Human Molecular Genetics, 2000, Vol. 9, No. 2 237-247
© 2000 Oxford University Press
Mutations affecting mRNA splicing are the most common molecular defects in patients with neurofibromatosis type 1
Medical and Molecular Genetics Center—IRO, Hospital Duran i Reynals, Avia. Castelldefels, Km 2.7, L’Hospitalet de Llobregat, 08907 Barcelona, Spain
Received 6 September 1999; Revised and Accepted 16 November 1999.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Neurofibromatosis type 1 (NF1) is one of the most common inherited disorders in humans and is caused by mutations in the NF1 gene. To date, the majority of the reported NF1 mutations are predicted to result in protein truncation, but very few studies have correlated the causative NF1 mutation with its effect at the mRNA level. We have applied a whole NF1 cDNA screening methodology to the study of 80 unrelated NF1 patients and have identified 44 different mutations, 32 being novel, in 52 of these patients. Mutations were detected in 87% of the familial cases, but in 51% of the sporadic ones. At least 15 of the 80 NF1 patients (19%) had recurrent mutations. The study shows that in 50% of the patients in whom the mutations were identified, these resulted in splicing alterations. Most of the splicing mutations did not involve the conserved AG/GT dinucleotides of the splice sites. One frameshift, two nonsense and two missense mutations were also responsible for alterations in mRNA splicing. The location and type of mutation within the NF1 gene, and its putative effect at the protein level, do not indicate any relationship to any specific clinical feature of NF1. The high proportion of aberrant spliced transcripts detected in NF1 patients stresses the importance of studying mutations at both the genomic and RNA level. It is possible that part of the clinical variability in NF1 could be due to mutations affecting mRNA splicing, which is the most common molecular defect in NF1.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Neurofibromatosis type 1 (NF1) (MIM 162200) is one of the most common inherited disorders in humans with a prevalence of ~1 in 3000 individuals. NF1 is an autosomal dominant disorder fully penetrant at the age of 5 years, but with a variable clinical expression, even among members of the same family. The main clinical features of NF1 are café au lait spots (CLSs), cutaneous neurofibromas and Lisch nodules (1,2). The NF1 gene was mapped to chromosome 17q11.2 and was positionally cloned in 1990 (3–5). It spans ~350 kb of genomic DNA, contains 60 exons (6,7) and is transcribed ubiquitously to an mRNA of 11–13 kb that encodes for a protein, neurofibromin, of 2818 amino acids (8). The only region of neurofibromin with a well-defined function and structure is a central domain with a high similarity to ras-specific GTPase-activating proteins (GAPs), called the GAP-related domain (GRD), which downregulates ras activity (9–11).
The NF1 gene has one of the highest mutation rates described for any human disorder (~1 x 10–4/gamete/generation), with the result that ~30–50% of NF1 cases represent new mutations (1,2). Despite the fact that the NF1 gene was identified ~9 years ago, information on the spectrum of mutations that cause NF1 is still very limited. So far, 255 different mutations (NNFF International NF1 Mutation Analysis Consortium, 29 March 1999; http://www.nf.org/nf1gene/nf1gene.home.html ) have been reported in the NF1 gene. The search for NF1 mutations has probably been hampered by: (i) the large size of the gene; (ii) the existence of homologous pseudogenes (12); and (iii) the lack of mutational hot spots. Several methods have been employed to search for NF1 mutations, but they have mainly been used for the analysis of a few exons (13–17). In addition, approaches that allow a rapid screening of the entire NF1 coding region have been employed (18–20). The protein truncation test (PTT) has been applied with an efficiency of ~70% (18,20), but PTT is a radioactive technique and is better performed using RNA from lymphoblastoid cell lines.
It has been described that a significant fraction (15%) of disease-causing mutations in mammalian genes affect mRNA splicing (21). Moreover, aberrations in NF1 pre-mRNA processing have been suggested to play an important role in the pathogenesis of NF1 (22). To date, the majority (60–70%) of NF1 mutations reported to the NNFF Consortium (29 March 1999) are predicted to result in protein truncation. Unfortunately, most of these NF1 mutations have been studied only in genomic DNA and their effect at the mRNA level has not been reported.
To gain knowledge on the spectrum of NF1 mutations in NF1 patients, we have developed a rapid and efficient strategy to screen the entire NF1 coding region. The method combines single-strand conformation polymorphism (SSCP) with heteroduplex (HD) analysis of NF1 cDNA (cDNA-SSCP/HD) fragments, followed by DNA sequencing. This method allows mutation analysis at both the genomic and mRNA level. By applying cDNA-SSCP/HD analysis to 80 unrelated NF1 patients, we have identified 44 different mutations and found that in 26 patients (50%) the disease is due to aberrant spliced transcripts. We have also tried to discern any possible correlation between genotype and phenotype.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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NF1 mutations detected by cDNA-SSCP/HD analysis
We have analyzed 80 unrelated patients with NF1, representing 61% sporadic and 39% familial cases. Mutations in the entire NF1 coding region were identified by RT–PCR followed by SSCP/HD analysis (Figs 1 and 2). Abnormal SSCP/HD mobility patterns were detected in 52 (65%) unrelated samples. In addition, several SSCP/HD patterns, which did not segregate with the disease or which were present in several subjects, were identified; these were considered as potential polymorphisms and were not characterized in this study. The abnormal SSCP/HD patterns were analyzed in the parents of the proband when available, allowing the detection of co-segregation with the disease. Mutations were identified for 87% of the familial cases, but for 51% of the sporadic ones. After identifying the NF1 alteration at the cDNA level, the underlying genomic mutation was characterized for each of the 52 unrelated patients. Forty-four different mutations were identified in these 52 patients, 32 of which have not been reported previously (Tables 1 and 2). Expression studies of the two NF1 alleles were performed in the RT–PCR fragment II1, ranging from exon 4b to exon 7, by analysis of an expressed polymorphic RsaI restriction site in exon 5 of the NF1 gene (23). This analysis was applied to nine NF1 individuals in whom the mutation was not detected and who were heterozygous at this polymorphic site. All the patients studied expressed the two NF1 alleles (data not shown).
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Eighteen different frameshift mutations were identified, all causing an altered reading frame probably leading to truncated proteins (Tables 1 and 2). Six different nonsense mutations were detected: R304X (910C
T); W599X (1797GA); R681X (2041CT), in two unrelated patients; S1140X (3419CG); R1513X (4537CT); Y2264X (6792CA), in two other unrelated patients.
Five missense mutations were detected: I117S (350TG); Y489C (1466AG), in three unrelated patients; G922S (2764GA); R1204W (3610CT); and L1425P (4274TC). R1204W and L1425P are located in the GRD domain. Mutations Y489C and G922S produce abnormal splicing of NF1 (see below). We rejected the hypothesis that the remaining three missense mutations found in this study were polymorphisms as they co-segregate with the disease and they were not detected in 200 control samples (unrelated unaffected relatives of the NF1 patients studied). Amino acids I117, R1204 and L1425 are conserved in mouse neurofibromin (24) and in Drosophila NF1 protein (25). L1425 is also conserved in RasGAP of Caenorhabditis elegans, but neither L1425 nor R1204 are conserved in RasGAPs of Bos taurus, Mus musculus, Rattus norvegicus, D.melanogaster, Sus scrofa and Saccharomyces cerevisiae (26).
A previously described (27) small in-frame deletion, 7096delAACTTT, which removed two amino acids (asparagine and phenylalanine) was identified in exon 39 of patient 97-113.
NF1 mutations producing splicing defects
Fourteen different mutations involving the consensus splice sites were identified (Tables 1–3). Of these, seven mutations inactivated the 5' splice site (5' ss), producing skipping of the corresponding exon [IVS4b+5GA, skipping 4b; IVS8+1GA, skipping 8; IVS10b+1GA, skipping 10b; IVS18+1GA, skipping 18; IVS19a+1GA, skipping 19a; 5546GA (R1849Q), skipping 29; and IVS37+2TG, skipping 37]; and one affected the 3' splice site (3' ss), also producing exon skipping (IVS16-1delGGTTT, skipping 17). The remaining six splice site mutations implied the utilization of cryptic splice sites, two resulting in the production of NF1 mRNAs lacking a portion of the coding sequence (IVS27b+1del9, 4704del69; IVS28+1GA, 5152del54), and four causing the insertion of intronic nucleotides in the coding sequence (IVS10a-9TA, 1392insUUUUUAG; IVS11-3CG, 1721ins42; IVS15-16AG, 2409ins15; IVS30+332AG, 5749ins177).
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Three unrelated patients presented the above-mentioned splice site mutation 5546G
A, which is also a missense (R1849Q) change with respect to the amino acid sequence. However, this mutation alters the last nucleotide of exon 29 of NF1, which has been considered highly conserved in the 5' ss (28), and causes skipping of exon 29.
Interestingly, one frameshift, two missense and two nonsense mutations were responsible for defects in mRNA processing. Frameshift mutation 3214del11 caused the utilization of a cryptic 5' ss, resulting in a deletion of 104 exonic nucleotides from cDNA position 3211. Two missense mutations introduced novel 5' ss inside the exon, producing the deletion of exonic nucleotides [1466AG (Y489C), causing 1466del62 as described (29), found in three unrelated patients; and 2764GA (G922S), causing 2761del90] (Fig. 3). Finally, exon skipping was induced by two nonsense mutations [910CT (R304X), skipping 7; and 6792CA (Y2264X), skipping 37, in two unrelated NF1 patients], as described (30,31).
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Overall, 10 of the 19 mutations producing splicing defects induced exon skipping (53%), whereas the remaining nine mutations involved the utilization of cryptic splice sites (47%) in the vicinity of or within the exon, producing an exon of altered size or transcripts with an abnormal supplementary exon. Twelve splicing mutations were due to nucleotide changes occurring at intronic regions (six being nucleotide substitutions of the conserved AG/GT dinucleotides of the splice sites), six alterations were found in the respective exon, and one deleted one intronic and four exonic nucleotides. Twelve of these mutations should lead to in-frame amino acid deletions or insertions, with a protein length of between 2760 and 2877 amino acids (Table 1). We have calculated the consensus values (CVs) for the splice sites involved in splicing mutations, which reflect the similarity of any one splice site to the consensus sequences (28) (Table 3). In all cases of skipping of NF1 exons, the CVs for the mutated splice sites (CVMs) were lower than those for the wild-type splice site sequences (CVNs) as found by Krawczak et al. (21). In the five cases where a novel splice site is created due to the mutation, the scores for the new site (CVAs) were higher or similar to the CVNs. Finally, in four cases in which the mutation altered the normal splice site, the cryptic site that was utilized had in general similar values to the mutated site. We have also calculated the CVs for the splice sites of exons 7 and 37, since mutations R304X and Y2264X produce skipping of exons 7 and 37, respectively. As also calculated by Hoffmeyer et al. (31), all the splice site CVs were within a normal range [exon 7: CVN (5' ss) = 80.1, CVN (3' ss) = 79.4; exon 37: CVN (5' ss) = 85.6, CVN (3' ss) = 87.5].
Recurrent mutations in the NF1 gene
Twenty-one of the 80 patients had mutations that were detected more than once in this study and/or were reported previously by other investigators (Table 1). Mutation 1466AG (Y489C) was identified in two sporadic and in one familial case; this mutation was recently found by Osborn and Upadhyaya (29). Mutation 5546GA (R1849Q) was identified in one familial and two sporadic cases, being described for the first time. Three other mutations [IVS30+332AG, R681X (2041CT) and 6792CA (Y2264X)] have each been detected in one familial and one sporadic case of NF1. However, mutation IVS15-16AG was identified in two unrelated families with the same NF1 haplotype, suggesting that they could be identical by descent. Finally, three additional mutations, previously described by other investigators, were found in three sporadic cases of NF1. Overall, at least 15 of the 80 NF1 patients (19%) had a total of eight different mutations that are recurrent.
Genotype/phenotype analysis
Our data suggest that there is no clear relationship between specific NF1 mutations and clinical features. However, several interesting findings were observed.
CLSs.
No relationship was found between the number of CLSs and the identified mutations in patients of similar age. For example, mutation 1466AG was found in patients with a wide variation in the number of CLSs (from 6 to >50) (Table 4). Similarly, mutations causing the same effect at the mRNA level (6792CA and IVS37+2TG, both producing skipping of exon 37) were present in patients with different numbers of CLSs (9 and >50) and similar ages (>40 years). However, we have also found patients with the same mutation (8134delAA) and a similar number of CLSs (Table 4).
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Cutaneous neurofibromas.
In general, there was no relationship between the number of neurofibromas and the identified mutations in individuals of similar age. However, none of the seven patients aged >20 years and harboring mutations causing in-frame deletions or insertions at the mRNA level (IVS10b+1GA; IVS11-3CG; 6792CA, in two related and two unrelated patients; IVS37+2TG) had more than seven cutaneous neurofibromas.
Plexiform neurofibromas.
Of 45 patients, for whom this clinical information was available, 7 had plexiform neurofibromas, with mutations located either within or outside the GRD (IVS4b+5GA; IVS10b+1GA; 1466AG; 3818delAT; IVS30+332AG; 8042insA). Two unrelated patients out of five with mutation 1466AG had plexiform neurofibromas (Table 4).
Scoliosis.
Of 40 patients with scoliosis data, 13 were affected but none had mutations within the GRD (IVS4b+5GA; IVS10b+1GA; 1466AG, in four unrelated patients; R681X; S1140X; IVS30+332AG; 5887/5888delA, in two related patients; IVS37+2TG; 8134delAA). Four patients with scoliosis had mutations involving exon 10b (1466AG; IVS10b+1GA) (Table 4).
Optic glioma.
This complication was found in 3 of 31 patients with information about this feature. All three patients had mutations that lead to a putative truncated NF1 protein (580delC; IVS10a-9TA; 5546GA).
Mental retardation.
Only one patient (92-152) was reported as having mental retardation. In contrast, another patient with the same mutation, R681X, had a good academic level.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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A large number of the mutations identified in this study cause alterations of the splicing of the NF1 mRNA. Whereas in 37% of the patients the identified mutation at the DNA level was predicted to affect mRNA splicing, this proportion was significantly increased to 50% when the effect of the mutation was studied at the mRNA level (Fig. 4). The putative consequences at the protein level of the detected mutations are NF1 proteins of abnormal size in 94% of patients. This high rate of detection of mutations that cause abnormally spliced transcripts is probably biased by the use of cDNA-SSCP/HD. Only 9% of the mutations reported to the NNFF International NF1 Genetic Analysis Consortium (29 March 1999) occur in NF1 intronic sequences, compared with 29% in our study. Osborn and Upadhyaya (29) have recently reported the characterization of 19 NF1 mutations at the genomic and RNA level and have found that ~20% cause splicing abnormalities. Interestingly, one frameshift (3214del11), two nonsense (R304X and Y2264X) and two missense (Y489C and G922S) mutations resulted in defective splicing of the NF1 transcript (Fig. 3), some of them described previously (29–31). Thus, it is likely that other reported mutations categorized as one mutation type could be considered splicing mutations if they were analyzed both at the mRNA and genomic levels. Similar findings have recently been reported for the ATM gene (32). Therefore, these results stress the importance of studying mutations at the genomic and RNA levels so as to clarify the primary effect of the mutation on mRNA processing, which could be different to that predicted solely at the genomic level.
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Of the 23 different single base pair substitutions identified here, 74% (17/23) correspond to transitions and 26% (6/23) to transversions. These proportions are similar to those reported previously (33). This higher rate of transitions compared with transversions can partially be attributed to the hypermutability of the CpG dinucleotide. Accordingly, five of these transitions occur within CpG dinucleotides, of which four are C
T and one GA. Some of the mutations involving small deletions and insertions could be explained by the model of ‘slipped mispairing’ that occurs during DNA replication, mediated by direct or indirect repeats as determinants of secondary structures, which act as mutation intermediates (33). For instance, mutation 717insTCCCACAG creates a tandem duplication; 1465insT creates an inverted repeat (CTtATAAG); another inverted repeat overlaps the deletion 6395del10 (TTTTTTGAGACTcagtctgacaGAGTTCT); 6593insT could be mediated by a symmetric element (GAGAtTATTCC). Slipped mispairing between two AACTTT repeats in tandem probably also accounts for the 7096delAACTTT mutation (27).
The identification of mutations in 52 of 80 patients (65%) using the cDNA-SSCP/HD method indicates that this approach is very powerful in the search for mutations in the NF1 gene and is extremely useful for the molecular diagnosis of NF1, especially for sporadic cases. Approximately 20% of the patients studied here have mutations that are recurrent; although mutation recurrence has previously been described in NF1 (34), the high proportion detected here was unexpected.
The efficiency of cDNA-SSCP/HD in mutation detection is similar to that previously obtained using the PTT (67%) (18). There are several advantages of cDNA-SSCP/HD over the PTT system, such as that it allows the detection of missense mutations and small in-frame insertions/deletions, and that it is a non-radioactive technique. Neither PTT nor cDNA-SSCP/HD detect deletions that span the regions of overlap of any two consecutive cDNA segments and deletions spanning the whole NF1 gene. However, putative deleted regions can potentially be identified by haplotype analysis using markers that may detect hemizygosity (35) or by FISH analysis using genomic clones that span the whole NF1 gene (36–39). Although deletions were partially excluded in the patients studied here by loss of heterozygosity (LOH) analysis, 8 of the 28 NF1 individuals in which the mutation was not detected by cDNA-SSCP/HD were homozygous for all the NF1 polymorphic markers analyzed and they could possibly bear intragenic NF1 deletions, although these could not be demonstrated due to sample limitations. Another problem in RNA methodology is that in some cases the mutation can result in mRNA instability. We have confirmed the expression of the two NF1 alleles in the nine patients where an NF1 mutation was not detected and who were heterozygous for the RsaI polymorphic site in exon 5 (23), but we cannot be sure that the allelic messages were equally represented since a quantitative analysis was not performed. Another possibility is that some of the unidentified mutations were located in the 3'-untranslated region (3'-UTR) and/or in the promoter (24,40). To date, no mutations have been reported in the promoter and only one in the NF1 3'-UTR (41).
One additional difficulty in mutation analysis of NF1 studying genomic regions is due to other sequences related to the NF1 gene, which are present in several human chromosomes (12). Mutation analysis using NF1 mRNA partially avoids this problem as most of these loci represent non-processed pseudogenes. When some of the mutations described here were characterized at the genomic level, we found that for some exons the primers designed by Purandare et al. (42) produced the co-amplification of the homologous pseudogene. To solve this problem, specific NF1 primers were designed (see Materials and Methods).
The different efficiency in the detection of mutations in familial cases (87%) versus sporadic ones (51%) could be attributed to mosaicism. Mosaicism seems to be present in the de novo cases of neurofibromatosis, as has been reported for NF1 (43–45) and NF2 (46). Thus, it is possible that some sporadic NF1 patients could be somatic mosaics with a low proportion or absence of the NF1 mutation in their lymphocytes. To overcome this problem, in the sporadic cases that are negative for mutation analysis in lymphocytes, it would be useful to search for mutations in NF1 target tissues such as neurofibromas, where the first- and second-hit mutations should be present (47).
The only genotype–phenotype correlation described so far in NF1 associates large deletions encompassing the whole NF1 gene with mental retardation and/or learning disabilities, mild facial dysmorphology and a large number of early-onset cutaneous neurofibromas (36–39,48). We have identified the NF1 mutation in only one patient with mental retardation. This low proportion of cases is probably due to the fact that patients bearing NF1 large deletions were already excluded before mutation analysis was performed (see Materials and Methods).
The information on the clinical features of the NF1 patients in whom mutations were identified suggests that the location and type of mutation within the NF1 gene and its putative effect at the protein level do not indicate any relationship to any specific clinical feature of NF1. However, since CLSs and neurofibromas are highly age dependent (49), their putative relationship with NF1 mutations should be analyzed in a large number of patients to allow comparisons between patients of similar ages. Notable findings detected here involve scoliosis with mutations in exon 10b, optic glioma with mutations causing putative truncated NF1, and a low number of neurofibromas in patients >20 years old with in-frame mutations. These findings will need further investigation, but the lack of relationship between phenotype and genotype strongly suggests that modifier genes probably play an important role in NF1 (49).
A large proportion of NF1 mutations have consequences for the correct splicing of the NF1 mRNA. In cystic fibrosis, it has been described that disease variability among patients carrying the same splicing mutation was associated with variable levels of aberrantly spliced cystic fibrosis transmembrane regulator (CFTR) transcripts (50). It is hypothesized that variations in the RNA splicing mechanism may lead to differential expression of the splicing mutation, and hence to different levels of the aberrantly spliced mRNA. Thus, it is tempting to postulate that splicing mutations, which we show here are predominant in NF1, could account for part of the clinical intrafamilial and interfamilial variability that is observed in NF1 patients carrying the same mutation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Patients and families
Eighty unrelated NF1 patients were studied, 31 being familial cases and 49 sporadic. Patients with large deletions in the NF1 gene, previously detected by LOH analysis (35), were excluded from this study. The NF1 diagnosis was based on the NIH Consensus Conference criteria (51). When available, blood samples of other family members were also obtained. All the participants were informed about the study and consent was obtained from all patients and their relatives.
RNA/DNA extraction
Total RNA was extracted from peripheral blood lymphocytes using the Tripure isolation reagent (Boehringer Mannheim, Mannheim, Germany), according to the manufacturer’s instructions. DNA was extracted by the ‘salting out’ method (52).
Reverse transcription and amplification of the NF1 coding region
RNA (2–5 µg) was reverse transcribed using 500 ng of random hexamers and 200 U of Superscript II reverse transcriptase (Gibco BRL, Life Technologies, Paisley, UK) in a 20 µl reaction volume using conditions recommended by the manufacturer. The entire NF1 cDNA was amplified in 10 overlapping fragments, ranging from 634 to 1262 bp, using primer sets designed by Hoffmeyer et al. (53). PCR reactions were performed with denaturation at 94°C for 3 min, 40 cycles of 94°C for 60 s, 58°C for 60 s and 74°C for 90 s, and a final extension at 74°C for 10 min under standard conditions, except for 3 mM MgCl2. The size of the PCR products was verified by electrophoresis in 2% agarose gels.
cDNA-SSCP/HD mutation analysis
RT–PCR products (1–4 µl of each) were mixed with 4 µl of denaturing buffer and heated at 95°C for 2 min. The samples were loaded on 10% polyacrylamide gels (CleanGel DNA Analysis kit; Pharmacia, Uppsala, Sweden). RT–PCR products, ranging from 634 to 996 bp (fragments I1, II1, I2, I3 and II5), were run for 2 h, and those ranging from 1034 to 1262 bp (fragments II2, II3, I4, II4 and I5) for 3 h, all at 600 V/15°C. SSCP/HD patterns were visualized by silver staining (Fig. 1B). When an abnormal pattern was detected, the corresponding RT–PCR was repeated to confirm that the anomalous pattern was reproducible, always comparing with controls (unaffected relatives of NF1 patients). Finally, the abnormal product was analyzed using an automatic sequencer (ABI PRISM 377; Perkin Elmer, Foster City, CA).
Characterization of NF1 mutations at the genomic level
Sequence changes identified at the cDNA level were confirmed at the genomic level by amplification and direct sequencing of the specific exon containing the mutation. When a skipped exon or the insertion or deletion of several nucleotides were detected at the cDNA level, then the corresponding exon and flanking intron boundaries were analyzed for potential point mutations. In most cases, the primers used for amplification and sequencing of individual exons were those described by Purandare et al. (42), except for exons 10b, 19b and 21 that also amplify pseudogenes being substituted for:
10bD2, 5'-CTACTATACCACACATTGGTAG-3', and
10bR2, 5'-TCAGGTATGATCAGAAAGG-3', for exon 10b;
19bD2, 5'-AACTTGAAAGATTCATGGTCTC-3', and
19bR2, 5'-TTTATGTTTTTTGGTGACTG-3', for exon 19b;
21D2, 5'-CATGTTAGTAAATTTGCATCTG-3', and
21R2, 5'-ATTTGCTATGTGCCAGGCAC-3', for exon 21.
Mutations that created or abolished a restriction site were confirmed by restriction enzyme analysis as described (54) (Fig. 2). Segregation analysis of the identified mutations verified the familial or sporadic nature of the mutation.
Expression analysis of the two NF1 alleles
RT–PCR fragment II1, which contains the RsaI polymorphic site in exon 5 (23), was analyzed in the nine samples that were heterozygous at this site and that were also negative for NF1 mutations, showing that the NF1 mRNA was stable.
Genotype/phenotype analysis
We compared the causative NF1 mutations with the clinical features of the patients. We analyzed the patients according to age, numbers of CLSs and neurofibromas, Lisch nodules, freckling, plexiform neurofibroma, scoliosis, optic glioma, mental retardation, hypertension, and the type/effect and location of the NF1 mutations.
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ACKNOWLEDGEMENTS |
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The authors wish to thank all the patients and family members who participated in this study, and all the clinicians for referring the families. We are indebted to M. Morell for technical help, to Y. Espinosa for advice with the technique and the manuscript, to A. Puig for sequencing and to A. Aviñó for synthesis of PCR primers. E.A. is a fellow of the Comissió Interdepartamental de Recerca i Innovació Tecnològica (CIRIT) of the Generalitat de Catalunya. This work was supported by grants from the Fondo de Investigaciones Sanitarias de la Seguridad Social (95-368 and 98-0992), the European Union (BIOMED BMH-4-CT96-1364), the Institut Català de la Salut and the Fundació Agust Pi i Sunyer/Marató de TV3.
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +34 93 2607775; Fax: +34 93 2607776; Email: estivill@iro.es
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REFERENCES |
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