LETTER TO THE EDITOR Improved assay for genotyping haemophilia A carriers with intron 22 dinucleotide repeat marker A. SAHA, S. MUKHERJEE and K. RAY Human Genetics and Genomics Division, Indian Institute of Chemical Biology, Kolkata, India Haemophilia A, the most common bleeding disorder caused by defects in the Factor VIII gene (FVIII), affects one in 5000 males worldwide. Due to the inherent instability of the gene, caused by the presence of multiple copies of the same genomic regions, de novo mutation accounts for 40–50% of the gene defects. In addition, heterogeneous muta- tions have been detected covering its entire length (186 kb). Thus, the mutation detection is an arduous job requiring a higher investment of time and money relative to marker-based carrier detection to control the disease. The availability of a relatively small number of PCR-based restriction fragment length polymorphism (RFLP) markers and linkage disequi- librium in some of them makes these markers less efficient for carrier-detection. On the contrary, Correspondence: Dr Kunal Ray, Human Genetics and Genomics Division, Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Jadavpur, Kolkata 700 032, India. Tel.: +91 33 2473 0350/3491/6793; fax: +91 33 2473 5197 or +91 33 2472 3967; e-mail: thisiskr@rediffmail.com or kunalray@vsnl.com Accepted after revision 7 December 2005 120 6400 (b) (a) 4800 3200 1600 0 0 1200 2400 3600 4800 0 1500 3000 4500 6000 160 144 144 148 148 146 146 Fig. 1. Genotyping using the Factor VIII gene intron 22 dinucleotide-repeat marker. Panel a, the primer pair used in the initial report [2] and the redesigned primer pair are shown by dotted and solid arrows respectively on the segment of the nucleotide sequence flanking the dinucleotide repeat region. Locations of the forward and reverse primers are indicated by arrows drawn above and below the sequence respectively. For PCR the forward primer was fluorescent labelled with 5¢-FAM. Panel b, typical chromatograms showing the allele sizes (indicated by arrowheads) in three heterozygous females. Smaller peaks preceding both the alleles are because of Ôstutter bandsÕ typically seen in PCR-amplified dinucleotide repeat markers irrespective of the method of analysis. 200 Ó 2006 Blackwell Publishing Ltd Haemophilia (2006), 12, 200–201 DOI: 10.1111/j.1365-2516.2006.01205.x