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


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Department of Pathology, Genetics Lab, Ram Manohar Lohia Institute of  Medical Sciences, Lucknow, Uttar Pradesh, India.
Praveen
P
Kumar
Department of Pathology, Genetics Lab, Ram Manohar Lohia Institute of  Medical Sciences, Lucknow, Uttar Pradesh, India drnuzhathusain@hotmail.com.
Nuzhat
N
Husain
Department of Pathology, Genetics Lab, Ram Manohar Lohia Institute of  Medical Sciences, Lucknow, Uttar Pradesh, India.
Priyanka
P
Soni
Department of Pathology, Genetics Lab, Ram Manohar Lohia Institute of  Medical Sciences, Lucknow, Uttar Pradesh, India.
Nuzhat Jahan
NJ
Faridi
Petroleum Toxicology Division, Indian Institute of Toxicology Research,  Lucknow, Uttar Pradesh, India.
Sudhir Kumar
SK
Goel
D041321:Chromosomes, Human, X / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods; D005169:Factor VIII / Q000235:genetics*; D005260:Female; D006467:Hemophilia A / Q000235:genetics*; D006801:Humans; D007438:Introns / Q000235:genetics*; D008297:Male; D016133:Polymerase Chain Reaction / Q000379:methods*; D057345:Sequence Inversion
C078147:F8 protein, human; D005169:Factor VIII
cDNA; factor VIII; intron 22 inversion; inverse PCR