Two Mutations in Exon XII of the Protein Sα Gene in Four Thrombophilic Families Resulting in Premature Stop Codons and Depressed Levels of Mutated mRNA

Summary

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 Sα gene. The present paper describes a part of this work, which deals with the identification and phenotypical presentation of two unique mutations in exon XII of the protein Sa gene in four of these families. The mutations were identified by SSCP screening followed by nucleotide sequence analysis or by direct nucleotide sequence analysis. The mutation found in one family (D) is a novel deletion of an A in either the codon for Gly⁴⁴⁸ (GGA) or Iie⁴⁴⁹ (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 hypermutable CG dinucleotide sequence converting Arg⁴¹⁰ (CGA) to Stop (1GA). All affected individuals are heterozygotes for their mutation and in each family the protein S genotype, the plasma protein S phenotype (not shown for Family J) and the clinical phenotype cosegregate. The two mutations can fully explain the abnormal protein S phenotype since premature stop codons are known to disrupt gene function of the mutated allele. Analysis of protein S mRNA from platelets showed that both mutations result in a marked reduction in the amount of protein S mRNA from the mutated alleles indicating that the mutations exert their deleterious effects on gene expression at the transcriptional level. The Arg⁴¹⁰ → Stop mutation in Families F, G and J is in all instances linked to a G at the site of a common neutral dimorphism in the codon for Pro⁶²⁶ (CCA/G) in exon XV. This indicates that the mutation in these families could have arisen in a common ancestor. The Arg⁴¹⁰ (CGA) → Stop (1GA) mutation is also seen in exon XII of the normal protein Sa gene. This gives rise to the speculation as to whether the mutation in the protein Sa gene is the result of an interaction with the protein Sβ gene leading to double homologous unequal crossing-over or gene conversion of a short DNA sequence. However, this is unlikely since none of the 7 other protein S β-specific nucleotides are present in the mutated exon XII sequence of the protein Sα gene. The common Arg⁵⁰⁶ → Gin Leiden mutation in coagulation factor V is not an additional risk factor for thrombosis in any of the four families studied.

• References

• 1 Dahlbäck B. Protein S and C4b-binding protein: Components involved in the regulation of the protein C anticoagulant system. Thromb Haemost 1991; 66: 49-61
• 2 Walker FJ. Protein S and thrombotic disease. Proc Soc Exp Biol Med 1992; 200: 285-295
• 3 Hillarp A, Hessing M, Dahlbäck B. Protein S binding in relation to the subunit composition of human C4b-binding protein. FEBS Lett 1989; 259: 53-56
• 4 Härdig Y, Rezaie A, Dahlbäck B. High affinity binding of human vitamin K-dependent protein S to a truncated recombinant β-chain of C4b-binding protein expressed in Escherichia coli. J Biol Chem 1993; 268: 3033-3036
• 5 Schmidel DK, Tatro AV, Phelps LG, Tomczak JA, Long GL. Organization of the human protein S genes. Biochemistry 1990; 29: 7845-7852
• 6 Ploos van Amstel HK, Reitsma PH, Van der Logt CPE, Bertina RM. Intron-exon organization of the active human protein S gene PSα and its pseudogene PSβ: Duplication and silencing during primate evolution. Biochemistry 1990; 29: 7853-7861
• 7 Edenbrandt C-M, Lundwall Å, Wydro R, Stenflo J. Molecular analysis of the gene for vitamin K dependent protein S and its pseudogene. Cloning and partial gene organization. Biochemistry 1990; 29: 7861-7868
• 8 Marchetti G, Legnani C, Patracchini P, Gemmati D, Ferrati M, Palareti G, Coccheri S, Bemardi F. Study of a protein S gene polymorphism at DNA and mRNA level in a family with symptomatic protein S deficiency. Br J Haematol 1993; 85: 173-175
• 9 Gómez E, Ledford MR, Pegelow CH, Reitsma PH, Bertina RM. Homozygous protein S deficiency due to a one base pair deletion that leads to a stop codon in exon III of the protein S gene. Thromb Haemost 1994; 71: 723-726
• 10 Gandrille S, Borgel D, Eschwege-Gufflet V, Aillaud M, Dreyfus M, Matheron C, Gaussem P, Abgrall JF, Jude B, Sié P, Toulon P, Aiach M. Identification of 15 different candidate causal point mutations and three polymorphisms in 19 patients with protein S deficiency using a scanning method for the analysis of the protein S active gene. Blood 1995; 85: 130-138
• 11 Formstone CJ, Wacey AI, Berg LP, Rahman S, Bevan D, Rowley M, Voke J, Bemardi F, Legnani C, Simioni P, Girolami A, Tuddenham EGD, Kakkar VV, Cooper DN. Detection and characterization of seven novel protein S (PROS) gene lesions: Evaluation of reverse transcript-polymerase chain reaction as a mutation screening strategy. Blood 1995; 86: 2632-2641
• 12 Mustafa S, Pabinger I, Mannhalter C. Protein S deficiency type I: Identification of point mutations in 9 of 10 families. Blood 1995; 86: 3444-3451
• 13 Yamazaki T, Hamaguchi M, Katsumi A, Kagami K, Kojima T, Takamatsu J, Saito H. A quantitative protein S deficiency associated with a novel nonsense mutation and markedly reduced levels of mutated mRNA. Thromb Haemost 1995; 74: 590-595
• 14 Hayashi T, Nishioka J, Shigekiyo T, Saito S, Suzuki K. Protein S Tokushima: Abnormal molecule with a substitution of Glu for Lys-155 in the second epidermal growth factor-like domain of protein S. Blood 1994; 83: 683-690
• 15 Bertina RM, Ploos van Amstel HK, Van Wijngaarden A, Coenen J, Leemhuis MP, Deutz-Teriouw PP, Van der Linden IK, Reitsma PH. Heerlen polymorphism of protein S, an immunologic polymorphism due to dimorphism of residue 460. Blood 1990; 76: 538-548
• 16 Gômez E, Poort SR, Bertina RM, Reitsma PH. Identification of eight point mutations in protein S deficiency type I - Analysis of 15 pedigrees. Thromb Haemost 1995; 73: 750-755
• 17 Duchemin J, Gandrille S, Borgel D, Feurgard P, Alhenc-Gelas M, Matheron C, Dreyfus M, Dupuy E, Juhan-Vague I, Aiach M. The Ser 460 to pro substitution of the protein S a (PROS1) gene is a frequent mutation associated with free protein S (type Ha) deficiency. Blood 1995; 86: 3436-3443
• 18 Reitsma PH, Ploos van Amstel HK, Bertina RM. Three novel mutations in five unrelated subjects with hereditary protein S deficiency type I. J Clin Invest 1994; 93: 486-492
• 19 Ploos van Amstel HK, Huisman MV, Reitsma PH, Ten Cate JW, Bertina RM. Partial protein S gene deletion in a family with hereditary thrombophilia. Blood 1989; 73: 479-483
• 20 Schmidel DK, Nelson RM, Broxson Jr EH, Comp PC, Marlar RA, Long GL. A 5.3-kb deletion including exon XIII of the protein S a gene occurs in two protein S-deficient families. Blood 1991; 77: 551-559
• 21 Borgel D, Gandrille S, Gouault-Heilmann M, Aiach M. First frameshift mutation in the active protein S gene associated with a quantitative hereditary deficiency. Blood Coagulation and Fibrinolysis 1994; 5: 593-600
• 22 Allaart CF, Briët E. Familial Venous Thrombophilia. In: Haemostasis and Thrombosis Bloom AL, Forbes CD, Thomas DP, Tuddenham EGD. eds. Churchill Livingstone, Edinburgh: 1994: 1349-1360
• 23 Zöller B, Garcia de Frutos P, Dahlbäck B. Evaluation of the relationship between protein S and C4b-binding protein isoforms in hereditary protein S deficiency demonstrating type I and type III deficiencies to be phenotypic variants of the same genetic disease. Blood 1995; 85: 3524-3531
• 24 Lind B, Van Solinge WW, Schwartz M, Thorsen S. Splice site mutation in the human protein C gene associated with venous thrombosis: Demonstration of exon skipping by ectopic transcript analysis. Blood 1993; 82: 2423-2432
• 25 Suomalainen A, Ciafaloni E, Koga Y, Peltonen L, Di Mauro S, Schon EA. Use of single strand conformation polymorphism analysis to detect point mutations in human mitochondrial DNA. J Neurol Sci 1992; 111: 222-226
• 26 Sambrook J, Fritsch EF, Maniatis T. Isolation of Cytoplasmatic RNA from Mammalian Cells. In: Molecular Cloning: A Laboratory Manual Cold Spring Harbor Laboratory Press; Cold Spring Harbor, New York: 1989
• 27 Diepstraten CM, Ploos van Amstel JK, Reitsma PH, Bertina RM. A CCA/CCG neutral dimorphism in the codon for Pro 626 of the human protein S gene PSa (PROS1). Nucleic Acids Res 1991; 19: 5091
• 28 Bertina RM, Koeleman BPC, Koster T, Rosendaal FR, Dirven RJ, De Ronde H, Van der Velden PA, Reitsma PH. Mutation in blood coagulation factor V associated with resistance to activated protein C. Nature 1994; 369: 64-67
• 29 Zöller B, Dahlbäck B. Linkage between inherited resistance to activated protein C and factor V gene mutation in venous thrombosis. Lancet 1994; 343: 1536-1538
• 30 Jenny RJ, Pittman DD, Toole JJ, Kriz RW, Aldape RA, Hewick RM, Kaufman RJ, Mann KG. Complete cDNA and derived amino acid sequence of human factor V. Proc Natl Acad Sci USA 1987; 84: 4846-4850
• 31 Dahlbäck B. Inherited thrombophilia: Resistance to activated protein C as a pathogenic factor of venous thromboembolism. Blood 1995; 85: 607-614
• 32 Koeleman BPC, Reitsma PH, Allaart CF, Bertina RM. Activated protein C resistance as an additional risk factor for thrombosis in protein C-deficient families. Blood 1994; 84: 1031-1035
• 33 Zöller B, Bemtsdotter A, Garcia de Frutos P, Dahlbäck B. Resistance to activated protein C as an additional genetic risk factor in hereditary deficiency of protein S. Blood 1995; 85: 3518-3523
• 34 Cooper DN, Krawczak M, Antonarakis SE. The Nature and Mechanisms of Human Gene Mutation. In: The Metabolic and Molecular Bases of Inherited Disease Scriver CR, Beaudet AL, Sly WS, Valle D. eds. McGraw-Hill, Inc; New York: 1995: 259-291
• 35 Barker GF, Beemon K. Rous sarcoma virus RNA stability requires an open reading frame in the gag gene and sequences downstream of the gag-pol junction. Mol Cell Biol 1994; 14: 1986-1996
• 36 Lim S-K, Sigmund C, Gross KW, Maquat LE. Nonsense codons in human β-globin mRNA result in the production of mRNA degradation products. Mol Cell Biol 1992; 12: 1149-1161
• 37 Urlaub G, Mitchell PJ, Ciudad CJ, Chasin LA. Nonsense mutations in the dihydrofolate reductase gene affect RNA processing. Mol Cell Biol 1989; 9: 2868-2880
• 38 Hagan KW, Ruiz-Echevarria MJ, Quan Y, Peltz SW. Characterization of cis-acting sequences and decay intermediates involved in nonsens-mediat-ed mRNA turnover. Mol Cell Biol 1995; 15: 809-823
• 39 Baserga SJ, Benz Jr EJ. β-Globin nonsense mutation: Deficient accumulation of mRNA occurs despite normal cytoplasmic stability. Proc Natl Acad Sci USA 1992; 89: 2935-2839
• 40 Dietz HC, Valle D, Francomano CA, Kendzior Jr RJ, Pyeritz RE, Cutting GR. The skipping of constitutive exons in vivo induced by nonsense ydmutations. Science 1993; 259: 680-683
• 41 Hull J, Shackleton S, Harris A. The stop mutation R553X in the CFTR gene results in exon skipping. Genomics 1994; 19: 362-364
• 42 Fojo SS, Lohse P, Parrott C, Baggio G, Gabelli C, Thomas F, Hoffman J, Brewer Jr HB. A nonsense mutation in the apolipoprotein C-IIPadova gene in a patient with apolipoprotein C-II deficiency. J Clin Invest 1989; 84: 1215-1219
• 43 Lehrman MA, Schneider WJ, Brown MS, Davis G, Elhammer A, Russell DW, Goldstein JL. The Lebanese allele at low density lipoprotein receptor locus. Nonsense mutation produces truncated receptor that is retained in endoplasmatic reticulum. J Biol Chem 1987; 262: 401-410
• 44 Rolfini R, Cabrini G. Nonsense mutation R1162X of cystic fibrosis transmembrane conductance regulator gene does not reduce messenger RNA expression in nasal epithelial tissue. J Clin Invest 1993; 92: 2683-2687
• 45 Ploos van Amstel HK, van der Zanden AL, Reitsma PH, Bertina RM. Human protein S cDNA encodes Phe-16 and Tyr 222 in the consensus sequences for the post-translational processing. FEBS Lett 1987; 222: 186-190