Molecular Basis for A Thrombophilic Patient's Combined Deficiencies in Proteins C and S.

Proteins C and S (PC and PS) are vitamin K-dependent plasma proteins with anticoagulant properties. Protein S functions as a non-enzymatic cofactor for activated protein C (APC). APC proteolytically degrades coagulation factors Va and VIIIa, thereby diminishing the activities of the prothrombinase and tenase complexes, respectively. The human PC gene (PROC) is located on the long arm of chromosome 2 (2q13-q14) and contains 9 exons which code for 461 amino acid residues. The human PS (PROS1) gene resides on chromosome 3 (3p11.1-q11.2) and contains 15 exons coding for 636 amino acid residues. Hereditary PS and PC deficiencies are both autosomal dominant disorders in which patients have diminished functional levels of the respective protein (usually ∼50% relative to normal controls). Clinically, this results in increased propensity toward thromboembolic disease, also known as thrombophilia. In this study, we describe a unique thrombophilic patient who has combined deficiencies in both proteins C and S. The objective of the study was to elucidate the precise genetic defect(s) causing these deficiencies.

Following purification of the patient's DNA from peripheral blood leukocytes, PCR amplifications were performed using oligonucleotide primers flanking all exons of the PROS1 and PROC genes. In addition, the 400bp region upstream of the first exon of PROS1 (corresponding to the promoter region) was also amplified by PCR. The PCR products were purified and their DNA sequences determined in both forward and reverse directions using the dye-terminator method. The resultant nucleotide sequences were compared with the PROS1 and PROC reference sequences [web address].

The patient was found to be heterozygous for a novel missense PROC gene mutation in exon 8, which codes in part for the proteolytic domain of protein C. The resulting ValàGly substitution of residue 221 is in close proximity to the catalytic triad, which could abrogate its enzymatic activity. Although no mutations were present in any of the patient's PROS1 exons, there was a novel heterozygous CàG nucleotide substitution in the PROS1 promoter region. This substitution is present within an Sp1 transcription factor binding site that is highly conserved among mammals. Therefore, this mutation could significantly diminish expression of the otherwise normal PROS1 gene, leading to the observed decreased protein S level. Both mutations are unreported in the literature and are not listed in the PROC and PROS1 mutation databases.

We have successfully identified two novel genetic defects leading to deficiencies of the anticoagulant proteins C and S in a patient with thrombophilia. Further studies are underway to confirm the suggested biochemical effects of the mutation on protein C and protein S gene expression.

No relevant conflicts of interest to declare.