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Factor IX Cardiff: a variant factor IX protein that shows abnormal activation is caused by an arginine to cysteine
M B Liddell1, I R Peake, S A Taylor
1Department of Haematology, University of Wales College of Medicine, Cardiff, U.K.
Insights
Researchers identified a genetic mutation in a patient with haemophilia B. This mutation, a C to T transition, alters a key amino acid, confirming faulty factor IX activation.
Area of Science:
- Biochemistry
- Genetics
- Hematology
Background:
- Haemophilia B is a genetic bleeding disorder caused by deficiency in coagulation factor IX.
- Cross-reacting material positive (CRM+) haemophilia B indicates the presence of factor IX antigen but impaired function.
- Understanding the molecular basis of CRM+ haemophilia B is crucial for diagnosis and treatment.
Purpose of the Study:
- To investigate the molecular defect in a CRM+ haemophilia B patient with aberrant factor IX activation.
- To identify the specific genetic mutation responsible for the observed functional defect.
Main Methods:
- Factor IX activation studies using barium chloride eluates and SDS-PAGE.
- Detection of factor IX antigenic material via Western blotting with radiolabeled antibodies.
- Polymerase chain reaction (PCR) amplification of exon 6 of the factor IX gene.
- DNA sequencing of the amplified fragment to identify mutations.
Main Results:
- Activation studies revealed a stable reaction product suggesting a light chain-activation intermediate in one CRM+ haemophilia B patient.
- PCR amplification yielded a 489-nucleotide DNA fragment from the patient's genomic DNA.
- DNA sequencing identified a single C to T transition at position 145, changing Arginine to Cysteine in the factor IX activation peptide.
Conclusions:
- The identified C to T transition results in an amino acid substitution (Arg145Cys) within the factor IX activation peptide.
- This mutation explains the faulty factor IX activation observed in the patient.
- The findings confirm the link between the genetic defect and the clinical phenotype of haemophilia B.
Abstract:
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.