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Letters to the Editor |
1 Laboratoire de, Génétique Moléculaire, Centre Hospitalier Universitaire, Henri Mondor AP-HP, Créteil, France, 2
INSERM U468, Créteil, France, 3
Service de Médecine F
tale, Institut de Puériculture de Paris, Paris, France, 4
Centre de Traitement des Hémophiles, Groupe Hospitalier Cochin, Saint Vincent de Paul, Paris, France, and 5
Centre de Diagnostic Prénatal, Hôpital Américain de Paris, Neuilly/Seine, France
aAddress correspondence to this author at: Service de Biochimie-Génétique, Laboratoire de Génétique Moléculaire, Centre Hospitalier Universitaire Henri Mondor, 51, av. du Mal-de-Lattre-de-Tassigny, 94010 CRETEIL Cedex, France. Fax 33-1-49-812-842; e-mail costa{at}im3.inserm.fr.
To the Editor:
Gene deletions are common events in various hereditary diseases, but conventional PCR often fails to detect such defects in heterozygous patients (1). This highlights the need for a suitable method, easily applicable in a diagnostic laboratory (2). Large deletions account for 
5% of cases of hemophilia, inherited as a recessive X-linked disease, making determination of carrier status difficult. It is of particular importance when a pregnant woman is carrying a male fetus. Prenatal testing that uses invasive methods of sample collection carries a risk of miscarriage or fetal injury, and is unnecessary if the woman is not a carrier. We designed a real-time quantitative PCR assay to establish heterozygous status for deletion carriers and to avoid such risky situations in a family in which a large deletion of the F9 gene was responsible for a severe form of hemophilia B.
After receiving informed consent, we prepared genomic DNA from a hemophiliac person and two females, a niece and a first cousin, from 200 µL of EDTA blood, using the High Pure PCR Template Preparation Kit (Roche Applied Science), according to the recommendations of the manufacturer.
Part of exon h of the F9 gene (Xq27) and part of exon 1 (used as reference) of the ß-globin gene (11p15) were amplified by real-time PCR from DNA obtained from the propositus, the two related female patients, unaffected males (n = 5), and female noncarriers (n = 6). Relative quantification was established by comparison with a range of serial dilutions of the female DNA. The sequences for the primers (MWG) are as follows: F9 forward, 5'-GTCAGTGGTCCCAAGTAGTC-3'; F9 reverse, 5'-AAGACATGTGGCTCGGTCAA-3'; ß-globin forward, 5'-TGCCAGAAGAGCCAAGGACA-3'; ß-globin reverse, 5'-CTCACCACCAACTTCATCCAC-3'. The primers were located in coding sequences if possible, avoiding the risk of reduced PCR efficiency attributable to a mismatch with the target sequence.
The reactions were carried out in a LightCycler instrument (Roche), with a reaction volume of 20 µL, using the FastStart DNA Master SYBR Green I® Kit (Roche), 3 mM MgCl2, 0.5 µM each primer, 50 ng of patient or control DNA, and 100, 20, and 4 ng of calibrators. PCR conditions were as follows : 95 °C for 8 min, 95 °C for 10 s, 60 °C for 10 s, and 72 °C for 15 s (45 cycles). Melting curve analysis was performed after PCR. Two calibration curves for each gene were generated with the female DNA calibrator. These curves were used to quantify unknown samples, and a F9:ß-globin concentration ratio was calculated. The results are summarized in Table 1
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As expected, the F9 gene copy number was correctly determined in control groups because it was two times higher in females than in males, demonstrating the reliability of the method to detect patients heterozygous for deletions because unaffected male samples mimic carriers for the gene deletion. Results in these two groups did not overlap, and the CV of the assay was 15% and 10% for the normal (two copies) or the deleted (one copy) samples, respectively. The first-cousin woman was pregnant, and she was carrying a male fetus. She was diagnosed as a carrier for the deletion by the gene dosage assay. The prenatal diagnosis, performed by amniocentesis, revealed that the fetus was affected, thus confirming the direct molecular determination of her status and the reliability of the method. When the method was applied for the niece, it revealed a normal result, establishing her noncarrier status.
The proposed rapid real-time PCR assay has several advantages. Real-time quantitative PCR using labeled probes has already been described, but the use of such probes, because the anomaly is often family specific, is particularly expensive (3)(4). Our strategy does not require specific hybridization probes and can avoid this additional cost. Because most of the deletions occurring in the F9 gene remove at least exon h, the assay was targeted to this region, and it can be applied to families in which F9 gene deletions are responsible for the disease. Furthermore, the method can be rapidly adapted to other genetic diseases.
References
The following articles in journals at HighWire Press have cited this article:
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  A. Vencesla, M. J. Barcelo, M. Baena, M. Quintana, M. Baiget, and E. F. Tizzano Marker and real-time quantitative analyses to confirm hemophilia B carrier diagnosis of a complete deletion of the F9 gene Haematologica, November 1, 2007; 92(11): 1583 - 1584. [Abstract] [Full Text] [PDF]
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