients suggest that the mutant and wild-type sequence are not present in equal amounts. This might reflect somatic mosaicism and is currently further investigated. All patients in whom we found no mutation are sporadic and low level somatic mosaicism may underly the failure to find a mutation. Alternatively, we may have missed the mutations as no technique is 100% sensitive or the mutations may reside in the exons 1 or 49 or in the 5' or 3' UTR that were not yet analysed. In the majority of cases the clinical features of NF1 are caused by haploinsufficiency due to a mutation leading to a PTC and rapid decay of the mutant RNA. In this study 6 missense mutations and/or small in frame deletions were identified that may exert their effect in a dominant-negative fashion. Another group of mutations that may produce some truncated neurofibromin are mutations that affect splicing. The frequency of splicing errors in the NF1 gene is very high (28%) compared to other genetic disorders or as can be expected by calculation of relative target sizes (Krawczak et al., 1992). Only a minority of splice mutations (4/19) were found at the invariant AG/GT dinucleotides and mutations at 3' ss were as frequent as at the 5' ss. 8 splice mutations induce in frame skipping of total exons or part of an exon and have the potential to be "leaky". As 4 of these splice mutations (S1765X, K2286N, C6792A and C6792G) form a stable mutant transcript it remains possible that a truncated neurofibromin is formed. The observed splicing defects provide an unusual opportunity to examine splice site competition and the sequence determinants of splice site selection. For some regions of the NF1 gene, we found exon-deleted transcripts in normal control persons. The presence of these transcripts was more pronounced in the RNA extracted from "aged" lymphocytes. Multiple alternatively spliced transcripts have been described for NF1 (Danglot et al., 1995; Suzuki et al., 1991 ; Cawthon et al., 1990; Park et al., 1998). The observation that other specific splice variants apparently are formed -albeit typically at low levels- if blood lymphocytes are not kept at physiological temperatures is intriguing. The results lend support to the hypothesis that epigenetic factors may contribute to the phenotypic variability in NF1 patients by altering the ratio of specific splice variants. The fact that exon-deleted products are easily detected in PTT assays indicates that caution is needed in the interpretation of a "positive" PTT result, until a credible underlying mutation in the genomic DNA is identified. In particular, it is ill advised to use the results of the PTT for diagnostic purposes if the mutation at the cDNA and genomic DNA can not be identified. Often clinical samples are delayed in transit and cDNA analysis of such samples may yield results that mimic splicing errors. We circumvented this problem by establishing EBV transformed cell cultures, however this is a time consuming and expensive step. Therefore, we are currently evaluating the efficiency and sensitivity of the PTT starting from short term cultures of phytohaemagglutinin stimulated lymphocytes. The combination of short term culture of stimulated lymphocytes and puromycin treatment may significantly decrease the time needed to identify in a reliable and sensitive way the mutations in the NF1 gene by PTT. The availability of a powerful mutation detection technology for the NF1 gene will allow to adress some longstanding questions such as i/ what is the contribution of the NF1 gene to segmental NF, gastrointestinal NF, familial spinal NF, familial cafe-au-lait spots, late- onset NF and to conditions related to NF1 but with additional features; ii/ do genotype- phenotype correlations in NF1 exist; iii/ what is the contribution of somatic mosaicism in sporadic NF1 cases. References Abernathy C, Rasmussen S, Stalker H, Zori R, Driscoll D, Williams C, Kousseff B and Wallace M. 1997. 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Table 1 Mutations detected by analysis of the whole coding region of the NF1 gene by PTT, HA, Cytogenetic and FISH analysis. a 00 sΩ O n 1 2 Numbering based on NF1 cDNA (Genbank M82814). + denotes that a truncated peptide was seen; - denotes that no truncated peptide was seen but mutation was identified by heteroduplex analysis (HA); (F1 to F5) denotes that the truncated peptide was seen after in vitro translation of RT- PCR fragments F1 to F5 using primers for amplifying NF1 cDNA as described by Heim et al (1995). F1 spans cDNA nt 1-1868, F2 1468-3583, F3 3217-5256, F4 4998-6987 and F5 6574-404. 3 For all 44 transitions is mentioned whether they change a G>A or C>T at a CpG dinucleotide. 4 Sporadic (S) or familial (F) patient. Abbrebiations: E exon/ IVS intervening sequence or intron; IF, inframe; OOF, out of frame; ss splice site; nt nucleotide; skip skipping; inactiv. inactivation; activ. activation; NA not applicable, fnyi: family not yet investigated (novel families: cfr red); * NF-037 and NF-038 are proven to be unrelated as the mutation resides on a different haplotype background in both families. TABLE 2: NF1 PRIMERS SEQUENCING c-DNA Name+Labe I AnnT° Position Sequence (5'-3') Oriqin NF.Icy 60 1 atggccgcgcacaggccggt own NF1 s1 cy 58 73 acaggacagcagaacaca Rina Wu NF1s2 cy 58 400 cttcggaattctgcctct Rina Wu NF1 s3 cy 58 713 ctgatatggctgaatgtg Rina Wu NF2s1 fl.cy 58 967 gcctgtgtcaaactgtgt Rina Wu NF2s2 fl,cy 58 1367 cacacccagcaatacgaa Rina Wu NF2s3,on, fl cy 58 1685 atcctgatgctcctgtag Rina Wu NF2s3b cy 55 1878 tttttacggggtaggatg own NF3s1 ,on, fl cy 60 2035 atttgccgacaagcccag Rina Wu NF3s2,on, fl cy 58 2308 actgcaggaaacactgag Rina Wu NF3s3,on, fl cy 58 2689 ggctgttgtccttaatgg Rina Wu NF4s1 ,on, fl cy 58 3001 tgcttgggaatatggtc Rina Wu NF4s1a,cy(H3b) 58 3229 gtttcacttctagctg gtct own NF4s1 b,on, cy 60 3376 caaacaggtggcaggaaacg own NF4s2 fl,cy 56 3558 ccttcaacaaggcacaga Rina Wu NF4s3 fl,cy 56 3756 actctaccaactgctctg Rina Wu NF5s1 fl.cy 55 4011 gaacctccttcagatgac Rina Wu NF5s3 fl.cy 55 4308 agaagaacatatgcggcc Rina Wu NF5s3b rev cy 58 4450 gcacattgccgtcacttatg own NF5s4 fl.cy 55 4658 ctaggcatcaggtacatg Rina Wu NF6s1 fl.cy 55 4957 gtctccgcagtctatatc Rina Wu NF6s1 b cy 60 5071 cctgggaaactggctgagca own NF6s2 fl.cy 55 5382 ggagtgtgaagccattgt Rina Wu NF6s3 fl.cy 55 5670 tagtaagacgctggcagc Rina Wu NF6s4 fl.cy 55 5934 ccttgggcagattacaga Rina Wu NF7s1 fl.cy 55 6195 gatgctgtccttcaacaa Rina Wu NF7s2 fl.cy 52 6544 gaaacagtcacagaagct Rina Wu NF7s2b,cy 57 6576 ggaggcatgcatgagagata own NF7s3 fl.cy 54 6841 cagccacttcttaataagg Rina Wu NF7s4 fl.cy 58 7134 gcatccttcacctgctatt Rina Wu NF8s1 fl.cy 55 7375 catggtgacccttcctat Rina Wu NF8s2 fl.cy 52 7742 atgttctcttggatgaag Rina Wu NF8s3 fl.cy 50 8119 gctgagcttattgttaag Rina Wu TABLE 3: NF1 Primers q-DNA Name+label Program PCR Length Sequence (5'-3') Sequence oriqin NFex1.1cy does not work cy-cccagcctccttgccaacgc Shen et al NFex1.2 gacccattccaccggcctgt NFex2x.1 95° 5' 100ng 340bp tttcaatggcaagtaagt own NFex2x.2 (95 o 45"-54°30"-72°30")> ( 10XCS gttatatccaaagtccaca own NFex2x.1cy 72°10" l UPItaq NFex2.1fl(nested) 25μl fluo-tttaaggataaactgtttacgtg own NFex3.1 HS 100ng tttcacttttcagatgtgtgttg Purandare et al NFex3.2 (95 o 1 '-60°ϊ -72°1 ")X35 5XDy 237bp tggtccacatctgtactttg NFex3.1cy 72° 10' 1 UtaqBRL 4° TV=25μl NFex4Ax.1 95° 5' 100ng 517bp ttaaatctaggtggtgtgt own 00 NFex4Ax.2 (95°45"-54 o 30"-72°30"))< 10XBoeh aaactcatttctctggag own 72°10" l UPItaq 4° 25μl NFex4B.1wal 95°5' 100ng ggcttcctgaagtgctgggat Walace M., pers. comm. NFex4B.2wal 95°45"-63 0 25"-72 0 25")X: 10XCS 305bp ccagtttggtgttctagttcagca Walace M., pers. comm. 72° 10' 1 UplTaq NFex4B.1Vfl (seq 60° 4° TV=25μl fluo-gtgagataccacacctgtccc Viskochil NFex4c.1 HS 100ng tttcctagcagacaactatcga Purandare et al NFex4c.2 (95°1 , -60 o 1 '-72°1 , )X45 5XDy 283bp catcaaaaaaaaaattttaataccag NFex4c.1fl 72° 10" 1 UtaqBRL 4° TV=25μl Name+label Program PCR Length Sequence (5'-3' Seguence origin NFex5.1fl HS 200ng fluo-catgtggttctttatttataggc Hoffmeyer et al NFex5.2 (95 0 1 , -52°1 , -72°1 , )X35 10XCS 1 13bp tcaatcgtatccttaccagcc 72° 10' 1 UtaqBRL (primers lay within exon) 4° TV=25μl NFex6.1 HS 100ng catgtttatcttttaaaaatgttgcc Purandare et al NFex6.2 (95 0 1 , -64°1 ' -72° T)X35 lOXBoeh 299bp ataatggaaataattttgccctcc 72° 10' 1 UtaqBRL 4° TV=25μl NFex7.1 HS 100ng tgctataatattagctacatctgg Purandare et al NFex7.2 (95 0 1'-58 0 1 ' -72°- l ')X35 5XDy 373bp cctatgaacttatcaacgaagag NFex7.1cy 72° 10" 1 UtaqBRL 4° TV=25μl . vO NFex7+8.1 HS 100ng tgctataatattagctacatctgg Purandare et al NFex7+8.2 (95°1 '-55 0 1 ' -72 c T)X35 lOXBoeh 880bp ctagtctttctgtttataaaggat 72° 10' 1 UtaqBRL 4° TV=25μl NFex9.1 br HS 100ng tccgctgtggctcagaacac Purandare et al NFex9.2br (95 0 1 '-63°1 ' -72° 1 ■ )X40 lOXBoeh 335bp agtagaagaggatgcacagcc 72° 10' 1 UtaqBRL 4° TV=25μl NFex9.1 95°5' 100ng tttgacctcatttgtattactgag NFex9.2 (95 o 1 '-60 o 1 '-72°1')X35 10XCS 249bp agaaccttttgaaaccaagagtg Purandare et al 72°10' l UPItaq 4° TV=25μl NFex10A.1 HS 100ng acgtaattttgtactttttcttcc Purandare et al 1995 NFex10A.2 (95 0 1 '-58 0 1' -72°1 ')X35 lOXBoeh 222bp caatagaaaggaggtgagattc NFex10A.1fl 72° 10' 1 UtaqBRL NFex10A.2cy 4° TV=25μl Name+label Program PCR Length Sequence (5'-3') Sequence oriqin NFex10C1 HS 100ng cttggtaccctttagcagtcac Purandare et al NFex10O2 (95°1 '-59°1 l '-72°1 l ')X35 5XDy 379bp ccttctttctccatggag NFex10C.1 cy 72° 10' 1 UtaqBRL 4° TN=25μl NFex11.1 HS 100ng gtactccagtgttatgtttacc Purandare et al 1995 NFex11.2 (95 0 1 , -55°1' -72°1 ')X40 5XDy 190bp taaagttgaaatttaaaaattaaagtac NFex1 1.1cy 72° 10' 1 UtaqBRL NFex11.2cy 4° TV=25μl NFex12A.1 HS 100ng acttgtattcattatgggagaatg MRC (Maynard) NFex12A.2 (95°1 '-60 o 1 ' -72°1 ')X35 5XDy 284bp agtaatctctcaccattaccattc NFex12A.2cy 72° 10' 1 UtaqBRL 4° TV=25μl σ NFex12B.1 HS 100ng tttctagtgaatctccttcaagt Purandare et al NFex12B.2 (95 0 1 '-59 0 1' -72° 1 ')X40 5XDy 382bp atgaaatttaccaaatttcattcag NFex12B.1 cy 72° 10' 1 UtaqBRL 4° TN=25μl ΝFex13X.1 cy 95°5' 100ng cy-gagttattgtatgcggagac own NFex13X.2 (95°1'-55 o 1 , -72°1 , )X30 10XCS,B,D 494bp ttgaatttcccctgtaaac own NFex13.1fl (seq bij °) 72°10' 1 UplTaq fluo-cacagtttattgcattgttagat Purandare et al 4° TV=25μl NFex14.1 HS 100ng tccttttgggtggagcttatc Purandare et al NFex14.2 (95°1 , -59 o r -72° 1 ')X35 lOXBoeh 286bp tatacttgtaatatgcacgtatc NFex14.1cy 72° 10' 1 UtaqBRL 4° TV=25μl Name+label Proqram PCR Length i Sequence (5'-3'. Sequence oriqin NFex15.1fl HS 100ng fluo-tgtgatcaggaatagcttttgaa Purandare et al NFex15.2 (95 o 1 '-57 o 1 , -72°1 , )X40 5XDy 276bp ttaacagataaaagtcaactttac 72° 10' 1 UtaqBRL 4° TV=25μl NFex16.1 HS 100ng tggataaagcataatttgtcaagt Purandare et al NFex16.2 (95°1 , -58 0 1 '-72°1 , )X35 5XDy 549bp tagagaaaggtgaaaaataagag NFex16.1cy 72° 10' 1 UtaqBRL NFex16seq cy 4° TV=25μl cy-ccagtcagtgaacgtaaggg own o NFex17.1 HS 100ng ctctgtgtgtttagatcagtca Purandare et al o z NFex17.2 (95°1 , -55°1 , -72°1 , )X35 lOXBoeh 319bp tttatcaattactaccagtatcag ■ π NFex17.1cy 72° 10' 1 UtaqBRL 55 4° 2 TV=25μl 5 NFex18.1 HS 200ng tagtaaggtagccagaagttgtgt MRC (Maynard) o NFex18.2 (95°1 , -60 o 1 , -72°1 , )X35 l OXBoeh 320bp atttacaaaaccctacattgctc z NFex18.1cy 72° 10' 1 UtaqBRL o o 4° TV=25μl NFex19A.1 HS 100ng tcatgtcacttaggttatctgg Purandare et al NFex19A.2 (95°1 , -53 o 1 '-72°1 , )X40 5XDy 272bp tgtaattaagtagttataactctc NFex19A.1Cy 72° 10" 1 UtaqBRL cy-tcatgtcacttaggttatctgg 4° TV=25μl NFex19B.1x 95°5' 100ng attaccttctccccatttga own NFex19B.2x (95°1 , -55 o 1 , -72°1 , )X30 lOXboeh 371 bp ggctttatttgctttttgc NFex19B.1xcy 72°10' 1 Upltaq NFex19B.2xcy 4° TV=25μl Name+label Program PCR Length Seguence (5'-3'l Seguence origin NFex20.1 HS 100ng ccaccctggctgattatcg Purandare et al NFex20.2 (95°1 '-62°1 ' -72 c '1')X35 10XDy 402bp taatttttgcttctcttacatgc NFex20.1cy 72° 10' 1 UtaqBRL 4° TV=25μl NFex20-21.1 95°5' 100ng ctatatcaggtaaaatcatgtccaac Fahsold et al NFex20-21.2 (95°1 '-60°1 '-72°1 , ) * 35 l OXboeh gatttgctatgtgccagggac 72°10' 1 Upltaq 4° TV=25μl NFex21xx.1 95° 5' 100ng gtcaaacttactcaatgcc own NFex21xx.2 (95°45"-54 o 30"-72°30")X 10XBoeh 542bp caaccacttccctacag own NFex21xx.1cy 72°10' l UPItaq NFex21x1.cy 4° 25μl cy-aactggcatgtaagagaag own σs tv5 NFex22.1 HS 100ng tgctactctttagcttcctac Purandare et al NFex22.2 (95°1 , -58°1 , -72°1 ')X35 10XCS 331 bp ccttaaaagaagacaatcagcc NFex22.1cy 72° 10' 1 UtaqBRL 4° TV=25μl NFex23AN.1 95°5' 100ng 382bp gattgggtctcaacatttc NFex23AN.2cy (95°r- ■ 57°1 ' -72° 1 ')X35 10XDy cy-aataggctgaagtgaagatantc own? 72°10' 1 UplTaq 4° TV=25μl NFex23.1.1 HS 100ng tttgtatcattcattttgtgtgta Purandare et al NFex23.1.2 (95 o 1 '-60 o 1 '-72°1 ')X35 5XDy 282bp aaaaacacggttctatgtgaaaag NFex23.1.1cy 72° 10' 1 UtaqBRL 4° TV=25μl Name+label Program PCR Length Seguence (5'-3') Sequence oriqin NFex23.2.1 HS 100ng cttaatgtctgtataagagtctc Purandare et al NFex23.2.2 (95 0 1 '-52 0 1' -72°1 ')X35 10XCS 268bp actttagattaataatggtaatctc NFex23.2.1cy 72° 10' 1 UtaqBRL 4° TV=25μl NFex24b.1 HS 100ng ttgaactctttgttttcatgtctt Purandare et al NFex24b.2 (95°1 , -57 0 1 ' -72° 1 ')X35 5XDy 267bp ggaatttaagatagctagattatc NFex24MRC, not ok 72° 10' 1 UtaqBRL MRC (Maynard) 4° TV=25μl NFex25b.1 HS 100ng aatataataattatatttgggaaggt Purandare et al NFex25b.2 (95°1 '-57 0 1 ' -72°1 ')X35 l OXBoeh 338bp gaaaatatttgattcaaacagagc NFex25b.1cy 72° 10' 1 UtaqBRL cy-aatataataattatatttgggaaggt 4° TV=25μl -ω NFex25L.1 HS 100ng cattttattatagcagatgtc own NFex25L.2 (95°45"-57 0 45"-72°45")X 5XDy 534bp acttacacaggaacttcat 72° 10' 1 UtaqBRL 4° TV=25μl NFex26.1fl HS 100ng fluo-gctttgtctaatgtcaagtcac Purandare et al NFex26.2 (95°1 , -58 o r -72°1 ')X35 5XDy 342bp ttaaacggagagtgttcactatc 72° 10' 1 UtaqBRL 4° TV=25μl NFex27A.1 HS 100ng gttacaagttaaagaaatgtgtag Purandare et al NFex27A.2 (95°30"-62°30"-72°30")X 10XBoeh 300bp ctaacaagtggcctggtggcaaac NFex27A.1fl 72° 5' 1 UtaqBRL 4° TV=25μl NFex27B.1fl does not work fluo-tttattgtttatccaattatagactt Purandare et al NFex27B.2 tcctgttaagtcaactgggaaaaac Name+label Proqram PCR Length Seguence (5'-3') Seguence origin NFex28 Aber.1 95°5' 200ng cactgctaataatctttgtcttttttgtc Abernathy NFex28 Aber.2 (95°1 '- 65°1 ' -72°1 ')X40 lOXBoeh 501 bp cgtttacaaaacacagactggaactta NFex28 Aber.1 cy 72°10' l UPItaq NFex28 Aber.2cy 4° TV=25μl NFex28.1fl(nested) ttccttaggttcaaaactggtca own NFex29L1 H.S. 100ng tacaatggtgggaactc own NFex29.L2 (95°1 '-56 0 45"-72 0 45")X3 10XCS 567bp atattaaggtagaggctgttt own NFex29L1cy 72°10' 1 UtaqBRL NFex29L2cy 4° TV=25μl NFex29.1cy(nested) cy-attcttctccacttcaccc MRC (Shen) NFex29.2cy(nested) cy-cccaaatcaaactgaagaga MRC NFex30x.1 95°5' 100ng 350bp tggaactataaggaaaaa own NFex30x.2cy (95°1 '-50°1 '-72°1 '30")X3 10XCS cy-aaagtcttcactggaaa own 72°10' l UPITaq NFex30.2fl(3)(nested) 4° TV=25μl NFex31.1 (4) HS 100ng ataattgttgatgtgattttcattg Cawthon NFex31.2fl(4) (93°1 '-63°1 '-72°1 '30")X3 10XKoh 424bp fluo-aattttgaaccagatgaagag NFex31.1cy 72° 5' 1 UtaqBRL _4° TV=25μl NFex32.1 HS 100ng ggtagagtgattaaaacatg NFex32.2 (95°1 , -51 °1 ' -72°1 ')X35 10XCS 220bp tatgctatagtacagaaggc Rina Wu NFex32.1cy 72° 10' 1 UtaqBRL 4° TV=25μl NFex33.1fl HS 100ng fluo-catatctgttttatcatcaggagg NFex33.2 (95°1 '-61 °1 -72°1 ')X35 5XDy 462bp aagtaaaatggagaaaggaactgg Cawthon NFex33.2cy 72° 10' 1 UtaqBRL 4° TV=25μl Name+label Program PCR Length Seguence ,5'-3') Sequence oriqin NFex34.1fl HS 10Ong fluo-caaaatgaaacatggaactttaga NFex34.2 (95°1 '-57 0 1 '-72°1 ')X35 5XDy 400bp taagcattaagtacaaatagcaca Cawthon 72° 10' 1 UtaqBRL 4° TV=25μl NFex35.1fl HS 10Ong fluo-agaagccaaaatgataagaa NFex35.2 (95°1 , -52 0 1 , -72°1 ')X35 1 OXCS 495bp acccaaagacaacaagag 72° 10' 1 UtaqBRL 4° TV=25μl NFex36.1 HS 100ng ggaccagtggacagaac own NFex36.2fl (95°1 , -55 0 1 '-72°1 , )X35 l OXBoeh 345bp fluo-atatgctttacaacttgagaa 72° 10" 1 UtaqBRL 4° =25μl NFex37.1 HS 10Ong tacattaagctagctaccaa own NFex37.2fl (95°1 , -54 0 1 , -72°1 ')X35 10XBoeh 460bp fluo-cgcttgagaacatactatcc σs 72° 10' 1 UtaqBRL 4° TV=25μl NFex38.1fl HS 10Ong fluo-ccagctaacagtgtctt own NFex38.2 (95°1 '-50 o 1 '-72°1 ')X35 10XBoeh 474bp aaggaaatatactcacaataa 72° 10' 1 UtaqBRL 4° TV=25μl NFex39.1fl HS 100ng fluo-gaacctaatcaaccatctc NFex39.2 (95°1 '-52 0 1 , -72°1 , )X35 5XDy 286bp ttgcatttaaagtaagacat 72° 10' 1 UtaqBRL 4° TV=25μl NFex40.1fl HS 100ng fluo-cctttccttgcagagttgtta NFex40.2 (95 0 1 '-57°1 '-72 0 1 ')X35 5XDy 371 bp caccactaaaggactagactgt 72° 10' 1 UtaqBRL 4° TV=25μl Name+label Proqram PCR Lenqt i Sequence (5'-3'. Sequence oriqin NFex41.1fl HS 100ng fluo-ttcatcctgttttaagtcacacttg NFex41.2 (95°1 , -61 o 1 '-72°1 , )X40 lOXBoeh 273bp ttgcctccattagttggaaaattg Abernathy et al NFex41.1 72° 10' 1 UtaqBRL ttcatcctgttttaagtcacacttg 4° TV=25μl NFex42.1fl HS 100ng fluo-cttggaaggagcaaacgatggttg NFex42.2 (95°1 '-61 0 1 '-72°1 ')X35 lOXBoeh 356bp caaaaactttgctacactgacatgg Abernathy et al 72°10' 1 UTaq BRL 4° TV=25μl NFex43.1fl HS 100ng f I uo-agacactgtagttaatgaacttgc NFex43.2 (95°1 , -60°1 , -72°1 ')X35 l OXBoeh 224bp catgtactctcccaccttattttc Abernathy et al NFex43.1 Cy 72° 5' Taq ST 1/20 cy-agacactgtagttaatgaacttgc 4° TV=25μl NFex44.1fl HS 100ng fluo-cacgttaattccctatcttgctgc F Shen et al NFex44.2 (95 0 1 '-65 o 1 '-72 o 1')X35 lOXBoeh 200bp taaaaatttgagggtgggggactc R ? S. 72° 5' Taq ST 1/20 4° TV=25μl NFex45.1fl HS 100ng fluo-catgaataggatacagtcttctac NFex45.2 (95°1 '-60 o 1 , -72°1 , )X35 lOXBoeh 269bp cacattactgggtaagcatttaac Abernathy NFex45.2fl 72° 5' 1 UtaqBRL NFex45.1bio 4° TV=25μl NFex46.1fl HS 100ng fluo-gggaatgtatattatgttttccac NFex46.2 (95 < -60°1'-72°1 ')X35 5XDy 275bp atgttaggaagttcatcaaccatc Abernathy NFex46.2cy 72° 5' 1 UtaqBRL 4° TV=25μl NFex47.1 HS 100ng ctgttacaattaaaagataccttgc MRC (Upadhyaya) NFex47.2 (95°1 '-60 o 1 '-72°2')X35 5XDy 185bp tgtgtgttcttaaagcaggcatac MRC (Upadhyaya) 72° 10' 1 UtaqBRL 4° TV=25μl Leqend Table 3: σs o T a ( / 0, C), annealing temperature for the primers during PCR Table 5: Mutations detected by analysis of the whole coding region of the NF1 gene by PTT and HA. t- ^J Numbering based on NF1 cDNA (Genbank M82814). + denotes that a truncated peptide was seen; - denotes that no truncated peptide ^ was seen but mutation was identified by heteroduplex analysis (HA); (F1 to F5) denotes that the truncated peptide was seen after in vitro translation of RT-PCR fragments F1 to F5 using primers for amplifying NF1 cDNA as described by Heim et al (1995). F1 spans cDNA nt 1- 1868, F2 1468-3583, F3 3217-5256, F4 4998-6987 and F5 6574-404. 3 For all 44 transitions is mentioned whether they change a G>A or C>T at a CpG dinucleotide. Sporadic (S) or familial (F) patient. Abbrebiations: E exon/ IVS intervening sequence or intron; IF, inframe; OOF, out of frame; ss splice site; nt nucleotide; skip skipping; inactiv. inactivation; activ. activation; NA not applicable Table 6: Comparison of the sensitivity to detect NFl mutations by direct cDNA cycle sequencing starting from Pmin and Pplus EBV cultures as measured by the ratio between mutant/wild -type peak height on sequencing chromatograms. 00 Table 7: Consensus values CV) and Splice Site Scores (SSS) of splice sites (ss) involved in splicin mutations in the NF1 ene CVN and CVM, consensus value normal and mutant; CVcry, consensus value of activated cryptic splice site; CVnov, consensus value of novel created splice site; ss, splice site; E, exon; upst., upstream;downst., downstream; NA, not applicable;./, no SSS can be obtained by SSPNN not even at the lowest threshold; capital letters denote nucleotides within exons and small letters within introns. 00 o

Claims: CLAIMS 1. Method for mutation analysis of the NF1 gene of a patient comprising the steps of: a) isolating peripheral blood lymphocytes of said patient, b) establishing an EBV transformed B-lymphoblastoid cell line with said peripheral blood lymphocytes of said patient, or short-term culturing of the blood lymphocytes by phytohaemaglutinin (PHA) stimulation, c) treatment of the EBV transformed B-lymphoblastoid cell line or short-term cultures with a protein synthesis inhibitor with a protein synthesis inhibitor, d) immediate extraction of RNA of cultures of said EBV transformed B-lymphoblastoid cell line, e) amplifying said RNA using suitable primers, and, f) obtaining peptide fragments by means of in vitro transcription/translation of said amplified fragments of step e). 2. Method according to any claim 1 wherein said protein synthesis inhibitor is puromycin, cycloheximide, actinomycinD or possible analogues. 3. Method according to any claims 1 to 2 wherein RNA is extracted within a time period of less then 48 hours at room temperature preferentially immediately after the protein synthesis inhibitor treatment of EBV transformed B-lymphoblastoid cell lines. 4. Method according to any of claims 1 to 3 wherein said amplification step is a polymerase chain reaction. 5. Method according to any of claim 1 to 4 wherein said RNA extracted in step d) as defined in claim 1 is total RNA. 6. Method according to any of claims 1 to 5 wherein step f) as defined in claim 1 is followed by a separation of said peptide fragments. 7. Method according to claim 6 wherein in case a truncated peptide is observed by means of protein separation, the amplified cDNA fragment obtained in step e) of claim 1 is analyzed by means of suitable primers allowing the characterization of some or all exons present in said cDNA fragment. 8. Method according to claim 7 wherein said analysis is performed by means of cycle sequencing of the suitable fragment by means of suitable primers. 9. Method according to any of the claims 1 to 8 wherein said primers of step e) are represented on Figure 2 or in any of the tables. 10. Method according to any of the claims 1 to 9 for mutation analysis of the NF1 gene of a patient involving the detection of a frame shift, missense or silent mutation. 1 1. Method according to any of the claims 1 to 9 wherein said primers are located in exon 4b, 7, 10a -10c, 13, 22, 23.2, 27a, 29, 37 or 39. 12. Method according to any of the claims 1 to 11 wherein said primers are labeled. 13. Method according to any of the claims 1 to 11 wherein said cDNA fragments are further analyzed by means of ALF or another (semi)-automated sequencing method. 14. Method according to any of claims 1 to 13 wherein in addition also DNA is extracted from said peripheral blood lymphocytes of said patient for further analysis of the genomic mutations present in the DNA of said patient. 15. Method according to claim 14 wherein said analysis is performed by means of heteroduplex analysis and/or single stranded conformation polymorphism analysis and/or conformation sensitive gelelectrophoresis (to detect aberrant migrating PCR fragments which are then further analyzed by cycle sequencing) or immediate cycle sequencing. 16. Method for mutation analysis of the NF1 gene of a patient involving the detection of a mutation in exon 7, 10a-10b-10c, 13, 23.2, 27a, 29 or 39. 17. Method for detecting at least one of the following specific mutations of the NF1 gene: K33K (99del105), C93Y (278G>A), C187Y (560G>A), R192X (574C>T), 603-604insT (idem), Q209X (625C>T), 819-821 delCCT (idem), 889-454del474nt (888del174), 987- 988insA (idem), 1261-19G>A (1260insTTTGTTTTTCTCTAGTC), W425X (1275G>A), R461 X (1381C>T), Y489C (1465del62), 1466insC (idem), 1527+5G>A (1392del135), E524X (1570G>T), 1605insA (idem), S536X (1607C>A), 1642-3C>G (1641 del80), 2305insT (idem), 2585insA (idem), 2836insT (idem), 2850+2del6 (2617del233), 2851 - 6del4 (2850del140), Q959X (2875C>T), Q963X (2887C>T), 2990+3A>C (2850del140), Y1044X (3132C>A), 3193delC (idem), V1093M (3277G>A/ 3274 del40), 3108-3C>G (3314del182), E1123X (3367G>T), 3457delCTCA (idem), Q1174X (3520C>T), 3704delA (idem), 3708+1 G>C (3496del212), 4026delG (idem), 4299delC (idem), Q1494X (4480C>T), 4515-2A>T (4515-14ins14/4515-17ins17), 4773-2A>T (4772del433/4772del293), 5033delG (idem), 5117delT (idem), R1849 (502del341/5205del544), S1755X (5264C>G), S1765X (5215del90/5294C>A), 5567delT (idem), 5798delC (idem), Q1966X (5896C>T), 6577delGAGgta (6364del215), R2237X (6709C>T), 6858G>C (6756del102), 7127-12T>A (7126del132/7127-10ins10), 7268delCA (idem), K2401X (7201 A>T), R2429X (7285C>T), 7884-7885delGT (idem), 8016delA (idem); with said letters and numbering referring to the mutation at the amino acid level which is mentioned first and the effect of said mutation at the mRNA level being mentioned between parentheses; or the genomic mutation t(14;17)(q32;q11.2) interrupting the NF1 gene. 18. Diagnostic kit comprising primers specifically amplifying the mutation region or positions as defined in claim 16 or 17. 19. Diagnostic kit comprising a probe specifically detecting a mutation region or position as defined in claim 16 or 17. 20. Method for identifying a compound correcting the defective structure of the mutated NF1 protein, coded by a mutated DNA as described in claim 17. 21. Model systems comprising an NF1 gene mutation as defined by claim 17 which can be used to screen for a therapeutic agent.

Inventors: CALLENS TOM, MESSIAEN LUDWINE, CALLENS, TOM, MESSIAEN, LUDWINE
Applicants: CALLENS TOM, MESSIAEN LUDWINE, UNIV GENT, CALLENS, TOM, MESSIAEN, LUDWINE, UNIVERSITEIT GENT
Application year: 2000
Main classification: C12Q1
IPC codes: C12N15, C12Q1