Volume 126, Issue 1, Pages 308-317 (January 2004) Hereditary persistence of α-fetoprotein is due to both proximal and distal hepatocyte nuclear factor-1 site mutations1  Youssef Alj, Maria Georgiakaki, Jean-François Savouret, FrÉdÉric Mal, Pierre Attali, Gilles Pelletier, Catherine Fourré, Edwin Milgrom, Catherine Buffet, Anne Guiochon-Mantel, Gabriel Perlemuter  Gastroenterology  Volume 126, Issue 1, Pages 308-317 (January 2004) DOI: 10.1053/j.gastro.2003.10.073

Figure 1 Family pedigrees. The number below each individual indicates AFP serum level (UI/mL). (A) Pedigree of family 1. (B) Pedigree of family 2. Gastroenterology 2004 126, 308-317DOI: (10.1053/j.gastro.2003.10.073)

Figure 2 Sequence of the AFP gene promoter in HPAFP. (A) Nucleotide sequence of the human AFP promoter gene between −182 bp and +112 bp. Sequence alignment of the −132/−33 region from family 1 and 2 with the WT sequence. Oligonucleotides TT1 and TT2 used in PCR are indicated above the corresponding sequence. The cap site is numbered +1. The potential HNF-1 binding sites (proximal and distal) and the potential CCAA(C/T) box are underlined. Mutations are indicated with an asterisk character (∗) below the corresponding amino acids. (B) Sequence alignment of the −76/−40 region of the human AFP promoter with the HNF-1 binding site consensus and the mutant sequence. Nucleotide changes between WT+ and mutant (Mut 55–65+) sequences are indicated with bold characters. Nucleotide changes from the HNF-1 binding site consensus are indicated with an asterisk character (∗). The substitution brings the sequence closer to the HNF-1 consensus sequence. The CCAAT box is underlined. Gastroenterology 2004 126, 308-317DOI: (10.1053/j.gastro.2003.10.073)

Figure 3 Binding of HNF-1α to AFP gene promoter was enhanced by the −55 and −65 mutations. Gel retardation competition analyses were performed with 30,000 cpm of 2 different probes and hepatoma cell (HepG2) extracts. The WT probe was incubated in the absence (lane 1) or the presence of increasing amounts (from 0.25 to 10 ng) of unlabeled double-stranded WT competitor (lanes 2–4). The Mut 55–65 probe was incubated in the absence (lane 5) or the presence of increasing amounts (from 0.25 to 10 ng) of unlabeled double-stranded WT competitor (lanes 6–8). The Mut 55–65 probe was preincubated with either 2 μL of a specific (S) anti-HNF1α antibody (Ab) (lane 11) or 2 μL of a nonspecific antibody (NS) as a control (lane 10). A supershift was observed only in the presence of the specific antibody. Gastroenterology 2004 126, 308-317DOI: (10.1053/j.gastro.2003.10.073)

Figure 4 The C−55 to A mutation strongly increased the affinity of AFP gene promoter for HNF-1α. (A) Gel retardation competition analyzed HepG2 cells extracts using consensus (Cs) sequence as a probe. The consensus sequence was incubated in the absence (lanes 1, 6, 11) or the presence of increasing amounts (from 0.25 to 50ng) of unlabeled double-stranded WT competitor (lanes 2–5), unlabeled double-stranded Mut 55–65 competitor (lanes 7–10) or increasing amounts of unlabeled double-stranded Mut 55 competitor (lanes 12–15). (B) Quantitative analysis of the HNF-1 binding. The intensity of the HNF-1-DNA retarded band was quantified by electronic autoradiography with Instant Imager (Packard Instrument Company, Meriden, CT). The band in the absence of competitor oligonucleotide represented HNF-1-Cs complex. Results were expressed in binding percentage relative to this complex, calculated for each amount of unlabeled competitor. WT, black curve, black squares; Mut 55, black dotted curve, black triangles; Mut 55–65, black curve, white diamonds. Gastroenterology 2004 126, 308-317DOI: (10.1053/j.gastro.2003.10.073)

Figure 5 Transcriptional activity of the WT and mutants constructs. Promoter activity from CAT vector, WT, and mutant AFP gene promoter sequences after transient transfection into (A) human hepatoma HepG2 cells and (B) mouse fibroblastic L cells. Results are expressed in CAT concentration relative to WT construct (mean ± SD). They are the mean of 3 independent experiments in triplicate. Gastroenterology 2004 126, 308-317DOI: (10.1053/j.gastro.2003.10.073)

Figure 6 Conservation of AFP gene promoter in different species. Multiple alignments of the –76/–40 region of the human, rat, mouse, chimp, gorilla, orangutan, and pig AFP promoter with the HNF-1 consensus binding site and the mutant. The 2 new identified mutations occurred in bases that are well conserved (underlined residue). Gastroenterology 2004 126, 308-317DOI: (10.1053/j.gastro.2003.10.073)