Figure 4

Expression analysis in the family patients carrying the QO _Porto mutation (II-1 and II-2) and the ones carrying both QO _Porto and QO _Madrid variants (II-3 and II-4). A) Schematic representation of the SERPINA1 gene. To amplify different transcripts some forward primers in exons 1A, 1B, and 1C and reverse primers in exons 2 and 5 were designed (arrows). B) RT-PCR amplification of mRNA using primers located in exon 1C and the reverse primers in exon 2 or exon 5, analyzed in normal hepatocytes (H), the AAT cases, and in a normal peripheral blood sample (PB). No expression products were found in cases II-3 and II-4 when using exon 1C primer. All the other cases showed a single band of 587 bp corresponding to expression products containing the exon 1C directly spliced to exon 2, or in the case of E1C-E5 the expression product corresponded to a fragment of 1318 bp with the exon 1C joined to all the coding exons (2 to 5). C) Fragments generated by amplification of expression products using primers in exon 1A and exon 2. All cases showed expression of a transcript including the exon 1A directly joined to exon 2. No other alternative splicing variants were found. D) Expression analysis using primers in exon 1B revealed multiple bands meaning that alternative splicing occurred between exons 1B and 1C. After cloning, we differentiated five different splicing forms, some showed the use of alternative splicing sites on exon 1B (3 and 4) previously described. One of the transcription species retained the intron 1B (5). Severe DAAT cases only express transcripts without exon 1C.