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Gen. Physiol. Biophys. (2003), 22, 243—253


Long Distance PCR in Detection of Inversion Mutations of F8C Gene in Hemophilia A Patients
H. Poláková1 , I. Zmetáková2 and Ľ. Kádasi1
1 2

Institute of Molecular Physiology and Genetics, Bratislava, Slovakia Comenius University, Faculty of Natural Sciences, Department of Molecular Biology, Bratislava, Slovakia

Abstract. In the presentpaper, the experience with detection of intron 22 inversion of F 8C gene in severe hemophilia A patients using a recently described long-distance PCR (LD-PCR) method was reported. To test the sensitivity and the specifity of the LD-PCR, analysis of 46 DNA samples of patients and their family members, previously tested by Southern hybridization, was performed. In addition, 16 DNA samples of severehemophilia A patients in which causative mutation was unknown, were included in analysis. Four-primers, P, Q, A&B, which are able to differentiate between the affected males with or without the inversion, and in female carriers, were used in LD-PCR. Two primers, P&Q, are located within the F 8C gene flanking int22h1. Two further primers, A&B, flank int22h2 and int22h3, extragene homologs of int22h1.Nine combinations of four primers were used to identify the optimal one. Four-primers (P, Q, A&B), three-primers (P, Q&B; P, A&B; A, B&Q; P, Q&A) and two-primers (A&B; P&Q; A&Q; P&B) PCR amplifications were performed in the hemophilia A patients as well as in obligate carriers DNA samples. Successful amplification required introduction of some modifications of the original protocol. The mostreproducible and uniform results were obtained using two-primers PCR, performed in four single reactions. Thus, a total of 46 DNA samples, 22 were hemizygous for inversion, 6 without the inversion, 14 carriers and 4 non-carriers of inversion. Perfect correlation between genotypes determined using Southern hybridization and LD-PCR was achieved. The optimalized two-primers LD-PCR protocol was used for analysisof 16 DNA samples of severe hemophilia A patients with unknown mutation. Ten cases of inversions and six cases without them were detected. Thus in additional 10 severe hemophilic patients DNA diagnosis was completed. The most successful and reproducible results were obtained performing four single LD-PCR reactions with combinations of two-primers A&B; P&Q; A&Q, and P&B in each DNA sample and thisapproach is recommended for routine using in clinical practice.
Correspondence to: Dr. Helena Poláková, Institute of Molecular Physiology and Genetics, Slovak Academy of Sciences, Vlárska 5, 833 34 Bratislava 37, Slovakia E-mail:


Poláková et al.

Key words: Hemophilia A — Inversion — Long distance – PCR — F 8C — DNA diagnosis Introduction Hemophilia A is anX-linked recessive bleeding disorder, caused by a deficiency in an activity of a coagulation factor VIIIC (F 8C) gene, affecting 1 in 5,000 to 10,000 male births (Antonarakis et al. 1985). F 8C gene (located at band Xq28) is 186 kb long and has 26 exons that code for the FVIII protein, consisting of 2332 amino acids (Gitschier et al. 1984). Unrelated patients were generally expected to carry differentmutations, and supporting this, a variety of F 8C gene defects has been described (Tuddenham et al. 1994; Antonarakis et al. 1995; HGMD 2003). Most of the mutations are private, they have been found in one or only in a few unrelated families. Recent studies have disclosed a large DNA inversion resulting from a homologous intrachromatid or intrachromosomal recombination between a 9.5 kb region inintron 22 (int22) of F 8C gene (int22h1) and one of two almost identical copies (int22h2 and int22h3) located about 500 and 600 kb telomeric to the F 8C gene (int22h2 and int22h3 are more than 99 % identical to each other). Recombination produces an inversion because the extragenic homologs are in the opposite orientation relative to int22h1 (Lakich et al. 1993; Naylor et al. 1993a,b, 1995). The...
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