MLPA assay in F8 gene mutation screening

Besides intron 22 inversion with a reported prevalence of 40–50% in patients with severe haemophilia A (HA) [1], and intron 1 inversion with a reported prevalence of about 2–5% [2], the gene alterations causing HA are spread throughout the gene [3]. Large deletions of one or more gene exons account for about 5% of all cases of severe HA [3]. Over the last few years, we characterized the mutation in a series of patients with severe HA, and traditional PCR failed to amplify one or more exons in four of the 126 patients. As mRNA was not available, we decided to search for further validation. Recently, multiplex ligation-dependent probe amplification (MLPA) has been broadly applied to gene mutation screening to detect exon deletions and duplications [4]. In an ongoing effort to make the technique easier to perform, modifications were made and new MLPA procedures were designed, including the one which contains probes for each of the 26 exons of the F8 gene (SALSA P178 FVIII MLPA kit; MRC-Amsterdam, The Netherlands). MLPA evaluation of the multiple amplification relies on the intensity ratio of sample to control, i.e. the relative quantity of each of the PCR products is proportional to the number of copies of the target sequence. Therefore, a ratio proximal or equal to zero indicates deletions of exon/s, a ratio close to 0.5 identifies the presence of the deletion in the heterozygous state. A ratio close to 1.5 identifies the duplication of the exon/s. We present results from a retrospective study that was carried out on patients in whom a deletion or duplication had previously been hypothesized on the basis of PCR alone. MLPA was also applied to the definition of carrier status in the female relatives of the same patients. In fact, the main obstacle to their counselling is the impossibility to demonstrate a heterozygous status because of the amplification of the exon/s that is/are present on the normal X chromosome. Real-time PCR offers the option of carrying out quantification of the amplified material, but simultaneous detection of the gene copy number requires critical consideration of the standardization and reference-curve protocols. In our retrospective study, we demonstrated that carrier status determination via MLPA is straightforward and correct. Multiplex ligation-dependent probe amplification product was performed according to the instructions of the manufacturer. In more detail, 100–150 ng of genomic DNA were denatured and hybridized with SALSA MLPA probes (60°C overnight). After ligation at 54°C for 15′, the samples were amplified using a Biometra T personal Thermocycler (Biometra, Göttingen, Germany). One and a half microlitre of PCR products was mixed with 0.3 μL of ROX-500 labelled internal size standard, separated by capillary electrophoresis on a Genetic Analyser 3130 (Applied Biosystem, Foster City, CA, USA), and analysed using the genemapper software (Applied Biosystem). DNA dosage was estimated using coffalyser software (version 6; MRC, Amsterdam, The Netherlands). In the presence of values suggesting deletion/insertion(s) or ambiguous values, MLPA analysis was repeated on independent samples. Traditional PCR on genomic DNA from a severe sporadic patient failed to amplify the first five exons: MLPA confirmed the conjectured deletion (Fig. 1, panel a). MLPA was applied on a DNA sample extracted from his mother thus allowing us to ascertain her heterozygous state (Fig. 1, panel b). To verify F8-MLPA applicability to DNA extracted from chorionic villi samples (CVS), we retrospectively examined the DNA from the CVS of her previous pregnancy. The deletion was clearly demonstrated in the foetus. MLPA assays in five samples. Panels (a) and (b) show the deletion of the first five exons in the hemizygous (patient 1) and the heterozygous state (patient's 1 mother: ratio 0.5 ± 0.1). Panels (c) and (d) depict the deletion of exon 23 in the hemizygous (patient 2) and the heterozygous state (patient's 2 daughter: ratio 0.5 ± 0.09). Panel (e) reports the exon 13 duplication in a male patient (ratio 1.8 ± 0.04). Y-axis, F8 copy number and standard deviation; X-axis, F8 exons from 1 to 26; arrows, deleted or duplicated regions. Classic PCR technique failed to amplify exon 23 in this severe case: MLPA detected the deletion (Fig. 1, panel c). The patient's daughter (obligate carrier) (Fig. 1, panel d) would have had the possibility to undergo prenatal diagnosis only via RFLPs analysis. MLPA, on the other hand, would offer direct visualization of the mutation. Exon 22 deletion was identified as the causative mutation in both patients. MLPA was adjuvant in identifying carrier status in a female relative of patient 4, thus avoiding performance of a time consuming laborious study. Multiplex ligation-dependent probe amplification efficiency was also tested in a series of patients from northern Italy where we found that the duplication of exon 13 is a frequent mutation [5]. The MLPA technique identified the duplication showing a sample/control ratio >1.4 in all positive patients. One example is shown in Fig. 1, panel e. On the basis of these results, we screened by the MLPA technique 10 never diagnosed severe HA who were negative for the two main intron inversions, and we found an exon 25 deletion in one of them. Therefore, on the basis of these data, we can draw some suggestions and conclusions: (i) the MLPA procedure can be used as a tool to confirm the presence of a deletion or a duplication in patients for whom an mRNA sample is not available; (ii) carrier status can easily be carried out using the MLPA technology and can be identified in females who are non-informative for the intragenic polymorphisms; and (iii) MLPA can be applied also to prenatal diagnosis. From a strictly technical point of view, MLPA can be performed at any laboratory where traditional PCR procedures and sequencing are part of the molecular biology routine. Its feasibility, reliability and moderate cost led us to introduce the technique in the gene screening of all patients we found to be negative for the intron 22 and intron 1 inversions, prior to starting the mutation identification on the entire gene. The Authors wish to thank Mrs Barbara Caruzzo for her excellent assistance in preparing the manuscript. The authors stated that they had no interests which might be perceived as posing a conflict or bias.