ALBERT

Description: Abstract 1302 Poster Board I-324 Combined vitamin K-dependent (VKD) coagulation factor deficiency is an autosomal recessive bleeding disorder associated with defects in either the γ-carboxylase (GGCX) which carboxylates VKD proteins to render them active or the vitamin K epoxide reductase (VKORC1) which supplies the reduced vitamin K cofactor required for carboxylation. Such deficiencies are rare, and due to mutations within either gene. Of note some mutations within the GGCX gene have recently been found associated with the pseudoxanthoma elasticum (PXE) syndrome, suggesting a role for GGCX in skin development. We report a new case of combined VKD coagulation factor deficiency resulting from two mutations in the GGCX gene, and the first identified in a French child, who exhibited impaired function in hemostatic VKD factors. The propositus exhibited bleeding at sites of venipuncture at birth, and at the age of 3 months was admitted in emergency for spontaneous multiple hematomas of the chest and thighs; recently, at the age of 1 year, he exhibited a hematoma of the wrist following a casual fall. Coagulation and VKD factors were low [PT (Control/patient): 12.8/〉100 sec; II : 3 % ; VII :2% ; X :3%] while the non-VKD factor V was normal (105%). Vitamin K infusion corrected the bleeding tendency as well as coagulation paramenters [PT (Control/patient): 12.8/14.8 sec; II :72% ; VII+X : 62%; factor V remained normal at 102%]. Family analysis revealed that both parents and one brother were unaffected, both clinically and biologically, but that a brother had died of an unexplained abdominal hemorrhage, in the neonatal period. Taken together the clinical family history is consistent with a recessive trait. No sign of PXE was found in the family. DNA sequence analysis of the propositus did not identify any mutations in the VKORC1 gene but revealed two new heterozygous mutations in the carboxylase gene: a G10233T transversion in exon 11 (G1565U in mRNA) that caused an W493C substitution and a C12078T transition in the last exon (15) (C2196U in mRNA) that caused a premature R704stop, presumably deleting the last 55 C-terminal amino acids. W493 is a highly conserved amino acid and its homozygous mutation (for S493) has recently been reported in a case of VKD coagulation factor deficiency associated with PXE. R704 mutation has never been reported previously. Family analysis showed that W493C was transmitted by the father, R704stop by the mother, that the deceased brother carried both mutations, while the unaffected brother carried neither. Mutational analysis was carried out by site-directed mutagenesis of the GGCX cDNA subsequently subcloned into the BacPak8 baculovirus-based vector, and GGCX was expressed in the virally-infected SF21 insect cell line. Protein expression level of the enzyme was assessed by western blotting, and enzymatic activity was evaluated by measuring the incorporation of [14]C-CO2 within the Boc-Glu-Glu-Leu-OMe peptide. No difference in expression level was detected for either mutant protein compared to the wild type protein. However, while no difference in molecular weight was seen between the W493C mutant and wild type on wertern blotting, the R704stop mutation generated a shorter form of carboxylase than wild type, consistent with the predicted shorter reading frame. W493C mutant exhibited a much lower activity than wild type, suggesting a functional role for W493. The activity of the R704stop mutant is currently being assessed, as well as the activity of the coexpressed mutants. Since PXE has been correlated with a W493S mutation, a role for the W493C mutation in future development of PXE in this young patient will be assessed during follow-up. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 4020 Poster Board III-956 Secretory platelet proteins such as the chemokine platelet factor 4 (PF4/CXCL4), are stored in platelet alpha granules from which they are released upon platelet activation. Defects in platelet storage of granule proteins lead to the hereditary bleeding disorder, grey platelet syndrome. The mechanisms of granule storage of platelet proteins remain unknown. We have shown that alpha granule storage of PF4 was dependent upon the 10 amino acid-long Leu41-Lys50 β-loop (El Golli et al, J Biol Chem 280:30329-35, 2005). CXCL4L1 (or PF4var) is a non-allelic variant of PF4, with which it shares high homology (67 identical residues over 70 of the mature sequence) including the same Leu41-Lys50 β-loop, and which is also present in human platelet alpha granules. Recently, Lasagni et al (Blood 109:4127-34, 2007), have shown that contrary to PF4 which follows the granule storage pathway, CXCL4L1 followed the constitutive secretory pathway. This was based on transfection studies in the HEK293 cell line, or on primary vascular cells including human microvascular endothelial cells (HMVECs), human coronary smooth muscle cells or T cells. In these conditions CXCL4L1 and PF4 exhibited distinct intracellular localizations as well as differential secretion: while release in the medium was spontaneous for CXCL4L1, it required agonist-mediated cell activation for PF4. The authors hypothesized that the differences in intracellular trafficking and compartmentalization of CXCL4L1 and PF4 may be due to their signal peptides, which exhibit significant sequence divergence (38%) compared to the mature sequences (4%). In contrast, PF4 and CXCL4L1 share 100% homology within the granule targeting Leu41-Lys50 β-loop. To test whether signal peptides or instead, the Leu41-Lys50 β-loop, specified alpha granule targeting in megakaryocytes, we compared PF4 and CXCL4L1 intracellular trafficking in the Dami cell line, a human megakaryocytic cell line (which stores proteins in alpha granules upon differentiation in the presence of the cytokine thrombopoietin and phorbol myristate acetate). PF4 and CXCL4L1, or constructs with swapped signal peptides and/or deleted Leu41-Lys50 β-loops were fused to the fluorescent reporter proteins GFP and mCherry, respectively, and their intracellular trafficking analyzed by laser scanning fluorescence microscopy. When cotransfected in the human megakaryocytic Dami cell line, PF4 and CXCL4L1 colocalized efficiently to the same granules (over 80%). Colocalization with the endogenous alpha granule resident protein von Willebrand factor was also observed. Deletion of the Leu41-Lys50 β-loop prevented alpha granule subcellular localization of both CXCL4L1 and PF4. Importantly, signal peptide swapping between PF4 and CXCL4L1 did not affect their final granule colocalization. Altogether these results suggest 1) that in a megakaryocytic context, PF4 and CXCL4L1 traffick along the same intracellular pathway, including the alpha granule storage pathway in megakaryocytes, 2) that the shared Leu41-Lys50 β-loop is essential to alpha granule storage of either protein, and 3) that the signal peptides of PF4 or CXCL4L1 do not appear to play any role in final alpha granule localization. The differential subcellular localization of PF4 and CXCL4L1 in non megakaryocytic contexts, if confirmed, would suggest that megakaryocytes possess storage mechanisms distinct from other cell types. Disclosures: No relevant conflicts of interest to declare.

Description: Combined deficiency in vitamin K-dependent (VKD) coagulation factors is an autosomal recessive bleeding disorder associated with defects in either the VKD carboxylase which converts Glus to Glas in VKD proteins to render them active or the vitamin K epoxide reductase (VKORC1) which supplies the reduced vitamin K cofactor required for carboxylation. Such defects are rare, and we now report the fourth case of deficiency caused by mutations in the carboxylase gene. The mutations were identified in a two year old Tunisian girl who exhibited impaired function in several VKD procoagulant and anticoagulant factors that was not restored by vitamin K administration. Sequence analysis of the propositus did not identify any mutations in the VKORC1 gene but, remarkably, revealed three heterozygous mutations in the carboxylase gene, D31N, W157R and T591K within exons 2, 4 and 13, respectively. None of these mutations have previously been reported. Family analysis showed that D31N and T591K were coallelic and transmitted by the mother while W157R was transmitted by the father. The mutations were not found in the genomes of 200 normal subjects, ruling out frequent polymorphisms. Mutational analysis indicated wild type activity for the D31N carboxylase. In contrast, the W157R and T591K enzymes had activities that were, respectively, 7% and 0% that of wild type carboxylase, and their compound heterozygosity can therefore account for defective carboxylation. Residues 157 and 591 are both highly evolutionarily conserved, and residue 157 lies within a region previously suggested to be important for carboxylase binding to VKD Glus or propeptide. However, the hydrophobic nature of this region and inability of vitamin K administration to restore VKD protein function alternatively suggests that residue 157 may be important for vitamin K binding.

Description: Hereditary combined vitamin K–dependent (VKD) coagulation factor deficiency is an autosomal recessive bleeding disorder associated with defects in either the γ-carboxylase, which carboxylates VKD proteins to render them active, or the vitamin K epoxide reductase (VKORC1), which supplies the reduced vitamin K cofactor required for carboxylation. Such deficiencies are rare, and we report the fourth case resulting from mutations in the carboxylase gene, identified in a Tunisian girl who exhibited impaired function in hemostatic VKD factors that was not restored by vitamin K administration. Sequence analysis of the proposita did not identify any mutations in the VKORC1 gene but, remarkably, revealed 3 heterozygous mutations in the carboxylase gene that caused the substitutions Asp31Asn, Trp157Arg, and Thr591Lys. None of these mutations have previously been reported. Family analysis showed that Asp31Asn and Thr591Lys were coallelic and maternally transmitted while Trp157Arg was transmitted by the father, and a genomic screen of 100 healthy individuals ruled out frequent polymorphisms. Mutational analysis indicated wild-type activity for the Asp31Asn carboxylase. In contrast, the respective Trp157Arg and Thr591Lys activities were 8% and 0% that of wild-type carboxylase, and their compound heterozygosity can therefore account for functional VKD factor deficiency. The implications for carboxylase mechanism are discussed.