Identification of a mutation that perturbs NF1 agene splicing using genomic DNA samples and a minigene assay

Abstract

N eurofibromatosis type 1 (NF1) is a common autosomal dominant genetic disease. In recent studies on the neurofibromatosis type 1 (NF1) gene neurofibromin, splicing abnormalities were seen in 30-50% of cases when RNA taken from cell lines was analysed.1 2 Unlike mutations that alter critical amino acids or generate premature stop codons, splicing abnormalities can be very hard to predict from sequence analysis alone. Apart from the two base pairs 5′ and 3′ of each exon, few of the nucleotides in regions critical for splicing are absolutely conserved. As a consequence, it can be very difficult to conclude that a sequence variation found in a patient will alter splicing and so represents a pathogenic mutation.This difficulty is well illustrated by a family with NF1 in which we recently identified a sequence variation. The three generation family is from the UK and meets NIH diagnostic criteria. The index case, at the age of 82, has classical features of NF1 including multiple café au lait macules, neurofibromas, and axillary and inguinal freckling. Her son was similarly affected and died in a road traffic accident. Her granddaughter additionally had macrocephaly and died aged 31 from a malignant nerve sheath tumour affecting the coeliac axis. Through sequencing all exons in the neurofibromin gene, as well as 50 base pairs of 5′ and 3′ intronic flanking sequence, only a single nucleotide change was found, consisting of a substitution of a cytidine for a guanosine 5 bp downstream of exon 3 (exon 3+5 G>C, fig 1). This variation was seen in the proband and the granddaughter and has not been found in 100 normal chromosomes (Mattocks et al, unpublished observations), consistent with it representing the disease causing mutation. The vicinity of the nucleotide change to the 5′ splice site of intervening sequence (IVS) 3 (figs 1 and 2A) suggests that it may interfere with the splicing of exon 3. However, there are examples of wild type sequences similar to the exon 3+5 G>C mutation in which the corresponding exon is spliced efficiently; for example within the normal NF1 gene IVS-1 and -7 share identical -1 to +5 sequence with the mutated IVS-3. We were therefore unable to predict on the basis of sequence analysis alone that this sequence change will Figure 1 Schematic representation of the α-globin fibronectin NF1 exon 3 minigene (black, shaded, and white boxes respectively, with intervening sequences (introns) shown as lines). The mutation is shown with exonic sequence in upper case and intronic sequence in lower case. Key points• Abnormalities of pre-mRNA splicing represent an important mechanism by which gene mutations cause disease.• Effects on splicing can be predicted from genomic DNA sequence analysis if mutations alter highly conserved canonical splicing signals. However, it is extremely difficult to predict the effects of changes in intronic and exonic sequences not obviously involved in the splicing process.• We present here an efficient and simple test using genomic DNA to construct a minigene and analyse the ef...

Systematic evaluation of the effect of common SNPs on pre-mRNA splicing

“…Furthermore, position +5 has recently gained great concern as a hot spot of disease-causing mutation. For example, a famous mutation at +5 of donor ss (IVS3+5GC, 5-GUAACU-3) and resultant exon 3 skipping was reported as a disease-causing mutation in the NF1 gene (Baralle et al, 2003). Also, exclusion of exon 2 at HMSD gene due to AS was completely controlled by an intronic SNP (rs9945924) at IVS+5 (Kawase et al, 2007).…”

“…Therefore, SNPSplicer screening tool was created to evaluate whether a potential site-specific splice effect is present in a given sequence. Clearly, minigene constructs provide a more defined and experimentally controlled system, which may need to be used for ultimate mechanistic clarification to determine the effects of SNPs on splicing (Niksic et al, 1999;Pagani et al, 2002;Baralle et al, 2003;Lewandowska et al, 2005). This classic mechanistic approach has certain limitations, especially low throughput, limited insert size, and thus incomplete detection of long-range effects.…”

Novel and recurrent mutations in theNF1 gene in Italian patients with neurofibromatosis type 1

“…However, unequal expression of both NF1 alleles is a common observation in NF1, and c.730-6A>C change was not detected in multiple controls. Thus, minigene assay (Baralle et al, 2003) is on going to test for the influence of both c.(730-6)A>C nucleotide change on the splicing process.…”

“…However, c.7907+5G>A change was not detected in patient's parents (originated de novo), and it was not found in 150 healthy subjects. Minigene assay (Baralle et al, 2003) is on going to test for the influence of c.7907+5G>A on the splicing process (data not shown). Heterozygosity is based on the number of patients.…”

Molecular analysis of Ceruloplasmin in a South African cohort presenting with oesophageal cancer

“…Further studies using the minigene system are warranted to determine what effect this variant has on protein expression in patients presenting with OC. 47 With regards to the iron parameter statistics, the serum iron, transferrin, ferritin and C-reactive protein levels (CRP) were found to be statistically significantly different between patient and control subjects regardless of gender (Table 5). Mean serum iron and mean transferrin levels were found to be higher in the control subjects relative to the OC patient group, whereas the mean ferritin and CRP levels were significantly higher in the patient group relative to the control group.…”