2 MUTATIONS IN EXON-XII OF THE PROTEIN S-ALPHA GENE IN 4 THROMBOPHILIC FAMILIES RESULTING IN PREMATURE STOP CODONS AND DEPRESSED LEVELS OF MUTATED MESSENGER-RNA

Sixteen Danish unrelated thrombophilic families with plasma protein S deficiency of type I (or III) are currently under investigation in our laboratory for defects in the protein S alpha gene, The present paper describes a part of this work, which deals with the identification an d phenotypical presentation of two unique mutations in exon XII of the protein S alpha gene in four of these families. The mutations were id entified by SSCP screening followed by nucleotide sequence analysis or by direct nucleotide sequence analysis. The mutation found in one fam ily (D) is a novel deletion of an A in either the codon for Gly(448) ( GGA) or Ile(449) (ATT) resulting in a frameshift and a premature stop codon at position 454. The other mutation shared by three families (F, G and J) is a previously reported C --> T transition within a hypermu table CG dinucleotide sequence converting Arg(410) (CGA) to Stop (TGA) . All affected individuals are heterozygotes for their mutation and in each family the protein S genotype, the plasma protein S phenotype (n ot shown for Family J) and the clinical phenotype cosegregate, The two mutations can fully explain the abnormal protein S phenotype since pr emature stop codons are known to disrupt gene function of the mutated allele. Analysis of protein S mRNA from platelets showed that both mut ations result in a marked reduction in the amount of protein S mRNA fr om the mutated alleles indicating that the mutations exert their delet erious effects on gene expression at the transcriptional level. The Ar g(410) --> Stop mutation in Families F, G and J is in all instances li nked to a G at the site of a common neutral dimorphism in the codon fo r Pro(626) (CCA-G) in exon XV. This indicates that the mutation in the se families could have arisen in a common ancestor. The Arg(410) (CGA) --> Stop (TGA) mutation is also seen in exon XII of the normal protei n S alpha gene, This gives rise to the speculation as to whether the m utation in the protein S alpha gene is the result of an interaction wi th the protein S beta gene leading to double homologous unequal crossi ng-over or gene conversion of a shea DNA sequence. However, this is un likely since none of the 7 other protein S beta-specific nucleotides a re present in the mutated exon XII sequence of the protein S alpha gen e. The common Arg(506) --> Gln Leiden mutation in coagulation factor V is not an additional risk factor for thrombosis in any of the four fa milies studied.