Molecular Genetic Analysis in Patients
with Inherited Thrombophilia and Antithrombin, Protein C or Protein S Deficiency
A. Pavlova, M. Lim-Eimer, M. Watzka, E. Lindhoff-Last,
B. Luxembourg, W. Kreuz, E. Rusicke, E. Seifried and J. Oldenburg
Introduction
Thrombophilia is a disorder associated with an increased tendency to either in- herited or acquired venous thrombosis. The three classic single-gene disorders causing inherited thromboembolic disease affect the coagulation inhibitors- anti- thrombin (AT), protein C (PC), and protein S (PS), each being responsible for roug- hly 5% of familial thromboses. The three genes are located on chromosome 1, 2 and 3, respectively (Fig. 1). The mature AT protein of 432 aa residues is encoded by exons 2 to 6 of AT gene [4]. Nine exons code the PC protein that comprises 461 aa resid- ues [2, 3]. The PS protein consists of 636 amino acids encoded by 15 exons [1].
Studies of the molecular basis of hereditary AT, PC and PS deficiencies showed a great variety of mutations occurring throughout the genes. The heterogeneity of the mutations hampered rapid genetic analysis in affected patients.
Materials and Methods
Twelve families – 2 with protein C, 1 with protein S, and 9 with antithrombin de- ficiency, have been examined. All the exons and the 5’flanking region of each of the three genes were amplified and the PCR samples screened by Denaturing High Performance Liquid Chromatography (DHPLC) followed by direct sequencing of the fragment showed abnormal pattern (Fig. 2).
Results and Discussion
By this approach we identified 9 mutations, 2 in PC, 1 in PS and 6 in AT, which have been found by examination of patients from eleven families with clinically expres- sed deficiencies of PC [1], PS [1] and AT [8]. In AT deficiency we found four mis- sense mutations (S116P, L99F, K114E, S162R) and one stop mutation (W49Stop).Two of the patients had one and the same mutation (S116P). All four missense mutati- ons affected the binding site of heparin and have been previously reported. In three of the families with AT deficiency no mutation has been found. The mutations found in PC and PS deficiencies patients were a 4 bp insertion (CCTG) and 1 mis- sense mutation (R230S) in exon 9 of the PC gene and an 1 bp (T) deletion in exon
I. Scharrer/W. Schramm (Ed.)
34thHemophilia Symposium Hamburg 2003
” Springer Medizin Verlag Heidelberg 2005
15 of the PS gene. Both, the deletion and the insertion led to a frame shift with a sub- sequent stop codon and therefore should be regarded as causative for the phenotype (Table 1) [5].
Conclusion
We conclude that the strategy of using DHPLC as a mutation screening method followed by direct sequencing of a single fragment with abnormal pattern re- presents rapid and reliable approach for the mutation analysis for AT , PC and PS genes.
Molecular Genetic Analysis in Patients 299
Exo n 9 Exo n2
Exon1 Exon2 Exon3 Exon4 Exon5 Exon 6 Exon7
Exon 4Exon 5
Exon 3 Exon 6 Exon 7 Exon 8
Protein S Gene Chromosome 3
Exon15 Exon12
Exon11 Exon10 Exon9 Exon8 Exon7 Exon6 Exon5 Exo n 4 Exon 3
Exon 2 Exon13 Exon14
Exon 1
exons - introns
Protein C Gene Chromosome 2
Antithrombin Gene Chromosome 1
Fig. 1. Schematical presentation of the chromosomal location and structure of AT, PC and PS genes
300 A. Pavlova et al.
Series 0
Time (Minutes)
7 6 5 4 3 2 1 8 7 6 5 4 3 2 1 0
+
Time (Minutes)
9 8 7 6 5 4 3 2 1 0 5 4 3 2 1 0
a
b
Absorbance (mV)Absorbance (mV)
Fig. 2a, b. a) DHPLC chromatograme of exon 15 of wild type protein S sample; b) DHPLC chro- matograme of exon 15 of mutated Protein S sample (T del 618)
Table 1. Overview of the detected mutations
gene number of type location
mutations of mutation of mutation
protein C 1 small insertion CCTG ins 372
1 missense mutation R 230 S
protein S 1 small deletion T del 618
antithrombin 5 stop codon W49Stop
missense mutation S116P missense mutation L99F missense mutation K114E missense mutation S162R
References
1. Schmidel DK, Tatro AV, Tomczak JV, Long GL. Organization of the human protein S genes.
Biochemistry. 1990; 29:7845
2. Foster Dc, Yoshitake S, Davie EW. The nucleotide sequence of the gene for human protein C.
ProcNatl Acad Sci USA 1985;82:467- 4677
3. Aiach M. et all. Complex association of protein C gene promoter polymorphism with circu- lating protein C levels and thrombotic risk. Arterioscler Thromb Vasc Biol. 1999 Jun;19(6):1573-6.
4. Lane DA, Kunz G, Olds RT, Thein SL. Molecular genetics of antithrombin deficiency. Blood Rev. 1996 Jun;10(2):59-74. Review.
5. Lane DA, Grant PJ. Role of hemostatic gene polymorphisms in venous and arterial throm- botic disease. Blood. 2000 Mar 1;95(5):1517-32. Review.
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