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Rapid Determination of -Fetoprotein Gene Promoter Mutations in Hereditary Persistence of -Fetoprotein

¹ Laboratoire d’Hormonologie et Biologie Moléculaire Hôpital Bicêtre AP-HP IFR93-Bicêtre, 78 rue du Général Leclerc, 94275 Le Kremlin-Bicêtre CEDEX, France

^aAuthor for correspondence. Fax 33-1-4521-2751; e-mail anne.mantel{at}bct.ap-hop-paris.fr.

To the Editor:

Hereditary persistence of -fetoprotein (HPAFP) is an autosomal inherited disorder in which AFP is persistent in adult life. Since the description of the first case in 1983 (1), this clinically benign disorder has been reported in 11 unrelated families [Ref. (2) and references therein]. The molecular mechanism has been identified in four unrelated families (2)(3)(4). It has been related to a –119G>A substitution in the distal hepatocyte nuclear factor-1 binding site of the AFP gene promoter in three families (2)(3)(4) and to a –55C>A substitution in the proximal hepatocyte nuclear factor-1 binding site of the AFP gene promoter in one (2). AFP is a widely used tumor marker. Although HPAFP is a rare disorder, it needs to be considered and differentiated from tumors to avoid inappropriate explorations and treatment decisions.

We present a simple test based on restriction fragment length polymorphism (RFLP) analysis for screening of HPAFP mutations. This test can be used in patients with increased AFP not accompanied by any obvious tumor or liver disease, and may avoid invasive studies. We have previously reported two unrelated families with HPAFP related to –119G>A and –55C>A substitutions (2). The –119G>A mutation creates a PshAI restriction site. A gain of this site in HPAFP patients with the –119G>A mutation produced two fragments on agarose gel electrophoresis compared with the one obtained from an individual without the substitution (Fig. 1A ). DNA isolated from four individuals of family 1 was tested for the presence of the –119G>A mutation by RFLP analysis. All of these patients also had increased AFP and were heterozygous for the RFLP (Fig. 1B ). The individual with a normal AFP concentration was homozygous for the wild-type (WT) allele. The –55C>A mutation abolished a SpeI restriction site. A loss of this site in HPAFP patients with the –55C>A mutation produced one fragment on agarose gel electrophoresis compared with the two fragments obtained from a control individual (Fig. 1C ).

View larger version (59K): [in this window] [in a new window]   Figure 1. RFLP screening of –119G>A and –55C>A mutations. (A), identification by RFLP analysis of the –119G>A mutation. Electrophoresis of the PshAI digest of PCR products from a control individual (lane 2) and the proband (lane 3). Lane 1, DNA ladder (X174DNA-HaeIII digest). The control shows one band, corresponding to the WT uncut allele (294 bp). The proband shows three bands, corresponding to the WT uncut allele and to the digested mutant allele (227 + 67 bp). (B), pedigree of family 1. Closed symbols indicate the individuals with increased serum AFP. Symbols with lines above them indicate examined individuals. and indicate deceased individuals. Arrow indicates the proband. The affected members show three bands (WT uncut and digested, mutant alleles). The unaffected member shows one band (WT uncut allele). (C), identification by RFLP analysis of the –55C>A mutation. Electrophoresis of the SpeI digest of PCR products from a control individual (lane 2) and the proband (lane 3). Lane 1, DNA ladder (X174DNA-HaeIII digest). The control shows two bands, corresponding to the WT digested allele (167 + 127 bp). The proband shows three bands, corresponding to the WT digested allele and to the uncut mutant allele (294 bp). (D), pedigree of family 2. The affected members show three bands (WT digested and uncut mutant allele). The unaffected members show two bands (WT digested allele). Symbols are the same as in panel B.

DNA isolated from seven individuals of family 2 was tested for the presence of the –55C>A mutation by RFLP analysis. All of the affected family members who had increased serum AFP were heterozygous for the RFLP (Fig. 1D ). The five individuals with normal AFP concentrations were homozygous for the WT allele.

Our RFLP analysis is potentially useful for direct detection of both mutations known to be associated with HPAFP (2)(3)(4). Detection of the –119G>A substitution by conformation-sensitive gel electrophoresis has recently been proposed (5). However, this method was designed for detection of the –119G>A substitution and not of the second reported mutation (–55C>A). Our RFLP test is inexpensive and simple to perform.

Acknowledgments

We thank S. Wenk for careful English proofreading of the text. We are grateful to the patients, to their families, and to the physicians, especially Dr. G. Perlemuter and Prof. C. Buffet.

References

1. Ferguson-Smith MA, May HM, O’Hare E, Aitken DA. Hereditary persistence of alphafetoprotein: a new autosomal dominant trait identified in an antenatal screening programme for spina bifida [Abstract]. J Med Genet 1983;20:458.
2. Alj Y, Georgiakaki M, Savouret JF, Mal F, Attali P, Pelletier G, et al. Hereditary persistence of -fetoprotein is due to both proximal and distal hepatocyte nuclear factor-1 site mutations. Gastroenterology 2004;126:308-317.
3. Blesa JR, Giner-Duran R, Vidal J, Lacalle ML, Catalan I, Bixquert M, et al. Report of hereditary persistence of -fetoprotein in a Spanish family: molecular basis and clinical concerns. J Hepatol 2003;38:541-544.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
4. McVey JH, Michaelides K, Hansen LP, Ferguson-Smith M, Tilghman S, Krumlauf R, et al. A GA substitution in an HNF I binding site in the human -fetoprotein gene is associated with hereditary persistence of -fetoprotein (HPAFP). Hum Mol Genet 1993;2:379-384.[Abstract/Free Full Text]
5. Blesa JR, Vidal J, Giner-Duran R, Lacalle ML, Catalan I, Hernandez-Yago J. Detection of hereditary persistence of -fetoprotein by conformation-sensitive gel electrophoresis analysis. Clin Chem 2003;49:2102-2103.[Free Full Text]

This Article Extract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Alj, Y. Articles by Guiochon-Mantel, A. Search for Related Content PubMed PubMed Citation Articles by Alj, Y. Articles by Guiochon-Mantel, A. Related Collections Molecular Diagnostics and Genetics