Identification of ten large deletions and one duplication in the F8 gene of eleven unrelated Iranian severe haemophilia A families using the multiplex ligation‐dependent probe amplification technique

The most common mutations in the F8 gene causing severe haemophilia A are the intron 22 and intron 1 inversions. In addition, a large number of other types of mutations in this gene may cause different types of severe, moderate or mild disease. Using conventional genomic DNA amplification followed by DNA sequencing fails to identify the causal mutation in a small number of haemophilia A patients which hampers the genetic counselling, carrier detection and prenatal diagnosis of such families, particularly those with severe disease. A recently described method of multiplex ligation-dependent probe amplification (MLPA) has become available, and several investigators have reported detection of a number of deletions and duplications in haemophilia patients using this technique [1–3]. This method relies on the relative variation in copy numbers of the target sequence in patients in comparison with the number of copies in the MLPA PCR products of normal controls, and it can identify large deletions or duplications in patients, and particularly in their female relatives. Since 2001, using a comprehensive stepwise approach for mutation detection, 469 unrelated Iranian haemophilia A patients, including 347 severe haemophilia A families have been investigated at the Iranian Comprehensive Haemophilia Care Centre (ICHCC). This approach which resulted in detection of a diverse group of mutations, including 125 novel mutations [4,5], included the assessment of intron 22 and intron 1 inversions, prescreening conformation sensitive gel electrophoresis (CSGE) followed by direct DNA sequencing of the detected defective exons and DNA sequencing of all of the F8 gene exons when no defective exon was detected by CSGE study. Other approaches such as investigation by mRNA were not available at ICHCC. In 10 families, when one or more exons did not amplify, the possibility of a large deletion was considered. In the remaining 1 family in which all exons were amplified and no heteroduplex formation was observed by CSGE and no mutation could still be discerned by direct sequencing, the possibility of exon duplication was suspected. Although some results have been available on these families from previous informative linkage analysis investigations, no definite mutation had been detected to confirm the carrier status of females in these families. In this letter, we present the results of MLPA investigations of 30 individuals from 11 unrelated families with severe haemophilia A. This study involved investigation of 10 patients with severe factor VIII:C (FVIII:C < 0.01 U mL) haemophilia A (8 probands and 2 affected uncles) and 20 relatives who were either investigated for confirmation of the mutation or had enquired about their carrier status. The members of these families were from different parts of Iran and there was no apparent relationship between any of the eleven pedigrees. Blood samples were collected from consenting patients and their relatives. FVIII:C was measured by a chromogenic assay, and FVIII inhibitors were monitored by the Nijmegen modification of the Bethesda method. Genomic DNA was extracted from anticoagulated peripheral blood according to the standard phenol-chloroform DNA extraction protocol. MLPA was performed following the manufacturer s instructions using SALSA MLPA kit p178 (MRC-Holland, Amsterdam, the Netherlands). Briefly, 500 ng of genomic DNA was denatured and then hybridized with SALSA MLPA probes at 60 C for 16 h. Following ligation at 54 C for 15 min, PCR was performed in a Gene Amp PCR system 9700 (Applied Biosystems, Foster City, CA, USA) using universal 6-FAM labelled primers supplied with the kit. Fluorescent amplification products were subsequently separated by capillary electrophoresis on an ABI 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) and analysed using the Genemapper V4.0 software. DNA copy number was estimated using Coffalyser V9.4, which quantifies the ratio of peak areas in test samples over the peak areas of normal controls in each target sequence. A ratio close to zero suggests the deletion of exon/s and a ratio close to 0.5 indicates the presence of deletion in the heterozygous state. In the case of hemizygous duplication of an exon, the ratio is doubled and in a heterozygous duplication state, it is near 1.5. MLPA was repeated several times on a second sample in individuals suspected of deletions, duplications or with ambiguous results. Where possible, the identified mutation in a family was confirmed on a second sample from the patient and authenticated in another affected family member. Results of all the investigations, including 10 large deletions and one duplication in the F8 gene, are summarized in Table 1 and examples of MLPA results for six families are presented in Fig. 1. Among 11 families studied, index patients of families 7, 8 and 9 had either died, or were not available for MLPA study consequently, their obligate carrier relatives were investigated instead. Families 2, 5, 6, 7, 8 and 9 were previously investigated by the Bcl I linkage analysis method, and in two informative families (7 and 8), the MLPA results were in agreement with linkage analysis findings. Although the combination of exon non-amplification together with linkage analysis results in these families could have been valuable for genetic counselling, demonstration of actual mutation segregation in the family provided a more precise direction for prenatal diagnosis, which removed any ambiguity in the status of female carriers. In other families with non-informative polymorphic markers, or in families with sporadic haemophilia who had waited for a definitive diagnosis for years, this methodology has proved invaluable and easy to perform. Multiplex ligation-dependent probe amplification has proved to be a relatively easy method to perform in laboratories with access to a DNA sequencer. Identification of the carrier status of females with deletions or duplications in families with non-informative polymorphic Correspondence: Saeed Reza Ghaffari, Department of Medical Genetics, Tehran University of Medical Sciences, Tehran, Iran. Tel.: +98 21 66935655; fax: +98 21 66933336; e-mail: saeed@ghaffari.org