Direct detection of factor IX gene deletions in Indian haemophiliacs by multiplex PCR

To the Editor: Haemophilia B is an X linked hereditary disorder caused by a wide variety of mutations distributed over the entire factor IX gene. The latest update of the database lists a total of 2511 mutations of which 896 are unique molecular events while the remaining are repeats (1). The acceptance rate of prenatal diagnosis and carrier detection in haemophilia families in India is very high and we have performed the same by the restriction fragment length polymorphism (RFLP) method (2). However, in case of haemophilia B, the informativeness of the five common polymorphic markers of the factor IX gene was only up to 87% (unpublished observation). We have earlier reported the efficacy of Southern Blotting technique using cDNA probes in picking up the mutations (3). However because of the poor efficiency, laborious and time consuming procedures, this could not be applied on a routine practice to the families. Various mutation screening methods like single strand conformation polymorphism (SSCP), denaturing gradient gel electrophoresis (DGGE), heteroduplex analysis, chemical mismatch cleavage are now available for the screening for the detection of mutations. However all these require sequencing analysis which is the gold standard for mutation identification. But because of the cost and time involved in these elaborate procedures, it has not become a routine application in many of the laboratories all over the world. Recently a multiplex polymerase chain reaction (PCR) for screening of deletions in the factor IX gene has been described (4). Using this protocol, we screened 86 severe haemophilia B patients (F IX < 0.01 U/mL) for the presence of partial or total deletions in the factor IX gene. Nine pairs of primers (Sigma-Genosys, The Woodlands, TX, USA) were used to encompass the entire factor IX gene including the coding regions, intron/exon splice junction, 5¢ and 3¢ untranslated regions. The amplified samples were run in a 8% polyacrylamide gel for the resolution of amplified fragments. For Southern Blotting technique, the restriction enzymes used were Taq1 and Bam H1 and the probes were Probe VIII, a 2.5 kb EcoR1/Hind III fragment of F IX gene (a generous gift from G.G. Brownlee, University of Oxford, Oxford, UK) and F IX c DNA, a 5.0 kb Eco R1/ Hha1 fragment (a gift from J.L. Mandel, INSERM, France). The probes were labeled with P dCTP and hybridized as per the standard protocol. Nine patients (10.5%) showed the presence of deletions of which three were sporadic and six familial. Three patients showed complete deletion of the factor IX gene and the remaining six patients showed partial deletions corresponding to different exons of the factor IX gene. Three patients showed deletions in exons 3 and 4; however all these three patients showed different haplotypes with the five common polymorphic markers of the factor IX gene (Fig. 1). These were further confirmed by Southern Blotting technique using cDNA and genomic DNA probes. In four families similar mutations were detected in one or more family members. None of these patients were positive for factor IX specific inhibitors (prevalence of inhibitors in non-deletional patients is 0.71%).