A Novel Splice Acceptor Site Mutation which Produces Multiple Splicing Abnormalities Resulting in Protein S Deficiency Type I

Summary

In an attempt to explore the molecular mechanisms for protein S deficiency, a patient with such a deficiency was examined at the DNA, RNA and protein levels. Nucleotide analyses revealed that the proband, the mother and the grandmother had a G → C substitution in the invariant AG dinucleotide at the splicing acceptor site of intron A/exon 2. This patient was heterozygous for this substitution and the mutant allele was inherited from the proband’s mother and grandmother. Reverse transcription-polymerase chain reaction analysis demonstrated several kinds of splicing abnormalities such as exon skipping and cryptic splicing, in addition to correct splicing. Semiquantitation of mRNA for the protein S gene revealed that the amount of the proband’s mRNA was reduced to 60% of normal. Thus, this mutation impaired the normal processing of mRNA for the protein S gene, resulting in the subject’s severe protein S deficiency.

• 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 Heeb MJ, Mesters RM, Tans G, Rosing J, Griffin JH. Binding of protein S to factor Va associated with inhibition of prothrombinase that is independent of activated protein C. J Biol Chem 1993; 268: 2872-7.
• 3 Heeb MJ, Rosing J, Bakker HM, Fernandez JA, Tans G, Griffin JH. Protein S binds to and inhibits factor Xa. Proc Natl Acad Sci USA 1994; 91: 2728-32.
• 4 Hackeng TM, van’t Veer C, Meijers JC, Bouma BN. Human protein S inhibits prothrombinase complex activity on endothelial cells and platelets via direct interactions with factor Va and Xa. J Biol Chem 1994; 269: 21051-8.
• 5 Dahlbäck B, Stenflo J. High molecular weight complex in human plasma between vitamin K-dependent protein S and complement C4b-binding protein. Proc Natl Acad Sci USA 1981; 78: 2512-6.
• 6 Dahlbäck B. Purification of human C4b-binding protein and formation of its complex with vitamin K-dependent protein S. Biochem J 1983; 209: 847-56.
• 7 Dahlbäck B. Inhibition of protein Ca cofactor function of human and bovine protein S by C4b-binding protein. J Biol Chem 1986; 261: 12022-7.
• 8 Nishioka J, Suzuki K. Inhibition of cofactor activity of protein S by a complex of protein S and C4b-binding protein: Evidence for inactive ternary complex formation between protein S, C4b-binding protein, and activated protein C. J Biol Chem 1990; 265: 9072-6.
• 9 Comp PC, Esmon CT. Recurrent venous thromboembolism in patients with a partial deficiency of protein S. N Engl J Med 1984; 311: 1525-8.
• 10 Kamiya T, Sugihara T, Ogata K, Saito H, Suzuki K, Nishioka J, Hashimoto S, Yamagata K. Inherited deficiency of protein S in a Japanese family with recurrent venous thrombosis: A study of three generations. Blood 1986; 67: 406-10.
• 11 Scientific and Standardization Committee of the International Society of Thrombosis and Heamostasis Subcommittee on protein C and protein S, Munchen, Germany, July 1992 Stuttgart, Germany, Schattauer.
• 12 Ploos van Amstel HK, Reitsma PH, van der Logt PE, Bertina RM. Intronexon organization of the active human protein S gene PS α and its pseudo-gene PS β: Duplication and silencing during primate evolution. Biochemistry 1990; 29: 7853-61.
• 13 Schmidel DK, Tatro AV, Phelps LG, Tomczak JA, Long GL. Organization of the human protein S genes. Biochemistry 1990; 29: 7845-52.
• 14 Gandrille S, Borgel D, Ireland H, Lane DA, Simmonds R, Reitsma PH, Mannhalter C, Pabinger I, Saito H, Suzuki K, Formstone C, Cooper DN, Espinosa Y, Sala N, Bernardi F, Aiach M. Protein S Deficiency: A Database of Mutations For the Plasma Coagulation Inhibitors Subcommittee of the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis. Thromb Haemost 1997; 77: 1201-14.
• 15 Sacchi E, Pinotti M, Marchetti G, Tagliabue L, Mannucci PM, Bernardi F. Protein S mRNA in patients with protein S deficiency. Thromb Haemost 1995; 73: 746-9.
• 16 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: 486-92.
• 17 Simmonds RE, Ireland H, Kunz G, Lane DA. and the Protein S Study group. Identification of 19 protein S gene mutations in patients with phenotypic protein S deficiency and thrombosis. Blood 1996; 88: 4195-204.
• 18 Yamazaki T, Katsumi A, Okamoto Y, Takafuta T, Tsuzuki S, Kagami K, Sugiura I, Kojima T, Fujimura K, Saito H. Two distinct novel splice site mutations in a compound heterozygous patient with protein S deficiency. Thromb Haemost 1997; 77: 14-20.
• 19 Mustafa S, Pabinger I, Mannhalter C. Protein S deficiency type I: identification of point mutations in 9 of 10 families. Blood 1995; 86: 3444-51.
• 20 Mustafa S, Pabinger I, Varadi K, Halbmayer WH, Lechner K, Schwarz HP, Fischer M, Mannhalter C. A hitherto unknown splice site defect in the protein S gene (PROS1): the mutation results in allelic exclusion type I and type III protein S deficiency. Br J Haemat 1997; 99: 298-300.
• 21 Mustafa S, Pabinger I, Mannhalter C. A frequent mutation in the protein S gene results in cryptic splicing. Br J Haemat 1997; 97: 555-7.
• 22 Formstone CJ, Wacey AI, Berg LP, Rahman S, Bevan D, Rowley M, Voke J, Bernardi 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 strategy. Blood 1995; 86: 2632-41.
• 23 Leroy-Matheron C, Gouault-Heilmann M, Aiach M, Gandrille S. A mutation of the active protein S gene leading to an EGF1-lacking protein in a family with qualitative (type II) deficiency. Blood 1998; 91: 4608-15.
• 24 Beauchamp NJ, Daly ME, Makris M, Preston FE, Peake IR. A novel mutation in intron K of the PROS1 gene causes aberrant RNA splicing and is a common cause of protein S deficiency in a UK thrombophilia cohort. Thromb Haemost 1998; 79: 1086-91.
• 25 Okamoto Y, Yamazaki T, Katsumi A, Kojima T, Takamatsu J, Nishida M, Saito H. A novel nonsense mutation associated with an exon skipping in a patient with hereditary protein S deficiency type I. Thromb Haemost 1996; 75: 877-82.
• 26 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680-5.
• 27 Chomczynski P, Sacchi N. Single step method of RNA isolation by acid guanidium thiocyanate-phenol-chlorophorm extraction. Anal Biochem 1987; 162: 156-9.
• 28 Diepstraten CM, Ploos van Amstel JK, Reitsma PH, Bertina RM. A CCA/CCG neutral polymorphism in the codon for Pro626 of the human protein S gene PS alpha (PROS1). Nucl Acid Res 1991; 19: 5091
• 29 Krawczak M, Reiss J, Copper DN. The mutational spectrum of single base-pair substitutions in mRNA splice junction of human genes: causes and consequences. Hum Genet 1992; 90: 41-51.
• 30 Kaler SG, Gallo LK, Proud VK, Percy AK, Mark Y, Segal NA, Goldstein DS, Holmes CS, Gahl WA. Occipital horn syndrome and a mild Menkes phenotype associated with splice site mutations at the MNK locus. Nat Genet 1994; 8: 195-202.
• 31 Treisman R, Orkin SH, Maniatis T. Specific transcription and RNA splicing defects in five cloned β-thalassemia genes. Nature 1983; 302: 591-6.
• 32 Arredondo-Vega FX, Santisteban I, Kelly S, Schlossman CM, Umetsu DT, Hershfield MS. Correct splicing despite mutation of the invariant first nucleotide of a 5’splice site: A possible basis for disparate clinical phenotypes in siblings with adenosine deaminase deficiency. Am J Hum Genet 1994; 54: 820-30.
• 33 Tee M, Lin D, Sugawara T, Holt JA, Guiguen Y, Buckinham B, Strauss JF, Miller WL. T->A transversion 11 bp from a splice acceptor site in the human gene for steroidogenic acute regulatory protein causes congenital lipid adrenal hyperplasia. Hum Mol Genet 1995; 4: 2299-305.
• 34 Nakai K, Sakamoto H. Construction of a novel database containing aberrant splicing mutations of mammalian genes. Gene 1994; 141: 171-7.
• 35 Oshima Y, Gotoh Y. Signals for the selection of a splice site pre-mRNA: Computer analysis of splice junction sequences and like sequences. J Mol Biol 1987; 195: 247-59.
• 36 Berg LP, Soria JM, Formstone CJ, Morell M, Kakkar VV, Estivill X, Sala N, Cooper DN. Aberrant RNA splicing of the protein C and protein S genes in healthy individuals. Blood Coag Fibrenol 1995; 6: 625-31.
• 37 Orkin SH, Sexton JP, Goff SC, Kazazian H. Inactivation of an Acceptor RNA Splice Site by a Short Deletion in β-Thalassemia. J Biol Chem 1983; 258: 7249-51.
• 38 Antonarakis SE, Irkin SH, Cheng T, Scott AF, Sexton JP, Trusko SP, Char-ache S, Kazazian HH. β-Thalassemia in American Black: Novel mutation in the “TATA” box and an acceptor splice site. Proc Natl Acad Sci USA 1984; 81: 1154-8.
• 39 Yu Q, Safavi F, Roberts R, Marian AJ. A variant β fibrinogen is a genetic risk factor for coronary artery disease and myocardial infarction. J Invest Med 1996; 44: 154-9.
• 40 Erikson P, Kallin B, van’t Hooft FM, Bavenholm P, Hamsten A. Allele-specific increase in basal transcription of the plasminogen-activator inhibitor 1 gene is associated with myocardial infarction. Proc Natl Acad Sci USA 1995; 92: 1851-5.
• 41 Mead TW, Brozovic M, Chakrabarti RR, Haines AP, Imeson JD, Mellows S, Miller GJ, North WRS, Stirling Y, Thompson SG. Haemostatic function and ischemic heart disease: principal results of the Northwick Park Heart Study. Lancet 1986; 533-7.
• 42 Humphries S, Temple A, Lane A, Green F, Cooper J. Low plasma levels of factor VIIc and antigen are more strongly associated with the 10base pair promoter (-323) insertion than the glutamine 353 variant. Thromb Haemost 1996; 75: 567-72.
• 43 Kanaji T, Okamura T, Osaki K, Kuroiwa M, Shimoda K, Hamasaki N, Niho Y. A common genetic polymorphism (46 C to T substitution) in the 5’-untranslated region of the coagulation factor XII gene is associated with low translation efficiency and decrease in plasma factor XII level. Blood 1998; 91: 2010-4.