Search

Mutations in LMAN1 (ERGIC-53)or MCFD2cause combined deficiency of factor V and factor VIII (F5F8D). LMAN1 and MCFD2 form a protein complex that functions as a cargo receptor ferrying FV and FVIII from the endoplasmic reticulum to the Golgi. In this study, we analyzed 10 previously reported and 10 new F5F8D families. Mutations in the LMAN1or MCFD2genes accounted for 15 of these families, including 3 alleles resulting in no LMAN1mRNA accumulation. Combined with our previous reports, we have identified LMAN1or MCFD2mutations as the causes of F5F8D in 71 of 76 families. Among the 5 families in which no mutations were identified, 3 were due to misdiagnosis, with the remaining 2 likely carrying LMAN1or MCFD2mutations that were missed by direct sequencing. Our results suggest that mutations in LMAN1and MCFD2may account for all cases of F5F8D. Immunoprecipitation and Western blot analysis detected a low level of LMAN1-MCFD2 complex in lymphoblasts derived from patients with missense mutations in LMAN1 (C475R) or MCFD2 (I136T), suggesting that complete loss of the complex may not be required for clinically significant reduction in FV and FVIII.

Mutations in LMAN1 (ERGIC-53) or MCFD2 cause combined deficiency of factor V and factor VIII (F5F8D). LMAN1 and MCFD2 form a protein complex that functions as a cargo receptor ferrying FV and FVIII from the endoplasmic reticulum to the Golgi. In this study, we analyzed 10 previously reported and 10 new F5F8D families. Mutations in the LMAN1 or MCFD2 genes accounted for 15 of these families, including 3 alleles resulting in no LMAN1 mRNA accumulation. Combined with our previous reports, we have identified LMAN1 or MCFD2 mutations as the causes of F5F8D in 71 of 76 families. Among the 5 families in which no mutations were identified, 3 were due to misdiagnosis, with the remaining 2 likely carrying LMAN1 or MCFD2 mutations that were missed by direct sequencing. Our results suggest that mutations in LMAN1 and MCFD2 may account for all cases of F5F8D. Immunoprecipitation and Western blot analysis detected a low level of LMAN1-MCFD2 complex in lymphoblasts derived from patients with missense mutations in LMAN1 (C475R) or MCFD2 (I136T), suggesting that complete loss of the complex may not be required for clinically significant reduction in FV and FVIII.

Zebrafish are a powerful vertebrate model system for the study of human disease as they share many molecular pathways with mammals. Of note, nearly all of the mammalian coagulation factors are also highly conserved in fish. As part of a whole genome ENU mutagenesis screen, we identified a mutant zebrafish which displayed intraventricular hemorrhage between 2–3 days post fertilization (dpf), which we named redhead. Using an F2 intercross strategy, we mapped this recessive mutant to a 100 kilobase interval on chromosome 2 and identified a splice site mutation in the gene for an ortholog of the p21-activated kinase Pak2. This IVS9 T+2A mutation renders greater than 90% of transcripts nonfunctional, resulting in a hypomorphic allele. Surveying zebrafish expressed sequence tag databases, we identified two Pak2 orthologs in zebrafish, designated Pak2a and Pak2b, with the redheadmutation in pak2a. Central nervous system (CNS) hemorrhage in redheadembryos was rescued by injection of wild type pak2amRNA. Morpholino knockdown of pak2ain wild type fish phenocopies the redheadmutant, but with an increase in penetrance and severity, including hydrocephalus and pericardial edema secondary to severe hemorrhage. Injection of either pak2aor pak2bmRNA was able to rescue this phenotype. In addition, pak2bknockdown worsened the bleeding phenotype in redheadembryos, with no effect on wild type fish. These results suggest a partial overlap in function between Pak2a and Pak2b. Confocal microscopy was performed at 2.5 dpf in pak2aknockdown embryos transgenic for Gata1-dsRED and Flk1-eGFP, distinctively labeling erythrocytes and endothelial cells, respectively. The sites and magnitude of hemorrhage were variable between individual embryos, but there were no obvious abnormalities in vessel patterning. In summary, we have identified a critical role for Pak2 in maintaining CNS vessel integrity in zebrafish, without apparent effects on other vascular beds. Further analysis of this signaling pathway in the vessel wall may provide novel insight into the mechanisms underlying vascular heterogeneity in mammals, and the tissue specific function of Pak2 in the CNS vasculature.