Hemophilia A (HA) is an X-linked hereditary disorder characterized by bleeding of variable severity through mild, moderate to severe owing to large range of mutations in the Factor VIII (F8) gene (Bowen [2002]). All kind of F8 mutations, except repeats, have been reported for HA, in total up to 2370 (Human Genome Mutation Database [2005]). A preliminary study was conducted in our lab for identification of mutations in F8 gene in Pakistani HA patients. Correlation of F8 mutations with clinical manifestation of HA patients was the main objective of the study. Blood samples were collected from 62 HA patients from all over the Pakistan and clinical history of all HA patients was recorded (only patients frequently visiting medical centers for the replacement of Factor VIII were selected for the study). Genomic DNA was extracted from whole blood by standard organic procedure. Specific primers (Figure 1) were designed using “Primer3” (http://biotools.umassmed.edu/bioapps/primer3_www.cgi) to amplify the coding region of F8 gene; amplified products were sequenced by ABI 310 and ABI 3100 sequencer (Applied Biosystems, Carlsbad, CA, USA). The sequencing results were visualized using “Chromas 2.33” software (Applied Biosystems) and mutations were detected using “BLAST” software available on the NCBI website (http://balst.ncbi.nlm.nih.gov/Blast.cgi). Three novel mutations (1 deletion; 2 point mutations) were detected in four sporadic HA patients, all from different ethnic backgrounds (Table 1). The deletion of T in exon 7 within the A1 domain represents a frame-shift change disrupting the protein structure and function, which result in severe manifestation of the disease. A missense point mutation in the A3 domain occurs in codon 1907 at nucleotide number 5720, replacing Serine with Isoleucine, and confers a moderate type of severity. It should be noted that Serine is a polar and acidic amino acid while Isoleucine is a nonpolar and basic amino acid. A nonsense point-mutation was found in two unrelated patients in the C3 domain (exon 26) and was correlated with moderate clinical findings. Beside these mutations, 27 common SNPs were also detected in F8 gene for the studied patients (Table 2). The allelic data and accession numbers of these SNPs were collected from Ensembl Genome Browser (Ensembl [2000]). The results of the study will form the basis not only for an enlarged study but also for diagnosis and genetic counseling of classical hemophilia in Pakistan.

Figure 1
figure 1

Primers used in the study.

Full size image
Table 1 Novel mutations in F8 gene
Full size table
Table 2 Common SNPs in F8 gene (exonic region)
Full size table