To further elucidate the molecular basis for hereditary thrombophilia, we screened the protein S active gene in 11 families with type I deficiency, using a strategy based on denaturating gradient gel electrophoresis (DGGE) of all the coding sequences. Fragments with an abnormal DGGE pattern were sequenced, and 5 novel mutations were identified in 8 families. The mutations were a 7-nucleotide deletion in exon II, a 4-nucleotide deletion in exon III, a T insertion in exon VII, a C to T transition transforming Leu 259 into Pro and a T to C transition transforming Cys 625 into Arg in 4 families. These mutations were the only sequence variations found in the propositus’ gene exons and co-segregated with the plasma phenotype. A total of 28 members of these 8 families were heterozygous for one of the 5 mutations. Twenty-four (58,5%) of the 41 deficient subjects over 18 years of age had clinical thrombophilia, whereas the 13 subjects under 18 were asymptomatic. Of the 28 subjects, 6 (21,5%) were also found to bear the factor V Arg 506 Gin mutation.
References
1
Lane DA,
Olds RJ,
Boisclair M,
Chowdhury V,
Thein SL,
Cooper DN,
Blajchman M,
Perry D,
Emmerich J,
Aiach M.
Antithrombin III mutation database: First update. Thromb Haemost 1993; 70: 361-369
2
Reitsma PH,
Poort SR,
Bemardi F,
Gandrille S,
Long GL,
Sala N,
Cooper DN.
Protein C deficiency: A database of mutations. Thromb Haemost 1993; 69: 77-84
3
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
4
Schmidel DK,
Nelson RM,
Broxson EH,
Comp PC,
Marlar RA,
Long GL.
A 5.3-kb deletion including exon XIII of the protein S Α-gene in two protein S-deficient families. Blood 1991; 77: 551-559
5
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
6
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
7
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
8
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 Coag Fibrinol 1994; 5: 593-600
9
Gémez 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
10
Gandrille S,
Borgel D,
Eschwege-Gufflet V,
Aillaud MF,
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
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
15
Dahlbäck B,
Stenflo J.
High molecular weight complex in human plasma between vitamin K-dependent protein S and complement component C4b-binding protein. Proc Natl Acad Sci USA 1981; 78: 2512-2516
19
Ploos van Amstel HK,
van der Zanden AL,
Bakker E,
Reitsma PH,
Bertina RM.
Two genes homologous with human protein S cDNA are located on chromosome 3. Thromb Haemost 1987; 58: 982-987
21
Ploos van Amstel HK,
Reitsma PH,
van der Logt PE,
Bertina RM.
Intron-exon organization of the active human protein S gene PSa and its pseudogene PSb: duplication and silencing during primate evolution. Biochemistry 1990; 29: 7853-7861
22
Edenbrandt C-M,
Lundwall A,
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
23
Gandrille S,
Greengard J,
Alhenc-Gelas M,
Juhan-Vague I,
Abgrall JF,
Jude B,
Griffin JH,
Aiach M.
the French Network on the behalf of INSERM. Molecular abnormalities responsible for protein C and protein S deficiencies. Incidence of activated protein C resistance due to Arg 506 Gin mutation in factor V in 113 unrelated symptomatic protein C deficient patients. Blood 1995; 86: 219-224
24
Beufé S,
Borg JY,
Vasse M,
Charbonnier F,
Moreau V,
Monconduit M,
Frebourg T.
Cosegregation of thrombosis with the factor V Q 506 mutation in an extended family with resistance to activated protein C. Br J Haematol 1995 (in press).
25
Comp PC,
Doray D,
Patton D,
Esmon CT.
An abnormal plasma distribution of protein S occurs in functional protein S deficiency. Blood 1986; 68: 504-508
27
Saiki RK,
Gelfand DH,
Stoffel S,
Scharf SJ,
Higuchi R,
Horn GT,
Mullis KB,
Erlich HA.
Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 1988; 239: 487-491
28
Lerman LS,
Silverstein K.
Computational simulation of DNA melting and its application to denaturing gradient gel electrophoresis. In:
Wu R.
(ed): Methods in Enzymology. New York: Academic Press; 1987: 482-501
29
Attree O,
Vidaud D,
Vidaud M,
Amselem S,
Lavergne JM,
Goossens M.
Mutations in the catalytic domain of human coagulation factor IX: rapid characterization by direct genomic sequencing of DNA fragments displaying an altered melting behavior. Genomics 1989; 4: 266-272
30
Gyllensten UB,
Erlich HA.
Generation of single-stranded DNA by the polymerase chain reaction and its application to direct sequencing of the HLA-DQA locus. Proc Natl Acad Sci USA 1988; 85: 7652-7656
31
Hoskins J,
Norman DK,
Beckmann RJ,
Long GL.
Cloning and characterization of human liver cDNA encoding a protein S precursor. Proc Natl Acad Sci USA 1987; 84: 349-353
32
Newton C,
Graham A,
Heptinstall L,
Powell S,
Summers C,
Kalshener N,
Smith J,
Markham A.
Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS). Nucleic Acids Res 1989 17. 2503-2516
33
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 PSΑ (PROS 1). Nucleic Acids Res 1991; 19: 5091
35
Sifers RN,
Brashears-Macatee S,
Kidd VJ,
Muensch H,
Woo SLC.
A frameshift mutation results in a truncated Α1-antitrypsin that is retained within the rough endoplasmic reticulum. J Biol Chem 1988; 263: 7330-7335
38
He X,
Dahlbäck B.
Molecular cloning, expression and functional characterization of rabbit anticoagulant vitamin K-dependent protein S. Eur J Biochem 1993; 217: 857-865
42
Kogan S,
Gitschier J.
Mutations and a polymorphism in the factor VIII gene discovered by denaturing gradient gel electrophoresis. Proc Natl Acad Sci USA 1990; 87: 2092-2096
43
Fanen P,
Ghanem N,
Vidaud M,
Besmond C,
Martin J,
Costes B,
Plassa F,
Goossens M.
Molecular characterization of cystic fibrosis: 16 novel mutations identified by analysis of the whole cystic fibrosis conductance transmembrane regulator (CFTR) coding regions and splice site junctions. Genomics 1992; 13: 770-776
44
Ferec C,
Audrezet MP,
Guilermit H,
Moullier P,
Quere I,
Verlingue C.
Detection of over 98% cystic fibrosis mutations in a Celtic population. Nature Genet 1992; 1: 188-191
45
Ghanem N,
Girodon E,
Vidaud M,
Martin J,
Fanen P,
Plassa F,
Goossens M.
A comprehensive scanning method for rapid detection of (3-globin gene mutations and polymorphisms. Hum Mut 1992; 1: 229-239
47
Gouault-Heilmann M,
Leroy-Matheron C,
Levent M.
Inherited protein S deficiency: Clinical manifestations and laboratory findings in 63 patients. Thromb Res 1994; 76: 269-279
48
Zöller B,
Svensson PJ,
He X,
Dahlbäck B.
Identification of the same factor V gene mutation in 47 out of 50 thrombosis-prone families with inherited resistance to activated protein C. J Clin Invest 1994; 94: 2521-2524
50
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
51
Koeleman BPC,
van Rumpt D,
Hamulyak K,
Reitsma PH,
Bertina RM.
Factor V Leiden: An additional risk factor for thrombosis in protein S deficient families ?. Thromb Haemost 1995; 74: 580-583
53
Koster T,
Blann AD,
Briet E,
Vandenbroucke JP,
Rosendaal FR.
Role of clotting factor VIII in effects of von Willebrand factor on occurence of deep-vein thrombosis. Lancet 1995; 345: 152-155
