Expression analysis of an FGFR2 IIIc 5' splice site mutation (1084+3A->G)

Sequence variations within splice sites may pose problems in the interpretation of their pathogenic effect, especially when these variations occur outside the highly conserved /gt (donor or 5′ site) and ag/ (acceptor or 3′ site) consensus dinucleotides that immediately flank most exons. A commonly used method to evaluate the probable effect of a sequence variation on splicing is to calculate the Shapiro-Senapathy (SS) score, which is based on the extended splice site consensus sequence.1–3 Here we present a sequence variation in a 5′ splice site of the gene encoding fibroblast growth factor receptor type 2 ( FGFR2 ) that maintains a consensus nucleotide at the variant position, but nevertheless causes a switch to the use of a cryptic 5′ splice site within the upstream (IIIc) exon. This variant is present in three generations of a family and manifests with mild features of Crouzon syndrome. Following informed consent blood samples were collected from members of the family pedigree shown in fig 1. DNA was isolated by proteinase K treatment and phenol chloroform extraction. RNA was also extracted from the blood of affected individual II-2 and cDNA synthesised by standard techniques. Figure 1  Clinical features of the family. (A) Pedigree showing transmission of the phenotype through three generations. (B) Facial appearance of family members (from left to right) II-3, II-5 (unaffected), III-3, and II-2 (Photograph with family’s permission) . Mutation screening of exons IIIa and IIIc of FGFR2 4 was undertaken in DNA from the proband using WAVE (3500HT; Transgenomic) denaturing high performance liquid chromatography.5 Sequencing was performed with Big Dye (version 3) on an ABI 3100 DNA sequencer. Oligonucleotide hybridisation was carried out on cDNA derived from individual II-2. Reverse transcriptase PCR (RT-PCR) using primers 4F and 9R located respectively in FGFR2 exons 6 and 12 (exon numbering according to Kan et …