ORIGINAL ARTICLE Women with bleeding disorders Heterozygous large deletions of Factor 8 gene in females identified by multiplex PCR-LC A. PAVLOVA,* T. FO ¨ RSTER,  D. DELEV,* J. SCHRO ¨ DER,* O. EL-MAARRI,* C. MU ¨ LLER-REIBLE   and J. OLDENBURG* *Institute of Experimental Haematology and Transfusion Medicine, University Clinic Bonn, Bonn; and  Institute for Human Genetics, University of Wu ¨ rzburg, Biocenter, Am Hubland, Wu ¨ rzburg, Germany Summary. Haemophilia A is the most common X-linked recessive bleeding disorder. In 5% of severely affected patients the mutations responsible for the disease are large deletions encompassing from one exon to the complete Factor 8 (F8) gene. Large deletions in a male haemophilic patient are easily detected by the absence of the corresponding PCR product. However, in female carriers, identification of the various heterozygous large deletions is difficult representing a major limitation to accurate carrier diagnosis. The deletion is masked by the presence of the second allele that serves as template for the PCR reaction. Quantitative PCR can differentiate between the presence of one or two alleles. Here we report an assay based on multiplex amplification of several exons of the F8 gene of various length and subsequent quantitative evaluation of the amplicons by liquid chromatogphy (LC). Using this approach we achieved an accurate classification of 16 female carriers and eight non-carriers for deletions in the F8 gene in 19 investigated families. One mother and one grandmother were classified as non-carriers, under- lining the high de novo mutation rate of large deletions in female germ cells. The large deletions in three families were confirmed by fluorescent in situ hybridization. In conclusion, the multiplex PCR-LC technique represents a rapid, simple and reliable method for detection of heterozygous large deletions in female carriers. Keywords: haemophilia A carriers, large deletions, liquid chromatogphy Introduction Haemophilia A (HA) is the most common, severe bleeding disorder affecting one in 5000 male new- borns. The X-linked disease is caused by a broad spectrum of mutations in the Factor 8 (F8) gene leading to an absent or dysfunctional protein. The F8 gene spans 186 kb on chromosome Xq28 and consists of 26 exons. Large deletions are not uncommon events in HA and occur in approximately 5% of patients with a severe form of the disease [1]. More than 100 unrelated patients with large dele- tions, encompassing one or more exons of the F8 gene, have been reported in the international F8 mutation database [2]. Because of the X-linked inheritance, female relatives of HA patients with large deletions are at risk for being carriers. There- fore, the availability of an accurate carrier test for the various heterozygous large deletions and appropriate genetic counselling are important for comprehensive care in haemophilia. Large deletions of F8 in males are readily detected by the absence of a PCR product of the deleted fragment. However, to identify large deletions of complete exons on the background of a normal allele – as it is the situation in a carrier – is challenging as the presence of the non-deleted allele masks the deletion in a standard PCR. Indirect gene analysis by classical segregation investigation of polymorphisms for tracing the F8 gene defects within a pedigree is limited because it requires examination of several family members, may be uninformative in approxi- mately 25% of potential carriers and cannot identify de novo large deletion that preferably originate in female germ cells [3–5]. Historically, the method of Correspondence: Prof. Dr. Med. Johannes Oldenburg, Institute of Experimental Haematology and Transfusion Medicine, University Clinic Bonn, Sigmund-Freud-Str. 25, Germany. Tel.: +49 228 287 5175; fax: +49 228 287 14783; e-mail: johannes.oldenburg@ukb.uni-bonn.de Accepted after revision 29 November 2007 Haemophilia (2008), 14, 599–606 DOI: 10.1111/j.1365-2516.2007.01629.x Ó 2008 The Authors Journal compilation Ó 2008 Blackwell Publishing Ltd 599