Severe Factor X Deficiency Research Articles

Second Trimester Antenatal Diagnosis in Rare Coagulation Factor Deficiencies

Prenatal diagnosis is sought after for those genetic disorders, whose management is not satisfactory either because of the outcome or owing to extreme cost involved in the management of the patients affected by a specific disorder. Severe hemophilia and homozygous thalassemia are the 2 disorders for which there is an increasing demand for prenatal diagnosis in India. Rare severe deficiencies of coagulation factor X (FX) and factor VII (FVII) may present with severe bleeding manifestations. Because of their rarity the laboratory offering prenatal diagnosis for severe hemophilia and thalassemia may not be in a position to provide genetic diagnosis in the fetus. In this communication, we describe 2 families, 1 with an index patient of severe FVII deficiency and the other with severe FX deficiency where successful prenatal diagnosis was given after cordocentesis between 17 and 19 weeks using a battery of coagulation factor assays. Follow-up studies were performed 3 to 4 months after delivery and the diagnoses were reconfirmed on these babies by a repeat factor assay for FX and FVII deficiency, respectively.

P1749 Detection of the Factor V Leiden mutation in septic patients

Factor X (FX) is a vitamin K-dependent plasma zymogen, which following activation to factor Xa (FXa), converts prothrombin to thrombin in the blood clotting cascade. It was recently demonstrated that a natural variant of FX carrying the Asp-185 deletion (FX-D185del, chymotrypsinogen numbering) was associated with mild bleeding in a patient with severe FX deficiency. In this study, we expressed FX-D185del in mammalian cells and characterized its properties in appropriate kinetic assays in purified systems. We discovered that while the FX variant can be normally activated by physiological activators; both amidolytic and proteolytic activities of the mutant are dramatically impaired. Interestingly, factor Va (FVa) significantly improved the proteolytic defect when the mutant protease was assembled into the prothrombinase complex. Thus, in contrast to > 50-fold catalytic defect in the absence of FVa, the variant activated prothrombin with only ~ 2.5-fold decreased catalytic efficiency in the presence of the cofactor. The FXa variant dramatically lost its susceptibility to inhibition by antithrombin and tissue factor pathway inhibitor, thus exhibiting ~ 2-3 orders of magnitude lower reactivity with the plasma inhibitors. Further studies revealed that Na+ no longer activates the variant protease, suggesting that the functionally important allosteric linkage between the Na+-binding and the P1-binding sites of the protease has been eliminated. These results suggest that the lower catalytic efficiency of FXa-D185del in the bleeding patient may be partially compensated by the loss of its reactivity with plasma inhibitors, possibly explaining the basis for the paradoxical severe FX deficiency with only mild bleeding tendency for this mutation.

A Novel 5bp Deletion Mutation in the Factor X (FX) Gene, Designated FX-Augusta, Causes Severe FX Deficiency Possibly by a Unique Mechanism Involving mRNAs that Lack Inframe Stop Codons.

Due to its position in the coagulation cascade, factor X (fX), a circulating zymogen is activated during hemostatic challenges to its serine protease fXa first via the extrinsic pathway's tissue factor/factor VIIa complex and next by the intrinsic Xase complex of factors Ixa and VIIIa on phospholipids membranes. Among the congenital factor abnormalities manifesting as hemorrhagic predispositions, fX deficiency states, which are often autosomal recessive and associated with consanguinity, are among the rarest. Since concurrent studies on the unrelated Stuart and Prower kindreds in the southeastern United States and London (England), respectively, led to the discovery of fX, it is also known as the Stuart-Prower factor. Only 59 distinct fX gene mutations have been described worldwide since its discovery in the 1950s. We report a novel mutation within the encoding structural locus that we have designated FX-Augusta. The phenotype and genotype of the proband, a 14 year-old African-American boy, were studied and we are currently in the process of obtaining samples from the patient's parents and siblings. The proband has experienced severe bleeding throughout his life with his first episode occurring three days after birth following circumcision. He experiences ~30 bleeds per year, mainly into joints and muscles, which occur spontaneously or following minor trauma. Although the patient appears to be mildly mentally retarded a formal workup has not occurred. On laboratory testing, a severely prolonged PT of 137.4 seconds and aPTT of 112.8 seconds were observed. While clotting-based fX activity (fX:C) levels were consistently <1% when using fX-deficient plasma and the PT-reagent, chromogenic factor X activity levels were ~5%. In light of these findings, we suspect the patient may have a CRM-reduced, or less-likely a CRM-positive, deficiency. However, the patient's fX antigen level (fX:Ag) has not yet been measured. Molecular analysis of the patient's fX gene by direct automated sequencing of PCR-amplicons representing ~200-bp of the promoter, all exonic and flanking intronic regions and ~200-bp of the flanking 3′-genomic DNA, disclosed a novel 5-bp deletion mutation (ATGCC) in exon 8 (del[1350–1354]; 1 is the transcription start site) that disrupts codons 477 and 478, and results in a frameshift. The patient is homozygous for this deletion and an additional 8-bp insertion mutation (TGCCGCCA) located downstream in the 3′-flanking genomic DNA between nucleotides 28376 and 28377 (GenBank #: AF503510). Previously, no insertions and only 4 small deletions have been reported for this gene. Interestingly, the wildtype fX mRNA has a 14-base 3′-UTR as the AUUAAA hexanucleotide polyadenylation element in the fX gene is located 1-bp upstream of the stop codon. Because the resultant frameshift predicts the use of an alternative stop codon 75 nucleotides downstream, 3′ of the in vivo polyadenylation site, this mutation may result in mature cytoplasmic transcripts that lack in-frame stop codons. To our knowledge this has not been reported. Since non-sense mediated decay has not been described for mutations that necessitate the use of downstream stop codons, if we are correct, translation of the mutant mRNAs would not terminate normally and the ribosome would translate the poly(A)-tail into a C-terminal poly-lysine stretch. We hypothesize that the mutant fX pre-mRNAs will be cleaved and polyadenylated immediately 3′ of nucleotide 28346, the same site utilized in vivo during 3′-end processing of the wildtype pre-mRNAs for the following reasons. First, there are no downstream signals capable of supporting 3′-end formation. Specifically, an examination of >1kb of the flanking 3′-genomic DNA failed to identify consensus AAUAAA polyadenylation signals or variants of this highly conserved element. Second, the native fX 3′-end signals appear to be “strong” or efficiently utilized, as no alternative polyadenylation was found in a comprehensive review of the fX cDNAs in the NCBI database, which included 6 full-length cDNAs and >20 3′-ESTs containing polyA-stretches on the 3′-end of high-quality fX mRNA sequence. Finally, neither the causative 5-bp deletion nor the 8-bp insertion disrupts these native 3′-end signals. Nevertheless, the molecular studies underway, including fX:Ag measurements, 3′-end characterization of the mutant mRNA via 3′-RACE and western blotting, should help to distinguish between the possible molecular mechanisms.

Blood Coagulation Factor X Deficiency Causes Partial Embryonic Lethality and Fatal Neonatal Bleeding in Mice

Mice with a total deficiency in blood coagulation Factor X (FX) were generated by targeted replacement of an 18-kb fragment of the FX gene, comprising all exons encoding the mature FX protein, with a neo(r) cassette. The genotype distribution among the offspring from heterozygous breeding pairs suggested that FX deficiency resulted in partial embryonic lethality, with approximately one-third of the FX-/- embryos dying around embryonic day (E) 11.5-12.5. Two of 44 non-resorbed FX-/- embryos analyzed at these stages showed signs of massive bleeding, one of which into the brain ventricles, but no histological defects in the vasculature of these embryos or their yolk sac were observed. The remainder of the FX-/- embryos appeared normal and survived to term, but the majority of neonates (90%) died within 5 days, most frequently from intraabdominal bleeding. The remaining FX-/- animals succumbed between postnatal day (P)5 and P20 with intraabdominal, subcutaneous, or intracranial bleeding or a combination thereof. The lethal phenotype of the FX-/- mice illustrates the importance of FX function in embryonic and postnatal survival and demonstrates that these mice serve as effective models of the bleeding disorders observed in severe FX deficiency in humans.