
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