Functional analysis of splicing mutations in exon 7 of NF1 gene

Irene Bottillo 1 2 Alessandro De Luca 1 2 Annalisa Schirinzi 1 2 Valentina Guida 2 Isabella Torrente 2 Stefano Calvieri 0 Cristina Gervasini 3 Lidia Larizza 3 Antonio Pizzuti 1 2 Bruno Dallapiccola 1 2 0 Department of Dermatology-Venereology and Plastic and Reconstructive Surgery, University of Rome "La Sapienza" , Rome , Italy 1 Department of Experimental Medicine and Pathology, University of Rome "La Sapienza" , Rome , Italy 2 IRCCS-CSS, San Giovanni Rotondo and CSS-Mendel Institute , Rome , Italy 3 Division of Medical Genetics, San Paolo School of Medicine, University of Milan , Milan , Italy Background: Neurofibromatosis type 1 is one of the most common autosomal dominant disorders, affecting about 1:3,500 individuals. NF1 exon 7 displays weakly defined exon-intron boundaries, and is particularly prone to missplicing. Methods: In this study we investigated the expression of exon 7 transcripts using bioinformatic identification of splicing regulatory sequences, and functional minigene analysis of four sequence changes [c.910C>T (R304X), c.945G>A/c.946C>A (Q315Q/L316M), c.1005T>C (N335N)] identified in exon 7 of three different NF1 patients. Results: Our results detected the presence of three exonic splicing enhancers (ESEs) and one putative exonic splicing silencer (ESS) element. The wild type minigene assay resulted in three alternative isoforms, including a transcript lacking NF1 exon 7 (NF1E7). Both the wild type and the mutated constructs shared NF1E7 in addition to the complete messenger, but displayed a different ratio between the two transcripts. In the presence of R304X and Q315Q/L316M mutations, the relative proportion between the different isoforms is shifted toward the expression of NF1E7, while in the presence of N335N variant, the NF1E7 expression is abolished. Conclusion: In conclusion, it appears mandatory to investigate the role of each nucleotide change within the NF1 coding sequence, since a significant proportion of NF1 exon 7 mutations affects premRNA splicing, by disrupting exonic splicing motifs and modifying the delicate balance between aberrantly and correctly spliced transcripts. - Background Alternative splicing, the process by which exons are included or excluded in the mature mRNA, is an important mechanism whereby different transcripts are generated from the same gene unit. In fact, most human genes are transcribed in multiple alternative mRNAs, according to different regulatory programs, resulting in functionally different protein isoforms [1]. In the best characterized models of vertebrate cell-specific alternative splicing, post-transcriptional regulation is tissue or developmental stage specific and may be mediated by intronic and exonic cis elements. These elements, which are important for the correct splice-site identification, can act by stimulating (exonic splicing enhancers, ESEs) or repressing (exonic splicing silencers, ESSs) the exon's splicing [2]. Neurofibromatosis type 1 (NF1, MIM#162200) is one of the most common autosomal dominant disorders, affecting about 1:3,500 individuals in all ethnic groups. The NF1 gene is approximately 280 kb in size and maps to chromosome 17q11.2 [3-5]. NF1 contains 60 exons, with an 11- to 13-kb transcript and an open reading frame coding for 2,818 amino acids [6]. The disease is fully penetrant and the diagnosis of NF1 is based on the clinical criteria recommended by NIH Consensus Conference (Stumpf, et al., 1988), which include multiple caf-au-lait spots, cutaneous or subcutaneous neurofibromas, plexiform neurofibromas, axillary or inguinal freckling, optic gliomas, and iris Lish nodules. Although NF1 mutations are distributed along the entire coding sequence, no genotype-phenotype correlation has been found so far [7], with the exception of the recurrent and atypical deletions underlying NF1 microdeletion syndrome [8]. The NF1 gene is ubiquitously expressed, and four normal in-frame NF1 splice isoforms are known, brain specific 9br isoform (30 bp) [9], exon 10a-2 isoform (45 bp) [10], exon 23a isoform (63 bp), which are expressed in all tissues at various levels [11], and the muscle specific exon 48a isoform (54 bp) [12]. In addition, several other alternative transcripts have been described, including ex29-, ex30-, ex29/30- and the N-isoform [13,14]. Mutation analysis has shown that approximately 50% of NF1 mutations result in splicing alterations [15-18]. In some cases, splicing mutations do not occur at the conserved AG/GT dinucleotides of the splice sites. For example, mutations leading to stop codons in exon 7 and 37 of NF1 gene have been reported to be involved in exon skipping [18-21]. In addition, mutational analysis of the NF1 gene disclosed several additional splice variants in which specific exons are skipped in fresh lymphocytes of unaffected persons, albeit typically at low levels [22,23]. A number of studies have also reported that some of these transcripts are more abundant when RNA from aged blood or from blood kept at non-physiological temperatures is analyzed [15,22,24,25]. The expression of an alternative transcript lacking exon 7 has been demonstrated [17,26]. Indeed, NF1 exon 7 displays weakly defined exon-intron boundaries, and is particularly prone to aberrant splicing. In the present study, we have used in silico and in vitro analysis to evaluate the functional consequences on gene expression of four nucleotide variants detected in NF1 exon 7, including a nonsense mutation (R304X), a missense mutation (L316M), and two silent changes (Q315Q and N335N). Since both Q315Q and L316M mutations were together in cis in the same patients [27], our analysis aim was to understand their effect together and independently. Methods DNA mutation analysis The nucleotide variants investigated in this study include a recurrent nonsense mutation [c.910C>T (R304X)] [22,27-29] and a novel silent change [c.1005T>C (N335N)] identified using denaturing high performance liquid chromatography (dHPLC) followed by bidirectional sequencing, as well as a silent change and a missense mutation occurring together in cis in the same patient [c.945G>A/c.946C>A (Q315Q/L316M)], and previously reported by us [27]. PCR conditions, amplicon length, and resolution temperatures for dHPLC analysis are reported elsewhere [28,30]. The N335N silent change was found in a two generation NF1 family (family NF-01) carrying another NF1 gene mutation, a frameshift deletion (c.476delC) in exon 4a. The silent change N335N was found in the proband (II-2) and her child (III-1), both affected by NF1, and in the proband's father (I-1) presenting out of NF1 clinical signs only three cutaneous neurofibromas. Frameshift mutation c.476delC was detected in the proband and her child, but not in the proband's father. Both N335N and c.476delC were not found in 200 healthy subjects. Microsatellite analysis performed using 10 markers tightly linked to the NF1 locus (D17S1873, D17S841, D17S1863, D17S635, D17S1166, I (...truncated)