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Combined factor V and VIII deficiency. Rare bleeding disorder described in 1954 by Oeri et al. Inheritance is autosomal recessive About 100 families identified worldwide Most frequent in Jews of Sephardic (Tunisian) and Middle Eastern origin Factor V and VIII activity of 5-30%
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Combined factor V and VIII deficiency • Rare bleeding disorder described in 1954 by Oeri et al. • Inheritance is autosomal recessive • About 100 families identified worldwide • Most frequent in Jews of Sephardic (Tunisian) and Middle Eastern origin • Factor V and VIII activity of 5-30% • Moderate to severe bleeding disorder
Factor VIII Factor V
Characteristics of factors V and VIII • Factor V and VIII have similar structure • Factor V and VIII get activated by thrombin • Activated factor V and VIII are inhibited by APC • Factor V and VIII have large B domains that are heavily glycosylated and released upon activation • Factor V gene was located to chromosome 1 • Factor VIII gene was located to chromosome X
Thrombin APC APC Activation and inhibition of factor V and factor VIII Thrombin
Looking for a new gene by homozygosity mapping • Disease is autosomal recessive • Several families, preferably several affected members and consanguinity
Looking for a new gene by homozygosity mapping • Disease is autosomal recessive • Several families, preferably several affected members and consanguinity • Homozygosity in cluster of markers with LOD score exceeding 3.0
Homozygosity search Not this time
Looking for a new gene by homozygosity mapping • Disease is autosomal recessive • Several families, preferable several affected members and consanguinity • Homozygosity in cluster of markers with LOD score exceeding 3.0 • Search for potential gene in data bases • Finding mutations associated with disease bearing alleles • Showing how the mutation affect function
Fishing a gene http://www.ensembl.org/
= LMANI Nichols et al. Cell 93:61-70,1998
Immunohistochemical staining of EBV transformed lymphocytes Anti ERGIC Control Tunisian patient Iraqi patient הוכחה סופית בחולים עם חוסר משולב של הפקטורים V ו VIII אין את החלבון ERGIC
ERGIC-53 (LMAN1) • Identified in 1988 as marker for Endoplasmatic Reticulum Golgi Intermediate Component • 53 kilo-daltons transmembrane protein • Hexamer of identical subunits • Homology to plant lectins (Leguminous mannose-binding lectin) • Mannose-selective binding
COP II COP I FV/FVIII LMAN1 MCFD2 Golgi ERGIC ER Mechanism of action
Lessons from FV and FVIII deficiency • If you find prolonged PT and PTT and low FV check also FVIII • Two mild deficiencies can result in moderate to severe phenotype • Regulatory genes can affect secretion of proteins with common properties • FV and FVIII levels of 5-30% indicate an additional pathway of trafficking for FV and FVIII