Patent Application: US-12856002-A

Abstract:
a method for mutation analysis of the neurofibromatosis 1 gene of a patient includes extracting dna from peripheral blood lymphocytes of the patient , establishing an ebv transformed b - lymphoblastoid cell line using lymphocytes from the patient , treating the ebv transformed b - lymphoblastoid cell line culture with puromycin , extracting rna from cultures of the cell line immediately , amplifying the rna using suitable primers , and obtaining peptide fragments by means of in vitro transcription / translation of the amplified fragments . the invention also relates to the identification of new hotspots and specific nf1 mutations . the invention also includes diagnostic kits for the detection of described specific mutations and hotspot domains , compounds correcting the structure of specific mutated nf1 proteins and in vitro and in vivo systems that may be used to screen for these therapeutic compounds .

Description:
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 ). 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 &# 39 ; s instructions . cdna was synthesized with 2 - 3 ug total rna using random hexamers ( amersham pharmacia biotech ) and 200 u superscript ii reverse transcriptase ( gibco brl ). 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 . 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 hmsh2 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 ( 10 cm 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 ( fig3 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 of 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 &# 39 ; 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 ( fig5 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 (& gt ; 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 . 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 . 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 . 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 ). 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 ′ ( seq id no 5 ) ( 400 - 418 ), 5 ′- ctgatatggctgaatgtg - 3 ′ ( seq id no 6 ) ( 719 - 736 ), 5 ′- gcctgtgtcaaactgtgt - 3 ′ ( seq id no 7 ) ( 967 - 984 ) and 5 ′- cacacccagcaatacgaa - 3 ′ ( seq id no 8 ) ( 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 . 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 . 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 ( fig1 b ). 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 ) ( fig1 a ). 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 / g t aag ) ( fig1 c ). analysis of the normal and mutant sequence using the program for splice site prediction by neural network ( http :// www - hgc . lbl . gov / projects / splice . html ) showed a 0 . 86 score for the normal exon 10b donor site ( gctttgt / gtaagtat ( seq id no 9 )) and a higher 0 . 97 score for the new donor site created by the missense mutation y489c ( agaagct / gtaagtat ( seq id no 10 )). 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 ( fig1 b ). 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 & gt ; 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 %. exhaustive mutation analysis of the nf1 gene allows the identification of 95 % of mutations and reveals a high frequency of unusual splicing defects . in this prospective study , 67 unrelated index patients seen at the departments of medical genetics of ghent university hospital , université de liège , vrije universiteit brussel and université 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 . 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 &# 39 ; 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 ). 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 × 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 . cdna was subjected to 20 cycles of pcr using the following primer pairs : 5 ′- fitc - ttgacttggtggatggttt - 3 ′ ( seq id no 11 ) ( cdna 749 - 777 ) and 5 ′- ttgagaatggcttacttgga - 3 ′ ( seq id no 12 ) ( cdna 1096 - 1077 ) for analysis of exon 7 ( e7 ) skipping ; 5 ′- fitc - gggcagataaagcagataat - 3 ′ ( seq id no 13 ) ( cdna 6721 - 6740 ) and 5 ′- ccggattgccataaatac - 3 ′ ( seq id no 14 ) ( 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 ). 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 . fruitfly . 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 ). 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 ). 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 ; abernathy 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 , me .) 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 . 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 ). 6 μg of genomic dna was digested with 30 u of ecori and bglii ( 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 ). 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 - 7101delaacttt or 2266delnf . 18 of the 44 ( 41 %) single base - pair substitutions were due to a c & gt ; t or g & gt ; 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 / nflgene / nflgene . 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 fig4 b . we compared the sensitivity of ptt and direct cycle sequencing in 13 ebv lymphoblastoid cell cultures treated with and without puromycin ( 6 , fig3 , 15 and 17 ). by ptt all truncated peptides from both types of cultures were discerned after 60 h 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 & lt ; 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 . mutations seem to be equally distributed along the gene . however , some exons may have a higher mutation density ( fig9 a ). 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 - 7101delaacttt ( fig9 b ). as nf1 haplotypes were different in both families carrying the mutation r1849q , recurrence can not be due to identity by descent . r304x , r440x , r461x , r1362x , r1513x , r1849q are c & gt ; t or g & gt ; a substitutions at cpg dinucleotides , which may explain their recurrence . there is no clue to explain the recurrence of 1466a & gt ; g ( y489c ) looking at the sequences surrounding this mutational hotspot . we found this mutation in { fraction ( 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 . we identified 6 genuine missense mutations or deletions of single amino acids , i . e . c93y , c187y , l847p , 2970 - 2972delaat and 7096 - 7101delaacttt . 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 ( fig1 ). however , this alteration did not segregate with the disorder within the family ( fig1 ). 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 . splicing errors were detected in { fraction ( 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 + 1g & gt ; t , 6577delgaggta , ivs26 - 2a & gt ; t and ivs27b - 2a & gt ; t . 6 mutations were at less conserved positions of the 5 ′ or 3 ′ splice site ( ss ): ivs16 + 3delaaagtg , r1849q , k2286n , ivs16 - 6delcut , ivs19b - 3c & gt ; g and ivs39 - 12t & gt ; 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 ( fig5 ). 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 , ivs16 - 6delcttt and ivs19b - 3c & gt ; g . only k2286n results in a transcript that , if translated , would leave the reading frame intact . remaining splice mutations had complex effects . ivs12a + 1g & gt ; t leads to skipping of both e11 and 12a . noteworthy , cv and sss for the normal 5 ′ and 3 ′ ss of e11a 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 & gt ; a ) results in transcripts lacking e29 ( ex29del ) and transcripts lacking both e29 + 30 ( ex { fraction ( 29 / 30 )} del ) in equal amounts . the same transcripts were found in a patient with mutation ivs29 + 1g & gt ; 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 & gt ; t ( fig1 ), ivs39 - 12t & gt ; a ( fig1 ) 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 ( fig1 ) and v1093m ( fig1 ) 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 ( a a / gtaa a t , 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 ( ⅗ ) or almost identical ( ⅖ ) 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 & gt ; 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 & gt ; a the mutant 3 ′ sequence ( gtttgtt a gttgtag / ggtacag ( seq id no 15 )) still has a high sss ( 0 . 99 ) yet apparently gets inactivated and a novel created 3 ′ ss at ivs39 - 12 ( gtttgtttgtttgtttgtt a g / tttttgtaggg ) 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 ( fig1 ) 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 ( fig1 ). 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 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 ( fig1 ). cycle sequencing of cdna from the pmin cultures showed unequal expression of the mutant transcript containing the nonsense codon ( fig1 ). semi - quantitative rt - pcr analysis of the transcripts showed that e7 skipping was present in a minor fraction of the transcripts ( 6 - 13 %; fig5 ). puromycin treatment did not alter the ratio of the e7 - skipped versus full - length transcript ( fig5 ). in ebv lymphoblastoid cell lines , ex7del transcripts were undetectable in normal control persons ( data not shown ) and in patients with the mutation y2264x ( fig5 ). 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 . in this study 67 unrelated typical nf1 patients were analysed using a cascade of complemenatry techniques and the mutation was identified in 64 patients (& gt ; 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 { fraction ( 6 / 67 )} patients ( 9 %). 18 out of 44 single base pair substitutions were due to a c & gt ; t or g & gt ; 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 ({ fraction ( 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 café - 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[ 0274 ] table 2 nf1 primers sequencing c - dna name + label annt ° position sequence ( 5 ′- 3 ′) origin nf . 1cy 60 1 atggccgcgcacaggccggt own ( seq id no 16 ) nf1s1 cy 58 73 acaggacagcagaacaca rina wu ( seq id no 17 ) nf1s2 cy 58 400 cttcggaattctgcctct rina wu ( seq id no 18 ) nf1s3 cy 58 713 ctgatatggctgaatgtg rina wu ( seq id no 19 ) nf2s1 fl , cy 58 967 gcctgtgtcaaactgtgt rina wu ( seq id no 20 ) nf2s2 fl , cy 58 1367 cacacccagcaatacgaa rina wu ( seq id no 21 ) nf2s3 , on , fl , cy 58 1685 atcctgatgctcctgtag rina wu ( seq id no 22 ) nf2s3b cy 55 1878 tttttacggggtaggatg own ( seq id no 23 ) nf3s1 , on , fl , cy 60 2035 atttgccgacaagcccag rina wu ( seq id no 24 ) nf3s2 , on , fl , cy 58 2308 actgcaggaaacactgag rina wu ( seq id no 25 ) nf3s3 , on , fl , cy 58 2689 ggctgttgtccttaatgg rina wu ( seq id no 26 ) nf4s1 , on , fl , cy 58 3001 tgcttgggaatatggtc rina wu ( seq id no 27 ) nf4s1a , cy ( h3b ) 58 3229 gtttcacttctagctggtct own ( seq id no 28 ) nf4s1b , on , cy 60 3376 caaacaggtggcaggaaacg own ( seq id no 29 ) nf4s2 fl , cy 56 3558 ccttcaacaaggcacaga rina wu ( seq id no 30 ) nf4s3 fl , cy 56 3756 actctaccaactgctctg rina wu ( seq id no 31 ) nf5s1 fl , cy 55 4011 gaacctccttcagatgac rina wu ( seq id no 32 ) nf5s3 fl , cy 55 4308 agaagaacatatgcggcc rina wu ( seq id no 33 ) nf5s3b rev cy 58 4450 gcacattgccgtcacttatg own ( seq id no 34 ) nf5s4 fl , cy 55 4658 ctaggcatcaggtacatg rina wu ( seq id no 35 ) nf6s1 fl , cy 55 4957 gtctccgcagtctatatc rina wu ( seq id no 36 ) nf6s1b cy 60 5071 cctgggaaactggctgagca own ( seq id no 37 ) nf6s2 fl , cy 55 5382 ggagtgtgaagccattgt rina wu ( seq id no 38 ) nf6s3 fl , cy 55 5670 tagtaagacgctggcagc rina wu ( seq id no 39 ) nf6s4 fl , cy 55 5934 ccttgggcagattacaga rina wu ( seq id no 40 ) nf7s1 fl , cy 55 6195 gatgctgtccttcaacaa rina wu ( seq id no 41 ) nf7s2 fl , cy 52 6544 gaaacagtcacagaagct rina wu ( seq id no 42 ) nf7s2b , cy 57 6576 ggaggcatgcatgagagata own ( seq id no 43 ) nf7s3 fl , cy 54 6841 cagccacttcttaataagg rina wu ( seq id no 44 ) nf7s4 fl , cy 58 7134 gcatccttcacctgctatt rina wu ( seq id no 45 ) nf8s1 fl , cy 55 7375 catggtgacccttcctat rina wu ( seq id no 46 ) nf8s2 fl , cy 52 7742 atgttctcttggatgaag rina wu ( seq id no 47 ) nf8s3 fl , cy 50 8119 gctgagcttattgttaag rina wu ( seq id no 48 ) [ 0275 ] table 3 nf1 primers g - dna : primers for amplification of all exons of the nf1 gene for ha name + la - bel program pcr length sequence ( 5 ′- 3 ′) sequence origin nfex1 . 1cy does not work cy - cccagcctccttgccaacgc shen et al ( seq id no 49 ) nfex1 . 2 gacccattccaccggcctgt ( seq id no 50 ) nfex2x . 1 95 ° 5 ′ 100 ng 340 bp tttcaatggcaagtaagt own ( seq id no 51 ) nfex2x . 2 ( 95 ° 45 ″- 54 ° 30 ″- 10 × cs gttatatccaaagtccaca own 72 ° 30 ″) × 35 ( seq id no 52 ) nfex2x . 1cy 72 ° 10 ′ 1upltaq nfex2 . 1fl 4 ° 25 μl fluo - tttaaggataaactgtt own ( nested ) ( seq id no 53 ) nfex3 . 1 hs 100 ng tttcacttttcagatgtgtgttg purandare et al ( seq id no 54 ) nfex3 . 2 ( 95 ° 1 ′- 60 ° 1 ′- 72 ° 1 ′) × 35 5 × dy 237 bp tggtccacatctgtactttg ( seq id no 55 ) nfex3 . 1cy 72 ° 10 ′ 1utaqbrl 4 ° tv = 25 μl nfex4ax . 1 95 ° 5 ′ 100 ng 517 bp ttaaatctaggtggtgtgt own ( seq id no 56 ) nfex4ax . 2 ( 95 ° 45 ″- 54 ° 30 ″- 10 × boeh aaactcatttctctggag own 72 ° 30 ″) × 35 ( seq id no 57 ) 72 ° 10 ′ 1upltaq 4 ° 25 μl nfex4b . 1wal 95 ° 5 ′ 100 ng ggcttcctgaagtgctgggat walace m ., pers . ( seq id no 58 ) comm . nfex4b . 2wal 95 ° 45 ″- 63 ° 25 ″- 10 × cs 305 bp ccagtttggtgttctagttcagca walace m ., pers . 72 ° 25 ″) × 35 ( seq id no 59 ) comm . 72 ° 10 ′ 1upltaq nfex4b . 1vfl 4 ° tv = 25μl fluo - gtgagataccacacctgtccc viskochil ( seq 60 °) ( seq id no 60 ) nfex4c . 1 hs 100 ng tttcctagcagacaactatcga purandare et al ( seq id no 61 ) nfex4c . 2 ( 95 ° 1 ′- 60 ° 1 ′- 72 ° 1 ′) × 45 5 × dy 283 bp catcaaaaaaaaaattttaataccag ( seq id no 62 ) nfex4c . 1fl 72 ° 10 ′ 1utaqbrl 4 ° tv = 25 μl nfex5 . 1fl hs 200 ng fluo - catgtggttctttatttataggc hoffmeyer et al ( seq id no 63 ) nfex5 . 2 ( 95 ° 1 ′- 52 ′ 1 ′- 72 ° 1 ′) × 35 10 × cs 113 bp tcaatcgtatccttaccagcc ( seq id no 64 ) 72 ° 10 ′ 1utaqbrl ( primers lay within exon ) 4 ° tv = 25 μl nfex6 . 1 hs 100 ng catgtttatcttttaaaaatgttgcc purandare et al ( seq id no 65 ) nfex6 . 2 ( 95 ° 1 ′- 64 ° 1 ′- 72 ° 1 ′) × 35 10 × boeh 299 bp ataatggaaataattttgccctcc ( seq id no 66 ) 72 ° 10 ′ 1utaqbrl 4 ° tv = 25 μl nfex7 . 1 hs 100 ng tgctataatattagctacatctgg purandare et al ( seq id no 67 ) nfex7 . 2 ( 95 ° 1 ′- 58 ° 1 ′- 72 ° 1 ′) × 35 5 × dy 373 bp cctatgaacttatcaacgaagag ( seq id no 68 ) nfex7 . 1cy 72 ° 10 ′ 1utaqbrl 4 ° tv = 25 μl nfex7 + 8 . 1 hs 100 ng tgctataatattagctacatctgg purandare et al ( seq id no 69 ) nfex7 + 8 . 2 ( 95 ° 1 ′- 55 ° 1 ′- 72 ° 1 ′) × 35 10 × boeh 880 bp ctagtctttctgtttataaaggat ( seq id no 70 ) 72 ° 10 ′ 1utaqbrl 4 ° tv = 25 μl nfex9 . 1br hs 100 ng tccgctgtggctcagaacac purandare et al ( seq id no 71 ) nfex9 . 2br ( 95 ° 1 ′- 63 ° 1 ′- 72 ° 1 ′) × 40 10 × boeh 335 bp agtagaagaggatgcacagcc ( seq id no 72 ) 72 ° 10 ′ 1utaqbrl 4 ° tv = 25 μl nfex9 . 1 95 ° 5 ′ 100 ng tttgacctcatttgtattactgag ( seq id no 73 ) nfex9 . 2 ( 95 ° 1 ′- 60 ° 1 ′- 72 ° 1 ′) × 35 10 × cs 249 bp agaaccttttgaaaccaagagtg purandare et al ( seq id no 74 ) 72 ° 10 ′ 1upltaq 4 ° tv = 25 μl nfex10a . 1 hs 100 ng acgtaattttgtactttttcttcc purandare et al ( seq id no 75 ) 1995 nfex10a . 2 ( 95 ° 1 ′- 58 ° 1 ′- 72 ° 1 ′) × 35 10 × boeh 222 bp caatagaaaggaggtgagattc ( seq id no 76 ) neex10a . 1fl 72 ° 10 ′ 1utaqbrl nfex10a . 2cy 4 ° tv = 25 μl nfex10c . 1 hs 100 ng cttggtaccctttagcagtcac purandare et al ( seq id no 77 ) nfex10c . 2 ( 95 ° 1 ′- 59 ° 1 ′- 72 ° 1 ′) × 35 5 × dy 379 bp ccttctttctccatggag ( seq id no 78 ) nfex10c . 1cy 72 ° 10 ′ 1utaqbrl 4 ° tv = 25 μl neex11 . 1 hs 100 ng gtactccagtgttatgtttacc purandare et al ( seq id no 79 ) 1995 nfex11 . 2 ( 95 ° 1 ′- 55 ° 1 ′- 72 ° 1 ′) × 40 5 × dy 190 bp taaagttgaaatttaaaaattaaagtac ( seq id no 80 ) nfex11 . 1cy 72 ° 10 ′ 1utaqbrl nfex11 . 2cy 4 ° tv = 25 μl nfex12a . 1 hs 100 ng acttgtattcattatgggagaatg mrc ( maynard ) ( seq id no 81 ) nfex12a . 2 ( 95 ° 1 ′- 60 ° 1 ′- 72 ° 1 ′) × 35 5 × dy 284 bp agtaatctctcaccattaccattc ( seq id no 82 ) nfex12a . 2cy 72 ° 10 ′ 1utaqbrl 4 ° tv = 25 μl nfex12b . 1 hs 100 ng tttctagtgaatctccttcaagt purandare et al ( seq id no 83 ) nfex12b . 2 ( 95 ° 1 ′- 59 ° 1 ′- 72 ° 1 ′) × 40 5 × dy 382 bp atgaaatttaccaaatttcattcag ( seq id no 84 ) nfex12b . 1cy 72 ° 10 ′ 1utaqbrl 4 ° tv = 25 μl nfex13x . 1cy 95 ° 5 ′ 100 ng cy - gagttattgtatgcggagac own ( seq id no 85 ) nfex13x . 2 ( 95 ° 1 ′- 55 ° 1 ′- 72 ° 1 ′) × 30 10 × cs , b , d 494 bp ttgaatttcccctgtaaac own ( seq id no 86 ) nfex13 . 1fl 72 ° 10 ′ 1upltaq fluo - cacagtttattgcattgttagat purandare et al ( seq bij °) ( seq id no 87 ) 4 ° tv = 25 μl nfex14 . 1 hs 100 ng tccttttgggtggagcttatc purandare et al ( seq id no 88 ) nfex14 . 2 ( 95 ° 1 ′- 59 ° 1 ′- 72 ° 1 ′) × 35 10 × boeh 286 bp tatacttgtaatatgcacgtatc ( seq id no 89 ) nfex14 . 1cy 72 ° 10 ′ 1utaqbrl 4 ° tv = 25 μl nfex15 . 1fl hs 100 ng fluo - tgtgatcaggaatagcttttgaa purandare et al ( seq id no 90 ) nfex15 . 2 ( 95 ° 1 ′- 57 ° 1 ′- 72 ° 1 ′) × 40 5 × dy 276 bp ttaacagataaaagtcaactttac ( seq id no 91 ) 72 ° 10 ′ 1utaqbrl 4 ° tv = 25 μl nfex16 . 1 hs 100 ng tggataaagcataatttgtcaagt purandare et al ( seq id no 92 ) nfex16 . 2 ( 95 ° 1 ′- 58 ° 1 ′- 72 ° 1 ′) × 35 5 × dy 549 bp tagagaaaggtgaaaaataagag ( seq id no 93 ) nfex16 . 1cy 72 ° 10 ′ 1utaqbrl nfex16seq 4 ° tv = 25 μl cy - ccagtcagtgacgtaaggg own cy ( seq id no 94 ) nfex17 . 1 hs 100 ng ctctgtgtgtttagatcagtca purandare et al ( seq id no 95 ) nfex17 . 2 ( 95 ° 1 ′- 55 ° 1 ′- 72 ° 1 ′) × 35 10 × boeh 319 bp tttatcaattactaccagtatcag ( seq id no 96 ) nfex17 . 1cy 72 ° 10 ′ 1utaqbrl 4 ° tv = 25 μl nfex18 . 1 hs 200 ng tagtaaggtagccagaagttgtgt mrc ( maynard ) ( seq id no 97 ) nfex18 . 2 ( 95 ° 1 ′- 60 ° 1 ′- 72 ° 1 ′) × 35 10 × boeh 320 bp atttacaaaaccctacattgctc ( seq id no 98 ) nfex18 . 1cy 72 ° 10 ′ 1utaqbrl 4 ° tv = 25 μl nfex19a . 1 hs 100 ng tcatgtcacttaggttatctgg purandare et al ( seq id no 99 ) nfex19a . 2 ( 95 ° 1 ′- 53 ° 1 ′- 72 ° 1 ′) × 40 5 × dy 272 bp tgtaattaagtagttataactctc ( seq id no 100 ) nfex19a . 1cy 72 ° 10 ′ 1utaqbrl cy - tcatgtcacttaggttatctgg ( seq id no 101 ) 4 ° tv = 25 μl nfex19b . 1x 95 ° 5 ′ 100 ng attaccttctccccatttga own ( seq id no 102 ) nfex19b . 2x ( 95 ° 1 ′- 55 ° 1 ′- 72 ° 1 ′) × 30 10 × boeh 371 bp ggctttatttgctttttgc ( seq id no 103 ) nfex19b . 1x 72 ° 10 ′ 1upltaq cy nfex19b . 2x 4 ° tv = 25 μl cy nfex20 . 1 hs 100 ng ccaccctggctgattatcg purandare et al ( seq id no 104 ) nfex20 . 2 ( 95 ° 1 ′- 62 ° 1 ′- 72 ° 1 ′) × 35 10 × dy 402 bp taatttttgcttctcttacatgc ( seq id no 105 ) nfex20 . 1cy 72 ° 10 ′ 1utaqbrl 4 ° tv = 25 μl nfex20 - 95 ° 5 ′ 100 ng ctatatcaggtaaaatcatgtccaac fahsold et al 21 . 1 ( seq id no 106 ) nfex20 - ( 95 ° 1 ′- 60 ° 1 ′- 72 ° 1 ′)* 35 10 × boeh gatttgctatgtgccagggac 21 . 2 ( seq id no 107 ) 72 ° 10 ′ 1upltaq 4 ° tv = 25 μl nfex21xx . 1 95 ° 5 ′ 100 ng gtcaaacttactcaatgcc own ( seq id no 108 ) nfex21xx . 2 ( 95 ° 45 ″- 54 ° 30 ″- 10 × boeh 542 bp caaccacttccctacag own 72 ° 30 ″) × 31 ( seq id no 109 ) nfex21xx . 1 72 ° 10 ′ 1upltaq cy nfex21x1 . cy 4 ° 25 μl cy - aactggcatgtaagagaag own ( seq id no 110 ) nfex22 . 1 hs 100 ng tgctactctttagcttcctac purandare et al ( seq id no 111 ) nfex22 . 2 ( 95 ° 1 ′- 58 ° 1 ′- 72 ° 1 ′) × 35 10 × cs 331 bp ccttaaaagaagacaatcagcc ( seq id no 112 ) nfex22 . 1cy 72 ° 10 ′ 1utaqbrl 4 ° tv = 25 μl nfex23an . 1 95 ° 5 ′ 100 ng 382 bp gattgggtctcaacatttc ( seq id no 113 ) nfex23an . 2 ( 95 ° 1 ′- 57 ° 1 ′- 72 ° 1 ′) × 35 10 × dy cy - aataggctgaagtgaagatantc own ? cy ( seq id no 114 ) 72 ° 10 ′ 1upltaq 4 ° tv = 25 μl nfex23 . 1 . 1 hs 100 ng tttgtatcattcattttgtgtgta purandare et al ( seq id no 115 ) nfex23 . 1 . 2 ( 95 ° 1 ′- 60 ° 1 ′- 72 ° 1 ′) × 35 5 × dy 282 bp aaaaacacggttctatgtgaaaag ( seq id no 116 ) nfex23 . 1 . 72 ° 10 ′ 1utaqbrl 1cy 4 ° tv = 25 μl nfex23 . 2 . 1 hs 100 ng cttaatgtctgtataagagtctc purandare et al ( seq id no 117 ) nfex23 . 2 . 2 ( 95 ° 1 ′- 52 ° 1 ′- 72 ° 1 ′) × 35 10 × cs 268 bp actttagattaataatggtaatctc ( seq id no 118 ) nfex23 . 2 . 1 72 ° 10 ′ 1utaqbrl cy 4 ° tv = 25 μl nfex24b . 1 hs 100 ng ttgaactctttgttttcatgtctt purandare et al ( seq id no 119 ) nfex24b . 2 ( 95 ° 1 ′- 57 ° 1 ′- 72 ° 1 ′) × 35 5 × dy 267 bp ggaatttaagatagctagattatc ( seq id no 120 ) nfex24mrc , 72 ° 10 ′ 1utaqbrl not ok 4 ° tv = 25 μl nfex25b . 1 hs 100 ng aatataataattatatttgggaaggt purandare et al ( seq id no 121 ) nfex25b . 2 ( 95 ° 1 ′- 57 ° 1 ′- 72 ° 1 ′) × 35 10 × boeh 338 bp gaaaatatttgattcaaacagagc ( seq id no 122 ) nfex25b . 1cy 72 ° 10 ′ 1utaqbrl cy - aatataataattatatttgggaaggt ( seq id no 123 ) 4 ° tv = 25 μl nfex25l . 1 hs 100 ng cattttattatagcagatgtc own ( seq id no 124 ) nfex25l . 2 ( 95 ° 45 ″- 57 ° 45 ″- 5 × dy 534 bp acttacacaggaacttcat 72 ° 45 ″) × 35 ( seq id no 125 ) 72 ° 10 ′ 1utaqbrl 4 ° tv = 25 μl nfex26 . 1fl hs 100 ng fluo - gctttgtctaatgtcaagtcac purandare et al ( seq id no 126 ) nfex26 . 2 ( 95 ° 1 ′- 58 ° 1 ′- 72 ° 1 ′) × 35 5 × dy 342 bp ttaaacggagagtgttcactatc ( seq id no 127 ) 72 ° 10 ′ 1utaqbrl 4 ° tv = 25 μl nfex27a . 1 hs 100 ng gttacaagttaaagaaatgtgtag purandare et al ( seq id no 128 ) nfex27a . 2 ( 95 ° 30 ″- 62 ° 30 ″- 10 × boeh 300 bp ctaacaagtggcctggtggcaaac 72 ° 30 ″) × 45 ( seq id no 129 ) nfex27a . 1fl 72 ° 5 ′ 1utaqbrl 4 ° tv = 25 μl nfex27b . 1fl does not work fluo - tttattgtttatccaattatagactt purandare et al ( seq id no 130 ) nfex27b . 2 tcctgttaagtcaactgggaaaaac ( seq id no 131 ) nfex28 aber 95 ° 5 ′ 200 ng cactgctaataatctttgtcttttttgtc abernathy . 1 ( seq id no 132 ) nfex28 aber ( 95 ° 1 ′- 65 ° 1 ′- 72 ° 1 ′) × 40 10 × boeh 501 bp cgtttacaaaacacagactggaactta . 2 ( seq id no 133 ) nfex28 aber 72 ° 10 ′ 1upltaq . 1cy nfex28 aber 4 ° tv = 25 μl . 2cy nfex28 . 1fl ttccttaggttcaaaactggtca own ( nested ) ( seq id no 134 ) nfex29l1 h . s . 100 ng tacaatggtgggaactc own ( seq id no 135 ) nfex29 . l2 ( 95 ° 1 ′- 56 ° 45 ″- 10 × cs 567 bp atattaaggtagaggctgttt own 72 ° 45 ″) × 35 ( seq id no 136 ) nfex29l1cy 72 ° 10 ′ 1utaqbrl nfex29l2cy 4 ° tv = 25 μl nfex29 . 1cy cy - attcttctccacttcaccc mrc ( shen ) ( nested ) ( seq id no 137 ) nfex29 . 2cy cy - cccaaatcaaactgaagaga mrc ( nested ) ( seq id no 138 ) nfex30x . 1 95 ° 5 ′ 100 ng 350 bp tggaactataaggaaaaa own ( seq id no 139 ) nfex30x . 2cy ( 95 ° 1 ′- 50 ° 1 ′- 10 × cs cy - aaagtcttcactggaaa own 72 ° 1 ′ 30 ″) × 30 ( seq id no 140 ) 72 ° 10 ′ 1upitaq nfex30 . 2fl 4 ° tv = 25 μl ( 3 )( nested ) nfex31 . 1 ( 4 ) hs 100 ng ataattgttgatgtgattttcattg cawthon ( seq id no 141 ) nfex31 . 2f1 ( 93 ° 1 ′- 63 ° 1 ′- 10 × koh 424 bp fluo - aattttgaaccagatgaagag ( 4 ) 72 ° 1 ′ 30 ″) × 31 ( seq id no 142 ) nfex31 . 1cy 72 ° 5 ′ 1utaqbrl 4 ° tv = 25 μl nfex32 . 1 hs 100 ng ggtagagtgattaaaacatg ( seq id no 143 ) nfex32 . 2 ( 95 ° 1 ′- 51 ° 1 ′- 72 ° 1 ′) × 35 10 × cs 220 bp tatgctatagtacagaaggc rina wu ( seq id no 144 ) nfex32 . 1cy 72 ° 10 ′ 1utaqbrl 4 ° tv = 25 μl nfex33 . 1fl hs 100 ng fluo - catatctgttttatcatcaggagg ( seq id no 145 ) nfex33 . 2 ( 95 ° 1 ′- 61 ° 1 ′- 72 ° 1 ′) × 35 5 × dy 462 bp aagtaaaatggagaaaggaactgg cawthon ( seq id no 146 ) nfex33 . 2cy 72 ° 10 ′ 1utaqbrl 4 ° tv = 25 μl nfex34 . 1fl hs 100 ng fluo - caaaatgaaacatggaactttaga ( seq id no 147 ) nfex34 . 2 ( 95 ° 1 ′- 57 ° 1 ′- 72 ° 1 ′) × 35 5 × dy 400 bp taagcattaagtacaaatagcaca cawthon ( seq id no 148 ) 72 ° 10 ′ 1utaqbrl 4 ° tv = 25 μl nfex35 . 1fl hs 100 ng fluo - agaagccaaaatgataagaa ( seq id no 149 ) nfex35 . 2 ( 95 ° 1 ′- 52 ° 1 ′- 72 ° 1 ′) × 35 10 × cs 495 bp acccaaagacaacaagag ( seq id no 150 ) 72 ° 10 ′ 1utaqbrl 4 ° tv = 25 μl nfex36 . 1 hs 100 ng ggaccagtggacagaac own ( seq id no 151 ) nfex36 . 2fl ( 95 ° 1 ′- 55 ° 1 ′- 72 ° 1 ′) × 35 10 × boeh 345 bp fluo - atatgctttacaacttgagaa ( seq id no 152 ) 72 ° 10 ′ 1utaqbrl 4 ° tv = 25 μl nfex37 . 1 hs 100 ng tacattaagctagctaccaa own ( seq id no 153 ) nfex37 . 2fl ( 95 ° 1 ′- 54 ° 1 ′- 72 ° 1 ′) × 35 10 × boeh 460 bp fluo - cgcttgagaacatactatcc ( seq id no 154 ) 72 ° 10 ′ 1utaqbrl 4 ° tv = 25 μl nfex38 . 1fl hs 100 ng fluo - ccagctaacagtgtctt own ( seq id no 155 ) nfex38 . 2 ( 95 ° 1 ′- 50 ° 1 ′- 72 ° 1 ′) × 35 10 × boeh 474 bp aaggaaatatactcacaataa ( seq id no 156 ) 72 ° 10 ′ 1utaqbrl 4 ° tv = 25 μl nfex39 . 1fl hs 100 ng fluo - gaacctaatcaaccatctc ( seq id no 157 ) nfex39 . 2 ( 95 ° 1 ′- 52 ° 1 ′- 72 ° 1 ′) × 35 5 × dy 286 bp ttgcatttaaagtaagacat ( seq id no 158 ) 72 ° 10 ′ 1utaqbrl 4 ° tv = 25 μl nfex40 . 1fl hs 100 ng fluo - cctttccttgcagagttgtta ( seq id no 159 ) nfex40 . 2 ( 95 ° 1 ′- 57 ° 1 ′- 72 ° 1 ′) × 35 5 × dy 371 bp caccactaaaggactagactgt ( seq id no 160 ) 72 ° 10 ′ 1utaqbrl 4 ° tv = 25 μl nfex41 . 1fl hs 100 ng fluo - ttcatcctgttttaagtcacacttg ( seq id no 161 ) nfex41 . 2 ( 95 ° 1 ′- 61 ° 1 ′- 72 ° 1 ′) × 40 10 × boeh 273 bp ttgcctccattagttggaaaattg abernathy et al ( seq id no 162 ) nfex41 . 1 72 ° 10 ′ 1utaqbrl ttcatcctgttttaagtcacacttg ( seq id no 163 ) 4 ° tv = 25 μl nfex42 . 1fl hs 100 ng fluo - cttggaaggagcaaacgatggttg ( seq id no 164 ) nfex42 . 2 ( 95 ° 1 ′- 61 ° 1 ′- 72 ° 1 ′) × 35 10 × boeh 356 bp caaaaactttgctacactgacatgg abernathy et al ( seq id no 165 ) 72 ° 10 ′ 1utaq brl 4 ° tv = 25 μl nfex43 . 1fl hs 100 ng fluo - agacactgtagttaatgaacttgc ( seq id no 166 ) nfex43 . 2 ( 95 ° 1 ′- 60 ° 1 ′- 72 ° 1 ′) × 35 10 × boeh 224 bp catgtactctcccaccttattttc abernathy et al ( seq id no 167 ) nfex43 . 1cy 72 ° 5 ′ taq st cy - agacactgtagttaatgaacttgc 1 / 20 ( seq id no 168 ) 4 ° tv = 25 μl nfex44 . 1fl hs 100 ng fluo - cacgttaattccctatcttgctgc f shen et al ( seq id no 169 ) nfex44 . 2 ( 95 ° 1 ′- 65 ° 1 ′- 72 ° 1 ′) × 35 10 × boeh 200 bp taaaaatttgagggtgggggactc r ? ( seq id no 170 ) 72 ° 5 ′ taq st 1 / 20 4 ° tv = 25 μl nfex45 . 1fl hs 100 ng fluo - catgaataggatacagtcttctac ( seq id no 171 ) nfex45 . 2 ( 95 ° 1 ′- 60 ° 1 ′- 72 ° 1 ′) × 35 10 × boeh 269 bp cacattactgggtaagcatttaac abernathy ( seq id no 172 ) nfex45 . 2fl 72 ° 5 ′ 1utaqbrl nfex45 . 1bio 4 ° tv = 25 μl nfex46 . 1fl hs 100 ng fluo - gggaatgtatattatgttttccac ( seq id no 173 ) nfex46 . 2 ( 95 ° 1 ′- 60 ° 1 ′- 72 ° 1 ′) × 35 5 × dy 275 bp atgttaggaagttcatcaaccatc abernathy ( seq id no 174 ) nfex46 . 2cy 72 ° 5 ′ 1utaqbrl 4 ° tv = 25 μl nfex47 . 1 hs 100 ng ctgttacaattaaaagataccttgc mrc ( upadhyaya ) ( seq id no 175 ) nfex47 . 2 ( 95 ° 1 ′- 60 ° 1 ′- 72 ° 2 ′) × 35 5 × dy 185 bp tgtgtgttcttaaagcaggcatac mrc ( upadhyaya ) ( seq id no 176 ) 72 ° 10 ′ 1utaqbrl 4 ° tv = 25 μl nfex48 . 1 hs 100 ng ttttggcttcagatggggatttac mrc ( upadhyaya ) ( seq id no 177 ) nfex48 . 2 ( 95 ° 1 ′- 65 ° 1 ′- 72 ° 1 ′) × 35 5 × dy 352 bp aagggaattcctaatgttggtgtc mrc ( upadhyaya ) ( seq id no 178 ) 72 ° 10 ′ 1utacbrl 4 ° tv = 25 μl nfex48a . 1fl hs 100 ng fluo - atctagtatctaattgtatttcacc li et al ( seq id no 179 ) nfex48a . 2 ( 95 ° 1 ′- 60 ° 1 ′- 72 ° 2 ′) × 40 5 × dy gcagactgagcttacagggac ( seq id no 180 ) 72 ° 10 ′ 1utaqbrl 4 ° tv = 25 μl legend labels fl fluoresceïn label cy cy5 label bio biothine label buffers dy dynazyme buffer 5x buffer contains 50 mm tris - hcl ( ph8 . 8 ) 250 mm kcl 0 . 5 % triton x - 100 7 . 5 mm mgcl 2 boeh boehringer buffer 10x buffer contains 100 mm tris hcl 15 mm mgcl 2 500 mm kcl ph 8 . 3 ( 20 ° c .) koh kohan buffer 10x buffer contains 166 mm ( nh 4 ) 2 so 4 670 mm tris - hcl ( ph8 . 8 ) 67 mm mgcl 2 100 mm β - mercaptoethanol 67 μm edta 1700 μg bsa / ml 5 % dmso cs ac - syvänen buffer 10x buffer contains 200 mm tris - hcl ( ph8 . 8 ) 15 mm mgcl 2 150mm ( nh 4 ) 2 so 4 1 % triton x - 100 0 . 1 % gelatin others hs hot start 95 ° c . 5 min , 80 ° c . 5 min while adding the polymerase 1 utaqbrl 1 unit of taq polymerase from brl 1upltaq 1 unit of platinum taq from brl references shen et al neurofibromatosis type 1 ( nf1 ): the search for mutations by pcr - heteroduplex analysis on hydrolink gels . hum mol gen , 1993 , vol2 , no 11 , 1861 - 1864 purandare et al identification of neurofibromatosis 1 ( nf1 ) homologous loci by direct sequencing , fluorescence in situ hybridization , and pcr amplification of somatic cell hybrids . genomics ; 1995 : 30 , 476 - 485 wallace et al personal communication sawada et al identification of nf1 mutation in both alleles of a dermal neurofibroma . nat genet ; 1996 sep : 14 ( 1 ): 110 - 2 hoffmeyer et al an rsal polymorphism in the transcribed region of the neurofibromatosis ( nf1 )- gene . hum genet ; 1994 : 93 : 481 - 482 fahsold et al minor lesion mutational spectrum of the nf1 gene does not explain its high mutability but points to a functional domain upstream of the gap - related domain . am j hum genet ; 2000 : 66 : 790 - 818 abernathy et al nf1 mutation analysis using a combined heteroduplex / sscp approach . hum mut 1997 ; 9 : 548 - 554 cawthon et al a major segment of the neurofibromatosis type 1 gene : cdna , genomic stucture , and point mutations . cell ; 1990 : 62 : 193 - 201 mrc medical research counsil , uk , hgmp primers database rina wu et al phd dissertation kul li et al genomic organization of the neurofibromatosis 1 gene ( nf1 ). genomics 1995 ; 25 : 9 - 18 [ 0276 ] table 4 ha - pcr primers and conditions primer sequences fragment exon ( 5 ′ to 3 ′) size t a (° c .) 1 2 tttcaatggcaagtaagt 340 54 gttatatccaaagtccaca 4a ttaaatctaggtggtgtgt 517 54 aaactcatttctctggag 13 gagttattgtatgcggagac 494 55 ttgaatttcccctgtaaac 19b attaccttctccccatttga 371 55 ggctttatttgctttttgc 21 gtcaaacttactcaatgcc 542 54 caaccacttccctacag 23a gattgggtctcaacatttc 382 57 aataggctgaagtgaagatantc 29 tacaatggtgggaactc 567 56 atattaaggtagaggctgttt 30 tggaactataaggaaaaa 350 50 aaagtcttcactggaaa 32 ggtagagtgattaaaacatg 220 51 tatgctatagtacagaaggc 35 agaagccaaaatgataagaa 495 52 acccaaagacaacaagag 36 ggaccagtggacagaac 345 55 atatgctttacaacttgagaa 37 tacattaagctagctaccaa 460 54 cgcttgagaacatactatcc 38 ccagctaacagtgtctt 474 50 aaggaaatatactcacaataa 39 gaacctaatcaaccatctc 286 52 ttgcatttaaagtaagacat 40 cctttccttgcagagttgtta 371 57 caccactaaaggactagactgt 44 cacgttaattccctatcttgctgc 200 65 taaaaatttgagggtgggggactc [ 0277 ] table 5 mutations detected by analysis of the whole coding region of the nf1 gene by ptt and ha ; analysis of 67 unrelated nf1 patients . codon patient genomic mutation 1 effect on cdna codon change number exon / intron nf - 004 247c & gt ; t 247c & gt ; t q & gt ; x 83 3 nf - 023 278g & gt ; a 278g & gt ; a c & gt ; y 93 3 nf - 036 560g & gt ; a 560g & gt ; a c & gt ; y 187 4b nf - 056 574c & gt ; t 574c & gt ; t r & gt ; x 192 4b nf - 029 603 - 604inst 603 - 604inst frameshift 202 4c nf - 027 910c & gt ; t 910c & gt ; t ; r & gt ; x 304 7 888del174 nf - 064 910c & gt ; t 910c & gt ; t ; r & gt ; x 304 7 888del174 nf - 024 987 - 988insa 987 - 988insa frameshift 330 7 nf - 048 1275g & gt ; a 1275g & gt ; a w & gt ; x 425 10a nf - 042 1318c & gt ; t 1318c & gt ; t r & gt ; x 440 10a nf - 057 1318c & gt ; t 1318c & gt ; t r & gt ; x 440 10a nf - 030 1381c & gt ; t 1381c & gt ; t r & gt ; x 461 10a nf - 026 1381c & gt ; t 1381c & gt ; t r & gt ; x 461 10a nf - 017 1466a & gt ; g 1465del62 y & gt ; c 489 10b nf - 012 1466a & gt ; g 1465del62 y & gt ; c 489 10b nf - 016 1465 - 1466insc 1466insc y & gt ; x 489 10b nf - 033 1570g & gt ; t 1570g & gt ; t e & gt ; x 524 10c nf - 052 1607c & gt ; a 1607c & gt ; a s & gt ; x 536 10c nf - 045 ivs12a + 1g & gt ; t 1641del204 none na ivs12a nf - 034 2033 - 2034insc 2033insc frameshift 678 13 nf - 035 2033 - 2034insc 2033insc frameshift 678 13 nf - 010 2540t & gt ; c 2540t & gt ; c l & gt ; p 847 16 nf - 051 ivs16 + 2del6 2617del233 frameshift na ivs16 nf - 028 ivs16 − 6del4 2850del140 frameshift na ivs16 nf - 014 2875c & gt ; t 2875c & gt ; t q & gt ; x 959 17 nf - 011 2887c & gt ; t 2887c & gt ; t q & gt ; x 963 17 nf - 001 2970 - 2972delaat 2970 - 2972delaat delm 991 17 nf - 053 3193delc 3193delc frameshift 1065 19a nf - 063 3277g & gt ; a 3277g & gt ; a , 3274del40 v & gt ; m ; 1093 19b frameshift nf - 025 ivs19b − 3c & gt ; g 3314del182 frameshift na ivs19b nf - 039 3367g & gt ; t 3367g & gt ; t e & gt ; x 1123 20 nf - 046 3520c & gt ; t 3520c & gt ; t q & gt ; x 1174 21 nf - 059 3826c & gt ; t 3826c & gt ; t r & gt ; x 1276 22 nf - 041 4084c & gt ; t 4084c & gt ; t r & gt ; x 1362 23 . 2 nf - 054 4084c & gt ; t 4084c & gt ; t r & gt ; x 1362 23 . 2 nf - 044 ivs26 − 2a & gt ; t 4515 - 14 ins 14 ; frameshift na ivs26 4515 - 17ins17 nf - 021 4537c & gt ; t 4537c & gt ; t r & gt ; x 1513 27a nf - 006 4537c & gt ; t 4537c & gt ; t r & gt ; x 1513 27a nf - 058 ivs27b − 2a & gt ; t 4772del433 ; 4772del293 frameshift na ivs27b nf - 049 5033delg 5033delg frameshift 1678 28 nf - 009 5264c & gt ; g 5264c & gt ; g s & gt ; x 1755 29 nf - 050 5294c & gt ; a 5215del90 ( 5294c & gt ; a ???) s & gt ; x 1765 29 nf - 037 5546g & gt ; a 5205del341 ; 5205del544 r & gt ; q 1849 29 nf - 038 5546g & gt ; a 5205del341 ; 5205del544 r & gt ; q 1849 29 nf - 047 5567delt 5567delt frameshift 1856 30 nf - 031 5798delc 5798delc frameshift 1933 31 nf - 060 5839c & gt ; t 5839c & gt ; t r & gt ; x 1947 31 nf - 043 5896c & gt ; t 5896c & gt ; t q & gt ; x 1966 31 nf - 015 6577delgaggta 6364del215 frameshift 2193 34 nf - 040 6709c & gt ; t 6709c & gt ; t r & gt ; x 2237 36 nf - 008 6789 - 6792delttac 6789 - 6792delttac frameshift 2263 37 nf - 019 6792c & gt ; a 6756del102 y & gt ; x 2264 37 nf - 003 6792c & gt ; g 6756del102 y & gt ; x 2264 37 nf - 032 6858g & gt ; c 6756del102 k & gt ; n 2286 37 nf - 013 7096 - 7101del6 7096 - 7101del6 delnf 2366 39 nf - 022 7096 - 7101del6 7096 - 7101del6 delnf 2366 39 nf - 005 ivs39 − 12t & gt ; a 7126del132 ; na na ivs39 7127 - 10ins10 nf - 002 7201a & gt ; t 7201a & gt ; t k & gt ; x 2401 40 nf - 055 7285c & gt ; t 7285c & gt ; t r & gt ; x 2429 41 nf - 061 7486c & gt ; t 7486c & gt ; t r & gt ; x 2496 42 nf - 020 7884 - 7885delgt 7884 - 7885delgt frameshift 2628 45 nf - 007 8016dela 8016dela frameshift 2672 46 ptt patient type / effect ( frag ) 2 cg 3 s / f 4 previously described nf - 004 nonsense +( f1 ) no f osborn et al ., 1999 nf - 023 missense −( ha ) no f novel nf - 036 missense −( ha ) no f novel nf - 056 nonsense +( f1 ) yes s fahsold et al ., in press nf - 029 frameshift +( f1 ) na f novel nf - 027 nonsense : truncation due to stopcodon ; +( f1 ) yes s hoffmeyer et al ., 1998 splice : intact 3 &# 39 ; and 5 &# 39 ; ss , no cryptic or novel ss , if skip e7 nf - 064 identical as nf - 027 +( f1 ) yes f hoffmeyer et al ., 1998 nf - 024 frameshift +( f1 ) na f novel nf - 048 nonsense +( f1 ) no s novel nf - 042 nonsense +( f1 ) yes s heim et al ., 1995 nf - 057 nonsense +( f1 ) yes s heim et al ., 1995 nf - 030 nonsense +( f1 ) yes s fahsold et al ., in press nf - 026 nonsense +( f1 ) yes s fahsold et al ., in press nf - 017 splice : intact wt 5 &# 39 ; ss , creation novel +( f1 ) no f messiaen et al ., 1999 5 &# 39 ; ss , skip last 62 nt e10b forms immediate stopcodon nf - 012 identical as nf - 017 +( f1 ) no s messiaen et al ., 1999 nf - 016 nonsense +( f1 ) na f novel nf - 033 nonsense +( f1 ) no s novel nf - 052 nonsense +( f1 ) no f novel nf - 045 splice : inactive 5 &# 39 ; ss , if skip e11 + 12a +( f2 ) no s abernathy et al ., 1997 nf - 034 frameshift +( f2 ) nao s heim et al ., 1995 nf - 035 frameshift +( f2 ) na s heim et al ., 1995 nf - 010 missense −( ha ) no f messiaen et al ., 1998 nf - 051 splice : inact 5 &# 39 ; ss , activ cryptic 5 &# 39 ; ss skip +( f2 ) na s novel last 233 nt e16 nf - 028 splice : inactiv 3 &# 39 ; ss , oof skip e17 +( f2 ) na f novel nf - 014 nonsense +( f2 ) no f novel nf - 011 nonsense +( f2 ) no f novel nf - 001 amino acid deletion −( ha ) na f shen et al ., 1993 nf - 053 frameshift +( f2 ) na s novel nf - 063 splice : intact wt 5 &# 39 ; ss , creation novel +( f2 ) no f novel 5 &# 39 ; ss , skip last 40 nt e19b nf - 025 splice : inactiv 3 &# 39 ; ss , oof skip e20 +( f2 ) no f novel nf - 039 nonsense +( f2 ) no s novel nf - 046 nonsense +( f2 ) no s novel nf - 059 nonsense +( f3 ) yes s heim et al ., 1995 nf - 041 nonsense +( f3 ) yes s upadhyaya et al ., 1997 nf - 054 nonsense +( f3 ) yes s upadhyaya et al ., 1997 nf - 044 splice : inactiv 3 &# 39 ; ss , activ 2 cryptic 3 &# 39 ; ss +( f3 ) no s novel sites leading to 2 different oof insertions nf - 021 nonsense +( f3 ) yes s side et al ., 1995 nf - 006 nonsense +( f3 ) yes s side et al ., 1995 nf - 058 splice : inactiv 3 &# 39 ; ss , activ cryptic 3 &# 39 ; ss , +( f3 ) no f novel oof skip e28 ; oof skip first 293 nt e28 nf - 049 frameshift +( f3 ) na f novel nf - 009 nonsense +( f4 ) no s novel nf - 050 splice : intact 3 &# 39 ; ss , creation novel 3 &# 39 ; ss , if +( f4 ) yes f novel skip first 90 nt e29 nf - 037 splice : inact 5 &# 39 ; ss , oof skip e29 and +( f4 ) yes f ars et al ., 2000 e29 + 30 nf - 038 identical as nf - 037 +( f4 ) yes f ars et al ., 2000 nf - 047 frameshift +( f4 ) na s novel nf - 031 frameshift +( f4 ) na s novel nf - 060 nonsense +( f4 ) yes f cawthon et al ., 1990 nf - 043 nonsense +( f4 ) no f novel nf - 015 splice : inactiv 5 &# 39 ; ss , oof skip e34 +( f4 ) na s novel nf - 040 nonsense +( f4 ) yes s fahsold et al ., in press nf - 008 frameshift +( f4 ) na s robinson et al ., 1995 nf - 019 splice : intact 3 &# 39 ; and 5 &# 39 ; ss , no cryptic or +( f4 ) no f messiaen et al ., 1997 novel ss , if skip e37 nf - 003 identical as nf - 019 +( f4 ) no f messiaen etal , 1997 nf - 032 splice : inactiv 5 &# 39 ; ss , if skip e37 +( f4 ) no f novel nf - 013 amino acid deletion −( ha ) na s abernathy et al ., 1994 nf - 022 amino acid deletion −( ha ) na f abernathy et al ., 1994 nf - 005 splice : inactiv 3 &# 39 ; ss , if skip e40 ; activ +( f5 ) no f novel cryptic 3 &# 39 ; ss , oof ins last 10 nt ivs39 nf - 002 nonsense +( f5 ) no s novel nf - 055 nonsense +( f5 ) yes s fahsold et al ., in press nf - 061 nonsense +( f5 ) yes s purandare et al ., 1994 nf - 020 frameshift +( f5 ) na s novel nf - 007 frameshift +( f5 ) na s novel [ 0278 ] table 6 comparison of the sensitivity of detecting 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 . with without puromycin puromycin ratio genomic ratio mutant / patient exon mutation mutant / wild - type wild - type nf - 056 4b r192x 0 . 51 0 . 80 nf - 064 7 r304x 0 . 34 1 . 00 nf - 057 10a r440x 0 . 60 1 . 00 nf - 012 10b y489c 0 . 60 1 . 00 nf - 016 10b 1466insc 0 . 65 1 . 00 nf - 033 10c e524x 0 . 35 1 . 00 nf - 035 13 2033insc 0 . 35 0 . 76 nf - 051 ivs16 2850 + 2deltaaagt 0 . 84 1 . 00 nf - 063 19b v1093m 0 . 58 0 . 92 nf - 039 20 e1123x 0 . 55 1 . 00 nf - 046 21 q1174x 0 . 32 0 . 72 nf - 002 40 k2401x 0 . 28 0 . 75 nf - 020 42 7884delgt 0 . 29 0 . 91 [ 0279 ] table 7 consensus values ( cv ) and splice site scores ( sss ) of splice sites ( ss ) involved in splicing mutations in the nf1 gene consensus values according to shapiro splice site scores and senepathy according to nnpss splice site cv cv cvc sss - sss - sss - ( mutation ) n m ry n m cry comment mutations at 5 &# 39 ; ss 6577delgaggta tg ( delgaggta ) tagaag 0 . 70 0 . 33 na 0 . 41 / na skipping e34 ivs12a + 1g & gt ; t ag ( g & gt ; t ) taagc 0 . 94 0 . 76 na 1 . 00 / na skipping e11 + e12a ivs16 + 2delaaagtg aggt ( delaaagtg ) ttct 0 . 81 0 . 64 0 . 82 0 . 92 0 . 12 0 . 95 activ . cryptic 5 &# 39 ; ss 233 nt upst . 5546g & gt ; a c ( g & gt ; a ) gtaggt 0 . 81 0 . 69 na 0 . 97 0 . 10 na skipping e29 / e29 + e30 ( r1849q ) 6858g & gt ; c a ( g & gt ; c ) gtaatt 0 . 86 0 . 72 na 0 . 99 0 . 73 na skipping e37 ( k2286n ) mutations at 3 &# 39 ; ss ivs26 − 2a & gt ; t tttgctgtatct ( a & gt ; t ) gg 0 . 82 0 . 66 ivs2 0 . 49 / ivs2 activ . cryptic 3 &# 39 ; ss 14 nt 6 - 6 - upst . and 17 nt upst . 14 : 0 . 84 14 : 0 . 92 ivs2 ivs2 6 - 6 - 17 :/ 17 : 0 . 81 ivs27b − 2a & gt ; t gtcattttcctt ( a & gt ; t ) gg 0 . 84 0 . 68 0 . 84 1 . 00 / 0 . 84 skipping e28 and activ . cryptic 3 &# 39 ; ss 293 nt downst ivs16 - 6delcttt tatttgttcttt ( delcttt ) agg 0 . 89 0 . 89 na 0 . 99 0 . 97 na skipping e17 ivs19bc & gt ; g tttttatttct ( c & gt ; g ) aga 0 . 96 0 . 84 na 0 . 96 0 . 04 na skipping e20 ivs39 − 12t & gt ; a tt ( t & gt ; a ) gttttttgtagg 0 . 90 0 . 89 0 . 79 1 . 00 0 . 99 0 . 92 skipping e40 and use of novel created 5 &# 39 ; ss 10 nt downst . splice site cv cv cvn sss - sss - sss - comment ( mutation ) n m ov n m nov creation of novel 5 &# 39 ; ss y489c ct ( a & gt ; g ) taagt 0 . 79 na 0 . 79 0 . 86 na 0 . 97 novel 5 &# 39 ; ss 62 nt upst . v1093m tggt ( g & gt ; a ) tgg 0 . 73 na 0 . 76 0 . 13 na 0 . 27 novel 5 &# 39 ; ss 40 nt upst . creation of novel 3 &# 39 ; ss s1765x aatgacatttattatgctt ( c & gt ; a ) gga 0 . 90 na 0 . 83 0 . 81 na 0 . 18 novel 3 &# 39 ; ss 90 nt downst . lys tyr ser thr asp asp phe asp gln arg ile leu tyr glu tyr leu lys tyr ser ser asp arg gly glu thr arg val leu tyr gln tyr leu