Conventional long PCR and I-PCR methods are work intensive, prolonged, and sometimes difficult to be standardize. The cDNA method is short, involves 3 short-segment amplifications, and is easy to reproduce.

Hemophilia A is an X-linked bleeding disorder resulting from heterogeneous mutations in the factor VIII (FVIII) gene. The disease is frequently caused by intron 22 inversion mutation in FVIII gene in approximately 50% cases and by intron 1 inversion (Inv1) in 2% to 5% cases with severe hemophilia A. Both inversion mutations occur due to intrachromosomal recombination between inverted repeats found in intronic sequences in upstream regions located telomeric to the FVIII gene and result in a severe phenotype. Frequency of Inv1 mutation varies in different populations. We have analyzed the frequency of the Inv1 in a group of 110 cases with Hemophilia A from North Indian Population and found 4 inversion-positive cases (3.6%). The Inv1 genotyping was further applied to analyze the hemophilia carrier status of the mother and proband's sister. Direct mutation detection strategy is more perfective in diagnosis of cases, carriers, and in prenatal diagnosis of hemophilia A.

The present study assessed the frequency of intron 22 inversion mutation (Inv 22) in north Indian population with a cost analysis of different methods used for Inv 22 detection. We assessed the frequency of intron 22 inversion mutation in a series of 181 cases with hemophilia A and also compared methods used for detection of the mutation including the long-distance PCR, Southern blot analysis, and inverse PCR in terms of cost, infrastructure, and technical input as well as turnaround time. The study group comprised 102 severe cases and 79 moderate cases of hemophilia A from a north Indian population of which 77 cases tested positive for Inv 22. The observed frequency of Inv22 mutation was 42.5%. Inv 22 resulted in a more severe phenotype and lower FVIII bioassay levels as compared to Inv 22 negative cases. Inv 22 positive cases also frequently presented with bleeding episodes at birth and the mean age for commencement of bleeding was lower (19 months) as compared to Inv-negative cases (50 months). The mean frequency of Inv 22 in cases with hemophilia A in a worldwide review is 44.25% of hemophilia A. Inv 22 can be conveniently detected by using the inverse PCR method. This technique is easy to standardize and lowest in cost.

Hemophilia A is a bleeding disorder caused by heterogeneous mutations of the factor VIII gene. A total of 60 unique mutations have been identified in exon 16. The current study was done with the objective of detecting small mutations in exon 16 of factor VIII gene in Indian cases with hemophilia A and to further analyze structural and functional alterations in protein structure. In all, 40 cases with mild and moderate hemophilia A, negative for intron 22 inversion mutations were screened with single-strand conformational polymorphism (SSCP) for point mutations in the exon 16 region. Two cases from unrelated families showed the presence of a missense mutation due to conversion of CGT to CAT at codon 1781 in which arginine was replaced by histidine residues, resulting in deficiency in A3 domain function. Small mutation detection can be achieved using a low-infrastructure SSCP-DNA sequencing protocol in developing countries. Protein modeling predicts structural and functional changes defining causative mutations.

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