Pathology (December 2014) 46(7), pp. 630–635

HAEMATOLOGY

Hereditary protein C deficiency caused by compound heterozygous mutants in two independent Chinese families YING-TING WU1,2,*, FEI YUE3,*, MIN WANG1, YE-LING LU1, JING DAI1, QIU-LAN DING1, HONG-LI WANG1, HUI-FEN CHEN2 AND XUE-FENG WANG1 1Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 2Department of Laboratory Medicine, Shanghai First Maternity and Infant Hospital, Tongji University School of Medicine, Shanghai, and 3Department of General Surgery, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China; *these authors contributed equally to

this study

Summary We report two compound heterozygous mutants that caused severe type I protein C (PC) deficiency in two independent Chinese families. PC antigen was determined by enzyme-linked immunosorbent assay (ELISA), and PC activity was measured by chromogenic assay. Genetic mutations were screened with polymerase chain reaction (PCR) followed by direct sequencing. PC mutants were transiently expressed in COS-7 cells for the evaluation of PC secretory activity and function. The subcellular location was visualised by immunofluorescence assay. The structural analysis of mutation was performed as well. Compound heterozygous mutations of Arg178Trp and Asp255His with reduced PC activity and antigen levels were identified in Proband 1, a 28-year-old male with deep vein thrombosis (DVT) and pulmonary embolism. The other mutations of Leu-34Pro and Thr295Ile with reduced PC activity and antigen levels were identified in Proband 2, a 19-year-old male with DVT. The PC activities with Arg178Trp, Asp255His, Leu-34Pro and Thr295Ile mutations decreased significantly. Immunofluorescence assay demonstrated that only trace amount of PC with novel Thr295Ile mutation was transported to the Golgi apparatus. Subsequent structural analysis indicated severe impairments of intracellular folding and secretion. The two rare compound heterozygous mutations could cause type I PC deficiency via impairment of secretory activity of PC. Key words: Compound heterozygous mutations, inherited thrombophilia, protein C deficiency, thrombosis. Received 19 February, revised 8 June, accepted 10 June 2014

INTRODUCTION Protein C (PC) pathway, one of the natural anticoagulant systems in vivo, plays an important role in the regulation of blood coagulation.1 PC, a vitamin K-dependent glycoprotein, is the key component of this pathway and circulates in the peripheral blood as a zymogen of an anticoagulant serine protease.2 PC can be activated by thrombin-thrombomodulin complex through the proteolytic removal of a 12-residue activation peptide from the amino terminus of the heavy chain. Activated PC (APC) exerts its anticoagulant function by inactivation of the blood coagulation cofactors Va and VIIIa in the Print ISSN 0031-3025/Online ISSN 1465-3931 DOI: 10.1097/PAT.0000000000000165

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presence of protein S. Human PC gene (PROC) is localised in chromosome 2q13-q14 and comprises nine exons that span over 11 kb genomic DNA.3 The physiological importance of PC has been highlighted by the discovery of families with PC deficiency, in which nucleotide variations in the PROC gene are associated with venous thromboembolic disease (VTE).4 PC deficiency is basically inherited in an autosomal dominant manner that is associated with an increased venous thrombosis risk by 5–10-fold.5,6 The prevalence of heterozygous PC deficiency is estimated at about 0.5% in the population and is associated with adult-onset VTE if other heritable or acquired VTE risk factors are present.4 In contrast, homozygote and compound heterozygote for deleterious mutations in the PROC gene are rare disorders (prevalence about 1 per 200,000–400,000 individuals) that present in early life with life-threatening and repeated venous thrombosis.7 Phenotypically, PC deficiency has been classified into two types. Type I deficiency, the most common, is characterised by reduction in both PC activity and antigen level due to the decrease of protein synthesis and/or stability. As for type II deficiency, PC activity is reduced more than its antigen level due to the synthesis of an abnormal PC molecule with reduced activity.8,9 The first report of inherited PC deficiency associated with VTE dates back to 1981.10 During the past several decades, a large number of mutations causing PC deficiency have been identified.4 Previously, our laboratory has reported several PC mutations, such as Cys64Trp, Phe139Val, Lys150 or Lys151 deletion mutation and Trp372Stop.11,12 The mutations responsible for compound heterozygote PC deficiency have been identified in only 23 patients.7 To accord with the distribution of mutation types in genetic diseases in general,13 more than 65% of the mutations identified in the PROC should be missense mutations. In most cases, little is known about their influence on protein biosynthesis, secretion, degradation and structure change. In the present study, we report two PROC compound heterozygous mutants associated with severe type I PC deficiency in two independent Chinese families. Furthermore, we used COS-7 cells expressing wild-type PC and mutated PC to investigate the mechanisms of the secretory activity and degradation of both wild-type and abnormal PC. In addition, structual analysis of the novel identified mutation was performed to elucidate the possible molecular mechanism.

2014 Royal College of Pathologists of Australasia

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HEREDITARY PC DEFICIENCY CAUSED BY COMPOUND HETEROZYGOUS MUTANTS

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PROC genetic analysis

MATERIALS AND METHODS Clinical cases Two pedigrees with inherited PC deficiency were studied. Proband 1, a 28-yearold male, was admitted for severe deep vein thrombosis (DVT) and mesenteric vein thrombosis that had been controlled by the application of both warfarin and heparin for only one week. He had a history of DVT and pulmonary embolism. Proband 2, a 19-year-old male, was diagnosed as DVT in both legs since 16 years of age. No thromboembolic episodes were noted in his family. The parents of both probands were non-consanguineous. The pedigrees of the two families are shown in Fig. 1.

For the probands, DNA fragments spanning all the exons including the exonintron boundaries of PROC were amplified by polymerase chain reaction (PCR) as reported previously.15 PCR products were then directly sequenced with a 3700 DNA Analyzer (Applied Biosystems, USA). Only corresponding PCR products in which the probands’ mutations were identified were amplified and sequenced for other family members. Assay of the Arg506Gln Leiden mutation in the coagulation factor V gene and the G20210A substitution in the prothrombin gene was performed as described.16 Nucleotide variations were identified by comparison with the published sequence by Foster et al.3 Construction of expression vectors and site-directed mutagenesis

Blood samples This investigation was approved by the Ethics Committee of Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China. Written informed consent was obtained from every participant. The blood samples were drawn for coagulation assays from the two probands and their family members without the administration of warfarin for more than 2 weeks. Venous blood samples were gently collected into tubes with 0.109 M sodium citrate in a mixture of nine parts blood to one anticoagulant (9:1 v/v). The platelet-poor plasma was prepared with two rounds of centrifugation at 2500  g for 20 min at 48C. Genomic DNA was extracted from the leukocyte fraction according to the standard phenol-chloroform protocol. Aliquots of plasma for functional analyses were stored at 808C before analysis.14

Coagulation assays Plasma PC antigen (PC:Ag) was determined by a sandwich enzyme-linked immunosorbent assay (ELISA). Rabbit anti-human PC polyclonal antibody (Dako, Denmark) was used as the capture antibody and horseradish peroxidise-conjugated rabbit anti-human PC antibody (Dako) as the detection antibody. PC activity (PC:A) was determined using the chromogenic end point assay (Dade Behring, Germany) and detected with a Sysmex CA 7000 coagulometer (Sysmex, Japan) according to the manufacturer’s instructions. Plasma acquired from 40 healthy individuals (20 males aged 22–48 years and 20 females aged 23–50 years) were pooled and used as a control plasma for screening tests. The relative levels of PC:A and PC:Ag compared to the values of that in pooled normal plasma were recorded.

D255H heterozygote I1

NA D255H and R178W heterozygote

A

II 1

The PC expression vector, pcDNA3.1(-)/PC wild type (PC wt) was constructed as described.16 PC wt was subjected to site-directed mutagenesis with QuikChange XL Site-Directed Mutagenesis kit (Stratagene, USA) for construction of pcDNA3.1(-)/PC L-34P, pcDNA3.1(-)/PC R178W, pcDNA3.1(-)/PC D255H and pcDNA3.1(-)/PC T295I. The plasmids were sequenced to confirm the presence of the mutations and to exclude PCR-induced errors. Cell culture, transfection and phenotype assays COS-7 cells were cultured in Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/ mL penicillin G and 100 mg/mL streptomycin in a 5% CO2 atmosphere at 378C. Transient expressions were performed with 1.5 mg of each construct (PC wt, PC L-34P, PC R178W, PC D255H and PC T295I) using PolyFect transfection reagent (Qiagen, Germany) in six-well plates as described.17 After 16 h, the culture medium was replaced with fresh serum-free medium supplemented with 10 mg/mL vitamin K1. Cells were cultured for another 24 h before the culture media were collected in pre-chilled tubes containing the protease inhibitor cocktail (Sigma, USA). Cell debris was removed by centrifugation. Proteins were extracted with M-PER mammalian protein extraction reagent (Pierce, USA) and with the presence of protease inhibitor cocktail. Media and cell lysates were measured for PC:Ag by ELISA. Four independent transfection experiments were performed. The pcDNA3.1(-) plasmid was used as a control. A luciferase expression vector pRL-SV40 (Promega, USA) was cotransfected to calibrate the variation due to transfection efficiency. Cellular localisation of wild type PC and mutant PC To examine the subcellular localisation of wild type PC and mutant PC, immunofluorescence was performed as described before.18 Briefly, transfected cells were cultured overnight in DMEM on poly-L-lysine coated cover slides, washed three times with 1x PBS, and then fixed in 4% paraformaldehyde. The cells were blocked and then incubated with rabbit anti-human Protein C polyclonal antibody followed by FITC-conjugated goat anti-rabbit IgG antibody (Dako). The cells were washed three times and then incubated with either endoplasmic reticulum (ER) marker Concanavalin A (Con A) (1 mg/mL) or Golgi marker wheat germ agglutinin (WGA) (1 mg/mL) (Molecular Probes, USA). The staining was analysed using LSM5 immunofluorescence confocal microscope (Zeiss, Germany). Structural analysis of mutant PC

I1

II 1

II 2

I2

II 3

I3

The structural analysis was conducted using the crystal structure of human Activated Protein C (APC, Protein Data Bank ID: 3F6U). Structural figures were generated with PyMOL (http://www.pymol.org; DeLano Scientific, USA).

RESULTS

II 4

Coagulation laboratory data

B

Normal male

T2951 heterozygote

Dead female

L-34P heterozygous

T295I and L-34P heterozygote

Fig. 1 Pedigrees of the two independent families. The black arrows indicate the probands. (A) Family pedigree of Proband 1. (B) Family pedigree of Proband 2. NA, not available.

Proband 1 demonstrated concordant reduction in both PC:Ag and PC:A. Corresponding values of his father were at the lower limit within the normal range. Similarly, Proband 2 showed drastically decreased PC:Ag and PC:A to only 20% of normal values. Meanwhile, some of his family members had decreased PC:Ag and PC:A without any symptoms. Other anticoagulation proteins such as protein S and antithrombin of these two pedigrees were normal (data not shown). The coagulation data of the two pedigrees was summarised in Table 1.

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632 Table 1

WU et al.

Pathology (2014), 46(7), December

Phenotypes and genotypes of the two families with PC deficiency Phenotype

Family 1 I1 II 1* Family 2 I1 I2 I3 II 1 II 2 II 3 II 4 III 1{ Normal range

Genotype

Age

APTT (s)

PT (s)

Fg (g/L)

PC:A (%)

PC:Ag (%)

Clinical symptoms

54 28

32.1 33.8

11.9 12.4

3.10 3.21

74 21

72 18

Asymptomatic VTE

70 73 68 48 45 43 42 16

43.7 36.0 31.9 34.9 33.6 30.4 29.5 32.6 25  45

12 12.2 11.2 12.1 12.4 10.9 10.6 11.5 10  16

5.75 2.58 4.13 2.86 2.07 2.01 3.22 4.07 24

39 60 48 74 44 43 55 21 70  140

36 50 36 86 33 34 34 20 70  130

Asymptomatic Asymptomatic Asymptomatic Asymptomatic Asymptomatic Asymptomatic Asymptomatic VTE Asymptomatic

Substitution of amino acids Arg178Trp – het Leu-34Pro – – het – – – het het –

Asp255His het het Thr295Ile het – – – het het – het –

*

Proband 1. Proband 2. APTT, activated partial thromboplastin time; Fg, fibrinogen; het, heterozygous mutation; PC, protein C; PC:A, protein C activity; PC:Ag, protein C antigen; PT, prothrombin time; s, seconds; VTE, venous thromboembolic disease.

{

PROC genotype analysis DNA sequencing of PROC identified compound heterozygous substitutions in the probands: 6245C>T and 8478G>C in Proband 1, and 26T>C and 8599C>T in Proband 2 (Fig. 2). The numbering system for PC is in accordance with Foster et al.3 The genetic mutations result in amino acid substitutions of Arg178Trp and Asp255His, Leu-34Pro and Thr295Ile in mature PC protein. The Asp255His substitution was only detected in the father (I1) of Proband 1 because samples from other family members were not available. The Thr295Ile substitution was also identified in the asymptomatic father of Proband 2 (II3) and in the paternal relatives II2 and III1. The Leu-34Pro substitution was identified in the asymptomatic mother (II4) and grandmother (I3) of Proband 2 (Table 1). Neither nucleotide variation was polymorphism after searching in the SNP databases (NCBI Entrez SNP database). Expression and analysis of the PC wild type and mutants In order to further study the relationship between genotype and molecular mechanism of PC deficiency observed in the probands, we transfected COS-7 cells with pcDNA3.1(-) vectors containing either wild type or mutated PC cDNA sequence. After being normalised to PC wt, ELISA results of cell lysates and media are shown in Table 2. PC mutants were normalised to PC wt whereas PC wt was set as 100%. PC mutants were expressed as the percentage of the PC (meanSD) produced by the PC wt. Immunofluorescence analysis of the PC wild type and PC Thr295Ile To investigate the subcellular localisation of wild type PC and the mutant PC protein in cells, immunofluorescence co-localisation assays were performed on transfected COS-7 cells expressing wild type PC and the novel identified mutant PC (Thr295Ile) using two-colour confocal microscopy. As shown in Fig. 3, the merged image (yellow colour) indicated the extensive presence of wild type PC in ER. Meanwhile, the fluorescent signal of wild type PC in the Golgi apparatus was weaker than that in ER. The mock transfected cells (insets in Fig. 3A) served as negative controls, demonstrating the absence

of PC and with the presence of ER and Golgi organelles, respectively. The PC with Thr295Ile mutant presented mostly in the ER with a very small amount in the Golgi apparatus (Fig. 3B), suggesting that only a trace amount of mutant PC was secreted. Crystal structure of mutant PC As demonstrated in the crystal structure (Fig. 4), the mutation of Thr295Ile localised on the strand of b-barrel in the C-terminal. The substitution of Thr by Ile with a bulkier aliphatic side chain may cause severe steric clashes in tightly packed PC, and the hydrogen bond between the hydroxyl of Thr295 side chain and the Ile319 in the main chain was disrupted as well.

DISCUSSION We characterised two compound PROC nucleotide variations in two independent Chinese probands who suffered severe VTE. Arg178Trp (6245C>T) and Asp255His (8478G>C) were found in Proband 1 who had severe thrombotic and related clinical symptoms. Following further investigation of his family members, we identified the same PROC mutation, Asp255His, in his father. His mother also suffered VTE. We did not get the mother’s blood sample because she died of cancer. In Proband 2, we also detected two nucleotide variations: Leu-34Pro (26T>C) and Thr295Ile (8599C>T) in Exon 2 and Exon 9, respectively. The proband showed a significant defect in PC anticoagulant activity. No other family members manifested any obvious symptom of thrombotic disorder. In addition to the mutations, three polymorphisms in the PROC promoter 5’ untranslated region at nucleotides -1654, -1641 and -1476 were also analysed.19 Both patients were found to be heterozygous for these promoter polymorphism haplotypes, C/G/T or T/A/A. The mean PC activity level of individuals with a homozygous C/G/T genotype is around 22% less than individuals with a homozygous T/A/A genotype.20 However, for the compound heterozygous individuals with severe PC deficiency reported in our study, there was no obvious relationship between phenotype and genotype. This was probably because heterozygous C/G/T genotype affected the PC activity levels much less than the homozygote did.

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HEREDITARY PC DEFICIENCY CAUSED BY COMPOUND HETEROZYGOUS MUTANTS A A

G

A

T

G

A

C

C

A

G

G

N

G

G

G

G

A

G

A C

A

G

C

Table 2 cells

PC:Ag in conditioned media and cell lysates of transfected COS-7

Plasmids A A

G

G

A

A

T

G

A

C

C

A

C

C

A

C

A

G

G

C

G

G

G

G

A G

A

C

A

C

A

T

G

PC wild type (wt) PC Leu-34Pro (L-34P) PC Thr295Ile (T295I) PC Arg178Trp (R178W) PC Asp255His (D255H) pcDNA3.1(-) Mock

C

6245C>T Arg178Trp A

G

A

C

N

C

A

A

C

A

A

G

C

633

Media (%)

Cell lysates (%)

100 6.99  1.83 34.00  6.49 7.62  1.53 57.33  15.85 0 0

100 8.72  6.87 61.57  23.63 44.38  13.39 38.05  15.72 0 0

G

PC, protein C; PC:Ag, protein C antigen.

A

A

G

G

A

C

C

C

A

B

C

A

G

C

A

A

T

G

A

C

A

T

C

G

G

G

8478G>C Asp255His T

A

C

C

A

C

A

A

C

A

A

C

G

G

C

C

C

C

T

C

C

T

C

C

T

T

G

C

N

G

T

T

C

G

T

G

C

T

G

T

T

C

G

T

G

G

C

N

C

C

T

C

G

T

G

A

C

C

26 T>C Leu-34Phe G

G

D

C

G

G

C

C

C

C

G

G

G

G

C

C

C

C

A

A

G

G

G

G

A

A

G

G

A

A C

C

C

T

C

G

T

G

A

C

8599 C>T Thr2951le

Fig. 2 Identification of the mutations in PROC of the probands by direct DNA sequencing. (A,B) Proband 1; (C,D) Proband 2. The position of mutated base is indicated with an arrow compared to normal sequence in the lower panel.

There was one novel mutation among the four single nucleotide mutations detected in this study. The Thr295Ile substitution was associated with type I PC deficiency showing parallel reduction of PC activity and antigen levels probably, and this might be caused by reduced synthesis, secretory activity or stability of the protein. Thr295 was located in the first catalytic domain of PC, which is not conserved in the other human vitamin-K-dependent serine proteases of coagulation. The substitution at residue Thr295 has not been reported previously, whereas the missense mutations in adjacent residue Val297Met and Thr298Lys or Met have been found in clinically symptomatic individuals with type I PC deficiency.21 Inspection of the APC structure, Thr295 is an internal residue, localised on a strand of the C-terminal b-barrel (Fig. 4). On closer inspection of the mutation site, it is clear that Thr295 demonstrates numerous close contacts with its surrounding residues Gln293, Val297, Ile319, Val321, Met358 and

Val369, which are located on the antiparallel b-sheet. Although the crystal structure shows that Thr295 is fully buried and far away from the glycosylation sites of PC (labelled by green balls in Fig. 4), the replacement of Thr295 by a bulkier aliphatic side chain of Ile would lead to serious steric clashes in such a tightly packed interior space of the protein. Additionally, the substitution disrupts a hydrogen bond between the hydroxyl of Thr295 side chain and main-chain atom from residue Ile319. Therefore, it is not surprising that replacement by Ile at position 295 might severely impair the intracellular folding and subsequent processes of secretion, resulting in the final low circulating level of PC due to impairing the local structural integrity and causing folding destabilisation of b-barrel. The other three missense mutations have been reported previously. Leu-34 is part of the signal sequence of protein C and necessary for transportation of the newly-synthesised peptide chain into the endoplasmic reticulum. The conformation of signal peptide and transportation of the polypeptide chain could be affected by the helix-breaking effect even though there was no change in the hydrophobicity of peptide when Leu was replaced by Pro.21 Three mutations at Arg178 have been reported so far, all resulting in type I deficiency (R178Q;22 R178P;23 R178W22). Arg178 was found at the surface of the PC molecule with positive charge interacts with the nearby Glu232, whose side chains were supposed to consist the calcium-binding loop in protein C. The calcium-binding loop would potentially misfold if the Arg178 side chains were not available to interact with Glu232.24 Loss of the ion pair would occur in the R178W mutant, which in addition had the unfavourable exposure of the new Trp to the solvent. Site-directed mutagenesis and expression studies could help to establish whether these mutations were detrimental. Our data provided strong evidence showing the impairment of the secretory processes of the PC mutants. The secretory activity of these mutated proteins reduced to 6.99%, 7.62%, 34.00% and 57.33% of wild type recombinant protein C, respectively. In transfected cells, synthetic amounts of PC mutants Thr295Ile, Arg178Trp and Asp255His were 61.57%, 44.38% and 38.05% of the wild type respectively (Table 2). Only 8.72% was produced in PC Leu-34Pro transfected cells (Table 2). Immunofluorescence assay showed that mutated PC Thr295Ile was located mainly in the pre-Golgi compartment, while wild type PC was mainly in the rough ER and secreted rapidly after modification at the Golgi apparatus.25 These data suggest that the mutation could affect the post-translational transportation through intracellular secretory compartments. In summary, four heterozygous mutations, Leu-34Pro, Thr295Ile, Arg178Trp and Asp255His in PROC, were identified in two independent Chinese families with venous

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634

WU et al.

Pathology (2014), 46(7), December

Wild type PC cells and mock cells (insets) Anti-PC

Golgi (anti-WGA)

Merge

Anti-PC

ER (anti-Con A)

Merge

A Thr2951le PC cells Anti-PC

Golgi (anti-WGA)

Merge

Anti-PC

ER (anti-Con A)

Merge

B Fig. 3 Co-localisation of PC wild type and the mutant in the ER and Golgi apparatus in the transfected COS-7 cells. Transfected cells expressing wild type and mutant PC are labelled with an anti-PC antibody (green) and either ER marker (Con A) or Golgi marker (WGA) (red). (A) Co-localisation of wild type PC with ER and the Golgi apparatus is indicated by the yellow colour in merged images. Insets (smaller figures): Mock transfected cells without PC, but with ER and Golgi apparatus. (B) Thr295Ile mutant co-localised with ER, but there was only a faint signal of co-localisation with the Golgi apparatus, demonstrating that only a trace amount of Thr295Ile PC was transferred from ER to the Golgi apparatus.

1319 V321 V297 T295 M358 Q293 V369

Fig. 4 Three-dimensional model of human Activated Protein C (APC). It highlights the mutation T295 (Thr295) identified in the current investigation (the mutated position is depicted in the red sphere, its spatial adjacent residues are in blue, the glycosylation sites are indicated by green balls). The light chain and heavy chain of protein C are represented as ribbon diagrams in colours magenta and cyan, respectively. On closer inspection (top right corner) T295 shows numerous close contacts with residues Q293, V297, I319, V321, M358 and V369. Hydrogen bond between T295 and I319 is indicated by yellow dashed lines.

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HEREDITARY PC DEFICIENCY CAUSED BY COMPOUND HETEROZYGOUS MUTANTS

thrombosis symptoms. Our data indicated that most of the mutant PC molecules were retained within the cells because their transportation was retarded in the intracellular secretory pathways. Thus, inherited protein C deficiency with these four mutations should be primarily caused by the impairment of protein C secretory activity from cells to plasma. Acknowledgements: We thank all members of these families for their participation in this study. Conflicts of interest and sources of funding: We thank the National Natural Science Foundation of China for the funding support (81170480, 81370621, 81201539). The authors state that there are no conflicts of interest to disclose. Address for correspondence: Professor X-F. Wang, Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, No.197, Rui Jin Er Road, Shanghai 200025, China. E-mail: wangxue [email protected]. Professor H-F. Chen, Department of Laboratory Medicine, Shanghai First Maternity and Infant Hospital, Tongji University School of Medicine, No.536, Chang Le Road, Shanghai 200040, China. E-mail: weedeng @sina.com

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Hereditary protein C deficiency caused by compound heterozygous mutants in two independent Chinese families.

We report two compound heterozygous mutants that caused severe type I protein C (PC) deficiency in two independent Chinese families.PC antigen was det...
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