Clinical manifestations and gene analysis of 18 cases of hereditary protein S deficiency / 中华血液学杂志

Objective: To analyze the clinical manifestations and molecular pathogenesis of 18 patients with inherited protein S (PS) deficiency. Methods: Eighteen patients with inherited PS deficiency who were admitted to the Institute of Hematology & Blood Diseases Hospital from June 2016 to February 2019 were analyzed: activity of protein C (PC) and antithrombin (AT) , PS activity were measured for phenotype diagnosis; high throughput sequencing (HTS) was used for screening of coagulation disease-related genes; Sanger sequencing was used to confirm candidate variants; Swiss-model was used for three-dimensional structure analysis. Results: The PS:C of 18 patients ranged from 12.5 to 48.2 U/dL. Among them, 16 cases developed deep vein thrombosis, including 2 cases each with mesenteric vein thrombosis and cerebral infarction, and 1 case each with pulmonary embolism and deep vein thrombosis during pregnancy. A total of 16 PROS1 gene mutations were detected, and 5 nonsense mutations (c.134_162del/p.Leu45*, c.847G>T/p.Glu283*, c.995_996delAT/p.Tyr332*, c.1359G> A/p.Trp453*, c.1474C>T/p.Gln492*) , 2 frameshift mutations (c.1460delG/p.Gla487Valfs*9 and c.1747_1750delAATC/p.Asn583Wfs*9) and 1 large fragment deletion (exon9 deletion) were reported for the first time. In addition, the PS:C of the deep vein thrombosis during pregnancy case was 55.2 U/dL carrying PROC gene c.565C>T/p.Arg189Trp mutation. Conclusion: The newly discovered gene mutations enriched the PROS1 gene mutation spectrum which associated with inherited PS deficiency.

Clinical phenotype and gene mutation analysis of 12 patients with hereditary protein C deficiency in different families / 中华血液学杂志

Objective: To investigate the molecular pathogenesis and clinical features of unrelated 12 patients with inherited coagulation protein C (PC) deficiency in Chinese population. Methods: The PC activity (PC:A) and PC antigen (PC:Ag) were detected by chromogenic substrate and enzyme linked immunosorbent assay, respectively. The nine exons and flanking sequences of the protein C (PROC) gene were amplified by polymerase chain reaction with direct sequencing, and the suspected mutations were validated by reverse sequencing (clone sequencing for deletion mutations) . Results: The PC:A of the 12 probands decreased significantly, ranging from 18% to 55%, and the PC:Ag of the 10 probands decreased significantly. Eleven mutations were found, out of which four mutations [c.383G>A (p.Gly128Asp) , c.997G>A (p.Ala291Thr) , c.1318C>T (p.Arg398Cys) , and c.532G>C (p.Leu278Pro) ] were discovered for the first time. Six mutations were in the serine protease domain, four mutations were located in epidermal growth factor (EGF) -like domains, and one mutation was located in activation peptide. There were two deletion mutations (p.Met364Trp fsX15 and p.Lys192del) , and the rest were missense mutations. Mutations p.Phe181Val and p.Arg189Trp were identified in three unrelated families. All mutations may be inherited, and consanguineous marriages were reported in two families. Among the probands, nine cases had venous thrombosis, two cases had poor pregnancy manifestations, and one case had purpura. Conclusion: Patients with PC deficiency caused by PROC gene defects are prone to venous thrombosis, especially when there are other thrombotic factors present at the same time.

Genetic and Clinical Characteristics of A Family with Combined PROC and PROS1 Genetic Variants / 中国实验血液学杂志

OBJECTIVE@#To test the anticoagulation functions, perform the genetic diagnosis and analyze the clinical characteristics in a family with combined heterozygous genetic variants of PROC and PROS1.@*METHODS@#Peripheral blood was collected from all the family members. Hematological phenotypes and activity of anticoagulant factors were analyzed. Target genes were amplified by PCR from DNA isolated from peripheral blood, and then were analyzed by Sanger DNA sequencing.@*RESULTS@#Many members in the family displayed the combined genetic variants in protein C and protein S, and six family members accompanied by deep venous thrombosis (DVT). The influences of genetic and secondary factors on the incidence of venous thrombosis in the family members were analyzed. The results showed that in this family, carriers of combined protein C and protein S gene defects had a higher incidence of VTE, but acquired factors still played a key role in the eventual thrombotic symptoms.@*CONCLUSION@#Venous thromboembolism (VTE) is a multifactorial disease, the combined genetic heterozygous mutations of protein C and S is an important genetic factor, and the clinical phenotype show a high heterogenicity, the secondary factors contribute to the VTE incidence.

Activated Protein C Anticoagulant System Dysfunction and Thrombophilia in Asia

Thrombophilia that is common among Caucasians is caused by genetic polymorphisms of coagulation factor V Leiden (R506Q) and prothrombin G20210A. Unlike that in Caucasians, thrombophilia that is common in the Japanese and Chinese involve dysfunction of the activated protein C (APC) anticoagulant system caused by abnormal protein S and protein C molecules. Approximately 50% of Japanese and Chinese individuals who develop venous thrombosis have reduced activities of protein S. The abnormal sites causing the protein S molecule abnormalities are distributed throughout the protein S gene, PROS1. One of the most common abnormalities is protein S Tokushima (K155E), which accounts for about 30% of the protein S molecule abnormalities in the Japanese. Whether APC dysfunction occurs in other Asian countries is an important aspect of mapping thrombophilia among Asians. International surveys using an accurate assay system are needed to determine this.

The role of autolysis loop in determining the specificity of coagulation proteases

We recently demonstrated that the substitution of the autolysis loop (residues 143 to 154 in the chymotrypsin numbering system) of activated protein C (APC) with the corresponding loop of factor Xa (fXa) renders the APC mutant (APC/fX143-154) susceptible to inhibition by antithrombin (AT) in the presence of pentasaccharide. Our recent results further indicated, that in addition to an improvement in the reactivity of APC/fX143-154 with AT, both the amidolytic and anti-factor Va activities of the mutant APC have also been significantly increased. Since the autolysis loop of APC is five residues longer than the autolysis loop of fXa, it could not be ascertained whether this loop in the mutant APC specifically interacts with the activated conformation of AT or if a shorter autolysis loop is responsible for a global improvement in the catalytic activity of the mutant protease. To answer this question, we prepared another APC mutant in which the autolysis loop of the protease was replaced with the corresponding loop of trypsin (APC/Tryp143-154). Unlike an ~500-fold improvement in the reactivity of APC/fX143-154 with AT in the presence of pentasaccharide, the reactivity of APC/Tryp143-154 with the serpin was improved ~10-fold. These results suggest that both the length and structure of residues of the autolysis loop are critical for the specificity of the coagulation protease interaction with AT. Further factor Va inactivation studies with the APC mutants revealed a similar role for the autolysis loop of APC in the interaction with its natural substrate.

A Novel Silent Substitution (C8516T) in Exon 9 of the Human PROC Gene

Protein C is a vitamin K dependent serine protease zymogen, which has a regulatory influence over the coagulation cascade via the inhibition of factors Va and VIIIa. Hereditary protein C deficiency is associated with an increased risk of thromboembolic disease. A multitude of families displaying protein C (PROC) gene defects have been reported, and a number of DNA sequence polymorphisms are known to occur in the PROC gene. We have identified a previously undescribed silent substitution (C8516T) by direct DNA sequencing in a Korean patient with thrombosis and protein C deficiency. In addition, a rare T allelic frequency (0.016) was determined in 123 patients with acquired or hereditary protein C deficiency.

A molecular model of a point mutation (Val297Met) in the serine protease domain of protein C

A heterozygous GTG to ATG (Val297Met) mutation was detected in a patient with inherited protein C deficiency and deep vein thrombosis. Cosegregation of the mutation with protein C deficiency was observed through a family pedigree study. Molecular models of the serine protease domains of wild type and mutant protein C were constructed by standard comparative method. Val 297 was found to be located in the hydrophobic core of the protein. Although the substitution of Met for Val does not greatly alter the hydrophobicity of the protein, it introduces a bulkier side chain, which yields steric hindrance between this residue and adjacent residues, such as Met364, Tyr393, Ile321, Ile323, and Val378. It seems that the Met can not fit into the tight packing into which it is trapped, thereby probably inducing misfolding and/or greater instability of the protein. Such misfolding and/or instability thereby eventually disturbs the catalytic triad, in consistent with the observed type I deficiency state.

Resistencia a la proteína C activada / Resistence to protein C activation

La trombosis es la causa más frecuente de muerte en el mundo occidental, en EUA por tromboembolismo pulmonar, trombosis coronaria y accidentes cerebrovasculares mueren 1.990.000/año. Aparecen 398.000 trombosis venosas profundas en paciente/año y abortos o pérdidas fetales por trombosis de la placenta. El sistema inhibidor de la coagulación está compuesto por la Antitrombina III(ATIII) cofactor II de la Heparina (CIIIHep), inhibudor de la vía del factor tisular (TFPI), la vía fibronolítica y el sistema anticoagulante de la proteína C, que posee el mayor número de trastornos genéticos asociados con trombosis venosa (TV). La trombina activa a la proteína C; (proteína C activada) (PCA) con la finalidad de clivar a los cofactores VIIIa y la Va.La proteína c recibe la potenciación de la proteína S. El factor V (FV) sinérgicamente con la S como cofactor de la PCA en la degradación del VIIIa. La PCA produce un efecto anticoagulante medible por APTT. La falla de éste, es la resistencia a la proteína C activada (RPCA) común en la población caucasiana. Se hereda en forma autosómica dominante. Algunos portadores nunca desarrollan trombosis, en otros el cuadro es recurrente a tempbrana edad. Se agrava la situación por la coexistencia de otros factores de riesgo congénitos o adquiridos : ingestión de anticonceptivos, embarazo, cáncer, traumas y cirugías. La mutación se describió en el síndrome HELLP , y en la preeclampsia con un 22 por ciento de positividades. El estudio de la anomalía por el test RPCA es de utilidad clínica pero solo el estudio del factor V LEIDEN por PCR permite el conocimiento de la alteración genética. No hay pautas firmes sobre tratamiento. Si la trombosis es recurrentes, puede llegarse a la terapéutica profiláctica por vida

Resistencia a la proteína C activada como causa de trombofilia / Resistance to activated protein C as a pathogenic factor of thrombophilia

En los últimos tres años han ocurrido avances notables en el esclarecimiento de las causas de la trombofilia. Hasta hace poco los estudios de laboratorio en pacientes con trombofilia familiar permitían esclarecer la causa sólo en el 5-10 por ciento de los casos, en los que se identifican deficiencia de proteína C de coagulación, de proteína S de coagulación, de antitrombina III, etc. La recien identificación de la resistencia de la proteína C activada (RPCa) ha cambiado este panorama: hasta el 50 por ciento de los pacientes con trombofilia familiar tienen el genotipo de la RPCa, mutación de nucleótido G por A en la posición 1691 del gen del factor V, lo que produce la mutación R-506-Q (tipo Leiden) de la molécula del factor V. Esta mutación codifica la síntesis de un factor V con actividad precoagulante normal pero "resistente" a la acción lítica de la proteína C activada; ocurre en proporciones variables entre 0 y 15 por ciento de la población general y en poblaciones nórdicas en el defecto genético relacionado con enfermedad más frecuente de todos. El fenotipo de l RPCa se estudia en el laboratorio por medio de una modificación de la medición del tiempo de tromboplastina parcial activada y puede presentarse en pacientes con el genotipo de la RPCa o ser secundario a enfermedades autoinmunes, hepatopatías, empleo de anovulatorios, embarazo, etc. El estudio de todo paciente con trombofilia debe incluir la investigación del fenotipo y en su caso, el genotipo de la RPCA. Este estudio, junto con las investigaciones de las actividades antigénica y procoagulante de las proteínas C, S y antitrombina III, permiten esclarecer la causa de la trombofilia familiar en 60-70 por ciento de los casos