Factor IX Cardiff: a variant factor IX protein that shows abnormal activation is caused by an arginine to cysteine substitution at position 145.
Crude barium chloride eluates prepared from 12 unrelated patients with cross-reacting material positive (CRM+) haemophilia B were activated with celite eluate, the reaction products resolved after reduction by 13% SDS-PAGE, and factor IX antigenic material detected by probing with radiolabelled immunopurified rabbit anti-factor IX antiserum followed by autoradiography. Out of the 12, one sample showed faulty activation with the production of a stable reaction product with a MW compatible with that of a putative light chain-activation intermediate. In order to confirm this, two oligonucleotide primers that bracketed exon 6 of the factor IX gene were constructed and used to prime a polymerase chain reaction on DNA isolated from the patient's peripheral blood leucocytes. A single 489 nucleotide DNA fragment was obtained, gel purified, subcloned into M13, and DNA sequencing carried out on both strands. A single C to T transition was discovered that changed the Arg residue at position 145, the first residue of the first bond in the activation peptide, to a Cys, a result that confirmed the inferences drawn from the activation studies.
Liddell
M B
MB
Department of Haematology, University of Wales College of Medicine, Cardiff, U.K.
Peake
I R
IR
Taylor
S A
SA
Lillicrap
D P
DP
Giddings
J C
JC
Bloom
A L
AL
D000941:Antigens / Q000032:analysis; D001120:Arginine; D001483:Base Sequence; D003001:Cloning, Molecular; D003545:Cysteine; D004247:DNA / Q000235:genetics*; D005091:Exons; D005164:Factor IX / Q000235:genetics*; D005786:Gene Expression Regulation; D002836:Hemophilia B / Q000235:genetics*; D006801:Humans; D008970:Molecular Weight
D000941:Antigens; C061130:factor IX Cardiff; D005164:Factor IX; D004247:DNA; D001120:Arginine; D003545:Cysteine