title: Functional characterization of twelve natural PROS1 mutations associated with anticoagulant protein S deficiency.
abstract: BACKGROUND
  The molecular mechanisms by which PROS1 mutations result in protein S deficiency are still unknown for many of the mutations, particularly for those that result in a premature termination codon. The aim of this study was to analyze the functional relevance on mRNA and protein expression of 12 natural PROS1 mutations associated with protein S deficiency.
  
  
  METHODS
  Five mutations were nonsense, three were small frameshift deletions, one was c.258,259AG>GT at the 3' end of exon 3, one was p.M640T and the last two were c.-7C>G and p.L15H, found in double heterozygosis as [c.-7C>G;44T>A]. The apparently neutral variant p.R233K was also analyzed. PROS1 cDNA was assessed by reverse transcriptase polymerase chain reaction of platelet mRNA. Expression of mutant proteins was determined by site-directed mutagenesis and analyses of transiently transfected PROS1 mutants in COS-7 cells.
  
  
  RESULTS
  Only cDNA from the normal allele was observed from the five nonsense mutations, the frameshift deletion c.1731delT and from c.258,259AG>GT. Both the normal and the mutated alleles were observed from [c.-7C>G;44T>A], c.187,188delTG and p.M640T. Transient expression analyses of PROS1 mutants whose mRNA was normally expressed revealed greatly reduced secretion of p.L15H and c.1272delA, mild secretion values of p.M640T and normal secretion levels of c.-7C>G and, as expected, p.R233K.
  
  
  CONCLUSIONS
  Whereas the main cause of quantitative protein S deficiency associated with missense mutations is defective synthesis, stability or secretion of the mutated protein, the main mechanism for the deficiency associated with mutations that generate a premature termination codon is not the synthesis of a truncated protein, but the exclusion of the mutated allele, probably by nonsense-mediated mRNA decay.
authors.affiliation: Center de Genètica Mèdica i Molecular, IDIBELL, L'Hospitalet de Llobregat, Barcelona.
authors.forename: Begoña
authors.initials: B
authors.lastname: Hurtado
authors.forename: Xavier
authors.initials: X
authors.lastname: Muñoz
authors.forename: Maria Carme
authors.initials: MC
authors.lastname: Mulero
authors.forename: Gemma
authors.initials: G
authors.lastname: Navarro
authors.forename: Pere
authors.initials: P
authors.lastname: Domènech
authors.forename: Pablo
authors.initials: P
authors.lastname: García de Frutos
authors.forename: Mercè
authors.initials: M
authors.lastname: Pérez-Riba
authors.forename: Núria
authors.initials: N
authors.lastname: Sala
mesh_terms: D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D018389:Codon, Nonsense; D016368:Frameshift Mutation; D005838:Genotype; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D020125:Mutation, Missense; D017293:Protein S / Q000096:biosynthesis / Q000235:genetics*; D018455:Protein S Deficiency / Q000235:genetics*; D020871:RNA Stability / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D017384:Sequence Deletion; D014162:Transfection
chemical_list: D018389:Codon, Nonsense; D017293:Protein S; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins