Identification of mutations in 15 Hungarian families with hereditary protein C deficiency.

Hereditary protein C deficiency represents about 2-9% of inherited thrombophilias. Our aim was to elucidate the molecular basis of protein C deficiency in 25 members of 15 Hungarian families with venous thromboembolic diseases and to identify hitherto undescribed genetical defects in the protein C (PROC) gene. The exons of the PROC gene were screened for mutations with the combination of polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE), and/or PCR and single-strand conformation polymorphism (SSCP) analysis. The amplified DNA fragments with aberrant migration during DGGE and SSCP were sequenced. Mutations were determined in the PROC gene in all of the examined families: one nonsense mutation, one rare frameshift deletion and nine different missense mutations, of which three were novel (1493 A-->G, 35Asp-->Gly; 6231 G-->A, 173Gly-->Glu; 8476 C-->T, 254Thr-->Ile). The combination of hereditary protein C deficiency with other hereditary thrombophilias was rather common. DGGE and SSCP were proved to be efficient and cost-effective screening methods in the genetic analysis of hereditary protein C deficiency.

The prothrombin nt20210 A allele as a risk factor for venous thromboembolism: detection of heterozygous and homozygous carriers by alternative methods.

The A allele of a G/A dimorphism at position nt20210 in the 3'-untranslated region of the prothrombin gene has been recently identified as a common risk factor for venous thrombosis. It is detected mostly by PCR amplification using a mutagenic primer and subsequent restriction analysis. We have applied two alternative methods--DGGE and SSCP-to analyse the nt20210 dimorphism in a series of 383 consecutive patients with deep venous thrombosis and in 144 controls. Both screening methods resulted in the unambiguous identification of carriers of the nt20210 A allele not only in heterozygotes but in two cases as well in homozygous state. The frequency of the A allele was 0.7% in the control group but 3.7% in the patient group, demonstrating a substantial increase in risk for venous thrombosis in carriers of the 20210 A allele. Both methods have the potential to detect other DNA sequence variations in the vicinity of the 20210 G/A dimorphism. This was demonstrated by the identification of a new mutation T20122G in the 3'-untranslated region of the prothrombin gene in a single patient.

[Screening methods in genetic diagnosis of hereditary protein C deficiency]. / Szürö módszerek alkalmazása a herediter protein C hiány genetikai diagnosztikájában.

Genomic analysis and detailed blood coagulation examinations of 22 family members of 18 families with repeatedly low protein C activity have been performed. Blood coagulation examinations: INR, fibrinogen, plasminogen, alpha-2-antiplasmin, lupus anticoagulant, APC resistance test, protein C activity and antigen, protein S activity and antithrombin activity. Genetic examinations: the presence of FII G20210A alle and FV:Q506 Leiden mutation were examined and for the mutation screening in the protein C gene combination of polymerase chain reaction (PCR) with denaturing gradient gelelectrophoresis (DGGE) or with single-strand conformation polymorphism (SSCP) analysis has been performed. The amplified DNA fragments with aberrant migration during DGGE and SSCP analysis were sequenced. Nine family members of seven families were identified carrying mutations in the protein C gene: one nonsense mutation in exon VII (Arg 157-Stop), two types of missense mutations in four patients in exon IXA (230 Arg-Lys, 254 Thr-Ile, the latter is a new mutation, Protein C Pécs), one missense mutation in two patients in exon IXB (325 Val-Ala), one missense mutation in exon IXC (359 Asp-Asn) and a rare frameshift deletion in exon IXC (364 Met-Trp, 378 Stop). Nine families were evaluated carrying no mutation in their protein C gene, but other genetic or blood coagulation disturbances have been identified, eight of them had borderline decrease in their protein C activity (60-70%). The presence of FV:Q506 mutation could be diagnosed in eight families (in 3 cases homozygous, in 5 cases heterozygous form), among them combination of the defects could be proved in three of the eight families: FV:Q506 Leiden mutation with antiphospholipoid antibodies in 2 families and the presence of Leiden mutation with prothrombin gene mutation in 1 family. Protein S deficiency in combination with prothrombin gene mutation has been identified in 1 family. There were 2 families where no genetic or blood coagulation alterations could be detected in the background of the repeatedly low protein C activity. Large deletions or insertions which are not detectable by our screening methods could not be excluded in these families and therefore sequencing of the total protein C gene had been performed with negative results. According to the literature and our experience the screening methods that were administered in this study are suitable for the detection of mutations in the protein C gene.