GENE-40330; No. of pages: 6; 4C: Gene xxx (2015) xxx–xxx
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Research paper
Compound heterozygous protein C deficiency in a family with venous thrombosis: Identification and in vitro study of p.Asp297His and p.Val420Leu mutations Hui Liu 1, Hua-Fang Wang 1, Liang Tang ⁎, Yan Yang, Qing-Yun Wang, Wei Zeng, Ying-Ying Wu, Zhi-Peng Cheng, Bei Hu, Tao Guo, Yu Hu ⁎ Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China Collaborative Innovation Center of Hematology, Huazhong University of Science and Technology, China
a r t i c l e
i n f o
Article history: Received 9 November 2014 Received in revised form 5 February 2015 Accepted 2 March 2015 Available online xxxx Keywords: Protein C deficiency Mutation Venous thromboembolism (VTE)
a b s t r a c t Hereditary protein C deficiency (PCD) is an autosomal inherited disorder associated with high risk for venous thromboembolism (VTE). This study aimed to explore the functional consequences of two missense mutations, p.Asp297His and p.Val420Ile, responsible for type I/II PCD and recurrent deep vein thrombosis (DVT) in a Chinese family. The plasma protein C activities (PC:A) of the proband and his sister were reduced to 4% and 5% of normal activity. However, protein C antigen (PC:Ag) concentrations were not equally decreased, with levels of 90.5% and 88.7%, respectively. Two missense mutations p.Asp297His and p.Val420Leu were identified in the protein C gene (PROC). The PC:A and PC:Ag levels in heterozygous state for p.Asp297His were 66% and 64.8%, whereas in heterozygous state for p.Val420Leu, these levels were 67% and 145%, respectively. Wild type (WT) and two mutant PROC cDNA expression plasmids were constructed and transfected into HEK 293T cells. Western blot analysis revealed that both p.Asp297His and p.Val420Leu showed a normal intracellular protein level. The extracellular protein level and specific activity of p.Asp297His were equally reduced to 37.7 ± 4.3% and 22.1 ± 2.5%, respectively. Mutant p.Val420Leu showed a relatively higher PC:Ag level and undetectable PC:A. Immunofluorescence staining revealed that WT and p.Val420Leu proteins were largely co-localized with both the protein disulfide isomerase (PDI) and cis–Golgi Marker (GM130), while the PC p.Asp297His mutant protein was mainly co-localized with PDI and much less co-localized with GM130. The thrombosis symptom in this family was associated with the two missense mutations in the PROC gene. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Protein C (PC) is a vitamin-K dependent serine protease zymogen synthesized mainly in the liver. Activated PC (APC) is generated upon the zymogen proteolysis between Arg169 and Leu170 on the surface of endothelial cells. Thrombin, thrombomodulin and endothelial protein C receptor (EPCR) play a role during the process (Wildhagen et al., 2011). APC exerts its anticoagulant function through inactivation of the blood coagulation factors FVa and FVIIIa in the presence of protein S (PS), Ca2+ and phospholipids (Griffin et al., 2007). PC is encoded by the protein C gene (PROC) on chromosome 2q13–q14, which is composed of 9 exons and spans about 11.2 kb Abbreviations: PCD, hereditary protein C deficiency; VTE, venous thromboembolism; DVT, deep vein thrombosis; PC, protein C; ERAD, ER associated degradation; PROC, protein C gene; APC, activated PC; LMWH, low molecular weight heparin; UFH, unfractionated heparin ⁎ Corresponding authors at: Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science & Technology, Wuhan, Hubei 430022, China. E-mail addresses: [email protected] (Y. Hu), [email protected] (L. Tang). 1 Equal contributions for these authors.
(Cooper et al., 2012). Hereditary protein C deficiency (PCD) is an autosomal inherited disorder with an increased risk of venous thrombosis (Vossen et al., 2004). Patients with undetectable protein C activity (PC:A) often present life-threatening thrombotic complications such as purpura fulminans and DIC during the neonatal period. Heterozygous PCD is often characterized by an increased risk of venous thrombosis in early adulthood (Ohga et al., 2013). Based on the functional and immunological protein C assays, PCD can be divided into two types. Type I deficiency is featured by a parallel reduction in concentration and function (CRM −), whereas type II is characterized by normal or increased concentration and reduced function (CRM +). Type II accounts for about 15% of symptomatic PCD (Faioni et al., 2000). After synthesizing, PC is subjected to several posttranslational modifications in the endoplasmic reticulum (ER) and the Golgi apparatus, and only correctly folded proteins are transported from the ER to Golgi and subsequently to the cell surface (Trombetta and Parodi, 2003). Previous studies have shown that mutations in PROC leading to type I PC deficiency are due to retention of the misfolding mutant proteins in the ER. Some of the studies also detect increased degradation by proteasomes; the latter is called ER associated degradation (ERAD)
http://dx.doi.org/10.1016/j.gene.2015.03.002 0378-1119/© 2015 Elsevier B.V. All rights reserved.
Please cite this article as: Liu, H., et al., Compound heterozygous protein C deficiency in a family with venous thrombosis: Identification and in vitro study of p.Asp297His and p.Val420Leu mutations..., Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.03.002
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H. Liu et al. / Gene xxx (2015) xxx–xxx
(Nishio et al., 2008; Tjeldhorn et al., 2010). The present work describes a family with compound heterozygous mutations in the PROC gene.
nih.gov/sites/entrez) with sequences from Pan troglodytes, Canis lupus familiaris, Bos taurus, Mus musculus, Rattus norvegicus, and Gallus gallus.
2. Materials and methods
2.6. Recombinant PC expression experiments
2.1. Clinical material
Functional effects of p.Asp297His and p.Val420Leu were analyzed by in vitro expression studies. To produce the PROC mutants (c.889GNC, c.1258GNT), mutagenesis was performed by high-fidelity PCR with the wild-type human PC open reading frame expression-ready clone (GeneCopoeia, Rockville, MD, USA) as a template. Mutagenic primer sequences are available upon request. Each plasmid was checked by sequencing the whole protein C cDNA to confirm the presence of the mutation and to exclude PCR-induced errors. HEK-293T cells were grown in DMEM (Gibco-BRL, Gaithersburg, MD, USA) supplemented with 10% FBS, 2 mM glutamine, 100 U mL−1 penicillin, and 100 μg mL−1 streptomycin, and maintained at 37 °C in 5% CO2. Approximately 70–80% confluent cells in six-well plates were co-transfected with 2.0 μg of each PROC construct (monk vector, wild type or mutant type) and 0.5 μg of the enhanced green fluorescent protein (EGFP, GeneCopoeia, Rockville, Maryland, USA) control vector using Lipofectamine LTX and Plus Reagent (Invitrogen, Carlsbad, California, USA). The EGFP was used for normalizing transfection efficiency. After 4 h transfection, the medium was changed to serum-free medium.
Subjects under study were the proband, his parents and two sisters. The proband suffered his first spontaneous attack of thrombosis at the age of 15 years affecting the right deep femoral vein. In the acute phase, the DVT was treated with low molecular weight heparin (LMWH) and followed by warfarin prescription, targeting an international normalized ratio of 2.0–3.0. Warfarin-induced skin necrosis and bleeding were not seen in this proband, but during the warfarin treatment the proband had several image-verified attacks of thrombosis affecting the left profound leg veins, the common iliac vein, the right internal jugular vein as well as a recurrence of thrombosis of his left profound femoral vein. The sister (III-2) had her first deep vein thrombosis in the right leg during pregnancy at the age of 20 years, and managed with LMWH and warfarin. She had no additional thrombosis attacks after the delivery. The blocked blood vessels of the proband and his sister (III-2) were not completely recanalized till now. For financial reasons, they refused to use new oral anticoagulants such as rivaroxaban and dabigatran. No history of thromboembolism was presented in other family members. 2.2. Sample collection and DNA extraction Peripheral blood samples were collected from the family members after obtaining written informed consent. The platelet-poor plasma (PPP) was stored at − 80 °C until assayed, and DNA was extracted from peripheral blood leukocytes according to the manufacturer's instructions (Bioteke, Beijing, China). 2.3. Ex vivo plasma measurements PC, PS, and antithrombin (AT) activities were assayed on a STA-R automated coagulation analyzer (Diagnostica Stago, France) using commercial reagents from Stago according to the manufacturers' recommendations. PC and AT activities were measured using a chromogenic substrate method. The activity of PS was evaluated using a clotting method. PC antigen (PC:Ag) was further tested by ELISA using the ZYMUTEST Protein C Kit (Hyphen Biomed, France). The normal ranges of these tests in our lab were established in 78 healthy subjects. 2.4. Molecular analysis After PCR amplification of the promoter region, nine exons, and the splicing regions of PROC, the PCR products were purified and directly sequenced (Applied Biosystems, USA). The results were compared with the reference sequences NM_000312.3 and NP_000303.1 in GenBank. The PCR primers and reaction conditions were available upon request. Novel variants were then screened in 50 healthy individuals with normal PC:A using direct sequencing. 2.5. In silico analysis of novel amino acid changes The possible impact of novel coding sequence changes on the structure and function of PC was assessed using three bioinformatics tools: Sorting Intolerant From Tolerant (SIFT, http://sift.jcvi.org), PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/) and MutationTaster (http:// www.mutationtaster.org/). The gene ID “5624”, the Protein ID “P04070” and ENSP ID “ENSP00000234071” were used. The conservation of the affected amino acids were further checked by multiple sequence alignment (HomoloGene, http://www.ncbi.nlm.
2.7. Expression levels of recombinant PC The cells were harvested 48 h after transfection and proteins were prepared by lysing transfected cells and ultrafiltering mediums, respectively, separating by 7.5% SDS-PAGE, and transferring to PVDF membranes. Protein C was detected by Western blot analysis using a rabbit polyclonal antibody against human PC (Hyphen Biomed, France), followed by HRP-conjugated anti-rabbit antibody, and ECL detection reagents (Amersham Biosciences, Piscataway, New Jersey, USA). The GAPDH served as control. After 72 h transfection, the secreted PC:Ag concentrations of the wild-type and mutant PC present in culture media were measured by ELISA described above (see “Ex vivo plasma measurements”). The total protein concentration was measured by Bio-Rad Dc Protein Assay (Bio-Rad, Hercules, CA). PC:Ag levels in cell lysates and culture medium were normalized against the total protein concentrations of the corresponding lysate samples. 2.8. Specific activity of recombinant PC After 48 h transfection, the mediums were collected without protease inhibitor and concentrated using ultrafiltration tubes (Millipore, USA). PC activities were tested as described (see “Ex vivo plasma measurements”). The relative PC specific activity (expressed as a percentage of wild type, set as 100%) was calculated as the ratio between the PC:A and PC:Ag level. 2.9. Immunofluorescence HEK-293T cells were grown on cover slips in the 24-well plate and transfected with 0.5 μg plasmid DNA. The cells were washed 3 times with PBS, fixed with 3.7% paraformaldehyde in PBS for half an hour at 4 °C, and permeated with 0.1% Triton-X (Sigma-Aldrich) in PBS for 20 min. Non-specific binding sites were blocked with 4% hydrogen peroxide for 20 min and 1% BSA for 20 min at room temperature. After removing the blocking solution, the cells were incubated with appropriate primary antibodies: polyclonal rabbit anti-human protein C antibody (Hyphen Biomed, France), mouse monoclonal anti-PDI (Abcam, UK) or mouse monoclonal anti-GM130 (Abcam, UK) diluted in PBS with 5% BSA. The cells were washed 3 times with PBS to remove unbound antibody and incubated in the dark for 1 h at 37 °C with Cy3 and FITC-conjugated secondary antibodies. The coverslips were removed from the well and mounted onto microscope slides after
Please cite this article as: Liu, H., et al., Compound heterozygous protein C deficiency in a family with venous thrombosis: Identification and in vitro study of p.Asp297His and p.Val420Leu mutations..., Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.03.002
H. Liu et al. / Gene xxx (2015) xxx–xxx
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Table 1 Patient demographics and laboratory results in family members. Subject
I-1 (father) I-2 (mother) II-1 (sister) II-2 (sister) II-3 (proband)
p.Asp297His (c.889GNC)
Normal Heterozygote Normal Heterozygote Heterozygote
p.Val420Leu (c.1258GNT)
Heterozygote Normal Normal Heterozygote Heterozygote
Plasma concentration
Symptoms
PC:A (%)
PC:Ag (%)
PS:A (%)
AT:A (%)
66 67 105 5 4
145 64.8 105.6 88.7 90.5
107 118 101 105 116
128 96 113 112 102
incubating in medium containing DAPI for 10 min. The slides were then treated with anti-fade solution and captured on an Olympus Fluoview FV1000 laser-scanning confocal microscope.
Asymptomatic Asymptomatic Asymptomatic Venous thrombosis during pregnancy at 20 years old Recurrent venous thrombosis, first onset at 15 years old
3.2. Bioinformatics prediction Both Asp297 and Val420 were totally conserved (Fig. 2), and were predicted to be disease-causing or residue damage by all online bioinformatics tools: Mutation Taster, PolyPhen2 and SIFT.
2.10. Statistics Continuous variables were expressed as means ± standard deviations or medians. The differences between two groups were analyzed with Student's t-test. Statistical analysis was performed with SPSS 13.0 (SPSS Inc., Chicago, IL, USA). For all analyses, the significance level was 0.05.
3. Results 3.1. Analysis of PROC genotype and plasma protein C phenotype Table 1 summarized the results of genotype and phenotype analyses of the investigated members in this family. PC:A levels of the proband and his sister (III-2) were reduced to 4% and 5%, but PC:Ag levels were not decreased, and remained at 90.5% and 88.7%, respectively. This condition also presented in their father with PC:A and PC:Ag of 67% and 145%, respectively. PC:A (66%) and antigen (64.8%) were equally reduced in their mother (I-2). The activities of PS and AT were all within normal range among the family members. Nucleotide sequence analysis demonstrated that the proband and his sister (III-2) were compound heterozygotes for a novel PROC mutation (p.Val420Leu) and a reported one (p.Asp297His) (Fig. 1). The parents were heterozygotes for either of the two mutations. The novel mutation was not detected in the control people.
3.3. Expression levels and specific activity of recombinant PC ELISA assays revealed that the antigen level of p.Asp297His decreased to 37.7 ± 4.3% of that of WT. However, p.Val420Leu antigen level was significantly higher than WT PC, at 152.1 ± 10.1% (Fig. 3A). Western blot analysis of the protein in cell lysates showed no difference in intracellular amounts between the two mutant types and the wild type PC (Fig. 3C). These results were adjusted by transfection efficiency using EGFP vector. Compared with WT, the specific activity of p.Asp297His was reduced to 22.1 ± 2.5%. Even with considerable antigen level, the activity of p.Val420Leu was undetectable (Fig. 3B). These data suggested that p.Asp297His represented a CRM − defect, whereas p.Val420Leu a CRM+ defect.
3.4. Immunofluorescence localization of wild type and mutant type PC Wild type and p.Val420Leu PC were largely co-localized with both the PDI and GM130, while the PC p.Asp297His mutant protein was mainly co-localized with PDI and much less co-localized with GM130 (Fig. 4). These results indicated that p.Asp297His was predominantly located in the ER and with less transportation to the Golgi, compared with WT and p.Val420Leu PC.
Fig. 1. The pedigree of the family with PCD. The index patient was indicated with an arrow. Half-solid symbols represent the individuals heterozygous for the p.Asp297His or the p.Val420Leu mutation, and solid symbols represent compound heterozygous for these two mutations. PC:A = PC activity, PC:Ag = PC antigen, PS:A = PS activity, AT:A = AT activity.
Please cite this article as: Liu, H., et al., Compound heterozygous protein C deficiency in a family with venous thrombosis: Identification and in vitro study of p.Asp297His and p.Val420Leu mutations..., Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.03.002
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Fig. 2. Multiple sequence alignment highlighting the affected amino acids among selected species. The conservation of the affected amino acids was evaluated in sequences from different species.
3.5. Analysis of other risk factors for bleeding and venous thrombosis The plasma of the proband and his sister (III-2) were analyzed for other haemostatic and thrombotic parameters that may predict an increased risk of venous thrombosis or bleeding tendency that may influence the clinical manifestation. These parameters were AT, PS, TM, fibrinogen, FV, FVII, FX, FVIII, FIX, FXI, FXII, vWF, TT and APTT. These parameters were all within normal range. 4. Discussion PC plays a critical role in anticoagulation, and PCD is one of the most severe risk factors for venous thrombosis. Patients with hereditary PCD have a high risk of VTE recurrence (Ozlu et al., 2008; Cooper et al., 2012). Therefore, patients with a severely reduced PC level due to mutation(s) within the PROC gene or combined with other genetic factors require lifelong anticoagulation treatment. The standard anticoagulation treatment during the acute phase of VTE involves unfractionated heparin (UFH) or LMWH. Warfarin is a widely used oral anticoagulant in the long-term treatment and prevention for VTE (Burgazli et al., 2013). Heterozygous PCD is found in 6% of families
with inherited thrombophilia, and 3% of patients with a first-time VTE in Caucasians (Wypasek and Undas, 2013). A retrospective study in a large cohort of families has shown that the annual incidence of first recurrence after a first episode of VTE was 6.6% (95% CI, 3.9–8.7) in PCD patients (Brouwer, J.L., Lijfering, W.M., et al., 2009). Our group has found that hereditary PCD plays a major role in thrombophilia in the Chinese population. The prevalences of PROC p.Arg189Trp and p.Lys192del in the Chinese VTE population were approximately 5% and 6%, respectively (Tang et al., 2012a,b). Recently, in addition, we analyzed 400 unrelated patients with VTE and found that 24 of the 400 individuals (6%) had a mutation in the PROC gene besides the two common variants. After 3 months' warfarin treatment, we observed different therapeutic effects between PCD and non-PCD DVT patients by venous ultrasonography. Most of the 376 un-PCD thrombosis patients could be managed by warfarin, and can be completely or partially recanalized, with blood being resumed to flow smoothly. But the recovery rate in PCD was significantly lower (3/24) (unpublished data). New oral anticoagulants targeting thrombin or factor Xa, including Dabigatran etexilate (Pradaxa®), Rivaroxaban (Xarelto®), and Apixaban (Eliquis®) are more expensive and have been introduced to these un-recovered DVT patients who can afford it (Alotaibi et al.,
Fig. 3. Expression levels and specific activities of wild and mutant types in HEK 293T cells. (A) The PC levels were measured using the ELISA kit as described in “Materials and methods”. (B) After concentrated conditional media (48 h after transfection), PC:Ag and PC:A were measured as described in “Materials and methods” (PC:A of p.V420L mutant can't be detected). The specific activities of recombinant proteins were determined by calculating the ratio between PC:A and PC:Ag. (C) 48 h after transfection, PC:Ag levels were measured by Western blot. GAPDH served as the internal control. WT = wild type PC. MT1 = p.Asp297His PC. MT2 = p.Val420Leu PC. Bars represent means ± SD of three independent experiments, each performed in duplicate. The mean value of wild type PC was set as 100%.
Please cite this article as: Liu, H., et al., Compound heterozygous protein C deficiency in a family with venous thrombosis: Identification and in vitro study of p.Asp297His and p.Val420Leu mutations..., Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.03.002
H. Liu et al. / Gene xxx (2015) xxx–xxx
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Fig. 4. Intracellular localization of WT and two mutant PCs. PC appears in red. PDI and GM130 appear in green. Overlaid image is yellow and corresponds to areas of co-localization of PC with ER and Golgi. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
2014; Arcelus et al., 2014). These new oral anticoagulants were more effective in managing thrombosis. Therefore, the diagnosis of PCD might influence clinical treatment protocols concerning the duration and type of anticoagulant treatment. Thus, identifying inherited PC defects is important. Besides the plasma PC:A and PC:Ag measurements, genetic analysis could make the diagnosis more accurate. Through the sequencing of the PROC gene, two missense mutations were detected. The importance of PC residues Asp297 and Val420 were underlined by its conservation in the PC sequence among studied species. The majority of mutations recorded in the Human Gene Mutation Database (HGMD) were type I deficiency, while the type II phenotype was rare. In the present study, the novel mutation (p.Val420Leu) reported resulted in reduced PC:A and relatively higher concentration (type II deficiency). Interestingly, several mutations near the Val420 residue have been reported to cause type II PCD, i.e. p.Trp422Cys, p.Trp422Gly, p.Gly423Ser, and p.Gly423Asp (Marchetti et al., 1993; Miyata et al., 1996; Levo et al., 2000; Rovida et al., 2007). Moreover, the heterozygotes of p.Gly423Ser also showed PC:A reduced to 58–65% (amidolytic activity) and 52–59% (clotting activity), and higher PC:Ag (144–156%). According to molecular modeling, amino acid Trp422 is located at the P1 specificity substrate-binding pocket of the serine protease and the Ser-Trp-Gly triplet therein is conserved in most serine proteases (Miyata et al., 1996). Mutations altering the conformation of the pocket affected the substrate recognition site and prevented the correct presentation of the scissile peptide bond to the catalytic residue (Rovida et al., 2007). We assumed that p.Val420Leu may lead to the type II deficiency by the same mechanism. The other mutation (p.Asp297His) has been reported in kindred from Korea. The index patient was also compound heterozygous for two missense mutations (p.Met406Ile and p.Asp297His) and was associated with late onset of thrombosis. Heterozygotes of p.Asp297His in Kim et al.'s study showed reduced PC:A and PC:Ag levels, 65% and 76%, respectively, which is consistent with our study (Kim et al., 2008). To further demonstrate that the genetic mutations were the cause of deficiency of PC, transient expression experiments of the wild type and the two mutant PC constructs were performed in HEK 293T cells. In vitro expression results revealed that p.Val420Leu impaired the function but not secretion of PC, while p.Asp297His influenced both the secretion and the function of PC. We hypothesized that the reduced PC in plasma of the proband and his family members were caused by p.Asp297His and p.Val420Leu mutations. The former impaired the protein secretion, leading to type I PC deficiency, and p.Val420Leu resulted in a non-
functional protein which was associated with type II deficiency. Taken together, compound heterozygous patients for these two mutations showed a severely reduced PC:A and nearly normal PC:Ag, i.e., the type II phenotype. The clinical consequences of a homozygous condition or compound heterozygosis will depend on the functional consequences of the underlying mutation(s). The significant heterogeneity of mutations, some with relevant functional consequences but others with mild or even negligible effects, may account for the different clinical manifestations. Severe congenital PCD with almost undetectable PC:A is a rare condition usually associated with purpura fulminans (PF) and DIC during the neonatal period (Pai et al., 2010; Unal et al., 2014). Most cases with homozygous or compound heterozygous PCD showed detectable PC:A and milder clinical manifestations as situations in our study, i.e. late onset of thrombotic symptoms (Iijima et al., 2010; Tjeldhorn et al., 2010; Yu et al., 2012). Moreover, additional factors and conditions may also influence the clinical manifestations. The case presented here is an excellent example as the proband has early and recurrent thrombosis. His sister (III-2), with the same mutations and similar levels of plasma PC:A and PC:Ag, only showed a thrombotic event during pregnancy. However, no differences in the intrinsic and extrinsic coagulation factors were detected between the proband and his sister. The risk for developing thrombosis among individuals with a genetic defect in PROC varies significantly and depends on multiple gene–gene or gene–environment interaction. Overall, this study describes compound heterozygous genetic defects in the PROC gene in a Chinese family associated with severe PC:A deficiency and recurrent late onset of deep vein thrombosis episodes. An in vitro functional study revealed that both p.Asp297His and p.Val420Leu were disease-causing mutations.
Conflict of interest The authors declare that they have no conflict of interest.
Acknowledgment This study was supported by grants from the National Natural Sciences Foundation of China (No. 81370622 and No. 81400099), and the Natural Science Foundation of Hubei Province (No. 2013CFB076).
Please cite this article as: Liu, H., et al., Compound heterozygous protein C deficiency in a family with venous thrombosis: Identification and in vitro study of p.Asp297His and p.Val420Leu mutations..., Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.03.002
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References Alotaibi, G., Alsaleh, K., Wu, C., McMurtry, M.S., 2014. Dabigatran, rivaroxaban and apixaban for extended venous thromboembolism treatment: network metaanalysis. Int. Angiol. 33, 301–308. Arcelus, J.I., Domenech, P., Fernandez-Capitan, M.D., Guijarro, R., Jimenez, D., Jimenez, S., Lozano, F.S., Monreal, M., Nieto, J.A., Paramo, J.A., 2014. Rivaroxaban in the treatment of venous thromboembolism and the prevention of recurrences: a practical approach. Clin. Appl. Thromb. Hemost. (in press). Brouwer, J.L., Lijfering, W.M., Ten Kate, M.K., Kluin-Nelemans, H.C., Veeger, N.J., van der Meer, J., 2009. High long-term absolute risk of recurrent venous thromboembolism in patients with hereditary deficiencies of protein S, protein C or antithrombin. Thromb. Haemost. 101, 93–99. Burgazli, K.M., Atmaca, N., Mericliler, M., Parahuleva, M., Erdogan, A., Daebritz, S.H., 2013. Deep vein thrombosis and novel oral anticoagulants: a clinical review. Eur. Rev. Med. Pharmacol. Sci. 17, 3123–3131. Cooper, P.C., Hill, M., Maclean, R.M., 2012. The phenotypic and genetic assessment of protein C deficiency. Int. J. Lab. Hematol. 34, 336–346. Faioni, E.M., Hermida, J., Rovida, E., Razzari, C., Asti, D., Zeinali, S., Mannucci, P.M., 2000. Type II protein C deficiency: identification and molecular modelling of two natural mutants with low anticoagulant and normal amidolytic activity. Br. J. Haematol. 108, 265–271. Griffin, J.H., Fernandez, J.A., Gale, A.J., Mosnier, L.O., 2007. Activated protein C. J. Thromb. Haemost. 5 (Suppl. 1), 73–80. Iijima, K., Nakamura, A., Kurokawa, H., Monobe, S., Nakagawa, M., 2010. A homozygous protein C deficiency (Lys 192 del) who developed venous thrombosis for the first time at adulthood. Thromb. Res. 125, 100–101. Kim, H.J., Kim, D.K., Koh, K.C., Kim, J.Y., Kim, S.H., 2008. Severe protein C deficiency from compound heterozygous mutations in the PROC gene in two Korean adult patients. Thromb. Res. 123, 412–417. Levo, A., Kuismanen, K., Holopainen, P., Vahtera, E., Rasi, V., Krusius, T., Partanen, J., 2000. Single founder mutation (W380G) in type II protein C deficiency in Finland. Thromb. Haemost. 84, 424–428. Marchetti, G., Patracchini, P., Gemmati, D., Castaman, G., Rodeghiero, F., Wacey, A., Cooper, D.N., Tuddenham, E.G., Bernardi, F., 1993. Symptomatic type II protein C deficiency caused by a missense mutation (Gly 381 → Ser) in the substrate-binding pocket. Br. J. Haematol. 84, 285–289. Miyata, T., Sakata, T., Zheng, Y.Z., Tsukamoto, H., Umeyama, H., Uchiyama, S., Ikusaka, M., Yoshioka, A., Imanaka, Y., Fujimura, H., Kambayashi, J., Kato, H., 1996. Genetic characterization of protein C deficiency in Japanese subjects using a rapid and nonradioactive method for single-stand conformational polymorphism analysis and a model building. Thromb. Haemost. 76, 302–311.
Nishio, M., Koyama, T., Nakahara, M., Egawa, N., Hirosawa, S., 2008. Proteasome degradation of protein C and plasmin inhibitor mutants. Thromb. Haemost. 100, 405–412. Ohga, S., Ishiguro, A., Takahashi, Y., Shima, M., Taki, M., Kaneko, M., Fukushima, K., Kang, D., Hara, T., 2013. Protein C deficiency as the major cause of thrombophilias in childhood. Pediatr. Int. 55, 267–271. Ozlu, F., Kyotani, M., Taskin, E., Ozcan, K., Kojima, T., Matsushita, T., Yapicioglu, H., Takagi, A., Sasmaz, I., Satar, M., Narli, N., 2008. A neonate with homozygous protein C deficiency with a homozygous Arg178Trp mutation. J. Pediatr. Hematol. Oncol. 30, 608–611. Pai, N., Shetty, S., Ghosh, K., 2010. Protein C (PROC) gene mutations in two Indian families with purpura fulminans. Ann. Hematol. 89, 835–836. Rovida, E., Merati, G., D'Ursi, P., Zanardelli, S., Marino, F., Fontana, G., Castaman, G., Faioni, E.M., 2007. Identification and computationally-based structural interpretation of naturally occurring variants of human protein C. Hum. Mutat. 28, 345–355. Tang, L., Guo, T., Yang, R., Mei, H., Wang, H., Lu, X., Yu, J., Wang, Q., Hu, Y., 2012a. Genetic background analysis of protein C deficiency demonstrates a recurrent mutation associated with venous thrombosis in Chinese population. PLoS One 7, e35773. Tang, L., Lu, X., Yu, J.M., Wang, Q.Y., Yang, R., Guo, T., Mei, H., Hu, Y., 2012b. PROC c.574_ 576del polymorphism: a common genetic risk factor for venous thrombosis in the Chinese population. J. Thromb. Haemost. 10, 2019–2026. Tjeldhorn, L., Iversen, N., Sandvig, K., Bergan, J., Sandset, P.M., Skretting, G., 2010. Functional characterization of the protein C A267T mutation: evidence for impaired secretion due to defective intracellular transport. BMC Cell Biol. 11, 67. Trombetta, E.S., Parodi, A.J., 2003. Quality control and protein folding in the secretory pathway. Annu. Rev. Cell Dev. Biol. 19, 649–676. Unal, S., Gumruk, F., Yigit, S., Tuncer, M., Tavil, B., Cil, O., Takci, S., Urata, M., Hotta, T., Kang, D., Cetin, M., 2014. A novel mutation in protein C gene (PROC) causing severe phenotype in neonatal period. Pediatr. Blood Cancer 61, 763–764. Vossen, C.Y., Conard, J., Fontcuberta, J., Makris, M., Van Der Meer, F.J., Pabinger, I., Palareti, G., Preston, F.E., Scharrer, I., Souto, J.C., Svensson, P., Walker, I.D., Rosendaal, F.R., 2004. Familial thrombophilia and lifetime risk of venous thrombosis. J. Thromb. Haemost. 2, 1526–1532. Wildhagen, K.C., Lutgens, E., Loubele, S.T., ten Cate, H., Nicolaes, G.A., 2011. The structure– function relationship of activated protein C. Lessons from natural and engineered mutations. Thromb. Haemost. 106, 1034–1045. Wypasek, E., Undas, A., 2013. Protein C and protein S deficiency — practical diagnostic issues. Adv. Clin. Exp. Med. 22, 459–467. Yu, T., Dai, J., Liu, H., Wang, J., Ding, Q., Wang, H., Wang, X., Fu, Q., 2012. Homozygous protein C deficiency with late onset venous thrombosis: identification and in vitro expression study of a novel Pro275Ser mutation. Pathology 44, 348–353.
Please cite this article as: Liu, H., et al., Compound heterozygous protein C deficiency in a family with venous thrombosis: Identification and in vitro study of p.Asp297His and p.Val420Leu mutations..., Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.03.002
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Research paper
Compound heterozygous protein C deficiency in a family with venous thrombosis: Identification and in vitro study of p.Asp297His and p.Val420Leu mutations Hui Liu 1, Hua-Fang Wang 1, Liang Tang ⁎, Yan Yang, Qing-Yun Wang, Wei Zeng, Ying-Ying Wu, Zhi-Peng Cheng, Bei Hu, Tao Guo, Yu Hu ⁎ Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China Collaborative Innovation Center of Hematology, Huazhong University of Science and Technology, China
a r t i c l e
i n f o
Article history: Received 9 November 2014 Received in revised form 5 February 2015 Accepted 2 March 2015 Available online xxxx Keywords: Protein C deficiency Mutation Venous thromboembolism (VTE)
a b s t r a c t Hereditary protein C deficiency (PCD) is an autosomal inherited disorder associated with high risk for venous thromboembolism (VTE). This study aimed to explore the functional consequences of two missense mutations, p.Asp297His and p.Val420Ile, responsible for type I/II PCD and recurrent deep vein thrombosis (DVT) in a Chinese family. The plasma protein C activities (PC:A) of the proband and his sister were reduced to 4% and 5% of normal activity. However, protein C antigen (PC:Ag) concentrations were not equally decreased, with levels of 90.5% and 88.7%, respectively. Two missense mutations p.Asp297His and p.Val420Leu were identified in the protein C gene (PROC). The PC:A and PC:Ag levels in heterozygous state for p.Asp297His were 66% and 64.8%, whereas in heterozygous state for p.Val420Leu, these levels were 67% and 145%, respectively. Wild type (WT) and two mutant PROC cDNA expression plasmids were constructed and transfected into HEK 293T cells. Western blot analysis revealed that both p.Asp297His and p.Val420Leu showed a normal intracellular protein level. The extracellular protein level and specific activity of p.Asp297His were equally reduced to 37.7 ± 4.3% and 22.1 ± 2.5%, respectively. Mutant p.Val420Leu showed a relatively higher PC:Ag level and undetectable PC:A. Immunofluorescence staining revealed that WT and p.Val420Leu proteins were largely co-localized with both the protein disulfide isomerase (PDI) and cis–Golgi Marker (GM130), while the PC p.Asp297His mutant protein was mainly co-localized with PDI and much less co-localized with GM130. The thrombosis symptom in this family was associated with the two missense mutations in the PROC gene. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Protein C (PC) is a vitamin-K dependent serine protease zymogen synthesized mainly in the liver. Activated PC (APC) is generated upon the zymogen proteolysis between Arg169 and Leu170 on the surface of endothelial cells. Thrombin, thrombomodulin and endothelial protein C receptor (EPCR) play a role during the process (Wildhagen et al., 2011). APC exerts its anticoagulant function through inactivation of the blood coagulation factors FVa and FVIIIa in the presence of protein S (PS), Ca2+ and phospholipids (Griffin et al., 2007). PC is encoded by the protein C gene (PROC) on chromosome 2q13–q14, which is composed of 9 exons and spans about 11.2 kb Abbreviations: PCD, hereditary protein C deficiency; VTE, venous thromboembolism; DVT, deep vein thrombosis; PC, protein C; ERAD, ER associated degradation; PROC, protein C gene; APC, activated PC; LMWH, low molecular weight heparin; UFH, unfractionated heparin ⁎ Corresponding authors at: Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science & Technology, Wuhan, Hubei 430022, China. E-mail addresses: [email protected] (Y. Hu), [email protected] (L. Tang). 1 Equal contributions for these authors.
(Cooper et al., 2012). Hereditary protein C deficiency (PCD) is an autosomal inherited disorder with an increased risk of venous thrombosis (Vossen et al., 2004). Patients with undetectable protein C activity (PC:A) often present life-threatening thrombotic complications such as purpura fulminans and DIC during the neonatal period. Heterozygous PCD is often characterized by an increased risk of venous thrombosis in early adulthood (Ohga et al., 2013). Based on the functional and immunological protein C assays, PCD can be divided into two types. Type I deficiency is featured by a parallel reduction in concentration and function (CRM −), whereas type II is characterized by normal or increased concentration and reduced function (CRM +). Type II accounts for about 15% of symptomatic PCD (Faioni et al., 2000). After synthesizing, PC is subjected to several posttranslational modifications in the endoplasmic reticulum (ER) and the Golgi apparatus, and only correctly folded proteins are transported from the ER to Golgi and subsequently to the cell surface (Trombetta and Parodi, 2003). Previous studies have shown that mutations in PROC leading to type I PC deficiency are due to retention of the misfolding mutant proteins in the ER. Some of the studies also detect increased degradation by proteasomes; the latter is called ER associated degradation (ERAD)
http://dx.doi.org/10.1016/j.gene.2015.03.002 0378-1119/© 2015 Elsevier B.V. All rights reserved.
Please cite this article as: Liu, H., et al., Compound heterozygous protein C deficiency in a family with venous thrombosis: Identification and in vitro study of p.Asp297His and p.Val420Leu mutations..., Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.03.002
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H. Liu et al. / Gene xxx (2015) xxx–xxx
(Nishio et al., 2008; Tjeldhorn et al., 2010). The present work describes a family with compound heterozygous mutations in the PROC gene.
nih.gov/sites/entrez) with sequences from Pan troglodytes, Canis lupus familiaris, Bos taurus, Mus musculus, Rattus norvegicus, and Gallus gallus.
2. Materials and methods
2.6. Recombinant PC expression experiments
2.1. Clinical material
Functional effects of p.Asp297His and p.Val420Leu were analyzed by in vitro expression studies. To produce the PROC mutants (c.889GNC, c.1258GNT), mutagenesis was performed by high-fidelity PCR with the wild-type human PC open reading frame expression-ready clone (GeneCopoeia, Rockville, MD, USA) as a template. Mutagenic primer sequences are available upon request. Each plasmid was checked by sequencing the whole protein C cDNA to confirm the presence of the mutation and to exclude PCR-induced errors. HEK-293T cells were grown in DMEM (Gibco-BRL, Gaithersburg, MD, USA) supplemented with 10% FBS, 2 mM glutamine, 100 U mL−1 penicillin, and 100 μg mL−1 streptomycin, and maintained at 37 °C in 5% CO2. Approximately 70–80% confluent cells in six-well plates were co-transfected with 2.0 μg of each PROC construct (monk vector, wild type or mutant type) and 0.5 μg of the enhanced green fluorescent protein (EGFP, GeneCopoeia, Rockville, Maryland, USA) control vector using Lipofectamine LTX and Plus Reagent (Invitrogen, Carlsbad, California, USA). The EGFP was used for normalizing transfection efficiency. After 4 h transfection, the medium was changed to serum-free medium.
Subjects under study were the proband, his parents and two sisters. The proband suffered his first spontaneous attack of thrombosis at the age of 15 years affecting the right deep femoral vein. In the acute phase, the DVT was treated with low molecular weight heparin (LMWH) and followed by warfarin prescription, targeting an international normalized ratio of 2.0–3.0. Warfarin-induced skin necrosis and bleeding were not seen in this proband, but during the warfarin treatment the proband had several image-verified attacks of thrombosis affecting the left profound leg veins, the common iliac vein, the right internal jugular vein as well as a recurrence of thrombosis of his left profound femoral vein. The sister (III-2) had her first deep vein thrombosis in the right leg during pregnancy at the age of 20 years, and managed with LMWH and warfarin. She had no additional thrombosis attacks after the delivery. The blocked blood vessels of the proband and his sister (III-2) were not completely recanalized till now. For financial reasons, they refused to use new oral anticoagulants such as rivaroxaban and dabigatran. No history of thromboembolism was presented in other family members. 2.2. Sample collection and DNA extraction Peripheral blood samples were collected from the family members after obtaining written informed consent. The platelet-poor plasma (PPP) was stored at − 80 °C until assayed, and DNA was extracted from peripheral blood leukocytes according to the manufacturer's instructions (Bioteke, Beijing, China). 2.3. Ex vivo plasma measurements PC, PS, and antithrombin (AT) activities were assayed on a STA-R automated coagulation analyzer (Diagnostica Stago, France) using commercial reagents from Stago according to the manufacturers' recommendations. PC and AT activities were measured using a chromogenic substrate method. The activity of PS was evaluated using a clotting method. PC antigen (PC:Ag) was further tested by ELISA using the ZYMUTEST Protein C Kit (Hyphen Biomed, France). The normal ranges of these tests in our lab were established in 78 healthy subjects. 2.4. Molecular analysis After PCR amplification of the promoter region, nine exons, and the splicing regions of PROC, the PCR products were purified and directly sequenced (Applied Biosystems, USA). The results were compared with the reference sequences NM_000312.3 and NP_000303.1 in GenBank. The PCR primers and reaction conditions were available upon request. Novel variants were then screened in 50 healthy individuals with normal PC:A using direct sequencing. 2.5. In silico analysis of novel amino acid changes The possible impact of novel coding sequence changes on the structure and function of PC was assessed using three bioinformatics tools: Sorting Intolerant From Tolerant (SIFT, http://sift.jcvi.org), PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/) and MutationTaster (http:// www.mutationtaster.org/). The gene ID “5624”, the Protein ID “P04070” and ENSP ID “ENSP00000234071” were used. The conservation of the affected amino acids were further checked by multiple sequence alignment (HomoloGene, http://www.ncbi.nlm.
2.7. Expression levels of recombinant PC The cells were harvested 48 h after transfection and proteins were prepared by lysing transfected cells and ultrafiltering mediums, respectively, separating by 7.5% SDS-PAGE, and transferring to PVDF membranes. Protein C was detected by Western blot analysis using a rabbit polyclonal antibody against human PC (Hyphen Biomed, France), followed by HRP-conjugated anti-rabbit antibody, and ECL detection reagents (Amersham Biosciences, Piscataway, New Jersey, USA). The GAPDH served as control. After 72 h transfection, the secreted PC:Ag concentrations of the wild-type and mutant PC present in culture media were measured by ELISA described above (see “Ex vivo plasma measurements”). The total protein concentration was measured by Bio-Rad Dc Protein Assay (Bio-Rad, Hercules, CA). PC:Ag levels in cell lysates and culture medium were normalized against the total protein concentrations of the corresponding lysate samples. 2.8. Specific activity of recombinant PC After 48 h transfection, the mediums were collected without protease inhibitor and concentrated using ultrafiltration tubes (Millipore, USA). PC activities were tested as described (see “Ex vivo plasma measurements”). The relative PC specific activity (expressed as a percentage of wild type, set as 100%) was calculated as the ratio between the PC:A and PC:Ag level. 2.9. Immunofluorescence HEK-293T cells were grown on cover slips in the 24-well plate and transfected with 0.5 μg plasmid DNA. The cells were washed 3 times with PBS, fixed with 3.7% paraformaldehyde in PBS for half an hour at 4 °C, and permeated with 0.1% Triton-X (Sigma-Aldrich) in PBS for 20 min. Non-specific binding sites were blocked with 4% hydrogen peroxide for 20 min and 1% BSA for 20 min at room temperature. After removing the blocking solution, the cells were incubated with appropriate primary antibodies: polyclonal rabbit anti-human protein C antibody (Hyphen Biomed, France), mouse monoclonal anti-PDI (Abcam, UK) or mouse monoclonal anti-GM130 (Abcam, UK) diluted in PBS with 5% BSA. The cells were washed 3 times with PBS to remove unbound antibody and incubated in the dark for 1 h at 37 °C with Cy3 and FITC-conjugated secondary antibodies. The coverslips were removed from the well and mounted onto microscope slides after
Please cite this article as: Liu, H., et al., Compound heterozygous protein C deficiency in a family with venous thrombosis: Identification and in vitro study of p.Asp297His and p.Val420Leu mutations..., Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.03.002
H. Liu et al. / Gene xxx (2015) xxx–xxx
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Table 1 Patient demographics and laboratory results in family members. Subject
I-1 (father) I-2 (mother) II-1 (sister) II-2 (sister) II-3 (proband)
p.Asp297His (c.889GNC)
Normal Heterozygote Normal Heterozygote Heterozygote
p.Val420Leu (c.1258GNT)
Heterozygote Normal Normal Heterozygote Heterozygote
Plasma concentration
Symptoms
PC:A (%)
PC:Ag (%)
PS:A (%)
AT:A (%)
66 67 105 5 4
145 64.8 105.6 88.7 90.5
107 118 101 105 116
128 96 113 112 102
incubating in medium containing DAPI for 10 min. The slides were then treated with anti-fade solution and captured on an Olympus Fluoview FV1000 laser-scanning confocal microscope.
Asymptomatic Asymptomatic Asymptomatic Venous thrombosis during pregnancy at 20 years old Recurrent venous thrombosis, first onset at 15 years old
3.2. Bioinformatics prediction Both Asp297 and Val420 were totally conserved (Fig. 2), and were predicted to be disease-causing or residue damage by all online bioinformatics tools: Mutation Taster, PolyPhen2 and SIFT.
2.10. Statistics Continuous variables were expressed as means ± standard deviations or medians. The differences between two groups were analyzed with Student's t-test. Statistical analysis was performed with SPSS 13.0 (SPSS Inc., Chicago, IL, USA). For all analyses, the significance level was 0.05.
3. Results 3.1. Analysis of PROC genotype and plasma protein C phenotype Table 1 summarized the results of genotype and phenotype analyses of the investigated members in this family. PC:A levels of the proband and his sister (III-2) were reduced to 4% and 5%, but PC:Ag levels were not decreased, and remained at 90.5% and 88.7%, respectively. This condition also presented in their father with PC:A and PC:Ag of 67% and 145%, respectively. PC:A (66%) and antigen (64.8%) were equally reduced in their mother (I-2). The activities of PS and AT were all within normal range among the family members. Nucleotide sequence analysis demonstrated that the proband and his sister (III-2) were compound heterozygotes for a novel PROC mutation (p.Val420Leu) and a reported one (p.Asp297His) (Fig. 1). The parents were heterozygotes for either of the two mutations. The novel mutation was not detected in the control people.
3.3. Expression levels and specific activity of recombinant PC ELISA assays revealed that the antigen level of p.Asp297His decreased to 37.7 ± 4.3% of that of WT. However, p.Val420Leu antigen level was significantly higher than WT PC, at 152.1 ± 10.1% (Fig. 3A). Western blot analysis of the protein in cell lysates showed no difference in intracellular amounts between the two mutant types and the wild type PC (Fig. 3C). These results were adjusted by transfection efficiency using EGFP vector. Compared with WT, the specific activity of p.Asp297His was reduced to 22.1 ± 2.5%. Even with considerable antigen level, the activity of p.Val420Leu was undetectable (Fig. 3B). These data suggested that p.Asp297His represented a CRM − defect, whereas p.Val420Leu a CRM+ defect.
3.4. Immunofluorescence localization of wild type and mutant type PC Wild type and p.Val420Leu PC were largely co-localized with both the PDI and GM130, while the PC p.Asp297His mutant protein was mainly co-localized with PDI and much less co-localized with GM130 (Fig. 4). These results indicated that p.Asp297His was predominantly located in the ER and with less transportation to the Golgi, compared with WT and p.Val420Leu PC.
Fig. 1. The pedigree of the family with PCD. The index patient was indicated with an arrow. Half-solid symbols represent the individuals heterozygous for the p.Asp297His or the p.Val420Leu mutation, and solid symbols represent compound heterozygous for these two mutations. PC:A = PC activity, PC:Ag = PC antigen, PS:A = PS activity, AT:A = AT activity.
Please cite this article as: Liu, H., et al., Compound heterozygous protein C deficiency in a family with venous thrombosis: Identification and in vitro study of p.Asp297His and p.Val420Leu mutations..., Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.03.002
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H. Liu et al. / Gene xxx (2015) xxx–xxx
Fig. 2. Multiple sequence alignment highlighting the affected amino acids among selected species. The conservation of the affected amino acids was evaluated in sequences from different species.
3.5. Analysis of other risk factors for bleeding and venous thrombosis The plasma of the proband and his sister (III-2) were analyzed for other haemostatic and thrombotic parameters that may predict an increased risk of venous thrombosis or bleeding tendency that may influence the clinical manifestation. These parameters were AT, PS, TM, fibrinogen, FV, FVII, FX, FVIII, FIX, FXI, FXII, vWF, TT and APTT. These parameters were all within normal range. 4. Discussion PC plays a critical role in anticoagulation, and PCD is one of the most severe risk factors for venous thrombosis. Patients with hereditary PCD have a high risk of VTE recurrence (Ozlu et al., 2008; Cooper et al., 2012). Therefore, patients with a severely reduced PC level due to mutation(s) within the PROC gene or combined with other genetic factors require lifelong anticoagulation treatment. The standard anticoagulation treatment during the acute phase of VTE involves unfractionated heparin (UFH) or LMWH. Warfarin is a widely used oral anticoagulant in the long-term treatment and prevention for VTE (Burgazli et al., 2013). Heterozygous PCD is found in 6% of families
with inherited thrombophilia, and 3% of patients with a first-time VTE in Caucasians (Wypasek and Undas, 2013). A retrospective study in a large cohort of families has shown that the annual incidence of first recurrence after a first episode of VTE was 6.6% (95% CI, 3.9–8.7) in PCD patients (Brouwer, J.L., Lijfering, W.M., et al., 2009). Our group has found that hereditary PCD plays a major role in thrombophilia in the Chinese population. The prevalences of PROC p.Arg189Trp and p.Lys192del in the Chinese VTE population were approximately 5% and 6%, respectively (Tang et al., 2012a,b). Recently, in addition, we analyzed 400 unrelated patients with VTE and found that 24 of the 400 individuals (6%) had a mutation in the PROC gene besides the two common variants. After 3 months' warfarin treatment, we observed different therapeutic effects between PCD and non-PCD DVT patients by venous ultrasonography. Most of the 376 un-PCD thrombosis patients could be managed by warfarin, and can be completely or partially recanalized, with blood being resumed to flow smoothly. But the recovery rate in PCD was significantly lower (3/24) (unpublished data). New oral anticoagulants targeting thrombin or factor Xa, including Dabigatran etexilate (Pradaxa®), Rivaroxaban (Xarelto®), and Apixaban (Eliquis®) are more expensive and have been introduced to these un-recovered DVT patients who can afford it (Alotaibi et al.,
Fig. 3. Expression levels and specific activities of wild and mutant types in HEK 293T cells. (A) The PC levels were measured using the ELISA kit as described in “Materials and methods”. (B) After concentrated conditional media (48 h after transfection), PC:Ag and PC:A were measured as described in “Materials and methods” (PC:A of p.V420L mutant can't be detected). The specific activities of recombinant proteins were determined by calculating the ratio between PC:A and PC:Ag. (C) 48 h after transfection, PC:Ag levels were measured by Western blot. GAPDH served as the internal control. WT = wild type PC. MT1 = p.Asp297His PC. MT2 = p.Val420Leu PC. Bars represent means ± SD of three independent experiments, each performed in duplicate. The mean value of wild type PC was set as 100%.
Please cite this article as: Liu, H., et al., Compound heterozygous protein C deficiency in a family with venous thrombosis: Identification and in vitro study of p.Asp297His and p.Val420Leu mutations..., Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.03.002
H. Liu et al. / Gene xxx (2015) xxx–xxx
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Fig. 4. Intracellular localization of WT and two mutant PCs. PC appears in red. PDI and GM130 appear in green. Overlaid image is yellow and corresponds to areas of co-localization of PC with ER and Golgi. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
2014; Arcelus et al., 2014). These new oral anticoagulants were more effective in managing thrombosis. Therefore, the diagnosis of PCD might influence clinical treatment protocols concerning the duration and type of anticoagulant treatment. Thus, identifying inherited PC defects is important. Besides the plasma PC:A and PC:Ag measurements, genetic analysis could make the diagnosis more accurate. Through the sequencing of the PROC gene, two missense mutations were detected. The importance of PC residues Asp297 and Val420 were underlined by its conservation in the PC sequence among studied species. The majority of mutations recorded in the Human Gene Mutation Database (HGMD) were type I deficiency, while the type II phenotype was rare. In the present study, the novel mutation (p.Val420Leu) reported resulted in reduced PC:A and relatively higher concentration (type II deficiency). Interestingly, several mutations near the Val420 residue have been reported to cause type II PCD, i.e. p.Trp422Cys, p.Trp422Gly, p.Gly423Ser, and p.Gly423Asp (Marchetti et al., 1993; Miyata et al., 1996; Levo et al., 2000; Rovida et al., 2007). Moreover, the heterozygotes of p.Gly423Ser also showed PC:A reduced to 58–65% (amidolytic activity) and 52–59% (clotting activity), and higher PC:Ag (144–156%). According to molecular modeling, amino acid Trp422 is located at the P1 specificity substrate-binding pocket of the serine protease and the Ser-Trp-Gly triplet therein is conserved in most serine proteases (Miyata et al., 1996). Mutations altering the conformation of the pocket affected the substrate recognition site and prevented the correct presentation of the scissile peptide bond to the catalytic residue (Rovida et al., 2007). We assumed that p.Val420Leu may lead to the type II deficiency by the same mechanism. The other mutation (p.Asp297His) has been reported in kindred from Korea. The index patient was also compound heterozygous for two missense mutations (p.Met406Ile and p.Asp297His) and was associated with late onset of thrombosis. Heterozygotes of p.Asp297His in Kim et al.'s study showed reduced PC:A and PC:Ag levels, 65% and 76%, respectively, which is consistent with our study (Kim et al., 2008). To further demonstrate that the genetic mutations were the cause of deficiency of PC, transient expression experiments of the wild type and the two mutant PC constructs were performed in HEK 293T cells. In vitro expression results revealed that p.Val420Leu impaired the function but not secretion of PC, while p.Asp297His influenced both the secretion and the function of PC. We hypothesized that the reduced PC in plasma of the proband and his family members were caused by p.Asp297His and p.Val420Leu mutations. The former impaired the protein secretion, leading to type I PC deficiency, and p.Val420Leu resulted in a non-
functional protein which was associated with type II deficiency. Taken together, compound heterozygous patients for these two mutations showed a severely reduced PC:A and nearly normal PC:Ag, i.e., the type II phenotype. The clinical consequences of a homozygous condition or compound heterozygosis will depend on the functional consequences of the underlying mutation(s). The significant heterogeneity of mutations, some with relevant functional consequences but others with mild or even negligible effects, may account for the different clinical manifestations. Severe congenital PCD with almost undetectable PC:A is a rare condition usually associated with purpura fulminans (PF) and DIC during the neonatal period (Pai et al., 2010; Unal et al., 2014). Most cases with homozygous or compound heterozygous PCD showed detectable PC:A and milder clinical manifestations as situations in our study, i.e. late onset of thrombotic symptoms (Iijima et al., 2010; Tjeldhorn et al., 2010; Yu et al., 2012). Moreover, additional factors and conditions may also influence the clinical manifestations. The case presented here is an excellent example as the proband has early and recurrent thrombosis. His sister (III-2), with the same mutations and similar levels of plasma PC:A and PC:Ag, only showed a thrombotic event during pregnancy. However, no differences in the intrinsic and extrinsic coagulation factors were detected between the proband and his sister. The risk for developing thrombosis among individuals with a genetic defect in PROC varies significantly and depends on multiple gene–gene or gene–environment interaction. Overall, this study describes compound heterozygous genetic defects in the PROC gene in a Chinese family associated with severe PC:A deficiency and recurrent late onset of deep vein thrombosis episodes. An in vitro functional study revealed that both p.Asp297His and p.Val420Leu were disease-causing mutations.
Conflict of interest The authors declare that they have no conflict of interest.
Acknowledgment This study was supported by grants from the National Natural Sciences Foundation of China (No. 81370622 and No. 81400099), and the Natural Science Foundation of Hubei Province (No. 2013CFB076).
Please cite this article as: Liu, H., et al., Compound heterozygous protein C deficiency in a family with venous thrombosis: Identification and in vitro study of p.Asp297His and p.Val420Leu mutations..., Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.03.002
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References Alotaibi, G., Alsaleh, K., Wu, C., McMurtry, M.S., 2014. Dabigatran, rivaroxaban and apixaban for extended venous thromboembolism treatment: network metaanalysis. Int. Angiol. 33, 301–308. Arcelus, J.I., Domenech, P., Fernandez-Capitan, M.D., Guijarro, R., Jimenez, D., Jimenez, S., Lozano, F.S., Monreal, M., Nieto, J.A., Paramo, J.A., 2014. Rivaroxaban in the treatment of venous thromboembolism and the prevention of recurrences: a practical approach. Clin. Appl. Thromb. Hemost. (in press). Brouwer, J.L., Lijfering, W.M., Ten Kate, M.K., Kluin-Nelemans, H.C., Veeger, N.J., van der Meer, J., 2009. High long-term absolute risk of recurrent venous thromboembolism in patients with hereditary deficiencies of protein S, protein C or antithrombin. Thromb. Haemost. 101, 93–99. Burgazli, K.M., Atmaca, N., Mericliler, M., Parahuleva, M., Erdogan, A., Daebritz, S.H., 2013. Deep vein thrombosis and novel oral anticoagulants: a clinical review. Eur. Rev. Med. Pharmacol. Sci. 17, 3123–3131. Cooper, P.C., Hill, M., Maclean, R.M., 2012. The phenotypic and genetic assessment of protein C deficiency. Int. J. Lab. Hematol. 34, 336–346. Faioni, E.M., Hermida, J., Rovida, E., Razzari, C., Asti, D., Zeinali, S., Mannucci, P.M., 2000. Type II protein C deficiency: identification and molecular modelling of two natural mutants with low anticoagulant and normal amidolytic activity. Br. J. Haematol. 108, 265–271. Griffin, J.H., Fernandez, J.A., Gale, A.J., Mosnier, L.O., 2007. Activated protein C. J. Thromb. Haemost. 5 (Suppl. 1), 73–80. Iijima, K., Nakamura, A., Kurokawa, H., Monobe, S., Nakagawa, M., 2010. A homozygous protein C deficiency (Lys 192 del) who developed venous thrombosis for the first time at adulthood. Thromb. Res. 125, 100–101. Kim, H.J., Kim, D.K., Koh, K.C., Kim, J.Y., Kim, S.H., 2008. Severe protein C deficiency from compound heterozygous mutations in the PROC gene in two Korean adult patients. Thromb. Res. 123, 412–417. Levo, A., Kuismanen, K., Holopainen, P., Vahtera, E., Rasi, V., Krusius, T., Partanen, J., 2000. Single founder mutation (W380G) in type II protein C deficiency in Finland. Thromb. Haemost. 84, 424–428. Marchetti, G., Patracchini, P., Gemmati, D., Castaman, G., Rodeghiero, F., Wacey, A., Cooper, D.N., Tuddenham, E.G., Bernardi, F., 1993. Symptomatic type II protein C deficiency caused by a missense mutation (Gly 381 → Ser) in the substrate-binding pocket. Br. J. Haematol. 84, 285–289. Miyata, T., Sakata, T., Zheng, Y.Z., Tsukamoto, H., Umeyama, H., Uchiyama, S., Ikusaka, M., Yoshioka, A., Imanaka, Y., Fujimura, H., Kambayashi, J., Kato, H., 1996. Genetic characterization of protein C deficiency in Japanese subjects using a rapid and nonradioactive method for single-stand conformational polymorphism analysis and a model building. Thromb. Haemost. 76, 302–311.
Nishio, M., Koyama, T., Nakahara, M., Egawa, N., Hirosawa, S., 2008. Proteasome degradation of protein C and plasmin inhibitor mutants. Thromb. Haemost. 100, 405–412. Ohga, S., Ishiguro, A., Takahashi, Y., Shima, M., Taki, M., Kaneko, M., Fukushima, K., Kang, D., Hara, T., 2013. Protein C deficiency as the major cause of thrombophilias in childhood. Pediatr. Int. 55, 267–271. Ozlu, F., Kyotani, M., Taskin, E., Ozcan, K., Kojima, T., Matsushita, T., Yapicioglu, H., Takagi, A., Sasmaz, I., Satar, M., Narli, N., 2008. A neonate with homozygous protein C deficiency with a homozygous Arg178Trp mutation. J. Pediatr. Hematol. Oncol. 30, 608–611. Pai, N., Shetty, S., Ghosh, K., 2010. Protein C (PROC) gene mutations in two Indian families with purpura fulminans. Ann. Hematol. 89, 835–836. Rovida, E., Merati, G., D'Ursi, P., Zanardelli, S., Marino, F., Fontana, G., Castaman, G., Faioni, E.M., 2007. Identification and computationally-based structural interpretation of naturally occurring variants of human protein C. Hum. Mutat. 28, 345–355. Tang, L., Guo, T., Yang, R., Mei, H., Wang, H., Lu, X., Yu, J., Wang, Q., Hu, Y., 2012a. Genetic background analysis of protein C deficiency demonstrates a recurrent mutation associated with venous thrombosis in Chinese population. PLoS One 7, e35773. Tang, L., Lu, X., Yu, J.M., Wang, Q.Y., Yang, R., Guo, T., Mei, H., Hu, Y., 2012b. PROC c.574_ 576del polymorphism: a common genetic risk factor for venous thrombosis in the Chinese population. J. Thromb. Haemost. 10, 2019–2026. Tjeldhorn, L., Iversen, N., Sandvig, K., Bergan, J., Sandset, P.M., Skretting, G., 2010. Functional characterization of the protein C A267T mutation: evidence for impaired secretion due to defective intracellular transport. BMC Cell Biol. 11, 67. Trombetta, E.S., Parodi, A.J., 2003. Quality control and protein folding in the secretory pathway. Annu. Rev. Cell Dev. Biol. 19, 649–676. Unal, S., Gumruk, F., Yigit, S., Tuncer, M., Tavil, B., Cil, O., Takci, S., Urata, M., Hotta, T., Kang, D., Cetin, M., 2014. A novel mutation in protein C gene (PROC) causing severe phenotype in neonatal period. Pediatr. Blood Cancer 61, 763–764. Vossen, C.Y., Conard, J., Fontcuberta, J., Makris, M., Van Der Meer, F.J., Pabinger, I., Palareti, G., Preston, F.E., Scharrer, I., Souto, J.C., Svensson, P., Walker, I.D., Rosendaal, F.R., 2004. Familial thrombophilia and lifetime risk of venous thrombosis. J. Thromb. Haemost. 2, 1526–1532. Wildhagen, K.C., Lutgens, E., Loubele, S.T., ten Cate, H., Nicolaes, G.A., 2011. The structure– function relationship of activated protein C. Lessons from natural and engineered mutations. Thromb. Haemost. 106, 1034–1045. Wypasek, E., Undas, A., 2013. Protein C and protein S deficiency — practical diagnostic issues. Adv. Clin. Exp. Med. 22, 459–467. Yu, T., Dai, J., Liu, H., Wang, J., Ding, Q., Wang, H., Wang, X., Fu, Q., 2012. Homozygous protein C deficiency with late onset venous thrombosis: identification and in vitro expression study of a novel Pro275Ser mutation. Pathology 44, 348–353.
Please cite this article as: Liu, H., et al., Compound heterozygous protein C deficiency in a family with venous thrombosis: Identification and in vitro study of p.Asp297His and p.Val420Leu mutations..., Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.03.002
