LAUSR

Various mutations are associated with haemophilia A. The mutational heterogeneity and the size of the gene complicate molecular studies [1]. It has been reported that approximately 45% of individuals with severe haemophilia A has an inversion in intron 22 of the FVIII gene [2]. The detection of mutations plays an important part in predicting treatment outcome, as FVIII mutations have been associated with FVIII inhibitor development [3,4]. Furthermore, mutation detection allows for carrier and prenatal diagnosis to be conducted [5]. The intron 22 inversion (inv22) was first detected by means of a Southern blot assay [2]. This technique proves to be accurate, but has the disadvantage of being time-consuming and labour intensive [6]. The availability of Southern blot reagents has also become a major obstacle. Newer methods such as long-distance polymerase chain reaction (LD-PCR) and inverse PCR (I-PCR) have been described to detect the inversion [7,8]. However, LD-PCR is reportedly difficult to standardize and is sensitive to reductions in DNA quality [6,9]. I-PCR involves three steps including restriction, self-ligation and standard PCR analysis. Although I-PCR seems to be reliable, it is time-consuming and requires multiple steps [6]. We had trouble implementing the I-PCR method in our laboratory at the Universitas Academic Hospital in Bloemfontein, South Africa. With the multiple steps involved, without a quality check step built into the method, troubleshooting proved to be almost impossible in our suboptimally resourced setting. DNA-based analysis of the inversion is also complicated by the size of the FVIII gene (186 kb), intron 22 (32.4 kb) and the homologous sequence involved in the inversion (9.5 kb) [10,11]. Therefore, our aim was to develop a method that is not only effective in detecting inv22 but also more rapid and cost-effective. It has been reported that individuals with the intron 22 inversion produce two FVIII exoncontaining mRNAs, namely FVIIII22I and FVIIIB respectively. These two FVIII exon-containing mRNAs express the full-length FVIII amino acid sequence, but are non-secretory polypeptide chains [12]. The amplification between the boundary of exons 22 and 23 of the FVIII gene in the mutant phenotype (intron 22 inversion) yields no product, as it is separated from each other [13]. However, it was found that the FVIIII22I transcript contained an additional exon, termed exon 23c. Sequence analysis revealed that exon 23c expressed 16 amino acids that were adjacent to the amino acids expressed for by exon 22 of the wild-type FVIII gene [12]. Subsequently, using this sequence, we designed primers that amplify a region that cover the splice site between the wild-type exon 22 and exon 23, as well as primers that amplify the splice site between the wild-type exon 22 and mutant exon 23c (Table 1). We selected two non-related severe haemophilia A patients (one black African and one Caucasian) who have been confirmed to be inv22 positive with Southern blot to set up the method. A normal healthy volunteer was used as control. Subsequently, we tested 10 severe haemophilia A patients with an unknown inv22 status, as well as two putative carriers also with an unknown status (mothers of patient 3 and patient 6 respectively). The fractionated white blood cells of each sample was stored in RNAlater (Ambion, Life Technologies, Waltham, Massachusetts, USA) solution and RNA was extracted using the RiboPure-Blood kit (Ambion, Life Technologies, Waltham, Massachusetts, USA). The mRNA was converted into cDNA using the High Capacity RNA-to-cDNA Kit (Applied Biosystems, Foster City, California, USA). Two separate PCR reactions were set up for each sample, one using wild-type exon 22 forward and exon 23 reverse primers, and one using a wild-type exon 22 forward and a mutant exon 23c reverse primer. A temperature gradient PCR was performed in order to determine the optimal annealing temperature (Data S1). The PCR product was loaded on a 2% agarose gel containing ethidium bromide for electrophoresis. After electrophoresis, the fragments were visualized under UV light using a Kodak Gel Logic 212 gel documentation system (Carestream Molecular Imaging, Rochester, NY, USA). Correspondence: Walter Janse van Rensburg, Department of Haematology and Cell Biology (G2), Faculty of Health Sciences, University of the Free State, 205 Nelson Mandela Drive, 9301 Bloemfontein, South Africa. Tel.: +27 51 405 3098; fax: +27 51 444 1036; e-mail: jan [email protected]