Sequence of EFNB1 splice site mutation c.406+2T > C and expression of EFNB1 transcript and protein in primary patient fibroblasts. (A) The mutation has been detected by direct sequencing of genomic DNA. Nucleotide exchange T > C in intron 2 at the splice donor site is indicated by an arrow. The major transcript expressed in patient fibroblasts was the wild type allele. (B) Wild type and mutant transcripts in patient fibroblasts (lane p) and control cell cultures (lanes pc1 and pc2) were determined by RT-PCR. The wild type RT-PCR product is indicated by an arrow, mutant RT-PCR products are indicated by arrowheads. Sequencing of the aberrant transcripts showed retention of intron 2 (generating a 1.2 kb RT-PCR product) or activation of a cryptic splice site in exon 2 (generating a 288 bp RT-PCR product). (C) Western blot analysis of ephrin-B1 expression in patient fibroblasts and control cell culture lysates showed an approximately 50 kDa protein (indicated by an arrow). No smaller truncated protein was detected in patient fibroblasts. Protein sizes were determined using Precision Plus Protein™ Standards Dual Color (BIO-RAD). (D) The sequence of exon 2 and 3 and part of intron 2 are shown. Coding sequences are shown in capital letters, flanking sequences of intron 2 are shown in small letters. The BfuAI site at the mutation site is underlined. Aberrant splicing is indicated by green letters, the cryptic splice junction is underlined. Premature termination codons (STOP) generated by aberrant splicing or retention of intron 2 are highlighted in red.