Figure 1

Del100 and del231 result from minor alternative splicing pathways and not from the presence of the W262X mutation. Total RNA extracted from different normal cell lines or cells of HTI patients was subjected to RT-PCR. Controls for FAH, del100 and del231 are displayed on the left to show that the PCR reactions are in the linear range. (A) FAH amplification from exons 6 to 14 in the lymphoblastoid cell lines. The two additional products (del100 and del231) seen in the homozygous cell line (W262X/W262X) are depicted below the gels. Exons are represented by boxes and introns by lines. Del100 has skipped exon 8 and as a consequence, the reading frame is shifted (W262X becomes G229E). A first PTC is located at the 3'end of exon 10 (270X). Del231 has skipped both exons 8 and 9, but without any change in the reading frame. (B) Primers designed to specifically amplify del100 and del231 span exon 7 to 9 or exon 7 to 10 junctions respectively (depicted in blue). Full-length, del100 or del231 cDNAs were cloned in pRC/CMV and were amplified to verify primer specificity to each alternative transcript. Both transcripts are present in normal cells (wild-type lymphoblasts or fibroblasts and HeLa cells) and human liver. Del100 is amplified in HTI cells with a splice mutation in exon 12 (IVS12/ IVS12) or two nonsense mutations in exon 13 (E357X/E364X). FAH was also amplified as a control in the different human cell lines.