New protocol for detection of intron 22 inversion mutation from cases with hemophilia A

Abstract

Background: Hemophilia A is a X-linked recessive bleeding disorder characterized by qualitative and quantitative deficiency of factor VIII resulting from heterogeneous mutations in the factor VIII gene located in the Xq28 region. Intron 22 inversion (Inv22) mutation is one of the major causes of the protein alteration in factor VIII; its frequency is 40% to 50% in severe patients. Long polymerase chain reaction (PCR) and inverse PCR (I-PCR) have been used for the detection of Inv22 mutation.

Objective: Development of new protocol for detection of Inv22 mutation.

Method: We have designed a new method for the detection of Inv22 mutation in complementary DNA (cDNA) of patients. Real-time PCR targeting exons 21 to 22, 22 to 23, and 23 to 24 of factor VIII gene were used in cases with hemophilia A. Samples that were inversion positive by this new method were cross-checked by the conventional I-PCR method. We observed that region between exons 22 and 23 could not be amplified, while in negative cases and controls a 480 bp product is obtained.

Result: The method was validated in 20 cases with severe hemophilia A by the new cDNA method, and 8 cases were inversion positive, whereas 12 were negative cases. The findings were confirmed by standard I-PCR method. Complete correlation was observed.

Conclusion: 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.

Keywords: cDNA; factor VIII; intron 22 inversion; inverse PCR.